Contraction Band Necrosis with Dephosphorylated Connexin 43 in Rat Myocardium after Daily Cocaine Administration

Contraction band necrosis (CBN) is a common abnormality found in the myocardium of cocaine abusers, but is rarely reported in experimental models of cocaine abuse. Connexin 43 (Cx43) is essential for cardiac intercellular communication and the propagation of CBN. Under stress or injury, cardiac Cx43 is dephosphorylated, which is related to cardiomyocyte dysfunction and pathogenesis, whereas adiponectin exerts beneficial effects in the myocardium. In this study, we explore the effects of cocaine on cardiac Cx43 in vivo. Rats were administered cocaine via the tail vein at 20 mg/kg/day for 14 days, and showed widespread CBN, microfocal myocarditis and myocardial fibrosis, corresponding to a dysfunction of cardiac mitochondria under increased oxidative stress. The increase in dephosphorylated cardiac Cx43 and its negative correlation with the myocardial distribution of CBN after cocaine administration were determined. In addition, apoptosis and necroptosis, as well as increased adiponectin levels, were observed in the myocardium after cocaine exposure. Accordingly, we found altered profiles of cardiac Cx43, CBN and its negative correlation with dephosphorylated cardiac Cx43, and the possible involvement of adiponectin in the myocardium after 14 days of cocaine administration. The latter might play a protective role in the cardiotoxicity of cocaine. The current findings would be beneficial for establishing novel therapeutic strategies in cocaine-induced cardiac consequences.


Introduction
Cocaine is well-established to induce serious cardiovascular damage [1,2]. The cardiotoxicity of cocaine derives from its potent blockage of sodium channels, persistent elevation of catecholamine levels, and disruption of cellular Ca2+ homeostasis [3]. These effects result in elevated myocardial oxygen demand while simultaneously reducing myocardial oxygen supply [4][5][6]. The cocaine-induced excessive catecholamine and anoxia in the myocardium lead to an overwhelming increase in the intramyocellular Ca 2+ concentration, which results in hypercontraction of the sarcomeres and subsequent myofibrillar rhexis, a condition known as contraction band necrosis (CBN, also referred to as coagulative myocytolysis) [7][8][9]. CBN appears as hypereosinophilic transverse bands in myocytes in isolated lesions or microscale clusters [10]. Instead of a pathognomonic feature, CBN can be observed in various conditions, including ischemic heart disease [11] and resuscitation [12]. It is considered one of the most common pathological findings in the myocardium of cocaine abusers [8]. The propagation of CBN in the myocardium is bound up with the potency of gap junctions, wherein cardiac connexin 43 (Cx43) is believed to play an essential role [13][14][15].
Cardiac Cx43 exists in myocardial gap junctions, which are primarily localized in intercalated discs [16,17]. In healthy myocardium, gap junctional Cx43 is in a phosphorylated state to enhance normal intercellular communication, which enables the exchange of electrical stimuli and synchronous myocardial contraction [18][19][20]. Sufficient evidence has been Int. J. Mol. Sci. 2022, 23, 11978 2 of 15 obtained from cardiac ischemia/reperfusion models to indicate the cardioprotective role of phosphorylated Cx43 [17], whereas under pathological conditions, dephosphorylated Cx43 undergoes lateral redistribution from the gap junctions and subsequent degradation, which results in the weakening of intercellular communication [21]. The dephosphorylation of cardiac Cx43 is related to cardiomyocyte apoptosis and myocardial fibrosis [22], as observed in arrhythmias, ischemic myocardial injury, and heart failure [23,24]. Similar cardiac consequences are observed under cocaine exposure, yet whether cardiac Cx43 plays a role in cocaine cardiotoxicity remains to be elucidated.
Adiponectin is a protein hormone that protects the cardiovascular system against stress and injury, as well as against numerous detrimental conditions, including atherogenesis, inflammation, and fibrosis [25][26][27]. AMP-activated protein kinase (AMPK) is a key mediator in most of these beneficial effects [28]. In addition, it has been determined that, in diabetic and septic models, adiponectin mitigates myocardial inflammation and dysfunction by regulating cardiac Cx43 expression [29,30]. However, the involvement of adiponectin in cocaine cardiotoxicity is still unknown.
Herein, we investigated the effects of cocaine on cardiac homeostasis using an in vivo model, in which rats were administered cocaine at a dose of 20 mg/kg/day via the tail vein for 14 days. We found widespread CBN and microfocal myocarditis and fibrosis in the myocardium after cocaine administration. Correspondingly, cardiac mitochondrial dysfunction and increased oxidative stress were observed. We also found an increase in dephosphorylated cardiac Cx43, and a negative correlation between CBN and dephosphorylated Cx43 in myocardial distribution after cocaine administration. Furthermore, the activation of pathways responsible for apoptosis and necroptosis, as well as the participation of adiponectin, were found in the myocardium after cocaine exposure. We believe that this is the first in vivo study revealing the alteration in the cardiac Cx43 profile and indicating the possible involvement of adiponectin in cardiomyocytes after cocaine administration. Taking advantage of the current findings, novel therapeutic strategies against cardiac consequences in cocaine addicts could be explored.

Pathological Changes in Myocardium after 14 Days of Cocaine Administration via Tail Vein
Sufficient evidence from in vivo animal models has shown a relationship between cardiovascular disorders and cocaine exposure via intraperitoneal administration [31,32]. Thus, Hematoxylin-eosin (H&E) and Elastica Masson-Goldner (EMG) staining were performed in order to assess the condition of the myocardium in our cocaine abuse model, applying tail vein administration ( Figure 1B-G). The H&E results revealed microfocal myocarditis in the myocardium of the cocaine group with eosinophil-dominated infiltration. Focal myocytolysis, swollen cardiomyocyte nuclei, and bright myocardial cytoplasm were found around the myocarditis regions ( Figure 1B,C). EMG staining of the myocardium from the cocaine group showed scattered perivascular fibrosis, with abundant green-stained collagen fibers surrounding the microvascular and infiltration into the interstitium ( Figure 1D,E). Furthermore, we observed widespread CBN-like hypereosinophilic transverse bands in myocardium from the cocaine group (Supplementary Figure S1), which we verified by Phosphotungstic Acid Hematoxylin (PTAH) staining [33]. PTAH staining verified CBN in the cocaine group by revealing extensive dense transverse contraction band lesions, accompanied by wavy myocardial fibers in the adjacent region ( Figure 1F,G). Collectively, the histological findings demonstrate the pathological condition of the myocardium after 14 days of cocaine administration via the tail vein. Filled arrowheads indicate cocaine administration. The open arrow indicates sample collection from sacrificed rats. (B) HE staining, (D) EMG staining, and (F) PTAH staining of the myocardium samples from the control group; (C) HE staining of the myocardium from the cocaine group, indicating myocarditis with eosinophil infiltration (arrowheads), focal myocytolysis, swollen cardiomyocyte nuclei, and bright myocardial cytoplasm around the myocarditis; (E) EMG staining of the myocardium from the cocaine group, showing perivascular fibrosis, abundant green-stained collagen fibers around the microvascular, and infiltration into the interstitium; (G) PTAH staining of the myocardium from the cocaine group, exhibiting broad transverse contraction band lesions (hollow arrowheads), and wavy myocardial fibers around the lesions. Scale bars = 100 μm.

Cardiac Mitochondrial Dysfunction and Elevated Oxidative Stress in Myocardium
Mitochondrial dysfunction and oxidative stress play prominent roles in the cardiotoxicity of cocaine. Therefore, we determined the effects of cocaine on cardiac mitochondria. Firstly, we examined the status of cardiac mitochondrial oxidative phosphorylation. Immunoblotting of mitochondrial oxidative phosphorylation (OXPHOS) subunit proteins and the outer mitochondrial membrane 20 (TOM20) protein revealed a general trend toward decreased expressions, with significant decreases found for the expressions of complex IV and V proteins ( Figure 2A). Complex IV is believed to play a central role in mito- (C) HE staining of the myocardium from the cocaine group, indicating myocarditis with eosinophil infiltration (arrowheads), focal myocytolysis, swollen cardiomyocyte nuclei, and bright myocardial cytoplasm around the myocarditis; (E) EMG staining of the myocardium from the cocaine group, showing perivascular fibrosis, abundant green-stained collagen fibers around the microvascular, and infiltration into the interstitium; (G) PTAH staining of the myocardium from the cocaine group, exhibiting broad transverse contraction band lesions (hollow arrowheads), and wavy myocardial fibers around the lesions. Scale bars = 100 µm.

Cardiac Mitochondrial Dysfunction and Elevated Oxidative Stress in Myocardium
Mitochondrial dysfunction and oxidative stress play prominent roles in the cardiotoxicity of cocaine. Therefore, we determined the effects of cocaine on cardiac mitochondria. Firstly, we examined the status of cardiac mitochondrial oxidative phosphorylation. Immunoblotting of mitochondrial oxidative phosphorylation (OXPHOS) subunit proteins and the outer mitochondrial membrane 20 (TOM20) protein revealed a general trend toward decreased expressions, with significant decreases found for the expressions of complex IV and V proteins ( Figure 2A). Complex IV is believed to play a central role in mitochondrial oxidative metabolism and oxidative stress [34]. Following the detection of transcripts, regulating complex IV determined significant downregulations of cytochrome c oxidase subunit I (MT-CO1) and cytochrome c oxidase subunit III (MT-CO3) ( Figure 2B). These represent an impairment of mitochondrial bioenergetic function by cocaine cardiotoxicity. In contrast, endogenous OXPHOS complex I activity assays showed significantly elevated complex I activity in the cocaine group as compared to the control group ( Figure 2C). Considering that the production of intracellular oxidative stress is mainly attributed to complex I [35], and that excessive complex I activity might lead to an elevation in the generation of reactive oxygen species (ROS), we examined the oxidative stress markers 4-hydroxy-2-nonenal (4-HNE) and 8-hydroxy-2 -deoxyguanosine (8-OHdG), which found apparently elevated oxidative stress in the myocardium of the cocaine group ( Figure 2D,E). These findings suggest cardiac mitochondrial dysfunction after 14 days of cocaine administration. chondrial oxidative metabolism and oxidative stress [34]. Following the detection of transcripts, regulating complex IV determined significant downregulations of cytochrome c oxidase subunit I (MT-CO1) and cytochrome c oxidase subunit III (MT-CO3) ( Figure 2B). These represent an impairment of mitochondrial bioenergetic function by cocaine cardiotoxicity. In contrast, endogenous OXPHOS complex I activity assays showed significantly elevated complex I activity in the cocaine group as compared to the control group ( Figure  2C). Considering that the production of intracellular oxidative stress is mainly attributed to complex I [35], and that excessive complex I activity might lead to an elevation in the generation of reactive oxygen species (ROS), we examined the oxidative stress markers 4hydroxy-2-nonenal (4-HNE) and 8-hydroxy-2'-deoxyguanosine (8-OHdG), which found apparently elevated oxidative stress in the myocardium of the cocaine group ( Figure  2D,E). These findings suggest cardiac mitochondrial dysfunction after 14 days of cocaine administration.

Electron Microscopic Analysis of Myocardium after 14 Days of Cocaine Administration via Tail Vein
Given the indications of mitochondrial damage by cocaine administration (Figure 2), transmission electron microscopy was performed for the direct observation of cardiac mitochondria. The nuclei of cardiomyocytes in the cocaine group showed lighter nucleoplasm and chromatin clumping as compared to the nuclei in the control group ( Figure  3A,B). This observation is in accordance with the nuclei of cardiomyocytes under stress or

Electron Microscopic Analysis of Myocardium after 14 Days of Cocaine Administration via Tail Vein
Given the indications of mitochondrial damage by cocaine administration (Figure 2), transmission electron microscopy was performed for the direct observation of cardiac mitochondria. The nuclei of cardiomyocytes in the cocaine group showed lighter nucleoplasm and chromatin clumping as compared to the nuclei in the control group ( Figure 3A,B). This observation is in accordance with the nuclei of cardiomyocytes under stress or injury, similarly indicating a pathological condition of the myocardium of the cocaine group. Moreover, compared to the normally arranged myofibers, as well as well-shaped and evenly sized mitochondria with intact matrices, as observed in the control group, the cocaine group presented with discontinuous and ruptured myofibers, irregularly shaped mitochondria with unclearly structured matrices, and highly dense dots within or around mitochondria, which reflects Ca 2+ overload ( Figure 3C,D). In addition, we found a significantly increased cardiac mitochondria number with a simultaneous remarkable reduction in the cardiac mitochondrial cross-sectional area, as observed for the longitude sections of myocardial fibers in the cocaine group ( Figure 3E,F). In general, the disrupted mitochondrial dynamics and mitochondrial fragmentation reflect dysfunction and injure cardiac mitochondria [36]. Therefore, we conclude that these observations confirm damage to cardiac mitochondria and cardiomyocytes after 14 days of cocaine administration. injury, similarly indicating a pathological condition of the myocardium of the cocaine group. Moreover, compared to the normally arranged myofibers, as well as well-shaped and evenly sized mitochondria with intact matrices, as observed in the control group, the cocaine group presented with discontinuous and ruptured myofibers, irregularly shaped mitochondria with unclearly structured matrices, and highly dense dots within or around mitochondria, which reflects Ca 2+ overload ( Figure 3C,D). In addition, we found a significantly increased cardiac mitochondria number with a simultaneous remarkable reduction in the cardiac mitochondrial cross-sectional area, as observed for the longitude sections of myocardial fibers in the cocaine group ( Figure 3E,F). In general, the disrupted mitochondrial dynamics and mitochondrial fragmentation reflect dysfunction and injure cardiac mitochondria [36]. Therefore, we conclude that these observations confirm damage to cardiac mitochondria and cardiomyocytes after 14 days of cocaine administration.

Dephosphorylated Cardiac Cx43 and Its Distribution in Myocardium
In view of the role of Cx43 in CBN propagation and cardiac pathogenesis, we investigated Cx43 expression in the myocardium. Immunoblotting of total cardiac Cx43 showed a significantly increased Cx43 level, with a decreased percentage of phosphorylated Cx43 (44-46 kDa) and an increased percentage of dephosphorylated Cx43 (41 kDa) in the cocaine group ( Figure 4A). The separate immunoblots for dephosphorylated Cx43 at serine 368 also showed an accordingly significant increase in the myocardium of the cocaine group, verifying the dephosphorylation of Cx43 after cocaine administration. This suggests an abnormal functioning of cardiac gap junctions after cocaine administration. The results of PTAH staining and immunochemistry of dephosphorylated Cx43 were compared in order to explore the association between CBN and Cx43 in the cardiotoxicity of cocaine ( Figure 4B-I). Intriguingly, the region staining positive for dephosphorylated Cx43 in the histochemistry results appeared to be negatively correlated with the highly-stained region in the PTAH results in the myocardium of the cocaine group ( Figure 4B,C). In the CBN region of the myocardium from the cocaine group, the staining seems to be restricted to the cytosol and nucleus, while in the areas adjacent to or absent from CBN, the dephosphorylated Cx43 staining was mostly observed at the intercalated discs ( Figure 4D-G). In addition, the level of dephosphorylated Cx43 staining was increased in the cocaine group as compared to the control, which is consistent with the immunoblot results showing an increased level of dephosphorylated Cx43 ( Figure 4G,I). The expression and distribution of cardiac Cx43 demonstrates the dephosphorylation of Cx43 and its negative connection with CBN in the myocardium after 14 days of cocaine administration. the longitudinal view of myofibrils. Each white and black bullet stands for the area of measured mitochondrion in control and cocaine group, respectively. Each bar represents the mean and S.D., *, p < 0.05, ***, p < 0.001.

Dephosphorylated Cardiac Cx43 and Its Distribution in Myocardium
In view of the role of Cx43 in CBN propagation and cardiac pathogenesis, we investigated Cx43 expression in the myocardium. Immunoblotting of total cardiac Cx43 showed a significantly increased Cx43 level, with a decreased percentage of phosphorylated Cx43 (44-46 kDa) and an increased percentage of dephosphorylated Cx43 (41 kDa) in the cocaine group ( Figure 4A). The separate immunoblots for dephosphorylated Cx43 at serine 368 also showed an accordingly significant increase in the myocardium of the cocaine group, verifying the dephosphorylation of Cx43 after cocaine administration. This suggests an abnormal functioning of cardiac gap junctions after cocaine administration. The results of PTAH staining and immunochemistry of dephosphorylated Cx43 were compared in order to explore the association between CBN and Cx43 in the cardiotoxicity of cocaine ( Figure 4B-I). Intriguingly, the region staining positive for dephosphorylated Cx43 in the histochemistry results appeared to be negatively correlated with the highlystained region in the PTAH results in the myocardium of the cocaine group ( Figure 4B,C). In the CBN region of the myocardium from the cocaine group, the staining seems to be restricted to the cytosol and nucleus, while in the areas adjacent to or absent from CBN, the dephosphorylated Cx43 staining was mostly observed at the intercalated discs ( Figure  4D-G). In addition, the level of dephosphorylated Cx43 staining was increased in the cocaine group as compared to the control, which is consistent with the immunoblot results showing an increased level of dephosphorylated Cx43 ( Figure 4G,I). The expression and distribution of cardiac Cx43 demonstrates the dephosphorylation of Cx43 and its negative connection with CBN in the myocardium after 14 days of cocaine administration.

Involvement of Apoptosis and Necroptosis in the Damage to the Myocardium Caused by Cocaine Administration
As mentioned in "Introduction", dephosphorylation of cardiac Cx43 leads to the apoptosis of cardiomyocytes. We therefore examined the expressions of apoptosis-related markers by immunoblotting. Protein kinase B (Akt) is an important intracellular regulator that inhibits apoptosis [37]. We found a downregulation of the proportion of the phosphorylated (activated) form of Akt at serine 473 in total Akt, along with increased expressions of the proapoptotic protein BCL2 associated X (Bax), and the activated apoptotic marker cleaved caspase-3 [38] in the myocardium of the cocaine group as compared to the control group ( Figure 5A). Furthermore, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) analysis also revealed apparently increased TUNEL-positive stained cardiomyocytes in the cocaine group ( Figure 5B). These findings indicate the occurrence of apoptosis in the myocardium after 14 days of cocaine administration via the tail vein.  In addition to apoptosis, there are many other pathways implicated in the damage to the myocardium. For example, Ca 2+ overload and oxidative stress can lead to cardiomyocyte death by either apoptotic or necrotic pathways [6]. Therefore, we also explored whether there are other cell death pathways active in the myocardium of the cocaine group, such as, for example, caspase-independent necroptosis [39]. Immunoblotting of necroptosis-related mediators revealed a significantly activated mixed lineage kinase domain-like protein (MLKL) yet only a slightly activated receptor-interacting protein (RIP) that was not statistically significant ( Figure 5C). Thus, in addition to apoptosis, the necroptosis machinery might also play some role in the damage to the myocardium caused by cocaine administration. Considering that necroptosis plays a more important role in inflammation than apoptosis, the incomplete development of necroptosis seems to be consistent with the limited inflammatory findings in the myocardium of the cocaine group (Figure 1).

Possible Involvement of Adiponectin in Protecting the Myocardium after Cocaine Administration
In order to obtain further insight into the changes in cellular status in the myocardium after cocaine treatment, we conducted a DNA microarray analysis. Table 1 lists the 10 genes that were most often upregulated in response to cocaine treatment. The microarray results revealed adiponectin (Adipoq) to be the most upregulated gene in the myocardium of the cocaine group (Table 1). Adiponectin has been shown to accumulate in the myocardium in response to stress or injury. As a result, we performed immunoblotting in order to examine the expressions of adiponectin and its important effector AMP-activated protein kinase (AMPK). The levels of both proteins were significantly increased in the cocaine group ( Figure 6), indicating the possible role of adiponectin in protecting the myocardium against cocaine toxicity. In the myocardium, adiponectin is mainly secreted by epicardial adipose tissue, with relatively limited amounts secreted by cardiomyocytes [40,41]. Our findings suggest that the increased level of adiponectin in the myocardium after 14 days of cocaine administration is mainly derived either from the epicardium or systematically, rather than from cardiomyocytes. dium after cocaine treatment, we conducted a DNA microarray analysis. Table 1 lists t 10 genes that were most often upregulated in response to cocaine treatment. The micro ray results revealed adiponectin (Adipoq) to be the most upregulated gene in the myoc dium of the cocaine group (Table 1). Adiponectin has been shown to accumulate in t myocardium in response to stress or injury. As a result, we performed immunoblotting order to examine the expressions of adiponectin and its important effector AMP-activat protein kinase (AMPK). The levels of both proteins were significantly increased in t cocaine group (Figure 6), indicating the possible role of adiponectin in protecting the m ocardium against cocaine toxicity. In the myocardium, adiponectin is mainly secreted epicardial adipose tissue, with relatively limited amounts secreted by cardiomyocy [40,41]. Our findings suggest that the increased level of adiponectin in the myocardiu after 14 days of cocaine administration is mainly derived either from the epicardium systematically, rather than from cardiomyocytes.

Discussion
Although CBN has been repeatedly reported among the postmortem findings in the myocardium of cocaine abusers, it is rarely reported in animal experiments of cocaine cardiotoxicity. In previous studies, CBN was induced separately by catecholamines or ischemia/reperfusion injury [10]. Ischemia/reperfusion-related CBN is associated with massive interstitial hemorrhaging with subsequent inflammatory infiltration [11]. In contrast, inflammation is not an inevitable event in catecholamine-induced CBN [42]. In our experiments, massive interstitial hemorrhaging and inflammation were not found in the myocardium after 14 days of cocaine administration via the tail vein. This suggests that cocaine-induced CBN is more likely attributable to catecholamine overload. Similarly, the presence of CBN and the absence of inflammatory infiltration have also been reported in heart samples from cocaine abusers [43]. The sparsely observed eosinophil-dominated myocarditis in the cocaine group is consistent with the characteristics of hypersensitivity myocarditis. Although not commonly seen, there are reports in which eosinophilic myocarditis was confirmed in cocaine dependents [1,43]. These might be related to the hypersensitive reaction towards cocaine or its metabolites. It is worth noting that intracellular calcium assay for cardiomyocytes will be helpful for better elucidation of the current findings, and is recommended for interested researchers.
Due to the short half-life of Cx43, most studies have been conducted in vivo or ex vivo, wherein the heart was harvested immediately (mostly less than 60 min) after preconditioning, in order to determine the instant status of Cx43 [15,44]. Although the hearts were harvested 24 h after the last administration in our experiments, we found an increased dephosphorylation of Cx43 under stress and injury that is consistent with previous findings [16]. This might reflect the persistent effect of cocaine or the relatively long-term cocaine administration in our experiments. It has been reported that myocardial damage induced by ischemia/reperfusion or diseases results in the rearrangement of Cx43, and is closely related to myocardial function [45]. Though it is fascinating to explore whether cocaine has a similar effect on cardiac Cx43, the rearrangement of Cx43 was not observed in our chronic cocaine administrated study. An acute administration model, either in vivo or ex vivo with instant sampling, may be more applicable to this point. There is evidence to suggest that the dephosphorylation of Cx43 contributes to arrhythmias and further cardiac injury [46]. In the myocardium of the cocaine group, we found increased intercalated, disc-localized, dephosphorylated Cx43 in CBN adjacent regions, which may play a role in cocaine-induced cardiac disorders. The current preliminary findings can serve as the basis of therapeutic strategies against cardiac consequences in cocaine addicts. Further examination of cardiac function will be beneficial for advancing our understanding of this point. It should also be noted that there are different phosphorylation sites of Cx43, whereas only serine 368 was determined in our study. Considering the interesting increased expression of total Cx43, the status of other phosphorylation sites of Cx43 would be worth pursuing in subsequent explorations. Aside from its canonical role, Cx43 is also detected in mito-chondria, where it is implicated in processes including respiration and ROS generation [47]. The link between cocaine-induced mitochondrial damage and the role of mitochondrial Cx43 is another topic which requires illumination [48]. Co-immunoprecipitation assay or immunoelectron microscopy should be appropriate techniques in this field [49].
Our DNA microarray-based attempt to seek gene expression alterations after cocaine administration resulted in the unexpected finding that adiponectin is the gene most affected by cocaine under our experimental conditions. The cardiac protective role of adiponectin-AMPK has been well established in various models, including cardiac ischemia/reperfusion injury, diabetes, and cardiac hypertrophy [50][51][52]. It has been reported that cocaine exposure increases systematic adiponectin levels [53]. In our previous study on short-term cocaine administration, mitochondrial fission was found to act as an early compensation mechanism against the cardiotoxicity of cocaine, prior to the occurrence of structural changes [54]. The beneficial effect of adiponectin is also considered an early event in cardiac pathogenesis [28]. Combined with our current findings, adiponectin may act protectively against cocaine cardiotoxicity during the early stages of cardiac pathogenesis. It is worth noticing that high molecular weight (multimeric) adiponectin is assumed to be the biologically active form [55], whereas the adiponectin observed in our experiments consisted of both multimeric and monomeric forms. This may limit our ability to interpret the increased adiponectin level within the heart in this study. An in-depth elucidation of the role of adiponectin in protecting against the cardiotoxicity of cocaine requires further study, for instance, by applying knockout mice or antagonists of adiponectin-related pathways. It should be noted that the limitation of this study is the relatively small sample size; a follow-up study based on a bigger population is being pursued.
In conclusion, we have demonstrated that the administration of 20 mg/kg/day cocaine to rats through the tail vein for 14 days results in disordered myocardium with widespread CBN, mitochondrial dysfunction, increased dephosphorylation of Cx43, involvement of apoptosis and necroptosis, and participation of cardiac protective adiponectin. Differences among individual models may be present; yet, our findings should be helpful for advancing understanding of the cardiotoxicity of relatively long-term exposure to cocaine, as well as establishing novel therapeutic strategies in this field.

Materials and Methods
All methods were carried out in accordance with relevant guidelines and regulations.

Animals and Cocaine Administration
Animal experiments were approved by the Institutional Animal Care and Use Committee of Tokyo Medical and Dental University, and in keeping with Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines and regulations. Eight-weekold male Sprague Dawley rats were randomly divided into 2 groups and administered saline (control group, n = 3) or cocaine (cocaine group, n = 4). The dose of cocaine was 20 mg/kg/day after dose conversions from humans to rats, in order to reproduce the typical serum concentrations approaching acute cocaine poisoning overdose in humans [56,57]. Cocaine hydrochloride (CAS: 50-36-2, Shionogi & Co., Ltd., Osaka, Japan) was dissolved in saline and administered intravenously through the tail vein once a day for 14 days. The same volume of saline was given via the tail vein for control group rats on the same schedule. Twenty-four hours after the last administration, the rats were sacrificed by intraperitoneal injection of an overdose of sodium pentobarbital (40 mg/kg). The hearts were collected immediately and placed in phosphate-buffered saline solution (PBS, 4 • C, pH 7.4). The left ventricles were then evenly separated for the following histological and biochemical analyses.

Histological Analysis and Immunohistochemical Staining
Sections of the left ventricular specimens (2.5 µm thickness) for microscopy were prepared from tissues fixed in 4% paraformaldehyde and embedded in paraffin. Hematoxylin-eosin (H&E), Elastica Masson-Goldner (EMG), and Phosphotungstic Acid Hematoxylin (PTAH) staining were conducted for histological analysis of the myocardium. Immunohistochemical staining of dephosphorylated Cx43 was performed. In brief, deparaffinized sections were heated for 5 min in a 10 mM citrate buffer (pH 6.0) for antigen retrieval. Next, the activity of endogenous peroxidase was quenched by applying 0.3% hydrogen peroxide. The sections were then incubated with mouse 8-OHdG monoclonal antibody (diluted 100-fold; JaICA, Shizuoka, Japan) or mouse Cx43 monoclonal antibody (diluted 100-fold; #13-8300, Thermo Fisher Scientific, Waltham, MA, USA) overnight at 4 • C. The following visualization of antigens was conducted applying Histofine Simple Stain MAX-PO (Multi) (Nichirei Biosciences Inc., Tokyo, Japan) and diaminobenzidine (Nichirei Biosciences Inc.) as the substrate. Controls for the immunostaining procedures were prepared by incubation in PBS in place of the primary antibody.

Transmission Electron Microscopy
Left ventricle specimens for transmission electron microscopy were prepared by washing the tissues in 0.1 M phosphate buffer (PB) and then fixing them with 2.5% glutaraldehyde and 4.5% paraformaldehyde in PB. The fixed specimens were then dehydrated and embedded in Epon epoxy resin after incubation in 1% osmium tetroxide for 2 h. Subsequently, ultrathin sections were prepared and stained with uranyl acetate and lead citrate. The sections were observed under a transmission electron microscope (H7100, Hitachi, Japan) and recorded using an AMT Advantage-HS CCD camera (AMT, Woburn, MA, USA). The number of mitochondria per field (magnitude at 10,000×) was recorded from 10 electron micrographs in each group. Cross-sectional areas of randomly selected mitochondria (n = 100 for each group) in longitude views of cardiac myofibrils at fields magnitude at 10,000× were counted using ImageJ software (ver1.53a).

Mitochondrial Electron Transport Chain Activity Detection
Left ventricle specimens for enzyme activity assays were immediately frozen at −80 • C. A Mitochondria Isolation Kit (ab110168, Abcam, Cambridge, UK) was used to extract mitochondria from the left ventricle. The protein concentrations of the extracts were determined and adjusted to 100 µg/mL by adding PBS. Mitochondrial oxidative phosphorylation (OXPHOS) Complex I enzyme activity was determined using a Complex I Enzyme Activity Assay Kit (ab109721, Abcam).

Real-Time Reverse Transcriptase-Mediated PCR Analysis
Real-time reverse transcriptase-mediated PCR analysis was performed as previously described [58]. The primers used are listed in Supplementary Table S1.

TUNEL Analysis
TUNEL analysis was performed using MEBSTAIN Apoptosis TUNEL Kit Direct (MLB International Corporation, New York, NY, USA). FITC and RHOD fluorescence were observed under a fluorescence microscope (DMi8, Leica).

Transcriptome Analysis
The total RNA of rats' left ventricle samples for transcriptome analysis was extracted using a TRIzol reagent (Thermo Fisher Scientific) and further purified using a RNeasy Mini Kit (Qiagen, MD, USA). The purified total RNA was examined for integrity on a BioAnalyzer (Agilent Technologies) and hybridized into ClariomS array (Thermo Fisher Scientific). The results were deposited in the GEO database (https://www.ncbi.nlm.nih. gov/geo/, accession number GSE200127, accessed on 7 April 2022). Microarray Data Analysis was conducted using Transcriptome Analysis Console (TAC) software (Thermo Fisher Scientific).

Statistical Analysis
Student's t-test was applied in order to assess statistical significance. p < 0.05 was considered significant. Statistical analysis was conducted using GraphPad Prism (Version 9.0.0, GraphPad Software, San Diego, CA, USA).