N-cadherin signals through Rac1 determine the localization of connexin 43 in cardiac myocytes.

It has been proposed that the formation of gap junction is influenced by adherens junction in cardiac myocytes. To examine whether signals through N-cadherin are involved in the distribution of connexin 43 (Cx43), the distribution of cell-cell adhesion molecules, N-cadherin and Cx43, was analyzed in aligned cardiac myocytes. To induce cell orientation running in parallel to tension direction, neonatal rat cardiac myocytes were plated for 3 hours and exposed to 20% cyclic stretch for 24 hours on silicone dishes. The aligned cells cultured for 0-5 days were immunostained with anti- N-cadherin or anti-Cx43 antibody. After cultivation for 3-5 days, following the accumulation of N-cadherin, Cx43 was localized at the longitudinal cell termini. Adenoviral gene transfer of dominant negative N-cadherin significantly attenuated the localization of Cx43 at the longitudinal cell termini, suggesting that Cx43 localization is regulated downstream of N-cadherin. In the process of Cx43 localization, Rho family proteins, RhoA and Rac1, were activated, but not Cdc42. RhoA and Rac1 activation was inhibited by the transfection of dominant negative N-cadherin, indicating that RhoA and Rac1 were activated by N-cadherin in the oriented cardiac myocytes. The inhibition of Rho family proteins by Rho GDI significantly attenuated the accumulation of Cx43, but not that of N-cadherin. Furthermore, the translocation of Cx43 to longitudinal cell termini was prevented by the inhibition of Rac1, but not RhoA. Collectively, these findings suggest that the localization of Cx43 was determined through the Rac1 pathway downstream of N-cadherin in cardiac myocytes.

N-cadherin-mediated cell adhesion determines the plasticity for cell alignment in response to mechanical stretch in cultured cardiomyocytes.

Mechanical stretch has been implicated as the growth stimuli in the heart. Physiologically, mechanical stretch is reported to contribute to the orientation of cardiomyocytes, though the molecular mechanism remains to be elucidated. This study was designed to make clear functional significances of N-cadherin in plasticity of cell alignment in response to mechanical stretch. Neonatal rat cardiomyocytes, cultured on silicone dishes, were subjected to artificial uniaxial cyclic stretch. Mechanical stretch was started at certain times (3-75h) after seeding and continued for 24h. Stretch stimulation in 3h after cultivation promoted cell orientation running parallel to tension direction. In contrast, cardiac myocytes fail to align when exposed to stretch 24-75h after cultivation. To address the importance of N-cadherin in the responsiveness to stretch, the expression and distribution of N-cadherin were analyzed. Immediately after seeding, N-cadherin showed dispersed distributions. During cultivation, N-cadherin localized to cell-cell contacts accompanied by the upregulation of its protein. Next, to investigate influence of cell-cell adhesion, cardiomyocytes cultured for 72h were replated by trypsin treatment and exposed to stretch 3h after replating. The cardiomyocytes replated by trypsinization were oriented in parallel to tension direction by mechanical stretch. Finally, adenoviral transfection of dominant-negative N-cadherin recovered the ability to exhibit cell orientation in response to stretch. Our results suggested that N-cadherin was involved in the oriented responses of cardiomyocytes induced by mechanical stretch.

Signals through gp130 upregulate Wnt5a and contribute to cell adhesion in cardiac myocytes.

Glycoprotein 130 (gp130), a common receptor of IL-6 family cytokines, plays critical roles in cardiac functions. Here, we demonstrate that the stimulation of gp130 with leukemia inhibitory factor (LIF) promoted cell adhesion in a cadherin-dependent manner in cultured cardiomyocytes. Wnt5a was upregulated by the stimulation of gp130 with IL-6 family cytokines, accompanied by N-cadherin protein upregulation. Wnt5a was not induced by LIF in cardiomyocytes expressing dominant-negative STAT3. Ablation of Wnt5a by antisense cDNA inhibited LIF-induced cell adhesion. Collectively, signals through gp130 upregulate Wnt5a through STAT3, promoting the N-cadherin-mediated cell adhesion.

Taurine inhibits apoptosis by preventing formation of the Apaf-1/caspase-9 apoptosome.

Cardiomyocyte apoptosis contributes to cell death during myocardial infarction. One of the factors that regulate the degree of apoptosis during ischemia is the amino acid taurine. To study the mechanism underlying the beneficial effect of taurine, we examined the interaction between taurine and mitochondria-mediated apoptosis using a simulated ischemia model with cultured rat neonatal cardiomyocytes sealed in closed flasks. Exposure to medium containing 20 mM taurine reduced the degree of apoptosis following periods of ischemia varying from 24 to 72 h. In the untreated group, simulated ischemia for 24 h led to mitochondrial depolarization accompanied by cytochrome c release. The apoptotic cascade was also activated, as evidenced by the activation of caspase-9 and -3. Taurine treatment had no effect on mitochondrial membrane potential and cytochrome c release; however, it inhibited ischemia-induced cleavage of caspase-9 and -3. Taurine loading also suppressed the formation of the Apaf-1/caspase-9 apoptosome and the interaction of caspase-9 with Apaf-1. These findings demonstrate that taurine effectively prevents myocardial ischemia-induced apoptosis by inhibiting the assembly of the Apaf-1/caspase-9 apoptosome.

Expression of taurine transporter is regulated through the TonE (tonicity-responsive element)/TonEBP (TonE-binding protein) pathway and contributes to cytoprotection in HepG2 cells.

In hypertonic environment, taurine accumulates in cells via activation of TauT (taurine transporter) as an adaptive regulation. Recent studies revealed that TonE (tonicity-responsive element)/TonEBP (TonE-binding protein) pathway regulated the expression of various molecules which protect cells against hypertonic stress. In the present study, we investigated the osmoregulatory mechanisms of TauT expression. TauT was up-regulated at both functional and transcriptional levels in HepG2 under hypertonic condition. The TonE site was identified in the promoter region of TauT gene. Reporter gene assay revealed that promoter activity was increased under hypertonic conditions, whereas deletion or mutation of TonE sequence abolished the induction of the promoter activity in response to hypertonicity. By using the reporter gene plasmids containing a TonE site of TauT promoter (p2xTonE-Luc), it was demonstrated that a TonE site was sufficient for the hypertonicity-mediated activation of TauT promoter. Importantly, co-transfection of TauT promoter gene plasmid with wild-type TonEBP expression vector enhanced promoter activity under isotonic conditions, whereas dominant-negative TonEBP abrogated the TauT promoter activity induced by hypertonicity. Finally, treatment with taurine prevented HepG2 cells from cell death induced by hypertonic medium. These findings suggested that induction of TauT by hypertonicity is mediated by the activation of the TonE/TonEBP pathway and confers resistance to hypertonic stress.

Minoxidil attenuates ischemia-induced apoptosis in cultured neonatal rat cardiomyocytes.

The effects of minoxidil (a mitochondrial K+(ATP) channel opener) on ischemia-induced necrosis and apoptosis were examined using a cardiomyocyte model of simulated ischemia, since mitochondrial K+(ATP) channel openers have been suggested to be involved in the mechanisms of cardioprotective action against ischemia/reperfusion injury. In the absence of minoxidil, simulated ischemia led to cellular release of creatine phosphokinase (CPK), morphologic degeneration, and beating cessation within 24 to 72 hours. Based on the Hoechst 33258 staining pattern, a significant number of cells placed in sealed flasks underwent apoptosis. Myocytes treated with 5 microM of minoxidil failed to alter the degree of ischemia-induced CPK loss for 48 to 72 hours. However, minoxidil treatment prevented the loss of beating function in many of the ischemic cells, and attenuated the decline in intracellular ATP content after a 48-hour ischemic incubation. The number of nuclear fragmentation was significantly reduced in minoxidil-treated cells after a 72-hour ischemic insult compared with untreated ischemic cells. This effect was blocked by the mitochondrial K+(ATP) channel antagonist 5-HD. The data suggest that minoxidil renders the cell resistant to ischemia-induced necrosis and apoptosis. The beneficial effects of minoxidil appear to be related to the opening of mitochondrial K+(ATP) channels.

Taurine prevents the ischemia-induced apoptosis in cultured neonatal rat cardiomyocytes through Akt/caspase-9 pathway.

Activated Akt kinase has been proposed as a central role in suppressing apoptosis by modulating the activities of Bcl-2 family proteins and/or caspase-9. To study the mechanism underlying the anti-apoptotic effect of taurine, the interaction between taurine and Akt/caspase-9 pathway was examined using a simulated ischemia model with cultured rat neonatal cardiomyocytes sealed in closed flasks. Taurine (20mM) treatment attenuated simulated ischemia-induced decline in the activity of Akt. Although taurine treatment had no effect on the expression of Bcl-2 in mitochondria and the level of cytosolic cytochrome c, it inhibited ischemia-induced cleavage of caspases 9 and 3. Moreover, adenovirus transfer of the dominant negative form of Akt objected taurine-mediated anti-apoptotic effects, cancelling the suppression of caspase-9 and caspase-3 activities by taurine. These findings provide the first evidence that taurine inhibits ischemia-induced apoptosis in cardiac myocytes with the increase in Akt activities, by inactivating caspase-9.

Taurine renders the cell resistant to ischemia-induced injury in cultured neonatal rat cardiomyocytes.

Taurine is found in very high concentration in the mammalian heart. Because chronic myocardial taurine loss produces myocardial injury, the effects of taurine supplementation on ischemia-induced necrosis and apoptosis were examined using a cardiomyocyte model of simulated ischemia. Neonatal rat heart cells were cultured for 24-72 h in a sealed flask, a condition that leads to simulated ischemia characterized by a decrease in the pH and oxygen content of the medium and a catabolite accumulation. The consequences of altered medium taurine on cellular apoptosis and necrosis were then evaluated. Exposure of cardiomyocytes to medium containing high extracellular concentrations of taurine (20 mM) significantly elevated intracellular taurine levels, reduced p53 content, and enhanced cellular Bcl-2 content. In the absence of taurine treatment, simulated ischemia led to cellular release of creatine phosphokinase (CPK), morphologic degeneration, and beating cessation by 24-72 h. Based on DNA ladder analysis and the Hoechst 33258 staining pattern, a significant number of cells placed in sealed flasks underwent apoptosis. CPK was lost from some of the cells during simulated ischemia. In contrast to the untreated ischemic cells, the cells that were incubated in medium supplemented with taurine exhibited significantly less ischemia-induced necrosis and apoptosis. The data suggest that taurine renders the cell resistant to ischemia-induced necrosis and apoptosis. The beneficial effects of taurine may be related to the elevation in cellular Bcl-2 content.