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...

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) Ca 2؉ 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 ␤-adrenergic agonists.The sarco(endo)plasmic reticulum (SR) 2 Ca 2ϩ ATPase (SERCA) plays a dominant role in transporting Ca 2ϩ into the SR during the contraction-relaxation cycle of the heart. The rate and amount of Ca 2ϩ transported into the SR determines both the rate of muscle relaxation and the SR Ca 2ϩ load available for the next cycle of contraction (1-4). It is well established that SERCA function is regulated by phospholamban (PLB), whose inhibitory effect is reversed by phosphorylation by protein kinase A and the calcium/calmodulin-dependent protein kinase (CAMKII) during adrenergic activation (5-7). Recent studies have shown that in addition to PLB, sarcolipin (SLN) could also play an important role in the regulation of SERCA pump activity (8 -12).SLN is a 31-amino acid protein expressed in both cardiac and skeletal muscle (11,(13)(14)(15). We have recently demonstrated that SLN is localized in the cardiac SR membrane, and its distribution pattern is similar to SERCA2a and PLB (11). SLN mRNA is differentially expressed in small as opposed to larger mammals. In rodents, SLN mRNA is abundant in the atria with very low levels in the ventricle and skeletal muscles (11,14,15). In contrast, in larger mammals including humans, SLN mRNA is abundant in fast-twitch skeletal muscle compared with atria and ventricle (13). SLN expression is developmentally regulated (11), and its expression levels are modified under certain pathological conditions of the muscle (16,17). Decreased expression of SLN mRNA has been shown in the atria of patients with atrial fibrillation (16). A recent study also showed that SLN mRNA was up-regulated ϳ50-fold in the hypertrophied ventricles of Nkx2-5-null mice (17). Structural similarities between SLN and PLB indicate that they are homolog...

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...

Striated muscle tropomyosin (TM) interacts with actin and the troponin complex to regulate calcium-mediated muscle contraction. Previous work by our laboratory established that ␣-and ␤-TM isoforms elicit physiological differences in sarcomeric performance. Heart myofilaments containing ␤-TM exhibit an increased sensitivity to calcium that is associated with a decrease in the rate of relaxation and a prolonged time of relaxation. To address whether the carboxyl-terminal, troponin T binding domain of ␤-TM is responsible for these physiological alterations, we exchanged the 27 terminal amino acids of ␣-TM (amino acids 258 -284) for the corresponding region in ␤-TM. Hearts of transgenic mice that express this chimeric TM protein exhibit significant decreases in their rates of contraction and relaxation when assessed by ex vivo work-performing cardiac analyses. There are increases in the time to peak pressure and a dramatic increase in end diastolic pressure. In myofilaments, this chimeric protein induces depression of maximum tension and ATPase rate, together with a significant decrease in sensitivity to calcium. Our data are the first to demonstrate that the TM isoformspecific carboxyl terminus is a critical determinant of sarcomere performance and calcium sensitivity in both the whole heart and in isolated myofilaments. Tropomyosin (TM),1 a ␣-helical coiled-coil dimer, plays an essential role in the regulation of contraction and relaxation of the sarcomere. TM regulates sarcomeric performance through its binding to actin and the troponin complex. During muscle relaxation when cytoplasmic levels of calcium are low, tropomyosin blocks the myosin-binding site on the filamentous striated muscle actin. Upon stimulation, calcium is released into the myofilament space from the sarcoplasmic reticulum, via the ryanodine receptor Ca 2ϩ channel, and from extracellular stores; this calcium binds to troponin C within the troponin complex, which causes a conformational change in the position of TM on actin, thereby exposing the myosin-binding site. Myosin binds to actin and triggers muscle contractile activity until the stimulation ceases, and calcium is re-sequestered into the sarcoplasmic reticulum.Recent studies (1-3) in our laboratory have demonstrated that there are functional differences among the three highly conserved striated muscle TM isoforms. The ␣-TM isoform is the predominant isoform in both skeletal and cardiac musculature. In the adult murine heart, this isoform constitutes ϳ98% of the total TM. The remaining 2% of TM is ␤-TM, which is 86% identical to ␣-TM. In the skeletal musculature, ␤-TM expression is much greater, with levels dependent upon the specific muscle. TPM 3 (TM 30sk, ␥-TM) is not expressed in the murine heart but is found in slow-twitch skeletal musculature, such as the soleus where it composes ϳ30% of all the TMs (4). The TPM 3 isoform is 93% identical to ␣-TM and is 86% identical to ␤-TM. Studies in our laboratory using transgenic mice that express ␤-TM in the heart demonstrate that ␤-TM increas...

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 α-, β-, and γ-tropomyosin isoforms; this identity ranges from 86% -91%. We employ transgenic mouse model systems that express the different tropomyosin isoforms or chimeric tropomyosin molecules specifically in the myocardium. Results show 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.

Rationale Myocardial ischemia-reperfusion (I/R) results in the generation of oxygen-derived free radicals and the accumulation of lipid peroxidation-derived unsaturated aldehydes. However, the contribution of aldehydes to myocardial I/R injury has not been assessed. Objective We tested the hypothesis that removal of aldehydes by glutathione S-transferase P (GSTP) diminishes I/R injury. Methods and Results In adult male C57BL/6 mouse hearts, Gstp1/2 was the most abundant GST transcript followed by Gsta4 and Gstm4.1, and GSTP activity was a significant fraction of the total GST activity. mGstp1/2 deletion reduced total GST activity, but no compensatory increase in GSTA and GSTM or major antioxidant enzymes was observed. Genetic deficiency of GSTP did not alter cardiac function, but in comparison with hearts from wild-type (WT) mice, the hearts isolated from GSTP-null mice were more sensitive to I/R injury. Disruption of the GSTP gene also increased infarct size after coronary occlusion in situ. Ischemia significantly increased acrolein in hearts, and GSTP deficiency induced significant deficits in the metabolism of the unsaturated aldehyde, acrolein, but not in the metabolism 4-hydroxy-trans-2-nonenal (HNE) or trans-2-hexanal; and, upon ischemia, the GSTP-null hearts accumulated more acrolein-modified proteins than WT hearts. GSTP-deficiency did not affect I/R-induced free radical generation, JNK activation or depletion of reduced glutathione. Acrolein-exposure induced a hyperpolarizing shift in INa, and acrolein-induced cell death was delayed by SN-6, a Na+/Ca++ exchange inhibitor. Cardiomyocytes isolated from GSTP-null hearts were more sensitive than WT myocytes to acrolein-induced protein crosslinking and cell death. Conclusions GSTP protects the heart from I/R injury by facilitating the detoxification of cytotoxic aldehydes such as acrolein.

Familial hypertrophic cardiomyopathy (FHC) is a disease caused by mutations in contractile proteins of the sarcomere. Our laboratory developed a mouse model of FHC with a mutation in the thin filament protein alpha-tropomyosin (TM) at amino acid 180 (Glu180Gly). The hearts of these mice exhibit dramatic systolic and diastolic dysfunction, and their myofilaments demonstrate increased calcium sensitivity. The mice also develop severe cardiac hypertrophy, with death ensuing by 6 mo. In an attempt to normalize calcium sensitivity in the cardiomyofilaments of the hypertrophic mice, we generated a chimeric alpha-/beta-TM protein that decreases calcium sensitivity in transgenic mouse cardiac myofilaments. By mating mice from these two models together, we tested the hypothesis that an attenuation of myofilament calcium sensitivity would modulate the severe physiological and pathological consequences of the FHC mutation. These double-transgenic mice "rescue" the hypertrophic phenotype by exhibiting a normal morphology with no pathological abnormalities. Physiological analyses of these rescued mice show improved cardiac function and normal myofilament calcium sensitivity. These results demonstrate that alterations in calcium response by modification of contractile proteins can prevent the pathological and physiological effects of this disease.

Tropomyosin (TM), an integral component of the thin filament, is encoded by three striated muscle isoforms: alpha-TM, beta-TM, and TPM 3. Although the alpha-TM and beta-TM isoforms are well characterized, less is known about the function of the TPM 3 isoform, which is predominantly found in the slow-twitch musculature of mammals. To determine its functional significance, we ectopically expressed this isoform in the hearts of transgenic mice. We generated six transgenic mouse lines that produce varying levels of TPM 3 message with ectopic TPM 3 protein accounting for 40-60% of the total striated muscle tropomyosin. The transgenic mice have normal life spans and exhibit no morphological abnormalities in their sarcomeres or hearts. However, there are significant functional alterations in cardiac performance. Physiological assessment of these mice by using closed-chest analyses and a work-performing model reveals a hyperdynamic effect on systolic and diastolic function. Analysis of detergent-extracted fiber bundles demonstrates a decreased sensitivity to Ca(2+) in force generation and a decrease in length-dependent Ca(2+) activation with no detectable change in interfilament spacing as determined by using X-ray diffraction. Our data are the first to demonstrate that TM isoforms can affect sarcomeric performance by decreasing sensitivity to Ca(2+) and influencing the length-dependent Ca(2+) activation.