GATA-4 and GATA-6 are similarly induced in the adult heart with pressure overload.

<p>A, Western blot analysis of GATA-4 and GATA-6 protein expression in the adult mouse heart at baseline or after pressure overload stimulation for the indicated times. A non-specific (n.s.) band from the GATA-6 western was used to show equal loading and running conditions. B, Relative luciferase units (RLU) from a plasmid containing the luciferase reporter fused to the b-type natriuretic peptide (BNP) promoter or a mutant promoter lacking functional GATA binding sites, which was co-transfected into neonatal rat cardiomyocytes with expression plasmids encoding GATA-4 (G4), GATA-6 (G6), or both together. *P<0.05 versus empty vector transfected control. N = 3. C, Heart weight normalized to body weight (HW/BW) of the indicated genotype of cardiac-specific transgenic mice expressing either GATA4, GATA6, or both together at 8 weeks of age.</p

Differentially regulated genes from microarray.

<p>Adult heart mRNA was collected from <i>Gata6^{fl/fl-βMHC-cre}</i> mice and compared against mRNA from <i>Gata6^fl/fl</i> to generate Affymetrix expression arrays of genes that were specifically changed in with deletion of <i>Gata6</i>. And identical protocol was used to analyze genes changed with deletion of <i>Gata4</i> from the heart. Afterwards the two arrays sets were cross compared to examine genes that might be more specific to GATA-4 or GATA-6 regulation, which is represented as the column “difference” showing normalized expression results. The values represent the difference between normalized GATA-4 expression over normalized GATA-6 expression, both of which are corrected to the common cre control and each respective floxed line control. The negative numbers represent downregulated genes. Gene names are shown in the left column and protein names are shown in the very next column.</p

qRT-PCR confirmation of differentially regulated genes.

<p>Adult heart mRNA was collected from the indicated lines of adult mice and subjected to qRT-PCR to analyze for differences in gene expression to verify or extend the Affymetrix results shown in Supplemental <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084591#pone-0084591-t001" target="_blank">Table 1</a>. Gene names are shown in the left column. Values are set relative to expression in <i>Gata6^fl/fl</i> or <i>Gata4^fl/fl</i> controls.</p

GATA-4 and GATA-6 play redundant roles in programming cardiac hypertrophy but not in adapting functional compensation.

<p>A, Ventricular weight to body weight (VW/BW) in the indicated genotypes of mice at 10–12 weeks of age following 2 weeks of TAC stimulation. The number of mice analyzed is shown in each bar of the graph. *P<0.05 versus <i>Gata4^fl/+ Gata6^fl/+</i>; #P<0.05 versus <i>Gata4^fl/+ Gata6^fl/+</i> with the βMHC-cre transgene. B, Fractional shortening (FS%) from the indicated mice at 10–12 weeks of age following sham or 2 weeks of TAC stimulation. The number of mice analyzed is shown in each bar of the graph. *P<0.05 versus <i>Gata4^fl/+ Gata6^fl/+</i> with the βMHC-cre transgene. C, VW/BW in the indicated groups of mice after 2 weeks of TAC stimulation at 10–12 weeks of age. The number of mice analyzed is shown in each bar of the graph. *P<0.05 versus <i>Gata6^fl/fl</i> tTA transgene; #P<0.05 versus <i>Gata6^{fl/fl-βMHC-cre}</i> tTA transgene. Abbreviations; tTA-G4, tetracycline transactivator transgene with the GATA-4 (G4) transgene; tTA-G6, tetracycline transactivator transgene with the GATA-6 (G6) transgene. D, Fractional shortening to measure ventricular performance of the same mice described in “C”. *P<0.05 versus <i>Gata6^fl/fl</i> tTA transgene; ^†P<0.05 versus <i>Gata6^{fl/fl-βMHC-cre}</i> tTA transgene; #P<0.05 versus <i>Gata6^{fl/fl-βMHC-cre}</i> tTA transgene or <i>Gata6^{fl/fl-βMHC-cre}</i> tTA transgene with GATA-4 transgene. E, F, Echocardiography measured left ventricular end diastolic dimension (LVED) and septal thickness in hearts of the indicated groups of mice after 2 weeks of TAC. G, Fractional shortening in <i>Gata4</i> heart-specific, deleted mice with the additional transgenes shown after 2 weeks of TAC stimulation. Number of mice analyzed is shown in the bars. *P<0.05 versus <i>Gata4^fl/fl</i> tTA transgene; #P<0.05 versus <i>Gata4^{fl/fl-βMHC-cre}</i> tTA transgene with the GATA-6 transgene. H, I, Western blot analysis for GATA-4 (G4) or GATA-6 (G6) protein from the hearts of the indicated mice. A non-specific (n.s.) band from each of the western blots was used to show equal loading and running conditions.</p

Cardiac-specific deletion of <i>Gata4</i> but not <i>Gata6</i> prevents compensatory angiogenesis in the hearts of mice.

<p>The graph shows immunohistological quantitation of capillaries in the left ventricle normalized to surrounding cardiomyocytes in the indicated groups of mice. TAC was performed for 2 weeks. Mice were 10–12 weeks of age at harvesting. Hearts from at least 4 mice were analyzed in each group. *P<0.05 versus <i>Gata6^fl/fl</i> or <i>Gata4^fl/fl</i> sham.</p

Nol3^-/-Sgcd^-/- mice have enhanced skeletal muscle pathology relative to Sgcd^-/- mice at 4 weeks of age.

<p>A, Muscle weights normalized to tibial length of gastrocnemius and quadriceps, and B, serum creatine kinase (CK) levels measured from wildtype (WT), Arc null (Nol3^-/-), Sgcd^-/-, or double null (Nol3^-/-Sgcd^-/-) mice. *P<0.05 vs WT; #P<0.05 vs Sgcd^-/-; N=8-15 per group. C, Images taken at 200x of Masson’s trichrome-stained sections of gastrocnemius and quadriceps from Sgcd^-/- and Nol3^-/-Sgcd^-/- mice. The blue areas are collagen and fibrotic. D, Quantitation of the muscle fibrotic area by Metamorph software. *P<0.05 vs WT; #P<0.05 vs Sgcd^-/-; N=5 per group. E, Quantitation of the number of fibers with central nucleation relative to total fiber number in all experimental groups for the muscles shown. *P<0.05 vs WT; #P<0.05 vs Sgcd^-/-; N=5 per group with the identical 4 quadrants of the muscle counted per group. F, Quantification of Evan’s blue dye (EBD) positive myofibers in gastrocnemius and quadriceps from Sgcd^-/- and Nol3^-/-Sgcd^-/- mice. #P<0.05 vs Sgcd^-/-; N=4 per group with the identical 4 quadrants of the muscle counted per group. G, Histological images at 200x of EBD in orange and wheat germ agglutinin conjugated to FITC (green), the latter of which shows the muscle membrane. </p

Apoptosis Repressor with a CARD Domain (ARC) Restrains Bax-Mediated Pathogenesis in Dystrophic Skeletal Muscle

<div><p>Myofiber wasting in muscular dystrophy has largely been ascribed to necrotic cell death, despite reports identifying apoptotic markers in dystrophic muscle. Here we set out to identify the contribution of canonical apoptotic pathways to skeletal muscle degeneration in muscular dystrophy by genetically deleting a known inhibitor of apoptosis, apoptosis repressor with a card domain (Arc), in dystrophic mouse models. <i>Nol3</i> (Arc protein) genetic deletion in the dystrophic <i>Sgcd</i> or <i>Lama2</i> null backgrounds showed exacerbated skeletal muscle pathology with decreased muscle performance compared with single null dystrophic littermate controls. The enhanced severity of the dystrophic phenotype associated with <i>Nol3</i> deletion was caspase independent but dependent on the mitochondria permeability transition pore (MPTP), as the inhibitor Debio-025 partially rescued skeletal muscle pathology in <i>Nol3</i>^<i>-/-</i><i>Sgcd</i>^<i>-/-</i> double targeted mice. Mechanistically, <i>Nol3</i>^<i>-/-</i><i>Sgcd</i>^<i>-/-</i> mice showed elevated total and mitochondrial Bax protein levels, as well as greater mitochondrial swelling, suggesting that Arc normally restrains the cell death effects of Bax in skeletal muscle. Indeed, knockdown of Arc in mouse embryonic fibroblasts caused an increased sensitivity to cell death that was fully blocked in <i>Bax Bak1</i> (genes encoding Bax and Bak) double null fibroblasts. Thus Arc deficiency in dystrophic muscle exacerbates disease pathogenesis due to a Bax-mediated sensitization of mitochondria-dependent death mechanisms.</p> </div

Treatment with the MPTP inhibitor Debio-025 reduces skeletal muscle pathology in Nol3^-/-Sgcd^-/- mice.

<p>A, Muscle weights normalized to body weight of gastrocnemius and quadriceps, and B, body weight measurements of Sgcd^-/- and Nol3^-/-Sgcd^-/- mice treated with vehicle or Debio-025 for 4 weeks. C, Quantification of the time to exhaustion as assessed by involuntary treadmill running measured from vehicle or Debio-025 treated Nol3^-/- Sgcd^-/- mice. D, Quantitation of fibrosis and associated E, histological images taken at 100x of Masson’s trichrome stained sections in gastrocnemius and quadriceps muscles from vehicle or Debio-025 treated Sgcd^-/- and Nol3^-/-Sgcd^-/- mice. *P<0.05 vs vehicle; #P<0.05 vs Sgcd^-/- + vehicle; †P<0.05 vs Nol3^-/-Sgcd^-/- + vehicle; N=5-7 per group. Representative absorbance (540 nm) readings from skeletal muscle derived mitochondria demonstrating the amount of mitochondrial F, swelling in response to Ca²⁺ and G, shrinkage in response to PEG from the indicated genotypes of mice. </p

Arc deficiency increases Bax expression and cell death.

<p>A, Western blot for Bak and Bax from quadriceps lysates of Nol3^-/-, Sgcd^-/-, and Nol3^-/-Sgcd^-/- mice. (β-tubulin serves as a loading control). B, Western blot for Bax and Bcl-X_L from mitochondrial protein fractions isolated from pooled hindlimb muscles of WT, Nol3^-/-, Sgcd^-/-, and Nol3^-/-Sgcd^-/- mice. (voltage-dependent anion channel (VDAC) serves as a mitochondrial protein loading control). C, Western blotting for Arc from lysates derived from WT and Bak^-/-Bak1^-/- SV40 transformed MEFs. Skeletal muscle lysates were included to show the enrichment of Arc in terminally differentiated cell types, while GAPDH serves as a protein loading control. D, Western blot for Bax and Arc in SV40 transformed MEFs infected with lentivirus expressing scrambled shRNA (con) or 3 different Bax-directed shRNAs. (GAPDH serves as a loading control). E, Western blot for Arc in SV40 transformed MEFs infected with lentivirus expressing either a scrambled shRNA or one of the Bax shRNAs and treated with proteosomal inhibitors Bortezomib or MG-132. WT MEFs are a control for normal endogenous Arc expression and GAPDH serves as a loading control. F, Western blot for Arc in SV40 transformed MEFs infected with a lentivirus expressing either a scrambled shRNA (con.) or shRNA directed against Arc. Western blots presented are quantified and statistically analyzed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082053#pone.0082053.s003" target="_blank">Figure S3A-G</a>. G, Quantification of dead cells by flow cytometry sorting for Annexin and PI positivity in the experimental groups shown, treated or untreated with staurosporin for 12 hours. *P<0.05 vs WT untreated. Experiment was run in triplicate.</p

Nol3^-/-Lama2^-/- mice have smaller skeletal muscles and more severe pathology.

<p>A, Muscle weights normalized to tibial length of gastrocnemius and quadriceps measured from WT, Nol3^-/-, Lama2^-/-, and Nol3^-/-Lama2^-/- mice. *P<0.05 vs WT; #P<0.05 vs Lama2^-/-; N=9 per group. B, Histological images taken at 200x of Masson’s trichrome stained sections of gastrocnemius, quadriceps and diaphragm from Lama2^-/- and Nol3^-/-Lama2^-/- mice.</p