In vitro Assessment of Cardiac Reprogramming by Measuring Cardiac Specific Calcium Flux with a GCaMP3 Reporter

Summary

We describe here, the establishment and application of an Tg(Myh6-cre)1Jmk/J /Gt(ROSA)26Sor^{tm38(CAG-GCaMP3)Hze}/J (referred to as αMHC-Cre/Rosa26A-Flox-Stop-Flox-GCaMP3 below) mouse reporter line for cardiac reprogramming assessment. Neonatal cardiac fibroblasts (NCFs) isolated from the mouse strain are converted into induced cardiomyocytes (iCMs), allowing for convenient and efficient evaluation of reprogramming efficiency and functional maturation of iCMs via calcium (Ca²⁺) flux.

Abstract

Cardiac reprogramming has become a potentially promising therapy to repair a damaged heart. By introducing multiple transcription factors, including Mef2c, Gata4, Tbx5 (MGT), fibroblasts can be reprogrammed into induced cardiomyocytes (iCMs). These iCMs, when generated in situ in an infarcted heart, integrate electrically and mechanically with the surrounding myocardium, leading to a reduction in scar size and an improvement in heart function. Because of the relatively low reprogramming efficiency, purity, and quality of the iCMs, characterization of iCMs remains a challenge. The currently used methods in this field, including flow cytometry, immunocytochemistry, and qPCR, mainly focus on cardiac-specific gene and protein expression but not on the functional maturation of iCMs. Triggered by action potentials, the opening of voltage-gated calcium channels in cardiomyocytes leads to a rapid influx of calcium into the cell. Therefore, quantifying the rate of calcium influx is a promising method to evaluate cardiomyocyte function. Here, the protocol introduces a method to evaluate iCM function by calcium (Ca²⁺) flux. An αMHC-Cre/Rosa26A-Flox-Stop-Flox-GCaMP3 mouse strain was established by crossing Tg(Myh6-cre)1Jmk/J (referred to as Myh6-Cre below) with Gt(ROSA)26Sor^{tm38(CAG-GCaMP3)Hze}/J (referred to as Rosa26A-Flox-Stop-Flox-GCaMP3 below) mice. Neonatal cardiac fibroblasts (NCFs) from P0-P2 neonatal mice were isolated and cultured in vitro, and a polycistronic construction of MGT was introduced to NCFs, which led to their reprogramming to iCMs. Because only successfully reprogrammed iCMs will express GCaMP3 reporter, the functional maturation of iCMs can be visually assessed by Ca²⁺ flux with fluorescence microscopy. Compared with un-reprogrammed NCFs, NCF-iCMs showed significant calcium transient flux and spontaneous contraction, similar to CMs. This protocol describes in detail the mouse strain establishment, isolation and selection of neonatal mice hearts, NCF isolation, production of retrovirus for cardiac reprogramming, iCM induction, the evaluation of iCM Ca²⁺ flux using our reporter line, and related statistical analysis and data presentation. It is expected that the methods described here will provide a valuable platform to assess the functional maturation of iCMs for cardiac reprogramming studies.

Introduction

Myocardial infarction (MI) is a severe disease worldwide. Cardiovascular diseases (CVDs) are the leading cause of death worldwide and account for approximately 18.6 million deaths in 2019^1,2. The total mortality of CVDs has decreased during the past half a century. However, this trend has been slowed or even reversed in some undeveloped countries¹, which calls for more effective treatments of CVDs. As one of the fatal manifestations of CVD, MI accounts for about half of all deaths attributed to CVDs in the United States². During the ischemia, with the blocking of....

Protocol

All experimental procedures and practices involving animals were approved by Institutional Animal Care & Use Committee at the University of Michigan. All experimental procedures and practices involving cell culture must be performed BSL2 Biological Safety Cabinet under sterile conditions. For the procedures and practices involving viruses, the guideline of the proper disposal of transfected cells, pipette tips, and tubes to avoid the risk of environmental and health hazards was followed.

1. Establishment of a Tg(Myh6-cre)1Jmk/J /Gt(ROSA)26Sor^{tm38(CAG-GCaMP3)Hze} /J (referred to as Myh6-Cre/Rosa26A-Flox-Stop-Flox-GCaMP3) mouse st....

Results

The experimental workflow to generate Myh6-Cre/Rosa26A-Flox-Stop-Flox-GCaMP3 mouse strain and the gene structure of the transgenic mice is shown in Figure 1. While the mouse strain is established, the pups' hearts were isolated and observed under a reverse fluorescence microscope to confirm the genotype. Hearts with correct genotype show Ca²⁺ flux synchronized with beating, visualized as GCaMP3 fluorescence, while no fluorescence was observed in control hearts (

Discussion

Evaluating iCMs function is necessary for the cardiac reprogramming field. In this manuscript, the protocol describes a Tg(Myh6-cre)1Jmk/J /Gt(ROSA)26Sor^{tm38(CAG-GCaMP3)Hze}/J mouse strain that has been established, how to use the NCFs isolated from the neonatal mice in this strain for the reprogramming to iCMs, and the evaluation of iCMs function by Ca²⁺ flux. This is a de novo method to evaluate iCMs functional maturation.

Several critical steps are important fo.......

Materials

Name	Company	Catalog Number	Comments
15 mL Conical Centrifuge Tubes	Thermo Fisher Scientific	14-959-70C
50mL Conical Centrifuge Tubes	Thermo Fisher Scientific	14-959-49A
6 Well Cell Culture Plates	Alkali Scientific	TP9006
A83-01	Stemgent	04–0014
All-in-One Fluorescence Microscope	Keyence	BZ-X800E	Inverted fluorescence microscope
B-27 Supplement (50X), serum free	Thermo Fisher Scientific	17504044
Blasticidin S HCl (10 mg/mL)	Thermo Fisher Scientific	A1113903
Bovine Serum Albumin (BSA) DNase- and Protease-free Powder	Thermo Fisher Scientific	BP9706100
CD90.2 MicroBeads, mouse	Miltenyi Biotec	130-049-101	Thy1.2 microbeads
Collagenase, Type 2	Thermo Fisher Scientific	NC9693955
Counting Chamber	Thermo Fisher Scientific	02-671-51B	Hemocytometer
DMEM, high glucose, no glutamine	Thermo Fisher Scientific	11960069
DPBS, calcium, magnesium	Thermo Fisher Scientific	14-040-133
Ethanol, 200 proof (100%)	Thermo Fisher Scientific	04-355-451
Ethylenediamine Tetraacetic Acid (Certified ACS)	Thermo Fisher Scientific	E478-500
Fetal Bovine Serum	Corning	35-010-CV
HBSS, calcium, magnesium, no phenol red	Thermo Fisher Scientific	14025092
IMDM media	Thermo Fisher Scientific	12440053
IX73 Inverted Microscope	Olympus	IX73P2F	Inverted fluorescence microscope
Lipofectamine 2000 Transfection Reagent	Thermo Fisher Scientific	11-668-019
LS Columns	Miltenyi Biotec	130-042-401
Medium 199, Earle's Salts	Thermo Fisher Scientific	11150059
MidiMACS Separator and Starting Kits	Miltenyi Biotec	130-042-302
Millex-HV Syringe Filter Unit, 0.45 µm, PVDF, 33 mm, gamma sterilized	Millipore Sigma	SLHV033RB
MM589			Obtained from Dr. Shaomeng Wang’s lab in University of Michigan
Opti-MEM I Reduced Serum Medium	Thermo Fisher Scientific	31-985-070
PBS, pH 7.4	Thermo Fisher Scientific	10-010-049
Penicillin-Streptomycin (10,000 U/mL)	Thermo Fisher Scientific	15140122
Platinum-E (Plat-E) Retroviral Packaging Cell Line	Cell Biolabs	RV-101
pMx-puro-MGT	Addgene	111809
Poly(ethylene glycol)	Millipore Sigma	P5413-1KG	PEG8000
Polybrene Infection / Transfection Reagent	Millipore Sigma	TR-1003-G
PTC-209	Sigma	SML1143–5MG
Puromycin Dihydrochloride	Thermo Fisher Scientific	A1113803
Recombinant Human IGF-I	Peprotech	100-11
RPMI 1640 Medium	Thermo Fisher Scientific	11875093
ST 16 Centrifuge Series	Thermo Fisher Scientific	75-004-381
Sterile Cell Strainers	Thermo Fisher Scientific	22-363-547	40 µm strainer
Surface Treated Tissue Culture Dishes	Thermo Fisher Scientific	FB012921
TE Buffer	Thermo Fisher Scientific	12090015
Trypan Blue solution	Millipore Sigma	T8154
Trypsin-EDTA (0.05%), phenol red	Thermo Fisher Scientific	25300054
Vortex Mixer	Thermo Fisher Scientific	02215365