L-type Cav1.3 channels regulate ryanodine receptor-dependent Ca2+ release during sino-atrial node pacemaker activity.

AIMS: Sino-atrial node (SAN) automaticity is an essential mechanism of heart rate generation that is still not completely understood. Recent studies highlighted the importance of intracellular Ca(2+) ([Ca(2+)]i) dynamics during SAN pacemaker activity. Nevertheless, the functional role of voltage-dependent L-type Ca(2+) channels in controlling SAN [Ca(2+)]i release is largely unexplored. Since Cav1.3 is the predominant L-type Ca(2+) channel isoform in SAN cells, we studied [Ca(2+)]i dynamics in isolated cells and ex vivo SAN preparations explanted from wild-type (WT) and Cav1.3 knockout (KO) mice (Cav1.3(-/-)). METHODS AND RESULTS: We found that Cav1.3 deficiency strongly impaired [Ca(2+)]i dynamics, reducing the frequency of local [Ca(2+)]i release events and preventing their synchronization. This impairment inhibited the generation of Ca(2+) transients and delayed spontaneous activity. We also used action potentials recorded in WT SAN cells as voltage-clamp commands for Cav1.3(-/-) cells. Although these experiments showed abolished Ca(2+) entry through L-type Ca(2+) channels in the diastolic depolarization range of KO SAN cells, their sarcoplasmic reticulum Ca(2+) load remained normal. ß-Adrenergic stimulation enhanced pacemaking of both genotypes, though, Cav1.3(-/-) SAN cells remained slower than WT. Conversely, we rescued pacemaker activity in Cav1.3(-/-) SAN cells and intact tissues through caffeine-mediated stimulation of Ca(2+)-induced Ca(2+) release. CONCLUSIONS: Cav1.3 channels play a critical role in the regulation of [Ca(2+)]i dynamics, providing an unanticipated mechanism for triggering local [Ca(2+)]i releases and thereby controlling pacemaker activity. Our study also provides an additional pathophysiological mechanism for congenital SAN dysfunction and heart block linked to Cav1.3 loss of function in humans.

Pancreatic alpha-cells from female mice undergo morphofunctional changes during compensatory adaptations of the endocrine pancreas to diet-induced obesity.

Obesity is frequently associated with insulin resistance. To compensate for this situation and maintain normoglycaemia, pancreatic beta-cells undergo several morphofunctional adaptations, which result in insulin hypersecretion and hyperinsulinaemia. However, no information exists about pancreatic alpha-cells during this compensatory stage of obesity. Here, we studied alpha-cells in mice fed a high-fat diet (HFD) for 12 weeks. These animals exhibited hyperinsulinaemia and normoglycaemia compared with control animals in addition to hypoglucagonaemia. While the in vivo response of glucagon to hypoglycaemia was preserved in the obese mice, the suppression of glucagon secretion during hyperglycaemia was impaired. Additionally, in vitro glucagon release at low glucose levels and glucagon content in isolated islets were decreased, while alpha-cell exocytosis remained unchanged. Assessment of morphological parameters revealed that alpha-cell area was reduced in the pancreas of the obese mice in association with alpha-cell hypotrophy, increased apoptosis and decreased proliferation. HFD feeding for 24 weeks led to significant deterioration in beta-cell function and glucose homeostasis. Under these conditions, the majority of alpha-cell changes were reversed and became comparable to controls. These findings indicate that pancreatic compensatory adaptations during obesity may also involve pancreatic alpha-cells. Additionally, defects in alpha-cell function during obesity may be implicated in progression to diabetes.

Rasgos no ventriculares, clínicos y funcionales de la mutación RyR2 R420Q causante de taquicardia ventricular polimórfica catecolaminérgica / Non-ventricular, clinical, and functional features of the ryr2 r420q mutation causing catecholaminergic polymorphic ventricular tachycardia

Introducción y objetivos: La taquicardia ventricular polimórfica catecolaminérgica es una enfermedad maligna que se debe a mutaciones en las proteínas que controlan la homeostasis del Ca2+. Aunque el fenotipo se caracteriza por arritmias ventriculares polimórficas desencadenadas por el estrés, no se han caracterizado plenamente las arritmias supraventriculares que en ocasiones las acompañan. Métodos: Veinticinco miembros de una familia española en la que había habido varias muertes súbitas fueron evaluados mediante electrocardiograma, pruebas de esfuerzo y prueba de desenmascaramiento con adrenalina opcionalmente. Se realizó secuenciación selectiva deRyR2 en un miembro afectado y un cribado en cascada al resto de la familia. Se generó la mutación RyR2R420Q en células HEK-293 mediante mutagénesis dirigida, con objeto de realizar estudios funcionales in vitro. Resultados: Las pruebas de esfuerzo desenmascararon taquicardia ventricular polimórfica catecolaminérgica en 8 familiares (sensibilidad del 89%; valor predictivo positivo del 100%; valor predictivo negativo del 93%), todos ellos portadores de una mutación heterocigota RyR2R420Q, que estaba presente también en el caso probando y en una chica joven sin prueba de esfuerzo, lo que corresponde a una penetrancia del 91% al final del seguimiento. Es de destacar que en los pacientes se identificó bradicardia sinusal, arritmias auriculares y de la unión y/u ondas U gigantes tras esfuerzo. Tras la permeabilización y en las células intactas, las células que expresaban RyR2R420Q mostraron un pico de liberación de Ca2+ menor que el de las célulasRyR2 no mutado o wild-type. Sin embargo, a una concentración de Ca2+ intracelular fisiológica, equivalente a la concentración citosólica diastólica, las células RyR2R420Q liberaban más Ca2+ y oscilaban con mayor rapidez que las células con RyR2 no mutado o wild-type. Conclusiones: La mutación missense RyR2R420Q se identificó en el extremo aminoterminal del gen RyR2 en esta familia muy sintomática. Es de destacar que esta mutación se asocia a bradicardia sinusal, arritmias auriculares y de la unión y ondas U gigantes. En conjunto, los estudios de expresión heteróloga funcional indican que la mutación RyR2R420Q causa un comportamiento aberrante del canal, con pérdida o ganancia de función, según cuál sea la concentración de Ca2+ intracelular citosólica

Introduction and objectives: Catecholaminergic polymorphic ventricular tachycardia is a malignant disease, due to mutations in proteins controlling Ca2+ homeostasis. While the phenotype is characterized by polymorphic ventricular arrhythmias under stress, supraventricular arrhythmias may occur and are not fully characterized. Methods: Twenty-five relatives from a Spanish family with several sudden deaths were evaluated with electrocardiogram, exercise testing, and optional epinephrine challenge. Selective RyR2 sequencing in an affected individual and cascade screening in the rest of the family was offered. The RyR2R420Q mutation was generated in HEK-293 cells using site-directed mutagenesis to conduct in vitro functional studies. Results: The exercise testing unmasked catecholaminergic polymorphic ventricular tachycardia in 8 relatives (sensitivity = 89%; positive predictive value = 100%; negative predictive value = 93%), all of them carrying the heterozygous RyR2R420Q mutation, which was also present in the proband and a young girl without exercise testing, a 91% penetrance at the end of the follow-up. Remarkably, sinus bradycardia, atrial and junctional arrhythmias, and/or giant post-effort U-waves were identified in patients. Upon permeabilization and in intact cells, the RyR2R420Q expressing cells showed a smaller peak of Ca2+ release than RyR2 wild-type cells. However, at physiologic intracellular Ca2+ concentration, equivalent to the diastolic cytosolic concentration, the RyR2R420Q released more Ca2+ and oscillated faster than RyR2 wild-type cells. Conclusions The missense RyR2R420Q mutation was identified in the N-terminus of the RyR2 gene in this highly symptomatic family. Remarkably, this mutation is associated with sinus bradycardia, atrial and junctional arrhythmias, and giant U-waves. Collectively, functional heterologous expression studies suggest that the RyR2R420Q behaves as an aberrant channel, as a loss- or gain-of-function mutation depending on cytosolic intracellular Ca2+ concentration

Enhanced glucose-induced intracellular signaling promotes insulin hypersecretion: pancreatic beta-cell functional adaptations in a model of genetic obesity and prediabetes.

Obesity is associated with insulin resistance and is known to be a risk factor for type-2 diabetes. In obese individuals, pancreatic beta-cells try to compensate for the increased insulin demand in order to maintain euglycemia. Most studies have reported that this adaptation is due to morphological changes. However, the involvement of beta-cell functional adaptations in this process needs to be clarified. For this purpose, we evaluated different key steps in the glucose-stimulated insulin secretion (GSIS) in intact islets from female ob/ob obese mice and lean controls. Obese mice showed increased body weight, insulin resistance, hyperinsulinemia, glucose intolerance and fed hyperglycemia. Islets from ob/ob mice exhibited increased glucose-induced mitochondrial activity, reflected by enhanced NAD(P)H production and mitochondrial membrane potential hyperpolarization. Perforated patch-clamp examination of beta-cells within intact islets revealed several alterations in the electrical activity such as increased firing frequency and higher sensitivity to low glucose concentrations. A higher intracellular Ca(2+) mobilization in response to glucose was also found in ob/ob islets. Additionally, they displayed a change in the oscillatory pattern and Ca(2+) signals at low glucose levels. Capacitance experiments in intact islets revealed increased exocytosis in individual ob/ob beta-cells. All these up-regulated processes led to increased GSIS. In contrast, we found a lack of beta-cell Ca(2+) signal coupling, which could be a manifestation of early defects that lead to beta-cell malfunction in the progression to diabetes. These findings indicate that beta-cell functional adaptations are an important process in the compensatory response to obesity.

Non-ventricular, Clinical, and Functional Features of the RyR2(R420Q) Mutation Causing Catecholaminergic Polymorphic Ventricular Tachycardia.

INTRODUCTION AND OBJECTIVES: Catecholaminergic polymorphic ventricular tachycardia is a malignant disease, due to mutations in proteins controlling Ca(2+) homeostasis. While the phenotype is characterized by polymorphic ventricular arrhythmias under stress, supraventricular arrhythmias may occur and are not fully characterized. METHODS: Twenty-five relatives from a Spanish family with several sudden deaths were evaluated with electrocardiogram, exercise testing, and optional epinephrine challenge. Selective RyR2 sequencing in an affected individual and cascade screening in the rest of the family was offered. The RyR2(R420Q) mutation was generated in HEK-293 cells using site-directed mutagenesis to conduct in vitro functional studies. RESULTS: The exercise testing unmasked catecholaminergic polymorphic ventricular tachycardia in 8 relatives (sensitivity = 89%; positive predictive value = 100%; negative predictive value = 93%), all of them carrying the heterozygous RyR2(R420Q) mutation, which was also present in the proband and a young girl without exercise testing, a 91% penetrance at the end of the follow-up. Remarkably, sinus bradycardia, atrial and junctional arrhythmias, and/or giant post-effort U-waves were identified in patients. Upon permeabilization and in intact cells, the RyR2(R420Q) expressing cells showed a smaller peak of Ca(2+) release than RyR2 wild-type cells. However, at physiologic intracellular Ca(2+) concentration, equivalent to the diastolic cytosolic concentration, the RyR2(R420Q) released more Ca(2+) and oscillated faster than RyR2 wild-type cells. CONCLUSIONS: The missense RyR2(R420Q) mutation was identified in the N-terminus of the RyR2 gene in this highly symptomatic family. Remarkably, this mutation is associated with sinus bradycardia, atrial and junctional arrhythmias, and giant U-waves. Collectively, functional heterologous expression studies suggest that the RyR2(R420Q) behaves as an aberrant channel, as a loss- or gain-of-function mutation depending on cytosolic intracellular Ca(2+) concentration.

Insulin hypersecretion in islets from diet-induced hyperinsulinemic obese female mice is associated with several functional adaptations in individual ß-cells.

Insulin resistance and hyperinsulinemia are generally associated with obesity. Obese nondiabetic individuals develop a compensatory ß-cell response to adjust insulin levels to the increased demand, maintaining euglycemia. Although several studies indicate that this compensation relies on structural changes, the existence of ß-cell functional adaptations is incompletely understood. Here, we fed female mice with a high-fat diet (HFD) for 12 weeks. These animals became obese, hyperinsulinemic, insulin-resistant, and mildly glucose-intolerant while fed, and fasting glycemia was comparable in HFD and control mice. Islets from HFD animals exhibited increased ß-cell mass and hypertrophy. Additionally, they had enhanced insulin gene expression and content and augmented glucose-induced insulin secretion. Electrophysiological examination of ß-cells from both groups showed no differences in KATP channel open probability and conductance. However, action potentials elicited by glucose had larger amplitude in obese mice. Glucose-induced Ca²âº signals in intact islets, in isolated ß-cells, and individual ß-cells within islets were also increased in HFD mice. Additionally, a higher proportion of glucose-responsive cells was present in obese mice. In contrast, whole-cell Ca²âº current densities were similar in both groups. Capacitance measurements showed that depolarization-evoked exocytosis was enhanced in HFD ß-cells compared with controls. Although this augment was not significant when capacitance increases of the whole ß-cell population were normalized to cell size, the exocytotic output varied significantly when ß-cells were distributed by size ranges. All these findings indicate that ß-cell functional adaptations are present in the islet compensatory response to obesity.

Paradoxical effect of increased diastolic Ca(2+) release and decreased sinoatrial node activity in a mouse model of catecholaminergic polymorphic ventricular tachycardia.

BACKGROUND: Catecholaminergic polymorphic ventricular tachycardia is characterized by stress-triggered syncope and sudden death. Patients with catecholaminergic polymorphic ventricular tachycardia manifest sinoatrial node (SAN) dysfunction, the mechanisms of which remain unexplored. METHODS AND RESULTS: We investigated SAN [Ca(2+)](i) handling in mice carrying the catecholaminergic polymorphic ventricular tachycardia-linked mutation of ryanodine receptor (RyR2(R4496C)) and their wild-type (WT) littermates. In vivo telemetric recordings showed impaired SAN automaticity in RyR2(R4496C) mice after isoproterenol injection, analogous to what was observed in catecholaminergic polymorphic ventricular tachycardia patients after exercise. Pacemaker activity was explored by measuring spontaneous [Ca(2+)](i) transients in SAN cells within the intact SAN by confocal microscopy. RyR2(R4496C) SAN presented significantly slower pacemaker activity and impaired chronotropic response under ß-adrenergic stimulation, accompanied by the appearance of pauses (in spontaneous [Ca(2+)](i) transients and action potentials) in 75% of the cases. Ca(2+) spark frequency was increased by 2-fold in RyR2(R4496C) SAN. Whole-cell patch-clamp experiments performed on isolated RyR2(R4496C) SAN cells showed that L-type Ca(2+) current (I(Ca,L)) density was reduced by ≈50%, an effect blunted by internal Ca(2+) buffering. Isoproterenol dramatically increased the frequency of Ca(2+) sparks and waves by ≈5 and ≈10-fold, respectively. Interestingly, the sarcoplasmic reticulum Ca(2+) content was significantly reduced in RyR2(R4496C) SAN cells in the presence of isoproterenol, which may contribute to stopping the "Ca(2+) clock" rhythm generation, originating SAN pauses. CONCLUSION: The increased activity of RyR2(R4496C) in SAN leads to an unanticipated decrease in SAN automaticity by a Ca(2+)-dependent decrease of I(Ca,L) and sarcoplasmic reticulum Ca(2+) depletion during diastole, identifying subcellular pathophysiological alterations contributing to the SAN dysfunction in catecholaminergic polymorphic ventricular tachycardia patients.

Role of leptin in the pancreatic ß-cell: effects and signaling pathways.

Leptin plays an important role in the control of food intake, energy expenditure, metabolism, and body weight. This hormone also has a key function in the regulation of glucose homeostasis. Although leptin acts through central and peripheral mechanisms to modulate glucose metabolism, the pancreatic ß-cell of the endocrine pancreas is a critical target of leptin actions. Leptin receptors are present in the ß-cell, and their activation directly inhibits insulin secretion from these endocrine cells. The effects of leptin on insulin occur also in the long term, since this hormone inhibits insulin gene expression as well. Additionally, ß-cell mass can be affected by leptin through changes in proliferation, apoptosis, or cell size. All these different functions in the ß-cell are triggered by leptin as a result of the large diversity of signaling pathways that this hormone is able to activate in the endocrine pancreas. Therefore, leptin can participate in glucose homeostasis owing to different levels of modulation of the pancreatic ß-cell population. Furthermore, it has been proposed that alterations in this level of regulation could contribute to the impairment of ß-cell function in obesity states. In the present review, we will discuss all these issues with special emphasis on the effects and pathways of leptin signaling in the pancreatic ß-cell.

RyRCa2+ leak limits cardiac Ca2+ window current overcoming the tonic effect of calmodulinin mice.

Ca(2+) mediates the functional coupling between L-type Ca(2+) channel (LTCC) and sarcoplasmic reticulum (SR) Ca(2+) release channel (ryanodine receptor, RyR), participating in key pathophysiological processes. This crosstalk manifests as the orthograde Ca(2+)-induced Ca(2+)-release (CICR) mechanism triggered by Ca(2+) influx, but also as the retrograde Ca(2+)-dependent inactivation (CDI) of LTCC, which depends on both Ca(2+) permeating through the LTCC itself and on SR Ca(2+) release through the RyR. This latter effect has been suggested to rely on local rather than global Ca(2+) signaling, which might parallel the nanodomain control of CDI carried out through calmodulin (CaM). Analyzing the CICR in catecholaminergic polymorphic ventricular tachycardia (CPVT) mice as a model of RyR-generated Ca(2+) leak, we evidence here that increased occurrence of the discrete local SR Ca(2+) releases through the RyRs (Ca(2+) sparks) cause a depolarizing shift in activation and a hyperpolarizing shift in isochronic inactivation of cardiac LTCC current resulting in the reduction of window current. Both increasing fast [Ca(2+)](i) buffer capacity or depleting SR Ca(2+) store blunted these changes, which could be reproduced in WT cells by RyRCa(2+) leak induced with Ryanodol and CaM inhibition.Our results unveiled a new paradigm for CaM-dependent effect on LTCC gating and further the nanodomain Ca(2+) control of LTCC, emphasizing the importance of spatio-temporal relationships between Ca(2+) signals and CaM function.

Sodium-calcium exchange is essential for effective triggering of calcium release in mouse heart.

In cardiac myocytes, excitation-contraction coupling depends upon sarcoplasmic reticular Ca2+ release triggered by Ca2+ influx through L-type Ca2+ channels. Although Na+-Ca2+ exchange (NCX) is essential for Ca2+ extrusion, its participation in the trigger process of excitation-contraction coupling is controversial. To investigate the role of NCX in triggering, we examined Ca2+ sparks in ventricular cardiomyocytes isolated from wild-type (WT) and cardiac-specific NCX knockout (KO) mice. Myocytes from young NCX KO mice are known to exhibit normal resting cytosolic Ca2+ and normal Ca2+ transients despite reduced L-type Ca2+ current. We loaded myocytes with fluo-3 to image Ca2+ sparks using confocal microscopy in line-scan mode. The frequency of spontaneous Ca2+ sparks was reduced in KO myocytes compared with WT. However, spark amplitude and width were increased in KO mice. Permeabilizing the myocytes with saponin eliminated differences between spontaneous sparks in WT and KO mice. These results suggest that sarcolemmal processes are responsible for the reduced spark frequency and increased spark width and amplitude in KO mice. When myocytes were loaded with 1 mM fluo-3 and 3 mM EGTA via the patch pipette to buffer diadic cleft Ca2+, the number of sparks triggered by action potentials was reduced by 60% in KO cells compared to WT cells, despite similar SR Ca2+ content in both cell types. When EGTA was omitted from the pipette solution, the number of sparks triggered in KO and WT myocytes was similar. Although the number of sparks was restored in KO cells, Ca2+ release was asynchronous. These results suggest that high subsarcolemmal Ca2+ is required to ensure synchronous triggering with short spark latency in the absence of NCX. In WT mice, high subsarcolemmal Ca2+ is not required for synchronous triggering, because NCX is capable of priming the diadic cleft with sufficient Ca2+ for normal triggering, even when subsarcolemmal Ca(2+) is lowered by EGTA. Thus, reducing subsarcolemmal Ca2+ with EGTA in NCX KO mice reveals the dependence of Ca2+ release on NCX.

Dysfunction of ouabain-induced cardiac contractility in mice with heart-specific ablation of Na,K-ATPase beta1-subunit.

Na,K-ATPase is composed of two essential alpha- and beta-subunits, both of which have multiple isoforms. Evidence indicates that the Na,K-ATPase enzymatic activity as well as its alpha(1), alpha(3) and beta(1) isoforms are reduced in the failing human heart. The catalytic alpha-subunit is the receptor for cardiac glycosides such as digitalis, used for the treatment of congestive heart failure. The role of the Na,K-ATPase beta(1)-subunit (Na,K-beta(1)) in cardiac function is not known. We used Cre/loxP technology to inactivate the Na,K-beta(1) gene exclusively in the ventricular cardiomyocytes. Animals with homozygous Na,K-beta(1) gene excision were born at the expected Mendelian ratio, grew into adulthood, and appeared to be healthy until 10 months of age. At 13-14 months, these mice had 13% higher heart/body weight ratios, and reduced contractility as revealed by echocardiography compared to their wild-type (WT) littermates. Pressure overload by transverse aortic constriction (TAC) in younger mice, resulted in compensated hypertrophy in WT mice, but decompensation in the Na,K-beta(1) KO mice. The young KO survivors of TAC exhibited decreased contractile function and mimicked the effects of the Na,K-beta(1) KO in older mice. Further, we show that intact hearts of Na,K-beta(1) KO anesthetized mice as well as isolated cardiomyocytes were insensitive to ouabain-induced positive inotropy. This insensitivity was associated with a reduction in NCX1, one of the proteins involved in regulating cardiac contractility. In conclusion, our results demonstrate that Na,K-beta(1) plays an essential role in regulating cardiac contractility and that its loss is associated with significant pathophysiology of the heart.

Myosin II contributes to fusion pore expansion during exocytosis.

During exocytosis, the fusion pore expands to allow release of neurotransmitters and hormones to the extracellular space. To understand the process of synaptic transmission, it is of outstanding importance to know the properties of the fusion pore and how these properties affect the release process. Many proteins have been implicated in vesicle fusion; however, there is little evidence for proteins involved in fusion pore expansion. Myosin II has been shown to participate in the transport of vesicles and, surprisingly, in the final phases of exocytosis, affecting the kinetics of catecholamine release in adrenal chromaffin cells as measured by amperometry. Here, we have studied single vesicle exocytosis in chromaffin cells overexpressing an unphosphorylatable form (T18AS19A RLC-GFP) of myosin II that produces an inactive protein by patch amperometry. This method allows direct determination of fusion pore expansion by measuring its conductance, whereas the release of catecholamines is recorded simultaneously by amperometry. Here we demonstrated that the fusion pore is of critical importance to control the release of catecholamines during single vesicle secretion in chromaffin cells. We proved that myosin II acts as a molecular motor on the fusion pore expansion by hindering its dilation when it lacks the phosphorylation sites.

Glycogen synthase kinase 3 activation is essential for the snake phospholipase A2 neurotoxin-induced secretion in chromaffin cells.

Neuroendocrine chromaffin cells were used to study the mechanism of the snake phospholipase A2 (PLA2) neurotoxin enhancement of exocytosis. Notexin, beta-bungarotoxin, taipoxin or textilotoxin enhanced the fast release of catecholamines elicited by flash photolysis of cytosolic caged calcium. Such an increase correlates with the capacity of these neurotoxins to cause fragmentation of the F-actin cortical barrier with subsequent accumulation of vesicles in the proximity of the plasma membrane. These PLA2 neurotoxins do not act via protein kinase C activation, which is known to promote F-actin fragmentation. Lithium, RO31-8220 and SB216763, three inhibitors of the glycogen synthase kinase 3, prevent both the alteration of the F-actin peripheral cortex and the enhancement of fast release elicited by these neurotoxins. In addition, glycogen synthase kinase 3 has been detected by immunolocalization in a membranous compartment of the chromaffin cell endoplasmic reticulum (ER). These results suggest that the activation of this enzyme plays a major role in the enhancement of exocytosis of the readily releasable granules caused by PLA2 neurotoxins in neuroendocrine chromaffin cells.

Calcium release domains in mammalian skeletal muscle studied with two-photon imaging and spot detection techniques.

The spatiotemporal characteristics of the Ca(2+) release process in mouse skeletal muscle were investigated in enzymatically dissociated fibers from flexor digitorum brevis (FDB) muscles, using a custom-made two-photon microscope with laser scanning imaging (TPLSM) and spot detection capabilities. A two-microelectrode configuration was used to electrically stimulate the muscle fibers, to record action potentials (APs), and to control their myoplasmic composition. We used 125 muM of the low-affinity Ca(2+) indicator Oregon green 488 BAPTA-5N (OGB-5N), and 5 or 10 mM of the Ca(2+) chelator EGTA (pCa 7) in order to arrest fiber contraction and to constrain changes in the [Ca(2+)] close to the release sites. Image and spot data showed that the resting distribution of OGB-5N fluorescence was homogeneous along the fiber, except for narrow peaks ( approximately 23% above the bulk fluorescence) centered at the Z-lines, as evidenced by their nonoverlapping localization with respect to di-8-ANEPPS staining of the transverse tubules (T-tubules). Using spot detection, localized Ca(2+) transients evoked by AP stimulation were recorded from adjacent longitudinal positions 100 nm apart. The largest and fastest DeltaF/F transients were detected at sites flanking the Z-lines and colocalized with T-tubules; the smallest and slowest were detected at the M-line, whereas transients at the Z-line showed intermediate features. Three-dimensional reconstructions demonstrate the creation of two AP-evoked Ca(2+) release domains per sarcomere, which flank the Z-line and colocalize with T-tubules. In the presence of 10 mM intracellular EGTA, these domains are formed in approximately 1.4 ms and dissipate within approximately 4 ms, after the peak of the AP. Their full-width at half-maximum (FWHM), measured at the time that Ca(2+) transients peaked at T-tubule locations, was 0.62 mum, similar to the 0.61 mum measured for di-8-ANEPPS profiles. Both these values exceed the limit of resolution of the optical system, but their similarity suggests that at high [EGTA] the Ca(2+) domains in adult mammalian muscle fibers are confined to Ca(2+) release sites located at the junctional sarcoplasmic reticulum (SR).

Quantitative evaluation of mammalian skeletal muscle as a heterologous protein expression system.

The production of mammalian proteins in sufficient quantity and quality for structural and functional studies is a major challenge in biology. Intrinsic limitations of yeast and bacterial expression systems preclude their use for the synthesis of a significant number of mammalian proteins. This creates the necessity of well-identified expression systems based on mammalian cells. In this paper, we demonstrate that adult mammalian skeletal muscle, transfected in vivo by electroporation with DNA plasmids, is an excellent heterologous mammalian protein expression system. By using the fluorescent protein EGFP as a model, it is shown that muscle fibers express, during the course of a few days, large amounts of authentic replicas of transgenic proteins. Yields of approximately 1mg/g of tissue were obtained, comparable to those of other expression systems. The involvement of adult mammalian cells assures an optimal environment for proper protein folding and processing. All these advantages complement a methodology that is universally accessible to biomedical investigators and simple to implement.

Real-time dynamics of the F-actin cytoskeleton during secretion from chromaffin cells.

Transmitted light images showed an intricate and dynamic cytoplasmic structural network in cultured bovine chromaffin cells observed under high magnification. These structures were sensitive to chemicals altering F-actin-myosin and colocalised with peripheral F-actin, beta-actin and myosin II. Interestingly, secretagogues induced a Ca2+-dependent, rapid (>10 second) and transitory (60-second cycle) disassembling of these cortical structures. The simultaneous formation of channel-like structures perpendicular to the plasmalemma conducting vesicles to the cell limits and open spaces devoid of F-actin in the cytoplasm were also observed. Vesicles moved using F-actin pathways and avoided diffusion in open, empty zones. These reorganisations representing F-actin transfer from the cortical barrier to the adjacent cytoplasmic area have been also confirmed by studying fluorescence changes in cells expressing GFP-beta-actin. Thus, these data support the function of F-actin-myosin II network acting simultaneously as a barrier and carrier system during secretion, and that transmitted light images could be used as an alternative to fluorescence in the study of cytoskeleton dynamics in neuroendocrine cells.

New roles of myosin II during vesicle transport and fusion in chromaffin cells.

Modified herpes virus (amplicons) were used to express myosin regulatory light chain (RLC) chimeras with green fluorescent protein (GFP) in cultured bovine chromaffin cells to study myosin II implication in secretion. After infection, RLC-GFP constructs were clearly identified in the cytoplasm and accumulated in the cortical region, forming a complex network that co-localized with cortical F-actin. Cells expressing wild type RLC-GFP maintained normal vesicle mobility, whereas cells expressing an unphosphorylatable form (T18A/S19A RLC-GFP) presented severe restrictions in granule movement as measured by individual tracking in dynamic confocal microscopy studies. Interestingly, the overexpression of this mutant form of RLC also affected the initial secretory burst elicited by either high K(+) or BaCl(2), as well as the secretion induced by fast release of calcium from caged compounds in individual cells. Moreover, T18A/S19A RLC-GFP-infected cells presented slower fusion kinetics of individual granules compared with controls as measured by analysis of amperometric spikes. Taken together, our results demonstrate the implication of myosin II in the transport of vesicles, and, surprisingly, in the final phases of exocytosis involving transitions affecting the activity of docked granules, and therefore uncovering a new role for this cytoskeletal element.

Differential participation of actin- and tubulin-based vesicle transport systems during secretion in bovine chromaffin cells.

The role of cytoskeletal elements in vesicle transport occurring during exocytosis was examined in adrenal medullary bovine chromaffin cells maintained in culture. Amperometric determination of depolarization-dependent catecholamine release from individual intact cells treated with actin or myosin inhibitors showed alterations in the fast and slow phases of secretion when compared with untreated cells. In contrast, microtubule disassemblers or stabilizers have a moderate effect on secretion, only affecting the release of slow secretory components. In experiments using confocal dynamic microscopy we have observed the drastic effect of actin and myosin inhibitors in abolishing vesicle movement throughout the cytoplasm, and the inhibition of granule mobility in deep perinuclear regions caused by the microtubule stabilizers. Following loss of mobility, vesicles were associated with filaments of F-actin or microtubules. In addition, the mobility of cortical vesicles was affected by actin-myosin inhibitors but not by microtubule inhibitors. The study of cortical cytoskeleton in living cells showed vesicles associated with dense tubular F-actin structures, with microtubules appearing as low density networks. These findings suggest that the distribution and density of both cytoskeletal elements in the cortical region may influence the recruitment of vesicle pools during secretion.

Taipoxin induces F-actin fragmentation and enhances release of catecholamines in bovine chromaffin cells.

Adrenomedullary bovine chromaffin cells were used to study the uptake and cellular effects of the phospholipase type A2 (PLA2) neurotoxin taipoxin in a neuroendocrine model. This toxin entered rapidly inside cultured cells. Within 1 h, taipoxin accumulated on the plasma membrane, independently of calcium presence, and caused fragmentation of the F-actin cytoskeleton. Toxin-induced cell death occurred after 24 h of incubation with the appearance of toxin containing large vesicles. Secretory experiments performed in cell populations showed an increased exocytosis in taipoxin-treated cells stimulated by depolarization or by incubation with the calcium-ionophore A23187. Like F-actin fragmentation, this effect is abolished by replacement of Ca2+ with Sr2+ during toxin incubation. The effect of taipoxin on exocytosis is not enhanced by latrunculin A, a F-actin disassembling drug altering secretion. Secretory studies in single taipoxin-treated cells using amperometry, showed an increase in the number of released vesicles without modification of the kinetic parameters of individual vesicle fusions. Taken together, these results suggest that taipoxin causes F-actin fragmentation and enhances secretion by redistribution of vesicles among secretory pools.

The role of myosin in vesicle transport during bovine chromaffin cell secretion.

Bovine adrenomedullary cells in culture have been used to study the role of myosin in vesicle transport during exocytosis. Amperometric determination of calcium-dependent catecholamine release from individual digitonin-permeabilized cells treated with 3 microM wortmannin or 20 mM 2,3-butanedione monoxime (BDM) and stimulated by continuous as well as repetitive calcium pulses showed alteration of slow phases of secretion when compared with control untreated cells. The specificity of these drugs for myosin inhibition was further supported by the use of peptide-18, a potent peptide affecting myosin light-chain kinase activity. These results were supported also by studying the impact of these myosin inhibitors on chromaffin granule mobility using direct visualization by dynamic confocal microscopy. Wortmannin and BDM affect drastically vesicle transport throughout the cell cytoplasm, including the region beneath the plasma membrane. Immunocytochemical studies demonstrate the presence of myosin types II and V in the cell periphery. The capability of antibodies to myosin II in abrogating the secretory response from populations of digitonin-permeabilized cells compared with the modest effect caused by anti-myosin V suggests that myosin II plays a fundamental role in the active transport of vesicles occurring in the sub-plasmalemmal area during chromaffin cell secretory activity.