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Although electrical cardioversion of atrial fibrillation (AF) is frequently performed, initial energy requirements for cardioversion of persistent AF is still a matter of debate. The aim of the study was to determine the efficacy of biphasic shocks for transthoracic cardioversion of persistent AF and to predict adequate initial energy. A prospective study enrolled 94 consecutive patients with persistent AF, who were referred for elective cardioversion with a biphasic waveform. The paddles were placed in the anterolateral position. A step-up protocol was used to estimate the cardioversion threshold. The initial shock energy was 50 J, with subsequent increments to 100, 200, and 300 J in the event of cardioversion failure. The mean age of the study group was of about 65 years (6 +/- 11 years) and a median duration of AF was 65 days (3-324). Sixty-two out of 94 patients were men, 55% of the study population was classified as having well-controlled hypertension. The overall success rate of cardioversion was 89%, with a mean 2.2 +/- 1.4 shocks, and effective J 217.8 +/- 113 delivered during repeated cardioversions. The success rate of low energy shocks: 50 and 50 +100 J was 51%. By logistic regression analysis the only independent predictor of success at low energy shock was shorter duration of AF (r =-0.51; P = 0.02). Patients with shorter duration of AF have a higher probability for successful cardioversion with low energy. In patients with longer AF duration, a 200 J shock should be considered for cardioversion as the initial energy. [ABSTRACT FROM AUTHOR]

Calcium (Ca 2+ ) release-activated Ca 2+ (CRAC) channels are a major route for Ca 2+ entry in eukaryotic cells. These channels are store operated, opening when the endoplasmic reticulum (ER) is depleted of Ca 2+ , and are composed of the ER Ca 2+ sensor protein STIM and the pore-forming plasma membrane subunit Orai. Recent years have heralded major strides in our understanding of the structure, gating, and function of the channels. Loss-of-function and gain-of-function mutants combined with RNAi knockdown strategies have revealed important roles for the channel in numerous human diseases, making the channel a clinically relevant target. Drugs targeting the channels generally lack specificity or exhibit poor efficacy in animal models. However, the landscape is changing, and CRAC channel blockers are now entering clinical trials. Here, we describe the key molecular and biological features of CRAC channels, consider various diseases associated with aberrant channel activity, and discuss targeting of the channels from a therapeutic perspective.

Key Points: Ca 2+ entry through Ca 2+  release-activated Ca 2+  channels activates numerous cellular responses. Under physiological conditions of weak intracellular Ca 2+ buffering, mitochondrial Ca 2+ uptake regulates CRAC channel activity. Knockdown of the mitochondrial Ca 2+ uniporter channel prevented the development of I CRAC in weak buffer but not when strong buffer was used instead. Removal of either extracellular or intra-pipette Na + had no effect on the selectivity, kinetics, amplitude, rectification or reversal potential of whole-cell CRAC current. Knockdown of the mitochondrial Na + -Ca 2+ exchanger did not prevent the development of I CRAC in strong or weak Ca 2+ buffer. Whole cell CRAC current is Ca 2+ -selective. Mitochondrial Ca 2+ channels, and not Na + -dependent transport, regulate CRAC channels under physiological conditions., Abstract: Ca 2+ entry through store-operated Ca 2+ release-activated Ca 2+ (CRAC) channels plays a central role in activation of a range of cellular responses over broad spatial and temporal bandwidths. Mitochondria, through their ability to take up cytosolic Ca 2+ , are important regulators of CRAC channel activity under physiological conditions of weak intracellular Ca 2+ buffering. The mitochondrial Ca 2+ transporter(s) that regulates CRAC channels is unclear and could involve the 40 kDa mitochondrial Ca 2+ uniporter (MCU) channel or the Na + -Ca 2+ -Li + exchanger (NCLX). Here, we have investigated the involvement of these mitochondrial Ca 2+ transporters in supporting the CRAC current (I CRAC ) under a range of conditions in RBL mast cells. Knockdown of the MCU channel impaired the activation of I CRAC under physiological conditions of weak intracellular Ca 2+ buffering. In strong Ca 2+ buffer, knockdown of the MCU channel did not inhibit I CRAC development demonstrating that mitochondria regulate CRAC channels under physiological conditions by buffering of cytosolic Ca 2+ via the MCU channel. Surprisingly, manipulations that altered extracellular Na + , cytosolic Na + or both failed to inhibit the development of I CRAC in either strong or weak intracellular Ca 2+ buffer. Knockdown of NCLX also did not affect I CRAC . Prolonged removal of external Na + also had no significant effect on store-operated Ca 2+ entry, on cytosolic Ca 2+ oscillations generated by receptor stimulation or on CRAC channel-driven gene expression. In the RBL mast cell, Ca 2+ flux through the MCU but not NCLX is indispensable for activation of I CRAC ., (© 2018 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

Analogues of the Ca 2+ -releasing intracellular messenger d- myo -inositol 1,4,5-trisphosphate [ 1 , Ins(1,4,5)P 3 ] are important synthetic targets. Replacement of the α-glucopyranosyl motif in the natural product mimic adenophostin 2 by d- chiro -inositol in d- chiro -inositol adenophostin 4 increased the potency. Similar modification of the non-nucleotide Ins(1,4,5)P 3 mimic ribophostin 6 may increase the activity. d- chiro -Inositol ribophostin 10 was synthesized by coupling as building blocks suitably protected ribose 12 with l-(+)-3- O -trifluoromethylsulfonyl-6- O - p -methoxybenzyl-1,2:4,5-di- O -isopropylidene- myo -inositol 11 . Separable diastereoisomeric 3- O -camphanate esters of (±)-6- O - p -methoxy-benzyl-1,2:4,5-di- O -isopropylidene- myo -inositol allowed the preparation of 11 . Selective trans -isopropylidene deprotection in coupled 13 , then monobenzylation gave separable regioisomers 15 and 16 . p -Methoxybenzyl group deprotection of 16 , phosphitylation/oxidation, then deprotection afforded 10 , which was a full agonist in Ca 2+ -release assays; its potency and binding affinity for Ins(1,4,5)P 3 R were similar to those of adenophostin. Both 4 and 10 elicited a store-operated Ca 2+ current I CRAC in patch-clamped cells, unlike Ins(1,4,5)P 3 consistent with resistance to metabolism. d- chiro -Inositol ribophostin is the most potent small-molecule Ins(1,4,5)P 3 receptor agonist without a nucleobase yet synthesized.

Store-operated Ca 2+ entry, involving endoplasmic reticulum Ca 2+ sensing STIM proteins and plasma membrane Orai1 channels, is a widespread and evolutionary conserved Ca 2+ influx pathway. This form of Ca 2+ influx occurs at discrete loci where peripheral endoplasmic reticulum juxtaposes the plasma membrane. Stimulation evokes numerous STIM1-Orai1 clusters but whether distinct signal transduction pathways require different cluster numbers is unknown. Here, we show that two Ca 2+ -dependent transcription factors, NFAT1 and c-fos, have different requirements for the number of STIM1-Orai1 clusters and on the Ca 2+ flux through them. NFAT activation requires fewer clusters and is more robustly activated than c-fos by low concentrations of agonist. For similar cluster numbers, transcription factor recruitment occurs sequentially, arising from intrinsic differences in Ca 2+ sensitivities. Variations in the number of STIM1-Orai1 clusters and Ca 2+ flux through them regulate the robustness of signalling to the nucleus whilst imparting a mechanism for selective recruitment of different Ca 2+ -dependent transcription factors.

Ca2+ entry into airway epithelia is important for activation of the NFAT family of transcription factors and expression of genes including epidermal growth factor that help orchestrate local inflammatory responses. However, the identity of epithelial Ca2+ channel that activates these transcriptional responses is unclear. In many other non-excitable cells, store-operated Ca2+ entry is a major route for Ca2+ influx and is mediated by STIM1 and Orai1 proteins. This study was performed to determine if store-operated Ca2+ channels were expressed in human bronchial epithelial cells and, if so, whether they coupled Ca2+ entry to gene expression. Cytoplasmic Ca2+ measurements, patch clamp recordings, RNAi knockdown and functional assays were used to identify and then investigate the role of these Ca2+ channels in activating the NFAT and c-fos pathways and EGF expression. STIM1 and Orai1 mRNA transcripts as well as proteins were robustly in epithelial cells and formed functional Ca2+ channels. Ca2+ entry through the channels activated expression of c-fos and EGF as well as an NFAT-dependent reporter gene. Store-operated Ca2+ entry was also important for epithelial cell migration in a scrape wound assay. These findings indicate that store-operated Ca2+ channels play an important role in stimulating airway epithelial cell gene expression and therefore comprise a novel potential therapeutic target for the treatment of chronic asthma and related airway disorders.

NFAT-dependent gene expression is essential for the development and function of the nervous, immune, and cardiovascular systems and kidney, bone, and skeletal muscle. Most NFAT protein resides in the cytoplasm because of extensive phosphorylation, which masks a nuclear localization sequence. Dephosphorylation by the Ca(2+)-calmodulin-activated protein phosphatase calcineurin triggers NFAT migration into the nucleus. In some cell types, NFAT can be activated by Ca(2+) nanodomains near open store-operated Orai1 and voltage-gated Ca(2+) channels in the plasma membrane. How local Ca(2+) near Orai1 is detected and whether other Orai channels utilize a similar mechanism remain unclear. Here, we report that the paralog Orai3 fails to activate NFAT. Orai1 is effective in activating gene expression via Ca(2+) nanodomains because it participates in a membrane-delimited signaling complex that forms after store depletion and brings calcineurin, via the scaffolding protein AKAP79, to calmodulin tethered to Orai1. By contrast, Orai3 interacts less well with AKAP79 after store depletion, rendering it ineffective in activating NFAT. A channel chimera of Orai3 with the N terminus of Orai1 was able to couple local Ca(2+) entry to NFAT activation, identifying the N-terminal domain of Orai1 as central to Ca(2+) nanodomain-transcription coupling. The formation of a store-dependent signaling complex at the plasma membrane provides for selective activation of a fundamental downstream response by Orai1., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

Stimulation of cells with physiological concentrations of calcium-mobilizing agonists often results in the generation of repetitive cytoplasmic Ca(2+) oscillations. Although oscillations arise from regenerative Ca(2+) release, they are sustained by store-operated Ca(2+) entry through Ca(2+) release-activated Ca(2+) (CRAC) channels. Here, we show that following stimulation of cysteinyl leukotriene type I receptors in rat basophilic leukemia (RBL)-1 cells, large amplitude Ca(2+) oscillations, CRAC channel activity, and downstream Ca(2+)-dependent nuclear factor of activated T cells (NFAT)-driven gene expression are all exclusively maintained by the endoplasmic reticulum Ca(2+) sensor stromal interaction molecule (STIM) 1. However, stimulation of tyrosine kinase-coupled FCεRI receptors evoked Ca(2+) oscillations and NFAT-dependent gene expression through recruitment of both STIM2 and STIM1. We conclude that different agonists activate different STIM proteins to sustain Ca(2+) signals and downstream responses.

Receptor desensitization is a universal mechanism to turn off a biological response; in this process, the ability of a physiological trigger to activate a cell is lost despite the continued presence of the stimulus. Receptor desensitization of G-protein-coupled receptors involves uncoupling of the receptor from its G-protein or second-messenger pathway followed by receptor internalization. G-protein-coupled cysteinyl leukotriene type I (CysLT1) receptors regulate immune-cell function and CysLT1 receptors are an established therapeutic target for allergies, including asthma. Desensitization of CysLT1 receptors arises predominantly from protein-kinase-C-dependent phosphorylation of three serine residues in the receptor carboxy terminus. Physiological concentrations of the receptor agonist leukotriene C(4) (LTC(4)) evoke repetitive cytoplasmic Ca(2+) oscillations, reflecting regenerative Ca(2+) release from stores, which is sustained by Ca(2+) entry through store-operated calcium-release-activated calcium (CRAC) channels. CRAC channels are tightly linked to expression of the transcription factor c-fos, a regulator of numerous genes important to cell growth and development. Here we show that abolishing leukotriene receptor desensitization suppresses agonist-driven gene expression in a rat cell line. Mechanistically, stimulation of non-desensitizing receptors evoked prolonged inositol-trisphosphate-mediated Ca(2+) release, which led to accelerated Ca(2+)-dependent slow inactivation of CRAC channels and a subsequent loss of excitation-transcription coupling. Hence, rather than serving to turn off a biological response, reversible desensitization of a Ca(2+) mobilizing receptor acts as an 'on' switch, sustaining long-term signalling in the immune system.

Store-operated Ca2+ channels in the plasma membrane (PM) are activated by the depletion of Ca2+ from the endoplasmic reticulum (ER) and constitute a widespread and highly conserved Ca2+ influx pathway. After store emptying, the ER Ca2+ sensor STIM1 forms multimers, which then migrate to ER-PM junctions where they activate the Ca2+ release-activated Ca2+ channel Orai1. Movement of an intracellular protein to such specialized sites where it gates an ion channel is without precedence, but the fundamental question of how STIM1 migrates remains unresolved. Here, we show that trafficking of STIM1 to ER-PM junctions and subsequent Ca2+ release-activated Ca2+ channel activity is impaired following mitochondrial depolarization. We identify the dynamin-related mitochondrial protein mitofusin 2, mutations of which causes the inherited neurodegenerative disease Charcot-Marie-Tooth IIa in humans, as an important component of this mechanism. Our results reveal a molecular mechanism whereby a mitochondrial fusion protein regulates protein trafficking across the endoplasmic reticulum and reveals a homeostatic mechanism whereby mitochondrial depolarization can inhibit store-operated Ca2+ entry, thereby reducing cellular Ca2+ overload.

Background: The diagnosis of the impaired left ventricle (LV) diastolic function during atrial fibrillation (AF) using traditional methods is very difficult. Natriuretic peptides seem to be useful for assessment of diastolic function in patients with AF., Aim: To evaluate the influence of LV diastolic dysfunction on natriuretic peptides concentrations and to assess the diagnostic value of atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) in patients with AF and impaired LV diastolic function., Methods: The study included 42 patients (23 males, 19 females), aged 58.6 +/- 8.2 years with nonvalvular persistent AF with preserved LV systolic function who were converted into sinus rhythm by DC cardioversion (CV) and maintained sinus rhythm for at least 30 days. Echocardiography (ECG), ANP, and BNP level measurements were taken at baseline 24 hours before CV and 24 hours and 30 days after CV. On the 30th day following CV in patients with sinus rhythm, Doppler ECG was performed to assess LV diastolic function., Results: Thirty days after CV, normal LV diastolic function in 15 patients and impaired diastolic function in 27 patients was diagnosed: 20 with impaired LV relaxation and seven with impaired LV compliance. During AF and 24 hours, and 30 days after sinus rhythm restoration, significantly higher ANP and BNP levels were observed in patients with LV diastolic dysfunction as compared to the subgroup with normal LV diastolic function. The average values of ANP during AF in patients with normal and impaired diastolic function were 167.3 +/- 70.1 pg/mL and 298.7 +/- 83.6 pg/mL, respectively (P < 0.001), and the average values of BNP in the above mentioned subgroups were 49.5 +/- 14.7 pg/mL and 145.6 +/- 49.6 pg/mL respectively (P < 0.001). While comparing the diagnostic value of both natriuretic peptides it was noted that BNP was a more specific and sensitive marker of impaired LV diastolic function. ANP value >220.7 pg/mL measured during AF identified patients with impaired LV diastolic function with 85% sensitivity and 90% specificity. BNP value >74.7 pg/mL proved 95% sensitive and 100% specific in the diagnosing of such a group., Conclusions: The increase of ANP/BNP concentration in patients with AF results not only from the presence of AF, but also reflects the impaired LV diastolic function. Natriuretic peptides, especially BNP, may be useful in diagnosing LV diastolic dysfunction in patients with AF.

Background: Acute myocarditis is one of the most challenging diagnoses in cardiology. It is a disease with variable clinical presentation, progression and outcome., Aim: To assess clinical characteristics and outcome of patients hospitalised with diagnosis of acute myocarditis from year 2006 to 2008., Methods: We analysed hospital files of consecutive 32 patients admitted to our hospital due to myocarditis. All demographic, clinical and laboratory data were analysed and compared between patients with acute or subacute myocarditis. After discharge the patients were followed for 8-24 months., Results: The majority of patients were males (84%) in a mean age of 33 years. Clinical and echocardiographic parameters improved in 25 (78%) of patients during hospital stay. During follow-up decreased left ventricular ejection fraction (LVEF) was observed more often in patients with subacute than acute myocarditis (mean LVEF values of 49 vs. 61%, respectively). Patients with a subacute form of the disease more frequently required chronic pharmacological therapy and more often retired from occupational activities., Conclusions: Diagnosis of myocarditis is still challenging. Careful history taking, serial laboratory, ECG and echocardiographic examinations are helpful in therapeutic decisions making and assessing prognosis. Patient with subacute myocarditis are more symptomatic than patients with acute myocarditis.

Venticular paced rhythm makes diagnosis of acute myocardial infarction difficult. We present a case of a 77-year-old woman with cardiac DDD pacemaker, suffering from diabetes, arterial hypertension and renal insufficiency. She was admitted to hospital due to heavy chest pain, radiated to neck and jaw and shortness of breath. The electrocardiogram recorded on admission showed ventricular paced rhythm and ST segment elevation > 0.5 mV, which fulfilled Sgarbossa's criteria for indetermined acute coronary syndrome in a patient with pacemaker. Troponin levels were not increased. Severe stenosis of left anterior descendent coronary artery was revealed in coronarography and percutaneus coronary angioplasty with stent implantation was performed immediately.

Background: Diastolic heart failure often coexists with atrial fibrillation (AF). Elevated plasma levels of natriuretic peptides are the left ventricular (LV) marker of diastolic dysfunction., Aim: To evaluate the influence of sinus rhythm restoration on ANP and BNP levels in patients with normal and impaired LV diastolic function., Methods: The study included 42 patients (19 men, 23 women), aged 58.6 +/- 8.2 years with non-valvular persistent AF with preserved LV systolic function who were successfully converted to sinus rhythm by DC cardioversion (CV) and maintained sinus rhythm for at least 30 days. On day 30 following CV in patients with sinus rhythm, Doppler echocardiography was performed to assess LV diastolic function. ECG, echocardiography, ANP and BNP plasma level measurements were made at baseline 24 h before CV and 24 h as well as 30 days after CV., Results: The average ANP level in the whole study group during AF was 254.9 +/- 79.9 pg/ml and the average BNP level was 113.6 +/- 49.1 pg/ml. There was an evident decrease in ANP/BNP serum concentration in all the patients after successful DC cardioversion. Measured on the 30th day after CV, ANP and BNP levels were 153.2 +/- 67.9 pg/ml and 61.9 +/- 25.1 pg/ml respectively (p < 0.001). Thirty days after CV normal LV diastolic function was diagnosed in 15 patients and in 27 patients impaired diastolic function: 20 with impaired LV relaxation and 7 with impaired LV compliance. The extent of natriuretic peptides drop was dependent on the LV diastolic function, being more substantial in the subgroup with impaired LV diastolic function. In the subgroup with LV diastolic dysfunction the average ANP serum concentration measured 30 days after conversion was reduced by 111.2 +/- 93.9 pg/ml (37%) (p < 0.001) and BNP level was reduced by 67.5 +/- 36.0 pg/ml (46%) (p < 0.001). In patients with normal diastolic function sinus rhythm restoration significantly influenced ANP level, while having no relevant effect on BNP plasma concentration. The average ANP reduction in this subgroup was 64.4 +/- 71.8 pg/ml (by 38%) and BNP reduction was 11.4 +/- 16.7 pg/ml (by 23%) (NS)., Conclusions: The drop in ANP and BNP plasma concentrations after conversion to sinus rhythm in patients with AF depends on the LV diastolic function. Restoration of sinus rhythm is associated with improvement of the heart's haemodynamics, especially in patients with impaired LV diastolic function, which may be inferred from the more pronounced decrease of BNP level after DC cardioversion in this subgroup, as compared to that with normal LV function.

Atrioventricular septal defect accounts for approximately 3% of all congenital cardiovascular malformations. We describe a case of a 34-year-old woman with inoperable sinus venosus atrial septal defect with severe pulmonary hypertension and symptoms of the Eisenmenger syndrome. Electrocardiography, chest radiography, transthoracic and transoesophageal echocardiography with and without contrast were performed and provided valuable information about haemodynamic consequences of this malformation.

Store-operated Ca2+ entry through CRAC channels is a major route for Ca2+ influx in non-excitable cells. Studies on store-operated channel selectivity using fluorescent dyes have found that the channels are impermeable to Ba2+. Furthermore, in such studies, agonists have been reported to increase Ba2+ influx, leading to the conclusion that additional Ca2+ entry pathways (permeable to Ba2+) co-exist with the Ba2+-impermeable store-operated channels. However, patch clamp experiments demonstrate that CRAC channels are permeable to Ba2+. We have addressed this paradox using fluorescence measurements and whole cell patch clamp recordings of ICRAC. In store-depleted cells loaded with fura 2, Ba2+ application results in a slower and smaller rise in fluorescence than is the case with Ca2+. Ba2+, unlike Ca2+, depolarises the membrane potential by approximately 40 mV, due to rapid block of an inwardly rectifying K+ current. Although Ba2+ permeates CRAC channels at very negative potentials in patch clamp recordings, Ba2+ permeation is steeply voltage-dependent. This combination of Ba2+-dependent depolarisation and voltage-dependent Ba2+ permeation accounts for the apparent lack of Ba2+ permeation through store-operated channels seen in fluorescence experiments. Our findings identify major limitations with the use of Ba2+ as a surrogate for Ca2+ in probing Ca2+ entry pathways and raise the possibility that some of the previous reports proposing multiple Ca2+ entry pathways based on Ba2+ entry into fura 2-loaded cells could be explained by voltage-dependent Ba2+ permeation through CRAC channels.

A rise in cytosolic Ca(2+) concentration is used as a key activation signal in virtually all animal cells, where it triggers a range of responses, including neurotransmitter release, muscle contraction, and cell growth and proliferation. A major route for Ca(2+) influx is through store-operated Ca(2+) channels. One important intracellular target for Ca(2+) entry through store-operated channels is the mitochondrion, which increases aerobic metabolism and ATP production after Ca(2+) uptake. Here, we reveal a novel feedback pathway whereby pyruvic acid, a critical rate-limiting substrate for mitochondrial respiration, increases store-operated entry by reducing inactivation of the channels. Importantly, the effects of pyruvic acid are manifest at physiologically relevant concentrations and membrane potentials. The reduction in the inactivation of calcium release-activated calcium (CRAC) channels by pyruvate is highly specific in that it is not mimicked by other intermediary metabolic acids, does not require its metabolism, is independent of its Ca(2+) buffering action, and does not involve mitochondrial Ca(2+) uptake or ATP production. These results reveal a new and direct link between intermediary metabolism and ion-channel gating and identify pyruvate as a potential signaling messenger linking energy demand to calcium-channel function.

Four patients with acute pulmonary embolism are presented. The diagnostic role of transesophageal echocardiography and spiral computerised tomography is underlined.

In many cell types, the emptying of intracellular Ca2+ stores results in the opening of store-operated Ca2+ channels in the plasma membrane. However, the nature of the signal that couples store content to the opening of these Ca2+ channels is unclear. One model proposes that the Ca2+ channels are initially stored in cytoplasmic vesicles but inserted into the plasma membrane upon store depletion via a regulated exocytoytic mechanism (vesicular fusion model). Using the whole-cell patch-clamp technique to measure the store-operated Ca2+ current ICRAC and the capacitance method to monitor vesicular fusion, an indicator of exocytosis, we have investigated the effects of interfering with regulated exocytosis on the ability of ICRAC to activate. We find that the recombinant protein alpha-SNAP1-285, an inhibitor of exocytosis in many systems, suppresses such fusion but has no impact on the activation of ICRAC. A variety of other manoeuvres that interfere with vesicle trafficking and exocytosis were also without effect on ICRAC. Impairing constitutive exocytosis with brefeldin A reduced the extent of ICRAC, but this effect was less pronounced when current density was considered instead. Activation of ICRAC can therefore be clearly dissociated from an exocytotic mechanism, a finding that is not easily reconcilable with the vesicular fusion model.

Store-operated Ca(2+) channels, which are activated by the emptying of intracellular Ca(2+) stores, provide one major route for Ca(2+) influx. Under physiological conditions of weak intracellular Ca(2+) buffering, the ubiquitous Ca(2+) releasing messenger InsP(3) usually fails to activate any store-operated Ca(2+) entry unless mitochondria are maintained in an energized state. Mitochondria rapidly take up Ca(2+) that has been released by InsP(3), enabling stores to empty sufficiently for store-operated channels to activate. Here, we report a novel role for mitochondria in regulating store-operated channels under physiological conditions. Mitochondrial depolarization suppresses store-operated Ca(2+) influx independently of how stores are depleted. This role for mitochondria is unrelated to their actions on promoting InsP(3)-sensitive store depletion, can be distinguished from Ca(2+)-dependent inactivation of the store-operated channels and does not involve changes in intracellular ATP, oxidants, cytosolic acidification, nitric oxide or the permeability transition pore, but is suppressed when mitochondrial Ca(2+) uptake is impaired. Our results suggest that mitochondria may have a more fundamental role in regulating store-operated influx and raise the possibility of bidirectional Ca(2+)-dependent crosstalk between mitochondria and store-operated Ca(2+) channels.

A patient with apical hypertrophic cardiomyopathy and myocardial infarction complicated by intramural haematoma of the left ventricle is presented. The angiographic examination confirmed atherosclerotic etiology of myocardial infarction. The other reasons of myocardial ischaemia in a hypertrophic cardiomyopathy are discussed, pointing at multi-factorial pathogenesis of myocardial infarction in this disease. Probable mechanisms of this rare complication and its prognostic value are also analysed.

Like voltage-operated Ca(2+) channels, store-operated CRAC channels become permeable to monovalent cations in the absence of external divalent cations. Using the whole-cell patch-clamp technique, we have characterized the permeation and selectivity properties of store-operated channels in the rat basophilic leukemia (RBL-1) cell line. Store depletion by dialysis with InsP(3) and 10 mM EGTA resulted in the rapid development of large inward currents in Na(+)- and Li(+)-based divalent-free solutions. Cs(+) permeated the channels poorly (P(Cs)/ P(Na)=0.01). Trimethylamine (TMA(+)), tetramethylammonium (TeMA(+)), tetraethylammonium (TEA(+)), N-methyl- D-glucamine (NMDG(+)) and TRIS(+) were not measurably permeant. NH(4)(+) was conducted well. We estimated the minimum pore diameter under divalent-free conditions to be between 0.32 nm and 0.55 nm. When cells were dialysed with buffered Ca(2+) solution and I(CRAC) activated by application of thapsigargin, P(Cs)/ P(Na) was still low (0.08). Outward currents through CRAC channels were carried by intracellular Na(+), K(+) and, to a much lesser extent, by Cs(+). Currents were unaffected by dialysis with Mg(2+)-free solution. The Na(+) current was inhibited by external Ca(2+) (half-maximal blocking concentration of 10 microM). This Ca(2+)-dependent block could be alleviated by hyperpolarization. The monovalent Na(+) current was voltage dependent, increasing as the holding potential depolarized above 0 mV. Our results suggest that CRAC channels in RBL-1 cells have a smaller pore diameter than voltage-operated Ca(2+) channels, discriminate between Group I cations, and differ markedly in their selectivity from CRAC channels reported in lymphocytes.

In non-excitable cells, the major Ca2+ entry pathway is the store-operated pathway in which emptying of intracellular Ca2+ stores activates Ca2+ channels in the plasma membrane. In many cell types, store-operated influx gives rise to a Ca2+-selective current called I(CRAC) (Ca2+ release-activated Ca2+ current). Using both the whole-cell patch clamp technique to measure I(CRAC) directly and fluorescent Ca2+ imaging, we have examined the role of the lipo-oxygenase pathway in the activation of store-operated Ca2+ entry in the RBL-1 rat basophilic leukaemia cell-line. Pretreatment with a variety of structurally distinct lipo-oxygenase inhibitors all reduced the extent of I(CRAC), whereas inhibition of the cyclo-oxygenase enzymes was without effect. The inhibition was still seen in the presence of the broad protein kinase blocker staurosporine, or when Na+ was used as the charge carrier through CRAC channels. The lipo-oxygenase blockers released Ca2+ from intracellular stores but this was not associated with subsequent Ca2+ entry. Lipo-oxygenase blockers also reduced both the amount of Ca2+ that could subsequently be released by the combination of thapsigargin and ionomycin in Ca2+-free solution and the Ca2+ influx component that occurred when external Ca2+ was re-admitted. The inhibitors were much less effective if applied after I(CRAC) had been activated. This inhibition of I(CRAC) could not be rescued by dialysis with 5(S)-hydroxyperoxyeicosa-6E,8Z,11Z,14Z,tetraenoic acid (5-HPETE), the first product of the 5-lipo-oxygenase pathway. Our findings indicate that exposure to pharmacological tools that inhibit the lipo-oxygenase enzymes all decrease the extent of activation of the current. Our results raise the possibility that a lipo-oxygenase might be involved in the activation of I(CRAC). Alternative explanations are also discussed.

In eukaryotic cells, activation of cell surface receptors that couple to the phosphoinositide pathway evokes a biphasic increase in intracellular free Ca2+ concentration: an initial transient phase reflecting Ca2+ release from intracellular stores, followed by a plateau phase due to Ca2+ influx. A major component of this Ca2+ influx is store-dependent and often can be measured directly as the Ca2+ release-activated Ca2+ current (I(CRAC)). Under physiological conditions of weak intracellular Ca2+ buffering, respiring mitochondria play a central role in store-operated Ca2+ influx. They determine whether macroscopic I(CRAC) activates or not, to what extent and for how long. Here we describe an additional role for energized mitochondria: they reduce the amount of inositol 1,4,5-trisphosphate (InsP3) that is required to activate I(CRAC). By increasing the sensitivity of store-operated influx to InsP3, respiring mitochondria will determine whether modest levels of stimulation are capable of evoking Ca2+ entry or not. Mitochondrial Ca2+ buffering therefore increases the dynamic range of concentrations over which the InsP3 is able to function as the physiological messenger that triggers the activation of store-operated Ca2+ influx.

One popular model for the activation of store-operated Ca2+ influx is the secretion-like coupling mechanism, in which peripheral endoplasmic reticulum moves to the plasma membrane upon store depletion thereby enabling inositol 1,4,5-trisphosphate (InsP3) receptors on the stores to bind to, and thus activate, store-operated Ca2+ channels. This movement is regulated by the underlying cytoskeleton. We have examined the validity of this mechanism for the activation of I(CRAC), the most widely distributed and best characterised store-operated Ca2+ current, in a model system, the RBL-1 rat basophilic cell line. Stabilisation of the peripheral cytoskeleton, disassembly of actin microfilaments and disaggregation of microtubules all consistently failed to alter the rate or extent of activation of I(CRAC). Rhodamine-phalloidin labelling was used wherever possible, and revealed that the cytoskeleton had been significantly modified by drug treatment. Interference with the cytoskeleton also failed to affect the intracellular calcium signal that occurred when external calcium was re-admitted to cells in which the calcium stores had been previously depleted by exposure to thapsigargin/ionomycin in calcium-free external solution. Application of positive pressure through the patch pipette separated the plasma membrane from underlying structures (cell ballooning). However, I(CRAC) was unaffected irrespective of whether cell ballooning occurred before or after depletion of stores. Pre-treatment with the membrane-permeable InsP3 receptor antagonist 2-APB blocked the activation of I(CRAC). However, intracellular dialysis with 2-APB failed to prevent I(CRAC) from activating, even at higher concentrations than those used extracellularly to achieve full block. Local application of 2-APB, once I(CRAC) had been activated, resulted in a rapid loss of the current at a rate similar to that seen with the rapid channel blocker La3+. Studies with the more conventional InsP3 receptor antagonist heparin revealed that occupation of the intracellular InsP3-sensitive receptors was not necessary for the activation or maintenance of I(CRAC). Similarly, the InsP3 receptor inhibitor caffeine failed to alter the rate or extent of activation of I(CRAC). Exposure to Li+, which reduces InsP3 levels by interfering with inositol monophosphatase, also failed to alter I(CRAC). Caffeine and Li+ did not affect the size of the intracellular Ca2+ signal that arose when external Ca2+ was re-admitted to cells which had been pre-exposed to thapsigargin/ionomycin in Ca2+-free external solution. Our findings demonstrate that the cytoskeleton does not seem to regulate calcium influx and that functional InsP3 receptors are not required for activation of I(CRAC). If the secretion-like coupling model indeed accounts for the activation of I(CRAC) in RBL-1 cells, then it needs to be revised significantly. Possible modifications to the model are discussed.

Whole-cell patch-clamp experiments were performed to examine the mechanism underlying the inability of intracellular Ins(1,4,5)P(3) to activate the Ca(2+) release-activated Ca(2+) current (I(CRAC)) in rat basophilic leukaemia (RBL)-1 cells under conditions of weak cytoplasmic Ca(2+) buffering. Dialysis with Ins(1,4,5)P(3) in weak Ca(2+) buffer did not activate any macroscopic I(CRAC) even after precautions had been taken to minimize the extent of Ca(2+) entry during the experiment. Following intracellular dialysis with Ins(1,4,5)P(3) for >150 s in weak buffer, external application of the sarcoplasmic/endoplasmic-reticulum Ca(2+)-ATPase (SERCA) pump blocker thapsigargin activated I(CRAC), and the current developed much more quickly than when thapsigargin was applied in the absence of Ins(1,4,5)P(3). This indicates that the Ins(1,4,5)P(3) receptors had not inactivated much over this timecourse. When external Ca(2+) was replaced by Ba(2+), Ins(1,4,5)P(3) still failed to generate any detectable I(CRAC) even though Ba(2+) permeates CRAC channels and is not taken up into the intracellular Ca(2+) stores. In strong Ca(2+) buffer, I(CRAC) could be activated by muscarinic-receptor stimulation, provided protein kinase C (PKC) was blocked. In weak buffer, however, as with Ins(1,4,5)P(3), stimulation of these receptors with carbachol did not activate I(CRAC) even after inhibition of PKC. The inability of Ins(1,4,5)P(3) to activate macroscopic I(CRAC) in weak Ca(2+) buffer was not altered by inhibition of Ca(2+)-dependent phosphorylation/dephosphorylation reactions. Our results suggest that the inability of Ins(1,4,5)P(3) to activate I(CRAC) under conditions of weak intracellular Ca(2+) buffering is not due to strong inactivation of the Ins(1,4,5)P(3) receptors. Instead, a futile Ca(2+) cycle across the stores seems to be occurring and SERCA pumps resequester sufficient Ca(2+) to ensure that the threshold for activation of macroscopic I(CRAC) has not been exceeded.

Three cases of patients with symptoms of digitalis overdosage were presented. The principal manifestations included complex supraventricular dysrhythmias and atrio-ventricular conduction disturbances. In the discussion a special attention was paid to digitalis dosage. Multiple factors influencing plasma concentration of digitalis including pharmacokinetics, bioavailability and drug interactions with glycosides were described. Short review of toxic manifestations of digitalis was made and the treatment of digitalis intoxication was outlined.

Tight-seal whole-cell patch-clamp experiments were carried out in order to investigate the effects of different holding potentials on the rate of development and amplitude of the Ca2+ release-activated Ca2+ current ICRAC in rat basophilic leukaemia (RBL-1) cells. ICRAC was monitored at -80 mV from fast voltage ramps, spanning 200 mV in 50 ms. At hyperpolarised potentials, the macroscopic CRAC conductance was lower than that seen at depolarised potentials. The conductance increased almost 5-fold over the voltage range -60 to +40 mV and was seen when the stores were depleted either by the combination of IP3 and thapsigargin in high Ca2+ buffer, or passively with 10 mM EGTA or BAPTA. The voltage-dependent conductance of the CRAC channels could not be fully accounted for by Ca2+-dependent fast inactivation, nor by other slower inhibitory mechanisms. It also did not seem to involve intracellular Mg2+ or the polycations spermine and spermidine. Voltage step relaxation experiments revealed that the voltage-dependent conductance changes developed and reversed slowly, with a time constant of several seconds at -60 mV. In the presence of physiological levels of intracellular Ca2+ buffers, ICRAC was barely detectable when cells were clamped at -60 mV and dialysed with IP3 and thapsigargin, but at 0 mV the current in low Ca2+ buffer was as large as that seen in high Ca2+ buffer. Our results suggest that CRAC channels exhibit slow voltage-dependent conductance changes which can triple the current amplitude over the physiological range of voltages normally encountered by these cells. The role of this conductance change and possible underlying mechanisms are discussed.

The subject of this study was a group of 757 patients hospitalized because of acute myocardial infarction in years 1992-1996, who survived the in-hospital course and were under observation for 2-6 years after the infarction. During the 14-18th day of the hospital stay they were made an echocardiographic test, including the ejection fraction (EF). The aim of this study was to define the influence of the ejection fraction value lowered below 40% on the long-term prognosis in patients after acute myocardial infarction. We compared two groups of patients; group I, consisting of 130 (17.2%) patients with EF lowered below 40% and group II, which included 627 patients with EF over 40%. To estimate the statistic significance we used the chi-square and t-Student test. The morbidity curves were made with the Kaplan-Meier method. The course of the myocardial infarction was much more grave in group I than in group II, what is confirmed by a more often anterior ventricular infarction and the quantity of dangerous complications which occurred during the in-hospital phase. The multi-factor regressive analysis showed that the ejection fraction lowered below 40% raises 2.47 times (95% confidence interval 1.50-4.07) (p < 0.001) the risk of death during the first year after myocardial infarction and nearly two times during the 5 year follow-up, compared to patients with a higher EF value. The influence of the EF value lowered below 40% on the creation of an infarction was not significant. The EF value lowered below 40% in patients after acute myocardial infarction was a significant risk factor in the long-term prognosis. More than 40% of deaths during the long-term prognosis in this group were caused by heart failure.

The subject of this trial were 243 patients with uncomplicated acute myocardial infarction, hospitalized in years 1992-1996, who were made an electrocardiographic exercise test in the second or third week of the in-hospital stay and whose further history in the 2-6 period after myocardial infarction (average follow-up time lasted for 4.0 +/- 1.9 years) was known. The aim of this trial was to determine the influence of the positive exercise test on the long-term prognosis after acute myocardial infarction in a group with uncomplicated acute myocardial infarction. The course of infarction and the frequency of cardiac events (cardiac death, reinfarction, revascularisation) occurrence in 78 patients with positive exercise test (group I) were compared with a group of 165 patients with negative exercise test (group II). Both groups were compared in respect to age, gender, history of myocardial infarction, risk factors and the course of the infarction in the in-hospital period. The multivariable logistic regression analyse showed that the positive exercise test did not have a statistically significant influence on the increase of post-hospital morbidity but it correlates with significantly more frequent use of the invasive treatment and reinfarction during the follow-up period in this group. Negative exercise test in patients with uncomplicated acute myocardial was a significant factor of the good long-term prognosis.

The risk of ischemic heart disease is connected with the definite mode of life. Improper nourishment, smoking, alcohol abuse, sedentary lifestyle and excessive mental stress cause disturbances leading to development of atherosclerosis. The change of the lifestyle may prevent from coronary heart disease and may play a main role in secondary prevention, making the prognosis after myocardial infarction much better. The epidemiological and clinical studies have shown the significance of particular risk factors reduction on survival after myocardial infarction and allowed to create the optimal preventive mode of life. Therefore the change of lifestyle should become the priority in the postinfarction therapy.

Acute pericarditis can be divided into primary pericarditis--of unknown etiology and secondary pericarditis--caused by earlier diagnosed disease. The diagnosis of acute pericarditis is based on clinical manifestations and accessory investigations, among which the most important is echocardiography. However, the etiologic diagnosis of pericarditis seems to be much more difficult, being ascertained in no more than 30%. It makes the causative treatment impossible in many cases. The good knowledge of epidemiology and symptoms of different pericardial diseases is important to make the proper choice of investigative procedures. At the beginning, usually less invasive investigations are planned to confirm etiologic diagnosis. Subsequently, more invasive procedures like pericardiocentesis, pericardiotomy and pericardial biopsy are reserved to explain the cause of pericarditis. The exceptions are cardiac tamponade and suspected purulent pericarditis, when pericardiocentesis and surgical drainage are made promptly in the therapeutic and diagnostic aim.