Magnetic Adjustment of Afterload in Engineered Heart Tissues

Summary

This protocol provides detailed methods describing the fabrication and implementation of a magnetics-based afterload tuning platform for engineered heart tissues.

Abstract

Afterload is known to drive the development of both physiological and pathological cardiac states. As such, studying the outcomes of altered afterload states could yield important insights into the mechanisms controlling these critical processes. However, an experimental technique for precisely fine-tuning afterload in heart tissue over time is currently lacking. Here, a newly developed magnetics-based technique for achieving this control in engineered heart tissues (EHTs) is described. In order to produce magnetically responsive EHTs (MR-EHTs), the tissues are mounted on hollow silicone posts, some of which contain small permanent magnets. A second set of permanent magnets is press-fit into an acrylic plate such that they are oriented with the same polarity and are axially-aligned with the post magnets. To adjust afterload, this plate of magnets is translated toward (higher afterload) or away (lower afterload) from the post magnets using a piezoelectric stage fitted with an encoder. The motion control software used to adjust stage positioning allows for the development of user-defined afterload regimens while the encoder ensures that the stage corrects for any inconsistencies in its location. This work describes the fabrication, calibration, and implementation of this system to enable the development of similar platforms in other labs around the world. Representative results from two separate experiments are included to exemplify the range of different studies that can be performed using this system.

Introduction

Afterload is the systolic load on the ventricle after it has begun to eject blood¹. During cardiac development, an appropriate afterload is of critical importance for cardiomyocyte maturation². In adulthood, low levels of ventricular afterload (e.g., in bedridden patients with high-level spinal cord injury³ or in very special cases like spaceflight⁴) can result in hypotrophy of the heart. Conversely, high afterload can lead to cardiac hypertrophy⁵. While cardiac hypertrophy in endurance athletes or pregnant women is considered beneficial and physiolo....

Protocol

1. Preparation of the Afterload Tuning Platform

NOTE: The steps involved in this portion of the protocol are not time-sensitive.

1. Manufacturing the magnetically responsive silicone racks
   NOTE: These racks serve as the culture platform for EHTs. Each EHT is suspended between two silicone posts, which impart afterload to the tissue. The degree of afterload is directly related to the stiffness of these posts. To enable magnetic afterload tuning, some of the posts need to be magnetically responsive.
   1. Acquire 24-well plate compatible racks of silicone posts (dimensions given in

Results

Magnet post stiffness quantification
A horizontally oriented magnetically responsive silicone post was mounted in a fixed position, and an axially aligned calibration magnet was placed at several defined distances (“magnet spacings”) from this post. Test loads of known weight were suspended from the end of the silicone post, causing the post to bend. This deflection was quantified optically. A linear relationship between the gravitational force of the test load and resulting post deflection was obser.......

Discussion

The protocol outlined herein describes a new technique for magnetically altering afterload in engineered heart tissues. This technique relies upon the use of a piezoelectric stage to translate a plate of strong magnets towards and away from magnetically responsive racks of silicone posts. The closer the two sets of magnets, the stronger the afterload experienced by the EHTs cultured on them.

There are several steps that are critical to the successful production and use of this system. While fa.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Cylindrical plate magnets	HKCM	9962-55184	h = 14 mm, d = 13 mm
Cylindrical post magnets	HKCM	9962-63571	h = 2 mm, d = 0.5 mm
Dental wire	Ormco	266-1316	d = 0.016 inches (0.406 mm)
GraphPad	GraphPad Software, La Jolla, California, USA	version 6.00 for Windows
Motion control software for piezo motor	Micronix USA		free download on manufacturer homepage
Motion controller for piezo motor	Micronix USA	MMC-100-01000
Optical contractility analysis platform	EHT technologies	A0001
Piezoelectric linear motor	Micronix USA	PPS-20-15206	fitted with linear optical encoder, incubator-environment compatible
Styrene Rod	Plastruct	MR-15	d = 0.015 inches (0.381 mm)
USB camera	Reichelt Elektronik	REFLECTA 66142