ABSTRACT
The possibility of rats mesenchymal stem cells (MSCs) modified with murine hyperpolarization-activated cyclic nucleotide-gated 2 (mHCN2) gene as biological pacemakers in vitro was studied.The cultured MSCs were transfected with pIRES2-EGFP plasmid carrying enhanced green fluorescent protein (EGFP) gene and mHCN2 gene.The identification using restriction enzyme and sequencing indicated that the mHCN2 gene was inserted to the pIRES2-EGFP.Green fluorescence could be seen in MSCs after transfection for 24-48 h.The expression of mHCN2 mRNA and protein in the transfected cells was detected by RT-PCR and Western blot,and the quantity of mHCN2 mRNA and protein expression in transfected MSCs was 5.31 times and 7.55 times higher than that of the non-transfected MSCs respectively (P＜0.05,P＜0.05).IHCN2 was recorded by whole-cell patch clamp method.The effect of Cs+,a specific blocker of pacemaker current,was measured after perfusion by patch clamp.The results of inward current indicated that there was no inward current recording in non-transfected MSCs and a large voltage-dependent inward and Cs+-sensitive current activated on hyperpolarizations presented in the transfected MSCs.IHCN2 was fully activated around -140 mV with an activation threshold of-60 mV.The midpoint (V50) was -95.1±0.9 mV (n=9).The study demonstrates that mHCN2 mRNA and protein can be expressed and the currents of HCN2 channels can be detected in genetically modified MSCs.The gene-modified MSCs present a novel method for pacemaker genes into the heart or other electrical syncytia.
ABSTRACT
Summary: The aim of this study was to determine if the potassium aspartate and magnesium (PAM) prevent reperfusion-induced ventricular arrhythmias (RIVA) in ischcmia-reperfusion (IR) rabbit heart. Thirty rabbits were randomly divided into control, ischemia and PAM groups. Arterially-perfused rabbit left ventricular preparations were made, and transmural ECG as well as action potentials from both endocardium and epicardium were simultaneously recorded in the whole process of all experiments. In control group rabbit ventricular wedge preparations were continuously perfused with Tyrode's solution, and in ischemia group and PAM groups the perfusion of Tyrode's solution was stopped for 30 min. Then the ischemia group was reperfused with Tyrode's solution and the PAM group with Tyrode's solution containing 2.42 mg/L PAM, respectively. ECG, QT interval, transmural repolarization dispersion (TDR) and action potentials from epicardium and endocardium were simultaneously recorded, and the RIVA of the wedge preparation was observed. Compared with control group, TDR and incidence of RIVA were significantly increased in ischemia group (P<0.05). The incidence of RIVA in control, ischemia and PAM group was 0/10, 9/10 and 1/10, respectively. Compared with ischemia group, TDR and incidence of RIVA were significantly reduced in PAM group (P<0.05). Potassium aspartate and magnesium significantly reduce TDR and prevent ventricular arrhythmia in ischemic rabbit heart.