Lack of Glutathione Peroxidase 1 Accelerates Cardiac-Specific Hypertrophy and Dysfunction in Angiotensin II Hypertension
Glutathione peroxidase 1 (Gpx1) plays an important role in cellular defense by converting hydrogen peroxide and organic hydroperoxides to nonreactive products, and Gpx1−/− mice, which are characterized by reduced tissue glutathione peroxidase activity, are known to exhibit enhanced oxidative stress. Peroxides participate in tissue injury, as well as the hypertrophy of cultured cells, yet the role of Gpx1 to prevent end organ damage in cardiovascular tissue is not clear. We postulated that Gpx1 deletion would potentiate both aortic and cardiac hypertrophy, as well as mean arterial blood pressure, in response to angiotensin II (AngII). Our results show that short-term AngII markedly increased left ventricular mass, myocyte cross-sectional area, and interventricular septum thickness and decreased shortening fraction in Gpx1−/− mice as compared with wild-type animals. On the other hand, AngII resulted in a similar increase in mean arterial blood pressure in wild-type and Gpx1−/− mice. Collagen deposition increased in response to AngII, but no differences were found between strains. Vascular hypertrophy increased to the same extent in Gpx1−/− and wild-type mice. Collectively, our results indicate that Gpx1 deficiency accelerates cardiac hypertrophy and dysfunction but has no effect on vascular hypertrophy and mean arterial blood pressure and suggest a major role for Gpx1 in cardiac dysfunction in AngII-dependent hypertension.
Left ventricular hypertrophy (LVH) is an important risk factor for coronary heart disease, heart failure, and stroke.1,2 LVH involves changes in myocardial architecture consisting of myocyte hypertrophy and perivascular and myocardial fibrosis, and there is a well-established link between LVH and high blood pressure. Factors such as age, sex, race, and stimulation of the renin-angiotensin-aldosterone system play important roles in the pathogenesis of LVH,3,4 and angiotensin-converting enzyme inhibitors, as well as angiotensin II (AngII) receptor antagonists, are effective in the reduction of cardiac hypertrophy.5,6 AngII is a prototypical stimulant of hydrogen peroxide (H2O2) and other reactive oxygen species (ROS) in cardiac and vascular cells,7 and H2O2 has been shown to be a potent signaling agent in cardiomyocytes and vascular smooth muscle, promoting their hypertrophy in vitro and in vivo.7–9
Glutathione peroxidase (Gpx) 1, a ubiquitous peroxidase isoform, is a selenium-dependent enzyme that reduces cellular peroxides via their conversion to water and other nonreactive products.10 In Gpx knockout mice (Gpx1−/−) tissue, Gpx activity is markedly reduced,11 and peroxide and ROS levels are elevated, which purportedly contribute to endothelial dysfunction and cardiac matrix deposition.12 Despite a well-established role of AngII to increase H2O2 in the heart and aorta, the potential for AngII hypertension and cardiac and vascular remodeling to be accelerated in Gpx1−/− mice has not been studied. In this study, we postulated that Gpx1 deletion would potentiate aortic and cardiac hypertrophy, as well as mean arterial blood pressure (MABP) elevation, in response to AngII. The novel findings described herein illustrate that Gpx1 deletion promotes cardiac-specific hypertrophy and dysfunction without affecting vascular hypertrophy or blood pressure. These results suggest an important role for Gpx1 in initial cardiac hypertrophy and dysfunction in response to AngII.
Male Gpx1−/− mice backcrossed to the C57Bl/6J background for >10 generations were kindly provided by Dr Ye-Shih Ho (Wayne State University) and bred at our institution. This study was approved by the Institutional Animal Care and Use Committee of Henry Ford Hospital and conforms to guidelines required by the National Institutes of Health.
Radiotelemetry of MABP
Eighteen- to 20-week–old mice were instrumented with transmitters as described previously13 (an expanded Methods section can be found in the online Data Supplement, please see http://hyper.ahajournals.org). MABP and heart rate were continuously recorded and reported as 24-hour mean±SEM.
Vehicle or AngII Infusion
Mice were anesthetized with Brevital (70 mg/kg IP) to allow for the SC implantation of osmotic minipumps (Alzet 1007D). Mice were implanted with minipumps infusing either vehicle or AngII (521 ng/kg · min SC) for 7 days.
Preparation of Tissue Samples
On day 7, mice were anesthetized and the heart stopped during diastole. Mice were perfusion fixed under pressure, and hearts and thoracic aortas removed. Total heart weight:body weight (THW:BW) ratio served as parameter of cardiac hypertrophy. The left ventricular (LV) middle section and the descending thoracic aorta were processed and sectioned for analyses.
Echocardiographic Evaluation of Cardiac Morphology and Function
LV wall thickness, dimensions, and shortening fraction (SF) were evaluated with a Doppler echocardiograph equipped with a linear transducer, as described previously.14
Measurements of Interventricular Septum and LV Mass
LV mass was calculated according to the following equation14:
where 1.055 is the specific gravity of the myocardium, IVSTd is diastolic interventricular septum thickness, LVDd is diastolic LV dimension, and PWT is diastolic posterior wall thickness.
Cardiac Chamber Dimensions
End-diastolic and end-systolic LV dimensions (LVDd and LVDs) and diastolic interventricular septum thickness were measured from the M-mode tracings.
LV Shortening Fraction
Myocyte Cross-Sectional Area and Interstitial Collagen Fraction
Six-micrometer heart sections were processed in Bouin fluid overnight, washed, and incubated with 0.1% picrosirius red. Twenty-one images of each left ventricle section were captured at ×400 magnification. Myocyte cross-sectional area (CSA; MCSA) and interstitial collagen fraction (ICF) were digitally recorded.
Histological Examination of Mouse Thoracic Aortic Cross-Sections for Measurement of Vascular Remodeling
Sections were stained with Masson Trichrome Accustain, as described previously.15 Cross-sectional area (CSA), thickness of the media, and external perimeter (Pe) were digitally measured. Lumen diameter (L) was calculated according to the formula L=2×[(Pe/2π)2−(CSA/π)]1/2. Remodeling was determined by comparing the ratio of medial thickness:lumen diameter.
Glutathione Peroxidase Activity and Protein Levels
Tissue homogenates were prepared in 50 mmol/L of potassium phosphate, containing 1 mmol/L of EDTA. Homogenates from aortic rings and heart tissue were centrifuged at 10 000g for 15 minutes at 4°C. Supernatants were assayed for protein and stored at −80°C. Glutathione peroxidase (Gpx) activity was determined using assay kit FR 17 from Oxford Biomedical Research following the manufacturer’s protocol. Gpx1 protein levels in heart homogenates were assessed by Western blots using rabbit antibody anti-Gpx1 (Abcam). Blots were probed with secondary antibody labeled with IRDye 800 and analyzed by the Odyssey Imager and its software (Li-Cor Biosciences).
Data are expressed as mean±SEM. Comparisons between groups were made by ANOVA, followed by the Hochberg method for multiple comparisons. A value of P<0.05 was considered statistically significant.
Body Weight and MABP
Mouse weights were ≈30 g and did not vary with time or among treatment groups (data not shown). Basal MABP was similar between strains and remained unchanged in vehicle groups of both strains throughout the study (Figure 1). AngII caused a significant and sustained increase in MABP. No significant difference was observed between Gpx1−/− and wild-type mice infused with AngII (Figure 1). Heart rate in beats per minute did not vary among treatment groups and strains of mice (wild-type: 705±6.3 bpm versus Gpx1−/−: 691±9.40 bpm for animals treated with vehicle, and wild-type: 711±8.33 bpm vs Gpx1−/−: 701±11.7 bpm for animals treated with AngII).
Cardiac Hypertrophy: Ratio of Total Heart:Body Weight
Large visible differences in heart size were observed in Gpx1−/− versus wild-type mice infused with AngII, as demonstrated by differences in heart cross-sections seen in Figure 2A through 2D. Figure 2A and 2B correspond with representative heart cross-sections from wild-type mice treated with vehicle (wild-type+vehicle) and Gpx1−/− mice treated with vehicle (Gpx1−/−+vehicle), respectively, showing no significant differences between the strains. Figure 2C and 2D correspond with representative heart cross-sections from wild-type mice treated with AngII (wild-type+AngII) and Gpx1−/− mice treated with AngII (Gpx1−/−+AngII), respectively, showing LVH in the heart from Gpx1−/−+AngII mice. THW:BW ratios in vehicle-treated wild-type and Gpx1−/− mice were similar (Figure 2E). THW:BW ratios from wild-type mice treated with AngII were not significantly larger than those in wild-type mice treated with vehicle. Gpx1−/−+AngII hearts, however, were significantly larger than Gpx1−/−+vehicle (P<0.05) and wild-type+AngII (21% larger; P<0.05; Figure 2E).
Echocardiographic Measurement of Cardiac Hypertrophy and Function
Diastolic Interventricular Septum
IVSTd at day 0 did not vary among treatment groups or strains (Figure 3A). In addition, IVSTd in vehicle-treated wild-type and Gpx1−/− mice did not increase from day 0 to 7. AngII treatment, however, significantly elevated IVSTd in the Gpx1−/− group (day 7) versus its vehicle control. This elevation was significantly greater than that observed in the AngII-treated wild-type group (P<0.05).
Measurements of LV Mass
At day 0, LV mass was not different among the treatment groups and strains (Figure 3B). LV mass did not change significantly in vehicle-treated wild-type and Gpx1−/− mice (days 0 to 7). AngII treatment resulted in elevation in LV mass in mice from both strains (P<0.05), and this increase in LV mass by AngII was significantly greater in Gpx1−/− versus wild-type groups at day 7 (P<0.05).
Cardiac Shortening Fraction
Cardiac SF did not vary significantly at day 0 among all of the groups (Figure 3C). SF did not change comparing day 7 with day 0 in wild-type+vehicle, wild-type+AngII, or Gpx1−/−+vehicle mice. SF was significantly reduced (13%) after 7 days in AngII-treated Gpx1−/− mice (P<0.05). Moreover, at day 7 there was a significantly lower SF in AngII-treated Gpx1−/− mice compared with wild-type+AngII and both vehicle-treated groups (P<0.05).
Measurements of Cardiac Chamber Dimensions
Baseline (day 0) LVDs or LVDd did not differ among the treatment groups. At 7 days, in vehicle-treated wild-type and Gpx1−/− mice, neither LVDs (wild-type: 1.15±0.03 mm versus Gpx1−/−: 1.16±0.03 mm) nor LVDd (wild-type: 2.41±0.06 mm versus Gpx1−/−: 2.42±0.05 mm) was significantly different. AngII had no significant effect on LVDd in either strain (2.45±0.10 versus 2.46±0.11 mm for wild-type and Gpx1−/−, respectively). Likewise, AngII had no effect on LVDs in wild-type versus Gpx1−/− mice (1.17±0.05 versus 1.29±0.10 mm, respectively).
MCSA and ICF
MCSA did not differ between vehicle-treated wild-type and Gpx1−/− mice (Figure 4A, 4B, and 4E). Ang II significantly increased MCSA in both strains (P<0.05; Figure 4C through 4E). Importantly, hypertrophy, as measured by MCSA, was greater in Gpx1−/− mice than in wild-type mice (P<0.05). Representative images showing interstitial collagen in each group are illustrated in Figure 5A through 5D. ICF was measured at 3.8±0.2% in wild-type+vehicle; AngII significantly increased ICF to 11.0±1.2% (P<0.001; Figure 5E). Similar collagen deposition was observed in Gpx1−/− mice. That is, ICF was 4.0±0.2% in Gpx1−/−+vehicle mice, and AngII elevated it to 12.0±2.0% (P<0.05; Figure 5E). The data showed no strain differences at basal conditions or after the addition of AngII.
Masson trichrome staining revealed similar aortic wall thickness/L ratio in wild-type and Gpx1−/− mice treated with vehicle (Figure 6, left). Ang II significantly increased wall thickness/L ratio compared with both vehicle groups with no difference between strains (P<0.001). Aortic medial CSA was similar between wild-type+vehicle and Gpx1−/−+vehicle mice (94.10±4.04 and 92.00±3.20×103 μm2, respectively; Figure 6, right). AngII significantly enhanced CSA in both wild-type and Gpx1−/− mice (133.00±8.23 and 115.00±6.33, respectively), with no significant difference between the strains.
Assessment of Gpx Activity and Protein Levels
Gpx activity was measured in aortas, as well as in heart homogenates. In aortas of wild-type mice, Gpx activity was 0.28±0.14 mU/mg in vehicle-treated animals, whereas it was 0.47±0.24 mU/mg in AngII-treated animals. In aortas of Gpx1−/− mice, the Gpx activity was below the detection limit of the assay under both treatment conditions. In the case of heart homogenates, Western blots were used to assess protein levels and to corroborate previously published findings showing a 10-fold decrease in Gpx1−/− compared with wild-type mice.11 As expected, the levels of Gpx1 in Gpx1−/− animals were undetectable compared with the robust levels in wild-type mice, both for vehicle or AngII-treated animals (Figure S1, available in the online Data Supplement). The data show a major reduction of vascular and cardiac Gpx1 from wild-type to knockout mice, corroborating previous findings by Ho et al,11 which demonstrated a 10-fold decrease in Gpx1 in Gpx1−/− mice compared with wild-type mice.
Gpx1 is suggested to play an important role in moderating H2O2 under pathological conditions.11 In nonstressed heterozygous Gpx1 knockout mice, elevated ROS and oxidative stress in cardiovascular tissue are associated with ROS-mediated endothelial dysfunction.12 Because AngII is a prototype stimulant of H2O2 in the heart and aorta, we hypothesized that, in AngII-infused mice, deletion of Gpx1 would potentiate blood pressure elevation and both cardiac and aortic hypertrophy. Previously, the role of Gpx1 in hypertension and its effects on cardiac and vascular remodeling had not been studied. Here, using a Gpx1-knockout mouse model characterized by suppressed levels of Gpx activity and protein,11 we provide anatomic (heart weight), morphological (LV mass and posterior wall thickness by echocardiography), and histological (MCSA) evidence of enhanced AngII-induced LV hypertrophy in Gpx1−/− mice, despite no difference in blood pressure between AngII-treated wild-type and Gpx1−/− mice. In contrast to the heart, AngII-induced hypertrophy was not enhanced in Gpx1−/− aortas. Furthermore, preliminary data indicated a rise in ANP levels in Gpx1−/− mice consistent with enhanced hypertrophy in this strain (data not shown). Taken together, our results reveal a novel observation of a unique cardiac hypertrophy and dysfunction in Gpx1−/− mice.
AngII increased MABP compared with vehicle-treated mice of both strains. In light of a reported endothelial dysfunction in Gpx1−/− mice12 and findings that Gpx1 deletion enhances AngII-induced impairments of vasodilatation,16 a greater elevation in MABP after AngII might have been predicted. However, no difference was observed between the strains, which is possibly attributable to the existence of countervailing mechanisms affecting blood pressure. Importantly, this absence of a blood pressure difference between strains highlights a critical dissociation among MABP, increased cardiac hypertrophy, and diminished function in response to AngII in Gpx1−/−. Thus, our observations challenge the notion that the effects of AngII on LV hypertrophy are simply pressor dependent.17
Measurements of Cardiac Hypertrophy and Function
THW:BW ratios did not increase significantly in wild-type animals after 7 days of AngII. This is not surprising given the short duration of AngII infusion. However, a significantly larger heart weight was observed in AngII-infused Gpx1−/− mice. This difference between strains was also clearly demonstrated histologically and echocardiographically. LV mass and MCSA were markedly higher in both AngII groups and further enhanced in Gpx1−/−. Furthermore, a significantly enhanced IVSTd was observed in Gpx1−/− mice, further supporting enhanced AngII susceptibility in Gpx1−/− versus wild-type mice.
To further test for an effect of Gpx1 knockout on cardiac function, changes in LVDs and LVDd were examined. We observed no changes in LVDd among the treatment groups. Although in wild-type mice LVDs did not change with AngII, there was a tendency for an increase in LVDs in Gpx1−/− treated with AngII. However, the data did not reach statistical significance. Despite this, we observed a 13% decrease in SF in Gpx1−/− mice treated with AngII. This decrease is considered a significant decrease in cardiac performance and might be viewed as a harbinger of more advanced dysfunction, highlighting the potentially important clinical significance of our findings. In fact, reduced Gpx1 activity in humans may be an important cardiac risk factor, because it has been shown that in patients with coronary heart disease that lower Gpx1 is associated with an increased risk of cardiovascular events.18
Myocardial fibrosis is a pathological condition associated with cardiac hypertrophy and dysfunction, and the ability of AngII to increase cardiac interstitial collagen via ROS has been reported.19 Thus, we also examined whether LV cardiac ICF is enhanced in Gpx1−/− mice and could explain the early change in SF. In contrast to the report by Forgione et al12 showing increased cardiac matrix in mice for which Gpx1 was partly compromised, we did not observe enhanced collagen deposition in Gpx1−/− mice. Two possible explanations for the lack of effect of AngII in our study include the following: increased ROS did not rise to levels capable of enhancing the effect of AngII on fibrosis, and/or longer periods of elevated ROS may be necessary to sustain such an enhancement. Moreover, genetic background differences of heterozygous versus homozygous knockouts, as well as their controls, could have contributed to the discrepancy.
Gpx1−/− mice treated with AngII exhibited a significantly decreased SF compared with controls. Theoretically, this functional change might be attributed to ROS-mediated hypertrophy leading to cardiac dysfunction and failure.20 However, in the present study, the rapid development of LV dysfunction in Gpx1−/− mice is likely to have resulted from a direct adverse cardiac cell effect of H2O2. In fact, previous studies showed that ROS including H2O2 accelerate contractile dysfunction of the heart.20,21 Detrimental effects of H2O2 include intracellular acidosis and electromechanical dysfunction, alterations in cardiac action potential, and contractile force inhibition.22,23 None of the differences observed can be attributed to changes in angiotensin II type 1 receptor, because its levels did not vary among treatment groups (Figure S2).
Two reports challenge the role of antioxidant defenses in cardiac dysfunction and/or heart failure. Dieterich et al24 report an increased catalase activity in human end-stage heart failure. These data may be interpreted to contradict our findings that reduced antioxidant defenses contribute to cardiac dysfunction. However, as the authors point out, increased catalase activity may compensate for elevated ROS at this late stage of the disease. A second study by Baumer et al reports the opposite.25 That is, catalase activity is reportedly decreased in end-stage heart failure, consistent with our hypothesis that decreased antioxidant protection leads to dysfunction. Importantly, the study did not examine catalase activity at earlier time points, and, thus, it is not possible to know whether compromised antioxidant defenses early in human disease contribute to heart failure. At this juncture, therefore, not enough evidence is available to solve this controversy. Moreover, caution should be taken in extrapolating findings in the mouse to human disease, especially considering that our findings are not related to chronic heart failure.
Lack of Effect of Gpx1 Deficiency on Vascular Remodeling
Vascular medial hypertrophy in response to AngII is mediated, in part, by ROS.15,26 Thus, we expected that in Gpx1−/− versus wild-type mice, AngII-induced medial hypertrophy would be enhanced. However, our data showed no difference in AngII-induced hypertrophy between the strains as assessed by wall:lumen ratio and CSA. These data are noteworthy because they highlight enhanced cardiac hypertrophy in Gpx1−/− mice in the absence of a change in vascular medial hypertrophy. The data indicate an accelerated cardiac response to Gpx1 deficiency or enhanced cardiac sensitivity to smaller ROS increases compared with aorta.
In conclusion, LVH and dysfunction were accelerated in AngII-treated Gpx1−/− mice. These effects were not associated with increases in collagen deposition and were independent of blood pressure levels. No changes in vascular hypertrophy were observed, indicating a greater role for Gpx1 in cardiac versus vascular protection. Our results demonstrate that Gpx1−/− mouse hearts are more susceptible to dysfunction and further support the significance of antioxidant defense by Gpx1 in cardiac remodeling and function.
Hypertension and ventricular hypertrophy are major risk factors for cardiac dysfunction and congestive heart failure. Oxidative stress is an important mechanism involved in cardiovascular disease. ROS trigger signaling pathways that lead to cell proliferation, dysfunction, and death, as well as release of proinflammatory mediators that promote cardiovascular injury. Under physiological conditions, ROS is controlled by intrinsic antioxidant systems, including Gpx1. In this study, we demonstrated that lack of Gpx1 promotes AngII-induced LVH, dilatation, and dysfunction, supporting a pathophysiological role of ROS in the heart. The data further support the concept that antioxidant therapy may benefit the heart. Large prospective and randomized clinical trials failed to demonstrate clinical benefits of antioxidants on blood pressure and end-organ damage. Although the reasons for these outcomes are not clear, there are noteworthy limitations to those studies including the doses and forms of antioxidants chosen, preexisting cardiovascular conditions, trial design, and other medications administered to enrolled patients. Future studies will require a more focused and rational approach to targeting ROS rather than emphasizing the use of scavengers, which may not be as efficacious or discriminating in their effects.
We thank Dr Imad Al Ghouleh for critical review of the article.
Sources of Funding
This work was supported by National Institutes of Health grants HL55425 and HL28982 and the Fund for Henry Ford Hospital. N.A. was also supported by American Heart Association Fellowship 0520056Z. X.-P.Y. was supported by National Institutes of Health grant HL078951. P.J.P. is an Established Investigator of the American Heart Association.
N.A. and X.-P.Y. contributed equally to this work.
The authors had full access to the data and take responsibility for its integrity. All of the authors have read and agree to the article as written.
- Received May 6, 2009.
- Revision received June 1, 2009.
- Accepted October 19, 2009.
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