Mitofusin-2 Is a Major Determinant of Oxidative Stress-mediated Heart Muscle Cell Apoptosis

Abstract

An inexorable loss of terminally differentiated heart muscle cells is a crucial causal factor for heart failure. Here, we have provided several lines of evidence to demonstrate that mitofusin-2 (Mfn-2; also called hyperplasia suppressor gene), a member of the mitofusin family, is a major determinant of oxidative stress-mediated cardiomyocyte apoptosis. First, oxidative stress with H 2 O 2 led to concurrent increases in Mfn-2 expression and apoptosis in cultured neonatal rat cardiomyocytes. Second, overexpression of Mfn-2 to a level similar to that induced by H 2 O 2 was sufficient to trigger myocyte apoptosis, which is associated with profound inhibition of Akt activation without altering ERK1/2 signaling. Third, Mfn-2 silencing inhibited oxidative stress-induced apoptosis in H9C2 cells, a cardiac muscle cell line. Furthermore, Mfn-2-induced myocyte apoptosis was abrogated by inhibition of caspase-9 (but not caspase-8) and by overexpression of Bcl-x L or enhanced activation of phosphatidylinositol 3-kinase-Akt, suggesting that inhibition of Akt signaling and activation of the mitochondrial death pathway are essentially involved in Mfn-2-induced heart muscle cell apoptosis. These results indicate that increased cardiac Mfn-2 expression is both necessary and sufficient for oxidative stress-induced heart muscle cell apoptosis, suggesting that Mfn-2 deregulation may be a crucial pathogenic element and a potential therapeutic target for heart failure.An imbalance between cell survival and cell death signaling pathways triggers either proliferative or degenerative disorders, diseases in which apoptosis (or programmed cell death) can be either abnormally blocked or undesirably activated. In this regard, Ras plays a central role in the regulation of cell fate, including cell proliferation, differentiation, senescence, and survival or apoptosis. In particular, Ras-mediated activation of Akt has emerged as a focal point for signal transduction pathways promoting cell survival, whereas Ras-activated ERK1/2 4 and Akt signaling cascades drive cell cycle progression (1-3). Thus, dysfunction of Ras signaling pathways has been implicated in a multitude of degenerative and proliferative diseases.Originally, Ras was identified as a viral oncogene because of its causal relationship with various cancers (3). Over the past decade, increasing evidence has, however, placed Ras signaling at the center of pathways for diverse cardiovascular diseases such as congestive heart failure, hypertensive vascular proliferative growth, and endothelial dysfunction (4). We have recently identified a powerful endogenous Ras inhibitor, mitofusin-2 (Mfn-2), also named hyperplasia suppressor gene because of its anti-proliferative effects (5). Although previous studies have shown that human Mfn-2 and its homologs localize to the mitochondrial outer membrane and play an essential role in mitochondrial fusion, thus regulating mitochondrial morphology and function (6 -11), we recently demonstrated that Mfn-2 profoundly suppresses cell growth and proliferat...

Signalling mechanisms underlying doxorubicin and Nox2 NADPH oxidase‐induced cardiomyopathy: involvement of mitofusin‐2

“…Similar studies have reported the effective use of siRNAs to knockdown both Nox and Mfn2 and inhibit ROS production and apoptosis in cardiac cells, including HL‐1 cardiomyocytes (Yeh et al, 2011), H9c2 cardiomyocytes, (Shen et al, 2007), cultured neonatal rat cardiomyocytes (Papanicolaou et al, 2011) and mouse ventricular cells (Moe et al, 2011). However, while this investigation found no changes in apoptosis in DOX‐treated HL‐1 cardiomyocytes in the presence of Nox2 or Mfn2 knockdown, it is possible that efficacy of transfection may have influenced the measured endpoint.…”

“…In agreement, a considerable number of studies identified in the Ingenuity Knowledge Base were found to support the idea that increased Mfn2 in cardiomyocytes predicted an adverse outcome, promoting cell death through apoptotic mechanisms. For example, Shen et al (2007) demonstrated that Mfn2 mediates oxidative stress‐induced apoptotic cell death in neonatal cardiomyocytes. Additionally, siRNA inhibition of Mfn2 prevented oxidative stress‐induced apoptotic cell death in H9c2 cardiomyocytes (Karbowski et al, 2002).…”

Astragalus polysaccharides suppress ICAM-1 and VCAM-1 expression in TNF-α-treated human vascular endothelial cells by blocking NF-κB activation

“…HUVECs were pretreated with APS for 4 h and then treated with the indicated concentrations of TNF-α for 24 h. All assays were performed in triplicate. The cells were incubated with 0.5 mg/mL 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenylterazolium bromide for 4 h, and the absorbance at 490 nm was measured, as described previously [14] . The MTT kit was purchased from Roche Applied Science (Indianapolis, Indiana).…”

“…Hoechst 33342 staining Nuclear fragmentation was detected by incubating fixed cells (70% alcohol and 30% acetone) in 10 mmol/L Hoechst 33342, as previously described [15] . A total of 500-700 cells in 10 randomly chosen fields from each dish were counted to determine the percentage of apoptotic nuclei.…”

Mitochondrial Dynamics and Apoptosis

“…Since ERK activation has been shown to mediate induction of apoptosis (Cheung and Slack 2004), Mfn2 may protect cells by inhibiting ERK signaling independent of Mfn2 mitochondrial fusion activity. Two recent studies have shown that overexpression of Mfn2 in heart muscle cells (Shen et al 2007) or vascular smooth muscle cells (Guo et al 2007) induces apoptosis through inhibition of Ras-PI3K-Akt signaling. Here, Mfn2 is suggested to act in a pro-apoptotic manner.…”