Abstract We010: In Vivo Partial Reprogramming of Cardiomyocytes to a Molecularly Rejuvenated State Ameliorates Cardiac Failure

Abstract

Introduction: i n vivo partial cell reprogramming through transient overexpression of Oct3/4 , Klf4 , Sox2 and cMyc (OKSM) transcription factors generates de-differentiated or molecularly rejuvenated cells that may contribute to tissue regeneration. However, not all cell types are equally amenable to reprogramming, and tools to induce cell type-specific reprogramming in vivo are scarce. Here, we generated cardiomyocyte-specific, “reprogrammable” mice (CM-OKSM) that enabled inducible OKSM expression specifically in this cell type, characterized by its remarkably poor regenerative capacity after birth. Using this model, we investigated if partial, in situ , cardiomyocyte reprogramming was feasible and able to enhance cardiac regeneration. Methods: To generate lineage-traceable CM-OKSM mice, we combined Cre recombination driven by a cardiomyocyte-specific promoter ( Myh6 ), conditional GFP labelling and conditional, doxycycline (dox)-inducible, control of OKSM expression. We administered 1 mg/ml dox in the drinking water of CM-OKSM mice, and investigated reprogramming at the molecular level (immunostaining, RT-qPCR, RNAseq, DNA methylation), histological level (H&E, electron microscopy) and functional level (teratoma assay, echocardiography). We compared dox administration for six cycles of a two-day ON/five-day OFF regime, and for eighteen continuous days, to investigate partial and full reprogramming to pluripotency, respectively. We conducted studies in young adult mice (8 to 12 weeks old) and on older mice (9 months old) with heart failure, to assess the effects of cardiomyocyte reprogramming in cardiac performance. Results and Discussion: In young CM-OKSM mice, adult cardiomyocytes can be fully reprogrammed to pluripotency in vivo by eighteen-day overexpression of OKSM. This was evidenced by significant upregulation of endogenous pluripotency markers, loss of sarcomere organization, cell de-differentiation, and the appearance of GFP+ teratomas with trilineage contribution. We then found that cyclic OKSM induction prevented reacquisition of pluripotency and tumorigenesis, both in young and old mice. In the latter, partial reprogramming of cardiomyocytes induced significant rejuvenation of their epigenetic age and halted the progression of congestive heart failure. Our results confirm that the OKSM cocktail can reprogram adult mouse cardiomyocytes in vivo and that partial reprogramming can contribute to functional improvements in cardiac disease.