Functional Analysis of a Troponin I (R145G) Mutation Associated with Familial Hypertrophic Cardiomyopathy

Abstract

Familial hypertrophic cardiomyopathy has been associated with several mutations in the gene encoding human cardiac troponin I (HCTnI). A missense mutation in the inhibitory region of TnI replaces an arginine residue at position 145 with a glycine and cosegregates with the disease. Results from several assays indicate that the inhibitory function of HCTnI R145G is significantly reduced. When HCTnI R145G was incorporated into whole troponin, Tn R145G (HCTnT⅐HCTnI R145G ⅐HCTnC), only partial inhibition of the actin-tropomyosin-myosin ATPase activity was observed in the absence of Ca 2؉ compared with wild type Tn (HCTnT⅐HCTnI⅐HCTnC). Maximal activation of actin-tropomyosin-myosin ATPase in the presence of Ca 2؉ was also decreased in Tn R145G when compared with Tn. Using skinned cardiac muscle fibers, we determined that in comparison with the wild type complex 1) the complex containing HCTnI R145G only inhibited 84% of Ca 2؉ -unregulated force, 2) the recovery of Ca 2؉ -activated force was decreased, and 3) there was a significant increase in the Ca 2؉ sensitivity of force development. Computer modeling of troponin C and I variables predicts that the primary defect in TnI caused by these mutations would lead to diastolic dysfunction. These results suggest that severe diastolic dysfunction and somewhat decreased contractility would be prominent clinical features and that hypertrophy could arise as a compensatory mechanism. Familial hypertrophic cardiomyopathy (FHC)1 has been linked to mutations in genes of nine different sarcomeric proteins. These mutations have been found in the genes for ␣-myosin heavy chain (1), cardiac myosin essential light chain and cardiac myosin regulatory light chain (2), ␣-tropomyosin, cardiac troponin T (TnT) (3), cardiac myosin-binding protein C (4, 5), troponin I (TnI) (6), ␣-actin (7), as well as titin (8), and possibly troponin C (TnC) (9). This disease has recently gained significant attention due to several highly publicized reports of sudden death and fainting spells in young athletes who were asymptomatic and otherwise healthy individuals. In general, patients with FHC demonstrate an increase in heart muscle mass and sometimes an irregular echocardiogram (10). Kimura et al. (6) reported five missense mutations in TnI, R145G, R145Q, R162W, G203S, and K206Q, that cosegregate with FHC (Fig. 1). Three other TnI FHC mutants (S199N, Lys-183 deletion, and an exon 8 deletion mutant encompassing the stop codon of the cardiac TnI gene) have recently been discovered (11, 12). Functionally TnI is the inhibitory subunit of the troponin (Tn) complex that controls the interaction between actin and myosin in a Ca 2ϩ -dependent manner (13-15). Studies using proteolytic fragments of fast skeletal TnI identified the central TnI sequence (residues 96 -116) as being responsible for its inhibitory activity. Residues 104 -115 of fast skeletal TnI (comparable to residues 136 -147 in cardiac TnI) formed the minimum sequence necessary for inhibition of muscle contraction (16 -19). Two of these mutations occ...

Different Functional Properties of Troponin T Mutants That Cause Dilated Cardiomyopathy

“…1A, porcine fibers exchanged and reconstituted with human Tn complex show a decrease in Ca 2ϩ sensitivity (⌬pCa 50 ϭ Ϫ0.14) when compared with untreated fibers. This result is consistent with what was previously observed in our laboratory (17,18). This difference in Ca 2ϩ sensitivity is due to the substitution of human Tn complex into the porcine fibers.…”

“…The functional consequences of TnT mutations causing FHC have been extensively characterized (12,17,(25)(26)(27). Previously, our laboratory and others have shown that FHC-causing mutations in TnT predominantly increase myofilament Ca 2ϩ sensitivity (12,25,28).…”

“…The experiment is performed after displacement of the endogenous Tn complex and replacement with HCWTnT or TnT mutants and reconstitution with the human cardiac TnI⅐TnC complex. This protocol is well established in this laboratory (12,17,18). Porcine muscle fiber bundles were mounted on a force transducer and treated with the pCa 8.0 relaxing solution containing 1% Triton X-100 for ϳ1 h. The composition of pCa 8.0 solution was 10 Ϫ8 M Ca 2ϩ , 1 mM Mg 2ϩ , 7 mM EGTA, 5 mM MgATP, 20 mM imidazole, pH 7.0, 20 mM creatinine phosphate, and 15 units/ml creatinine phosphokinase; ionic strength ϭ 150 mM.…”

Molecular genetics of familial hypertrophic cardiomyopathy (FHC)

“…Their results demonstrated that most of the FHC-linked cTnI mutations did affect the regulatory processes involving the TnI3 molecule, and that at least five mutations (p.R145G, p.R145Q, p.R162W, K183del, and p.K206Q) increased the Ca 2ϩ sensitivity of cardiac muscle contraction (Takahashi-Yanaga et al 2001). The R145G mutation has been shown to result in severe diastolic dysfunction and somewhat decreased contractility leading to hypertrophy as a compensatory mechanism (Lang et al 2002). The K183del is the most common mutation found in this gene.…”

Allograftic bone marrow-derived mesenchymal stem cells transplanted into heart infarcted model of rabbit to renovate infarcted heart

“…cTn I is only expressed in cardiomyocyte, and is not expressed in the skeletal muscle. Therefore, cTn I is the antigen of cardiomyocyte specificity (Lang et al, 2002). This experiment used BrdUrd labelling transplant cell and sites at the cell nucleus, used cTn I as the specificity antigen of cardiomyocyte and sites at cytoplasm.…”