Echocardiographic parameters in mice after 8 weeks of rAAV6 treatment.

<p>Echocardiographic parameters in mice after 8 weeks of rAAV6 treatment.</p

Comparison of rAAV6-PLCβ1-mediated gene expression changes to those caused by neonatal overexpression of Gαq or PKCα.

<p><b>A</b>. Venn diagram demonstrating limited overlap of 748 mRNAs regulated at FDR<0.1 in a comparison of Gαq transgenic mice vs WT [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158317#pone.0158317.ref024" target="_blank">24</a>] and 29 mRNAs regulated at FDR<0.1 in a comparison of PKCα transgenic mice vs tet-off (TO) controls [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158317#pone.0158317.ref027" target="_blank">27</a>]. <b>B</b>. 761 unique mRNAs are displayed, from a composite of 15 mRNAs regulated at FDR<0.1 in a comparison of PLCβ1a or PLCβ1b vs blank, 748 mRNAs regulated at FDR<0.1 in a comparison of Gqα transgenic mice vs WT [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158317#pone.0158317.ref024" target="_blank">24</a>] and 29 mRNAs regulated at FDR<0.1 in a comparison of PKCα transgenic mice vs tet-off (TO) controls [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158317#pone.0158317.ref027" target="_blank">27</a>]. Unsupervised hierarchical clustering of relative mRNA abundances (rows) and of individual hearts (columns) was performed using Euclidean distance with average linkage. Colors represent log₂ ratios for each individual heart vs the mean of the appropriate controls (red is upregulated, blue is downregulated); the color range spans -2.0 to +2.0 in log₂ scale (from 4-fold downregulation to 4-fold upregulation in linear scale).</p

Canonical markers of cardiac hypertrophy in response to PLCβ1a or PLCβ1b expression.

<p>mRNAs are displayed according to expression calculated from mRNA-sequencing (FPKM, fragments per kilobase of exon per million mapped fragments). <i>Red</i>, PLCβ1b; <i>blue</i>, PLCβ1a; <i>black</i>, blank control. Values are mean ± SEM, n = 6. No comparisons to blank control were significant at FDR<0.1 (according to DESeq). Acta1, α-skeletal actin; Actc1, α-cardiac actin; Atp2a2, SERCA2a; Myh6, α-myosin heavy chain; Myh7, β-myosin heavy chain; Nppa, atrial natriuretic peptide; Nppb, natriuretic peptide B; Pln, phospholamban.</p

mRNAs regulated at FDR<0.1 by either or both of PLCβ1a or PLCβ1b expression.

<p>15 mRNAs are displayed according to expression calculated from mRNA-sequencing (FPKM). <i>Red</i>, PLCβ1b; <i>blue</i>, PLCβ1a; <i>black</i>, blank control. Values are mean ± SEM, n = 6. Blue lines with asterisk indicate false discovery rate (FDR) <0.1 between PLCβ1a and blank (according to DESeq); red lines with asterisk indicate FDR <0.1 between PLCβ1b and blank. Full gene descriptions and assignment to Gene Ontology categories are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158317#pone.0158317.s002" target="_blank">S1 Table</a>.</p

Identification of miR-34 regulatory networks in settings of disease and antimiR-therapy: Implications for treating cardiac pathology and other diseases

<p>Expression of the miR-34 family (miR-34a, -34b, -34c) is elevated in settings of heart disease, and inhibition with antimiR-34a/antimiR-34 has emerged as a promising therapeutic strategy. Under chronic cardiac disease settings, targeting the entire miR-34 family is more effective than targeting miR-34a alone. The identification of transcription factor (TF)-miRNA regulatory networks has added complexity to understanding the therapeutic potential of miRNA-based therapies. Here, we sought to determine whether antimiR-34 targets secondary miRNAs via TFs which could contribute to antimiR-34-mediated protection. Using miRNA-Seq we identified differentially regulated miRNAs in hearts from mice with cardiac pathology due to transverse aortic constriction (TAC), and focused on miRNAs which were also regulated by antimiR-34. Two clusters of stress-responsive miRNAs were classified as “pathological” and “cardioprotective,” respectively. Using ChIPBase we identified 45 TF binding sites on the promoters of “pathological” and “cardioprotective” miRNAs, and 5 represented direct targets of miR-34, with the capacity to regulate other miRNAs. Knockdown studies in a cardiomyoblast cell line demonstrated that expression of 2 “pathological” miRNAs (let-7e, miR-31) was regulated by one of the identified TFs. Furthermore, by qPCR we confirmed that expression of let-7e and miR-31 was lower in hearts from antimiR-34 treated TAC mice; this may explain why targeting the entire miR-34 family is more effective than targeting miR-34a alone. Finally, we showed that Acsl4 (a common target of miR-34, let-7e and miR-31) was increased in hearts from TAC antimiR-34 treated mice. In summary, antimiR-34 regulates the expression of other miRNAs and this has implications for drug development.</p

Long-Term Overexpression of Hsp70 Does Not Protect against Cardiac Dysfunction and Adverse Remodeling in a MURC Transgenic Mouse Model with Chronic Heart Failure and Atrial Fibrillation

<div><p>Previous animal studies had shown that increasing heat shock protein 70 (Hsp70) using a transgenic, gene therapy or pharmacological approach provided cardiac protection in models of acute cardiac stress. Furthermore, clinical studies had reported associations between Hsp70 levels and protection against atrial fibrillation (AF). AF is the most common cardiac arrhythmia presenting in cardiology clinics and is associated with increased rates of heart failure and stroke. Improved therapies for AF and heart failure are urgently required. Despite promising observations in animal studies which targeted Hsp70, we recently reported that increasing Hsp70 was unable to attenuate cardiac dysfunction and pathology in a mouse model which develops heart failure and intermittent AF. Given our somewhat unexpected finding and the extensive literature suggesting Hsp70 provides cardiac protection, it was considered important to assess whether Hsp70 could provide protection in another mouse model of heart failure and AF. The aim of the current study was to determine whether increasing Hsp70 could attenuate adverse cardiac remodeling, cardiac dysfunction and episodes of arrhythmia in a mouse model of heart failure and AF due to overexpression of Muscle-Restricted Coiled-Coil (MURC). Cardiac function and pathology were assessed in mice at approximately 12 months of age. We report here, that chronic overexpression of Hsp70 was unable to provide protection against cardiac dysfunction, conduction abnormalities, fibrosis or characteristic molecular markers of the failing heart. In summary, elevated Hsp70 may provide protection in acute cardiac stress settings, but appears insufficient to protect the heart under chronic cardiac disease conditions.</p></div

Cardiac morphology in MURC and MURC-Hsp70 Tg mouse models.

<p><b>(A)</b> Transverse sections of hearts highlighting dilated chambers in MURC-Hsp70 Tg mice compared to MURC Tg mice. LV = left ventricle, RV = right ventricle. Scale bar = 1 mm. <b>(B)</b> Graph of atria weight/tibia length (AW/TL). N = 3–5 per group. *P<0.05 vs. Ntg, †P<0.05 vs. MURC. The dotted line reflects normal AW/TL for Ntg mice at about 3–4 months of age. <b>(C)</b> qPCR analysis of Collagen 3 <i>(Col3a1)</i> relative to <i>Hprt1</i> in atria. N = 3–5 per group. *P≤0.05 vs. Ntg (One way ANOVA with Fisher’s posthoc test), ^P<0.05 vs. Ntg (unpaired t-test).</p

Analysis of cardiac fibrosis and collagen gene expression in MURC and MURC-Hsp70 Tg mouse models.

<p><b>(A)</b> Representative LV cross-sections stained with Masson’s trichrome and quantification of LV fibrosis in Ntg, MURC and MURC-Hsp70 Tg mice. Scale = 200 μM. N = 3–5 per group. *P<0.05 vs. Ntg. <b>(B)</b> qPCR analysis of Collagen 1 <i>(Col1α1)</i>, Collagen 3 <i>(Col3a1)</i> and Connective tissue growth factor <i>(Ctgf)</i> relative to <i>Hprt1</i>. N = 3–5 per group. *P<0.05 vs. Ntg (unpaired t-test), ^P<0.05 vs. Ntg (Mann Whitney nonparametric test). <b>(C)</b> Representative Western blots and quantification of collagen 1 (Col1α1) in ventricles of Ntg, MURC and MURC-Hsp70 Tg mice. Arrow indicates band quantified. N = 3–5 per group. *P<0.05 vs. Ntg.</p

Transgenic overexpression of HSP70 did not attenuate cardiac dysfunction and electrophysiology abnormalities.

<p><b>(A)</b> Quantification of LVEDD, LVESD and FS in 12 month old Ntg, MURC and MURC-Hsp70 Tg mice. N = 3–5 per group. *P<0.05 vs. Ntg, **P<0.0005 vs. Ntg, †P<0.05. Lower right: Representative M-mode echocardiograms in 12 month old Ntg, MURC and MURC-Hsp70 Tg mice. <b>(B)</b> Representative ECG traces in 12 month old Ntg, MURC and MURC-Hsp70 Tg mice. Arrows highlight clear P-waves. <b>(C)</b> Quantification of R amplitude and PR interval in Ntg, MURC and MURC-Hsp70 Tg mice. N = 3–5 per group. *P<0.05 vs. Ntg, †P≤0.05. <b>(D)</b> Representative heart rate (HR) variability traces and quantification of time in arrhythmia in 12 month old Ntg, MURC and MURC-Hsp70 Tg mice. N = 3–5 per group. *P<0.05 vs. Ntg (One Way ANOVA with Fisher’s posthoc test), †P<0.05, P = 0.06 (Mann-Whitney nonparametric t-test).</p

RHOA and HSP70 is upregulated in MURC-Hsp70 Tg mice.

<p><b>(A)</b> Representative Western blots and quantification of active RHOA relative to total RHOA (sum of active and inactive RHOA) in ventricles of Ntg, MURC and MURC-Hsp70 Tg mice. N = 3 per group. *P<0.05 vs. Ntg. Representative Western blots and quantification of HSP70 in <b>(B)</b> ventricles and <b>(C)</b> atria of MURC and MURC-Hsp70 Tg mice. N = 3–5 per group. *P<0.05 vs. MURC.</p