N-Acetylcysteine, an ROS Inhibitor, Alleviates the Pathophysiology of Hyperthyroidism-Induced Cardiomyopathy via the ROS/Ca2+ Pathway

Hyperthyroidism is common and can induce cardiomyopathy, but there is no effective therapeutic strategy. The purpose of this study was to investigate the molecular mechanism of hyperthyroidism-induced cardiomyopathy (HTC) and the effect of N-acetylcysteine (NAC), an ROS inhibitor, on the pathophysiology of HTC in vivo and in vitro. Compared with those in the control groups in vivo and in vitro, TT3 and TT4 were significantly increased, the structure of myocardial cells was enlarged and disordered, and interstitial fibrosis and the apoptosis of myocardial cells were markedly increased in the L-Thy group. The ROS and inflammatory response were increased in the hyperthyroidism group. In the NAC group, the contents of TT3 and TT4 were decreased, the myocardial cell structure was slightly disturbed, fibrosis and apoptosis were significantly reduced, and the ROS level and inflammatory response were significantly reduced. Interestingly, L-Thy decreased the viability of fibroblasts and H9c2 cells, suggesting that L-Thy-induced fibrosis was not caused by the proliferation of fibroblasts. The molecular mechanism of HTC could be explained by the fact that L-Thy could cause cardiac hypertrophy, inflammation, and fibrosis by regulating the Ca2+/calpain/Rcan1-dependent signalling pathway, the Ca2+/Rcan1/NF-κB/p65-dependent signalling pathway, and the Ca2+/ROS/Bcl-2/caspase-3-dependent signalling pathway. In conclusion, NAC can alleviate the pathophysiology of hyperthyroidism-induced cardiomyopathy, probably by regulating the ROS/Ca2+-dependent pathway.


Introduction
Hyperthyroidism refers to the persistently high-functioning state of the thyroid gland, which synthesises and releases too much thyroxine, thus resulting in clinical symptoms such as thyrotoxicosis [1]. It has been reported that thyroxine can exert several effects on the cardiovascular system and influence arrhythmias, cardiac hypertrophy, and congestive heart failure [1,2]. Cardiomyopathy triggered by hyperthyroidism is called hyperthyroidisminduced cardiomyopathy (HTC), and cardiac hypertrophy is the most documented cardiomyopathy following hyperthyroidism in experimental animals and clinical practice [3,4]. Thyroid hormone-induced cardiac hypertrophy is described as a relative ventricular hypertrophy encompassing the whole heart and is linked with contractile abnormalities in both the right and left ventricles.

High L-Thy Induced the Occurrence of Cardiac Structural and Functional Changes in Mice
First, mice were treated with L-thyroxine (L-Thy) by intraperitoneal injection daily for 4 weeks (Material and Methods) [13,14]. From the second week, L-Thy-induced mice were lighter than controls (23.55 ± 0.29 vs. 25.04 ± 0.48 g, p = 0.0076), and the difference in weight of both groups increased at the 4th week (24.25 ± 0.28 vs. 27.29 ± 0.54 g, p < 0.0001, Figure 1A). Second, after 4 weeks of induction with L-Thy, the RR and PR interval times were both decreased in the L-Thy-treated mouse group, while the QT interval time was increased in the L-Thy-treated mouse group ( Figure 1B,C). The serum levels of TT3 and TT4 indicated that the levels of TT3 and TT4 in the L-Thy group were significantly higher than those in the control group at the 2nd and 4th weeks ( Figure 1D). Meanwhile, the heart weight was significantly higher in the L-Thy group than in the control group ( Figure 1E). Additionally, L-Thy-treated mice showed a significant increase in the left ventricular hypertrophy parameters, and LVAW and LVPW coincided with a significant reduction in the LV cardiac function markers, EF, and FS as compared to controls ( Figure 1F).
At the same time, HE and WGA staining revealed that the L-Thy group had myocardial hypertrophy (Figure 2A,B). Moreover, Masson staining revealed that the L-Thy group had obvious fibrosis ( Figure 2C), and TUNEL staining revealed that the L-Thy group had a higher cardiomyocyte apoptosis percentage ( Figure 2D). Together, L-Thy could induce cardiomyopathy that was characterised by cardiac structural and functional changes, to meet mechanistic research needs.  At the same time, HE and WGA staining revealed that the L-Thy group had myocardial hypertrophy (Figure 2A,B). Moreover, Masson staining revealed that the L-Thy group had obvious fibrosis ( Figure 2C), and TUNEL staining revealed that the L-Thy group had a higher cardiomyocyte apoptosis percentage ( Figure 2D). Together, L-Thy could induce cardiomyopathy that was characterised by cardiac structural and functional changes, to meet mechanistic research needs.

Increased ROS and Inflammatory Gene Expression Was Higher in the Hyperthyroidism-Induced Cardiomyopathy Group
It was reported that L-Thy increases the oxidative stress response, leading to a weakened antioxidative capacity of the myocardial cells and accelerated cell damage [3]. In this study, quantitative real time-polymerase chain reaction (qRT-PCR) and Western blot (WB) showed that Sod1 and Sod2 mRNA expression was lower in the L-Thy group, suggesting that ROS expression was higher in the L-Thy group ( Figure 3A and Figure S1). In addition, it has been reported that an inflammatory response occurs in hyperthyroidism-induced cardiomyopathy. In our study, we detected the expression levels of Tnf-α, Il-1β, and Il-6 in myocardial tissues, which are key indicators of the proinflammatory response. The results showed that compared with those in the control group, the mRNA expression levels of Tnf-α, Il-1β, and Il-6 in the hyperthyroidism group were significantly increased compared with those in the controls ( Figure 3B and Figure S1). This suggested that L-Thy would induce the cardiac inflammatory response and oxygen free radical production. study, quantitative real time-polymerase chain reaction (qRT-PCR) and Western blot (WB) showed that Sod1 and Sod2 mRNA expression was lower in the L-Thy group, suggesting that ROS expression was higher in the L-Thy group ( Figures 3A and S1). In addition, it has been reported that an inflammatory response occurs in hyperthyroidism-induced cardiomyopathy. In our study, we detected the expression levels of Tnf-α, Il-1β, and Il-6 in myocardial tissues, which are key indicators of the proinflammatory response. The results showed that compared with those in the control group, the mRNA expression levels of Tnf-α, Il-1β, and Il-6 in the hyperthyroidism group were significantly increased compared with those in the controls ( Figures 3B and S1). This suggested that L-Thy would induce the cardiac inflammatory response and oxygen free radical production.

Hyperthyroidism-Induced Cardiomyopathy Cell Model for Mechanistic Investigation
To further elucidate the mechanism, we built a cell model. The H9c2 cell line was also treated with 2 μM L-Thy [13], and we found that L-Thy decreased the viability of H9c2 cells, as detected by the CCK-8 assay ( Figure 4A). To explain why fibrosis increases in L-Thy-induced cardiomyopathy ( Figure 2C), we also treated the rat cardiac fibroblasts (RCF) with L-Thy. The results showed that L-Thy also decreased the cell viability of fibroblasts, indicating that fibrosis was not caused by the proliferation of fibroblasts treated with L-Thy in L-Thy-induced cardiomyopathy ( Figure 4B). To verify the apoptosis and

Hyperthyroidism-Induced Cardiomyopathy Cell Model for Mechanistic Investigation
To further elucidate the mechanism, we built a cell model. The H9c2 cell line was also treated with 2 µM L-Thy [13], and we found that L-Thy decreased the viability of H9c2 cells, as detected by the CCK-8 assay ( Figure 4A). To explain why fibrosis increases in L-Thy-induced cardiomyopathy ( Figure 2C), we also treated the rat cardiac fibroblasts (RCF) with L-Thy. The results showed that L-Thy also decreased the cell viability of fibroblasts, indicating that fibrosis was not caused by the proliferation of fibroblasts treated with L-Thy in L-Thy-induced cardiomyopathy ( Figure 4B). To verify the apoptosis and necrosis of H9c2 cells, flow cytometry was used to detect that the necrosis and apoptosis percentages of the H9c2 cell line were higher in the L-Thy group than in the control group ( Figure 4C). In addition, the immunofluorescence of α-actin results suggested that the cell size of L-Thy-treated H9c2 cells was larger than that of control cells ( Figure 4D).
Biomolecules 2022, 12, x FOR PEER REVIEW 6 of 20 necrosis of H9c2 cells, flow cytometry was used to detect that the necrosis and apoptosis percentages of the H9c2 cell line were higher in the L-Thy group than in the control group ( Figure 4C). In addition, the immunofluorescence of α-actin results suggested that the cell size of L-Thy-treated H9c2 cells was larger than that of control cells ( Figure 4D).

ROS/Ca 2+ Pathway and Inflammatory Gene Expression in a Hyperthyroidism-Induced Cardiomyopathy Cell Model, Consistent with the Performance of the Animal Model
Furthermore, the ROS level was detected by flow cytometry, which showed that the ROS level in the L-Thy group was higher than that in the control ( Figure 5A), and this was consistent with the animal model ( Figure 3A). Similarly, Sod1 and Sod2 mRNA expression was lower in the L-Thy group ( Figure 5B). Due to the relationship of ROS and Ca 2+ in cardiovascular disease, we detected Ca 2+ by immunofluorescence of Rhod-4, which suggested that Ca 2+ increased in the L-Thy group ( Figure 5C). At same time, the cell model also indicated that L-Thy could induce a cardiac inflammatory response in which Il-1β, Tnf-α, and Il-6 mRNA expressions were increased in the L-Thy-treated H9c2 cell line ( Figure 5D). Together, cell lines and animal models suggested that the ROS/Ca 2+ pathway contributed to L-Thy-induced cardiomyopathy.
Furthermore, the ROS level was detected by flow cytometry, which showed that the ROS level in the L-Thy group was higher than that in the control ( Figure 5A), and this was consistent with the animal model ( Figure 3A). Similarly, Sod1 and Sod2 mRNA expression was lower in the L-Thy group ( Figure 5B). Due to the relationship of ROS and Ca 2+ in cardiovascular disease, we detected Ca 2+ by immunofluorescence of Rhod-4, which suggested that Ca 2+ increased in the L-Thy group ( Figure 5C). At same time, the cell model also indicated that L-Thy could induce a cardiac inflammatory response in which Il-1β, Tnf-α, and Il-6 mRNA expressions were increased in the L-Thy-treated H9c2 cell line (Figure 5D). Together, cell lines and animal models suggested that the ROS/Ca 2+ pathway contributed to L-Thy-induced cardiomyopathy.

NAC Could Relieve L-Thy-Induced Myocardial Hypertrophy and the Cardiac ROS/Ca 2+ Pathway
To verify the role of the ROS/Ca 2+ pathway in L-Thy-induced cardiomyopathy, we used a small molecular ROS inhibitor, NAC [15], to treat the L-Thy-treated H9c2 cell line ( Figure 6A). First, we evaluated different concentrations to identify the most appropriate one, finding that there was a dose-response effect at first, but later, when the concentration of NAC increased from 6 mM to 8 mM, there was no significant change in cell viability (p = 0.4451) ( Figure 6B). Then, we compared the time of giving treatment before disease and after disease. We found that the effect of treatment was better than prevention at a concentration of 6 mM (p = 0.0138) ( Figure 6C). Furthermore, NAC could relieve myocardial necrosis and the apoptosis percentage ( Figure 6D,E). According to the immunofluorescence of α-actin, NAC significantly relieved L-Thy-induced myocardial hypertrophy ( Figure 6F,G). NAC, an ROS inhibitor, could relieve L-Thy-induced myocardial hypertrophy in the H9c2 cell line. Furthermore, we detected the expression of ROS/Ca 2+ pathway components during NAC treatment of L-Thy-induced myocardial changes. ROS levels were decreased, while Sod1 and Sod2 mRNA expression were increased in the NAC-treated L-Thy group ( Figure 7A,B). The immunofluorescence results showed that the ROS/Ca 2+ pathway could be inhibited by NAC in L-Thy-induced myocardial hypertrophy ( Figure 7C-E). Moreover, the inflammatory response was also inhibited, which indicated that the L-Thy-induced cardiac inflammatory response was regulated by the ROS/Ca 2+ pathway ( Figure 7F). Together, NAC could relieve L-Thy-induced myocardial hypertrophy and inflammation via the ROS/Ca 2+ pathway in vitro.  The intracellular ROS and calcium contents were expressed by the fluorescence intensity of DCFH-DA and Rhod-4, respectively. Fluorescence intensity was quantified by the ImageJ plugin 3D Object Counter [16,17]. (F) The mRNA levels of Il-6, Il-1, and Tnf-α were measured by qRT-PCR. */# p < 0.05, **/## p < 0.01, ***/### p < 0.001, and ****/#### p < 0.0001.

NAC Attenuated the Phenotype of Hyperthyroidism-Induced Cardiomyopathy In Vi
According to in vitro experiments, NAC relieved L-Thy-induced myocardia physiological changes via the ROS/Ca 2+ pathway. Furthermore, NAC was applied L-Thy-induced cardiomyopathy to investigate whether inhibition of the ROS/Ca way could attenuate the phenotype of hyperthyroidism-induced cardiomyopath 1 week of intraperitoneal injection of NAC in the L-Thy-induced cardiomyopathy we found that NAC could attenuate the loss of body weight, ratio of HW to B content of TT3 and TT4 ( Figure 9A,B). In addition, after NAC treatment, the my hypertrophy of diseased mice decreased, and cardiac function improved comp

NAC Attenuated the Phenotype of Hyperthyroidism-Induced Cardiomyopathy In Vivo
According to in vitro experiments, NAC relieved L-Thy-induced myocardial pathophysiological changes via the ROS/Ca 2+ pathway. Furthermore, NAC was applied to treat L-Thy-induced cardiomyopathy to investigate whether inhibition of the ROS/Ca 2+ pathway could attenuate the phenotype of hyperthyroidism-induced cardiomyopathy. After 1 week of intraperitoneal injection of NAC in the L-Thy-induced cardiomyopathy model, we found that NAC could attenuate the loss of body weight, ratio of HW to BW, and content of TT3 and TT4 ( Figure 9A,B). In addition, after NAC treatment, the myocardial hypertrophy of diseased mice decreased, and cardiac function improved compared to before treatment ( Figure 9C). Moreover, NAC could attenuate myocardial hypertrophy, in which LVAW and LVPW thickness were decreased in the L-Thy + NAC group compared with the L-Thy group, and cardiac function, in which LVEF and FS increased in the L-Thy + NAC group compared with that of the L-Thy group ( Figure 9D). In addition, morphological assessment of the cardiac tissue of L-Thy + NAC-treated mice showed normal myocardial size, less fibrosis, a lower apoptosis percentage, and lower ROS levels than L-Thy-induced cardiomyopathy mice ( Figure 10). Together, inhibition of the ROS/Ca 2+ pathway was the potential target of hyperthyroidism-induced cardiomyopathy. before treatment ( Figure 9C). Moreover, NAC could attenuate myocardial hypertrophy, in which LVAW and LVPW thickness were decreased in the L-Thy + NAC group compared with the L-Thy group, and cardiac function, in which LVEF and FS increased in the L-Thy + NAC group compared with that of the L-Thy group ( Figure 9D). In addition, morphological assessment of the cardiac tissue of L-Thy + NAC-treated mice showed normal myocardial size, less fibrosis, a lower apoptosis percentage, and lower ROS levels than L-Thy-induced cardiomyopathy mice ( Figure 10). Together, inhibition of the ROS/Ca 2+ pathway was the potential target of hyperthyroidism-induced cardiomyopathy.

Discussion
The results reported in our study suggest that hyperthyroidism induced by chronic administration of L-Thy in mice and the H9c2 cell line was an independent factor that affected cardiac structure and function and then led to adverse remodelling, fibrosis, and inflammation. Meanwhile, our data suggested that these effects may be mediated by the ROS/Ca 2+ pathway. Interestingly, scavenging ROS with NAC protected against L-Thy-induced impairment in cardiac function and associated cardiac damage, by alleviating all the pro-oxidant, inflammatory, and fibrotic events. We were surprised to find that in animal experiments, NAC could not only improve the cardiac function and pathology of HTC by reducing ROS but also reduce the contents of TT3 and TT4 in serum. There are two pathways that NAC plays roles in the pathogenesis of HTC: one is that NAC reduces ROS and, thus, improves the cardiac function and pathology, and the other is that NAC reduces the content of TT3 and TT4 via the decreased level of ROS and, thus, improves

Discussion
The results reported in our study suggest that hyperthyroidism induced by chronic administration of L-Thy in mice and the H9c2 cell line was an independent factor that affected cardiac structure and function and then led to adverse remodelling, fibrosis, and inflammation. Meanwhile, our data suggested that these effects may be mediated by the ROS/Ca 2+ pathway. Interestingly, scavenging ROS with NAC protected against L-Thyinduced impairment in cardiac function and associated cardiac damage, by alleviating all the pro-oxidant, inflammatory, and fibrotic events. We were surprised to find that in animal experiments, NAC could not only improve the cardiac function and pathology of HTC by reducing ROS but also reduce the contents of TT3 and TT4 in serum. There are two pathways that NAC plays roles in the pathogenesis of HTC: one is that NAC reduces ROS and, thus, improves the cardiac function and pathology, and the other is that NAC reduces the content of TT3 and TT4 via the decreased level of ROS and, thus, improves the cardiac function and pathology of HTC. Then, in the cell model, we found that NAC could ameliorate myocardial injury by reducing ROS but not reduce TT3 and TT4 levels. ROS have been documented to promote hyperthyroidism [19], so we can speculate that NAC reduces the levels of TT3 and TT4 via the decreased level of ROS.
Administration of L-Thy by intraperitoneal injection remained the best strategy to induce hyperthyroidism to induce cardiomyopathy in rodents [13,20]. In our study, we evaluated our animal and cell models from different aspects, such as serum thyroxine levels, body weight, heart mass, cardiac structure and function, and histopathologic alterations. Combining these results, the animal and cell model simulated the process of heart disease caused by hyperthyroidism, which could be applied to investigate the molecular mechanism. As shown in Figure 11, an increase in Cav1.3 induces an increase in the cytosolic Ca 2+ concentration of H9c2 cells, thereby increasing the activation of Ca 2+ -dependent signalling, Rcan1, calcineurin, and calpain, which subsequently upregulates the gene expression of hypertrophy markers and upregulates the gene expression of inflammatory cytokines by activating the NF-κB/p65-dependent signalling pathway [21][22][23][24][25][26][27][28], causing cardiac hypertrophy and inflammation. Meanwhile, the elevation of Gal-3, RhoA, and Rock further increased ROS [27][28][29][30][31][32], subsequently regulating the Bcl-2/caspase-3-dependent apoptotic signalling pathway [33,34], thereby leading to cell rupture and fibrosis. Untreated hyperthyroidism is associated with increased cardiovascular morbidity and mortality [37]. Under this circumstance, the clinical question arises of whether to administer prophylactic drugs to treat hyperthyroidism-associated cardiac injury. We found that prophylactic administration was not beneficial for hyperthyroidism-induced In our study, ROS played critical roles in HTC, consistent with a previous report that showed that ROS were the central mechanism of hyperthyroidism-induced cardiomyopathy that was characteristic of myocardial hypertrophy, apoptosis, inflammation, and arrhythmia [20]. ROS and Ca 2+ are versatile signalling molecules that coordinate physiological and pathophysiological processes [8]. In our study, we found that the ROS/Ca 2+ pathway contributed to the pathogenesis of HTC. This result suggested that the Ca 2+ antagonist nifedipine could be applied for treating HTC patients in situations where the application value of Ca 2+ antagonists in HTC is controversial [35,36]. In the future, more clinical studies should be conducted to investigate how to use Ca 2+ antagonists to treat HTC. Besides, the detailed mechanism of L-Thy-ROS/Ca 2+ signalling pathway in HTC was not elucidated, which is the limitation of this study.
Untreated hyperthyroidism is associated with increased cardiovascular morbidity and mortality [37]. Under this circumstance, the clinical question arises of whether to administer prophylactic drugs to treat hyperthyroidism-associated cardiac injury. We found that prophylactic administration was not beneficial for hyperthyroidism-induced cardiomyopathy ( Figure 6C). Therefore, prophylactic drugs are not recommended to protect the heart in hyperthyroidism patients.

Conclusions
Our data showed that cardiac structural and functional changes were related to the ROS/Ca 2+ pathway. In addition, these data showed that thyroid hormones would induce ROS overproduction and then induce Ca 2+ release to cause myocardial hypertrophy, apoptosis, fibrosis, and inflammation, while inhibition of ROS could regulate the ROS/Ca 2+ signalling pathways to alleviate HTC.

Hyperthyroid Cardiomyopathy Model Induction
Eight-week-old male C57BL/6 mice (Vital River Laboratories, Beijing, China) were maintained in a specific pathogen-free facility and provided a normal diet and drinking water. After 1 week of adaptive feeding, the mice were intraperitoneally injected with 100 µg/kg L-Thy (Merck, Darmstadt, Germany) compatible in solution with sodium carboxymethylcellulose (C4888, Sigma, Shanghai, China) every day. The mice were examined with echocardiography and euthanised on Day 28. Meanwhile, the weight and heart rate of the mice were measured. This investigation was approved by the Animal Ethics Committee of Fuwai Hospital (FW-2022-0015).
When the cell density reached approximately 60%, H9c2 cells were incubated with 2 µM L-Thy at 37 • C for 24 h.

Experimental Grouping
These experimental animals were randomly divided into three groups: the control group, L-Thy + PBS group, and L-Thy + NAC group. The control group was intraperitoneally injected with PBS for 35 days. In the L-Thy + PBS group and L-Thy + NAC group, mice were injected intraperitoneally with L-Thy for 28 days. In the L-Thy + NAC group, after the hyperthyroid cardiomyopathy model was established, 200 mg/kg NAC (HY-B0215, MCE, Shanghai, China) was continuously injected for 7 days. In addition, the L-Thy + PBS group was given the same amount of PBS daily.
H9c2 cells in the logarithmic growth phase were plated. The control group was cultured in normal medium, and the L-Thy group was treated with 2 µM L-Thy for 24 h. In addition, the NAC group was treated with 6 mM NAC for 6 h. In the L-Thy + NAC group, H9c2 cells were previously incubated with L-Thy and then maintained in NAC-containing medium for 6 h.

Haematoxylin-Eosin (HE) and Masson Staining
After cardiac perfusion, the hearts were removed quickly from the mice, wiped softly with clean absorbent paper, and cut in half horizontally with a knife. Half of the heart tissues were taken using paraformaldehyde, and the remaining tissues were stored at −80 • C.
The fixed mouse heart tissues were cut into 4 µm thick sections, stained in accordance with the instructions of the kit, sealed with a sealing liquid, and finally observed with a pathologic scanner (Zeiss, Oberkochen, Germany).

TUNEL Assay
Paraffin-embedded heart sections were deparaffinised, rehydrated, permeated, and antigen-retrieved, in accordance with the instructions of the TUNEL assay kit (11684795910, Roche, Shanghai, China). Sections were detected by the pathologic scanner.

Cell Counting Kit-8 (CCK-8) Assay
Cells inoculated in a 96-well plate were incubated with L-Thy at different time points and then not treated or treated with NAC at different concentrations (0 mM, 2 mM, 4 mM, 6 mM, 8 mM) for the next day. Otherwise, the cells were not pretreated or pretreated with NAC, and L-Thy was added. Then, CCK-8 reagent (CCK8-500, Hanbio, Shanghai, China) was added to the cells in the dark. After incubation at 37 • C for 3 h, the plate was detected at 450 nm by a microplate reader.

Flow Cytometry
To detect ROS levels, H9c2 cells were prepared as a single-cell suspension, and DCFH-DA was added, followed by incubation at 37 • C for 30 min in the dark. Following centrifugation, the supernatant was discarded, 10% FBS was added, and the cells were incubated for 20 min. After centrifugation, the supernatant was replaced with an appropriate amount of cold PBS.
To analyse apoptosis, H9c2 cells were trypsinised, centrifuged, collected, and washed with cold PBS to concentrate to 1 × 10 5 cells/mL. A 100-ul sample solution was mixed with 5 µL of annexin V-FITC (V13242, AV-FITC, Thermo Fisher Scientific, Shanghai, China) and 5 µL of propidium iodide (V13242, PI, Thermo Fisher Scientific, Shanghai, China), then incubated for 15 min at room temperature, and finally washed with cold PBS.

Quantitative Real Time-Polymerase Chain Reaction (qRT-PCR)
Total RNA was extracted from the heart tissues stored at −80 • C and H9c2 cells in each group by TRIzol (15596018, Thermo Fisher Scientific, Shanghai, China). After quantification using a NanoDrop, 2 µg of total RNA was reverse-transcribed into complementary deoxyribose nucleic acid cDNA. The transcribed cDNA was analysed by real-time PCR with SYBR-Green dye (4472908, Thermo Fisher Scientific, Shanghai, China). The relative expression of a target gene was calculated using the 2 −∆∆ct method. The primers used are shown in Table 1.

Data Analysis
The images and flow cytometry results were analysed with ImageJ software and FlowJo software, respectively. All data are presented as the mean ± SD. Differences between two groups were analysed using Student's t-test. Comparisons between multiple groups were performed using one-way ANOVA followed by a post hoc test (least significant difference). p < 0.05 was considered to indicate a statistically significant difference.

Supplementary Materials:
The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/biom12091195/s1. Figure S1: The expression of SOD and TNF-α protein in hyperthyroidism-induced cardiomyopathy; Figure S2: The expression of proteins about signalling pathways in L-Thy induced phenotypes; Figure S3: Calcium antagonists (Isradipine) reduce heart muscle damage induced by high thyroid hormones.