Toll4 (TLR4) expression in cardiac myocytes in normal and failing myocardium

Abstract

Expression of innate immune response proteins, including IL-1beta, TNF, and the cytokine-inducible isoform of nitric oxide synthase (iNOS), have been documented in the hearts of humans and experimental animals with heart failure regardless of etiology, although the proximal events leading to their expression are unknown. Noting that expression of a human homologue of Drosophila Toll, a proximal innate immunity transmembrane signaling protein in the fly, now termed human Toll-like receptor 4 (hTLR4), appeared to be relatively high in the heart, we examined TLR4 mRNA and protein abundance in isolated cellular constituents of cardiac muscle and in normal and abnormal murine, rat, and human myocardium. TLR4 expression levels in cardiac myocytes and in coronary microvascular endothelial cells could be enhanced by either LPS or IL-1beta, an effect inhibited by the oxygen radical scavenger PDTC. Transfection of a constitutively active TLR4 construct, CD4/hTLR4, resulted in activation of a nuclear factor-kappaB reporter construct, but not of an AP-1 or an iNOS reporter construct, in cardiac myocytes. In normal murine, rat, and human myocardium, TLR4 expression was diffuse, and presumably cytoplasmic, in cardiac myocytes. However, in remodeling murine myocardium remote from sites of ischemic injury and in heart tissue from patients with idiopathic dilated cardiomyopathy, focal areas of intense TLR4 staining were observed in juxtaposed regions of 2 or more adjacent myocytes; this staining was not observed in control myocardium. Increased expression and signaling by TLR4, and perhaps other Toll homologues, may contribute to the activation of innate immunity in injured myocardium.

Figure 1

Alignment of the hTLR4 and rTLR4 sequences. A full-length rTLR4 cDNA was sequenced by RACE PCR from a rat cDNA library using the hTLR4 sequence to design primers. The full-length rTLR4 contains 3,385 bp with an open reading frame of 2,508 bp, encoding a protein of 836 amino acids. The hTLR4 and rTLR4 sequences exhibited an overall 84% homology in DNA sequences (top, rTLR4; bottom, hTLR4; alignment by an algorithm: http://vega.igh.cnrs.fr; : = amino acid identity; . = conservative substitution).

Figure 2

Expression of rTLR4 in cardiac myocytes and coronary microvascular endothelial cells. Representative Northern (a) and Western (b) blots for TLR4 in homogenized rat heart tissue. (c and d) Representative Northern blot and aggregate data from 5 independent experiments for basal levels of TLR4 mRNA abundance in ARVMs, NRVMs, and CMECs in primary cultures after normalization to GAPDH (**P ≤ 0.01). An approximately 5-fold higher basal level of TLR4 mRNA was present in the endothelial cell preparation compared with the cardiac myocytes.

Figure 3

Time course of rTLR4 expression in response to LPS. Primary cultures of confluent CMECs (a) and NRVMs (b) were exposed to 10 μg/mL LPS for the periods indicated. Cumulative data from 5 independent experiments for both cell types are shown (*P ≤ 0.05). Data have been normalized to GAPDH mRNA content. The rTLR4 mRNA content at 24 hours was defined as 1 arbitrary unit.

Figure 4

Role of NF-κB in regulating the expression of rTLR4 mRNA. CMECs and NRVMs were treated as indicated in the text for 24 hours with either LPS or cytokines, with and without the NF-κB inhibitor PDTC. The Northern blots shown are representative of 4 independent experiments.

Figure 5

PDTC inhibits upregulation of rTLR4 expression in vitro in response to IL-1β or LPS. CMECs and NRVMs were exposed to LPS or cytokines in the presence or absence of PDTC as indicated. This Western blot is representative of 3 independent experiments and illustrates a 96-kDa band consistent with TLR4. CMECs were treated for 36 hours, and NRVMs for 48 hours, with LPS, cytokines, or the NF-κB inhibitor PDTC, as indicated.

Figure 6

EMSAs of NF-κB and AP-1 in response to LPS and IL-1β. EMSAs were also performed on nuclear extracts from CMECs (a) and NRVMs (b) for NF-κB– and AP-1–mediated signaling. The data shown are representative of 3 experiments.

Figure 7

CD4/TLR4 activates NF-κB in cardiac myocytes. A constitutively active TLR4 construct, consisting of CD4 linked to the hTLR4 transmembrane and TIR domains, was transfected into NRVM primary cultures at concentrations of 0.01, 0.1, and 0.5 μg/well, and the extent of activation of an NF-κB–luciferase construct was measured. Data are shown as fold induction of luciferase activity over that of mock-transfected (control) myocytes, with the average activity of mock-transfected cells arbitrarily given as 1. The data are from 32 independent experiments (**P < 0.01 vs. control).

Figure 8

rTLR4 mRNA expression in murine ventricular muscle after myocardial infarction. rTLR4 mRNA levels in apical ventricular muscle of sham-operated and infarcted animals are shown after normalization to GAPDH (*P < 0.05). Approximately 2.5-fold higher mRNA was present in infarcted animals compared with sham-operated animals at day 4 after the procedure.

Figure 9

Immunohistochemical analysis of TLR4 in rat, murine, and human cardiac muscle. Photomicrographs are shown for primary isolates of ventricular myocytes isolated from adult rat hearts (×400) 24 hours after isolation, stained with a polyclonal Toll antibody targeted to a TLR4-specific epitope adjacent to the cytoplasmic TIR domain of hTLR4. Absorption with the peptide used to generate the primary antibody (Anti-Toll + peptide) and substitution of the primary antibody with a control rabbit antiserum (Control serum) were used as controls. Both normal murine cardiac muscle (×200), as shown here, and sham-operated murine cardiac muscle and normal rat cardiac muscle (not shown) exhibited diffuse, homogeneous myocyte staining. However, cardiac myocytes adjacent to an area of ischemic injury (Infarct) in the mouse exhibited intense sarcolemmal staining for TLR4. Note the absence of significant staining of infiltrating inflammatory cells (×400). Finally, in samples from both humans with a dilated cardiomyopathy (bottom left panel) and in remodeling murine ventricular muscle remote from the site of ischemic injury (not shown), intensely stained focal expression of TLR4 was observed in adjacent regions of 2 or more juxtaposed cardiac myocytes (×600). This intense focal staining pattern for TLR4 was not observed in sections of normal human myocardium (not shown).