ACTIN-TITIN INTERACTION IN CARDIAC MYOFIBRILS - PROBING A PHYSIOLOGICAL-ROLE

The high stiffness of relaxed cardiac myofibrils is explainable mainly by the expression of a short-length titin (connectin), the giant elas tic protein of the vertebrate myofibrillar cytoskeleton. However, addi tional molecular features could account for this high stiffness, such as interaction between titin and actin, which has previously been repo rted in vitro. To probe this finding for a possible physiological sign ificance, isolated myofibrils from rat heart were subjected to selecti ve removal of actin filaments by a calcium-independent gelsolin fragme nt, and the ''passive'' stiffness of the specimens was recorded. Upon actin extraction, stiffness decreased by nearly 60%, and to a similar degree after high-salt extraction of thick filaments. Thus actin-titin association indeed contributes to the stiffness of resting cardiac mu scle. To identify possible sites of association, we employed a combina tion of different techniques. Immunofluorescence microscopy revealed t hat actin extraction increased the extensibility of the previously sti ff Z-disc-flanking titin region. Actin-titin interaction within this r egion was confirmed in in vitro cosedimentation assays, in which multi module recombinant titin fragments were tested for their ability to in teract with F-actin. By contrast, such assays showed no actin-titin-bi nding propensity for sarcomeric regions outside the Z-disc comb. Accor dingly, the results of mechanical measurements demonstrated that compe tition with native titin by recombinant titin fragments from Z-disc-re mote, I-band or A-band regions did not affect passive myofibril stiffn ess. These results indicate that it is actin-titin association near th e Z-disc, but not along the remainder of the sarcomere, that helps to anchor the titin molecule at its N-terminus and maintain a high stiffn ess of the relaxed cardiac myofibril.