Manipulating Senescence of Cardiac Fibroblasts In Vivo and In Vitro to Investigate Arrhythmogenic Effect of Senescence

Abstract

Cellular senescence is a stress response characterized by irreversible cell cycle arrest, secretion of inflammatory cytokines and growth factors termed the “senescence associated secretory phenotype” (SASP), and resistance to apoptosis. Senescence plays beneficial roles in tumor suppression, wound healing, and development in young animals, but is thought to underlie aging‐associated pathology in numerous tissues, mainly via the SASP. Our previous work in the aged rabbit myocardial infarction (MI) model demonstrated an increase and persistence of senescence, which we hypothesize to drive increased risk of arrhythmogenesis with age post‐MI. Here, we investigate the molecular mechanisms by which we believe senescence drives arrhythmogenesis. Specifically, we hypothesize that inflammatory cytokines in the SASP locally produced by senescent myofibroblasts acts on border zone myocytes to disrupt cellular‐level electrophysiology. Concordantly, we hypothesize that genetically or pharmacologically eliminating senescent cells post‐MI will mitigate arrhythmogenesis in vivo and in vitro, and that genetically increasing senescence post‐MI in a mouse Cre‐Lox model will increase arrhythmogenesis.Here, we show that commercially‐available aged human cardiac fibroblasts and primary adult rabbit cardiac fibroblasts can be induced into senescence via treatment with low doses of Etoposide in a time‐dependent manner. Additionally, via a senolytic drug screening, we found that high doses of Dasatinib, Quercetin, and Navitoclax effectively eliminate senescent cells but not non‐senescent cells. However, we found combinations of Dasatinib and Quercetin or Dasatinib and Venetoclax did not perform better than either drug in solo. Additionally, we verify that calcium linescan imaging as well as patch clamp measurements of the IKr current can be measured in primary 3‐week‐old rabbit cardiomyocytes.Using genetically altered mice to conditionally knock out the pro‐senescence p53 gene in myofibroblasts under control of the Periostin gene (p53 CKO mice), we find that p53 CKO mice have significantly less senescence post‐MI compared to littermate controls, and the effect is stronger in females compared to males. We also report that fibroblast‐specific knockout of the MDM2 gene in the same condition is embryonic lethal, as Periostin is expressed in the developing valve leaflets and endocardial cushion cells, where senescence plays a vital role in pruning transient fetal structures.In conclusion, we demonstrate methods to genetically and pharmacologically manipulate senescence in vivo and in vitro, which will allow us to investigate the molecular mechanisms through which senescence might play a role in promoting arrhythmogenesis with age post‐MI.Support or Funding InformationThe authors would like to acknowledge our supporting NIH funding sources, R21 grant 1R21AG049608‐01 and R01 grant R01HL139467