Dedifferentiation, Proliferation, and Redifferentiation of Adult Mammalian Cardiomyocytes After Ischemic Injury

Abstract

Background: Adult mammalian hearts have a limited ability to generate new cardiomyocytes. Proliferation of existing adult cardiomyocytes (ACMs) is a potential source of new cardiomyocytes. Understanding the fundamental biology of ACM proliferation could be of great clinical significance for treating myocardial infarction (MI). We aim to understand the process and regulation of ACM proliferation and its role in new cardiomyocyte formation of post-MI mouse hearts.

Methods: β-Actin-green fluorescent protein transgenic mice and fate-mapping Myh6-MerCreMer-tdTomato/lacZ mice were used to trace the fate of ACMs. In a coculture system with neonatal rat ventricular myocytes, ACM proliferation was documented with clear evidence of cytokinesis observed with time-lapse imaging. Cardiomyocyte proliferation in the adult mouse post-MI heart was detected by cell cycle markers and 5-ethynyl-2-deoxyuridine incorporation analysis. Echocardiography was used to measure cardiac function, and histology was performed to determine infarction size.

Results: In vitro, mononucleated and bi/multinucleated ACMs were able to proliferate at a similar rate (7.0%) in the coculture. Dedifferentiation proceeded ACM proliferation, which was followed by redifferentiation. Redifferentiation was essential to endow the daughter cells with cardiomyocyte contractile function. Intercellular propagation of Ca2+ from contracting neonatal rat ventricular myocytes into ACM daughter cells was required to activate the Ca2+-dependent calcineurin-nuclear factor of activated T-cell signaling pathway to induce ACM redifferentiation. The properties of neonatal rat ventricular myocyte Ca2+ transients influenced the rate of ACM redifferentiation. Hypoxia impaired the function of gap junctions by dephosphorylating its component protein connexin 43, the major mediator of intercellular Ca2+ propagation between cardiomyocytes, thereby impairing ACM redifferentiation. In vivo, ACM proliferation was found primarily in the MI border zone. An ischemia-resistant connexin 43 mutant enhanced the redifferentiation of ACM-derived new cardiomyocytes after MI and improved cardiac function.

Conclusions: Mature ACMs can reenter the cell cycle and form new cardiomyocytes through a 3-step process: dedifferentiation, proliferation, and redifferentiation. Intercellular Ca2+ signal from neighboring functioning cardiomyocytes through gap junctions induces the redifferentiation process. This novel mechanism contributes to new cardiomyocyte formation in post-MI hearts in mammals.

Keywords: cell dedifferentiation; cellular proliferation; myocardial infarction; myocytes, cardiac; redifferentiation.

© 2017 American Heart Association, Inc.

Figures

Figure 1. ACM dedifferentiation, proliferation and redifferentiation in-vitro

Freshly isolated ACMs from β-actin-GFP mice were co-cultured with NRVMs for 7 days, and the ACMs remodeled over time. A: Identification of ACM dedifferentiation. A1: Representative images (A1a) and quantification (A1b) of ACMs that lost the contractile protein cTnI at day 3 after co-culture. Arrow “a” indicates an ACM without cTnI expression, while arrow “b” indicates a neighboring NRVM. A2: Representative images (A2a) and quantification (A2b) of ACMs expressing dedifferentiation marker Runx1. A3: Representative images (A3a) and quantification (A3b) of ACMs expressing dedifferentiation marker Dab2. B: Identification of ACM proliferation. B1: Representative images (B1a) and quantification (B1b) of ACMs expressing proliferation marker Ki67. B2: Representative images (B2a) and quantification (B2b) of ACMs expressing proliferation marker PH3. C: Identification of ACM redifferentiation. C1: Representative images of an ACM regained organized sarcomere at day 7 after co-culture. C2: Quantification of ACMs with organized sarcomeres or spontaneous beating out of total survived ACMs. N=12; * p<0.05 vs. ACMs at day 1; # p<0.05 vs. ACMs at day 3; & p<0.05 vs. % of spontaneous beating at day 3. Scale bars represent 20μm.

Figure 2. ACM proliferation with cytokinesis in-vitro

Freshly isolated ACMs from β-actin-GFP transgenic mice were co-cultured with NRVMs for 7 days. Every ACM was traced with a time-lapse video microscopy, and only the division events with completed cytokinesis were counted as ACM proliferation. A: The morphological remodeling of ACMs in the co-culture system observed with a time-lapse video microscopy. A bi-nucleated ACM indicated with red arrow became spherical and lost organized contractile apparatus during the first 3 days of co-culture, and then proliferated into several daughter cells that assumed a NRVM-like shape over the next few days. White arrow indicates the nuclei (labeled with DRAQ5™ Fluorescent Probe) of the ACM that underwent cytokinesis at the beginning of co-culture. The proliferation process of this ACM can be found in Supplemental Video 1. B: The cell fate of ACMs during the 7 days co-culture with NRVMs. The rates of cell death, survival and proliferation were quantified. C: The proliferation rates of mononucleated and bi/multi-nucleated ACMs. D: The surface area of ACMs that underwent proliferation and those without proliferation. E: The percent composition of proliferated ACMs. A total number of 38 ACM proliferation events with clearly visible nuclei were quantified. F: The cell division patterns of ACMs with different nuclei number. G: The characteristics of the daughter cells after ACM cytokinesis immunostained with cTnI. G1: The percentage of ACM progeny with different properties. G2: Representative images showing both of the ACM derived daughter cells lost cTnI expression. G3: Representative images showing one ACM derived daughter cell maintained sarcomeric structure while the other lost cTnI. G4: Representative images showing both of the ACM derived daughter cells regained sarcomeric structure. The proliferation process of the ACMs shown in G2-4 can be found in Supplemental Videos 2–4. For A–G, scale bars represent 50μm.

Figure 3. Cx43 mediated intercellular transmission of Ca2+ influence the ACM redifferentiation

A: The percentage of sarcomere+ ACMs in co-culture at day 7 with Ca2+ blocker BAPTA-AM and thapsigargin. B: The property of Ca2+ transients in ACMs cocultured with NRVMs over-expressing Ca2+ regulating protein SERCA2a or NCX1. The graph bars represent [Ca2+]i transient amplitude (change from diastolic [Ca2+]i to peak systolic [Ca2+]i) of redifferentiated ACMs and their coupled NRVMs. C: The percentage of ACMs with sarcomere+ (C1) or spontaneous beating (C2) in co-cultures with NRVMs overexpressing SERCA2a or NCX1 at day 7. D: The percentage of sarcomere+ ACMs in co-cultures at day 7 with Cx43 manipulation. E: The percentage of sarcomere+ ACMs in co-cultures at day 7 with Cx43 manipulation under hypoxia. N=8–10; * p<0.05 vs. control or scramble or vector; # p<0.05 vs. vector+hypoxia.

Figure 4. Intracellular Ca2+ dependent calcineurin-NFAT signaling pathways regulate ACM redifferentiation

A–B: The role of Ca2+ dependent calcineurin-NFAT signaling pathway in ACM redifferentiation. The percentage of ACMs with organized sarcomere (A) and spontaneous beating (B) were quantified at 7 days post-co-culture. The cells were pretreated with mouse specific siRNAs against calcineurin, NFATc3 and MEF2C, or calcineurin inhibitor CsA, respectively. N=6; * p<0.05 vs. Scramble; # p<0.05 vs. DMSO. C: Representative images of nuclear translocation of NFATc3 in ACMs at day 5 post-co-culture. The cells were pretreated with mouse specific siRNAs of Cx43 or scramble sequence as a control. Scale bars represent 20 μm.

Figure 5. Dedifferentiation, proliferation and redifferentiation of ACMs in Myh6-MerCreMer-lacZ mice subjected to MI

A: Representative images of ACMs expressing dedifferentiation markers Runx1 (A1) or Dab2 (A2) in the infarct border zone. B: Representative images of ACMs expressing proliferation markers Ki67 (B1) and PH3 (B2). C: Representative images of a mononuclear lacZ+/EdU+ ACM isolated from hearts 3 weeks after MI. The organized sarcomere was clearly seen in the ACM. For A–C, scale bars represent 20 μm.

Figure 6. Cardiac AAV9-Cx43-S3E (ischemia resistant Cx43 mutant) therapy promotes ACM proliferation and redifferentiation and improves cardiac function in the post-MI heart

AAV9-Cx43-S3E or AAV9-vector was administrated 3 days after MI. A: Altered Cx43 phosphorylation in post-MI mice with AAV9-Cx43-S3E treatment. Western blot analysis of whole-cell lysates prepared from left ventricles of mice 6 weeks post-MI. Cx43 lysates are shown in various major phosphorylated forms of Cx43 (P0, P1, P2, P3) with the indicated treatments, probed with polyclonal pan-Cx43 antibody. B: The representative images (B1) and quantification (B2) of heart infarct size 6 weeks post-MI. C: The cardiac structure and function were assessed by serially echocardiography. Left ventricle internal diameter in systole (LVIDs, C1), left ventricular ejection fraction (LVEF, C2) and fraction shortening (LVFS, C3) at 2–6 weeks post-MI were presented. D: ACM proliferation and redifferentiation detected by EdU incorporation analysis at 3 weeks post-MI. Total EdU+/sarcomere+/lacZ+ cells (D1) and mononuclear EdU+/sarcomere+/lacZ+ cells (D2) were quantified. N=10–14; * p<0.05 vs. Sham; # p<0.05 vs. AAV9-vector.