Methods in Molecular Biology 1299 Gary R. Skuse Maureen C. Ferran Editors Cardio- myocytes Methods and Protocols M M B ETHODS IN OLECULAR IOLOGY SeriesEditor JohnM.Walker School of Lifeand MedicalSciences University ofHertfordshire Hatfield, Hertfordshire,AL109AB,UK For further volumes: http://www.springer.com/series/7651 Cardiomyocytes Methods and Protocols Edited by Gary R. Skuse and Maureen C. Ferran Rochester Institute of Technology, Rochester, NY, USA Editors GaryR.Skuse MaureenC.Ferran RochesterInstituteofTechnology RochesterInstituteofTechnology Rochester,NY,USA Rochester,NY,USA ISSN1064-3745 ISSN1940-6029 (electronic) MethodsinMolecularBiology ISBN978-1-4939-2571-1 ISBN978-1-4939-2572-8 (eBook) DOI10.1007/978-1-4939-2572-8 LibraryofCongressControlNumber:2015935077 SpringerNewYorkHeidelbergDordrechtLondon #SpringerScience+BusinessMediaNewYork2015 Thisworkissubjecttocopyright.AllrightsarereservedbythePublisher,whetherthewholeorpartofthematerialis concerned,specificallytherightsoftranslation,reprinting,reuseofillustrations,recitation,broadcasting,reproduction onmicrofilmsorinanyotherphysicalway,andtransmissionorinformationstorageandretrieval,electronicadaptation, computersoftware,orbysimilarordissimilarmethodologynowknownorhereafterdeveloped. Theuseofgeneraldescriptivenames,registerednames,trademarks,servicemarks,etc.inthispublicationdoesnot imply,evenintheabsenceofaspecificstatement,thatsuchnamesareexemptfromtherelevantprotectivelawsand regulationsandthereforefreeforgeneraluse. Thepublisher,theauthorsandtheeditorsaresafetoassumethattheadviceandinformationinthisbookarebelievedto betrueandaccurateatthedateofpublication.Neitherthepublishernortheauthorsortheeditorsgiveawarranty, expressorimplied,withrespecttothematerialcontainedhereinorforanyerrorsoromissionsthatmayhavebeenmade. CoverIllustration: CytivaTMPlusCardiomyocytes:ThedeconvolvedImagewasacquiredonDeltaVisionOMXby AngelaWilliams(GEHealthcare).After14daysofgrowththecellswerestainedforcardiacTroponinI(Red)and a-Actinin(Green) Printedonacid-freepaper HumanaPressisabrandofSpringer SpringerScience+BusinessMediaLLCNewYorkispartofSpringerScience+BusinessMedia(www.springer.com) Preface Heartdiseaseisresponsibleforuntoldglobalmorbidityandmortality.Traditionalmedical approaches to the treatment of heart disease often strive to ameliorate damage and to prevent future damage, but they cannot reverse what has already happened. This is especially apparent in the most extreme instances where heart transplantation is used to replacetheentireorgan.Unfortunately,thesupplyofdonorheartscannotkeeppacewith the demand; something must be done to enable us to repair damaged cardiac tissue and generatewholeorganswhenneeded.Despitethefactthatdeathratesfromcoronaryheart diseasearefalling,heartdiseaseremainstheleadingcauseofdeathworldwide. Thisvolume,Cardiomyocytes:MethodsandProtocols,hasbeenassembledforscientists interested in basic and applied biomedical research directed toward understanding the development, genetics, and function of cardiomyocytes. The methods and protocols contained herein address cell culture techniques, cardiomyocyte differentiation and redif- ferentiation, experimental induction of cardiomyopathies, introducing genes into cardio- myocytes,genomicapproachestotheunderstandingofcardiomyocytes,cryopreservation ofneonatalcardiomyocytes,andmodelingofcardiomyocytefunction.Amongthechapters of this work, readers will find complimentary areas of cardiomyocyte science that, taken together,shouldinformindividualswithabroadrangeofinterests. Thesecollectedcontributionswerewrittenbycurrentandnascentleadersinthefield of cardiomyocyte biology. Together the authors have provided a wealth of methods that canbeusedtofurtherexplorethemanyaspectsofcardiomyocytebiologythatweneedto understand in order to better grasp the development and function of these cells and to developthenextgenerationofeffectivetherapies.Thechaptersareorganizedthematically with regard to cardiovascular disease, modelling of cardiomyocytes function, isolation of cells,induceddifferentiationofcellsintocardiomyocytes,genetransferintocardiacmyo- cytes,geneexpressionanalysis,andtheapplicationofnext-generationsequencingtoward furtheringour understandingofcardiovasculardisease. Of course it is impossible to include contributions from every researcher who is contributing to this important field. Instead we have compiled a collection of chapters thattogetherrepresentsomeoftheleadingandpotentiallymostimpactfulwork.Wehope youfindtheminformativeandusefulinyourownlaboratories. Rochester,NY,USA GaryR.Skuse MaureenC.Ferran v Contents Preface.................................................................... v Contributors............................................................... ix 1 GeneratingPrimaryCulturesofMurineCardiacMyocytes andCardiacFibroblaststoStudyViralMyocarditis ........................ 1 BarbaraSherry 2 EnrichmentofCardiomyocytesinPrimaryCulturesofMurine NeonatalHearts....................................................... 17 SreejitParameswaran,RajalakshmiSanthakumar, PrasannaVidyasekar,andRamaS.Verma 3 DeepSequencingofCardiacMicroRNA-mRNAInteractomes inClinicalandExperimentalCardiomyopathy ............................ 27 ScotJ.MatkovichandGeraldW.DornII 4 Next-GenerationSequencingTechnologyintheGenetics ofCardiovascularDisease............................................... 51 CeciliaVecoli 5 ComputationalCardiacElectrophysiology:ImplementingMathematical ModelsofCardiomyocytestoSimulateActionPotentialsoftheHeart....... 65 MichaelM.BellandElizabethM.Cherry 6 MethodsofMyofibrillogenesisModeling................................. 75 NancyK.DrewandAnnaGrosberg 7 UsingtheMechanicalBidomainModeltoAnalyzetheBiomechanical BehaviorofCardiomyocytes............................................ 93 BradleyJ.Roth 8 FabricationofaMyocardialPatchwithCellsDifferentiated fromHuman-InducedPluripotentStemCells ............................ 103 LeiYe,JoydeepBasu,andJianyiZhang 9 EfficientDifferentiationofCardiomyocytesfromHumanPluripotent StemCellswithGrowthFactors......................................... 115 RajneeshJha,Ren-HeXu,andChunhuiXu 10 Isolation,Culturing,andCharacterizationofCardiacMuscleCells fromNonhumanPrimateHeartTissue .................................. 133 StevenM.HoynowskiandJohnW.Ludlow 11 MouseEmbryonicStemCell-DerivedCardiacMyocytes inaCellCultureDish.................................................. 145 CarleyGlass,ReetuSingla,AnshuArora,andDinenderK.Singla 12 CryopreservationofNeonatalCardiomyocytes............................ 153 AdamC.Vandergriff,M.TaylorHensley,andKeCheng 13 EvaluationofSarcomericOrganizationinHumanPluripotent StemCell-DerivedCardiomyocytes...................................... 161 ChrishanJ.A.RamachandraandWinstonShim vii viii Contents 14 ElectrotonicCoupledMetabolicPurificationofChickCardiomyocytes ...... 167 WinstonShim,HaiyangYu,K.P.MyuMaiJa, MuhammadParasuram,KeePahLim,andPhilipWong 15 GeneTransferintoCardiacMyocytes.................................... 177 SarahE.LangandMargaretV.Westfall 16 Analysisof4DMyocardialWallMotionDuringEarlyStages ofChickHeartDevelopment........................................... 191 MadelineMidgettandSandraRugonyi Index..................................................................... 213 Contributors ANSHUARORA (cid:1) BiomolecularScienceCenter,BurnettSchoolofBiomedicalSciences, CollegeofMedicine,UniversityofCentralFlorida,Orlando,FL,USA JOYDEEPBASU (cid:1) TissueEngineeringandAssayDevelopment,Zen-Bio,Inc.,Research TrianglePark,NCUSA;ProcessResearchandTranslation,Tengion,Inc., Winston-Salem,NC,USA MICHAELM.BELL (cid:1) SchoolofMathematicalSciences,RochesterInstituteofTechnology, Rochester,NY,USA KECHENG (cid:1) DepartmentofMolecularBiomedicalSciences,CollegeofVeterinaryMedicine, NorthCarolinaStateUniversity,Raleigh,NC,USA;UNC/NCSUJointDepartmentof BiomedicalEngineering,ChapelHill,NC,USA ELIZABETHM.CHERRY (cid:1) SchoolofMathematicalSciences,RochesterInstituteofTechnology, Rochester,NY,USA GERALD W.DORN II (cid:1) Center forPharmacogenomics,DepartmentofInternalMedicine, WashingtonUniversitySchoolofMedicine,St.Louis,MO,USA NANCY K.DREW (cid:1) TheEdwardsLifesciencesCenter forAdvancedCardiovascular Technology,Irvine,CA,USA CARLEYGLASS (cid:1) BiomolecularScienceCenter,BurnettSchoolofBiomedicalSciences, CollegeofMedicine,UniversityofCentralFlorida,Orlando,FL,USA ANNAGROSBERG (cid:1) TheEdwardsLifesciencesCenter forAdvancedCardiovascular Technology,Irvine,CA,USA M.TAYLORHENSLEY (cid:1) DepartmentofMolecularBiomedicalSciences,CollegeofVeterinary Medicine,NorthCarolinaStateUniversity,Raleigh,NC,USA STEVENM.HOYNOWSKI (cid:1) ZenBio,Inc.,ResearchTrianglePark,NC,USA K.P.MYUMAIJA (cid:1) StemCellLaboratory,NationalHeartResearchInstituteSingapore (NHRIS),NationalHeartCentreSingapore(NHCS),Singapore,Singapore RAJNEESHJHA (cid:1) DepartmentofPediatrics,EmoryUniversitySchoolofMedicineand Children’sHealthcareofAtlanta,Atlanta,GA,USA SARAH E.LANG (cid:1) PrograminCellularandMolecularBiology,UniversityofMichigan, AnnArbor,MI,USA KEEPAHLIM (cid:1) StemCellLaboratory,NationalHeartResearchInstituteSingapore (NHRIS),NationalHeartCentreSingapore(NHCS),Singapore,Singapore JOHNW.LUDLOW (cid:1) RegenerativeMedicine,ZenBio,Inc.,ResearchTrianglePark,NC, USA SCOTJ.MATKOVICH (cid:1) Center forPharmacogenomics,DepartmentofInternalMedicine, WashingtonUniversitySchoolofMedicine,St.Louis,MO,USA MADELINEMIDGETT (cid:1) DepartmentofBiomedicalEngineering,OregonHealth&Science University,Portland,OR,USA SREEJITPARAMESWARAN (cid:1) StemCellandMolecularBiologyLaboratory, DepartmentofBiotechnology,IndianInstituteofTechnology,Chennai, TamilNadu,India MUHAMMADPARASURAM (cid:1) StemCellLaboratory,NationalHeartResearchInstitute (NHRIS)Singapore,NationalHeartCentreSingapore (NHCS),Singapore,Singapore ix