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In vitro motility assay of atrial and ventricular myosin from pig
C Svensson1, I Morano, A Arner
1Department of Physiology and Neuroscience, Lund University, Sweden.
Abstract:
The role of myosin isoforms in determining contractile filament velocity in the atrium and ventricle of the pig heart was studied by measuring the motion of fluorescently labeled actin over myosin (in vitro motility assay). A rapid and relatively simple method for purification of myosin from small tissue samples was used. The relative extent of light chain-2 phosphorylation was about 30% in both atrial and ventricular myosin extracts. Although the extracted myosin was not free from contaminating proteins, mainly actin, the mean velocity at optimal pH and 32 degrees C of both atrial (3.3 microns/s) and ventricular (2.3 microns/s) myosin were similar to those obtained using extensively purified myosin. The filament sliding velocities using isolated myosin and actin are lower than those estimated from previously published experiments on skinned fiber preparations, which might reflect an influence on sliding velocity by the filament organization or regulatory proteins in the muscle fiber. However, the ratio between velocities of atrial and ventricular myosin was similar in the motility assay (1.5) and muscle fiber experiments (1.6), which might suggest that these two methods reflect the same fundamental processes in cardiac contraction and that the difference in filament sliding velocity between the atrium and ventricle of the pig heart is determined my their myosin isoforms.
Insights
Pig heart myosin isoforms influence cardiac muscle contraction speed. Atrial and ventricular myosin showed similar velocities in vitro, suggesting isoform differences drive distinct heart chamber functions.
Area of Science:
- Cardiology
- Biochemistry
- Muscle Physiology
Background:
- Cardiac muscle contraction relies on myosin's interaction with actin filaments.
- Myosin isoforms are known to influence contractile properties.
- Understanding differences between atrial and ventricular myosin is crucial for cardiac function.
Purpose of the Study:
- To investigate the role of specific myosin isoforms in determining contractile filament velocity in pig cardiac atria and ventricles.
- To compare the in vitro motility of atrial and ventricular myosin.
Main Methods:
- Developed a rapid myosin purification method from small cardiac tissue samples.
- Utilized an in vitro motility assay measuring fluorescently labeled actin filament motion over immobilized myosin.
- Assessed relative light chain-2 phosphorylation levels in myosin extracts.
Main Results:
- Mean filament sliding velocities for atrial (3.3 microns/s) and ventricular (2.3 microns/s) myosin were determined.
- The ratio of atrial to ventricular myosin velocity in the motility assay (1.5) closely matched ratios from muscle fiber experiments (1.6).
- Phosphorylation levels of light chain-2 were similar (~30%) in both atrial and ventricular myosin.
Conclusions:
- Myosin isoforms are the primary determinant of the difference in filament sliding velocity between pig atrial and ventricular cardiac muscle.
- The in vitro motility assay reflects fundamental processes observed in intact muscle fibers.
- Despite lower absolute velocities in vitro, the relative differences between cardiac chambers are preserved, supporting the role of myosin isoforms.