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A novel technique for assessing physiological platelet function in the mouse
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ABSTRACTS / Journal of Molecular and Cellular Cardiology 40 (2006) 920 – 1015
004. 1H NMR-based metabonomic analysis of patients with exercise-induced myocardial ischemia I. Barba, D. Garcı´a-Dorado, G. de Leon, J. Candell, E. Martı´n, J. Soler-Soler. Laboratorio de Cardiologı´a Experimental, Servicio de Cardiologı´a, Hospital Universitari Vall dHebron, Barcelona, Spain Background: NMR spectroscopy-based metabolomics has been recently proposed to allow stratification of coronary artery disease and detection of ischemia. We aimed to develop a 1H NMR based metabolomic approach capable of predicting the occurrence of exercise-induced ischemia. Methods and results: One milliliter of plasma was obtained just prior to a stress SPECT test in 31 patients with suspected effort angina and without prior myocardial infarction. NMR spectra were acquired with a CPMG sequence with an effective delay of 136 ms and digitized in bins of equal width. PLS-DA discriminant analysis was performed using SIMCA-P software. Seventeen patients showed myocardial perfusion defects that disappeared at rest and 14 did not. Both groups had similar age and clinical profile except for more smokers and diabetics in the ischemia group, and attained a similar peak heart rate. The first component (t1) of the PLS-DA analysis provided a significative discrimination between ischemic and non-ischemic patients. t1 was >0 was observed in 11 of 14 patients with and t1 <0 in 14 of 17 patients without effort induced ischemia. The main contributor to the discrimination was a higher level of lactate in the ischemia group. Conclusion: 1H NMR based metabonomic analysis of plasma samples may predict exercise ischemia. Increased systemic lactate may indicate the presence of some degree of ischemia at rest in patients with effort angina. doi:10.1016/j.yjmcc.2006.03.019
005. A novel technique for assessing physiological platelet function in the mouse C. Tymvios a, W. Paul b, C.P. Page b, M. Emerson a. a Comparative Medicine Section, Division of Biomedical Sciences, Imperial College London, SW7 2AZ, UK. b Sackler Institute of Pulmonary Pharmacology, Kings College London, SE1 1NL, UK Platelets are central to haemostasis and inflammation as well as thrombogenesis, the underlying event in occlusive vascular diseases such as myocardial infarction and stroke. Pathophysiological platelet activity is regulated both by signalling events within the platelet and non-platelet factors such as blood flow and endothelial status. There is a need therefore for measuring platelet function in situ. We have developed a non-invasive method for platelet monitoring in real-time in transgenic mice. Platelets are isolated from donor mice, radiolabelled with 111 Indium Oxine and infused into anaesthetised recipient mice. Platelet-associated counts are monitored in the pulmonary vasculature by placing a 1-cm crystal scintillation probe over the thoracic area. Intravenous injection of the platelet agonist
ADP (4– 400 Ag/kg) induced transient, dose-dependent platelet accumulation in the pulmonary vasculature indicated by a 14 to 39% increase in counts. The platelet disaggregation phase was also recorded and counts returned to basal levels within 40 s. ADP had no effect on circulating erythrocyte levels indicating a platelet-specific effect. We therefore describe a novel method for assessing platelet function in transgenic mice. The model allows assessment of platelet function in intact mice and will be useful in determining the molecular basis of pathophysiological platelet function and as a model of platelet thromboembolism. doi:10.1016/j.yjmcc.2006.03.020
006. Reduced phospholamban phosphorylation is responsible for impaired myocardial relaxation in nNOS/ mice Yin Hua Zhang, Mei Hua Zhang, Claire E. Sears, Euan A. Ashley, Barbara Casadei. Department of Cardiovascular Medicine, University of Oxford, John Radcliffe Hospital, Oxford, UK nNOS gene deletion (nNOS/) is associated with impaired myocardial relaxation both in vivo and in isolated myocytes. Here we evaluated the mechanisms responsible for these findings in LV myocytes from nNOS/ mice and their wild type littermates (WT). Myocyte relaxation (TR50) and the decay of [Ca2+]i transient (Tau) were significantly prolonged in field-stimulated (3 Hz, 35 -C) or voltage-clamped (25 ms from 70 to 20 mV) nNOS/ myocytes (in ms, TR50: 34.1 T 0.7 vs. 27.3 T 0.5 in WT, P < 0.05; Tau: 93.8 T 3.5 vs. 78.1 T 4.1 in WT, P < 0.01). Disabling the sarcoplasmic reticulum (SR) with thapsigargin (10 AM) abolished differences in TR50 between nNOS/ and WT mice; similarly, the Tau of the caffeine-induced [Ca2+]i transient did not differ between groups, suggesting that a slower reuptake of Ca2+ into the SR may be responsible for impaired relaxation in nNOS/ myocytes. Immunoblotting showed a modest reduction in total phospholamban (PLB) in nNOS/; however, further experiments revealed that a larger proportion of PLB was unphosphorylated in the nNOS/ myocardium (e.g., Ser16-PLB/PLB ratio: 0.48 T 0.05 in nNOS/ vs. 0.74 T 0.03 in WT, P = 0.01). Furthermore, acute inhibition of nNOS with L-VNIO (100 AM) or SMTC (107M) prolonged TR50 and the decay of [Ca2+]i transient, and caused a significant reduction in PLB phosphorylation without affecting total PLB. eNOS gene deletion did not affect PLB phosphorylation or myocyte relaxation, suggesting that the NO-mediated autocrine effects on relaxation and PLB phosphorylation are nNOS-specific. These findings suggest that nNOS-derived NO regulates myocardial relaxation by promoting PLB phosphorylation and the rate of SR Ca2+ reuptake in the murine myocardium. doi:10.1016/j.yjmcc.2006.03.021
ABSTRACTS / Journal of Molecular and Cellular Cardiology 40 (2006) 920 – 1015
004. 1H NMR-based metabonomic analysis of patients with exercise-induced myocardial ischemia I. Barba, D. Garcı´a-Dorado, G. de Leon, J. Candell, E. Martı´n, J. Soler-Soler. Laboratorio de Cardiologı´a Experimental, Servicio de Cardiologı´a, Hospital Universitari Vall dHebron, Barcelona, Spain Background: NMR spectroscopy-based metabolomics has been recently proposed to allow stratification of coronary artery disease and detection of ischemia. We aimed to develop a 1H NMR based metabolomic approach capable of predicting the occurrence of exercise-induced ischemia. Methods and results: One milliliter of plasma was obtained just prior to a stress SPECT test in 31 patients with suspected effort angina and without prior myocardial infarction. NMR spectra were acquired with a CPMG sequence with an effective delay of 136 ms and digitized in bins of equal width. PLS-DA discriminant analysis was performed using SIMCA-P software. Seventeen patients showed myocardial perfusion defects that disappeared at rest and 14 did not. Both groups had similar age and clinical profile except for more smokers and diabetics in the ischemia group, and attained a similar peak heart rate. The first component (t1) of the PLS-DA analysis provided a significative discrimination between ischemic and non-ischemic patients. t1 was >0 was observed in 11 of 14 patients with and t1 <0 in 14 of 17 patients without effort induced ischemia. The main contributor to the discrimination was a higher level of lactate in the ischemia group. Conclusion: 1H NMR based metabonomic analysis of plasma samples may predict exercise ischemia. Increased systemic lactate may indicate the presence of some degree of ischemia at rest in patients with effort angina. doi:10.1016/j.yjmcc.2006.03.019
005. A novel technique for assessing physiological platelet function in the mouse C. Tymvios a, W. Paul b, C.P. Page b, M. Emerson a. a Comparative Medicine Section, Division of Biomedical Sciences, Imperial College London, SW7 2AZ, UK. b Sackler Institute of Pulmonary Pharmacology, Kings College London, SE1 1NL, UK Platelets are central to haemostasis and inflammation as well as thrombogenesis, the underlying event in occlusive vascular diseases such as myocardial infarction and stroke. Pathophysiological platelet activity is regulated both by signalling events within the platelet and non-platelet factors such as blood flow and endothelial status. There is a need therefore for measuring platelet function in situ. We have developed a non-invasive method for platelet monitoring in real-time in transgenic mice. Platelets are isolated from donor mice, radiolabelled with 111 Indium Oxine and infused into anaesthetised recipient mice. Platelet-associated counts are monitored in the pulmonary vasculature by placing a 1-cm crystal scintillation probe over the thoracic area. Intravenous injection of the platelet agonist
ADP (4– 400 Ag/kg) induced transient, dose-dependent platelet accumulation in the pulmonary vasculature indicated by a 14 to 39% increase in counts. The platelet disaggregation phase was also recorded and counts returned to basal levels within 40 s. ADP had no effect on circulating erythrocyte levels indicating a platelet-specific effect. We therefore describe a novel method for assessing platelet function in transgenic mice. The model allows assessment of platelet function in intact mice and will be useful in determining the molecular basis of pathophysiological platelet function and as a model of platelet thromboembolism. doi:10.1016/j.yjmcc.2006.03.020
006. Reduced phospholamban phosphorylation is responsible for impaired myocardial relaxation in nNOS/ mice Yin Hua Zhang, Mei Hua Zhang, Claire E. Sears, Euan A. Ashley, Barbara Casadei. Department of Cardiovascular Medicine, University of Oxford, John Radcliffe Hospital, Oxford, UK nNOS gene deletion (nNOS/) is associated with impaired myocardial relaxation both in vivo and in isolated myocytes. Here we evaluated the mechanisms responsible for these findings in LV myocytes from nNOS/ mice and their wild type littermates (WT). Myocyte relaxation (TR50) and the decay of [Ca2+]i transient (Tau) were significantly prolonged in field-stimulated (3 Hz, 35 -C) or voltage-clamped (25 ms from 70 to 20 mV) nNOS/ myocytes (in ms, TR50: 34.1 T 0.7 vs. 27.3 T 0.5 in WT, P < 0.05; Tau: 93.8 T 3.5 vs. 78.1 T 4.1 in WT, P < 0.01). Disabling the sarcoplasmic reticulum (SR) with thapsigargin (10 AM) abolished differences in TR50 between nNOS/ and WT mice; similarly, the Tau of the caffeine-induced [Ca2+]i transient did not differ between groups, suggesting that a slower reuptake of Ca2+ into the SR may be responsible for impaired relaxation in nNOS/ myocytes. Immunoblotting showed a modest reduction in total phospholamban (PLB) in nNOS/; however, further experiments revealed that a larger proportion of PLB was unphosphorylated in the nNOS/ myocardium (e.g., Ser16-PLB/PLB ratio: 0.48 T 0.05 in nNOS/ vs. 0.74 T 0.03 in WT, P = 0.01). Furthermore, acute inhibition of nNOS with L-VNIO (100 AM) or SMTC (107M) prolonged TR50 and the decay of [Ca2+]i transient, and caused a significant reduction in PLB phosphorylation without affecting total PLB. eNOS gene deletion did not affect PLB phosphorylation or myocyte relaxation, suggesting that the NO-mediated autocrine effects on relaxation and PLB phosphorylation are nNOS-specific. These findings suggest that nNOS-derived NO regulates myocardial relaxation by promoting PLB phosphorylation and the rate of SR Ca2+ reuptake in the murine myocardium. doi:10.1016/j.yjmcc.2006.03.021