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. 2023 Jan 10;147(2):183-186.
doi: 10.1161/CIRCULATIONAHA.122.061018. Epub 2023 Jan 9.

Increasing Mononuclear Diploid Cardiomyocytes by Loss of E2F Transcription Factor 7/8 Fails to Improve Cardiac Regeneration After Infarct

Zhe Yu #  1 , Lunfeng Zhang #  1 , Paola Cattaneo  2   3   4   5 , Nuno Guimarães-Camboa  4   5 , Xi Fang  6 , Yusu Gu  6 , Kirk L Peterson  6 , Julius Bogomolovas  6 , Cecilia Cuitino  7 , Gustavo W Leone  8 , Ju Chen  6 , Sylvia M Evans  1   6   9
Affiliations

Increasing Mononuclear Diploid Cardiomyocytes by Loss of E2F Transcription Factor 7/8 Fails to Improve Cardiac Regeneration After Infarct

Zhe Yu et al. Circulation. .
No abstract available

Keywords: E2f7; E2f8; heart regeneration; mononuclear diploid cardiomyocytes; polyploidization; proliferation.

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Figures

Figure.
Figure.. Increased frequency of mononuclear diploid CMs fails to promote cardiac regeneration after MI.
A, Representative images of major types of cardiomyocytes (CMs) with different nucleation and ploidy levels. CM nuclei and sarcomeric F-actin were labeled by DAPI and phalloidin, respectively. Scale bar, 10 μm. B, XMLC2–78dKO mice exhibited a significantly increased percentage of mononuclear CMs in the left ventricle (LV), right ventricle (RV), and interventricular septum (IVS; 5.1% to 14% in LV; 17% in mice used by Patterson et al). No differences in sarcomere structure or size of CMs were observed (data not shown). C, Ploidy levels of mononuclear CMs as determined by integrated fluorescence intensity of DAPI. Significantly increased mononuclear diploid (MND) CMs were observed in the LV, RV, and IVS of mutants (0.5% to 5.1% in the LV). CMs were isolated from 8-week-old mice (n=5 for each; 200 CMs per animal). D, Echocardiography data showed that 8-week-old XMLC2–78dKO mice had normal cardiac function (n=40 for each). E, Regional radial strain analyses showed no significant differences in cardiac function between XMLC2–78dKO mice and controls on day 2 (n=4 for each sham; n=15 for control myocardial infarction [MI]; n=16 for 78dKO MI) and day 7 or 28 (n=8 for control sham; n=20 for control MI; n=7 for 78dKO sham; n=24 for 78dKO MI) after MI. F, Representative images of sham and MI hearts in both XMLC2–78dKO mice and controls. Hearts were sectioned every 500 μm from suture. Sirius red staining was performed to label the infarct zone (IZ). Scale bar, 1 mm. G, XMLC2–78dKO mice and controls had similar infarct sizes on day 28 (n=6 for each) and day 42 (n=7 for control; n=10 for 78dKO) after MI. Infarct size was calculated as total infarct circumference divided by total LV circumference from all sections. H, Schematic of experimental protocol. Mice were injected once a day with 200 μg EdU from days 10 to 18 after MI. Hearts were harvested on day 28 after MI and either sectioned or border zone (BZ)/IZ CM isolation was performed. I, Representative images of EdU+ CM nuclei in sections of BZ/IZ. CM nuclei were labeled by Tnnt2 RNA scope intronic probes. Scale bar, 100 μm. J, Significantly increased numbers of EdU+ CM nuclei were observed in sections of BZ/IZ of XMLC2–78dKO mice (n=4 for each sham; n=6 for each MI; total count of CM nuclei in control/78dKO MI hearts: 14 099/14 390 in BZ, 2462/2582 in IZ, 4536/6497 in LV, 8822/10 092 in RV, and 8595/10 666 in IVS; total count of CM nuclei in control/78dKO sham hearts: 7595/7769 in LV, 9018/8878 in RV, and 9639/10 014 in IVS). Coimmunostaining with Ki67 and PCM1 confirmed increased cell-cycle reentry in sections of BZ/IZ of XMLC2–78dKO mice (data not shown). K, Representative images of major types of EdU+ CMs with different nucleation and ploidy levels isolated from BZ/IZ tissue. Scale bar, 10 μm. L, Mononuclear CMs were overrepresented in EdU+ CMs relative to their baseline frequencies (see B) for both control and XMLC2–78dKO mice (n=7 for each; total count of mononuclear/binuclear EdU+ CMs: 95/102 in controls; 592/56 in 78dKO mice). M, EdU+ mononuclear CMs were predominantly polyploid in XMLC2–78dKO mice and controls. Increased frequency of 4n+ mononuclear CMs was observed in controls compared with mutants. N, Distribution of ploidy levels in EdU+ binuclear CMs was similar in XMLC2–78dKO mice and controls. O, For pulse labeling, each mouse was given a single injection of 200 μg EdU on day 13 after MI (maximal EdU incorporation; data not shown). Hearts were harvested 1 hour later, and BZ/IZ CMs were isolated. P, Comparison of frequencies of ploidy categories of mononuclear CMs at baseline (a) and after EdU pulse labeling (b; n=6 for each; total EdU+ mononuclear CMs: 14 in controls; 40 in 78dKO mice) suggested that MND CMs preferentially incorporated EdU. Comparison of frequencies of ploidy categories of mononuclear CMs after pulse labeling (b) and on day 28 (M) suggested that preferentially labeled MND CMs became polyploid. Proliferation of initially labeled MND CMs would be expected to result in increased frequency of EdU-labeled MND CMs on day 28, which was not observed. Instead, decreased frequency of EdU-labeled MND CMs and increased frequency of EdU-labeled mononuclear polyploid CMs were observed on day 28, thus supporting polyploidization of EdU+ MND CMs. P (a), Distinct representation of C to facilitate comparison with M/P(b). Q, Similar comparison as in P but for binuclear CMs. EdU pulse labeling (b; total EdU+ binuclear CMs: 25 in controls; 9 in 78dKO mice) suggested that EdU incorporation by binuclear CMs was likely to result in polyploidization. R, Schematic summary. XMLC2–78dKO mice exhibited a 10-fold baseline increase in MND CMs. After MI, EdU was preferentially incorporated by MND CMs in BZ/IZ. Proliferation of EdU+ MND CMs would result in increased frequency of EdU+ MND CMs, which was not observed. Polyploidization of EdU+ MND CMs would result in increased frequency of EdU+ mononuclear polyploid CMs, which was observed, thus supporting polyploidization rather than proliferation. Consistent with these results, we found that 99% of mononuclear CMs in both controls and mutants had perinuclear PCM1 (inability to proliferate; data not shown). No improvement in cardiac function or reduction in infarct scar was observed between controls and mutants after MI. Data are presented as mean±SD. To determine statistical significance, negative binomial regression was performed for B, C, J, and L; Conway-Maxwell-Poisson regression was performed for M, N, P, and Q; Student t test was used for D; and mixed ANOVA and 2-way ANOVA were used for E and G, respectively. Conway-Maxwell-Poisson regression was performed with the mpcmp package in R. Other statistics were performed by SPSS 28. Graphs were generated with GraphPad Prism 9. EdU+ 4n+ indicates EdU+ nuclei with ploidy level higher than 4n; EDV, end-diastolic volume; EF, ejection fraction; ESV, end-systolic volume; FS, fractional shortening; LVIDd, left ventricular internal diameter at end diastole; LVIDs, left ventricular internal diameter at end systole; LVPWd, left ventricular posterior wall end diastole; and RZ, remote zone. All mice were males and maintained on FVB/NJ background. *P<0.05. **P<0.01. ***P<0.001.
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References

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