Nanodysferlins support membrane repair and binding to TRIM72/MG53 but do not localize to t-tubules or stabilize Ca2+ signaling.

Mutations in the DYSF gene, encoding the protein dysferlin, lead to several forms of muscular dystrophy. In healthy skeletal muscle, dysferlin concentrates in the transverse tubules and is involved in repairing the sarcolemma and stabilizing Ca2+ signaling after membrane disruption. The DYSF gene encodes 7-8 C2 domains, several Fer and Dysf domains, and a C-terminal transmembrane sequence. Because its coding sequence is too large to package in adeno-associated virus, the full-length sequence is not amenable to current gene delivery methods. Thus, we have examined smaller versions of dysferlin, termed "nanodysferlins," designed to eliminate several C2 domains, specifically C2 domains D, E, and F; B, D, and E; and B, D, E, and F. We also generated a variant by replacing eight amino acids in C2G in the nanodysferlin missing domains D through F. We electroporated dysferlin-null A/J mouse myofibers with Venus fusion constructs of these variants, or as untagged nanodysferlins together with GFP, to mark transfected fibers We found that, although these nanodysferlins failed to concentrate in transverse tubules, three of them supported membrane repair after laser wounding while all four bound the membrane repair protein, TRIM72/MG53, similar to WT dysferlin. By contrast, they failed to suppress Ca2+ waves after myofibers were injured by mild hypoosmotic shock. Our results suggest that the internal C2 domains of dysferlin are required for normal t-tubule localization and Ca2+ signaling and that membrane repair does not require these C2 domains.

TRP channels: a provocative rationalization for local Ca2+ control in arterial tone development.

Arterial networks are controlled by the consolidated output of stimuli that set "how much" (magnitude) and "where" (distribution) blood flow is delivered. While notable changes in magnitude are tied to network wide responses, altered distribution often arises from focal changes in tone, whose mechanistic foundation remains unclear. We propose herein a framework of focal vasomotor contractility being controlled by pharmacomechanical coupling and the generation of Ca2+ waves via the sarcoplasmic reticulum. We argue the latter is sustained by receptor operated, transient receptor potential (TRP) channels through direct extracellular Ca2+ influx or indirect Na+ influx, reversing the Na+/Ca2+ exchanger. We view this focal regulatory mechanism as complementary, but not redundant with, electromechanical coupling in the precision tuning of blood flow delivery.

Cracking the Endothelial Calcium (Ca2+) Code: A Matter of Timing and Spacing.

A monolayer of endothelial cells lines the innermost surface of all blood vessels, thereby coming into close contact with every region of the body and perceiving signals deriving from both the bloodstream and parenchymal tissues. An increase in intracellular Ca2+ concentration ([Ca2+]i) is the main mechanism whereby vascular endothelial cells integrate the information conveyed by local and circulating cues. Herein, we describe the dynamics and spatial distribution of endothelial Ca2+ signals to understand how an array of spatially restricted (at both the subcellular and cellular levels) Ca2+ signals is exploited by the vascular intima to fulfill this complex task. We then illustrate how local endothelial Ca2+ signals affect the most appropriate vascular function and are integrated to transmit this information to more distant sites to maintain cardiovascular homeostasis. Vasorelaxation and sprouting angiogenesis were selected as an example of functions that are finely tuned by the variable spatio-temporal profile endothelial Ca2+ signals. We further highlighted how distinct Ca2+ signatures regulate the different phases of vasculogenesis, i.e., proliferation and migration, in circulating endothelial precursors.

Mitochondrial Calcium Waves by Electrical Stimulation in Cultured Hippocampal Neurons.

Mitochondria are critical to cellular Ca2+ homeostasis via the sequestering of cytosolic Ca2+ in the mitochondrial matrix. Mitochondrial Ca2+ buffering regulates neuronal activity and neuronal death by shaping cytosolic and presynaptic Ca2+ or controlling energy metabolism. Dysfunction in mitochondrial Ca2+ buffering has been implicated in psychological and neurological disorders. Ca2+ wave propagation refers to the spreading of Ca2+ for buffering and maintaining the associated rise in Ca2+ concentration. We investigated mitochondrial Ca2+ waves in hippocampal neurons using genetically encoded Ca2+ indicators. Neurons transfected with mito-GCaMP5G, mito-RCaMP1h, and CEPIA3mt exhibited evidence of mitochondrial Ca2+ waves with electrical stimulation. These waves were observed with 200 action potentials at 40 Hz or 20 Hz but not with lower frequencies or fewer action potentials. The application of inhibitors of mitochondrial calcium uniporter and oxidative phosphorylation suppressed mitochondrial Ca2+ waves. However, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors and N-methyl-d-aspartate receptor blockade had no effect on mitochondrial Ca2+ wave were propagation. The Ca2+ waves were not observed in endoplasmic reticula, presynaptic terminals, or cytosol in association with electrical stimulation of 200 action potentials at 40 Hz. These results offer novel insights into the mechanisms underlying mitochondrial Ca2+ buffering and the molecular basis of mitochondrial Ca2+ waves in neurons in response to electrical stimulation.

The C2 domains of dysferlin: roles in membrane localization, Ca2+ signalling and sarcolemmal repair.

Dysferlin is an integral membrane protein of the transverse tubules of skeletal muscle that is mutated or absent in limb girdle muscular dystrophy 2B and Miyoshi myopathy. Here we examine the role of dysferlin's seven C2 domains, C2A through C2G, in membrane repair and Ca2+ release, as well as in targeting dysferlin to the transverse tubules of skeletal muscle. We report that deletion of either domain C2A or C2B inhibits membrane repair completely, whereas deletion of C2C, C2D, C2E, C2F or C2G causes partial loss of membrane repair that is exacerbated in the absence of extracellular Ca2+ . Deletion of C2C, C2D, C2E, C2F or C2G also causes significant changes in Ca2+ release, measured as the amplitude of the Ca2+ transient before or after hypo-osmotic shock and the appearance of Ca2+ waves. Most deletants accumulate in endoplasmic reticulum. Only the C2A domain can be deleted without affecting dysferlin trafficking to transverse tubules, but Dysf-ΔC2A fails to support normal Ca2+ signalling after hypo-osmotic shock. Our data suggest that (i) every C2 domain contributes to repair; (ii) all C2 domains except C2B regulate Ca2+ signalling; (iii) transverse tubule localization is insufficient for normal Ca2+ signalling; and (iv) Ca2+ dependence of repair is mediated by C2C through C2G. Thus, dysferlin's C2 domains have distinct functions in Ca2+ signalling and sarcolemmal membrane repair and may play distinct roles in skeletal muscle. KEY POINTS: Dysferlin, a transmembrane protein containing seven C2 domains, C2A through C2G, concentrates in transverse tubules of skeletal muscle, where it stabilizes voltage-induced Ca2+ transients and participates in sarcolemmal membrane repair. Each of dysferlin's C2 domains except C2B regulate Ca2+ signalling. Localization of dysferlin variants to the transverse tubules is not sufficient to support normal Ca2+ signalling or membrane repair. Each of dysferlin's C2 domains contributes to sarcolemmal membrane repair. The Ca2+ dependence of membrane repair is mediated by C2C through C2G. Dysferlin's C2 domains therefore have distinct functions in Ca2+ signalling and sarcolemmal membrane repair.

Mitochondria regulate TRPV4-mediated release of ATP.

BACKGROUND AND PURPOSE: Ca2+ influx via TRPV4 channels triggers Ca2+ release from the IP3 -sensitive internal store to generate repetitive oscillations. Although mitochondria are acknowledged regulators of IP3 -mediated Ca2+ release, how TRPV4-mediated Ca2+ signals are regulated by mitochondria is unknown. We show that depolarised mitochondria switch TRPV4 signalling from relying on Ca2+ -induced Ca2+ release at IP3 receptors to being independent of Ca2+ influx and instead mediated by ATP release via pannexins. EXPERIMENTAL APPROACH: TRPV4-evoked Ca2+ signals were individually examined in hundreds of cells in the endothelium of rat mesenteric resistance arteries using the indicator Cal520. KEY RESULTS: TRPV4 activation with GSK1016790A (GSK) generated repetitive Ca2+ oscillations that required Ca2+ influx. However, when the mitochondrial membrane potential was depolarised, by the uncoupler CCCP or complex I inhibitor rotenone, TRPV4 activation generated large propagating, multicellular, Ca2+ waves in the absence of external Ca2+ . The ATP synthase inhibitor oligomycin did not potentiate TRPV4-mediated Ca2+ signals. GSK-evoked Ca2+ waves, when mitochondria were depolarised, were blocked by the TRPV4 channel blocker HC067047, the SERCA inhibitor cyclopiazonic acid, the PLC blocker U73122 and the inositol trisphosphate receptor blocker caffeine. The Ca2+ waves were also inhibited by the extracellular ATP blockers suramin and apyrase and the pannexin blocker probenecid. CONCLUSION AND IMPLICATIONS: These results highlight a previously unknown role of mitochondria in shaping TRPV4-mediated Ca2+ signalling by facilitating ATP release. When mitochondria are depolarised, TRPV4-mediated release of ATP via pannexin channels activates plasma membrane purinergic receptors to trigger IP3 -evoked Ca2+ release.

Glial ER and GAP junction mediated Ca2+ waves are crucial to maintain normal brain excitability.

Astrocytes play key roles in regulating multiple aspects of neuronal function from invertebrates to humans and display Ca2+ fluctuations that are heterogeneously distributed throughout different cellular microdomains. Changes in Ca2+ dynamics represent a key mechanism for how astrocytes modulate neuronal activity. An unresolved issue is the origin and contribution of specific glial Ca2+ signaling components at distinct astrocytic domains to neuronal physiology and brain function. The Drosophila model system offers a simple nervous system that is highly amenable to cell-specific genetic manipulations to characterize the role of glial Ca2+ signaling. Here we identify a role for ER store-operated Ca2+ entry (SOCE) pathway in perineurial glia (PG), a glial population that contributes to the Drosophila blood-brain barrier. We show that PG cells display diverse Ca2+ activity that varies based on their locale within the brain. Ca2+ signaling in PG cells does not require extracellular Ca2+ and is blocked by inhibition of SOCE, Ryanodine receptors, or gap junctions. Disruption of these components triggers stimuli-induced seizure-like episodes. These findings indicate that Ca2+ release from internal stores and its propagation between neighboring glial cells via gap junctions are essential for maintaining normal nervous system function.

Rotavirus research: 2014-2020.

Rotaviruses are major causes of acute gastroenteritis in infants and young children worldwide and also cause disease in the young of many other mammalian and of avian species. During the recent 5-6 years rotavirus research has benefitted in a major way from the establishment of plasmid only-based reverse genetics systems, the creation of human and other mammalian intestinal enteroids, and from the wide application of structural biology (cryo-electron microscopy, cryo-EM tomography) and complementary biophysical approaches. All of these have permitted to gain new insights into structure-function relationships of rotaviruses and their interactions with the host. This review follows different stages of the viral replication cycle and summarizes highlights of structure-function studies of rotavirus-encoded proteins (both structural and non-structural), molecular mechanisms of viral replication including involvement of cellular proteins and lipids, the spectrum of viral genomic and antigenic diversity, progress in understanding of innate and acquired immune responses, and further developments of prevention of rotavirus-associated disease.

The functional significance of redox-mediated intersubunit cross-linking in regulation of human type 2 ryanodine receptor.

The type 2 ryanodine receptor (RyR2) plays a key role in the cardiac intracellular calcium (Ca2+) regulation. We have previously shown that oxidative stress activates RyR2 in rabbit cardiomyocytes by promoting the formation of disulfide bonds between neighboring RyR2 subunits. However, the functional significance of this redox modification for human RyR2 (hRyR2) remains largely unknown. Here, we studied the redox regulation of hRyR2 in HEK293 cells transiently expressing the ryr2 gene. Analysis of hRyR2 cross-linking and of the redox-GFP readout response to diamide oxidation revealed that hRyR2 cysteines involved in the intersubunit cross-linking are highly sensitive to oxidative stress. In parallel experiments, the effect of diamide on endoplasmic reticulum (ER) Ca2+ release was studied in cells co-transfected with hRyR2, ER Ca2+ pump (SERCA2a) and the ER-targeted Ca2+ sensor R-CEPIA1er. Expression of hRyR2 and SERCA2a produced "cardiac-like" Ca2+ waves due to spontaneous hRyR2 activation. Incubation with diamide caused a fast decline of the luminal ER Ca2+ (or ER Ca2+ load) followed by the cessation of Ca2+ waves. The maximal effect of diamide on ER Ca2+ load and Ca2+ waves positively correlates with the maximum level of hRyR2 cross-linking, indicating a functional significance of this redox modification. Furthermore, the level of hRyR2 cross-linking positively correlates with the degree of calmodulin (CaM) dissociation from the hRyR2 complex. In skeletal muscle RyR (RyR1), cysteine 3635 (C3635) is viewed as dominantly responsible for the redox regulation of the channel. Here, we showed that the corresponding cysteine 3602 (C3602) in hRyR2 does not participate in intersubunit cross-linking and plays a limited role in the hRyR2 regulation by CaM during oxidative stress. Collectively, these results suggest that redox-mediated intersubunit cross-linking is an important regulator of hRyR2 function under pathological conditions associated with oxidative stress.

Mechanisms of artemether toxicity on single cardiomyocytes and protective effect of nanoencapsulation.

BACKGROUND AND PURPOSE: The artemisinin derivative, artemether, has antimalarial activity with potential neurotoxic and cardiotoxic effects. Artemether in nanocapsules (NC-ATM) is more efficient than free artemether for reducing parasitaemia and increasing survival of Plasmodium berghei-infected mice. NCs also prevent prolongation of the QT interval of the ECG. Here, we assessed cellular cardiotoxicity of artemether and how this toxicity was prevented by nanoencapsulation. EXPERIMENTAL APPROACH: Mice were treated with NC-ATM orally (120 mg·kg-1 twice daily) for 4 days. Other mice received free artemether, blank NCs, and vehicle for comparison. We measured single-cell contraction, intracellular Ca2+ transient using fluorescent Indo-1AM Ca2+ dye, and electrical activity using the patch-clamp technique in freshly isolated left ventricular myocytes. The acute effect of free artemether was also tested on cardiomyocytes of untreated animals. KEY RESULTS: Artemether prolonged action potentials (AP) upon acute exposure (at 0.1, 1, and 10 µM) of cardiomyocytes from untreated mice or after in vivo treatment. This prolongation was unrelated to blockade of K+ currents, increased Ca2+ currents or promotion of a sustained Na+ current. AP lengthening was abolished by the NCX inhibitor SEA-0400. Artemether promoted irregular Ca2+ transients during pacing and spontaneous Ca2+ events during resting periods. NC-ATM prevented all effects. Blank NCs had no effects compared with vehicle. CONCLUSION AND IMPLICATIONS: Artemether induced NCX-dependent AP lengthening (explaining QTc prolongation) and disrupted Ca2+ handling, both effects increasing pro-arrhythmogenic risks. NCs prevented these adverse effects, providing a safe alternative to the use of artemether alone, especially to treat malaria.

Effect of carvedilol on atrial excitation-contraction coupling, Ca2+ release, and arrhythmogenicity.

Carvedilol is an FDA-approved ß-blocker commonly used for treatment of high blood pressure, congestive heart failure, and cardiac tachyarrhythmias, including atrial fibrillation. We investigated at the cellular level the mechanisms through which carvedilol interferes with sarcoplasmic reticulum (SR) Ca2+ release during excitation-contraction coupling (ECC) in single rabbit atrial myocytes. Carvedilol caused concentration-dependent (1-10 µM) failure of SR Ca2+ release. Failure of ECC and Ca2+ release was the result of dose-dependent inhibition of voltage-gated Na+ (INa) and L-type Ca2+ (ICa) currents that are responsible for the rapid depolarization phase of the cardiac action potential (AP) and the initiation of Ca2+-induced Ca2+ release from the SR, respectively. Carvedilol (1 µM) led to AP duration shortening, AP failures, and peak INa inhibition by ~80%, whereas ICa was not markedly affected. Carvedilol (10 µM) blocked INa almost completely and reduced ICa by ~40%. No effect on Ca2+-transient amplitude, ICa, and INa was observed in control experiments with the ß-blocker metoprolol, suggesting that the carvedilol effect on ECC is unlikely the result of its ß-blocking property. The effects of carvedilol (1 µM) on subcellular SR Ca2+ release was spatially inhomogeneous, where a selective inhibition of peripheral subsarcolemmal Ca2+ release from the junctional SR accounted for the cell-averaged reduction in Ca2+-transient amplitude. Furthermore, carvedilol significantly reduced the probability of spontaneous arrhythmogenic Ca2+ waves without changes of SR Ca2+ load. The data suggest a profound antiarrhythmic action of carvedilol in atrial myocytes resulting from an inhibitory effect on the SR Ca2+ release channel.NEW & NOTEWORTHY Here we show that the clinically widely used ß-blocker carvedilol has profound effects on Ca2+ signaling and ion currents, but also antiarrhythmic effects in adult atrial myocytes. Carvedilol inhibits sodium and calcium currents and leads to failure of ECC but also prevents spontaneous Ca2+ release from cellular sarcoplasmic reticulum (SR) Ca2+ stores in form of arrhythmogenic Ca2+ waves. The antiarrhythmic effect occurs by carvedilol acting directly on the SR ryanodine receptor Ca2+ release channel.

Mechanisms of malignancy in glioblastoma cells are linked to mitochondrial Ca2+ uniporter upregulation and higher intracellular Ca2+ levels.

Glioblastoma (GBM) is one of the most malignant brain tumours and, despite advances in treatment modalities, it remains largely incurable. Ca2+ regulation and dynamics play crucial roles in different aspects of cancer, but they have never been investigated in detail in GBM. Here, we report that spontaneous Ca2+ waves in GBM cells cause unusual intracellular Ca2+ ([Ca2+]i) elevations (>1 µM), often propagating through tumour microtubes (TMs) connecting adjacent cells. This unusual [Ca2+]i elevation is not associated with the induction of cell death and is concomitant with overexpression of mitochondrial Ca2+ uniporter (MCU). We show that MCU silencing decreases proliferation and alters [Ca2+]i dynamics in U87 GBM cells, while MCU overexpression increases [Ca2+]i elevation in human astrocytes (HAs). These results suggest that changes in the expression level of MCU, a protein involved in intracellular Ca2+ regulation, influences GBM cell proliferation, contributing to GBM malignancy.This article has an associated First Person interview with the first author of the paper.

Coupling of the Na+/K+-ATPase to Ankyrin B controls Na+/Ca2+ exchanger activity in cardiomyocytes.

AIMS: Ankyrin B (AnkB) is an adaptor protein that assembles Na+/K+-ATPase (NKA) and Na+/Ca2+ exchanger (NCX) in the AnkB macromolecular complex. Loss-of-function mutations in AnkB cause the AnkB syndrome in humans, characterized by ventricular arrhythmias and sudden cardiac death. It is unclear to what extent NKA binding to AnkB allows regulation of local Na+ and Ca2+ domains and hence NCX activity. METHODS AND RESULTS: To investigate the role of NKA binding to AnkB in cardiomyocytes, we synthesized a disruptor peptide (MAB peptide) and its AnkB binding ability was verified by pulldown experiments. As opposed to control, the correlation between NKA and NCX currents was abolished in adult rat ventricular myocytes dialyzed with MAB peptide, as well as in cardiomyocytes from AnkB+/- mice. Disruption of NKA from AnkB (with MAB peptide) increased NCX-sensed cytosolic Na+ concentration, reduced Ca2+ extrusion through NCX, and increased frequency of Ca2+ sparks and Ca2+ waves without concomitant increase in Ca2+ transient amplitude or SR Ca2+ load, suggesting an effect in local Ca2+ domains. Selective inhibition of the NKAα2 isoform abolished both the correlation between NKA and NCX currents and the increased rate of Ca2+ sparks and waves following NKA/AnkB disruption, suggesting that an AnkB/NKAα2/NCX domain controls Ca2+ fluxes in cardiomyocytes. CONCLUSION: NKA binding to AnkB allows ion regulation in a local domain, and acute disruption of the NKA/AnkB interaction using disruptor peptides lead to increased rate of Ca2+ sparks and waves. The functional effects were mediated through the NKAα2 isoform. Disruption of the AnkB/NKA/NCX domain could be an important pathophysiological mechanism in the AnkB syndrome.

Estimation of stochastic behaviour in cardiac myocytes: I. Ca2+ movements inside the cytosol and sarcoplasmic reticulum on curvilinear domains.

Since the discovery of Ca2+ sparks and their stochastic behaviour in cardiac myocytes, models have focused on the inclusion of stochasticity in their studies. While most models pay much attention to the stochastic modelling of cytosolic Ca2+ concentration the coupling of Ca2+ sparks and blinks in a stochastic model has not been explored fully. The cell morphology in in silico studies in the past is assumed to be Cartesian, spherical or cylindrical. The application on curvilinear grids can easily address certain restrictions posed by such grid set up and provide more realistic cell morphology. In this paper, we present a stochastic reaction-diffusion model that couples Ca2+ sparks and blinks in realistic shapes of cells in curvilinear domains. Methodology: Transformation of the model was performed to the curvilinear coordinate system. The set of equations is used to produce Ca2+ waves initiated from sparks and blinks. A non-buffered and non-dyed version as well as a buffered and dyed version of these equations were studied in light of observing the dynamics on the two different systems. For comparison, results for both the Cartesian and curvilinear grids are provided. Results and conclusions: A successful demonstration of the application of curvilinear grids serving as basis for future developments.

Smooth muscle gap-junctions allow propagation of intercellular Ca2+ waves and vasoconstriction due to Ca2+ based action potentials in rat mesenteric resistance arteries.

The role of vascular gap junctions in the conduction of intercellular Ca2+ and vasoconstriction along small resistance arteries is not entirely understood. Some depolarizing agents trigger conducted vasoconstriction while others only evoke a local depolarization. Here we use a novel technique to investigate the temporal and spatial relationship between intercellular Ca2+ signals generated by smooth muscle action potentials (APs) and vasoconstriction in mesenteric resistance arteries (MA). Pulses of exogenous KCl to depolarize the downstream end (T1) of a 3 mm long artery increased intracellular Ca2+ associated with vasoconstriction. The spatial spread and amplitude of both depended on the duration of the pulse, with only a restricted non-conducting vasoconstriction to a 1 s pulse. While blocking smooth muscle cell (SMC) K+ channels with TEA and activating L-type voltage-gated Ca2+ channels (VGCCs) with BayK 8644 spread was dramatically facilitated, so the 1 s pulse evoked intercellular Ca2+ waves and vasoconstriction that spread along an entire artery segment 3000 µm long. Ca2+ waves spread as nifedipine-sensitive Ca2+ spikes due to SMC action potentials, and evoked vasoconstriction. Both intercellular Ca2+ and vasoconstriction spread at circa 3 mm s-1 and were independent of the endothelium. The spread but not the generation of Ca2+ spikes was reversibly blocked by the gap junction inhibitor 18ß-GA. Thus, smooth muscle gap junctions enable depolarization to spread along resistance arteries, and once regenerative Ca2+-based APs occur, spread along the entire length of an artery followed by widespread vasoconstriction.

Wound healing of human embryonic stem cell-derived retinal pigment epithelial cells is affected by maturation stage.

BACKGROUND: Wound healing of retinal pigment epithelium (RPE) is a complex process that may take place in common age-related macular degeneration eye disease. The purpose of this study was to evaluate whether wounding and wound healing has an effect on Ca2+ dynamics in human embryonic stem cell (hESC)-RPEs cultured different periods of time. METHODS: The 9-day-cultured or 28-day-cultured hESC-RPEs from two different cell lines were wounded and the dynamics of spontaneous and mechanically induced intracellular Ca2+ activity was measured with live-cell Ca2+ imaging either immediately or 7 days after wounding. The healing time and speed were analyzed with time-lapse bright field microscopy. The Ca2+ activity and healing speed were analysed with image analysis. In addition the extracellular matrix deposition was assessed with confocal microscopy. RESULTS: The Ca2+ dynamics in hESC-RPE monolayers differed depending on the culture time: 9-day-cultured cells had higher number of cells with spontaneous Ca2+ activity close to freshly wounded edge compared to control areas, whereas in 28-day-cultured cells there was no difference in wounded and control areas. The 28-day-cultured, wounded and 7-day-healed hESC-RPEs produced wide-spreading intercellular Ca2+ waves upon mechanical stimulation, while in controls propagation was restricted. Most importantly, both wave spreading and spontaneous Ca2+ activity of cells within the healed area, as well as the cell morphology of 28-day-cultured, wounded and thereafter 7-day-healed areas resembled the 9-day-cultured hESC-RPEs. CONCLUSIONS: This acquired knowledge about Ca2+ dynamics of wounded hESC-RPE monolayers is important for understanding the dynamics of RPE wound healing, and could offer a reliable functionality test for RPE cells. The data presented in here suggests that assessment of Ca2+ dynamics analysed with image analysis could be used as a reliable non-invasive functionality test for RPE cells.

Ca2+ signaling, apoptosis and autophagy in the developing cochlea: Milestones to hearing acquisition.

In mammals, the sense of hearing arises through a complex sequence of morphogenetic events that drive the sculpting of the auditory sensory epithelium into its terminally functional three-dimensional shape. While the majority of the underlying mechanisms remain unknown, it has become increasingly clear that Ca2+ signaling is at center stage and plays numerous fundamental roles both in the sensory hair cells and in the matrix of non-sensory, epithelial and supporting cells, which embed them and are tightly interconnected by a dense network of gap junctions formed by connexin 26 (Cx26) and connexin 30 (Cx30) protein subunits. In this review, we discuss the intricate interplay between Ca2+ signaling, connexin expression and function, apoptosis and autophagy in the crucial steps that lead to hearing acquisition.

Triggered intracellular calcium waves in dog and human left atrial myocytes from normal and failing hearts.

AIMS: Abnormal intracellular Ca2+ cycling contributes to triggered activity and arrhythmias in the heart. We investigated the properties and underlying mechanisms for systolic triggered Ca2+ waves in left atria from normal and failing dog hearts. METHODS AND RESULTS: Intracellular Ca2+ cycling was studied using confocal microscopy during rapid pacing of atrial myocytes (36 °C) isolated from normal and failing canine hearts (ventricular tachypacing model). In normal atrial myocytes (NAMs), Ca2+ waves developed during rapid pacing at rates ≥ 3.3 Hz and immediately disappeared upon cessation of pacing despite high sarcoplasmic reticulum (SR) load. In heart failure atrial myocytes (HFAMs), triggered Ca2+ waves (TCWs) developed at a higher incidence at slower rates. Because of their timing, TCW development relies upon action potential (AP)-evoked Ca2+ entry. The distribution of Ca2+ wave latencies indicated two populations of waves, with early events representing TCWs and late events representing conventional spontaneous Ca2+ waves. Latency analysis also demonstrated that TCWs arise after junctional Ca2+ release has occurred and spread to non-junctional (cell core) SR. TCWs also occurred in intact dog atrium and in myocytes from humans and pigs. ß-adrenergic stimulation increased Ca2+ release and abolished TCWs in NAMs but was ineffective in HFAMs making this a potentially effective adaptive mechanism in normals but potentially arrhythmogenic in HF. Block of Ca-calmodulin kinase II also abolished TCWs, suggesting a role in TCW formation. Pharmacological manoeuvres that increased Ca2+ release suppressed TCWs as did interventions that decreased Ca2+ release but these also severely reduced excitation-contraction coupling. CONCLUSION: TCWs develop during the atrial AP and thus could affect AP duration, producing repolarization gradients and creating a substrate for reentry, particularly in HF where they develop at slower rates and a higher incidence. TCWs may represent a mechanism for the initiation of atrial fibrillation particularly in HF.

The role of spatial organization of Ca2+ release sites in the generation of arrhythmogenic diastolic Ca2+ release in myocytes from failing hearts.

In heart failure (HF), dysregulated cardiac ryanodine receptors (RyR2) contribute to the generation of diastolic Ca2+ waves (DCWs), thereby predisposing adrenergically stressed failing hearts to life-threatening arrhythmias. However, the specific cellular, subcellular, and molecular defects that account for cardiac arrhythmia in HF remain to be elucidated. Patch-clamp techniques and confocal Ca2+ imaging were applied to study spatially defined Ca2+ handling in ventricular myocytes isolated from normal (control) and failing canine hearts. Based on their activation time upon electrical stimulation, Ca2+ release sites were categorized as coupled, located in close proximity to the sarcolemmal Ca2+ channels, and uncoupled, the Ca2+ channel-free non-junctional Ca2+ release units. In control myocytes, stimulation of ß-adrenergic receptors with isoproterenol (Iso) resulted in a preferential increase in Ca2+ spark rate at uncoupled sites. This site-specific effect of Iso was eliminated by the phosphatase inhibitor okadaic acid, which caused similar facilitation of Ca2+ sparks at coupled and uncoupled sites. Iso-challenged HF myocytes exhibited increased predisposition to DCWs compared to control myocytes. In addition, the overall frequency of Ca2+ sparks was increased in HF cells due to preferential stimulation of coupled sites. Furthermore, coupled sites exhibited accelerated recovery from functional refractoriness in HF myocytes compared to control myocytes. Spatially resolved subcellular Ca2+ mapping revealed that DCWs predominantly originated from coupled sites. Inhibition of CaMKII suppressed DCWs and prevented preferential stimulation of coupled sites in Iso-challenged HF myocytes. These results suggest that CaMKII- (and phosphatase)-dependent dysregulation of junctional Ca2+ release sites contributes to Ca2+-dependent arrhythmogenesis in HF.