Differential Expression of Cardiac Titin Isoforms and Modulation of Cellular Stiffness

Abstract

Abstract-Extension of the I-band segment of titin gives rise to part of the diastolic force of cardiac muscle. Previous studies of human cardiac titin transcripts suggested a series of differential splicing events in the I-band segment of titin leading to the so-called N2A and N2B isoform transcripts. Here we investigated titin expression at the protein level in a wide range of mammalian species. Results indicate that the myocardium coexpresses 2 distinct titin isoforms: a smaller isoform containing the N2B element only (N2B titin) and a larger isoform with both the N2B and N2A elements (N2BA titin). The 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 Ϸ0.05 in mouse and Ϸ1.5 in pig. Differences in the expression ratio were also found between atria and ventricles and between different layers of the ventricular wall. Immunofluorescence experiments with isoform-specific antibodies suggest that coexpression of these isoforms takes place at the single-myocyte level. The diastolic properties of single cardiac myocytes isolated from various species expressing high levels of the small (rat and mouse) or large (pig) titin isoform were studied. On average, pig myocytes are significantly less stiff than mouse and rat myocytes. Gel analysis indicates that this result cannot be explained by varying amounts of titin in mouse and pig myocardium. Rather, low stiffness of pig myocytes 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. Implications of our findings to cardiac function are discussed. (Circ Res. 2000;86:59-67.)Key Words: compliance Ⅲ passive tension Ⅲ diastolic force Ⅲ mechanical properties Ⅲ myocyte Ⅲ connectin D uring diastole, the myocardium stretches and passive force (F) is generated. A major contributor to this force is the giant protein titin, spanning the half-sarcomere from the Z-band to the M-line. When sarcomere length (SL) increases during diastole, the I-band region of titin extends and force develops. The shape of F-SL relation of titin is expected to influence ventricular filling during diastole and ventricular emptying during systole. In addition to influencing ventricular filling, titin also helps to maintain the structural integrity of the contracting sarcomere (for reviews, see References 1-5).The force of titin arises from its extensible I-band region, which consists of 2 main segment types: (1) a segment rich in proline (P), glutamate (E), valine (V), and lysine (K) residues (the so-called PEVK segment) and (2) serially linked immunoglobulin (Ig)-like domains (tandem Ig segments) flanking this PEVK segment. 6 Several titin isoforms are now known, all of which contain PEVK and tandem Ig segments. In addition to these common segments, the extensible region of the N2B isoform contains the N2B element (3 Ig domains and a 572-residue unique sequence),...

Mutation that dramatically alters rat titin isoform expression and cardiomyocyte passive tension

“…In cardiac muscle such tension correlates with the relative ratio of the longer N2BA isoform species to that of the shorter N2B [11,40]. It was anticipated that myocytes from the homozygous mutants, with their giant sized titin, would have reduced passive tension compared to wild type, and this was confirmed (Figure 7).…”

“…The heart contains two major isoform classes, N2B and N2BA, with the former being shorter in length [9]. The ratio of amounts of these two classes varies between species [11] with small mammals having a predominance of the smaller N2B isoform while large mammals, including humans, have a more equal representation of both classes. The isoform ratio changes in rats during development starting with a single large N2BA species at the embryonic stage and proceeding to the adult pattern by 20 days after birth [12][13][14].…”

Nuances of electrophoresis study of titin/connectin

“…Using human and animal skeletal muscle myosin heavy chain (205 kDa) and, nebulin (770-890 kDa), as well as the N2A titin isoform (∼3600 kDa and 3700 kDa) of rabbit and human soleus as standards Krüger et al 1991;Granzier and Wang 1993;Prado et al 2005), we estimated that the NT have a Mr of ∼3.8-3.9 × 10 6 (Vikhlyantsev and Podlubnaya 2006). Expression of titin isoforms with these molecular weights is not excluded (Bang et al 2001;Guo et al 2010Guo et al , 2012Li et al 2012), but titin aggregates in gels could not be excluded either (Granzier and Wang 1993;Cazorla et al 2000;Warren et al 2003a). Assuming that molecular masses of titin aggregates should considerably exceed 3800-3900 kDa, we decided to find out more about the differences in electrophoretic mobility of the observed bands.…”

Genome-wide linkage scan for exercise stroke volume and cardiac output in the HERITAGE Family Study

“…Titin is a structural protein in striated muscle cells and is a major determinant of elastic properties of muscle fibers. Consequently, titin seems to be a major contributor to the diastolic force of myocardium, and differential expression of titin isoforms has been suggested to contribute to the elastic diversity of atrial and ventricular myofibrils (9,13). Furthermore, a mutation in the titin gene has been proposed to contribute to some forms of familial hypertrophic cardiomyopathies (29).…”