Heart Regeneration

       Cardiac disease or injury frequently leads to heart failure because the mammal heart cannot regenerate damaged myocardial tissue. The non-regeneration is primarily driven by the inability to proliferate in cardiomyocytes. On the other hand, cardiomyocytes are robustly proliferative during the final weeks of fetal development. Cardiomyocytes may continue proliferating up to seven days after birth. So, when myocardial infarction (MI) is induced on postnatal day (P) 1 in newborn mice, the animals recovered completely by P28 with no decline in cardiac function and negligible myocardial scarring. Furthermore, this recovery ability is virtually dismissed if MI is induced on P7 and later time points.

Left ventricle heart sections from injured pigs, induced on P1 and P14, were obtained on day 30 after MI induction. Left panel: Sirius Red and Fast Green to identify the scarred (red) and unscarred regions (green). Right panel: the live images of the heart sections showing LV anterior wall thickness. They demonstrate cardiac regeneration following P1 injury, but this ability dismisses if the injury is induced on P14.

       Our lab is among the first team who experimented with and verified heart regeneration in pigs, a large-animal model very similar to the human heart. The gradually-declined regenerative capability was strongly associated with the lower rate of pig cardiomyocyte proliferation. We found that after birth, pig cardiomyocytes still exercised the cell cycle, which explained why they proliferate. However, they stopped cell cycling a few days after birth, therefore, unable to proliferate, and the heart failed to regenerate.

The proliferation marker expression (Ki67) was evaluated via immunofluorescent staining in paraformaldehyde-fixed heart sections; cardiomyocytes were visualized by staining for cardiac troponin T (cTnT), and nuclei were labeled with anti-Nkx2.5 antibodies and counterstained with DAPI. The colocalization among Ki67 (red), cTnT (green), anti-Nkx2.5 antibodies (white), and DAPI (blue) is significantly higher in the injured-heart-P1 group (top) than the injured-heart-P14 group. The results show that cardiomyocytes are significantly more proliferative in the P1 group.

       Furthermore, we found that if the neonatal pigs undergo a cardiac injury on P1, they will not only completely recover on P28 but also recover after a second MI injury induced on P28. Thus, we were the first lab to extend the mammal heart regeneration windows from seven to at least twenty-eight days after birth. Analyzing the genetic data in these pigs’ cardiomyocytes, we discovered that important cell cycle regulators were significantly elevated after the neonatal P1 injury. Then, when they recover on P28, although cardiomyocytes did not need cell cycling, a number of these regulators, particularly TBX5, TBX20, and ERBB4, maintain high expression. Also, these regulators are required in heart development during the fetal and early-postnatal periods.

       Collectively, these provocative observations suggest that it may be possible to reactivate the cell cycle in cardiomyocytes of adult mammalian hearts, which could lead to the development of transformative new therapies for the treatment of myocardial disease.

Our newly-established pig heart regeneration model using a double-injury experiment, which extends the regeneration windows at least 4 weeks after birth. Top: the sematic overview of this new model. First, the hearts underwent Apical Resection (AR) on neonatal day P1. Then the hearts will regenerate and recover on P28 (4 week after). Then, on P28, a second MI injury was induced on P28 in these AR pigs (AR-MI); also, wild-type pigs (without AR on P1) also underwent MI on P28 (MI only) for comparison. On P56 (another four week), these heart tissues were collected. Bottom: live-image of these tissues on P56. As expected, the MI-only clearly shows a large scar area (white); meanwhile, the AR-MI does not, which indicate regeneration.

Read more

– Zhu W, Zhang E, Zhao M, Chong Z, Fan C, Tang Y, Hunter JD, Borovjagin AV, Walcott GP, Chen JY, Qin G, Zhang J. Regenerative Potential of Neonatal Porcine Hearts. Circulation. 2018 Dec 11;138(24):2809-2816. Epub 2018 July 20. PMID: 30030418; PMCID: PMC6301098

– Zhao M, Zhang E, Wei Y, Zhou Y, Walcott GP, Zhang J. Apical Resection Prolongs the Cell Cycle Activity and Promotes Myocardial Regeneration after LV Injury in Neonatal Pig.  Circulation 2020;142:913–916

– Nguyen, T.M., Wei, Y., Nakada, Y., Zhou, Y. and Zhang, J., Cardiomyocyte cell-cycle regulation in neonatal large mammals: Single Nucleus RNA-sequencing Data analysis via an Artificial-intelligence–based pipeline. Frontiers in Bioengineering and Biotechnology, p.972.

– Nakada, Y., Zhou, Y., Gong, W., Zhang, E.Y., Skie, E., Nguyen, T., Wei, Y., Zhao, M., Chen, W., Sun, J. and Raza, S.N., 2022. Single Nucleus Transcriptomics: Apical Resection in Newborn Pigs Extends the Time Window of Cardiomyocyte Proliferation and Myocardial Regeneration. Circulation, 145(23), pp.1744-1747.

– Steinhauser ML and Lee RT. Regeneration of the heart. EMBO Mol Med. 2011;3:701-12.