BPS2026 - Unexplained and reversible Z-line widening in isolated myofibrils at long sarcomere lengths

The sarcomeric Z-line is an organized lattice composed primarily of α-actinin providing anchor points for actin filaments and titin. The Z-line transmits both passive and active force between adjacent sarcomeres. Although changes to Z-line width have been reported in disordered muscle, wild-type skeletal muscle Z-lines are typically 100–180 nm at physiological lengths, depending on fiber-type. Recent observations in our lab, however, show extreme expansion of the Z-line (∼up to 1 μm) at long sarcomere lengths (>3.6 μm) in isolated myofibrils stretched passively. Since the Z-line is not known as an elastic structure, and its expansion has not been characterized, we sought to explain this phenomenon. First, we characterized Z-line expansion in isolated rabbit psoas (type II) and soleus (type I) myofibrils. Myofibrils were stretched using a needle operated by piezoelectric motor, force was measured optically using a custom nano-lever, and Z-line width tracked using α-actinin immunolabelling. In psoas and soleus myofibrils, significant Z-line expansion occurred above sarcomere lengths of 3.6 μm; upon shortening, Z-lines returned to original widths. We noted significantly more Z-line expansion in psoas (type II) myofibrils compared to soleus (type I) myofibrils, likely due to increased α-actinin layers in the type I Z-line. After Z-line expansion, when myofibrils were shortened, both psoas and soleus recapitulated original force-extension curves, indicating that damage to myofilaments had not occurred. Second, we tested Z-line expansion in rabbit psoas myofibrils after titin cleavage using partial trypsin digestion. Titin cleavage eliminated virtually all Z-disk expansion, establishing titin-transmitted force is essential for Z-line expansion to occur. Third, we performed electron microscopy to better characterize widened Z-lines in psoas and soleus myofibrils. Our findings suggest for the first time that Z-lines of skeletal muscle may expand at long sarcomere lengths, potentially acting as an injury prevention mechanism.