Name: visualization of cell cycle variations and determination of nucleation in postnatal cardiomyocytes
Uploaded: 2017-02-24T14:00:00
Description: Watch this Scientific Journal Video about Visualization of Cell Cycle Variations and Determination of Nucleation in Postnatal Cardiomyocytes at JoVE.com

We thank S. Gr&#252;nberg (Bonn, Germany) and P. Freitag (Bonn, Germany) for their technical assistance.

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
<ol>
	<li>Postnatal cardiomyocyte dissociation
	<ol>
		<li>Pre-experimental preparations
		<ol>
			<li>Prepare culture media (IMDM, 1% Penicill...

<ol>
	<li>Bergmann, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+cardiomyocyte+renewal+in+humans.">Evidence for cardiomyocyte renewal in humans.</a> Science. 324 (5923), 98-102 (2009).</li><li>Raulf, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenic+systems+for+unequivocal+identification+of+cardiac+myocyte+nuclei+and+analysis+of+cardiomyocyte+cell+cycle+status.">Transgenic systems for unequivocal identification of cardiac myocyte nuclei and analysis of cardiomyocyte cell cycle status.</a> Basic Res.Cardiol. 110 (3), 33 (2015).</li><li>Field, C. M., Alberts, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anillin,+a+contractile+ring+protein+that+cycles+from+the+nucleus+to+the+cell+cortex.">Anillin, a contractile ring protein that cycles from the nucleus to the cell cortex.</a> J.Cell Biol. 131 (1), 165-178 (1995).</li><li>Hesse, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+visualization+of+cell+division+using+high-resolution+imaging+of+M-phase+of+the+cell+cycle.">Direct visualization of cell division using high-resolution imaging of M-phase of the cell cycle.</a> Nat.Commun. 3, 1076 (2012).</li><li>Eulalio, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+screening+identifies+miRNAs+inducing+cardiac+regeneration.">Functional screening identifies miRNAs inducing cardiac regeneration.</a> Nature. 492 (7429), 376-381 (2012).</li><li>Di, S. V., Giacca, M., Capogrossi, M. C., Crescenzi, M., Martelli, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Knockdown+of+cyclin-dependent+kinase+inhibitors+induces+cardiomyocyte+re-entry+in+the+cell+cycle.">Knockdown of cyclin-dependent kinase inhibitors induces cardiomyocyte re-entry in the cell cycle.</a> J.Biol.Chem. 286 (10), 8644-8654 (2011).</li><li>Hosoda, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clonality+of+mouse+and+human+cardiomyogenesis+in+vivo.">Clonality of mouse and human cardiomyogenesis in vivo.</a> Proc.Natl.Acad.Sci.U.S.A. 106 (40), 17169-17174 (2009).</li><li>Soonpaa, M. H., Field, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+cardiomyocyte+DNA+synthesis+in+normal+and+injured+adult+mouse+hearts.">Assessment of cardiomyocyte DNA synthesis in normal and injured adult mouse hearts.</a> Am.J.Physiol. 272 (1 Pt 2), H220-H226 (1997).</li><li>Ang, K. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Limitations+of+conventional+approaches+to+identify+myocyte+nuclei+in+histologic+sections+of+the+heart.">Limitations of conventional approaches to identify myocyte nuclei in histologic sections of the heart.</a> Am.J.Physiol Cell Physiol. 298 (6), C1603-C1609 (2010).</li><li>Engel, F. B., Schebesta, M., Keating, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anillin+localization+defect+in+cardiomyocyte+binucleation.">Anillin localization defect in cardiomyocyte binucleation.</a> J Mol Cell Cardiol. 41 (4), 601-612 (2006).</li><li>Bergmann, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+Cell+Generation+and+Turnover+in+the+Human+Heart.">Dynamics of Cell Generation and Turnover in the Human Heart.</a> Cell. 161 (7), 1566-1575 (2015).</li></ol>

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...

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 and acytokinetic mitosis, which result in polyploid and multinucleated cells rather than in cell division. To this end, the interpretation of antibody staining against common cell cycle markers is not conclusive in all cases.
Here, we present methods for the straight-forward recognition of mouse cardiomyocytes and their nuclearity in native isolated cells and thick tissue sections at postnatal and adult stages by the unequivocal identification of their nuclei. For that purpose, a transgenic mouse line with cardiomyocyte-specific expression of a fusion protein consisting of human histone H2B and mCherry under the control of the Myh6 promoter (Myh6-H2B-mCh) was used2. Cross-breeding this mouse line with a transgenic proliferation indicator mouse line, in which the expression of an eGFP-anillin fusion protein is under the control of the ubiquitous chicken actin promoter with a CMV enhancer (CAG-eGFP-anillin), allows for the determination of the cell cycle status. The scaffolding protein anillin is specifically expressed in cell-cycle active cells3, and its differential subcellular localization during the cell cycle allows for live-tracking cell-cycle progression with a high resolution of M-Phase4. Therefore, the double transgenic mice can be used to discriminate between proliferating cardiomyocytes and those that undergo cell-cycle variations. This proves especially useful in screening for proliferation-inducing substances in vitro.

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			<td height="21" style="height:21px;width:383px;">10 cm petri dish</td>
			<td style="width:331px;">Sarstedt</td>
			<td style="width:119px;">821472</td>
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			<td style="width:119px;">352360</td>
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			<td style="width:331px;">Sigma-Aldrich</td>
			<td style="width:119px;">B0753</td>
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			<td height="21" style="height:21px;">G20x1 &#189; injection cannula, Sterican</td>
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			<td>4657519</td>
			<td></td>
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			<td height="21" style="height:21px;width:383px;">20 gauge needle</td>
			<td>Becton Dickinson GmbH</td>
			<td style="width:119px;">301300</td>
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			<td height="21" style="height:21px;width:383px;">24-well plates</td>
			<td style="width:331px;">Becton Dickinson GmbH/Falcon</td>
			<td style="width:119px;">353047</td>
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			<td height="21" style="height:21px;width:383px;">2-Methyl-butane</td>
			<td style="width:331px;">Carl Roth GmbH + Co. KG</td>
			<td style="width:119px;">3927.1</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:383px;">37% formaldehyde solution</td>
			<td style="width:331px;">AppliChem GmbH&#160;</td>
			<td>A0936,1000</td>
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			<td height="21" style="height:21px;width:383px;">3-way stopcock</td>
			<td style="width:331px;">B. Braun Medical Inc.</td>
			<td style="width:119px;">16494C</td>
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			<td height="21" style="height:21px;width:383px;">50 ml syringe</td>
			<td style="width:331px;">B. Braun Medical Inc.</td>
			<td style="width:119px;">8728810F</td>
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			<td height="21" style="height:21px;width:383px;">70% ethanol</td>
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			<td style="width:119px;">27669</td>
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			<td height="21" style="height:21px;width:383px;">Alexa-Fluor-conjugated secondary antibody</td>
			<td>Jackson ImmunoResearch</td>
			<td style="width:119px;">115-605-205</td>
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			<td height="21" style="height:21px;width:383px;">Alpha-Aktinin EA-53, Mouse IgG</td>
			<td style="width:331px;">Sigma-Aldrich, Steinheim</td>
			<td style="width:119px;">A7811</td>
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			<td height="21" style="height:21px;width:383px;">CaCl</td>
			<td style="width:331px;">Sigma-Aldrich</td>
			<td style="width:119px;">C4901</td>
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			<td style="width:119px;">675986</td>
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			<td height="21" style="height:21px;width:383px;">Collagenase B</td>
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			<td height="21" style="height:21px;width:383px;">confocal microscope Eclipse Ti-E</td>
			<td style="width:331px;">Nikon</td>
			<td style="width:119px;"></td>
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			<td style="width:119px;"></td>
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			<td height="21" style="height:21px;">donkey serum</td>
			<td>Jackson Immuno Research, Suffolk, GB</td>
			<td style="width:119px;">017-000-121</td>
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			<td height="21" style="height:21px;width:383px;">Dulbecco&#39;s Phosphate Buffered Saline</td>
			<td style="width:331px;">Sigma-Aldrich</td>
			<td style="width:119px;">D8537</td>
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			<td height="30" style="height:30px;width:383px;">EDTA</td>
			<td style="width:331px;">Sigma-Aldrich</td>
			<td style="width:119px;">E4884</td>
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			<td height="21" style="height:21px;">fetal calf serum</td>
			<td>PromoCell, Heidelberg</td>
			<td style="width:119px;"></td>
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			<td height="21" style="height:21px;">Formaldehyde solution (4%)</td>
			<td>PanReac AppliChem</td>
			<td style="width:119px;">A3697</td>
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			<td height="25" style="height:25px;">Gelatine from porcine skin, Type A</td>
			<td>Sigma-Aldrich, Steinheim</td>
			<td style="width:119px;">G2500</td>
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			<td height="21" style="height:21px;width:383px;">glass coverslips</td>
			<td style="width:331px;">VWR</td>
			<td style="width:119px;">631-0146</td>
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			<td height="21" style="height:21px;width:383px;">Glucose</td>
			<td style="width:331px;">Sigma-Aldrich</td>
			<td style="width:119px;">G7021</td>
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			<td height="21" style="height:21px;width:383px;">Heidelberger extension tube</td>
			<td>IMPROMEDIFORM GmbH</td>
			<td style="width:119px;">MF 1833</td>
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			<td height="21" style="height:21px;width:383px;">Heparin-Natrium</td>
			<td style="width:331px;">Ratiopharm</td>
			<td style="width:119px;">5394.02.00</td>
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			<td height="21" style="height:21px;width:383px;">HEPES</td>
			<td style="width:331px;">Sigma-Aldrich</td>
			<td style="width:119px;">H3375</td>
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			<td height="21" style="height:21px;width:383px;">HistoBond microscope slides</td>
			<td style="width:331px;">Marienfeld</td>
			<td style="width:119px;">0810000</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Hoechst 33342 (1mg/ml)</td>
			<td>Sigma Aldrich, Taufkirchen</td>
			<td style="width:119px;">B2261</td>
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			<td height="21" style="height:21px;width:383px;">Insulin syringe</td>
			<td>Becton Dickinson GmbH</td>
			<td style="width:119px;">300334</td>
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			<td height="21" style="height:21px;">Iscove&#8217;s ModifiedDulbecco&#8217;s Medium (IMDM)</td>
			<td>Gibco/Life Technologies, Darmstadt</td>
			<td>21980-032</td>
			<td></td>
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			<td height="21" style="height:21px;width:383px;">KCl</td>
			<td style="width:331px;">Sigma-Aldrich</td>
			<td style="width:119px;">P9333</td>
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			<td height="21" style="height:21px;width:383px;">Laminin</td>
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			<td height="21" style="height:21px;">Laser Scanning Mikroskop Eclipse Ti</td>
			<td>Nikoninstruments, D&#252;sseldorf</td>
			<td style="width:119px;"></td>
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			<td height="21" style="height:21px;">Lipofectamine RNAiMAX</td>
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			<td>13778075</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:383px;">Mouse IgG Cy5 (donkey)</td>
			<td style="width:331px;">Jackson ImmunoResearch</td>
			<td>715-175-151</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:383px;">MGCl</td>
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			<td height="21" style="height:21px;width:383px;">microcentrifuge tube</td>
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			<td style="width:119px;">72690</td>
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			<td height="21" style="height:21px;width:383px;">Mini shaker</td>
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			<td>12620-940</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">mirVana miRNA mimic, hsa-miR199a-3p</td>
			<td style="width:331px;">Ambion/Thermo Fischer Scientific</td>
			<td>4464066</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">Biopsy Mold</td>
			<td style="width:331px;">Sakura Finetek/ VWR</td>
			<td>4565</td>
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			<td height="21" style="height:21px;width:383px;">M-slide 8-well ibiTreat</td>
			<td style="width:331px;">ibidi</td>
			<td style="width:119px;">80826</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:383px;">NaCl</td>
			<td style="width:331px;">Sigma-Aldrich</td>
			<td style="width:119px;">S9888</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">NaOH</td>
			<td style="width:331px;">Merck Millipore</td>
			<td style="width:119px;">567530</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">negative control(scrambled RNA)</td>
			<td style="width:331px;">Ambion/Thermo Fischer Scientific</td>
			<td>AM4611</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Neonatal Heart Dissociation Kit</td>
			<td>Miltenyi Biotech, Bergisch Gladbach</td>
			<td style="width:119px;">130-098-373</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NIS Elements AR 4.12.01-4.30.02-64bit</td>
			<td>Nikoninstruments, D&#252;sseldorf</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Non essential amino acids, NEAA</td>
			<td>Gibco/Life Technologies, Darmstad</td>
			<td>11140-035</td>
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			<td height="21" style="height:21px;width:383px;">Opti-MEM, Reduced Serum Medium</td>
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			<td style="width:331px;">Ambion/Thermo Fischer Scientific</td>
			<td>4390771</td>
			<td></td>
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			<td style="width:331px;">Ambion/Thermo Fischer Scientific</td>
			<td>4390771</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin/Streptomycin</td>
			<td>Gibco/Life Technologies, Darmstadt</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphate buffered saline (PBS)</td>
			<td>Sigma-Aldrich, Steinheim</td>
			<td>14190-094</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:383px;">Polyvinyl alcohol mounting medium with DABCO&#174;, antifading</td>
			<td style="width:331px;">Sigma-Aldrich</td>
			<td style="width:119px;">10981</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:383px;">RNase A</td>
			<td style="width:331px;">Qiagen</td>
			<td>1007885</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RNaseZap</td>
			<td>Invitrogen/Life Technologies, Darmstadt</td>
			<td style="width:119px;">AM9780</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:383px;">sample containers</td>
			<td style="width:331px;">Vitlab</td>
			<td style="width:119px;">80731</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:383px;">Serological pipette</td>
			<td style="width:331px;">Greiner</td>
			<td style="width:119px;">607180</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">software NIS Elements</td>
			<td style="width:331px;">Nikon</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">Sucrose</td>
			<td style="width:331px;">Sigma-Aldrich</td>
			<td style="width:119px;">S0389</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:383px;">Tissue-Tek O.C.T. Compound</td>
			<td style="width:331px;">Sakura Finetek/ VWR</td>
			<td>25608-930</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">ToPro3 iodide (642/661)</td>
			<td style="width:331px;">Molecular probes/ThermoFisher Scientific</td>
			<td style="width:119px;">T3605</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">Tris</td>
			<td style="width:331px;">Sigma-Aldrich</td>
			<td style="width:119px;">T1503</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">Triton X</td>
			<td style="width:331px;">Fluka</td>
			<td style="width:119px;">93418</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">Triton X-100</td>
			<td style="width:331px;">Fluka</td>
			<td style="width:119px;">93418</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">Trypsin</td>
			<td style="width:331px;">Sigma-Aldrich</td>
			<td style="width:119px;">T1426</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">Wheat germ agglutinine (WGA) Fluorescein labeled</td>
			<td>Vector Laboratories</td>
			<td>VEC-FL-1021-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">&#945;-actinin antibody</td>
			<td style="width:331px;">Sigma-Aldrich</td>
			<td style="width:119px;">A7811</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#946;-Mercaptoethanol</td>
			<td>Sigma-Aldrich, Steinheim</td>
			<td>M3148</td>
			<td></td>
		</tr>
	</tbody>
</table>

visualization of cell cycle variations and determination of nucleation in postnatal cardiomyocytes

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.

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</...

Watch this Scientific Journal Video about Visualization of Cell Cycle Variations and Determination of Nucleation in Postnatal Cardiomyocytes at JoVE.com

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

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.

Cardiomyocytes are prone to variations of the cell cycle, such as endoreduplication (continuing rounds of DNA synthesis without karyokinesis and ...

The overall goal of this procedure is to visualize cell cycle activity in postnatal cardiomyocytes and to determine their nuclearity. This method can help answer key questions in the cardiac regeneration field, such as does a cardiomyocyte really divide or does it just increase its ploidy or become multi-nucleated? The main advantages of this technique are that the M phase of the cell cycle can be visualized in high spatiotemporal resolution and that cardiomyocyte nuclei can be identified by fluorescence.Demonstrating the procedure will be Patricia Freitag, a technician from our laboratory. If using non-homozygous breeding pairs, first identify the transgenic hearts via fluorescent microscopy to check for their EGFP analin and H2B-mCherry expression. EGFP analyin nuclear signals can be most easily detected at the edges of the atria by focusing through its tissue layers.To isolate the cardiomyocytes, place the appropriate number of hearts in 1.5 milliliter reaction tubes containing one milliliter of enzyme mix from a neonatal heart dissociation kit and cut the hearts into small pieces with scissors. Then, transfer the solution into 15 milliliter reaction tubes. Place the tube in an almost horizontal position at 37 degrees celsius for 15 minutes, mixing the tissue five to 10 times with a five milliliter pipette at the end of the incubation.At the end of the third digestion, stop the reaction with 7.5 milliliters of medium two and filter the cell suspension through a 70 micron cell strainer. Rinse the cell strainer with three milliliters of medium two, pooling the wash with the collected cells and collect the cells by centrifugation. Re-suspend the pellet in 500 microliters of medium one for counting.Then, seed one times ten to the fourth cells per well in 120 microliters of medium one in a 96-well flat-bottom plate and centrifuge the cells before their overnight culture in a cell culture incubator. The next day, most of the cardiomyocytes should be beating. Before beginning the transfection, wipe the bench and all of the transfection materials with RNAse decontamination solution to remove any RNAse.When all of the materials are ready, add 20 microliters of freshly prepared siRNA mix to each well and return the cells to the incubator for 48 hours. After two days, replace the supernatant in each well with 120 microliters of medium one and return the cells to the incubator for at least another 24 hours. At the end of the incubation, wash the cells one time with PBS, followed by fixation in 4%formaldehyde solution for 20 minutes.To analyze the cells by confocal video microscopy, transfer the plate to the confocal microscope stage and start the imaging software. Open the A1plus settings and check the channel one, two, three and four boxes. In the pull-down menu, set channel one to DAPI, channel two to eGFP, channel three to RFP, and channel four to Cy5.Click the channel voltage and use the slide bars to set the voltage to 80 for each channel. Use the slide bars to set the offset to zero and click on the home button to set the pinhole to the home position. Set the scan size to 1024 by 1024 pixels in the pull-down menu.Click optimize to open the XYZ size setup window and check the perfect voxel box under the suggested step Z.Select the 20x objective and adjust the laser intensities until the picture is neither under-nor overexposed. When all of the parameters have been set, open the acquire menu. Select scan large image then choose current position is at top left corner under area.Then set the number of fields in X and Y to three by three and click scan. To define the number of cardiomyocyte nuclei, click measure, manual measurement, and counts. Select the nuclei with H2B-mCherry signals, thereby marking and counting them.Then, select the alpha-actinin positive cells to determine the number of cardiomyocytes. To count the G1, S, G2 phase cardiomyocytes with nuclear EGFP analin signals, select measure, manual measurement, and counts. Mark the EGFP analin and H2B-mCherry expressing nuclei with clicking and manually count the cardiomyocytes with the mitosis-specific EGFP analin signals such as contractile rings and mid-bodies.Then, click measure, manual measurement, and counts and click the cardiomyocytes with mitosis-specific EGFP analin signals, cytoplasmatic signals, contractile rings, and mid-bodies. Compared to the negative controls, the miRNA and siRNA transfected cardiomyocytes demonstrate an induction of cell cycle activity. Cardiomyocytes that perform endoreduplication exhibit an exclusively nuclear EGFP analin expression, whereas cardiomyocytes undergoing cell division display EGFP analin expression in M phase typical localizations.After enzymatic digestion of the heart tissue at the Langendorff apparatus, the atria and ventricles can be mechanically separated and analyzed independently, allowing the visualization of H2B-mCherry transgenic ventricular cardiomyocytes with a high number of H2B-mCherry positive bi-nucleated cardiomyocytes. By contrast, the majority of atrial cardiomyocytes are mono-nucleated. Enzymatic digestion does not result in a 100%single-cell population revealing a pattern of cross-striation by alpha-actinin staining, further facilitating the discrimination between bi-nucleated cardiomyocytes as a continuous pattern of cross-striation and cell doublets.To analyze the bi-nucleation index of cardiomyocytes in thick slices, it is necessary to manually scroll through the stack as the nuclei do not necessarily lay within one Z-plane, with wheat germ agglutinin staining permitting the detection of the cell borders. 3D reconstructions of fixed slices of adult transgenic mouse hearts allow the detection and automated counting of hooks, stained, and H2B-mCherry positive nuclei to determine the proportion of cardiomyocyte nuclei under physiological conditions within the tissue. Once mastered, the heart dissociation can be completed in two to three hours if it is performed properly.While attempting this procedure, it is important to remember to work continuously to maintain the viability of the cells throughout the experiment. Following this procedure, it is possible to screen microRNAs or other small substances for their proliferation-inducing effects in postnatal cardiomyocytes.

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