Animal Models and "Omics" Technologies for Identification of Novel Biomarkers and Drug Targets to Prevent Heart Failure

Abstract

It is now accepted that heart failure (HF) is a complex multifunctional disease rather than simply a hemodynamic dysfunction. Despite its complexity, stressed cardiomyocytes often follow conserved patterns of structural remodelling in order to adapt, survive, and regenerate. When cardiac adaptations cannot cope with mechanical, ischemic, and metabolic loads efficiently or become chronically activated, as, for example, after infection, then the ongoing structural remodelling and dedifferentiation often lead to compromised pump function and patient death. It is, therefore, of major importance to understand key events in the progression from a compensatory left ventricular (LV) systolic dysfunction to a decompensatory LV systolic dysfunction and HF. To achieve this, various animal models in combination with an "omics" toolbox can be used. These approaches will ultimately lead to the identification of an arsenal of biomarkers and therapeutic targets which have the potential to shape the medicine of the future.

Figure 1

End-systolic midventricular short axis and long axis MRI frames of healthy and failing hearts from humans and mice. (a) MRI images from a healthy control (Con) and patients with aortic stenosis (HT), myocardial infarction (MI), and idiopathic dilated cardiomyopathy (DCM). (b) MRI images from mice 4 weeks after transaortic constriction (HT), 3 weeks after LAD ligation (LAD), and 6-month-old mice with a cardiac restricted overexpression of MCP-1 (iDCM). A healthy control animal is also shown (Con). Note the increase in size and ventricular mass of HT hearts while increases in heart size after MI and after development of (i) DCM are associated with ventricular thinning. Explanations are given in the text. (c) Electron microscopy pictures show different degrees of sarcomeric degeneration in patients with dilated cardiomyopathy (Con versus DCM1 and DCM2; EF < 30%). Human MRI images and electron micrographs were kindly provided by Professor Georg Bachmann and by Dr. Viktoria Polyakova, respectively. Mouse MRI images are adapted and modified from the “Venia legendi” work of J. Pöling.

Figure 2

The design of an “omics” technology research platform consisting of complementary core facilities for the future of a personalized medicine (adapted and modified from the “Venia legendi” work of J. Pöling, 2013). (a) Multiplex systems, Western blot, and confocal microscopy are antibody based methods. The Western blot shows a stained membrane resolving 1 μL of two serum samples (2&3) and 1 μL of pericardial fluid (PCF) from a patient with HF and high myocardial level of oncostatin M (size marker was run parallel in 1). A strong signal of TIMP-1 was detected in PCF after antibody hybridization of the membrane and subsequent chemiluminescence detection (WB). When targets are not known the separation of proteins lysates by 2-dimensional gel electrophoresis (2DE) reveals thousands of not yet defined protein spots after silver staining (proteome analysis). Here, a cytoplasmic cardiac 2DE of a 3-day-old rat is shown. Then, gels are scanned and compared and a computer based software program identifies regulated spots. (4) The protein spot is excised and identified by mass spectrometry combined with database searches. (1) In addition RNA might be extracted from the same sample and expression levels of ten thousands of genes can be simultaneously analyzed on DNA microarrays (transcriptome analysis). (5) Confocal microscopy verifies observations and provides further information about the protein. A confocal image shows FGF23-positive cardiomyocytes in a patient with aortic stenosis and high myocardial level of oncostatin M. (b) ANP, BNP, interleukin-6, and FGF-23 were identified as oncostatin M-regulated biomarkers on this platform. Further potential biomarkers such as radixin and moesin are indicated.

Figure 3

Cardiomyocytes respond to stress by membranous translocation of ERM proteins. (a) Fluorescence micrographs of freshly isolated adult rat cardiomyocytes (4 hours) show different degrees of ezrin translocation (yellow arrows). Ezrin is usually located at the intercalated disc (white arrows) but upon translocation it is detected laterally of the membrane. (b) Fluorescence micrographs demonstrate massive translocation of ezrin. Ezrin is part of the cell blebs which are, when occurring to this extent, characteristic for dying cells. (c) Fluorescence micrographs show increases and accumulation of ezrin in cell extensions of IGF-1 stimulated adult cardiomyocytes after seven days. Note that serum shows also some effects on ezrin localization in control cultures (Con). (d) Fluorescence images of oncostatin M receptor-β siRNA treated adult rat cardiomyocytes (OSM + siOβ) in culture demonstrate successful interruption of OSM induced remodeling after 7 days. Con indicates albumin treated control cultures.

Figure 4

Spatial distribution of ezrin during adaptation and HF. (a) Longitudinal sections of the mouse myocardium 1 month after transaortic constriction (TAC). In sham operated animals ezrin shows a regular appearance at the intercalated disc, which is disturbed to a variable degree in mice after TAC (yellow circles). In the fluorescence micrographs cardiomyocytes show different degrees of ezrin translocation in cardiomyocytes (white arrows (TAC1) and oval circles (TAC2)). These animals recover after the release of constriction. (b) Fluorescence micrographs demonstrate massive lateral accumulation of ezrin in patients with end-stage HF. This pattern of moesin and radixin labeling corresponds with that previously described [14].