β‐adrenergic‐mediated dynamic augmentation of sarcolemmal Ca(V)1.2 clustering and co‐operativity in ventricular myocytes

KEY POINTS: Prevailing dogma holds that activation of the β‐adrenergic receptor/cAMP/protein kinase A signalling pathway leads to enhanced L‐type Ca(V)1.2 channel activity, resulting in increased Ca(2+) influx into ventricular myocytes and a positive inotropic response. However, the full mechanistic and molecular details underlying this phenomenon are incompletely understood. Ca(V)1.2 channel clusters decorate T‐tubule sarcolemmas of ventricular myocytes. Within clusters, nanometer proximity between channels permits Ca(2+)‐dependent co‐operative gating behaviour mediated by physical interactions between adjacent channel C‐terminal tails. We report that stimulation of cardiomyocytes with isoproterenol, evokes dynamic, protein kinase A‐dependent augmentation of Ca(V)1.2 channel abundance along cardiomyocyte T‐tubules, resulting in the appearance of channel ‘super‐clusters’, and enhanced channel co‐operativity that amplifies Ca(2+) influx. On the basis of these data, we suggest a new model in which a sub‐sarcolemmal pool of pre‐synthesized Ca(V)1.2 channels resides in cardiomyocytes and can be mobilized to the membrane in times of high haemodynamic or metabolic demand, to tune excitation–contraction coupling. ABSTRACT: Voltage‐dependent L‐type Ca(V)1.2 channels play an indispensable role in cardiac excitation–contraction coupling. Activation of the β‐adrenergic receptor (βAR)/cAMP/protein kinase A (PKA) signalling pathway leads to enhanced Ca(V)1.2 activity, resulting in increased Ca(2+) influx into ventricular myocytes and a positive inotropic response. Ca(V)1.2 channels exhibit a clustered distribution along the T‐tubule sarcolemma of ventricular myocytes where nanometer proximity between channels permits Ca(2+)‐dependent co‐operative gating behaviour mediated by dynamic, physical, allosteric interactions between adjacent channel C‐terminal tails. This amplifies Ca(2+) influx and augments myocyte Ca(2+) transient and contraction amplitudes. We investigated whether βAR signalling could alter Ca(V)1.2 channel clustering to facilitate co‐operative channel interactions and elevate Ca(2+) influx in ventricular myocytes. Bimolecular fluorescence complementation experiments reveal that the βAR agonist, isoproterenol (ISO), promotes enhanced Ca(V)1.2–Ca(V)1.2 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(V)1.2 ‘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(V)1.2 channel abundance after ISO application. Optical and electrophysiological single channel recordings confirm that these newly inserted channels are functional and contribute to overt co‐operative gating behaviour of Ca(V)1.2 channels in ISO stimulated myocytes. The results of the present study reveal a new facet of βAR‐mediated regulation of Ca(V)1.2 channels in the heart and support the novel concept that a pre‐synthesized pool of sub‐sarcolemmal Ca(V)1.2 channel‐containing vesicles/endosomes resides in cardiomyocytes and can be mobilized to the sarcolemma to tune excitation–contraction coupling to meet metabolic and/or haemodynamic demands.