Thesis title: Effects of autophagy modulation on survival and phenotype of primitive cardiac stromal cells
Ischaemic heart disease leads to maladaptive remodelling and progressive heart failure (HF). Despite advancements in medical treatments, new therapies against HF progression are needed in order to reduce its morbidity and mortality burden. The natural history of the disease after myocardial infarction includes cardiomyocyte death, followed by scar formation operated by collagen-depositing interstitial cells. Cardiac interstitial and stromal cells embrace a variety of different phenotypes that play a primary role in cardiac homeostasis, injury, and repair mainly through paracrine action. Autophagy is a degradation process of damaged intracellular components that can be either cytoprotective or trigger cell death. Autophagy can sustain cardiomyocyte survival during ischemia, but its role in cardiac stromal cells (CSCs) biology still needs elucidation.
This thesis project aimed at investigating whether autophagy enhancement may exert a protective and anti-fibrotic effect on a population of murine CSCs under conditions of metabolic stress.
METHODS AND DESIGN.
CSCs were isolated from atrial explants of four-week old C57BL/6J mice, and selected through spontaneous spheroid culture (cardiospheres) for a primitive phenotype. We have optimized pharmacological and genetic tools for autophagy modulation in this model: trehalose (disaccharide known to activate autophagy) or TAT-Beclin (cell permeable autophagy-inducer peptide) treatments; adeno-vector transduction of the autophagy promoter ATG7 (Ad-ATG7); transfection of a small interfering RNA against ATG7 (siATG7). CSCs have been subjected to acute nutrient deprivation for 32h, or acute and chronic hyperglycemia (HG; 50mM glucose).
First, we have investigated in vitro the effects of autophagy modulation on CSC viability and phenotype after nutrient deprivation. Results have shown significantly higher CSC viability after stress associated with autophagy activation (by Ad-ATG7 or trehalose), and, conversely, increased cell death after autophagy inhibition through siATG7. Moreover, pre-treatment with trehalose significantly preserved the Sca1+/CD90+/DDR2- fraction of CSCs with cardiogenic potential, while reducing the Sca1-/CD90-/DDR2+ fibroblastoid fraction after stress.
Next we tested whether intermediate autophagy activation (by Ad-ATG7 or TAT-beclin at day 3) preserves CSC cardiogenic immunophenotype and paracrine properties when exposed to 7 days of HG. We detected a significant reduction of the autophagic flux after HG, associated to reduction of the Sca1+ cardiogenic fraction, but this phenotypic shift could not be opposed by intermediate autophagy modulation. Nonetheless, screening of conditioned media after 7 days of HG revealed a consistent reduction in the secretion of several pro-inflammatory cytokines and chemokines in Ad-ATG7-transduced CSCs.
We then investigated whether previous exposure to HG could impair CSC resistance to stress, and whether autophagy modulation may be protective. Results showed that exposure to HG significantly reduced CSC viability after nutrient deprivation, while autophagy activation during stress could significantly preserve subsequent CSC survival.
Overall, results of this thesis project suggest that autophagy enhancement may represent a useful tool:
1) to oppose stromal pro-fibrotic shift during HF progression and dysmetabolic conditions;
2) to possibly increase the efficacy of cardiac cell therapy with endogenous primitive populations by improving cell survival and enrichment in beneficial cardiogenic cells.