Regenerative potential of epicardium-derived extracellular vesicles mediated by conserved miRNA transfer

Abstract

Aims After a myocardial infarction, the adult human heart lacks sufficient regenerative capacity to restore lost tissue, leading to heart failure progression. Finding novel ways to reprogram adult cardiomyocytes into a regenerative state is a major therapeutic goal. The epicardium, the outermost layer of the heart, contributes cardiovascular cell types to the forming heart and is a source of trophic signals to promote heart muscle growth during embryonic development. The epicardium is also essential for heart regeneration in zebrafish and neonatal mice and can be reactivated after injury in adult hearts to improve outcome. A recently identified mechanism of cell-cell communication and signalling is that mediated by extracellular vesicles (EVs). Here, we aimed to investigate epicardial signalling via EV release in response to cardiac injury and as a means to optimise cardiac repair and regeneration. Methods and results We isolated epicardial EVs from mouse and human sources and targeted the cardiomyocyte population. Epicardial EVs enhanced proliferation in H9C2 cells and in primary neonatal murine cardiomyocytes in vitro and promoted cell cycle re-entry when injected into the injured area of infarcted neonatal hearts. These EVs also enhanced regeneration in cryo-injured engineered human myocardium (EHM) as a novel model of human myocardial injury. Deep RNA-sequencing of epicardial EV cargo revealed conserved microRNAs (miRs) between human and mouse-epicardial-derived exosomes, and the effects on cell cycle re-entry were recapitulated by administration of cargo miR-30a, miR-100, miR-27a and miR-30e to human stem cell-derived cardiomyocytes and cryo-injured EHM constructs. Conclusion Here we describe the first characterisation of epicardial EV secretion, which can signal to promote proliferation of cardiomyocytes in infarcted mouse hearts and in a human model of myocardial injury, resulting in enhanced contractile function. Analysis of exosome cargo in mouse and human identified conserved pro-regenerative microRNAs which in combination recapitulated the therapeutic effects of promoting cardiomyocyte proliferation. Translational perspective Coronary artery disease leading to myocardial infarction (“heart attack”) is a major cause of death worldwide . After a heart attack, the myocardium is unable to regenerate, therefore, the heart remains permanently scarred and unable to carry out its function, which eventually leads to heart failure. Current therapies are palliative, aimed at unburdening the failing heart, or heart transplantation. Reprogramming adult cardiomyocytes to a proliferative state, via the use of non-inmunogenic EVs is a potential therapeutic approach to restore lost heart muscle, resolve scarring and prevent heart failure progression.

A New Role for Extracellular Vesicles in Cardiac Tissue Engineering and Regenerative Medicine

“…Untreated tissues still showed some increase in CM proliferation 3 days after injury, but contractile force never recovered and continued to decline through 7 days. [ 72 ] These data supported the findings of previous experiments performed in vivo and in 2D cultures. Additionally, viral transfection of EHM to overexpress miR‐30a or miR‐100 both reproduced the same 7‐day postinjury recovery of force that was observed for EPDC EV treatment, supporting the suggestion that these miRNA species had an important mechanistic role in the therapeutic efficacy of epicardial EVs.…”

“…[ 11 ] Another study by Villa del Campo et al found that epicardial EVs enhanced cell cycle re‐entry as a mechanism of functional recovery in cryoinjured in vitro cardiac tissues, mediated by the action of miR‐30a, miR‐100, miR‐27a, and miR‐30e. [ 72 ]…”

“…In the most recent study published in 2021, Villa del Campo et al applied a model of cryoinjured engineered heart tissue as part of their study on the effects and mechanisms of epicardial EVs on cardiac functional recovery postinjury. [ 72 ] Using a circular engineered human myocardium (EHM) model suspended around flexible pillars that was pioneered by the Zimmerman lab, cardiac injury was induced via treatment with liquid nitrogen. EVs isolated from primary human epicardium‐derived cell (EPDC) cultures via ultracentrifugation were applied to EHM tissue cultures postinjury.…”

“…EVs isolated from primary human epicardium‐derived cell (EPDC) cultures via ultracentrifugation were applied to EHM tissue cultures postinjury. [ 72 ] It was observed that EV‐treated tissues expressed an increase in CM cell cycle re‐entry and proliferation 3 days after treatment, which manifested as a complete recovery of contractile force to preinjury levels after 7 days. Untreated tissues still showed some increase in CM proliferation 3 days after injury, but contractile force never recovered and continued to decline through 7 days.…”

Mesenchymal stem cells from biology to therapy

“…EVs are nanoscale, membranous particles secreted from cells, containing diverse cargo including nucleic acids, such as miRNA, and proteins. It has been shown that EVs mediate cell-to-cell communication by shuttling biomolecules to influence the microenvironment [67][68][69][70]. Given that the function of EVs is to act as signalling particles for surrounding cells, it follows that the signals carried by the EVs could be harnessed to deliver desirable biological factors to target cells, affording them innate therapeutic utility (Figure 2).…”