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

Abstract 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. </jats:sec

Cholinergic regulation of cardiac pacemaker activity by l-type cav1.3 channels

Abstract Introduction The cholinergic regulation of heart rate (HR) is mediated by acetylcholine (ACh)-dependent activation of M2-receptors (M2R). Activated M2R promote release of the βγ-subunit of G-proteins to directly gate GIRK1/4 channels (underlying the cardiac IKACh current), while αi-subunits inhibit adenylate cyclase (AC) activity. AC inhibition reduces the intracellular concentration of cAMP, decreasing the activity of ion channels involved in pacemaking, including “funny” f-(HCN4) and L-type Cav1.3 calcium channels. Purpose To determine the importance of L-type Cav1.3 channels in the cholinergic regulation of heart rate. Methods We recorded the frequency and the position of the pacemaker leading site in ex vivo sinus nodes and the HR of isolated Langendorff perfused hearts of mice in control or during ACh perfusion. We used control wild type (WT) mice, and five genetically modified mouse models: Cav1.3 knockout (KO, ablated Cav1.3-mediated L-type current), GIRK4KO (ablated IKACh current), HCN4-CNBD (selective deletion of cAMP-dependent regulation of HCN4), GIRK4KO/HCN4-CNBD and GIRK4KO/Cav1.3KO. Results Data from optical mapping experiments showed that, under basal conditions, perfusion of 3 μM ACh significantly reduced the frequency of action potentials in WT (44%), HCN4-CNBD (38%), Cav1.3KO (65%) and GIRK4KO (8%) isolated mouse sinus node tissues. ACh application did not significantly affect the frequency of action potentials recorded in tissue from GIRK4KO/HCN4-CNBD and GIRK4KO/Cav1.3KO animals. Furthermore, in all the sinus node tissues tested, regardless of the genotypes, ACh shifted the pacemaker leading site from its normal position by at least 0.7 mm. Upon stimulation of the β-adrenergic pathway by Isoproterenol, to reproduce conditions of accentuated antagonism, 3μM ACh reduced HR in isolated hearts from WT (43.8%), HCN4-CNBD (38.7%), Cav1.3KO (25,4%), GIRK4KO (16.9%) and GIRK4KO/HCN4-CNBD (16.4%) mice. No significant HR reduction was recorded in hearts from GIRK4KO/Cav1.3KO animals. Conclusion Our data indicate that L-type Cav1.3 channels are involved in cholinergic regulation of heart rate in mice. In addition, when the intracellular concentration of cAMP is elevated (i.e. under conditions of accentuated antagonism), the cholinergic regulation of sinus node pacemaking is predominantly ensured by Cav1.3 and KACh channels. Funding Acknowledgement Type of funding source: Foundation. Main funding source(s): “Fondation pour la recherche medicale” FRM </jats:sec

Cholinergic regulation of cardiac pacemaker activity by L-type Cav1.3 channels

Abstract Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): Fondation Recherche Médicale Introduction The cholinergic regulation of heart rate (HR) is mediated by acetylcholine (ACh)-dependent activation of M2-receptors (M2R). Activated M2R promote release of the βγ-subunit of G-proteins to directly gate GIRK1/4 channels (underlying the cardiac IKACh current), while αi-subunits inhibit adenylate cyclase (AC) activity. AC inhibition reduces the intracellular concentration of cAMP, decreasing the activity of ion channels involved in pacemaking, including "funny" f-(HCN4) and L-type Cav1.3 calcium channels. Purpose To determine the role of L-type Cav1.3 channels in cholinergic regulation of heart rate. Methods We recorded the frequency of activation and position of pacemaker leading site in ex vivo sinus nodes and the HR of isolated Langendorff perfused hearts of mice at baseline or during ACh perfusion.  We used control wild type (WT) mice, and five genetically modified mouse models: Cav1.3 knockout (KO, ablated Cav1.3-mediated L-type current), GIRK4KO (ablated IKACh current), HCN4-CNBD (selective deletion of cAMP-dependent regulation of HCN4), GIRK4KO/HCN4-CNBD and GIRK4KO/Cav1.3KO. We performed in vivo telemetric recordings of heart rate (HR) in WT and GIRK4KO/Cav1.3KO animals. Results Data from optical mapping experiments showed that, under basal conditions, perfusion of 3 μM ACh significantly reduced the frequency of action potentials in WT (44%), HCN4-CNBD (38%), Cav1.3KO (65%) and GIRK4KO (8%) isolated mouse sinus node tissues. ACh application did not significantly affect the frequency of action potentials recorded in tissue from GIRK4KO/HCN4-CNBD and GIRK4KO/Cav1.3KO animals. Furthermore, in all sinus nodes tested, regardless of genotype, ACh shifted the pacemaker leading site from its normal position by at least 0.7 mm. Upon stimulation of the β-adrenergic pathway by Isoproterenol, to reproduce conditions of accentuated antagonism, 3µM ACh reduced HR in isolated hearts from WT (43.8%), HCN4-CNBD (38.7%), Cav1.3KO (25,4%), GIRK4KO (16.9%) and GIRK4KO/HCN4-CNBD (16.4%) mice. No significant HR reduction was recorded in hearts from GIRK4KO/Cav1.3KO animals. In vivo data indicate that HR reduction induced by combined injection of Hexamethonium ( a Nicotinic acetylcholine receptor blocker) with Carbamoylcholine (CCH, M2 receptor agonist) or with 2-Chloro-N6-Cyclopentyladenosine (CCPA, A1 receptor agonist) is higher in WT than in GIRK4KO/Cav1.3KO animals (68% vs 48% CCH, and 79% vs 62% CCPA, respectively). Conclusion Our data indicate that L-type Cav1.3 channels are involved in cholinergic regulation of heart rate in mice. In addition, when the intracellular concentration of cAMP is elevated (i.e. under conditions of accentuated antagonism), cholinergic regulation of sinus node pacemaking is reliant on Cav1.3 and KACh channels. </jats:sec

Synthesis of DnaK and GroEL in Escherichia coli cells exposed to different magnetic field signals.

The effects of extremely low frequency magnetic field (ELF-MF)(1 mT, 50 Hz) on the heat shock protein (HSP) synthesis in Escherichia coli were investigated. Two magnetic field signals were studied: sinusoidal (SMF) and pulsed square wave (PMF). It was found that bacteria exposed to SMF showed a significantly higher level of DnaK and GroEL proteins as compared to sham-exposed bacteria as revealed by Western blot, whereas a lower level was observed after PMF exposure. Similar results were obtained when bacterial cells were exposed to heat shock (HS) after ELF-MF exposure: again SMF and PMF resulted in an increase and in a reduction of HSP amount in comparison with sham control, respectively. In conclusion, the MF influences the synthesis of HSPs in E. coli in a way that critically depends on the signal characteristics