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Redox-sensitive intermediates mediate angiotensin II-induced p38 MAP kinase activation, AP-1 binding activity, and TGF-ß expression in adult ventricular cardiomyocytes ¹

Physiologisches Institut, Universität Giessen, Germany

²Correspondence: Physiologisches Institut, Aulweg 129, D-35392 Giessen, Germany. E-mail: Klaus-Dieter.Schlueter{at}physiologie.med.uni-giessen.de

SPECIFIC AIMS

Transforming growth factor ß (TGF-ß) seems to play a key role in processes involved in the adaptation of the heart to increased afterload and at the transition from compensated myocardial hypertrophy to heart failure. Another key player in responses of the heart to hemodynamic overload is the renin-angiotensin system. Both factors seems to operate together during the progression of myocardial hypertrophy by induction of ventricular expression of TGF-ß. In this study, we asked: by which intracellular signals does angiotensin II induce the expression of TGF-ß in adult ventricular cardiomyocytes?

PRINCIPAL FINDINGS

1. Angiotensin II induces TGF-ß mRNA expression on isolated adult cardiomyocytes
Isolated ventricular cardiomyocytes were cultured under unloaded conditions and remained mechanical quiescent throughout the experiments. Angiotensin II caused a concentration-dependent increase in TGF-ß mRNA (EC₅₀: 519 pmol/l). Maximal effects were observed at 100 nmol/l. At this concentration, angiotensin II increased TGF-ß mRNA 2.9-fold, but not in the presence of saralasin, an angiotensin II receptor antagonist.

2. Angiotensin II-dependent induction of TGF-ß depends on AP-1 activation
To investigate whether the angiotensin II-dependent activation of TGF-ß expression depends on activation of the transcription factor AP-1, gel retardation assays were performed to show that angiotensin II enlarged AP-1 binding activity in ventricular cardiomyocytes. Transfection of cardiomyocytes with ‘decoys’ (corresponding to the AP-1 binding site) completely abolished both effects evoked by angiotensin II: induction of AP-1 binding activity and TGF-ß mRNA.

3. p38-MAP kinase activation is part of the signaling pathway leading to TGF-ß mRNA expression
In the next set of experiments, we tested whether p38 MAP kinase activation is required for the induction of TGF-ß mRNA by angiotensin II. Angiotensin II evoked an activation of p38- MAP kinase within 60 min. This activation was inhibited by either bisindolylmaleimide, an inhibitor of protein kinase C activation, or SB202190, an inhibitor of p38-MAP kinase activation. Both inhibitors also antagonized the effect of angiotensin II on TGF-ß mRNA expression (Fig. 1 ).

View larger version (25K): [in this window] [in a new window]   Figure 1. TGF-ß mRNA expression was determined by RT-PCR analysis 20 h after incubation with angiotensin (Ang, 100 nmol/l), Ang and bisindolylmaleimide (BIM, 5 µmol/l), angiotensin and SB202190 (SB, 1 µmol/l), phorbol myristate acetate (PMA, 100 nmol/l), PMA and BIM, or PMA and SB. Data are normalized to basal values of untreated cells cultured for the same period. Data are mean ± SE from n = 4 experiments. *P < 0.05 vs. control (C).

4. Involvement of redox-sensitive signaling in angiotensin II-dependent activation of p38-MAP kinase
We further investigated whether oxygen radicals are involved in angiotensin II-dependent activation of p38-MAP kinase, because redox-sensitive steps have been shown to be involved in angiotensin II signaling in various cell types, but not on adult cardiomyocytes. Angiotensin II-dependent activation of p38-MAP kinase was inhibited by antioxidants like N-acetyl cystein and ascorbic acid. It was also inhibited by diphenyleneiodonium chloride (DPI), a specific inhibitor of flavoprotein-containing enzymes. In cells transfected with antisense oligonucleotides directed against phox22 and nox, two distinct components of the active smooth muscle type NAD(P)H-oxidase complex, angiotensin II was unable to induce p38-MAP kinase (Fig. 2 ). Activation of a smooth muscle like NAD(P)H-oxidase by angiotensin II was shown directly on ventricular cardiomyocytes by oxidation of NADPH and NADH, which is in accordance with a smooth muscle like NAD(P)H-oxidase. In contrast, angiotensin II increased the oxidation of NADPH but not NADH on isolated coronary endothelial cells, which is in accordance with previous characterizations of the endothelial-like NAD(P)H-oxidase, which is distinct from the smooth muscle type.

View larger version (28K): [in this window] [in a new window]   Figure 2. p38-MAP kinase activation in cardiomyocytes incubated for 60 min with angiotensin II (Ang, 100 nmol/l), Ang and diphenyleneiodonium chloride (DPI, 10 µmol/l), or in cells pretreated for 20 h with oligonucleotides corresponding to the translational initiation site of phox22 and nox in either the sense (S) or antisense (AS) mode, and stimulated thereafter with Ang. p38-MAP kinase activation is expressed as the ratio of phosphorylated to nonphosphorylated p38-MAP kinase relative to the ratio in untreated control cells. Data are mean ± SE from n = 4 cultures. *P < 0.05 vs. control.

Finally, we investigated whether intracellular steps involved in the angiotensin II-dependent activation of TGF-ß expression, i.e., p38-MAP kinase and protein kinase C, are located downstream or upstream of the redox-sensitive signaling pathway. However, neither SB202190 nor bisindolylmaleimide, used to antagonize the activation of the two kinases, were able to inhibit the angiotensin II-dependent activation of NAD(P)H-oxidase. Direct activation of protein kinase C by phorbol myristate acetate did not activate NAD(P)H-oxidase on ventricular cardiomyocytes either. Transfection of cardiomyocytes with antisense oligonucleotides directed against phox22 attenuated NADH oxidation induced by angiotensin II.

CONCLUSIONS

Myocardial hypertrophy is an adaptive process of the heart to withstand increased afterload, i.e., caused by elevated blood pressure. This period of compensation, which may last for weeks in rodents and months to years in humans, is often followed by a transition to heart failure. At the transition to heart failure, ventricular expression of TGF-ß increases. Angiotensin II is known to play a key role during the progression of heart failure, although the exact mechanism by which angiotensin II promotes this process in unclear. Since angiotensin II is known to induce TGF-ß expression in various tissues by multiple pathways, we studied whether angiotensin II is able to induce TGF-ß mRNA in isolated cardiomyocytes and investigated intracellular key steps involved in this process. As outlined in Fig. 3 , angiotensin II induces TGF-ß expression via an activation of a NAD(P)H-oxidase system, and subsequent activation of protein kinase C, p38-MAP kinase, and AP-1 binding activity. Some of the intracellular signaling steps characterized in this study for adult ventricular cardiomyocytes have been found before to be involved in angiotensin II-dependent induction of TGF-ß. For example, an angiotensin II-dependent activation of AP-1 binding activity has been found on smooth muscle cells. It has already been suggested for other cells types that AP-1 may be part of the angiotensin II-dependent control of gene regulation, but evidence has been indirect, based on the observation that angiotensin II activates c-fos expression, which can become part of the AP-1 complex. It should be noted that many transcription factors may share the same binding site; some may not have been characterized yet. The decoy approach applied here is a powerful tool to identify a specific role for AP-1 binding, as it removes all trans-factors from the AP-1 binding sequence. Our study shows that binding of transcription factors at the AP-1 binding site is an indispensable step in the induction of TGF-ß mRNA by angiotensin II in cardiomyocytes.

View larger version (20K): [in this window] [in a new window]   Figure 3. Schematic diagram of the postulated intracellular signaling pathway by which angiotensin II induces the expression of TGF-ß1 in adult ventricular cardiomyocytes. Angiotensin II (Ang II) binds to a specific angiotensin receptor subtype (AT1), which leads to the activation of a NAD(P)H-oxidase that shares similarities with the smooth muscle type enzyme. Subsequently, protein kinase C (PKC) and p38-MAP kinase are activated, and finally, transcription factors binding at the AP-1 binding site. Participation of the suggested pathways was demonstrated by use of the angiotensin receptor blockers losartan and saralasin, inhibitors of NAD(P)H-like enzymes (DPI), transfection of cardiomyocytes with antisense oligonucleotides to down-regulate phox22 and nox expression, inhibitors of p38-MAP kinase activation (SB202190), or transfection of cardiomyocytes with double-stranded oligonucleotides (decoy) directed against the binding site of the AP-1 transcription factors.

Among possible upstream elements involved in the activation of AP-1 binding activity, we focused on p38-MAP kinase, as this was recently shown to be required for angiotensin II-dependent c-fos activation, which is part of the transcription factor family binding to the AP-1 site. Our study shows that angiotensin II activates p38-MAP kinase in a protein kinase C-dependent way. This finding agrees with recent reports on ischemic preconditioning revealing that under these conditions, p38-MAP kinase in the rat heart is activated in a protein kinase C-dependent manner.

Since it is well known from other cell types that p38-MAP kinase may be activated by redox-sensitive signaling pathways that are activated by angiotensin II, we tried to identify such a pathway in adult ventricular cardiomyocytes for which such a pathway has not previously been shown. Pharmacological evidence for such an interaction comes from experiments in which we show that angiotensin II-dependent activation of p38-MAP kinase is inhibited by antioxidants and DPI, an inhibitor of flavoprotein-containing enzymes. This already suggests that NAD(P)H-oxidase-like enzymes are involved in this process. More specific evidence for a participation of a NAD(P)H-oxidase in the redox-sensitive pathway leading to an angiotensin II-dependent activation of p38-MAP kinase comes from experiments in which cardiomyocytes were transfected with phox22 and nox antisense oligonucleotides. Furthermore, angiotensin II stimulates oxidation of either NADPH or NADH, again suggesting an involvement of a smooth muscle like NAD(P)H-oxidase in the effects described.

In summary, our study describes for the first time important intracellular signaling steps involved in the angiotensin II-dependent induction of TGF-ß expression, a critical event implied in the transition from stable hypertrophy to heart failure. We show an activation of a NAD(P)H-oxidase by angiotensin II on adult ventricular cardiomyocytes. These observations argue for the hypothesis that a reduced oxidant redox potential contributes to the progression of heart failure, because under these conditions the initial steps leading to induction of TGF-ß expression would be more sensitive.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0827fje; to cite this article, use FASEB J. (August 17, 2001) 10.1096/fj.00-0827fje

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