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1.
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
3.
J Biol Chem ; 275(16): 12129-35, 2000 Apr 21.
Article in English | MEDLINE | ID: mdl-10766848

ABSTRACT

Phospholamban is a phosphoprotein in the cardiac sarcoplasmic reticulum (SR) which regulates the apparent Ca(2+) affinity of the SR Ca(2+)-ATPase (SERCA2). To determine the levels of phospholamban which are associated with maximal inhibition of SERCA2, several lines of transgenic mice were generated which expressed increasing levels of a non-phosphorylatable form of phospholamban (S16A,T17A) specifically in the heart. This mutant form of phospholamban was chosen to prevent phosphorylation as a compensatory mechanism in vivo. Quantitative immunoblotting revealed increased phospholamban protein levels of 1.8-, 2.6-, 3.7-, and 4.7-fold in transgenic hearts compared with wild types. There were no changes in the expression levels of SERCA2, calsequestrin, calreticulin, and ryanodine receptor. Assessment of SR Ca(2+) uptake in hearts of transgenic mice indicated increases in the inhibition of the affinity of SERCA2 for Ca(2+) with increased phospholamban expression. Maximal inhibition was obtained at phospholamban expression levels of 2.6-fold or higher. Transgenic hearts with functional saturation in phospholamban:SERCA2 (>/=2.6:1) exhibited increases in beta-myosin heavy chain expression, associated with cardiac hypertrophy. These findings demonstrate that overexpression of a non-phosphorylatable form of phospholamban in transgenic mouse hearts resulted in saturation of the functional phospholamban:SERCA2 ratio at 2.6:1 and suggest that approximately 40% of the SR Ca(2+) pumps are functionally regulated by phospholamban in vivo.


Subject(s)
Calcium-Binding Proteins/pharmacology , Calcium-Transporting ATPases/metabolism , Calcium/metabolism , Myocardium/metabolism , Animals , Blotting, Western , Calcium-Binding Proteins/metabolism , Calreticulin , Calsequestrin/metabolism , Heart/drug effects , Kinetics , Mice , Mice, Transgenic , Mutagenesis, Site-Directed , Phosphorylation , Ribonucleoproteins/metabolism , Ryanodine Receptor Calcium Release Channel/metabolism , Sarcoplasmic Reticulum/metabolism
4.
J Mol Cell Cardiol ; 31(9): 1725-37, 1999 Sep.
Article in English | MEDLINE | ID: mdl-10471356

ABSTRACT

The aim of the present study was to determine the effects of beta -adrenergic stimulation in wild-type and phospholamban-deficient mouse hearts with altered thyroid conditions. Hypothyroidism was associated with significant decreases in heart/body weight ratio in wild-type and phospholamban-deficient mice, whereas hyperthyroidism was associated with significant increases in heart/body weight ratio in both groups. Hypothyroid hearts of wild-type and phospholamban-deficient mice exhibited similar increases in beta -myosin heavy chain protein levels and decreases in alpha -myosin heavy chain protein levels. In hyperthyroidism, there were increases in the alpha -myosin heavy chain protein levels and these were similar in wild-type and phospholamban-deficient hearts. There were no detectable levels of beta -myosin heavy chain protein in the hyperthyroid hearts. The relative tissue level of phospholamban in wild-type hearts was increased (133%, P<0.01) in hypothyroidism, and decreased (69%, P<0.01) in hyperthyroidism, when compared to euthyroid controls (100%). Similar increases and decreases in SR Ca(2+)-ATPase protein levels were observed between phospholamban-deficient and wild-type hearts in hyperthyroidism and hypothyroidism, respectively. The basal contractile state of wild-type and phospholamban-deficient hearts was significantly depressed in hypothyroidism. On the other hand, the basal contractile state of wild-type and phospholamban-deficient hearts was significantly increased in hyperthyroidism. During beta -agonist stimulation of wild-type hearts, the responses in the rates of contraction and relaxation were highest in the hypothyroid group, followed by the euthyroid, and lastly by the hyperthyroid groups. There was a close linear correlation between the magnitude of the contractile parameter responses and the phospholamban/SERCA2 ratios in these hearts. However, the phospholamban-deficient hypothyroid, euthyroid, and hyperthyroid hearts did not exhibit any responses to isoproterenol, indicating that the alterations in the thyroid states of these hearts do not influence the effects of isoproterenol on cardiac function. These findings suggest that phospholamban is an important regulator of the heart's responses to beta -adrenergic stimulation under various thyroid states.


Subject(s)
Calcium-Binding Proteins/physiology , Heart/drug effects , Hemodynamics/drug effects , Hyperthyroidism/physiopathology , Hypothyroidism/physiopathology , Isoproterenol/pharmacology , Myosin Heavy Chains/genetics , Thyroid Gland/physiology , Animals , Blood Pressure/drug effects , Calcium-Binding Proteins/deficiency , Calcium-Binding Proteins/genetics , Coronary Circulation/drug effects , Female , Gene Expression Regulation/drug effects , Gene Expression Regulation/physiology , Heart/physiology , Heart/physiopathology , Male , Mice , Mice, Knockout , Myocardial Contraction/drug effects , Myocardium/metabolism , Protein Isoforms/genetics , Thyroid Hormones/physiology
5.
Circ Res ; 83(6): 608-13, 1998 Sep 21.
Article in English | MEDLINE | ID: mdl-9742056

ABSTRACT

Alterations in the expression levels of the sarcoplasmic reticulum (SR) Ca2+-ATPase and its regulator, phospholamban, have been implicated in the effects of thyroxine hormone on cardiac function. To determine the role of phospholamban in these effects, hypothyroidism and hyperthyroidism were induced in phospholamban-deficient mice and their isogenic wild types. Hypothyroidism resulted in significant decreases of left ventricular contractility, which could be moderately stimulated by increases in preload or afterload, in both phospholamban-deficient and wild-type mice. However, the basal contractile parameters in hypothyroid phospholamban-deficient hearts were at least as high as those exhibited by hyperthyroid wild-type hearts. In hyperthyroidism, there was no further enhancement of the hyperdynamic contractile parameters in phospholamban-deficient hearts, although the wild-type hearts exhibited significantly increased contractile function compared with their respective euthyroid groups. Furthermore, increases in preload or afterload did not enhance contractility in either phospholamban-deficient or wild-type hyperthyroid hearts. Examination of the relative tissue levels of cardiac SR Ca2+-ATPase revealed increases in hyperthyroidism and decreases in hypothyroidism compared with euthyroidism, and these changes were similar between phospholamban-deficient and wild-type hearts. An opposite trend was observed for phospholamban expression levels in the wild-type group, which were depressed in hyperthyroid hearts but increased in hypothyroid hearts. These findings indicate that (1) thyroid hormones induce similar changes in the cardiac SR Ca2+-ATPase levels in either the presence or absence of phospholamban, (2) the thyroxine-induced increases in SR Ca2+-ATPase levels are not associated with any further stimulation of the hyperdynamic cardiac function in phospholamban-deficient mice, and (3) the decreased contractile parameters in hypothyroid phospholamban-deficient hearts associated with decreases in SR Ca2+-ATPase levels and myosin heavy chain isoform switches are at least as high as those of the stimulated hyperthyroid wild-type hearts. Thus, alterations in the phospholamban level or its activity may be a critical determinant of the contractile responses to altered thyroid states in the mammalian heart.


Subject(s)
Calcium-Binding Proteins/deficiency , Myocardium/chemistry , Thyroid Hormones/pharmacology , Animals , Calcium-Transporting ATPases/pharmacology , Female , Hypothyroidism/metabolism , Male , Mice , Myocardial Contraction/drug effects , Ventricular Function/physiology
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