Visualization of Cell Cycle Variations and Determination of Nucleation in Postnatal Cardiomyocytes

Summary

To distinguish cell division from cell cycle variations in cardiomyocytes, we present protocols using two transgenic mouse lines: Myh6-H2B-mCh transgenic mice, for the unequivocal identification of cardiomyocyte nuclei, and CAG-eGFP-anillin mice, for distinguishing cell division from cell cycle variations.

Abstract

Cardiomyocytes are prone to variations of the cell cycle, such as endoreduplication (continuing rounds of DNA synthesis without karyokinesis and cytokinesis) and acytokinetic mitosis (karyokinesis but no cytokinesis). Such atypical cell cycle variations result in polyploid and multinucleated cells rather than in cell division. Therefore, to determine cardiac turnover and regeneration, it is of crucial importance to correctly identify cardiomyocyte nuclei, the number of nuclei per cell, and their cell cycle status. This is especially true for the use of nuclear markers for identifying cell cycle activity, such as thymidine analogues Ki-67, PCNA, or pHH3. Here, we present methods for recognizing cardiomyocytes and their nuclearity and for determining their cell cycle activity. We use two published transgenic systems: the Myh6-H2B-mCh transgenic mouse line, for the unequivocal identification of cardiomyocyte nuclei, and the CAG-eGFP-anillin mouse line, for distinguishing cell division from cell cycle variations. Combined together, these two systems ease the study of cardiac regeneration and plasticity.

Introduction

The correct identification of cardiomyocyte nuclei and the cell cycle status is of crucial importance for the determination of cardiac muscle turnover and regeneration. This is especially true for the use of nuclear markers, such as pHH3, Ki-67, or Thymidine analogs, for identifying cell cycle activity. As the proliferative capacity of adult mammalian cardiomyocytes is very small¹, a false identification of a nucleus positive for a proliferation marker of a cardiomyocyte nucleus could make a crucial difference in the outcome of a proliferation assay. Moreover, cardiomyocytes are prone to variations in the cell cycle, such as endoreduplication a....

Protocol

All procedures of this protocol involving animals were in accordance with the ethical standards of the University of Bonn and complied with the guidelines from Directive 2010/63/EU of the European Parliament on the protection of animals used for scientific purposes.

1. In Vitro Visualization of Cell Cycle Activity in Postnatal Cardiomyocytes

1. Postnatal cardiomyocyte dissociation
   1. Pre-experimental preparations
      1. Prepare culture media (IMDM, 1% Penicillin/Streptomycin, 1% non-essential amino acids, 0.1% β-Mercaptoethanol); medium 1: add FCS to 2%; medium 2: add FCS to 20%.
      2. Coat a 96-....

Results

In order to analyze the effects of siRNAs/miRNAs on the cell cycle activity of postnatal cardiomyocytes in vitro, cardiomyocytes of double-transgenic Myh6-H2B-mCh/CAG-eGFP-anillin mice were isolated on postnatal day 3 (P3) and transfected with cell cycle activity-inducing miR199⁵, siRNA p27, and siRNA Fzr1. Compared to the negative control (Figure 1A), the pictures of miR199- (Figure 1B) and siRNA p27- (Figure 1C

Discussion

There is a controversy over whether cardiomyocytes are able to reenter the cell cycle and divide after injury and during tissue homeostasis. Values for the basic turnover of cardiomyocytes have been given in the range between 1%¹ and 80%⁷. Also after a cardiac lesion, the induction of cell cycle activity and the generation of new cardiomyocytes has been reported in the border zone, with values between 0.0083%⁸ and 25 - 40%⁷. T.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
10 cm Petri dish	Sarstedt	821472
100 µm cell strainer	Becton Dickinson GmbH/Falcon	352360
2,3-Butanedione monoxime (BDM)	Sigma-Aldrich	B0753
G20x1 ½ injection cannula, Sterican	Braun, Melsungen	4657519
20 gauge needle	Becton Dickinson GmbH	301300
24-well plates	Becton Dickinson GmbH/Falcon	353047
2-Methyl-butane	Carl Roth GmbH + Co. KG	3927.1
37% formaldehyde solution	AppliChem GmbH	A0936,1000
3-way stopcock	B. Braun Medical Inc.	16494C
50 mL syringe	B. Braun Medical Inc.	8728810F
70% ethanol	Otto Fischar GmbH	27669
Alexa-Fluor-conjugated secondary antibody	Jackson ImmunoResearch	115-605-205
Alpha-Aktinin EA-53, Mouse IgG	Sigma-Aldrich, Steinheim	A7811
CaCl2	Sigma-Aldrich	C4901
Cell Culture Microplate, 96 Well, Half Area	Greiner bio-one	675986
Collagenase B	Roche	11088815001
confocal microscope Eclipse Ti-E	Nikon
cryostat CM 3050S	Leica
donkey serum	Jackson Immuno Research, Suffolk, GB	017-000-121
Dulbecco's Phosphate Buffered Saline	Sigma-Aldrich	D8537
EDTA	Sigma-Aldrich	E4884
fetal calf serum	PromoCell, Heidelberg
Formaldehyde solution (4%)	PanReac AppliChem	A3697
Gelatine from porcine skin, Type A	Sigma-Aldrich, Steinheim	G2500
glass coverslips	VWR	631-0146
Glucose	Sigma-Aldrich	G7021
Heidelberger extension tube	IMPROMEDIFORM GmbH	MF 1833
Heparin-Natrium	Ratiopharm	5394.02.00
HEPES	Sigma-Aldrich	H3375
HistoBond microscope slides	Marienfeld	0810000
Hoechst 33342 (1 mg/mL)	Sigma Aldrich, Taufkirchen	B2261
Insulin syringe	Becton Dickinson GmbH	300334
Iscove’s ModifiedDulbecco’s Medium (IMDM)	Gibco/Life Technologies, Darmstadt	21980-032
KCl	Sigma-Aldrich	P9333
Laminin	Corning	354221
Laser Scanning Mikroskop Eclipse Ti	Nikoninstruments, Düsseldorf
Lipofectamine RNAiMAX	Invitrogen/Life Technologies, Darmstadt	13778075
Mouse IgG Cy5 (donkey)	Jackson ImmunoResearch	715-175-151
MgCl2	Sigma-Aldrich	M8266
microcentrifuge tube	Sarstedt	72690
Mini shaker	VWR	12620-940
mirVana miRNA mimic, hsa-miR199a-3p	Ambion/Thermo Fischer Scientific	4464066
Biopsy Mold	Sakura Finetek/ VWR	4565
M-slide 8-well ibiTreat	ibidi	80826
NaCl	Sigma-Aldrich	S9888
NaOH	Merck Millipore	567530
negative control(scrambled RNA)	Ambion/Thermo Fischer Scientific	AM4611
Neonatal Heart Dissociation Kit	Miltenyi Biotech, Bergisch Gladbach	130-098-373
NIS Elements AR 4.12.01-4.30.02-64bit	Nikoninstruments, Düsseldorf
Non essential amino acids, NEAA	Gibco/Life Technologies, Darmstad	11140-035
Opti-MEM, Reduced Serum Medium	Gibco	51985-026
P21-siRNA	Ambion/Thermo Fischer Scientific	4390771
P27-siRNA	Ambion/Thermo Fischer Scientific	4390771
Penicillin/Streptomycin	Gibco/Life Technologies, Darmstadt	15140-122
Phosphate buffered saline (PBS)	Sigma-Aldrich, Steinheim	14190-094
Polyvinyl alcohol mounting medium with DABCO®, antifading	Sigma-Aldrich	10981
RNase A	Qiagen	1007885
RNaseZap	Invitrogen/Life Technologies, Darmstadt	AM9780
sample containers	Vitlab	80731
Serological pipette	Greiner	607180
software NIS Elements	Nikon
Sucrose	Sigma-Aldrich	S0389
Tissue-Tek O.C.T. Compound	Sakura Finetek/ VWR	25608-930
ToPro3 iodide (642/661)	Molecular probes/ThermoFisher Scientific	T3605
Tris	Sigma-Aldrich	T1503
Triton X	Fluka	93418
Triton X-100	Fluka	93418
Trypsin	Sigma-Aldrich	T1426
Wheat germ agglutinin (WGA) Fluorescein labeled	Vector Laboratories	VEC-FL-1021-5
α-actinin antibody	Sigma-Aldrich	A7811
β-Mercaptoethanol	Sigma-Aldrich, Steinheim	M3148