Enantiomeric dissection of the effects of the inotropic agent, EMD 53998, in single cardiac myocytes

The effects of the thiadiazinone derivative, 5-[1-(3,4-dimethoxybenzoyl)- 1,2,3,4-tetrahydrochinolin-6-yl]-6-methyl-3,6-dihydro-2H-1,3,4-thiadiazin-2- on (EMD 53998), and of its (+)EMD 57033 and (-)EMD 57439 enantiomers, were tested on the contractile properties and cytosolic [Ca²⁺] ([Ca²⁺](i)) transients of single intact guinea pig cardiac myocytes. Cells were loaded with the ester form of the fluorescent probe, indo-1, and bathed in a N-2- hydroxyethyl-piperazine-N'-2-ethanesulfonic acid-buffered solution at 25°C (1 mM of CaCl₂, 1 Hz stimulation rate). All three substances exerted a pronounced increase in twitch amplitude: the maximal effect of the racemate (380% of control value) was approximately the sum of the effects of its two enantiomers (186 and 236% of control value for the (+)- and (-)-enantiomer, respectively). The [Ca²⁺](i) transient, measured as the 410-to-490 nm indo- 1 fluorescence ratio transient after excitation, was increased by the racemate and its (-)-enantiomer (172 and 152% of control value, respectively), but was not increased by the (+)-enantiomer. The racemate and the (-)-enantiomer, but not the (+)-enantiomer, markedly reduced the contraction duration and [Ca²⁺](i) transient duration. In unstimulated cells resting length was significantly reduced by the (+)-enantiomer, and this was accompanied by a decrease in indo-1 fluorescence; the (-)- enantiomer had no effect on either parameter. In the presence of 2,3 butanedione monoxime (BDM), which markedly reduces twitch amplitude by inhibiting cross-bridge mechanics, addition of the (+)-enantiomer restored the twitch contraction to above the pre-BDM level without augmenting the [Ca²⁺](i) transient. In contrast, the (-)-enantiomer failed to reverse the BDM-induced contractile depression, even though it caused a significant increase of the [Ca²⁺](i) transient. Thus, in intact cells the positive inotropic effect of EMD 53998 is due to specific properties of its enantiomers: the (-)-enantiomer has adenosine 3',5'-cyclic monophosphate- like effects (increase in amplitude and reduction of [Ca²⁺](i) transient and contraction durations), whereas the (+)-enantiomer enhances the myofilament-Ca²⁺ interaction.