Journal: Nature Communications

Article Title: NF-κB inhibition rescues cardiac function by remodeling calcium genes in a Duchenne muscular dystrophy model

doi: 10.1038/s41467-018-05910-1

Figure Lengend Snippet: NF-κB causes heart dysfunction in mdx mice. a EMSA performed on wild-type (wt) and mdx hearts (left panel). Supershift EMSA performed on mdx hearts using specific antibodies for p65 and p50, and IgG as a control (Right panel). Arrowheads indicate shifted bands. b Western blots performed on whole heart lysates and probed for phosphorylated p65-ser536 (phospho-p65), p65, p50, and α-tubulin (used as a loading control). c Representative images of H&E and phosho-p65 staining prepared from 1-year old heart sections. Boxed regions appear as magnified images in neighboring panels. Scale bar = 50 μm. d cardiac output, e stroke volume, f ejection fraction ( p = 0.686), g end diastolic diameter, and h end diastolic volume assessed by echocardiogram on 13–14-month-old mice ( n = 4 wt; 8 mdx IKKβf/f ; 5 mdx HRTΔIKKβ ). i End-diastolic pressure volume relationship (EDPVR) assessed by ventricular pressure-volume relationship analysis on 13–14-month old mice ( n = 7 wt; 18 mdx IKKβf/f ; 12 mdx HRTΔIKKβ ). j Developed force measured from isolated multicellular cardiac muscles of 7-month old mice in response to β-adrenergic stimulation with isoproterenol ( n = 7 wt; 7 mdx IKKβf/f ; 9 mdx HRTΔIKKβ ; 3 NBD treated ( mdx -NBD); p = 0.278). k Relaxation time (RT 90 ) after isoproterenol stimulation in multicellular cardiac muscles ( n = same as J). l, m Ventricular pressure-volume relationship measurements after dobutamine administration on 13–14-month-old mice. l Maximal heart rate (HR) ( n = 5 wt; 8 mdx IKKβf/f ; 7 mdx HRTΔIKKβ ) and ( m ) Tau (isovolumetric relaxation) ( n = 5 wt; 7 mdx IKKβf/f ; 4 mdx HRTΔIKKβ ; p = 0.027 but multiple comparisons test did not detect differences between groups). Data expressed as means ± SEM with bars and plungers and individual data points with dots. * p < 0.05 relative to wt and # p < 0.05 relative to mdx IKKβf/f by d – i , k – m 1-way ANOVA followed by Tukey multiple comparison test where appropriate, j 2-way repeated measures ANOVA. Main effects for genotype/treatment

Article Snippet: For supershifts, extracts were incubated with the following antibodies: 4 μl p50 (114 Santa Cruz) or IgG or 0.5 μl p65 (Rockland) prior to loading on the gel.

Techniques: Control, Western Blot, Staining, Isolation, Muscles, Comparison

Journal: Nature Communications

Article Title: NF-κB inhibition rescues cardiac function by remodeling calcium genes in a Duchenne muscular dystrophy model

doi: 10.1038/s41467-018-05910-1

Figure Lengend Snippet: Cardiomyocyte NF-κB ablation normalizes calcium handling and increases gene expression. a Statistically significant gene categories from microarray analysis identified using Gene Set Enrichment Analysis. Heatmaps represent genes identified in annotations. b Calcium transient amplitude measured from cardiomyocytes isolated from 7–8-month old mice ( n = 38 wt; 43 mdx IKKβf/f ; 35 mdx HRTΔIKKβ cardiomyocytes). c Depiction of individual microarray genes that were up- and down-regulated in mdx HRTΔIKKβ relative to mdx IKKβf/f hearts. Genes shown in red are ≥ 1.5-fold upregulated and those in blue are ≥ 1.5-fold downregulated. d – g qPCR analysis of Slc8a1 expression. RNA isolated from d 6–7-month-old hearts ( n = 5 wt; 5 mdx IKKβf/f ; 4 mdx HRTΔIKKβ ), e mouse embryonic fibroblasts (MEFs) that were wt ( p65 +/+ ) or null ( p65 −/− ) for p65 ( n = 5). f C2C12 myotubes expressing empty vector as control (CT) or IκBα super repressor (SR) ( n = 6 CT; 8 SR), and g MEFs untreated (CT) or treated with TNF ( n = 4). Data expressed as means ± SEM with bars and plungers and individual data points with dots. * p < 0.05 relative to respective control and # p < 0.05 relative to mdx IKKβf/f by b, d 1-way ANOVA followed by Tukey multiple comparison test and e – g 2-tailed Student’s t test

Article Snippet: For supershifts, extracts were incubated with the following antibodies: 4 μl p50 (114 Santa Cruz) or IgG or 0.5 μl p65 (Rockland) prior to loading on the gel.

Techniques: Gene Expression, Microarray, Isolation, Expressing, Plasmid Preparation, Control, Comparison

Journal: Nature Communications

Article Title: NF-κB inhibition rescues cardiac function by remodeling calcium genes in a Duchenne muscular dystrophy model

doi: 10.1038/s41467-018-05910-1

Figure Lengend Snippet: Cardiomyocyte NF-κB ablation causes global H3K27ac enrichment in mdx hearts. a Venn diagram pie chart depicting the H3K27ac ChIP-seq annotation within specified regions of the genome from mouse hearts. b Genome-wide distribution of H3K27ac binding loci relative to transcription start sites (TSS). c – d Genome-wide fragment density showing potential overlap of c . ChIP-seq histone marks across peaks from our ChIP-seq performed in mdx HRTΔIKKβ hearts and d our ChIP-seqs across peaks from p65 ChIP-seq. e Network showing the top 15 Gene Ontology clusters identified from differentially enriched genes in the mdx HRTΔIKKβ when compared to mdx IKKβf/f H3K27 regions. The most significantly enriched pathway for each cluster is labeled as the representative term for that group. f Gene expression analyzed by qPCR on total RNA isolated from hearts ( n = Rcan1 : 5 wt; 6 mdx IKKβf/f ; 6 mdx HRTΔIKKβ ; Cacna1h : 5 for all genotypes; Camk4 5 wt; 6 mdx IKKβf/f ; 5 mdx HRTΔIKKβ ). g – h ChIP performed with an H3K27ac antibody and qPCR analysis was used to detect enrichment on denoted genes. DNA extracted from g mouse hearts ( n = 3) or h control and TNF treated MEFs ( n = 4 Slc8a1 and Rcan1 and 3 Cacna1h and Camk4 ). Genes were expressed as a ratio. Dotted line represents level of enrichment equal to g mdx IKKβf/f hearts and h control MEFs. Bars represent ( g ) enrichment in hearts and h depletion in TNF treated MEFs. f Data expressed using box and whiskers plots. The central line in the boxes is the median value; the lower and upper boundaries of the boxes represent the lower and upper quartiles, respectively; the lower and upper whiskers represent the minimum and maximum values, respectively. Individual data points are plotted with dots. g – h Data expressed as means ± SEM with bars and plungers and individual data points with dots. f * p < 0.05 wt and # p < 0.05 mdx IKKβf/f , by 1-way ANOVA followed by Tukey multiple comparison test. g – h * p < 0.05 mdx IKKβf/f or untreated MEFs by 2-tailed Student’s t test

Article Snippet: For supershifts, extracts were incubated with the following antibodies: 4 μl p50 (114 Santa Cruz) or IgG or 0.5 μl p65 (Rockland) prior to loading on the gel.

Techniques: ChIP-sequencing, Genome Wide, Binding Assay, Labeling, Gene Expression, Isolation, Control, Comparison

Journal: Nature Communications

Article Title: NF-κB inhibition rescues cardiac function by remodeling calcium genes in a Duchenne muscular dystrophy model

doi: 10.1038/s41467-018-05910-1

Figure Lengend Snippet: CTCF, SIN3A, and HDAC1 mediate a less permissive chromatin conformation on calcium genes upon NF-κB activation. a Motif analysis performed on genes identified as having both p65 ChIP-seq peaks and H3K27ac mdx HRTΔIKKβ ChIP-seq regions. b Pie graph representing the percentage of genes with CTCF motifs up- and downregulated in the microarray (relative to mdx hearts with intact NF-κB). c ChIP-seq data derived from genome-wide fragment density analysis showing potential overlap of CTCF peaks with p65 peaks. d The same analysis as in c except p65 peaks were split between two groups either containing or lacking an NF-κB consensus motif. e – f ChIP performed with a CTCF antibody and qPCR analysis was used to detect enrichment on denoted genes. DNA extracted from e mdx IKKβf/f and mdx HRTΔIKKβ hearts ( n = 4 Slc8a1 ; 5 Rcan1 ; 3 cacna1h ; 2 Camk4 ) and ( f ) control and TNF treated MEFs ( n = 4 except n = 2 Camk4 ), g – h The same analyses were performed as in e, f . DNA extracted from mdx IKKβf/f and mdx HRTΔIKKβ hearts and ChIP performed with a ( g ) SIN3A antibody ( n = 3 Slc8a1 and Cacna1h ( p = 0.7); n = 4 Rcan1 and Camk4 ) ( h ) HDAC1 antibody ( n = 3 except n = 4 Slc8a1 ). e – h Enrichment on different genes were plotted as a ratio. Dotted line represents level of enrichment equal to e, g – h mdx IKKβf/f hearts and ( f ) control MEFs. Bars represent e, g – h depletion in mdx HRTΔIKKβ hearts and f enrichment in TNF treated MEFs. i Gene expression analyzed by qPCR on total RNA isolated from HL-1 cardiomyocytes ( n = 4). j Heatmaps showing ChIP-seq fragment densities of SIN3A and HDAC1 surrounding p65 peaks, showing potential overlap. Left panel includes genes upregulated and right panel includes genes downregulated in the microarray. e – h Data expressed as means ± SEM with bars and plungers and individual data points with dots. ( i ) Data expressed using box and whiskers plots. The central line in the boxes is the median value; the lower and upper boundaries of the boxes represent the lower and upper quartiles, respectively; the lower and upper whiskers represent the minimum and maximum values, respectively. Individual data points are plotted with dots. e – h * p < 0.05 mdx IKKβf/f or untreated by 2-tailed Student’s t test. i * p < 0.05 CT and # p < 0.05 TSA by 1-way ANOVA followed by Tukey multiple comparison test

Article Snippet: For supershifts, extracts were incubated with the following antibodies: 4 μl p50 (114 Santa Cruz) or IgG or 0.5 μl p65 (Rockland) prior to loading on the gel.

Techniques: Activation Assay, ChIP-sequencing, Microarray, Derivative Assay, Genome Wide, Control, Gene Expression, Isolation, Comparison