Use of liquid chromatography-mass spectrometry (LC-MS) to detect substances of nanomolar concentration in the coronary effluent of isolated perfused hearts

作者：Ward Marie Louise^*; Shen Xin; Greenwood David R

来源：Progress in Biophysics and Molecular Biology, 2014, 115(2-3): 270-278.

DOI：10.1016/j.pbiomolbio.2014.07.005

• Stretch
• Trabeculae
• Slow force response
• Tissue exudates
• Liquid chromatography mass spectrometry

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 Ca2+ 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 Ca2+ 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 Ca2+ 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).

• 出版日期2014-8