Expression of Normally Repressed Myosin Heavy Chain 7b in the Mammalian Heart Induces Dilated Cardiomyopathy

In mammals, muscle contraction is controlled by a family of 10 sarcomeric myosin motors. The expression of one of its members, MYH7b, is regulated by alternative splicing, and while the protein is restricted to specialized muscles such as extraocular muscles or muscle spindles, RNA that cannot encode protein is expressed in most skeletal muscles and in the heart. Remarkably, birds and snakes express MYH7b protein in both heart and skeletal muscles. This observation suggests that in the mammalian heart, the motor activity of MYH7b may only be needed during development since its expression is prevented in adult tissue, possibly because it could promote disease by unbalancing myocardial contractility. Methods and Results We have analyzed MYH7b null mice to determine the potential role of MYH7b during cardiac development and also generated transgenic mice with cardiac myocyte expression of MYH7b protein to measure its impact on cardiomyocyte function and contractility. We found that MYH7b null mice are born at expected Mendelian ratios and do not have a baseline cardiac phenotype as adults. In contrast, transgenic cardiac MYH7b protein expression induced early cardiac dilation in males with significantly increased left ventricular mass in both sexes. Cardiac dilation is progressive, leading to early cardiac dysfunction in males, but later dysfunction in females. Conclusions The data presented show that the expression of MYH7b protein in the mammalian heart has been inhibited during the evolution of mammals most likely to prevent the development of a severe cardiomyopathy that is sexually dimorphic.</p

Distinct effects of two hearing loss-associated mutations in the sarcomeric myosin MYH7b

For decades, sarcomeric myosin heavy chain proteins were assumed to be restricted to striated muscle where they function as molecular motors that contract muscle. However, MYH7b, an evolutionarily ancient member of this myosin family, has been detected in mammalian nonmuscle tissues, and mutations in MYH7b are linked to hereditary hearing loss in compound heterozygous patients. These mutations are the first associated with hearing loss rather than a muscle pathology, and because there are no homologous mutations in other myosin isoforms, their functional effects were unknown. We generated recombinant human MYH7b harboring the D515N or R1651Q hearing loss-associated mutation and studied their effects on motor activity and structural and assembly properties, respectively. The D515N mutation had no effect on steady-state actin-activated ATPase rate or load-dependent detachment kinetics, but increased actin sliding velocity due to an increased displacement during the myosin working stroke. Furthermore, we found that the D515N mutation caused an increase in the proportion of myosin heads that occupy the disordered-relaxed state, meaning more myosin heads are available to interact with actin. Although we found no impact of the R1651Q mutation on myosin rod secondary structure or solubility, we observed a striking aggregation phenotype when this mutation was introduced into nonmuscle cells. Our results suggest that each mutation independently affects MYH7b function and structure. Together, these results provide the foundation for further study of a role for MYH7b outside of the sarcomere

Interplay between Exonic Splicing Enhancers, mRNA Processing, and mRNA Surveillance in the Dystrophic Mdx Mouse

BACKGROUND: Pre-mRNA splicing, the removal of introns from RNA, takes place within the spliceosome, a macromolecular complex composed of five small nuclear RNAs and a large number of associated proteins. Spliceosome assembly is modulated by the 5′ and 3′ splice site consensus sequences situated at the ends of each intron, as well as by exonic and intronic splicing enhancers/silencers recognized by SR and hnRNP proteins. Nonsense mutations introducing a premature termination codon (PTC) often result in the activation of cellular quality control systems that reduce mRNA levels or alter the mRNA splicing pattern. The mdx mouse, a commonly used genetic model for Duchenne muscular dystrophy (DMD), lacks dystrophin by virtue of a premature termination codon (PTC) in exon 23 that also severely reduces the level of dystrophin mRNA. However, the effect of the mutation on dystrophin RNA processing has not yet been described. METHODOLOGY/PRINCIPAL FINDING: Using combinations of different biochemical and cellular assays, we found that the mdx mutation partially disrupts a multisite exonic splicing enhancer (ESE) that is recognized by a 40 kDa SR protein. In spite of the presence of an inefficient intron 22 3′ splice site containing the rare GAG triplet, the mdx mutation does not activate nonsense-associated altered splicing (NAS), but induces exclusively nonsense-mediated mRNA decay (NMD). Functional binding sites for SR proteins were also identified in exon 22 and 24, and in vitro experiments show that SR proteins can mediate direct association between exon 22, 23, and 24. CONCLUSIONS/SIGNIFICANCE: Our findings highlight the complex crosstalk between trans-acting factors, cis-elements and the RNA surveillance machinery occurring during dystrophin mRNA processing. Moreover, they suggest that dystrophin exon–exon interactions could play an important role in preventing mdx exon 23 skipping, as well as in facilitating the pairing of committed splice sites

SR proteins bind exon 23 enhancer elements.

<p>³²P labeled D90 RNA was incubated at 30°C for 10 min with purified SR proteins prepared from calf thymus as previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000427#pone.0000427-Zahler1" target="_blank">[63]</a>. Binding reactions performed in the absence or in the presence of increasing concentrations of cold competitor RNAs (with molar excess of 30, 60, 90 and 120 fold, as indicated by the gradients above the gel lanes) were fractionated on a 4% non-denaturing polyacrylamide gel. The schematic representation of the RNAs is depicted as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000427#pone-0000427-g001" target="_blank">Figure 1</a>. The positions of free RNA and bound complexes are shown. Comparable results were obtained in two independent experiments.</p

Exon 23 contains purine-rich elements that activate splicing of a heterologous substrate containing a weak 3′ splice site.

<p>The sequence of the first 90 nucleotides of dystrophin exon 23 is shown at the top of the figure. Arrows identify the natural mutation (C→T) occurring in the <i>mdx</i> mouse and the mutations that disrupt the purine-rich region R1. The borders of the M1 element are defined by the homology (12 of 12 positions) shared with the human dystrophin exon 23. Different regions of dystrohin exon 23 were inserted in the second exon of Dpy2, an enhancer-dependent mRNA containing a weak pyrimidine stretch <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000427#pone.0000427-Dye1" target="_blank">[60]</a>. The structure of the chimeric pre-mRNA substrates subjected to <i>in vitro</i> splicing reactions is shown at the bottom right of the figure. White boxes indicate Dpy2 exons, black boxes indicate different regions of the dystrophin exon 23, dashed boxes indicate portions of exon 23 not included in the substrates, white vertical lines indicate the <i>mdx</i> and R1 mutations. The gray box in Dpy2E identifies the two splicing enhancers found in the α-tropomyosin exon 2 gene <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000427#pone.0000427-Dye1" target="_blank">[60]</a>. The structures and the mobility of the products and intermediates of splicing are shown on the sides of the gel. D172-212Δ RNA was used as a positive control for D1-40Δ to rule out any exon size inhibitory effect on splicing. Asterisks indicate the position of the spliced RNAs. Splicing reactions, carried out for 70 min at 30°C, were resolved on a 12% denaturing polyacrylamide gel. Comparable results were obtained in three independent experiments.</p

UV cross-linking of a 40 kDa SR protein to exon 23.

<p>A) Different ³²P labeled RNAs represented as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000427#pone-0000427-g001" target="_blank">Figure 1</a> were incubated at 30°C for 10 min with purified SR proteins. Following UV cross-linking and RNAse A/T1 digestion, ³²P labeled proteins were analyzed by SDS-PAGE. Molecular weight markers are shown on the left. B) Cross-linking of ³²P labeled D90 RNA was performed in the presence of increasing amounts of cold competitor RNAs (10 and 30 fold molar excess, as indicated by the gradients above the gel lanes). C) Coomassie–stained SDS-PAGE separated SR proteins (7 µg). Comparable results were obtained in three independent experiments.</p

SR proteins interact with dystrophin exon 22 and 24.

<p>A) Different SR proteins associate with exons 22 and 24. ³²P labeled exon 22 and 24 RNAs, incubated with SR proteins were subjected to UV-crosslinking as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000427#pone-0000427-g003" target="_blank">Figure 3</a>. Molecular weight markers are shown at the left; exon 22A and 24A correspond to the antisense RNAs used as controls. B) Exon 24, but not exon 22 activates splicing of the enhancer-dependent Dpy2 RNA. Different regions of dystrohin exon 22 (145 nt) and 24 (113 nt) were cloned in the second exon of the enhancer-dependent Dpy2 substrate. The structure of the chimeric constructs subjected to <i>in vitro</i> splicing reactions is shown on the right of the panel, with black boxes indicating exon 22 and 24 and dashed boxes the portion of the corresponding exon not included in the pre-mRNA. A indicates the anti-sense orientation of each region, while numbers after the slash indicate their nucleotide length. Dpy2 and Dpy2E are defined as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000427#pone-0000427-g001" target="_blank">Figure 1</a>. The structures and the mobility of the products and intermediates of splicing are shown on both sides of the panel. Asterisks indicate the position of the spliced RNAs. The molecular weigh of the band detected below the D24/60 RNA precursor does not correspond to the expected splicing product. <i>In vitro</i> splicing reactions were carried out as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000427#pone-0000427-g001" target="_blank">Figure 1</a>. Comparable results were obtained in two independent experiments. C) Schematic representation of SR binding sites within dystrophin exon 22 and 24 computed by ESEfinder <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000427#pone.0000427-Cartegni3" target="_blank">[61]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000427#pone.0000427-Smith3" target="_blank">[62]</a>.</p

Splicing of dystrophin mini-genes <i>in vivo.</i>

<p>Cos 7 cells were transfected with plasmids expressing different portions of the genomic region of the dystrophin gene spanning from exon 22 to exon 23/24. Northern blot analysis was carried out on total RNA prepared 24 hr after transfection, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000427#s4" target="_blank">Materials and Methods</a>. Band intensities, quantified by phosphorimager analysis, were normalized for transfection efficiency and RNA recovery to the level of the co-expressed neomycin mRNA (neo). Normalized values were expressed as a percentage of wt mRNAs that were defined as 100. The schematic structure of the substrates is shown on the right part of both panels A and B. Dystrophin exons are identified as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000427#pone-0000427-g001" target="_blank">Figure 1</a>. β-globin exons are shown as gray boxes while the 5′ and 3′ UTRs as white thinner boxes. (A) Two exon mini-genes carrying dystrophin exons 22 and 23. (B) Three exon mini-genes carrying dystrophin exon 22 and 23 and 24, chimeric constructs, and the β-globin system (β−globin wt and β−globin 39 carrying a nonsense mutation at codon 39) used as a reference <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000427#pone.0000427-LykkeAndersen1" target="_blank">[81]</a>. Comparable results were obtained in three independent experiments. (C) Left panel: autoradiogram of a representative quantitative RT-PCR performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000427#s4" target="_blank">Materials and Methods</a>. The upper bands correspond to RT-PCR products from dystrophin mRNA obtained with primers located in exons 22 and 24; the lower bands correspond to the neomycin gene used as an internal control. −and+lanes correspond to reactions carried out in absence or presence of reverse transcriptase. Right panel: non-quantitative RT-PCR performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000427#s4" target="_blank">Materials and Methods</a>. Mobility of the predicted splicing products is shown on the right. The PCR product deriving from skipping of exon 23 was not detected.</p