Blebbistatin extends culture life of adult mouse cardiac myocytes and allows efficient and stable transgene expression.
The characterization of cellular phenotypes of heart disorders can be achieved by isolating cardiac myocytes from mouse models or genetically modifying wild-type cells in culture. However, adult mouse cardiac myocytes show extremely low tolerance to isolation and primary culture conditions. Previous studies indicate that 2,3-butanedione monoximine (BDM), a nonspecific excitation-contraction coupling inhibitor, can improve the viability of isolated adult mouse cardiac myocytes. The mechanisms of the beneficial and unwanted nonspecific actions of BDM on cardiac myocytes are not understood. To understand what contributes to murine adult cardiac myocyte stability in primary culture and improve this model system for experimental use, the specific myosin II inhibitor blebbistatin was explored as a media supplement to inhibit mouse myocyte contraction. Enzymatically isolated adult mouse cardiac myocytes were cultured with blebbistatin or BDM as a media supplement. Micromolar concentrations of blebbistatin significantly increased the viability, membrane integrity, and morphology of adult cardiac myocytes compared with cells treated with previously described 10 mM BDM. Cells treated with blebbistatin also showed efficient adenovirus gene transfer and stable transgene expression, and unlike BDM, blebbistatin does not appear to interfere with cell adhesion. Higher concentrations of BDM actually worsened myocyte membrane integrity and transgene expression. Therefore, the specific inhibition of myosin II activity by blebbistatin has significant beneficial effects on the long-term viability of adult mouse cardiac myocytes. Furthermore, the unwanted effects of BDM on adult mouse cardiac myocytes, perhaps due to its nonspecific activities or action as a chemical phosphatase, can be avoided by using blebbistatin.
Kabaeva
Zhyldyz
Z
Dept. of Molecular and Integrative Physiology, University of Michigan, 7623A Medical Science II, Ann Arbor, MI 48109-0622, USA.
Zhao
Mei
M
Michele
Daniel E
DE
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D002448:Cell Adhesion / Q000187:drug effects; D018929:Cell Culture Techniques; D002462:Cell Membrane / Q000187:drug effects; D048430:Cell Shape / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D003931:Diacetyl / Q000031:analogs & derivatives* / Q000494:pharmacology / Q000633:toxicity; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology* / Q000633:toxicity; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D006576:Heterocyclic Compounds, 4 or More Rings / Q000494:pharmacology*; D051379:Mice; D008810:Mice, Inbred C57BL; D009200:Myocardial Contraction / Q000187:drug effects*; D032383:Myocytes, Cardiac / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism; D024462:Myosin Type II / Q000037:antagonists & inhibitors* / Q000378:metabolism; D013997:Time Factors; D014162:Transfection; D019076:Transgenes
D004791:Enzyme Inhibitors; D006576:Heterocyclic Compounds, 4 or More Rings; D049452:Green Fluorescent Proteins; C004717:diacetylmonoxime; C472645:blebbistatin; D024462:Myosin Type II; D003931:Diacetyl