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

Haas S, Jahnke HG, Moerbt N, von Bergen M, Aharinejad S, Andrukhova O, Robitzki AA - PLoS ONE (2012)

Bottom Line: Therefore, there is a high demand for suitable model systems with the capability to simulate ischemic and cardiotoxic effects in vitro, under defined conditions.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.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Biological-Biochemical Processing Technology, Center for Biotechnology and Biomedicine, Universität Leipzig, Leipzig, Germany.

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.

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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.
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pone-0031669-g002: 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.

Mentions: Changes in protein expression profile after 8 h of induced ischemia and after 16 h of ischemia-reperfusion were analysed using the DIGE 2-DE approaches (Fig. 2). We detected 1435 protein spots in the ischemic HL-1 proteome by 2-DE and quantified the expression of ischemic HL-1 cells with pI between 3 and 10 using Delta 2D software, version 3.6, yielding 644 spots displaying a difference in protein expression stronger than 2-fold (p<0.05). In general, we detected more repressed (441) than induced protein spots (203) after 8 h incubation in ischemic buffer (Fig. 2A). Comparison between ischemic samples and controls showed a clear clustered Heatmap according to the experiments and a high similarity between the three independent replicates (Fig. S2). By using an unsupervised approach comprising all spots the gels were clustered into the correct groups. Changes in expression profile after revitalization in comparison to the control were reduced to a minimum, indicating a regeneration of HL-1 cells during re-incubation in Claycomb-medium and an approximation to the control state (Fig. 2B). Alike expression profile of 8 h samples, we detected more repressed than induced protein spots in revitalized HL-1 cardiomyocytes. By tryptic digest and MS analysis of the peptides in order to create a proteome map of HL-1 cells, we identified 81 differentially expressed proteins from ischemic HL-1 cells (Table S2) and 14 protein spots, which were differentially expressed after reperfusion-time using MALDI-MS (Table S3). In detail, identified proteins are involved in cell death signalling (death associated protein kinase 2, granzyme K), oxidative stress regulation (stress induced phosphoproteine 1), protein quality control (HSPA2), cell structure organisation or metabolism related proteins (Fig. 2C, Table 1).


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

Haas S, Jahnke HG, Moerbt N, von Bergen M, Aharinejad S, Andrukhova O, Robitzki AA - PLoS ONE (2012)

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.
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Related In: Results  -  Collection

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pone-0031669-g002: 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.
Mentions: Changes in protein expression profile after 8 h of induced ischemia and after 16 h of ischemia-reperfusion were analysed using the DIGE 2-DE approaches (Fig. 2). We detected 1435 protein spots in the ischemic HL-1 proteome by 2-DE and quantified the expression of ischemic HL-1 cells with pI between 3 and 10 using Delta 2D software, version 3.6, yielding 644 spots displaying a difference in protein expression stronger than 2-fold (p<0.05). In general, we detected more repressed (441) than induced protein spots (203) after 8 h incubation in ischemic buffer (Fig. 2A). Comparison between ischemic samples and controls showed a clear clustered Heatmap according to the experiments and a high similarity between the three independent replicates (Fig. S2). By using an unsupervised approach comprising all spots the gels were clustered into the correct groups. Changes in expression profile after revitalization in comparison to the control were reduced to a minimum, indicating a regeneration of HL-1 cells during re-incubation in Claycomb-medium and an approximation to the control state (Fig. 2B). Alike expression profile of 8 h samples, we detected more repressed than induced protein spots in revitalized HL-1 cardiomyocytes. By tryptic digest and MS analysis of the peptides in order to create a proteome map of HL-1 cells, we identified 81 differentially expressed proteins from ischemic HL-1 cells (Table S2) and 14 protein spots, which were differentially expressed after reperfusion-time using MALDI-MS (Table S3). In detail, identified proteins are involved in cell death signalling (death associated protein kinase 2, granzyme K), oxidative stress regulation (stress induced phosphoproteine 1), protein quality control (HSPA2), cell structure organisation or metabolism related proteins (Fig. 2C, Table 1).

Bottom Line: Therefore, there is a high demand for suitable model systems with the capability to simulate ischemic and cardiotoxic effects in vitro, under defined conditions.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.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Biological-Biochemical Processing Technology, Center for Biotechnology and Biomedicine, Universität Leipzig, Leipzig, Germany.

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.

Show MeSH
Related in: MedlinePlus