Heat Shock Protein 90 Transfection Reduces Ischemia-Reperfusion–Induced Myocardial Dysfunction via Reciprocal Endothelial NO Synthase Serine 1177 Phosphorylation and Threonine 495 Dephosphorylation

Abstract

Objectives-The interaction of the heat shock protein 90 (Hsp90) with the endothelial NO synthase (eNOS) has been shown to account for a sustained production of NO in vitro. Here, we examined whether overexpression of Hsp90 in a pig model of cardiac infarct could preserve the myocardium from the deleterious effects of ischemia-reperfusion. Methods and Results-Percutaneous liposome-based gene transfer was performed by retroinfusion of the anterior interventricular vein before left anterior descending occlusion and reperfusion. We found that recombinant Hsp90 expression in the ischemic region of the heart led to a 33% reduction in infarct size and prevented the increase in postischemic left ventricular end diastolic pressure observed in mock-transfected animals. Regional myocardial function, assessed by subendocardial segment shortening in the infarct region, was increased in Hsp90-transfected animals at baseline and after pacing. All these effects were completely abrogated by administration of the NOS inhibitor N G -nitro-L-arginine methyl ester. We further documented in vivo and in cultured endothelial cells that the cardioprotective effects of Hsp90 were associated to its capacity to act as an adaptor for both the kinase Akt and the phosphatase calcineurin, thereby promoting eNOS serine 1177 phosphorylation and threonine 495 dephosphorylation, respectively. Conclusions-Hsp90 is a promising target to enhance NO formation in vivo, which may efficiently reduce myocardial reperfusion injury. Key Words: Hsp90 Ⅲ nitric oxide Ⅲ eNOS Ⅲ calcineurin Ⅲ Akt M yocardial ischemia caused by an occluded coronary artery is treated clinically by rapid re-establishment of perfusion, which is currently viewed as uniquely effective in preventing myocyte cell death. Nevertheless, a body of evidence documents that reperfusion also leads to organ dysfunction, including endothelial and microcirculatory disturbance, arrhythmias, and myocardial stunning. 1 Among the multitude of strategies aiming to prevent ischemiareperfusion (I/R)-induced myocardial detriments, overexpression of the endothelial NO synthase (eNOS) isoform in the myocyte 2 or endothelial 3 compartment has proven recently to be cardioprotective. The endothelial-targeted enhancement of eNOS activity appears particularly promising because it induces vasorelaxation, inhibits platelet aggregation as well as leukocyte recruitment, and antagonizes microcirculatory disturbances. 4 Yet, increase in NO formation by endothelium can exert beneficial effects on the cardiomyocyte compartment, preserving myocyte function by preventing necrosis, 5 apoptosis 6,7 as well as reducing myocyte vulnerability via induction of hibernation. 8 However, the therapeutically desired increase in endothelial NO production does not necessarily require an increase in eNOS abundance because it may also evolve from specific post-translational modifications of endogenously expressed eNOS. Accordingly, the cardioprotective effects of insulin, 6 corticosteroids, 9 and vascular endothelial growth factor (VEGF)...

Src-mediated Phosphorylation of Hsp90 in Response to Vascular Endothelial Growth Factor (VEGF) Is Required for VEGF Receptor-2 Signaling to Endothelial NO Synthase

“…In addition to a direct allosteric effect of Hsp90 on eNOS, it is thought that binding of Hsp90 to Akt prevents the phosphatase 2A-mediated dephosphorylation of Thr 308 of Akt, which maintains its positive functional effects on eNOS catalytic activity (Sato et al, 2000;Takahashi and Mendelsohn, 2003). Furthermore, Hsp90 has been proposed to facilitate the calcineurin-dependent dephosphorylation of Thr495 of eNOS, which contributes to enzyme activation (Kupatt et al, 2004). Hyperphosphorylation of serine and/or threonine residues on Hsp90 has been shown to negatively regulate Hsp90's role in the conformational maturation of oncogenic signaling proteins, including ErbB2, c-Src, Akt, and Raf-1 (Schulte et al, 1995;Mimnaugh et al, 1995;Xu et al, 2001;Zhao et al, 2001;Basso et al, 2002).…”

Overexpression of the muscle-specific protein, melusin, protects from cardiac ischemia/reperfusion injury

“…Given that melusin binds to HSP90 and is co-expressed with HSPs [57], we evaluated the expression of HSP90, an ATP-dependent chaperone known to have a cardio-protective role in I/R [35]. Interestingly, as shown in Figure 3 …”

Cardiac gene therapy in large animals: bridge from bench to bedside

“…In some studies, DNA complexed with liposome was employed to improve the transfection efficiency. 18,91,93 Viral vectors Adenovirus has been the most frequently employed viral vector in large animal cardiac gene delivery studies. This vector efficiently transduces both dividing and non-dividing cells with significantly higher efficiency than plasmid DNA.…”