Familial hypertrophic cardiomyopathy mutations in troponin I (K183Δ, G203S, K206Q) enhance filament sliding

Abstract

A major cause of familial hypertrophic cardiomyopathy (FHC) is dominant mutations in cardiac sarcomeric genes. Linkage studies identified FHC-related mutations in the COOH terminus of cardiac troponin I (cTnI), a region with unknown function in Ca(2+) regulation of the heart. Using in vitro assays with recombinant rat troponin subunits, we tested the hypothesis that mutations K183Delta, G203S, and K206Q in cTnI affect Ca(2+) regulation. All three mutants enhanced Ca(2+) sensitivity and maximum speed (s(max)) of filament sliding of in vitro motility assays. Enhanced s(max) (pCa 5) was observed with rabbit skeletal and rat cardiac (alpha-MHC or beta-MHC) heavy meromyosin (HMM). We developed a passive exchange method for replacing endogenous cTn in permeabilized rat cardiac trabeculae. Ca(2+) sensitivity and maximum isometric force did not differ between preparations exchanged with cTn(cTnI,K206Q) or wild-type cTn. In both trabeculae and motility assays, there was no loss of inhibition at pCa 9. These results are consistent with COOH terminus of TnI modulating actomyosin kinetics during unloaded sliding, but not during isometric force generation, and implicate enhanced cross-bridge cycling in the cTnI-related pathway(s) to hypertrophy.

Interaction Between Troponin and Myosin Enhances Contractile Activity of Myosin in Cardiac Muscle

“…Many FHC mutations have been identified in Tn as well as the thick filament proteins. The Tn-myosin interaction may be relevant to some of the hyper-Ca 2þ -sensitivity and changes in cross-bridge kinetics seen in cTn and/or myosin mutations implicated as causative agents of FHC (Kö hler et al, 2003;Gomes and Potter, 2004). It is possible that the interaction site (for either cTn or myosin) is essential for normal cardiac muscle regulation, so mutations at or near these sites could ultimately be relevant to development of cardiac hypertrophy.…”

“…Rabbit skeletal or porcine cardiac F-actin was fluorescently labeled with rhodamine phalloidin (RhPh). Immediately before motility assays with regulatory proteins, cTn, cTm, or equimolar concentrations of both cTn and cTm were applied to labeled F-actin in the flow cell as described for thin filaments (Kö hler et al, 2003;Liang et al, 2003;Schoffstall et al, 2006a); cTn, cTm, or cTn and cTm were also added to motility buffers. Solution composition for assays with thin filaments was calculated as described (Kö hler et al, 2003;Liang et al, 2003;Schoffstall et al, 2006a) and consisted of 2 mM MgATP, 1 mM Mg 2þ , 10 mM ethylene glycol tetraacetic acid (EGTA), sufficient Ca(CH 3 COO) 2 to achieve the desired pCa (pCa 9-4), 50 mM K þ , 15 mM Na þ , 20 mM 3-(N-morpholino) propanesulfonic acid (MOPS), pH 7.00 at 308C, 0.3% methyl cellulose, and ionic strength was adjusted to 0.085 mM with TrisOH/CH 3 COOH).…”

“…During the cardiac cycle, MD binds actin at low [Ca 2þ ] and is thought to maintain Tm in the closed position, thus preventing strong actomyosin interactions. A number of DNA mutations that lead to familial hypertrophic cardiomyopathy (FHC) are clustered in the MD (Gomes and Potter, 2004), and some of these mutations further enhance maximum V f for thin filaments (Kö hler et al, 2003). A direct interaction of MD with myosin could be significant to clinical pathology of FHC.…”

Troponin I Mutations R146G and R21C Alter Cardiac Troponin Function, Contractile Properties, and Modulation by Protein Kinase A (PKA)-mediated Phosphorylation

“…Proteins, cTnC Labeling, cTnI Phosphorylation, and cTn Complex Reconstitution-WT rat cTnC, cTnI, and cTnT in the pET24 vectors were constructed and expressed as described previously (33). cTnC C35S , cTnI…”

The Δ14 Mutation of Human Cardiac Troponin T Enhances ATPase Activity and Alters the Cooperative Binding of S1-ADP to Regulated Actin

“…Mechanical measurements with rat cardiac trabeculae were conducted as described earlier (45). Force-pCa relations were measured before and after exchange of the reconstituted troponin complex (sk TnC and TnI, hc TnT).…”