Browsing School, Graduate by Author "Abraham, Roselle"
Protein Expression Differences in Hypertrophic Cardiomyopathy Transgenic MiceShah, Mitsu Rakesh; Abraham, Roselle (2015)Hypertrophic Cardiomyopathy (HCM) is the most common genetic cardiac disease, occurring in 1 out of every 500 individuals. Lacking concrete treatment and cure, the underlying mechanisms and normal pathogenesis of the disease is extremely understudied. HCM is characterized by various clinical phenotypes such as left ventricular hypertrophy, myocyte disarray, and fibrosis. These phenotypes arise from protein mutations of the cardiac sarcomere; however the direct pathway of how these mutations lead to variable phenotypes needs to be elucidated. Abnormalities in energy and calcium handling have been studied in experimental models and have shown to be a consequence of causal mutations, leading to HCM. This study focuses on transgenic mice containing the R403Q mutation of the Myosin Heavy Chain and R92W mutation of Troponin T. AMPK, CamKII, and MCU relative protein expression levels were studied, as these proteins play a role in energy and calcium ion regulation, facilitating proper signaling under normal conditions. Methods: Each transgenic mouse heart of age 5 weeks and 24 weeks was harvested and homogenized in 2% Triton-Ripa buffer. Protein from whole heart homogenate was isolated by centrifugation and subjected to Western Immunoblotting. Results: In vitro studies have shown protein expression differences in all three transgenic mouse models at both age 5 weeks and 24 weeks. Total target protein, AMPK, CamKII, and MCU were equally expressed in all three models; however there was a 25% increase in phosphorylated AMPK protein expression in R403Q-MHC transgenic mice than R92W-TnT transgenic mice of age 5 weeks and 24 weeks. Phosphorylated CamKII had 38% increase in protein expression in R92W-TnT transgenic mice than R403Q-MHC transgenic mice of age 5 weeks. Phosphorylated MCU had 13% increase in protein expression in R92W-TnT transgenic mice of age 5 weeks and 26% increase in R92W-TnT transgenic mice of age 24 weeks than R403Q-MHC transgenic mice of age 5 weeks and 24 weeks. Although pCamKII lacked expression in transgenic mice of age 24 weeks, this result can lead us to further investigations to gain additional insight. Conclusion: The R403Q mutation of MHC results in abnormal energy caused by conditions of low ATP, while the R92W mutation of the TnT results in high calcium sensitivity by an increase of calcium ions. Results confirm that there is disruption in energy and calcium handling due to the respective mutations compared to that of control. At 5 weeks, in the early development of disease, pAMPK protein was expressed 15% lower than at 24 weeks, showing more energy stress at a later stage of disease. However, the opposite holds true for pCamKII, as it was expressed 100% higher at age of 5 weeks, as it lacked expression at age of 24 weeks. This shows that there may be abnormal calcium handling at an early developing stage of disease, but not when the disease is well developed.