Recent Advances in Mitochondrial Fission/Fusion-Targeted Therapy in Doxorubicin-Induced Cardiotoxicity

Doxorubicin (DOX) has been recognized as one of the most effective chemotherapies and extensively used in the clinical settings of human cancer. However, DOX-mediated cardiotoxicity is known to compromise the clinical effectiveness of chemotherapy, resulting in cardiomyopathy and heart failure. Recently, accumulation of dysfunctional mitochondria via alteration of the mitochondrial fission/fusion dynamic processes has been identified as a potential mechanism underlying DOX cardiotoxicity. DOX-induced excessive fission in conjunction with impaired fusion could severely promote mitochondrial fragmentation and cardiomyocyte death, while modulation of mitochondrial dynamic proteins using either fission inhibitors (e.g., Mdivi-1) or fusion promoters (e.g., M1) can provide cardioprotection against DOX-induced cardiotoxicity. In this review, we focus particularly on the roles of mitochondrial dynamic pathways and the current advanced therapies in mitochondrial dynamics-targeted anti-cardiotoxicity of DOX. This review summarizes all the novel insights into the development of anti-cardiotoxic effects of DOX via the targeting of mitochondrial dynamic pathways, thereby encouraging and guiding future clinical investigations to focus on the potential application of mitochondrial dynamic modulators in the setting of DOX-induced cardiotoxicity.


Introduction
In 2040, the number of cancer cases is expected to hit 28.4 million, a 47 percent increase from 2020 [1]. Due to current medical advances in early cancer diagnosis and effective cancer treatment, the number of cancer survivors is projected to reach 22.1 million by the year 2030 [2]. However, the increasing survival of cancer patients is accompanied by an increasing number of cardiovascular issues attributable to chemotherapies [3,4]. Doxorubicin (DOX), which originally belonged to the class of drugs known as anthracyclines, is a potent cytotoxic chemotherapy drug that is frequently administered in the treatment of cancer. DOX is often used to treat solid tumors and hematological malignancies in both children and adults [3]. Unfortunately, clinical use of DOX is associated with dosedependent and cumulative cardiotoxicity, which may result in a range of cardiovascular outcomes [4]. Oxidative stress is the most frequently documented molecular mechanism associated with DOX-induced cardiotoxicity (DIC) via altering redox status at the level of biological macromolecules. DOX primarily damages mitochondria by interacting with cardiolipin, a unique phospholipid of the inner mitochondrial membrane that helps stabilize oxidative phosphorylation (OXPHOS) complexes [5]. Mechanistically, the accumulation of DOX in the mitochondria causes the uncoupling of OXPHOS complexes, resulting in a decrease in ATP synthesis and an increase in the AMP/ATP ratio, which activates AMPKmediated cardiomyocyte injury [5]. Moreover, DOX-induced mitochondrial dysfunction disrupts intracellular calcium homeostasis and has been demonstrated to diminish mitochondrial membrane potential, open the mPTP, and increase reactive oxygen species (ROS) production, resulting in mitochondrial dysfunction and endothelial damage [6]. Since DIC has a complex pathophysiology involving several molecular pathways, including oxidative stress, autophagy, and inflammation, emerging evidence clearly suggests that mitochondrial dynamics play a crucial role in the development of DIC.
Mitochondria are crucially responsible for the regulation of the production of ROS, energy metabolism, cell death signaling, and calcium homeostasis in cardiomyocytes [7][8][9]. Mitochondria are vulnerable to damage; however, the processes involved in the dynamics of the mitochondria, including fission and fusion, are essential for the preservation of mitochondrial function. Mitochondrial dynamics, including mitochondrial fission and fusion, are regulated by guanosine triphosphatases (GTPases) in the dynamin family [7][8][9]. The proteins in the outer mitochondrial membrane, Mitofusin 1/2 (Mfn1/2), and the inner mitochondrial membrane, Optic atrophy 1 (Opa1), facilitate mitochondrial fusion, while Dynamin-related protein 1 (Drp1) binds to its receptor proteins in the outer mitochondrial membrane, including mitochondrial fission protein 1 (MTFP1), mitochondrial fission factor (MFF), and mitochondrial fission 1 protein (Fis1), to regulate mitochondrial fission [10]. The fission activity of Drp1 can be modulated by multiple posttranslational modifications, such as phosphorylation, SUMOylation, ubiquitylation, and S-nitrosylation [11]. Phosphorylation of Drp1 (p-Drp1) is the most studied posttranslational modification regulating mitochondrial fission, which occurs at two serine residues, including serine-616 (p-Drp1 ser616 ) and serine-637 (p-Drp1 ser637 ). For p-Drp1 ser616 , it has been associated with increased Drp1 activity to promote Drp1 coordinating fission and fragmentation via the mitosis inducer and cyclin B1-cyclin dependent kinase (cyclin B1-CDK1), whereas phosphorylation by calcium calmodulin-dependent kinase (CamK) links fission to intracellular calcium signaling [11]. Unlike p-Drp1 ser616 , p-Drp1 ser637 is phosphorylated by the cAMPdependent protein kinase A (PKA), which inhibits mitochondrial fission through impairing Drp1 GTPase activity and preventing the translocation of Drp1 to mitochondria [12]. It has been shown that dephosphorylation of serine-637 by calcineurin appears to increase Drp1 activation and recruitment to the mitochondria, thus promoting fission. As described previously, an imbalance between mitochondrial fission and fusion is a key factor in the development of DIC. Drp1-mediated mitochondrial fission and fragmentation are greatly increased in conditions of DIC, which are associated with increased oxidative stress and ROS-mediated cardiomyocyte death. In contrast, mitochondrial fusion-related proteins, including Mfn1/2 and Opa1, were found to be downregulated throughout the course of DIC [13][14][15][16]. According to the results of prior studies, DIC pathogenesis may be effectively prevented by the modulation of mitochondrial dynamics through blocking fission or stimulating fusion [16][17][18][19][20].
Currently, DIC continues to be the most extensively researched form of antitumor drug-induced toxicity. DOX can disturb the equilibrium of mitochondrial dynamics and cause mitochondrial dysfunction. Alterations in mitochondrial quality control may also play a crucial role in the initiation and progression of DIC. Potential techniques for the prevention and treatment of DIC have been developed over the last decade by targeting mitochondrial fission/fusion homeostasis [16][17][18][19][20]. In this review, we discuss the information pertaining to the roles of mitochondrial dynamics in DIC, as well as the current breakthroughs in mitochondrial fission/fusion-targeted treatment in DIC, including the molecular signaling pathways, physiological functions, and pathological significance of DIC. This review provides insights into the anti-cardiotoxic effects which occur as a result of DOX by targeting mitochondrial dynamic pathways, the proposal being that mitochondrial fission/fusion-targeted therapy could be beneficial in preventing DIC. mitochondrial dynamics via an increase in fission and decrease in fusion could be associated with the aggravation of the severity of DIC through multiple molecular signaling pathways, including mitochondrial dysfunction, impaired autophagy and mitophagy, the promotion of oxidative stress, inflammation, and programmed cardiomyocyte death, which eventually lead to a reduction in cell viability and elevated cytotoxicity [17][18][19][20][26][27][28][29][30][31][32][33][34][35][36]. These findings suggested that DOX altered the balance of mitochondrial dynamic by inhibiting mitochondrial fusion and promoting mitochondrial fission which result in DIC pathogenesis. The roles of mitochondrial dynamics in DIC from in vitro reports are summarized in Table 1. Regarding the roles of mitochondrial dynamics in DIC as determined by in vivo studies, the accumulation of DOX is known to induce cardiomyocyte death via oxidative stress, inflammation, and mitochondria-dependent pathways [7,8]. The lowest accumulative dose of DOX (2.25 mg/kg) was administered in white pig models via intracoronary injection (IC). This dose of DOX was shown to upregulate the expression of fission and autophagy proteins, as well as promote fragmentation of mitochondria with severe morphological abnormalities, resulting in fibrosis and LVEF depression [44]. In mouse models, an accumulation of DOX between 12-30 mg/kg administered via intraperitoneal injection (IP) was shown to cause fragmentation of mitochondria via increased activation of mitochondrial fission, as indicated by elevation of the expression of the proteins Drp1, p-Drp1 ser616 , Fis1, and MIEF2 and a decrease of p-Drp1 ser637 [20,28,29,36,37,39,42,45,46]. These dosages of DOX also significantly depressed mitochondrial fusion via reducing the expression of Mfn1/2 and Opa1 in those mouse models. In rat models, a wide range of DOX at 12, 12.5, 14, 15, and 18 mg/kg accumulative dosages effectively increased mitochondrial fission with impaired fusion during DIC progression [13][14][15][16]30,34,43,[47][48][49]. Accordingly, administration of therapeutically effective doses could enhance fission but blunt fusion, which eventually results in fragmentation of mitochondria during progression of DIC. In addition, the precise mechanisms linking DIC to mitochondrial dynamics, specifically mitochondrial dysfunction, modification of autophagy and mitophagy, oxidative stress, inflammation, and programmed cell death have been proposed as mechanisms to provoke cardiac impairments in both morphological and functional remodeling [13][14][15][16]30,34,43,[47][48][49]. Therefore, findings from in vivo models suggested that DOX induced an imbalance in mitochondrial dynamics by blocking mitochondrial fusion, encouraging mitochondrial fission, and causing DOX cardiotoxicity, as comprehensively summarized in Table 2. The molecular insights into DIC and mitochondrial dynamics are illustrated in Figure 1. Regarding the roles of mitochondrial dynamics in DIC as determined by in vivo studies, the accumulation of DOX is known to induce cardiomyocyte death via oxidative stress, inflammation, and mitochondria-dependent pathways [7,8]. The lowest accumulative dose of DOX (2.25 mg/kg) was administered in white pig models via intracoronary injection (IC). This dose of DOX was shown to upregulate the expression of fission and autophagy proteins, as well as promote fragmentation of mitochondria with severe morphological abnormalities, resulting in fibrosis and LVEF depression [44]. In mouse models, an accumulation of DOX between 12-30 mg/kg administered via intraperitoneal injection (IP) was shown to cause fragmentation of mitochondria via increased activation of mitochondrial fission, as indicated by elevation of the expression of the proteins Drp1, p-Drp1 ser616 , Fis1, and MIEF2 and a decrease of p-Drp1 ser637 [20,28,29,36,37,39,42,45,46]. These dosages of DOX also significantly depressed mitochondrial fusion via reducing the expression of Mfn1/2 and Opa1 in those mouse models. In rat models, a wide range of DOX at 12, 12.5, 14, 15, and 18 mg/kg accumulative dosages effectively increased mitochondrial fission with impaired fusion during DIC progression [13][14][15][16]30,34,43,[47][48][49]. Accordingly, administration of therapeutically effective doses could enhance fission but blunt fusion, which eventually results in fragmentation of mitochondria during progression of DIC. In addition, the precise mechanisms linking DIC to mitochondrial dynamics, specifically mitochondrial dysfunction, modification of autophagy and mitophagy, oxidative stress, inflammation, and programmed cell death have been proposed as mechanisms to provoke cardiac impairments in both morphological and functional remodeling [13][14][15][16]30,34,43,[47][48][49]. Therefore, findings from in vivo models suggested that DOX induced an imbalance in mitochondrial dynamics by blocking mitochondrial fusion, encouraging mitochondrial fission, and causing DOX cardiotoxicity, as comprehensively summarized in Table 2. The molecular insights into DIC and mitochondrial dynamics are illustrated in Figure 1. and inhibition of p-Drp1 ser637 and impairs mitochondrial fusion via downregulation of Mfn1/2 and Opa1, resulting in mitochondrial dynamic imbalance, dysfunction, and fragmentation. These mechanisms could lead to increased oxidative stress, inflammation, impaired autophagy and mitophagy, Figure 1. Molecular insights into the association between DOX-induced cardiotoxicity and mitochondrial dynamics pathways. DOX induces mitochondrial fission via the promotion of p-Drp1 ser616 and inhibition of p-Drp1 ser637 and impairs mitochondrial fusion via downregulation of Mfn1/2 and Opa1, resulting in mitochondrial dynamic imbalance, dysfunction, and fragmentation. These mechanisms could lead to increased oxidative stress, inflammation, impaired autophagy and mitophagy, and cardiomyocyte apoptosis, leading to cardiac dysfunction. Figure created with BioRender.com. DOX: doxorubicin; Drp1: dynamin-related protein 1; MFF: mitochondrial fission factor; Mfn1: mitofusin 1; Mfn2: mitofusin 2; MTFP1: mitochondrial fission process 1; Opa1: optic atrophy type 1; p-Drp1 ser616 : phosphorylation of dynamin-related protein 1 at serine 616; p-Drp1 ser637 : phosphorylation of dynamin-related protein 1 at serine 637.

Roles of Pharmacological Interventions Targeting Mitochondrial Fission/Fusion Therapy in DOX-Induced Cardiotoxicity: A Report from Recent In Vitro and In Vivo Studies
Up to 90 percent of the ATP necessary for sufficient cardiac contractility is produced by mitochondria in cardiomyocytes, and mitochondrial dynamics plays a key role in cardiac homeostasis [53][54][55]. DOX causes cardiomyocyte damage by modifying the structure and function of mitochondria, which is coupled with a change in mitochondrial homeostasis and dysregulation of mitochondrial fission/fusion. DOX promotes excessive generation of ROS in mitochondria and impairs mitochondrial morphology and function, resulting in the death of cardiac cells [13][14][15][16]30,34,43,[47][48][49]. In addition, it has been shown that DOX induces mitochondrial fragmentation through the elevation of p-Drp1 ser616 and reduction of Mfn1/2 and Opa1, which accelerate mitochondrial-dependent cardiomyocyte apoptotic death [13][14][15][16]30,34,43,[47][48][49]. Consequently, it is necessary to confirm the targeting of mitochondrial fusion and fission proteins as potential therapeutic targets for protection against DIC by restoring the balance of mitochondrial dynamics.
As shown in Tables 3 and 4, numerous in vitro and in vivo preclinical investigations have verified the cardioprotective impact of pharmacologically targeted mitochondrial fission/fusion treatment against DIC. Firstly, Mdivi-1 (mitochondrial division inhibitor), a putative Drp1 inhibitor, is a widely used small molecule that inhibits Drp1-dependent fission, causes the elongation of mitochondria, and reduces injury [27,56]. It has been shown that DOX treatment substantially reduced cell viability along with increased lactate dehydrogenase (LDH) levels in H9c2 cardiomyocytes, and also that the cytotoxic effects of DOX were blunted by 5.0 µM Mdivi-1 co-treatment for 48 h [16]. Moreover, increased cardiomyocyte apoptosis and p-Drp1 ser 616 post DOX stimulation could be effectively alleviated by 1-µM Mdivi-1 pre-treatment for 30 min in H9c2 cells [28]. In the AC16 human cardiomyocyte cell line, Mdivi-1 co-treatment could prevent DOX-induced overproduction of mitochondrial superoxides and mitochondrial dysfunction by inhibiting mitophagy and preserving mitochondrial biogenesis [18]. The anti-DIC effects of Mdivi-1 have also been revealed in an in vivo study using Male Wistar rats. Co-treatment with Mdivi-1 at 1.2 mg/kg for 30 days markedly reduced DOX-induced mitochondrial dysfunction, oxidative stress, inflammation, and apoptosis, leading to improved cardiac function via modulation of mitochondrial fission/fusion proteins [16]. These results imply that suppressing fission via Mdivi-1 may be a potential therapeutic target for mitigating the cytotoxic effects of DOX.
In addition to Mdivi-1, Klotho, another anti-aging protein, has also been shown to contribute to human aging [20]. Klotho regulates energy metabolism, stress resistance, antioxidation, and calcium and mineral homeostasis. In DIC, pre-treatment with 0.1 µg/mL Klotho for 24 h significantly reduced DOX-induced p-Drp1 ser 616 -mediated apoptosis in H9c2 cardiomyocytes. As in DOX-treated mice, Klotho (0.01 mg/kg, every 48 h for 4 weeks, IP) also suppressed p-Drp1 ser616 protein, cardiac cell death, and improved cardiac function [20]. Loulu flowers (LLF), the inflorescence of Rhaponticum uniflorum (L.) DC. (R. uniflorum), a member of the Compositae family, has frequently been used in treatment of various cardiovascular diseases (CVDs) [35]. Pre-treatment with LLF (200 µg/mL, 2 h) attenuated the DOX-induced aberrant expression of mitochondrial fusion/fission proteins via promotion of Opa1 and Mfn1 along with inhibition of MFF and Fis1 in H9c2 cells. LLF treatment increased cell viability while decreasing ROS production, maintaining mitochondrial membrane integrity, suppressing apoptosis, and inhibiting the DOX-induced activation of inflammation signaling in H9c2 cells [35].
Neuraminidase 1 (NEU1) inhibitor or oseltamivir (OSE) also protected against DIC via suppression of Drp1-dependent mitophagy [34]. NEU1, a glycosidases responsible for the removal of terminal sialic acid from glycoproteins and glycolipids, is the most abundantly expressed glycosidases in the heart, and is implicated in a number of CVDs. Pre-treatment with 10 µM OSE for 2 h could suppress Drp1-dependent mitochondrial fission and PTEN-induced kinase 1 (PINK1)/Parkin pathway-mediated mitophagy and alleviate cellular apoptosis in H9c2 cells. These cardioprotective effects of the NEU1 inhibitor against DIC were also confirmed in a rat model using OSE (20 mg/kg, 31 days, PO) [34]. Shenmai injection (SMI) is a patented traditional Chinese medicine derived from Panax ginseng and Ophiopogon japonicus, which are extensively used to treat CVDs. SMI has been shown to prevent DOX-induced excessive mitochondrial fission and insufficient mitochondrial fusion in H9c2 cardiomyocytes by increasing the ratio of L-Opa1 to S-Opa1, AMPK phosphorylation, and p-Drp1 ser637 [33]. Furthermore, co-treatment with Liensinine, a newly discovered mitophagy inhibitor, was shown to reduce p-Drp1 ser616 and inhibit mitochondrial fragmentation, oxidative stress, mitophagy, apoptosis, and improve mitochondrial function in cases of DOX-induced NMVMs injury [38].
Melatonin is known to influence mitochondrial homeostasis and function [30]. Pretreatment with melatonin (10 µM, 24 h) followed by DOX exposure decreased mitochondrial fragmentation and increased ATP production, resulting in preservation of H9c2 rat cardiomyoblast viability. Similarly, in a rat model, a specific dose of melatonin (6 mg/kg) given for 14 days effectively decreased Drp1-Fis1-mediated fission and apoptosis and increased Mfn1/2-mediated fusion, cellular ATP levels, and mitochondrial biogenesis, contributing to improved cardiac function [30]. A novel angiotensin receptor, neprilysin inhibitor LCZ696, has been beneficial in treating individuals with heart failure. Pre-treatment with 20 µM LCZ696 for 30 min significantly inhibited DOX-activated Drp1 and its ser616 phosphorylation protein, thus decreasing cardiomyocyte apoptosis [28]. Vitamin D, acting as an antioxidant, was also shown to prevent DIC in a mouse in a triple-negative breast cancer model (TNBC). Pretreatment with vitamin D3 (11,500 IU/kg, 14 days) reduced p-Drp1 ser616associated oxidative stress and apoptosis, resulting in improved cardiac function in TNBC mice [45].
The managing of a balance between cardiac sympathetic and parasympathetic activity by acetylcholine receptor (AChR) agonists has been shown to be associated with mitochondrial function, cellular oxidative balance, and immunomodulation in both healthy and pathological conditions. Both an a7nAChR agonist (PNU-282987, 3 mg/kg, daily for 30 days, IP) and a mAChR agonist (bethanechol, 12 mg/kg, daily for 30 days, IP) promoted Mfn1/2-induced mitochondrial fusion and inhibited Drp1-induced mitochondrial fission, preventing DOX-induced autophagy and mitophagy [15]. In a similar manner, Luteolin (20 µM, 24 h, co-treatment) ameliorated DOX-induced toxicity in H9c2 cells via inhibition of mitochondrial fission, mitochondrial dysfunction, and apoptosis [32].
Pharmacologically induced mitochondrial fusion (M1) or hydrazone M1 has been shown to be cardioprotective in a variety of cardiovascular settings, most notably DIC. M1 dose-dependently induces mitochondrial elongation, with a requirement for basal fusion activity from Mfn1, Mfn2, or Opa1 proteins [16,27,57]. It has been strongly verified that co-treatment of DOX with M1 (2 mg/kg, daily for 30 days, IP) markedly mitigated mitochondrial dysfunction, oxidative stress, inflammation, and apoptosis, leading to improved cardiac function via modulation of mitochondrial fission/fusion proteins in a rat model. M1 (5 µM, 48h) increased cell viability while decreasing cytotoxicity in H9c2 cells following the DOX challenge [16].
Paeonol (Pae) is a natural antioxidant made from the root bark of Paeonia suffruticosa. It has been approved by the FDA in China to treat diseases that cause inflammation and pain [43]. It has been demonstrated that Pae inhibits ischemia-induced cardiomyocyte apoptosis via suppressing ROS, and has the ability to protect against DIC. Pae effectively enhanced Mfn2-mediated mitochondrial fusion by activating the transcription factor Stat3, which restored mitochondrial function and cardiac performance both in vivo (150 mg/kg/day, PO, post-treatment) and in vitro (50 µM, 24 h, co-treatment) under DOX conditions [43]. In addition, Honokiol (HKL), an activator of SIRT3, has been shown to increase the activation of SIRT3 and Mfn1/Opa1-mediated fusion preventing DOX-induced ROS production, mitochondrial damage, and cell death in rat neonatal cardiomyocytes (10 µM, 24 h, co-treatment). Treatment with HKL (0.2 mg/kg, daily, 45 days, IP) successfully blocked DIC in mice via promoting Mfn1/Opa1-mediated fusion, reducing mitochondrial DNA damage, and improving mitochondrial function, leading to amelioration of cardiac dysfunction [37]. Additionally, last but not least, flavonoids of Selaginella tamariscina (P.Beauv.) Spring (TFST) have been shown to prevent DIC by enhancing Mfn2mediated fusion, leading to alleviation of mitochondrial dysfunction and endoplasmic reticulum stress by activating Mfn2/PERK in an in vivo mouse model (70-140 mg/kg, 8 days, PO) [46]. Taken together, targeting mitochondrial fission and fusion could be a novel potential strategy for cancer patients undergoing DOX-based chemotherapy. Roles of pharmacological interventions targeting mitochondrial fission/fusion therapy in DIC are illustrated in Figure 2. model. M1 (5 μM, 48h) increased cell viability while decreasing cytotoxicity in H9c2 cells following the DOX challenge [16]. Paeonol (Pae) is a natural antioxidant made from the root bark of Paeonia suffruticosa. It has been approved by the FDA in China to treat diseases that cause inflammation and pain [43]. It has been demonstrated that Pae inhibits ischemia-induced cardiomyocyte apoptosis via suppressing ROS, and has the ability to protect against DIC. Pae effectively enhanced Mfn2-mediated mitochondrial fusion by activating the transcription factor Stat3, which restored mitochondrial function and cardiac performance both in vivo (150 mg/kg/day, PO, post-treatment) and in vitro (50 μM, 24 h, co-treatment) under DOX conditions [43]. In addition, Honokiol (HKL), an activator of SIRT3, has been shown to increase the activation of SIRT3 and Mfn1/Opa1-mediated fusion preventing DOX-induced ROS production, mitochondrial damage, and cell death in rat neonatal cardiomyocytes (10 μM, 24 h, co-treatment). Treatment with HKL (0.2 mg/kg, daily, 45 days, IP) successfully blocked DIC in mice via promoting Mfn1/Opa1-mediated fusion, reducing mitochondrial DNA damage, and improving mitochondrial function, leading to amelioration of cardiac dysfunction [37]. Additionally, last but not least, flavonoids of Selaginella tamariscina (P.Beauv.) Spring (TFST) have been shown to prevent DIC by enhancing Mfn2mediated fusion, leading to alleviation of mitochondrial dysfunction and endoplasmic reticulum stress by activating Mfn2/PERK in an in vivo mouse model (70-140 mg/kg, 8 days, PO) [46]. Taken together, targeting mitochondrial fission and fusion could be a novel potential strategy for cancer patients undergoing DOX-based chemotherapy. Roles of pharmacological interventions targeting mitochondrial fission/fusion therapy in DIC are illustrated in Figure 2. The underlying mechanisms of anti-DIC include prevention of DOX-induced mitochondrial fission via the promotion of p-Drp1 ser616 and inhibition of p-Drp1 ser637 , and impaired mitochondrial fusion via downregulation of Mfn1/2 and Opa1, resulting in mitochondrial dynamic imbalance, dysfunction, and fragmentation. It also decreases ROS production, lipid peroxidation, inflammation, cardiomyocyte apoptosis, and impairments of autophagy and mitophagy, leading to reduced mitochondrial and cellular injury.

Roles of Non-Pharmacological Interventions Targeting Mitochondrial Fission/Fusion Therapy in DOX-Induced Cardiotoxicity: A Report from Recent In Vitro and In Vivo Studies
In addition to pharmacological interventions, the roles of non-pharmacological therapies targeting mitochondrial fission and fusion in DOX cardiotoxicity have been reported. Both in vitro and in vivo research findings regarding those non-pharmacological interventions are summarized in Tables 5 and 6. Primarily, Drp1 knockdown has been shown to prevent Drp1-dependent mitochondrial fragmentation, mitophagy flux, and H9c2 cell death. Drp1-deficient mice were consistently rescued from DOX-induced mitochondrial fragmentation, mitochondrial degradation by the lysosomes, and myocardial injury, providing strong evidence for a role for Drp1-associated mitochondrial fragmentation in DIC [36].
It has been claimed that Foxo3a inhibits DOX-induced mitochondrial fission and apoptosis in cardiomyocytes. This is due to Foxo3a being downregulated in cardiomyocytes and in the mouse heart in response to DOX administration [42]. Cardiac specific Foxo3a transgenic mice showed a reduction in mitochondrial dynamics protein of 49 kDa (MIEF2)-mediated mitochondrial fission, apoptosis, and cardiotoxicity upon DOX exposure. Knockdown of MIEF2 reduced DOX-induced mitochondrial fission and apoptosis in cardiomyocytes and in vivo. Additionally, knockdown of MIEF2 protected the heart from DOX-induced cardiotoxicity [42]. Additionally, cardiomyocyte mitochondrial dynamicrelated lncRNA 1 (CMDL-1) is markedly downregulated in cardiomyocytes by the DOX challenge, which targets the Drp1 protein. Lentiviral overexpression of CMDL-1 for 24 h prevented DOX-induced p-Drp1 ser637 -mediated mitochondrial fission and apoptosis in H9c2 cardiomyocytes [31]. Likewise, loss of Rubicon, an inhibitory interacting partner of autophagy protein UVRAG, ameliorated DOX-induced cardiotoxicity through enhancement of Opa1-mediated mitochondrial fusion and the improvement of autophagic flux and mitophagy in Rubicon-deficient mice [52].
Heme oxygenase-1 (HO-1) is a transcriptional stress response gene that is increased in pathological conditions of the heart and other organ systems [50]. Mice that overexpressed human HO-1 were rescued from DOX-induced dilated cardiomyopathy, cardiac remodeling, and infiltration of CD11b. Cardiac-specific HO-1 overexpression reduced DOX-mediated dilatation of the sarcoplasmic reticulum and mitochondrial disorganization, including a reduction in both mitochondrial fragmentation and increased numbers of damaged mitochondria in autophagic vacuoles. Overexpression of HO-1 increased NRF1, PGC1-α, and TFAM protein expression, which accelerated mitochondrial biogenesis. HO-1 overexpression also reduced Fis1 upregulation and enhanced Mfn1 and Mfn2 expression. PINK1 and Parkin, mitophagy pathway mediators, were similarly prevented from altering mitochondrial dynamics [50]. Moreover, early implantation of mitochondria (Mito) or exogenous mitochondrial administration into the left myocardium could effectively protect the heart against DOX-induced dilated cardiomyopathy in rats. Mito (500 µg/rat intramyocardial injection) effectively inhibited Drp1-mediated fission and promoted Mfn2-mediated fusion, reducing oxidative stress, autophagy, apoptosis, and mitochondrial damage, leading to preservation of LVEF and myocardial remodeling in DIC rats [48].
In addition, an increase in Mfn2 levels in NRVMs cardiomyocytes via a CRISPR activation plasmid (48 h, pre-treatment) attenuated the DOX-induced increase in mitochondrial fission and prevented mitochondrial ROS production, thereby preventing DOX-induced apoptosis of cardiomyocytes [41]. In DOX-treated cardiomyocytes, restoration of Mfn2mediated mitochondrial fusion with adenoviruses encoding the Mfn2 gene (Ad-Mfn2) (48 h, pre-treatment) increased mitochondrial oxidative metabolism, decreased cellular injury and apoptosis, and inhibited mitochondria-derived oxidative stress. Transgenic mice with cardiac-specific Mfn2 exhibited preserved mitochondrial fusion and diminished myocardial damage when treated with DOX [39]. Targeting Mfn2-mediated mitochondrial fusion may therefore provide a double therapeutic benefit in DOX-based chemotherapy by simultaneously preventing DIC.
In pig models, remote ischemic conditioning (RIPC) applied immediately before each DOX injection resulted in preservation of cardiac contractility with significantly higher longterm left ventricular ejection fraction (LVEF) and less cardiac fibrosis through prevention of mitochondrial fragmentation and dysregulated autophagy [44]. Endurance treadmill training (TM) and voluntary free wheel activity (FW) also prevented DOX-induced mPTP opening and apoptosis, mitochondrial dynamic alterations, and increases in autophagy and mitophagy signaling [49]. In summary, inhibition of mitochondrial fission and promotion of fusion hold potential in translating a targeted mitochondrial therapy for DIC into a new clinical setting. The roles of non-pharmacological interventions targeting mitochondrial fission/fusion therapy in DIC are illustrated in Figure 3. In pig models, remote ischemic conditioning (RIPC) applied immediately before each DOX injection resulted in preservation of cardiac contractility with significantly higher long-term left ventricular ejection fraction (LVEF) and less cardiac fibrosis through prevention of mitochondrial fragmentation and dysregulated autophagy [44]. Endurance treadmill training (TM) and voluntary free wheel activity (FW) also prevented DOX-induced mPTP opening and apoptosis, mitochondrial dynamic alterations, and increases in autophagy and mitophagy signaling [49]. In summary, inhibition of mitochondrial fission and promotion of fusion hold potential in translating a targeted mitochondrial therapy for DIC into a new clinical setting. The roles of non-pharmacological interventions targeting mitochondrial fission/fusion therapy in DIC are illustrated in Figure 3.     Restoration of Mfn2-mediated mitochondrial fusion reduced cardiac dysfunction, cellular injury/apoptosis, and mitochondria-derived oxidative stress in DOX-treated mice. [39]

Existing Controversial Reports on Mitochondrial Fission/Fusion-Targeted Therapy in DOX-Induced Cardiotoxicity
As mentioned above, inhibiting fission via downregulation of the Drp1 protein or promoting fusion via upregulation of the Mfn1/2 and Opa1 proteins has been shown to be anti-DIC in both in vitro and in vivo experiments. However, there are some conflicting findings regarding mitochondrial fission/fusion-targeted therapy in DIC. It has been demonstrated that luteolin, a natural product that is extracted from vegetables and fruits, possesses properties that are anti-oxidative, anti-tumorigenic, and anti-inflammatory [32]. Luteolin application in adult mouse cardiomyocytes or AMCM cells at a concentration of 10 M for a period of 24 h as a co-treatment overtly alleviated DOX-induced cardiomyocyte contractile dysfunction, inhibited apoptosis, accumulation of ROS, and loss of mitochondrial membrane potential via promotion of mitochondrial autophagy in association with facilitating p-Drp1 ser616 , with reduced mitochondrial elongation, and upregulation of transcription factor EB (TFEB) expression [32] (Table 3). Likewise, motorized treadmill exercise (speed of 13-15 m/min for 60 min per day for 4 weeks after treatment) prevented DOX-induced apoptosis and mitigated tissue damage via an increase in mitophagy flux, an increase in Drp1-mitochondrial fission, and a decrease in fusion markers (Opa1 and Mfn2) [51] (Table 6). Although the major findings from these two studies demonstrated that promoting fission via upregulation of the Drp1 protein or inhibiting fusion via downregulation of the Mfn2 and Opa1 proteins exhibited anti-DIC efficacy, further studies are still required to validate the protective role of the promotion of Drp1-mediated fission and the inhibition of Mfn1/Mfn2/Opa1-mediated fusion against DIC conditions.

Conclusions
Although DOX is one of the most widely used chemotherapeutic treatments effective in the increase in the survival of cancer patients, DIC remains a key limiting factor in the use of this drug. Therapies that target the mitochondrial dynamic pathways have appeared as possible preventative and therapeutic options for DIC. Although mitochondrial fission/fusion-targeted remedies could be cardioprotective regimens to protect against DOX's life threatening cardiotoxic effects, limited clinical evidence is available. Future clinical investigations are needed to warrant the use of these mitochondrial dynamictargeted interventions in a clinical setting.