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

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.


Introduction
Pulmonary arterial hypertension (PAH) is a complex disease resulted from the interplay of several biological and environmental processes leading to pulmonary vasculature remodeling, therefore pulmonary hypertension [1]. Consequently, the low-pressure, thin-walled, crescent-shaped RV has to overcome structural changes to accomplish its function and pump against such an increased afterload [2]. Therefore, RV hypertrophy is a necessary adaptation to preserve RVpulmonary arterial coupling by decreasing RV wall tension and increasing RV cardiomyocyte force-generating capacity [2]. Consequently, in early stages, it emerges as an adaptative remodeling, while at end stage of the disease, it becomes a maladaptive remodeling [2]. Despite the publication of 41 randomized clinical trials in the past 25 years and the regulatory approval of multiple drugs delivered by four administration routes [3], there is no drug focused on improving RV performance and/or reducing inflammation [4]. Despite currently available therapies PAH patients remain significant morbidity and mortality [1]. A polyphenol from the stilbene family, 3,5,4′-trihydroxystilbene resveratrol (RES), has drawn researchers' attention by its cardioprotective activity in other cardiovascular diseases [5]. Although RES acts as a pleiotropic agent in several conditions, it has an intrinsic antioxidant capacity, as well as an ability to regulate membrane receptors, kinases, and other enzymes [6][7][8]. In PAH models, RES improves lung functioning through its antiproliferative [9], antioxidant [10], and anti-inflammatory properties [11]. More precisely, RES activates sirtuins, a relevant group of deacetylases that participate in the regulation of numerous cellular processes [12]. In the heart, sirtuin activation has been linked to the prevention of hypertrophy [13] and energetic dysfunction [14,15].
Previously, we found the prevention of RV hypertrophy and cardiac fibrosis by RES in PAH, accompanied by a decrease in the RV acetylation profile [16]. However, the connection between these two mechanisms remains unclear. Progression to RV failure has been linked to mitochondrial dysfunction [17,18], as PAH generates a disruption in the mitochondrial structure [17], decreasing its oxidative capability [19] and diminishing ATP production [17,20]. The compromised cardiac energetics impairs RV contractility by reducing creatine kinase expression [21], a key component in transferring energy to myofilaments. Thus, protecting mitochondrial function with cyclosporine A (CsA), which blocks mPTP opening by interacting with cyclophilin D (CypD), prevents mitochondrial disruption in PAH and preserves RV function [22]. CypD hyperacetylation is an essential trigger of mPTP opening [23], and its contribution to mitochondrial dysfunction and heart failure has been established in animal models [24] and humans [15]. Notably, CypD acetylation is regulated by SIRT3 [23,24], a sirtuin stimulated by RES [12], which is associated with the lossof-function polymorphism found in PAH patients [25].
The search for additional therapeutic options for more effective PAH management has led to the development and approval of new drugs [26]; however, the available treatments focus on exclusively in managing pulmonary alterations [27]. Gaining a basic understanding of RV alterations through dysfunction, RV failure, and mechanisms that delay these changes may be helpful in developing new therapeutic strategies to improve PAH prognosis. Therefore, the aim of this study is to evaluate the extent of SIRT3 activation in the cardioprotection conferred by RES in the RV of a MC-induced PAH model.

Materials and Methods
2.1. Reagents. All reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA), unless otherwise stated.

Murine
Model of Pulmonary Arterial Hypertension. PAH was induced in male Sprague-Dawley rats (Bioinvert, Edo. de México, MX) weighing >300 g by a single MC (PHL8925) dose (60 mg/kg, subcutaneous) diluted in dimethylsulfoxide (DMSO, 472301), as previously reported [16]. The control group was treated with equivalent volume of DMSO. Animals were kept at 25°C with 12 h light/dark cycle. Water and food were given ad libitum. A group of MC-injected animals was treated with RES dissolved in water (20 mg/kg/day, intra gastric) during day 1 to 42 after MC injection (PAH-RES) [16]. The other groups were given equivalent volume of water (intragastric). All animals were observed for general appearance and respiratory symptomatology. A group of control animals treated with RES during 42 days (20 mg/kg/day, intragastric) was evaluated.
2.3. Histologic Preparations. As reported previously [16], after injection of sodium heparin (1000 U/kg), animals were anesthetized with 5% sevoflurane and the heart and lungs were removed to be fixed in 4% (wt/vol) paraformaldehyde in PBS at room temperature for more than 2 hours. Afterwards, tissues were embedded in paraffin and stained with hematoxylin/eosin (H&E) and Masson's trichrome. An Imager Z1 Zeiss microscope with an AxioCam HRm was used, and images were processed with the AxioVision software. Micrograph from the whole Masson's trichrome stained slides was taken at 2.5x; then, the image was decomposed in more than 7 fields at 5x. Fibrotic index was assessed by quantification of blue and red pixels, using ImageJ (http:// imagej.nih.gov/ij/, NIH, Bethesda, MD, USA); a blue%/red% ratio was made. Two blinded analysts performed the analysis, and three different fields were analyzed. H&E micrograph from the papillary muscles was used to quantify cardiomyocyte area at 10x. An object carrier with a capacity for 7 slides was used to analyze all slides with their respective batches. Arterioles with smooth muscular medial layer proliferation were quantified in seven random fields of lung H&E micrograph, to analyze its diameter, and the occlusion vessel of 100 μm was selected. Occlusion was obtained by averaging more than seven measurements of the medial layer thickness.

Cell Shortening and Ca 2+ Handling in Intact
Cardiomyocytes. Cell shortening was evaluated in 0.5 Hz paced Ca 2+ transient records, where the scanned line was longer than the cell length, following a previous report [29]. Briefly, a rectangular region comprising both cellular edges was selected and a threshold was set to distinguish the intracellular from the extracellular space, converting the image into binary. The cell border of the resulting binary image was compared to the border on the original record to ensure a close fit. Cell shortening parameters evaluated were time to peak shortening from fully cell rest length and time to 50% of relaxation from maximal shortening. ImageJ software (http:// imagej.nih.gov/ij/, NIH, Bethesda, MD, USA) was used to process images. Intracellular Ca 2+ signaling was measured as previously reported [28,30].  [32]. After washing with a fluorophore-free and Ca 2+ -free solution, 2D images (1024 × 1024 pixels, 400 Hz, 1 μm section thickness) were taken using 543 nm excitation and 555-700 nm emission window. Results were normalized to the CTRL group as a percentage. The degree of mitochondrion polarization was then expressed as TMRE intensity per cell. As a negative control, cells were perfused with 0.8 μM cyanide m-chlorophenyl hydrazone (CCCP) (C2759) during 10 min (data not shown).

Mitochondrial
Isolation. Heart mitochondrial fractions were obtained according to the method described previously [33]. Briefly, the heart was isolated and placed in an ice-cold buffer (SHE), containing (mM) the following: 250 sucrose, 10 HEPES and 1 EGTA, and pH 7.3. After, only RV tissue was digested for 10 min using 0.12 mg of protease in cold SHE buffer and centrifuged at 800 x g. The homogenates were centrifuged at 10000 x g for 10 min, and then the resulting pellet was suspended in SH buffer containing (mM) the following: 250 sucrose, 10
2.14. Immunoprecipitation. As described before [15], isolated mitochondria from right ventricles (1 mg) were solubilized in buffer containing (in mM) the following: 150 NaCl, 1 EGTA, Igepal 1%, 20 Tris-HCl, pH 7.2, and protease inhibitor cocktail (Roche). Afterwards, they were clarified of endogenous IgG and incubated with 2 μg of mouse anti-CypD (ab110324, Abcam, UK), or the isotype IgG as control, 1 hour at 4°C in a rotator. The immunoprecipitation complexes were captured by adding 50% activated slurry of Protein G Sepharose beads (GE) to the solubilized protein and incubated overnight at 4°C in a rotator. Beads were centrifuged and washed thrice. Complexes were eluted in SDS-loading buffer prior to electrophoretic separation and subsequent western blot analysis.

Statistics.
Data is presented as the mean ± SEM. Statistical analysis and graphs were performed using GraphPad Prism software (V.5.01; La Jolla, CA, USA). Data were analyzed by one-way ANOVA or two-tailed Student's t-test;to compare the groups, Dunn's post hoc test was performed when appropriate. Statistical significance was set at p < 0:05.

RES Preserves Right Ventricular Function with a Limited
Effect on Lung Vasculature. The PAH model requires 28-42 days to develop phenotypic changes in the lungs and heart [37]. Previously, it has been shown that heart alterations caused by MC target the RV and RES treatment improve RV function with a limited protective effect on pulmonary architecture [16]. In addition, rats treated only with RES showed no differences compared to the control animals (without MC) for lung morphological parameters such avessel lumen diameter, number of muscular arteries, luminal occlusion, and RV histological characteristics (Supplemental Figure 1).

RES Prevents Contractility Alterations and Improves Ca 2+
Handling. RV myocyte function was evaluated by characterizing cell contraction and Ca 2+ dynamics to assess alterations in excitation-contraction coupling (ECC). Cell shortening was less efficient in the PAH group since time to peak shortening (Figure 1(b)) and half relaxation (Figure 1(c)) were 82% and 41% slower, respectively. RES treatment accelerated the time to peak shortening by 22% in PAH (Figure 1(b)) and 4 Oxidative Medicine and Cellular Longevity   Oxidative Medicine and Cellular Longevity maintained the CTRL group time to half relaxation (Figure 1(c)), indicating that RES treatment improved cell relaxation in PAH.
RV myocytes were challenged after isoprenaline (ISO, 100 nM) perfusion to induce the β-adrenergic response (β-AR), a highly energy-dependent state (Supplemental. Fig. 2). All groups were capable to increase Ca 2+ transient amplitude and to reduce T 50% . However, cell contraction failed to comply in the PAH group, while RES-treated PAH prevented this Ca 2+ transient and cell contractility mismatch.
The involvement of oxidative stress in mitochondria permeability transitioning was also evaluated. As shown in Figure 3(b), there was an increase in membrane peroxidation concomitant with 48% decrease in aconitase activity in the PAH group indicating an increased oxidant environment within the mitochondria. However, other antioxidant enzymes and oxidation in protein or DNA did not change in the PAH group (Figure 3). RES treatment maintained the same level of aconitase activity as CTRL (97%;  Oxidative Medicine and Cellular Longevity Figure 3(e)) and increased mitochondrial superoxide dismutase (SOD) by 36% compared to the PAH group (Figure 3(g)), which is a direct target of SIRT3.

RES Decreases the Acetylation of CypD.
To evaluate the extent of sirtuin activation by RES, the mitochondrial protein acetylation profile, SIRT3 expression, and acetylation of CypD were assessed. In Figure 4(a), the protein acetylation profiles of isolated mitochondria show a 59% increase in acetylated proteins in the PAH group, including a threefold increase in CypD acetylation (Figure 4(c)). Protein acetylation was decreased by 13% in the PAH-RES group (p = 0:5303 vs. PAH); however, SIRT3 was 96% overexpressed (Figure 4(b)). Importantly, CypD was a critical component of mitochondria permeability transitioning, as shown by the significant decrease of 51% (p = 0:0581, vs. PAH) in acetylation in the PAH-RES group (Figure 4(c)), indicating the importance of SIRT3 in reducing mitochondrial permeability transitioning. There were no significant betweengroup differences in SIRT1 gene expression (CTRL = 0:89 ±

Discussion
The cardiovascular protective actions of RES in PAH have been reported in RV function [16,38]; however, the mechanisms involved have not been fully described. According to our results, PAH causes an energetic dysfunction in the RV myocyte by promoting the opening of the mPTP due to CypD hyperacetylation. The decreased energy supply impairs the highly energy-demanding processes involved in myocyte functions, such as cellular relaxation, by hampering SERCA activity and delaying myofibrils' unbinding. Treating this model with RES stimulates and overexpresses SIRT3, which prevents mPTP opening by acting directly on CypD deacetylation. Preserving cellular energetics also preserves myocyte contraction and relaxation.  Oxidative Medicine and Cellular Longevity RV myocyte functioning was evaluated using confocal microscopy by assessing intracellular Ca 2+ signaling and cell shortening. Characterization of the cellular contractionrelaxation cycle showed that PAH has a negative impact on the overall contraction-relaxation dynamic. Regardless, no alterations in SR Ca 2+ release synchronicity during Ca 2+ transient; it takes longer to accomplish maximal shortening. Treating PAH with RES decreased time to maximal shortening possibly by increased myofilament Ca 2+ sensitivity [39,40]; however, it does not fully normalize this parameter. Interestingly, relaxation dynamics showed the most significant changes. PAH considerably prolongs cell relaxation, while PAH under RES treatment keeps the relaxation rate similar to the CTRL group, a finding that has not been previously reported.
Accordingly, major alteration in intracellular Ca 2+ transient caused by PAH was a decreased SERCA activity caused by a decreased SERCA/PLB ratio. However, both increased [41] and decreased [42] SERCA activities have been reported, as well as no change in SERCA and PLB expression [41]. Although RES treatment did not change the SERCA/PLB ratio, it did increase intracellular Ca 2+ removal, thus SERCA activity.
This behavior was replicated after stimulating the β-AR, a highly energy-demanding condition [43,44]. PAH showed the highest transient amplitude but the lowest cell shortening, while RES preserved the proportionality of Ca 2+ released and cell contraction.
This effect might be related to a mismatch in the ECEC since it has been documented that creatine kinase (CK) expression is diminished in failing RV caused by PAH due to a decreased ATP supply [21,45,46]. Failing to meet the energetic demand, myofilament cross-bridge cycling is inhibited in finding shorter sarcomere lengths [21], compromising myocyte function. In this regard, the preservation of ATP production by RES preserves CK activity and an efficient ATP supply to SERCA, displaying improved cell contraction and relaxation.
Additionally, PAH did not modify Ca 2+ spark frequency, indicating intact RyR activity [47], unlike previous reports [41,42]. RES treatment also showed no changes in spark frequency and amplitude.

RES Preserves Mitochondrial Functioning and Integrity.
Since major alterations caused by PAH reside among highly energy-dependent mechanisms, cell excitation and contraction must be tightly linked to the energy supply to ensure the proper functioning of the myocyte as a whole in an ECEC process. Since mitochondrial dysfunction has been identified as one of the mechanisms underlying heart failure [18,19,22,46], mitochondrial activity was evaluated in this model. In isolated myocytes, PAH compromises ATP production by decreasing ΔΨm; the electrochemical force needed for ATP production, decreasing the oxidative phosphorylation rate (state 3). Decreased phosphorylation response have been reported in RV by PAH [48][49][50]. The cardioprotective effects of RES on mitochondria have been described in several models [51][52][53]. Similarly, RES treatment preserved ΔΨm and respiratory chain activity in PAH. Even though the results obtained by mitochondrial respiration and fragility are evident when stimulating complex II, these same aspects have yet to be evaluated under NADH-dependent respiration.
Mitochondrial function preservation by RES in PAH could be related to decrease mitochondrial fragility. The loss of mitochondrial integrity by mPTP formation has been described as a main contributor to ventricular dysfunction in the right and left ventricles [22,54,55]. Most importantly, preventing mPTP opening using CsA has been shown to reduce RV dysfunction in PAH [22]. Mitochondrial Ca 2+ overload in PAH, complemented by an increased oxidative environment within the mitochondria, makes them more prone to permeability transition [54,56]; however, unlike other reports [57], no critical signs of proteins being modified by oxidative stress were observed, indicating that the antioxidant system is capable of managing oxidative stress caused by PAH in this model.
Although evident impairment in ΔΨm was found in this study, outright evidence from reduced cellular bioenergetics in this PAH model would directly measure ATP levels and evaluate the phosphocreatine system. However, it is well established that the ATP phosphorylation is particularly sensitive to decreases of mitochondrial membrane potential; for every 14 mV decrease in proton-motive force (equivalent to ΔΨm), the ATP/ADP ratio decreases by 10-fold [58]. Also, the mitochondrial membrane potential has been described as a crucial factor to generate rotational torque by the F 0 nanomotor [59,60]. Thus, a decrease in this electrochemical force may impede ATP production. In this regard, the integrity of ΔΨm in PAH+RES RV myocytes suggests a capable, energetic system to sustain the cardiac ECC.

RES Promotes the Deacetylation of CypD by SIRT3
Overexpression. One of the mechanisms through which RES bestows its protective effects is the activation of deacetylases. In pulmonary arterial smooth muscle cells (PASMC), the protective effect of RES has been linked to expression and activity modulation of the cytosolic deacetylase SIRT1 [61,62]. Similarly, an increase in acetylated proteins was found in PAH RV myocytes [16,38], and the cardiac protection of RES through SIRT1 activation has been reported in different conditions [38,[63][64][65]. Since PAH's effect is on mitochondrial function and activity, the expression of SIRT3 and mitochondrial acetylome were evaluated to determine the extent of its involvement.
The mitochondrial deacetylase SIRT3, which regulates mitochondrial function [66][67][68][69], is modulated by RES [12], and the rs11246020 polymorphism, associated with a~30% loss of function, has been found to be overrepresented in patients with idiopathic PAH [25]. PAH showed an increase in the protein acetylation profile from mitochondria, similar to reports in PASMC [25]. RES treatment increased SIRT3 expression almost 3.5-fold; however, the acetylation profile did not decrease significantly from PAH levels. Regardless of this discrepancy, an important direct target of SIRT3, cyclophilin D (CypD), decreased by almost half its hyperacetylation, diminishing the mitochondria's proneness to mPTP opening [15,23], thus preserving the cellular energetic state ( Figure 5).
Furthermore, SIRT3 activity has been linked to the prevention of myocardial dysfunction. Treatment with honokiol, a potent SIRT3 activator, prevents and reverses ventricular hypertrophy [70] and preserves mitochondrial integrity and energetic capability [70,71]. The mechanisms involved still need to be elucidated; however, it has been found that SIRT3 prevents mitochondrial Ca 2+ overload by regulating mitochondrial Ca 2+ uptake, regulating the expression of mitochondrial Ca 2+ uniporter and its main regulator MICU1. These data are relevant because it was established that MCU and the MICU1 promoter are regulated by SIRT3-dependent histone acetylation [72]. However, no change in MCU gene expression was found in this model.
While SIRT3 activity has been identified as being involved in cardiovascular protection [73][74][75][76][77], it is not the only mechanism through which RES acts as a cardioprotective agent. Other sirtuins and enzymes, such as SIRT1, SIRT5, and AMPK [5,6,78], contribute to modulating the activation or repression of a wide range of cellular components [5,6,78]. Furthermore, PAH pathogenesis is not caused by a single modification; according to the widely accepted "multiple-hit" hypothesis: inflammation, genetic determinants, and environmental factors are needed to develop the disease [79,80].
Another important mechanism involved in modulating mitochondrial functioning that we did not explore thoroughly in this study is mitochondrial dynamics. The deacetylation of OPA1 by SIRT3 has been described as a regulator of mitochondrial dynamics and plays a role in maintaining a competent mitochondrial population in the heart when it is under pathological stress [81]. While previous reports did not find changes in the gene expression of proteins involved in mitochondrial dynamics and biogenesis in PAH [82], it has been reported that RES promotes mitochondrial fission [83]. This phenomenon is needed to preserve mitochondrial function [84] and may be involved in ΔΨm preservation by RES in this model. However, the implications of mitochondrial dynamics in the progression of RV failure still need to be addressed; this phenomenon could be an interesting area of research in the future to elucidate new therapeutic targets in PAH. 10 Oxidative Medicine and Cellular Longevity Although we found a strong relationship between preservation of mitochondrial function and CypD acetylation via SIRT3, loss and gain of function experiments can assess its definitive role in this model. The availability of deficient-SIRT3 mice faces the challenge of PHA model development, since it has been reported that mouse PAH models may not develop pulmonary hypertension, RV hypertrophy, and RV failure regardless pulmonary artery remodeling; besides, some of the models show spontaneous reverse of pulmonary hypertension once the inductor is not present [85,86].

Clinical Implications.
This study suggests that mitochondrial protection and cellular energetic preservation could be possible therapeutic targets to improve the PAH phenotype. The currently approved PAH medications decrease vascular remodeling by restoring the balance between vasoactive and vasodilator mediators [87]. However, researchers have begun to focus on mitochondrial disorders to develop new strategies. Molecules that prevent and reverse mitochondrial bioenergetic abnormalities in PASMC are being explored due to their ability to promote apoptosis, thus decreasing pulmonary vascular remodeling [88][89][90][91] and, in RV fibroblasts, reducing cardiac fibrosis in PAH [92]. Unfortunately, cardioprotective effects on RV muscle cells have not been explored. Although RES' usefulness in treating cardiovascular diseases has been observed as an overall beneficial effect in diverse clinical trials, further research is needed to ensure sustained effectiveness in patients since its major disadvantage is its poor bioavailability [93]. No published studies have evaluated RES use in PAH patients. Finally, ECEC disruption is a phenomenon found in the right and left heart failure. Describing the underlying mechanism and identifying shared targets may be useful to recognize therapeutic strategies that can help to improve heart failure prognosis and care.

Conclusions
The present work assessed the alterations in RV myocytes caused by PAH and RES' protection. For the first time, we showed that the development of PAH causes a mismatch in the ECEC: compromised mitochondrial functioning and altered ATP synthesis resulted in deficient cell relaxation and disrupted cell contraction. RES protects mitochondria integrity by decreasing CypD hyperacetylation and increasing SIRT3 expression and activity, preventing mPTP opening, and preserving the ΔΨm. Maintaining cellular energetics preserves ECEC, ensuring the proper functioning of the myocyte as a whole. Understanding the mechanisms involved in the protection that RES confers on the RV in PAH could facilitate the development of new adjuvant therapies that improve the daily lives of PAH patients.

Data Availability
The data used to support the findings of this study are available from the corresponding author upon request.

Disclosure
This work was submitted in partial fulfilment of the requirements for the PhD degree (JBR) for the Doctorate in Biotechnology, Tecnológico de Monterrey.  Table 1: qPCR primer sequences hypoxanthine phosphoribosyltransferase 1 (HPRT), Na + /Ca 2+ exchanger (NCX), sirtuin 1 (Sirt1), sirtuin 5 (Sirt5), and mitochondria calcium uniplex (MCU). HPRT was used as a housekeeping gene. Supplementary Figure 1: heart and lung structural comparison between control animals and RES-treated control animals. Pooled data of muscularized arteries (A), luminal diameter (B), and occlusion (C) in the lung tissue (H&E, 10x). Bar graph depicting the myocyte area (D) in RV. Data was normalized to CTRL mean values. All data are presented as the mean ± SEM. * p < 0:05 vs. CTRL calculated by a t-test followed by the Mann-Whitney test. Supplementary