Myofilament Force Production and Calcium Sensitivity: Cardiac slices 100-150 um thick are demembranated with detergent, sculpted to ~125 um width and attached to aluminum T-clips, which are then attached to a length motor and force transducer as shown in Figure 3A. Sarcomere length is often set to 2.2 um, and isometric force will be recorded at incrementally increasing calcium concentrations. This isometric force is often normalized to maximum force, as illustrated with an example in Figure 3B, to provide an indicator of calcium sensitivity.
We typically examine two samples from each heart and 6-12 hearts per condition, such as genotype. Characteristics reported include minimum tension (i.e., resting tension as the prescribed sarcomere length), maximum tension development, pCa50, Hill coefficient and sensitivity to sarcomere length.
The influence of phosphorylation by PKA on calcium sensitivity can be assessed by incubation in catalytic domain of PKA as illustrated in Figure 3B.
