Telomerase/myocardin expressing mesenchymal cells induce survival and cardiovascular markers in cardiac stromal cells undergoing ischaemia/reperfusion

Abstract Cardiac stromal cells (CSCs) contain a pool of cells with supportive and paracrine functions. Various types of mesenchymal stromal cells (MSCs) can influence CSCs in the cardiac niche through their paracrine activity. Ischaemia/reperfusion (I/R) leads to cell death and reduction of the paracrine activity of CSCs. The forced co‐expression of telomerase reverse transcriptase (TERT) and myocardin (MYOCD), known to potentiate anti‐apoptotic, pro‐survival and pro‐angiogenic activities of MSCs isolated from the adipose tissue (AT‐MSCs), may increase CSC survival, favouring their paracrine activities. We aimed at investigating the hypothesis that CSCs feature improved resistance to simulated I/R (SI/R) and increased commitment towards the cardiovascular lineage when preconditioned with conditioned media (CM) or extracellular vesicles (EV) released from AT‐MSCs overexpressing TERT and MYOCD (T/M AT‐MSCs). Murine CSCs were isolated with the cardiosphere (CSps) isolation technique. T/M AT‐MSCs and their secretome improved spontaneous intracellular calcium changes and ryanodine receptor expression in aged CSps. The cytoprotective effect of AT‐MSCs was tested in CSCs subjected to SI/R. SI/R induced cell death as compared to normoxia (28 ± 4 vs 10 ± 3%, P = .02). Pre‐treatment with CM (15 ± 2, P = .02) or with the EV‐enriched fraction (10 ± 1%, P = .02) obtained from mock‐transduced AT‐MSCs in normoxia reduced cell death after SI/R. The effect was more pronounced with CM (7 ± 1%, P = .01) or the EV‐enriched fraction (2 ± 1%, P = .01) obtained from T/M AT‐MSCs subjected to SI/R. In parallel, we observed lower expression of the apoptosis marker cleaved caspase‐3 and higher expression of cardiac and vascular markers eNOS, sarcomeric α‐actinin and cardiac actin. The T/M AT‐MSCs secretome exerts a cytoprotective effect and promotes development of CSCs undergoing SI/R towards a cardiovascular phenotype.


| INTRODUC TI ON
Heart failure (HF), with or without myocardial ischaemia, is a leading cause of death worldwide. 1 HF can be attributed, at least in part, to the limited ability of the heart to repair or regenerate the damaged myocardium. 2 7 The crosstalk between cardiomyocytes and CSCs plays a fundamental role for repair processes after cardiac damage through the release of growth factors, pro-angiogenic factors and the regulation of cardiac metabolism. [8][9][10] Exposure to ischaemia and reperfusion (I/R) accelerates apoptosis and necrosis of cardiomyocytes and CSCs, hampering their crosstalk and repairing functions, and ultimately favours the development of HF. [11][12][13] Cardiac microenvironments housing CSCs, known as cardiac niches, provide CSCs with regulatory signals, including oxygen tension, essential for their maintenance, proliferation and differentiation. 14 A number of studies have recently shown that mesenchymal stromal cells (MSCs) of different origins can influence cardiac niche microenvironment through their paracrine activity. 15,16 The combination of CSCs and MSCs (the so-called combo-approach) is an example of a multipronged cell therapy attempt to cardiac repair. 2,3,17,18 By virtue of their intense paracrine activities, MSCs can influence the phenotype and paracrine activity of CSCs. A pool of such MSCs with paracrine activity has been described in the adipose tissue (AT). 19,20 AT-MSCs thus represent an interesting source of cells for cardiac repair, being able to improve heart function in the infarcted area mainly through paracrine action and soluble factors. 3 As a result, the two cell populations-AT-MSCs and CSCsmight cooperate if combined, 21 establishing an interaction mediated by their respective microenvironments, thus providing relevant biological advantages towards cardiac repair. Two proteins that may promote cell survival are telomerase reverse transcriptase (TERT), an antisenescence protein, 10,22 and myocardin (MYOCD), a promyogenic transcription factor with anti-apoptotic and pro-angiogenic activities. 11 The forced co-expression of TERT and MYOCD, known to potentiate anti-apoptotic, pro-survival and pro-angiogenic activities in AT-MSCs, 23  reported below is provided in the Appendix S1.

| Isolation and culture of adipose tissue-derived mesenchymal stromal cells
Twelve-month-old male C57BL/6 mice (Charles River Laboratories) were anesthetized by inhalation of 2%-5% isoflurane in oxygen and killed. Adipose tissue-derived mesenchymal stromal cells (AT-MSCs) were then isolated from the peri-epididymal visceral adipose tissue by using a modified version of a previously described protocol, as described in the Appendix S1 19 The vascular stromal fraction was plated, and AT-MSCs selected based on their adherence to plastic.
Before transduction, AT-MSCs were cultured and subsequently characterized at both passages ≤3 and >3 to assess their expression of markers of MSCs, progenitor endothelial cells, pericytes, and smooth muscle cells. 24 markers eNOS, sarcomeric α-actinin and cardiac actin. The T/M AT-MSCs secretome exerts a cytoprotective effect and promotes development of CSCs undergoing SI/R towards a cardiovascular phenotype.

K E Y W O R D S
adipose tissue-derived mesenchymal stromal cells, cardiac stromal cells, extracellular vesicles, myocardin, simulated ischaemia-reperfusion, telomerase

| cDNA cloning and expression vector constructs of telomerase and myocardin
Full-length cDNAs for human telomerase (TERT, 3.6 kb, Genebank accession number NM_198253.2) and human myocardin (MYOCD) isoform 1 (3.1 kb, Genebank accession number NM_153604.1) were amplified via polymerase chain reaction, subcloned and cloned into the pLenti-TOPO cloning vector (Invitrogen), as previously described. 24 A more detailed explanation of the methods reported below is provided in the Appendix S1.

| Isolation of cardiospheres and cardiospherederived cardiac stromal cells
One-week-old neonatal mice, 6-week-old adult mice and 1-year-old C57BL/6 mice were anesthetized by inhalation of 2-5% isoflurane in oxygen and killed. Cardiac stromal cells (CSCs) were isolated from hearts trough the cardiosphere (CSp) isolation technique. 26 A more detailed explanation of the methods reported below is provided in the Appendix S1.

| Western analyses
Total protein extracts of CSCs and AT-MSCs were isolated in ice-cold radioimmunoprecipitation buffer (Sigma-Aldrich). A more detailed explanation of the methods is provided in the Appendix S1.  conditions was found to be >90%-95%. The conditioned medium containing EV was transferred to 50-mL centrifuge tubes (Thermo Fisher Scientific) and centrifuged at 2500 × g at 4°C for 5 minutes to remove cells and cellular debris. Isolation of the EV-enriched fraction by ultrafiltration and fluorescent labelling of EVs was performed as described in the Appendix S1. EV-enriched fraction quantitation and size distribution analyses were performed by nanoparticle tracking analysis ( Figure S1). EV-enriched fractions were further characterized by the lipid-to-protein ratio ( Figure S1) and immunoblotting for the expression of CD63 and ALG-2-interacting protein X (ALIX). 23

| Analysis of adipose tissue-mesenchymal stromal cell-induced cardiac stromal cell cytoprotection in simulated ischaemia/reperfusion
The cytoprotective effect of AT-MSCs on CSCs was tested in CSCs subjected to the previously described SI/R experiments, with cycles of hypoxia-reoxygenation ( Figure S2). 27 CSCs were first pre- To simulate ischaemic conditions, cells were incubated in a hypoxic solution (in mM: NaCl 119, KCl 5.4, MgSO 4 1.3, NaH 2 PO 4 1.2, HEPES 5, MgCl 2 0.5, CaCl 2 0.9, Na-lactate 20, BSA 0.1%, 310 mOsm/L, pH = 6.4) and exposed to a constant flow of a mixture of 95% N 2 and 5% CO 2 for 2.5 hours at 37°C. Either normoxic or SI treatments were followed by 2.5 hours of treatment with differentiating medium and in a 37°C incubator with 95% air and 5% CO 2 prior to harvesting. A more detailed explanation of the methods is provided in the Appendix S1.

| Statistical analysis
Data are expressed as mean ± standard deviation (SD). Two-group comparisons were performed by using the Student t test for unpaired values. Multiple-group comparisons were performed by using analysis of variance and the Gabriel or Tukey Honestly Significant Difference (HSD) post hoc tests to determine statistical significance within and between groups. P values <.05 were considered statistically significant.

| Adipose tissue-mesenchymal stromal cells overexpressing TERT and MYOCD and their secretome improve spontaneous intracellular calcium changes of aged cardiospheres in vitro
Interactions between AT-MSCs and CSps were initially analysed in an in vitro co-culture system. Young CSps obtained and cultured from neonatal, adult (6-month-old) and aged (1-year-old) C57BL/6 mice grew well and colonized in regular media and morphologically showed a growth pattern of typical stromal cells ( Figure 1A).  10 mg/mL). CSCs were then exposed to normoxic or SI/R conditions ( Figure 4). We found that CSCs cultured in the SI/R condition had significantly higher mortality than CSCs cultured in normoxic conditions (cell death: 28 ± 4 vs 10 ± 3%, P = .02, n = 3 independent experiments, 10 replicates each) ( Figure 4A). However, when CSCs were pre-treated with CM from mock-transduced AT-MSCs before being cultured in SI/R, cell mortality was decreased (% dead cells: These results indicate that the promyogenic factors of the secretome resided primarily in the EV-free fraction.

| D ISCUSS I ON
We