Beta-1 adrenergic receptors modulate pacemaker activity of mouse sino-atrial myocytes through L-type Cav1.3 channels

Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Doctoral program in Biology and Biotechnologies b1- and b2- adrenergic receptors (ARs) are co-expressed in different regions of the heart. The b2/ b1 expression ratio is higher in the sino-atrial node (SAN) than in atria and ventricles, but the specific contribution of either type of receptor to modulation of pacemaker activity is still not well established. Specific stimulation of b2-ARs in rabbit SAN myocytes is associated with a positive shift of the pacemaker "funny" current (If) activation curve. However, previous studies showed that L-type Cav1.3 channels play an important role in the generation of cardiac pacemaker activity by contributing to the diastolic depolarization (DD) in SAN myocytes. Since Cav1.3 channels are positively regulated by b-ARs activation2, we investigated which is the main b-ARs isoform that modulates Cav1.3-mediated ICaL andthe pacemaker activity of SAN myocytes. To address this point, we recorded spontaneous activity and Cav1.3-mediated ICaL from mouse SAN myocytes. We found that the positive chronotropic effect of the non-selective b-AR agonist isoproterenol (ISO, 0.1mM) was decreased by the b1-ARs antagonist CGP-20712 (0.3mM) and the b2-ARs antagonist ICI-118,551 (1mM) by -18% and -9%, respectively. Perfusion of CGP-20712 strongly reduced the positive chronotropic effect induced by ISO. Finally, we recorded Cav1.3-mediated L-type currents in presence of the b1-ARs antagonist. CGP-20712 reduced the basal Cav1.3-mediated ICaL. Furthermore, the increase in Cav1.3-mediated ICaL by isoproterenol was abolished during b1-ARs inhibition by CGP-20712. In conclusion, these preliminary data show that b1- and b2-ARs differently modulate the spontaneous activity of mouse SAN myocytes. In addition, b1-ARs play a predominant role in the adrenergic regulation of L-type Cav1.3 channels to increase pacemaker activity. Future studies will be performed to clarify the role of b2-ARs antagonist on Cav1.3-mediated ICaL and the functional relationships between b-ARs and Cav1.3 channels.

SLMAP-3 is downregulated in human dilated ventricles and its overexpression promotes cardiomyocyte response to adrenergic stimuli by increasing intracellular calcium

Structural dilation of cardiomyocytes (CMs) imposes a decline in cardiac performance that precipitates cardiac failure and sudden death. Since membrane proteins are implicated in dilated cardiomyopathy and heart failure, we evaluated the expression of the sarcolemmal membrane-associated protein (SLMAP) in dilated cardiomyopathy and its effect on CM contraction. We found that all 3 SLMAP isoforms (SLMAP-1, -2, and -3) are expressed in CMs and are downregulated in human dilated ventricles. Knockdown of SLMAPs in cultured CMs transduced with recombinant adeno-associated viral particles releasing SLMAP-shRNA precipitated reduced spontaneous contractile rate that was not fully recovered in SLMAP-depleted CMs challenged with isoproterenol (ISO), thus phenotypically mimicking heart failure performance. Interestingly, the overexpression of the SLMAP-3 full-length isoform induced a positive chronotropic effect in CMs that was more pronounced in response to ISO insult (vs. ISO-treated naïve CMs). Confocal live imaging showed that H9c2 cardiac myoblasts overexpressing SLMAP-3 exhibit a higher intracellular calcium transient peak when treated with ISO (vs. ISO-treated cells carrying a control adeno-associated viral particle). Proteomics revealed that SLMAP-3 interacts with the regulator of CM contraction, striatin. Collectively, our data demonstrate that SLMAP-3 is a novel regulator of CM contraction rate and their response to adrenergic stimuli. Loss of SLMAPs phenotypically mimics cardiac failure and crystallizes SLMAPs as predictive of dilated cardiomyopathy and heart failure.

Pharmacological Evaluation of the Effects of Phenylcarbamic Acid Derivatives on Cardiovascular Functions in Rats

Four phenylcarbamic acid derivatives, (1-(4-fluorophenyl)- 4-[3-(4-methoxyphenylcarbamoyloxy)-2-hydroxypropyl]piperazinium chloride (1), (1-(2-methylphenyl)-4-[3-(4-methoxyphenylcarbamoyloxy)- 2-hydroxypropyl]piperazinium chloride) (2), (1-(2-methylphenyl)-4-[3-(4-ethoxyphenylcarbamoyloxy)- 2-hydroxypropyl]piperazinium chloride) (3) and (1-(3-trifluoromethylphenyl)-4-[3-(4-methoxyphenylcarbamoyloxy)- 2-hydroxypropyl]piperazinium chloride) (4) were investigated for their ability to affect various cardiovascular functions and to establish their chemical structure-biological activity relationship. The compounds were evaluated for their antiarrhythmic efficacy using ouabain-induced rhythm disturbances and the ability to inhibit the positive chronotropic effect of isoproterenol in isolated atria of Wistar rats. Electrocardiogram (ECG) parameters in isolated hearts of spontaneously hypertensive rats (SHR) perfused according to the Langendorff method and ability to decrease phenylephrine- -induced contraction of the aortic strips after repeated administration of the compounds were also analyzed. Only compound 3 delayed significantly the evaluated parameter of arrhythmogenicity and was able to antagonize the isoproterenol- induced positive chronotropic effect in normotensive rats’ atria. Similarly, in SHR rats, only compound 3 was able to decrease heart frequency significantly without influencing the duration of QT (time between the start of the Q wave and the end of the T wave) and QTc (frequency corrected QT) intervals. The evaluated endothelial function was improved after administration of compound 2. Fluorine-containing structures (1 and 4) were less effective compared to 2´-methylphenylpiperazine derivatives (2 and 3). The latter two compounds showed suitable efficacy, which supported their use for futher pharmacological research.

Metabolic and functional consequences of cytosolic 5′-nucleotidase-IA overexpression in neonatal rat cardiomyocytes

Adenosine exerts a spectrum of energy-preserving actions on the heart negative chronotropic effects. The pathways leading to adenosine formation have remained controversial. In particular, although cytosolic 5′-nucleotidases can catalyze adenosine formation in cardiomyocytes, their contribution to the actions of adenosine has not been documented previously. We recently cloned two closely related AMP-preferring cytosolic 5′-nucleotidases (cN-IA and -IB); the A form predominates in the heart. In this study, we overexpressed pigeon cN-IA in neonatal rat cardiomyocytes using an adenovirus. cN-IA overexpression increased adenosine formation and release into the medium caused by simulated hypoxia and by isoproterenol in the absence and presence of inhibitors of adenosine metabolism. Adenosine release was not affected by an ecto-5′-nucleotidase inhibitor, α,β-methylene-ADP, but was affected by a nucleoside transporter, dipyridamole. The positive chronotropic effect of isoproterenol (130 ±3 vs. 100 ±4 beats/min) was inhibited (107 ±3 vs. 94 ±3 beats/min) in cells overexpressing cN-IA, and this was reversed by the addition of the adenosine receptor antagonist 8-( p-sulfophenyl)theophilline (120 ± 3 vs. 90 ± 4 beats/min). Our results demonstrate that overexpressed cN-IA can be sufficiently active in cardiomyocytes to generate physiologically effective concentrations of adenosine at its receptors.

Bradykinin induced a positive chronotropic effect via stimulation of T- and L-type calcium currents in heart cells

Using Fluo-3 calcium dye confocal microscopy and spontaneously contracting embryonic chick heart cells, bradykinin (10–10 M) was found to induce positive chronotropic effects by increasing the frequency of the transient increase of cytosolic and nuclear free Ca2+. Pretreatment of the cells with either B1 or B2 receptor antagonists (R126 and R817, respectively) completely prevented bradykinin (BK) induced positive chronotropic effects on spontaneously contracting single heart cells. Using the whole-cell voltage clamp technique and ionic substitution to separate the different ionic current species, our results showed that BK (10–6 M) had no effect on fast Na+ inward current and delayed outward potassium current. However, both L- and T-type Ca2+ currents were found to be increased by BK in a dose-dependent manner (10–10–10–7 M). The effects of BK on T- and L-type Ca2+ currents were partially blocked by the B1 receptor antagonist [Leu8]des-Arg9-BK (R592) (10–7 M) and completely reversed by the B2 receptor antagonist D-Arg[Hyp3,D-Phe7,Leu8]BK (R-588) (10–7 M) or pretreatment with pertussis toxin (PTX). These results demonstrate that BK induced a positive chronotropic effect via stimulation of T- and L-type Ca2+ currents in heart cells mainly via stimulation of B2 receptor coupled to PTX-sensitive G-proteins. The increase of both types of Ca2+ current by BK in heart cells may explain the positive inotropic and chronotropic effects of this hormone.Key words: chick heart cells, bradykinin, B1 receptor, B2 receptor, Ca2+, Na+, K+ currents.

Autonomic control of heart rate during forced activity and digestion in the snakeBoa constrictor

SUMMARYReptiles, particularly snakes, exhibit large and quantitatively similar increments in metabolic rate during muscular exercise and following a meal, when they are apparently inactive. The cardiovascular responses are similar during these two states, but the underlying autonomic control of the heart remains unknown. We describe both adrenergic and cholinergic tonus on the heart during rest, during enforced activity and during digestion (24–36 h after ingestion of 30 % of their body mass) in the snake Boa constrictor. The snakes were equipped with an arterial catheter for measurements of blood pressure and heart rate, and autonomic tonus was determined following infusion of the β-adrenergic antagonist propranolol (3 mg kg–1) and the muscarinic cholinoceptor antagonist atropine (3 mg kg–1).The mean heart rate of fasting animals at rest was 26.4±1.4 min–1, and this increased to 36.1±1.4 min–1 (means ± s.e.m.; N=8) following double autonomic block (atropine and propranolol). The calculated cholinergic and adrenergic tones were 60.1±9.3 % and 19.8±2.2 %, respectively. Heart rate increased to 61.4±1.5 min–1 during enforced activity, and this response was significantly reduced by propranolol (maximum values of 35.8±1.6 min–1), but unaffected by atropine. The cholinergic and adrenergic tones were 2.6±2.2 and 41.3±1.9 % during activity, respectively. Double autonomic block virtually abolished tachycardia associated with enforced activity (heart rate increased significantly from 36.1±1.4 to 37.6±1.3 min–1), indicating that non-adrenergic, non-cholinergic effectors are not involved in regulating heart rate during activity. Blood pressure also increased during activity.Digestion was accompanied by an increase in heart rate from 25.6±1.3 to 47.7±2.2 min–1 (N=8). In these animals, heart rate decreased to 44.2±2.7 min–1 following propranolol infusion and increased to 53.9±1.8 min–1 after infusion of atropine, resulting in small cholinergic and adrenergic tones (6.0±3.5 and 11.1±1.1 %, respectively). The heart rate of digesting snakes was 47.0±1.0 min–1 after double autonomic blockade, which is significantly higher than the value of 36.1±1.4 min–1 in double-blocked fasting animals at rest. Therefore, it appears that some other factor exerts a positive chronotropic effect during digestion, and we propose that this factor may be a circulating regulatory peptide, possibly liberated from the gastrointestinal system in response to the presence of food.

Possible Involvement of Cyclic Adenosine Monophosphate–independent Mechanism in the Positive Chronotropic Effect of Norepinephrine in the Isolated Guinea Pig Right Atrium

Background Although both positive chronotropic and inotropic effects of beta-adrenergic stimulation are thought to be mediated by cyclic adenosine 3'5'-monophosphate, phosphodiesterase III inhibitors such as amrinone and milrinone potentiate the positive inotropic effect of catecholamines with minimum influence on the heart rate in clinical setting. The aim of the current study was to compare the positive chronotropic effect of norepinephrine with that of forskolin to elucidate whether cyclic adenosine monophosphate is relevant to the chronotropic effect of norepinephrine. Methods Concentration-response curves for the positive chronotropic effects of norepinephrine and forskolin on the spontaneously beating right atria of guinea pigs were determined in the absence and presence of phosphodiesterase inhibitors or ion channel inhibitors. In some experiments, the left atria driven electrically were used to determine the positive inotropic effect of norepinephrine. Results Norepinephrine and forskolin increased the beating rate in a concentration-dependent manner. The positive chronotropic effect of forskolin was potentiated by amrinone and 3-isobutyl-1-methylxanthine, whereas the positive chronotropic effect of norepinephrine was not potentiated by the phosphodiesterase inhibitors. In contrast, the positive inotropic effect of norepinephrine was potentiated by amrinone. The hyperpolarization-activated inward current inhibitor cesium chloride and L-type voltage-dependent Ca2+ current inhibitor verapamil suppressed the chronotropic effect of norepinephrine, whereas these inhibitors did not affect the chronotropic effect of forskolin. Conclusion Norepinephrine increases the spontaneously beating rate by a different mechanism from that of forskolin, suggesting that cyclic adenosine monophosphate is causally unrelated to the positive chronotropic effect of norepinephrine in the guinea pig heart.