Mitochondrial Permeability Transition Pore: The Cardiovascular Disease’s Molecular Achilles Heel

Cardiovascular diseases (CVDs) remain the leading cause of morbidity and mortality worldwide, with mitochondrial dysfunction increasingly recognized as a pivotal contributor to their pathogenesis. Among the mitochondrial mechanisms implicated in cardiac injury, the mitochondrial permeability transition pore (mPTP) has emerged as a central player in the regulation of cell fate during ischemia–reperfusion events [1]. Upon reperfusion following myocardial infarction, oxidative stress, calcium overload, and ATP depletion converge to trigger mPTP opening, resulting in mitochondrial depolarization, bioenergetic collapse, and activation of regulated cell death pathways.

This mitochondrial crisis affects various cardiac cell types, including cardiomyocytes, endothelial cells, and fibroblasts, differently, contributing to complex pathological remodelling and expansion of the infarct zone. Key mitochondrial processes, such as impaired mitophagy, disrupted dynamics, and excessive reactive oxygen species (ROS) production, further exacerbate tissue damage and compromise cardiac function [2].

Recent advances in mitochondrial biology have unveiled promising therapeutic strategies aimed at modulating mPTP activity, enhancing mitochondrial quality control, and even exploring mitochondrial transplantation. These approaches hold the potential to mitigate myocardial injury, improve cardiac outcomes, and redefine therapeutic paradigms in CVD management. However, challenges remain, including targeted drug delivery, safety, and patient-specific variability. Continued research into mitochondrial-targeted therapies and precision medicine will be essential to translate these insights into effective clinical interventions.

Regulated cell death (RCD) is a maladaptation of cardiomyocytes in heart diseases with various degrees of regulation in multifaceted molecular events [3]. Profound and long-lasting effects of RCD occur in cardiomyocytes, remaining largely refractory to regenerative cues [4]. Cellular energy homeostasis controlled by mitochondria is compromised in the presence of reduced oxygen availability. As a consequence, mitochondrial dysfunction arises from this pathological condition, triggering a cascade of molecular events that compromise cardiac functions [5]. Tissues with high energy demands, such as the heart, suffer bioenergetic failure during hypoxia or ischemic conditions. The alteration of mitochondrial function is caused by the disruption of oxidative phosphorylation (OXPHOS) dependent on oxygen deprivation. Impaired mitochondria can decrease ATP production, whereas mitochondrial calcium homeostasis is often dysregulated, and reactive oxygen species (ROS) are generated by mitochondrial electron transport chain inhibition [6]. This critical triangle promotes deleterious cellular pathology leading to the formation of the mPTP [7]. The collapse of the electrochemical gradient of protons through the inner mitochondrial membrane (IMM) blocks OXPHOS, whereas ions with small molecules efflux-influx across the IMM are not regulated. Uncontrolled water uptake into the mitochondrial matrix, a consequence of mPTP opening, leads to mitochondrial swelling [8]. This swelling causes the outer mitochondrial membrane and/or IMM to rupture to release mitochondrial proteins and causing organelle damage, ultimately resulting in regulated cell death. mPTP formation plays a crucial role in the progression of irreversible damage to cardiovascular cells. The nature of this biological effect has a negative effect on the bioenergetics of mitochondria [9,10]. The permeability transition of the IMM, when an abrupt elevation of intramitochondrial Ca² occurs, is mediated by the opening of mPTP, which permits the passage of solutes with a molecular mass up to approximately 1.5 kDa [11]. The component and structure of mPTP is not fully understood, even if different evidence suggests the adenine nucleotide translocator (ANT) and, in the last 10 years, with strong evidence, the involvement of F₁F_O-ATPase in mPTP formation [12,13,14]. Among all the protein components of the mPTP, only cyclophilin D (CyPD) is the protein characterized by having a regulatory role that influences the pore opening [15]. Recently, ATAD3 has been established as the first essential component of the mPTP [16]. Redox state of thiol groups can modulate the formation of mPTP and sulfhydryl reagents, as diamide and phenylarsine oxide are mPTP activators, whereas dibromobimane is an inhibitor [17]. Other amino acids play a key role in mPTP regulation. Protonation of the specific residue of Hys at pH 6.5 has an inhibitory effect on mPTP and can be relieved by carbethoxylation with diethylpyrocarbonate [18]. Conversely, phenylglyoxal, a known compound to cause post-translational modifications of Arg residues, stimulates the mPTP opening [19,20]. Typical inhibitors of mPTP, such as ADP and cations as Mg²⁺, Sr²⁺, Ba²⁺, and Gd³⁺, which are also recognized as substrates and cofactors of F₁F_O-ATPase, block mPTP formation [21,22]. Atractylate (ATR) or carboxyatractylate (CATR) and bongkrekate (BKA) are proposed to determine the mPTP opening and mPTP closure, respectively [23]. Moreover, ATR and CATR lock ANT in the c-state (nucleotide binding site facing the cytosol), whereas BKA stabilizes it in the m-state (nucleotide binding site facing the matrix). In addition to this, by considering the natural ligand ATP and ADP of ANT, the adenine nucleotide exerts selective inhibition on the mPTP opening. ANT may, therefore, have a role as a component of mPTP [24]. Pharmacological compounds are used to block the pore opening in pathological conditions. Cyclosporin A is the most commonly used inhibitor of mPTP by targeting the CyPD [25]. However, different small synthetic molecules or natural molecules targeting the F₁F_O-ATPase have been proposed in different drug discovery studies to elucidate the mechanisms governing mPTP opening [26,27,28,29,30,31,32,33,34,35,36,37,38].

The conditions used to open the mPTP can selectively activate different pore populations or sensitivities, all characterized by a Ca²⁺-induced channel facilitating Ca²⁺ release. The methods used to detect mPTP activity can make the interpretation of pore formation sensitive to type of assay performed. Direct assessments of mPTP opening include calcium retention capacity and monitoring of transmembrane potential by using fluorescent dyes, mitochondrial swelling assays evaluating optical density linked to osmotic collapse of mitochondria, and electrophysiological measurements detecting the ionic currents dependent on membrane permeability [39].

mPTP opening appears to be a pivotal molecular event in the pathological condition, triggering several RCD events following myocardial infarction (MI). For this reason, mPTP desentization is a critical process in ischaemic pre- and postconditioning. Specific inhibition of mPTP opening during reperfusion after acute MI offers significant antinecrotic and antiapoptotic protection [40]. The occurrence and progression of cardiovascular diseases (CVDs) associated with mitochondrial dysfunction are due to a bioenergetics imbalance of mitochondria. Particularly, overload of Ca²⁺ in mitochondria, which disrupts its homeostasis, triggers mPTP opening leading to excessive ROS production, responsible for oxidative stress-induced damage [41,42]. As a consequence of impaired mitochondrial function, the mPTP formation occurs to support pathological changes in the cardiovascular system. The knowledge about the mPTP formation in CVDs is a critical point guiding the development of innovative treatments to improve outcomes and quality of human health (Figure 1). This review provides directions for the mPTP mechanism involved in CVDs as a potential therapeutic target and interventions for the prevention and treatment of mitochondrial dysfunction in CVDs.

The irreversible damage may occur primarily upon myocardial reperfusion. In this specific context, ischemia/reperfusion (I/R) injury refers to a phenomenon in which the restoration of blood flow may paradoxically result in additional damage. As a protective intervention, ischaemic postconditioning, acting on repeated interruptions of blood flow (ischemia) during the early phase of reperfusion, following a period of sustained ischemia, serves as a protective intervention that can mitigate tissue damage resulting from ischaemia–reperfusion injury [43]. Pharmacological inhibition of the mPTP during reperfusion may serve as a significant adjunct therapy to dramatically reduce infarct size [44]. mPTP opening following acute myocardial infarction is a key event attenuating myocardial apoptosis and necrosis. Cardioprotection has been investigated with a non-immunosuppressive CyPD inhibitor identified as NIM811, which confers cardioprotection when administered at reperfusion. The specific binding to the CyPD, without interaction with cytosolic CyPA, which causes interference within the cellular survival/death pathways, can protect the ischemic heart in in vivo model of acute myocardial infarction [40]. To mitigate lethal reperfusion injury, postconditioning can be used as a potent cardioprotective strategy that relies on brief periods of I/R to limit the damage caused by reperfusion after a prolonged heart attack.

Emerging evidence considers the critical role of mPTP in the mechanism of postconditioning [45]. The complex phenomenon of reperfusion injury encompasses several detrimental biological effects. Prolonged ischemia leads to a progressive breakdown of ionic homeostasis, resulting in intracellular accumulation of Na⁺ and Ca²⁺, ATP depletion, and the onset of ischemic contracture. During ischemia, glycolysis causes a gradual increase in acidosis. Cardiomyocytes counteract the low pH through the Na⁺/H⁺ exchanger, which drives Na⁺ influx. However, due to ATP decreases, Na⁺/K⁺-ATPase cannot regulate intracellular Na⁺ levels. Consequently, the Na⁺/Ca²⁺ exchanger operates in reverse mode, expelling Na⁺ and promoting cytosolic Ca²⁺ overload [45,46,47]. Moreover, Ca²⁺ and Na⁺ play an important role in cells as mediators of second messengers or membrane potential, respectively. Ca²⁺ involved in mPTP opening can be supported by Na⁺ stimulation with ROS production that triggers the mPTP phenomenon. In addition to this, Na⁺ accumulation, behaving like a second messenger, modifies the fluidity of the IMM to control OXPHOS activity and ROS generation [48]. Na⁺ signalling is linked to cell acidification driven by glycolytic activation during ischemia. The resulting decrease in mitochondrial matrix pH triggers the release of free Ca²⁺ from calcium phosphate deposits and simultaneously activates the mitochondrial Na⁺/Ca²⁺ exchanger. This exchanger facilitates Na⁺ entry into the matrix through both electroneutral and electrogenic exchange mechanisms, a process governed by the mitochondrial membrane potential under ischemic conditions. Na⁺ excess interacts with phospholipids of IMM, reducing its fluidity. This decrease in membrane fluidity limits the mobility of free ubiquinone between Complex II and Complex III, while leaving its movement within respiratory supercomplexes unaffected [49]. The alteration of electron transport in respiratory complexes leads to the formation of superoxide at Complex III. Therefore, Na⁺ regulates OXPHOS activity and redox signalling, profoundly impacting cellular metabolism under anaerobic conditions [48]. This dysregulation leads to excessive ROS generation and mitochondrial Ca²⁺ overload, ultimately triggering mPTP opening, the main cause of lethal reperfusion injury [50].

In addition to this, postconditioning also emerges as a promising strategy to mitigate lethal reperfusion injury, and accumulating evidence points toward mPTP as a central player in this process. The temporal coincidence between reperfusion and mPTP opening, the modulation of its activity by genetic and/or pharmacological interventions, and its established role in postconditioning collectively support the hypothesis that mPTP inhibition contributes to postconditioning-induced cardioprotection. Future studies should aim to clarify the precise molecular mechanisms linking postconditioning stimuli to mPTP regulation, as this could open new therapeutic avenues for limiting I/R injury [45]. In lethal reperfusion injury, a powerful anti-ischemic protection is provided with post-conditioning blocks of the mPTP [51].

Recent studies have further elucidated the molecular involvement of necroptosis, a regulated form of cell death distinct from apoptosis, as a key contributor to MI and I/R injury. The results highlight its role as a promising target for cardioprotective strategies. Necroptosis is characterized by its pro-inflammatory properties, which worsen fibrosis, myocardial injury, and unfavourable cardiac remodelling. The regulatory proteins eceptor-interacting protein kinase 3 (RIPK3)/mixed lineage kinase domain-like protein (MLKL), which mediate inflammation and cell death, are essential to this process [52]. According to recent data, calcium/calmodulin-dependent protein kinase II (CaMKII), as a substrate for RIPK3, forms together with mPTP a signalling axis depicted as a crucial mechanism in necroptosis. By blocking the RIPK3-CaMKII-mPTP pathway, it may have cardioprotective effects. Therefore, focusing on necroptosis is a potentially effective treatment approach to reduce cardiac damage, improve recovery, and possibly avoid heart failure after MI [53,54].

On balance, upon reperfusion, the abrupt restoration of physiological pH, and oxygen or ischaemic accumulation of succinate [55] triggers a burst of mitochondrial respiration and ROS generation (Figure 2).

This oxidative stress, combined with the pre-existing Ca²⁺ overload and ATP depletion, creates a permissive environment for the opening of mPTP. The mPTP, as a non-specific channel that spans the IMM, when open, leads to mitochondrial depolarization, matrix swelling, rupture of the outer membrane, and release of pro-apoptotic factors such as cytochrome c. These events culminate in necrotic and apoptotic cell death, significantly contributing to infarct size.

Mitochondrial dysfunction in cardiovascular diseases does not affect all myocardial cell types uniformly. The heart is composed of various cell populations, including cardiomyocytes, endothelial cells, fibroblasts, and immune cells, each with distinct metabolic profiles and mitochondrial dependencies. The pathomechanism of mitochondrial dysfunction varies across these cell types, contributing to the complexity of cardiac pathology. Key pathological features, which can be identified in the mPTP opening, Ca²⁺ overload, membrane potential disruption, mitochondrial fission, and impaired mitophagy, drive mitochondrial-dependent regulated cell death, with cell-type-specific manifestations that collectively determine the extent of myocardial injury and remodelling [56] (Figure 3).

Mitochondrial homeostasis is essential for maintaining cardiac function, as mitochondria regulate energy production, redox balance, Ca²⁺ signalling, and cell survival. Disruption of these processes leads to the progression of CVDs such as heart failure and I/R injury [73]. This dysregulation extends to core processes of mitochondrial quality control, encompassing dynamic regulation through fusion and fission, maintenance of organelle mass via biogenesis, and turnover through selective clearance via mitophagy. Furthermore, the altered function of mitochondria, as the main cause of ROS production, promotes pro-inflammatory and pro-thrombotic states, where the release of oxidized lipid particles and mitochondrial DNA into the cytoplasm acts as a DAMP, potentially triggering innate immune responses [70,74].

Innovative therapeutic strategies are emerging to restore mitochondrial function and improve patient outcomes. These include pharmacological agents targeting mitochondrial dynamics and bioenergetics, mitochondria-targeted antioxidants, and modulators of mitophagy and mPTP opening [9,58]. Novel approaches such as mitochondrial transplantation, mitochondrial transfer, and gene therapy aim to replace or repair damaged mitochondria, while lifestyle interventions like structured exercise have shown efficacy in enhancing mitochondrial biogenesis and quality control. Collectively, these strategies hold promise for reducing morbidity and improving quality of life in patients with CVDs by targeting the central role of mitochondria in cardiac health [75].

Recent findings highlight novel therapeutic strategies focusing on the modulation of mitochondrial bioenergetics, mitochondrial dynamics and quality control mechanisms, such as mitophagy. However, emerging research has expanded the understanding of mitochondrial-derived signals in cardiac pathophysiology and introduced innovative approaches like mitochondrial transplantation. Transfer therapies of mitochondria may enhance cardiac function after MI and attenuate left ventricular remodelling by reducing fibrosis, limiting apoptosis, and promoting angiogenesis [76]. Nevertheless, several limitations constrain this approach, the most significant being the relatively rapid loss of cardiomyocytes following MI. On balance, targeting mitochondrial pathways represents a highly promising therapeutic strategy for managing CVDs.

However, translating mPTP-targeted interventions into clinical practice remains challenging. Limitations include incomplete understanding of pore structure and regulation in human myocardium, variability in patient response, and potential off-target effects due to the ubiquitous role of mitochondria in multiple tissues. The possibility of disrupting physiological permeability transition, which is crucial in regular Ca²⁺ signalling and metabolic adaptation, raises safety concerns. Furthermore, long-term inhibition of mPTP could predispose to abnormal mitochondrial dynamics or impaired mitophagy. To improve patient stratification, emerging biomarkers such as circulating mtDNA [77], cardiolipin oxidation products, and mitochondrial Ca²⁺ load are being investigated as indicators of mitochondrial stress and therapeutic responsiveness [78]. Addressing these challenges through rigorous preclinical validation and biomarker-driven clinical trials will be essential for successful therapeutic translation.

Beyond these concepts, several mitochondria-directed therapies are advancing from preclinical work into early clinical evaluation (Table 1).

Mitochondria-targeted antioxidants aim to preserve respiratory chain function and limit ROS-driven damage, whereas small molecules that modulate mitochondrial dysfunction, mitophagy, or mPTP opening seek to restore mitochondrial quality control and stress tolerance [29,85]. In parallel, innovative approaches such as mitochondrial transplantation, mitochondrial transfer via extracellular vesicles, and gene- or RNA-based strategies targeting mitochondrial regulators are being explored as means to directly repair or replace dysfunctional organelles [86,87]. Collectively, these emerging interventions could complement guideline-directed therapies and enable more precise, mechanism-based management of CVDs.

CVDs remain the leading cause of morbidity and mortality worldwide, with mitochondrial dysfunction recognized as a central contributor to their pathogenesis. Recent advances have deepened our understanding of mitochondrial roles in cardiac bioenergetics, signalling, and quality control, while highlighting innovative therapeutic strategies such as modulation of mitophagy and regulated cell death, mPTP-dependent, and mitochondrial-targeted drugs. To build on promising preclinical and early clinical results, it is essential to overcome key challenges, including targeted delivery, safety, and patient variability. Continued research into mitochondrial biology and drug discovery strategies will be essential to develop effective, personalized therapies that improve outcomes and quality of life in patients with CVDs.