A novel bioreactor platform to apply dynamic load to neonatal lamb living myocardial slices for the study of the regenerative properties of the heart
Authors: Downing B , Nicastro L , Smith J , Hayman D , Pitoulis F , Connolly D , Terracciano C M N 
Published: November 2023

Abstract

Background
The inability of the myocardium to regenerate is a determining factor in heart failure (HF).Platforms that model cardiac disease require to approximate the in vivo environment and related factors.One such factor is mechanical load, and in vitro, application of this has been by static, isometric systems that mimic preload only.Application of both preload and afterload, however, better recapitulates the cardiac cycle.Mechanical load is relevant in neonatal tissue, where the perinatal period sees changes to the haemodynamics, and a loss to the CM's regenerative capacity.MyoLoop is a novel bioreactor that can apply dynamic loading conditions upon living myocardial slices (LMS) by using a 3-element Windkessel model, producing dynamically changing afterload, simulating pressure-volume (P/V) loops.Here we apply physiologically relevant pre- and afterload to neonatal lamb tissue and establish an in vitro method studying the effects of changing load on the regenerative properties of the heart.
Hypothesis
The aim of this study is to investigate the feasibility of the MyoLoop system to modulate contractility in neonatal LMS, in comparison to LMS under isometric preload.

Methods
300 µm-thick LMS were obtained from the left ventricle of neonatal lambs (12-17hrs after birth). Mechanical load was applied isometrically by stretching LMS by 117.5% of their slack length, to recapitulate physiological preload. To reproduce dynamic afterload, LMS were placed in the MyoLoop and stretched at physiological preload (117.5%) but exposed to a dynamic physiological afterload, achieved by shortening of a linear actuator arm attached to the LMS. After 48 hours culture, maximum force produced by the slices under static preload was assessed using a force transducer and compared to the force production and P/V loop measurements recorded from the MyoLoop.

Results
Isometrically loaded slices at 0hrs demonstrated large force amplitude of 41±5.7mN/mm2(mean± SEM, n=6),compared to a lower range of force at 24 hrs(10.9±5.07mN/mm2; mean±SEM, n=5) and even weaker forces at 48hrs(2.42±0.84mN/mm2; n=8).There was a gradual decrease in time-to-peak (TTP) after 48hrs,from 264±0.01ms (mean±SEM, n=6) to 240±0.03ms (mean±SEM, n=8),with an shortening also seen in the relaxation parameters TT50% and TT90%. In contrast, LMS cultured in MyoLoop demonstrated an increase in force amplitude of 25.2% from 0-48hrs,and an increase in TTP and TT50% and TT90% decay(n=2).Additionally, LMS cultured within the MyoLoop system demonstrated viability at 72hrs, producing P/V loops and maintaining contractility at 12.6% above 0 hrs contraction.

Conclusions
The dynamic loading of neonatal LMS through the MyoLoop maintained LMS contractility over 48hrs, compared with isometrically loaded LMS. This highlights the importance of modelling both physiological pre-and afterload in vitro. Further work will elucidate the effects of changing dynamic mechanical load on the regenerative properties of the neonatal heart.