Role of protein phosphatases in regulation of cardiac inotropy and relaxation

Boknı́k, Peter; Khorchidi, S; Bodor, G.; Huke, Sabine; Knapp, Jörg; Linck, Bettina; Lüss, Hartmut; Müller, Frank; Schmitz, Wilhelm; Neumann, Joachim

doi:10.1152/ajpheart.2001.280.2.h786

Cited by 62 publications

(28 citation statements)

References 25 publications

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“…Phosphorylation at both sites, Ser 16 and Thr 17 , relieves the inhibitory effect of PLB on SERCA and enhances SR Ca 2ϩ uptake, although the exact contribution of each phosphorylation site and its specific effect on heart function is not clear (3,6,9). Interestingly, in SERCA2 (ϩ/Ϫ) mice we found an increase in the phosphorylation status of PLB.…”

Section: Discussionmentioning

confidence: 66%

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Altered dose response to β-agonists in SERCA1a-expressing hearts ex vivo and in vivo

Huke

Prasad

Nieman

et al. 2002

American Journal of Physiology-Heart and Circulatory Physiology

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. Altered dose response to ␤-agonists in SERCA1a-expressing hearts ex vivo and in vivo. Am J Physiol Heart Circ Physiol 283: H958-H965, 2002; 10.1152/ ajpheart.00078.2002. -In this study we evaluated the contractile characteristics of sarco(endo)plasmic reticulum Ca 2ϩ -ATPase (SERCA)1a-expressing hearts ex vivo and in vivo and in particular their response to ␤-adrenergic stimulation. Analysis of isolated, work-performing hearts revealed that transgenic (TG) hearts develop much higher maximal rates of contraction and relaxation than wild-type (WT) hearts. Addition of isoproterenol only moderately increased the maximal rate of relaxation (ϩ20%) but did not increase contractility or decrease relaxation time in TG hearts. Perfusion with varied buffer Ca 2ϩ concentrations indicated an altered dose response to Ca 2ϩ . In vivo TG hearts displayed fairly higher maximal rates of contraction (ϩ 25%) but unchanged relaxation parameters and a blunted but significant response to dobutamine. Our study also shows that the phospholamban (PLB) level was decreased (Ϫ40%) and its phosphorylation status modified in TG hearts. This study clearly demonstrates that increases in SERCA protein level alter the ␤-adrenergic response and affect the phosphorylation of PLB. Interestingly, the overall cardiac function in the live animal is only slightly enhanced, suggesting that (neuro)hormonal regulations may play an important role in controlling in vivo heart function. transgenic mice; contractility; sarco(endo)plasmic reticulum calcium adenosinetriphosphatase; phospholamban SEVERAL STUDIES HAVE SHOWN that sarco(endo)plasmic reticulum Ca 2ϩ -ATPase (SERCA) activity is decreased in animal models of cardiac hypertrophy and failure as well as in human heart failure (11). This decrease in sarcoplasmic reticulum (SR) Ca 2ϩ transport function was thought to contribute to abnormalities in Ca 2ϩ handling and contractile dysfunction in failing hearts (14). Adenoviral gene transfer of SERCA is currently being explored as a new therapeutic approach for the treatment of heart failure (7,(26)(27)(28)(29).Recent studies have shown that the fast-twitch skeletal muscle isoform SERCA1 has a higher Ca 2ϩ uptake activity than the cardiac isoform SERCA2a because of a higher Ca 2ϩ turnover rate when expressed in cardiac myocytes (34). To test whether SERCA1a can functionally substitute for SERCA2a and increase cardiac contractility, we have developed a transgenic (TG) mouse model expressing SERCA1a in the heart (16,19,23). In this model, SERCA1a expression resulted in a 2.5-fold increase in total SERCA pump protein level, but the expression of the endogenous SERCA2a isoform was reduced to ϳ50% in TG hearts. As a consequence 20% of the total number of SERCA pumps in TG hearts were type 2a whereas the majority of 80% were type 1a pumps. The contractility of isolated TG hearts was clearly increased despite the reduced expression of SERCA2a (23). SERCA1a pump expression was well tolerated without any signs of hypertrophy or other pathophysiological changes in the...

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Section: Discussionmentioning

confidence: 66%

“…Thus the PLB-to-SERCA ratio remains unchanged (15). Although this idea is not supported by findings in failing hearts (3,11), this mechanism might be functional solely in nonfailing hearts.…”

Section: Discussionmentioning

confidence: 85%

Altered dose response to β-agonists in SERCA1a-expressing hearts ex vivo and in vivo

Huke

Prasad

Nieman

et al. 2002

American Journal of Physiology-Heart and Circulatory Physiology

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“…CaMKII autophosphorylation was only observed when phosphatase 1 was inhibited, but then pacing strongly enhanced autophosphorylation. The increase in CaMKII autophosphorylation is likely due to direct inhibition of phosphatases dephosphorylating CaMKII, but it is also possible that phosphatase inhibition enhances CaMKII activation by increasing [Ca 2+ ] i (via an increase in the phosphorylation level of Ca 2+ -regulatory proteins; [38][39][40] We also show however that in the presence of okadaic acid the RyR Ser-2814 was the first site significantly increased in response to pacing, not global CaMKII Thr-287. This does not rule out that CaMKII specifically associated with the RyR does become significantly transiently activated and/or autophosphorylated sooner, especially when considering that the local [Ca 2+ ] around the RyR in the junctional cleft might reach very high concentrations during each excitation-contraction cycle [41][42][43].…”

Section: Does Pacing Activate Camkii and Increase Autophosphorylation?mentioning

confidence: 62%

Temporal dissociation of frequency-dependent acceleration of relaxation and protein phosphorylation by CaMKII

Huke

Bers

2007

Journal of Molecular and Cellular Cardiology

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Frequency-dependent acceleration of relaxation (FDAR) is an important intrinsic mechanism that allows for diastolic filling of the ventricle at higher heart rates, yet its molecular mechanism is still not understood. Previous studies showed that FDAR is dependent on functional sarcoplasmic reticulum (SR) and can be abolished by phosphatase or by Ca/CaM kinase (CaMKII) inhibition. Additionally, CaMKII activity/autophosphorylation has been shown to be frequency-dependent. Thus, we tested the hypothesis that CaMKII phosphorylation of SR Ca 2+ -handling proteins (Phospholamban (PLB), Ca 2+ release channel (RyR)) mediates FDAR. Here we show that FDAR occurs abruptly in fluo-4 loaded isolated rat ventricular myocytes when frequency is raised from 0.1 to 2 Hz. The effect is essentially complete within four beats (2 s) with the τ of [Ca 2+ ] i decline decreasing by 42±3%. While there is a detectable increase in PLB Thr-17 and RyR Ser-2814 phosphorylation, the increase is quantitatively small (PLB<5%, RyR~8%) and the time-course is clearly delayed with regard to FDAR. The low substrate phosphorylation indicates that pacing of myocytes only mildly activates CaMKII and consistent with this CaMKIIδ autophosphorylation did not increase with pacing alone. However, in the presence of phosphatase 1 inhibition pacing triggered a net-increase in autophosphorylated CaMKII and also greatly enhanced PLB and RyR phosphorylation. We conclude that FDAR does not rely on phosphorylation of PLB or RyR. Even though CaMKII does become activated when myocytes are paced, phosphatases immediately antagonize CaMKII action, limit substrate phosphorylation and also prevent sustained CaMKII autophosphorylation (thereby suppressing global CaMKII effects).

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“…Radioligand Binding Assay-Cardiac ventricular membranes were prepared, and binding assays were performed as published (7) using the nonselective ␤-AR ligand (Ϯ)-[ SDS-PAGE and Quantitative Immunoblotting-Preparation of LV homogenates, electrophoresis on polyacrylamide gels, transfer onto nitrocellulose membranes, and immunological detection of total PLB (not differentiating between phosphorylated and nonphosphorylated forms), PLB phosphorylated at Ser 16 , PLB phosphorylated at Thr 17 , SERCA2, calsequestrin, junctin, total CREB, and phosphorylated CREB were performed as described (7,12,13). We thank Dr. L. R. Jones (Indianapolis, IN) for providing SERCA2, calsequestrin, and junctin antibodies.…”

Section: Methodsmentioning

confidence: 99%

Heart-directed Expression of a Human Cardiac Isoform of cAMP-Response Element Modulator in Transgenic Mice

Müller

Lewin

Baba

et al. 2005

Journal of Biological Chemistry

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The transcriptional activation mediated by cAMP-response element (CRE) and transcription factors of the CRE-binding protein (CREB)/CRE modulator (CREM) family represents an important mechanism of cAMP-dependent gene regulation possibly implicated in detrimental effects of chronic ␤-adrenergic stimulation in end-stage heart failure. We studied the cardiac role of CREM in transgenic mice with heart-directed expression of CREM-Ib⌬C-X, a human cardiac CREM isoform. Transgenic mice displayed atrial enlargement with atrial and ventricular hypertrophy, developed atrial fibrillation, and died prematurely. In vivo hemodynamic assessment revealed increased contractility of transgenic left ventricles probably due to a selective up-regulation of SERCA2, the cardiac Ca 2؉ -ATPase of the sarcoplasmic reticulum. In transgenic ventricles, reduced phosphorylation of phospholamban and of the CREB was associated with increased activity of serine-threonine protein phosphatase 1. The density of ␤ 1 -adrenoreceptor was increased, and messenger RNAs encoding transcription factor dHAND and small G-protein RhoB were decreased in transgenic hearts as compared with wild-type controls. Our results indicate that heart-directed expression of CREM-Ib⌬C-X leads to complex cardiac alterations, suggesting CREM as a central regulator of cardiac morphology, function, and gene expression.The transcriptional activation mediated by the cAMP-response element (CRE) 1 and transcription factors of the CREbinding protein (CREB)/CRE modulator (CREM) family represents an important mechanism of cAMP-responsive gene control (1). CREB and CREM bind as homo-or heterodimers to the CRE, a palindromic consensus element in gene promoters of numerous target genes. One mechanism of CRE-mediated transcriptional activation is the cAMP-dependent protein kinase A (PKA)-dependent phosphorylation of a critical serine in activating isoforms of CREB or CREM, finally leading to activation of the transcriptional complex (2). Inhibitory CREM or CREB isoforms lack functional domains that mediate transcriptional activation or regulation by phosphorylation. Those repressors bind to the CRE as homodimers or as heterodimers in combination with other activating or inhibitory isoforms and suppress transcriptional activation by displacing functionally active dimers from the CRE.Several studies suggested that CRE-mediated transcriptional regulation plays an important role in cardiac gene regulation contributing to the pathophysiology of heart failure: (i) CREB and CREM are both expressed in human heart (3, 4); (ii) transgenic mice with heart-directed expression of a nonphosphorylatable, dominant-negative CREB isoform (dnCREB) (5) or of ATF3 (6), another repressor of CRE-mediated transcriptional activation, developed cardiac hypertrophy and signs of heart failure; and (iii) CREM-deficient mice (general knockout) displayed left ventricular dysfunction in the absence of hypertrophy and premature death (7,8).Here, we tested the role of CREM-Ib⌬C-X, a CREM isoform previously isolated from ...

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Role of protein phosphatases in regulation of cardiac inotropy and relaxation

Cited by 62 publications

References 25 publications

Altered dose response to β-agonists in SERCA1a-expressing hearts ex vivo and in vivo

Altered dose response to β-agonists in SERCA1a-expressing hearts ex vivo and in vivo

Temporal dissociation of frequency-dependent acceleration of relaxation and protein phosphorylation by CaMKII

Heart-directed Expression of a Human Cardiac Isoform of cAMP-Response Element Modulator in Transgenic Mice

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