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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online September 4, 2003 as doi:10.1096/fj.03-0331fje. |
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Department of Cardiology and Angiology, Medizinische Hochschule Hannover, Germany;
* Department of Cardiology, Medical School of Bialystok, Poland; and
Department of Biochemistry, Christian-Albrechts-Universität zu Kiel, Germany
2Correspondence: Kardiologie und Angiologie, Medizinische Hochschule Hannover, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany. E-mail: drexler.helmut{at}mh-hannover.de
SPECIFIC AIMS
Circulating levels of Interleukin-6 (IL-6) are elevated postmyocardial infarction (MI) and heart failure and are associated with increased morbidity and mortality. IL-6 is expressed in the myocardium post-MI, suggesting a pathophysiologic role for IL-6 in ischemia and infarction, but not establishing a functional role of endogenous IL-6 in heart failure and postinfarction. To explore the role of endogenous IL-6 on infarct size, LV remodeling, and mortality after myocardial infarction, we used IL-6 knockout mice (IL-6-/-) and wild-type controls (WT) in an in vivo model of experimental infarction by permanent coronary artery ligation.
PRINCIPAL FINDINGS
1. No long-term differences in survival rates of IL-6-/- and WT mice after myocardial infarction
There was no difference in the survival rates of IL-6-/- and WT mice during the 6 wk follow-up post-MI: 40 IL-6-/- animals (66.7%) and 34 WT mice (63.0%) survived the surgical procedure. The intra- and early post-MI mortality was similar. The rate of LV rupture between day 3 and 7 post-MI was also similar (IL-6-/- 14%, n=8; WT 13%, n=7) (Fig. 1
).
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2. No differences in infarct size
Six weeks after MI, the mean infarct size was comparable in both groups of surviving mice (IL6-/- 41.2±2.6%, WT 38.3±4.0%, ns). All mice that died within 6 wk revealed comparable infarct size in postmortem analysis (IL-6-/-: n=20, 52.3±5%, WT: n=20, 47.4±7%). Twenty-four hours after induction of permanent ischemia, no differences in infarct size related to area at risk were observed as assessed by Evans blue and TTC staining. At this time the proportion of infarct related to the area at risk was >95% in both genotypes (IL-6-/- 0.97±0.01 n=4; WT 0.97±0.01; n=4). The area at risk was comparable in both groups (IL-6-/- 0.53±0.04; WT 0.48±0.03).
3. Significant compensatory hypertrophy and LV-dilation after myocardial infarction without differences between IL-6-/- and WT mice
After in situ fixation, infarcted mice showed a significant increase in LV mass as indicated by increased heart weight indexed for body weight, increased septal thickness, and increased myocyte cross sectional area. No differences were observed between MI IL-6-/- and MI WT, even when related to infarct size. The observed hypertrophy in infarcted mice was associated with significant LV dilation, without differences between genotypes.
4. Significant LV-dysfunction after myocardial infarction without differences between IL-6-/- and WT mice
Six weeks post-MI mice exhibited significant LV dysfunction compared with sham-operated controls by in vivo application of a conductance catheter. No differences were detected between MI IL-6-/- and MI WT. Consistent with the dilation 6 wk post-MI observed in the morphometry, significant increase in LV dimension in systole and diastole was noted. EF was reduced post-MI to similar extent in IL-6-/- and wild type animals; similarly, dP/dtmax and dP/dtmin. LV end diastolic pressure (LVEDP) was significantly elevated post-MI compared with sham-operated mice. There were no significant differences in heart rate between the individual groups.
5. The lack of IL-6 is probably compensated by the redundancy within the IL-6 system and factors activating the JAK/STAT pathway by alternative receptors
By signaling through the receptors IL-6 R
and gp130, a subunit used by all IL-6 cytokine family members, IL-6 activates the JAK/STAT pathway, which is considered to represent an important signaling pathway for IL-6-mediated cellular effects. Gene expression of IL-6, IL-6 R, gp130, and STAT3 phosphorylation of the noninfarcted septal myocardium post-MI was analyzed. IL-6 transcription was induced 12 h after permanent coronary artery occlusion in the remote and infarcted myocardium of infarcted WT, but was absent in IL-6-deficient mice. In sham-operated WT mice, no IL-6 expression was detected by Northern blot. Persistent myocardial IL-6 R expression was detected at the mRNA and protein level by PCR and immunohistochemistry as well as Western blot in sham-operated and infarcted mice of both genotypes. Expression of gp130 was similar in all four groups (WT and MI WT, IL-6-/- and MI IL6-/-). MI resulted in a marked STAT3 phosphorylation in both remote and infarcted LV without differences between genotypes, suggesting compensation for the loss of IL-6 by other cytokines of the IL-6 family in IL-6-/- mice. Therefore, we analyzed expression of LIF and CT-1 at the protein level post-MI. A moderate increase in LIF protein in remote myocardium of IL-6-/- hearts was observed 48 h post-MI (+40% compared with WT). The expression of CT-1 post-MI was similar in IL-6-/- or WT mice.
Numerous factors are induced in the setting of myocardial infarction that may activate the JAK/STAT pathway, thereby compensating for the lack of IL-6 in our knockout model. We found a marked induction of angiotensin II (Ang II) at the protein level in LV myocardium of IL-6-/- mice compared with WT mice prior to intervention. The increased expression of Ang II in IL-6-/- was still detectable post-MI in both the remote and infarcted LV myocardium. Under baseline conditions and postinfarction, Ang II receptor 1 (AT1R) expression was increased in LV myocardium of IL-6-/- mice compared with WT (Fig. 2
).
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No differences in the expression of cardiac protective genes (eNOS and SOD) were found between IL-6-/- and WT mice. We detected decreased LV expression of eNOS after induction of experimental myocardial infarction at protein level in IL-6-/- and WT mice. SOD protein expression was similar in IL-6-/- and WT at baseline without alteration postmyocardial infarction.
6. No effect of exogenous interleukin-6 on infarct size
Since the lack of IL-6 was not associated with a persistent difference in infarct size, we analyzed whether exogenous IL-6 may influence the infarct size post-MI in WT mice. Exogenous IL-6 given i.v. prior to infarct induction did not affect infarct size at 24 h postcoronary occlusion, even though marked STAT3 phosphorylation after IL-6 infusion at 15 and 180 min was detectable (Infarct/Area at Risk: WT 0.97±0.01; WT+IL-6 0.99±0.02).
CONCLUSIONS AND SIGNIFICANCE
The present study demonstrates that targeted deletion of the IL-6 gene does not affect infarct size, LV remodeling, LV function, or mortality during long-term follow-up after permanent coronary occlusion. These observations suggest that IL-6 is not required for the response of the myocardium to ischemic injury and long-term adaptation of the left ventricle postinfarction. Vice versa, IL-6 does not appear to provide protective effects early after infarction, since the administration of IL-6, although resulting in substantial STAT3 activation, did not exert beneficial effects.
The lack of effect of IL-6 on LV structure, function, and outcome post-MI may be surprising given the recent clinical observations of a strong association between IL-6 and the severity or prognosis of chronic heart failure.
Moreover, recent studies have shown the important role of IL-6 in the setting of transient cardiac ischemia leading to neutrophil dependent myocyte injury. Initial studies comparing permanent occlusion with transient ischemia, followed by reperfusion, demonstrated that early after onset of ischemia the induction of IL-6 was dependent on reperfusion. Deten et al. recently reported significantly increasing levels of IL-6 mRNA as early as 3 h postpermanent coronary occlusion in rats peaking at 6 h, raising the possibility that IL-6 may play a role in permanent ischemia as well as in transient ischemia. Nevertheless, similar to our observations, Clark et al. recently demonstrated that the extent of central nervous system injury was similar in mice lacking IL-6 and in matched controls after transient ischemia.
The lack of effect in IL-6-/- mice cannot be attributed to the absence of IL-6 R in the myocardium since we clearly found IL-6 R expression in sham-operated and infarcted animals of both genotypes by immunohistochemistry, Western blot, and PCR. Moreover, exogenous IL-6, which can activate gp130 only in the presence of the IL-6R, caused a robust activation of STAT3, supporting the functional coupling of IL-6/IL-6R to STAT3.
IL-6 is only one member of a large IL-6 family that includes LIF, CT-1, OSM, CNTF, IL-11, and NNT-1, which share the common receptor subunit gp130 for signaling. Activation of gp130 leads to JAK/STAT, MAP-kinase, and PI3-kinase pathway activation. Activation of these signaling pathways is critically involved in the transcription of hypertrophic and cytoprotective genes as well as genes involved in the regulation of the extracellular matrix. In our present study, STAT3 was shown to be phosphorylated 12 h post-MI in IL-6-/- mice to the same extent as in WT mice. This finding suggests that the lack of IL-6-dependent activation of this signaling pathway was compensated for by other factors (i.e., members of the IL-6 family) (Fig. 3
). In fact, we observed that LIF protein expression was increased 48 h post-MI in IL-6-/- compared with WT mice. There is evidence that, similar to IL-6, LIF confers hypertrophic and cytoprotective responses. Thus, it is tempting to speculate that this cytokine may represent a factor that may compensate for the lack of IL-6. Angiotensin II has also been shown to activate the JAK/STAT pathway. Numerous studies have shown that angiotensin II generation and release is increased post-MI. The JAK/STAT pathway is activated by pressure overload and stretch of cardiomyocytes, possibly related in part to angiotensin II. We found a marked induction of Ang II and its AT1R in LV myocardium of IL-6-/- compared with WT mice at baseline and post-MI. This induction in IL-6-/- mice supports the notion that Ang II may in part activate the JAK/STAT pathway in a compensatory manner in mice lacking IL-6.
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While IL-6 has been shown to be cytoprotective in the heart, our results suggest that the lack of endogenous IL-6 does not play a significant role post-MI. Conversely, administration of exogenous IL-6 injected before induction of MI in WT mice did not influence infarct size, despite marked STAT3 phosphorylation.
We cannot exclude that longer follow-up of IL-6-/- mice may reveal subtle alterations in cardiac function, structure, and survival. However, given our consistent observations for remodeling indices, LV function, and survival, it appears unlikely that IL-6 is playing a major role in the adaptation of the LV to myocardial infarction in the long term. Our results support the notion that the lack of IL-6 in the mouse model is compensated for by the redundancy within the IL-6 system (by other cytokines of the IL-6 family or even other factors such as Ang II). Thus, it is conceivable that increased levels of IL-6 post-MI and heart failure may represent more of a marker rather than a mediator of outcome post-MI and failure. Elevated levels of IL-6 in permanent ischemia post-MI may reflect a response to the extent of the underlying injury and therefore represent a useful prognostic marker similar to IL-6 in sepsis.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0331fje; doi: 10.1096/fj.03-0331fje 
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