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1.
Neth Heart J ; 27(2): 64-69, 2019 Feb.
Article in English | MEDLINE | ID: mdl-30547415

ABSTRACT

Pathogenic mutations in the phospholamban (PLN) gene may give rise to inherited cardiomyopathies due to its role in calcium homeostasis. Several PLN mutations have been identified, with the R14del mutation being the most prevalent cardiomyopathy-related mutation in the Netherlands. It is present in patients diagnosed with arrhythmogenic cardiomyopathy as well as dilated cardiomyopathy. Awareness of the phenotype of this PLN mutation is of great importance, since many carriers remain to be identified. Patients with the R14del mutation are characterised by older age at onset, low-voltage electrocardiograms and a high frequency of ventricular arrhythmias. Additionally, these patients have a poor prognosis often with left ventricular dysfunction and early-onset heart failure. Therefore, when there is a suspicion of a PLN mutation, cardiac and genetic screening is strongly recommended.

2.
J Physiol ; 594(11): 3005-30, 2016 06 01.
Article in English | MEDLINE | ID: mdl-26695843

ABSTRACT

KEY POINTS: Mice with Ca(2+) -calmodulin-dependent protein kinase (CaMKII) constitutive pseudo-phosphorylation of the ryanodine receptor RyR2 at Ser2814 (S2814D(+/+) mice) exhibit a higher open probability of RyR2, higher sarcoplasmic reticulum (SR) Ca(2+) leak in diastole and increased propensity to arrhythmias under stress conditions. We generated phospholamban (PLN)-deficient S2814D(+/+) knock-in mice by crossing two colonies, S2814D(+/+) and PLNKO mice, to test the hypothesis that PLN ablation can prevent the propensity to arrhythmias of S2814D(+/+) mice. PLN ablation partially rescues the altered intracellular Ca(2+) dynamics of S2814D(+/+) hearts and myocytes, but enhances SR Ca(2+) sparks and leak on confocal microscopy. PLN ablation diminishes ventricular arrhythmias promoted by CaMKII phosphorylation of S2814 on RyR2. PLN ablation aborts the arrhythmogenic SR Ca(2+) waves of S2814D(+/+) and transforms them into non-propagating events. A mathematical human myocyte model replicates these results and predicts the increase in SR Ca(2+) uptake required to prevent the arrhythmias induced by a CaMKII-dependent leaky RyR2. ABSTRACT: Mice with constitutive pseudo-phosphorylation at Ser2814-RyR2 (S2814D(+/+) ) have increased propensity to arrhythmias under ß-adrenergic stress conditions. Although abnormal Ca(2+) release from the sarcoplasmic reticulum (SR) has been linked to arrhythmogenesis, the role played by SR Ca(2+) uptake remains controversial. We tested the hypothesis that an increase in SR Ca(2+) uptake is able to rescue the increased arrhythmia propensity of S2814D(+/+) mice. We generated phospholamban (PLN)-deficient/S2814D(+/+) knock-in mice by crossing two colonies, S2814D(+/+) and PLNKO mice (SD(+/+) /KO). SD(+/+) /KO myocytes exhibited both increased SR Ca(2+) uptake seen in PLN knock-out (PLNKO) myocytes and diminished SR Ca(2+) load (relative to PLNKO), a characteristic of S2814D(+/+) myocytes. Ventricular arrhythmias evoked by catecholaminergic challenge (caffeine/adrenaline) in S2814D(+/+) mice in vivo or programmed electric stimulation and high extracellular Ca(2+) in S2814D(+) /(-) hearts ex vivo were significantly diminished by PLN ablation. At the myocyte level, PLN ablation converted the arrhythmogenic Ca(2+) waves evoked by high extracellular Ca(2+) provocation in S2814D(+/+) mice into non-propagated Ca(2+) mini-waves on confocal microscopy. Myocyte Ca(2+) waves, typical of S2814D(+/+) mice, could be evoked in SD(+/+) /KO cells by partially inhibiting SERCA2a. A mathematical human myocyte model replicated these results and allowed for predicting the increase in SR Ca(2+) uptake required to prevent the arrhythmias induced by a Ca(2+) -calmodulin-dependent protein kinase (CaMKII)-dependent leaky RyR2. Our results demonstrate that increasing SR Ca(2+) uptake by PLN ablation can prevent the arrhythmic events triggered by SR Ca(2+) leak due to CaMKII-dependent phosphorylation of the RyR2-S2814 site and underscore the benefits of increasing SERCA2a activity on SR Ca(2+) -triggered arrhythmias.


Subject(s)
Arrhythmias, Cardiac/metabolism , Calcium-Binding Proteins/deficiency , Calcium-Calmodulin-Dependent Protein Kinase Type 2/metabolism , Ryanodine Receptor Calcium Release Channel/metabolism , Action Potentials/physiology , Animals , Arrhythmias, Cardiac/genetics , Arrhythmias, Cardiac/physiopathology , Calcium/metabolism , Calcium-Binding Proteins/genetics , Calcium-Calmodulin-Dependent Protein Kinase Type 2/genetics , Gene Knock-In Techniques , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Myocytes, Cardiac/metabolism , Myocytes, Cardiac/physiology , Phosphorylation/physiology , Ryanodine Receptor Calcium Release Channel/genetics
3.
J Mol Cell Cardiol ; 60: 151-60, 2013 Jul.
Article in English | MEDLINE | ID: mdl-23632046

ABSTRACT

Nebulette (NEBL) is a sarcomeric Z-disk protein involved in mechanosensing and force generation via its interaction with actin and tropomyosin-troponin complex. Genetic abnormalities in NEBL lead to dilated cardiomyopathy (DCM) in humans and animal models. The objectives of this study are to determine the earliest preclinical mechanical changes in the myocardium and define underlying molecular mechanisms by which NEBL mutations lead to cardiac dysfunction. We examined cardiac function in 3-month-old non-transgenic (non-Tg) and transgenic (Tg) mice (WT-Tg, G202R-Tg, A592E-Tg) by cardiac magnetic resonance (CMR) imaging. Contractility and calcium transients were measured in isolated cardiomyocytes. A592E-Tg mice exhibited enhanced in vivo twist and untwisting rate compared to control groups. Ex vivo analysis of A592E-Tg cardiomyocytes showed blunted calcium decay response to isoproterenol. CMR imaging of G202R-Tg mice demonstrated reduced torsion compared to non-Tg and WT-Tg, but conserved twist and untwisting rate after correcting for geometric changes. Ex vivo analysis of G202R-Tg cardiomyocytes showed elevated calcium decay at baseline and a conserved contractile response to isoproterenol stress. Protein analysis showed decreased α-actinin and connexin43, and increased cardiac troponin I phosphorylation at baseline in G202R-Tg, providing a molecular mechanism for enhanced ex vivo calcium decay. Ultrastructurally, G202R-Tg cardiomyocytes exhibited increased I-band and sarcomere length, desmosomal separation, and enlarged t-tubules. A592E-Tg cardiomyocytes also showed abnormal ultrastructural changes and desmin downregulation. This study showed distinct effects of NEBL mutations on sarcomere ultrastructure, cellular contractile function, and calcium homeostasis in preclinical DCM in vivo. We suggest that these abnormalities correlate with detectable myocardial wall motion patterns.


Subject(s)
Calcium Signaling , Cardiomegaly/metabolism , Cytoskeletal Proteins/metabolism , Heart Defects, Congenital/metabolism , LIM Domain Proteins/metabolism , Mutation , Myocardium/metabolism , Myocytes, Cardiac/metabolism , Actinin/genetics , Actinin/metabolism , Animals , Cardiomegaly/genetics , Cardiomegaly/pathology , Cytoskeletal Proteins/genetics , Heart Defects, Congenital/genetics , Heart Defects, Congenital/pathology , LIM Domain Proteins/genetics , Mice , Mice, Transgenic , Myocardial Contraction/genetics , Myocardium/pathology , Myocytes, Cardiac/pathology , Sarcomeres/genetics , Sarcomeres/metabolism , Sarcomeres/pathology
4.
Subcell Biochem ; 45: 523-37, 2007.
Article in English | MEDLINE | ID: mdl-18193651

ABSTRACT

Regulation of Calcium (Ca) cycling by the sarcoplasmic reticulum (SR) underlies the control of cardiac contraction during excitation-contraction (E-C) coupling. Moreover, alterations in E-C coupling occurring in cardiac hypertrophy and heart failure are characterized by abnormal Ca-cycling through the SR network. A large body of evidence points to the central role of: a) SERCA and its regulator phospholamban (PLN) in the modulation of cardiac relaxation; b) calsequestrin in the regulation of SR Ca-load; and c) the ryanodine receptor (RyR) Ca-channel in the control of SR Ca-release. The levels or activity of these key Ca-handling proteins are altered in cardiomyopathies, and these changes have been linked to the deteriorated cardiac function and remodeling. Furthermore, genetic variants in these SR Ca-cycling proteins have been identified, which may predispose to heart failure or fatal arrhythmias. This chapter concentrates on the pivotal role of SR Ca-cycling proteins in health and disease with specific emphasis on their recently reported genetic modifiers.


Subject(s)
Calcium/physiology , Cardiomyopathies/physiopathology , Calcium Signaling , Calcium-Binding Proteins/genetics , Calcium-Binding Proteins/physiology , Calcium-Calmodulin-Dependent Protein Kinases/physiology , Calsequestrin/genetics , Cardiomyopathies/genetics , Heart Failure/genetics , Heart Failure/physiopathology , Humans , Mutation , Receptors, Adrenergic, beta/physiology , Ryanodine Receptor Calcium Release Channel/physiology , Sarcoplasmic Reticulum/physiology , Sarcoplasmic Reticulum Calcium-Transporting ATPases/genetics , Sarcoplasmic Reticulum Calcium-Transporting ATPases/physiology
5.
Braz J Med Biol Res ; 39(5): 563-72, 2006 May.
Article in English | MEDLINE | ID: mdl-16648892

ABSTRACT

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, beta-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 beta-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.


Subject(s)
Acidosis/metabolism , Calcium-Binding Proteins/metabolism , Myocardial Stunning/metabolism , Sarcoplasmic Reticulum Calcium-Transporting ATPases/metabolism , Threonine/metabolism , Acidosis/physiopathology , Animals , Calcium-Binding Proteins/physiology , Myocardial Contraction/physiology , Myocardial Stunning/physiopathology , Phosphorylation , Threonine/physiology
6.
Braz. j. med. biol. res ; 39(5): 563-572, May 2006. ilus, graf
Article in English | LILACS | ID: lil-425791

ABSTRACT

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.


Subject(s)
Animals , Acidosis/metabolism , Calcium-Binding Proteins/metabolism , Myocardial Stunning/metabolism , Sarcoplasmic Reticulum Calcium-Transporting ATPases/metabolism , Threonine/metabolism , Acidosis/physiopathology , Calcium-Binding Proteins/physiology , Myocardial Contraction/physiology , Myocardial Stunning/physiopathology , Phosphorylation , Threonine/physiology
7.
Arch Mal Coeur Vaiss ; 98(12): 1239-43, 2005 Dec.
Article in English | MEDLINE | ID: mdl-16435604

ABSTRACT

Our knowledge and understanding of the normal and diseased heart has advanced significantly over the past decade. Evidence indicates that several signaling pathways involved in the induction of cardiac disease and heart failure are associated with abnormal calcium handling by the sarcoplasmic reticulum proteins: calcium-ATPase pump and phospholamban. Indeed, the failing heart is characterized by impaired removal of cytosolic calcium, reduced loading of the cardiac sarcoplasmic reticulum, and defective calcium release, culminating in impairment of cardiac diastolic and systolic function. This review summarizes studies which highlight the key role of the sarcoplasmic reticulum proteins, calcium-ATPase pump and phospholamban, in the regulation of cardiac function; the significance of the phospholamban interaction with the calcium-ATPase pump through transgenic animal models; the recent findings of the inhbitor-1 of protein phosphatase-1 as a new potential therapeutic agent in heart failure; and finally, the discoveries of human phospholamban mutations leading to disease states.


Subject(s)
Calcium-Binding Proteins/metabolism , Heart Failure/metabolism , Myocardial Contraction , Animals , Calcium/metabolism , Calcium-Binding Proteins/genetics , Calcium-Transporting ATPases/metabolism , Enzyme Inhibitors/therapeutic use , Heart Failure/drug therapy , Heart Failure/genetics , Heart Failure/physiopathology , Humans , Myocardium/metabolism , Proteins/therapeutic use , Sarcoplasmic Reticulum/metabolism , Sarcoplasmic Reticulum Calcium-Transporting ATPases
8.
Am J Physiol Heart Circ Physiol ; 285(3): H1198-205, 2003 Sep.
Article in English | MEDLINE | ID: mdl-12763747

ABSTRACT

Phosphorylation of phospholamban (PLB) at Ser16 (protein kinase A site) and at Thr17 [Ca2+/calmodulin kinase II (CaMKII) site] increases sarcoplasmic reticulum Ca2+ uptake and myocardial contractility and relaxation. In perfused rat hearts submitted to ischemia-reperfusion, we previously showed an ischemia-induced Ser16 phosphorylation that was dependent on beta-adrenergic stimulation and an ischemia and reperfusion-induced Thr17 phosphorylation that was dependent on Ca2+ influx. To elucidate the relationship between these two PLB phosphorylation sites and postischemic mechanical recovery, rat hearts were submitted to ischemia-reperfusion in the absence and presence of the CaMKII inhibitor KN-93 (1 microM) or the beta-adrenergic blocker dl-propranolol (1 microM). KN-93 diminished the reperfusion-induced Thr17 phosphorylation and depressed the recovery of contraction and relaxation after ischemia. dl-Propranolol decreased the ischemia-induced Ser16 phosphorylation but failed to modify the contractile recovery. To obtain further insights into the functional role of the two PLB phosphorylation sites in postischemic mechanical recovery, transgenic mice expressing wild-type PLB (PLB-WT) or PLB mutants in which either Thr17 or Ser16 were replaced by Ala (PLB-T17A and PLB-S16A, respectively) into the PLB-null background were used. Both PLB mutants showed a lower contractile recovery than PLB-WT. However, this recovery was significantly impaired all along reperfusion in PLB-T17A, whereas it was depressed only at the beginning of reperfusion in PLB-S16A. Moreover, the recovery of relaxation was delayed in PLB-T17A, whereas it did not change in PLB-S16A, compared with PLB-WT. These findings indicate that, although both PLB phosphorylation sites are involved in the mechanical recovery after ischemia, Thr17 appears to play a major role.


Subject(s)
Calcium-Binding Proteins/genetics , Calcium-Binding Proteins/metabolism , Myocardial Stunning/metabolism , Myocardial Stunning/physiopathology , Amino Acid Substitution , Animals , Male , Mice , Mice, Inbred BALB C , Mice, Transgenic , Myocardial Contraction/physiology , Myocardial Reperfusion Injury/metabolism , Myocardial Reperfusion Injury/physiopathology , Phosphorylation , Rats , Rats, Wistar
9.
Novartis Found Symp ; 246: 228-38; discussion 238-43, 272-6, 2002.
Article in English | MEDLINE | ID: mdl-12164311

ABSTRACT

Smooth muscle Ca2+ handling is of major importance to understanding its function. A new approach utilizes molecular biology to develop transgenic mouse models in which the protein constituents of the various Ca2+ regulatory subsystems have been altered. Gene-targeted or gene-ablated (knockout) mice have been reported for the sarcoplasmic reticulum (SR) Ca2+ pump isoforms SERCA2, SERCA2a and SERCA3, the plasma membrane Ca2+ pump isoforms, PMCA1, PMCA2 and PMCA4, and the SR-associated protein, phospholamban (PLB), an inhibitor of SERCA2. A mouse line carrying a transgene for the smooth muscle specific expression of PLB has been reported. Evidence from studies using these mice combined with the classical pharmacological approaches has provided new insight into the relative role of the SR. We review this field with particular emphasis on PLB, since its modulation of SR function and smooth muscle contractility has the largest database. PLB via modulation of SERCA can play a major role in regulation of both phasic and tonic smooth muscle contractility. The use of transgenic mice has yielded surprises ,uch as PLB modulation of endothelial cell Ca2+ homeostasis, and the demonstration that PLB is the major site for A-kinase-mediated relaxation of mouse bladder. The use of these gene-altered models has provided evidence clearly implicating a major role for the SR in modulating smooth muscle Ca2+ and contractility, with the caveat that this modulation is tissue specific.


Subject(s)
Muscle Contraction/physiology , Muscle, Smooth/physiology , Sarcoplasmic Reticulum/physiology , Animals , Calcium Signaling/physiology , Calcium-Binding Proteins/deficiency , Calcium-Binding Proteins/genetics , Calcium-Binding Proteins/physiology , Calcium-Transporting ATPases/genetics , Mice , Mice, Knockout , Mice, Transgenic , Sarcoplasmic Reticulum Calcium-Transporting ATPases
10.
J Physiol ; 535(Pt 3): 867-78, 2001 Sep 15.
Article in English | MEDLINE | ID: mdl-11559781

ABSTRACT

1. Phospholamban (PLB) is an inhibitor of the sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA). Its presence and/or functional significance in contractility of bladder, a smooth muscle tissue particularly dependent on SR function, is unknown. We investigated this by measuring the effects of carbachol (CCh) on force and [Ca2+]i in bladder from mice in which the PLB gene was ablated (PLB-KO mice). In the PLB-KO bladder, the maximum increases in [Ca2+]i and force were significantly decreased (41.5 and 47.4 % of WT), and the EC50 values increased. 2. Inhibition of SERCA with cyclopiazonic acid (CPA) abolished these differences between WT and PLB-KO bladder, localizing the effects to the SR. 3. To determine whether these effects were specific to PLB, we generated mice with smooth-muscle-specific expression of PLB (PLB-SMOE mice), using the SMP8 alpha-actin promoter. Western blot analysis of PLB-SMOE mice showed approximately an eightfold overexpression of PLB while SERCA was downregulated 12-fold. 4. In PLB-SMOE bladders, in contrast, the response of [Ca2+]i and force to CCh was significantly increased and the EC50 values were decreased. CPA had little affect on the CCh-induced increases in [Ca2+]i and force in PLB-SMOE bladder. 5. These results show that alteration of the PLB:SERCA ratio can significantly modulate smooth muscle [Ca2+]i. Importantly, our data show that PLB can play a major role in modulation of bladder contractility.


Subject(s)
Calcium-Binding Proteins/physiology , Urinary Bladder/physiology , Animals , Blotting, Western , Calcium/metabolism , Carbachol/antagonists & inhibitors , Carbachol/pharmacology , Down-Regulation/physiology , In Vitro Techniques , Isometric Contraction/drug effects , Isometric Contraction/physiology , Mice , Mice, Knockout , Mice, Transgenic , Muscarinic Agonists/pharmacology , Muscle Contraction/drug effects , Muscle Contraction/physiology , Sarcoplasmic Reticulum/drug effects , Sarcoplasmic Reticulum/metabolism , Urinary Bladder/drug effects
12.
J Physiol ; 534(Pt. 2): 357-66, 2001 Jul 15.
Article in English | MEDLINE | ID: mdl-11454956

ABSTRACT

1. The Ca(2+) sensitivity of smooth muscle contractility is modulated via regulation of phosphatase activity. Protein phosphatase inhibitor-1 (I-1) is the classic type-1 phosphatase inhibitor, but its presence and role in cAMP-dependent protein kinase (PKA) modulation of smooth muscle is unclear. To address the relevance of I-1 in vivo, we investigated smooth muscle function in a mouse model lacking the I-1 protein (I-1((-/-)) mice). 2. Significant amounts of I-1 protein were detected in the wild-type (WT) mouse aorta and could be phosphorylated by PKA, as indicated by (32)P-labelled aortic extracts from WT mice. 3. Despite the significant presence of I-1 in WT aorta, phenylephrine and KCl concentration- isometric force relations in the presence or absence of the PKA pathway activator isoproterenol (isoprenaline) were unchanged compared to I-1((-/-)) aorta. cGMP-dependent protein kinase (PKG) relaxation pathways were also not different. Consistent with these findings, dephosphorylation rates of the 20 kDa myosin light chains (MLC(20)), measured in aortic extracts, were nearly identical between WT and I-1((-/-)) mice. 4. In the portal vein, I-1 protein ablation was associated with a significant (P < 0.05) rightward shift in the EC(50) of isoproterenol relaxation (EC(50) = 10.4 +/- 1.4 nM) compared to the WT value (EC(50) = 3.5 +/- 0.2 nM). Contraction in response to acetylcholine as well as Ca(2+) sensitivity were similar between WT and I-1((-/-)) aorta. 5. Despite the prevalence of I-1 and its activation by PKA in the aorta, I-1 does not appear to play a significant role in contractile or relaxant responses to any pharmacomechanical or electromechanical agonists used. I-1 may play a role as a fine-tuning mechanism involved in regulating portal vein responsiveness to beta-adrenergic agonists.


Subject(s)
Carrier Proteins , Intracellular Signaling Peptides and Proteins , Muscle, Smooth, Vascular/enzymology , Phosphoprotein Phosphatases/metabolism , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Acetylcholine/pharmacology , Animals , Aorta/enzymology , Calcium/pharmacology , Colforsin/pharmacology , Mice , Mice, Inbred C57BL , Mice, Knockout , Myosin Light Chains/metabolism , Myosin-Light-Chain Phosphatase , Nitroprusside/pharmacology , Phenylephrine/pharmacology , Phosphorylation , Portal Vein/enzymology , Potassium Chloride/pharmacology , Receptors, Adrenergic, beta/metabolism , Vasoconstriction/drug effects , Vasoconstriction/physiology , Vasoconstrictor Agents/pharmacology , Vasodilation/drug effects , Vasodilation/physiology , Vasodilator Agents/pharmacology
13.
J Mol Cell Cardiol ; 33(7): 1345-53, 2001 Jul.
Article in English | MEDLINE | ID: mdl-11437540

ABSTRACT

Gender has recently been implicated as an important modulator of cardiovascular disease. However, it is not known how gender may specifically influence the Ca2+-handling deficits that characterize the depressed cardiac contractility of human heart failure. To elucidate the contributory role of gender to sarcoplasmic reticulum (SR) Ca2+ cycling alterations, the protein levels of SR Ca2+-ATPase (SERCA), phospholamban, and calsequestrin, as well as the site-specific phospholamban phosphorylation status, were quantified in a mixed gender population of failing (n=14) and donor (n=15) myocardia. The apparent affinity (EC50) and the maximal velocity (Vmax) of SR Ca2+-uptake were also determined to lend functional significance to any observed protein alterations. Phospholamban and calsequestrin levels were not altered; however, SERCA protein levels were significantly reduced in failing hearts. Additionally, phospholamban phosphorylation (serine-16 and threonine-17 sites) and myocardial cAMP content were both attenuated. The alterations in SR protein levels were also accompanied by a decreased V(max)and an increased EC50 (diminished apparent affinity) of SR Ca2+-uptake for Ca2+ in failing myocardia. Myocardial protein levels and Ca2+ uptake parameters were then analyzed with respect to gender, which revealed that the decreases in phosphorylated serine-16 were specific to male failing hearts, reflecting increases in the EC50 values of SR Ca2+-uptake for Ca2+, compared to donor males. These findings suggest that although decreased SERCA protein and phospholamban phosphorylation levels contribute to depressed SR Ca2+-uptake and left ventricular function in heart failure, the specific subcellular alterations which underlie these effects may not be uniform with respect to gender.


Subject(s)
Calcium-Transporting ATPases/metabolism , Calcium/metabolism , Heart Failure/physiopathology , Myocardial Contraction/physiology , Sarcoplasmic Reticulum/metabolism , Adult , Calcium-Binding Proteins/metabolism , Calsequestrin/metabolism , Cyclic AMP/metabolism , Female , Humans , In Vitro Techniques , Male , Middle Aged , Sex Factors
14.
J Mol Cell Cardiol ; 33(5): 1031-40, 2001 May.
Article in English | MEDLINE | ID: mdl-11343424

ABSTRACT

J. P. Slack, I. L. Grupp, R. Dash, D. Holder, A. Schmidt, M. J. Gerst, T. Tamura, C. Tilgmann, P. F. James, R. Johnson, A. M. Gerdes and E. G. Kranias. The Enhanced Contractility of the Phospholamban-deficient Mouse Heart Persists with Aging. Journal of Molecular and Cellular Cardiology (2001) 33, 1031-1040. Phospholamban ablation in the mouse is associated with significant increases in cardiac contractility. To determine whether this hyperdynamic function persists through the aging process, a longitudinal examination of age-matched phospholamban-deficient and wild-type mice was employed. Kaplan-Meier survival curves indicated no significant differences between phospholamban-deficient and wild-type mice over the first year. Examination of cardiac function revealed significant increases in the rates of contraction (+dP/dt) and relaxation (-dP/dt) in phospholamban-deficient hearts compared with their wild-type counterparts at 3, 6, 12, 18 and 24 months of age. Quantitative immunoblotting indicated that the expression levels of the sarcoplasmic reticulum Ca(2+)-ATPase were not altered in wild-type hearts, while they were significantly decreased at 12 months (40%) and 18 months (20%) in phospholamban-deficient hearts. These findings on the persistence of hyperdynamic cardiac function over the long term suggest that phospholamban may constitute an important target for treatment in heart disease.


Subject(s)
Aging , Calcium-Binding Proteins/genetics , Calcium-Binding Proteins/physiology , Heart/physiology , Myocardial Contraction , Myocardium/metabolism , Age Factors , Animals , Blotting, Western , Echocardiography , Female , Genotype , Immunoblotting , Male , Mice , Time Factors
15.
J Biol Chem ; 276(26): 24145-52, 2001 Jun 29.
Article in English | MEDLINE | ID: mdl-11328820

ABSTRACT

To determine whether selective impairment of cardiac sarcoplasmic reticulum (SR) Ca(2+) transport may drive the progressive functional deterioration leading to heart failure, transgenic mice, overexpressing a phospholamban Val(49) --> Gly mutant (2-fold), which is a superinhibitor of SR Ca(2+)-ATPase affinity for Ca(2+), were generated, and their cardiac phenotype was examined longitudinally. At 3 months of age, the increased EC(50) level of SR Ca(2+) uptake for Ca(2+) (0.67 +/- 0.09 microm) resulted in significantly higher depression of cardiomyocyte rates of shortening (57%), relengthening (31%), and prolongation of the Ca(2+) signal decay time (165%) than overexpression (2-fold) of wild type phospholamban (68%, 64%, and 125%, respectively), compared with controls (100%). Echocardiography also revealed significantly depressed function and impaired beta-adrenergic responses in mutant hearts. The depressed contractile parameters were associated with left ventricular remodeling, recapitulation of fetal gene expression, and hypertrophy, which progressed to dilated cardiomyopathy with interstitial tissue fibrosis and death by 6 months in males. Females also had ventricular hypertrophy at 3 months but exhibited normal systolic function up to 12 months of age. These results suggest a causal relationship between defective SR Ca(2+) cycling and cardiac remodeling leading to heart failure, with a gender-dependent influence on the time course of these alterations.


Subject(s)
Calcium-Binding Proteins/physiology , Heart Failure/etiology , Myocardial Contraction , Sarcoplasmic Reticulum/physiology , Aging , Animals , Calcium/metabolism , Calcium Channels, L-Type/physiology , Calcium-Binding Proteins/genetics , Calcium-Transporting ATPases/antagonists & inhibitors , Cardiomegaly/etiology , Cardiomegaly/metabolism , Cardiomegaly/physiopathology , Cells, Cultured , Echocardiography , Female , Heart Failure/metabolism , Heart Failure/physiopathology , Male , Mice , Mice, Transgenic , Myocardium/metabolism , Myocardium/pathology , Point Mutation , Sarcoplasmic Reticulum Calcium-Transporting ATPases , Sex Factors , Survival Rate
16.
J Clin Invest ; 107(8): 967-74, 2001 Apr.
Article in English | MEDLINE | ID: mdl-11306600

ABSTRACT

The medical treatment of chronic heart failure has undergone a dramatic transition in the past decade. Short-term approaches for altering hemodynamics have given way to long-term, reparative strategies, including beta-adrenergic receptor (betaAR) blockade. This was once viewed as counterintuitive, because acute administration causes myocardial depression. Cardiac myocytes from failing hearts show changes in betaAR signaling and excitation-contraction coupling that can impair cardiac contractility, but the role of these abnormalities in the progression of heart failure is controversial. We therefore tested the impact of different manipulations that increase contractility on the progression of cardiac dysfunction in a mouse model of hypertrophic cardiomyopathy. High-level overexpression of the beta(2)AR caused rapidly progressive cardiac failure in this model. In contrast, phospholamban ablation prevented systolic dysfunction and exercise intolerance, but not hypertrophy, in hypertrophic cardiomyopathy mice. Cardiac expression of a peptide inhibitor of the betaAR kinase 1 not only prevented systolic dysfunction and exercise intolerance but also decreased cardiac remodeling and hypertrophic gene expression. These three manipulations of cardiac contractility had distinct effects on disease progression, suggesting that selective modulation of particular aspects of betaAR signaling or excitation-contraction coupling can provide therapeutic benefit.


Subject(s)
Calcium Signaling , Cardiomyopathy, Hypertrophic/physiopathology , Receptors, Adrenergic, beta-2/metabolism , Actins/genetics , Animals , Atrial Natriuretic Factor/genetics , Biomarkers , Calcium/metabolism , Calcium-Binding Proteins/metabolism , Cardiomyopathy, Hypertrophic/metabolism , Cardiomyopathy, Hypertrophic/pathology , Cyclic AMP-Dependent Protein Kinases/antagonists & inhibitors , Cyclic AMP-Dependent Protein Kinases/genetics , Disease Models, Animal , Disease Progression , Female , Gene Expression , Heart Failure/pathology , Male , Mice , Mice, Transgenic , Motor Activity , Myocardium/metabolism , Myocardium/pathology , Myosin Heavy Chains/genetics , Receptors, Adrenergic, beta-2/genetics , beta-Adrenergic Receptor Kinases
17.
Circulation ; 103(6): 889-96, 2001 Feb 13.
Article in English | MEDLINE | ID: mdl-11171800

ABSTRACT

BACKGROUND: Relieving the inhibition of sarcoplasmic reticular function by phospholamban is a major target of beta-adrenergic stimulation. Chronic beta-adrenergic receptor activity has been suggested to be detrimental, on the basis of transgenic overexpression of the receptor or its signaling effectors. However, it is not known whether physiological levels of sympathetic tone, in the absence of preexisting heart failure, are similarly detrimental. METHODS AND RESULTS: Transgenic mice overexpressing phospholamban at 4-fold normal levels were generated, and at 3 months, they exhibited mildly depressed ventricular contractility without heart failure. As expected, transgenic cardiomyocyte mechanics and calcium kinetics were depressed, but isoproterenol reversed the inhibitory effects of phospholamban on these parameters. In vivo cardiac function was substantially depressed by propranolol administration, suggesting enhanced sympathetic tone. Indeed, plasma norepinephrine levels and the phosphorylation status of phospholamban were elevated, reflecting increased adrenergic drive in transgenic hearts. On aging, the chronic enhancement of adrenergic tone was associated with a desensitization of adenylyl cyclase (which intensified the inhibitory effects of phospholamban), the development of overt heart failure, and a premature mortality. CONCLUSIONS: The unique interaction between phospholamban and increased adrenergic drive, elucidated herein, provides the first evidence that compensatory increases in catecholamine stimulation can, even in the absence of preexisting heart failure, be a primary causative factor in the development of cardiomyopathy and early mortality.


Subject(s)
Aging , Calcium-Binding Proteins/metabolism , Cardiomyopathies/etiology , Receptors, Adrenergic, beta/metabolism , Adenylyl Cyclases/metabolism , Adrenergic beta-Antagonists/pharmacology , Animals , Calcium-Binding Proteins/biosynthesis , Calcium-Binding Proteins/genetics , Cardiomyopathies/blood , Cardiomyopathies/mortality , Echocardiography , Heart Failure/etiology , Heart Failure/mortality , Isoproterenol/pharmacology , Mice , Mice, Transgenic , Myocardium/metabolism , Myocardium/pathology , Norepinephrine/blood , Phosphorylation , Propranolol/pharmacology , Ventricular Function, Left
18.
J Biol Chem ; 276(12): 9392-9, 2001 Mar 23.
Article in English | MEDLINE | ID: mdl-11115498

ABSTRACT

Cardiac-specific overexpression of murine cardiac calsequestrin results in depressed cardiac contractile parameters, low Ca(2+)-induced Ca(2+) release from sarcoplasmic reticulum (SR) and cardiac hypertrophy in transgenic mice. To test the hypothesis that inhibition of phospholamban activity may rescue some of these phenotypic alterations, the calsequestrin overexpressing mice were cross-bred with phospholamban-knockout mice. Phospholamban ablation in calsequestrin overexpressing mice led to reversal of the depressed cardiac contractile parameters in Langendorff-perfused hearts or in vivo. This was associated with increases of SR Ca(2+) storage, assessed by caffeine-induced Na(+)-Ca(2+) exchanger currents. The inactivation time of the L-type Ca(2+) current (I(Ca)), which has an inverse correlation with Ca(2+)-induced SR Ca(2+) release, and the relation between the peak current density and half-inactivation time were also normalized, indicating a restoration in the ability of I(Ca) to trigger SR Ca(2+) release. The prolonged action potentials in calsequestrin overexpressing cardiomyocytes also reversed to normal upon phospholamban ablation. Furthermore, ablation of phospholamban restored the expression levels of atrial natriuretic factor and alpha-skeletal actin mRNA as well as ventricular myocyte size. These results indicate that attenuation of phospholamban function may prevent or overcome functional and remodeling defects in hypertrophied hearts.


Subject(s)
Calsequestrin/metabolism , Cardiomegaly , Myocardial Contraction , Myocardium/metabolism , 1-Methyl-3-isobutylxanthine/pharmacology , Animals , Atrial Natriuretic Factor/biosynthesis , Calcium-Binding Proteins/genetics , Calcium-Binding Proteins/physiology , Heart/drug effects , Immunohistochemistry , Isoproterenol/pharmacology , Mice , Mice, Knockout , Myocardium/cytology , Patch-Clamp Techniques , Sarcoplasmic Reticulum/metabolism
19.
J Biol Chem ; 276(10): 7195-201, 2001 Mar 09.
Article in English | MEDLINE | ID: mdl-11087739

ABSTRACT

We tested the hypothesis that increased Sarcoplasmic reticulum (SR) Ca content ([Ca](SRT)) in phospholamban knockout mice (PLB-KO) is because of increased SR Ca pump efficiency defined by the steady-state SR [Ca] gradient. The time course of thapsigargin-sensitive ATP-dependent (45)Ca influx into and efflux out of cardiac SR vesicles from PLB-KO and wild-type (WT) mice was measured at 100 nm free [Ca]. We found that PLB decreased the initial SR Ca uptake rate (0.13 versus 0.31 nmol/mg/s) and decreased steady-state (45)Ca content (0.9 versus 4.1 nmol/mg protein). Furthermore, at similar total SR [Ca], the pump-mediated Ca efflux rate was higher in WT (0.065 versus 0.037 nmol/mg/s). The pump-independent leak rate constant (k(leak)) was also measured at 100 nm free [Ca]. The results indicate that k(leak) was < 1% of pump-mediated backflux and was not different among nonpentameric mutant PLB (PLB-C41F), WT pentameric PLB (same expression level), and PLB-KO. Therefore differences in passive SR Ca leak cannot be the cause of the higher thapsigargin-sensitive Ca efflux from the WT membranes. We conclude that the decreased total SR [Ca] in WT mice is caused by decreased SR Ca influx rate, an increased Ca-pump backflux, and unaltered leak. Based upon both thermodynamic and kinetic analysis, we conclude that PLB decreases the energetic efficiency of the SR Ca pump.


Subject(s)
Calcium-Binding Proteins/metabolism , Calcium-Transporting ATPases/metabolism , Calcium/metabolism , Enzyme Inhibitors/metabolism , Myocardium/cytology , Myocardium/metabolism , Sarcoplasmic Reticulum/metabolism , Animals , Calcium/pharmacokinetics , Kinetics , Mice , Mice, Knockout , Protein Binding , Thapsigargin/pharmacology , Thermodynamics , Time Factors
20.
Basic Res Cardiol ; 96(6): 636-44, 2001 Nov.
Article in English | MEDLINE | ID: mdl-11770083

ABSTRACT

Calsequestrin is a sarcoplasmic reticulum protein, which plays a predominant role in diastolic Ca2+-storage in the mammalian heart. The present study was designed to define the gene structure, developmental and tissue specific expression of the murine, cardiac isoform of calsequestrin. Two sets of genomic libraries (lambda phage and PAC) were screened using the mouse cardiac calsequestrin cDNA, and several overlapping clones were isolated. These clones were characterized using restriction enzyme digestion, Southern blotting and partial sequencing. The cardiac calsequestrin gene consists of 11 exons and its 5' flanking region is characterized by the presence of a TATA-like box, muscle specific promoter elements such as 7 E-boxes, 1 MEF-2, 1 MCBF and 1 Repeat (musS) motifs, as well as several muscle non-specific transcriptional elements (AP-2A, NRE1, NRE2, p53, Spel and TFI-IIA). Expression of the cardiac isoform of calsequestrin was first detected on day 11 pre-birth and approached adult levels by day 4 post-birth. Expression of cardiac calsequestrin was also detected in adult fast-twitch skeletal muscle, thyroid, testis and epididymis tissues. This genomic characterization of cardiac calsequestrin may form the basis for further evaluation of its regulatory role in Ca2+ homeostasis and contractility in the murine heart.


Subject(s)
Calcium/metabolism , Calsequestrin/genetics , Myocardium/metabolism , 5' Untranslated Regions/genetics , Animals , Base Sequence , Calsequestrin/metabolism , Gene Expression Regulation, Developmental , Mice , Molecular Sequence Data , Myocardial Contraction/physiology , Promoter Regions, Genetic/physiology , Restriction Mapping , Sarcoplasmic Reticulum/metabolism
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