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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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Related in: MedlinePlus

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.
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pone-0031669-g001: 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.

Mentions: With the objective to develop an in vitro culture model for cardiac ischemia, we established a disease model of using the spontaneous contractile cardiac HL-1 cell line, were pathological effects of ischemia and ischemia-reperfusion injury can be simulated. For induction of ischemia in vitro, the standard used Claycomb-medium was replaced by a nutrient deficiency medium additionally contained 25 µM of hydrogen peroxide to enhance the oxidative stress [21]. Microscopic investigations of ischemic cell layers show a degeneration of the connected cell structure with increasing incubation time in ischemic buffer (Fig. 1). Single cells switched over to a stage of early cell death and detached from the monolayer within the first hours of ischemia. Furthermore, decreasing of contraction rate and power could be observed in ischemic HL-1 cultures using electrophysiological field potential measurement (Fig. S1), whereas control cells showed no interferences in monolayer structure and contractile behaviour. To investigate effects caused by ischemia-reperfusion, a revitalization period was initiated by medium exchange with standard Claycomb-medium and cells retained for 16 h. After revitalization time cell culture display a regeneration of aggrieved cells by the rebuild of a complex monolayer. In addition, we observed a revival of cardiac contraction.


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)

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3285183&req=5

pone-0031669-g001: 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.
Mentions: With the objective to develop an in vitro culture model for cardiac ischemia, we established a disease model of using the spontaneous contractile cardiac HL-1 cell line, were pathological effects of ischemia and ischemia-reperfusion injury can be simulated. For induction of ischemia in vitro, the standard used Claycomb-medium was replaced by a nutrient deficiency medium additionally contained 25 µM of hydrogen peroxide to enhance the oxidative stress [21]. Microscopic investigations of ischemic cell layers show a degeneration of the connected cell structure with increasing incubation time in ischemic buffer (Fig. 1). Single cells switched over to a stage of early cell death and detached from the monolayer within the first hours of ischemia. Furthermore, decreasing of contraction rate and power could be observed in ischemic HL-1 cultures using electrophysiological field potential measurement (Fig. S1), whereas control cells showed no interferences in monolayer structure and contractile behaviour. To investigate effects caused by ischemia-reperfusion, a revitalization period was initiated by medium exchange with standard Claycomb-medium and cells retained for 16 h. After revitalization time cell culture display a regeneration of aggrieved cells by the rebuild of a complex monolayer. In addition, we observed a revival of cardiac contraction.

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