Molecular profiling of dilated cardiomyopathy that progresses to heart failure

Abstract

Dilated cardiomyopathy (DCM) is defined by progressive functional and structural changes. We performed RNA-seq at different stages of disease to define molecular signaling in the progression from pre-DCM hearts to DCM and overt heart failure (HF) using a genetic model of DCM (phospholamban missense mutation, PLN^R9C/+). Pre-DCM hearts were phenotypically normal yet displayed proliferation of nonmyocytes (59% relative increase vs. WT, P = 8 × 10^-4) and activation of proinflammatory signaling with notable cardiomyocyte-specific induction of a subset of profibrotic cytokines including TGFβ2 and TGFβ3. These changes progressed through DCM and HF, resulting in substantial fibrosis (17.6% of left ventricle [LV] vs. WT, P = 6 × 10^-33). Cardiomyocytes displayed a marked shift in metabolic gene transcription: downregulation of aerobic respiration and subsequent upregulation of glucose utilization, changes coincident with attenuated expression of PPARα and PPARγ coactivators -1α (PGC1α) and -1β, and increased expression of the metabolic regulator T-box transcription factor 15 (Tbx15). Comparing DCM transcriptional profiles with those in hypertrophic cardiomyopathy (HCM) revealed similar and distinct molecular mechanisms. Our data suggest that cardiomyocyte-specific cytokine expression, early fibroblast activation, and the shift in metabolic gene expression are hallmarks of cardiomyopathy progression. Notably, key components of these profibrotic and metabolic networks were disease specific and distinguish DCM from HCM.

Figure 1. PLN^R9C/+ mice develop increased cardiac fibrosis and nonmyocyte cell proliferation with disease progression.

(A) Light microscopy (scale bars: 100 μm) of Masson’s trichrome–stained LV tissue at 8 weeks (pre-DCM), 18 weeks (DCM), and 22 weeks (overt HF) demonstrates progressive cardiac fibrosis in PLN^R9C/+ hearts compared with age-matched WT animals. Data quantitated were individual LV slices (2 per slide) at 10 levels (apex to base) from n = 3 mice per group; 2-tailed Student’s t test. (B) Confocal microscopy (scale bars: 75 μm) and quantification of LV sections from hearts labeled with BrdU demonstrating nonmyocyte proliferation both pre-DCM and with overt HF. BrdU (magenta), wheat germ agglutinin (WGA; green), and nuclear DAPI (blue). Cells were counted from individual LV slices at 10 levels (apex to base) from n = 3 mice per group. Greater than 25,000 nuclei were counted per experiment; 2-tailed Student’s t test.

Figure 2. Activation of the cardiac stress response in PLN^R9C/+ mice with disease progression.

(A) Progressive and marked increase in atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) mRNA levels with disease progression in PLN^R9C/+ mice (P < 1 × 10^–300 vs. WT at all disease stages). (B) Progressive increase in 4-and-a-half LIM domains protein 1 (Fhl1) mRNA levels with disease progression in PLN^R9C/+ mice (P < 1× 10^–300 vs. WT at all disease stages). (C) Reduction in the ratio of adult (α-MHC/Myh6) to fetal (β-MHC/Myh7) myosin heavy chain gene expression with disease progression in PLN^R9C/+ mice. RNA from n = 3 mice was pooled prior to RNA-seq. Bayesian P value corrected for multiple hypothesis testing (10).

Figure 3. Differential gene expression at distinct stages of disease in nonmyocytes and myocytes from PLN^R9C/+ hearts.

(A) Differences in nonmyocyte gene expression progress steadily with worsening disease in LV tissue, with nearly a quarter of differentially expressed genes common to all stages. (B) Few differentially expressed genes were unique to cardiomyocytes before DCM, and only 12% of differentially expressed genes were common to all stages. More significant changes were noted with onset of phenotypic disease.

Figure 4. Increased expression of TGFβ in PLN^R9C/+ mice.

(A) All TGFβ isoforms were predicted to be activated at all 3 stages of disease by upstream regulator analysis. Z-score reflects both the confidence and direction of the inferred activation state (P < 1 × 10^–6 for all analyses). (B) TGFβ gene expression was induced in PLN^R9C/+ LV tissue at all stages of disease (n = 3 mice pooled prior to RNA-seq). (C) Isolated nonmyocyte and cardiomyocyte cells (n = 6 mice pooled prior to RNA-seq) at 18 weeks (DCM) showed that TGFβ genes were predominantly expressed in WT nonmyocytes, with modest increases in Tgfb2 and Tgfb3 expression in PLN^R9C/+ nonmyocytes. In contrast, Tgfb2 and Tgfb3 were strongly induced in cardiomyocytes of PLN^R9C/+ mice with DCM. Tgfb1 levels do not change significantly in either cell compartment. Bayesian P value corrected for multiple hypothesis testing (10).

Figure 5. Heat maps of metabolic genes demonstrate marked dysregulation of myocyte metabolism.

(A) Mitochondrial genes controlling oxidative phosphorylation were significantly downregulated with the onset of DCM. (B) There was also reduced expression of genes governing fatty acid β oxidation and the citric acid cycle with the onset of DCM. (C) By contrast, genes associated with glucose utilization became dysregulated with the onset of DCM, and their expression was progressively increased with overt HF. Data plotted are fold-change values versus age-matched WT controls (n = 3 mice pooled prior to RNA-seq).

Figure 6. Downregulation of PPAR signaling in PLN^R9C/+ with development of DCM.

(A) Upstream regulator analysis predicted transcription factors that were activated (gray, Z-score > 2) or inhibited (black, Z-score < –2) in PLN^R9C/+ cardiomyocytes with DCM. (B) PPAR pathway genes and key cofactors were downregulated in PLN^R9C/+ mice with development of DCM (*P < 0.001). Data plotted as natural log (ln) of fold-change (n = 3 mice pooled prior to RNA-seq). Bayesian P value corrected for multiple hypothesis testing (10). (C) PPARα and RXRα downregulation is predicted to affect a number of downstream metabolic pathways. Genes and pathways regulated by PPARα/RXRα that are downregulated (red) or upregulated (green) in PLN^R9C/+ mice with DCM are highlighted. Dashed arrows indicate signaling networks not shown in detail. Adapted from Ingenuity canonical pathway PPARα/RXRα activation. †, representative cell surface receptors that signal to PPARα/RXRα.

Figure 7. Transcriptional comparisons of DCM and HCM.

(A) Enriched (FDR < 0.01) Ingenuity canonical pathways showed a predominance of inflammatory and cell remodeling pathways in nonmyocytes with perturbed cardiomyocyte metabolic pathways in both DCM and HCM. (B) Few differentially expressed genes in nonmyocytes and cardiomyocytes were common to both DCM and HCM. (C) TLR genes were upregulated in DCM but not in HCM. (D) Genes controlling mitochondrial oxidative phosphorylation and (E) those controlling fatty acid oxidation and the citric acid cycle were significantly downregulated, while (F) genes governing glucose metabolism were upregulated with the onset of DCM or HCM. Data plotted in heat maps are fold-change values versus age-matched WT controls (n = 3 mice pooled prior to RNA-seq).