reoxygenation

J Mol

172

Cell

TOXICITY

Cardiol

21

(Supplement

OF CYCLOSPORIN

L. Ver Donck,

G. Kober,

II)

A ON

E. Mutschler,

(1989)

ISOLATED

RAT

M. Kaltenbach.

MYOCYTES. H. G. Olbrich, University Hospital Frankfurt,

S. Dols, FRG.

The acute cardiotoxicity of Cyclosporin A (CsA) was studied in isolated calcium-tolerant cardiac myocytes from adult rats. In a first series (n = 7) myocytes were incubated with CsA concentrations ranging from 1 ug/ml up to 50 ug/ml. Morphological changes of untreated cells, cells treated with CsA and cells treated with the solvent of CsA (Tween 80) were evaluated 8h and 16h following incubation. After 8h and 16h of incubation, 92 f 4.3% and 72 f: 8.7% of the respective non-treated control cells were still rod-shaped. CsA, however, in a concentration of 5 ug/ml decreased the number of rodshaped cells (79 f 3.2% at Sh, 51 f 3.5% at 16h; p < 0.02) as compared to the solvent (94 + 3.5% at 8h, 76 f 5.8% at 16h). This effect became more pronounced with higher CsA concentrations. Ultrastructural investigation on the localization of calcium showed an accumulation of intramitochondrial calcium deposits in hypercontracted cells. In a second series (n = 7), myocytes were incubated 16 hours with 15 ug/ml of CsA in a Ca-containing (1 mM) and Ca-free medium (lOA M EGTA) resulting in a higher percentage of rod-shaped cells in the Ca-free medium (62 ?I 3.3%) as compared to the medium containing Ca (25 f 2.8%; p < 0.02). It is concluded that CsA has a direct cardiotoxic effect which appears to be Ca-dependent. It is further concluded that isolated cardiac myocytes are a suitable model to study this effect.

173

PROPRANOLOL PROTECTS AGAINST PEROXIDATlVE INJURY AND LOSSES OF VlARILlTY OF MYOCYTES EXPOSED TO ANOXJA/REOXYGENATION. J.H. Kramer, I.T. Mak, A.M. Freedman and W.B. Weglicki. Dept. of Medicine, Division of Experimental Medicine, The George Washington University, Washington, DC. 20037 Our previous studies demonstrated a protective effect of propranolol (PRO) on adult canine myocytes subjected to an exogenous free radical generating system. In the present study, we examined the protective actions of @propranolol and the more water-soluble beta-blocker, atenolol, on freshly isolated myocytes (l.5x106/mI) exposed to 30 min anoxia (A: 95%N2/5%COz) and 20 min reoxygenation (R: 95%02/5%CO$. Changes in peroxide formation (thiobarbituric acid reactive products or T&l&P), cellular viability (trypan blue exclusion), ultrastructure (TEM) and release of lactate dehydrogenase (LDH) were monitored in cell samples incubated under control (95%02/5%CO2) and the A/R conditions described; or in samples exposed to 10 min preincubation with either drug (2OO~M) prior to the experimental incubations. Atenolol caused a small but insignificant improvement (13%) in cellular viability with little effect on morphology, TBAR-P or LDH release following A/R. By contrast, PRO prevented the increases in TBAR-P and LDH release, diminished rhe loss of cellular viability and reduced the u&structural injury associated with A/R. PRO had no effect on control parameters. Table values are means of 4-9 TBAR-P Viability LDH Release studies. (% cont.) (net %total) (% time 0) These findings are consistent with our previous studies using an exogenous source of Control 100 2.9 free radicals; this suggests that the ___ % PRO-mediated protective effect provided to 1:: 5.6 %R A/R + PRO 105 2.4 SE myocytes subjected to A/R may be due to the drug’s anti-radical properties.

174ULTRASTRUCTURAL CHANGES BY HYDROGEN PEROXIDE (H202) TO CULTURED MYOCYTES. K. Aida, Department of Pathology and Laboratory Medicine, T. Onodera, T. Oguro, M. Ashraf. University of Cincinnati, Cincinnati, Ohio, USA. H202 has been implicated as one of the oxygen metabolites which take part in the post-ischemic reperfusion injury. In this study, we evaluated the direct effects of Myocytes were incubated for 60 minexogenous H202 on cultured adult rat myocytes. utes with 600 pM or 300 PM of H202 and then processed for light and electron microsAltered cell membrane permeability was evaluated by adding 0.1% trypan blue copy. and 5 mM lanthanum (La+++). The myocytes in the control group excluded trypan blue Even with the maximum dosage of 600 pM H202, and La'++ and retained their rod shape. about 70% of the myocytes were rod shaped. However, all the myocytes incubated with either 300 pM or 600 PM H202 were stained by trypan blue, irrespective of their shape In the electron microscope, the bleb formation of the cell membrane (rod or round). was seen in all the myocytes which had swollen mitochondria with disrupted cristae. The deposits of La++" were observed in the cytoplasm of myocytes, especially beneath These data suggest that 1) H202 causes severe the cell membrane which had blebs. myocyte membrane damage, resulting in increased cell membrane permeability; 2) lipid peroxidation suggested by membrane bleb formation may play a role in the injury by the majority of myocytes to round shaped, although the Hz0 ; 3) H202 does not alter Supported by NIH grant HL23597. ccl f membranes are severely damaged. S.58