Cryopreserved neonatal clonetice rat cardiac myocytes in cluture display hypertrophy to non-hemodynamic factors

Lonza Lonza Cryopreserved Neonatal Clonetics@ Rat Cardiac Myocytesin Culture Display Hypertrophy to Non-hemodynamicFactors： New Research and Development Opportunities By Anthony Krantis， Ph.D.， Alex Chichirau， MSc.， and Teresa Tam*， MSc. Cryopreserved neonatal Clonetics@ Rat Ventricular Cardiac Myocytes can be easily thawed and cultured to give physiologi-cally normal in vitro cardiac cell test beds， ideal for a spectrumofassays. Freshly prepared neonatal rat ventricular cardiac myocytesmaintained in culture are viable test beds for examining ques-tions of cardiac cell connectivity， activity and pharmacology[Chlopikova et al.， 2001]. Furthermore， modeling cardiac tox-icity， hypertrophy， anoxic and ischemic states， cellular growthandfunction has been well studied in cardiac cell culture prepa-rations [Estevez et al.，2000； Grynberg et al.， 1986； Huang2002； Jovanovic et al.， 1996； Long et al.， 1997； Severs et al.，1991； Wang et al.， 1999]. We recently demonstrated that neonatal rat ventricular cardiacmyocytes can be cryopreserved， stored frozen and thenthawed for cell culture application. The thawed neonatal rat ven-tricular cardiac myocytes display normal morphology andphysiological viability in long term cell culture. Typically， within24 hours of thaw and plating， the cardiac myocytes sponta-neously beat， with syncytial beating of the entire culture evi-dent bu 48 hours. These characteristics remain consistentacross 6 to 96-well plate culture formats. The cardiac myocytesdisplay robust electrical and contractile activity with normalpharmacology to cardioactive and gap junctional drug chal-lenge. Activity and gap-junctional connectivity of the cardiacmyocytes is sustained for more than 40 days in culture. The availability of high quality， batch tested， cryopreservedneonatal Clonetics@ Rat Ventricular Cardiac Myocytes， whichcan be simply thawed and cultured， represents a significantadvantage for the laboratory， eliminating animal handling/dis-section and difficult cardiac tissue preparation. In addition， theability to control when and where the cardiac myocyte culturecan be undertaken streamlines the research workflow andspeeds up R&D programs. We report here the results of further study of cryopreservedneonatal rat ventricular cardiac myocytes to determine theirresponsiveness in cultureto hallenge wwithpositivechronotropic compounds， as well as determination of whethermyocardial hypertrophy can be evaluated using cultures pre-pared with cryopreserved cardiac myocytes. The ability offreshly cultured neonatal ventricular cardiac myocytes to dis- play hypertrophy following challenge with non-hemodynamicfactors is known [Simpson et al.， 1982； Nakamura et al.，1998].Therefore， we chose to study non-hemodynamic hypertrophyusing Angiotensin Il and Isoproterenol. MATERIALS AND METHODS Cardiac myocyte cell culture All experiments were conducted with cardiac cell cultures es-tablished using vials of cryopreserved Clonetics RatVentricular Cardiac Myocytes [neonatal [P1-3]， purified to≥85%； Lonza]. Cells were thawed following the manufacturer’sprotocol， then DMEM/F12+FBS+HS+ Pen/Strep + Hepes cul-ture medium was added and cells plated according to the re-quirements of each experimental protocol. For all experiments，the cultures were changed to fresh medium 4 hours after plat-ing and then again at 2 days in vitro， with media changes every2 days thereafter. Unless otherwise stated， all cultures weremaintained with BrdU in the culture medium to minimize fi-broblast proliferation. Morphology For morphological assessment， the thawed cardiac myocyteswere plated on coated coverslips in 24-well plates at 5 x105/well or in 24-well plates without coverslips for up to 14days. Cultures were fixed and stained for immunohistochemi-cal examination of the sarcomeric protein a-actinin [anti-o-actinin， Sigma]， the gap junction connexin 43 [anti-Cx43，Zymed Laboratories]， and the nuclei stain， Hoechst [Sigma]. Functional assessment using multielectrode arrays Vials of cryopreserved cardiac myocytes were prepared for cul-ture as described above and plated on 60-electrode formatMulti Electrode Arrays [MEA； Multi Channels Systems， Reutlin-gen， Germany] at a concentration of 1.5x 106 cells/MEA. Cellbeating [beats/min] was recorded by observation of the indi-vidual cultures. The cultures were also analyzed daily for spon-taneous spike activity and individual network properties in longterm culture [up to 7 days]. Separate MEA cultures were usedto assess cardiac myocyte network responsiveness to the pos-itive chronotropic agent， Angiotensin II [Ang II]. Recordingswere obtained using a MEA60 System [Multi Channel Systems，Reutlingen， Germany]. Recordings were performed at 37℃ andsignals were simultaneously sampled at 25 kHz， visualized andstored using the software MCRack [Multi Channel Systems，Reutlingen， Germany]. Files were converted into Axoscope Bi-nary File format and displayed using Axoscope software [Mol-ecular Devices]. The data were graphed as mean ± SEM. Cell growth and hypertrophy Cardiac myocytes were thawed and plated onto 6-well plates.The cell growth medium employed for our studies contains 7.5%[v/v] FBS and 7.5% [v/v] horse serum. Sera is known to causeincreased cardiac cell size [Simpson et al.， 1982]， therefore weexamined whether this effect was evident in our cultures. Todetermine the effect of serum， we compared the cell size of car-diac myocytes at day 1 vs. day 12 in culture. Briefly， 5 ml of acell suspension of thawed cardiac myocytes was plated intoeach of 3 wells [2x106/well] of a 6-well plate. At different timepoints during culture， wells were trypsinized using a standardprotocol. A 10 pl suspension oftrypsinized cells was added ontoeach glass coverslip and covered with a microruled Cellattice'Slide「Nexcelom Biosciencel]. CCellattice""is an optically smoothplastic with microscopic identification and measurement mark-ers.The combination of numbers，letters， and tick marks iden-tifies each 25 pm in a 10 x 10 mm area of the coverslip. Thisallows cell size to be easily measured， with a high degree of ac-curacy. Photomicrographs of the plated cells were used formeasurement of the cells from the microruled grids. In parallel experiments， cryopreserved cardiac myocytes werethawed and a 1 ml suspension [4x 105/ml] was plated in eachof 3 wells of a 24-well plate. These cultures were used to test forhypertrophy following treatment with Ang lI or Isoproterenol.The cultures were maintained for 7 days using our standardgrowth medium [containing sera]. The first medium change[95%] was performed 4 hours after plating， using fresh mediumwith 200 pM BrdU. On day 3， the medium was replaced com-pletely with fresh medium without BrdU. On day 5， a 50%medium change was performed using fresh medium [withoutBrdU] and drug [100 nM Ang Il or 1 pM Isoproterenol] wasadded. Control cultures were maintained in medium withoutdrug and without BrdU. On day ?， the cultures were fixed andstained with the monoclonal antibody， anti-c-actinin [Sigma].Data analysis： 5-7 images [32X] for each of the conditions[control； Ang ll； Isoproterenol] were taken. The images were Fig. 1. Cardiac myocyte morphology and gap junctionlocalization. Cryopreservedneonatal rat ventricular cardiac myocytes can be easily thawed and plated to giverobust long term cardiac cell cultures. In this high magnification micrograph ofcardiac myocytes at 7 days in culture， typical anti-o-actinin staining [green]ofthe sarcomeres is clearly evident. The gap junctions which are critical for the two-dimensional cardiac myocyte syncytium are revealed by the bright red im-munostaining of the gap junction protein， connexin 43，localized as puncta at theinterfaces of the cardiac myocytes. Nuclei [purple] are stained with Hoechst. then processed using AxioVision software and individual cellswere contoured [5-12 cells per condition] to determine the cellsurface area. Measurements were averaged， normalized to con-trol cell average surface area and graphed as mean ± SEM. RESULTS The cryopreserved neonatal rat ventricular cardiac myocytesthrived in culture and displayed normal morphology and gapjunction localization， as shown in Fig. 1. Contractile [beating]activity was evident by 24 hours and robust physiological con-nectivity was apparent by observation of the whole culturebeating as a syncytium within 48 hours [not shown here]. Thissynchronous beating was reflected in the MEA recordings ofelectrical activity which showed the activity time-locked [syn-chronous] across all electrodes. Spike activity and beating Fig. 2. MEA recording of cardiac myocyte culture electrical activity. The three frame panel represents recordings from the same single electrode of a 64-electrodearray-based cardiac myocyte culture. Observed from left to right， the frames represent days 1-3 respectively. The negative and positive components of the spikepotential progressively increase in amplitude. synchrony was maintained in long-term culture. The culturesdispayed electrical activity from day 1， as shown in the MEAelectrode output panel of Fig. 2. In this example recording froma single electrode of the MEA， the field potential recorded day1 through day 3 shows a typical progressive increase in spikeamplitude. 8 Fig. 3. Frequency of cardiac myocyte activity in long term culture. Electrical ac-tivity [correlating contractile activity] was summated and averaged daily acrossall 64 electrodes of the MEA-based cultures. Recordings of electrical activity (which correlated with con-traction frequency] across all electrodes， were measured dailyfor 4 days and again at day 7 [Fig. 3]. By day 2， activity fre-quency was 100.5±13.5， rising significantly to a peak of 157±28 at day 3. At day 4， the activity recovered to the day 2 level.At day ?， activity was lower but not significantly different fromday4. To determine whether these cultures were responsive tochronot opic stimulation， we tested Ang ll and Isoproterenol.The graphed data of Fig. 4 shows the increase in frequencyabove baseline with application of different concentrations ofAng ll and Isoproterenol. This action was concentration de-pendent. Hypertrophy Studies using freshly prepared neonatal Clonetics@ Rat Ven-tricular Cardiac Myocyte cultures [Simpson et al.， 1982] reportthat cardiac myocyte hypertrophy [increased cell size] can beinduced by regulating the amount of serum in the culturemedium. We observed similar effects of serum in the cryopre-served neonatal rat ventricular cardiac myocyte cells grown instandard medium containing serum. These cells display in-creased size with time in culture {Fig.5]. The diameter of thetrypsinized cardiac myocytes can be easily determined fromthe microruled surface which is clearly visible in the two setsof images. By day 12[bottom panel]， the average cell diameterincreased by 55% to 38 pm compared to the day 1 [top panel]average of 21 um. For the study of drug-ind uced hypertrophy， we used the samemedium conditions across control and treatment cultures，thereby controlling for any serum effect on cell size. The ability to undergo hypertrophy was examined by exposingcardiac myocyte cultures maintained in standard medium， toeither Ang I [100nM] or Isoproterenol[1 pM][Fig. 6]. Cultureswere challenged with drug at day 5 and the cultures left untilday 7， when they were washed， fixed and immunostained withanti-o-actinin. The cultures were then examined microscopi-cally to allow surface area calculation using image software. Atthese concentrations， the positive chronotropic agents werecomparable [50% and 58% respectively] in their ability to in-duce hypertrophy. Fig. 4. Concentration dependent actions of Ang Il and Isoproterenol on cardiacmyocyte electrical activity. Electrical activity [correlating contractile activity]was summated and averaged across all 64 electrodes of the MEA-based cultureswith and without exposure to drug. DISCUSSION Cryopreserved neonatal Clonetics@ Rat Ventricular Cardiac My-ocytes are ideal for developing primary cell cultures for analy-sis of structure and function. In this study， we confirm andextend our previous report on the sensitivity of these culturesto pharmacological challenge and show that the profile of de-velopment of electrical activity and connectivity in these cul-tures is consistent with that seen in cultures of freshly preparedneonatal rat ventricular cardiac myocytes reported by Rother-melet al. [2005]. In their multielectrode array studies， they ob-served the peak amplitude of the spike potentials to increase 38 um 30，35um Fig. 5. Cardiac myocyte growth in culture medium containing serum. Five sets of micrographs of cardiac myocytes trypsinized at day 1 [top panel] and at day 12[bottom panel] can be seen in relief against the microruled grid. Each micrograph shows representative populations of cell sizes as indicated. At day 12， the cardiacmyocytes showed a large increase in average size. in intensity in long term culture up to 5 days. We observed a sim-ilar profile in spike potential amplitude. We also found the elec-trical activity， which is correlated with contractions， peaked atday 3 and by day 7， returned to a lower stable rate. This profile ofactivity is comparable to that reported by Rothermel et al.[20051. Our cardiac myocyte cultures also responded with a similar in-crease and concentration dependence in activity [contrac-tions] to challenge with Ang II [Rothermel et al.， 2005]. We alsofound our cultured cardiac myocytes to display a concentra-tion-dependent increase in activity to Isoproterenol， compara-ble to that reported by Rothermel et al. [2005]. Taken togetherwith our previous report of MEA-based cultures of cryopre-served cardiac myocytes displaying concentration dependentresponses to cholinergic and adrenergic drugs， these results Fig. 6. Chronotropic drug treatment induces hypertrophy in cultured cardiacmyocytes. The cardiac myocyte culture was exposed to either Ang ll or Isopro-terenol at day 5 and the drug remained in the medium until day 7 when the cul-tures were washed， fixed and immunostained to determine cell surface area byimage analysis.Data were normalized and compared to untreated cultures [con-trol] run in parallel. show that cryopreservation has not affected the spontaneouscontractile capacity and chronotropic sensitivity of the neona-tal rat ventricular cardiac myocytes. Further evidence that cryopreserved cardiac myocytes behavenormally in culture was demonstrated when we examined thecell growth profile and hypertrophic sensitivity to long term ex-posure to Ang ll or Isoproterenol. These chronotropic agents areknown to cause hypertrophy in freshly prepared neonatal ratheart cultures [Nakamura et al.， 1998；Simpson et al.，1982].In our study， treatment of rat heart cultures prepared using cry-opreserved neonatal ventricular cardiac myocytes， with thesame concentration of Ang ⅡI[100 nM] and for the same dura-tion as that used by Nakamura et al.， caused a similar level ofcardiac myocyte enlargement. We also found Isoproterenol [atthe same concentration used by Simpson et al.， 1982] causedhypertrophy and at a level almost equivalent to that obtainedwith Ang Il. These results reinforce the proposal that cryopreserved neona-tal Clonetics° Rat Ventricular Cardiac Myocytes display normalstructure， connectivity， sensitivity and physiology whenthawed and grown in long term culture. The ease of use of thesecardiac myocytes for extracellular electrophysiological exami-nation in multielectrode array cultures (‘cardiac chip'] providesthe researcher with an excellent cardiac screening assay. Fur-thermore， the similarity in sensitivity and profile to inductionof hypertrophy compared to freshly prepared cardiac myocytecultures broadens the scop eof the application of these cells inthe study of cardiac function in health and disease. *Teresa Tam， MSc. is a recent graduate of the University of OttawaCellular and Molecular Medicine Graduate program. Teresa’s researchwas funded by a grant from Health Canada. Chlopcikova， S.， Psotová， J.， Miketova， P. [2001] Neonatal rat cardiomyocytes- Amodel for the study of morphological， biochemical and electrophysiological char-acteristics of the heart.Biomed.Papers. 145： 49-55. Estevez， M. D.， Wolf， A.， Schramm， U. [2000]. Effect of PSC 833， Verapamil and Amio-darone on Adriamycin toxicity in cultured rat cardiomyocytes. 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[1998]Inhibitory effects of antioxidants on neonatal rat cardiac myocyte hypertrophy in-duced by Tumor Necrosis Factor-o and Angiotensin II. Circulation 98：794-799. Rothermel A.， Kurz R.， Ruffer M.， Weigel W.， Jahnke H-G.， Sedello A.K.， Stepan H.，Faber R.， Schulze-Forster K.， Robitzki A.A. [2005] Cells on a chip-the use of electricproperties for highly sensitive monitoring of blood-derived factors involved in An-giotensin Il type 1 receptor signaling. Cell. Physiol. Biochem. 16：51-58. Severs， N. J.，Twist， V. W.， Powel， T. [1991] Acute effects of Adriamycin on the macromole-cular organization of the cardiac muscle cell plasma membrane. Cardioscience. 2：35-45. Simpson P， McGrath A.， Savion S. [1982] Myocyte hypertrophy in neonatal rat heartcultures and its regulation by serum and by catecholamines. Circ.Res. 51：787-801. Wang， G.， Schuschke， D. A.， Kang，Y. J. [1999] Metallothione in over expressing neona-tal mouse cardiomyocytes are resistant to H202. Am. J. Physiol. 276：H167-H175. ORDERING INFORMATION Cat. No. Description Size R-CM-561 Rat Cardiac Myocytes ≥4 million cells Meaningful Targets Efficiently TransfectRelevant Cells 800 Nucleofection@ enables RNAi library screens inphysiologically relevant cell types； e.g. for studyinggenes involved in angiogenesis in HUVEC cells. Up to 99% efficiency for siRNA oligonucleotidesUp to 90% efficiency with shRNA vectors96-well Shuttle Device for reproducibletransfection from 1 to 96 reactions at a time Relative Viability[% of siCONTROL" [Thermo Fisher Scientific，Inc.]] Example of a validated hit [COPB2] identified by a primarysiRNAlibrary screen in HUVEC using Nucleofection@.Datagenerated in collaboration with Dharmacon/ThermoFisher Scientific Inc. Discover more at www.lonza.com/screening www.lonza.com