Abstract Body: Introduction: Dysregulated cardiomyocyte (CM) intercalated disc (ICD) function is associated with arrhythmias and heart failure development. Zonula Occludens (ZO) 1 stabilizes the ICD, and its CM-specific loss causes cardiac conduction abnormalities, including high-grade AV block. However, ZO1 loss triggers compensatory ZO2 upregulation in ventricular CMs. We therefore hypothesize that the combined loss of CM ZO1 and ZO2 causes a destabilized ICD, ventricular dysfunction and remodeling. This study aims to evaluate cardiac structure and function at baseline and under hemodynamic stress to identify ZO-related pathways.

Methods: A tamoxifen-inducible MerCreMer model was used to generate a CM-specific ZO1 and ZO2 knockout (ZO1,2cKO). Cardiac assessment was performed through echocardiography, histology, and immunofluorescence for six months. ZO1,2cKO mice also underwent transverse aortic constriction (TAC) and further evaluated. Molecular studies, including western blot and qPCR, identified altered mechanisms.

Results: The 6-month baseline assessment of ZO1,2cKO (n=12-20) revealed a 15% reduction in ejection fraction (EF) (±2.4%, p<0.0001), a 1mm (±0.23, p<0.0001) decrease in systolic relative wall thickness and a concurrent 10uL (±1.7, p<0.0001) increase in left ventricular volume. This was supported by a 11.5% (±4%, p=0.0337) increase in fibrosis, eccentric hypertrophy and a 30-fold upregulation of Nppa indicating cardiomyocyte stress. Confocal evaluation of ICD structure following ZO1,2cKO showed destabilized expression of ICD components including connexin 43, sodium channel protein type 5 subunit alpha. Following baseline characterization, the ZO1,2cKO mice were subjected to a 4-week hemodynamic challenge via TAC and results suggest an accelerated more prominent deterioration of function exhibiting a 20% reduction in EF with concomitant remodeling. Further bulk RNA sequencing will reveal key dysregulated pathways associated with ZO1,2cKO.

In conclusion, our study highlights an underrecognized role for ZO1 and ZO2 in the stabilization of the ICD in CMs which is necessary for maintenance of normal cardiac function and structural integrity. Ongoing research aims to define their mechanistic role in the ICD and myocardium.