SCRMC Featured Researcher Fall 2018

Key populations may illuminate causes of hypertrophic cardiomyopathy


Cardiomyocytes from human induced pluripotent stem cells in the Ralphe lab

By Jessica Knackert, SCRMC editorial intern

Stem cell research at the University of Wisconsin-Madison is realizing its potential to change the fields of health and medicine. Every day, campus scientists are conducting exciting new research, and much of this research is featured during the SCRMC campus stem cell seminars held every Tuesday at the Discovery Building. To kick off the fall semester, Chief of Pediatric Cardiology and Pediatric Heart Program Director J. Carter Ralphe presented his lab’s studies on inherited forms of cardiomyopathy, specifically hypertrophic cardiomyopathy (HCM).

J. Carter Ralphe, M.D. (UW Health)

HCM is a disease that causes a thickening or enlarging of muscle in the heart, making it difficult for the heart to pump blood to the rest of the body. It often goes undiagnosed, but an unfortunate few who do display symptoms can suffer from shortness of breath, irregular heartbeats, and even possible heart failure.

Every 1 in 200 people carry a genetic mutation related to this condition, but Ralphe’s lab focuses on proving the pathogenicity, or ability to cause disease, of a mutation in hopes of understanding how each ultimately leads to the disease. A wide range of mutations may be responsible, but Ralphe’s studies primarily focus on mutations in cardiac myosin binding protein C (cMyBP-C), as these are considered a leading cause of familial HCM.

In previous years, Ralphe’s lab has resorted to a functional model with mice test subjects. Myocytes, or muscle cells, have been isolated from mouse hearts deficient in cMyBP-C and made into muscle strips. Researchers then produce a contraction, or twitch, in the cells similar to the contraction the heart makes when pumping blood, and study how the loss of cMyBp-C affects function. They then use a virus to introduce cMyBP-C carrying mutations that are suspected to cause HCM in order to test the effects.

Unfortunately, Ralphe found that studying only mice would not bring his lab over the finish line in regard to proving pathogenicity. The fact that the mouse heart beats 600 times per minute, nearly 10 times that of the human heart, limits the ability to translate data collected from mice to people with HCM. Ralphe decided to take his research in a new direction with the use of human induced pluripotent stem cells (iPS cells) and gene editing.

iPS cells are adult stems cells reprogrammed to act as pluripotent stem cells, meaning they have the capability to develop into a wide variety of cell types in the body. These cells can be used to grow human heart tissue that can then be studied in the lab. But how well do these lab-grown tissues represent real tissue found in our own bodies? In Ralphe’s lab, a three-dimensional environment is used, as it was with the mice. This living heart tissue model promotes a higher level of cell maturation that makes the engineered construct very similar to actual human tissue.

With these tissue constructs, the researchers can also utilize gene editing using the common CRISPR-Cas9 approach. With this method, specific DNA sequences are targeted to either introduce or fix mutations within the genes of the cardiomyocytes formed from the iPS cells. Experiments are then performed using these cultures to examine how the edited changes impact contractile function.

While the tools now available to researchers are helping define the direct impact of specific mutations, they are not helping to understand the enormous variation in disease severity and timing of onset. It is widely assumed that some other factor, either environmental or genetic, exerts a modifying effect on HCM. Ralphe has chosen to focus on the potential role of genetic modifiers to influence the severity of HCM. A genetic modifier is a variant in a gene carried in the population that in and of itself does not cause any disease, but when carried in conjunction with a primary HCM-mutation, changes or modifies the disease severity. Identification of potential genetic modifiers is difficult – akin to finding a small needle in a very large haystack.

Ralphe’s transition to using iPS cells may seem to be the ideal solution, but there are still some complications. Subjects would have to be drawn in groups of three from the same family that include an individual without the mutation and two individuals with the mutation, one with HCM and one without. For the data collected to be considered reliable, multiple triads would need to be drawn from the same family. However, this situation is extremely difficult to find.

“HCM is a tough genetic disease to study because there are hundreds of different mutations, and no way to predict who will have a more severe disease.  A large population of people carrying the same primary HCM mutation is needed. Access to a large enough population to make real progress is very difficult,” Ralphe commented when describing how to go about collecting enough data with human subjects to understand the disease. Fortunately, there is a population right in Wisconsin that proves to be the perfect group for drawing conclusions.

Ralphe went on to describe how the growing Amish population in the state may be an ideal community to partner with on these studies, as 1 out of every 20 individuals carries a cMyBP-C mutation. With generally larger family sizes and relative genetic isolation of many generations, Amish families could offer valuable data on how genetic modifiers influence the severity of HCM. The results from these studies would be potentially applicable to the general public and could possibly extend to other forms of cardiomyopathy. The development of a genetic screening tool that could predict who among HCM mutation carriers is more likely to develop an actual disease and who is not would be extremely beneficial to the health of the Amish population. As  Ralphe explained, the costs of lifelong screening to monitor for the development of disease is high and the restrictions on life activities can be severe.  For those without healthcare coverage, as is common in the Old Order Amish community, these health care costs can add up quickly. Any studies leading to a rational risk assessment for disease that can reduce the need for unnecessary testing would be especially beneficial to this population, he explained.

For funding his studies, Ralphe has turned to a surprising source in the U.S. Department of Defense. As it turns out, Ralphe explained, “Anybody who is genotype positive is excluded from military participation, and there is a relatively large population of people who develop HCM while they’re in the military, or afterwards, who need care. Unfortunately, there are also recruits every year in apparent peak health who drop dead suddenly. Not many, but one is too many.” The military is very determined to understand the causes of this condition to help reduce the risk for disease in service members by offering possible ways to predict or mitigate HCM. At the end of September, Ralphe’s lab sent a proposal to the Department of Defense in hopes of possible collaboration.

Ralphe’s HCM research is currently funded by the UW-Madison Office of the Vice Chancellor for Research and Graduate Education and also the National Insitutes of Health, National Heart, Lung and Blood Institute.