Protein Expression Differences in Hypertrophic Cardiomyopathy Transgenic Mice
Authors: Shah Mitsu R 
Published: November 2023

Hypertrophic Cardiomyopathy (HCM) is the most common genetic cardiac disease, occurring in 1 out of every 500 individuals. The pathologic hallmarks of HCM are myocyte hypertrophy, myocyte disarray, fibrosis and arteriolar remodeling. Majority of HCM patients have mutations in genes coding for sarcomeric proteins. However, the mechanisms whereby the mutations lead to myocyte hypertrophy are not fully elucidated. Despite successes in animal models, there is no known therapy that can prevent the cardiac phenotype in HCM patients. We hypothesized that differences in pathophysiology of the cardiac phenotype in HCM could underlie failure of clinical trials in HCM patients. Hence, in this study, we investigated 2 transgenic mouse models of HCM, namely the R403Q mutation in the alpha Myosin Heavy Chain gene (R403Q‐MHC) and the R92W mutation in the Troponin T gene (R92W‐TnT). Since energetic deficits and abnormalities in calcium handling are 2 postulated mechanisms underlying the cardiac phenotypes in HCM, we investigated expression levels of AMP‐Activated Protein Kinase (AMPK), Ca2+/Calmodulin‐Dependent Protein Kinase II (CamKII) and the mitochondrial calcium uniporter (MCU) in these 2 mouse models using western immunoblotting.

Methods

Transgenic mice of age 5 weeks and 24 weeks containing the R403Q‐MHC and R92W‐TnT HCM mutations were euthanized and each heart 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, R403Q‐MHC, R92W‐TnT, and wild type, 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 (pCamKII) 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 testing other protein expression differences to gain additional insight of underlying mechanisms how mutations lead to myocyte hypertrophy.

Conclusion

R403Q‐MHC results in abnormal energy caused by conditions of low ATP, while R92W‐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, phosphorylated AMPK 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.

Support or Funding Information

This research was supported by the John Taylor Babbitt (JTB) Foundation to Roselle Abraham.