Effects of troponin C isoform on the action of the cardiotonic agent EMD 57033

The effects of the cardiotonic potentiator EMD 57033 on different TnC (troponin C) isoforms were investigated. Endogenous skeletal TnC was extracted from glycerinated, permeabilized rabbit psoas fibres and replaced with either purified native rabbit psoas TnC (fast TnC) or human recombinant cTnC (cardiac TnC) (3 mg/ml in relaxing solution for 30 min). In both conditions, 10 μM EMD 57033 increased maximal calcium-activated force (Pmax) and gave a leftward shift in the pCa–tension curve. With cTnC, the increase in Pmax was much greater (228%) compared with the effect seen for fast TnC (137%), which was the same as that in unextracted control fibres. When the whole troponin was replaced rather than just TnC, the effects of EMD 57033 on fibres replaced with cTn were the same as with the cTnC subunit alone, except that the force at low Ca2+ concentrations was not increased as much. If TnC was only partially extracted, it was found that the degree of extraction did not influence the effect of EMD 57033, except when force was decreased to below 10% of the pre-extraction Pmax. Dynamic stiffness was not altered by EMD 57033 in any of the preparations. The rate of tension recovery following a release–restretch method (ktr) was decreased by EMD 57033. We conclude that EMD 57033 acts by a rate-modulating effect, and that the quantitative response of this effect is dependent on the TnC isoform present.

Propane-jet freezing of muscle fibers for freeze fracture

Recent advances in freezing techniques have intensified interest in freeze fracture for the study of nonmembranous cellular structures. We describe here a method for freezing single skinned (demembranated) muscle fibers in a dual propane jet freezing device.The freezing apparatus we used, developed by J. Gilkey and A. Staehelin (Univ. of Colo.), is based on the design by Müller. To adapt this device to studies of skinned relaxed and activated muscle fibers, we built the sample holder shown in Figure 1. The holder is separable into two parts (at the arrow) to allow mounting of fibers under relaxing solution. A modified Balzers freeze fracture support, with the back hollowed out and a groove milled in the top (figure 2) is placed between the hooks. A skinned frog semitendinosus fiber (F) is mounted over the support, between the fixed hook (right) and the moving hook (left). Fiber length is adjusted by moving the lever (L). To activate samples, the holder is removed from the bath and several drops of activating solution (relaxing solution plus calcium) are flooded over the fiber.

Passive Stiffness of Rat Cardiac Myocytes

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.

Compositional studies of myofibrils from rabbit striated muscle.

The localization of high-molecular-weight (80,000-200,000-daltons) proteins in the sarcomere of striated muscle has been studied by coordinated electron-microscopic and sodium dodecyl sulfate (SDS) gel electrophoretic analysis of native myofilaments and extracted and digested myofibrils. Methods were developed for the isolation of thick and thin filaments and of uncontracted myofibrils which are devoid of endoproteases and membrane fragments. Treatment of crude myofibrils with 0.5% Triton X-100 results in the release of a 110,000-dalton component without affecting the myofibrillar structure. Extraction of uncontracted myofibrils with a relaxing solution of high ionic strength results in the complete disappearance of the A band and M line. In this extract, five other protein bands in addition to myosin are resolved on SDS gels: bands M 1 (190,000 daltons) and M 2 (170,000 daltons), which are suggested to be components of the M line; M 3 (150,000 daltons), a degradation product; and a doublet M 4, M 5 (140,000 daltons), thick-filament protein having the same mobility as C protein. Extraction of myofibrils with 0.15% deoxycholate, previously shown to remove Z-line density, releases a doublet Z 1, Z 2 (90,000 daltons) with the same mobility as alpha-actinin, as well as proteins of 60,000 daltons and less, and small amounts of M 1, M 2, M 4, and M 5; these proteins were not extracted with 0.5% Triton X-100. The C, M-line, and Z-line proteins and/or their binding to myofibrils are very sensitive to tryptic digestion, whereas the M 3 (150,000 daltons) component and an additional band at 110,000 daltons are products of proteolysis. Gentle treatment of myofibrils with an ATP relaxing solution results in the release of thick and thin myofilaments which can be pelleted by 100,000-g centrifugation. These myofilaments lack M-and Z-line structure when examined with the electron microscope, and their electrophoretograms are devoid of the M 1, M 2, Z 1, and Z 2 bands. The M 4, M 5 (C-protein doublet), and M 3 bands, however, remain associated with the filaments.

Calcium regulation of the contractile state of isolated mammalian fibroblast cytoplasm

Calcium-dependent contractions have been induced in fresh, naked cytoplasm of L-929 fibroblasts using physiological solutions (rigor, relaxing and contracting) similar to those designed to control the contractile state of vertebrate striated muscle. Free access of solutions to the cytoplasm was achieved by popping and stripping the plasma membrane from cells using 7–10 strokes of a Dounce homogenizer. Contracting solution (free Ca2+ 7 X 10(−5) M; with added MgATP) applied locally from a micropipette to cells popped in rigor (free Ca2+ less than 10(−8) M) or relaxing (free Ca2+ less than 10(−8) M; with added MgATP) solutions induced symmetrical contractions of unstretched cytoplasm and directional shortening of stretched cytoplasm. The contractions produced 12–18% shortenings and were complete in 1–3 s. The cytoplasm could be cycled repeatedly through the contracted state from the relaxed state. Exogenous MgATP was required for the Ca2+-dependent contractions. At low free Ca2+ concentrations (less than 10(−8) M), MgATP had a marked plasticizing effect on the cytoplasm. Thus cytoplasm prepared in relaxing solution was less cohesive and more easily deformed than cytoplasm prepared in rigor solution. When induced to contract, relaxed cytoplasm showed a loss of plasticity. Using this criterion, the threshold concentration of free Ca2+ for contraction was determined to lie between 7 X 10(−8) M and 5 X 10(−7) M.