Arg972 Insulin receptor substrate-1 is associated with decreased serum angiotensin-converting enzyme 2 levels in acute myocardial infarction patients: in vivo and in vitro evidence
© Liu et al.; licensee BioMed Central Ltd. 2013
Received: 16 August 2013
Accepted: 2 October 2013
Published: 17 October 2013
Activation of the renin-angiotensin system (RAS) plays a critical role in the pathophysiology of myocardial infarction (MI) and the development of heart failure. Both angiotensin-converting enzyme 2 (ACE2) and insulin/insulin receptor substrate-1 (IRS-1) show cardioprotective effects after acute MI. The Arg972 IRS-1 polymorphism is associated with diminished activity of insulin. In the present study, we explored the association among Arg972 IRS-1, acute MI, and serum levels of ACE2.
A total of 711 subjects, including 351 subjects with first-time acute MI and 360 subjects without a history of MI were genotyped for Arg972 IRS-1 polymorphism. Serum levels of ACE2 and MI severity scores were determined. Primary human cardiomyocytes with overexpression of wild type IRS-1 or Arg972 IRS-1 or knockdown of endogenous IRS-1 were exposed to normoxia and hypoxia, and the expression levels of ACE2 were determined.
The serum ACE2 level was significantly increased in acute MI patients compared with that of non-MI controls. Compared with wild type IRS-1 carriers, Arg972 IRS-1 carriers exhibited decreased serum ACE2 levels and increased MI severity scores after MI. Our in vitro data demonstrate that impairment of insulin/IRS-1/PI3K signaling by overexpression of Arg972-IRS-1, knockdown of endogenous IRS-1, or PI3K inhibitor can abolish hypoxia-induced IRS-1-associated PI3K activity and ACE2 expression in human cardiomyocytes, which suggests a causal relationship between Arg972-IRS-1 and decreased serum ACE2 levels in acute MI patients. Our in vitro data also indicate that insulin/IRS-1/PI3K signaling is required for ACE2 expression in cardiomyocytes, and that hypoxia can enhance the induction effect of insulin/IRS-1/PI3K signaling on ACE2 expression in cardiomyocytes.
This study provides the first evidence of crosstalk between insulin/IRS-1/PI3K signaling and RAS after acute MI, thereby adding fresh insights into the pathophysiology and treatment of acute MI.
KeywordsInsulin receptor substrate-1 Gene polymorphism Angiotensin-converting enzyme 2 Acute myocardial infarction
Activation of the renin-angiotensin system (RAS) plays a critical role in the pathophysiology of myocardial infarction (MI) and the development of heart failure . RAS blockade with angiotensin-converting enzyme (ACE) inhibitors improves cardiac remodeling and outcomes in both experimental models of MI as well as in humans . ACE metabolizes angiotensin (Ang) I to form Ang II, which is of key importance in the pathophysiology of the RAS in the heart . Recently, ACE2, a new member of the RAS, was found to function as a negative regulator of the Ang system by metabolizing Ang II to a putatively protective peptide Ang-(1–7) with high efficiency . ACE2 is present in the heart, and a reduction in its expression is associated with enhanced cardiac hypertrophy and reduced pumping ability [4, 5]. Although ACE2 was initially localized exclusively in cardiac endothelial cells, more recent studies demonstrate ACE2 immunoreactivity in both the endothelial and smooth muscle cells of myocardial vessels as well as in cardiomyocytes [4, 5]. After MI, there is significant activation of cardiac ACE2 in rats and humans, which acts to combat the adverse effects of an activated cardiac RAS . Other evidence for a cardioprotective role for ACE2 arises from studies in ACE2-knockout mice where the loss of ACE2 facilitates adverse post-MI ventricular remodeling, and studies showing that ACE2 overexpression in MI rats improves cardiac contractility and remodeling [6, 7]. It has been reported that ACE2 serum activity rises during the first week following acute MI and that ACE2 activation may be a compensatory mechanism in advanced heart failure .
Insulin has beneficial effects on cardiomyocyte function and survival, including protection against acute ischemic injury . Insulin signaling can promote glucose uptake, glycolytic flux and glucose oxidation, and improve ischemic tolerance of the heart [10–12]. The protective effects of insulin on the heart are mediated via activation of a signaling pathway involving the insulin receptor, insulin receptor substrate-1 (IRS-1), and phosphoinositide-3 kinase (PI3K) [9, 13, 14]. Previous studies have demonstrated that a common polymorphism in the IRS-1 gene, the Arg972 IRS-1 polymorphism, in which a Gly/Arg substitution takes place at codon 972 (Arg972), is associated with impaired IRS-1 ability to activate the downstream factor phosphatidylinositol-3 kinase (PI3K), leading to diminished activity of insulin [15, 16].
Results from one of our pilot studies suggested that the Arg972 IRS-1 polymorphism might be associated with ACE2 activation in MI patients. In the present study, we explored the association among Arg972 IRS-1, acute MI, and serum levels of ACE2 in a relatively large sample of acute MI patients, with an aim to identify potential interaction between insulin/IRS-1 signaling and RAS in the pathophysiology of acute MI.
Between July 2010 and October 2012, 351 subjects with first-time acute MI (mean age 62.7±15.9 years; range, 27–78 years) were recruited to this study at the Second Xiangya Hospital of Central South University. Inclusion criteria were: (1) patients who had undergone a first attack of ST-elevation myocardial infarction (STEMI); (2) verbally communicable; (3) those who agreed to participate in the study. Subjects without a history of MI (mean age 61.2±13.5 years; range, 25–75 years) were enrolled as controls (n=360). Subjects with congenital heart diseases were excluded. Blood samples were drawn on day 7 post MI from the acute MI subjects, and on day 1 from the controls after they consented to participate in the study. All blood samples were subject to ELISA assays for serum ACE2 levels. This study was approved by the Ethics Committee of the Second Xiangya Hospital, Central South University. Written informed consents were obtained from all participants before the start of the study.
The Arg972 IRS-1 polymorphism was identified by restriction fragment length polymorphism as previously described . Three single nucleotide polymorphisms (SNPs), 1075A/G (rs1978124), 8790A/G (rs2285666) and 16854G/C (rs4646142) were selected as proxies to study ACE2 polymorphisms as previously described .
MI severity score
We used the Predicting Risk of Death in Cardiac Disease Tool (PREDICT) score to create an index of severity as previously described . It was used to provide a simple, long-term admission-day prognostic score for patients hospitalized for MI or unstable angina. Score components include shock (0 to 4 points), clinical history (MI, stroke, angina; 0 to 2 points), age (0 to 3 points), ECG findings (0 to 3 points), congestive heart failure (0 to 3 points), and Charlson Comorbidity Index (0 to 6 points) for a maximum severity score of 21 points .
Plasmids and reagents
A fragment of human genomic DNA containing the entire coding sequence of IRS-1 was cloned and ligated into pcDNA3.1 expression vector (pcDNA-WT-IRS-1) as previously described . The pcDNA-Arg972-IRS-1 expression vector was constructed as previously described . Superfect™ transfection reagent was purchased from Qiagen (Valencia, CA, USA). TRIzol reagent for RNA isolation, and the SYBR Green Master Mix were purchased from Invitrogen (Carlsbad, CA, USA) and PE Applied Biosystems (Foster City, CA, USA), respectively. IRS-1 (sc-29376-V) short hairpin RNA (shRNA) lentiviral particles, control shRNA lentiviral particles-A (sc-108080), and anti-ACE2 (sc-73668) antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). ACE2 (human) ELISA Kit (AG-45A-0022EK-KI01) was purchased from Adipogen (San Diego, CA, USA). SensoLyte® 520 TACE (α-Secretase) activity assay kit (72085) was purchased from AnaSpec (Fremont, CA, USA). Puromycin, LY294002 and all other chemicals of reagent grade were purchased from Sigma (St. Louis, MO, USA).
Cell culture, lentiviral transduction, transfection of IRS-1 cDNAs, and application of hypoxia treatment
Human adult cardiomyocytes (#6210) and cardiomyocyte medium (#6210) were purchased from ScienCell Research Laboratories (Carlsbad, CA, USA). Lentiviral transduction was performed in human cardiomyocytes. The IRS-1 shRNA lentiviral particles contain expression constructs encoding target-specific 19–25 nt (plus hairpin) shRNA designed to specifically knock down IRS-1 gene expression. The control shRNA lentiviral particles contain a scrambled shRNA sequence that will not lead to degradation of any cellular mRNA, and was used as a negative control for IRS-1 shRNA lentiviral particles. Pools of stable transductants were generated via selection with puromycin (5 μg/mL) by the manufacturer’s protocol (Santa Cruz Biotechnology).
Human cardiomyocytes were stably transfected with empty pcDNA3.1, pcDNA-WT-IRS-1 or pcDNA-Arg972-IRS-1 plasmids using Superfect™ transfection reagent (Qiagen) according to the manufacturer's instructions. Pools of stable transfectants were generated via selection with puromycin (5 μg/ml) by the manufacturer’s protocol. For hypoxia treatment, cells were respectively kept at 1% O2 for 24 hours in serum-free medium with or without 1 mg/L insulin .
Real-time quantitative reverse transcription PCR
RNA from human cardiomyocytes were prepared using TRIzol reagent followed by purification with TURBO DNA-free System (Ambion, Austin, TX, USA). The cDNAs were synthesized using SuperScript II reverse transcriptase (Invitrogen). Real-time quantitative PCR was performed on an Abi-Prism 7700 Sequence Detection System (Applied Biosystems), using the fluorescent dye SYBR Green Master Mix (PE Applied Biosystems) as described by the manufacturer. The results were normalized against that of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in the same sample. The primers used are as follows: for ACE2, 5′-GCAGCTAAGTATAATGGTTCTCTG -3′ (forward) and 5′-AGTGTTCCACCCCACAAAA -3′ (reverse); for GAPDH, 5′-GTCAGTGGTGGACCTGACCT-3′ (forward) and 5′-TGCTGTAGCCAAATTCGTTG-3′ (reverse). Each experiment was repeated for three times in triplicates.
Western blot analysis
Human cardiomyocytes were lysed in 250 μ l of 2× SDS loading buffer (62.5 mM TrisHCl, pH 6.8, 2% SDS, 25% glycerol, 0.01% bromphenol blue, 5% 2-mercaptoethanol), and incubated at 95 °C for 10 min. Equal amount of proteins (100 μg) for each sample were separated by 8-15% SDS-polyacrylamide gel and blotted onto a polyvinylidene difluoride microporous membrane (Millipore, Billerica, MA, USA). Membranes were incubated for 1 hour with a 1:1000 dilution of primary antibody, and then washed and revealed using secondary antibodies with horseradish peroxidase conjugate (1:5000, 1 hour). Peroxidase was revealed with a GE Healthcare ECL kit. Proteins were quantified before being loaded onto the gel.
PI3K Activity assay
IRS-1-associated PI3K activities were determined as previously described . Briefly, 700 μg of total protein was immunoprecipitated with anti-IRS-1 antibody (Santa Cruz Biotechnology Inc.), and kinase activity was detected by the appearance of radiolabeled 32P-labeled phosphatidylinositol 3-phosphate ([32P]PI-3-P) after TLC as described . Autoradiographic signals were quantitated using NIH Image version 1.63.
Statistical analyses were carried out using SPSS15.0 (IBM, Chicago, IL, USA). All continuous variable values were expressed as Mean±SD. Comparison of means between two independent groups was performed with student t-tests. Comparison of serum ACE2 levels before and after acute MI in the same genotype group was performed with paired t-tests. Comparison of serum ACE2 levels after acute MI between two genotype groups was performed with analysis of covariance using serum ACE2 levels before acute MI as a covariate. Comparisons of means among multiple groups were performed with one-way ANOVA followed by post hoc pairwise comparisons using Tukey's tests. Categorical variables were compared with Chi-square tests. The main effect of and interaction among Arg972 IRS-1 and MI on serum ACE2 levels were analyzed with ANOVA. The significance level of this study was set at a two-tailed α=0.05.
Characteristics of study subjects
Acute MI (n=351)
Age Group (years) n(%)
29.7 ± 9.2
28.6 ± 9.7
Total Cholesterol (mmol/L)
6.0 ± 3.5
5.6 ± 2.6
LDL- Cholesterol (mmol/L)
3.9 ± 2.3
3.5 ± 1.8
HDL- Cholesterol (mmol/L)
1.1 ± 0.7
1.2 ± 0.9
2.4 ± 1.5
2.1 ± 1.2
Systolic Blood Pressure (mmHg)
133.5 ± 19.2
131.4 ± 14.5
Diastolic Blood Pressure (mmHg)
86.2 ± 13.6
84.5 ± 11.5
Fasting Glucose (mmol/L)
5.4 ± 2.1
5.1 ± 2.4
Fasting Insulin (μU/mL)
18.3 ± 7.4
17.3 ± 6.9
Association between arg 972 irs-1 polymorphism and acute myocardial infarction (mi) in total subjects and by gender
Serum angiotensin-converting enzyme 2 (ace2) levels and myocardial infarction (mi) scores in control and acute mi subjects
Serum ACE2 level (ng/mL)
MI severity score
ANOVA analysis of the main effect of and interaction between arg 972 irs-1 and acute myocardial infarction (mi) on serum angiotensin-converting enzyme 2 (ace2) levels
Acute MI × Genotype
Partial Eta squared
Partial Eta squared
Partial Eta squared
Serum ACE2 Levels
Serum angiotensin-converting enzyme 2 (ace2) levels before and after acute myocardial infarction (mi)
Serum ACE2 level (ng/mL)
Before acute MI
After acute MI
Relative mrna levels of angiotensin-converting enzyme 2 (ace2) in human cardiomyocytes under normoxia and hypoxia with or without insulin treatment
Secreted/soluble angiotensin-converting enzyme 2 (ace2) levels in culture media of human cardiomyocytes under normoxia and hypoxia with or without insulin treatment
In the present study, we found that Arg972 IRS-1 was associated with decreased serum ACE2 levels in acute MI patients. In vitro data revealed that in the presence of insulin, Arg972 IRS-1 inhibits ACE2 expression in human cardiomyocytes under hypoxia.
A previous study showed that serum ACE2 activity significantly increased from baseline to 7 days in STEMI patients . On day 7, a significant direct correlation was observed between ACE2 activity and infarct size and left ventricular function index . Thus, in the present study, we measured serum ACE2 levels on day 7 after acute MI. Our preliminary studies with non-STEMI subjects showed inconsistent serum ACE2 levels. Therefore, only STEMI subjects were enrolled in the present study.
ACE2 is expressed in the heart and reportedly plays a protective role during MI [1, 4, 5, 9]. Increasing evidence show that the expression of cardiac ACE2 is increased after MI, which acts to combat the adverse effects of an activated cardiac RAS and therefore may be a compensatory mechanism in MI [1, 4, 8]. In agreement with this, in subjects who had blood samples collected before and after MI, we observed a significant increase in the ACE2 serum level after MI. Arg972-IRS-1 carriers had significantly lower serum ACE2 levels and higher MI severity scores compared with wild type-IRS-1 carriers after MI, confirming the cardioprotective role of increased ACE2 levels after MI. The findings also suggest an important role for insulin/IRS-1 signaling in the increased ACE2 expression after MI, since Arg972-IRS-1 is associated with impaired IRS-1 ability to activate PI3K, which leads to diminished activity of insulin [15, 16]. We demonstrated in vitro that a shedding mechanism was not involved in the effects of Arg972 IRS-1 on ACE2 expression in cardiomyocytes, suggesting that insulin/IRS-1/PI3K signaling directly targets ACE2 expression.
Myocardial hypoxia is a major factor in the pathophysiology of MI and is thought to be a prime determinant of the progression to heart failure . Thus, in subsequent in vitro experiments to investigate the potential causal relationship between Arg972 IRS-1 and ACE2 expression, we exposed primary human cardiomyocytes to hypoxia. In agreement with the in vivo data, ACE2 expression at both the mRNA and the protein/soluble protein levels was increased under hypoxia compared with that under normoxia, which was abolished by overexpression of Arg972 IRS-1, knock down of endogenous IRS-1, or PI3K inhibitor.
Insulin signaling can improve ischemic tolerance of the heart via activation of a signaling pathway involving the insulin receptor, insulin receptor substrate-1 (IRS-1), and PI3K [9–14, 26]. Our in vitro data indicate that hypoxia can significantly enhance the IRS-1-associated PI3K activity and thereby insulin/IRS-1/PI3K signaling in cardiomyocytes. Our western blot analysis results showed that ACE2 was expressed at a constitutively low level in cardiomyocytes in normoxia. This may explain why overexpression of Arg972 IRS-1 had no significant effect on ACE2 expression in cardiomyocytes in normoxia, where a small amount of endogenous wild type IRS-1 in the cells may well suffice for basal level of insulin/IRS-1/PI3K signaling and ACE2 expression despite the competition of Arg972 IRS-1. When the IRS-1-associated PI3K activity and the need for wild type IRS-1 were significantly increased by hypoxia in cardiomyocytes, however, the competition of overexpressed Arg972 IRS-1 would manifest significant inhibitory effect on insulin/IRS-1/PI3K signaling and thereby ACE2 expression. This may explain for why only Arg972 IRS-1 carriers with acute MI showed decreased serum ACE2 levels.
Recent discoveries on the influence of local tissue RAS in the skeletal muscle, heart, vasculature, adipocytes, and pancreas have led to an improved understanding of how activated tissue RAS influences the development of insulin resistance and diabetes in humans . Our study suggests that insulin/IRS-1/PI3K signaling is involved in regulating local RAS in the heart, which may be a novel topic in both the fields of cardiology and diabetology.
There were several limitations of the present study: (1) The increased serum ACE2 levels in acute MI subjects could be from multiple sources such as vascular smooth muscle cells, endothelial cells, and cardiomyocytes. We only used cardiomyocytes in vitro to identify the causal relationship between Arg972 IRS-1 and serum ACE2 levels in acute MI subjects. The increased level of secreted/soluble ACE2 in the culture media of cardiomyocytes under hypoxia did suggest that cardiomyocyte-secreted ACE2 could at least partially account for the increased serum ACE2 levels in acute MI patients. However, it would be interesting in future studies to explore the effects of insulin/IRS-1 signaling on ACE2 expression in other cell types. (2) There are many other pathophysiological factors involved in acute MI besides hypoxia. We only explored the effect of insulin/IRS-1 signaling on ACE2 expression under hypoxia and normoxia. It would be interesting to investigate how insulin/IRS-1 signaling regulates ACE2 expression under the influence of other conditions involved in acute MI (e.g. increased cardiomyocyte apoptosis and inflammatory responses, etc.). Nevertheless, based on in vivo and in vitro data, we have shown a potential causal relationship between Arg972 IRS-1 and serum ACE2 levels in acute MI patients. Our findings suggest that insulin/IRS-1/PI3K signaling exerts cardioprotective effects after acute MI at least partially through increased ACE2 expression; in agreement with this, Arg972 IRS-1 impairs insulin/IRS-1 signaling and results in decreased ACE2 expression after acute MI, which leads to more severe MI. Thus, insulin/IRS-1/PI3K signaling and ACE2 could be potential new targets for acute MI therapy. In addition, our study suggest that special attention should be paid to acute MI patients carrying Arg972 IRS-1, for they tend to have more severe MI and poorer prognosis than those carrying wild type IRS-1.
In conclusion, our in vivo data indicate that Arg972-IRS-1 is associated with decreased serum ACE2 levels in acute MI patients. Our in vitro data demonstrate that impairment of insulin/IRS-1/PI3K signaling by overexpression of Arg972-IRS-1, knockdown of endogenous IRS-1, or PI3K inhibitor can abolish hypoxia-induced IRS-1-associated PI3K activity and ACE2 expression in human cardiomyocytes, which suggests a causal relationship between Arg972-IRS-1 and decreased serum ACE2 levels in acute MI patients. Our in vitro data also indicate that insulin/IRS-1/PI3K signaling is required for ACE2 expression in cardiomyocytes, and that hypoxia can markedly enhance the induction effect of insulin/IRS-1/PI3K signaling on ACE2 expression in cardiomyocytes. This study provides the first evidence of crosstalk between insulin/IRS-1/PI3K signaling and RAS after acute MI, thereby adding fresh insights into the pathophysiology and treatment of acute MI.
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