Skinned Cardiac Muscle Fiber Contractility

Thin bundles of the left ventricular wall were teased/cut out and chemically skinned using a protocol described by Lu et al. (15 (link)). The samples were stored in 50% glycerol at −20°C until analysis. Thin preparations (diameter of about 200 μm, length about 1 mm) were prepared and glued using cellulose acetate glue between two carbon pins, one attached to a micrometer screw for length adjustment, and the other to a AE801 force transducer (Kronex, Oakland, CA, USA). The samples were stretched to about 1.3 × slack length, and kept in 0.5 ml Perspex baths at 22°C. Solutions and procedures were as described previously (16 (link)). After 30 min in relaxing [pCa = −log₁₀([Ca²⁺]) 9] solution with 1% Triton X-100 and a further brief washout in relaxing solution, the preparations were activated at pCa 4.7 to determine initial maximal force responses. Thereafter, the muscles were relaxed (pCa 9) to determine residual tension and activated at increasing Ca²⁺ levels (pCa 6.6, 6.0, 5.7, and 4.7). The force at each Ca²⁺ level was normalized to the initial maximal force response. The force (F) and [Ca²⁺] (C) data of each sample was analyzed by fitting a hyperbolic function [F = R + M × C^h/(C^h + EC₅₀^h)], where the fitted parameter R corresponds to the residual tension at pCa 9, M the maximal response, EC₅₀ the concentration giving half-maximal tension and h the Hill steepness coefficient.
To examine the effects of the Mavacamten (MYK-461), skinned fibers were activated at pCa 4.7 and exposed to increasing concentrations of MYK-461. Composition of the solutions for skinned fibers were calculated as described by Fabiato (17 (link)). MYK-461 was purchased from Selleckchem (Planegg, Germany), and dissolved in DMSO.