Dynamic regulation of ryanodine receptor type 1 (RyR1) channel activity by Homer 1

Homer, a family of scaffolding proteins originally identified in neurons, is also expressed in skeletal muscle. Previous studies showed that splice variants of Homer 1 (H1) amplify the gain of the ryanodine receptor type 1 (RyR1) channel complex. Using [3H]ryanodine ([3H]Ry) to probe the conformational state of RyR1, the actions of long- and short-forms of H1 are examined singly and in combination. At < or =200 nM, H1 long-forms (H1b or H1c possessing coiled-coil (CC) domains) and short-forms (H1a or H1EVH1 lacking CC domains) enhance specific [3H]Ry binding to RyR1. However, at a concentration > 200 nM, either H1 form completely inhibited [3H]Ry binding. Importantly, the combinations of H1c+H1EVH1, or H1b+H1a acted in an additive manner to enhance or inhibit [3H]Ry-binding activity. H1a and H1c individually or in combination produced the same dynamic pattern in regulating purified RyR1 channels reconstituted in planar lipid bilayers. In combination, their net action on RyR1 channels depends on total concentrations of H1. These data provide a mechanism by which constitutively and transiently expressed H1 forms can tightly regulate RyR1 channel activity in response to changing levels of expression and degradation of H1 proteins.     

Personal recollections on the discovery of the ryanodine receptors of muscle

The intracellular Ca²⁺ release channels are indispensable molecular machinery in practically all eukaryotic cells of multicellular animals. They serve a key role in cell signaling by way of Ca²⁺ as a second messenger. In response to a signaling event, the channels release Ca²⁺ from intracellular stores. The resulting rise in cytoplasmic Ca²⁺ concentration triggers the cell to carry out its specialized role, after which the intracellular Ca²⁺ concentration must be reduced so that the signaling event can again be repeated. There are two types of intracellular Ca²⁺ release channels, i.e., the ryanodine receptors and the inositol triphosphate receptors. My focus in this minireview is to present a personal account, from the vantage point our laboratory, of the discovery, isolation, and characterization of the ryanodine receptors from mammalian muscle. There are three isoforms: ryanodine receptor 1 (RyR1), first isolated from rabbit fast twitch skeletal muscle; ryanodine receptor 2 (RyR2), first isolated from dog heart; and ryanodine receptor 3 (RyR3), first isolated from bovine diaphragm muscle. The ryanodine receptors are the largest channel structures known. The RyR isoforms are very similar albeit with important differences. Natural mutations in humans in these receptors have already been associated with a number of muscle diseases.     

Characterization of recombinant skeletal muscle (Ser-2843) and cardiac muscle (Ser-2809) ryanodine receptor phosphorylation mutants

Phosphorylation of the skeletal muscle (RyR1) and cardiac muscle (RyR2) ryanodine receptors has been reported to modulate channel activity. Abnormally high phosphorylation levels (hyperphosphorylation) at Ser-2843 in RyR1 and Ser-2809 in RyR2 and dissociation of FK506-binding proteins from the receptors have been implicated as one of the causes of altered calcium homeostasis observed during human heart failure. Using site-directed mutagenesis, we prepared recombinant RyR1 and RyR2 mutant receptors mimicking constitutively phosphorylated and dephosphorylated channels carrying a Ser/Asp (RyR1-S2843D and RyR2-S2809D) and Ser/Ala (RyR1-S2843A and RyR2-S2809A) substitution, respectively. Following transient expression in human embryonic kidney 293 cells, the effects of Ca2+, Mg2+, and ATP on channel function were determined using single channel and [3H]ryanodine binding measurements. In both assays, neither the skeletal nor cardiac mutants showed significant differences compared with wild type. Similarly essentially identical caffeine responses were observed in Ca2+ imaging measurements. Co-immunoprecipitation and Western blot analysis showed comparable binding of FK506-binding proteins to wild type and mutant receptors. Finally metabolic labeling experiments showed that the cardiac ryanodine receptor was phosphorylated at additional sites. Taken together, the results did not support the view that phosphorylation of a single site (RyR1-Ser-2843 and RyR2-Ser-2809) substantially changes RyR1 and RyR2 channel function.     

Effects of supplementing two levels of magnesium aspartate and transportation stress on pork quality and gene expression of micro-calpain and calpastatin of finishing pigs

The objective of this study was to investigate the effects of supplementing swine finishing diets with two levels of magnesium aspartate (MgAsp) and short-term transportation stress on blood parameters, pork quality and the mRNA abundance of p-calpain and calpastatin in muscles of finishing pigs. Thirty-six crossbred finishing pigs (mean BW 90 kg) were assigned randomly to 0, 1000, or 2000 mg supplemental Mg from MgAsp per kg of diet for five days before slaughter. Then six pigs from each dietary treatment were subjected either to no transportation stress (NTS) or 2 h of transportation stress (TS). Transportation stress resulted in higher concentrations (p < 0.01) of serum calcium, glucose and cortisol, lower pH (p < 0.01), higher Warner-Bratzler shear force (WBSF) (p < 0.05) and higher calpastatin mRNA abundance (p = 0.05) of longissimus muscle (LM) compared with NTS treatments. Supplementation of MgAsp in TS treatments increased serum Mg concentration (p < 0.05) at 2000 mg of Mg/kg, reduced drip loss (p < 0.05) and improved pork quality colour (p < 0.05) at 2000 mg of Mg/kg, and decreased 1-day and 3-day WBSF (p < 0.05) at 1000 mg of Mg/kg compared with TS treatments fed the control diet. It is concluded that supplementation of MgAsp improves water-holding capacity and pork colour, and alleviates the negative effects of transportation stress on meat tenderness.     

Thyroid hormone-induced overexpression of functional ryanodine receptors in the rabbit heart

Modifications in the Ca(2+)-uptake and -release functions of the sarcoplasmic reticulum (SR) may be a major component of the mechanisms underlying thyroid state-dependent alterations in heart rate, myocardial contractility, and metabolism. We investigated the influence of hyperthyroid state on the expression and functional properties of the ryanodine receptor (RyR), a major protein in the junctional SR (JSR), which mediates Ca(2+) release to trigger muscle contraction. Experiments were performed using homogenates and JSR vesicles derived from ventricular myocardium of euthyroid and hyperthyroid rabbits. Hyperthyroidism, with attendant cardiac hypertrophy, was induced by the injection of L-thyroxine (200 microg/kg body wt) daily for 7 days. Western blotting analysis using cardiac RyR-specific antibody revealed a significant increase (>50%) in the relative amount of RyR in the hyperthyroid compared with euthyroid rabbits. Ca(2+)-dependent, high-affinity [(3)H]ryanodine binding was also significantly greater ( approximately 40%) in JSR from hyperthyroid rabbits. The Ca(2+ )sensitivity of [(3)H]ryanodine binding and the dissociation constant for [(3)H]ryanodine did not differ significantly between euthyroid and hyperthyroid hearts. Measurement of Ca(2+)-release rates from passively Ca(2+)-preloaded JSR vesicles and assessment of the effect of RyR-Ca(2+)-release channel (CRC) blockade on active Ca(2+)-uptake rates revealed significantly enhanced (>2-fold) CRC activity in the hyperthyroid, compared with euthyroid, JSR. These results demonstrate overexpression of functional RyR in thyroid hormone-induced cardiac hypertrophy. Relative abundance of RyR may be responsible, in part, for the changes in SR Ca(2+) release, cytosolic Ca(2+) transient, and cardiac systolic function associated with thyroid hormone-induced cardiac hypertrophy.     

Expressional changes of RyR1 and RyR2 in PC12 cells after induction of apoptosis

Ca2+ released from endoplasmic reticulum through ryanodine receptors (RyRs) and inositol 1,4,5-trisphosphate receptors (IP3Rs) can trigger apoptotic or necrotic pathways in cooperation with proapoptotic and/or prosurvival proteins, as those of Bcl-2 family. In such regulatory pathways expressional modulation of these Ca2+ transporters could also be expected. Therefore, our aim was to determine the expressional changes of RyR1 and RyR2 after experimental induction of apoptosis in PC12 cells. Our results showed significant decrease of RyR1 and RyR2 expressions, while caspase-3 and Bax expression significantly increased. We conclude that induction of apoptosis in PC12 cells could result in RyR expression down regulation.     

Properties of ryanodine receptor in rat muscles submitted to unloaded conditions

Unloading of skeletal muscles by hindlimb unweighting is known to induce muscle atrophy and a shift toward faster contractile properties associated with an increase in the expression of fast contractile proteins, particularly in slow soleus muscles. Contractile properties suggest that slow soleus muscles acquire SR properties close to those of a faster one. We studied the expression and properties of the sarcoplasmic reticulum calcium release (RyR) channels in soleus and gastrocnemius muscles of rats submitted to hindlimb unloading (HU). An increase in RyR1 and a slight decrease in RyR3 expression was detected in atrophied soleus muscles only after 4 weeks of HU. No variation appeared in fast muscles. [(3)H]Ryanodine binding experiments showed that HU neither increased the affinity of the receptors for [(3)H]ryanodine nor changed the caffeine sensitivity of [(3)H]ryanodine binding. Our results suggested that not only RyR1 but also RyR3 expression can be regulated by muscle activity and innervation in soleus muscle. The changes in the RyR expression in slow fibers suggested a transformation of the SR from a slow to a fast phenotype.     

Characterization of [(3)H]ryanodine binding sites in mammalian lung

The ryanodine-sensitive calcium channels, also called ryanodine receptors, are intracellular Ca(2+)-release channels that have been shown to bind the neutral plant alkaloid ryanodine with nanomolar affinity. The activity of the skeletal muscle (RyR1), cardiac muscle (RyR2), and brain (RyR3) ryanodine receptor isoforms have been shown to be highly regulated by physiological factors including pH, temperature, and ionic strength; endogenous compounds including Ca(2+), Mg(2+), and adenosine triphosphate (ATP); and pharmacological agents including caffeine, ruthenium red, and neomycin. RyR3 is reportedly expressed in diverse tissues including lung; however, specific [(3)H]ryanodine binding sites in mammalian lung tissue have not been characterized. In this study, hamster lung ryanodine binding proteins were shown to specifically bind [(3)H]ryanodine with an affinity similar to that of RyR isoforms found in other tissues and this binding was shown to be sensitive to Ca(2+) concentration, stimulation by caffeine and spermine, and inhibition by Mg(2+), ruthenium red, and neomycin. The solubilized, intact ryanodine binding protein from hamster lung demonstrated approximately the same 30S sedimentation coefficient as RyR1 and RyR2, but a putative ryanodine receptor subunit from hamster lung was not found to cross-react with antibodies specific for the three known isoforms. We conclude that the hamster lung ryanodine binding protein demonstrates sedimentation and binding characteristics that are similar to those of the known RyR isoforms, but may exhibit antigenic dissimilarity from the typical RyR isoforms found in muscle and brain.     

Intracellular Ca2+ release through ryanodine receptors contributes to AMPA receptor-mediated mitochondrial dysfunction and ER stress in oligodendrocytes

Overactivation of ionotropic glutamate receptors in oligodendrocytes induces cytosolic Ca²⁺ overload and excitotoxic death, a process that contributes to demyelination and multiple sclerosis. Excitotoxic insults cause well-characterized mitochondrial alterations and endoplasmic reticulum (ER) dysfunction, which is not fully understood. In this study, we analyzed the contribution of ER-Ca²⁺ release through ryanodine receptors (RyRs) and inositol triphosphate receptors (IP₃Rs) to excitotoxicity in oligodendrocytes in vitro. First, we observed that oligodendrocytes express all previously characterized RyRs and IP₃Rs. Blockade of Ca²⁺-induced Ca²⁺ release by TMB-8 following α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) receptor-mediated insults attenuated both oligodendrocyte death and cytosolic Ca²⁺ overload. In turn, RyR inhibition by ryanodine reduced as well the Ca²⁺ overload whereas IP₃R inhibition was ineffective. Furthermore, AMPA-triggered mitochondrial membrane depolarization, oxidative stress and activation of caspase-3, which in all instances was diminished by RyR inhibition. In addition, we observed that AMPA induced an ER stress response as revealed by α subunit of the eukaryotic initiation factor 2α phosphorylation, overexpression of GRP chaperones and RyR-dependent cleavage of caspase-12. Finally, attenuating ER stress with salubrinal protected oligodendrocytes from AMPA excitotoxicity. Together, these results show that Ca²⁺ release through RyRs contributes to cytosolic Ca²⁺ overload, mitochondrial dysfunction, ER stress and cell death following AMPA receptor-mediated excitotoxicity in oligodendrocytes.     

Up‐regulation of the type 3 ryanodine receptor is neuroprotective in the TgCRND8 mouse model of Alzheimer’s disease

The cellular pathology of Alzheimer’s disease is progressive and protracted leading eventually to considerable neuronal death. The underlying mechanisms of the pathology are complex but changes in the control of intracellular Ca²⁺ are believed to contribute to the demise of neurons. In this study, we investigated the functional consequences of an increase in the expression of the type 3 isoform of the ryanodine receptor (RyR3). We found that although cortical neurons from TgCRND8 mice secreted significantly more amyloid beta protein and showed significantly increased RyR3 expression, they were no more sensitive to cell stress than non‐transgenic neurons. Furthermore, despite increased intracellular Ca²⁺ release in response to ryanodine, we found that basal Ca²⁺, K⁺‐evoked Ca²⁺ responses, and capacitative Ca²⁺ entry were no different in TgCRND8 neurons compared with non‐transgenic neurons. Therefore, as RyR3 up‐regulation did not affect neuronal health or global Ca²⁺ homeostasis, we investigated the effect of reducing RyR3 expression using small interfering RNA. Surprisingly, a reduction of RyR3 expression in TgCRND8, but not in non‐transgenic, neurons increased neuronal death. These data reveal a new role for RyR3 and indicate a novel potential therapeutic target to delay or prevent the progression of Alzheimer’s disease.     

Functional properties of ryanodine receptors from rat dorsal root ganglia

The properties of ryanodine receptors (RyRs) from rat dorsal root ganglia (DRGs) have been studied. The density of RyRs (Bmax) determined by [3H]ryanodine binding was 63 fmol/mg protein with a dissociation constant (Kd) of 1.5 nM. [3H]Ryanodine binding increased with caffeine, decreased with ruthenium red and tetracaine, and was insensitive to millimolar concentrations of Mg2+ or Ca2+. DRG RyRs reconstituted in planar lipid bilayers were Ca2+-dependent and displayed the classical long-lived subconductance state in response to ryanodine; however, unlike cardiac and skeletal RyRs, they lacked Ca2+-dependent inactivation. Antibodies against RyR3, but not against RyR1 or RyR2, detected DRG RyRs. Thus, DRG RyRs are immunologically related to RyR3, but their lack of divalent cation inhibition is unique among RyR subtypes.     

Expression and functional activity of ryanodine receptors (RyRs) during skeletal muscle development

Two isoforms of ryanodine receptors are expressed in skeletal muscles, RyR1 and RyR3. We investigated the relative level of expression of RyRs in developing murine skeletal muscles using [3H]ryanodine binding and immunoprecipitation experiments. In the diaphragm RyR3 accounted for 11% of total RyRs in 5-day-old mice and for 3% of total RyRs in 60-day-old mice. In hindlimb muscles, RyR3 accounted for 3% and 1% of total RyRs in 5-day-old and adult mice, respectively. The activity of RyR1 channels in native microsomal vesicles from murine muscles was found to be as low as 35% of that measured after CHAPS exposure, while no inhibition was observed for RyR3. CHAPS sensitivity of recombinant RyR1 and RyR3 expressed in HEK293 cells was also investigated. The activity of recombinant RyR1 but not RyR3 channels was found to be inhibited in native conditions, suggesting that this property may not be dependent on a muscle environment.     

Evidence for a role of C-terminus in Ca(2+) inactivation of skeletal muscle Ca(2+) release channel (ryanodine receptor).

Six chimeras of the skeletal muscle (RyR1) and cardiac muscle (RyR2) Ca(2+) release channels (ryanodine receptors) previously used to identify RyR1 dihydropyridine receptor interactions [Nakai et al. (1998) J. Biol. Chem. 273, 13403] were expressed in HEK293 cells to assess their Ca(2+) dependence in [(3)H]ryanodine binding and single channel measurements. The results indicate that the C-terminal one-fourth has a major role in Ca(2+) activation and inactivation of RyR1. Further, our results show that replacement of RyR1 regions with corresponding RyR2 regions can result in loss and/or reduction of [(3)H]ryanodine binding affinity while maintaining channel activity.     

Expression levels of RyR1 and RyR3 control resting free Ca2+ in skeletal muscle

To better understand the role of the transient expression of ryanodine receptor (RyR) type 3 (RyR3) on Ca²⁺ homeostasis during the development of skeletal muscle, we have analyzed the effect of expression levels of RyR3 and RyR1 on the overall physiology of cultured myotubes and muscle fibers. Dyspedic myotubes were infected with RyR1 or RyR3 containing virions at 0.2, 0.4, 1.0, and 4.0 moieties of infection (MOI), and analysis of their pattern of expression, caffeine sensitivity, and resting free Ca²⁺ concentration ([Ca²⁺]_r) was performed. Although increased MOI resulted in increased expression of each receptor isoform, it did not significantly affect the immunopattern of RyRs or the expression levels of calsequestrin, triadin, or FKBP-12. Interestingly, myotubes expressing RyR3 always had significantly higher [Ca²⁺]_r and lower caffeine EC₅₀ than did cells expressing RyR1. Although some of the increased sensitivity of RyR3 to caffeine could be attributed to the higher [Ca²⁺]_r in RyR3-expressing cells, studies of [³H]ryanodine binding demonstrated intrinsic differences in caffeine sensitivity between RyR1 and RyR3. Tibialis anterior (TA) muscle fibers at different stages of postnatal development exhibited a transient increase in [Ca²⁺]_r coordinately with their level of RyR3 expression. Similarly, adult soleus fibers, which also express RyR3, had higher [Ca²⁺]_r than did adult TA fibers, which exclusively express RyR1. These data show that in skeletal muscle, RyR3 increases [Ca²⁺]_r more than RyR1 does at any expression level. These data suggest that the coexpression of RyR1 and RyR3 at different levels may constitute a novel mechanism by which to regulate [Ca²⁺]_r in skeletal muscle. ryanodine receptor; calcium release; ryanodine binding; muscle fibers     

Type-3 ryanodine receptors mediate hypoxia-, but not neurotransmitter-induced calcium release and contraction in pulmonary artery smooth muscle cells

In this study we examined the expression of RyR subtypes and the role of RyRs in neurotransmitter- and hypoxia-induced Ca2+ release and contraction in pulmonary artery smooth muscle cells (PASMCs). Under perforated patch clamp conditions, maximal activation of RyRs with caffeine or inositol triphosphate receptors (IP3Rs) with noradrenaline induced equivalent increases in [Ca2+]i and Ca2+-activated Cl- currents in freshly isolated rat PASMCs. Following maximal IP3-induced Ca2+ release, neither caffeine nor chloro-m-cresol induced a response, whereas prior application of caffeine or chloro-m-cresol blocked IP3-induced Ca2+ release. In cultured human PASMCs, which lack functional expression of RyRs, caffeine failed to affect ATP-induced increases in [Ca2+]i in the presence and absence of extracellular Ca2+. The RyR antagonists ruthenium red, ryanodine, tetracaine, and dantrolene greatly inhibited submaximal noradrenaline- and hypoxia-induced Ca2+ release and contraction in freshly isolated rat PASMCs, but did not affect ATP-induced Ca2+ release in cultured human PASMCs. Real-time quantitative RT-PCR and immunofluorescence staining indicated similar expression of all three RyR subtypes (RyR1, RyR2, and RyR3) in freshly isolated rat PASMCs. In freshly isolated PASMCs from RyR3 knockout (RyR3-/-) mice, hypoxia-induced, but not submaximal noradrenaline-induced, Ca2+ release and contraction were significantly reduced. Ruthenium red and tetracaine can further inhibit hypoxic increase in [Ca2+]i in RyR3-/- mouse PASMCs. Collectively, our data suggest that (a) RyRs play an important role in submaximal noradrenaline- and hypoxia-induced Ca2+ release and contraction; (b) all three subtype RyRs are expressed; and (c) RyR3 gene knockout significantly inhibits hypoxia-, but not submaximal noradrenaline-induced Ca2+ and contractile responses in PASMCs.     

Characterization of RyR1-slow, a ryanodine receptor specific to slow-twitch skeletal muscle

Two distinct skeletal muscle ryanodine receptors (RyR1s) are expressed in a fiber type-specific manner in fish skeletal muscle (11). In this study, we compare [(3)H]ryanodine binding and single channel activity of RyR1-slow from fish slow-twitch skeletal muscle with RyR1-fast and RyR3 isolated from fast-twitch skeletal muscle. Scatchard plots indicate that RyR1-slow has a lower affinity for [(3)H]ryanodine when compared with RyR1-fast. In single channel recordings, RyR1-slow and RyR1-fast had similar slope conductances. However, the maximum open probability (P(o)) of RyR1-slow was threefold less than the maximum P(o) of RyR1-fast. Single channel studies also revealed the presence of two populations of RyRs in tuna fast-twitch muscle (RyR1-fast and RyR3). RyR3 had the highest P(o) of all the RyR channels and displayed less inhibition at millimolar Ca(2+). The addition of 5 mM Mg-ATP or 2.5 mM beta, gamma-methyleneadenosine 5'-triphosphate (AMP-PCP) to the channels increased the P(o) and [(3)H]ryanodine binding of both RyR1s but also caused a shift in the Ca(2+) dependency curve of RyR1-slow such that Ca(2+)-dependent inactivation was attenuated. [(3)H]ryanodine binding data also showed that Mg(2+)-dependent inhibition of RyR1-slow was reduced in the presence of AMP-PCP. These results indicate differences in the physiological properties of RyRs in fish slow- and fast-twitch skeletal muscle, which may contribute to differences in the way intracellular Ca(2+) is regulated in these muscle types.     

Interplay between ryanodine and IP3 receptors in ATP-stimulated mouse luteinized-granulosa cells

In mouse luteinized-granulosa cells (MGLC), ATP induces an increase in intracellular Ca2+ concentration by stimulating phospholipase C (PLC) associated with purinergic receptors, leading to production of inositol 1,4,5-trisphosphate (IP3) and subsequent release of Ca2+ from intracellular stores. In this study, we examined the cross-talk between the ryanodine receptors (RyR) and IP3 receptors (IP3R) in response to ATP in MGLC. Specifically, the effect of RyR modulators on ATP response was examined. The results showed that ATP-induced intracellular calcium elevation was abolished by inhibitors of the RyR, such as dantrolene (25 microM) and ryanodine (80 microM). When the MGLC were stimulated with activators of RyR, 2 microM ryanodine and 10 mM caffeine, the ATP-elicited response was decreased. These actions were independent of IP3 production stimulated by ATP. Hence, ATP-induced intracellular Ca2+ mobilization involves the coordinated action of both types of calcium release channels (CRCs). Using fluorescent probes, it was shown that IP3R is uniformly distributed throughout the cell; in contrast, RyR is mainly found around the nuclei. It is concluded that the IP3R and the RyR are functionally associated, and both play a role in the pattern of Ca2+ increase observed during purinergic stimulation of MGLC. This coupling may provide a highly efficient amplification mechanism for ATP stimulation of Ca2+ mobilization.     

Pigs' aggressive temperament affects pre-slaughter mixing aggression, stress and meat quality

Pre-slaughter stress has a negative impact on animal welfare and on meat quality. Aggressive behaviour when pigs are mixed together for transportation to, or on arrival at, the abattoir is an important factor in pre-slaughter stress. Aggressiveness of pigs varies between individuals in the population, and this study investigated its effects on stress and meat quality at slaughter. We mixed pigs at a young age to identify individuals of high (H) or low (L) aggressive temperament using the previously validated approach of lesion scoring. To contrast extremes of social stress single-sex groups of eight pigs were mixed according to their aggressiveness in HH, HL or LL combinations or left unmixed (U) prior to transport and slaughter (n = 271). Each treatment was replicated in at least two groups in each of four slaughter batches. Mixing per se had little effect, but mixed groups composed of aggressive pigs (HH) had more carcass skin lesions and higher levels of plasma cortisol at slaughter and had loin muscle samples with higher pH at 24 h, and lower redness (a*) and yellowness (b*) compared to the other treatments. Females had higher levels of plasma cortisol at slaughter, a more rapid decline in pH post-slaughter and greater lean content of meat. Lactate and creatine kinase (CK) levels and meat pH were affected by the interaction of sex and treatment. Genetic factors, dam and sire line composition, and halothane locus (ryanodine receptor 1, RYR1) genotype, also affected a number of production and meat quality parameters as expected. Additionally, 'commercially normal' levels of social stress were studied in four further slaughter batches with no manipulation of group composition (n = 313). In these pigs, the proportion of unfamiliar pigs and group size of lairage groups explained limited variation in lesion scores at slaughter, but earlier aggressiveness did not. High numbers of skin lesions on the carcass were associated with high levels of cortisol and lactate and low glucose at slaughter, but not with meat quality measures. When stress and meat quality measures were compared for all pigs, high lactate was associated with low early pH and high drip loss, while high cortisol and CK were associated with high pH at 24 h and changes in meat colour. In conclusion, mixing pigs of above average aggressiveness resulted in greater aggression and stress, and changes in meat quality parameters, consistent with the effects of pre-slaughter stress on muscle chemistry.     

Ryanodine receptor type-1 (RyR1) expression and protein S-nitrosylation pattern in human soleus myofibres following bed rest and exercise countermeasure

The ryanodine receptor type-I (RyR1) is one key player of the excitation-contraction coupling (E-CC) machinery. However, RyR1 expression in human skeletal muscle disuse and plasticity changes are not well documented. We studied the expression and the functional modifications of RyR1 following prolonged bed rest (BR) without and with exercise countermeasure (Resistive Vibration Exercise, RVE). Soleus biopsies were taken from a non-trained control (BR-CTRL) and trained (BR-RVE) group (each n = 10) before and after BR. In BR-CTRL group, a fibre type-specific immunopattern of RyR1 (type-I < type-II) was documented, and RyR1 immunofluorescence intensity and protein expression together with [(3)H]ryanodine binding were decreased after BR. In BR-RVE group, RyR1 immunosignals were increased and fiber type specificity was no longer present. RyR1 protein expression was unchanged, whereas [(3)H]ryanodine binding increased after BR. Confocal and biochemical analysis confirmed subcellular co-localisation and protein-protein interaction of RyR1 with nitric oxide (NO)-synthase type-1 (NOS1). S-nitrosylation of RyR1 was increased in BR-CTRLpost only, suggesting a reduction of RyR1 open channel probability by nitrosylation mechanisms following prolonged disuse. We conclude that following extended body deconditioning in bed rest, RVE countermeasure maintained normal RyR1 expression and nitrosylation patterns required for adequate E-CC in human performance control.     

Functional expression of recombinant type 1 ryanodine receptor in insect cells

We have investigated the biochemical properties of the rabbit ryanodine receptor type 1 (RyR1) from skeletal muscle functionally expressed in insect sf 21 cells infected with recombinant baculovirus. Equilibrium [3H]ryanodine binding assays applied to total membrane fractions from sf 21 cells expressing recombinant RyR1 showed a non-hyperbolic saturation curve (Hill coefficient = 2.1). The [3H]ryanodine binding was enhanced by 1 mM AMP-PCP and 10 mM caffeine, whereas 10 mM Mg(2+) and 5 microM ruthenium red reduced the specific binding. The dependence of [3H]ryanodine binding on ionic strength showed positive cooperativity (Hill coefficient = 2.2) with a plateau at 1 M KCl. The recombinant RyR1 showed a bell-shaped [3H]ryanodine binding curve when free [Ca(2+)] was increased, with an optimal concentration around 100 microM.Confocal microscopy studies using the Ca(2+) ATPase selective inhibitor, thapsigargin coupled to fluorescein and ryanodine coupled to Texas red demonstrated that the recombinant RyR1 and the Ca(2+) ATPase co-localize to the same intracellular membrane. No significant RyR1 fluorescence was observed at the plasma membrane.Fluo-4-loaded sf 21 cells expressing recombinant RyR1 responded to activating-low ryanodine concentrations (100 nM) or caffeine (10 mM) with a sharp rise in intracellular Ca2 followed by a sustained phase, in contrast, sf 21 cells expressing the human bradykinin type 2 receptor did not respond to ryanodine or caffeine.These results demonstrate the expression of recombinant RyR1 in sf 21 cells with functional properties similar to what has been previously reported for native RyR1 in mammalian tissues, however, some differences were observed in [3H]ryanodine binding assays compared to native rabbit RyR1. Hence, the baculovirus expression system provides a generous source of protein to accomplish structure-function studies and an excellent model to assess functional properties of wild type and mutant RyR1.