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Research ArticleCardiovascular

Cytosolic Ca2+–Induced Apoptosis in Rat Cardiomyocytes via Mitochondrial NO-cGMP-Protein Kinase G Pathway

Kazuhiko Seya, Kyoichi Ono, Susumu Fujisawa, Ken Okumura, Shigeru Motomura and Ken-Ichi Furukawa
Journal of Pharmacology and Experimental Therapeutics January 2013, 344 (1) 77-84; DOI: https://doi.org/10.1124/jpet.112.198176
Kazuhiko Seya
Department of Pharmacology, Hirosaki University Graduate School of Medicine, Hirosaki, Japan (K.S., S.M., K-I.F.); Department of Cell Physiology, Akita University Graduate School of Medicine, Akita, Japan (K.On., S.F.); Department of Cardiology, Respiratory Medicine and Nephrology, Hirosaki University Graduate School of Medicine, Hirosaki, Japan (K.Ok.)
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Kyoichi Ono
Department of Pharmacology, Hirosaki University Graduate School of Medicine, Hirosaki, Japan (K.S., S.M., K-I.F.); Department of Cell Physiology, Akita University Graduate School of Medicine, Akita, Japan (K.On., S.F.); Department of Cardiology, Respiratory Medicine and Nephrology, Hirosaki University Graduate School of Medicine, Hirosaki, Japan (K.Ok.)
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Susumu Fujisawa
Department of Pharmacology, Hirosaki University Graduate School of Medicine, Hirosaki, Japan (K.S., S.M., K-I.F.); Department of Cell Physiology, Akita University Graduate School of Medicine, Akita, Japan (K.On., S.F.); Department of Cardiology, Respiratory Medicine and Nephrology, Hirosaki University Graduate School of Medicine, Hirosaki, Japan (K.Ok.)
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Ken Okumura
Department of Pharmacology, Hirosaki University Graduate School of Medicine, Hirosaki, Japan (K.S., S.M., K-I.F.); Department of Cell Physiology, Akita University Graduate School of Medicine, Akita, Japan (K.On., S.F.); Department of Cardiology, Respiratory Medicine and Nephrology, Hirosaki University Graduate School of Medicine, Hirosaki, Japan (K.Ok.)
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Shigeru Motomura
Department of Pharmacology, Hirosaki University Graduate School of Medicine, Hirosaki, Japan (K.S., S.M., K-I.F.); Department of Cell Physiology, Akita University Graduate School of Medicine, Akita, Japan (K.On., S.F.); Department of Cardiology, Respiratory Medicine and Nephrology, Hirosaki University Graduate School of Medicine, Hirosaki, Japan (K.Ok.)
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Ken-Ichi Furukawa
Department of Pharmacology, Hirosaki University Graduate School of Medicine, Hirosaki, Japan (K.S., S.M., K-I.F.); Department of Cell Physiology, Akita University Graduate School of Medicine, Akita, Japan (K.On., S.F.); Department of Cardiology, Respiratory Medicine and Nephrology, Hirosaki University Graduate School of Medicine, Hirosaki, Japan (K.Ok.)
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Abstract

Previously, we showed that in adult rat cardiomyocytes, nitric oxide (NO) donors stimulate mitochondrial cGMP production, followed by cytochrome c release, independently of the mitochondrial permeable transition pore. We investigated whether mitochondrial cGMP-induced cytochrome c release from cardiac mitochondria is Ca2+-sensitive. Mitochondria and primary cultured cardiomyocytes were prepared from left ventricles of male Wistar rats. The cytosolic Ca2+ concentration was adjusted with Ca2+-EGTA buffers. Cytochrome c released from mitochondria was measured by Western blotting. Cardiomyocyte apoptosis was assessed by Annexin V staining. Cytochrome c release from cardiac mitochondria was evoked by buffered Ca2+ (1 μM); this was inhibited by NO-cGMP pathway inhibitors such as NG-monomethyl-l-arginine monoacetate (inhibitor of NO synthase), 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (NO scavenger), 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, NO-sensitive guanylyl cyclase inhibitor) and voltage-dependent anion channel (VDAC) inhibitor, 4,4′-diisothiocyano-2,2′-disulfonic acid stilbene, but not by cyclosporin A (mitochondrial permeable transition pore inhibitor). Furthermore, this release was significantly and dose dependently inhibited by 0.3–3 μM KT5823 (protein kinase G inhibitor). At the cellular level, intracellular perfusion of cardiomyocytes with buffered Ca2+ (1 μM) also induced apoptosis, which was inhibited in the presence of ODQ. A membrane-permeable cGMP analog, 8-Br-cGMP, but not cGMP itself, mimicked buffered Ca2+ actions in both cardiac mitochondria and cardiomyocytes. We further confirmed an increase in protein kinase G activity by adding cGMP in mitochondrial protein fraction. Our results suggest that mitochondrial NO-cGMP pathway-induced cytochrome c release from cardiac mitochondria, triggered by increased cytosolic Ca2+, occurs through VDAC via the stimulation of an undiscovered mitochondrial protein kinase G.

Footnotes

  • This work was supported in part by grants of Aomori Bank for young researchers; a Grant-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan [Grant-in-Aid 20590245]; the Hirosaki University Educational Improvement and Promotional Aid; and the Fund of the Northern Tohoku National Three Universities Cooperation Promotion Research Project.

  • ↵Embedded ImageThis article has supplemental material available at jpet.aspetjournals.org.

  • dx.doi.org/10.1124/jpet.112.198176.

  • Received July 5, 2012.
  • Accepted October 25, 2012.
  • Copyright © 2013 by The American Society for Pharmacology and Experimental Therapeutics
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Journal of Pharmacology and Experimental Therapeutics: 344 (1)
Journal of Pharmacology and Experimental Therapeutics
Vol. 344, Issue 1
1 Jan 2013
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Research ArticleCardiovascular

Mitochondrial NO-cGMP-PKG–Induced Cardiac Apoptosis

Kazuhiko Seya, Kyoichi Ono, Susumu Fujisawa, Ken Okumura, Shigeru Motomura and Ken-Ichi Furukawa
Journal of Pharmacology and Experimental Therapeutics January 1, 2013, 344 (1) 77-84; DOI: https://doi.org/10.1124/jpet.112.198176

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Research ArticleCardiovascular

Mitochondrial NO-cGMP-PKG–Induced Cardiac Apoptosis

Kazuhiko Seya, Kyoichi Ono, Susumu Fujisawa, Ken Okumura, Shigeru Motomura and Ken-Ichi Furukawa
Journal of Pharmacology and Experimental Therapeutics January 1, 2013, 344 (1) 77-84; DOI: https://doi.org/10.1124/jpet.112.198176
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