BACKGROUNDTransient receptor potential vanilloid 4 (TRPV4), a calcium (Ca2+) permeable channel, is upregulated during myocardial ischemia reperfusion (IR). Although TRPV4 inhibition shows cardioprotective effects, its impact on arrhythmogenesis remains unclear.OBJECTIVEThis study aimed to evaluate the anti-arrhythmic effects of TRPV4 inhibition, using the TRPV4 antagonist GSK2193874 (GSK219) and TRPV4-knockout (TRPV4-/-) mice, following IR.METHODSurface electrocardiogram (ECG) and optical mapping recordings were performed during 15 minutes of global ischemia and 10 minutes of reperfusion in Langendorff-perfused mouse hearts. Ca2+ sparks were detected using confocal microscopy, and protein expression was analyzed by Western blot.RESULTSGSK219 or TRPV4 deletion significantly decreased the incidence and duration of ventricular tachycardia (VT) during reperfusion. TRPV4 inhibition shortened Ca2+ transient (CaT) recovery, suppressed CaT alternans, and decreased Ca2+ leak without affecting IR-induced the prolongation of action potential duration (APD) and APD alternations. Activation of TRPV4 by GSK101790A (GSK101) increased arrhythmias susceptibility and Ca2+ leak. Moreover, GSK101 prolonged the CaT recovery and promoted CaT alternans, which were greatly avoided by pretreatment with CaMKII inhibitor. Interestingly, IR or GSK101 markedly increased the phosphorylation of CaMKII, ryanodine receptors, and phospholamban, which was significay blocked by TRPV4 inhibition.CONCLUSIONTRPV4 inhibition exerts antiarrhythmic effects post-IR by modulating CaMKII-dependent Ca2+ handling abnormalities, reducing CaT alternans and Ca2+ leak, without affecting APD.