Isolation, electron microscopic imaging, and 3-D visualization of native cardiac thin myofilaments

An increasing number of cardiac diseases are currently pinpointed to reside at the level of the thin myofilaments (e.g., cardiomyopathies, reperfusion injury). Hence the aim of our study was to develop a new method for the is olation of mammalian thin myofilaments suitable for subsequent high-resolut ion electron microscopic imaging. Native cardiac thin myofilaments were ext racted from glycerinated porcine myocardial tissue in the presence of prote ase inhibitors. Separation of thick and thin myofilaments was achieved by a ddition of ATP and several centrifugation steps. Negative staining and subs equent conventional and scanning transmission electron microscopy (STEM) of thin myofilaments permitted visualization of molecular details; unlike con ventional preparations of thin myofilaments, our method reveals the F-actin moiety and allows direct recognition of thin myofilament-associated porcin e cardiac troponin complexes, They appear as "bulges" at regular intervals of similar to 36 nm along the actin filaments. Protein analysis using SDS-p olyacrylamide gel electrophoresis revealed that only similar to 20% troponi n I was lost during the isolation procedure. In a further step, 3-D helical reconstructions were calculated using STEM darkfield images. These 3-D rec onstructions will allow further characterization of molecular details, and they will be useful for directly visualizing molecular alterations related to diseased cardiac thin myofilaments (e.g., reperfusion injury, alteration s of Ca2+-mediated tropomyosin switch). (C) 1999 Academic Press.