Resveratrol Prevents Right Ventricle Dysfunction, Calcium Mishandling, and Energetic Failure via SIRT3 Stimulation in Pulmonary Arterial Hypertension

Figures

• 

  Figure 1. Characterization of cell contraction and Ca2+ dynamics in isolated RV myocytes. (a) Representative profile of cellular shortening. Average of time to peak shortening (b) (CTRL: n = 20 cells; PAH: n = 21 cells; PAH+RES: n = 45) and time to 50% relaxation (c) (CTRL: n = 18 cells; PAH: n = 17 cells; PAH+RES: n = 16). (d) Representative florescence profile of Ca2+ transient. Pooled data from Ca2+ transient amplitude (e) and T50% (f) (CTRL: n = 16 cells; PAH: n = 53 cells; PAH+RES: n = 69 cells). (g) Representative images and pooled data of western blot to the SERCA2/PLB ratio; GAPDH was used as a loading control (n = 4). (h) Representative line scan images of treated groups; surface plots from selected sparks (doted square) are above; shown below are line profiles from 2 μm regions of the selected spark (black marks in line scan images). Pooled data of Ca2+ spark frequency (i) and amplitude (j) (CTRL: n = 46 cells, 3 animals; PAH: n = 55 cells, 3 animals; PAH+RES: n = 71 cells, 5 animals). CTRL (solid black line), pulmonary arterial hypertension (PAH, dotted line), and PAH treated with resveratrol (PAH+RES, solid gray line). CTRL: control; PAH: pulmonary arterial hypertension;…

• 

  Figure 2. Characterization of mitochondrial function from RV. Representative images (a) and pooled data (b) from ΔΨm in isolated RV myocytes (CTRL: n = 66 cells and 3 animals; PAH: n = 59 cells and 3 animals; PAH+RES: n = 25 cells and 2 animals). Mitochondrial respiratory states (c, d) and respiratory control ratio (e) of isolated mitochondria preparations (CTRL: n = 6, PAH: n = 5, PAH+RES: n = 6). (f) CRC from isolated mitochondria, in left image each arrow represents a 10 nmol CaCl2 bolus (CTRL: n = 8, PAH: n = 5, PAH+RES: n = 4; ap < 0.05 vs. PAH, calculated by a 2-tailed t-test). CTRL: control; PAH: pulmonary arterial hypertension; PAH+RES: PAH treated with resveratrol. All data are presented as the mean ± SEM. ∗p < 0.05 vs. CTRL; ap < 0.05 vs. PAH, calculated by 1-way ANOVA.

• 

  Figure 3. Oxidative stress markers in RV. Pooled data from free thiol groups in isolated mitochondria (a). Average of protein carbonylation (b), TBARS (c), and 8-OHdG/total DNA ratio in RV tissue (d). Enzymatic activity of aconitase (e), catalase (f), manganese superoxide dismutase (Mn-SOD) (g), and copper-zinc superoxide dismutase (CuZn-SOD) (h). Control (CTRL: n = 4-7); pulmonary arterial hypertension (PAH, n = 5-9); PAH treated with resveratrol (PAH+RES, n = 6-7). All data are presented as the mean ± SEM. ∗p < 0.05 vs. CTRL; ap < 0.05 vs. PAH, calculated by 1-way ANOVA.

• 

  Figure 4. Protein acetylation and SIRT3 expression in RV. Representative image and pooled data of western blot to acetylated lysine (Ac-Lys) profile from isolated mitochondria (a) (CTRL: n = 5, PAH: n = 4, PAH+RES: n = 6). ∗p < 0.05 vs. CTRL unpaired 2-tailed t-test. (b) Representative images and pooled data of western blot from isolated mitochondria against SIRT3; cytochrome c oxidase subunit 4 (COX-4) was used as a loading control (CTRL: n = 6, PAH: n = 3, PAH+RES: n = 4). (c) Immunoprecipitation (IP) of cyclophilin D (CypD) followed by immunoblot analysis against Ac-Lys; acetylation signal was normalized to total CypD (CTRL: n = 3, PAH: n = 3, PAH+RES: n = 3). Control (CTRL); pulmonary arterial hypertension (PAH); PAH treated with resveratrol (PAH+RES). All data are presented as the mean ± SEM. ∗p < 0.05 vs. CTRL, ap < 0.05 vs. PAH, calculated by 1-way ANOVA.

• 

  Figure 5. Proposed mechanism of RES cardioprotection. Resveratrol stimulates SIRT3 activation and expression, which deacetylates CypD preventing mPTP formation and preserving ΔΨm, thus increasing ATP synthesis. The preserved mitochondrial function promotes a high-energy demand process to occur, such as cellular relaxation and SERCA activity, preventing excitation-contraction-energetic coupling mismatch. RES: resveratrol; LTCC: L-type Ca2+ channel; RyR: ryanodine receptor; SERCA2: sarco-endoplasmic reticulum Ca2+-ATPase; PLB: phospholamban; mPTP: mitochondrial permeability and transition pore; CypD: cyclophilin D; Ac: acetyl-group; SIRT3: sirtuin 3; ΔΨm: mitochondrial membrane potential. Created with http://BioRender.com.