Cardiac contractile dysfunction during acute hyperglycemia due to impairment of SERCA by polyol pathway-mediated oxidative stress

Abstract

Hyperglycemia is an indication of poor outcome for heart attack patients, even for nondiabetic patients with stress-induced hyperglycemia. Previous studies showed that inhibition of aldose reductase, the first and rate-limiting enzyme of the polyol pathway, attenuated contractile dysfunction in diabetic animals, but the mechanism is unclear. We therefore wanted to find out whether the polyol pathway also contributes to acute hyperglycemia-induced cardiac contractile dysfunction, and determine the mechanism involved. Rat hearts were isolated and retrogradely perfused with Krebs buffer containing either normal or high concentrations of glucose for 2 h. Short exposure to high-glucose medium led to contractile dysfunction as indicated by decreased -dP/dt(max), as well as elevation in left ventricular end-diastolic pressure. Cardiomyocytes incubated in high-glucose medium showed abnormal Ca2+ signaling, most likely because of decreased activity of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) inactivated by oxidative stress. Inhibition of aldose reductase or sorbitol dehydrogenase, the second enzyme in the polyol pathway, ameliorated contractile dysfunction, attenuated oxidative stress, and normalized Ca2+ signaling and SERCA activity caused by high glucose, indicating that the polyol pathway is the major contributor to acute hyperglycemia-induced oxidative stress leading to the inactivation of SERCA and contractile dysfunction.

Fig. 1.

Changes in levels of sorbitol (A) and fructose (B) in hearts perfused with normal [N; control (Ctrl)] or high glucose (HG) medium. ARI, aldose reductase inhibitor; SDI, sorbitol dehydrogenase (SDH) inhibitor. Data are means ± SD (n = 6). *P < 0.05, ***P < 0.001, vs. corresponding groups.

Fig. 2.

A: lactate-to-pyruvate (L/P) ratio indicating NADH-to-NAD⁺ ratio in hearts perfused with normal (Ctrl), HG medium, HG + ARI, HG + SDI, and HG + niacin. Data are means ± SD (n = 13). ***P < 0.001 vs. Ctrl group. B: effect of acute hyperglycemia on GSH level in normal, ARI-treated, and SDI-treated perfused rat hearts. Data are means ± SD (n = 9). **P < 0.01 vs. HG group; #P < 0.05 vs. SDI group.

Fig. 3.

A: left ventricular superoxide content in hearts perfused with normal (Ctrl), HG medium, HG + ARI, HG + SDI, HG + niacin, and HG + Tiron. RLU, relative light units. Data are means ± SD (n = 5). *P < 0.05, **P < 0.01, ***P < 0.001 vs. Ctrl group; ##P < 0.01 vs. HG + Tiron group. The level of nitrotyrosine was assessed by the immunostaining in left ventricles from isolated perfused rat hearts (n = 4). Representative staining (n = 6) was taken from normal (B), high glucose (C), mannitol (D), ARI-treated (E), SDI-treated (F), and niacin-treated (G) groups. Scale bar, 50 μm.

Fig. 4.

Effect of acute hyperglycemia on time to peak (TP; A), decay time (DT; B), peak amplitude (PA; C), and resting level (D) of electrically induced [Ca²⁺]_i (E[Ca²⁺]_i) transients of normal, ARI-treated, SDI-treated, and NAD⁺-treated cardiomyocytes. Data are means ± SD (n = 6 cells from 5 rats in each group). ***P < 0.001 vs. HG group.

Fig. 5.

A: effect of acute hyperglycemia on the activities of ryanodine (RyR) and sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA) in normal, ARI-treated, and SDI-treated perfused rat hearts. Data are means ± SD (n = 4 vesicles from 5 rats in each group). *P < 0.05 vs. HG group. B: effect of acute hyperglycemia on the protein expression of RyR and SERCA in normal, ARI-treated, and SDI-treated perfused rat hearts. Data are means ± SD (n = 4 vesicles from 5 rats in each group).

Fig. 6.

A: effect of acute hyperglycemia on the level of tyrosine nitration on SERCA in sham-operated (SH), ARI-treated, and SDI-treated hearts isolated from rats. For detection of nitrotyrosine in SERCA protein, an immunoprecipitate (IP) was obtained using anti-SERCA2 antibody and then immunoblotted (IB) with anti-SERCA2 antibody or anti-nitrotyrosine antibody. Data are means ± SD (n = 3). ***P < 0.001, **P < 0.01 vs. HG group. B: effect of acute hyperglycemia on the level of GSH on SERCA in SH, ARI-treated, and SDI-treated hearts isolated from rats. An IP was obtained using anti-SERCA2 antibody and then immunoblotted with anti-GSH antibody. Data are means ± SD (n = 3). *P < 0.05, **P < 0.01 vs. HG group; #P < 0.01 vs. SH group.

Fig. 7.

Effect of acute hyperglycemia on the level of SERCAC674-SO₃H, inactivated SERCA in normal, ARI-treated, and SDI-treated hearts isolated from rats (n = 3). Representative immunohistochemical staining was taken from normal (A), high glucose (B), ARI-treated (C), and SDI-treated (D) groups. Scale bar, 50 μm. E: quantitative analysis of SERCAC674-SO₃H staining in normal, ARI-treated, and SDI-treated hearts isolated from rats (n = 3). Data are means ± SD (n = 3). ***P < 0.001 vs. HG group.