Passive Stiffness of Rat Cardiac Myocytes

Abstract

Intact single cells were isolated from adult rat hearts by enzymatic digestion and suspended in 0.25 mM Ca++ Tyrode’s solution. Quiescent, clearly striated rodlike cells were selected for study of the elastic properties of the cells at various stages of membrane and myofilament extraction. Selected cells were placed in a relaxing solution (pCa + 9, 10 mn EGTA) and then each end gently pulled into the tip of a closely fitting suction micropipette for attachment to a force transducer and length perturbation driver. This procedure was performed in low Ca++ to prevent Ca++ loading of the cell during attachment and at room temperature to prevent chemical skinning of the cell [1]. Stiffness was measured by applying a 5-Hz sinusoidal length perturbation (5 percent L0) to one end of the cell while measuring the induced tension change at the other. The ratio of sinusoidal tension change to applied length change (stiffness) was determined for each cell over a length range of about 1–1.3 L0 before removal of the contractile filaments and up to 3.0 L0 after treatment with 0.6 M KI. The stiffness-length relation was measured first in relaxing solution and then in 0.25 mM Ca++ Tyrode’s. If spontaneous contractions or contracture occurred the cell was rejected. If the cell remained quiescent and relaxed it was treated again with relaxing solution and 1 percent Triton X-100 to remove the membranes. The stiffness-length relation was again measured and then the cell was superfused with 0.47 M KCl/10 mM pyrophosphate solution to remove the myosin filaments. The stiffness-length relation was again determined and the cell finally perfused with 0.6 M KI to remove all the contractile filaments. A rodlike, faintly striated structure remained at this point whose stiffness could still be measured. In cells which remained quiescent during the entire extraction procedure and did not develop contracture the following results were obtained. In the relaxing solution and in 0.25 mM Ca++ the stiffness-length relation was similar to that of rat papillary muscle [2]. When the cell membranes were removed with detergent a transient increase in stiffness sometimes occurred which declined within a few minutes to a level near that in the relaxing solution. With KC1 treatment the stiffness declined variably to about half its control value. Immediately upon treatment of the cell with KI solution the major striation pattern disappeared and stiffness fell dramatically. Also the cell became highly extensible such that is could be reversibly extended in length to 2.5–3 L0 with the faintly striated pattern uniformly following the extension. At 3 L0 the sinusoidally measured stiffness was about equal to that of the intact cell at L0. These data indicate that a significant source of the high resting stiffness of rat heart muscle resides within the muscle cells and is dependent to a large extent on the presence of the myofilaments [3]. Also, a measurable stiffness remains in the cells after contractile filament extraction, which may be attributable to the cytoskeletal intermediate filaments.