Expression of cardiac copper chaperone encoding genes and their correlation with cardiac function parameters in goats

Copper deficiency (CuD) is a common cause of oxidative cardiac tissue damage in ruminants. The expression of copper chaperone (Cu-Ch) encoding genes enables an in-depth understanding of copper-associated disorders, but no previous studies have been undertaken to highlight Cu-Ch disturbances in heart tissue in ruminants due to CuD. The current study aimed to investigate the Cu-Ch mRNA expression in the heart of goats after experimental CuD and highlight their relationship with the cardiac measurements. Eleven male goats were enrolled in this study and divided into the control group (n = 4) and CuD group (n = 7), which received copper-reducing dietary regimes for 7 months. Heart function was evaluated by electrocardiography and echocardiography, and at the end of the experiment, all animals were sacrificed and the cardiac tissues were collected for histopathology and quantitative mRNA expression by real-time PCR. In the treatment group, cardiac measurements revealed increased preload and the existence of cardiac dilatation, and significant cardiac tissue damage by histopathology. Also, the relative mRNA expression of Cu-Ch encoding genes; ATP7A, CTr1, LOX, COX17, as well as ceruloplasmin (CP), troponin I3 (TNNI3), glutathione peroxidase (GPX1), and matrix metalloprotease inhibitor (MMPI1) genes were significantly down-regulated in CuD group. There was a significant correlation between investigated genes and some cardiac function measurements; meanwhile, a significant inverse correlation was observed between histopathological score and ATP7B, CTr1, LOX, and COX17. In conclusion, this study revealed that CuD induces cardiac dilatation and alters the mRNA expression of Cu-Ch genes, in addition to TNNI3, GPX1, and MMPI1 that are considered key factors in clinically undetectable CuD-induced cardiac damage in goats which necessitate further studies for feasibility as biomarkers.


Introduction
Copper (Cu) has crucial importance for various physiological processes because of being an essential cofactor in numerous enzymatic activities. These activities including, but not limited to, iron metabolism, erythropoiesis, energy production, collagen formation, hormone biosynthesis, and antioxidant defense against peroxidative damage (Sinclair and Mackenzie 2013). The safety margin of Cu content in the diet of ruminants is comparatively narrow hence dietary copper deficiency (CuD) due to antagonist elements such as sulfur and molybdenum and hepatic toxicity following Cu supplementation is a matter of challenge during ration formulation (López-Alonso and Miranda 2020). In the biological systems, Cu has unique electrochemical properties and is regulated by a distinctive transport and traffic system known as metallochaperones or copper chaperone proteins (Cu-Ch) which helps in delivering Cu to the target site without inflicting harm or becoming impound in unplanned sites. These chaperones include Cu transporter protein (CTr1), ATPases 7 alpha and beta (ATP7A, ATP7B), chaperone for superoxide dismutase (CCS), cytochrome c oxidase 17 (COX17) and lysyl oxidase (LOX). These chaperones assists Cu uptake, export, and intracellular compartmentalization, and much of these chaperones have been learned from studies on isolated proteins, knockout or mutant mice, and cell culture systems. The expression of genes encoding these proteins is guarded by certain transcriptional regulators known as metalloregulatory proteins (Fry et al. 2013;Prohaska and Gybina 2004;Reyes-Caballero et al. 2011).
Copper has a potent cardiovascular health relevance because of its incorporation as a cofactor in antioxidant superoxide dismutase, lysyl oxidase, and mitochondrial cytochrome c-oxidase. The requirement of Cu in the cardiac muscles is relatively high to maintain high energy requirement which is necessary for continuous muscle contraction, antioxidant function, and other critical functions (Fukai et al. 2018;Kim et al. 2010). Studies revealed that CuD could be a leading cause of adverse cardiac events including ischemic and hypertrophic disorders (DiNicolantonio et al. 2018;Li et al. 2018;Liu et al. 2018). At the molecular basis, CuD alters the myocardial gene expression including inflammatory cytokines as well as other factors which are involved in contractility, calcium cycling, fibrosis as well as ATP synthesis in the cardiac tissues (DiNicolantonio et al. 2018). Thus, during heart failure, the low level of left ventricular Cu concentration could aggravate the development of cardiac dysfunction due to extensive changes in myofibrillar and mitochondrial morphology and distribution (Zhang et al. 2014).
Ruminants have a well-adapted mechanism to accommodate dietary Cu restriction via reducing its biliary excretion (López-Alonso and Miranda 2020). This mechanism is important to alleviate the effect of hypocuprosis; however, these animals become more vulnerable to hepatic overloading when excess Cu is available, particularly in cattle and sheep (Suttle 2012). On the contrary, goats are more tolerant to higher dietary Cu concentrations than sheep without any signs of Cu toxicity (Huang et al. 2013) owing to the lower hepatic Cu uptake (Solaiman et al. 2001;Zervas et al. 1990), but this also notoriously increases the goat's susceptibility to clinical hypocuprosis (Ivan et al. 1990;Silva et al. 2014). Various clinical disorders including skin and coat abnormalities, anemia, reduced animal productivity, and locomotor disturbances are repeatedly observed in small ruminants with CuD (Smith 2014;Vázquez-Armijo et al. 2011). Despite clinical studies focusing on cardiac damage in ruminants due to CuD are limited, cardiac ultrastructural and biochemical alterations in calves (Olivares et al. 2019) as well as disturbances in heart measurements during CuD in goats (Mandour et al. 2021) have been recently studied.
Characterization of Cu metabolism and homeostasis in mice (Lee et al. 2000), humans (Lee et al. 2002), and cattle (Hepburn et al. 2009) have been established. However, little is known about the selective genetic basis of Cu chaperones in the ruminant's heart. To our knowledge, there is no study exploring the relationship between cardiac measurements and the expression of Cu chaperone encoding genes in any farm animals. Therefore, the current experiment aimed to investigate the changes in Cu-Ch mRNA expression in CuD-induced cardiac damage and highlight the relationship between the expression level and cardiac measurements as well as the histopathological scoring in goats.

Animals and experimental design
The experimental procedures were followed by the recommendations of the Animal Experimental Committee held in the Tokyo University of Agriculture and Technology, Japan . Eleven male Shiba goats, 2-3 years old and weight 45 ± 5 kg were included. All animals were healthy upon clinical examination and hematobiochemical profile. Goats received alfalfa hay cubes (Edinburg, USA) as a basal diet twice per day to meet the nutritional needs for maintenance throughout the experiment (NRC 2007) which provides 11.0, 0.5, and 0.5 mg/kg DM of copper, molybdenum, and sulfur, respectively. Chemical analysis of the diet was performed using standard methods and illustrated in the supplementary table (1). After adequate dietary adaptation to individual feeding, goats were divided into two groups; the control group (n = 4 goats) which received the normal diet, and the CuD group (n = 7) in which Cu-reducing compounds ammonium sulfate (3 gm/kg DM) and sodium molybdate (40 mg/kg DM) were added to the diet (Moeini et al. 2008) until ensuring clinical copper deficiency after 7 months. Heparinized jugular venous blood samples were collected by disposable vacutainers (Venoject II, Terumo, Tokyo, Japan) and used for hematobiochemical analysis. The hematological analysis using automatic cell counter (Nihon Kohden Celltac Alpha MEK-6400, Tokyo, Japan), plasma trace elements concentration (copper, iron and molybdenum) measured by inductively coupled plasma mass spectrometry (ICP-MS) using dry ashing technique (Mandour et al. 2020a), and the activity of plasma enzymes including superoxide dismutase (SOD), ceruloplasmin (CP), aspartate transaminase (AST), creatine phosphokinase (CPK), lactate dehydrogenase(LDH) measured by IDEXX vet test (IDEXX Vet test 8008®, 111 Inc., Westbrook, Maine, U.S.A) were evaluated.
CuD was confirmed clinically based on the observed deteriorative changes of the skin and hair coat in all goats from the treated group ( Supplementary Fig. 1) in addition to anemia, reduced plasma copper level, increased plasma molybdenum concentrations, and reduced superoxide dismutase and ceruloplasmin activities. The increased CPK, AST, and LDH activities suspect cardiac involvement ( Table 1).

Assessment of cardiac functions
The electrocardiography (ECG) from the base-apex lead was carried out using Cardisuny (Cardisuny, M-E 8000 BP, Fukuda, Japan). The ECG-derived data includes wave durations measured as milliseconds (ms), wave voltage expressed as millivolt (mv), as well as cardiac intervals (PR, RR, ST, QT) derived from the standard ECG waves (P, QRS, and T) according to Koether et al. (2016). In standing position, right side two-dimensional (2D) and short-axis M-mode echocardiography were performed using the ultrasound machine (EUB-7500, Hitachi Medical Corporation, Tokyo, Japan) equipped with a sector probe (3-7 MHz; Model EUP-L65; Hitachi Medical Corporation, Tokyo, Japan). A detailed description of the preparation and recording procedures was previously described (Mandour et al. 2020b). All measurements were carried out according to Boon (2011). The left atrial area in systole (LAAs) was obtained from 2D in longaxis view. The interventricular septum diameters in diastole and systole (IVSd, IVSs), left ventricular free wall diameters in diastole and systole (LVPWd, LVPWs), left ventricular internal diameter in diastole and systole (LVIDd, LVIDs), stroke volume (SV), cardiac output (CO), and right ventricular internal-diastolic diameter (RVIDd) were obtained by M-mode from the short-axis view at the papillary muscle level. The left ventricular end-diastolic volume (EDV) and end-systolic volume (ESV) were calculated using Teichholz's formula as follow (Boon 2011): left ventricular ejection fraction (EF) and Fractional shortening (FS) were calculated as follow: The aortic root diameter (Ao), left atrium diameter in late systole (LADs), left atrial diameter: Aortic diameter ratio (LA/Ao), and pulmonary artery diameter (PA) were obtained by 2D-mode from the short-axis view at the level of the aorta and main pulmonary artery. The mean of the measured variables was obtained from 5 repeated heart cycles.
In our previous report we evaluated the longitudinal changes in cardiac function parameters during CuD in goats by ECG and echocardiography at three different times interval; once at the baseline, and twice (at 5 months and 7 months) after the existence of CuD (Mandour et al. 2021); however, in this study, the echocardiography and electrocardiography were done in the two groups twice before euthanasia and the data were averaged for accurate correlation with the gene expressions.  Mean ± SD of hematological and biochemical parameters in the investigated groups after 7 months of the dietary regime to confirm the existence of copper deficiency (P < 0.05). Hb, hemoglobin; RBCs, red blood cell count; PCV, packed cell volume; WBCs, leukocytic count; Plat, platelet count; Cu, copper; Fe, iron; Mo, molybdenum; SOD, superoxide dismutase; CP, ceruloplasmin; AST, aspartate aminotransferase; CPK, creatine phosphokinase; LDH, lactate dehydrogenase. The reference range was provided by Smith (2014) Variables Unit Control (n = 4) CuD (n = 7) P value Reference range Hb mg/dl 12.2 ± 0.1 9.9 ± 0.8 0.033 * 8.0-12.0 RBCs 10 6 12.5 ± 0.5 8.

Euthanasia, sample collection and histopathology
At the end of the 7 th months, all goats were euthanized using a combination of xylazine premedication (0.05 mg/Kg BW, xylazine hydrochloride, Fujita-Pharm, Japan), followed by pentobarbital (30 mg/kg, pentobarbital, Somuno Pentil injection®, Kyoritsu Seiyaku Corporation, Tokyo, Japan). For histopathology, formalin-preserved specimens that were collected from the left ventricular free wall were dehydrated by ethanol and xylene, blocked in paraffin wax, sectioned at 4 μm, and underwent H&E staining. A semi-quantitative histopathologic scoring was carried out blindly depending on the level of tissue destruction, the intensity of inflammatory cells, fibrosis, and loss of striations (Suvarna et al. 2018). Tissue specimens from different parts of the heart were collected, washed with phosphate buffer saline, and properly kept at -80 C° for further analysis.

Analysis of cardiac tissue trace elements
Cardiac tissue samples from each animal were collected from the LV for analysis of Cu, iron (Fe), molybdenum (Mo) and zinc (Zn) concentrations. Specimens were finely chopped, dried overnight at 100 C° in a hot air oven, finely grounded by mortar and pestle, and 100 mg of the mixture was digested with concentrated nitric acid (Suzuki et al. 2007). After filtration, the resultant solution was diluted with milli-Q water and analyzed by ICP-MS using the previously mentioned procedures (Mandour et al. 2020a). ICP-MS setting conditions were illustrated in the supplementary table (2).

Quantitative mRNA expression
Cardiac tissue samples were finely minced, and 0.1 mg of the tissues were used for the total RNA extraction by Isogen II reagent (Nippon gene, Tokyo, Japan) according to the manufacturer's instructions. The RNA was normalized at 300 ng/ul using Tris EDTA (TE). Complementary cDNA was created using PrimeScript 1 st cDNA kit (Takara Bio Inc., Otsu, Japan). The mRNA expression of six Cu chaperone encoding genes (CTr1, LOX1, COX17, CCS, ATP7A, ATP7B), antioxidant encoding genes (glutathione peroxidase 1, GPX1; superoxide dismutase 1, SOD1), ceruloplasmin (CP), matrix metalloproteinase inhibitor 1 (MMPI1) for cardiac damage and cardiac troponin I3 (TNNI3) for myocardial protein marker were examined. The oligonucleotide primers were designed using web-based Primer3 software (http:// prime r3. wi.m 119 it.edu/). Details of the target genes and sequences  Table 2. Conventional PCR and agarose gel electrophoresis were carried out to identify the expression specificity of the target mRNA with the used primer sequences. For real-time PCR, the PCR reactions were carried out in a 10 μl volume using Ex TaqR Hot Start version containing SYBR-Green I (Takara, Kusatsu, Shiga, Japan) with a real-time PCR system (Applied biosystem, Foster City, CA) using the following conditions: 95 C ○ for 30 s, followed by 40 cycles of 95 C ○ for 5 s, 60 C ○ for 30 s and then a dissociation protocol. The relative expression level of each target mRNA was calculated using the 2-ΔΔ CT method in which all values were normalized to the housekeeping gene β-actin.

Statistical analysis
All data were analyzed through unpaired t-test for nonparametric data using Mann Whitney test with a probability of P < 0.05 using Graphpad prism 6 (GraphPad Software, San Diego, CA, USA). The data are expressed as Mean ± SD. Multiple Person's correlation analysis was done between the relative mRNA expressions in the cardiac tissue and element concentration (Cu, Mo), cardiac parameters, and histopathological scoring.

Changes in cardiac function parameters
The obtained echocardiography from the right parasternal long-axis and short-axis views are illustrated in Fig. 1. Table 3 Figure 3 summarizes the results of SOD1, CP, GPX1, MMP1 and TNNI3 mRNA expressions in the heart. The data showed that CP, GPX1, MMPI1, and TNNI3 expressions were significantly reduced (P = 0.0290, 0.0136, 0.0189 and 0.0189), respectively in the CuD group compared with the control, whilst SOD1 was reduced but not statistically significant (P = 0.3065). The reference range of the measured parameters obtained by ECG from the B-A lead (Sudo et al. 1979) and the echocardiographic variables recorded by the 2D and M-mode (Leroux et al. 2012) were provided.
IVSd interventricular septum diameter in diastole, LVIDd left ventricular (LV) internal diameter in diastole, LVFWd LV free wall diameter in diastole, IVSs interventricular septum diameter in systole, LVIDs LV internal diameter in systole, LVFWs LV free wall diameter in systole, EDV end-diastolic volume of LV, ESV end-systolic volume of LV, EF ejection fraction, FS fraction shortening, LADs left atrial diameter in systole, AoD aortic diameter, LA/Ao the ratio between left atrium to aortic diameters, LAAs left atrial area in systole, PA pulmonary artery diameter * P < 0.05, ** P < 0.01

Effect of cardiac copper and molybdenum concentrations on the investigated mRNA expressions
In the CuD group, the level of cardiac tissue Cu was reduced compared with the control group (P < 0.05); meanwhile, Mo concentration was significantly increased (P < 0.01). Cardiac zinc and iron concentration showed no change (Supplementary table 3). Pearson's correlation between cardiac Cu and Mo concentrations and the mRNA expression level of the targeted genes are summarized in Table 4. The obtained results revealed no significant correlation between cardiac Cu concentration and encoding gene expressions (P > 0.05). Table 5A illustrates the correlation between ECG parameters and mRNA expression in cardiac tissues. SOD1 showed a significantly strong negative correlation with P-wave time (r = -0.83, P = 0.042). Besides, GPX1 showed a significantly strong negative correlation with ST-segment (r = -0.86, P = 0.036). Cardiac CP and COX17 were strongly and positively correlated with both T-wave time (r = 0.82, 0.87; P < 0.05) and T-wave voltage (r = 0.90, 0.83; P < 0.05), respectively; meanwhile, the TNNI3 showed a significant strong positive correlation with T-wave duration (r = 0.86, P = 0.032).    LOX expression (r = 0.88, P = 0.024) were reported. Also, the RVIDd showed a significant strong positive correlation with CTr1 (r = -0.84, P = 0.041). ATP7A showed an inverse correlation with LVIDd, LVIDs, EDV, and ESV.

Correlation with the histopathological score
Unlike the histopathological findings obtained from cardiac tissue sections upon H&E staining from the control goats that showed normal histological architecture with longitudinally striated branching, nucleated, and anastomosing myocardial fibers (Fig. 4a), the CuD group showed cardiac tissue damage detected as myocardial degeneration and congestion of the intermuscular tissue. The intercellular spaces were increased due to collagen deposition between the myocardial cells. Also, cardiomyocyte degeneration, focal coagulative necrosis manifested as pallor of the myocardium, and loss of myocardial striations with extensive lymphocytic cell infiltrations were detected. Fibrous tissue deposition and atrophied myofibrils were also observed in the CuD group (Fig. 4b, c, d, e, f). The overall score of cardiac tissue damage in the CuD group was significantly higher than the control group (2.78 ± 0.32 vs 0.8 ± 0.2 respectively; P = 0.011). There was a significant negative correlation between histopathological score and mRNA expression of ATP7B (r = -0.735, P = 0.038), CTr1 (r = -0.765, P = 0.027), LOX (r = -0.744, P = 0.034), COX17 (r = -0.829, P = 0.11), and TNN13 (r = -0.756, P = 0.030), while MMPI1 was trend (r = -0.688, P = 0.059). CCS, ATP7A, SOD, CP, and GPX showed no significant correlation with the histopathological score.

Discussion
Copper deficiency is a grave nutritional disorder not only because it is widely spread in ruminants but also due to its associated secondary multi-organ disorders. The problem constitutes a nutritional challenge in grazing ruminants particularly, but not limited to, tropical regions where the soil is deficient in Cu or excessive Cu antagonisms are found with high levels (López-Alonso and Miranda 2020). In the current study, we conducted a dietary Cu restriction protocol to reduce Cu availability by creating a chemical reaction between sulfur and molybdenum in the ruminal fluid (Gould and Kendall 2011;Scheiber et al. 2013). We aimed to highlight, for the first time, the disruption in Cu-Ch mRNA expression in the cardiac tissues in Shiba goats, a native Japanese non-seasonal breeding breed that is considered to be a good model for studying the blood flow and ruminant animal physiology (Mandour et al. 2020a;Samir et al. 2020), to find out whether CuD is correlated with cardiac measurements and histopathological scoring.
In the current work, the ECG has been evaluated from the base apex lead only. This is a common and suitable lead to investigate arrhythmias in farm animals since it provides easily detectable waves and minimizes movement-related erroneous measurements (Fakour et al. 2013;Mousavi et al. 2007;Radostits et al. 2006;Rezakhani et al. 2004). Besides, the echocardiography has been evaluated only from the right side and without Doppler evaluation of the flow tracts because of the difficulty to obtain Doppler alignment in the standing position. This has been previously attributed to the presence of gases-filled rumen (Leroux et al. 2012) in addition to the large-sized goats included in this study compared with goats used in another report (Mandour et al. 2020b).
Previously, we investigated the longitudinal disruption of cardiac measurements in experimentally-induced CuD in goats throughout different time intervals (Mandour et al. 2021); however, in this study, we measured the cardiac parameters after confirming CuD-associated cardiac involvement to correlate the data with Cu-Ch mRNA expressions. The treatment group showed dilated LV which was indicated by increased QRS time, LV internal diameter in diastole, LV end-diastolic volume, stroke volume and cardiac output, in addition to increased left atrium size. These changes have been attributed to increasing preload, which induced cardiac remodeling as dilatation to accommodate the increased blood volume (Turner et al. 2002;Yamaya et al. 1997). The non-significant decline in EF and FS indicated that the CuDassociated cardiac damage was not severe enough to induce cardiomyopathy and systolic dysfunction in goats. This finding enforces that silent change in cardiac function occurs in goats that could tolerate the adverse effect of CuD without cardiovascular symptoms. In contrast, dramatic cardiac Fig. 4 H&E staining of histopathological examination of the cardiac tissues from goats in the control showed the normal cardiac architecture and striation (a), and cardiac tissue damage in goat from the copper deficiency (CuD) group (b,c,d,e,f). b, cardiac muscles degeneration (arrowhead) and congestion of intermuscular tissue (arrow); c, focal intermuscular edema (stars) with inflammatory cells and degeneration (arrows, X 400); d, myocardial degeneration and necrosis (arrow) with extensive lymphocytic infiltrations (arrowhead, X 400); e, intermuscular edema and increased intermuscular spaces (stars) and myocardial atrophy (arrow, X 400); f, extensive fibrosis and collagen deposition (stars) and destruction of the surrounding myocardium (arrows, X 400) tissue damage in copper-deficient goats was detected that could be explained by the increased oxidative damage secondary to a reduction in Cu level that in turn leads to lower SOD, CP and GPX expression (McDowell 2003;Olivares et al. 2019) and promoting ischemic changes in the cardiac tissue (DiNicolantonio et al. 2018). Copper is known to comprise copious cardiovascular-relevant functions to withstand the high-energy demand necessary for maintaining continuous heart contractility. Therefore, during CuD, there is a deprivation of Cu and improper activity of Cudependent metalloenzymes which further influence collagen cross-linkage, energy production, and antioxidant defense mechanism (Smith 2014;Vázquez-Armijo et al. 2011).
Copper homeostasis within the cell is mediated by Cu-Ch proteins to ensure adequate Cu absorption and intracellular distribution (Fry et al. 2013;Prohaska & Gybina 2004). Our results showed significant down-regulation in the relative expression of CP, ATP7A, CTr1, LOX and COX17 in cardiac tissues of the CuD group compared with the control; meanwhile, SOD1, CCS and ATP7B were not significantly reduced. There is a shortage of data in ruminants regarding the effect of CuD on cardiac Cu regulation genes. Nevertheless, our results were consistent with previously reported data in other species (Dermauw et al. 2014;Getz et al. 2011;Han et al. 2009Han et al. , 2012Hepburn et al. 2009). Simmental cattle, a highly susceptible breed to CuD, showed reduced mRNA expression of COX17, CTr1 and ATP7A in duodenal tissue, placenta, and liver compared with the nondeficient breed (Fry 2011;Fry et al. 2013). Also, hepatic Cu-regulating genes in Cu-deficient cross-breed cattle were less than the non-deficient Zebu cattle (Dermauw et al. 2014). Furthermore, Cardiac COX1 and COX4 proteins were down-regulated in Cu-deficient rats (Getz et al. 2011). Despite the above-mentioned studies were focused on cattle and rats, their data indicate that CuD down-regulates the Cu-regulating genes in duodenal and hepatic tissues. Thereafter, the same effect could exist in the goat's heart. Rather than Cu-Ch mRNA encoding genes, in the present study, the GPX1, MMPI1 and TNNI3 mRNA expressions were significantly reduced in cardiac tissues of Cu-deficient goats. Experimental CuD in cattle reduces hepatic GPX1 in which selenium level was also reduced (Dermauw et al. 2014).
The expression level of inflammatory cytokines in addition to fibrosis regulating factors was previously linked to CuD-related cardiac damage in laboratory animals (DiNicolantonio et al. 2018). In the current study, upon histopathological examination, we observed cardiac tissue damage and fibrosis as well as increased histopathological scoring in CuD goats. These findings revealed cardiac tissue damage and suggest impaired microcirculation and oxidative myocardial damage. This could be explained by the combination of reduced Cu concentration, together with reduced SOD1, CP and GPX expression in the cardiac tissues. Also, for further illustration, Gunja-Smith et al. (1996) suggested that the down-regulation of MMPI1 results in the accumulation of weak cross-linked collagen fibers and cardiac dilatation. The cross-linkage of the collagen is the function of the Cu-dependent enzyme lysyl oxidase (López et al. 2010) which is regulated by LOX chaperone that was reduced in the current study. Therefore, it can be stated that down-regulation of LOX and MMPI1 in CuD aggravates cardiac tissue damage by deposition of abnormal extracellular matrix. Furthermore, TNNI3, a gene encoding cardiac sarcomere troponin I protein, was examined as a regulating gene for cardiac proteins necessary for the proper assembly of the cardiomyocytes. Changes in TNNI3 are a well-known cause of cardiomyopathy (Mirza et al. 2005;Rai et al. 2009) and its expression showed down-regulation in cardiac muscles of type2 diabetes (Howarth et al. 2011) in which Cu plays an important role in its development due to defective myocellular Cu regulation (Zhang et al. 2014(Zhang et al. , 2020. In our study, the correlation between Cu concentration and the investigated genes in the cardiac tissues revealed no significant correlation. A variable correlation between pulmonary artery Cu concentration and mRNA expression of Cu regulating genes was also observed in goats, cattle, and pigs (Dermauw et al. 2014;Han et al. 2012) in which the authors suggested that these genes are regulated differently among species.
To the best of our knowledge, this is the first study to demonstrate the correlation between Cu-regulating genes as well as CP, SOD1, GPX1, TNNI3 and MMPI1 in the cardiac tissue and cardiac parameters in goats. Even though our findings indicated variable correlations with heart function measurements, the observed significant correlation of certain mRNA encoding gene expression and specific cardiac measurements enforced our observation of impacted cardiac functions under CuD due to disturbances at the molecular level. This finding was further confirmed by the significant negative correlation of the investigated genes and the histopathological score.
Some limitations in the current study should be considered. It was difficult to assess the left apical four-chamber view and therefore the Doppler assessment did not carry out. The low population in the present study is another limitation. The present study did not evaluate the effect of different levels of Cu status and the response of cardiac tissue to copper supplements. However, our findings warranted further study in the same area of specialization to prove the direct link between copper chaperone encoding gene expression and cardiac dysfunction caused by CuD in ruminants.

Conclusion
In this study, CuD significantly down-regulated the cardiac tissue mRNA expressions of copper chaperones in addition to SOD1, CP, GPX1, MMPI1 and TNNI3 and some of them were negatively correlated with the degree of cardiac tissue damage. Disturbances at the molecular level in CuD aggravate heart tissue damage and induce tissue remodeling in goats even though the cardiovascular symptoms were not clear. The usefulness of Cu chaperones as biomarkers in clinically undetectable CuD-related heart damage in ruminants warranted further studies.