**1.2. Mitochondrial Ca2+ transport and mitochondrial membrane permeability transition (MPT)**

Ca2+ modulates several metabolic pathways through transient changes in its free concentrations in different cell compartments [50, 51]. In order to fulfill these physiological roles, Ca2+ movements across cell membranes are driven directly or indirectly by ATP hydrolysis. Therefore, defects in processes that supply cellular ATP may lead to deregulation in Ca2+ signaling that may compromise cell functioning, redox balance, and mitochondrial membrane permeability transition (MPT) [51, 52]. In this review, we briefly describe how mitochondrial Ca2+ load promotes MPT [53].

MPT is characterized by the opening of a high conductance, nonspecific proteinaceous pore, the PTP. It was first described by Hunter and collaborators [54] and then demonstrated by Vercesi's group to be dependent on redox imbalance promoted either by thiol oxidants or oxidative stress [55]. Matrix Ca2+ participates in at least two steps in the process of PTP opening: (a) stimulates superoxide generation by mitochondria and (b) binds to membrane sites exposing specific buried thiols to the oxidants (**Figure 1**) [55]. Accordingly, Ca2+ binding to cardiolipin alters mitochondria inner membrane lipid organization characterized by increased lipid packing and domain formation. As a consequence, the electron transfer along the respiratory complexes is impaired favoring superoxide generation [56].

Robust data has provided evidence that PTP opening is a main step in the mitochondrial pathway leading to cell death either by apoptosis or necrosis [57, 58], and is a major cause of cell death under a variety of pathophysiological conditions, including ischemia/reperfusion injury, traumatic brain injury, neurodegenerative diseases, metabolic diseases, muscular dystrophy, and drug toxicity [59–67].

Since mitochondrial Ca2+ overload stimulates superoxide generation and MPT, the mechanisms of Ca2+ transport by mitochondria will be outlined next. The inner mitochondrial membrane possesses three different carriers for Ca2+ influx and efflux [68]. A mitochondrial calcium uniporter (MCU) located in the inner membrane mediates the influx of Ca2+ down its electrochemical gradient without coupling Ca2+ transport to the flux of another ion. This mechanism was discovered in the 1960s [69, 70], but the molecular nature of the channel was only recently identified [71, 72]. Ca2+ release from mitochondria occurs via Ca2+/3Na<sup>+</sup> or a Ca2+/2H+ exchangers [73–75] depending on the tissue [68, 76].

The high loads of matrix Ca2+ that stimulate ROS production in mitochondria [55] appear to be associated with either dysregulation of cellular Ca2+ homeostasis or regulated release from endo(sarco)plasmic reticulum [77–79] (**Figure 1**). Under both conditions, the opening of the PTP can occur allowing for the movements of molecules up to 1.5 KDa. The entry of solutes and water to the matrix causes large amplitude mitochondrial swelling. These conditions disrupt both the electrochemical proton potential and oxidative phosphorylation [23, 55]. When PTP opens in a large number of mitochondria, cell death occurs by necrosis due to the lack of ATP, and when PTP is limited to a small number of mitochondria, apoptosis is triggered by the release of cytochrome c [80]. Anti-apoptotic proteins (members of Bcl-2 family) or cyclosporine A inhibits the opening of PTP [81, 82]. Evidence has been provided that high intracellular Ca2+ levels and ROS have additive effects in the process of PTP opening [23, 53, 55, 83–88].

of inorganic phosphate [53, 55, 83, 85, 91], and Bcl-2 family proteins [81, 82] participate in PTP modulation. The close location of mitochondria and the endoplasmic reticulum (ER) [75] permits mitochondria to take up large amounts of Ca2+ that are released from the ER. This process seems to be controlled via a redox-regulated cross talk between mitochondria and ER that is mediated by NADPH oxidases [36]. Such redox interactions may link PTP opening to the induction of Ca2+ signals specifically for cell death [26]. Considering the understanding on how Ca2+ and ROS act synergistically in the mechanism of PTP opening, it should be emphasized that mitochondria are more susceptible to MPT when their antioxidant systems are exhausted, especially due to an oxidized state of NADPH and GSH [55]. Accordingly, mitochondria isolated from mice deficient in nicotinamide nucleotide transhydrogenase (NNT), which cannot sustain NADPH in the reduced state, present defective antioxidant capacity and increased susceptibility to MPT [92, 93]. Thus, MPT can be induced by pro-oxidants and prevented or even

**Figure 1.** Statins triggers mitochondrial oxidative stress and calcium-dependent permeability transition. Statins diminishes the respiratory capacity at the level of complexes I, II and III of the respiratory chain, increasing superoxide generation

sulfhydryl-disulfide transitions, a process involved in PTP opening. Statins also impair cellular Ca2+ homeostasis, inducing

via VDAC and MCU channels, leading to its accumulation in mitochondrial matrix. Ca2+ binds to membrane sites exposing

of ROS and mitochondrial Ca2+ overload, PTP may open and trigger cell death. In addition, a decrease in the levels of CoQ10 that acts as an electron carrier and antioxidant also occurs due to inhibition of the mevalonate pathway by statins. The

specific buried thiols to the oxidants and also impairs mitochondrial respiration, increasing O<sup>2</sup>

antioxidants CoQ10, L-carnitine and creatine prevent PTP opening induced by statins.

). The Fe-S clusters present in these respiratory complexes are vulnerable to superoxide attack, thus inhibiting their activity and diminishing their resistance to Ca2+ induced MPT. Superoxide is dismutated in hydrogen peroxide (H<sup>2</sup>

O2

Mitochondrial Oxidative Stress and Calcium-Dependent Permeability Transition are Key Players…

R and increasing cytosolic Ca2+ levels. Thus, mitochondria uptake the excessive cytosolic Ca2+

O2 ).

**.**- formation. The association

can induce (directly or indirectly) membrane protein

http://dx.doi.org/10.5772/intechopen.71610

389

reversed by antioxidants [85, 86, 94, 95].

When not metabolized by mitochondrial antioxidant systems, H2

(O<sup>2</sup> **.**-

Ca2+ release from the ER via IP<sup>3</sup>

It is well recognized that mitochondrial Ca2+ is essential for PTP opening [54, 55, 89, 90], whereas oxidative modifications of inner membrane protein thiols, oxidative stress, presence Mitochondrial Oxidative Stress and Calcium-Dependent Permeability Transition are Key Players… http://dx.doi.org/10.5772/intechopen.71610 389

**1.2. Mitochondrial Ca2+ transport and mitochondrial membrane permeability** 

complexes is impaired favoring superoxide generation [56].

exchangers [73–75] depending on the tissue [68, 76].

Ca2+ modulates several metabolic pathways through transient changes in its free concentrations in different cell compartments [50, 51]. In order to fulfill these physiological roles, Ca2+ movements across cell membranes are driven directly or indirectly by ATP hydrolysis. Therefore, defects in processes that supply cellular ATP may lead to deregulation in Ca2+ signaling that may compromise cell functioning, redox balance, and mitochondrial membrane permeability transition (MPT) [51, 52]. In this review, we briefly describe how mitochondrial Ca2+ load promotes MPT [53].

MPT is characterized by the opening of a high conductance, nonspecific proteinaceous pore, the PTP. It was first described by Hunter and collaborators [54] and then demonstrated by Vercesi's group to be dependent on redox imbalance promoted either by thiol oxidants or oxidative stress [55]. Matrix Ca2+ participates in at least two steps in the process of PTP opening: (a) stimulates superoxide generation by mitochondria and (b) binds to membrane sites exposing specific buried thiols to the oxidants (**Figure 1**) [55]. Accordingly, Ca2+ binding to cardiolipin alters mitochondria inner membrane lipid organization characterized by increased lipid packing and domain formation. As a consequence, the electron transfer along the respiratory

Robust data has provided evidence that PTP opening is a main step in the mitochondrial pathway leading to cell death either by apoptosis or necrosis [57, 58], and is a major cause of cell death under a variety of pathophysiological conditions, including ischemia/reperfusion injury, traumatic brain injury, neurodegenerative diseases, metabolic diseases, muscular dystrophy,

Since mitochondrial Ca2+ overload stimulates superoxide generation and MPT, the mechanisms of Ca2+ transport by mitochondria will be outlined next. The inner mitochondrial membrane possesses three different carriers for Ca2+ influx and efflux [68]. A mitochondrial calcium uniporter (MCU) located in the inner membrane mediates the influx of Ca2+ down its electrochemical gradient without coupling Ca2+ transport to the flux of another ion. This mechanism was discovered in the 1960s [69, 70], but the molecular nature of the channel was only recently identified [71, 72]. Ca2+ release from mitochondria occurs via Ca2+/3Na<sup>+</sup>

The high loads of matrix Ca2+ that stimulate ROS production in mitochondria [55] appear to be associated with either dysregulation of cellular Ca2+ homeostasis or regulated release from endo(sarco)plasmic reticulum [77–79] (**Figure 1**). Under both conditions, the opening of the PTP can occur allowing for the movements of molecules up to 1.5 KDa. The entry of solutes and water to the matrix causes large amplitude mitochondrial swelling. These conditions disrupt both the electrochemical proton potential and oxidative phosphorylation [23, 55]. When PTP opens in a large number of mitochondria, cell death occurs by necrosis due to the lack of ATP, and when PTP is limited to a small number of mitochondria, apoptosis is triggered by the release of cytochrome c [80]. Anti-apoptotic proteins (members of Bcl-2 family) or cyclosporine A inhibits the opening of PTP [81, 82]. Evidence has been provided that high intracellular Ca2+

levels and ROS have additive effects in the process of PTP opening [23, 53, 55, 83–88].

It is well recognized that mitochondrial Ca2+ is essential for PTP opening [54, 55, 89, 90], whereas oxidative modifications of inner membrane protein thiols, oxidative stress, presence

or a

**transition (MPT)**

388 Mitochondrial Diseases

and drug toxicity [59–67].

Ca2+/2H+

**Figure 1.** Statins triggers mitochondrial oxidative stress and calcium-dependent permeability transition. Statins diminishes the respiratory capacity at the level of complexes I, II and III of the respiratory chain, increasing superoxide generation (O<sup>2</sup> **.**- ). The Fe-S clusters present in these respiratory complexes are vulnerable to superoxide attack, thus inhibiting their activity and diminishing their resistance to Ca2+ induced MPT. Superoxide is dismutated in hydrogen peroxide (H<sup>2</sup> O2 ). When not metabolized by mitochondrial antioxidant systems, H2 O2 can induce (directly or indirectly) membrane protein sulfhydryl-disulfide transitions, a process involved in PTP opening. Statins also impair cellular Ca2+ homeostasis, inducing Ca2+ release from the ER via IP<sup>3</sup> R and increasing cytosolic Ca2+ levels. Thus, mitochondria uptake the excessive cytosolic Ca2+ via VDAC and MCU channels, leading to its accumulation in mitochondrial matrix. Ca2+ binds to membrane sites exposing specific buried thiols to the oxidants and also impairs mitochondrial respiration, increasing O<sup>2</sup> **.**- formation. The association of ROS and mitochondrial Ca2+ overload, PTP may open and trigger cell death. In addition, a decrease in the levels of CoQ10 that acts as an electron carrier and antioxidant also occurs due to inhibition of the mevalonate pathway by statins. The antioxidants CoQ10, L-carnitine and creatine prevent PTP opening induced by statins.

of inorganic phosphate [53, 55, 83, 85, 91], and Bcl-2 family proteins [81, 82] participate in PTP modulation. The close location of mitochondria and the endoplasmic reticulum (ER) [75] permits mitochondria to take up large amounts of Ca2+ that are released from the ER. This process seems to be controlled via a redox-regulated cross talk between mitochondria and ER that is mediated by NADPH oxidases [36]. Such redox interactions may link PTP opening to the induction of Ca2+ signals specifically for cell death [26]. Considering the understanding on how Ca2+ and ROS act synergistically in the mechanism of PTP opening, it should be emphasized that mitochondria are more susceptible to MPT when their antioxidant systems are exhausted, especially due to an oxidized state of NADPH and GSH [55]. Accordingly, mitochondria isolated from mice deficient in nicotinamide nucleotide transhydrogenase (NNT), which cannot sustain NADPH in the reduced state, present defective antioxidant capacity and increased susceptibility to MPT [92, 93]. Thus, MPT can be induced by pro-oxidants and prevented or even reversed by antioxidants [85, 86, 94, 95].
