This study stress the possible occurrence of noxious effects caused by exposure to the recommended daily dose, tolerable upper intake dose and twice the tolerable upper intake level of copper on cardiac contractility. Copper exposure did not change weight gain, vascular effects leading to pressure overload, or promoted cardiac hypertrophy in the animals at any dose evaluated. The amount equivalent to 10 mg of copper/day for humans of 70 kg, although considered safe by regulatory agencies, reduced the cardiac force generation, the time derivatives of force, the response to extracellular calcium, the β-adrenergic response, the postrest contraction and tetanic contractions. Similar effects were obtained after exposure to an amount equivalent to 20 mg/day of copper in adults, which is considered twice the tolerable upper intake levels for this metal. The amount equivalent to 0.9 mg/day of copper, despite causing changes in some of the regulatory mechanisms of cardiac contractility, was not able to reduce the force generation of papillary muscles.
It was observed that the body weight gain during the 4 weeks of exposure was similar among the experimental groups that received 13 µg, 0.14 mg or 0.28 mg/kg/day of copper, corroborating a study by Naseri et al. [31] and Mattiolli et al. [32], in which steers that received copper supplementation up to the maximum recommended level showed no change in body mass gain.
To investigate whether the cardiac effects were not due to other factors, such as vascular effects leading to pressure overload, we tested the copper effects on aortic segments. As the aorta is a vessel that readily reflects the effects of metals producing oxidative stress [33–35], the fact that these three doses did not alter vascular reactivity reinforces the idea that the effects found in this study are restricted to the heart only. Therefore, we did not proceed with any vascular studies because no changes were found.
Cardiac hypertrophy is one of the most serious risk factor for cardiovascular disease. In our findings, exposure to or above the recommended levels of copper was not able to change the weight of the right and left ventricles or the papillary muscles of rats. We know that copper acts as a cofactor of cytochrome c oxidase and lysyl oxidase, and increased exposure to copper improves the activity of these enzymes [36]. Zheng et al. [36] reported that increased cytochrome c oxidase activity prevents cardiac hypertrophy. Rodrigues and González [37] reported that an increase in copper concentration improves the function of lysyl oxidase, maintaining myocardial extracellular matrix homeostasis through the oxidation of collagen and elastin chains and preventing the occurrence of cardiac hypertrophy.
The evaluation of force, time to peak tension, time to 90% relaxation, and positive and negative time derivatives of force suggest that exposure to the tolerable upper intake levels and twice the tolerable upper intake levels of copper were able to reduce inotropism and affect the temporal parameters of contraction. According to our data, these effects seem to be related to reduced extracellular calcium influx through the sarcolemma and reduced activity of cardiac contractile proteins [38]. We know that increased exposure to copper is capable of impairing the contractility of papillary muscles and their regulatory mechanisms due to the increased formation of reactive oxygen species (ROS) [39]. Other studies have described the occurrence of changes in cardiac function in situations of increased exposure and/or elevated blood concentration of copper, but they do not explain the underlying mechanisms responsible for these effects [40–43].
Copper leads to the formation of free radicals from the chemical reactions of Fenton and Haber-Weiss [44]. Previous reports have shown that oxidative stress development is the main mechanism by which copper impairs cardiac contractility [45–51]. Based on this, we performed a detailed assessment of each of the regulatory mechanisms of cardiac contractility to describe a model of cardiotoxicity induced by exposure to increasing levels of copper. We used the recommended daily doses, tolerable upper intake levels, and twice the tolerable upper intake levels.
Calcium influx assessed through postrest contraction decreased after exposure to 0.14 mg/kg/day copper, while the inotropic response to extracellular calcium decreased in groups of animals exposed to all doses evaluated. These results suggest that copper reduces calcium influx through L-type calcium channels. Previous studies evaluating exposure to toxic metals such as iron and lead showed that ROS impairs the mechanisms that make calcium available for myocardial contractility, including calcium influx through sarcolemmal L-type channels [52, 53]
Another analysis of our study was to assess whether exposure to copper doses could influence the response to a β-adrenergic agonist. The β-adrenergic agonist promotes a series of second messenger-mediated effects on cardiomyocytes that culminate in phosphorylation of phospholamban (PLBp) and L-type calcium channels, resulting in greater permeability to extracellular calcium and increasing calcium reuptake by SERCA, resulting in increased force of contraction and accelerated relaxation. This response was significantly diminished in papillary muscles from animals exposed to the recommended daily doses, tolerable upper intake levels, and twice the tolerable upper intake levels. Kaneko et al. [54] investigated the effects of ROS on α- and β-adrenergic receptors in rat hearts, which suggested that free radicals are able to modify β-adrenergic receptors and downregulate them, causing a decrease in the inotropic response.
Tetanic contractions were used to assess the cardiac contractile machinery with the dysfunctional sarcoplasmic reticulum; thus, the contraction becomes dependent on the influx of calcium and the sensitivity of contractile proteins [24, 53]. The developed peak and plateau forces of the tetanic contractions of the LV papillary muscles were impaired by exposure to doses of 13 µg, 0.14 mg and 0.28 mg/kg/day of copper. A previous study by Filetti et al. [39] also showed a reduction in tetanic contraction after acute exposure to high concentrations of copper, which in this case was related, at least in part, to a reduction in myosin ATPase activity. In fact, Moreira et al. [55] evaluated the effects of other toxic metals and showed the ability of divalent metals to bind to the SH radicals of the myosin molecule, reducing ATP hydrolysis and consequently reducing contraction. Furthermore, other studies have demonstrated the ability of copper to bind to SH radicals in other body tissues [56, 57].
It has already been shown that oxidative stress is the main mechanism by which copper impairs cardiac contractility [45–51] thus, the effects of antioxidant treatment with EWH during copper exposure were evaluated. Although a dose of 13 µg/kg/day of copper altered some regulatory mechanisms of cardiac contraction, the force of contraction remained stable, while in the groups exposed to doses of 0.14 and 0.28 mg/kg/day of copper, the force of contraction was reduced. In addition, a dose of 0.14 mg/kg/day of copper is considered safe and easy to obtain in exposed populations, so the evaluation of the antioxidant role of EWH was performed only in the group that received the equivalent of 10 mg/day of copper in humans.
The presence of tyrosine and phenylalanine in EWH is related to the neutralization of free radicals [58]. In addition, previous studies have shown that EWH is able to reduce peroxidation, one of the mechanisms that causes lipid damage, in addition to increasing SOD expression and reducing NADPH oxidase expression [18–20, 59]. Finally, EWH can be considered an ingredient in functional foods and can be used as an additional therapy in the treatment of cardiovascular toxicity promoted by copper.