Oxysterol, 5α-cholestan-3-one, modulates a contractile response to ß2-adrenoceptor stimulation in the mouse atria: Involvement of NO signaling.

AIM: Atrial ß2-adrenoceptors provide an important mechanism for regulation of cardiac function and changes in their downstream signaling are involved in processes underlying heart disorders. We have investigated the mechanism by which the cholesterol metabolite 5α-cholestan-3-one (5ɑCh3) modulates inotropic effect of ß2-adrenoceptor agonist fenoterol. MAIN METHODS: Atria from mice were electrically stimulated and changes in contraction amplitude in response to fenoterol were studied in 5ɑCh3-pretreated samples. Intracellular Ca2+ and NO levels were estimated using fluorescent dyes Fluo-4 and DAF-FM, respectively. KEY FINDINGS: By itself 5αCh3 that appears in the circulation under some pathological conditions had a negligible influence on contraction, Ca2+-transient and NO production. However, pretreatment with 5αCh3 markedly attenuated the positive inotropic effect of fenoterol which was accompanied by an increase in the NO synthesis. Unexpectedly, the oxysterol also augmented an enhancement of Ca2+-transient amplitude in response to fenoterol. Under conditions of a pharmacological inhibition of Gi-protein/Akt/NO synthase/protein kinase G signaling, 5αCh3 augmented the inotropic effect of fenoterol. Herein, Akt antagonist suppressed the increase in NO production, while inhibition of NO synthesis did not modify the increased amplitude of the Ca2+-transient. Along similar lines, enrichment of plasma membranes with cholesterol reduced the stimulatory effect of 5αCh3 on ß2-adrenoceptor-evoked NO production, but not on the Ca2+-transient amplitude, leading to an elevation of the positive inotropic response to fenoterol. SIGNIFICANCE: These data suggest that 5ɑCh3 potentiates the effect of pharmacological ß2-adrenoceptor activation on both NO production and Ca2+ transient via independent mechanisms, thereby affecting the positive inotropy.

Cholesterol regulates contractility and inotropic response to ß2-adrenoceptor agonist in the mouse atria: Involvement of Gi-protein-Akt-NO-pathway.

Majority of cardiac ß2-adrenoceptors is located in cholesterol-rich microdomains. Here, we have investigated the underlying mechanisms by which a slight to moderate cholesterol depletion with methyl-ß-cyclodextrin (MßCD, 1 and 5mM) interferes with contractility and inotropic effect of ß2-adrenergic agonist (fenoterol, 50µM) in the mouse atria. Treatment with MßCD itself increased amplitude of Ca2+ transient but did not change the contraction amplitude due to a clamping action of elevated NO. Cholesterol depletion significantly attenuated the positive inotropic response to fenoterol which is accompanied by increase in NO generation and decrease in Ca2+ transient. Influence of 1mM MßCD on the fenoterol-driven changes in both contractility and NO level was strongly attenuated by inhibition of Gi-protein (pertussis toxin), Akt (Akt 1/2 kinase inhibitor) or NO-synthase (L-NAME). After exposure to 5mM MßCD, pertussis toxin or Akt inhibitor could recover the ß2-agonist effects on contractility, NO production and Ca2+ transient, while L-NAME only reduced NO level. An adenylyl cyclase activator (forskolin, 50nM) had no influence on the MßCD-induced changes in the ß2-agonist effects. Obtained results suggest that slight cholesterol depletion upregulates Gi-protein/Akt/NO-synthase signaling that attenuates the positive inotropic response to ß2-adrenergic stimulation without altering the Ca2+ transient. Whilst moderate cholesterol depletion additionally could suppress the enhancement of the Ca2+ transient amplitude caused by the ß2-adrenergic agonist administration in Gi-protein/Akt-dependent but NO-independent manner.

ß2-adrenoceptor agonist-evoked reactive oxygen species generation in mouse atria: implication in delayed inotropic effect.

Fenoterol, a ß2-adrenoceptor agonist, has anti-apoptotic action in cardiomyocytes and induces a specific pattern of downstream signaling. We have previously reported that exposure to fenoterol (5 µM) results in a delayed positive inotropic effect which is related to changes in both Ca2+ transient and NO. Here, the changes in reactive oxygen species (ROS) production in response to the fenoterol administration and the involvement of ROS in effect of this agonist on contractility were investigated in mouse isolated atria. Stimulation of ß2-adrenoceptor increases a level of extracellular ROS, while intracellular ROS level rises only after removal of fenoterol from the bath. NADPH-oxidase inhibitor (apocynin) prevents the increase in ROS production and the Nox2 isoform is immunofluorescently colocalized with ß2-adrenoceptor at the atrial myocytes. Treatments with antioxidants (N-acetyl-L-cysteine, NADPH inhibitors, exogenous catalases) significantly inhibit the fenoterol induced increase in the contraction amplitude, probably by attenuating Ca2+ transient and up-regulating NO production. ROS generated in a ß2-adrenoceptor-dependent manner can potentiate the activity of some Ca2+ channels. Indeed, inhibition of ryanodine receptors, TRPV-or L-type Ca2+- channels shows a similar efficacy in reduction of positive inotropic effect of both fenoterol and H2O2. In addition, detection of mitochondrial ROS indicates that fenoterol triggers a slow increase in ROS which is prevented by rotenone, but rotenone has no impact on the inotropic effect of fenoterol. We suggest that stimulation of ß2-adrenoceptor with fenoterol causes the activation of NADPH-oxidase and after the agonist removal extracellularly generated ROS penetrates into the cell, increasing the atrial contractions probably via Ca2+ channels.