Thapsigargin decreases the Na+- Ca2 + exchanger mediated Ca2 + entry in pig coronary artery smooth muscle

Na⁺- Ca^2 + exchanger (NCX) has been proposed to play a role in refilling the sarco/endoplasmic reticulum (SER) Ca^2 + pool along with the SER Ca^2 + pump (SERCA). Here, SERCA inhibitor thapsigargin was used to determine the effects of SER Ca^2 + depletion on NCX–SERCA interactions in smooth muscle cells cultured from pig coronary artery. The cells were Na⁺-loaded and then placed in either a Na⁺-containing or in a Na⁺-substituted solution. Subsequently, the difference in Ca^2 + entry between the two groups was examined and defined as the NCX mediated Ca^2 + entry. The NCX mediated Ca^2 + entry in the smooth muscle cells was monitored using two methods: Ca^2 +sensitive fluorescence dye Fluo-4 and radioactive Ca^2 +. Ca^2 +-entry was greater in the Na⁺-substituted cells than in the Na⁺-containing cells when measured by either method. This difference was established to be NCX-mediated as it was sensitive to the NCX inhibitors. Thapsigargin diminished the NCX mediated Ca^2 + entry as determined by either method. Immunofluorescence confocal microscopy was used to determine the co-localization of NCX1 and subsarcolemmal SERCA2 in the cells incubated in the Na⁺-substituted solution with or without thapsigargin. SER Ca^2 + depletion with thapsigargin increased the co-localization between NCX1 and the subsarcolemmal SERCA2. Thus, inhibition of SERCA2 leads to blockade of constant Ca^2 + entry through NCX1 and also increases proximity between NCX1 and SERCA2. This blockade of Ca^2 + entry may protect the cells against Ca^2 +-overload during ischemia–reperfusion when SERCA2 is known to be damaged.     

Spatially confined diffusion of calcium in dendrites of hippocampal neurons revealed by flash photolysis of caged calcium

The extent of diffusion of a locally evoked calcium surge in dendrites of cultured hippocampal neurons was studied by flash photolysis of caged EGTA. Cells were transfected with pDsRed for visualization, preincubated with caged NP-EGTA (AM) and Fluo-4 (AM) at room temperature and imaged in a PASCAL confocal microscope. Pulses of UV laser light within an active sphere of about 1 μm² produced a rise of Fluo-4 fluorescence transients in dendrites which peaked at 1 ms and decayed exponentially with a fast (8–10 ms) time constant. A slower decay component was uncovered following incubation with thapsigargin. Lateral diffusion of [Ca²⁺]i did not vary significantly among different size dendrites being symmetric and reaching about 3–3.5 μm at a diffusion rate of 0.8 μm/ms on both sides of the photolysis center. Fluo-4 was also replaced by the membrane-bound Fluo-NOMO (AM) or by the ‘heavy’ Calcium Green dextran (CGd) loaded through a patch pipette. Similar rates of diffusion were found in these cases, indicating that the diffusion is not of the dye complexed to calcium but of genuine free calcium ions. Interestingly, presence of a dendritic spine at the focus of photolysis significantly reduced [Ca²⁺]i spread while the focal transient remained unaffected. Finally, [Ca²⁺]i diffused about twice as far from the photolysis sphere in glass tubes of a similar diameter to that of a dendrite, indicating that intrinsic calcium uptake mechanisms in the dendrite determine the diffusion of calcium away from its original site of rise.     

Homer1 knockdown protects dopamine neurons through regulating calcium homeostasis in an in vitro model of Parkinson's disease

Homer1 protein is an important scaffold protein at postsynaptic density and has been demonstrated to play a central role in calcium signaling in the central nervous system. The aim of this study was to investigate the effects of Homer1 knockdown on MPP⁺ induced neuronal injury in cultured dopamine (DA) neurons. We found that down-regulating Homer1 expression with specific small interfering RNA (siRNA) significantly suppressed LDH release, reduced Propidium iodide (PI) or Hoechst staining, increased the number of tyrosine hydroxylase (TH) positive cells and DA uptake, and attenuated apoptotic and necrotic cell death after MPP⁺ injury. Homer1 knockdown decreased intracellular reactive oxygen species (ROS) generation through inhibition of intracellular calcium overload, but did not affect the endogenous antioxidant enzyme activities. Calcium imaging was used to examine the changes of intracellular Ca^2 + concentration ([Ca^2 +]_cyt) and Ca^2 + in endoplasmic reticulum (ER) ([Ca^2 +]_ER), and the results showed that Homer1 siRNA transfection attenuated ER Ca^2 + release up to 120 min after MPP⁺ injury. Furthermore, decrease of [Ca^2 +]_cyt induced by Homer1 knockdown in MPP⁺ treated neurons was further enhanced by NMDA receptor antagonists MK-801 and AP-5, but not canonical transient receptor potential (TRPC) channel antagonist SKF-96365. l-type calcium antagonist isradipine but not nimodipine further inhibited intracellular calcium overload after MPP⁺ insult in Homer1 down-regulated neurons. These results suggest that Homer1 knockdown has protective effects against neuronal injury in in vitro PD model by reducing calcium overload mediated ROS generation, and this protection may be dependent at least in part on the regulatory effects on the function of calcium channels in both plasma membrane and ER.     

Calcium signalling in Drosophila photoreceptors measured with GCaMP6f

Drosophila phototransduction is mediated by phospholipase C leading to activation of cation channels (TRP and TRPL) in the 30000 microvilli forming the light-absorbing rhabdomere. The channels mediate massive Ca²⁺ influx in response to light, but whether Ca²⁺ is released from internal stores remains controversial. We generated flies expressing GCaMP6f in their photoreceptors and measured Ca²⁺ signals from dissociated cells, as well as in vivo by imaging rhabdomeres in intact flies. In response to brief flashes, GCaMP6f signals had latencies of 10–25 ms, reached 50% F_max with ∼1200 effectively absorbed photons and saturated (ΔF/F₀ ∼ 10–20) with 10000–30000 photons. In Ca²⁺ free bath, smaller (ΔF/F₀ ∼4), long latency (∼200 ms) light-induced Ca²⁺ rises were still detectable. These were unaffected in InsP₃ receptor mutants, but virtually eliminated when Na⁺ was also omitted from the bath, or in trpl;trp mutants lacking light-sensitive channels. Ca²⁺ free rises were also eliminated in Na⁺/Ca²⁺ exchanger mutants, but greatly accelerated in flies over-expressing the exchanger. These results show that Ca²⁺ free rises are strictly dependent on Na⁺ influx and activity of the exchanger, suggesting they reflect re-equilibration of Na⁺/Ca²⁺ exchange across plasma or intracellular membranes following massive Na⁺ influx. Any tiny Ca²⁺ free rise remaining without exchanger activity was equivalent to <10 nM (ΔF/F₀ ∼0.1), and unlikely to play any role in phototransduction.     

Temperature-induced labelling of Fluo-3 AM selectively yields brighter nucleus in adherent cells

Fluo-3 is widely used to study cell calcium. Two traditional approaches: (1) direct injection and (2) Fluo-3 acetoxymethyl ester (AM) loading, often bring conflicting results in cytoplasmic calcium ([Ca²⁺]_c) and nuclear calcium ([Ca²⁺]_n) imaging. AM loading usually yields a darker nucleus than in cytoplasm, while direct injection always induces a brighter nucleus which is more responsive to [Ca²⁺]_n detection. In this work, we detailedly investigated the effects of loading and de-esterification temperatures on the fluorescence intensity of Fluo-3 in response to [Ca²⁺]_n and [Ca²⁺]_c in adherent cells, including osteoblast, HeLa and BV2 cells. Interestingly, it showed that fluorescence intensity of nucleus in osteoblast cells was about two times larger than that of cytoplasm when cells were loaded with Fluo-3 AM at 4 °C and allowed a subsequent step for de-esterification at 20 °C. Brighter nuclei were also acquired in HeLa and BV2 cells using the same experimental condition. Furthermore, loading time and adhesion quality of cells had effect on fluorescence intensity. Taken together, cold loading and room temperature de-esterification treatment of Fluo-3 AM selectively yielded brighter nucleus in adherent cells.     

Mechanism of dopamine D2 receptor-induced Ca2 + release in PC-12 cells

Intracellular Ca^2 + levels are tightly regulated in the neuronal system. The loss of Ca^2 + homeostasis is associated with many neurological diseases and neuropsychiatric disorders such as Parkinson's, Alzheimer's, and schizophrenia. We investigated the mechanisms involved in intracellular Ca^2 + signaling in PC-12 cells. The stimulation of NGF-differentiated PC-12 cells with 3 μM ATP caused an early Ca^2 + release followed by a delayed Ca^2 + release. The delayed Ca^2 + release was dependent on prior ATP priming and on dopamine secretion by PC-12 cells. Delayed Ca^2 + release was abolished in the presence of spiperone, suggesting that it is due to the activation of D2 dopamine receptors (D2R) by dopamine secreted by PC-12 cells. This was shown to be independent of PKA activation but dependent on PLC activity. An endocytosis step was required for inducing the delayed Ca^2 + release. Given the importance of calcyon in clathrin-mediated endocytosis, we verified the role of this protein in the delayed Ca^2 + release phenomenon. siRNA targeting of calcyon blocked the delayed Ca^2 + release, decreased ATP-evoked IP₃R-mediated Ca^2 + release, and impaired subsequent Ca^2 + oscillations. Our results suggested that calcyon is involved in an unknown mechanism that causes a delayed IP₃R-mediated Ca^2 + release in PC-12 cells. In schizophrenia, Ca^2 + dysregulation may depend on the upregulation of calcyon, which maintains elevated Ca^2 + levels as well as dopamine signaling.     

The use of transgenic mouse models to reveal the functions of Ca2 + buffer proteins in excitable cells

BackgroundCytosolic Ca^2 + buffers are members of the large family of Ca^2 +-binding proteins and are essential components of the Ca^2 + signaling toolkit implicated in the precise regulation of intracellular Ca^2 + signals. Their physiological role in excitable cells has been investigated in vivo by analyzing the phenotype of mice either lacking one of the Ca^2 + buffers or mice with ectopic expression.Scope of ReviewIn this review, results obtained with knockout mice for the three most prominent Ca^2 + buffers, parvalbumin, calbindin-D28k and calretinin are summarized.Major ConclusionsThe absence of Ca^2 + buffers in specific neuron subpopulations, and for parvalbumin additionally in fast-twitch muscles, leads to Ca^2 + buffer-specific changes in intracellular Ca^2 + signals. This affects the excitation–contraction cycle in parvalbumin-deficient muscles, and in Ca^2 + buffer-deficient neurons, properties associated with synaptic transmission (e.g. short-term modulation), excitability and network oscillations are altered. These findings have not only resulted in a better understanding of the physiological function of Ca^2 + buffers, but have revealed that the absence of Ca^2 + signaling toolkit components leads to protein-and neuron-specific adaptive/homeostatic changes that also include changes in neuron morphology (e.g. altered spine morphology, changes in mitochondria content) and network properties.General SignificanceThe complex phenotype of Ca^2 + buffer knockout mice arises from the direct effect of these proteins on Ca^2 + signaling and moreover from the homeostatic mechanisms induced in these mice. For a better mechanistic understanding of neurological diseases linked to disturbed/altered Ca^2 + signaling, a global view on Ca^2 + signaling is expected to lead to new avenues for specific therapies. This article is part of a Special Issue entitled Biochemical, biophysical and genetic approaches to intracellular calcium signaling.     

Total internal reflectance fluorescence imaging of genetically engineered ryanodine receptor-targeted Ca2+ probes in rat ventricular myocytes

The details of cardiac Ca²⁺ signaling within the dyadic junction remain unclear because of limitations in rapid spatial imaging techniques, and availability of Ca²⁺ probes localized to dyadic junctions. To critically monitor ryanodine receptors’ (RyR2) Ca²⁺ nano-domains, we combined the use of genetically engineered RyR2-targeted pericam probes, (FKBP-YCaMP, K_d = 150 nM, or FKBP-GCaMP6, K_d = 240 nM) with rapid total internal reflectance fluorescence (TIRF) microscopy (resolution, ∼80 nm). The punctate z-line patterns of FKBP,²-targeted probes overlapped those of RyR2 antibodies and sharply contrasted to the images of probes targeted to sarcoplasmic reticulum (SERCA2a/PLB), or cytosolic Fluo-4 images. FKBP-YCaMP signals were too small (∼20%) and too slow (2–3 s) to detect Ca²⁺ sparks, but the probe was effective in marking where Fluo-4 Ca²⁺ sparks developed. FKBP-GCaMP6, on the other hand, produced rapidly decaying Ca²⁺ signals that: a) had faster kinetics and activated synchronous with I_Ca³ but were of variable size at different z-lines and b) were accompanied by spatially confined spontaneous Ca²⁺ sparks, originating from a subset of eager sites. The frequency of spontaneously occurring sparks was lower in FKBP-GCaMP6 infected myocytes as compared to Fluo-4 dialyzed myocytes, but isoproterenol enhanced their frequency more effectively than in Fluo-4 dialyzed cells. Nevertheless, isoproterenol failed to dissociate FKBP-GCaMP6 from the z-lines. The data suggests that FKBP-GCaMP6 binds predominantly to junctional RyR2s and has sufficient on-rate efficiency as to monitor the released Ca²⁺ in individual dyadic clefts, and supports the idea that β-adrenergic agonists may modulate the stabilizing effects of native FKBP on RyR2.     

How do reactive oxygen species and calcium trigger mitochondrial membrane permeabilisation?

BackgroundMitochondrial membrane permeabilisation (MMP) is classically considered as a point of no return in several forms of cell death and is involved in numerous diseases such as cancer, neurodegenerative disorders or ischemia/reperfusion injuries. Many studies established that reactive oxygen species (ROS) and Ca^2 + were the prominent inducers of MMP. However, the mechanisms connecting ROS and Ca^2 + to the players of MMP are still a matter of debate.Scope of reviewThe aim of this review is to summarise the various studies related to the mechanisms of ROS- and Ca^2 +-induced MMP. Several lines of evidence suggest that ROS and Ca^2 + cooperate to induce MMP but the molecular details of the ROS–Ca^2 +-MMP network remain controversial. We then discuss recent data depicting this topic.Major conclusionsCytotoxic stimuli may be transduced within the cell by ROS and Ca^2 + increases. In most models, Ca^2 + and ROS can cooperate to induce MMP. Moreover, several data suggest that MMP increases mitochondrial Ca^2 + and ROS which therefore amplify the cytotoxic signal. Intriguingly, many reports have identified players of MMP as direct ROS targets. On the contrary, direct targets of Ca^2 + remain elusive. At the same time, the mechanisms by which mitochondrial Ca^2 + overload induces ROS generation are well documented. Upon these observations, we hypothesise that Ca^2 + cannot directly induce MMP and requires ROS production as a mandatory step.General significanceGiven the importance of Ca^2 +- and ROS-induced MMP in diseases, we expect that a better understanding of this process will lead to the development of novel therapies.     

Glucagon-like peptide-1 regulates calcium homeostasis and electrophysiological activities of HL-1 cardiomyocytes

Glucagon like-peptide-1 (GLP-1) is an incretin hormone with antidiabetic effects through stimulating insulin secretion, β cell neogenesis, satiety sensation, and inhibiting glucagon secretion. Administration of GLP-1 provides cardioprotective effects through attenuating cardiac inflammation and insulin resistance. GLP-1 also modulates the heart rate and systolic pressure, which suggests that GLP-1 may have cardiac electrical effects. Therefore, the purposes of this study were to evaluate whether GLP-1 has direct cardiac effects and identify the underlying mechanisms. Patch clamp, confocal microscopy with Fluo-3 fluorescence, and Western blot analyses were used to evaluate the electrophysiological characteristics, calcium homeostasis, and calcium regulatory proteins in HL-1 atrial myocytes with and without GLP-1 (1 and 10 nM) incubation for 24 h. GLP-1 (1 and 10 nM) and control cells had similar action potential durations. However, GLP-1 at 10 nM significantly increased calcium transients and sarcoplasmic reticular Ca²⁺ contents. Compared to the control, GLP-1 (10 nM)—treated cells significantly decreased phosphorylation of the ryanodine receptor at S2814 and total phospholamban, but there were similar protein levels of sarcoplasmic reticular Ca²⁺-ATPase and the sodium–calcium exchanger. Moreover, exendin (9–39) amide (a GLP-1 receptor antagonist, 10 nM) attenuated GLP-1-mediated effects on total SR content and phosphorylated ryanodine receptor S2814. This study demonstrates GLP-1 may regulate HL-1 cell arrhythmogenesis through modulating calcium handling proteins.     

The effects of calcium sulphate on growth,membrane stability and nutrient uptake of tomato plants grown under salt stress

A pot experiment was carried out with tomato (Lycopersicon esculentum Mill.) cv. “Target F1” in a mixture of peat, perlite, and sand (1:1:1) to investigate the effects of supplementary calcium sulphate on plants grown at high NaCl concentration (75 mM). The treatments were: (i) control (C), nutrient solution alone; (ii) salt treatment (C + S), 75 mM NaCl; (iii) salt plus calcium treatment 1 (C + S + Ca1), 75 mM NaCl plus additional mixture of 2.5 mM CaSO₄ in nutrient solution; (iv) salt plus calcium treatment 2 (C + S + Ca2), 75 mM NaCl plus additional mixture of 5 mM CaSO₄ in nutrient solution. The plants grown under salt stress produced low dry matter, fruit weight, and relative water content than those grown in standard nutrient solution. Supplemental calcium sulphate added to nutrient solution containing salt significantly improved growth and physiological variables affected by salt stress (e.g. plant growth, fruit yield, and membrane permeability) and also increased leaf K⁺, Ca²⁺, and N in tomato plants. The effects of supplemental CaSO₄ in maintaining membrane permeability, increasing concentrations of Ca²⁺, N, and K⁺ and reducing concentration of Na⁺ (because of cation competition in root zone) in leaves could offer an economical and simple solution to tomato crop production problems caused by high salinity.     

Simultaneous application of salicylic acid and calcium improves salt tolerance in two contrasting tomato (Solanum lycopersicum) cultivars

Soil salinity is one of the most important environmental factors responsible for serious agricultural problems. Tomato salt tolerance may be improved by genetic selection and by the use of adapted physiological tools. The aim of this study was to investigate the impact of exogenous application of salicylic acid (SA 0.01 mM) and calcium sulphate (CaSO₄ 5 mM), singly or in combination, on plant growth, photosynthetic pigments, nutritional behaviour and some metabolic parameters (total chlorophyll, carotenoids, soluble sugars, proline and lipid peroxidation) of two tomato cultivars (cv. Super Marmande and cv. Red River) exposed to salt stress (100 mM NaCl). Application of 100 mM NaCl reduced plant growth, total chlorophyll and carotenoid contents. Salt stress also induced an accumulation of Na⁺, a decrease in K⁺ and Ca^2 + concentration and root sugar level, an increase in malondialdehyde (MDA) and proline concentration. Deleterious impact of salinity was related to modification in ion content rather than modification in the plant water status. Exogenous application of SA or Ca alone improved plant behaviour in the presence of NaCl. Nevertheless, the best results in terms of growth, photosynthetic pigment concentrations and mineral nutrition (limitation of Na⁺ accumulation and maintenance of K⁺ and Ca^2 + content) were obtained in response to the combined SA + Ca treatment. Although the involved physiological parameters varied depending on the considered cultivar, our results suggest that Ca^2 + and SA may interact to reduce the stress experienced by the plant in the presence of NaCl.     

The polybasic lysine-rich domain of plasma membrane-resident STIM1 is essential for the modulation of store-operated divalent cation entry by extracellular calcium

STIM1 acts as an endoplasmic reticulum Ca^2 + sensor that communicates the filling state of the intracellular stores to the store-operated channels. In addition, STIM1 is expressed in the plasma membrane, with the Ca^2 + binding EF-hand motif facing the extracellular medium; however, its role sensing extracellular Ca^2 + concentrations in store-operated Ca^2 + entry (SOCE), as well as the underlying mechanism remains unclear. Here we report that divalent cation entry stimulated by thapsigargin (TG) is attenuated by extracellular Ca^2 + in a concentration-dependent manner. Expression of the Ca^2 +-binding defective STIM1(D76A) mutant did not alter the surface expression of STIM1 but abolishes the regulation of divalent cation entry by extracellular Ca^2 +. Orai1 and TRPC1 have been shown to play a major role in SOCE. Expression of the STIM1(D76A) mutant did not alter Orai1 phosphoserine content. TRPC1 silencing significantly attenuated TG-induced Mn^2 + entry. Expression of the STIM1(K684,685E) mutant impaired the association of plasma membrane STIM1 with TRPC1, as well as the regulation of TG-induced divalent cation entry by extracellular Ca^2 +, which suggests that TRPC1 might be involved in the regulation of divalent cation entry by extracellular Ca^2 + mediated by plasma membrane-resident STIM1. Expression of the STIM1(D76A) or STIM1(K684,685E) mutants reduced store-operated divalent cation entry and resulted in loss of dependence on the extracellular Ca^2 + concentration, providing evidence for a functional role of plasma membrane-resident STIM1 in the regulation of store-operated divalent cation entry, which at least involves the EF-hand motif and the C-terminal polybasic lysine-rich domain.     

Stress-induced dissociations between intracellular calcium signaling and insulin secretion in pancreatic islets

In healthy pancreatic islets, glucose-stimulated changes in intracellular calcium ([Ca²⁺]_i) provide a reasonable reflection of the patterns and relative amounts of insulin secretion. We report that [Ca²⁺]_i in islets under stress, however, dissociates with insulin release in different ways for different stressors. Islets were exposed for 48 h to a variety of stressors: cytokines (low-grade inflammation), 28 mM glucose (28G, glucotoxicity), free fatty acids (FFAs, lipotoxicity), thapsigargin (ER stress), or rotenone (mitochondrial stress). We then measured [Ca²⁺]_i and insulin release in parallel studies. Islets exposed to all stressors except rotenone displayed significantly elevated [Ca²⁺]_i in low glucose, however, increased insulin secretion was only observed for 28G due to increased nifedipine-sensitive calcium-channel flux. Following 3–11 mM glucose stimulation, all stressors substantially reduced the peak glucose-stimulated [Ca²⁺]_i response (first phase). Thapsigargin and cytokines also substantially impacted aspects of calcium influx and ER calcium handling. Stressors did not significantly impact insulin secretion in 11 mM glucose for any stressor, although FFAs showed a borderline reduction, which contributed to a significant decrease in the stimulation index (11:3 mM glucose) observed for FFAs and also for 28G. We also clamped [Ca²⁺]_i using 30 mM KCl + 250 μM diazoxide to test the amplifying pathway. Only rotenone-treated islets showed a robust increase in 3–11 mM glucose-stimulated insulin secretion under clamped conditions, suggesting that low-level mitochondrial stress might activate the metabolic amplifying pathway. We conclude that different stressors dissociate [Ca²⁺]_i from insulin secretion differently: ER stressors (thapsigargin, cytokines) primarily affect [Ca²⁺]_i but not conventional insulin secretion and ‘metabolic’ stressors (FFAs, 28G, rotenone) impacted insulin secretion.     

Influence of urea and calcium dosage on the effectiveness of bacterially induced carbonate precipitation on limestone

Bacterially induced carbonate precipitation has been explored for the protection and consolidation of ornamental stone. Attempts to improve the efficiency of this biodeposition process were primarily focused on the microbial aspects, i.e. type of microorganism and metabolic pathway. In this study, the influence of the chemical parameters, i.e. concentration of calcium salts and urea, on the effectiveness of the biodeposition treatment has been examined. The amount of calcium carbonate that can be precipitated in the stone is conditioned both by the amount of cells retained in the stone and the concentration of urea and calcium used. From sonication experiments, a good consolidation was observed for limestone prisms treated with a calcium dosage of 17 g Ca²⁺ m^?2 with no improvement at higher concentrations. For limestone prisms of 4 cm × 2 cm × 1 cm, the biodeposition treatment resulted in a 63% lower weight loss upon sonication compared to untreated specimens. The waterproofing effect was observed to increase with increasing calcium dosages. While for a calcium dosage of 17 g Ca²⁺ m^?2 the water absorption was similar to that of untreated specimens, concentrations of 67 g Ca²⁺ m^?2 resulted in a 50% decrease of the rate of water absorption. For calcium dosages higher than 34 g Ca²⁺ m^?2 a significant change in the visual aspect (ΔE > 6) of the treated stones could be observed. Overall, the urea/calcium chloride-based biodeposition treatment attained a protective performance comparable with that of the commonly used ethylsilicates.     

Alterations in ryanodine receptors and related proteins in heart failure

Sarcoplasmic reticulum (SR) Ca^2 + release plays an essential role in mediating cardiac myocyte contraction. Depolarization of the plasma membrane results in influx of Ca^2 + through l-type Ca^2 + channels (LTCCs) that in turn triggers efflux of Ca^2 + from the SR through ryanodine receptor type-2 channels (RyR2). This process known as Ca^2 +-induced Ca^2 +release (CICR) occurs within the dyadic region, where the adjacent transverse (T)-tubules and SR membranes allow RyR2 clusters to release SR Ca^2 + following Ca^2 + influx through adjacent LTCCs. SR Ca^2 + released during systole binds to troponin-C and initiates actin–myosin cross-bridging, leading to muscle contraction. During diastole, the cytosolic Ca^2 + concentration is restored by the resequestration of Ca^2 + into the SR by SR/ER Ca^2 +-ATPase (SERCA2a) and by the extrusion of Ca^2 + via the Na⁺/Ca^2 +-exchanger (NCX1). This whole process, entitled excitation–contraction (EC) coupling, is highly coordinated and determines the force of contraction, providing a link between the electrical and mechanical activities of cardiac muscle. In response to heart failure (HF), the heart undergoes maladaptive changes that result in depressed intracellular Ca^2 + cycling and decreased SR Ca^2 + concentrations. As a result, the amplitude of CICR is reduced resulting in less force production during EC coupling. In this review, we discuss the specific proteins that alter the regulation of Ca^2 + during HF. In particular, we will focus on defects in RyR2-mediated SR Ca^2 + release. This article is part of a Special Issue entitled: Heart failure pathogenesis and emerging diagnostic and therapeutic interventions.     

Calcium and mitochondrial metabolism in ceramide-induced cardiomyocyte death

Ceramides are important intermediates in the biosynthesis and degradation of sphingolipids that regulate numerous cellular processes, including cell cycle progression, cell growth, differentiation and death. In cardiomyocytes, ceramides induce apoptosis by decreasing mitochondrial membrane potential and promoting cytochrome-c release. Ca^2 + overload is a common feature of all types of cell death. The aim of this study was to determine the effect of ceramides on cytoplasmic Ca^2 + levels, mitochondrial function and cardiomyocyte death. Our data show that C₂-ceramide induces apoptosis and necrosis in cultured cardiomyocytes by a mechanism involving increased Ca^2 + influx, mitochondrial network fragmentation and loss of the mitochondrial Ca^2 + buffer capacity. These biochemical events increase cytosolic Ca^2 + levels and trigger cardiomyocyte death via the activation of calpains.