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?-adrenergic-mediated dynamic augmentation of sarcolemmal CaV 1.2 clustering and co-operativity in ventricular myocytes.

ABSTRACT: KEY POINTS:Prevailing dogma holds that activation of the ?-adrenergic receptor/cAMP/protein kinase A signalling pathway leads to enhanced L-type CaV 1.2 channel activity, resulting in increased Ca2+ influx into ventricular myocytes and a positive inotropic response. However, the full mechanistic and molecular details underlying this phenomenon are incompletely understood. CaV 1.2 channel clusters decorate T-tubule sarcolemmas of ventricular myocytes. Within clusters, nanometer proximity between channels permits Ca2+ -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 CaV 1.2 channel abundance along cardiomyocyte T-tubules, resulting in the appearance of channel 'super-clusters', and enhanced channel co-operativity that amplifies Ca2+ influx. On the basis of these data, we suggest a new model in which a sub-sarcolemmal pool of pre-synthesized CaV 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 CaV 1.2 channels play an indispensable role in cardiac excitation-contraction coupling. Activation of the ?-adrenergic receptor (?AR)/cAMP/protein kinase A (PKA) signalling pathway leads to enhanced CaV 1.2 activity, resulting in increased Ca2+ influx into ventricular myocytes and a positive inotropic response. CaV 1.2 channels exhibit a clustered distribution along the T-tubule sarcolemma of ventricular myocytes where nanometer proximity between channels permits Ca2+ -dependent co-operative 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 ?AR signalling could alter CaV 1.2 channel clustering to facilitate co-operative channel interactions and elevate Ca2+ influx in ventricular myocytes. Bimolecular fluorescence complementation experiments reveal that the ?AR agonist, isoproterenol (ISO), promotes enhanced CaV 1.2-CaV 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 CaV 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 CaV 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 CaV 1.2 channels in ISO stimulated myocytes. The results of the present study reveal a new facet of ?AR-mediated regulation of CaV 1.2 channels in the heart and support the novel concept that a pre-synthesized pool of sub-sarcolemmal CaV 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.

SUBMITTER: Ito DW

PROVIDER: S-EPMC6462464 | biostudies-literature | 2019 Apr

REPOSITORIES: biostudies-literature

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β-adrenergic-mediated dynamic augmentation of sarcolemmal Ca<sub>V</sub> 1.2 clustering and co-operativity in ventricular myocytes.

Ito Danica W DW Hannigan Karen I KI Ghosh Debapriya D Xu Bing B Del Villar Silvia G SG Xiang Yang K YK Dickson Eamonn J EJ Navedo Manuel F MF Dixon Rose E RE

The Journal of physiology 20190312 8

<h4>Key points</h4>Prevailing dogma holds that activation of the β-adrenergic receptor/cAMP/protein kinase A signalling pathway leads to enhanced L-type Ca<sub>V</sub> 1.2 channel activity, resulting in increased Ca<sup>2+</sup> influx into ventricular myocytes and a positive inotropic response. However, the full mechanistic and molecular details underlying this phenomenon are incompletely understood. Ca<sub>V</sub> 1.2 channel clusters decorate T-tubule sarcolemmas of ventricular myocytes. With ...[more]

PMID: 30714156

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Project description:RationaleChanging activity of cardiac CaV1.2 channels under basal conditions, during sympathetic activation, and in heart failure is a major determinant of cardiac physiology and pathophysiology. Although cardiac CaV1.2 channels are prominently upregulated via activation of PKA (protein kinase A), essential molecular details remained stubbornly enigmatic.ObjectiveThe primary goal of this study was to determine how various factors converging at the CaV1.2 I-II loop interact to regulate channel activity under basal conditions, during β-adrenergic stimulation, and in heart failure.Methods and resultsWe generated transgenic mice with expression of CaV1.2 α1C subunits with (1) mutations ablating interaction between α1C and β-subunits, (2) flexibility-inducing polyglycine substitutions in the I-II loop (GGG-α1C), or (3) introduction of the alternatively spliced 25-amino acid exon 9* mimicking a splice variant of α1C upregulated in the hypertrophied heart. Introducing 3 glycine residues that disrupt a rigid IS6-α-interaction domain helix markedly reduced basal open probability despite intact binding of CaVβ to α1C I-II loop and eliminated β-adrenergic agonist stimulation of CaV1.2 current. In contrast, introduction of the exon 9* splice variant in the α1C I-II loop, which is increased in ventricles of patients with end-stage heart failure, increased basal open probability but did not attenuate stimulatory response to β-adrenergic agonists when reconstituted heterologously with β2B and Rad or transgenically expressed in cardiomyocytes.ConclusionsCa2+ channel activity is dynamically modulated under basal conditions, during β-adrenergic stimulation, and in heart failure by mechanisms converging at the α1C I-II loop. CaVβ binding to α1C stabilizes an increased channel open probability gating mode by a mechanism that requires an intact rigid linker between the β-subunit binding site in the I-II loop and the channel pore. Release of Rad-mediated inhibition of Ca2+ channel activity by β-adrenergic agonists/PKA also requires this rigid linker and β-binding to α1C.

Project description:KEY POINTS:Small-conductance Ca2+ -activated K+ (SK) channels expressed in ventricular myocytes are dormant in health, yet become functional in cardiac disease. SK channels are voltage independent and their gating is controlled by intracellular [Ca2+ ] in a biphasic manner. Submicromolar [Ca2+ ] activates the channel via constitutively-bound calmodulin, whereas higher [Ca2+ ] exerts inhibitory effect during depolarization. Using a rat model of cardiac hypertrophy induced by thoracic aortic banding, we found that functional upregulation of SK2 channels in hypertrophic rat ventricular cardiomyocytes is driven by protein kinase A (PKA) phosphorylation. Using site-directed mutagenesis, we identified serine-465 as the site conferring PKA-dependent effects on SK2 channel function. PKA phosphorylation attenuates ISK rectification by reducing the Ca2+ /voltage-dependent inhibition of SK channels without changing their sensitivity to activating submicromolar [Ca2+ ]i . This mechanism underlies the functional recruitment of SK channels not only in cardiac disease, but also in normal physiology, contributing to repolarization under conditions of enhanced adrenergic drive. ABSTRACT:Small-conductance Ca2+ -activated K+ (SK) channels expressed in ventricular myocytes (VMs) are dormant in health, yet become functional in cardiac disease. We aimed to test the hypothesis that post-translational modification of SK channels under conditions accompanied by enhanced adrenergic drive plays a central role in disease-related activation of the channels. We investigated this phenomenon using a rat model of hypertrophy induced by thoracic aortic banding (TAB). Western blot analysis using anti-pan-serine/threonine antibodies demonstrated enhanced phosphorylation of immunoprecipitated SK2 channels in VMs from TAB rats vs. Shams, which was reversible by incubation of the VMs with PKA inhibitor H89 (1 ?mol L-1 ). Patch clamped VMs under basal conditions from TABs but not Shams exhibited outward current sensitive to the specific SK inhibitor apamin (100 nmol L-1 ), which was eliminated by inhibition of PKA (1 ?mol L-1 ). Beta-adrenergic stimulation (isoproterenol, 100 nmol L-1 ) evoked ISK in VMs from Shams, resulting in shortening of action potentials in VMs and ex vivo optically mapped Sham hearts. Using adenoviral gene transfer, wild-type and mutant SK2 channels were overexpressed in adult rat VMs, revealing serine-465 as the site that elicits PKA-dependent phosphorylation effects on SK2 channel function. Concurrent confocal Ca2+ imaging experiments established that PKA phosphorylation lessens rectification of ISK via reduction Ca2+ /voltage-dependent inhibition of the channels at high [Ca2+ ] without affecting their sensitivity to activation by Ca2+ in the submicromolar range. In conclusion, upregulation of SK channels in diseased VMs is mediated by hyperadrenergic drive in cardiac hypertrophy, with functional effects on the channel conferred by PKA-dependent phosphorylation at serine-465.

Project description:The balance of Ca2+ influx and efflux regulates the Ca2+ load of cardiac myocytes, a process known as autoregulation. Previous work has shown that Ca2+ influx, via L-type Ca2+ current (ICa), and efflux, via the Na+/Ca2+ exchanger (NCX), occur predominantly at t-tubules; however, the role of t-tubules in autoregulation is unknown. Therefore, we investigated the sarcolemmal distribution of ICa and NCX current (INCX), and autoregulation, in mouse ventricular myocytes using whole cell voltage-clamp and simultaneous Ca2+ measurements in intact and detubulated (DT) cells. In contrast to the rat, INCX was located predominantly at the surface membrane, and the hysteresis between INCX and Ca2+ observed in intact myocytes was preserved after detubulation. Immunostaining showed both NCX and ryanodine receptors (RyRs) at the t-tubules and surface membrane, consistent with colocalization of NCX and RyRs at both sites. Unlike INCX, ICa was found predominantly in the t-tubules. Recovery of the Ca2+ transient amplitude to steady state (autoregulation) after application of 200 µM or 10 mM caffeine was slower in DT cells than in intact cells. However, during application of 200 µM caffeine to increase sarcoplasmic reticulum (SR) Ca2+ release, DT and intact cells recovered at the same rate. It appears likely that this asymmetric response to changes in SR Ca2+ release is a consequence of the distribution of ICa, which is reduced in DT cells and is required to refill the SR after depletion, and NCX, which is little affected by detubulation, remaining available to remove Ca2+ when SR Ca2+ release is increased.NEW & NOTEWORTHY This study shows that in contrast to the rat, mouse ventricular Na+/Ca2+ exchange current density is lower in the t-tubules than in the surface sarcolemma and Ca2+ current is predominantly located in the t-tubules. As a consequence, the t-tubules play a role in recovery (autoregulation) from reduced, but not increased, sarcoplasmic reticulum Ca2+ release.

Dataset Information

?-adrenergic-mediated dynamic augmentation of sarcolemmal CaV 1.2 clustering and co-operativity in ventricular myocytes.

Publications

β-adrenergic-mediated dynamic augmentation of sarcolemmal Ca<sub>V</sub> 1.2 clustering and co-operativity in ventricular myocytes.

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