Analysis of obscurin protein expression in tissue samples.

<p>Left: Identification of obscurin in cardiac myofibrils by 2% agarose/2% PAA/SDS gel electrophoresis. Left, SYPRO Ruby protein stain, Right Western blot Ob59 antibody. Centre: Obscurin expression in myofibrils. Western blot of 4–18% gradient SDS-PAGE. Left: membrane stained with MemCode protein stain, right: probed with Ob59 antibody. For quantification obscurin was normalised to α-actinin. Right: Identification of obscurin in whole heart muscle homogenates. Top, MemCode protein stain; bottom, Western blot with Ob59 antibody.</p

DataSheet1.PDF

<p>The inherited cardiomyopathies, hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM) are relatively common, potentially life-threatening and currently untreatable. Mutations are often in the contractile proteins of cardiac muscle and cause abnormal Ca²⁺ regulation via troponin. HCM is usually linked to higher myofilament Ca²⁺-sensitivity whilst in both HCM and DCM mutant tissue there is often an uncoupling of the relationship between troponin I (TnI) phosphorylation by PKA and modulation of myofilament Ca²⁺-sensitivity, essential for normal responses to adrenaline. The adrenergic response is blunted, and this may predispose the heart to failure under stress. At present there are no compounds or interventions that can prevent or treat sarcomere cardiomyopathies. There is a need for novel therapies that act at a more fundamental level to affect the disease process. We demonstrated that epigallocatechin-3 gallate (EGCG) was found to be capable of restoring the coupled relationship between Ca²⁺-sensitivity and TnI phosphorylation in mutant thin filaments to normal in vitro, independent of the mutation (15 mutations tested). We have labeled this property “re-coupling.” The action of EGCG in vitro to reverse the abnormality caused by myopathic mutations would appear to be an ideal pharmaceutical profile for treatment of inherited HCM and DCM but EGCG is known to be promiscuous in vivo and is thus unsuitable as a therapeutic drug. We therefore investigated whether other structurally related compounds can re-couple myofilaments without these off-target effects. We used the quantitative in vitro motility assay to screen 40 compounds, related to C-terminal Hsp90 inhibitors, and found 23 that can re-couple mutant myofilaments. There is no correlation between re-couplers and Hsp90 inhibitors. The Ca²⁺-sensitivity shift due to TnI phosphorylation was restored to 2.2 ± 0.01-fold (n = 19) compared to 2.0 ± 0.24-fold (n = 7) in wild-type thin filaments. Many of these compounds were either pure re-couplers or pure desensitizers, indicating these properties are independent; moreover, re-coupling ability could be lost with small changes of compound structure, indicating the possibility of specificity. Small molecules that can re-couple may have therapeutic potential.</p><p>HIGHLIGHTS</p><p>- Inherited cardiomyopathies are common diseases that are currently untreatable at a fundamental level and therefore finding a small molecule treatment is highly desirable.</p><p>- We have identified a molecular level dysfunction common to nearly all mutations: uncoupling of the relationship between troponin I phosphorylation and modulation of myofilament Ca²⁺-sensitivity, essential for normal responses to adrenaline.</p><p>- We have identified a new class of drugs that are capable of both reducing Ca²⁺-sensitivity and/or recouping the relationship between troponin I phosphorylation and Ca²⁺-sensitivity.</p><p>- The re-coupling phenomenon can be explained on the basis of a single mechanism that is testable.</p><p>- Measurements with a wide range of small molecules of varying structures can indicate the critical molecular features required for recoupling and allows the prediction of other potential re-couplers.</p><p></p

Quantification of Obscurin content of human cardiac myofibrils.

<p>A. 4–18% SDS-PAGE of muscle myofibril fraction. Western blot reversibly stained for total protein by MemCode and then probed with Obscurin antibody Ob59. Obscurin band is normalized to the α-actinin band as a loading control and further normalized to D21, present in every slab gel to control for inter-gel variation. B. Relative obscurin protein level normalised to D21. D4, D14, and D20 are compared with 7 DCM samples without obscurin mutations, 6 donor heart samples and 3 myectomy samples (described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138568#pone.0138568.s001" target="_blank">S1 File</a> Figs A, B). Data from 22 slab gels similar to that in 2A pooled. The bars represent sem for replicate measurements on the same sample.</p

Clinical and genetic data of the 4 heart samples with potentially disease-causing <i>OBSCN</i> mutations.

<p>The locations of the mutations in the Obscurin B domain diagram are shown, based on the model of Ackerman et al. 2014 (ref 14) (Fig E in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138568#pone.0138568.s001" target="_blank">S1 File</a>)</p

Immunofluorescence images of donor and <i>OBSCN</i> mutation- containing human heart tissue sections labelled with Ob59 antibody (green) and anti- α-actinin or anti-myomesin (red).

<p>The monochrome panels show the obscurin immunofluorescence on its own.</p