Background

After heart attacks, generating healthy myocardial tissue by reversing scar tissue will trigger changes in cardiology and regenerative medicine. In the laboratory, scientists have proved that it is possible to convert cardiac fibroblasts (cicatricial tissue cells) into cardiomyocytes, but it is not easy to sort out details of how to process and it is difficult to apply this approach in Clinical practice or even other basic research projects.

Now, in a new study, researchers from the University of North Carolina in the United States combined microfluidic single-cell RNA sequencing with mathematical modeling, genetic methods, and chemical methods to describe the molecular changes that progressively occur during cell fate transformation from fibroblast to cardiomyocyte. With the leadership of Dr. Li Qian, an assistant professor of medicine, pathology and laboratory medicine at the University of North Carolina, researchers succeeded in reconstructing the path chosen by individual fibroblasts in the process, moreover, they identified the molecular pathways and key regulators that play an important role during the conversion of fibroblasts to cardiomyocytes. Relevant findings were published online in the Journal of Nature.

They initiated the direct cardiac reprogramming approach and has optimized this approach over the past few years. As a promising method of cardiac regeneration and disease modeling, it involves the direct conversion of non-cardiomyocytes in the heart into induced cardiomyocytes (iCMs), which are very similar to endogenous cardiomyocytes. Just like any other reprogramming processes, many cells under reprogramming will not reprogram simultaneously, which means that this is an "asynchronous" process. The conversion will take place in a different time period so that the cell population always contains non-transformed cells, partially reprogrammed cells, and fully reprogrammed cells at any stage of the process. In other words, cell reprogramming is "heterogeneous," making it difficult to analyze with traditional methods.

In this new study, researchers address two major issues which involve asynchronous programming and heterogeneous cell populations by leveraging microfluidic single-cell RNA sequencing. They analyzed global transcriptome changes during the fate transformation from fibroblasts to iCMs. With mathematical algorithms, these researchers identified subpopulations of cells with different molecular characteristics during this reprogramming process. Subsequently, they reconstructed the path of iCM formation based on simulation and experimental verification. These pathways provide them an unprecedented high-resolution roadmap to study this cellular transformation mechanism in the future.

These findings will play an important role in the clinical, such as, it is known that cardiac fibroblasts around an impaired area will be activated immediately after a heart attack and get highly proliferated, but the capacity of proliferation will decrease over time, which makes it important to figure out how to utilize fibroblasts in different cell cycle states during and after a heart attack should extend the range of cell reprogramming applications and optimize cell transformation results.

Qian and her team revealed the pathway between cell proliferation and cell reprogramming. They also provide experimental evidence to confirm that changing the initial cell cycle status of fibroblasts alters new cardiomyocyte formation results. It is found that molecular features of fibroblast subsets were repressed to varying degrees during reprogramming, suggesting a difference in reprogramming sensitivity of cells.

Interestingly, this reprogramming sensitivity is consistent with cardiomyocyte differentiation during cardiac development. It seems that the cell population appears to be more robust against these changes during the early stages of cardiac development, suggests that recent epigenetic memory of cells may be more easily erased, making it easier to convert fibroblast subpopulations with these epigenetic traits into cardiomyocytes.

With further analyzing global gene expression changes during reprogramming, these researchers unexpectedly identified down-regulation of factors involved in mRNA processing and splicing. Qian and her team continued to carry out a detailed functional analysis of the top priority candidate factor, the splicing factor Ptbp1. Some earlier evidence suggests that it is a key hurdle for fibroblasts to acquire cardiomyocyte-specific splicing patterns and studies by Qian and her team further confirmed that removing Ptbp1 promoted more iCM production.

Further quantitative analysis revealed that the expression for reprogramming of individual (that is Mef2, Gata4, Tbx5, and DsRed, expressing these four reprogramming factors with fibroblasts will turn them into iCMs) has a strong correlation with the progress of reprogramming of individual fibroblasts, and that will result in the discovery of new surface markers enriched for iCM.

Qian claimed that the interdisciplinary approach in this paper is very powerful to identify previously unrecognized functions or mechanisms, meanwhile, it will help better understand the nature of the cell and the progression of the disease. She also said, ‘ Eventually, This approach may not only benefit heart disease patients but also benefit patients with cancer, diabetes, neurological diseases and other diseases.’ We will also look forward to the further application of this approach.