Coenzyme Q10 prevents oxidative stress and fibrosis in isoprenaline induced cardiac remodeling in aged rats

Cardiovascular disease (CVD) became epidemic currently and responsible for the increased mortality and morbidity. At the beginning of the 21^st centuries one in every three persons of the world population dies from heart attack, and strokes. Despite all hype about the new drug development, the global cardiovascular epidemic continues relentlessly. WHO information predicted that nearly 23.6 million people will die from CVD each year by 2030 [1]. Cardiac remodeling is usually characterized by expression of genome, changes of molecular, cellular and interstitial state, which ultimately results in changes in size, shape and function of the heart. This is one of the common events that occurred in many cardiovascular disease including myocardial infarction (MI), aortic stenosis, hypertension, myocarditis, idiopathic dilated cardiomyopathy. Cardiac hypertrophy is a typical type of cardiac remodeling [2], where not only the crucial cardiac cell myocyte, but also other components like fibroblasts, collagens, coronary vasculature, interstitium are involved in remodeling process which finally causes ischemia, apoptosis and cell necrosis [3]. Cardiomyocyte hypertrophy is commonly found in aging heart and contributes to the development of congestive heart failure [4]. Stimulation of the sympathetic nervous system is a common attribute in cardiovascular disease [5] and age-related changes in autonomic nervous system function may cause heart-rate variability and increase in resting heart rate. Prolong overstimulation of the β-adrenergic receptor (β-AR) by different stressors causes cardiac hypertrophy, myocardial infarction, strokes, heart failure, coronary artery disease in response to catecholamine and are also responsible for oxidative stress in heart and coronary artery [6]. Auto oxidation of catecholamine generate catecholamine-o-quinones, aminochromes, adrenochrome are thought to be responsible for catecholamine related cardio toxicity [7]. Even previous study have supported that patients with chronic heart failure showed poor survival rate when treated with β-adrenergic agonists [8]. Subcutaneous administration of isoprenaline (ISO), which mimics the β-adrenergic receptor activity, can produce myocardial necrosis [9]. Cardiac hypertrophy induced by isoprenaline is a dependable, consistent and well characterized prototype correlated with arrhythmias, myocyte loss and fibrosis with advancement to heart failure [10].

Oxidative stress and inflammatory responses are one of the underlying mechanisms of myocardial remodeling in aged heart [11]. Excess production of reactive oxygen species and inflammatory cytokines may cause a change in extracellular matrix by activating matrix metalloproteinase (MMP) ultimately results in collagen synthesis and myocardial fibrosis in aged heart [12, 13]. Cardiac apoptosis is another crucial factor to hypertrophic remodeling and cell dysfunction where, ROS stimulate cellular apoptosis signaling kinase-1 and over expression of kinase-1 activates nuclear factor Kappa B (NF-κB) to stimulate hypertrophy [14]. Increasing evidences have suggested that ISO treatment at high doses increases myocardial oxidative stress, pro-inflammatory cytokine synthesis, and stimulate mitogen-activated protein kinases [6, 15]. Functional hypoxia and ischemia, metabolism alterations, coronary insufficiency, energy depletion, calcium overload are some other possible mechanism by which ISO induced cardiotoxicity [16].

Many natural products and dietary supplements containing antioxidant properties demonstrated ROS scavenge in the ISO treated rats [17]. Coenzyme Q10 (Co-Q10), the only synthesized antioxidant in human body is the first drug to improve heart mortality by decreasing all cases of mortality by half [18]. Coenzyme Q10 or ubiquinone is a naturally occurring lipid soluble benzoquinone can be obtained from consumption of meat, poultry, fish, vegetables and fruits [19], a key element for adenosine triphosphate synthesis of the mitochondrial respiratory chain [20]. Co-Q10 can provide protection for membrane phospholipids, mitochondrial membrane protein, and low-density lipoprotein against oxidative damage [21]. Co-Q10 treatment is helpful for lowering pro-inflammatory cytokines and blood viscosity in patients with heart failure and coronary artery disease. It can also improve ischemia and reperfusion injury of coronary revascularization [22]. In addition to its antioxidant activity, Co-Q10 is also responsible for intracellular energy production, responsible for improvement of endothelial dysfunction as well as important for activating mitochondrial uncoupling proteins [23]. Considering the cardioprotective effect of antioxidants, the following investigation was conducted to evaluate the effect of coenzyme Q10 against isoprenaline induced cardiac remodeling in rats.

Aging is a retrogressive process that is kindred with ongoing accumulation of detrimental changes with time, results in reduction of physiological such as cardiovascular, renal, neurological, endocrine function with the increase of chances of disease and death. With advancing age one of the most debilitating is loss of myocardial function. Decreased cardiac elasticity and inability to respond of changes in pressure to the arterial system mainly influence the heart function associated with aging [31]. Cardiac tissue is mainly postmiotic and oxidative damage is most pervasive because of its high dependency on oxidative phosphorylation to derive energy [32]. Altered inflammatory mediator expression and inability to respond of senescent cells to growth factors are important mechanism of age related adverse cardiac remodeling [33]. Besides, in aging and ischemic disease, ROS are responsible for peroxidation of mitochondrial phospholipids. Mitochondrial respiratory chain is mainly responsible for production of these ROS free radicals [34]. In this study, cardiac remodeling in rats was induced by subcutaneous injection of isoprenaline. At low doses, catecholamine exerts positive inotropic effect that is beneficial for heart function. But high doses of isoprenaline causes energy depletion of heart resulting in biochemical and structural changes of cardiomyocyte [16]. This study showed that isoprenaline induced cardiac hypertrophy by increasing left ventricular wet weight. Isoprenaline administration also raised the level of different oxidative stress markers and lowered the level of antioxidant from plasma and heart. Colossal amount of infiltrating cell and fibrosis was found on infract area induced by isoprenaline. In mitochondria, 80% oxygen consumption is occurred and this very compartment is mainly responsible for production of O₂ ^.- and H₂O₂ [23]. The mitochondrial element coenzyme Q10 (Co-Q10) or ubiquinone has been used as a dietary supplement for improving health condition by eradicating free radicals [32]. Several previous studies have revealed the beneficial effect of CoQ10 in aging [35–37]. Previous studies showed that CoQ10 level was found low in patients with cardiovascular disease [18, 19]. Generally, CoQ10 or ubiquinone in its reduced form acts as an antioxidant and lowers different inflammatory markers like TNF-α and IL-6 [38]. This research suggests that treatment with CoQ10 helps to minimize oxidative stress, inflammatory cell infiltration and fibrosis in heart in isoprenaline treated rats.

Damage to cardiac tissue induced by different β-adrenergic receptor agonist like isoprenaline increases the level of ALT, AST, and ALP enzymes [39]. Myocardial necrosis induced by ISO treatment increases ALT, AST and ALP activities are observed in plasma. In our study, CoQ10 significantly inhibit the increase of AST and ALP. Previous study also supported that treatment with CoQ10 can reduce these enzyme activities from plasma [40]. Creatine kinase-MB (CK-MB) is a superior and special marker, serves as an indicator of myocardial necrosis [41]. Elevation of CK-MB activity because of ISO treatment is observed in plasma of aged rats [42]. In this study, treatment with CoQ10 inhibited the elevation of CK-MB activity in plasma. This study is also supported by previous finding which also showed CoQ10 can reduce CK-MB activity in isoprenaline induced cardiotoxicity and cardiac hypertrophy in rats [43].

Oxidative stress emanating from subcutaneously isoprenaline injection is mediated mainly through β-adrenergic receptor stimulation which rapidly generates ROS by spontaneous oxidation of catecholamine. Isoprenaline administration results in increase calcium overload and myocardial dysfunction in cardiomyocytes [44]. Increased lipid peroxidation is an important biomarker indicative of elevated oxidative stress. Alterations of balance between prooxidant and antioxidant defense is thought to be the main mechanism behind lipid peroxidation where, ATP is converted to hypoxanthine and uric acid respectively [45]. Myocardial phospholipids membrane peroxidation causes permeability change and intracellular calcium overload results in cardiomyocytes damage in the heart [46]. Catecholamine induced increase in MDA level in myocardial tissue was reported in previous study [47]. Increased malondialdehyde (MDA, product of lipid peroxidation) content was increased in ISO treated rats that were attenuated by CoQ10 in our study. Besides, another novel oxidative stress marker AOPP (Advanced Oxidation Protein Products) level was high in ISO treated group which was lowered significantly by CoQ10 treatment. Nitric oxide is an another reactive molecule that can control cardiovascular homeostasis, myocyte growth, function and remodeling [14]. Uncoupled NO-synthase is the main contributor of ROS generation which ultimately results in endothelial dysfunction [48]. In cardiac hypertrophy induced by isoprenaline, increased iNOS expression in left ventricle and increased level of NO production in heart was reported associated with increased apoptosis [10]. Mainly, activation of β-adrenergic receptor is the main mechanism of up regulation of iNOS [49]. This excess nitric oxide concentration can react with superoxide radical and can form peroxynitrite (˙ONOO-) a highly toxic reactive species capable of activating cytotoxic process including, protein oxidation, lipid peroxidation and nitration ultimately results in myocardial injury [14]. In this study, nitric oxide level was also found to be elevated in ISO treated rats whereas, mitochondrial component CoQ10 successfully normalized the elevation of nitric oxide level. Increased heart wet weight was observed in ISO treatment which was normalized by CoQ10 treatment. Indeed, Akt signaling induces physiological hypertrophic responses important for controlling cardiac structure and function. Previous study proved that cardioprotective effect of CoQ10 lies on preservation of Akt signaling where coenzymeQ10 can protect mice from pathological cardiac hypertrophy [50]. CoQ10 can efficaciously reduce alpha-tocopheroxyl radical to alpha-tocopherol. Thus, having the property of terminating pro-oxidant radical, CoQ10 can regenerate the active form of vitamin E [34].

Antioxidant contrives the defense mechanism capable of decelerating or averting the unstable free radicals from initiating toxic effects that simultaneously causes tissue damage. The prime endogenous antioxidants such as SOD (superoxide dismutase), CAT (catalase), GSH (reduced glutathione) are responsible for trapping free radicals where, SOD catalyses the conversion of O₂ ^.- to H₂O₂ and H₂O; CAT converts H₂O₂ to H₂O and O₂; glutathione reduces H₂O₂ to H₂O. Myocardial injury induced by ISO treatment increases lipid per oxidation which in turn reduces these enzyme levels [51, 52]. This study also showed significant reduction of CAT activity and GSH level in ISO treated rat both in plasma and heart compared to control group. CoQ10 treatment restored the GSH level significantly.

ISO is considered as a model compound that causes cardiac inflammation and fibrosis. It has been postulate that this beta adrenergic agonist results in hypoxic condition in cardiac tissue as a result of uncontrolled oxidative metabolism in myocyte. The imbalance of energy, in addition with a number of complex biochemical (altered calcium flux, stimulation of the adenyl cyclase system, aggregation of platelets, and formation of reactive oxygen species) [53] and structural changes (alterations in membrane permeability) [54] & [55], appear to contribute to the pathogenesis of the myocyte damage [56]. The area of the heart most susceptible to hypoxia caused by tachycardia appears to be the left ventricular subendocardium [53]. Recently, changes in iNOS expression have also been associated with ISO-induced cardiotoxicity [57]. Myocyte damage observed following exposure to ISO includes both apoptosis and necrosis [58]. Cardiac inflammation by ISO was observed by histological evaluation which demonstrated significant endomyocardial eosinophilic infiltration and areas of myocyte necrosis [59]. On the other hand myocardial fibrosis was identified by the appearance of thick, coarse groups of interstitial collagen fibrils associated with a few necrotic myocytes [60]. This study showed that administering ISO resulted in the massive mono nuclear cells infiltration as well as deposition of interstitial collagen fibrils on histological analysis of cardiac and kidney tissues which strongly supported by previous findings [61]. Both scenarios were reversed after the treatment with CoQ10.

Kidney dysfunction may also contribute to the development of cardio-vascular complications [61, 62]. Isoprenaline induced renal complications and fibrosis was also reported in animal studies. Recent evidence also suggests that ISO administration increase cardio-renal fibrosis which was ameliorated by sympathetic nerve system (SNS) inhibition by denervation procedure [62]. This study suggests that Co-Q10 administration prevents inflammation and fibrosis in kidney of ISO treated rats. Improved cardiac and kidney tissues structure due to Co-Q10 administration could be relied on the improvement of oxidative stress and restoration of antioxidant defence in tissues which is also observed in other studies as well [63, 64].

The results of this study indicate that the coenzyme Q10 treatment provides significant cardiac and renoprotective effect against ISO administered rats. Coenzyme Q10 inhibits cardio-renal fibrogenesis through reducing oxidative stress and preventing inflammatory cells infiltration in tissues of ISO treated rats. Thus targeting mitochondrial function would be a possible way of preventing oxidative stress related disorders in future.