Calsarcin-2 deficiency increases exercise capacity in mice through calcineurin/NFAT activation

The composition of skeletal muscle, in terms of the relative number of slow- and fast-twitch fibers, is tightly regulated to enable an organism to respond and adapt to changing physical demands. The phosphatase calcineurin and its downstream targets, transcription factors of the nuclear factor of activated T cells (NFAT) family, play a critical role in this process by promoting the formation of slow-twitch, oxidative fibers. Calcineurin binds to calsarcins, a family of striated muscle–specific proteins of the sarcomeric Z-disc. We show here that mice deficient in calsarcin-2, which is expressed exclusively by fast-twitch muscle and encoded by the myozenin 1 (Myoz1) gene, have substantially reduced body weight and fast-twitch muscle mass in the absence of an overt myopathic phenotype. Additionally, Myoz1 KO mice displayed markedly improved performance and enhanced running distances in exercise studies. Analysis of fiber type composition of calsarcin-2–deficient skeletal muscles showed a switch toward slow-twitch, oxidative fibers. Reporter assays in cultured myoblasts indicated an inhibitory role for calsarcin-2 on calcineurin, and Myoz1 KO mice exhibited both an excess of NFAT activity and an increase in expression of regulator of calcineurin 1-4 (RCAN1-4), indicating enhanced calcineurin signaling in vivo. Taken together, these results suggest that calsarcin-2 modulates exercise performance in vivo through regulation of calcineurin/NFAT activity and subsequent alteration of the fiber type composition of skeletal muscle.

The ubiquitously expressed serine/threonine phosphatase calcineurin plays a critical role in the remodeling of striated muscle tissue (reviewed in ref. 1). Low amplitude, sustained calcium waves induce calcium binding to calmodulin, which in turn associates with the regulatory calcineurin B subunit, thereby activating calcineurin A (2). Activated calcineurin A dephosphorylates transcription factors of the nuclear factor of activated T cells (NFAT) family, leading to their nuclear translocation. In skeletal muscle, nuclear NFATs associate with other transcription factors such as myocyte enhancer factor 2 (MEF2) and activate a characteristic Ca²⁺-dependent gene expression program (1, 3, 4). The activation of calcineurin/NFAT specifically induces a muscle fiber type switch toward a slow-twitch and oxidative phenotype, with an increase in expression of a subset of genes associated with type I myofibers, such as myoglobin and troponin I slow (5, 6), allowing for sustained and fatigue-resistant muscle activity. In contrast, high-frequency nerve activity promotes the formation of fast-twitch, glycolytic type IIb fibers, thereby facilitating quick contractions with rapid fatigability. In genetically engineered mice, forced overexpression of calcineurin in skeletal muscle induces an increase in the number of slow-twitch muscle fibers (7), while genetic disruption of calcineurin Aα or Aβ leads to a significant decrease in slow-twitch, oxidative fibers. Likewise, skeletal muscle–specific calcineurin B1–null mice fail to upregulate slow-twitch fibers upon increased workload (8).

In an attempt to identify novel muscle-specific modulators of calcineurin activity, we previously performed a yeast 2-hybrid assay using the regulatory subunit of calcineurin A as bait. With this approach, we discovered a family of striated muscle–specific, calcineurin-binding proteins, the calsarcins (9), which localize to the Z-disc of the sarcomere (reviewed in ref. 10). The calsarcin family consists of 3 members, with calsarcin-2 (also termed “myozenin 1” [Myoz1; ref. 11], or “FATZ” [ref. 12]) and calsarcin-3 being exclusively expressed in fast-twitch fibers of skeletal muscle tissue (9, 13). In contrast, calsarcin-1 is expressed in the adult heart and in slow-twitch fibers of skeletal muscle. Calsarcins are hallmarked by a multitude of Z-disc interaction partners that, in addition to calcineurin, include α-actinin, LIM domain–binding 3 (LDB3, also known as Cypher, ZASP, and Oracle), Telethonin/T-cap, γ-filamin (9, 13), and myotilin (14). Targeted ablation of the Myoz2 allele sensitized calsarcin-1–deficient mouse hearts to pressure overload and chronic calcineurin activation with excessive hypertrophy and subsequent cardiomyopathy (15), while transgenic overexpression of calsarcin-1 protected mouse hearts from angiotensin II–induced cardiac hypertrophy (16). In slow-twitch skeletal muscle, the lack of calsarcin-1 led to an increase in calcineurin activity associated with an expansion of type I muscle fibers (15).

To determine the function of calsarcins in fast-twitch skeletal muscle in vivo, we generated mice with genetic ablation of the fast-twitch muscle isoform calsarcin-2. Compared with WT mice, calsarcin-2–deficient mice displayed a significantly reduced body weight as well as a decreased fast-twitch muscle mass. Muscle atrophy does not account for the reduced muscle weight, as Myoz1 KO mice displayed markedly increased running distances in voluntary running as well as in a forced treadmill setting. Fiber type composition analyses of skeletal muscles revealed a significant switch toward oxidative fibers, with a substantial increase in type IIA fibers in fast-twitch muscles, consistent with an increase in calcineurin activity. In fact, skeletal muscles of mice lacking calsarcin-2 showed a significant excess of calcineurin activity compared with WT controls. Our data suggest an essential role for calsarcin-2 in the regulation of fiber type composition as well as exercise tolerance of skeletal muscle, likely via modulation of calcineurin-dependent signaling.

Generation of Myoz1 mutant mice. To analyze the function of calsarcin-2 in vivo, we generated mice with germline deletion of the Myoz1 allele by replacing exons 2 (which contains the translational start) and 3 with a neomycin resistance cassette and a lacZ reporter gene (Figure 1A). Breeding of Myoz1 mutant mice resulted in the expected Mendelian ratios of offspring. Genotyping was performed by Southern blot analysis and/or PCR (Figure 1B). The absence of calsarcin-2 expression was confirmed in Myoz1 KO mice by western blot analysis using protein homogenates of the gastrocnemius, a predominantly fast-twitch muscle. As expected, WT mice expressed calsarcin-2 protein, while no calsarcin-2 protein was detectable in Myoz1 KO mice (Figure 1C). The in vivo expression pattern of the Myoz1 gene was determined using β-galactosidase staining to detect the lacZ reporter under the control of the endogenous Myoz1 promoter (Figure 1D). These experiments revealed strong expression of Myoz1 in predominantly fast-twitch muscles, such as white vastus and gastrocnemius. In contrast, the soleus, a predominantly slow-twitch muscle, showed low Myoz1 expression confined to a subset of myofibers. The adult myocardium was devoid of Myoz1 expression, consistent with our previous observations (9).

Figure 1

Generation of Myoz1 mutant mice. (A) Schematic protein structure of calsarcin-2 highlights the calsarcin homology domain (CHD), which is highly conserved between calsarcin isoforms, the α-actinin binding domain (ABD), and the C terminus, which is highly homologous throughout the calsarcin family. The targeting vector and the targeted mouse Myoz1 allele are shown. Homologous recombination resulted in the deletion of exon 2 and 3 in the Myoz1 gene and their replacement with a neomycin-lacZ reporter cassette. CS2, calsarcin-2. (B) Southern blot analysis of EcoRI-digested genomic DNA of the F1 generation of Myoz1^–/+ mice. The 3.5-kb band represents the WT allele, and the 2.5-kb band corresponds to the targeted allele. (C) Western blot of muscle protein homogenate (50 and 150 μg) from gastrocnemius of WT and Myoz1 KO mice. (D) β-Galactosidase staining of tissues from WT and Myoz1 KO animals. The calsarcin-2–deficient tissues showed strong staining in predominantly fast-twitch fiber–containing muscles (e.g., white vastus [WV], gastrocnemius [gastroc]). The soleus, a predominantly slow-twitch muscle, displayed staining in a minor subset of fibers. The heart showed no significant expression of Myoz1. Original magnification, ×4. (E) H&E staining (top) and van Giesson staining (bottom) of gastrocnemius from Myoz1 KO mice and WT littermates. Scale bars: 50 μm. (F) Electron microscopy photographs of gastrocnemius (original magnification, ×19,000). (G) Quantification of 100 Z-disc widths from n = 4 mice per genotype. ^‡P < 0.001.

No obvious signs of myopathy were detected in the skeletal muscle of Myoz1-deficient mice by light microscopy (Figure 1E). However, by electron microscopy we found a widening of Z-discs (Figure 1, F and G), similar to previous findings in hearts of Myoz2-deficient mice (15).

Myoz1-deficient mice display a reduction in body weight and fast-twitch muscle mass. Myoz1 KO mice were viable, fertile, showed no overt abnormalities, and had a normal life span of greater than 20 months without premature death. However, morphometric analyses revealed a significant reduction in body weight in Myoz1 KO mice (P = 0.0017; Figure 2A). This effect remained significant when normalizing body weight to tibia length, thereby excluding a global growth retardation phenotype (P = 0.018; Figure 2B). Subsequent analyses of the gross morphology of skinned hindlimbs of Myoz1 KO mice uncovered a considerably slimmer shape of the calf compared with WT (Figure 2C). Of note, the silhouette of the calf is chiefly modeled by the gastrocnemius, whereas the soleus only marginally contributes to the shape of the calf. To further characterize this morphological difference, we weighed individual hindlimb muscles. The gastrocnemius of Myoz1 KO animals showed a 24.4% weight reduction compared with the gastrocnemius of WT littermates (P < 0.0001; Figure 2D). Normalization to body weight or tibia length (P = 0.0066; Figure 2E) did not change the level of significance. Interestingly, when comparing the weight of the soleus, a slow-twitch muscle with low expression levels of calsarcin-2, no difference was observed between Myoz1 KO and WT mice (Figure 2, F and G).

Figure 2

Reduced body weight and fast-twitch muscle mass in Myoz1-deficient mice. (A) Significant reduction in body weight of Myoz1 KO mice (n = 13–15) and (B) in body weight normalized to tibia length in Myoz1 KO mice (n = 6). (C) Morphology of skinned hindlimbs of Myoz1 KO mouse, revealing a slimmer shape of the calf in Myoz1 KO mice compared with WT littermate. Original magnification, ×4. (D) Weight of gastrocnemius in Myoz1 KO mice compared with WT littermates (n = 13–15). (E) Gastrocnemius mass normalized to tibia length (n = 6). (F) Weight of soleus in Myoz1 KO mice compared with WT controls. (G) Soleus weight normalized to tibia length. *P < 0.05, ^†P < 0.01, ^‡P < 0.001.

Lack of enhanced muscle atrophy in Myoz1-deficient mice. A common cause of muscle weight loss is atrophy, a process which accounts for the progressive muscle loss during immobilization, denervation or severe systemic diseases like AIDS, heart failure, or cancer (17). To test whether atrophy accounted for the reduced muscle weight observed in Myoz1 KO animals, we performed denervation experiments by severing the sciatic nerve, which leads to progressive atrophy of the gastrocnemius, soleus, and tibialis anterior, as measured by a reduction in muscle mass. As expected, both Myoz1 KO and WT mice showed substantial atrophy upon denervation (Figure 3A), but no substantial differences in the relative degree of atrophy were detectable between KO and WT mice. The relative gastrocnemius weight loss in Myoz1 KO mice after 7 days was 32.3%, while WT littermates lost 26.9% in muscle weight. After 14 days, Myoz1 KO mice lost 48.5% of gastrocnemius weight compared with 47.0% in WT muscle (Figure 3B). Weight loss of the soleus of Myoz1 KO and WT mice also did not differ significantly (Figure 3C). After one week, Myoz1 KO mice had lost 25.5% of muscle mass, compared with 21.6% in WT mice. After 2 weeks, the soleus weight loss was 35.7% in Myoz1 KO, while the soleus of similarly treated WT animals lost 30.3% of its weight. Thus, following denervation, mice lacking calsarcin-2 showed no differential degree of atrophy compared with WT mice in either fast-twitch and slow-twitch muscles. Hence, a marked contribution of atrophic processes to the baseline weight loss of muscles lacking calsarcin-2 appears unlikely.

Figure 3

Lack of enhanced muscle atrophy in Myoz1-deficient mice. (A) Following denervation (den) or sham operation, atrophy of the hindlimb muscles was observed in both WT and Myoz1 KO mice after 2 weeks Original magnification, ×4. (B and C) Weight of gastrocnemius (B) and soleus (C) showed no significant differences between Myoz1 KO mice and WT littermates after 1 or 2 weeks following denervation (n = 5 each).

Ablation of calsarcin-2 increases endurance capacity. To determine whether ablation of calsarcin-2 leads to impaired muscle function, we analyzed intact diaphragm muscle strips for fatigability and tension power (Figure 4, A and B). Calsarcin-2 is highly expressed in the diaphragm. Surprisingly, both analyses showed greater resistance to fatigue as well as greater tension power upon tetanic stimulations in Myoz1 KO muscles. To further analyze this finding in vivo, we compared the voluntary running behavior of Myoz1 KO mice with that of their WT littermates. Mice were provided unlimited access to running wheels, and after 1 week of acclimatization, data were acquired for 14 days. Unexpectedly, Myoz1 KO mice ran considerably longer distances per day (P = 0.033; Figure 4C) and reached higher average speeds (P = 0.036; Figure 4D) compared with WT littermates. Of note, even after 14 days of running, animals lacking calsarcin-2 still displayed a strong tendency toward reduced body weight (P = 0.061; Figure 4E). Similar to the observations in sedentary mice, the mass of the gastrocnemius remained significantly reduced (P = 0.0001; Figure 4F) independent of normalization to body weight (P = 0.002; Figure 4G). Again, the weight of the soleus was comparable in both Myoz1 KO and WT control mice (data not shown).

Figure 4

Ablation of calsarcin-2 increases endurance capacity following voluntary running wheel exercise. Diaphragm muscle strip analyses of Myoz1 KO mice compared with WT controls showed (A) less fatigability (–13.8%; P < 0.0001) (100 Hz/250 ms) and (B) greater tension power upon tetanic stimulations (+13.4%; P = 0.011). (C) Increases were observed in daily running distances (n = 8 each) and (D) average speeds (n = 8 per group) of Myoz1 KO mice compared with WT littermates. Changes were also observed in (E) body weight (n = 8), (F) gastrocnemius weight (n = 8), and (G) gastrocnemius mass normalized to body weight (n = 8) of Myoz1 KO mice and WT littermates after 14 days of running. *P < 0.05, ^†P < 0.01, ^‡P < 0.001.

Given that endurance is fundamentally based on the available amount of oxidative capacity, these findings suggest that Myoz1-deficient mice display an increased oxidative capacity in their skeletal muscles. To further test this hypothesis, we used a treadmill to run mice to exhaustion. Mice were forced to run according to a defined protocol (10% incline, 10 minutes at 10 m/min, with an increase of an additional 2 m/min every 2 minutes [+0.033 m/s]). This experimental design is known to rapidly reach the point at which the oxidative threshold is exceeded (18). Consistently, Myoz1 KO mice displayed a significantly extended endurance capacity compared with WT littermates (P = 0.027; Figure 5A). The Myoz1 KO mice ran 1,511 seconds (25.2 minutes) to exhaustion, while WT mice only ran for a mean of 982 seconds (16.4 minutes). This enhanced performance corresponds to a distance of 435.8 m in Myoz1 KO mice compared with 248.7 m in WT mice (P = 0.027; Figure 5B) and to an energy expenditure of 24.8 joules and 13.9 joules, respectively (P = 0.024; Figure 5C). We conclude that lack of calsarcin-2 markedly increases the endurance capacity of mice under both voluntary and forced conditions.

Figure 5

Ablation of calsarcin-2 increases endurance capacity after treadmill running. (A) Myoz1 KO mice ran 1,511 seconds (25.2 min) to exhaustion, while WT mice ran for a mean of 982 seconds (16.4 min, n = 5–6). (B) Myoz1 KO mice ran a distance of 435.8 m, compared with WT mice, which ran 248.7 m (n = 5–6). (C) Myoz1 KO mice spent 24.8 joules while running, compared with WT littermates, which used 13.9 joules (n = 5–6). *P < 0.05.

Myoz1 KO mice display a fiber type shift toward slow-twitch oxidative fibers. Skeletal muscles are composed of a spectrum of different fibers, ranging from ultra-fast glycolytic type IIb fibers to slow, oxidative, fatigue-resistant type I fibers. The range between these extreme fiber types is complemented by IIx and IIa fibers, which reveal an intermediary phenotype. While fiber types I and IIa exhibit an oxidative metabolism, type IIx and IIb fibers bear glycolytic capacities (1, 19, 20). To elucidate the morphological and molecular basis of the increased endurance capacity of Myoz1 KO mice, we analyzed the fiber composition of gastrocnemius (predominantly fast-twitch) and soleus (predominantly slow-twitch) muscles.

Interestingly, Myoz1 KO mice exhibited a considerable excess of oxidative type IIa fibers in the gastrocnemius (Figure 6A). When quantifying and normalizing this effect per mm², we found a 56.2% increase of type IIa fibers in Myoz1 KO mice compared with WT littermates (P = 0.01; Figure 6B), while the cross-sectional area of individual type IIa fibers remained unchanged (Figure 6C).

Figure 6

Fiber type shift seen in skeletal muscle of Myoz1 KO mice. (A) Immuno­fluorescence histochemistry for type IIa fibers using SC-71 antibody (green, FITC-labeled lectin). (B) An increase of 56.2% was found in the number of type IIa fibers/mm² in the gastrocnemius of Myoz1 KO mice compared with WT littermates (n = 6–7 animals). (C) The cross-sectional area of type IIa fibers remained unchanged. (D) SDH staining of gastrocnemius showed an increase of SDH-positive fibers in Myoz1 KO mice compared with WT controls. (E) Quantification of SDH-positive fibers shows a 2.58-fold increase in Myoz1 KO mice compared with WT (n = 4 mice per group). (F) Immunofluorescence histochemistry for type IIb fibers using BF-F3 antibody (green, FITC-labeled lectin). (G) The number of type IIb fibers/mm² increased in Myoz1 KO mice. (H) A reduction was found in the cross-sectional area of type IIb fibers (n = 105–178 cells). (I) Immunofluorescence histochemistry for type I fibers using NOQ7.5.4D antibody. (J) The number of type I fibers/mm² increased in the soleus of Myoz1 KO mice compared with WT littermates (n = 4–6 mice). (K) Cross-sectional area of type I fibers increased in the soleus in Myoz1 KO mice compared with WT controls (n = 143–260 fibers). Scale bars: 100 μm. *P < 0.05, ^†P < 0.01, ^‡P < 0.001.

We examined whether the number of fibers staining positive for succinate dehydrogenase (SDH) also differed between Myoz1 KO and WT controls. SDH is localized to the inner membrane of the mitochondria and takes part in the Krebs cycle. Therefore, it can be used as an indicator for oxidative fibers (Supplemental Figure 1; supplemental material available online with this article; doi: 10.1172/JCI36277DS1). Consistent with the markedly increased number of type IIa fibers, the content of fibers staining positive for SDH was significantly higher in the gastrocnemius of Myoz1 KO mice (Figure 6D). When quantifying SDH-positive fibers, we found a 2.6-fold increase in Myoz1 KO mice (P = 0.008; Figure 6E), confirming an enhanced oxidative capacity in calsarcin-2–deficient gastrocnemius muscles.

Conversely, the glycolytic fast-twitch and fatigue-susceptible type IIb fibers (Figure 6F) were increased in number per mm² (P = 0.0007; Figure 6G) but substantially reduced in size (P = 0.0001; Figure 6H). The mean total muscle cross-sectional area of gastrocnemius muscle was reduced as well (78.7%, P < 0.05; Supplemental Figure 2). Thus, the increase in number per mm² can be explained by a compaction of muscle fibers, while the total amount of type IIb fibers remained unchanged. Immunofluorescence analyses of soleus muscle (Figure 6I) revealed a highly significant increase of 68.1% in the number of type I fibers in Myoz1 KO mice (P = 0.007; Figure 6J). Interestingly, the gain of slow-twitch fibers was further augmented by the finding that Myoz1 KO type I fibers also featured a pronounced increase (24.6%) in cross-sectional area (P < 0.0001; Figure 6K), while the number of type IIa fibers remained unchanged (data not shown). Taken together, these findings show that the absence of calsarcin-2 leads to a marked fiber type shift towards oxidative, slow-twitch fibers, either via an increase of type IIa fibers (in fast-twitch muscles) or of type I fibers (in slow-twitch muscles).

Calsarcin-2 inhibits calcineurin activity. The phosphatase calcineurin acts as a key regulator of fiber type specificity by promoting expression of a slow-twitch and oxidative phenotype with an increase of a subset of genes, which are associated with type I and type IIa myofibers (5, 6). We have previously shown that the absence of another member of the calsarcin family, calsarcin-1, which is exclusively expressed in slow-twitch muscle fibers and the heart (9), leads to an excess of calcineurin activity in these tissues (15). Since calsarcin-1 and calsarcin-2 both bind to calcineurin, we tested whether calsarcin-2 modulates calcineurin activity in C2C12 myoblasts stably transfected with an NFAT-responsive luciferase reporter (NFAT-luc). These cells were cotransfected with full-length (non–constitutively active) calcineurin A, an NFAT expression plasmid, and increasing dosages of calsarcin-2. Subsequently, cells were incubated with the known calcineurin stimulators PMA and ionomycin. PMA and ionomycin induced NFAT reporter activity 5.0-fold (P < 0.001; Figure 7A). In contrast, adding increasing amounts of calsarcin-2 completely abrogated the increase in reporter gene activity to values even lower than the unstimulated baseline level (75.8% of the baseline value), suggesting that calsarcin-2 inhibits calcineurin/NFAT signaling. Moreover, calsarcin-2 also inhibits baseline calcineurin/NFAT activity in C2C12 cells (Supplemental Figure 3).

Figure 7

Calsarcin-2 inhibits calcineurin activity in vitro and in vivo. C2C12 cells containing an NFAT reporter plasmid (NFATluc) were transfected with (A) full-length calcineurin A (CnA), an NFAT expression plasmid, and an increasing amount of calsarcin-2 or (B) constitutively active calcineurin (ΔC) and NFAT expression plasmid, resulting in induction of the NFAT-luciferase reporter (P < 0.01). (A) After transfection, cells underwent treatment with the calcineurin stimulators PMA and ionomycin (PMA+Iono). PMA and ionomycin strongly induced NFAT reporter activity, while increasing amounts of calsarcin-2 completely abrogated the increase in reporter gene activity. (B) The presence of calsarcin-2 inhibited the calcineurin activity in a dose-dependent and significant manner. (C) Western blot analysis (left) and densitometric quantification (right) showing induction of the endogenous calcineurin-responsive Rcan1-4 gene in Myoz1 KO mice and (D) an increase of nuclear NFATc4 in the gastrocnemius of Myoz1 KO mice compared with that of WT littermates. *P < 0.05, ^†P < 0.01, ^‡P < 0.001.

To further corroborate these data, we examined whether calsarcin-2 is also capable of inhibiting constitutively activated calcineurin. NFAT-luc reporter cells were transfected with cDNAs encoding for constitutively active calcineurin (ΔC; ref. 21) and NFAT, resulting in a significant induction of the luciferase reporter (P < 0.01; Figure 7B). Again, the addition of calsarcin-2 completely inhibited the activation of the reporter gene.

Increased calcineurin activity in skeletal muscles of Myoz1 KO mice. Given the results from the in vitro experiments, we hypothesized that the loss of calsarcin-2 might cause an increase in calcineurin activity in vivo, which in turn would provide an explanation for the observed fiber type shift. To test this assumption, we examined the regulation of a calcineurin-dependent gene, regulator of calcineurin 1 (Rcan1; formerly termed modulatory calcineurin interacting protein [Mcip1] or Down syndrome candidate region 1 [Dscr1]; ref. 22), in gastrocnemius. Since the expression of the Rcan isoform 1-4 is tightly controlled by a cluster of 15 NFAT binding sites, it represents a sensitive endogenous calcineurin reporter gene (23). Consistent with an increase in calcineurin activity, Rcan1-4 was markedly induced in Myoz1 KO mice (Figure 7C). Densitometric quantification of the western blot revealed a 4.0-fold upregulation of Rcan1-4 (P = 0.02; Figure 7C).

Another hallmark of calcineurin signaling is the dephosphorylation and consequent nuclear translocation of its downstream targets, transcription factors of the NFAT family (24). In Myoz1 KO mice, the amount of nuclear NFATc4 was significantly increased (P = 0.017; Figure 7D). Densitometric analysis showed a significantly greater abundance, 158.1%, of nuclear NFATc4 in Myoz1 KO gastrocnemius compared with that of WT (Figure 7D). Taken together, these data support the notion that calsarcin-2 represents an endogenous inhibitor of calcineurin/NFAT signaling. In turn, when calsarcin-2 is absent, calcineurin activity is enhanced.

Enhanced muscle regeneration in Myoz1-deficient mice. Recently it was reported that activated calcineurin has beneficial effects on skeletal muscle regeneration in vivo (25), while pharmacological inhibition of calcineurin led to a delayed regeneration process (26, 27). Given our hypothesis that deficiency for calsarcin-2 leads to release of inhibition of calcineurin activity, one might predict enhanced muscle regeneration in Myoz1 KO mice. We thus subjected Myoz1-deficient and WT mice to unilateral cardiotoxin injection into the tibialis anterior, with the contralateral muscle serving as a control. After 4 days, Myoz1 KO muscles already showed enhanced regeneration, such as the formation of new muscles fibers (Figure 8A) and an increase in cell proliferation as measured by BrdU incorporation (n = 4 animals, P = 0.015; Figure 8, B and C). After 6 days, this effect was still observed (Figure 8D). We consistently detected a significantly attenuated weight loss of tibialis anterior muscle in Myoz1 KO compared with WT mice (71.2% in WT vs. 87.4% in KO mice, P = 0.029; Figure 8E). Moreover, Myoz1 KO mice showed a higher cell surface area recovery (42.2% in WT vs. 64.8% in Myoz1 KO mice, P < 0.0001; Figure 8F) and a decreased area of degenerated tissue (43.9% in WT vs. 32.7% in KO mice, P = 0.05; Figure 8G). Taken together, these data show that Myoz1 KO mice have an enhanced capacity for skeletal muscle regeneration, similar to other mouse models with enhanced calcineurin activity.

Figure 8

Enhanced regeneration in Myoz1 KO mice after cardiotoxin injury. Calsarcin-2–deficient mice and WT controls were subjected to unilateral cardiotoxin injection into the tibialis anterior. (A) After 4 days, Myoz1 KO muscles showed enhanced formation of new muscles fibers, as well as an increase in cell proliferation as measured by BrdU incorporation (B and C). After 6 days, this effect could still be observed (D). (E) Weight loss of tibialis anterior muscle was attenuated in Myoz1 KO compared with WT mice. Myoz1 KO mice showed a higher cell surface area recovery (F) and a decreased area of degenerated tissue (G) compared with WT mice. *P < 0.05, ^‡P < 0.001. Scale bars: 10 μm.

The results of this study show that calsarcin-2 negatively modulates calcineurin activity both in vitro and in vivo. Specifically, the genetic ablation of calsarcin-2 induced a significant fiber type shift toward more oxidative fibers with enhanced endurance (Figure 9). Based on these and earlier findings (9, 13, 15), we propose that calsarcin-2 serves a critical role in the regulation of muscle fiber composition and exercise performance in vivo.

Figure 9

Schematic model depicting calsarcin-2 inhibition of calcineurin activity. In WT skeletal muscle, calsarcin-2 is localized to the Z-disc and inhibits calcineurin activity. Phosphorylated NFAT remains in the cytoplasm. In Myoz1 KO skeletal muscle, calcineurin dephosphorylates NFAT with consequent nuclear translocation and activation of the calcium-dependent gene program.

A key finding in the current study is that calsarcin-2–deficient mice run faster and greater distances than their WT littermates. This “endurance phenotype” is accompanied by a reduction of body weight and fast-twitch muscle mass. Such a phenomenon has been observed in only a few other mouse models. For example, mice overexpressing the PPAR coactivators 1α and β (PGC-1α and -β; refs. 18, 28) as well as PPARδ (29) share the ability to strongly induce mitochondrial biogenesis and gene expression. Muscle-confined PGC-1α overexpression predominantly induced slow-twitch, oxidative type I and IIa fibers (28). Yet this overexpression cannot be solely responsible for type I fiber formation, since whole body PGC-1α–deficient mice show a normal distribution of fiber types (30). In contrast, PGC-1β, a close relative of PGC-1α, drives the formation of fast-twitch but oxidative IIx fibers without increasing type I fibers (18). Similarly, activated PPARδ, which is known to associate with PGC-1, specifically increases type I fibers (29).

In Myoz1-deficient mice, the fiber type and endurance phenotype appear to be mediated via the phosphatase calcineurin, which, besides the PGC-1/PPAR axis, determines muscle fiber specificity. Activated calcineurin promotes a fiber type shift toward slower, more oxidative fibers (1, 3, 4, 7, 8, 31). Here we show that calsarcin-2 inhibits calcineurin activity in muscle cells in vitro. Conversely, the lack of calsarcin-2 in striated muscles led to a significant increase in calcineurin/NFAT activity. Interestingly, Myoz1-deficient mice also reveal enhanced muscle regeneration after myotoxic injury, similar to mice overexpressing calcineurin in striated muscle–specific fashion (25).

Consistent with the effects observed in mice with muscle-specific overexpression of activated calcineurin (7), the Myoz1-deficient skeletal muscle displays an increase in slow-twitch, oxidative fibers. However, the phenotype of Myoz1-deficient mice reveals additional features, such as an increase in type I fibers in the slow-twitch soleus muscle, although calsarcin-2 is not expressed in these fibers. This effect might be mediated by a shift from type II fibers, which account for approximately 40%–60% of the soleus (32). In contrast, in fast-twitch fiber–dominant muscles such as the gastrocnemius, the myofiber transition was confined to type IIa fibers without an increased switch toward type I fibers. Likewise, the fast-twitch muscle weight reduction resembled the phenotype observed in the gastrocnemius from calcineurin transgenic mice (33). In Myoz1 KO mice, the gastrocnemius showed no significant increase of type I fibers compared with WT mice. In contrast, in mice overexpressing activated calcineurin in a muscle-specific fashion, both the number of type I fibers and the weight of the soleus increased (33).

Upregulation of slow, oxidative genes in skeletal muscle promotes fatigue resistance and produces an endurance phenotype (19, 29, 34, 35). Recently we showed that overexpression of activated MEF2 (MEF2C-VP16), a transcription factor that has been shown to associate with NFAT (36), increases the number of slow-twitch fibers as well as the slow-twitch fiber–specific contractile proteins, troponin I and myoglobin. Similar to the Myoz1-deficient mice, the MEF2C-VP16 transgenic mice displayed an increase in running time and distance, suggesting that activation of MEF2 is sufficient to enhance skeletal muscle oxidative capacity (37). In addition, in transgenic mice, skeletal muscle overexpression of PKD1, a kinase that phosphorylates class II HDACs and releases repression of MEF2 activity, resulted in an increase in oxidative gene expression and fatigue resistance (38). These studies show that the activation of MEF2, a transcription factor, promotes slow-twitch, oxidative gene expression, resulting in fatigue-resistant skeletal muscle.

Although calcineurin is known to induce the slow-twitch muscle program, to our knowledge it has not been previously shown that an animal model with augmented calcineurin activity actually displays an increase in endurance capacity. Interestingly, the only other mouse model in which a loss-of-function approach led to an increase in exercise capacity is the genetic ablation of another Z-disc protein, α–actinin-3 (34). Similar to Myoz1-deficient mice, mice lacking actinin-3 display a significant increase in endurance and performance, accompanied by an increase in oxidative metabolism in skeletal muscle. While the exact molecular mechanism of the shift toward a more oxidative metabolism in the latter model remains elusive, it is noteworthy that a human polymorphism in the actinin-3 gene results in the complete absence of this protein in about 18% of the healthy population (39). Remarkably, the lack of actinin-3 is significantly associated with an elite athlete status in humans (40). Similar to calsarcin-2, the expression of α–actinin-3 is restricted to type II skeletal muscle fibers (41). Moreover, α–actinin-3 directly interacts with calsarcin-2 (9). It is thus intriguing to speculate that the effects of α–actinin-3 deficiency on exercise performance are mediated, at least in part, via altered calcineurin/calsarcin/NFAT signaling. While no differences in α–actinin-2 and -3 expression levels were observed in Myoz1-deficient animals (Supplemental Figure 4), it is conceivable that calsarcin-2 acts downstream of α–actinin-3. Nevertheless, we cannot exclude that calsarcin-2 as well as α–actinin-3 also modulate other molecular pathways.

In summary, we conclude that calsarcin-2 modulates exercise performance in vivo via regulation of calcineurin/NFAT activity and subsequently the fiber type composition of skeletal muscle. Elimination of calsarcin-2, an endogenous calcineurin inhibitor, from skeletal muscle increases sarcomeric calcineurin activity and results in enhanced exercise capability.

View Supplemental data

We thank Ulrike ϖhl and Jutta Krebs for excellent technical assistance and Jose Cabrera for help with the graphics. We are indebted to Alan Beggs for the kind gift of the isoform-specific antibodies. E.N. Olson was supported by grants from the NIH, the Donald W. Reynolds Cardiovascular Clinical Research Center, and the Robert A. Welch Foundation. N. Frey and D. Frank were supported by a grant from the DFG (Fr1289/4-1).

Address correspondence to: Norbert Frey, Department of Internal Medicine III, University of Heidelberg, Im Neuenheimer Feld 410, D-69120 Heidelberg, Germany. Phone: 49-6221-566877; Fax: 49-6221-564866; E-mail: norbert.frey@med.uni-heidelberg.de. Or to: Eric N. Olson, Department of Molecular Biology, University of Texas Medical Center, 5323 Harry Hines Blvd., Dallas, Texas 75390-9148, USA. Phone: (214) 648-1187; Fax: (214) 648-1196; E-mail: eric.olson@utsouthwestern.edu.

Nonstandard abbreviations used: MEF2, myocyte enhancer factor 2; Myoz1, myozenin 1; NFAT, nuclear factor of activated T cells; RCAN1, regulator of calcineurin 1; SDH, succinate dehydrogenase.

Conflict of interest: The authors have declared that no conflict of interest exists.

Reference information: J. Clin. Invest.118:3598–3608 (2008). doi:10.1172/JCI36277

Tomasa Barrientos’s present address is: Department of Pharmacology and Cancer Biology, Duke University, Durham, North Carolina, USA.

Norbert Frey, Derk Frank, and Stefanie Lippl contributed equally to this work.

See the related article at Maintenance of cardiac energy metabolism by histone deacetylase 3 in mice.

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