Abstract
Studies from our lab have shown that decreasing myocardial G protein–coupled receptor kinase 2 (GRK2) activity and expression can prevent heart failure progression after myocardial infarction. Since GRK2 appears to also act as a pro-death kinase in myocytes, we investigated the effect of cardiomyocyte-specific GRK2 ablation on the acute response to cardiac ischemia/reperfusion (I/R) injury. To do this we utilized two independent lines of GRK2 knockout (KO) mice where the GRK2 gene was deleted in only cardiomyocytes either constitutively at birth or in an inducible manner that occurred in adult mice prior to I/R. These GRK2 KO mice and appropriate control mice were subjected to a sham procedure or 30 min of myocardial ischemia via coronary artery ligation followed by 24 hrs reperfusion. Echocardiography and hemodynamic measurements showed significantly improved post-I/R cardiac function in both GRK2 KO lines, which correlated with smaller infarct sizes in GRK2 KO mice compared to controls. Moreover, there was significantly less TUNEL positive myocytes, less caspase-3, and -9 but not caspase-8 activities in GRK2 KO mice compared to control mice after I/R injury. Of note, we found that lowering cardiac GRK2 expression was associated with significantly lower cytosolic cytochrome C levels in both lines of GRK2 KO mice after I/R compared to corresponding control animals. Mechanistically, the anti-apoptotic effects of lowering GRK2 expression were accompanied by increased levels of Bcl-2, Bcl-xl, and increased activation of Akt after I/R injury. These findings were reproduced in vitro in cultured cardiomyocytes and GRK2 mRNA silencing. Therefore, lowering GRK2 expression in cardiomyocytes limits I/R-induced injury and improves post-ischemia recovery by decreasing myocyte apoptosis at least partially via Akt/Bcl-2 mediated mitochondrial protection and implicates mitochondrial-dependent actions, solidifying GRK2 as a pro-death kinase in the heart.
Highlights
The myocyte death that follows acute myocardial ischemia and subsequent reperfusion (I/R) injury is a major factor contributing to high mortality and morbidity in ischemic heart disease
Studies targeting the role of G protein–coupled receptor kinase 2 (GRK2) in I/R injury were performed with two independent lines of cardiac-specific GRK2 KO mice: a constitutive GRK2KO line and an inducible GRK2 KO line where GRK2 was deleted in adulthood using Tmx
To investigate the impact of lowered cardiomyocyte GRK2 levels on ischemic injury, we first measured in vivo cardiac function using echocardiography and terminal hemodynamics at 24 hrs post-I/R in both lines of GRK2 KO mice along with their respective controls (MCM for GRK2iKO and GRK2fl/fl for GRK2KO)
Summary
The myocyte death that follows acute myocardial ischemia and subsequent reperfusion (I/R) injury is a major factor contributing to high mortality and morbidity in ischemic heart disease. The ‘‘extrinsic’’ pathway is triggered by the binding of ligands, such as tumor necrosis factor and Fas, to their cognate receptors to induce receptor clustering and the formation of a death-inducing signaling complex (DISC) [6,7]. This complex recruits multiple procaspase-8 molecules via an adaptor molecule FADD (Fas-associated death domain protein), resulting in the activation of caspase-8 and downstream caspase-3 [6]. The ‘‘intrinsic’’ pathway utilizes mitochondria to produce cell death through opening of the mitochondrial permeability transition pore (mPTP), triggering the sudden release of cytochrome C and other proteins from the intermembrane space of mitochondria into the cytosol [8]. Released cytochrome C facilitates formation of the ‘‘apoptosome’’ complex, which results in caspase-9 activation and subsequent activation of caspase-3, the final effector of apoptosis [5,9]
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