MODULATION OF MYOFILAMENT CA2+ SENSITIVITY AS A POSITIVE INOTROPIC INTERVENTION

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A persistent challenge has been the development of substances that increase myocardial contractility via an enhancement of myofilament responsiveness to Ca2+ rather than by increasing the extent of cellular Ca2+ loading. The diazinone derivative, EMD 53998 (designed by E. Merck, Darmstadt, Germany), increases the peak force and shift leftward the pCa-force relationship in skinned myocardial fibers; and in cell homogenates it exhibits phosphodiesterase (PDE) inhibitory activity. However, the potency of myofilament sensitization relative to that of PDE inhibition is greater than for any known substance. The effects of EMD 53998, and of its (+), EMD 57033 and (-), EMD 57439, enantiomers, were tested on the contractile properties and Cai transients of single, intact, guinea pig and dog cardiac myocytes. Cells were loaded with the fluorescent dye, indo-1, and bathed in a Hepes buffer at 25 degrees C. Our aim was to ascertain whether the optical enantiomers could separate the effect mediated through PDE inhibition from that obtained via an increased myofilament responsiveness to Ca2+. All three substances exerted a pronounced increase in twitch amplitude: the maximal effect of the racemate was approximately the sum of the effects of its two enantiomers. The Cai transient, measured as the 410/490 nm indo-1 fluorescence ratio transient, was increased by the racemate and its (-) enantiomer, but not by the (+)-enantiomer. 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. Qualitatively similar effects were obtained in experiments with intact dog cardiac cells. The molecular mechanism of the effect of the (+)-enantiomer was further studied in dog cardiac myofibrils. EMD 57033 stimulates the ATPase activity in myofibrils in which troponin-tropomyosin have been extracted, but does not affect Ca2+ binding to isolated troponin C. Furthermore, in a motility assay containing isolated actin and myosin, but devoid of Ca2+ and regulatory proteins, the substance increases the velocity of actin motion along myosin. Thus, in intact cells the (+)-enantiomer of EMD 53998 has direct myofilament effects which are not simply due to "Ca2+- sensitization" (i.e. effects downstream to Ca2+-binding to troponin C and regulatory protein modulation of thin filament activation); rather an effect on acto-myosin interaction, i.e. the cross-bridge is likely involved.