This work was funded by grants from Deutsche Forschungsgemeinschaft (DFG), Fritz-Thyssen-Stiftung and Charit&eacute;/MDC.

1. Animal Anesthesia and Organ Harvesting
<ol>
 <li>Anaesthetize the mouse by intraperitoneal (i.p.) injection of Ketamine (200 mg/kg BW) and Xylazine (20 mg/kg BW).</li>
 <li>To anticoagulate inject 250 IU Heparin i.p. to avoid blood clotting and thrombus formation.</li>
 <li>Wait until deep narcosis is reached, which is characterized by areflexia. To check for areflexia, test corneal reflex by gently touching the cornea or test flight reflex by tail pinching.</li>
 <li>Transfer the mouse onto operating table and fix it in supine position.</li>
 <li>Incise the skin and the abdominal wall below the xiphoid and perform clamshell thoracotomy: Extend the cut to both sides along the costal arch and subsequently cut ribs in the medial axillary line, deflect the rib cage upwards.</li>
 <li>Open pericardium, locate great vessels. Gently press heart caudal to better display the aorta. Clamp the aorta using forceps.</li>
 <li>Place the heart in concavity of a pair of scissors and dissect all connecting vessels with one single cut. Make sure to preserve a large enough part of the ascending aorta for Langendorff cannulation.</li>
 <li>Transfer excised heart immediately to a Petri dish filled with ice cold and pre-oxygenized solution 1 (for solutions, see Table 1).</li>
</ol>
2. Preparation of Heart and Langendorff Perfusion
<ol>
 <li>Cannulate the aorta with a 1.8F steel cannula. Make sure to avoid air embolism.</li>
 <li>Fixate aorta on the cannula with a surgical suture and flush coronaries with 1 ml of solution 1.</li>
 <li>Connect cannula with a Langendorff apparatus.</li>
 <li>Make sure time from thoracotomy to Langendorff cannulation does not exceed 120 sec to avoid extended ischemia/reperfusion injury to the myocardium.</li>
 <li>Perfuse heart with 10 ml of Ca2+ free solution 2 (4 ml/min).</li>
 <li>Perfuse heart with collagenase solution 3 for 8 min (4 ml/min).</li>
</ol>
3. Microsurgical Dissociation of Cardiac Chambers
<ol>
 <li>Transfer heart into a pre-warmed 100-mm Petri dish containing low Ca2+ solution 4.</li>
 <li>Carefully remove aortic and other non-cardiac tissue with scissors and discard it.</li>
 <li>Separate atria and ventricles and continue with each chamber separately. Keep cells immersed in solution 4. Use small volumes (less than 5 ml).</li>
</ol>
4. Further Dissociation of Cardiomyocytes
<ol>
 <li>Atrial cardiomyocytes:
 <ol>
 <li>To individualize atrial cardiomyocytes transfer the atria into a separate pre-warmed 100-mm culture dish and dissociate the tissue through gently pulling it apart with fine forceps. Ensure an almost complete dissociation of the tissue.</li>
 <li>Use a 1 ml pipette with an enlarged fire-polished plastic pipette tip to suspend the cells in 1 ml of solution 5 for 5 min.</li>
 <li>Separate the cells from debris by using a cell filter (200 &mu;m mesh size).</li>
 <li>Add 5 ml solution 5 to the cell suspension and centrifuge for 2 min at 16 x g at room temperature.</li>
 <li>The following steps are operated under a cell culture hood. Discard the supernatant and re-suspended the pellet in 5 ml of solution 6.</li>
 <li>After sedimentation by gravity for 10 min in a 15 ml tube, centrifuge for 1 min at 16 x g at room temperature. Remove the supernatant. Re-suspend the cells depending on their quantity in 1-5 ml of solution 6.</li>
 </ol>
 </li>
 <li>Ventricular cardiomyocytes:
 <ol>
 <li>Dissect the left ventricular region of interest with fine forceps in 5 ml of solution 4.</li>
 <li>Suspend cells by gently pipetting until most of the cells are separated. Transfer the cell solution after filtering (200 &mu;m mesh size) into a 50 ml tube, add a volume of 25 ml.</li>
 <li>Centrifuge for 2 min at 16 x g at room temperature.</li>
 <li>The following steps are operated under a cell culture hood. Remove supernatant, re-suspend the pellet in 25 ml of solution 6 and allow sedimentation of the cells for 10 min.</li>
 <li>Count the cells and remove the supernatant, add 25 - 50 ml solution 6 on the cells.</li>
 </ol>
 </li>
</ol>
5. Preparation of Cardiomyocytes for Cellular Electrophysiology, Biochemical or IF Studies
<ol>
 <li>For electrophysiology studies, keep the myocytes in solution 6 in a 50 ml tube and inhibit sedimentation.</li>
 <li>For biochemical studies, e.g. calcium imaging, plate myocytes on laminin coated cell culture dish (final concentration 20 &mu;g/ml laminin in PBS).</li>
 <li>For immunofluorescence staining prepare a cell culture dish plate with glass cover slips and coated with laminin solution (final concentration 50 &mu;g/ml laminin in PBS).
 <ol>
 <li>Remove the solution before plating myocytes. Plate the cells and control the cell density by using a microscope.</li>
 <li>Let the myocytes adhere to cover slip for 1 hr at 37 &deg;C in 2 % CO2, remove the solution and start immediately with a standard staining procedure protocol.</li>
 <li>Incubate with fixative, e.g. 4% PFA in PBS (pH 7.5) for 10 min at room temperature and follow with three PBS washing steps for 5 min each.</li>
 <li>To permeabilize the cells and to inhibit unspecific antibody binding incubate the myocytes with 10 % serum, 0.3% Triton, 0.2% BSA in PBS for 30 min at room temperature.</li>
 <li>Incubate with the primary antibody for 1 hr at 37 &deg;C and wash as described before.</li>
 <li>Incubate with the secondary antibody for 1 hr at room temperature. To counterstain the nuclei and &alpha;-actinin use DAPI and fluorochrome conjugated phalloidin (Alexa Fluor 488, Invitrogen).</li>
 <li>After washing, transfer the glass cover slips carefully on silane treated microscope slides and embed cells in fluorescence mounting medium.</li>
 </ol>
 </li>
</ol>
6. Cellular Electrophysiology
<ol>
 <li>Perform Whole-cell patch-clamp recordings on freshly isolated atrial and ventricular cardiomyocytes at room temperature.</li>
 <li>Transfer healthy appearing cardiomyocytes into perfusion chamber filled with a defined volume of extracellular bath solution. 117 mM NaCl, 4 mM KCl, 1 mM KH2PO4, 4 mM NaHCO3, 1.7 mM MgCl2, 3 mM CoCl2, 10 mM HEPES, 10 mM glucose, and 0.02 mM Tetrodotoxin (TTX), (pH 7.4). Use NaOH for pH value adjustment.</li>
 <li>Use proper patch clamp equipment (i.e. IX71 inverted microscope, a Multiclamp 700B Amplifier, a Digidata 1440A acquisition system and PC with pClamp10.3 software (Molecular Devices)).</li>
 <li>Use patch pipettes with resistances of 3-5 M&Omega; when filled with intracellular solution containing 130 mM KCl, 2 mM MgCl2, 11 mM HEPES, 11 mM EGTA, 5 mM Na2ATP, 0.4 mM Na2GTP, 5 mM Na2CP and 4.9 mM CaCl2 (pH 7.2). Use KOH for pH value adjustment.</li>
 <li>Evoke outward K+ currents during 4.5-sec voltage steps to test potentials between -60 and +50 mV in 10-mV increments from a holding potential of -70 mV after a 20-msec prepulse to -40 mV.</li>
 <li>Make sure leak currents are always &lt;100 pA.</li>
 <li>For dissection of different K+ currents use specific inhibitors such as 4-aminopyridine (4-AP; ICN Biomedicals, Irvine, CA, USA), Heteropodatoxin 2 (HpTx2; Alomone) or Tetraethylammonium (TEA; Sigma). Stock solutions should be prepared in extracellular bath solution, and applied directly to the closest possible vicinity of the cell via a microtip after "baseline" recordings. Equilibration should be allowed for 2-3 min before "drug" recordings.</li>
 <li>For analysis normalize current amplitudes in individual cells to cell size (whole-cell membrane capacitance). Analyze data offline by using pClamp10.3 software (Molecular Devices) or comparable software.</li>
</ol>

<ol>
	<li>Abbott, G. W., Goldstein, S. A., Sesti, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Do+all+voltage-gated+potassium+channels+use+MiRPs?.">Do all voltage-gated potassium channels use MiRPs?.</a> Circ. Res. 88, 981-983 (2001).</li><li>Abbott, G. W., Sesti, F., Splawski, I., Buck, M. E., Lehmann, M. H., Timothy, K. W., Keating, M. T., Goldstein, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MiRP1+forms+IKr+potassium+channels+with+HERG+and+is+associated+with+cardiac+arrhythmia.">MiRP1 forms IKr potassium channels with HERG and is associated with cardiac arrhythmia.</a> Cell. 97, 175-187 (1999).</li><li>Barry, D. M., Trimmer, J. S., Merlie, J. P., Nerbonne, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+expression+of+voltage-gated+K++channel+subunits+in+adult+rat+heart.+Relation+to+functional+K++channels?.">Differential expression of voltage-gated K+ channel subunits in adult rat heart. Relation to functional K+ channels?.</a> Circ. Res. 77, 361-369 (1995).</li><li>Benjamin, E. J., Wolf, P. A., D'Agostino, R. B., Silbershatz, H., Kannel, W. B., Levy, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+atrial+fibrillation+on+the+risk+of+death:+the+Framingham+Heart+Study.">Impact of atrial fibrillation on the risk of death: the Framingham Heart Study.</a> Circulation. 98, 946-952 (1998).</li><li>Bou-Abboud, E., Nerbonne, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+correlates+of+the+calcium-independent,+depolarization-activated+K++currents+in+rat+atrial+myocytes.">Molecular correlates of the calcium-independent, depolarization-activated K+ currents in rat atrial myocytes.</a> J. Physiol. 517 (Pt 2), 407-420 (1999).</li><li>Gaborit, N., Steenman, M., Lamirault, G., Le, M. N., Le, B. S., Lande, G., Leger, J., Charpentier, F., Christ, T., Dobrev, D., Escande, D., Nattel, S., Demolombe, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+atrial+ion+channel+and+transporter+subunit+gene-expression+remodeling+associated+with+valvular+heart+disease+and+atrial+fibrillation.">Human atrial ion channel and transporter subunit gene-expression remodeling associated with valvular heart disease and atrial fibrillation.</a> Circulation. 112, 471-481 (2005).</li><li>Guo, W., Xu, H., London, B., Nerbonne, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+basis+of+transient+outward+K++current+diversity+in+mouse+ventricular+myocytes.">Molecular basis of transient outward K+ current diversity in mouse ventricular myocytes.</a> J. Physiol. 521 (Pt 3), 587-599 (1999).</li><li>Lundby, A., Ravn, L. S., Svendsen, J. H., Hauns, S., Olesen, S. P., Schmitt, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=KCNE3+mutation+V17M+identified+in+a+patient+with+lone+atrial+fibrillation.">KCNE3 mutation V17M identified in a patient with lone atrial fibrillation.</a> Cell Physiol. Biochem. 21, 47-54 (2008).</li><li>Ma, K. J., Li, N., Teng, S. Y., Zhang, Y. H., Sun, Q., Gu, D. F., Pu, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+KCNQ1+current+by+atrial+fibrillation-associated+KCNE4+(145E/D)+gene+polymorphism.">Modulation of KCNQ1 current by atrial fibrillation-associated KCNE4 (145E/D) gene polymorphism.</a> Chin Med. J. (Engl.). 120, 150-154 (2007).</li><li>McCrossan, Z. A., Abbott, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+MinK-related+peptides.">The MinK-related peptides.</a> Neuropharmacology. 47, 787-821 (2004).</li><li>Pongs, O., Schwarz, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ancillary+subunits+associated+with+voltage-dependent+K++channels.">Ancillary subunits associated with voltage-dependent K+ channels.</a> Physiol. Rev. 90, 755-796 (2010).</li><li>Ravn, L. S., Aizawa, Y., Pollevick, G. D., Hofman-Bang, J., Cordeiro, J. M., Dixen, U., Jensen, G., Wu, Y., Burashnikov, E., Haunso, S., Guerchicoff, A., Hu, D., Svendsen, J. H., Christiansen, M., Antzelevitch, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gain+of+function+in+IKs+secondary+to+a+mutation+in+KCNE5+associated+with+atrial+fibrillation.">Gain of function in IKs secondary to a mutation in KCNE5 associated with atrial fibrillation.</a> Heart Rhythm. 5, 427-435 (2008).</li><li>Ravn, L. S., Hofman-Bang, J., Dixen, U., Larsen, S. O., Jensen, G., Haunso, S., Svendsen, J. H., Christiansen, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relation+of+97T+polymorphism+in+KCNE5+to+risk+of+atrial+fibrillation.">Relation of 97T polymorphism in KCNE5 to risk of atrial fibrillation.</a> Am. J. Cardiol. 96, 405-407 (2005).</li><li>Roepke, T. K., Abbott, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacogenetics+and+cardiac+ion+channels.">Pharmacogenetics and cardiac ion channels.</a> Vascul. Pharmacol. 44, 90-106 (2006).</li><li>Roepke, T. K., Kontogeorgis, A., Ovanez, C., Xu, X., Young, J. B., Purtell, K., Goldstein, P. A., Christini, D. J., Peters, N. S., Akar, F. G., Gutstein, D. E., Lerner, D. J., Abbott, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+deletion+of+kcne2+impairs+ventricular+repolarization+via+disruption+of+I(K,slow1)+and+I(to,f).">Targeted deletion of kcne2 impairs ventricular repolarization via disruption of I(K,slow1) and I(to,f).</a> FASEB J. 22, 3648-3660 (2008).</li><li>Sesti, F., Abbott, G. W., Wei, J., Murray, K. T., Saksena, S., Schwartz, P. J., Priori, S. G., Roden, D. M., George, A. L., Goldstein, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+common+polymorphism+associated+with+antibiotic-induced+cardiac+arrhythmia.">A common polymorphism associated with antibiotic-induced cardiac arrhythmia.</a> Proc. Natl. Acad. Sci. U.S.A. 97, 10613-10618 (2000).</li><li>Temple, J., Frias, P., Rottman, J., Yang, T., Wu, Y., Verheijck, E. E., Zhang, W., Siprachanh, C., Kanki, H., Atkinson, J. B., King, P., Anderson, M. E., Kupershmidt, S., Roden, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atrial+fibrillation+in+KCNE1-null+mice.">Atrial fibrillation in KCNE1-null mice.</a> Circ. Res. 97, 62-69 (2005).</li><li>Yang, Y., Xia, M., Jin, Q., Bendahhou, S., Shi, J., Chen, Y., Liang, B., Lin, J., Liu, Y., Liu, B., 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=Identification+of+a+KCNE2+gain-of-function+mutation+in+patients+with+familial+atrial+fibrillation.">Identification of a KCNE2 gain-of-function mutation in patients with familial atrial fibrillation.</a> Am. J. Hum. Genet. 75, 899-905 (2004).</li><li>Zeng, Z. Y., Pu, J. L., Tan, C., Teng, S. Y., Chen, J. H., Su, S. Y., Zhou, X. Y., Zhang, S., Li, Y. S., Wang, F. Z., 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=[The+association+of+single+nucleotide+polymorphism+of+slow+delayed+rectifier+K++channel+genes+with+atrial+fibrillation+in+Han+nationality+Chinese].">[The association of single nucleotide polymorphism of slow delayed rectifier K+ channel genes with atrial fibrillation in Han nationality Chinese].</a> Zhonghua Xin. Xue. Guan. Bing. Za Zhi. 33, 987-991 (2005).</li><li>Zhang, M., Jiang, M., Tseng, G. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=minK-related+peptide+1+associates+with+Kv4.2+and+modulates+its+gating+function:+potential+role+as+beta+subunit+of+cardiac+transient+outward+channel?+Circ.">minK-related peptide 1 associates with Kv4.2 and modulates its gating function: potential role as beta subunit of cardiac transient outward channel? Circ.</a> Circ. Res. 88, 1012-1019 (2001).</li></ol>

We have nothing to disclose.

With the growing development of genetically engineered mouse strains to study cardiac function and cardiac pathology related to gene deletion there is also an increasing interest in specialized methods to study effects of the specific gene deletion in vitro. In our laboratory we study the roles of a family of Kv channel ancillary subunits on cardiac repolarization. The KCNE genes comprise a family of 5 genes (KCNE1-5) that play important roles in human ventricular and atrial repolarization <sup...

<table border="1">
 <tbody>
 <tr>
 <td>Name of the Reagent</td>
 <td>Company</td>
 </tr>
 <tr>
 <td>Tetrodotoxin</td>
 <td>Alomone</td>
 </tr>
 <tr>
 <td>4-aminopyridine</td>
 <td>ICN Biomedicals</td>
 </tr>
 <tr>
 <td>Heteropodatoxin 2</td>
 <td>Alomone</td>
 </tr>
 <tr>
 <td>Tetraethylammonium</td>
 <td>Sigma Chemicals</td>
 </tr>
 <tr>
 <td>Collagenase Type 2</td>
 <td>Worthington</td>
 </tr>
 </tbody>
</table>

isolation and kv channel recordings in murine atrial and ventricular cardiomyocytes

KCNE genes encode for a small family of Kv channel ancillary subunits that form heteromeric complexes with Kv channel alpha subunits to modify their functional properties. Mutations in KCNE genes have been found in patients with cardiac arrhythmias such as the long QT syndrome and/or atrial fibrillation. However, the precise molecular pathophysiology that leads to these diseases remains elusive. In previous studies the electrophysiological properties of the disease causing mutations in these genes have mostly been studied in heterologous expression systems and we cannot be sure if the reported effects can directly be translated into native cardiomyocytes. In our laboratory we therefore use a different approach. We directly study the effects of KCNE gene deletion in isolated cardiomyocytes from knockout mice by cellular electrophysiology - a unique technique that we describe in this issue of the Journal of Visualized Experiments. The hearts from genetically engineered KCNE mice are rapidly excised and mounted onto a Langendorff apparatus by aortic cannulation. Free Ca2+ in the myocardium is bound by EGTA, and dissociation of cardiac myocytes is then achieved by retrograde perfusion of the coronary arteries with a specialized low Ca2+ buffer containing collagenase. Atria, free right ventricular wall and the left ventricle can then be separated by microsurgical techniques. Calcium is then slowly added back to isolated cardiomyocytes in a multiple step comprising washing procedure. Atrial and ventricular cardiomyocytes of healthy appearance with no spontaneous contractions are then immediately subjected to electrophysiological analyses by patch clamp technique or other biochemical analyses within the first 6 hours following isolation.

Isolation of adult murine cardiomyocytes from genetically engineered mice to study the function of specific genes of interest in vitro has become a powerful tool to further understand cardiac pathophysiology. This method is currently used by only a small but increasing number of basic science laboratories worldwide. However, isolation of adult ventricular murine cardiomyocytes can be tricky and needs to be done thoroughly and repetitively by experienced hands. Figure 1 shows freshly isolated exe...

Watch this Scientific Journal Video about Isolation and Kv Channel Recordings in Murine Atrial and Ventricular Cardiomyocytes at JoVE.com

Isolation and Kv Channel Recordings in Murine Atrial and Ventricular Cardiomyocytes

Kv channel dysfunction is associated with cardiac arrhythmias. In order to study the molecular mechanisms that lead to such arrhythmias we utilize a systematic protocol for isolation of atrial and ventricular cardiomyocytes from Kv channel ancillary subunit knockout mice. Isolated cardiomyocytes can then immediately be used for cellular electrophysiological studies, biochemical or immunofluorescence (IF) assays.

KCNE genes encode for a small family of Kv channel ancillary subunits that form heteromeric complexes with Kv channel alpha subunits to modify their ...

isolation-kv-channel-recordings-murine-atrial-ventricular

Research

JoVE Journal

Biology

13.3K visualizações.  Charité Medical Faculty and Max-Delbrück Center for Molecular Medicine (MDC). Kv canal disfunção está associada a arritmias cardíacas. Para estudar os mecanismos moleculares que conduzem à ocorrência de arritmias tais que utilizam um protocolo sistemático para o isolamento dos cardiomiócitos atriais e ventriculares do canal Kv auxiliares ratinhos knockout de subunidade. Cardiomiócitos isolados podem então ser imediatamente utilizados para celulares estudos eletrofisiológicos, bioquímicos ou imunofluorescência (IF) ensaios.

Vídeo: Isolation and Kv Channel Recordings in Murine Atrial and Ventricular Cardiomyocytes

Phenotypic Analysis and Isolation of Murine Hematopoietic Stem Cells and Lineage-committed Progenitors

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs constitute a minute cell population of pluripotent cells capable of generating all blood cell lineages for a life-time1. The molecular dissection of HSCs homeostasis in the bone marrow has important implications in hematopoiesis, oncology and regenerative medicine. We describe the labeling protocol with fluorescent antibodies and the electronic gating procedure in flow cytometry to score hematopoietic progenitor subsets and HSCs distribution in individual mice (Fig. 1). In addition, we describe a method to extensively enrich hematopoietic progenitors as well as long-term (LT) and short term (ST) reconstituting HSCs from pooled bone marrow cell suspensions by magnetic enrichment of cells expressing c-Kit. The resulting cell preparation can be used to sort selected subsets for in vitro and in vivo functional studies (Fig. 2).
Both trabecular osteoblasts2,3 and sinusoidal endothelium4 constitute functional niches supporting HSCs in the bone marrow. Several mechanisms in the osteoblastic niche, including a subset of N-cadherin+ osteoblasts3 and interaction of the receptor tyrosine kinase Tie2 expressed in HSCs with its ligand angiopoietin-15 concur in determining HSCs quiescence. "Hibernation" in the bone marrow is crucial to protect HSCs from replication and eventual exhaustion upon excessive cycling activity6. Exogenous stimuli acting on cells of the innate immune system such as Toll-like receptor ligands7 and interferon-&alpha;6 can also induce proliferation and differentiation of HSCs into lineage committed progenitors. Recently, a population of dormant mouse HSCs within the lin- c-Kit+ Sca-1+ CD150+ CD48- CD34- population has been described8. Sorting of cells based on CD34 expression from the hematopoietic progenitors-enriched cell suspension as described here allows the isolation of both quiescent self-renewing LT-HSCs and ST-HSCs9. A similar procedure based on depletion of lineage positive cells and sorting of LT-HSC with CD48 and Flk2 antibodies has been previously described10. In the present report we provide a protocol for the phenotypic characterization and ex vivo cell cycle analysis of hematopoietic progenitors, which can be useful for monitoring hematopoiesis in different physiological and pathological conditions. Moreover, we describe a FACS sorting procedure for HSCs, which can be used to define factors and mechanisms regulating their self-renewal, expansion and differentiation in cell biology and signal transduction assays as well as for transplantation.

A method to analyse the distribution of bone marrow hematopoietic progenitors in flow cytometry as well as to efficiently isolate highly purified hematopoietic stem cells (HSCs) is described. The isolation procedure is essentially based on magnetic enrichment of c-Kit+ cells and cell sorting to purify HSCs for cellular and molecular studies.

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs ...

Isolation and Culture of Neonatal Mouse Cardiomyocytes

Cultured neonatal cardiomyocytes have long been used to study myofibrillogenesis and myofibrillar functions. Cultured cardiomyocytes allow for easy investigation and manipulation of biochemical pathways, and their effect on the biomechanical properties of spontaneously beating cardiomyocytes.	
The following 2-day protocol describes the isolation and culture of neonatal mouse cardiomyocytes. We show how to easily dissect hearts from neonates, dissociate the cardiac tissue and enrich cardiomyocytes from the cardiac cell-population. We discuss the usage of different enzyme mixes for cell-dissociation, and their effects on cell-viability. The isolated cardiomyocytes can be subsequently used for a variety of morphological, electrophysiological, biochemical, cell-biological or biomechanical assays. We optimized the protocol for robustness and reproducibility, by using only commercially available solutions and enzyme mixes that show little lot-to-lot variability. We also address common problems associated with the isolation and culture of cardiomyocytes, and offer a variety of options for the optimization of isolation and culture conditions.

Primary mouse cardiomyocyte cultures are one of the pivotal tools for the investigation of myofibrillar organization and function. The following protocol describes the isolation and culture of primary cardiomyocytes from neonatal mouse hearts. The resulting cardiomyocyte cultures may be subsequently used for a variety of biomechanical, biochemical and cell-biological assays.

Cultured neonatal cardiomyocytes have long been used to study  myofibrillogenesis and myofibrillar functions. Cultured cardiomyocytes allow  for easy ...

Reconstitution of a Kv Channel into Lipid Membranes for Structural and Functional Studies

To study the lipid-protein interaction in a reductionistic fashion, it is necessary to incorporate the membrane proteins into membranes of well-defined lipid composition. We are studying the lipid-dependent gating effects in a prototype voltage-gated potassium (Kv) channel, and have worked out detailed procedures to reconstitute the channels into different membrane systems. Our reconstitution procedures take consideration of both detergent-induced fusion of vesicles and the fusion of protein/detergent micelles with the lipid/detergent mixed micelles as well as the importance of reaching an equilibrium distribution of lipids among the protein/detergent/lipid and the detergent/lipid mixed micelles. Our data suggested that the insertion of the channels in the lipid vesicles is relatively random in orientations, and the reconstitution efficiency is so high that no detectable protein aggregates were seen in fractionation experiments. We have utilized the reconstituted channels to determine the conformational states of the channels in different lipids, record electrical activities of a small number of channels incorporated in planar lipid bilayers, screen for conformation-specific ligands from a phage-displayed peptide library, and support the growth of 2D crystals of the channels in membranes. The reconstitution procedures described here may be adapted for studying other membrane proteins in lipid bilayers, especially for the investigation of the lipid effects on the eukaryotic voltage-gated ion channels.

Procedures for complete reconstitution of a prototype voltage-gated potassium channel into lipid membranes are described. The reconstituted channels are suitable for biochemical assays, electrical recordings, ligand screening and electron crystallographic studies. These methods may have general applications to the structural and functional studies of other membrane proteins.

To study the lipid-protein interaction in a reductionistic fashion, it is necessary to incorporate the membrane proteins into membranes of well-defined ...

Isolation and Physiological Analysis of Mouse Cardiomyocytes

Cardiomyocytes, the workhorse cell of the heart, contain exquisitely organized cytoskeletal and contractile elements that generate the contractile force used to pump blood. Individual cardiomyocytes were first isolated over 40 years ago in order to better study the physiology and structure of heart muscle. Techniques have rapidly improved to include enzymatic digestion via coronary perfusion. More recently, analyzing the contractility and calcium flux of isolated myocytes has provided a vital tool in the cellular and sub-cellular analysis of heart failure. Echocardiography and EKGs provide information about the heart at an organ level only. Cardiomyocyte cell culture systems exist, but cells lack physiologically essential structures such as organized sarcomeres and t-tubules required for myocyte function within the heart. In the protocol presented here, cardiomyocytes are isolated via Langendorff perfusion. The heart is removed from the mouse, mounted via the aorta to a cannula, perfused with digestion enzymes, and cells are introduced to increasing calcium concentrations. Edge and sarcomere detection software is used to analyze contractility, and a calcium binding fluorescent dye is used to visualize calcium transients of electrically paced cardiomyocytes; increasing understanding of the role cellular changes play in heart dysfunction. Traditionally used to test drug effects on cardiomyocytes, we employ this system to compare myocytes from WT mice and mice with a mutation that causes dilated cardiomyopathy. This protocol is unique in its comparison of live cells from mice with known heart function and known genetics. Many experimental conditions are reliably compared, including genetic or environmental manipulation, infection, drug treatment, and more. Beyond physiologic data, isolated cardiomyocytes are easily fixed and stained for cytoskeletal elements. Isolating cardiomyocytes via perfusion is an extremely versatile method, useful in studying cellular changes that accompany or lead to heart failure in a variety of experimental conditions.

Individual cardiomyocytes from wild type and mutant mice can be isolated from the heart in order to study their contractility and calcium transients. This allows characterization of the contribution of cellular dysfunction to heart dysfunction from any cause.

Cardiomyocytes, the workhorse cell of the heart, contain exquisitely organized cytoskeletal and contractile elements that generate the contractile force ...

Isolation and Culture of Adult Mouse Cardiomyocytes for Cell Signaling and in vitro Cardiac Hypertrophy

Technological advances have made genetically modified mice, including transgenic and gene knockout mice, an essential tool in many research fields. Adult cardiomyocytes are widely accepted as a good model for cardiac cellular physiology and pathophysiology, as well as for pharmaceutical intervention. Genetically modified mice preclude the need for complicated cardiomyocyte infection processes to generate the desired genotype, which are inefficient due to cardiomyocytes&#8217; terminal differentiation. Isolation and culture of high quantity and quality functional cardiomyocytes will dramatically benefit cardiovascular research and provide an important tool for cell signaling transduction research and drug development. Here, we describe a well-established method for isolation of adult mouse cardiomyocytes that can be implemented with little training. The mouse heart is excised and cannulated to an isolated heart system, then perfused with a calcium-free and high potassium buffer followed by type II collagenase digestion in Langendorff retrograde perfusion mode. This protocol yields a consistent result for the collection of functional adult mouse cardiomyocytes from a variety of genetically modified mice.

Isolation and Culture of Adult Mouse Cardiomyocytes for Cell Signaling and in vitro Cardiac Hypertrophy

We describe a reliable method for isolation of adult mouse cardiomyocytes. This protocol yields a consistent result for the culture of functional adult cardiomyocytes from a variety of genetically modified mice.

Technological advances have made genetically modified mice, including transgenic and gene knockout mice, an essential tool in many research fields. Adult ...

One-channel Cell-attached Patch-clamp Recording

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate depends on properties intrinsic to each channel protein as well as the mechanism by which it is generated and controlled and represents an important area of current research. Information about the operational dynamics of ion channel proteins can be obtained by observing long stretches of current produced by a single molecule. Described here is a protocol for obtaining one-channel cell-attached patch-clamp current recordings for a ligand gated ion channel, the NMDA receptor, expressed heterologously in HEK293 cells or natively in cortical neurons. Also provided are instructions on how to adapt the method to other ion channels of interest by presenting the example of the mechano-sensitive channel PIEZO1. This method can provide data regarding the channel&#8217;s conductance properties and the temporal sequence of open-closed conformations that make up the channel&#8217;s activation mechanism, thus helping to understand their functions in health and disease.

Described here is a procedure for obtaining long stretches of current recording from one ion channel with the cell-attached patch-clamp technique. This method allows for observing, in real time, the pattern of open-close channel conformations that underlie the biological signal. These data inform about channel properties in undisturbed biological membranes.

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate ...

Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of transporters. The phosphorylation and nucleotide dependent chloride channel activity of CFTR has been frequently studied in whole cell systems and as single channels in excised membrane patches. Many Cystic Fibrosis-causing mutations have been shown to alter this activity. While a small number of purification protocols have been published, a fast reconstitution method that retains channel activity and a suitable method for studying population channel activity in a purified system have been lacking. Here rapid methods are described for purification and functional reconstitution of the full-length CFTR protein into proteoliposomes of defined lipid composition that retains activity as a regulated halide channel. This reconstitution method together with a novel flux-based assay of channel activity is a suitable system for studying the population channel properties of wild type CFTR and the disease-causing mutants F508del- and G551D-CFTR. Specifically, the method has utility in studying the direct effects of phosphorylation, nucleotides and small molecules such as potentiators and inhibitors on CFTR channel activity. The methods are also amenable to the study of other membrane channels/transporters for anionic substrates.

Described here is a rapid and effective procedure for functional reconstitution of purified wild-type and mutant CFTR protein that preserves activity for this chloride channel, which is defective in Cystic Fibrosis. Iodide efflux from reconstituted proteoliposomes mediated by CFTR allows studies of channel activity and the effects of small molecules.

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of ...

Voltage and Calcium Dual Channel Optical Mapping of Cultured HL-1 Atrial Myocyte Monolayer

Optical mapping has proven to be a valuable technique to detect cardiac electrical activity on both intact ex vivo hearts and in cultured myocyte monolayers. HL-1 cells have been widely used as a 2-Dimensional cellular model for studying diverse aspects of cardiac physiology. However, it has been a great challenge to optically map calcium (Ca) transients and action potentials simultaneously from the same field of view in a cultured HL-1 atrial cell monolayer. This is because special handling and care is required to prepare healthy cells that can be electrically captured and optically mapped. Therefore, we have developed an optimal working protocol for dual channel optical mapping. In this manuscript, we have described in detail how to perform the dual channel optical mapping experiment. This protocol is a useful tool to enhance the understanding of action potential propagation and Ca kinetics in arrhythmia development.

This article describes the technique used to perform dual channel optical mapping in cultured HL-1 atrial cell monolayers. This unique protocol allows the simultaneous visualization of both calcium (Ca) and voltage (Vm) activity in the same area for the detailed detection and analysis of electrophysiological properties of culture monolayers.

Optical mapping has proven to be a valuable technique to detect cardiac electrical activity on both intact ex vivo hearts and in cultured myocyte ...

Isolation, Culture and Transduction of Adult Mouse Cardiomyocytes

Cultured cardiomyocytes can be used to study cardiomyocyte biology using techniques that are complementary to in vivo systems. For example, the purity and accessibility of in vitro culture enables fine control over biochemical analyses, live imaging, and electrophysiology. Long-term culture of cardiomyocytes offers access to additional experimental approaches that cannot be completed in short term cultures. For example, the in vitro investigation of dedifferentiation, cell cycle re-entry, and cell division has thus far largely been restricted to rat cardiomyocytes, which appear to be more robust in long-term culture. However, the availability of a rich toolset of transgenic mouse lines and well-developed disease models make mouse systems attractive for cardiac research. Although several reports exist of adult mouse cardiomyocyte isolation, few studies demonstrate their long-term culture. Presented here, is a step-by-step method for the isolation and long-term culture of adult mouse cardiomyocytes. First, retrograde Langendorff perfusion is used to efficiently digest the heart with proteases, followed by gravity sedimentation purification. After a period of dedifferentiation following isolation, the cells gradually attach to the culture and can be cultured for weeks. Adenovirus cell lysate is used to efficiently transduce the isolated cardiomyocytes. These methods provide a simple, yet powerful model system to study cardiac biology.

This protocol describes a step-by-step method for the reproducible isolation and long-term culture of adult mouse cardiomyocytes with high yield, purity, and viability.

Cultured cardiomyocytes can be used to study cardiomyocyte biology using techniques that are complementary to in vivo systems. For example, the purity and ...

Subtype-specific Optical Action Potential Recordings in Human Induced Pluripotent Stem Cell-derived Ventricular Cardiomyocytes

Cardiomyocytes generated from human induced pluripotent stem cells (iPSC-CMs) are an emerging tool in cardiovascular research. Rather than being a homogenous population of cells, the iPSC-CMs generated by current differentiation protocols represent a mixture of cells with ventricular-, atrial-, and nodal-like phenotypes, which complicates phenotypic analyses. Here, a method to optically record action potentials specifically from ventricular-like iPSC-CMs is presented. This is achieved by lentiviral transduction with a construct in which a genetically-encoded voltage indicator is under the control of a ventricular-specific promoter element. When iPSC-CMs are transduced with this construct, the voltage sensor is expressed exclusively in ventricular-like cells, enabling subtype-specific optical membrane potential recordings using time-lapse fluorescence microscopy.

Here we present a method to optically image action potentials, specifically in ventricular-like induced pluripotent stem cell-derived cardiomyocytes. The method is based on the promoter-driven expression of a voltage-sensitive fluorescent protein.