Role of troponin T in disease

Several striated muscle myopathies have been directly linked to mutations in contractile and associated proteins. Troponin T (TnT) is one of the three subunits that form troponin (Tn) which together with tropomyosin is responsible for the regulation of striated muscle contraction. All three subunits of cardiac Tn as well as tropomyosin have been associated with hypertrophic cardiomyopathy (HCM). However, TnT accounts for most of the mutations that cause HCM in these regulatory proteins. To date 30 mutations have been identified in the cardiac TnT (CTnT) gene that results in familial HCM (FHC). The CTnT gene has also been associated with familial dilated cardiomyopathy (DCM). CTnT deficiency is lethal due to impaired cardiac development. A recessive nonsense mutation in the gene encoding slow skeletal TnT has been associated with an unusual, severe form of nemaline myopathy among the Old Order Amish. How each mutation leads to the diverse clinical symptoms associated with FHC, DCM or nemaline myopathy is unclear. However, the use of animal model systems, in particular transgenic mice, has significantly increased our knowledge of normal and myopathic muscle physiology. In this review, we focus on the role of TnT in muscle physiology and disease. (Mol Cell Biochem 263: 115–129, 2004)     

Selective deletion of the NH2-terminal variable region of cardiac troponin T in ischemia reperfusion by myofibril-associated mu-calpain cleavage

The structure of the NH2-terminal region of troponin T (TnT) is hypervariable among the muscle type-specific isoforms and is also regulated by alternative RNA splicing. This region does not contain binding sites for other thin filament proteins, but alteration of its structure affects the Ca2+ regulation of muscle contraction. Here we report a truncated cardiac TnT produced during myocardial ischemia reperfusion. Amino acid sequencing and protein fragment reconstruction determined that it is generated by a posttranslational modification selectively removing the NH2-terminal variable region and preserving the conserved core structure of TnT. Triton X-100 extraction of cardiac muscle fibers promoted production of the NH2-terminal truncated cardiac TnT (cTnT-ND), indicating a myofibril-associated proteolytic activity. Mu-calpain is a myofibril-associated protease and is known to degrade TnT. Supporting a role of mu-calpain in producing cTnT-ND in myocardial ischemia reperfusion, calpain inhibitors decreased the level of cTnT-ND in Triton-extracted myofibrils. Mu-calpain treatment of the cardiac myofibril and troponin complex specifically reproduced cTnT-ND. In contrast, mu-calpain treatment of isolated cardiac TnT resulted in nonspecific degradation, suggesting that this structural modification is relevant to physiological structures of the myofilament. Triton X-100 treatment of transgenic mouse cardiac myofibrils overexpressing fast skeletal muscle TnT produced similar NH2-terminal truncations of the endogenous and exogenous TnT, despite different amino acid sequences at the cleavage site. With the functional consequences of removing the NH2-terminal variable region of TnT, the mu-calpain-mediated proteolytic modification of TnT may act as an acute mechanism to adjust muscle contractility under stress conditions.     

Organ culture of adult amphibian heart: a fine structural analysis.

Pieces of hearts from adult newts were cultured up to 2 months. Within 7 days of culture, approximately 37% of the cardiac explants were attached to the substrate and more than 33% of the attached explants and approximately 15% of the unattached explants established pulsation rates ranging from 3 to 67 beats/min. The control and cultured explants were processed at weekly intervals for electron microscopy. The diameter of the control cardiac muscle cells ranged approximately 3-5 micron. The cell surface was provided with microvilli. The intercellular spaces ranged approximately 150-500 A. The intercalated discs lacked the step-like courses observed in the mammalian cardiac muscle. Sarcoplasmic reticulum was scanty. Desmosomal-dense materials were frequently continuous with the Z-bands of both control and cultured cardiac muscle cells. The transverse tubular system and gap junction were absent in newt ventricles. The functional implications of these characterisitics are discussed. At the end of 1 week of culture, the surfaces of the explants were covered by one or more layers of non-muscle cells, and the core of the explants consisted mostly of cardiac muscle cells. In a few cardiac muscle cells the myofibrillar organization was disrupted, resulting in the distribution of scattered patches of myofibrils and free myofilaments in the sarcoplasm. A small number of intact muscle cells contained a considerable number of dense granules in the sarcoplasm. At 15 days in culture, a large number of muscle cells showed structural features reminiscent of embryonic cardiac muscle cells. These cells possessed patches of myofibrils, scattered myofilaments and scanty sarcoplasmic reticulum along with other cellular organelles and inclusions. Several of these altered cardiac muscle cells contained mitotic figures. The cardiac explants maintained the initial beating rate until the end of 2 months of culture, except for the 11% of the explants which stopped beating. By 3-4 weeks in culture, most of the cardiac muscle cells possessed the altered cell morphology mentioned above. The explants after 60 days in culture became more flattened than the earlier explants. The intact cardiac muscle cells were rare, and the cores of the explants were mostly occupied by the altered cardiac muscle cells. It is evident from our studies that the cardiac muscle cells have undergone dedifferentiation in long-term culture, and that this dedifferentiation process has yet had no effect in the maintenance of contractility of the explants. Furthermore, these dedifferentiated cardiac muscle cells are capable of DNA synthesis and mitosis.     

Myofibril organisation and mitosis in cultured cardiac muscle cells.

Isolated cardiac muscle cells grown in vitro have been studied with respect to their ability to contract spontaneously and maintain myofibrillar organisation during division. These cells do not round up to undergo mitosis; division is achieved by the cell pinching itself in two in a selected area. This adaptation minimises disturbance to cell attachment sites and to myofibrils running between them. We correlated this with the persistence of beating during division and the maintenance of myofibrils with intact Z bands, even in close proximity to the nucleus, through division in many cells. Cessation of beating and disorganisation of myofibrils are therefore not prerequisites for division of cardiac muscle cells, as reported previously.     

Alterations of tension-dependent ATP utilization in a transgenic rat model of hypertrophic cardiomyopathy

Although it is established that familial hypertrophic cardiomyopathy (FHC) is caused by mutations in several sarcomeric proteins, including cardiac troponin T (TnT), its pathogenesis is still not completely understood. Previously, we established a transgenic rat model of FHC expressing a human TnT molecule with a truncation mutation (DEL-TnT). This study investigated whether contractile dysfunction and electrical vulnerability observed in DEL-TnT rats might be due to alterations of intracellular Ca(2+) homeostasis, myofibrillar Ca(2+) sensitivity, and/or myofibrillar ATP utilization. Simultaneous measurements of the force of contraction and intracellular Ca(2+) transients were performed in right ventricular trabeculae of DEL-TnT hearts at 0.25 and 1.0 Hz. Rats expressing wild-type human TnT as well as nontransgenic rats served as controls. In addition, calcium-dependent ATPase activity and tension development were investigated in skinned cardiac muscle fibers. Force of contraction was significantly decreased in DEL-TnT compared with nontransgenic rats and TnT. Time parameters of Ca(2+) transients were unchanged at 0.25 Hz but prolonged at 1.0 Hz in DEL-TnT. The amplitude of the fura-2 transient was similar in all groups investigated, whereas diastolic and systolic fura-2 ratios were found elevated in rats expressing nontruncated human troponin T. In DEL-TnT rats, myofibrillar Ca(2+)-dependent tension development as well as Ca(2+) sensitivity of tension were significantly decreased, whereas tension-dependent ATP consumption ("tension cost") was markedly increased. Thus, a C-terminal truncation of the cardiac TnT molecule impairs the force-generating capacity of the cycling cross-bridges resulting in increased tension-dependent ATP utilization. Taken together, our data support the hypothesis of energy compromise as a contributing factor in the pathogenesis of FHC.     

Molecular and immunohistochemical analyses of cardiac troponin T during cardiac development in the Mexican axolotl, Ambystoma mexicanum

The Mexican axolotl, Ambystoma mexicanum, is an excellent animal model for studying heart development because it carries a naturally occurring recessive genetic mutation, designated gene c, for cardiac nonfunction. The double recessive mutants (c/c) fail to form organized myofibrils in the cardiac myoblasts resulting in hearts that fail to beat. Tropomyosin expression patterns have been studied in detail and show dramatically decreased expression in the hearts of homozygous mutant embryos. Because of the direct interaction between tropomyosin and troponin T (TnT), and the crucial functions of TnT in the regulation of striated muscle contraction, we have expanded our studies on this animal model to characterize the expression of the TnT gene in cardiac muscle throughout normal axolotl development as well as in mutant axolotls. In addition, we have succeeded in cloning the full-length cardiac troponin T (cTnT) cDNA from axolotl hearts. Confocal microscopy has shown a substantial, but reduced, expression of TnT protein in the mutant hearts when compared to normal during embryonic development.     

A troponin T mutation that causes infantile restrictive cardiomyopathy increases Ca2+ sensitivity of force development and impairs the inhibitory properties of troponin

Restrictive cardiomyopathy (RCM) is a rare disorder characterized by impaired ventricular filling with decreased diastolic volume. We are reporting the functional effects of the first cardiac troponin T (CTnT) mutation linked to infantile RCM resulting from a de novo deletion mutation of glutamic acid 96. The mutation was introduced into adult and fetal isoforms of human cardiac TnT (HCTnT3-DeltaE96 and HCTnT1-DeltaE106, respectively) and studied with either cardiac troponin I (CTnI) or slow skeletal troponin I (SSTnI). Skinned cardiac fiber measurements showed a large leftward shift in the Ca(2+) sensitivity of force development with no differences in the maximal force. HCTnT1-DeltaE106 showed a significant increase in the activation of actomyosin ATPase with either CTnI or SSTnI, whereas HCTnT3-DeltaE96 was only able to increase the ATPase activity with CTnI. Both mutants showed an impaired ability to inhibit the ATPase activity. The capacity of the CTnI.CTnC and SSTnI.CTnC complexes to fully relax the fibers after TnT displacement was also compromised. Experiments performed using fetal troponin isoforms showed a less severe impact compared with the adult isoforms, which is consistent with the cardioprotective role of SSTnI and the rapid onset of RCM after birth following the isoform switch. These data indicate that troponin mutations related to RCM may have specific functional phenotypes, including large leftward shifts in the Ca(2+) sensitivity and impaired abilities to inhibit ATPase and to relax skinned fibers. All of this would account for and contribute to the severe diastolic dysfunction seen in RCM.     

Contractile activity and cell-cell contact regulate myofibrillar organization in cultured cardiac myocytes

Adult feline ventricular myocytes cultured on a laminin-coated substratum reestablish intercellular junctions, yet disassemble their myofibrils. Immunofluorescence microscopy reveals that these non- beating heart cells lack vinculin-positive focal adhesions; moreover, intercellular junctions are also devoid of vinculin. When these quiescent myocytes are stimulated to contract with the beta-adrenergic agonist, isoproterenol, extensive vinculin-positive focal adhesions and intercellular junctions emerge. If solitary myocytes are stimulated to beat, an elaborate series of vinculin-positive focal adhesions develop which appear to parallel the reassembly of myofibrils. In cultures where neighboring myocytes reestablish cell-cell contact, myofibrils appear to reassemble from the fascia adherens rather than focal contacts. Activation of beating is accompanied by a significant reduction in the rate of total and cytoskeletal protein synthesis; in fact, myofibrillar reassembly, redevelopment of focal adhesions and fascia adherens junctions require no protein synthesis for at least 24 h, implying the existence of an assembly competent pool of cytoskeletal proteins. Maturation of the fasciae adherens and the appearance of vinculin within Z-line/costameres, does require de novo synthesis of new cytoskeletal proteins. Changes in cytoskeletal protein turnover appear dependent on beta agonist-induced cAMP production, but myofibrillar reassembly is a cAMP-independent event. Such observations suggest that mechanical forces, in the guise of contractile activity, regulate vinculin distribution and myofibrillar order in cultured adult feline heart cells.     

Co-expression of skeletal and cardiac troponin T decreases mouse cardiac function

In contrast to skeletal muscles that simultaneously express multiple troponin T (TnT) isoforms, normal adult human cardiac muscle contains a single isoform of cardiac TnT. To understand the significance of myocardial TnT homogeneity, we examined the effect of TnT heterogeneity on heart function. Transgenic mouse hearts overexpressing a fast skeletal muscle TnT together with the endogenous cardiac TnT was investigated in vivo and ex vivo as an experimental system of concurrent presence of two classes of TnT in the adult cardiac muscle. This model of myocardial TnT heterogeneity produced pathogenic phenotypes: echocardiograph imaging detected age-progressive reductions of cardiac function; in vivo left ventricular pressure analysis showed decreased myocardial contractility; ex vivo analysis of isolated working heart preparations confirmed an intrinsic decrease of cardiac function in the absence of neurohumoral influence. The transgenic mice also showed chronic myocardial hypertrophy and degeneration. The dominantly negative effects of introducing a fast TnT into the cardiac thin filaments to produce two classes of Ca(2+) regulatory units in the adult myocardium suggest that TnT heterogeneity decreases contractile function by disrupting the synchronized action during ventricular contraction that is normally activated as an electrophysiological syncytium.     

Truncation by Glu180 nonsense mutation results in complete loss of slow skeletal muscle troponin T in a lethal nemaline myopathy

A lethal form of nemaline myopathy, named "Amish Nemaline Myopathy" (ANM), is linked to a nonsense mutation at codon Glu180 in the slow skeletal muscle troponin T (TnT) gene. We found that neither the intact nor the truncated slow TnT protein was present in the muscle of patients with ANM. The complete loss of slow TnT is consistent with the observed recessive pattern of inheritance of the disease and indicates a critical role of the COOH-terminal T2 domain in the integration of TnT into myofibrils. Expression of slow and fast isoforms of TnT is fiber-type specific. The lack of slow TnT results in selective atrophy of type 1 fibers. Slow TnT confers a higher Ca2+ sensitivity than does fast TnT in single fiber contractility assays. Despite the lack of slow TnT, individuals with ANM have normal muscle power at birth. The postnatal onset and infantile progression of ANM correspond to a down-regulation of cardiac and embryonic splice variants of fast TnT in normal developing human skeletal muscle, suggesting that the fetal TnT isoforms complement slow TnT. These results lay the foundation for understanding the molecular pathophysiology and the potential targeted therapy of ANM.     

Protein kinase A–dependent modulation of Ca2+ sensitivity in cardiac and fast skeletal muscles after reconstitution with cardiac troponin

Protein kinase A (PKA)-dependent phosphorylation of troponin (Tn)I represents a major physiological mechanism during β-adrenergic stimulation in myocardium for the reduction of myofibrillar Ca²⁺ sensitivity via weakening of the interaction with TnC. By taking advantage of thin filament reconstitution, we directly investigated whether or not PKA-dependent phosphorylation of cardiac TnI (cTnI) decreases Ca²⁺ sensitivity in different types of muscle: cardiac (porcine ventricular) and fast skeletal (rabbit psoas) muscles. PKA enhanced phosphorylation of cTnI at Ser23/24 in skinned cardiac muscle and decreased Ca²⁺ sensitivity, of which the effects were confirmed after reconstitution with the cardiac Tn complex (cTn) or the hybrid Tn complex (designated as PCRF; fast skeletal TnT with cTnI and cTnC). Reconstitution of cardiac muscle with the fast skeletal Tn complex (sTn) not only increased Ca²⁺ sensitivity, but also abolished the Ca²⁺-desensitizing effect of PKA, supporting the view that the phosphorylation of cTnI, but not that of other myofibrillar proteins, such as myosin-binding protein C, primarily underlies the PKA-induced Ca²⁺ desensitization in cardiac muscle. Reconstitution of fast skeletal muscle with cTn decreased Ca²⁺ sensitivity, and PKA further decreased Ca²⁺ sensitivity, which was almost completely restored to the original level upon subsequent reconstitution with sTn. The essentially same result was obtained when fast skeletal muscle was reconstituted with PCRF. It is therefore suggested that the PKA-dependent phosphorylation or dephosphorylation of cTnI universally modulates Ca²⁺ sensitivity associated with cTnC in the striated muscle sarcomere, independent of the TnT isoform.     

Effect of substitution of troponin C in cardiac myofibrils with skeletal troponin C or calmodulin on the Ca2+- and Sr2+-sensitive ATPase activity

Troponin C was removed almost completely from the porcine cardiac myofibrils by the same extraction procedure using CDTA as that previously reported for the rabbit skeletal myofibrils (Morimoto, S. & Ohtsuki, I. (1987) J. Biochem. 101, 291-301), and the effects of substitution of troponin C in cardiac myofibrils with rabbit skeletal troponin C or bovine brain calmodulin were examined. While the ATPase activity of intact cardiac myofibrils or cardiac troponin C-reconstituted cardiac myofibrils was activated at only a little higher concentration of Sr2+ than Ca2+, the skeletal troponin C-substituted cardiac myofibrils, as well as intact rabbit skeletal myofibrils, required more than 10 times higher concentration of Sr2+ than Ca2+ for activation of the myofibrillar ATPase activity. However, the concentrations of Ca2+ and Sr2+ required for the activation of the ATPase activity of the skeletal troponin C-substituted cardiac myofibrils were both about 5 times higher than those of intact skeletal myofibrils. The skeletal troponin C-substituted cardiac myofibrils, as well as intact skeletal myofibrils, also showed higher cooperativity in the Ca2+-activation of the ATPase activity than intact or cardiac troponin C-reconstituted cardiac myofibrils. The ATPase activity of calmodulin-substituted cardiac myofibrils was activated at a several times lower concentration of Ca2+ or Sr2+ than that of calmodulin-substituted skeletal myofibrils, while the ratios of the concentration of Sr2+ to Ca2+ required for activation were almost the same in both cases.(ABSTRACT TRUNCATED AT 250 WORDS)     

Troponin T isoforms modulate calcium dependence of the kinetics of the cross-bridge cycle: studies using a transgenic mouse line

Alternative splicing of troponin T (TnT) in striated muscle during development results in expression of different isoforms, with the splicing of a 5(') exon of TnT resulting in the expression of low-molecular-weight basic adult TnT isoforms and high-molecular-weight acidic embryonic TnT isoforms. Although other differences exist, the main differences between cardiac TnT (cTnT) and fast skeletal muscle TnT (fTnT) are in the NH(2) terminus, with fTnT being less acidic than cTnT. A transgenic mouse line expressing chicken fTnT in the heart was used to investigate the functional significance of TnT NH(2)-terminal charge differences on cardiac muscle contractility. The rates of force redevelopment (k(tr)) at four levels of Ca(2+) activation were recorded for skinned left ventricular trabeculae from control and transgenic mice. The k(tr) vs Ca(2+) relationship was different in control mice and transgenic mice, suggesting that the structure of TnT, and possibly the NH(2)-terminal region, is involved in determining the kinetics of cross-bridge cycle. These results suggest that isoform shifts in TnT may be an important molecular mechanism for determining the Ca(2+) dependence of cardiac muscle contractility.     

DNA synthesis, mitosis, and differentiation in cardiac myogenesis

Cardiac muscle cells obtained by trypsinizing 5-day chick embryonic heart were cultured as single cells in separate culture dishes. Using this technique, problems of heterotypic cell interactions, “overgrowth” of one cell type, etc., are eliminated. Experiments performed on these single cell cultures show that the muscle cells in the embryonic chick hearts differ in morphology, including content of cross-striated myofibrils; in ability to synthesize DNA and undergo mitosis; and in frequency of contraction. Contracting cells containing cross-striated myofibrils undergo mitosis in vitro, giving rise to spontaneously beating daughter cells. These daughter cells contain cytoplasmic fibrils, which bind fluorescein-labeled antimyosin immediately after cytokinesis. Some cardiac muscle cells from 5-day heart do not divide in culture; the rest undergo 1–5 doublings. This preliminary investigation suggests that the new muscle cells formed during cardiac growth are derived from mitotically active “overtly” differentiated cardiac muscle cells.     

Effects of troponin T mutations in familial hypertrophic cardiomyopathy on regulatory functions of other troponin subunits.

We have previously shown that mutations in troponin T (TnT), which is associated with familial hypertrophic cardiomyopathy (HCM), cause an increase in the Ca(2+) sensitivity and a potentiation of cardiac muscle contraction. To gain further insight into the patho-physiological role of these mutations, four mutations (Arg92Gln, Phe110Ile, Glu244Asp, Arg278Cys) were introduced into recombinant human cardiac TnT, and the mutants were exchanged into isolated porcine cardiac myofibrils. The effects of mutations were tested on maximal ATPase activity, the inhibitory function of troponin I (TnI) in the absence of troponin C (TnC), and the neutralizing function of TnC. Arg92Gln, Phe110Ile, and Glu244Asp markedly impaired the inhibitory function of TnI. Arg278Cys also impaired the inhibitory function of TnI, but the effect was much smaller. Phe110Ile and Glu244Asp markedly enhanced the neutralizing function of TnC and potentiated the maximum ATPase activity. Arg92Gln and Arg278Cys only slightly enhanced the neutralizing function of TnC, and they conferred no potentiation on the maximum ATPase activity. These results indicate that mutations in TnT impair multiple processes of Ca(2+) regulation by troponin, and there are marked differences in the degree of impairment from mutation to mutation.     

Troponin T core structure and the regulatory NH2-terminal variable region

The conserved central and COOH-terminal regions of troponin T (TnT) interact with troponin C, troponin I, and tropomyosin to regulate striated muscle contraction. Phylogenic data show that the NH2-terminal region has evolved as an addition to the conserved core structure of TnT. This NH2-terminal region does not bind other thin filament proteins, and its sequence is hypervariable between fiber type and developmental isoforms. Previous studies have demonstrated that NH2-terminal modifications alter the COOH-terminal conformation of TnT and thin filament Ca2+-activation, yet the functional core structure of TnT and the mechanism of NH2-terminal modulation are not well understood. To define the TnT core structure and investigate the regulatory role of the NH2-terminal variable region, we investigated two classes of model TnT molecules: (1) NH2-terminal truncated cardiac TnT and (2) chimera proteins consisting of an acidic or basic skeletal muscle TnT NH2-terminus spliced to the cardiac TnT core. Deletion of the TnT hypervariable NH2-terminus preserved binding to troponin I and tropomyosin and sustained cardiac muscle contraction in the heart of transgenic mice. Further deletion of the conserved central region diminished binding to tropomyosin. The reintroduction of differently charged NH2-terminal domains in the chimeric molecules produced long-range conformational changes in the central and COOH-terminal regions to alter troponin I and tropomyosin binding. Similar NH2-terminal charge effects are demonstrated in naturally occurring cardiac TnT isoforms, indicating a physiological significance. These results suggest that the hypervariable NH2-terminal region modulates the conformation and function of the TnT core structure to fine-tune muscle contractility.     

Background to the discovery of troponin and Setsuro Ebashi's contribution to our knowledge of the mechanism of relaxation in striated muscle

The discovery of the actomyosin system provided for the first time a model system that enabled the study of the role of the muscle protein components in the contraction and relaxation cycle to be undertaken. It soon became apparent that ATP was essential for both processes but progress really began when it became clear that components both in the myofibrillar and sarcoplasmic fractions were involved in relaxation. After it was apparent that a trace of calcium was required for the activation of the MgATPase of the myofibrils it was shown that an active calcium pump was located in the sarcoplasmic reticulum. The report by Ebashi in 1963 that a new myofibrillar protein, troponin, was the target for calcium opened up the investigation of the calcium control of the MgATPase. Troponin was shown to be a complex of troponin C, I and T, each protein being under individual genetic control and existing in isoforms specific for the muscle type. The unique forms of troponin I and T in cardiac muscle make them the biomarkers of choice for cardiac injury.     

Two-dimensional electrophoresis of troponin complex with nonequilibrium pH gradient-sodium dodecyl sulfate polyacrylamide slab gel

A two-dimensional electrophoresis procedure for the separation and analysis of troponin subunits is described in which the protein solution supplemented with 50 mM each of both glutamic and aspartic acids is subjected to nonequilibrium pH gradient electrophoresis in the first dimension. Complete dissolution and gelation of the sample with agarose are essential for analysis of constituent proteins of cardiac myofibrils. Electrophoresis in the first dimension gel is carried out for a relatively short time, 2-3 h. In combination with sodium dodecyl sulfate slab gel electrophoresis (second dimension), three subunits, troponin T, troponin I, and troponin C, of dog cardiac troponin-tropomyosin complex and myofibrils can be simultaneously analyzed quantitatively on a slab gel. The contents of troponin and tropomyosin of cardiac myofibrils were 275 +/- 34 pmol/mg of myofibrillar protein. The molar ratio of troponin T, troponin I, troponin C, and tropomyosin was close to 1 : 1 : 1 : 1 in troponin-tropomyosin complex and myofibrils.     

An R111C polymorphism in wild turkey cardiac troponin I accompanying the dilated cardiomyopathy-related abnormal splicing variant of cardiac troponin T with potentially compensatory effects

Cardiac muscle contraction is regulated by Ca(2+) through the troponin complex consisting of three subunits: troponin C (TnC), troponin T (TnT), and troponin I (TnI). We reported previously that the abnormal splicing of cardiac TnT in turkeys with dilated cardiomyopathy resulted in a greater binding affinity to TnI. In the present study, we characterized a polymorphism of cardiac TnI in the heart of wild turkeys. cDNA cloning and sequencing of the novel turkey cardiac TnI revealed a single amino acid substitution, R111C. Arg(111) in avian cardiac TnI corresponds to a Lys in mammals. This residue is conserved in cardiac and skeletal muscle TnIs across the vertebrate phylum, implying a functional importance. In the partial crystal structure of cardiac troponin, this amino acid resides in an alpha-helix that directly contacts with TnT. Structural modeling indicates that the substitution of Cys for Arg or Lys at this position would not disrupt the global structure of troponin. To evaluate the functional significance of the different size and charge between the Arg and Cys side chains, protein-binding assays using purified turkey cardiac TnI expressed in Escherichia coli were performed. The results show that the R111C substitution lowered binding affinity to TnT, which is potentially compensatory to the increased TnI-binding affinity of the cardiomyopathy-related cardiac TnT splicing variant. Therefore, the fixation of the cardiac TnI Cys(111) allele in the wild turkey population and the corresponding functional effect reflect an increased fitness value, suggesting a novel target for the treatment of TnT myopathies.     

Adult mammalian cardiac muscle cells in culture

Adult rat cardiac muscle cells were isolated from the ventricle by a retrograde perfusion technique through the aorta (Nag and Zak, 1979). These single, isolated cardiac muscle cells were cultured for 4 weeks. Throughout the culture period, a small number of muscle cells retained their cylindrical shape, while the rest exhibited alterations in shape and size assuming a flattened body of irregular shape with pseudopodia-like processes and thereby resembling embryonic/neonatal cardiac muscle cells in culture. Transmission electron microscopy revealed that the cylindrical muscle cells contained compactly arranged myofibrils and cellular organelles, similar to those of freshly isolated and in vivo cells. A few irregularly shaped cardiac muscle cells were similar to the cylindrical cells in their internal structural organization. Most of the irregular cells exhibited less myofibrillar content than that of the freshly dissociated and in vivo cells. Myofibrils in the irregular cells were widely spaced and myofilament of some of the myofibrils were loosely bunched. In addition, scattered patches of myofibrils and free myofilaments were observed in many of these cells. The internal structural organization of these irregularly shaped cardiac muscle cells closely resembled the embryonic and neonatal cardiac muscle cells in vitro and in vivo. Most of the muscle cells in culture continued to contract spontaneously, and electron microscope studies clearly indicated that they underwent dedifferentiation. Autoradiography studies demonstrated that the cylindrical and irregularly shaped cardiac muscle cells underwent DNA synthesis and cell division in culture.