Asynchronous Ca2 Research Articles

Loss-of-function W4645R mutation in the RyR2-caffeine binding site: implications for synchrony and arrhythmogenesis

AimsPrevious studies have identified RyR2 W4645R mutation, located in the caffeine-binding site, to associate with CPVT1 pathology. Caffeine binding to its site is thought to displace the carboxyl-terminal domain to Ca2+-binding, allowing the tryptophan residue (W4645) to regulate Ca2+ sensitivity of RyR2. To gain insights into regulation of RyR2 Ca2+-binding and its interaction with caffeine-binding site, we introduced W4645R-RyR2 point mutation via CRISPR/Cas9 gene-editing in human induced pluripotent stem cell-derived cardiomyocytes (hiPSCCMs) and characterized their Ca2+-signaling phenotype compared to WT hiPSCCMs. Methods and ResultsW4645R-RyR2 cardiomyocytes had: (1) no significant change in ICa magnitude or voltage-dependence; (2) slightly reduced CICR; (3) altered relaxation kinetics of Ca2+-transients with no change in isoproterenol sensitivity; (4) complete loss of caffeine-triggered Ca2+ release; (5) larger SR Ca2+ leak resulting in 40 % lower SR Ca2+ content, as determined by myocytes’ response to 4-CmC; (6) lower incidence of calcium sparks and asynchronous spontaneous SR Ca2+ releases. ConclusionsW4645R-RyR2 mutation induces loss of caffeine-triggered SR Ca2+ release and enhances SR Ca2+ leak that underlie asynchronous spontaneous Ca2+ releases, triggering arrhythmia and impairing cardiac function.

Functional nitrergic innervation of smooth muscle structures in the mucosa of pig lower urinary tract.

Neurally released nitric oxide (NO) functions as an inhibitory neurotransmitter of urethral but not detrusor smooth muscles while relaxing bladder vasculature and muscularis mucosae (MM). Here, the distribution of nitrergic nerves was examined in the mucosa of pig lower urinary tract using immunohistochemistry, and their vasodilatory functions were studied by measuring arteriolar diameter changes. Properties of smooth muscle cells in the lamina propria (SMC-LP) of urethra and trigone were also investigated using florescence Ca2+ imaging. In the bladder mucosa, neuronal nitric oxide synthase (nNOS)-immunoreactive nitrergic fibres projected to suburothelial arterioles and venules. Perivascular nitrergic nerves were intermingled with but distinct from tyrosine hydroxylase (TH)-immunoreactive sympathetic or calcitonin gene-related peptide (CGRP)-immunoreactive afferent nerves. MM receive a nitrergic but not sympathetic or afferent innervation. In the mucosa of urethra and trigone, nitrergic nerves were in close apposition with sympathetic or afferent nerves around suburothelial vasculature but did not project to SMC-LP. In suburothelial arterioles of bladder and urethra, N ω-nitro-L-arginine (L-NA, 100μM), an NOS inhibitor, enhanced electrical field stimulation (EFS)-induced sympathetic vasoconstrictions, while tadalafil (10nM), a phosphodiesterase type 5 (PDE5) inhibitor, suppressed the vasoconstrictions. SMC-LP developed asynchronous spontaneous Ca2+ transients without responding to EFS. The spontaneous Ca2+ transients were enhanced by acetylcholine (1μM) and diminished by noradrenaline (1μM) but not SIN-1 (10μM), an NO donor. In the lower urinary tract mucosa, perivascular nitrergic nerves appear to counteract the sympathetic vasoconstriction to maintain the mucosal circulation. Bladder MM but not SMC-LP receive an inhibitory nitrergic innervation.

Expression and distribution of synaptotagmin family members in the zebrafish retina.

Synaptotagmins belong to a large family of proteins. Although various synaptotagmins have been implicated as Ca2+ sensors for vesicle replenishment and release at conventional synapses, their roles at retinal ribbon synapses remain incompletely understood. Zebrafish is a widely used experimental model for retinal research. We therefore investigated the homology between human, rat, mouse, and zebrafish synaptotagmins 1-10 using a bioinformatics approach. We also characterized the expression and distribution of various synaptotagmin (syt) genes in the zebrafish retina using RT-PCR, qPCR, and in situhybridization, focusing on the family members whose products likely underlie Ca2+ -dependent exocytosis in the central nervous system (synaptotagmins 1, 2, 5, and 7). Most zebrafish synaptotagmins are well conserved and can be grouped in the same classes as mammalian synaptotagmins, based on crucial amino acid residues needed for coordinating Ca2+ binding and determining phospholipid binding affinity. The only exception is synaptotagmin 1b, which lacks 34 amino acid residues in the C2B domain and is therefore unlikely to bind Ca2+ there. Additionally, the products of zebrafish syt5a and syt5b genes share identity with mammalian class 1 and 5 synaptotagmins. Zebrafish syt1, syt2, syt5, and syt7 paralogues are found in the zebrafish brain, eye, and retina, excepting syt1b, which is only present in the brain. The complementary expression pattern of the remaining paralogues in the retina suggests that syt1a and syt5a may underlie synchronous release and syt7a and syt7b may mediate asynchronous release or other Ca2+ -dependent processes in different retinal neurons.

High spatial and temporal resolution Ca2+ imaging of myocardial strips from human, pig and rat.

Ca2+ handling within cardiac myocytes underpins coordinated contractile function within the beating heart. This protocol enables high spatial and temporal Ca2+ imaging of ex vivo multicellular myocardial strips. The endocardial surface is retained, and strips of 150-300-µm thickness are dissected, loaded with Ca2+ indicators and mounted within 1.5 h. A list of the equipment and reagents used and the key methodological aspects allowing the use of this technique on strips from any chamber of the mammalian heart are described. We have successfully used this protocol on human, pig and rat biopsy samples. On use of this protocol with intact endocardial endothelium, we demonstrated that the myocytes develop asynchronous spontaneous Ca2+ events, which can be ablated by electrically evoked Ca2+ transients, and subsequently redevelop spontaneously after cessation of stimulation. This protocol thus offers a rapid and reliable method for studying the Ca2+ signaling underpinning cardiomyocyte contraction, in both healthy and diseased tissue.

RyR2 Gain-of-Function and Not So Sudden Cardiac Death

RyR2 Gain-of-Function and Not So Sudden Cardiac Death

CA2+ Handling in Non-Failing Hypertrophic Cardiomyocytes Subjected to Inotropic Interventions

Impaired force development during ventricular hypertrophy has been associated with structural and functional alterations of Ca2+ handling proteins. Previous findings in whole hearts and multicellular trabeculae from a rat model of right ventricular (RV) hypertrophy showed spontaneous contractile activity and asynchronous Ca2+ release during inotropic interventions. Our aim was to investigate whether these changes occurred at the single cell level. Male Wistar rats were injected with 60 mg kg−1 of monocrotaline (MCT, n=3) or saline as control (CON, n=4). Four weeks post injection, hearts were isolated and enzymatically digested to yield rod-shaped quiescent cardiomyocytes. Aliquots of RV myocytes were collected and measurements of cell shortening and intracellular Ca2+ (fura-2AM, 340/380 ratio) were made in response to inotropic interventions (increased stimulation frequency and ß-adrenergic stimulation). Application of 20mM caffeine revealed Ca2+ content of the sarcoplasmic reticulum (SR). A separate group of cardiomyocytes were fixed and key proteins were labelled with antibodies for subsequent confocal imaging. MCT myocytes had the largest cross-sectional area (4338 ± 292µm2) versus CON (3170 ± 262µm2, P=0.01), although no difference in cell shortening was observed in response to 1 Hz stimulation. MCT myocytes had larger Ca2+ transients at all stimulation frequencies (P= 0.004) and increased spontaneous activity during ß-adrenergic stimulation. No difference between groups was found in Ca2+ store content, although the time constant of caffeine transient decay was prolonged in MCT (17.3 ± 1.5s) versus CON (13.7 ± 0.6s, P=0.05). Although there was no difference in shortening or SR Ca2+ content, there was evidence of changes in Ca2+ handling in MCT myocytes during inotropic stimulation. Furthermore, hypertrophic MCT myocytes exhibited slower trans-sarcolemmal Ca2+ removal during 20mM caffeine, potentially as a result of disorganized T-tubular arrangement and/or reduced Na+/Ca2+ exchanger abundance.

Interaction of the Joining Region in Junctophilin-2 With the L-Type Ca2+ Channel Is Pivotal for Cardiac Dyad Assembly and Intracellular Ca2+ Dynamics.

Ca2+-induced Ca2+ release (CICR) in normal hearts requires close approximation of L-type calcium channels (LTCCs) within the transverse tubules (T-tubules) and RyR (ryanodine receptors) within the junctional sarcoplasmic reticulum. CICR is disrupted in cardiac hypertrophy and heart failure, which is associated with loss of T-tubules and disruption of cardiac dyads. In these conditions, LTCCs are redistributed from the T-tubules to disrupt CICR. The molecular mechanism responsible for LTCCs recruitment to and from the T-tubules is not well known. JPH (junctophilin) 2 enables close association between T-tubules and the junctional sarcoplasmic reticulum to ensure efficient CICR. JPH2 has a so-called joining region that is located near domains that interact with T-tubular plasma membrane, where LTCCs are housed. The idea that this joining region directly interacts with LTCCs and contributes to LTCC recruitment to T-tubules is unknown. To determine if the joining region in JPH2 recruits LTCCs to T-tubules through direct molecular interaction in cardiomyocytes to enable efficient CICR. Modified abundance of JPH2 and redistribution of LTCC were studied in left ventricular hypertrophy in vivo and in cultured adult feline and rat ventricular myocytes. Protein-protein interaction studies showed that the joining region in JPH2 interacts with LTCC-α1C subunit and causes LTCCs distribution to the dyads, where they colocalize with RyRs. A JPH2 with induced mutations in the joining region (mutPG1JPH2) caused T-tubule remodeling and dyad loss, showing that an interaction between LTCC and JPH2 is crucial for T-tubule stabilization. mutPG1JPH2 caused asynchronous Ca2+-release with impaired excitation-contraction coupling after β-adrenergic stimulation. The disturbed Ca2+ regulation in mutPG1JPH2 overexpressing myocytes caused calcium/calmodulin-dependent kinase II activation and altered myocyte bioenergetics. The interaction between LTCC and the joining region in JPH2 facilitates dyad assembly and maintains normal CICR in cardiomyocytes.

Sarcoplasmic reticulum calcium mishandling: central tenet in heart failure?

Excitation-contraction coupling links excitation of the sarcolemmal surface membrane to mechanical contraction. In the heart this link is established via a Ca2+-induced Ca2+ release process, which, following sarcolemmal depolarisation, prompts Ca2+ release from the sarcoplasmic reticulum (SR)though the ryanodine receptor (RyR2). This substantially raises the cytoplasmic Ca2+ concentration to trigger systole. In diastole, Ca2+ is removed from the cytoplasm, primarily via the sarcoplasmic-endoplasmic reticulum Ca2+-dependent ATPase (SERCA) pump on the SRmembrane, returning Ca2+ to the SR store. Ca2+ movement across the SR is thus fundamental to the systole/diastole cycle and plays an essential role in maintaining cardiac contractile function. Altered SR Ca2+ homeostasis (due to disrupted Ca2+ release, storage, and reuptake pathways) is a central tenet of heart failure and contributes to depressed contractility, impaired relaxation, and propensity to arrhythmia. This review will focus on the molecular mechanisms that underlie asynchronous Ca2+ cycling around the SR in the failing heart. Further, this review will illustrate that the combined effects of expression changes and disruptions to RyR2 and SERCA2a regulatory pathways are critical to the pathogenesis of heart failure.

A novel intramuscular Interstitial Cell of Cajal is a candidate for generating pacemaker activity in the mouse internal anal sphincter

The internal anal sphincter (IAS) generates phasic contractions and tone. Slow waves (SWs) produced by interstitial cells of Cajal (ICC) underlie phasic contractions in other gastrointestinal regions. SWs are also present in the IAS where only intramuscular ICC (ICC-IM) are found, however the evidence linking ICC-IM to SWs is limited. This study examined the possible relationship between ICC-IM and SWs by recording Ca2+ transients in mice expressing a genetically-encoded Ca2+-indicator in ICC (Kit-Cre-GCaMP6f). A role for L-type Ca2+ channels (CavL) and anoctamin 1 (ANO1) was tested since each is essential for SW and tone generation. Two distinct ICC-IM populations were identified. Type I cells (36% of total) displayed localised asynchronous Ca2+ transients not dependent on CavL or ANO1; properties typical of ICC-IM mediating neural responses in other gastrointestinal regions. A second novel sub-type, i.e., Type II cells (64% of total) generated rhythmic, global Ca2+ transients at the SW frequency that were synchronised with neighbouring Type II cells and were abolished following blockade of either CavL or ANO1. Thus, the spatiotemporal characteristics of Type II cells and their dependence upon CavL and ANO1 all suggest that these cells are viable candidates for the generation of SWs and tone in the IAS.

A Novel Asynchronous CA Neuron Model: Design of Neuron-Like Nonlinear Responses Based on Novel Bifurcation Theory of Asynchronous Sequential Logic Circuit

This paper presents a novel asynchronous sequential logic circuit neuron model, the nonlinear dynamics of which is described by an asynchronous cellular automaton. As a novel theoretical analysis tool for the model, a continuous-discrete hybrid Poincare map is derived. The map reveals that the presented model can reproduce fundamental dynamic properties and typical bifurcation mechanisms of a biologically plausible neuron model. Using the bifurcation analysis results, systematic design methods of the presented model are proposed. It is shown that the design methods enable the presented model to reproduce typical nonlinear response curves of neurons as well as their underlying bifurcation mechanisms. In addition, the model is implemented in a field programmable gate array and experiments validate its operation. It is shown that the presented model can be implemented by fewer circuit elements and consumes lower power compared to typical conventional neuron models.

Intercellular calcium waves integrate hormonal control of glucose output in the intact liver.

Key points Sympathetic outflow and circulating glucogenic hormones both regulate liver function by increasing cytosolic calcium, although how these calcium signals are integrated at the tissue level is currently unknown.We show that stimulation of hepatic nerve fibres or perfusing the liver with physiological concentrations of vasopressin only will evoke localized cytosolic calcium oscillations and modest increases in hepatic glucose production.The combination of these stimuli acted synergistically to convert localized and asynchronous calcium responses into co‐ordinated intercellular calcium waves that spread throughout the liver lobule and elicited a synergistic increase in hepatic glucose production.The results obtained in the present study demonstrate that subthreshold levels of one hormone can create an excitable medium across the liver lobule, which allows global propagation of calcium signals in response to local sympathetic innervation and integration of metabolic regulation by multiple hormones. This enables the liver lobules to respond as functional units to produce full‐strength metabolic output at physiological levels of hormone. Glucogenic hormones, including catecholamines and vasopressin, induce frequency‐modulated cytosolic Ca2+ oscillations in hepatocytes, and these propagate as intercellular Ca2+ waves via gap junctions in the intact liver. We investigated the role of co‐ordinated Ca2+ waves as a mechanism for integrating multiple endocrine and neuroendocrine inputs to control hepatic glucose production in perfused rat liver. Sympathetic nerve stimulation elicited localized Ca2+ increases that were restricted to hepatocytes in the periportal zone. During perfusion with subthreshold vasopressin, sympathetic stimulation converted asynchronous Ca2+ signals in a limited number of hepatocytes into co‐ordinated intercellular Ca2+ waves that propagated across entire lobules. A similar synergism was observed between physiological concentrations of glucagon and vasopressin, where glucagon also facilitated the recruitment of hepatocytes into a Ca2+ wave. Hepatic glucose production was significantly higher with intralobular Ca2+ waves. We propose that inositol 1,4,5‐trisphosphate (IP3)‐dependent Ca2+ signalling gives rise to an excitable medium across the functional syncytium of the hepatic lobule, co‐ordinating and amplifying the metabolic responses to multiple hormonal inputs.

Role of K+ channels in maintaining the synchrony of spontaneous Ca2+ transients in the mural cells of rat rectal submucosal arterioles.

Mural cells in precapillary arterioles (PCAs) generate spontaneous Ca2+ transients primarily arising from the periodic release of Ca2+ from sarcoendoplasmic reticulum (SR/ER). The Ca2+ release induces Ca2+-activated chloride channel (CaCC)-dependent depolarisations that spread to neighbouring mural cells to develop the synchrony of their Ca2+ transients. Here, we explored the roles of K+ channels in maintaining the synchrony of spontaneous Ca2+ transients. Intracellular Ca2+ dynamics in mural cells were visualised by Cal-520 fluorescence Ca2+ imaging in the submucosal PCAs of rat rectum. Increasing extracellular K+ concentration ([K+]o) from 5.9 to 29.7mM converted synchronous spontaneous Ca2+ transients into asynchronous, high-frequency Ca2+ transients. Similarly, the blockade of inward rectifier K+ (Kir) channels with Ba2+ (50μM) or Kv7 voltage-dependent K+ (Kv7) channels with XE 991 (10μM) disrupted the synchrony of spontaneous Ca2+ transients, while the blockers for large-, intermediate- or small-conductance Ca2+-activated K+ channels had no effect. Kir2.1 immunoreactivity was detected in the arteriolar endothelium but not mural cells. In the PCAs that had been pretreated with XE 991 or Ba2+, nifedipine (1μM) attenuated the asynchronous Ca2+ transients but failed to restore their synchrony. In contrast, levcromakalim, an ATP-sensitive K+ channel opener, restored the synchronous Ca2+ transients. Thus, constitutively active Kv7 and Kir channels appear to be involved in maintaining the relatively hyperpolarised membrane of mural cells. The hyperpolarised membrane prevents depolarisation-induced 'premature' Ca2+ transients to ensure sufficient SR/ER Ca2+ refilling that is required for regenerative Ca2+ release resulting in synchronous Ca2+ transients amongst the mural cells.

Quantitative tests reveal that microtubules tune the healthy heart but underlie arrhythmias in pathology.

Microtubule (MT) mechanotransduction links diastolic stretch to generation of NADPH oxidase 2 (NOX2)-dependent reactive oxygen species (ROS), signals we term X-ROS. While stretch-elicited X-ROS primes intracellular calcium (Ca2+) channels for synchronized activation in the healthy heart, the dysregulated excess in this pathway underscores asynchronous Ca2+ release and arrhythmia. Here, we expanded our existing computational models of Ca2+ signalling in heart to include MT-dependent mechanotransduction through X-ROS. Informed by new focused experimental tests to properly constrain our model, we quantify the role of X-ROS on excitation-contraction coupling in healthy and pathological conditions. This approach allowed for a mechanistic investigation that revealed new insights into X-ROS signalling in disease including changes in MT network density and post-translational modifications (PTMs), elevated NOX2 expression, altered Ca2+ release dynamics (i.e. Ca2+ sparks and Ca2+ waves), how NOX2 is activated by and responds to stretch, and finally the degree to which normalizing X-ROS can prevent Ca2+-dependent arrhythmias.

Pacemaker Mechanisms Driving Pyeloureteric Peristalsis: Modulatory Role of Interstitial Cells.

The peristaltic pressure waves in the renal pelvis that propel urine expressed by the kidney into the ureter towards the bladder have long been considered to be 'myogenic', being little affected by blockers of nerve conduction or autonomic neurotransmission, but sustained by the intrinsic release of prostaglandins and sensory neurotransmitters. In uni-papilla mammals, the funnel-shaped renal pelvis consists of a lumen-forming urothelium and a stromal layer enveloped by a plexus of 'typical' smooth muscle cells (TSMCs), in multi-papillae kidneys a number of minor and major calyces fuse into a large renal pelvis. Electron microscopic, electrophysiological and Ca2+ imaging studies have established that the pacemaker cells driving pyeloureteric peristalsis are likely to be morphologically distinct 'atypical' smooth muscle cells (ASMCs) that fire Ca2+ transients and spontaneous transient depolarizations (STDs) which trigger propagating nifedipine-sensitive action potentials and Ca2+ waves in the TSMC layer. In uni-calyceal kidneys, ASMCs predominately locate on the serosal surface of the proximal renal pelvis while in multi-papillae kidneys they locate within the sub-urothelial space. 'Fibroblast-like' interstitial cells (ICs) located in the sub-urothelial space or adventitia are a mixed population of cells, having regional and species-dependent expression of various Cl-, K+, Ca2+ and cationic channels. ICs display asynchronous Ca2+ transients that periodically synchronize into bursts that accelerate ASMC Ca2+ transient firing. This review presents current knowledge of the architecture of the proximal renal pelvis, the role Ca2+ plays in renal pelvis peristalsis and the mechanisms by which ICs may sustain/accelerate ASMC pacemaking.

Interplay Between Sub-Cellular Alterations of Calcium Release and T-Tubular Defects in Cardiac Diseases.

Asynchronous Ca2+ release promotes non-homogeneous myofilament activation, leading to mechanical dysfunction, as well as initiation of propagated calcium waves and arrhythmias. Recent advances in microscopy techniques have allowed for optical recordings of local Ca2+ fluxes and action potentials from multiple sub-cellular domains within cardiac cells with unprecedented spatial and temporal resolution. Since then, sub-cellular local information of the spatio-temporal relationship between Ca2+ release and action potential propagation have been unlocked, providing novel mechanistic insights in cardiac excitation-contraction coupling (ECC). Here, we review the promising perspectives arouse from repeatedly probing Ca2+ release at the same sub-cellular location while simultaneously probing multiple locations at the same time within a single cardiac cell. We also compare the results obtained in three different rodent models of cardiac diseases, highlighting disease-specific mechanisms. Slower local Ca2+ release has been observed in regions with defective action potential conduction in diseased cardiac cells. Moreover, significant increment of Ca2+ variability (both in time and in space) has been found in diseased cardiac cells but does not directly correlate with local electrical defects nor with the degree of structural aberrations of the cellular membrane system, suggesting a role for other players of the ECC machinery. We finally explore exciting opportunities provided by the technology for studying different cardiomyocyte populations, as well as for dissecting the mechanisms responsible for subcellular spatio-temporal variability of Ca2+ release.

Spontaneous Ca2+ transients in rat pulmonary vein cardiomyocytes are increased in frequency and become more synchronous following electrical stimulation

The pulmonary veins have an external sleeve of cardiomyocytes that are a widely recognised source of ectopic electrical activity that can lead to atrial fibrillation. Although the mechanisms behind this activity are currently unknown, changes in intracellular calcium (Ca2+) signalling are purported to play a role. Therefore, the intracellular Ca2+ concentration was monitored in the pulmonary vein using fluo-4 and epifluorescence microscopy. Electrical field stimulation evoked a synchronous rise in Ca2+ in neighbouring cardiomyocytes; asynchronous spontaneous Ca2+ transients between electrical stimuli were also present. Immediately following termination of electrical field stimulation at 3 Hz or greater, the frequency of the spontaneous Ca2+ transients was increased from 0.45 ± 0.06 Hz under basal conditions to between 0.59 ± 0.05 and 0.65 ± 0.06 Hz (P < 0.001). Increasing the extracellular Ca2+ concentration enhanced this effect, with the frequency of spontaneous Ca2+ transients increasing from 0.45 ± 0.05 Hz to between 0.75 ± 0.06 and 0.94 ± 0.09 Hz after electrical stimulation at 3 to 9 Hz (P < 0.001), and this was accompanied by a significant increase in the velocity of Ca2+ transients that manifested as waves. Moreover, in the presence of high extracellular Ca2+, the spontaneous Ca2+ transients occurred more synchronously in the initial few seconds following electrical stimulation. The ryanodine receptors, which are the source of spontaneous Ca2+ transients in pulmonary vein cardiomyocytes, were found to be arranged in a striated pattern in the cell interior, as well as along the periphery of cell. Furthermore, labelling the sarcolemma with di-4-ANEPPS showed that over 90% of pulmonary vein cardiomyocytes possessed T-tubules. These findings demonstrate that the frequency of spontaneous Ca2+ transients in the rat pulmonary vein are increased following higher rates of electrical stimulation and increasing the extracellular Ca2+ concentration.

A universal Brownian cellular automaton with 3 states and 2 rules

This paper presents a 3-state asynchronous CA that requires merely two transition rules to achieve computational universality. This universality is achieved by embedding Priese’s delay-insensitive circuit elements, called the E-element and the K-element, on the cell space of a so-called Brownian CA, which is an asynchronous CA containing local configurations that conduct a random walk in the circuit topology.

Calcium-Dependent Arrhythmogenic Foci Created by Weakly Coupled Myocytes in the Failing Heart.

Intercellular uncoupling and Ca2+ (Ca) mishandling can initiate triggered ventricular arrhythmias. Spontaneous Ca release activates inward current which depolarizes membrane potential (Vm) and can trigger action potentials in isolated myocytes. However, cell-cell coupling in intact hearts limits local depolarization and may protect hearts from this arrhythmogenic mechanism. Traditional optical mapping lacks the spatial resolution to assess coupling of individual myocytes. We investigate local intercellular coupling in Ca-induced depolarization in intact hearts, using confocal microscopy to measure local Vm and intracellular [Ca] simultaneously. We used isolated Langendorff-perfused hearts from control (CTL) and heart failure (HF) mice (HF induced by transaortic constriction). In CTL hearts, 1.4% of myocytes were poorly synchronized with neighboring cells and exhibited asynchronous (AS) Ca transients. These AS myocytes were much more frequent in HF (10.8% of myocytes, P<0.05 versus CTL). Local Ca waves depolarized Vm in HF but not CTL hearts, suggesting weaker gap junction coupling in HF-AS versus CTL-AS myocytes. Cell-cell coupling was assessed by calcein fluorescence recovery after photobleach during intracellular [Ca] recording. All regions in CTL hearts exhibited faster calcein diffusion than in HF, with HF-AS myocyte being slowest. In HF-AS, enhancing gap junction conductance (with rotigaptide) increased coupling and suppressed Vm depolarization during Ca waves. Conversely, in CTL hearts, gap junction inhibition (carbenoxolone) decreased coupling and allowed Ca wave-induced depolarizations. Synchronization of Ca wave initiation and triggered action potentials were observed in HF hearts and computational models. Well-coupled CTL myocytes are effectively voltage-clamped during Ca waves, protecting the heart from triggered arrhythmias. Spontaneous Ca waves are much more common in HF myocytes and these AS myocytes are also poorly coupled, enabling local Ca-induced inward current of sufficient source strength to overcome a weakened current sink to depolarize Vm and trigger action potentials.

Synchronous circadian voltage rhythms with asynchronous calcium rhythms in the suprachiasmatic nucleus

The suprachiasmatic nucleus (SCN), the master circadian clock, contains a network composed of multiple types of neurons which are thought to form a hierarchical and multioscillator system. The molecular clock machinery in SCN neurons drives membrane excitability and sends time cue signals to various brain regions and peripheral organs. However, how and at what time of the day these neurons transmit output signals remain largely unknown. Here, we successfully visualized circadian voltage rhythms optically for many days using a genetically encoded voltage sensor, ArcLightD. Unexpectedly, the voltage rhythms are synchronized across the entire SCN network of cultured slices, whereas simultaneously recorded Ca2+ rhythms are topologically specific to the dorsal and ventral regions. We further found that the temporal order of these two rhythms is cell-type specific: The Ca2+ rhythms phase-lead the voltage rhythms in AVP neurons but Ca2+ and voltage rhythms are nearly in phase in VIP neurons. We confirmed that circadian firing rhythms are also synchronous and are coupled with the voltage rhythms. These results indicate that SCN networks with asynchronous Ca2+ rhythms produce coherent voltage rhythms.

Synaptotagmin-1- and Synaptotagmin-7-Dependent Fusion Mechanisms Target Synaptic Vesicles to Kinetically Distinct Endocytic Pathways

Synaptic vesicle recycling is essential for maintaining normal synaptic function. The coupling of exocytosis and endocytosis is assumed to be Ca2+ dependent, but the exact role of Ca2+ and its key effector synaptotagmin-1 (syt1) in regulation of endocytosis is poorly understood. Here, we probed the role of syt1 in single- as well as multi-vesicle endocytic events using high-resolution optical recordings. Our experiments showed that the slowed endocytosis phenotype previously reported after syt1 loss of function can also be triggered by other manipulations that promote asynchronous release such as Sr2+ substitution and complexin loss of function. The link between asynchronous release and slowed endocytosis was due to selective targeting of fused synaptic vesicles toward slow retrieval by the asynchronous release Ca2+ sensor synaptotagmin-7. In contrast, after single synaptic vesicle fusion, syt1 acted as an essential determinant of synaptic vesicle endocytosis time course by delaying the kinetics of vesicle retrieval in response to increasing Ca2+ levels.