Automated analysis of smooth muscle calcium sparks in line-scan recordings of intact arteries

Abstract

Activation of ryanodine receptors on the sarcoplasmic reticulum are well known to cause subcellular rapid Ca2+ events that are commonly referred to as Ca2+ sparks. These Ca2+ spark events regulate vascular reactivity through activation of potassium channels on the plasma membrane. Intracellular Ca2+ signals are commonly examined using fluorescent imaging approaches using high speed confocal microscopes. Over the past two decades, analysis of these events has advanced from visual observation and manual analysis by trained observers to more automated approaches. The purpose of the current study was to test whether fully automated approaches could accurately detect Ca2+ sparks in line-scan images and analyze spatial and temporal aspects to the events. This evaluation was addressed by re-analyzing a published “gold standard” dataset, where original analysis was performed through experimenter guided analysis of Ca2+ signals from recordings made in pulmonary arterial myocytes of en face arterial preparations. Ca2+ sparks were detected and analyzed automatically post-hoc using a customized Ca2+ spark analysis program, Sparklab 5.8. Increasing the threshold for detection led to incremental decreases in the percentage of cells with activity and the firing frequency. Raising the threshold also impacted the PPV, sensitivity and false discovery rate, which was examined through comparison to visually observed Ca2+ spark events. Interestingly, changing the threshold had little impact on spatial and temporal aspects of the Ca2+ spark events. Overall, automated Ca2+ spark detection and analysis with Sparklab 5.8 is expected to greatly improve experimental workflow and can be well optimized with low false discovery rates and reasonable levels of sensitivity. NIH R01HL155295, R01HL149608 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.