Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 14-Day Trial for You and Your Team.

Learn More →

Early onset muscle weakness and disruption of muscle proteins in mouse models of spinal muscular atrophy

Early onset muscle weakness and disruption of muscle proteins in mouse models of spinal muscular... Background: The childhood neuromuscular disease spinal muscular atrophy (SMA) is caused by mutations or deletions of the survival motor neuron (SMN1) gene. Although SMA has traditionally been considered a motor neuron disease, the muscle-specific requirement for SMN has never been fully defined. Therefore, the purpose of this study was to investigate muscle defects in mouse models of SMA. −/− Methods: We have taken advantage of two different mouse models of SMA, the severe Smn ;SMN2 mice and the 2B/− less severe Smn mice. We have measured the maximal force produced from control muscles and those of SMA model mice by direct stimulation using an ex vivo apparatus. Immunofluorescence and immunoblot experiments were performed to uncover muscle defects in mouse models of SMA. Means from control and SMA model mice samples were compared using an analysis of variance test and Student’s t tests. −/− Results: We report that tibialis anterior (TA) muscles of phenotype stage Smn ;SMN2 mice generate 39% less maximal force than muscles from control mice, independently of aberrant motor neuron signal transmission. In −/− addition, during muscle fatigue, the Smn ;SMN2 muscle shows early onset and increased unstimulated force compared with controls. Moreover, we demonstrate a significant decrease in force production in muscles from pre- −/− 2B/− symptomatic Smn ;SMN2 and Smn mice, indicating that muscle weakness is an early event occurring prior to any overt motor neuron loss and muscle denervation. Muscle weakness in mouse models of SMA was associated with a delay in the transition from neonatal to adult isoforms of proteins important for proper muscle contractions, such as ryanodine receptors and sodium channels. Immunoblot analyses of extracts from hindlimb skeletal muscle 2+ revealed aberrant levels of the sarcoplasmic reticulum Ca ATPase. Conclusions: The findings from this study reveal a delay in the appearance of mature isoforms of proteins important for muscle contractions, as well as muscle weakness early in the disease etiology, thus highlighting the contributions of skeletal muscle defects to the SMA phenotype. Keywords: Motor neuron disease, Skeletal muscle, Sodium channels, Ryanodine receptors, SERCA, Spinal muscular atrophy, Survival motor neuron * Correspondence: rkothary@ohri.ca Ottawa Hospital Research Institute, Regenerative Medicine Program, 501 Smyth Road, Ottawa, ON K1H 8L6, Canada Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, ON K1H 8M5, Canada Full list of author information is available at the end of the article © 2013 Boyer et al.; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Boyer et al. Skeletal Muscle 2013, 3:24 Page 2 of 13 http://www.skeletalmusclejournal.com/content/3/1/24 Background studies demonstrating a lack of increase in myofiber size With an overall carrier frequency of 1:40, spinal muscu- and the increased levels of embryonic and neonatal lar atrophy (SMA) is a major leading genetic cause of myosin heavy chain (MHC) isoforms [18-20]. However, infant deaths, affecting 1 in 6,000 to 10,000 births [1-3]. it is not known whether or how impaired muscle growth Spinal muscular atrophy is an autosomal recessive dis- contributes to muscle weakness in SMA, since at present order traditionally classified into different types based on no comprehensive analysis has been performed relating the clinical severity of the symptoms [4]. In 1995, the to muscle force production in mouse models of SMA. SMA-determining gene was identified and named Here, we show previously unreported pathophysio- −/− ‘survival motor neuron’ (SMN) [5]. This gene is located logical muscle defects in severe (Smn ;SMN2) and less 2B/− on chromosome 5q13 in humans, in a region containing severe (Smn ) mouse models of SMA. We report pro- an inverted duplication of 500 kilobase pairs. This nounced muscle weakness in these mice. These observa- results in two virtually identical copies of the SMN gene; tions were associated with altered expression of proteins SMN1 and SMN2 [5-8]. that are developmentally regulated and are important for In the mouse, the Smn gene is present as a single copy, proper physiological muscle function. Furthermore, we and homozygous loss of function leads to a pre- show that muscle weakness is an early feature, observed implantation lethality [9]. However, when the Smn knock- prior to any overt motor neuron loss and muscle de- out is coupled with low levels of human SMN expressed nervation in mouse models of SMA. Thus, we conclude from a SMN2 transgene, a severe phenotype approximat- that muscle defects contribute to the phenotype in SMA −/− ing type I SMA is observed in Smn ;SMN2 mice [10]. mouse models. Uncovering skeletal muscle defects in Since this original discovery, several other mouse models the context of SMA is of the utmost importance to bet- of SMA have been generated, including a milder model ter understand the SMA phenotype and for the develop- 2B/− termed Smn . These latter mice do not harbor the ment of targeted therapeutics. SMN2 transgene but rather harbor one null allele and a second allele with a 3-nucleotide substitution in the Methods exonic splice enhancer of exon 7 of the mouse Smn gene Mouse models 2B/- −/− (2B mutation) [11]. Smn model mice display a milder The Smn ;SMN2 (Jackson Labs, Bar Harbor, ME, USA) 2B/− SMA phenotype, owing to slightly higher Smn protein and Smn [12] mice were housed and cared for levels than the severe model [12]. according to the Canadian Council on Animal Care guide- Motor neuron cell loss and muscle denervation are lines and the University of Ottawa Animal Care Commit- considered two pathological hallmarks of SMA. Exactly tee protocols. Tissues from pre-symptomatic mice were −/− how SMN depletion leads to motor neuron degeneration collected at postnatal day (P) 2 for severe Smn ;SMN2 2B/− is unclear and remains the focus of intense research. In mice, and P9 for Smn mice. Tissues were also col- −/− addition, recent advances in the field have highlighted lected from phenotype stages at P5 for Smn ;SMN2 2B/− the involvement of other tissues in the pathophysiology and P21 for Smn mice. Muscles used for RNA and of SMA, of which skeletal muscle appears to be an protein analysis were flash frozen in liquid nitrogen and important candidate [4,13]. stored at −80°C. In Drosophila, Smn was discovered to be a sarcomeric protein interacting with α-actinin, a cross-linking pro- Hindlimb denervation tein that stabilizes actin microfilaments [14]. Walker and Denervation surgeries were performed in accordance with colleagues later confirmed these findings and specifically the guidelines set by the Canadian Council on Animal identified Smn as a Z-disc component in skeletal and Care. Young mice (P14) were anaesthetized by inhalation cardiac muscle of mice [15]. At present, the function of of isoflurane. Experimental denervation was achieved by Smn at this adhesion site is unknown but Smn is likely revealing the sciatic nerve and removing 2 to 3 mm of the to have a specific function other than snRNP biogenesis nerve in the thigh section to cease neural input and pre- in muscle [15]. An Smn interacting protein screen in vent nerve regrowth. A sham procedure was performed in C2C12 myoblasts suggests that the function of Smn in parallel to serve as control; this consisted of exposing the muscle is dynamic and probably differs during varying mice to identical experimental conditions except for cut- stages of myogenesis based on its protein interactome ting the nerve. The TA muscles were collected and flash [16]. A proteomic screen performed by Mutsaers et al. frozen from denervated and sham operated mice one and identified an increase in proteins involved in pro- seven days following surgery. −/− grammed cell death in pre-symptomatic Smn ;SMN2 mice [17]. Several reports have highlighted the possibil- Immunoblotting ity of delayed myogenesis in mouse models of SMA. The Total tissue lysate extract was obtained by grinding flash basis of this notion comes from muscle morphological frozen tissues in a liquid nitrogen pre-cooled mortar and Boyer et al. Skeletal Muscle 2013, 3:24 Page 3 of 13 http://www.skeletalmusclejournal.com/content/3/1/24 pestle. The concentration of each sample was deter- it was calculated as the difference in the baseline 5 ms be- mined by Bradford assay. Samples were subjected to fore a contraction and the baseline 5 ms before fatigue was sodium dodecyl sulfate polyacrylamide gel electrophor- elicited. Muscle weight and length were used to calculate esis and examined by immunoblot, as previously de- the cross-sectional area of the muscle that was used to scribed [16]. Primary antibodies used were: calsequestrin normalize force measurements in each experiment. (Abcam, Toronto, ON, Canada), glyceraldehyde-3- phosphate dehydrogenase (GAPDH, Abcam, Toronto, ON, RNA isolation Canada), Na 1.4 (Alomone, Jerusalem, Israel), Na 1.5 Total RNA was isolated from skeletal muscle tissue using v v (Alomone, Jerusalem, Israel), nuclear factor 1 (Abcam, a homogenizer and the RNeasy kit (Qiagen, Toronto, ON, 2+ Toronto, ON, Canada), sarcoplasmic reticulum Ca Canada) according to the manufacturer’s instructions. ATPase (SERCA1a, Cell Signaling, Danvers, MA, USA), RNA samples were treated with DNase (gDNA wipeout and zinc-finger E box-binding protein (ZEB) (Novus Bio- buffer, Qiagen, Toronto, ON, Canada) to eliminate DNA logicals, Littleton, CO, USA). Signals were detected using contamination and concentrations were determined using enhanced chemiluminescence (Thermo, Florence, KY, a Nanophotometer spectrophotometer (MBI Lab Equip- USA). Densitometric analyses were performed using ImageJ ment, Dorval, QC, Canada). software (NIH). Immunoblot data were normalized to GAPDH levels to control for possible loading differences. Reverse-transcription polymerase chain reaction (RT-PCR) RNA was reverse-transcribed using the quantitect reverse- Force measurements and fatigue protocol transcription kit (Qiagen, Toronto, ON, Canada). Primer The TA muscles were dissected from P2 and P5 control sequences and PCR conditions used to detect the spliced −/− and severe Smn ;SMN2 mice, and from P9 control and variants of the ryanodine receptor RyR1 gene were identi- 2B/− Smn mice. Muscles were constantly immersed in cal to those previously described [21]. A negative control physiological saline solution containing 118.5 mM NaCl, in which water was added instead of cDNA was prepared 4.7 mM KCl, 2.4 mM CaCl ,3.1 mM MgCl ,25mM in parallel for every PCR. Quantification of the RT-PCR 2 2 NaHCO ,2mM NaH PO ,and 5.5 mMD-glucose.Solu- results was achieved using ImageJ software. 3 2 4 tions were continuously bubbled with 95% O ,5%CO for 2 2 a pH of 7.4. Solutions containing 30 μM of tubocurarine Immunofluorescence hydrochloride pentahydrate (Sigma, Oakville, ON, Canada) Neuromuscular junction (NMJ) immunofluorescence and were prepared by adding the appropriate amount directly quantification was performed as described previously [22]. to the physiological solution. The flow of physiological so- Post-synaptic acetylcholine receptors were labeled with lution below and above muscles was maintained at a total α-bungarotoxin (Molecular Probes, Burlington, ON, of 15 ml/min and a temperature of 37°C. Tetanic contrac- Canada) while the pre-synaptic terminal was labeled with tions were elicited with electrical stimulations applied anti-neurofilament and anti-synaptic vesicle protein 2 across two platinum wires (4 mm apart) located on oppos- (both from Developmental Studies Hybridoma Bank, Iowa ite sides of the muscle. Electrodes were connected to a City, IA, USA). All secondary antibodies were purchased Grass S88 stimulator and a Grass SIU5 isolation unit from Jackson Labs. Immunofluorescence images were cap- (Grass Technologies/Astro-Med Inc., Warwick, RI, USA). tured using a Zeiss Confocal microscope (LSM 510 Tetanic contractions were elicited with 200 ms trains of Meta DuoScan, Toronto, ON, Canada). For each 0.3 ms, 12 V (supramaximal voltage) pulses at a frequency muscle, four to six fields of view were quantified and a of 200 Hz. For all experiments, muscle length was adjusted total of counted endplates ranging between 99 and 263 to achieve maximal force production and muscles were were included per animal in the analysis. allowed a 30 min equilibration period during which a tet- anic contraction was elicited every second. Maximal force Histological analysis production was determined by increasing frequencies from The lumbar (L1 and L2) region of the spinal cord was −/− 1 to 200 Hz. Muscles were then fatigued by increasing the collected from control, pre-symptomatic Smn ;SMN2 2B/− contraction rate to one contraction per second for 180 s. and Smn mice. Tissues were fixed in 4% paraformal- Twitch (obtained when stimulated with one square pulse) dehyde for 24 hrs, embedded in paraffin, cut into sec- or tetanic force was defined as the force that developed tions (10 μm) and stained with hematoxylin and eosin during stimulation and was calculated as the difference be- (H & E). Motor neurons were identified by their shape tween the maximum force during contraction and the and size within the ventral horn region of the spinal force measured 5 ms before the contraction. Unstimulated cord. Motor neuron quantification was performed on force was defined as the force generated by muscles in the every fifth section within the L1 and L2 region on three absence of electrical stimulation and was observed during mice from each genotype. Histological analyses were also fatigue when muscles failed to relax between contractions; performed on cross-sections (10 μm) from frozen TA Boyer et al. Skeletal Muscle 2013, 3:24 Page 4 of 13 http://www.skeletalmusclejournal.com/content/3/1/24 −/− 2B/− muscles of P2 Smn ;SMN2,and P9 Smn mice. Sec- less maximum peak tetanic force, as measured at 200 Hz tions were stained with H & E using a standard protocol, (Figure 1A,B). images were taken with a Zeiss Axioplan2 microscope, Aberrant NMJ morphology and function have previ- and the myofiber area was calculated using ImageJ soft- ously been highlighted in several mouse models of SMA ware. Approximately one thousand fibers were counted [20,22-24]. In isolated muscle preparations, many fibers for each genotype analyzed. receive an indirect stimulation via the remaining nerve stump. Although it has previously been demonstrated that the TA muscle is fully innervated in SMA model Statistical analyses mice [20,25], one possible explanation for the observed Data are presented as the mean ± standard error of the decrease in force is that the applied stimulus enters the mean. Analysis of variance (Statistical Analysis Software residual nerve before reaching the myofibers. Thus, if Institute Inc., Cary, NC, USA) was used to determine poorly functioning NMJs were present in the prepar- significance in the fatigue data. A Student’s t test was ation, it would negatively impact the force because fewer performed using MS Excel to compare the means of all fibers would be stimulated. To address this possibility, other data. Significance was set at P < 0.05. we measured the maximal force production in muscles from control and mutant mice in the presence or ab- Results sence of tubocurarine, which blocks acetylcholine recep- −/− Skeletal muscle weakness in Smn ;SMN2 mice tors. Should aberrant NMJs negatively affect TA muscle To date, no physiological study has been performed on force production, it would be expected that the relative −/− muscles from severe SMA model mice. To this end, we force produced by muscles from Smn ;SMN2 mice have analyzed the twitch and peak tetanic force pro- would be equal to the control values in the presence of −/− duced by direct stimulation of TA muscles of Smn ; tubocurarine. The force production of control muscles SMN2 mice and control littermates at P5. To account was not affected by the presence of tubocurarine nor did −/− for variations in muscle size, all force values were nor- we observe an increase in force production in Smn ; malized to muscle cross-sectional area. Compared with SMN2 muscles after the addition of tubocurarine to the −/− control muscles, Smn ;SMN2 mice produced 47% less preparation compared with non-treated muscles (data not twitch force, as measured after one stimulation, and 39% shown). Furthermore, in five independent experiments, −/− −/− Figure 1 Muscle weakness in muscle from Smn ;SMN2 mice. (A) TA muscle preparations from P5 Smn ;SMN2 mice and control littermates −/− were used to assess tetanic and twitch force normalized to the muscle cross-sectional area. P5 Smn ;SMN2 TA muscles produce significantly less −/− twitch force than control littermates. (B) Reduction in normalized maximal peak tetanic force in P5 Smn ;SMN2 TA muscle compared with −/− controls. (C) Administration of tubocurarine to block NMJs did not influence relative force production in Smn ;SMN2 muscles. In five −/− independent experiments, Smn ;SMN2 mice produced less force than controls following treatment with tubocurarine. (D) Similar relative force −/− decreases in Smn ;SMN2 and control mice during fatigue elicited with one tetanic contraction every second for 3 min. Peak tetanic forces are expressed as a percentage of the pre-fatigue force. (E) Unstimulated force occurred when muscles failed to relax between contractions and is −/− expressed as a percentage of the pre-fatigue peak tetanic force. During a fatigue protocol, P5 Smn ;SMN2 TA muscles show increased −/− unstimulated force compared with controls. (F) Unstimulated force appeared much sooner in Smn ;SMN2 TA muscles than in controls. NMJ, neuromuscular junction; TA, tibialis anterior; N = 5 or 6; *, P < 0.05. Boyer et al. Skeletal Muscle 2013, 3:24 Page 5 of 13 http://www.skeletalmusclejournal.com/content/3/1/24 −/− the force production from Smn ;SMN2 muscle was Although we observed no overt motor neuron loss or lower than that of controls (Figure 1C). These data show denervation in both models at pre-symptomatic stage, we that at the phenotype stage, aberrant NMJs did not im- observed a significant decrease in peak tetanic force. TA −/− −/− pact Smn ;SMN2 TA ex vivo muscle force production muscles from P2 Smn ;SMN2 mice produced 67% lower andthatmechanismsof stimuluspropagation might be peak tetanic force than control littermates (Figure 3A). TA −/− 2B/− compromised in muscles from Smn ;SMN2 mice. muscles from P9 Smn mice produced 61% lower peak tetanic force than control littermates (Figure 3C). The −/− Smn ;SMN2 muscles respond abnormally to induced peak forces were normalized to the cross-sectional area of muscle fatigue each muscle; however, at this stage, we did not observe −/− To determine whether Smn ;SMN2 muscles respond any significant difference in mean fiber area between 2B/− −/− differently to muscle fatigue, we measured the decline in mutant and control muscle in either Smn or Smn ; force with repeated tetanic stimulation for 180 s. The SMN2 mice (Figure 3B,D). Taken together, these data −/− decrease in peak tetanic force recorded in Smn ;SMN2 demonstrate that in two different mouse models of SMA, muscles was similar to control littermates (Figure 1D). muscle weakness is an early feature, occurring prior to During the fatigue protocol, we also measured the un- any overt motor neuron loss and denervation. stimulated force, which is defined as the force measured 100 ms before a contraction is elicited. The TA muscles Decreased expression of mature ryanodine receptor 1 −/− of both control and Smn ;SMN2 P5 mice generated an transcripts in muscle from SMA model mice increase in unstimulated force as they failed to com- The results of our physiology experiments led us to in- pletely relax between contractions (Figure 1E). However, vestigate possible causes for the decrease in force pro- −/− −/− the Smn ;SMN2 muscle produced significantly more duction from Smn ;SMN2 muscle. During a muscle unstimulated force than control counterparts. The un- contraction, calcium is released from the sarcoplasmic stimulated force of control TA muscles had a mean time reticulum to the sarcomere to allow for the actin-myosin of appearance at 133 s and was equivalent to 4.4% of the cross-bridge cycling. The calcium release is mediated by pre-fatigue tetanic force by the end of the protocol ryanodine receptor 1 (RyR1), which is the predominant −/− (Figure 1E,F). However, for the P5 Smn ;SMN2 TA ryanodine receptor expressed in mature muscle [26]. muscles, the average time of appearance started signifi- Several splice variants of the RyR1 gene exist. For ex- cantly sooner, i.e. at 49 s, with a final mean of 8.2% ample, one variant is called ASI and is expressed without (Figure 1E,F). Therefore, our results suggest the pres- exon 70 [ASI (−)] in neonatal muscle and transitions to −/− ence of a defect in Smn ;SMN2 muscles, resulting in an alternatively spliced variant that includes exon 70 in an inability to recover from muscle fatigue over time. mature skeletal muscle [ASI (+)] [27]. The second RyR1 splice variant is ASII, which is further spliced to exclude −/− Pre-symptomatic muscle weakness in Smn ;SMN2 and exon 83 in immature muscle [ASII (−)] or to include 2B/− Smn mice that exon in mature muscle [ASII (+)] [21,27]. Using Whilst we observed a significant decrease in force pro- PCR primers designed to target the mature and imma- −/− duction in muscles from phenotypic Smn ;SMN2 mice, ture variants, we assessed the RyR1 transcripts in hind- which was independent of aberrant nerve transmission limb skeletal muscle RNA extracts from mouse models in the ex vivo preparations, it remains possible that the of SMA and in controls. A time course analysis demon- muscle weakness observed could be attributed to motor strates the predominant expression of ASII (+) in mature neuron degeneration occurring prior to the stage of our muscle (P21) over the ASII (−) variant in wild type mice analyses. We therefore assessed the peak tetanic force in (Figure 4A). At P5, the predominant ASII isoform in −/− pre-symptomatic mice. For this, we have extended our control mice was ASII (+), while in Smn ;SMN2 muscle −/− 2B/− analysis to include both Smn ;SMN2 and Smn there was a relative increase in the proportion of the neo- mouse models. This analysis was performed at pre- natal variant ASII (−) over ASII (+) (Figure 4B middle −/− phenotypic time point of P2 and P9 in Smn ;SMN2 panel and Figure 4D). In control P21 mice, the adult ASII 2B/− and Smn model mice, respectively. To confirm that (+) variant was predominant relative to the neonatal ASII 2B/− these time points preceded neurodegenerative events, (−) variant, whereas in Smn mice both variants were we assessed motor neuron number and NMJ integrity. expressed at similar levels (Figure 4C middle panel and −/− 2B/− At P2 in Smn ;SMN2 mice, and P9 in Smn mice, Figure 4D). there was no difference in the number of motor neuron Using the same approach, we assessed the transcript cell bodies compared with controls (Figure 2A,D). Fur- levels of ASI using primers targeting both the neonatal thermore, the percentage of fully occupied endplates was and adult ASI splice variant. We did not observe any dif- unchanged between each mouse model of SMA and the ference in neonatal versus adult transcript levels of the −/− respective controls (Figure 2E,H). ASI variant for the Smn ;SMN2 mice (Figure 4B upper Boyer et al. Skeletal Muscle 2013, 3:24 Page 6 of 13 http://www.skeletalmusclejournal.com/content/3/1/24 Figure 2 (See legend on next page.) Boyer et al. Skeletal Muscle 2013, 3:24 Page 7 of 13 http://www.skeletalmusclejournal.com/content/3/1/24 (See figure on previous page.) −/− 2B/− Figure 2 Normal motor neuron counts and NMJ integrity in P2 Smn ;SMN2 and P9 Smn mice. (A, B) Representative images of H & E −/− staining of motor neurons in the ventral horn region of the L1 and L2 spinal cord region of P2 control and Smn ;SMN2 mice, and P9 control 2B/− and Smn mice. Scale bar = 50 μm. (C, D) Quantification of motor neuron cell body number within the ventral horn of the lumbar (L1 and L2) −/− 2B/− region of the spinal cord for control and pre-symptomatic Smn ;SMN2 and Smn mice. (E, F) Representative images showing fully intact −/− 2B/− NMJs from TA muscles of control and pre-symptomatic Smn ;SMN2 (E) and Smn (F) mice. Post-synaptic acetylcholine receptors were labeled with α-bungarotoxin (red) while the pre-synaptic terminal was labeled with anti-NF (green) and anti-SV2 (green). Scale bar = 20 μm. −/− (G, H) Quantification of the percentage of fully occupied endplates revealed no difference between control and pre-symptomatic Smn ;SMN2 2B/− and Smn mice. NF, neurofilament; NMJ, neuromuscular junction; SV2, synaptic vesicle protein 2; N = 3 for all experiments. 2B/− panel and Figure 4D). In P21 Smn mice, however, the splicing pattern of the RyR1 ASII variants in denervation neonatal ASI (−) transcript was the predominant variant compared with control samples, either one day or seven expressed, while in control muscles, the ASI (+) transcript days post-denervation (Figure 4E,F). These results support was the major ASI variant expressed (Figure 4C upper the hypothesis that the changes in RyR1 splicing pattern panel and Figure 4D). Collectively, the aberrant expression in muscles from the mouse models of SMA are not attrib- pattern of the RyR1 transcripts suggests a delay in muscle utable to pre-synaptic pathology and are therefore poten- development in mouse models of SMA. tially reflective of a muscle developmental defect. Muscle denervation at the NMJ is a pathological fea- ture observed in mouse models of SMA. Although there Altered sodium channel levels in SMA mice are generally low levels of denervation in the hindlimb In excitable cell types, such as neurons and myocytes, muscles of SMA model mice [28], we investigated whether sodium channels propagate the action potential. Sodium denervation would influence the expression the RyR1 channel expression is a developmentally regulated process transcript. To do so, we experimentally denervated mus- in which an isoform switch, Na 1.5 to Na 1.4, occurs dur- v v cles of 2-week-old wild type mice and examined the ex- ing postnatal development in the mouse [29,30]. Na 1.4, pression of RyR1 in denervated samples compared with the predominant sodium channel isoform in adult skeletal sham controls. We did not detect any changes in the muscle [31], must be expressed at the correct time point −/− 2B/− −/− Figure 3 Pre-symptomatic muscle weakness in Smn ;SMN2 and Smn mice. (A) P2 Smn ;SMN2 force measurements revealed a 67% decrease in maximal tetanic force production compared with controls. Force data were normalized to the muscle cross-sectional area. (B) The 2B/− average peak tetanic force was reduced by 61% in P9 pre-symptomatic Smn TA muscles compared with control littermates. (C) Mean fiber −/− 2B/− area of P2 TA muscles from Smn ;SMN2 and control mice. (D) Average fiber cross-sectional area for P9 Smn and control TA muscle. N = 3 for all experiments. *, P < 0.05. Boyer et al. Skeletal Muscle 2013, 3:24 Page 8 of 13 http://www.skeletalmusclejournal.com/content/3/1/24 Figure 4 Delayed expression of adult RyR1 mRNA splice variant in muscles from mouse models of SMA. (A) RT-PCR on RNA from hindlimb muscle from wild type mice with primers directed against ASII (+) and ASII (−). GAPDH served as a loading control to confirm equivalence of starting cDNA levels . Note that relative ratio of ASII (+) to ASII (−) increases from P2 to P21. (B) RT-PCR results demonstrated no −/− change in the expression of ASI (+) and ASI (−) variants in control and Smn ;SMN2 samples at P5 (upper panel). However, there was decreased −/− expression of ASII (+) and sustained expression of ASII (−) in muscle samples from P5 Smn ;SMN2 compared with controls (middle panel). GAPDH served as a loading control. N = 5 for each genotype. (C) In control P21 mice, we observed increased expression of ASI (+) transcripts 2B/− relative to ASI (−) transcripts. However in Smn mice, the relative ratio of ASI (+) to ASI (−) transcripts was decreased (upper panel). 2B/− Furthermore, for the ASII variant, we observed the presence of a single transcript [ASII (+)] in P21 control samples, while in Smn samples, we observed a decrease in ASII (+) transcripts compared with controls. The ASII (−) variant was also now apparent (middle panel). GAPDH served as a −/− loading control. N = 5 for each variant. (D) Quantification of RT-PCR data show significant changes in the ASII+/ASII − ratio in Smn ;SMN2 samples compared with controls. The relative levels of adult and neonatal RYR1 isoforms was significantly altered for both the ASI and ASII 2B/− variants in Smn animals compared with controls. (E,F) The relative levels of adult and neonatal ASII RyR1 transcript variants are not altered in P14 mice one (E) and seven (F) days post-denervation compared with sham operated mice. N =3. during development to fulfill its role. A delay in expression the predominant isoform expressed in the adult heart of the Na 1.4 isoform can negatively impact muscle force and in early stages of skeletal muscle development [30]. production [32]. These results suggest that muscle development is de- As expected, we observed a robust increase in Na 1.4 layed in SMA model mice and that development is se- −/− levels in wild type muscle during postnatal development verely impaired, especially in Smn ;SMN2 mice, where from P2 to P21 (Figure 5A). Interestingly, in two inde- both Na isoform levels are decreased. pendent mouse models of SMA, there is a decrease in To gain a better understanding of how Na 1.4 is mis- the levels of Na 1.4 compared with control mice. Speci- regulated in SMA mice, we assessed the status of proteins −/− fically, in P5 Smn ;SMN2 mice, Na 1.4 and Na 1.5 known to regulate sodium channel expression. Hebert and v v levels were significantly decreased in hindlimb skeletal colleagues [32] have previously demonstrated that the muscle compared with control counterparts (Figure 5B). transcription factor NF1 is recruited to the Na 1.4 gene 2B/− Similarly, in muscle from phenotype stage P21 Smn promoter by myogenic regulatory factors to enhance its mice, there was a decrease in Na 1.4 levels compared expression. We did not observe any differences in the 2B/− with controls (Figure 5C). In addition to the decrease in levels of NF1 in muscle from P21 Smn mice compared Na 1.4, we observed an increase in Na 1.5 levels in with controls (Figure 5D). Another transcription factor, v v 2B/− Smn muscle (Figure 5C). Sodium channel Na 1.5 is ZEB, is a Na 1.4 repressor. As with NF1, we did not v v Boyer et al. Skeletal Muscle 2013, 3:24 Page 9 of 13 http://www.skeletalmusclejournal.com/content/3/1/24 Figure 5 Na 1.4 protein levels are decreased in muscles from mouse models of SMA. (A) Immunoblot analysis using muscle lysate from P2, P5, P9, and P21 wild type mice. Na 1.4 protein levels increase during postnatal muscle development and form the predominant sodium channel expressed in mature skeletal muscle. GAPDH served as a loading control (N = 3). (B) Representative immunoblot with quantification, showing a decrease in levels of −/− sodium channel Na 1.4 and Na 1.5 in P5 Smn ;SMN2 hindlimb v v muscle compared with controls (N = 3). (C) Quantification of 2B/− immunoblot analyses in P21 Smn and control hindlimb muscles revealed a decrease in Na 1.4 levels. Early in postnatal muscle development, the Na 1.5 sodium channel isoform is the most 2B/− predominant. In P21 Smn mice, the protein levels of Na 1.5 are higher than in controls (N = 3). (D) The protein level of the Na 1.4 2B/− positive regulator, NF1, is not altered in muscles from P21 Smn mice. Similarly, no change was detected in the protein levels of the Na 1.4 repressor ZEB. (E) Expression of sodium channel Na 1.4 in v v control sham and denervated samples 1 and 7 days post- denervation was assessed by immunoblot (N = 3). A decrease in the levels of Na 1.4 in muscle was noted at 7 days post-denervation. *, P < 0.05; **, P < 0.01. 2B/− observe any change in ZEB levels in muscle from Smn mice (Figure 5D). We next investigated whether Na 1.4 expression was influenced by experimental denervation. There was no change in Na 1.4 levels one day post-denervation (Figure 5E). However, a significant decrease was ob- served seven days following denervation, in agreement with previous studies [33,34]. Therefore, although the muscles used in the Na 1.4 expression analysis are not morphologically denervated, we cannot rule out the possibility that functional synaptic defects at the NMJ influence sodium channel expression in muscles from mouse models of SMA. −/− SERCA1a protein expression is altered in Smn ;SMN2 mice One possible mechanism that can cause increased un- stimulated force production is an incomplete removal of 2+ Ca from the sarcoplasm because of decreased levels of 2+ the Ca ATPase pump. The protein responsible for the 2+ Ca uptake following a muscle contraction is the sarco- 2+ plasmic reticulum Ca ATPase (SERCA), of which SERCA1a is the predominant isoform found in fast- twitch muscles, such as the TA muscle [35]. The protein expression of SERCA1a is developmentally regulated. It peaksbyP9and dropsslightlyatP21 (Figure6A).Im- munoblot analysis revealed a decrease in SERCA1a pro- −/− tein levels in hindlimb skeletal muscles from P5 Smn ; SMN2 mice compared with control samples (Figure 6B). Interestingly, levels of calsequestrin, a protein that binds 2+ and stores Ca in the sarcoplasmic reticulum, was un- −/− changed in Smn ;SMN2 muscle compared with con- 2+ trols (Figure 6B), indicating that a Ca handling defect was likely limited to the sarcoplasmic reticulum pump. Boyer et al. Skeletal Muscle 2013, 3:24 Page 10 of 13 http://www.skeletalmusclejournal.com/content/3/1/24 −/− Figure 6 SERCA1a protein level is altered in muscles from Smn ;SMN2 mice. (A) Whole muscle lysate was collected from P2, P5, P9, and P21 wild type mice and immunoblot analysis was performed to assess SERCA1a protein levels. SERCA1a levels increase over time and peak at P9 −/− (N = 3). (B) Immunoblot with quantification showing a decrease in SERCA1a, but not calsequestrin, in hindlimb muscle from P5 Smn ;SMN2 mice compared with control (N = 3). (C) Immunoblots were performed on muscle lysates collected from experimentally denervated (DEN) and sham operated (SHAM) muscle. No change in SERCA1a levels was observed. N =3, *, P < 0.05. −/− Next, we measured the influence of denervation on muscle force from pre-symptomatic Smn ;SMN2 and 2B/− SERCA1a protein levels. Protein lysate from gastrocne- Smn mice prior to any overt motor neuron loss and mius muscles was collected from denervated and sham denervation, although we cannot rule out the influence operated mice. SERCA1a protein levels were unchanged of a functional deficit within the motor neurons. It in skeletal muscle from denervated mice compared with should be noted, however, that our physiological results controls (Figure 6C). This again supports the hypothesis were normalized to the cross-sectional area of each that the observed decrease in SERCA1a in muscle from muscle tested. Therefore, the overt decrease in muscle −/− −/− Smn ;SMN2 mice could be due to a muscle develop- size observed in P5 Smn ;SMN2 mice cannot explain mental defect. the decrease in force production, per se. In addition, our experiments performed on pre-symptomatic mice allow us to rule out the possibility that smaller myofibers are Discussion the reason for the decrease in relative force production, Here, we show that in two mouse models of SMA, since no significant difference was observed in muscle muscle weakness occurs early, being evident prior to any size between pre-symptomatic and control mice. How- overt physical denervation and motor neuron loss. This ever, the maturity of the muscle may influence force physiological defect was associated with delayed expres- production, irrespective of size. As we have observed a sion of mature isoforms of proteins important for muscle decrease in the mature isoforms of a number of muscle function. Our results therefore point to muscle weakness proteins, we suggest that a decrease in muscle maturity coupled with delayed muscle development and provide −/− 2B/− in P2 Smn ;SMN2 and P9 Smn mice could contrib- new insight into the pathophysiology underlying SMA. ute to a marked decrease in force production. This work highlights the potential of muscle as a thera- peutic target and warrants further work to identify muscle directed strategies to increase muscle force production. Delayed expression of mature isoforms of muscle function proteins in mouse models of SMA Several groups have indirectly demonstrated impaired Muscle weakness in SMA mice muscle growth in mouse models of SMA by measuring We have employed an ex vivo method in which the the cross-sectional area of developing myofibers [18-20]. muscle is excised and placed in a chamber where it can These analyses suggest that shortly after birth, muscle be directly stimulated to contract. By doing so, we re- development is significantly impaired. During postnatal duce the negative contribution that degenerating motor muscle development, as myotubes grow to become neurons might have in eliciting a contraction, with the myofibers, a switch in expression from neonatal to adult caveat that there may still be functional defects preced- protein isoforms occurs for many muscle function pro- ing the analysis. We show a decrease in normalized peak −/− teins. A delay in this switch could compromise muscle tetanic force in muscle from phenotype stage Smn ; maturation and function. Such might be the case with SMN2 mice. Importantly, we show a similar decrease in Boyer et al. Skeletal Muscle 2013, 3:24 Page 11 of 13 http://www.skeletalmusclejournal.com/content/3/1/24 the expression of MHC, in which the embryonic and that of Na 1.4 decreases in denervated muscle. Indeed, perinatal MHC isoforms are predominantly expressed in we observed a decrease in Na 1.4 levels in experimen- muscle from SMA model mice [19,20]. Therefore, we tally denervated muscles (day 7), as well as in muscles hypothesized that several other proteins important for from both SMA mouse models studied. As such, we generating muscle contractions could be aberrantly cannot rule out the possibility that the mis-regulation of expressed, with juvenile isoforms predominating rather Na 1.4 is due to denervation in muscle from the symp- than adult ones, which could lead to muscle weakness in tomatic mice. mouse models of SMA. We focused on proteins that are The expression of Na 1.4 is positively regulated by the directly involved in the regulation of muscle contraction, transcription factor NF1 and is repressed by the tran- that is, proteins important for calcium regulation and ac- scription factor ZEB [32]. We did not observe any differ- tion potential propagation. ences in the expression of these two transcription 2B/− factors in Smn mice. The recruitment of the NF1 RyR1 expression in muscle from mouse models of SMA protein to the Na 1.4 promoter is mediated through two Results from our RT-PCR analysis revealed a delay in transcription factors that are important for muscle dif- the expression of the mature RyR1 splice variants in ferentiation, namely myogenin and muscle-specific regu- skeletal muscle from mouse models of SMA. In pheno- latory factor 4 (MRF4). It can be envisaged that a delay 2B/− type stage Smn mice, we observed a mis-regulation in the expression of myogenic regulatory factors, such as of both the ASI and ASII alternatively spliced variants. myogenin and MRF4, or others even more up-stream of −/− At P5 in the Smn ;SMN2 model, a change in expres- myogenin and MRF4, may explain the deferred Na 1.4 sion was evident for the ASII variant but not the ASI. expression in SMA mice. During development, the transition from ASII (−)to ASII (+) begins at P0 and is complete by P21 [27]. For −/− the ASI variant, the transition from the neonatal ASI (−) Decreased SERCA1a expression in Smn ;SMN2 mice to the adult ASI (+) form begins only at P8. Therefore, The results from our fatigue protocol demonstrate an in- the timing of the ASI transition probably explains why crease in unstimulated force and a decrease in the time 2B/− −/− we observed the delay in P21 Smn mice but not in of unstimulated force onset in Smn ;SMN2 mice. This −/− 2+ P5 Smn ;SMN2 mice. The functional studies performed observation may be indicative of a defect in Ca uptake by Kimura et al. demonstrate that neonatal RyR1 is less from the sarcomere to the sarcoplasmic reticulum, active than adult RyR1, as it binds ryanodine with less which is supported by the muscle intrinsic decrease in 2+ −/− affinity than the adult form, and therefore releases less cal- levels of the SERCA1a Ca pump in muscles of Smn ; 2+ cium [21]. Thus, the persistent expression of the neonatal SMN2 mice. Defects in Ca handling have previously RyR1 variants in mouse models of SMA probably leads to been reported in mouse models of muscular dystrophies 2+ 2+ decreased Ca release from the sarcoplasmic reticulum [21,36]. Specifically, defects related to Ca uptake and to the sarcomere, and subsequently results in weaker SERCA1 function have been described in a mouse model muscle contractions. of Duchenne’s muscular dystrophy [37]. Indeed, the overexpression of SERCA1 in skeletal muscles led to ro- Sodium channel expression in muscle from mouse bust improvements in muscle function and attenuated models of SMA muscle pathology in mouse models of muscular dys- In skeletal muscle, action potentials are generated and trophy [38]. Furthermore, RyR1 splicing defects resulting propagated by voltage-gated sodium channels. Na 1.4 is in the expression of the neonatal variants contribute to the predominant pore-conducting channel in adult the pathogenesis of the neuromuscular disease myotonic muscle. Its expression significantly increases in mice in dystrophy type 1 [21]. Thus, the defects we report in the first two weeks after birth [29,31]. Here we show muscles from SMA model mice are reminiscent of those that Na 1.4 levels are decreased in muscles from two dif- that occur in other muscle diseases. ferent mouse models of SMA. This may explain in part the lower force generation, since there would have been an insufficient number of available Na 1.4 channels to Conclusions generate action potentials during a train. Furthermore, In summary, we have demonstrated early and profound this period after birth coincides with a period of dra- muscle weakness, and aberrant expression of muscle pro- matic muscle growth, and Na 1.5 is the major sodium teins in two different mouse models of SMA, which may channel expressed during early muscle development. contribute to the SMA phenotype. Our results provide Upon denervation of skeletal muscle, the expression of significant insight into muscle defects in SMA pathophysi- sodium channels reverts to that which occurs during de- ology and suggest that including skeletal muscle as a velopment [31]. The expression of Na 1.5 increases and therapeutic target in SMA is warranted. v Boyer et al. Skeletal Muscle 2013, 3:24 Page 12 of 13 http://www.skeletalmusclejournal.com/content/3/1/24 Abbreviations 9. Schrank B, Gotz R, Gunnersen JM, Ure JM, Toyka KV, Smith AG, Sendtner M: GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; H & E: Hematoxylin Inactivation of the survival motor neuron gene, a candidate gene for and eosin; MHC: Myosin heavy chain; MRF4: Muscle-specific regulatory factor human spinal muscular atrophy, leads to massive cell death in early 4; NF: Neurofilament; NF1: Nuclear factor 1; NMJ: Neuromuscular junction; mouse embryos. Proc Natl Acad Sci USA 1997, 94(18):9920–9925. P: Postnatal day; PCR: Polymerase chain reaction; RT-PCR: Reverse- 10. Monani UR, Sendtner M, Coovert DD, Parsons DW, Andreassi C, Le TT, transcription polymerase chain reaction; RyR1: Ryanodine receptor 1; Jablonka S, Schrank B, Rossoll W, Prior TW, Morris GE, Burghes AH: The SERCA: Sarcoplasmic reticulum Ca2+ ATPase; SMA: Spinal muscular atrophy; human centromeric survival motor neuron gene (SMN2) rescues −/− SMN: Survival motor neuron; SV2: Synaptic vesicle protein 2; TA: Tibialis embryonic lethality in Smn mice and results in a mouse with spinal anterior; ZEB: Zinc-finger E box-binding protein. muscular atrophy. Hum Mol Genet 2000, 9(3):333–339. 11. Hammond SM, Gogliotti RG, Rao V, Beauvais A, Kothary R, DiDonato CJ: Mouse survival motor neuron alleles that mimic SMN2 splicing and are Competing interests inducible rescue embryonic lethality early in development but not late. The authors declared that they have no competing interests. PLoS One 2010, 5(12):e15887. 12. Bowerman M, Murray LM, Beauvais A, Pinheiro B, Kothary R: A critical smn Authors’ contributions threshold in mice dictates onset of an intermediate spinal muscular JGB and RK conceived and designed the project. JGB performed and atrophy phenotype associated with a distinct neuromuscular junction analyzed most of the experiments and was assisted by KS for Figures 1 and pathology. Neuromuscul Disord 2012, 22(3):263–276. 3, and by YDR for panels A and C of Figure 2. LMM performed experiments 13. Hamilton G, Gillingwater TH: Spinal muscular atrophy: going beyond the and analyzed the data in panels E-H of Figure 2. JMR supervised KS and motor neuron. Trends Mol Med 2013, 19(1):40–50. helped analyze the data in Figure 1. JGB wrote the paper and RK revised and 14. Rajendra TK, Gonsalvez GB, Walker MP, Shpargel KB, Salz HK, Matera AG: A edited it. All authors read and approved the final manuscript. Drosophila melanogaster model of spinal muscular atrophy reveals a function for SMN in striated muscle. J Cell Biol 2007, 176(6):831–841. 15. Walker MP, Rajendra TK, Saieva L, Fuentes JL, Pellizzoni L, Matera AG: SMN Acknowledgements complex localizes to the sarcomeric Z-disc and is a proteolytic target of We thank Marc-Olivier Deguise for excellent technical assistance. This project calpain. Hum Mol Genet 2008, 17(21):3399–3410. was funded by grants from the Canadian Institutes of Health Research (CIHR) and The Muscular Dystrophy Association (USA) to RK. JGB is a recipient of a 16. Shafey D, Boyer JG, Bhanot K, Kothary R: Identification of novel interacting Frederick Banting and Charles Best CIHR Doctoral Research Award, LMM is a protein partners of SMN using tandem affinity purification. J Proteome recipient of a Multiple Sclerosis Society of Canada Postdoctoral Fellowship, Res 2010, 9(4):1659–1669. and RK is a recipient of a University Health Research Chair from the 17. Mutsaers CA, Wishart TM, Lamont DJ, Riessland M, Schreml J, Comley LH, University of Ottawa. Murray LM, Parson SH, Lochmüller H, Wirth B, Talbot K, Gillingwater TH: Reversible molecular pathology of skeletal muscle in spinal muscular Author details atrophy. Hum Mol Genet 2011, 20(22):4334–4344. Ottawa Hospital Research Institute, Regenerative Medicine Program, 501 18. Dachs E, Hereu M, Piedrafita L, Casanovas A, Caldero J, Esquerda JE: Smyth Road, Ottawa, ON K1H 8L6, Canada. Department of Cellular and Defective neuromuscular junction organization and postnatal Molecular Medicine, University of Ottawa, Ottawa, ON K1H 8M5, Canada. myogenesis in mice with severe spinal muscular atrophy. J Neuropathol Department of Medicine, University of Ottawa, Ottawa, ON K1H 8M5, Exp Neurol 2011, 70(6):444–461. Canada. 19. Lee YI, Mikesh M, Smith I, Rimer M, Thompson W: Muscles in a mouse model of spinal muscular atrophy show profound defects in neuromuscular Received: 18 July 2013 Accepted: 26 September 2013 development even in the absence of failure in neuromuscular transmission Published: 11 October 2013 or loss of motor neurons. Dev Biol 2011, 356(2):432–444. 20. Kong L, Wang X, Choe DW, Polley M, Burnett BG, Bosch-Marce M, Griffin JW, Rich MM, Sumner CJ: Impaired synaptic vesicle release and immaturity of References neuromuscular junctions in spinal muscular atrophy mice. J Neurosci 1. Prior TW, Snyder PJ, Rink BD, Pearl DK, Pyatt RE, Mihal DC, Conlan T, 2009, 29(3):842–851. Schmalz B, Montgomery L, Ziegler K, Noonan C, Hashimoto S, Garner S: 21. Kimura T, Nakamori M, Lueck JD, Pouliquin P, Aoike F, Fujimura H, Dirksen Newborn and carrier screening for spinal muscular atrophy. Am J Med RT, Takahashi MP, Dulhunty AF, Sakoda S: Altered mRNA splicing of the Genet A 2010, 152A(7):1608–1616. skeletal muscle ryanodine receptor and sarcoplasmic/endoplasmic 2. Pearn J: Incidence, prevalence, and gene frequency studies of chronic reticulum Ca2 -ATPase in myotonic dystrophy type 1. Hum Mol Genet childhood spinal muscular atrophy. J Med Genet 1978, 15(6):409–413. 2005, 14(15):2189–2200. 3. Ogino S, Leonard DG, Rennert H, Ewens WJ, Wilson RB: Genetic risk 22. Murray LM, Comley LH, Thomson D, Parkinson N, Talbot K, Gillingwater TH: assessment in carrier testing for spinal muscular atrophy. Am J Med Selective vulnerability of motor neurons and dissociation of pre- and Genet 2002, 110(4):301–307. post-synaptic pathology at the neuromuscular junction in mouse 4. Boyer JG, Bowerman M, Kothary R: The many faces of SMN: deciphering models of spinal muscular atrophy. Hum Mol Genet 2008, 17(7):949–962. the function critical to spinal muscular atrophy pathogenesis. 23. Kariya S, Park GH, Maeno-Hikichi Y, Leykekhman O, Lutz C, Arkovitz MS, Future Neurol 2010, 5(6):873–890. Landmesser LT, Monani UR: Reduced SMN protein impairs maturation of 5. Lefebvre S, Bürglen L, Reboullet S, Clermont O, Burlet P, Viollet L, Benichou the neuromuscular junctions in mouse models of spinal muscular B, Cruaud C, Millasseau P, Zeviani M, Le Paslier D, Frézal J, Cohen D, atrophy. Hum Mol Genet 2008, 17(16):2552–2569. Weissenbach J, Munnich A, Melki J: Identification and characterization of a 24. Murray LM, Beauvais A, Bhanot K, Kothary R: Defects in neuromuscular spinal muscular atrophy-determining gene. Cell 1995, 80(1):155–165. B/− junction remodelling in the Smn2 mouse model of spinal muscular 6. Brzustowicz LM, Merette C, Kleyn PW, Lehner T, Castilla LH, Penchaszadeh atrophy. Neurobiol Dis 2012, 49C:57–67. GK, Das K, Munsat TL, Ott J, Gilliam TC: Assessment of nonallelic genetic 25. Bowerman M, Beauvais A, Anderson CL, Kothary R: Rho-kinase inactivation heterogeneity of chronic (type II and III) spinal muscular atrophy. prolongs survival of an intermediate SMA mouse model. Hum Mol Genet Hum Hered 1993, 43(6):380–387. 2010, 19(8):1468–1478. 7. Melki J, Abdelhak S, Sheth P, Bachelot MF, Burlet P, Marcadet A, Aicardi J, 26. Van Petegem F: Ryanodine receptors: structure and function. J Biol Chem Barois A, Carriere JP, Fardeau M, Fontan D, Ponsot G, Billette T, Angelini C, 2012, 287(38):31624–31632. Barbosa C, Ferriere G, Lanzi G, Ottolini A, Babron MC, Cohen D, Hanauer A, 27. Futatsugi A, Kuwajima G, Mikoshiba K: Tissue-specific and developmentally Clerget-Darpoux F, Lathrop M, Munnich A, Frezal J: Gene for chronic regulated alternative splicing in mouse skeletal muscle ryanodine proximal spinal muscular atrophies maps to chromosome 5q. receptor mRNA. Biochem J 1995, 305(Pt 2):373–378. Nature 1990, 344(6268):767–768. 28. Ling KK, Gibbs RM, Feng Z, Ko CP: Severe neuromuscular denervation of 8. Rochette CF, Gilbert N, Simard LR: SMN gene duplication and the clinically relevant muscles in a mouse model of spinal muscular atrophy. emergence of the SMN2 gene occurred in distinct hominids: SMN2 is Hum Mol Genet 2012, 21(1):185–195. unique to Homo sapiens. Hum Genet 2001, 108(3):255–266. Boyer et al. Skeletal Muscle 2013, 3:24 Page 13 of 13 http://www.skeletalmusclejournal.com/content/3/1/24 29. David M, Martinez-Marmol R, Gonzalez T, Felipe A, Valenzuela C: Differential regulation of Na(v)β subunits during myogenesis. Biochem Biophys Res Commun 2008, 368(3):761–766. 30. Morel J, Rannou F, Talarmin H, Giroux-Metges MA, Pennec JP, Dorange G, Gueret G: Sodium channel Na(V)1.5 expression is enhanced in cultured adult rat skeletal muscle fibers. J Membr Biol 2010, 235(2):109–119. 31. Kallen RG, Cohen SA, Barchi RL: Structure, function and expression of voltage-dependent sodium channels. Mol Neurobiol 1993, 7(3–4):383–428. 32. Hebert SL, Simmons C, Thompson AL, Zorc CS, Blalock EM, Kraner SD: Basic helix-loop-helix factors recruit nuclear factor I to enhance expression of the NaV 1.4 Na channel gene. Biochim Biophys Acta 2007, 1769(11–12):649–658. 33. Lupa MT, Krzemien DM, Schaller KL, Caldwell JH: Expression and distribution of sodium channels in short- and long-term denervated rodent skeletal muscles. J Physiol 1995, 483(Pt 1):109–118. 34. Rich MM, Kraner SD, Barchi RL: Altered gene expression in steroid-treated denervated muscle. Neurobiol Dis 1999, 6(6):515–522. 35. Beard NA, Laver DR, Dulhunty AF: Calsequestrin and the calcium release channel of skeletal and cardiac muscle. Prog Biophys Mol Biol 2004, 85(1):33–69. 36. Millay DP, Goonasekera SA, Sargent MA, Maillet M, Aronow BJ, Molkentin JD: Calcium influx is sufficient to induce muscular dystrophy through a TRPC-dependent mechanism. Proc Natl Acad Sci USA 2009, 106(45):19023–19028. 37. Divet A, Huchet-Cadiou C: Sarcoplasmic reticulum function in slow- and fast-twitch skeletal muscles from mdx mice. Pflugers Arch 2002, 444(5):634–643. 38. Goonasekera SA, Lam CK, Millay DP, Sargent MA, Hajjar RJ, Kranias EG, Molkentin JD: Mitigation of muscular dystrophy in mice by SERCA overexpression in skeletal muscle. J Clin Invest 2011, 121(3):1044–1052. doi:10.1186/2044-5040-3-24 Cite this article as: Boyer et al.: Early onset muscle weakness and disruption of muscle proteins in mouse models of spinal muscular atrophy. Skeletal Muscle 2013 3:24. Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Skeletal Muscle Springer Journals

Early onset muscle weakness and disruption of muscle proteins in mouse models of spinal muscular atrophy

Loading next page...
 
/lp/springer-journals/early-onset-muscle-weakness-and-disruption-of-muscle-proteins-in-mouse-mTbQnyuGMB
Publisher
Springer Journals
Copyright
Copyright © 2013 by Boyer et al.; licensee BioMed Central Ltd.
Subject
Life Sciences; Cell Biology; Developmental Biology; Biochemistry, general; Systems Biology; Biotechnology
eISSN
2044-5040
DOI
10.1186/2044-5040-3-24
pmid
24119341
Publisher site
See Article on Publisher Site

Abstract

Background: The childhood neuromuscular disease spinal muscular atrophy (SMA) is caused by mutations or deletions of the survival motor neuron (SMN1) gene. Although SMA has traditionally been considered a motor neuron disease, the muscle-specific requirement for SMN has never been fully defined. Therefore, the purpose of this study was to investigate muscle defects in mouse models of SMA. −/− Methods: We have taken advantage of two different mouse models of SMA, the severe Smn ;SMN2 mice and the 2B/− less severe Smn mice. We have measured the maximal force produced from control muscles and those of SMA model mice by direct stimulation using an ex vivo apparatus. Immunofluorescence and immunoblot experiments were performed to uncover muscle defects in mouse models of SMA. Means from control and SMA model mice samples were compared using an analysis of variance test and Student’s t tests. −/− Results: We report that tibialis anterior (TA) muscles of phenotype stage Smn ;SMN2 mice generate 39% less maximal force than muscles from control mice, independently of aberrant motor neuron signal transmission. In −/− addition, during muscle fatigue, the Smn ;SMN2 muscle shows early onset and increased unstimulated force compared with controls. Moreover, we demonstrate a significant decrease in force production in muscles from pre- −/− 2B/− symptomatic Smn ;SMN2 and Smn mice, indicating that muscle weakness is an early event occurring prior to any overt motor neuron loss and muscle denervation. Muscle weakness in mouse models of SMA was associated with a delay in the transition from neonatal to adult isoforms of proteins important for proper muscle contractions, such as ryanodine receptors and sodium channels. Immunoblot analyses of extracts from hindlimb skeletal muscle 2+ revealed aberrant levels of the sarcoplasmic reticulum Ca ATPase. Conclusions: The findings from this study reveal a delay in the appearance of mature isoforms of proteins important for muscle contractions, as well as muscle weakness early in the disease etiology, thus highlighting the contributions of skeletal muscle defects to the SMA phenotype. Keywords: Motor neuron disease, Skeletal muscle, Sodium channels, Ryanodine receptors, SERCA, Spinal muscular atrophy, Survival motor neuron * Correspondence: rkothary@ohri.ca Ottawa Hospital Research Institute, Regenerative Medicine Program, 501 Smyth Road, Ottawa, ON K1H 8L6, Canada Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, ON K1H 8M5, Canada Full list of author information is available at the end of the article © 2013 Boyer et al.; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Boyer et al. Skeletal Muscle 2013, 3:24 Page 2 of 13 http://www.skeletalmusclejournal.com/content/3/1/24 Background studies demonstrating a lack of increase in myofiber size With an overall carrier frequency of 1:40, spinal muscu- and the increased levels of embryonic and neonatal lar atrophy (SMA) is a major leading genetic cause of myosin heavy chain (MHC) isoforms [18-20]. However, infant deaths, affecting 1 in 6,000 to 10,000 births [1-3]. it is not known whether or how impaired muscle growth Spinal muscular atrophy is an autosomal recessive dis- contributes to muscle weakness in SMA, since at present order traditionally classified into different types based on no comprehensive analysis has been performed relating the clinical severity of the symptoms [4]. In 1995, the to muscle force production in mouse models of SMA. SMA-determining gene was identified and named Here, we show previously unreported pathophysio- −/− ‘survival motor neuron’ (SMN) [5]. This gene is located logical muscle defects in severe (Smn ;SMN2) and less 2B/− on chromosome 5q13 in humans, in a region containing severe (Smn ) mouse models of SMA. We report pro- an inverted duplication of 500 kilobase pairs. This nounced muscle weakness in these mice. These observa- results in two virtually identical copies of the SMN gene; tions were associated with altered expression of proteins SMN1 and SMN2 [5-8]. that are developmentally regulated and are important for In the mouse, the Smn gene is present as a single copy, proper physiological muscle function. Furthermore, we and homozygous loss of function leads to a pre- show that muscle weakness is an early feature, observed implantation lethality [9]. However, when the Smn knock- prior to any overt motor neuron loss and muscle de- out is coupled with low levels of human SMN expressed nervation in mouse models of SMA. Thus, we conclude from a SMN2 transgene, a severe phenotype approximat- that muscle defects contribute to the phenotype in SMA −/− ing type I SMA is observed in Smn ;SMN2 mice [10]. mouse models. Uncovering skeletal muscle defects in Since this original discovery, several other mouse models the context of SMA is of the utmost importance to bet- of SMA have been generated, including a milder model ter understand the SMA phenotype and for the develop- 2B/− termed Smn . These latter mice do not harbor the ment of targeted therapeutics. SMN2 transgene but rather harbor one null allele and a second allele with a 3-nucleotide substitution in the Methods exonic splice enhancer of exon 7 of the mouse Smn gene Mouse models 2B/- −/− (2B mutation) [11]. Smn model mice display a milder The Smn ;SMN2 (Jackson Labs, Bar Harbor, ME, USA) 2B/− SMA phenotype, owing to slightly higher Smn protein and Smn [12] mice were housed and cared for levels than the severe model [12]. according to the Canadian Council on Animal Care guide- Motor neuron cell loss and muscle denervation are lines and the University of Ottawa Animal Care Commit- considered two pathological hallmarks of SMA. Exactly tee protocols. Tissues from pre-symptomatic mice were −/− how SMN depletion leads to motor neuron degeneration collected at postnatal day (P) 2 for severe Smn ;SMN2 2B/− is unclear and remains the focus of intense research. In mice, and P9 for Smn mice. Tissues were also col- −/− addition, recent advances in the field have highlighted lected from phenotype stages at P5 for Smn ;SMN2 2B/− the involvement of other tissues in the pathophysiology and P21 for Smn mice. Muscles used for RNA and of SMA, of which skeletal muscle appears to be an protein analysis were flash frozen in liquid nitrogen and important candidate [4,13]. stored at −80°C. In Drosophila, Smn was discovered to be a sarcomeric protein interacting with α-actinin, a cross-linking pro- Hindlimb denervation tein that stabilizes actin microfilaments [14]. Walker and Denervation surgeries were performed in accordance with colleagues later confirmed these findings and specifically the guidelines set by the Canadian Council on Animal identified Smn as a Z-disc component in skeletal and Care. Young mice (P14) were anaesthetized by inhalation cardiac muscle of mice [15]. At present, the function of of isoflurane. Experimental denervation was achieved by Smn at this adhesion site is unknown but Smn is likely revealing the sciatic nerve and removing 2 to 3 mm of the to have a specific function other than snRNP biogenesis nerve in the thigh section to cease neural input and pre- in muscle [15]. An Smn interacting protein screen in vent nerve regrowth. A sham procedure was performed in C2C12 myoblasts suggests that the function of Smn in parallel to serve as control; this consisted of exposing the muscle is dynamic and probably differs during varying mice to identical experimental conditions except for cut- stages of myogenesis based on its protein interactome ting the nerve. The TA muscles were collected and flash [16]. A proteomic screen performed by Mutsaers et al. frozen from denervated and sham operated mice one and identified an increase in proteins involved in pro- seven days following surgery. −/− grammed cell death in pre-symptomatic Smn ;SMN2 mice [17]. Several reports have highlighted the possibil- Immunoblotting ity of delayed myogenesis in mouse models of SMA. The Total tissue lysate extract was obtained by grinding flash basis of this notion comes from muscle morphological frozen tissues in a liquid nitrogen pre-cooled mortar and Boyer et al. Skeletal Muscle 2013, 3:24 Page 3 of 13 http://www.skeletalmusclejournal.com/content/3/1/24 pestle. The concentration of each sample was deter- it was calculated as the difference in the baseline 5 ms be- mined by Bradford assay. Samples were subjected to fore a contraction and the baseline 5 ms before fatigue was sodium dodecyl sulfate polyacrylamide gel electrophor- elicited. Muscle weight and length were used to calculate esis and examined by immunoblot, as previously de- the cross-sectional area of the muscle that was used to scribed [16]. Primary antibodies used were: calsequestrin normalize force measurements in each experiment. (Abcam, Toronto, ON, Canada), glyceraldehyde-3- phosphate dehydrogenase (GAPDH, Abcam, Toronto, ON, RNA isolation Canada), Na 1.4 (Alomone, Jerusalem, Israel), Na 1.5 Total RNA was isolated from skeletal muscle tissue using v v (Alomone, Jerusalem, Israel), nuclear factor 1 (Abcam, a homogenizer and the RNeasy kit (Qiagen, Toronto, ON, 2+ Toronto, ON, Canada), sarcoplasmic reticulum Ca Canada) according to the manufacturer’s instructions. ATPase (SERCA1a, Cell Signaling, Danvers, MA, USA), RNA samples were treated with DNase (gDNA wipeout and zinc-finger E box-binding protein (ZEB) (Novus Bio- buffer, Qiagen, Toronto, ON, Canada) to eliminate DNA logicals, Littleton, CO, USA). Signals were detected using contamination and concentrations were determined using enhanced chemiluminescence (Thermo, Florence, KY, a Nanophotometer spectrophotometer (MBI Lab Equip- USA). Densitometric analyses were performed using ImageJ ment, Dorval, QC, Canada). software (NIH). Immunoblot data were normalized to GAPDH levels to control for possible loading differences. Reverse-transcription polymerase chain reaction (RT-PCR) RNA was reverse-transcribed using the quantitect reverse- Force measurements and fatigue protocol transcription kit (Qiagen, Toronto, ON, Canada). Primer The TA muscles were dissected from P2 and P5 control sequences and PCR conditions used to detect the spliced −/− and severe Smn ;SMN2 mice, and from P9 control and variants of the ryanodine receptor RyR1 gene were identi- 2B/− Smn mice. Muscles were constantly immersed in cal to those previously described [21]. A negative control physiological saline solution containing 118.5 mM NaCl, in which water was added instead of cDNA was prepared 4.7 mM KCl, 2.4 mM CaCl ,3.1 mM MgCl ,25mM in parallel for every PCR. Quantification of the RT-PCR 2 2 NaHCO ,2mM NaH PO ,and 5.5 mMD-glucose.Solu- results was achieved using ImageJ software. 3 2 4 tions were continuously bubbled with 95% O ,5%CO for 2 2 a pH of 7.4. Solutions containing 30 μM of tubocurarine Immunofluorescence hydrochloride pentahydrate (Sigma, Oakville, ON, Canada) Neuromuscular junction (NMJ) immunofluorescence and were prepared by adding the appropriate amount directly quantification was performed as described previously [22]. to the physiological solution. The flow of physiological so- Post-synaptic acetylcholine receptors were labeled with lution below and above muscles was maintained at a total α-bungarotoxin (Molecular Probes, Burlington, ON, of 15 ml/min and a temperature of 37°C. Tetanic contrac- Canada) while the pre-synaptic terminal was labeled with tions were elicited with electrical stimulations applied anti-neurofilament and anti-synaptic vesicle protein 2 across two platinum wires (4 mm apart) located on oppos- (both from Developmental Studies Hybridoma Bank, Iowa ite sides of the muscle. Electrodes were connected to a City, IA, USA). All secondary antibodies were purchased Grass S88 stimulator and a Grass SIU5 isolation unit from Jackson Labs. Immunofluorescence images were cap- (Grass Technologies/Astro-Med Inc., Warwick, RI, USA). tured using a Zeiss Confocal microscope (LSM 510 Tetanic contractions were elicited with 200 ms trains of Meta DuoScan, Toronto, ON, Canada). For each 0.3 ms, 12 V (supramaximal voltage) pulses at a frequency muscle, four to six fields of view were quantified and a of 200 Hz. For all experiments, muscle length was adjusted total of counted endplates ranging between 99 and 263 to achieve maximal force production and muscles were were included per animal in the analysis. allowed a 30 min equilibration period during which a tet- anic contraction was elicited every second. Maximal force Histological analysis production was determined by increasing frequencies from The lumbar (L1 and L2) region of the spinal cord was −/− 1 to 200 Hz. Muscles were then fatigued by increasing the collected from control, pre-symptomatic Smn ;SMN2 2B/− contraction rate to one contraction per second for 180 s. and Smn mice. Tissues were fixed in 4% paraformal- Twitch (obtained when stimulated with one square pulse) dehyde for 24 hrs, embedded in paraffin, cut into sec- or tetanic force was defined as the force that developed tions (10 μm) and stained with hematoxylin and eosin during stimulation and was calculated as the difference be- (H & E). Motor neurons were identified by their shape tween the maximum force during contraction and the and size within the ventral horn region of the spinal force measured 5 ms before the contraction. Unstimulated cord. Motor neuron quantification was performed on force was defined as the force generated by muscles in the every fifth section within the L1 and L2 region on three absence of electrical stimulation and was observed during mice from each genotype. Histological analyses were also fatigue when muscles failed to relax between contractions; performed on cross-sections (10 μm) from frozen TA Boyer et al. Skeletal Muscle 2013, 3:24 Page 4 of 13 http://www.skeletalmusclejournal.com/content/3/1/24 −/− 2B/− muscles of P2 Smn ;SMN2,and P9 Smn mice. Sec- less maximum peak tetanic force, as measured at 200 Hz tions were stained with H & E using a standard protocol, (Figure 1A,B). images were taken with a Zeiss Axioplan2 microscope, Aberrant NMJ morphology and function have previ- and the myofiber area was calculated using ImageJ soft- ously been highlighted in several mouse models of SMA ware. Approximately one thousand fibers were counted [20,22-24]. In isolated muscle preparations, many fibers for each genotype analyzed. receive an indirect stimulation via the remaining nerve stump. Although it has previously been demonstrated that the TA muscle is fully innervated in SMA model Statistical analyses mice [20,25], one possible explanation for the observed Data are presented as the mean ± standard error of the decrease in force is that the applied stimulus enters the mean. Analysis of variance (Statistical Analysis Software residual nerve before reaching the myofibers. Thus, if Institute Inc., Cary, NC, USA) was used to determine poorly functioning NMJs were present in the prepar- significance in the fatigue data. A Student’s t test was ation, it would negatively impact the force because fewer performed using MS Excel to compare the means of all fibers would be stimulated. To address this possibility, other data. Significance was set at P < 0.05. we measured the maximal force production in muscles from control and mutant mice in the presence or ab- Results sence of tubocurarine, which blocks acetylcholine recep- −/− Skeletal muscle weakness in Smn ;SMN2 mice tors. Should aberrant NMJs negatively affect TA muscle To date, no physiological study has been performed on force production, it would be expected that the relative −/− muscles from severe SMA model mice. To this end, we force produced by muscles from Smn ;SMN2 mice have analyzed the twitch and peak tetanic force pro- would be equal to the control values in the presence of −/− duced by direct stimulation of TA muscles of Smn ; tubocurarine. The force production of control muscles SMN2 mice and control littermates at P5. To account was not affected by the presence of tubocurarine nor did −/− for variations in muscle size, all force values were nor- we observe an increase in force production in Smn ; malized to muscle cross-sectional area. Compared with SMN2 muscles after the addition of tubocurarine to the −/− control muscles, Smn ;SMN2 mice produced 47% less preparation compared with non-treated muscles (data not twitch force, as measured after one stimulation, and 39% shown). Furthermore, in five independent experiments, −/− −/− Figure 1 Muscle weakness in muscle from Smn ;SMN2 mice. (A) TA muscle preparations from P5 Smn ;SMN2 mice and control littermates −/− were used to assess tetanic and twitch force normalized to the muscle cross-sectional area. P5 Smn ;SMN2 TA muscles produce significantly less −/− twitch force than control littermates. (B) Reduction in normalized maximal peak tetanic force in P5 Smn ;SMN2 TA muscle compared with −/− controls. (C) Administration of tubocurarine to block NMJs did not influence relative force production in Smn ;SMN2 muscles. In five −/− independent experiments, Smn ;SMN2 mice produced less force than controls following treatment with tubocurarine. (D) Similar relative force −/− decreases in Smn ;SMN2 and control mice during fatigue elicited with one tetanic contraction every second for 3 min. Peak tetanic forces are expressed as a percentage of the pre-fatigue force. (E) Unstimulated force occurred when muscles failed to relax between contractions and is −/− expressed as a percentage of the pre-fatigue peak tetanic force. During a fatigue protocol, P5 Smn ;SMN2 TA muscles show increased −/− unstimulated force compared with controls. (F) Unstimulated force appeared much sooner in Smn ;SMN2 TA muscles than in controls. NMJ, neuromuscular junction; TA, tibialis anterior; N = 5 or 6; *, P < 0.05. Boyer et al. Skeletal Muscle 2013, 3:24 Page 5 of 13 http://www.skeletalmusclejournal.com/content/3/1/24 −/− the force production from Smn ;SMN2 muscle was Although we observed no overt motor neuron loss or lower than that of controls (Figure 1C). These data show denervation in both models at pre-symptomatic stage, we that at the phenotype stage, aberrant NMJs did not im- observed a significant decrease in peak tetanic force. TA −/− −/− pact Smn ;SMN2 TA ex vivo muscle force production muscles from P2 Smn ;SMN2 mice produced 67% lower andthatmechanismsof stimuluspropagation might be peak tetanic force than control littermates (Figure 3A). TA −/− 2B/− compromised in muscles from Smn ;SMN2 mice. muscles from P9 Smn mice produced 61% lower peak tetanic force than control littermates (Figure 3C). The −/− Smn ;SMN2 muscles respond abnormally to induced peak forces were normalized to the cross-sectional area of muscle fatigue each muscle; however, at this stage, we did not observe −/− To determine whether Smn ;SMN2 muscles respond any significant difference in mean fiber area between 2B/− −/− differently to muscle fatigue, we measured the decline in mutant and control muscle in either Smn or Smn ; force with repeated tetanic stimulation for 180 s. The SMN2 mice (Figure 3B,D). Taken together, these data −/− decrease in peak tetanic force recorded in Smn ;SMN2 demonstrate that in two different mouse models of SMA, muscles was similar to control littermates (Figure 1D). muscle weakness is an early feature, occurring prior to During the fatigue protocol, we also measured the un- any overt motor neuron loss and denervation. stimulated force, which is defined as the force measured 100 ms before a contraction is elicited. The TA muscles Decreased expression of mature ryanodine receptor 1 −/− of both control and Smn ;SMN2 P5 mice generated an transcripts in muscle from SMA model mice increase in unstimulated force as they failed to com- The results of our physiology experiments led us to in- pletely relax between contractions (Figure 1E). However, vestigate possible causes for the decrease in force pro- −/− −/− the Smn ;SMN2 muscle produced significantly more duction from Smn ;SMN2 muscle. During a muscle unstimulated force than control counterparts. The un- contraction, calcium is released from the sarcoplasmic stimulated force of control TA muscles had a mean time reticulum to the sarcomere to allow for the actin-myosin of appearance at 133 s and was equivalent to 4.4% of the cross-bridge cycling. The calcium release is mediated by pre-fatigue tetanic force by the end of the protocol ryanodine receptor 1 (RyR1), which is the predominant −/− (Figure 1E,F). However, for the P5 Smn ;SMN2 TA ryanodine receptor expressed in mature muscle [26]. muscles, the average time of appearance started signifi- Several splice variants of the RyR1 gene exist. For ex- cantly sooner, i.e. at 49 s, with a final mean of 8.2% ample, one variant is called ASI and is expressed without (Figure 1E,F). Therefore, our results suggest the pres- exon 70 [ASI (−)] in neonatal muscle and transitions to −/− ence of a defect in Smn ;SMN2 muscles, resulting in an alternatively spliced variant that includes exon 70 in an inability to recover from muscle fatigue over time. mature skeletal muscle [ASI (+)] [27]. The second RyR1 splice variant is ASII, which is further spliced to exclude −/− Pre-symptomatic muscle weakness in Smn ;SMN2 and exon 83 in immature muscle [ASII (−)] or to include 2B/− Smn mice that exon in mature muscle [ASII (+)] [21,27]. Using Whilst we observed a significant decrease in force pro- PCR primers designed to target the mature and imma- −/− duction in muscles from phenotypic Smn ;SMN2 mice, ture variants, we assessed the RyR1 transcripts in hind- which was independent of aberrant nerve transmission limb skeletal muscle RNA extracts from mouse models in the ex vivo preparations, it remains possible that the of SMA and in controls. A time course analysis demon- muscle weakness observed could be attributed to motor strates the predominant expression of ASII (+) in mature neuron degeneration occurring prior to the stage of our muscle (P21) over the ASII (−) variant in wild type mice analyses. We therefore assessed the peak tetanic force in (Figure 4A). At P5, the predominant ASII isoform in −/− pre-symptomatic mice. For this, we have extended our control mice was ASII (+), while in Smn ;SMN2 muscle −/− 2B/− analysis to include both Smn ;SMN2 and Smn there was a relative increase in the proportion of the neo- mouse models. This analysis was performed at pre- natal variant ASII (−) over ASII (+) (Figure 4B middle −/− phenotypic time point of P2 and P9 in Smn ;SMN2 panel and Figure 4D). In control P21 mice, the adult ASII 2B/− and Smn model mice, respectively. To confirm that (+) variant was predominant relative to the neonatal ASII 2B/− these time points preceded neurodegenerative events, (−) variant, whereas in Smn mice both variants were we assessed motor neuron number and NMJ integrity. expressed at similar levels (Figure 4C middle panel and −/− 2B/− At P2 in Smn ;SMN2 mice, and P9 in Smn mice, Figure 4D). there was no difference in the number of motor neuron Using the same approach, we assessed the transcript cell bodies compared with controls (Figure 2A,D). Fur- levels of ASI using primers targeting both the neonatal thermore, the percentage of fully occupied endplates was and adult ASI splice variant. We did not observe any dif- unchanged between each mouse model of SMA and the ference in neonatal versus adult transcript levels of the −/− respective controls (Figure 2E,H). ASI variant for the Smn ;SMN2 mice (Figure 4B upper Boyer et al. Skeletal Muscle 2013, 3:24 Page 6 of 13 http://www.skeletalmusclejournal.com/content/3/1/24 Figure 2 (See legend on next page.) Boyer et al. Skeletal Muscle 2013, 3:24 Page 7 of 13 http://www.skeletalmusclejournal.com/content/3/1/24 (See figure on previous page.) −/− 2B/− Figure 2 Normal motor neuron counts and NMJ integrity in P2 Smn ;SMN2 and P9 Smn mice. (A, B) Representative images of H & E −/− staining of motor neurons in the ventral horn region of the L1 and L2 spinal cord region of P2 control and Smn ;SMN2 mice, and P9 control 2B/− and Smn mice. Scale bar = 50 μm. (C, D) Quantification of motor neuron cell body number within the ventral horn of the lumbar (L1 and L2) −/− 2B/− region of the spinal cord for control and pre-symptomatic Smn ;SMN2 and Smn mice. (E, F) Representative images showing fully intact −/− 2B/− NMJs from TA muscles of control and pre-symptomatic Smn ;SMN2 (E) and Smn (F) mice. Post-synaptic acetylcholine receptors were labeled with α-bungarotoxin (red) while the pre-synaptic terminal was labeled with anti-NF (green) and anti-SV2 (green). Scale bar = 20 μm. −/− (G, H) Quantification of the percentage of fully occupied endplates revealed no difference between control and pre-symptomatic Smn ;SMN2 2B/− and Smn mice. NF, neurofilament; NMJ, neuromuscular junction; SV2, synaptic vesicle protein 2; N = 3 for all experiments. 2B/− panel and Figure 4D). In P21 Smn mice, however, the splicing pattern of the RyR1 ASII variants in denervation neonatal ASI (−) transcript was the predominant variant compared with control samples, either one day or seven expressed, while in control muscles, the ASI (+) transcript days post-denervation (Figure 4E,F). These results support was the major ASI variant expressed (Figure 4C upper the hypothesis that the changes in RyR1 splicing pattern panel and Figure 4D). Collectively, the aberrant expression in muscles from the mouse models of SMA are not attrib- pattern of the RyR1 transcripts suggests a delay in muscle utable to pre-synaptic pathology and are therefore poten- development in mouse models of SMA. tially reflective of a muscle developmental defect. Muscle denervation at the NMJ is a pathological fea- ture observed in mouse models of SMA. Although there Altered sodium channel levels in SMA mice are generally low levels of denervation in the hindlimb In excitable cell types, such as neurons and myocytes, muscles of SMA model mice [28], we investigated whether sodium channels propagate the action potential. Sodium denervation would influence the expression the RyR1 channel expression is a developmentally regulated process transcript. To do so, we experimentally denervated mus- in which an isoform switch, Na 1.5 to Na 1.4, occurs dur- v v cles of 2-week-old wild type mice and examined the ex- ing postnatal development in the mouse [29,30]. Na 1.4, pression of RyR1 in denervated samples compared with the predominant sodium channel isoform in adult skeletal sham controls. We did not detect any changes in the muscle [31], must be expressed at the correct time point −/− 2B/− −/− Figure 3 Pre-symptomatic muscle weakness in Smn ;SMN2 and Smn mice. (A) P2 Smn ;SMN2 force measurements revealed a 67% decrease in maximal tetanic force production compared with controls. Force data were normalized to the muscle cross-sectional area. (B) The 2B/− average peak tetanic force was reduced by 61% in P9 pre-symptomatic Smn TA muscles compared with control littermates. (C) Mean fiber −/− 2B/− area of P2 TA muscles from Smn ;SMN2 and control mice. (D) Average fiber cross-sectional area for P9 Smn and control TA muscle. N = 3 for all experiments. *, P < 0.05. Boyer et al. Skeletal Muscle 2013, 3:24 Page 8 of 13 http://www.skeletalmusclejournal.com/content/3/1/24 Figure 4 Delayed expression of adult RyR1 mRNA splice variant in muscles from mouse models of SMA. (A) RT-PCR on RNA from hindlimb muscle from wild type mice with primers directed against ASII (+) and ASII (−). GAPDH served as a loading control to confirm equivalence of starting cDNA levels . Note that relative ratio of ASII (+) to ASII (−) increases from P2 to P21. (B) RT-PCR results demonstrated no −/− change in the expression of ASI (+) and ASI (−) variants in control and Smn ;SMN2 samples at P5 (upper panel). However, there was decreased −/− expression of ASII (+) and sustained expression of ASII (−) in muscle samples from P5 Smn ;SMN2 compared with controls (middle panel). GAPDH served as a loading control. N = 5 for each genotype. (C) In control P21 mice, we observed increased expression of ASI (+) transcripts 2B/− relative to ASI (−) transcripts. However in Smn mice, the relative ratio of ASI (+) to ASI (−) transcripts was decreased (upper panel). 2B/− Furthermore, for the ASII variant, we observed the presence of a single transcript [ASII (+)] in P21 control samples, while in Smn samples, we observed a decrease in ASII (+) transcripts compared with controls. The ASII (−) variant was also now apparent (middle panel). GAPDH served as a −/− loading control. N = 5 for each variant. (D) Quantification of RT-PCR data show significant changes in the ASII+/ASII − ratio in Smn ;SMN2 samples compared with controls. The relative levels of adult and neonatal RYR1 isoforms was significantly altered for both the ASI and ASII 2B/− variants in Smn animals compared with controls. (E,F) The relative levels of adult and neonatal ASII RyR1 transcript variants are not altered in P14 mice one (E) and seven (F) days post-denervation compared with sham operated mice. N =3. during development to fulfill its role. A delay in expression the predominant isoform expressed in the adult heart of the Na 1.4 isoform can negatively impact muscle force and in early stages of skeletal muscle development [30]. production [32]. These results suggest that muscle development is de- As expected, we observed a robust increase in Na 1.4 layed in SMA model mice and that development is se- −/− levels in wild type muscle during postnatal development verely impaired, especially in Smn ;SMN2 mice, where from P2 to P21 (Figure 5A). Interestingly, in two inde- both Na isoform levels are decreased. pendent mouse models of SMA, there is a decrease in To gain a better understanding of how Na 1.4 is mis- the levels of Na 1.4 compared with control mice. Speci- regulated in SMA mice, we assessed the status of proteins −/− fically, in P5 Smn ;SMN2 mice, Na 1.4 and Na 1.5 known to regulate sodium channel expression. Hebert and v v levels were significantly decreased in hindlimb skeletal colleagues [32] have previously demonstrated that the muscle compared with control counterparts (Figure 5B). transcription factor NF1 is recruited to the Na 1.4 gene 2B/− Similarly, in muscle from phenotype stage P21 Smn promoter by myogenic regulatory factors to enhance its mice, there was a decrease in Na 1.4 levels compared expression. We did not observe any differences in the 2B/− with controls (Figure 5C). In addition to the decrease in levels of NF1 in muscle from P21 Smn mice compared Na 1.4, we observed an increase in Na 1.5 levels in with controls (Figure 5D). Another transcription factor, v v 2B/− Smn muscle (Figure 5C). Sodium channel Na 1.5 is ZEB, is a Na 1.4 repressor. As with NF1, we did not v v Boyer et al. Skeletal Muscle 2013, 3:24 Page 9 of 13 http://www.skeletalmusclejournal.com/content/3/1/24 Figure 5 Na 1.4 protein levels are decreased in muscles from mouse models of SMA. (A) Immunoblot analysis using muscle lysate from P2, P5, P9, and P21 wild type mice. Na 1.4 protein levels increase during postnatal muscle development and form the predominant sodium channel expressed in mature skeletal muscle. GAPDH served as a loading control (N = 3). (B) Representative immunoblot with quantification, showing a decrease in levels of −/− sodium channel Na 1.4 and Na 1.5 in P5 Smn ;SMN2 hindlimb v v muscle compared with controls (N = 3). (C) Quantification of 2B/− immunoblot analyses in P21 Smn and control hindlimb muscles revealed a decrease in Na 1.4 levels. Early in postnatal muscle development, the Na 1.5 sodium channel isoform is the most 2B/− predominant. In P21 Smn mice, the protein levels of Na 1.5 are higher than in controls (N = 3). (D) The protein level of the Na 1.4 2B/− positive regulator, NF1, is not altered in muscles from P21 Smn mice. Similarly, no change was detected in the protein levels of the Na 1.4 repressor ZEB. (E) Expression of sodium channel Na 1.4 in v v control sham and denervated samples 1 and 7 days post- denervation was assessed by immunoblot (N = 3). A decrease in the levels of Na 1.4 in muscle was noted at 7 days post-denervation. *, P < 0.05; **, P < 0.01. 2B/− observe any change in ZEB levels in muscle from Smn mice (Figure 5D). We next investigated whether Na 1.4 expression was influenced by experimental denervation. There was no change in Na 1.4 levels one day post-denervation (Figure 5E). However, a significant decrease was ob- served seven days following denervation, in agreement with previous studies [33,34]. Therefore, although the muscles used in the Na 1.4 expression analysis are not morphologically denervated, we cannot rule out the possibility that functional synaptic defects at the NMJ influence sodium channel expression in muscles from mouse models of SMA. −/− SERCA1a protein expression is altered in Smn ;SMN2 mice One possible mechanism that can cause increased un- stimulated force production is an incomplete removal of 2+ Ca from the sarcoplasm because of decreased levels of 2+ the Ca ATPase pump. The protein responsible for the 2+ Ca uptake following a muscle contraction is the sarco- 2+ plasmic reticulum Ca ATPase (SERCA), of which SERCA1a is the predominant isoform found in fast- twitch muscles, such as the TA muscle [35]. The protein expression of SERCA1a is developmentally regulated. It peaksbyP9and dropsslightlyatP21 (Figure6A).Im- munoblot analysis revealed a decrease in SERCA1a pro- −/− tein levels in hindlimb skeletal muscles from P5 Smn ; SMN2 mice compared with control samples (Figure 6B). Interestingly, levels of calsequestrin, a protein that binds 2+ and stores Ca in the sarcoplasmic reticulum, was un- −/− changed in Smn ;SMN2 muscle compared with con- 2+ trols (Figure 6B), indicating that a Ca handling defect was likely limited to the sarcoplasmic reticulum pump. Boyer et al. Skeletal Muscle 2013, 3:24 Page 10 of 13 http://www.skeletalmusclejournal.com/content/3/1/24 −/− Figure 6 SERCA1a protein level is altered in muscles from Smn ;SMN2 mice. (A) Whole muscle lysate was collected from P2, P5, P9, and P21 wild type mice and immunoblot analysis was performed to assess SERCA1a protein levels. SERCA1a levels increase over time and peak at P9 −/− (N = 3). (B) Immunoblot with quantification showing a decrease in SERCA1a, but not calsequestrin, in hindlimb muscle from P5 Smn ;SMN2 mice compared with control (N = 3). (C) Immunoblots were performed on muscle lysates collected from experimentally denervated (DEN) and sham operated (SHAM) muscle. No change in SERCA1a levels was observed. N =3, *, P < 0.05. −/− Next, we measured the influence of denervation on muscle force from pre-symptomatic Smn ;SMN2 and 2B/− SERCA1a protein levels. Protein lysate from gastrocne- Smn mice prior to any overt motor neuron loss and mius muscles was collected from denervated and sham denervation, although we cannot rule out the influence operated mice. SERCA1a protein levels were unchanged of a functional deficit within the motor neurons. It in skeletal muscle from denervated mice compared with should be noted, however, that our physiological results controls (Figure 6C). This again supports the hypothesis were normalized to the cross-sectional area of each that the observed decrease in SERCA1a in muscle from muscle tested. Therefore, the overt decrease in muscle −/− −/− Smn ;SMN2 mice could be due to a muscle develop- size observed in P5 Smn ;SMN2 mice cannot explain mental defect. the decrease in force production, per se. In addition, our experiments performed on pre-symptomatic mice allow us to rule out the possibility that smaller myofibers are Discussion the reason for the decrease in relative force production, Here, we show that in two mouse models of SMA, since no significant difference was observed in muscle muscle weakness occurs early, being evident prior to any size between pre-symptomatic and control mice. How- overt physical denervation and motor neuron loss. This ever, the maturity of the muscle may influence force physiological defect was associated with delayed expres- production, irrespective of size. As we have observed a sion of mature isoforms of proteins important for muscle decrease in the mature isoforms of a number of muscle function. Our results therefore point to muscle weakness proteins, we suggest that a decrease in muscle maturity coupled with delayed muscle development and provide −/− 2B/− in P2 Smn ;SMN2 and P9 Smn mice could contrib- new insight into the pathophysiology underlying SMA. ute to a marked decrease in force production. This work highlights the potential of muscle as a thera- peutic target and warrants further work to identify muscle directed strategies to increase muscle force production. Delayed expression of mature isoforms of muscle function proteins in mouse models of SMA Several groups have indirectly demonstrated impaired Muscle weakness in SMA mice muscle growth in mouse models of SMA by measuring We have employed an ex vivo method in which the the cross-sectional area of developing myofibers [18-20]. muscle is excised and placed in a chamber where it can These analyses suggest that shortly after birth, muscle be directly stimulated to contract. By doing so, we re- development is significantly impaired. During postnatal duce the negative contribution that degenerating motor muscle development, as myotubes grow to become neurons might have in eliciting a contraction, with the myofibers, a switch in expression from neonatal to adult caveat that there may still be functional defects preced- protein isoforms occurs for many muscle function pro- ing the analysis. We show a decrease in normalized peak −/− teins. A delay in this switch could compromise muscle tetanic force in muscle from phenotype stage Smn ; maturation and function. Such might be the case with SMN2 mice. Importantly, we show a similar decrease in Boyer et al. Skeletal Muscle 2013, 3:24 Page 11 of 13 http://www.skeletalmusclejournal.com/content/3/1/24 the expression of MHC, in which the embryonic and that of Na 1.4 decreases in denervated muscle. Indeed, perinatal MHC isoforms are predominantly expressed in we observed a decrease in Na 1.4 levels in experimen- muscle from SMA model mice [19,20]. Therefore, we tally denervated muscles (day 7), as well as in muscles hypothesized that several other proteins important for from both SMA mouse models studied. As such, we generating muscle contractions could be aberrantly cannot rule out the possibility that the mis-regulation of expressed, with juvenile isoforms predominating rather Na 1.4 is due to denervation in muscle from the symp- than adult ones, which could lead to muscle weakness in tomatic mice. mouse models of SMA. We focused on proteins that are The expression of Na 1.4 is positively regulated by the directly involved in the regulation of muscle contraction, transcription factor NF1 and is repressed by the tran- that is, proteins important for calcium regulation and ac- scription factor ZEB [32]. We did not observe any differ- tion potential propagation. ences in the expression of these two transcription 2B/− factors in Smn mice. The recruitment of the NF1 RyR1 expression in muscle from mouse models of SMA protein to the Na 1.4 promoter is mediated through two Results from our RT-PCR analysis revealed a delay in transcription factors that are important for muscle dif- the expression of the mature RyR1 splice variants in ferentiation, namely myogenin and muscle-specific regu- skeletal muscle from mouse models of SMA. In pheno- latory factor 4 (MRF4). It can be envisaged that a delay 2B/− type stage Smn mice, we observed a mis-regulation in the expression of myogenic regulatory factors, such as of both the ASI and ASII alternatively spliced variants. myogenin and MRF4, or others even more up-stream of −/− At P5 in the Smn ;SMN2 model, a change in expres- myogenin and MRF4, may explain the deferred Na 1.4 sion was evident for the ASII variant but not the ASI. expression in SMA mice. During development, the transition from ASII (−)to ASII (+) begins at P0 and is complete by P21 [27]. For −/− the ASI variant, the transition from the neonatal ASI (−) Decreased SERCA1a expression in Smn ;SMN2 mice to the adult ASI (+) form begins only at P8. Therefore, The results from our fatigue protocol demonstrate an in- the timing of the ASI transition probably explains why crease in unstimulated force and a decrease in the time 2B/− −/− we observed the delay in P21 Smn mice but not in of unstimulated force onset in Smn ;SMN2 mice. This −/− 2+ P5 Smn ;SMN2 mice. The functional studies performed observation may be indicative of a defect in Ca uptake by Kimura et al. demonstrate that neonatal RyR1 is less from the sarcomere to the sarcoplasmic reticulum, active than adult RyR1, as it binds ryanodine with less which is supported by the muscle intrinsic decrease in 2+ −/− affinity than the adult form, and therefore releases less cal- levels of the SERCA1a Ca pump in muscles of Smn ; 2+ cium [21]. Thus, the persistent expression of the neonatal SMN2 mice. Defects in Ca handling have previously RyR1 variants in mouse models of SMA probably leads to been reported in mouse models of muscular dystrophies 2+ 2+ decreased Ca release from the sarcoplasmic reticulum [21,36]. Specifically, defects related to Ca uptake and to the sarcomere, and subsequently results in weaker SERCA1 function have been described in a mouse model muscle contractions. of Duchenne’s muscular dystrophy [37]. Indeed, the overexpression of SERCA1 in skeletal muscles led to ro- Sodium channel expression in muscle from mouse bust improvements in muscle function and attenuated models of SMA muscle pathology in mouse models of muscular dys- In skeletal muscle, action potentials are generated and trophy [38]. Furthermore, RyR1 splicing defects resulting propagated by voltage-gated sodium channels. Na 1.4 is in the expression of the neonatal variants contribute to the predominant pore-conducting channel in adult the pathogenesis of the neuromuscular disease myotonic muscle. Its expression significantly increases in mice in dystrophy type 1 [21]. Thus, the defects we report in the first two weeks after birth [29,31]. Here we show muscles from SMA model mice are reminiscent of those that Na 1.4 levels are decreased in muscles from two dif- that occur in other muscle diseases. ferent mouse models of SMA. This may explain in part the lower force generation, since there would have been an insufficient number of available Na 1.4 channels to Conclusions generate action potentials during a train. Furthermore, In summary, we have demonstrated early and profound this period after birth coincides with a period of dra- muscle weakness, and aberrant expression of muscle pro- matic muscle growth, and Na 1.5 is the major sodium teins in two different mouse models of SMA, which may channel expressed during early muscle development. contribute to the SMA phenotype. Our results provide Upon denervation of skeletal muscle, the expression of significant insight into muscle defects in SMA pathophysi- sodium channels reverts to that which occurs during de- ology and suggest that including skeletal muscle as a velopment [31]. The expression of Na 1.5 increases and therapeutic target in SMA is warranted. v Boyer et al. Skeletal Muscle 2013, 3:24 Page 12 of 13 http://www.skeletalmusclejournal.com/content/3/1/24 Abbreviations 9. Schrank B, Gotz R, Gunnersen JM, Ure JM, Toyka KV, Smith AG, Sendtner M: GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; H & E: Hematoxylin Inactivation of the survival motor neuron gene, a candidate gene for and eosin; MHC: Myosin heavy chain; MRF4: Muscle-specific regulatory factor human spinal muscular atrophy, leads to massive cell death in early 4; NF: Neurofilament; NF1: Nuclear factor 1; NMJ: Neuromuscular junction; mouse embryos. Proc Natl Acad Sci USA 1997, 94(18):9920–9925. P: Postnatal day; PCR: Polymerase chain reaction; RT-PCR: Reverse- 10. Monani UR, Sendtner M, Coovert DD, Parsons DW, Andreassi C, Le TT, transcription polymerase chain reaction; RyR1: Ryanodine receptor 1; Jablonka S, Schrank B, Rossoll W, Prior TW, Morris GE, Burghes AH: The SERCA: Sarcoplasmic reticulum Ca2+ ATPase; SMA: Spinal muscular atrophy; human centromeric survival motor neuron gene (SMN2) rescues −/− SMN: Survival motor neuron; SV2: Synaptic vesicle protein 2; TA: Tibialis embryonic lethality in Smn mice and results in a mouse with spinal anterior; ZEB: Zinc-finger E box-binding protein. muscular atrophy. Hum Mol Genet 2000, 9(3):333–339. 11. Hammond SM, Gogliotti RG, Rao V, Beauvais A, Kothary R, DiDonato CJ: Mouse survival motor neuron alleles that mimic SMN2 splicing and are Competing interests inducible rescue embryonic lethality early in development but not late. The authors declared that they have no competing interests. PLoS One 2010, 5(12):e15887. 12. Bowerman M, Murray LM, Beauvais A, Pinheiro B, Kothary R: A critical smn Authors’ contributions threshold in mice dictates onset of an intermediate spinal muscular JGB and RK conceived and designed the project. JGB performed and atrophy phenotype associated with a distinct neuromuscular junction analyzed most of the experiments and was assisted by KS for Figures 1 and pathology. Neuromuscul Disord 2012, 22(3):263–276. 3, and by YDR for panels A and C of Figure 2. LMM performed experiments 13. Hamilton G, Gillingwater TH: Spinal muscular atrophy: going beyond the and analyzed the data in panels E-H of Figure 2. JMR supervised KS and motor neuron. Trends Mol Med 2013, 19(1):40–50. helped analyze the data in Figure 1. JGB wrote the paper and RK revised and 14. Rajendra TK, Gonsalvez GB, Walker MP, Shpargel KB, Salz HK, Matera AG: A edited it. All authors read and approved the final manuscript. Drosophila melanogaster model of spinal muscular atrophy reveals a function for SMN in striated muscle. J Cell Biol 2007, 176(6):831–841. 15. Walker MP, Rajendra TK, Saieva L, Fuentes JL, Pellizzoni L, Matera AG: SMN Acknowledgements complex localizes to the sarcomeric Z-disc and is a proteolytic target of We thank Marc-Olivier Deguise for excellent technical assistance. This project calpain. Hum Mol Genet 2008, 17(21):3399–3410. was funded by grants from the Canadian Institutes of Health Research (CIHR) and The Muscular Dystrophy Association (USA) to RK. JGB is a recipient of a 16. Shafey D, Boyer JG, Bhanot K, Kothary R: Identification of novel interacting Frederick Banting and Charles Best CIHR Doctoral Research Award, LMM is a protein partners of SMN using tandem affinity purification. J Proteome recipient of a Multiple Sclerosis Society of Canada Postdoctoral Fellowship, Res 2010, 9(4):1659–1669. and RK is a recipient of a University Health Research Chair from the 17. Mutsaers CA, Wishart TM, Lamont DJ, Riessland M, Schreml J, Comley LH, University of Ottawa. Murray LM, Parson SH, Lochmüller H, Wirth B, Talbot K, Gillingwater TH: Reversible molecular pathology of skeletal muscle in spinal muscular Author details atrophy. Hum Mol Genet 2011, 20(22):4334–4344. Ottawa Hospital Research Institute, Regenerative Medicine Program, 501 18. Dachs E, Hereu M, Piedrafita L, Casanovas A, Caldero J, Esquerda JE: Smyth Road, Ottawa, ON K1H 8L6, Canada. Department of Cellular and Defective neuromuscular junction organization and postnatal Molecular Medicine, University of Ottawa, Ottawa, ON K1H 8M5, Canada. myogenesis in mice with severe spinal muscular atrophy. J Neuropathol Department of Medicine, University of Ottawa, Ottawa, ON K1H 8M5, Exp Neurol 2011, 70(6):444–461. Canada. 19. Lee YI, Mikesh M, Smith I, Rimer M, Thompson W: Muscles in a mouse model of spinal muscular atrophy show profound defects in neuromuscular Received: 18 July 2013 Accepted: 26 September 2013 development even in the absence of failure in neuromuscular transmission Published: 11 October 2013 or loss of motor neurons. Dev Biol 2011, 356(2):432–444. 20. Kong L, Wang X, Choe DW, Polley M, Burnett BG, Bosch-Marce M, Griffin JW, Rich MM, Sumner CJ: Impaired synaptic vesicle release and immaturity of References neuromuscular junctions in spinal muscular atrophy mice. J Neurosci 1. Prior TW, Snyder PJ, Rink BD, Pearl DK, Pyatt RE, Mihal DC, Conlan T, 2009, 29(3):842–851. Schmalz B, Montgomery L, Ziegler K, Noonan C, Hashimoto S, Garner S: 21. Kimura T, Nakamori M, Lueck JD, Pouliquin P, Aoike F, Fujimura H, Dirksen Newborn and carrier screening for spinal muscular atrophy. Am J Med RT, Takahashi MP, Dulhunty AF, Sakoda S: Altered mRNA splicing of the Genet A 2010, 152A(7):1608–1616. skeletal muscle ryanodine receptor and sarcoplasmic/endoplasmic 2. Pearn J: Incidence, prevalence, and gene frequency studies of chronic reticulum Ca2 -ATPase in myotonic dystrophy type 1. Hum Mol Genet childhood spinal muscular atrophy. J Med Genet 1978, 15(6):409–413. 2005, 14(15):2189–2200. 3. Ogino S, Leonard DG, Rennert H, Ewens WJ, Wilson RB: Genetic risk 22. Murray LM, Comley LH, Thomson D, Parkinson N, Talbot K, Gillingwater TH: assessment in carrier testing for spinal muscular atrophy. Am J Med Selective vulnerability of motor neurons and dissociation of pre- and Genet 2002, 110(4):301–307. post-synaptic pathology at the neuromuscular junction in mouse 4. Boyer JG, Bowerman M, Kothary R: The many faces of SMN: deciphering models of spinal muscular atrophy. Hum Mol Genet 2008, 17(7):949–962. the function critical to spinal muscular atrophy pathogenesis. 23. Kariya S, Park GH, Maeno-Hikichi Y, Leykekhman O, Lutz C, Arkovitz MS, Future Neurol 2010, 5(6):873–890. Landmesser LT, Monani UR: Reduced SMN protein impairs maturation of 5. Lefebvre S, Bürglen L, Reboullet S, Clermont O, Burlet P, Viollet L, Benichou the neuromuscular junctions in mouse models of spinal muscular B, Cruaud C, Millasseau P, Zeviani M, Le Paslier D, Frézal J, Cohen D, atrophy. Hum Mol Genet 2008, 17(16):2552–2569. Weissenbach J, Munnich A, Melki J: Identification and characterization of a 24. Murray LM, Beauvais A, Bhanot K, Kothary R: Defects in neuromuscular spinal muscular atrophy-determining gene. Cell 1995, 80(1):155–165. B/− junction remodelling in the Smn2 mouse model of spinal muscular 6. Brzustowicz LM, Merette C, Kleyn PW, Lehner T, Castilla LH, Penchaszadeh atrophy. Neurobiol Dis 2012, 49C:57–67. GK, Das K, Munsat TL, Ott J, Gilliam TC: Assessment of nonallelic genetic 25. Bowerman M, Beauvais A, Anderson CL, Kothary R: Rho-kinase inactivation heterogeneity of chronic (type II and III) spinal muscular atrophy. prolongs survival of an intermediate SMA mouse model. Hum Mol Genet Hum Hered 1993, 43(6):380–387. 2010, 19(8):1468–1478. 7. Melki J, Abdelhak S, Sheth P, Bachelot MF, Burlet P, Marcadet A, Aicardi J, 26. Van Petegem F: Ryanodine receptors: structure and function. J Biol Chem Barois A, Carriere JP, Fardeau M, Fontan D, Ponsot G, Billette T, Angelini C, 2012, 287(38):31624–31632. Barbosa C, Ferriere G, Lanzi G, Ottolini A, Babron MC, Cohen D, Hanauer A, 27. Futatsugi A, Kuwajima G, Mikoshiba K: Tissue-specific and developmentally Clerget-Darpoux F, Lathrop M, Munnich A, Frezal J: Gene for chronic regulated alternative splicing in mouse skeletal muscle ryanodine proximal spinal muscular atrophies maps to chromosome 5q. receptor mRNA. Biochem J 1995, 305(Pt 2):373–378. Nature 1990, 344(6268):767–768. 28. Ling KK, Gibbs RM, Feng Z, Ko CP: Severe neuromuscular denervation of 8. Rochette CF, Gilbert N, Simard LR: SMN gene duplication and the clinically relevant muscles in a mouse model of spinal muscular atrophy. emergence of the SMN2 gene occurred in distinct hominids: SMN2 is Hum Mol Genet 2012, 21(1):185–195. unique to Homo sapiens. Hum Genet 2001, 108(3):255–266. Boyer et al. Skeletal Muscle 2013, 3:24 Page 13 of 13 http://www.skeletalmusclejournal.com/content/3/1/24 29. David M, Martinez-Marmol R, Gonzalez T, Felipe A, Valenzuela C: Differential regulation of Na(v)β subunits during myogenesis. Biochem Biophys Res Commun 2008, 368(3):761–766. 30. Morel J, Rannou F, Talarmin H, Giroux-Metges MA, Pennec JP, Dorange G, Gueret G: Sodium channel Na(V)1.5 expression is enhanced in cultured adult rat skeletal muscle fibers. J Membr Biol 2010, 235(2):109–119. 31. Kallen RG, Cohen SA, Barchi RL: Structure, function and expression of voltage-dependent sodium channels. Mol Neurobiol 1993, 7(3–4):383–428. 32. Hebert SL, Simmons C, Thompson AL, Zorc CS, Blalock EM, Kraner SD: Basic helix-loop-helix factors recruit nuclear factor I to enhance expression of the NaV 1.4 Na channel gene. Biochim Biophys Acta 2007, 1769(11–12):649–658. 33. Lupa MT, Krzemien DM, Schaller KL, Caldwell JH: Expression and distribution of sodium channels in short- and long-term denervated rodent skeletal muscles. J Physiol 1995, 483(Pt 1):109–118. 34. Rich MM, Kraner SD, Barchi RL: Altered gene expression in steroid-treated denervated muscle. Neurobiol Dis 1999, 6(6):515–522. 35. Beard NA, Laver DR, Dulhunty AF: Calsequestrin and the calcium release channel of skeletal and cardiac muscle. Prog Biophys Mol Biol 2004, 85(1):33–69. 36. Millay DP, Goonasekera SA, Sargent MA, Maillet M, Aronow BJ, Molkentin JD: Calcium influx is sufficient to induce muscular dystrophy through a TRPC-dependent mechanism. Proc Natl Acad Sci USA 2009, 106(45):19023–19028. 37. Divet A, Huchet-Cadiou C: Sarcoplasmic reticulum function in slow- and fast-twitch skeletal muscles from mdx mice. Pflugers Arch 2002, 444(5):634–643. 38. Goonasekera SA, Lam CK, Millay DP, Sargent MA, Hajjar RJ, Kranias EG, Molkentin JD: Mitigation of muscular dystrophy in mice by SERCA overexpression in skeletal muscle. J Clin Invest 2011, 121(3):1044–1052. doi:10.1186/2044-5040-3-24 Cite this article as: Boyer et al.: Early onset muscle weakness and disruption of muscle proteins in mouse models of spinal muscular atrophy. Skeletal Muscle 2013 3:24. Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit

Journal

Skeletal MuscleSpringer Journals

Published: Oct 11, 2013

References