DIGE proteome analysis reveals suitability of ischemic cardiac in vitro model for studying cellular response to acute ischemia and regeneration

Abstract

Proteomic analysis of myocardial tissue from patient population is suited to yield insights into cellular and molecular mechanisms taking place in cardiovascular diseases. However, it has been limited by small sized biopsies and complicated by high variances between patients. Therefore, there is a high demand for suitable model systems with the capability to simulate ischemic and cardiotoxic effects in vitro, under defined conditions. In this context, we established an in vitro ischemia/reperfusion cardiac disease model based on the contractile HL-1 cell line. To identify pathways involved in the cellular alterations induced by ischemia and thereby defining disease-specific biomarkers and potential target structures for new drug candidates we used fluorescence 2D-difference gel electrophoresis. By comparing spot density changes in ischemic and reperfusion samples we detected several protein spots that were differentially abundant. Using MALDI-TOF/TOF-MS and ESI-MS the proteins were identified and subsequently grouped by functionality. Most prominent were changes in apoptosis signalling, cell structure and energy-metabolism. Alterations were confirmed by analysis of human biopsies from patients with ischemic cardiomyopathy.With the establishment of our in vitro disease model for ischemia injury target identification via proteomic research becomes independent from rare human material and will create new possibilities in cardiac research.

Figure 1. Analysis of ischemia induced changes in the cellular structure of HL-1 cardiomyocytes.

While control and reperfused cells show a vital cell layer consists of contractile HL-1 cardiomyocytes and an intact cytoskeleton structure, 8 h after ischemia-induction cells die off and detached from the layer-assembly, indicated by reflecting microscopic structures. Furthermore contractility of HL-1 cardiomyocytes was stopped. Immunocytochemical staining of this phase offered a degradation of the cytoskeleton filaments alpha-actinin, desmin and cadherin, which were labelled with Cy™-antibodies (green). Nuclei were stained using Sytox®-orange. Scale bar microscopic images = 25 µm, fluorescence images = 50 µm.

Figure 2. Proteome changes in HL-1 cardiomyocyte cell line with induction of ischemic conditions and after reperfusion.

In total 360 µg of cytosolic proteins of ischemia-induced HL-1 cells (A) and reperfused HL-1 cells (B) were separated by DIGE-2-DE (18 cm IPG stripes 3–10 non-linear, 10% acrylamide/bisacrylamide). C) Gel image regions of differentially expressed proteins following ischemia induction. Abbreviations are gene names. The details of identifications are shown in Table S1 and Table S2.

Figure 3. Cytosolic HL-1 cardiomyocyte proteins showing changes in expression after cellular exposure to ischemic conditions.

(A) For four identified pathways verification was done by western blot analysis of two proteins per pathway. Shown is the expression in HL-1 cardiomyocytes after induction of ischemia (8 h) and after reperfusion (24 h) from control (C) and treated cells (I). (B) All protein expression levels were calculated relative to the housekeeping protein histone H3 and three to six experiments were statistical analysed.

Figure 4. Apoptosis and proliferation analysis of ischemic HL-1 cardiomyocytes.

Induced ischemia resulted in an increased apoptosis and decrease of proliferation. (A) For quantification apoptotic cells were stained using the TUNEL-assay, proliferating cells were labelled with EdU using the Click-iT™-assay and analysed via flow cytometer. For comparison of different time points, proliferation values were normalized to controls of the corresponding time point (n = 3). (B) Immunocytochemical staining of TUNEL positive apoptotic HL-1 cardiomyocytes (red) and EdU positive proliferating cells (green) in control cells, ischemic cells and reperfused cells. Total cell number obtained by a DAPI stain (blue). Scale bar = 100 µm.

Figure 5. Proteome map of human biopsy samples from patients with ischemic cardiomyopathy.

(Identification data are shown in Table S4 and Table S5). In total 360 µg protein of male (A) and female (B) biopsy samples were separated by DIGE-2-DE (18 cm IPG stripes 3–10 non-linear, 10% acrylamide/bisacrylamide).

Figure 6. Gel image regions of differentially expressed proteins following ischemic cardiomyopathy in male and female biopsies.

(A) Abbreviations are gene names. Regions are emphasized in the DIGE-2D-gel images in Figure 5. (B) Relative Expression of HSP70 (left) and HSP90 (right) in male and female biopsies by western blot analysis, normalized to control samples.