Species variations in cDNA sequence and exon splicing patterns in the extensible I-band region of cardiac titin: relation to passive tension

Greaser, Marion L.; Berri, Mustapha; Warren, Chad M.; Mozdziak, Paul E.

doi:10.1007/978-94-010-0147-2_8

Cited by 11 publications

(25 citation statements)

References 33 publications

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“…Our previous studies have shown that the alternatively splicing patterns in the PEVK region of the N2BA isoforms were extremely heterogeneous [12,25,27]. Ten separate clones from a single PCR reaction using human cDNA were found to have different alternatively spliced patterns [25].…”

Section: Discussionmentioning

confidence: 95%

“…Previous studies with human and rat titin cDNA have outlined the splicing patterns for several of the major isoforms [8][9][10]12,25,27]. Most of the titin splicing occurs in the PEVK region of the I band and in exons coding for a series of Ig domains that are on the Z line side of the PEVK (the so called middle Ig region; see Figure 1 [27]). The largest isoform described to date is that from human soleus with a size of approximately 3.7 MDa [8].…”

Section: Discussionmentioning

confidence: 99%

“…Samples were obtained from wild type (Wt), heterozygote (Ht), and homozygote (Hm) mutant animals at ages 1, 12, 88, and 500 days. Primers (Supplement Table 1) were designed from nucleotide sequences previously determined for rat cardiac N2BA and rat genomic GenBank Accession Number AF525411 [27] and AC098426.4 respectively. Products were obtained with primers from exon 109 to 225 (includes the full PEVK region) or exon 115 to 225.…”

Section: Cdna Cloning and Sequencingmentioning

confidence: 99%

“…Rat titin exons were numbered using the homologous numbers from the human sequence. Further details of the methods for RNA extraction, cDNA synthesis, RT-PCR, and sequence analysis have been described in prior publications [26][27].…”

Section: Cdna Cloning and Sequencingmentioning

confidence: 99%

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

Greaser

Warren

Esbona

et al. 2008

Journal of Molecular and Cellular Cardiology

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102

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Titin is a very large alternatively spliced protein that performs multiple functions in heart and skeletal muscle. A rat strain is described with an autosomal dominant mutation that alters the isoform expression of titin. While wild type animals go through a developmental program where the 3.0 MDalton N2B becomes the major isoform expressed by two to three weeks after birth (~85%), the appearance of the N2B is markedly delayed in heterozygotes and never reaches more than 50% of the titin in the adult. Homozygote mutants express a giant titin of the N2BA isoform type (3.9 MDalton) that persists as the primary titin species through ages of more than one and half years. The mutation does not affect the isoform switching of troponin T, a protein that also is alternatively spliced with developmental changes. The basis for the apparently greater size of the giant titin in homozygous mutants was not determined, but additional length was not due to inclusion of sequence from larger numbers of PEVK exons or the Novex III exon. Passive tension measurements using isolated cardiomyocytes from homozygous mutants showed that cells could be stretched to sarcomere lengths greater than 4 µm without breakage. This novel rat model should be useful for exploring the potential role of titin in the Frank-Starling relationship and mechano-sensing/signaling mechanisms.

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Section: Discussionmentioning

confidence: 95%

Section: Discussionmentioning

confidence: 99%

Section: Cdna Cloning and Sequencingmentioning

confidence: 99%

Section: Cdna Cloning and Sequencingmentioning

confidence: 99%

See 2 more Smart Citations

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

Greaser

Warren

Esbona

et al. 2008

Journal of Molecular and Cellular Cardiology

Self Cite

102

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“…We further demonstrated that S469 can also be phosphorylated by PKA. As the length and amino acid composition of the N2-Bus differ among mammalian species, 36 it remains to be seen whether PKG (and PKA) phosphorylates the N2-Bus at the same and/or different sites in other species. By screening the N2-Bus sequence of rat cardiac titin, 36 we found a sequence motif (QKTS) at position 556 to 559 of the N2-Bus that is very similar to the sequence motif (AKTS) at residues 466 to 469 of the human N2-Bus.…”

Section: Discussionmentioning

confidence: 99%

Protein Kinase G Modulates Human Myocardial Passive Stiffness by Phosphorylation of the Titin Springs

Krüger

Kötter

Grützner

et al. 2009

Circulation Research

352

273

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Abstract-The sarcomeric titin springs influence myocardial distensibility and passive stiffness. Titin isoform composition and protein kinase (PK)A-dependent titin phosphorylation are variables contributing to diastolic heart function. However, diastolic tone, relaxation speed, and left ventricular extensibility are also altered by PKG activation. We used back-phosphorylation assays to determine whether PKG can phosphorylate titin and affect titin-based stiffness in skinned myofibers and isolated myofibrils. PKG in the presence of 8-pCPT-cGMP (cGMP) phosphorylated the 2 main cardiac titin isoforms, N2BA and N2B, in human and canine left ventricles. In human myofibers/myofibrils dephosphorylated before mechanical analysis, passive stiffness dropped 10% to 20% on application of cGMP-PKG. Autoradiography and anti-phosphoserine blotting of recombinant human I-band titin domains established that PKG phosphorylates the N2-B and N2-A domains of titin. Using site-directed mutagenesis, serine residue S469 near the COOH terminus of the cardiac N2-B-unique sequence (N2-Bus) was identified as a PKG and PKA phosphorylation site. To address the mechanism of the PKG effect on titin stiffness, single-molecule atomic force microscopy force-extension experiments were performed on engineered N2-Bus-containing constructs. The presence of cGMP-PKG increased the bending rigidity of the N2-Bus to a degree that explained the overall PKG-mediated decrease in cardiomyofibrillar stiffness. Thus, the mechanically relevant site of PKG-induced titin phosphorylation is most likely in the N2-Bus; phosphorylation of other titin sites could affect protein-protein interactions. The results suggest that reducing titin stiffness by PKG-dependent phosphorylation of the N2-Bus can benefit diastolic function. Failing human hearts revealed a deficit for basal titin phosphorylation compared to donor hearts, which may contribute to diastolic dysfunction in heart failure. Key Words: cGMP Ⅲ nitric oxide Ⅲ diastolic function Ⅲ connectin Ⅲ passive tension M yocardial and chamber diastolic function are influenced by chamber geometry, hypertrophy, the extracellular matrix and the sarcomeric titin springs. Titins are giant proteins which exist in the heart in 2 main isoforms coexpressed in sarcomeres: a shorter, stiffer N2B-titin (3.0 MDa) and longer, more compliant N2BA isoforms (3.2 to 3.7 MDa). Differential expression of these isoforms is related to alternate gene splicing affecting the functionally elastic titin region, which is confined to the sarcomeric I-band. 1 The springy titin segment comprises regions of serially linked immunoglobulin-like (Ig) domains separated by a cardiacspecific N2-B domain and a so-called PEVK segment (rich in proline, glutamate, valine, and lysine residues). The N2BA isoforms additionally have an N2-A domain and contain more Ig domains and PEVK-rich modules compared to the N2B isoform.Differential expression of titin isoforms determines passive stiffness of the sarcomere. 2,3 A low ratio of N2BA:N2B isoforms is found in sarcome...

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Nebulin isoforms of extraocular muscle

Moncman

Andrade

2006

Cell Tissue Res

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The extraocular muscles (EOMs), which are responsible for reflexive and voluntary eye movements, have many unique biochemical, physiological, and ultrastructural features that set them apart from other skeletal muscles. For example, rodent EOMs lack M-lines and express EOM-specific myosin heavy chain (MYH13) and alpha-cardiac myosin heavy chain. Recent gene-expression profiling studies indicate the presence of other cardiac-specific proteins in adult EOMs. This interesting mixture of myofibrillar and cytoskeletal proteins poses the questions as to whether nebulette, as opposed to nebulin, might be expressed in EOM, and what isoforms of titin are expressed in the EOM. We have performed gel electrophoresis and immunological analyses to determine the titin and nebulin isoforms expressed in the EOM. We have found that the mass of the titin isoforms expressed in the EOM most closely resemble those found in the skeletal muscles tested, viz., the soleus and extensor digitorum longus (EDL). We also demonstrate that, although the EOM expresses cardiac isoforms of myosin, it does not express nebulette and contains a nebulin isoform with a mass consistent with that found in the prototypical fast hindlimb muscle EDL.

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Species variations in cDNA sequence and exon splicing patterns in the extensible I-band region of cardiac titin: relation to passive tension

Cited by 11 publications

References 33 publications

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

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

Protein Kinase G Modulates Human Myocardial Passive Stiffness by Phosphorylation of the Titin Springs

Nebulin isoforms of extraocular muscle

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