Mouse CEC were isolated with a modified protocol based on an online resource (http://vrd.bwh.harvard.edu/ ) and a publication [39 (link)]. Briefly, mouse hearts were dissected and minced into small pieces. After the digestion of the heart using Collagenase I (Worthington), cells were washed and incubated with Dynabeads conjugated with anti-CD31 antibody (Thermo Fisher). The beads with CEC were washed several times and cultured in a mouse endothelial culture medium (Cell Biologics). When confluent, cells were purified with Dynabeads conjugated with anti-Mouse CD102 (ICAM2) antibody. Each animal was used separate isolations, and cells less than passage 6 were used for the experiments.
For loss of function PGC-1α studies, we treated miR-21+/+ or miR-21−/− mouse CEC with either vehicle or a PGC-1α inhibitor SR-18292 (SR, MedChemExpress) at 20 μM for 72 h. SR-18292 induces PGC-1α acetylation [59 (link)]. Conversely, for the gain of function PGC-1α studies, we overexpressed PGC-1α in WT CEC using adenoviral Ad-PGC-1α and control Ad-GFP (generously provided by Dr. Pere Puigserver from Harvard Medical School) [65 (link)]. CEC were transduced with adenovirus at a multiplicity of infection (MOI) of 50 and treated with high glucose (25.5 mM) for 72 h.
For loss of function PGC-1α studies, we treated miR-21+/+ or miR-21−/− mouse CEC with either vehicle or a PGC-1α inhibitor SR-18292 (SR, MedChemExpress) at 20 μM for 72 h. SR-18292 induces PGC-1α acetylation [59 (link)]. Conversely, for the gain of function PGC-1α studies, we overexpressed PGC-1α in WT CEC using adenoviral Ad-PGC-1α and control Ad-GFP (generously provided by Dr. Pere Puigserver from Harvard Medical School) [65 (link)]. CEC were transduced with adenovirus at a multiplicity of infection (MOI) of 50 and treated with high glucose (25.5 mM) for 72 h.
