Ganapathy Jagatheesan

Dilated Cardiomyopathy Mutant Tropomyosin Mice Develop Cardiac Dysfunction With Significantly Decreased Fractional Shortening and Myofilament Calcium Sensitivity

Mutations in striated muscle &agr;-tropomyosin (&agr;-TM), an essential thin filament protein, cause both dilated cardiomyopathy (DCM) and familial hypertrophic cardiomyopathy. Two distinct point mutations within &agr;-tropomyosin are associated with the development of DCM in humans: Glu40Lys and Glu54Lys. To investigate the functional consequences of &agr;-TM mutations associated with DCM, we generated transgenic mice that express mutant &agr;-TM (Glu54Lys) in the adult heart. Results showed that an increase in transgenic protein expression led to a reciprocal decrease in endogenous &agr;-TM levels, with total myofilament TM protein levels remaining unaltered. Histological and morphological analyses revealed development of DCM with progression to heart failure and frequently death by 6 months. Echocardiographic analyses confirmed the dilated phenotype of the heart with a significant decrease in the left ventricular fractional shortening. Work-performing heart analyses showed significantly impaired systolic, and diastolic functions and the force measurements of cardiac myofibers revealed that the myofilaments had significantly decreased Ca2+ sensitivity and tension generation. Real-time RT-PCR quantification demonstrated an increased expression of &bgr;-myosin heavy chain, brain natriuretic peptide, and skeletal actin and a decreased expression of the Ca2+ handling proteins sarcoplasmic reticulum Ca2+-ATPase and ryanodine receptor. Furthermore, our study also indicates that the &agr;-TM54 mutation decreases tropomyosin flexibility, which may influence actin binding and myofilament Ca2+ sensitivity. The pathological and physiological phenotypes exhibited by these mice are consistent with those seen in human DCM and heart failure. As such, this is the first mouse model in which a mutation in a sarcomeric thin filament protein, specifically TM, leads to DCM.

Targeted Overexpression of Sarcolipin in the Mouse Heart Decreases Sarcoplasmic Reticulum Calcium Transport and Cardiac Contractility

The role of sarcolipin (SLN) in cardiac physiology was critically evaluated by generating a transgenic (TG) mouse model in which the SLN to sarco(endoplasmic)reticulum (SR) Ca2+ ATPase (SERCA) ratio was increased in the ventricle. Overexpression of SLN decreases SR calcium transport function and results in decreased calcium transient amplitude and rate of relaxation. SLN TG hearts exhibit a significant decrease in rates of contraction and relaxation when assessed by ex vivo work-performing heart preparations. Similar results were also observed with muscle preparations and myocytes from SLN TG ventricles. Interestingly, the inhibitory effect of SLN was partially relieved upon high dose of isoproterenol treatment and stimulation at high frequency. Biochemical analyses show that an increase in SLN level does not affect PLB levels, monomer to pentamer ratio, or its phosphorylation status. No compensatory changes were seen in the expression of other calcium-handling proteins. These studies suggest that the SLN effect on SERCA pump is direct and is not mediated through increased monomerization of PLB or by a change in PLB phosphorylation status. We conclude that SLN is a novel regulator of SERCA pump activity, and its inhibitory effect can be reversed by β-adrenergic agonists.

Molecular and Functional Characterization of a Novel Cardiac-Specific Human Tropomyosin Isoform

Background— Tropomyosin (TM), an essential actin-binding protein, is central to the control of calcium-regulated striated muscle contraction. Although TPM1&agr; (also called &agr;-TM) is the predominant TM isoform in human hearts, the precise TM isoform composition remains unclear. Methods and Results— In this study, we quantified for the first time the levels of striated muscle TM isoforms in human heart, including a novel isoform called TPM1&kgr;. By developing a TPM1&kgr;-specific antibody, we found that the TPM1&kgr; protein is expressed and incorporated into organized myofibrils in hearts and that its level is increased in human dilated cardiomyopathy and heart failure. To investigate the role of TPM1&kgr; in sarcomeric function, we generated transgenic mice overexpressing cardiac-specific TPM1&kgr;. Incorporation of increased levels of TPM1&kgr; protein in myofilaments leads to dilated cardiomyopathy. Physiological alterations include decreased fractional shortening, systolic and diastolic dysfunction, and decreased myofilament calcium sensitivity with no change in maximum developed tension. Additional biophysical studies demonstrate less structural stability and weaker actin-binding affinity of TPM1&kgr; compared with TPM1&agr;. Conclusions— This functional analysis of TPM1&kgr; provides a possible mechanism for the consequences of the TM isoform switch observed in dilated cardiomyopathy and heart failure patients.

Investigations into tropomyosin function using mouse models

Tropomyosin plays a key role in controlling calcium regulated sarcomeric contraction through its interactions with actin and the troponin complex. The focus of this review is on striated muscle tropomyosin isoforms and the in vivo approach we have taken to define the functional differences among these isoforms in regulating cardiac physiology. In addition, we address specific regions within tropomyosin that differ among the isoforms to impart differences in the physiological performance of muscle and the sarcomere itself. There is a high degree of amino acid identity among the three striated muscle alpha-, beta-, and gamma-tropomyosin isoforms; this identity ranges from 86% to 91%. We employ transgenic mouse model systems that express the different tropomyosin isoforms or chimeric tropomyosin molecules specifically in the myocardium. Results show that the three isoforms differentially regulate the rates of cardiac contraction and relaxation, along with conferring differences in myofilament calcium sensitivity and sarcomere tension development. We also found the putative troponin T binding regions of tropomyosin (amino acids 175-190 and 258-284) appear to a play significant role in imparting these physiological differences that are observed during cardiac and sarcomeric contraction/relaxation. In addition, we have successfully used chimeric tropomyosin molecules to rescue cardiomyopathic diseased mice by normalizing sarcomeric performance. These studies illustrate not only the importance of tropomyosin structure and function for understanding muscle physiology, but also demonstrate how this information can potentially be used for gene therapy.

The role of tropomyosin in heart disease.

Cardiovascular disease is the number one cause of mortality in the Western world, with heart failure representing one of the fastest growing subgroups over the past decade. Heart failure, the progressive loss of cardiac contractile performance resulting in an inability to pump an adequate supply of systemic blood, affects an estimated 5 million Americans with estimated medical costs of

Neonatal gene transfer of Serca2a delays onset of hypertrophic remodeling and improves function in familial hypertrophic cardiomyopathy.

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Striated muscle tropomyosin isoforms differentially regulate cardiac performance and myofilament calcium sensitivity

50 billion per year1. A number of common disease disease stimuli can induce heart failure, including hypertension, myocardial infarction, ischemia associated with coronary artery disease, congenital malformation, familial hypertrophic and dilated cardiomyopathies and diabetic cardiomyopathy. Systolic and diastolic dysfunction is common in patients suffering from coronary artery disease and hypertension and is a main cause of heart failure. Hypertrophic growth of cardiomyocytes also occurs in many forms of heart failure and may contribute to the pathogenesis of the failure state2.

The Function of Normal and Familial Hypertrophic Cardiomyopathy-Associated Tropomyosin

Familial hypertrophic cardiomyopathy (FHC) is an autosomal dominant genetic disorder linked to numerous mutations in the sarcomeric proteins. The clinical presentation of FHC is highly variable, but it is a major cause of sudden cardiac death in young adults with no specific treatments. We tested the hypothesis that early intervention in Ca(2+) regulation may prevent pathological hypertrophy and improve cardiac function in a FHC displaying increased myofilament sensitivity to Ca(2+) and diastolic dysfunction. A transgenic (TG) mouse model of FHC with a mutation in tropomyosin at position 180 was employed. Adenoviral-Serca2a (Ad.Ser) was injected into the left ventricle of 1-day-old non-transgenic (NTG) and TG mice. Ad.LacZ was injected as a control. Serca2a protein expression was significantly increased in NTG and TG hearts injected with Ad.Ser for up to 6 weeks. Compared to TG-Ad.LacZ hearts, the TG-Ad.Ser hearts showed improved whole heart morphology. Moreover, there was a significant decline in ANF and β-MHC expression. Developed force in isolated papillary muscle from 2- to 3-week-old TG-Ad.Ser hearts was higher and the response to isoproterenol (ISO) improved compared to TG-Ad.LacZ muscles. In situ hemodynamic measurements showed that by 3 months the TG-Ad.Ser hearts also had a significantly improved response to ISO compared to TG-Ad.LacZ hearts. The present study strongly suggests that Serca2a expression should be considered as a potential target for gene therapy in FHC. Moreover, our data imply that development of FHC can be successfully delayed if therapies are started shortly after birth.

Rescue of tropomyosin-induced familial hypertrophic cardiomyopathy mice by transgenesis

Tropomyosin (TM) plays a central role in calcium mediated striated muscle contraction. There are three muscle TM isoforms: α-TM, β-TM, and γ-TM. α-TM is the predominant cardiac and skeletal muscle isoform. β-TM is expressed in skeletal and embryonic cardiac muscle. γ-TM is expressed in slow-twitch musculature, but is not found in the heart. Our previous work established that muscle TM isoforms confer different physiological properties to the cardiac sarcomere. To determine whether one of these isoforms is dominant in dictating its functional properties, we generated single and double transgenic mice expressing β-TM and/or γ-TM in the heart, in addition to the endogenously expressed α-TM. Results show significant TM protein expression in the βγ-DTG hearts: α-TM: 36%, β-TM: 32%, and γ-TM: 32%. These βγ-DTG mice do not develop pathological abnormalities; however, they exhibit a hyper contractile phenotype with decreased myofilament calcium sensitivity, similar to γ-TM transgenic hearts. Biophysical studies indicate that γ-TM is more rigid than either α-TM or β-TM. This is the first report showing that with approximately equivalent levels of expression within the same tissue, there is a functional dominance of γ-TM over α-TM or β-TM in regulating physiological performance of the striated muscle sarcomere. In addition to the effect expression of γ-TM has on Ca2+ activation of the cardiac myofilaments, our data demonstrates an effect on cooperative activation of the thin filament by strongly bound rigor cross-bridges. This is significant in relation to current ideas on the control mechanism of the steep relation between Ca2+ and tension.

Microarray analysis of gene expression during early stages of mild and severe cardiac hypertrophy

This review will focus on the role of sarcomeric tropomyosin during the assembly and function of thin filaments in striated muscle. Particular emphasis will be devoted to how mutations in familial hypertrophic cardiomyopathy influence these processes. Due to the complex and extensive nature of this topic, this review will not address the role of tropomyosin in the cytoskeleton; however, excellent articles on this topic have addressed this area (Lin et al., 1988; Hegmann et al., 1989).