title: Isolation and physiological analysis of mouse cardiomyocytes.
abstract: 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.
authors.affiliation: Department of Medicine, Vanderbilt University.
authors.forename: Gretchen M
authors.initials: GM
authors.lastname: Roth
authors.affiliation: Department of Medicine, Vanderbilt University; Department of Cell and Developmental Biology, Vanderbilt University; david.bader@vanderbilt.edu.
authors.forename: David M
authors.initials: DM
authors.lastname: Bader
authors.affiliation: Department of Cell and Developmental Biology, Vanderbilt University.
authors.forename: Elise R
authors.initials: ER
authors.lastname: Pfaltzgraff
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism; D051379:Mice; D009200:Myocardial Contraction / Q000502:physiology; D032383:Myocytes, Cardiac / Q000166:cytology / Q000378:metabolism / Q000502:physiology*
chemical_list: D002118:Calcium