Calcium-tolerant rabbit cardiomyocytes were isolated using retrograde aortic perfusion with a nominally calcium-free, collagenase buffer. In vitro ischemic preconditioning was induced by a 10-min episode of ischemic pelleting, followed by a 15-min post-incubation and a prolonged period of ischemic pelleting. Injury was assessed by determination of cell contracture and trypan blue permeability following hypotonic swelling and correlated with metabolic assays of lactate and adenine nucleotides. The protein phosphatase PP1/2A inhibitor calyculin A and PP2A-selective fostriecin protected isolated rabbit cardiomyocytes from lethal injury after a 10-min pre-incubation and when added late into ischemic pellets after a delay of 75 min. At the time of late drug addition, cells were severely ATP-depleted and in rigor contracture. Protection with Calyculin A from 1 nM to 1 μM was dose-related. Cells pre-incubated with 10 nM to 10 μM fostriecin 10 min prior to ischemic pelleting were protected with an EC50 approximating 71 nM, implying protection at a PP2A-selective dose. The selective protein kinase C inhibitor, calphostin C, blocked ischemic preconditioning protection but not protection from 1 μM calyculin A. Protection of severely ischemic cardiomyocytes following protein phosphatase inhibition appears not to require PKC activity or ATP conservation. Pre-incubation of cells with calyculin A induced high levels of phosphorylation in p38 mitogen activated protein kinase (MAPK), as compared to the ischemia-induced phosphorylation observed in the untreated group only at 30 min of ischemia, providing evidence of protein phosphatase activity in cardiomyocytes. Pharmacological protection in late ischemia has been demonstrated, but the mechanism of protection is undetermined.
| Identifer | oai:union.ndltd.org:ETSU/oai:dc.etsu.edu:etsu-works-13268 |
| Date | 01 January 1998 |
| Creators | Armstrong, Stephen C., Gao, W., Lane, J. R., Ganote, C. E. |
| Publisher | Digital Commons @ East Tennessee State University |
| Source Sets | East Tennessee State University |
| Detected Language | English |
| Type | text |
| Source | ETSU Faculty Works |
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