The ligand specificity of transforming growth factor beta (TGF␤) in vivo in mouse cardiac cushion epithelial-to-mesenchymal transition (EMT) is poorly understood. To elucidate the function of TGF␤ in cushion EMT, we analyzed Tgfb1 ؊/؊ , Tgfb2 ؊/؊ , and Tgfb3 ؊/؊ mice between embryonic day (E) 9.5 and E14.5 using both in vitro and in vivo approaches. Atrioventricular (AV) canal collagen gel assays at E9.5 indicated normal EMT in both Tgfb1 ؊/؊ and Tgfb3 ؊/؊ mice. However, analysis of Tgfb2 ؊/؊ AV explants at E9.5 and E10.5 indicated that EMT, but not cushion cell proliferation, was initially delayed but later remained persistent. This was concordant with the observation that Tgfb2 ؊/؊ embryos, and not Tgfb1 ؊/؊ or Tgfb3

Background-Tropomyosin (TM), an essential actin-binding protein, is central to the control of calcium-regulated striated muscle contraction. Although TPM1␣ (also called ␣-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. By developing a TPM1-specific antibody, we found that the TPM1 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 in sarcomeric function, we generated transgenic mice overexpressing cardiac-specific TPM1. Incorporation of increased levels of TPM1 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 compared with TPM1␣. Conclusions-This functional analysis of TPM1 provides a possible mechanism for the consequences of the TM isoform switch observed in dilated cardiomyopathy and heart failure patients. (Circulation. 2010;121:410-418.)Key Words: cardiomyopathy Ⅲ contractility Ⅲ heart failure Ⅲ myocardial contraction T he heart adapts to different challenges presented by an array of mechanical, hormonal, and nutritional signals in the process of maintaining its circulatory function. Isoform switching of sarcomeric proteins is 1 mode the heart uses to adapt to those challenges, along with alterations in the relative abundance and phosphorylation status of contractile and regulatory proteins. 1 These changes in isoform expression and phosphorylation status also play an essential role during cardiac development and in response to cardiac hypertrophy and heart failure (HF). Although sarcomeric protein isoforms are subject to developmental regulation, cardiomyopathy and HF primarily elicit changes in thick filament protein isoforms. 2 The only thin filament protein to change isoform expression in the failing human heart is troponin T. 3,4 Furthermore, altered phosphorylation of troponin I, myosin binding protein C, and other sarcomeric proteins has dramatic effects on cardiac function in the failing human myocardium. 5 Editorial see p 351 Clinical Perspective on p 418To understand the specific role of another thin filament protein, tropomyosin (TM), in the normal and the pathological heart, it is essential to define the TM isoform expression profile. Tropomyosins comprise a family of actin-binding proteins encoded by 4 different genes (TPM1, TPM2, TPM3, and TPM4). Each gene uses alternative splicing, alternative promoters, and differential processing to encode multiple striated muscle, smooth muscle, and cytoskeletal transcripts. For example, the TPM1...

Abstract-Mutations in striated muscle ␣-tropomyosin (␣-TM), an essential thin filament protein, cause both dilated cardiomyopathy (DCM) and familial hypertrophic cardiomyopathy. Two distinct point mutations within ␣-tropomyosin are associated with the development of DCM in humans: Glu40Lys and Glu54Lys. To investigate the functional consequences of ␣-TM mutations associated with DCM, we generated transgenic mice that express mutant ␣-TM (Glu54Lys) in the adult heart. Results showed that an increase in transgenic protein expression led to a reciprocal decrease in endogenous ␣-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 Ca 2ϩ sensitivity and tension generation. Real-time RT-PCR quantification demonstrated an increased expression of ␤-myosin heavy chain, brain natriuretic peptide, and skeletal actin and a decreased expression of the Ca 2ϩ handling proteins sarcoplasmic reticulum Ca 2ϩ -ATPase and ryanodine receptor. Furthermore, our study also indicates that the ␣-TM54 mutation decreases tropomyosin flexibility, which may influence actin binding and myofilament Ca 2ϩ 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. Key Words: mouse model Ⅲ transgenic Ⅲ myocardial contractility Ⅲ thin filament T ropomyosin (TM) is an ␣ helical coiled-coil fibrous protein that binds actin filaments providing structural stability and modulation of filament function. In striated muscle, TM along with the troponin complex regulates Ca 2ϩ -mediated actin-myosin crossbridges. Numerous mutations in many of the contractile proteins of the cardiac sarcomere have been associated with dilated and hypertrophic cardiomyopathy, where the myocardial performance is compromised. In humans, 2 dilated cardiomyopathy (DCM)-associated mutations (Glu54Lys and Glu40Lys) have been identified in ␣-tropomyosin (␣-TM) (or TPM1), 1 in contrast to the 8 distinct mutations in the same gene that are associated with familial hypertrophic cardiomyopathy (FHC). 2 The DCM mutations in ␣-TM are located in a region (amino acids 40 to 100) where half of the reported human FHC mutations occur (Glu62Gln, Ala63Val, Lys70Thr, Val95Ala); this region does not interact with troponin (Tn)T.Protein-modeling studies on the TM filaments harboring Glu54Lys and Glu40Lys substitutions show that both of them create a strong local increase in the positive cha...