Faculty Bibliography

Increased cardiac contractility during the fight-or-flight response is caused by β-adrenergic augmentation of Ca_V1.2 voltage-gated calcium channels^1-4. However, this augmentation persists in transgenic murine hearts expressing mutant Ca_V1.2 α_1C and β subunits that can no longer be phosphorylated by protein kinase A-an essential downstream mediator of β-adrenergic signalling-suggesting that non-channel factors are also required. Here we identify the mechanism by which β-adrenergic agonists stimulate voltage-gated calcium channels. We express α_1C or β_2B subunits conjugated to ascorbate peroxidase⁵ in mouse hearts, and use multiplexed quantitative proteomics^6,7 to track hundreds of proteins in the proximity of Ca_V1.2. We observe that the calcium-channel inhibitor Rad^8,9, a monomeric G protein, is enriched in the Ca_V1.2 microenvironment but is depleted during β-adrenergic stimulation. Phosphorylation by protein kinase A of specific serine residues on Rad decreases its affinity for β subunits and relieves constitutive inhibition of Ca_V1.2, observed as an increase in channel open probability. Expression of Rad or its homologue Rem in HEK293T cells also imparts stimulation of Ca_V1.3 and Ca_V2.2 by protein kinase A, revealing an evolutionarily conserved mechanism that confers adrenergic modulation upon voltage-gated calcium channels.

Ca2+ channel β-subunit interactions with pore-forming α-subunits are long-thought to be obligatory for channel trafficking to the cell surface and for tuning of basal biophysical properties in many tissues. Unexpectedly, we demonstrate that transgenic expression of mutant α1C subunits lacking capacity to bind CaVβ can traffic to the sarcolemma in adult cardiomyocytes in vivo and sustain normal excitation-contraction coupling. However, these β-less Ca2+ channels cannot be stimulated by β-adrenergic pathway agonists, and thus adrenergic augmentation of contractility is markedly impaired in isolated cardiomyocytes and in hearts. Similarly, viral-mediated expression of a β-subunit-sequestering peptide sharply curtailed β-adrenergic stimulation of WT Ca2+ channels, identifying an approach to specifically modulate β-adrenergic regulation of cardiac contractility. Our data demonstrate that β subunits are required for β-adrenergic regulation of CaV1.2 channels and positive inotropy in the heart, but are dispensable for CaV1.2 trafficking to the adult cardiomyocyte cell surface, and for basal function and excitation-contraction coupling.

Regulation of intracellular calcium (Ca²⁺) is critical in all cell types. The ryanodine receptor (RyR), an intracellular Ca²⁺ release channel located on the sarco/endoplasmic reticulum (SR/ER), releases Ca²⁺ from intracellular stores to activate critical functions including muscle contraction and neurotransmitter release. Dysfunctional RyR-mediated Ca²⁺ handling has been implicated in the pathogenesis of inherited and non-inherited conditions including heart failure, cardiac arrhythmias, skeletal myopathies, diabetes, and neurodegenerative diseases. Here we have reviewed the evidence linking human disorders to RyR dysfunction and describe novel approaches to RyR-targeted therapeutics.

BACKGROUND:handling because of leaky RyR channels in CHF. METHODS:stores within the endoplasmic reticulum. RESULTS:leak was significantly reduced in mice treated with the Rycal S107. Patients with CHF treated with left-ventricular assist devices exhibited a heterogeneous response. CONCLUSIONS:handling and systemic sympathetic burden, presenting a novel biomarker for monitoring response to pharmacological and mechanical CHF therapy.

Research over the past two decades has implicated dysfunction of the ryanodine receptor (RyR), a Ca(2+) release channel on the sarcoplasmic reticulum (SR) required for excitation-contraction (EC) coupling, in the pathogenesis of cardiac and skeletal myopathies. These discoveries have led to the development of novel drugs, screening tools, and research methods. The patents associated with these advances tell the story of the initial discovery of RyRs as a target for plant alkaloids, to their central role in cardiac and skeletal muscle excitation-contraction coupling, and ongoing clinical trials with a novel class of drugs called RycalsTM that inhibit pathological intracellular Ca(2+) leak. Additionally, these patents highlight questions, controversies, and future directions of the RyR field.

During the classic "fight-or-flight" stress response, sympathetic nervous system activation leads to catecholamine release, which increases heart rate and contractility, resulting in enhanced cardiac output. Catecholamines bind to β-adrenergic receptors, causing cAMP generation and activation of PKA, which phosphorylates multiple targets in cardiac muscle, including the cardiac ryanodine receptor/calcium release channel (RyR2) required for muscle contraction. PKA phosphorylation of RyR2 enhances channel activity by sensitizing the channel to cytosolic calcium (Ca²+). Here, we found that mice harboring RyR2 channels that cannot be PKA phosphorylated (referred to herein as RyR2-S2808A+/+ mice) exhibited blunted heart rate and cardiac contractile responses to catecholamines (isoproterenol). The isoproterenol-induced enhancement of ventricular myocyte Ca²+ transients and fractional shortening (contraction) and the spontaneous beating rate of sinoatrial nodal cells were all blunted in RyR2-S2808A+/+ mice. The blunted cardiac response to catecholamines in RyR2-S2808A+/+ mice resulted in impaired exercise capacity. RyR2-S2808A+/+ mice were protected against chronic catecholaminergic-induced cardiac dysfunction. These studies identify what we believe to be new roles for PKA phosphorylation of RyR2 in both the heart rate and contractile responses to acute catecholaminergic stimulation.

Increased sarcoplasmic reticulum (SR) Ca2+ leak via the cardiac ryanodine receptor/calcium release channel (RyR2) is thought to play a role in heart failure (HF) progression. Inhibition of this leak is an emerging therapeutic strategy. To explore the role of chronic PKA phosphorylation of RyR2 in HF pathogenesis and treatment, we generated a knockin mouse with aspartic acid replacing serine 2808 (mice are referred to herein as RyR2-S2808D+/+ mice). This mutation mimics constitutive PKA hyperphosphorylation of RyR2, which causes depletion of the stabilizing subunit FKBP12.6 (also known as calstabin2), resulting in leaky RyR2. RyR2-S2808D+/+ mice developed age-dependent cardiomyopathy, elevated RyR2 oxidation and nitrosylation, reduced SR Ca2+ store content, and increased diastolic SR Ca2+ leak. After myocardial infarction, RyR2-S2808D+/+ mice exhibited increased mortality compared with WT littermates. Treatment with S107, a 1,4-benzothiazepine derivative that stabilizes RyR2-calstabin2 interactions, inhibited the RyR2-mediated diastolic SR Ca2+ leak and reduced HF progression in WT and RyR2-S2808D+/+ mice. In contrast, β-adrenergic receptor blockers improved cardiac function in WT but not in RyR2-S2808D+/+ mice.Thus, chronic PKA hyperphosphorylation of RyR2 results in a diastolic leak that causes cardiac dysfunction. Reversing PKA hyperphosphorylation of RyR2 is an important mechanism underlying the therapeutic action of β-blocker therapy in HF.

The force frequency relationship (FFR), first described by Bowditch 139 years ago as the observation that myocardial contractility increases proportionally with increasing heart rate, is an important mediator of enhanced cardiac output during exercise. Individuals with heart failure have defective positive FFR that impairs their cardiac function in response to stress, and the degree of positive FFR deficiency correlates with heart failure progression. We have identified a mechanism for FFR involving heart rate dependent phosphorylation of the major cardiac sarcoplasmic reticulum calcium release channel/ryanodine receptor (RyR2), at Ser2814, by calcium/calmodulin-dependent serine/threonine kinase-delta (CaMKIIdelta). Mice engineered with an RyR2-S2814A mutation have RyR2 channels that cannot be phosphorylated by CaMKIIdelta, and exhibit a blunted positive FFR. Ex vivo hearts from RyR2-S2814A mice also have blunted positive FFR, and cardiomyocytes isolated from the RyR2-S2814A mice exhibit impaired rate-dependent enhancement of cytosolic calcium levels and fractional shortening. The cardiac RyR2 macromolecular complexes isolated from murine and human failing hearts have reduced CaMKIIdelta levels. These data indicate that CaMKIIdelta phosphorylation of RyR2 plays an important role in mediating positive FFR in the heart, and that defective regulation of RyR2 by CaMKIIdelta-mediated phosphorylation is associated with the loss of positive FFR in failing hearts.