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Abstract
Angiotensin-converting enzyme 2 (ACE2) plays a critical role against myocardial infarction (MI). We hypothesized that activation of intrinsic ACE2 would be protective against ischemia-induced cardiac pathophysiology. Diminazene aceturate (DIZE), a small molecule ACE2 activator, has been used to evaluate this hypothesis. DIZE (15 mg/kg per day, s.c.) was injected 2 days before MI surgery and continued throughout the study period. MI rats showed a 62% decrease in fractional shortening (%; control, 51.1±3.2; DIZE alone, 52.1±3.2; MI, 19.1±3.0), a 55% decrease in contractility (dP/dtmax mm Hg/s; control, 9480±425.3; DIZE alone, 9585±597.4; MI, 4251±657.7), and a 27% increase in ventricular hypertrophy (mg/mm; control, 26.5±1.5; DIZE alone, 26.9±1.4; MI, 33.4±1.1). DIZE attenuated the MI-induced decrease in fractional shortening by 89%, improved dP/dtmax by 92%, and reversed ventricular hypertrophy by 18%. MI also significantly increased ACE and angiotensin type 1 receptor levels but decreased ACE2 activity by 40% (control, 246.2±25.1; DIZE alone, 254.2±20.6; MI, 148.9±29.2; RFU/min), which was reversed by DIZE treatment. Thus, DIZE treatment decreased the infarct area, attenuated LV remodeling post-MI, and restored normal balance of the cardiac renin–angiotensin system. In addition, DIZE treatment increased circulating endothelial progenitor cells, increased engraftment of cardiac progenitor cells, and decreased inflammatory cells in peri-infarct cardiac regions. All of the beneficial effects associated with DIZE treatment were abolished by C-16, an ACE2 inhibitor. Collectively, DIZE and DIZE-like small molecules may represent promising new therapeutic agents for MI.
Introduction
The renin–angiotensin system (RAS) plays a critical role in maintaining cardiovascular homeostasis. Angiotensin-converting enzyme 2 (ACE2) metabolizes angiotensin II (AngII) into the heptapeptide angiotensin-(1–7) (Ang-[1–7]),1,2 thereby maintaining a balance between the deleterious axis (ACE/AngII/angiotensin type 1 receptor [AT1R]) and the vasoprotective axis (ACE2/Ang-(1–7)/Mas receptor [MasR]) of the RAS. ACE inhibitors and angiotensin receptor blockers are leading pharmacotherapeutic interventions for cardiovascular diseases.3–5 It has been demonstrated that the use of these agents to inhibit the deleterious axis of the RAS leads to an upregulation of ACE2.6
ACE2 modulates cardiac function and could be a potential therapeutic target for cardiovascular diseases, on the basis of the following evidences: (1) ACE2 and Ang-(1–7) levels are increased as a compensatory mechanism in the heart after myocardial infarction (MI) in both humans and rats7–9; (2) we and others have demonstrated that cardiac overexpression of ACE2 renders protection against ischemia-induced cardiac pathophysiology10,11; (3) overexpression of Ang-(1–7) protects the heart from the ischemic-induced cardiac dysfunction12; (4) we have previously demonstrated that a small molecule, XNT (1-[(2-dimethylamino) ethylamino]-4-(hydroxymethyl)-7-[(4-methylphenyl) sulfonyl oxy]-9H-xanthene-9-one), possessing the potential to activate ACE2, prevents cardiac remodeling in spontaneously hypertensive rats.13 Collectively, these findings have led several groups to propose that administration of recombinant ACE2 could be a potential therapeutic strategies for the treatment of heart failure.14,15
Recently, diminazene aceturate (DIZE), another US Food and Drug Administration–listed small molecule used as an antitrypanosomal drug, has been shown to exert off-target effects of activating ACE2.16 We have recently demonstrated that DIZE treatment renders cardioprotective effects and improves angiogenic progenitor cell functions in rat model of pulmonary hypertension.17 This complied with the fact that current standard treatments for MI cannot efficiently improve cardiac function and prevent further damage to the myocardium. We decided to evaluate the effects of DIZE on ischemic-induced cardiac pathophysiology. Our observations demonstrate that DIZE treatment prevents MI-induced cardiac pathophysiology, thus providing us an opportunity to explore the therapeutic potential of this compound in ischemic heart disease and heart failure.
Material and Methods
All experimental protocols are presented in the Methods in the online-only Data Supplement.
Results
DIZE Treatment Prevents the MI-Induced Left Ventricular Dysfunction, and C-16 Abolishes the Protective Effects of DIZE
MI caused a 62% reduction in fractional shortening (Figure 1A) and a 27% increase in ventricular hypertrophy (Figure 1B), which were significantly prevented by DIZE treatment. Measurement of cardiac hemodynamic functions in MI animals revealed higher left ventricular end diastolic pressure and decreased dP/dtmax and dP/dtmin compared with normal and DIZE controls, which were restored by DIZE treatment (Figure 1C–1E). We had hypothesized that these beneficial effects of DIZE are associated with the activation of endogenous cardiac ACE2. Therefore, we coadministered C-16, a selective ACE2 inhibitor,17 along with DIZE. Four weeks of cotreatment with C-16 abolished the improvements in cardiac function produced by DIZE (Figure 1A–1E). In contrast to these beneficial effects on cardiac pathophysiology, DIZE treatment did not alter systolic blood pressure (Figure 1F).
Diminazene aceturate (DIZE) treatment prevents the development of myocardial infarction (MI)–induced heart failure and associated cardiac hypertrophy. Measurement of (A) fractional shortening and (B) ventricular hypertrophy reflected by the ratio of ventricular weight to tibia length (VW/TL), (C) dP/dtmax, (D) dP/dtmin, (E) left ventricular end diastolic pressure (LVEDP), and (F) systolic blood pressure (SBP). Data represent mean±SEM. *MI (M), MI+DIZE (M+D), and MI+DIZE+C16 (M+D+C) vs Control (Con) and DIZE (D); #M+D vs M and M+D+C;**M and M+D+C vs Con, D, and M+D; P<0.001.
DIZE Treatment Alters Cardiac RAS Levels
mRNA levels of ACE and ACE2 and protein levels of AT1R and MasR were measured to determine whether DIZE treatment shifts the balance from deleterious to vasoprotective axis of the RAS. Cardiac ACE mRNA increased by 2-fold, whereas ACE2 mRNA decreased 2-fold in MI animals (Figure 2A and 2B). DIZE treatment reduced ACE mRNA and significantly elevated ACE2 mRNA in the MI animals. As a result, the ratio of the mRNA levels of ACE2/ACE was 2-fold higher in DIZE-treated MI hearts (Figure 2C). MI induced 2-fold increase in ACE activity, although this activity in DIZE-treated MI hearts was not significantly different from the control and DIZE-treated hearts (Figure 2D). In contrast, ACE2 activity was modestly decreased in the peri-infarct area of the MI hearts, and DIZE treatment resulted in 2.5-fold increase in this activity (Figure 2E). Plasma ACE2 activity was slightly lowered in MI animals and significantly increased by DIZE treatment, which was abolished by C-16 (Figure 2F). Moreover, Western blot analysis demonstrated a 1.9-fold increase in AT1R protein levels (Figure 2G) and a moderate increase in MasR (Figure 2H) in the MI group. DIZE treatment significantly inhibited the increase in AT1R. These changes induced by DIZE were blocked by treatment with C-16. However, DIZE treatment did not significantly alter the increase in MasR compared with the MI group.
Diminazene aceturate (DIZE) modulates cardiac renin–angiotensin system. Relative change in cardiac mRNA levels of the angiotensin-converting enzyme (ACE; A) and ACE2 (B) genes, ACE2/ACE ratio (C), cardiac ACE activity (D), cardiac ACE2 activity (E), and plasma ACE2 activity (F). Representative Western blot showing protein expression of AT1R (G) and Mas receptor (MasR) (H). Data represent mean±SEM. **MI (M) and MI+DIZE+C16 (M+D+C) vs control (Con), D, and M+D; *M, M+D, and M+D+C vs Con and D; #M+D vs M and M+D+C; ***M+D vs Con, M, and M+D+C; %D vs M and M+D+C; P<0.05. AT1R indicates angiotensin type 1 receptor.
DIZE Treatment Increases Numbers of Cardiac Progenitor Cells and Circulating Endothelial Progenitor Cells
Our next objective was to determine whether the reparative capacities of DIZE are because of its effects on progenitor cells. Thus, we determined the levels of circulating endothelial progenitor cells (EPCs) and Islet-1 (a marker for cardiac progenitor cells [CPCs]). We observed a 2-fold increase in the CPCs in the peri-infarct region of the DIZE-treated ischemic heart (Figure 3A and 3B). This increase in the number of CPCs suggests a higher regenerative potential in the hearts from DIZE-treated animals. Moreover, the number of circulating EPCs was also increased by 48% in the DIZE-treated animals over that observed in the MI group (Figure 3C). Cotreatment of DIZE animals with C-16 abolished both increases in the CPCs and in the EPCs.
Diminazene aceturate (DIZE) increases the number of cardiac progenitor cells in the peri-infarct area of the heart and circulating rat endothelial progenitor cell (EPCs). Islet-1 is predominantly found in the nuclei. Representative immunofluorescence images of Islet-1+ cells (red fluorescence, indicated by yellow arrowhead). The nuclei were stained with DAPI (4',6-diamidino-2-phenylindol; blue fluorescence). Islet-1 level was also quantified by Western blot (A and B). The number of rat EPCs (CD4−/5−/8−/90+) in the blood was also measured using flow cytometry (C). Data are expressed as mean±SEM; **MI (M) and MI+DIZE+C16 (M+D+C) vs control (Con), D, and M+D; $M+D vs M+D+C; P<0.05. Scale bar, 20 μm.
DIZE Treatment Decreases Infiltration of Macrophages in the Peri-Infarct Area of the Heart and Modulates Proinflammatory Cytokines
Infiltration of macrophage in the peri-infarct area of the heart is associated with MI.18 Thus, we examined whether DIZE treatment would influence this process. DIZE treatment significantly prevented the infiltration of CD68+ cells (a marker for macrophages; Figure 4, top panel). Western blot analysis showed that CD68 immunoreactivity in MI hearts was increased by 46%, which was completely attenuated by DIZE treatment (Figure 4A and 4B). Interleukin-1 beta (IL-1β) and tumor necrosis factor alpha (TNF-α) mRNA levels were examined next to determine whether the beneficial effects of DIZE on cardiac hemodynamic were associated with changes in the proinflammatory cytokines. As expected, MI resulted in 2.5- and 3-fold increases in IL-1β and TNF-α, respectively (Figure 4C and 4D). DIZE treatment alone did not affect these cytokines in control animals. However, DIZE treatment in the MI animals prevented the increase in these cytokines, which further supported the anti-inflammatory effects of DIZE. C-16 blocked all the anti-inflammatory effects of DIZE (Figure 4).
Diminazene aceturate (DIZE) decreases the number of infiltrated macrophage and modulates cardiac proinflammatory cytokines. Representative immunofluorescence images of CD68 antibody–positive cells (red fluorescence) and nuclei (blue). CD68 was quantified by Western blot (A and B). Relative mRNA levels of IL-1β (C) and TNF-α (D). Data are expressed as mean±SEM; **MI (M) and MI+DIZE+C16 (M+D+C) vs control (Con), D, and M+D; P<0.05. Scale bar, 20 μm.
DIZE Treatment Decreases Infarct Area and MI-Induced Apoptosis
DIZE treatment decreased the infarct size by 50% (Figure S1A in the online-only Data Supplement). Next, we examined whether DIZE exerts antiapoptotic effects in response to ischemia. Heart sections of the MI group showed 6-fold increase in terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining, which was prevented by DIZE treatment (Figure S1B). C-16 treatment abolished all the effects produced by DIZE (Figure S1).
DIZE Treatment Prevents the MI-Induced Loss of Capillary Density
A significant reduction (1-fold) in capillary density was observed in MI hearts. DIZE treatment alone did not alter capillary density; however, DIZE treatment in the MI animals significantly prevented the decrease in the capillary density of the MI group (Figure S2).
Discussion
Our study is significant in that it provides evidence that DIZE, an ACE2 activator offers impressive protection against MI by preserving fractional shortening, attenuating the increase in LVEDP, preventing the dysfunctional changes in dP/dt, and decreasing infarct size and apoptosis. This treatment also modulates the RAS in a positive manner, reduces inflammatory processes, and increases the engraftment of CPCs and circulating EPCs. All these beneficial effects were blocked by C-16.
The imbalance in the RAS as a result of an increase in the vasodeleterious (ACE/AngII/AT1R) axis and a decrease in the vasoprotective (ACE2/Ang-(1–7)/MasR) axis has been proposed to be critical in the development and establishment of cardiac pathophysiology.19,20 ACE2 expression is upregulated at the beginning of the MI as a compensatory mechanism8 and then significantly decreased at heart failure stage.21 Because DIZE is administered before the injury is induced, it protects heart function and thus prevents the ACE2 downregulation. It is also possible that DIZE modulates ACE2 expression and acts in a positive feedback manner because DIZE has been found to be able to bind DNA.22 Thus, we hypothesize that restoring this imbalance by the activation of endogenous ACE2 could provide protection against cardiac damage. This study provides evidence in support of this concept and confirms the protective role of the vasoprotective axis because all the dysfunctional and pathological parameters were prevented by the ACE2 activator.
It has been shown that ACE2 overexpression in the human umbilical vein endothelial cells leads to an increase in tube formation and can promote endothelial cell migration in vitro, and ACE2 can promote capillary formation and neovessel maturation in vivo.23,24 DIZE, as an ACE2 activator, may also promote angiogenesis and exert its effects via Ang-(1–7). Ang-(1–7) also has been shown to directly act on hematopoietic progenitor cells and modulate their function.25,26 In the present study, DIZE causes an increase in CPCs homing to the peri-infarct area of the ischemic heart, increases circulating EPCs, and restores capillary density, all of which are correlated with an improvement of cardiac function. Our study and others indicate that the ACE2/Ang-(1–7)/MasR axis induces mechanisms that are pivotal for regenerative therapy, and further studies on the signaling pathways activated by DIZE in progenitor cells are warranted.
Elevated levels of proinflammatory cytokines, such as IL-1β and TNF-α, contribute to the development and progression of ischemic injury.27,28 The ACE2/Ang-(1–7)/MasR axis possesses an anti-inflammatory role.29 Our observation that DIZE prevents increases in proinflammatory cytokines, and macrophage cells further supports the importance of this compound in influencing many pathological events, directly and indirectly. This is supported by our previous observations that overexpression of Ang-(1–7) and DIZE treatment decreases pulmonary proinflammatory cytokines in models of lung disease.17,30
In summary, our study illustrates that DIZE treatment preserves cardiac function and attenuates cardiac remodeling post-MI. The beneficial effects of DIZE may be a result of preventing the imbalance of the RAS observed post-MI, subsequently modulating various proinflammatory cytokines associated with MI injury, decreasing the infiltration of macrophages associated with cardiac ischemia, and increasing the homing of CPCs and mobilizing EPCs.
Finally, it is pertinent to point out that we have used DIZE as a compound to test this proof of concept. DIZE has been shown to exert toxic effects in some studies31–33 and may not be the pharmacodynamically ideal molecule for translational studies. Nonetheless, the study is relevant in that it provides evidence that the chemical structure resides in DIZE has the potential to activate ACE2, provide beneficial outcomes in MI-induced cardiac pathophysiology, and form the basis to develop structure similar and safe compounds.
Perspectives
Recently, Oudit and Penninger14 have suggested that recombinant ACE2 can be used as a novel therapy for heart failure. However, the application of recombinant ACE2 protein for MI is limited by its peptide nature and possible metabolic degradation if administrated orally. The use of synthetic small molecules, such as DIZE, or other compounds that are ACE2 activators, could circumvent these issues. Thus, current observations support the concept that targeting of the cardiac ACE2/Ang-(1–7)/MasR axis using a small molecule could hold novel therapeutic strategy in the treatment of MI and its associated complications.
Acknowledgments
We acknowledge Marda Jorgensen and Neal A. Benson for their technical assistance and help with immunohistochemistry and flow cytometry.
Sources of Funding
This work was supported by National Institutes of Health grants HL056921 and HL102033 awarded to M.K. Raizada and M.J. Katovich.
Disclosures
None.
Footnotes
The online-only Data Supplement is available with this article at http://hyper.ahajournals.org/lookup/suppl/doi:10.1161/HYPERTENSIONAHA.113.01337/-/DC1.
- Received March 4, 2013.
- Revision received March 22, 2013.
- Accepted July 22, 2013.
- © 2013 American Heart Association, Inc.
References
Novelty and Significance
What Is New?
This study demonstrates, for the first time, that chronic administration of ACE2 activator (diminazene aceturate [DIZE]) attenuated the myocardial infarction–induced cardiac dysfunction.
DIZE increased the number of circulating endothelial progenitor cells and cardiac progenitor cells and decreased the macrophage infiltration in the ischemic myocardium, which reveals a previously unidentified role of DIZE in modulating progenitor cells and the regulation of inflammation.
What Is Relevant?
It is significant that chronic activation of endogenous ACE2 by DIZE or as such attenuated MI-induced cardiac pathophysiology, which provides a new therapeutic approach for ischemic heart diseases and related cardiovascular disorders.
Summary
DIZE attenuated MI-induced cardiac pathophysiology through modulating the gene expression of the RAS and inflammatory cytokines, increasing EPCs and CPCs, and suppressing macrophage infiltration.
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