Polyol pathway impairs the function of SERCA and RyR in ischemic-reperfused rat hearts by increasing oxidative modifications of these proteins

Abstract

A number of studies have shown that the polyol pathway, consisting of aldose reductase (AR) and sorbitol dehydrogenase (SDH), contributes to ischemia-reperfusion (I/R)-induced myocardial infarction due to depletion of ATP. In this report we show that the polyol pathway in I/R heart also contributes to the impairment of sacro/endoplasmic reticulum Ca(2+)-ATPase (SERCA) and ryanodine receptor (RyR), two key players in Ca(2+) signaling that regulate cardiac contraction. Rat hearts were isolated and retrogradely perfused with either Krebs' buffer containing 1 microM AR inhibitor, zopolrestat, or 200 nM SDH inhibitor, CP-170,711, and challenged by 30 min of regional ischemia and 45 min of reperfusion. We found that post-ischemic contractile function of the isolated perfused hearts was improved by pharmacological inhibition of the polyol pathway. I/R-induced contractile dysfunction is most likely due to impairment in Ca(2+) signaling and the activities of SERCA and RyR. All these abnormalities were significantly ameliorated by treatment with ARI or SDI. We showed that the polyol pathway activities increase the level of peroxynitrite, which enhances the tyrosine nitration of SERCA and irreversibly modifies it to form SERCAC674-SO(3)H. This leads to reduced level of S-glutathiolated SERCA, contributing to its inactivation. The polyol pathway activities also deplete the level of GSH, leading to decreased active RyR, the S-glutathiolated RyR. Thus, in I/R heart, inhibition of polyol pathway improved the function of SERCA and RyR by protecting them from irreversible oxidation.

Figure 1

A. Changes in levels of sorbitol in the isolated hearts challenged with 30 minutes of ischemia (Ischemia) and followed by 45 minutes of reperfusion (IR). Within each group some were injected with saline (Saline), some treated with ARI (ARI) and some treated with SDI (SDI). Data are means ± SE (n=5). ***p<0.001 & **p<0.01, compared with SDH-inhibited IR group. B. Changes in levels of fructose the isolated hearts challenged with 30 minutes of ischemia (Ischemia) and followed by 45 minutes of reperfusion (IR). Within each group, some were injected with saline (saline), some treated with ARI (ARI) and some treated with SDI (SDI). Data are means ± SE (n=4). ***p<0.001 & **p<0.01, compared with control group after reperfusion.

Figure 2

Effect of ischemic insults on (A) LVSP, (B) LVEDP, (C & D) ±dP/dt_max and (E) LVDP in control (vehicle group), ARI-treated and SDI-treated hearts from rats. All these parameters were obtained from isolated perfused hearts in preischemic conditions (stabilization), during ischemia and then during reperfusion. Data are means ± SE (n=5). ***p<0.001, **p<0.01 & *p<0.05, compared with corresponding ARI group; ^###p<0.001, ^##p<0.01 & ^#p<0.05, compared with corresponding SDI group. (F) LDH release from SH (sham-operated), vehicle, ARI-treated and SDI-treated hearts from rats during post-ischemic reperfusion (IR). Data are means ± SE (n=4). ^###p<0.001, ^##p<0.01, compared with SH group; *p<0.05, compared with vehicle group with I/R.

Figure 3

A. Effect of ischemic insults on GSH level in sham-operated (SH), ARI-treated and SDI-treated hearts isolated from rats. GSH level was measured in the heart homogenates after I/R. Data are means ± SE (n=8). **p<0.01 & *p<0.05, compared with I/R group. B. Effect of ischemic insults on superoxide level in SH, ARI-treated and SDI-treated hearts isolated from rats. Superoxide level was measured by lucigenin-elicited chemiluminescence after I/R. Data are means ± SE (n=6). ***p<0.001, **p<0.01 & *p<0.05, compared with normal group; ^$$$p<0.001, ^$$p<0.01 & ^$p<0.05, compared with I/R+Tiron group; ^###p<0.001, ^##p<0.01 & ^#p<0.05, compared with I/R group.

Figure 4

Effect of ischemic insult on nitrotyrosine level in left ventricles from normal, ARI-treated and SDI-treated rat hearts. Representative staining with sham operation was taken from (A) normal, (B) ARI-treated and (C) SDI-treated group at 40X magnification; Representative staining challenged by I/R was taken from (D) normal, (E) ARI-treated & (F) SDI-treated groups (n=6).

Figure 5

Changes in the (A) peak amplitude, (B) basal cytosolic Ca²⁺ level, (C) time-to-peak and (D) decay time of E[Ca²⁺]_i transients of normal, ARI-treated and SDI-treated cardiomyocytes subjected to I/R. Data are means ± SE (n=6 cells from 5 rats in each group). ***p<0.001, **p<0.01 & *p<0.05, compared with corresponding normal group. (E) The representative tracings of electrically stimulated intracellular [Ca²⁺] transients of normal, ARI-treated & SDI-treated cardiomyocytes subjected to I/R.

Figure 6

Activities of cardiac RyR and SERCA in the normal, ARI-treated and SDI-treated groups subjected to ischemia (A & B) and reperfusion (C & D). Data are means ± SE (n=5). ***p<0.001, **p<0.01 & *p<0.05, compared with corresponding I/R group.

Figure 7

A. Effect of ischemic insult on the level of tyrosine nitration on SERCA2 in sham-operated (SH), ARI & SDI-treated hearts isolated from rats. For detection of nitrotyrosine in SERCA2 protein, (A, upper) an immunoprecipitate (IP) was obtained using anti-SERCA2 antibody and then immunoblotted with anti-SERCA2 antibody. (A, lower) An IP was obtained using anti-SERCA2 antibody and then immunoblotted with anti-nitrotyrosine antibody. B. Quantitative analysis of nitrotyrosine by scanning densitometry. Data are means ± SE (n=3). ***p <0.001 & **p<0.01, compared with I/R group; ^#p<0.01, compared with SDI group. C. Effect of ischemic insult on the level of glutathiolation on SERCA2 in sham-operated (SH), ARI & SDI-treated hearts isolated from rats. (C, lower) An IP was obtained using anti-SERCA2 antibody and then immunoblotted with anti-GSH antibody. D. Quantitative analysis of GSH by scanning densitometry. Data are means ± SE (n=3). **p<0.01 & *p<0.05, compared with I/R group; ^#p<0.01, compared with SH group.

Figure 8

Immunohistochemical staining with anti-SERCA C674-SO₃H of left ventricles from post-ischemic reperfused rat hearts. Representative staining was taken from (A) normal, (B) I/R, (C) I/R+ARI, (D) I/R+SDI group and (E) non-specific IgG control.

Figure 9

A. Effect of ischemic insult on the level of glutathiolation on RyR in sham-operated (SH), ARI & SDI-treated hearts isolated from rats. For detection of GSH in RyR protein, (A, upper) an immunoprecipitate (IP) was obtained using anti-RyR antibody and then immunoblotted with anti-RyR antibody. (A, lower) An IP was obtained using anti-RyR antibody and then immunoblotted with anti-GSH antibody. B. Quantitative analysis of GSH by scanning densitometry. Data are means ± SE (n=3). **p<0.01 & *p<0.05, compared with I/R group; ^#p<0.01, compared with SDI group.