title: Mitochondrial respiratory inhibition by 2,3-butanedione monoxime (BDM): implications for culturing isolated mouse ventricular cardiomyocytes.
abstract: Experiments in isolated ventricular cardiomyocytes have greatly facilitated the study of cellular and subcellular physiology in the heart. However, the isolation and culture of high-quality adult murine ventricular cardiomyocytes can be technically challenging. In most experimental protocols, the culture of viable adult murine cardiomyocytes for prolonged time periods is achieved with the addition of the myosin II ATPase inhibitors blebbistatin and/or 2,3-butanedione monoxime (BDM). These drugs are added to increase cell viability and life span by inhibiting spontaneous cardiomyocyte contraction, thereby preventing calcium overload, cell hypercontracture, and cell death. While the addition of BDM has been reported to prolong the life span of isolated adult murine cardiomyocytes, it is also associated with several off-target effects. Here, we report a novel off-target effect, in which BDM inhibits mitochondrial respiration by acting directly on the electron transport chain to reduce cell viability. In contrast, when cells were cultured with blebbistatin alone, cells survived for longer, and no metabolic off-target effects were observed. Based on these novel observations, we recommend that culture media for isolated mouse ventricular cardiomyocytes should be supplemented with blebbistatin alone, as BDM has the potential to affect mitochondrial respiration and cell viability, effects which may impact adversely on subsequent experiments.
authors.affiliation: The Hatter Cardiovascular Institute, Institute of Cardiovascular Science, University College London Hospital & Medical School, London, UK andrew.r.hall@ucl.ac.uk.
authors.forename: Andrew R
authors.initials: AR
authors.lastname: Hall
authors.affiliation: The Hatter Cardiovascular Institute, Institute of Cardiovascular Science, University College London Hospital & Medical School, London, UK Cardiovascular and Metabolic Disorders Program, Duke-National University of Singapore, Singapore, Singapore National Heart Research Institute Singapore, National Heart Centre Singapore, Singapore, Singapore.
authors.forename: Derek J
authors.initials: DJ
authors.lastname: Hausenloy
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods; D019069:Cell Respiration / Q000187:drug effects / Q000502:physiology; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D003931:Diacetyl / Q000031:analogs & derivatives* / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D051379:Mice; D008810:Mice, Inbred C57BL; D008929:Mitochondria, Heart / Q000187:drug effects* / Q000378:metabolism*; D032383:Myocytes, Cardiac / Q000187:drug effects* / Q000378:metabolism*
chemical_list: D004791:Enzyme Inhibitors; C004717:diacetylmonoxime; D003931:Diacetyl
keywords: BDM; murine cardiomyocytes; respiration