Muthu Periasamy

Alterations in sarcoplasmic reticulum gene expression in human heart failure. A possible mechanism for alterations in systolic and diastolic properties of the failing myocardium.

Recent studies have shown that intracellular Ca2+ handling is abnormal in the myocardium of patients with end-stage heart failure. Muscles from the failing hearts showed a prolonged Ca2+ transient and a diminished capacity to restore a low resting Ca2+ level during diastole. Accordingly, we examined whether this defect in Ca2+ transport function is due to alterations in sarcoplasmic reticulum gene expression. We determined the messenger RNA (mRNA) levels of sarcoplasmic reticulum Ca2+ transport proteins in failing human hearts from 17 cardiac transplant recipients with a diagnosis of dilated cardiomyopathy, primary pulmonary hypertension, or ischemic heart disease. The expression levels of each mRNA were compared with each other and then correlated with that of atrial natriuretic factor (ANF) mRNA in the failing ventricle. The mRNA levels for the calcium release channel (ryanodine receptor, RYR2), Ca2+ uptake pump (Ca(2+)-ATPase, SERCA2 isoform), and phospholamban differed significantly between heart samples but showed an inverse relation with that of ventricular ANF mRNA. In contrast, calsequestrin mRNA levels remained unchanged in these failing hearts. In addition, beta-myosin and alpha-cardiac actin mRNA levels also showed an inverse relation with ANF mRNA levels. These changes were observed in both right and left ventricles of hearts with congestive heart failure due to dilated cardiomyopathy, primary pulmonary hypertension, or ischemic heart disease. The results are consistent with the hypothesis that abnormal calcium handling in the sarcoplasmic reticulum of failing hearts is due to the altered expression of the genes encoding sarcoplasmic reticulum proteins.

Smooth muscle myosin heavy chain exclusively marks the smooth muscle lineage during mouse embryogenesis.

We cloned a portion of the mouse smooth muscle myosin heavy chain (SM-MHC) cDNA and analyzed its mRNA expression in adult tissues, several cell lines, and developing mouse embryos to determine the suitability of the SM-MHC promoter as a tool for identifying smooth muscle-specific transcription factors and to define the spatial and temporal pattern of smooth muscle differentiation during mouse development. RNase protection assays showed SM-MHC mRNA in adult aorta, intestine, lung, stomach, and uterus, with little or no signal in brain, heart, kidney, liver, skeletal muscle, spleen, and testes. From an analysis of 14 different cell lines, including endothelial cells, fibroblasts, and rhabdomyosarcomas, we failed to detect any SM-MHC mRNA; all of the cell lines induced to differentiate also showed no detectable SM-MHC. In situ hybridization of staged mouse embryos first revealed SM-MHC transcripts in the early developing aorta at 10.5 days post coitum (dpc). No hybridization signal was demonstrated beyond the aorta and its arches until 12.5 to 13.5 dpc, when SM-MHC mRNA appeared in smooth muscle cells (SMCs) of the developing gut and lungs as well as peripheral blood vessels. By 17.5 dpc, SM-MHC transcripts had accumulated in esophagus, bladder, and ureters. Except for blood vessels, no SM-MHC transcripts were ever observed in developing brain, heart, or skeletal muscle. These results indicate that smooth muscle myogenesis begins by 10.5 days of embryonic development in the mouse and establish SM-MHC as a highly specific marker for the SMC lineage. The SM-MHC promoter should therefore serve as a useful model for defining the mechanisms that govern SMC transcription during development and disease.

Human smooth muscle myosin heavy chain isoforms as molecular markers for vascular development and atherosclerosis.

Smooth muscle myosin heavy chains (MHCs) exist in multiple isoforms. Rabbit smooth muscles contain at least three types of MHC isoforms: SM1 (204 kD), SM2 (200 kD), and SMemb (200 kD). SM1 and SM2 are specific to smooth muscles, but SMemb is a nonmuscle-type MHC abundantly expressed in the embryonic aorta. We recently reported that these three MHC isoforms are differentially expressed in rabbit during normal vascular development and in experimental arteriosclerosis and atherosclerosis. The purpose of this study was to clarify whether expression of human smooth muscle MHC isoforms is regulated in developing arteries and in atherosclerotic lesions. To accomplish this, we have isolated and characterized three cDNA clones from human smooth muscle: SMHC94 (SM1), SMHC93 (SM2), and HSME6 (SMemb). The expression of SM2 mRNA in the fetal aorta was significantly lower as compared with SM1 mRNA, but the ratio of SM2 to SM1 mRNA was increased after birth. SMemb mRNA in the aorta was decreased after birth but appeared to be increased in the aged. To further examine the MHC expression at the histological level, we have developed three antibodies against human SM1, SM2, and SMemb using the isoform-specific sequences of the carboxyl terminal end. Immunohistologically, SM1 was constitutively positive from the fetal stage to adulthood in the apparently normal media of the aorta and coronary arteries, whereas SM2 was negative in fetal arteries of the early gestational stage. In human, unlike rabbit, aorta or coronary arteries, SMemb was detected even in the adult. However, smaller-sized arteries, like the vasa vasorum of the aorta or intramyocardial coronary arterioles, were negative for SMemb. Diffuse intimal thickening in the major coronary arteries was found to be composed of smooth muscles, reacting equally to three antibodies for MHC isoforms, but reactivities with anti-SM2 antibody were reduced with aging. With progression of atherosclerosis, intimal smooth muscles diminished the expression of not only SM2 but also SM1, whereas alpha-smooth muscle actin was well preserved. We conclude from these results that smooth muscle MHC isoforms are important molecular markers for studying human vascular smooth muscle cell differentiation as well as the cellular mechanisms of atherosclerosis.

SERCA pump isoforms: Their role in calcium transport and disease

The sarcoendoplasmic reticulum (SR) calcium transport ATPase (SERCA) is a pump that transports calcium ions from the cytoplasm into the SR. It is present in both animal and plant cells, although knowledge of SERCA in the latter is scant. The pump shares the catalytic properties of ion‐motive ATPases of the P‐type family, but has distinctive regulation properties. The SERCA pump is encoded by a family of three genes, SERCA1, 2, and 3, that are highly conserved but localized on different chromosomes. The SERCA isoform diversity is dramatically enhanced by alternative splicing of the transcripts, occurring mainly at the COOH‐terminal. At present, more than 10 different SERCA isoforms have been detected at the protein level. These isoforms exhibit both tissue and developmental specificity, suggesting that they contribute to unique physiological properties of the tissue in which they are expressed. The function of the SERCA pump is modulated by the endogenous molecules phospholamban (PLB) and sarcolipin (SLN), expressed in cardiac and skeletal muscles. The mechanism of action of PLB on SERCA is well characterized, whereas that of SLN is only beginning to be understood. Because the SERCA pump plays a major role in muscle contraction, a number of investigations have focused on understanding its role in cardiac and skeletal muscle disease. These studies document that SERCA pump expression and activity are decreased in aging and in a variety of pathophysiological conditions including heart failure. Recently, SERCA pump gene transfer was shown to be effective in restoring contractile function in failing heart muscle, thus emphasizing its importance in muscle physiology and its potential use as a therapeutic agent. Muscle Nerve, 2007

Sarcolipin is a newly identified regulator of muscle-based thermogenesis in mammals

The role of skeletal muscle in nonshivering thermogenesis (NST) is not well understood. Here we show that sarcolipin (Sln), a newly identified regulator of the sarco/endoplasmic reticulum Ca2+-ATPase (Serca) pump, is necessary for muscle-based thermogenesis. When challenged to acute cold (4 °C), Sln−/− mice were not able to maintain their core body temperature (37 °C) and developed hypothermia. Surgical ablation of brown adipose tissue and functional knockdown of Ucp1 allowed us to highlight the role of muscle in NST. Overexpression of Sln in the Sln-null background fully restored muscle-based thermogenesis, suggesting that Sln is the basis for Serca-mediated heat production. We show that ryanodine receptor 1 (Ryr1)-mediated Ca2+ leak is an important mechanism for Serca-activated heat generation. Here we present data to suggest that Sln can continue to interact with Serca in the presence of Ca2+, which can promote uncoupling of the Serca pump and cause futile cycling. We further show that loss of Sln predisposes mice to diet-induced obesity, which suggests that Sln-mediated NST is recruited during metabolic overload. These data collectively suggest that SLN is an important mediator of muscle thermogenesis and whole-body energy metabolism.

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.

Troponin I gene expression during human cardiac development and in end-stage heart failure.

Recent reports have demonstrated the presence of two isoforms of troponin I in the human fetal heart, namely, cardiac troponin I and slow skeletal muscle troponin I. Structural and physiological considerations indicate that these isoforms would confer differing contractile properties on the myocardium, particularly on the phosphorylation-mediated regulation of contractility by adrenergic agonists. We have investigated the developmental expression of these isoforms in the human heart from 9 weeks of gestation to 9 months of postnatal life, using Western blots revealed with troponin I antibodies to detect troponin protein isoforms and Northern blots to detect the corresponding mRNAs. The results show the following: 1) Slow skeletal muscle troponin I is the predominant isoform throughout fetal life. 2) After birth, the slow skeletal isoform is lost, with cardiac troponin I being the only isoform detectable by 9 months of postnatal development. 3) The protein isoforms and their corresponding mRNAs follow the same pattern of accumulation, suggesting that the transition in troponin expression is regulated at the level of gene transcription. The developmental transition in troponin I isoform content has implications for contractility of the fetal and postnatal myocardium. We further analyzed right and left ventricular muscle samples from 17 hearts in end-stage heart failure resulting from pulmonary hypertension, ischemic heart disease, or dilated cardiomyopathy. Cardiac troponin I mRNA remained abundant in each case, and slow skeletal muscle troponin I mRNA was not detectable in any of sample. We conclude that alterations in troponin I isoform content do not therefore contribute to the altered contractile characteristics of the adult failing ventricle.

Effect of thyroid hormone on the expression of mRNA encoding sarcoplasmic reticulum proteins.

The purpose of this study was to determine the expression of genes encoding various sarcoplasmic reticulum components that are functionally coupled with calcium release, uptake, and storage function during cardiac hypertrophy induced by thyroid hormone. Hyperthyroidism was induced in two groups of rabbits by the injection of 200 micrograms/kg L-thyroxine (T4) daily for 4 days (T4-4-day group) and 8 days (T4-8-day group). Hypothyroidism was induced in another group of rabbits by adding 0.8 mg/ml propylthiouracil to the drinking water for 4 weeks. The relative expression level of mRNA encoding different sarcoplasmic reticulum proteins was determined by RNA slot blot and Northern blot analysis. In hyperthyroid hearts, the steady-state level of cardiac ryanodine receptor mRNA and sarcoplasmic reticulum cardiac/slow-twitch Ca(2+)-ATPase mRNA were both increased to 147% (T4-4-day group) and 186% (T4-8-day group) of control, respectively, but decreased to 71% and 75%, respectively, in hypothyroid ventricles. The mRNA level for phospholamban was decreased in both hyperthyroidism (T4-8-day group, 72%) and hypothyroidism (77%) in these hearts. On the other hand, calsequestrin mRNA levels did not change in hyperthyroid and hypothyroid ventricles. In accord with the changes in Ca(2+)-ATPase mRNA levels, the Ca(2+)-ATPase protein was increased to 199% (T4-8-day group) in hyperthyroid ventricles and decreased to 86% of control in hypothyroid ventricles. The expression levels of ryanodine receptor, Ca(2+)-ATPase, phospholamban, and calsequestrin mRNAs were similarly altered in skeletal muscle tissues from hyperthyroid and hypothyroid rabbits. These results indicate that the mRNA levels of sarcoplasmic reticulum proteins responsible for calcium release and calcium uptake are coordinately regulated in response to changes in thyroid hormone level in both heart and skeletal muscle. These changes in mRNA level should lead to changes in protein levels and thus to altered calcium release and uptake in the chronic stages of hyperthyroidism and hypothyroidism.

Resveratrol, an activator of SIRT1, upregulates sarcoplasmic calcium ATPase and improves cardiac function in diabetic cardiomyopathy.

Reduced sarcoplasmic calcium ATPase (SERCA2a) expression has been shown to play a significant role in the cardiac dysfunction in diabetic cardiomyopathy. The mechanism of SERCA2a repression is, however, not known. This study was designed to examine the effect of resveratrol (RSV), a potent activator of SIRT1, on cardiac function and SERCA2a expression in chronic type 1 diabetes. Adult male mice were injected with streptozotocin (STZ) and fed with either a regular diet or a diet enriched with RSV. STZ administration produced progressive decline in cardiac function, associated with markedly reduced SERCA2a and SIRT1 protein levels and increased collagen deposition; RSV treatment to these mice had a tremendous beneficial effect both in terms of improving SERCA2a expression and on cardiac function. In cultured cardiomyocytes, RSV restored SERCA2 promoter activity, which was otherwise highly repressed in high-glucose media. Protective effects of RSV were found to be dependent on its ability to activate Silent information regulator (SIRT) 1. In cardiomyocytes, overexpression of SIRT1 was found sufficient to activate SERCA2 promoter in a dose-dependent manner. In contrast, pretreatment of cardiomyocytes with SIRT1 antagonist, splitomycin, blocked these beneficial effects of RSV. In addition, SIRT1 knockout (+/-) mice were also found to be more sensitive to STZ-induced decline in SERCA2a mRNA. The data demonstrate that, in chronic diabetes, 1) the enzymatic activity of cardiac SIRT1 is reduced, which contributes to reduced expression of SERCA2a and 2) through activation of SIRT1, RSV enhances expression of SERCA2a and improves cardiac function.

Ablation of a Ca2+‐activated K+ channel (SK2 channel) results in action potential prolongation in atrial myocytes and atrial fibrillation

Small conductance Ca2+‐activated K+ channels (SK channels) have been reported in excitable cells, where they aid in integrating changes in intracellular Ca2+(Ca2+i) with membrane potential. We have recently reported the functional existence of SK2 channels in human and mouse cardiac myocytes. Moreover, we have found that the channel is predominantly expressed in atria compared to the ventricular myocytes. We hypothesize that knockout of SK2 channels may be sufficient to disrupt the intricate balance of the inward and outward currents during repolarization in atrial myocytes. We further predict that knockout of SK2 channels may predispose the atria to tachy‐arrhythmias due to the fact that the late phase of the cardiac action potential is highly susceptible to aberrant excitation. We take advantage of a mouse model with genetic knockout of the SK2 channel gene. In vivo and in vitro electrophysiological studies were performed to probe the functional roles of SK2 channels in the heart. Whole‐cell patch‐clamp techniques show a significant prolongation of the action potential duration prominently in late cardiac repolarization in atrial myocytes from the heterozygous and homozygous null mutant animals. Morover, in vivo electrophysiological recordings show inducible atrial fibrillation in the null mutant mice but not wild‐type animals. No ventricular arrhythmias are detected in the null mutant mice or wild‐type animals. In summary, our data support the important functional roles of SK2 channels in cardiac repolarization in atrial myocytes. Genetic knockout of the SK2 channels results in the delay in cardiac repolarization and atrial arrhythmias.