The role of CaMKII regulation of phospholamban activity in heart disease

Phospholamban (PLN) is a phosphoprotein in cardiac sarcoplasmic reticulum (SR) that is a reversible regulator of the Ca(2)(+)-ATPase (SERCA2a) activity and cardiac contractility. Dephosphorylated PLN inhibits SERCA2a and PLN phosphorylation, at either Ser(16) by PKA or Thr(17) by Ca(2)(+)-calmodulin-dependent protein kinase (CaMKII), reverses this inhibition. Through this mechanism, PLN is a key modulator of SR Ca(2)(+) uptake, Ca(2)(+) load, contractility, and relaxation. PLN phosphorylation is also the main determinant of β1-adrenergic responses in the heart. Although phosphorylation of Thr(17) by CaMKII contributes to this effect, its role is subordinate to the PKA-dependent increase in cytosolic Ca(2)(+), necessary to activate CaMKII. Furthermore, the effects of PLN and its phosphorylation on cardiac function are subject to additional regulation by its interacting partners, the anti-apoptotic HAX-1 protein and Gm or the anchoring unit of protein phosphatase 1. Regulation of PLN activity by this multimeric complex becomes even more important in pathological conditions, characterized by aberrant Ca(2)(+)-cycling. In this scenario, CaMKII-dependent PLN phosphorylation has been associated with protective effects in both acidosis and ischemia/reperfusion. However, the beneficial effects of increasing SR Ca(2)(+) uptake through PLN phosphorylation may be lost or even become deleterious, when these occur in association with alterations in SR Ca(2)(+) leak. Moreover, a major characteristic in human and experimental heart failure (HF) is depressed SR Ca(2)(+) uptake, associated with decreased SERCA2a levels and dephosphorylation of PLN, leading to decreased SR Ca(2)(+) load and impaired contractility. Thus, the strategy of altering SERCA2a and/or PLN levels or activity to restore perturbed SR Ca(2)(+) uptake is a potential therapeutic tool for HF treatment. We will review here the role of CaMKII-dependent phosphorylation of PLN at Thr(17) on cardiac function under physiological and pathological conditions

Role of phospholamban phosphorylation on Thr17 in cardiac physiological and pathological conditions

The sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA2a) is under the control of a closely associated SR protein named phospholamban (PLN). Dephosphorylated PLN inhibits the SR Ca2+ pump, whereas phosphorylation of PLN, at either Ser16 by PKA or Thr17 by calmodulin-dependent protein kinase II (CaMKII), reverses this inhibition, thus increasing SERCA2a activity and the rate of Ca2+ uptake by the SR. This would in turn lead to an increase in the velocity of relaxation, SR Ca 2+ load, and myocardial contractility. Thus, PLN is a major determinant of cardiac contractility and relaxation. Although in the intact heart, β-adrenoceptor stimulation results in phosphorylation of PLN at both Ser16 and Thr17 residues, the role of Thr17 site has long remained equivocal. In this review, we attempt to highlight the signaling cascade and the physiological relevance of the phosphorylation of this residue in the heart under both physiological and pathological situations.Facultad de Ciencias Médica

Role of phospholamban phosphorylation on Thr17 in cardiac physiological and pathological conditions

The sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA2a) is under the control of a closely associated SR protein named phospholamban (PLN). Dephosphorylated PLN inhibits the SR Ca2+ pump, whereas phosphorylation of PLN, at either Ser16 by PKA or Thr17 by calmodulin-dependent protein kinase II (CaMKII), reverses this inhibition, thus increasing SERCA2a activity and the rate of Ca2+ uptake by the SR. This would in turn lead to an increase in the velocity of relaxation, SR Ca 2+ load, and myocardial contractility. Thus, PLN is a major determinant of cardiac contractility and relaxation. Although in the intact heart, β-adrenoceptor stimulation results in phosphorylation of PLN at both Ser16 and Thr17 residues, the role of Thr17 site has long remained equivocal. In this review, we attempt to highlight the signaling cascade and the physiological relevance of the phosphorylation of this residue in the heart under both physiological and pathological situations.Facultad de Ciencias Médica

Phospholamban phosphorylation sites enhance the recovery of intracellular Ca2+ after perfusion arrest in isolated, perfused mouse heart

Objective: To investigate the importance of the phosphorylation of Ser16 and Thr17 sites of phospholamban (PLN) on intracellular Ca2+ (Cai2+) handling and contractile recovery of the stunned myocardium. Methods: Cai2+ (Rhod-2, pulsed local-field fluorescence microscopy) and contractility (isovolumic left ventricular developed pressure, LVDP) were simultaneously measured in Langendorff perfused hearts from transgenic mice expressing either intact PLN (PLN-WT) or PLN with both phosphorylation sites mutated to Ala (PLN-DM), subjected to 12 min of global ischemia followed by a reperfusion period of 30 min. Results: Pre-ischemic values of Cai2+ and LVDP were similar in both groups. In PLN-WT, a transient increase in Thr17 phosphorylation at early reperfusion preceded a recovery of Ca2+ transient amplitude, virtually completed by the end of reperfusion. LVDP at 30 min reperfusion was 67.9 ± 7.6% of pre-ischemic values, n = 14. In contrast, in PLN-DM, there was a poor recovery of Cai2+ transient amplitude and LVDP was significantly lower (28.3 ± 6.7%, n = 11, 30 min reperfusion) than in PLN-WT hearts. Although myofilament Ca2+ responsiveness and troponin I (TnI) degradation did not differ between groups, the episodes of mechanical alternans, typical of Cai2+ overload, were significantly prolonged in PLN-DM vs. PLN-WT hearts. Conclusions: PLN phosphorylation appears to be crucial for the mechanical and Cai2+ recovery during stunning and protective against the mechanical abnormalities typical of Cai2+ overload. The importance of PLN phosphorylation would primarily reside in the Thr17 residue, which is phosphorylated during the critical early phase of reperfusion. Our results emphasize that, although ablation of PLN phosphorylation does not affect basal contractility, it does alter Ca2+ handling and mechanical performance under stress situations.Facultad de Ciencias Médica

Phospholamban Regulates Nuclear Ca2+ Stores and Inositol 1,4,5-Trisphosphate Mediated Nuclear Ca2+ Cycling in Cardiomyocytes

AIMS: Phospholamban (PLB) is the key regulator of the cardiac Ca2+ pump (SERCA2a)-mediated sarcoplasmic reticulum Ca2+ stores. We recently reported that PLB is highly concentrated in the nuclear envelope (NE) from where it can modulate perinuclear Ca2+ handling of the cardiomyocytes (CMs). Since inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) mediates nuclear Ca2+ release, we examined whether the nuclear pool of PLB regulates IP3-induced nuclear Ca2+ handling. METHODS AND RESULTS: Fluo-4 based confocal Ca2+ imaging was performed to measure Ca2+ dynamics across both nucleus and cytosol in saponin-permeabilized CMs isolated from wild-type (WT) or PLB-knockout (PLB-KO) mice. At diastolic intracellular Ca2+ ([Ca2+]i = 100 nM), the Fab fragment of the monoclonal PLB antibody (anti-PLB Fab) facilitated the formation and increased the length of spontaneous Ca2+ waves (SCWs) originating from the nuclear region in CMs from WT but not from PLB-KO mice. We next examined nuclear Ca2+ activities at basal condition and after sequential addition of IP3, anti-PLB Fab, and the IP3R inhibitor 2-aminoethoxydiphenyl borate (2-APB) at a series of [Ca2+]i. In WT mice, at 10 nM [Ca2+]i where ryanodine receptor (RyR2) based spontaneous Ca2+ sparks rarely occurred, IP3 increased fluorescence amplitude (F/F0) of overall nuclear region to 1.19 ± 0.02. Subsequent addition of anti-PLB Fab significantly decreased F/F0 to 1.09 ± 0.02. At 50 nM [Ca2+]i, anti-PLB Fab not only decreased the overall nuclear F/F0 previously elevated by IP3, but also increased the amplitude and duration of spark-like nuclear Ca2+ release events. These nuclear Ca2+ releases were blocked by 2-APB. At 100 nM [Ca2+]i, IP3 induced short SCWs originating from nucleus. Anti-PLB Fab transformed those short waves into long SCWs with propagation from the nucleus into the cytosol. In contrast, neither nuclear nor cytosolic Ca2+ dynamics was affected by anti-PLB Fab in CMs from PLB-KO mice in all these conditions. Furthermore, in WT CMs pretreated with RyR2 blocker tetracaine, IP3 and anti-PLB Fab still increased the magnitude of nuclear Ca2+ release but failed to regenerate SCWs. Finally, anti-PLB Fab increased low Ca2+ affinity mag-fluo 4 fluorescence intensity in the lumen of NE of nuclei isolated from WT but not in PLB-KO mice. CONCLUSION: PLB regulates nuclear Ca2+ handling. By increasing Ca2+ uptake into lumen of the NE and perhaps other perinuclear membranes, the acute reversal of PLB inhibition decreases global Ca2+ concentration at rest in the nucleoplasm, and increases Ca2+ release into the nucleus, through mechanisms involving IP3R and RyR2 in the vicinity

Role of phosphorylation of Thr17 residue of phospholamban in mechanical recovery during hypercapnic acidosis

Objectives: To assess the time course of phosphorylation of phospholamban residues, the underlying mechanisms determining these phosphorylations, and their functional impact on the mechanical recovery during acidosis. Methods: Langendorff perfused rat hearts were submitted to 30 min of hypercapnic acidosis. Contractility, relaxation, and phosphorylation of phospholamban residues, immunodetected by specific antibodies, were determined. Results: Acidosis produced a mechanical impairment followed by a spontaneous recovery, most of which occurred within the first 3 min of acidosis (early recovery). During this period, contractility and relaxation recovered by 67±9% and 77±11%, respectively, from its maximal depression, together with an increase in the Ca2+-calmodulin-dependent protein kinase II (CaMKII)-dependent phosphorylation of Thr17. The CaMKII inhibitor KN-93, at 1, 5 and 10 μM, decreased Thr17 phosphorylation to basal levels and produced a similar impairment of the early relaxation recovery (50%). However, only 5 and 10 μM KN-93 inhibited the early contractile recovery and completely blunted the late mechanical recovery. Inhibition of the reverse mode of the Na+/Ca2+ exchanger by KB-R7943 decreased Thr17 phosphorylation but accelerated the early contractile recovery. Conclusions: CaMKII-dependent Thr17 phosphorylation significantly increased at the beginning of acidosis, is responsible for 50% of the early relaxation recovery, and is linked to the activation of the reverse Na+/Ca2+ mode. The early contractile recovery and the late mechanical recovery are dependent on CaMKII but independent of the phosphorylation of the Thr17 residue of phospholamban. The reverse Na+/Ca2+ mode has an additional negative effect that opposes the early mechanical recovery.Facultad de Ciencias MédicasCentro de Investigaciones Cardiovasculare

Role of phosphorylation of Thr17 residue of phospholamban in mechanical recovery during hypercapnic acidosis

Objectives: To assess the time course of phosphorylation of phospholamban residues, the underlying mechanisms determining these phosphorylations, and their functional impact on the mechanical recovery during acidosis. Methods: Langendorff perfused rat hearts were submitted to 30 min of hypercapnic acidosis. Contractility, relaxation, and phosphorylation of phospholamban residues, immunodetected by specific antibodies, were determined. Results: Acidosis produced a mechanical impairment followed by a spontaneous recovery, most of which occurred within the first 3 min of acidosis (early recovery). During this period, contractility and relaxation recovered by 67±9% and 77±11%, respectively, from its maximal depression, together with an increase in the Ca2+-calmodulin-dependent protein kinase II (CaMKII)-dependent phosphorylation of Thr17. The CaMKII inhibitor KN-93, at 1, 5 and 10 μM, decreased Thr17 phosphorylation to basal levels and produced a similar impairment of the early relaxation recovery (50%). However, only 5 and 10 μM KN-93 inhibited the early contractile recovery and completely blunted the late mechanical recovery. Inhibition of the reverse mode of the Na+/Ca2+ exchanger by KB-R7943 decreased Thr17 phosphorylation but accelerated the early contractile recovery. Conclusions: CaMKII-dependent Thr17 phosphorylation significantly increased at the beginning of acidosis, is responsible for 50% of the early relaxation recovery, and is linked to the activation of the reverse Na+/Ca2+ mode. The early contractile recovery and the late mechanical recovery are dependent on CaMKII but independent of the phosphorylation of the Thr17 residue of phospholamban. The reverse Na+/Ca2+ mode has an additional negative effect that opposes the early mechanical recovery.Facultad de Ciencias MédicasCentro de Investigaciones Cardiovasculare

Role of phospholamban phosphorylation on Thr17 in cardiac physiological and pathological conditions

The sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA2a) is under the control of a closely associated SR protein named phospholamban (PLN). Dephosphorylated PLN inhibits the SR Ca2+ pump, whereas phosphorylation of PLN, at either Ser16 by PKA or Thr17 by calmodulin-dependent protein kinase II (CaMKII), reverses this inhibition, thus increasing SERCA2a activity and the rate of Ca2+ uptake by the SR. This would in turn lead to an increase in the velocity of relaxation, SR Ca 2+ load, and myocardial contractility. Thus, PLN is a major determinant of cardiac contractility and relaxation. Although in the intact heart, β-adrenoceptor stimulation results in phosphorylation of PLN at both Ser16 and Thr17 residues, the role of Thr17 site has long remained equivocal. In this review, we attempt to highlight the signaling cascade and the physiological relevance of the phosphorylation of this residue in the heart under both physiological and pathological situations.Facultad de Ciencias Médica

Ankyrin-B Syndrome: Enhanced Cardiac Function Balanced by Risk of Cardiac Death and Premature Senescence

Here we report the unexpected finding that specific human ANK2 variants represent a new example of balanced human variants. The prevalence of certain ANK2 (encodes ankyrin-B) variants range from 2 percent of European individuals to 8 percent in individuals from West Africa. Ankyrin-B variants associated with severe human arrhythmia phenotypes (eg E1425G, V1516D, R1788W) were rare in the general population. Variants associated with less severe clinical and in vitro phenotypes were unexpectedly common. Studies with the ankyrin-B+/− mouse reveal both benefits of enhanced cardiac contractility, as well as costs in earlier senescence and reduced lifespan. Together these findings suggest a constellation of traits that we term “ankyrin-B syndrome”, which may contribute to both aging-related disorders and enhanced cardiac function