Simultaneous Electrical and Mechanical Stimulation to Enhance Cells' Cardiomyogenic Potential

Summary

Here we present a protocol for training a cell population using electrical and mechanical stimuli emulating cardiac physiology. This electromechanical stimulation enhances the cardiomyogenic potential of the treated cells and is a promising strategy for further cell therapy, disease modeling, and drug screening.

Abstract

Cardiovascular diseases are the leading cause of death in developed countries. Consequently, the demand for effective cardiac cell therapies has motivated researchers in the stem cell and bioengineering fields to develop in vitro high-fidelity human myocardium for both basic research and clinical applications. However, the immature phenotype of cardiac cells is a limitation on obtaining tissues that functionally mimic the adult myocardium, which is mainly characterized by mechanical and electrical signals. Thus, the purpose of this protocol is to prepare and mature the target cell population through electromechanical stimulation, recapitulating physiological parameters. Cardiac tissue engineering is evolving toward more biological approaches, and strategies based on biophysical stimuli, thus, are gaining momentum. The device developed for this purpose is unique and allows individual or simultaneous electrical and mechanical stimulation, carefully characterized and validated. In addition, although the methodology has been optimized for this stimulator and a specific cell population, it can easily be adapted to other devices and cell lines. The results here offer evidence of the increased cardiac commitment of the cell population after electromechanical stimulation. Electromechanically stimulated cells show an increased expression of main cardiac markers, including early, structural, and calcium-regulating genes. This cell conditioning could be useful for further regenerative cell therapy, disease modeling, and high-throughput drug screening.

Introduction

Heart function is based on the coupling of electrical excitation and mechanical contraction. Briefly, cardiomyocyte intercellular junctions permit electrical signal propagation to produce almost synchronous contractions of the heart that pump blood systemically and through the pulmonary system. Cardiac cells, thus, undergo both electrical and mechanical forces that regulate gene expression and cellular function. Accordingly, many groups have attempted to develop culture platforms that mimic the cardiac physiological environment to understand the role of mechanical and electrical stimulation on cardiac development, function, and maturation. In vitro electrical....

Protocol

This study uses human cardiac ATDPCs from patient samples. Their use has been approved by the local ethics committee, and all patients gave informed consent. The study protocol conforms to the principles outlined in the Declaration of Helsinki.

1. Preparations

1. Autoclave two tweezers, 12 platinum PTFE electrodes for electrical stimulation, and some paper towels, at 121 °C for 20 min.
2. Sterilize 12 PDMS custom-made constructs (Figure 1

Discussion

Electromechanical stimulation appears to be a safe alternative for preparing cells for a hostile cardiac environment and enhancing their cardiac commitment. Here, a protocol described for cardiac progenitor cells increased the expression of main cardiac markers and was reported to be beneficial for their next implantation on infarcted murine myocardium³⁰. In general, electromechanically stimulated cardiac ATDPCs increased the expression of genes related to early, structural, and calcium regulation.......

Materials

Name	Company	Catalog Number	Comments
Stimulator
nickel plated neodymium magnets	Supermagnete	Q-10-10-05-N
nickel-plated neodymium magnets	Supermagnete	Q-06-04-02-HN
polydimethylsiloxane (PDMS) SYLGAR 184 Silicone Elastomer Kit	Dow Corning Corp	184
ruled diffraction grating (1250 grooves/mm)	Newport	05RG150-1250-2
Motor controller	Faulhaber	MCLM-3006-S
Labview	National Instruments
Cell culture
phosphate-buffered saline (PBS)	Gibco	70013-065
0.05% trypsin-EDTA	Gibco	25300-120
35 mm cell culture dish	BD Falcon	45353001
fetal bovine serum (FBS)	Gibco	10270-106
L-Glutamine 200 mM, 100x	Gibco	25030-024
Penicilina/Streptomicine, 10.000 U/mL	Gibco	15140-122
Minimum essential medium eagle (alfa-MEM)	Sigma	M4526-24x500ML
Protein & RNA analyses
protease inhibitor cocktail	Sigma	P8340
QIAzol Lysis Reagent	Qiagen	79306
AllPrep RNA/Protein Kit	Qiagen	50980404
Rneasy mini kit	Qiagen	74104
iTaq Universal Probes One-Step Kit	Bio-Rad Laboratories	172-5140
Random hexamers	Qiagen	79236
TaqMan PreAmp MasterMix 2X	Applied Biosystems	4391128
TaqMan Universal PCR MasterMix	Applied Biosystems	4324018
Immunostaining
10% formalin	Sigma	HT-501128-4L
horse serum	Sigma	H1138
Triton X-100	Sigma	X100-500ML
Bovine Serum Albumina (BSA)	Sigma	A7906-100G
PARAFILM	Sigma	P6543
4',6-diamidino-2-phenylindole (DAPI)	Sigma	D9542
Phalloidin Alexa 568	Invitrogen	A12380
sodium azide	Sigma	S8032-100g
Hoechst 33342	Sigma	14533
Connexin-43 rabbit primary antibody	Sigma	C6219 lot#061M4823
sarcomeric α-actinin mouse primary antibody	Sigma	A7811 lot#080M4864
GATA-4 goat primary antibody	R&D	AF2606 VAZ0515101
MEF2 rabbit primary antibody	Santa Cruz	sc-313 lot#E0611
SERCA2 goat primary antibody	Santa Cruz	sc-8095 lot#D2709
Cy3 secondary antibody	Jackson ImmunoResearch	711-165-152
Cy3 secondary antibody	Jackson ImmunoResearch	715-165-151
Cy3 secondary antibody	Jackson ImmunoResearch	712-165-150
Cy2 secondary antibody	Jackson ImmunoResearch	715-225-150
Cy2 secondary antibody	Jackson ImmunoResearch	711-225-152
Cy2 secondary antibody	Jackson ImmunoResearch	705-225-147