Rationale: The giant protein titin plays key roles in myofilament assembly and determines the passive mechanical properties of the sarcomere. The cardiac titin molecule has 2 mayor elastic elements, the N2B and the PEVK region. Both have been suggested to determine the elastic properties of the heart with loss of function data only available for the N2B region. Objective: The purpose of this study was to investigate the contribution of titin's proline-glutamate-valine-lysine (PEVK) region to biomechanics and growth of the heart. Methods and Results: We removed a portion of the PEVK segment (exons 219 to 225; 282 aa) that corresponds to the PEVK element of N2B titin, the main cardiac titin isoform. Adult homozygous PEVK knockout (KO) mice developed diastolic dysfunction, as determined by pressure-volume loops, echocardiography, isolated heart experiments, and muscle mechanics. Immunoelectron microscopy revealed increased strain of the N2B element, a spring region retained in the PEVK-KO. Interestingly, the PEVK-KO mice had hypertrophied hearts with an induction of the hypertrophy and fetal gene response that includes upregulation of FHL proteins. This contrasts the cardiac atrophy phenotype with decreased FHL2 levels that result from the deletion of the N2B element. Key Words: diastole Ⅲ connectin Ⅲ hypertrophy Ⅲ compliance Ⅲ FHL T itin is the largest protein in mammals and forms a continuous elastic filament along the myofibril (reviewed in 1 ). Because of its enormous size, titin is a prominent target for mutations that give rise to diseases such as familial dilated cardiomyopathy and muscular dystrophy. 2,3 Titin's extensible region resides in the I-band of the sarcomere and consists of immunoglobulin (Ig)-like domains arranged in tandem, the heart specific N2B element, and the prolineglutamate-valine-lysine (PEVK) element. 4 The PEVK element is thought to function as a largely unfolded polypeptide that extends at low force levels and that thereby provides an important source of elasticity at physiological sarcomere lengths. [5][6][7] Unlike the 1-exon heart specific N2B element, the titin gene contains 112 PEVK exons that are differentially expressed between muscle types. 8 Of these PEVK exons, 219 to 225 are expressed in the so-called N2B titin isoform, that constitutes the dominant cardiac isoform in the left ventricle of a wide range of species, including rodents and human. 9 Here we generated a mouse deficient in titin's exons 219 to 225 that results in a deletion of the c-terminal PEVK region (282 aa) and determined its role in cardiac function using echocardiography, in vivo pressure-volume loops, isolated heart physiology, muscle mechanics, immunoelectron microscopy, and expression analysis. We investigated the hypertrophy phenotype and studied members of the four-and-a-half LIM family involved in atrophy/hypertrophy signaling-FHL1 and FHL2. 10,11 Our results reveal the strong effect of the PEVK element on diastolic function but also that the role of the PEVK extends beyond that of a mechanical spr...

We investigated the contractile phenotype of skeletal muscle deficient in the titin’s M-band exons MEx1 and MEx2 (KO) by using the cre-lox recombination system and a multidisciplinary physiological approach to study skeletal muscle contractile performance. At a maximal tetanic stimulation frequency, intact KO EDL muscle was able to produce wildtype levels of force. However, at submaximal stimulation frequency force was reduced in KO mice giving rise to a rightward shift of the force-frequency curve. This rightward shift of the force-frequency curve could not be explained by altered sarcoplasmic reticulum Ca2+ handling as indicated by analysis of Ca2+ transients in intact myofibers and expression of Ca2+ handling proteins, but can be explained by the reduced myofilament Ca2+ sensitivity of force generation that we found. Western blotting experiments suggested that the excision of titin exons MEx1 and MEx2 did not result in major changes in expression of titin M-band binding proteins or phosphorylation level of the thin filament regulatory proteins, but rather in a shift towards expression of slow isoforms of the thick filament-associated protein myosin binding protein-C (MyBP-C). Extraction of MyBP-C from skinned muscle normalized myofilament Ca2+ sensitivity of KO EDL muscle. Thus, our data suggest that the M-band region of titin affects the expression of genes involved in the regulation of skeletal muscle contraction.

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