SK Current, Expressed During the Development and Regeneration of Chick Hair Cells, Contributes to the Patterning of Spontaneous Action Potentials

Chick hair cells display calcium (Ca2+)-sensitive spontaneous action potentials during development and regeneration. The role of this activity is unclear but thought to be involved in establishing proper synaptic connections and tonotopic maps, both of which are instrumental to normal hearing. Using an electrophysiological approach, this work investigated the functional expression of Ca2+-sensitive potassium [IK(Ca)] currents and their role in spontaneous electrical activity in the developing and regenerating hair cells (HCs) in the chick basilar papilla. The main IK(Ca) in developing and regenerating chick HCs is an SK current, based on its sensitivity to apamin. Analysis of the functional expression of SK current showed that most dramatic changes occurred between E8 and E16. Specifically, there is a developmental downregulation of the SK current after E16. The SK current gating was very sensitive to the availability of intracellular Ca2+ but showed very little sensitivity to T-type voltage-gated Ca2+ channels, which are one of the hallmarks of developing and regenerating hair cells. Additionally, apamin reduced the frequency of spontaneous electrical activity in HCs, suggesting that SK current participates in patterning the spontaneous electrical activity of HCs.

The natural compound TMYX reduces SAN cells rate by antagonizing the cAMP modulation of f-channels

Tongmai Yangxin (TMYX), is a complex compound of a Traditional Chinese Medicine (TCM) used to treat several cardiac rhythm disorders; however, no information regarding its mechanism of action is available. In this study we provide a detailed characterization of the effects of TMYX on the electrical activity of pacemaker cells and unravel its mechanism of action. Single-cell electrophysiology revealed that TMYX elicits a reversible and dose-dependent (2/6 mg/ml) slowing of spontaneous action potentials rate (-20.8/-50.2%) by a selective reduction of the diastolic phase (-50.1/-76.0%). This action is mediated by a negative shift of the If activation curve (-6.7/-11.9 mV) and is caused by a reduction of the cAMP-induced stimulation of pacemaker channels. We provide evidence that TMYX acts by directly antagonizes the cAMP-induced allosteric modulation of the pacemaker channels. Noticeably, this mechanism functionally resembles the pharmacological actions of muscarinic stimulation or β-blockers, but it does not require generalized changes in cytoplasmic cAMP levels thus ensuring a selective action on rate. In agreement with a competitive inhibition mechanism, TMYX exerts its maximal antagonistic action at submaximal cAMP concentrations and then progressively becomes less effective thus ensuring a full contribution of If to pacemaker rate during high metabolic demand and sympathetic stimulation.

Acute Genetic Ablation of Cardiac Sodium/Calcium Exchange in Adult Mice: Implications for Cardiomyocyte Calcium Regulation, Cardioprotection, and Arrhythmia

Background Sodium‐calcium (Ca 2+ ) exchanger isoform 1 (NCX1) is the dominant Ca 2+ efflux mechanism in cardiomyocytes and is critical to maintaining Ca 2+ homeostasis during excitation‐contraction coupling. NCX1 activity has been implicated in the pathogenesis of cardiovascular diseases, but a lack of specific NCX1 blockers complicates experimental interpretation. Our aim was to develop a tamoxifen‐inducible NCX1 knockout (KO) mouse to investigate compensatory adaptations of acute ablation of NCX1 on excitation‐contraction coupling and intracellular Ca 2+ regulation, and to examine whether acute KO of NCX1 confers resistance to triggered arrhythmia and ischemia/reperfusion injury. Methods and Results We used the α‐myosin heavy chain promoter (Myh6)‐MerCreMer promoter to create a tamoxifen‐inducible cardiac‐specific NCX1 KO mouse. Within 1 week of tamoxifen injection, NCX1 protein expression and current were dramatically reduced. Diastolic Ca 2+ increased despite adaptive reductions in Ca 2+ current and action potential duration and compensatory increases in excitation‐contraction coupling gain, sarcoplasmic reticulum Ca 2+ ATPase 2 and plasma membrane Ca2+ ATPase. As these adaptations progressed over 4 weeks, diastolic Ca 2+ normalized and SR Ca 2+ load increased. Left ventricular function remained normal, but mild fibrosis and hypertrophy developed. Transcriptomics revealed modification of cardiovascular‐related gene networks including cell growth and fibrosis. NCX1 KO reduced spontaneous action potentials triggered by delayed afterdepolarizations and reduced scar size in response to ischemia/reperfusion. Conclusions Tamoxifen‐inducible NCX1 KO mice adapt to acute genetic ablation of NCX1 by reducing Ca 2+ influx, increasing alternative Ca 2+ efflux pathways, and increasing excitation‐contraction coupling gain to maintain contractility at the cost of mild Ca 2+ ‐activated hypertrophy and fibrosis and decreased survival. Nevertheless, KO myocytes are protected against spontaneous action potentials and ischemia/reperfusion injury.

The increase of extracellular Ca2+ from physiological concentrations to hypercalcemia impairs sino-atrial automaticity

Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): ESC FRM Lefoulon Delalande Aims To investigate whether extracellular hypercalcemia alters the conduction through L-type Ca2+ channels (LTCCs), impairing the pacemaker activity of the heart. Introduction In the sino-atrial node (SAN), membrane currents and the dynamics of intracellular Ca2+ ([Ca2+]i) generate the pacemaker activity of the heart. SAN dysfunctions (SNDs) harm heart automaticity and have been associated with abnormal dynamics of [Ca2+]i. The LTCCs, Cav1.2 and Cav1.3 carry the main Ca2+ influx of SAN cells, which is necessary to sustain [Ca2+]i dynamics. Modified extracellular Ca2+ ([Ca2+]o) could alter Ca2+ influx through these channels. For example, cancer and hyperparathyroidism can raise [Ca2+]o, causing an extracellular hypercalcemia that could alter [Ca2+]i dynamics and impair SAN activity and heart automaticity. Methods and results To test this hypothesis, we measured contractions, [Ca2+]i release and L-type Ca2+ current (ICa,L) in spontaneous cells of the murine SAN. Then, we recorded rate and propagation of the spontaneous action potentials (APs) generated by the SAN tissue ex-vivo. In spontaneously beating SAN cells, we observed that the modification of [Ca2+]o affected [Ca2+]i and cell contractility through changes of ICa,L. In particular, the increase of [Ca2+]o dysregulated pacemaker activity, likely through excessive Ca2+ influx mediated by Cav1.2. [Ca2+]o increase to hypercalcemia induced arrhythmia also in the intact SAN tissues, activating ectopic leading regions of pacemaking and impairing conduction towards the atria. Conclusions Hypercalcemia causes excessive Cav1.2-mediated Ca2+ influx, which alters [Ca2+]I leading to pacemaker impairment. Modulation of LTCC may reduce pacemaker dysfunctions, preventing SND progression.

Id2 Represses Aldosterone-Stimulated Cardiac T-Type Calcium Channels Expression

Aldosterone excess is a cardiovascular risk factor. Aldosterone can directly stimulate an electrical remodeling of cardiomyocytes leading to cardiac arrhythmia and hypertrophy. L-type and T-type voltage-gated calcium (Ca2+) channels expression are increased by aldosterone in cardiomyocytes. To further understand the regulation of these channels expression, we studied the role of a transcriptional repressor, the inhibitor of differentiation/DNA binding protein 2 (Id2). We found that aldosterone inhibited the expression of Id2 in neonatal rat cardiomyocytes and in the heart of adult mice. When Id2 was overexpressed in cardiomyocytes, we observed a reduction in the spontaneous action potentials rate and an arrest in aldosterone-stimulated rate increase. Accordingly, Id2 siRNA knockdown increased this rate. We also observed that CaV1.2 (L-type Ca2+ channel) or CaV3.1, and CaV3.2 (T-type Ca2+ channels) mRNA expression levels and Ca2+ currents were affected by Id2 presence. These observations were further corroborated in a heart specific Id2- transgenic mice. Taken together, our results suggest that Id2 functions as a transcriptional repressor for L- and T-type Ca2+ channels, particularly CaV3.1, in cardiomyocytes and its expression is controlled by aldosterone. We propose that Id2 might contributes to a protective mechanism in cardiomyocytes preventing the presence of channels associated with a pathological state.

Expression and functions of N-type Cav2.2 and T-type Cav3.1 channels in rat vasopressin neurons under normotonic conditions

Arginine vasopressin (AVP) neurons play essential roles in sensing the change in systemic osmolarity and regulating AVP release from their neuronal terminals to maintain the plasma osmolarity. AVP exocytosis depends on the Ca2+ entry via voltage-gated Ca2+ channels (VGCCs) in AVP neurons. In this study, suppression by siRNA-mediated knockdown and pharmacological sensitivity of VGCC currents evidenced molecular and functional expression of N-type Cav2.2 and T-type Cav3.1 in AVP neurons under normotonic conditions. Also, both the Cav2.2 and Cav3.1 currents were found to be sensitive to flufenamic acid (FFA). TTX-insensitive spontaneous action potentials were suppressed by FFA and T-type VGCC blocker Ni2+. However, Cav2.2-selective ω-conotoxin GVIA failed to suppress the firing activity. Taken together, it is concluded that Cav2.2 and Cav3.1 are molecularly and functionally expressed and both are sensitive to FFA in unstimulated rat AVP neurons. Also, it is suggested that Cav3.1 is primarily involved in their action potential generation.