Effects of Small Molecules Mimicking cTnI Phosphorylation on the Hypertrophic Cardiomyopathy-Causing R92L-cTnT Mutation

Abstract

The cardiac sarcomere is the contractile unit of the heart. It is composed of regulatory filaments: thick filament, thin filament and titin. Titin acts as a molecular spring, keeping the thick filament centered in the sarcomere. The thick filament is primarily composed of myosin, which acts as the motor protein for sarcomere contraction. The thin filament (TF) is composed of the proteins actin, tropomyosin (TPM), and the troponin (Tn) complex (consisting of troponin C, T, and I). The thin filament is responsible for the regulation of calcium-sensitive contraction. Ca2+ binding to cTnC results in conformational changes in the TF that allow for actomyosin cross-bridges, generating force during contraction. Mutations in sarcomeric proteins have been linked to genetic cardiomyopathy forms such as hypertrophic cardiomyopathy (HCM). Impaired left ventricular relaxation and dysregulated Ca2+ homeostasis are common in HCM, often contributing to diastolic dysfunction. The Arg92Leu (R92L) mutation is a missense substitution in the gene TNNT2, that encodes for cTnT. This mutation is an HCM-causing mutation resulting in left ventricular hypertrophy and impaired diastolic function due to allosterically mediated changes to protein flexibility and cTnT binding properties to tropomyosin. Protein kinase A (PKA) mediated phosphorylation of cTnI-S23S24 has been shown to be important to early sarcomeric relaxation, and the phosphorylation potential of cTnI is reduced in the R92L-cTnT mutation. The R92L-cTnT mutation also has decreased rates of calcium dissociation from cTnI compared to wildtype. Mimicking cTnI phosphorylation increases the rate of calcium dissociation in both wildtype and R92L-cTnT reconstituted protein and mouse models. A sequence of screens was designed to identify small molecules that result in time-resolved fluorescence structural changes, as well as changes in the stopped-flow calcium dissociation kinetics. The stopped-flow calcium dissociation kinetics screen was conducted on a fully reconstituted cardiac thin filament, using bacterially-expressed protein based on the human sequences. The Selleck and ACDD Diversity drug libraries underwent structural and stopped-flow Ca2+ dissociation kinetics screening, and compounds that increased the percent change of stopped-flow Ca2+ dissociation kinetics were selected as candidates to undergo further screening. We hypothesize that the effect on the percent change in Ca2+ dissociation rates due to individual small molecules will translate from in vitro to ex vivo studies. NADH-coupled myofibril ATPase was used to assess the effects of the small molecules on the R92L-cTnT myofibrils, which have increased actomyosin interactions at baseline. The two small molecule candidates with the most significant decreases in ATPase rate were then tested on isolated R92L-cTnT cardiomyocytes for analysis on a more complex ex vivo system level. Data collected on isolated myofibrils and cardiomyocytes will provide information regarding how the small molecules affect the impaired function caused by the R92L-cTnT mutation, with the possibility of identifying candidates to proceed to in vivo studies on mouse models. Results of these studies will provide information about the validity of the primary structural and the secondary calcium dissociation kinetics screens as a method identify small molecules that bind to the thin filament and modulate ATPase activity.