Differential expression of cardiac titin isoforms and modulation of cellular stiffness

Extension of the I-band segment of titin gives rise to part of the diastoli c force of cardiac muscle. Previous studies of human cardiac titin transcri pts suggested a series of differential splicing events in the I-band segmen t of titin leading to the so-called N2A and N2B isoform transcripts. Here w e investigated titin expression at the protein level in a wide range of mam malian species. Results indicate that the myocardium coexpresses 2 distinct titin isoforms: a smaller isoform containing the N2B element only (N2B tit in) and a larger isoform with both the N2B and N2A elements (N2BA titin). T he expression ratio of large N2BA to small N2B titin isoforms was found to vary greatly in different species; eg, in the left ventricle the ratio is a pproximate to 0.05 in mouse and approximate to 1.5 in pig. Differences in t he expression ratio were also found between atria and ventricles and betwee n different layers of the ventricular wall. Immunofluorescence experiments with isoform-specific antibodies suggest that coexpression of these isoform s takes place at the single-myocyte level. The diastolic properties of sing le cardiac myocytes isolated from various species expressing high levels of the small (rat and mouse) or large (pig) titin isoform were studied. On av erage, pig myocytes are significantly less stiff than mouse and rat myocyte s. Gel analysis indicates that this result cannot be explained by varying a mounts of titin in mouse and pig myocardium. Rather, low stiffness of pig m yocytes can be explained by its high expression level of the large isoform: the longer extensible region of this isoform results in a lower fractional extension for a given sarcomere length and hence a lower force. Implicatio ns of our findings to cardiac function are discussed.