Role In Pacemaking Research Articles

Resonance in the Mouse Ventral Tegmental Area Dopaminergic Network Induced by Regular and Poisson Distributed Optogenetic Stimulation in-vitro.

Neurons in many brain regions exhibit spontaneous, intrinsic rhythmic firing activity. This rhythmic firing activity may determine the way in which these neurons respond to extrinsic synaptic inputs. We hypothesized that neurons should be most responsive to inputs at the frequency of the intrinsic oscillation frequency. We addressed this question in the ventral tegmental area (VTA), a dopaminergic nucleus in the midbrain. VTA neurons have a unique propensity to exhibit spontaneous intrinsic rhythmic activity in the 1–5 Hz frequency range, which persists in the in-vitro brain slice, and form a network of weakly coupled oscillators. Here, we combine in-vitro simultaneous recording of action potentials from a 60 channel multi-electrode-array with cell-type-specific optogenetic stimulation of the VTA dopamine neurons. We investigated how VTA neurons respond to wide-band stochastic (Poisson input) as well as regular laser pulses. Strong synchrony was induced between the laser input and the spike timing of the neurons, both for regular pulse trains and Poisson pulse trains. For rhythmically pulsed input, the neurons demonstrated resonant behavior with the strongest phase locking at their intrinsic oscillation frequency, but also at half and double the intrinsic oscillation frequency. Stochastic Poisson pulse stimulation provided a more effective stimulation of the entire population, yet we observed resonance at lower frequencies (approximately half the oscillation frequency) than the neurons' intrinsic oscillation frequency. The non-linear filter characteristics of dopamine neurons could allow the VTA to predict precisely timed future rewards based on past sensory inputs, a crucial component of reward prediction error signaling. In addition, these filter characteristics could contribute to a pacemaker role for the VTA in synchronizing activity with other regions like the prefrontal cortex and the hippocampus.

Na/Ca exchange in the atrium: Role in sinoatrial node pacemaking and excitation-contraction coupling

Na/Ca exchange is the dominant calcium (Ca) efflux mechanism in cardiac myocytes. Although our knowledge of exchanger function (NCX1 in the heart) was originally established using biochemical and electrophysiological tools such as cardiac sarcolemmal vesicles and the giant patch technique [1–4], many advances in our understanding of the physiological/pathophysiological roles of NCX1 in the heart have been obtained using a suite of genetically modified mice. Early mouse studies focused on modification of expression levels of NCX1 in the ventricles, with transgenic overexpressors, global NCX1 knockout (KO) mice (which were embryonic lethal if homozygous), and finally ventricular-specific NCX1 KO [5–12]. We found, to our surprise, that ventricular cardiomyocytes lacking NCX1 can survive and function by engaging a clever set of adaptations to minimize Ca entry, while maintaining contractile function through an increase in excitation-contraction (EC) coupling gain [5,6,13]. Having studied ventricular NCX1 ablation in detail, we more recently focused on elucidating the role of NCX1 in the atria through altering NCX1 expression. Using a novel atrial-specific NCX1 KO mouse, we found unexpected changes in atrial cell morphology and calcium handling, together with dramatic alterations in the function of sinoatrial node (SAN) pacemaker activity. In this review, we will discuss these findings and their implications for cardiac disease.

Beta-Band Resonance and Intrinsic Oscillations in a Biophysically Detailed Model of the Subthalamic Nucleus-Globus Pallidus Network.

Increased beta-band oscillatory activity in the basal ganglia network is associated with Parkinsonian motor symptoms and is suppressed with medication and deep brain stimulation (DBS). The origins of the beta-band oscillations, however, remains unclear with both intrinsic oscillations arising within the subthalamic nucleus (STN)—external globus pallidus (GPe) network and exogenous beta-activity, originating outside the network, proposed as potential sources of the pathological activity. The aim of this study was to explore the relative contribution of autonomous oscillations and exogenous oscillatory inputs in the generation of pathological oscillatory activity in a biophysically detailed model of the parkinsonian STN-GPe network. The network model accounts for the integration of synaptic currents and their interaction with intrinsic membrane currents in dendritic structures within the STN and GPe. The model was used to investigate the development of beta-band synchrony and bursting within the STN-GPe network by changing the balance of excitation and inhibition in both nuclei, and by adding exogenous oscillatory inputs with varying phase relationships through the hyperdirect cortico-subthalamic and indirect striato-pallidal pathways. The model showed an intrinsic susceptibility to beta-band oscillations that was manifest in weak autonomously generated oscillations within the STN-GPe network and in selective amplification of exogenous beta-band synaptic inputs near the network's endogenous oscillation frequency. The frequency at which this resonance peak occurred was determined by the net level of excitatory drive to the network. Intrinsic or endogenously generated oscillations were too weak to support a pacemaker role for the STN-GPe network, however, they were considerably amplified by sparse cortical beta inputs and were further amplified by striatal beta inputs that promoted anti-phase firing of the cortex and GPe, resulting in maximum transient inhibition of STN neurons. The model elucidates a mechanism of cortical patterning of the STN-GPe network through feedback inhibition whereby intrinsic susceptibility to beta-band oscillations can lead to phase locked spiking under parkinsonian conditions. These results point to resonance of endogenous oscillations with exogenous patterning of the STN-GPe network as a mechanism of pathological synchronization, and a role for the pallido-striatal feedback loop in amplifying beta oscillations.

Clinically Focused Review of Diagnosis and Treatment of Cardiovascular Implantable Electronic Device Infections

Context: The role of cardiac pacemakers (PPMs), implantable cardioverter-defibrillators (ICDs), and cardiac resynchronization therapy (CRT) devices has increasingly prominent in cardiac disease management in the last 50 years. Evidence Acquisition: We reviewed the literature for cardiovascular implantable electronic device (CIED)-related infections, their diagnosis, treatment, complications, and long-term treatment. Results: The most common signs of CIED infection include inflammatory changes in the generator pocket area. Evidence of dehiscence of the infected skin due to percutaneous exposure to the generator or even its leads is commonly reported. Timely diagnosis and subsequent treatment of patients with possible CIED infections are vital, which could otherwise affect prognosis. In the case of CIED-related infections close to the surface of the pocket site without the direct involvement of the device, removal is not deemed necessary. The oral administration of antibiotics with antistaphylococcal activity is suggested for 7 to 10 days. If there is the infection of bloodstream or endocarditis, the extraction of the entire system is considered mandatory along with antimicrobial therapy. Patients who suffer from CIED infections and cannot be subjected to device removal using percutaneous or surgical methods must be given long-term antimicrobial suppressive therapy. Such cases are those who are expected to have a short-term life expectancy or do not accept device removal. Conclusions: CIED infection is life-threatening. Early diagnosis is crucial to the survival of the patients. The use of antibiotics and a conservative approach without the CIED system removal may not be enough to cure this condition.

Ano1 mediates pressure-sensitive contraction frequency changes in mouse lymphatic collecting vessels.

Lymphatic collecting vessels exhibit spontaneous contractions with a pressure-dependent contraction frequency. The initiation of contraction has been proposed to be mediated by the activity of a Ca2+-activated Cl- channel (CaCC). Here, we show that the canonical CaCC Anoctamin 1 (Ano1, TMEM16a) plays an important role in lymphatic smooth muscle pacemaking. We find that isolated murine lymphatic muscle cells express Ano1, and demonstrate functional CaCC currents that can be inhibited by the Ano1 inhibitor benzbromarone. These currents are absent in lymphatic muscle cells from Cre transgenic mouse lines targeted for Ano1 genetic deletion in smooth muscle. We additionally show that loss of functional Ano1 in murine inguinal-axillary lymphatic vessels, whether through genetic manipulation or pharmacological inhibition, results in an impairment of the pressure-frequency relationship that is attributable to a hyperpolarized resting membrane potential and a significantly depressed diastolic depolarization rate preceding each action potential. These changes are accompanied by alterations in action potential shape and duration, and a reduced duration but increased amplitude of the action potential-induced global "Ca2+ flashes" that precede lymphatic contractions. These findings suggest that an excitatory Cl- current provided by Ano1 is critical for mediating the pressure-sensitive contractile response and is a major component of the murine lymphatic action potential.

Abstract 17303: Permanent Cardiac Pacemaker Implant Improves Sleep Habit

Background: Permanent cardiac pacemaker is indicated in patients with symptomatic bradycardia. Its role in sleep disorder is unknown, however some patient report improvement in their sleep habit after permanent cardiac pacemaker implant. We examined the role of permanent cardiac pacemaker in relation to patients’ sleeping habits. Methods: Total of 174 patients who agreed to participate in the study were included (21 Implantable Cardioverter Defibrillator (ICD) and 153 pacemaker implants). The ICD group was used as the control group. Patients with bi-ventricular pacemaker implants for cardiac re-synchronization therapy were excluded from the study. Questionnaires about the sleep habit were evaluated before and after the device implant and analyzed using paired T test and Chi square test. Results: The indications for permanent pacemaker therapy were high grade AV block (33%), sick sinus syndrome (64%) and syncope (1%). Indications of ICD were primary or secondary sudden cardiac death prevention without history of known symptomatic bradycardia (100%). Mean Ventricular pacing was 40.39% (interquartile range: 97%) of the time in the pacemaker group. There were no baseline differences in sleep duration and quality of sleep in both the ICD and pacemaker group prior to device implant. Improved sleep habit (46% in pacemaker vs 23% in the ICD group, P=0.04) and better quality of sleep (44% in pacemaker vs 19% in ICD, P=0.01) were reported after device implant. The patients in pacemaker group reported much improvement in sleep duration (average by 30 minutes in Pacemaker vs 15 minutes in ICD, P=0.002). Conclusions: Patients who had permanent pacemaker implant for symptomatic bradycardia showed improved sleep quality and duration than the patients with ICD implant for primary or secondary sudden cardiac death prevention. Mechanisms of improved sleep habit in pacemaker group are unclear, but possibly associated with improved cardiac output with consistent heat rate during sleep from the pacemaker. Further large-scale studies are needed to validate this concept.

Tricuspid Valve Dysfunction Caused by Right Ventricular Leads.

Tricuspid regurgitation is increasingly recognized as a clinically significant valvular condition. The role of multiple pacemaker and implantable cardiac defibrillator leads in distortion of the valve structure and the risk of trauma to the valve and subvalvular apparatus with lead extraction contribute to the development of tricuspid regurgitation (TR). There is a clinical imperative to better understand the optimal way to diagnose lead-related TR, risk factors for the development of TR, and optimal strategies to mitigate this problem.

Regulation of the RyR channel gating by Ca2+ and Mg2.

Ryanodine receptors (RyRs) are the Ca2+ release channels in the sarcoplasmic reticulum in striated muscle which play an important role in excitation-contraction coupling and cardiac pacemaking. Single channel recordings have revealed a wealth of information about ligand regulation of RyRs from mammalian skeletal and cardiac muscle (RyR1 and RyR2, respectively). RyR subunit has a Ca2+ activation site located in the luminal and cytoplasmic domains of the RyR. These sites synergistically feed into a common gating mechanism for channel activation by luminal and cytoplasmic Ca2+. RyRs also possess two inhibitory sites in their cytoplasmic domains with Ca2+ affinities of the order of 1μM and 1mM. Magnesium competes with Ca2+ at these sites to inhibit RyRs and this plays an important role in modulating their Ca2+-dependent activity in muscle. This review focuses on how these sites lead to RyR modulation by Ca2+ and Mg2+ and how these mechanisms control Ca2+ release in excitation-contraction coupling and cardiac pacemaking.

Heterogeneous functional expression of the sustained inward Na+ current in guinea pig sinoatrial node cells.

The sustained inward Na+ current (I st) identified in the sinoatrial node (SAN) cell has been suggested to play a pivotal role in cardiac pacemaking. However, the composition of cells in the SAN is heterogeneous and cell-to-cell variability in the magnitude of I st remains to be fully characterized. The present study investigated the current density of I st in morphologically different types of pacemaker cells dissociated from guinea pig SAN. I st was preferentially detected in spontaneously active spindle or spider-shaped cells, but was less well expressed in larger-sized elongated spindle-type cells and practically absent in clearly striated atrial-like cells, despite clear expression of the funny current (I f). The current density of I st in spindle and spider cells varied from 0.7 to 1.6 pA pF-1 and was significantly reduced in non-beating cells with similar morphologies. By linear regression analysis, we identified a positive correlation between the current densities of I st and the L-type Ca2+ current (I Ca,L), which was specifically observed in spindle and spider cells. These cells exhibited a more negative voltage for half maximal I Ca,L activation than atrial-like cells, suggesting a variable ratio between CaV1.2- and CaV1.3-mediated I Ca,L in SAN cells. Consistent single-cell transcript measurements confirmed a higher relative expression of CaV1.3, which activates at more negative potentials, in spindle cells than in atrial-like cells. Taken together, these results can be interpreted as indicating that I st plays a specific role in primary pacemaker cells and that its presence is closely correlated with functional levels of CaV1.3-mediated I Ca,L.

The Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels: from Biophysics to Pharmacology of a Unique Family of Ion Channels.

Hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels are important members of the voltage-gated pore loop channels family. They show unique features: they open at hyperpolarizing potential, carry a mixed Na/K current, and are regulated by cyclic nucleotides. Four different isoforms have been cloned (HCN1-4) that can assemble to form homo- or heterotetramers, characterized by different biophysical properties. These proteins are widely distributed throughout the body and involved in different physiologic processes, the most important being the generation of spontaneous electrical activity in the heart and the regulation of synaptic transmission in the brain. Their role in heart rate, neuronal pacemaking, dendritic integration, learning and memory, and visual and pain perceptions has been extensively studied; these channels have been found also in some peripheral tissues, where their functions still need to be fully elucidated. Genetic defects and altered expression of HCN channels are linked to several pathologies, which makes these proteins attractive targets for translational research; at the moment only one drug (ivabradine), which specifically blocks the hyperpolarization-activated current, is clinically available. This review discusses current knowledge about HCN channels, starting from their biophysical properties, origin, and developmental features, to (patho)physiologic role in different tissues and pharmacological modulation, ending with their present and future relevance as drug targets.

Memory in a fractional-order cardiomyocyte model alters properties of alternans and spontaneous activity

Cardiac memory is the dependence of electrical activity on the prior history of one or more system state variables, including transmembrane potential (Vm), ionic current gating, and ion concentrations. While prior work has represented memory either phenomenologically or with biophysical detail, in this study, we consider an intermediate approach of a minimal three-variable cardiomyocyte model, modified with fractional-order dynamics, i.e., a differential equation of order between 0 and 1, to account for history-dependence. Memory is represented via both capacitive memory, due to fractional-order Vm dynamics, that arises due to non-ideal behavior of membrane capacitance; and ionic current gating memory, due to fractional-order gating variable dynamics, that arises due to gating history-dependence. We perform simulations for varying Vm and gating variable fractional-orders and pacing cycle length and measure action potential duration (APD) and incidence of alternans, loss of capture, and spontaneous activity. In the absence of ionic current gating memory, we find that capacitive memory, i.e., decreased Vm fractional-order, typically shortens APD, suppresses alternans, and decreases the minimum cycle length (MCL) for loss of capture. However, in the presence of ionic current gating memory, capacitive memory can prolong APD, promote alternans, and increase MCL. Further, we find that reduced Vm fractional order (typically less than 0.75) can drive phase 4 depolarizations that promote spontaneous activity. Collectively, our results demonstrate that memory reproduced by a fractional-order model can play a role in alternans formation and pacemaking, and in general, can greatly increase the range of electrophysiological characteristics exhibited by a minimal model.

The pacemaker role of thalamic reticular nucleus in controlling spike-wave discharges and spindles.

Absence epilepsy, characterized by 2-4 Hz spike-wave discharges (SWDs), can be caused by pathological interactions within the thalamocortical system. Cortical spindling oscillations are also demonstrated to involve the oscillatory thalamocortical rhythms generated by the synaptic circuitry of the thalamus and cortex. This implies that SWDs and spindling oscillations can share the common thalamocortical mechanism. Additionally, the thalamic reticular nucleus (RE) is hypothesized to regulate the onsets and propagations of both the epileptic SWDs and sleep spindles. Based on the proposed single-compartment thalamocortical neural field model, we firstly investigate the stimulation effect of RE on the initiations, terminations, and transitions of SWDs. It is shown that the activations and deactivations of RE triggered by single-pulse stimuli can drive the cortical subsystem to behave as the experimentally observed onsets and self-abatements of SWDs, as well as the transitions from 2-spike and wave discharges (2-SWDs) to SWDs. In particular, with increasing inhibition from RE to the specific relay nucleus (TC), rich transition behaviors in cortex can be obtained through the upstream projection path, RE→TC→Cortex. Although some of the complex dynamical patterns can be expected from the earlier single compartment thalamocortical model, the effect of brain network topology on the emergence of SWDs and spindles, as well as the transitions between them, has not been fully investigated. We thereby develop a spatially extended 3-compartment coupled network model with open-/closed-end connective configurations, to investigate the spatiotemporal effect of RE on the SWDs and spindles. Results show that the degrees of activations of RE1 can induce the rich spatiotemporal evolution properties including the propagations from SWDs to spindles within different compartments and the transitions between them, through the RE1→TC1→Cortex1 and Cortex1→Cortex2→Cortex3 projecting paths, respectively. Overall, those results imply that RE possesses the pacemaker function in controlling SWDs and spindling oscillations, which computationally provide causal support for the involvement of RE in absence seizures and sleep spindles.

HCN2 ion channels: basic science opens up possibilities for therapeutic intervention in neuropathic pain.

Nociception - the ability to detect painful stimuli - is an invaluable sense that warns against present or imminent damage. In patients with chronic pain, however, this warning signal persists in the absence of any genuine threat and affects all aspects of everyday life. Neuropathic pain, a form of chronic pain caused by damage to sensory nerves themselves, is dishearteningly refractory to drugs that may work in other types of pain and is a major unmet medical need begging for novel analgesics. Hyperpolarisation-activated cyclic nucleotide (HCN)-modulated ion channels are best known for their fundamental pacemaker role in the heart; here, we review data demonstrating that the HCN2 isoform acts in an analogous way as a 'pacemaker for pain', in that its activity in nociceptive neurons is critical for the maintenance of electrical activity and for the sensation of chronic pain in pathological pain states. Pharmacological block or genetic deletion of HCN2 in sensory neurons provides robust pain relief in a variety of animal models of inflammatory and neuropathic pain, without any effect on normal sensation of acute pain. We discuss the implications of these findings for our understanding of neuropathic pain pathogenesis, and we outline possible future opportunities for the development of efficacious and safe pharmacotherapies in a range of chronic pain syndromes.

Temporary Transvenous Pacemaker Implantation during Carotid Endarterectomy in Patients with Trifascicular Block

The risk of severe perioperative bradyarrhythmias in patients with chronic left bundle branch, or bifascicular block, and an additional first-degree atrioventricular block undergoing carotid endarterectomy (CEA) has never been specifically addressed. In this study, we aimed to investigate whether these conduction abnormalities entail an increased risk of hemodynamic compromise during CEA and the role of temporary transvenous pacemaker (TTVPM) implantation as a prophylactic measure in this subgroup of patients. Between June 2006 and June 2013, 37 CEAs were performed in 31 patients (29 men, mean age 76±6years), in whom a TTVPM was implanted for a trifascicular block. Thirty-seven concurrent, consecutive patients operated for other vascular pathologies also with a prophylactic TTVPM for an asymptomatic trifascicular block served as controls. Adverse events were considered: pacemaker activation, block progression, bradycardia ≤40 beats/min, and asystole. Study and control groups were overall comparable. No perioperative mortality was recorded. All patients undergoing CEA were asymptomatic for syncope preoperatively. Among them, in 34 cases, indication for TTVPM was based on preoperative EKG, and in 4, a pacemaker activation was recorded. Three additional patients were also included in the study group in whom TTVPM was implanted due to the occurrence of adverse advents, and not prophylactically. In 2 of these, severe bradycardia with eventual asystole occurred intraoperatively. In both cases, the procedure was discontinued and rescheduled for the following day after a TTVPM was implanted. In the last additional case, the patient had a block progression on day 1 after an uneventful CEA and was emergently treated with a TTVPM. Overall, 7 adverse events were recorded in the study group, and none in the control group (P<0.011). Morbidity in the CEA group also included 1 myocardial infarction, 1 minor stroke, 1 surgical revision for cervical hematoma, 1 new-onset atrial fibrillation, and 1 femoral artery pseudoaneurysm. In our experience, TTVPM implantation was a clinically useful adjunct in patients with trifascicular block submitted to CEA, as compared with other vascular surgical procedures. However, the risks inherent to CEA in this subgroup of patients suggest that surgical treatment may not be warranted for those with asymptomatic carotid disease.

Current understanding of the pathophysiological mechanisms responsible for inappropriate sinus tachycardia: role of the If "funny" current.

Together with the afferent branches of the autonomic nervous system, the sinoatrial node (SAN) forms a functional unit whose function is to fire rhythmic action potentials at a rate optimal for coping with the metabolic needs of the body. Dysfunctional behavior of this complex unit may thus result in SAN rhythm disorders. Among these disorders, there is the inappropriate sinus tachycardia (IST) which occurs when an unjustified fast SAN rate is present. We here present a critical review of the role of pacemaker f/HCN channels in cardiac rhythm generation and modulation and their involvement in IST. Recent evidence demonstrates that a familial form of IST is associated with a gain-of-function mutation in the HCN4 pacemaker channel (R524Q) which confers an increased sensitivity to the second messenger cAMP, a key mediator in sympathetic modulation. This finding is consistent with the general view that hypersympathetic tone is one of the causes of IST and introduces the novel concept of defective funny channel-dependent tachyarrhythmias.

TRPC3 regulates the automaticity of embryonic stem cell-derived cardiomyocytes

BackgroundCardiac pacemaking is a complex phenomenon that is not completely understood. Canonical transient receptor potential isoform 3 (TRPC3) channel is a cation channel that permeates both Ca2+ and Na+. TRPC3 was previously found to express in adult cardiomyocytes. However, its role in cardiac pacemaking is unexplored. Here we used mouse embryonic stem cell-derived cardiomyocytes (mESC-CMs) to investigate whether TRPC3 regulates the spontaneous automaticity and the underlying mechanism involved. Methods and resultsImmunocytochemistry results showed that TRPC3 is expressed at the T-tubules of mESC-CMs. Whole-cell patch clamping showed that single mESC-CMs contain TRPC3 current. Confocal Ca2+ imaging showed that the TRPC3-specific blocker Pyr3 decreased Ca2+ transients and local Ca2+ release (LCR) of mESC-CMs. Combined current and voltage clamp recordings from the same cell showed that reducing the TRPC3 current, either by Pyr3 or a dominant negative (loss-of-function) construct of TRPC3, decreased the pacemaker activity of mESC-CMs as reflected by a decrease in action potential rate, a depolarized maximum diastolic potential and a decrease in slope of phase 4 diastolic depolarization. Furthermore, decreasing the TRPC3 current diminished, while increasing the TRPC3 current augmented the sodium–calcium exchanger (NCX) current in mESC-CMs. Lastly, decrease in TRPC3 current decreased the phosphorylation of ryanodine receptor isoform 2 at Ser2809 and phospholamban at Thr17. ConclusionsTRPC3 positively regulates diastolic depolarization of spontaneous action potential by increasing LCR and NCX current and therefore is an important determinant in pacemaking of mESC-CMs.

Clustering and phase synchronization in populations of coupled phase oscillators

In many species daily rhythms are endogenously generated by groups of coupled neurons that play the role of a circadian pacemaker. The adaptation of the circadian clock to environmental and seasonal changes has been proposed to be regulated by a dual oscillator system. In order to gain insight into this model, we analyzed the synchronization properties of two fully coupled groups of Kuramoto oscillators. Each group has an internal coupling parameter and the interaction between the two groups can be controlled by two parameters allowing for symmetric or non-symmetric coupling. We show that even for such a simple model counterintuitive behaviours take place, such as a global decrease in synchrony when the coupling between the groups is increased. Through a detailed analysis of the local synchronization processes we explain this behaviour.

R-Type Voltage-Gated Ca²⁺ Channels in Cardiac and Neuronal Rhythmogenesis.

During the past decades, an increasing number of ion channel and transporter types have been identified acting together to produce cardiac and neuronal pacemaker action potentials. The basis of pacemaker activity was understood in more detail by using single-microelectrode recordings on cells isolated from pacemaker regions. Meanwhile, this powerful technique was complemented by computer modeling and recombinant technologies, including gene inactivation of ion channels and transporters, which may be involved in the generation of the electrical activity of pacemaker cells. Several genes of the voltage-gated Ca(2+) channel (VGCC) family have been ablated, and their role in cardiac and neuronal pacemaking is compared in the present summary, focusing on the role of murine R-type voltage-gated Ca(2+) channels encoded by cacna1e and expressing the ion conducting subunit Cav2.3.

Pacemaker role of pericytes in generating synchronized spontaneous Ca2+ transients in the myenteric microvasculature of the guinea-pig gastric antrum

Properties of spontaneous Ca2+ transients in the myenteric microvasculature of the guinea-pig stomach were investigated. Specifically, we explored the spatio-temporal origin of Ca2+ transients and the role of voltage-dependent Ca2+ channels (VDCCs) in their intercellular synchrony using fluorescence Ca2+ imaging and immunohistochemistry.The microvasculature generated spontaneous Ca2+ transients that were independent of both Ca2+ transients in interstitial cells of Cajal (ICC) and neural activity. Spontaneous Ca2+ transients were highly synchronous along the length of microvasculature, and appeared to be initiated in pericytes and spread to arteriolar smooth muscle cells (SMCs). In most cases, the generation or synchrony of Ca2+ transients was not affected by blockers of L-type VDCCs. In nifedipine-treated preparations, synchronous spontaneous Ca2+ transients were readily blocked by Ni2+, mibefradil or ML216, blockers for T-type VDCCs. These blockers also suppressed the known T-type VDCC dependent component of ICC Ca2+ transients or slow waves. Spontaneous Ca2+ transients were also suppressed by caffeine, tetracaine or cyclopiazonic acid (CPA). After the blockade of both L- and T-type VDCCs, asynchronous Ca2+ transients were generated in pericytes on precapillary arterioles and/or capillaries but not in arteriolar SMCs, and were abolished by CPA or nominally Ca2+ free solution.Together these data indicate that pericytes in the myenteric microvasculature may act as the origin of synchronous spontaneous Ca2+ transients. Pericyte Ca2+ transients arise from Ca2+ release from the sarco-endoplasmic reticulum and the opening of T-type Ca2+ VDCCs is required for their synchrony and propagation to arteriolar SMCs.

Store-operated calcium entry and the localization of STIM1 and Orai1 proteins in isolated mouse sinoatrial node cells.

In many non-excitable and excitable cells, store-operated calcium entry (SOCE) represents an additional pathway for calcium entry upon Ca2+ store depletion. In a previous study, we demonstrated SOCE activity in intact mouse cardiac pacemaker tissue, specifically from sinoatrial node (SAN) tissue. However, store content as a key determinant of SOCE activity is difficult to measure in intact SAN tissue. Therefore, to investigate the interaction between SOCE and store content and its role in cardiac pacemaking, it is necessary to investigate SOCE activity in single cardiac pacemaker cells. Furthermore, recent studies in other tissues have identified two new proteins involved in SOCE, stromal interacting molecule (STIM), which is an ER Ca2+ sensor, and the surface membrane channel Orai, a prototypic gene encoding for SOCE. However, whether STIM and Orai are expressed in native pacemaker cells is still unknown. In this current study, we examined SOCE activity in single firing pacemaker cells isolated from mouse sinoatrial node tissue. We found a significant rise in Ca2+ entry in response to Ca2+ store depletion. SOCE blockers reduced the amplitude and frequency of spontaneous Ca2+ transients and reduced Ca2+ store content. We demonstrated for the first time that STIM and Orai are expressed in pacemaker cells. After store depletion, STIM1 redistributed to the cell periphery and showed increased co-localization with surface membrane located Orai1, indicating a possible involvement of these proteins in SOCE activity in native cardiac pacemaker cells. These results suggest the novel concept that SOCE plays a functional role in regulating intracellular Ca2+ of cardiac pacemaker cells.