Cardioprotection And Mechanisms Of Minocycline During Acute Myocardial Ischemia

Objectives:1 To investigate the effect of high mobility group box 1 protein (HMGB1) on the cell viability and the apoptosis of neonatal rat ventricular myocytes.2 To investigate the role of HMGB1 in the cardioprotection of minocycline during myocardial ischemia and reperfusion (I/R) in rats..3 To investigate the effects of minocycline on the activation kinetics of L-type calcium current (ICa-L) in single ventricular myocyte.4 To investigate the effect and mechanism of minocycline on ischemia-induced ventricular arrhythmias in rats..Methods:1 Neonatal rat ventricular myocytes were prepared and then cultured with recombinant HMGB1. Cell viability and cell apoptosis wwere measured by using a CCK-8 and annexin V-FITC apoptosis detection kit, respectively.2 Anesthetized male rats were once treated with minocycline (45 mg/kg, i.p.) 1 h before ischemia, and then subjected to ischemia for 30 min followed by reperfusion for 4 h. The lactate dehydrogenase (LDH), creatine kinase (CK), malondialdehyde (MDA) and superoxide dismutase (SOD) were measured. Infarct size were measured by TTC and the myocardial tissue apoptosis was assessed by TUNNEL assay. HMGB1 expression was assessed by Western blot.3 The whole-cell patch-clamp technique was used to investigate the effects of Minocycline on ICa-L in acutely isolated ventricular myocytes of adult rat.4 Anesthetized male rats were once treated with minocycline (45 mg/kg, i.p.) 1 h before ischemia in both the absence and/or presence of LY294002 (0.3 mg/kg, i.v., a PI3K inhibitor) and 5-hydroxydecanoic acid [5-HD,10 mg/kg, i.v., a specific of mitochondrial ATP-sensitive potassium (KATp) channels inhibitor] which were once injected 10 min before ischemia. And then subjected to ischemia for 30 min. Ventricular arrhythmias were assessed.Results:1 The cell viability of myocardial cell cultured with HMGB1 was decreased in a dose-dependent manner. When the concentration of HMGB 1 was more than 100 ng/ml, the cell viability of myocardial cell was significantly decreased compared to that in the control group (p<0.05). When the concentration of HMGB1 was more than 1 ng/ml, HMGB1 could promote the apoptosis of neonatal myocytes in a dose-dependent manner.2 After 4 h reperfusion, minocycline could significantly decrease the infarct size, myocardium apoptosis and the levels of LDH and CK (all p<0.05). Minocycline could significantly inhibit the increase in the MDA level and the decrease in the SOD level (both p<0.05). Meanwhile, minocycline could also significantly inhibit the HMGB1 expression during myocardial I/R.3 Minocycline inhibited the current density of ICa-L, and the decreased rates of the peak ICa-L were 11%±5.86%,54.79%±6.32%,71.73%±5.29%at 10umol/L,50umol/L, 250umol/L group, respectively. The current-voltage curve was shifted upwards, and the steady-state curve was shifted to right by minocycline in the concentration of 50umol/L, 250umol/L, which also prolonged the recovery time from inactivation.4 During the 30-minute ischemia, minocycline reduced markedly the incidence of ventricular fibrillation (VF) (p<0.05); however, there was no significant difference in ventricular tachycardia (VT) (p>0.05). The duration of VT+VF, number of VT+VF episodes and severity of arrhythmias were all significantly reduced by minocycline compared to those in myocardial ischemia group (all p<0.05). Administration of LY294002 or 5-HD could abolish the effects of minocycline on VF, duration of VT+ VF, number of VT+VF episodes and severity of arrhythmias (all p<0.05).Conclusions:1 In vitro, HMGB1 could significantly decrease the cell viability and promote the apoptosis of neonatal myocytes in a dose-dependent manner.2 Minocycline could protect against myocardial ischemia and reperfusion injury and myocardial tissue apoptosis by inhibiting HMGB1 expression.3 Minocycline could inhibit Ica-L in a dose-dependent manner to prevent intercellular calcium overload, which may contribute to its cardioprotection effect.4 Minocycline could attenuate ischemia-induced ventricular arrhythmias in rats which may be involved in PI3K/Akt signaling pathway, mitochondrial KATP channels and L-type Ca2+channels.