Figure 1. Increased miR-195 suppresses SIRT3 expression and increases acetylation.

1A. miR-195 abundance in human cardiac tissue was assessed by qRT-PCR, which detected a 3.04-fold increase (p<0.05) in patients with HF. Control n=5, HF n=11. Cardiac miR-195 abundance in mouse model was assessed by qRT-PCR, which detected a 2.01-fold (p<0.05) and 2.09-fold elevation of the mature miR-195 in TAC HF mice and MI HF mice, compared to TAC Sham and MI Sham mice respectively. miR-191 was used as normalization. 1B. Prediction of SIRT3 as a target of miR-195. The prediction was conducted by using data from microRNA target prediction Systems (www.microrna.org). 1C. Luciferase assay confirmed miR-195 association at SIRT3 mRNA 3’UTR. Constructs carrying the SIRT3 3’ UTR or not (vector) were co-transfected with scramble miR control or miR-195 precursor in AC16 cells. The ratios of the firefly/Renilla values for the SIRT3 construct relative to the vector construct were shown. The firefly/Renilla values were calculated from three independent samples. Errors represent the SD derived from three independent experiments and p<0.05. 1D. miR-195 overexpression induced global protein acetylation. Cells were transfected with overexpression vector containing miR-195 precursor or scramble miR control, and the whole cell lysates were prepared. Global acetylation level and SIRT3 expression were analyzed by western blot and GAPDH was used as loading control. 1E. Silencing of miR-195 rescues SIRT3 expression in response to Ang II stimulation. AC16 cells were treated with Ang II or/and anti-miR-195 or miRNA inhibitor negative control. Whole cell lysates were prepared and the SIRT3 expression was analyzed by western blot and GAPDH was used as loading control. 1F. PDH complex and ATP synthase α subunit were more acetylation in cells overexpressing miR-195. A representative Kac immunoprecipitation (IP) reaction from three independent assays was shown. Cell lysates prepared from miR-195/scramble control miR transfected cell were subjected to IP assay using anti-Acetylated-Lysine (anti-Kac). Equivalent amounts of the pellets (IP) were analyzed by western blotting. 10% of the cell lysate used in the IP reaction was shown as input. 1G. miR-195 overexpression resulted in 24% decrease in PDH activity. Errors represent the SD derived from three independent experiments and p<0.01. 1H. miR-195 overexpression resulted in 38% decrease in ATP synthase activity. Errors represent the SD derived from three independent experiments and p<0.05. 1I. Basal Raspiration, ATP turnover, H+ leak and respiratory capacity were all significantly decreased in AC16 cells overexpression miR-195. The OCR was measured as described before. Measurements were made in triplicate (mean ans s.d.), and results were indicative of three independent experiments (p<0.05 or p<0.01). * represents p<0.05, ** represents p<0.01.