Cross-talk between cAMP and Ca2+ signalling in cardiac pacemaker cells involves IP3-evoked Ca2+ release and stimulation of adenylyl cyclase 1

Atrial arrhythmias, such as atrial fibrillation (AF), are a major mortality risk and a leading cause of stroke. The IP3 signalling pathway has been proposed as an atrial specific target for AF therapy, and atrial IP3 signalling has been linked to the activation of calcium sensitive adenylyl cyclases AC1 and AC8. Here we investigated the involvement of AC1 in the response of intact mouse atrial tissue and isolated guinea pig atrial and sinoatrial node (SAN) cells to the α-adrenoceptor agonist phenylephrine (PE) using the selective AC1 inhibitor ST034307. The maximum rate change of spontaneously beating mouse right atrial tissue exposed to PE was reduced from 14.46 % to 8.17% (P = 0.005) in the presence of 1 μM ST034307, whereas the increase in tension generated in paced left atrial tissue in the presence of PE was not inhibited by ST034307 (Control = 14.20 %, ST034307 = 16.32 %; P > 0.05). Experiments were performed using isolated guinea pig atrial and SAN cells loaded with Fluo-5F-AM to record changes in calcium transient amplitude (CaT) generated by 10μM PE in the presence and absence of 1μM ST034307. ST034307 significantly reduced the beating rate of SAN cells (0.34-fold decrease; P = 0.004), but did not result in an inhibition of CaT amplitude increase in response to PE in atrial cells. The results presented here demonstrate the involvement of AC1 in the downstream response of atrial pacemaker activity to α-adrenoreceptor stimulation and IP3R calcium release.

Synus node dysfunction in heart failure is characterized by reduced CaMKII signaling

Heart failure (HF) is a complex syndrome in which death rates are over 50%. The main cause of death among HF patients is pump failure and ventricular arrhythmias, but severe bradycardia is also a common cause of sudden cardiac death, pointing to sinoatrial node (SAN) dysfunction. SAN pacemaker activity is regulated by voltage-clock and Ca2+-clock mechanisms and, although voltage-clock dysfunction in SAN has been largely proved in HF, Ca2+-clock dysfunction mechanisms in SAN remains undiscovered. Here, we used a HF model in mice with transverse aortic constriction (TAC) and using telemetry saw slower heart rhythm under autonomic nervous system blockade. Then, using confocal microscopy we analyzed Ca2+ handling in HF SAN tissue and found that intracellular Ca2+ transient rates were slower in addition to less frequency of Ca2+ sparks than in SHAM SAN tissue. Next, we studied protein expression of key excitation–contraction coupling proteins and found reduced expression of the Na+/Ca2+ exchanger and reduced phosphorylated status of ryanodine receptor and phospholamban in the CaMKII sites for the SAN in TAC mice. Finally, the application of the CaMKII inhibitor KN93 caused less effect in slowing the Ca2+ transient rates in HF SAN tissue, confirming the reduced CaMKII activation. In conclusion, our data demonstrate a reduction in CaMKII activation in the Ca2+-clock function of the SAN tissue in a mouse model of HF.

Synus node dysfunction in heart failure is characterized by reduced CaMKII signaling

Heart failure (HF) is a complex syndrome in which death rates are >50%. The main causes of death among HF patients are pump failure and ventricular arrhythmias, but severe bradycardia is also a common cause of sudden cardiac death, pointing to sinoatrial node (SAN) dysfunction. SAN pacemaker activity is regulated by voltage-clock and Ca2+-clock mechanisms and, although voltage-clock dysfunction in SAN has been largely proved in HF, Ca2+-clock dysfunction mechanisms in SAN remains unraveled. Here, we used an HF model in mice with transverse aortic constriction (TAC) and, using telemetry, saw slower heart rhythm under autonomic nervous system blockade. Then, by confocal microscopy, we analyzed Ca2+ handling in HF SAN tissue and found that intracellular Ca2+ transients rate were slower together with less frequency of Ca2+ sparks than in SHAM SAN tissue. Next, we studied protein expression of key excitation–contraction coupling proteins and found reduced expression of the Na+/Ca2+ exchanger and reduced phosphorylated status of ryanodine receptor and phospholamban in the CaMKII sites for the SAN in TAC mice. Finally, the application of the CaMKII inhibitor, KN93, caused less effect in slowing the Ca2+ transient rates in HF SAN tissue, confirming the reduced CaMKII activation. In conclusion, our data demonstrates a reduction in CaMKII activation in the Ca2+-clock function of the SAN tissue in a mouse model of HF.

Hypoglycaemia combined with mild hypokalaemia reduces the heart rate and causes abnormal pacemaker activity in a computational model of a human sinoatrial cell

Low blood glucose, hypoglycaemia, has been implicated as a possible contributing factor to sudden cardiac death (SCD) in people with diabetes but it is challenging to investigate in clinical studies. We hypothesized the effects of hypoglycaemia on the sinoatrial node (SAN) in the heart to be a candidate mechanism and adapted a computational model of the human SAN action potential developed by Fabbri et al. , to investigate the effects of hypoglycaemia on the pacemaker rate. Using Latin hypercube sampling, we combined the effects of low glucose (LG) on the human ether-a-go-go-related gene channel with reduced blood potassium, hypokalaemia, and added sympathetic and parasympathetic stimulus. We showed that hypoglycaemia on its own causes a small decrease in heart rate but there was also a marked decrease in heart rate when combined with hypokalaemia. The effect of the sympathetic stimulus was diminished, causing a smaller increase in heart rate, with LG and hypokalaemia compared to normoglycaemia. By contrast, the effect of the parasympathetic stimulus was enhanced, causing a greater decrease in heart rate. We therefore demonstrate a potential mechanistic explanation for hypoglycaemia-induced bradycardia and show that sinus arrest is a plausible mechanism for SCD in people with diabetes.

Desarrollo de un simulador de la corriente funny del nodo sinusal para la enseñanza-aprendizaje

Las células del nodo sinusal generan la principal actividad eléctrica marcapaso del corazón. La actividad marcapaso se debe a la presencia de la corriente funny (If). Esta corriente iónica es entrante y se activa con la hiperpolarización en rangos de voltaje presentes durante la fase de despolarización diastólica; contrario a la mayoría de las corrientes iónicas que se activan con la despolarización. El canal funny es permeable a iones Na+ y K+. Generalmente, en el curso de biofísica a nivel de licenciatura los alumnos conocen algunos canales dependientes de voltaje del tipo de Hodgkin y Huxley que se activan con la despolarización. Sin embargo, no se estudia ningún canal que se active con una hiperpolarización. En este trabajo se diseñó y desarrolló un simulador para el estudio y comprensión de la corriente funny presente en el nodo sinusal del conejo. El simulador fue programado en lenguaje Visual Basic ver. 6.0 para ambiente Windows® de XP a Windows® 10. El usuario puede realizar los experimentos con la técnica de fijación de voltaje, modificar las variables y concentraciones de Na+ y K+ externos y observar el efecto en la amplitud de If y la cinética de la curva I-V. Se recomienda su uso como material didáctico de apoyo durante los cursos de biofísica y fisiología en una sala de cómputo o a distancia en cualquier computadora personal con recursos mínimos. The sinus node cells generate the main electric pacemaker activity of the heart. The pacemaker activity is due to the presence of the current funny (If). This ionic current is incoming and is activated with hypolarization in voltage ranges present during the diastolic depolarization phase, contrary to most ionic currents that are activated by depolarization. The funny channel is permeable to Na+ and K+ ions. Generally, students enrolled in the biophysics course at the undergraduate level are familiar with some voltage-gated channels of the Hodgkin and Huxley type. However, no channel that is activated by hiperpolarization is studied. In this work, a simulator was designed and developed for the study and understanding of the funny current present in the sinus node of rabbits. The simulator was programmed in Visual Basic Language ver. 6.0 for Windows® environment from XP to Windows® 10. The user can perform the experiments with the voltage clamp technique, modify the variables and concentrations of external Na+ and K+ and observe the effect on the amplitude of If and the kinetics of the curve I-V. It is recommended to be used as a support didactic material during biophysics and physiology courses in a computer room or remotely on any personal computer with minimal resources.

Intrinsic regulators of the action potential waveform control dopamine release to shape behavior

Despite the widely known role of dopamine in reinforcement learning, how the patterns of dopamine release that are critical to the acquisition, performance, and extinction of conditioned responses are generated is poorly resolved. Here, we demonstrate that the coordinated actions of two ion channels, Kv4.3 and BKCa1.1, control the pattern of dopamine release on different time scales to regulate separate phases of reinforced behavior in mice. Inactivation of Kv4.3 in VTA dopamine neurons increases pacemaker activity and excitability that is associated with increased ramping prior to lever press in a learned instrumental response paradigm. Loss of Kv4.3 enhanced performance of the learned response and facilitated extinction. In contrast, loss of BKCa1.1 increased burst firing and phasic dopamine release that enhanced learning of an instrumental response. Inactivation of BKCa1.1 increased the reward prediction error that was associated with an enhanced extinction burst in early extinction training. These data demonstrate that temporally distinct patterns of dopamine release are regulated by the intrinsic properties of the cell to shape behavior.

Structural Identification of the Pacemaker Cells and Expression of Hyperpolarization-Activated Cyclic Nucleotide-Gated (HCN) Channels in the Heart of the Wild Atlantic Cod, Gadus morhua (Linnaeus, 1758)

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are proteins that contain highly conserved functional domains and sequence motifs that are correlated with their unique biophysical activities, to regulate cardiac pacemaker activity and synaptic transmission. These pacemaker proteins have been studied in mammalian species, but little is known now about their heart distribution in lower vertebrates and c-AMP modulation. Here, we characterized the pacemaker system in the heart of the wild Atlantic cod (Gadus morhua), with respect to primary pacemaker molecular markers. Special focus is given to the structural, ultrastructural and molecular characterization of the pacemaker domain, through the expression of HCN channel genes and the immunohistochemistry of HCN isoforms, including the location of intracardiac neurons that are adjacent to the sinoatrial region of the heart. Similarly to zebrafish and mammals, these neurons are immunoreactive to ChAT, VAChT and nNOS. It has been shown that cardiac pacemaking can be modulated by sympathetic and parasympathetic pathways, and the existence of intracardiac neurons projecting back to the central nervous system provide a plausible link between them.