Abstract 19216: Optical Magnetic Twist Cytometry to Study the Effects of Desmin Cytoskeleton Modifications in an in vitro Model of Cardiac Hypertrophy
Background: Desmin is the major protein component of the intermediate filaments (IF) cytoskeleton in the cardiac myocyte. Desmin phosphorylation modulates IF lattice formation and correlates with disease in canine and human models of heart failure (HF). In order to investigate the contribution of desmin phosphorylation to cell biophysics as a potential mechanism of contractile dysfunction and HF we employed optical magnetic twist cytometry (OMTC) to measure the elastic and frictional properties of cultured neonatal rat ventricular myocytes (NRVMs), under both basal conditions and after hypertrophy was induced by endothelin-1 (ET). We also characterized desmin phosphorylation by tandem mass spectrometry (MS2) and identified glycogen synthase kinase 3 (GSK3) as a modulator of desmin phosphorylation.
Methods and Results: Freshly isolated NRVMs were incubated with RGD-coated magnetic beads, which avidly bind to the integrin receptors, localized at focal adhesions. The beads integrate with the cytoskeleton and can be magnetized and twisted using magnetic fields to measure cell stiffness and friction. In NRVMs, ET treatment alone (10 nM, 48 hrs) induced a 23%±3.0SE (n≥230, p=0.008) increase in cell stiffness in the presence of the contraction uncoupler heptanol (2 mM). Depolymerization of the other cytoskeleton components (microfilaments and microtubules) with selective inhibitors (cytochalasin D and colchicine, respectively, both 10μ M, 5min) was also achieved. Finally, we confirmed desmin phosphorylation at Ser-27 in NRVMs by MS2 and identified a mediator of desmin phosphorylation by phos-tag gels combined with a GSK3-specific inhibitor (BIO, 2μ M).
Conclusion: OMTC was employed for the first time, to our knowledge, in NRVMs inaugurating the application of a powerful biophysical tool to the investigation of a well established in vitro model for cardiac hypertrophy. Desmin cytoskeleton was also monitored and a new signaling pathway for its phosphorylation identified which was associated with increased cell stiffness.
- © 2012 by American Heart Association, Inc.