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

Abstract

Titin is a very large alternatively spliced protein that performs multiple functions in heart and skeletal muscles. 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 MDa N2B becomes the major isoform expressed by two to three weeks after birth (approximately 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 MDa) that persists as the primary titin species through ages of more than one and a half years. The mutation does not affect the isoform switching of troponin T, a protein that is also alternatively spliced with developmental changes. The basis for the apparently greater size of the giant titin in homozygous mutants was not determined, but the 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 mum 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.

Figure 1

Pedigree for the rat mutation that alters titin alternative splicing.

Figure 2

SDS agarose gels of rat ventricle. A. Samples from a one day old litter of rat pups. Wt – wild type; Ht – heterozygotes. Wild type animals have three major titin bands in addition to the T2, a titin proteolytic degradation band. Heterozygotes have primarily a single band equivalent to or slightly larger than the large neonatal titin (N2BA-N1). RS – rat soleus; DV – dog ventricle. B. Age comparisons of wild type and heterozygotes. All the samples were run on the same gel except the one year wild type. 16F – sixteen day fetal. C. Off spring (12 days of age) from a cross of two heterozygotes. Three different phenotypes were present – the wild type and heterozygote patterns as in (A) and a new larger sized, slower migrating band that came from potential homozygotes (Hm). Standards contained human soleus (Hu Sol – 3.7 MDa) and adult rat soleus (primarily N2B – 3.0 MDa). D. Developmental patterns of titin isoforms in homozygotes from 1 to 590 days of age.

Figure 3

Sizes of different regions of the heart between wild type (Wt), heterozygotes (Ht), and homozygote mutants (Hm). The left ventricle weight includes both the septum and the free wall. Only the atrial sizes were significantly different between the homozygous mutants and the other genetic groups. Wt, N=6; Ht, N=7; Hm, N=6.

Figure 4

Western blots with an antibody against troponin T of wild type (Wt) and homozygote mutant (Hm) ventricle from animals 1 to 31 days of age. The upper fetal type is gradually replaced by the lower adult form. The transition time course is similar in both groups of rats.

Figure 5

Comparisons of amino acid length of the PEVK in wild type, heterozygotes, and homozygotes at various ages. Error bars depict standard deviations. Differences between mutation status groups are not statistically significant at 88 or 500 days of age.

Figure 6

Western blot with antibody against Novex III. Wild type and homozygote samples were prepared from animals at 10 day, 49 day, and adult (> 1 year) of age. Left – Western blot; right – silver stained companion gel with the same sample lanes and protein loads.

Figure 7

Resting tension comparisons between single enzymatically isolated cardiomyocytes from wild type and homozygote mutant ventricles. Note that homozygote mutant cells could be stretched to at least 4 µm without breakage.

Figure 8

Light micrograph patterns of wild type (left pair) and homozygous mutant (right pair). Myocytes. Cells were stretched by 5% above their passive length (upper) or by 25% (lower). While the sarcomere patterns become significantly obscured by stretch of 25% to a sarcomere length of 2.25 µm in wild type cells, sarcomere patterns were still prominent when stretched to 2.7 µm in the homozygote mutants.