Dual Regulation Mechanisms for Ca2+-Activated and Stretch-Activated Forces in Asynchronous Flight Muscle of Insect: Mechanical and X-Ray Evidence

Abstract

During flight of insects, their flight muscles (IFMs) are often endothermically maintained at an optimal temperature. In the case of bumblebee, this temperature is ∼42°C At saturating [Ca2+], glycerinated bumblebee IFM fibers develop large Ca2+-activated (CA) force of ∼50 kPa above 20°C, as well as stretch-activated (SA) force. Below the critical temperature of 15°C, the CA force is sharply suppressed. Surprisingly, the SA force is not suppressed even at 5°C. This suggests that the inhibition of CA force at low temperatures is not due to myosin inactivation, but it is an issue of regulation. The CA force-pCa curve remains sigmoidal at lower temperatures, and the pCa50 value is only slightly affected, indicating that myosin is unable to develop large CA force even if troponin is saturated with Ca2+. The mechanism for temperature-dependent CA force regulation is further investigated by X-ray diffraction, by recording semi-static patterns in the stretch and release phases of a repeated stretch-release protocol. At 5°C, the 2nd actin layer line (ALL) is increased from 5% at rest (relative to 6th ALL) to 19% in the release phase, indicating that the myosin binding sites on actin are almost fully open even when the CA force is suppressed. In the stretch phase, the 2nd ALL is further enhanced to 26%. At 20°C, the 2nd ALL intensity is enhanced to 25 and 29% in the release and stretch phases, respectively. These results suggest that a pathway after thin-filament activation is blocked at low temperatures, and SA force could be regulated independently of this pathway. The critical temperature for CA force development is also found in other insects, such as a true bug Nezara (T=20-25°C), but not in a giant waterbug Lethocerus.