*3.5. Mitochondrial Permeability Transition Pore Complex (PTPC)*

Dissipation of the mitochondrial membrane potential, which is the signal for PINK1 and Parkin-dependent mitophagy, is usually caused by permeabilization of the IMM in a phenomenon called mitochondrial permeability transition (MPT). MPT occurs when proteinaceous channels, by a still partially unknown entity, open across the IMM and OMM, which causes uncontrolled fluxes of solutes and thereby osmotic stress, mitochondrial swelling, and pro-apoptotic factor release into the cytosol [148–150]. Years of intense research found strong similarities between ATP synthase and PTPC, and, in fact, the latter is currently considered a molecular rearrangement of the dimeric form of ATP synthase into monomers with contributions from a plethora of additional proteins with either structural [151] or modulatory properties [152–154], as observed following exposure to stressful conditions [150]. The alteration of the ATP synthase structure (in the putative PTPC conformation) exerts major effects on the disturbance of mitochondrial energy and respiration and hence on cardiac performance. Currently, only little information is available regarding the involvement of the PTPC in HF, and, thus, knowledge on its cause/effect consequentiality remains elusive. Of note, as mentioned above, metabolic changes in HF (especially lipids accumulation) might first contribute to membranes' permeability and membrane potential dissipation, triggering the opening of the PTPC [155].

The activation of mitochondrial phospholipases in the failing heart reportedly prompts the formation of toxic metabolites in a Ca2+-dependent manner. These hydroxyeicosatetraenoic acids induce PTPC opening by Ca2+ overload, which further worsens the HF scenario and increases the percentage of nonfunctional mitochondria and cell death [156]. This final endpoint was also suggested by independent groups: The final stages of HF are characterized by increased oxidative stress, increased diastolic Ca2+ overload, and episodes of ischemia, all of which prompt abrupt PTPC opening [157]. The pharmacological inhibition of the PTPC, such as by the use of cyclosporin A (CsA), reduced PTPC opening, the dissipation of mitochondrial potential and respiratory deficits, ameliorate the adverse conditions surrounding HF [158].

Another crucial determinant in PTPC opening is ROS. Major sources of ROS in the heart are caused by ETC function, nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (Noxs), and uncoupled NO synthases (NOS). Evidence about the importance in limiting oxidative stress in the heart comes from in vivo studies using transgenic mice with impaired levels of intracellular antioxidants, such as SOD2 [159,160], peroxiredoxins [161], glutathione peroxidase [162], and thioredoxin reductase [163]. In all cases, the alteration of the antioxidant expression correlated with the extent of oxidative stress, with important implications in cardiovascular diseases [164]. In the same way, PTPC opening is also dependent on the action of scavenger enzymes in the cell, especially on the glutathione content and function [165]. This phenomenon was in principle analyzed in single cardiomyocytes and described as a vicious cycle of ROS-induced ROS release (RIRR) [165,166] in which the PTPC opening (triggered by ROS) is essential to generate a ROS burst in contiguous mitochondria, and is associated with a significant dissipation of their membrane potential. The use of selective inhibitors, such as rotenone for complex I of ETC and bongkrekic acid for PTPC, inhibited RIRR, providing the proof of evidence that the ROS burst derives from mitochondria and is generated solely by PTPC opening consequences [165].

Further research on the modulation of PTPC in the failing heart might lead to the discovery of new therapeutic approaches for the treatment of symptoms and regression.

#### *3.6. Calcium Cycling and Handling*

Ca2+ plays an important role in the pathophysiology of the heart [1,167,168]. Its homeostasis is considered an essential mediator in regulating ECC and modulating systolic and diastolic function. Here, Ca2+ transduces action potentials in mechanical force. In healthy hearts, the action potential depolarizes the sarcolemma, allowing the passage of low amounts of Ca2+ from the extracellular space to the cytosol via L-type calcium channels (LTCCs). As consequence, the magnitude of Ca2+ action increases, due to the induced and highly synchronized Ca2+ sparks from the RyRs of SR in the whole cell [169,170]. After systole, Ca2+ should be removed from the cytosol to guarantee the correct muscle relaxation and, thus, the diastolic phase. SERCA2a, PMCA, and NCX are deputed to do this [171] in an ATP-dependent manner in the first two cases, evidencing further roles and the importance ascribed to ATP in muscle relaxation before a new EC cycle. All these proteinaceous channels are highly regulated by cytosolic proteins which often contribute to the end of the EC coupling but also to pathological states.

SR membranes are closely juxtaposed to mitochondria giving rise to a subcellular compartment named as mitochondria-associated membranes (MAMs) which, in lieu of material exchange, signal transduction and metabolic regulation [172,173]. During ECC it was demonstrated that Ca2+ fluctuations occur in mitochondria after SR Ca2+ release [174] but they remain controversial if they take part in the regulation of cytosolic Ca2+ levels, as it was estimated that mitochondria can handle up to 15% of cytosolic Ca2+, compared to other extrusion systems [175]. However, mitochondrial Ca2+ uptake has great repercussions on cardiomyocytes fate; in healthy bodies, by ensuring bioenergetics and cardiac contractility [9,10,176], and in disease bodies, where an overload can induce the PTPC opening upon I/R and a vicious cycling of ROS generation, oxidation of RyR2, and SR Ca2+ leak in HF, associated with mitochondrial fragmentation and dysfunction [177]. Mitochondrial Ca2+ dysregulation was reported also as defects in MAMs' morphology where the distance among contact sites between SR and mitochondria is increased in stages immediately preceding HF such as hypertrophy, chronic noradrenaline stimulation, and aging [178,179].

Myocytes from failing hearts are characterized by reduced and slower cytosolic Ca2+ transients ([Ca2+]<sup>i</sup> ); indeed, SR Ca2+ storage and release are impaired (Figure 2). Although this feature is mainly associated with a reduction in the SERCA2a protein amount, which becomes significantly downregulated in failing hearts compared with nonfailing hearts, its regulation might also play a crucial role because serine 16 phospholamban phosphorylation is reportedly reduced accordingly [16]. Moreover, increased Ca2+ leakage from the SR was explored as a secondary cause due to the dissociation of FKBP12.6 from RyR2 following protein kinase A (PKA)-dependent hyperphosphorylation at serine 2808 [180,181] (Figure 2). RyR2 expression does not change in HF but assumes aberrant gating based on the previously mentioned model.

Confirming the undoubtedly essential function of SERCA2a, the restoration of its expression with adeno-associated (AAV) vectors was classified as successful gene therapy in preclinical models of HF [17,182]. Recently, multiple ongoing clinical trials (as reviewed in [183]) are studying the use of AAV to improve SERCA2a expression and function. In addition, pharmacological treatments, such as the use of Istaroxime [184], stimulate SERCA2a and completely restore Ca2+ cycling.

The recorded elevation in cytosolic Ca2+ load is also caused by an altered influx from the extracellular milieu. Indeed, pathological SR Ca2+ depression activates the function of LTCCs and store-operated calcium entry (SOCE), which rewire a notable amount of extracellular Ca2+ into the cytosol [185] (Figure 2).

Concomitant with this finding, an additional molecular mechanism was observed in some models of HF, including humans: An increase in NCX function attempts to remove excess cytosolic Ca2+ to compensate for inefficient SERCA2a function and also further

depresses SR calcium [186,187]. In contrast, other studies found that NCX is impaired by working in a reverse mode due to high [Na<sup>+</sup> ] conditions [188]. These combinatorial effects lead to a complete change in the spatiotemporal fluxes of intracellular Ca2+, which reflect a markedly defective systolic contraction and diastolic relaxation of the heart [189].

**Figure 2.** Calcium cycling in the failing heart. The figure summarizes calcium cycling in the normal heart and how it changes in the failing heart. Proteins responsible for these alterations are highlighted in red (e.g., SERCA2a expression and molecular regulation; RyR2 aberrant gating), and the pathways to which they refer are described in the text. The importance of ATP in muscle contraction and relaxion is also depicted. On the right, a graphical abstract highlighting biogenesis and fusion-fission machinery in HF.
