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

Abstract

The effects of the thiadiazinone derivative, 5-[1-(3,4-dimethoxybenzoyl)-1,2,3,4-tetrahydrochinolin-6-yl]-6-met hyl-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 degrees 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.

Interaction of Cardiotonic Thiadiazinone Derivatives with Cardiac Troponin C

“…Thiadiazinone derivative EMD 57033 1 (1,2), developed by Pharmaceutical Research, E. Merck (Darmstadt, Germany), is a novel cardiotonic agent that induces positive inotropy in intact cardiac myocytes without a significant effect on [Ca 2ϩ ] i transients (1)(2)(3)(4)(5)(6)(7). The pharmacological effects of the compound are thought to be mediated by enhanced responsiveness of the contractile filaments to Ca 2ϩ (2)(3)(4)(5)(6)(7).…”

“…The pharmacological effects of the compound are thought to be mediated by enhanced responsiveness of the contractile filaments to Ca 2ϩ (2)(3)(4)(5)(6)(7). EMD 57033 has been found to increase Ca 2ϩ sensitivity of both myofibrillar ATPase (2, 6) and force development by skinned muscle fibers (2).…”

“…The compound appears to have a direct effect on actin/myosin interactions (6). It has also been hypothesized that EMD 57033 may bind cardiac troponin C and increase its affinity for Ca 2ϩ (4,7). Troponin C (TnC) is the Ca 2ϩ binding subunit of troponin, a hetero-trimeric protein containing, in addition, inhibitory subunit (TnI) and tropomyosin binding subunit.…”

The effects of the inotropic agent EMD 57033 on activation and relaxation kinetics in frog skinned skeletal muscle

“…Whereas EMD 57439 raises resting intracellular Ca 2+ , EMD 57033 has no effect on the size or time course of the Ca 2+ transient in either guinea-pig skinned myocytes [12] or intact canine myocytes [37,40]. In intact ferret papillary muscle, both the size and the time course of the Ca 2+ transient were reduced by EMD 57033 [41].…”

“…It increases maximal calcium-activated tension (P max ) in a variety of intact and skinned preparations [2,3,26], without increasing the size or time course of the Ca 2+ transient [10,12,26,39].…”