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Neonatal rat heart cell cultures are popular research models in cardiovascular investigations. A major disadvantage is the variable contribution of non-muscle cells to the cultures. As biochemical and pharmacological quantities are generally measured in homogenised cultures, we looked for a method to calculate numbers of muscle cells and non-muscle cells per culture after homogenisation. By means of a model based on the presence of one diploid nucleus per myocyte and per non-muscle cell, a nuclear DNA content of 7.5 pg, and a constant ratio of DNA content and sum of the lactate dehydrogenase isoenzymes LDH-4 and LDH-5 (DNA/LDH4+5 = 11.6±1.5 μg·U−1)in non-muscle cells, we calculated that in 21 neonatal rat heart cell cultures, cultured for 0–6 days, the number of muscle cells was 1.5 × 106 per culture, independent of time; and the number of non-muscle cells was low at day 0 (1.6 × 105 per culture), increasing to 4 × 106 per culture at day 6. Based on a time dependent increase in lactate dehydrogenase content per muscle cell we showed that muscle cells in culture underwent progressive hypertrophy: in 6 days myocyte volume increased fourfold. Thus, by measurement of DNA content and the activities of lactate dehydrogenase and its isoenzymes in a homogenised culture the cellular composition of the culture can be assessed quantitatively.

Objective: The aim was to find effective protection of myocytes against peroxide induced damage in terms of preservation of contractile activity, protection against lipid peroxidation, and protection against cell death. Methods: The components of the glutathione redox cycle, the production of malondialdehyde, cell contractions, and enzyme release from myocytes were measured in cultured neonatal rat heart cells before and after administration of cumene hydroperoxide, 80 µmol·litre–1. The protective action was tested of (1) glucose (10 mmol·litre) which stimulates the production of <$O_SCPCAP>NADPH<$C_SCPCAP>; (2) Trolox-C (0.16 mmol·litre–1) which is a water soluble analogue of a tocopherol and a scavenger of free radicals; and (3) <$O_SCPCAP>GSSG<$C_SCPCAP> (0.6 mmol·litre–1) which increases the intracellular concentrations of <$O_SCPCAP>GSH<$C_SCPCAP> and <$O_SCPCAP>GSSG<$C_SCPCAP>. Results: Although the three substances tested were equally effective in reducing the formation of malondialdehyde, exogenous <$O_SCPCAP>GSSG<$C_SCPCAP> afforded only slight protection against cumene hydroperoxide induced cell death, whereas glucose and Trolox-C were highly effective protectors. The depressant effect of cumene hydroperoxide on beating frequency was not influenced by preincubation with <$O_SCPCAP>GSSG<$C_SCPCAP>, nor by coadministration of glucose, but Trolox-C was able to diminish the negative chronotropic action of cumene hydroperoxide. Conclusions: Effective protection against cumene hydroperoxide induced lipid peroxidation is not associated per se with effective protection against cumene hydroperoxide induced loss of beating frequency and cell death.

Spontaneously beating monolayer cultures of neonatal rat heart cells were first exposed to depletion of oxygen and metabolic substrates for 1 to 7 h (anoxia). Subsequently the cultures were resupplied with oxygen and substrates (reoxygenation). The release of -hydroxybutyrate dehydrogenase (HBDH) from the cells, the extent of necrosis, and the changes in spontaneous contractile activity were measured. HBDH release was observed to start after 1 h of anoxia and to increase to 84% of intracellular HBDH activity after 7 h of anoxia. Reoxygenation of anoxic heart cells is associated with accelerated HBDH release (oxygen paradox). The activity of HBDH released by cardiac cells, the number of necrotic cells and the impairment of beating capacity of the cells depend on the duration of the anoxic period in a similar way. This study demonstrates that cardiac cell death can be assessed quantitatively and reliably by the measurement of the activity of HBDH released by the cells.