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

Abstract

Pulmonary arterial hypertension (PAH) is characterized by pulmonary vessel remodeling; however, its severity and impact on survival depend on right ventricular (RV) failure. Resveratrol (RES), a polyphenol found in red wine, exhibits cardioprotective effects on RV dysfunction in PAH. However, most literature has focused on RES protective effect on lung vasculature; recent finding indicates that RES has a cardioprotective effect independent of pulmonary arterial pressure on RV dysfunction, although the underlying mechanism in RV has not been determined. Therefore, this study is aimed at evaluating sirtuin-3 (SIRT3) modulation by RES in RV using a monocrotaline- (MC-) induced PAH rat model. Myocyte function was evaluated by confocal microscopy as cell contractility, calcium signaling, and mitochondrial membrane potential (ΔΨm); cell energetics was assessed by high-resolution respirometry, and western blot and immunoprecipitation evaluated posttranslational modifications. PAH significantly affects mitochondrial function in RV; PAH is prone to mitochondrial permeability transition pore (mPTP) opening, thus decreasing the mitochondrial membrane potential. The compromised cellular energetics affects cardiomyocyte function by decreasing sarco-endoplasmic reticulum Ca²⁺-ATPase (SERCA) activity and delaying myofilament unbinding, disrupting cell relaxation. RES partially protects mitochondrial integrity by deacetylating cyclophilin-D, a critical component of the mPTP, increasing SIRT3 expression and activity and preventing mPTP opening. The preserved energetic capability rescues cell relaxation by maintaining SERCA activity. Avoiding Ca²⁺ transient and cell contractility mismatch by preserving mitochondrial function describes, for the first time, impairment in excitation-contraction-energetics coupling in RV failure. These results highlight the importance of mitochondrial energetics and mPTP in PAH.

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 T_50% (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; 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; ^cp < 0.05 vs. CTRL, calculated by a 2-tailed t-test.

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.