An embryonic CaVβ1 isoform promotes muscle mass maintenance via GDF5 signaling in adult mouse

CaVβ1E boosts downstream growth differentiation factor 5 signaling to counteract muscle mass loss in denervated or aged mouse muscles. Counteracting aging muscles The mechanisms mediating age-related muscle atrophy are not completely understood. Now, Traoré et al. used mouse models to elucidate the mechanisms mediating age-related muscle atrophy. The authors first showed that muscle denervation triggered compensatory mechanisms to counteract excessive muscle loss; after denervation, muscle cells increased the expression of an embryonic isoform of the voltage-gated calcium channel (CaVβ1E) to limit muscle loss. The expression of CaVβ1E was reduced in aged muscles in mice. Moreover, CaVβ1E expression in skeletal muscles correlated with muscle mass in a cohort of aged individuals. The results suggest that targeting CaVβ1E could contribute to prevent muscle atrophy associated with aging and disuse. Deciphering the mechanisms that govern skeletal muscle plasticity is essential to understand its pathophysiological processes, including age-related sarcopenia. The voltage-gated calcium channel CaV1.1 has a central role in excitation-contraction coupling (ECC), raising the possibility that it may also initiate the adaptive response to changes during muscle activity. Here, we revealed the existence of a gene transcription switch of the CaV1.1 β subunit (CaVβ1) that is dependent on the innervation state of the muscle in mice. In a mouse model of sciatic denervation, we showed increased expression of an embryonic isoform of the subunit that we called CaVβ1E. CaVβ1E boosts downstream growth differentiation factor 5 (GDF5) signaling to counteract muscle loss after denervation in mice. We further reported that aged mouse muscle expressed lower quantity of CaVβ1E compared with young muscle, displaying an altered GDF5-dependent response to denervation. Conversely, CaVβ1E overexpression improved mass wasting in aging muscle in mice by increasing GDF5 expression. We also identified the human CaVβ1E analogous and show a correlation between CaVβ1E expression in human muscles and age-related muscle mass decline. These results suggest that strategies targeting CaVβ1E or GDF5 might be effective in reducing muscle mass loss in aging.


INTRODUCTION
A decrease in electrical activity, such as during neuromuscular disease, disuse, or aging, can cause massive muscle atrophy (1). Disuse atrophy after muscle denervation or immobilization is characterized by the activation of a compensatory response to counteract mass loss. In particular, the induction of the growth differentiation factor 5 (GDF5)/ SMAD4 (an acronym from the fusion of Caenorhabditis elegans Sma genes and the Drosophila Mad, Mothers against decapentaplegic) pathway is essential not only for avoiding excessive muscle mass loss but also for promoting reinnervation after nerve crush (2,3).
Aging muscles are characterized by progressive loss of mass and strength (4), suggesting an impairment of compensatory mechanisms. Currently, the best approaches to maintain aged muscle function and size are caloric restriction (limits autophagy and DNA damage) and exercise (restores muscle activity) (5)(6)(7)(8)(9)). Yet, the evidence that muscle stimulation improves mass maintenance suggests that proteins sensing sarcolemmal depolarization might be suitable candidates for triggering a compensatory response. Among the five subunits of the L-type Ca 2+ channel CaV1.1, the CaV1s subunit displays voltage sensor activity (10,11). The intracellular subunit of the complex, CaV1 (encoded by the Cacnb1 gene), plays a role in excitationcontraction coupling (ECC) by targeting CaV1s to the membrane and regulating its activity (12). Currently, CaV1A is considered as the skeletal muscle-specific isoform (13). Furthermore, CaV1 can be anchored to membrane, free in the cytoplasm, or located to the nucleus of proliferating muscle cells where it acts as regulator of transcription factors (14). CaV1 is essential for the development of embryonic mouse muscles and for the prepatterning and development of neuromuscular junctions (NMJs), independently of ECC (14)(15)(16). Yet, no ECC-independent pathway involving CaV1 is known in adult muscle.
Here, we provided the link between electrical activity sensing protein and muscle adaptation that compensates for atrophy. First, we demonstrated how the innervation state of skeletal muscle regulates the CaV1 isoform transition epigenetically. We showed that CaV1D is the constitutively expressed isoform in adult mouse muscle, whereas CaV1E is the embryonic variant up-regulated upon impairment of electrical activity. Second, we highlighted CaV1E as a major player in limiting muscle mass loss in mice through its ability to trigger GDF5 signaling in denervated muscle. In addition, we revealed that muscle aging is associated with altered CaV1E/GDF5 axis and that overexpression of CaV1E drives the pathway necessary to counterbalance age-related muscle atrophy in rodents. Last, we provided evidence of a human hCaV1E isoform expressed in adult muscle and tightly linked to muscle decline in aging.

Embryonic CaV1E expression in adult mouse muscle
Resection of mouse sciatic nerve is a suitable model for measuring the response of adult skeletal muscle to alterations in activity (1,17). To establish whether the expression of Cacnb1 could change in this model, we quantified its mRNA by reverse transcription quantitative polymerase chain reaction (RT-qPCR) using primers in exons 2 and 3. We observed a time-dependent increase in the amplification of this region in denervated tibialis anterior muscles (TAs) compared with innervated control muscles [day zero (D0)] (Fig. 1A). Western blot of CaV1 revealed the appearance of an extra 70-kDa band whose intensity increased with time after denervation, whereas the intensity of the band around 55 kDa was unchanged (Fig. 1, B and C).  (40)  , n = 6 mice per group; (C) and (I), n = 3 independent experiments]; *P < 0.05, ***P < 0.001 (ordinary one-way ANOVA, Dunnett's test). (G) Means ± SEM (n = 3 independent experiments); ***P < 0.001 (ordinary two-way, Sidak's test).

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The Cacnb1 gene (GSMG0007319) has 14 exons that can be spliced to give six transcript variants (Fig. 1D). To identify potential Cacnb1 splicing events occurring in denervated muscle, we performed a genome-wide transcriptomic analysis at the exon level on RNA extracted from innervated and denervated mouse TAs. We found 1022 differentially regulated alternative splicing events (from 706 distinct genes) (data file S1). Among these, Cacnb1 displayed a first exon splicing event, showing that the transcript started in a putative noncoding sequence at the 5′ end of exon 3 in innervated muscles. In denervated muscles, another transcript of Cacnb1 starting at exon 1 was found to be up-regulated ( Fig. 1E and table S1), implying the transcription of two different splicing isoforms. RT-PCR confirmed that in innervated TAs, the Cacnb1 open reading frame is at the 5′ end of exon 3 (ATG1). By contrast, in denervated muscles, two Cacnb1 transcripts were expressed: one starting at exon 1 (ATG2) and another at ATG1-exon 3 (Fig. 1, F and G).
By searching Cacnb1 variants in the National Center for Biotechnology Information database, we found that Cacnb1-D is the only one starting immediately upstream the exon 3. On the basis of these results, our data suggest that the specific CaV1 isoform expressed in the adult mouse skeletal muscle is CaV1D and not CaV1A, as expected (14). Moreover, the size of the extra CaV1 band appearing in denervated muscle suggested that it could be CaV1B or CaV1E (66 and 70 kDa, respectively). However, CaV1 antibody targeting a central peptide of the protein [AbCaV1: sc-25689 (H-50) raised against amino acids 211 to 260 L-type CaV1A of human origin, NM_199247-NP_954855] recognized only CaV1E in mouse. To validate which isoform was up-regulated after denervation, we designed specific primers matching the two sequences at the level of the 3′ end of exon 13 for Cacnb1-D and exon 14 for Cacnb1-E. RT-PCR data confirmed that only Cacnb1-E increased in denervated muscle (Fig. 1, H and I), confirming protein expression data.
Several embryonic proteins are induced after muscle denervation (3,(21)(22)(23)(24)(25). We wondered whether CaV1E could be an embryonic isoform. RT-PCR (primers matching exon 14) (Fig. 1K) and RT-qPCR (primers in exons 2 and 3 for Cacnb1-E and exon 13 for Cacnb1-D) revealed that Cacnb1-E is the specific variant in embryonic and neonatal muscles, whereas Cacnb1-D is not expressed in the embryo ( fig. S1, A and B). The Cacnb1-E transcript in embryonic muscle started at exon 1 (fig. S1, C and D) as Cacnb1-E expressed in denervated adult muscle. Western blotting showed the same bands in denervated adult and embryonic muscle protein extracts using AbCaV1 (Fig. 1L) or an antibody specific to CaV1E (fig. S1E). Last, we analyzed whether CaV1D and CaV1E could have different localization in muscle fiber. Immunofluorescence analysis of innervated and denervated muscle sections and isolated fibers with AbCaV1 showed that CaV1 staining and triadic localization most likely reflected expression of CaV1D ( fig. S2, A to C). In contrast, CaV1E-specific staining was distributed at the Z-lines ( fig. S2, D to F). Furthermore, CaV1E-specific staining localized mostly at the nuclei, consistently with the presence of a nuclear localization signal predicted by cNLS mapper, and its intensity increased in denervated muscle compared with CaV1 staining ( fig. S2, G to L).
In conclusion, our results show that an alternative first exon splicing is the source of the differential expression of mouse adult and embryonic Cacnb1 variants. CaV1D, not CaV1A, is expressed in innervated adult skeletal muscle, whereas embryonic muscle expresses CaV1E. CaV1E is low in innervated adult mouse muscle, but denervation specifically increases its expression. Moreover, CaV1D and CaV1E display different intracellular locations in adult muscle fibers.

CaV1E and GDF5 signaling activation after denervation
To evaluate whether CaV1E has a role in disuse atrophy, we injected into mouse TA an adeno-associated virus (AAV) vector carrying a short hairpin RNA targeting a sequence in Cacnb1 exon 2 (AAV-ShCaV1E) (26) and thereby abolishing specifically Cacnb1-E. Two months after injection, CaV1E expression induced after denervation was decreased by ~90% (Fig. 2 , C to F). This suggested a role for CaV1E in preserving disused muscle mass. The GDF5 pathway has been shown to be essential for limiting muscle loss under atrophic conditions (2). We thus evaluated Gdf5 expression in the absence of CaV1E and found a significant (P < 0.0001) reduction in the denervation-induced Gdf5 increase (Fig. 2G). In contrast, Gdf8 or Bmp7 transcription, which could be both implicated in increased atrophy (27,28), was not modified upon CaV1E ablation (Fig. 2, H and I). Furthermore, all components of the GDF5 pathway, SMAD1/5 phosphorylation, SMAD4 nuclear translocation, and Id-2 transcription (29,30), were inhibited with CaV1E downregulation, suggesting the positive control of GDF5 signaling by CaV1E (Fig. 2, J to O). However, Id-1 expression was not modified after denervation in scrambled (Scra)-or AAV-ShCaV1E-treated muscle (Fig. 2N), suggesting that its activation might be very weak or transient (2).
CaV1 has been shown to inhibit myogenin signaling during myoblast proliferation (14); yet, there are no data about this regulation in adult muscle. In innervated or denervated TAs, we found no change in transcription of Myogenin after CaV1E knockdown ( fig. S3G) and no alterations in Fbxo32, MuRF1 expression ( fig. S3, H and I), indicating that the myogenin pathway is unlikely to participate in the increased atrophy after CaV1E down-regulation. However, Chrna1 transcription after denervation was significantly (P < 0.0001) reduced in the absence of CaV1E, suggesting its involvement in modulating genes associated with end-plate formation, independently of myogenin. A previous study showed that in proliferating muscle cells, canonical and noncanonical DNA E-box sequences (CANNTG and CANNNTG) of several promoter regions could be targeted by CaV1 (14). Analysis of the 100 most differentially regulated genes in innervated and denervated TAs revealed the presence of one or more E-box sequences in the promoter of all these genes (table S2). However, among them, only Gdf5 and myogenin had a demonstrable role in muscle mass homeostasis (2,31).
To further analyze the role of CaV1E on Gdf5 expression, we used the myogenic cell line C2C12 as tool in vitro.  S4, G and H), mimicking its effect in vivo. We then measured in C2C12 the consequence of CaV1E down-regulation on the promoter activity of Gdf5. Thus, the sequence from −312 to the Gdf5 transcription start site, which contains one CANNTG and two CANNNTG E-boxes, was cloned upstream of firefly luciferase in herpes simplex virus thymidine kinase (HSVTK)-Luc3′-modified plasmid and transfected into C2C12 cells. Firefly/Renilla signal increased during differentiation, reflecting Gdf5 promoter activation, and this effect was abolished by the ShCaV1E (Fig. 2P). These data indicated that CaV1E targets the Gdf5 promoter, consistently with the effect observed in vivo and in vitro.
CaV1E seems to precede CaV1D appearance in the embryo and in C2C12. We thus checked whether Cacnb1-E and To confirm that the increased atrophy upon CaV1E downregulation was dependent on inadequate GDF5 activation, we over-

Aging muscles: A key role for CaV1E
During aging, skeletal muscle shows denervation-like signs and progressive muscle wasting (4,32). This suggests that impairment in compensatory response might occur. However, little is known about CaV1 expression and function (26), and nothing is known about Gdf5 amount in aging muscle.
Because our data indicate the involvement of CaV1E via Gdf5 in muscle maintenance, we investigated its role in age-related muscle wasting. In C57bl/6 mice, we observed significant (P < 0.0001) TA muscle mass loss at 78 weeks relative to adult mice at 12 weeks (Fig. 3A). In addition, Cacnb1-E basal expression was significantly lower in TAs of 78-week-old compared with 12-week-old mice (P < 0.0001) (Fig. 3B), whereas Cacnb1-D transcription did not change (Fig. 3C). To evaluate whether the cross-talk between Cacnb1-E and Gdf5 would also be affected, we quantified these transcripts after denervation in TAs from 12-, 52-, and 78-week-old mice. Although CaV1E expression increased in denervated young muscle, the upregulation of transcript and protein in response to denervation was impaired since 52 weeks of age (Fig. 3, D, G, and H). Consequently, the Gdf5 increase was reduced (Fig. 3F), affecting SMAD1/5 phosphorylation (Fig. 3, J and K). Id-1 remained unchanged (Fig. 3L), whereas Id-2 transcription followed the altered Gdf5 induction (Fig. 3M). CaV1D expression did not change (Fig. 3, E, G, and I).
We explored some possible causes responsible for decreasing CaV1E during aging. Measuring the expression of NMJ components Chrna1, Chrne, Chrng, and Musk in 78-week-old mice muscles, we could not detect changes indicating end-plate alterations at this age (Fig. 4, A to D). Similarly, we checked whether age-related modifications in fiber-type composition occurred, maybe explaining CaV1E down-regulation. Expression of myosin heavy chain (MyHC) isotypes IIA and IIX decreased significantly during aging (P = 0.006 and 0.013, respectively), as reported (9,33,34), whereas MyHC-I and MyHC-IIB did not change between 12 and 78 weeks of age (Fig. 4, E to H). To elucidate whether CaV1E expression could be associated to fiber-type modifications, we analyzed its amount in MyHC-I-and MyHC-II-positive fibers by immunofluorescence. In young muscle, CaV1E was expressed at higher intensity in MyHC-IIA-and MyHC-IIX-positive fibers, although in old muscle, it was decreased in all fiber types (Fig. 4, I to K).
If CaV1E increased upon lack of nerve activity, for example, denervation, modifications in muscle activity could potentially affect basal Cacnb1-E transcription. Hence, we measured Cacnb1-E mRNA after acute exercise of young and old mice (35,36). Exercise training restored Cacnb1-E transcription in old muscles but not in young (Fig. 4L), with Cacnb1-D expression unaffected (Fig. 4M), suggesting that increased muscle activity is able to rapidly normalize Cacnb1-E transcript only when it is decreased. However, MyHC-IIA and MyHC-IIX expressions were not restored, implying that contractile activity could regulate Cacnb1-E expression independently of type IIA-IIX fiber abundance (Fig. 4, N and O).
To evaluate whether CaV1E or GDF5 overexpression might mitigate age-related muscle mass loss, we injected AAV vectors carrying CaV1E or GDF5 into the TA of 78-to 80-week-old mice. A strong overexpression of CaV1E and GDF5 was observed 3 months after injection (Fig. 5, A, B, and D; and fig. S6A), without modifying Cacnb1-D expression (Fig. 5C and fig. S6C), yet increasing expression of each other reciprocally ( Fig. 5D and fig. S6B). The up-regulation of Cacnb1-E in old muscle activated GDF5 signaling, as measured by SMAD1/5 phosphorylation and SMAD4 nuclear translocation. (Fig. 5, E to G). The increase in Gdf5 either by CaV1E or by its own overexpression induced Id-2 transcription, with no changes in Id-1, confirming a weaker or transient response of this factor to GDF5 (Fig. 5, H and I; and fig. S6, D and E). In addition, the rise of GDF5 signaling after Cacnb1-E or Gdf5 overexpression was associated with preservation of aged muscle mass (Fig. 5J) and a gain of specific force (Fig. 5K) compared with control old muscles.
Subsequently, we investigated whether CaV1E overexpression could affect muscle mass of young mice (12 weeks). Ectopic expression of CaV1E ( fig. S7, A, D, and E) had no effect on Cacnb1-D, yet it induced a slight increase in Gdf5 expression ( fig. S7, B to E). However, GDF5 signaling, measured by Id-1 and Id-2 transcription, was not or poorly activated ( fig. S7, F and G). Consequently, muscle mass and the response to denervation were not different between Scra-and CaV1E-treated TAs of young mice ( fig. S7, H to L). We also overexpressed GDF5 in young TAs ( fig. S8, A, D, and E), which induced Cacnb1-E transcription in innervated TAs compared with scrambled ( fig. S8, B, D, and E) without affecting Cacnb1-D expression (fig. S8C). Nevertheless, GDF5 overexpression and its activated signaling, measured as Id-1 and Id-2 transcription ( fig. S8, F and G), increased mostly innervated muscle mass ( fig. S8, H to L). Overall, these data show that the loss of CaV1E/GDF5 cross-talk observed during aging may be critical (H, I, and K) Means ± SEM (n = 3; Western blots used for quantification are showed in raw data); *P < 0.05, ** P < 0.01, and ***P < 0.001 (ordinary one-way ANOVA, Sidak's test).

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for muscle wasting, and its rescue counteracts the process of agerelated muscle decline.

Human muscle: A new CaVE isoform implicated in skeletal muscle aging
Given the apparent importance of CaV1E in mouse skeletal muscle, we wondered whether an analogous mechanism might be conserved in humans and, thus, if another unidentified CACNB1 isoform was expressed in adult human skeletal muscle. Three human CACNB1 (hCACNB1) variants have been identified corresponding to the mouse isoforms A (specific of skeletal muscle), B, and C (Fig. 6A) (18)(19)(20)37). Human mRNA extracted from one quadriceps and two fascia lata (FL) muscle biopsies of healthy adult participants together with human mRNA extracted from the cervical SC (table S2), as positive control for hCACNB1-B (18)(19)(20), were probed for exons 13 and 5 to 9. Amplification of the sequence in exon 13 showed that all muscles expressed hCACNB1-A. As in mouse muscle, amplification of the region between exons 5 and 9 demonstrated that only a 380 bp corresponding to the putative hCACNB1-A appeared and that the exon 7B-containing isoform, hCACNB1-B (245 bp), was solely expressed in SC. Furthermore, no transcription of hCACNB1-C, which also has the short 7B exon, was found in muscle (Fig. 6B). We further checked whether human muscle could express an hCACNB1 transcript analogous to mouse Cacnb1-E. Amplification of the region in exon 14 revealed that the human muscles expressed the previously unidentified variant that we have called hCACNB1-E (Fig. 6C). This isoform corresponded to the predicted XM_006722072.2 variant, having a codon start (ATG2) upstream the exon 3 (Fig. 6, A and C). Western blot and immunofluorescence experiments confirmed its expression in two different human FLs (Fig. 6, D and E), with localization similar to that of mouse CaV1E (fig. S2, E and F).
Because we found that the altered CaV1E/GDF5 axis was associated to muscle wasting during aging in mice, we compared characteristics indicating muscle mass (lean mass percentage) and function (power)  (Table 1). (H and I) Distribution of (H) hCACNB1-E or (I) hCACNB1-A expression in human quadriceps biopsies from healthy young and aged volunteers (Table 1). (J) Linear regression between hCACNB1-E expression and lean mass percentage in human quadriceps biopsies from healthy young and aged volunteers (Table 1). (K) Distribution of hGDF5 (pink triangles, left y axis) and hCACNB1-E (blue circles, right y axis) expression in human quadriceps biopsies from healthy aged volunteers (Table 1) having increasing lean mass percentage. Dotted black line indicates the average of lean mass percentage of the young group. (F to I) Means ± SEM (young, n = 8; aged, n = 17); **P < 0.01, ***P < 0.001 [independent samples t test (two-tailed)]. (38,39) in a cohort of healthy young (20 to 42 years) and aged (70 to 81 years) volunteers included in a previous study (Table 1) (39). The aged group displayed significantly lower lean mass and power than the young group (P = 0.003 and P < 0.0001, respectively) (Fig. 6, F and G). We then measured the hCACNB1-E transcript and found a significant reduction (P = 0.0045) of its expression in the aged group (Fig. 6H), whereas hCACNB1-A transcription did not differ between groups (Fig. 6I). A low hCACNB1-E expression was also associated with a low lean mass percentage (Fig. 6J). We detected a weak amount of hGDF5 in muscle biopsies; however, in aged muscle samples, we could associate low expression of both hCACNB1-E and hGDF5 with low lean mass percentage. In addition, participants with higher lean mass percentage displayed higher expression of both hCACNB1-E and hGDF5 (Fig. 6K). Overall these results suggest that the CaV1E/GDF5 axis in compensatory response and aging ( fig. S9) might be a conserved mechanism between mice and humans.

DISCUSSION
In adult skeletal muscle, the mechanism connecting electrical activity sensing and changes in gene expression is unclear. Here, we showed that denervation boosts the expression of embryonic CaV1E isoform, which mediates transcriptional reprogramming in adult muscle. Many years ago, CaV1 isoforms were identified in humans (19,20,40), in rats (18), and, less clearly, in mice (13). We anticipated that the 55-kDa band corresponded to CaV1A, a previously identified muscle-specific isoform with this molecular weight (18,19). However, we speculated that the 70-kDa band could be a previously unappreciated CaV1 isoform. Our results revealed that CaV1D, not CaV1A, is the main constitutive isoform specific to normal adult mouse muscle and that CaV1D and CaV1E have distinct roles. In silico analysis using the cNLS mapper confirmed the presence of a nuclear localization signal at the N terminus of CaV1E but not in CaV1D. CaVA is not expressed in adult mouse muscle and C2C12; however, its N terminus is putatively identical with those of CaVE, suggesting that the mechanism by which it translocates to the nucleus is the same described in past publications (14,41). This finding also implicates CaVD in regulating calcium channel assembly in the cytoplasm, whereas CaVE could have a role in both modulating nuclear transcription factors and cooperating with cytoskeletal or T tubules at the Z-lines, as demonstrated for others proteins (42,43). We also discovered that CaV1E is the main CaV1 isoform in proliferating and differentiating C2C12 cells, whereas CaV1D and CaV1A isoforms are undetectable. Previous studies reporting expression, transcriptional activity, and nuclear localization of CaV1A in muscle fibers (41), muscle precursors, and C2C12 cells (14) were likely evaluating the as-yet-undiscovered CaV1E. An issue of our study could be deciphering mechanisms underlying the transition between CaV1E and CaV1D during myogenesis and identifying molecular factors regulating the implicated splicing events.
The major finding of this work is the central role of CaV1E in maintaining muscle mass. GDF5 is one of the main regulators of muscle mass homeostasis in response to disuse atrophy (2); however, few studies report progress on modulators of this factor (3,44,45). Our data validate previously described roles of GDF5 (2,46) and add a positive upstream player controlling its signaling. CaV1E expression is needed for the activation of the Gdf5 promoter, probably by acting on specific E-box sequences, as described for other target genes (14).
In addition, we demonstrated that a compromised CaV1E/GDF5 axis during aging is associated to muscle wasting. In our study, CaV1E overexpression led to increased GDF5 expression and activation of its pathway, improving age-related muscle decline. By contrast, CaV1E overexpression did not affect muscle mass in young mice. This suggests that endogenous CaV1E induction after nerve withdrawal is sufficient to trigger the maximal compensatory response. In addition, when muscles express a physiological amount of GDF5, feedback mechanisms could be activated to prevent hypertrophy. GDF5 exerted hypertrophic effects mostly on innervated muscle, suggesting that nerve is important for its trophic signal. In this hypothesis, GDF5 could stimulate pre-and/or postsynaptic NMJ partners, improving neurotransmission. Thus, increased GDF5 signaling, induced by overexpression of CaV1E or GDF5 itself, might counteract age-related muscle decline also by preventing the associated denervation occurring later. CaV1 has been shown as important for correct synaptic patterning in the embryo (15). Although a role for GDF5 in this process has not been established yet, its relevance to mediate reinnervation in adult muscle is proven (3). We also demonstrated that an hCACNB1-E variant is expressed in human muscle, indicating that mechanisms governing skeletal muscle homeostasis might be conserved across mammalian species. Although the hCACNB1-E transcript is slightly different compared with mouse Cacnb1-E, we observed a similar localization and hypothesize the same function. hCACNB1-E expression was higher in young compared with aged human muscle with reduced lean mass and power, strongly suggesting a defect in compensatory mechanism contributing to age-related muscle mass loss. Despite weak transcription of hGDF5 in undamaged young human muscle, we demonstrated an association between its expression and muscle mass.
Here, we identified CaV1E as "sensor" of electrical activity defect, which could be a central player, upstream of all these factors.
Age-related muscle decline is a life-threatening condition (49) affecting a large population, which results in progressive loss of autonomy, increased mortality associated to frailty, and risk of falls in elderly individuals (50). GDF5 or GDF5-like molecules can be considered as potential therapeutic compounds not only to prevent muscle mass wasting during senescence but also, in a wider application, to ameliorate consequences of neuromuscular defects. Last, deciphering the fine regulation of CaV1E expression and GDF5 signaling would open a promising therapeutic field that will contribute to increasing the quality of life.
The study has some limitations. The events altering the correct basal expression of CaV1E in aging muscle remain unresolved. Previous studies have reported a link between elevated CaV1A and age-related muscle weakness (26,51,52), but Cacnb1 transcripts were not fully characterized nor antibodies used were well defined. CaV1E expression is higher in young type IIA-IIX fibers compared with IIB. The fiber type II decrease occurring during aging may thus have a role in CaV1E reduction. Still, other mechanisms, dependent on muscle activity per se, modifying DNA methylation or mitochondrial metabolism, could participate in altering the CaV1E/GDF5 axis.

Study design
The primary objective of this study was to decipher the link between alteration of electrical activity and skeletal muscle mass maintenance. We focused on the role of CaV1, a subunit of the voltage sensor CaV1.1. RNA sequencing (RNAseq) data of innervated and denervated TAs led to the identification of an embryonic variant, CaV1E, up-regulated in mouse muscle by denervation. Validation of RNAseq data and characterization of CaV1 isoforms in mouse and human muscles have been performed by RT-PCR, qPCR, Western blot, and immunostaining experiments. To investigate the role of CaV1E in muscle mass homeostasis in mouse, we knocked down its local expression in vivo by AAV-shRNA. We quantified atrophy by measuring muscle weight and fiber size distribution. To determine fiber size, images were processed with a machine learning algorithm (Weka) under Fiji for accurate segmentation. Muscle histology was performed by hematoxylin and eosin and Sirius red staining. To evaluate the effect of CaV1E down-regulation on the transcriptional activity of the Gdf5 promoter, we performed a luciferase reporter-based assay in C2C12. To establish the effect of CaV1E or GDF5 in vivo on muscle mass, we overexpressed them in mouse by local AAV-gene transfer. To characterize the expression of CaV1 isoforms in humans, we used muscle biopsies of a cohort of healthy young and aged volunteers (39). Body composition and muscle function of volunteers included in the study (39) had been measured previously and provided for correlation analysis with hCACNB1-E. We estimated the sample size on the basis of known variability of assays. All mouse experiments were performed twice. Outliers not following normal distribution of samples were removed on the basis of Grubb's test. All experiments using animals and cells were done in a nonblinded manner, yet investigators were blinded to allocation in using human samples for RT-qPCR experiments.

Statistical analysis
For comparison between two groups, two-tailed paired and unpaired Student's t tests were performed to calculate P values and to determine statistically significant differences (significance was for P < 0.05, as detailed in the figure legends). For comparison among more than two groups, ordinary one-or two-way analysis of variance (ANOVA) tests followed by the appropriate multiple comparison tests (as detailed in the figure legends) were performed. All experiments have been done twice with the same results. All statistical analyses were performed with the GraphPad Prism 7 software.
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