Combination of angiotensin-(1–7) with perindopril is better than single therapy in ameliorating diabetic cardiomyopathy

We recently found that overexpression of angiotensin (Ang)-converting enzyme 2, which metabolizes Ang-II to Ang-(1–7) and Ang-I to Ang-(1–9), may improve left ventricular remodeling in diabetic cardiomyopathy. Here we aimed to test whether chronic infusion of Ang-(1–7) can dose-dependently ameliorate left ventricular remodeling and function in a rat model of diabetic cardiomyopathy and whether the combination of Ang-(1–7) and Ang-converting enzyme inhibition may be superior to single therapy. Our results showed that Ang-(1–7) treatment dose-dependently ameliorated left ventricular remodeling and dysfunction in diabetic rats by attenuating myocardial fibrosis, myocardial hypertrophy and myocyte apoptosis via both the Mas receptor and angiotensin II type 2 receptor. Furthermore, combining Ang-(1–7) with perindopril provided additional cardioprotection relative to single therapy. Ang-(1–7) administration provides a novel and promising approach for treatment of diabetic cardiomyopathy.

vitro whether chronic infusion of Ang-(1-7) may dose-dependently ameliorate LV remodeling and function in a rat model of DCM, and whether Ang-(1-7) and ACE inhibition combined may be superior to single therapy.

Methods
Please see the Online Appendix for details.
Ethics statement. All experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication, 8th Edition, 2011). The Institutional Animal Care and Use Committee at Qilu Hospital, Shandong University approved the experiments.
Blood pressure and blood glucose measurement. Heart rate, systolic blood pressure, diastolic blood pressure and mean arterial pressure were measured before and after treatment by use of a noninvasive tail-cuff device (Softron BP-98A; Softron, Tokyo) as described previously 2 . Fasting blood glucose level was analyzed by use of the Bayer 1650 blood chemistry analyzer (Bayer, Tarrytown, NY).
Echocardiographic and hemodynamic measurement. Echocardiographic and hemodynamic measurement was performed before and after treatment as described 2,5 with modifications.
Histology. We used 4-mm paraffin-embedded tissue sections for hematoxylin and eosin and Masson trichrome staining to assess tissue architecture and interstitial and perivascular fibrosis.
Transmission electron microscopy (TEM). After hearts were excised, fresh LV tissue was quickly cut into 1-mm cubes and underwent standard block preparation for TEM.
Real-time RT-PCR. The mRNA levels of genes were determined as described 2 and their relative levels were quantified by the 2 2DDCT method, with b-actin as the endogenous reference gene. Primer sequences are listed in Supplementary Table 1.
ACE and ACE2 activities. ACE and ACE2 activities were determined with assays based on internally quenched fluorescent substrates.
Activity of a disintegrin and metalloproteinase 17 (ADAM17). Activity of ADAM17 (also called tumor necrosis factor-a-converting enzyme), was determined by use of the SensoLyte 520 ADAM17 Activity Assay Kit Fluorimetric (AnaSpec, San Jose, CA).
Isolation and culture of neonatal rat cardiac fibroblasts and myocytes. Neonatal rat cardiac fibroblasts and myocytes were isolated and cultured as described 15 with modification. 3 H-proline incorporation assay. Collagen synthesis of cultured cardiac fibroblasts was measured by 3 H-proline incorporation as described 5 with modifications.
ELISA. ELISA was used to measure protein levels of soluble collagen I and III and transforming growth factor (TGF)-b1 in the medium of cardiac fibroblasts.
Assessment of cardiomyocyte hypertrophy. The cross-sectional area of myocytes in cardiac sections was measured by staining with Alexa Fluor 488-conjugated wheat germ agglutinin (Invitrogen, Carlsbad, CA) and the surface area of cultured cardiomyocytes was determined by immunostaining with a rabbit polyclonal antibody against myosin heavy chain (Santa Cruz Biotechnology, Santa Cruz, CA).
Detection and quantitation of apoptosis. Apoptotic cells in tissue sections were quantified by terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling (TUNEL). Apoptosis of cultured cardiomyocytes was evaluated by double immunofluorescence for myosin heavy chain and TUNEL.
Dihydroethidium fluorescence and lucigenin-enhanced chemiluminescence. The oxidative fluorescent dye dihydroethidium was used to measure superoxide (O 2 2 ) levels in myocardial frozen sections and cultured cardiac fibroblasts and myocytes 8 . NADPH oxidase activity in myocardial tissues and cardiac fibroblasts and myocytes was quantified by lucigenin-enhanced chemiluminescence as describe8.
Western blot, immunohistochemistry, and immunocytochemistry. Western blot, immunohistochemistry, and immunocytochemistry were performed with standard methods.
Statistical analysis. SPSS v11.5 (SPSS Inc., Chicago, IL) was used for statistical analysis. Continuous data are expressed as mean 6 SEM and compared by one-way ANOVA, followed by Tukey-Kramer post-hoc test and independent samples t test. P , 0.05 was considered statistically significant.

Results
Ang-(1-7) has no effect on blood glucose level. One week after streptozotocin injection, fasting blood glucose level was markedly elevated in the model group and remained high until the end of the experiment, and its level did not differ between the mock group and the 7 treatment groups at the end of weeks 12 (Supplementary  Table 2) and 16 (Supplementary Table 3). No apparent side effects were observed in any treatment group.
Ang-(1-7) prevents LV dysfunction. At 12 weeks after streptozotocin injection, rats in the mock group showed decreased LV ejection fraction, fractional shortening, E/A, E'/A', maximal LV systolic pressure and 6dp/dt and increased LV end-systolic diameter, LV end-diastolic diameter and LV end-diastolic pressure as compared with the control group (Supplementary Tables 4 and 5). These measurements were dose-dependently improved by 4-week treatment with Ang-(1-7), and the salutary effects were enhanced by co-administration of perindopril and largely offset by A779 or PD123319 (Supplementary Fig. 1 and Tables 1 and 2). However, Ang-(1-7) had no significant effect on blood pressure and heart rate in DCM rats (Supplementary Table 3).
Ang-(1-7) improves LV ultrastructural abnormalities. TEM revealed clear sarcomeres and Z lines and apparently normal-sized mitochondria, with normal numbers, in the LV myocytes of the control group but significant swelling and disruption of mitochondria, as well as myofibril disarray, in the LV myocardium of the mock group (Fig. 1A). However, these abnormalities in LV ultrastructure were markedly improved by Ang-(1-7) or perindopril treatment. The effects of Ang-(1-7) on LV ultrastructural abnormalities were blocked in part by co-administration of A779 or PD123319.
Ang-(1-7) suppresses myocardial fibrosis. In the mock group, the collagen volume fraction (CVF) and ratio of perivascular collagen area to luminal area (PVCA/LA) increased by 2.9-and 3.1-fold, respectively, as compared with the control group, and these values were significantly reduced by the Ang-(1-7) treatment at the end of week 16. Moreover, CVF and PVCA/LA were greatly decreased with combined Ang-(1-7) and perindopril treatment relative to single therapy with Ang-(1-7) or perindopril. However, the beneficial effects of Ang-(1-7) on CVF and PVCA/LA were completely reversed by A779 treatment (Fig. 1A-C).
As compared with the mock group, Ang-(1-7) treatment dosedependently reduced mRNA expression of fibrosis-associated genes, including fibronectin-1 (Fig. 1D), collagen I-a1 (Fig. 1E) and TGF-b1 ( Fig. 2A), as well as the ratio of collagen I-a1 to III-a1 (Fig. 1G). Similar effects were observed in the perindopril group. Again, these Ang-(1-7)-induced effects were completely reversed by A779 and partially blocked by PD123319 (Figs. 1D, E, G, and 2F). Immunohistochemistry revealed that the contents of collagen I and III and the ratio of collagen I to III were significantly higher in the mock than control group (Fig. 1H-K). Compared with the mock group, Ang-(1-7) treatment reduced the content of collagen I and ratio of collagen I to III (Fig. 1H, I, and K) but not content of collagen III ( Fig. 1H and J). The effect of Ang-(1-7) on collagen I expression was reversed completely by A779 and partially by PD123319.     In addition, Ang-(1-7) treatment dose-dependently inhibited activation of ERK1/2 and p38-MAPK and TGF-b1 protein expression as compared with mock treatment (Fig. 2B-E). The inhibitory effects of Ang-(1-7) on ERK1/2 and p38-MAPK activation and TGF-b1 expression were completely reversed by A779 and partially by PD123319 ( Fig. 2G-K).
Ang-(1-7) attenuates myocardial hypertrophy. Intraventricular septal thickness (IVSth), LV posterior wall thickness (LVPWth) and ratio of heart weight to body weight were significantly higher in the mock than control group (Table 1 and Fig. 3A and C). Treatment with Ang-(1-7) dose-dependently reduced IVSth, LVPWth and the ratio of heart weight to body weight compared  with the mock group. Notably, combined treatment with Ang-(1-7) and perindopril further reduced IVSth, LVPWth and ratio of heart weight to body weight as compared with single treatment. However, the effects of Ang-(1-7) on myocardial hypertrophy were largely reversed by co-administration of PD123319 or A779.
Cardiomyocyte cross-sectional areas were significantly larger in the mock than control group ( Fig. 3B and D), which indicates myocardial hypertrophy in DCM rats. In contrast, Ang-(1-7)-treated groups showed a dose-dependent decrease in the cardiomyocyte cross-sectional areas as compared with the mock group, and this effect was enhanced by co-administration of perindopril and reversed by PD123319 or A779. Similarly, the mRNA expression of brain natriuretic peptide and b-myosin heavy chain as markers of cardiac hypertrophy was dose-dependently reduced by Ang-(1-7) treatment, which was also enhanced by co-administration of perindopril and completely reversed by PD123319 ( Fig. 3E and F).
Ang-(1-7) inhibits cardiac apoptosis. Rats in the mock group showed prominent cardiac apoptosis, as indicated by significantly increased proportion of TUNEL-positive cells ( Fig. 4A and B), abnormal morphology of myocyte nuclei by TEM (Fig. 4C), increased mRNA and protein expression of Bax and ratio of Bax to Bcl-2 (Fig. 4D, F-H, and J) and significantly decreased mRNA and protein expression of Bcl-2 ( Fig. 4E, G, and I), all of which were ameliorated by treatment with Ang-(1-7) at 800 ng?kg 21 ?min 21 . Of note, Ang-(1-7) at 800 ng?kg 21 ?min 21 improved features of cardiomyocyte nuclei (Fig. 4C). The effects of Ang-(1-7) on cardiac apoptosis were completely reversed by PD123319 and partially blocked by A779 (Fig. 4A, B, and K-R).
Ang-(1-7) ameliorates myocardial oxidative stress and inflammation. Consistent with a previous report that oxidative stress may be a pivotal mechanism of high glucose-mediated cardiovascular injury 16 , we found greater O 2 2 production and NADPH oxidase activation in myocardia of the mock than control group. Ang-(1-7) treatment dose-dependently attenuated and the combined Ang-(1-7) and perindopril normalized O 2 production and NADPH oxidase activation (Supplementary Fig. 2A and B). These effects were offset by co-administration of A779 or PD123319.
On immunohistochemical staining of myocardia, the protein expression of IL-1b, IL-6 and MCP-1 was greater in the mock than control group (Supplementary Fig. 2A and C). Ang-(1-7) treatment dose-dependently attenuated and combined treatment of Ang-(1-7) and perindopril normalized the expression of IL-1b, IL-6 and MCP-1. These inhibitory effects were reversed by co-administration with A779 or PD123319.
Ang-(1-7) reduces collagen synthesis of cardiac fibroblasts. Highglucose stimulation significantly increased types I and III collagen content in the cultured media of cardiac fibroblasts (Supplementary Fig. 3A and B), which was inhibited by Ang-(1-7) treatment timeand dose-dependently ( Supplementary Fig. 3A-D) as was the ratio of collagen I to III (Supplementary Fig. 3G). These inhibitory effects of Ang-(1-7) were enhanced by combined treatment with perindopril and completely reversed by A779 (Supplementary Fig. 3E-G).
Ang-(1-7) inhibits proliferation, differentiation and oxidative stress of cardiac fibroblasts. High glucose stimulation significantly augmented fibroblast proliferation and differentiation into myofibroblasts ( Fig. 5A-C). Ang-(1-7) significantly decreased fibroblast proliferation and differentiation over 72-hr culture versus highglucose treatment alone; this effect of Ang-(1-7) was completely reversed by A779. Moreover, combined treatment with Ang-(1-7) and perindopril significantly inhibited fibroblast proliferation and differentiation relative to Ang-(1-7) or perindopril alone. O 2 2 level and NADPH oxidase activity were increased by highglucose stimulation in cultured cardiac fibroblasts; the upregulated NADPH oxidase activation was attenuated by Ang-(1-7) for 72 hr (Fig. 5A, D, and E). Combined incubation with Ang-(1-7) and perindopril normalized the O 2 2 level and NADPH oxidase activity in cardiac fibroblasts. In addition, the ability of Ang-(1-7) to attenuate oxidative stress of cardiac fibroblasts induced by high glucose was partially blunted by co-administration of A779 or PD123319. Ang-(1-7) inhibits ERK1/2 and p38-MAPK phosphorylation and TGF-b1 expression in cardiac fibroblasts. The ratio of phosphorylated to total protein expression of ERK1/2 and p38-MAPK in cardiac fibroblasts was markedly lower after Ang-(1-7) treatment than high-glucose treatment (Fig. 5F-H). Likewise, the protein expression level of TGF-b1 in cardiac fibroblasts was lower with Ang-(1-7) and high glucose than with high glucose alone ( Fig. 5F and I).
Ang-(1-7) suppresses fibroblast-myocyte communication. The protein expression of collagen I and III and TGF-b1 was significantly higher in fibroblasts 1 non-treated myocytes than fibroblasts alone. In contrast, the protein expression of collagen I and III and TGF-b1 were lower in fibroblasts 1 Ang-(1-7)treated myocytes than fibroblasts 1 non-treated myocytes ( Supplementary Fig. 4A-C).
The protein expression of collagen I and III and TGF-b1 was substantially higher in fibroblasts 1 non-treated myocytes than fibroblasts alone. In contrast, the protein levels of collagen I and III and TGF-b1 were lower in fibroblasts 1 Ang-(1-7)-treated myocytes than fibroblasts 1 non-treated myocytes ( Supplementary Fig. 4D-F).
The inhibitory effects of Ang-(1-7) on collagen and TGF-b1 production induced by fibroblast-myocyte communication were partially blocked by co-administration of A779 or PD123319.
Ang-(1-7) prevents cardiomyocyte hypertrophy, apoptosis and oxidative stress via MasR and AT 2 R. In in vitro experiments, high glucose significantly increased cardiomyocyte size and apoptosis rate as compared with normal glucose (Supplementary Fig. 5A, B and C). Ang-(1-7) treatment normalized cardiomyocyte size and apoptosis rate as compared with high-glucose treatment alone, which were abrogated by co-treatment with A779 or PD123319. Neither A779 nor PD123319 alone altered the size and apoptosis rate of highglucose-incubated cardiomyocytes. O 2 2 level and NADPH oxidase activity were higher with high glucose than normal-glucose treatment ( Supplementary Fig. 5D and E). Ang-(1-7) treatment with or without administration of perindopril normalized high glucose-induced O 2 2 production and NADPH oxidase activation in cardiac myocytes. In addition, the ability of Ang-(1-7) to attenuate oxidative stress of cardiac myocytes after high-glucose stimulation was partially blocked by co-administration of A779 or PD123319.
Ang-(1-7) downregulates AT 1 R and upregulates AT 2 R in vivo and in vitro. In vivo, AT 1 R expression was significantly increased and AT 2 R expression was decreased with mock than control treatment. These effects were largely attenuated by high-dose Ang-(1-7) (Fig. 6A-C). In vitro, high glucose induced higher AT 1 R expression and lower AT 2 R expression than normal glucose in both cardiac fibroblasts and myocytes, which was reversed by Ang-(1-7) treatment (Fig. 6E-G and I-K). Nevertheless, Ang-(1-7) did not affect MasR expression in rat hearts (Fig. 6A and D) or cardiac fibroblasts ( Fig. 6E and H) and myocytes ( Fig. 6I and L) cultured in high-glucose medium.
The effect of Ang-(1-7) on AT 1 R expression in rat hearts and cultured cardiac fibroblasts was completely inhibited with co-admin-istration of A779 or PD123319, and that in cardiac myocytes was reversed by A779 but not by PD123319. The effect of Ang-(1-7) on AT 2 R expression in rat hearts was completely reversed by PD123319 and partially by A779; that in cardiac fibroblasts was completely blocked by PD123319 but not altered by A779; and that in cardiac myocytes was totally inhibited by A779 or PD123319.

Discussion
The major finding of the present study was that chronic Ang-(1-7) treatment protected against LV remodeling and dysfunction in DCM rats without altering body weight, blood pressure, heart rate or blood glucose level. The cellular mechanisms may involve decreased fibroblast proliferation and differentiation into myofibroblasts, attenuated mitochondria swelling, myofibril disarray, hypertrophy and apoptosis of cardiomyocytes, as well as inhibited fibroblast-myocyte communication. The molecular mechanisms may involve reduced inflammatory cytokine expression, oxidative stress and collagen synthesis, inhibited ERK1/2 and p38-MAPK phosphorylation and TGF-b1 expression, upregulated ACE2 activity and Ang-(1-9) level, as well as a complex interaction of MasR, AT 2 R and AT 1 R (Supplementary Fig. 9). Furthermore, Ang-(1-7) combined with perindopril provided additional cardioprotection, a finding of important clinical implications in the development of a novel therapeutic regimen for DCM.
DCM is characterized by cardiac fibrosis, myocardial hypertrophy and myocyte apoptosis; the most salient pathological feature of DCM is the accumulation of collagen in the extracellular matrix. A wealth of evidence has confirmed the importance of RAS in collagen production in DCM. Therefore, most recent studies have focused on discovering novel therapeutic targets from RAS 3 . Although ACE inhibitors or AT 1 R antagonists have been found effective in preventing diabetic complications, recent studies revealed that AT 1 R antagonists can inhibit the effects of only extracellular Ang-II 3 ; however, intracellular Ang-II is an important mediator of collagen production in DCM. ACE inhibitors can block Ang-II synthesis catalyzed by ACE in cardiac fibroblasts but not that catalyzed by chymase in cardiac myocytes 3,17 . Thus, ACE inhibitors and AT 1 R antagonists can only partially inhibit RAS activities in DCM. In contrast, Ang-(1-7) has powerful anti-oxidative stress effects opposite to those of Ang-II in both fibroblasts and myocytes.
In the pathological process of myocardial fibrosis, cardiac fibroblasts play an important role by differentiating into myofibroblasts, which may induce fibroblast proliferation. Myofibroblasts, which are absent in the normal myocardium, are characterized by acquisition of a-smooth muscle actin expression and enhanced collagen synthesis and constitute the major cellular source of collagen production in myocardial fibrosis 18 . Thus, inhibition of the differentiation of cardiac fibroblasts to myofibroblasts is considered a key target for anti-fibrosis therapy. In addition, recent studies found that after high glucose or Ang-II stimulation, myocytes may secrete active TGF-b that may induce collagen synthesis in fibroblasts 5,19 , which suggests that the cross-talk between myocytes and fibroblasts may play an important role in the pathogenesis of DCM. Inhibition of the crosstalk between myocytes and fibroblasts is an indispensable mechanism underlying the therapeutic effects of Ang-(1-7) in DCM.
The molecular mechanisms of cardiac fibrosis are complex and involve a cascade of intracellular signaling pathways. TGF-b1 is a key pro-fibrotic cytokine markedly elevated in experimental DCM. The finding that Ang-(1-7) inhibited the expression of TGF-b1 in this study indicates that suppression of the TGF-b1 pathway underlies the anti-fibrotic effects of Ang-(1-7) in DCM. Previous studies implicated ERK1/2 and p38-MAPK pathways in cardiac fibrosis and hypertrophy 20,21 and our study demonstrated that Ang-(1-7) significantly inhibited both phosphorylation of ERK1/2 and p38-MAPK in diabetic hearts and high glucose-induced activation of ERK1/2 and p38-MAPK in cardiac fibroblasts, which agreed with recent reports in other disease models [22][23][24] . Recently, emerging evidence suggests that the effects of Ang-(1-7) may be mediated by activation of some other phosphatases including Src homology 2-containing protein-tyrosine phosphatase-1, phosphatase and tensin homologue, and dual-specificity phosphatase 1 22,23,25 .
Whether inflammation plays a role in the pathogenesis of DCM remains disputed. Accumulating evidence suggests that increased oxidative stress coupled with activation of downstream pro-inflammatory pathways leads to LV remodeling and dysfunction in DCM 2 , and other evidence suggests that DCM is different from other types of cardiomyopathy in the lack of an inflammatory response 26 . In the current study, we found that enhanced O 2 2 generation, NADPH oxidase activation and proinflammatory cytokine expression in the mock group was inhibited by Ang-(1-7) treatment. Therefore, oxidative stress and inflammation may take part in the pathogenesis of DCM, which was significantly attenuated by Ang- (1-7) treatment.
In the present study, we examined the impact of Ang-(1-7) on the expression of AT 1 R, AT 2 R and MasR. Previous studies showed that upregulated AT 1 R and downregulated AT 2 R promoted cardiac fibrosis and hypertrophy 27,28 . We found that AT 1 R expression was significantly increased and AT 2 R expression was decreased in the mock group; these effects were largely reversed by Ang-(1-7) treatment. The mechanism underlying the effects of Ang-(1-7) on AT 1 R is unclear but may involve the complex interactions among AT 1 R, AT 2 R and MasR, because Ang-(1-7) had no effect on Ang-II levels and the effect of Ang-(1-7) on AT 1 R expression was inhibited by A779 or PD123319 in vivo and in vitro. The effects of Ang-(1-7) on cardiac fibrosis were completely reversed by A779 and partially blocked by PD123319, whereas those on myocardial hypertrophy and myocyte apoptosis were completely reversed by PD123319 and partially blocked by A779. Similar to Ang-II, with two receptors, Ang-(1-7) may exert its cardioprotective effects via both MasR and AT 2 R, and different receptor subtypes may mediate different biological effects. Although Ang-(1-7) appears to act via the MasR in vitro 29 , this selectivity is lost in vivo 30 . One recent study showed that Ang-(1-7) may act via both AT 2 R and MasR because both PD123319 and A779 abrogated Ang-(1-7)-induced vasoprotection 31 , which lends support to our results.
We found that combining exogenous Ang-(1-7) with perindopril significantly increased plasma and myocardial levels of Ang-(1-7) and provided more cardioprotection than single therapy, probably because perindopril reduces Ang-II generation and inhibits conversion of Ang-(1-7) into inactive Ang-(1-5) 13 . A recent study demonstrated that long-term AT 2 R activation increased renal ACE2 activity 32 . Our results showed that exogenous Ang-(1-7) treatment significantly increased myocardial ACE2 activity and Ang-(1-9) level, possibly via its effect on AT 2 R, and the activated ACE2 may catalyze Ang-II into Ang-(1-7), thus forming a positive feedback. Ang-II was recently found to induce ACE2 shedding by promoting ADAM17 activity as a positive feedback mechanism whereby Ang-II facilitates the loss of its negative regulator, ACE2 33 . Thus, the discrepancy between the increased myocardial ACE2 activity and decreased plasma ACE2 activity with perindopril treatment might be attributed to the reduced ACE2 shedding due to downregulated ADAM17 activity after inhibited generation of Ang-II. However, Ang-(1-7) had no effect on myocardial ACE activity and Ang-II level and plasma ADAM17 activity, suggestive of a negative impact of Ang-(1-7) on ACE2 shedding. Therefore, increased ACE2 activity by Ang-(1-7) and decreased ACE2 shedding by ACE inhibition might offer another advantage of combined therapy versus single therapy.
Several clinical studies reported that elevated plasma ACE2 activity as a compensatory response was associated with increased severity of myocardial dysfunction and was an independent predictor of adverse clinical events [34][35][36] . Moreover, we recently found that plasma Ang-(1-7) level was independently associated with LV remodeling and dysfunction in diabetic patients 37 and could also predict myocardial salvage after reperfusion treatment for acute myocardial infarction 38 . Therefore, plasma Ang-(1-7) may be a biomarker for www.nature.com/scientificreports LV function and a predictor for long-term myocardial remodeling under disease conditions. Whether the association of Ang-(1-7) and LV function is attributed to the inhibitory effect of Ang-(1-7) on LV remodeling merits future study.
Study limitations. The STZ-induced diabetic model has important limitations and our results need to be validated in genetically manipulated diabetic models. Also, although we found that Ang-(1-7) treatment was associated with downregulated AT 1 R, upregulated AT 2 R, attenuated oxidative stress and inflammation, and inhibited fibroblast-myocyte communication, the key mechanism mediating these effects remains obscure and require further investigation. Future research should also focus on the relative AT 1 R-, AT 2 R-and MasR-binding affinities of Ang-(1-7) in the heart.

Conclusions
Chronic Ang-(1-7) treatment ameliorated LV remodeling and dysfunction in DCM via multiple mechanisms involving attenuated inflammation, oxidative stress and collagen synthesis, inhibited ERK1/2 and p38-MAPK signaling and TGF-b1 expression, upregulated ACE2 activity and Ang-(1-9) level as well as complex interactions of MasR, AT 2 R and AT 1 R. Furthermore, combining Ang-(1-7) with perindopril provided additional cardioprotection. Thus, Ang-(1-7) administration may be a novel and promising approach to the treatment of DCM.