Abstract
Isolated intact myocytes can be used to investigate contractile mechanisms and to screen new therapeutic compounds. These experiments typically require euthanizing an animal and isolating fresh cells each day or analyzing cultured myocytes, which quickly lose their rod-shaped morphology. Recent data suggest that the viability of canine myocytes can be prolonged using low temperature and N-benzyl-p-toluene sulfonamide (an inhibitor of skeletal myosin ATPase). We performed similar studies in rat myocytes in order to test whether the cardiac myosin ATPase inhibitors 2,3-Butanedione monoxime (BDM) and blebbistatin help to maintain cell-level function over multiple days. Myocytes were isolated from rats and separated into batches that were stored at 4°C in a HEPES-buffered solution that contained 0.5 mmol L-1 Ca2+ and (1) no myosin ATPase inhibitors; (2) 10 mmol L-1 BDM; or (3) 3 μmol L-1 blebbistatin. Functional viability of myocytes was assessed up to 3 days after the isolation by measuring calcium transients and unloaded shortening profiles induced by electrical stimuli in inhibitor-free Tyrode's solution. Cells stored without myosin ATPase inhibitors had altered morphology (fewer rod-shaped cells, shorter diastolic sarcomere lengths, and membrane blebbing) and were not viable for contractile assays after 24 h. Cells stored in BDM maintained morphology and contractile function for 48 h. Storage in blebbistatin maintained cell morphology for 72 h but inhibited contractility. These data show that storing cells with myosin ATPase inhibitors can extend the viability of myocytes that will be used for functional assays. This may help to refine and reduce the use of animals in experiments.
Document Type
Article
Publication Date
6-26-2015
Digital Object Identifier (DOI)
http://dx.doi.org/10.14814/phy2.12445
Funding Information
This study was supported by the American Heart Association (Scientist Development Grant 14SDG20100063) to CSC and the National Institutes of Health (R01 HL090749) to KSC and CTSA UL1TR000117.
Repository Citation
Chung, Charles S.; Mechas, Charles; and Campbell, Kenneth S., "Myocyte Contractility Can Be Maintained by Storing Cells with the Myosin ATPase Inhibitor 2,3 Butanedione Monoxime" (2015). Physiology Faculty Publications. 73.
https://uknowledge.uky.edu/physiology_facpub/73
Notes/Citation Information
Published in Physiological Reports, v. 3, no. 6, article e12445, p. 1-9.
© 2015 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.
This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.