High-intensity interval training improves mitochondrial function and attenuates cardiomyocytes damage in ischemia-reperfusion

Abstract

Ischemic heart disease remains a leading cause of global mortality and morbidity, underscoring the urgent need for effective therapies particularly for patients recovering from ischemic cardiac events. High-intensity interval training (HIIT) has emerged as a promising non-pharmacological intervention with substantial cardioprotective potential. Both clinical studies and animal models demonstrate that HIIT attenuates cardiac damage induced by ischemia–reperfusion injury. However, the precise cellular and molecular mechanisms underlying these benefits remain incompletely elucidated. Mitochondria play a pivotal yet dual role in ischemia–reperfusion injury, serving as central regulators of both cell survival and death pathways. Although HIIT is recognized to modulate mitochondrial function, its specific impact on cardiac susceptibility to ischemic injury requires further clarification. This review synthesizes current evidence on the beneficial effects of HIIT on cardiac mitochondrial function, with a focus on the mechanisms through which it confers cardioprotection. We examine how HIIT may enhance mitochondrial resilience by activating critical signaling pathways that mitigate ischemia–reperfusion injury. Despite significant advances, key knowledge gaps persist. This review emphasizes the necessity of further research to fully unravel HIIT’s cardioprotective potential and its role in promoting mitochondrial adaptation under ischemic stress.

1. Introduction

Cardiovascular disease (CVD) remains a growing public health concern, significantly impairing physical function, increasing the risk of mortality, and healthcare costs. Despite ongoing efforts to alleviate its impact, CVD continues to be the leading cause of death worldwide, with its prevalence remaining alarmingly high. In 2019, CVDs accounted for 17.9 million deaths, representing 32 % of all global mortality. One of the most detrimental consequences of ischemia–reperfusion injury (IRI) is its effect on cardiomyocyte mitochondria. During ischemia, oxygen and respiratory substrates deprivation inhibit oxidative phosphorylation, resulting in mitochondrial membrane potential collapse, swelling, and Ca²⁺ overload. These pathological changes trigger cell necrosis, cytochrome c release, and membrane disintegration. Reperfusion further exacerbates this damage by generating oxidative stress, reactive oxygen species (ROS), and mitochondrial dysfunction. Mitochondria play a central role in mediating pathways leading to both necrotic and apoptotic cell death [1]. Consequently, mitochondrial-targeted therapies have emerged as a critical focus for addressing the role of mitochondrial dysfunction in the etiology and progression of IRI [2].

High-intensity interval training (HIIT) is characterized by alternating bouts of high-intensity aerobic activity performed at or near maximum oxygen consumption with periods of light recovery exercise or rest. It has gained as a powerful non-pharmacological therapy for cardiac rehabilitation [3]. HIIT has been shown to improve cardiac function and survival in patients with ischemic cardiac disease while reducing CVD risk factors [4]. Whether implemented as a standalone intervention or integrated into broader cardiac rehabilitation program, HIIT has demonstrated significant benefits in mitigating long-term disability, enhancing cardiac function capacity, preventing left ventricular remodeling, and alleviating cardiac symptoms. Evidence suggests that four days of HIIT per week effectively minimize cardiac tissue damage during ischemia and reperfusion in both human and rodent models [5]. Comparisons between HIIT and moderate continuous training (MCT) have further highlighted the superior effects of HIIT in improving cardiac function [6].

Exercise and hypoxia are known to enhance mitochondrial biogenesis and turnover. The electron transport chain (ETC) not only drives ATP production but also facilitates the repair of damaged mitochondrial sites through oxygen consumption. However, many cardiovascular disorders, including atherosclerosis, reperfusion damage, and cardiomyopathy, disrupt mitochondrial turnover [7]. Zhang et al., 2024 demonstrated in a mouse model that HIIT significantly lessens myocardial IRI, underscoring its therapeutic potential. While HIIT has shown promising cardioprotective effects, the underlying cellular and molecular mechanisms, particularly those involving mitochondrial function, remain incompletely understood. Despite compelling evidence supporting its benefits in improving cardiac function and reducing ischemic damage, the precise ways in which HIIT influences mitochondrial health during IRI require further investigation. Understanding these mechanisms is essential for developing targeted interventions to minimize myocardial damage and enhance recovery in individuals with ischemic cardiac disease. The objective of this review is to critically examine the protective effects of HIIT on cardiac mitochondrial function during IRI. Specifically, this review will explore the physiological and molecular mechanisms underlying HIIT-induced cardioprotection, focusing on its role in restoring mitochondrial health and reducing myocardial damage during IRI.

2. Mitochondrial involvement in cardiac ischemia-reperfusion injury

Mitochondria are vital organelles within eukaryotic cells, playing a central role in energy generation and stress response regulation. Their critical involvement in cardiac IRI has been extensively studied [8]. This section outlines key concepts essential for understanding mitochondrial roles in IRI, setting the foundation for subsequent discussions.

2.1. Structure and function of cardiac mitochondria

Under normal physiological conditions, the structure and function of cardiac mitochondria are well-characterized [9]. Cardiomyocytes contain two distinct mitochondria subtypes: subsarcolemmal (SSM) and intermyofibrillar (IFM) mitochondria. These subtypes differ in location, morphology, and functional roles, including respiratory and Ca²⁺ retention capacities. IFM are located between myofibril bundles near the sarcoplasmic reticulum (SR) Ca²⁺ release sites. These mitochondria generally exhibit consistent sizes and predominantly tubular shapes [10]. In contrast, SSM are positioned closer to the sarcolemma and display greater variability in shape and size. This morphological diversity correlates with functional differences. For example, Boengler, K., et al., (2009) found that connexin 43 (GJA1) is exclusively localized to the inner membrane of SSM, suggesting a role in regulating processes such as respiration, ROS production, and ATP synthesis at complex I of the ETC [1,11,12].

Mature cardiomyocytes rely on mitochondria to generate approximately 90 % of their ATP, with these organelles occupying about 30 % of the cell volume. The heart’s rhythmic contractions demand a continuous, efficient supply of energy, which mitochondria provide. Beyond energy production, mitochondria regulate Ca²⁺ homeostasis, ROS generation, and cell death processes [13]. Consequently, mitochondria are integral to both normal cardiac function and pathological conditions. Mitochondria generate cellular energy through oxidative phosphorylation (OX-PHOS), where substrates from the cytosol to fuel the tricarboxylic acid (TCA) cycle, producing reduced nicotinamide adenine dinucleotide (NADH) and reduced flavin adenine dinucleotide (FADH2). These molecules power the ETC, which establishes the mitochondrial membrane potential (ΔΨm) and pH gradient (ΔpHm). These gradients drive ATP synthesis via ATP synthase (complex V). Approximately 60–70 % of ATP supports cardiac contraction, while 30–40 % is utilized by the sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase (SERCA) and other ion pumps [14].

Mitochondria also play a critical role in cellular Ca²⁺ homeostasis, modulating numerous signaling pathways and maintaining the balance between function and dysfunction. During increased cardiac workload, intracellular Ca²⁺ levels rise, facilitating excitation–contraction coupling. Mitochondria remove cytosolic Ca²⁺ via the mitochondrial Ca²⁺ uniporter (MCU), a selective Ca²⁺ channel. Elevated mitochondrial Ca²⁺ levels activate enzymes in the Krebs cycle, ATP synthase, and adenine nucleotide translocator, enhancing OX-PHOS [15,16]. However, excessive Ca²⁺ accumulation can lead to pathological outcomes, including ROS overproduction, ΔΨm disruption, mitochondrial swelling, cytochrome C release, and apoptosis [17].

Moreover, mitochondria are the primary intracellular sources of ROS, which are by-products of the ETC [1]. Under normal conditions, ROS levels are tightly regulated by enzymatic antioxidant systems, ensuring localized redox signaling [18]. In pathological states, respiratory chain dysfunction can cause excessive ROS production. Moderate ROS levels can trigger stress responses and apoptosis by increasing mitochondrial membrane permeability and modulating Bcl2 family protein expression [19].

2.2. Mitochondrial dynamics in ischemia-reperfusion injury: mechanisms and therapeutic targets

Given the heart’s dependence on aerobic metabolism, cardiac cells are particularly vulnerable to oxygen and substrate depletion, making mitochondrial function critical in IRI. As previously discussed, mitochondria are central to both cell survival and death. Although the role of mitochondria in IRI has been extensively reviewed [8], this section aims to provide a concise summary of their crucial involvement in IR processes.

Current evidence suggests that cell death following IR is predominantly driven by the activation of the mitochondrial permeability transition pore (mPTP). This activation causes bioenergetic failure, membrane destabilization, and the release of pro-apoptotic factors [20]. During reperfusion, mPTP activation is intricately regulated by the interplay between ROS generation and Ca²⁺ overload [21,22], as shown in Point 5, Fig. 1. Excessive mitochondrial Ca²⁺ accumulation significantly increases the likelihood of mPTP activation, resulting in the collapse of ΔΨm, inhibition of mitochondrial respiration, and eventual cell death [23]. Elevated mitochondrial matrix ROS levels exacerbate mPTP activation [24], as illustrated in Point 4, Fig. 1.

Fig. 1.

Mitochondrial function in the heart during ischemia and reperfusion injury. Lactic acid buildup and intracellular acidosis occur when oxygen delivery is interrupted during cardiac ischemia. This disruption halts oxidative phosphorylation (OX-PHOS) and shifts the cell's energy metabolism to anaerobic glycolysis (1). As a result, cellular processes are compromised, and the pH decreases. To counteract the acidosis, the Na+/H+ exchange extrudes H+, leading to cellular Na+ overload. In response, the 2Na+/Ca²⁺ exchange (NCX) is activated in reversed mode to export the excess Na+, which leads to an excess of Ca²⁺ in the cytosol (2). As ischemia progresses, ATP levels gradually deplete, impairing ion pumps and exacerbating the intracellular Ca²⁺ buildup. During both ischemia and reperfusion, mitochondrial Ca²⁺ accumulation further elevates cytosolic Ca²⁺ levels. Low ATP production is mainly a result of OX-PHOS suppression, which is linked to a decreased mitochondrial membrane potential (Δψm). During ischemia, mPTP channels remain closed due to both the reduced mitochondrial potential and acidic pH (3). However, upon reperfusion, the rapid restoration of OX-PHOS and Δψm leads to excessive Ca²⁺ accumulation in the mitochondria, significantly increasing ROS production (4). The combined effects of ROS generation and Ca²⁺ overload interact to trigger mPTP opening (5). This in turn results in Δψm collapse, suppression of mitochondrial respiration, mitochondrial swelling, dysfunction, and ultimately cell death (6).

Elevated mitochondrial matrix [Ca²⁺] and ROS levels during reperfusion disrupt the function of various Ca²⁺ channels, pumps, and exchangers, highlighting their interdependence [22]. The collapse of ΔΨm further amplifies mitochondrial ROS production through a process known as “ROS-induced ROS release” [25]. Recent studies have identified reverse electron transport at complex I as a key mechanism driving excessive ROS generation during the initial stages of reperfusion [26]. During ischemia, succinate accumulates electrons in the absence of oxygen. Upon reperfusion, the rapid re-oxidation of succinate-by-succinate dehydrogenase initiates reverse electron transport at mitochondrial complex I. Notably, inhibiting complex I with rotenone reduces ROS levels, oxidative damage, and IRI [27]. Similarly, reducing succinate accumulation during ischemia using dimethyl malonate has been shown to lower ROS production upon reperfusion [28].

Mitochondria play dual roles in IRI, mediating both cell death and survival mechanisms. Numerous survivals signaling pathways converge on mitochondria, underscoring their critical role in the cellular response to injury. Ischemic conditioning protects the heart from IR injury by activating the reperfusion injury salvage kinase (RISK) and survivor activating factor enhancement (SAFE) pathways, which converge on mitochondria to inhibit mPTP activation and promote cell survival [29]. Targeting mPTP activation during reperfusion remains a promising cardioprotective strategy. In mice, pharmacological inhibition or genetic deletion of cyclophilin D effectively prevents mPTP activation during IR, leading to reduced infarct size [30]. Cyclosporine A, an immunosuppressant, inhibits mPTP activation by binding to cyclophilin D [24]. Although this approach showed efficacy in experimental models, it failed to reduce mortality or the risk of cardiac failure in patients receiving cyclosporine A before percutaneous coronary intervention [31]. This highlights the need for further research into the mechanisms underlying IRI and cardioprotection. By addressing the complex interplay between ROS, Ca²⁺, and mitochondrial pathways, future strategies may identify novel interventions to mitigate IR-induced cardiac damage and improve clinical outcomes.

3. Key pathophysiological mechanisms and contributing factors in IRI

IRI arises from a complex interplay of extracellular, intracellular, and mechanical mechanisms [32,33], intricately linked to inflammatory responses. Within this context, the coronary circulation functions as both a contributor to and a target of these pathological processes. IRI disrupts physiological homeostasis and significantly influences post–ST-segment elevation myocardial infarction (STEMI) left ventricular remodeling, thereby heightening the risk of arrhythmias and contributing to the progression toward heart failure. This section aims to examine several of the most promising therapeutic strategies, recognizing that an effective clinical approach may require simultaneous targeting of multiple pathways [32].

Recent findings highlight the central role of the NOD-like receptor protein 3 (NLRP3) inflammasome in the pathogenesis of IRI. Histological analyses have revealed the presence of NLRP3 aggregates during the early phases of acute myocardial infarction (AMI), particularly within cardiomyocytes and endothelial cells located in the ischemic core and border zones. Subsequent pathological changes include leukocyte infiltration and fibroblast activation. As inflammation resolves, NLRP3 expression becomes more localized, primarily within isolated cardiomyocytes and fibroblasts [34].

Various regulated forms of cell death contribute to the development of IRI, including pyroptosis, necrosis, apoptosis, ferroptosis, and autophagy, each of which exacerbates inflammation and may promote pyroptotic activity [35]. Among these, pyroptosis is a distinctive inflammatory form of programmed cell death marked by the formation of membrane pores in cardiomyocytes, the release of proinflammatory cytokines, and eventual cell lysis [36]. This process typically occurs in response to sterile inflammation triggered by endogenous cellular damage or pathogenic signals [32]. Mechanistically, pyroptosis is initiated when injured cardiomyocytes activate the NLRP3 inflammasome by recruiting the apoptosis-associated speck-like protein containing a CARD (ASC) and procaspase-1. This leads to the formation of a multiprotein inflammasome complex that activates caspase-1, which in turn cleaves gasdermin D (GSDMD), the key effector molecule responsible for pore formation and pyroptotic cell death [37].

Conversely, functional autophagy, another regulated form of cell death, can suppress NLRP3 activation and limit cytokine release. However, impairment in autophagy or mitophagy pathways may result in incomplete clearance of damaged organelles, thereby activating NLRP3 and aggravating myocardial injury [38]. The primary mechanisms implicated in MIRI and closely associated with NLRP3 activation include excessive production of ROS, intracellular calcium overload, opening of the mPTP, endothelial dysfunction, and sustained inflammation.

3.1. ATP and mitochondrial function

Ischemia arises when blood supply becomes insufficient to meet the metabolic demands required for normal cellular function, leading to deficiencies in oxygen, glucose, and other essential substrates. Metabolic derangements commence during the ischemic phase, initiating a cascade of pathological changes. Initially, mitochondrial anaerobic glycolysis facilitates glycogen breakdown, generating two adenosine triphosphate (ATP) molecules per glucose unit while concurrently producing lactic acid. The resultant decline in tissue pH subsequently exerts negative feedback, further inhibiting ATP synthesis. Thereafter, ATP undergoes sequential degradation into adenosine diphosphate (ADP), adenosine monophosphate (AMP), and inosine monophosphate (IMP), followed by further catabolism into adenosine, inosine, hypoxanthine, and xanthine.

At the cellular level, the cessation of ATP production impairs the function of ATP-dependent ionic pumps, notably the Na+/K+-ATPase and Ca²⁺-ATPase, leading to the dissipation of transmembrane electrochemical gradients. This failure results in intracellular sodium accumulation, which osmotically drives water influx and subsequent cellular edema as the cell attempts to maintain osmotic equilibrium. Concurrently, potassium ions efflux from the cell into the extracellular space to compensate for the ionic imbalance. Simultaneously, Ca²⁺ is released from mitochondrial stores into both the cytosol and extracellular matrix. This Ca²⁺ influx activates Ca²⁺-dependent proteases, particularly calpain, which catalyzes the conversion of xanthine dehydrogenase to xanthine oxidase. Furthermore, ischemia induces phospholipase activation, promoting membrane phospholipid degradation and consequent elevation of circulating free fatty acids.

3.2. ROS-mediated damage in ischemia-reperfusion

ROS serve as critical mediators of tissue damage in IRI through multiple mechanisms. During the ischemic phase, ATP degradation generates hypoxanthine. Upon reperfusion, molecular oxygen becomes available, enabling xanthine oxidase to catalyze the conversion of hypoxanthine into uric acid, while simultaneously releasing the highly reactive superoxide anion (O₂⁻). Superoxide is further metabolized into hydrogen peroxide (H₂O₂) and the hydroxyl radical (OH^•), both of which contribute to oxidative stress.

A major consequence of hydroxyl radical formation is lipid peroxidation, which disrupts cell membrane integrity, leading to the release of proinflammatory eicosanoids, loss of cellular permeability, and eventual cell death. Additionally, ROS activate endothelial cells during IRI, upregulating the transcription factor NF-κB. This activation induces the expression of adhesion molecules, including E-selection, vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), and endothelial-leukocyte adhesion molecule-1 (ELAM-1), as well as prothrombotic and proinflammatory mediators such as plasminogen activator inhibitor-1 (PAI-1), tissue factor, and interleukin-8 (IL-8). These molecules facilitate critical interactions between neutrophils and the endothelium, a process that will be examined in greater detail in subsequent sections.

Emerging evidence suggests that HIIT may mitigate IRI by targeting oxidative stress pathways. Superoxide anions (O₂^•−), detectable in ischemic muscle and venous effluent of reperfused limbs [39], contribute not only to local tissue damage but also to remote organ dysfunction during skeletal muscle IRI. The widespread distribution of xanthine oxidase across tissues implies variable susceptibility to oxidant-mediated injury, a process potentially modulated by HIIT-induced mitochondrial adaptations. Pre-ischemic administration of allopurinol, a xanthine oxidase inhibitor, reduces O₂^•− production and attenuates IRI severity in animal models (skeletal muscle, brain, and gut), supporting the mechanistic role of oxidative stress in tissue damage. Clinically, allopurinol shows promise in reducing postoperative cardiac dysfunction and arrhythmias after coronary artery bypass grafting [40], though larger trials are needed. Notably, HIIT’s ability to enhance mitochondrial efficiency and antioxidant capacity may similarly protect cardiomyocytes by: Reducing ROS generation through improved electron transport chain function, upregulating endogenous antioxidants (e.g., superoxide dismutase), Preserving xanthine dehydrogenase (antioxidant form) over xanthine oxidase (pro-oxidant form). While direct studies on HIIT and xanthine oxidase modulation remain limited, its documented effects on mitochondrial biogenesis and redox homeostasis suggest synergistic cardioprotective potential against IRI.

3.3. Calcium overload and pyroptosis in IRI

IRI disrupts calcium handling mechanisms, leading to impaired calcium homeostasis and an abnormal elevation in intracellular calcium concentrations. This calcium overload significantly affects mitochondrial function by reducing ATP levels, altering mitochondrial membrane potential, and promoting the opening of the mPTP. A strong association has been established between calcium overload, NLRP3 inflammasome activation, and pyroptosis in various tissues, including the myocardium [37]. In the context of IRI, calcium overload acts as a pro-oxidative stimulus, exacerbating oxidative stress and facilitating inflammasome formation [37]. For instance, Mo et al. [41] demonstrated that calcium overload induces pyroptosis in a hypoxia/reperfusion model of adult rat cardiomyocytes through activation of the NLRP3/caspase-1 signaling pathway.

3.4. Role of mPTP and ROS in Mitochondria-Induced cardiomyocyte death

Mitochondria are essential organelles responsible for cellular energy production through oxidative phosphorylation [42]. Following ischemia, reduced oxygen availability, along with the generation of ROS during the early phases of reperfusion as well as the phenomenon of ROS-induced ROS release collectively contribute to mitochondrial damage [43]. These stressors play a central role in triggering cell death via the irreversible opening of the mPTP. mPTP opening leads to a decline in oxidative phosphorylation efficiency, a reduction in mitochondrial membrane potential, and a substantial decrease in ATP synthesis. This cascade of events induces mitochondrial osmotic stress, culminating in outer mitochondrial membrane rupture and promoting various forms of cardiomyocyte death, including pyroptosis [36].

3.5. Endothelial dysfunction, MVO, and inflammatory signaling in IRI

Endothelial dysfunction plays a pivotal role in the pathophysiology of IRI and is characterized by diminished nitric oxide (NO) production alongside elevated expression of adhesion molecules. These alterations facilitate leukocyte adhesion to the endothelial lining, followed by leukocyte infiltration into the myocardial tissue. Collectively, these events contribute to the development of IRI and are closely associated with the phenomenon known as “no-reflow.” Notably, regions exhibiting no-reflow and microvascular obstruction (MVO) are typically confined to myocardial areas exhibiting clear necrosis. MVO arises primarily during reperfusion and is attributed to NO deficiency and paradoxical production of reactive nitrogen species (RNS) [44]. Clinically, MVO manifests as coronary no-reflow and is primarily driven by vasoconstriction and the formation of microthrombi within the lumens of small blood vessels [45,46].

Both MVO and cardiomyocyte death are associated with common pathological features, including intracellular and interstitial edema, intravascular platelet and erythrocyte aggregation, and early inflammatory responses. However, the precise contributions of these factors to each phenomenon may differ. Given that the causal relationship between MVO and cardiomyocyte death remains uncertain, these processes should be regarded as distinct yet closely interconnected, potentially arising from shared underlying mechanisms [47]. Furthermore, endothelial dysfunction is also marked by the activation of nuclear factor kappa-B (NFκB) and other transcription factors, which drive the upregulation of cell adhesion molecules [45]. Importantly, NFκB plays a critical role in the priming and activation of the NLRP3 inflammasome, further linking endothelial dysfunction to inflammatory signaling cascades implicated in IRI [48].

3.6. Inflammation

Early reperfusion contributes to inflammatory injury by activating mast cells and neutrophils, whose secreted products function as chemoattractant, recruiting additional leukocytes to the site of injury [48]. Among the mediators involved, cathepsins lysosomal proteases with diverse pathophysiological roles have recently garnered significant scientific interest. Specifically, cathepsin G, a key modulator of neutrophil chemoattractant activity, induces morphological alterations in cardiomyocytes, disrupting focal adhesions and intracellular junctions [48]. Notably, pharmacological inhibition of cathepsin G using dual cathepsin G and chymase inhibitor has been shown to reduce mortality associated with IRI as shown in Fig. 2a, Fig. 2b [49].

Fig. 2a.

Depicts the sequence of myocardial ischemia, reperfusion injury, and subsequent cell death. Ischemia causes metabolic stress and ATP depletion, leading to various forms of cell death. Reperfusion, while restoring blood flow, triggers ROS production, inflammation, and calcium overload. CMR imaging at 3–5 days and 6 months reveals infarct size and scar formation, respectively.

Fig. 2b.

HIIT enhances cardiac metabolic processes and the heart’s response to stress. The trained heart's improved ability to maintain energy balance during post-ischemic reperfusion may help preserve mPTP activation, potentially boosting ATP synthesis via F1F0-ATP synthase. Moreover, HIIT-induced activation of sarcoKATP and/or mitoKATP channels promotes cellular and mitochondrial hyperpolarization, offering protection during ischemia–reperfusion events.

4. Cardioprotection via ischemic conditioning

Murry et al. [50] first described in 1986 that a protocol involving four cycles of 5-minute occlusions of the circumflex coronary artery, each separated by 5 min of reperfusion, followed by a sustained 40-minute occlusion (termed “index ischemia”) significantly reduced IRI in the dog heart. This cardioprotective phenomenon was termed ischemic preconditioning (PreC). Subsequently, in 2003, Zhao et al. [51] demonstrated that three episodes of 30 s of reperfusion followed by 30 s of ischemia, administered immediately after a 60-minute coronary occlusion, markedly attenuated reperfusion injury in a canine model. This approach was defined as ischemic postconditioning (PostC). It became evident that the later the initiation of the first PostC ischemic episode, the less the degree of cardioprotection observed. The identification of ischemic PostC provided compelling evidence supporting the existence of a distinct reperfusion injury phase [52]. Currently, it is widely accepted that the protective effects of both PreC and PostC are mediated through signaling pathways that modulate mitochondrial function, specifically by inhibiting mitochondria-mediated cell death pathways. Thus, elucidating the mechanisms of cardioprotection is inherently tied to understanding how mitochondria regulate cell survival and death.

The protective outcomes observed with PostC are comparable in magnitude to those induced by the well-established PreC approach [53]. PostC has demonstrated beneficial effects across multiple cardiac tissue compartments, including cardiomyocytes and endothelial cells, offering protection against a spectrum of pathophysiological processes such as necrosis, apoptosis, contractile dysfunction, arrhythmias, and microvascular obstruction or no-reflow phenomena. The underlying mechanisms through which PostC mitigates reperfusion injury are highly complex. They encompass both physiological processes such as delayed re-alkalinization of intracellular pH, release of endogenous autacoids, redox-sensitive signaling events, and the dynamic regulation of ion channels and molecular mechanisms involving the activation of intracellular kinase cascades, notably those comprising the RISK (Reperfusion Injury Salvage Kinase) and SAFE (Survivor Activating Factor Enhancement) pathways, which modulate a variety of downstream cellular targets [54,55].

The concept of PostC has underscored the critical therapeutic window at the onset of myocardial reperfusion, which holds significant clinical relevance, particularly for patients presenting with AMI. Its potential for clinical translation has led to rapid application in both ST-elevation myocardial infarction (STEMI) patients undergoing primary percutaneous coronary intervention (PCI) and those undergoing on-pump cardiac surgery [56]. Extensive investigation into the signaling pathways that underlie PreC and PostC has revealed a network of cardioprotective cascades that transmit signals from the sarcolemma to the mitochondria, many of which are shared between the two conditioning strategies. Both PreC and PostC initiate the activation of these pathways during the early moments of reperfusion following the index ischemic event [57]. Of particular interest are the cGMP/PKG pathway, the RISK pathway encompassing kinases such as Akt and ERK1/2 and more recently, the SAFE pathway, which contributes to PostC-mediated protection through activation of tumor necrosis factor-alpha (TNF-α), its type-2 receptor, Janus kinase (JAK), and signal transducer and activator of transcription-3 (STAT-3). These signaling pathways ultimately converge at the mitochondria, where they regulate mitochondrial stability and function via multiple kinases, including glycogen synthase kinase-3β (GSK-3β), Bcl-2 family proteins (Bcl-2, Bax, Bad), and protein kinase C epsilon (PKCε).

As previously discussed, the opening of the mPTP has been observed at the onset of reperfusion and is primarily attributed to mitochondrial matrix Ca²⁺ overload and/or increased levels of ROS. Consequently, it is generally considered that cardioprotective signaling mitigates mPTP opening not through direct modulation of its structural components, but rather by reducing matrix Ca²⁺ concentrations and ROS accumulation. Notably, redox signaling and acidosis during the initial moments of reperfusion represent two critical cardioprotective mechanisms active during both ischemic PreC and PostC. These mechanisms may exert their effects directly by limiting mPTP opening and subsequently initiating signaling cascades that, although initiated during ischemia, may also be pivotal during reperfusion injury [58,59].

Furthermore, the phenomenon of reversible contractile dysfunction following myocardial ischemia–reperfusion, commonly referred to as myocardial stunning, is recognized as a manifestation of oxidative stress [60]. Although the potential role of ROS-induced ROS release (RIRR) in stunning remains to be fully elucidated, there is compelling evidence supporting the deleterious impact of an early ROS burst. For example, the administration of ROS scavengers such as MPG and phenanthroline at the onset of reperfusion significantly reduced cell death in embryonic chick cardiomyocytes, suggesting a causal role of early reperfusion-generated ROS in cellular injury [61]. Conversely, antioxidant therapy using a combination of catalase and superoxide dismutase (SOD) during reperfusion failed to reduce infarct size in vivo dog heart models but did attenuate microvascular damage and the no-reflow phenomenon [62]. In contrast, reperfusion combined with SOD administration was effective in limiting myocardial infarct size in a closed-chest pig model [63]. Thus, the key point is that the effects of ROS/RNS can vary from beneficial to harmful, including reversible or irreversible damage, depending primarily on their concentration, temporal dynamics, and subcellular localization.

5. Exercise training and cardioprotection mechanisms in ischemia-reperfusion injury

It is well established that exercise training confers cardioprotection against IRI; however, despite numerous proposed mechanisms, a definitive unifying theory has yet to be identified [64,65]. The extent of this cardioprotective effect appears to be proportional to the level of physical activity, and although not yet conclusively verified, HIIT seems to confer superior protection compared to moderate-intensity continuous training (MICT) [66,67]. Clinical outcomes following AMI, including the risk of progression to heart failure, are strongly associated with the extent of cardiomyocyte (CM) death [68]. IR exacerbates myocardial injury following AMI, and both in vivo and ex vivo animal models of IR demonstrate that exercise training can reduce IR-induced CM death by approximately 30–40 % [69,70]. The observation that exercise-mediated protection is preserved in ex vivo hearts implies that intrinsic cellular and molecular adaptations within the myocardium contribute to this effect.

The cellular consequences of IR have been thoroughly characterized [64,69] and include a reduction in ATP levels, increased reliance on glycolysis, decreased pH, enhanced generation ROS, activation of Ca²⁺-dependent proteases, impaired function of energy-dependent ion pumps, and elevations in both intracellular (iCa²⁺) and mitochondrial Ca²⁺ (mCa²⁺), all of which may compromise cardiac function. While elevated ROS levels are a recognized contributor to IR injury, the cardioprotective effects of exercise are thought to involve enhanced antioxidant defenses that mitigate oxidative damage to key proteins such as myofilaments and the SR [64]. Although ROS production plays a significant role in IR injury, dysregulation of iCa²⁺ homeostasis leading to mCa²⁺ overload and subsequent apoptosis is likely a central mechanism underlying IR-induced myocardial damage.

Supporting this hypothesis, evidence indicates that activation of the sarcolemmal ATP-sensitive potassium (sKATP) channel is essential for exercise-induced cardioprotection during IR, with upregulation and activation of this channel constituting a key adaptive response to exercise training [71,72]. Notably, genetic deletion or pharmacological inhibition of the sKATP channel abrogates the cardioprotective benefits conferred by exercise training [71,72]. It is presumed that in trained hearts, early and rapid sarcolemmal repolarization facilitated by outward K+ current through the sKATP channel leads to closure of L-type Ca²⁺ channels, thereby limiting Ca²⁺ influx and mitigating injury [73]. Animal studies suggest that exercise may elicit more pronounced protection against IR injury in females compared to males, potentially due to a higher incorporation of sKATP channel subunits into the sarcolemma [74]. This sex-specific effect has been proposed to involve protein kinase C-ξ (PKCξ) signaling, with its influence attenuated by both ovariectomy and pharmacological PKCξ inhibition [75].

Regular physical exercise is a potent physiological stressor that induces a protective phenotype in the heart, enhancing its resilience against IRI and other cardiovascular damage. This cardioprotective effect is mediated through multiple cellular and molecular mechanisms, including the activation of radical defense systems and modulation of key signaling molecules [33].

Exercise enhances the heart's antioxidant capacity by upregulating enzymes such as superoxide dismutase (SOD), catalase, and glutathione peroxidase. These enzymes mitigate oxidative stress by scavenging ROS, which are pivotal in IRI. For instance, endurance training increases MnSOD activity, reducing infarct size and improving post-ischemic recovery [33]. Additionally, exercise-induced ROS, when produced in moderation, act as signaling molecules to trigger preconditioning pathways, further bolstering cardioprotection [64].

Exercise stimulates endothelial nitric oxide synthase (eNOS), increasing NO bioavailability. NO plays a dual role in cardioprotection: it improves coronary vasodilation and inhibits mitochondrial permeability transition pore (mPTP) opening during reperfusion, thereby reducing cell death [33]. Studies in animal models demonstrate that NO donors mimic ischemic preconditioning, highlighting its central role in exercise-induced protection [68].

Contracting skeletal muscles release metabolites (e.g., adenosine, bradykinin, opioids) and myokines (e.g., interleukin-6) during exercise. These substances mimic remote ischemic preconditioning, activating cardioprotective pathways such as RISK (Reperfusion Injury Salvage Kinases) and SAFE (Survivor Activating Factor Enhancement) without direct cardiac ischemia [53]. For example, delta-opioid receptor activation by exercise reduces infarct size in animal models [76].

Exercise enhances mitochondrial resilience by inhibiting mPTP opening and improving calcium handling, thus preserving ATP synthesis during I/R. Training also reduces monoamine oxidase activity, lowering ROS production and apoptosis [77].

6. Ischemic preconditioning, postconditioning, and exercise training in cardioprotection

In addition to exercise training, brief episodes of ischemia and reperfusion administered prior to deleterious ischemia–reperfusion (ischemic preconditioning, IPC) or at the onset of reperfusion (postconditioning, POC) have been demonstrated to attenuate IR-induced CM death [50,71,74]. The phenomenon of IPC was first described by Murry et al. in 1986 [50], while Zhao et al. subsequently identified POC and conducted comparative analyses with IPC [78]. Unlike exercise, both IPC and POC confer only transient cardioprotection; therefore, despite their clinical relevance, these modalities are not practical for limiting myocardial damage following AMI. Although the precise mechanisms underlying IPC and POC remain incompletely elucidated, it is plausible that certain signaling pathways, molecular substrates, and enzymatic processes activated by exercise are also engaged by IPC and POC.

Frasier et al. [75] proposed that while exercise and IPC share some mechanistic features, they differ in key aspects; specifically, IPC appears to involve activation of the PI3K-Akt-GSK3β pathway, whereas increases in phosphorylated Akt (pAkt) and phosphorylated GSK3β (pGSK3β) do not contribute to exercise-induced cardioprotection against IRI. However, this distinction may be premature, as other evidence suggests that TRN can elevate pAkt and pGSK3β levels [79]. Consequently, protection may be mediated by pGSK3β increasing the open probability of the sarcolemmal ATP-sensitive potassium (sKATP) channel and by inhibiting inositol 1,4,5-trisphosphate receptor (IP3R)-mediated calcium release from the endoplasmic reticulum (ER), thereby preventing mitochondrial Ca²⁺ (mCa²⁺) overload [80]. This enhancement of sKATP channel activity may be further supported by exercise-induced upregulation of brain-derived neurotrophic factor (BDNF) [81]. Supporting this notion, BDNF knockout mice exhibit increased CM death and left ventricular dysfunction following IR compared to wild-type controls [82]. It is well recognized that IPC and exercise share overlapping signaling pathways, and the observation that combining IPC and exercise does not confer additive cardioprotection implies convergence on common protective targets against IRI.

7. Exercise-induced cardioprotection: evidence from animal and human models

One of the key mechanisms through which physical exercise may confer cardioprotection is by mimicking ischemic preconditioning (IP). IP is a well-documented phenomenon in which brief, non-lethal episodes of ischemia enhance the myocardium’s resistance to subsequent, more severe ischemic insults [83]. First identified in canine models by Murry et al., IP was shown to significantly reduce infarct size independent of collateral blood flow development [83]. Since this initial discovery, the infarct-sparing effects of IP have been replicated across multiple mammalian species, including rats, mice, rabbits, swine, and goats [84]. Beyond limiting infarct size, IP has also been demonstrated to attenuate ischemia–reperfusion injury and improve vascular and coronary reactivity [84]. These findings suggest that exercise-induced cardioprotection may, in part, operate through analogous adaptive responses triggered by IP.

Notably, accumulating evidence suggests that exercise can elicit cardioprotective effects resembling those of IP, even in the absence of prior ischemic stimuli. Unlike classical IP, exercise-induced protection does not require direct myocardial ischemia, yet it appears to trigger similar adaptive mechanisms. This phenomenon has been well-documented in numerous animal studies, demonstrating that regular physical activity can confer robust resistance to ischemic injury [85]. However, in humans, direct evidence remains limited, with most support derived from indirect observations [85]. These findings highlight the potential for exercise to serve as a non-invasive alternative to IP, though further research is needed to clarify its translational relevance in clinical settings.

7.1. Animal studies

Consistent reductions in infarct size following exercise training have been repeatedly demonstrated in rats and mice. This protective effect, resembling IP, has been observed with various exercise modalities, including long- and short-term endurance training, resistance training, interval training, and even a single bout of exercise [86,87].

Interestingly, studies in dogs have shown that tachycardia-induced acceleration of myocardial metabolism can confer cardioprotection like IP, even in the absence of ischemia [88]. This suggests that an increased heart rate alone can precondition the heart by enhancing myocardial metabolic activity. However, the infarct-sparing effect of exercise-induced tachycardia was found to be more pronounced than that elicited by tachycardia alone, indicating that additional exercise-related stimuli beyond metabolic activation contribute to IP-like protection [89].

Exercise has also been shown to mitigate ischemia/reperfusion injury. Experiments using isolated hearts from trained rats demonstrated superior recovery of cardiac function after global ischemia compared to sedentary controls [90]. These findings align with other studies reporting that endurance training enhances myocardial performance during ischemia/reperfusion [91], further supporting the concept that exercise mimics and extends the protective mechanisms of IP.

In summary, substantial evidence supports the notion that IP-like cardioprotection can be achieved through non-ischemic means, particularly via exercise or tachycardia, across multiple mammalian species. These findings establish a robust scientific foundation for the concept that exercise serves as an effective strategy for inducing cardiac conditioning.

7.2. Human studies

While direct experimental induction of ischemia is not feasible in human studies, indirect evidence suggests exercise may precondition the human heart. The “warm-up phenomenon” unstable angina patients characterized by improved exercise tolerance and reduced ischemic signs during subsequent efforts has been proposed as a clinical manifestation of preconditioning [90]. This protective effect, associated with attenuated myocardial stunning and reduced oxygen demand [92], persists for 24–48 h, aligning with the second window of protection (SWOP) observed in ischemic preconditioning (98). Improved myocardial performance has been confirmed via thallium scintigraphy and hemodynamic measurements, with protective effects following a biphasic pattern similar to SWOP [93]. However, one study failed to replicate exercise-induced SWOP [94], and these observations are limited to patients with coronary disease, where protection requires exercise-induced ischemia. Notably, preconditioning effects were absent in healthy subjects undergoing maximal exercise, suggesting ischemia may be necessary for detectable protection.

Crucially, no studies have examined whether submaximal, non-ischemic exercise training confers preconditioning benefits in humans. Retrospective data associate higher pre-infarction physical activity with improved outcomes, but the intensity and duration thresholds for exercise-induced protection remain undefined. Prospective studies are needed to clarify these parameters and establish whether non-ischemic exercise can mimic preconditioning in healthy or at-risk populations [95].

7.3. Mechanisms of exercise-induced cardioprotection

Animal studies consistently demonstrate that exercise training reduces myocardial necrosis and improves function following ischemia. While the precise mechanisms remain incompletely understood, several key cellular adaptations have been identified. Exercise enhances expression of sarcolemmal ATP-sensitive K+ (KATP) channels, known mediators of classical IP [96]. Pharmacological blockade studies in rats and dogs confirm the essential role of both sarcolemmal and mitochondrial KATP channels in exercise-induced protection [94], though their relative contributions require further clarification.

Exercise stimulates production of cardioprotective metabolites including adenosine, bradykinin, and opioids [89]. These substances, typically associated with ischemic conditions, accumulate during moderate exercise, potentially explaining how exercise can trigger IP-like effects without ischemia. Notably, submaximal exercise in rats induced protection through protein kinase C (PKC)-mediated mechanisms [97], demonstrating ischemia-independent activation of IP pathways. NO also plays a central role, with exercise increasing production via shear stress. Mouse studies reveal that both endothelial and inducible NOS isoforms are required for exercise-mediated protection, while canine studies show NO's dual-phase cardioprotective action [98].

The antioxidant system represents another important mechanism. Exercise upregulates myocardial manganese SOD and extracellular SOD [99], enhancing resistance to ischemia–reperfusion injury. However, the role of other antioxidants appears limited [100], reflecting the complex, concentration-dependent effects of reactive oxygen species [101]. Heat shock proteins (HSPs), particularly HSP72 and HSP70, increase with exercise and may contribute to protection [101], though their temporal expression patterns don't fully correlate with the duration of cardioprotection [89].

Mitochondrial adaptations are particularly significant. Exercise promotes a resistant mitochondrial phenotype characterized by reduced ROS production, decreased mitochondrial permeability transition pore opening, and enhanced calcium tolerance [102]. Proteomic studies reveal exercise-induced changes in metabolic proteins that may improve ischemia tolerance [103], though direct evidence linking these changes to protection remains limited.

Lipid mediators including ceramide, sphingosine-1-phosphate [104], and platelet-activating factors may participate in exercise-induced protection. Notably, exercise restores IP effectiveness in aged hearts by modulating polyamine metabolism, addressing a key limitation of classical IP [105].

The concept of remote preconditioning suggests humoral factors from exercising skeletal muscle may induce cardioprotection. Myokines like IL-6 [106] and opioids appear particularly important, with plasma from exercised humans demonstrating protective effects in isolated hearts [107]. Neural mechanisms may also contribute, though evidence remains preliminary [108].

Collectively, these findings indicate that exercise-induced cardioprotection involves multiple integrated mechanisms, including activation of survival pathways like RISK (Akt, PKCε, ERK1/2). The convergence of extracardiac and intracardiac factors creates a robust protective phenotype resembling classical IP [108]. While substantial progress has been made, important questions remain regarding the relative contributions of specific mechanisms and their translation to human physiology.

8. Cardiac mitochondrial adaptation and cardiometabolic benefits of HIIT

HIIT, characterized by alternating short bursts of high-intensity activity with periods of rest or lower-intensity exercise, has gained significant attention for its potential benefits in enhancing exercise tolerance, prognosis, and cardiomyogenesis in patients with CVD [109]. HIIT has emerged as a potent modulator of cardiac mitochondrial function, with studies highlighting its capacity to maintain mitochondrial integrity with age. This makes HIIT a viable exercise option for patients with IR injuries [110]. HIIT demonstrates the ability to enhance mitochondrial function and stability, thereby mitigating cardiomyocyte apoptosis [111]. Moreover, HIIT has shown efficacy in countering high-fat diet-induced mitochondrial dysfunction, as evidenced by improvements in mitochondrial morphology and vascular density [112]. Enhanced mitochondrial capacity and ATP production rates following HIIT further indicate improved mitochondrial function. Additionally, HIIT promotes mitophagy activation and prevents protein disassembly and misfolding, thereby preserving mitochondrial integrity [113]. Collectively, these findings underscore the potential of HIIT to elicit favorable adaptations in cardiac mitochondria, with implications for enhancing overall cardiometabolic health.

The rhythmic contraction of the heart necessitates high energy expenditure, especially during HIIT, leading to escalated energy demands. To meet these demands, mitochondrial ATP production increases, as mitochondria constitute over 30 % of cardiomyocyte volume and generate more than 30 kg of ATP daily [114]. The metabolic profile of a HIIT-trained heart significantly differs from that of a sedentary heart. A trained heart exhibits heightened fatty acid and glucose oxidation rates and reduced glycolysis rates [115]. These metabolic adaptations enable the heart to better respond to acute stress by upregulating AMP-activated protein kinase (AMPK), peroxisome proliferator-activated receptor-coactivator-1 (PGC-1α), and Phosphatidylinositol 3-kinase (PI3K), thereby promoting fatty acid and glucose oxidation, glucose uptake, and mitochondrial biogenesis [116]. Although HIIT elevates ROS production in the heart as by-products of the ETC, controlled ROS generation during exercise is believed to trigger beneficial adaptations in the cardiomyocytes as illustrated in Fig. 2a, Fig. 2b.

Over the past decade, multiple studies have demonstrated that mitochondrial adaptations are key mediators of the cardiovascular benefits of exercise. Exercise improves cardiac mitochondrial oxidative phosphorylation [117], promotes ROS clearance, and increases mitochondria number, size, and turnover of mitochondria in the heart [118]. These exercise-induced improvements in myocardial antioxidant status and Ca²⁺ homeostasis are thought to underline cardioprotection from IRI [119]. Although these studies did not focus on HIIT, the findings are relevant as they highlight the potential for exercise-induced mitochondrial adaptation. These findings collectively suggest that HIIT represents a promising strategy for improving cardiac mitochondrial function, with potential therapeutic implications for managing CVD and related metabolic disorders.

8.1. HIIT for ischemia-reperfusion injury: mechanisms and evidence

HIIT is emerging as a promising addition to standard therapies for IRI, showing potential in improving various physiological and functional outcomes. HIIT has demonstrated efficacy in mitigating sarcopenia [120]. A randomized crossover trial compared acute HIIT (four 4-minute high-intensity intervals at 70 % maximal capacity alternating with 4 min at 30 %) versus moderate-intensity continuous training (MICT) [121]. Studies show that HIIT can enhance aerobic capacity, reduce insulin resistance, and improve glucose metabolism, making it a time-efficient approach for diabetes management [122]. Both the Korean Diabetes Association (KDA) and the American Diabetes Association (ADA) recommend HIIT for individuals who are physically capable and have limited time [123].

One study explored the cardioprotective effects of HIIT versus ischemic preconditioning (IPC) in rat myocardial ischemia–reperfusion [77]. The study sought to evaluate whether HIIT and IPC could reduce myocardial injury [124]. Additionally, research indicates that exercise training, including HIIT, can affect Ca²⁺ transport, reducing hypertension-induced myocardial injury in mice [125]. This was demonstrated by analyzing an exercise response model of hypertension-induced myocardial injury in mice using multiomics data [125].

Moreover, HIIT has shown promise in improving vascular reactivity and skeletal muscle perfusion in older adults [126]. A study hypothesized that HIIT would enhance muscle microvascular blood flow and vascular reactivity to acute contractile activity in older adults [126]. In the context of spinal cord injury (SCI), research aims to summarize the training parameters and effects of HIIT, as inactivity in SCI patients can further deteriorate cardiorespiratory function and muscle strength [127]. Studies also show that HIIT leads to an increase in Brain-Derived Neurotrophic Factor (BDNF), Interleukin-6 (IL-6), Interleukin-10 (IL-10), Irisin, and Osteocalcin (OC), promoting comprehensive abilities of the organism [128].

While HIIT demonstrates potential benefits, it is important to consider injury incidence associated with this training [129]. Researchers can advise patients on ways to mitigate potential risks prior to participation [129]. Meta-analysis of different studies compared the effects of MICT and HIIT. The pooled results showed a negative Hedge's g value of −0.308 with a 95 % CI of −1.440 to 0.824, suggesting a slight overall favor towards HIIT, although the wide confidence interval indicates some uncertainty in this estimate [130].

8.2. Energetic metabolic responses to HIIT training

Cardiac mitochondria possess remarkable versatility in utilizing various substrates, earning them the moniker “metabolic omnivores.” Fatty acids, carbohydrates, ketones, and amino acids all play vital roles in fulfilling the heart’s elevated energy requirements. At rest, fatty acid oxidation predominates, contributing approximately 40–60 % of ATP production, while carbohydrate metabolism accounts for the remaining 20–40 %. During HIIT, heightened adipose tissue lipolysis and muscle glycolysis elevate circulating levels of fatty acids and lactate, which provide essential fuel to meet the heart’s augmented energy demands. An increase in circulating lactate is a crucial component of the cardiac response to HIIT for two main reasons: First during HIIT, lactate’s contribution to overall oxidative metabolism becomes predominant [131]. Lactate functions as an anaplerotic substrate, potentially enhancing fatty acid oxidation in the heart [132].

Repeated sessions of exercise, such as HIIT, induce specific metabolic adaptations that may enhance the cardiac response to metabolic energetic stress. While the effects of HIIT on skeletal muscle energetic metabolism have been extensively documented and reviewed [133], its impact on the cardiac muscle remains somewhat ambiguous. A strong correlation exists between mitochondrial density and cardiac workload in cardiac myocytes [134]. This suggests that HIIT induces mitochondrial biogenesis in the cardiac tissues, both in healthy and pathological conditions [135]. HIIT activates peroxisome proliferator-activated receptor alpha (PPARα), leading to elevated expression of genes responsible for fatty acid oxidation. These include fatty acid transport protein 1 (FATP1), carnitine palmitoyltransferase I (CPTI), acyl carrier protein (ACP), and medium-chain acyl-CoA dehydrogenase (MCAD) [136].

Additionally, HIIT enhances cardiac lactate utilization. High intensity trained cardiomyocytes exhibit increased expression of the lactate transporter monocarboxylate transporter 1 (MCT1) [137]. Each HIIT session also increases the ATP/AMP ratio, activating AMPK in the heart [138]. AMPK activation impacts various proteins crucial for cardiac energy metabolism. Specifically, it facilitates Glucose transporter 4 (GLUT4) translocation to the cellular membrane, enhances glucose uptake by cardiomyocytes, and boosts glucose oxidation through increased 6-phosphofructo-2-kinase (PFK2) activity [139,140].

Recent studies suggest that HIIT enhances the expression of ETC components in mitochondria and promotes their assembly into super complexes in skeletal muscle, representing an innovative adaptive response to heightened energy demands [141]. The assembly of mitochondrial super complexes in skeletal muscles in response to HIIT also reduces ROS production and mitigates mitochondrial oxidative damage [142]. However, comparable data regarding the myocardium is currently lacking. HIIT has demonstrated potential in improving mitochondrial oxidative capacity in patients with IRI. Enhanced mitochondrial function in skeletal muscles and platelets through aerobic exercise using HIIT protocols has been observed [143]. Despite these promising findings, the specific mechanisms and metabolic adaptations in cardiac tissue remain an area requiring further investigation.

8.3. Dual role of ROS in cardiomyocytes adaptation to HIIT

HIIT exerts profound effects on cardiomyocyte function by modulating ROS production and activating antioxidant defense systems. The elevated ROS levels observed during HIIT result from increased oxygen consumption and activation of inflammatory pathways. This transient ROS production represents a natural physiological response to the stress induced by HIIT. However, the body’s antioxidant defense system strives to regulate ROS levels and minimize oxidative damage. If the delicate balance between ROS production and antioxidant defenses is disrupted, acute oxidative stress may occur, potentially resulting in cellular injury. The body’s robust antioxidant system plays a pivotal role in maintaining this balance and protecting against oxidative stress triggered by HIIT [144].

HIIT-induced mechanical and metabolic stress in the heart stimulates the production of ROS and reactive nitrogen species (RNS) [145,146]. This initial burst of ROS/RNS, observed following a single bout of HIIT, is believed to trigger many of the adaptive mechanisms associated with repeated training. Interestingly, the benefits of HIIT on cardiomyocytes are diminished when combined with antioxidant supplementation, suggesting that ROS play a dual role in both stress and adaptation [147].

Research by Muthusamy, V.R., et al., (2012) highlights the activation of nuclear factor erythroid 2-related factor 2 (Nrf2), as a key initiator of molecular adaptations to HIIT [148]. Nrf2, a critical regulator of antioxidant enzymes and the cellular stress response, is activated by HIIT-induced ROS. Although traditionally regarded as harmful, ROS are now recognized for their role in cell signaling. Notably, even a single bout of HIIT elevates Nrf2 gene expression in cardiomyocytes. In Nrf2 knockout (KO) mice, HIIT-induced activation of the antioxidant response element is absent, resulting in no significant increases in key antioxidant enzymes like SOD1, SOD2, catalase, and GPx, despite similar ROS levels compared to wild-type mice. This indicates that the anticipated response to ROS depends on Nrf2 signaling [149].

HIIT enhances mitochondrial glutathione (GSH) and manganese superoxide dismutase (MnSOD) levels in the myocardium and upregulates cytosolic copper-zinc superoxide dismutase (CuZnSOD) and catalase through an Nrf2-dependent mechanism. These adaptations are believed to contribute to the cardioprotective effects of HIIT. The activation of Nrf2 in cardiac tissue following acute exercise is closely linked to ROS production mediated by NOX4, a mitochondrial enzyme highly expressed in cardiac cells [150,151]. Hancock M. et al., (2018) proposed that mitochondrial NOX4 is essential for Nrf2 activation during HIIT. Importantly, the role of Nrf2-ARE signaling in HIIT-induced adaptations has been validated in human skeletal muscle, where mitochondrial H2O2 levels correlate with the response [152]. HIIT also influences cytokine activity, with elevated levels of TNF-α and IL-1β contributing to the adaptive antioxidant response in cardiomyocytes. Neutralizing these cytokines with antibodies nullifies the activation of MnSOD following a single bout of HIIT [153]. Interestingly, administration of TNF-α mimics the effects of HIIT, while pre-treatment with the antioxidant MPG reduces the adaptive response. Nuclear factor kappa B (NFκB), another regulator of stress responses, is well-documented in skeletal muscles as a trigger for training adaptations, including the induction of antioxidant enzymes and nitric oxide (NO) synthase [154]. In cardiomyocytes, however, its role remains less clear. While acute HIIT activates myocardial NFκB in rats [155], prolonged training reduces cardiac NFκB levels and increases IκB, an inhibitor that maintains NFκB in its inactive state [156]. In conclusion, ROS production during HIIT initiates a complex antioxidant response, mediated either through Nrf2-dependent or independent mechanisms. Repeated HIIT enhances the capacity of cardiomyocytes to endure oxidative stress, contributing to the cardioprotective adaptations associated with this training modality.

8.4. Mitochondria as a key contributor to post-ischemia reperfusion injury

Mitochondria are key contributors to post-IRI, with damage to the mitochondrial ETC primarily occurring primarily during the ischemic phase. During reperfusion, tissues with ischemia-damaged mitochondria experience dysfunction-driven injuries, characterized by excessive ROS production and Ca²⁺ dysregulation [157]. These processes culminate in the opening of mPTP, followed by membrane permeation, mitochondrial swelling, structural disruption, and the release of pro-apoptotic factors [158].

The first adverse event during the initial phase of reperfusion is a burst ROS, including mitochondrial ROS [159]. Superoxide, primarily produced in complexes I and III of the mitochondrial ETC, and to a lesser extent in complex II, directly triggers oxidative damage [76]. It can also lead to the formation of harmful species such as H2O2. Increased ROS generation facilitates mPTP opening, which leads to apoptosis. Moreover, ROS-mediated mitochondrial damage causes oxidation of lipids, proteins, and DNA, releasing damage-associated patterns (DAMPs). The mitochondrial damage exacerbates ischemia-induced injuries, activating both extrinsic and intrinsic cell death pathways [160]. Suppressing the pathogenic cascade following ischemia by limiting mitochondrial ROS formation during the initial moments of reperfusion is critical. Numerous researchers have investigated antioxidant methods due to mitochondrial oxidative stress being a primary cause of IRI [161]. Conventional antioxidants have shown limited efficacy. However, mitochondria-targeted antioxidants, such as MitoQ, have demonstrated potential in mitigating cardiac failure and mitochondrial dysfunction following IR in rats [162].

Higher antioxidant levels resulting from HIIT have been shown to reduce myocardial injury during ischemia/reperfusion. HIIT enhances the activity of mitochondrial antioxidant enzymes including MnSOD, CuZnSOD, and glutathione peroxide (GPx), while reducing the mitochondrial oxidants [163]. This adaptation protects cardiac SSM and IFM against oxidative damage and IR-induced changes in OX-PHOS [164]. Kemi OJ et al., (2010) demonstrated that cardiac mitochondria in Fischer rats engaged in voluntary wheel running produced less H2O2 compared to their sedentary counterparts. This reduction was observed in both IFM and SSM and was confirmed through proteomic analysis [165]. HIIT-induced alterations in SSM and IFM promote a cardioprotective phenotype. Specifically, HIIT decreases monoamine oxidase, a major ROS generator, and increases the expression of peroxiredoxin III, an H2O2-scavaging enzyme exclusive to mitochondria in SSM.

Increased MnSOD activity is a primary mediator of HIIT's cardioprotective effects. Suppression of MnSOD expressions with antisense oligodeoxyribonucleotide in HIIT-trained heart reduced but did not eliminate the cardioprotective benefits, suggesting additional mechanisms are involved [166]. Another crucial component of ROS detoxification is the mitochondrial glutathione (GSH) pool. Although physical exertion does not significantly alter the GSH/GSSG ratio or total GSH levels, HIIT-trained hearts exhibit better GSH replenishment following IR [167]. The positive impact of HIIT on glutathione reductase activity is significant, as blocking this enzyme with BCNU negates HIIT-induced cardioprotection [168]. Furthermore, GSH recovery after reoxygenation in isolated cardiomyocytes correlates with the mitochondria’s ability to maintain ΔΨm [169].

Extensive evidence indicates that complex I serves is the primary site of oxidative damage during IR episodes, demonstrating notable sensitivity to these conditions [170]. Starnes, J.W et al., (2007) reported that an 8-week HIIT regimen significantly reduced ROS production within cardiac mitochondria by inducing specific adaptations in complex I. However, while HIIT mitigates ROS production, it does not enhance mitochondrial resilience against Ca²⁺ overload [170]. Notably, the study observed that rotenone, a complex I inhibitor, eliminated differences in H2O2 production between exercised and sedentary hearts [170]. Overall, HIIT plays a significant role in regulating ROS production and maintaining the balance between ROS generation and scavenging, contributing notably but not exclusively to cardioprotection. Further investigation is needed to elucidate the exact mechanisms of ROS effects on cardiomyocytes function and mitochondrial integrity.

9. Exploring HIIT cardioprotective mechanisms

HIIT has demonstrated promising effects on mitochondrial function, particularly in how mitochondria adapt to acute stress, contributing to cardioprotection. Despite its significance, the precise pathways through which HIIT mediates these effects remain unclear. This section explores potential mechanisms through which mitochondria may contribute to HIIT-induced cardioprotection as shown in Fig. 3.

Fig. 3.

Possible mechanisms of cardioprotection induced by HIIT. Regulating cytosolic Ca²⁺ concentration, particularly during ischemia/reperfusion (IR), is a critical factor in HIIT-induced cardioprotection. HIIT enhances the ability of cardiac cells to maintain calcium homeostasis and safeguard key Ca²⁺-handling proteins, such as SERCA, during IR. Mitigating mitochondrial Ca²⁺ overload, which triggers ROS production and mPTP activation, is essential for preserving cell viability. Studies propose that HIIT-trained hearts demonstrate reduced sensitivity to elevated Ca²⁺ load and delayed mPTP activation, alongside improved mitochondrial antioxidant enzyme activity. These adaptive responses contribute to the cardioprotective effects of HIIT.

9.1. Mitochondrial mechanisms and HIIT-induced cardioprotection

During ischemia, reduced ATP levels impair critical pump proteins responsible for regulating cardiomyocyte ion homeostasis. This impairment leads to increased intracellular Ca²⁺, excessive ROS production, activation of calpain, contractile dysfunction, and ultimately cell death. Restoring ATP synthesis is therefore essential for cardiac functional recovery during post-IR.

HIIT enhances the expression of mitochondrial creatine kinase, tricarboxylic acid (TCA) cycle proteins, and respiratory chain components. These adaptations improve cardiac functional recovery, ion homeostasis, and mitochondrial oxidative capacity, both qualitatively and quantitatively [162]. However, the direct correlation between mitochondrial bioenergetics and HIIT-induced cardioprotection remains inconclusive [72]. Alleman, R.J et al., (2016) observed that HIIT-trained animals demonstrated superior recovery of mitochondrial energetics, as indicated by higher oxygen consumption rates following anoxia/reoxygenation compared to sedentary controls [74]. Additionally, the ratio of oxygen consumption to hydrogen peroxide production in HIIT-trained animals was twice as high as in sedentary ones, suggesting reduced ROS production. HIIT also mitigates changes in the respiratory control ratio during in vitro anoxia-reoxygenation, Tao, L., et al., (2015) reported that ischemia in mice caused downregulation of PPAR-α, PPAR-γ, and enzymes involved in fatty acid metabolism [171]. In contrast, HIIT following myocardial infarction reduced alterations in glucose transporter 1 (Glut-1) and glycogen synthase 1 (Gsy1) expression while promoting mitochondrial biogenesis [172]. These findings may explain the accelerated recovery of mitochondrial energetics during reperfusion, highlighting the roles of intact β-oxidation, enhanced mitochondrial biogenesis, and connectivity in supporting cardiomyocytes adaptation to IR. During reperfusion, mPTP activation can hinder ATP synthesis recovery, as the F1F0-ATP synthase hydrolyzes ATP instead of synthesizing it, ultimately causing cell death [173,174]. The impact of HIIT on mPTP activation remains a topic of debate. Some studies suggest that HIIT reduces mPTP stimulation during IR, potentially enhancing mitochondrial energetics and minimizing reperfusion arrhythmias [74,175]. Further research is needed to clarify the relationship between HIIT and mPTP activation.

KATP channels, located in the sarcolemma (sarcoKATP) and mitochondria (mitoKATP) of cardiomyocytes, act as energy sensors essential for cardiomyocyte function. Their activation before ischemia confers cardioprotection by shortening the ventricular action potential and protecting the myocardium. The opening of sarcoKATP channels may also activate mitoKATP channels, providing mitochondrial protection under ischemic conditions [176]. Evidence suggests that HIIT modulates sarcoKATP channels, contributing to cardioprotection; however, the role of mitoKATP channels in HIIT-induced cardioprotection requires further investigation [72,177]. While significant progress has been made in understanding the mitochondrial mechanisms underlying HIIT-induced cardioprotection, many aspects remain to be elucidated. Further studies should aim to address these gaps to fully unravel the cardioprotective potential of HIIT.

9.2. Regulation of mitochondrial Ca²⁺ homeostasis

In addition to causing myofilament contact and sarcomere shortening during excitation–contraction coupling (ECC), the Ca²⁺ transient helps to regulate mitochondrial activity. Mitochondrial calcium uniporter (MCU) channels facilitate the transfer of calcium ions from the cytosol to the mitochondria, maintaining the equilibrium of Ca²⁺ levels between the mitochondria and the cytosol. At physiological levels, Ca²⁺ can promote a variety of metabolic activities, including activating mitochondrial dehydrogenases, making its uptake by mitochondria vital. However, higher concentrations of calcium ions can be harmful, initiating processes that lead to cell death (apoptosis and necrosis). Controlling the cytosolic calcium ion concentration, particularly in the absence of iron, may play a significant role in HIIT-induced cardioprotection. To the best of our knowledge, no research has investigated how HIIT affects MCU expression and heart mitochondrial calcium uptake. Nine weeks of exercise have been shown to increase MCU level in human skeletal muscles [178]. It is reasonable to speculate that HIIT impact on important Ca²⁺-handling proteins may help regulate mitochondrial Ca²⁺ load considering the balance between cytosolic and mitochondrial Ca²⁺ concentrations. HIIT accelerates [Ca²⁺]i transient decline, partially due to increased SERCA2a levels and more effective coupling of the L-type Ca²⁺ current to RyR2 Ca²⁺ release [179].

The influence of HIIT Ca²⁺-handling proteins may enhance Ca²⁺ homeostasis and protect the heart during IR. The capacity of HIIT to increase cardiac SERCA2a levels is important for cardiac protection during IR [73,180]. Furthermore, the degradation of Ca²⁺-handling proteins, such as SERCA2a [90], is reduced in the exercise-trained heart because HIIT can limit calpain activation [180], and ROS-dependent protein carbonylation during IR [41]. These positive effects help prevent Ca²⁺ overloading in the mitochondrial matrix during IR and improve Ca²⁺-handling regulation during post-IR. Direct evidence remains lacking.

It is possible to hypothesize that a trained heart may better resist calcium overloading during IR, potentially avoiding calcium-dependent mitochondrial mPTP activation. However, conflicting and limited data exist. For example, Magalhães, J. et al., (2013) found that mitochondria isolated from exercised cardiac tissues exhibit delayed swelling in response to Ca²⁺. Increased mitochondrial Ca²⁺ retention capacity (CRC) is another benefit of physical activity, suggesting delayed mPTP activation in response to Ca²⁺ excess [181]. This has been observed in the cardiac mitochondria of healthy and ob/ob mice [182,183], and in skeletal muscle under chronic critical ischemia treated with antioxidant therapy [184]. These findings are partly attributed to higher levels of GSK3β phosphorylation in exercised mice [184]. Conversely, da Silva et al., (1985) reported that exercise training reduces mitochondrial ROS generation but does not enhance mitochondrial resistance to Ca²⁺ overload. Furthermore, in rats with type 1 diabetes, exercise training lowers mitochondrial CRC, which can only [185]. Lastly, exercise training lowers mitochondrial CRC in the hearts of rats with type 1 diabetes, but it can only improve with combined exercise training and insulin therapy [185].

Recent evidence links mitochondrial Ca²⁺ content to the physical connections between mitochondria and the ER/SR. Co-sedimentation of ER particles with mitochondria and electron microscopy findings demonstrate these physical interactions [186]. Proteins such as the voltage-dependent anion-selective channel protein (VDAC) in mitochondria, inositol 1,4,5-trisphosphate receptors (IP3R) on the ER, and GRP75, which regulate ER-mitochondrial Ca²⁺ transfer, play essential roles in mitochondria-associated membranes (MAMs). Ca²⁺ signaling is critical for ER/SR-mitochondria interactions. Pharmacological treatment or genetic ablation of MCU reduces mitochondrial Ca²⁺ entry, thereby lowering necroptosis and apoptosis, particularly by mitigating calcium imbalance, mitochondrial potential changes, ROS generation, and mPTP opening [[187], [188], [189]]. According to Zhou et al., (2018) ER–mitochondrial interactions influence autophagy, Ca²⁺ imbalance, mitochondrial fission, ROS generation and apoptosis during IR. However, no research has explored how HIIT affects MAMs. Investigating the effects of HIIT on MAMs and mitochondrial function during IR represents an original and promising research avenue [104,190,191].

In conclusion, HIIT affects cardiac cells’ ability to regulate Ca²⁺ homeostasis and preserve important Ca²⁺-handling proteins during IR. This may protect the heart by preventing mitochondrial Ca²⁺ overload, but direct evidence is limited. Conflicting findings exist regarding high intensity trained hearts’ mitochondrial sensitivity to Ca²⁺ overloading. Furthermore, MAMs represent a novel target for understanding the cardioprotective effects of HIIT. More research is required to elucidate the interplay between mitochondrial Ca²⁺ homeostasis in HIIT-induced cardioprotection.

10. Conclusions and perspectives

HIIT has emerged as a promising strategy to mitigate cardiovascular risk factors and enhance cardiac resistance to IRI. However, pinpointing the precise molecular mechanisms underlying HIIT-induced cardioprotection remains a complex and ongoing challenge. This review explores the potential connections between mitochondrial function and signaling cascades activated by HIIT. Mitochondria are central to cardioprotective processes, acting both as contributors to and targets of cell death during IRI. Despite significant advancements, numerous questions remain unresolved, and critical gaps in our understanding persist. HIIT significantly impacts Ca²⁺ homeostasis, which may, in turn, enhance mitochondrial resilience to IR damage. However, the direct effects of HIIT on cardiac mitochondrial activity remain poorly understood. Further investigation is needed to elucidate how HIIT influences various aspects of mitochondrial function and regulation key areas of focus include: (i) the dynamics and movement of the mitochondrial network; (ii) the mechanisms controlling mitochondrial Ca²⁺-handling and the activation of the mPTP; (iii) the complex interactions between inflammation and mitochondria; and (iv) the links between HIIT-induced redox signaling and mitochondrial function. These areas of study hold promise for uncovering novel cardioprotective mechanisms and developing innovative therapeutic approaches for cardiovascular disease.

Author contributions

Z.W., M.A., and R.C. developed the idea for the review and outlined its content. M.A., S.F., and S.S. authored the article, with contributions from Z.W., M.A, and R.C., Z.W. reviewed the article prior to submission. All authors have read and approved the final version of the manuscript.

Institutional review board statement

Not applicable.

Informed consent statement

Not Applicable.

CRediT authorship contribution statement

Zhan Wei: Writing – review & editing, Funding acquisition. Mujahid Ahmad: Writing – original draft. Rongzhi Chen: Writing – original draft, Conceptualization. Sana Fatima: Writing – review & editing. Shahab Shah: Writing – review & editing, Visualization.

Funding

This research was self-funded by the authors.

Declaration of competing interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Zhan Wei reports financial support was provided by Centre of Research, Education, Innovation, and Intervention in Sport (CIFI2D), Faculty of Sport, University of Porto, Portugal. Zhan Wei reports a relationship with Centre of Research, Education, Innovation, and Intervention in Sport (CIFI2D), Faculty of Sport, University of Porto, Portugal that includes: employment and funding grants. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability

Data will be made available on request.