Contractile deactivation and uncoupling of crossbridges. Effects of 2,3-butanedione monoxime on mammalian myocardium.

Abstract

We investigated the effects of 1 and 3 mM 2,3-butanedione monoxime (BDM, diacetyl monoxime) on excitation and contraction of cardiac muscle in several types of preparations at various levels of organization. We selected a concentration of BDM that was not expected to affect sarcolemmal calcium flux and action potential duration in cardiac tissue. Two indicators were used to record intracellular calcium. Aequorin, a bioluminescent calcium indicator, was used in studies with ferret papillary muscle preparations, and fura-2, a fluorescent calcium indicator, was used in studies with guinea pig cardiac myocytes. In both cases, addition of BDM resulted in a reduction of peak intracellular calcium released from the sarcoplasmic reticulum and a reduction of peak twitch force. The duration of the action potential of isolated myocytes was slightly abbreviated in the presence of BDM. In studies on the calcium current in the myocytes, addition of BDM was associated with reduced calcium current at any potential. Peak calcium current was reduced by 7.9±1% in the presence of BDM. In tetanized ferret papillary muscles, BDM reduced maximal calcium-activated force by 30±5% and increased the calcium ion concentration required for half-maximal force by 0.1±0.01 ,uM. The Hill coefficient was reduced from 5.00±0.11 to 3.40+0.20. Maximal shortening velocity of ferret papillary muscles was increased in the presence of BDM from 1.55+±0.24 to 2.04±0.33 mm/sec. Ca2' binding to troponin C in skinned fiber preparations from guinea pig, bovine, and canine hearts was unaffected by addition of up to 10 mM BDM. Our results indicate that BDM affects both calcium availability and responsiveness of the myofilaments to Ca2 . Uncoupling of contractile activation from excitation may also result from altered crossbridge kinetics. (Circulation Research 1991;69:1280-1292 A n agent that apparently uncouples cardiac excitation from contraction,' 2, paradox,3 intracellular calcium mobilization,4 and crossbridge kinetics5-7 and to inhibit contractile function.8 BDM has been shown to affect force-pCa relations in chemically skinned fiber preparations in which the sarcoplasmic reticulum (SR) and sarcolemma have been extracted.9"10 There are conflicting reports as to whether BDM affects slow inward calcium current (Ilc), thereby inducing a negative inotropic effect.10-12 Previous studies have been unable to rule out possible effects of BDM on SR calcium handling.2"10 Because the SR is the predominant intracellular organelle that supplies calcium for activation of force in cardiac muscle, the effect of BDM on this organelle is pivotal in potentially explaining its mechanism of action.Studies using this compound rely on a full understanding of its mechanism of action, yet the mechanism by which BDM produces force inhibition remains unclear. This study was designed to provide greater insight into the mechanisms of contractile deactivation in the presence of BDM. We investi-

Cross-Bridge-Dependent Change in Ca2+ Sensitivity is Involved in the Negative Inotropic Effect of Nifedipine in Aequorin-Injected Ferret Ventricular Muscles

“…An inhibitor of the active cross-bridge, BDM, 15,18 showed the similar effect on the slope of the extra-Ca 2+ -tension relation (Fig 4) to that in Nif, which also supports our hypothesis. Thus, Nif exerts its negative inotropic effect on the contraction of ventricular muscles directly through the upstream mechanism as well as through the cross-bridge (downstream)-dependent change in the Ca 2+ affinity of TnC (central).…”

“…15,18 In these experiments, we measured the extra-Ca 2+ in the solution containing 8 2+ ]o, and the slope of the extra-Ca 2+ -tension relation was measured. Three mmol/L BDM was used because the downstream mechanism was selectively inhibited without influencing SR, 18 which would antagonize the effect of BAY on the downstream mechanism.…”

Cardiac troponin T mutations result in allele-specific phenotypes in a mouse model for hypertrophic cardiomyopathy

“…Subsequent video image analysis of ventricular myocytes incubated in the presence and absence of BDM yielded several very interesting results. BDM is known to disrupt force-producing cross-bridge formation in mammalian myocardium (32). An important finding of this analysis was that the mean area of R92Q myocytes was approximately 18% smaller than the areas of non-Tg or WT-Myc myocytes in the presence or absence of BDM (Table 1).…”

Glycolytic inhibition: effects on diastolic relaxation and intracellular calcium handling in hypertrophied rat ventricular myocytes.

“…To investigate whether increased diastolic cell position in hypertrophied myocytes exposed to 2DG is due to irreversible Ca2+ -independent rigor or Ca2+ -dependent crossbridge cycling, a separate experiment was performed in hypertrophied myocytes (n = 3) using BDM, which uncouples contractile activity from Ca2+ transients (26,27). Hypertrophied myocytes were first superfused with the same solution used for baseline superfusion in the 2DG protocol.…”