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Skeletal, cardiac, and respiratory muscle function and histopathology in the P448Lneo− mouse model of FKRP-deficient muscular dystrophy

Skeletal, cardiac, and respiratory muscle function and histopathology in the P448Lneo− mouse... Background: Fukutin-related protein (FKRP) mutations are the most common cause of dystroglycanopathies known to cause both limb girdle and congenital muscular dystrophy. The P448Lneo− mouse model has a knock-in mutation in the FKRP gene and develops skeletal, respiratory, and cardiac muscle disease. Methods: We studied the natural history of the P448Lneo− mouse model over 9 months and the effects of twice weekly treadmill running. Forelimb and hindlimb grip strength (Columbus Instruments) and overall activity (Omnitech Electronics) assessed skeletal muscle function. Echocardiography was performed using VisualSonics Vevo 770 (FujiFilm VisualSonics). Plethysmography was performed using whole body system (ADInstruments). Histological evaluations included quantification of inflammation, fibrosis, central nucleation, and fiber size variation. Results: P448Lneo− mice had significantly increased normalized tissue weights compared to controls at 9 months of age for the heart, gastrocnemius, soleus, tibialis anterior, quadriceps, and triceps. There were no significant differences seen in forelimb or hindlimb grip strength or activity monitoring in P448Lneo− mice with or without exercise compared to controls. Skeletal muscles demonstrated increased inflammation, fibrosis, central nucleation, and variation in fiber size compared to controls (p < 0.05) and worsened with exercise. Plethysmography showed significant differences in respiratory rates and decreased tidal and minute volumes in P448Lneo− mice (p < 0.01). There was increased fibrosis in the diaphragm compared to controls (p < 0.01). Echocardiography demonstrated decreased systolic function in 9-month- old mutant mice (p < 0.01). There was increased myocardial wall thickness and mass (p < 0.001) with increased fibrosis in 9-month-old P448Lneo− mice compared to controls (p < 0.05). mRNA expression for natriuretic peptide type A (Nppa) was significantly increased in P448Lneo− mice compared to controls at 6 months (p < 0.05) and for natriuretic peptide type B (Nppb) at 6 and 9 months of age (p <0.05). Conclusions: FKRP-deficient P448Lneo− mice demonstrate significant deficits in cardiac and respiratory functions compared to control mice, and this is associated with increased inflammation and fibrosis. This study provides new functional outcome measures for preclinical trials of FKRP-related muscular dystrophies. Keywords: Limb-girdle muscular dystrophy, Congenital muscular dystrophy, Fukutin related protein (FKRP), P448Lneo− mice, Echocardiography, Plethysmography, Preclinical trials * Correspondence: cspurney@childrensnational.org Children’s National Heart Institute, Center for Genetic Medicine Research, Children’s National Health System, Washington, DC, USA Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Yu et al. Skeletal Muscle (2018) 8:13 Page 2 of 16 Tyr307Asn Background Neo that demonstrated reduced levels of Muscular dystrophies are a heterogeneous group of FKRP transcript. However, the mutant mice died soon disorders characterized by progressive muscle weak- after birth and were therefore not useful for ness and can also affect the respiratory, cardiac, and experimental therapy development [13]. Mouse central nervous systems. The clinical phenotype and models with the common mutation L276I were prognosis vary significantly making the diagnosis and created by several groups [14–16]. However, treatment different for each disease. However, the dystrophic phenotype of the mutant mice is very mild continued identification of specific genetic causes for with clearly observable pathology only after 6 months the more common muscular dystrophies has led to a of age without significant involvement of respiratory better understanding of disease pathogenesis and new and cardiac muscles, apparently milder than therapeutic strategies [1]. phenotype in patients with the same homozygous One pathogenic mechanism of muscular dystrophies mutations. Another reported FKRP mutant mouse lies in the disruption of the dystrophin-glycoprotein model contains P448L mutation associated with complex (DGC) [2]. The DGC is responsible for linking congenital muscular dystrophy type 1C (MDC1C) in the sarcolemmal membrane with the extracellular matrix clinic [8, 17]. The knock-in FKRP P448L (with neo (ECM) and transmitting contraction forces to maintain cassette removed, referred as P448Lneo− mouse) muscle cell membrane integrity [3]. The DGC is com- homozygous mouse has been reported with a severe posed of multiple proteins including dystrophin, dystro- phenotype consistent with severe LGMD2I, but milder glycans, multiple sarcoglycans, dystrobrevin, laminin, than MDC1C as almost all newborn mice survive and and collagens. Without even one of these proteins, have a life-span of more than 1 year with near nor- membranes can tear and activate multiple pathogenic mal breeding capacity [18]. Also important, the pathways that lead to cell death. mouse was reported to show involvement of respira- Alpha-dystroglycan (α-DG) is one component of the tory and cardiac muscles with progressive fibrosis DCG and interacts with proteins in the ECM includ- [16]. Blaeser et al. (2016) examined the P448Lneo− ing laminin, perlecan, agrin, neurexin using glycosyl- mouse and demonstrated increased diaphragmatic fi- ated O-mannose sugar moieties [4]. Defective brosis and decreased cardiac function by 12 months glycosylation of α-DG is the pathogenic basis of sev- of age [19]. This pattern of phenotype represents well eral muscular dystrophy subtypes known as dystrogly- the clinic manifestation of dystroglycanopathies, as a canopathies, including limb-girdle muscular dystrophy proportion of the patient population is associated (LGMD) and congenital muscular dystrophy (CMD). with pulmonary and cardiac disease. Clinically, Pane These subtypes demonstrate heterogeneous pheno- et al. (2012) described cardiac involvement in 6% and types that can range from early presentations with pulmonary involvement in 12% of patients with con- severe eye and brain disease to more mild skeletal genital muscular dystrophies [20]. And significant car- muscle diseasein older patients.Morethan17genes diac disease can be seen in LGMD 2I, even leading to are involved in the pathogenesis including POMT1, cardiac transplantation [21–23]. More recently, a POMT2, POMGnT, FKRP, Fukutin,and LARGE acting study by Maricelli et al. also demonstrated cardiac as glycosyl-transferases in the O-mannosylation of α- dysfunction with and without exercise [24]. We con- DG [5]. The severity of disease is thought to be re- sider the P448Lneo− mouse highly relevant and valu- lated to the effect of each mutation on degree of able for developing experimental therapies to FKRP glycosylation and laminin binding ability [6]. FKRP is dystroglycanopathy. Therefore, validation of the skel- a gene that encodes fukutin-related protein and its etal muscle phenotype and further characterization of mutations cause dystroglycanopathies of both LGMD respiratory and cardiac muscle are essential. and CMD phenotypes as well as muscle-eye-brain and Walker-Warburg syndrome [7–11]. FKRP has recently Methods been demonstrated as a ribitol 5-phosphate transfer- Animal care ase in the synthesis pathway of laminin binding gly- This study was carried out in strict accordance with can of α-DG [12]. the recommendations in the Guide for the Care and Multiple mouse models were developed to study the Use of Laboratory Animals of the National Institutes role of FKRP and experimental therapies. These of Health. All experiments were performed in accord- models show a range of phenotypes consistent with ance with Children’s National Health System IACUC human FKRP diseases. In general, the severity of the approved protocol #30432. P448Lneo− homozygous reported mouse models follows the same trend as the male mice were generated in McColl Lockwood severity observed in patients with the same mutations. Laboratory (Charlotte, NC) and rederived and Ackroyd et al. (2009) developed the model FKRP- imported from Jackson Laboratory (Bar Harbor, ME) Yu et al. Skeletal Muscle (2018) 8:13 Page 3 of 16 Table 1 Timeline of experimental procedures performed on groups A, B, C, D of P448Lneo− and control mice. Groups A, B, and D were composed of P448Lneo− (n = 8) and control (n = 8) mice. Group C was composed of P448Lneo− (n = 8), exercised P448Lneo− (n = 12), and control (n = 8) mice. Group A was studied at 1 month of age. Group B was studied at 2 months of age. Group C was studied every month until age 6 months of age. A group of P448Lneo− mice underwent exercise treadmill running until 6 months of age. Control mice did not undergo exercise testing. Group D was studied until 9 months of age Group A B C D Timeline (months of age) 1 2 1 2 34569 Treadmill exercise xx xxxx (2×/week only P448Lneo− mice) Body weight x x x x xxxxx Grip strength test x x xxxxx Digiscan activity x x xxxxx Echocardiography x x x Plethysmography x x x Serum creatinine kinase x x x x Histology x x x x Fibrosis x x x x [18]. Age-matched male C57BL/6J (referred to as con- pathology becomes detectable. Group B was studied at trol, C57,or BL6) mice were purchased from Jackson 2monthsofage.Group Cwas studiedeverymonth Laboratory. Animals were ear tagged prior to group until 6 months of age. A 1-month interval was chosen assignment and were housed in cages of standard di- with the aim to identify the peak of muscle degener- mension on ground corn cob bedding mixed with a ation and severity as the disease progresses. A separate soft recycled shredded paper (nesting material) called group of P448Lneo− mice underwent exercise tread- Tek Fresh. The animals were housed in a temperature mill running until 6 months of age. Control mice did controlled (20–24 °C) colony room with a 12-h light/ not undergo exercise testing. Group D was studied at dark cycle and received mouse chow and water ad 9 months of age when both histological and functional libitum. No animals were euthanized prior to reaching data have already shown severe and detectable defects. end of study criteria. Table 1 shows the timing of different testing for each group. Experimental procedure Groups A, B, and D were composed of P448Lneo− Treadmill (n = 8) and control (n = 8) mice. Group C was com- The mice were placed on the treadmill (Columbus In- posed of P448Lneo− (n = 8), exercised P448Lneo− struments, Columbus, OH) twice a week, one per lane (n = 12), and control (n = 8) mice. Group A was for 30 min running at 12 m per minute speed per studied at 1 month of age when the skeletal muscle TREAT-NMD SOP for chronic exercise protocol in Table 2 Body, muscle, and organ weights normalized by body weight in P448Lneo− (FKRP) and control (BL6) mice showing significant differences at 6 and 9 months of age Weight 1 month 2 months 6 months 9 months (n =8) BL6 FKRP BL6 FKRP BL6 FKRP BL6 FKRP Body (g) 19 ± 1.2 19.6 ± 0.9 21.9 ± 1.5 22.3 ± 0.83 28 ± 1.7 30 ± 1.2 33 ± 6.0 31 ± 1.7 −6 GAS/BW(10 ) 6.2 ± 0.5 6.3 ± 0.3 6.4 ± 0.4 6.5 ± 0.4 5.4 ± 1 6.0 ± 0.2*** 5.0 ± 0.2 6.3 ± 0.5*** −7 Sol/BW(10 ) 3.7 ± 0.3 3.7 ± 0.2 3.8 ± 0.9 3.6 ± 0.5 3.0 ± 0.4 3.5 ± 0.2* 3.3 ± 0.3 4.1 ± 0.4** −6 TA/BW(10 ) 2.4 ± 0.4 2.3 ± 0.2 2.3 ± 0.1 2.3 ± 0.2 1.8 ± 0.2 2.2 ± 0.2*** 1.4 ± 0.2 2.0 ± 0.3*** −6 Triceps/BW(10 ) 4.1 ± 0.4 4.4 ± 0.4 4.4 ± 0.6 4.3 ± 0.4 3.4 ± 0.6 4.5 ± 0.3*** 3.3 ± 0.6 5.4 ± 0.9*** −6 Quad/BW(10 ) 7.0 ± 0.2 7.3 ± 1.1 5.7 ± 0.8 6.0 ± 0.8 5.3 ± 0.8 6.5 ± 0.9* 5.0 ± 0.9 6.3 ± 0.4** −6 Heart/BW(10 ) 5.0 ± 0.2 5.3 ± 0.5 5.1 ± 0.2 5.2 ± 0.4 4.3 ± 0.3 4.4 ± 0.2 3.9 ± 0.5 4.4 ± 0.4* −6 Brain/BW(10 ) 21 ± 1.2 20 ± 3.6 20 ± 1.4 19 ± 1 16 ± 1.2 15 ± 0.5* 14.5 ± 2.8 14.2 ± 0.1 *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; using t test when compared to BL6 control mice at same age. Data presented as mean ± SD. GAS gastrocnemius, Sol soleus, TA tibialis anterior, Quad quadriceps, BW body weight Yu et al. Skeletal Muscle (2018) 8:13 Page 4 of 16 Table 3 Body, muscle, and organ weights normalized by body weights among groups of P448Lneo− (FKRP) without and with treadmill exercise (FKRP-treadmill) and control (BL6) mice at 6 months of age Measurement BL6 control FKRP FKRP-treadmill Significantly different groups (adjusted p values using ANOVA N Mean ± SD N Mean ± SD N Mean ± SD followed by post hoc analysis) Body (g) 8 28 ± 1.7 8 30 ± 1.2 12 29 ± 1.8 NS −6 GAS/BW(10 ) 8 5.4 ± 0.3 8 6.0 ± 0.2 12 6.4 ± 0.7 BL6 vs FKRP: p < 0.05, BL6 vs FKRP-treadmill: p < 0.001 −7 Sol/BW(10 ) 8 3.0 ± 0.4 8 3.5 ± 0.2 12 3.4 ± 0.3 BL6 vs FKRP: p < 0.05, BL6 vs FKRP-treadmill: p < 0.05. −6 TA/BW(10 ) 8 1.8 ± 0.2 8 2.2 ± 0.2 12 2.4 ± 0.3 BL6 vs FKRP: p < 0.01, BL6 vs FKRP-treadmill: p < 0.001 −6 Triceps/BW(10 ) 8 3.4 ± 0.6 8 4.5 ± 0.3 12 5.2 ± 0.7 BL6 vs FKRP: p < 0.01, BL6 vs FKRP-treadmill: p < 0.0001 FKRP vs FKRP-treadmill: p < 0.05 −6 Quad/BW(10 ) 8 5.3 ± 0.8 8 6.5 ± 0.9 12 7.0 ± 1.3 BL6 vs FKRP: p < 0.05, BL6 vs FKRP-treadmill: p < 0.01 −6 Heart/BW(10 ) 8 4.3 ± 0.3 8 4.4 ± 0.2 12 4.4 ± 0.5 NS −6 Brain/BW(10 ) 8 16 ± 1.2 8 15 ± 0.5 12 16 ± 1.2 NS GAS gastrocnemius, Sol soleus, TA tibialis anterior, Quad quadriceps, BW body weight, NS not significant dystrophic mice (http://www.treat-nmd.eu/research/pre- of KGF (kilogram-force) and normalized to bodyweights clinical/dmd-sops/). If a mouse rested at the end of the as “KGF/kg.” lane, the animal would be gently pushed back onto the treadmill surface to restart running. The treadmill tests Locomotor activity started on mice at approximately 1 month of age and Locomotor activity was measured using an open-field continued until 6 months of age. During the weeks of digiscan apparatus (Omnitech Electronics, Columbus, measurements including grip strength, activity monitor, OH). Total distance, horizontal activity, and vertical ac- echo, and plethysmography, treadmill running was tivity were recorded every 10 min for 1 h as described avoided. previously [26, 27]. As with the grip strength, the activity data were collected in the morning hours over a 4-day Grip strength period and the mice were trained in the open field ap- Forelimb grip strength was measured by a grip strength paratus prior to the trial [25]. meter (Columbus Instruments, Columbus, OH). The animal was held so that only the forelimb paws grasped Echocardiography the specially designed mouse flat mesh assembly and the Echocardiography was performed and quantitative mea- mouse was pulled back until their grip was broken. The surements were made offline using analytic software force transducer retained the peak force reached when (FujiFilm VisualSonics, Toronto, Ontario, Canada) as the animal’s grip was broken, and this was recorded previously described [25]. Measurements included vessel from a digital display. For hindlimb strength, an angled diameters, ventricular chamber size, and blood flow vel- mesh assembly was used. Mice were allowed to rest on ocities and timing across the atrioventricular and semi- the angled mesh assembly, facing away from the meter lunar valves. M-mode images were used to measure left with its hindlimbs at least one-half of the way down the ventricular (LV) chamber sizes and wall thicknesses. Per- length of the mesh. The mouse tail was pulled directly cent shortening fraction (SF) and ejection fraction (EF) toward the meter and parallel to the mesh assembly. were calculated from M-mode measurements. Myocar- During this procedure, the mice resist by grasping the dial performance index (MPI) was also calculated from mesh with all four limbs. Pulling toward the meter was Doppler measurements. continued until the hindlimbs released from the mesh assembly. Five successful hindlimb and forelimb strength Plethysmography measurements within 2 min were recorded. The max- The whole body plethysmography system (ADInstru- imum values were used for analysis. The grip strength ments, St. Paul, MN) utilized a custom mouse cham- measurements were collected in the morning hours over ber developed by the Research Instrument Shop at a 5-day period. The mice were trained on the grip theUniversityofPennsylvaniatominimizedead strength meter before the trial [25]. Forelimb and hind- space. Other components in the system included the limb maximal muscle strength were obtained as values spirometer (ML141), respiratory flow head (MLTL1), Yu et al. Skeletal Muscle (2018) 8:13 Page 5 of 16 Fig. 1 Normalized grip strengths for P448Lneo− and control mice. Panel a shows no significant differences in normalized forelimb grip strength (kilogram force per kilogram; KGF/kg) at 9 months of age in P448Lneo− (FKRP) and control (BL6) mice. Panel b shows no significant differences in normalized hindlimb grip strength (kilogram force per kilogram; KGF/kg) at 9 months of age in FKRP and control mice. Panel c shows normalized forelimb and panel d shows normalized hindlimb grip strengths from 1 to 9 months of age for FKRP mice, exercised FKRP mice (FKRP-treadmill; 1–6 months only), and control mice with no significant differences and the PowerLab 4/30 with LabChart software. The was recorded for 10 min. For data analysis, values for mouse was brought to the measurement room respiratory rate, tidal volume (TV), minute ventilation 15 min before the start of the measurement session (MV), TV normalized by body weight (TV/BW), and to recover from the transportation and new environ- MV normalized by body weight (MV/BW) were re- ment stresses. The spirometer was calibrated every corded using LabChart software. time the hardware was powered on to read in terms of flow (ml/s) rather than pressure (mv).Calibration of Blood collection the plethysmography was performed with 1 ml of air Blood samples were taken via retro-orbital bleeding injected into the animal chamber to correlate the when the animals were euthanized and the serum col- injected volume (ml) with the differential pressure lected was used for creatinine kinase levels. (mv) measured in the chamber by integration. A 700 ml/min flow of dry air through the chambers was Tissue collection and histological evaluations constantly delivered to avoid CO and water accumu- Animals were sacrificed via inhaled carbon dioxide and lation and to maintain a constant temperature. The cervical dislocation, and tissue samples were obtained. All mouse was weighed and placed into the chamber first tissue samples were weighed using the Mettler ToLedo to acclimate for 15 min then the respiratory flow data scale (Columbus, OH) prior to processing. Skeletal Yu et al. Skeletal Muscle (2018) 8:13 Page 6 of 16 Fig. 2 Inflammation levels (foci/mm2) in P448Lneo− (FKRP) mice gastrocnemius (panel a), quadriceps (panel b), and triceps (panel c)at 1, 2, 6, and 9 months of age compared to controls (BL6) and serum creatinine kinase (CK) at 9 months of age (panel d). Significant increases in inflammation are seen with a peak at 2 months of age. Serum CK levels are significantly increased at 1, 6, and 9 months. Data presented as mean ± standard deviation;*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 using t test when compared to BL6 control mice at same age; ###p < 0.001 using two-tailed Mann-Whitney nonparametric test when compared to BL6 control mice at same age muscles (gastrocnemius, tibialis anterior, soleus, triceps, Inc. (Germantown, MD). The tissues were magnified and quadriceps) from one side of the animal, half the dia- under a light microscope at an objective of 10 x and phragm, half the heart, and whole brain were stored in digital images obtained using computer software (Olym- formalin. The contralateral or other half of muscles were pus C.A.S.T. Stereology System, Olympus America Inc., snapped frozen in isopentane cooled in liquid nitrogen Center Valley, PA). These digital images were processed and stored at − 80 °C for further analysis. Slides were pre- using ImageJ (NIH) with additional threshold color pared and stained by Histoserv Inc. (Gaithersburg, MD). plug-ins to process jpeg images. Pixels corresponding to Histological evaluations were performed in a blinded the area stained in red were normalized to the total pixel manner using coded slides. One transverse tissue section area of the tissue image, and the results were expressed per muscle per animal was analyzed. Whole muscle digital as percent of fibrotic area. images of the tissues were taken at × 20 using NanoZoo- mer slide scanner (Hamamatsu Inc., Bridgewater, NJ) and mRNA expression analysis were opened using NDP.view2 software. Each tissue sec- Snapped frozen hearts from 2-, 6-, and 9-month-old tion was analyzed throughout its entire area. The total FKRP and BL6 control mice were collected into tubes number of inflammation foci (an interstitial group of 10 with 1 mL of TRIzol and homogenized. Total RNA smaller inflammatory cell dark blue nuclei in a high- was isolated and washed and the RNA yield and pur- power field) was quantified. The entire tissue section area ity was determined using a NanoDrop 2000 microvo- was measured (mm ), and all counts were normalized to lume spectrophotometer (ThermoFisher). cDNA was the tissue area. The parameters including percentage of generated using the high-capacity cDNA reverse tran- fibers with central nucleation and fiber diameter were scription kit (Applied Biosystems, cat #4368813). The measured using MetaMorph Microscopy Automation and cDNA was added to TaqMan universal PCR master Image Analysis Software on paraffin sections of mix (Applied Biosystems, cat #4304437), and the fol- gastrocnemius, triceps, quadriceps, and diaphragm. lowing TaqMan Gene Expression Assays (Applied Bio- systems): Nppa, Nppb, and Fn1. GAPDH was used as Quantification of fibrosis the reference gene. Real-type PCR was performed Paraffin sections of gastrocnemius, diaphragm, and heart using the CFX384 Touch Real-Time PCR Detection tissue were stained with picrosirius red by Histoserv, System and associated software (Bio-Rad). Yu et al. Skeletal Muscle (2018) 8:13 Page 7 of 16 Statistical analysis of age, exercise had no significant effects on normalized Data is presented as mean ± standard deviation (SD). Nor- forelimb or hindlimb grip strength (Fig. 1). mality of each phenotype was tested using both the Shapiro-Wilk normality test and visual inspection of his- Activity monitor tograms except for percent central nucleation and fiber There were no significant differences between BL6 and diameter size as there are only three total samples. All P448Lneo− in horizontal and vertical activity, movement tested phenotypes were normally distributed except in- time, rest time, and total distance. While exercised mice flammation in quadriceps of 1- and 9-month-old BL6,and showed decreased activity compared to unexercised 6months old P448Lneo− excised mice, inflammation in P448Lneo− and BL6 mice, the differences were not sig- gastrocnemius of 2-, 6-, and 9-month-old BL6,and 6- nificant (Additional file 1: Figure S1). month-old P448Lneo− excised mice, and inflammation in Triceps of 2-, 6-, and 9-month-old BL6 mice. For normally Inflammation distributed parameters, comparisons were made among Analysis of the skeletal muscle including the gastrocne- 6 –month-old BL6, P448Lneo−,and P448Lneo− excised mius, quadriceps and triceps showed an increase in in- mice using analysis of the variance (ANOVA) followed by flammatory foci at 1 month of age in P448Lneo− mice Tukey multiple comparison analysis. A single t test was compared to control mice (Figs. 2 and 3). This difference used to compare the BL6 control group to the P448Lneo− increases by 2 to 4 fold to a maximum inflammation at group. RT-PCR data were normalized to the 2-month-old 2 months of age. The maximum amount of inflamma- BL6 control group and are presented as fold change. For tion was noted in the quadriceps muscle. The inflam- abnormally distributed parameters, comparisons were matory infiltrates then decreased at both 6 and made among 6-month-old BL6, P448Lneo−,and 9 months in all 3 muscles. In P448Lneo− mice exer- P448Lneo− excised mice using Kruskal-Wallis test cised until 6 months of age, the inflammatory infil- followed by Dunn’s multiple comparison analysis and a trates increase from 1.8 to 2.2 folds compared to two-tailed Mann-Whitney test was used to compare the unexercised P448Lneo− mice (Table 4; Fig. 3). BL6 control group to the P448Lneo− group. A value of p < 0.05 was considered statistically significant. Fibrosis No significant differences in percent fibrosis in the quad- Results riceps or triceps between P448Lneo− and controls were Body, organ, and muscle weights seen at 1, 2, 6, or 9 months of age (Additional file 2: No significant differences were seen in total body weight Table S1). P448Lneo− exercised mice showed signifi- between BL6 (control) and P448Lneo− mice (Table 2). cantly increased percent fibrosis in the quadriceps mus- There was a significant difference in brain weight nor- cles at 6 months of age compared to unexercised malized to body weight at 6 months of age (p < 0.05). P448Lneo− mice (p < 0.05) and controls (p < 0.001; The P448Lneo− heart showed significantly increased Table 4; Fig. 3). There were no significant differences in mass when normalized to body weight compared to BL6 the gastrocnemius and triceps between the 2 mice at 9 months of age (p < 0.05). The skeletal muscles groups (data not shown). gastrocnemius, soleus, tibialis anterior, quadriceps, and the triceps from mutant mice all demonstrated signifi- Percent central nucleation cantly increased mass normalized to body weight com- Control BL6 mice showed between 0.2 and 1.4% central pared to BL6 at 6 and 9 months of age (Table 2). nucleation in the quadriceps, gastrocnemius, and tri- P448Lneo− mice exercised on the treadmill showed an ceps from 1 to 9 months. P448Lneo− mice showed per- increase in normalized muscle weight compared to con- cent central nucleation of 9.6% at 1 month, 56% at trols for the soleus, tibialis anterior, triceps, and quadri- 6 months, and 60.4% at 9 months (Table 4; ceps muscles (p < 0.05; Table 3). Exercised P448Lneo− Additional file 2: Table S1). The percent central nucle- mice also demonstrated a higher normalized muscle ation in the quadriceps and triceps were increased in 6- weight for the triceps compared to unexercised month-old exercised P448Lneo− mice compared to un- P448Lneo− mice (p < 0.05). exercised mice while the quadriceps decreased slightly (Table 4). There were differences in the variation (SD Skeletal muscle of percent central nucleation for each mouse) of Grip strength P448Lneo− and control mice (Additional file 2:Table No significant differences in normalized forelimb or S1) and P448Lneo− exercised mice showed increased hindlimb grip strength were seen at 9 months of age be- SD of percent central nucleation in the triceps com- tween control and P448Lneo− mice (Fig. 1). At 6 months pared to controls at 6 months of age (Table 4). Yu et al. Skeletal Muscle (2018) 8:13 Page 8 of 16 Fig. 3 Histology images showing inflammation (hematoxylin and eosin staining at 20x) and fibrosis (picrosirius red staining at 10x) in the quadriceps of P448Lneo− (FKRP), exercised P448Lneo− (FKRP-treadmill), and control (BL6) mice. Panels a–d show inflammation in BL6 mice at 1, 2, 6, and 9 months of age. Panels e–h show inflammation in FKRP mice at 1, 2, 6, and 9 months of age. Panel i shows inflammation in FKRP- treadmill mice at 6 months of age. Panels j–l show fibrosis in BL6, FKRP, and FKRP-treadmill at 6 months of age Fiber diameter Respiratory muscle Figure 4 shows the fiber diameters of the quadriceps mus- Plethysmography cles for P448Lneo− mice and controls at 1, 6, and 9 months P448Lneo− mice demonstrated a reduced decline in re- of age. There was no difference in the average fiber size of spiratory rate over time compared to BL6 controls at P448Lneo− and control BL6 mice at 1and 6monthsof age. 9 months of age (p < 0.001; Fig. 5). P448Lneo− mice also At 9 months of age, P448Lneo− mice have smaller average showed significantly decreased tidal volumes (p < 0.001), fiber diameter compared to control BL6 (Additional file 2: normalized tidal volumes (p < 0.01), and minute volumes Table S1). There was a greater variation in fiber sizes (SD (p < 0.001) compared to BL6 controls at 6 and 9 months of fiber size for each mouse) in P448Lneo− mice at 1 and of age. There were significant differences in plethys- 6 months of age compared to control BL6 (Additional file 2: mography measures at 6 months that were improved Table S1). There were no differences in fiber size, but un- in exercised P448Lneo− mice compared to unexer- exercised and exercised P448Lneo− mice showed increased cised P448Lneo− including tidal volume (p < 0.001), SD of fiber size for each mouse compared to controls in minute volume (p < 0.01), and normalized minute volume the quadriceps, gastrocnemius, and triceps (Table 4). (p < 0.01; Fig. 5). Serum creatinine kinase (CK) Inflammation Serum CK was significantly increased in P448Lneo− The diaphragm of P448Lneo− mice showed the most in- mice compared to control BL6 at 1, 6, and 9 months flammatory infiltrates at 1 month of age (p < 0.001; Figs. of age (p <0.05; Fig. 2). At 6 months of age, exer- 6 and 7). The infiltrates decreased but remained signifi- cised P448Lneo− mice showed significantly increased cant compared to controls from 2 to 9 months of age serum CK levels compared to BL6 controls (p < 0.05; (p < 0.0.01). Exercised P448Lneo− mice showed signifi- Table 4). cant inflammation that was increased compared to Yu et al. Skeletal Muscle (2018) 8:13 Page 9 of 16 Table 4 Histological analyses for skeletal muscles and serum creatinine kinase levels among groups of P448Lneo− (FKRP) without and with treadmill exercise (FKRP-treadmill) and control (BL6) mice at 6 months of age Measurement BL6 control FKRP FKRP-treadmill Significantly different groups (adjusted p values using ANOVA N Mean ± SD N Mean ± SD N Mean ± SD followed by post hoc analysis) Inflammation GAS 8 0.03 ± 0.03 8 0.5 ± 0.4 12 0.9 ± 0.3 #BL6 vs FKRP-treadmill: p < 0.0001 (foci/mm ) Quad 8 0.05 ± 0.04 8 0.6 ± 0.2 12 1.1 ± 0.5 #BL6 vs FKRP: p < 0.01, #BL6 vs FKRP-treadmill: p < 0.0001 Triceps 8 0.04 ± 0.06 8 0.6 ± 0.4 12 1.1 ± 0.3 #BL6 vs FKRP: p < 0.01, #BL6 vs FKRP-treadmill: p < 0.0001 % fibrosis Quad 8 0.29 ± 0.07 8 0.41 ± 0.18 12 0.61 ± 0.15 BL6 vs FKRP-treadmill: p < 0.001, FKRP vs FKRP-treadmill: p < 0.05 GAS 3 0.47 ± 0.23 3 46.18 ± 5.4 3 38.44 ± 5.1 NP % central nucleation Quad 3 0.2 ± 0.3 3 56.0 ± 1.9 3 51.1 ± 2.1 NP Triceps 3 1.44 ± 1.11 3 65.71 ± 1.1 3 75.16 ± 8.4 NP GAS 3 34.27 ± 0.7 3 37.9 ± 3.6 3 42.7 ± 2.6 NP Fiber diameter size (μm) Quad 3 48.2 ± 5.5 3 44.8 ± 1.2 3 49.7 ± 1.6 NP Triceps 3 37.75 ± 1.2 3 37.03 ± 1.8 3 43.70 ± 2.0 NP GAS 3 0.5 ± 0.2 3 7.6 ± 5.4 3 10.7 ± 5.1 NP SD of % central nucleation Quad 3 0.4 ± 0.5 3 8.9 ± 3.9 3 12.6 ± 4.4 NP Triceps 3 1.28 ± 1.1 3 15.94 ± 1.1 3 8.17 ± 8.4 NP GAS 3 1.93 ± 0.7 3 8.5 ± 3.6 3 8.73 ± 2.6 NP SD of fiber size Quad 3 11.8 ± 1.3 3 20.5 ± 1.8 3 21.5 ± 1.9 NP Triceps 3 1.93 ± 1.2 3 4.8 ± 1.8 3 6.96 ± 2.0 NP Serum creatinine kinase(μ/l) 8 254 ± 131 7 749 ± 405 12 947 ± 575 BL6 VS. FKRP-treadmill: p < 0.05 Kruskal-Wallis test followed by Dunn’s multiple comparison test used. Statistical measures not performed on measures with N =3. GAS gastrocnemius, Quad quadriceps, SD standard deviation, NP not performed unexercised P448Lneo− mice and BL6 controls at nucleation for each mouse) among P448Lneo− exercised, 6 months of age (p < 0.05; Table 5). unexercised, and control mice (Table 5; Additional file 3: Table S2). Percent central nucleation diaphragm Control BL6 mice showed between 2.3 and 3.7% central Diaphragm fiber diameter nucleation in the diaphragm from 1 to 9 months of age. Figure 8 shows the fiber diameters of the diaphragm 1-month-old P448Lneo− mice showed 4.4% central nu- muscle for P448Lneo− mice and BL6 controls at 1, 6, cleation, which increased to 34% at 9 months old (Add- and 9 months of age. No differences were seen in aver- itional file 3: Table S2). 6-month-old unexercised age fiber size in the mutant mice compared to controls P448Lneo− mice showed 25% central nucleation, and in the diaphragm, but there was greater variation in fiber this increased to 44% in exercised mice (Table 5). There sizes (SD of fiber size for each mouse) in P448Lneo− is no difference in the variation (SD of percent central mice at 1 and 6 months of age compared to controls Fig. 4 Percent number of muscle fiber diameter sizes (μm) in the quadriceps muscle in P448Lneo− and control (C57) mice at 1(panel a), 6 (panel b), and 9 (panel c) months of age. Error bars indicate standard deviation Yu et al. Skeletal Muscle (2018) 8:13 Page 10 of 16 Fig. 5 Plethysmography results in P448Lneo− (FKRP) mice, exercised P448Lneo− mice (FKRP-treadmill), and controls (BL6) at 2, 6, and 9 months of age. Respiratory rates (panel a) are significantly less in control mice compared to FKRP. Tidal volume (panel b) and normalized tidal volume (panel c) are significantly increased in controls. Minute volume (panel d) and normalized minute volume (panel e) show significant changes only at 6 months. FKRP-treadmill mice only measured at 2 and 6 months. ****p < 0.0001 between BL6 and FKRP; ‡‡‡p < 0.001, ‡‡‡‡p < 0.0001, and p < 0.001 between BL6 and FKRP/FKRP-treadmill; ^p < 0.05 among all groups. Data presented as mean ± standard deviation Fig. 6 Significantly increased diaphragm inflammation (panel a) and fibrosis (panel b) are seen in P448Lneo− mice (FKRP) compared to controls (BL6) at 2, 6, and 9 months of age. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 compared to BL6 control at same age. Data presented as mean ± standard deviation Yu et al. Skeletal Muscle (2018) 8:13 Page 11 of 16 Fig. 7 Inflammation (hematoxylin and eosin staining at 20x) and fibrosis (picrosirius red staining at 10x) in diaphragm in P448Lneo− (FKRP) and control (BL6) mice. Panels a–d show inflammation in the diaphragm of BL6 mice at 1, 2, 6, and 9 months of age. Panels e–h show inflammation in the diaphragm of FKRP mice at 1, 2, 6, and 9 months of age. Panels i–l show fibrosis in the diaphragm of BL6 mice at 1, 2, 6, and 9 months of age. Panels m–p shows fibrosis in the diaphragm of FKRP mice at 1, 2, 6, and 9 months of age Table 5 Histological analyses of the diaphragm among groups of P448Lneo− (FKRP) with and without treadmill exercise (FKRP-treadmill) and control (BL6) mice at 6 months of age Measurement BL6 control FKRP FKRP-treadmill Significantly different groups (adjusted p values using ANOVA N Mean ± SD N Mean ± SD N Mean ± SD followed by post hoc analysis) Inflammation (foci/mm ) 8 0.5 ± 0.3 8 4.0 ± 0.8 12 8.7 ± 2.8 BL6 vs FKRP: p < 0.01, BL6 vs FKRP-treadmill: p < 0.0001 FKRP vs FKRP-treadmill: p < 0.0001 % fibrosis 8 2.0 ± 0.7 8 13.7 ± 3.8 12 13.4 ± 2.2 BL6 vs FKRP: p < 0.0001, BL6 vs FKRP-treadmill: p < 0.0001 % central nucleation 3 2.4 ± 0.4 3 24.8 ± 2.0 3 44 ± 9.4 NP Fiber diameter size (μm) 3 24.6 ± 2.6 3 21.3 ± 1.2 3 26.7 ± 2.5 NP SD of % central nucleation for each mouse 3 1.6 ± 1.2 3 4.9 ± 3.3 3 7.7 ± 2.3 NP SD of fiber size for each mouse 3 4.5 ± 0.8 3 6.3 ± 0.2 3 8.1 ± 0.2 NP Statistical analyss not performed on measures with N =3. SD standard deviation, NP not performed Yu et al. Skeletal Muscle (2018) 8:13 Page 12 of 16 Fig. 8 Percent number of muscle fiber diameter sizes (μm) in the diaphragm muscle in P448Lneo− and control (C57) mice at 1(panel a), 6 (panel b), and 9 (panel c) months of age. Error bars indicate standard deviation (Additional file 3: Table S2) and between unexercised Myocardial fibrosis and exercised P448Lneo− mice at 6 months of age There were no significant differences in myocardial fi- (Table 5). brosis at 1, 2, and 6 months of age between P448Lneo− mice compared to controls. There was significantly in- creased myocardial percent fibrosis at 9 months of age Diaphragm fibrosis in P448Lneo− mice (0.69 ± 0.24) compared to controls There were no significant differences in percent fibrosis of (0.38 ± 0.17; p < 0.05; Fig. 9). the diaphragm at 1 month of age; however, there was signifi- cantly increased percent fibrosis in the diaphragms of 2-, 6- Myocardial mRNA expression (unexercised and exercised), and 9-month-old P448Lneo− mRNA expression for natriuretic peptide type A (Nppa) mice compared to controls (p < 0.01; Table 5;Fig. 6). was significantly increased in P448Lneo− mice compared to controls at 6 months (p < 0.05) and for natriuretic Cardiac muscle peptide type B (Nppb)at6(p < 0.05) and 9 months of Echocardiography age (p < 0.01; Fig. 10). There were no differences in Echocardiographic data collected at 2 and 6 months mRNA expression of fibronectin 1 (Fn1) between were not significantly different between P448Lneo− mice P448Lneo− and control mice at all ages (Fig. 10). and controls except for heart rates (Additional file 4: Table S3). At 9 months of age, there was significantly de- Discussion creased systolic function measured via SF in the mutant In this study, we further phenotyped the P448Lneo− mice (29 ± 2%) compared to controls (31 ± 1%; p < 0.01; mouse model of FKRP-related limb girdle muscular dys- Fig. 9). The left ventricular internal diameter in diastole trophy. One important aspect of this study is to better measured in the parasternal short axis was smaller in understand and validate cardiac muscle disease in the P448Lneo− mice compared to controls and corre- FKRP mutant mouse. Earlier studies reported mild ef- sponded to smaller left ventricular volume in diastole fects of the disease on the histology and functions of the and a significantly decreased left ventricular stroke vol- cardiac muscle. Blaeser et al. [19] reported an EF 49 ± ume (p < 0.01). The myocardial thickness of the left ven- 5% in P448Lneo− and 55 ± 9% in BL6 control mice at tricular infero-posterior wall was significantly increased 10 months of age, although this difference was not sta- in P448Lneo− mice compared to controls (p < 0.0001), tistically significant. However, Blaeser et al. did find a and this corresponded with a significantly increased left significant difference in EF between P448Lneo− (55 ± ventricular mass in the mutant mice (p < 0.001; Fig. 9; 5%) and BL6 (62 ± 7%) at the age of 6 months [19]. Additional file 4: Table S3). The myocardial performance Maricelli et al. (2017) also demonstrated decreased EF index (MPI) was also noted to be significantly increased and SF at 6 months of age in male and female P448Lneo in P448Lneo− mice compared to controls at 9 months of − mice compared to controls, with female mice demon- age. This was related to a significantly decreased isovolu- strating more significant deficits [24]. In this current mic relaxation time (IVRT) seen in the mutant mice (12. study, we demonstrated significant decrease in systolic 1 ms versus 15.3 ms in controls; p < 0.04). This increase cardiac function in P448Lneo− male mice compared to may be related to decreased ventricular compliance. BL6 at 9 months of age. P448Lneo− mice had a SF of Heart rates at 6 months of exercised P448Lneo− mice 29% compared to 31% in controls (p < 0.01). This corre- (463 ± 40 beats per minute; BPM) and unexercised sponds to an EF of 56% in P448Lneo− mice compared to P448Lneo− (461 ± 27 BPM) were significantly increased 60% in controls. However, we show no significant differ- compared to controls (433 ± 29 BPM; p < 0.05). ences in cardiac function at 6 months of age. We also Yu et al. Skeletal Muscle (2018) 8:13 Page 13 of 16 Fig. 9 Cardiac phenotypes in P448Lneo− (FKRP) and control (BL6) mice. At 9 months of age, there was significantly decreased systolic function measured via fractional shortening percent (FS%; panel a) and ejection fraction (EF%; panel b)in P448Lneo− mice compared to controls (p < 0.01). FKRP-treadmill mice were only measured at 2 and 6 months. FKRP mice showed significantly increased left ventricular anterior wall (LVAW) thickness at 6 and 9 months of age (panel c). Left ventricular posterior wall (LVPW) thickness was significantly increased in FKRP mice at 9 months (panel d). Panel e is an echo image in the parasternal short axis showing the M-mode tracing for a 9-month-old BL6 control mouse. The left ventricular internal diameter in diastole measured 4.18 mm. Panel f is an echo image in the parasternal short axis showing the M-mode image for a 9-month-old FKRP mouse. The left ventricular internal diameter in diastole measured 3.86 mm. FKRP mice showed a smaller left ventricular internal diameter in diastole at 9 months of age. Picrosirius red staining of the left ventricle (panel g 10x; panel h 20x) of a control mouse at 9 months of age shows no significant collagen staining. Picrosirius red staining of the left ventricle (panel i 10x; panel j 20x) of a FKRP mouse at 9 months of age shows patchy, diffuse collagen staining. There was significantly increased cardiac fibrosis in 9-month-old FKRP mice compared to controls demonstrated increased myocardial wall thickness and showed approximately twice the amount of fibrosis in left ventricular mass at 9 months of age in P448Lneo− P448Lneo− mice compared to controls at 9 months of mice associated with increased mRNA expression of age. The increasing myocardial fibrosis with age likely Nppa and Nppb [28]. leads to worsening systolic function as these mice get Histopathology demonstrated an increase in myocardial older. Data from all studies are therefore consistent indi- fibrosis. Blaeser et al. showed patchy myocardial fibrosis cating that lack of functional glycosylation of a-DG results that was 4% of measured area at 6 months of age and in- in a mild but progressive degeneration and fibrosis in the creased to about 6% at 12 months of age, compared to ap- cardiac muscle. This leads to a clear trend of decrease in proximately 1% in BL6 controls [19]. The current study cardiac systolic function. However, demonstration of sig- also demonstrated an increase in myocardial fibrosis and nificance in cardiac function between normal and mutant Yu et al. Skeletal Muscle (2018) 8:13 Page 14 of 16 Fig. 10 Real-time PCR of Nppa (panel a), Nppb (panel b), and Fn1 (panel c) for P448Lneo− (FKRP) and control (BL6) mice at 2, 6, and 9 months of age. Fold-changes are shown relative to 2-month-old control mice. Data are presented as mean and error bars denote SD for n =4–6per group. * represents a significant difference between age-matched FKRP and BL6 mice, # represents a significant difference across age for FKRP mice mice is dependent on age, method of detection, and likely plethysmography and associated pathologic changes in requires a larger cohort size. the diaphragm make the P448Lneo− a strong model for Respiratory disease is seen in the clinical spectrum of respiratory disease in FKRP-related LGMD. FKRP-mediated LGMD [29]. This was also demonstrated We did not demonstrate any significant functional dif- in the P448Lneo− mouse model. Blaeser et al. demon- ferences in muscle strength or activity in unexercised strated significant pathology in the diaphragm starting at P448Lneo− mice compared to controls. This is likely re- 6 weeks of age. By 6 months of age, there were large lated to the significant evidence of skeletal muscle regen- areas of inflammatory infiltration. By 10 and 12 months eration present in the mouse model. Blaeser et al. of age, the area of fibrotic tissue increased to approxi- showed that all limb skeletal muscles had severe degen- mately 60% with the majority of fibers demonstrating eration (necrotic fibers) and regeneration (central nucle- central nucleation [19]. An earlier study also showed se- ation) as a predominant feature with relatively limited vere pathology in the diaphragm with clear variation in fibrosis [19]. Cycles of muscle degeneration and regener- fiber size, the presence of necrotic fibers and central nu- ation were clearly indicated by the significant variation cleation (17.6%) [16]. Maricelli et al. also showed in fiber size and central nucleation in more than 37% of changes in central nucleation of the diaphragm at the muscle fibers [16]. We also demonstrated increased 3 months of age [24]. The current study confirms that regeneration by percent central nucleation in the quadri- the decreased normalized tidal and minute volumes at 6 ceps of P448Lneo− mice at 2, 6 (both unexercised and and 9 months of age correspond with increased inflam- exercised), and 9 months of age. Interestingly, fibrosis mation and fibrosis in the diaphragm. We also show a was limited in skeletal muscle (quadriceps and triceps) functional decline in respiration with age. Interestingly, of the unexercised mice, but was significantly increased P448Lneo− mice demonstrated a reduced decline in re- in exercised mice. Maricelli et al. used a modified exer- spiratory rate over time. This is likely related to the fact cise protocol, based on studies from Rocco et al. [30], that older mice have reduced tidal volumes, and they which included two sessions where mice exercised to ex- can maintain higher respiratory rates for their activity haustion. This protocol elicited both functional and due to the compensatory effort by the remaining mus- histological change in exercised mice including de- cles. However, respiratory rates in more severe dys- creased grip strength, short time to exhaustion, in- trophic phenotypes, and perhaps also patients lacking creased fibrosis in the diaphragm, and increased serum regeneration capacity, will likely decrease more signifi- CK levels of P448Lneo− mice compared to unexercised cantly with age. Interestingly, exercised P448Lneo− mice mice and controls [24, 30]. While an optimal exercise showed less tidal and minute volume loss compared to protocol is not yet known, degree of exercise is clearly unexercised mice. This may be again related to exercise- important to the course of disease progression, and the related compensatory regeneration in the diaphragm in- P448Lneo− mice provide a model for such further dicated by significant increase in central nucleation analysis. (44%) compared to unexercised mice (25%). Other po- tential factors, not evaluated in this study, including pul- Conclusions monary inflammation and vascular function could also This study provides more comprehensive outcome mea- be involved. Exercised mice showed significantly in- sures for the P448Lneo− mouse model of FKRP defi- creased diaphragm muscle inflammation compared to ciency. The study shows significant decrease in cardiac unexercised mice. This could lead to a more dramatic function at 9 months of age. This study is the first to decrease in respiratory function at an older age; further provide respiratory function data demonstrating signifi- studies are needed. The functional parameters of cantly decreased tidal and minute volumes in the mouse Yu et al. Skeletal Muscle (2018) 8:13 Page 15 of 16 model at 6 and 9 months of age. A chronic exercise Availability of data and materials All data generated or analyzed during this study are included in this protocol demonstrated increased skeletal muscle fibrosis, published article except for some histological data which is available upon but improved respiratory function at 6 months of age in request from the corresponding author. mutant mice. Further studies are needed to better Authors’ contributions understand the complexities of exercise on muscle path- QY collected and analyzed the functional and histological data. MM ology and disease progression. The results provide new collected and analyzed histological and biochemical data. NL collected and data on outcome measures for future preclinical drug analyzed the histological and biochemical data. AF collected and analyzed biochemical data. RR collected and analyzed the functional data. AB was trials using the P448Lneo− mouse as a model system for involved in the study design, and collected and analyzed the histological FKRP deficiency muscular dystrophy. data. EF collected and analyzed the histological data. QL was involved in the study design and data analysis. KN was involved in the study design and data analysis. CS was involved in the study design, data analysis, and a major Additional files contributor to writing manuscript. All authors read and approved the final manuscript. Additional file 1: Figure S1. Behavioral activity monitoring in P448Lneo− (FKRP), exercised P448Lneo− (FKRP-treadmill), and control (BL6)mice from 1 Ethics approval to 9 months of age. FKRP-treadmill mice were only measured until 6 months This study was carried out in strict accordance with the recommendations in of age. Panel A: vertical activity (VACTV) data; panel B: horizontal activity the Guide for the Care and Use of Laboratory Animals of the National (HACTV) data; panel C: total distance traveled (cm) during session (TOTDIST) Institutes of Health. All experiments were performed in accordance with data; panel D: time (sec, seconds) spent in movement (MOVTIME); panel E: Children’s National Medical Center IACUC approved protocol #30432. time (sec, seconds) spent resting (RESTIME). No significant differences were seen between groups for all measures. (TIFF 136 kb) Consent for publication Not applicable. Additional file 2: Table S1. Histological analyses for skeletal muscles and serum creatinine kinase levels in P448Lneo− (FKRP) and control Competing interests (BL6) mice at 1, 2, 6, and 9 months of age. (DOCX 16 kb) The authors declare that they have no competing interests. Additional file 3: Table S2. Histological analyses of the diaphragm in P448Lneo− (FKRP) and control (BL6) mice at 1, 2, 6, and 9 months of age. (DOCX 15 kb) Publisher’sNote Springer Nature remains neutral with regard to jurisdictional claims in Additional file 4: Table S3. Echocardiography results for P448Lneo− (FKRP) published maps and institutional affiliations. and control (BL6) mice at 2, 6, and 9 months of age showing increased cardiac hypertrophy and decreased systolic function at 9 months of age. Author details (DOCX 15 kb) Center for Genetic Medicine Research, Children’s Research Institute, Children’s National Health System, Washington, DC, USA. School of Abbreviations Pharmacy and Pharmaceutical Sciences, Binghamton University, State ANOVA: analysis of the variance; BL6: C57BL/6J control mice; bpm: Beats per University of New York, Binghamton, NY, USA. Department of Oncology, minute; BPM: Breaths per minute; BW: Body weight; C57: C57BL/6J control Ruijing Hospital, School of Medicine, Shanghai Jiao Tong University, mice; cDNA: Complementary deoxyribonucleic acid; CK: Creatinine kinase; Shanghai, China. McColl-Lockwood Laboratory for Muscular Dystrophy CMD: Congenital muscular dystrophies; CO: Cardiac output; DGC: Dystrophin- Research, Department of Neurology, Carolinas Healthcare System, Charlotte, glycoprotein complex; ECM: Extracellular matrix; EF: Ejection fraction; NC, USA. Children’s National Heart Institute, Center for Genetic Medicine FKRP: Fukutin-related protein; Fn1: Fibronectin 1; GADPH: Glyceraldehyde Research, Children’s National Health System, Washington, DC, USA. 3-phosphate dehydrogenase; GAS: Gastrocnemius; HR: Heart rate; IACUC: Institutional Animal Care and Use Committee; LGMD: Limb-girdle Received: 17 August 2017 Accepted: 20 March 2018 muscular dystrophy; LV mass cor: Left ventricular mass corrected; LV: Left ventricular; LVAW, d: Left ventricular anterior wall thickness in diastole; LVID, d: Left ventricular internal dimension in diastole; LVPW, d: Left ventricular References posterior wall thickness in diastole; LVVol, d: Left ventricular volume in 1. Mercuri E, Muntoni F. Muscular dystrophies. Lancet. 2013;381:845–60. diastole; MPI: Myocardial performance index; mRNA: Messenger ribonucleic 2. Kanagawa M, Toda T. The genetic and molecular basis of muscular acid; MV: Minute ventilation; NIH: National Institutes of Health; dystrophy: roles of cell-matrix linkage in the pathogenesis. J Hum Genet. Nppa: Natriuretic peptide type a; Nppb: Natriuretic peptide type b; NS: Not 2006;51:915–26. significant; PCR: Polymerase chain reaction; Quad: Quadriceps; 3. Ervasti JM, Campbell KP. Membrane organization of the dystrophin- RNA: Ribonucleic acid; SD: Standard deviation; SF: Shortening fraction; glycoprotein complex. Cell. 1991;66:1121–31. Sol: Soleus; SV: Stroke volume; TA: Tibialis anterior; Tri: Triceps; TV: Tidal 4. Taniguchi-Ikeda M, Morioka I, Iijima K, Toda T. Mechanistic aspects of the volume; α-DG: Alpha-dystroglycan formation of alpha-dystroglycan and therapeutic research for the treatment of alpha-dystroglycanopathy: a review. Mol Asp Med. 2016;51:115–24. Acknowledgements 5. Falsaperla R, Pratico AD, Ruggieri M, Parano E, Rizzo R, Corsello G, Vitaliti G, Not applicable. Pavone P. 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J Appl Physiol (1985). 2017; � Our selector tool helps you to find the most relevant journal 123(5):1126–38. � We provide round the clock customer support 25. Spurney CF, Gordish-Dressman H, Guerron AD, Sali A, Pandey GS, Rawat R, � Convenient online submission Van Der Meulen JH, Cha HJ, Pistilli EE, Partridge TA, et al. Preclinical drug trials in the mdx mouse: assessment of reliable and sensitive outcome � Thorough peer review measures. Muscle Nerve. 2009;39:591–602. � Inclusion in PubMed and all major indexing services 26. Nagaraju K, Raben N, Loeffler L, Parker T, Rochon PJ, Lee E, Danning C, � Maximum visibility for your research Wada R, Thompson C, Bahtiyar G, et al. Conditional up-regulation of MHC class I in skeletal muscle leads to self-sustaining autoimmune myositis and Submit your manuscript at myositis-specific autoantibodies. 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Skeletal, cardiac, and respiratory muscle function and histopathology in the P448Lneo− mouse model of FKRP-deficient muscular dystrophy

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Springer Journals
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Copyright © 2018 by The Author(s).
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Life Sciences; Cell Biology; Developmental Biology; Biochemistry, general; Systems Biology; Biotechnology
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2044-5040
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10.1186/s13395-018-0158-x
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29625576
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Abstract

Background: Fukutin-related protein (FKRP) mutations are the most common cause of dystroglycanopathies known to cause both limb girdle and congenital muscular dystrophy. The P448Lneo− mouse model has a knock-in mutation in the FKRP gene and develops skeletal, respiratory, and cardiac muscle disease. Methods: We studied the natural history of the P448Lneo− mouse model over 9 months and the effects of twice weekly treadmill running. Forelimb and hindlimb grip strength (Columbus Instruments) and overall activity (Omnitech Electronics) assessed skeletal muscle function. Echocardiography was performed using VisualSonics Vevo 770 (FujiFilm VisualSonics). Plethysmography was performed using whole body system (ADInstruments). Histological evaluations included quantification of inflammation, fibrosis, central nucleation, and fiber size variation. Results: P448Lneo− mice had significantly increased normalized tissue weights compared to controls at 9 months of age for the heart, gastrocnemius, soleus, tibialis anterior, quadriceps, and triceps. There were no significant differences seen in forelimb or hindlimb grip strength or activity monitoring in P448Lneo− mice with or without exercise compared to controls. Skeletal muscles demonstrated increased inflammation, fibrosis, central nucleation, and variation in fiber size compared to controls (p < 0.05) and worsened with exercise. Plethysmography showed significant differences in respiratory rates and decreased tidal and minute volumes in P448Lneo− mice (p < 0.01). There was increased fibrosis in the diaphragm compared to controls (p < 0.01). Echocardiography demonstrated decreased systolic function in 9-month- old mutant mice (p < 0.01). There was increased myocardial wall thickness and mass (p < 0.001) with increased fibrosis in 9-month-old P448Lneo− mice compared to controls (p < 0.05). mRNA expression for natriuretic peptide type A (Nppa) was significantly increased in P448Lneo− mice compared to controls at 6 months (p < 0.05) and for natriuretic peptide type B (Nppb) at 6 and 9 months of age (p <0.05). Conclusions: FKRP-deficient P448Lneo− mice demonstrate significant deficits in cardiac and respiratory functions compared to control mice, and this is associated with increased inflammation and fibrosis. This study provides new functional outcome measures for preclinical trials of FKRP-related muscular dystrophies. Keywords: Limb-girdle muscular dystrophy, Congenital muscular dystrophy, Fukutin related protein (FKRP), P448Lneo− mice, Echocardiography, Plethysmography, Preclinical trials * Correspondence: cspurney@childrensnational.org Children’s National Heart Institute, Center for Genetic Medicine Research, Children’s National Health System, Washington, DC, USA Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Yu et al. Skeletal Muscle (2018) 8:13 Page 2 of 16 Tyr307Asn Background Neo that demonstrated reduced levels of Muscular dystrophies are a heterogeneous group of FKRP transcript. However, the mutant mice died soon disorders characterized by progressive muscle weak- after birth and were therefore not useful for ness and can also affect the respiratory, cardiac, and experimental therapy development [13]. Mouse central nervous systems. The clinical phenotype and models with the common mutation L276I were prognosis vary significantly making the diagnosis and created by several groups [14–16]. However, treatment different for each disease. However, the dystrophic phenotype of the mutant mice is very mild continued identification of specific genetic causes for with clearly observable pathology only after 6 months the more common muscular dystrophies has led to a of age without significant involvement of respiratory better understanding of disease pathogenesis and new and cardiac muscles, apparently milder than therapeutic strategies [1]. phenotype in patients with the same homozygous One pathogenic mechanism of muscular dystrophies mutations. Another reported FKRP mutant mouse lies in the disruption of the dystrophin-glycoprotein model contains P448L mutation associated with complex (DGC) [2]. The DGC is responsible for linking congenital muscular dystrophy type 1C (MDC1C) in the sarcolemmal membrane with the extracellular matrix clinic [8, 17]. The knock-in FKRP P448L (with neo (ECM) and transmitting contraction forces to maintain cassette removed, referred as P448Lneo− mouse) muscle cell membrane integrity [3]. The DGC is com- homozygous mouse has been reported with a severe posed of multiple proteins including dystrophin, dystro- phenotype consistent with severe LGMD2I, but milder glycans, multiple sarcoglycans, dystrobrevin, laminin, than MDC1C as almost all newborn mice survive and and collagens. Without even one of these proteins, have a life-span of more than 1 year with near nor- membranes can tear and activate multiple pathogenic mal breeding capacity [18]. Also important, the pathways that lead to cell death. mouse was reported to show involvement of respira- Alpha-dystroglycan (α-DG) is one component of the tory and cardiac muscles with progressive fibrosis DCG and interacts with proteins in the ECM includ- [16]. Blaeser et al. (2016) examined the P448Lneo− ing laminin, perlecan, agrin, neurexin using glycosyl- mouse and demonstrated increased diaphragmatic fi- ated O-mannose sugar moieties [4]. Defective brosis and decreased cardiac function by 12 months glycosylation of α-DG is the pathogenic basis of sev- of age [19]. This pattern of phenotype represents well eral muscular dystrophy subtypes known as dystrogly- the clinic manifestation of dystroglycanopathies, as a canopathies, including limb-girdle muscular dystrophy proportion of the patient population is associated (LGMD) and congenital muscular dystrophy (CMD). with pulmonary and cardiac disease. Clinically, Pane These subtypes demonstrate heterogeneous pheno- et al. (2012) described cardiac involvement in 6% and types that can range from early presentations with pulmonary involvement in 12% of patients with con- severe eye and brain disease to more mild skeletal genital muscular dystrophies [20]. And significant car- muscle diseasein older patients.Morethan17genes diac disease can be seen in LGMD 2I, even leading to are involved in the pathogenesis including POMT1, cardiac transplantation [21–23]. More recently, a POMT2, POMGnT, FKRP, Fukutin,and LARGE acting study by Maricelli et al. also demonstrated cardiac as glycosyl-transferases in the O-mannosylation of α- dysfunction with and without exercise [24]. We con- DG [5]. The severity of disease is thought to be re- sider the P448Lneo− mouse highly relevant and valu- lated to the effect of each mutation on degree of able for developing experimental therapies to FKRP glycosylation and laminin binding ability [6]. FKRP is dystroglycanopathy. Therefore, validation of the skel- a gene that encodes fukutin-related protein and its etal muscle phenotype and further characterization of mutations cause dystroglycanopathies of both LGMD respiratory and cardiac muscle are essential. and CMD phenotypes as well as muscle-eye-brain and Walker-Warburg syndrome [7–11]. FKRP has recently Methods been demonstrated as a ribitol 5-phosphate transfer- Animal care ase in the synthesis pathway of laminin binding gly- This study was carried out in strict accordance with can of α-DG [12]. the recommendations in the Guide for the Care and Multiple mouse models were developed to study the Use of Laboratory Animals of the National Institutes role of FKRP and experimental therapies. These of Health. All experiments were performed in accord- models show a range of phenotypes consistent with ance with Children’s National Health System IACUC human FKRP diseases. In general, the severity of the approved protocol #30432. P448Lneo− homozygous reported mouse models follows the same trend as the male mice were generated in McColl Lockwood severity observed in patients with the same mutations. Laboratory (Charlotte, NC) and rederived and Ackroyd et al. (2009) developed the model FKRP- imported from Jackson Laboratory (Bar Harbor, ME) Yu et al. Skeletal Muscle (2018) 8:13 Page 3 of 16 Table 1 Timeline of experimental procedures performed on groups A, B, C, D of P448Lneo− and control mice. Groups A, B, and D were composed of P448Lneo− (n = 8) and control (n = 8) mice. Group C was composed of P448Lneo− (n = 8), exercised P448Lneo− (n = 12), and control (n = 8) mice. Group A was studied at 1 month of age. Group B was studied at 2 months of age. Group C was studied every month until age 6 months of age. A group of P448Lneo− mice underwent exercise treadmill running until 6 months of age. Control mice did not undergo exercise testing. Group D was studied until 9 months of age Group A B C D Timeline (months of age) 1 2 1 2 34569 Treadmill exercise xx xxxx (2×/week only P448Lneo− mice) Body weight x x x x xxxxx Grip strength test x x xxxxx Digiscan activity x x xxxxx Echocardiography x x x Plethysmography x x x Serum creatinine kinase x x x x Histology x x x x Fibrosis x x x x [18]. Age-matched male C57BL/6J (referred to as con- pathology becomes detectable. Group B was studied at trol, C57,or BL6) mice were purchased from Jackson 2monthsofage.Group Cwas studiedeverymonth Laboratory. Animals were ear tagged prior to group until 6 months of age. A 1-month interval was chosen assignment and were housed in cages of standard di- with the aim to identify the peak of muscle degener- mension on ground corn cob bedding mixed with a ation and severity as the disease progresses. A separate soft recycled shredded paper (nesting material) called group of P448Lneo− mice underwent exercise tread- Tek Fresh. The animals were housed in a temperature mill running until 6 months of age. Control mice did controlled (20–24 °C) colony room with a 12-h light/ not undergo exercise testing. Group D was studied at dark cycle and received mouse chow and water ad 9 months of age when both histological and functional libitum. No animals were euthanized prior to reaching data have already shown severe and detectable defects. end of study criteria. Table 1 shows the timing of different testing for each group. Experimental procedure Groups A, B, and D were composed of P448Lneo− Treadmill (n = 8) and control (n = 8) mice. Group C was com- The mice were placed on the treadmill (Columbus In- posed of P448Lneo− (n = 8), exercised P448Lneo− struments, Columbus, OH) twice a week, one per lane (n = 12), and control (n = 8) mice. Group A was for 30 min running at 12 m per minute speed per studied at 1 month of age when the skeletal muscle TREAT-NMD SOP for chronic exercise protocol in Table 2 Body, muscle, and organ weights normalized by body weight in P448Lneo− (FKRP) and control (BL6) mice showing significant differences at 6 and 9 months of age Weight 1 month 2 months 6 months 9 months (n =8) BL6 FKRP BL6 FKRP BL6 FKRP BL6 FKRP Body (g) 19 ± 1.2 19.6 ± 0.9 21.9 ± 1.5 22.3 ± 0.83 28 ± 1.7 30 ± 1.2 33 ± 6.0 31 ± 1.7 −6 GAS/BW(10 ) 6.2 ± 0.5 6.3 ± 0.3 6.4 ± 0.4 6.5 ± 0.4 5.4 ± 1 6.0 ± 0.2*** 5.0 ± 0.2 6.3 ± 0.5*** −7 Sol/BW(10 ) 3.7 ± 0.3 3.7 ± 0.2 3.8 ± 0.9 3.6 ± 0.5 3.0 ± 0.4 3.5 ± 0.2* 3.3 ± 0.3 4.1 ± 0.4** −6 TA/BW(10 ) 2.4 ± 0.4 2.3 ± 0.2 2.3 ± 0.1 2.3 ± 0.2 1.8 ± 0.2 2.2 ± 0.2*** 1.4 ± 0.2 2.0 ± 0.3*** −6 Triceps/BW(10 ) 4.1 ± 0.4 4.4 ± 0.4 4.4 ± 0.6 4.3 ± 0.4 3.4 ± 0.6 4.5 ± 0.3*** 3.3 ± 0.6 5.4 ± 0.9*** −6 Quad/BW(10 ) 7.0 ± 0.2 7.3 ± 1.1 5.7 ± 0.8 6.0 ± 0.8 5.3 ± 0.8 6.5 ± 0.9* 5.0 ± 0.9 6.3 ± 0.4** −6 Heart/BW(10 ) 5.0 ± 0.2 5.3 ± 0.5 5.1 ± 0.2 5.2 ± 0.4 4.3 ± 0.3 4.4 ± 0.2 3.9 ± 0.5 4.4 ± 0.4* −6 Brain/BW(10 ) 21 ± 1.2 20 ± 3.6 20 ± 1.4 19 ± 1 16 ± 1.2 15 ± 0.5* 14.5 ± 2.8 14.2 ± 0.1 *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; using t test when compared to BL6 control mice at same age. Data presented as mean ± SD. GAS gastrocnemius, Sol soleus, TA tibialis anterior, Quad quadriceps, BW body weight Yu et al. Skeletal Muscle (2018) 8:13 Page 4 of 16 Table 3 Body, muscle, and organ weights normalized by body weights among groups of P448Lneo− (FKRP) without and with treadmill exercise (FKRP-treadmill) and control (BL6) mice at 6 months of age Measurement BL6 control FKRP FKRP-treadmill Significantly different groups (adjusted p values using ANOVA N Mean ± SD N Mean ± SD N Mean ± SD followed by post hoc analysis) Body (g) 8 28 ± 1.7 8 30 ± 1.2 12 29 ± 1.8 NS −6 GAS/BW(10 ) 8 5.4 ± 0.3 8 6.0 ± 0.2 12 6.4 ± 0.7 BL6 vs FKRP: p < 0.05, BL6 vs FKRP-treadmill: p < 0.001 −7 Sol/BW(10 ) 8 3.0 ± 0.4 8 3.5 ± 0.2 12 3.4 ± 0.3 BL6 vs FKRP: p < 0.05, BL6 vs FKRP-treadmill: p < 0.05. −6 TA/BW(10 ) 8 1.8 ± 0.2 8 2.2 ± 0.2 12 2.4 ± 0.3 BL6 vs FKRP: p < 0.01, BL6 vs FKRP-treadmill: p < 0.001 −6 Triceps/BW(10 ) 8 3.4 ± 0.6 8 4.5 ± 0.3 12 5.2 ± 0.7 BL6 vs FKRP: p < 0.01, BL6 vs FKRP-treadmill: p < 0.0001 FKRP vs FKRP-treadmill: p < 0.05 −6 Quad/BW(10 ) 8 5.3 ± 0.8 8 6.5 ± 0.9 12 7.0 ± 1.3 BL6 vs FKRP: p < 0.05, BL6 vs FKRP-treadmill: p < 0.01 −6 Heart/BW(10 ) 8 4.3 ± 0.3 8 4.4 ± 0.2 12 4.4 ± 0.5 NS −6 Brain/BW(10 ) 8 16 ± 1.2 8 15 ± 0.5 12 16 ± 1.2 NS GAS gastrocnemius, Sol soleus, TA tibialis anterior, Quad quadriceps, BW body weight, NS not significant dystrophic mice (http://www.treat-nmd.eu/research/pre- of KGF (kilogram-force) and normalized to bodyweights clinical/dmd-sops/). If a mouse rested at the end of the as “KGF/kg.” lane, the animal would be gently pushed back onto the treadmill surface to restart running. The treadmill tests Locomotor activity started on mice at approximately 1 month of age and Locomotor activity was measured using an open-field continued until 6 months of age. During the weeks of digiscan apparatus (Omnitech Electronics, Columbus, measurements including grip strength, activity monitor, OH). Total distance, horizontal activity, and vertical ac- echo, and plethysmography, treadmill running was tivity were recorded every 10 min for 1 h as described avoided. previously [26, 27]. As with the grip strength, the activity data were collected in the morning hours over a 4-day Grip strength period and the mice were trained in the open field ap- Forelimb grip strength was measured by a grip strength paratus prior to the trial [25]. meter (Columbus Instruments, Columbus, OH). The animal was held so that only the forelimb paws grasped Echocardiography the specially designed mouse flat mesh assembly and the Echocardiography was performed and quantitative mea- mouse was pulled back until their grip was broken. The surements were made offline using analytic software force transducer retained the peak force reached when (FujiFilm VisualSonics, Toronto, Ontario, Canada) as the animal’s grip was broken, and this was recorded previously described [25]. Measurements included vessel from a digital display. For hindlimb strength, an angled diameters, ventricular chamber size, and blood flow vel- mesh assembly was used. Mice were allowed to rest on ocities and timing across the atrioventricular and semi- the angled mesh assembly, facing away from the meter lunar valves. M-mode images were used to measure left with its hindlimbs at least one-half of the way down the ventricular (LV) chamber sizes and wall thicknesses. Per- length of the mesh. The mouse tail was pulled directly cent shortening fraction (SF) and ejection fraction (EF) toward the meter and parallel to the mesh assembly. were calculated from M-mode measurements. Myocar- During this procedure, the mice resist by grasping the dial performance index (MPI) was also calculated from mesh with all four limbs. Pulling toward the meter was Doppler measurements. continued until the hindlimbs released from the mesh assembly. Five successful hindlimb and forelimb strength Plethysmography measurements within 2 min were recorded. The max- The whole body plethysmography system (ADInstru- imum values were used for analysis. The grip strength ments, St. Paul, MN) utilized a custom mouse cham- measurements were collected in the morning hours over ber developed by the Research Instrument Shop at a 5-day period. The mice were trained on the grip theUniversityofPennsylvaniatominimizedead strength meter before the trial [25]. Forelimb and hind- space. Other components in the system included the limb maximal muscle strength were obtained as values spirometer (ML141), respiratory flow head (MLTL1), Yu et al. Skeletal Muscle (2018) 8:13 Page 5 of 16 Fig. 1 Normalized grip strengths for P448Lneo− and control mice. Panel a shows no significant differences in normalized forelimb grip strength (kilogram force per kilogram; KGF/kg) at 9 months of age in P448Lneo− (FKRP) and control (BL6) mice. Panel b shows no significant differences in normalized hindlimb grip strength (kilogram force per kilogram; KGF/kg) at 9 months of age in FKRP and control mice. Panel c shows normalized forelimb and panel d shows normalized hindlimb grip strengths from 1 to 9 months of age for FKRP mice, exercised FKRP mice (FKRP-treadmill; 1–6 months only), and control mice with no significant differences and the PowerLab 4/30 with LabChart software. The was recorded for 10 min. For data analysis, values for mouse was brought to the measurement room respiratory rate, tidal volume (TV), minute ventilation 15 min before the start of the measurement session (MV), TV normalized by body weight (TV/BW), and to recover from the transportation and new environ- MV normalized by body weight (MV/BW) were re- ment stresses. The spirometer was calibrated every corded using LabChart software. time the hardware was powered on to read in terms of flow (ml/s) rather than pressure (mv).Calibration of Blood collection the plethysmography was performed with 1 ml of air Blood samples were taken via retro-orbital bleeding injected into the animal chamber to correlate the when the animals were euthanized and the serum col- injected volume (ml) with the differential pressure lected was used for creatinine kinase levels. (mv) measured in the chamber by integration. A 700 ml/min flow of dry air through the chambers was Tissue collection and histological evaluations constantly delivered to avoid CO and water accumu- Animals were sacrificed via inhaled carbon dioxide and lation and to maintain a constant temperature. The cervical dislocation, and tissue samples were obtained. All mouse was weighed and placed into the chamber first tissue samples were weighed using the Mettler ToLedo to acclimate for 15 min then the respiratory flow data scale (Columbus, OH) prior to processing. Skeletal Yu et al. Skeletal Muscle (2018) 8:13 Page 6 of 16 Fig. 2 Inflammation levels (foci/mm2) in P448Lneo− (FKRP) mice gastrocnemius (panel a), quadriceps (panel b), and triceps (panel c)at 1, 2, 6, and 9 months of age compared to controls (BL6) and serum creatinine kinase (CK) at 9 months of age (panel d). Significant increases in inflammation are seen with a peak at 2 months of age. Serum CK levels are significantly increased at 1, 6, and 9 months. Data presented as mean ± standard deviation;*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 using t test when compared to BL6 control mice at same age; ###p < 0.001 using two-tailed Mann-Whitney nonparametric test when compared to BL6 control mice at same age muscles (gastrocnemius, tibialis anterior, soleus, triceps, Inc. (Germantown, MD). The tissues were magnified and quadriceps) from one side of the animal, half the dia- under a light microscope at an objective of 10 x and phragm, half the heart, and whole brain were stored in digital images obtained using computer software (Olym- formalin. The contralateral or other half of muscles were pus C.A.S.T. Stereology System, Olympus America Inc., snapped frozen in isopentane cooled in liquid nitrogen Center Valley, PA). These digital images were processed and stored at − 80 °C for further analysis. Slides were pre- using ImageJ (NIH) with additional threshold color pared and stained by Histoserv Inc. (Gaithersburg, MD). plug-ins to process jpeg images. Pixels corresponding to Histological evaluations were performed in a blinded the area stained in red were normalized to the total pixel manner using coded slides. One transverse tissue section area of the tissue image, and the results were expressed per muscle per animal was analyzed. Whole muscle digital as percent of fibrotic area. images of the tissues were taken at × 20 using NanoZoo- mer slide scanner (Hamamatsu Inc., Bridgewater, NJ) and mRNA expression analysis were opened using NDP.view2 software. Each tissue sec- Snapped frozen hearts from 2-, 6-, and 9-month-old tion was analyzed throughout its entire area. The total FKRP and BL6 control mice were collected into tubes number of inflammation foci (an interstitial group of 10 with 1 mL of TRIzol and homogenized. Total RNA smaller inflammatory cell dark blue nuclei in a high- was isolated and washed and the RNA yield and pur- power field) was quantified. The entire tissue section area ity was determined using a NanoDrop 2000 microvo- was measured (mm ), and all counts were normalized to lume spectrophotometer (ThermoFisher). cDNA was the tissue area. The parameters including percentage of generated using the high-capacity cDNA reverse tran- fibers with central nucleation and fiber diameter were scription kit (Applied Biosystems, cat #4368813). The measured using MetaMorph Microscopy Automation and cDNA was added to TaqMan universal PCR master Image Analysis Software on paraffin sections of mix (Applied Biosystems, cat #4304437), and the fol- gastrocnemius, triceps, quadriceps, and diaphragm. lowing TaqMan Gene Expression Assays (Applied Bio- systems): Nppa, Nppb, and Fn1. GAPDH was used as Quantification of fibrosis the reference gene. Real-type PCR was performed Paraffin sections of gastrocnemius, diaphragm, and heart using the CFX384 Touch Real-Time PCR Detection tissue were stained with picrosirius red by Histoserv, System and associated software (Bio-Rad). Yu et al. Skeletal Muscle (2018) 8:13 Page 7 of 16 Statistical analysis of age, exercise had no significant effects on normalized Data is presented as mean ± standard deviation (SD). Nor- forelimb or hindlimb grip strength (Fig. 1). mality of each phenotype was tested using both the Shapiro-Wilk normality test and visual inspection of his- Activity monitor tograms except for percent central nucleation and fiber There were no significant differences between BL6 and diameter size as there are only three total samples. All P448Lneo− in horizontal and vertical activity, movement tested phenotypes were normally distributed except in- time, rest time, and total distance. While exercised mice flammation in quadriceps of 1- and 9-month-old BL6,and showed decreased activity compared to unexercised 6months old P448Lneo− excised mice, inflammation in P448Lneo− and BL6 mice, the differences were not sig- gastrocnemius of 2-, 6-, and 9-month-old BL6,and 6- nificant (Additional file 1: Figure S1). month-old P448Lneo− excised mice, and inflammation in Triceps of 2-, 6-, and 9-month-old BL6 mice. For normally Inflammation distributed parameters, comparisons were made among Analysis of the skeletal muscle including the gastrocne- 6 –month-old BL6, P448Lneo−,and P448Lneo− excised mius, quadriceps and triceps showed an increase in in- mice using analysis of the variance (ANOVA) followed by flammatory foci at 1 month of age in P448Lneo− mice Tukey multiple comparison analysis. A single t test was compared to control mice (Figs. 2 and 3). This difference used to compare the BL6 control group to the P448Lneo− increases by 2 to 4 fold to a maximum inflammation at group. RT-PCR data were normalized to the 2-month-old 2 months of age. The maximum amount of inflamma- BL6 control group and are presented as fold change. For tion was noted in the quadriceps muscle. The inflam- abnormally distributed parameters, comparisons were matory infiltrates then decreased at both 6 and made among 6-month-old BL6, P448Lneo−,and 9 months in all 3 muscles. In P448Lneo− mice exer- P448Lneo− excised mice using Kruskal-Wallis test cised until 6 months of age, the inflammatory infil- followed by Dunn’s multiple comparison analysis and a trates increase from 1.8 to 2.2 folds compared to two-tailed Mann-Whitney test was used to compare the unexercised P448Lneo− mice (Table 4; Fig. 3). BL6 control group to the P448Lneo− group. A value of p < 0.05 was considered statistically significant. Fibrosis No significant differences in percent fibrosis in the quad- Results riceps or triceps between P448Lneo− and controls were Body, organ, and muscle weights seen at 1, 2, 6, or 9 months of age (Additional file 2: No significant differences were seen in total body weight Table S1). P448Lneo− exercised mice showed signifi- between BL6 (control) and P448Lneo− mice (Table 2). cantly increased percent fibrosis in the quadriceps mus- There was a significant difference in brain weight nor- cles at 6 months of age compared to unexercised malized to body weight at 6 months of age (p < 0.05). P448Lneo− mice (p < 0.05) and controls (p < 0.001; The P448Lneo− heart showed significantly increased Table 4; Fig. 3). There were no significant differences in mass when normalized to body weight compared to BL6 the gastrocnemius and triceps between the 2 mice at 9 months of age (p < 0.05). The skeletal muscles groups (data not shown). gastrocnemius, soleus, tibialis anterior, quadriceps, and the triceps from mutant mice all demonstrated signifi- Percent central nucleation cantly increased mass normalized to body weight com- Control BL6 mice showed between 0.2 and 1.4% central pared to BL6 at 6 and 9 months of age (Table 2). nucleation in the quadriceps, gastrocnemius, and tri- P448Lneo− mice exercised on the treadmill showed an ceps from 1 to 9 months. P448Lneo− mice showed per- increase in normalized muscle weight compared to con- cent central nucleation of 9.6% at 1 month, 56% at trols for the soleus, tibialis anterior, triceps, and quadri- 6 months, and 60.4% at 9 months (Table 4; ceps muscles (p < 0.05; Table 3). Exercised P448Lneo− Additional file 2: Table S1). The percent central nucle- mice also demonstrated a higher normalized muscle ation in the quadriceps and triceps were increased in 6- weight for the triceps compared to unexercised month-old exercised P448Lneo− mice compared to un- P448Lneo− mice (p < 0.05). exercised mice while the quadriceps decreased slightly (Table 4). There were differences in the variation (SD Skeletal muscle of percent central nucleation for each mouse) of Grip strength P448Lneo− and control mice (Additional file 2:Table No significant differences in normalized forelimb or S1) and P448Lneo− exercised mice showed increased hindlimb grip strength were seen at 9 months of age be- SD of percent central nucleation in the triceps com- tween control and P448Lneo− mice (Fig. 1). At 6 months pared to controls at 6 months of age (Table 4). Yu et al. Skeletal Muscle (2018) 8:13 Page 8 of 16 Fig. 3 Histology images showing inflammation (hematoxylin and eosin staining at 20x) and fibrosis (picrosirius red staining at 10x) in the quadriceps of P448Lneo− (FKRP), exercised P448Lneo− (FKRP-treadmill), and control (BL6) mice. Panels a–d show inflammation in BL6 mice at 1, 2, 6, and 9 months of age. Panels e–h show inflammation in FKRP mice at 1, 2, 6, and 9 months of age. Panel i shows inflammation in FKRP- treadmill mice at 6 months of age. Panels j–l show fibrosis in BL6, FKRP, and FKRP-treadmill at 6 months of age Fiber diameter Respiratory muscle Figure 4 shows the fiber diameters of the quadriceps mus- Plethysmography cles for P448Lneo− mice and controls at 1, 6, and 9 months P448Lneo− mice demonstrated a reduced decline in re- of age. There was no difference in the average fiber size of spiratory rate over time compared to BL6 controls at P448Lneo− and control BL6 mice at 1and 6monthsof age. 9 months of age (p < 0.001; Fig. 5). P448Lneo− mice also At 9 months of age, P448Lneo− mice have smaller average showed significantly decreased tidal volumes (p < 0.001), fiber diameter compared to control BL6 (Additional file 2: normalized tidal volumes (p < 0.01), and minute volumes Table S1). There was a greater variation in fiber sizes (SD (p < 0.001) compared to BL6 controls at 6 and 9 months of fiber size for each mouse) in P448Lneo− mice at 1 and of age. There were significant differences in plethys- 6 months of age compared to control BL6 (Additional file 2: mography measures at 6 months that were improved Table S1). There were no differences in fiber size, but un- in exercised P448Lneo− mice compared to unexer- exercised and exercised P448Lneo− mice showed increased cised P448Lneo− including tidal volume (p < 0.001), SD of fiber size for each mouse compared to controls in minute volume (p < 0.01), and normalized minute volume the quadriceps, gastrocnemius, and triceps (Table 4). (p < 0.01; Fig. 5). Serum creatinine kinase (CK) Inflammation Serum CK was significantly increased in P448Lneo− The diaphragm of P448Lneo− mice showed the most in- mice compared to control BL6 at 1, 6, and 9 months flammatory infiltrates at 1 month of age (p < 0.001; Figs. of age (p <0.05; Fig. 2). At 6 months of age, exer- 6 and 7). The infiltrates decreased but remained signifi- cised P448Lneo− mice showed significantly increased cant compared to controls from 2 to 9 months of age serum CK levels compared to BL6 controls (p < 0.05; (p < 0.0.01). Exercised P448Lneo− mice showed signifi- Table 4). cant inflammation that was increased compared to Yu et al. Skeletal Muscle (2018) 8:13 Page 9 of 16 Table 4 Histological analyses for skeletal muscles and serum creatinine kinase levels among groups of P448Lneo− (FKRP) without and with treadmill exercise (FKRP-treadmill) and control (BL6) mice at 6 months of age Measurement BL6 control FKRP FKRP-treadmill Significantly different groups (adjusted p values using ANOVA N Mean ± SD N Mean ± SD N Mean ± SD followed by post hoc analysis) Inflammation GAS 8 0.03 ± 0.03 8 0.5 ± 0.4 12 0.9 ± 0.3 #BL6 vs FKRP-treadmill: p < 0.0001 (foci/mm ) Quad 8 0.05 ± 0.04 8 0.6 ± 0.2 12 1.1 ± 0.5 #BL6 vs FKRP: p < 0.01, #BL6 vs FKRP-treadmill: p < 0.0001 Triceps 8 0.04 ± 0.06 8 0.6 ± 0.4 12 1.1 ± 0.3 #BL6 vs FKRP: p < 0.01, #BL6 vs FKRP-treadmill: p < 0.0001 % fibrosis Quad 8 0.29 ± 0.07 8 0.41 ± 0.18 12 0.61 ± 0.15 BL6 vs FKRP-treadmill: p < 0.001, FKRP vs FKRP-treadmill: p < 0.05 GAS 3 0.47 ± 0.23 3 46.18 ± 5.4 3 38.44 ± 5.1 NP % central nucleation Quad 3 0.2 ± 0.3 3 56.0 ± 1.9 3 51.1 ± 2.1 NP Triceps 3 1.44 ± 1.11 3 65.71 ± 1.1 3 75.16 ± 8.4 NP GAS 3 34.27 ± 0.7 3 37.9 ± 3.6 3 42.7 ± 2.6 NP Fiber diameter size (μm) Quad 3 48.2 ± 5.5 3 44.8 ± 1.2 3 49.7 ± 1.6 NP Triceps 3 37.75 ± 1.2 3 37.03 ± 1.8 3 43.70 ± 2.0 NP GAS 3 0.5 ± 0.2 3 7.6 ± 5.4 3 10.7 ± 5.1 NP SD of % central nucleation Quad 3 0.4 ± 0.5 3 8.9 ± 3.9 3 12.6 ± 4.4 NP Triceps 3 1.28 ± 1.1 3 15.94 ± 1.1 3 8.17 ± 8.4 NP GAS 3 1.93 ± 0.7 3 8.5 ± 3.6 3 8.73 ± 2.6 NP SD of fiber size Quad 3 11.8 ± 1.3 3 20.5 ± 1.8 3 21.5 ± 1.9 NP Triceps 3 1.93 ± 1.2 3 4.8 ± 1.8 3 6.96 ± 2.0 NP Serum creatinine kinase(μ/l) 8 254 ± 131 7 749 ± 405 12 947 ± 575 BL6 VS. FKRP-treadmill: p < 0.05 Kruskal-Wallis test followed by Dunn’s multiple comparison test used. Statistical measures not performed on measures with N =3. GAS gastrocnemius, Quad quadriceps, SD standard deviation, NP not performed unexercised P448Lneo− mice and BL6 controls at nucleation for each mouse) among P448Lneo− exercised, 6 months of age (p < 0.05; Table 5). unexercised, and control mice (Table 5; Additional file 3: Table S2). Percent central nucleation diaphragm Control BL6 mice showed between 2.3 and 3.7% central Diaphragm fiber diameter nucleation in the diaphragm from 1 to 9 months of age. Figure 8 shows the fiber diameters of the diaphragm 1-month-old P448Lneo− mice showed 4.4% central nu- muscle for P448Lneo− mice and BL6 controls at 1, 6, cleation, which increased to 34% at 9 months old (Add- and 9 months of age. No differences were seen in aver- itional file 3: Table S2). 6-month-old unexercised age fiber size in the mutant mice compared to controls P448Lneo− mice showed 25% central nucleation, and in the diaphragm, but there was greater variation in fiber this increased to 44% in exercised mice (Table 5). There sizes (SD of fiber size for each mouse) in P448Lneo− is no difference in the variation (SD of percent central mice at 1 and 6 months of age compared to controls Fig. 4 Percent number of muscle fiber diameter sizes (μm) in the quadriceps muscle in P448Lneo− and control (C57) mice at 1(panel a), 6 (panel b), and 9 (panel c) months of age. Error bars indicate standard deviation Yu et al. Skeletal Muscle (2018) 8:13 Page 10 of 16 Fig. 5 Plethysmography results in P448Lneo− (FKRP) mice, exercised P448Lneo− mice (FKRP-treadmill), and controls (BL6) at 2, 6, and 9 months of age. Respiratory rates (panel a) are significantly less in control mice compared to FKRP. Tidal volume (panel b) and normalized tidal volume (panel c) are significantly increased in controls. Minute volume (panel d) and normalized minute volume (panel e) show significant changes only at 6 months. FKRP-treadmill mice only measured at 2 and 6 months. ****p < 0.0001 between BL6 and FKRP; ‡‡‡p < 0.001, ‡‡‡‡p < 0.0001, and p < 0.001 between BL6 and FKRP/FKRP-treadmill; ^p < 0.05 among all groups. Data presented as mean ± standard deviation Fig. 6 Significantly increased diaphragm inflammation (panel a) and fibrosis (panel b) are seen in P448Lneo− mice (FKRP) compared to controls (BL6) at 2, 6, and 9 months of age. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 compared to BL6 control at same age. Data presented as mean ± standard deviation Yu et al. Skeletal Muscle (2018) 8:13 Page 11 of 16 Fig. 7 Inflammation (hematoxylin and eosin staining at 20x) and fibrosis (picrosirius red staining at 10x) in diaphragm in P448Lneo− (FKRP) and control (BL6) mice. Panels a–d show inflammation in the diaphragm of BL6 mice at 1, 2, 6, and 9 months of age. Panels e–h show inflammation in the diaphragm of FKRP mice at 1, 2, 6, and 9 months of age. Panels i–l show fibrosis in the diaphragm of BL6 mice at 1, 2, 6, and 9 months of age. Panels m–p shows fibrosis in the diaphragm of FKRP mice at 1, 2, 6, and 9 months of age Table 5 Histological analyses of the diaphragm among groups of P448Lneo− (FKRP) with and without treadmill exercise (FKRP-treadmill) and control (BL6) mice at 6 months of age Measurement BL6 control FKRP FKRP-treadmill Significantly different groups (adjusted p values using ANOVA N Mean ± SD N Mean ± SD N Mean ± SD followed by post hoc analysis) Inflammation (foci/mm ) 8 0.5 ± 0.3 8 4.0 ± 0.8 12 8.7 ± 2.8 BL6 vs FKRP: p < 0.01, BL6 vs FKRP-treadmill: p < 0.0001 FKRP vs FKRP-treadmill: p < 0.0001 % fibrosis 8 2.0 ± 0.7 8 13.7 ± 3.8 12 13.4 ± 2.2 BL6 vs FKRP: p < 0.0001, BL6 vs FKRP-treadmill: p < 0.0001 % central nucleation 3 2.4 ± 0.4 3 24.8 ± 2.0 3 44 ± 9.4 NP Fiber diameter size (μm) 3 24.6 ± 2.6 3 21.3 ± 1.2 3 26.7 ± 2.5 NP SD of % central nucleation for each mouse 3 1.6 ± 1.2 3 4.9 ± 3.3 3 7.7 ± 2.3 NP SD of fiber size for each mouse 3 4.5 ± 0.8 3 6.3 ± 0.2 3 8.1 ± 0.2 NP Statistical analyss not performed on measures with N =3. SD standard deviation, NP not performed Yu et al. Skeletal Muscle (2018) 8:13 Page 12 of 16 Fig. 8 Percent number of muscle fiber diameter sizes (μm) in the diaphragm muscle in P448Lneo− and control (C57) mice at 1(panel a), 6 (panel b), and 9 (panel c) months of age. Error bars indicate standard deviation (Additional file 3: Table S2) and between unexercised Myocardial fibrosis and exercised P448Lneo− mice at 6 months of age There were no significant differences in myocardial fi- (Table 5). brosis at 1, 2, and 6 months of age between P448Lneo− mice compared to controls. There was significantly in- creased myocardial percent fibrosis at 9 months of age Diaphragm fibrosis in P448Lneo− mice (0.69 ± 0.24) compared to controls There were no significant differences in percent fibrosis of (0.38 ± 0.17; p < 0.05; Fig. 9). the diaphragm at 1 month of age; however, there was signifi- cantly increased percent fibrosis in the diaphragms of 2-, 6- Myocardial mRNA expression (unexercised and exercised), and 9-month-old P448Lneo− mRNA expression for natriuretic peptide type A (Nppa) mice compared to controls (p < 0.01; Table 5;Fig. 6). was significantly increased in P448Lneo− mice compared to controls at 6 months (p < 0.05) and for natriuretic Cardiac muscle peptide type B (Nppb)at6(p < 0.05) and 9 months of Echocardiography age (p < 0.01; Fig. 10). There were no differences in Echocardiographic data collected at 2 and 6 months mRNA expression of fibronectin 1 (Fn1) between were not significantly different between P448Lneo− mice P448Lneo− and control mice at all ages (Fig. 10). and controls except for heart rates (Additional file 4: Table S3). At 9 months of age, there was significantly de- Discussion creased systolic function measured via SF in the mutant In this study, we further phenotyped the P448Lneo− mice (29 ± 2%) compared to controls (31 ± 1%; p < 0.01; mouse model of FKRP-related limb girdle muscular dys- Fig. 9). The left ventricular internal diameter in diastole trophy. One important aspect of this study is to better measured in the parasternal short axis was smaller in understand and validate cardiac muscle disease in the P448Lneo− mice compared to controls and corre- FKRP mutant mouse. Earlier studies reported mild ef- sponded to smaller left ventricular volume in diastole fects of the disease on the histology and functions of the and a significantly decreased left ventricular stroke vol- cardiac muscle. Blaeser et al. [19] reported an EF 49 ± ume (p < 0.01). The myocardial thickness of the left ven- 5% in P448Lneo− and 55 ± 9% in BL6 control mice at tricular infero-posterior wall was significantly increased 10 months of age, although this difference was not sta- in P448Lneo− mice compared to controls (p < 0.0001), tistically significant. However, Blaeser et al. did find a and this corresponded with a significantly increased left significant difference in EF between P448Lneo− (55 ± ventricular mass in the mutant mice (p < 0.001; Fig. 9; 5%) and BL6 (62 ± 7%) at the age of 6 months [19]. Additional file 4: Table S3). The myocardial performance Maricelli et al. (2017) also demonstrated decreased EF index (MPI) was also noted to be significantly increased and SF at 6 months of age in male and female P448Lneo in P448Lneo− mice compared to controls at 9 months of − mice compared to controls, with female mice demon- age. This was related to a significantly decreased isovolu- strating more significant deficits [24]. In this current mic relaxation time (IVRT) seen in the mutant mice (12. study, we demonstrated significant decrease in systolic 1 ms versus 15.3 ms in controls; p < 0.04). This increase cardiac function in P448Lneo− male mice compared to may be related to decreased ventricular compliance. BL6 at 9 months of age. P448Lneo− mice had a SF of Heart rates at 6 months of exercised P448Lneo− mice 29% compared to 31% in controls (p < 0.01). This corre- (463 ± 40 beats per minute; BPM) and unexercised sponds to an EF of 56% in P448Lneo− mice compared to P448Lneo− (461 ± 27 BPM) were significantly increased 60% in controls. However, we show no significant differ- compared to controls (433 ± 29 BPM; p < 0.05). ences in cardiac function at 6 months of age. We also Yu et al. Skeletal Muscle (2018) 8:13 Page 13 of 16 Fig. 9 Cardiac phenotypes in P448Lneo− (FKRP) and control (BL6) mice. At 9 months of age, there was significantly decreased systolic function measured via fractional shortening percent (FS%; panel a) and ejection fraction (EF%; panel b)in P448Lneo− mice compared to controls (p < 0.01). FKRP-treadmill mice were only measured at 2 and 6 months. FKRP mice showed significantly increased left ventricular anterior wall (LVAW) thickness at 6 and 9 months of age (panel c). Left ventricular posterior wall (LVPW) thickness was significantly increased in FKRP mice at 9 months (panel d). Panel e is an echo image in the parasternal short axis showing the M-mode tracing for a 9-month-old BL6 control mouse. The left ventricular internal diameter in diastole measured 4.18 mm. Panel f is an echo image in the parasternal short axis showing the M-mode image for a 9-month-old FKRP mouse. The left ventricular internal diameter in diastole measured 3.86 mm. FKRP mice showed a smaller left ventricular internal diameter in diastole at 9 months of age. Picrosirius red staining of the left ventricle (panel g 10x; panel h 20x) of a control mouse at 9 months of age shows no significant collagen staining. Picrosirius red staining of the left ventricle (panel i 10x; panel j 20x) of a FKRP mouse at 9 months of age shows patchy, diffuse collagen staining. There was significantly increased cardiac fibrosis in 9-month-old FKRP mice compared to controls demonstrated increased myocardial wall thickness and showed approximately twice the amount of fibrosis in left ventricular mass at 9 months of age in P448Lneo− P448Lneo− mice compared to controls at 9 months of mice associated with increased mRNA expression of age. The increasing myocardial fibrosis with age likely Nppa and Nppb [28]. leads to worsening systolic function as these mice get Histopathology demonstrated an increase in myocardial older. Data from all studies are therefore consistent indi- fibrosis. Blaeser et al. showed patchy myocardial fibrosis cating that lack of functional glycosylation of a-DG results that was 4% of measured area at 6 months of age and in- in a mild but progressive degeneration and fibrosis in the creased to about 6% at 12 months of age, compared to ap- cardiac muscle. This leads to a clear trend of decrease in proximately 1% in BL6 controls [19]. The current study cardiac systolic function. However, demonstration of sig- also demonstrated an increase in myocardial fibrosis and nificance in cardiac function between normal and mutant Yu et al. Skeletal Muscle (2018) 8:13 Page 14 of 16 Fig. 10 Real-time PCR of Nppa (panel a), Nppb (panel b), and Fn1 (panel c) for P448Lneo− (FKRP) and control (BL6) mice at 2, 6, and 9 months of age. Fold-changes are shown relative to 2-month-old control mice. Data are presented as mean and error bars denote SD for n =4–6per group. * represents a significant difference between age-matched FKRP and BL6 mice, # represents a significant difference across age for FKRP mice mice is dependent on age, method of detection, and likely plethysmography and associated pathologic changes in requires a larger cohort size. the diaphragm make the P448Lneo− a strong model for Respiratory disease is seen in the clinical spectrum of respiratory disease in FKRP-related LGMD. FKRP-mediated LGMD [29]. This was also demonstrated We did not demonstrate any significant functional dif- in the P448Lneo− mouse model. Blaeser et al. demon- ferences in muscle strength or activity in unexercised strated significant pathology in the diaphragm starting at P448Lneo− mice compared to controls. This is likely re- 6 weeks of age. By 6 months of age, there were large lated to the significant evidence of skeletal muscle regen- areas of inflammatory infiltration. By 10 and 12 months eration present in the mouse model. Blaeser et al. of age, the area of fibrotic tissue increased to approxi- showed that all limb skeletal muscles had severe degen- mately 60% with the majority of fibers demonstrating eration (necrotic fibers) and regeneration (central nucle- central nucleation [19]. An earlier study also showed se- ation) as a predominant feature with relatively limited vere pathology in the diaphragm with clear variation in fibrosis [19]. Cycles of muscle degeneration and regener- fiber size, the presence of necrotic fibers and central nu- ation were clearly indicated by the significant variation cleation (17.6%) [16]. Maricelli et al. also showed in fiber size and central nucleation in more than 37% of changes in central nucleation of the diaphragm at the muscle fibers [16]. We also demonstrated increased 3 months of age [24]. The current study confirms that regeneration by percent central nucleation in the quadri- the decreased normalized tidal and minute volumes at 6 ceps of P448Lneo− mice at 2, 6 (both unexercised and and 9 months of age correspond with increased inflam- exercised), and 9 months of age. Interestingly, fibrosis mation and fibrosis in the diaphragm. We also show a was limited in skeletal muscle (quadriceps and triceps) functional decline in respiration with age. Interestingly, of the unexercised mice, but was significantly increased P448Lneo− mice demonstrated a reduced decline in re- in exercised mice. Maricelli et al. used a modified exer- spiratory rate over time. This is likely related to the fact cise protocol, based on studies from Rocco et al. [30], that older mice have reduced tidal volumes, and they which included two sessions where mice exercised to ex- can maintain higher respiratory rates for their activity haustion. This protocol elicited both functional and due to the compensatory effort by the remaining mus- histological change in exercised mice including de- cles. However, respiratory rates in more severe dys- creased grip strength, short time to exhaustion, in- trophic phenotypes, and perhaps also patients lacking creased fibrosis in the diaphragm, and increased serum regeneration capacity, will likely decrease more signifi- CK levels of P448Lneo− mice compared to unexercised cantly with age. Interestingly, exercised P448Lneo− mice mice and controls [24, 30]. While an optimal exercise showed less tidal and minute volume loss compared to protocol is not yet known, degree of exercise is clearly unexercised mice. This may be again related to exercise- important to the course of disease progression, and the related compensatory regeneration in the diaphragm in- P448Lneo− mice provide a model for such further dicated by significant increase in central nucleation analysis. (44%) compared to unexercised mice (25%). Other po- tential factors, not evaluated in this study, including pul- Conclusions monary inflammation and vascular function could also This study provides more comprehensive outcome mea- be involved. Exercised mice showed significantly in- sures for the P448Lneo− mouse model of FKRP defi- creased diaphragm muscle inflammation compared to ciency. The study shows significant decrease in cardiac unexercised mice. This could lead to a more dramatic function at 9 months of age. This study is the first to decrease in respiratory function at an older age; further provide respiratory function data demonstrating signifi- studies are needed. The functional parameters of cantly decreased tidal and minute volumes in the mouse Yu et al. Skeletal Muscle (2018) 8:13 Page 15 of 16 model at 6 and 9 months of age. A chronic exercise Availability of data and materials All data generated or analyzed during this study are included in this protocol demonstrated increased skeletal muscle fibrosis, published article except for some histological data which is available upon but improved respiratory function at 6 months of age in request from the corresponding author. mutant mice. Further studies are needed to better Authors’ contributions understand the complexities of exercise on muscle path- QY collected and analyzed the functional and histological data. MM ology and disease progression. The results provide new collected and analyzed histological and biochemical data. NL collected and data on outcome measures for future preclinical drug analyzed the histological and biochemical data. AF collected and analyzed biochemical data. RR collected and analyzed the functional data. AB was trials using the P448Lneo− mouse as a model system for involved in the study design, and collected and analyzed the histological FKRP deficiency muscular dystrophy. data. EF collected and analyzed the histological data. QL was involved in the study design and data analysis. KN was involved in the study design and data analysis. CS was involved in the study design, data analysis, and a major Additional files contributor to writing manuscript. All authors read and approved the final manuscript. Additional file 1: Figure S1. Behavioral activity monitoring in P448Lneo− (FKRP), exercised P448Lneo− (FKRP-treadmill), and control (BL6)mice from 1 Ethics approval to 9 months of age. FKRP-treadmill mice were only measured until 6 months This study was carried out in strict accordance with the recommendations in of age. Panel A: vertical activity (VACTV) data; panel B: horizontal activity the Guide for the Care and Use of Laboratory Animals of the National (HACTV) data; panel C: total distance traveled (cm) during session (TOTDIST) Institutes of Health. All experiments were performed in accordance with data; panel D: time (sec, seconds) spent in movement (MOVTIME); panel E: Children’s National Medical Center IACUC approved protocol #30432. time (sec, seconds) spent resting (RESTIME). No significant differences were seen between groups for all measures. (TIFF 136 kb) Consent for publication Not applicable. Additional file 2: Table S1. Histological analyses for skeletal muscles and serum creatinine kinase levels in P448Lneo− (FKRP) and control Competing interests (BL6) mice at 1, 2, 6, and 9 months of age. (DOCX 16 kb) The authors declare that they have no competing interests. Additional file 3: Table S2. Histological analyses of the diaphragm in P448Lneo− (FKRP) and control (BL6) mice at 1, 2, 6, and 9 months of age. (DOCX 15 kb) Publisher’sNote Springer Nature remains neutral with regard to jurisdictional claims in Additional file 4: Table S3. Echocardiography results for P448Lneo− (FKRP) published maps and institutional affiliations. and control (BL6) mice at 2, 6, and 9 months of age showing increased cardiac hypertrophy and decreased systolic function at 9 months of age. Author details (DOCX 15 kb) Center for Genetic Medicine Research, Children’s Research Institute, Children’s National Health System, Washington, DC, USA. School of Abbreviations Pharmacy and Pharmaceutical Sciences, Binghamton University, State ANOVA: analysis of the variance; BL6: C57BL/6J control mice; bpm: Beats per University of New York, Binghamton, NY, USA. Department of Oncology, minute; BPM: Breaths per minute; BW: Body weight; C57: C57BL/6J control Ruijing Hospital, School of Medicine, Shanghai Jiao Tong University, mice; cDNA: Complementary deoxyribonucleic acid; CK: Creatinine kinase; Shanghai, China. McColl-Lockwood Laboratory for Muscular Dystrophy CMD: Congenital muscular dystrophies; CO: Cardiac output; DGC: Dystrophin- Research, Department of Neurology, Carolinas Healthcare System, Charlotte, glycoprotein complex; ECM: Extracellular matrix; EF: Ejection fraction; NC, USA. Children’s National Heart Institute, Center for Genetic Medicine FKRP: Fukutin-related protein; Fn1: Fibronectin 1; GADPH: Glyceraldehyde Research, Children’s National Health System, Washington, DC, USA. 3-phosphate dehydrogenase; GAS: Gastrocnemius; HR: Heart rate; IACUC: Institutional Animal Care and Use Committee; LGMD: Limb-girdle Received: 17 August 2017 Accepted: 20 March 2018 muscular dystrophy; LV mass cor: Left ventricular mass corrected; LV: Left ventricular; LVAW, d: Left ventricular anterior wall thickness in diastole; LVID, d: Left ventricular internal dimension in diastole; LVPW, d: Left ventricular References posterior wall thickness in diastole; LVVol, d: Left ventricular volume in 1. Mercuri E, Muntoni F. Muscular dystrophies. Lancet. 2013;381:845–60. diastole; MPI: Myocardial performance index; mRNA: Messenger ribonucleic 2. Kanagawa M, Toda T. The genetic and molecular basis of muscular acid; MV: Minute ventilation; NIH: National Institutes of Health; dystrophy: roles of cell-matrix linkage in the pathogenesis. J Hum Genet. Nppa: Natriuretic peptide type a; Nppb: Natriuretic peptide type b; NS: Not 2006;51:915–26. significant; PCR: Polymerase chain reaction; Quad: Quadriceps; 3. Ervasti JM, Campbell KP. Membrane organization of the dystrophin- RNA: Ribonucleic acid; SD: Standard deviation; SF: Shortening fraction; glycoprotein complex. Cell. 1991;66:1121–31. Sol: Soleus; SV: Stroke volume; TA: Tibialis anterior; Tri: Triceps; TV: Tidal 4. Taniguchi-Ikeda M, Morioka I, Iijima K, Toda T. Mechanistic aspects of the volume; α-DG: Alpha-dystroglycan formation of alpha-dystroglycan and therapeutic research for the treatment of alpha-dystroglycanopathy: a review. Mol Asp Med. 2016;51:115–24. Acknowledgements 5. Falsaperla R, Pratico AD, Ruggieri M, Parano E, Rizzo R, Corsello G, Vitaliti G, Not applicable. Pavone P. Congenital muscular dystrophy: from muscle to brain. Ital J Pediatr. 2016;42:78. Funding 6. Godfrey C, Clement E, Mein R, Brockington M, Smith J, Talim B, Straub V, This work was supported by a grant from the LGMD2i Research Fund; the Robb S, Quinlivan R, Feng L, et al. Refining genotype phenotype Carolinas Muscular Dystrophy Research Endowment at the Carolinas correlations in muscular dystrophies with defective glycosylation of HealthCare Foundation, Charlotte NC; National Institutes of Health NICHD dystroglycan. Brain. 2007;130:2725–35. 5U54HD071601; National Institutes of Health NCRR K26 OD011171; National 7. Brockington M, Yuva Y, Prandini P, Brown SC, Torelli S, Benson MA, Institutes of Health NIAMS P50AR060836 and R56NS097229; NIAID Herrmann R, Anderson LV, Bashir R, Burgunder JM, et al. 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J Appl Physiol (1985). 2017; � Our selector tool helps you to find the most relevant journal 123(5):1126–38. � We provide round the clock customer support 25. Spurney CF, Gordish-Dressman H, Guerron AD, Sali A, Pandey GS, Rawat R, � Convenient online submission Van Der Meulen JH, Cha HJ, Pistilli EE, Partridge TA, et al. Preclinical drug trials in the mdx mouse: assessment of reliable and sensitive outcome � Thorough peer review measures. Muscle Nerve. 2009;39:591–602. � Inclusion in PubMed and all major indexing services 26. Nagaraju K, Raben N, Loeffler L, Parker T, Rochon PJ, Lee E, Danning C, � Maximum visibility for your research Wada R, Thompson C, Bahtiyar G, et al. Conditional up-regulation of MHC class I in skeletal muscle leads to self-sustaining autoimmune myositis and Submit your manuscript at myositis-specific autoantibodies. Proc Natl Acad Sci U S A. 2000;97:9209–14. www.biomedcentral.com/submit

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Skeletal MuscleSpringer Journals

Published: Apr 6, 2018

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