Calcium mishandling in Duchenne dystrophic muscle suggested that dystrophin, a membrane-associated cytoskeleton protein, might regulate calcium signaling cascade such as calcium influx pathway. It was previously shown that abnormal calcium entries involve uncontrolled stretch-activated currents and store-operated Ca2+ currents supported by TRPC1 channels. Moreover, our recent work demonstrated that reintroduction of minidystrophin in dystrophic myotubes restores normal capacitative calcium entries (CCEs). However, until now, no molecular link between the dystrophin complex and calcium entry channels has been described. This study is the first to show by coimmunoprecipitation assays the molecular association of TRPC1 with dystrophin and alpha1-syntrophin in muscle cells. TRPC1 was also associated with alpha1-syntrophin in dystrophic muscle cells independently of dystrophin. Furthermore, glutathione S-transferase (GST) pull-down assays showed that TRPC1 binds to the alpha1-syntrophin PDZ domain. Transfected recombinant alpha1-syntrophin formed a complex with TRPC1 channels and restored normal CCEs in dystrophic muscle cells. We suggest that normal regulation of CCEs in skeletal muscle depends on the association between TRPC1 channels and alpha1-syntrophin that may anchor the store-operated channels to the dystrophin-associated protein complex (DAPC). The loss of this molecular association could participate in the calcium alterations observed in dystrophic muscle cells. This study provides a new model for the regulation of calcium influx by interaction with the scaffold of the DAPC in muscle cells.

The dystrophin-associated protein complex (DAPC) is essential for skeletal muscle, and the lack of dystrophin in Duchenne muscular dystrophy results in a reduction of DAPC components such as syntrophins and in fiber necrosis. By anchoring various molecules, the syntrophins may confer a role in cell signaling to the DAPC. Calcium disorders and abnormally elevated cation influx in dystrophic muscle cells have suggested that the DAPC regulates some sarcolemmal cationic channels. We demonstrated previously that minidystrophin and ␣1-syntrophin restore normal cation entry in dystrophin-deficient myotubes and that sarcolemmal TRPC1 channels associate with dystrophin and the bound PDZ domain of ␣1-syntrophin. This study shows that small interfering RNA (siRNA) silencing of ␣1-syntrophin dysregulated cation influx in myotubes. Moreover, deletion of the PDZcontaining domain prevented restoration of normal cation entry by ␣1-syntrophin transfection in dystrophin-deficient myotubes. TRPC1 and TRPC4 channels are expressed at the sarcolemma of muscle cells; forced expression or siRNA silencing showed that cation influx regulated by ␣1-syntrophin is supported by TRPC1 and TRPC4. A molecular association was found between TRPC1 and TRPC4 channels and the ␣1-syntrophin-dystrophin complex. TRPC1 and TRPC4 channels may form sarcolemmal channels anchored to the DAPC, and ␣1-syntrophin is necessary to maintain the normal regulation of TRPC-supported cation entry in skeletal muscle. Cation channels with DAPC form a signaling complex that modulates cation entry and may be crucial for normal calcium homeostasis in skeletal muscles.

Store-operated Ca2؉ entry (SOCE) has emerged as an important mechanism in cardiac pathology. However, the signals that up-regulate SOCE in the heart remain unexplored. Clinical trials have emphasized the beneficial role of mineralocorticoid receptor (MR) signaling blockade in heart failure and associated arrhythmias. Accumulated evidence suggests that the mineralocorticoid hormone aldosterone, through activation of its receptor, MR, might be a key regulator of Ca 2؉ influx in cardiomyocytes. We thus assessed whether and how SOCE involving transient receptor potential canonical (TRPC) and Orai1 channels are regulated by aldosterone/MR in neonatal rat ventricular cardiomyocytes. Molecular screening using qRT-PCR and Western blotting demonstrated that aldosterone treatment for 24 h specifically increased the mRNA and/or protein levels of Orai1, TRPC1, -C4, -C5, and stromal interaction molecule 1 through MR activation. These effects were correlated with a specific enhancement of SOCE activities sensitive to store-operated channel inhibitors (SKF-96365 and BTP2) and to a potent Orai1 blocker (S66) and were prevented by TRPC1, -C4, and Orai1 dominant negative mutants or TRPC5 siRNA. A mechanistic approach showed that up-regulation of serum-and glucocorticoid-regulated kinase 1 mRNA expression by aldosterone is involved in enhanced SOCE. Functionally, 24-h aldosterone-enhanced SOCE is associated with increased diastolic [Ca 2؉ ] i , which is blunted by store-operated channel inhibitors. Our study provides the first evidence that aldosterone promotes TRPC1-, -C4-, -C5-, and Orai1-mediated SOCE in cardiomyocytes through an MR and serum-and glucocorticoid-regulated kinase 1 pathway.The last discovered Ca 2ϩ channel family, transient receptor potential canonical (TRPC) 2 and Orai1 channels, is widely expressed, but the role of these channels and the modulation of their expression are not completely understood. Notably, the dysregulation of these important mediators of Ca 2ϩ -dependent signal transduction has been involved in a broad range of human diseases such as adenocarcinomas, type 2 diabetes and diabetic nephropathy, human proteinuric kidney disease focal and segmental glomerulosclerosis, severe immunodeficiency, congenital myopathy, chronic pulmonary disease, and ectodermal dysplasia (1, 2). Although predominantly thought of in the context of non-excitable cells, store-operated Ca 2ϩ entry (SOCE) by store-operated channels (SOCs) has recently emerged as a potential mechanism to alter Ca 2ϩ in the diseased cardiomyocyte. Although still under debate, the prime candidate proteins for SOCs encompass the TRPC channel(s) as non-selective cation channels as well as Orai1, the Ca 2ϩ selective pore-forming unit of the Ca 2ϩ release-activated Ca 2ϩ channel (3). Stromal interaction molecule 1 (STIM1) serves as a Ca 2ϩ sensor in the endoplasmic reticulum/sarcoplasmic reticulum (SR), clustering proximal to the plasma membrane to activate Orai1 (4) and TRPC channel(s) (5) when Ca 2ϩ stores are depleted. The seven isoforms of the T...

These results give new insights into the key role that TRPC channels, via interaction with the Cav1.2 channel, play in regulation of cardiac pacemaking, conduction, ventricular activity, and contractility during cardiogenesis.

BACKGROUND: Monoallelic mutations in the gene encoding bone morphogenetic protein receptor 2 (Bmpr2) are the main genetic risk factor for heritable pulmonary arterial hypertension (PAH) with incomplete penetrance. Several Bmpr2 transgenic mice have been reported to develop mild spontaneous PAH. In this study, we examined whether rats with the Bmpr2 mutation were susceptible to developing more severe PAH. METHODS: The zinc finger nuclease method was used to establish rat lines with mutations in the Bmpr2 gene. These rats were then characterized at the hemodynamic, histological, electrophysiological, and molecular levels. RESULTS: Rats with a monoallelic deletion of 71 bp in exon 1 (Δ71 rats) showed decreased BMPRII expression and phosphorylated SMAD1/5/9 levels. Δ71 Rats develop age-dependent spontaneous PAH with a low penetrance (16%-27%), similar to that in humans. Δ71 Rats were more susceptible to hypoxia-induced pulmonary hypertension than wild-type rats. Δ71 Rats exhibited progressive pulmonary vascular remodeling associated with a proproliferative phenotype and showed lower pulmonary microvascular density than wild-type rats. Organ bath studies revealed severe alteration of pulmonary artery contraction and relaxation associated with potassium channel subfamily K member 3 (KCNK3) dysfunction. High levels of perivascular fibrillar collagen and pulmonary interleukin-6 overexpression discriminated rats that developed spontaneous PAH and rats that did not develop spontaneous PAH. Finally, detailed assessments of cardiomyocytes demonstrated alterations in morphology, calcium (Ca 2+), and cell contractility specific to the right ventricle; these changes could explain the lower cardiac output of Δ71 rats. Indeed, adult right ventricular cardiomyocytes from Δ71 rats exhibited a smaller diameter, decreased sensitivity of sarcomeres to Ca 2+ , decreased [Ca 2+ ] transient amplitude, reduced sarcoplasmic reticulum Ca 2+ content, and short action potential duration compared with right ventricular cardiomyocytes from wild-type rats. CONCLUSIONS: We characterized the first Bmpr2 mutant rats and showed some of the critical cellular and molecular dysfunctions described in human PAH. We also identified the heart as an unexpected but potential target organ of Bmpr2 mutations. Thus, this new genetic rat model represents a promising tool to study the pathogenesis of PAH.

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Store-operated Ca2؉ channels (SOCs) are voltage-independent Ca 2؉ channels activated upon depletion of the endoplasmic reticulum Ca 2؉ stores. Early studies suggest the contribution of such channels to Ca 2؉ homeostasis in insulin-secreting pancreatic ␤-cells. However, their composition and contribution to glucose-stimulated insulin secretion (GSIS) remains unclear. In this study, endoplasmic reticulum Ca 2؉ depletion triggered by acetylcholine (ACh) or thapsigargin stimulated the formation of a ternary complex composed of Orai1, TRPC1, and STIM1, the key proteins involved in the formation of SOCs. Ca 2؉ imaging further revealed that Orai1 and TRPC1 are required to form functional SOCs and that these channels are activated by STIM1 in response to thapsigargin or ACh. Pharmacological SOCs inhibition or dominant negative blockade of Orai1 or TRPC1 using the specific pore mutants Orai1-E106D and TRPC1-F562A impaired GSIS in rat ␤-cells and fully blocked the potentiating effect of ACh on secretion. In contrast, pharmacological or dominant negative blockade of TRPC3 had no effect on extracellular Ca 2؉ entry and GSIS. Finally, we observed that prolonged exposure to supraphysiological glucose concentration impaired SOCs function without altering the expression levels of STIM1, Orai1, and TRPC1. We conclude that Orai1 and TRPC1, which form SOCs regulated by STIM1, play a key role in the effect of ACh on GSIS, a process that may be impaired in type 2 diabetes.

Pulmonary arterial hypertension (PAH) is a multifactorial and severe disease without curative therapies. PAH pathobiology involves altered pulmonary arterial tone, endothelial dysfunction, distal pulmonary vessel remodeling, and inflammation, which could all depend on ion channel activities (K+, Ca2+, Na+ and Cl−). This review focuses on ion channels in the pulmonary vasculature and discusses their pathophysiological contribution to PAH as well as their therapeutic potential in PAH.