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Murine Isolated Heart Model of Myocardial Stunning Associated with Cardioplegic Arrest

The following protocol is of use to evaluate impaired cardiac function or myocardial stunning following moderate ischemic insults. The technique is useful ...

Brown University

We previously demonstrated that myocardial p38 mitogen-activated protein kinase (MAPK) and heat shock protein 27 (HSP27) are phosphorylated following cardioplegic arrest in patients undergoing cardiac surgery and correlate with reduced cardiac function. The following studies were performed to determine whether inhibition of p38 MAPK and/or overexpression of nonphosphorylatable HSP27 improves cardiac function following cardioplegic arrest. Langendorff-perfused isolated rat hearts were subjected to 2 h of intermittent cold cardioplegia followed by 30 min of reperfusion. Hearts were treated with (CP+SB) or without (CP) the p38 MAPK inhibitor SB-203580 (5 μM) supplied in the cardioplegia. Sham-treated hearts served as controls. In separate experiments, isolated rat ventricular myocytes infected with either green fluorescent protein (GFP) or a nonphosphorylatable HSP27 mutant (3A-HSP27) were subjected to 3 h of cold hypoxic cardioplegia and simulated reperfusion (CP) followed by video microscopy and length change measurements. Baseline parameters of cardiac function were similar between groups [left ventricular developed pressure (LVDP), 119 ± 4.9 mmHg; positive and negative first derivatives of LV pressure (± dP/dt), 3,139 ± 245 and 2, 314 ± 110 mmHg/s]. CP resulted in reduced cardiac function (LVDP, 72.2 ± 5.8 mmHg; ± dP/dt, 2,076 ± 231 and -1,317 ± 156 mmHg/s) compared with baseline. Treatment with 5 μM SB-203580 significantly improved CP-induced cardiac function (LVDP, 101.9 ± 0 mmHg; ± dP/dt, 2,836 ± 163 and -2,108 ± 120 mmHg/s; P = 0.03, 0.01, and 0.04, CP+SB vs. CP). Inhibition of p38 MAPK significantly lowered CP-induced p38 MAPK, HSP27, and αB-crystallin (cryAB) phosphorylation. In vitro CP decreased myocyte length changes from 10.3 ± 1.5% (GFP) to 5.7 ± 0.8% (GFP+CP). Infection with 3A-HSP27 completely rescued CP-induced decreased myocyte contraction (11.1 ± 1.0%). However, infection with 3A-HSP27 did not block the endogenous HSP27 response. We conclude that inhibition of p38 MAPK and subsequent HSP27 and cryAB phosphorylation and/or overexpression of nonphosphorylatable HSP27 significantly improves cardiac performance following cardioplegic arrest. Modulation of HSP27 phosphorylation may improve myocardial stunning following cardiac surgery.

p38 MAPK-dependent small HSP27 and αB-crystallin phosphorylation in regulation of myocardial function following cardioplegic arrest.

Cardioplegia and cardiopulmonary bypass (CP/CPB) subjects myocardium to complex injurious stimuli that can result in cardiomyocyte and vascular contractile abnormalities. Rottlerin, originally identified as a delta-protein kinase C inhibitor, has a number of known additional effects that may be beneficial in the setting of CP/CPB. We tested the hypothesis that rottlerin mitigates deleterious effects associated with CP/CPB.

Rottlerin increases cardiac contractile performance and coronary perfusion through BKCa++ channel activation after cold cardioplegic arrest in isolated hearts.

Age-associated decline in cardiovascular function is believed to occur from the deleterious effects of reactive oxygen species (ROS). However, failure of recent clinical trials using antioxidants in patients with cardiovascular disease, and the recent findings showing paradoxical role for NADPH oxidase-derived ROS in endothelial function challenge this long-held notion against ROS. Here, we examine the effects of endothelium-specific conditional increase in ROS on coronary endothelial function. We have generated a novel binary (Tet-ON/OFF) conditional transgenic mouse (Tet-Nox2:VE-Cad-tTA) that induces endothelial cell (EC)-specific overexpression of Nox2/gp91 (NADPH oxidase) and 1.8?0.42-fold increase in EC-ROS upon tetracycline withdrawal (Tet-OFF). We examined ROS effects on EC signaling and function. First, we demonstrate that endothelium-dependent coronary vasodilation was significantly improved in Tet-OFF Nox2 compared to Tet-ON (control) littermates. Using EC isolated from mouse heart, we show that endogenous ROS increased eNOS activation and nitric oxide (NO) synthesis through activation of the survival kinase AMPK. Coronary vasodilation in Tet-OFF Nox2 animals was CaMKK?-AMPK-dependent. Finally, we demonstrate that AMPK activation induced autophagy and thus, protected ECs from oxidant-induced cell death. Together, these findings suggest that increased ROS levels, often associated with cardiovascular conditions in advanced age, play a protective role in endothelial homeostasis by inducing AMPK-eNOS axis.

Oxidative stress improves coronary endothelial function through activation of the pro-survival kinase AMPK.

Activation of large conductance calcium-activated potassium (BKCa) channels is cardioprotective for ischemic injury and can enhance vasorelaxation. Rottlerin has recently been identified as a potent BKCa activator. We demonstrated that rottlerin improves cardiac function and increases coronary flow when used as a cardioplegia additive in rat and mouse models of cardioplegic arrest and reperfusion. In this study we examined the effectiveness and specificity of the putative BKCa activator rottlerin on vascular reactivity in response to specific contractile and dilatory agonists.

Rottlerin-induced BKCa channel activation impairs specific contractile responses and promotes vasodilation.

Mitochondrial BKCa channels are thought to provide protection during ischemic insults in the heart. Rottlerin (a.k.a mallotoxin) has been implicated as a potent BKCa activator. The purpose of this study was twofold: 1) investigate the efficacy of BKCa channel activation as a cardioprotective strategy during ischemic cardioplegic arrest and reperfusion (CP/R) and 2) assess the specificity of rottlerin for BKCa channels.

Brenda Cordeiro

Cardiothoracic Surgery Research Center

Brown University

Murine Isolated Heart Model of Myocardial Stunning Associated with Cardioplegic Arrest

Brenda Cordeiro

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Brown University

Murine Isolated Heart Model of Myocardial Stunning Associated with Cardioplegic Arrest

Cardiothoracic Surgery Research Center