Simulation-based training improves process times in acute stroke care (STREAM).

BACKGROUND: The objective of the STREAM Trial was to evaluate the effect of simulation training on process times in acute stroke care. METHODS: The multicenter prospective interventional STREAM Trial was conducted between 10/2017 and 04/2019 at seven tertiary care neurocenters in Germany with a pre- and post-interventional observation phase. We recorded patient characteristics, acute stroke care process times, stroke team composition and simulation experience for consecutive direct-to-center patients receiving intravenous thrombolysis (IVT) and/or endovascular therapy (EVT). The intervention consisted of a composite intervention centered around stroke-specific in situ simulation training. Primary outcome measure was the 'door-to-needle' time (DTN) for IVT. Secondary outcome measures included process times of EVT and measures taken to streamline the pre-existing treatment algorithm. RESULTS: The effect of the STREAM intervention on the process times of all acute stroke operations was neutral. However, secondary analyses showed a DTN reduction of 5 min from 38 min pre-intervention (interquartile range [IQR] 25-43 min) to 33 min (IQR 23-39 min, p = 0.03) post-intervention achieved by simulation-experienced stroke teams. Concerning EVT, we found significantly shorter door-to-groin times in patients who were treated by teams with simulation experience as compared to simulation-naive teams in the post-interventional phase (-21 min, simulation-naive: 95 min, IQR 69-111 vs. simulation-experienced: 74 min, IQR 51-92, p = 0.04). CONCLUSION: An intervention combining workflow refinement and simulation-based stroke team training has the potential to improve process times in acute stroke care.

Role of the Cysteine 81 Residue of Macrophage Migration Inhibitory Factor as a Molecular Redox Switch.

Macrophage migration inhibitory factor (MIF) is a pro-inflammatory and tumor-promoting cytokine that occurs in two redox-dependent immunologically distinct conformational isoforms. The disease-related structural isoform of MIF (oxMIF) can be specifically and predominantly detected in the circulation of patients with inflammatory diseases and in tumor tissue, whereas the ubiquitously expressed isoform of MIF (redMIF) is abundantly expressed in healthy and diseased subjects. In this article, we report that cysteine 81 within MIF serves as a "switch cysteine" for the conversion of redMIF to oxMIF. Modulating cysteine 81 by thiol reactive agents leads to significant structural rearrangements of the protein, resulting in a decreased ß-sheet content and an increased random coil content, but maintaining the trimeric quaternary structure. This conformational change in the MIF molecule enables binding of oxMIF-specific antibodies BaxB01 and BaxM159, which showed beneficial activity in animal models of inflammation and cancer. Crystal structure analysis of the MIF-derived EPCALCS peptide, bound in its oxMIF-like conformation by the Fab fragment of BaxB01, revealed that this peptide adopts a curved conformation, making the central thiol protein oxidoreductase motif competent to undergo disulfide shuffling. We conclude that redMIF might reflect a latent zymogenic form of MIF, and formation of oxMIF leads to a physiologically relevant, i.e., enzymatically active, state.

Translating bioinformatics in oncology: guilt-by-profiling analysis and identification of KIF18B and CDCA3 as novel driver genes in carcinogenesis.

MOTIVATION: Co-regulated genes are not identified in traditional microarray analyses, but may theoretically be closely functionally linked [guilt-by-association (GBA), guilt-by-profiling]. Thus, bioinformatics procedures for guilt-by-profiling/association analysis have yet to be applied to large-scale cancer biology. We analyzed 2158 full cancer transcriptomes from 163 diverse cancer entities in regard of their similarity of gene expression, using Pearson's correlation coefficient (CC). Subsequently, 428 highly co-regulated genes (|CC| ≥ 0.8) were clustered unsupervised to obtain small co-regulated networks. A major subnetwork containing 61 closely co-regulated genes showed highly significant enrichment of cancer bio-functions. All genes except kinesin family member 18B (KIF18B) and cell division cycle associated 3 (CDCA3) were of confirmed relevance for tumor biology. Therefore, we independently analyzed their differential regulation in multiple tumors and found severe deregulation in liver, breast, lung, ovarian and kidney cancers, thus proving our GBA hypothesis. Overexpression of KIF18B and CDCA3 in hepatoma cells and subsequent microarray analysis revealed significant deregulation of central cell cycle regulatory genes. Consistently, RT-PCR and proliferation assay confirmed the role of both genes in cell cycle progression. Finally, the prognostic significance of the identified KIF18B- and CDCA3-dependent predictors (P = 0.01, P = 0.04) was demonstrated in three independent HCC cohorts and several other tumors. In summary, we proved the efficacy of large-scale guilt-by-profiling/association strategies in oncology. We identified two novel oncogenes and functionally characterized them. The strong prognostic importance of downstream predictors for HCC and many other tumors indicates the clinical relevance of our findings. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Femoral cannulation: a safe vascular access option for cardiopulmonary bypass in minimally invasive cardiac surgery.

Femoral cannulation during cardiopulmonary bypass has become a common approach for many cardiac procedures and serves as an important access option, especially during minimally invasive cardiac surgery. Opponents, however, argue that there is significant risk, including site-specific and overall morbidity, which makes the use of this modality dangerous compared to conventional aortoatrial cannulation techniques. We analyzed our institutional experience to elucidate the safety and efficacy of femoral cannulation. All data were collected from a single hospital's cardiac surgery database. A total of 346 cardiac surgeries were evaluated from September 2012 to September 2013, of which 85/346 (24.6%) utilized a minimally invasive approach. Of the 346 operations performed, 72/346 (20.8%) utilized femoral cannulation while 274/346 (79.2%) used aortoatrial cannulation. Stroke occurred in 1/72 (1.39%) after femoral cannulation, specifically, in a conventional sternotomy patient, while it occurred in 6/274 (2.19%) [p=0.67] after aortoatrial cannulation. When comparing postoperative complications between the femoral cannulation and aortoatrial cannulation groups, the rates of atrial fibrillation [10/72 (13.9%) versus 46/274 (16.8%), p=0.55], renal failure [2/72 (2.78%) versus 11/274 (4.01%), p=0.62], prolonged ventilation time [4/72 (5.56%) versus 27/274 (9.85%), p=0.26] and re-operation for bleeding [3/72 (4.17%) versus 13/274 (4.74%), p=0.84] showed no significant difference. Selective femoral cannulation provides a safe alternative to aortoatrial cannulation for cardiopulmonary bypass and is especially important when performing minimally invasive cardiac surgery. When comparing aortoatrial and femoral cannulation, we found no significant difference in the postoperative complication rates and overall mortality.

Computed tomographies and cancer risk in children: a literature overview of CT practices, risk estimations and an epidemiologic cohort study proposal.

Radiation protection is a topic of great public concern and of many scientific investigations, because ionizing radiation is an established risk factor for leukaemia and many solid tumours. Exposure of the public to ionizing radiation includes exposure to background radiation, as well as medical and occupational exposures. A large fraction of the exposure from diagnostic procedures comes from medical imaging. Computed tomography (CT) is the major single contributor of diagnostic radiation exposure. An increase in the use of CTs has been reported over the last decades in many countries. Children have smaller bodies and lower shielding capacities, factors that affect the individual organ doses due to medical imaging. Several risk models have been applied to estimate the cancer burden caused by ionizing radiation from CT. All models predict higher risks for cancer among children exposed to CT as compared to adults. However, the cancer risk associated with CT has not been assessed directly in epidemiological studies. Here, plans are described to conduct an historical cohort study to investigate the cancer incidence in paediatric patients exposed to CT before the age of 15 in Germany. Patients will be recruited from radiology departments of several hospitals. Their individual exposure will be recorded, and time-dependent cumulative organ doses will be calculated. Follow-up for cancer incidence via the German Childhood Cancer Registry will allow computation of standardized incidence ratios using population-based incidence rates for childhood cancer. Dose-response modelling and analyses for subgroups of children based on the indication for and the result of the CT will be performed.

PROSE: Prospective Randomized Trial of the On-X Mechanical Prosthesis and the St Jude Medical Mechanical Prosthesis Evaluation: Part 2: Study results-prostheses, positions, and economic development.

Objectives: The Prospective Randomized On-X Mechanical Prosthesis Versus St Jude Medical Mechanical Prosthesis Evaluation (PROSE) trial purpose was to investigate whether a current-generation mechanical prosthesis (On-X; On-X Life Technologies/Artivion Inc) reduced the incidence of thromboembolic-related complications compared with a previous-generation mechanical prosthesis (St Jude Medical Mechanical Prosthesis; Abbott/St Jude Medical). This second report documents the valve-related complications by individual prostheses and by Western and Developing populations. Methods: The PROSE trial study was conducted in 28 worldwide centers and incorporated 855 subjects randomized between 2003 and 2016. The study enrollment was discontinued on August 31, 2016. The study protocol, and analyses of 10 demographic variables and 24 risk factors were published in detail in 2021. Results: The total patient population (N = 855) included patients receiving an On-X valve (n = 462) and a St Jude Medical valve (n = 393). The overall freedom evaluation showed no differences at 5 years between the prostheses for thromboembolism or for valve thrombosis. There were also no differences in mortality. There were several differences between Developing and Western populations. The freedom relations at 5 years for mortality favored Western over Developing populations. Valve thrombosis was differentiated by position and site: aortic < mitral (P = .007) and Western < Developing (P = .005). In the mitral position there were no cases in Western populations, whereas there were 8 in Developing populations (P = .217). Conclusions: The On-X valve and St Jude Medical valve performed equally well in the study with no differences found. The only differentiation occurred with valve thrombosis in the mitral position more than the aortic position and occurring in Developing more than Western populations. The occurrence of valve thrombosis was also related to a younger population possibly due to anticoagulation compliance based on record review.

In vitro sortagging of an antibody fab fragment: overcoming unproductive reactions of sortase with water and lysine side chains.

Sortase A from Staphylococcus aureus attracts growing interest for its use in biotechnological protein modification. This enzyme binds to a short signal sequence at the C terminus of a target protein, cleaves it by formation of an acyl-enzyme intermediate, and subsequently attaches an oligoglycine with a peptide bond. In this work, we explored its usability for the modification of the L19 Fab fragment (specific for fibronectin ED-B), a promising candidate for antibody-based cancer therapy. The Fab fragment was expressed with a sortase signal sequence attached to its light chain, and was successfully modified with a fluorescent oligoglycine probe in good yield. Our interest focused on performance under conditions of limited oligoglycine concentrations. Two unproductive side reactions of sortase were observed. The first was hydrolysis of the acyl-enzyme intermediate; in the second, sortase accepted the ε-amino group of lysine as substrate, thereby resulting in polypeptide crosslinking. In case of the L19 Fab fragment, it led to the covalent connection of the heavy and light chains. Both side reactions were effectively suppressed by sufficient concentrations of the oligoglycine probe.

PROSE: Prospective Randomized Trial of the On-X Mechanical Prosthesis and the St Jude Medical Mechanical Prosthesis Evaluation : Part 1(Patient Dynamics): Preoperative demographics and preoperative and operative risk factors.

OBJECTIVES: The PROSE trial purpose is to investigate whether the incidence of thromboembolic-related complications is reduced with a current generation mechanical prosthesis (On-X Life Technologies/CryoLife Inc.-On-X) compared with a previous generation mechanical prosthesis (St Jude Medical-SJM). The primary purpose of the initial report is to document the preoperative demographics, and the preoperative and operative risk factors by individual prosthesis and by Western and Developing populations. METHODS: The PROSE study was conducted in 28 worldwide centres and incorporated 855 subjects randomized between 2003 and 2016. The study enrollment was discontinued on August 31, 2016. The preoperative demographics incorporated age, gender, functional class, etiology, prosthetic degeneration, primary rhythm, primary valve lesion, weight, height, BSA and BMI. The preoperative and operative evaluation incorporated 24 risk factors. RESULTS: The total patient population (855) incorporated On-X population (462) and the St Jude Medical population (393). There was no significant difference of any of the preoperative demographics between the On-X and SJM groups. The preoperative and operative risk factors evaluation showed there was no significant difference between the On-X and St Jude Medical populations. The preoperative and operative risk factors by valve position (aortic and mitral) also documented no differentiation. The dominant preoperative demographics of the Western world population were older age, male gender, sinus rhythm, aortic stenosis, congenital aortic lesion, and mitral regurgitation. The dominant demographics of the Developing world population were rheumatic etiology, atrial fibrillation, aortic regurgitation, mixed aortic lesions, mitral stenosis and mixed mitral lesions. The Developing world group had only one significant risk factor, congestive heart failure. The majority of the preoperative and operative risk factors were significant in the Western world population. CONCLUSIONS: The preoperative demographics do not differentiate the prostheses but do differentiate the Western and Developing world populations. The preoperative and operative risk factors do not differentiate the prostheses BUT do differentiate the Western and Developing world populations.

Hypoxia inducible factor-1 improves the actions of positive inotropic agents in stunned cardiac myocytes.

1. In the present study, we tested hypothesis that upregulation of hypoxia-inducible factor-1 (HIF-1) would improve the actions of positive inotropic agents in cardiac myocytes after simulated ischaemia-reperfusion (I/R). 2. Hypoxia-inducible factor-1α was upregulated with deferoxamine (150 mg/kg per day for 2 days). Rabbit cardiac myocytes were subjected to simulated ischaemia (15 min, 95% N(2)-5% CO2) and reperfusion (re-oxygenation) and compared with control myocytes. Cell contraction and calcium transients were measured at baseline and after forskolin (10(-7) and 10(-6) mol/L) or ouabain (10(-5) and 10(-4) mol/L). 3. Under control conditions, high-dose forskolin and ouabain increased percentage shortening by 20 and 18%, respectively. Deferoxamine-treated control myocytes responded similarly. In stunned myocytes, forskolin and ouabain did not significantly increase shortening (increases of 8% and 9%, respectively). Deferoxamine restored the effects of forskolin (+26%) and ouabain (+28%) in stunning. The results for maximum shortening and relaxation rates were similar. The increased calcium transients caused by forskolin and ouabain were also depressed in stunned myocytes, but were maintained by HIF-1 upregulation. 4. These results suggest that simulated I/R impaired the functional and calcium transient effects of positive inotropic agents. Upregulation of HIF-1 protects cardiac myocyte function after I/R by maintaining calcium release.

SERCA inhibition limits the functional effects of cyclic GMP in both control and hypertrophic cardiac myocytes.

The negative functional effects of cyclic GMP are controlled by the sarcoplasmic reticulum calcium-ATPase (SERCA). The effects of cyclic GMP are blunted in cardiac hypertrophy. We tested the hypothesis that the interaction between cyclic GMP and SERCA would be reduced in hypertrophic cardiac myocytes. Myocytes were isolated from 7 control and 7 renal-hypertensive hypertrophic rabbits. Control and hypertrophic myocytes received 8-bromo-cGMP (8-Br-cGMP; 10(-7), 10(-6), 10(-5) mol/l), the SERCA blocker thapsigargin (10(-8) mol/l) followed by 8-Br-cGMP, or the SERCA blocker, cyclopiazonic acid (CPA; 10(-7) mol/l) followed by 8-Br-cGMP. Percent shortening and maximal rate of shortening and relaxation were recorded using a video edge detector. Changes in cytosolic Ca2+ were assessed in fura 2-loaded myocytes. In controls, 8-Br-cGMP caused a significant 36% decrease in percent shortening from 5.8 +/- 0.4 to 3.7 +/- 0.3%. Thapsigargin and CPA did not affect basal control or hypertrophic myocyte function. When 8-Br-cGMP was given following thapsigargin or CPA, the negative effects of 8-Br-cGMP on control myocyte function were reduced. In hypertrophic myocytes, 8-Br-cGMP caused a smaller but significant 17% decrease in percent shortening from 4.7 +/- 0.2 to 3.9 +/- 0.1%. When 8-Br-cGMP was given following thapsigargin or CPA, no significant changes occurred in hypertrophic cell function. Intracellular Ca2+ transients responded in a similar manner to changes in cell function in control and hypertrophic myocytes. These results show that the effects of cyclic GMP were reduced in hypertrophic myocytes, but this was not related to SERCA. In presence of SERCA inhibitors, the responses to cyclic GMP were blunted in hypertrophic as well as control myocytes.

Conducting effective tailgate trainings.

The California Department of Health Services' Occupational Health Branch and others have identified the construction industry as being at high risk for injuries, illnesses, and fatalities. Effective tailgate trainings (brief job site safety meetings) can be a powerful tool to promote hazard awareness and safe work practices. The authors found that many contractors and supervisors conducted ineffective tailgate trainings. They developed the BuildSafe California Project to assist contractors to have more effective programs by holding 25 training-of-trainers sessions reaching 1,525 participants. The needs assessment, intervention, and evaluation results from the first 18 trainings are presented. Eighty-six percent of the participants found the program "very helpful." Participants used the materials and made improvements in the quality and frequency of trainings. Supervisors must be skilled at conducting tailgate trainings as part of their responsibilities. There is a serious need to provide more culturally appropriate safety training in a workforce increasingly made up of Latino workers.

Hypoxia inducible factor-1 improves the actions of nitric oxide and natriuretic peptides after simulated ischemia-reperfusion.

Ischemia-reperfusion reduces the negative functional effects of cyclic GMP in cardiac myocytes. In this study, we tested the hypothesis that upregulation of hypoxic inducible factor-1 (HIF-1) would improve the actions of cyclic GMP signaling following simulated ischemia-reperfusion. HIF-1 alpha was increased with deferoxamine (150 mg/kg for 2 days). Rabbit cardiac myocytes were subjected to simulated ischemia [15 min 95% N(2)-5% CO(2)] and reperfusion [reoxygenation] to produce myocyte stunning. Cell function was measured utilizing a video-edge detector. Shortening was examined at baseline and after brain natriuretic peptide (BNP, 10(-8), 10(-7)M) or S-nitroso-N-acetyl-penicillamine (SNAP, 10(-6), 10(-5)M) followed by KT5823 (cyclic GMP protein kinase inhibitor, 10(-6)M). Kinase activity was measured via a protein phosphorylation assay. Under control conditions, BNP (-30%) and SNAP (-41%) reduced percent shortening, while KT5823 partially restored function (+18%). Deferoxamine treated control myocytes responded similarly. In stunned myocytes, BNP (-21%) and SNAP (-25%) reduced shortening less and KT5823 did not increase function (+2%). Deferoxamine increased the effects of BNP (-38%) and SNAP (-41%) in stunning and restored the effects of KT5823 (+12%). The cyclic GMP protein kinase increased phosphorylation of several proteins in control HIF-1 +/- cells. Phosphorylation was reduced in stunned cells and was restored in deferoxamine treated stunned cells. This study demonstrated that simulated ischemia-reperfusion reduced the negative functional effects of increasing cyclic GMP and this was related to reduced effects of the cyclic GMP protein kinase. Increased HIF-1 alpha protects the functional effects of cyclic GMP thorough maintenance of cyclic GMP protein kinase activity after ischemic-reperfusion.

T4-induced cardiac hypertrophy disrupts cyclic GMP mediated responses to brain natriuretic peptide in rabbit myocardium.

Brain natriuretic peptide (BNP) affects the regulation of myocardial metabolism through the production of cGMP and these effects may be altered by cardiac hypertrophy. We tested the hypothesis that BNP would cause decreased metabolism and function in the heart and cardiac myocytes by increasing cGMP and that these effects would be disrupted after thyroxine-induced cardiac hypertrophy (T4). Open-chest control and T4 rabbits were instrumented to determine local effects of epicardial BNP (10(-3) M). Function of isolated cardiac myocytes was examined with BNP (10(-8)-10(-7) M) with or without KT5823 (10(-6) M, cGMP protein kinase inhibitor). Cyclic GMP levels were measured in myocytes. In open-chest controls, O2 consumption was reduced in the BNP area of the subepicardium (6.6+/-1.3 ml O2/min/100 g versus 8.9+/-1.4 ml O2/min/100 g) and subendocardium (9.4+/-1.3 versus 11.3+/-0.99). In T4 animals, functional and metabolic rates were higher than controls, but there was no difference between BNP-treated and untreated areas. In isolated control myocytes, BNP (10(-7) M) reduced percent shortening (PSH) from 6.5+/-0.6 to 4.3+/-0.4%. With KT5823 there was no effect of BNP on PSH. In T4 myocytes, BNP had no effect on PSH. In control myocytes, BNP caused cGMP levels to rise from 279+/-8 to 584+/-14 fmol/10(5) cells. In T4 myocytes, baseline cGMP levels were lower (117+/-2 l) and were not significantly increased by BNP. Thus, BNP caused decreased metabolism and function while increasing cGMP in control. These effects were lost after T4 due to lack of cGMP production. These data indicated that the effects of BNP on heart function operated through a cGMP-dependent mechanism, and that this mechanism was disrupted in T4-induced cardiac hypertrophy.

Importance of ryanodine receptors in effects of cyclic GMP is reduced in thyroxine-induced cardiac hypertrophy.

We tested the hypothesis that the negative functional effects of cyclic GMP were mediated by ryanodine receptors, and that these effects would be reduced in thyroxine (thyroxine, 0.5 mg/kg/day, 16 days)-induced hypertrophic myocytes. Using rabbit ventricular myocytes from control (n=9) and thyroxine (n=9) hearts, percent cell shortening (%) and maximum rate of contraction and relaxation were determined using a video edge detector at baseline and after 10(-6), 10(-5) M 8-bromo-cyclic GMP. Dantrolene 10(-6) M, ryanodine receptor inhibitor, was added alone or after 8-Br-cGMP treatment. Changes in cytosolic Ca(2+) concentration were assessed in fura-2-loaded control and thyroxine myocytes. 8-Br-cGMP caused a significant decrease in percent shortening, from 5.3+/-0.9% to 3.9+/-0.6% at 10(-5 )M in control, and 3.4+/-0.3% to 2.6+/-0.4% in thyroxine myocytes. Dantrolene significantly decreased percent shortening from 4.5+/-0.8% to 3.7+/-0.1% in control and from 3.7+/-0.1% to 2.8+/-0.3% in thyroxine myocytes. In 8-Br-cGMP treated control myocytes, dantrolene did not significantly change myocyte contractility, which suggested that cyclic GMP acted on ryanodine receptors. However, in 8-Br-cGMP treated thyroxine myocytes, dantrolene further reduced myocyte contractility implying that the interaction of cyclic GMP and ryanodine receptors appeared to be interrupted in thyroxine myocytes. Maximum rate of contraction data were consistent with the percent cell shortening data and Ca(2+) transients changed similarly to myocyte contractility. We conclude that effects of cyclic GMP on myocytes contractility were partially mediated though interaction with ryanodine receptors and the subsequent decrease in cytosolic calcium levels. This interaction was reduced in thyroxine hypertrophic myocytes.

Chronic nitric oxide synthase blockade desensitizes the heart to the negative metabolic effects of nitric oxide.

The consequences of chronic nitric oxide synthase (NOS) blockade on the myocardial metabolic and guanylyl cyclase stimulatory effects of exogenous nitric oxide (NO) were determined. Thirty-three anesthetized open-chest rabbits were randomized into four groups: control, NO donor S-nitroso-N-acetyl-penicillamine (SNAP, 10(-4 )M), NOS blocking agent N(G)-nitro-L-arginine methyl ester (L-NAME, 20 mg/kg/day) for 10 days followed by a 24 hour washout and L-NAME for 10 days followed by a 24 hour washout plus SNAP. Myocardial O(2) consumption was determined from coronary flow (microspheres) and O(2) extraction (microspectrophotometry). Cyclic GMP and guanylyl cyclase activity were determined by radioimmunoassay. There were no baseline metabolic, functional or hemodynamic differences between control and L-NAME treated rabbits. SNAP in controls caused a reduction in O(2) consumption (SNAP 5.9+/-0.6 vs. control 8.4+/-0.8 ml O(2)/min/100 g) and a rise in cyclic GMP (SNAP 18.3+/-3.8 vs. control 10.4+/-0.9 pmol/g). After chronic L-NAME treatment, SNAP caused no significant changes in O(2) consumption (SNAP 7.1+/-0.8 vs. control 6.4+/-0.7) or cyclic GMP (SNAP 14.2+/-1.8 vs. control 12.1+/-1.3). In controls, guanylyl cyclase activity was significantly stimulated by SNAP (216.7+/-20.0 SNAP vs. 34.4+/-2.5 pmol/mg/min base), while this increase was blunted after L-NAME (115.9+/-24.5 SNAP vs. 24.9+/-4.7 base). These results demonstrated that chronic NOS blockade followed by washout blunts the response to exogenous NO, with little effect on cyclic GMP or myocardial O(2) consumption. This was related to reduced guanylyl cyclase activity after chronic L-NAME. These results suggest that, unlike many receptor systems, the NO-cyclic GMP signal transduction system becomes downregulated upon chronic inhibition.

Cyclic GMP signaling and regulation of SERCA activity during cardiac myocyte contraction.

We tested the hypothesis that cGMP-induced reductions in cardiac myocyte function were related to activation of the sarcoplasmic reticulum Ca2+-ATPase (SERCA) and cGMP-dependent phosphorylation of phospholamban. Ventricular myocyte function was measured using a video edge detector (n = 11 rabbits). Thapsigargin (TG) or cyclopiazonic acid (CPA) were used to inhibit SERCA. 8-Bromo-cGMP was added at 10(-6), 10(-5) M followed by TG 10(-8) M or KT5823 (cGMP-protein kinase inhibitor, 10(-6) M) prior to TG or CPA. Cyclic GMP-dependent protein phosphorylation and immunoblotting with anti-phospholamban antibody were examined. TG 10(-8) M significantly increased percent shortening (from 6.6+/-0.7 to 9.1+/-1.3%). Cyclic GMP 10(-5) M significantly decreased cell shortening from 9.3+/-0.9 to 5.1+/-0.6%. This was partially reversed by KT5823 (5.1+/-0.6 to 8.2+/-1.4%) suggesting that negative functional effects of cGMP were partially through the cGMP-dependent protein kinase. Addition of TG after cGMP also reduced the negative effects of cGMP on myocyte shortening suggesting involvement of SERCA in cGMP signaling. TG after cGMP and KT5823 treatment did not alter myocyte contractility (8.2+/-1.4 to 7.2+/-1.3%). CPA had similar effects as those of TG. Protein phosphorylation and immunoblotting showed that phospholamban was a target of the cGMP protein kinase. These results indicated that the cyclic GMP-induced reductions in myocyte function were partially mediated through the action of SERCA. It further suggested that cGMP signaling affects myocyte function through phosphorylation of phospholamban which regulates SERCA activity.

Diabetes mellitus increases short-term mortality and morbidity in patients undergoing coronary artery bypass graft surgery.

OBJECTIVES: The aim of this study was to determine the impact of diabetes mellitus (DM) on short-term mortality and morbidity in patients undergoing coronary artery bypass surgery (CABG). BACKGROUND: Diabetes mellitus is present in approximately 20% to 30% of patients undergoing CABG, and the impact of diabetes on short-term outcome is unclear. METHODS: We performed a retrospective cohort study in 434 hospitals from North America. The study population included 146,786 patients undergoing CABG during 1997: 41,663 patients with DM and 105,123 without DM. The primary outcome was 30-day mortality. Secondary outcomes were in-hospital morbidity, infections and composite outcomes of mortality or morbidity and mortality or infection. RESULTS: The 30-day mortality was 3.7% in patients with DM and 2.7% in those without DM; the unadjusted odds ratio was 1.40 (95% confidence interval [CI], 1.31 to 1.49). After adjusting for other baseline risk factors, the overall adjusted odds ratio for diabetics was 1.23 (95% CI, 1.15 to 1.32). Patients treated with oral hypoglycemic medications had adjusted odds ratio 1.13; 95% CI, 1.04 to 1.23, whereas those on insulin had an adjusted odds ratio 1.39; 95% CI, 1.27 to 1.52. Morbidity, infections and the composite outcomes occurred more commonly in diabetic patients and were associated with an adjusted risk about 35% higher in diabetics than nondiabetics, particularly among insulin-treated diabetics (adjusted risk between 1.5 to 1.61). CONCLUSIONS: Diabetes mellitus is an important risk factor for mortality and morbidity among those undergoing CABG. Research is needed to determine if good control of glucose levels during the perioperative time period improves outcome.

Atrial natriuretic peptide reverses the negative functional effects of stunning in rabbit myocardium.

We tested the hypothesis that atrial natriuretic peptide (ANP) would decrease both the effects of myocardial stunning and oxygen consumption in rabbit hearts. In two groups of anesthetized open-chest rabbits, myocardial stunning was produced by two 15 min occlusions of the left anterior descending (LAD) artery separated by 15 min of reperfusion. Either ANP (0.2 mg) or vehicle (lactated Ringers) was then injected into the affected area of the left ventricle. In a third group, ANP was injected into the LAD region of non-stunned rabbits. Hemodynamic (heart rate, aortic and left ventricular pressures) and functional (wall thickening (WT), delay of onset of WT, and rate of WT) parameters were measured. Coronary blood flow (microspheres) and O2 extraction (microspectrophotometry) were used to determine myocardial O2 consumption. Stunning was demonstrated by an increase in the time delay to contraction and depressed WT. In the control group, baseline delay to contraction was 25+/-7 ms, and this increased to 84+/-16 following stunning and vehicle administration. In the ANP group, baseline delay was 20+/-6 at baseline and after stunning and ANP administration it was 30+/-7. Wall thickening decreased by approximately 30% with stunning and vehicle but only 8% in the ANP treated hearts. Stunning did not affect regional O2 consumption (6.0+/-1.1 stunned vs. 7.4+/-1.2 mlO2/min/100g non-stunned). ANP administration did not affect O2 consumption (7.3+/-1.7 stunned vs. 6.4+/-1.0 non-stunned). We therefore concluded that ANP administration reversed the effects of stunning without alteration in local O2 consumption in stunned myocardium.

Effects of natriuretic peptides on ventricular myocyte contraction and role of cyclic GMP signaling.

Natriuretic peptides, including atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP) act through different receptors and at different potencies to affect cardiac myocyte function. We tested the hypothesis that these three peptides would differentially reduce cardiomyocyte function through their effects on the cyclic GMP signaling pathway. Rabbit ventricular myocytes were isolated and stimulated by electrical field stimulation. Cell function was measured using a video edge detector. ANP BNP or CNP at 10(-9), 10(-8), 10(-7) M were added to the myocytes. Intracellular cyclic GMP was determined using a radioimmunoassay in the absence or presence of ANP, BNP or CNP. All natriuretic peptides decreased myocyte contractility in a similar concentration dependent manner. Myocyte percentage shortening was significantly decreased with all peptides at 10(-7) M compared with baseline (ANP from 5.4+/-0.4 to 3.9+/-0.2%; BNP from 5.0+/-0.2 to 3.5+/-0.1%; CNP from 5.6+/-0.3 to 4.0+/-0.3%). Maximum rate of shortening and relaxation were also decreased similarly and significantly. Intracellular cyclic GMP was significantly increased in myocytes treated with ANP, BNP or CNP (Baseline 1.0+/-0.2, ANP 2.1+/-0.2, BNP 2.3+/-0.3, CNP 2.0+/-0.2 pmol/10(5) myocytes). Furthermore, inhibition of the cyclic GMP protein kinase with KT5823 caused a reversal in the functional effects of CNP. We concluded that all natriuretic peptides had similar negative effects on ventricular myocyte function and their effects were accompanied by increased cyclic GMP. Blockade the effect of CNP by a cyclic GMP protein kinase inhibitor demonstrated that effects were mediated through the cyclic GMP signaling pathway.

Differential effects of cGMP produced by soluble and particulate guanylyl cyclase on mouse ventricular myocytes.

Particulate guanylyl cyclase (pGC) and soluble guanylyl cyclase (sGC) are cGMP-generation systems distributed in different intracellular locations. Our aim was to test the hypothesis that the functional effects of cGMP produced by pGC and sGC on contraction and Ca2+ transients would differ in ventricular myocytes. We measured myocyte shortening from adult mice using a video edge-detector and investigated the functional changes after stimulating pGC with C-type natriuretic peptide (CNP; 10(-8) M and 10(-7) M) or sGC with S-nitroso-N-acetyl-penicillamine (SNAP; nitric oxide donor; 10(-6) M and 10(-5) M). Significant concentration-dependent decreases in percentage shortening (PCS), maximal rate of shortening (RSmax), and relaxation (RRmax) were produced by CNP. To a similar degree, SNAP concentration-dependently reduced PCS, RSmax, and RRmax. The addition of Rp-8-[(4-chlorophenyl)thio]-cGMPS triethylamine (cGMP-dependent protein kinase inhibitor; 5 x 10(-6) M) or erythro-9-(2-hydroxy-3-nonyl) adenine (cGMP-stimulated cAMP phosphodiesterase inhibitor; 10(-5) M) reduced the responses induced by CNP or SNAP, suggesting that their actions were through cGMP-mediated pathways. While SNAP significantly increased intracellular cGMP concentration by 57%, CNP had little effect on cGMP production. We also found that CNP markedly decreased the amplitude of Ca2+ transients while SNAP had little effect, suggesting the cGMP generated by sGC may decrease myofilament Ca2+ sensitivity. The small amount of cGMP generated by pGC had a major effect in reducing Ca2+ level. This study suggested the existence of compartmentalization for cGMP in ventricular myocytes.