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Expression of MuRF1 or MuRF2 is essential for the induction of skeletal muscle atrophy and dysfunction in a murine pulmonary hypertension model

Expression of MuRF1 or MuRF2 is essential for the induction of skeletal muscle atrophy and... Background: Pulmonary hypertension leads to right ventricular heart failure and ultimately to cardiac cachexia. Cardiac cachexia induces skeletal muscles atrophy and contractile dysfunction. MAFbx and MuRF1 are two key proteins that have been implicated in chronic muscle atrophy of several wasting states. Methods: Monocrotaline (MCT) was injected over eight weeks into mice to establish pulmonary hypertension as a murine model for cardiac cachexia. The effects on skeletal muscle atrophy, myofiber force, and selected muscle proteins were evaluated in wild-type (WT), MuRF1, and MuRF2-KO mice by determining muscle weights, in vitro muscle force and enzyme activities in soleus and tibialis anterior (TA) muscle. Results: In WT, MCT treatment induced wasting of soleus and TA mass, loss of myofiber force, and depletion of citrate synthase (CS), creatine kinase (CK), and malate dehydrogenase (MDH) (all key metabolic enzymes). This suggests that the murine MCT model is useful to mimic peripheral myopathies as found in human cardiac cachexia. In MuRF1 and MuRF2-KO mice, soleus and TA muscles were protected from atrophy, contractile dysfunction, while metabolic enzymes were not lowered in MuRF1 or MuRF2-KO mice. Furthermore, MuRF2 expression was lower in MuRF1KO mice when compared to C57BL/6 mice. Conclusions: In addition to MuRF1, inactivation of MuRF2 also provides a potent protection from peripheral myopathy in cardiac cachexia. The protection of metabolic enzymes in both MuRF1KO and MuRF2KO mice as well as the dependence of MuRF2 expression on MuRF1 suggests intimate relationships between MuRF1 and MuRF2 during muscle atrophy signaling. Keywords: Cardiac cachexia, Pulmonary hypertension, Muscle atrophy, Myofibrillar proteins, MuRF1 and MuRF2, Muscle energy metabolism Background atrophy occurs as a result of changes in the balance Skeletal muscle mass adapts rapidly to activity by either between anabolic and catabolic processes and in many clin- activating hypertrophic or atrophic pathways. Muscle ical conditions, like chronic heart failure [1–3], limb immobilization [4, 5], mechanical ventilation [6, 7], sepsis [8], diabetes [9], and advanced aging [10]skeletalmuscle * Correspondence: volker.adams@mailbox.tu-dresden.de Laboratory of Molecular and Experimental Cardiology, TU Dresden, Heart mass is lost, leading to muscle weakness, inactivity and Center Dresden, Dresden, Germany increased mortality. The activation of both the autophagic/ Full list of author information is available at the end of the article © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Nguyen et al. Skeletal Muscle (2020) 10:12 Page 2 of 10 lysosomal proteolysis and the ubiquitin proteasome system cardiac cachexia monocrotaline (MCT) was subcutane- (UPS) are recognized to play important roles in the protein ously injected weekly at a concentration of 600 mg/kg breakdown. Especially the UPS system and relevant ubiqui- into either C57/BL6 (WT, n = 9), MuRF-1 (n = 11), and tin E3-ligases are discussed as potential targets to modulate MuRF-2 (n = 9) knockout animals for 8 weeks. Control skeletal muscle atrophy. Performing transcript profiling in animals of each group received the same volume of sa- −/− −/− several atrophy models identified MuRF1 and MAFbx as line (C57/Bl6 n = 12; MuRF-1 n = 11; MuRF-2 n = ubiquitin E3 ligases only expressed in heart and skeletal 11). Body weight was recorded every week for each ani- muscle [11]. MuRF1 belongs to a family of MuRF proteins mal. Animals were exposed to identical conditions under consisting of MuRF1, MuRF2 and MuRF3 [12]. MuRF1 a 12:12 h light/dark cycle with food and water provided knockout animal’s exhibit resistance towards the develop- ad libitum. Mice were sacrificed following deep ment of skeletal muscle atrophy [11, 13]and when sub- anesthetization with i.p. administration of fentanyl (0.05 jected to chronic pressure overload the animals developed mg/kg), medetomidine (0.5 mg/kg), midazolam (5 mg/ massive cardiac hypertrophy [14]. MuRF2 seems to be in- kg), and ketamine (100 mg/kg). At sacrifice, the heart volved in sarcomere formation [15] and intracellular signal- and lungs were dissected, cleaned, blotted dry, and ing in cardiomyocytes by decreasing serum response weighed, with the heart fixed in 4% PBS-buffered forma- transcription factor (SRF) during mechanical inactivity [16]. lin. The left tibialis anterior (TA) and soleus (SO) Furthermore, MuRF2 in mononuclear cells attenuates muscle were dissected, weighed, and fixed in 4% PBS- LPS-induced macrophage activation by inhibiting the buffered formalin, while the remaining muscle portions generation of inflammatory cytokines [17]. MuRF3 were immediately frozen in liquid N for molecular ana- binds to microtubules helping to develop a network lysis. Muscle wet weights were normalized to tibia resistant to depolarization [18], plays a role in myosin length, which allowed a fair comparison of relative protein quality control [19] and protects against dia- changes in muscle mass between all groups due to betic cardiomyopathy [20]. differences in body weight. Studies analyzing the myocardium of knockout All experiments and procedures were approved by the (KO) animals suggest that MuRF1 and MuRF2 play a local Animal Research Council, University of Leipzig redundant role in regulating developmental physio- and the Landesbehörde Sachsen (TVV 40/16). logic hypertrophy [21]. Synergistic cooperation be- tween MuRF1 and MuRF2 is further supported by the Contractile function observation that MuRF1/MuRF2 double KO animals The SO of the right leg was dissected to allow in vitro present a fulminant cardiac phenotype (74% early contractile function to be assessed using a length- postnatal lethality with acute heart failure), whereas controlled lever system (301B, Aurora Scientific Inc., single knockout animals are healthy with normal life Aurora, Canada), as previously described [25, 26]. span and myocardial functionality [22]. This cooper- Briefly, a muscle bundle was mounted vertically in a ation among MuRFs and with other atrogenes like buffer-filled organ bath (~ 22 °C), set at optimal length, MAFbx was also documented in MuRF1 KO mice and after 15 min was stimulated over a force-frequency undergoing denervation [23]oraging [24], where a protocol between 1 and 300 Hz (600 mA; 500 ms train significant upregulation of MAFbx was observed when duration; 0.25 ms pulse width). Force (N) was normal- compared to wild-type animals. A molecular explan- ized to muscle cross-sectional area (CSA; cm ) by divid- ation for the cooperation between MuRF1 and ing muscle mass (g) by the product of L (cm) and MuRF2 may be their shared recognition of 35 or estimated muscle density (1.06), which allowed specific more protein targets [22]. These cooperative effects of force in N/cm to be calculated. MuRF1 and MuRF2 are mainly shown in the myocar- dium but less data are available for skeletal muscle Tissue analyses remodeling in experimental heart failure. Therefore, Histology the aim of the present study was to induce heart fail- The heart (medial section), the SO, and TA muscle were ure in MuRF1 and MuRF2 knockout mice and com- embedded in paraffin; 3 μm sections were obtained, pare the development of muscle atrophy and which where mounted on slides and stained with dysfunction to wild-type littermates. hematoxylin and eosin. Sections were than captured as images on a computer connected to a microscope and Methods and materials subsequently evaluated using Analysis software (Analysis Animals and study design 3.0, Olympus Soft Imaging Solutions GmbH, Münster, The mice used in this study are all on a clean C57/BL6 Germany). As recently described [25], RV wall thickness background. Details on the gene inactivation of MuRF1 (in μm) was determined from the mean of 10 individual and MuRF2 are described in Witt et al. [22]. To induce measurements distributed along the free ventricular wall, Nguyen et al. Skeletal Muscle (2020) 10:12 Page 3 of 10 while mean fibre CSA (in μm ) of the soleus and TA ventricular hypertrophy (Fig. 1c; p for all groups < 0.01). was evaluated after assessment of approximately 300– Cachexia was evident after 8 weeks in WT mice with 500 fibers per animal. regardstotheir wholebodyweights:While control animals increased body weights by 15%, MCT mice Western blot analysis developed a 9% reduction in body weights during For western blot analyses, frozen TA was homogenized the 8-week study period (Fig. 1d, p = 0.001). Lung in Relax buffer (90 mmol/L HEPES, 126 mmol/L potas- and heart tissues in MuRF1 and MuRF2 KO mice sium chloride, 36 mmol/L sodium chloride, 1 mmol/L responded to MCT injection similar as WT mice, magnesium chloride, 50 mmol/L EGTA, 8 mmol/L ATP, and total lung and heart eights, as well as RV thick- 10 mmol/L creatine phosphate, pH 7.4) containing a ness were augmented at week 8 (Fig. 1a–c). How- protease inhibitor mix (Inhibitor mix M, Serva, Heidel- ever, the effects on body weights differed in the WT, −/− berg, Germany), sonicated, and centrifuged at 16,000xg MuRF1, and MuRF2 KO groups: MuRF1 animals for 5 min. Protein concentration of the supernatant was lost 7% of body weight (p =0.09MCT vs.NaCl −/− determined (BCA assay, Pierce, Bonn, Germany) and ali- treatment), whereas MuRF2 lost 4% (no statistical quots (5–20 μg) were separated by SDS-polyacrylamide significance when compared to NaCl-treated coun- gel electrophoresis. Proteins were transferred to a polyvi- terparts; Fig. 1d). nylidene fluoride membrane (PVDF) and incubated overnight at 4 °C with the following primary antibodies: TA and SO muscles differ in their response to MCT in WT, MuRF1 (1/1000, Abcam, Cambridge, UK), MuRF2 (1: MuRF1 and MuRF2 KO mice 1.600, Myomedix GmbH, Neckargemünd, Germany). Quantifying muscle weight of the tibialis anterior (Fig. 2a) Membranes were subsequently incubated with a horse- and soleus muscle (Fig. 2b) in wild-type and in MuRF1KO radish peroxidase-conjugated secondary antibody and and MuRF2KO mice, a significant higher muscle weight specific bands visualized by enzymatic chemilumines- was evident in MuRF1KO and MuRF2KO mice when cence (Super Signal West Pico, Thermo Fisher Scientific compared to C57BL/6 animals. In accordance with our Inc., Bonn, Germany) and densitometry quantified using recent study [25], the whole body weight losses of WT a 1D scan software package (Scanalytics Inc., Rockville, mice underlies a progressing muscle atrophy during the USA). Blots were then normalized to the loading control eight week MCT treatment. Different degrees of muscle GAPDH (1/30000; HyTest Ltd, Turku, Finland). All data weight losses are present in TA and SO: while TA and SO are presented as fold change relative to control. lost about 10% total wet weights (Fig. 2c, d) (TA 10% loss, soleus 11% loss), the fiber cross-sectional area (CSA) were Enzyme activity measurements even markedly lower (TA 32% lowered CSA; SO 15% low- TA was homogenized in Relax buffer and aliquots were ered CSA) (Fig. 2e, f). In contrast, inactivation of either used for enzyme activity measurements. Enzyme activ- MuRF1 or MuRF2 protected mice from MCT induced at- ities for citrate synthase (CS, EC 2.3.3.1), creatine kinase rophy features both with regards to muscle wet weight (EC 2.7.3.2), and malate dehydrogenase (EC 1.1.1.37) (Fig. 2c, d) and fiber CSA (Fig. 2e, f). No significant differ- were measured spectrophotometrically as described in ences between the NaCl and MCT groups were observed detail [27, 28]. Enzyme activity data are presented as the in any of the MuRF1KO and MuRF2KO SO or TA muscle fold change vs. control. measures. Statistical analyses MCT-induced SO muscle force depletion is prevented by Data are presented as mean ± SEM. Unpaired t test was MuRF1 or MuRF2 inactivation used to compare groups, while two-way repeated mea- Next, we compared contractile properties of isolated sures ANOVA followed by Bonferroni post hoc test was muscle bundles from MCT-stressed and control mice used to assess contractile function (GraphPad Prism). by our force-frequency protocol (see “Methods” sec- Significance was accepted as p < 0.05. tion above). Consistent with our earlier results [25], treatment of C57Bl/6 mice with MCT for 8 weeks Results resulted in a 16% loss of absolute SO myofiber force Comparison of cachexia response to MCT stress in WT, (Fig. 3a; p < 0.01). However, no change was apparent MuRF1, MuRF2 KO mice after normalization to muscle mass in terms of spe- Weekly injections of MCT into WT mice are suitable to cific myofiber force (Fig. 3b). establish a chronic cardiac cachexia condition as previ- In vitro comparisons of SO muscle contractility ously described (see [25, 29]): MCT treatment of WT between non-MCT-treated C57BL/6, MuRF1KO and animals for 8 weeks resulted in increased lung weight MuRF2KO mice revealed a significant higher absolute (Fig. 1a), increased heart weight (Fig. 1b), and right force in both knockout strains (C57BL/6 24.5 ± 0.6 g; Nguyen et al. Skeletal Muscle (2020) 10:12 Page 4 of 10 MuRF1KO 27.6 ± 1.1 g p < 0.05 vs. C57BL/6; these atrogenes was not seen in MuRF1 and MuRF2KO MuRF2KO 28.2 ± 1.4 g p <0.05vs. C57BL6). How- animals. Interestingly, even a downregulation of MafBx ever, after normalization to muscle mass, specific was noted in MuRF2KO animals when treated with force was not different the different strains (C57BL/6 MCT (Fig. 4a). Finally, we tested if the expression of 2 2 27.7 ± 0.5 N/cm ; MuRF1KO 27.8 ± 0.9 N/cm ; MuRF1 influences the expression of MuRF2. For this, MuRF2KO 28.5 ± 0.6 N/cm ). Intriguingly, MCT we determined the expression of MuRF2 in TA muscle treatment for 8 weeks in did not have an impact on from MuRF1KO animals. Intriguingly, gene inactivation absolute or specific muscle forces in both MuRF1KO of MuRF1 also markedly lowered the expression of (Fig. 3c, d) and in MuRF2KO mice (Fig. 3e, f). MuRF2 (see Fig. 4d for comparison of MuRF2 levels in TA of MuRF1 KO and C57BL/6 WT animals). Impact of MCT treatment on atrophy-associated protein expression Depletion of energy delivering enzymes by MCT Next, during MCT stress we assessed the protein expres- treatment and rescue from this by MuRF1 and 2 gene sion of MafBx and MuRF1 (two key atrogin factors). We inactivation included MuRF2 as its gene inactivation also protect In our recent study [25] on MCT induced cardiac cach- from atrophy and wasting (see Figs. 1, 2, and 3). exia, we noted a depletion of enzymatic activities that The treatment of C57BL/6 mice with MCT for a generate ATP in myocytes. We therefore determined period of 8 weeks resulted in a significant increased again the activity of these enzymes under MCT-stress expression of MafBx (Fig. 4a), MuRF1 (Fig. 4b), and but included also muscle extracts from MuRF1 and 2 MuRF2 (Fig. 4c). This MCT-induced upregulation of KO mice. Consistent with our recent study [25], Fig. 1 Physical characteristics of NaCl- or monocrotaline (MCT)-treated C57BL/6 wild-type animals or MuRF1 and MuRF2 knockout animals. When compared to the NaCl-treated animals, the administration of MCT to the animals had significant effects on final body weight (a), lung weight (b), heart weight (c), and the thickness of the right ventricle (d) independent of the phenotype. Data are presented as mean ± SEM Nguyen et al. Skeletal Muscle (2020) 10:12 Page 5 of 10 treatment of C57BL6 animals with MCT resulted in muscle dysfunction in a right ventricular heart failure reduced enzyme activities for CS (Fig. 5a), creatine kin- setting that mimics human heart failure during chronic −/− ase (Fig. 5b), and MDH (Fig. 5c). In contrast, MuRF1 pulmonary hypertension. The results of the present animals showed an upregulation of these enzymes that study can be summarized as follows: (1) the develop- are connected to mitochondrial energy metabolism in ment of heart failure is associated with muscle atrophy −/− muscle. In MuRF2 animals, MCT did not alter and muscle dysfunction (loss of absolute force). This is enzyme activities of CS, CK, and MDH in TA muscles not observed in MuRF1KO and MuRF2KO animals. (2) (Fig. 5a–c). Muscle mass is already higher in the MuRF1KO and MuRF2KO animals when compared to WT mice inde- Discussion pendent of heart failure induction, (3) muscle atrophy Skeletal muscle atrophy occurs frequently in a variety of induced by MCT goes along with the activation of diseases, including tumor, chronic heart failure, diabetes, MuRF1, MuRF2, and MAFbx, and a downregulation of sepsis, and mechanical ventilation, contributing to a re- enzymes involved in mitochondrial energy production duced muscle function and reduced quality of life. The and energy transfer, (4) the expression of MuRF1 influ- understanding of molecular mechanisms and the rele- ences also the expression of MuRF2. vance of specific proteins for the development of muscle dysfunction/muscle atrophy is essential for developing Importance of MuRF1 and MuRF2 for muscle atrophy in effective treatment strategies. In the present study we in- cardiac cachexia vestigated in mouse models the roles of MuRF1 and Cardiac cachexia and the development of heart failure MuRF2 for the development of muscle atrophy and are often associated with skeletal muscle atrophy, being Fig. 2 Skeletal muscle wet weight (normalized to tibia length) and cross-sectional area for soleus and tibialis anterior (TA). Muscle wet weight for the TA (a) and soleus (b) is significantly increased in both knockout animals (MuRF1KO and MuRF2KO) when compared to C57BL/6 mice. Administration of monocrotaline (MCT) to the animals resulted in a reduced muscle wet weight of the TA (c) and soleus muscle (d) in the C57BL/ 6 animals, whereas MCT had no effect on muscle wet weight in the MuRF1KO and MuRF2KO animals (c, d). In addition, also the cross-sectional area (CSA) of TA (e) and soleus (f) was reduced in C57BL/6 whereas this reduction was not seen in MuRF1KO and MuRF2KO mice. Data are presented as mean ± SEM Nguyen et al. Skeletal Muscle (2020) 10:12 Page 6 of 10 an independent prognostic marker for survival [30–33]. application of monocrotaline to mice resulted in the de- Therefore, understanding the molecular pathways acti- velopment of pulmonary hypertension and right ven- vated and resulting in muscle atrophy are potential tar- tricular hypertrophy, evident by the increased thickness gets to develop specific treatment strategies to fight of the right ventricular wall. Inducing cardiac cachexia muscle loss and modulate morbidity and mortality. Dif- in C57Bl6 mice resulted in muscle atrophy as described ferential transcriptional profiling has identified MuRF1 in the current literature [25, 29, 37]. Giving monocrota- and MAFBx as markers for muscle atrophy [11, 34] and line to either MuRF1 or MuRF2 knockout animals this their genetic deletion resulted in muscle sparing induction of muscle atrophy was not evident. This following hind-limb unloading [35], denervation [11], or clearly shows for the first time that not only MuRF1 is glucocorticoid treatment [36]. In the present study, we essential for the induction of muscle atrophy, but also used monocrotaline to induce muscle atrophy. The the expression of MuRF2 is critical for the development Fig. 3 In vitro skeletal muscle function of the soleus muscle determined in C57BL/6 (a, b), MuRF1KO (c, d), and MuRF2KO (e, f) animals. Muscle force is shown as absolute force (a, c, e) and as specific force (b, d, f). Monocrotaline significantly impaired absolute muscle force only in C57BL6 animals (a) but not in MuRF1KO (c) and MuRF2KO mice (e). Monocrotaline had no effect on muscle specific force. Data are presented as mean ± SEM. Control animals are depicted in black squares and solid line whereas MCT-treated animals are shown in triangles and dotted lines Nguyen et al. Skeletal Muscle (2020) 10:12 Page 7 of 10 of muscle atrophy. Furthermore there seems to exist a transcription [16]. Our data here point to joint roles cooperation or a cross-talk between MuRF1 and MuRF2 in energy metabolism as another important pathway since the deletion of MuRF1 resulted, without induction affected by both MuRF1 and MuRF2. Interestingly, of muscle atrophy, in a lower expression of MuRF2. This the MCT-induced reduction of enzymes involved in is in line with a recent observation by Silva et al. describ- mitochondrial energy production is different in ing that MuRF1 directed siRNAs also knock-down ex- MuRF1 and MuRF2 KO animals. This may point to- pression of MuRF2 mRNA expression in cultured wards an interesting divergence in the mechanisms myotubes [38]. These findings point to an intimate con- underlying the actions of MuRF1 and MuRF2 in this nectivity between both MuRF1 and MuRF2. cachexia model for inducing muscle dysfunction. The The relevance of MuRF1 and MuRF2 for modulat- different role with respect to enzymes involved in en- ing muscle mass and muscle function is furthermore ergy production is supported by the observation by supported by our observation that the absolute Willis and colleagues [39] who observed in MuRF1 muscle forceissignificantlyhigherin the MuRF1and transgenic animals (specific overexpression in the MuRF2 knockout animals. The molecular pathways heart) a significantly reduced CK activity. Applying a controlled by MuRF1/2 leading to muscle atrophy yeast two hybrid screen to identify specific MuRF1 therefore warrant more studies with regard to coop- and MuRF2 targets Witt and colleagues [40]reported erativity and their signaling interrelationships: While that mainly myofibrillar proteins are targets for both MuRF1 ablation has been extensively studied in the MuRF1 and MuRF2 whereas the situation for en- context of myofibrillar protein degradation, MuRF2 zymes involved in energy production is less clear. was implicated in nuclear strength-regulated Nevertheless, more research is necessary to clarify the Fig. 4 Protein expression of atrophy related proteins in the TA muscle C57BL/6, MuRF1KO, and MuRF2KO mice. MAFbx (a), MuRF1 (b), and MuRF2 (c) protein expression was quantified in NaCl- or monocrotaline-treated animals. To test for cooperativity between MuRF1 and MuRF2, MuRF2 expression was assessed also in C57BL6 MuRF1KO mice (d). Data are presented as mean ± SEM and representative blots are depicted. In the representative blot KO = MuRF-1 KO mice; WT = C57BL/6 mice Nguyen et al. Skeletal Muscle (2020) 10:12 Page 8 of 10 Fig. 5 Enzymatic activity of citrate synthase (a), creatine kinase (b), and malate dehydrogenase (c) in the TA muscle C57BL/6, MuRF1KO, and MuRF2KO mice treated either with NaCl or monocrotaline. Data are presented as mean ± SEM Nguyen et al. Skeletal Muscle (2020) 10:12 Page 9 of 10 exact role of MuRF1 and MuRF2 and their interaction Funding TSB supported by NIRG, MRC UK (MR/S025472/1) and Heart Research UK in inducing muscle atrophy and muscle dysfunction. Translational Project Grant (TRP16/19). We are grateful to the Foundation Leducq (network 13CVD04) for generous support, to the European Union’s Horizon 2020 research and innovation programme (grant agreement no. Clinical considerations 645648 “Muscle Stress Relief”). The results discussed here were obtained using well established and previously extensively characterized KO Availability of data and materials The datasets used and/or analyzed during the current study are available models for MuRF1 and 2 [22, 40]. The results of the from the corresponding author on reasonable request. present investigation suggest that modulating MuRF1 and/or MuRF2 expression may be an attractive approach Ethics approval in the future to influence the development of muscle at- All experiments and procedures were approved by the local Animal Research Council, University of Leipzig and the Landesbehörde Sachsen (TVV rophy in cardiac cachexia. Unfortunately, it will be im- 40/16). portant not to completely inhibit the activity of both MuRF1 and MuRF2, because MuRF1/MuRF2 double Competing interests knockout animals display a severe phenotype including The authors declare that they have no competing interests. severe cardiac hypertrophy massively reduced left ven- Author details tricular ejection fraction and signs of heart failure [21, University Clinic of Cardiology, Heart Center Leipzig, Leipzig, Germany. 22]. A recently developed and tested MuRF1/2 inhibitor 2 3 School of Biomedical Sciences, University of Leeds, Leeds, UK. Laboratory of from our group prevented the development of muscle Molecular and Experimental Cardiology, TU Dresden, Heart Center Dresden, Dresden, Germany. Medical Faculty Mannheim, University of Heidelberg, atrophy and exhibited no severe side effects and was well Heidelberg, Germany. Myomedix GmbH, Neckargemünd, Germany. tolerated. One possible explanation for its “side-effect”- free action is probably due to the fact that the inhibitor Received: 6 January 2020 Accepted: 13 April 2020 was screened to inhibit the interaction of MuRF1 with its target proteins but leaving its activity intact. More References emphasis seems to be warranted for further drug devel- 1. von Haehling S, Steinbeck L, Doehner W, Springer J, Anker SD. Muscle opment or chemical modulation of the described small wasting in heart failure: An overview. Int J Biochem Cell Biol. 2013;45:2257– molecule and for testing in other models of muscle 2. Suzuki T, Palus S, Springer J. Skeletal muscle wasting in chronic heart failure. wasting. ESC heart failure. 2018;5:1099–107. 3. Lavine KJ, Sierra OL. Skeletal Muscle Inflammation and Atrophy in Heart Failure. Heart Fail Rev. 2017;22:179–89. Conclusion 4. Jones SW, Hill RJ, Krasney PA, O’Conner B, Peirce N, Greenhaff PL. 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Baehr LM, Furlow JD, Bodine SC. Muscle sparing in muscle RING finger 1 null mice: response to synthetic glucocorticoids. J Physiol. 2011;589:4759– http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Skeletal Muscle Springer Journals

Expression of MuRF1 or MuRF2 is essential for the induction of skeletal muscle atrophy and dysfunction in a murine pulmonary hypertension model

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Abstract

Background: Pulmonary hypertension leads to right ventricular heart failure and ultimately to cardiac cachexia. Cardiac cachexia induces skeletal muscles atrophy and contractile dysfunction. MAFbx and MuRF1 are two key proteins that have been implicated in chronic muscle atrophy of several wasting states. Methods: Monocrotaline (MCT) was injected over eight weeks into mice to establish pulmonary hypertension as a murine model for cardiac cachexia. The effects on skeletal muscle atrophy, myofiber force, and selected muscle proteins were evaluated in wild-type (WT), MuRF1, and MuRF2-KO mice by determining muscle weights, in vitro muscle force and enzyme activities in soleus and tibialis anterior (TA) muscle. Results: In WT, MCT treatment induced wasting of soleus and TA mass, loss of myofiber force, and depletion of citrate synthase (CS), creatine kinase (CK), and malate dehydrogenase (MDH) (all key metabolic enzymes). This suggests that the murine MCT model is useful to mimic peripheral myopathies as found in human cardiac cachexia. In MuRF1 and MuRF2-KO mice, soleus and TA muscles were protected from atrophy, contractile dysfunction, while metabolic enzymes were not lowered in MuRF1 or MuRF2-KO mice. Furthermore, MuRF2 expression was lower in MuRF1KO mice when compared to C57BL/6 mice. Conclusions: In addition to MuRF1, inactivation of MuRF2 also provides a potent protection from peripheral myopathy in cardiac cachexia. The protection of metabolic enzymes in both MuRF1KO and MuRF2KO mice as well as the dependence of MuRF2 expression on MuRF1 suggests intimate relationships between MuRF1 and MuRF2 during muscle atrophy signaling. Keywords: Cardiac cachexia, Pulmonary hypertension, Muscle atrophy, Myofibrillar proteins, MuRF1 and MuRF2, Muscle energy metabolism Background atrophy occurs as a result of changes in the balance Skeletal muscle mass adapts rapidly to activity by either between anabolic and catabolic processes and in many clin- activating hypertrophic or atrophic pathways. Muscle ical conditions, like chronic heart failure [1–3], limb immobilization [4, 5], mechanical ventilation [6, 7], sepsis [8], diabetes [9], and advanced aging [10]skeletalmuscle * Correspondence: volker.adams@mailbox.tu-dresden.de Laboratory of Molecular and Experimental Cardiology, TU Dresden, Heart mass is lost, leading to muscle weakness, inactivity and Center Dresden, Dresden, Germany increased mortality. The activation of both the autophagic/ Full list of author information is available at the end of the article © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Nguyen et al. Skeletal Muscle (2020) 10:12 Page 2 of 10 lysosomal proteolysis and the ubiquitin proteasome system cardiac cachexia monocrotaline (MCT) was subcutane- (UPS) are recognized to play important roles in the protein ously injected weekly at a concentration of 600 mg/kg breakdown. Especially the UPS system and relevant ubiqui- into either C57/BL6 (WT, n = 9), MuRF-1 (n = 11), and tin E3-ligases are discussed as potential targets to modulate MuRF-2 (n = 9) knockout animals for 8 weeks. Control skeletal muscle atrophy. Performing transcript profiling in animals of each group received the same volume of sa- −/− −/− several atrophy models identified MuRF1 and MAFbx as line (C57/Bl6 n = 12; MuRF-1 n = 11; MuRF-2 n = ubiquitin E3 ligases only expressed in heart and skeletal 11). Body weight was recorded every week for each ani- muscle [11]. MuRF1 belongs to a family of MuRF proteins mal. Animals were exposed to identical conditions under consisting of MuRF1, MuRF2 and MuRF3 [12]. MuRF1 a 12:12 h light/dark cycle with food and water provided knockout animal’s exhibit resistance towards the develop- ad libitum. Mice were sacrificed following deep ment of skeletal muscle atrophy [11, 13]and when sub- anesthetization with i.p. administration of fentanyl (0.05 jected to chronic pressure overload the animals developed mg/kg), medetomidine (0.5 mg/kg), midazolam (5 mg/ massive cardiac hypertrophy [14]. MuRF2 seems to be in- kg), and ketamine (100 mg/kg). At sacrifice, the heart volved in sarcomere formation [15] and intracellular signal- and lungs were dissected, cleaned, blotted dry, and ing in cardiomyocytes by decreasing serum response weighed, with the heart fixed in 4% PBS-buffered forma- transcription factor (SRF) during mechanical inactivity [16]. lin. The left tibialis anterior (TA) and soleus (SO) Furthermore, MuRF2 in mononuclear cells attenuates muscle were dissected, weighed, and fixed in 4% PBS- LPS-induced macrophage activation by inhibiting the buffered formalin, while the remaining muscle portions generation of inflammatory cytokines [17]. MuRF3 were immediately frozen in liquid N for molecular ana- binds to microtubules helping to develop a network lysis. Muscle wet weights were normalized to tibia resistant to depolarization [18], plays a role in myosin length, which allowed a fair comparison of relative protein quality control [19] and protects against dia- changes in muscle mass between all groups due to betic cardiomyopathy [20]. differences in body weight. Studies analyzing the myocardium of knockout All experiments and procedures were approved by the (KO) animals suggest that MuRF1 and MuRF2 play a local Animal Research Council, University of Leipzig redundant role in regulating developmental physio- and the Landesbehörde Sachsen (TVV 40/16). logic hypertrophy [21]. Synergistic cooperation be- tween MuRF1 and MuRF2 is further supported by the Contractile function observation that MuRF1/MuRF2 double KO animals The SO of the right leg was dissected to allow in vitro present a fulminant cardiac phenotype (74% early contractile function to be assessed using a length- postnatal lethality with acute heart failure), whereas controlled lever system (301B, Aurora Scientific Inc., single knockout animals are healthy with normal life Aurora, Canada), as previously described [25, 26]. span and myocardial functionality [22]. This cooper- Briefly, a muscle bundle was mounted vertically in a ation among MuRFs and with other atrogenes like buffer-filled organ bath (~ 22 °C), set at optimal length, MAFbx was also documented in MuRF1 KO mice and after 15 min was stimulated over a force-frequency undergoing denervation [23]oraging [24], where a protocol between 1 and 300 Hz (600 mA; 500 ms train significant upregulation of MAFbx was observed when duration; 0.25 ms pulse width). Force (N) was normal- compared to wild-type animals. A molecular explan- ized to muscle cross-sectional area (CSA; cm ) by divid- ation for the cooperation between MuRF1 and ing muscle mass (g) by the product of L (cm) and MuRF2 may be their shared recognition of 35 or estimated muscle density (1.06), which allowed specific more protein targets [22]. These cooperative effects of force in N/cm to be calculated. MuRF1 and MuRF2 are mainly shown in the myocar- dium but less data are available for skeletal muscle Tissue analyses remodeling in experimental heart failure. Therefore, Histology the aim of the present study was to induce heart fail- The heart (medial section), the SO, and TA muscle were ure in MuRF1 and MuRF2 knockout mice and com- embedded in paraffin; 3 μm sections were obtained, pare the development of muscle atrophy and which where mounted on slides and stained with dysfunction to wild-type littermates. hematoxylin and eosin. Sections were than captured as images on a computer connected to a microscope and Methods and materials subsequently evaluated using Analysis software (Analysis Animals and study design 3.0, Olympus Soft Imaging Solutions GmbH, Münster, The mice used in this study are all on a clean C57/BL6 Germany). As recently described [25], RV wall thickness background. Details on the gene inactivation of MuRF1 (in μm) was determined from the mean of 10 individual and MuRF2 are described in Witt et al. [22]. To induce measurements distributed along the free ventricular wall, Nguyen et al. Skeletal Muscle (2020) 10:12 Page 3 of 10 while mean fibre CSA (in μm ) of the soleus and TA ventricular hypertrophy (Fig. 1c; p for all groups < 0.01). was evaluated after assessment of approximately 300– Cachexia was evident after 8 weeks in WT mice with 500 fibers per animal. regardstotheir wholebodyweights:While control animals increased body weights by 15%, MCT mice Western blot analysis developed a 9% reduction in body weights during For western blot analyses, frozen TA was homogenized the 8-week study period (Fig. 1d, p = 0.001). Lung in Relax buffer (90 mmol/L HEPES, 126 mmol/L potas- and heart tissues in MuRF1 and MuRF2 KO mice sium chloride, 36 mmol/L sodium chloride, 1 mmol/L responded to MCT injection similar as WT mice, magnesium chloride, 50 mmol/L EGTA, 8 mmol/L ATP, and total lung and heart eights, as well as RV thick- 10 mmol/L creatine phosphate, pH 7.4) containing a ness were augmented at week 8 (Fig. 1a–c). How- protease inhibitor mix (Inhibitor mix M, Serva, Heidel- ever, the effects on body weights differed in the WT, −/− berg, Germany), sonicated, and centrifuged at 16,000xg MuRF1, and MuRF2 KO groups: MuRF1 animals for 5 min. Protein concentration of the supernatant was lost 7% of body weight (p =0.09MCT vs.NaCl −/− determined (BCA assay, Pierce, Bonn, Germany) and ali- treatment), whereas MuRF2 lost 4% (no statistical quots (5–20 μg) were separated by SDS-polyacrylamide significance when compared to NaCl-treated coun- gel electrophoresis. Proteins were transferred to a polyvi- terparts; Fig. 1d). nylidene fluoride membrane (PVDF) and incubated overnight at 4 °C with the following primary antibodies: TA and SO muscles differ in their response to MCT in WT, MuRF1 (1/1000, Abcam, Cambridge, UK), MuRF2 (1: MuRF1 and MuRF2 KO mice 1.600, Myomedix GmbH, Neckargemünd, Germany). Quantifying muscle weight of the tibialis anterior (Fig. 2a) Membranes were subsequently incubated with a horse- and soleus muscle (Fig. 2b) in wild-type and in MuRF1KO radish peroxidase-conjugated secondary antibody and and MuRF2KO mice, a significant higher muscle weight specific bands visualized by enzymatic chemilumines- was evident in MuRF1KO and MuRF2KO mice when cence (Super Signal West Pico, Thermo Fisher Scientific compared to C57BL/6 animals. In accordance with our Inc., Bonn, Germany) and densitometry quantified using recent study [25], the whole body weight losses of WT a 1D scan software package (Scanalytics Inc., Rockville, mice underlies a progressing muscle atrophy during the USA). Blots were then normalized to the loading control eight week MCT treatment. Different degrees of muscle GAPDH (1/30000; HyTest Ltd, Turku, Finland). All data weight losses are present in TA and SO: while TA and SO are presented as fold change relative to control. lost about 10% total wet weights (Fig. 2c, d) (TA 10% loss, soleus 11% loss), the fiber cross-sectional area (CSA) were Enzyme activity measurements even markedly lower (TA 32% lowered CSA; SO 15% low- TA was homogenized in Relax buffer and aliquots were ered CSA) (Fig. 2e, f). In contrast, inactivation of either used for enzyme activity measurements. Enzyme activ- MuRF1 or MuRF2 protected mice from MCT induced at- ities for citrate synthase (CS, EC 2.3.3.1), creatine kinase rophy features both with regards to muscle wet weight (EC 2.7.3.2), and malate dehydrogenase (EC 1.1.1.37) (Fig. 2c, d) and fiber CSA (Fig. 2e, f). No significant differ- were measured spectrophotometrically as described in ences between the NaCl and MCT groups were observed detail [27, 28]. Enzyme activity data are presented as the in any of the MuRF1KO and MuRF2KO SO or TA muscle fold change vs. control. measures. Statistical analyses MCT-induced SO muscle force depletion is prevented by Data are presented as mean ± SEM. Unpaired t test was MuRF1 or MuRF2 inactivation used to compare groups, while two-way repeated mea- Next, we compared contractile properties of isolated sures ANOVA followed by Bonferroni post hoc test was muscle bundles from MCT-stressed and control mice used to assess contractile function (GraphPad Prism). by our force-frequency protocol (see “Methods” sec- Significance was accepted as p < 0.05. tion above). Consistent with our earlier results [25], treatment of C57Bl/6 mice with MCT for 8 weeks Results resulted in a 16% loss of absolute SO myofiber force Comparison of cachexia response to MCT stress in WT, (Fig. 3a; p < 0.01). However, no change was apparent MuRF1, MuRF2 KO mice after normalization to muscle mass in terms of spe- Weekly injections of MCT into WT mice are suitable to cific myofiber force (Fig. 3b). establish a chronic cardiac cachexia condition as previ- In vitro comparisons of SO muscle contractility ously described (see [25, 29]): MCT treatment of WT between non-MCT-treated C57BL/6, MuRF1KO and animals for 8 weeks resulted in increased lung weight MuRF2KO mice revealed a significant higher absolute (Fig. 1a), increased heart weight (Fig. 1b), and right force in both knockout strains (C57BL/6 24.5 ± 0.6 g; Nguyen et al. Skeletal Muscle (2020) 10:12 Page 4 of 10 MuRF1KO 27.6 ± 1.1 g p < 0.05 vs. C57BL/6; these atrogenes was not seen in MuRF1 and MuRF2KO MuRF2KO 28.2 ± 1.4 g p <0.05vs. C57BL6). How- animals. Interestingly, even a downregulation of MafBx ever, after normalization to muscle mass, specific was noted in MuRF2KO animals when treated with force was not different the different strains (C57BL/6 MCT (Fig. 4a). Finally, we tested if the expression of 2 2 27.7 ± 0.5 N/cm ; MuRF1KO 27.8 ± 0.9 N/cm ; MuRF1 influences the expression of MuRF2. For this, MuRF2KO 28.5 ± 0.6 N/cm ). Intriguingly, MCT we determined the expression of MuRF2 in TA muscle treatment for 8 weeks in did not have an impact on from MuRF1KO animals. Intriguingly, gene inactivation absolute or specific muscle forces in both MuRF1KO of MuRF1 also markedly lowered the expression of (Fig. 3c, d) and in MuRF2KO mice (Fig. 3e, f). MuRF2 (see Fig. 4d for comparison of MuRF2 levels in TA of MuRF1 KO and C57BL/6 WT animals). Impact of MCT treatment on atrophy-associated protein expression Depletion of energy delivering enzymes by MCT Next, during MCT stress we assessed the protein expres- treatment and rescue from this by MuRF1 and 2 gene sion of MafBx and MuRF1 (two key atrogin factors). We inactivation included MuRF2 as its gene inactivation also protect In our recent study [25] on MCT induced cardiac cach- from atrophy and wasting (see Figs. 1, 2, and 3). exia, we noted a depletion of enzymatic activities that The treatment of C57BL/6 mice with MCT for a generate ATP in myocytes. We therefore determined period of 8 weeks resulted in a significant increased again the activity of these enzymes under MCT-stress expression of MafBx (Fig. 4a), MuRF1 (Fig. 4b), and but included also muscle extracts from MuRF1 and 2 MuRF2 (Fig. 4c). This MCT-induced upregulation of KO mice. Consistent with our recent study [25], Fig. 1 Physical characteristics of NaCl- or monocrotaline (MCT)-treated C57BL/6 wild-type animals or MuRF1 and MuRF2 knockout animals. When compared to the NaCl-treated animals, the administration of MCT to the animals had significant effects on final body weight (a), lung weight (b), heart weight (c), and the thickness of the right ventricle (d) independent of the phenotype. Data are presented as mean ± SEM Nguyen et al. Skeletal Muscle (2020) 10:12 Page 5 of 10 treatment of C57BL6 animals with MCT resulted in muscle dysfunction in a right ventricular heart failure reduced enzyme activities for CS (Fig. 5a), creatine kin- setting that mimics human heart failure during chronic −/− ase (Fig. 5b), and MDH (Fig. 5c). In contrast, MuRF1 pulmonary hypertension. The results of the present animals showed an upregulation of these enzymes that study can be summarized as follows: (1) the develop- are connected to mitochondrial energy metabolism in ment of heart failure is associated with muscle atrophy −/− muscle. In MuRF2 animals, MCT did not alter and muscle dysfunction (loss of absolute force). This is enzyme activities of CS, CK, and MDH in TA muscles not observed in MuRF1KO and MuRF2KO animals. (2) (Fig. 5a–c). Muscle mass is already higher in the MuRF1KO and MuRF2KO animals when compared to WT mice inde- Discussion pendent of heart failure induction, (3) muscle atrophy Skeletal muscle atrophy occurs frequently in a variety of induced by MCT goes along with the activation of diseases, including tumor, chronic heart failure, diabetes, MuRF1, MuRF2, and MAFbx, and a downregulation of sepsis, and mechanical ventilation, contributing to a re- enzymes involved in mitochondrial energy production duced muscle function and reduced quality of life. The and energy transfer, (4) the expression of MuRF1 influ- understanding of molecular mechanisms and the rele- ences also the expression of MuRF2. vance of specific proteins for the development of muscle dysfunction/muscle atrophy is essential for developing Importance of MuRF1 and MuRF2 for muscle atrophy in effective treatment strategies. In the present study we in- cardiac cachexia vestigated in mouse models the roles of MuRF1 and Cardiac cachexia and the development of heart failure MuRF2 for the development of muscle atrophy and are often associated with skeletal muscle atrophy, being Fig. 2 Skeletal muscle wet weight (normalized to tibia length) and cross-sectional area for soleus and tibialis anterior (TA). Muscle wet weight for the TA (a) and soleus (b) is significantly increased in both knockout animals (MuRF1KO and MuRF2KO) when compared to C57BL/6 mice. Administration of monocrotaline (MCT) to the animals resulted in a reduced muscle wet weight of the TA (c) and soleus muscle (d) in the C57BL/ 6 animals, whereas MCT had no effect on muscle wet weight in the MuRF1KO and MuRF2KO animals (c, d). In addition, also the cross-sectional area (CSA) of TA (e) and soleus (f) was reduced in C57BL/6 whereas this reduction was not seen in MuRF1KO and MuRF2KO mice. Data are presented as mean ± SEM Nguyen et al. Skeletal Muscle (2020) 10:12 Page 6 of 10 an independent prognostic marker for survival [30–33]. application of monocrotaline to mice resulted in the de- Therefore, understanding the molecular pathways acti- velopment of pulmonary hypertension and right ven- vated and resulting in muscle atrophy are potential tar- tricular hypertrophy, evident by the increased thickness gets to develop specific treatment strategies to fight of the right ventricular wall. Inducing cardiac cachexia muscle loss and modulate morbidity and mortality. Dif- in C57Bl6 mice resulted in muscle atrophy as described ferential transcriptional profiling has identified MuRF1 in the current literature [25, 29, 37]. Giving monocrota- and MAFBx as markers for muscle atrophy [11, 34] and line to either MuRF1 or MuRF2 knockout animals this their genetic deletion resulted in muscle sparing induction of muscle atrophy was not evident. This following hind-limb unloading [35], denervation [11], or clearly shows for the first time that not only MuRF1 is glucocorticoid treatment [36]. In the present study, we essential for the induction of muscle atrophy, but also used monocrotaline to induce muscle atrophy. The the expression of MuRF2 is critical for the development Fig. 3 In vitro skeletal muscle function of the soleus muscle determined in C57BL/6 (a, b), MuRF1KO (c, d), and MuRF2KO (e, f) animals. Muscle force is shown as absolute force (a, c, e) and as specific force (b, d, f). Monocrotaline significantly impaired absolute muscle force only in C57BL6 animals (a) but not in MuRF1KO (c) and MuRF2KO mice (e). Monocrotaline had no effect on muscle specific force. Data are presented as mean ± SEM. Control animals are depicted in black squares and solid line whereas MCT-treated animals are shown in triangles and dotted lines Nguyen et al. Skeletal Muscle (2020) 10:12 Page 7 of 10 of muscle atrophy. Furthermore there seems to exist a transcription [16]. Our data here point to joint roles cooperation or a cross-talk between MuRF1 and MuRF2 in energy metabolism as another important pathway since the deletion of MuRF1 resulted, without induction affected by both MuRF1 and MuRF2. Interestingly, of muscle atrophy, in a lower expression of MuRF2. This the MCT-induced reduction of enzymes involved in is in line with a recent observation by Silva et al. describ- mitochondrial energy production is different in ing that MuRF1 directed siRNAs also knock-down ex- MuRF1 and MuRF2 KO animals. This may point to- pression of MuRF2 mRNA expression in cultured wards an interesting divergence in the mechanisms myotubes [38]. These findings point to an intimate con- underlying the actions of MuRF1 and MuRF2 in this nectivity between both MuRF1 and MuRF2. cachexia model for inducing muscle dysfunction. The The relevance of MuRF1 and MuRF2 for modulat- different role with respect to enzymes involved in en- ing muscle mass and muscle function is furthermore ergy production is supported by the observation by supported by our observation that the absolute Willis and colleagues [39] who observed in MuRF1 muscle forceissignificantlyhigherin the MuRF1and transgenic animals (specific overexpression in the MuRF2 knockout animals. The molecular pathways heart) a significantly reduced CK activity. Applying a controlled by MuRF1/2 leading to muscle atrophy yeast two hybrid screen to identify specific MuRF1 therefore warrant more studies with regard to coop- and MuRF2 targets Witt and colleagues [40]reported erativity and their signaling interrelationships: While that mainly myofibrillar proteins are targets for both MuRF1 ablation has been extensively studied in the MuRF1 and MuRF2 whereas the situation for en- context of myofibrillar protein degradation, MuRF2 zymes involved in energy production is less clear. was implicated in nuclear strength-regulated Nevertheless, more research is necessary to clarify the Fig. 4 Protein expression of atrophy related proteins in the TA muscle C57BL/6, MuRF1KO, and MuRF2KO mice. MAFbx (a), MuRF1 (b), and MuRF2 (c) protein expression was quantified in NaCl- or monocrotaline-treated animals. To test for cooperativity between MuRF1 and MuRF2, MuRF2 expression was assessed also in C57BL6 MuRF1KO mice (d). Data are presented as mean ± SEM and representative blots are depicted. In the representative blot KO = MuRF-1 KO mice; WT = C57BL/6 mice Nguyen et al. Skeletal Muscle (2020) 10:12 Page 8 of 10 Fig. 5 Enzymatic activity of citrate synthase (a), creatine kinase (b), and malate dehydrogenase (c) in the TA muscle C57BL/6, MuRF1KO, and MuRF2KO mice treated either with NaCl or monocrotaline. Data are presented as mean ± SEM Nguyen et al. Skeletal Muscle (2020) 10:12 Page 9 of 10 exact role of MuRF1 and MuRF2 and their interaction Funding TSB supported by NIRG, MRC UK (MR/S025472/1) and Heart Research UK in inducing muscle atrophy and muscle dysfunction. Translational Project Grant (TRP16/19). We are grateful to the Foundation Leducq (network 13CVD04) for generous support, to the European Union’s Horizon 2020 research and innovation programme (grant agreement no. Clinical considerations 645648 “Muscle Stress Relief”). The results discussed here were obtained using well established and previously extensively characterized KO Availability of data and materials The datasets used and/or analyzed during the current study are available models for MuRF1 and 2 [22, 40]. The results of the from the corresponding author on reasonable request. present investigation suggest that modulating MuRF1 and/or MuRF2 expression may be an attractive approach Ethics approval in the future to influence the development of muscle at- All experiments and procedures were approved by the local Animal Research Council, University of Leipzig and the Landesbehörde Sachsen (TVV rophy in cardiac cachexia. Unfortunately, it will be im- 40/16). portant not to completely inhibit the activity of both MuRF1 and MuRF2, because MuRF1/MuRF2 double Competing interests knockout animals display a severe phenotype including The authors declare that they have no competing interests. severe cardiac hypertrophy massively reduced left ven- Author details tricular ejection fraction and signs of heart failure [21, University Clinic of Cardiology, Heart Center Leipzig, Leipzig, Germany. 22]. A recently developed and tested MuRF1/2 inhibitor 2 3 School of Biomedical Sciences, University of Leeds, Leeds, UK. Laboratory of from our group prevented the development of muscle Molecular and Experimental Cardiology, TU Dresden, Heart Center Dresden, Dresden, Germany. Medical Faculty Mannheim, University of Heidelberg, atrophy and exhibited no severe side effects and was well Heidelberg, Germany. Myomedix GmbH, Neckargemünd, Germany. tolerated. One possible explanation for its “side-effect”- free action is probably due to the fact that the inhibitor Received: 6 January 2020 Accepted: 13 April 2020 was screened to inhibit the interaction of MuRF1 with its target proteins but leaving its activity intact. More References emphasis seems to be warranted for further drug devel- 1. von Haehling S, Steinbeck L, Doehner W, Springer J, Anker SD. Muscle opment or chemical modulation of the described small wasting in heart failure: An overview. Int J Biochem Cell Biol. 2013;45:2257– molecule and for testing in other models of muscle 2. Suzuki T, Palus S, Springer J. Skeletal muscle wasting in chronic heart failure. wasting. ESC heart failure. 2018;5:1099–107. 3. Lavine KJ, Sierra OL. Skeletal Muscle Inflammation and Atrophy in Heart Failure. Heart Fail Rev. 2017;22:179–89. Conclusion 4. Jones SW, Hill RJ, Krasney PA, O’Conner B, Peirce N, Greenhaff PL. Dissue atrophy and exercise rehabilitation in humans profoundly affects the In the present study, we show for the first time that in expression of genes associated with the regulation of skeletal muscle mass. addition to MuRF1, inactivation of MuRF2 also provides FASEB J. 2004;18:1025–7. a potent protection from peripheral myopathy and skel- 5. Marimuthu K, Murton AJ, Greenhaff PL. Mechanisms regulating muscle mass during disuse atrophy and rehabilitation in humans. J Appl Physiol. 2010; etal muscle dysfunction in cardiac cachexia. The protec- 110:555–60. tion of metabolic enzymes in both MuRF1KO and 6. Johnson RW, Ng KWP, Dietz AR, Hartman ME, Baty JD, Hasan N, et al. MuRF2KO mice as well as the dependence of MuRF2 Muscle atrophy in mechanically-ventilated critically ill children. 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Published: Apr 27, 2020

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