*2.5. Determination of Collagen Content by Hydroxyproline Measurement*

The hydroxyproline content in the left ventricle tissue was estimated by the spectrophotometric method, as described previously [23]. Samples were hydrolyzed in 6 M HCl for 3 h at 130 ◦C. Dried samples were treated at room temperature with chloramine T in the acetate–citrate buffer (pH 6.0), and the reaction was stopped after 20 min by adding Ehrlich's reagent solution. It was followed by incubation at 65 ◦C for 15 min. The concentration of hydroxyproline was measured spectrophotometrically at 550 nm and expressed in mg per total weight of the left ventricle [22].

#### *2.6. Determination of Myocardial Protein Levels by Western Blotting*

Frozen left ventricular tissue was powdered in liquid nitrogen and homogenized in SB20 lysis buffer (20% SDS, 10 mmol/L EDTA and 100 mmol/L Tris, pH 6.8). To analyze the active form of collagen-1, the samples were prepared in Laemmli buffer without 2-mercaptoethanol and loaded onto gels without denaturation. For other proteins, the tissue lysate was diluted in the Laemmli sample buffer and boiled for 5 min, and an equal amount of protein was loaded in each well, followed by separation on SDS-PAGE 10% bis-acrylamide gels at a constant voltage of 120 V (Mini-Protean TetraCell, Bio-Rad, Hercules, CA, USA), as previously described [17]. Subsequently, the proteins were transferred to a nitrocellulose membrane (0.2-μm pore size, Advantec, Tokyo, Japan) and blocked for 4 h with 5% fat-free milk in Tris-buffered saline containing 0.1% Tween 20 (TBST). The membrane was incubated overnight with the primary antibodies listed in Table 1. The membrane was subsequently washed in TBST and incubated for 1 h with a horseradish peroxidase-linked secondary anti-rabbit antibody (1:2000, 7074S, Cell Signaling Technology, Denver, CO, USA) and anti-mouse antibody (1:2000, 7076C, Cell Signaling Technology, Denver, CO, USA). The enhanced luminol-based chemiluminescent was used for visualization of the proteins and the quantification of the relevant bands was assessed densitometrically using Carestream Molecular Imaging Software (version 5.0, Carestream Health, New Haven, CT, USA) and normalized to GAPDH.

#### *2.7. Statistical Evaluation*

Differences between groups were evaluated using one-way analysis of variance (ANOVA) and Bonferroni's multiple comparison test. The Kolmogorov–Smirnov normality test was used to examine whether variables are normally distributed. Data were expressed as means ± standard deviations (SD); *p* < 0.05 was considered to be statistically significant.


**Table 1.** List of primary antibodies used for the Western blot detection of proteins.

## **3. Results**

*3.1. Biometric, Blood Samples and Cardiac Left Ventricular Tissue Parameters of Experimental Rats*

Comparing to the systolic blood pressure in normotensive WKY rats (114.2 ± 17 mmHg in males and 99.5 ± 11 mmHg in females), this parameter was significantly elevated in either sex of the wild type SHR (190.0 ± 7 mmHg in males and 179.6 ± 12 mmHg in females) and in hairless SHR<sup>M</sup> (182.9 ± 6 mmHg in males and 169.7 ± 9 mmHg in females). As shown in Table 2, wild type SHR males exhibited higher body weights when compared to SHRM, whereas there was no difference in body weights between these two strains in the females. The heart and left ventricular weights did not noticeably differ between SHR and SHRM, regardless of the sex, but both cardiac parameters were significantly higher when compared to normotensive WKY. Hyperthyroidism resulted in a decrease in body weight in SHR males and, to a lesser extent, in SHRM males but not in females. There were no significant differences in the myocardial TBARS levels between wild type SHR and hairless SHRM or in comparing to WKY rats, but there was a tendency to increase due to hyperthyroidism in both SHR strains. The myocardial levels of GSH did not differ significantly among the experimental groups, regardless of the strain and sex.

As shown in Table 2, the serum total T3 and T4 concentrations were similar in wild type SHR and hairless SHRM males, but in SHR females, the T3 concentrations were a bit higher in comparison with SHR<sup>M</sup> females. While comparing to WKY rats, the serum T3 concentrations were significantly higher in both SHR strains, regardless of the sex. The serum triglycerides, total cholesterol, HDL and LDL cholesterol did not significantly differ in SHR vs. SHRM, but these parameters were lower in both SHR strains compared to WKY rats. There were no significant differences in the atherogenic index, expressed as the TC/HDL ratio, among the groups.


**Table 2.** General characteristics of the experimental rats.

BW—body weight, HW—heart weight, LVW—left ventricular weight, TBARS—thiobarbituric acid reactive substances in the left ventricle, GSH—reduced glutathione in the left ventricle, T3—total triiodothyronine, T4 total thyroxine, TG—plasma triglycerides, TC—total cholesterol, HDL—high-density lipoprotein-cholesterol, LDL—low-density lipoprotein-cholesterol, WKY—Wistar Kyoto normotensive control rats, SHR—wild type spontaneously hypertensive rats, TH—hyperthyroid rats, HY—hypothyroid rats, SHRM—hairless spontaneously hypertensive rats, n = 5 per group. Data are presented as means <sup>±</sup> SD, <sup>a</sup> *<sup>p</sup>* < 0.05 vs. WKY, <sup>b</sup> *<sup>p</sup>* < 0.05 vs. SHR and <sup>e</sup> *<sup>p</sup>* < 0.05 vs. SHRM. One-way ANOVA and Bonferroni's multiple comparison tests were used as the statistical method.

#### *3.2. Myocardial Histology and Capillary Enzyme Histochemistry*

Hematoxylin–eosin staining (Figure 1) revealed that, compared to WKY rats, the left ventricular tissue of both wild type SHR and SHR<sup>M</sup> of either sex exhibited focal areas infiltrated with polymorphonuclears. This histopathological feature persisted regardless of the hyper- or hypothyroid status. Besides, Van Gieson staining for collagen deposition (Figure 2) revealed interstitial and perivascular fibrosis in both SHR strains males and females but to lesser extent in SHRM. Of note, the representative microscopic images indicate the most pronounced area of fibrosis detected in each experimental group of rats. The hypothyroid status enhanced the myocardial fibrosis in both SHR strains.

Histochemical determination of the activity of alkaline phosphatase (AP) that points out the function and myocardial density of the arterial part of capillaries is demonstrated in Figure 3. Alterations of the capillary network AP activity may reflect the myocardial adaptation to hypertension to maintain the function of a structurally remodeled and fibrotic heart. Compared to normotensive WKY rats, the AP activity was significantly increased in both wild type SHR and SHRM, regardless of the sex, and was enhanced due to hypothyroidism in males and females of both SHR strains, as assessed by a quantitative image analysis.

**Figure 1.** Hematoxylin–eosin staining revealed the infiltration of polymorphonuclears (asterisks) in all SHR groups. WKY—Wistar Kyoto normotensive control rats, SHR—wild type spontaneously hypertensive rats, TH—hyperthyroid rats, HY—hypothyroid rats and SHRM—hairless spontaneously hypertensive rats, n = 5 per group. Scale bar represents 200 μm.

**Figure 2.** Van Gieson staining shows the collagen deposition (pink) of various degrees in all SHR groups with massive patchy fibrosis in hypothyroid rats. WKY—Wistar Kyoto euthyroid control rats, SHR—wild type spontaneously hypertensive rats, TH—hyperthyroid rats, HY—hypothyroid rats and SHRM—hairless spontaneously hypertensive rats, n = 5 per group. Scale bar represents 200 μm.

**Figure 3.** Histochemical demonstration of the alkaline phosphatase (AP) activity (blue) in endothelial cells of the arterial portion of capillaries and quantification of the intensity of the reaction. Note the enhanced AP activity in SHRM males compared to the wild type strain, as well as in response to the hypothyroid status. WKY—Wistar Kyoto normotensive control rats, SHR—wild type spontaneously hypertensive rats, TH—hyperthyroid rats, HY—hypothyroid rats and SHRM—hairless spontaneously hypertensive rats, n = 5 per group. Scale bar represents 200 μm. Data are presented as means ± SD, <sup>a</sup> *p* < 0.05 vs. WKY and <sup>b</sup> *p* < 0.05 vs. SHR. One-way ANOVA and Bonferroni's multiple comparison test were used as the statistical method.

The activity of dipeptidyl peptidase-4 (DPP4), that points out the function and density of the venous part of capillaries, is demonstrated in Figure 4. Enhanced DPP4 activity is considered detrimental in pathophysiological conditions due to the implication in collagen metabolism and proinflammatory signaling. A quantitative image analysis revealed that the DPP4 activity was significantly lower in SHRM regardless of the sex when compared to normotensive WKY rats or wild type SHR. Hyperthyroidism resulted in a decrease of DPP4 activity, while an increase was observed in hypothyroidism in both wild type SHR and SHRM, regardless of the sex.

#### *3.3. Myocardial Protein Levels of Cx43 and Its Variants*

The expression level of the Cx43 protein was significantly reduced in the left ventricle of SHR males and females, as well as in SHRM males (Figure 5), compared to normotensive WKY rats. However, compared to wild type SHR males and females, the protein levels of Cx43 were significantly higher in sex-matched SHRM. The hypothyroid status enhanced the Cx43 levels in wild type SHR, as well as in SHRM, and the increase was more pronounced in females than in males of both SHR strains. The hyperthyroid status resulted in a decrease of Cx43 in SHR<sup>M</sup> males while not in wild type SHR males, but an increase was demonstrated in SHR females and. to a lesser extent, in SHRM females.

**Figure 4.** Histochemical demonstration of dipeptidyl peptidase-4 (DPP4) activity (red) in endothelial cells of the venous portion of the capillary network and quantification of the intensity of the reaction. Note the significant decrease of DPP4 activity in SHRM compared to SHR, regardless of the sex, as well as in response to the hyperthyroid status. WKY—Wistar Kyoto normotensive control rats, SHR—wild type spontaneously hypertensive rats, TH—hyperthyroid rats, HY—hypothyroid rats and SHRM—hairless spontaneously hypertensive, n = 5 per group. Scale bar represents 200 μm. Data are presented as means <sup>±</sup> SD, <sup>a</sup> *<sup>p</sup>* < 0.05 vs. WKY, <sup>b</sup> *<sup>p</sup>* < 0.05 vs. SHR, <sup>e</sup> *<sup>p</sup>* < 0.05 vs. SHRM. One-way ANOVA and Bonferroni's multiple comparison test were used as the statistical method.

**Figure 5.** Myocardial protein levels of Cx43 assessed by Western blots. Note the significant decrease of the Cx43 levels in the hypertensive strains compared to normotensive WKY but, to a lesser extent, in hairless SHRM males and females. The hypothyroid status enhanced the Cx43 protein levels in both SHR strains. WKY—Wistar Kyoto normotensive control rats, SHR—wild type spontaneously hypertensive rats, TH—hyperthyroid rats, HY—hypothyroid rats and SHRM—hairless spontaneously hypertensive rats, n = 5 per group. Data are presented as means <sup>±</sup> SD, <sup>a</sup> *<sup>p</sup>* < 0.05 vs. WKY, <sup>b</sup> *p* < 0.05 vs. SHR, <sup>e</sup> *p* < 0.05 vs. SHRM. One-way ANOVA and Bonferroni's multiple comparison test were used as the statistical method.

The levels of expression of the Cx43 variant phosphorylated at serine368 (pCx43368), which, is associated with a reduced channel conductivity and the Cx43 variant phosphorylated at serine279 (pCx43279) that hampers channel conductivity are shown in Figure 6. Compared to WKY rats, the expression of pCx43<sup>368</sup> was significantly decreased in wild type SHR males and females but increased in SHRM, apparently in males. The hypothyroid status enhanced the pCx43368 expression only in wild type SHR, regardless of the sex, while not in SHRM. On the contrary, the hyperthyroid status reduced the pCx43368 protein levels only in SHR<sup>M</sup> males. There were no significant changes in the pCx43279 protein abundance either in wild type SHR or SHRM, regardless of the sex, when compared to normotensive WKY rats. Both the hyperthyroid and hypothyroid status reduced the pCx43279 protein levels significantly in SHR<sup>M</sup> males but not in females.

**Figure 6.** Protein levels of Cx43 variants pCx43368 (**A**) and pCx43279 (**B**) assessed by the Western blot analysis. Note the reduced levels of pCx43368 in wild type SHR males compared to WKY but not in hairless SHRM males. WKY—Wistar Kyoto normotensive control rats, SHR—wild type spontaneously hypertensive rats, TH—hyperthyroid rats, HY—hypothyroid rats and SHRM—hairless spontaneously hypertensive rats, n = 5 in each group. Data are presented as means <sup>±</sup> SD, <sup>a</sup> *<sup>p</sup>* < 0.05 vs. WKY, <sup>b</sup> *p* < 0.05 vs. SHR and <sup>e</sup> *p* < 0.05 vs. SHRM. One-way ANOVA and Bonferroni's multiple comparison test were used as the statistical method.

#### *3.4. Myocardial Topology of Cx43 and Quantification of Its Abnormal Distribution*

The cardiomyocyte distribution of Cx43 in experimental rats is demonstrated in Figure 7. The obvious prevalent distribution of Cx43 is detected at the gap junction plaques of the intercalated discs in normotensive WKY rats. Besides this obvious end-to-end pattern, there was enhanced immunofluorescence labeling of Cx43 at the lateral sides of the cardiomyocytes (side-to-side pattern) in both SHR strains, regardless of the sex and thyroid status. A quantitative image analysis of Cx43-positive labeling revealed a significant increase of Cx43 at the lateral sides of the cardiomyocytes in wild type SHR males and females but to a lesser extent in SHRM, regardless of the sex. Moreover, there was a tendency to increase the lateral distribution of Cx43 due to the hyperthyroid status, apparently in females of both SHR strains. In the context of lateral Cx43 distribution, the use of Triton X-100 soluble and Triton X-100 insoluble portions analysis [24] could provide information about the integrity of the gap junction plaques.

**Figure 7.** Detection of the myocardial topology of Cx43 using immunofluorescence labeling. Double arrows represent the colocalization of Cx43 (green) with cadherin (red) at the intercalated discs of the cardiomyocytes, and simple arrows show Cx43 localization on the lateral sides of the cardiomyocytes. The graphs represent the quantification of lateral Cx43. Note the abnormal lateral localization is suppressed in male SHRM compared to SHR. WKY—Wistar Kyoto normotensive control rats, SHR—wild type spontaneously hypertensive rats, TH—hyperthyroid rats, HY—hypothyroid rats and SHRM—hairless spontaneously hypertensive rats, n = 5 per group. Scale bar represents 400 μm. Data are presented as means <sup>±</sup> SD, <sup>a</sup> *<sup>p</sup>* < 0.05 vs. WKY. One-way ANOVA and Bonferroni's multiple comparison test were used as the statistical method.

#### *3.5. Myocardial Protein Levels and Topology of Cx43 Interacting Protein, β-Catenin*

β-catenin is an adhesive junction protein interacting with Cx43 and impacting channel assembly and function [25]. Immunolabeling of β-catenin is confined to the intercalated disc junctions (end-to-end pattern), as shown in Figure 8. There was no difference in the topology of β-catenin among the experimental groups of rats. While the Western blotting analysis revealed (Figure 8) that, compared to WKY rats, the protein levels of β-catenin exhibited a tendency to be lower in wild type SHR but higher in SHRM (significantly in females). The hyperthyroid status increased the β-catenin protein levels in wild type SHR males and females but not in SHRM. The hypothyroid status exhibited a tendency to decrease in the β-catenin protein levels in males while not in females of both SHR strains. Nevertheless, regarding the Cx43 function, it would be useful to explore active vs. inactive forms of β-catenin.

**Figure 8.** Detection of the myocardial topology in β-catenin (green) using immunofluorescence labeling. Graphs represent quantification of protein levels of β-catenin determined by Western blot analysis. Note the enhanced β-catenin in hairless SHRM males and, to a lesser extent, in females compared to the wild type strain. WKY—Wistar Kyoto normotensive control rats, SHR—wild type spontaneously hypertensive rats, TH—hyperthyroid rats, HY—hypothyroid rats, SHRM—hairless spontaneously hypertensive rats, n = 5 per group. Scale bar represents 400 μm. Data are presented as means <sup>±</sup> SD, <sup>b</sup> *<sup>p</sup>* < 0.05 vs. SHR. One-way ANOVA and Bonferroni's multiple comparison test were used as the statistical method.

#### *3.6. Myocardial Expression of Cx43 Interacting Protein Kinases*

Cx43 interacting protein kinases by the phosphorylation of the Cx43 impact channel's function and inter-myocyte communication [26,27]. Accordingly, PKCε, PKG and MAPK42/44 attenuate, while PKA and Akt kinase facilitate the Cx43 channel conductivity. The expression levels of the assessed protein kinases are demonstrated in Figure 9. Compared to wild type SHR, the protein levels of PKCε were increased in SHR<sup>M</sup> males, as well as due to the hypothyroid status in both SHRM and wild type SHR, regardless of the sex, while the expression of PKG and MAPK42/44 was not significantly altered in SHR<sup>M</sup> males vs. wild type SHR or in response to the altered thyroid status. However, the MAPK42/44 expression was increased in SHR<sup>M</sup> females compared to wild type SHR. The expression of PKA was lower in SHR<sup>M</sup> males vs. wild type SHR but enhanced due to the hypothyroid or hyperthyroid status. There were no changes in PKA expression among the experimental rat groups in the females. Moreover, there was no significant difference in the expression of Akt kinase between the SHR<sup>M</sup> and wild type SHR groups, while an increase of Akt kinase protein due to the hypothyroid status was detected in males but not in females of both SHR strains. Demonstrated changes of the protein kinase expression may have an impact on Cx43 phosphorylation and function. Therefore, their interaction with Cx43 (coimmunoprecipitation), along with its phosphorylated status, is challenging to explore.

**Figure 9.** Myocardial levels of the Cx43 interacting protein kinases assessed by Western blotting. Note an increase of PKCε in hairless SHR<sup>M</sup> males compared to the wild type strain, as well as in response to a hypothyroid status, regardless of the strain and sex. WKY—Wistar Kyoto normotensive control rats, SHR—wild type spontaneously hypertensive rats, TH—hyperthyroid rats, HY—hypothyroid rats and SHRM—hairless spontaneously hypertensive rats, n = 5 per group. Data are presented as means <sup>±</sup> SD, <sup>a</sup> *<sup>p</sup>* < 0.05 vs. WKY, <sup>b</sup> *<sup>p</sup>* < 0.05 vs. SHR and <sup>e</sup> *<sup>p</sup>* < 0.05 vs. SHRM. One-way ANOVA and Bonferroni's multiple comparison test were used as the statistical method.

## *3.7. Myocardial Level of Profibrotic Markers TGF-β1, SMAD2/3, Collagen-1 and Hydroxyproline*

As shown in Figure 10, the protein expression of TGF-β1 was significantly higher in wild type SHR males but not in SHRM, compared to normotensive WKY rats. While TGF-β1 expression was not altered in females of both SHR strains when compared to WKY rats. A significant elevation of TGF-β1 was observed only in SHR<sup>M</sup> females in response to the hypothyroid status.

**Figure 10.** Myocardial protein levels of profibrotic markers TGF-β1, SMAD2/3 and Collagen-1 determined by Western blotting, as well as the levels of hydroxyproline. Note the reduced levels of these proteins in hairless SHRM compared to wild type SHR, apparently in males. WKY—Wistar Kyoto normotensive control rats, SHR—wild type spontaneously hypertensive rats, TH—hyperthyroid rats, HY—hypothyroid rats and SHRM—hairless spontaneously hypertensive rats, n = 5 per group. Data are presented as means <sup>±</sup> SD, <sup>a</sup> *<sup>p</sup>* < 0.05 vs. WKY, <sup>b</sup> *<sup>p</sup>* < 0.05 vs. SHR and <sup>e</sup> *<sup>p</sup>* < 0.05 vs. SHRM. One-way ANOVA and Bonferroni's multiple comparison tests were used as the statistical method.

In parallel, there was an increase of SMAD2/3 expression in wild type SHR (significantly in males) but not in SHRM, regardless of the sex, when compared to WKY rats (Figure 10). The hypothyroid status increased the SMAD2/3 expression in wild type males (SHR), while the hyperthyroid status decreased it in SHRM.

Moreover, there was a significant increase of collagen-1 (Figure 10) in wild type SHR males, as well as females, but not so much in SHR<sup>M</sup> when compared to WKY rats. The hypothyroid status highly increased the content of collagen-1 in wild type SHR males and females but much less in SHRM.

The hydroxyproline levels (Figure 10) were significantly increased in wild type SHR males but not in SHRM when compared to WKY rats. However, the hypothyroid status highly increased the hydroxyproline content in both SHR strains, regardless of the sex.

#### *3.8. Myocardial Protein Levels of β1, β2 and β3-Adrenergic Receptors (AR)*

The adrenergic stimulation is important for cold acclimation-elicited non-shivering thermogenesis and the cardiac-specific overexpression of β3-AR hampers hypertrophy and myocardial fibrosis [28,29]. According to our analyses (Figure 11), the expression of cardiac-dominant β1-AR was significantly decreased in wild type SHR, as well as in SHRM males and females, when compared to normotensive WKY rats. There was a tendency to increase the expression of β1-AR in response to the hypothyroid status in wild type SHR, as well as in SHR<sup>M</sup> males. Like β1-AR, the expression of β2-AR was decreased in males regardless of the SHR strain when compared to WKY rats, while the hyperthyroid status

resulted in an increase of β2-AR expression in wild type SHR, as well as in SHRM males. Compared to WKY rats, there were no differences in β3-AR expression among the males, unlike hairless SHR<sup>M</sup> females that exhibited an increased expression of β3-AR.

**Figure 11.** Myocardial protein levels of the β-adrenergic receptors (AR) determined by Western blotting. Note the reduced protein levels of β1-AR and β2-AR in both the SHR and SHRM strains, compared to normotensive WKY rats, as well as an increase of the β3-AR protein in hairless SHRM females compared to wild type SHR. WKY—Wistar Kyoto normotensive control rats, SHR—wild type spontaneously hypertensive rats, TH—hyperthyroid rats, HY—hypothyroid rats and SHRM—hairless spontaneously hypertensive rats, n = 5 per group. Data are presented as means <sup>±</sup> SD, <sup>a</sup> *<sup>p</sup>* < 0.05 vs. WKY, <sup>b</sup> *p* < 0.05 vs. SHR. One-way ANOVA and Bonferroni's multiple comparison test were used as the statistical method.
