Cardiomyocyte cell cycle dynamics and proliferation revealed through cardiac-specific transgenesis of fluorescent ubiquitinated cell cycle indicator (FUCCI)

Abstract

Rationale: Understanding and manipulating the cardiomyocyte cell cycle has been the focus of decades of research, however the ultimate goal of activating mitotic activity in adult mammalian cardiomyocytes remains elusive and controversial. The relentless pursuit of controlling cardiomyocyte mitosis has been complicated and obfuscated by a multitude of indices used as evidence of cardiomyocyte cell cycle activity that lack clear identification of cardiomyocyte "proliferation" versus cell cycle progression, endoreplication, endomitosis, and even DNA damage. Unambiguous appreciation of the complexity of cardiomyocyte replication that avoids oversimplification and misinterpretation is desperately needed.

Objective: Track cardiomyocyte cell cycle activity and authenticate fidelity of proliferation markers as indicators of de novo cardiomyogenesis in post-mitotic cardiomyocytes.

Methods and results: Cardiomyocytes expressing the FUCCI construct driven by the α-myosin heavy chain promoter were readily and uniformly detected through the myocardium of transgenic mice. Cardiomyocyte cell cycle activity peaks at postnatal day 2 and rapidly declines thereafter with almost all cardiomyocytes arrested at the G1/S cell cycle transition. Myocardial infarction injury in adult hearts prompts transient small increases in myocytes progressing through cell cycle without concurrent mitotic activity, indicating lack of cardiomyogenesis. In comparison, cardiomyogenic activity during early postnatal development correlated with coincidence of FUCCI and cKit⁺ cells that were undetectable in the adult myocardium.

Conclusions: Cardiomyocyte-specific expression of Fluorescence Ubiquitination-based Cell Cycle Indicators (FUCCI) reveals previously unappreciated aspects of cardiomyocyte cell cycle arrest and biological activity in postnatal development and in response to pathologic damage. Compared to many other methods and model systems, the FUCCI transgenic (FUCCI-Tg) mouse represents a valuable tool to unambiguously track cell cycle and proliferation of the entire cardiomyocyte population in the adult murine heart. FUCCI-Tg provides a desperately needed novel approach in the armamentarium of tools to validate cardiomyocyte proliferative activity that will reveal cell cycle progression, discriminate between cycle progression, DNA replication, and proliferation, and provide important insight for enhancing cardiomyocyte proliferation in the context of adult myocardial tissue.

Keywords: Cardiomyocyte; Cell-cycle; FUCCI; Myocardial infarct; Regeneration.

Fig. 1.. Markers of division and cell-cycle status.

FUCCI fluorescence mKO (red) presents in G1 phase, and AzG (green) presents during S/G2/M phases, where during the G1/S transition both fluorescence (mKO/AzG) present simultaneously and merge into a yellow color. BrdU or Edu, both thymidine analogs incorporate into DNA during synthesis (cyan). Phosphorylated Histone 3 (pHH3) is responsible for chromatin condensation and is thus present during G2 through M phase (magenta). Nuclear antigen Ki67 is present from G1 to M phase (emerald). PCNA is presents between G1 and G2 phase in response to DNA synthesis (burgundy). Anillin plays a role in creating the cleavage furrow formation and begin to accumulate in late G2 through late M phase (blue). Aurora B plays a role in mitosis, present from G2 through M phase (sand).

Fig. 2.. FUCCI-Tg expression is specific to cardiomyocytes.

(A) Schematic of transgenic mouse production, n=250 embryos injected, n=28 pups screened for transgene integration, n=1 founder line established. (B) PCR analysis confirm transgene integration in genomic DNA. (C) Immunoblot analysis of founder organs demonstrate cardiac specificity of transgenes. (D-E) Representative images of isolated P2 (D) and P90 (E) mouse cardiomyocytes express FUCCI fluorescence of AzG (green, D’, E’), mKO (red, D”, E”), scale bar 100μm. (F-G) Representative images of nuclear AzG (green) and mKO (red) native fluorescence visualized in α-sarcomeric actinin (αSA, blue) positive P2 (F) and P90 (G) isolated cardiomyocytes, respectively. Scale bar 20μm

Fig. 3.. Postnatal cardiomyocyte cell cycle progression transitions toward arrest at G1/S within days after birth.

(A) Schematic of developmental time-point isolation, single BrdU injection (150mg/kg) given 2 hours prior to harvest. (B-F) Merged representative confocal images showing AzG and mKO expression in cardiomyocytes during postnatal development visualized by immunostaining and confocal microscopy in cardiac tissue sections at P2 (B), P7 (C), P14 (D), P21 (E), and P90 (F); AzG (green, B’-F’), mKO (red, B”-F”), pHH3 (magenta, B”‘-F”‘), BrdU (cyan, B”‘-F”‘), Sytox (white, B”“-F”“) and cardiac Troponin I (blue, B”“-F”“), Scale bar 10μm, n=3–5 hearts per time point. (G) Quantification of percent of BrdU⁺ cardiomyocytes in all cardiomyocytes counted peaks at P2 and decreases thereafter, * P <0.05 vs. P0. (H) Quantification of percent of cardiomyocyte nuclei in different cell cycle phases shows G0(mKO⁻/AzG⁻), G1(mKO⁺) and S/G2/M (AzG⁺). G0: φP<0.05, φφP<0.001, φφφP<0.0001 vs. P0; *P<0.05, ***P<0.001 vs. P7, δP<0.05 vs. P21. G1: σP<0.05 vs. P7, ΔP<0.05 vs. P0. ### P<0.0001 vs. P2. S/G2/M: ψP<0.05 vs. P7, &P<0.001 vs. P0, $$$ P<0.0001 vs. P2. (I) Quantification of percent of cardiomyocyte nuclei in G1/S transition of the cell cycle, ***P<0.0001 vs. P2. (J) Representative immunoblot of whole heart lysates indicate AzG/mKOA/Vinculin protein expression from P2 to P90 show increased protein expression with age. (K) Quantitation of AzG (left) and mKO (right) protein expression relative to loading Vinculin vs P2. *P<0.05, **P<0.001, ***P<0.0001 vs P2. n = 4055 (P0), 1953 (P2), 7536 (P7), 2861 (P14), 4765 (P21), 1873 (P90) CM nuclei from 6 hearts per time point. One-way ANOVA, Tukey’s post hoc test.

Fig. 4.. Border zone cardiomyocytes exhibit signs of cell cycle re-entry but fail to show new cardiomyocyte formation.

(A) Schematic timeline showing MI and daily BrdU pulse (50mg/kg). (B, D) Representative confocal images of sham tissue sections at 14 and 21 days post operation. (C) Border zone (BZ) cardiomyocytes at 14dpi show AzG expression (yellow arrowhead); BrdU incorporation is restricted to interstitial population. (E) BZ cardiomyocytes at 21dpi show mKO⁺/AzG⁺/BrdU⁺ (yellow arrowhead) and mKO⁺/BrdU⁺ (open arrowhead). Native fluorescence AzG (green, C’, E’), mKO (red, C’, E’), and immunolabeled for cardiac Troponin I (blue, C”, E”), BrdU (magenta, C’, E’), and Sytox (white, C”, E”) in (B-E) scale bar 20μm. (F) Quantification of percent BrdU⁺ cardiomyocytes in all cardiomyocytes counted, n = 4 BrdU⁺CM in 1,802 CM at 14dpi, n=85 BrdU⁺CM in 6,705 CM at 21 dpi. (G) Percent CM nuclei at GO, G1, S/G2/M phase in tissue sections at 7, 10, 14 and 21dpi. G0: ##P<0.001, ###P<0.0001 vs. P90. S/G2/M: *P<0.05, **P<0.001 vs. P90. (H) Percent CM nuclei at G1/S interface; *P<0.05, **P<0.001, ***P<0.0001 vs. P90. n=3351 (P90), 2116 (sham), 987 (7dpi), 936 (10dpi), 1299 (14dpi), 1235 (21dpi) CM nuclei counted from 9 sections along IZ/BZ for each heart. One-way ANOVA, Tukey’s post hoc test.

Fig. 5.. Amplifying progenitors express AzG in vivo.

Representative confocal images of early postnatal development tissue sections P2 (A) and P7 (B, C) respectively, showing native fluorescence of AzG (green, A’”, B”), mKO (red, A”-C”), and immunolabeling for cKit (cyan, A”, B’”, C’”), pHH3 (magenta, A”‘-C”‘), sytox (white, A”“-C”“) and tropomyosin (blue, A”“-C”“), scale bar 50μm in A, B, C; 10μm in A’-C’. Yellow arrow shows cKit⁺/AzG⁺/pHH3⁺ amplifying cardiac progenitor in P2 cardiac sections (A), and AzG⁺/cTnl⁺ immature cardiomyocyte in P7 section (C).