CCN1-Induced Cellular Senescence Promotes Heart Regeneration

◼ regeneration C ellular senescence plays important roles in a variety of physiological and pathological processes. 1However, whether it is associated with neonatal heart regeneration 2 remains unknown.We performed apical resection (AR) at postnatal day (P) 1 and collected hearts for senescence-associated β-galactosidase staining at different time points after AR (Figure [A]).Senescent cells (SCs) were identified in the periresected regions at P3, P7, and P14, whereas no SCs were detected in the fully restored hearts at P21 (Figure [A]).Because β-galactosidase is not exclusively expressed in SCs, we also collected apices and periresected regions from sham and injured hearts, respectively, at P7 and examined the mRNA levels of Trp53, Cdkn1a, p16INK4a, and several genes associated with the senescence-associated secretory phenotype including IL-1a, IL-6, Ccl2, PAI1, Vegfc, and Mmp2.All these genes were upregulated in the periresected regions at P7 (data not shown).Taken together, AR induced senescence in neonatal hearts, and these SCs disappeared when the hearts were fully restored.To investigate the role of SCs in neonatal heart regeneration, we administered neonatal mice with the senolytic drug ABT263 after AR (Figure [B]). 3The ABT263-treated hearts had fewer SCs in the periresected regions at P8 and were not fully restored at P22, showing evidence of fibrotic scars (Figure [C and D]).Immunostaining revealed that the percentage of proliferating cardiomyocytes was decreased and fibroblast proliferation was increased in the periresected regions of ABT263-treated hearts at P8 (Figure [E and F] and data not shown).Next, we performed AR on Trp53 knockout mice that are deficient in SCs.The Trp53 -/-hearts had fewer SCs in the periresected regions at P7 and were not fully regenerated at P22, showing apparent scars (Figure [G and H]).Cardiomyocyte proliferation was impaired and fibroblast expansion was enhanced in the periresected regions of Trp53 -/-hearts at P8 (Figure [I and J] and data not shown).Thus, clearance of SCs by ABT263 or inactivation of senescence by Trp53 knockout significantly inhibited neonatal heart regeneration after AR, indicating critical roles of senescence in neonatal heart regeneration.Activated fibroblasts (myofibroblasts) highly express periostin (POSTN).We used Postn-CreER; R26-loxp-stop-loxp-tdTomato (R26-tdTomato) to label myofibroblasts after AR and found CDKN1A + myofibroblasts in the periresected regions at P8 (Figure [K]).Senescence-associated β-galactosidase activity was also detected in PDGFRa + fibroblasts isolated from AR-treated hearts (data not shown).These results suggest that some fibroblasts are senescent post-AR.Therefore, we crossed Postn-CreER;Trp53 fl/+ with Trp53 fl/fl , performed AR at P1, and injected tamoxifen at P3 and P6 to delete Trp53 in myofibroblasts.The mutant hearts had fewer SCs at P7 and were not fully restored at P22 (Figure [L and M]).Cardiomyocyte proliferation was inhibited, and fibroblast proliferation was increased in the periresected regions of mutants at P8 (Figure [N and O] and data not shown).