Basic residues within the cardiac troponin T C terminus are required for full inhibition of muscle contraction and limit activation by calcium.

Striated muscle is activated by myosin- and actin-linked processes, with the latter being regulated through changes in the position of tropomyosin relative to the actin surface. The C-terminal region of cardiac troponin T (TnT), a tropomyosin-associated protein, is required for full TnT inactivation at low Ca2+ and for limiting its activation at saturating Ca2+ Here, we investigated whether basic residues in this TnT region are involved in these activities, whether the TnT C terminus undergoes Ca2+-dependent conformational changes, and whether these residues affect cardiac muscle contraction. We generated a human cardiac TnT variant in which we replaced seven C-terminal Lys and Arg residues with Ala and added a Cys residue at either position 289 or 275 to affix a fluorescent probe. At pCa 3.7, actin filaments containing high-alanine TnT had an elevated ATPase rate like that obtained when the last TnT 14 residues were deleted. Acrylodan-tropomyosin fluorescence changes and S1-actin binding kinetics revealed that at pCa 8, the high-alanine TnT-containing filaments did not enter the first inactive state. FRET analyses indicated that the C-terminal TnT region approached Cys-190 of tropomyosin as actin filaments transitioned to the inactive B state; that transition was abolished with high-alanine TnT. High-alanine TnT-containing cardiac muscle preparations had increased Ca2+ sensitivity of both steady-state isometric force and sinusoidal stiffness as well as increased maximum steady-state isometric force and sinusoidal stiffness. We conclude that C-terminal basic residues in cardiac TnT are critical for the regulation of cardiac muscle contraction.

Cardiac Troponin T in Healthy Full-Term Infants.

In this prospective cohort study of healthy full-term infants, we hypothesized that high-sensitivity cardiac troponin T (hs-cTnT) would be elevated in cord blood, compared with adult reference values, and that it would further increase over the first days of age. Cardiac troponin T has been shown to be significantly increased in healthy full-term newborns compared with adult reference values, but there is no established reference range. Most studies of cTnT in newborns have been performed before the introduction of high-sensitivity cTnT (hs-cTnT) assay. We conducted a study including 158 full-term newborns, at Stockholm South General Hospital. High-sensitivity cTnT was analyzed in umbilical cord blood and at 2-5 days of age. Median hs-cTnT (interquartile range) in cord blood was 34(26-44) ng/L; 99th percentile 88 ng/L. Median hs-cTnT at 2-5 days of age was 92(54-158) ng/L; 99th percentile 664 ng/L. We conclude that hs-cTnT is elevated in cord blood in healthy, full-term newborn infants compared with adult reference values, and that it increases significantly during the first days of life. Our findings further underline the need of caution when using hs-cTnT as a measurement of cardiac impact in newborns.

TNNT2 Gene Polymorphisms are Associated with Susceptibility to Idiopathic Dilated Cardiomyopathy in Kazak and Han Chinese.

BACKGROUND: Dilated cardiomyopathy (DCM) is characterized by left ventricular enlargement, systolic dysfunction, and heart failure. Both genetic and non-genetic factors have been linked to DCM pathogenesis. Familial DCM (FDCM) accounts for 20%-50% of all DCM cases, highlighting the importance of genetics in pathogenesis. Indeed, more than 40 DCM-associated genes have been identified, including the gene encoding cardiac troponin T type-2 (TNNT2). We examined polymorphisms of the TNNT2 gene in idiopathic DCM (IDCM) patients of Kazak and Han ethnicity compared with healthy Kazak and Han controls. MATERIAL AND METHODS: Peripheral blood samples were collected from 180 patients with IDCM (90 Kazak and 90 Han), and 180 healthy controls (90 Kazak and 90 Han). PCR was used to amplify 15 exons and nearby introns of the TNNT2 gene. The amplified products were sequenced and compared to the standard sequence in PubMed by BLAST and CHROMAS software, to identify mutation sites. RESULTS: Results from Kazak and Han IDCM patients were complied for Hardy-Weinberg equilibrium analysis. There was a significant difference in the genotype distribution (χ2=6.67, P=0.015) and allele frequency (χ2=5.71, P=0.017) between Kazaks with IDCM and Kazak controls of SNP rs3729547. There was also a difference in the genotype distribution (χ2=6.62, P=0.036) and allele frequency (χ2=4.91, P=0.018) between Han with IDCM and Han controls. The TNNT2 gene polymorphism loci rs3729547 may be associated with the IDCM onset in Kazak and Han patients (OR=2.5, 95% CI: 1.233~5.068). CONCLUSIONS: The TNNT2 polymorphisms might play an important role in susceptibility to DCM in Xinjiang Kazak and Han patients.

Tirasemtiv amplifies skeletal muscle response to nerve activation in humans.

INTRODUCTION: In this study we tested the hypothesis that tirasemtiv, a selective fast skeletal muscle troponin activator that sensitizes the sarcomere to calcium, could amplify the response of muscle to neuromuscular input in humans. METHODS: Healthy men received tirasemtiv and placebo in a randomized, double-blind, 4-period, crossover design. The deep fibular nerve was stimulated transcutaneously to activate the tibialis anterior muscle and produce dorsiflexion of the foot. The force-frequency relationship of tibialis anterior dorsiflexion was assessed after dosing. RESULTS: Tirasemtiv increased force produced by the tibialis anterior in a dose-, concentration-, and frequency-dependent manner with the largest increases [up to 24.5% (SE 3.1), P < 0.0001] produced at subtetanic nerve stimulation frequencies (10 Hz). CONCLUSIONS: The data confirm that tirasemtiv amplifies the response of skeletal muscle to nerve input in humans. This outcome provides support for further studies of tirasemtiv as a potential therapy in conditions marked by diminished neuromuscular input.

The functional properties of human slow skeletal troponin T isoforms in cardiac muscle regulation.

Human slow skeletal troponin T (HSSTnT) shares a high degree of homology with cardiac TnT (CTnT). Although the presence of HSSTnT has not been confirmed in the heart at the protein level, detectable levels of HSSTnT mRNA have been found. Whether HSSTnT isoforms are expressed transiently remains unknown. Because transient re-expression of HSSTnT may be a potential mechanism of regulating function, we explored the effect of HSSTnT on the regulation of cardiac muscle. At least three HSSTnT isoforms have been found to exist in slow skeletal muscle: HSSTnT1 (+exons 5 and 12), HSSTnT2 (+exon 5, -exon 12), and HSSTnT3 (-exons 5 and 12). Another isoform, HSSTnT hypothetical (Hyp) (-exon 5, +exon 12), has only been found at the mRNA level. Compared with HCTnT3 (adult isoform), Tn complexes containing HSSTnT1, -2, and -3 did not alter the actomyosin ATPase activation and inhibition in the presence and absence of Ca(2+), respectively. HSSTnTHyp was not evaluated as it did not form a Tn complex under a variety of conditions. Porcine papillary skinned fibers displaced with HSSTnT1, -2, or -3 and reconstituted with human cardiac troponin I and troponin C (HCTnI·TnC) complex showed a decrease in the Ca(2+) sensitivity of force development and an increase in maximal recovered force (HSSTnT1 and -3) compared with HCTnT3. In contrast, HSSTnTHyp showed an increase in the Ca(2+) sensitivity of force development. This suggests that re- or overexpression of specific SSTnT isoforms might have therapeutic potential in the failing heart because they increase the maximal force of contraction. In addition, circular dichroism and proteolytic digestion experiments revealed structural differences between HSSTnT isoforms and HCTnT3 and that HSSTnT1 is more susceptible to calpain and trypsin proteolysis than the other HSSTnTs. Overall, HSSTnT isoforms despite being homologues of CTnT may display distinct functional properties in muscle regulation.

Cardiac troponin T is essential in sarcomere assembly and cardiac contractility.

Mutations of the gene (TNNT2) encoding the thin-filament contractile protein cardiac troponin T are responsible for 15% of all cases of familial hypertrophic cardiomyopathy, the leading cause of sudden death in young athletes. Mutant proteins are thought to act through a dominant-negative mode that impairs function of heart muscle. TNNT2 mutations can also lead to dilated cardiomyopathy, a leading cause of heart failure. Despite the importance of cardiac troponin T in human disease, its loss-of-function phenotype has not been described. We show that the zebrafish silent heart (sih) mutation affects the gene tnnt2. We characterize two mutated alleles of sih that severely reduce tnnt2 expression: one affects mRNA splicing, and the other affects gene transcription. Tnnt2, together with alpha-tropomyosin (Tpma) and cardiac troponins C and I (Tnni3), forms a calcium-sensitive regulatory complex within sarcomeres. Unexpectedly, in addition to loss of Tnnt2 expression in sih mutant hearts, we observed a significant reduction in Tpma and Tnni3, and consequently, severe sarcomere defects. This interdependence of thin-filament protein expression led us to postulate that some mutations in tnnt2 may trigger misregulation of thin-filament protein expression, resulting in sarcomere loss and myocyte disarray, the life-threatening hallmarks of TNNT2 mutations in mice and humans.

Myosin-driven rescue of contractile reserve and energetics in mouse hearts bearing familial hypertrophic cardiomyopathy-associated mutant troponin T is mutation-specific.

The thin filament protein troponin T (TnT) is a regulator of sarcomere function. Whole heart energetics and contractile reserve are compromised in transgenic mice bearing missense mutations at R92 within the tropomyosin-binding domain of cTnT, despite being distal to the ATP hydrolysis domain of myosin. These mutations are associated with familial hypertrophic cardiomyopathy (FHC). Here we test the hypothesis that genetically replacing murine αα-MyHC with murine ßß-MyHC in hearts bearing the R92Q cTnT mutation, a particularly lethal FHC-associated mutation, leads to sufficiently large perturbations in sarcomere function to rescue whole heart energetics and decrease the cost of contraction. By comparing R92Q cTnT and R92L cTnT mutant hearts, we also test whether any rescue is mutation-specific. We defined the energetic state of the isolated perfused heart using (31)P-NMR spectroscopy while simultaneously measuring contractile performance at four work states. We found that the cost of increasing contraction in intact mouse hearts with R92Q cTnT depends on the type of myosin present in the thick filament. We also found that the salutary effect of this manoeuvre is mutation-specific, demonstrating the major regulatory role of cTnT on sarcomere function at the whole heart level.

Identification of cardiomyocyte nuclei and assessment of ploidy for the analysis of cell turnover.

Assays to quantify myocardial renewal rely on the accurate identification of cardiomyocyte nuclei. We previously ¹4C birth dated human cardiomyocytes based on the nuclear localization of cTroponins T and I. A recent report by Kajstura et al. suggested that cTroponin I is only localized to the nucleus in a senescent subpopulation of cardiomyocytes, implying that ¹4C birth dating of cTroponin T and I positive cell populations underestimates cardiomyocyte renewal in humans. We show here that the isolation of cell nuclei from the heart by flow cytometry with antibodies against cardiac Troponins T and I, as well as pericentriolar material 1 (PCM-1), allows for isolation of close to all cardiomyocyte nuclei, based on ploidy and marker expression. We also present a reassessment of cardiomyocyte ploidy, which has important implications for the analysis of cell turnover, and iododeoxyuridine (IdU) incorporation data. These data provide the foundation for reliable analysis of cardiomyocyte turnover in humans.

Myofibrillogenesis in the developing zebrafish heart: A functional study of tnnt2.

Various hypotheses have been proposed to explain the molecule processes of sarcomere assembly, partially due to the lack of systematic genetic studies of sarcomeric genes in an in vivo model. Towards the goal of developing zebrafish as a vertebrate model for this purpose, we characterized myofibrillogenesis in a developing zebrafish heart and went on to examine the functions of cardiac troponin T (tnnt2). We found that sarcomere assembly in zebrafish heart was initiated from a non-striated actin filament network at the perimembrane region, whereas sarcomeric myosin is independently assembled into thick filaments of variable length before integrating into the thin filament network. Compared to Z-discs that are initially aligned to form shorter periodic dots and expanded longitudinally at a later time, M-lines assemble later and have a constant length. Depletion of full-length tnnt2 disrupted the striation of thin filaments and Z-bodies, which sequentially affects the striation of thick filaments and M-lines. Conversely, truncation of a C-terminal troponin complex-binding domain did not affect the striation of these sarcomere sub-structures, but resulted in reduced cardiomyocyte size. In summary, our data indicates that zebrafish are a valuable in vivo model for studying both myofibrillogenesis and sarcomere-based cardiac diseases.

Targeted disruption of the cardiac troponin T gene causes sarcomere disassembly and defects in heartbeat within the early mouse embryo.

Cardiac troponin T (cTnT) is a component of the troponin (Tn) complex in cardiac myocytes, and plays a regulatory role in cardiac muscle contraction by anchoring two other Tn components, troponin I (TnI) and troponin C, to tropomyosin (Tm) on the thin filaments. In order to determine the in vivo function of cTnT, we created a null cTnT allele in the mouse TNNT2 locus. In cTnT-deficient (cTnT(-/-)) cardiac myocytes, the thick and thin filaments and alpha-actinin-positive Z-disk-like structures were not assembled into sarcomere, causing early embryonic lethality due to a lack of heartbeats. TnI was dissociated from Tm in the thin filaments without cTnT. In spite of loss of Tn on the thin filaments, the cTnT(-/-) cardiac myocytes showed regular Ca(2+)-transients. These findings indicate that cTnT plays a critical role in sarcomere assembly during myofibrillogenesis in the embryonic heart, and also indicate that the membrane excitation and intracellular Ca(2+) handling systems develop independently of the contractile system. In contrast, heterozygous cTnT(+/-) mice had a normal life span with no structural and functional abnormalities in their hearts, suggesting that haploinsufficiency could not be a potential cause of cardiomyopathies, known to be associated with a variety of mutations in the TNNT2 locus.

Structure-function relationships of molluscan troponin T revealed by limited proteolysis.

Molluscan troponin regulates muscle contraction through a novel Ca(2+)-dependent activating mechanism associated with Ca(2+)-binding to the C-terminal domain of troponin C. To elucidate the further details of this regulation, we performed limited chymotryptic digestion of the troponin complex from akazara scallop striated muscle. The results indicated that troponin T is very susceptible to the protease, compared to troponin C or troponin I. The cleavage occurred at the C-terminal extension, producing an N-terminal 33-kDa fragment and a C-terminal 6-kDa fragment. This extension is conserved in various invertebrate troponin T proteins, but not in vertebrate troponin T. A ternary complex composed of the 33-kDa fragment of troponin T, troponin I, and troponin C could be separated from the 6-kDa troponin T fragment by gel filtration. This complex did not show any Ca(2+)-dependent activation of the Mg-ATPase activity of rabbit-actomyosin-scallop-tropomyosin. In addition, the actin-tropomyosin-binding affinity of this complex was significantly decreased with increasing Ca(2+) concentration. These results indicate that the C-terminal extension of molluscan troponin T plays a role in anchoring the troponin complex to actin-tropomyosin filaments and is essential for regulation.

Nonmyofilament-associated troponin T fragments induce apoptosis.

Troponin T (TnT) is a striated muscle-specific protein and an abundant component of the myofilaments. Nonmyofilament-associated TnT is rapidly degraded in myocytes, implying an importance in the maintenance of the cellular environment. However, if the level of nonmyofilament-associated TnT or TnT fragments exceeds the degradation capacity, it may cause cytotoxicity. To investigate this hypothesis, we constructed bicistronic vectors to express different portions of TnT polypeptide chain, together with nonfusion green fluorescent protein as a tracer for the transfection. Cytotoxicity of the TnT fragments was studied through forced expression in C(2)C(12) myoblasts and human embryonic kidney-293 nonmuscle cells and examination of the viability of the transfected cells. The results demonstrated that, in the absence of myofilaments, the conserved COOH-terminal and middle fragments of TnT were highly effective on inducing cell death via apoptosis, whereas the NH(2)-terminal variable region was not. As combined effects, nonmyofilament-associated intact cardiac TnT and a COOH-terminal truncated slow TnT fragment found in Amish nemaline myopathy exhibited intermediate cytotoxicity. A particular significance of this finding is that peak releases of TnT or TnT fragments from decomposition of a large number of myofibrils in acute myocardial infarction may breach the cellular protection of proteolytic degradation and result in apoptosis as a potential cause for the loss of cardiomyocytes.

Ca2+-dependent muscle dysfunction caused by mutation of the Caenorhabditis elegans troponin T-1 gene.

We have investigated the functions of troponin T (CeTnT-1) in Caenorhabditis elegans embryonic body wall muscle. TnT tethers troponin I (TnI) and troponin C (TnC) to the thin filament via tropomyosin (Tm), and TnT/Tm regulates the activation and inhibition of myosin-actin interaction in response to changes in intracellular [Ca2+]. Loss of CeTnT-1 function causes aberrant muscle trembling and tearing of muscle cells from their exoskeletal attachment sites (Myers, C.D., P.-Y. Goh, T. StC. Allen, E.A. Bucher, and T. Bogaert. 1996. J. Cell Biol. 132:1061-1077). We hypothesized that muscle tearing is a consequence of excessive force generation resulting from defective tethering of Tn complex proteins. Biochemical studies suggest that such defective tethering would result in either (a) Ca2+-independent activation, due to lack of Tn complex binding and consequent lack of inhibition, or (b) delayed reestablishment of TnI/TnC binding to the thin filament after Ca2+ activation and consequent abnormal duration of force. Analyses of animals doubly mutant for CeTnT-1 and for genes required for Ca2+ signaling support that CeTnT-1 phenotypes are dependent on Ca2+ signaling, thus supporting the second model and providing new in vivo evidence that full inhibition of thin filaments in low [Ca2+] does not require TnT.

Effect of Training Load on Post-Exercise Cardiac Troponin T Elevations in Young Soccer Players.

Training load (TL) metrics are usually assessed to estimate the individual, physiological and psychological, acute, and adaptive responses to training. Cardiac troponins (cTn) reflect myocardial damage and are routinely analyzed for the clinical diagnosis of myocardial injury. The association between TL and post-exercise cTn elevations is scarcely investigated in young athletes, especially after playing common team sports such as soccer. The objective of this study was to assess the relationship between TL measurements during a small-sided soccer game and the subsequent increase in cTn in young players. Twenty male soccer players (age 11.9 ± 2 years, height 151 ± 13 cm, weight 43 ± 13 kg) were monitored during a 5 × 5 small-sided game and had blood samples drawn before, immediately after, and 3 h after exercise for a posterior analysis of high-sensitivity cardiac troponin T (hs-cTnT). Internal, external, and mixed metrics of TL were obtained from the rating of perceived exertion (RPE), heart rate (HR), and GPS player tracking. The results show that the concentration of hs-cTnT peaked at 3 h post-exercise in all participants. The magnitude of hs-cTnT elevation was mainly explained by the exercise duration in the maximal heart rate zone (Maximum Probability of Effect (MPE) = 92.5%), time in the high-speed zone (MPE = 90.4 %), and distance in the high-speed zone (MPE = 90.45%). Our results support the idea that common metrics of TL in soccer, easily obtained using player tracking systems, are strongly associated with the release of hs-cTnT in children and adolescents.

Identification of a contractile deficit in adult cardiac myocytes expressing hypertrophic cardiomyopathy-associated mutant troponin T proteins.

The direct effects of expressing hypertrophic cardiomyopathy-associated (HCM-associated) mutant troponin T (TnT) proteins on the force generation of single adult cardiac myocytes have not been established. Replication-defective recombinant adenovirus vectors were generated for gene transfer of HCM-associated I79N and R92Q mutant cardiac TnT cDNAs into fully differentiated adult cardiac myocytes in primary culture. We tested the hypothesis that the mutant TnT proteins would be expressed and incorporated into the cardiac sarcomere and would behave as dominant-negative proteins to directly alter calcium-activated force generation at the level of the single cardiac myocyte. Interestingly, under identical experimental conditions, the ectopic expression of the mutant TnTs was significantly less ( approximately 8% of total) than that obtained with expression of wild-type TnT ( approximately 35%) in the myocytes. Confocal imaging of immunolabeled TnT showed a regular periodic pattern of localization of ectopic mutant TnT that was not different than that in normal controls, suggesting that mutant TnT incorporation had no deleterious effects on sarcomeric architecture. Direct measurements of isometric force production in single cardiac myocytes demonstrated marked desensitization of submaximal calcium-activated tension, with unchanged maximum tension generation in mutant TnT-expressing myocytes compared with control myocytes. Collectively, these results demonstrate an impaired expression of the mutant protein and a disabling of cardiac contraction in the submaximal range of myoplasmic calcium concentrations. Our functional results suggest that development of new pharmacological, chemical, or genetic approaches to sensitize the thin-filament regulatory protein system could ameliorate force deficits associated with expression of I79N and R92Q in adult cardiac myocytes.

Myofibrillar troponin exists in three states and there is signal transduction along skeletal myofibrillar thin filaments.

Activation of striated muscle contraction is a highly cooperative signal transduction process converting calcium binding by troponin C (TnC) into interactions between thin and thick filaments. Once calcium is bound, transduction involves changes in protein interactions along the thin filament. The process is thought to involve three different states of actin-tropomyosin (Tm) resulting from changes in troponin's (Tn) interaction with actin-Tm: a blocked (B) state preventing myosin interaction, a closed (C) state allowing weak myosin interactions and favored by calcium binding to Tn, and an open or M state allowing strong myosin interactions. This was tested by measuring the apparent rate of Tn dissociation from rigor skeletal myofibrils using labeled Tn exchange. The location and rate of exchange of Tn or its subunits were measured by high-resolution fluorescence microscopy and image analysis. Three different rates of Tn exchange were observed that were dependent on calcium concentration and strong cross-bridge binding that strongly support the three-state model. The rate of Tn dissociation in the non-overlap region was 200-fold faster at pCa 4 (C-state region) than at pCa 9 (B-state region). When Tn contained engineered TnC mutants with weakened regulatory TnI interactions, the apparent exchange rate at pCa 4 in the non-overlap region increased proportionately with TnI-TnC regulatory affinity. This suggests that the mechanism of calcium enhancement of the rate of Tn dissociation is by favoring a TnI-TnC interaction over a TnI-actin-Tm interaction. At pCa 9, the rate of Tn dissociation in the overlap region (M-state region) was 100-fold faster than the non-overlap region (B-state region) suggesting that strong cross-bridges increase the rate of Tn dissociation. At pCa 4, the rate of Tn dissociation was twofold faster in the non-overlap region (C-state region) than the overlap region (M-state region) that likely involved a strong cross-bridge influence on TnT's interaction with actin-Tm. At sub-maximal calcium (pCa 6.2-5.8), there was a long-range influence of the strong cross-bridge on Tn to enhance its dissociation rate, tens of nanometers from the strong cross-bridge. These observations suggest that the three different states of actin-Tm are associated with three different states of Tn. They also support a model in which strong cross-bridges shift the regulatory equilibrium from a TnI-actin-Tm interaction to a TnC-TnI interaction that likely enhances calcium binding by TnC.

Troponin T and histological characteristics of rat myocardial infarction induced by isoproterenol.

In our investigation,we used short-time model of myocardial infarction of rats induced by high dose of isoproterenol (ISP). We investigated cardiac troponin T blood level (cTnT) and histological characteristics of rat myocardium. ISP, single, intraperitoneal dose 250 mg/kg was given to male, adult, Wistar rats (n=12). Rats were distributed depending on their body weight in subgroups: ISP I (BW 260-280g) and ISP II (BW 250-400g).Control group (n=9) was treated with intraperitoneal dose of 0,95% NaCl. Cardiac TnT was measured by electrochemiluminiscence (ECLA) sandwich immunoassay in rat serum 4 hours after ISP application. Rats' hearts were dissected and examined by qualitative histological method (HE). Statistical significance was set at 0,05. There was significant difference in cTnT of ISP II (p=0,0001) vs. control and ISP I (p<0,05) vs. control. Significant difference was between ISP I and ISP II subgroups (p<0.001). The accent of histological changes of myocardium was on nuclei of cell. Cells showed acidophilic changes and nuclei disappearance as signs of coagulative necrosis development. Extensivity of histological changes were different between ISP I and ISP II subgroup. Used dose of ISP induced development of myocardial necrosis in rats. Subendocardial portion of myocardium was more vulnerability than subepicardial portion. Rats of ISP II had more extensive histological changes than these in ISP I. Administered doses of ISP enabled cTnT utilization as a marker of myocardial necrosis.

Overexpression of miRNA-9 Generates Muscle Hypercontraction Through Translational Repression of Troponin-T in Drosophila melanogaster Indirect Flight Muscles.

MicroRNAs (miRNAs) are small noncoding endogenous RNAs, typically 21-23 nucleotides long, that regulate gene expression, usually post-transcriptionally, by binding to the 3'-UTR of target mRNA, thus blocking translation. The expression of several miRNAs is significantly altered during cardiac hypertrophy, myocardial ischemia, fibrosis, heart failure, and other cardiac myopathies. Recent studies have implicated miRNA-9 (miR-9) in myocardial hypertrophy. However, a detailed mechanism remains obscure. In this study, we have addressed the roles of miR-9 in muscle development and function using a genetically tractable model system, the indirect flight muscles (IFMs) of Drosophila melanogaster Bioinformatics analysis identified 135 potential miR-9a targets, of which 27 genes were associated with Drosophila muscle development. Troponin-T (TnT) was identified as major structural gene target of miR-9a. We show that flies overexpressing miR-9a in the IFMs have abnormal wing position and are flightless. These flies also exhibit a loss of muscle integrity and sarcomeric organization causing an abnormal muscle condition known as "hypercontraction." Additionally, miR-9a overexpression resulted in the reduction of TnT protein levels while transcript levels were unaffected. Furthermore, muscle abnormalities associated with miR-9a overexpression were completely rescued by overexpression of TnT transgenes which lacked the miR-9a binding site. These findings indicate that miR-9a interacts with the 3'-UTR of the TnT mRNA and downregulates the TnT protein levels by translational repression. The reduction in TnT levels leads to a cooperative downregulation of other thin filament structural proteins. Our findings have implications for understanding the cellular pathophysiology of cardiomyopathies associated with miR-9 overexpression.

Myocardial regulatory proteins and heart failure.

Cardiac troponin T (cTnT) and cardiac troponin I (cTnI) are considered to be the most specific and sensitive biochemical markers of myocardial damage. Troponins have been studied in a wide range of clinical settings, including heart failure; however, there are few data on the role of regulatory proteins in the pathogenesis of heart failure, although a few interesting hypotheses have been proposed. A considerable body of evidence favours the view that alteration of the myocardial thin filament is the primary event leading to defective contractility of the failing myocardium, while the changes in Ca(2+) handling are a compensatory response. A better understanding of the role of regulatory proteins under different physiological and pathological conditions could lead to new therapeutic approaches in heart failure. Recently, calcium sensitisation has been proposed as a novel method by which cardiac performance may be enhanced via an increase in the affinity of troponin C for calcium but without affecting intracellular calcium concentration. To date, the only calcium sensitizer used in clinical practice is levosimendan.

Porcine cardiac myocyte power output is increased after chronic exercise training.

Chronic exercise training increases the functional capacity of the heart, perhaps by increased myocyte contractile function, as has been observed in rodent exercise models. We examined whether cardiac myocyte function is enhanced after chronic exercise training in Yucatan miniature swine, whose heart characteristics are similar to humans. Animals were designated as either sedentary (Sed), i.e., cage confined, or exercise trained (Ex), i.e., underwent 16-20 wk of progressive treadmill training. Exercise training efficacy was shown with significantly increased heart weight-to-body weight ratios, skeletal muscle citrate synthase activity, and exercise tolerance. Force-velocity properties were measured by attaching skinned cardiac myocytes between a force transducer and position motor, and shortening velocities were measured over a range of loads during maximal Ca2+ activation. Myocytes (n = 9) from nine Ex pigs had comparable force production but a approximately 30% increase in peak power output compared with myocytes (n = 8) from eight Sed. Interestingly, Ex myofibrillar samples also had higher baseline PKA-induced phosphorylation levels of cardiac troponin I, which may contribute to the increase in power. Overall, these results suggest that enhanced power-generating capacity of porcine cardiac myofibrils contributes to improved cardiac function after chronic exercise training.