Muscle fiber shortening is obtained by actin filaments
sliding over the myosin filaments. Our theory of the sarcomere contraction
suggests that XB dynamics is determined by two distinct, slow and fast,
kinetics, and the myosin head performs several stroke steps over the actin
filament per hydrolysis of a single ATP molecule. Tracking the isolated single
actin filament trajectory over scattered isolated myosin heads provides insight
into the mechanisms that determines the actin-myosin cross-bridge (XB)
dynamics. The actin filament is labeled by fluorescent dye and the data is
acquired by utilizing photomultiplier. The study developed the image analysis
algorithms for quantifying the dynamics of actin propulsion in these motility
assays studies. The image analysis software was developed using Matlab, and
included the following steps: object detection, object tracking, backbone axis
identification, measurement of actin filament length, path recognition, and
calculations of the local velocity at various points along the filaments. The
image sets examined in this study contained 7 actin filaments. The mean
velocity for the actin filaments was 14.86 ± 3.5mm/s. This velocity is significantly faster than expected
velocity based on the rate of ATP. The filament lengths were constant, within
the available spatial resolution. The results establish the hypothesis that the
myosin head performs several stroke steps per hydrolysis of a single ATP
molecule, and supports the two-kinetic theory of muscle contraction. The
algorithm can be used for exploring the actin-myosin dynamics from various
specimens, and for providing diagnostic test for differentiating between
various cardiac diseases (cardiomyopathies) based on the actin-myosin
phenotype.