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1.
Minerva Cardioangiol ; 51(6): 609-18, 2003 Dec.
Article in English | MEDLINE | ID: mdl-14676746

ABSTRACT

Doppler indices of transmitral flow are used commonly to assess noninvasively LV diastolic function in man and in large animals. This review examines echocardiographic indices of LV diastole (focusing on color M-mode) in mice with abnormal LV relaxation and hypertrophic cardiomyopathy based on genetic alterations of phospholamban and alpha-tropomyosin, respectively. Phospholamban (PLB) reversibly inhibits the sarcoplasmic reticulum Ca(2+) ATPase and is a crucial regulator of myocardial relaxation; tropomyosin is a contractile protein that plays a critical role in regulating contractile activity vis-à-vis interactions with the actin and troponin complex. Accordingly, diastolic function was assessed in PLB knockout mice (PLB/KO) and age-matched transgenic mice expressing a mutant, superinhibiting form of PLB (PLB/N27A), and in mice with cardiac-specific expression a mutant a-tropomyosin (TM-180). Transmitral Doppler flow indexes suggested impaired diastolic filling in the PLB/N27A mice, but improved LV diastolic function in the PLB/KO mice and TM-180 mutants. However, the propagation velocity of early flow into the LV cavity, measured by color M-mode Doppler, confirmed the expected direction of altered LV relaxation in each mouse model. We conclude that transmitral filling patterns and color M-mode flow propagation velocity reflect changes in myocardial relaxation in genetically engineered mice, and may be useful tools to characterize LV diastolic function in other mouse models of disease.


Subject(s)
Diastole , Echocardiography, Doppler, Color , Animals , Animals, Genetically Modified , Calcium-Binding Proteins/genetics , Disease Models, Animal , Mice , Mutation , Tropomyosin/genetics , Ventricular Dysfunction, Left/genetics
2.
Lab Invest ; 81(11): 1527-36, 2001 Nov.
Article in English | MEDLINE | ID: mdl-11706060

ABSTRACT

Highly active antiretroviral therapy (HAART) is implicated in cardiomyopathy (CM) and in elevated plasma lactate (LA) in AIDS through mechanisms of mitochondrial dysfunction. To determine mitochondrial events from HAART in vivo, 8-week-old hemizygous transgenic AIDS mice (NL4-3Delta gag/pol; TG) and wild-type FVB/n littermates were treated with the HAART combination of zidovudine, lamivudine, and indinavir or vehicle control for 10 days or 35 days. At termination of the experiments, mice underwent echocardiography, quantitation of abundance of molecular markers of CM (ventricular mRNA encoding atrial natriuretic factor [ANF] and sarcoplasmic calcium ATPase [SERCA2]), and determination of plasma LA. Myocardial histologic features were analyzed semiquantitatively and results were confirmed by transmission electron microscopy. After 35 days in the TG + HAART cohort, left ventricular mass increased 160% by echocardiography. Molecularly, ANF mRNA increased 250% and SERCA2 mRNA decreased 57%. Biochemically, LA was elevated (8.5 +/- 2.0 mM). Pathologically, granular cytoplasmic changes were found in cardiac myocytes, indicating enlarged, damaged mitochondria. Findings were confirmed ultrastructurally. No changes were found in other cohorts. After 10 days, only ANF was elevated, and only in the TG + HAART cohort. Results show that cumulative HAART caused mitochondrial CM with elevated LA in AIDS transgenic mice.


Subject(s)
Acquired Immunodeficiency Syndrome/drug therapy , Antiretroviral Therapy, Highly Active/adverse effects , Cardiomyopathies/chemically induced , Lactic Acid/blood , Acquired Immunodeficiency Syndrome/blood , Acquired Immunodeficiency Syndrome/genetics , Animals , Anti-HIV Agents/adverse effects , Atrial Natriuretic Factor/genetics , Calcium-Transporting ATPases/genetics , Cardiomyopathies/pathology , Cardiomyopathies/virology , Drug Therapy, Combination , Echocardiography , Gene Expression , HIV Protease Inhibitors/adverse effects , Indinavir/adverse effects , Lamivudine/adverse effects , Mice , Mice, Transgenic , Microscopy, Electron , Myocardium/pathology , Myocardium/ultrastructure , RNA, Messenger/analysis , Sarcoplasmic Reticulum Calcium-Transporting ATPases , Zidovudine/adverse effects
3.
Trends Cardiovasc Med ; 11(2): 82-9, 2001 Feb.
Article in English | MEDLINE | ID: mdl-11530297

ABSTRACT

Cardiovascular diseases such as hypertension, atherosclerosis, cardiac hypertrophy homocysteinemia and arrhythmias impose great health, social and financial costs. Some of these diseases are single gene traits that segregate in a simple Mendelian manner. Most are genetically complex, however, and result from combinations of large numbers of genes (polygenic and epistatic traits) or from interactions between genetic and environmental factors (multifactorial traits). Insights into the genetic control of these diseases could lead to improved diagnosis and treatment as well as a deeper understanding of basic physiological processes.


Subject(s)
Mice/genetics , Animals , Blood Pressure/genetics , Blood Pressure/physiology , Cardiovascular Diseases/genetics , Cardiovascular Diseases/physiopathology , Disease Models, Animal , Heart Rate/genetics , Heart Rate/physiology , Humans , Phenotype , Ventricular Function, Left/genetics , Ventricular Function, Left/physiology
4.
J Mol Cell Cardiol ; 33(9): 1637-48, 2001 Sep.
Article in English | MEDLINE | ID: mdl-11549343

ABSTRACT

Activation of members of the mitogen-activated protein (MAP) kinase family and their downstream effectors has been proposed to play a key role in the pathogenesis of cell survival, ischaemic preconditioning, cardiac hypertrophy and heart failure. This study investigated the responses of Src kinase and multiple MAP kinases during the transition from compensated pressure-overload hypertrophy to decompensated congestive heart failure. Extracellular signal-regulated protein kinase (ERK) 1/2, p38, and Src were activated by chronic pressure-overload and their activity was sustained for 8 weeks after aortic banding. In contrast, while p90 ribosomal S6 kinase (90RSK) and big MAP kinase 1 (BMK1) were activated in compensated hypertrophy, their activities were significantly decreased in hearts with heart failure. No changes were found in C-Jun NH2 terminal kinase (JNK) activity after aortic banding. These data suggest that differential activation of MAP kinase family members may contribute to the transition from compensated to decompensated hypertrophy. We also examined acute effects of mechanical stretch on the activation of these kinases in normal and hypertrophied hearts. In the isolated coronary-perfused heart, a balloon in the left ventricle was inflated to achieve minimum end-diastolic pressure of 25 mmHg for 10-20 min. In normal guinea pig hearts, stretch activated ERK1/2, p90RSK, p38, Src, and BMK1 but not JNK. However in hypertrophied hearts, further activation of these kinases was not observed by acute mechanical stretch. Mechanical stretch-induced activation of ERK1/2 and p38 kinase in normal hearts was attenuated significantly by a protein kinase C inhibitor, chelerythrine. We demonstrate that ERK1/2, p90RSK, p38, Src, and BMK1 are activated by chronic pressure-overload and by acute mechanical stretch. These data suggest that Src, BMK1 and p90RSK play a role as novel signal transduction pathways leading to cardiac hypertrophy. In addition, the differential inhibition of p90RSK and BMK1 in hearts with congestive heart failure suggests the specific role of these two kinases to maintain cardiac function under chronic pressure-overload.


Subject(s)
Cardiomegaly/enzymology , Heart Failure/enzymology , Heart/physiology , Mitogen-Activated Protein Kinases/metabolism , Myocardium/enzymology , Stress, Mechanical , src-Family Kinases/metabolism , Alkaloids , Animals , Aorta/surgery , Benzophenanthridines , Blood Pressure/physiology , Cardiomegaly/physiopathology , Enzyme Activation , Enzyme Inhibitors/pharmacology , Guinea Pigs , Heart/drug effects , Heart Failure/physiopathology , Immunoblotting , In Vitro Techniques , Male , Phenanthridines/pharmacology , Ribosomal Protein S6 Kinases/metabolism , Time Factors
7.
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
8.
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
9.
J Mol Cell Cardiol ; 33(1): 27-35, 2001 Jan.
Article in English | MEDLINE | ID: mdl-11133220

ABSTRACT

Gain- and loss-of-function strategies using transgenic over-expression and targeted ablation of candidate genes in in the mouse have provided important mechanistic insights into cardiovascular development, physiology and disease. An essential, but challenging step is the functional analysis of the resultant phenotype. The methods described in this review permit the study of integrated cardiovascular physiology in the adult mouse. A critical review of the available in vivo methods that assay cardiac volume (echocardiography, conductance volumetry, sonomicrometry, magnetic resonance imaging) pressure (micromanometers), flow (Doppler echocardiography), and bioelectricity (electrophysiologic studies) are presented.


Subject(s)
Cardiovascular Diseases/pathology , Mice, Mutant Strains , Mice, Transgenic , Phenotype , Age Factors , Angiography/methods , Angiography/veterinary , Animals , Blood Pressure Determination/instrumentation , Blood Pressure Determination/methods , Blood Pressure Determination/veterinary , Cardiomyopathies/diagnostic imaging , Cardiomyopathies/genetics , Cardiomyopathies/pathology , Cardiovascular Diseases/diagnostic imaging , Cardiovascular Diseases/genetics , Disease Models, Animal , Echocardiography/instrumentation , Echocardiography/methods , Echocardiography/veterinary , Echocardiography, Doppler/instrumentation , Echocardiography, Doppler/veterinary , Electric Conductivity , Electrophysiology/instrumentation , Electrophysiology/methods , Heart Diseases/diagnostic imaging , Heart Diseases/genetics , Heart Diseases/pathology , Heart Function Tests/instrumentation , Heart Function Tests/methods , Heart Function Tests/veterinary , Heart Ventricles/diagnostic imaging , Heart Ventricles/pathology , Magnetic Resonance Imaging/instrumentation , Magnetic Resonance Imaging/methods , Magnetic Resonance Imaging/veterinary , Mice , Mice, Inbred A , Mice, Inbred C57BL , Mice, Mutant Strains/genetics , Mice, Transgenic/genetics , Myocardium/pathology , Organ Size , Plethysmography/veterinary , Reference Values , Species Specificity , Ventricular Function, Left
10.
Comp Med ; 51(1): 26-9, 2001 Feb.
Article in English | MEDLINE | ID: mdl-11926298

ABSTRACT

BACKGROUND AND PURPOSE: Pentobarbital and ketamine-xylazine anesthesia in mice result in markedly decreased left ventricular fractional shortening and cardiac output. However, to the authors' knowledge, the effect of short-acting, alcohol-based anesthesia on these parameters is unknown. METHODS: Fifteen mice (FVB/N, C57Bl/6J, A/J, n = 5 each) underwent high-resolution (15 MHz) 2-dimensional-directed M-mode echocardiography before and after undergoing 2.5% tribromoethanol anesthesia (0.01 ml/g of body weight). RESULTS: Tribromoethanol anesthesia resulted in significant heart rate slowing (29%) and left ventricular enlargement (20%), and a more modest (12%) reduction in left ventricular fractional shortening. Cardiac output was unchanged. The differences in left ventricular function between conscious and tribromoethanol studies were similar for each of the three strains of mice. CONCLUSIONS: Tribromoethanol anesthesia induced only modest effects on M-mode estimates of basal cardiac function and did not influence cardiac output. The effects to tribromoethanol anesthesia were similar among three commonly used mice strains.


Subject(s)
Anesthetics/pharmacology , Ethanol/analogs & derivatives , Ethanol/pharmacology , Ventricular Function, Left/drug effects , Anesthesia , Animals , Cardiac Output/drug effects , Echocardiography , Female , Heart Rate/drug effects , Male , Mice , Mice, Inbred A , Mice, Inbred C57BL , Species Specificity
11.
Echocardiography ; 18(8): 685-8, 2001 Nov.
Article in English | MEDLINE | ID: mdl-11801211

ABSTRACT

We report on a patient with obstructive sleep apnea in whom percutaneous transmyocardial revascularization was complicated by hemopericardium. Absence of phasic variation in the transmitral and transtricuspid Doppler inflow velocities during apneic episodes and marked variation during spontaneous respiration were identified with a respirometer, and helped us identify a novel false-negative cause of flow velocity paradoxus.


Subject(s)
Blood Flow Velocity/physiology , Pericardial Effusion/diagnosis , Pericardial Effusion/etiology , Sleep Apnea, Obstructive/complications , Sleep Apnea, Obstructive/diagnosis , Aged , Echocardiography, Doppler , False Negative Reactions , Humans , Male , Pericardial Effusion/physiopathology , Sleep Apnea, Obstructive/physiopathology
13.
J Card Fail ; 7(4): 342-7, 2001 Dec.
Article in English | MEDLINE | ID: mdl-11782858

ABSTRACT

BACKGROUND: The renin-angiotensin system is involved in cardiac remodeling. In contrast to the well-recognized salutary effects of angiotensin-converting enzyme inhibition, the value of angiotensin II type I (AT(1))-receptor blockade on left ventricular (LV) hypertrophy and dysfunction is controversial. METHODS AND RESULTS: Descending thoracic aorta-banded and sham-operated guinea pigs were given either losartan (30 mg x kg(-1) x day(-1) intraperitoneally) or vehicle for 8 weeks (n = 7 in each group). LV end-diastolic and end-systolic dimensions and wall thicknesses were measured echocardiographically, and LV fractional shortening, relative wall thickness, and LV mass normalized by body weight were calculated. Isolated heart function (Langendorff perfusion) was studied 8 weeks after surgery, and LV performance was assessed by maximum LV pressure and +/-dP/dt normalized by LV mass. Eight weeks after banding guinea pigs developed concentric LV hypertrophy and had decreased maximum LV pressure and +/-dP/dt normalized by LV mass; LV end-diastolic dimension and LV fractional shortening were unchanged. In band-operated guinea pigs treatment with losartan had no significant effects on any of these measurements. CONCLUSIONS: In guinea pigs with descending aortic banding, treatment with losartan for 8 weeks neither attenuates progression of pressure overload hypertrophy nor significantly improves impaired mass-normalized pressure-derived indices of LV contraction and relaxation.


Subject(s)
Angiotensin Receptor Antagonists , Hypertrophy, Left Ventricular/drug therapy , Receptors, Angiotensin/therapeutic use , Ventricular Pressure/drug effects , Ventricular Remodeling/drug effects , Animals , Blood Pressure/drug effects , Disease Models, Animal , Echocardiography , Guinea Pigs , Heart/drug effects , Losartan/antagonists & inhibitors , Losartan/therapeutic use , Lung/blood supply , Lung/drug effects , Male , Models, Cardiovascular , Organ Size/drug effects , Receptor, Angiotensin, Type 1 , Treatment Outcome , Ventricular Function, Left/drug effects
14.
J Am Coll Cardiol ; 36(5): 1713-9, 2000 Nov 01.
Article in English | MEDLINE | ID: mdl-11079681

ABSTRACT

OBJECTIVES: The purpose of this study was to understand the significance of an effective atrial systole and the interactions between atrial and ventricular function. BACKGROUND: The significance of atrial function is controversial, particularly in the setting of left ventricular (LV) dysfunction. METHODS: Serial, rapid pacing in five dogs that had undergone radiofrequency ablation and implantation of right atrial and ventricular pacemakers produced reversible atrial and ventricular dysfunction (alone and in combination). Atrial function (echocardiograph-determined transmitral diastolic flow, left atrial appendage emptying, and pulmonary venous flow), cardiac output, and right heart pressures were measured at matched paced heart rates of 80 beats/min. RESULTS: Isolated rapid atrial pacing (LV ejection fraction approximately 60%) decreased atrial booster pump in the body and appendage of the left atrium, but increased the conduit function of the left atrium. Isolated LV dysfunction (LV ejection fraction approximately 34%) increased atrial booster pump function. The decreased atrial booster pump function in animals with combined atrial and ventricular dysfunction was incompletely compensated by the redistribution of the reservoir and conduit functions of the left atrium. As a result, cardiac output decreased and right heart pressures increased only after superimposed pacing. CONCLUSIONS: In the presence of a normal left ventricle (LV), atrial failure has little effect on cardiac output and right heart pressures because of compensatory conduit function, but when early LV dysfunction coexists, changes in reservoir and conduit functions are insufficient to compensate for an impairment of atrial contraction.


Subject(s)
Heart Atria/diagnostic imaging , Heart Atria/physiopathology , Hemodynamics , Myocardial Contraction , Ventricular Dysfunction, Left/diagnostic imaging , Ventricular Dysfunction, Left/physiopathology , Animals , Dogs , Ultrasonography, Doppler
15.
J Mol Cell Cardiol ; 32(9): 1735-44, 2000 Sep.
Article in English | MEDLINE | ID: mdl-10966834

ABSTRACT

Cardiac-specific overexpression of calsequestrin has been shown to result in significant decreases in contractile parameters and intracellular Ca(2+)transients in vitro. Therefore, the purpose of the present study was to determine the effects of calsequestrin overexpression on basal cardiac function and the force-frequency relation in vivo. Calsequestrin overexpression mice (CSQ-OE, n=20) and their isogenic controls (WT) were studied with an integrative approach using transthoracic echocardiography, stress-shortening relations, and invasive hemodynamics in intact closed-chest mice. M-mode echocardiography indicated that calsequestrin overexpression resulted in concentric hypertrophy (+52%) and an increase in LV ejection phase indices. However, mean end-systolic stress-shortening coordinates revealed that at matched end-systolic wall-stress, fractional shortening was depressed in CSQ-OE mice. This was confirmed by depressed indices of LV isovolumic contraction and relaxation in CSQ-OE v. WT mice. Furthermore, overexpression of calsequestrin resulted in a downward and leftward shift of the biphasic force-frequency relation; thus, the critical heart (HR(crit)) was significantly lower in calsequestrin-overexpression mice (264+/-15 bpm) than in wild-type controls (365+/-21 bpm). Surprisingly, calsequestrin overexpression was associated with the induction of pulsus alternans in every animal (at an average heart rate of 428+/-26 bpm), whereas none of the wild-type controls displayed this phenomenon. We conclude that: (i) although increased levels of calsequestrin result in decreased myocardial contractility and a depressed force-frequency relation, LV wall stress is reduced and chamber function is normal, and (ii) an increase in SR Ca(2+)storage capacity induces pulsus alternans in the intact anesthetized mouse.


Subject(s)
Calsequestrin/physiology , Hypertrophy, Left Ventricular/etiology , Animals , Echocardiography , Gene Expression , Hemodynamics , Hypertrophy, Left Ventricular/physiopathology , Mice , Mice, Transgenic , Myocardial Contraction
16.
Circ Res ; 86(12): 1218-23, 2000 Jun 23.
Article in English | MEDLINE | ID: mdl-10864911

ABSTRACT

To test the hypothesis that activation of the protein kinase C (PKC) epsilon isoform leads to cardiac hypertrophy without failure, we studied transgenic mice with cardiac-specific overexpression of a constitutively active mutant of the PKCepsilon isoform driven by an alpha-myosin heavy chain promoter. In transgenic mice, the protein level of PKCepsilon in heart tissue was increased 9-fold. There was a 6-fold increase of the membrane/cytosol ratio, and PKC activity in the membrane fraction was 4.2-fold compared with wild-type mice. The heart weight was increased by 28%, and upregulation of the mRNA for beta-myosin heavy chain and alpha-skeletal actin was observed in transgenic mouse hearts. Echocardiography demonstrated increased anterior and posterior wall thickness with normal left ventricular function and dimensions, indicating concentric cardiac hypertrophy. Isolated cardiomyocyte mechanical function was slightly decreased, and Ca(2+) signals were markedly depressed in transgenic mice, suggesting that myofilament sensitivity to Ca(2+) was increased. No differences were observed in either the levels of cardiac Ca(2+)-handling proteins or the degree of cardiac regulatory protein phosphorylation between wild-type and transgenic mice. Unlike mice with PKCbeta(2) overexpression, transgenic mice with cardiac-specific overexpression of the active PKCepsilon mutant demonstrated concentric hypertrophy with normal in vivo cardiac function. Thus, PKC isoforms may play differential functional roles in cardiac hypertrophy and failure.


Subject(s)
Cardiomegaly/etiology , Isoenzymes/metabolism , Protein Kinase C/metabolism , Animals , Calcium/metabolism , Cardiomegaly/diagnostic imaging , Cardiomegaly/genetics , Cardiomegaly/pathology , Cell Separation , Echocardiography , Gene Expression , Heart/physiopathology , Homeostasis , Lung/physiology , Mice , Mice, Transgenic/genetics , Mice, Transgenic/physiology , Muscle Proteins/metabolism , Muscle Proteins/physiology , Myocardium/metabolism , Myocardium/pathology , Organ Size , Phosphorylation , Protein Kinase C-epsilon
17.
J Biol Chem ; 275(20): 14985-91, 2000 May 19.
Article in English | MEDLINE | ID: mdl-10809743

ABSTRACT

Transgenic mice were generated with cardiac-specific overexpression of the monomeric, dominant-acting, superinhibitory L37A and I40A mutant forms of phospholamban (PLN), and their phenotypes were compared with wild-type (wt) mice or 2-fold overexpressors of wt PLN (wtOE). The level of PLN monomer in cardiac microsomes was increased 11-13-fold, and the apparent affinity of the sarco(endo)plasmic reticulum Ca(2+)-ATPase for Ca(2+) was decreased from pCa 6.22 in wt or 6.12 in wtOE to 5.81 in L37A and 5.72 in I40A. Basal physiological parameters, measured in isolated myocytes, indicated a significant reduction in the rates of shortening (+dL/dt) and relengthening (-dL/dt). Hemodynamic measurements indicated that peak systolic pressure was unaffected but that pressure changes (+dP/dt and -dP/dt) were lowered significantly in both mutant lines, and relaxation time (tau) was also lengthened significantly. Echocardiography for both mutants showed depressed systolic function and an increase in left ventricular mass of over 1.4-fold. Significant decreases in left ventricular shortening fraction and velocity of circumferential shortening and increases in ejection time were corrected by isoproterenol. The use of antibodies specific against Ser(16)- and Thr(17)-PLN peptides showed that phosphorylation of both pentameric and monomeric PLN were increased between 1.2- and 2.4-fold in both the L37A and I40A lines but not in the wtOE line. These observations show that overexpression of superinhibitory mutant forms of PLN causes depression of contractile parameters with induction of cardiac hypertrophy, as assessed with echocardiography.


Subject(s)
Calcium-Binding Proteins/genetics , Calcium-Binding Proteins/metabolism , Calcium-Transporting ATPases/metabolism , Hemodynamics , Myocardial Contraction/physiology , Myocardium/metabolism , Amino Acid Substitution , Animals , Blood Pressure , Calcium/metabolism , Echocardiography , Major Histocompatibility Complex , Mice , Mice, Transgenic , Mutagenesis, Site-Directed , Point Mutation , Rabbits , Sarcoplasmic Reticulum/enzymology , Systole , Ventricular Function, Left
18.
J Biol Chem ; 275(14): 10538-44, 2000 Apr 07.
Article in English | MEDLINE | ID: mdl-10744747

ABSTRACT

Phospholamban is a regulator of the Ca(2+) affinity of the cardiac sarcoplasmic reticulum Ca(2+) ATPase (SERCA2a) and of cardiac contractility. In vitro expression studies have shown that several mutant phospholamban monomers are superinhibitory, suggesting that monomeric phospholamban is the active species. However, a phospholamban Asn(27) --> Ala (N27A) mutant, which maintained a normal pentamer to monomer ratio, was shown to act as a superinhibitor of SERCA2a Ca(2+) affinity. To determine whether the pentameric N27A mutant is superinhibitory in vivo, transgenic mice with cardiac-specific overexpression of mutant phospholamban were generated. Quantitative immunoblotting revealed a 61 +/- 6% increase in total phospholamban in mutant hearts, with 90% of the overexpressed protein being pentameric. The EC(50) value for Ca(2+) dependence of Ca(2+) uptake was 0.69 +/- 0.07 microM in mutant hearts, compared with 0.29 +/- 0.02 microM in wild-type hearts or 0. 43 +/- 0.03 microM in hearts overexpressing wild-type PLB by 2-fold. Myocytes from phospholamban N27A mutant hearts also exhibited more depressed contractile parameters than wild-type phospholamban overexpressing cells. The shortening fraction was 52%, rates of shortening and relengthening were 46% and 38% respectively, and time for 80% decay of the Ca(2+) signal was 146%, compared with wild-types (100%). Langendorff-perfused mutant hearts also demonstrated depressed contractile parameters. Furthermore, in vivo echocardiography showed a depression in the ratio of early to late diastolic transmitral velocity and a 79% prolongation of the isovolumic relaxation time. Isoproterenol stimulation did not fully relieve the depressed contractile parameters at the cellular, organ, and intact animal levels. Thus, pentameric phospholamban N27A mutant can act as a superinhibitor of the affinity of SERCA2a for Ca(2+) and of cardiac contractility in vivo.


Subject(s)
Calcium-Binding Proteins/genetics , Calcium-Binding Proteins/metabolism , Calcium-Transporting ATPases/metabolism , Heart/physiology , Amino Acid Substitution , Animals , Calcium/metabolism , Calcium-Binding Proteins/chemistry , Cell Line , Humans , Kinetics , Macromolecular Substances , Mice , Mice, Transgenic , Myocardium/metabolism , Organ Specificity , Oxalates/pharmacology , Rabbits , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Sarcoplasmic Reticulum/drug effects , Sarcoplasmic Reticulum/metabolism , Transfection
19.
Am J Physiol Heart Circ Physiol ; 278(3): H958-63, 2000 Mar.
Article in English | MEDLINE | ID: mdl-10710365

ABSTRACT

Mechanical and relaxation restitution represent the restoration of contractile force and relaxation, respectively, in premature beats having progressively longer extrasystolic intervals (ESI); these phenomena are related to intracellular activator Ca(2+) by poorly defined mechanisms. We tested the hypothesis that the level of phospholamban [which modulates the affinity of the sarcoplasmic reticulum (SR) Ca(2+)-ATPase for Ca(2+), and thus the SR Ca(2+) load] may be an important determinant of both mechanical and relaxation restitution. Five mice with ablation of the phospholamban (PLB) gene (PLBKO), eight isogenic wild-type controls (129SvJ), eleven mice with PLB overexpression (PLBOE), and nine isogenic wild-type (FVB/N) controls were anesthetized and instrumented with a 1.4-Fr Millar catheter in the left ventricle and a 1-Fr pacemaker in the right atrium. At a cycle length of 200 ms, extrastimuli with increasing ESI were introduced, and the peak rates of left ventricular isovolumic contraction (+/-dP/dt(max)) were normalized and fit to monoexponential equations. In a subset, the protocols were repeated after ryanodine (4 ng/g) was administered to deplete SR Ca(2+) stores. The time constant of mechanical restitution in PLBKO was significantly shorter [6.3 +/- 1.2 (SE) vs. 47.7 +/- 7.6 ms] and began earlier (50 +/- 10 vs. 70 +/- 19 ms) than in 129SvJ. In contrast, the time constant of mechnical restitution was significantly longer (80.3 +/- 7.6 vs. 54.1 +/- 9.2 ms) in PLBOE than in FVB/N. The time constant of relaxation restitution was less in PLBKO than in 129SvJ (26.2 +/- 9.9 vs. 44.6 +/- 3.3, P < 0.05) but was similar in PLBOE and FVB/N (21.1 +/- 6.3 vs. 20.5 +/- 5.7 ms). Intravenous ryanodine decreased significantly the time constants of mechanical restitution in PLBOE, 129SvJ, and FVB/N but was lethal in PLBKO. In contrast, ryanodine increased the time constant of relaxation restitution. Thus 1) the phospholamban level is a critical determinant of mechanical restitution and (to a lesser extent) relaxation restitution in these transgenic models, and 2) ryanodine differentially affects mechanical and relaxation restitution. Furthermore, our data suggest a dissociation of processes within the SR that govern contraction and relaxation.


Subject(s)
Calcium/metabolism , Myocardial Contraction/physiology , Sarcoplasmic Reticulum/physiology , Animals , Biomechanical Phenomena , Blood Pressure , Calcium-Binding Proteins/deficiency , Calcium-Binding Proteins/genetics , Calcium-Binding Proteins/physiology , Heart Rate , Mice , Mice, Knockout , Ryanodine/pharmacology , Sarcoplasmic Reticulum/drug effects
20.
Cardiovasc Res ; 45(4): 883-8, 2000 Mar.
Article in English | MEDLINE | ID: mdl-10728414

ABSTRACT

OBJECTIVE: Left ventricular assist device support mechanically unloads the failing ventricle with resultant improvement in cardiac geometry and function in patients with end-stage heart failure. Activation of the G alpha q signaling pathway, including protein kinase C, appears to be involved in the progression of heart failure. Similarly down-regulation of Ca2+ cycling proteins may contribute to contractile depression in this clinical syndrome. Thus we examined whether protein kinase C activation and decreased Ca2+ cycling protein levels could be reversed by left ventricular assist device support. METHODS: Left ventricular myocardial specimens were obtained from seven patients during placement of left ventricular assist device and heart transplantation. We examined changes in protein levels of G alpha q, phospholipase C beta 1, regulators of G protein signaling (RGS), sarcoplasmic reticulum Ca2+ ATPase, phospholamban and translocation of protein kinase C isoforms (alpha, beta 1, and beta 2). RESULTS: The paired pre- and post-left ventricular assist device samples revealed that RGS2, a selective inhibitor of G alpha q, was decreased (P < 0.01), while the status of G alpha q, phospholipase C beta 1, RGS3 and RGS4 were unchanged after left ventricular assist device implantation. Translocation of protein kinase C isoforms remained unchanged. Left ventricular assist device support increased sarcoplasmic reticulum Ca2+ ATPase protein level (P < 0.01), while phospholamban abundance was unchanged. CONCLUSIONS: We conclude that altered protein expression and stoichiometry of the major cardiomyocyte Ca2+ cycling proteins rather than reduced phospholipase C beta 1 activation may contribute to improved mechanical function produced by left ventricular assist device support in human heart failure.


Subject(s)
GTP-Binding Proteins/metabolism , Heart Failure/metabolism , Heart-Assist Devices , Myocardium/metabolism , Protein Kinase C/metabolism , RGS Proteins/metabolism , Adolescent , Adult , Calcium-Binding Proteins/metabolism , Calcium-Transporting ATPases/metabolism , Case-Control Studies , Female , Heart Failure/therapy , Humans , Immunoblotting , Isoenzymes/metabolism , Male , Middle Aged , Sarcoplasmic Reticulum/drug effects , Sarcoplasmic Reticulum/enzymology , Signal Transduction/physiology , Statistics, Nonparametric
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