Catecholamine treatment induces hypercontractile left ventricular function
Left ventricular function after IsoPE treatment and after recovery (Fig. 1A) was assessed by echocardiography (Fig. 1B-E, Table 1). IsoPE treated mice showed a gain in heart rate (IsoPE 522 ± 15 bpm vs. CTRL 317 ± 6 bpm, p < 0.001, Fig. 1C) and left ventricular ejection fraction (IsoPE 69 ± 2 % vs. CTRL 45 ± 2 %, p < 0.001, Fig. 1D), resulting in an increased cardiac index (Fig. 1E). Within two weeks after removal of the minipump, cardiac function returned to baseline with no significant difference between REC and CTRL (Fig. 1B-E).
Left ventricular structural remodeling after catecholamine treatment
Despite no signs of overt heart failure, substantial structural remodeling of the heart could be observed in IsoPE treated mice. The extent of left ventricular interstitial fibrosis, as determined by the sirius red positive area, was 3.1 ± 0.3 % in untreated mice, 6.0 ± 0.9 % after IsoPE treatment, and 4.1 ± 0.3 % after recovery (Fig. 2A-B). Heart weight to tibia length ratio increased from 6.1 ± 0.2 mg/mm in untreated mice to 7.3 ± 0.1 mg/mm after IsoPE treatment (IsoPE vs. CTRL, p < 0.001), and returned to 6.4 ± 0.1 mg/mm after recovery (REC vs. CTRL, p < 0.001) (Fig. 2C). Cardiomyocyte cross sectional area was 310 ± 16 µm² in untreated mice, 422 ± 27 µm² after IsoPE treatment, and 367 ± 4 µm² after recovery (Fig. 2D-E). mRNA expression of marker genes for cardiac fibrosis (biglycan, connective tissue growth factor), inflammation (lysozyme 2), or hypertrophy (myosin heavy chain beta) was significantly increased after IsoPE treatment compared to untreated controls and returned to baseline after recovery (Fig. 2F-I).
Catecholamine-induced gene expression in cardiomyocytes and cell-cell communication
To assess the impact of catecholamine treatment on cardiomyocyte gene expression, mRNA expression in isolated cardiomyocyte nuclei was determined by RNA-seq (Fig. 3A-B). After IsoPE treatment, 295 genes were differentially expressed compared to untreated mice (210 upregulated, 85 downregulated; fold change > 2, q < 0.05; Fig. 3C, Suppl. Table S2). These included typical markers of cardiac remodeling such as atrial natriuretic peptide precursor B (2.4-fold up; Fig. 3D) or connective tissue growth factor (6.5-fold up; Fig. 3E). Gene ontology analysis revealed an enrichment of genes related to biological processes such as cellular response to transforming growth factor stimulus, vasculature development, or extracellular matrix organization among the differentially expressed genes (Fig. 3F). As these processes typically involve non-myocyte cells in the heart [32] we predicted cardiomyocyte to non-myocyte interactions after IsoPE treatment. Among the genes that were differentially expressed in IsoPE vs. CTRL, we found 32 secreted ligands with their corresponding receptors being expressed in endothelial cells, fibroblasts, monocytes, or macrophages (Fig. 3G) [31, 45].
Regulation of catecholamine-induced gene expression
Gene expression is regulated by transcription factors binding to distal regulatory regions (enhancers) within the chromatin that physically interact with the promoter region of their target genes (Fig. 4A). We applied a previously published dataset of regulatory regions in cardiomyocytes identified by DNA methylation-guided annotation [31] and linked these regions to IsoPE-induced differential gene expression (Fig. 4B). Genes that were differentially expressed after IsoPE treatment showed a strong enrichment of binding motifs for myocyte enhancer factor-2 (Mef2) and GATA family members in their associated regulatory regions (Suppl. File S1). We intersected the list of enriched binding motifs with mRNA expression of the respective transcription factors (Fig. 4C). Among the upregulated transcription factors with corresponding binding motif enriched were AP-1 family members Jun (Jun, 3.8-fold up, Junb 5.3-fold up) and Fos (Fos, 8.0-fold up; Fosb 2.2-fold up) (Fig. 4D-E). We predicted 115 genes among the 295 differentially expressed genes in IsoPE to be direct Jun or Fos target genes (Suppl. Table S2). Gene ontology analysis linked these genes to biological processes that are related to cardiac remodeling (Fig. 4F).
Reversibility of catecholamine-induced gene expression
After two weeks recovery period, IsoPE-induced gene expression largely returned to baseline. 280 (94.9 %) and 11 (3.7 %) out of 295 differentially expressed genes showed full or partial recovery, respectively (Fig. 5A, Suppl. Fig. S1; Suppl. Table S2). Only 4 genes (1.4 %) remained dysregulated after recovery (Peg3, Cyp26b1, Gm38031, Pnpla3) when compared to untreated controls. 4 genes (Thbs1, Slc41a3, Arntl, Garnl3) that have not been regulated in IsoPE were differentially expressed in recovered mice compared to control (Fig. 5A). Quantitative recovery was reflected by mean absolute value of fold change (log2) versus untreated control which was 1.4 after IsoPE and 0.2 after recovery (p < 0.001; Fig. 5B).
We analyzed the impact of catecholamine treatment on the expression of adrenergic receptors and components of related signaling pathways in cardiomyocytes (Fig. 5C). The expression of the main receptors for isoprenaline and phenylephrine in cardiomyocytes, β1-adrenergic receptor (Adrab1, 1.4-fold down) and α1-adrenergic receptors (Adra1a, 1.5-fold down; Adra1b, 1.6-fold down), was moderately downregulated after IsoPE treatment. We observed changes in expression of G-protein coupled receptor kinases GRK3 and GRK5, but not GRK2 (Grk3, 2.1-fold up; Grk5, 2.1-fold up; Adrbk1/Grk2 1.0-fold), regulators of G-protein signaling RGS1, RGS2, and RGS7 (Rgs1, 2.0-fold up; Rgs2, 2.1-fold up; Rgs7, 2.7-fold down), and A-kinase anchoring protein 5 (Akap5, 2.5-fold down). In addition, expression of endothelin-1 (Edn1, 2.4-fold up) and its receptors (Ednra, 2.0-fold up; Ednrb, 1.8-fold up) was induced in IsoPE treated animals. All the before mentioned changes returned to baseline after recovery (Suppl. Table S2).