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.
Highlights
Intact myocytes are frequently used to investigate the molecular mechanisms that underlie contraction and to screen potential therapies for cardiac disease (King et al 2011; Malik and Morgan 2011; Aronson and Krum 2012; Campbell et al 2013; Chung and Campbell 2013)
Isolated myocytes that were tolerant to calcium had well-defined edges, clear striation patterns, and a a 2015 The Authors
Cells that were stored in either 10 mmol LÀ1 Butanedione monoxime (BDM) or 3 lmol LÀ1 blebbistatin had sharper cell edges, more defined striation patterns, and were more likely to be rod-shaped (Fig. 3) than cells stored without myosin ATPase inhibitors
Summary
Intact myocytes are frequently used to investigate the molecular mechanisms that underlie contraction and to screen potential therapies for cardiac disease (King et al 2011; Malik and Morgan 2011; Aronson and Krum 2012; Campbell et al 2013; Chung and Campbell 2013). Many experiments can be performed using myocytes that have been cultured but these cells typically exhibit altered morphology (e.g., nonrectangular shape and misaligned sarcomeres) that make it difficult to assess contractility. Groups studying how mutations and/or drugs alter calcium transients and cell shortening often isolate fresh cells on a nearly daily basis
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