Calcium Machinery Research Articles

Mitochondrial mechanosensor in cardiovascular diseases.

The role of mitochondria in cardiac tissue is of utmost importance due to the dynamic nature of the heart and its energetic demands, necessary to assure its proper beating function. Recently, other important mitochondrial roles have been discovered, namely its contribution to intracellular calcium handling in normal and pathological myocardium. Novel investigations support the fact that during the progression toward heart failure, mitochondrial calcium machinery is compromised due to its morphological, structural and biochemical modifications resulting in facilitated arrhythmogenesis and heart failure development. The interaction between mitochondria and sarcomere directly affect cardiomyocyte excitation-contraction and is also involved in mechano-transduction through the cytoskeletal proteins that tether together the mitochondria and the sarcoplasmic reticulum. The focus of this review is to briefly elucidate the role of mitochondria as (mechano) sensors in the heart.

Phagosomal removal of fungal melanin reprograms macrophage metabolism to promote antifungal immunity

In response to infection, macrophages adapt their metabolism rapidly to enhance glycolysis and fuel specialized antimicrobial effector functions. Here we show that fungal melanin is an essential molecule required for the metabolic rewiring of macrophages during infection with the fungal pathogen Aspergillus fumigatus. Using pharmacological and genetic tools, we reveal a molecular link between calcium sequestration by melanin inside the phagosome and induction of glycolysis required for efficient innate immune responses. By remodeling the intracellular calcium machinery and impairing signaling via calmodulin, melanin drives an immunometabolic signaling axis towards glycolysis with activation of hypoxia-inducible factor 1 subunit alpha (HIF-1α) and phagosomal recruitment of mammalian target of rapamycin (mTOR). These data demonstrate a pivotal mechanism in the immunometabolic regulation of macrophages during fungal infection and highlight the metabolic repurposing of immune cells as a potential therapeutic strategy.

Calcium Mobilization in Endothelial Cell Functions.

Endothelial cells (ECs) constitute the innermost layer that lines all blood vessels from the larger arteries and veins to the smallest capillaries, including the lymphatic vessels. Despite the histological classification of endothelium of a simple epithelium and its homogeneous morphological appearance throughout the vascular system, ECs, instead, are extremely heterogeneous both structurally and functionally. The different arrangement of cell junctions between ECs and the local organization of the basal membrane generate different type of endothelium with different permeability features and functions. Continuous, fenestrated and discontinuous endothelia are distributed based on the specific function carried out by the organs. It is thought that a large number ECs functions and their responses to extracellular cues depend on changes in intracellular concentrations of calcium ion ([Ca2+]i). The extremely complex calcium machinery includes plasma membrane bound channels as well as intracellular receptors distributed in distinct cytosolic compartments that act jointly to maintain a physiological [Ca2+]i, which is crucial for triggering many cellular mechanisms. Here, we first survey the overall notions related to intracellular Ca2+ mobilization and later highlight the involvement of this second messenger in crucial ECs functions with the aim at stimulating further investigation that link Ca2+ mobilization to ECs in health and disease.

Phosphoproteomics reveals conserved exercise-stimulated signaling and AMPK regulation of store-operated calcium entry.

Exercise stimulates cellular and physiological adaptations that are associated with widespread health benefits. To uncover conserved protein phosphorylation events underlying this adaptive response, we performed mass spectrometry-based phosphoproteomic analyses of skeletal muscle from two widely used rodent models: treadmill running in mice and insitu muscle contraction in rats. We overlaid these phosphoproteomic signatures with cycling in humans to identify common cross-species phosphosite responses, as well as unique model-specific regulation. We identified >22,000 phosphosites, revealing orthologous protein phosphorylation and overlapping signaling pathways regulated by exercise. This included two conserved phosphosites on stromal interaction molecule 1 (STIM1), which we validate as AMPK substrates. Furthermore, we demonstrate that AMPK-mediated phosphorylation of STIM1 negatively regulates store-operated calcium entry, and this is beneficial for exercise in Drosophila. This integrated cross-species resource of exercise-regulated signaling in human, mouse, and rat skeletal muscle has uncovered conserved networks and unraveled crosstalk between AMPK and intracellular calcium flux.

Jack bean urease modulates neurotransmitter release at insect neuromuscular junctions

BackgroundPlants have developed a vast range of mechanisms to compete with phytophagous insects, including entomotoxic proteins such as ureases. The legume Canavalia ensiformis produces several urease isoforms, of which the more abundant is called Jack Bean Urease (JBU). Previews work has demonstrated the potential insecticidal effects of JBU, by mechanisms so far not entirely elucidated. In this work, we investigated the mechanisms involved in the JBU-induced activity upon neurotransmitter release on insect neuromuscular junctions. MethodsElectrophysiological recordings of nerve and muscle action potentials, and calcium imaging bioassays were employed. Results and conclusionJBU (0.28 mg/animal/day) in Locusta migratoria 2nd instar through feeding and injection did not induce lethality, although it did result in a reduction of 20% in the weight gain at the end of 168 h (n = 9, p ≤ 0.05). JBU (0.014 and 0.14 mg) injected direct into the locust hind leg induced a dose and time-dependent decrease in the amplitude of muscle action potentials, with a maximum decrease of 70% in the amplitude at the highest dose (n = 5, p ≤ 0.05). At the same doses JBU did not alter the amplitude of action potentials evoked from motor neurons. Using Drosophila 3rd instar larvae neuromuscular preparations, JBU (10−7 M) increased the occurrence of miniature Excitatory Junctional Potentials (mEJPs) in the presence of 1 mM CaCl2 (n = 5, p ≤ 0.05). In low calcium (0.4 mM) assays, JBU (10−7 M) was not able to modulate the occurrence of the events. In Ca2+-free conditions, with EGTA or CoCl2, JBU induced a significant decrease in the occurrence of mEPJs (n = 5, p ≤ 0.05). Injected into the 3rd abdominal ganglion of Nauphoeta cinerea cockroaches, JBU (1 μM) induced a significant increase in Ca2+ influx (n = 7, p ≤ 0.01), similar to that seen for high KCl (35 mM) condition. Taken together the results confirm a direct action of JBU upon insect neuromuscular junctions and possibly central synapses, probably by disrupting the calcium machinery in the pre-synaptic region of the neurons.

Severe DCM phenotype of patient harboring RBM20 mutation S635A can be modeled by patient-specific induced pluripotent stem cell-derived cardiomyocytes

The ability to generate patient-specific induced pluripotent stem cells (iPSCs) provides a unique opportunity for modeling heart disease in vitro. In this study, we generated iPSCs from a patient with dilated cardiomyopathy (DCM) caused by a missense mutation S635A in RNA-binding motif protein 20 (RBM20) and investigated the functionality and cell biology of cardiomyocytes (CMs) derived from patient-specific iPSCs (RBM20-iPSCs). The RBM20-iPSC-CMs showed abnormal distribution of sarcomeric α-actinin and defective calcium handling compared to control-iPSC-CMs, suggesting disorganized myofilament structure and altered calcium machinery in CMs of the RBM20 patient. Engineered heart muscles (EHMs) from RBM20-iPSC-CMs showed that not only active force generation was impaired in RBM20-EHMs but also passive stress of the tissue was decreased, suggesting a higher visco-elasticity of RBM20-EHMs. Furthermore, we observed a reduced titin (TTN) N2B-isoform expression in RBM20-iPSC-CMs by demonstrating a reduction of exon skipping in the PEVK region of TTN and an inhibition of TTN isoform switch. In contrast, in control-iPSC-CMs both TTN isoforms N2B and N2BA were expressed, indicating that the TTN isoform switch occurs already during early cardiogenesis. Using next generation RNA sequencing, we mapped transcriptome and splicing target profiles of RBM20-iPSC-CMs and identified different cardiac gene networks in response to the analyzed RBM20 mutation in cardiac-specific processes. These findings shed the first light on molecular mechanisms of RBM20-dependent pathological cardiac remodeling leading to DCM. Our data demonstrate that iPSC-CMs coupled with EHMs provide a powerful tool for evaluating disease-relevant functional defects and for a deeper mechanistic understanding of alternative splicing-related cardiac diseases.

Altered Mitochondrial Metabolism and Mechanosensation in the Failing Heart: Focus on Intracellular Calcium Signaling.

The heart consists of millions of cells, namely cardiomyocytes, which are highly organized in terms of structure and function, at both macroscale and microscale levels. Such meticulous organization is imperative for assuring the physiological pump-function of the heart. One of the key players for the electrical and mechanical synchronization and contraction is the calcium ion via the well-known calcium-induced calcium release process. In cardiovascular diseases, the structural organization is lost, resulting in morphological, electrical, and metabolic remodeling owing the imbalance of the calcium handling and promoting heart failure and arrhythmias. Recently, attention has been focused on the role of mitochondria, which seem to jeopardize these events by misbalancing the calcium processes. In this review, we highlight our recent findings, especially the role of mitochondria (dys)function in failing cardiomyocytes with respect to the calcium machinery.

Studying dyadic structure–function relationships: a review of current modeling approaches and new insights into Ca2+ (mis)handling

Excitation–contraction coupling in cardiac myocytes requires calcium influx through L-type calcium channels in the sarcolemma, which gates calcium release through sarcoplasmic reticulum ryanodine receptors in a process known as calcium-induced calcium release, producing a myoplasmic calcium transient and enabling cardiomyocyte contraction. The spatio-temporal dynamics of calcium release, buffering, and reuptake into the sarcoplasmic reticulum play a central role in excitation–contraction coupling in both normal and diseased cardiac myocytes. However, further quantitative understanding of these cells’ calcium machinery and the study of mechanisms that underlie both normal cardiac function and calcium-dependent etiologies in heart disease requires accurate knowledge of cardiac ultrastructure, protein distribution and subcellular function. As current imaging techniques are limited in spatial resolution, limiting insight into changes in calcium handling, computational models of excitation–contraction coupling have been increasingly employed to probe these structure–function relationships. This review will focus on the development of structural models of cardiac calcium dynamics at the subcellular level, orienting the reader broadly towards the development of models of subcellular calcium handling in cardiomyocytes. Specific focus will be given to progress in recent years in terms of multi-scale modeling employing resolved spatial models of subcellular calcium machinery. A review of the state-of-the-art will be followed by a review of emergent insights into calcium-dependent etiologies in heart disease and, finally, we will offer a perspective on future directions for related computational modeling and simulation efforts.

Calcium homeostasis and signaling in fungi and their relevance forpathogenicity of yeasts and filamentous fungi

Though fungi show peculiarities in the purposes and specific traits of calcium signaling pathways, the general scheme and the most important players are well conserved if compared to higher eukaryotes. This provides a powerful opportunity either to investigate shared features using yeast as a model or to exploit fungal specificities as potential targets for antifungal therapies. The sequenced genomes from yeast Saccharomyces cerevisiae, Schizosaccharomyces pombe and the filamentous fungus Neurospora crassa were already published more than ten years ago. More recently the genome sequences of filamentous fungi of Aspergillus genus, some of which threatening pathogens, and dimorphic fungi Ustilago maydis were published, giving the chance to identify several proteins involved in calcium signaling based on their homology to yeast or mammalian counterparts. Nonetheless, unidentified calcium transporters are still present in these organisms which await to be molecularly characterized. Despite the relative simplicity in yeast calcium machinery and the availability of sophisticated molecular tools, in the last years, a number of new actors have been identified, albeit not yet fully characterized. This review will try to describe the state of the art in calcium channels and calcium signaling knowledge in yeast, with particular attention to the relevance of this knowledge with respect to pathological fungi.

Cisplatin-Induced Cachexia in rats Causes Alterations in Skeletal Muscle Calcium Homeostasis

Cachexia is a wasting condition associated with cancer types and, at the same time, is a serious and dose-limiting effect of cancer chemotherapy. Skeletal muscle loss is the main characteristic of cachexia and the primary cause of function impairment, fatigue and respiratory complications. Because calcium homeostasis is a biomarker of muscle function, here we assessed whether it is altered in cachetic muscles by using fura-2 cytofluorimetric and electrophysiological techniques in the rat model of cisplatin-induced cachexia. We showed that besides a significant reduction of the muscle weight and fiber diameter, indexes of an overt muscle atrophy, cachetic EDL fast-twitch fibers are characterized by a significant 56% increase of resting intracellular calcium, [Ca2+]i, compared to control rats. Moreover the amplitude of the calcium transient induced by caffeine and depolarizing high potassium solution was significantly reduced in cisplatin-treated rats. Importantly, changes of some calcium-dependent functional outcomes, such as an increase of the latency of the action potential and a decrease of resting chloride conductance, also occurred in cachetic EDL muscles, thus indicating that the cachexia-induced alteration of calcium homeostasis influences muscle functionality. To gain insight into the molecular mechanism responsible for calcium handling alteration, we are currently investigating the expression levels of the genes related to calcium machinery in cachetic muscles. Our findings provide the first direct evidence of a calcium homeostasis dysregulation in cachexia, thus contributing to the clarification of the mechanisms underlying the muscle wasting and to the identification of druggable targets for the treatment of this invalidating syndrome.

A Multiscale Hybrid Model for Pro-angiogenic Calcium Signals in a Vascular Endothelial Cell

Cytosolic calcium machinery is one of the principal signaling mechanisms by which endothelial cells (ECs) respond to external stimuli during several biological processes, including vascular progression in both physiological and pathological conditions. Low concentrations of angiogenic factors (such as VEGF) activate in fact complex pathways involving, among others, second messengers arachidonic acid (AA) and nitric oxide (NO), which in turn control the activity of plasma membrane calcium channels. The subsequent increase in the intracellular level of the ion regulates fundamental biophysical properties of ECs (such as elasticity, intrinsic motility, and chemical strength), enhancing their migratory capacity. Previously, a number of continuous models have represented cytosolic calcium dynamics, while EC migration in angiogenesis has been separately approached with discrete, lattice-based techniques. These two components are here integrated and interfaced to provide a multiscale and hybrid Cellular Potts Model (CPM), where the phenomenology of a motile EC is realistically mediated by its calcium-dependent subcellular events. The model, based on a realistic 3-D cell morphology with a nuclear and a cytosolic region, is set with known biochemical and electrophysiological data. In particular, the resulting simulations are able to reproduce and describe the polarization process, typical of stimulated vascular cells, in various experimental conditions. Moreover, by analyzing the mutual interactions between multilevel biochemical and biomechanical aspects, our study investigates ways to inhibit cell migration: such strategies have in fact the potential to result in pharmacological interventions useful to disrupt malignant vascular progression.

Endothelial Calcium Machinery and Angiogenesis: Understanding Physiology to Interfere with Pathology

Endothelial cells (ECs) play a pivotal role in physiological and altered tissue neovascularization. They face multiple morphological, biochemical and functional changes during the different phases of angiogenesis, under the regulation of a great number of proangiogenic and antiangiogenic signals, including soluble and insoluble factors, cell-cell and cell-matrix interactions. ECs mutual contacts (and also interactions with other cell types, such as pericytes and smooth vascular muscle cells), motility, proliferation, apoptosis and differentiation are all calcium-dependent events finely tuned in space and time. Most of the angiogenic-related peptidic factors (VEGF, bFGF and others) promote an increase of cytosolic free calcium concentration in ECs, giving rise to calcium-activated intracellular cascades engaged in the different steps of the angiogenic process. A better knowledge of such signals could allow to set new diagnostic and therapeutical approaches aimed to interfere with altered neovascularization, particularly during cancer progression. This review reports the state of the art about endothelial angiogenic-related calcium signaling and discusses the most attractive perspectives for the future.

1,4,5-Inositol Trisphosphate-Operated Intracellular Ca2+ Stores and Angiotensin-II/Endothelin-1 Signaling Pathway Are Functional in Human Embryonic Stem Cell-Derived Cardiomyocytes

On the basis of previous findings suggesting that in human embryonic stem cell-derived cardiomyocytes (hESC-CM) the sarcoplasmic reticulum Ca(2+)-induced release of calcium machinery is either absent or immature, in the present study we tested the hypothesis that hESC-CM contain fully functional 1,4,5-inositol trisphosphate (1,4,5-IP(3))-operated intracellular Ca(2+) ([Ca(2+)](i)) stores that can be mobilized upon appropriate physiological stimuli. To test this hypothesis we investigated the effects of angiotensin-II (AT-II) and endothelin-1 (ET-1), which activate the 1,4,5-IP(3) pathway, on [Ca(2+)](i) transients and contractions in beating clusters of hESC-CM. Our major findings were that in paced hESC-CM both AT-II and ET-1 (10(-9) to 10(-7) M) increased the contraction amplitude and the maximal rates of contraction and relaxation. In addition, AT-II (10(-9) to 10(-7) M) increased the [Ca(2+)](i) transient amplitude. The involvement of 1,4,5-IP(3)-dependent intracellular Ca(2+) release in the inotropic effect of AT-II was supported by the findings that (a) hESC-CM express AT-II, ET-1, and 1,4,5-IP(3) receptors determined by immunofluorescence staining, and (b) the effects of AT-II were blocked by 2 microM 2-aminoethoxyphenyl borate (a 1,4,5-IP(3) receptor blocker) and U73122 (a phospholipase C blocker). In conclusion, these findings demonstrate for the first time that hESC-CM exhibit functional AT-II and ET-1 signaling pathways, as well as 1,4,5-IP(3)-operated releasable Ca(2+) stores.

Calbindin Independence of Calcium Transport in Developing Teeth Contradicts the Calcium Ferry Dogma

Cytosolic calcium-binding proteins termed calbindins are widely regarded as a key component of the machinery used to transport calcium safely across cells. Acting as mobile buffers, calbindins are thought to ferry calcium in bulk and simultaneously protect against its potentially cytotoxic effects. Here, we contradict this dogma by showing that teeth and bones were produced normally in null mutant mice lacking calbindin(28kDa). Structural analysis of dental enamel, the development of which depends critically on active calcium transport, showed that mineralization was unaffected in calbindin(28kDa)-null mutants. An unchanged rate of calcium transport was verified by measurements of (45)Ca incorporation into developing teeth in vivo. In enamel-forming cells, the absence of calbindin(28kDa) was not compensated by other cytosolic calcium-binding proteins as detectable by (45)Ca overlay, two-dimensional gel, and equilibrium binding analyses. Despite a 33% decrease in cytosolic buffer capacity, cytotoxicity was not evident in either the null mutant enamel or its formative cells. This is the first definitive evidence that calbindins are not required for active calcium transport, either as ferries or as facilitative buffers. Moreover, in challenging the broader notion of a cytosolic route for calcium, the findings support an alternative paradigm involving passage via calcium-tolerant organelles.