Cobalt-Protoporphyrin Improves Heart Function by Blunting Oxidative Stress and Restoring NO Synthase Equilibrium in an Animal Model of Experimental Diabetes

Abstract

Myocardial dysfunction and coronary macro/microvascular alterations are the hallmarks of diabetic cardiomyopathy and are ascribed to increased oxidative stress and altered nitric oxide synthase (NOS) activity. We hypothesize that pre-treatment by cobalt-protoporphyrin IX (CoPP) ameliorates both myocardial function and coronary circulation in streptozotocin (STZ)-induced diabetic rats. Isolated hearts from diabetic rats in Langendorff configuration displayed lower left ventricular function and higher coronary resistance (CR) compared to hearts from control animals. CoPP treatment of diabetic animals (0.3 mg/100 g body weight i.p., once a week for 3 weeks) significantly increased all the contractile/relaxation indexes (p < 0.01), while decreasing CR (p < 0.01). CoPP enhanced HO-1 protein levels and reduced oxidative stress in diabetic animals, as indicated by the significant (p < 0.05) decrease in heart % GSSG, [Formula: see text] and malondialdehyde (MDA) levels. CoPP increased adiponectin levels and phosphorylation of AKT and AMPK and reversed the eNOS/iNOS expression imbalance observed in the untreated diabetic heart. Furthermore, after CoPP treatment, a rise in malonyl-CoA as well as a decrease in acetyl-CoA was observed in diabetic hearts. In this experimental model of diabetic cardiomyopathy, CoPP treatment improved both cardiac function and coronary flow by blunting oxidative stress, restoring eNOS/iNOS expression balance and increasing HO-1 levels, thereby favoring improvement in both endothelial function and insulin sensitivity.

Keywords: AMPK; cardiac contractility; coronary microcirculation; diabetes; nitric oxide.

Figure 1

After 30 min of stabilization, dP/dt_max, and dP/dt_min, rate-pressure product (RPP), and levels of coronary resistance (CR) in control rats (C, n = 20), diabetic rats (D, n = 15), and diabetic rats treated for 3 weeks with CoPP (D + CoPP, both n = 16). Data are expressed as means ± SEM. *p < 0.05 vs. other groups; ^#p < 0.05 vs. CoPP-treated animals.

Figure 2

HO-1/HO-2 expression and HO activity in C, D, and D + CoPP rat hearts. Heart samples were subjected to Western blotting for the determination of HO-1/HO-2 protein. A representative blot is shown. Densitometric analysis of HO-1 vs. actin is shown and expressed as means ± SEM of three independent experiments. HO activity was determined by bilirubin formation (see Materials and Methods). *p < 0.05 vs. other groups.

Figure 3

Cardiac tissue levels of (A) % GSSG [GSSG/(GSH+GSSG) ×100], (B) ${\text{O}}_2^{-}$ and (C) malonyldialdehyde (MDA) measured in hearts from C, D, and D + CoPP rats. (D) Representative Western blot and densitometric analysis of 3-nitrotyrosine (3-NT) expression in cardiac tissue of C, D, and D+CoPP rats. Data are expressed as means ± SEM of three independent determinations *p < 0.05 vs. other groups.

Figure 4

Representative Western blot and densitometric analysis of eNOS, p-eNOS, iNOS, from C, D, and D+CoPP rat hearts. Quantitative densitometry evaluation of iNOS and p-eNOS/eNOS ratio from three independent experiments is shown. Data are expressed as means ± SEM of three independent determinations ^#p < 0.05 vs. C; *p < 0.05 vs. D.

Figure 5

Representative Western blot and densitometric analysis of cardiac expression of pAKt, Akt, pAMPK, AMPK, and adiponectin. Data are expressed as means ± SEM of three independent determinations of pAkt/Akt and pAMPK/AMPK ratios as well as of adiponectin/actin ratio. *p < 0.05 vs. other groups; ^#p < 0.01 vs. C.

Figure 6

Effect of CoPP on malonyl-CoA and acetyl-CoA in hearts isolated from C, D, and D + CoPP animals. Data are expressed as means ± SEM of three independent determinations ^#p < 0.01 D vs. other groups; *p < 0.05 vs. other groups

Figure 7

Diagrammatic representation of the major changes observed in diabetic heart after CoPP treatment as compared to untreated hearts. Black arrows represent results demonstrated in this paper and, blue arrows represent previously shown work and/or hypothesis drawn from the literature.