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

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 Ca 21 -sensitive. Mitochondria and primary cultured cardiomyocytes were prepared from left ventricles of male Wistar rats. The cytosolic Ca 21 concentration was adjusted with Ca 21 -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 Ca 21 (1 mM); this was inhibited by NO-cGMP pathway inhibitors such as N G -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, 4,49-diisothiocyano-2,29-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 mM KT5823 (protein kinase G inhibitor). At the cellular level, intracellular perfusion of cardiomyocytes with buffered Ca 21 (1 mM) 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 Ca 21 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 Ca 21 , occurs through VDAC via the stimulation of an undiscovered mitochondrial protein kinase G.

The mitochondrial NO‐synthase/guanylate cyclase/protein kinase G signaling system underpins the dual effects of nitric oxide on mitochondrial respiration and opening of the permeability transition pore

“…All these data indicate that the elements of Ca 2+ ‐dependent mtNOS/guanylate cyclase (GC)/PKG signaling system (mtNOS‐SS) may operate in mitochondria. However, according to the results presented by Seya and coauthors , S ‐nitrosoacetylpenacillamine (SNAP) or 8‐Bromo‐cGMP stimulated calcium‐dependent CytC release and apoptosis, while the inhibitors of NOS, GC, and PKG prevented these effects. These results contradict the generally admitted prosurvival action of cytosolic NOS/GC/PKG1 signaling system involved in the prevention of mPTP opening and cell death .…”

“…However, potential intramitochondrial mechanisms of protection may also include some signaling chains involved in calcium and NO interplay. Recently, Seya and coauthors discovered that cardiac mitochondrial protein fraction possesses protein kinase G (PKG) activity and provides cGMP synthesis triggered by NO donors . Besides, the hydrolysis of cGMP by mitochondrial cyclic nucleotide phosphodiesterase PDE2A was demonstrated in brain and liver mitochondria in independent experiments .…”

Bidirectional action of nitric oxide on mitochondrial respiration and permeability transition pore induced by calcium and palmitoylcarnitine

“…All these data indicate that the elements Ca 2+ -dependent mtNOS/ GC/PKG-signaling system (mtNOS-SS) may operate in mitochondria. However, according to 4 the results presented by Seya and coauthors [23], SNAP or 8-Bromo-cGMP induced calciumdependent CytC release and apoptosis, while the inhibitors of NOS, GC, and PKG prevented these effects. These results contradict to the generally admitted pro-survival action of cytosolic NOS/GC/PKG1-SS directed at the prevention of MPTP opening and cell death [26][27][28][29].…”

“…However, potential intramitochondrial mechanisms of protection may also include some signaling chains involved in calcium and NO interplay. Recently Seya and coauthors discovered that cardiac mitochondrial protein fraction possesses PKG activity [23] and provides cGMP synthesis triggered by NO donors [24]. Besides, the hydrolysis of cGMP by mitochondrial cyclic nucleotide phosphodiesterase PDE2A was demonstrated in brain and liver mitochondria in independent experiments [25].…”

Burn-Induced Cardiac Mitochondrial Dysfunction via Interruption of the PDE5A-cGMP-PKG Pathway

“…PKG1α arbitrates the down-stream effects that are involved in keeping the contractile force of cardiomyocytes and phosphorylates serine and threonine residues on numerous cytosolic proteins [13]. Seya et al found that the NO-cGMP-PKG pathway might play a role in ischemic pre-conditioning and antioxidant cardiac protection via cardiac mitochondria [14], even though the precise mechanism remains to be discovered. PDE5 inhibitors, such as sildenafil (SIL), prolong the effect of NO, a potent vasodilator, and cGMP, causing increased vasodilation and smooth muscle tone regulation [15].…”