Speg interactions that regulate the stability of excitation-contraction coupling protein complexes in triads and dyads.

Here we show that striated muscle preferentially expressed protein kinase α (Spegα) maintains cardiac function in hearts with Spegß deficiency. Speg is required for stability of excitation-contraction coupling (ECC) complexes and interacts with esterase D (Esd), Cardiomyopathy-Associated Protein 5 (Cmya5), and Fibronectin Type III and SPRY Domain Containing 2 (Fsd2) in cardiac and skeletal muscle. Mice with a sequence encoding a V5/HA tag inserted into the first exon of the Speg gene (HA-Speg mice) display a >90% decrease in Spegß but Spegα is expressed at ~50% of normal levels. Mice deficient in both Spegα and Speg ß (Speg KO mice) develop a severe dilated cardiomyopathy and muscle weakness and atrophy, but HA-Speg mice display mild muscle weakness with no cardiac involvement. Spegα in HA-Speg mice suppresses Ca2+ leak, proteolytic cleavage of Jph2, and disruption of transverse tubules. Despite it's low levels, HA-Spegß immunoprecipitation identified Esd, Cmya5 and Fsd2 as Spegß binding partners that localize to triads and dyads to stabilize ECC complexes. This study suggests that Spegα and Spegß display functional redundancy, identifies Esd, Cmya5 and Fsd2 as components of both cardiac dyads and skeletal muscle triads and lays the groundwork for the identification of new therapeutic targets for centronuclear myopathy.

Pathological mechanisms of vacuolar aggregate myopathy arising from a Casq1 mutation.

Mice with a mutation (D244G, DG) in calsequestrin 1 (CASQ1), analogous to a human mutation in CASQ1 associated with a delayed onset human myopathy (vacuolar aggregate myopathy), display a progressive myopathy characterized by decreased activity, decreased ability of fast twitch muscles to generate force and low body weight after one year of age. The DG mutation causes CASQ1 to partially dissociate from the junctional sarcoplasmic reticulum (SR) and accumulate in the endoplasmic reticulum (ER). Decreased junctional CASQ1 reduces SR Ca2+ release. Muscles from older DG mice display ER stress, ER expansion, increased mTOR signaling, inadequate clearance of aggregated proteins by the proteasomes, and elevation of protein aggregates and lysosomes. This study suggests that the myopathy associated with the D244G mutation in CASQ1 is driven by CASQ1 mislocalization, reduced SR Ca2+ release, CASQ1 misfolding/aggregation and ER stress. The subsequent maladaptive increase in protein synthesis and decreased protein aggregate clearance are likely to contribute to disease progression.

Untargeted metabolomics profiling of skeletal muscle samples from malignant hyperthermia susceptible patients.

PURPOSE: Malignant hyperthermia (MH) is a potentially fatal hypermetabolic condition triggered by certain anesthetics and caused by defective calcium homeostasis in skeletal muscle cells. Recent evidence has revealed impairment of various biochemical pathways in MH-susceptible patients in the absence of anesthetics. We hypothesized that clinical differences between MH-susceptible and control individuals are reflected in measurable differences in myoplasmic metabolites. METHODS: We performed metabolomic profiling of skeletal muscle samples from MH-negative (control) individuals and MH-susceptible patients undergoing muscle biopsy for diagnosis of MH susceptibility. Cellular metabolites were extracted from 33 fresh and 87 frozen human muscle samples using solid phase microextraction and Metabolon® untargeted biochemical profiling platforms, respectively. Ultra-performance liquid chromatography-high resolution mass spectrometry was used for metabolite identification and validation, followed by analysis of differences in metabolites between the MH-susceptible and MH-negative groups. RESULTS: Significant fold-change differences between the MH-susceptible and control groups in metabolites from various pathways were found (P value range: 0.009 to < 0.001). These included accumulation of long chain acylcarnitines, diacylglycerols, phosphoenolpyruvate, histidine pathway metabolites, lysophosphatidylcholine, oxidative stress markers, and phosphoinositols, as well as decreased levels of monoacylglycerols. The results from both analytical platforms were in agreement. CONCLUSION: This metabolomics study indicates a shift from utilization of carbohydrates towards lipids for energy production in MH-susceptible individuals. This shift may result in inefficiency of beta-oxidation, and increased muscle protein turnover, oxidative stress, and/or lysophosphatidylcholine levels.

RéSUMé: OBJECTIF : L'hyperthermie maligne (HM) est une condition hypermétabolique potentiellement mortelle déclenchée par certains agents anesthésiques et causée par une homéostasie calcique perturbée des cellules musculaires squelettiques. Des données probantes récentes ont mis en lumière une atteinte de diverses voies biochimiques chez les patients susceptibles à l'HM en l'absence d'anesthésiques. Nous avons émis l'hypothèse que les différences cliniques entre les individus susceptibles à l'HM et des témoins se refléteraient dans des différences mesurables de métabolites myoplasmiques. MéTHODE : Nous avons réalisé un profilage métabolomique d'échantillons de muscles squelettiques provenant de personnes négatives à l'HM (témoins) et de patients susceptibles à l'HM subissant une biopsie musculaire dans le but de poser un diagnostic de susceptibilité à l'HM. Les métabolites cellulaires ont été extraits de 33 échantillons de muscles humains frais et de 87 échantillons congelés à l'aide d'une microextraction en phase solide et des plateformes de profilage biochimique non ciblées Metabolon®, respectivement. La chromatographie en phase liquide à haute performance et la spectrométrie de masse à haute résolution ont été utilisées pour l'identification et la validation des métabolites, puis suivies d'une analyse des différences dans les métabolites entre les groupes susceptibles à l'HM et les groupes négatifs à l'HM. RéSULTATS : Des différences significatives ont été observées entre les groupes susceptibles à l'HM et les groupes témoins dans les métabolites issus de diverses voies (P : de 0,009 à < 0,001). Ces différences comprenaient l'accumulation d'acylcarnitines à longue chaîne, de diacylglycérols, de phosphoénolpyruvate, de métabolites de la voie d'histidine, de lysophosphatidylcholine, de marqueurs de stress oxydatif, et de phosphoinositols, aussi bien que des taux réduits de monoacylglycérols. Les résultats des deux plateformes analytiques concordaient. CONCLUSION : Cette étude métabolomique indique un changement de l'utilisation des glucides vers les lipides pour la production d'énergie chez les personnes susceptibles à l'HM. Ce changement pourrait entraîner une inefficacité de la bêta-oxydation, ainsi qu'une augmentation du renouvellement des protéines musculaires, du stress oxydatif, et/ou des taux de lysophosphatidylcholine.

MyoSight-semi-automated image analysis of skeletal muscle cross sections.

BACKGROUND: Manual analysis of cross-sectional area, fiber-type distribution, and total and centralized nuclei in skeletal muscle cross sections is tedious and time consuming, necessitating an accurate, automated method of analysis. While several excellent programs are available, our analyses of skeletal muscle disease models suggest the need for additional features and flexibility to adequately describe disease pathology. We introduce a new semi-automated analysis program, MyoSight, which is designed to facilitate image analysis of skeletal muscle cross sections and provide additional flexibility in the analyses. RESULTS: We describe staining and imaging methods that generate high-quality images of immunofluorescent-labelled cross sections from mouse skeletal muscle. Using these methods, we can analyze up to 5 different fluorophores in a single image, allowing simultaneous analyses of perinuclei, central nuclei, fiber size, and fiber-type distribution. MyoSight displays high reproducibility among users, and the data generated are in close agreement with data obtained from manual analyses of cross-sectional area (CSA), fiber number, fiber-type distribution, and number and localization of myonuclei. Furthermore, MyoSight clearly delineates changes in these parameters in muscle sections from a mouse model of Duchenne muscular dystrophy (mdx). CONCLUSIONS: MyoSight is a new program based on an algorithm that can be optimized by the user to obtain highly accurate fiber size, fiber-type identification, and perinuclei and central nuclei per fiber measurements. MyoSight combines features available separately in other programs, is user friendly, and provides visual outputs that allow the user to confirm the accuracy of the analyses and correct any inaccuracies. We present MyoSight as a new program to facilitate the analyses of fiber type and CSA changes arising from injury, disease, exercise, and therapeutic interventions.

Adaptive thermogenesis enhances the life-threatening response to heat in mice with an Ryr1 mutation.

Mutations in the skeletal muscle Ca2+ release channel, the type 1 ryanodine receptor (RYR1), cause malignant hyperthermia susceptibility (MHS) and a life-threatening sensitivity to heat, which is most severe in children. Mice with an MHS-associated mutation in Ryr1 (Y524S, YS) display lethal muscle contractures in response to heat. Here we show that the heat response in the YS mice is exacerbated by brown fat adaptive thermogenesis. In addition, the YS mice have more brown adipose tissue thermogenic capacity than their littermate controls. Blood lactate levels are elevated in both heat-sensitive MHS patients with RYR1 mutations and YS mice due to Ca2+ driven increases in muscle metabolism. Lactate increases brown adipogenesis in both mouse and human brown preadipocytes. This study suggests that simple lifestyle modifications such as avoiding extreme temperatures and maintaining thermoneutrality could decrease the risk of life-threatening responses to heat and exercise in individuals with RYR1 pathogenic variants.

TRPV1 variants impair intracellular Ca2+ signaling and may confer susceptibility to malignant hyperthermia.

PURPOSE: Malignant hyperthermia (MH) is a pharmacogenetic disorder arising from uncontrolled muscle calcium release due to an abnormality in the sarcoplasmic reticulum (SR) calcium-release mechanism triggered by halogenated inhalational anesthetics. However, the molecular mechanisms involved are still incomplete. METHODS: We aimed to identify transient receptor potential vanilloid 1 (TRPV1) variants within the entire coding sequence in patients who developed sensitivity to MH of unknown etiology. In vitro and in vivo functional studies were performed in heterologous expression system, trpv1-/- mice, and a murine model of human MH. RESULTS: We identified TRPV1 variants in two patients and their heterologous expression in muscles of trpv1-/- mice strongly enhanced calcium release from SR upon halogenated anesthetic stimulation, suggesting they could be responsible for the MH phenotype. We confirmed the in vivo significance by using mice with a knock-in mutation (Y524S) in the type I ryanodine receptor (Ryr1), a mutation analogous to the Y522S mutation associated with MH in humans. We showed that the TRPV1 antagonist capsazepine slows the heat-induced hypermetabolic response in this model. CONCLUSION: We propose that TRPV1 contributes to MH and could represent an actionable therapeutic target for prevention of the pathology and also be responsible for MH sensitivity when mutated.

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 chemical chaperone improves muscle function in mice with a RyR1 mutation.

Mutations in the RYR1 gene cause severe myopathies. Mice with an I4895T mutation in the type 1 ryanodine receptor/Ca2+ release channel (RyR1) display muscle weakness and atrophy, but the underlying mechanisms are unclear. Here we show that the I4895T mutation in RyR1 decreases the amplitude of the sarcoplasmic reticulum (SR) Ca2+ transient, resting cytosolic Ca2+ levels, muscle triadin content and calsequestrin (CSQ) localization to the junctional SR, and increases endoplasmic reticulum (ER) stress/unfolded protein response (UPR) and mitochondrial ROS production. Treatment of mice carrying the I4895T mutation with a chemical chaperone, sodium 4-phenylbutyrate (4PBA), reduces ER stress/UPR and improves muscle function, but does not restore SR Ca2+ transients in I4895T fibres to wild type levels, suggesting that decreased SR Ca2+ release is not the major driver of the myopathy. These findings suggest that 4PBA, an FDA-approved drug, has potential as a therapeutic intervention for RyR1 myopathies that are associated with ER stress.

Ca(2+) permeation and/or binding to CaV1.1 fine-tunes skeletal muscle Ca(2+) signaling to sustain muscle function.

BACKGROUND: Ca(2+) influx through CaV1.1 is not required for skeletal muscle excitation-contraction coupling, but whether Ca(2+) permeation through CaV1.1 during sustained muscle activity plays a functional role in mammalian skeletal muscle has not been assessed. METHODS: We generated a mouse with a Ca(2+) binding and/or permeation defect in the voltage-dependent Ca(2+) channel, CaV1.1, and used Ca(2+) imaging, western blotting, immunohistochemistry, proximity ligation assays, SUnSET analysis of protein synthesis, and Ca(2+) imaging techniques to define pathways modulated by Ca(2+) binding and/or permeation of CaV1.1. We also assessed fiber type distributions, cross-sectional area, and force frequency and fatigue in isolated muscles. RESULTS: Using mice with a pore mutation in CaV1.1 required for Ca(2+) binding and/or permeation (E1014K, EK), we demonstrate that CaV1.1 opening is coupled to CaMKII activation and refilling of sarcoplasmic reticulum Ca(2+) stores during sustained activity. Decreases in these Ca(2+)-dependent enzyme activities alter downstream signaling pathways (Ras/Erk/mTORC1) that lead to decreased muscle protein synthesis. The physiological consequences of the permeation and/or Ca(2+) binding defect in CaV1.1 are increased fatigue, decreased fiber size, and increased Type IIb fibers. CONCLUSIONS: While not essential for excitation-contraction coupling, Ca(2+) binding and/or permeation via the CaV1.1 pore plays an important modulatory role in muscle performance.

The RNA-binding protein Rbfox1 regulates splicing required for skeletal muscle structure and function.

The Rbfox family of RNA-binding proteins is highly conserved with established roles in alternative splicing (AS) regulation. High-throughput studies aimed at understanding transcriptome remodeling have revealed skeletal muscle as displaying one of the largest number of AS events. This finding is consistent with requirements for tissue-specific protein isoforms needed to sustain muscle-specific functions. Rbfox1 is abundant in vertebrate brain, heart and skeletal muscle. Genome-wide genetic approaches have linked the Rbfox1 gene to autism, and a brain-specific knockout mouse revealed a critical role for this splicing regulator in neuronal function. Moreover, a Caenorhabditis elegans Rbfox1 homolog regulates muscle-specific splicing. To determine the role of Rbfox1 in muscle function, we developed a conditional knockout mouse model to specifically delete Rbfox1 in adult tissue. We show that Rbfox1 is required for muscle function but a >70% loss of Rbfox1 in satellite cells does not disrupt muscle regeneration. Deep sequencing identified aberrant splicing of multiple genes including those encoding myofibrillar and cytoskeletal proteins, and proteins that regulate calcium handling. Ultrastructure analysis of Rbfox1(-/-) muscle by electron microscopy revealed abundant tubular aggregates. Immunostaining showed mislocalization of the sarcoplasmic reticulum proteins Serca1 and Ryr1 in a pattern indicative of colocalization with the tubular aggregates. Consistent with mislocalization of Serca1 and Ryr1, calcium handling was drastically altered in Rbfox1(-/-) muscle. Moreover, muscle function was significantly impaired in Rbfox1(-/-) muscle as indicated by decreased force generation. These results demonstrate that Rbfox1 regulates a network of AS events required to maintain multiple aspects of muscle physiology.

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.

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.

Ligands for FKBP12 increase Ca2+ influx and protein synthesis to improve skeletal muscle function.

Rapamycin at high doses (2-10 mg/kg body weight) inhibits mammalian target of rapamycin complex 1 (mTORC1) and protein synthesis in mice. In contrast, low doses of rapamycin (10 µg/kg) increase mTORC1 activity and protein synthesis in skeletal muscle. Similar changes are found with SLF (synthetic ligand for FKBP12, which does not inhibit mTORC1) and in mice with a skeletal muscle-specific FKBP12 deficiency. These interventions also increase Ca(2+) influx to enhance refilling of sarcoplasmic reticulum Ca(2+) stores, slow muscle fatigue, and increase running endurance without negatively impacting cardiac function. FKBP12 deficiency or longer treatments with low dose rapamycin or SLF increase the percentage of type I fibers, further adding to fatigue resistance. We demonstrate that FKBP12 and its ligands impact multiple aspects of muscle function.

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.

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.

Alvimopan addition to a standard perioperative recovery pathway.

BACKGROUND AND OBJECTIVES: Alvimopan, a peripherally acting mu-opioid receptor antagonist, decreased time to gastrointestinal recovery and hospital length of stay in open bowel resection patients in Phase 3 trials. However, the benefit in laparoscopic colectomy patients remains unclear. METHODS: A retrospective case series review was performed to study addition of alvimopan to a well-established standard perioperative recovery pathway for elective laparoscopic colectomy. The main outcome measures were length of stay and incidence of charted postoperative ileus. Wilcoxon and chi-square tests were used to calculate P values for length of stay and postoperative ileus endpoints, respectively. RESULTS: Demographic/baseline characteristics from the 101 alvimopan and 64 pre-alvimopan control patients were generally comparable. Mean length of stay in the alvimopan group was 1.55 days shorter (alvimopan, 2.81±0.95 days; control, 4.36±2.4 days; P<.0001). The proportion of patients with postoperative ileus was lower in the alvimopan group (alvimopan, 2%; control, 20%; P<.0001). CONCLUSION: In this case series, addition of alvimopan to a standard perioperative recovery pathway decreased length of stay and incidence of postoperative ileus for elective uncomplicated laparoscopic colectomy. The improvement in the mean length of stay for patients who receive alvimopan is a step forward in achieving a fasttrack surgery model for elective laparoscopic colectomies.

Unique isoform-specific properties of calsequestrin in the heart and skeletal muscle.

Calcium signaling in myocytes is dependent on the cardiac ryanodine receptor (RyR2) calcium release channel and the calcium buffering protein in the sarcoplasmic reticulum, cardiac calsequestrin (CSQ2). The overall properties of CSQ2 and its regulation of RyR2 have not been explored in detail or directly compared with skeletal CSQ1 and its regulation of the skeletal RyR1, with physiological ionic strength and Ca(2+) concentrations. We find that there are major differences between the two isoforms under these physiological conditions. Ca(2+) binding to CSQ2 is 50% lower than to CSQ1. Only approximately 30% of CSQ2 is bound to cardiac junctional face membrane (JFM), compared with approximately 70% of CSQ1 and the ratio of CSQ2 to RyR2 is only 50% of the CSQ1/RyR1 ratio. Chemical crosslinking shows that CSQ2 is mostly monomer/dimer, while CSQ1 is mostly polymerized. In single channel lipid bilayer experiments, CSQ2 monomers and/or dimers increase the open probability of both RyR1 and RyR2 channels, while CSQ1 polymers decrease the activity of RyR1. We speculate that CSQ2 facilitates high rates of Ca(2+) release through RyR2 during systole, while CSQ1 curtails RyR1 opening in response to a single action potential to maintain Ca(2+) and allow repeated Ca(2+) release and graded activation with increased stimulation frequency.