50% of mortalities attributable to cardiac causes are accounted for by ventricular tachyarrhythmias occurring either independently or as a result of other underlying heart diseases. Yet, despite the enormity of this problem, the identification of pathophysiological mechanisms contributing to ventricular tachyarrhythmias as well as the development of safe and effective antiarrhythmic agents remain elusive1,2. The aim of the present study is to determine the role and underlying mechanisms for P21 activated kinase 1(Pak1) in ventricular arrhythmogenesis under stress conditions in mice carrying a ventricular cardiomyocytes specific deletion of Pak1 mice (Pak1cko) and control mice (Pak1f/f). 10-12 week old mice were subjected to either acute treatment of isoprenaline (ISO, 5-50nM) or chronic treatment with ISO at concentration 10mg/kg/day for 14-days delivered by mini-osmotic pump. The in vivo, ex vivo electrocardiography (ECG) and monophasic action potential (MAP) were recorded by Chart5 program. At both in vivo, ex vivo conditions, Pak1cko mice, but not control PAK1f/f mice, showed high occurrences (4/12 and 8/12 respectively) of ventricular arrhythmias induced by programmed electrical stimulation (PES) protocols, particularly at high stimulation frequencies. At cellular level, action potential (AP) alternans, delayed after-depolarization typed APs and spontaneous APs were frequently observed from PAK1cko myocytes, but not from control Pak1f/f myocytes. The involvement of Pak1 in Ca2+ handling was further investigated at cellular level. Dysfunctional Ca2+ handling was observed in Pak1cko myocytes in contrast to control Pak1f/f myocytes, in particular, under ISO stress conditions, these include alterations of SR content, the kinetics of NCX, SERCA, SR refilling and the diastolic cytosolic Ca2+. Molecular characterisation demonstrated that SERCA2 protein and mRNA levels were increased in Pak1f/f hearts under ISO stress condition, however, such effect was abrogated in Pak1cko hearts. Further in vitro experiments on primary neonatal cardiomyocytes demonstrated that SERCA2 expression is regulated by Pak1 through activation of serum response factor, a transcription factor that has previously been linked to cardiac hypertrophy and development3. In conclusion, Pak1 plays a crucial role in regulating cardiac electrical function and Ca2+ handling in preventing stress associated ventricular arrhythmogenesis.