Introduction. L-Type calcium channels (LTCC) are an essential element in the excitation-contraction coupling of cardiomyocytes (CMs). Recently, we have shown that the regulation of the LTCC activity by proximity coupling mechanisms occurs only via β₂AR, but not β₁AR, with the requirement of caveolin 3 (Cav3) to form complexes (1). We hypothesise that this mechanism can play a role in tuning the LTCC response by β₂ARs under sympathetic stimulation. This study aims to investigates how sympathetic innervation regulates CM adrenergic responses, the effect of innervation on the formation of an LTCC/β₂ARs/Cav3 complex(es), and what impact innervation has in driving arrhythmogenic events.

Methods. CMs and sympathetic neurons (SNs) were isolated from neonatal rat pups and cocultured for periods of 3-4 and 7-10 days. Methods ranged from contraction analysis and Ca²⁺ transient with either β₁AR or β₂AR antagonists by high-throughput multicellular CytoCypher, scanning ion conductance microscopy (SICM), smart patch-clamp, and immunostaining confocal imaging. All experiments were performed under Imperial College London ethical guidelines.

Results. CytoCypher contraction analysis shows how the role of innervation matures over time in culture.  At 4 days, innervated CMs show no remarkable differences from non-innervated. However, at 8 days, innervated CMs shows a higher peak of contraction (0.332±SEM, n=19) than non-innervated (0.175±SEM, n=22). Interestingly, using β₁AR and β₂AR blockers on contracting cells show an overall decrease in time to peak and time to baseline in both innervated and non-innervated, suggesting a competitive role between βAR in our culture conditions. Single channel patch clamp analysis suggest that innervation doubles the open probability of LTCCs (innervated: 0.089, n=4, p=0.0571 vs non-innervated: 0.046, n=4). Through immunostaining of LTCC and Cav3 proteins, we show the presence of LTCC/Cav3 complexes, with a higher co-localisation coefficient on innervated CMs than non-innervated (p=0.0076). Arrhythmia analysis under nicotine stimulation showed that only innervated CMs under β₂AR blockage are more susceptible to arrhythmias than any other group tested at both time points (4-day, n=4-5, p=0.0005; and 8-day, n=3-4 p<0.0001 versus control).  

Conclusion. Our preliminary results suggest that β₂AR plays an important role in the control of CM contraction by sympathetic innervation. Specifically, our results suggest that innervated CMs may have a higher number of LTCC complexes associated with Cav3 and β₂AR, and they may be essential in mitigating CM susceptibility to arrhythmias.