title: Use of liquid chromatography-mass spectrometry (LC-MS) to detect substances of nanomolar concentration in the coronary effluent of isolated perfused hearts.
abstract: When the heart is stretched there is an abrupt increase in force that coincides with the stretch that is then followed by a second phase that develops slowly over a period of minutes, known as the "slow force response", or "SFR". The SFR is accompanied by a slow increase in the Ca(2+) transients, but the underlying cellular mechanisms are still debated. We therefore devised a simple bioassay system to investigate whether stretch released autocrine/paracrine factors that could explain the stretch-dependent Ca(2+) influx during the SFR. Our analysis was based around a metabolomics approach using tandem mass spectrometry whereby secreted compounds were analysed by liquid chromatography mass spectrometry (LC-MS/MS) at high resolution and accurate mass. Our bioassay consisted of two isolated cardiac preparations in series. A sustained stretch was applied to an "upstream" Langendorff-perfused heart, whilst monitoring the performance of an un-stretched, "downstream" trabecula perfused with coronary effluent collected from the "upstream" heart. Switching from "un-stretched" to "stretched" coronary effluent increased the amplitude of the Ca(2+) transients (340/380) fura-2 ratio) in the "downstream" trabeculae from 0.87 ± 0.04 to 0.94 ± 0.05 (p < 0.05, n = 4), and active stress (mN mm(-2)) from 15.4 ± 1.5 to 19.2 ± 1.9 (p < 0.01, n = 13), confirming the stretch-dependent release of one or more positively inotropic factor(s). To identify the factor, coronary effluent samples were concentrated by Solid Phase Extraction (SPE) treatment, lyophilised, then analysed using a Thermo Finnigan LTQ FT-ICR hybrid mass spectrometer. Differential profile data was obtained initially followed by targeting specific compounds at elevated levels in the stretched coronary effluent samples. Quantitative and statistical analyses of the differential LC-MS/MS results were then carried out using SIEVE 2.1, statistical software capable of performing differential analysis of peptides and small molecules. A number of substances were identified that were different between stretched and unstretched effluents (p < 0.01).
authors: Department of Physiology, Faculty of Medicine and Health Sciences, University of Auckland, Private Bag 92019, Auckland, New Zealand. Electronic address: m.ward@auckland.ac.nz.
authors: Marie-Louise
authors: ML
authors: Ward
authors: Department of Physiology, Faculty of Medicine and Health Sciences, University of Auckland, Private Bag 92019, Auckland, New Zealand.
authors: Xin
authors: X
authors: Shen
authors: School of Biological Sciences, Faculty of Science, University of Auckland, Private Bag 92019, Auckland, New Zealand.
authors: David R
authors: DR
authors: Greenwood
mesh_terms: D000818:Animals; D001826:Body Fluids / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling / Q000502:physiology; D002853:Chromatography, Liquid / Q000379:methods*; D005260:Female; D066298:In Vitro Techniques; D008297:Male; D013058:Mass Spectrometry / Q000379:methods*; D008832:Microchemistry / Q000379:methods; D009200:Myocardial Contraction / Q000502:physiology*; D036103:Nanotechnology / Q000379:methods; D010477:Perfusion; D051381:Rats; D017208:Rats, Wistar; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013314:Stress, Mechanical; D016277:Ventricular Function, Left / Q000502:physiology*
chemical_list: D002118:Calcium
keywords: Liquid chromatography mass spectrometry; Slow force response; Stretch; Tissue exudates; Trabeculae