Defining cardiac stability during Langendorff perfusion

Abstract

The Langendorff heart preparation is a valuable ex vivo technique for studying cardiac electrophysiology. For the past century it has been used in toxicology studies, drug testing, and disease modeling and has contributed significantly to understanding cardiac physiology, development, and pathological states. This study aimed to investigate the stability and reproducibility of the modern Langendorff heart preparation under various experimental conditions and to establish cardiac metric standards with corresponding exclusion criteria. Isolated hearts were extracted from adult Sprague Dawley rats and retrograde perfused with a modified Krebs-Henseleit buffer. A cohort of isolated hearts were used to establish a control baseline of multiple physiological parameters measured over time (e.g., electrocardiogram, flow rate, electrophysiology, electrical and calcium dynamics). These control baseline values were then compared to hearts exposed to the most common media additives in Langendorff studies (e.g., blebbistatin(−/−), bovine serum albumin, and voltage and calcium indicator dye). Over a three-hour period, cardiac metrics were monitored in stable Langendorff-perfused hearts. The addition of blebbistatin, DMSO or dyes did not change EP or ECG measurements as compared to control baseline. The most significant drift in all the EP values was ventricular effective refractory period (VERP). With or without blebbistatin, VERP increased from 40 to 60ms over the course of three hours. In that time heart rate and flow also decreased slightly. Blebbistatin was associated with an increased action potential duration but did not alter calcium transient duration. It was also observed that 1% circulating bovine serum albumin quenched the rh237 voltage dye signal 80% as measured by signal to noise. Constant pressure Langendorff-perfusion with a crystalloid buffer provides a reliable and reproducible model for cardiac electrophysiology research with a few caveats. The total length of study should be carefully considered to account for adaptations in cardiac physiology during longer perfusion times. As an excitation-contraction uncoupler, blebbistatin reduces motion artifact but delays action potential duration. Although BSA is used to minimize cardiac edema, it interferes with membrane bound voltage dyes. This study sets physiological parameters as a useful guide for future cardiac studies that use the isolated heart as a research model. American Physiological Society, R01HD108839 to N.G.P. This is the full abstract presented at the American Physiology Summit 2024 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.