Possibly affecting at least 1.5 million Americans today, cardiomyopathy greatly contributes to the reign of heart disease as the number one killer in the United States. Consisting of four main disease manifestations (hypertrophic, dilated, restrictive, and arrythmogenic right ventricular), this heterogeneous group of myocardium diseases can cause an array of dysfunction often leading to heart failure related disability or death. Interestingly, cardiomyopathy has been associated to sarcomeric variants. Therefore, the goal of my dissertation research was to elucidate calcium sensitivity in the attenuation of two mutations with opposing cardiomyopathy phenotypes, to dive deeper into a possible treatment method for a strong calcium sensitizing cardiomyopathy mutation, and to further classify transcriptomic as well as sex differences observed within our cardiomyopathy mouse model. In order to test the hypothesis that two mutations with opposing effects on myofilament calcium sensitivity, a cardiomyopathy indicator, could attenuate disease manifestation, we interbred the HCM-associated cTnT-I79N mouse model and the DCM-associated cTnT-R141W+/- mouse model which exhibit calcium sensitivity and de-sensitivity, respectively. We analyzed the skinned cardiac muscle preparation, echocardiography, fibrosis content, heart weight, and ex vivo pacing of the progeny of these two breeding pairs. Overall, we found that attenuation was possible for the progeny with a lessened calcium sensitivity in cardiac preparations, attenuated hemodynamics, lessened fibrosis content, and complete ablation of arrhythmia and electrocardiogram QRS widening. With the previous study pinpointing calcium sensitivity as a promising venue for hypertrophic cardiomyopathy treatment exploration, we looked to the main calcium sensor subunit of troponin and the HCM cTnC-A8V mouse model that had been previously characterized in our lab for more answers. Importantly, the previous study provided a proof of concept that a cardiomyopathy phenotype could be attenuated in utero as we were studying the bred progeny of our mutant mice pairing. However, in a true human experience, a patient would be diagnosed post disease manifestation. To mimic this scenario we chose to design an experiment of hypertrophic cardiomyopathy “treatment” post disease manifestation. Multiple studies have elucidated the role of phosphorylated regulatory light chain (RLC) plays in enhancing contraction. Furthermore, cardiac myosin light chain kinase (cMLCK) has also been extensively studied in its dedicated role as an RLC phosphorylator. Pinpointing cMLCK as a viable target for therapeutic treatment of a calcium sensitizing HCM mutation, we decided to breed a conditional cMLCK knock-out mouse with our cTnC-A8V mouse model. This proved a conditional cMLCK knock-out cTnC-A8V mouse, which we could allow to progress through natural disease and choose when to “treat” the effects of the mutation by knocking out a main RLC phosphorylator. In effect, this would test the hypothesis that a lessening of myosin head availability to bind, by the dephosphorylation of myosin heads, would therefore decrease contraction in a mouse model whose hypertrophy is majorly impacted by its hyper-contractile phenotype due to a genetically heightened calcium sensitivity. To study the effects of this experimental set-up analysis was done on echocardiography, heart weight to tibia length ratios, histopathology, fibrosis quantification, Pressure-volume loops, protein quantification through western blots, and RNA sequencing, before and after treatment. This project is currently underway. The last experiment is a branch off of the cTnC-A8V experiment, which specifically views the transcriptomic profile of the HCM mouse model to discover dimorphisms. After analyzing the echocardiography of some of the cTnC-A8V mice, slightly noticeable sexual differences seemed hidden in the morphology. Sure enough this became evident in analysis. Therefore we ran RNA sequencing on male and female cTnC-A8V mice and their controls to further explore this sexual dimorphism. This study uncovered multiple avenues for further HCM exploration and may explain many of the physical differences we see between the male and female HCM hearts. This experiment has concluded and is currently being reviewed for publication. Overall, calcium sensitivity and its role in contraction, stress, and thus, hypertrophy, plays an important role in cardiomyopathy and can be attenuated. The HCM cTnC-A8V mutation exhibits sex differences especially in metabolism and hypertrophy response. Furthermore, cMLCK has proved itself a promising therapeutic target for HCM.
