BPS2026 - Broken clocks and intranodal conduction shifts underlie sinoatrial node dysfunction in cardiomyocyte-specific ZO1 knockout mice

Electrical coupling and cell adhesion among pacemaker cells are critical for sinoatrial node (SAN) impulse generation and conduction to the surrounding tissue. The junctional protein zona occludens-1 (ZO1) is an intracellular scaffolding protein crucial for the formation and maintenance of tight junctions. Recent studies using ZO1 knockout (KO) mice have shown that ZO1 is essential for conduction across the atrioventricular node (AVN). However, the role of ZO1 in SAN automaticity was not studied despite evidence of SAN dysfunction in KO. Here, we produced cardiomyocyte-specific KO (cKO) of ZO1 by crossing ZO1 floxed (fx) mice with αMyosin heavy chain (αMHC) nuclear Cre mice. Isolated cardiomyocyte protein showed complete loss of ZO1 in cKO samples compared with control (ZO1 fx/fx mice). ZO1 cKO mice exhibited enlargement of atria and SAN compared to control mice but no reduction in systolic function by echocardiogram. Surface ECG recordings in KO mice demonstrated sinus bradycardia and sinus pauses together with variable conduction across the AV node. In addition to reduced frequency, action potential (AP) recording using optical mapping indicated a shift in the SAN leading region and reduced conduction velocity in cKO. Electrophysiological recording using the perforated patch-clamp technique showed reduced frequency of spontaneous APs and reduced diastolic depolarization rate in cKO compared to control. Voltage clamp measurements confirmed decreased I f in ZO1cKO, consistent with decreased HCN4 RNA expression. In Cal-520AM loaded SAN cells, spontaneous local calcium (Ca) release and Ca transients were reduced in amplitude and frequency in ZO1cKO compared to control. These results indicate defects in intranodal conduction as well as components of both the membrane clock and the Ca clock in ZO1 cKO mice, and suggest that cardiomyocyte ZO1 is essential for normal SAN automaticity.