Cardiac diseases associated with mutations in Tn subunits include hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM) and restrictive cardiomyopathy (RCM). Altered calcium handling in these diseases is evidenced by changes in the Ca2+ sensitivity of contraction. Mutations were generated to increase/ decrease the Ca2+ sensitivity of skinned fibers, and create the classified effects of DCM, HCM and RCM. This study mimicked the changes in Ca2+ sensitivity and relaxation properties of the muscle to determine if this was sufficient to recreate the disease. Four mutants (A23Q, S37G, V44Q, L48Q) were identified with RCM-like properties; a large increase in Ca2+ sensitivity, increased basal force and loss of ATPase inhibition. Two mutations were identified (E40A, I61Q) with DCM properties; decreased Ca2+ sensitivity in skinned fibers, decreased force recovery (%), and decreased activation of the ATPase at high Ca2+ levels (pCa 6-4). Also, the functional effects of four newly identified cTnC mutations associated with HCM were reported. Three of these HCM mutations A8V, C84Y, and D145E displayed HCM characteristics, increased Ca2+ sensitivity in skinned fibers and ATPase and A8V and D145E increased the force recovery. Only, D145E significantly increased the ATPase activation of the reconstituted thin filament. Also, Ca2+ affinity measurements using IAANS fluorescence were performed. No significant changes were found for E134D. The C84Y IAANS fluorescence measurements revealed that cTnC Ca2+ affinity of the cTn complex was unaltered. The Ca2+ affinity increased for D145E in isolated cTnC and the cTn complex, however in the regulated thin filament (RTF) with myosin subfragment-1 (S1) and rigor crossbridges the Ca2+ affinity values were similar to the fiber Ca2+ sensitivity. For A8V, the RTF significantly increased the Ca2+ affinity, and addition of S1 and rigor crossbridges caused the values to parallel the Ca2+ sensitivity values. In conclusion, direct and indirect protein-protein interactions contribute to the enhanced Ca2+ sensitivity of the HCM mutants. The cTnC mutant screen allowed selection of mutations that mimic the disease states: S37G (RCM) and, E40A (DCM); A8V (HCM) from the patient study for analysis in knock-in mice for futures studies to determine if these disease states can be recapitulated in vivo.

Three novel mutations (G159D, L29Q and E59D/D75Y) in cardiac troponin C (CTnC) associate their clinical outcomes with a given cardiomyopathy. Current paradigms propose that sarcomeric mutations associated with dilated cardiomyopathy (DCM) decrease the myofilament calcium sensitivity while those associated with hypertrophic (HCM) cardiomyopathy increase it. Therefore, we incorporated the mutant CTnCs into skinned cardiac muscle in order to determine if their effects on the calcium regulation of tension and ATPase activity coincide with the current paradigms and phenotypic outcomes. This required the development of new calculator programs to solve complex ionic equilibria to more accurately buffer and expand the free calcium range of our test solutions. In accordance with the DCM paradigms, our result show that G159D and E59D/D75Y CTnC decrease the myofilament calcium sensitivity and force generating capabilities which would likely increase the rate of muscle relaxation and weaken the contractile force of the myocardium. Alternatively, the lack of myofilament change from L29Q CTnC (associated with HCM) may explain why the only proband is seemingly unaffected. Notably, the changes in the calcium sensitivity of tension (in fibers) do not necessarily occur in the isolated CTnC and vice versa. These counter-intuitive findings are justified through a transition in calcium affinity occurring at the level of cardiac troponin (CTn) and higher, implying that the true effects of these mutations become apparent as the hierarchal level of the myofilament increases. Despite these limitations, the regulated thin filament (RTF) retains its role as the calcium regulatory unit and best indicates a mutation's ability to sensitize (+) or desensitize (-) the muscle to calcium. Since multiple forms of cardiomyopathies exist, the identification of new drugs that sensitize (+) or desensitize (-) the calcium sensitivity could potentially reverse (+ or -) these aberrant changes in myofilament sensitivity. Therefore, we have developed an RTF mediated High Throughput Screening assay to identify compounds in libraries of molecules that can specifically modulate the calcium sensitivity of cardiac contraction. The knowledge gained from these studies will help us and others to uncover new pharmacological agents for the investigation and treatments of cardiomyopathies, hypertension and other forms of cardiovascular diseases.