Activin A directly impairs human cardiomyocyte contractile function indicating a potential role in heart failure development

Figures

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  FIGURE 1. Activin A overexpression in mice produced cardiac dysfunction. Comparison of physical and molecular characteristics in control and Activin A–treated mice. (A) In vivo circulating levels of Activin A. (B) Heart-to-body weight ratio. (C) Circulating NT-proANP. (D) Gene expression in LV + S tissue. (E) Pressure-volume loop analyses. Statistical analyses were performed using an unpaired two-tailed t-test. *P < 0.05, **P < 0.01. ActA, Activin A; ESPVR, end systolic pressure–volume relationship; HDD, hydrodynamic delivery; LV + S, left ventricle and septum; NT-proANP, N-terminal pro-atrial natriuretic peptide.

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  FIGURE 2. Activin A induces SMAD3 phosphorylation and downstream signaling in hiPSC-CMs. (A) Automated western blot data for the effect of Activin A plus anti–Activin A antibody or an isotype control on SMAD2/3 phosphorylation. (B) RNA sequencing data for expression of FSTL3, and SERPINE1 (PAI1) expression following exposure to 1 nM Activin A. One-way ANOVA was used to determine significance. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. ActA, Activin A; hiPSC-CM, human-induced pluripotent stem cell-derived cardiomyocyte; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; TPM, transcripts per million.

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  FIGURE 3. Activin A exposure time course on hiPSC-CM function. (A) The effect of different concentrations of Activin A on hiPSC-CM contractile impedance amplitude. (B,C) The impact of Activin A inhibition on Activin A–induced contractile dysfunction in hiPSC-CMs: dose-dependent effect on contractile impedance amplitude (B) and contractile parameters at both intrinsic and normalized beat rate (C). (D) Representative traces at both intrinsic beat rate and paced at 1 Hz hiPSC-CM, induced pluripotent stem cell-derived CM. Vertical lines indicate media changes in panels (A,B). One-way ANOVA was used to determine significance. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. ActA, Activin A; CM, cardiomyocyte; hiPSC-CM, human-induced pluripotent stem cell-derived cardiomyocyte.

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  FIGURE 4. IL-1β induces Activin A gene expression in cardiac fibroblasts. (A) The effect of inflammatory cytokines, IL-1β, IL-6, and oncostatin M on gene expression in cardiac fibroblasts. (B) The concentration of Activin A in control- and IL-1β-treated cells. (C) The effect of IL-1β on induced pluripotent stem cell-derived CM contractility (vertical lines indicate media changes). One-way ANOVA was used to determine significance. ****P < 0.0001. ActA, Activin A; CM, cardiomyocyte.

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  FIGURE 5. Activin A–dependent gene signature in hiPSC-CMs. (A) Significantly enriched pathways in hiPSC-CMs treated with acute (24 h) and chronic Activin A. All pathways were upregulated with positive normalized enrichment scores except glycogen metabolism and cardiac conduction. Cardiac conduction and signaling by TGF-β family members were only significant (P < 0.05) at 24 h. (B) Heatmap of leading-edge gene expression in cardiac conduction and signaling by TGF-β family members. Although both pathways were only significant after acute Activin A exposure (24 h), a similar pattern of gene expression was observed in chronic exposure. (C) TPM values for a subset of cardiac genes highlighted in the heat map. One-way ANOVA was used to determine significance. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. ActA, Activin A; hiPSC-CM, human-induced pluripotent stem cell-derived cardiomyocyte; ns, not significant; TGF-β, transforming growth factor-β; TPM, transcripts per million.

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  FIGURE 6. Effect of Activin A on myocardial electrophysiology and calcium transients. The effect of Activin A or Activin A plus inhibitory antibody on (A,B) electrophysiology and (C,D) calcium transients in induced pluripotent stem cell–derived cardiomyocytes. One-way ANOVA was used to determine significance. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. ActA, Activin A; BPM, beats per minute; FLIPR, fluorescent imaging plate reader; FPD, field potential duration; IPSC, induced pluripotent stem cells; RFU, relative fluorescence units.

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  FIGURE 7. Effect of Activin A on contractile function and arrhythmia in HECTs. (A) Effect of Activin A on cardiac rhythm in HECTs for spontaneous beat rate (beating in the absence of pacing) during the full treatment period and at 8 days post treatment. (B) Effect of Activin A, MSTN (GDF8), and GDF11 on cardiac rhythm in HECTs for spontaneous beat rate during treatment and at 8 days post treatment. (C,D) impact of Activin A and preventive Activin A inhibition on HECT contraction kinetics on day 20 and the reversal by applying anti–Activin A starting on day 20 to day 34. (E,F) Impact of Activin A, Activin A inhibition, MSTN (GDF8), and GDF11 on HECT contraction kinetics on day 20. Shading on D denotes the 95% confidence interval. One-way ANOVA was used to determine significance. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. ActA, Activin A; GDF, growth differentiation factor; HECT, human engineered cardiac tissue; MSTN, myostatin.