Visualizing Actin Packing and the Effects of Actin Attachment on Lipid Membrane Viscosity Using Molecular Rotors.

The actin cytoskeleton and its elaborate interplay with the plasma membrane participate in and control numerous biological processes in eukaryotic cells. Malfunction of actin networks and changes in their dynamics are related to various diseases, from actin myopathies to uncontrolled cell growth and tumorigenesis. Importantly, the biophysical and mechanical properties of actin and its assemblies are deeply intertwined with the biological functions of the cytoskeleton. Novel tools to study actin and its associated biophysical features are, therefore, of prime importance. Here we develop a new approach which exploits fluorescence lifetime imaging microscopy (FLIM) and environmentally sensitive fluorophores termed molecular rotors, acting as quantitative microviscosity sensors, to monitor dynamic viscoelastic properties of both actin structures and lipid membranes. In order to reproduce a minimal actin cortex in synthetic cell models, we encapsulated and attached actin networks to the lipid bilayer of giant unilamellar vesicles (GUVs). Using a cyanine-based molecular rotor, DiSC2(3), we show that different types of actin bundles are characterized by distinct packing, which can be spatially resolved using FLIM. Similarly, we show that a lipid bilayer-localized molecular rotor can monitor the effects of attaching cross-linked actin networks to the lipid membrane, revealing an increase in membrane viscosity upon actin attachment. Our approach bypasses constraints associated with existing methods for actin imaging, many of which lack the capability for direct visualization of biophysical properties. It can therefore contribute to a deeper understanding of the role that actin plays in fundamental biological processes and help elucidate the underlying biophysics of actin-related diseases.

Building Synthetic Cells─From the Technology Infrastructure to Cellular Entities.

The de novo construction of a living organism is a compelling vision. Despite the astonishing technologies developed to modify living cells, building a functioning cell "from scratch" has yet to be accomplished. The pursuit of this goal alone has─and will─yield scientific insights affecting fields as diverse as cell biology, biotechnology, medicine, and astrobiology. Multiple approaches have aimed to create biochemical systems manifesting common characteristics of life, such as compartmentalization, metabolism, and replication and the derived features, evolution, responsiveness to stimuli, and directed movement. Significant achievements in synthesizing each of these criteria have been made, individually and in limited combinations. Here, we review these efforts, distinguish different approaches, and highlight bottlenecks in the current research. We look ahead at what work remains to be accomplished and propose a "roadmap" with key milestones to achieve the vision of building cells from molecular parts.

Association of glucagon-like peptide receptor 1 agonist therapy with the presence of gastric contents in fasting patients undergoing endoscopy under anesthesia care: a historical cohort study.

PURPOSE: We aimed to estimate the association of glucagon-like peptide 1 (GLP-1) receptor agonist therapy with the incidence of endoscopically visible gastric contents after preprocedural fasting. METHODS: We reviewed the records of esophagogastroduodenoscopy (EGD) performed at our institution between 2019 and 2023 and determined the presence of residual gastric contents from the procedure notes and saved images. We compared patients taking GLP-1 agonists at the time of the procedure (GLP group, 90 procedures) with patients who started GLP-1 agonist therapy within 1,000 days after undergoing EGD (control, 102 procedures). We excluded emergent procedures without fasting, combined EGD/colonoscopy procedures, and patients with known gastroparesis or previous gastric surgery. We estimated the association between GLP-1 agonist therapy and residual gastric contents with a confounder-adjusted generalized linear mixed effect model. RESULTS: Compared with controls, the GLP cohort had a higher age, American Society of Anesthesiologists' Physical Status, and incidence of nausea and diabetes mellitus. Body mass index and fasting duration were comparable between groups. Visible gastric content was documented in 17 procedures in the GLP group (19%) and in five procedures in the control group (5%), with an associated confounder adjusted odds ratio of 5.8 (95% confidence interval, 1.7 to 19.3; P = 0.004). There were five instances of emergent endotracheal intubation in the GLP group vs one case in control and one case of pulmonary aspiration vs none in control. CONCLUSIONS: In fasting patients, GLP-1 agonist therapy was associated with an increased incidence of residual gastric contents, potentially posing an additional risk of periprocedural pulmonary aspiration.

RéSUMé: OBJECTIF: Notre objectif était d'estimer l'association d'un traitement par agonistes des récepteurs du peptide-1 de type glucagon (glucagon-like peptide 1, GLP-1) avec l'incidence de contenu gastrique visible par endoscopie malgré le jeûne préopératoire. MéTHODE: Nous avons examiné les dossiers des œsophagogastroduodénoscopies (OGD) réalisées dans notre établissement entre 2019 et 2023 et déterminé la présence de contenu gastrique résiduel à partir des notes d'intervention et des images enregistrées. Nous avons comparé les patient·es prenant des agonistes du GLP-1 au moment de l'intervention (groupe GLP, 90 procédures) avec les patient·es qui ont commencé un traitement par agonistes du GLP-1 dans les 1000 jours suivant l'OGD (groupe témoin, 102 procédures). Nous avons exclu les procédures d'urgence sans jeûne, les procédures combinées OGD/coloscopie et les patient·es présentant une gastroparésie connue ou une chirurgie gastrique antérieure. Nous avons estimé l'association entre le traitement par agonistes du récepteur GLP-1 et le contenu gastrique résiduel à l'aide d'un modèle linéaire généralisé à effets mixtes ajusté en fonction des facteurs de confusion. RéSULTATS: Par rapport aux témoins, la cohorte GLP était plus âgée, de statut physique selon l'American Society of Anesthesiologists plus élevé et présentait une incidence plus élevée de nausées et de diabète. L'indice de masse corporelle et la durée du jeûne étaient comparables entre les groupes. Du contenu gastrique visible a été documenté dans 17 procédures dans le groupe GLP (19 %) et dans cinq procédures dans le groupe témoin (5 %), avec un rapport de cotes ajusté associé de 5,8 (intervalle de confiance à 95 %, 1,7 à 19,3; P = 0,004). Il y a eu cinq cas d'intubation endotrachéale urgente dans le groupe GLP vs un cas dans le groupe témoin et un cas d'aspiration pulmonaire vs aucun dans le groupe témoin. CONCLUSION: Chez la patientèle à jeun, le traitement par agonistes des récepteurs du GLP-1 a été associé à une incidence accrue de contenu gastrique résiduel, ce qui pourrait entraîner un risque supplémentaire d'aspiration pulmonaire périprocédurale.

Semaglutide, delayed gastric emptying, and intraoperative pulmonary aspiration: a case report.

PURPOSE: We report a case in which the use of semaglutide for weight loss was associated with delayed gastric emptying and intraoperative pulmonary aspiration of gastric contents. CLINICAL FEATURES: A 42-yr-old patient with Barrett's esophagus underwent repeat upper gastrointestinal endoscopy and ablation of dysplastic mucosa. Two months earlier, the patient had started weekly injections of semaglutide for weight loss. Despite having fasted for 18 hr, and differing from the findings of prior procedures, endoscopy revealed substantial gastric content, which was suctioned before endotracheal intubation. Food remains were removed from the trachea and bronchi using bronchoscopy. The patient was extubated four hours later and remained asymptomatic. CONCLUSION: Patients using semaglutide and other glucagon-like peptide 1 agonists for weight management may require specific precautions during induction of anesthesia to prevent pulmonary aspiration of gastric contents.

RéSUMé: OBJECTIF: Nous rapportons un cas dans lequel l'utilisation de sémaglutide à des fins de perte de poids a été associée à un retard de vidange gastrique et à une aspiration pulmonaire peropératoire du contenu gastrique. CARACTéRISTIQUES CLINIQUES: Un patient de 42 ans souffrant d'un œsophage de Barrett a subi une cinquième endoscopie gastro-intestinale supérieure avec ablation de la muqueuse dysplasique. Deux mois plus tôt, le patient avait commencé à recevoir des injections hebdomadaires de sémaglutide pour perdre du poids. Bien qu'à jeun depuis 18 heures et à la différence des évaluations lors des interventions antérieures, l'endoscopie a révélé un contenu gastrique important, qui a été aspiré avant l'intubation endotrachéale. Les restes de nourriture ont été retirés de la trachée et des bronches par bronchoscopie. Le patient a été extubé quatre heures plus tard et est demeuré asymptomatique. CONCLUSION: Les patients utilisant du sémaglutide et d'autres agonistes du peptide analog au glucagon-1 pour la gestion du poids pourraient nécessiter des précautions spécifiques lors de l'induction de l'anesthésie pour empêcher l'aspiration pulmonaire du contenu gastrique.

CARDIOMYOCYTE REPROGRAMMING IN ANIMAL MODELS OF SEPTIC SHOCK.

ABSTRACT: Cardiomyocyte reprogramming plays a pivotal role in sepsis-induced cardiomyopathy through the induction or overexpression of several factors and enzymes, ultimately leading to the characteristic decrease in cardiac contractility. The initial trigger is the binding of LPS to TLR-2, -3, -4, and -9 and of proinflammatory cytokines, such as TNF, IL-1, and IL-6, to their respective receptors. This induces the nuclear translocation of nuclear factors, such as NF-κB, via activation of MyD88, TRIF, IRAK, and MAPKs. Among the latter, ROS- and estrogen-dependent p38 and ERK 1/2 are proinflammatory, whereas JNK may play antagonistic, anti-inflammatory roles. Nuclear factors induce the synthesis of cytokines, which can amplify the inflammatory signal in a paracrine fashion, and of several effector enzymes, such as NOS-2, NOX-1, and others, which are ultimately responsible for the degradation of cardiomyocyte contractility. In parallel, the downregulation of enzymes involved in oxidative phosphorylation causes metabolic reprogramming, followed by a decrease in ATP production and the release of fragmented mitochondrial DNA, which may augment the process in a positive feedback loop. Other mediators, such as NO, ROS, the enzymes PI3K and Akt, and adrenergic stimulation may play regulatory roles, but not all signaling pathways that mediate cardiac dysfunction of sepsis do that by regulating reprogramming. Transcription may be globally modulated by miRs, which exert protective or amplifying effects. For all these mechanisms, differentiating between modulation of cardiomyocyte reprogramming versus systemic inflammation has been an ongoing but worthwhile experimental challenge.

MECHANISMS OF CARDIAC DYSFUNCTION IN SEPSIS.

ABSTRACT: Studies in animal models of sepsis have elucidated an intricate network of signaling pathways that lead to the dysregulation of myocardial Ca 2+ handling and subsequently to a decrease in cardiac contractile force, in a sex- and model-dependent manner. After challenge with a lethal dose of LPS, male animals show a decrease in cellular Ca 2+ transients (ΔCa i ), with intact myofilament function, whereas female animals show myofilament dysfunction, with intact ΔCa i . Male mice challenged with a low, nonlethal dose of LPS also develop myofilament desensitization, with intact ΔCa i . In the cecal ligation and puncture (CLP) model, the causative mechanisms seem similar to those in the LPS model in male mice and are unknown in female subjects. ΔCa i decrease in male mice is primarily due to redox-dependent inhibition of sarco/endoplasmic reticulum Ca 2+ ATP-ase (SERCA). Reactive oxygen species (ROS) are overproduced by dysregulated mitochondria and the enzymes NADPH/NADH oxidase, cyclooxygenase, and xanthine oxidase. In addition to inhibiting SERCA, ROS amplify cardiomyocyte cytokine production and mitochondrial dysfunction, making the process self-propagating. In contrast, female animals may exhibit a natural redox resilience. Myofilament dysfunction is due to hyperphosphorylation of troponin I, troponin T cleavage by caspase-3, and overproduction of cGMP by NO-activated soluble guanylate cyclase. Depleted, dysfunctional, or uncoupled mitochondria likely synthesize less ATP in both sexes, but the role of energy deficit is not clear. NO produced by NO synthase (NOS)-3 and mitochondrial NOSs, protein kinases and phosphatases, the processes of autophagy and sarco/endoplasmic reticulum stress, and ß-adrenergic insensitivity may also play currently uncertain roles.

Distribution of Triclocarban in Natural Aqueous Systems Amended with Carbon Nanomaterials.

In this work, the sorption of TCC in natural aqueous solutions with different chemical compositions, on amended sediments with carbon nanomaterials such as MWCNTs and C60, pristine and weathered is presented. The variation of the temperature, the composition of the natural water, the compositions of the sediment were studied, the results were correlated for a better understanding of the possible effects of the carbon nanomaterials in the environment.

Investigations on chemical composition and natural radioactivity levels from salt water and peloid used in pelotherapy from the Techirghiol Lake, Romania.

The work presents the historical evolution, objectives, goals, concepts, chemical and radiometric methods, results and conclusions for salt waters and natural peloids used in pelotherapy. This study assesses chemical composition, natural radioactivity concentrations and the radiological hazard in peloid and salt water samples, from ten places in the Techirghiol Lake from Romania. Pelotherapy is a very important procedure, and thus, the materials used for this purpose must be well characterized to guaranty safety use. Concentrations of elements such as Sr, Ba, Mn, Fe, Sb, Zn, Cu, Pb, Ti, Ni, Cr, As have been measured using ICP-OES analytical technique. The natural radionuclides such as 238U, 226Ra, 232Th and 40K have been determined by gamma-ray spectrometry. The average activity concentrations were of 0.48 ± 0.10 Bq/kg for 238U, 0.60 ± 0.10 Bq/kg for 226Ra, 0.30 ± 0.08 Bq/kg for 232Th and 17.5 ± 1.3 Bq/kg for 40K for salt water samples. Also, the mean activity concentrations for peloids were: 5.70 ± 1.00 Bq/kg for 238U, 6.85 ± 1.60 Bq/kg for 232Th, 15.3 ± 3.7 Bq/kg for 226Ra and 95.8 ± 5.5 Bq/kg for 40K. The results from this study contribute to the identification of possible contaminants in the salt water and peloid, and their association with the potential ecological and human health risk. In this context, of using salt water and peloid in a relatively long treatment period, several radiological indices have been calculated, to determine if the radionuclide's content can be also harmful to human health. The assessment indicates that humans are not exposed to concentrations of metal contaminants higher than the international recommended values.

Loneliness and perceived social support in pregnancy and early postpartum of mothers living with HIV in Ontario, Canada.

The HIV Mothering Study (n = 72) was a prospective, observational, cohort study exploring psychosocial experiences and needs of WLWHIV in pregnancy and postpartum. We performed quantitative analysis of determinants of loneliness (UCLA Loneliness Scale) and lower perceived social support (SS) (Medical Outcomes Study-Social Support Survey). The hypothesized determinants included: age, years with HIV, racism (Everyday Discrimination Scale), depression (Edinburgh Postnatal Depression Scale [EPDS]), nadir CD4 (<200 cells/µL), tertiary vs. community HIV care, and marital status. The median age was 33 (IQR = 30-37); 65.3% were African/Caribbean/Black. Multivariable analyses revealed associations between marital status and perceived social support (ß = -16.48, p < 0.0001), and this association was also seen with change over time (p = 0.02). Variables associated with SS that did not change over time were: income, EDS racism, EPDS score. Significant associations with loneliness were seen with the same variables associated with SS. Variables associated with loneliness that also changed over time were: EDS Racism (ß = 0.22, p = 0.0005, and over time p = 0.003), and EPDS score (ß = 0.74, p < 0.0001), and over time (p = 0.0211). Variables associated with loneliness but that did not change over time were: marital status and income. This analysis provides clinicians with prenatal risk factors which may be associated with increase loneliness and lower SS during pregnancy and postpartum: marital status, income, racism and depression.

Myocardial Redox Hormesis Protects the Heart of Female Mice in Sepsis.

Mice challenged with lipopolysaccharide develop cardiomyopathy in a sex and redox-dependent fashion. Here we extended these studies to the cecal ligation and puncture (CLP) model.We compared male and female FVB mice (wild type, WT) and transgenic littermates overexpressing myocardial catalase (CAT). CLP induced 100% mortality within 4 days, with similar mortality rates in male and female WT and CAT mice. 24 h after CLP, isolated (Langendorff) perfused hearts showed depressed contractility in WT male mice, but not in male CAT or female WT and CAT mice. In WT male mice, CLP induced a depression of cardiomyocyte sarcomere shortening (ΔSS) and calcium transients (ΔCai), and the inhibition of the sarcoplasmic reticulum Ca ATPase (SERCA). These deficits were associated with overexpression of NADPH-dependent oxidase (NOX)-1, NOX-2, and cyclooxygenase 2 (COX-2), and were partially prevented in male CAT mice. Female WT mice showed unchanged ΔSS, ΔCai, and SERCA function after CLP. At baseline, female WT mice showed partially depressed ΔSS, ΔCai, and SERCA function, as compared with male WT mice, which were associated with NOX-1 overexpression and were prevented in CAT female mice.In conclusion, in male WT mice, septic shock induces myocardial NOX-1, NOX-2, and COX-2, and redox-dependent dysregulation of myocardial Ca transporters. Female WT mice are resistant to CLP-induced cardiomyopathy, despite increased NOX-1 and COX-2 expression, suggesting increased antioxidant capacity. Female resistance occurred in association with NOX-1 overexpression and signs of increased oxidative signaling at baseline, indicating the presence of a protective myocardial redox hormesis mechanism.

Changes in V-ATPase subunits of human urinary exosomes reflect the renal response to acute acid/alkali loading and the defects in distal renal tubular acidosis.

In the kidney, final urinary acidification is achieved by V-ATPases expressed in type A intercalated cells. The B1 subunit of the V-ATPase is required for maximal urinary acidification, while the role of the homologous B2 subunit is less clear. Here we examined the effect of acute acid/alkali loading in humans on B1 and B2 subunit abundance in urinary exosomes in normal individuals and of acid loading in patients with distal renal tubular acidosis (dRTA). Specificities of B1 and B2 subunit antibodies were verified by yeast heterologously expressing human B1 and B2 subunits, and murine wild-type and B1-deleted kidney lysates. Acute ammonium chloride loading elicited systemic acidemia, a drop in urinary pH, and increased urinary ammonium excretion. Nadir urinary pH was achieved at four to five hours, and exosomal B1 abundance was significantly increased at two through six hours after ammonium chloride loading. After acute equimolar sodium bicarbonate loading, blood and urinary pH rose rapidly, with a concomitant reduction of exosomal B1 abundance within two hours, which remained lower throughout the test. In contrast, no change in exosomal B2 abundance was found following acid or alkali loading. In patients with inherited or acquired distal RTA, the urinary B1 subunit was extremely low or undetectable and did not respond to acid loading in urine, whereas no change in B2 subunit was found. Thus, both B1 and B2 subunits of the V-ATPase are detectable in human urinary exosomes, and acid and alkali loading or distal RTA cause changes in the B1 but not B2 subunit abundance in urinary exosomes.

Up-regulation of Intracellular Calcium Handling Underlies the Recovery of Endotoxemic Cardiomyopathy in Mice.

BACKGROUND: In surviving patients, sepsis-induced cardiomyopathy is spontaneously reversible. In the absence of any experimental data, it is generally thought that cardiac recovery in sepsis simply follows the remission of systemic inflammation. Here the authors aimed to identify the myocardial mechanisms underlying cardiac recovery in endotoxemic mice. METHODS: Male C57BL/6 mice were challenged with lipopolysaccharide (7 µg/g, intraperitoneally) and followed for 12 days. The authors assessed survival, cardiac function by echocardiography, sarcomere shortening, and calcium transients (with fura-2-acetoxymethyl ester) in electrically paced cardiomyocytes (5 Hz, 37°C) and myocardial protein expression by immunoblotting. RESULTS: Left ventricular ejection fraction, cardiomyocyte sarcomere shortening, and calcium transients were depressed 12 h after lipopolysaccharide challenge, started to recover by 24 h (day 1), and were back to baseline at day 3. The recovery of calcium transients at day 3 was associated with the up-regulation of the sarcoplasmic reticulum calcium pump to 139 ± 19% (mean ± SD) of baseline and phospholamban down-regulation to 35 ± 20% of baseline. At day 6, calcium transients were increased to 123 ± 31% of baseline, associated with increased sarcoplasmic reticulum calcium load (to 126 ± 32% of baseline, as measured with caffeine) and inhibition of sodium/calcium exchange (to 48 ± 12% of baseline). CONCLUSIONS: In mice surviving lipopolysaccharide challenge, the natural recovery of cardiac contractility was associated with the up-regulation of cardiomyocyte calcium handling above baseline levels, indicating the presence of an active myocardial recovery process, which included sarcoplasmic reticulum calcium pump activation, the down-regulation of phospholamban, and sodium/calcium exchange inhibition.

Distinct Myocardial Mechanisms Underlie Cardiac Dysfunction in Endotoxemic Male and Female Mice.

In male mice, sepsis-induced cardiomyopathy develops as a result of dysregulation of myocardial calcium (Ca) handling, leading to depressed cellular Ca transients (ΔCai). ΔCai depression is partially due to inhibition of sarcoplasmic reticulum Ca ATP-ase (SERCA) via oxidative modifications, which are partially opposed by cGMP generated by the enzyme soluble guanylyl cyclase (sGC). Whether similar mechanisms underlie sepsis-induced cardiomyopathy in female mice is unknown.Male and female C57Bl/6J mice (WT), and mice deficient in the sGC α1 subunit activity (sGCα1), were challenged with lipopolysaccharide (LPS, ip). LPS induced mouse death and cardiomyopathy (manifested as the depression of left ventricular ejection fraction by echocardiography) to a similar degree in WT male, WT female, and sGCα1 male mice, but significantly less in sGCα1 female mice. We measured sarcomere shortening and ΔCai in isolated, externally paced cardiomyocytes, at 37°C. LPS depressed sarcomere shortening in both WT male and female mice. Consistent with previous findings, in male mice, LPS induced a decrease in ΔCai (to 30 ±â€Š2% of baseline) and SERCA inhibition (manifested as the prolongation of the time constant of Ca decay, τCa, to 150 ±â€Š5% of baseline). In contrast, in female mice, the depression of sarcomere shortening induced by LPS occurred in the absence of any change in ΔCai, or SERCA activity. This suggested that, in female mice, the causative mechanism lies downstream of the Ca transients, such as a decrease in myofilament sensitivity for Ca. The depression of sarcomere shortening shortening after LPS was less severe in female sGCα1 mice than in WT female mice, indicating that cGMP partially mediates cardiomyocyte dysfunction.These results suggest, therefore, that LPS-induced cardiomyopathy develops through distinct sex-specific myocardial mechanisms. While in males LPS induces sGC-independent decrease in ΔCai, in female mice LPS acts downstream of ΔCai, possibly via sGC-dependent myofilament dysfunction.

Dysregulation of intracellular calcium transporters in animal models of sepsis-induced cardiomyopathy.

Sepsis-induced cardiomyopathy (SIC) develops as the result of myocardial calcium (Ca) dysregulation. Here we reviewed all published studies that quantified the dysfunction of intracellular Ca transporters and the myofilaments in animal models of SIC. Cardiomyocytes isolated from septic animals showed, invariably, a decreased twitch amplitude, which is frequently caused by a decrease in the amplitude of cellular Ca transients (ΔCai) and sarcoplasmic reticulum (SR) Ca load (CaSR). Underlying these deficits, the L-type Ca channel is downregulated, through mechanisms that may involve adrenomedullin-mediated redox signaling. The SR Ca pump is also inhibited, through oxidative modifications (sulfonylation) of one reactive thiol group (on Cys) and/or modulation of phospholamban. Diastolic Ca leak of ryanodine receptors is frequently increased. In contrast, Na/Ca exchange inhibition may play a partially compensatory role by increasing CaSR and ΔCai. The action potential is usually shortened. Myofilaments show a bidirectional regulation, with decreased Ca sensitivity in milder forms of disease (due to troponin I hyperphosphorylation) and an increase (redox mediated) in more severe forms. Most deficits occurred similarly in two different disease models, induced by either intraperitoneal administration of bacterial lipopolysaccharide or cecal ligation and puncture. In conclusion, substantial cumulative evidence implicates various Ca transporters and the myofilaments in SIC pathology. What is less clear, however, are the identity and interplay of the signaling pathways that are responsible for Ca transporters dysfunction. With few exceptions, all studies we found used solely male animals. Identifying sex differences in Ca dysregulation in SIC becomes, therefore, another priority.

Lipopolysaccharide and cytokines inhibit rat cardiomyocyte contractility in vitro.

BACKGROUND: Sepsis-induced cardiomyopathy (SIC) is thought to be the result of detrimental effects of inflammatory mediators on the cardiac muscle. Here we studied the effects of prolonged (24 ± 4 h) exposure of adult rat ventricular myocytes (ARVM) to bacterial lipopolysaccharide (LPS) and inflammatory cytokines tumor necrosis factor (TNF) and interleukins-1 (IL-1) and IL-6. MATERIALS AND METHODS: We measured sarcomere shortening (SS) and cellular calcium (Ca(2+)) transients (ΔCai, with fura-2 AM) in isolated cardiomyocytes externally paced at 5 Hz at 37°C. RESULTS: SS decreased after incubation with LPS (100 µg/mL), IL-1 (100 ng/mL), and IL-6 (30 ng/mL), but not with lesser doses of these mediators, or TNF (10-100 ng/mL). A combination of LPS (100 µg/mL), TNF, IL-1, and IL-6 (each 100 ng/mL; i.e., "Cytomix-100") induced a maximal decrease in SS and ΔCai. Sarcoplasmic reticulum (SR) Ca(2+) load (CaSR, measured with caffeine) was unchanged by Cytomix-100; however, SR fractional release (ΔCai/CaSR) was decreased. Underlying these effects, Ca(2+) influx into the cell (via L-type Ca(2+) channels, LTCC) and Ca(2+) extrusion via Na(+)/Ca(2+) exchange were decreased by Cytomix-100. SR Ca(2+) pump (SERCA) (SR Ca(2+) ATPase) was not affected. CONCLUSIONS: Prolonged exposure of ARVM to a mixture of LPS and inflammatory cytokines inhibits cell contractility. The effect is mediated by the inhibition of Ca(2+) influx via LTCC, and partially opposed by the inhibition of Na(+)/Ca(2+) exchange. Because both mechanisms are commonly seen in animal models of SIC, we conclude that prolonged challenge with Cytomix-100 of ARVM may represent an accurate in vitro model for SIC.