Cardiac Slice Intracellular Calcium Regulation: As a companion to contraction-relaxation function, intact cardiac slices can be loaded with CalRed-AM, a calcium indicator dye. After a sufficient incubation period to allow loading of dye and de-esterification, the slice will be placed between a force transducer and length motor (Figure 2A). Diastolic sarcomere length will be initially established at 2.2 um. Slices will be exposed to excitation light of 490 nm and resulting fluorescence will be recorded at 525 nm and 650 nm. The ratio of intensities 525/650 will provide a measure of intracellular calcium concentration. Figure 2B shows an example set of Cal Red signals measured in a contracting cardiac slice.
These measures of calcium regulation are important to this study to demonstrate that functional phenotype of under investigation is not due to either direct or compensatory effects on calcium regulation or is possibly partially due to effects on calcium regulation.
We typically prepare at least two slices per heart and examine at least one slice per heart at four sarcomere lengths between 1.9-2.3 um. Characteristics reported will include ratio (i.e., resting calcium at the prescribed sarcomere length), peak ratio, rates of ratio rise and ratio decline, and exponential tau decay constant.
