Identification of a protein phosphatase-1/phospholamban complex that is regulated by cAMP-dependent phosphorylation

In human and experimental heart failure, the activity of the type 1 phosphatase is significantly increased, associated with dephosphorylation of phospholamban, inhibition of the sarco(endo)plasmic reticulum Ca2+ transport ATPase (SERCA2a) and depressed function. In the current study, we investigated the molecular mechanisms controlling protein phosphatase-1 activity. Using recombinant proteins and complementary in vitro binding studies, we identified a multi-protein complex centered on protein phosphatase-1 that includes its muscle specific glycogen-targeting subunit GM and substrate phospholamban. GM interacts directly with phospholamban and this association is mediated by the cytosolic regions of the proteins. Our findings suggest the involvement of GM in mediating formation of the phosphatase-1/GM/phospholamban complex through the direct and independent interactions of GM with both protein phosphatase-1 and phospholamban. Importantly, the protein phosphatase-1/GM/ phospholamban complex dissociates upon protein kinase A phosphorylation, indicating its significance in the β-adrenergic signalling axis. Moreover, protein phosphatase-1 activity is regulated by two binding partners, inhibitor-1 and the small heat shock protein 20, Hsp20. Indeed, human genetic variants of inhibitor-1 (G147D) or Hsp20 (P20L) result in reduced binding and inhibition of protein phosphatase-1, suggesting aberrant enzymatic regulation in human carriers. These findings provide insights into the mechanisms underlying fine-tuned regulation of protein phosphatase-1 and its impact on the SERCA2/phospholamban interactome in cardiac function

Histidine-rich calcium binding protein: The new regulator of sarcoplasmic reticulum calcium cycling

The histidine-rich calcium binding protein (HRC) is a novel regulator of sarcoplasmic reticulum (SR) Ca2+-uptake, storage and release. Residing in the SR lumen, HRC binds Ca2+ with high capacity but low affinity. In vitro phosphorylation of HRC affects ryanodine affinity of the ryanodine receptor (RyR), suggesting a functional role of HRC on SR Ca2+-release. Indeed, acute HRC overexpression in isolated rodent cardiomyocytes decreases Ca2+-induced Ca2+-release, increases SR Ca2+-load, and impairs contractility. The HRC effects on RyR may be regulated by the Ca2+-sensitivity of its interaction with triadin. However, HRC also affects the SR Ca2+-ATPase, as shown by HRC overexpression in transgenic mouse hearts, which resulted in reduced SR Ca2+-uptake rates, cardiac remodeling and hypertrophy. In fact, in vitro generated evidence suggests that HRC directly interacts with SR Ca2+-ATPase2, supporting a dual role of HRC in Ca2+-homeostasis: regulation of both SR Ca2+-uptake and Ca2+-release. Furthermore, HRC plays an important role in myocyte differentiation and in antiapoptotic cardioprotection against ischemia/reperfusion induced cardiac injury. Interestingly, HRC has been linked with familiar cardiac conduction disease and an HRC polymorphism was shown to associate with malignant ventricular arrhythmias in the background of idiopathic dilated cardiomyopathy. This review summarizes studies, which have established the critical role of HRC in Ca2+-homeostasis, suggesting its importance in cardiac physiology and pathophysiology. © 2010 Elsevier Ltd

Pharmacogenetically tailored treatments for heart disease

Heart disease represents the primary cause of death worldwide, with mortality rates being predicted to remain constant within the next couple of decades. Cardiac disease treatment currently includes the administration of drugs, predominantly aiming at improving heart performance, through controlling heart rhythm, blood pressure, as well as reducing cholesterol and blood clotting. Despite, however, the medical advances that have led towards a better understanding of heart disease pathophysiology and the development of new therapeutic approaches, the degree of success of the available drug therapies varies among patients. Polymorphisms in a number of genes have been shown to result in differences in pharmacokinetics, pharmacodynamics and drug metabolism and have therefore been associated with response to drug treatment. The occurrence of adverse drug reactions represents another factor influencing the outcome of therapeutic treatments. While the influence of genetic polymorphisms in patient's response to heart disease drugs is being unveiled, the rapidly evolving field of pharmacogenetics is promising to aid clinicians in choosing the best suited drug/dose for each patient and the pharmaceutical companies in the design of better targeted, more effective new chemical compounds. In the near future individualized, targeted therapy will become part of clinical care routine maximizing patient therapeutic benefits and minimizing risks of adverse effects. © 2010 Bentham Science Publishers Ltd

The Histidine-Rich Calcium Binding Protein in Regulation of Cardiac Rhythmicity

Sudden unexpected cardiac death (SCD) accounts for up to half of all-cause mortality of heart failure patients. Standardized cardiology tools such as electrocardiography, cardiac imaging, electrophysiological and serum biomarkers cannot accurately predict which patients are at risk of life-threatening arrhythmic episodes. Recently, a common variant of the histidine-rich calcium binding protein (HRC), the Ser96Ala, was identified as a potent biomarker of malignant arrhythmia triggering in these patients. HRC has been shown to be involved in the regulation of cardiac sarcoplasmic reticulum (SR) Ca2+ cycling, by binding and storing Ca2+ in the SR, as well as interacting with the SR Ca2+ uptake and release complexes. The underlying mechanisms, elucidated by studies at the molecular, biochemical, cellular and intact animal levels, indicate that transversion of Ser96 to Ala results in abolishment of an HRC phosphorylation site by Fam20C kinase and dysregulation of SR Ca2+ cycling. This is mediated through aberrant SR Ca2+ release by the ryanodine receptor (RyR2) quaternary complex, due to the impaired HRC/triadin interaction, and depressed SR Ca2+ uptake by the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA2) pump, due to the impaired HRC/SERCA2 interaction. Pharmacological intervention with KN-93, an inhibitor of Ca2+/calmodulin-dependent protein kinase II (CaMKII), in the HRC Ser96Ala mouse model, reduced the occurrence of malignant cardiac arrhythmias. Herein, we summarize the current evidence on the pivotal role of HRC in the regulation of cardiac rhythmicity and the importance of HRC Ser96Ala as a genetic modifier for arrhythmias in the setting of heart failure. Copyright © 2018 Arvanitis, Vafiadaki, Johnson, Kranias and Sanoudou

The cardioprotective pka-mediated hsp20 phosphorylation modulates protein associations regulating cytoskeletal dynamics

The cytoskeleton has a primary role in cardiomyocyte function, including the response to mechanical stimuli and injury. The small heat shock protein 20 (Hsp20) conveys protective effects in cardiac muscle that are linked to serine-16 (Ser16) Hsp20 phosphorylation by stress-induced PKA, but the link between Hsp20 and the cytoskeleton remains poorly understood. Herein, we demonstrate a physical and functional interaction of Hsp20 with the cytoskeletal protein 14-3-3. We show that, upon phosphorylation at Ser16, Hsp20 translocates from the cytosol to the cytoskeleton where it binds to 14-3-3. This leads to dissociation of 14-3-3 from the F-actin depolymerization regulator cofilin-2 (CFL2) and enhanced F-actin depolymerization. Importantly, we demonstrate that the P20L Hsp20 mutation associated with dilated cardiomyopathy exhibits reduced physical interaction with 14-3-3 due to diminished Ser16 phosphorylation, with subsequent failure to translocate to the cytoskeleton and inability to disassemble the 14-3-3/CFL2 complex. The topological sequestration of Hsp20 P20L ultimately results in impaired regulation of F-actin dynamics, an effect implicated in loss of cytoskeletal integrity and amelioration of the cardioprotective functions of Hsp20. These findings underscore the significance of Hsp20 phosphorylation in the regulation of actin cytoskeleton dynamics, with important implications in cardiac muscle physiology and pathophysiology. © 2020 by the authors. Licensee MDPI, Basel, Switzerland

Calcium/calmodulin-dependent protein kinase II (CaMKII) inhibition ameliorates arrhythmias elicited by junctin ablation under stress conditions

Background Aberrant calcium signaling is considered one of the key mechanisms contributing to arrhythmias, especially in the context of heart failure. In human heart failure, there is significant down-regulation of the sarcoplasmic reticulum (SR) protein junctin, and junctin deficiency in mice is associated with stress-induced arrhythmias. Objective The purpose of this study was to determine whether the increased SR Ca2+ leak and arrhythmias associated with junctin ablation may be associated with increased calcium/calmodulin-dependent protein kinase II (CaMKII) activity and phosphorylation of the cardiac ryanodine receptor (RyR2) and whether pharmacologic inhibition of CaMKII activity may prevent these arrhythmias. Methods Using a combination of biochemical, cellular, and in vivo approaches, we tested the ability of KN-93 to reverse aberrant CaMKII phosphorylation of RyR2. Specifically, we performed protein phosphorylation analysis, in vitro cardiomyocyte contractility and Ca2+ kinetics, and in vivo ECG analysis in junctin-deficient mice. Results In the absence of junctin, RyR2 channels displayed CaMKII-dependent hyperphosphorylation. Notably, CaMKII inhibition by KN-93 reduced the in vivo incidence of stress-induced ventricular tachycardia by 65% in junctin null mice. At the cardiomyocyte level, KN-93 reduced the percentage of junctin null cells exhibiting spontaneous Ca2+ aftertransients and aftercontractions under stress conditions by 35% and 37%, respectively. At the molecular level, KN-93 blunted the CaMKII-mediated hyperphosphorylation of RyR2 and phospholamban under stress conditions. Conclusion Our data suggest that CaMKII inhibition is effective in preventing arrhythmogenesis in the setting of junctin ablation through modulation of both SR Ca2+ release and uptake. Thus, it merits further investigation as promising molecular therapy. © 2015 Published by Elsevier Inc. on behalf of Heart Rhythm Society

The importance of the Thr17 residue of phospholamban as a phosphorylation site under physiological and pathological conditions

The sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA2a) is under the control of an SR protein named phospholamban (PLN). Dephosphorylated PLN inhibits SERCA2a, whereas phosphorylation of PLN at either the Ser16 site by PKA or the Thr17 site by CaMKII reverses this inhibition, thus increasing SERCA2a activity and the rate of Ca2+ uptake by the SR. This leads to an increase in the velocity of relaxation, SR Ca2+ load and myocardial contractility. In the intact heart, ß-adrenoceptor stimulation results in phosphorylation of PLN at both Ser16 and Thr17 residues. Phosphorylation of the Thr17 residue requires both stimulation of the CaMKII signaling pathways and inhibition of PP1, the major phosphatase that dephosphorylates PLN. These two prerequisites appear to be fulfilled by ß-adrenoceptor stimulation, which as a result of PKA activation, triggers the activation of CaMKII by increasing intracellular Ca2+, and inhibits PP1. Several pathological situations such as ischemia-reperfusion injury or hypercapnic acidosis provide the required conditions for the phosphorylation of the Thr17 residue of PLN, independently of the increase in PKA activity, i.e., increased intracellular Ca2+ and acidosis-induced phosphatase inhibition. Our results indicated that PLN was phosphorylated at Thr17 at the onset of reflow and immediately after hypercapnia was established, and that this phosphorylation contributes to the mechanical recovery after both the ischemic and acidic insults. Studies on transgenic mice with Thr17 mutated to Ala (PLN-T17A) are consistent with these results. Thus, phosphorylation of the Thr17 residue of PLN probably participates in a protective mechanism that favors Ca2+ handling and limits intracellular Ca2+ overload in pathological situations