Abstract 380: Myocardial Angiotensin Metabolism Analysis Using Matrix-Assisted Laser Desorption Ionization (MALDI) Imaging

Abstract

Mass spectrometric (MS) methods were used to study angiotensin (Ang) metabolism in mouse heart. The focus was on Ang II, Ang-(1-7) and Ang III. Goals were: 1) develop and optimize new technique for in situ assessment of Ang II processing within the myocardium using matrix-assisted laser desorption ionization (MALDI) imaging and 2) to validate results by in vitro MALDI MS enzyme assay as well as specific enzyme inhibitors. This newly developed imaging method has the advantage of allowing for regional spatial visualization and quantification of the formed peptides within the myocardium. Frozen mouse heart sections (12 μm) were incubated with 5 μL of 10-1000 ng/μL Ang II for 5-15 min at 37 °C. The main formed peptides, Ang III and Ang-(1-7), were identified by MALDI-TOF/TOF. Ang-(1-7) was concentrated in the ventricle while Ang III was detected throughout the heart. For the MALDI MS enzyme assay, left ventricular (LV) homogenate was incubated with Ang II at 37 °C. The reaction mixture was spotted on target plate and peptides were detected using MS. Results showed that Ang peptide formation was greatest at 100 ng/μL Ang II with short incubation period. Ang III formation was predominant with Ang III/ Ang II peak intensity 63%, whereas Ang (1-7)/ Ang II peak intensity was 22%. Ang III generation was blocked by the APA inhibitor, glutamate phosphonate (Glu-P), in a concentration dependent manner. The ACE2 inhibitor, MLN-4760, and the PCP/PEP inhibitor, Z-pro-prolinal, reduced myocardial Ang (1-7) formation by 66% (from 70.02 to 23.8 and from 112.7 to 38.3), respectively. Interestingly, blockade of APA by Glu-P caused a 4 fold increase in Ang (1-7)/Ang II ratio. Results validated MALDI imaging for the in situ assessment of Ang II metabolizing enzymes. Ang II is mainly metabolized by APA in the myocardium while ACE2 is the main enzyme responsible for Ang-(1-7) formation from Ang II in the LV. This is the first report on localization of Ang II processing enzymes in the heart using MALDI imaging. This procedure can be exploited for the development of new therapeutic avenues that can be used to target the devastating Ang II effects within the myocardium.