Cardiomyocyte-specific overexpression of an active form of Rac predisposes the heart to increased myocardial stunning and ischemia-reperfusion injury

Abstract

The GTP-binding protein Rac regulates diverse cellular functions including activation of NADPH oxidase, a major source of superoxide production (O(2)(·-)). Rac1-mediated NADPH oxidase activation is increased after myocardial infarction (MI) and heart failure both in animals and humans; however, the impact of increased myocardial Rac on impending ischemia-reperfusion (I/R) is unknown. A novel transgenic mouse model with cardiac-specific overexpression of constitutively active mutant form of Zea maize Rac D (ZmRacD) gene has been reported with increased myocardial Rac-GTPase activity and O(2)(·-) generation. The goal of the present study was to determine signaling pathways related to increased myocardial ZmRacD and to what extent hearts with increased ZmRacD proteins are susceptible to I/R injury. The effect of myocardial I/R was examined in young adult wild-type (WT) and ZmRacD transgenic (TG) mice. In vitro reversible myocardial I/R for postischemic cardiac function and in vivo regional myocardial I/R for MI were performed. Following 20-min global ischemia and 45-min reperfusion, postischemic cardiac contractile function and heart rate were significantly reduced in TG hearts compared with WT hearts. Importantly, acute regional myocardial I/R (30-min ischemia and 24-h reperfusion) caused significantly larger MI in TG mice compared with WT mice. Western blot analysis of cardiac homogenates revealed that increased myocardial ZmRacD gene expression is associated with concomitant increased levels of NADPH oxidase subunit gp91(phox), O(2)(·-), and P(21)-activated kinase. Thus these findings provide direct evidence that increased levels of active myocardial Rac renders the heart susceptible to increased postischemic contractile dysfunction and MI following acute I/R.

Fig. 1.

Verification of ZmRacD gene and protein expression, and myocardial O₂^·− generation in wild-type (WT) and ZmRacD transgenic (TG) mice. A: PCR analysis with tail sample from offspring of TG breeders. Genomic DNA with specific primer for the transgene shows distinct band of ZmRacD transgene in the TG mice only, but not in the WT mice. (+), positive control; (−), negative control. B: representative Western blots and densitometric ratios for total myocardial Rac proteins in WT and TG mice where WT was assigned as 100%. Values are means ± SE; n = 6/group. *P < 0.05 vs. WT. C: representative images of myocardial O₂^·− generation with DHE in frozen sections and bar graphs for mean fluorescence intensity in arbitrary units. Total intensity in each heart was normalized where WT was assigned as 100%. Values are means ± SE; n = 4–6/group. ***P < 0.001 vs. WT. Magnification, 40×.

Fig. 2.

Postischemic myocardial function in isolated hearts subjected to 20-min global ischemia and 45-min reperfusion. Time course for the recovery of postischemic left ventricular (LV) systolic pressure (LVSP), LV end-diastolic pressure (LVEDP), LV developed pressure (LVDP), and cardiac contractile index as rate-pressure product (RPP) in WT and TG hearts. LVSP (A), LVDP (C), and RPP (D) are expressed as percentage of preischemic (PI) value, and LVEDP (B) is expressed as mmHg. IS, 20-min ischemia. Values are means ± SE. The differences between WT and TG hearts are highly significant with *P < 0.05. n = 6/group.

Fig. 3.

Measurements of infarct size in hearts subjected to 30-min in vivo regional myocardial ischemia and 24-h reperfusion. A: blue dye was infused to visualize the nonrisk area and TTC-staining was performed to visualize the infarcted area (IA). B: percentages of LV area at risk (AAR) over LV (AAR/LV) and IA over the LV AAR (IA/AAR). Values are means ± SE. *P < 0.05 vs. WT. n = 5/group.

Fig. 4.

Quantification of myocardial proteins in WT and TG hearts. Representative Western blots and densitometric ratios for gp91^phox (A) and PAK1 (B) in total cardiac homogenates of WT and TG mice. Each blot was normalized to internal control (GAPDH), and then expressed as percentage of WT where WT was assigned as 100%. Values are means ± SE. *P < 0.05, **P < 0.01 vs. WT. n = 6/group.

Fig. 5.

Cascade of events by which ischemia-perfusion (I/R)-induced stimulation of Rac-mediated signaling pathways may influence postischemic myocardial contractile function and infarction. E-C coupling, excitation-contraction coupling.