Poricoic acid a inhibits mitochondrial dysfunction in myocardial infarction by activating SIRT3

Abstract

Acute myocardial infarction (MI) is the most severe clinical manifestation of ischemic heart disease. Despite this, the mechanisms that disrupt mitochondrial homeostasis and contribute to cardiomyocyte loss during MI are poorly understood, emphasizing the urgent need for new therapeutic interventions. Poricoic acid A (PAA), the principal active component of pachymaria, possesses a range of pharmacological effects. However, the specific role and mechanisms by which PAA addresses mitochondrial dysfunction in MI remain unclear. This study aims to elucidate the impact of PAA on MI and uncover its potential regulatory mechanisms. We developed MI cell models using mouse primary cardiomyocytes incubated in a Forma Steri-Cult chamber containing 1% oxygen, 94% nitrogen, and 5% carbon dioxide. Our results demonstrate that PAA significantly improves cardiomyocyte injury in hypoxia-induced mouse primary cardiomyocytes. Furthermore, PAA activates the AMP-activated protein kinase/peroxisome proliferator-activated receptor gamma coactivator 1-alpha/Sirtuin 3 (AMPK/PGC-1α/SIRT3) signaling pathway in hypoxia-induced mouse primary cardiomyocytes. PAA enhances the oxidative stress response in hypoxia-induced mouse primary cardiomyocytes by activating SIRT3. Additionally, it improves mitochondrial dysfunction in these cardiomyocytes and reduces apoptosis by activating SIRT3. In summary, PAA inhibits mitochondrial dysfunction associated with MI by activating SIRT3, indicating its promise as a therapeutic agent for MI.

Introduction

Acute myocardial infarction (MI) is the most severe clinical manifestation of ischemic heart disease, characterized by the ischemia of myocardial cells and subsequent irreversible cell death.⁽¹⁾ Despite significant advancements in reperfusion strategies, evidence-based treatments, and healthcare logistics, patients who experience extensive tissue damage during the acute phase continue to face a high mortality rate and may eventually develop heart failure.^(1,2) The degree of myocardial cell loss in the early stages of MI is directly linked to the subsequent prognosis. During the progression of MI, mitochondrial dysfunction and energy depletion occur early in the process of heart injury.^(3,4) This metabolic dysfunction within the mitochondria poses a significant threat to the survival of cardiomyocytes.⁽⁵⁾ However, the mechanisms that lead to the disruption of mitochondrial homeostasis and the subsequent loss of cardiomyocytes during MI remain inadequately understood. Therefore, developing new therapeutic agents to combat this disease is essential.

Natural products are emerging as promising candidates for the treatment of MI. Tuckahoe, an edible and medicinal substance, contains a wealth of beneficial compounds that may be effective in managing MI.⁽⁶⁾ Poricoic acid A (PAA) is a white powder derived from the dried sclerotia of Poria cocos (Schw.) Wolf and it serves as the primary active component of pachygyria.⁽⁷⁾ PAA possesses a wide range of pharmacological effects, including diuretic, swelling, anti-inflammatory, antibacterial, and antioxidant properties.^(8–10) As a tetracyclic triterpenoid, PAA has been shown to upregulate Sirtuin 3 (Sirt3), significantly reducing renal fibrosis and supporting renal function.⁽¹⁰⁾ Previous studies have confirmed that PAA can enhance angiogenesis and facilitate myocardial regeneration following MI by modulating the AMP-activated protein kinase (AMPK)/mTOR pathway to induce autophagy.⁽¹¹⁾ However, the specific role and mechanism by which PAA addresses mitochondrial dysfunction in acute MI remains to be fully elucidated.

It is widely acknowledged that mitochondrial oxidative stress and apoptosis are integral components of the pathological mechanism underlying MI.⁽¹²⁾ Oxidative stress is characterized by an imbalance between the production of reactive oxygen species (ROS) and the body’s antioxidant defenses. The inhibition of oxidative stress is beneficial for alleviating MI.⁽¹³⁾ Furthermore, apoptosis is a critical downstream mediator of oxidative stress, playing an essential role in the progression of MI.⁽¹⁴⁾ Recent studies have highlighted the vital role of the AMPK/SIRT3 pathway in regulating mitochondrial oxidative stress.⁽¹⁵⁾ AMPK functions as a vital energy sensor and is essential in managing myocardial ischemia and protecting MI by alleviating oxidative stress and apoptosis within cardiac tissue.⁽¹⁶⁾ The activation of AMPK reduces the production of mitochondrial ROS by enhancing peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) expression. In addition, PGC-1α acts as an upstream regulator, influencing the expression of SIRT3, which is essential for maintaining mitochondrial homeostasis and regulating oxidative stress and apoptosis.⁽¹⁷⁾ When the AMPK/PGC-1α/SIRT3 pathway is downregulated, it can result in mitochondrial dysfunction and worsening myocardial injury following MI. Consequently, activating the AMPK/PGC-1α/SIRT3 pathway may effectively reduce oxidative stress and apoptosis in the heart after an MI.⁽¹⁸⁾

In this study, we aim to elucidate the effects of PAA on MI and uncover its potential regulatory mechanisms. Our findings indicate that PAA inhibits mitochondrial dysfunction in MI by activating SIRT3. Consequently, PAA may serve as a promising therapeutic agent in managing MI.

Materials and Methods

Cell culture and hypoxia model

Mouse primary cardiomyocytes were sourced from the American Type Culture Collection (ATCC, Manassas, VA) and cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) at 37°C in a humidified incubator with 5% CO₂. To establish the hypoxia injury model, the cells were exposed to hypoxic conditions (1% O₂, 94% N₂, and 5% CO₂) for 6 ‍h. To inhibit SIRT3 activity, 3-TYP (10 ‍μM; Beyotime, Beijing, China) and Compound C (1 ‍μM; Sigma, St. Louis, MO) were used in the cellular experiments. The cells were treated with 3-TYP for 48 ‍h under hypoxic conditions in the presence or absence of PAA. This concentration was selected based on prior studies and was optimized to effectively inhibit SIRT3 activity without causing significant cytotoxicity. SIRT3 was depleted by transfecting the cells with its siRNA (Riobio, Guangzhou, China) using Lipofectamine 2000 (Invitrogen, Waltham, MA) for 48 ‍h.

Cell viability and cytotoxicity assays

The viability of mouse primary cardiomyocytes was measured using the CCK-8 assay (C0038; Beyotime). Mouse primary cardiomyocytes were treated with varying concentrations of PAA (0, 5, 10, 20, and 40 ‍μM) for 48 ‍h under hypoxic conditions. Cytotoxicity was evaluated by measuring LDH release using the LDH Cytotoxicity Assay Kit (C0016; Beyotime). Absorbance was measured at 450 ‍nm using a microplate reader.

Mitochondrial function and ROS

Mitochondrial membrane potential was evaluated using the JC-1 Mitochondrial Membrane Potential Assay Kit (C2006; Beyotime). Following hypoxia treatment and PAA administration, the cells were stained with JC-1 dye and analyzed using a fluorescence microscope (Zeiss, Oberkochen, Germany). ROS levels were measured using MitoSOX Red (M36008; Thermo Fisher Scientific, Waltham, MA). MDA levels were assessed using respective assay kits from Beyotime (S0131).

Immunoblot assay

Proteins were loaded onto SDS-PAGE gels and transferred onto PVDF membranes (IPVH00010; Millipore, Burlington, MA). The membranes were then blocked with 5% non-fat milk in TBST and incubated overnight at 4°C with primary antibodies (Anti-Bax, ab32503; Anti-cleaved Caspase-3, ab32042; Anti-AMPK, ab32047; Anti-p-AMPK, ab92701; Anti-PGC-1α, ab313559; Anti-SIRT3, ab86671; Abcam, Cambridge, UK). Following this, HRP-conjugated secondary antibodies were applied. Bands were visualized using an enhanced chemiluminescence (ECL) kit (P0018; Beyotime).

TUNEL assay

Apoptosis was evaluated using the TUNEL assay (In Situ Cell Death Detection Kit; Roche, Basel, Switzerland). The cells were first fixed with 4% paraformaldehyde and subsequently permeabilized with 0.1% Triton X-100. They were then incubated with the TUNEL reaction mixture at 37°C for 1 ‍h. After washing, the nuclei were counterstained with DAPI, which facilitated the visualization of TUNEL-positive cells under a fluorescence microscope (Zeiss). The percentage of apoptotic cells was calculated by counting the TUNEL-positive cells relative to the total number of DAPI-stained nuclei in five randomly selected fields per group.

Statistical analysis

All data were presented as mean ± SD. Statistical analyses were performed using GraphPad Prism 8.0 (GraphPad, San Diego, CA). Group differences were analyzed using one-way ANOVA, followed by Tukey’s post hoc test. A p value of <0.05 was considered statistically significant.

Results

PAA improves cardiomyocyte injury in hypoxia-induced mouse primary cardiomyocytes

To evaluate the protective effects of PAA on cardiomyocytes subjected to hypoxic injury, we treated mouse primary cardiomyocytes with varying concentrations of PAA (0, 5, 10, 20, and 40 ‍μM) for 48 ‍h. The results from the CCK-8 assays revealed that lower concentrations of PAA (5 and 10 ‍μM) had modest effects on the viability of the mouse primary cardiomyocytes (Fig. 1A). In contrast, higher concentrations of PAA (20 and 40 ‍μM) suppressed cell viability (Fig. 1A). Moreover, the CCK-8 assay indicated a significant increase in cell viability in the PAA-treated groups compared to the hypoxia-only group, with the 10 ‍μM concentration showing maximal protection (Fig. 1B). Additionally, the release of lactate dehydrogenase (LDH), an indicator of cellular damage, was markedly reduced in the PAA-treated cells compared to those in the untreated hypoxia group (Fig. 1C). These results suggest that PAA is effective in mitigating hypoxia-induced cardiomyocyte injury.

Fig. 1.

PAA improves cardiomyocyte injury in hypoxia-induced mouse primary cardiomyocytes. (A) CCK-8 assays showed the viability of mouse primary cardiomyocytes upon the treatment of PAA at the concentration of 5, 10, 20, and 40 ‍μM for 48 ‍h. The OD450 value was measured. ***p<0.001. (B) CCK-8 assays showed the viability of mouse primary cardiomyocytes upon hypoxia and the treatment of PAA at the concentration of 5 and 10 ‍μM for 48 ‍h. The OD450 value was measured. (C) LDH assays showed the cytotoxicity of mouse primary cardiomyocytes upon hypoxia and the treatment of PAA at the concentration of 5 and 10 ‍μM for 48 ‍h. The LDH levels were measured. ***p<0.001, hypoxia vs control, ^&&p<0.01, ^&&&p<0.001, hypoxia + PAA vs hypoxia.

PAA activates the AMPK/PGC-1α/SIRT3 axis in hypoxia-induced mouse primary cardiomyocytes

We explored the underlying mechanism by which PAA mitigates cardiomyocyte injury in hypoxia-induced mouse primary cardiomyocytes. Specifically, we examined its effect on the AMPK/PGC-1α/SIRT3 axis, a key pathway involved in mitochondrial function and ROS process. Immunoblot analysis revealed that hypoxia suppressed the phosphorylation levels of AMPK and the expression levels of PGC-1α (Fig. 2A). However, PAA treatment effectively reversed these changes, restoring both phosphorylation levels of AMPK and the expression levels of PGC-1α in hypoxia-induced mouse primary cardiomyocytes (Fig. 2A). While hypoxia reduced SIRT3 expression in mouse primary cardiomyocytes, PAA treatment significantly increased SIRT3 expression in hypoxia-induced mouse primary cardiomyocytes (Fig. 2B, up). Additionally, administration of the AMPK inhibitor, Compound C, reversed the upregulation of PGC-1α and SIRT3 in hypoxia-induced mouse primary cardiomyocytes upon PAA treatment (Fig. 2B, down). Therefore, we conclude that PAA activates the AMPK/PGC-1α/SIRT3 axis in hypoxia-induced mouse primary cardiomyocytes.

Fig. 2.

PAA activates the AMPK/PGC-1α/SIRT3 axis in hypoxia-induced mouse primary cardiomyocytes. (A) Immunoblot showed the expression of AMPK and PGC-1α and phosphorylation levels of AMPK in mouse primary cardiomyocytes upon hypoxia and the treatment of PAA at the concentration of 5 and 10 ‍μM for 48 ‍h. (B) Immunoblot showed the expression of SIRT3 in mouse primary cardiomyocytes upon hypoxia and the treatment of PAA at the concentration of 5 and 10 ‍μM for 48 ‍h (up). Immunoblot showed the expression of SIRT3 and in mouse primary cardiomyocytes upon the indicated treatment for 48 ‍h (down). ***p<0.001, hypoxia vs control, ^&p<0.05, ^&&p<0.01, ^&&&p<0.001, hypoxia + PAA vs hypoxia.

PAA improves oxidative stress in hypoxia-induced mouse primary cardiomyocytes by activating SIRT3

To examine the effects of PAA on mitochondrial function under hypoxic conditions, we evaluated several mitochondrial parameters in mouse primary cardiomyocytes. We first detected the MDA levels, which serve as indicators of ROS levels. Our results showed that hypoxia significantly increased MDA levels in mouse primary cardiomyocytes. However, PAA treatment further decreased MDA levels upon hypoxia treatment (Fig. 3A). Importantly, 3-TYP acts as a specific inhibitor of SIRT3. We observed that treatment with 3-TYP or depletion of SIRT3 reversed the decrease in MDA levels caused by PAA treatment in hypoxia cells (Fig. 3A). Additionally, immunostaining for mitochondrial superoxide (mitoSOX) demonstrated a significant reduction in ROS levels in PAA-treated cells subjected to hypoxia stimulation, indicating a decrease in oxidative stress (Fig. 3B). Similarly, 3-TYP treatment or depletion of SIRT3 led to an increase in the ROS levels in mouse primary cardiomyocytes upon hypoxia and PAA treatment (Fig. 3B). In summary, PAA improves oxidative stress in hypoxia-induced mouse primary cardiomyocytes by activating SIRT3.

Fig. 3.

PAA improves oxidative stress in hypoxia-induced mouse primary cardiomyocytes by activating SIRT3. (A) The levels of MDA were measured in mouse primary cardiomyocytes upon hypoxia and the treatment of PAA at the concentration of 5 and 10 ‍μM or 3-TYP, or the depletion of SIRT3 for 48 ‍h. (B) MitoSOX staining showed the ROS levels in mouse primary cardiomyocytes upon hypoxia and the treatment of PAA at the concentration of 5 and 10 ‍μM or 3-TYP, or the depletion of SIRT3 for 48 ‍h. ***p<0.001, hypoxia vs control, ^&&p<0.01, ^&&&p<0.001, hypoxia + PAA vs hypoxia, ^$$$p<0.001, hypoxia + PAA + 3-TYP vs hypoxia + PAA.

PAA improves mitochondrial dysfunction in hypoxia-induced mouse primary cardiomyocytes by activating SIRT3

We investigated the effects of PAA on the mitochondrial function of mouse primary cardiomyocytes upon hypoxia treatment. JC-1 staining demonstrated that hypoxia stimulation diminishes the mitochondrial membrane potential, whereas PAA preserves it, as evidenced by a higher red/green fluorescence intensity ratio in hypoxia-induced cells (Fig. 4). Notably, 3-TYP treatment or depletion of SIRT3 further reduces the mitochondrial membrane potential, indicating the development of mitochondrial dysfunction (Fig. 4). Therefore, PAA improves mitochondrial dysfunction in hypoxia-induced mouse primary cardiomyocytes by activating SIRT3.

Fig. 4.

PAA improves mitochondrial dysfunction of hypoxia-induced mouse primary cardiomyocytes by activating SIRT3. JC-1 staining showed the mitochondrial dysfunction degree in mouse primary cardiomyocytes upon hypoxia and the treatment of PAA at the concentration of 5 and 10 ‍μM or 3-TYP, or the depletion of SIRT3 for 48 ‍h. Red panel indicates aggregate. Green panel indicates monomer. Scale bar, 50 ‍μm.

PAA suppresses apoptosis in hypoxia-induced mouse primary cardiomyocytes by activating SIRT3

We examine the effects of PAA on apoptosis in hypoxia-induced mouse primary cardiomyocytes and investigate the underlying mechanism. Flow cytometry analysis indicated a marked increase in cell apoptosis in hypoxia-induced mouse primary cardiomyocytes. Conversely, PAA treatment significantly decreased apoptosis rates, as demonstrated by lower levels of Bax and cleaved caspase-3 observed in immunoblots (Fig. 5A and B). Notably, the suppression of SIRT3 through 3-TYP or the depletion of SIRT3 further elevated apoptosis rates in PAA-treated cells subjected to hypoxia (Fig. 5A). In addition, 3-TYP treatment or depletion of SIRT3 resulted in increased levels of Bax and cleaved caspase-3 in PAA-treated cells following hypoxia stimulation, suggesting a promotion of apoptosis (Fig. 5B). Therefore, PAA suppresses the apoptosis of hypoxia-induced mouse primary cardiomyocytes by activating SIRT3.

Fig. 5.

PAA suppresses the apoptosis of hypoxia-induced mouse primary cardiomyocytes by activating SIRT3. (A) TUNEL showed the apoptosis levels of mouse primary cardiomyocytes upon hypoxia and the treatment of PAA at the concentration of 5 and 10 ‍μM or 3-TYP, or the depletion of SIRT3 for 48 ‍h. The percentage of apoptosis cells was measured. (B) Immunoblot showed the expression of cleaved caspase-3 and Bax in mouse primary cardiomyocytes upon hypoxia and the treatment of PAA at the concentration of 5 and 10 ‍μM or 3-TYP, or the depletion of SIRT3 for 48 ‍h. ***p<0.001, hypoxia vs control, ^&&p<0.01, ^&&&p<0.001, hypoxia + PAA vs hypoxia, ^$$p<0.01, ^$$$p<0.001, hypoxia + PAA + 3-TYP vs hypoxia + PAA.

Discussion

Acute MI continues to be one of the foremost causes of mortality worldwide, highlighting the pressing need for the development of new treatments for this condition. Mitochondrial dysfunction and ROS play pivotal roles in the pathogenesis of MI. Mitochondrial dysfunction disrupts the energy supply to cardiomyocytes, compromising their survival, while excessive ROS production further aggravates cellular injury and stimulates apoptosis.⁽¹⁹⁾ Preserving mitochondrial function is vital for cardio-protection in patients with MI patients, and reducing ROS can significantly lower the risk of cardiomyocyte death.⁽²⁰⁾ Our study confirms that targeting mitochondrial function and modulating ROS levels may present promising therapeutic strategies for MI. We found that PAA markedly mitigates hypoxia-induced cardiomyocyte injury by activating SIRT3. Natural products could play a significant role in regulating mitochondrial function and oxidative stress in the treatment of MI.

Poria cocos has a long history of use in traditional medicine and is esteemed for its diverse pharmacological properties, including anti-inflammatory, antioxidant, and anticancer effects.⁽²¹⁾ These properties suggest that compounds derived from Poria cocos may offer therapeutic potential for various diseases, particularly cardiovascular conditions. PAA is a key active component, specifically a tetracyclic triterpenoid, known for its protective effects across various pathological settings.⁽¹¹⁾ Our study further reveals that PAA provides significant protection to hypoxia-induced cardiomyocytes by regulating mitochondrial function and alleviating oxidative stress. These findings highlight PAA’s potential as a promising candidate for the treatment of MI.

Our in vitro models clearly demonstrate PAA’s protective effects on mitochondrial function. Our findings indicate that PAA preserves mitochondrial membrane potential, reduces ROS production, and maintains mitochondrial function under hypoxic conditions through the activation of SIRT3. This suggests that PAA can mitigate hypoxia-induced injury in cardiomyocytes by regulating mitochondrial activity and lowering oxidative stress. Based on our findings, PAA holds significant promise as a potential therapeutic agent for MI by targeting mitochondrial function and ROS levels.

SIRT3 plays a vital role in mitochondrial regulation and oxidative defense.⁽²²⁾ In our study, we further examined the role of SIRT3 using its inhibitor, 3-TYP, which confirmed its critical importance in MI. Inhibition of SIRT3 significantly increased ROS levels exacerbated mitochondrial dysfunction, and promoted apoptosis in cardiomyocytes.⁽²³⁾ These findings underscore SIRT3 as a key regulator of mitochondrial health and a potential therapeutic target for MI. Modulating SIRT3, as suggested by our results, could offer new avenues for developing treatments to enhance mitochondrial function and reduce oxidative stress in patients with MI.

The AMPK/PGC-1α/SIRT3 axis plays an essential role in the regulation of mitochondrial function and the production of ROS. Our study illustrates that PAA activates this pathway, enhancing antioxidant capacity and preserving mitochondrial function in hypoxia-induced cardiomyocytes. The activation of this pathway has been shown to provide cardio-protection by decreasing oxidative stress and apoptosis during MI. Several drugs have been identified that target the AMPK/PGC-1α/SIRT3 signaling cascade to slow the progression of MI.^(24,25) Our findings reinforce this concept, demonstrating that PAA effectively alleviated MI by modulating this pathway, thereby emphasizing the therapeutic potential of natural products for the treatment of MI through mitochondrial intervention.

Moreover, our study demonstrates that SIRT3 significantly decreases hypoxia-induced mitochondrial ROS production. This process involves both the induction of antioxidant enzyme expression and the activation of these enzymes via deacetylation. Previous studies have established that SIRT3 deacetylates and activates mitochondrial superoxide dismutase 2 (SOD2) and catalase, enhancing their enzymatic activity to neutralize ROS effectively. Furthermore, SIRT3-mediated deacetylation can improve the stability and functionality of other mitochondrial proteins essential for maintaining redox balance. In line with these mechanisms, our findings suggest that PAA treatment enhances SIRT3 activity, resulting in improved mitochondrial antioxidant defenses in hypoxic conditions. This dual role of SIRT3, regulating both the expression and activity of antioxidant enzymes, emphasizes its central role in combating oxidative stress during MI.

The exact mechanism through which PAA activates AMPK is still a subject of ongoing research. While our findings demonstrate that PAA significantly increases the phosphorylation of AMPK (p-AMPK) under hypoxic conditions, the precise target of PAA in this process is still not fully understood. One plausible explanation is that PAA may activate AMPK via upstream kinases such as LKB1 or CaMKKβ, both of which are involved in the phosphorylation of AMPK at Thr172. These kinases are triggered by cellular stress, including oxidative stress and energy depletion, which are present in the hypoxic conditions used in our study. In addition, the advantageous effects of PAA on mitochondrial function may also contribute to AMPK activation. By promoting mitochondrial health and reducing ROS production, PAA has the potential to restore cellular energy balance, thereby indirectly facilitating AMPK activation. This cascade of events could enhance AMPK phosphorylation, activating downstream pathways, including PGC-1α and SIRT3, which play a role in the protective effects observed in our study.

Our study provides robust evidence of PAA’s cardioprotective effects; however, several limitations necessitate further investigation. First, although our in vitro results are promising, these findings need to be validated in animal models and clinical settings. Second, a deeper exploration of the molecular mechanisms underlying PAA is required to comprehend its role in the treatment of MI fully. Additionally, it is essential to address the safety profile and potential side effects of PAA in future research.

Conclusion

In conclusion, this study represents the first systematic investigation into the protective effects of PAA against hypoxia-induced cardiomyocyte injury. It emphasizes PAA’s role in regulating mitochondrial function and ROS via activating the AMPK/PGC-1α/SIRT3 pathway. These findings establish a robust foundation for developing PAA as a potential therapeutic agent for MI and offer new insights into the application of natural products in cardiovascular disease treatment.

Author Contributions

JY designed the study, completed the experiment and supervised the data collection, QJ analyzed the data, interpreted the data. ZL prepare the manuscript for publication and reviewed the draft of the manuscript. All authors have read and approved the manuscript.

Availability of Data and Materials

All data generated or analyzed during this study are included in this published article.

The datasets used and/or analyzed during the present study are available from the corresponding author on reasonable request.

Conflict of Interest

No potential conflicts of interest were disclosed.