Rapid electrical stimulation of contraction modulates gap junction protein in neonatal rat cultured cardiomyocytes
Involvement of mitogen-activated protein kinases and effects of angiotensin ii-receptor antagonist
Noriko Inoue, MD*,
Tomoko Ohkusa, MD, PhD*,
Tomoko Nao, MD, PhD*,
Jong-Kook Lee, MD, PhD ,
Tomo Matsumoto, MD*,
Yuji Hisamatsu, MD, PhD*,
Takashi Satoh, MD*,
Masafumi Yano, MD, PhD*,
Kenji Yasui, MD, PhD,
Itsuo Kodama, MD, PhD and
Masunori Matsuzaki, MD, PhD, FACC*,*
* Division of Cardiovascular Medicine, Department of Medical Bioregulation, Yamaguchi University Graduate School of Medicine, Yamaguchi, Japan
Department of Circulation, Research Institute of Environmental Medicine, Nagoya University, Nagoya, Japan
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Figure 1 Quantitative analysis of connexin (Cx)43 protein and messenger ribonucleic acid (mRNA) levels in cardiomyocytes subjected to rapid electrical stimulation. (A) Connexin43 protein levels estimated by Western analysis; Cx43 amount was normalized to control protein (mean ± SD, n = 8). *p < 0.05; **p < 0.01 vs. baseline. (B) Connexin43 mRNA levels estimated by reverse transcription-polymerase chain reaction. Connexin43 mRNA amount was normalized to GAPDH (means ± SD, n = 8). **p < 0.01 vs. baseline.
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Figure 2 Confocal images of connexin (Cx)43 immunolabeling in cultured cardiomyocytes. (A) A picuture of cultured cardiomyocytes and representative immunofluorescence images of Cx43 before (0 min) and after rapid electrical stimulation for 60 min and 90 min. (B) The proportion of total cell area occupied by Cx43 immunoreactive signal (means ± SD, n = 8). **p < 0.01 vs. 0 min (baseline).
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Figure 3 Angiotensin II (AngII) secretion and production by cultured cardiomyocytes. (A) Angiotensin II content in the culture media (means ± SD, n = 8). **p < 0.01 vs. 0 min (baseline). (B) Angiotensin II content of cardiomyocytes (Western analysis). Values were normalized to the baseline (mean ± SD, n = 8). *p < 0.05 vs. baseline.
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Figure 4 Changes of three mitogen-activated protein kinases (MAPKs) and their phosphorylated forms in response to rapid electrical stimulation (Western analysis). (A) Total extracellular signal-regulated protein kinases (ERK) and phospho-ERK (p-ERK). (B) Total c-Jun NH2-terminal kinases (JNK) and phospho-JNK (p-JNK). (C) Total p38 MAPKs and phospho-p-38 MAPKs (p-p38). Values were normalized to their baseline (mean ± SD, n = 8). *p < 0.05; **p < 0.01 vs. baseline.
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Figure 5 Prevention of rapid electrical stimulation (RES)-induced connexin (Cx)43 upregulation by losartan. (A) Connexin43 protein levels before (0 min) and after RES for 60 min and 90 min in the absence (solid bars) and presence (hatched bars) of 100 nmol/l losartan (Western analysis). Connexin43 amount was normalized to control protein (means ± SD, n = 8). (B) Connexin43 messenger ribonucleic acid (mRNA) levels before (0 min) and after RES for 60 min and 90 min (reverse transcription-polymerase chain reaction). Connexin43 mRNA amount was normalized to GAPDH (mean ± SD, n = 8). (C) Immunofluorescence signals of Cx43 before (0 min) and after RES for 30 to 90 min. (Top) Representative confocal images. (Bottom) The proportion of total cell area occupied by Cx43 immunoreactive signal. Values were normalized to baseline (mean ± SD, n = 8). **p < 0.01 vs. baseline.
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Figure 6 Prevention of rapid electrical stimulation (RES)-induced activation of mitogen-activated protein kinase (MAPK) by losartan. (A) Phospho-extracellular signal-regulated protein kinases (ERK) (p-ERK) levels before (0 min) and after RES for 60 min and 90 min in the absence (solid bars) and presence (hatched bars) of 100 nmol/l losartan. (B) Phospho-c-Jun NH2-terminal kinases (JNK) (p-JNK) levels. (C) Phospho-p38 MAPKs (p-p38) levels. The amount of each protein was normalized to the baseline without losartan (mean ± SD, n = 8). *p < 0.05; **p < 0.01 vs. baseline.
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Figure 7 Prevention of rapid electrical stimulation (RES)-induced upregulation of connexin (Cx)43 by specific mitogen-activated protein kinase (MAPK) inhibitors. Connexin43 proteins levels before (0 min) and after RES for 60 min in the absence and presence of 50 µmol/l PD98059 (a MAPK/extracellular signal-regulated protein kinase inhibitor), 50 µmol/l SP600125 (a selective c-Jun NH2-terminal kinases [JNK] inhibitor), and 10 µmol/l SB203580 (a p38 and JNK2 inhibitor). Values were normalized to baseline in the control medium (mean ± SD, n = 8). *p < 0.05; **p < 0.01 vs. baseline.
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Figure 8 Multielectrode extracellular potential mapping during propagation of excitation. (A) Representative electrograms recorded from the 64 terminals. (Arrows) Stimulus artifacts. (B and C) Isochrone maps of activation time in a culture dish before (control) and after rapid electrical stimulation (RES) for 90 min in the absence of losartan. (D) Isochrone map of activation time in another culture dish after RES for 90 min in the presence of 100 nM losartan. ST = stimulation point; * = stimulation points.
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Figure 9 Effects of rapid electrical stimulation (RES) on the conduction velocity. Conduction velocity from left to right was measured along the midline in the observation area before and 15 to 90 min after RES. Values (means ± SD) obtained in the absence (open circle, n = 7 to 9) and presence of 100 nM losartan (solid circle, n = 7 to 9) were plotted against time after the initiation of RES. *p < 0.05 vs. baseline value before RES.
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