Contrasting Roles of NADPH Oxidase Isoforms in Pressure-Overload Versus Angiotensin II–Induced Cardiac Hypertrophy
Increased production of reactive oxygen species (ROS) is implicated in the development of left ventricular hypertrophy (LVH). Phagocyte-type NADPH oxidases are major cardiovascular sources of ROS, and recent data indicate a pivotal role of a gp91phox-containing NADPH oxidase in angiotensin II (Ang II)–induced LVH. We investigated the role of this oxidase in pressure-overload LVH. gp91phox−/− mice and matched controls underwent chronic Ang II infusion or aortic constriction. Ang II–induced increases in NADPH oxidase activity, atrial natriuretic factor (ANF) expression, and cardiac mass were inhibited in gp91phox−/− mice, whereas aortic constriction-induced increases in cardiac mass and ANF expression were not inhibited. However, aortic constriction increased cardiac NADPH oxidase activity in both gp91phox−/− and wild-type mice. Myocardial expression of an alternative gp91phox isoform, Nox4, was upregulated after aortic constriction in gp91phox−/− mice. The antioxidant, N-acetyl-cysteine, inhibited pressure-overload–induced LVH in both gp91phox−/− and wild-type mice. These data suggest a differential response of the cardiac Nox isoforms, gp91phox and Nox4, to Ang II versus pressure overload.
An increase in reactive oxygen species (ROS) production is implicated in left ventricular hypertrophy (LVH) pathophysiology.1 For example, cardiomyocyte hypertrophy in response to angiotensin II (Ang II), tumor necrosis factor-α (TNF-α), or mechanical stretch involves increased ROS production.2,3⇓ In vivo LVH induced by aortic banding is also inhibited by antioxidants.4 However, the sources of ROS generation in the hypertrophying heart remain poorly defined.
Recently, phagocyte-type NADPH oxidases have emerged as major ROS sources in the cardiovascular system.5 NADPH oxidase activity is increased by stimuli such as Ang II, TNF-α, and cyclical load, and NADPH oxidases are implicated in Ang II–induced vascular smooth muscle (VSM) hypertrophy, endothelial dysfunction, and atherosclerosis.5 The prototypic NADPH oxidase comprises a membrane-bound p22phox/gp91phox heterodimer and 4 regulatory subunits, p40phox, p47phox, p67phox, and rac1.6 Several gp91phox homologues (termed Noxs) have recently been identified7; however, a gp91phox (or Nox2)–containing oxidase is known to be expressed in endothelium, fibroblasts, and cardiomyocytes.5,8⇓
We previously reported that NADPH oxidase activity increases during development of pressure-overload LVH in guinea pigs.8 NADPH oxidases have been implicated in cardiomyocyte hypertrophic signaling in response to Ang II or α-adrenergic agonists.9,10⇓ Recently, we demonstrated that in vivo cardiac hypertrophy induced by subpressor Ang II infusion was inhibited in gp91phox−/− mice.11 Although Ang II plays a role in development of in vivo pressure-overload LVH, several other stimuli including intracavitary pressure per se and additional neurohumoral factors are involved. This study investigated the role of a gp91phox-containing NADPH oxidase in the development of pressure-overload–induced LVH.
Materials and Methods
A detailed Materials and Methods section can be found in the online data supplement available at http://www.circresaha.org.
Male gp91phox−/− and matched wild-type mice underwent (1) subcutaneous Ang II or saline infusion by osmotic minipump or (2) suprarenal abdominal aortic constriction or sham constriction. See the online data supplement for body weights.
Hemodynamics and Hypertrophy
Systolic blood pressure (tail-cuff plethysmography) was lower in conscious gp91phox−/− mice than wild-type (123±3.5 versus 132±2.2 mm Hg; n>6 each) as reported previously.11 We previously showed that subpressor Ang II infusion increased cardiac mass by 5% in gp91phox−/− mice versus 20% in wild-type.11 To ensure that these differences were not due to the lower basal blood pressure in gp91phox−/− mice, the present study used pressor Ang II infusion (1.1 mg · kg−1 · d−1), which increased blood pressure to 138±6.0 mm Hg after 6 days in gp91phox−/− mice, a level similar to baseline levels in wild-type mice. Nevertheless, heart/body weight ratio increased by only ≈4% in gp91phox−/− mice (n=6; P=NS) (Figure 1A). In wild-types, pressor Ang II increased blood pressure to 162±9.8 mm Hg (P<0.05 versus baseline) and heart/body weight ratio by ≈20% (P<0.05; n=6).
Aortic constriction caused similar increases in systolic blood pressure (measured invasively 1 week postoperatively) in anesthetized wild-type mice (134.6±6.0 versus 90±5.2 mm Hg; P<0.001; n=4 each) and gp91phox−/− mice (135.1±8.2 versus 81±2.3 mm Hg; P<0.001; n=4 each). It should be noted that these values are not directly comparable to those in Ang II–infused animals because the latter were undertaken in conscious animals (by tail-cuff) whereas in banded animals tail-cuff pressure would be distal to the site of aortic constriction.
Two weeks of pressure overload induced similar increases in LV/body weight ratio in wild-type and gp91phox−/− mice (Figure 1B) (n=6 per group). Cardiomyocyte areas in transverse heart sections were also similarly increased in wild-type (+69.8±3.7%) and gp91phox−/− mice (+54.7±8.5%) after aortic banding (online Figure 1A; n=6 per group). There were no significant differences between groups in lung weights. Similar data were found after 1 week of aortic constriction (not shown).
Molecular Markers of LVH
Ang II infusion increased atrial natriuretic factor (ANF) mRNA expression (assayed by real-time PCR) in wild-type but not gp91phox−/− hearts (online Figure 1B; n=4 per group). Likewise, Ang II–induced increases in β-myosin heavy chain (β-MHC) mRNA were significantly lower in gp91phox−/− mice compared with wild-type (3.1±1.9 versus 6.6±2.0-fold; P<0.05; n=4 per group).
However, aortic banding induced similar increases in ANF expression in wild-type and gp91phox−/− mice (online Figure 1B; n=6 per group). β-MHC mRNA increased 5.5-fold in wild-type and 8.0-fold in gp91phox−/− mice after banding (n=6).
NADPH Oxidase Activity
Ang II infusion significantly increased oxidase activity in wild-type but not gp91phox−/− mice (Figure 2A) (n=6 per group). Surprisingly, aortic constriction significantly elevated NADPH oxidase activity in both wild-type and gp91phox−/− mice (Figure 2A) (n=6 per group). NADPH-dependent superoxide production was abolished by diphenyleneiodonium and Tiron in all groups and was inhibited by apocynin but was unaffected by NG-nitro-l-arginine methyl ester (L-NAME) or rotenone (data for gp91phox−/− mice shown in online Figure 2).
To explore the basis for NADPH oxidase activity in gp91phox−/− hearts, we investigated the expression of alternative Nox isoforms. Nox1 mRNA levels were exceedingly low in all experimental groups (not shown). Nox2 (gp91phox) mRNA was undetectable in gp91phox−/− hearts, and levels were unaltered by either Ang II or pressure overload in wild-type hearts (not shown).
Basal Nox4 mRNA levels were similar in wild-type and gp91phox−/− hearts. Aortic constriction significantly increased Nox4 mRNA in gp91phox−/− hearts but not wild-types (Figure 2B; n≥6 per group). Nox4 protein expression also increased after aortic constriction in gp91phox−/− mice (Figures 2C and 2D; n=6 per group). Ang II infusion nonsignificantly increased Nox4 mRNA in wild-type and had no effect in gp91phox−/− hearts (Figure 2B; n=4 per group).
Effects of N-Acetyl-Cysteine (NAC)
Chronic NAC treatment (500 mg · kg−1 · d−1 in drinking water for 7 days) markedly reduced the aortic banding–induced increase in LV/body weight ratio in both wild-type mice and gp91phox−/− mice (Table; n≥6 per group). NAC had no significant effect in sham-operated mice (Table). Blood pressure was unaltered by NAC (data not shown).
The major finding of this study is a differential involvement of Nox2 and Nox4 in Ang II versus pressure overload–induced cardiac hypertrophy. We previously reported that LVH induced by subpressor Ang II infusion was inhibited in gp91phox−/− mice.11 Since gp91phox−/− mice have lower baseline blood pressures than wild-type,11 it is theoretically possible that this may in part have contributed to the above differences. However, in the present study, a modest pressor dose of Ang II, which raised blood pressure in gp91phox−/− mice to similar levels as baseline pressure in wild-types, failed to induce significant hypertrophy in gp91phox−/− mice. This finding supports the conclusion that Nox2 is essential for development of Ang II–induced cardiac hypertrophy. In marked contrast, Nox2 was not essential for development of pressure-overload LVH since aortic constriction had similar effects in gp91phox−/− and wild-type mice. Importantly, the degree of pressure overload after aortic constriction (assessed by invasive blood pressure measurement) was similar in both groups.
Surprisingly, pressure overload induced significant increases in NADPH oxidase activity not only in wild-type but also gp91phox−/− hearts. NADPH-dependent superoxide production was inhibited by diphenyleneiodonium and significantly reduced by apocynin but unaffected by NO synthase inhibition or rotenone, consistent with it originating predominantly from an NADPH oxidase. These data suggested the presence of an alternative Nox isoform. Indeed, significant Nox4 mRNA and protein expression were detected in both wild-type and gp91phox−/− hearts. Furthermore, myocardial Nox4 mRNA and protein levels increased significantly after aortic constriction in gp91phox−/− mice. Previous studies3,4⇓ suggest an involvement of ROS in pressure-overload hypertrophy, with inhibition of hypertrophy by antioxidants. Interestingly, in the present study, an ROS scavenger, NAC, significantly reduced aortic banding–induced LVH in both wild-type and gp91phox−/− mice. Taken together, these results (1) are consistent with involvement of a Nox4-containing NADPH oxidase in pressure-overload hypertrophy, (2) indicate that Nox4 cannot compensate for Nox2 with respect to the cardiac response to Ang II.
Nox4 was first identified in kidney and postulated to play a role in oxygen sensing. Recently, however, a coexpression of Nox1 and Nox4 was reported in rat aortic VSM.7 Nox1 is implicated in VSM Ang II signaling, but, interestingly, Nox4 could not compensate for Nox1 with respect to Ang II–induced VSM ROS production.7 This is analogous to results of the present study where Nox4 could not compensate for Nox2 in terms of the response to Ang II in gp91phox−/− hearts. Increased activity of the Nox2-containing oxidase can involve either posttranslational protein modifications and/or increased gene expression of component subunits.5,6⇓ The mechanisms of activation of Nox4-containing NADPH oxidases are as yet unknown, but it is possible that the increase in Nox4 expression may have contributed to increased oxidase activity after pressure overload.
This study is the first to report the presence of Nox4 in murine myocardium. The results presented indicate a differential regulation of myocardial Nox2- and Nox4-containing oxidases. Nox2 is absolutely required for the response to Ang II, and Nox4 cannot compensate for the absence of Nox2 in this setting. However, Nox2 is not essential for the response to pressure overload. Our results suggest that Nox4 may be involved in the cardiac response to pressure overload; however, confirmation of such a role will require studies using specific molecular perturbations to dissect out the relative roles of different Nox isoforms.
This work was supported by the British Heart Foundation.
↵*Both authors contributed equally to this study.
Original received December 16, 2002; first resubmission received May 1, 2003; second resubmission received September 10, 2003; accepted September 25, 2003.
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