Activation of GPER limits adverse changes to Ca2+ signalling and arrhythmogenic activity in cardiomyocytes of ovariectomised guinea pigs

Background Post-menopausal women have an enhanced risk of developing cardiovascular disease and disturbances of cardiac rhythm, generally attributed to declining oestrogen levels during menopause. In an animal model that mimics menopause, the long-term withdrawal of oestrogen dysregulated Ca2+ signalling and increased the formation of a pro-arrhythmic substrate. Selective targeting of the membrane bound G-protein coupled oestrogen receptor 1 (GPER) eliminated such arrhythmogenic activity associated with the loss of oestrogen. Purpose We aim to assess the “cardioprotective” role of GPER in response to oestrogen withdrawal. Methods Ovariectomy (OVx) or sham surgeries were conducted on female guinea pigs. Left ventricular cardiomyocytes were isolated 150-days post-operatively for experimental use. GPER expression was quantified by western blot. Myocytes were incubated in solutions containing GPER agonist G-1 for >2h before recording some electrophysiological and Ca2+ regulatory parameters. Results GPER expression was 32% higher in OVx. OVx cardiomyocytes had prolonged action potential duration (APD) compared with sham and in the presence of G-1, APD90 shortened by 12% and 22% in sham and OVx, respectively. G-1 reduced early after depolarisation (EAD) formation by >99% in OVx. OVx cells had larger sarcoplasmic reticulum (SR) Ca2+ content (by 13%), compared with sham. While G-1 had little effect on SR content, it reduced Ca2+ transient amplitude (by 40%), SR fractional release (by 11%) and sarcomere shortening (by 29%) in OVx cells. The frequency of occurrence of spontaneous Ca2+ waves evoked by periods of rapid stimulation reduced by 40% and wave-free survival time prolonged in OVx cells incubated with G-1. Conclusions In the hearts of an animal species whose electrophysiology and intracellular Ca2+ regulation is akin to humans, we show that following oestrogen deficiency, GPER expression increased and its activation induces negative inotropic responses in cardiomyocytes. It limits the adverse changes to Ca2+ signalling and induces anti-arrhythmogenic behaviours in OVx. FUNDunding Acknowledgement Type of funding sources: Private grant(s) and/or Sponsorship. Main funding source(s): British Heart Foundation

ALPHA(1)-ADRENOCEPTORS ACTIVATION DECREASES MYOCARDIAL CONTRACTILITY IN NEWBORN RATS

Alpha(1)-adrenergic receptors (α1-AR) are found in cardiomyocytes, endothelial cells, and smooth muscle cells of humans and animals. Despite the fact that α1-AR make up 10% of the total number of adrenergic receptors, these receptors also involved in the regulation of inotropic and chronotropic functions of the heart. According to some scientists, the effects of α1-AR activation are not required for the basal contractile function of the heart while other group of researchers believe that α1-AR can be considered as cardioprotective targets; in particular, it is postulated that the α1A-subtype of adrenergic receptors can provide significant inotropic support in cardiac pathologies. This study was carried out on 6-7-day-old outbred newborn rat pups to evaluate the effect of alpha(1)-adrenoceptors activation on the myocardial contractility in newborn rats. For this, Alpha1-adrenergic receptors were stimulated by the pharmacological drug methoxamine at concentrations of 10-9-10-6 mol and the reaction of the contractile force of the strips of myocardium ventricles and heart atria in response to the agonist was investigated. Results of study revealed that stimulation of alpha1-adrenergic receptors, regardless of the methoxamine concentration, led to a negative inotropic reaction of the myocardium of atria and ventricles of newborn rat pups. This study showed unidirectional inotropic responses on rat atrial and ventricular myocardium in response to α1-adrenergic receptors stimulation. Methoxamine smoothly reduces the contractile force of the strips of myocardium atria and ventricles. At the same time, the concentration dependence on the inotropic reaction of the myocardium was observed. Results of study suggested that probably α1-adrenergic receptors along with the main regulators β-adrenergic receptors carry out fine tuning of the heart activity.

Abstract 12677: Impaired Cardiomyocyte Contractility to Angiotensin-(1-12) in a Humanized Angiotensinogen Model of Hypertension

Background: Angiotensin-(1-12) [Ang-(1-12)] is a chymase-dependent source for Angiotensin II (Ang II) inotropic activity that may be impaired in a model of sustained hypertension with high cardiac Ang II content due to insertion of the human angiotensinogen (AGT) gene in the rat genome. Accordingly, we evaluated the effects of Ang-(1-12) and Ang II on myocyte contractility and [Ca 2+ ] i regulation in 9 adult male transgenic rats expressing the human AGT gene [TGR(hAGT)L1623)] and 9 SD controls. Methods: We compared LV myocyte contraction, relaxation and [Ca 2+ ] i transient ([Ca 2+ ] iT ) responses to Ang-(1-12) (4x10 -6 M) and Ang II (10 -6 M) in freshly isolated LV myocytes. Results: Myocyte contraction (dL/dt max , 109.6 vs 127.9 μm/s), relaxation (dR/dt max , 95.3 vs 107.5 μm/s) and [Ca 2+ ] iT (0.15 vs 0.24) were depressed in TGR(hAGT)L1623 rats compared to SD controls. Moreover, cell contractile and [Ca 2+ ] iT responses following exposure to Ang-(1-12) or Ang II were markedly blunted. In SD myocytes, versus baseline, Ang II or Ang-(1-12) superfusion produced significant increases in dL/dt max [Ang II: 44% vs Ang-(1-12): 34%], dR/dt max (33% vs 26%) and [Ca 2+ ] iT (31% vs 25%). Importantly, the magnitude of the responses to the two agents in TGR(hAGT)L1623 myocytes was significantly reduced. Versus the changes in SD myocytes, Ang-(1-12) caused significantly less increases in dL/dt max (22%), dR/dt max (16%) and [Ca 2+ ] iT (15%) in TGR(hAGT)L1623 myocytes . Ang II also caused similar significantly attenuated increases in dL/dt max (27%), dR/dt max (25%) and [Ca 2+ ] iT (23%). The Ang-(1-12)-induced inotropic effects were completely prevented in the presence of the inhibitory cAMP analog, Rp-cAMPS (10 –4 M, 2 hours) in both SD and TGR(hAGT)L1623 myocytes, but were further augmented only intransgenic rats after incubation of myocytes with the G i inhibitor, pertussis toxin (PTX, 2 μg/ml, 36°C, 5 hours). Conclusions: Ang-(1-12) stimulates LV myocyte contractile function and [Ca 2+ ] iT in both SD and TGR(hAGT)L1623 rats. Furthermore, we now show that the blunted inotropic responses to Ang-(1-12) and Ang II in rats expressing the human AGT gene is mediated through a cAMP-dependent mechanism that is coupled to both stimulatory G and inhibitory PTX-sensitive G proteins.

Impact of phosphodiesterases PDE3 and PDE4 on 5-hydroxytryptamine receptor4-mediated increase of cAMP in human atrial fibrillation

Atrial fibrillation (AF)–associated remodeling includes contractile dysfunction whose reasons are only partially resolved. Serotonin (5-HT) increases contractile force and causes arrhythmias in atrial trabeculae from patients in sinus rhythm (SR). In persistent atrial fibrillation (peAF), the force responses to 5-HT are blunted and arrhythmic effects are abolished. Since force but not arrhythmic responses to 5-HT in peAF could be restored by PDE3 + PDE4 inhibition, we sought to perform real-time measurements of cAMP to understand whether peAF alters PDE3 + PDE4-mediated compartmentation of 5-HT4 receptor-cAMP responses. Isolated human atrial myocytes from patients in SR, with paroxysmal AF (paAF) or peAF, were adenovirally transduced to express the FRET-based cAMP sensor Epac1-camps. Forty-eight hours later, cAMP responses to 5-HT (100 μM) were measured in the absence or concomitant presence of the PDE3 inhibitor cilostamide (0.3 μM) and the PDE4 inhibitor rolipram (1 μM). We successfully established real-time cAMP imaging in AF myocytes. 5-HT increased cAMP in SR, paAF, and peAF, but in line with previous findings on contractility, this increase was considerably smaller in peAF than in SR or paAF. The maximal cAMP response to forskolin (10 μM) was preserved in all groups. The diminished cAMP response to 5-HT in peAF was recovered by preincubation with cilostamide + rolipram. We uncovered a significantly diminished cAMP response to 5-HT4 receptor stimulation which may explain the blunted 5-HT inotropic responses observed in peAF. Since both cAMP and force responses but not arrhythmic responses were recovered after concomitant inhibition of PDE3 + PDE4, they might be regulated in different subcellular microdomains.

THE CORRELATION BETWEEN MOTOR QUALITIES AND HEART PUMPING FUNCTION RESERVES IN YOUNG ATHLETES

Aim. The purpose of the article is to study the patterns of forming heart pumping function reserves in schoolchildren in the conditions of hypo- and hyperkinesia. Materials and methods. The heart pumping function was assessed with the help of Ergoracer cycle ergometer (Kettler) and the load adjusted using ERGO-KONZEPT software developed for this equipment. Reserves were established by the difference between the indicators of the heart pumping function: heart rate, stroke volume, minute blood flow at maximum load and at rest. The peculiarities of heart pumping function reserves were studied in the conditions of adaptation to different movement modes in schoolchildren aged 7–18 years. Results. It was established that heart reserves in terms of heart contractions increase with age mainly by lowering the frequency at rest. Heart reserves in terms of stroke volume and minute blood flow also increase with age, especially in trained persons. The nature of changes in the reserves of minute blood flow coincides with the pattern of changes in heart reserves in terms of stroke volume. Conclusion. The study of heart reserves allowed to find the peculiarities of changes in the chronotropic and inotropic responses of the heart, which were established as the difference between heart rate frequency, stroke volume, and minute blood flow at maximum stress and the data obtained at relative rest during adaptation to different movement modes.