Dilated cardiomyopathy (DCM) is one of the most common causes of heart failure and is often associated with mutations in sarcomeric proteins. The sarcomere is the basic contractile unit of the heart and depends on Ca²⁺ for force generation. Two sarcomeric protein mutations, D230N in tropomyosin and I61Q in cardiac troponin C, have been shown to decrease force production and Ca²⁺ sensitivity of the sarcomere, leading to DCM. We are investigating if the small molecule, Danicamtiv, is effective at improving Ca²⁺ sensitivity in D230N and I61Q mouse models. Danicamtiv binds to myosin and has been previously shown to enhance force production capacity in cardiac muscles. Using demembranated D230N and I61Q tissue positioned between a force transducer and length controlling motor, we move the tissue between different solutions of varying Ca²⁺ concentrations in the presence and absence of Danicamtiv. We experimentally determine Ca²⁺ sensitivity for each solution by calculating the calcium concentration (pCa = -log[Ca²⁺]) required for half maximal force (pCa50). Additionally, we measure the rate at which the tissue is able to redevelop force (ktr). By exposing each tissue preparation to physiological solutions both with and without Danicamtiv, we can determine how maximal force, pCa50, and ktr are affected. Preliminary results suggest that Danicamtiv increases pCa50 in both D230N and I61Q tissues, and also decreases ktr by about half. Data gathered in this project will contribute to our understanding of the mechanism by which Danicamtiv improves cardiac function. In the future, these data will also help build and strengthen computer models of the sarcomere that can predict the effects of different mutations or small molecules on force production and contractile kinetics.

Cardiomyopathies are diseases of the heart characterized by structurally and functionally abnormal cardiac tissue and can be caused by non-genetic or genetic causes. Genetic cardiomyopathies are the most common genetic cardiac condition, affecting 1 in 250 to 500. The two most common types of genetic cardiomyopathies are hypertrophic (HCM) and dilated (DCM). HCM is characterized by a thickening of the heart muscle. This thickening can lead to a blockage in the blood flow and cardiac relaxation abnormalities. DCM is pathologized by a weakening in the cardiac muscle, leading to a lengthening and thinning in the muscle. My research focuses on determining human specific mechanisms of DCM and HCM, specifically on determining the early developmental phenotypes of the cells that lead to downstream pathologies. I particularly emphasize how changes in sarcomere function lead to HCM and DCM using human induced pluripotent stem cell cardiomyocytes (hiPSC-CM). We have shown that sarcomeric mutations alter the amount of tension integrated over time (TTI) and those variations in TTI are predictive of HCM and DCM. I am generating two mutant hiPSC-CM lines, L48Q and I61Q, using CRISPR/Cas9. These mutations are both in the sarcomere, more specifically in cardiac troponin C (cTnC). They alter the calcium binding properties of cTnC. I have optimized the polymerase chain reactions in order to make the sequencing data clean for validation. I have generated the I61Q line and am working on the L48Q line. After validating the lines, I will differentiate them to cardiomyocytes in order to study the cellular mechanisms involved. I will then use IonOptix to test early vs. late calcium transience, cell contractility, and cell size. There are currently no treatments that address the contractile abnormalities present in HCM or DCM. My research will allow for greater understanding of these mechanisms which will inform potential therapies.