Extraction of Sub-microscopic Ca Fluxes from Blurred and Noisy Fluorescent Indicator Images with a Detailed Model Fitting Approach
The release of Ca from intracellular stores is key to cardiac muscle function; however, the molecular control of intracellular Ca release remains unclear. Depletion of the intracellular Ca store (sarcoplasmic reticulum, SR) may play an important role, but the ability to measure local SR Ca with fluorescent Ca indicators is limited by the microscope optical resolution and properties of the indicator. This leads to an uncertain degree of spatio-temporal blurring, which is not easily corrected (by deconvolution methods) due to the low signal-to-noise ratio of the recorded signals. In this study, a 3D computer model was constructed to calculate local Ca fluxes and consequent dye signals, which were then blurred by a measured microscope point spread function. Parameter fitting was employed to adjust a release basis function until the model output fitted recorded (2D) Ca spark data. This ‘forward method’ allowed us to obtain estimates of the time-course of Ca release flux and depletion within the sub-microscopic local SR associated with a number of Ca sparks. While variability in focal position relative to Ca spark sites causes more out-of-focus events to have smaller calculated fluxes (and less SR depletion), the average SR depletion was to 20±10% (s.d.) of the resting level. This focus problem implies that the actual SR depletion is likely to be larger and the five largest depletions analyzed were to 8±6% of the resting level. This profound depletion limits SR release flux during a Ca spark, which peaked at 8±3 pA and declined with a half time of 7±2 ms. By comparison, RyR open probability declined more slowly, suggesting release termination is dominated by neither SR Ca depletion nor intrinsic RyR gating, but results from an interaction of these processes. Calcium levels inside myocytes regulate the heart's force of contraction. Calcium is released from the primary intracellular store called the sarcoplasmic reticulum. Calcium release was directly observed as ‘calcium sparks’ using fluorescent calcium indicators inside the cell. More recently, calcium levels inside the store have been measured as calcium ‘blinks’. These suggest that some depletion of store calcium occurs during cell excitation; however, the actual extent of depletion is made uncertain by the complex sarcoplasmic reticulum shape, dye saturation and optical properties of the microscope. While previous studies have assumed idealized microscope properties, we measured microscope blurring and applied it to a computer model of calcium movements inside the cell. In this model, calcium release was adjusted to match the simulated blurred calcium signals to experimental results. The calculations show that the depth of local sarcoplasmic reticulum calcium depletion is much greater than inferred from calcium blinks and that the time-course of calcium release is affected by this depletion. An estimate for the time-course of gating of the ion channels that regulate calcium release inside the cell was also calculated. We suggest that the time-course of SR Ca release arises from a complex interaction of Ca depletion and channel gating.