The ABA-LANCL1/2 Hormone-Receptors System Protects H9c2 Cardiomyocytes from Hypoxia-Induced Mitochondrial Injury via an AMPK- and NO-Mediated Mechanism

Abstract

Abscisic acid (ABA) regulates plant responses to stress, partly via NO. In mammals, ABA stimulates NO production by innate immune cells and keratinocytes, glucose uptake and mitochondrial respiration by skeletal myocytes and improves blood glucose homeostasis through its receptors LANCL1 and LANCL2. We hypothesized a role for the ABA-LANCL1/2 system in cardiomyocyte protection from hypoxia via NO. The effect of ABA and of the silencing or overexpression of LANCL1 and LANCL2 were investigated in H9c2 rat cardiomyoblasts under normoxia or hypoxia/reoxygenation. In H9c2, hypoxia induced ABA release, and ABA stimulated NO production. ABA increased the survival of H9c2 to hypoxia, and L-NAME, an inhibitor of NO synthase (NOS), abrogated this effect. ABA also increased glucose uptake and NADPH levels and increased phosphorylation of Akt, AMPK and eNOS. Overexpression or silencing of LANCL1/2 significantly increased or decreased, respectively, transcription, expression and phosphorylation of AMPK, Akt and eNOS; transcription of NAMPT, Sirt1 and the arginine transporter. The mitochondrial proton gradient and cell vitality increased in LANCL1/2-overexpressing vs. -silenced cells after hypoxia/reoxygenation, and L-NAME abrogated this difference. These results implicate the ABA-LANCL1/2 hormone-receptor system in NO-mediated cardiomyocyte protection against hypoxia.

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“…Cytochalasin B: Dissolve 5 mg of cytochalasin B and 250 µL of DMSO (dimethyl sulfoxide) as a stock solution (20 µg/µL). As described in [ 22 ], add 5 µg of cytochalasin B per mL of culture medium. Add 0.25 µL from the stock solution per mL of culture medium.…”

The ABA/LANCL Hormone/Receptor System in the Control of Glycemia, of Cardiomyocyte Energy Metabolism, and in Neuroprotection: A New Ally in the Treatment of Diabetes Mellitus?

“…Interestingly, after the brain, the heart is the organ with the highest expression levels of LANCL1/2. In particular, LANCL1 expression in the heart is among the highest in non-neurological tissues and is approximately 4-times higher compared with LANCL2 expression [ 34 ]. Indeed, an important role for the LANCL proteins in cardiomyocyte mitochondrial function has recently been discovered.…”

“…The ultimate stressor for an eminently aerobic tissue such as the heart is hypoxia; indeed, the culture of rat H9c2 cardiomyocytes under hypoxic conditions results in ABA release and ABA in turn stimulates NO production [ 34 ]. ABA also stimulates cardiomyocyte glucose uptake and oxidation, increased O 2 consumption under normoxia, and conservation of the mitochondrial proton gradient (ΔΨ) after hypoxia.…”

“…Stimulation by ABA of eNOS in H9c2 cells indeed occurs via both AMPK- and Akt-dependent phosphorylation of eNOS. In addition to increasing eNOS transcription, protein expression and post-translational activation, ABA also increases the expression of the mitochondrial arginine transporter and of the regulatory enzyme for synthesis of tetrahydrobiopterine, a coenzyme required for the synthesis of NO from arginine, and reduces the expression of arginase, an enzyme competing with NOS for its substrate arginine [ 34 ].…”

Amino acid motifs for the identification of novel protein interactants

“…In muscle cells for instance, LANCL1 has been shown to bind ABA and trigger glucose uptake [75] . Both LANCL1 and LANCL2 activate glucose transporters GLUT4 and GLUT1, and the signaling proteins in the AMPK/PGC-1α/Sirt1 pathway, stimulating respiration in mitochondria, while also protecting mitochondria of cardiomyocytes from hypoxia-induced injury through AMPK- and nitric oxide (NO)-mediated mechanisms [75] , [81] . Previously, LANCL2 has already been shown to bind ABA on the membrane of human granulocytes which is necessary for ABA signal transduction in granulocytes and in insulinoma cells [74] .…”