Lmcd1 expression is high in skeletal muscle but decreases with disease and aging

To identify novel potential regulators of skeletal muscle mass and strength, we analyzed gene expression data from muscle of patients with diverse muscle pathologies (Fig. 1b). From this analysis, we identified Lmcd1 (Fig. 1a), which showed significantly decreased expression in diseases such as Duchenne muscular dystrophy and myotonic dystrophy (Fig. 1b). To determine how the skeletal muscle expression levels of LMCD1 compare with other organs, we screened a panel of tissues from 2-month-old mice by qRT-PCR. Results are shown as expression relative to the liver, the tissue with the lowest Lmcd1 mRNA levels (Fig. 1c). We observed that although Lmcd1 is robustly expressed in the heart (110.5-fold vs. liver), its highest expression is in skeletal muscle (727.8- and 612.1-fold in quadriceps and gastrocnemius, respectively, and 1546.9-fold in tibialis anterioris—TA). This is in agreement with what has been observed in human tissues by Northern blotting [16]. We could also observe significant Lmcd1 expression in brown adipose tissue but low levels in white adipose depots (Fig. 1c). Although there are no differences at sedentary levels, after combined resistance and endurance exercise training [12], LMCD1 expression increases in human skeletal muscle. However, this effect is abrogated in older subjects (18–30 vs. 65–80 years old; Fig. 1d). On the other hand, older mice (24 months vs. 6 months [11]; showed significantly decreased Lmcd1 expression in skeletal muscle (Fig. 1e), which was not observed in older human subjects (Fig. 1d). Interestingly, in a skeletal muscle gene expression data set obtained from tumor-bearing mice, LMCD1 expression decreases during the first weeks of cancer cachexia [17].

LMCD1 induces skeletal muscle hypertrophy in vivo

To assess if increasing LMCD1 levels is sufficient to induce changes in skeletal muscle mass, we transiently expressed it in mouse gastrocnemius muscle. To this end, a control adenoviral vector (expressing green fluorescent protein, GFP) was delivered to one limb, while the contralateral muscle was transduced with an adenovirus expressing Lmcd1 and GFP from two separate and independent promoters, which allows to determine transduction efficiency without using fusion proteins (Fig. 1f). Within 7-days post-injection, we observed an increase in protein synthesis determined by puromycin incorporation into neo-synthesized proteins, compared to the GFP-injected contralateral gastrocnemius (Fig. 1g). Ectopic Lmcd1 expression resulted in an increase in the gastrocnemius muscle mass (Fig. 1h). This was also translated in an increase in the ratio of total protein/genomic DNA and in a modest increase in the percentage of fibers with higher cross-sectional area, compared to the control, GFP-transduced gastrocnemius (Fig. 1i and Additional file 3: Figure S1a). Altogether, those results indicate that LMCD1 induces hypertrophy with an increase in protein synthesis. We next evaluated if these changes were reflected in the expression of genes related to skeletal muscle mass maintenance. In line with the observed LMCD1-induced hypertrophy, we did observe a modest induction of Pgc-1α4 and Igf-1 expression and a decrease in myostatin and muscle RING-finger protein-1 (Murf1) mRNA levels (Fig. 1j). In addition, Lmcd1 overexpression increased the expression of myosin heavy chain (Myhc) type I, IIa, and IIx with a decrease in IIb (Fig. 1k). However, the magnitude of these effects suggests that these changes in gene expression might be consequence of signaling pathways activated by LMCD1, as opposed to direct effects on gene transcription. No major changes were observed in the expression of genes related to SR-stress, thick and thin filaments, and Z-disc proteins, after LMCD1 overexpression (Additional file 3: Figure S1b–e).

LMCD1 increases specific force with less SR Ca2+ release, and resistance to fatigue

To assess if skeletal muscle hypertrophy induced by LMCD1 was translated into an increase in force, we transduced the flexor digitorum brevis (FDB) muscle of one mouse hind paw with the control GFP adenovirus and the contralateral muscle with the LMCD1 adenovirus (Fig. 2a). Seven days post-injection, FDB muscles overexpressing Lmcd1 showed an increase in muscle mass and diameter of individual fibers (Fig. 2b–c), compared with the contralateral GFP-transduced FDB muscle. Remarkably, when measuring specific force (absolute force normalized by the cross-sectional area of the fiber) and SR Ca2+ release in individual fibers, Lmcd1 overexpression induced an increase in specific force with less SR Ca2+ release compared with GFP-control muscle for the different frequencies analyzed (Fig. 2d–f). Interestingly, when measuring fatigue, Lmcd1 overexpression showed fatigue resistance with no decline in force or SR Ca2+ release after 50 contractions (Fig. 2g–h). To confirm that the levels of SR Ca2+ are not different from control conditions, we performed a caffeine treatment before and after the fatigue protocol (Fig. 2i). Caffeine treatment is often used to determine the pool of Ca2+ in the SR since it induces its release. SR Ca2+ did not change with Lmcd1 overexpression but increased after a fatigue protocol, suggesting that LMCD1 has a protective effect against fatigue in skeletal muscle fibers by increasing the reuptake of SR Ca2+.

Fig. 2 LMCD1 increases fiber diameter, force, and resistance to fatigue. a Schematic representation of Green Fluorescent Protein (GFP) and LMCD1 adenovirus injection in each flexor digitorum brevis (FDB) muscle from 14-day-old SCID mice. b FDB mass normalized by mouse body weight after 7 days of GFP (control) or Lmcd1 expression (n = 6). c Isolated fiber diameter and representative microscopy image (× 200) in mice treated as in (b) (n = 10 mice, 15–18 fibers). Scale bar = 30 μm. d Force normalized by cross-sectional area (specific force) for the different frequencies tested (15–150 Hz) at 1-min intervals in mice treated as in (b) (n = 10 mice, 15–18 fibers). e Myoplasmic free Ca2+ concentrations for the different frequencies tested (15–150 Hz) at 1-min intervals in mice treated as in (b) (n = 10 mice, 15–18 fibers). f Mean value for specific force and myoplasmic free Ca2+ concentration for the different frequencies tested (15–150 Hz) at 1-min intervals, in mice treated as in (b) (n = 10 mice, 15–18 fibers). g Percentage of force relative to the first contraction. Peak force was measured at 70 Hz tetani of 350-ms duration given at 2-s intervals for 50 contractions in mice treated as in (b) (n = 10 mice, 15–18 fibers). h Percentage of myoplasmic free Ca2+ concentration normalized for the first contraction. Myoplasmic free Ca2+ concentration was measured as in (g) (n = 10 mice, 15–18 fibers). i Myoplasmic free Ca2+ concentrations at 150 Hz with caffeine treatment before and after fatigue protocol as in (g) (n = 10). Data is shown as mean ± SEM and *p < 0.05; **p < 0.01 Full size image

In vivo Lmcd1 silencing impairs SR Ca2+ handling and decreases force but does not reduce skeletal muscle mass

To evaluate if loss of LMCD1 expression resulted in skeletal muscle atrophy and reduced force, we performed loss-of-function experiments using an adenoviral-encoded shRNA against Lmcd1, which allowed an almost 60% reduction in LMCD1 levels (Additional file 3: Figure S2a). Although we did not observe significant differences in the gastrocnemius mass upon Lmcd1 silencing (Fig. 3a), we could still determine a decrease in protein synthesis/puromycin incorporation into newly synthesized proteins when compared to a scrambled shRNA control (Fig. 3b). For functional measurements, we used isolated FDB fibers with Lmcd1 knockdown vs. control (as depicted in Fig. 2a). Interestingly, despite no changes in FDB fiber diameter upon Lmcd1 silencing (Fig. 3c), those fibers produced lower specific force without significant changes in SR Ca2+ release (compared to the scrambled shRNA control fibers) (Fig. 3d–f). The fatigue phenotype was also similar to control (Fig. 3g). In fact, specific force is lower in shLmcd1 fibers than in shRNA control fibers (Fig. 3d), suggesting the importance of LMCD1 for force maintenance. In addition, caffeine treatment before and after the fatigue protocol (Fig. 3h), showed no changes in SR Ca2+ upon Lmcd1 silencing. These results confirmed the crucial effect of LMCD1 in force and Ca2+ handling together with fatigue resistance.

Fig. 3 Lmcd1 silencing results in reduced protein synthesis and decreased intrinsic force, without muscle atrophy. a Gastrocnemius mass normalized by mouse body weight 7 days after intramuscular delivery of a scrambled/control shRNA or a shLmcd1 (n = 6). b In vivo muscle protein synthesis by puromycin incorporation, and corresponding quantification (n = 6). c Isolated fiber diameter and representative microscopy image (× 200) in mice treated as in (a) (n = 10). Scale bar = 30 μm. d Force normalized for the cross-sectional area (specific force) for the different frequency tested (15–150 Hz) at 1-min interval in mice treated as in (a) (n = 10). e Myoplasmic free Ca2+ concentration for the different frequency tested (15–150 Hz) at 1-min interval in mice treated as in (a) (n = 10). f Mean value for specific force and myoplasmic free Ca2+ concentration for the different frequencies tested (15–150 Hz) at 1 min intervals in mice treated as in (a) (n = 10). g Percentage of force relative to the first contraction. Peak force was measured at 70 Hz tetani of 350 ms duration given at 2-s intervals for 50 contractions in mice treated as in (a) (n = 10). h Myoplasmic free Ca2+ concentrations at 150 Hz with caffeine treatment before and after fatigue protocol as in (g) (n = 10). Data is shown as mean ± SEM and *p < 0.05; **p < 0.01 Full size image

LMCD1-induced kinase activity profiling in mouse primary myotubes

To determine the molecular mechanism by which LMCD1 increases muscle hypertrophy, increased force, and calcium handling, we used mouse primary myotube cultures. To increase the levels of LMCD1, fully differentiated myotubes were transduced as above (i.e., LMCD1 vs GFP adenovirus). To understand the molecular effects of LMCD1, general kinase activities were evaluated using PamChip® kinase activity arrays. Following phosphorylation patterns of the peptides, we could determine that several kinase activities were altered in the presence of LMCD1 overexpression. Interestingly, when consolidating the top 35 kinases, we could determine that some were involved in protein synthesis (RPS6KA3, RPS6KA4, RPS6KA6, RPS6KC1) and calcium signaling (CAMK2B, CAMK4). These results confirmed our previous findings of increased protein synthesis and calcium handling (Fig. 4a, b and Additional file 2: Table S2). To better visualize LMCD1 signaling pathways, we used the Kyoto Encyclopedia of Genes and Genomes (KEGG), a database resource for understanding high-level functions in biological systems. Some of the top pathways were the mitogen-activated protein kinases (MAPKs) and the mammalian target of rapamycin (mTOR) (Fig. 4c), which are closely involved in protein synthesis and regulation of S6 kinase [18, 19]. To visually represent biological pathways in full mechanistic detail, we took into account the reactome signaling pathways (Fig. 4d). This software allowed us to confirm the regulation of S6 kinase in the KEGG signaling pathways by presenting also MAP kinase activation and AKT targets phosphorylation. In addition, one of the top signaling pathways presented by the reactome analysis was the cAMP-response-element-binding protein (CREB) phosphorylation. CREB activation occurs, for example, after calcium and β-adrenergic stimulation and it has been shown to induce anabolic changes that drive myofiber hypertrophy in vivo and in vitro [20]. Finally, we also organized the top kinases accordingly with the Uniprot Keywords, which can be used to retrieve subsets of protein entries or to generate indexes of entries based on functional, structural, or other categories (Fig. 4e). Using this approach, we could determine that the top kinases are involved, for instance, in cytoskeleton remodeling and calmodulin-binding. Since these kinase pathways could suggest that LMCD1 affects myoblast fusion, and although all experiments were performed in fully differentiated myotubes, we determined the myoblast fusion index, but no changes were observed between the two groups (GFP–75.9 ± 6.5%; LMCD1–74.7 ± 7.6%). Altogether, the kinase activity assay indicated that LMCD1 changes kinase activities that are responsible for the effects observed of increased protein synthesis, force and calcium handling.

Fig. 4 Lmcd1 overexpression alters several kinase activities in mouse primary myotubes related to skeletal muscle function. a Top 35 kinases interactions that significantly changed in a kinase activity assay in differentiated primary mouse myotubes after 2 days transduction with GFP (control) or LMCD1 adenovirus (n = 4). b Top 35 kinases scores, taking into account specificity and selectivity, that significantly changed in a kinase activity assay in myotubes transduced as in (a) (n = 4). c Top Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathways of the top kinases that significantly changed in a kinase activity assay in myotubes transduced as in (a) (n = 4). d Top Reactome signaling pathways of the top kinases that significantly changed in a kinase activity assay in myotubes transduced as in (a) (n = 4). e Top Uniprot keywords of the top kinases that significantly changed in a kinase activity assay in myotubes transduced as in (a) (n = 4). EPHA1 Ephrin type-A receptor 1, CDK18 cyclin-dependent kinase 18, CDK11A cyclin-dependent kinase 11A, PRKCZ protein kinase C zeta, RPS6KA3 ribosomal protein S6 kinase A3, CDK17 cyclin-dependent kinase 17, CHEK1 checkpoint kinase 1, PRKDC protein kinase DNA-activated catalytic subunit, GSK3A glycogen synthase kinase-3 alpha, GSK3B glycogen synthase kinase-3 beta, EIF2AK3 eukaryotic translation initiation factor 2-alpha kinase 3, IKBKB inhibitor of nuclear factor kappa B kinase subunit beta, CSNK1A1 casein kinase 1 alpha 1, CDK6 cyclin-dependent 6, RPS6KC1 ribosomal protein S6 kinase C1, HIPK4 homeodomain interacting protein kinase 4, CAMK2B calcium/calmodulin-dependent protein kinase II beta, MAPK11 mitogen-activated protein kinase 11, TBK1 tumor necrosis factor receptor-associated factor binding kinase 1, NRP2 neuropilin 2, DAPK2 death associated protein kinase 2, ICK intestinal cell kinase, RPS6KA4 ribosomal protein S6 kinase A4, GRK1 G protein-coupled receptor kinase 1, IKBKE inhibitor of nuclear factor kappa B kinase subunit epsilon, RPS6KA6 ribosomal protein S6 kinase A6, MAP2 K7 dual specificity mitogen-activated protein kinase 7, CAMK4 calcium/calmodulin-dependent protein kinase type IV, CDKL4 cyclin-dependent kinase Like 4, PLK2 polo like kinase 2, PLK3 polo like kinase 3, AURKB aurora kinase B, FoxO forkhead box, mTOR mammalian target of rapamycin Full size image

LMCD1 regulates protein synthesis in mouse primary myotubes

To confirm the mechanistic aspects of LMCD1 action determined by the kinase activity assay, we first confirmed whether we could obtain the same effects in vitro as we observed in vivo regarding hypertrophy and protein synthesis. For that, we used fully differentiated myotubes transduced with LMCD1 or GFP adenovirus. As seen in vivo, Lmcd1 overexpression resulted in an increase in myotube size (approx. 40%) (Fig. 5a). We also observed an increase in the ratio of total protein/genomic DNA (Fig. 5b), and in puromycin incorporation into newly synthesized proteins (Fig. 5c), when compared to control myotubes. In agreement with our in vivo results (Fig. 1i), LMCD1 induced a slight increase in myotube Pgc-1α4 and Igf-1 expression, and a decrease in myostatin and Murf1 mRNA levels (Fig. 5d). In addition, we could also determine an increase in the expression of Myhc type I, IIa, and IIx with a decrease in IIb expression (Fig. 5e). When we transduced differentiated mouse primary myotubes with the Lmcd1 shRNA adenovirus, we achieved almost 70% reduction in LMCD1 protein levels (Fig. 5f). Despite this decrease in LMCD1 levels, we did not observe any differences in myotube size or total protein/genomic DNA ratio (Fig. 5g–h), confirming the same results seen in vivo (Fig. 3a–b). Moreover, Lmcd1 silencing did not change the expression of several skeletal muscle genes (Fig. 5j and Additional file 3: Figure S2b–e). LMCD1 silencing led to a decrease in MyhcIIx expression, without changes in the other Myhc types (Fig. 5i). In addition, some of the genes involved in the regulation of SR stress were also decreased after LMCD1 silencing (Additional file 3: Figure S2b).

Fig. 5 Lmcd1 induces protein synthesis and myotube hypertrophy in mouse primary myotubes. a Representative microscopy image of differentiated mouse primary myotubes 2 days after transduction with GFP (control) or LMCD1 adenovirus (× 200; n = 8; scale bar = 100 μm) and corresponding myotube diameter measurements. b Total protein/genomic DNA ratio determined in myotubes transduced as in (a) (n = 8). c Protein synthesis determination by puromycin incorporation in differentiated primary mouse myotubes transduced as in (a), and respective quantification (n = 8). d qRT-PCR determination of peroxisome proliferator-activated receptor gamma coactivator-1alpha1 (Pgc-1α1), Pgc-1α4, myostatin, insulin-like growth factor 1 (Igf-1), atrogin, and muscle RING-finger protein-1 (Murf1; n = 8) in differentiated primary mouse myotubes transduced as in (a). e qRT-PCR of myosin heavy chain (Myhc) I, IIa, IIb, and IIx (n = 8) in differentiated primary mouse myotubes transduced as in (a). f Western blot of LMCD1 protein expression in differentiated primary mouse myotubes after 2 days transduction of scrambled shRNA or shLmcd1 adenovirus (n = 8). g Representative microscopy image of differentiated mouse primary myotubes (× 200; n = 8; scale bar = 100 μm) transduced as in (f). h Total protein/genomic DNA ratio in differentiated primary mouse myotubes transduced as in (f) (n = 8). j qRT-PCR for Lmcd1, Pgc-1α1, Pgc-1α4, myostatin, Igf-1, atrogin, and Murf1 (n = 8) in differentiated primary mouse myotubes transduced as in (f). i qRT-PCR of MyhcI, IIa, IIb, and IIx (n = 8) in differentiated primary mouse myotubes transduced as in (f). Data is shown as mean ± SD and *p < 0.05; **p < 0.01; ***p < 0.001 Full size image

LMCD1 regulates AKT/S6K signaling and myotube hypertrophy independently of 4E-BP1

Since the effects of increased protein synthesis and hypertrophy were observed in vivo and in vitro, next we determined if changes in S6 kinase, AKT phosphorylation, and calcium signaling were present in fully differentiated myotubes transduced either with LMCD1 or GFP adenovirus, as indicated by the kinase activity assay (Fig. 4). When we overexpressed LMCD1 in mouse primary myotubes, it increased the total protein and the phosphorylation levels of AKT and S6 (1.6-, 1.4-fold expression for total levels, respectively, Fig. 6a and Additional file 3: Figure S3a). This was also in agreement with our kinase activity assay (Fig. 4), that showed an increase in the S6 and AKT signaling. On the other hand, LMCD1 reduced total and phosphorylated levels of 4E-binding protein 1 (4E-BP1) (0.7-fold expression for total levels, Fig. 6a and Additional file 3: Figure S3a). These results support an increase in protein synthesis, which was already indicated by the kinase activity assay (Fig. 4), the determination of puromycin incorporation into newly synthetized proteins and increased total protein/genomic DNA ratio. Since LMCD1 improved Ca2+ handling, next we checked the Ca2+ signaling pathway. LMCD1 expression increased calcineurin A and B protein levels (1.4- and 1.2-fold, respectively), with slight changes in the expression of Ca2+ regulators or channel genes, specially the Na+/Ca2+ and Na+/K+/Ca2+ exchangers (Fig. 6b and Additional file 3: Figure S3a–c). Our next aim was to connect the increased protein synthesis and the Ca2+ signaling pathway. Indeed, calcineurin activation can lead to PTEN-mediated AKT dephosphorylation. Since P-Ser473AKT levels are increased in the presence of LMCD1, we determined PTEN levels but saw no significant changes in its protein levels (Fig. 6c and Additional file 3: Figure S3d). The other link between calcineurin and AKT activation is through the IGF1/IRS1 (insulin receptor substrate 1) axis, which can induce the phosphorylation of AKT, mTOR and finally protein synthesis. Accordingly, after LMCD1 expression, we observed an increase in mTOR signaling (Fig. 4c) and in IRS1 phosphorylation (Fig. 6d and Additional file 3: Figure S3e), indicating that this might be the molecular connection between increased calcineurin and protein synthesis. This was also corroborated by our kinase activity assay, that showed an increase in insulin signaling-related activities (Fig. 4d).

Fig. 6 Lmcd1 increases S6 and AKT phosphorylation as well as calcineurin expression while silencing of Lmcd1 has the opposite effect. a Representative western blot for AKT Ser473 phosphorylation, total AKT, S6 Ser235 phosphorylation, total S6, 4E-BP1 Thr37phosphorylation, and total 4E-BP1, normalized for α-tubulin expression, in differentiated primary mouse myotubes 2 days after transduction with GFP (control) or LMCD1 adenovirus (n = 8). b Representative Western blot for calcineurin A and calcineurin B, normalized for α-tubulin expression, in differentiated myotubes transduced as in (a) (n = 8). c Representative western blot for PTEN normalized for α-tubulin expression in differentiated myotubes transduced as in (a) (n = 6). d Representative Western blot for insulin receptor substrate 1 (IRS1) Tyr612 phosphorylation normalized for α-tubulin expression in differentiated myotubes transduced as in (a) (n = 4). e Representative Western blot for AKT phosphorylation Ser473, total AKT, S6 phosphorylation Ser235, and total S6, normalized for α-tubulin expression, in differentiated primary mouse myotubes after 2 days transduction of scrambled shRNA or shLmcd1 adenovirus (n = 8). f Representative western blot for calcineurin A and calcineurin B, normalized for α-tubulin expression, in differentiated primary mouse myotubes transduced as in (e) (n = 8) Full size image

Since we observed small effects on gene expression, our next step was to determine the cellular localization of LMCD1. For that, we performed immunochemistry in differentiated mouse primary myotubes. As expected, LMCD1 has a predominantly cytosolic localization in myotubes (Additional file 3: Figure S3f). In addition, Lmcd1 overexpression in differentiated mouse primary myotubes did not induce any major changes in several skeletal muscle-related genes (Additional file 3: Figure S3 g–k), suggesting it acts primarily through cellular signaling mechanisms (as opposed to direct regulation of gene expression).

Interestingly, after silencing Lmcd1, there was a decrease in S6 and AKT total and phosphorylation (0.9-, 0.7-fold expression for total levels, respectively, Fig. 6e and Additional file 3: Figure S3 l) as well as in the levels of calcineurin A and B (Fig. 6f and Additional file 3: Figure S3 l), suggesting that Lmcd1 silencing induces a decrease in the rate of protein synthesis and Ca2+ handling which reduces force but is not enough to reduce the muscle/myotube size. By reducing protein synthesis and Ca2+, the reduction in LMCD1 might contribute to the decrease in genes related to SR stress.

Calcineurin inhibition impairs LMCD1 activity in vivo

To determine if calcineurin is downstream of LMCD1 in the induction of skeletal muscle hypertrophy, we ectopically expressed Lmcd1 in the gastrocnemius muscle (or GFP in the contralateral limb, as before), and treated those mice with cyclosporine A (CsA). Strikingly, treatment with CsA completely abolished the LMCD1-mediated increase in muscle mass (Fig. 7a) and decreased protein synthesis (Fig. 7b). In addition, FDB fibers overexpressing Lmcd1 and treated with CsA showed a higher Ca2+ requirement to achieve the same specific force as the control fibers (Fig. 7c–e), with a fatigue phenotype similar with the control fibers (Fig. 7f–g). Those results were similar to the ones obtained when silencing Lmcd1 confirming the role of calcineurin in LMCD1 actions regulating skeletal muscle hypertrophy, force, and intracellular calcium handling.

Fig. 7 LMCD1 depends on calcineurin signaling to increase protein synthesis, force and calcium handling. a Gastrocnemius mass normalized by body weight after 7 days of GFP (control) or Lmcd1 expression and cyclosporine A (CsA) i.p. injection (n = 6). b In vivo muscle protein synthesis determination by puromycin incorporation in mice treated as in (a), and respective quantification (n = 6). c Force normalized for the cross-sectional area (specific force) for the different frequency tested (15–150 Hz) at 1-min interval in mice treated as in (a) (n = 10). d Myoplasmic free Ca2+ concentration for the different frequency tested (15–150 Hz) at 1-min interval in mice treated as in (a) (n = 10). e Specific force and myoplasmic free Ca2+ concentration for the mean of the fibers for the different frequencies tested (15–150 Hz) at 1-min intervals. Mice treated as in (a) (n = 10). f Percentage of force relative to the first contraction. Peak force was measured at 70 Hz tetani of 350 ms duration given at 2-s intervals for 50 contractions in mice treated as in (a) (n = 10). g Percentage of myoplasic free Ca2+ relative to the first contraction. Peak myoplasmic free Ca2+ concentration was measured at 70 Hz tetani of 350-ms duration given at 2-s intervals for 50 contractions in mice treated as in (a) (n = 10). Data is shown as mean ± SEM and *p < 0.05 Full size image

LMCD1 interacts with Calcineurin to inhibit MLN activity and improve Ca2+ handling

The effects of LMCD1 on Ca2+ handling we observed are reminiscent of what has been previously reported for Mln-knockout (KO) mice, which show better exercise performance and no changes in gene expression related to SERCA and ryanodine receptor 1. Since MLN has been shown to negatively regulate SERCA pump activity [10], we investigated if LMCD1 affects Mln expression. We observed that Lmcd1 expression does indeed decrease Mln transcript levels (Fig. 8a). In addition, Mln expression is increased upon Lmcd1 silencing, further highlighting the strong connection between these two proteins (Fig. 8b). Furthermore, Lmcd1 expression was sufficient to decrease the activity of a luciferase reporter vector containing a Mln gene promoter fragment [10], (Fig. 8c). This effect was abrogated when calcineurin was silenced (Fig. 8c), suggesting that LMCD1 together with calcineurin regulate MLN expression and calcium handling. Together, our results indicate that LMCD1 is an important regulator of muscle mass and function. Through a series of gain- and loss-of-function studies, we show that LMCD1 controls protein synthesis, muscle fiber size, and increases specific force and Ca2+ handling.