Antiarrhythmic Effects of Melatonin and Omega-3 Are Linked with Protection of Myocardial Cx43 Topology and Suppression of Fibrosis in Catecholamine Stressed Normotensive and Hypertensive Rats

Figure 1. Design of the experiment and mortality rate: ISO was applied during 7 days in dose 7, 15, 20, 30 mg/kg (s.c.) to S and W rats. Drinking of melatonin and feeding with omega-3 was realized from the 1st until the 67th day. The highest mortality was registered after the last injection of ISO on day 7. W-Wistar rats; Wi-isoproterenol injured W; Wim-melatonin treated Wi; Wio-omacor treated Wi; S-spontaneously hypertensive rats; Si-isoproterenol injured S; Sim-melatonin treated Si; Sio-omacor treated Si.

Figure 2. The threshold to induce persistent VF was lower in spontaneously hypertensive rat hearts (S) compared to Wistar rat (W) heart and reduced in W rats in response to ISO (Wi) while not in S. Treatment of ISO exposed rats with either melatonin (Wim, Sim) or omega-3 resulted (Wio, Sio) in a significant increase of VF threshold in both strains. Values are the mean ± SD of 6 rats in each group. * p < 0.05 compared with W; # p < 0.05 compared with S; • p < 0.05 compared to Wi vs. Wim, Wio/Si vs. Sim, Sio.

Figure 3. Diffuse ISO-induced myocardial injury characterized by necrotic and atrophied cardiomyocytes (asterisks) as well as by infiltration with basophils in Wistar rats (Wi) and Spontaneously hypertensive rats (Si) comparing to controls (W, S). Note reduced foci of injury after treatment of ISO exposed rats to melatonin (Wim, Sim) or omega-3 (Wio, Sio). Haematoxylin-eosin staining, bar 200 μm.

Figure 4. Reliable histochemical demonstration of SDH activity revealed its absence in necrotic cardiomyocytes (asterisks) and reduced activity in surrounding atrophied myocytes in ISO-exposed Wistar (Wi) and Spontaneously hypertensive rat (Si) hearts. Note apparent preservation of SDH activity in viable cardiomyocytes (arrows) and reduced areas of injury (asterisks) in ISO-exposed rats after treatment with melatonin (Wim, Sim) or omega-3 (Wio, Sio). The intensity of staining in controls (W, S) corresponds to normal SDH activity. Bar 200 μm.

Figure 5. The myocardial topology of Cx43 immunofluorescence labeling. Note absence of Cx43 in areas of injury (F) and disordered distribution of Cx43 in surrounding viable cardiomyocytes in ISO-exposed heart (Wi, Si) compared to controls (W) with a regular polar distribution of Cx43 confined to intercalated discs (long arrows) and lateral sides of the cardiomyocytes (short arrows) that is more frequent in Spontaneously hypertensive rat (S) hearts. Abnormal distribution of Cx43 was attenuated by treatment with melatonin (Wim, Sim) and omega-3 (Wio, Sio). Quantitative image analysis revealed enhanced Cx43 positive signals in ISO-exposed rats that were suppressed by treatment (upper graphs). Importantly, the treatment of ISO exposed rats by melatonin and omega-3 significantly suppressed laterally distributed Cx43 (bottom graphs). * p < 0.05 compared with W; • p < 0.05 compared to Wi vs. Wim, Wio/Si vs. Sim, Sio. Bar 100 um.

Figure 6. Western blot demonstration of Cx43 protein levels (A), its functional phosphorylated forms (B) and PKCε protein levels (C) in experimental rats. Note reduced levels of Cx43 in S compared to W rats and increase of Cx43 (p < 0.05 in W) along with PKCε in ISO-stressed rats of both strains. In contrast, treatment by melatonin and omega-3 prevented an increase of these parameters in W rat and to a lesser extent in S hearts. W-Wistar rats; Wi-isoproterenol injured W; Wim-melatonin treated Wi; Wio-omacor treated Wi; S-spontaneously hypertensive rats; Si-isoproterenol injured S; Sim-melatonin treated Si; Sio-omacor treated Si. Values are the mean ± SD of 10 rats in each group. * p < 0.05 compared with W; # p < 0.05 compared with S; • p < 0.05 compared to Wi vs. Wim, Wio/Si vs. Sim, Sio.

Figure 7. Van Gieson staining demonstrated abnormal collagen deposition (arrows) and a significant increase in ISO-stressed Wistar (Wi) and Spontaneously hypertensive rat hearts (Si) compared to control rat hearts (W, S). The treatment with melatonin (Wim, Sim) and omega-3 (Wio, Sio) suppressed the ISO-induced changes in both strains. * p < 0.05 compared with W; # p < 0.05 compared with S; • p < 0.05 compared to Wi vs. Wim, Wio/Si vs. Sim, Sio. Bar 200 μm.

Figure 8. Reliable histochemical demonstration of AP activity (arrows) confined to endothelial cells of capillaries in control Wistar (W) as well as Spontaneously hypertensive rat (S) hearts. In addition, AP activity was increased in the foci of injury in ISO-stressed W rat hearts (Wi) as well as S rat (Si) hearts. Note reduced injured areas with AF activity due to treatment of ISO-stressed rats with melatonin (Wim, Sim) and omega-3 (Wio, Sio). Findings of quantitative image analyses are plotted on graphs. * p < 0.05 compared with W; # p < 0.05 compared with S; • p < 0.05 compared to Wi vs. Wim, Wio/Si vs. Sim, Sio. Bar 200 μm.

Figure 9. Demonstration of examined hydroxyproline (A), collagen-1 (B), SMAD2/3 (C), and TGF-β (D) in experimental rats. There is an increase of hydroxyproline and collagen-1 content paralleled with an increase of SMAD2/3 and TGF-β in ISO-stressed Wistar rat (Wi) and Spontaneously hypertensive rat (Si) hearts. Treatments with melatonin (Wim, Sim) and omega-3 (Sim, Sio) suppressed ISO-induced changes in both strains. Note strain-related differences in ISO actions as well as in effects of treatment. Myocardial MMP-2 activity (E) was lower in S compared to W rats and reduced in Wi while increased in Si. Treatment did not alter MMP-2 activity compared to ISO exposed rats (Wi, Si) or controls (W, S). Values are the mean ± SD of 10 rats in each group. * p < 0.05 compared with W; # p < 0.05 compared with S; • p < 0.05 compared to Wi vs. Wim, Wio/Si vs. Sim, Sio.

Figure 10. Demonstration of examined myocardial markers modulating redox state, TBARS (A), Keap1 (B), SOD1 (C), and SOD2 (D) in experimental rats. There were no changes in TBARS among the groups. The protein level of Keap1 was lower in the spontaneously hypertensive rat (S) compared to Wistar rat (W) hearts and reduced in response to ISO in W rats (Wi) while not in S (Si). Treatment with melatonin and omega-3 did not affect this parameter regardless of the strain (Wim, Wio, Sim, Sio). Protein levels of SOD1 and SOD2 were lower in S compared to W rat hearts. ISO exposure resulted in suppression of SOD isoforms in Wistar rats (Wi) but not in S (Si). There was a tendency to increase SOD1 and SOD2 in both ISO-exposed rat strains by treatment either with melatonin or omega-3. Values are the mean ± SD of 10 rats in each group. * p < 0.05 compared with W; # p < 0.05 compared with S; • p < 0.05 compared to Wi vs. Wim, Wio/Si vs. Sim, Sio.