A crucial role of activin A-mediated growth hormone suppression in mouse and human heart failure

Abstract

Infusion of bone marrow-derived mononuclear cells (BMMNC) has been reported to ameliorate cardiac dysfunction after acute myocardial infarction. In this study, we investigated whether infusion of BMMNC is also effective for non-ischemic heart failure model mice and the underlying mechanisms. Intravenous infusion of BMMNC showed transient cardioprotective effects on animal models with dilated cardiomyopathy (DCM) without their engraftment in heart, suggesting that BMMNC infusion improves cardiac function via humoral factors rather than their differentiation into cardiomyocytes. Using conditioned media from sorted BMMNC, we found that the cardioprotective effects were mediated by growth hormone (GH) secreted from myeloid (Gr-1(+)) cells and the effects was partially mediated by signal transducer and activator of transcription 3 in cardiomyocytes. On the other hand, the GH expression in Gr-1(+) cells was significantly downregulated in DCM mice compared with that in healthy control, suggesting that the environmental cue in heart failure might suppress the Gr-1(+) cells function. Activin A was upregulated in the serum of DCM models and induced downregulation of GH levels in Gr-1(+) cells and serum. Furthermore, humoral factors upregulated in heart failure including angiotensin II upregulated activin A in peripheral blood mononuclear cells (PBMNC) via activation of NFκB. Similarly, serum activin A levels were also significantly higher in DCM patients with heart failure than in healthy subjects and the GH levels in conditioned medium from PBMNC of DCM patients were lower than that in healthy subjects. Inhibition of activin A increased serum GH levels and improved cardiac function of DCM model mice. These results suggest that activin A causes heart failure by suppressing GH activity and that inhibition of activin A might become a novel strategy for the treatment of heart failure.

Figure 1. Transgenic overexpression of EGFRdn in the heart causes progressive heart failure.

(A) Schematic representation of the cDNA construct used to generate EGFRdn mice. The construct contains an αMHC promoter, human EGFRdn cDNA and a human growth hormone polyadenylation signal (Hgh-pA). (B) Kaplan-Meier survival curves for wild-type (n = 62) and EGFRdn (L2–5, n = 19; L9–12, n = 21) mice, showing a significant reduction in the survival rates in EGFRdn mice (log rank test, P<0.0001). (C) Gross morphology of whole hearts (upper panels) and longitudinal sections (lower panels) of hearts from wild-type and EGFRdn mice (L9–12) at 6 weeks of age. Ao, aorta; LA, left atrium; LV, left ventricle; RA, right atrium; RV, right ventricle. Scale bars: 2 mm. (D) Heart-to-body weight ratios (HW/BW) of wild-type (n = 9) and EGFRdn (L9–12, n = 7) mice at 6 weeks of age. *P<0.01. (E) Echocardiographic analysis. The upper photographs show representative M-mode images. The lower graphs show the left ventricular diastolic and systolic dimensions and FS of 8 week-old EGFRdn mice (L9–12) (n = 23) and age-matched wild-type mice (n = 10). LVDd, left ventricular diastolic dimension; LVDs, left ventricular systolic dimension. Data are means ± s.e.m.

Figure 2. BMMNC infusion transiently improved the cardiac function of DCM mice.

(A) Echocardiographic analysis. Transient improvements of FS were observed at 3 d in the BMMNC-treated group, but not the control (PBS) group, in EGFRdn mice (left), and at 3 and 7 d in DOX-treated mice (right). *p<0.05 versus PBS (n = 8 per group). (B) Repeated-infusion experiments. BMMNC were infused every 2 weeks. A similar pattern of improvement in FS was observed after each infusion. *p<0.05 versus PBS (n = 8 per group). (C–E) Immunohistochemical analysis. (C) Left, the number of GFP-positive BMMNC in peripheral blood (n = 3). Right, photomicrographs of peripheral blood. Nuclei were stained with Hoechst 33258 (blue). Scale bars, 75 µm. (D) Images of the spleen 3 d after infusion. Many GFP-positive cells were observed in the spleen (lower photographs). Upper photographs, negative control. Nuclei were stained with Hoechst (blue color). Scale bars, 25 µm. (E) No GFP-positive cells were observed in any organs. Upper photographs, negative control. Middle and lower photographs, images taken at 3 and 14 d, respectively, after infusion. The vessels were stained with smooth muscle cell actin (red). Nuclei were stained with Hoechst 33258 (blue). The photographs of muscle are merged fluorescent and phase-contrast images. Scale bars, 75 µm. Data are means ± s.e.m.

Figure 3. BMMNC-derived CM directly affects cardiomyocyte contractility.

(A) Cell shortening and the beating rate of neonatal rat cardiomyocytes were significantly increased after exposure to CM from BMMNC compared with the control (n = 26 cells per group). The left and right graphs show the results at 30 min and at 12 h after treatment, respectively. Upper graph, cell shortening. Lower graph, beating rate. (B) CM from Gr-1(+) cells improved the cell shortening and increased the beating rate similar to that achieved by CM from BMMNC (n = 27 per group). (C, D) Effects of CM from Gr-1 cells on cardiac function in vivo. (C) Echocardiographic analysis (n = 7). The infusion of CM from Gr-1(+) cells significantly improved the FS of DOX mice at 1 and 3 d. (D) Infusion of CM from Gr-1(+) cells significantly improved the +dp/dt of DOX mice at 1 d, in vivo (n = 7). n.s., not significant. Data are means ± s.e.m.

Figure 4. Analysis of secreted factors.

(A) CM from Gr-1(+) cells from wild-type mice significantly improved the cell shortening and increased the beating rate in neonatal rat cardiomyocytes, as compared with CM from Gr-1(+) cells from EGFRdn mice. Left graph, cell shortening (n = 24 cells per group). Right graph, beating rate (n = 24 cells per group). (B) Quantitative RT-PCR analysis of GH mRNA in Gr-1(+) cells isolated from wild-type mice and EGFRdn mice (n = 4). (C, D) GH concentrations in (C) CM from Gr-1(+) cells isolated from wild-type mice and EGFRdn mice (n = 4) and (D) CM from PBMNC isolated from healthy (n = 11) and DCM subjects (n = 10). (E) GH concentration in serum from several mouse models of heart failure (n = 4). Data are means ± s.e.m.

Figure 5. GH mediates the cardioprotective effects of Gr-1(+) cell-derived CM.

(A) Pegvisomant (PEG) treatment inhibited the Gr-1(+) cell CM-mediated improvements cardiomyocyte cell shortening and beating rate at 30 min and at 12 h after treatment (n = 27 cells per group). Left graphs, cell shortening; right graphs, beating rate. (B) Anti-IGF-1 antibody failed to affect the Gr-1(+) cell CM-mediated improvements in cell shortening or beating rate at 30 min or at 12 h after treatment (n = 23 cells per group). Left graphs, cell shortening; right graphs, beating rate. (C) GH and CM from Gr-1(+) cells phosphorylated Akt, Erk, Jak2, Stat3/5 and PKA in cardiomyocytes (n = 3), which was inhibited by pegvisomant (n = 3). (D) GH (500 pg/ml) and CM from Gr-1(+) cells increased the cAMP concentration in cardiomyocytes (n = 5), which was inhibited by pegvisomant (n = 5). (E, F) Cardiac function analysis by echocardiography (upper graphs, n = 8) and catheterization (lower graphs, n = 8). Pegvisomant (E), but not anti-IGF-1 antibody (F), inhibited the improvements in FS and +dp/dt elicited by the infusion of CM from Gr-1(+) cells. *p<0.05 (n = 8). (G) Serum GH concentrations in DOX mice treated with CM from Gr-1(+) cells (n = 4 per group). The infusion of CM from Gr-1(+) cells from wild-type mice increased the serum GH concentration at 1 d, but not at 5 d. Data are means ± s.e.m.

Figure 6. Regulatory mechanisms of GH in heart failure.

(A) The serum activin A concentration was higher in EGFRdn mice (left, n = 5) and in DCM patients (right, n = 10) than in wild-type mice (n = 5) and healthy subjects (n = 11). (B) Activin A downregulated GH mRNA expression in Gr-1(+) cells and GH protein levels in Gr-1(+) cell CM. Left graph, GH protein concentration; middle photographs, representative semi-quantitative RT-PCR images; right graph, GH mRNA expression (n = 3). (C, D) AngII upregulated activin A secretion (C, n = 4) and phosphorylated NFκB expression (D, n = 5) in wild-type PBMNC. (D) Left graph, total NFκB; right graph, phosphorylated NFκB. (E) Inhibition of NFκB [50 µM; NFκB p65 (Ser276) inhibitory peptide] suppressed AngII (10 µM)-mediated upregulation of activin A in CM derived from wild-type PBMNC (n = 5). Isotype peptide was used as control. (F) The GH concentration in CM from EGFRdn Gr-1(+) cells (n = 5) was significantly increased by treatment with an anti-activin A antibody (n = 5). (G) Effects of anti-activin A antibody treatment on cell shortening and the beating rate of cardiomyocytes induced by CM from Gr-1(+) cells isolated from EGFRdn mice (n = 18 cells per group). (H) Treatment with the anti-activin A antibody improved the cardiac function of EGFRdn mice. Left graph, echocardiography (n = 7). Middle graph, miller catheter results (n = 7). Right graph, serum GH concentration in EGFRdn mice after antibody treatment (n = 7). Data are means ± s.e.m. (I) Proposed mechanism underlying impaired GH expression by activin A in heart failure.