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.