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.

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.

Mechanosensitive channels in striated muscle and the cardiovascular system: not quite a stretch anymore.

Stretch-activated or mechanosensitive channels transduce mechanical forces into ion fluxes across the cell membrane. These channels have been implicated in several aspects of cardiovascular physiology including regulation of blood pressure, vasoreactivity, and cardiac arrhythmias, as well as the adverse remodeling associated with cardiac hypertrophy and heart failure. This review discusses mechanosensitive channels in skeletal muscle and the cardiovascular system and their role in disease pathogenesis. We describe the regulation of gating of mechanosensitive channels including direct mechanisms and indirect activation by signaling pathways, as well as the influence on activation of these channels by the underlying cytoskeleton and scaffolding proteins. We then focus on the role of transient receptor potential channels, several of which have been implicated as mechanosensitive channels, in the pathogenesis of adverse cardiac remodeling and as potential therapeutic targets in the treatment of heart 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.

Bap31 enhances the endoplasmic reticulum export and quality control of human class I MHC molecules.

The assembly of class I MHC molecules and their export from the endoplasmic reticulum (ER) is governed by chaperones and accessory proteins. We present evidence that the putative cargo receptor protein Bap31 participates in the transport and the quality control of human class I molecules. Transfection of the human adenocarcinoma cell line HeLa with yellow fluorescent protein-Bap31 chimeras increased surface levels of class I in a dose-dependent manner, by as much as 3.7-fold. The increase in surface class I resulted from an increase in the rate of export of newly synthesized class I molecules to the cell surface and from an increase in the stability of the exported molecules. We propose that Bap31 performs quality control on class I molecules in two distinct phases: first, by exporting peptide-loaded class I molecules to the ER/Golgi intermediate compartment, and second, by retrieving class I molecules that have lost peptides in the acidic post-ER environment. This function of Bap31 is conditional or redundant, because we find that Bap31 deficiency does not reduce surface class I levels. Overexpression of the Bap31 homolog, Bap29, decreases surface class levels in HeLa, indicating that it does not substitute for Bap31.

Studies of Ca2+ ATPase (SERCA) inhibition.

The Ca(2+) transport ATPase of intracellular membranes (SERCA) can be inhibited by a series of chemical compounds such as Thapsigargin (TG), 2,5-di(tert-butyl)hydroquinone (DBHQ) and 1,3-dibromo-2,4,6-tris (methyl-isothio-uronium) benzene (Br(2)-TITU). These compounds have specific binding sites in the ATPase protein, and different mechanisms of inhibition. On the other hand, SERCA gene silencing offers a convenient and specific method for suppression of SERCA activity in cells. The physiological and pharmacological implications of SERCA inhibition are discussed.

Dependence of exogenous SERCA gene expression on coxsackie adenovirus receptor levels in neonatal and adult cardiac myocytes.

We demonstrate that the efficiency of adenovirus-assisted exogenous Ca(2+) ATPase (SERCA) and reporter (EGFP) gene expression is much higher in primary cultures of myocytes from neonatal rat hearts, than in primary cultures of myocytes from adult rat hearts. In this respect, the neonatal myocytes behave similarly to the established COS-1 cell line. This difference is related to the level of coxsackie adenovirus receptor (CAR) that affects cell penetration and expression level of exogenous genes, and explains variations in the observed consequences of exposure to adenovirus vector carrying SERCA cDNA. Awareness of these differences should be highly advantageous in complementary studies of exogenous gene expression in neonatal and adult myocytes. It should also be advantageous in evaluating conditions yielding optimal ratios of functional benefits over possible toxic effects upon exogenous SERCA gene delivery to cardiac muscle.

Poly(C) synthesis by class I and class II CCA-adding enzymes.

The CCA-adding enzymes [ATP(CTP):tRNA nucleotidyl transferases], which catalyze synthesis of the conserved CCA sequence to the tRNA 3' end, are divided into two classes. Recent studies show that the class II Escherichia coli CCA-adding enzyme synthesizes poly(C) when incubated with CTP alone, but switches to synthesize CCA when incubated with both CTP and ATP. Because the poly(C) activity can shed important light on the mechanism of the untemplated synthesis of CCA, it is important to determine if this activity is also present in the class I CCA enzymes, which differ from the class II enzymes by significant sequence divergence. We show here that two members of the class I family, the archaeal Sulfolobus shibatae and Methanococcus jannaschii CCA-adding enzymes, are also capable of poly(C) synthesis. These two class I enzymes catalyze poly(C) synthesis and display a response of kinetic parameters to the presence of ATP similar to that of the class II E. coli enzyme. Thus, despite extensive sequence diversification, members of both classes employ common strategies of nucleotide addition, suggesting conservation of a mechanism in the development of specificity for CCA. For the E. coli enzyme, discrimination of poly(C) from CCA synthesis in the intact tRNA and in the acceptor-TPsiC domain is achieved by the same kinetic strategy, and a mutation that preferentially affects addition of A76 but not poly(C) has been identified. Additionally, we show that enzymes of both classes exhibit a processing activity that removes nucleotides in the 3' to 5' direction to as far as position 74.