33 Generation of Novel Human Induced Pluripotent Stem Cell lines to study Proteinopathy in Cardiomyocytes

Faculty Mentor: Rajeshwary Ghosh (Nutrition and Integrative Physiology, University of Utah)

Cardiovascular disease (CVD) is a major problem in the United States and is one of the leading causes of death with over 300,000 deaths annually (1). This is a grave concern but is specifically detrimental to the African American community. The age of onset is much younger than the typical 50 and over age range. 1 in 100 Black Males and Females are expected to develop CVD before the age of 50 (2). This is likely due to social factors as well as genetic determinants. Not only are members of the black community more likely to develop heart disease, but they are also 30% more likely to die from heart disease than non-Hispanic whites.

Cardiac Proteinopathy is a common feature of different types of heart failure. Cardiac proteinopathy is a protein conformational disorder which results in the accumulation of misfolded and mutant proteins in the heart. An example of cardiac protienopathy in cells is the Crystalline ab r120g model. Crystallin (CryAB) is a small heat shock protein which acts as a chaperone and participates in intermediate filament formation and maintenance. A missense mutation in CryAB, R120G, causes the formation of dense protein aggregates and is linked to familial desminopathy. The accumulation of R120G causes progressive muscle weakness, cataracts, and cardiac disorders like cardiomyopathy leading to disability and death.

Human Induced Pluripotent Stem cells (hiPSC) sourced from human fibroblasts, can be reprogrammed into new cell types under appropriate growth conditions. This ethically sourced approach was chosen over other models as hiPSC derived cardiomyocytes (hiPSC-CM) can faithfully recapitulate a human disease phenotype. We aim to generate a human induced pluripotent stem cell (hiPSC) line harboring the CryABWT or CryABR120G gene that can be differentiated into cardiomyocytes. Both CryAB wild-type (WT) and R120G mutant genes were knocked-in to the genome of hiPSCs. They were then cultured and differentiated into cardiomyocytes. More specifically, these cells were grown in vitronectin-coated plates and maintained in Essential 8 (E8) and Stem Flex (SF) media. Cells were differentiated using the stem cell cardiomyocyte differentiation media over 24-day period. The hiPSC-CM were then treated with doxycycline to induce WT or R120G gene expression.

Quantitative polymerase chain reaction (qPCR) was used to validate differentiation of hiPSCs into cardiomyocytes by determining the mRNA expression of several cardiomyocyte-related genes (i.e., Tbx5, Actn1, Cryab, Hcn4, etc.) Next, immunocytochemistry was employed to determine the levels of the induced proteins: crystallin and mCherry. The qPCR results show increased expression of several cardiomyocyte markers in the differentiated cells vs. the non-differentiated cells. Confocal imaging revealed increased expression of both mCherry and CryAB protein aggregates in mutant R120G line but not in the WT cells. While the WT cell lines indicated increased expression of both mCherry and CryAB, the extent of mCherry or CryAB protein expression was lower in the mutant line. Ongoing experiments involve culturing the mutant lines in Matrigel-coated plates with mTeSR Plus media to achieve higher expression of the proteins. Our novel WT or R120G hiPSC-CM cells will serve as an important tool in the study of clearing mutant protein aggregates.

References

1. Centers for Disease Control and Prevention. (2023, May 15). Heart disease facts. Centers for Disease Control and Prevention. https://www.cdc.gov/heartdisease/facts.htm#:~:text=Heart%20disease%20is%20the%2 0leading,groups%20in%20the%20United%20States.&text=One%20person%20dies%2 0every%2033,United%20States%20from%20cardiovascular%20disease.

2. U.S. Department of Health and Human Services. (2015, July 2). Heart failure before age 50 more common in black people. National Institutes of Health. https://www.nih.gov/news-events/nih-research-matters/heart-failure-age-50-more-common-black-
people#:~:text=According%20to%20a%20new%20study,rate%20than%20for%20white%20people