Induction of Native Cardiomyocyte Cell Cycle Re-Entry by Transplanted Cells as a Novel Mechanism of Therapeutic Regeneration in the Heart

Abstract

BackgroundThe extent of cardiomyocyte (CM) turnover in adult mammals remains unclear, while controversy surrounds the putative mechanisms of CM replenishment.HypothesisUsing a genetic fate mapping approach and objective methods of quantification, we investigated the ability of resident adult CMs to re-enter the cell cycle in the normal, infarcted and cell-treated heart.Methods & ResultsTamoxifen pulsing of bitransgenic MerCreMer-ZEG mice (aMHC promoter driving tamoxifen-inducible Cre), resulted in efficient (∼80%) and specific labeling of CMs by GFP. Mice were then subjected to: a) sham surgery, b) MI, and c) MI followed by cardiosphere-derived cell (CDC) injection. Mice were pulsed with BrdU daily for up to 5 weeks. CMs, isolated by Langendorff enzymatic dissociation, underwent FACS-sorting for GFP (∼98.9% GFP+, with minimal contamination by non-CMs) and subsequent flow cytometric analysis for BrdU and Ki67. The normal adult mouse heart contains a small pool of cycling resident CMs (∼0.08%/week, projecting to a CM turnover of ∼1.3-4%/yr), which roughly doubles during the first 3 weeks after MI. Transplantation of CDCs further dramatically upregulates the percentage of cycling host CMs (to ∼0.7%/week) (Fig 1), while boosting heart function and increasing viable myocardium. Immunocytochemistry of isolated CMs confirmed the flow cytometry results, while PCR microarray analysis showed upregulation of several genes associated with cell-cycle progression in resident CMs post MI and CDC therapy. Cycling CMs are smaller, more mononucleated and primarily located in the border zone. The observed phenomena could not be explained by stem cell differentiation, CM polyploidization, bi/multinucleation, cell fusion or DNA repair. Quantitative estimates indicate that CM proliferation accounts for ∼18% of the salutary effect of CDC therapy.Conclusions BackgroundThe extent of cardiomyocyte (CM) turnover in adult mammals remains unclear, while controversy surrounds the putative mechanisms of CM replenishment. The extent of cardiomyocyte (CM) turnover in adult mammals remains unclear, while controversy surrounds the putative mechanisms of CM replenishment. HypothesisUsing a genetic fate mapping approach and objective methods of quantification, we investigated the ability of resident adult CMs to re-enter the cell cycle in the normal, infarcted and cell-treated heart. Using a genetic fate mapping approach and objective methods of quantification, we investigated the ability of resident adult CMs to re-enter the cell cycle in the normal, infarcted and cell-treated heart. Methods & ResultsTamoxifen pulsing of bitransgenic MerCreMer-ZEG mice (aMHC promoter driving tamoxifen-inducible Cre), resulted in efficient (∼80%) and specific labeling of CMs by GFP. Mice were then subjected to: a) sham surgery, b) MI, and c) MI followed by cardiosphere-derived cell (CDC) injection. Mice were pulsed with BrdU daily for up to 5 weeks. CMs, isolated by Langendorff enzymatic dissociation, underwent FACS-sorting for GFP (∼98.9% GFP+, with minimal contamination by non-CMs) and subsequent flow cytometric analysis for BrdU and Ki67. The normal adult mouse heart contains a small pool of cycling resident CMs (∼0.08%/week, projecting to a CM turnover of ∼1.3-4%/yr), which roughly doubles during the first 3 weeks after MI. Transplantation of CDCs further dramatically upregulates the percentage of cycling host CMs (to ∼0.7%/week) (Fig 1), while boosting heart function and increasing viable myocardium. Immunocytochemistry of isolated CMs confirmed the flow cytometry results, while PCR microarray analysis showed upregulation of several genes associated with cell-cycle progression in resident CMs post MI and CDC therapy. Cycling CMs are smaller, more mononucleated and primarily located in the border zone. The observed phenomena could not be explained by stem cell differentiation, CM polyploidization, bi/multinucleation, cell fusion or DNA repair. Quantitative estimates indicate that CM proliferation accounts for ∼18% of the salutary effect of CDC therapy. Tamoxifen pulsing of bitransgenic MerCreMer-ZEG mice (aMHC promoter driving tamoxifen-inducible Cre), resulted in efficient (∼80%) and specific labeling of CMs by GFP. Mice were then subjected to: a) sham surgery, b) MI, and c) MI followed by cardiosphere-derived cell (CDC) injection. Mice were pulsed with BrdU daily for up to 5 weeks. CMs, isolated by Langendorff enzymatic dissociation, underwent FACS-sorting for GFP (∼98.9% GFP+, with minimal contamination by non-CMs) and subsequent flow cytometric analysis for BrdU and Ki67. The normal adult mouse heart contains a small pool of cycling resident CMs (∼0.08%/week, projecting to a CM turnover of ∼1.3-4%/yr), which roughly doubles during the first 3 weeks after MI. Transplantation of CDCs further dramatically upregulates the percentage of cycling host CMs (to ∼0.7%/week) (Fig 1), while boosting heart function and increasing viable myocardium. Immunocytochemistry of isolated CMs confirmed the flow cytometry results, while PCR microarray analysis showed upregulation of several genes associated with cell-cycle progression in resident CMs post MI and CDC therapy. Cycling CMs are smaller, more mononucleated and primarily located in the border zone. The observed phenomena could not be explained by stem cell differentiation, CM polyploidization, bi/multinucleation, cell fusion or DNA repair. Quantitative estimates indicate that CM proliferation accounts for ∼18% of the salutary effect of CDC therapy. Conclusions