IL-6 loss causes ventricular dysfunction, fibrosis, reduced capillary density, and dramatically alters the cell populations of the developing and adult heart

Abstract

Interleukin-6 (IL-6) is a pleiotropic cytokine responsible for many different processes including the regulation of cell growth, apoptosis, differentiation, and survival in various cell types and organs, including the heart. Recent studies have indicated that IL-6 is a critical component in the cell-cell communication between myocytes and cardiac fibroblasts. In this study, we examined the effects of IL-6 deficiency on the cardiac cell populations, cardiac function, and interactions between the cells of the heart, specifically cardiac fibroblasts and myocytes. To examine the effects of IL-6 loss on cardiac function, we used the IL-6(-/-) mouse. IL-6 deficiency caused severe cardiac dilatation, increased accumulation of interstitial collagen, and altered expression of the adhesion protein periostin. In addition, flow cytometric analyses demonstrated dramatic alterations in the cardiac cell populations of IL-6(-/-) mice compared with wild-type littermates. We observed a marked increase in the cardiac fibroblast population in IL-6(-/-) mice, whereas a concomitant decrease was observed in the other cardiac cell populations examined. Moreover, we observed increased cell proliferation and apoptosis in the developing IL-6(-/-) heart. Additionally, we observed a significant decrease in the capillary density of IL-6(-/-) hearts. To elucidate the role of IL-6 in the interactions between cardiac fibroblasts and myocytes, we performed in vitro studies and demonstrated that IL-6 deficiency attenuated the activation of the STAT3 pathway and VEGF production. Taken together, these data demonstrate that a loss of IL-6 causes cardiac dysfunction by shifting the cardiac cell populations, altering the extracellular matrix, and disrupting critical cell-cell interactions.

Fig. 1.

Interleukin-6 (IL-6) loss causes cardiac abnormities. A and B: representative images of adult wild-type (WT) and IL-6^−/− hearts, respectively. C and D: representative 100-μm sections of left (LV) and right ventricular (RV) chambers of WT and IL-6^−/− identically fixed hearts, respectively. E: analysis of heart weight (HW) normalized to body weight (BW). F: analysis of HW compared with tibia length (TL). Scale bars set to 1 mm. N = 5 animals per condition. *P < 0.05 vs. WT.

Fig. 2.

Ventricular collagen levels in IL-6^−/− mice. A and B: representative Masson's trichrome-stained sections from WT and IL-6^−/− LV chambers, respectively. C: quantification of collagen content in the LV and RV free walls and intraventricular septa (IVS). D: quantification of cardiac collagen content via hydroxyproline analysis in adult mice. N = 4–6 animals per condition. #P < 0.001 and *P < 0.05 vs. WT.

Fig. 3.

Periostin levels in IL-6^−/− mice. A and B: total heart periostin levels as determined by Western blot analysis and densitometry. Representative blot shown. C: total heart periostin mRNA levels as determined by real-time PCR analyses. D and E: analysis of cardiac fibroblast-myocyte adhesion in vitro. The total number of adherent cardiac fibroblasts plated onto myocytes (Myo) is shown. Cardiac fibroblasts or myocytes were isolated from neonatal WT and IL-6^−/− hearts, and different combinations were plated as indicated. Con, conditioned. N = 5–7 animals per condition. *P < 0.05 vs. WT.

Fig. 4.

Cardiac cell populations of IL-6^−/− mice. A: representative flow cytometry experiment. B: cell populations of the WT and IL-6^−/− heart as determined by flow cytometry analysis of cells labeled with immunospecific markers conjugated to quantum dot. #P < 0.001 and *P < 0.05 vs. WT. N = 3 to 4 animals per condition. DDR2, discoidin domain receptor-2; α-MHC, α-myosin heavy chain; VSMC, vascular smooth muscle cell; α-SMA, α-smooth muscle actin.

Fig. 5.

Analysis of proliferation and apoptosis in IL-6^−/− mice. A: analyses of proliferation by phospho-histone H3, tropomyosin, and wheat germ agglutinin staining in neonatal day-5 WT and IL-6^−/− hearts. B: analyses of apoptosis via cleaved poly(ADP-ribose) polymerase (cleaved PARP), tropomyosin, and wheat germ agglutinin staining in day-5 hearts from WT and IL-6^−/− mice. N = 5 experiments. #P < 0.01.

Fig. 6.

Cardioangiography analysis. A and B: representative cardiac microsphere angiography images from WT and IL-6^−/− mice, respectively. C: capillary density for WT and IL-6^−/− hearts. N = 3 to 4 experiments. *P < 0.05. Scale bar equals 50 μm.

Fig. 7.

Analysis of fibroblast-myocyte signaling interactions. A: representative Western blot analyses of STAT3, MAPK p42/44, and AKT activation of in vitro coculture analyses. B: ELISA analyses of fold increase of VEGF levels secreted in the media of cardiac fibroblasts-myocyte cocultures at 24 h. KO, knockout; Rc, receptor. N = 3 experiments. *P < 0.05.