Reprogramming Fibroblasts into New Heart Cells

Researchers reported important molecular details that could lead to the development of new heart cells. In a study (“Initiating Events in Direct Cardiomyocyte Reprogramming”) published in Cell Reports, two labs at the University of North Carolina-Chapel Hill UNC and a team at Princeton University reprogrammed fibroblasts into new and healthy heart muscle cells and recorded changes that appear to be necessary for this reprogramming.

“Direct reprogramming of fibroblasts into cardiomyocyte-like cells (iCM) holds great potential for heart regeneration and disease modeling and may lead to future therapeutic applications. Currently, application of this technology is limited by our lack of understanding of the molecular mechanisms that drive direct iCM reprogramming. Using a quantitative mass spectrometry-based proteomic approach, we identified the temporal global changes in protein abundance that occur during initial phases of iCM reprogramming,” write the investigators.

“Collectively, our results show systematic and temporally distinct alterations in levels of specific functional classes of proteins during the initiating steps of reprogramming including extracellular matrix proteins, translation factors, and chromatin-binding proteins. We have constructed protein relational networks associated with the initial transition of a fibroblast into an iCM. These findings demonstrate the presence of an orchestrated series of temporal steps associated with dynamic changes in protein abundance in a defined group of protein pathways during the initiating events of direct reprogramming.”

“From these studies we may be able to define pathways to increase the efficiency of fibroblast reprogramming,” said senior author Frank Conlon, Ph.D., professor of genetics in the UNC School of Medicine and professor of biology in the UNC College of Arts and Sciences.

Heart disease occurs in part because cardiomyocytes have a limited ability to proliferate and replace damaged heart muscle. Scientists have been experimenting with techniques to transform fibroblasts—collagen-making cells that are abundant in the heart—into new cardiomyocytes. They have shown that they can make this therapeutic cell-reprogramming process work in the diseased hearts of lab mice and improve heart function. But the process isn't as efficient as it needs to be for clinical use, and scientists are still learning why.

“The application of this technology has been limited by our lack of understanding of the molecular mechanisms driving this direct reprogramming process,” said Dr. Conlon, who is also a member of the UNC McAllister Heart Institute.

For this study, Dr. Conlon's lab—in collaboration with the UNC McAllister Heart Institute lab of Li Qian, Ph.D., and the Princeton lab of Ileana Cristea, Ph.D.—employed advanced techniques to map changes in protein levels in fibroblasts as they underwent reprogramming into cardiomyocytes.

First, they triggered the reprogramming using a technique based on one Dr. Qian developed in 2012. They exposed fibroblasts to an engineered retrovirus that enters the cells and starts producing three key transcription factor proteins, which effectively reprogram gene expression in the cells, causing the cells to turn into cardiomyocytes within a few days.

The researchers examined the levels of thousands of distinct proteins in the cells during the three-day transformation from fibroblasts to cardiomyocytes. In so doing, said Dr. Conlon, “We revealed a carefully orchestrated series of molecular events.”

The data suggest that the reprogramming process kicked off at about 48 hours after the viruses entered the fibroblasts and significantly affected the abundance of 23 classes of protein, he added.

One of the most striking changes was a sharp rise in the level of Agrin, a protein that has been found to promote repair processes in damaged hearts. Agrin also inhibits another signaling pathway called  Hippo, known to be involved in regulating organ size. This finding raises the possibility that inhibition of Hippo signaling is needed for cardiomyocyte reprogramming.

Future studies will determine which of these myriad changes does indeed drive reprogramming, and more importantly which changes can be enhanced to improve reprogramming efficiency.