Increased cardiac contractility during fight-or-flight response is caused by β-adrenergic augmentation of Ca V 1.2 channels 1-4. In transgenic murine hearts expressing fully PKA phosphorylation-site-deficient mutant Ca V 1.2 α 1C and β subunits, this regulation persists, implying involvement of extra-channel factors. Here, we identify the mechanism by which β-adrenergic agonists stimulate voltage-gated Ca 2+ channels. We expressed α 1C or β 2B subunits conjugated to ascorbate-peroxidase 5 in mouse hearts and used multiplexed, quantitative proteomics 6,7 to track hundreds of proteins in proximity of Ca V 1.2. We observed that the Ca 2+ channel inhibitor Rad 8,9 , a monomeric G-protein, is enriched in the Ca V 1.2 micro-environment but is depleted during β-adrenergic stimulation. PKA-catalyzed phosphorylation of specific Ser residues on Rad decreases its affinity for auxiliary β-Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

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Statistics. Results are mean ± SEM. For multiple group comparisons, 1-way ANOVA followed by multiple comparison testing was performed. For comparisons between 2 groups, an unpaired Student's t test was used. Statistical analyses were performed using Prism 6 (Graphpad Software). Differences were considered statistically significant at P values less than 0.05. Data availability. The data and study materials will be made available to other researchers for purposes of reproducing the results or replicating the procedure. Study approval. The Institutional Animal Care and Use Committee at Columbia University approved all animal experiments.

Currently available skin grafts and skin substitute for healing following third-degree burn injuries is fraught with complications, often resulting in long-term physical and psychological sequelae. Synthetic treatment that can promote wound healing in a regenerative fashion would provide an off-the-shelf, non-immunogenic strategy to improve clinical care of severe burn wounds. Here, we demonstrate vulnerary efficacy and accelerated healing mechanism of dextran-based hydrogel in third-degree porcine burn model. The model was optimized to allow examination of the hydrogel treatment for clinical translation and its regenerative response mechanisms. Hydrogel treatment accelerated third-degree burn wound healing by rapid wound closure, improved reepithelialization, enhanced extracellular matrix remodeling, and greater nerve reinnervation, compared to the dressing treated group. These effects appear to be mediated through the ability of the hydrogel to facilitate a rapid but brief initial inflammatory response that coherently stimulates neovascularization within the granulation tissue during the first week of treatment, followed by an efficient vascular regression to promote a regenerative healing process. Our results suggest that the dextran-based hydrogels may substantially improve healing quality and reduce skin grafting incidents and thus pave the way for clinical studies to improve the care of severe burn injury patients.

Rationale: Changing activity of cardiac Ca V 1.2 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 up-regulated via activation of protein kinase A, essential molecular details remained stubbornly enigmatic. Objective: The primary goal of this study was to determine how various factors converging at the Ca V 1.2 I-II loop interact to regulate channel activity under basal conditions, during β-adrenergic stimulation, and in heart failure. Methods and Results: We generated transgenic mice with expression of Ca V 1.2 α 1C subunits with: 1) mutations ablating interaction between α 1C and β subunits; 2) flexibility-inducing polyglycine substitutions in the I-II loop (GGG-α 1C ); or 3) introduction of the alternatively spliced 25-amino acid exon 9* mimicking a splice variant of α 1C up-regulated in the hypertrophied heart. Introducing three glycine residues that disrupt a rigid IS6-AID helix markedly reduced basal open probability despite intact binding of Ca V β to α 1C I-II loop, and eliminated β-adrenergic agonist stimulation of Ca V 1.2 current. In contrast, introduction of the exon 9* splice variant in α 1C 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 β 2B and Rad or transgenically expressed in cardiomyocytes. Conclusions: Ca 2+ channel activity is dynamically modulated under basal conditions, during β-adrenergic stimulation, and in heart failure by mechanisms converging at the α 1C I-II loop. Ca V β binding to α 1C 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 Ca 2+ channel activity by β-adrenergic agonists/PKA also requires this rigid linker and β binding to α 1C .

Each heartbeat is initiated by the action potential, an electrical signal that depolarizes the plasma membrane and activates a cycle of calcium influx via voltage-gated calcium channels, calcium release via ryanodine receptors, and calcium reuptake and efflux via calcium-ATPase pumps and sodium-calcium exchangers. Agonists of the sympathetic nervous system bind to adrenergic receptors in cardiomyocytes, which, via cascading signal transduction pathways and protein kinase A (PKA), increase the heart rate (chronotropy), the strength of myocardial contraction (inotropy), and the rate of myocardial relaxation (lusitropy). These effects correlate with increased intracellular concentration of calcium, which is required for the augmentation of cardiomyocyte contraction. Despite extensive investigations, the molecular mechanisms underlying sympathetic nervous system regulation of calcium influx in cardiomyocytes have remained elusive over the last 40 years. Recent studies have uncovered the mechanisms underlying this fundamental biologic process, namely that PKA phosphorylates a calcium channel inhibitor, Rad, thereby releasing inhibition and increasing calcium influx. Here, we describe an updated model for how signals from adrenergic agonists are transduced to stimulate calcium influx and contractility in the heart. Expected final online publication date for the Annual Review of Physiology, Volume 84 is February 2022. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Fight-or-flight responses involve β-adrenergic-induced increases in heart rate and contractile force. In the present study, we uncover the primary mechanism underlying the heart’s innate contractile reserve. We show that four protein kinase A (PKA)-phosphorylated residues in Rad, a calcium channel inhibitor, are crucial for controlling basal calcium current and essential for β-adrenergic augmentation of calcium influx in cardiomyocytes. Even with intact PKA signaling to other proteins modulating calcium handling, preventing adrenergic activation of calcium channels in Rad-phosphosite-mutant mice (4SA-Rad) has profound physiological effects: reduced heart rate with increased pauses, reduced basal contractility, near-complete attenuation of β-adrenergic contractile response and diminished exercise capacity. Conversely, expression of mutant calcium-channel β-subunits that cannot bind 4SA-Rad is sufficient to enhance basal calcium influx and contractility to adrenergically augmented levels of wild-type mice, rescuing the failing heart phenotype of 4SA-Rad mice. Hence, disruption of interactions between Rad and calcium channels constitutes the foundation toward next-generation therapeutics specifically enhancing cardiac contractility.

in approximately 1.7 superhelical turns. How this nucleosome-decorated DNA further folds into higher level arrangements has been a subject of intensive study since the discovery of the nucleosome. Models used to account for different experimental properties of chromatin often disagree with one another. We have recently found that these discrepancies may arise from the choice of nucleosome core particle (NCP) structure used in the model. Here we present a survey of the precise DNA folding around the histone core in all high-resolution nucleosome structures currently available in the Protein Data Bank. The subtle differences seen among the different NCP structures lead to significant changes in simulated chromatin fibers. For example, the slight changes in DNA folding between certain models have the same effect on chromatin structure as changing the nucleosome spacing by 2-3 base pairs. Structural changes of this nature alter the patterns of nucleosome association and convert the simulated chromatin structures between compact and extended forms. Consideration of the different nucleosomal DNA pathways takes account of the sedimentation coefficients extracted from ultracentrifugation studies, as well as the long-range communication between regulatory proteins studied under physiological conditions, providing a simple mechanism for modulating DNA accessibility.