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Cardiac cytoskeleton is modulated by human recombinant Vasostatin 1
ABSTRACTS / Journal of Molecular and Cellular Cardiology 42 (2007) S102–S124
To test our hypothesis that interaction of ECM protein and integrin would alter the mechanical properties in cardiomyocytes, we measured the unbinding force and cell stiffness of the ECM protein, fibronectin (FN)–integrin interactions on mouse cardiomyocytes using atomic force microscopy (AFM). In the un-stimulated condition, total adhesion events and binding probabilities between FN and cardiomyocytes were selectively blocked by 59% or 50% in the cells pretreated with α5/β1 integrin antibodies or RGD-containing peptides (10 cells/group; p < 0.05). The FN-α5β1 unbinding force (represented as detachment of FN from α5β1, 36.5 ± 0.3 pN) was not significantly changed by any other pre-treatments. The cell stiffness measured was 28.2 ± 0.2 kPa. FN-integrin adhesion events, adhesion probability and stiffness were not inhibited by α3 integrin antibody or RAD-containing peptide. In the 1 Hz stimulated cells, the total adhesion events of FN–α5β1 interaction was 15% less in the contraction phase than that in the relaxation phase. The binding probability was not significantly different between both conditions. The cell stiffness and the unbinding force during the contraction phase of the myocyte were larger when compared to that of during relaxation. Our results provide the first evidence that FN interacts with α5β1 integrin and these interactions are dynamic in nature during the contractile cycles of the cardiomyocytes. Keywords: Integrins; Mechanotransduction; Cardiomyocytes doi:10.1016/j.yjmcc.2007.03.279
Cardiac cytoskeleton is modulated by human recombinant Vasostatin 1 T. Angelone, R. Mazza, C. Mannarino, E. Filice, S. Barbieri, Y. Goumon, G. Zummo, M.-H. Metz-Boutigue, B. Tota. University of Calabria (Italy) and INSERM575 University of Strasbourg, France Cytoskeleton is an important scaffold in cardiac myocytes which provides structural support and compartmentalisation of intracellular components. It is implicated in cardiac pathologies including hypertophy and failure, playing a key role in the determinism of contractile and diastolic dysfunctions. Chromogranin A (CGA) and its derived peptides have revealed themselves as novel cardiovascular modulators. In humans, normal CGA levels are of 0.5 nmol/l and considerably increase in several pathologies. In cardiac failure, the increase represents a prognostic indicator of mortality. We found on the unstimulated rat heart that the CGA1–76 N-terminal fragment (Vasostatin 1; VS-1) depresses contractility and counteracts the β-adrenergic (Isoproterenol; ISO)-dependent positive inotropism with a functional non competitive antagonism (Cerra et al., 2006). This study analysed the influence of VS-1 on cytoskeleton. Using fluorescent phalloidin, we showed on rat cardiac H9C2 cells, that VS-1 (10 nM ÷ 10 μM) stimulates actin polymerization. This effect is absent after treatment with both ISO and the inert peptide Chromogranin B. On the isolated Langendorff perfused rat heart VS-1-mediated inotropy is abolished by either
S121
Cytochalasin-D (actin polymerisation inhibitor), or Wortmannin (inhibitor of PI3-K/Akt transduction cascade), or W-7 (calciumcalmodulin antagonist). Taken together these data strongly suggest a role of VS-1 in modulating actin microfilaments network. Keywords: Cytoskeleton; Cardiomyocytes; Isolated rat heart doi:10.1016/j.yjmcc.2007.03.280
The rate of loss of T-tubules in cultured adult ventricular myocytes is species-dependent Davor Pavlovic, Linda M. McLatchie, Michael J. Shattock. Cardiovascular Division, Kings College London In adult ventricular myocytes, the T-tubule network represents a complex system of interconnected membranes and is integrally involved in excitation–contraction (E–C) coupling. However, in adult cells in short-term quiescent culture, and in tachycardia-induced heart failure, T-tubules are lost leading to changes in EC coupling. In this study we compared the rate of detubulation of adult mouse and rat ventricular myocytes over a 72-h culture period. T-tubule density was measured in two ways (i) using di8-ANEPP staining and confocal microscopy, and (ii) as whole-cell capacitance in voltage-clamped myocytes. In rat adult ventricular myocytes T-tubular density, as assessed optically, was significantly reduced by 30 ± 5%, compared to control (time = 0), following 48 h in culture (mean ± sem; n = 14) and was further reduced to 56 ± 6% of control after 72 h in culture (n = 10). Whole-cell capacitance was also significantly reduced at 72 h compared to control cells. In contrast, optical assessment showed no significant loss of T-tubules in mouse myocytes after 48 h in culture, significant reduction (by 21 ± 4% of control) was only seen after 72 h in culture (n = 8). In mouse myocytes, whole-cell capacitance was not significantly reduced after 72 h culture. Thus, the rate of T-tubule loss in adult ventricular myocytes in quiescent culture varies between rat and mouse. This has implications for the use of these cells when studying EC coupling. While the maintenance of T-tubular structures in adult mouse cells over 72 h in culture may mean that short term culture remains a useful technique in mouse cells, whether E–C coupling mechanisms remain intact needs to be determined. Keywords: T-tubules; Ventricular myocytes; Cell culture doi:10.1016/j.yjmcc.2007.03.281
Electron tomography reveals the structure of the C-zone in striated muscle Pradeep Luther*, Hanspeter Winkler^ , Kenneth Taylor^ , Roger Craig#, Raul Padron#, Jun Liu¶. *Imperial College London, UK. ^University of Florida, USA. #University of Massachusetts, USA. ¶NIH
To test our hypothesis that interaction of ECM protein and integrin would alter the mechanical properties in cardiomyocytes, we measured the unbinding force and cell stiffness of the ECM protein, fibronectin (FN)–integrin interactions on mouse cardiomyocytes using atomic force microscopy (AFM). In the un-stimulated condition, total adhesion events and binding probabilities between FN and cardiomyocytes were selectively blocked by 59% or 50% in the cells pretreated with α5/β1 integrin antibodies or RGD-containing peptides (10 cells/group; p < 0.05). The FN-α5β1 unbinding force (represented as detachment of FN from α5β1, 36.5 ± 0.3 pN) was not significantly changed by any other pre-treatments. The cell stiffness measured was 28.2 ± 0.2 kPa. FN-integrin adhesion events, adhesion probability and stiffness were not inhibited by α3 integrin antibody or RAD-containing peptide. In the 1 Hz stimulated cells, the total adhesion events of FN–α5β1 interaction was 15% less in the contraction phase than that in the relaxation phase. The binding probability was not significantly different between both conditions. The cell stiffness and the unbinding force during the contraction phase of the myocyte were larger when compared to that of during relaxation. Our results provide the first evidence that FN interacts with α5β1 integrin and these interactions are dynamic in nature during the contractile cycles of the cardiomyocytes. Keywords: Integrins; Mechanotransduction; Cardiomyocytes doi:10.1016/j.yjmcc.2007.03.279
Cardiac cytoskeleton is modulated by human recombinant Vasostatin 1 T. Angelone, R. Mazza, C. Mannarino, E. Filice, S. Barbieri, Y. Goumon, G. Zummo, M.-H. Metz-Boutigue, B. Tota. University of Calabria (Italy) and INSERM575 University of Strasbourg, France Cytoskeleton is an important scaffold in cardiac myocytes which provides structural support and compartmentalisation of intracellular components. It is implicated in cardiac pathologies including hypertophy and failure, playing a key role in the determinism of contractile and diastolic dysfunctions. Chromogranin A (CGA) and its derived peptides have revealed themselves as novel cardiovascular modulators. In humans, normal CGA levels are of 0.5 nmol/l and considerably increase in several pathologies. In cardiac failure, the increase represents a prognostic indicator of mortality. We found on the unstimulated rat heart that the CGA1–76 N-terminal fragment (Vasostatin 1; VS-1) depresses contractility and counteracts the β-adrenergic (Isoproterenol; ISO)-dependent positive inotropism with a functional non competitive antagonism (Cerra et al., 2006). This study analysed the influence of VS-1 on cytoskeleton. Using fluorescent phalloidin, we showed on rat cardiac H9C2 cells, that VS-1 (10 nM ÷ 10 μM) stimulates actin polymerization. This effect is absent after treatment with both ISO and the inert peptide Chromogranin B. On the isolated Langendorff perfused rat heart VS-1-mediated inotropy is abolished by either
S121
Cytochalasin-D (actin polymerisation inhibitor), or Wortmannin (inhibitor of PI3-K/Akt transduction cascade), or W-7 (calciumcalmodulin antagonist). Taken together these data strongly suggest a role of VS-1 in modulating actin microfilaments network. Keywords: Cytoskeleton; Cardiomyocytes; Isolated rat heart doi:10.1016/j.yjmcc.2007.03.280
The rate of loss of T-tubules in cultured adult ventricular myocytes is species-dependent Davor Pavlovic, Linda M. McLatchie, Michael J. Shattock. Cardiovascular Division, Kings College London In adult ventricular myocytes, the T-tubule network represents a complex system of interconnected membranes and is integrally involved in excitation–contraction (E–C) coupling. However, in adult cells in short-term quiescent culture, and in tachycardia-induced heart failure, T-tubules are lost leading to changes in EC coupling. In this study we compared the rate of detubulation of adult mouse and rat ventricular myocytes over a 72-h culture period. T-tubule density was measured in two ways (i) using di8-ANEPP staining and confocal microscopy, and (ii) as whole-cell capacitance in voltage-clamped myocytes. In rat adult ventricular myocytes T-tubular density, as assessed optically, was significantly reduced by 30 ± 5%, compared to control (time = 0), following 48 h in culture (mean ± sem; n = 14) and was further reduced to 56 ± 6% of control after 72 h in culture (n = 10). Whole-cell capacitance was also significantly reduced at 72 h compared to control cells. In contrast, optical assessment showed no significant loss of T-tubules in mouse myocytes after 48 h in culture, significant reduction (by 21 ± 4% of control) was only seen after 72 h in culture (n = 8). In mouse myocytes, whole-cell capacitance was not significantly reduced after 72 h culture. Thus, the rate of T-tubule loss in adult ventricular myocytes in quiescent culture varies between rat and mouse. This has implications for the use of these cells when studying EC coupling. While the maintenance of T-tubular structures in adult mouse cells over 72 h in culture may mean that short term culture remains a useful technique in mouse cells, whether E–C coupling mechanisms remain intact needs to be determined. Keywords: T-tubules; Ventricular myocytes; Cell culture doi:10.1016/j.yjmcc.2007.03.281
Electron tomography reveals the structure of the C-zone in striated muscle Pradeep Luther*, Hanspeter Winkler^ , Kenneth Taylor^ , Roger Craig#, Raul Padron#, Jun Liu¶. *Imperial College London, UK. ^University of Florida, USA. #University of Massachusetts, USA. ¶NIH