There are several key findings of this study. First, TNFR1- and TNFR2-dependent signaling had unique effects on post-infarction remodeling in vivo, such that TNFR1 aggravated, whereas TNFR2 ameliorated, chamber remodeling and hypertrophy. Second, the impact on cardiac mechanics and survival were more complex: whereas TNFR1- and TNFR2-responses magnified and alleviated, respectively, LV systolic dysfunction, signaling through both receptors was necessary to increase post-infarction mortality (due to myocardial rupture) and to induce diastolic dysfunction. Third, TNFR1- and TNFR2-induced remodeling responses were accompanied by exacerbation and moderation of cardiac inflammation as assessed by NF-κB activation, inflammatory cytokine expression, p38 MAPK phosphorylation, and macrophage infiltration. Fourth, in H9c2 cardiomyocytes, TNFR1 augmented whereas TNFR2 moderated NF-κB activation and sustained NF-κB activation was pro-apoptotic in a TNFR1-dependent manner. Fifth, TNFR1 was pro-apoptotic and TNFR2 anti-apoptotic in the failing heart in vivo, whereas signaling via both receptors cooperatively augmented oxidative stress. Taken together, we have demonstrated complex pathophysiological responses in HF specific to each TNFR that are related in large part to disparate, opposing effects on NF-κB, inflammatory activation, and apoptosis. Analogous dichotomous TNFR-mediated responses in human HF may therefore help explain the unexpectedly negative results of clinical trials of global TNF blockade.
Although the “cytokine hypothesis” posits a uniformly detrimental effect of TNF in HF, TNF has bimodal effects on contractility [17
] and is cardioprotective during acute stress [6
]. As shown in and , TNF, TNFR1, and TNFR2 are all upregulated during the progression of remodeling in murine HF, indicating uniform enhancement of TNF signaling. This contrasts with end-stage human HF where TNF levels are high but both TNFRs are downregulated [18
]. Exacerbation of LV remodeling in TNFR2−/− HF mice occurred despite similar degrees of upregulation of both TNF and TNFR1, suggesting that unique cardioprotective benefits are referable to TNFR2 in HF. Moreover, amelioration of remodeling in TNFR1−/− HF mice occurred without an increase in TNFR2 expression and despite persistent (though attenuated) TNF upregulation, suggesting that detrimental biological responses in HF are uniquely referable to TNFR1. Thus, our results demonstrate that TNFR1 promotes detrimental remodeling whereas TNFR2 is cardioprotective in HF with regard to chamber remodeling, systolic dysfunction, and hypertrophy.
These generalized effects on post-infarction remodeling notwithstanding, the complex functional interrelationship between the TNFRs in HF is evidenced by the cooperative, rather than divergent, effects of TNFR1 and TNFR2 on LV diastolic performance and survival, as loss of signaling via either TNFR improved diastolic function and mortality post-infarction. Prior studies have established that the most prevalent cause of death following infarction in mice is LV rupture (usually within the first week), that TNF directly contributes to cardiac rupture, and that this event is related to activation of MMPs, particularly MMP-2 and MMP-9, in the heart [15
]. MMP-2 and MMP-9 activities increase by day 3, peak at day 7, and remain elevated to day 28 post-infarction [19
]. In our study, early LV rupture was prevented in both TNFR1−/− and TNFR2−/− HF mice, with both groups exhibiting less infarct and non-infarct zone MMP-2 and MMP-9 expression at 28 days as compared to WT HF. This suggested that analogous MMP modulation with loss of either TNFR1 or TNFR2 function was also occurring at earlier time points after infarction, offering one potential mechanism for the reduced mortality in TNFR1−/− and TNFR2−/− mice. Hence, joint functionality of both TNF receptors was required for LV rupture to occur in the early post-infarction period. Notably, this mortality benefit was independent of the subsequent effects of TNFR1 and TNFR2 on LV remodeling. However, we speculate that the divergent TNFR-specific effects on progressive LV remodeling would secondarily impact mortality over extended periods of time after scar stabilization.
As there was improved global remodeling in TNFR1−/− HF, accompanying improvements in diastolic function would be expected with TNFR1 deficiency. Indeed, there were generalized reductions in CTGF expression and cardiac fibrosis in TNFR1−/− HF hearts, which would favorably influence LV diastolic properties. More difficult to reconcile is the maintenance of diastolic function in TNFR2−/− HF mice despite worsening of chamber remodeling. As LV rupture was abrogated in these mice, these effects may be related to improved scar mechanics and/or border zone stability. Indeed, although the overall extent of cardiac fibrosis was similar in TNFR2−/− and WT HF, there was greater border zone collagen deposition that can potentially better resist rupture and favorably influence diastolic performance. However, it is important to recognize that the degree and distribution of myocardial fibrosis may itself also be influenced by altered global/regional wall stress, and whether the changes in connective tissue composition are a cause or consequence of altered chamber diastolic properties and wall stress is difficult to resolve with our experimental design.
A key finding of our study is that TNFR1 and TNFR2 had directionally opposite effects on NF-κB and inflammation in HF, and that these events contributed to the differences in LV remodeling. TNFR1 recruits adaptor proteins via its death domain to trigger TRAF2-dependent signaling that activates NF-κB, JNK, and p38 MAPK [9
]. TNFR2 can also activate NF-κB, JNK, and p38 MAPK via direct TRAF2 binding. TNF can also induce apoptosis via either TNFR and trigger the generation of reactive oxygen species (ROS) [9
]. We observed robust myocardial NF-κB activation in HF that was due almost entirely to p65. The failing heart also exhibited significant p38 and JNK2 activation, both of which have significant pro-inflammatory effects [21
], upregulation of pro-inflammatory TNF, IL-1β, IL-6 and anti-inflammatory IL-10, and enhanced tissue infiltration of activated macrophages, albeit at low absolute levels. Hence, there was a pro-inflammatory state in WT HF, consistent with prior studies [1
]. In TNFR1−/− HF there was attenuation of NF-κB activation, p38 and JNK phosphorylation, and TNF, IL-1β, IL-6 and IL-10 expression as compared to WT HF, and no significant activated macrophage infiltration as compared to TNFR1−/− sham. In contrast, TNFR2−/− HF hearts exhibited greater NF-κB activation, p38 MAPK phosphorylation, and IL-1β and IL-6 expression, and less anti-inflammatory IL-10 expression compared to WT HF, and greater activated macrophage infiltration than TNFR1−/− HF. Thus, our data establish that in chronic HF, TNFR1 is proinflammatory whereas TNFR2 is anti-inflammatory. Moreover, the sharp divergence of TNFR1 and TNFR2 effects on downstream mediators suggests that although acute signaling via the TNFRs may overlap significantly, TNFR crosstalk is much less prominent in chronic HF, leading to dichotomous downstream TNF responses.
Although NF-κB is chronically activated in HF [24
], whether this is protective or detrimental is unclear. In addition to stimulating inflammation, NF-κB upregulates both anti-apoptotic and pro-apoptotic genes [9
], and can potentially induce either survival or death. Our cell studies indicate that p65 and/or p50 overexpression is pro-apoptotic in H9c2 cardiomyocytes via a mechanism that appears independent of changes in classical pro-and anti-apoptotic gene expression. Moreover, analogous to in vivo
HF, TNFR1 increased whereas TNFR2 blunted NF-κB activation. Importantly, the pro-apoptotic effects of NF-κB overexpression required TNF elaboration and concomitant TNFR1 signaling, but was not modified by TNFR2 overexpression. As HF is characterized by increases in both TNF/TNFRs and NF-κB, analogous functional interrelationships between TNFR1 and NF-κB may also occur in the failing heart. Indeed, evaluation of apoptotic rates revealed that in TNFR1−/− HF, attenuated NF-κB activation was paralleled by reduced myocardial apoptosis as compared to WT HF, whereas the opposite response was seen in TNFR2−/− HF. Augmented myocardial TNF expression has been shown to increase oxidative protein modifications in the heart [26
]. However, oxidative stress, as indexed by protein-MDA adducts, was equally reduced in both TNFR1−/− and TNFR2−/− HF, suggesting that the changes in cell survival were not simply epiphenomena accompanying global directional changes in remodeling. Hence, sustained changes in NF-κB activation are likely to underlie many of the divergent remodeling responses related to each TNFR. Indeed, recent studies indicate that post-infarction remodeling is attenuated in p50 null mice [27
]. However, as our data show that NF-κB in the murine failing heart is almost entirely p65, further studies are required to define the relevance of these findings.
Our results extend as well as contrast with recent work in this area by others [29
]. Ramani et al [29
] also reported improved remodeling and survival in TNFR1−/− mice post-infarction over WT, but no differences in TNF and IL-1β expression. Recently, after our original presentation of these data [31
], Monden et al [30
] reported that TNFR1 ablation improved but TNFR2 ablation exacerbated post-infarct remodeling and IL-1β and IL-6 expression. Although these general conclusions are the same, there are also significant differences from our study, which establishes more complex effects of TNFR1 and TNFR2 in HF. Monden et al did not observe a post-infarction mortality benefit in TNFR2−/− mice or differences in LV rupture in either TNFR1−/− or TNFR2−/− mice. Moreover, we observed multifaceted hemodynamic responses in our study, with improved LV diastolic performance in TNFR2−/− HF mice despite exaggerated structural remodeling. Also, unlike our results demonstrating a pro-hypertrophic and pro-fibrotic effect of TNFR1 in the failing heart, Monden et al reported no effects of TNFR1 on these parameters. While the reasons for these conflicting results are not fully clear, potential explanations include the older age of the mice and greater degrees of HF in WT mice (which exhibited a two-fold higher LVEDP) in our study, and perhaps an analytical approach that afforded greater discrimination of subtler differences between the genotypes. Further studies will be needed to resolve this. Most importantly, however, we provide novel mechanistic data that link in vivo remodeling to the primary downstream signaling pathways activated by TNF in the failing heart (particularly NF-kB), as well as to alterations in apoptosis and oxidative stress, and characterize the interrelationship between TNFR1, TNFR2, NF-kB, and cell survival. Indeed, our results indicate for the first time an opposing relationship between TNFR1 and TNFR2 and the activation of NF-kB in HF, and help provide a more comprehensive and mechanistic basis for TNFR-specific remodeling responses.
In summary, TNF induces dichotomous effects in HF that are directly referable to its two membrane receptors, and occur (at least in part) as a result of disparate effects on the critical downstream mediator NF-κB, inflammatory signaling responses, and apoptosis. The overall balance between these opposing receptor-specific responses in turn determines the ultimate impact of TNF on the HF phenotype. Hence, these results provide a potential explanation for the failure of the anti-TNF clinical trials, and, as a corollary, suggest that selective targeting of the individual TNFRs (TNFR1 blockade and/or TNFR2 augmentation) represents a better therapeutic approach in HF.
Clinical Impact Commentary
Despite the seminal observation that tumor necrosis factor-α (TNF) is an important mediator of pathological left ventricular remodeling in heart failure (HF), this discovery has not resulted in the development of new, effective treatments. On the contrary, the unexpected failure of clinical trials of global TNF blockade cast doubt as to the precise roles of inflammatory activation in general and of TNF in particular in the progression of chronic HF. As there are two cell-surface receptors for TNF (TNFR1 and TNFR2), we evaluated the remodeling responses specifically referable to each TNF receptor in chronic ischemic HF in vivo using TNFR1 and TNFR2 null mice. Our results indicate that TNF induces dichotomous effects in HF such that TNFR1 aggravated, whereas TNFR2 ameliorated, chamber remodeling and hypertrophy. Moreover, these effects occurred, at least in part, due to divergent effects on the activation of the downstream signaling mediator nuclear factor-κB, the regulation of inflammatory cytokines, and the induction of apoptosis: TNFR1 exacerbated, whereas TNFR2 ameliorated, these events. These results suggest that the overall balance between these opposing receptor-specific responses determines the ultimate impact of TNF on the HF phenotype, and that analogous TNF receptor-specific effects in human HF should be considered when developing anti-TNF therapies. Dichotomous TNFR-specific effects may also provide one explanation for the failure of the anti-TNF clinical trials. Selective targeting of the individual TNFRs (TNFR1 blockade and/or TNFR2 augmentation) may represent a better therapeutic approach in HF.