Research ArticleHEART DISEASE

Deregulated Ca2+ cycling underlies the development of arrhythmia and heart disease due to mutant obscurin

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Science Advances  07 Jun 2017:
Vol. 3, no. 6, e1603081
DOI: 10.1126/sciadv.1603081
  • Fig. 1 Examination of the hearts of 1-year-old homozygous female knock-in mice under sedentary conditions.

    (A) Immunoblotting using antibodies to the NH2 and COOH termini of obscurins and protein lysates prepared from left ventricles of wild-type (WT) and knock-in (KI) hearts indicated similar expression levels of wild-type and mutant obscurins. Please note that obscurin A is the major giant isoform expressed in the heart, which is shown in our blots, whereas obscurin B is present in low amounts and is only detectable after longer exposure. The expression levels of obscurin A were quantified and found to be unaltered; n = 3 animals per group, two to three replica blots per heart; t test, P = 0.629; error bar represents SEM; heat shock protein 90 (HSP90)α/β served as loading and normalization control. (B and B′) Immunofluorescence combined with confocal optics and antibodies to the COOH terminus demonstrated that mutant obscurins containing the R4344Q substitution are incorporated normally into sarcomeric M-bands in knock-in left ventricles; obscurins are shown in red, and α-actinin, which is a Z-disk marker, is shown in green; scale bar, 5 μm. (C and C′) Gross morphology of wild-type and knock-in hearts showed that they have comparable sizes; scale bar, 3 mm. (D and D′) Transmission electron micrographs of wild-type and knock-in left ventricles revealed no major structural alterations; scale bar, 500 nm. (E and E′) Masson’s trichrome staining of wild-type and knock-in left ventricle sections illustrating the presence of peripheral fibrosis (arrow) in the latter; scale bar, 100 μm. (F) A trend of increased hydroxyproline content was found in the apices of knock-in left ventricles compared with wild-type tissue, which, however, did not reach statistical significance (t test, P = 0.202; n = 4 animals per group, three replica assays per heart).

  • Fig. 2 Evaluation of the expression levels, phosphorylation status, and subcellular distribution of Ca2+-cycling proteins in 1-year-old homozygous female knock-in hearts.

    (A) Representative immunoblots and relative quantification of the expression levels of major Ca2+-handling proteins showed that the amounts of SERCA2 are significantly increased (~10%), whereas the amounts of pentameric (but not monomeric) PLN are significantly reduced (~15%) in knock-in compared to wild-type hearts; t test, *P < 0.05; error bars represent SEM; n = 3 animals per group, two replica blots per heart. HSP90α/β served as loading and normalization control. (B to G′) Immunolabeling of freshly isolated cardiomyocytes from wild-type and knock-in hearts with antibodies to Ca2+-cycling proteins did not show any apparent changes in their localization; scale bar, 5 μm; note that all images from wild-type and knock-in animals were obtained using the same laser power and microscope settings. (H) Representative immunoblots and relative quantification of the phosphorylation levels of select Ca2+-cycling proteins using antibodies to phospho-Ser2808 and phospho-Ser2814 of RyR2, phospho-Ser38 of SERCA2, and phospho-Ser16 of PLN did not reveal any differences between wild-type and knock-in hearts. Phosphorylation levels were normalized to total levels for each protein; t test, P > 0.05; error bars represent SEM; n = 3 animals per group, two replica blots per heart. As in (A), HSP90α/β served as loading and normalization control.

  • Fig. 3 Enhanced SERCA2 activity and increased contractility were observed in 1-year-old homozygous knock-in female hearts.

    (A) Representative tracing of electrically stimulated and caffeine-induced Ca2+ transients in isolated wild-type ventricular myocytes. The final electrically stimulated and caffeine-induced Ca2+ transients were fit to a single exponential decay to calculate systolic (CaT) and caffeine (Caff) decay rates, respectively. A.U., arbitrary units. (B and C) Average traces of Ca2+ transients (B) revealed increased Ca2+ release during systolic contractions (C) in isolated ventricular myocytes from 1-year-old knock-in mice. (B′ and C′) Normalization of Ca2+ transients (B′) showed a faster decay rate (C′) in knock-in cardiomyocytes. (C″ to D″) Knock-in cardiomyocytes exhibited increased Ca2+ release after caffeine-induced contractions compared to wild-type cells (C″), indicating that more Ca2+ is stored in the SR. However, fractional release of Ca2+ during systolic contraction (D) and Ca2+ decay rates in the presence of caffeine (D′) remained unaltered, suggesting no clear change in RyR2 sensitivity or NCX activity. Consistent with this, calculations showed that SERCA2 activity is significantly increased (~30%) in knock-in hearts (D″). (E to F′) Isolated knock-in cardiomyocytes exhibit enhanced contractility (E and F) and accelerated contractility kinetics compared to wild-type cells, as indicated by their averaged (E) and normalized (E′) tracings of contractions that showed faster peak contraction and relaxation velocities (F′); t test, ***P < 0.001; error bars represent SEM; n = 3 animals per group, 40 cardiomyocytes per group. SL, sarcomeric length.

  • Fig. 4 One-year-old knock-in animals develop ventricular arrhythmia under sedentary conditions.

    (A to A″) ECG tracings of 1-year-old wild-type (A) and homozygous obscurin knock-in (A′) female mice indicated that obscurin knock-in mice exhibit faster heart rate compared to wild-type mice (A″); t test, ***P < 0.001 (P = 0.00047); error bars represent SEM; wild-type, n = 4 animals; knock-in, n = 5 animals. bpm, beats per minute. (B) Knock-in but not wild-type mice exhibit frequent episodes of PVC during the 50-min monitoring period. (B′) Notably, one knock-in mouse also displayed incidents of bigeminy and trigeminy following a PVC episode. (C) An extreme case of a knock-in mouse developed spontaneous ventricular tachycardia that lasted throughout the 50-min monitoring period.

  • Fig. 5 Homozygous female knock-in animals exhibit enlarged hearts and extensive fibrosis and calcification 8 weeks after TAC surgery.

    Two-month-old wild-type and knock-in female mice were subjected to either sham or TAC surgery and evaluated 8 weeks later. (A) Representative immunoblots of protein lysates prepared from left ventricles of sham- or TAC-subjected wild-type and knock-in animals using antibodies to the NH2 and COOH termini of obscurins. Relative quantification revealed increased levels of obscurins in the knock-in–TAC hearts due to up-regulation of obscurin B (asterisk). HSP90α/β served as loading control; n = 3 animals per group, two to three replica blots per heart; t test, *P < 0.05, #P < 0.005; error bars represent SEM. (B to B‴) Immunofluorescence combined with confocal optics and antibodies to the COOH terminus of obscurins demonstrated that their localization is unaltered in knock-in left ventricles subjected to sham or TAC surgery; obscurins occupying M-bands are shown in red, and α-actinin, which is a Z-disk marker, is shown in green; scale bar, 5 μm. (C to C‴) Gross evaluation of wild-type and knock-in hearts 8 weeks after sham or TAC surgery indicated that the KI-TAC hearts are notably enlarged; scale bar, 2 mm. (D to D‴) H&E staining of cardiac sections from left ventricles revealed no obvious structural abnormalities in any group; scale bar, 50 μm. (E and E′) Masson’s trichrome staining of sections from left ventricles of wild-type–TAC and knock-in–TAC hearts revealed the presence of excessive interstitial fibrosis in the latter; scale bar, 50 μm. (F) Left ventricles from knock-in–TAC animals contained significantly increased amounts of hydroxyproline compared to left ventricles from wild-type–TAC animals; t test, *P < 0.01; error bars represent SEM; n = 3 animals per group, three repeats per heart. (G to G″) von Kossa staining of left ventricles demonstrated the presence of more prominent peripheral (G′) and interstitial (G″) dystrophic calcification in knock-in–TAC versus wild-type–TAC (G) hearts; scale bar, 25 μm; 40× objective.

  • Fig. 6 Mutant obscurin Ig58 exhibits distorted electrostatic topology and target binding characteristics.

    (A) An ensemble of the 20 best NMR structures of wild-type Ig58, colored by β strands A to G. (B) Overlay of the NMR and x-ray structure of wild-type Ig58. (C) Top: Chemical shift differences between mutant and wild-type Ig58 are mapped onto the wild-type Ig58 structure. Residues that exhibit greater than twofold and threefold the average NMR chemical shift change are colored yellow and red, respectively. The location of the R4344Q mutation is colored purple. Bottom: The residue-by-residue chemical shift differences between mutant and wild-type Ig58 HSQC spectra. (D) Electron density of the residues surrounding R4344 (in wild-type Ig58) involved in extensive charge-charge interactions. (E) Molecular dynamics plot of the side-chain N–O distances between surface residues of Ig58, showing long-lived electrostatic interactions. (F) The presence of the R4344Q mutation enhances the ability of Ig58 to interact with PLN. Equivalent amounts of wild-type and mutant glutathione S-transferase (GST)–tagged Ig58 as well as control GST-protein were bound to glutathione matrices (top) and incubated with protein homogenates prepared from adult cardiac muscle. Both wild-type and mutant GST-Ig58 but not control GST-protein efficiently adsorbed the monomeric form of native PLN. Remarkably, mutant GST-Ig58 carrying the R4344Q mutation did so more efficiently than wild-type protein (~10-fold higher). Moreover, neither wild-type nor mutant GST-Ig58 was able to retain other Ca2+-cycling proteins examined, including RyR2, SERCA2, Hax1, and sAnk1.5, further demonstrating the specific interaction between the wild-type and mutant Ig58 domain with PLN.

  • Fig. 7 The R4344Q substitution allows tighter binding of PLN to Ig58 of obscurins.

    YASARA modeling of the interaction between Ig58 and PLN suggests that the bulky side chain of R4344 hinders PLN docking onto the surface of Ig58 (left), whereas the smaller side chain of the R4344Q substitution allows more significant PLN-Ig58 electrostatic interactions to occur (right). The obscurin Ig58 domain is colored green, and the cytoplasmic domain of PLN is colored yellow, with the R4344Q mutation on Ig58 colored purple.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/3/6/e1603081/DC1

    fig. S1. Generation of obscurin knock-in mice carrying the R4344Q mutation in Ig58.

    fig. S2. Sarcomeric organization is unaltered in 1-year-old homozygous knock-in female mice.

    table S1. Morphometric and echocardiographic analyses of sedentary wild-type and homozygous knock-in hearts.

    table S2. Morphometric and echocardiographic analyses of sham- and TAC-subjected wild-type and knock-in hearts.

    table S3. NMR-derived restraints and statistics of 20 NMR structures of wild-type Ig58.

    table S4. Statistics of wild-type Ig58 crystal diffraction.

    table S5. List of primers used for confirmation of gene targeting, animal genotyping, and site-directed mutagenesis.

  • Supplementary Materials

    This PDF file includes:

    • fig. S1. Generation of obscurin knock-in mice carrying the R4344Q mutation in Ig58.
    • fig. S2. Sarcomeric organization is unaltered in 1-year-old homozygous knock-in female mice.
    • table S1. Morphometric and echocardiographic analyses of sedentary wild-type and homozygous knock-in hearts.
    • table S2. Morphometric and echocardiographic analyses of sham- and TAC-subjected wild-type and knock-in hearts.
    • table S3. NMR-derived restraints and statistics of 20 NMR structures of wild-type Ig58.
    • table S4. Statistics of wild-type Ig58 crystal diffraction.
    • table S5. List of primers used for confirmation of gene targeting, animal genotyping, and site-directed mutagenesis.

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