Role of uncoupling protein 3 in ischemia-reperfusion injury, arrhythmias, and preconditioning

Abstract

Overexpression of mitochondrial uncoupling proteins (UCPs) attenuates ischemia-reperfusion (I/R) injury in cultured cardiomyocytes. However, it is not known whether UCPs play an essential role in cardioprotection in the intact heart. This study evaluated the cardioprotective efficacy of UCPs against I/R injury and characterized the mechanism of UCP-mediated protection in addition to the role of UCPs in ischemic preconditioning (IPC). Cardiac UCP3 knockout (UCP3(-/-)) and wild-type (WT) mice hearts were subjected to ex vivo and in vivo models of I/R injury and IPC. Isolated UCP3(-/-) mouse hearts were retrogradely perfused and found to have poorer recovery of left ventricular function compared with WT hearts under I/R conditions. In vivo occlusion of the left coronary artery resulted in twofold larger infarcts in UCP3(-/-) mice compared with WT mice. Moreover, the incidence of in vivo I/R arrhythmias was higher in UCP3(-/-) mice. Myocardial energetics were significantly impaired with I/R, as reflected by a decreased ATP content and an increase in the AMP-to-ATP ratio. UCP3(-/-) hearts generated more reactive oxygen species (ROS) than WT hearts during I/R. Pretreatment of UCP3(-/-) hearts with the pharmacological uncoupling agent carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone improved postischemic functional recovery. Also the protective efficacy of IPC was abolished in UCP3(-/-) mice. We conclude that UCP3 plays a critical role in cardioprotection against I/R injury and the IPC phenomenon. There is increased myocardial vulnerability to I/R injury in hearts lacking UCP3. The mechanisms of UCP3-mediated cardioprotection include regulation of myocardial energetics and ROS generation by UCP3 during I/R.

Keywords: energetics; mitochondria.

Fig. 1.

Effects of ischemia and reperfusion (I/R) on left ventricular function. The overexpression of uncoupling protein (UCP) 2 and UCP3 (hUCP2/3) in hearts results in better postischemic recovery of function compared with wild-type (WT) hearts (A). Left ventricular contractile functional recovery was similar in WT and UCP2^−/− hearts following I/R (A). However, the recovery of function was significantly lower in UCP3^−/− hearts compared with WT hearts (A). Representative M-mode echocardiograms from WT and UCP3^−/− mice and echocardiographic analysis of left ventricular size and function, which reveals comparable cardiac function and structure in UCP3^−/− and WT hearts in vivo (B). Myocardial architecture on hematoxylin- and eosin-stained sections was grossly similar in WT and UCP3^−/− hearts (C). RPP, rate pressure product; LVEDD, LV end diastolic dimension; LVESD, LV end systolic dimension; FS, fractional shortening; LVPW, LV posterior wall. *P < 0.05 compared with WT.

Fig. 2.

In vivo myocardial infarction following left coronary artery (LCA) ligation in WT and UCP3^−/− animals. ST-segment elevation, indicative of myocardial infarction, was observed during 20 min of ischemia in WT and UCP3^−/− mice, which improved during reperfusion (A). Representative cross sections of WT and UCP3^−/− hearts stained with triphenyltetrazolium chloride and Evan's blue dye depict infarct area (IA) and area at risk (AAR), respectively. UCP3^−/− hearts had greater IA than WT hearts following I/R (B), whereas the AAR was comparable in the two groups (C). Thus, the IA to AAR was higher in UCP3^−/− hearts (D). Also, UCP3^−/− hearts released more troponin I following ischemia and I/R injury (E) as a result of further myocardial necrosis. *P < 0.05 compared with WT.

Fig. 3.

There was a strong trend toward a greater incidence of I/R arrhythmias in UCP3^−/− mice compared with WT mice (P = 0.0689; A). Various types of arrhythmia, including atrioventricular block, premature ventricular contractions, couplets, triplets, nonsustained ventricular tachycardia, and idioventricular rhythm, were observed during I/R injury (A). The heart rate response to I/R stress was blunted in UCP3^−/− mice compared with WT mice (B). Thus, both impulse conduction and formation are impaired in UCP3^−/− hearts. *P < 0.05 compared with baseline.

Fig. 4.

I/R injury causes more severe disruption in myocardial energetics and greater reactive oxygen species (ROS) generation in UCP3^−/− hearts. The myocardial content of ATP in I/R injury was significantly reduced in both WT and UCP3^−/− hearts, whereas the decline in ATP production was more severe in UCP3^−/− hearts than in WT hearts (A). Consequently, metabolic stress, as reflected by the AMP-to-ATP ratio, was significantly higher in UCP3^−/− hearts than in WT hearts (B). Following 20 min of ischemia, UCP3^−/− hearts produced more ROS in the myocardium as shown by higher lucigenin-derived chemiluminescence (LDCL; C). RLU, relative light units. *P < 0.05 compared with WT. †P < 0.05 compared with control.

Fig. 5.

Treatment with the pharmacological uncoupler carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP; 100 nM) before I/R improves left ventricle (LV) functional recovery in UCP3^−/− hearts compared with untreated hearts. *P < 0.05 compared with 0 FCCP.

Fig. 6.

The role of UCP3 in ischemic preconditioning (IPC) was evaluated in the isolated working perfused heart subjected to severe ischemia with 3 cycles of 4 min of no-flow ischemia (Isc or I) followed with 4 min of reperfusion (Rep or R) before 30 min of ischemia and 30 min reperfusion (top). Postischemic contractile function was significantly improved by IPC in WT hearts, whereas there was no postischemic recovery of function with IPC in UCP3^−/− hearts (A). In addition, there were less troponin I release (B) and greater ATP content in WT hearts with IPC compared with UCP3^−/− hearts (C). Thus, IPC is abolished in UCP3^−/− hearts. *P < 0.01 compared with I/R and UCP3^−/−. †P < 0.05 compared with I/R and UCP3^−/−. ‡P < 0.05 compared with I/R.