Protein phosphatase 5 regulates titin phosphorylation and function at a sarcomere-associated mechanosensor complex in cardiomyocytes.

Serine/threonine protein phosphatase 5 (PP5) is ubiquitously expressed in eukaryotic cells; however, its function in cardiomyocytes is unknown. Under basal conditions, PP5 is autoinhibited, but enzymatic activity rises upon binding of specific factors, such as the chaperone Hsp90. Here we show that PP5 binds and dephosphorylates the elastic N2B-unique sequence (N2Bus) of titin in cardiomyocytes. Using various binding and phosphorylation tests, cell-culture manipulation, and transgenic mouse hearts, we demonstrate that PP5 associates with N2Bus in vitro and in sarcomeres and is antagonistic to several protein kinases, which phosphorylate N2Bus and lower titin-based passive tension. PP5 is pathologically elevated and likely contributes to hypo-phosphorylation of N2Bus in failing human hearts. Furthermore, Hsp90-activated PP5 interacts with components of a sarcomeric, N2Bus-associated, mechanosensor complex, and blocks mitogen-activated protein-kinase signaling in this complex. Our work establishes PP5 as a compartmentalized, well-controlled phosphatase in cardiomyocytes, which regulates titin properties and kinase signaling at the myofilaments

P424Short-term ACE Inhibition upregulates cardiac expression of SERCA2a and protects against ventricular arrhythmias in healthy rats

Introduction: Chronic angiotensin converting enzyme inhibitor (ACEIs) treatment can suppress arrhythmogenesis. To examine whether the effect is more immediate and independent of suppression of pathological remodelling, we tested the antiarrhythmic effect of short-term ACE inhibition in healthy normotensive rats. Methods and results: Wistar rats were administered with enalaprilat (ENA, i.p., 5 mg/kg every 12 h) or vehicle (CON) for two weeks. Cellular shortening was measured in isolated, electrically paced cardiomyocytes. Standard 12-lead electrocardiography was performed and, hearts of anesthetized open-chest rats were subjected to 6-min ischemia followed by 10-minute reperfusion to examine susceptibility to ventricular arrhythmias. Expressions of calcium regulating proteins (SERCA2a, cardiac sarco/endoplasmic reticulum Ca2+-ATPase; CSQ, calsequestrin; TRD, triadin; PLB, phospholamban; FKBP12.6, FK506-binding protein) were measured by Western blot and mRNA levels of L-type calcium channel (Cacna1c), ryanodine receptor (Ryr2) and potassium channels Kcnh2 and Kcnq1 were measured by qRT-PCR. ENA decreased systolic as well as diastolic blood pressure (by 20%, and by 31%, respectively, for both P<0.05) but enhanced shortening of cardiomyocytes at basal conditions (by 34%, P<0.05) and under beta-adrenergic stimulation (by 73%, P<0.05). Enalaprilat shortened QTc interval duration (CON: 78±1 ms vs. ENA: 72±2 ms; P<0.05) and significantly decreased the total duration of ventricular fibrillations (VF) and the number of VF episodes (P<0.05). Reduction in arrhythmogenesis was associated with a pronounced upregulation of SERCA2a and increased Cacna1c mRNA levels. Conclusion: Short-term ACEI treatment can provide protection against I/R injury-induced ventricular arrhythmias in healthy myocardium and this effect is associated with increased SERCA2a expression. CON ENA Calcium regulating proteins SERCA2a 100±20 304±13* CSQ 100±6 105±7 TRD 100±16 117±10 PLB 100±9 109±16 FKBP12 100±12 93±

Protein phosphatase 2A affects myofilament contractility in non-failing but not in failing human myocardium

Protein phosphatase (PP) type 2A is a multifunctional serine/threonine phosphatase that is involved in cardiac excitation–contraction coupling. The PP2A core enzyme is a dimer, consisting of a catalytic C and a scaffolding A subunit, which is targeted to several cardiac proteins by a regulatory B subunit. At present, it is controversial whether PP2A and its subunits play a critical role in end-stage human heart failure. Here we report that the application of purified PP2AC significantly increased the Ca2+-sensitivity (ΔpCa50 = 0.05 ± 0.01) of the contractile apparatus in isolated skinned myocytes of non-failing (NF) hearts. A higher phosphorylation of troponin I (cTnI) was found at protein kinase A sites (Ser23/24) in NF compared to failing myocardium. The basal Ca2+-responsiveness of myofilaments was enhanced in myocytes of ischemic (ICM, ΔpCa50 = 0.10 ± 0.03) and dilated (DCM, ΔpCa50 = 0.06 ± 0.04) cardiomyopathy compared to NF. However, in contrast to NF myocytes the treatment with PP2AC did not shift force-pCa relationships in failing myocytes. The higher basal Ca2+-sensitivity in failing myocytes coincided with a reduced protein expression of PP2AC in left ventricular tissue from patients suffering from ICM and DCM (by 50 and 56% compared to NF, respectively). However, PP2A activity was unchanged in failing hearts despite an increase of both total PP and PP1 activity. The expression of PP2AB56α was also decreased by 51 and 62% in ICM and DCM compared to NF, respectively. The phosphorylation of cTnI at Ser23/24 was reduced by 66 and 49% in ICM and DCM compared to NF hearts, respectively. Our results demonstrate that PP2A increases myofilament Ca2+-sensitivity in NF human hearts, most likely via cTnI dephosphorylation. This effect is not present in failing hearts, probably due to the lower baseline cTnI phosphorylation in failing compared to non-failing hearts

Multiple Potential Molecular Contributors to Atrial Hypocontractility Caused by Atrial Tachycardia Remodeling in Dogs

Background-Atrial fibrillation impairs atrial contractility, inducing atrial stunning that promotes thromboembolic stroke. Action potential (AP)-prolonging drugs are reported to normalize atrial hypocontractility caused by atrial tachycardia remodeling (ATR). Here, we addressed the role of AP duration (APD) changes in ATR-induced hypocontractility. Methods and Results-ATR (7-day tachypacing) decreased APD (perforated patch recording) by approximate to 50%, atrial contractility (echocardiography, cardiomyocyte video edge detection), and [Ca2+](i) transients. ATR AP waveforms suppressed [Ca2+](i) transients and cell shortening of control cardiomyocytes; whereas control AP waveforms improved [Ca2+](i) transients and cell shortening in ATR cells. However, ATR cardiomyocytes clamped with the same control AP waveform had approximate to 60% smaller [Ca2+](i) transients and cell shortening than control cells. We therefore sought additional mechanisms of contractile impairment. Whole-cell voltage clamp revealed reduced I-CaL; I-CaL inhibition superimposed on ATR APs further suppressed [Ca2+](i) transients in control cells. Confocal microscopy indicated ATR-impaired propagation of the Ca2+ release signal to the cell center in association with loss of t-tubular structures. Myofilament function studies in skinned permeabilized cardiomyocytes showed altered Ca2+ sensitivity and force redevelopment in ATR, possibly due to hypophosphorylation of myosin-binding protein C and myosin light-chain protein 2a (immunoblot). Hypophosphorylation was related to multiple phosphorylation system abnormalities where protein kinase A regulatory subunits were downregulated, whereas autophosphorylation and expression of Ca2+-calmodulin-dependent protein kinase II delta and protein phosphatase 1 activity were enhanced. Recovery of [Ca2+](i) transients and cell shortening occurred in parallel after ATR cessation. Conclusions-Shortening of APD contributes to hypocontractility induced by 1-week ATR but accounts for it only partially. Additional contractility-suppressing mechanisms include I-CaL current reduction, impaired subcellular Ca2+ signal transmission, and altered myofilament function associated with abnormal myosin and myosin-associated protein phosphorylation. The complex mechanistic basis of the atrial hypocontractility associated with AF argues for upstream therapeutic targeting rather than interventions directed toward specific downstream pathophysiological derangements. (Circ Arrhythm Electrophysiol. 2010;3:530-541.

Expression and regulation of type 2A protein phosphatases and alpha4 signalling in cardiac health and hypertrophy

Abstract Cardiac physiology and hypertrophy are regulated by the phosphorylation status of many proteins, which is partly controlled by a poorly defined type 2A protein phosphatase-alpha4 intracellular signalling axis. Quantitative PCR analysis revealed that mRNA levels of the type 2A catalytic subunits were differentially expressed in H9c2 cardiomyocytes (PP2ACb[PP2ACa[PP4C[PP6C), NRVM (PP2ACb[PP2ACa = PP4C = PP6C), and adult rat ventricular myocytes (PP2ACa[ PP2ACb[PP6C[PP4C). Western analysis confirmed that all type 2A catalytic subunits were expressed in H9c2 cardiomyocytes; however, PP4C protein was absent in adult myocytes and only detectable following 26S proteasome inhibition. Short-term knockdown of alpha4 protein expression attenuated expression of all type 2A catalytic subunits. Pressure overload-induced left ventricular (LV) hypertrophy was associated with an increase in both PP2AC and alpha4 protein expression. Although PP6C expression was unchanged, expression of PP6C regulatory subunits (1) Sit4-associated protein 1 (SAP1) and (2) ankyrin repeat domain (ANKRD) 28 and 44 proteins was elevated, whereas SAP2 expression was reduced in hypertrophied LV tissue. Co-immunoprecipitation studies demonstrated that the interaction between alpha4 and PP2AC or PP6C subunits was either unchanged or reduced in hypertrophied LV tissue, respectively. Phosphorylation status of phospholemman (Ser63 and Ser68) was significantly increased by knockdown of PP2ACa, PP2ACb, or PP4C protein expression. DNA damage assessed by histone H2A.X phosphorylation (cH2A.X) in hypertrophied tissue remained unchanged. However, exposure of cardiomyocytes to H2O2 increased levels of cH2A.X which was unaffected by knockdown of PP6C expression, but was abolished by the short-term knockdown of alpha4 expression. This study illustrates the significance and altered activity of the type 2A protein phosphatase-alpha4 complex in healthy and hypertrophied myocardium

Ouabain treatment is associated with upregulation of phosphatase inhibitor-1 and Na+/Ca2+-exchanger and β-adrenergic sensitization in rat hearts

Cardiac glycosides are widely used in the treatment of congestive heart failure. While the mechanism of the positive inotropic effect after acute application of cardiac glycosides is explained by blockade of the Na +/K+-pump, little is known about consequences of a prolonged therapy. Here male Wistar rats were treated for 4 days with continuous infusions of ouabain (6.5mg/kg/day) or 0.9% NaCl (control) via osmotic minipumps. Electrically driven (1Hz, 35°C) papillary muscles from ouabain-treated rats exhibited shorter relaxation time (-15%) and a twofold increase in the sensitivity for the positive inotropic effect of isoprenaline. The density and affinity of β1- and β2- adrenoceptors as well as mRNA and protein levels of stimulatory (G sα) and inhibitory (Giα-2, G iα-3) G-proteins were unaffected by ouabain. Similarly, SR-Ca2+-ATPase 2A, phospholamban, ryanodine-receptor expression as well as the oxalate-stimulated 45Ca-uptake of membrane vesicles remained unchanged. However, mRNA abundance of the protein phosphatase inhibitor-1 (I-1) and the Na+/Ca2+-exchanger (NCX) were increased by 52% and 26%, respectively. I-1 plays an amplifier role in cardiac signaling. Downregulation of I-1 in human heart failure is associated with desensitization of the β-adrenergic signaling pathway. The present data suggest that the ouabain-induced increase in I-1 expression might be at least partly responsible for the increased isoprenaline sensitivity and increased expression of NCX for the accelerated relaxation after chronic ouabain in this model. © 2004 Elsevier Inc. All rights reserved.link_to_subscribed_fulltex