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.
ABSTRACT 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.
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ABSTRACT: We previously reported a point mutation substituting Cys for Arg111 in the highly conserved troponin T (TnT)-contacting helix of cardiac troponin I (cTnI) in wild turkey hearts (Biesiadecki et al., J Biol Chem 279:13825-32, 2004). This dominantly negative TnI-TnT interface mutation decreases the binding affinity of cTnI for TnT, impairs diastolic function, and blunts β-adrenergic response of cardiac muscle (Wei et al., J Biol Chem 285:27806-16, 2010). Here we further investigated cellular phenotypes of transgenic mouse cardiomyocytes expressing the equivalent mutation cTnI-K118C. Functional studies were performed on single adult cardiomyocytes after recovering in short-term-culture from isolation stress. The amplitude of contraction and the velocities of shortening and re-lengthening were lower in cTnI-K118C cardiomyocytes than wild type controls. Intracellular Ca2+ transient was slower in cTnI-K118C cardiomyocytes than that in wild type cells. cTnI-K118C cardiomyocytes also showed weaker β-adrenergic response. The resting length of cTnI-K118C cardiomyocytes was significantly longer than that of age-matched wild type cells with no difference in cell width. The resting sarcomere length was not longer but slightly shorter in cTnI-K118C cardiomyocytes than that in wild type cells, indicating longitudinal addition of sarcomeres. More tri- and quadri-nuclei cardiomyocytes were found in TnI-K118C than that in wild type hearts, suggesting increased nuclear divisions. Whole genome mRNA array and Western blots detected an increased expression of leukemia inhibitory factor receptor β in the hearts of 2 months old cTnI-K118C mice, suggesting a signaling pathway responsible for the potent effect of cTnI-K118C mutation on causing early remodeling in cardiomyocytes.American journal of physiology. Cell physiology. 06/2014;
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ABSTRACT: The binding of Ca2 + to troponin C (TnC) in the troponin complex is a critical step regulating the thin filament, the actin-myosin interaction and cardiac contraction. Phosphorylation of the troponin complex is a key regulatory mechanism to match cardiac contraction to demand. Here we demonstrate phosphorylation of the troponin I (TnI) subunit is simultaneously increased at Ser-150 and Ser-23/24 during in vivo myocardial ischemia. Myocardial ischemia decreases intracellular pH resulting in depressed binding of Ca2 + to TnC and impaired contraction. To determine the pathological relevance of simultaneous TnI phosphorylation we measured individual TnI Ser-150 (S150D), Ser-23/24 (S23/24D) and combined (S23/24/150D) pseudo-phosphorylation effects on thin filament regulation at acidic pH similar to that in myocardial ischemia. Results demonstrate that while acidic pH decreased thin filament Ca2 + binding to TnC regardless of TnI composition, TnI S150D attenuated this decrease rendering it similar to non-phosphorylated TnI at normal pH. The dissociation of Ca2 + from TnC was unaltered by pH such that TnI S150D remained slow, S23/24D remained accelerated and the combined S23/24/150D remained accelerated. This effect of the combined TnI Ser-150 and Ser-23/24 pseudo-phosphorylation to maintain Ca2 + binding while accelerating Ca2 + dissociation represents the first post-translational modification of troponin by phosphorylation to both accelerate thin filament deactivation and maintain Ca2 + sensitive activation. These data suggest TnI Ser-150 phosphorylation attenuation of the pH-dependent decrease in Ca2 + sensitivity and its combination with Ser-23/24 phosphorylation to maintain accelerated thin filament deactivation may impart an adaptive role to preserve contraction during acidic ischemia pH without slowing relaxation.Journal of Molecular and Cellular Cardiology 01/2014; · 5.15 Impact Factor
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ABSTRACT: Troponin plays a central role in regulating the contraction and relaxation of vertebrate striated muscles. This review focuses on the isoform gene regulation, alternative RNA splicing, and posttranslational modifications of troponin subunits in cardiac development and adaptation. Transcriptional and posttranscriptional regulations such as phosphorylation and proteolysis modifications, and structure-function relationships of troponin subunit proteins are summarized. The physiological and pathophysiological significances are discussed for impacts on cardiac muscle contractility, heart function, and adaptations in health and diseases.Frontiers in Physiology 01/2014; 5:165.