Single-molecule tracking of fluorescently labeled ATP turnover in porcine cardiac myofibrils. (A) Schematic representation of a single sarcomere. P-, C-, and D-zones of the thick filament are labeled accordingly. Alexa 488–labeled α-actinin is denoted in magenta, while Cy3-ATP binding to the thick filament is represented as cyan circles. (B) Enlarged section of a representative porcine cardiac LV myofibril as observed following the overlay of 561-nm illuminated Cy3-ATP (cyan) and 488-nm illuminated α-actinin–containing Z-disks (magenta). (C) Distribution of sarcomere length for untreated porcine myofibrils with a mean of 1.82 µm ± 0.04 SD (N = 3 pigs, 647 sarcomeres) at 21°C. Distribution of sarcomere lengths for PKA-treated samples can be found in Fig. S2. The red dotted line indicates the threshold for the smallest sarcomere that was analyzed for all datasets. (D) Cumulative residence time histograms showing the distribution of ATP attachment event durations in untreated porcine cardiac myofibrils (circles; N = 3 pigs, n = 3,638 events) and PKA-treated porcine cardiac myofibrils (squares; N = 3 pigs, n = 1,463 events). Dashed lines show triple-exponential fits. Note the frequency axis is plotted on a logarithmic scale.