Introduction
Depression is a mood illness marked by feelings of melancholy, worry, insomnia, and suicide ideation. Trazodone (TRZ) is a serotonin receptor type 2 (5-HT2) antagonist and reuptake inhibitor that has been clinically approved since 2001. Despite the fact that TRZ is a pharmacologically approved treatment for insomnia, it has a long list of documented side effects, including headaches, dizziness, hypotension, syncope, and feeling off balance problems [
1]. Also, previous studies reported cardiovascular adverse effects such as electrocardiogram (ECG) irregularities (prolonged PR interval & long QT syndrome), postural hypotension, and cardiac arrhythmias were induced at therapeutic and even subtherapeutic doses of TRZ [
2]. However, the molecular pathogenesis and mechanisms by which TRZ exert its cardiac side effects still not fully understood.
One of the most prevalent mechanisms of drug-induced cardiotoxicity is the generation of reactive oxygen species (ROS) and free radicals. The production of reactive oxygen species (ROS) regulates the expression of many cytokines, including tumor necrosis factor (TNF-α), which serves as an essential inflammatory biomarker for cardiotoxicity [
3,
4] Therefore, the inhibition of TNF-α through the administration of cardioprotective agents could have a potential therapeutic role.
Autophagy is one of the most recently discovered main cell death mechanisms, since it begins with the destruction and recycling of proteins and organelles, which are subsequently sequestered into double-membrane vesicles known as autophagosomes [
5,
6]. Recent studies reported more than 30 autophagy-related (ATG) genes that stimulate autophagy through coding proteins essential for induction, maturation, and recycling of autophagosomes [
7,
8] Furthermore, autophagy has been found to play a critical role in cardiac homeostasis mechanisms. The inhibition of autophagy gene 5 (ATG5) resulting in myocardial dysfunction, as well as inactivation of disruption of ATG6 (also known as Beclin1) accelerating heart failure in cardiomyopathic mice [
3,
9]. Additionally in heart failure patients, autophagic myocyte death was observed. Cardiomyocytes are differentiated cells that lose their proliferative potential as a result of lethal injury such as necrosis, apoptosis, or autophagy, which causes heart malfunction and worsens cardiac failure [
7].
l-carnitine (LC) (β-hydroxy-γ-N-trimethylaminobutyric acid) is required for the transport of long-chain fatty acids into mitochondrial matrix to start β-oxidation of fatty acids which is the primary energy source in the myocardium[
10]. Previous studies demonstrated that LC has antioxidant, anti-inflammatory, and antiapoptotic properties which could be of value in protection against cardiotoxicity [
11,
12]. The purpose of this study was to investigate the molecular mechanisms that explain
l-carnitine's protection against TRZ-induced cardiotoxicity by examining its effects on autophagy, inflammation, and oxidative stress.
Discussion
Cardiovascular side effects can arise as a result of acute or chronic pharmacological treatment, and they might affect the mechanical functioning of the myocardium and/or structure (e.g., morphological damage or loss of cellular/subcellular components of the heart or vasculature [
24]. The cardiovascular adverse effects and pathological cardiovascular findings of TRZ were investigated and compared to
l-carnitine combination with TRZ in the present study.
Trazodone, a tetracyclic SARI, has been linked to cardiovascular problems in multiple studies. In the heart, it has the potential to block calcium, potassium, and sodium channels. Trazodone can also cause drug-induced long QT syndrome, which can cause life-threatening cardiac arrhythmias in persons with mutations in cardiac ionic channel genes [
25]. TRZ prolonged PR interval has also been linked to hypertrophy and other structural abnormalities like as cardiomyopathy [
1].
Myocardial damage leads myocytes to release biomarkers like AST into the bloodstream, albeit it does not have total specificity to the myocardium. Also, CK-MB had a short half-life, indicating acute myocardial injury. In addition, cardiac troponin T, is one of the most important specific indicators of myocardial ischemic injury [
26,
27]. The current investigation found that TRZ administration elevated serum CK-MB, AST, and cTnI levels, as well as a substantial increase in cardiac necrosis and myolysis in rat hearts. Previous studies support our findings that TRZ treatment induces myocardial ischemia [
1,
28].
l-carnitine is a crucial component of energy metabolism and blood glucose control.
l-carnitine helps transport long-chain fatty acids through the inner mitochondrial membrane, where they become a key substrate in energy generation and are degraded through beta-oxidation in muscle tissue and the heart's myocardium. Because the myocardium prefers to metabolize long-chain fatty acids for energy,
l-carnitine becomes especially vital in the heart [
29].
l-carnitine helps eliminate toxins from inside the mitochondria, mediates oxidative stress, inhibits fatty acid ester formation during ischemia events, and prevents cardiac cell death [
30]. Herein, the dose of 200 mg/kg
l-carnitine administered for 4 weeks significantly modulated TRZ cardiotoxicity; serum levels of CK-MB, AST, and cTnI levels were significantly decreased.
Clinical studies have shown,
l-carnitine supplementation can enhance myocardial fat metabolism due to the increased demand for free fatty acids and their metabolites and thus creates a beneficial effect on myocardial function [
31,
32]. Besides, therapeutic potentials of
l-carnitine have been investigated in many rat models, for instances, in tilmicosin-induced cardiotoxicity [
33], and imatinib-induced cardiotoxicity [
34].
In the heart, reactive oxygen species are produced during normal cellular functions of mitochondria during oxidative phosphorylation as well as enzymatic reactions catalyzed by xanthine oxidase, NAD(P)H oxidases, and cytochrome P450 [
35]. As the oxidative status of the cardiac tissue was assessed following TRZ treatment, a diminished catalase activity and increased MDA levels were reported, as well as a decrease in total thiol levels, when compared to the control, LC, and LC + TRZ groups. As a result, it's possible that the observed cardiac toxicity was accompanied with oxidative stress in cardiac tissue, which was alleviated by
l-carnitine delivery. Also, in vitro studies showed that TRZ and its reactive metabolite caused oxidative stress by increasing MDA levels and depletion of glutathione [
36].
l-carnitine, on the other hand, restored oxidative stress by lowering ROS and increasing endogenous antioxidant levels. In a dose-dependent manner,
l-carnitine has radical scavenging capabilities and metal chelating activity [
37].
In the TRZ-treated group, histological examination indicated a change in the shape of cardiac cells, as well as evidence of localized necrosis and inflammatory cell infiltration. Increased oxidative stress in cardiac tissue can lead to structural alterations like cardiomyopathy, cardiac hypertrophy, development of interstitial cardiac fibrosis, endothelial dysfunction, and contractile protein failure [
38]. However,
l-carnitine treatment successfully protected the heart as indicated by the improvement of the histopathological parameters.
During cellular stress, ATG5, a protein essential for the creation of the autophagy precursor, is cleaved by a cysteine protease and plays a key role in the initiation of autophagy. ATG12 is required for the development of autophagosomes. This protein undergoes ATP-dependent conjugation to ATG5, which is mediated by ATG7 and ATG10. Following that, the ATG12-ATG5 complex interacts with ATG16L1, generating an ATG12-ATG5-ATG16L1 conjugate that is required for effective Atg8/LC3 conjugation to phagophore membranes [
39]. However, Beclin-1, which normally binds to Bcl-2 and inhibits autophagy, is competitively displaced, increasing the autophagic process under autophagy-inducing conditions [
40]. P62 also increases aggresome formation and autophagy activation, as well as protecting cardiomyocytes from proteotoxic stress [
41].
Following autophagy induction, the autophagy proteins ATG7, ATG3, and the ATG12-ATG5-ATG16L1 complex covalently conjugate MAP1LC3-I to phosphatidylethanolamine (PE) to generate MAP1LC3-II, which is then attracted to the inner and outer surface of autophagosomal membranes via PE. As a result of the development of MAP1LC3-positive puncta, autophagosomes can be easily detected immunohistochemistry in tissue sections [
42]. Herein, the present study showed high expression of autophagosomal LC3II in cardiac tissues of TRZ-treated groups. However, LC treatment ameliorated this effect.
Cardiomyocytes, being postmitotic cells with high rates of energy consumption and an insatiable demand for ATP, have a plethora of mitochondria and are thus especially vulnerable to damaged or dysfunctional mitochondria. Cellular functions worsen as ROS accumulate, which is followed by autophagy disruption [
43]. Herein, the present study showed that the expression of the Beclin1 and ATG5 genes, as well as the level of the p62 protein, were significantly higher in the hearts of rats given TRZ for 4 weeks compared to the control group. When compared to the TRZ-treated group,
l-carnitine administration significantly reduced the effect of TRZ and reduced autophagy gene expression of Beclin1 and ATG5, as well as p62 protein levels. These effects could be explained by the increased oxidative stress situation caused by TRZ and controlled by
l-carnitine 's antioxidant and scavenging abilities [
13].
The current study showed increased gene expression of cardiac TNF-α following TRZ treatment. However, LC downregulated cardiac TNF-α gene expression. These results were in consistence with previous reports [
37,
44,
45]. TNF-α is a pro-inflammatory cytokine produced by all cardiac cells in response to stress in order to initiate an inflammatory response. TNF-α binds to its receptor that have a death domain then recruited the death domain associated proteins that binds to caspase-8. P62 could be bind to polyubiquitinated casp-8 and recruits casp-8 to the autophagosome membranes. In heart illnesses such dilated cardiomyopathy, myocardial infarction, and left ventricular pressure overload, circulating and cardiac TNF- levels are high. As a result, TNF- has been linked to the development of ventricular remodeling and cardiac dysfunction in infarcted hearts [
44,
46].
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