The increase in non-cross-bridge forces after stretch of activated striated muscle is related to titin isoforms

Abstract

Skeletal muscles present a non-cross-bridge increase in sarcomere stiffness and tension on Ca(2+) activation, referred to as static stiffness and static tension, respectively. It has been hypothesized that this increase in tension is caused by Ca(2+)-dependent changes in the properties of titin molecules. To verify this hypothesis, we investigated the static tension in muscles containing different titin isoforms. Permeabilized myofibrils were isolated from the psoas, soleus, and heart ventricle from the rabbit, and tested in pCa 9.0 and pCa 4.5, before and after extraction of troponin C, thin filaments, and treatment with the actomyosin inhibitor blebbistatin. The myofibrils were tested with stretches of different amplitudes in sarcomere lengths varying between 1.93 and 3.37 μm for the psoas, 2.68 and 4.21 μm for the soleus, and 1.51 and 2.86 μm for the ventricle. Using gel electrophoresis, we confirmed that the three muscles tested have different titin isoforms. The static tension was present in psoas and soleus myofibrils, but not in ventricle myofibrils, and higher in psoas myofibrils than in soleus myofibrils. These results suggest that the increase in the static tension is directly associated with Ca(2+)-dependent change in titin properties and not associated with changes in titin-actin interactions.

Keywords: force enhancement; myofibrils; myosin; static tension.

Fig. 1.

Pictures of a myofibril isolated from the rabbit psoas muscle before (A) and after (B) treatment with gelsolin. The myofibrils were stained with fluorescent phalloidin, which binds to the actin filaments. Note that the A bands are larger and more visible before gelsolin treatment. C: contractile response of a myofibril isolated from the soleus muscle before and after treatment with rigor-EDTA, gelsolin, and blebbistatin (BB). Before BB, the myofibril produced an isometric contraction on activation (pCa 4.5), with a resulting force that is similar to that observed in previous studies. After treatment with rigor-EDTA, gelsolin, and BB, the myofibrils did not produce substantial active force on activation. Myofibril cross-sectional area: 0.62 μm²; sarcomere length (SL) during activation: 2.6 μm.

Fig. 2.

Four superimposed contractions produced by myofibrils isolated from the psoas (A), soleus (B), and ventricle (C) muscles. Black traces: isometric contractions and passive stretches performed in pCa 4.5 and 9.0, respectively. Red traces: stretches produced after full development in pCa 4.5. Blue traces: stretches applied in pCa 4.5 after myofibrils were treated with rigor-EDTA, gelsolin, and BB. Note the high signal-to-noise ratio in the force traces, which allows the detection of small force differences during the experiments. During the stretches in pCa 4.5, the force increased substantially, and after the stretch the force decreased to attain a steady state that was higher than the force obtained during isometric contractions at a similar length. During the stretches in pCa 4.5 after the myofibrils were treated with rigor-EDTA, gelsolin, and BB, the force was higher than that produced during stretches in pCa 9.0 for the psoas and soleus muscles, but not for the ventricle muscle.

Fig. 3.

Consecutive stretches performed with myofibrils isolated from the psoas (A), soleus (B), and ventricle (C) muscles in pCa 4.5 after treatment with rigor-EDTA, gelsolin, and BB, and pCa 9.0. An increase in Ca²⁺ concentration caused an increase in forces produced by the myofibrils during and after stretches of psoas and soleus myofibrils, but not in ventricle myofibrils.

Fig. 4.

Passive force-SL relation for all myofibrils investigated in this study, before and after treatment with rigor-EDTA, gelsolin, and BB, in pCa 9.0 and pCa 4.5. There is an increase in the forces and an upward shift in the force-SL relation in myofibrils isolated from the psoas and soleus muscles, but not in myofibrils isolated from ventricle muscles.