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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., (© 2024. Canadian Anesthesiologists' Society.)

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., (© 2023. Canadian Anesthesiologists' Society.)

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 β-adrenergic insensitivity may also play currently uncertain roles., Competing Interests: The author reports no conflicts of interest., (Copyright © 2022 by the Shock Society.)

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., Competing Interests: The author reports no conflicts of interest., (Copyright © 2022 by the Shock Society.)

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.

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.

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 ± 2% of baseline) and SERCA inhibition (manifested as the prolongation of the time constant of Ca decay, τCa, to 150 ± 5% 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., Competing Interests: None.

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., (Copyright © 2015 Elsevier Inc. All rights reserved.)

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.

The goal of this study was to identify the cellular mechanisms responsible for cardiac dysfunction in endotoxemic mice. We aimed to differentiate the roles of cGMP [produced by soluble guanylyl cyclase (sGC)] versus oxidative posttranslational modifications of Ca(2+) transporters. C57BL/6 mice [wild-type (WT) mice] were administered lipopolysaccharide (LPS; 25 μg/g ip) and euthanized 12 h later. Cardiomyocyte sarcomere shortening and Ca(2+) transients (ΔCai) were depressed in LPS-challenged mice versus baseline. The time constant of Ca(2+) decay (τCa) was prolonged, and sarcoplasmic reticulum Ca(2+) load (CaSR) was depressed in LPS-challenged mice (vs. baseline), indicating decreased activity of sarco(endo)plasmic Ca(2+)-ATPase (SERCA). L-type Ca(2+) channel current (ICa,L) was also decreased after LPS challenge, whereas Na(+)/Ca(2+) exchange activity, ryanodine receptors leak flux, or myofilament sensitivity for Ca(2+) were unchanged. All Ca(2+)-handling abnormalities induced by LPS (the decrease in sarcomere shortening, ΔCai, CaSR, ICa,L, and τCa prolongation) were more pronounced in mice deficient in the sGC main isoform (sGCα1(-/-) mice) versus WT mice. LPS did not alter the protein expression of SERCA and phospholamban in either genotype. After LPS, phospholamban phosphorylation at Ser(16) and Thr(17) was unchanged in WT mice and was increased in sGCα1(-/-) mice. LPS caused sulphonylation of SERCA Cys(674) (as measured immunohistochemically and supported by iodoacetamide labeling), which was greater in sGCα1(-/-) versus WT mice. Taken together, these results suggest that cardiac Ca(2+) dysregulation in endotoxemic mice is mediated by a decrease in L-type Ca(2+) channel function and oxidative posttranslational modifications of SERCA Cys(674), with the latter (at least) being opposed by sGC-released cGMP.

Tracheal resection and reconstruction (TRR) is the treatment of choice for most patients with tracheal stenosis or tracheal tumors. Anesthesia for TRR offers distinct challenges, especially for the less experienced practitioner. This article explores the preoperative assessment, strategies for induction and emergence from anesthesia, the essential coordination between the surgical and anesthesia teams during airway excision and anastomosis, and postoperative care. The most common complications are reviewed. Targeted readership is practitioners with less extensive experience in managing airway surgery cases. As such, the article focuses first on the most common proximal tracheal resection. Final sections discuss specific considerations for more complicated cases., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Objectives: The authors analyzed surgical cases in which electrocardiographic (ECG) signs of cardiac ischemia were noted to be precipitated by increases in heart rate (ie, heart rate-related ECG changes [REC]). The authors aimed to find REC incidence, specificity for coronary artery disease (CAD), and the outcome associated with different management strategies., Design: A retrospective review., Setting: A university hospital, tertiary care., Participants: Patients undergoing surgery under anesthesia., Interventions: A chart review., Measurements: The authors searched 158,252 anesthesia electronic records for comments noting REC (ie, ST-segment or T-wave changes). After excluding cases with potentially confounding conditions (eg, hypotension, hyperkalemia, and so on), 26 cases were analyzed., Results: REC commonly was precipitated by anesthesia-related events (ie, intubation, extubation, and treatment of bradycardia). In 24 cases, REC was managed by prompt heart rate reduction using β-blocker agents, opioids, and/or cardioversion in the addition to the removal of stimulus. Only 1 case had a copy of the ECG printed. Two cases were aborted, 1 was shortened and 23 proceeded without change. Postoperative troponin T levels were checked, and cardiology consultation was obtained in selected cases and led to further cardiac evaluation in 6 cases. Postoperative myocardial infarction developed in only 1 patient in whom the ECG changes were allowed to persist throughout the case., Conclusions: This incidence of reported REC was much lower than the previously reported incidence of ischemia-related ECG changes, suggesting that the largest proportion of events go unnoticed. In many patients, subsequent cardiology workup did not confirm the existence of clinically significant CAD., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Paraplegia caused by a spinal cord infarction (SCI) is a devastating perioperative complication, most often associated with aortic and spine surgery. We present two other clinical scenarios in which perioperative SCI may occur. They happened during surgical procedures performed with epidural anesthesia, in the presence of several specific risk factors such as spinal stenosis, vascular disease, intraoperative hypotension, or the use of epinephrine in the local anesthetic solution. Second, SCI may occur during episodes of postoperative hypotension in patients with a history of aortic aneurysms.

Sodium/calcium (Na+/Ca2+) exchange (NCX) overexpression is common to human heart failure and heart failure in many animal models, but its specific contribution to the cellular Ca2+ ([Ca2+]i) handling deficit is unclear. Here, we investigate the effects of exchange inhibitory peptide (XIP) on Ca2+ handling in myocytes isolated from canine tachycardic pacing-induced failing hearts. Whole-cell patch-clamped left ventricular myocytes from failing hearts (F) showed a 52% decrease in steady-state sarcoplasmic reticulum (SR) Ca2+ load and a 44% reduction in the amplitude of the [Ca2+]i transient, as compared with myocytes from normal hearts (N). Intracellular application of XIP (30 micromol/L) normalized the [Ca2+]i transient amplitude in F (3.86-fold increase), concomitant with a similar increase in SR Ca2+ load. The degree of NCX inhibition at this concentration of XIP was 27% and was selective for NCX: L-type Ca2+ currents and plasmalemmal Ca2+ pumps were not affected. XIP also indirectly improved the rate of [Ca2+]i removal at steady-state, secondary to Ca2+-dependent activation of SR Ca2+ uptake. The findings indicate that in the failing heart cell, NCX inhibition can improve SR Ca2+ load by shifting the balance of Ca2+ fluxes away from trans-sarcolemmal efflux toward SR accumulation. Hence, inhibition of the Ca2+ efflux mode of the exchanger could potentially be an effective therapeutic strategy for improving contractility in congestive heart failure.

The Na(+)/Ca(2+) exchanger (NCX), a surface membrane antiporter, is the primary pathway for Ca(2+) efflux from the cardiac cell and a determinant of both the electrical and contractile state of the heart. Enhanced expression of NCX has recently been recognised as one of the molecular mechanisms that contributes to reduced Ca(2+) release, impaired contractility and an increased risk of arrhythmias during the development of cardiac hypertrophy and failure. The NCX has also been implicated in the mechanism of arrhythmias and cellular injury associated with ischaemia and reperfusion. Hence, NCX blockade represents a potential therapeutic strategy for treating cardiac disease, however, its reversibility and electrogenic properties must be taken into consideration when predicting the outcome. NCX inhibition has been demonstrated to be protective against ischaemic injury and to have a positive inotropic and antiarrhythmic effect in failing heart cells. However, progress has been impaired by the absence of clinically useful agents. Two drugs, KB-R7943 and SEA-0400, have been developed as NCX blockers but both lack specificity. Selective peptide inhibitors have been well characterised but are active only when delivered to the intracellular space. Gene therapy strategies may circumvent the latter problem in the future. This review discusses the effects of NCX blockade, supporting its potential as a new cardiovascular therapeutic strategy.

Increased Na+-Ca2+ exchange (NCX) activity in heart failure and hypertrophy may compensate for depressed sarcoplasmic reticular Ca2+ uptake, provide inotropic support through reverse-mode Ca2+ entry, and/or deplete intracellular Ca2+ stores. NCX is electrogenic and depends on Na+ and Ca2+ transmembrane gradients, making it difficult to predict its effect on the action potential (AP). Here, we examine the effect of [Na+]i on the AP in myocytes from normal and pacing-induced failing canine hearts and estimate the direction of the NCX driving force using simultaneously recorded APs and Ca2+ transients. AP duration shortened with increasing [Na+]i and was correlated with a shift in the reversal point of the NCX driving force. At [Na+]i > or =10 mmol/L, outward NCX current during the plateau facilitated repolarization, whereas at 5 mmol/L [Na+]i, NCX had a depolarizing effect, confirmed by partially inhibiting NCX with exchange inhibitory peptide. Exchange inhibitory peptide shortened the AP duration at 5 mmol/L [Na+]i and prolonged it at [Na+]i > or =10 mmol/L. With K+ currents blocked, total membrane current was outward during the late plateau of an AP clamp at 10 mmol/L [Na+]i and became inward close to the predicted reversal point for the NCX driving force. The results were reproduced using a computer model. These results indicate that NCX plays an important role in shaping the AP of the canine myocyte, helping it to repolarize at high [Na+]i, especially in the failing heart, but contributing a depolarizing, potentially arrhythmogenic, influence at low [Na+]i.

Subcellular localization of nitric oxide (NO) synthases with effector molecules is an important regulatory mechanism for NO signalling. In the heart, NO inhibits L-type Ca2+ channels but stimulates sarcoplasmic reticulum (SR) Ca2+ release, leading to variable effects on myocardial contractility. Here we show that spatial confinement of specific NO synthase isoforms regulates this process. Endothelial NO synthase (NOS3) localizes to caveolae, where compartmentalization with beta-adrenergic receptors and L-type Ca2+ channels allows NO to inhibit beta-adrenergic-induced inotropy. Neuronal NO synthase (NOS1), however, is targeted to cardiac SR. NO stimulation of SR Ca2+ release via the ryanodine receptor (RyR) in vitro, suggests that NOS1 has an opposite, facilitative effect on contractility. We demonstrate that NOS1-deficient mice have suppressed inotropic response, whereas NOS3-deficient mice have enhanced contractility, owing to corresponding changes in SR Ca2+ release. Both NOS1-/- and NOS3-/- mice develop age-related hypertrophy, although only NOS3-/- mice are hypertensive. NOS1/3-/- double knockout mice have suppressed beta-adrenergic responses and an additive phenotype of marked ventricular remodelling. Thus, NOS1 and NOS3 mediate independent, and in some cases opposite, effects on cardiac structure and function.

Background: Altered excitation-contraction (E-C) coupling in canine pacing-induced heart failure involves decreased sarcoplasmic reticulum (SR) Ca uptake and enhanced Na/Ca exchange, which could be expected to decrease SR Ca content (Ca(SR)) and may explain the reduced intracellular Ca (Ca(i)) transient. Studies in other failure models have suggested that the intrinsic coupling between L-type Ca current (I:(Ca,L)) and SR Ca release is reduced without a change in SR Ca load. The present study investigates whether Ca(SR) and/or coupling is altered in midmyocardial myocytes from failing canine hearts (F)., Methods and Results: Myocytes were indo-1-loaded via patch pipette (37 degrees C), and Ca(i) transients were elicited with voltage-clamp steps applied at various frequencies. I(Ca,L) density was not significantly decreased in F, but steady-state Ca(i) transients were reduced to 20% to 40% of normal myocytes (N). Ca(SR), measured by integrating Na/Ca exchange currents during caffeine-induced release, was profoundly decreased in F, to 15% to 25% of N. When Ca(SR) was normalized in F by preloading in 5 mmol/L external Ca before a test pulse at 2 mmol/L Ca, a normal-amplitude Ca(i) transient was elicited. E-C coupling gain was dependent on Ca(SR) but was affected similarly in both groups, indicating that intrinsic coupling is unaltered in F., Conclusions: A decrease in Ca(SR) is sufficient to explain the diminished Ca(i) transients in F, without a change in the effectiveness of coupling. Therefore, therapeutic approaches that increase Ca(SR) may be able to fully correct the Ca handling deficit in heart failure.

Defective excitation-contraction coupling in heart failure is generally associated with both a reduction in sarcoplasmic reticulum (SR) Ca(2+) uptake and a greater dependence on transsarcolemmal Na(+)-Ca(2+) exchange (NCX) for Ca(2+) removal. Although a relative increase in NCX is expected when SR function is impaired, few and contradictory studies have addressed whether there is an absolute increase in NCX activity. The present study examines in detail NCX density and function in left ventricular midmyocardial myocytes isolated from normal or tachycardic pacing-induced failing canine hearts. No change of NCX current density was evident in myocytes from failing hearts when intracellular Ca(2+) ([Ca(2+)](i)) was buffered to 200 nmol/L. However, when [Ca(2+)](i) was minimally buffered with 50 micromol/L indo-1, Ca(2+) extrusion via NCX during caffeine application was doubled in failing versus normal cells. In other voltage-clamp experiments in which SR uptake was blocked with thapsigargin, both reverse-mode and forward-mode NCX currents and Ca(2+) transport were increased >2-fold in failing cells. These results suggest that, in addition to a relative increase in NCX function as a consequence of defective SR Ca(2+) uptake, there is an absolute increase in NCX function that depends on [Ca(2+)](i) in the failing heart.

The characteristics of nickel (Ni) block of L-type Ca current (I(Ca, L)) were studied in whole cell patch-clamped guinea pig cardiac myocytes at 37 degrees C in the absence and presence of 100 microM cAMP in the pipette solution. Ni block of peak I(Ca,L) had a dissociation constant (K(d)) of 0.33 +/- 0.03 mM in the absence of cAMP, whereas in the presence of cAMP, the K(d) was 0.53 +/- 0.05 mM (P = 0.006). Ni blocked Ca entry via Ca channels (measured as I(Ca, L) integral over 50 ms) with similar kinetics (K(d) of 0.35 +/- 0.03 mM in cAMP-free solution and 0.30 +/- 0.02 mM in solution with cAMP, P = not significant). Under both conditions, 5 mM Ni produced a maximal block that was complete for the first pulse after application. Ni block of I(Ca,L) was largely use independent. Ni (0. 5 mM) induced a positive shift (4 to 6 mV) in the activation curve of I(Ca,L). The block of I(Ca,L) by 0.5 mM Ni was independent of prepulse membrane potential (over the range of -120 to -40 mV). Ni (0.5 mM) also induced a significant shift in I(Ca,L) inactivation: by 6 mV negative in cAMP-free solution and by 4 mV positive in cells dialyzed with 100 microM cAMP. These data suggest that, in addition to blocking channel conductance by binding to a site in the channel pore, Ni may bind to a second site that influences the voltage-dependent gating of the L-type Ca channel. They also suggest that Ca channel phosphorylation causes a conformational change that alters some effects of Ni. The results may be relevant to excitation-contraction coupling studies, which have employed internal cAMP dialysis, and where Ni has been used to block I(Ca,L) and Ca entry into cardiac cells.

In heart muscle, strong evidence shows that excitation-contraction coupling involves Ca-induced Ca-release. However, under some conditions, single heart cells show Ca release and contraction which is not correlated with Ca entry via the Ca channel, suggesting a second Ca-independent release mechanism. Similar observations were made in early, pioneering studies using voltage-clamped multi-cellular preparations. We review the influence that experimental preparations and conditions have had on excitation-contraction coupling theory over the last 20 years.

In many mammalian tissue types an integral membrane protein--the sodium/calcium (Na/Ca) exchanger--plays a key role in intracellular Ca homeostasis, and evidence suggests that Na/Ca exchange function can be modulated by cAMP-dependent phosphorylation. External Nickel (Ni) ions are used widely to inhibit the exchange but little is known about the mode of Ni action. In guinea-pig ventricular myocytes, we investigated inhibition of Na/Ca exchange by external Ni under phosphorylated (cells dialysed with cAMP) and non-phosphorylated conditions. Ventricular myocytes were isolated from adult guinea-pig hearts, recordings were made at 37 degrees C using the whole-cell patch clamp technique. Internal and external solutions were used which allowed Na/Ca exchange current (INaCa) to be measured during a descending voltage ramp protocol (+80 to -120 mV) applied from a holding potential of -40 mV. The application of 10 mM Ni caused a maximal block of INaCa since inhibition was identical to that when a Na- and Ca-free (0Na/0Ca) solution was superfused externally. Kinetics of Ni-block of INaCa were assessed using applications of different external [Ni] to cells dialysed internally with cAMP-free and 100 microM cAMP-containing solutions. At +60 mV, Ni inhibited INaCa in cells dialysed with a cAMP-free solution with a dissociation constant (KD) of 0.29 +/- 0.03 mM and the data were fitted with a Hill coefficient of 0.89 +/- 0.07 (n = 9 cells). In cells dialysed with 100 microM cAMP the exchange was inhibited by Ni with a KD of 0.16 +/- 0.05 mM, the Hill coefficient was 0.82 +/- 0.16 (n = 6-7 cells). The KD and Hill coefficient values obtained in cells dialysed with cAMP-free and cAMP-containing solutions were not significantly different. Inhibition of INaCa by Ni did not appear to be voltage-dependent, was maximal within 3-4 s of application and was rapidly reversible. With cAMP-free internal dialysate, inhibition was 'mixed' showing competition with external Ca and a degree of non-competitive block. With 100 microM cAMP the inhibition appeared to be more non-competitive. We conclude that, under these experimental conditions, a concentration of external Ni of 10 mM is sufficient to produce maximal inhibition of INaCa in guinea-pig cardiac cells.

It is widely believed that Ca release from the sarcoplasmic reticulum (SR) in heart muscle is due to "Ca-induced Ca-release" (CICR), triggered by transmembrane Ca entry. However, in intact guinea-pig cells or cells dialysed with cAMP there may be an additional mechanism - SR release may be activated directly by membrane depolarisation without Ca entry. The first objective of the present study was to investigate whether this "voltage-activated Ca release" (VACR) mechanism is present across species such as rabbit, rat and guinea-pig. The second objective was to characterise the dependence of a VACR mechanism on internal [cAMP]. Membrane current was measured with the whole-cell patch-clamp technique, intracellular [Ca] was monitored with Fura-2 (or a combination of Fluo-3/SNARF-1). Rapid changes of superfusate (within 100 ms) were made using a system which maintained cell temperature at 37 degrees C. We used a train of conditioning pulses to ensure a standard SR load before each test pulse. In rabbit myocytes dialysed with 100 microM cAMP, 89.6 +/- 7.0% of the control intracellular Ca (Cai) transient was still elicited by depolarisation during a switch to 5 mM Ni, which blocked pathways for Ca entry. This suggested that rabbit myocytes possess a VACR mechanism. The percentage of control Cai transient elicited by depolarisation in the presence of 5 mM Ni (i.e. magnitude of VACR) increased in a graded fashion with the pipette [cAMP] between zero and 100 microM. In rat myocytes dialysed with 50 microM cAMP, 64.4 +/- 6.2% of SR release was activated by depolarisation in the presence of 5 mM Ni, suggesting the presence of a VACR mechanism. The extent to which VACR triggered SR release increased with the pipette [cAMP] between zero and 50 microM. In guinea-pig myocytes dialysed with 100 microM cAMP, 74.6 +/- 3.6% of the control Cai transient was elicited by depolarisation in the presence of 5 mM Ni. The degree to which VACR triggered SR release was also graded with the pipette [cAMP] between zero and 100 microM. It therefore appears that each of the three species might possess a VACR mechanism which can be modulated by the internal [cAMP]. This may reflect an effect of cAMP to phosphorylate key proteins involved in excitation-contraction coupling. Under normal physiological conditions with a basal [cAMP] between 2 and 20 microM, VACR may play a role in triggering SR release. The role of VACR may increase under conditions which increase internal [cAMP].

A number of clinical cardiac disorders may be associated with a rise of the intracellular Na concentration (Na(i)) in heart muscle. A clear example is digitalis toxicity, in which excessive inhibition of the Na/K pump causes the Na(i) concentration to become raised above the normal level. Especially in digitalis toxicity, but also in many other situations, the rise of Na(i) may be an important (or contributory) cause of increased cardiac arrhythmias. In this review, we consider the mechanisms by which a raised Na(i) may cause cardiac arrhythmias. First, we describe the factors that regulate Na(i), and we demonstrate that the equilibrium level of Na(i) is determined by a balance between Na entry into the cell, and Na extrusion from the cell. A number of mechanisms are responsible for Na entry into the cell, whereas the Na/K pump appears to be the main mechanism for Na extrusion. We then consider the processes by which an increased level of Nai might contribute to cardiac arrhythmias. A rise of Na(i) is well known to result in an increase of intracellular Ca, via the important and influential Na/Ca exchange mechanism in the cell membrane of cardiac muscle cells. A rise of intracellular Ca modulates the activity of a number of sarcolemmal ion channels and affects release of intracellular Ca from the sarcoplasmic reticulum, all of which might be involved in causing arrhythmia. It is possible that the increase in contractile force that results from the rise of intracellular Ca may initiate or exacerbate arrhythmia, since this will increase wall stress and energy demands in the ventricle, and an increase in wall stress may be arrhythmogenic. In addition, the rise of Na(i) is anticipated to modulate directly a number of ion channels and to affect the regulation of intracellular pH, which also may be involved in causing arrhythmia. We also present experiments in this review, carried out on the working rat heart preparation, which suggest that a rise of Na(i) causes an increase of wall stress-induced arrhythmia in this model. In addition, we have investigated the effect on wall stress-induced arrhythmia of maneuvers that might be anticipated to change intracellular Ca, and this has allowed identification of some of the factors involved in causing arrhythmia in the working rat heart.

We investigated the effect of external Cd2+ on the Na/Ca exchange and the L-type Ca channel current (ICa,L) in whole cell patch-clamped rabbit ventricular myocytes at 36 degrees C. After the interfering ion channels and the Na/K pump were blocked, the exchange current was measured as the membrane current that was inhibited by 5 mM nickel. External Cd2+ inhibited Na/Ca exchange with a dissociation constant (KD) of 320.6 +/- 12.4 microM and a Hill coefficient of 1.5 +/- 0.09 (n = 13 cells) and ICa,L with a KD of 2.14 +/- 0.15 microM and a Hill coefficient of 0.74 +/- 0.03 (n = 11 cells). We observed some overlap in the Cd2+ concentration that blocked each mechanism. Cd2+ (100-500 microM) is used commonly to block ICa,L completely. However, 100 microM Cd2+ also inhibits 20% of the Na/Ca exchange activity, whereas 500 microM Cd2+ inhibits 60%.

We investigated the effect in rabbit ventricular myocytes of "FRCRCFa", a newly developed peptide inhibitor of the Na/Ca exchange. Myocytes were whole-cell patch clamped and experiments were carried out at 36 degrees C. The Na/Ca exchange was measured selectively, by blocking interfering ion channel currents and the Na/K pump, as the membrane current which could be inhibited by 5 mM nickel (Ni; a known blocker of the Na/Ca exchange). Increasing concentrations of FRCRCFa dialysed into the cell from the patch-pipette inhibited the Na/Ca exchange current. The dose/response curve could be fitted by a function for co-operative ligand binding, which predicted a KD for FRCRCFa-mediated inhibition of 22.7 +/- 3.7 nM, with a Hill coefficient of 0.61 +/- 0.06. Pipette FRCRCFa concentrations of 1 micro;M and above were sufficient to cause complete inhibition of Na/Ca exchange current. The inhibitory effect of FRCRCFa was independent of membrane potential and relatively selective: 10 micro;M FRCRCFa dialysed into the cell had no effect on the L-type Ca current and delayed rectifier and inward rectifier K currents. Thus FRCRCFa appears to be a potent and relatively selective inhibitor of the Na/Ca exchange in intact cardiac myocytes, and may be of value for studies of the Na/Ca exchange.