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, 120 (2), 408-21

Heart Failure Causes Cholinergic Transdifferentiation of Cardiac Sympathetic Nerves via gp130-signaling Cytokines in Rodents

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Heart Failure Causes Cholinergic Transdifferentiation of Cardiac Sympathetic Nerves via gp130-signaling Cytokines in Rodents

Hideaki Kanazawa et al. J Clin Invest.

Abstract

Although several cytokines and neurotrophic factors induce sympathetic neurons to transdifferentiate into cholinergic neurons in vitro, the physiological and pathophysiological roles of this remain unknown. During congestive heart failure (CHF), sympathetic neural tone is upregulated, but there is a paradoxical reduction in norepinephrine synthesis and reuptake in the cardiac sympathetic nervous system (SNS). Here we examined whether cholinergic transdifferentiation can occur in the cardiac SNS in rodent models of CHF and investigated the underlying molecular mechanism(s) using genetically modified mice. We used Dahl salt-sensitive rats to model CHF and found that, upon CHF induction, the cardiac SNS clearly acquired cholinergic characteristics. Of the various cholinergic differentiation factors, leukemia inhibitory factor (LIF) and cardiotrophin-1 were strongly upregulated in the ventricles of rats with CHF. Further, LIF and cardiotrophin-1 secreted from cultured failing rat cardiomyocytes induced cholinergic transdifferentiation in cultured sympathetic neurons, and this process was reversed by siRNAs targeting Lif and cardiotrophin-1. Consistent with the data in rats, heart-specific overexpression of LIF in mice caused cholinergic transdifferentiation in the cardiac SNS. Further, SNS-specific targeting of the gene encoding the gp130 subunit of the receptor for LIF and cardiotrophin-1 in mice prevented CHF-induced cholinergic transdifferentiation. Cholinergic transdifferentiation was also observed in the cardiac SNS of autopsied patients with CHF. Thus, CHF causes target-dependent cholinergic transdifferentiation of the cardiac SNS via gp130-signaling cytokines secreted from the failing myocardium.

Figures

Figure 1
Figure 1. Distribution of sympathetic and parasympathetic nerves in the rat heart.
(A) Immunofluorescence staining for TH, CHT (green), and α-actinin (red) in the rat LV. TH+ nerves were more abundant in the subepicardial layer (epi) than the subendocardial layer (endo). The arrow indicates sympathetic nerves at the epicardial surface. No CHT+ nerves were observed at the epicardial surface, and CHT+ nerves were more abundant in the subendocardial layer. Higher-magnification views of the boxed regions are shown in the insets. (B) Quantitative analysis of TH+ nerve area of LVs (n = 5). (C) Quantitative analysis of CHT+ nerve area of LVs (n = 5). (D) Representative immunostaining for TH (red), ChAT (green), and Toto3 (blue) in rat stellate ganglia and cardiac ganglia. A ChAT+ neuron (arrow, top row) was surrounded by TH+ neurons in the stellate ganglia. A few TH+ cells (arrow, bottom row) were observed in cardiac ganglia. Higher-magnification views of the boxed regions in the third column are shown in the fourth column. Representative data are shown in each panel. *P < 0.01. Scale bars: 100 μm (A); 50 μm (A, insets, and D).
Figure 2
Figure 2. Cardiac nerve fibers and stellate ganglia neurons in DS rat ventricles have both catecholaminergic and cholinergic activity.
(A) Confocal images of double immunofluorescence staining for TH (red) and CHT (green) in LVs at the epicardium in DR and DS rats. Numbers of CHT+ nerve fibers (arrowheads) were increased, and some nerves coexpressed TH. Higher-magnification views of the boxed regions in the third column are shown in the fourth column. Representative data are shown in each panel. (B and C) Quantitative analysis of the TH+/NF+ and CHT+/NF+ area ratios in LVs at the epicardium (n = 5). (D) Double immunostaining with TH (red) and ChAT (green) of the stellate ganglia in DR and DS rats. Note that DS rats had more ChAT+ neurons. Some cells coexpressed TH (arrowheads), some did not (arrow). (E) Quantitative analysis of the ChAT+/TH+ ratio in the stellate ganglia (n = 4). (F) Cht1 expression in the stellate ganglia of DR and DS rats, determined by qRT-PCR (n = 4). (G) VAChT expression in the stellate ganglia of DR and DS rats, determined by qRT-PCR (n = 4). *P < 0.01. Scale bars: 20 μm (A); 100 μm (D); 5 μm (A, zoom); 50 μm (D, zoom).
Figure 3
Figure 3. Cardiac sympathetic nerve fibers acquire cholinergic activity in the ventricles of rats with CHF.
(A) BDA injection into the left stellate ganglion (left panel); a stereomicroscopic image of the stellate ganglion (middle panel); and BDA after injection, visualized as green signals (right panel) are shown. SG, stellate ganglia. (B) Epicardial sympathetic nerves in the DS rat LV. BDA (green, arrowheads) was transported to the cardiac sympathetic nerve fascicules. Some fibers coexpressed TH (red, arrowheads), but others did not (arrow). BDA+ nerve fibers (green, arrowheads) coexpressed CHT, indicating CHT+ nerve fibers were derived from sympathetic nerves. Both cross and longitudinal sections are shown. (C) Transmission electron micrographs of the nerve endings in DR and DS rat LVs. Black arrows indicate SGVs in sympathetic nerve endings (SN). White arrows indicate SAGVs in parasympathetic nerve endings (PSN). (D) Immunoelectron micrographs of nerve endings in DR and DS rat LVs. Rats were injected with AF64A (cholinergic nerve fiber toxin), and the sections were immunostained with TH and silver-enhanced nanogold (NG) particles (red arrows). AF64A induced vacuoles (blue asterisks) and degeneration (blue star). The black arrows indicate SGVs. A higher-magnification view of the boxed region is shown in the inset. Representative data are shown in each panel. Scale bars: 2 mm (A, middle panel); 100 μm (A, right panel); 50 μm (B, left cross section panel and both longitudinal sections); 20 μm (B, right cross section panel); 0.5 μm (C and D); 10 μm (B, insets); 0.1 μm (D, inset).
Figure 4
Figure 4. Cardiomyocytes secrete cholinergic differentiation factors that can induce neurotransmitter switching in cultured sympathetic neurons.
(A) Expression of IL-6 family cytokines (Lif, Ctf1, cardiotrophin-like cytokine [Clc], ciliary neurotrophic factor [Cntf]), neurotrophins (nerve growth factor [Ngf], brain-derived neurotrophic factor [Bdnf], neurotrophin-3 [Ntf3]), Gdnf, angiotensinogen (Agt), and angiotensin-converting enzyme (Ace) mRNA in DR and DS rat ventricles was determined by qRT-PCR (n = 4–6). (B) Primary cultured cardiomyocytes were stimulated with Ang II, H2O2, doxorubicin (DOX), NE, ceramide (Cer), hypoxia (5% O2, Hypo), or 20% mechanical stretch. Expression of Lif and Ctf1 mRNA was investigated using qRT-PCR (n = 4). (C) Expression of IL-6 family cytokines and neurotrophins in Ang II–CMs were determined by qRT-PCR (n = 4). (D) Effect of unstimulated and Ang II–CM-conditioned media on the levels of expression of Th, Cht1, and VAChT in cultured sympathetic neurons (n = 3). (E) Effect of LIF on the levels of expression of Th, Cht1, and VAChT in cultured sympathetic neurons (n = 3). RT-PCR analysis of Chat and VAChT in cultured sympathetic neurons stimulated with LIF. (F) Immunocytochemical staining for TH, ChAT, and Toto3 in cultured sympathetic neurons. (G) Effect of preincubation of siRNA on the expression of Lif and Ctf1 on VAChT in cultured sympathetic neurons. Representative data are shown in each panel. *P < 0.01, **P < 0.05 compared with control (B); *P < 0.01; **P < 0.05 (A, CE, and G). Scale bars: 100 μm.
Figure 5
Figure 5. Cardiac-specific overexpression of LIF induces neurotransmitter switching in the cardiac sympathetic nervous system.
(A) Schema of generation of LifαMHC-Cre mice using the Cre-loxP system. (B) RT-PCR and Western blot analysis of Lif mRNA expression in LifαMHC-Cre mice (line 10). (C) Quantitative analysis of LIF expression in lines 3 and 10. (D) Double immunofluorescence staining for α-actinin (red), TH, or CHT (green) in WT and LifαMHC-Cre hearts (line 10). TH+ nerves were decreased in LifαMHC-Cre hearts. In contrast, CHT+ nerves were increased in LifαMHC-Cre hearts. A higher-magnification view of the boxed region is shown in the inset. (E) Quantitative analysis of TH+ and CHT+ nerve areas in the hearts of WT, αMHC-Cre, and Lif-loxP mice and 2 lines of LifαMHC-Cre mice (n = 5). (F) NE content was lower in the ventricles of LifαMHC-Cre mice (line 10) than in those of WT, αMHC-Cre, and Lif-loxP littermates or line 3 mice. (n = 5). (G) Representative immunostaining for TH (red), ChAT (green), and Toto3 (blue) in WT and LifαMHC-Cre stellate ganglia (line 10). (H) Quantitative analysis of ChAT+/TH+ ratios in the stellate ganglia in WT, αMHC-Cre, Lif-loxP, and LifαMHC-Cre (lines 3 and 10) mice (n = 3). Representative data are shown in each panel. *P < 0.01; **P < 0.05. Scale bars: 50 μm (D); 100 μm (G); 25 μm (D, inset).
Figure 6
Figure 6. Conditional gp130 gene targeting in sympathetic nerves prevents heart failure–induced cholinergic transdifferentiation.
(A) Immunofluorescent staining in the LV, stellate ganglia, and adrenal grand (AG) of EGFPDBH-Cre mice. Green and blue signals indicate GFP and Toto3 (nucleus), respectively. (B) Quantitative analysis of the percentage of EGFP+ neurons in EGFPDBH-Cre mice (n = 3). (C) Conditional gp130 gene-targeted mice (gp130DBH-Cre mice) and control mice (gp130flox/flox mice) were subjected to heart failure according to the TAC or hypoxia models. Immunofluorescent staining with anti-TH (red) or anti-CHT (green) antibody was performed on failing or control myocardium. (D) Quantitative analysis of the TH+/NF+ and CHT+/NF+ area ratios in LVs and RVs is indicated (n = 4). (E) gp130DBH-Cre and gp130flox/flox mice were subjected to heart failure, and immunofluorescent staining with TH (red) and ChAT (green) was performed on the stellate ganglia. (F) Quantitative analysis of the ChAT+/TH+ ratio in stellate ganglia is shown (n = 4). Higher-magnification views of the boxed regions are shown in the insets (A, C, and E). Scale bars: 100 μm (A and E); 20 μm (C and insets in A and E); 5 μm (C, insets). *P < 0.01; **P < 0.05.
Figure 7
Figure 7. Cardiac function and Kaplan-Meier analysis of gp130flox/flox and gp130DBH-Cre mice under chronic hypoxia.
(A) Measurement of the RVSP under chronic hypoxia at 8 weeks. (B) Measurement of the heart rate under chronic hypoxia at 8 weeks. (C) Representative recordings of maximum (max) RVdP/dt under chronic hypoxia at 8 weeks. (D) Quantitative analysis of maximum RVdP/dt under control conditions and under chronic hypoxia at 8 weeks. (E) Kaplan-Meier analysis of the cumulative survival under control conditions and under chronic hypoxia. *P < 0.01; **P < 0.05.
Figure 8
Figure 8. Histological analysis of human sample.
(A) H&E and Masson trichrome staining of the LV and cardiac sympathetic nerves in the control group. SN, sympathetic nerve; H-E low, H&E staining with low magnification; H-E high, H&E staining with high magnification. (B) H&E and Masson trichrome staining of the LV and cardiac sympathetic nerves in the heart failure group. (C) Nissl staining of stellate ganglia in the control and CHF patients. (D) Quantitative analysis of stellate ganglia neuron numbers in the control and heart failure group (n = 5). Representative data are shown in each panel. Scale bars: 100 μm (A and B); 200 μm (C).
Figure 9
Figure 9. Neurotransmitter switching in the human cardiac sympathetic nervous system.
(A) Representative immunostaining for TH (red) and CHT (green) in LVs at the epicardial sites in control and heart failure patients. Cross and longitudinal sections are shown. The longitudinal image is composed of 3 serial images. The heart failure group has fewer TH+ nerves and markedly more CHT+ nerves than the control group. In the ventricle of heart failure patients, some CHT+ nerves coexpress TH (arrowhead). The longitudinal section also revealed sympathetic nerves coexpressing TH and CHT. Higher-magnification views of the boxed regions are shown in the insets (right panels). (B) Quantitative analysis of TH+ and CHT+ nerve areas in LV (n = 4). (C) Representative immunostaining for TH (red) and ChAT (green) in the stellate ganglia of control and heart failure patients. Arrows indicate ChAT+ cells, and arrowheads indicate TH+/ChAT+ neurons. The heart failure group has fewer TH+ cells and more ChAT+ cells than the control group. (D) Quantification of TH+ and ChAT+ cells per total number of neurons in the stellate ganglia (n = 5). Representative data are shown in each panel. *P < 0.01. Scale bars: 10 μm (A, insets); 50 μm (A and inset in D); 100 μm (C).

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