N-acetylcysteine with low-dose estrogen reduces cardiac ischemia-reperfusion injury

in Journal of Endocrinology
Correspondence should be addressed to N Chattipakorn: nchattip@gmail.com
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Myocardial damage and mitochondrial dysfunction caused by cardiac ischemia-reperfusion (I/R) injury are intensified by endogenous estrogen deprivation. Although N-acetylcysteine (NAC) exerted cardioprotective effects, its benefits when used in combination with hormone therapy are unknown. We tested the hypothesis that a combination of NAC with low-dose estrogen improves cardiometabolic function and protects cardiac mitochondria against I/R injury, to a similar extent to regular-dose estrogen treatment, in estrogen-deprived rats. Female Wistar rats had a bilateral ovariectomy (OVX) or sham operation. Twelve weeks after the operation, OVX rats were treated with regular-dose estrogen (E; 50 µg/kg/day), low-dose estrogen (e; 25 µg/kg/day), NAC (N; 100 mg/kg/day) or combined low-dose estradiol with NAC (eN) for 4 weeks (n = 6/group). Metabolic parameters, echocardiography, heart rate variability and then cardiac I/R protocol involving 30-min coronary artery ligation, followed by 120-min reperfusion, were performed. OVX rats had increased body weight, visceral fat, fasting plasma glucose, HOMA-IR index, triglycerides, cholesterol and LDL levels (P < 0.05 vs sham). Only OVX-E and OVX-eN had a similarly improved HOMA-IR index. LVEF was increased in all treatment groups, but HRV was restored only by OVX-E and OVX-eN. After I/R, myocardial infarct size was decreased in both OVX-E and OVX-eN groups. OVX-E and OVX-eN rats similarly had a reduced mitochondrial ROS level and increased mitochondrial membrane potential in the ischemic myocardium. In conclusion, combined NAC with low-dose estrogen and regular-dose estrogen therapy similarly improve cardiometabolic function, prevent cardiac mitochondrial dysfunction and reduces the infarct size in estrogen-deprived rats with cardiac I/R injury.

 

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    Effect of estrogen deprivation and the treatments on cardiac systolic and diastolic function. (A) M-mode of echocardiography in parasternal short axis view; (B) left ventricular ejection fraction (%LVEF); (C) left ventricular internal diameter in systole (LVIDs); (D) left ventricular posterior wall thickness in systole (LVPWs); (E) pulsed-wave Doppler shows mitral inflow in apical four-chamber view; (F) mitral valve E/A ratio and (G) mitral valve deceleration time in sham rats (sham), ovariectomized (OVX) rats and OVX rats treated with normal-dose estrogen (OVX-E), low-dose estrogen (OVX-e), NAC (OVX-N) or combined low-dose estrogen with NAC (OVX-eN). Values are mean ± s.e.m. for six rats per group. *P < 0.05 vs sham and P < 0.05 vs OVX. A full colour version of this figure is available at https://doi.org/10.1530/JOE-19-0108.

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    Effect of estrogen deprivation and the treatments on heart rate variability. (A) LF/HF ratio and (B) resting heart rate in sham rats (sham), ovariectomized (OVX) rats and OVX rats treated with normal-dose estrogen (OVX-E), low-dose estrogen (OVX-e), NAC (OVX-N) or combined low-dose estrogen with NAC (OVX-eN). LF is low-frequency power (LF; 0.15–1.5 Hz, in ms2), and HF is high-frequency power (HF; 1.5–5 Hz, in ms2). Values are mean ± s.e.m. for six rats per group. *P < 0.05 vs sham, and P < 0.05 vs OVX.

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    The changes of cardiac performance during cardiac I/R protocol. Data from P-V loop study demonstrate the changes of cardiac parameters during baseline (BL), ischemia (I) and reperfusion (R) of cardiac I/R procedure. (A) Stroke volume per body weight (SV/BW); (B) left ventricular ejection fraction (%LVEF); (C) left ventricular end-systolic pressure (LVESP); (D) left ventricular end-diastolic pressure (LVEDP); (E and F) maximal and minimal slope of the diastolic pressure decrement (dP/dtMax and dP/dtMin). Values are mean ± s.e.m. for 5–6 rats per group. *P < 0.05 vs BL, P < 0.05 vs sham and P < 0.05 vs OVX. Sham, sham rats; OVX, ovariectomized rats; OVX-E, OVX rats treated with normal-dose estrogen; OVX-e, OVX rats treated with low-dose estrogen; OVX-N, OVX rats treated with NAC; OVX-eN, OVX rats treated with combined low-dose estrogen with NAC. A full colour version of this figure is available at https://doi.org/10.1530/JOE-19-0108.

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    Cardiac arrhythmia score, time to 1st VT/VF, mortality rate and cardiac p-Cx43 expression, after treatment with E, e, N or eN for 4 weeks. (A) Arrhythmia score during ischemia and reperfusion periods; (B) time to 1st VT/VF following ischemia induction; (C) representative western blot bands and the level of p-Cx43 Ser368 per total Cx43 presented in level of ischemic/remote area; (D) mortality rate during cardiac I/R condition presented in percentage from total rats in the group. Values are mean ± s.e.m. for six rats per group. *P < 0.05 vs sham and P < 0.05 vs OVX. Sham, Sham rats; OVX, ovariectomized rats; OVX-E, OVX rats treated with normal-dose estrogen; OVX-e, OVX rats treated with low-dose estrogen; OVX-N, OVX rats treated with NAC; OVX-eN, OVX rats treated with combined low-dose estrogen with NAC.

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    Levels of infarct size and cardiac expression of apoptotic and anti-apoptotic proteins, after treatment with E, e, N or eN for 4 weeks. (A) Percentage of myocardial infarct size per area at risk (AAR) and the representatives of the infarcted myocardium. (B) Level of malondialdehyde (MDA) in ischemic tissue (I) normalized by the MDA level of remote (R) tissues. (C) Expression levels of the anti-apoptotic protein Bcl-2 and (D) the apoptotic protein Bax from ischemic area normalized by the level of those in remote area (I/R ratio). Values are mean ± s.e.m. for six rats per group. *P < 0.05 vs sham and P < 0.05 vs OVX. Sham, Sham rats; OVX, ovariectomized rats; OVX-E, OVX rats treated with normal-dose estrogen; OVX-e, OVX rats treated with low-dose estrogen; OVX-N, OVX rats treated with NAC; OVX-eN, OVX rats treated with combined low-dose estrogen with NAC. A full colour version of this figure is available at https://doi.org/10.1530/JOE-19-0108.

  • View in gallery

    Cardiac mitochondrial function in estrogen-deprived rats after treatment with E, e, N or eN for 4 weeks. (A) Mitochondrial ROS level; (B) mitochondrial membrane potential change (∆Ψ) and (C) absorbance intensity of mitochondria from ischemic myocardium normalized by those from the remote area. (D) Transmission electron microscopy illustrates cardiac mitochondria morphology from ischemic myocardium of rats in each group. Values are mean ± s.e.m. for six rats per group. *P < 0.05 vs sham and P < 0.05 vs OVX. Sham, Sham rats; OVX, ovariectomized rats; OVX-E, OVX rats treated with normal-dose estrogen; OVX-e, OVX rats treated with low-dose estrogen; OVX-N, OVX rats treated with NAC; OVX-eN, OVX rats treated with combined low-dose estrogen with NAC.

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