Expression and roles of N-type Ca channel in cardiaomyocytes

Background Voltage dependent Ca channels are divided to L-, T-, N-, P/Q-, and R-types, and N-type Ca channel (NCC) is mainly expressed in nerve terminal and regulates neurotransmitter release. Recently, NCC has been reported to express in adrenal gland and renal tubular cells. We examined whether NCC is expressed in cardiac myocytes and if so, the roles of this channel. Methods Expression of NCC mRNA and protein in cardiomyocytes were assessed by quantitative real time PCR and Western blot analysis using neonatal rat cultured cardiomyocytes, infant, and adult rat hearts. Expression site of NCC in cardiomyocytes was examined by confocal imaging of immunofluorescent staining. The roles of NCC in physiological Ca transient in neonatal myocytes were examined using fluorescence imaging of Fluo4, an intracellular Ca indicator. To examine the effects of pathological condition, such as heart failure and ischemia-reperfusion, on NCC expression, cultured cardiomyocytes were treated with norepinephrine (10 μmol/L, 24 hours) or subjected to 5 hours of hypoxia followed by 30 minutes of reoxygenation. In addition, adult rats were subjected to myocardial infarction by ligating the left anterior coronary artery. Lethal myocyte injury was examined by LDH activity in culture medium and myocyte apoptosis was examined by nuclear staining with DAPI and caspase 3 activity. To clarify the roles of NCC in neonatal myocytes in these pathological conditions, we examine the effect of ω-conotoxin, a selective NCC blocker. Results NCC mRNA and protein were expressed in neonatal cardiomyocytes. Immunocytochemical staining showed NCC was expressed in myocyte plasma membrane. During physiological spontaneous beating, ω-conotoxin did not affect beating rate and intra cellular Ca transient, suggesting that the roles of NCC on physiological beating are little. After birth level of NCC mRNA expression in cardiac tissue gradually decreased within 2 weeks and low level of mRNA expressed continuously in adult cardiac tissue. However, in pathological condition, mRNA and protein levels of NCC in non-infarcted region were increased 4 weeks after myocardial infarction. In addition, hypoxia-reoxygenation and norepinephrine administration increased LDH release and myocyte apoptosis in association with increase in NCC expression in neonatal cultured myocytes. ω-conotoxin significantly suppressed hypoxia/reoxygenation- and norepinephrine-induced LDH release and caspase 3 activation. Conclusion NCC is expressed in neonatal cardiac myocytes and the expression level was decreased after birth. Pathological condition, such as ischemic heart disease and heart failure, upregulated NCC expression in cardiomyocytes and NCC exacerbated lethal myocyte injury, while roles of NCC in physiological beating are little. FUNDunding Acknowledgement Type of funding sources: None.

Garlic extract diallyl sulfide protects against dilated cardiomyopathy through inhibition of oxidative stress and apoptosis in mice

Background: Diallyl sulfide (DAS) is an active ingredient in garlic that is induced when the garlic is chopped and ground. DAS has been found to act as a competitive inhibitor of CYP2E1, which is a member of the cytochrome P450 enzyme family and catalyses the metabolism of various substrates.CYP2E1 is upregulated in multiple heart diseases and causes damage mainly through the production of ROS. In mice, increased CYP2E1 expression induces cardiac myocyte apoptosis, and CYP2E1 knockdown can attenuate the pathological development of DCM. Nevertheless, Targeted inhibition of CYP2E1 for the treatment of dilated cardiomyopathy (DCM) remain limited. The aim of this study was to investigate the therapeutic effect of DAS on cardiomyopathy and its possible molecular mechanisms, and provide new clues and approaches for the clinical treatment of cardiomyopathy and heart failure.Methods: Echocardiography were performed to identify mouse heart function and structure. histological analysis and RT-PCR were conducted to investigate that improved Myocardial morphology and fibrosis and measurement of reactive oxygen species (ROS) and tunel Assay were used to detected DAS inhibit ROS production and myocyte apoptosis in cTnTR141W DCM mice. Western Blot were performed to investigate the mechanism of apoptosis pathway.Results: Diallyl sulfide (DAS), a competitive inhibitor of CYP2E1, improves the typical DCM phenotype, including chamber dilation, wall thinning, fibrosis, poor myofibril organization and decreased ventricular blood ejection, by inhibiting ROS production and myocyte apoptosis in cTnTR141W DCM mice.Conclusions: Our results suggest that inhibition of CYP2E1 might be a valuable therapeutic strategy to control the development of heart diseases associated with CYP2E1 overexpression. Moreover, the development of DAS analogues with superior inhibitory properties and lower substrate potential for CYP2E1 might be beneficial for patients with heart disease.

Exogenous ubiquitin attenuates hypoxia/reoxygenation-induced cardiac myocyte apoptosis via the involvement of CXCR4 and modulation of mitochondrial homeostasis

Exogenous ubiquitin (UB) plays a protective role in β-adrenergic receptor-stimulated and ischemia/reperfusion (I/R)-induced myocardial remodeling. Here, we report that UB treatment inhibits hypoxia/reoxygenation (H/R)-induced apoptosis in adult rat ventricular myocytes (ARVMs). The activation of Akt was elevated, whereas the activation of glycogen synthase kinase-3β was reduced in UB-treated cells post-H/R. The level of oxidative stress was lower, whereas the number of ARVMs with polarized mitochondria was significantly greater in the UB-treated samples. ARVMs express CXCR4 with majority of CXCR4 localized in the membrane fraction. CXCR4 antagonism using AMD3100, and siRNA-mediated knockdown of CXCR4 negated the protective effects of UB. Two mutated UB proteins (unable to bind CXCR4) had no effect on H/R-induced apoptosis, activation of Akt and GSK-3β, or oxidative stress. UB treatment enhanced mitochondrial biogenesis, and inhibition of mitochondrial fission using mdivi1 inhibited H/R-induced apoptosis. Ex vivo, UB treatment significantly decreased infarct size and improved functional recovery of the heart following global I/R. Activation of caspase-9, a key player of the mitochondrial death pathway, was significantly lower in UB-treated hearts post-I/R. UB, most likely acting via CXCR4, plays a protective role in H/R-induced myocyte apoptosis and myocardial I/R injury via modulation of mitochondrial homeostasis and the mitochondrial death pathway of apoptosis.

The Long Noncoding RNA Hotair Regulates Oxidative Stress and Cardiac Myocyte Apoptosis during Ischemia-Reperfusion Injury

Oxidative stress and subsequent cardiac myocyte apoptosis play central roles in the initiation and progression of myocardial ischemia-reperfusion (I/R) injury. Homeobox transcript antisense intergenic RNA (Hotair) was previously implicated in various heart diseases, yet its role in myocardial I/R injury has not been clearly demonstrated. Mice with cardiac-restricted knockdown or overexpression of Hotair were exposed to I/R surgery. H9c2 cells were cultured and subjected to hypoxia/reoxygenation (H/R) stimulation to further verify the role and underlying mechanisms of Hotair in vitro. Histological examination, molecular detection, and functional parameters were determined in vivo and in vitro. In response to I/R or H/R treatment, Hotair expression was increased in a bromodomain-containing protein 4-dependent manner. Cardiac-restricted knockdown of Hotair exacerbated, whereas Hotair overexpression prevented I/R-induced oxidative stress, cardiac myocyte apoptosis, and cardiac dysfunction. Mechanistically, we observed that Hotair exerted its beneficial effects via activating AMP-activated protein kinase alpha (AMPKα). Further detection revealed that Hotair activated AMPKα through regulating the enhancer of zeste homolog 2/microRNA-451/calcium-binding protein 39 (EZH2/miR-451/Cab39) axis. We provide the evidence that endogenous lncRNA Hotair is an essential negative regulator for oxidative stress and cardiac myocyte apoptosis in myocardial I/R injury, which is dependent on AMPKα activation via the EZH2/miR-451/Cab39 axis.