Abstract-Changes in membrane tension resulting from membrane stretch represent one of the key elements in blood flow regulation in vascular smooth muscle. However, the molecular mechanisms involved in the regulation of membrane stretch remain unclear. In this study, we provide evidence that a vanilloid receptor (TRPV) homologue, TRPV2 is expressed in vascular smooth muscle cells, and demonstrate that it can be activated by membrane stretch. Cell swelling caused by hypotonic solutions activated a nonselective cation channel current (NSCC) and elevated intracellular Ca 2ϩ ([Ca 2ϩ ] i ) in freshly isolated cells from mouse aorta. Both of these signals were blocked by ruthenium red, an effective blocker of TRPVs. The absence of external Ca 2ϩ abolished this increase in [Ca 2ϩ ] i caused by the hypotonic stimulation and reduced the activation of NSCC. Significant immunoreactivity to mouse TRPV2 protein was detected in single mouse aortic myocytes. Moreover, the expression of TRPV2 was found in mesenteric and basilar arterial myocytes. Treatment of mouse aorta with TRPV2 antisense oligonucleotides resulted in suppression of hypotonic stimulationinduced activation of NSCC and elevation of [Ca 2ϩ ] i as well as marked inhibition of TRPV2 protein expression. In Chinese hamster ovary K1 (CHO) cells transfected with TRPV2 cDNA (TRPV2-CHO), application of membrane stretch through the recording pipette and hypotonic stimulation consistently activated single NSCC. Moreover, stretch of TRPV2-CHO cells cultured on an elastic silicon membrane significantly elevated [Ca 2ϩ ] i . These results provide a strong basis for our purpose that endogenous TRPV2 in mouse vascular myocytes functions as a novel and important stretch sensor in vascular smooth muscles. Key Words: TRPV2 Ⅲ vanilloid receptor Ⅲ mouse aorta Ⅲ membrane stretch Ⅲ vascular smooth muscle D etection of mechanical stimuli is essential for diverse biological functions including audition, touch, and maintenance of vascular myogenic tone. In the latter, elevation of intravascular pressure depolarizes vascular smooth muscle cells via membrane stretch. 1,2 This depolarization activates voltage-dependent L-type Ca 2ϩ channels (VDCC) and increases [Ca 2ϩ ] i , resulting in vasoconstriction and/or myogenic tone. 3 A large component of the elevation of [Ca 2ϩ ] i by myogenic tone can be inhibited by blockers of VDCC. However, some components are resistant to these agents, and instead can be accounted for by a separate nonselective cation channel that is permeable to Ca 2ϩ and also is activated by the intravascular pressure. 4,5 Because stretchactivated channels play obligatory roles in regulation of the myogenic tone, extensive studies have been performed to identify the molecular entity of these channels. Originally, a yeast MID1 gene product (MID1) was shown to be a eukaryotic stretch-activated channel, because CHO cells expressing MID1 responded to membrane stretch. 6 A member of the transient receptor potential channels (TRP), specifically TRPC6, is sensitive ...

Disruption of the dystrophin–glycoprotein complex caused by genetic defects of dystrophin or sarcoglycans results in muscular dystrophy and/or cardiomyopathy in humans and animal models. However, the key early molecular events leading to myocyte degeneration remain elusive. Here, we observed that the growth factor–regulated channel (GRC), which belongs to the transient receptor potential channel family, is elevated in the sarcolemma of skeletal and/or cardiac muscle in dystrophic human patients and animal models deficient in dystrophin or δ-sarcoglycan. However, total cell GRC does not differ markedly between normal and dystrophic muscles. Analysis of the properties of myotubes prepared from δ-sarcoglycan–deficient BIO14.6 hamsters revealed that GRC is activated in response to myocyte stretch and is responsible for enhanced Ca2+ influx and resultant cell damage as measured by creatine phosphokinase efflux. We found that cell stretch increases GRC translocation to the sarcolemma, which requires entry of external Ca2+. Consistent with these findings, cardiac-specific expression of GRC in a transgenic mouse model produced cardiomyopathy due to Ca2+ overloading, with disease expression roughly parallel to sarcolemmal GRC levels. The results suggest that GRC is a key player in the pathogenesis of myocyte degeneration caused by dystrophin–glycoprotein complex disruption.

Abstract-Activation of the sarcolemmal Na ϩ /H ϩ exchanger (NHE)1 is increasingly documented as a process involved in cardiac hypertrophy and heart failure. However, whether NHE1 activation alone is sufficient to induce such remodeling remains unknown. We generated transgenic mice that overexpress a human NHE1 with high activity in hearts. The hearts of these mice developed cardiac hypertrophy, contractile dysfunction, and heart failure. In isolated transgenic myocytes, intracellular pH was elevated in Hepes buffer but not in physiological bicarbonate buffer, yet intracellular Na ϩ concentrations were higher under both conditions. In addition, both diastolic and systolic Ca 2ϩ levels were increased as a consequence of Na ϩ -induced Ca 2ϩ overload; this was accompanied by enhanced sarcoplasmic reticulum Ca 2ϩ loading via Ca 2ϩ /calmodulin-dependent protein kinase (CaMK)II-dependent phosphorylation of phospholamban. Negative force-frequency dependence was observed with preservation of high Ca 2ϩ , suggesting a decrease in myofibril Ca 2ϩ sensitivity. Furthermore, the Ca 2ϩ -dependent prohypertrophic molecules calcineurin and CaMKII were highly activated in transgenic hearts. These effects observed in vivo and in vitro were largely prevented by the NHE1 inhibitor cariporide. Interestingly, overexpression of NHE1 in neonatal rat ventricular myocytes induced cariporide-sensitive nuclear translocation of NFAT (nuclear factor of activated T cells) and nuclear export of histone deacetylase 4, suggesting that increased Na ϩ /H ϩ exchange activity can alter hypertrophy-associated gene expression. However, in transgenic myocytes, contrary to exclusive translocation of histone deacetylase 4, NFAT only partially translocated to nucleus, possibly because of marked activation of p38, a negative regulator of NFAT signaling. We conclude that activation of NHE1 is sufficient to initiate cardiac hypertrophy and heart failure mainly through activation of CaMKII-histone deacetylase pathway. Key Words: Na ϩ /H ϩ exchanger Ⅲ Na ϩ and Ca 2ϩ overload Ⅲ cardiac remodeling Ⅲ CaMKII-HDAC pathway Ⅲ calcineurin-NFAT pathway I ntracellular Na ϩ levels are regulated by a network of ion channels and transporters. 1 In the myocardium, Na ϩ homeostasis is closely linked to intracellular Ca 2ϩ handling via the Na ϩ /Ca 2ϩ exchanger (NCX), the principal mechanism for Ca 2ϩ efflux from cardiomyocytes. Na ϩ dysfunction alters Ca 2ϩ homeostasis, thereby contributing to the pathogenesis of heart failure (HF) in animal models and in humans. The sarcolemmal Na ϩ /H ϩ exchanger (NHE)1 is a major Na ϩ influx pathway that also serves as a powerful acid extrusion system. NHE1 couples H ϩ efflux to Na ϩ influx in a 1:1 stoichiometry under the driving force of a Na ϩ gradient formed by the Na ϩ pump. Thus, enhanced NHE activity leads to elevated intracellular Na ϩ concentration ([Na ϩ ] i ) and cytoplasmic alkalinization. NHE1 activity is controlled by intracellular pH (pH i ) and numerous other factors, such as hormones, catecholamines, and mechanical stimuli...

Muscular dystrophy is a severe degenerative disorder of skeletal muscle characterized by progressive muscle weakness. One subgroup of this disease is caused by a defect in the gene encoding one of the components of the dystrophin-glycoprotein complex, resulting in a significant disruption of membrane integrity and/or stability and, consequently, a sustained increase in the cytosolic Ca(2+) concentration ([Ca(2+)](i)). In the present study, we demonstrate that muscular dystrophy is ameliorated in two animal models, dystrophin-deficient mdx mice and delta-sarcoglycan-deficient BIO14.6 hamsters by dominant-negative inhibition of the transient receptor potential cation channel, TRPV2, a principal candidate for Ca(2+)-entry pathways. When transgenic (Tg) mice expressing a TRPV2 mutant in muscle were crossed with mdx mice, the [Ca(2+)](i) increase in muscle fibers was reduced by dominant-negative inhibition of endogenous TRPV2. Furthermore, histological, biochemical and physiological indices characterizing dystrophic pathology, such as an increased number of central nuclei and fiber size variability/fibrosis/apoptosis, elevated serum creatine kinase levels, and reduced muscle performance, were all ameliorated in the mdx/Tg mice. Similar beneficial effects were also observed in the muscles of BIO14.6 hamsters infected with adenovirus carrying mutant TRPV2. We propose that TRPV2 is a principal Ca(2+)-entry route leading to a sustained [Ca(2+)](i) increase and muscle degeneration, and that it is a promising therapeutic target for the treatment of muscular dystrophy.

The rat L6 skeletal muscle cell line was used to study expression of the dystrophin-containing glycoprotein complex and its interaction with the integrin system involved in the cell-matrix adhesion reaction. A complex of dystrophin and its associated proteins was fully expressed in L6 myotubes, from which anti-dystrophin or anti-␣-sarcoglycan co-precipitated integrin ␣ 5 ␤ 1 and other focal adhesion-associated proteins vinculin, talin, paxillin, and focal adhesion kinase. Immunostaining and confocal microscopy revealed that dystrophin, ␣-sarcoglycan, integrin ␣ 5 ␤ 1 , and vinculin exhibited overlapping distribution in the sarcolemma, especially at focal adhesion-like, spotty structures. Adhesion of cells to fibronectin-or collagen type I-coated dishes resulted in induction of tyrosine phosphorylation of ␣-and ␥-sarcoglycans but not ␤-sarcoglycan. The same proteins were also tyrosine-phosphorylated when L6 cells in suspension were exposed to Arg-Gly-Asp-Ser peptide. All of these tyrosine phosphorylations were inhibited by herbimycin A. On the other hand, treatment of L6 myotubes with ␣-and ␥-sarcoglycan antisense oligodeoxynucleotides resulted in complete disappearance of ␣-and ␥-sarcoglycans and in significant reduction of levels of the associated focal adhesion proteins, which caused about 50% reduction of cell adhesion. These results indicate the existence of bidirectional communication between the dystrophin-containing complex and the integrin adhesion system in cultured L6 myocytes.