Inhibition of Senescence-Associated Genes Rb1 and Meis2 in Adult Cardiomyocytes Results in Cell Cycle Reentry and Cardiac Repair Post-Myocardial Infarction

Abstract

Background Myocardial infarction results in a large-scale cardiomyocyte loss and heart failure due to subsequent pathological remodeling. Whereas zebrafish and neonatal mice have evident cardiomyocyte expansion following injury, adult mammalian cardiomyocytes are principally nonproliferative. Despite historical presumptions of stem cell-mediated cardiac regeneration, numerous recent studies using advanced lineage-tracing methods demonstrated that the only source of cardiomyocyte renewal originates from the extant myocardium; thus, the augmented proliferation of preexisting adult cardiomyocytes remains a leading therapeutic approach toward cardiac regeneration. In the present study we investigate the significance of suppressing cell cycle inhibitors Rb1 and Meis2 to promote adult cardiomyocyte reentry to the cell cycle. Methods and Results In vitro experiments with small interfering RNA-mediated simultaneous knockdown of Rb1 and Meis2 in both adult rat cardiomyocytes, isolated from 12-week-old Fischer rats, and human induced pluripotent stem cell-derived cardiomyocytes showed a significant increase in cell number, a decrease in cell size, and an increase in mononucleated cardiomyocytes. In vivo, a hydrogel-based delivery method for small interfering RNA-mediated silencing of Rb1 and Meis2 is utilized following myocardial infarction. Immunofluorescent imaging analysis revealed a significant increase in proliferation markers 5-ethynyl-2'-deoxyuridine, PH3, KI67, and Aurora B in adult cardiomyocytes as well as improved cell survivability with the additional benefit of enhanced peri-infarct angiogenesis. Together, this intervention resulted in a reduced infarct size and improved cardiac function post-myocardial infarction. Conclusions Silencing of senescence-inducing pathways in adult cardiomyocytes via inhibition of Rb1 and Meis2 results in marked cardiomyocyte proliferation and increased protection of cardiac function in the setting of ischemic injury.

Keywords: adult cardiomyocytes; angiogenesis; cardioprotection; cardiovascular disease; induced cell cycle reentry; microRNA; myocardial infarction.

Figure 1

Simultaneous inhibition of Rb1 and Meis2 leads to ACM proliferation. A, The expression analysis of Rb1 and Meis2 was performed through Western blot showing a significant increase in the expression of Rb1 and Meis2 in ACM when compared with NRCM. N=3 rats per group. B, Schematic representation of in vitro experimental design, illustrating ACM isolation, transfection with Cel‐miR‐67 (control) and siRNA‐cocktail on day 1, followed by ACM proliferation and survival assays on day 7. C, Representative immunoblots for Rb1 and Meis2. Immunoblotting was performed with cell lysate from ACMs, transfected with siRNA‐cocktail and control. D and E, Densitometric analysis showed regulation in the expression of cell cycle regulators in the siRNA‐cocktail–transfected group in comparison to control. F and G, Representative immunostained images of ACMs showing an increase in cell number in the siRNA‐cocktail–transfected group on day 7 after transfection vs control. H, Quantification of ACM size shows a significant decrease in ACM size in the siRNA‐cocktail–transfected group vs control. Immunostaining shows a significant increase in (I and J) EdU‐positive ACMs, (K and L) KI67‐positive ACMs, (M and N) PH3‐positive ACMs, and (O and P) Aurora B–positive ACMs in siRNA‐cocktail–transfected groups vs control. We used ACMs, isolated from adult rats (≈12 weeks old) for all in vitro experiments. N=3 rats, n=8 experimental replicates each, and n=3 images each. Scale bar=100 μm or as cited in the image. Panels in White rectangles represent respective enlarged sections. Yellow arrows indicate the ACMs labeled with the indicated proliferative markers. ACM indicates adult cardiomyocyte; DAPI, 4′,6‐diamidino‐2‐phenylindole; EdU, 5‐ethynyl‐2′‐deoxyuridine; NRCM, neonatal rat cardiomyocyte; PH3, phosphor histone 3; siRNA, short interfering RNA; TnI, Troponin I. Results are presented as mean±SEM; *P≤0.05. P≤0.05 was considered statistically significant.

Figure 2

Simultaneous inhibition of Rb1 and Meis2 leads to hiPSc‐CM proliferation. Representative immunostained images of hiPSc‐CM showing a significant increase in (A and B) EdU‐positive hiPSc‐CM, (C and D) KI67‐positive hiPSc‐CM, and (E and F) Aurora B positive hiPSc‐CM in the siRNA‐cocktail–transfected group on day 5 after transfection vs control. Scale bar=100 μm. Panels in dashed rectangles represent respective enlarged sections. n=8 experimental replicates per group, and n=3 images each. Arb indicates Aurora B; DAPI, 4′,6‐diamidino‐2‐phenylindole; EdU, 5‐ethynyl‐2′‐deoxyuridine; hiPSc‐CM, human induced pluripotent stem cell–derived cardiomyocytes; SEM, standard error of the mean; siRNA, short interfering RNA; TnI, Troponin I. Results are presented as mean±SEM; *P≤0.05. P≤0.05 was considered statistically significant.

Figure 3

Hydrogel‐mediated delivery of siRNA‐cocktail leads to ACM proliferation in adult hearts post‐MI. A, Schematic representation of in vivo experimental design, illustrating LAD ligation, intramyocardial injection of hydrogel, and hydrogel+siRNA‐cocktail. Animals were observed for 21 days; echocardiography was performed at day 3 and day 21, followed by euthanasia and sample collection for molecular and histochemical analysis. B, Representative images for WGA staining (green) and troponin I (red) immunostaining. C, Quantification showing a moderate but significant decrease in ACM size in the siRNA‐cocktail–transfected group when compared with controls. N=4 rats per group, n=5 nonserial sections were imaged from each, and n>100 cardiomyocytes quantified from each confocal image. D, Representative immunostaining images for TnI (green), KI67 (red), and DAPI (blue). E, Bar graphs show a significant increase in KI67‐positive ACMs in the siRNA‐cocktail–transfected group vs control. F and G, Representative images and bar graphs for immunostaining with Aurora B, showing a significant increase in Aurora B–positive ACMs in the siRNA‐cocktail–transfected group vs control. In vivo analysis was performed in adult male rats (≈12 weeks old). N=4 rats per group, n=5 nonserial sections were imaged each, and n=8 confocal images from different regions quantified each. Scale bar=50 μm. ACM indicates adult cardiomyocyte; DAPI, 4′,6‐diamidino‐2‐phenylindole; LAD, left anterior descending coronary artery; MI, myocardial infarction; si, short interfering [RNA]; TnI, Troponin I; WGA, wheat germ agglutinin. Results are presented as mean±SEM; *P≤0.05. P≤0.05 was considered as statistically significant.

Figure 4

Hydrogel‐mediated delivery of siRNA‐cocktail leads to increased vascularization in adult animals post‐MI. A, Representative immunostaining images for blood vessel density in different study groups: the inner endothelial layer is visualized by von Willebrand factor (vWF) staining (red), whereas the outer layer is visualized by smooth muscle actin (SMA) staining (green). B, Quantification of blood vessel density shows a significant increase in the siRNA‐cocktail–treated group when compared with controls. N=4 rats per group, n=5 nonserial sections were imaged from each, and n=3 confocal images from different regions quantified each. C, Schematic representation of tube formation assay, illustrating ACM transfection and conditioned medium (CM) isolation, which was used to culture HUVECs and the subsequent tube formation assay. D, Representative bright‐field images for endothelial tube formation assay in HUVECs illustrate the increase in total number of tubes, total tube length, and number of branching points in the siRNA‐cocktail group vs control. E‐G, Quantification of endothelial tube formation was performed by Wimasis image analysis tool (2017; WimTube: Tube Formation Assay Image Analysis Solution, Release 4.0; Onimagin Technologies, Córdoba, Spain). Significant increases in total number of tubes, total tube length, and number of branching points were observed. Images represent N=3 rats, n=6 experimental replicates each, and n=5 images quantified each. Scale bar=100 μm. ACM indicates adult cardiomyocytes; HUVEC, human umbilical vein endothelial cells; siRNA, short interfering RNA; SMA, α‐smooth muscle actin; vWF, von Willebrand factor. Results are presented as mean±SEM; *P≤0.05. P≤0.05 was considered statistically significant.

Figure 5

Hydrogel‐mediated delivery of siRNA‐cocktail leads to a reduction in infarct size and improved cardiac function post‐MI. A, No significant difference was observed in heart weight–to–body weight ratio between the groups. B and C, Representative images for Masson trichrome staining show a significant reduction in infarct size after siRNA‐cocktail treatment in adult animals post‐MI. N=6 rats per group; n=5 nonserial sections were imaged each. D through F, Representative echocardiography images of heart function analysis at the long axis and M‐mode. G through L, Quantification of echocardiography shows significantly improved EF, CO, LVIDs, radial strain, radial strain rate, and radial displacement in the siRNA‐cocktail–treated group in comparison to controls. In vivo analysis of cardiac function was performed in adult male rats (≈12 weeks old). Results represent N=6 rats per group. BW indicates body weight; CO, cardiac output; EF, ejection fraction; HW, heart weight; LVIDd, left ventricular internal dimension, diastole (mm); LVIDs, left ventricular internal dimension, systole (mm); NS, nonsignificant; siRNA, short interfering RNA. Results are presented as mean±SEM; *P≤0.05. NS=P≥0.05. P≤0.05 was considered as statistically significant.

Figure 6

Simultaneous inhibition of Rb1 and Meis2 leads to ACM cell cycle progression and cardioprotection post‐MI. A, Schematic representation of siRNA‐mediated simultaneous inhibition of Rb1 and Meis2, showing induced entry to S phase by siRb1‐mediated silencing Rb1. Mies2 inhibition facilitates mitosis, which collectively leads to induced ACM proliferation. The differential expression of Aurora B, β‐catenin, cyclin D1, and p16 are complementary to the induced ACM proliferation after Rb1 and Meis2 inhibition. B, The cascade of events that occur when an ischemic injury to the heart that leads to heart failure is significantly reversed or prevented by an Rb1 and Meis2 knockdown approach. More significant than ACM proliferation, protection against cardiomyocyte death and cardiomyocyte‐mediated enhanced angiogenesis protect the heart from progressive myocardial loss, reduce fibrosis, and restore function in an adult animal post‐MI. ACM indicates adult cardiomyocytes; MI, myocardial infarction; si‐, short interfering [RNA].