Modelling of calcium handling in airway myocytes

Airway myocytes are the primary effectors of airway reactivity which modulates airway resistance and hence ventilation. Stimulation of airway myocytes results in an increase in the cytosolic Ca²⁺ concentration ([Ca²⁺]_i) and the subsequent activation of the contractile apparatus. Many contractile agonists, including acetylcholine, induce [Ca²⁺]_i increase via Ca²⁺ release from the sarcoplasmic reticulum through InsP₃ receptors. Several models have been developed to explain the characteristics of InsP₃-induced [Ca²⁺]_i responses, in particular Ca²⁺ oscillations. The article reviews the modelling of the major structures implicated in intracellular Ca²⁺ handling, i.e., InsP₃ receptors, SERCAs, mitochondria and Ca²⁺-binding cytosolic proteins. We developed theoretical models specifically dedicated to the airway myocyte which include the major mechanisms responsible for intracellular Ca²⁺ handling identified in these cells. These biocomputations pointed out the importance of the relative proportion of InsP₃ receptor isoforms and the respective role of the different mechanisms responsible for cytosolic Ca²⁺ clearance in the pattern of [Ca²⁺]_i variations. We have developed a theoretical model of membrane conductances that predicts the variations in membrane potential and extracellular Ca²⁺ influx. Stimulation of this model by simulated increase in [Ca²⁺]_i predicts membrane depolarisation, but not great enough to trigger a significant opening of voltage-dependant Ca²⁺ channels. This may explain why airway contraction induced by cholinergic stimulation does not greatly depend on extracellular calcium. The development of such models of airway myocytes is important for the understanding of the cellular mechanisms of airway reactivity and their possible modulation by pharmacological agents.     

Simultaneous study of mechanobiology and calcium dynamics on hESC‐derived cardiomyocytes clusters

Calcium ions act like ubiquitous second messengers in a wide amount of cellular processes. In cardiac myocytes, Ca²⁺ handling regulates the mechanical contraction necessary to the heart pump function. The field of intracellular and intercellular Ca²⁺ handling, employing in vitro models of cardiomyocytes, has become a cornerstone to understand the role and adaptation of calcium signalling in healthy and diseased hearts. Comprehensive in vitro systems and cell‐based biosensors are powerful tools to enrich and speed up cardiac phenotypic and drug response evaluation. We have implemented a combined setup to measure contractility and calcium waves in human embryonic stem cells‐derived cardiomyocyte 3D clusters, obtained from embryoid body differentiation. A combination of atomic force microscopy to monitor cardiac contractility, and sensitive fast scientific complementary metal‐oxide‐semiconductor camera for epifluorescence video recording, provided correlated signals in real time. To speed up the integrated data processing, we tested several post‐processing algorithms, to improve the automatic detection of relevant functional parameters. The validation of our proposed method was assessed by caffeine stimulation (10mM) and detection/characterization of the induced cardiac response. We successfully report the first simultaneous recording of cardiac contractility and calcium waves on the described cardiac 3D models. The drug stimulation confirmed the automatic detection capabilities of the used algorithms, measuring expected physiological response, such as elongation of contraction time and Ca²⁺ cytosolic persistence, increased calcium basal fluorescence, and transient peaks. These results contribute to the implementation of novel, integrated, high‐information, and reliable experimental systems for cardiac models and drug evaluation.     

Enhanced ryanodine receptor-mediated calcium leak determines reduced sarcoplasmic reticulum calcium content in chronic canine heart failure

In this study, we investigated the role of elevated sarcoplasmic reticulum (SR) Ca²⁺ leak through ryanodine receptors (RyR2s) in heart failure (HF)-related abnormalities of intracellular Ca²⁺ handling, using a canine model of chronic HF. The cytosolic Ca²⁺ transients were reduced in amplitude and slowed in duration in HF myocytes compared with control, changes paralleled by a dramatic reduction in the total SR Ca²⁺ content. Direct measurements of [Ca²⁺]_SR in both intact and permeabilized cardiac myocytes demonstrated that SR luminal [Ca²⁺] is markedly lowered in HF, suggesting that alterations in Ca²⁺ transport rather than fractional SR volume reduction accounts for the diminished Ca²⁺ release capacity of SR in HF. SR Ca²⁺ ATPase (SERCA2)-mediated SR Ca²⁺ uptake rate was not significantly altered, and Na⁺/Ca²⁺ exchange activity was accelerated in HF myocytes. At the same time, SR Ca²⁺ leak, measured directly as a loss of [Ca²⁺]_SR after inhibition of SERCA2 by thapsigargin, was markedly enhanced in HF myocytes. Moreover, the reduced [Ca²⁺]_SR in HF myocytes could be nearly completely restored by the RyR2 channel blocker ruthenium red. The effects of HF on cytosolic and SR luminal Ca²⁺ signals could be reasonably well mimicked by the RyR2 channel agonist caffeine. Taken together, these results suggest that RyR2-mediated SR Ca²⁺ leak is a major factor in the abnormal intracellular Ca²⁺ handling that critically contributes to the reduced SR Ca²⁺ content of failing cardiomyocytes.     

Analysis of EF-hand-containing proteins in <Emphasis Type="Italic">Arabidopsis</Emphasis>

Background  

In plants, calcium (Ca²⁺) has emerged as an important messenger mediating the action of many hormonal and environmental signals, including biotic and abiotic stresses. Many different signals raise cytosolic calcium concentration ([Ca²⁺]_cyt), which in turn is thought to regulate cellular and developmental processes via Ca²⁺-binding proteins. Three out of the four classes of Ca²⁺-binding proteins in plants contain Ca²⁺-binding EF-hand motif(s). This motif is a conserved helix-loop-helix structure that can bind a single Ca²⁺ ion. To identify all EF-hand-containing proteins in Arabidopsis, we analyzed its completed genome sequence for genes encoding EF-hand-containing proteins.     

Differences of Ca2+ handling properties in identified central olfactory neurons of the antennal lobe

Information processing in neurons depends on highly localized Ca²⁺ signals. The spatial and temporal dynamics of these signals are determined by a variety of cellular parameters including the calcium influx, calcium buffering and calcium extrusion. Our long-term goal is to better understand how intracellular Ca²⁺ dynamics are controlled and contribute to information processing in defined interneurons of the insect olfactory system. The latter has served as an excellent model to study general mechanisms of olfaction. Using patch-clamp recordings and fast optical imaging in combination with the ‘added buffer approach’, we analyzed the Ca²⁺ handling properties of different identified neuron types in Periplaneta americana's olfactory system. Our focus was on two types of local interneurons (LNs) with significant differences in intrinsic electrophysiological properties: (1) spiking LNs that generate ‘normal’ Na⁺ driven action potentials and (2) non-spiking LNs that do not express voltage-activated Na⁺ channels. We found that the distinct electrophysiological properties from different types of central olfactory interneurons are strongly correlated with their cell specific calcium handling properties: non-spiking LNs, in which Ca²⁺ is the only cation that enters the cell to contribute to membrane depolarization, had the highest endogenous Ca²⁺ binding ratio and Ca²⁺ extrusion rate.     

Calcium Movement in Cardiac Mitochondria

Existing theory suggests that mitochondria act as significant, dynamic buffers of cytosolic calcium ([Ca²⁺]_i) in heart. These buffers can remove up to one-third of the Ca²⁺ that enters the cytosol during the [Ca²⁺]_i transients that underlie contractions. However, few quantitative experiments have been presented to test this hypothesis. Here, we investigate the influence of Ca²⁺ movement across the inner mitochondrial membrane during both subcellular and global cellular cytosolic Ca²⁺ signals (i.e., Ca²⁺ sparks and [Ca²⁺]_i transients, respectively) in isolated rat cardiomyocytes. By rapidly turning off the mitochondria using depolarization of the inner mitochondrial membrane potential (ΔΨ_m), the role of the mitochondria in buffering cytosolic Ca²⁺ signals was investigated. We show here that rapid loss of ΔΨ_m leads to no significant changes in cytosolic Ca²⁺ signals. Second, we make direct measurements of mitochondrial [Ca²⁺] ([Ca²⁺]_m) using a mitochondrially targeted Ca²⁺ probe (MityCam) and these data suggest that [Ca²⁺]_m is near the [Ca²⁺]_i level (∼100 nM) under quiescent conditions. These two findings indicate that although the mitochondrial matrix is fully buffer-capable under quiescent conditions, it does not function as a significant dynamic buffer during physiological Ca²⁺ signaling. Finally, quantitative analysis using a computational model of mitochondrial Ca²⁺ cycling suggests that mitochondrial Ca²⁺ uptake would need to be at least ∼100-fold greater than the current estimates of Ca²⁺ influx for mitochondria to influence measurably cytosolic [Ca²⁺] signals under physiological conditions. Combined, these experiments and computational investigations show that mitochondrial Ca²⁺ uptake does not significantly alter cytosolic Ca²⁺ signals under normal conditions and indicates that mitochondria do not act as important dynamic buffers of [Ca²⁺]_i under physiological conditions in heart.     

Regulation of sarcoplasmic reticulum Ca2+ release by cytosolic glutathione in rabbit ventricular myocytes

Of the major cellular antioxidant defenses, glutathione (GSH) is particularly important in maintaining the cytosolic redox potential. Whereas the healthy myocardium is maintained at a highly reduced redox state, it has been proposed that oxidation of GSH can affect the dynamics of Ca²⁺-induced Ca²⁺ release. In this study, we used multiple approaches to define the effects of oxidized glutathione (GSSG) on ryanodine receptor (RyR)-mediated Ca²⁺ release in rabbit ventricular myocytes. To investigate the role of GSSG on sarcoplasmic reticulum (SR) Ca²⁺ release induced by the action potential, we used the thiol-specific oxidant diamide to increase intracellular GSSG in intact myocytes. To more directly assess the effect of GSSG on RyR activity, we introduced GSSG within the cytosol of permeabilized myocytes. RyR-mediated Ca²⁺ release from the SR was significantly enhanced in the presence of GSSG. This resulted in decreased steady-state diastolic [Ca²⁺]_SR, increased SR Ca²⁺ fractional release, and increased spark- and non-spark-mediated SR Ca²⁺ leak. Single-channel recordings from RyR’s incorporated into lipid bilayers revealed that GSSG significantly increased RyR activity. Moreover, oxidation of RyR in the form of intersubunit crosslinking was present in intact myocytes treated with diamide and permeabilized myocytes treated with GSSG. Blocking RyR crosslinking with the alkylating agent N-ethylmaleimide prevented depletion of SR Ca²⁺ load induced by diamide. These findings suggest that elevated cytosolic GSSG enhances SR Ca²⁺ leak due to redox-dependent intersubunit RyR crosslinking. This effect can contribute to abnormal SR Ca²⁺ handling during periods of oxidative stress.     

Regulation of neuronal energy metabolism by calcium: Role of MCU and Aralar/malate-aspartate shuttle

Calcium is a major regulator of cellular metabolism. Calcium controls mitochondrial respiration, and calcium signaling is used to meet cellular energetic demands through energy production in the organelle. Although it has been widely assumed that Ca²⁺-actions require its uptake by mitochondrial calcium uniporter (MCU), alternative pathways modulated by cytosolic Ca²⁺ have been recently proposed. Recent findings have indicated a role for cytosolic Ca²⁺ signals acting on mitochondrial NADH shuttles in the control of cellular metabolism in neurons using glucose as fuel. It has been demonstrated that AGC1/Aralar, the component of the malate/aspartate shuttle (MAS) regulated by cytosolic Ca²⁺, participates in the maintenance of basal respiration exerted through Ca²⁺-fluxes between ER and mitochondria, whereas mitochondrial Ca²⁺-uptake by MCU does not contribute. Aralar/MAS pathway, activated by small cytosolic Ca²⁺ signals, provides in fact substrates, redox equivalents and pyruvate, fueling respiration. Upon activation and increases in workload, neurons upregulate OxPhos, cytosolic pyruvate production and glycolysis, together with glucose uptake, in a Ca²⁺-dependent way, and part of this upregulation is via Ca²⁺ signaling. Both MCU and Aralar/MAS contribute to OxPhos upregulation, Aralar/MAS playing a major role, especially at small and submaximal workloads. Ca²⁺ activation of Aralar/MAS, by increasing cytosolic NAD⁺/NADH provides Ca²⁺-dependent increases in glycolysis and cytosolic pyruvate production priming respiration as a feed-forward mechanism in response to workload. Thus, except for glucose uptake, these processes are dependent on Aralar/MAS, whereas MCU is the relevant target for Ca²⁺ signaling when MAS is bypassed, by using pyruvate or β-hydroxybutyrate as substrates.     

Calcium and ATP handling in human NADH:Ubiquinone oxidoreductase deficiency

Proper cell functioning requires precise coordination between mitochondrial ATP production and local energy demand. Ionic calcium (Ca²⁺) plays a central role in this coupling because it activates mitochondrial oxidative phosphorylation (OXPHOS) during hormonal and electrical cell stimulation. To determine how mitochondrial dysfunction affects cytosolic and mitochondrial Ca²⁺/ATP handling, we performed life-cell quantification of these parameters in fibroblast cell lines derived from healthy subjects and patients with isolated deficiency of the first OXPHOS complex (CI). In resting patient cells, CI deficiency was associated with a normal mitochondrial ([ATP]_m) and cytosolic ([ATP]_c) ATP concentration, a normal cytosolic Ca²⁺ concentration ([Ca²⁺]_c), but a reduced Ca²⁺ content of the endoplasmic reticulum (ER). Furthermore, cellular NAD(P)H levels were increased, mitochondrial membrane potential was slightly depolarized, reactive oxygen species (ROS) levels were elevated and mitochondrial shape was altered. Upon stimulation with bradykinin (Bk), the peak increases in [Ca²⁺]_c, mitochondrial Ca²⁺ concentration ([Ca²⁺]_m), [ATP]_c and [ATP]_m were reduced in patient cells. In agreement with these results, ATP-dependent Ca²⁺ removal from the cytosol was slower. Here, we review the interconnection between cytosolic, endoplasmic reticular and mitochondrial Ca²⁺ and ATP handling, and summarize our findings in patient fibroblasts in an integrative model.     

Calcium Movement in Cardiac Mitochondria

Existing theory suggests that mitochondria act as significant, dynamic buffers of cytosolic calcium ([Ca²⁺]_i) in heart. These buffers can remove up to one-third of the Ca²⁺ that enters the cytosol during the [Ca²⁺]_i transients that underlie contractions. However, few quantitative experiments have been presented to test this hypothesis. Here, we investigate the influence of Ca²⁺ movement across the inner mitochondrial membrane during both subcellular and global cellular cytosolic Ca²⁺ signals (i.e., Ca²⁺ sparks and [Ca²⁺]_i transients, respectively) in isolated rat cardiomyocytes. By rapidly turning off the mitochondria using depolarization of the inner mitochondrial membrane potential (ΔΨ_m), the role of the mitochondria in buffering cytosolic Ca²⁺ signals was investigated. We show here that rapid loss of ΔΨ_m leads to no significant changes in cytosolic Ca²⁺ signals. Second, we make direct measurements of mitochondrial [Ca²⁺] ([Ca²⁺]_m) using a mitochondrially targeted Ca²⁺ probe (MityCam) and these data suggest that [Ca²⁺]_m is near the [Ca²⁺]_i level (∼100 nM) under quiescent conditions. These two findings indicate that although the mitochondrial matrix is fully buffer-capable under quiescent conditions, it does not function as a significant dynamic buffer during physiological Ca²⁺ signaling. Finally, quantitative analysis using a computational model of mitochondrial Ca²⁺ cycling suggests that mitochondrial Ca²⁺ uptake would need to be at least ∼100-fold greater than the current estimates of Ca²⁺ influx for mitochondria to influence measurably cytosolic [Ca²⁺] signals under physiological conditions. Combined, these experiments and computational investigations show that mitochondrial Ca²⁺ uptake does not significantly alter cytosolic Ca²⁺ signals under normal conditions and indicates that mitochondria do not act as important dynamic buffers of [Ca²⁺]_i under physiological conditions in heart.     

Mitochondria in cardiomyocyte Ca signaling

Ca²⁺ signaling is of vital importance to cardiac cell function and plays an important role in heart failure. It is based on sarcolemmal, sarcoplasmic reticulum and mitochondrial Ca²⁺ cycling. While the first two are well characterized, the latter remains unclear, controversial and technically challenging.In mammalian cardiac myocytes, Ca²⁺ influx through L-type calcium channels in the sarcolemmal membrane triggers Ca²⁺ release from the nearby junctional sarcoplasmic reticulum to produce Ca²⁺ sparks. When this triggering is synchronized by the cardiac action potential, a global [Ca²⁺]_i transient arises from coordinated Ca²⁺ release events. The ends of intermyofibrillar mitochondria are located within 20 nm of the junctional sarcoplasmic reticulum and thereby experience a high local [Ca²⁺] during the Ca²⁺ release process. Both local and global Ca²⁺ signals may thus influence calcium signaling in mitochondria and, reciprocally, mitochondria may contribute to the local control of calcium signaling. In addition to the intermyofibrillar mitochondria, morphologically distinct mitochondria are also located in the perinuclear and subsarcolemmal regions of the cardiomyocyte and thus experience a different local [Ca²⁺].Here we review the literature in regard to several issues of broad interest: (1) the ultrastructural basis for mitochondrion – sarcoplasmic reticulum cross-signaling; (2) mechanisms of sarcoplasmic reticulum signaling; (3) mitochondrial calcium signaling; and (4) the possible interplay of calcium signaling between the sarcoplasmic reticulum and adjacent mitochondria.Finally, this review discusses experimental findings and mathematical models of cardiac calcium signaling between the sarcoplasmic reticulum and mitochondria, identifies weaknesses in these models, and suggests strategies and approaches for future investigations.     

Calcium dynamics in the ventricular myocytes of SERCA2 knockout mice: A modeling study

We describe a simulation study of Ca²⁺ dynamics in mice with cardiomyocyte-specific conditional excision of the sarco(endo)plasmic reticulum calcium ATPase (SERCA) gene, using an experimental data-driven biophysically-based modeling framework. Previously, we reported a moderately impaired heart function measured in mice at 4 weeks after SERCA2 gene deletion (knockout (KO)), along with a >95% reduction in the level of SERCA2 protein. We also reported enhanced Ca²⁺ flux through the L-type Ca²⁺ channels and the Na⁺/Ca²⁺ exchanger in ventricular myocytes isolated from these mice, compared to the control Serca2^flox/flox mice (flox-flox (FF)). In the current study, a mathematical model-based analysis was applied to enable further quantitative investigation into changes in the Ca²⁺ handling mechanisms in these KO cardiomyocytes. Model parameterization based on a wide range of experimental measurements showed a 67% reduction in SERCA activity and an over threefold increase in the activity of the Na⁺/Ca²⁺ exchanger. The FF and KO models were then validated against experimentally measured [Ca²⁺]_i transients and experimentally estimated sarco(endo)plasmic reticulum (SR) function. Simulation results were in quantitative agreement with experimental measurements, confirming that sustained [Ca²⁺]_i transients could be maintained in the KO cardiomyocytes despite severely impaired SERCA function. In silico analysis shows that diastolic [Ca²⁺]_i rises sharply with progressive reductions in SERCA activity at physiologically relevant pacing frequencies. Furthermore, an analysis of the roles of the compensatory mechanisms revealed that the major combined effect of the compensatory mechanisms is to lower diastolic [Ca²⁺]_i. Finally, by using a comprehensive sensitivity analysis of the role of all cellular calcium handling mechanisms, we show that the combination of upregulation of the Na⁺/Ca²⁺ exchanger and increased L-type Ca²⁺ current is the most effective means to maintain diastolic and systolic calcium levels after loss of SERCA function.     

Mitochondrial Calcium Signaling Driven by the IP3 Receptor

Many agonists bring about their effects on cellular functions through a rise incytosolic [Ca²⁺]([Ca²⁺]_c) mediated by the second messenger inositol 1,4,5-trisphosphate (IP₃). Imaging studiesof single cells have demonstrated that [Ca²⁺]_c signals display cell specific spatiotemporalorganization that is established by coordinated activation of IP₃ receptor Ca²⁺ channels.Evidence emerges that cytosolic calcium signals elicited by activation of the IP₃ receptors areefficiently transmitted to the mitochondria. An important function of mitochondrial calciumsignals is to activate the Ca²⁺-sensitive mitochondrial dehydrogenases, and thereby to meetdemands for increased energy in stimulated cells. Activation of the permeability transitionpore (PTP) by mitochondrial calcium signals may also be involved in the control of cell death.Furthermore, mitochondrial Ca²⁺ transport appears to modulate the spatiotemporal organizationof [Ca²⁺]_c responses evoked by IP₃ and so mitochondria may be important in cytosolic calciumsignaling as well. This paper summarizes recent research to elucidate the mechanisms andsignificance of IP₃-dependent mitochondrial calcium signaling.     

Role of phospholipases in cytosolic calcium overload and cardiomyocytes death in the presence of activated fatty acid derivatives

Ten to fifty micromoles of palmitoyl-L-carnitine (PC) or myristoyl-D,L-carnitine (MC) evoke a high-amplitude elevation of cytosolic calcium level ([Ca²⁺]_i), hypercontraction and cell death in the primary culture of rat ventricular myocytes. The lag period of this effect varies within 2–8 min and depends on the mitochondrial capacity to accumulate Ca²⁺. Maximal level of Ca²⁺, attainable at the end of the lag period, depends on calcium concentration in the external medium and is mediated by plasma membrane nonspecific permeability. Preincubation of cardiomyocytes with the inhibitors of phospholipase C, cytosolic phospholipase A₂ and/or Ca²⁺/calmodulin-dependent protein kinase II prevents cell death, increases lag period duration and reduces maximal [Ca²⁺]_i. Both PC and MC, even at low concentrations (1–5 μM), dramatically increase the frequency of Ca²⁺-sparks and Ca²⁺-waves in cardiomyocytes and promote the formation of sustained microdomains with elevated calcium concentration. We discuss possible mechanisms of Ca²⁺-microdomain formation, where the “vicious circle” of Ca²⁺-dependent phospholipases activation may arise. The “vicious circle” with combined autocatalytic action of Ca²⁺-dependent phospholipases may be implicated in hydrolysis of membrane phosphatidylcholine and subsequent induction of nonselective permeability for Na⁺ and Ca²⁺ (lipid pore).     

Dietary Omega-3 Fatty Acids Promote Arrhythmogenic Remodeling of Cellular Ca2+ Handling in a Postinfarction Model of Sudden Cardiac Death

It has been proposed that dietary omega-3 polyunsaturated fatty acids (n-3 PUFAs) can reduce the risk of ventricular arrhythmias in post-MI patients. Abnormal Ca²⁺ handling has been implicated in the genesis of post-MI ventricular arrhythmias. Therefore, we tested the hypothesis that dietary n-3 PUFAs alter the vulnerability of ventricular myocytes to cellular arrhythmia by stabilizing intracellular Ca²⁺ cycling. To test this hypothesis, we used a canine model of post-MI ventricular fibrillation (VF) and assigned the animals to either placebo (1 g/day corn oil) or n-3 PUFAs (1-4 g/day) groups. Using Ca²⁺ imaging techniques, we examined the intracellular Ca²⁺ handling in myocytes isolated from post-MI hearts resistant (VF-) and susceptible (VF+) to VF. Frequency of occurrence of diastolic Ca²⁺ waves (DCWs) in VF+ myocytes from placebo group was significantly higher than in placebo-treated VF- myocytes. n-3 PUFA treatment did not decrease frequency of DCWs in VF+ myocytes. In contrast, VF- myocytes from the n-3 PUFA group had a significantly higher frequency of DCWs than myocytes from the placebo group. In addition, n-3 PUFA treatment increased beat-to-beat alterations in the amplitude of Ca²⁺ transients (Ca²⁺ alternans) in VF- myocytes. These n-3 PUFAs effects in VF- myocytes were associated with an increased Ca²⁺ spark frequency and reduced sarcoplasmic reticulum Ca²⁺ content, indicative of increased activity of ryanodine receptors. Thus, dietary n-3 PUFAs do not alleviate intracellular Ca²⁺ cycling remodeling in myocytes isolated from post-MI VF+ hearts. Furthermore, dietary n-3 PUFAs increase vulnerability of ventricular myocytes to cellular arrhythmia in post-MI VF- hearts by destabilizing intracellular Ca²⁺ handling.     

Bcl-2 family in inter-organelle modulation of calcium signaling; roles in bioenergetics and cell survival

Bcl-2 family proteins, known for their apoptosis functioning at the mitochondria, have been shown to localize to other cellular compartments to mediate calcium (Ca²⁺) signals. Since the proper supply of Ca²⁺ in cells serves as an important mechanism for cellular survival and bioenergetics, we propose an integrating role for Bcl-2 family proteins in modulating Ca²⁺ signaling. The endoplasmic reticulum (ER) is the main Ca²⁺ storage for the cell and Bcl-2 family proteins competitively regulate its Ca²⁺ concentration. Bcl-2 family proteins also regulate the flux of Ca²⁺ from the ER by physically interacting with inositol 1,4,5-trisphosphate receptors (IP₃Rs) to mediate their opening. Type 1 IP₃Rs reside at the bulk ER to coordinate cytosolic Ca²⁺ signals, while type 3 IP₃Rs reside at mitochondria-associated ER membrane (MAM) to facilitate mitochondrial Ca²⁺ uptake. In healthy cells, mitochondrial Ca²⁺ drives pyruvate into the citric acid (TCA) cycle to facilitate ATP production, while a continuous accumulation of Ca²⁺ can trigger the release of cytochrome c, thus initiating apoptosis. Since multiple organelles and Bcl-2 family proteins are involved in Ca²⁺ signaling, we aim to clarify the role that Bcl-2 family proteins play in facilitating Ca²⁺ signaling and how mitochondrial Ca²⁺ is relevant in both bioenergetics and apoptosis. We also explore how these insights could be useful in controlling bioenergetics in apoptosis-resistant cell lines.     

Mechanisms by which cytoplasmic calcium wave propagation and alternans are generated in cardiac atrial myocytes lacking T-tubules-insights from a simulation study

This study investigated the mechanisms underlying the propagation of cytoplasmic calcium waves and the genesis of systolic Ca²⁺ alternans in cardiac myocytes lacking transverse tubules (t-tubules). These correspond to atrial cells of either small mammals or large mammals that have lost their t-tubules due to disease-induced structural remodeling (e.g., atrial fibrillation). A mathematical model was developed for a cluster of ryanodine receptors distributed on the cross section of a cell that was divided into 13 elements with a spatial resolution of 2 μm. Due to the absence of t-tubules, L-type Ca²⁺ channels were only located in the peripheral elements close to the cell-membrane surface and produced Ca²⁺ signals that propagated toward central elements by triggering successive Ca²⁺-induced Ca²⁺ release (CICR) via Ca²⁺ diffusion between adjacent elements. Under control conditions, the Ca²⁺ signals did not fully propagate to the central region of the cell. However, with modulation of several factors responsible for Ca²⁺ handling, such as the L-type Ca²⁺ channels (Ca²⁺ influx), SERCA pumps (sarcoplasmic reticulum (SR) Ca²⁺ uptake), and ryanodine receptors (SR Ca²⁺ release), Ca²⁺ wave propagation to the center of the cell could occur. These simulation results are consistent with previous experimental data from atrial cells of small mammals. The model further reveals that spatially functional heterogeneity in Ca²⁺ diffusion within the cell produced a steep relationship between the SR Ca²⁺ content and the cytoplasmic Ca²⁺ concentration. This played an important role in the genesis of Ca²⁺ alternans that were more obvious in central than in peripheral elements. Possible association between the occurrence of Ca²⁺ alternans and the model parameters of Ca²⁺ handling was comprehensively explored in a wide range of one- and two-parameter spaces. In addition, the model revealed a spontaneous second Ca²⁺ release in response to a single voltage stimulus pulse with SR Ca²⁺ overloading and augmented Ca²⁺ influx. This study provides what to our knowledge are new insights into the genesis of Ca²⁺ alternans and spontaneous second Ca²⁺ release in cardiac myocytes that lack t-tubules.     

PGC-1α accelerates cytosolic Ca clearance without disturbing Ca homeostasis in cardiac myocytes

Energy metabolism and Ca²⁺ handling serve critical roles in cardiac physiology and pathophysiology. Peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1α) is a multi-functional coactivator that is involved in the regulation of cardiac mitochondrial functional capacity and cellular energy metabolism. However, the regulation of PGC-1α in cardiac Ca²⁺ signaling has not been fully elucidated. To address this issue, we combined confocal line-scan imaging with off-line imaging processing to characterize calcium signaling in cultured adult rat ventricular myocytes expressing PGC-1α via adenoviral transduction. Our data shows that overexpressing PGC-1α improved myocyte contractility without increasing the amplitude of Ca²⁺ transients, suggesting that myofilament sensitivity to Ca²⁺ increased. Interestingly, the decay kinetics of global Ca²⁺ transients and Ca²⁺ waves accelerated in PGC-1α-expressing cells, but the decay rate of caffeine-elicited Ca²⁺ transients showed no significant change. This suggests that sarcoplasmic reticulum (SR) Ca²⁺-ATPase (SERCA2a), but not Na⁺/Ca²⁺ exchange (NCX) contribute to PGC-1α-induced cytosolic Ca²⁺ clearance. Furthermore, PGC-1α induced the expression of SERCA2a in cultured cardiac myocytes. Importantly, overexpressing PGC-1α did not disturb cardiac Ca²⁺ homeostasis, because SR Ca²⁺ load and the propensity for Ca²⁺ waves remained unchanged. These data suggest that PGC-1α can ameliorate cardiac Ca²⁺ cycling and improve cardiac work output in response to physiological stress. Unraveling the PGC-1α-calcium handing pathway sheds new light on the role of PGC-1α in the therapy of cardiac diseases.     

Cytosolic calcium and pH signaling in plants under salinity stress

Calcium is one of the essential nutrients for growth and development of plants. It is an important component of various structures in cell wall and membranes. Besides some fundamental roles under normal condition, calcium functions as a major secondary-messenger molecule in plants under different developmental cues and various stress conditions including salinity stress. Also changes in cytosolic pH, pH_cyt, either individually, or in coordination with changes in cytosolic Ca²⁺ concentration, [Ca²⁺]_cyt, evoke a wide range of cellular functions in plants including signal transduction in plant-defense responses against stresses. It is believed that salinity stress, like other stresses, is perceived at cell membrane, either extra cellular or intracellular, which then triggers an intracellular-signaling cascade including the generation of secondary messenger molecules like Ca²⁺ and protons. The variety and complexity of Ca²⁺ and pH signaling result from the nature of the stresses as well as the tolerance level of the plant species against that specific stress. The nature of changes in [Ca²⁺]_cyt concentration, in terms of amplitude, frequency and duration, is likely very important for decoding the specific downstream responses for salinity stress tolerance in planta. It has been observed that the signatures of [Ca²⁺]_cyt and pH differ in various studies reported so far depending on the techniques used to measure them, and also depending on the plant organs where they are measured, such as root, shoot tissues or cells. This review describes the recent advances about the changes in [Ca²⁺]_cyt and pH_cyt at both cellular and whole-plant levels under salinity stress condition, and in various salinity-tolerant and -sensitive plant species.Key words: cytosolic calcium, ionic toxicity, osmotic stress, pH, salinity stress, salt tolerance, signaling