Cardiac myosin binding protein‐C (cardiac MyBP‐C, cardiac C protein) belongs to a family of proteins implicated in both regulatory and structural functions of striated muscle. For the cardiac isoform, regulatory phosphorylation in vivo by cAMP‐dependent protein kinase (PKA) upon adrenergic stimulation is linked to modulation of cardiac contraction. The sequence of human cardiac MyBP‐C now reveals regulatory motifs specific for this isoform. Site‐directed mutagenesis identifies a LAGGGRRIS loop in the N‐terminal region of cardiac MyBP‐C as the key substrate site for phosphorylation by both PKA and a calmodulin‐dependent protein kinase associated with the native protein. Phosphorylation of two further sites by PKA is induced by phosphorylation of this isoform‐specific site. This phosphorylation switch can be mimicked by aspartic acid instead of phosphoserine. Cardiac MyBP‐C is therefore specifically equipped with sensors for adrenergic regulation of cardiac contraction, possibly implicating cardiac MyBP‐C in cardiac disease. The gene coding for cardiac MyBP‐C has been assigned to the chromosomal location 11p11.2 in humans, and is therefore in a region of physical linkage to subsets of familial hypertrophic cardiomyopathy (FHC). This makes cardiac MyBP‐C a candidate gene for chromosome 11‐associated FHC.

Titins are megadalton-sized filamentous polypeptides of vertebrate striated muscle. The I-band region of titin underlies the myofibrillar passive tension response to stretch. Here, we show how titins with highly diverse I-band structures and elastic properties are expressed from a single gene. The differentially expressed tandem-Ig, PEVK, and N2B spring elements of titin are coded by 158 exons, which are contained within a 106-kb genomic segment and are all subject to tissue-specific skipping events. In ventricular heart muscle, exons 101 kb apart are joined, leading to the exclusion of 155 exons and the expression of a 2.97-MDa cardiac titin N2B isoform. The atria of mammalian hearts also express larger titins by the exclusion of 90 to 100 exons (cardiac N2BA titin with 3.3 MDa). In the soleus and psoas skeletal muscles, different exon-skipping pathways produce titin transcripts that code for 3.7- and 3.35-MDa titin isoforms, respectively. Mechanical and structural studies indicate that the exon-skipping pathways modulate the fractional extensions of the tandem Ig and PEVK segments, thereby influencing myofibrillar elasticity. Within the mammalian heart, expression of different levels of N2B and N2BA titins likely contributes to the elastic diversity of atrial and ventricular myofibrils.

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),...