Adult murine cardiomyocytes exhibit regenerative activity with cell cycle reentry through STAT3 in the healing process of myocarditis

Mammalian cardiomyocytes substantially lose proliferative capacity immediately after birth, limiting adult heart regeneration after injury. However, clinical myocarditis appears to be self-limiting with tissue-reparative properties. Here, we investigated the molecular mechanisms underlying the recovery from myocarditis with regard to cardiomyocyte proliferation using an experimental autoimmune myocarditis (EAM) model. Three weeks after EAM induction (EAM3w), cardiac tissue displayed infiltration of inflammatory cells with cardiomyocyte apoptosis. However, by EAM5w, the myocardial damage was remarkably attenuated, associated with an increase in cardiomyocytes that were positively stained with cell cycle markers at EAM3w. Cardiomyocyte fate mapping study revealed that the proliferating cardiomyocytes primarily derived from pre-existing cardiomyocytes. Signal transducer and activator of transcription 3 (STAT3) was robustly activated in cardiomyocytes during inflammation, accompanied by induction of interleukin-6 family cytokines. Cardiomyocyte-specific ablation of STAT3 gene suppressed the frequency of cycling cardiomyocytes in the recovery period without influencing inflammatory status, resulting in impaired tissue repair and cardiac dysfunction. Finally, microarray analysis revealed that the expression of regeneration-related genes, metallothioneins and clusterin, in cardiomyocytes was decreased by STAT3 gene deletion. These data show that adult mammalian cardiomyocytes restore regenerative capacity with cell cycle reentry through STAT3 as the heart recovers from myocarditis-induced cardiac damage.


Capillary density quantification
Heart sections were fixed with acetone for 15 min. The endogenous peroxidase was silenced with 0.3% H 2 O 2 in methanol, followed by blocking with 3% bovine serum albumin (BSA).
Anti-CD31 primary antibody and biotin-linked secondary antibody were applied at 1/200 in sequence. Vectastain ABC kit (Vector Laboratories) and DAKO® Liquid DAB + (DAKO) were used for staining. The number of CD31 + cells was counted by a researcher who was blinded to the experimental condition, and capillary density at post-inflamed regions was calculated by dividing the number by the area of post-inflamed regions.

IL-11 administration
IL-11 (0.5 μg in 200 μL PBS) was intravenously administered in mice at EAM3w, followed by four times of intraperitoneal BrdU administration. The heart sections were collected 24 hours after the last BrdU injection and subjected to immunofluorescence staining.

Picrosirius red staining
Picrosirius red staining was performed using Picro-Sirius Red Stain Kit (Scytek) according to manufacturer's protocol. Briefly, frozen heart sections were fixed with 4% PFA for 15 min.
After rinsing in distilled water, the sections were incubated with Picro-sirius Red Solution for 60 min, followed by two quick rinses in 0.5% Acetic Acid Solution. Dehydration in absolute ethanol was performed prior to clearing and mounting. Figure S1. Cardiac tissue was restored at EAM5w.

Supplementary
The entire HE-stained cross sections of EAM0w, EAM3w and EAM5w hearts are shown.

Supplementary Figure S2. Cardiomyocytes were efficiently labeled with eYFP.
Cardiomyocytes were labeled with eYFP in advance to EAM induction, as described in Methods. Heart sections were immunostained for cTnI and YFP at EAM3w. Scale bar: 50 μm. (a) Heart sections from PBS-or IL-11-treated mice at EAM3w were immunostained for Ki-67 and MHC. Ki-67 + MHC + cells in the inflamed region were counted and the percentage of Ki-67 + MHC + cells in MHC + cells is shown. n=8 mice for PBS; 6 mice for IL-11. (b) Heart sections from PBS-or IL-11-treated mice at EAM3w were immunostained for Aurora B and cTnI. Aurora B + cTnI + cells in the inflamed region were counted and the percentage of Aurora B + cTnI + cells in cTnI + cells is shown. n=8 mice for PBS; 6 mice for IL-11. (c) BrdU incorporation assay was performed for PBS-or IL-11-treated mice at EAM3w. Heart sections were immunostained for BrdU and MHC 24 hours after the last BrdU injection. BrdU + MHC + cells in the inflamed region were counted and the percentage of BrdU + MHC + cells in MHC + cells is shown. n=8 mice for PBS; 6 mice for IL-11. Welch's t-test. * P<0.05. Data are shown as mean ± s.d.

Supplementary Figure S10. Rigid collagen fiber was observed in the injured area of STAT3cKO hearts at EAM5w.
Picrosirius red staining was performed for heart sections of STAT3fl/fl and STAT3cKO mice at EAM5w. Scale bar: 100 μm.

Supplementary Figure S11. Meis1 expression was unchanged in EAM model.
Cardiomyocytes were isolated at EAM0w and EAM3w, and meis1 mRNA expression was quantified by quantitative RT-PCR. The expression of meis1 was normalized to that of gapdh and shown as fold increase relative to 0w. n=7 mice for 0w; 6 mice for 3w. Welch's t-test.
Data are shown as mean ± s.d.