A familial hypertrophic cardiomyopathy alpha-tropomyosin mutation causes severe cardiac hypertrophy and death in mice.

Tropomyosin, an essential component of the sarcomere, regulates muscle contraction through Ca(2+)-mediated activation. Familial hypertrophic cardiomyopathy (FHC) is caused by mutations in numerous cardiac sarcomeric proteins, including myosin heavy and light chains, actin, troponin T and I, myosin binding protein C, and alpha-tropomyosin. This study developed transgenic mouse lines that encode an FHC mutation in alpha-tropomyosin; this mutation is an amino acid substitution at codon 180 (Glu180Gly) which occurs in a troponin T binding region. Non-transgenic and control mice expressing wild-type alpha-tropomyosin demonstrate no morphological or physiological changes. Expression of exogenous mutant tropomyosin leads to a concomitant decrease in endogenous alpha-tropomyosin without altering the expression of other contractile proteins. Histological analysis shows that initial pathological changes, which include ventricular concentric hypertrophy, fibrosis and atrial enlargement, are detected within 1 month. The disease-associated changes progressively increase and result in death between 4 and 5 months. Physiological analyses of the FHC mice using echocardiography, work-performing heart analyses, and force measurements of cardiac myofibers, demonstrate dramatic functional differences in diastolic performance and increased sensitivity to calcium. This report demonstrates that mutations in alpha-tropomyosin can be severely disruptive of sarcomeric function, which consequently triggers a dramatic hypertrophic response that culminates in lethality.

Altered SR protein expression associated with contractile dysfunction in diabetic rat hearts.

The goal of this study was to examine whether alteration of sarcoplasmic reticulum (SR) protein levels is associated with early-onset diastolic and late-onset systolic dysfunction in streptozotocin (STZ)-induced diabetic rat hearts. Four-week diabetic rat hearts exhibited slow relaxation, whereas 6-wk diabetic rat hearts exhibited slow and depressed contraction. Total phospholamban level was increased, and phosphorylated level was decreased in 4- and 6-wk diabetic rat hearts. Sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA2) protein level was unchanged in 4-wk but decreased in 6-wk diabetic rat hearts. Only the apparent affinity of SR Ca2+ uptake for Ca2+ was decreased in 4-wk diabetic rat hearts, but the apparent affinity and the maximum rate was decreased in 6-wk diabetic rat hearts. Insulin treatment of the diabetic rats normalized SR protein expression and function. It was concluded that an increase in nonphosphorylated phospholamban and a decrease in the apparent affinity of SR Ca2+ pump for Ca2+ are associated with early-onset diastolic dysfunction and decreases in SERCA2 protein level and apparent affinity and maximum velocity of SR Ca2+ pump are associated with late-onset systolic dysfunction in diabetic rats.

The enhanced contractility of the phospholamban-deficient mouse heart persists with aging.

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.

Progression from hypertrophic to dilated cardiomyopathy in mice that express a mutant myosin transgene.

A mouse model of hypertrophic cardiomyopathy (HCM) was created by expression of a cardiac alpha-myosin transgene including the R(403)Q mutation and a deletion of a segment of the actin-binding domain. HCM mice show early histopathology and hypertrophy, with progressive hypertrophy in females and ventricular dilation in older males. To test the hypothesis that dilated cardiomyopathy (DCM) is part of the pathological spectrum of HCM, we studied chamber morphology, exercise tolerance, hemodynamics, isolated heart function, adrenergic sensitivity, and embryonic gene expression in 8- to 11-mo-old male transgenic animals. Significantly impaired exercise tolerance and both systolic and diastolic dysfunction were seen in vivo. Contraction and relaxation parameters of isolated hearts were also decreased, and lusitropic responsiveness to the beta-adrenergic agonist isoproterenol was modestly reduced. Myocardial levels of the G protein-coupled beta-adrenergic receptor kinase 1 (beta-ARK1) were increased by more than twofold over controls, and total beta-ARK1 activity was also significantly elevated. Induction of fetal gene expression was also observed in transgenic hearts. We conclude that transgenic male animals have undergone cardiac decompensation resulting in a DCM phenotype. This supports the idea that HCM and DCM may be part of a pathological continuum rather than independent diseases.

Overexpression of SERCA2b in the heart leads to an increase in sarcoplasmic reticulum calcium transport function and increased cardiac contractility.

The sarcoplasmic reticulum calcium ATPase SERCA2b is an alternate isoform encoded by the SERCA2 gene. SERCA2b is expressed ubiquitously and has a higher Ca(2+) affinity compared with SERCA2a. We made transgenic mice that overexpress the rat SERCA2b cDNA in the heart. SERCA2b mRNA level was approximately approximately 20-fold higher than endogenous SERCA2b mRNA in transgenic hearts. SERCA2b protein was increased 8-10-fold in the heart, whereas SERCA2a mRNA/protein level remained unchanged. Confocal microscopy showed that SERCA2b is localized preferentially around the T-tubules of the SR, whereas SERCA2a isoform is distributed both transversely and longitudinally in the SR membrane. Calcium-dependent calcium uptake measurements showed that the maximal velocity of Ca(2+) uptake was not changed, but the apparent pump affinity for Ca(2+) (K(0.5)) was increased in SERCA2b transgenic mice (0.199 +/- 0.011 micrometer) compared with wild-type control mice (0.269 +/- 0.012 micrometer, p < 0.01). Work-performing heart preparations showed that SERCA2b transgenic hearts had a higher rates of contraction and relaxation, shorter time to peak pressure and half-time for relaxation than wild-type hearts. These data show that SERCA2b is associated in a subcompartment within the sarcoplasmic reticulum of cardiac myocytes. Overexpression of SERCA2b leads to an increase in SR calcium transport function and increased cardiac contractility, suggesting that SERCA2b plays a highly specialized role in regulating the beat-to-beat contraction of the heart.

Cardiac dysfunction occurs in the HIV-1 transgenic mouse treated with zidovudine.

Cardiomyopathy in AIDS is an increasingly important clinical problem. Mechanisms of AIDS cardiomyopathy were explored using AIDS transgenic mice that express replication-incompetent HIV-1 (NL4-3delta gag/pol). Transgenic and FVB/n mice (n = 3 to 6 per cohort) received water ad libitum with and without zidovudine (3'-azido-2',3'-deoxythymidine; AZT; 0.7 mg/ml) for 21 or 35 days. After 21 days, echocardiographic studies were performed and abundance of mRNA for cardiac sarcoplasmic reticulum calcium ATPase (SERCA2), sodium calcium exchanger (NCX1), and atrial natriuretic factor were determined individually using Northern analysis of extracts of left ventricles. After 35 days, contractile function and relaxation were analyzed in isolated work-performing hearts. Histopathological and ultrastructural (transmission electron microscopy) changes were identified. After 21 days, molecular indicators of cardiac dysfunction were found. Depressed SERCA2 and increased atrial natriuretic factor mRNA abundance occurred in left ventricles from AZT-treated transgenic mice. NCX1 abundance was unchanged. Eccentric left ventricle hypertrophy was determined echocardiographically. After 35 days, cardiac dysfunction was worst in AZT-treated and AZT-untreated transgenic mice. Decreases in the first derivative of the maximal change in left ventricle systolic pressure with respect to time (+dP/dt) occurred in transgenic mice with and without AZT. Increased half-time of relaxation and ventricular relaxation (-dP/dt) occurred in AZT-treated and -untreated transgenic mice. Increased time to peak pressure was found only in AZT-treated transgenic mice. In AZT-treated FVB/n mice, -dP/dt was decreased. Ultrastructurally, mitochondrial destruction was most pronounced in AZT-treated transgenic mice, but also was found in AZT-treated FVB/n mice. Transgenic mice that express HIV-1 demonstrate cardiac dysfunction. AZT treatment of FVB/n mice causes mitochondrial ultrastructural alterations that are similar to those in other species. In transgenic mice, AZT treatment worsens molecular and ultrastructural features of cardiomyopathy. HIV-1 constructs and AZT each contribute to cardiac dysfunction in this murine model of AIDS cardiomyopathy.

The effect of isoproterenol on phospholamban-deficient mouse hearts with altered thyroid conditions.

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.

Mouse model of a familial hypertrophic cardiomyopathy mutation in alpha-tropomyosin manifests cardiac dysfunction.

To investigate the functional consequences of a tropomyosin (TM) mutation associated with familial hypertrophic cardiomyopathy (FHC), we generated transgenic mice that express mutant alpha-TM in the adult heart. The missense mutation, which results in the substitution of asparagine for aspartic acid at amino acid position 175, occurs in a troponin T binding region of TM. S1 nuclease mapping and Western blot analyses demonstrate that increased expression of the alpha-TM 175 transgene in different lines causes a concomitant decrease in levels of endogenous alpha-TM mRNA and protein expression. In vivo physiological analyses show a severe impairment of both contractility and relaxation in hearts of the FHC mice, with a significant change in left ventricular fractional shortening. Myofilaments that contain alpha-TM 175 demonstrate an increased activation of the thin filament through enhanced Ca2+ sensitivity of steady-state force. Histological analyses show patchy areas of mild ventricular myocyte disorganization and hypertrophy, with occasional thrombi formation in the left atria. Thus, the FHC alpha-TM transgenic mouse can serve as a model system for the examination of pathological and physiological alterations imparted through aberrant TM isoforms.

Cardiac-specific overexpression of the alpha(1) subunit of the L-type voltage-dependent Ca(2+) channel in transgenic mice. Loss of isoproterenol-induced contraction.

The L-type voltage-dependent calcium channel (L-VDCC) regulates calcium influx in cardiac myocytes. Activation of the beta-adrenergic receptor (betaAR) pathway causes phosphorylation of the L-VDCC and that in turn increases Ca(2+) influx. Targeted expression of the L-VDCC alpha(1) subunit in transgenic (Tg) mouse ventricles resulted in marked blunting of the betaAR pathway. Inotropic and lusitropic responses to isoproterenol and forskolin in Tg hearts were significantly reduced. Likewise, Ca(2+) current augmentation induced by iso- proterenol and forskolin was markedly depressed in Tg cardiomyocytes. Despite no change in betaAR number, isoproterenol-stimulated adenylyl cyclase activity was absent in Tg membranes and NaF and forskolin responses were reduced. We postulate an important pathway for regulation of the betaAR by Ca(2+) channels.

Identification of a specific role for the Na,K-ATPase alpha 2 isoform as a regulator of calcium in the heart.

It is well accepted that inhibition of the Na,K-ATPase in the heart, through effects on the Na/Ca exchanger, raises the intracellular Ca2+ concentration and strengthens cardiac contraction. However, the contribution that individual isoforms make to this calcium regulatory role is unknown. Assessing the phenotypes of mouse hearts with genetically reduced levels of Na,K-ATPase alpha 1 or alpha 2 isoforms clearly demonstrates different functional roles for these isoforms in vivo. Heterozygous alpha 2 hearts are hypercontractile as a result of increased calcium transients during the contractile cycle. In contrast, heterozygous alpha 1 hearts are hypocontractile. The different functional roles of these two isoforms are further demonstrated since inhibition of the alpha 2 isoform with ouabain increases the contractility of heterozygous alpha 1 hearts. These results definitively illustrate a specific role for the alpha 2 Na,K-ATPase isoform in Ca2+ signaling during cardiac contraction.

Protection against hypoxia-reoxygenation in the absence of poly (ADP-ribose) synthetase in isolated working hearts.

Peroxynitrite and hydroxyl radical are reactive oxidants produced during myocardial reperfusion injury. They have been shown to induce dysfunction in cardiac myocytes, in part, via the activation of the nuclear enzyme poly (ADP-ribose) synthetase (PARS). These oxidants can trigger DNA single strand breakage, which triggers PARS activation, resulting in cellular NAD+ and ATP depletion and cytotoxicity. Recent work has demonstrated that hypoxia-reoxygenation of cardiac myocytes in vitro also causes peroxynitrite formation, PARS activation and cytotoxicity. In the present study, using hearts from genetically engineered mice lacking PARS, we have investigated whether the absence of PARS alters the functional response to hypoxia reoxygenation. Isolated work-performing mouse hearts were stabilized under the same loading condition (cardiac minute work of 250 mmHg x ml/min, an afterload of 50 mmHg aortic pressure and similar venous return of 5 ml/min, resulting in the same preload). After 30 min equilibration the hearts were subjected to 30 min hypoxia followed by 30 min of reoxygenation. At the end of the reoxygenation, in hearts from wild-type animals, there was a significant suppression in the rate of intraventricular pressure development (+dP/dt) from 3523 to 2907 mmHg. There was also a significant suppression in the rate of relaxation (-dP/dt) in the wild-type hearts from 3123 to 2168 mmHg. The time to peak pressure (TPP) increased from 0.48 to 0.59 ms/mmHg and the half-time of relaxation (RT1/2) increased from 0.59 to 0.74 ms/mmHg. In contrast, in the hearts from the PARS knockout animals, no significant suppression of +dP/dt (from 3654 to 3419 mmHg), and no significant increase in the TPP (from 0.462 to 0.448 ms/mmHg) were found, and the decrease in -dP/dt was partially ameliorated (from 3399 to 2687 mmHg) as well as the half-time of relaxation (from 0.507 to 0.55 ms/mmHg) when compared to the response to the wild-type hearts. The current data demonstrate that the reoxygenation induced suppression of the myocardial contractility is dependent on the functional integrity of PARS.

Targeted overexpression of the sarcoplasmic reticulum Ca2+-ATPase increases cardiac contractility in transgenic mouse hearts.

Cardiac hypertrophy and heart failure are known to be associated with a reduction in Ca2+-ATPase pump levels of the sarcoplasmic reticulum (SR). To determine whether, and to what extent, alterations in Ca2+ pump numbers can affect contraction and relaxation parameters of the heart, we have overexpressed the cardiac SR Ca2+-ATPase specifically in the mouse heart using the alpha-myosin heavy chain promoter. Analysis of 2 independent transgenic lines demonstrated that sarco(endo)plasmic reticulum Ca2+-ATPase isoform (SERCA2a) mRNA levels were increased 3.88+/-0. 4-fold and 7.90+/-0.2-fold over those of the control mice. SERCA2a protein levels were increased by 1.31+/-0.05-fold and 1.54+/-0. 05-fold in these lines despite high levels of mRNA, suggesting that complex regulatory mechanisms may determine the SERCA2a pump levels. The maximum velocity of Ca2+ uptake (Vmax) was increased by 37%, demonstrating that increased pump levels result in increased SR Ca2+ uptake function. However, the apparent affinity of the SR Ca2+-ATPase for Ca2+ remains unchanged in transgenic hearts. To evaluate the effects of overexpression of the SR Ca2+ pump on cardiac contractility, we used the isolated perfused work-performing heart model. The transgenic hearts showed significantly higher myocardial contractile function, as indicated by increased maximal rates of pressure development for contraction (+dP/dt) and relaxation (-dP/dt), together with shortening of the normalized time to peak pressure and time to half relaxation. Measurements of intracellular free calcium concentration and contractile force in trabeculae revealed a doubling of Ca2+ transient amplitude, with a concomitant boost in contractility. The present study demonstrates that increases in SERCA2a pump levels can directly enhance contractile function of the heart by increasing SR Ca2+ transport.

Enhanced myocardial contractility and increased Ca2+ transport function in transgenic hearts expressing the fast-twitch skeletal muscle sarcoplasmic reticulum Ca2+-ATPase.

In this study, we investigated whether the fast-twitch skeletal muscle sarco(endo)plasmic reticulum Ca2+ transport pump (SERCA1a) can functionally substitute the cardiac SERCA2a isoform and how its overexpression affects cardiac contractility. For this purpose, we generated transgenic (TG) mice that specifically overexpress SERCA1a in the heart, using the cardiac-specific alpha-myosin heavy chain promoter. Ectopic expression of SERCA1a resulted in a 2.5-fold increase in the amount of total SERCA protein. At the same time, the level of the endogenous SERCA2a protein was decreased by 50%, whereas the level of other muscle proteins, including calsequestrin, phospholamban, actin, and tropomyosin, remained unchanged. The steady-state level of SERCA phosphoenzyme intermediate was increased 2.5-fold, and the maximal velocity of Ca2+ uptake was increased 1.7-fold in TG hearts, demonstrating that the overexpressed protein is functional. Although the basal cytosolic calcium signal was decreased by 38% in TG cardiomyocytes, the amplitude of cytosolic calcium signal was increased by 71.8%. The rate of calcium resequestration was also increased in TG myocytes, which was reflected by a 51.6% decrease in the normalized time to 80% decay of calcium signal. This resulted in considerably increased peak rates of myocyte shortening and relengthening (50.0% and 66.6%, respectively). Cardiac functional analysis using isolated work-performing heart preparations revealed significantly faster rates of contraction and relaxation in TG hearts (41.9% and 39.5%, respectively). The time to peak pressure and the time to half-relaxation were shorter (29.1% and 32.7%, respectively). In conclusion, our study demonstrates that the SERCA1a pump can functionally substitute endogenous SERCA2a, and its overexpression significantly enhances Ca2+ transport and contractile function of the myocardium. These results also demonstrate that the SERCA pump level is a critical determinant of cardiac contractility.

Thyroid hormone-induced alterations in phospholamban-deficient mouse hearts.

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.

Overexpression of alpha1B-adrenergic receptor induces left ventricular dysfunction in the absence of hypertrophy.

The stimulation of cardiac alpha1-adrenergic receptors (AR) modulates the heart's inotropic response and plays a role in the induction of cardiomyocyte hypertrophy. We have analyzed transgenic mouse lines overexpressing a wild-type alpha1B-AR specifically in the heart. Basal level systolic and diastolic left ventricular (LV) contractile function was depressed both in the anesthetized closed-chest mouse and the perfused working-heart preparation. Intrinsic LV function was further characterized under controlled preload and afterload conditions using the perfusion model. Contractile parameters were restored by chronic treatment with the alpha-AR antagonist prazosin. In ventricular function curves, the load-dependent force increases (length-tension effects) remained intact, although the transgenic curve was shifted to lower levels. The basal level contractile deficits were paralleled by a decrease in calcium transients in isolated LV cardiomyocytes. LV function comparable to controls was restored by isoproterenol stimulation. The physiological changes occurred in the absence of cardiomyocyte hypertrophy. This transgenic model will be useful for studying the potential role of alpha1-AR in cardiac contractility and hypertrophy.

Beta-tropomyosin overexpression induces severe cardiac abnormalities.

Tropomyosin, a coiled-coil dimer, stabilizes actin filaments and is central to the control of calcium-regulated striated muscle contraction. Striated muscle-specific alpha-tropomyosin is the predominant isoform in cardiac muscle, with low levels of beta-tropomyosin restricted to fetal development in the mouse. To understand the functional role of various tropomyosin isoforms during myofilament activation and regulation in the intact sarcomere, we generated transgenic mice that overexpress striated muscle-specific beta-tropomyosin in the adult heart. Our earlier results succinctly demonstrate that overexpression of beta-tropomyosin in the hearts of transgenic mice decreases endogeneous alpha-tropomyosin levels while altering diastolic function of the myocardium. To explore further the significance of altering the alpha- to beta-tropomyosin isoform ratio in developing murine myocardium, we generated transgenic mice which express beta-tropomyosin at high levels in the heart. The data show that higher levels of beta-tropomyosin expression are lethal with death ensuing between 10-14 days postnatally. A detailed histological analysis demonstrates that the hearts of these mice exhibit several pathological abnormalities, including thrombus formation in the lumen of both atria and in the subendocardium of the left ventricle. Other changes include atrial enlargement and fibrosis, and diffuse myocytolysis, Physiological analyses using ventricular muscle strip preparations from these mice reveal that both myocardial contraction and relaxation parameters are severely impaired. Thus, these results firmly demonstrate an essential difference in tropomyosin isoform function in physiologically regulating cardiac performance.

Phospholamban modulates murine atrial contractile parameters and responses to beta-adrenergic agonists.

Phospholamban gene transcript levels are much lower in murine atria as compared to murine ventricles and this reduced phospholamban expression has been suggested to result in enhanced atrial contractile parameters. To delineate the functional role of phospholamban in murine atrium, the contractile parameters of isolated muscles from phospholamban knockout and cardiac-specific phospholamban overexpression mice along with their isogenic wild-type controls were evaluated. Assessment of the times (ms) to peak tension development and to half-relaxation of developed tension, as well as the rates (mg/s) of tension development and relaxation in paced atrial muscles, revealed that phospholamban ablation was associated with enhanced rates of relaxation with no significant effect on contraction rate, while phospholamban overexpression (three-fold) was associated with depressed rates of both contraction and relaxation. Isoproterenol stimulation resulted in significant increases in the rates of developed tension and relaxation in both phospholamban deficient and phospholamban overexpression atria, indicating that the beta-adrenergic pathway was functional in these muscles. These findings suggest that phospholamban is an important modulator of atrial contractility and its responses to beta-adrenergic agonists.

Ectopic expression of phospholamban in fast-twitch skeletal muscle alters sarcoplasmic reticulum Ca2+ transport and muscle relaxation.

There are three isoforms of the sarcoplasmic reticulum Ca2+-ATPase; they are known as SERCA1, SERCA2, and SERCA3. Phospholamban is present in tissues that express the SERCA2 isoform and is an inhibitor of the affinity of SERCA2 for calcium. In vitro reconstitution and cell culture expression studies have shown that phospholamban can also regulate SERCA1, the fast-twitch skeletal muscle isoform. To determine whether regulation of SERCA1 by phospholamban can be of physiological relevance, we generated transgenic mice that ectopically express phospholamban in fast-twitch skeletal muscle, a tissue normally devoid of phospholamban. Ectopic expression of phospholamban was associated with a decrease in the affinity of SERCA1 for calcium. Assessment of isometric twitch contractions of intact fast-twitch skeletal muscles revealed depressed rates of relaxation in transgenic mice compared with wild-type cohorts. Furthermore, the prolongation of muscle relaxation appeared to correlate with the levels of phospholamban expressed in two transgenic mouse lines. These findings indicate that ectopic expression of phospholamban in fast-twitch skeletal muscle is associated with inhibition of SERCA1 activity and decreased relaxation rates of this muscle.

Phospholamban ablation enhances relaxation in the murine soleus.

Phospholamban (PLB) is expressed in slow-twitch skeletal, cardiac, and smooth muscles. Several studies have indicated that it is an important regulator of basal contractility and the stimulatory responses to isoproterenol in the mammalian heart. To determine whether PLB is also a key modulator of slow-twitch skeletal muscle contractility, we examined isometric twitch contractions of isolated, intact soleus muscles from wild-type (WT) and PLB-deficient mice in parallel. Soleus muscles from PLB-deficient mice exhibited a significant (25%) decrease in the time to half relaxation, with no change in contraction time compared with WT soleus muscles. The observed enhancement of relaxation in the PLB-deficient soleus was not associated with alterations in the protein levels of either the sarcoplasmic reticular Ca(2+)-adenosinetriphosphatase or the ryanodine receptor. Examination of the effects of isoproterenol on the twitch kinetics of these muscles revealed 1) no effect on the contraction times of either WT or PLB-deficient muscles and 2) a significant decrease in the half relaxation time of the WT soleus, whereas this parameter remained unchanged in the PLB-deficient muscle. Furthermore, with maximal isoproterenol stimulation, the half relaxation time of the WT soleus was similar to that of the nonstimulated PLB-deficient soleus. These results suggest that PLB is a key determinant of relaxation in slow-twitch skeletal muscle under basal conditions and during isoproterenol stimulation.

The relative phospholamban and SERCA2 ratio: a critical determinant of myocardial contractility.

Phospholamban is a regulatory phosphoprotein which modulates the active transport of Ca2+ by the cardiac sarcoplasmic reticular Ca(2+)-ATPase enzyme (SERCA2) into the lumen of the sarcoplasmic reticulum. Phospholamban, which is a reversible inhibitor of SERCA2, represses the enzyme's activity, and this inhibition is relieved upon phosphorylation of phospholamban in response to beta-adrenergic stimulation. In this way, phospholamban is an important regulator of SERCA2-mediated myocardial relaxation during diastole. This report centers on the hypothesis that the relative levels of phospholamban: SERCA2 in cardiac muscle plays an important role in the muscle's overall contractility status. This hypothesis was tested by comparing the contractile parameters of: a) murine atrial and ventricular muscles, which differentially express phospholamban, and b) murine wild-type and phospholamban knock-out hearts. These comparisons revealed that atrial muscles, which have a 4.2-fold lower phospholamban: SERCA2 ratio than ventricular muscles, exhibited rates of force development and relaxation of tension, which were three-fold faster that these parameters for ventricular muscles. Similar comparisons were made via analyses of left-ventricular pressure development recorded for isolated, work-performing hearts from wild-type and phospholamban knock-out mice. In these studies, hearts from phospholamban knock-out mice, which were devoid of phospholamban, exhibited enhanced parameters of left-ventricular contractility in comparison to wild-type hearts. These results suggest that the relative phospholamban: SERCA2 ratio is critical in the regulation of myocardial contractility and alterations in this ratio may contribute to the functional deterioration observed during heart failure.