Insights into the frank-starling behaviour and mechanical vulnerability of isolated adult cardiomyocytes: a single-cell work-loop contractility study

Background Understanding cellular mechanisms driving heart failure (HF) progression requires characterisation of cardiomyocyte mechanics under varying loading conditions. While traditional isometric experiments offer limited insight into the dynamic cardiac function, work-loops models provide a more physiologically relevant framework for studying cardiac mechanics in-vitro. We employ a work-loop technique to examine myocyte mechanics under both physiological and pathological load, investigating the Frank-Starling relationship at a cellular level and the impact of high mechanical stress on cardiomyocyte function and structure.

Methods Isolated adult rat ventricular myocytes (n=15) were attached to a piezo-translator and force transducer (IonOptix, Milton, USA) and subjected to both isometric and workloop contractions at increasing sarcomere lengths (preload) at 1 Hz. Work-loops were configured with a preload of 10% and afterload of 50% of developed isometric force. Developed force and stroke work were averaged from ten stable traces. A high-preload protocol (paced at 3 Hz) was used to simulate volume overloaded conditions. Stroke work (area within the work-loop) was continuously monitored. Isometric force and cell morphology were measured pre- and post-experiment. Bathing solution was carefully replaced without disrupting cell position to assess for potential recovery.

Results Myocytes exhibited exponential force augmentation with stretch, with a mean 3.45-fold (±0.58 SD) increase, but stretching beyond 2.2µM led to cell instability and death (figure 1A). Similarly, stroke work increased significantly from 1.7µm to 2.15µM, with a 5.42-fold (±0.96SD) rise from baseline (figure 1B). However, stable workloops could not be maintained at 2.2µM due to pro-arrhythmic behaviour or cell detachment from vigorous force-length changes. No optimal length plateau was demonstrated, unlike multicellular or in-vivo preparations. Under sustained high-preload conditions, significant mechanical deterioration was observed (30.3% force reduction. p=0.0012) within 8–10 minutes of stressed work-loops, accompanied by membrane blebbing and cellular distortion (figure 2). Buffer replacement or cell rest did not restore cellular function, indicating irreversible damage under pathologically high preload conditions.