Ion channel gating in cardiac ryanodine receptors from the arrhythmic RyR2-P2328S mouse.

Mutations in the cardiac ryanodine receptor Ca2+ release channel (RyR2) can cause deadly ventricular arrhythmias and atrial fibrillation (AF). The RyR2-P2328S mutation produces catecholaminergic polymorphic ventricular tachycardia (CPVT) and AF in hearts from homozygous RyR2P2328S/P2328S (denoted RyR2S/S) mice. We have now examined P2328S RyR2 channels from RyR2S/S hearts. The activity of wild-type (WT) and P2328S RyR2 channels was similar at a cytoplasmic [Ca2+] of 1 mM, but P2328S RyR2 was significantly more active than WT at a cytoplasmic [Ca2+] of 1 µM. This was associated with a >10-fold shift in the half maximal activation concentration (AC50) for Ca2+ activation, from ∼3.5 µM Ca2+ in WT RyR2 to ∼320 nM in P2328S channels and an unexpected >1000-fold shift in the half maximal inhibitory concentration (IC50) for inactivation from ∼50 mM in WT channels to ≤7 µM in P2328S channels, which is into systolic [Ca2+] levels. Unexpectedly, the shift in Ca2+ activation was not associated with changes in sub-conductance activity, S2806 or S2814 phosphorylation or the level of FKBP12 (also known as FKBP1A) bound to the channels. The changes in channel activity seen with the P2328S mutation correlate with altered Ca2+ homeostasis in myocytes from RyR2S/S mice and the CPVT and AF phenotypes.This article has an associated First Person interview with the first author of the paper.

Dietary Intake and Gastrointestinal Integrity in Runners Undertaking High-Intensity Exercise in the Heat.

Gastrointestinal disturbances are one of the most common issues for endurance athletes during training and competition in the heat. The relationship between typical dietary intake or nutritional interventions and perturbations in or maintenance of gut integrity is unclear. Twelve well-trained male endurance athletes (peak oxygen consumption = 61.4 ± 7.0 ml·kg-1·min-1) completed two trials in a randomized order in 35 °C (heat) and 21 °C (thermoneutral) conditions and kept a detailed nutritional diary for eight consecutive days between the two trials. The treadmill running trials consisted of 15 min at 60% peak oxygen consumption, 15 min at 75% peak oxygen consumption, followed by 8 × 1-min high-intensity efforts. Venous blood samples were taken at the baseline, at the end of each of the three exercise stages, and 1 hr postexercise to measure gut integrity and the permeability biomarker concentration for intestinal fatty-acid-binding protein, lipopolysaccharide, and lipopolysaccharide-binding protein. The runners self-reported gut symptoms 1 hr postexercise and 3 days postexercise. The heat condition induced large (45-370%) increases in intestinal fatty-acid-binding protein, lipopolysaccharide-binding protein, and lipopolysaccharide concentrations compared with the baseline, but induced mild gastrointestinal symptoms. Carbohydrate and polyunsaturated fat intake 24 hr preexercise were associated with less lipopolysaccharide translocation. Protein, carbohydrate, total fat, and polyunsaturated fat intake (8 days) were positively associated with the percentage increase of intestinal fatty-acid-binding protein in both conditions (range of correlations, 95% confidence interval = .62-.93 [.02, .98]). Typical nutrition intake partly explained increases in biomarkers and the attenuation of symptoms induced by moderate- and high-intensity exercise under both heat and thermoneutral conditions.

Calmodulin inhibition of human RyR2 channels requires phosphorylation of RyR2-S2808 or RyR2-S2814.

Calmodulin (CaM) is a Ca-binding protein that binds to, and can directly inhibit cardiac ryanodine receptor calcium release channels (RyR2). Animal studies have shown that RyR2 hyperphosphorylation reduces CaM binding to RyR2 in failing hearts, but data are lacking on how CaM regulates human RyR2 and how this regulation is affected by RyR2 phosphorylation. Physiological concentrations of CaM (100 nM) inhibited the diastolic activity of RyR2 isolated from failing human hearts by ~50% but had no effect on RyR2 from healthy human hearts. Using FRET between donor-FKBP12.6 and acceptor-CaM bound to RyR2, we determined that CaM binds to RyR2 from healthy human heart with a Kd = 121 ±â€¯14 nM. Ex-vivo phosphorylation/dephosphorylation experiments suggested that the divergent CaM regulation of healthy and failing human RyR2 was caused by differences in RyR2 phosphorylation by protein kinase A and Ca-CaM-dependent kinase II. Ca2+-spark measurements in murine cardiomyocytes harbouring RyR2 phosphomimetic or phosphoablated mutants at S2814 and S2808 suggest that phosphorylation of residues corresponding to either human RyR2-S2808 or S2814 is both necessary and sufficient for RyR2 regulation by CaM. Our results challenge the current concept that CaM universally functions as a canonical inhibitor of RyR2 across species. Rather, CaM's biological action on human RyR2 appears to be more nuanced, with inhibitory activity only on phosphorylated RyR2 channels, which occurs during exercise or in patients with heart failure.

Association of FK506 binding proteins with RyR channels - effect of CLIC2 binding on sub-conductance opening and FKBP binding.

Ryanodine receptor (RyR) Ca2+ channels are central to striated muscle function and influence signalling in neurons and other cell types. Beneficially low RyR activity and maximum conductance opening may be stabilised when RyRs bind to FK506 binding proteins (FKBPs) and destabilised by FKBP dissociation, with submaximal opening during RyR hyperactivity associated with myopathies and neurological disorders. However, the correlation with submaximal opening is debated and quantitative evidence is lacking. Here, we have measured altered FKBP binding to RyRs and submaximal activity with addition of wild-type (WT) CLIC2, an inhibitory RyR ligand, or its H101Q mutant that hyperactivates RyRs, which probably causes cardiac and intellectual abnormalities. The proportion of sub-conductance opening increases with WT and H101Q CLIC2 and is correlated with reduced FKBP-RyR association. The sub-conductance opening reduces RyR currents in the presence of WT CLIC2. In contrast, sub-conductance openings contribute to excess RyR 'leak' with H101Q CLIC2. There are significant FKBP and RyR isoform-specific actions of CLIC2, rapamycin and FK506 on FKBP-RyR association. The results show that FKBPs do influence RyR gating and would contribute to excess Ca2+ release in this CLIC2 RyR channelopathy.

The Anthracycline Metabolite Doxorubicinol Abolishes RyR2 Sensitivity to Physiological Changes in Luminal Ca2+ through an Interaction with Calsequestrin.

The chemotherapeutic anthracycline metabolite doxorubicinol (doxOL) has been shown to interact with and disrupt the function of the cardiac ryanodine receptor Ca2+ release channel (RyR2) in the sarcoplasmic reticulum (SR) membrane and the SR Ca2+ binding protein calsequestrin 2 (CSQ2). Normal increases in RyR2 activity in response to increasing diastolic SR [Ca2+] are influenced by CSQ2 and are disrupted in arrhythmic conditions. Therefore, we explored the action of doxOL on RyR2's response to changes in luminal [Ca2+] seen during diastole. DoxOL abolished the increase in RyR2 activity when luminal Ca2+ was increased from 0.1 to 1.5 mM. This was not due to RyR2 oxidation, but depended entirely on the presence of CSQ2 in the RyR2 complex. DoxOL binding to CSQ2 reduced both the Ca2+ binding capacity of CSQ2 (by 48%-58%) and its aggregation, and lowered CSQ2 association with the RyR2 complex by 67%-77%. Each of these effects on CSQ2, and the lost RyR2 response to changes in luminal [Ca2+], was duplicated by exposing native RyR2 channels to subphysiologic (≤1.0 µM) luminal [Ca2+]. We suggest that doxOL and low luminal Ca2+ both disrupt the CSQ2 polymer, and that the association of the monomeric protein with the RyR2 complex shifts the increase in RyR2 activity with increasing luminal [Ca2+] away from the physiologic [Ca2+] range. Subsequently, these changes may render the channel insensitive to changes of luminal Ca2+ that occur through the cardiac cycle. The altered interactions between CSQ2, triadin, and/or junctin and RyR2 may produce an arrhythmogenic substrate in anthracycline-induced cardiotoxicity.

A new cytoplasmic interaction between junctin and ryanodine receptor Ca2+ release channels.

Junctin, a non-catalytic splice variant encoded by the aspartate-ß-hydroxylase (Asph) gene, is inserted into the membrane of the sarcoplasmic reticulum (SR) Ca(2+) store where it modifies Ca(2+) signalling in the heart and skeletal muscle through its regulation of ryanodine receptor (RyR) Ca(2+) release channels. Junctin is required for normal muscle function as its knockout leads to abnormal Ca(2+) signalling, muscle dysfunction and cardiac arrhythmia. However, the nature of the molecular interaction between junctin and RyRs is largely unknown and was assumed to occur only in the SR lumen. We find that there is substantial binding of RyRs to full junctin, and the junctin luminal and, unexpectedly, cytoplasmic domains. Binding of these different junctin domains had distinct effects on RyR1 and RyR2 activity: full junctin in the luminal solution increased RyR channel activity by ∼threefold, the C-terminal luminal interaction inhibited RyR channel activity by ∼50%, and the N-terminal cytoplasmic binding produced an ∼fivefold increase in RyR activity. The cytoplasmic interaction between junctin and RyR is required for luminal binding to replicate the influence of full junctin on RyR1 and RyR2 activity. The C-terminal domain of junctin binds to residues including the S1-S2 linker of RyR1 and N-terminal domain of junctin binds between RyR1 residues 1078 and 2156.

Core skeletal muscle ryanodine receptor calcium release complex.

The core skeletal muscle ryanodine receptor (RyR1) calcium release complex extends through three compartments of the muscle fibre, linking the extracellular environment through the cytoplasmic junctional gap to the lumen of the internal sarcoplasmic reticulum (SR) calcium store. The protein complex is essential for skeletal excitation-contraction (EC)-coupling and skeletal muscle function. Its importance is highlighted by perinatal death if any one of the EC-coupling components are missing and by myopathies associated with mutation of any of the proteins. The proteins essential for EC-coupling include the DHPR α1S subunit in the transverse tubule membrane, the DHPR ß1a subunit in the cytosol and the RyR1 ion channel in the SR membrane. The other core proteins are triadin and junctin and calsequestrin, associated mainly with SR. These SR proteins are not essential for survival but exert structural and functional influences that modify the gain of EC-coupling and maintain normal muscle function. This review summarises our current knowledge of the individual protein/protein interactions within the core complex and their overall contribution to EC-coupling. We highlight significant areas that provide a continuing challenge for the field. Additional important components of the Ca2+ release complex, such as FKBP12, calmodulin, S100A1 and Stac3 are identified and reviewed elsewhere.

Cardiac ryanodine receptor activation by a high Ca²âº store load is reversed in a reducing cytoplasmic redox environment.

Here, we report the impact of redox potential on isolated cardiac ryanodine receptor (RyR2) channel activity and its response to physiological changes in luminal [Ca(2+)]. Basal leak from the sarcoplasmic reticulum is required for normal Ca(2+) handling, but excess diastolic Ca(2+) leak attributed to oxidative stress is thought to lower the threshold of RyR2 for spontaneous sarcoplasmic reticulum Ca(2+) release, thus inducing arrhythmia in pathological situations. Therefore, we examined the RyR2 response to luminal [Ca(2+)] under reducing or oxidising cytoplasmic redox conditions. Unexpectedly, as luminal [Ca(2+)] increased from 0.1 to 1.5 mM, RyR2 activity declined when pretreated with cytoplasmic 1 mM DTT or buffered with GSH∶GSSG to a normal reduced cytoplasmic redox potential (-220 mV). Conversely, with 20 µM cytoplasmic 4,4'-DTDP or buffering of the redox potential to an oxidising value (-180 mV), RyR2 activity increased with increasing luminal [Ca(2+)]. The luminal redox potential was constant at -180 mV in each case. These responses to luminal [Ca(2+)] were maintained with cytoplasmic 2 mM Na2ATP or 5 mM MgATP (1 mM free Mg(2+)). Overall, the results suggest that the redox potential in the RyR2 junctional microdomain is normally more oxidised than that of the bulk cytoplasm.

Multiple actions of phi-LITX-Lw1a on ryanodine receptors reveal a functional link between scorpion DDH and ICK toxins.

We recently reported the isolation of a scorpion toxin named U1-liotoxin-Lw1a (U1-LITX-Lw1a) that adopts an unusual 3D fold termed the disulfide-directed hairpin (DDH) motif, which is the proposed evolutionary structural precursor of the three-disulfide-containing inhibitor cystine knot (ICK) motif found widely in animals and plants. Here we reveal that U1-LITX-Lw1a targets and activates the mammalian ryanodine receptor intracellular calcium release channel (RyR) with high (fM) potency and provides a functional link between DDH and ICK scorpion toxins. Moreover, U1-LITX-Lw1a, now described as ϕ-liotoxin-Lw1a (ϕ-LITX-Lw1a), has a similar mode of action on RyRs as scorpion calcines, although with significantly greater potency, inducing full channel openings at lower (fM) toxin concentrations whereas at higher pM concentrations increasing the frequency and duration of channel openings to a submaximal state. In addition, we show that the C-terminal residue of ϕ-LITX-Lw1a is crucial for the increase in full receptor openings but not for the increase in receptor subconductance opening, thereby supporting the two-binding-site hypothesis of scorpion toxins on RyRs. ϕ-LITX-Lw1a has potential both as a pharmacological tool and as a lead molecule for the treatment of human diseases that involve RyRs, such as malignant hyperthermia and polymorphic ventricular tachycardia.

Adverse effects of doxorubicin and its metabolic product on cardiac RyR2 and SERCA2A.

The use of anthracycline chemotherapeutic drugs is restricted owing to potentially fatal cardiotoxic side effects. It has been hypothesized that anthracycline metabolites have a primary role in this cardiac dysfunction; however, information on the molecular interactions of these compounds in the heart is scarce. Here we provide novel evidence that doxorubicin and its metabolite, doxorubicinol, bind to the cardiac ryanodine receptor (RyR2) and to the sarco/endoplasmic reticulum Ca(2+) ATPase (SERCA2A) and deleteriously alter their activity. Both drugs (0.01 µM-2.5 µM) activated single RyR2 channels, and this was reversed by drug washout. Both drugs caused a secondary inhibition of RyR2 activity that was not reversed by drug washout. Preincubation with the reducing agent dithiothreitol (DTT, 1 mM) prevented drug-induced inhibition of channel activity. Doxorubicin and doxorubicinol reduced the abundance of thiol groups on RyR2, further indicating that oxidation reactions may be involved in the actions of the compounds. Ca(2+) uptake into sarcoplasmic reticulum vesicles by SERCA2A was inhibited by doxorubicinol, but not doxorubicin. Unexpectedly, in the presence of DTT, doxorubicinol enhanced the rate of Ca(2+) uptake by SERCA2A. Together the evidence provided here shows that doxorubicin and doxorubicinol interact with RyR2 and SERCA2A in similar ways, but that the metabolite acts with greater efficacy than the parent compound. Both compounds modify RyR2 and SERCA2A activity by binding to the proteins and also act via thiol oxidation to disrupt SR Ca(2+) handling. These actions would have severe consequences on cardiomyocyte function and contribute to clinical symptoms of acute anthracycline cardiotoxicity.

Proteins within the intracellular calcium store determine cardiac RyR channel activity and cardiac output.

SUMMARY: The contractile function of the heart requires the release of Ca(2+) from intracellular Ca(2+) stores in the sarcoplasmic reticulum (SR) of cardiac muscle cells. The efficacy of Ca(2+) release depends on the amount of Ca(2+) loaded into the Ca(2+) store and the way in which this 'Ca(2+) load' influences the activity of the cardiac ryanodine receptor Ca(2+) release channel (RyR2). The effects of the Ca(2+) load on Ca(2+) release through RyR2 are facilitated by: (i) the sensitivity of RyR2 itself to luminal Ca(2+) concentrations; and (ii) interactions between the cardiac Ca(2+) -binding protein calsequestrin (CSQ) 2 and RyR2, transmitted through the 'anchoring' proteins junctin and/or triadin. Mutations in RyR2 are linked to catecholaminergic polymorphic ventricular tachycardia (CPVT) and sudden cardiac death. The tachycardia is associated with changes in the sensitivity of RyR2 to luminal Ca(2+) . Triadin-, junctin- or CSQ-null animals survive, but their longevity and ability to tolerate stress is compromised. These studies reveal the importance of the proteins in normal muscle function, but do not reveal the molecular nature of their functional interactions, which must be defined before changes in the proteins leading to CPVT and heart disease can be understood. Herein, we discuss known interactions between the RyR, triadin, junctin and CSQ with emphasis on the cardiac isoforms of the proteins. Where there is little known about the cardiac isoforms, we discuss evidence from skeletal isoforms.

Short-Term Very High Carbohydrate Diet and Gut-Training Have Minor Effects on Gastrointestinal Status and Performance in Highly Trained Endurance Athletes.

We implemented a multi-pronged strategy (MAX) involving chronic (2 weeks high carbohydrate [CHO] diet + gut-training) and acute (CHO loading + 90 g·h−1 CHO during exercise) strategies to promote endogenous and exogenous CHO availability, compared with strategies reflecting lower ranges of current guidelines (CON) in two groups of athletes. Nineteen elite male race walkers (MAX: 9; CON:10) undertook a 26 km race-walking session before and after the respective interventions to investigate gastrointestinal function (absorption capacity), integrity (epithelial injury), and symptoms (GIS). We observed considerable individual variability in responses, resulting in a statistically significant (p < 0.001) yet likely clinically insignificant increase (Δ 736 pg·mL−1) in I-FABP after exercise across all trials, with no significant differences in breath H2 across exercise (p = 0.970). MAX was associated with increased GIS in the second half of the exercise, especially in upper GIS (p < 0.01). Eighteen highly trained male and female distance runners (MAX: 10; CON: 8) then completed a 35 km run (28 km steady-state + 7 km time-trial) supported by either a slightly modified MAX or CON strategy. Inter-individual variability was observed, without major differences in epithelial cell intestinal fatty acid binding protein (I-FABP) or GIS, due to exercise, trial, or group, despite the 3-fold increase in exercise CHO intake in MAX post-intervention. The tight-junction (claudin-3) response decreased in both groups from pre- to post-intervention. Groups achieved a similar performance improvement from pre- to post-intervention (CON = 39 s [95 CI 15−63 s]; MAX = 36 s [13−59 s]; p = 0.002). Although this suggests that further increases in CHO availability above current guidelines do not confer additional advantages, limitations in our study execution (e.g., confounding loss of BM in several individuals despite a live-in training camp environment and significant increases in aerobic capacity due to intensified training) may have masked small differences. Therefore, athletes should meet the minimum CHO guidelines for training and competition goals, noting that, with practice, increased CHO intake can be tolerated, and may contribute to performance outcomes.

The ß(1a) subunit of the skeletal DHPR binds to skeletal RyR1 and activates the channel via its 35-residue C-terminal tail.

Although it has been suggested that the C-terminal tail of the ß(1a) subunit of the skeletal dihyropyridine receptor (DHPR) may contribute to voltage-activated Ca(2+) release in skeletal muscle by interacting with the skeletal ryanodine receptor (RyR1), a direct functional interaction between the two proteins has not been demonstrated previously. Such an interaction is reported here. A peptide with the sequence of the C-terminal 35 residues of ß(1a) bound to RyR1 in affinity chromatography. The full-length ß(1a) subunit and the C-terminal peptide increased [(3)H]ryanodine binding and RyR1 channel activity with an AC(50) of 450-600 pM under optimal conditions. The effect of the peptide was dependent on cytoplasmic Ca(2+), ATP, and Mg(2+) concentrations. There was no effect of the peptide when channel activity was very low as a result of Mg(2+) inhibition or addition of 100 nM Ca(2+) (without ATP). Maximum increases were seen with 1-10 µM Ca(2+), in the absence of Mg(2+) inhibition. A control peptide with the C-terminal 35 residues in a scrambled sequence did not bind to RyR1 or alter [(3)H]ryanodine binding or channel activity. This high-affinity in vitro functional interaction between the C-terminal 35 residues of the DHPR ß(1a) subunit and RyR1 may support an in vivo function of ß(1a) during voltage-activated Ca(2+) release.

Multiple actions of the anthracycline daunorubicin on cardiac ryanodine receptors.

Our aim was to examine the molecular basis for acute effects of the anthracycline daunorubicin on cardiac ryanodine receptor (RyR2) channels and cardiac calsequestrin (CSQ2). Cardiotoxic effects of anthracyclines preclude their chemotherapeutic use in patients with pre-existing heart conditions. To address this significant problem, the mechanisms of anthracycline toxicity must be defined but at present are poorly understood. RyR2 channel activity was assessed by measuring Ca²âº release from cardiac sarcoplasmic reticulum vesicles and by examining single RyR2 channels inserted into artificial lipid bilayers. We show that 0.5 to 10 µM daunorubicin increases the activity of RyR2 channels after 5 to 10 min and that activity then declines to very low levels when channels are exposed to daunorubicin concentrations of ≥ 2.5 µM for a further 10 to 20 min. Extensive dissection of these effects shows for the first time that the activation results from a redox-independent binding of daunorubicin to the RyR2 complex. Novel data include the demonstration of daunorubicin binding to RyR2. We provide compelling evidence that RyR2 channel inhibition is due to the oxidation of free SH groups. The oxidation reaction is prevented by the presence of 1 mM dithiothreitol. We also present novel data showing that CSQ2 modifies the response of RyR2 to daunorubicin, but that the response of RyR2 is not dependent on daunorubicin binding to CSQ2. We suggest that binding of daunorubicin to RyR2 and CSQ2, and oxidation of RyR2, are all likely to contribute to anthracycline-induced cardiotoxicity during chemotherapy.

Quantification of calsequestrin 2 (CSQ2) in sheep cardiac muscle and Ca2+-binding protein changes in CSQ2 knockout mice.

Calsequestrin 2 (CSQ2) is generally regarded as the primary Ca2+-buffering molecule present inside the sarcoplasmic reticulum (SR) in cardiac cells, but findings from CSQ2 knockout experiments raise major questions about its role and necessity. This study determined the absolute amount of CSQ2 present in cardiac ventricular muscle to gauge its likely influence on SR free Ca2+ concentration ([Ca2+]) and maximal Ca2+ capacity. Ventricular tissue from hearts of freshly killed sheep was examined by SDS-PAGE without any fractionation, and CSQ2 was detected by Western blotting; this method avoided the >90% loss of CSQ2 occurring with usual fractionation procedures. Band intensities were compared against those for purified CSQ2 run on the same blots. Fidelity of quantification was verified by demonstrating that CSQ2 added to homogenates was detected with equal efficacy as purified CSQ2 alone. Ventricular tissue from sheep (n=8) contained 24±2 µmol CSQ2/kg wet wt. Total Ca2+ content of the ventricular tissue, measured by atomic absorption spectroscopy, was 430±20 µmol/kg (with SR Ca2+ likely<250 µmol/kg) and displayed a linear correlation with CSQ2 content, with gradient of ∼10 Ca2+ per CSQ2. The large amount of CSQ2 bestows the SR with a high theoretical maximal Ca2+-binding capacity (∼1 mmol Ca2+/kg ventricular tissue, assuming a maximum of ∼40 Ca2+ per CSQ2) and would keep free [Ca2+] within the SR relatively low, energetically favoring Ca2+ uptake and reducing SR leak. In mice with CSQ2 ablated, histidine-rich Ca2+-binding protein was upregulated ∼35% in ventricular tissue, possibly in compensation.

Running at Increasing Intensities in the Heat Induces Transient Gut Perturbations.

PURPOSE: The risk of exercise-induced endotoxemia is increased in the heat and is primarily attributable to changes in gut permeability resulting in the translocation of lipopolysaccharides (LPS) into the circulation. The purpose of this study was to quantify the acute changes in gut permeability and LPS translocation during submaximal continuous and high-intensity interval exercise under heat stress. METHODS: A total of 12 well-trained male runners (age 37 [7] y, maximal oxygen uptake [VO2max] 61.0 [6.8] mL·min-1·kg-1) undertook 2 treadmill runs of 2 × 15-minutes at 60% and 75% VO2max and up to 8 × 1-minutes at 95% VO2max in HOT (34°C, 68% relative humidity) and COOL (18°C, 57% relative humidity) conditions. Venous blood samples were collected at the baseline, following each running intensity, and 1 hour postexercise. Blood samples were analyzed for markers of intestinal permeability (LPS, LPS binding protein, and intestinal fatty acid-binding protein). RESULTS: The increase in LPS binding protein following each exercise intensity in the HOT condition was 4% (5.3 µg·mL-1, 2.4-8.4; mean, 95% confidence interval, P < .001), 32% (4.6 µg·mL-1, 1.8-7.4; P = .002), and 30% (3.0 µg·mL-1, 0.03-5.9; P = .047) greater than in the COOL condition. LPS was 69% higher than baseline following running at 75% VO2max in the HOT condition (0.2 endotoxin units·mL-1, 0.1-0.4; P = .011). Intestinal fatty acid-binding protein increased 43% (2.1 ng·mL-1, 0.1-4.2; P = .04) 1 hour postexercise in HOT compared with the COOL condition. CONCLUSIONS: Small increases in LPS concentration during exercise in the heat and subsequent increases in intestinal fatty acid-binding protein and LPS binding protein indicate a capacity to tolerate acute, transient intestinal disturbance in well-trained endurance runners.

Sarcoplasmic reticulum calcium mishandling: central tenet in heart failure?

Excitation-contraction coupling links excitation of the sarcolemmal surface membrane to mechanical contraction. In the heart this link is established via a Ca2+-induced Ca2+ release process, which, following sarcolemmal depolarisation, prompts Ca2+ release from the sarcoplasmic reticulum (SR) though the ryanodine receptor (RyR2). This substantially raises the cytoplasmic Ca2+ concentration to trigger systole. In diastole, Ca2+ is removed from the cytoplasm, primarily via the sarcoplasmic-endoplasmic reticulum Ca2+-dependent ATPase (SERCA) pump on the SR membrane, returning Ca2+ to the SR store. Ca2+ movement across the SR is thus fundamental to the systole/diastole cycle and plays an essential role in maintaining cardiac contractile function. Altered SR Ca2+ homeostasis (due to disrupted Ca2+ release, storage, and reuptake pathways) is a central tenet of heart failure and contributes to depressed contractility, impaired relaxation, and propensity to arrhythmia. This review will focus on the molecular mechanisms that underlie asynchronous Ca2+ cycling around the SR in the failing heart. Further, this review will illustrate that the combined effects of expression changes and disruptions to RyR2 and SERCA2a regulatory pathways are critical to the pathogenesis of heart failure.

Calsequestrin content and SERCA determine normal and maximal Ca2+ storage levels in sarcoplasmic reticulum of fast- and slow-twitch fibres of rat.

Whilst calsequestrin (CSQ) is widely recognized as the primary Ca2+ buffer in the sarcoplasmic reticulum (SR) in skeletal muscle fibres, its total buffering capacity and importance have come into question. This study quantified the absolute amount of CSQ isoform 1 (CSQ1, the primary isoform) present in rat extensor digitorum longus (EDL) and soleus fibres, and related this to their endogenous and maximal SR Ca2+ content. Using Western blotting, the entire constituents of minute samples of muscle homogenates or segments of individual muscle fibres were compared with known amounts of purified CSQ1. The fidelity of the analysis was proven by examining the relative signal intensity when mixing muscle samples and purified CSQ1. The CSQ1 contents of EDL fibres, almost exclusively type II fibres, and soleus type I fibres [SOL (I)] were, respectively, 36 +/- 2 and 10 +/- 1 micromol (l fibre volume)(-1), quantitatively accounting for the maximal SR Ca2+ content of each. Soleus type II [SOL (II)] fibres (approximately 20% of soleus fibres) had an intermediate amount of CSQ1. Every SOL (I) fibre examined also contained some CSQ isoform 2 (CSQ2), which was absent in every EDL and other type II fibre except for trace amounts in one case. Every EDL and other type II fibre had a high density of SERCA1, the fast-twitch muscle sarco(endo)plasmic reticulum Ca2+-ATPase isoform, whereas there was virtually no SERCA1 in any SOL (I) fibre. Maximal SR Ca2+ content measured in skinned fibres increased with CSQ1 content, and the ratio of endogenous to maximal Ca2+ content was inversely correlated with CSQ1 content. The relative SR Ca2+ content that could be maintained in resting cytoplasmic conditions was found to be much lower in EDL fibres than in SOL (I) fibres (approximately 20 versus >60%). Leakage of Ca2+ from the SR in EDL fibres could be substantially reduced with a SR Ca2+ pump blocker and increased by adding creatine to buffer cytoplasmic [ADP] at a higher level, both results indicating that at least part of the Ca2+ leakage occurred through SERCA. It is concluded that CSQ1 plays an important role in EDL muscle fibres by providing a large total pool of releasable Ca2+ in the SR whilst maintaining free [Ca2+] in the SR at sufficiently low levels that Ca2+ leakage through the high density of SERCA1 pumps does not metabolically compromise muscle function.