P5373Mechanical heterogeneity across the left ventricular wall - a study using intact multicellular preparations
Authors: Pitoulis F , Perbellini F , Harding S E , De Tombe P , Terracciano C M 
Published: October 2019

Abstract

Introduction
The importance of transmural heterogeneity for left ventricular (LV) function is well recognised. Mid-wall systolic shortening is a better predictor of cardiovascular morbidity than ejection fraction. While variation in electrical properties is well-documented, transmural mechanical differences remain poorly characterised. Most studies are based on isolated cells or permeabilised preparations, with limited data acquired in living multicellular myocardial preparations.

Purpose
Here, we test the hypothesis that heterogeneity in intrinsic mechanical properties exists across the LV wall. We use a novel intact organotypic preparation, myocardial slices, from different layers of the ventricular wall.

Methods
300μm-thick living tangential myocardial slices with preserved structure and function were sequentially obtained from LVs of adult male Sprague-Dawley rats using a high precision vibrating microtome. Each slice corresponded to a different layer of the LV wall. Slices from endo-, mid- and epicardium were used (Fig 1C).
To rule out transmural differences in Ca2+ -response, force-Ca2+ experiments were first performed on slices at 2.1μm sarcomere length (SL) with a half-log [Ca2+] (10–3.5 to 10–2.0 M) in the supernatant. SL-tension experiments were then conducted by measuring steady state force of isometrically twitching slices at 2.00-, 2.10-, 2.20-, 2.25-, 2.35-, & 2.40μm SL in Tyrode solution containing the EC50 [Ca2+], determined from the force-Ca2+ experiments. Analysis of co-variance and two-way ANOVA with Turkey post-hoc were used for statistical comparisons.

Results
Force-Ca2+ data were fit with a variable hill-slope. As no differences in the EC50 of endo-, mid-, and epi- slices (n=6, 5, 4) were found, length-tension experiments were performed at a common EC50 [Ca2+] (10–2.54 M).
Increasing SL increased developed tension in all slices. However, mid- generated significantly greater tension than endo- at 2.25-, 2.35-, & 2.40μm SL but not epi- slices (p=0.006, 0.045, 0.048 respectively) (Fig 1A). A linear regression was fit to the SL-active tension data of the different layers. Epi- had significantly steeper slope than endo- (p=0.0137) suggesting greater ability to respond to stretch (n=6, 5, 6) (Fig 1B). Increasing SL increased passive tension in all slices. Epi- was significantly stiffer than endo- at 2.35- & 2.40μm SL (p=0.023, 0.0002 respectively). Mid- was also significantly stiffer than endo- at 2.40μm SL (p=0.0007) (n=6, 5, 6) (Fig 1A).
Figure 1. A: SL-Tension; B: Slopes; C: Schematic

Conclusions
Here, we demonstrate for the first time the use of myocardial slices for investigation of transmural mechanics in intact adult cardiac tissue. We show that both active and passive mechanical properties differ across the LV wall. Coupled with transmural electrical differences, mechanical heterogeneity may act to orchestrate the normal operation of the whole heart. In disease, loss of heterogeneity may contribute to impaired LV function and accelerate clinical deterioration.

Acknowledgement/Funding
British Heart Foundation (MBBS PhD Studentship FS/18/37/33642)