Calcium sensitivity during staircase with sequential incompletely fused contractions.

Abstract

Activity dependent potentiation is thought to result from phosphorylation of the regulatory light chains of myosin, increasing Ca²⁺ sensitivity. Yet, Ca²⁺ sensitivity decreases early in a period of intermittent contractions. The purpose of this study was to investigate the early change in Ca²⁺ sensitivity during intermittent submaximal tetanic contractions. Flexor digitorum brevis muscle fibres were dissected from mice after cervical disarticulation. Fibres were superfused with Tyrode solution at 32 °C. Length was set to yield maximal tetanic force. Indo-1 was microinjected into fibres and allowed to dissipate for 30 min. Fluorescence was measured at 405 and 495 nm wavelength and the ratio was used to estimate [Ca²⁺]. A control force-Ca²⁺ relationship was determined with stimulation over a range of frequencies, yielding constants for slope, max force, and half-maximal [Ca²⁺] (pCa^2 +₅₀). Data were collected for sequential contractions at 40 Hz at 2 s intervals. Active force decreased over the first 1-4 contractions then increased. A force-pCa²⁺ curve was fit to each contraction, using the control values for the Hill slope and max force by adjusting pCa²⁺₅₀ until the curve passed through the target contraction. Data are presented for three contractions for each fibre: first, maximum shift to the right, and last contraction. There was a significant shift to the right for pCa²⁺₅₀ (decreased Ca²⁺ sensitivity), usually early in the series of intermittent contractions, then pCa^2 +₅₀ shifted to the left, but remained significantly different from the control value. Although potentiation is associated with increased Ca²⁺ sensitivity, this increase begins only after Ca²⁺ sensitivity has decreased and, in most cases, Ca²⁺ sensitivity does not increase above the control level.