Proarrhythmic atrial ectopy associated with heart sympathetic innervation dysfunctions is specific for murine B6CBAF1 hybrid strain.

A lot of animal models are developed with aim to advance in atrial fibrillation (AF) understanding. The hybrid B6CBAF1 mice are used extensively as a background to create manifestation of various diseases, however, their atrial electrophysiology, autonomic sympathetic innervation of the heart and potential for AF investigation is poorly characterized. In the present study we used ECG and microelectrode recordings from multicellular atrial preparations to reveal attributes of atrial electrical activity in B6CBAF1. Also, experiments with a fluorescent false monoamine neurotransmitter and glyoxylic acid-based staining were carried out to characterize functionally and morphologically catecholaminergic innervation of the B6CBAF1 atria. Atrial myocardium of B6CBAF1 is highly prone to ectopic automaticity and exhibits abnormal spontaneous action potential accompanied by multiple postdepolarizations that result in proarrhythmic triggered activity unlike two parental C57Bl/6 and CBA strains. In vivo experiments revealed that B6CBAF1 hybrids are more susceptible to the norepinephrine induced AF. Also, sympathetic nerve terminals are partially dysfunctional in B6CBAF1 revealing lower ability to accumulate and release neurotransmitters unlike two parental strains. The analysis of the heart rate variability revealed suppressed sympathetic component of the autonomic heart control in B6CBAF1. The organization of sympathetic innervation is very similar morphologically in all three murine strains however the abundance of non-bifurcated catecholamine-positive fibers in B6CBAF1 was increased. These results suggest that B6CBAF1 mice exhibit enhanced intrinsic atrial proarrhythmicity, while the abnormalities of sympathetic neurotransmitter cycling probably underlie disturbed autonomic heart control.

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.