Background:
Ca²⁺ influx via L-type calcium channels (LTCCs) triggers muscle contraction in the heart and controls action potential duration. Alterations in the density or function of L-type Ca²⁺ channels have been implicated in various cardiovascular diseases, including atrial fibrillation, heart failure, and ischemic heart disease. Gain-of-function mutations in LTCCs are linked to prolonged QT interval and ventricular arrhythmia, as in Timothy syndrome. Here, we used FPL 64176 as a LTCC activator, which increases channel conductance and shifts voltage-dependent activation to more negative potentials.

Method:
12-16 weeks old C57Bl/6J mice were used in all experiments. The voltage-sensitive dye di-8-ANEPPS and blebbistatin as an excitation-contraction uncoupler were perfused in a Langendorff setup of mouse heart. Hearts were then cut parallel to the left ventricle free wall into 350 μm thick slices. The slices were moved to a custom-made chamber and incubated with circulating tyrode solution keeping them at 37 ºC. The ventricle slices were electrically stimulated with a pace cycle length of 300 ms and action potentials were optically mapped at 256×256 spatial pixel resolution. We modified a LTCC mathematical model to reflect the impact of FPL and incorporated it into a mouse ventricular cell model to simulate action potentials upon LTCC gain of function.

Results:
Action potentials in heart slices showed stable temporal characteristics over 5 hours, making it an appropriate system to investigate the kinetics of compounds effects. Application of FPL increases action potential duration gradually at 50% repolarization (APD₅₀) over time with a rising plateau phase. Further, higher concentrations of FPL elicited early after depolarizations (EAD) and repolarization failure (Fig. 1). Our mathematical modeling approach of single mouse cardiomyocytes was able to resemble the experimental data.

Figure 1. FPL increases APD₅₀ and triggers EADs. (a) APD₅₀ analysis and overlay of action potentials over time before and after applying 500 μm FPL. (b) quantification of steady-state APD₅₀ (n=10). (c) EAD formation at high concentration of FPL (1 μm) and respective activation time maps.

Conclusion- We conclude that gain of function of ltcc currents prolongs apd and triggers ead, thereby increasing the probability of cardiac arrhythmia.