**6.3. Bioenergetics dysfunction induced by doxorubicin**

effect of DOX on mitochondria has been reported to appear when it destroys mtDNA [44], mainly developing mtDNA deletion. The prevalence of the elimination has been reported to be between 33 and 80% at a low and high dose of DOX, respectively [10]. When DOX oxidizes mtDNA, mitochondria can no longer produce high-energy substrate, resulting from destroying to reproduce mtDNA [10]. This alternation is explained by DNA repair and elimination of damage to the genomic material, which changes or eliminates the protein function. Alternation or disappearance of mitochondrial protein function elevates ROS formation as well [13]. At this moment, we should take time to diagnose DOX's chronic cardiotoxic effect, e.g., heart failure, dilated cardiomyopathy, and congestive heart failure [10]. Moreover, mitochondrial complex I activity has been claimed to inhibit isolated mitochondria from cardiac tissue, but not hepatic tissue by chronic DOX therapy for 28 weeks. This notion has given rise to the thought that the drug's toxicity in mitochondria is cardioselective [10]. One study suggested that endurance exercises reduce DOX toxicity based on modulation of state 3 alternation at mitochondria. Also, the study reported that apoptosis induced by DOX could be counteracted by endurance exercises giving rise to a decline in apoptotic factors, such as Bax or Bax/Bcl-2 ratio. Moreover, DOX alters the ultrastructure of heart tissue by mitochondrial destruction, including damaging cristae and vacuoles, and causing distension and abnormal size and shape [80]. Mainly, the outer mitochondrial membrane plays a role in the transduction of signals, e.g., apoptotic [82]. Endurance exercises have been suggested to reverse the ultrastructural alternation, e.g., a rise in glycogen storage, and enhance cytosolic and mitochondrial sodium oxide dismutase. Due mostly to the sensitive oxidative stress of MPT, elevation of antioxidant by endurance exercises

**Figure 5.** The effect of doxorubicin and its derivate on the electron transport system and mitochondrial energy

leads to decreased apoptosis induced by DOX [80].

production. Modified from Govender et al. [34].

344 Mitochondrial Diseases

Mitochondria have sources of bioenergetics and ROS as well. This is why mitochondria become the target of multiple factors such as drugs, including DOX, and environmental compounds. PGC-1α plays an essential role in the regulation of mitochondrial function, including production of bioenergy and energy homeostasis. PGC-1α can regulate gene transcription, including mitochondrial services such as NRF-1, PPARa, and ERRa, which modulate and are divided into three groups of metabolic enzymes in the TCA cycle: antioxidant enzymes, other mitochondrial protein components of the ETC complexes, and mitochondrial transcription factor A (TFAM) [38].

When mitochondrial dysfunction occurs, i.e., enhancing ROS, reduction of ATP synthesis, a complex transcriptional network, including PGC-1α can be triggered to maintain cellular homeostasis. PGC-1α can transcript three groups of genes, which are mentioned above. Although PGC-1α's effect on three groups is suggested to have a minor impact at a low mitochondrial toxic concentration to alter ROS and ATP production, its effect on the groups is essential at a high level of mitochondrial toxins. It is reported that DOX's toxic effect is related to increasing mitochondrial ROS production and the most destructive impact of DOX appears in cardiac tissue due to its accumulation in cardiac cells [38].

It is well known that heart tissue contains a high cell volume of mitochondria, almost 35% due to the requirement of energy supply because of maintaining the contraction function of the tissue [15]. The heart produces energy requirement by using β-oxidation of fatty acid in mitochondria. The cardiotoxicity of DOX might be related to swelling and destroy bioenergy from the organelle and myofibril of cardiac tissue. DOX also increases oxidative stress, resulting in enhancing mitochondria dysfunction. The other mechanism of DOX on mitochondrial malfunction is reported to be associated with the dissipation of mitochondrial ETC at different levels. So, NADH and succinate oxidase in cardiac tissue have been shown to be blocked by DOX treatment. Also, DOX separates complex I from ETC, resulting in the elevation of oxidative damage by producing semiquinone free radicals. Moreover, DOX might inhibit stage-3 and stage-4 respiration. The other mechanism for blocking mitochondrial function is related to the prevention of Mg-dependent F0F1-ATPase in muscle, including heart and skeletal muscle. The other mechanism associates with DOX's structure since DOX has a high affinity to bind cardiolipin, which is one of lipids and locates at the inner mitochondrial membranes. DOX's toxicity on mitochondria is related to its dose and is time dependent. Mitochondrial toxicity of DOX has been observed from 2 to 13 weeks at a low dose. Moreover, mitochondria play an essential role in the regulation of calcium homeostasis. Under normal physiological conditions, there is little impact of mitochondria on calcium homeostasis. However, the mitochondrial function of calcium homeostasis is essential under pathological circumstances to decrease cytosolic calcium. So, calcium efflux into mitochondria depolarizes MMP. DOX's effect on calcium homeostasis at mitochondria is based on inhibition of the inward calcium flux and exaggeration of the release of calcium from mitochondria by swelling of the mitochondria–calcium-dependent pathways through MPT, resulting in enhancing calcium concentration at the cytosol. Furthermore, DOX at a low dose gives rise to a decline in the calcium storage capacity of mitochondria. DOX attenuates mitochondria calcium storage capacity exaggerated at its high dose, which means it is related to dose. Eventually, the effects of DOX lead to the dissipation of MMP. The mitochondrial permeability pore is highly sensitive to MMP and regulated by the redox status of mitochondria. DOX also opens MPT in the manufacture of ROS through complex I, which is mentioned above (**Figure 5**). Thus, DOX's MPT effect might be based on the prevention of ANT, an element of the MPT pore complex. It is interesting to note that DOX-treated cardiomyocytes have fewer ATP levels (~30%) vs. normal cardiomyocytes [15].

Some compensating mechanism is recommended to modulate energy supply to maintain cellular function, e.g., CK or AMPK. Studies have shown how to decrease ATP and phosphocreatine (PCr). One μM DOX concentration has indicated a decline of ~50% ATP production within 24 h. Why is the reduction essential to utilize around 90% ATP synthesized by mitochondria? When mitochondrial function is destroyed, heart or tissue function is automatically affected by low energy supply. DOX's mitochondrial effect is shown to change the ultrastructure, swelling, and oxidative capacity as well. Although DOX inhibits state 3, state 4 is activated by the drug. DOX is highly bound to cardiolipin, one of the anionic phospholipids from the inner membrane of mitochondria. When DOX binds to cardiolipin, enzymes from respiration and oxidation, e.g., cytochrome-*c*, might be inactive due to the alternation of the lipid environment. After binding cardiolipin to DOX, cardiolipin-related proteins such as cytochrome-*c* and mitochondrial creatine kinase (MtCK) are released from the mitochondrial inner membrane to the cytosol. Besides the indirect effect of that enzyme, DOX also has a direct effect on these enzymes. The mitochondrial impact of DOX can amplify its toxic effect by using the target organelle's enzyme system. DOX inactivates NADH dehydrogenase, cytochrome P-450 reductase, and xanthine oxidase. Moreover, DOX tends to accumulate in nuclei and mitochondria vs. plasma. The heart mainly utilizes fatty acid to generate energy by β-oxidation. So, DOX also destroys β-oxidation by inhibiting of consumption of palmitate, a long chain fatty acid, through impairment of carnitine palmitoyltransferase I (CPTI) and/or its substrate L-carnitine. DOX elevates glycolysis as a compensatory response to a decline in fatty acid oxidation. However, some studies' results show a decrease in both fatty acid and glucose oxidation by using cell line and rat models. Why glucose utilization is decreased after DOX treatment is explained by two theories. One is that DOX might reduce glucose supply. It is reported that DOX treatment initially increases glycolysis (~50%; within 1 h of exposure to the drug), but later depresses it sharply. The second explanation is that DOX reduces phosphofructokinase (PFK) activity, one of the rate-limiting enzymes of glycolysis. So, one reason for DOX's low-energy generation is that it disrupts cell metabolism tissue. The other reason is the effects of DOX's CK system. There are two CK isoforms in the heart: cytosolic and mitochondrial (MtCK). CK can easily produce PCr from creatine. Transformation of creatine to PCr closely binds between energy generation and utilization. MtCK, an octameric, is accompanied by ANT) and VDAC (porin), so transforming energy by the generation of oxidative phosphorylation to the cytoplasm. However, cytosolic CK isoform (MM-, MB-, BBCK), a dimeric, links to both energy manufacture by glycolysis and energy consumption, including

actomyosin ATPase at myofibrils, the Ca2+-ATPase at the SR, Na<sup>+</sup>

lemma [43]. DOX inactivates the entire CK system, especially MtCK. MtCK's effects dissociate its structure from octamer to dimer, resulting in dissociation from the mitochondrial inner membrane. Moreover, cardiac MtCK has been reported to be more sensitive to DOX than ubiquitous MtCK, leading to selective toxicity in heart tissue. The inactivation of DOX on MtCK has been indicated to be linked to the drug's dose. At a low dose below 100 μM, MtCK became inactive because of DOX's redox modification from its cysteine residues. Its high dose, however, depresses MtCK due to ROS production. Furthermore, DOX and MtCK have been indicated to have a common feature, i.e., they tend to attach to an inner mitochondrial membrane. So, the feature provides high DOX concentration around the MtCK. Additionally,

, and K+

Mitochondrial Dysfunction Associated with Doxorubicin http://dx.doi.org/10.5772/intechopen.80284 347


It is suggested that ROS produced by mitochondria has a critical role in DOX's cardiotoxicity. Hepatic tissue plays a role in detoxification by the cytochrome P450 enzyme in the ER, which also occurs in DOX's redox cycling. However, this bioreductive activation of DOX by cytochrome P450 in the heart is much less than in the liver. So, ROS production and DOX redox cycling in the ER in the heart are negligible [13]. Because it is continuously working, the heart needs ATP by β-oxidation in mitochondria. Therefore, cardiomyocytes contain tremendous mitochondrial density (around 25–30%) vs. the other cell types. This is why ROS production by mitochondria is significant compared to the other compartments of cardiomyocytes in heart tissue [13]. DOX has been reported to have high affinity to bind to cardiolipin from inner mitochondria. Also, DOX inhibits complexes II–IV from ETS [13]. Another suggestion is that DOX affects complex I, II, III, and IV [42]. DOX can also alter mitochondrial membrane organization but not physically interact with mitochondrial enzymes. According to isolated mitochondria and in vivo studies, DOX can capture an electron from complex I, resulting in a decline in oxidative phosphorylation and elevated oxygen consumption [13].

DOX elevates the nonphosphorylating rate of oxygen consumption (state 4) and decreases phosphorylation-linked oxygen consumption (state 3). Therefore, DOX triggers the manufacture of superoxide. Also, these impacts lead to declining ATP synthesis. Energy stress induced by DOX eventually prefers other pathways for producing ATP, e.g., glycolysis. However, glycolytic ATP does not have sufficient energy to maintain cell function. The metabolic switch is a requirement to make alternations such as enhancing glucose transporter type 1 (GLUT1) transportation to plasmalemma. So, glucose uptake increases by using GLUT1 within 1 h of DOX treatment [13].

Some compensating mechanism is recommended to modulate energy supply to maintain cellular function, e.g., CK or AMPK. Studies have shown how to decrease ATP and phosphocreatine (PCr). One μM DOX concentration has indicated a decline of ~50% ATP production within 24 h. Why is the reduction essential to utilize around 90% ATP synthesized by mitochondria? When mitochondrial function is destroyed, heart or tissue function is automatically affected by low energy supply. DOX's mitochondrial effect is shown to change the ultrastructure, swelling, and oxidative capacity as well. Although DOX inhibits state 3, state 4 is activated by the drug. DOX is highly bound to cardiolipin, one of the anionic phospholipids from the inner membrane of mitochondria. When DOX binds to cardiolipin, enzymes from respiration and oxidation, e.g., cytochrome-*c*, might be inactive due to the alternation of the lipid environment. After binding cardiolipin to DOX, cardiolipin-related proteins such as cytochrome-*c* and mitochondrial creatine kinase (MtCK) are released from the mitochondrial inner membrane to the cytosol. Besides the indirect effect of that enzyme, DOX also has a direct effect on these enzymes. The mitochondrial impact of DOX can amplify its toxic effect by using the target organelle's enzyme system. DOX inactivates NADH dehydrogenase, cytochrome P-450 reductase, and xanthine oxidase. Moreover, DOX tends to accumulate in nuclei and mitochondria vs. plasma. The heart mainly utilizes fatty acid to generate energy by β-oxidation. So, DOX also destroys β-oxidation by inhibiting of consumption of palmitate, a long chain fatty acid, through impairment of carnitine palmitoyltransferase I (CPTI) and/or its substrate L-carnitine. DOX elevates glycolysis as a compensatory response to a decline in fatty acid oxidation. However, some studies' results show a decrease in both fatty acid and glucose oxidation by using cell line and rat models. Why glucose utilization is decreased after DOX treatment is explained by two theories. One is that DOX might reduce glucose supply. It is reported that DOX treatment initially increases glycolysis (~50%; within 1 h of exposure to the drug), but later depresses it sharply. The second explanation is that DOX reduces phosphofructokinase (PFK) activity, one of the rate-limiting enzymes of glycolysis. So, one reason for DOX's low-energy generation is that it disrupts cell metabolism tissue. The other reason is the effects of DOX's CK system. There are two CK isoforms in the heart: cytosolic and mitochondrial (MtCK). CK can easily produce PCr from creatine. Transformation of creatine to PCr closely binds between energy generation and utilization. MtCK, an octameric, is accompanied by ANT) and VDAC (porin), so transforming energy by the generation of oxidative phosphorylation to the cytoplasm. However, cytosolic CK isoform (MM-, MB-, BBCK), a dimeric, links to both energy manufacture by glycolysis and energy consumption, including actomyosin ATPase at myofibrils, the Ca2+-ATPase at the SR, Na<sup>+</sup> , and K+ -ATPase in the sarcolemma [43]. DOX inactivates the entire CK system, especially MtCK. MtCK's effects dissociate its structure from octamer to dimer, resulting in dissociation from the mitochondrial inner membrane. Moreover, cardiac MtCK has been reported to be more sensitive to DOX than ubiquitous MtCK, leading to selective toxicity in heart tissue. The inactivation of DOX on MtCK has been indicated to be linked to the drug's dose. At a low dose below 100 μM, MtCK became inactive because of DOX's redox modification from its cysteine residues. Its high dose, however, depresses MtCK due to ROS production. Furthermore, DOX and MtCK have been indicated to have a common feature, i.e., they tend to attach to an inner mitochondrial membrane. So, the feature provides high DOX concentration around the MtCK. Additionally,

skeletal muscle. The other mechanism associates with DOX's structure since DOX has a high affinity to bind cardiolipin, which is one of lipids and locates at the inner mitochondrial membranes. DOX's toxicity on mitochondria is related to its dose and is time dependent. Mitochondrial toxicity of DOX has been observed from 2 to 13 weeks at a low dose. Moreover, mitochondria play an essential role in the regulation of calcium homeostasis. Under normal physiological conditions, there is little impact of mitochondria on calcium homeostasis. However, the mitochondrial function of calcium homeostasis is essential under pathological circumstances to decrease cytosolic calcium. So, calcium efflux into mitochondria depolarizes MMP. DOX's effect on calcium homeostasis at mitochondria is based on inhibition of the inward calcium flux and exaggeration of the release of calcium from mitochondria by swelling of the mitochondria–calcium-dependent pathways through MPT, resulting in enhancing calcium concentration at the cytosol. Furthermore, DOX at a low dose gives rise to a decline in the calcium storage capacity of mitochondria. DOX attenuates mitochondria calcium storage capacity exaggerated at its high dose, which means it is related to dose. Eventually, the effects of DOX lead to the dissipation of MMP. The mitochondrial permeability pore is highly sensitive to MMP and regulated by the redox status of mitochondria. DOX also opens MPT in the manufacture of ROS through complex I, which is mentioned above (**Figure 5**). Thus, DOX's MPT effect might be based on the prevention of ANT, an element of the MPT pore complex. It is interesting to note that DOX-treated cardiomyocytes have fewer ATP levels (~30%) vs.

It is suggested that ROS produced by mitochondria has a critical role in DOX's cardiotoxicity. Hepatic tissue plays a role in detoxification by the cytochrome P450 enzyme in the ER, which also occurs in DOX's redox cycling. However, this bioreductive activation of DOX by cytochrome P450 in the heart is much less than in the liver. So, ROS production and DOX redox cycling in the ER in the heart are negligible [13]. Because it is continuously working, the heart needs ATP by β-oxidation in mitochondria. Therefore, cardiomyocytes contain tremendous mitochondrial density (around 25–30%) vs. the other cell types. This is why ROS production by mitochondria is significant compared to the other compartments of cardiomyocytes in heart tissue [13]. DOX has been reported to have high affinity to bind to cardiolipin from inner mitochondria. Also, DOX inhibits complexes II–IV from ETS [13]. Another suggestion is that DOX affects complex I, II, III, and IV [42]. DOX can also alter mitochondrial membrane organization but not physically interact with mitochondrial enzymes. According to isolated mitochondria and in vivo studies, DOX can capture an electron from complex I, resulting in a

DOX elevates the nonphosphorylating rate of oxygen consumption (state 4) and decreases phosphorylation-linked oxygen consumption (state 3). Therefore, DOX triggers the manufacture of superoxide. Also, these impacts lead to declining ATP synthesis. Energy stress induced by DOX eventually prefers other pathways for producing ATP, e.g., glycolysis. However, glycolytic ATP does not have sufficient energy to maintain cell function. The metabolic switch is a requirement to make alternations such as enhancing glucose transporter type 1 (GLUT1) transportation to plasmalemma. So, glucose uptake increases by using GLUT1 within 1 h of

decline in oxidative phosphorylation and elevated oxygen consumption [13].

normal cardiomyocytes [15].

346 Mitochondrial Diseases

DOX treatment [13].

when DOX is activated by peroxidase/H2 O2 , CK inhibition via DOX accelerates. The inhibition link to oxidative and nitrous stress means that CK is very vulnerable to the redox status of cells. Even a μM DOX concentration has been reported to lead to dimerization of MtCK and augments inhibition and dimerization at a 20 μM concentration. Also, it is indicated that total CK activity has been noticed to reduce (by nearly 20%) for DOX treatment compared to 20 μM concentrations. Even under this circumstance, CK can still maintain its function due to a compensatory mechanism that causes to reduce muscle-type CK (MCK) (a myofibrillar isoform) and elevate brain-type CK (BCK; a fetal isoform) that is raised by heart failure or cardiac hypertrophy. It is important to know that CK shift is reported to be within 1 h at 2 μM DOX treatment. So, CK system dysfunction might probably participate in DOX-mediated heart failure. MtCK inhibition by dimerization not only causes energy transfer from mitochondria to the cytosol but also affects the mitochondrial respiratory chain. Moreover, inhibition destroys the three-modal interaction between MtCK, ANT, and DAC, which means that MtCK plays a role in MPT. So, destruction of modal interaction could trigger apoptosis as well. Besides programmed cell death, myofibrillar CK functionally integrates with the sarcoplasmic Ca2+ pump (SERCA). When a CK defect occurs, cytosolic Ca2+ balance is destroyed, leading to defects in contraction and relaxation coupling due to Ca2+ accumulation. Ca2+accumulation could trigger apoptosis as well. This is why dysfunction of CK causes innate apoptosis in two ways. When energy disruption occurs such as CK dysfunction, AMPK is activated to regain energy balance. AMPK is one of the sensory energy proteins that compensates for shifting from ATP to ADP and/or AMP. It means that AMPK is highly sensitive to a ratio of AMP/ ATP and oxidative stress as well. Under energy stress, AMPK changes the metabolic activity of cells to increase ATP synthesis by elevation of fatty acid oxidation, glycolysis, and a decline in ATP utilization. All these processes are crucial to surviving cells to maintain protein, lipid, and carbohydrate manufacture. It is reported that DOX inhibits AMPK, resulting in energy stress. In a study by using isolated heart, DOX at 2 μM, which is the plasma peak value of the patients treated with the drug, was reported to cause to plume AMPK and acetyl-CoA carboxylase proteins after 1-h perfusion. Therefore, further study is needed to evaluate the mechanism. However, it is suggested that DOX causes energy and oxidative stress in both reactive and nitrogen stress. AMPK inhibition means that DOX leads to a change in metabolic activity of cells by a decline in fatty acid oxidant. How the fatty acid oxidant decreases relates to enhancing acetyl-CoA carboxylase, resulting in CPTI by malonyl-CoA, eventually leading to a decrease in mitochondrial fatty acid oxidation. Besides the decline of mitochondrial fatty acid oxidation, AMPK inhibition also causes to reduce glycolysis by decreasing of PFK and glucose uptake as well. Under physiological conditions, energy stress is expected to activate AMPK [43].One study showed that AMPK, glucose, and fatty acid is related to gene and protein expressions, and acetyl-CoA carboxylase have been decreased by DOX in males more than in females. AMPK is also a crucial function for cardiolipin synthesis and remodeling. By AMPK, PGC-1α/β modulates cardiolipin synthesis as well [36].

tissue [84]. The heart produces an energy requirement by using the β-oxidation of fatty acid in mitochondria [15]. Adequate ATP production is not just significant to maintain contractile function, but is also crucial for protein synthesis, controlling the protein quality function of ER, cytoskeletal function, and to clear the cellular waste from lysosomes. This is why DOX destroys energy production systems and has been reported to destroy the protein degrada-

Mitochondrial Dysfunction Associated with Doxorubicin http://dx.doi.org/10.5772/intechopen.80284 349

The impact of DOX on bioenergetics and oxidative stress might partially be associated with its structure because it has a high affinity to bind to cardiolipin, which is one of the lipids and locates at the inner mitochondrial membrane [13, 43]. ROS production by mitochondria might be a significant contributor to the drug's toxicity in heart tissue [13]. This is why mitochondria is one of the targets of multiple factors such as drugs including DOX and environmental compounds. There are defined control systems that maintain healthy, functional mitochondria. One of the systems is PGC-1α, which plays an essential role in the regulation of mitochondrial function, including production of bioenergy and energy homeostasis. PGC-1α can regulate gene transcription, including mitochondrial functions such as NRF-1, PPARa, and ERRa, which modulate and divided three groups in metabolic enzymes in the TCA cycle: antioxidant enzymes, other mitochondrial protein components of the ETC complexes, and TFAM [38]. When mitochondrial dysfunction occurs, i.e., enhancing ROS and reducing ATP synthesis, a complex transcriptional network, including PGC-1α, can be triggered to maintain cellular homeostasis. PGC-1α can transcribe three groups of genes, which are mentioned above. Although PGC-1α has an effect on three groups it is suggested to have a minor impact at a low mitochondrial toxic concentration to alter ROS and ATP production; its effect on the groups is important at high concentration of mitochondrial toxins. It is reported that DOX's toxic effect is related to increasing mitochondrial ROS production and the most destructive impact of DOX can appear in cardiac tissue due to its accumulation in cardiac cells [38]. One study has noticed that DOX led to a decrease in PGC-1α and its related genes, including NRF1, TFAM, SOD2, CS, VDAC, and COXIV. PGC-1α is phosphorylated by AMPK, and is also modulated by acetylation by SIRT1. The posttranslational modification of PGC-1α is a potent mechanism for mitochondrial function by oxidative stress and apoptosis. So, SIRT1 is suggested to decline mitochondrial dysfunction and cardiotoxicity induced by DOX [16]. The other control system is SIRT-3 which is one of NAD-dependent deacetylases and places at mitochondria. Deacetylase plays a crucial role in maintaining healthy mitochondrial function by deacetylation of metabolic, apoptotic, and ROS-production enzymes. Also, SIRT-3 has been shown to enhance Foxo-3a, which is associated with an antioxidant mechanism. Thus, SIRT-3 closes the MPT by blocking Cyp D activity. SIRT-3 has a modulating effect on cardiac hypertrophy via strengthening the LKB1 event, which is one of the upstream kinases of AMPK. One study has reported that SIRT-3 is decreased by DOX treatment due to the rise in ROS production and mitochondrial dysfunction. So, reducing SIRT-3 leads to elevation to ROS and HIF1α stabilization, which is an essential factor to shift metabolism from β-oxidation of fatty acid to glycolysis (the Warburg effect) [85]. Further, the inhibition of protein or slicing

of the HIF1α gene has been suggested to decrease drug resistance against DOX [86].

It is well known that the heart can produce ATP from fatty acid. However, metabolic shift is developed from fatty acid to glucose due to a compensating energy demand under pathologic

tion function, resulting in overwhelming the ER and mitochondria [4].

#### **6.4. Doxorubicin's effect on myocardial energy metabolism**

It is well known that heart tissue contains a high cell volume of mitochondria nearly (25–35%) [3, 15] due to the requirement of energy supply to maintain the contraction function of the tissue [84]. The heart produces an energy requirement by using the β-oxidation of fatty acid in mitochondria [15]. Adequate ATP production is not just significant to maintain contractile function, but is also crucial for protein synthesis, controlling the protein quality function of ER, cytoskeletal function, and to clear the cellular waste from lysosomes. This is why DOX destroys energy production systems and has been reported to destroy the protein degradation function, resulting in overwhelming the ER and mitochondria [4].

when DOX is activated by peroxidase/H2

348 Mitochondrial Diseases

AMPK, PGC-1α/β modulates cardiolipin synthesis as well [36].

**6.4. Doxorubicin's effect on myocardial energy metabolism**

It is well known that heart tissue contains a high cell volume of mitochondria nearly (25–35%) [3, 15] due to the requirement of energy supply to maintain the contraction function of the

O2

tion link to oxidative and nitrous stress means that CK is very vulnerable to the redox status of cells. Even a μM DOX concentration has been reported to lead to dimerization of MtCK and augments inhibition and dimerization at a 20 μM concentration. Also, it is indicated that total CK activity has been noticed to reduce (by nearly 20%) for DOX treatment compared to 20 μM concentrations. Even under this circumstance, CK can still maintain its function due to a compensatory mechanism that causes to reduce muscle-type CK (MCK) (a myofibrillar isoform) and elevate brain-type CK (BCK; a fetal isoform) that is raised by heart failure or cardiac hypertrophy. It is important to know that CK shift is reported to be within 1 h at 2 μM DOX treatment. So, CK system dysfunction might probably participate in DOX-mediated heart failure. MtCK inhibition by dimerization not only causes energy transfer from mitochondria to the cytosol but also affects the mitochondrial respiratory chain. Moreover, inhibition destroys the three-modal interaction between MtCK, ANT, and DAC, which means that MtCK plays a role in MPT. So, destruction of modal interaction could trigger apoptosis as well. Besides programmed cell death, myofibrillar CK functionally integrates with the sarcoplasmic Ca2+ pump (SERCA). When a CK defect occurs, cytosolic Ca2+ balance is destroyed, leading to defects in contraction and relaxation coupling due to Ca2+ accumulation. Ca2+accumulation could trigger apoptosis as well. This is why dysfunction of CK causes innate apoptosis in two ways. When energy disruption occurs such as CK dysfunction, AMPK is activated to regain energy balance. AMPK is one of the sensory energy proteins that compensates for shifting from ATP to ADP and/or AMP. It means that AMPK is highly sensitive to a ratio of AMP/ ATP and oxidative stress as well. Under energy stress, AMPK changes the metabolic activity of cells to increase ATP synthesis by elevation of fatty acid oxidation, glycolysis, and a decline in ATP utilization. All these processes are crucial to surviving cells to maintain protein, lipid, and carbohydrate manufacture. It is reported that DOX inhibits AMPK, resulting in energy stress. In a study by using isolated heart, DOX at 2 μM, which is the plasma peak value of the patients treated with the drug, was reported to cause to plume AMPK and acetyl-CoA carboxylase proteins after 1-h perfusion. Therefore, further study is needed to evaluate the mechanism. However, it is suggested that DOX causes energy and oxidative stress in both reactive and nitrogen stress. AMPK inhibition means that DOX leads to a change in metabolic activity of cells by a decline in fatty acid oxidant. How the fatty acid oxidant decreases relates to enhancing acetyl-CoA carboxylase, resulting in CPTI by malonyl-CoA, eventually leading to a decrease in mitochondrial fatty acid oxidation. Besides the decline of mitochondrial fatty acid oxidation, AMPK inhibition also causes to reduce glycolysis by decreasing of PFK and glucose uptake as well. Under physiological conditions, energy stress is expected to activate AMPK [43].One study showed that AMPK, glucose, and fatty acid is related to gene and protein expressions, and acetyl-CoA carboxylase have been decreased by DOX in males more than in females. AMPK is also a crucial function for cardiolipin synthesis and remodeling. By

, CK inhibition via DOX accelerates. The inhibi-

The impact of DOX on bioenergetics and oxidative stress might partially be associated with its structure because it has a high affinity to bind to cardiolipin, which is one of the lipids and locates at the inner mitochondrial membrane [13, 43]. ROS production by mitochondria might be a significant contributor to the drug's toxicity in heart tissue [13]. This is why mitochondria is one of the targets of multiple factors such as drugs including DOX and environmental compounds. There are defined control systems that maintain healthy, functional mitochondria. One of the systems is PGC-1α, which plays an essential role in the regulation of mitochondrial function, including production of bioenergy and energy homeostasis. PGC-1α can regulate gene transcription, including mitochondrial functions such as NRF-1, PPARa, and ERRa, which modulate and divided three groups in metabolic enzymes in the TCA cycle: antioxidant enzymes, other mitochondrial protein components of the ETC complexes, and TFAM [38]. When mitochondrial dysfunction occurs, i.e., enhancing ROS and reducing ATP synthesis, a complex transcriptional network, including PGC-1α, can be triggered to maintain cellular homeostasis. PGC-1α can transcribe three groups of genes, which are mentioned above. Although PGC-1α has an effect on three groups it is suggested to have a minor impact at a low mitochondrial toxic concentration to alter ROS and ATP production; its effect on the groups is important at high concentration of mitochondrial toxins. It is reported that DOX's toxic effect is related to increasing mitochondrial ROS production and the most destructive impact of DOX can appear in cardiac tissue due to its accumulation in cardiac cells [38]. One study has noticed that DOX led to a decrease in PGC-1α and its related genes, including NRF1, TFAM, SOD2, CS, VDAC, and COXIV. PGC-1α is phosphorylated by AMPK, and is also modulated by acetylation by SIRT1. The posttranslational modification of PGC-1α is a potent mechanism for mitochondrial function by oxidative stress and apoptosis. So, SIRT1 is suggested to decline mitochondrial dysfunction and cardiotoxicity induced by DOX [16]. The other control system is SIRT-3 which is one of NAD-dependent deacetylases and places at mitochondria. Deacetylase plays a crucial role in maintaining healthy mitochondrial function by deacetylation of metabolic, apoptotic, and ROS-production enzymes. Also, SIRT-3 has been shown to enhance Foxo-3a, which is associated with an antioxidant mechanism. Thus, SIRT-3 closes the MPT by blocking Cyp D activity. SIRT-3 has a modulating effect on cardiac hypertrophy via strengthening the LKB1 event, which is one of the upstream kinases of AMPK. One study has reported that SIRT-3 is decreased by DOX treatment due to the rise in ROS production and mitochondrial dysfunction. So, reducing SIRT-3 leads to elevation to ROS and HIF1α stabilization, which is an essential factor to shift metabolism from β-oxidation of fatty acid to glycolysis (the Warburg effect) [85]. Further, the inhibition of protein or slicing of the HIF1α gene has been suggested to decrease drug resistance against DOX [86].

It is well known that the heart can produce ATP from fatty acid. However, metabolic shift is developed from fatty acid to glucose due to a compensating energy demand under pathologic conditions. So, the expression of peroxisome proliferator-activated receptor gamma is also affected by the drug, resulting in decline in adipogenesis and destruction of glucose intake via glucose transporter type 4 (GLUT4) [34]. In contrast, according to previous study results, 1 h after 1 μM of DOX was given to cultured adult rat cardiac cells, sarcomeric titin protein was reported significantly to degrade via the calpain-dependent mechanism. DOX increases glucose uptake by GLUT1 into plasma membrane [24], which is a requirement of the metabolic switch in the first hours of treatment [13]. However, it is reported that GLUT4 was not affected by DOX [24]. Also, DOX impairs PFK, which is a rate-limiting glycolytic flux [24, 34]. Energy stress induced by DOX eventually prefers the other pathways for producing ATP, e.g., glycolysis. However, glycolytic ATP does not have sufficient energy to maintain cell function [13]. The cardiotoxicity of DOX might relate to swelling and destroy bioenergy from the organelle and myofibril of cardiac tissue. DOX also increases oxidative stress, resulting in enhancing mitochondrial dysfunction [15].

The other mechanism of energy dysfunction due to DOX treatment is AMPK destruction [87]. It is well known that the heart mainly utilizes fatty acid to generate energy by β-oxidation [43]. DOX inhibits fatty acid β-oxidation and myocardial function as well, but enhances glucose intake though AMPK phosphorylation [88]. AMPK inhibition means that DOX leads to a change in the metabolic activity of cells by declining fatty acid oxidant, particularly palmitate consumption. How the fatty acid oxidant decreases relates to enhancing acetyl-CoA carboxylase by AMPK inhibition, resulting in a decline in CPTI and/or its substrate L-carnitine by malonyl-CoA, eventually leading to a decrease in mitochondrial fatty acid oxidation [43]. Under physiological conditions, energy stress activates AMPK [43]. DOX elevates glycolysis as a compensatory response to a decline in fatty acid oxidation. However, some study results showed a decrease in both fatty acid and glucose oxidation by using cell line and rat models. Why glucose utilization is reduced after DOX treatment is explained by two theories. One is that DOX might minimize glucose supply. It is reported that DOX treatment initially increases glycolysis (~50%; within 1 h of exposure to the drug), but later causes it to depress sharply. The second explanation is that DOX leads to reduced PFK activity, one of the rate-limiting

Mitochondrial Dysfunction Associated with Doxorubicin http://dx.doi.org/10.5772/intechopen.80284 351

When energy disruption occurs such as CK dysfunction, AMPK is activated to regain energy balance. AMPK is one of the sensory energy proteins that compensates for shifting from ATP to ADP and/or AMP. It means AMPK is highly sensitive to a ratio of AMP/ATP and oxidative stress. Under energy stress, AMPK changes the metabolic activity of cells to increase ATP synthesis by elevation of fatty acid oxidation, glycolysis, and a decline in ATP utilization. All these processes are crucial to the surviving cell by maintaining proteins, lipids, and the manufacture of carbohydrate. It is reported that DOX inhibits AMPK, resulting in energy stress [43]. Another study noticed that AMPK can be inactivated with a 2 μM concentration of DOX. This explains how DOX can change substrate utilization to produce energy [24]. There is no clearly understood process as to how AMPK is inhibited. Therefore, further study is needed to evaluate the mechanism. However, it is suggested that DOX causes energy and oxidative stress in both reactive and nitrogen stress [43]. Kinase is also a crucial function in cardiolipin synthesis and remodeling. By AMPK, PGC-1α/β modulates cardiolipin synthesis as well [36]. Moreover, DOX could destroy desmin interaction with mitochondria, resulting in triggering apoptosis [89]. When our knowledge of AMPK, cardiolipin, DOX, and PGC1α/β are superimposed, it can easily be understood that DOX-induced cardiac mitochondrial toxicity is more complex and multifactorial. Metabolic dysfunction induced by DOX might also relate to gender [36]. One pathway of DOX's low-energy generation disrupts cell metabolism tissue. DOX destroys CK as an energy shuttle and storage system, AMPK as an energy-sensing and signaling system [24], and the channel of ATP and PCr from mitochondria to the cytosol. PCr and creatine can regulate the ATP/ADP ratio [14]. This is why the mechanism of DOX's mitochondrial energy dysfunction can be explained by DOX's cardiac cell metabolism CK system effects [43]. CK in the heart has two isoforms. One is located free at the cytosol (cytosolic CK (cCK)), and the other is bound to sarcoplasmic or mitochondrial membranes [14, 43]. cCK has two subtypes: muscle-type MCK and brain-type BCK. Also, MtCK has two subtypes known as sarcomeric MtCK (sMtCK) that exist only in the heart and skeletal muscles and ubiquitous MtCK (uMtCK) that is present in other organs and tissues, such as the brain, spermatozoa,

enzymes of glycolysis [43].

The other mechanism of DOX on mitochondrial dysfunction is reported to be associated with dissipation of mitochondrial ETC at different levels. So, NADH and succinate oxidase in cardiac tissue has been shown to be blocked by DOX treatment. Also, DOX separates complex I from ETC, resulting in elevated oxidative damage by producing semiquinone free radicals [15]. Also, DOX inhibits complexes II–IV from ETS [13]. DOX disrupts complex I, III, and IV, and is especially susceptible to complex I and IV [34]. Specifically, DOX decreases the content of the complex I NDUFB8 subunit and the ATP synthase ATP5A subunit [87]. The opinion of others is that DOX can alter mitochondrial membrane organization but not physical interaction with any mitochondrial enzymes [13]. In other words, DOX affects all complexes from I to IV [42]. There is a discrepancy between DOX's effects on the stage of respiration. One of the studies suggested that DOX might inhibit state 3 and state 4 respiration [15]. Although others have reported that DOX impedes state 3, which is phosphorylation-linked oxygen consumption, it is activated by the drug [13, 43]. Therefore, DOX triggers superoxide manufacturing. Also, this impact led to declining ATP synthesis [13]. The other mechanism for blocking the mitochondrial function is related to prevention of Mg-dependent F0F1-ATPase in muscle, including heart and skeletal muscle [15].

The ATP pool of cardiac tissue is reported to be 5 mmol/kg wet heart weight. When the demand for energy in the heart is increased, PCr (concentration around 10 mmol/kg wet heart weight) can compensate for the requirement. PCr can be transformed by CK, one of the energy reservoir regulators [34]. Interestingly, total ATP decline due to DOX treatment on cardiomyocytes vs. normal, healthy cardiomyocytes is reported to be ~30% [15]. Some compensating mechanism is recommended to modulate energy supply to maintain cellular function, e.g., CK or AMPK. Studies have shown that ATP and/or PCr decrease by using the drug. One μM DOX concentration has indicated a decline of ~50% ATP production within 24 h [43]. PCr is destroyed by DOX treatment due to the accumulation of ferrous iron by the drug. So, DOX declines both ATP and PCr. Children treated with DOX for 4 years have been reported to have decreased PCr/ATP ratios of around 20% [34]. Why the decline is vital to utilize around 90% ATP synthesized by mitochondria is because heart or tissue function is automatically affected by low energy supply when mitochondrial function is destroyed [43].

The other mechanism of energy dysfunction due to DOX treatment is AMPK destruction [87]. It is well known that the heart mainly utilizes fatty acid to generate energy by β-oxidation [43]. DOX inhibits fatty acid β-oxidation and myocardial function as well, but enhances glucose intake though AMPK phosphorylation [88]. AMPK inhibition means that DOX leads to a change in the metabolic activity of cells by declining fatty acid oxidant, particularly palmitate consumption. How the fatty acid oxidant decreases relates to enhancing acetyl-CoA carboxylase by AMPK inhibition, resulting in a decline in CPTI and/or its substrate L-carnitine by malonyl-CoA, eventually leading to a decrease in mitochondrial fatty acid oxidation [43]. Under physiological conditions, energy stress activates AMPK [43]. DOX elevates glycolysis as a compensatory response to a decline in fatty acid oxidation. However, some study results showed a decrease in both fatty acid and glucose oxidation by using cell line and rat models. Why glucose utilization is reduced after DOX treatment is explained by two theories. One is that DOX might minimize glucose supply. It is reported that DOX treatment initially increases glycolysis (~50%; within 1 h of exposure to the drug), but later causes it to depress sharply. The second explanation is that DOX leads to reduced PFK activity, one of the rate-limiting enzymes of glycolysis [43].

conditions. So, the expression of peroxisome proliferator-activated receptor gamma is also affected by the drug, resulting in decline in adipogenesis and destruction of glucose intake via glucose transporter type 4 (GLUT4) [34]. In contrast, according to previous study results, 1 h after 1 μM of DOX was given to cultured adult rat cardiac cells, sarcomeric titin protein was reported significantly to degrade via the calpain-dependent mechanism. DOX increases glucose uptake by GLUT1 into plasma membrane [24], which is a requirement of the metabolic switch in the first hours of treatment [13]. However, it is reported that GLUT4 was not affected by DOX [24]. Also, DOX impairs PFK, which is a rate-limiting glycolytic flux [24, 34]. Energy stress induced by DOX eventually prefers the other pathways for producing ATP, e.g., glycolysis. However, glycolytic ATP does not have sufficient energy to maintain cell function [13]. The cardiotoxicity of DOX might relate to swelling and destroy bioenergy from the organelle and myofibril of cardiac tissue. DOX also increases oxidative stress, resulting in

The other mechanism of DOX on mitochondrial dysfunction is reported to be associated with dissipation of mitochondrial ETC at different levels. So, NADH and succinate oxidase in cardiac tissue has been shown to be blocked by DOX treatment. Also, DOX separates complex I from ETC, resulting in elevated oxidative damage by producing semiquinone free radicals [15]. Also, DOX inhibits complexes II–IV from ETS [13]. DOX disrupts complex I, III, and IV, and is especially susceptible to complex I and IV [34]. Specifically, DOX decreases the content of the complex I NDUFB8 subunit and the ATP synthase ATP5A subunit [87]. The opinion of others is that DOX can alter mitochondrial membrane organization but not physical interaction with any mitochondrial enzymes [13]. In other words, DOX affects all complexes from I to IV [42]. There is a discrepancy between DOX's effects on the stage of respiration. One of the studies suggested that DOX might inhibit state 3 and state 4 respiration [15]. Although others have reported that DOX impedes state 3, which is phosphorylation-linked oxygen consumption, it is activated by the drug [13, 43]. Therefore, DOX triggers superoxide manufacturing. Also, this impact led to declining ATP synthesis [13]. The other mechanism for blocking the mitochondrial function is related to prevention of Mg-dependent F0F1-ATPase in muscle,

The ATP pool of cardiac tissue is reported to be 5 mmol/kg wet heart weight. When the demand for energy in the heart is increased, PCr (concentration around 10 mmol/kg wet heart weight) can compensate for the requirement. PCr can be transformed by CK, one of the energy reservoir regulators [34]. Interestingly, total ATP decline due to DOX treatment on cardiomyocytes vs. normal, healthy cardiomyocytes is reported to be ~30% [15]. Some compensating mechanism is recommended to modulate energy supply to maintain cellular function, e.g., CK or AMPK. Studies have shown that ATP and/or PCr decrease by using the drug. One μM DOX concentration has indicated a decline of ~50% ATP production within 24 h [43]. PCr is destroyed by DOX treatment due to the accumulation of ferrous iron by the drug. So, DOX declines both ATP and PCr. Children treated with DOX for 4 years have been reported to have decreased PCr/ATP ratios of around 20% [34]. Why the decline is vital to utilize around 90% ATP synthesized by mitochondria is because heart or tissue function is automatically affected by low energy supply when mitochondrial function is

enhancing mitochondrial dysfunction [15].

350 Mitochondrial Diseases

including heart and skeletal muscle [15].

destroyed [43].

When energy disruption occurs such as CK dysfunction, AMPK is activated to regain energy balance. AMPK is one of the sensory energy proteins that compensates for shifting from ATP to ADP and/or AMP. It means AMPK is highly sensitive to a ratio of AMP/ATP and oxidative stress. Under energy stress, AMPK changes the metabolic activity of cells to increase ATP synthesis by elevation of fatty acid oxidation, glycolysis, and a decline in ATP utilization. All these processes are crucial to the surviving cell by maintaining proteins, lipids, and the manufacture of carbohydrate. It is reported that DOX inhibits AMPK, resulting in energy stress [43]. Another study noticed that AMPK can be inactivated with a 2 μM concentration of DOX. This explains how DOX can change substrate utilization to produce energy [24]. There is no clearly understood process as to how AMPK is inhibited. Therefore, further study is needed to evaluate the mechanism. However, it is suggested that DOX causes energy and oxidative stress in both reactive and nitrogen stress [43]. Kinase is also a crucial function in cardiolipin synthesis and remodeling. By AMPK, PGC-1α/β modulates cardiolipin synthesis as well [36]. Moreover, DOX could destroy desmin interaction with mitochondria, resulting in triggering apoptosis [89]. When our knowledge of AMPK, cardiolipin, DOX, and PGC1α/β are superimposed, it can easily be understood that DOX-induced cardiac mitochondrial toxicity is more complex and multifactorial. Metabolic dysfunction induced by DOX might also relate to gender [36].

One pathway of DOX's low-energy generation disrupts cell metabolism tissue. DOX destroys CK as an energy shuttle and storage system, AMPK as an energy-sensing and signaling system [24], and the channel of ATP and PCr from mitochondria to the cytosol. PCr and creatine can regulate the ATP/ADP ratio [14]. This is why the mechanism of DOX's mitochondrial energy dysfunction can be explained by DOX's cardiac cell metabolism CK system effects [43]. CK in the heart has two isoforms. One is located free at the cytosol (cytosolic CK (cCK)), and the other is bound to sarcoplasmic or mitochondrial membranes [14, 43]. cCK has two subtypes: muscle-type MCK and brain-type BCK. Also, MtCK has two subtypes known as sarcomeric MtCK (sMtCK) that exist only in the heart and skeletal muscles and ubiquitous MtCK (uMtCK) that is present in other organs and tissues, such as the brain, spermatozoa, and skin. Cardiac cCK has two forms as a homodimer (MMCK and/or BBCK) or heterodimer (MBCK), which is a cardiac-specific form [24]. According to this knowledge, it is said that MBCK can usually be determined as an indicator of a heart attack. sMtCK is the mainly octameric form [24] and places the outer intermembrane space and mitochondrial cristae between membranal protein ANT in the inner membrane and VDAC in the outer layer [24]). MtCK has high affinity to cardiolipin [14] and the outer surface of the inner mitochondrial membrane [24]. It must not be forgotten that DOX has a great relationship with cardiolipin. So, one of DOX's targets is MtCK. Moreover, DOX oxidizes MtCK at cysteine residues. Besides oxidation, DOX leads to inactivation of MtCK, resulting in enhanced embryonic CK isoform expression [14].

Besides the CK shuttle, the malate–aspartate shuttle (MAS) has a role in declining traffic equivalents between mitochondria and the cytosol. Moreover, TCA and MAS have demonstrated to be associated with each other physically. This interaction provides a direct reason for metabolic alternation of the mitochondrial matrix to the cytosol. MAS suppression in the heart has been proposed to reduce mitochondrial respiration before cardiac damage, thereby declining oxidative injury. A cancer cell produces energy by glycolysis known as the Warburg effect. So, it is suggested that MAS inhibition might be an excellent candidate for overt DOX

Mitochondrial Dysfunction Associated with Doxorubicin http://dx.doi.org/10.5772/intechopen.80284 353

DOX directly affects oxidative phosphorylation enzymes, e.g., NADH dehydrogenase, Rieske iron sulfur protein, succinate dehydrogenase, cyclooxygenase, CK, carnitine palmitoyltransferase, fatty acid β-oxidation-related enzymes, as well as the translocation of phosphate and pyruvate to the mitochondrial matrix. DOX reduces fatty acid β-oxidation by blocking fatty acid transfer protein to mitochondria, resulting in an increase in the pyruvate dehydrogenase complex, which is a rate-limiting enzyme of glycolysis. The other effect of DOX metabolism is that the triosephosphate isomerase enzyme essential for glucose metabolism can be inhibited by the treatment [14]. The anthracyline causes mitochondrial dysfunction, e.g., displacing α-enolase from mitochondria

It is impossible to ignore DOX therapy from cancer patients' treatment due to its inevitable chemotherapeutic efficiency on a variety of cancers. Unfortunately, there is limited knowledge available on DOX's cardiotoxicity, particularly mitochondriopathy. This is why the molecular clarifying mechanism of DOX's myocardial and mitochondrial toxicities will hopefully overcome the side effects and increase the survival rate of cancer patients as well. Therefore, further studies are needed to evaluate the detrimental effects of DOX on mitochondria to

[30]. ATP synthesis is decreased in both the cytosol and mitochondria by DOX [90].

\* and Eylem Taskin3

1 Department of Biophysics, Faculty of Medicine, Omer Halisdemir University, Nigde,

2 Department of Biology, Faculty of Science and Letters, Adiyaman University, Adiyaman,

3 Department of Physiology, Faculty of Medicine, Omer Halisdemir University, Nigde,

toxicity, without affecting the anticancer drug's effects [29].

restore its limited utilization in cancer patients' therapy.

\*Address all correspondence to: ysevgiler@protonmail.com

, Yusuf Sevgiler<sup>2</sup>

**7. Conclusion**

**Author details**

Celal Guven1

Turkey

Turkey

Turkey

MtCK can efficiently produce PCr from creatine [24, 43]. Transformation of creatine to PCr firmly binds between energy generation and utilization. MtCK, an octameric, accompanies ANT, and VDAC (porin) [43]. ANT can transfer ADP to matrix space. Then, ADP resynthesizes ATP through oxidative phosphorylation. However, PCr can be sent to the cytosol via VDAC [24]. PCr is utilized by cCK to maintain the subcellular local ATP/ADP ratio [24].

Although DOX inactivates all CK, MtCK is especially destroyed [43] by the drug through dissociation of its structure from octamer to dimer [24, 43] or infusion of binding MtCK at mitochondrial membranes, such as cardiolipin [24]. Moreover, cardiac MtCK has been reported to be more sensitive to DOX than uMtCK, leading to selective toxicity in heart tissue. The inactivation of MtCK by DOX is linked to the drug's dosage. At a low dose below 100 μM, MtCK's inactivation occurs because of DOX's redox modification from its cysteine residues. Its high treatment, however, depresses MtCK due to ROS production. Furthermore, DOX and MtCK have been indicated to have a standard feature, they tend to attach an inner mitochondrial membrane, providing high DOX concentration around the MtCK. Additionally, when DOX is activated by peroxidase/H2 O2 , CK inhibition via DOX accelerates. The inhibition is linked to oxidative and nitrous stress, which means that CK is very vulnerable to the redox status of cells. Even a 2 μM DOX concentration has been reported to lead to dimerization of MtCK (ordinarily octameric), and augment the inhibition and dimerization at a 20 μM intensity. Also, it is indicated that total CK activity has been noticed to reduce (by nearly 20%) for DOX treatment concentrated at 20 μM. Under this circumstance, CK has still been maintaining its function due to a compensatory mechanism, which reduces MCK (a myofibrillar isoform) and high BCK (a fetal isoform) that is elevated by heart failure or cardiac hypertrophy. It is vital to know that CK shift is reported to be within 1 h at 2 μM of DOX. So, CK system dysfunction might probably participate in DOX-mediated heart failure. MtCK inhibition by dimerization not only causes energy transfer from mitochondria to the cytosol but also influences the mitochondrial respiratory chain. Moreover, this inhibition destroys the three-modal interaction between MtCK, ANT, and VDAC, which means that MtCK plays a role in MPT. So, damage to the modal interaction could first trigger apoptosis. Besides programmed cell death, myofibrillar CK functionally integrates with SERCA. When a CK defect occurs, cytosolic Ca2+ balance is destroyed, leading to defects in contraction and relaxation coupling due to Ca2+ accumulation. Ca2+accumulation could also trigger apoptosis. This is why dysfunction of CK causes innate apoptosis in two ways [43].

Besides the CK shuttle, the malate–aspartate shuttle (MAS) has a role in declining traffic equivalents between mitochondria and the cytosol. Moreover, TCA and MAS have demonstrated to be associated with each other physically. This interaction provides a direct reason for metabolic alternation of the mitochondrial matrix to the cytosol. MAS suppression in the heart has been proposed to reduce mitochondrial respiration before cardiac damage, thereby declining oxidative injury. A cancer cell produces energy by glycolysis known as the Warburg effect. So, it is suggested that MAS inhibition might be an excellent candidate for overt DOX toxicity, without affecting the anticancer drug's effects [29].

DOX directly affects oxidative phosphorylation enzymes, e.g., NADH dehydrogenase, Rieske iron sulfur protein, succinate dehydrogenase, cyclooxygenase, CK, carnitine palmitoyltransferase, fatty acid β-oxidation-related enzymes, as well as the translocation of phosphate and pyruvate to the mitochondrial matrix. DOX reduces fatty acid β-oxidation by blocking fatty acid transfer protein to mitochondria, resulting in an increase in the pyruvate dehydrogenase complex, which is a rate-limiting enzyme of glycolysis. The other effect of DOX metabolism is that the triosephosphate isomerase enzyme essential for glucose metabolism can be inhibited by the treatment [14]. The anthracyline causes mitochondrial dysfunction, e.g., displacing α-enolase from mitochondria [30]. ATP synthesis is decreased in both the cytosol and mitochondria by DOX [90].
