An Overview of Alamadine/MrgD signaling and its role in cardiomyocytes

Abstract

The renin-angiotensin system (RAS) is a classical hormonal system involved in a myriad of cardiovascular functions. This system is composed of many different peptides that act in the heart through different receptors. One of the most important of these peptides is Angiotensin II, which in pathological conditions triggers a set of actions that lead to heart failure. On the other hand, another RAS peptide, Angiotensin-(1–7) is well known to develop powerful therapeutic effects in many forms of cardiac diseases. In the last decade, two new components of RAS were described, the heptapeptide Alamandine and its receptor, the Mas-related G protein-coupled receptor member D (MrgD). Since then, great effort was made to characterize their physiological and pathological function in the heart. In this review, we summarize the latest insights about the actions of Alamandine/MrgD axis in the heart, with particular emphasis in the cardiomyocyte. More specifically, we focused on their anti-hypertrophic and contractility effects, and the related molecular events activated in the cardiomyocyte.

Introduction

The renin-angiotensin system (RAS) plays a critical role in the maintenance of the cardiovascular function. The history of RAS began in 1898 with the discovery of renin by Tigerstedt and Bergman (1, 2). Almost 40 years later, two independent groups found a substance with pressor activity secreted by the kidneys of animals made hypertensive by the Goldblatt technique (1–3). Both groups went on to describe a new molecule in the renal vein blood of ischemic kidneys. In 1958 this molecule was named angiotensin (3).

Originally, the RAS was best known for its role as a powerful modulator of blood pressure and electrolyte balance. However, decades of subsequent research describe the RAS as a much more complex system composed of a series of enzymes, peptides and receptors, acting in diverse tissues, including the heart. Because of this complexity, it can be usefully divided into two branches: classical RAS and non-classical or counter regulatory RAS. The classical RAS pathway is formed by Angiotensin-Converting Enzyme (ACE)/Angiotensin II (AngII)/Angiotensin II receptor type 1 (AT₁R). The non-classical or counter-regulatory axis is composed of Angiotensin-Converting Enzyme 2 (ACE2)/ Angiotensin-(1–7) (Ang-(1–7))/Mas receptor (MasR). In addition, Angiotensin II receptor type 2 (AT₂R) and Angiotensin-(1–9) also participate in the counter-regulatory RAS axis (4).

Pre-clinical and clinical studies have shown that overstimulation of the classical RAS pathway is implicated in the pathogenesis of heart failure. For instance, activation of Ang II/AT₁R axis induces cardiac oxidative stress (5) and apoptosis (6), cardiac hypertrophy (7), and myocardial fibrosis (8), all of which can lead to heart failure. Importantly, antagonists of AT₁R are widely used in clinical practice in the management of heart failure patients (9, 10).

Starting in the 1980s, a number of new RAS metabolites and receptors have been discovered and form what is current known as the non-classical RAS pathway. In general terms, this pathway opposes the actions of the classical RAS pathway in the cardiovascular system. The major non-classical RAS peptide described is Ang-(1–7), which acts through the Mas receptor to promote diverse cardioprotective effects (11–13). Then, in 2013, a second peptide was identified and named alamandine (14). Alamandine is a heptapeptide that shares some similarities with Ang-(1–7) and acts through Mas-related G protein-coupled receptor member D (MrgD). In this review, we discuss the actions of alamandine/MrgD in the heart. An emphasis is placed on the anti-hypertrophic and contractile effects of alamandine, and the signaling pathways it activates in cardiomyocytes.

Discovery, and sources of alamandine

Alamandine is a heptapeptide (Ala-Arg-Val-Tyr-Ile-His-Pro) discovered and characterized by Robson Santos’s group (14). Its presence has been detected in human blood and the rat heart, and as described so far, it can be synthesized by two pathways: (i) catalytic hydrolysis of the octapeptide Angiotensin A by angiotensin-converting enzyme 2 (ACE2) or (ii) decarboxylation of Ang-(1–7) N-terminal aspartate amino acid residue by an aspartate decarboxylase (AD). A schematic representation of the enzymatic pathways involved in the production of alamandine is presented in Figure 1A and its molecular structure is represented in Figure 1B.

Figure 1. Biosynthetic pathway and molecular structure of alamandine.

A. Enzymatic pathway involved in the alamadine generation. Alamandine can be formed by two main pathways described so far: (i) catalytic hydrolysis of the octapeptide Angiotensin A by angiotensin-converting enzyme 2 (ACE2) or by (ii) decarboxylation of Angiotensin-(1–7) N-terminal aspartate residue by an aspartate decarboxylase (AD). In turn, Angiotensin A and Ang-(1–7) are active metabolites of Ang II (45, 46). B. Molecular structure of alamandine.

Lautner et al. (2013), detected alamandine production in the isolated rat heart after Ang-(1–7) perfusion, suggesting that the heart contains the enzymatic machinery necessary to synthesize alamandine (14). However, the cellular compartment responsible for alamandine production is still unknown. It has been reported that cardiac levels of RAS peptides change under pathological conditions. For example, Averill et al. (2002) have shown an increase in Ang-(1–7) expression in cardiomyocytes from rats subjected to coronary artery ligation-induced heart failure (15). Likewise, it was shown that the clinical course of heart failure in human patients is associated with a progressive increase in Ang II formation in the heart (16). Although, it is not clear how cardiac alamandine production responds to pathological insults, given that levels of Ang-(1–7) and Ang II seem to be altered under pathological conditions, it is likely that alamandine levels are altered too (Figure 1A). For now, Lautner et al. (2013) described that nephropathic patients present increased plasmatic concentration of alamandine (14). On the other hand, in patients with idiopathic pulmonary fibrosis, the alamandine plasma concentration was lower than in controls (17). With this idea in mind, future studies are needed to investigate local cardiac alamandine production in different models of heart disease, and how it relates to plasmatic levels.

Expression and signaling of MrgD in the heart

Initial studies demonstrated the presence and functionality of the MrgD receptor in nociceptive sensory neurons (18, 19). Recent data extended these findings and revealed the MrgD presence in the cardiovascular system (14, 20–25). This section summarizes much of the known data on MrgD identification and expression in the heart. Moreover, we highlight evidence that cardiac MrgD expression is responsive to different pathological stimuli.

In 2013, MrgD was identified as a receptor for alamandine by using a combination of an in vivo pharmacological approach and in vitro experiments with MrgD-transfected cells (14). In mice aortic rings the authors showed that alamandine induces endothelial-dependent vasorelaxation (14), while microinjection of alamandine in caudal and rostral ventrolateral medulla modulates arterial pressure. Under both conditions a MasR antagonist, A-779, failed to block vasorelaxation and modulatory effects of alamandine in pressure control. Consistent with this finding, alamandine-induced vasorelaxation was preserved in aortic rings of MasR-deficient mice. These interesting observations indicated that alamandine and Ang-(1–7) activate distinct receptors, despite the high structural similarity shared between them (Figure 1A). Indeed, alamandine action was blocked by D-Pro⁷-Ang-(1–7), an antagonist of receptors Mas and MrgD (14). By using chinese hamster ovary (CHO) cells stably transfected with MrgD, the authors showed that alamandine induced nitric oxide (NO) production, which was not observed in MasR-transfected CHO cells (14). Taken together, these data provide important information supporting the idea that alamandine signals through MrgD receptor. However, Tetzner et al challenged the idea that alamandine signals only through MrgD receptors (26). By working with primary endothelial and mesangial cells and performing measurements of intracellular cAMP as a readout of alamandine-induced signaling, the authors showed that alamandine promoted a dose dependent increase in cAMP, which was blocked by D-PRO, A-779 and PD123319. Experiments performed in HEK cells with gain or loss of Mas and MrgD receptors supported the data obtained with the receptor blockers. Future studies are needed in order to confirm these observations.

The identification of MrgD as a putative receptor of alamandine, led to studies that confirmed its presence in the heart. Liu et al. (2018) showed the expression of MrgD in the rat heart by Western-blot (20). Additionally, Oliveira et al. (2018) observed the presence of MrgD in the cell membrane and in the perinuclear and nuclear regions of neonatal cardiomyocytes by immunofluorescence (21). We extended these findings by detecting MrgD mRNA in neonatal and adult cardiomyocytes (22), and by using FAM-alamandine (FAM-ALA) to label ventricular myocytes. Incubation of cardiomyocytes with FAM-ALA resulted in a clear staining pattern, which was absent in the presence of the MrgD antagonist D-Pro⁷-Ang-(1–7). Confirming the MrgD as a binding site for alamandine, FAM-ALA labeling was markedly reduced in ventricular myocytes from MrgD-deficient (MrgD^−/−) mice (22). Importantly, FAM-ALA staining was preserved in cardiomyocytes from MasR-deficient mice. Together, these findings not only demonstrate the presence of MrgD receptor at both neonatal and adult stages but also established this receptor as a functional binding site for alamandine in the heart.

Previous evidence has shown that different members of RAS receptor family may change their expression pattern in response to different pathological conditions (27, 28). Likewise, Liu et al. (2018) showed that MrgD expression is increased in the heart of spontaneously hypertensive rats (SHR) (20). Consistent with this finding, we showed an increase in MrgD abundance in ventricular myocytes from another model of hypertension, the TGR (mREN2)27 rat (23). In addition, it has shown that Ang II treatment induced MrgD expression in different cell types and tissue, among these neonatal rat cardiomyocytes (NRCMs) (20), mice thoracic aorta and rat vascular smooth muscle cells (VSMCs) (24). Overall, these data show that MrgD expression is upregulated under pathological conditions, suggesting a protective role during disease development. Contrary to this idea, Silva et al. (2021) reported a reduction in cardiac MrgD expression following 2 weeks of transverse aortic constriction (TAC) in mice (25). In this case, the authors assessed MrgD expression at the compensated stage of TAC. It would be desirable to find whether MrgD levels are altered during the heart failure stage, when the hemodynamic overload becomes maladaptive. Therefore, more studies are necessary to better understand how the MrgD responds to different physiological and pathological stimuli.

Anti-hypertrophic effect of Alamandine/MrgD axis

Cardiac hypertrophy is an adaptive response that accompanies many forms of heart disease. On a cellular level is characterized by an increase in the size of cardiomyocytes, and enhanced protein synthesis induced by hemodynamic overload. While initially adaptive to maintain cardiac function, sustained hypertrophic stimulation can lead to systolic dysfunction and heart failure (29). Growing experimental evidence points to an anti-hypertrophic effect of alamandine that activates various cellular targets in the cardiac cell. In this section, we provide an up-to-date overview of the current knowledge and point out potential signaling pathways involved in this beneficial effect.

Jesus et al. (2018) provided the first evidence for an anti-hypertrophic role of alamandine in cardiomyocytes (22). By using the NRCM in vitro model system, we showed that alamandine prevented Ang II-induced cellular hypertrophy and upregulation of myosin heavy chain 7 (Myh7) expression (Figure 3). This anti-hypertrophic effect was mediated by MrgD receptor since it was blocked by D-Pro⁷-Ang-(1–7), whereas the MasR antagonist, A-779, had no effect. Importantly, β-alanine, a MrgD agonist, recapitulated alamandine anti-hypertrophic actions in NRCMs (22). The finding that alamandine signals through MrgD to induce anti-hypertrophic actions in cardiac cells, independently from MasR, could be therapeutically relevant. In this context, a differential expression pattern of MrgD versus MasR can assure more effective therapeutic potential for alamandine over Ang-(1–7) depending on the pathophysiological condition. This interesting possibility awaits clarification.

Figure 3. Signal transduction mechanisms mediating the actions of alamandine/MrgD axis in the cardiomyocyte.

(Left) In neonatal cardiomyocytes, alamandine promotes AMPKα phosphorylation [1], which culminates in NO generation (22). Activation of this pathway is involved in the anti-hypertrophic effect of alamandine/MrgD axis in AngII treated neonatal cardiomyocytes (22). Additional evidence suggests that PKA (20) participates in the alamandine anti-hypertrophic effect. Moreover, considering data from the literature showing that alamandine prevents ERK1/2 activation that occurs in response to different cardiac insult, it is likely that the modulation of this pathway contributes to the alamandine anti-hypertrophic action (23,25,34). Moreover, alamandine treatment reduces AngII-induced transcription of stress markers Myh7, Anp and Bnp in neonatal cardiomyocytes. (Right) The current knowledge about alamandine downstream signaling in adult ventricular myocytes shows that alamandine via MrgD activates the NO/CaMKII pathway to restore Ca²⁺ transient and contractility parameters of mREN cells back to normal [2]. Whether other receptors are activated by alamandine in the cardiac myocyte to modulate Ca²⁺ and ultimately contraction is still unknown [3]. In addition, genetic deletion of MrgD leads to contractility dysfunction in ventricular myocytes [4]. The participation of MrgE in alamandine effects in the cardiomyocyte is unknown [5]. There are no studies that explore the possible interaction between MrgD and others receptors in the cardiac myocyte [6].

The molecular mechanism involved in alamandine anti-hypertrophic effect in Ang II treated NRCMs included AMPK (AMP-activated protein kinase)/NO signaling, since compound C (an AMPK inhibitor) and L-NAME (L-N^G-Nitro arginine methyl ester, a non-selective NO synthase inhibitor) prevented alamandine actions (Figure 3). AMPK is a multifunctional protein involved in the regulation of several cellular functions (30). In particular, AMPK decreases protein synthesis by inhibiting eEF2 (Eukaryotic elongation factor-2) activity, which might explain, at least in part, its anti-hypertrophic effect (31). Further evidence supporting an anti-hypertrophic action of alamandine was provided by Silva et al. (2021) (25) by showing that oral treatment of mice with an inclusion compound of alamandine/β-hydroxypropyl cyclodextrin (HPβCD) prevented the cardiac hypertrophy induced by TAC in mice. Beneficial effects of alamandine/HPβCD complex in the TAC model also included antifibrotic and antioxidant effects (25). Perhaps more importantly, mice subjected to TAC showed a reduction in AMPKα phosphorylation in the heart, which was prevented by alamandine/HPβCD treatment. This finding provides additional evidence for AMPK as a downstream target of alamandine signaling in the heart. Our in vitro experiments using ventricular myocytes from TGR (mREN2)27 hypertensive rats also suggest regulation of AMPKα activation by alamandine. By treating mREN ventricular myocytes acutely with alamandine (100 nmol/L, 15 minutes), we found an increase in AMPKα phosphorylation, confirming its activation by alamandine (Figure 2A–B).

Figure 2. Alamandine treatment regulates AMPKα and ERK1/2 phosphorylation in cardiomyocytes from TGR (mREN2)27 hypertensive rat.

A. Representative cartoon of the methodology used. Ventricular myocyte isolation and western blotting were performed as previously described (23). Heterozygous TGR (mREN2)27 rat (male, 10–14 weeks old) was obtained from the breeding colony established at the animal facility of the Laboratory of Hypertension, Institute of Biological Sciences, Universidade Federal de Minas Gerais, Brazil. Experiments were performed according to protocols approved by the Institutional Animal Care and Use Committee at UFMG and in accordance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals. Rats were euthanized via rapid decapitation. Cardiomyocytes were incubated with alamandine (100 nmol/L) for 15 minutes and then, we assessed AMPKα and ERK1/2 phosphorylation by western-blot. B-C. Top: representative western blotting. Bottom: Bar graph shows an increase in AMPKα phosphorylation at Thr¹⁷² (Cell Signaling Technology, Cat# 2535, RRID: AB_331250) (B) and a decrease in ERK1/2 phosphorylation (Cell Signaling Technology, Cat# 4370, RRID: AB_2315112) (C) in response to short-term alamandine treatment (100 nmol/L, 15 minutes). GAPDH (Santa Cruz Biotechnology, Cat# sc-32233, RRID: AB_627679) was used as a loading control. n = number of independent experiments. Data are expressed as mean ± SE. *p < 0.05 when compared with CTR untreated mREN cells by Unpaired Student’s t test.

Further evidence of alamandine anti-hypertrophic action was provided by Liu et al. (2018) working with SHR rats treated with alamandine for 6 weeks. Alamandine treatment of SHR rats induced a reduction in left ventricle mass evaluated by echocardiography and a decrease in cardiac gross morphometry (20). This result was confirmed at the cellular level by a reduction in the cardiomyocyte cross-sectional area and by in vitro experiments showing that alamandine prevented Ang II-induced cellular hypertrophy (20). Supporting these findings, alamandine prevented Ang II-induced upregulation in atrial natriuretic peptide (Anp) and B-type natriuretic peptide (Bnp) transcript levels (20). In the same study, the authors observed that cardiac expression of protein kinase A (PKA) increased in SHR hearts and in H9c2 cells treated with Ang II (Figure 3). In both cases, alamandine treatment prevented the upregulation of PKA expression (20). Considering the importance of PKA during the development of cardiac pathology (32), it is very likely that the modulation of this pathway contributes to the alamandine anti-hypertrophic action. Other studies using different cells and tissues also support PKA as a downstream target of the alamandine/MrgD pathway (33, 34), but in these studies alamandine induced PKA activation. More experiments will be necessary to address the involvement of PKA in the alamandine/MrgD pathway in different tissues and physiopathological conditions.

Another important signal transduction pathway that has been associated with alamandine in the heart is the extracellular signal-regulated kinase 1/2 (ERK1/2). Accordingly, Silva et al. (2021) observed that cyclodextrin-encapsulated alamandine prevented the increase of ERK1/2 phosphorylation induced by TAC (25). Likewise, in another study, alamandine prevented lipopolysaccharide-induced ERK1/2 phosphorylation in neonatal cardiomyocytes (35). Similarly, we found that alamandine decreases ERK1/2 phosphorylation in the TGR (mREN2)27 cardiomyocytes (23) (Figure 2C). ERK1/2 is a member of the MAP (mitogen-activated protein) kinase family, which also included p38, and JNK, among others. Activation of this family has been linked to the induction of a hypertrophic program in the cardiac cell (36). Given the evidence that ERK1/2 is a target of alamandine in the heart, it is important to investigate whether its regulation by alamandine is involved in its anti-hypertrophic effect.

In summary, accumulating evidence supports the idea that Alamandine/MrgD axis plays anti-hypertrophic actions. Potential downstream targets participating in this effect are AMPK, PKA, and ERK1/2. For the future, more in vivo studies using different models of cardiac disorders with hypertrophic remodeling are needed to consolidate this potential therapeutic effect and elucidate the molecular mechanism underlying these effects. Figure 3 summarizes the main pathways related to the anti-hypertrophic effect of alamandine described so far.

Contractility effect of Alamandine

The heart is the organ responsible for pumping blood throughout the body, and the cardiomyocyte is responsible for generating contraction force. This is possible due to its unique features that make the cardiomyocyte a powerful contractile cell. In this section, we review studies linking alamandine/MrgD axis to myocardial contractility in different models of cardiac injury.

Hekmat et al. (2017) investigated the effect of alamandine on cardiovascular parameters of normotensive (Sprague-Dawley) and two-kidney, one clip (2K1C) hypertensive rats (37). In normotensive rats, intravenous infusion of alamandine induced an increase in left-ventricular systolic pressure (LVSP) and maximum rate of pressure change in the left ventricle (LV dP/dt(max)). Yet, alamandine infusion decreased left ventricular end-diastolic pressure (LVEDP) in normotensives rats. Importantly, these cardiovascular effects were mediated by AT₁R, since losartan blocked these alterations (37). In hypertensive rats, alamandine produced a biphasic effect, which consisted of an initial increase in LVSP and LV dP/dt(max) parameters, mediated by AT₁R, followed by a decrease in these parameters, with the latter blocked by PD123319, an antagonist of AT₂R and MrgD. Moreover, alamandine induced a decrease in LVEDP during the infusion period in hypertensive rats (37). In another study, using the same 2K1C hypertension model but at later hypertension stage, Hekmat et al. (2019) showed that 2-weeks of alamandine infusion increased the maximum rate of pressure change in the LV and decreased LVSP and LVEDP values (38). In addition, Park et al. (2018) showed that alamandine pre-treatment improved the postischemic LVDP and LV ±dP/dT parameters in Sprague-Dawley rats (39). This change was accompanied by a reduction in the infarct size induced by the ischemia-reperfusion. Also, PD 123319 and D-Pro⁷-Ang-(1–7) blocked these effects.

The study of Hekmat et al. (2017) suggests that alamandine actions could be mediated by receptors other than MrgD (37). In line with this idea, Valenzuela et al. (2021) have shown binding of alamandine to the new Mas-related G protein-coupled receptor, member E (MrgE) in rat brain mitochondria (40). Experiments in HEK293 cells transfected with MrgE receptor not only supported the binding of alamandine to MrgE receptor, but also assessed the functionality of this response by showing an increase in mitochondrial NO production induced by alamandine (Figure 3). This study opens new avenues for further understanding alamandine signaling and the complexity of its binding partners. Whether MrgE receptor is expressed in the heart, or more specifically in the cardiac mitochondria, as well as its responsiveness to alamandine are unanswered questions that deserve future attention. Finally, given the knowledge that Mas receptor interacts physically and functionally with other receptors, e.g. MasR-AT2R (41) and MasR-D2R (Dopamine D2 Receptor) (42), it is of utmost importance to investigate whether MrgD shows similar interactions with other receptors and their influence in alamandine signaling in the cardiomyocyte. Figure 3 summarizes this controversial topic that deserves further attention.

By using a well-established model of doxorubicin (DOX)-induced cardiomyopathy in rats, Hekmat et al. (2021) observed that alamandine treatment via mini-osmotic pumps prevented several aspects of DOX-induced cardiac dysfunction, including contractile defects, oxidative stress, and increase in inflammatory cytokines (43). Using Wistar Kyoto (normotensive) and SHR rats, Liu et al. (2018) demonstrated that 6-weeks subcutaneous infusion of alamandine does not alter ejection fraction (EF) and fractional shortening (FS) in normotensive rats. However, alamandine treatment ameliorated these two parameters in hypertensive rats (20). Work from our group investigated the contractility effect of alamandine at cellular level in ventricular myocytes isolated from Sprague-Dawley (normotensive) and TGR (mREN2)27 (hypertensive) rats (23), and confirmed the findings of Liu et al. (2018); alamandine improved myocyte fractional shortening of mREN myocytes, with no effects on control Sprague-Dawley cells (Figure 3). Mechanistically, the increase in cardiomyocyte shortening fraction induced by alamandine in mREN myocytes was attributed to an enhanced peak of Ca²⁺ transient, attenuated Ca²⁺ spark rate, and faster Ca²⁺ reuptake to the sarcoplasmic reticulum. The contractility effect of alamandine was mediated by MrgD in a mechanism dependent on NO-induced CaMKII (Calmodulin Kinase II) activation (23). The activation of CaMKII by alamandine was also indirectly observed by Silva et al. (2021) (25). In this study, TAC mice treated with alamandine/HPβCD complex showed an increase in phospholamban (PLN) phosphorylation at Thr17 residue when compared to cardiac samples from TAC mice. PLN is a protein involved in the regulation of Ca²⁺ reuptake to the sarcoplasmic reticulum by SERCA (sarcoendoplasmic reticulum calcium ATPase), and a well-known target of CaMKII phosphorylation at Thr17 residue (44).

The impact of MrgD ablation on cardiac function was evaluated by Oliveira et al. (2019) (21). For this, we compared cardiac function of wild-type and MrgD^−/− mice in vivo and in vitro by using echocardiography and isolated ventricular myocytes, respectively. Interestingly, MrgD^−/− mice presented dilated cardiomyopathy with a remarkable decrease in cardiac function. This contractile dysfunction was also observed in isolated cardiomyocytes from MrgD^−/− mice, which displayed a decrease in fractional shortening when compared to wild-type (21) (Figure 3). Whether this reduction in contractility seen in MrgD^−/− mice is a direct effect of MrgD deletion in the cardiac myocyte or indirect consequences of its ablation during the animal development is a topic that awaits investigation.

Taken together, studies that evaluated the contractility effects of alamandine in different models of cardiac injury have described that this peptide ameliorates cardiac function in several disease conditions. Undoubtedly, more data are needed to clarify how alamandine/MrgD axis modulates excitation-contraction coupling in the cardiomyocytes.

Future prospects

In summary, our current understanding of alamandine and its MrgD receptor indicates a cardioprotective role for this new axis. Growing evidence supports the anti-hypertrophic and contractile effects of alamandine/MrgD on cardiomyocytes from disease models of cardiac dysfunction which resemble those that occur in response to Ang-(1–7)/MasR. Future work in this regard should explore synergistic or additive effects of alamandine and Ang-(1–7) in the cardiac myocyte, and characterize the differential expression pattern of MasR and MrgD in models of cardiac disease, which could confer important therapeutic implications.

List of Abbreviations

RAS

renin-angiotensin system

HF

heart failure

ACE

angiotensin-converting enzyme

Ang II

Angiotensin II

AT₁R

angiotensin II receptor type-1

Ang-(1–7)

Angiotensin-(1–7)

MasR

Mas receptor

MrgD

Mas-related G protein-coupled receptor member D

ACE2

angiotensin-converting enzyme 2

AD

aspartate decarboxylase

CHO

chinese hamster ovary

IPF

idiopathic pulmonary fibrosis

NO

nitric oxide

FAM-ALA

FAM-labeled-alamandine

MrgD^−/−

MrgD-deficient mice

VCMCs

vascular smooth muscle cells

NRCMs

neonatal rat cardiomyocytes

TAC

transverse aortic constriction

AMPKα

Adenosine Monophosphate-activated Protein Kinase α

L-NAME

L-N^G-Nitro arginine methyl ester

eEF2

Eukaryotic elongation factor-2

Myh7

Myosin Heavy Chain 7

Anp

atrial natriuretic peptide

Bnp

B-type natriuretic peptide

SHRs

spontaneously hypertensive rats

PKA

protein kinase A

HPβCD

hydroxypropyl-β-cyclodextrin

MAPKs

mitogen-activated protein kinase

ERK1/2

extracellular signal-regulated kinase 1/2

2K1C

two-kidney, one clip

SD

Sprague-Dawley rat

LVSP

left-ventricular systolic pressure

LV dP/dt(max)

maximum rate of pressure change in the left ventricle

LVEDP

left ventricular end-diastolic pressure

D2R

dopamine D2 receptor

MrgE

Mas-related G protein-coupled receptor, member E

AT₂R

angiotensin II receptor type-2

EF

ejection fraction

FS

fractional shortening

CaMKII

calmodulin kinase II

SERCA

sarcoendoplasmic reticulum calcium ATPase