FUCCI-Based Live Imaging Platform Reveals Cell Cycle Dynamics and Identifies Pro-proliferative Compounds in Human iPSC-Derived Cardiomyocytes

Abstract

One of the major goals in cardiac regeneration research is to replace lost ventricular tissue with new cardiomyocytes. However, cardiomyocyte proliferation drops to low levels in neonatal hearts and is no longer efficient in compensating for the loss of functional myocardium in heart disease. We generated a human induced pluripotent stem cell (iPSC)-derived cardiomyocyte-specific cell cycle indicator system (TNNT2-FUCCI) to characterize regular and aberrant cardiomyocyte cycle dynamics. We visualized cell cycle progression in TNNT2-FUCCI and found G2 cycle arrest in endoreplicating cardiomyocytes. Moreover, we devised a live-cell compound screening platform to identify pro-proliferative drug candidates. We found that the alpha-adrenergic receptor agonist clonidine induced cardiomyocyte proliferation in vitro and increased cardiomyocyte cell cycle entry in neonatal mice. In conclusion, the TNNT2-FUCCI system is a versatile tool to characterize cardiomyocyte cell cycle dynamics and identify pro-proliferative candidates with regenerative potential in the mammalian heart.

Keywords: cardiomyocyte; cell cycle activity; cell cycle indicator; clonidine; fluorescence ubiquitination cell cycle indicator; induced pluripotent stem cell; polyploidy.

Figure 1

Generation and validation of TNNT2-FUCCI in hiPSC-derived CMs. (A) Schematic representation showing TNNT2-FUCCI reporter cell line generation and differentiation towards cardiomyocytes. Upon differentiation into cardiomyocytes, the FUCCI signal became visible at Day 6. Arrowheads indicate FUCCI⁺ nuclei. Scale bar, 20 μm. (B) Immunocytochemistry showing cardiomyocytes at Day 30 of differentiation. Arrowheads indicate cardiomyocytes (TNNT2⁺) that were FUCCI⁺, demonstrating the specificity of the FUCCI reporter. Scale bar, 20 μm. (C) Imaging flow cytometry shows TNNT2⁺ cells expressing nuclear mVenus and/or mCherry, indicating cardiomyocyte specificity of the TNNT2-FUCCI system.

Figure 2

Quantification of cell cycle duration using live cell imaging of hiPSC-derived CMs. (A) Pie chart showing the percentage of non-cycling and cycling cardiomyocytes (n = 570) collected from 20 movies. (B–D) left panels show oscillation patterns of FUCCI fluorescence measured by live imaging in cardiomyocytes undergoing (B) cell division (n = 9), (C) multinucleation (n = 9), and (D) polyploidization (n = 5). Data show mean intensity ± SEM. Right panels show images of selected intervals of the movies. (E) Quantification of the total duration of the combined S, G2, and M phases shows a significant increase in cell cycle duration in cardiomyocytes undergoing polyploidization (n = 5) compared to multinucleation (n = 9) and polyploidization (n = 9). Data show mean intensity ± SEM. P-value was determined by one-way ANOVA. **p < 0.01, ***p < 0.001.

Figure 3

Live-image-based TNNT2-FUCCI hiPSC-derived cardiomyocyte screen identifies compounds that induce cell cycle activity. (A) Schematic drawing of the screening experiment. hiPSC-derived CMs were seeded in 96-well plates and stimulated with 94 compounds of an autophagy-related compound library at a concentration of 25 μM. FUCCI signal was documented at 24 h, 48 h, and 72 h. (B) The effect of each compound is shown as the percentage of mVenus⁺ nuclei relative to the control at the 48 h time point. Results are from one screen with three replicate wells on separate plates. Error bars represent STDEV between the triplicates. Six compounds that increased the percentage of mVenus⁺ cardiomyocyte nuclei were subjected to further validation, these are indicated with colors. (C) Six compounds were further tested in mNCMs at three different concentrations (2.5 nM, 250 nM and 25 μM). Cell cycle activity was validated by EdU incorporation and subsequent detection at 72 h. The experiment was repeated three times, each with three replicates wells. Values are mean ± SEM. P-values were determined by one-way ANOVA. *p < 0.05, ***p < 0.001, ****p < 0.0001. (D) Representative immunocytochemistry images of untreated and clonidine-treated mNCMs with EdU incorporation detected in magenta and cardiac troponin I in green. Scale bar, 20 μm.

Figure 4

Effect of clonidine on cell cycle activity and proliferation in human and mouse cardiomyocytes (A) Quantification of Ki-67⁺ hiPSC-derived CMs after 72 h of clonidine treatment. P-values were determined by unpaired t-test. Datapoints represent four individual wells from one experiment. (B,C) Cell cycle activity induced by clonidine did not show any changes in (B) nuclear ploidy or (C) binucleation. Datapoints represent four individual wells with 11 fields of view analyzed per well. P-values were determined by two-way ANOVA. (D,E) AurKB⁺ midbodies, indicative of late phase cytokinesis, increased significantly in clonidine-treated hiPSC-derived CMs. Datapoint represent three individual wells. Scale bar, 20 μm. P-values were determined by unpaired t-test. (F) Clonidine significantly increased the percentage of Ki-67⁺ mNCMs. Datapoints represent six individual wells, P-values were determined by unpaired t-test. (G) Quantification showed an increase in nuclear ploidy in mNCMs treated with clonidine. Datapoints represent six individual wells. P-values were determined by two-way ANOVA. (H) Ratios of binucleated mNCMs were not altered by clonidine treatment. P-values were determined by two-way ANOVA. Values are mean ± SEM. *P < 0.05, **p < 0.01, ***p < 0.001. Datapoints represent nine wells with three fields of view analyzed for each well.

Figure 5

Clonidine induces cell cycle activity in neonatal mice. (A) Neonatal mice were subjected to four days (P1-P5) of clonidine (60 ng/pup) and EdU treatment (20mg/kg). Pups were sacrificed and hearts were collected at P7. (B,C) Quantification of EdU⁺ cardiomyocyte (PCM-1⁺) nuclei in sectioned mouse hearts showed an increase in EdU incorporation in clonidine-treated neonatal mice. Three sites per heart section were imaged and a minimum of 100 cardiomyocytes nuclei were analyzed per sample. Each point represents a biological replicate, clonidine n = 8, untreated n = 4. Values are mean ± SEM. P-value was determined by unpaired t-test. *P < 0.05. Scale bar, 50 μm. (D) Shows compilation of individual representative images of digested P7 cardiomyocytes stained with α-actinin and connexin-43. Scale bar, 20 μm. (E) Quantification of mono and binucleated cardiomyocytes with clonidine treatment (n = 8) and untreated (n = 3). At least 100 cardiomyocytes per biological replicate were analyzed. Values are mean ± SEM. P-value was determined by unpaired t-test. *P < 0.05. (F) Quantification of nuclear ploidy on cardiomyocytes with clonidine treatment (n = 8) and untreated (n = 4) shows no significant difference. At least 422 cardiomyocyte nuclei were analyzed per mouse.