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.
SourceAvailable from: Genaro A Ramirez-Correa[Show abstract] [Hide abstract]
ABSTRACT: Aims: Troponin I variant Pro82Ser (cTnIP82S) was initially considered a disease-causing mutation, however, later studies suggested the contrary. We tested the hypothesis of whether a causal link exists between cTnIP82S and cardiac structural and functional remodeling, such as during aging or chronic pressure-overload. Methods and results: A cardiac specific transgenic (Tg) mouse model of cTnIP82S was created to test this hypothesis. During aging Tg cTnIP82S displayed diastolic dysfunction, characterized by longer isovolumetric relaxation time (IVRT), impaired ejection and relaxation time. In young, Tg mice In vivo pressure-volume loops and intact trabecular preparations revealed normal cardiac contractility at baseline. However, upon β-adrenergic stimulation, a blunted contractile reserve and no hastening in left ventricle relaxation were evident in vivo, whereas in isolated muscles Ca(2+) transients amplitude isoproterenol dose-response was blunted. In addition, when exposed to chronic pressure-overload, Tg mice show exacerbated hypertrophy and decreased contractility when compared to age-matched non-transgenic (NTg) littermates. At the molecular level, this mutation significantly impairs myofilament cooperative activation. Importantly, this occurs in the absence of alterations in TnI or MyBP-C phosphorylation. The cTnIP82S variant occurs near a region of interactions with TnT, therefore structural changes in this region could explain its meaningful effects on myofilament cooperativity. Conclusions: Our data indicate that cTnIP82S mutation modifies age-dependent diastolic dysfunction, and impairs overall contractility after β-adrenergic stimulation or chronic pressure overload. Thus, cTnIP82S variant should be regarded as a disease-modifying factor for dysfunction and adverse remodeling with aging and chronic pressure overload.Journal of Applied Physiology 10/2014; 118(2). DOI:10.1152/japplphysiol.00463.2014 · 3.43 Impact Factor
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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.AJP Cell Physiology 06/2014; 307(4). DOI:10.1152/ajpcell.00053.2014 · 3.67 Impact Factor
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ABSTRACT: The α-helix in troponin I (TnI) at the interface with troponin T (TnT) is a highly conserved structure. A point mutation in this region, A116G, was found in human cardiac TnI in a case of cardiomyopathy. An adjacent dominantly negative mutation found in turkey cardiac TnI (K111C, equivalent to K117C in human and K118C in mouse) decreased diastolic function and blunted beta-adrenergic response in transgenic mice. To investigate the functional importance of the TnI-TnT interface and pathological impact of the cardiac TnI mutations, we engineered K118C and A117G mutations in mouse cardiac TnI for functional studies. Despite their adjacent locations, A117G substitution results in faster mobility of cardiac TnI in SDS-PAGE whereas K118C decreases gel mobility, indicating significant and distinct changes in overall protein conformation. Consistently, monoclonal antibody epitope analysis demonstrated distinct local and remote conformational alterations in the two mutant proteins. Protein binding assays showed that K118C, but not A117G, decreased the relative binding affinity of cardiac TnI for TnT. K118C mutation decreased binding affinity for troponin C in a Ca2+-dependent manner, whereas A117G had a similar but less profound effect. Protein kinase A phosphorylation or truncation to remove the cardiac specific N-terminal extension of cardiac TnI resulted in similar conformational changes in the region interfacing with TnT and minimized the functional impacts of the mutations. The data demonstrate potent conformational and functional impacts of the TnT-interfacing helix in TnI and suggest a role of the N-terminal extension of cardiac TnI in modulating TnI-TnT interface functions.01/2015; 80. DOI:10.1016/j.fob.2015.01.001