Biochemical and Biophysical Research Communications
Regulator of G-protein signaling 5 protects cardiomyocytes against apoptosis during in vitro cardiac ischemia-reperfusion in mice by inhibiting both JNK1/2 and P38 signaling pathways
Introduction
Ischemic heart disease is one of the most common diseases in modern society. Ischemic myocardium can be salvaged by vascular recanalization therapy using thrombolytic, percutaneous coronary intervention (PCI) or surgery approaches, but its benefit is attenuated by injury that can occur during reperfusion [1], especially for multivessel coronary artery disease, aortic aneurysm dissecting into coronary artery, sudden cardiac arrest, and heart transplantation. In vitro and in vivo experimental results have confirmed that cardiac myocyte death during ischemia-reperfusion (IR) is related to not only necrosis but also apoptosis [2], [3]. Apoptosis is not only the main form of cardiac myocyte death, especially in the early phase of acute myocardial infarction (AMI), but is also more exacerbated in the reperfused myocardium [4]. Previous studies have stated that active caspase-3 is an potent executioner of apoptosis, and B cell lymphoma/lewkmia-2 (Bcl-2) protein inhibits apoptosis, while Bcl-2 Associated X protein (Bax) protein induces apoptosis [5], [6], [7]. Consequently, for the development of new therapeutic methods it is important to aim mainly at rescuing apoptotic cardiomyocytes during cardiac IR.
Many extracellular signals are transduced into cells via membrane-binding receptors linked with heterotrimeric guanine nucleotide-binding proteins (G proteins) [8]. Previous studies have identified numerous G protein effectors, comprising two classes of the mitogen-activated protein kinases (MAPKs): p38 MAPK and c-Jun NH2-terminal kinase (JNK)/stress-activated protein kinase. It is generally known that signaling pathways such as JNK1/2 and p38 promote cardiac IR-induced apoptosis [9], [10], however the molecular mechanisms involved in these pathways have not been completely elucidated. These pathways are activated by stimulating G protein–coupled receptors (GPCRs) which are regulated by both G(q) and G(i/o) proteins under control of the regulator of G-protein signaling (RGS) proteins [8], [11]. As a negative regulator of GPCR-regulated signaling, RGS5 serves as a guanosine triphosphatase – activating protein (GAP) to inhibit many Gαq- and Gαi-mediated signaling pathways, including endothelin-1 and angiotensin II (Ang II). Reported from others have demonstrated the role of RGS5 in wound healing and reproductive cycle maintenance [12], [13]. In cardiovascular system, RGS5 is highly expressed in different cell types of murine and human adult heart [14], and actively involves in angiogenesis, and vascular pericyte maturation, the pathogenesis of hypertension, atherosclerosis, and heart failure [15], [16].
Our previous studies strongly indicate that RGS5 genetic deletion in mice induced prolongation of myocardium repolarization and occurrence of ventricular, atrial tachyarrhythmia, and that RGS5 also was protective against cardiac hypertrophy and fibrosis in response to biomechanical stress induced by pressure overload [17], [18], [19]. However, the effect of RGS5 on cardiomyocyte apoptosis during cardiac IR has not been fully elucidated. More importantly, the benefit of vascular recanalization therapy can be attenuated by injury that can occur during reperfusion in clinical practice [1] and more and more people are receiving PCI and surgery during acute myocardial ischemia or infarction, making reperfusion injury more common than before. Therefore, we purse this study particularly focusing on the role of RGS5 during IR.
The current study found that overexpression of the human RGS5 gene in the hearts of transgenic mice protected cardiomyocytes against apoptosis during cardiac IR through inhibition of both JNK1/2 and p38 signaling pathways, with the opposite effects in RGS5-knockout mice. Our experimental results indicate that RGS5 is a critical protein in the regulation of cardiomyocyte apoptosis during cardiac IR in mice.
Section snippets
Materials
The specific rabbit polyclonal antibodies against p38, phospho-p38, JNK, phospho-JNK, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were purchased from Cell Signaling Technology (CST, Waltham, MA, USA). The anti-RGS5 antibody with reactivity with both mouse and human RGS5 was purchased from Abcam (Cambridge, MA, USA). SB203580 (SB), a p38 MAPK inhibitor, was purchased from Beyotime (Shanghai, China) (Sun et al., 2012a). SP600125 (SP), a JNK inhibitor, was purchased from Kinasechem
Effect of RGS5 on cardiomyocyte apoptosis during cardiac IR
In order to define the impact of RGS5 expression on IR-induced cardiomyocyte apoptosis, RGS5-TG mice, KO mice, and RGS5-WT mice were used for perfusion of isolated hearts under either IR or control conditions. We found that IR–induced cardiomyocyte apoptosis was significantly enhanced among all mice compared with controls (Fig. 1). We also found that during IR, TG mice showed inhibition of cardiomyocyte apoptosis, as indicated by an obvious reduction in nuclear atrophy or chromatin condensation
Discussion
Increasing evidence from both clinical observations and animal experiments indicate that apoptosis plays a pivotal role in myocardial IR injury. Also, the reduction in cardiomyocyte numbers due to apoptosis is a major mechanism involved in the development of cardiac failure during myocardial infarction and IR [25]. Several studies have shown that RGS5 has significant effects on blood vessels and the hearts, and mediates these actions through inactivation of Gα(q) and Gα(i) to negatively
Conflict of interest
The authors declare no conflict of interest.
Acknowledgments
This study was funded by National Natural Science Foundation of China Grants (81270249), Hubei Provincial Natural Science Foundation of China Grants (2013CFA059), and Hubei Provincial commonweal Foundation of China Grants (2013BCB013). Yu Liu, Teng Wang, Siwei Song, Yanhong Tang, Hongliang Li, and Congxin Huang are gratefully acknowledged for providing technology help.
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