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Angiotensin-(1-7) Prevents Cardiomyocyte Pathological Remodeling Through a Nitric Oxide/Guanosine 3′,5′-Cyclic Monophosphate–Dependent Pathway

Originally published 2010;55:153–160


The renin-angiotensin (Ang) system plays a pivotal role in the pathogenesis of cardiovascular disease, with Ang II being the major effector of this system. Multiple lines of evidence have shown that Ang-(1-7) exerts cardioprotective effects in the heart by counterregulating Ang II actions. The questions that remain are how and where Ang-(1-7) exerts its effects. By using a combination of molecular biology, confocal microscopy, and a transgenic rat model with increased levels of circulating Ang-(1-7) (TGR[A1-7]3292), we evaluated the signaling pathways involved in Ang-(1-7) cardioprotection against Ang II–induced pathological remodeling in ventricular cardiomyocytes. Rats were infused with Ang II for 2 weeks. We found that ventricular myocytes from TGR(A1-7)3292 rats are protected from Ang II pathological remodeling characterized by Ca2+ signaling dysfunction, hypertrophic fetal gene expression, glycogen synthase kinase 3β inactivation, and nuclear factor of activated T-cells nuclear accumulation. Moreover, cardiomyocytes from TGR(A1-7)3292 rats infused with Ang II presented increased expression levels of neuronal NO synthase. To provide a signaling pathway involved in the beneficial effects of Ang-(1-7), we treated neonatal cardiomyocytes with Ang-(1-7) and Ang II for 36 hours. Treatment of cardiomyocytes with Ang-(1-7) prevented Ang II–induced hypertrophy by modulating calcineurin/nuclear factor of activated T-cell signaling cascade. Importantly, antihypertrophic effects of Ang-(1-7) on Ang II–treated cardiomyocytes were prevented by NG-nitro-l-arginine methyl ester and 1H-1,2,4oxadiazolo4,2-aquinoxalin-1-one, suggesting that these effects are mediated by NO/cGMP. Taken together, these data reveal a key role for NO/cGMP as a mediator of Ang-(1-7) beneficial effects in cardiac cells.

The renin-angiotensin (Ang) system plays a pivotal role in the pathogenesis of cardiovascular diseases. In this context, strategies directed to inhibit the Ang-converting enzyme or to block the corresponding Ang II receptors are used extensively in clinical practice, highlighting the importance of Ang II as the major effector of this system.1 Of particular importance is the heptapeptide Ang-(1-7) that has shown a key role in the regulation of the cardiovascular system2 and as an oppositor of Ang II actions in the heart. Briefly, Ang-(1-7) decreased the incidence and duration of ischemia-reperfusion arrhythmias,3 improved the postischemic contractile function in isolated perfused rat hearts, and prevented Ang II–induced cardiac remodeling.4 Recently, Mercure et al5 reported that a transgenic mice model with targeted overproduction of Ang-(1-7) in the heart was protected from Ang II–induced cardiac remodeling, corroborating the hypothesis that Ang-(1-7) is an important modulator of heart function. Overall, these findings highlight the role of Ang-(1-7) as a regulator of cardiac function; however, the specific signaling pathways underlying these beneficial effects in ventricular cardiomyocytes have not been investigated.

Previously, we have demonstrated that Ang-(1-7)/Mas axis activates a phosphatidylinositol 3′-kinase-protein kinase B (Akt) pathway leading to endothelial NO synthase (eNOS) activation and NO generation in ventricular cardiomyocytes,6 demonstrating that Ang-(1-7) actions occur directly in ventricular cells. In this study, our first goal was to investigate whether Ang-(1-7) antipathological remodeling in the heart was mirrored by changes in ventricular cardiomyocytes by using a transgenic rat with increased circulating Ang-(1-7) levels, TGR(A1-7)32927 (TG), chronically infused with Ang II. Next we sought to determine the signaling pathway involved in Ang-(1-7) antiremodeling effects in the cardiomyocytes. Here we showed that Ang-(1-7) cardioprotective effects in the heart were paralleled by changes in ventricular cardiomyocytes that involved alteration of hypertrophic signaling pathways and Ca2+ handling. Moreover, we show evidence that Ang-(1-7) cardioprotective effects against Ang II are mediated by an NO/cGMP signaling pathway.



Neonatal cardiomyocytes isolated from 4- to 5-day–old male Sprague-Dawley (SD) rats were used in this study. We also used 19 male SD and 21 male transgenic rats that overexpress Ang-(1-7) in plasma, and TG were obtained from the transgenic animal facilities of the Federal University of Minas Gerais Laboratory of Hypertension. Three- to 4-month–old SD and TG rats were used in this study. All of the experimental protocols conform to the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by our institution. Phenotypic data about this transgenic rats were already published.7

Primary Culture of Cardiomyocytes

Neonatal cardiomyocytes were isolated from hearts of 4- to 5-day–old SD rats, as described previously.8 Cells were treated with peptides or pharmacological inhibitors for 36 hours, then the cells were fixed with paraformaldehyde 4%, and immunofluorescence experiments were performed.

Freshly Isolated Ventricular Cardiomyocytes and Ca2+ Recording

Adult ventricular myocytes were freshly isolated and stored in DMEM (Sigma) until they were used (within 6 hours), as described previously.9 Intracellular Ca2+ ([Ca2+]i) imaging experiments were performed in Fluo-4/am loaded-cardiomyocytes.10 Images were obtained using the Zeiss Meta Confocal Microscope (Zeiss Germany) from Cemel (Biological Sciences Institute, Federal University of Minas Gerais).

Statistical Analysis

Data are presented as mean±SEM. Sample comparisons were performed using Student t test, 1-way or 2-way ANOVA, as appropriate. In all of the statistical tests, P<0.05 was used as a measure of statistical significance.

For an expanded Methods section, please see the online Data Supplement at


Ventricular Cardiomyocytes From TG Rats Are Protected From Ang II–Induced Cardiac Remodeling

To investigate whether rats with increased Ang-(1-7) plasma levels are protected from Ang II–induced cardiac remodeling, we chronically infused SD controls and TG rats with Ang II (6 μg/kg per day) for 2 weeks. As expected, Ang II infusion led to a significant increase in systolic blood pressure in SD rats (Figure 1A); however, this increase in blood pressure was blunted in TG rats infused with Ang II. Cardiac hypertrophy was evident in SD rats infused with Ang II, in contrast with TG rats that presented no cardiac hypertrophy, as shown in Figure 1B. To investigate whether Ang-(1-7) protective effects were paralleled by molecular changes in ventricular cardiomyocytes, we performed experiments in freshly isolated ventricular cells. In agreement with the data showing cardiac hypertrophy in SD Ang II–infused rats, measurements of cardiomyocyte area indicated that ventricular myocytes from SD rats undergo hypertrophy in response to Ang II infusion (Figure S1, available in the online Data Supplement at In contrast, cardiomyocytes from TG rats were protected from Ang II hypertrophic effects. Moreover, transcripts encoding atrial natriuretic factor, B-type natriuretic peptide, and β-myosin heavy chain were upregulated in ventricular myocytes from Ang II–infused SD rats. Ang II–induced changes in cardiac fetal genes were not observed in TG rats (Figure 1C). Because Ang II has been shown to cause a significant increase in TGF-β,11 we also assessed TGF-β mRNA levels in cardiomyocyte samples. TGF-β levels were similar in cardiomyocytes from all of the groups, except in cells from SD rats treated with Ang II that showed a significant increase in TGF-β transcripts (Figure S2).

Figure 1. TG rats are protected against Ang II–induced cardiac remodeling. A, Blood pressure recordings from TG and SD control rats. Rats were either sham operated or implanted with Ang-II–infusing osmotic minipumps for 2 weeks. A significant increase in blood pressure was observed in SD rats infused with Ang II. n=data from ≥4 rats per group. B, SD rats showed a significant hypertrophic response on Ang II stimulation, whereas TG rats were largely resistant against cardiac hypertrophy response. n=number of hearts. C, Lack of hypertrophic fetal gene program upregulation in TG rats on Ang II stimulation. ANF indicates atrial natriuretic factor; BNP, B-type natriuretic peptide; β-MHC, β-myosin heavy chain. mRNA levels were measured by real-time PCR. n=data from ≥5 independent experiments. D, Significant reduction in peak Ca2+ transient amplitude is observed in ventricular cardiomyocytes from SD rats infused with Ang II. n=number of cells analyzed. *P<0.05 when compared with other groups.

Changes in [Ca2+]i are a major feature in heart failure and myocyte pathology. To investigate whether Ang-(1-7) cardioprotective effects were related to changes in Ca2+ handling in ventricular cells, we examined [Ca2+]i in Fluo-4–loaded cardiomyocytes. Ventricular cardiomyocytes from SD-Ang II rats presented a significantly reduced peak Ca2+ transient. The amplitude of the [Ca2+]i transient was reduced by 26% in SD-Ang II cardiomyocytes when compared with SD cells (Figure 1D), in contrast to cells from Ang II–infused TG rats that showed preserved Ca2+ responses.

The calcineurin/nuclear factor of activated T cells (NFAT) signaling pathway in particular is considered an important contributor to Ang II maladaptative remodeling.12–14 A major pathway regulated by calcineurin is that involving the transcription factor NFAT, which, on dephosphorylation, translocates to the nucleus and eventually results in the transcription of a number of hypertrophic genes leading to cardiac hypertrophy.15 To directly address whether Ang-(1-7) blunts Ang II pathological remodeling by modulating calcineurin-NFAT signaling in cardiac cells, we investigated NFAT localization in cytoplasmic and nuclear fractions of ventricular cardiomyocytes by Western blotting. The results showed that Ang II treatment of SD rats caused accumulation of NFAT in the nuclear fraction of ventricular cardiomyocytes (Figure 2A). This feature was not observed in ventricular cells from Ang II–infused TG rats, confirming the ability of Ang-(1-7) to suppress hypertrophic activation in these cells. Similar levels of NFAT were found in the nuclear fraction of ventricular cardiomyocytes from SD and TG rats (Figure 2A). The activity of the Ca2+-calcineurin-NFAT signaling pathway in cardiac myocytes is tightly controlled at multiple levels, and inhibitory mechanisms upstream and downstream from calcineurin have been described.16 Glycogen synthase kinase 3β (GSK3β), in particular, is considered a potent inhibitor of this pathway downstream of calcineurin. In the nucleus, GSK3β phosphorylates conserved serines in the N-terminal regulatory region of NFAT proteins, thereby promoting their nuclear export.17 Moreover, GSK3β has been shown to regulate hypertrophy development by restraining gene expression. To gain further insight into the mechanism by which Ang-(1-7) protects ventricular cardiomyocytes from Ang II–induced remodeling, we next assessed GSK3β expression levels in nuclear fractions. Chronic infusion of Ang II induced a significant increase in GSK3β phosphorylation levels, leading to enzyme inactivation (Figure 2B). This effect was not observed in cardiomyocytes from TG rats treated with Ang II.

Figure 2. Cardioprotective signaling by Ang-(1-7) in ventricular cardiomyocytes. A through D, Top, Representative Western blot. Bottom, Averaged-densitometry. Histone-3 or GAPDH expression levels were used as loading controls. A, Significant increase in nuclear NFAT was found in ventricular cardiomyocytes from SD rats infused with Ang II. Ang-(1-7) prevented Ang II–induced NFAT nuclear accumulation in TG rats. B, Increased GSK3β phosphorylation levels were observed in the nuclear fraction of cardiomyocytes from Ang II–treated SD rats. C, eNOS protein levels were unaltered in cardiomyocytes from SD and TG rats treated or not with Ang II. D, nNOS levels were significantly increased in ventricular cardiomyocytes from TG rats treated or not with Ang II, when compared with SD rats. n indicates the number of cardiomyocyte homogenates. *P<0.05 when compared with other groups. #P<0.05 when compared with SD group treated or not with Ang II.

Given the potential protective effect triggered by eNOS activation in cardiac cells18 and our previous finding that eNOS is a downstream mediator of Ang-(1-7) signaling pathway in cardiomyocytes, we hypothesized that these effects of Ang-(1-7) on cardiac remodeling could be related to changes in eNOS. To investigate this issue, we assessed eNOS expression levels in cardiomyocyte homogenates. Immunoblots revealed similar eNOS protein levels in cardiomyocytes from SD and TG rats infused with Ang II (Figure 2C). Other than eNOS, neuronal NO synthase (nNOS) also leads to NO production in the heart and has been shown to participate in cardiac protection.19,20Figure 2D shows pronounced changes in nNOS in cardiomyocytes from TG rats when compared with cells from SD rats treated or not with Ang II. Moreover, nNOS expression was also higher in cardiomyocytes from Ang II–infused TG rats. Taken together, our data show strong evidence that the Ang-(1-7) protective effect in the heart is paralleled by activation of antipathological remodeling pathways in ventricular cardiomyocytes.

Ang-(1-7) Blunts Cellular Hypertrophy Triggered by Ang II in Neonatal Cardiomyocytes

The lack of effect of Ang II on blood pressure in TG rats raised the question of whether cardioprotective signaling observed in these rats was the sole result of reduced mechanical overload. To make this distinction and to provide the mechanism by which Ang-(1-7) exerts its cardioprotective effects in cardiac cells we treated neonatal rat cardiac myocytes (NRCMs) with Ang II and Ang-(1-7) or a combination of both peptides for 36 hours. Figure 3A shows representative immunofluorescence images from α-actinin–stained NRCMs. Neonatal cells treated with Ang II (100 nmol/L) demonstrated an increased cell surface area of 65%, whereas treatment with Ang-(1-7) alone did not significantly alter the cell surface area of NRCMs (Figure 3B). In contrast, treatment of the cells with 100 nmol/L of Ang-(1-7) significantly reduced Ang II–induced hypertrophy. To verify whether Ang-(1-7) antihypertrophic actions occurred via receptor Mas, cells were treated with the Mas antagonist A-779 (1 μmol/L). As shown in Figure 3B, A-779 reversed the antihypertrophic effects of Ang-(1-7) in Ang II–treated cells.

Figure 3. Ang-(1-7) suppresses Ang II–induced hypertrophy in NRCMs. A, Representative confocal images showing α-actinin–stained NRCMs from control (CT) cells, Ang-(1-7)–treated cells (100 nmol/L), Ang II–treated cells (100 nmol/L), cells treated with Ang II and Ang-(1-7) (100 nmol/L), and cells treated with Ang II and Ang-(1-7) (100 nmol/L) in the presence of Mas antagonist A-779 (10 μmol/L) for 36 hours. Bar: 10 μm. B, Averaged bar graph shows a significant increase in cell area induced by Ang II and prevention by Ang-(1-7) when compared with control. Abolition of Ang-(1-7) antihypertrophic effects was observed in the presence of the Mas antagonist, A-779. n indicates the number of cardiomyocytes analyzed. *P<0.05 when compared with control, Ang-(1-7), and Ang-(1–7)+Ang II–treated cells.

Circumstantial evidence exist between Ang-(1-7)–mediated cardioprotective effects and NO generation. To investigate whether the effects of Ang-(1-7) on cellular remodeling were related to changes in NO, we recorded NO basal levels in 4-amino-5-methylamino-2,7-dichlorofluorescein diacetate–loaded NRCMs. Higher NO production was observed in NRCMs treated with Ang-(1-7) for 36 hours (Figure S3). Treatment with a combination of Ang-(1-7) and Ang II also led to a significant increase in NO levels; nevertheless, NO production under this condition remained lower than that observed in the presence of Ang-(1-7) alone. To further establish an NO-dependent mechanism for Ang-(1-7) antihypertrophic effects, NRCMs were treated with an NO synthase inhibitor, NG-nitro-l-arginine methyl ester (l-NAME; 1 μmol/L), and cellular area was measured. Quantification of these results indicated that l-NAME blunted Ang-(1-7) effects in Ang II–treated neonatal cells (Figure 4A and 4B), suggesting that NO was involved in the antihypertrophic effects of Ang-(1-7) in NRCMs. NO signals in cells directly or indirectly through a cGMP-dependent signaling pathway.20 Involvement of cGMP on Ang-(1-7) cardioprotective effects was investigated in cells treated with a guanylyl cyclase inhibitor, 1H-1,2,4oxadiazolo4,2-aquinoxalin-1-one (ODQ; 50 μmol/L). As shown in Figure 4, ODQ blunted Ang-(1-7) antihypertrophic effects.

Figure 4. Inhibition of NO/cGMP production prevents Ang-(1-7) effects on cellular hypertrophy. A, Representative confocal images showing α-actinin stained NRCMs. Bar: 10 μm. B, Averaged-bar graph showing an increase in cellular area. Antihypertrophic effects of Ang-(1-7) on Ang II–treated cells are prevented by l-NAME (1 μmol/L), and ODQ (50 μmol/L). n indicates the number of cardiomyocytes analyzed. *P<0.05 when compared with control and Ang II+Ang-(1-7)–treated cells.

Ang-(1-7) Acts via NO/cGMP to Prevent Ang II–Induced NFAT Translocation in Cardiomyocytes

To gain further insight into how Ang-(1-7) exerts its antihypertrophic effects, we investigated the activity of calcineurin/NFAT signaling in cardiomyocytes treated with Ang II by investigating the localization of NFAT in the cells. Figure 5A shows representative confocal images of NRCMs stained with an anti-NFAT antibody. Consistent with previous findings,21 Ang II stimulation of cardiac myocytes led to increased nuclear NFAT localization, as shown in immunofluorescence images (Figure 5A) and in a bar graph (Figure 5B). The Ang II–induced NFAT translocation was completely suppressed by Ang-(1-7). Ang-(1-7) treatment alone had no effect on NFAT localization in the cell. As shown in Figure 5, the addition of A-779 to the culture medium prevented Ang-(1-7) effects on NFAT localization in Ang II–treated cardiomyocytes. To investigate the involvement of NO and cGMP in this cardioprotective effect of Ang-(1-7), we treated the cells with l-NAME or ODQ (Figure 6A and 6B). Both l-NAME and ODQ significantly blunted Ang-(1-7) effects on NFAT localization in Ang II–treated cells (Figure 6A and 6B). Collectively these data show that Ang-(1-7) activates an antihypertrophic signaling pathway mediated by NO/cGMP signaling to counterregulate Ang II effects in cardiac cells.

Figure 5. Regulation of NFAT subcellular localization by Ang-(1-7) in NRCMs. A, Representative images of neonatal cardiomyocytes stained with anti-NFAT (green) are displayed. The nucleus was stained with 4′,6-diamidino-2-phenylindole (blue), and the merged image is presented on the right. B, Averaged bar graph. In Ang II–treated NRCMs, we observed increased nuclear NFAT accumulation. Ang-(1-7) prevented Ang II–induced NFAT translocation to the nucleus. Ang-(1-7) suppression of NFAT translocation was blunted in the presence of the Mas antagonist, A-779. Each group represents data from ≥60 cells.

Figure 6. Ang-(1-7) effects on NFAT localization are mediated by NO/cGMP. A, Representative confocal images are displayed. Bar: 10 μm. B, Averaged bar graph shows that Ang II induces NFAT translocation from cytosol to the nucleus when compared with control, and its effect is prevented by Ang-(1-7). The Ang-(1-7) effect on NFAT localization was blunted by l-NAME and ODQ. Each group represents data from ≥75 cells.


Multiple lines of evidence have shown that Ang-(1-7) exerts cardioprotective effects in the heart. The question that remains is where and how Ang-(1-7) exerts its effects. Here we show that Ang-(1-7) can act directly on cardiomyocytes to suppress maladaptative cardiac remodeling induced by Ang II by activating an NO/cGMP signaling pathway. More specifically, Ang-(1-7) blunted Ang II effects by inhibiting the calcineurin/NFAT signaling cascade.

Cardioprotective Signaling by Ang-(1-7) in Ventricular Myocytes

Grobe et al4 have shown that Ang-(1-7) infusion protected the heart from Ang II deleterious effects, such as hypertrophy and fibrosis. In line with these findings, a recent study5 reported that chronic overproduction of Ang-(1-7) in the heart of TG mice reduced Ang II–induced cardiac remodeling through a direct effect on the heart. These observations are further supported by our finding that TG rats are protected from Ang II–induced maladaptative remodeling. To extend these findings, we have identified an array of molecular features that occur in ventricular cardiomyocytes from Ang II– infused TG rats that parallel the protective effects of Ang-(1-7) in the heart. First, we found that ventricular cardiomyocytes from TG rats were protected from Ca2+ signaling dysfunction induced by Ang II. These data, in particular, contrast to our previous findings showing that acute Ang-(1-7) treatment failed to alter Ca2+-transient parameters in ventricular myocytes,6 suggesting that Ang-(1-7) effects on Ca2+ handling observed in cardiomyocytes from TG rats infused with Ang II result from indirect changes in other signaling pathways. Moreover, we found that increased Ang-(1-7) levels reduced NFAT accumulation in the nucleus and prevented GSK3β inactivation induced by Ang II. In cardiomyocytes, NFAT is a potent regulator of prohypertrophic gene transcription,22 and activation of this signaling pathway plays a pivotal role in the development of cardiac hypertrophy induced by various causes. Although we cannot attribute Ang-(1-7) antiremodeling effects in ventricular cardiomyocytes of TG rats only to calcineurin/NFAT signaling suppression, once Ang II activates a complex series of signaling cascades, the role of the calcineurin/NFAT signaling pathway in the Ang II–induced maladaptative stress is well documented. Indeed, NFATc3-null mice demonstrated attenuated Ang II–induced cardiac hypertrophy, suggesting an important contribution of this signaling pathway to Ang II effects.23

On the other hand, catalytically active GSK3β restrains gene expression and prevents hypertrophic growth of cardiac myocytes by inhibition of transcriptional regulators, including NFAT.24 The ability of Ang-(1-7) to prevent GSK3β phosphorylation by Ang II may represent another mechanism by which Ang-(1-7) exerts its cardioprotective effects.

How Does Ang-(1-7) Regulate the Calcineurin/NFAT Cascade?

Several studies have shown that cGMP-dependent protein kinase (PKG) signaling suppresses calcium-calcineurin-NFAT activation at multiple levels.16,22 In cardiomyocytes, Ca2+ activation of calcineurin results in the dephosphorylation of cytoplasmic NFAT promoting its translocation into the nucleus resulting in expression of a hypertrophic gene program. It has been proposed that inhibitory effects of the NO-cGMP-PKG pathway upstream from calcineurin are mediated by inhibition of the L-type Ca2+ channel, because PKG has shown to modulate this channel. Hsu et al25 have shown recently that mice deficient in the calcineurin catalytic subunit Aβ develop less hypertrophy in response to pressure overload than their wild-type counterparts, and this hypertrophy is inhibited by enhancing PKG activity via PDE5A inhibitors. These data show important evidence that antihypertrophic effects of PKG signaling are mediated by mechanisms other than calcineurin/NFAT inhibition.

Here we have shown that Ang-(1-7) antiremodeling effects are mediated, at least in part, through cGMP production. Because PKG has been identified as a prime downstream target mediating the antihypertrophic effects of NO and cGMP,16 it is reasonable to speculate that Ang-(1-7) leads to PKG activation and consequent modulation of calcineurin/NFAT signaling in cardiomyocytes. Additional experiments are necessary to elucidate the exact role of Ang-(1-7)–derived cGMP in protecting against calcineurin/NFAT cascade activation.

How Do Ang-(1-7) and Ang II Signaling Pathways Cross-Talk in Cardiac Cells?

Although our results support a major role for the NO/cGMP pathway in the antihypertrophic effects of Ang-(1-7) against Ang II cellular maladaptative remodeling, where exactly these 2 signaling pathways cross-talk is still unknown. Our hypothesis is that this cross-talk occurs at multiple points. Ang II acts via Ang II type 1 receptors through 2 major signal transduction pathways; one is associated with Gα12/13 and the other with Gαq.12 Recently, Takimoto et al26 have shown a critical role for regulator of G-protein signaling 2 as a brake against the Gαq/phospholipase Cβ pathway in the early cardiac stress response to pressure overload. Regulator of G-protein signaling 2 activation is mediated by cGMP, suggesting a possible mechanism by which Ang-(1-7) via cGMP antagonizes Ang II actions in cardiac cells.

Besides calcineurin/NFAT signaling, it still remains to be determined whether Ang-(1-7) antagonizes other Gq-linked pathways, such as calmodulin-dependent kinase II (CaMKII) and extracellular signal–regulated kinase 1/2, in ventricular cells. Ang II–induced oxidation leads to CaMKII activation and subsequent apoptosis in cardiomyocytes.27 In cardiac cells, CaMKII is involved in coupling excitation and contraction through the regulation of a variety of Ca2+ handling proteins. Moreover, constitutively active CaMKII is elevated in a number of pathological conditions of the heart,28 and it is an important downstream mediator of Ang II effects.29 Tallant et al30 reported that Ang-(1-7) significantly reduced serum-stimulated extracellular signal–regulated kinase 1/2 mitogen-activated protein kinase activity in cardiomyocytes. In contrast, another study did not observe an inhibitory effect of Ang-(1-7) on extracellular signal–regulated kinase 1/2 activation in response to Ang II by using TG mice with chronic overproduction of Ang-(1-7) in the heart.5 The reason for these differences is not clear and awaits clarification.

Origin of NO in Cardiac Cells Under Ang-(1-7) Stimulus

On the basis of the present findings, the mechanism of Ang-(1-7) antipathological remodeling involves NO generation in vitro. Because formation of reactive oxygen species in combination with a low NO bioavailability predisposes for cardiac damage, it is reasonable to speculate that NO plays a role in Ang-(1-7) modulation of Ang II pathological remodeling and hypertrophy in vivo. We have previously identified eNOS as a downstream mediator of Ang-(1-7) signaling pathway in ventricular cells.6 Intriguingly, we have not detected changes in eNOS expression levels in cardiomyocytes from TG rats. In fact, this lack of change is in line with our previous findings showing no significant change in eNOS levels in ventricular myocytes from Mas−/− mice.6 Whether eNOS activity is altered in vivo and its contribution as a source of Ang-(1-7) derived NO remain to be clarified. Other than eNOS, nNOS has also been linked to an antihypertrophic response in the heart.31,32 Interestingly, we observed marked upregulation of nNOS in cells from TG rats infused with Ang II. In keeping with this finding, unpublished data from our laboratory showed reduced nNOS expression in Mas−/− cardiomyocytes (M.F. Dias-Peixoto, R.A.S.S., and S.G., unpublished data, 2009), suggesting an important association between the Ang-(1-7)/Mas axis and nNOS signaling in ventricular cardiomyocytes. A previous study has shown that nNOS overexpression within cardiomyocytes correlates with enhanced cardiac contractility by modulating Ca2+ cycling in the cell.33 Moreover, Casadei34 suggested that nNOS-derived NO may delay the development of heart failure after myocardial infarction. Increased nNOS-derived NO was also found in the failing human heart.19 Therefore, it remains to be elucidated whether nNOS-derived NO contributes significantly to the cardioprotective effects of Ang-(1-7) in vivo.


Ang-(1-7) has been reported to have a pivotal role in the regulation of the cardiovascular system by counterregulating Ang II actions. We now report that Ang-(1-7) can act directly on cardiomyocytes to suppress Ang II–induced maladaptative remodeling by activating an NO/cGMP signaling pathway. More specifically, Ang-(1-7) exerts its effects by inhibiting the prohypertrophic calcineurin/NFAT signaling cascade. Although calcineurin/NFAT signaling plays an important role in Ang II–induced maladaptative stress, Ang II is known to activate a complex series of signaling cascades, suggesting that the cross-talk between these 2 peptides occurs at multiple levels. It still remains to be determined how these 2 peptides interact at the cellular level and the implications of this molecular cross-talk for cardiac disease development.

Sources of Funding

This work was supported by grants from Deutscher Akademischer Austauschdienst (PROBRAL program of DAAD/CAPES), Conselho Nacional de Desenvolvimento Científico e Tecnológico (to S.G. and R.A.S.S.), and Fundação de Amparo à Pesquisa do Estado de Minas Gerais (to S.G.). E.R.M.G. is a recipient of the Conselho Nacional de Desenvolvimento Científico e Tecnológico PhD fellowship at the Postgraduation Program in Biological Science: Physiology and Pharmacology at the Federal University of Minas Gerais.




Correspondence to Silvia Guatimosim, Institute of Biological Sciences, Federal University of Minas Gerais, Av Antônio Carlos 6627, Belo Horizonte, MG-CEP: 31270-901 Brazil. E-mail


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