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.

Keywords: adipose tissue-derived mesenchymal stromal cells; cardiac stromal cells; extracellular vesicles; myocardin; simulated ischaemia-reperfusion; telomerase.

FIGURE 1

A, Representative images of cardiospheres (CSps), observed in Nomarski interference contrast, isolated from hearts of neonatal (a), 6‐month‐old (b) and 1‐year‐old (c) C57BL/6 mice. B, EV‐enriched fraction taken up by cardiosphere‐derived stromal cells (CSCs). PKH26‐stained EV‐enriched fraction (red) harvested from conditioned media of mock‐transduced adipose tissue‐derived mesenchymal stromal cells (AT‐MSCs) or AT‐MSCs overexpressing TERT and MYOCD (T/M‐MSCs) are taken up by CSCs within 72 h of treatment

FIGURE 2

In vitro effects of AT‐MSCs overexpressing TERT and MYOCD and their secretome on sarcomeric α‐actinin expression in CSps from aged (1‐year‐old) C57BL/6 mice. A, Representative images of CSps co‐cultured with AT‐MSCs acquired in Nomarski interference contrast. B, Representative confocal microscopy images of sarcomeric α‐actinin expression (green) in the cardiospheres (CSps), according to treatment groups. DAPI (blue) was used for nuclear counterstaining. Images were taken in the peripheral area of CSps

FIGURE 3

In vitro effects of AT‐MSCs overexpressing TERT and MYOCD and their secretome on intracellular Ca²⁺ spikes and ryanodine receptor levels in CSps from aged (1‐year‐old) C57BL/6 mice. A, Representative intracellular Ca²⁺ spikes in neonatal CSps, aged CSps, according to treatment groups. B, Representative Western blot showing the levels of ryanodine receptor (RyR) in aged CSps according to treatment groups. GAPDH levels were assessed as a loading control. Results presented as mean ± standard deviation, n = 3 independent experiments. *P < .05 aged CSps+mock AT‐MSCs vs aged CSps; **P < .01 wild‐type AT‐MSCs vs aged CSps; °°P < .01 T/M AT‐MSCs vs wild‐type AT‐MSCs or mock‐transduced AT‐MSCs; °°P < .01 aged CSps+CM T/M AT‐MSC vs aged CSps. Legend: CM, conditioned media; AT‐MSCs, mock‐transduced adipose tissue‐derived mesenchymal stromal cells; AT‐MSCs overexpressing TERT and MYOCD (T/M AT‐MSCs); GAPDH, glyceraldehyde 3‐phosphate dehydrogenase

FIGURE 4

A, Representative microphotographs showing the effect of the secretome of AT‐MSC overexpressing TERT and MYOCD (T/M AT‐MSC) and its extracellular vesicle (EV)‐enriched fraction on viability of aged cardiosphere‐derived stromal cells (CSCs) from 1‐year‐old murine hearts in a simulated model of ischaemia/reperfusion (SI/R). The conditioned medium (CM) or EV‐enriched fraction were harvested from 72 h cultures of mock‐transduced AT‐MSCs or T/M AT‐MSCs. CM or EV‐enriched fraction were applied 72 h before simulated ischaemia/reperfusion (SI/R) or normoxia conditions. Round cells represent the proportion of cells detached from the surface of the plates in each culture conditions. B, Effect of treatments on cardiosphere‐derived stromal cells (CSCs) viability. Cell viability was measured with the Trypan blue assay. *P < .05 CM or EV‐treated vs basal medium CSCs; °P < .05 EV‐treated vs CM‐treated; ^# P < .05 CM or EV T/M AT‐MSCs‐treated vs CM or EV mock‐transduced AT‐MSCs; § < .05 vs normoxia

FIGURE 5

Effects of the secretome of AT‐MSC overexpressing TERT and MYOCD and their EV‐enriched fraction on aged CDCs in a simulated model of ischaemia/reperfusion. A‐C, Effect of treatments on the expression of cell survival (phosphorylated AKT) normalized for AKT and apoptosis (cleaved caspase‐3) markers A, endothelial marker (endothelial nitric oxide synthase, eNOS) B, and cardiomyocyte markers (cardiac actin and sarcomeric α‐actinin) C, in CSCs. Graphs are based on densitometric analysis of protein bands in Western blots. GAPDH levels were assessed as loading controls for cleaved caspase‐3, cardiac actin and sarcomeric α‐actinin, whereas beta‐actin levels were assessed as loading control for eNOS. Results presented are mean ± standard deviation, n = 3 independent experiments. A‐C:* P < .05 CM‐ or EV‐treated vs basal medium; °P <.05 EV‐treated vs CM‐treated; ^# P < .05 CM or EV T/M AT‐MSCs‐treated vs CM or EV mock‐transduced AT‐MSCs; ^§ P <.05 SI/R vs normoxia. Legend: EV −1, extracellular vesicle‐enriched fraction 0.1 mg/mL; EV‐2, extracellular vesicle‐enriched fraction 1 mg/mL; EV‐3, extracellular vesicle‐enriched fraction 10 mg/mL