β‐adrenergic‐mediated dynamic augmentation of sarcolemmal CaV1.2 clustering and cooperativity in ventricular myocytes

Abstract

Voltage‐dependent L‐type Ca_V1.2 channels play an indispensable role in cardiac excitation‐contraction (EC) coupling. Activation of the β‐adrenergic receptor (βAR)/cAMP/PKA signaling pathway leads to enhanced Ca_V1.2 activity resulting in increased Ca²⁺ influx into ventricular myocytes, and a positive inotropic response. Ca_V1.2 channels exhibit a clustered distribution along the T‐tubule sarcolemma of ventricular myocytes where nanometer proximity between channels permits Ca²⁺‐dependent cooperative gating behaviour mediated by dynamic, physical, allosteric interactions between adjacent channel C‐terminal tails. This amplifies Ca²⁺ influx and augments myocyte Ca²⁺ transient and contraction amplitudes. We investigated whether βAR signaling could alter Ca_V1.2 channel clustering to facilitate cooperative channel interactions and elevate Ca²⁺ influx in ventricular myocytes. Bimolecular fluorescence complementation experiments reveal that the βAR agonist, isoproterenol (ISO), promotes enhanced Ca_V1.2‐Ca_V1.2 physical interactions. Super‐resolution nanoscopy and dynamic channel tracking indicate that these interactions are expedited by enhanced spatial proximity between channels, resulting in the appearance of Ca_V1.2 'super‐clusters' along the z‐lines of ISO‐stimulated cardiomyocytes. The mechanism that leads to super‐cluster formation involves rapid, dynamic augmentation of sarcolemmal Ca_V1.2 channel abundance after ISO application. Optical and electrophysiological single channel recordings confirm that these newly inserted channels are functional and contribute to overt cooperative gating behaviour of Ca_V1.2 channels in ISO stimulated myocytes. Our study reveals a new facet of βAR‐mediated regulation of Ca_V1.2 channels in the heart and supports the novel concept that a pre‐synthesized pool of sub‐sarcolemmal Ca_V1.2 channel‐containing vesicles/endosomes reside in cardiomyocytes and can be mobilized to the sarcolemma to tune EC‐coupling to meet metabolic and/or hemodynamic demands.

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