Role Of Transcription Factor ISL-1 In Inducing Ventricular Myocytes To Pacing - Like Cells And Its Mechanism

The normal heartbeat originates within the sinoatrial node(SAN), which is a specialized structure containing less than 10000 pacemaker cells responsible for pulse generation. Once the SAN fails, the billions of working cardiomyocytes downstream of it will work dysfunctionally leading to lethal bradycardia. Biological pacing has emerged as a new therapy for bradycardia without certain inevitable shortcomings of electronic pacemakers. Pacemaker-like cells could be induced by transcription factor-mediated reprogramming. Transcription factor ISL-1 is required for SAN development as a key upstream regulator and plays a fundamental role in the maintaining of pacemaker function of SAN. Here, we explore the expression of ISL-1 in the adult mouse heart and investigate whether ISL-1 is able to reprogramme terminally differentiated working cardiomyocytes into pacemaker-like cells.The present study was divided into three parts. First, location of mouse SAN area was verified and pacemaker cells were isolated, cell morphology and whole-cell electrophysiology recordings were performed; second, by qPCR, western blot and immunofluorescence, ISL-1 expression were detected in mouse heart and compared among SAN, atrium and ventricle; finally, with the construction of ISL-1 adenovirues, ISL-1 was overexpressed in neonatal rat ventricular cardiomyocytes(NRCMs) and adult mouse ventricles to investigate the possibility of conversion to pacemaker-like cells.First, mouse SAN area was located by the borders of the crista terminalis, the interatrial septum, and the superior and inferior vena cavae. In isolated mouse SAN cells, spontaneous action potential could be detected. With hyperpolarizing voltage clamp pulses, the If current was recorded. These results served as the positive control in the following experiments. In the second part, we observed that ISL-1 was highly expressed in the SAN of adult mouse heart, while hardly no expression was detected in ventricle either in mRNA or protein level. Immunohistochemical staining for ISL1 demonstrated that ISL1 protein was expressed in SAN and mainly focused in nuclei. In the final part, ISL-1 adenoviruses were constructed as vetors. Within days of in vitro ISL-1 adenoviruses transduction, NRCMs increased in spontaneous electrical firing and had the morphological features characteristic of SAN cells. In vivo, focal ISL-1 adenovirus transfer in the mouse ventricle yielded frequent ventricular premature contraction(PVC) in ECG under anesthesia and atrioventricular dissociation with a faster ventricular rhythm was observed when given isoproterenol. Expression analysis revealed a switch from the working myocardial expression profile to that of the pacemaker myocardium.In conclusion, ISL-1 is mainly expressed in adult mouse SAN. Transcription factor ISL-1 reprograms terminally differentiated working cardiomyocytes and induces important pacemaker properties. The creation of ISL-1 induced pacemaker-like cells opens new prospects for biological pacemakers.