Studies of interaction of intracellular signalling and metabolic pathways under inhibition of mitochondrial aconitase with fluoroacetate

Mitochondrial aconitase has been shown to be inactivated by a spectrum of substances or critical states. Fluoroacetate (FA) is the most known toxic agent inhibiting aconitase. The biochemistry of toxic action of FA is rather well understood, though no effective therapy has been proposed for the past six decades. In order to reveal novel approaches for possible antidotes to be developed, experiments were performed with rat liver mitochondria, Ehrlich ascite tumor cells and cardiomyocytes, exposed to FA or fluorocitrate in vitro. The effect of FA developed at much higher concentrations in comparison with fluorocitrate and was dependent upon respiratory substrates in experiments with mitochondria: with pyruvate, FA induced a slow oxidation and/or leak of pyridine nucleotides and inhibition of respiration. Oxidation of pyridine nucleotides was prevented by incubation of mitochondria with cyclosporin A. Studies of the pyridine nucleotides level and calcium response generated in Ehrlich ascite tumor cells under activation with ATP also revealed a loss of pyridine nucleotides from mitochondria resulting in a shift in the balance of mitochondrial and cytosolic NAD(P)H under exposure to FA. An increase of cytosolic [Ca2+] was observed in the cell lines exposed to FA and is explained by activation of plasma membrane calcium channels; this mechanism, could have an impact on amplitude and rate of Ca2+ waves in cardiomyocytes. Highlighting the reciprocal relationship between intracellular pyridine nucleotides and calcium balance, we discuss metabolic pathway modulation in the context of probable development of an effective therapy for FA poisoning and other inhibitors of aconitase.     

Control of the pyridine nucleotide-linked Ca release from mitochondria by respiratory substrates

Oxidation of mitochondrial pyridine nucleotides followed by their hydrolysis promotes Ca²⁺ release from intact liver mitochondria. In most of the previous studies oxidation was achieved with pro-oxidants which were added to mitochondria respiring on succinate in the presence of rotenone, a site I-specific inhibitor of the respiratory chain. Here we investigate pro-oxidant dependent and independent Ca²⁺ release from mitochondria when respiration is supported either by the NAD⁺-linked substrate β-hydroxybutyrate, or by succinate. In the presence, as well as in the absence, of the pro-oxidant t-butylhydroperoxide mitochondria retain Ca²⁺ much better with succinate than with β-hydroxybutyrate, as respiratory substrate. When Ca²⁺ release is induced by t-butylhydroperoxide succinate-supported Ca²⁺ retention is impeded by rotenone. Ca²⁺ release (pro-oxidant dependent or independent) is paralleled by oxidation and hydrolysis of intramitochondrial pyridine nucleotides, and Ca²⁺ retention is paralleled by reduction of pyridine nucleotides. It is concluded that the pyridine nucleotide-linked Ca²⁺ release from mitochondria can be controlled by respiratory substrates which regulate the intramitochondrial hydrolysis of oxidized pyridine nucleotides.     

Destabilization of the cytosolic calcium level and the death of cardiomyocytes in the presence of derivatives of long-chain fatty acids

By means of fluorescent microscopy, long-chain fatty acid derivatives, myristoylcarnitine and palmitoylcarnitine, were shown to exert the most toxic effect on rat ventricular cardiomyocytes. The addition of 20–50 μM acylcarnitines increased calcium concentration in cytoplasm ([Ca²⁺]_i) and caused cell death after a lag-period of 4–8 min. This effect was independent of extracellular calcium level and Ca²⁺ inhibitors of L-type channels. Free myristic and palmitic acids at concentrations of 300–500 μM had little effect on [Ca²⁺]_i within 30 min. We suggest that the toxic effect is due to the activation of calcium channels of sarcoplasmic reticulum by acylcarnitines and/or arising acyl-CoA. Mitochondria play a role of calcium-buffer system under these conditions. The calcium capacity of the buffer determines the duration of the lag-period. Phosphate increases the calcium capacity of mitochondria and the lag-period. In the presence of rotenone and oligomycin, the elevation of [Ca²⁺]_i after the addition of acylcarnitines occurs without the lag-period. The exhaustion of the mitochondrial calcium-buffer capacity or significant depolarization of mitochondria leads to a rapid release of calcium from mitochondria and cell death. Thus, the activation of reticular calcium channels is the main reason of the toxicity of myristoylcarnitine and palmitoylcarnitine.     

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.     

Diversity of mitochondrial Ca signaling in rat neonatal cardiomyocytes: Evidence from a genetically directed Ca probe,mitycam-E31Q

I_Ca-gated Ca²⁺ release (CICR) from the cardiac SR is the main mechanism mediating the rise of cytosolic Ca²⁺, but the extent to which mitochondria contribute to the overall Ca²⁺ signaling remains controversial. To examine the possible role of mitochondria in Ca²⁺ signaling, we developed a low affinity mitochondrial Ca²⁺ probe, mitycam-E31Q (300–500 MOI, 48–72 h) and used it in conjunction with Fura-2AM to obtain simultaneous TIRF images of mitochondrial and cytosolic Ca²⁺ in cultured neonatal rat cardiomyocytes. Mitycam-E31Q staining of adult feline cardiomyocytes showed the typical mitochondrial longitudinal fluorescent bandings similar to that of TMRE staining, while neonatal rat cardiomyocytes had a disorganized tubular or punctuate appearance. Caffeine puffs produced rapid increases in cytosolic Ca²⁺ while simultaneously measured global mitycam-E31Q signals decreased more slowly (increased mitochondrial Ca²⁺) before decaying to baseline levels. Similar, but oscillating mitycam-E31Q signals were seen in spontaneously pacing cells. Withdrawal of Na⁺ increased global cytosolic and mitochondrial Ca²⁺ signals in one population of mitochondria, but unexpectedly decreased it (release of Ca²⁺) in another mitochondrial population. Such mitochondrial Ca²⁺ release signals were seen not only during long lasting Na⁺ withdrawal, but also when Ca²⁺ loaded cells were exposed to caffeine-puffs, and during spontaneous rhythmic beating. Thus, mitochondrial Ca²⁺ transients appear to activate with a delay following the cytosolic rise of Ca²⁺ and show diversity in subpopulations of mitochondria that could contribute to the plasticity of mitochondrial Ca²⁺ signaling.     

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.     

ADP requirement for prevention by a cytosolic factor of Mg2+ and Ca2+ release from rat liver mitochondria

One hundred micromolar Ca²⁺ added to rat liver mitochondria induces a transient uptake of Ca²⁺ plus a rapid efflux of the mitochondrial Mg²⁺. Addition of a cytosolic molecule, cytosolic metabolic factor, to mitochondria prevents the efflux of the two divalent cations. ADP is required for this cytosolic metabolic factor action. This requirement for ADP is specific as it is shown by experiments with traps for nucleotides and inhibitors of the translocase. The implication of cytosolic metabolic factor in the mitochondrial regulation process is discussed.     

Ca release from mitochondria induced by prooxidants

A variety of chemically different prooxidants causes Ca²⁺ release from mitochondria. The prooxidant-induced Ca²⁺ release occurs from intact mitochondria via a route which is physiologically relevant and may be regulated by protein ADP-ribosylation. When the released Ca²⁺ is excessively cycled by mitochondria they are damaged. This leads to uncoupling, a decreased ATP supply, and a decreased ability of mitochondria to retain Ca²⁺. Excessive Ca²⁺ cycling by mitochondria will deprive cells of ATP. As a result, Ca²⁺ ATPases of the endoplasmic (sarcoplasmic) reticulum and the plasma membrane are stopped. The rising cytosolic Ca²⁺ level cannot be counterbalanced due to damage of mitochondria which, under normoxic conditions, act as safety device against increased cytosolic Ca²⁺. It is proposed that prooxidants are toxic because they impair the ability of mitochondria to retain Ca²⁺.     

Cardiac ryanodine receptor N-terminal region biosensors identify novel inhibitors via FRET-based high-throughput screening

The N-terminal region (NTR) of ryanodine receptor (RyR) channels is critical for the regulation of Ca²⁺ release during excitation–contraction (EC) coupling in muscle. The NTR hosts numerous mutations linked to skeletal (RyR1) and cardiac (RyR2) myopathies, highlighting its potential as a therapeutic target. Here, we constructed two biosensors by labeling the mouse RyR2 NTR at domains A, B, and C with FRET pairs. Using fluorescence lifetime (FLT) detection of intramolecular FRET signal, we developed high-throughput screening (HTS) assays with these biosensors to identify small-molecule RyR modulators. We then screened a small validation library and identified several hits. Hits with saturable FRET dose–response profiles and previously unreported effects on RyR were further tested using [³H]ryanodine binding to isolated sarcoplasmic reticulum vesicles to determine effects on intact RyR opening in its natural membrane. We identified three novel inhibitors of both RyR1 and RyR2 and two RyR1-selective inhibitors effective at nanomolar Ca²⁺. Two of these hits activated RyR1 only at micromolar Ca²⁺, highlighting them as potential enhancers of excitation–contraction coupling. To determine whether such hits can inhibit RyR leak in muscle, we further focused on one, an FDA-approved natural antibiotic, fusidic acid (FA). In skinned skeletal myofibers and permeabilized cardiomyocytes, FA inhibited RyR leak with no detrimental effect on skeletal myofiber excitation–contraction coupling. However, in intact cardiomyocytes, FA induced arrhythmogenic Ca²⁺ transients, a cautionary observation for a compound with an otherwise solid safety record. These results indicate that HTS campaigns using the NTR biosensor can identify compounds with therapeutic potential.     

ZUR ENERGIEBEDÜRFTIGEN Ca2+-AKKUMULATION IN MITOCHONDRIEN AUS RATTENHIRN

—(1) The properties of a preparation of functional intact rat brain mitochondria are described and the resulting fraction is enzymatically characterized. (2) These mitochondria are able to accumulate Ca²⁺ in a respiration-dependent reaction without additions of P_i and/or adenine nucleotides. (3) As criteria the increased acidity of the incubation medium accompanying the energy-dependent Ca²⁺ accumulation, its inhibition by rotenon and Antimycin A, the acceleration of respiration and the redox change of the pyridine nucleotides were recorded and Ca²⁺ accumulation by the mitochondria was determined by complexemetric methods. (4) The maximum Ca²⁺ accumulation by rat brain mitochondria amounts to only 25 per cent of that by rat liver mitochondria under similar conditions (on the base per mg protein); after addition of 1 mm -ADP (without P_i) the comparable value was about 50 per cent.     

The influence of probiotics and probiotic product on respiration of mitochondria and intracellular calcium signal in cells of cardiovascular system

The influence of lactobacilli and new probiotic product on mitochondrial energetics of rat heart mitochondria and on dynamics of intracellular calcium concentration ([Ca²⁺]_i) of cardiomyocytes and rat aortic smooth muscle cells was investigated. Respiration of mitochondra was estimated polarographically. [Ca²⁺]_i was measured using fluorescent calcium indicator Fura 2 AM and calcium imaging system. The application of lactobacilli (5 × 10⁷ CFU/mL) was shown to increase [Ca²⁺]_i in cardiomyocytes, thereby increasing myocardial contractility. On the other hand, application of lactobacilli reduced thapsigargin-induced calcium influx in smooth rat aortic muscle, thus exhibiting some hypotensive effect. It was shown that probiotic product stimulated mitochondria respiration and exerted a mild uncoupling effect on electronic transport and oxidative phosphorylation in mitochondria. In cardiomyocytes and in smooth muscles probiotic product increased [Ca²⁺]_i and consequent increase in contractility of blood vessels and myocardium. It is supposed that the probiotic product can be effectively applied at the endotoxic shock, when contractility of blood vessels in response to vasoconstrictor agents is suppressed.     

线粒体和细胞内钙自稳平衡

线粒体对胞浆钙信号调节作用的研究已经历较长时间.近年,随着研究方法和技术的不断改进,发现在绝大多数生理条件下,线粒体都能参与胞内钙通信过程.线粒体可感受其周围钙微区的存在从而摄取钙,又可以通过钠-钙交换和大分子孔道将钙释放出来,因此可以调节胞浆钙信号的时空特性,影响相关的细胞功能.但是,由于技术上的局限性,目前的研究仍然存在模糊不清和自相矛盾之处,有待于进一步研究.     

Palmitic acid methyl ester induces cardiac hypertrophy through activating the GPR receptor-mediated changes of intracellular calcium concentrations and mitochondrial functions

Myocardial hypertrophy is associated with a significant increase in intracellular Ca²⁺, which can be induced by long-chain fatty acid. Palmitic acid methyl ester (PAME), a fatty acid ester released from adipose tissue, superior cervical ganglion, and retina, has been found to have anti-inflammation, antifibrosis, and peripheral vasodilation effects. However, the effects of PAME on cardiomyocytes are still unclear. The aim of this study was to determine whether PAME could disrupt the intracellular Ca²⁺ balance, leading to cardiomyocyte hypertrophy. Neonatal rat cardiomyocytes were treated with various concentrations (10–100 μM) of PAME for 1–4 days. Cytosolic Ca²⁺ and mitochondrial Ca²⁺ concentrations were examined using Fura-2 AM and Rhod-2, respectively. After treatment with PAME for 4 days, mitochondrial Ca²⁺, an indicator of the state of mitochondrial permeability transition pore (MPTP), and cell death were monitored by flow cytometric analysis. ATP levels were detected using the ATP assay kit. Cardiomyocyte hypertrophy was analyzed by measuring the cardiac hypertrophy biomarker and cell area using quantitative real time-polymerase chain reaction, Western Blot analysis and immunofluorescence analysis. Our results show that PAME concentration- and time-dependently increased cytosolic and mitochondria Ca²⁺ through the mitochondrial calcium uniporter. Moreover, treatment with PAME for 4 days caused MPTP opening, thereby reducing ATP production and enhancing reactive oxygen species (ROS) generation, and finally led to cardiomyocyte hypertrophy. These effects caused by PAME treatment were attenuated by the G-protein coupled receptor 40 (GPR40) inhibitor. In conclusion, PAME impaired mitochondrial function, which in turn led to cardiomyocyte hypertrophy through increasing the mitochondrial Ca²⁺ levels mediated by activating the GPR40 signaling pathway.     

Oxidative modification sensitizes mitochondrial apoptosis-inducing factor to calpain-mediated processing

Although processing of mitochondrial apoptosis-inducing factor (AIF) is essential for its function during apoptosis in most cell types, the detailed mechanisms of AIF cleavage remain elusive. Recent findings indicate that the proteolytic process is Ca²⁺-dependent and that it is mediated by a calpain located in the mitochondrial intermembrane space. We can now report that, in addition to a sustained intracellular Ca²⁺ elevation, enhanced formation of reactive oxygen species (ROS) is a prerequisite step for AIF to be cleaved and released from mitochondria in staurosporine-treated cells. These events occurred independent of the redox state of the mitochondria and were not influenced by binding of pyridine nucleotides to AIF. Chelation of cytosolic Ca²⁺ by BAPTA/AM suppressed the elevation of both Ca²⁺ and ROS, suggesting that the Ca²⁺ rise was the most upstream signal required for AIF processing. We could further show that the stimulated ROS production leads to oxidative modification (carbonylation) of AIF, which markedly increases its rate of cleavage by calpain. Accordingly, pretreatment of the cells with antioxidants blocked AIF carbonylation, as well as its subsequent cleavage and release from the mitochondria. Combined, our data provide evidence that ROS-mediated, posttranslational modification of AIF is critical for its cleavage by calpain and thus for AIF-mediated cell death.     

The interplay between mitochondria and store-operated Ca2+ entry: Emerging insights into cardiac diseases

Store-operated Ca²⁺ entry (SOCE) machinery, including Orai channels, TRPCs, and STIM1, is key to cellular calcium homeostasis. The following characteristics of mitochondria are involved in the physiological and pathological regulation of cells: mitochondria mediate calcium uptake through calcium uniporters; mitochondria are regulated by mitochondrial dynamic related proteins (OPA1, MFN1/2, and DRP1) and form mitochondrial networks through continuous fission and fusion; mitochondria supply NADH to the electron transport chain through the Krebs cycle to produce ATP; under stress, mitochondria will produce excessive reactive oxygen species to regulate mitochondria-endoplasmic reticulum interactions and the related signalling pathways. Both SOCE and mitochondria play critical roles in mediating cardiac hypertrophy, diabetic cardiomyopathy, and cardiac ischaemia-reperfusion injury. All the mitochondrial characteristics mentioned above are determinants of SOCE activity, and vice versa. Ca²⁺ signalling dictates the reciprocal regulation between mitochondria and SOCE under the specific pathological conditions of cardiomyocytes. The coupling of mitochondria and SOCE is essential for various pathophysiological processes in the heart. Herein, we review the research focussing on the reciprocal regulation between mitochondria and SOCE and provide potential interplay patterns in cardiac diseases.     

Secretory products of guinea pig epicardial fat induce insulin resistance and impair primary adult rat cardiomyocyte function

Epicardial adipose tissue (EAT) has been implicated in the development of heart disease. Nonetheless, the crosstalk between factors secreted from EAT and cardiomyocytes has not been studied. Here, we examined the effect of factors secreted from EAT on contractile function and insulin signalling in primary rat cardiomocytes. EAT and subcutaneous adipose tissue (SAT) were isolated from guinea pigs fed a high-fat (HFD) or standard diet. HFD feeding for 6 months induced glucose intolerance, and decreased fractional shortening and ejection fraction (all P < 0.05). Conditioned media (CM) generated from EAT and SAT explants were subjected to cytokine profiling using antibody arrays, or incubated with cardiomyocytes to assess the effects on insulin action and contractile function. Eleven factors were differentially secreted by EAT when compared to SAT. Furthermore, secretion of 30 factors by EAT was affected by HFD feeding. Most prominently, activin A-immunoreactivity was 6.4-fold higher in CM from HFD versus standard diet-fed animals and, 2-fold higher in EAT versus SAT. In cardiomyocytes, CM from EAT of HFD-fed animals increased SMAD2-phosphorylation, a marker for activin A-signalling, decreased sarcoplasmic-endoplasmic reticulum calcium ATPase 2a expression, and reduced insulin-mediated phosphorylation of Akt-Ser473 versus CM from SAT and standard diet-fed animals. Finally, CM from EAT of HFD-fed animals as compared to CM from the other groups markedly reduced sarcomere shortening and cytosolic Ca(2+) fluxes in cardiomyocytes. These data provide evidence for an interaction between factors secreted from EAT and cardiomyocyte function.     

A Simulation Study of Calcium Dynamics Features Caused by Exchange between the Cytosol and Organellar Stores of Neurons

The objects of the study were single-compartment mathematical models corresponding to a fragment of the dendrite of a cerebellar Purkinje neuron. The fragments contained the mitochondria (model 1) or a cistern of the endoplasmic reticulum, ER (model 2), functioning as calcium stores. With simulating single excitatory synaptic actions, we examined the dependence of the dynamics of intracellular Ca²⁺ levels on the maximum rate of Ca²⁺ exchange between the cytosol and these stores, as well as on the intensity of the diffusion flow into adjacent organelle-free regions. The plasma membrane of the compartment had ion channels (including those of the synaptic current) and the calcium pump characteristic of the mentioned neurons. The model equations took into account Ca²⁺ exchange between the cytosol, extracellular environment, and organellar stores, as well as the diffusion process. In model 1, the mitochondria exchanged Ca²⁺ with the cytosol through the uniporter and sodium-calcium exchanger; mitochondrial processes, such as the tricarboxylic acid cycle and aerobic cellular respiration, were also included. In model 2, the ER membrane had the calcium pump, leak channels, and channels of calcium-induced and inositol-3-phosphate-dependent Ca²⁺ release. The stores (mitochondria or ER) occupied 36% of the total volume of the compartment. An increase in the maximum rate of calcium exchange with the stores led to a proportional decrease in the peak Ca²⁺ concentrations in the cytosol ([Ca²⁺]_i), more pronounced in the case of the ER; the Ca²⁺ concentration in both types of stores increased significantly. Due to the higher storage rate, the ER was able to absorb several times greater amounts of Ca²⁺ than the mitochondria did. With smaller diffusion flux (e.g., similarly to the case of diffusion from a larger-sized head into the neck of the dendritic spine), the intensity of cytosolic transients increased at fixed kinetics of flux exchange with the stores. Therefore, the organellar stores can significantly modulate not only the intensity but also the time course of changes in the intracellular Ca²⁺ levels.     

Autonomous activation of CaMKII exacerbates diastolic calcium leak during beta-adrenergic stimulation in cardiomyocytes of metabolic syndrome rats

Autonomous Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) activation induces abnormal diastolic Ca²⁺ leak, which leads to triggered arrhythmias in a wide range of cardiovascular diseases, including diabetic cardiomyopathy. In hyperglycemia, Ca²⁺ handling alterations can be aggravated under stress conditions via the β-adrenergic signaling pathway, which also involves CaMKII activation. However, little is known about intracellular Ca²⁺ handling disturbances under β-adrenergic stimulation in cardiomyocytes of the prediabetic metabolic syndrome (MetS) model with obesity, and the participation of CaMKII in these alterations.MetS was induced in male Wistar rats by administering 30 % sucrose in drinking water for 16 weeks. Fluo 3-loaded MetS cardiomyocytes exhibited augmented diastolic Ca²⁺ leak (in the form of spontaneous Ca²⁺ waves) under basal conditions and that Ca²⁺ leakage was exacerbated by isoproterenol (ISO, 100 nM). At the molecular level, [³H]-ryanodine binding and basal phosphorylation of cardiac ryanodine receptor (RyR2) at Ser2814, a CaMKII site, were increased in heart homogenates of MetS rats with no changes in RyR2 expression. These alterations were not further augmented by Isoproterenol. SERCA pump activity was augmented 48 % in MetS hearts before β-adrenergic stimuli, which is associated to augmented PLN phosphorylation at T17, a target of CaMKII. In MetS hearts. CaMKII auto-phosphorylation (T287) was increased by 80 %. The augmented diastolic Ca²⁺ leak was prevented by CaMKII inhibition with AIP. In conclusion, CaMKII autonomous activation in cardiomyocytes of MetS rats with central obesity significantly contributes to abnormal diastolic Ca²⁺ leak, increasing the propensity for β-adrenergic receptor-driven lethal arrhythmias.     

Role of mitochondria in the operation of calcium signaling system in heat-stressed plants

Mild heat stress induces the expression of heat shock proteins (HSPs) that protect plants from death during damaging heat treatments. It was assumed that the appearance in the cell of denatured proteins triggers the expression of HSP; however, recent results show that protein denaturation is not a prerequisite for this process. In this work we discuss a hypothetical mechanism for activation under heat stress of HSP expression promoted by short-term elevation of cytosolic Ca²⁺ level. According to our hypothesis, a prolonged elevation of Ca²⁺ has a negative influence on HSP expression. Therefore, calcium is transported from the cytosol into intracellular compartments, including mitochondria. The Ca²⁺ entry into mitochondria is accompanied by hyperpolarization of the inner mitochondrial membrane and by the increased production of reactive oxygen species (ROS). The increased ROS production contributes to the activation of HSP expression under mild heat stress but leads to plant death under severe heat shock. Thus, mitochondria and, possibly, other organelles play the crucial role in determining life or death fate of heat-treated plant cells by controlling the cytosolic Ca²⁺ content and ROS production.