Cardiovascular diseases rank first among the causes of death worldwide (32%), according to the WHO [1], with 85% of these deaths related to heart attacks and strokes. Despite modern treatment methods, including primary percutaneous coronary intervention and coronary artery bypass grafting, chronic total occlusion (CTO) of the left coronary artery remains a common form of coronary artery disease (15–30%) [2, 3]. Studying myocardial cellular changes under CTO conditions is therefore of fundamental importance, as it is key to assessing long-term myocardial viability and developing therapeutic strategies aimed at preventing the progression of heart failure. Increasing evidence indicates that mitochondrial dysfunction in cardiomyocytes (CMCs) plays a central role in remodeling following myocardial infarction (MI) [4, 5, 6].

Mitochondria are essential for CMC survival and function, supplying most of the cellular adenosine triphosphate (ATP) through oxidative phosphorylation and regulating key cell death pathways [7, 8]. Importantly, mitochondria in CMCs are not homogeneous but consist of distinct subpopulations with specific localizations and functional roles [9, 10]. Intermyofibrillar mitochondria (IFM) are located between the myofibrils and are thought to primarily supply ATP for the contractile activity of CMCs. Subsarcolemmal mitochondria (SSM), located beneath the sarcolemma, regulate ion transport and membrane-related processes. Perinuclear mitochondria (PNM), clustered around the nucleus, contribute to transcriptional regulation, signaling, and mitochondrial biogenesis. Numerous studies have demonstrated that these mitochondrial subpopulations respond differently to myocardial injury [11, 12, 13, 14, 15, 16]. Studying these differences is essential because the heterogeneous responses of mitochondrial subpopulations help explain how MI affects multiple aspects of CMC physiology, from contractile function to ion homeostasis and gene regulation. Moreover, therapeutic strategies may need to be tailored to target specific subpopulations. For example, inhibitors of mitochondrial permeability transition pore opening or mitochondria-targeted antioxidants may preferentially protect IFM and SSM, whereas activators of mitophagy and biogenesis may be particularly beneficial for PNM [7, 17]. Thus, investigating morpho-functional changes in distinct mitochondrial subpopulations after MI is critical not only for understanding the mechanisms of post-infarction remodeling but also for identifying precise therapeutic targets that may enhance cardioprotection and improve clinical outcomes.

Most existing research has focused on mitochondrial changes during the acute phase of MI or acute ischemia-reperfusion injury [18, 19, 20]. Far fewer studies have examined alterations in mitochondrial morphology and function in CMCs following CTO of the left coronary artery [21, 22], and virtually no studies have specifically addressed changes across the distinct mitochondrial subpopulations under this condition. The limited available data, derived from different experimental models, are inconsistent. For example, in a rat model of heart failure induced by transverse aortic constriction, IFM exhibited reduced mitochondrial content and respiratory capacity, whereas SSM function was only modestly affected [23]. In contrast, a microembolization-induced heart failure dog model showed no differences between SSM and IFM in oxidative phosphorylation or electron transport chain organization [14]. In a mouse model of pressure overload, PNM were more susceptible than IFM to mitochondrial membrane depolarization, reactive oxygen species (ROS) generation, and impaired Ca²⁺ uptake [16]. Notably, no previous study has compared all three CMC mitochondrial subpopulations simultaneously, leaving a significant gap in understanding subacute and chronic remodeling.

In the present study, we focused on the 2-week time point after MI, which corresponds to the subacute phase of post-infarction remodeling, when granulation tissue transitions into a forming scar and CMCs undergo pronounced yet still reversible structural and metabolic adaptations [24, 25, 26, 27]. We aimed to provide a detailed analysis of mitochondrial subpopulation remodeling at this biologically important stage. Focusing on this time window enables the identification of subpopulation-specific mitochondrial vulnerabilities during a period in which remodeling is substantial but remains potentially reversible, thereby offering insights into mechanisms that may shape the trajectory toward chronic heart failure. Accordingly, we investigated both morphological and functional alterations in all three mitochondrial subpopulations (IFM, SSM, PNM) two weeks after MI induced by CTO of the left coronary artery in a rat model.

Following MI, the heart undergoes a dynamic remodeling process that includes necrosis, inflammation, granulation tissue formation, and ultimately mature scar development. Each stage is accompanied by characteristic structural and functional changes at the tissue, cellular, and molecular levels within the surviving myocardium, and these changes largely determine whether the heart recovers or progresses toward heart failure. Mitochondria play a central role in this process by supplying the energy required for CMC contraction and by regulating cell death pathways and redox homeostasis [36]. The dynamics of mitochondrial morphology have been shown to play a critical role in post-infarction remodeling and represent potential therapeutic targets [37]. Mitochondrial dynamic proteins, including Drp1, Fis1, Mfn1/2, and OPA1, regulate fission and fusion processes, thereby maintaining mitochondrial structure and function and enabling CMCs to adapt to stress conditions [6].

In the early phase following MI, within hours to a few days, CMC mitochondria experience significant fragmentation, swelling, loss of cristae integrity, bioenergetic failure, excessive ROS production, and the initiation of cell death pathways [6, 10, 18, 38, 39]. In contrast, studies investigating the later stages of post-infarction remodeling, occurring weeks to months after MI, report more complex changes, including increased mitochondrial functional heterogeneity, the accumulation of damaged mitochondria, and the activation of compensatory mechanisms such as mitochondrial biogenesis induction, selective mitophagy [6, 40, 41], and compensatory mitochondrial hypertrophy [42]. In our previous studies, we showed that six months after permanent coronary occlusion in rats, heart failure was associated with an increased mitochondrial volume density in the peri-infarction zone and enlarged mitochondria in the intact myocardium, accompanied by CMC hypertrophy [42, 43]. Simultaneously, the overall mitochondrial membrane potential of CMCs remained reduced compared to controls [30], likely reflecting alterations in the pathological architecture of CMC cristae, which were less tightly packed and exhibited more irregular morphology.

Despite this knowledge, the subacute phase of remodeling, typically spanning two to four weeks post-infarction, remains less well characterized [6, 44]. This period is particularly critical as it represents a transitional window during which the balance between mitochondrial injury and adaptive responses may significantly influence the progression toward heart failure with reduced ejection fraction or mitigate the extent of ischemic myocardium [24, 25, 26]. In our previous works, we demonstrated that during the scar maturation process two weeks after experimental MI, a complex hypotrophic response of CMCs is observed throughout the entire volume of the LV, including the remote intact regions and border zone adjacent to the infarct [42, 43]. This hypotrophy, manifested by reduced dry mass and cell size, was accompanied by a decrease in average mitochondrial area across all subpopulations, suggesting a potential functional relationship between the condition of the contractile apparatus and the level of energy metabolism in CMCs. In the present study, we aimed to determine which mitochondrial subpopulations are most severely affected at this stage of myocardial remodeling, because different mitochondrial subpopulations have different vulnerabilities to ischemic injury and can undergo different changes during scar formation [9].

We found that two weeks after post-infarction LV remodeling, CMCs exhibit a significant reduction in overall mitochondrial activity accompanied by a marked increase in intracellular ROS levels (Fig. 3). Similar alterations have been widely reported in post-infarction myocardium across various stages of ischemia-reperfusion [18, 19, 45, 46], as well as during subacute and chronic myocardial damage [20, 21]. Because IFMs constitute the dominant mitochondrial subpopulation, representing approximately 80% of all CMC mitochondria [47], the overall decrease in integral fluorescence intensity is likely driven primarily by a reduction in their membrane potential. However, the magnitude of membrane potential loss may differ among mitochondrial subpopulations. By comparing the average fluorescence intensities of SSMs and PNMs relative to IFMs under normal conditions and after MI, we observed that the membrane potentials of the SSMs and PNMs decreased by approximately 30% more than that of IFMs (Fig. 4A,C). Morphological analyses further revealed that the sizes of SSMs and PNMs were reduced to a greater extent than the sizes of IFMs (Fig. 5C,D). Thus, the size and activity of individual mitochondria in the perinuclear and subsarcolemmal subpopulations declined more profoundly than those of the intermyofibrillar subpopulation.

The existing literature on this topic remains highly contradictory. Lesnefsky et al. [12], using an isolated buffer-perfused rabbit heart model, reported that after 45 min of ischemia, oxidative phosphorylation was impaired in SSMs, but not in IFMs. Similarly, Leistner et al. [48], employing an isolated rat heart model of ischemia-reperfusion, observed more pronounced damage in SSMs. By contrast, in a rat model of heart failure induced by transverse aortic constriction, IFMs displayed a loss of mitochondrial content and respiratory capacity, whereas SSMs were only mildly affected [23]. A subsequent study by the same group, using an acute pre-clinical porcine model of ischemia-reperfusion, found no significant functional differences between IFMs and PNMs, despite clear evidence of global mitochondrial dysfunction, including impaired ATP production, altered state 3 and state 4 respiration, and reduced calcium retention capacity [15]. Studies examining PNMs specifically are relatively limited. However, Voglhuber et al. [16], using a mouse model of pressure overload, demonstrated that PNMs were more susceptible than IFMs to mitochondrial membrane depolarization, elevated ROS generation, and impaired calcium uptake. Overall, despite inconsistencies across experimental models and species, the majority of studies indicate that all mitochondrial subpopulations are affected by ischemic injury, with SSMs and PNMs often exhibiting more severe dysfunction than IFMs. These observations are in line with our findings.

However, a reduction in the size and functional activity of individual mitochondria can potentially be compensated by an increase in their number, thereby maintaining the required level of cellular energy. For this reason, one of the most informative morphometric indicators is mitochondrial volume density, which reflects the proportion of the CMC volume occupied by mitochondria. In our study, we detected a decrease in the volume density of SSMs and IFMs (Fig. 5E), whereas no significant changes were found in the volume density of PNMs. The proximity of PNMs to the nucleus may facilitate a more rapid restoration of fragmented and degraded mitochondria compared with other regions of the CMC cytoplasm, partially compensating for the reduction in activity and size of individual mitochondria within this subpopulation. Consequently, the average brightness of perinuclear regions of the CMC cytoplasm decreased in the MI group proportionally to the decrease observed in regions containing the contractile apparatus and IFMs. In contrast, the average brightness of submembrane regions containing SSMs declined more markedly than that of intermyofibrillar regions (Fig. 4B). Thus, based on our data, SSMs appear to be the most vulnerable mitochondrial subpopulation at studied stage of myocardial remodeling, as they exhibit a simultaneous reduction in membrane potential, mitochondrial area, and volume density, changes that collectively contribute to diminished overall energy production in these cytoplasmic regions.

During the post-infarction period, SSMs and PNMs in CMCs may be particularly susceptible due to their localization and regulatory dependencies. In both populations, mitochondrial dynamics proteins such as DRP1 and OPA1 play a critical role. Stress-induced translocation of DRP1 and its imbalance with OPA1 promote excessive fragmentation, especially in mitochondria closely linked to nuclear and sarcolemmal stress pathways [6, 16, 49]. The increased vulnerability of SSMs in post-infarction CMCs may result from persistent oxidative stress, chronic Ca²⁺ overload, and structural remodeling. Due to their proximity to $\beta$-adrenergic and AT1 receptors, SSMs are exposed to sustained neurohumoral activation, which can enhance ROS generation [50, 51]. Impaired calcium handling may increase the likelihood of mitochondrial permeability transition pore opening, promoting dysfunction [52, 53]. Holmuhamedov et al. [54] demonstrate that SSMs are inherently more sensitive to Ca²⁺-dependent metabolic stress than IFMs. Lower antioxidant capacity of SSMs and loosely organized cristae may further predispose them to injury [9, 13]. Structural alterations, including loss of transverse tubules and disrupted sarcoplasmic reticulum–mitochondria contacts, along with potential suppression of PGC-1$\alpha$ signaling, and impaired mitophagy, may hinder mitochondrial renewal, leading to the accumulation of dysfunctional SSMs [55, 56, 57]. Collectively, these factors render SSMs particularly susceptible during the late post-infarction period.

Further investigation of the underlying regulatory mechanisms, including the signaling pathways controlling mitochondrial dynamics, mitophagy and mitochondrial biogenesis, calcium homeostasis and interactions with sarcolemmal and nuclear domains on subacute stage of postinfarction remodeling, will enhance understanding of different mitochondrial subpopulations remodeling processes and guide the identification of potential therapeutic targets.

Morphofunctional analysis of intermyofibrillar, subsarcolemmal, and perinuclear mitochondrial subpopulations in LV CMCs under conditions of CTO of the left coronary artery revealed a decrease in both the activity and size of these organelles during the subacute stage of the disease. Among them, SSMs emerged as the most sensitive mitochondrial subpopulation to ischemic injury. Thus, the comparative assessment of mitochondrial damage performed in this study expands current knowledge of mitochondrial remodeling in CMCs during the subacute phase of MI, and serves as preliminary research supporting the development of therapeutic approaches to limit scar expansion.

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

SAF: validation, formal analysis, visualization, writing — original draft, writing — review and editing. AVS: methodology, investigation, writing — review and editing. GAS: methodology, investigation, writing — review and editing. MLV: methodology, investigation, visualization, writing — review and editing. EVB: conceptualization, methodology, data curation, investigation, validation, supervision, funding acquisition, formal analysis, visualization, project administration, resources, writing — original draft, writing — review and editing. All authors read and approved the final manuscript. All authors have participated sufficiently in the work and agreed to be accountable for all aspects of the work.

All animal experiments were conducted in accordance with the Treaty of Lisbon amending the Treaty on European Union and the Treaty establishing the European Community, which entered into force on 1 December 2009. The study was approved by the Ethical Committee for Animal Research of the Institute of Cytology of the Russian Academy of Science (Protocol No. 3; date of approval 28 June 2016) and the Animal Care and Use Committee at the L.A. Orbeli Institute of Physiology NAS RA (Protocol No. 07.02.2025/1; date of approval 19 April 2025).

We would like to express our gratitude to Mr. Yury Filippov for his assistance with the analysis of rat ECGs.

These studies have been supported by the Higher Education and Science Committee of the Republic of Armenia, grant # 23/2PostDoc-1F001, and the ERA Chair Project (NAR-SAR-IPH) funded by the European Union.

The authors declare no conflict of interest.

Supplementary material associated with this article can be found, in the online version, at https://doi.org/10.31083/FBL47046.