**4. Metabolic Preconditioning**

Adaptation of the heart to altered metabolic conditions allows the maintenance of its function. It allows the heart to meet the requirements of the body effectively [84]. One of the most commonly used experimental models for the induction of metabolic preconditioning (MPC) is streptozotocin-induced diabetes mellitus with positive structural and metabolic changes present during its acute phase [84–88]. The acute stage of diabetes is characterized by the inhibition of insulin secretion and decreased signaling of insulin receptors in target cells [89]. After the seven days following streptozotocin administration, changes induced by diabetes mellitus are fully developed, without side complication characteristics for the chronic stage of the disease [90]. The acute phase of diabetes mellitus persists for the next three weeks [89].

The chronic phase of streptozotocin diabetes mellitus can be induced by a longer administration of streptozotocin, i.e., for more than 60 days. It is characterized by many complications, such as neuropathy, retinopathy, nephropathy, microangiopathy, etc. [91].

Despite the fact that diabetes mellitus causes extensive changes in the structure and function of cardiomyocytes, its impact is not necessarily entirely harmful. A short-term exposure of the heart to a high glucose concentration or diabetes mellitus has proven beneficial effects against ischemic insult [92,93]. The first evidence of a compensation effect due to diabetes was presented by a study pointing out a better recovery of contractility of a diabetic heart after an I/R injury [94].

The acute phase of MPC induced by diabetes is characterized not only by metabolic changes [84], but also by positively affecting the heart efficiency and its sensitivity to pathological stimuli, remodeling the cardiomyocyte membrane, as well as cardiac mitochondria [95,96]. Indeed, remodeling has a central role in maintaining or repairing the heart tissue [97].

#### **5. Involvement of Mitochondrial Connexin 43 in Cardioprotection**

It has been well established that the modulation of membrane channel protein "connexin 43" (Cx43), the most abundant connexin in the heart [98], could have various cardioprotective effects [99–102]. The association of six subunits of Cx43 results in the formation of hemichannel "connexon" [103]. After transportation in secretory vesicles to the plasma membrane, two opposing connexons from adjacent cells create the "Cx channel". Thousands of Cx43 channels aggregate into gap junction plaques at the intercalated disks [104,105]. This direct connection between two adjacent cells provides electrical and metabolic cell-to-cell coupling [106]. Cx43 hemichannels are not only precursors for Cx43 channels, but can also exist as non-junctional hemichannels at the plasma membrane and can contribute to volume regulation, to the release of ATP and NAD+ from the cytosol, and the activation of cell survival pathways [107]. In addition to predominantly localized Cx43 at the intercalated disks, 4% Cx43 is present in mitochondria due to translocation from cardiomyocytes [108,109].

MtCx43 in the cardiomyocytes is situated in the inner mitochondrial membrane (IMM) of subsarcolemmal mitochondria (SSM) where it forms an mtCx43 hemichannel [110,111]. MtCx43 import to the IMM of SSM is mediated by the interaction between Cx43 with the heat shock protein 90 (HSP90) and translocase of the outer membrane 20 [109]. The physiological role of mtCx43 is not fully clarified, but some studies support its involvement in the regulation of K<sup>+</sup> fluxes [112], mitochondrial respiration [113], oxygen consumption [111,112], mitochondrial redox state [114], and in mitochondrial Ca2+ homeostasis [115] (Figure 1). In this context, it is understandable that mtCx43 is attributed to cardioprotection.

**Figure 1.** Diagram of signaling pathways affecting mitochondrial permeability transition pores (mPTP) regulation in preconditioned and pathological myocardium. For details, see the text.

The implication of mtCx43 in the cardioprotective pathway of IPC has been mostly elucidated [109,111,116]. The protein level of mtCx43 very rapidly increased in response to IPC and was maintained for at least 90 min in a pig model of IPC [108], whereas attenuation of mtCx43 was associated with lost IPC cardioprotection [108]. Evidence that mtCx43 is implicated in this mechanism was also demonstrated in an experiment by Heinzel et al. in 2015, where pharmacological preconditioning by diazoxide mediated by gating of mitochondrial ATP sensitive potassium channels (KATP) and protein kinase C activation [117] was repealed in cardiomyocytes isolated from mice with a reduced Cx43 level. Mitochondrial KATP channels have also a key role in mitochondrial physiology and potential effects on several pathological processes, thanks to their involvement in cellular energetic status by regulation of organelle volume and function [118].

Indeed, diazoxide affects the generation of ROS necessary in low amounts as trigger molecules of IPC [119]. In the experimental model of Cx43-deficient mice, nitric oxide (NO) production was significantly lower compared to wild type control mice [120]. Increased S-nitrosation of mtCx43 by IPC elevated mitochondrial permeability and subsequently ROS formation [121]. Moreover, diazoxide modulates the opening of the mPTP [122]. The relationship between mtCx43 and mPTP has been elucidated. In this study, pharmacological inhibition of mtCx43 induced opening of mPTP in SSM by increased levels of Ca2+ [123]. Another study with IPC abolishment in which reduction of mtCx43 was induced by geldanamycin (which prevents translocation of Cx43 to mitochondria by blocking the HSP90 dependent pathway) confirmed that only mtCx43 is implicated in this cardioprotection [124]. MtCx43 can also be implicated in cardioprotection by interaction with proteins related to mitochondrial fraction and metabolism. MtCx43 interacts with the apoptosis inducing factor (AIF) involved in oxidative phosphorylation and redox control. Interestingly, AIF deficient mice had the same pattern of changes in ROS generation and mitochondrial complex 1 activity as Cx43 deficient mice [113]. In cardiomyocytes with overexpression of Cx43, only complex I respiration was increased, while complex II remained unchanged [113]. A close relationship between mtCx43 and with anti- and pro-apoptosis markers Bcl-2 and Bax was observed. In this experiment, elevated levels of mtCx43 were accompanied by the upregulation of Bcl-2 and inhibition of Bax in the cardiac mitochondria after hypoxic postconditioning [125].

#### **6. The Role of Cardiolipin in Heart Mitochondrial Signaling**

Cardiolipin, as a unique phospholipid, is an important component of the IMM [126,127]. It is a relevant indicator of mitochondrial membrane fluidity damage [128]. Due to the localization of respiratory enzymes and oxidative phosphorylation in the IMM, maintaining a positive membrane fluidity remodeling is essential to ensure the bioenergetic processes of the cell [95,129–131] (Figure 1). The fluidity of the mitochondrial membrane is an important part of endogenous protective mechanisms, especially in pathological conditions such as diabetes mellitus [34], ischemia-reperfusion damage [132,133], and hypercholesterolemia [130]. Maintaining membrane fluidity under load conditions at the control level improves ATP transport from the mitochondrial matrix to the cytosol of cardiomyocytes [130]. The sustainability of phospholipid composition in the mitochondrial membrane results in the proper mitochondrial function and structure, phospholipid metabolism, and energy transport [134].

Changes in the composition of the cardiolipin structure, content, and acyl chain are associated with mitochondrial dysfunction in the tissues of certain pathophysiological conditions, including apoptosis [125], ischemia [135], I/R [136], in various stages of thyroid disease [137,138], diabetes mellitus [139], aging [140], and heart failure [135,141].

Cardiolipin is an oxidatively sensitive phospholipid, particularly to ROS [142], due to a high content of unsaturated fatty acids [143]. Oxidative damage of cardiolipin negatively a ffects the biochemical function of mitochondrial membranes [127], which is reflected in the alteration of the membrane fluidity, ion permeability, as well as the structure and function of the electron transport chain. These alterations lead to a reduced oxidative phosphorylation e fficacy of mitochondria [144,145].

Cardiolipin contributes to the protein function in the IMM and maintains the integrity and flow of the electron transport chain, including anionic carriers and respiratory chain complexes [146,147]. Cardiolipin is specifically required for electron transfer in mitochondria respiratory chain complex I [136]. Respiratory complex III of the mammalian chain contains bound cardiolipin molecules that are essential for the enzyme activity [148]. ROS induced oxidative damage of cardiolipin in mitochondria may be responsible for the observed defect in the activity of complex III [149]. Similarly, complex IV contains tightly bound cardiolipin whose removal results in a change of its structure and function [150].

Today, many diseases in which mitochondrial dysfunction has been associated with cardiolipin peroxidation have been described [151]. It seems that a high concentration of Ca2+ has a negative impact on mitochondrial function related to the cardiolipin peroxidation. A high concentration of Ca2+ together with cardiolipin peroxidation participates in mPTP opening [127]. It has been suggested the cardiolipin associated with the adenine nucleotide translocator (ANT) may be the site at which Ca2+ binds and activates mPTP opening. Binding of Ca2+ to ANT surrounding cardiolipins enhances the mobility of ANT-Cys56, which could be a potential pathway of Ca2+ for induction of mPTP opening [152] (Figure 1).

The accumulation of oxidized cardiolipin in the outer mitochondrial membrane (OMM) contributes to mPTP opening, which is also accompanied by the release of cytochrome c (Cyt c) from mitochondria into the cytosol [153]. The role of cardiolipin in Cyt c releasing from mitochondria seems to be very important in the process of apoptosis [154,155].

Cardiolipin is required to maintain the proper function of ATP synthase and facilitate its rotation, which is supported by the transmembrane proton gradient [156,157]. Cardiolipin is involved in mPTP control via affecting the function of ATP synthase [158,159]. Positive mitochondrial membrane remodeling is associated with an increased membrane fluidity, as well as increased mitochondrial ATP synthase activity in streptozotocin induced pseudohypoxic acute diabetic conditions [160].

#### **7. The Role of Mitochondrial Permeability Transition Pores in Signaling Processes of Cardioprotection**

Substantial evidence has revealed that the mPTP are associated with the signaling pathway of cardioprotective models and seem to be an end-effector of cardioprotection [161,162]. It has been shown that the inhibition of mPTP opening not only provides a protective strategy against reperfusion injury [163], but is also a key point in cardioprotective mechanisms such as IPC or MPC [164–166] (Figure 1). The cardioprotective effect of RPC has been associated with the inhibition of mPTP formation [44]. Transient mPTP opening, which allows the release of Ca2+ from the mitochondria into the matrix, appears to be a key mechanism in MPC [167].

Under physiological conditions, the mPTP are closed or not present. Their opening is associated with postischemic reperfusion, when the perturbations in intracellular Ca2+ homeostasis, ROS accumulation, and a reduction of mitochondrial membrane potential (Δψ) are characteristic [168,169]. The massive opening of mPTP results in an increase in IMM permeability and the entry of metabolites into the mitochondrial matrix, which leads to mitochondrial swelling, collapse of Δψ, reduction in the efficiency of ATP production by uncoupling the electron transport system from oxidative phosphorylation [170–172], and cell death [10,168]. mPTP remain closed due to low intracellular pH (<7.0) during ischemia, but they are opened during the first minutes of reperfusion associated with the normalization of pH, which causes irreversible heart damage [162,173,174]. Although mPTP are associated with mitochondrial damage and cell death, transient mPTP opening represents one of the physiological processes that is used in the mitochondria of healthy cells [170]. In the heart, transient mPTP opening during preconditioning could be a protective tool that ensures a physiological role during damage [175]. It is believed that transient mPTP opening releases Ca2+ from the mitochondrial matrix to maintain mitochondrial homeostasis. Transient mPTP opening is also associated with a temporary increase in ROS as signaling molecules [176].

Increased mPTP production has also been reported in the experimental model of acute diabetes mellitus [177]. The increased formation of mPTP is presented as a compensating mechanism that facilitates the transfer of ATP molecules from the mitochondria into the cytosol, where energy supply is currently needed. Residual mitochondrial ATP production due to its increased cytosolic transfer has been shown to be adequate to maintain sufficient levels of adenine nucleotides in acute diabetic myocardium [34]. The inhibition of mPTP opening may also be achieved by pharmacological drugs. The development of inhibitors, except of a prototype compound such as cyclosporine A (CsA), is limited by side effects and a low therapeutic efficacy [178]. Similarly, there is evidence of a protective mechanism of mPTP inhibition against cancer cell survival and proliferation. mPTP has become a promising strategy for improving cancer therapies [179].

In studies by Heather et al., SSM was adapted to hypoxic conditions and thus mitochondria acquired increased resistance to oxidative damage under conditions of limited oxygen supply. These hypoxia mediated changes induced functional adaptation of mitochondria to a certain dose of stress, resembling the mechanism of the preconditioning effect [180].

mPTP represent a protein complex whose molecular composition remains unexplained. The new knowledge about the structure and regulation of this mitochondrial pore comes annually. The hypothesis about the nature of mPTP suggests that their number is increasing after a conformational change in ATP synthase after binding of Ca2+ [181,182]. This change should lead to the opening of the hidden megachannel. It has been discussed whether the role of ATP synthase can be changed from a key energy producing enzyme to an energy dissipating channel that leads to cell death [182]. ATP synthase, together with a phosphate carrier protein (PiC) and ANT, is organized into supramolecular units called synthasomes, which increases the efficiency of ATP production [14,183]. Cyclophilin D (CypD) regulates mPTP, as well as the dynamics of the synthasome, depending on the bioenergy state of mitochondria [184]. Cardiolipin oxidation can disrupt the interactions between the components of the ATP synthasome, which can cause destabilization in this supercomplex, thereby promoting mPTP opening [185].

However, a study by Carroll et al. denied the idea of ATP synthase as the main structural component of mPTP. mPTP opening also occurred after deletion of selected ATP synthase subunits after Ca2+ overload. Based on these findings, the authors unlikely considered that ATP synthase and its subunits are involved in the mPTP structure [186]. Interestingly, new findings confirmed the participation of ANT in the mPTP structure. Results achieved by Karch et al. supported the idea about ANT dependent mPTP activity, which is regulated by CypD. ANT dependent mPTP is activated in response to higher mitochondrial matrix Ca2+ levels, which means independently of CypD [187]. Many question marks hang over the mPTP structure, again.

#### **8. Unregulated Mitochondrial Permeability Transition Pore Opening**

The prolonged mPTP opening results in disruption of the mitochondrial ultrastructure, halting of mitochondrial energy, and ATP synthesis, resulting in a variety of diseases, currently without successful treatment [188]. The unregulated mPTP opening and the consequent oxidative damage are considered as the major mechanisms of mitochondrial energetic dysfunction, which ultimately lead to cell death [8,189]. mPTP opening has an impact on the release of Cyt c from mitochondria, which is associated with pathophysiological situations such as I/R injury, aging, and other degenerative diseases [127]. One of the independent risk factors that may cause structural, molecular, and biochemical changes is aging. Aging increases CypD expression and its interaction with ATP synthase leading to a higher risk of mPTP opening. Thus, mPTP are important factors controlling mitochondrial function affected by aging [190]. Cardioprotective mechanisms, such as preconditioning, could be also impaired by aging and lead to defects in protective cell signaling. In a study by Griecsova et al., the efficacy of preconditioning was attenuated in mature adult rats in contrast with younger animals. Increasing age caused the decrease of heart ischemic tolerance, as well as changes in cellular expression of proteins involved in the protective signaling [191]. Age related disorders are also associated with increased ROS production and dysregulation of intracellular Ca2+ levels, resulting in mPTP opening [192]. mPTP are opened during reperfusion after previous ischemic injury of the heart, leading to myocardial damage [193]. Likewise, disruption of Ca2+ homeostasis in addition to myocardial I/R injury also occur in neurodegenerative diseases that lead to mPTP opening [194]. Chronic diseases such as diabetes or hypertension cause changes in mitochondrial bioenergetics manifested by inhibition of respiratory chain complex activity, increased proton leakage from the IMM, increased ROS production, and Ca2+ overload resulting in mPTP opening [8,172,195]. It has been found that the prevention of mPTP opening by mPTP inhibitors would be beneficial in a wide range of therapeutically challenging diseases. Therefore, significant effort is being made to develop mPTP specific inhibitors that would overcome the major disadvantages of CsA. Further studies are needed to progress from research to therapeutics [188,196].
