| Background and Aim:The incidence of malignant ventricular arrhythmia and sudden cardiac death increases in heart failure which impairs the quality of the patients’life and even threatens their lives. Recent studies have shown that the increased incidence of heart arrhythmia in heart failure is closely related to the dysfunction of repolarization reserve, in which rapidly activating delayed rectifier potassium current (IKr) and slowly activating delayed rectifier potassium current (IKr) play most important roles in mitigating excessive extension of action potential duration (APD). On one hand, via rabbit heart failure model established by abdominal aorta coarctation, we investigated not only the mRNA expression of KCNH2and KCNQ1in ventricular myocytes in HF, but also the relationship among action potential (AP),IKr channel and IKs channel, in order to research the mechanism of repolarization reserve change in rabbit heart failure model. On the other hand, to research the mechanisms of the interaction between the two important channels in repolarization reserve, we transfected KCNH2and KCNQ1into HEK293cells and investigated the expression of mRNA and protein of these two genes and the KCNQ1current changes. This study provides experimental and theoretical evidences for the mechanism of malignant ventricular arrhythmia and sudden cardiac death in heart failure.Methods:Twenty-four rabbits were divided randomly into sham operation (Sham) and heart failure (HF) groups, twelve in each. Abdominal aortic coarctation was performed in group HF. After surgery, each rabbit was raised for twelve weeks. And general physical conditions, signs and various indicators of heart function under cardiac ultrasound were evaluated. Then hearts were removed from alive rabbits, and ventricular myocytes were isolated by enzymatic method. Total cellular RNA was extracted, and RT-qPCR was performed to compare the differences of KCNH2and KCNQ1between HF and Sham groups in the level of mRNA. Action potential (AP),IKr and IKs were recorded by whole-cell patch clamp technique with or without drugs dofetilide (Dof) and293B. Action potential duration at50%repolarization (APD50), action potential duration at90%repolarization (APD90), action potential amplitude (APA) and resting membrane potential (RMP) were all compared in different conditions. According to the transient transfection of different genes, HEK293cells were divided into seven groups:KCNQ1(Ql), WT-KCNH2(WT), T618I-KCNH2(gain of function mutation, T618I), V535M-KCNH2(loss of function mutation, V535M), and co-transfection KCNQ1with WT-KCNH2(Q1+WT), T618I-KCNH2(Q1+T618I), and V535M-KCNH2(Q1+V535M) groups. After gene transfection, HEK293cells were incubated for48hours. Then total RNA and protein were extracted, detected by RT-qPCR and Western blot, respectively. KCNQ1current changes induced by different functional KCNH2were recording by whole-cell patch clamp technique.Results:(1) Compared with group Sham, mRNA expressions of KCNH2and KCNQ1in ventricular myocytes of the rabbits with HF were decreased (P<0.05).(2) APD50and APD90were prolonged significantly in ventricular myocytes in group HF (P<0.05). The prolongation of APD50and APD90in HF group with both Dof and293B were more significant than those of using only Dof or without any drug (P<0.05).(3) APA and RMP in groups Sham or HF, with or without Dof and293B, had no significant differences (P>0.05).(4) Compared with group Sham, the density of IKr current was reduced in group HF (P<0.05). These effects were aggravated when Dof was used (P<0.05).(5) In groups Sham and HF, the density of IKr tail current (IKr,tail) increased along with the increment of test potentials,IKr,tail in group HF was lower than that in group Sham at the same test potential. And IKr,tail was inhibited by using Dof in both groups.(6) Compared with group Sham, the density of IKs current was reduced in group HF (P<0.05). And the effect was significantly diminished when Dof was used (P<0.05), but still lower than Sham. (7) The relative mRNA expression of KCNQ1in groups with co-transfection of different functional KCNH2was significantly increased than that in group Q1(P<0.05). Among three groups with co-transfection of KCNQ1and KCNH2, the relative mRNA expression of KCNQ1was highest in group Q1+V535M and lowest in group Q1+T618I. The relative mRNA expression of different functional KCNH2was increased with co-transfection of KCNQ1.(8) Compared with Q1, the relative protein expression of KCNQ1was increased when KCNQ1was co-transfected with different functional KCNH2(P<0.05). Among co-transfection groups, the relative protein expression of KCNQ1was higher in Q1+V535M group and lower in Q1+T618I than Q1+WT. Co-transfection of KCNQ1increased the relative protein expression of different functional KCNH2.(9) With co-transfenction of WT-KCNH2increased the KCNQ1current densities from152.8±7.5pA/pF to209.3±9.1pA/pF (P<0.05). Compared with group Q1, the KCNQ1current density in group Q1+T618I was slightly increased to169.2±6.7pA/pF (P<0.05), and the KCNQ1current density in group Q1+V535M was much more increased and up to253.5±13.6pA/pF (P<0.05).Conclusion:(1) Heart failure reduced the relative mRNA expression of KCNH2and KCNQ1.(2) APD was prolonged and the densities of IKr and IKs current were lower in ventricular myocytes of rabbits with heart failure, which impaired the repolarization reserve. The manifestations were getting worse by inhibiting IKr and IKs.(3) KCNQ1and KCNH2in three functional forms increased the relative mRNA expression of each other.(4) Analogously, different functional KCNH2and KCNQ1also increased the relative protein expression of each other.(5) KCNH2significantly increased KCNQ1current density, and the KCNQ1current density in group Q1+V535M was increased most among three co-transfection groups. |
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