Yuko Iwata

TRPV2 Is a Component of Osmotically Sensitive Cation Channels in Murine Aortic Myocytes

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 Ca2+ ([Ca2+]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 Ca2+ abolished this increase in [Ca2+]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 stimulation-induced activation of NSCC and elevation of [Ca2+]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 [Ca2+]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.

A novel mechanism of myocyte degeneration involving the Ca2+-permeable growth factor–regulated channel

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.

Bidirectional Signaling between Sarcoglycans and the Integrin Adhesion System in Cultured L6 Myocytes

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-α-sarcoglycan co-precipitated integrin α5β1 and other focal adhesion-associated proteins vinculin, talin, paxillin, and focal adhesion kinase. Immunostaining and confocal microscopy revealed that dystrophin, α-sarcoglycan, integrin α5β1, 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 α- and γ-sarcoglycans but not β-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 α- and γ-sarcoglycan antisense oligodeoxynucleotides resulted in complete disappearance of α- and γ-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.

Activation of Na+/H+ Exchanger 1 Is Sufficient to Generate Ca2+ Signals That Induce Cardiac Hypertrophy and Heart Failure

Activation of the sarcolemmal Na+/H+ 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+ concentrations were higher under both conditions. In addition, both diastolic and systolic Ca2+ levels were increased as a consequence of Na+-induced Ca2+ overload; this was accompanied by enhanced sarcoplasmic reticulum Ca2+ loading via Ca2+/calmodulin-dependent protein kinase (CaMK)II-dependent phosphorylation of phospholamban. Negative force–frequency dependence was observed with preservation of high Ca2+, suggesting a decrease in myofibril Ca2+ sensitivity. Furthermore, the Ca2+-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+/H+ 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.

Dominant-negative inhibition of Ca2+ influx via TRPV2 ameliorates muscular dystrophy in animal models

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.

Defective association of dystrophin with sarcolemmal glycoproteins in the cardiomyopathic hamster heart

In ventricular muscle from 30‐ to 60‐day‐old Bio 14.6 cardiomyopathic hamsters, dystrophin‐associated glycoproteins of 43, 50 and 150 kDa are markedly reduced in abundance. In particular, the 50‐kDa glycoprotein is totally deficient in the sareolemma of myopathic ventricular myocytes as revealed by immunofluorescence microscopy. The dystrophin‐glycoprotein complex formation is defective in the cardiomyopathic hamster heart, because dystrophin and the glycoproteins behave independently when digitonin‐solubilized ventricular homogenates are fractionated on wheat germ agglutinin beads or anti‐dystrophin immunoaffinity beads.

Essential role of TRPV2 ion channel in the sensitivity of dystrophic muscle to eccentric contractions.

Duchenne myopathy is a lethal disease due to the absence of dystrophin, a cytoskeletal protein. Muscles from dystrophin‐deficient mice (mdx) typically present an exaggerated susceptibility to eccentric work characterized by an important force drop and an increased membrane permeability consecutive to repeated lengthening contractions. The present study shows that mdx muscles are largely protected from eccentric work‐induced damage by overexpressing a dominant negative mutant of TRPV2 ion channel. This observation points out the role of TRPV2 channel in the physiopathology of Duchenne muscular dystrophy.

Global metabolomic analysis of heart tissue in a hamster model for dilated cardiomyopathy

Dilated cardiomyopathy (DCM), a common cause of heart failure, is characterized by cardiac dilation and reduced left ventricular ejection fraction, but the underlying mechanisms remain unclear. To investigate the mechanistic basis, we performed global metabolomic analysis of myocardial tissues from the left ventricles of J2N-k cardiomyopathic hamsters. This model exhibits symptoms similar to those of human DCM, owing to the deletion of the δ-sarcoglycan gene. Charged and lipid metabolites were measured by capillary electrophoresis mass spectrometry (MS) and liquid chromatography MS(/MS), respectively, and J2N-k hamsters were compared with J2N-n healthy controls at 4 (presymptomatic phase) and 16weeks (symptomatic) of age. Disturbances in membrane phospholipid homeostasis were initiated during the presymptomatic phase. Significantly different levels of charged metabolites, occurring mainly in the symptomatic phase, were mapped to primary metabolic pathways. Reduced levels of metabolites in glycolysis, the pentose phosphate pathway, and the tricarboxylic acid cycle, together with large decreases in major triacylglycerol levels, suggested that decreased energy production leads to cardiac contractile dysfunction in the symptomatic phase. A mild reduction in glutathione and a compensatory increase in ophthalmate levels suggest increased oxidative stress in diseased tissues, which was confirmed by histochemical staining. Increased levels of 4 eicosanoids, including prostaglandin (PG) E2 and 6-keto-PGF1α, in the symptomatic phase suggested activation of the protective response pathways. These results provide mechanistic insights into DCM pathogenesis and may help identify new targets for therapeutic intervention and diagnosis.

Blockade of sarcolemmal TRPV2 accumulation inhibits progression of dilated cardiomyopathy

AIMS Dilated cardiomyopathy (DCM) is a severe disorder defined by ventricular dilation and contractile dysfunction. Abnormal Ca(2+) handling is hypothesized to play a critical pathological role in DCM progression. The transient receptor potential vanilloid 2 (TRPV2) has been previously suggested as a candidate pathway for enhanced Ca(2+) entry. Here, we examined the sarcolemmal accumulation of TRPV2 in various heart-failure model animals and DCM patients, and assessed whether presently available inhibitory tools against TRPV2 ameliorate DCM symptoms. METHODS AND RESULTS Immunological and cell physiological analyses revealed that TRPV2 is highly concentrated and activated in the ventricular sarcolemma of DCM patients and three animal models-δ-sarcoglycan-deficient hamsters (J2N-k), transgenic mice over-expressing sialytransferase (4C30), and doxorubicin (DOX)-induced DCM mice. Over-expression of the amino-terminal (NT) domain of TRPV2 could block the plasma membrane accumulation and influx of Ca(2+) via TRPV2. Transgenic (Tg) or adenoviral expression of the NT domain in DCM animals caused effective removal of sarcolemmal TRPV2 along with reduction in the phosphorylation of calmodulin-dependent protein kinase II (CaMKII) and reactive oxygen species (ROS) production, which were activated in DCM; further, it prevented ventricular dilation and fibrosis, ameliorated contractile dysfunction in DCM, and improved survival of the affected animals. The TRPV2 inhibitor tranilast markedly suppressed DCM progression. CONCLUSION Sarcolemmal TRPV2 accumulation appears to have considerable pathological impact on DCM progression, and blockade of this channel may be a promising therapeutic strategy for treating advanced heart failure.

Syntrophin is an actin-binding protein the cellular localization of which is regulated through cytoskeletal reorganization in skeletal muscle cells.

We have characterized the interaction of syntrophin with F-actin. Subcellular fractionation of cardiac and skeletal muscle tissues showed that alpha-, beta1- and beta2-syntrophins were present in the soluble and the membrane fraction. Syntrophins are known to bind to the dystrophin-glycoprotein complex (DGC), but since the DGC is not present in the soluble fraction, it was concluded that some syntrophin did not associate with the DGC. Native syntrophins purified from the soluble fraction and recombinant syntrophins were both able to bind to F-actin, and binding occurred through several sites on syntrophin, including the second pleckstrin homology domain and the unique carboxyl-terminal domain. Syntrophin was also able to inhibit actin-activated myosin ATPase activity and actomyosin super-precipitation. alpha-Syntrophin co-localized with cortical F-actin fibers when expressed in Chinese hamster ovary cells, and deletion of the actin-binding region abolished co-localization. Most of exogenous or endogenous syntrophin also co-localized with stress fibers in endothelial and smooth muscle (A7r5) cells. However, syntrophins were mostly localized in the cytosol of serum-starved C2C12 or primary cultured skeletal muscle myotubes, and translocated to the membrane upon treatment with lysophosphatidic acid or the actin-stabilizing agent jasplakinolide. The actin-depolymerizing agent latrunculin-B abolished this syntrophin translocation. These findings suggest that syntrophin is an actin-binding protein the subcellular localization of which is regulated through cytoskeletal reorganization.