Cardiac Cx43 and ECM Responses to Altered Thyroid Status Are Blunted in Spontaneously Hypertensive versus Normotensive Rats

Abstract

Heart function and its susceptibility to arrhythmias are modulated by thyroid hormones (THs) but the responsiveness of hypertensive individuals to thyroid dysfunction is elusive. We aimed to explore the effect of altered thyroid status on crucial factors affecting synchronized heart function, i.e., connexin-43 (Cx43) and extracellular matrix proteins (ECM), in spontaneously hypertensive rats (SHRs) compared to normotensive Wistar Kyoto rats (WKRs). Basal levels of circulating THs were similar in both strains. Hyperthyroid state (HT) was induced by injection of T3 (0.15 mg/kg b.w. for eight weeks) and hypothyroid state (HY) by the administration of methimazol (0.05% for eight weeks). The possible benefit of omega-3 polyunsaturated fatty acids (Omacor, 200 mg/kg for eight weeks) intake was examined as well. Reduced levels of Cx43 in SHRs were unaffected by alterations in THs, unlike WKRs, in which levels of Cx43 and its phosphorylated form at serine368 were decreased in the HT state and increased in the HY state. This specific Cx43 phosphorylation, attributed to enhanced protein kinase C-epsilon signaling, was also increased in HY SHRs. Altered thyroid status did not show significant differences in markers of ECM or collagen deposition in SHRs. WKRs exhibited a decrease in levels of profibrotic transforming growth factor β1 and SMAD2/3 in HT and an increase in HY, along with enhanced interstitial collagen. Short-term intake of omega-3 polyunsaturated fatty acids did not affect any targeted proteins significantly. Key findings suggest that myocardial Cx43 and ECM responses to altered thyroid status are blunted in SHRs compared to WKRs. However, enhanced phosphorylation of Cx43 at serine368 in hypothyroid SHRs might be associated with preservation of intercellular coupling and alleviation of the propensity of the heart to malignant arrhythmias.

Keywords: connexin-43; extracellular matrix; heart; spontaneously hypertensive rats; thyroid hormones.

Figure 1

Myocardial expression of CXA1 mRNA normalized to hypoxanthine guanine phosphoribosyl transferase (HPRT) level (A), and protein abundance of total myocardial connexin-43 (Cx43) normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein level (B,C) in the male normotensive and hypertensive rats with altered thyroid status with or without feeding with Omacor. W—Wistar Kyoto euthyroid control rats; o—fed with Omacor; HT—hyperthyroid rats; HY—hypothyroid rats; S—spontaneously hypertensive rats; n = 6 in each group. Data are presented as means ± SD; * p < 0.05 vs. W; ♠ p < 0.05 HT vs. S-HT/ HY vs. S-HY.

Figure 2

Myocardial protein abundance of the phosphorylated form of connexin-43 (Cx43) specifically on Serine368 (A,B) and non-phosphorylated form of Cx43 (C,D) normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein level in the male normotensive and hypertensive rats with altered thyroid status with or without feeding with Omacor. W—Wistar Kyoto euthyroid control rats; o—fed with Omacor; HT—hyperthyroid rats; HY—hypothyroid rats; S—spontaneously hypertensive rats; n = 6 in each group. Data are presented as means ± SD; * p < 0.05 vs. W; ♣ p < 0.05 vs. S; # p < 0.05 untreated vs. treated with Omacor; ♠ p < 0.05 HT vs. S-HT/ HY vs. S-HY.

Figure 3

Topology of myocardial connexin-43 (Cx43, green) colocalized with N-cadherin (red) in the male normotensive rats with altered thyroid status with or without feeding with Omacor; W—Wistar Kyoto euthyroid control rats; o—fed with Omacor; HT—hyperthyroid rats; HY—hypothyroid rats. Note conventional immunofluorescence labeling of Cx43 and N-cadherin at the gap junctions (long arrows) and Cx43 on lateral surfaces of the cardiomyocytes (short arrows). Objective 40×, Scale bar 10 µm.

Figure 4

Topology of myocardial connexin-43 (Cx43, green) colocalized with N-cadherin (red) in the male hypertensive rats with altered thyroid status with or without feeding with Omacor; S—spontaneously hypertensive rats; o—fed with Omacor; HT—hyperthyroid rats; HY—hypothyroid rats. Note conventional immunofluorescence labeling of Cx43 and N-cadherin at the gap junctions (long arrows) and Cx43 on lateral surfaces of the cardiomyocytes (short arrows). Objective 40×. Scale bar 10 µm.

Figure 5

Myocardial expression of protein kinase C epsilon (PKC-ε) mRNA normalized to hypoxanthine guanine phosphoribosyl transferase (HPRT) level (A), and protein abundance of myocardial PKC-ε normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein level (B,C) in the male normotensive and hypertensive rats with altered thyroid status with or without feeding with Omacor. W—Wistar Kyoto euthyroid control rats; o—fed with Omacor; HT—hyperthyroid rats; HY—hypothyroid rats; S—spontaneously hypertensive rats; n = 6 in each group. Data are presented as means ± SD; * p < 0.05 vs. W; ♠ p < 0.05 HT vs. S-HT/ HY vs. S-HY.

Figure 6

Protein levels of myocardial transforming growth factor β (TGF-β1; A,B) and TGF transcription factor (SMAD2/3; C,D) normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein level in the male normotensive and hypertensive rats with altered thyroid status with or without feeding with Omacor. W—Wistar Kyoto euthyroid control rats; o—fed with Omacor; HT—hyperthyroid rats; HY—hypothyroid rats; S—spontaneously hypertensive rats; n = 6 in each group. Data are presented as means ± SD; * p < 0.05 vs. W; ♠ p < 0.05 HT vs. S-HT/ HY vs. S-HY.

Figure 7

Protein abundance of myocardial matrix metalloproteinase-2 (MMP-2) normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein level in the male normotensive and hypertensive rats with altered thyroid status with or without feeding with Omacor. A 72 kDa pro-MMP-2 (A,B) and 63 kDa active MMP-2 (A, C) are shown. W—Wistar Kyoto euthyroid control rats; o—fed with Omacor; HT—hyperthyroid rats; HY—hypothyroid rats; S—spontaneously hypertensive rats; n = 6 in each group. Data are presented as means ± SD; * p < 0.05 vs. W; ♠ p < 0.05 HT vs. S-HT/ HY vs. S-HY.

Figure 8

Zymography of matrix metalloproteinase-2 (MMP-2) and quantitative analysis of MMP-2 activity in the male normotensive and hypertensive rats with altered thyroid status with or without feeding with Omacor. 72 kDa pro-MMP-2 (A,B); 63 kDa active MMP-2 (A,C); W—Wistar Kyoto euthyroid control rats; o—fed with Omacor; HT—hyperthyroid rats; HY—hypothyroid rats; S—spontaneously hypertensive rats; n = 6 in each group. Data are presented as means ± SD; * p < 0.05 vs. W; ♠ p < 0.05 HT vs. S-HT/ HY vs. S-HY.

Figure 9

Representative images of van Gieson staining and quantitative analysis of collagen deposition in the male normotensive rats with altered thyroid status, fed with or without Omacor. Interstitial fibrosis—long arrows; Perivascular fibrosis—short arrows. W—Wistar Kyoto euthyroid control rats; o—fed with Omacor; HT—hyperthyroid rats; HY—hypothyroid rats. Objective 40×. Scale bar 10 µm. Data are presented as means ± SD; * p < 0.05 vs. W; # p < 0.05 untreated vs. treated with Omacor.

Figure 10

Representative images of van Gieson staining and quantitative analysis of collagen deposition in the male hypertensive rats with altered thyroid status, fed with or without Omacor. Interstitial fibrosis—long arrows; perivascular fibrosis—short arrows. S—spontaneously hypertensive rats; o—fed with Omacor; HT—hyperthyroid rats; HY—hypothyroid rats. Objective 40×. Scale bar 10 µm. Data are presented as means ± SD.