Truncations of the titin Z-disc predispose to a heart failure with preserved ejection phenotype in the context of pressure overload - Fig 3

<p>Vascular density and arteriole density were evaluated in cryo-sections eight months after TAC from the <b>(A1-A3)</b> WT-Sham, <b>(B1-B3)</b> Z-Sham, <b>(C1-C3)</b> WT-TAC, and <b>(D1-D3)</b> Z-TAC groups via immunofluorescent staining for CD31 and SMA. <b>(E)</b> Total vessel density was determined by counting CD31+ vascular structures, and <b>(F)</b> arteriole density was determined by counting vascular structure that co-expressed CD31 and SMA. (*: p<0.05. Bar = 50 μm).</p

Cardiac function at 8 months after surgery as assessed by echocardiogram.

<p>Cardiac function at 8 months after surgery as assessed by echocardiogram.</p

Truncations of the titin Z-disc predispose to a heart failure with preserved ejection phenotype in the context of pressure overload - Fig 1

<p>Rat LV function assessed by PV loop. LV systolic function indexes: <b>(A)</b> Left ventricular ejection fraction (LVEF), <b>(B)</b> dp/dt_max. LV diastolic function indexes: <b>(C)</b> dp/dt_min and <b>(D)</b> Tau. (*: p<0.05; **: p<0.01; ***: p<0.001.</p

Rat heart weight/bodyweight eight months post-surgery.

<p>Representative pictures of Sirus Red/Fast Green Collage staining to visualize fibrosis in rat heart of WT-Sham <b>(A)</b>, Z-Sham <b>(B)</b>, WT-TAC <b>(C)</b>, and Z-TAC <b>(D)</b> groups. <b>(E)</b> Quantification of LV fibrosis using Sirus red/Fast green stained rat cardiac tissue sections. <b>(F)</b> Quantification of total collagen in rat myocardium. Gene expression levels of rat collagen type I alpha 1 chain (Col1a1) <b>(G)</b>, collagen type III alpha 1 chain (Col3a1) <b>(H)</b>, atrial natriuretic peptide (ANP) <b>(I)</b>, and natriuretic peptide B (NppB) <b>(J)</b> in rat myocardium. (*: p<0.05; **: p<0.01; ***: p<0.001. Bar = 2000 μm).</p

Truncations of the titin Z-disc predispose to a heart failure with preserved ejection phenotype in the context of pressure overload - Fig 4

<p>TUNEL staining of rat cardiac tissue to identify apoptotic cells. TUNEL⁺ nuclei in WT-Sham <b>(A1)</b>, Z-Sham <b>(B1)</b>, WT-TAC <b>(C1)</b>, and Z-TAC <b>(D1)</b> rat hearts. The same sections were count-stained with α-sarcomere actin (α-SA) to show the localization of TUNEL+ nuclei in rat cardiac tissue <b>(A2-D2)</b>. <b>(E)</b> Quantification of TUNEL⁺ cardiac cells per LV section. Representative pictures of a TUNEL⁺ nucleus <b>(F1)</b> located in a cardiomyocyte that was stained for α-SA expression <b>(F2)</b>. <b>(G)</b> Quantification of TUNEL⁺ cardiomyocytes per LV section. (*: p<0.05; **: p<0.01; ***: p<0.001. Bar = 50 μm).</p

Comparison of bench-top NGS platforms.

*<p>316 scale chip; ** average.</p

Sequencing coverage of each gene.

<p>The coverage of the protein-coding sequence of each gene of interest is tabulated, as a percentage, for a range of sequencing depths (≥1x, 10x, 20x, 30x, 50x and 100x reads).</p

Characteristics of six genes included in the assay.

<p>LRG: locus reference genomic (<a href="http://www.lrg-sequence.org" target="_blank">http://www.lrg-sequence.org</a>), LQT: Long QT syndrome, CPVT: catecholaminergic polymorphic ventricular tachycardia.</p

Coverage of KCNQ1 and KCNH2 for the two platforms.

<p>Mean depth of coverage for 15 samples is shown for two genes on a log scale. Regions of no coverage therefore have negative values. The blue lines indicate local GC content (calculated with a 50 bp sliding window). Regions consistently missed have high GC content, with similar patterns for both platforms. KCNQ1 exons 1 & 8 and KCNH2 exons 1, 4 & 12 are difficult to sequence. A cartoon of the exon structure is shown beneath each panel. Plus (+) and minus (-) denote gene strand. Plots for all genes are shown in Supporting Information Figure S5. a.) MiSeq b.) Ion Torrent PGM.</p

Coverage of target genes.

<p>a. The percentage of each gene that is captured and sequenced (at least one read) is shown for each platform (MiSeq in red, PGM in black), for 15 samples; Three genes were consistently fully sequenced. Coverage of KCNQ1 and KCNH2 was more variable: KCNQ1 and KCNE1 were fully covered in the best performing samples, while the best performance on KCNH2 covered >97% of the gene. b. Mean sequencing depth across each gene, for 15 samples. Quartiles are shown. There is significant intra- and inter- sample variability.</p