Abstract 17282: Cytoskeletal Protein 4.1R is Required for Normal Cardiac Myocyte Membrane Structure and Stiffness.
Protein 4.1R is a member of the spectrin-associated cytoskeleton family which promotes mechanical stability of the cell membrane and interacts with membrane ion channels in red blood cells (RBC). We previously reported that myocytes from 4.1R deficient mouse (4.1RKO) hearts had multiple electrophysiological changes including increased calcium spark frequency and dysynchronous and prolonged calcium transients. Such changes have been associated with alterations to the t-tubule system of ventricular cardiomyocytes in the pathogenesis of multiple disease states (e.g. heart failure and arrhythmia). Whether cytoskeletal protein 4.1R plays a role in determining t-tubular structure and membrane stiffness in cardiac muscle is unknown. Here we investigated the structure and mechanical properties of the cell membrane of ventricular cardiomyocytes isolated from 4.1RKO and wild-type (WT) mice. The t-tubules were stained with Di-8-ANEPPS and imaged using confocal microscopy. To analyse the regularity of the t-tubules, we calculated the Fourier transform of the t-tubule striations. 4.1RKO myocytes had a significant disruption of the t-tubule system with a reduced power of the dominant frequency (WT: 2.86 X 107 n=29 vs 4.1RKO: 1.69 X 107 n=44, p=0.0005). However, the total density of Di-8-ANEPPS staining was unchanged between WT and 4.1RKO (WT: 65.54 ± 3.7 % n=29 vs. 4.1RKO: 63.23 ± 3.4 % n=44, ns). This indicates that t-tubules are not reduced in amount but irregularly distributed in 4.1RKO myocytes. As protein 4.1R deficient RBCs have altered membrane stiffness we also tested the hypothesis that 4.1R deficient cardiomyocytes might have different membrane mechanical properties. We used the scanning ion conductance microscope to measure the displacement of the cell surface per unit of force. 4.1RKO cells had significantly softer membranes than WT cells (WT:0.03±0.003 µm/kPa n=16 vs. 4.1RKO: 0.06±0.01 µm/kPa n=11). These results suggest that the electrophysiological abnormalities reported in 4.1R deficient cardiomyocytes may be partially caused by alterations of cell membrane structure and mechanical properties and may have a role during cardiac disease.
- © 2010 by American Heart Association, Inc.