The heart is the first organ to form during the developmental stage of mammals, providing blood support for life activities throughout the body. Although heart function is so critical, mature cardiomyocytes generally do not have the ability to proliferate, leading to irreversible heart damage, so many scientists around the world are exploring how to restore the regenerative capacity of the heart [1][2].

Currently, the most important concern in the study of the regenerative capacity of the heart is: how to allow mature cardiomyocytes to proliferate cellularly again. Since cardiomyocytes can beat spontaneously, if newly proliferated cardiomyocytes can replace damaged cells, then it is likely that repair of cardiac function will be achieved.

In order to find a way to proliferate cardiomyocytes, Jingwei Xiong's research group from School of Future Technology, Peking University, recently published an online paper entitled "A small-molecule cocktail promotes mammalian cardiomyocyte proliferation and proliferation" in the international journal Cell Stem Cell. A small-molecule cocktail promotes mammalian cardiomyocyte proliferation and heart regeneration". In this study, the authors identified a mixture of several small molecules that enable adult cardiomyocytes to proliferate, thereby promoting cardiac repair.

Research idea

Screening and testing were performed through an in vitro-in vivo-ex vivo process by:

1. first find alternative bioactive substances through a large-scale in vitro screening;

2. then to experimentally test these biologically active small molecules in animals in vivo;

3. finally, the activated cells are cultured and analyzed by single cell sequencing in an in vitro environment.

The main studies in the article are summarized as follows:

1. combinations of drug small molecules can stimulate cardiomyocyte multiplication

To find ways to get cardiomyocytes to proliferate, the researchers first screened thousands of drug small molecules in hopes of finding drugs that stimulate cardiomyocyte proliferation. To screen for drugs that work well, the authors utilized two different systems for screening drug small molecules:

One is the FUCCI system, which is used to perform a primary screening of alternative drugs to see if they can "rejuvenate" cardiomyocytes - "rejuvenation" is the only way to restore proliferation.

The other is the MADM system, which is used to re-screen these FUCCI screened alternatives to see which drugs can restore the ability of cardiomyocytes to "rejuvenate".

Using these two screening systems, the authors identified 13 small molecules that were shown in simple experiments to promote the proliferation of cardiomyocytes. Next, the authors randomly combined these 13 alternative molecules in pairs and, with the help of a computer algorithm, identified 10 combinations of small molecules that were likely to be more effective.

To verify the effectiveness of these combinations, the authors further did FUCCI and MADM screening on these 10 combinations above. Finally, 5 small molecules (collectively 5SM) were found to significantly promote cardiomyocyte division and cardiac regeneration in adult mice and rats. And, a series of pathological experiments also demonstrated that 5SM could promote cardiomyocyte proliferation in myocardial infarcted hearts.

2. Combinations of drug small molecules can reactivate cardiac function

To further investigate how 5SM promotes cardiomyocyte proliferation, the authors first performed RNA-seq and ATAC-seq, and found that 5SM can induce mature cardiomyocytes to dedifferentiate and revert to non-mature cardiomyocytes, thus gaining the ability to divide and proliferate again.

During this process, many genes related to glycolysis and dedifferentiation are activated and actively expressed, contributing to the "rejuvenation" of cardiomyocytes.

3. Single-cell sequencing reveals cardiomyocyte value-added pathways

Now that genes related to proliferation and dedifferentiation have been identified, a new question arises: How do these genes change the fate of cardiomyocytes to achieve "rejuvenation"? To answer this question, the authors isolated cardiomyocytes from rat ventricles, cultured them in vitro and treated them with 5SM, and then performed single-cell transcriptome sequencing on the treated cardiomyocytes. Analysis of the single-cell sequencing results led the authors to identify a pathway for cardiomyocyte fate transition:

In the 5SM-treated cardiomyocytes, in addition to the CM1 isoform, which would normally be present in cardiomyocytes, there were two isoforms, CM2 and CM4, that would not be present in untreated cardiomyocytes. Moreover, in terms of the pathway of developmental differentiation, cardiomyocytes will shift again to the proliferative phase along the pathway C1-C2-C4 under the stimulation of 5SM.

(Note: The top left panel illustrates that six cardiomyocyte (CM) subpopulations were identified by single-cell sequencing analysis, and the proximity of these subpopulations to each other represents the high similarity of gene expression in different subpopulations of cells, with the closer the more similar. In the figure, CM1, CM2, and CM4 are in adjacent positions, indicating that their gene expression is relatively similar. (The top right figure illustrates that CM1, CM2, and CM4 are in a developmental pathway, a process that occurs from early to late.)

This process can be thought of as the reactivation of cardiomyocytes. After activation, mature cardiomyocytes will re-enter the division cycle and provide a new source of cells for the damaged heart, leading to cardiac repair.

Summary

This article obtained a set of drug small molecule combinations capable of promoting cardiomyocyte proliferation through the screening of small molecule drugs, and investigated the mechanism of drug initiation by single-cell sequencing, with sufficient evidence chain and obvious application value. With the help of single-cell sequencing technology, this study has advanced the bottlenecked cardiac regenerative medicine by a big step forward, bringing a boon to the treatment of heart attack-related diseases.

References

[1] Fernández-Ruiz I. Rejuvenating cardiomyocytes facilitates heart regeneration. Nat Rev Cardiol. 2021, 18(12):807.
[2] Sadek H, Olson EN. Toward the Goal of Human Heart Regeneration. Cell Stem Cell. 2020, 26 (1):7-16.
[3] Garbern JC, Lee RT. Heart regeneration: 20 years of progress and renewed optimism. Dev Cell. 2022, 57(4):424-439.