The functional significance of the actin-binding region at the N terminus of the cardiac myosin essential light chain (ELC) remains elusive. In a previous experiment, the endogenous ventricular ELC was replaced with a protein containing a 10-amino acid deletion at positions 5-14 (ELC1v⌬5-14, referred to as 1v⌬5-14), a region that interacts with actin (Sanbe, A., Gulick, J., Fewell, J., and Robbins, J. (2001) J. Biol. Chem. 276, 32682-32686). 1v⌬5-14 mice showed no discernable mutant phenotype in skinned ventricular strips. However, because the myofilament lattice swells upon skinning, the mutant phenotype may have been concealed by the inability of the ELC to reach the actin-binding site. Using the same mouse model, we repeated earlier measurements and performed additional experiments on skinned strips osmotically compressed to the intact lattice spacing as determined by x-ray diffraction. 1v⌬5-14 mice exhibited decreased maximum isometric tension without a change in calcium sensitivity. The decreased force was most evident in 5-6-month-old mice compared with 13-15-month-old mice and may account for the greater ventricular wall thickness in young 1v⌬5-14 mice compared with age-matched controls. No differences were observed in unloaded shortening velocity at maximum calcium activation. However, 1v⌬5-14 mice exhibited a significant difference in the frequency at which minimum complex modulus amplitude occurred, indicating a change in cross-bridge kinetics. We hypothesize that the ELC N-terminal extension interaction with actin inhibits the reversal of the power stroke, thereby increasing isometric force. Our results strongly suggest that an interaction between residues 5-14 of the ELC N terminus and the C-terminal residues of actin enhances cardiac performance.

There is increasing evidence that cardiac glycosides act through mechanisms distinct from inhibition of the sodium pump but which may contribute to their cardiac actions. To more fully define differences between agents indicative of multiple sites of action, we studied changes in contractility and action potential (AP) configuration in cat ventricular myocytes produced by six cardiac glycosides (ouabain, ouabagenin, dihydroouabain, actodigin, digoxin, and resibufogenin). AP shortening was observed only with ouabain and actodigin. There was extensive inotropic variability between agents, with some giving full inotropic effects before automaticity occurred whereas others produced minimal inotropy before toxicity. AP shortening was not a result of alterations in calcium current or the inward rectifier potassium current, but correlated with an increase in steady-state outward current (I ss ), which was sensitive to KB-R7943, a Na ϩ -Ca 2ϩ exchange (NCX) inhibitor.Interestingly, I ss was observed following exposure to ouabain and dihydroouabain, suggesting that an additional mechanism is operative with dihydroouabain that prevents AP shortening.Further investigation into differences in inotropy between ouabagenin, dihydroouabain and ouabain revealed almost identical responses under AP voltage clamp. Thus all agents appear to act on the sodium pump and thereby secondarily increase the outward reverse mode NCX current, but the extent of AP duration shortening and positive inotropy elicited by each agent is limited by development of their toxic actions. The quantitative differences between cardiac glycosides suggest that mechanisms independent of sodium pump inhibition may result from an altered threshold for calcium overload possibly involving direct or indirect effects on calcium release from the sarcoplasmic reticulum.

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Mean intracellular Na+ activity (aNai) was measured in rat left atrial muscle stimulated at increasing frequencies between 0 and 12 Hz. Low-pass filtered signals from conventional and ion-selective microelectrodes were used to determine aNai. Preparations were bathed in a low Ca2+ (0.1 mM) Krebs-Henseleit buffer containing 1.0 mM Mn2+ to abolish contractile motion and permit stable impalements. Under these conditions, aNai increased progressively with frequency from 5.8 +/- 1.5 mM at 0 Hz to a maximum of 12.7 +/- 2.1 mM, which was observed at 10, 11, or 12 Hz. Further increases in frequency exceeded the effective refractory period, and aNai tended to decrease. These data suggest that aNai can be approximately doubled in rat atrial muscle by increasing the depolarization rate from 0 to 10-12 Hz, a range that has been shown to elicit a two- to three-fold elevation in Na+-pump activity in similar preparations.

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