STIM1-Ca(2+) signaling is required for the hypertrophic growth of skeletal muscle in mice.

Immediately after birth, skeletal muscle must undergo an enormous period of growth and differentiation that is coordinated by several intertwined growth signaling pathways. How these pathways are integrated remains unclear but is likely to involve skeletal muscle contractile activity and calcium (Ca(2+)) signaling. Here, we show that Ca(2+) signaling governed by stromal interaction molecule 1 (STIM1) plays a central role in the integration of signaling and, therefore, muscle growth and differentiation. Conditional deletion of STIM1 from the skeletal muscle of mice (mSTIM1(-/-) mice) leads to profound growth delay, reduced myonuclear proliferation, and perinatal lethality. We show that muscle fibers of neonatal mSTIM1(-/-) mice cannot support the activity-dependent Ca(2+) transients evoked by tonic neurostimulation, even though excitation contraction coupling (ECC) remains unperturbed. In addition, disruption of tonic Ca(2+) signaling in muscle fibers attenuates downstream muscle growth signaling, such as that of calcineurin, mitogen-activated protein (MAP) kinases, extracellular signal-regulated kinase 1 and 2 (ERK1/2), and AKT. Based on our findings, we propose a model wherein STIM1-mediated store-operated calcium entry (SOCE) governs the Ca(2+) signaling required for cellular processes that are necessary for neonatal muscle growth and differentiation.

Dynamic regulation of sarcoplasmic reticulum Ca(2+) stores by stromal interaction molecule 1 and sarcolipin during muscle differentiation.

During muscle development, the sarco/endoplasmic reticulum (SR/ER) undergoes remodeling to establish a specialized internal Ca(2+) store for muscle contraction. We hypothesized that store operated Ca(2+) entry (SOCE) is required to fill Ca(2+) stores and is, therefore, critical to creating a mature SR/ER. Stromal interaction molecule 1 (STIM1) functions as a sensor of internal Ca(2+) store content and an activator of SOCE channels. Myocytes lacking STIM1 display reduced SR Ca(2+) content and altered expression of key SR proteins. Sarcolipin (SLN), an inhibitor of the SR calcium pump, was markedly increased in the muscle of mutant STIM1 mice. SLN opposes the actions of STIM1 by limiting SOCE, reducing SR Ca(2+) content and delaying muscle differentiation. During mouse muscle development SLN is highly expressed in embryonic muscle, while the expression of STIM1 is up-regulated postnatally. These results suggest that SOCE regulates SR/ER specialization and that SLN and STIM1 act in opposing fashions to govern SOCE during myogenesis.

TRPC6 enhances angiotensin II-induced albuminuria.

Mutations in the canonical transient receptor potential cation channel 6 (TRPC6) are responsible for familial forms of adult onset focal segmental glomerulosclerosis (FSGS). The mechanisms by which TRPC6 mutations cause kidney disease are not well understood. We used TRPC6-deficient mice to examine the function of TRPC6 in the kidney. We found that adult TRPC6-deficient mice had BP and albumin excretion rates similar to wild-type animals. Glomerular histomorphology revealed no abnormalities on both light and electron microscopy. To determine whether the absence of TRPC6 would alter susceptibility to hypertension and renal injury, we infused mice with angiotensin II continuously for 28 days. Although both groups developed similar levels of hypertension, TRPC6-deficient mice had significantly less albuminuria, especially during the early phase of the infusion; this suggested that TRPC6 adversely influences the glomerular filter. We used whole-cell patch-clamp recording to measure cell-membrane currents in primary cultures of podocytes from both wild-type and TRPC6-deficient mice. In podocytes from wild-type mice, angiotensin II and a direct activator of TRPC6 both augmented cell-membrane currents; TRPC6 deficiency abrogated these increases in current magnitude. Our findings suggest that TRPC6 promotes albuminuria, perhaps by promoting angiotensin II-dependent increases in Ca(2+), suggesting that TRPC6 blockade may be therapeutically beneficial in proteinuric kidney disease.

TRPC1 channels are critical for hypertrophic signaling in the heart.

RATIONALE: Cardiac muscle adapts to increase workload by altering cardiomyocyte size and function resulting in cardiac hypertrophy. G protein-coupled receptor signaling is known to govern the hypertrophic response through the regulation of ion channel activity and downstream signaling in failing cardiomyocytes. OBJECTIVE: Transient receptor potential canonical (TRPC) channels are G protein-coupled receptor operated channels previously implicated in cardiac hypertrophy. Our objective of this study is to better understand how TRPC channels influence cardiomyocyte calcium signaling. METHODS AND RESULTS: Here, we used whole cell patch clamp of adult cardiomyocytes to show upregulation of a nonselective cation current reminiscent of TRPC channels subjected to pressure overload. This TRPC current corresponds to the increased TRPC channel expression noted in hearts of mice subjected to pressure overload. Importantly, we show that mice lacking TRPC1 channels are missing this putative TRPC current. Moreover, Trpc1(-)(/)(-) mice fail to manifest evidence of maladaptive cardiac hypertrophy and maintain preserved cardiac function when subjected to hemodynamic stress and neurohormonal excess. In addition, we provide a mechanistic basis for the protection conferred to Trpc1(-)(/)(-) mice as mechanosensitive signaling through calcineurin/NFAT, mTOR and Akt is altered in Trpc1(-)(/)(-) mice. CONCLUSIONS: From these studies, we suggest that TRPC1 channels are critical for the adaptation to biomechanical stress and TRPC dysregulation leads to maladaptive cardiac hypertrophy and failure.

STIM1 signalling controls store-operated calcium entry required for development and contractile function in skeletal muscle.

It is now well established that stromal interaction molecule 1 (STIM1) is the calcium sensor of endoplasmic reticulum stores required to activate store-operated calcium entry (SOC) channels at the surface of non-excitable cells. However, little is known about STIM1 in excitable cells, such as striated muscle, where the complement of calcium regulatory molecules is rather disparate from that of non-excitable cells. Here, we show that STIM1 is expressed in both myotubes and adult skeletal muscle. Myotubes lacking functional STIM1 fail to show SOC and fatigue rapidly. Moreover, mice lacking functional STIM1 die perinatally from a skeletal myopathy. In addition, STIM1 haploinsufficiency confers a contractile defect only under conditions where rapid refilling of stores would be needed. These findings provide insight into the role of STIM1 in skeletal muscle and suggest that STIM1 has a universal role as an ER/SR calcium sensor in both excitable and non-excitable cells.

Mice lacking Homer 1 exhibit a skeletal myopathy characterized by abnormal transient receptor potential channel activity.

Transient receptor potential (TRP) channels are nonselective cation channels, several of which are expressed in striated muscle. Because the scaffolding protein Homer 1 has been implicated in TRP channel regulation, we hypothesized that Homer proteins play a significant role in skeletal muscle function. Mice lacking Homer 1 exhibited a myopathy characterized by decreased muscle fiber cross-sectional area and decreased skeletal muscle force generation. Homer 1 knockout myotubes displayed increased basal current density and spontaneous cation influx. This spontaneous cation influx in Homer 1 knockout myotubes was blocked by reexpression of Homer 1b, but not Homer 1a, and by gene silencing of TRPC1. Moreover, diminished Homer 1 expression in mouse models of Duchenne's muscular dystrophy suggests that loss of Homer 1 scaffolding of TRP channels may contribute to the increased stretch-activated channel activity observed in mdx myofibers. These findings provide direct evidence that Homer 1 functions as an important scaffold for TRP channels and regulates mechanotransduction in skeletal muscle.

Cardiac arterial pole alignment is sensitive to FGF8 signaling in the pharynx.

Morphogenesis of the cardiac arterial pole is dependent on addition of myocardium and smooth muscle from the secondary heart field and septation by cardiac neural crest cells. Cardiac neural crest ablation results in persistent truncus arteriosus and failure of addition of myocardium from the secondary heart field leading to malalignment of the arterial pole with the ventricles. Previously, we have shown that elevated FGF signaling after neural crest ablation causes depressed Ca2+ transients in the primary heart tube. We hypothesized that neural crest ablation results in elevated FGF8 signaling in the caudal pharynx that disrupts secondary heart field development. In this study, we show that FGF8 signaling is elevated in the caudal pharynx after cardiac neural crest ablation. In addition, treatment of cardiac neural crest-ablated embryos with FGF8b blocking antibody or an FGF receptor blocker rescues secondary heart field myocardial development in a time- and dose-dependent manner. Interestingly, reduction of FGF8 signaling in normal embryos disrupts myocardial secondary heart field development, resulting in arterial pole malalignment. These results indicate that the secondary heart field myocardium is particularly sensitive to FGF8 signaling for normal conotruncal development, and further, that cardiac neural crest cells modulate FGF8 signaling in the caudal pharynx.

Calcium buffering and excitation-contraction coupling in developing avian myocardium.

This report provides a detailed analysis of developmental changes in cytoplasmic free calcium (Ca(2+)) buffering and excitation-contraction coupling in embryonic chick ventricular myocytes. The peak magnitude of field-stimulated Ca(2+) transients declined by 41% between embryonic day (ED) 5 and 15, with most of the decline occurring between ED5 and 11. This was due primarily to a decrease in Ca(2+) currents. Sarcoplasmic reticulum (SR) Ca(2+) content increased 14-fold from ED5 to 15. Ca(2+) transients in voltage-clamped myocytes after blockade of SR function permitted computation of the fast Ca buffer power of the cytosol as expressed as generalized values of B(max) and K(D). B(max) rose with development whereas K(D) did not change significantly. The computed SR Ca(2+) contribution to the Ca(2+) transient and gain factor for Ca(2+)-induced Ca(2+) release increased markedly between ED5 and 11 and slightly thereafter. These results paralleled the maturation of SR and peripheral couplings reported by others and demonstrated a strong relationship between structure and function in development of excitation-contraction coupling. Modeling of buffer power from estimates of the major cytosolic Ca binding moieties yielded a B(max) and K(D) in reasonable agreement with experiment. From ED5 to 15, troponin C was the major Ca(2+) binding moiety, followed by SR and calmodulin.

T-type Ca2+ current contribution to Ca2+-induced Ca2+ release in developing myocardium.

In normal adult-ventricular myocardium, Ca2+-induced Ca2+ release (CICR) from the sarcoplasmic reticulum (SR) is activated via Ca2+ entry through L-type Ca2+ channels. However, embryonic-ventricular myocytes have a prominent T-type Ca2+ current (ICa,T). In this study, the contribution of ICa,T to CICR was determined in chick-ventricular development. Electrically stimulated Ca2+ transients were examined in myocytes loaded with fura-2 and Ca2+ currents with perforated patch-clamp. The results show that the magnitudes of the Ca2+ transient, L-type Ca2+ current (ICa,L) and ICa,T, decline with development with the majority of the decline of transients and ICa,L occurring between embryonic day (ED) 5 and 11. Compared to controls, the magnitude of the Ca2+ transient in the presence of nifedipine was reduced by 41% at ED5, 77% at ED11, and 78% at ED15. These results demonstrated that the overall contribution of ICa,T to the transient was greatest at ED5, while ICa,L was predominate at ED11 and 15. This indicated a decline in the contribution of ICa,T to the Ca2+ transient with development. Nifedipine plus caffeine was added to deplete the SR of Ca2+ and eliminate the occurrence of CICR due to ICa,T. Under these conditions, the transients were further reduced at all three developmental ages, which indicated that a portion of the Ca2+ transients present after just nifedipine addition was due to CICR stimulated by ICa,T. These results indicate that Ca2+ entry via T-type channels plays a significant role in excitation-contraction coupling in the developing heart that includes stimulation of CICR.

Localization, macromolecular associations, and function of the small heat shock-related protein HSP20 in rat heart.

BACKGROUND: The small heat shock proteins HSP20, HSP25, alphaB-crystallin, and myotonic dystrophy kinase binding protein (MKBP) may regulate dynamic changes in the cytoskeleton. For example, the phosphorylation of HSP20 has been associated with relaxation of vascular smooth muscle. This study examined the function of HSP20 in heart muscle. METHODS AND RESULTS: Western blotting identified immunoreactive HSP20, alphaB-crystallin, and MKBP in rat heart homogenates. Subcellular fractionation demonstrated that HSP20, alphaB-crystallin, and MKBP were predominantly in cytosolic fractions. Chromatography with molecular sieving columns revealed that HSP20 and alphaB-crystallin were associated in an aggregate of approximately 200 kDa, and alphaB-crystallin coimmunoprecipitated with HSP20. Immunofluorescence microscopy demonstrated that the pattern of HSP20, alphaB-crystallin, and actin staining was predominantly in transverse bands. Treatment with sodium nitroprusside led to increases in the phosphorylation of HSP20, as determined with 2-dimensional immunoblots. Incubation of transiently permeabilized myocytes with phosphopeptide analogues of HSP20 led to an increase in the rate of shortening. The increased shortening rate was associated with an increase in the rate of lengthening and a more rapid decay of the calcium transient. CONCLUSIONS: HSP20 is associated with alphaB-crystallin, possibly at the level of the actin sarcomere. Phosphorylated HSP20 increases myocyte shortening rate through increases in calcium uptake and more rapid lengthening.