Human Angiotensinogen Gene Research Articles

Increased CaMKII activation and contrast changes of cardiac β1-and β3-Adrenergic signaling pathways in a humanized angiotensinogen model of hypertension

AimsUpregulation of Ca2+/calmodulin-dependent protein kinase II (CaMKII) contributes to the pathogenesis of cardiovascular disease, including hypertension. Transgenic rats expressing the human angiotensinogen gene [TGR (hAGT)L1623] are a new novel humanized model of hypertension that associates with declines in cardiac contractile function and β-adrenergic receptor (AR) reserve. The molecular mechanisms are unclear. We tested the hypothesis that in TGR (hAGT)L1623 rats, left ventricular (LV) myocyte CaMKIIδ and β3-AR are upregulated, but β1-AR is down-regulated, which are important causes of cardiac dysfunction and β-AR desensitization. Main methodsWe compared LV myocyte CaMKIIδ, CaMKIIδ phosphorylation (at Thr287) (pCaMKIIδ), and β1-and β3-AR expressions and determined myocyte functional and [Ca2+]I transient ([Ca2+]iT) responses to β-AR stimulation with and without pretreatment of myocytes using an inhibitor of CaMKII, KN-93 (10−6 M, 30 min) in male Sprague Dawley (SD; N = 10) control and TGR (hAGT)L1623 (N = 10) adult rats. Key findingsHypertension in TGR (hAGT)L1623 rats was accompanied by significantly increased LV myocyte β3-AR protein levels and reduced β1-AR protein levels. CaMKIIδ phosphorylation (at Thr287), pCaMKIIδ was significantly increased by 35%. These changes were followed by significantly reduced basal cell contraction (dL/dtmax), relaxation (dR/dtmax), and [Ca2+]iT. Isoproterenol (10−8 M) produced significantly smaller increases in dL/dtmax, dR/dtmax, and [Ca2+]iT. Moreover, only in TGR (hAGT)L1623 rats, pretreatment of LV myocytes with KN-93 (10−6 M, 30 min) fully restored normal basal and isoproterenol-stimulated myocyte contraction, relaxation, and [Ca2+]iT. SignificanceLV myocyte CaMKIIδ overactivation with associated contrast changes in β3-AR and β1-AR may be the key molecular mechanism for the abnormal contractile phenotype and β-AR desensitization in this humanized model of hypertension.

Abstract 430: Epigenome Editing Of The AGT Gene As A Therapeutic Approach For Cardiovascular Disorders

Overactivation of the renin-angiotensin system contributes to many common and rare cardiovascular diseases such as hypertension, atherosclerosis, and genetic thoracic aortopathies. Current pharmacologic therapies that target this pathway have been proven safe and effective, yet they require individuals with these conditions to adhere to daily medications. CRISPR-Cas9-mediated hepatic inactivation of the angiotensinogen ( AGT ) gene, the precursor of all angiotensin peptides, could be a promising “one and done” therapeutic approach for these disorders. However, disruption of AGT by Cas9 nuclease would result in irreversible changes to the genome and would likely have negative consequences if targeted to organs outside of the liver. CRISPRoff is a CRISPR-based epigenome editing tool consisting of catalytically inactive Cas9 fused to a DNA methyltransferase, as well as a guide RNA (gRNA) designed to target a genomic promoter of interest. Targeting to a gene promoter can result in DNA methylation and subsequent silencing of gene expression, a phenomenon that has the potential to be reversed by CRISPRon, a catalytically inactive Cas9 fused to a demethylase that could be targeted to the same site using the same gRNA. We hypothesized that CRISPRoff can be used to efficiently and durably silence AGT expression by methylation of the AGT promoter. To test this, we screened HuH-7 hepatoma cells with CRISPRoff and either individual gRNAs or dual combinations of gRNAs targeting the AGT promoter. We measured AGT mRNA expression by RT-qPCR and selected the two combinations of gRNAs that resulted in the greatest decrease in AGT expression for methylation analysis. We found that AGT expression was decreased by ≈50% in treated cells, and this correlated with substantially increased methylation throughout the AGT promoter. A long-term in vitro study to assess the durability of this methylation is underway. We are also generating a humanized AGT mouse model to test whether these CRISPRoff gRNAs can silence the human AGT gene in vivo. These results provide a proof of concept of CRISPRoff for silencing of AGT expression as a therapeutic approach for common and rare cardiovascular disorders.

Abstract 124: Angiotensin-(1-12) Immunoneutralization: A New Approach To Hypertension Treatment

There is a critical need to identify new therapeutic targets and treatments, given emerging data documenting the limited effectiveness of current antihypertensive drugs to have a marked impact in preventing target organ damage and cardiovascular death. While monoclonal antibodies (mAb) are revolutionizing the treatment of human diseases, including severe hypercholesterolemia, their applicability to long-term hypertension control remains unexplored. Identification of angiotensin-(1-12) [Ang-(1–12)] as a non-renin-dependent substrate for Ang II generation in rodents and humans led us to engineer a mAb directed against the C-terminus of human Ang-(1–12) [h-Ang-(1-12)] sequence. We utilized a model of hypertension expressing the human angiotensinogen (AGT) gene in the rat genome [TGR(hAGT)L1623 rats] to address the effectiveness of endogenous h-Ang-(1–12) immunoneutralization in controlling the elevated blood pressure and reversing hypertension-induced target organ damage. Acute delivery of the h-Ang-(1–12) mAb (30 mg/Kg, i.v.) in anesthetized hypertensive transgenic rats induced a prompt arterial pressure fall (M ± SE: -18 ± 6/-16 ± 3 mm Hg, n=7; p< 0.001) in the absence of corresponding heart rate changes. The antihypertensive effect of the h-Ang-(1-12) mAb triggered a rise in plasma renin concentrations (from 107 ± 24 to 381 ± 260 ng/mL/h) that was inversely correlated with the fall in mean arterial pressure (r= -0.92, p< 0.001). Prior intravenous delivery of aliskiren fumarate (3-5 mg/Kg, i.v.) while inducing a fall in arterial pressure did not mask the antihypertensive action of the h-Ang-(1-12) mAb. Extension of these acute effects of the h-Ang-(1-12) mAb to awake instrumented transgenic hypertensive rats given consecutive daily doses of the mAb (30 mg/Kg, i.m. x 4 -5 days, n=7) documented a trend for significant statistical drops in daytime blood pressure and no changes in heart rate. We conclude that Ang-(1–12) is an endogenous Ang II forming substrate participating in the control of arterial pressure in a humanized model of high blood pressure and that selective immunoneutralization of this alternate substrate is a promising and novel therapeutic approach to treat hypertension and prevent its adverse cardiovascular sequelae.

Immunoneutralization of human angiotensin-(1-12) with a monoclonal antibody in a humanized model of hypertension

We engineered a monoclonal antibody (mAb) against the human C-terminus of angiotensin-(1–12) [h-Ang-(1–12)] and performed a biochemical characterization in concert with direct in vivo and ex vivo (carotid artery strips) assessments of h-Ang-(1–12) vasoconstrictor activity in 78 (36 females) transgenic rats expressing the human angiotensinogen gene [TGR(hAGT)L1623] and 26 (10 female) Sprague Dawley (SD) controls. The mAb shows high specificity in neutralizing angiotensin II formation from h-Ang-(1–12) and did not cross-react with human and rat angiotensins. Changes in arterial pressure and heart rate in Inactin® hydrate anesthetized rats were measured before and after h-Ang-(1–12) injections [dose range: 75–300 pmol/kg i.v.] prior to and 30–60 minutes after administration of the h-Ang-(1–12) mAb. Neutralization of circulating Ang-(1–12) inhibited the pressor action of h-Ang-(1–12), prevented Ang-(1–12) constrictor responses in carotid artery rings in both SD and TGR(hAGT)L1623 rats, and caused a fall in the arterial pressure of male and female transgenic rats. The Ang-(1–12) mAb did not affect the response of comparable dose-related pressor responses to Ang II, pre-immune IgG, or the rat sequence of Ang-(1–12). This h-Ang-(1–12) mAb can effectively suppress the pressor actions of the substrate in the circulation of hypertensive rats or in carotid artery strips from both SD and transgenic rats. The demonstration that this Ang-(1–12) mAb by itself, induced a fall in arterial pressure in transgenic hypertensive rats supports further exploring the potential abilities of Ang-(1–12) mAb in the treatment of hypertension.

Kidney Injury Caused by Preeclamptic Pregnancy Recovers Postpartum in a Transgenic Rat Model.

Preeclampsia (PE) is characterized by the onset of hypertension (≥140/90 mmHg) and presence of proteinuria (>300 mg/L/24 h urine) or other maternal organ dysfunctions. During human PE, renal injuries have been observed. Some studies suggest that women with PE diagnosis have an increased risk to develop renal diseases later in life. However, in human studies PE as a single cause of this development cannot be investigated. Here, we aimed to investigate the effect of PE on postpartum renal damage in an established transgenic PE rat model. Female rats harboring the human-angiotensinogen gene develop a preeclamptic phenotype after mating with male rats harboring the human-renin gene, but are normotensive before and after pregnancy. During pregnancy PE rats developed mild tubular and glomerular changes assessed by histologic analysis, increased gene expression of renal damage markers such as kidney injury marker 1 and connective-tissue growth factor, and albuminuria compared to female wild-type rats (WT). However, four weeks postpartum, most PE-related renal pathologies were absent, including albuminuria and elevated biomarker expression. Only mild enlargement of the glomerular tuft could be detected. Overall, the glomerular and tubular function were affected during pregnancy in the transgenic PE rat. However, almost all these pathologies observed during PE recovered postpartum.

Abstract 12731: Acute Calcium/Calmodulin-Dependent Protein Kinase II Inhibition Augments Cardiomyocyte Contractility and Improves β-Adrenergic Regulation in Experimental Hypertension of Transgenic Rats Expressing Human Angiotensinogen Gene

Background: Previously we showed that rats expressing the human angiotensinogen (AGT) gene (hAogen) exhibit sustained hypertension and cardiac dysfunction, associated with increased cardiac angiotensin II (Ang II). Recent evidence indicates that Ang II increases CaMKII activation which plays a crucial role in ANG II-related pathological processes associated with hypertension, hypertrophy and cardiac dysfunction. Although CaMKII inhibitors are being developed as safe and effective therapeutic agents, their mechanism of action remains to be fully understood. We tested the hypothesis that in hAogen rats, CaMKII is upregulated. Increased CaMKII activation may produce direct depression in LV myocyte basal function and β-adrenergic reserve. Acute inactivation of CaMKII with KN93 would have beneficial actions in hAogen. Methods: We compared LV myocyte CaMKIIδ expression and activity and myocyte functional performance in 10 Sprague Dawley (SD) control and 10 hAogen male rats. In hAogen, we further assessed myocyte contractile and [Ca 2+ ] i transient ([Ca 2+ ] iT ) responses to β-AR stimulation by isoproterenol (ISO, 10 -8 M) with and without pretreatment of myocytes with KN93 (10 -6 M, 30 min). Results: Versus SD, in hAogen myocytes, the CaMKIIδ protein levels (19%, 0.50 vs 0.42) and CaMKIIδ phosphorylation (at Thr 287 ) (35%, 0.27 vs 0.20) were significantly increased. These changes were followed by significantly reduced cell contraction (dL/dtmax, 104.9 vs 138.4 μm/s), relaxation (dR/dtmax, 93.0 vs 104.2 μm/s) and [Ca 2+ ] iT (0.15 vs 0.20). ISO-stimulated increases in dL/dtmax (43% vs 62%), dR/dtmax (32% vs 49%) and [Ca 2+ ] iT (19% vs 29%) were also significantly reduced. Moreover, in hAogen myocytes, pretreatment with KN93 markedly improved myocyte basal contraction (134.4 μm/s), relaxation (107.4 μm/s) and [Ca 2+ ] iT (0.20). ISO-induced increases in dL/dtmax (60%), dR/dtmax (50%), and [Ca 2+ ] iT (31%) were also significantly augmented. Conclusions: In rats harboring the human AGT gene, upregulation of CaMKIIδ has adverse functional effects. Acute endogenous CaMKII activation exacerbates LV myocyte dysfunction and β-AR desensitization. Inhibition of CaMKII has a significant beneficial impact in this established humanized model of hypertension.

Abstract 12677: Impaired Cardiomyocyte Contractility to Angiotensin-(1-12) in a Humanized Angiotensinogen Model of Hypertension

Background: Angiotensin-(1-12) [Ang-(1-12)] is a chymase-dependent source for Angiotensin II (Ang II) inotropic activity that may be impaired in a model of sustained hypertension with high cardiac Ang II content due to insertion of the human angiotensinogen (AGT) gene in the rat genome. Accordingly, we evaluated the effects of Ang-(1-12) and Ang II on myocyte contractility and [Ca 2+ ] i regulation in 9 adult male transgenic rats expressing the human AGT gene [TGR(hAGT)L1623)] and 9 SD controls. Methods: We compared LV myocyte contraction, relaxation and [Ca 2+ ] i transient ([Ca 2+ ] iT ) responses to Ang-(1-12) (4x10 -6 M) and Ang II (10 -6 M) in freshly isolated LV myocytes. Results: Myocyte contraction (dL/dt max , 109.6 vs 127.9 μm/s), relaxation (dR/dt max , 95.3 vs 107.5 μm/s) and [Ca 2+ ] iT (0.15 vs 0.24) were depressed in TGR(hAGT)L1623 rats compared to SD controls. Moreover, cell contractile and [Ca 2+ ] iT responses following exposure to Ang-(1-12) or Ang II were markedly blunted. In SD myocytes, versus baseline, Ang II or Ang-(1-12) superfusion produced significant increases in dL/dt max [Ang II: 44% vs Ang-(1-12): 34%], dR/dt max (33% vs 26%) and [Ca 2+ ] iT (31% vs 25%). Importantly, the magnitude of the responses to the two agents in TGR(hAGT)L1623 myocytes was significantly reduced. Versus the changes in SD myocytes, Ang-(1-12) caused significantly less increases in dL/dt max (22%), dR/dt max (16%) and [Ca 2+ ] iT (15%) in TGR(hAGT)L1623 myocytes . Ang II also caused similar significantly attenuated increases in dL/dt max (27%), dR/dt max (25%) and [Ca 2+ ] iT (23%). The Ang-(1-12)-induced inotropic effects were completely prevented in the presence of the inhibitory cAMP analog, Rp-cAMPS (10 –4 M, 2 hours) in both SD and TGR(hAGT)L1623 myocytes, but were further augmented only intransgenic rats after incubation of myocytes with the G i inhibitor, pertussis toxin (PTX, 2 μg/ml, 36°C, 5 hours). Conclusions: Ang-(1-12) stimulates LV myocyte contractile function and [Ca 2+ ] iT in both SD and TGR(hAGT)L1623 rats. Furthermore, we now show that the blunted inotropic responses to Ang-(1-12) and Ang II in rats expressing the human AGT gene is mediated through a cAMP-dependent mechanism that is coupled to both stimulatory G and inhibitory PTX-sensitive G proteins.

Abstract 12717: Enhance Negative Modulation of Beta 3 -Adrenergic Stimulation on Cardiomyocyte Functional Performance, [Ca 2+ ] i Regulation and β-Adrenergic Reserve in Rats Expressing Human Angiotensinogen Gene

Background: Transgenic rats expressing the human angiotensinogen gene [TGR (hAogen) 1623] (hAogen) exhibit sustained hypertension and systolic dysfunction associated with altered cardiac renin-angiotensin system and β- adrenergic receptor (AR) signal transduction systems. We have shown previously, in contrast to β 1 - and β 2 -ARs, β 3 -ARs are linked to G i proteins, and stimulation of β 3 -ARs inhibits cardiac contraction and relaxation. Hypertension is associated with increased cardiac expression of G i proteins. This may alter β 3 -AR stimulation and play an important role in the cardiac functional impairment of this humanized model of hypertension. However, the alteration and functional effect of β 3 -AR activation in this model are unknown. We tested the hypothesis that high cardiac tissue Ang II content in hAogen may cause an up-regulation of cardiac β 3 -AR, which exacerbates myocyte dysfunction and impairs calcium regulation, thereby directly contributing to the cardiac dysfunction. Methods: We compared LV myocyte β 3 -AR expression and myocyte functional and [Ca 2+ ] i transient ([Ca 2+ ] iT ) responses to β- and β 3 -AR stimulation in myocytes obtained from 8 Sprague Dawley (SD) control and 8 hAogen male adult rats. Results: Versus SD, in hAGT rats, basal myocyte contraction (dL/dt max , 107.1 vs 138.8 μm/s), relaxation (dR/dt max , 94.0 vs 103.9 μm/s) and [Ca 2+ ] iT (0.15 vs 0.21) were depressed. A non-selective β-AR agonist, isoproterenol (ISO) (10 -8 M) produced significantly smaller increases in dL/dt max , (42% vs. 61%), dR / dtmax (35% vs. 51%), and [Ca 2+ ] iT (20% vs. 30%). Moreover, versus SD, in hAogen rats, LV myocyte β 3 -AR protein level increased by 32% (0.29 vs 0.22) with resulted significantly altered myocyte functional response to β 3 -AR stimulation. Compared with the changes in SD myocytes, in hAogen myocytes, BRL (10 -8 M) produced a significantly greater decreases in dL/dt max , (27% vs 12%), dR/dt max (28% vs 11%), and [Ca 2+ ] iT (17% vs 10%). Conclusions: TGR (hAogen) 1623, this humanized model of hypertension is associated with an up-regulation of LV myocyte β 3 -AR, which enhances β 3 -AR-caused inhibition of myocyte contractile, relaxation and [Ca 2+ ] iT and exacerbates β-AR desensitization, thereby directly contributing to the cardiac dysfunction.

Abstract P212: Kidney Injury Caused By Preeclamptic Pregnancy Improves Postpartum In A Transgenic Rat Model

Preeclamptic pregnancies involve mild renal injuries. However, there has been evidence that women with a history of preeclampsia (PE) have an increased risk to develop kidney disease in association to high blood pressure later in life. This study aims to characterize renal injury during pregnancy and postpartum in an established transgenic rat model for PE. Female Sprague-Dawley rats transgenic for the human angiotensinogen gene develop a PE phenotype in pregnancy, including cardiac remodeling, when mated with male rats harboring the human renin gene. Postpartum, blood pressure restores but cardiac remodeling persists. We hypothesize that PE during pregnancy mediates kidney injuries but does not fully restore after ending of the high blood pressure (postpartum). The renal alterations were analyzed by histological staining, gene expression and urine analysis. PE rats have elevated mean arterial blood pressure during pregnancy (PE d19 148.4 ± 20.7 mmHg) compared to normotensive values in control animals (WT d19 105.3 ± 4.6 mmHg). During PE increased expression of kidney injury marker 1 ( Kim-1/18S : PE d21 4.14 ± 3.26; WT d21 0.08 ± 0.02), neutrophil gelatinase associated lipocalin 2 ( Ngal/18S : PE d21 1.64 ± 0.83; WT d21 0.59 ± 0.28) and connective-tissue growth factor ( Ctgf/18S: PE d21 1.51 ± 0.46; WT d21 0.92 ± 0.32) were detected. Kidneys of PE rats showed mild glomerular (PAS-positive glomerular area PE d21 86.2 ± 4.4%; WT d21 79.1 ± 3.2%) and tubular changes during PE pregnancy resulting in albuminuria (albumin/creatinine ratio PE d19 2193.8 ± 1878.2; WT d19 290.4 ± 252.0). However, 4 weeks after pregnancy (approx. 2 years in humans) most of the PE related renal damages were absent including albuminuria and elevated expression of biomarkers ( Kim-1/18S : PE d50 0.09 ± 0.05; WT d50 0.23 ± 0.13; Ctgf/18S : PE d50 0.78 ± 0.25; WT d50 0.8 ± 0.25; Ngal/18S : PE d50 0.37 ± 0.17; WT 50 0.47 ± 0.11). Only mild enlargement of glomerular tuft area (PE d50 7523.8 ± 418.7 μm 2 ; WT d50 7058.4 ± 198.8 μm 2 ) was detected. Overall, the glomerular and tubular injuries are present during pregnancy in this transgenic PE rat. Most restore postpartum, speculating long-term kidney failure observed in humans is associated to hypertension and additional cardiovascular events.

Computational analyses prioritize and reveal the deleterious nsSNPs in human angiotensinogen gene

Angiotensinogen (AGT) is a key component of renin-angiotensin-aldosterone system (RAAS), which plays central role in blood pressure homeostasis. Association of AGT polymorphisms have been investigated in different ethnic populations in variety of cardiovascular and non-cardiovascular conditions. In this study, 354 non-synonymous SNPs (nsSNPs) of AGT were evaluated to predict damaging and structurally important variants. Majority of the deleterious nsSNPs occurred in the evolutionary conserved regions. Several of these nsSNPs were found to affect post-translational modifications like methylation, glycosylation, phosphorylation, ubiquitination etc. Structural evaluations predicted 19 variants as destabilizing and some of them were also predicted to destabilize the renin-AGT interaction. Therefore, the present computational investigation predicted pathogenic and functionally important variants of human AGT gene. The study has also shown that AGT deregulation is associated with survival outcome in patients with gastric and breast cancer, using microarray gene expression profile. Furthermore, the computationally screened nsSNPs can be analyzed in population based genotyping studies and may help futuristic drug development in the area of AGT pharmacogenomics.

Statins Reverse Postpartum Cardiovascular Dysfunction in a Rat Model of Preeclampsia.

Preeclampsia is associated with increased cardiovascular long-term risk; however, the underlying functional and structural mechanisms are unknown. We investigated maternal cardiac alterations after preeclampsia. Female rats harboring the human angiotensinogen gene [TGR(hAogen)L1623] develop a preeclamptic phenotype with hypertension and albuminuria during pregnancy when mated with male rats bearing the human renin gene [TGR(hRen)L10J] but behave physiologically normal before and after pregnancy. Furthermore, rats were treated with pravastatin. We tested the hypothesis that statins are a potential therapeutic intervention to reduce cardiovascular alterations due to simulated preeclamptic pregnancy. Although hypertension persists for only 8 days in pregnancy, former preeclampsia rats exhibit significant cardiac hypertrophy 28 days after pregnancy observed in both speckle tracking echocardiography and histological staining. In addition, fibrosis and capillary rarefaction was evident. Pravastatin treatment ameliorated the remodeling and improved cardiac output postpartum. Preeclamptic pregnancy induces irreversible structural changes of cardiac hypertrophy and fibrosis, which can be moderated by pravastatin treatment. This pathological cardiac remodeling might be involved in increased cardiovascular risk in later life.

Activation of the Human Angiotensin-(1-12)-Chymase Pathway in Rats With Human Angiotensinogen Gene Transcripts.

Angiotensin-(1-12) [Ang-(1-12)], an alternate substrate for tissue angiotensin II (Ang II) formation, underscores the importance of alternative renin-independent pathway(s) for the generation of angiotensins. Since renin enzymatic activity is species-specific, a transgenic model of hypertension due to insertion of the human angiotensinogen (AGT) gene in Sprague Dawley rats allowed for characterizing the contribution of a non-renin dependent mechanism for Ang II actions in their blood and heart tissue. With this in mind, we investigated whether TGR(hAGT)L1623 transgenic rats express the human sequence of Ang-(1-12) before and following a 2-week oral therapy with the type I Ang II receptor (AT1-R) antagonist valsartan. Plasma and cardiac expression of angiotensins, plasma renin activity, cardiac angiotensinogen, and chymase protein and the enzymatic activities of chymase, angiotensin converting enzyme (ACE) and ACE2 were determined in TGR(hAGT)L1623 rats given vehicle or valsartan. The antihypertensive effect of valsartan after 14-day treatment was associated with reduced left ventricular wall thickness and augmented plasma concentrations of angiotensin I (Ang I) and Ang II; rat and human concentrations of angiotensinogen or Ang-(1-12) did not change. On the other hand, AT1-R blockade produced a 55% rise in left ventricular content of human Ang-(1-12) concentration and no changes in rat cardiac Ang-(1-12) levels. Mass-Spectroscopy analysis of left ventricular Ang II content confirmed a >4-fold increase in cardiac Ang II content in transgenic rats given vehicle; a tendency for decreased cardiac Ang II content following valsartan treatment did not achieve statistical significance. Cardiac chymase and ACE2 activities, significantly higher than ACE activity in TGR(hAGT)L1623 rats, were not altered by blockade of AT1-R. We conclude that this humanized model of angiotensinogen-dependent hypertension expresses the human sequence of Ang-(1-12) in plasma and cardiac tissue and responds to blockade of AT1-R with further increases in the human form of cardiac Ang-(1-12). Since rat renin has no hydrolytic activity on human angiotensinogen, the study confirms and expands knowledge of the importance of renin-independent mechanisms as a source for Ang II pathological actions.

Angiotensin-(1–12)/chymase axis modulates cardiomyocyte L-type calcium currents in rats expressing human angiotensinogen

BackgroundActivation of the intracrine renin angiotensin systems (RAS) is increasingly recognized as contributing to human pathologies, yet non-canonical renin-independent mechanisms for angiotensin II (Ang II) biosynthesis remain controversial. Direct Ang II generation from angiotensin-(1–12) [Ang-(1–12)] by chymase is an essential intracrine source for regulation of cardiac function. Using a transgenic rat model that overexpresses the human angiotensinogen gene [TGR(hAGT)L1623] and displays increased cardiac Ang II levels, this study aimed to provide evidence for intracrine activation of L-type calcium currents (ICa-L) mediated by the Ang-(1–12)/chymase axis. Methods and resultsOn patch clamp, ICa-L density was significantly higher in TGR(hAGT)L1623 (−6.4 ± 0.3 pA/pF) compared to Sprague Dawley (SD) cardiomyocytes (−4.8, ± 0.5 pA/pF). Intracellular administration of Ang II and Ang-(1–12) elicited a ICa-L increase in both SD and TGR(hAGT)L1623 cardiomyocytes, albeit blunted in transgenic cells. ICa-L activation by intracellular Ang II and Ang-(1–12) was abolished by the specific Ang II type 1 receptor blocker E−3174. Co-administration of a chymase inhibitor prevented activation of ICa-L by Ang-(1–12). Confocal micrographs revealed abundant chymase (mast cell protease 5) immunoreactive protein in SD and TGR(hAGT)L1623 cardiomyocytes. ConclusionsOur data demonstrate the existence in cardiomyocytes of a calcium channel modulatory activity responsive to Ang II generated by the Ang-(1–12)/chymase axis that signals via intracellular receptors. Chronically elevated Ang II in TGR(hAGT)L1623 hearts leading to increased intracellular calcium through ICa-L suggests that activation of this Ang-(1–12)/chymase-governed cardiac intracrine RAS may contribute to the pathological phenotypes observed in the humanized model of chronic hypertension and cardiac hypertrophy.

Abstract P126: Statines Reduce Cardiac Dysfunction After Preeclamptic Pregnancy

Introduction: Former preeclamptic women have an increased risk of cardiovascular disease in later life. However, the underlying functional and structural mechanisms are still unknown. Objective: We tested the hypothesis that pravastatin has a beneficial effect on the remodeling of the maternal heart after preeclamptic pregnancy and thus reduces the long-term cardiovascular risk. Methods: Female rats harboring the human angiotensinogen gene [TGR(hAogen)L1623] developed a preeclamptic phenotype during pregnancy when mated with male rats carrying the human renin gene [TGR(hRen)L10J], but behaved physiologically normal postpartum. To investigate a possible intervention, we treated preeclamptic dams with pravastatin for four weeks after birth, which was started already during pregnancy. Cardiac structure and function was compared by speckle tracking echocardiography, gene expression analysis, and immune staining of the maternal heart. Results: Due to pravastatin treatment, former preeclamptic rats reduced signs of cardiac hypertrophy (perimeter cardiomyocytes 65.3±0.3 vs. 60.5±0.5 μm), fibrosis (fibronectin 3.9±0.3 vs. 1.8±0.2 %) and improved cardiac microcirculation (CD31+ cells 219.0±4.7 vs. 269.1±8.6). In addition, cardiac output was increased postpartum by statin treatment (ejection fraction 57.2±1.2 vs. 61.0±1.9; global longitudinal strain -14.5±0.6 vs. -19.8±1.0 %; heartrate 355.0±8.7 vs. 328.6±8.3 bpm). Analysis of maternal blood serum showed a reduction of brain natriuretic peptide (BNP 0.39±0.05 vs. 0.33±0.01 ng/ml) and soluble fms-like tyrosine kinase 1 (sFlt1 0.69±0.02 vs. 0.51±0.02 ng/ml) even four weeks after birth. Fetuses at the time of birth also benefited from treatment (brain liver ratio 0.93±0.03 vs. 0.82±0.01; heart body ratio 7.14±0.09 vs. 6.80±0.12). Discussion: Former preeclamptic rats showed significant cardiac hypertrophy in combination with fibrosis and capillary rarefaction. These irreversible structural changes can be reduced by pravastatin treatment. We could not detect any harmful influence of statins on the fetus. Values mean±SEM, PE vs. PE+pravastatin

A polymorphism in intron I of the human angiotensinogen gene (hAGT) affects binding by HNF3 and hAGT expression and increases blood pressure in mice

Angiotensinogen (AGT) is the precursor of one of the most potent vasoconstrictors, peptide angiotensin II. Genome-wide association studies have shown that two A/G polymorphisms (rs2493134 and rs2004776), located at +507 and +1164 in intron I of the human AGT (hAGT) gene, are associated with hypertension. Polymorphisms of the AGT gene result in two main haplotypes. Hap-I contains the variants -217A, -6A, +507G, and +1164A and is pro-hypertensive, whereas Hap-II contains the variants -217G, -6G, +507A, and +1164G and does not affect blood pressure. The nucleotide sequence of intron I of the hAGT gene containing the +1164A variant has a stronger homology with the hepatocyte nuclear factor 3 (HNF3)-binding site than +1164G. Here we found that an oligonucleotide containing +1164A binds HNF3β more strongly than +1164G and that Hap-I-containing reporter gene constructs have increased basal and HNF3- and glucocorticoid-induced promoter activity in transiently transfected liver and kidney cells. Using a knock-in approach at the hypoxanthine-guanine phosphoribosyltransferase locus, we generated a transgenic mouse model containing the human renin (hREN) gene and either Hap-I or Hap-II. We show that transgenic animals containing Hap-I have increased blood pressure compared with those containing Hap-II. Moreover, the transcription factors glucocorticoid receptor, CCAAT enhancer-binding protein β, and HNF3β bound more strongly to chromatin obtained from the liver of transgenic animals containing Hap-I than to liver chromatin from Hap-II-containing animals. These findings suggest that, unlike Hap-II variants, Hap-I variants of the hAGT gene have increased transcription rates, resulting in elevated blood pressure.

58. A peptide antagonist in a genetic rat angiotensinogen × renin model of preeclampsia

Introduction Preeclampsia is a pregnancy-related disorder characterized by hypertension and excess protein excretion in the urine. It is the most common hypertensive disease of pregnancy and affects about 5–8% of pregnancies. The Renin-Angiotensin-System (RAS) is involved in the pathogenesis of the disease; however, treatment with a RAS-blocker is contraindicated due to fetal toxicity. Objective The rat angiotensinogen renin cross model has been described as a rodent model that recapitulates many of the changes observed in human preeclampsia. This experimental rat model of preeclampsia will be used to test the hypothesis that peptide antagonists targeting the RAS system impact both maternal and fetal outcomes. Methods Sprague-Dawley (SD) rats harboring the human angiotensinogen (hAGT) gene [SD-Tg(hAGT) L1623] were mated with male SD rats bearing the human renin (hREN) gene [SD-Tg(hRen) L10J] to produce a model of preeclampsia (PE) in the dams. These pregnant dams developed hypertension on day 13 of pregnancy (plug-recognition was assigned as day 1) and albuminuria. At day 15 of gestation when the animals were already hypertensive, a RAS peptide antagonist was administered via mini-osmotic pumps sc. Results The major finding of the study was that a peptide antagonist targeting RAS ameliorated the preeclamptic phenotype. In the transgenic model, treatment led to a reduction of blood pressure and urinary albumin excretion. The improvement of intrauterine growth retardation (IUGR) indicates an improved fetal development. Discussion RAS peptide antagonist ameliorates the preeclamptic phenotype of the rat model due to the reduction of blood pressure and albuminuria. Furthermore an improvement of the fetal outcome was observed by an amelioration of the IUGR.

Abstract P377: Effect of Salt Diet on Haplotype Specific Expression of Human Angiotensinogen Gene

Human Angiotensinogen gene (hAGT) is an important component of the Renin- Angiotensin System. The 2.5 Kb promoter of the hAGT gene has ten polymorphisms that form two haplotype (Hap) blocks. Hap-I containing -6A, -20A, -217A, -532T, -793A, -1074T, -1178G, -1561T, -1562C, and -1670A is associated with increased blood pressure whereas Hap-II containing -6G, -20A, -217G, -532C, -793G, -1074G, -1178A, -1561G, -1562G, and -1670G is associated with normal blood pressure in human subjects. In order to understand the role of these SNPs in the transcriptional regulation, we have generated novel transgenic mice with the hAGT gene, targeted to the mHPRT locus with either Hap I or II variants. In the present study, we have examined the effect of high salt diet on the hAGT gene expression and transcriptional regulation in the 8 weeks old male transgenic mice containing these two haplotypes. Quantitative real time PCR analysis revealed an increase in hAGT expression in the liver of Hap-I as compared to Hap-II in basal condition (4.09 fold), Hap-I high salt vs Hap-I basal (2.15 fold), Hap-II high salt vs Hap-II basal (1.9 fold) and Hap-I high salt vs Hap-II high salt (2.47 fold) with p<0.05 in these transgenic mice. Immunoblot analysis also indicated the upregulation of the hAGT in plasma in all the study groups, viz; Hap-I compared to that of Hap-II (2.5 fold), Hap-I high salt vs Hap-I basal (1.9 fold) and Hap-II high salt vs Hap-II basal (1.7 fold) with p<0.05. Our studies show that the transgenic mice fed high salt diet have increased mRNA levels in the liver for the transcription factors HNF1, FOXO1, FOXA1 and MR (4, 3.13, 3.93 and 5.4 fold increase respectively; p<0.05). CHIP assay also complements the above observations. Our results strongly suggest that SNPs in Hap-I promote increased transcription and expression of the hAGT gene in multiple tissues, with resultant elevation of plasma hAGT levels. Our studies would provide new insights into the susceptibility of individuals to salt induced hypertension and explain the novel paradigm in salt induced transcriptional regulation of human angiotensinogen gene.

Abstract 116: Epigenetic Regulation of Human Angiotensinogen (hAGT) Gene by SNPs.

Hypertension is a complex disease caused by a combination of genetic and environmental factors. Renin angiotensin system (RAS) dysfunction leads to essential hypertension. Polymorphisms in the AGT gene increase RAS activity and cause blood pressure elevation. We and others have shown that SNPs in the promoter of hAGT gene can be arranged mainly in two haplotypes. Variants -1670A, -1562C, and -1561T always occur with -6A and form the haplotype-l (Hap-l), while variants -1670G, -1562G, and -1561G always occur with -6G and constitute haplotype-ll (Hap-ll). We have made transgenic animals containing these two haplotype blocks and shown that Haplotype-I is the risk haplotype and increases hAGT expression in TG animals. We hypothesized that these SNPs, when present together as Hap-I or Hap-II in transgenic mice (TG) may lead to haplotype dependent DNA methylation of CpG sites in the promoter of the hAGT gene. Methylation patterns may modulate gene transcription at an epigenetic level and this, in turn, could be dependent on individual polymorphisms. Hypo-methylation is associated with higher gene expression and hyper methylation is associated with lower gene expression. We have found that the number of methylated CpG sites in the hAGT promoter is decreased after high salt (HS) treatment in the liver of Hap-I TG animals as compared to the liver of Hap-I animals. In the liver of Hap-I animals, the hAGT promoter CpG sites are methylated at -460,-434,-386,-261,+42, whereas after high salt treatment, the promoter DNA methylation is observed at -460,-386,+65. On the other hand methylation of hAGT promoter CpG sites in the liver of Hap-II are increased as compared to the liver of Hap-II after high salt treatment. In the liver of Hap-II animals, the CpG sites are methylated at -386,-346,+65, whereas after high salt treatment, methylate sites are observed at -386,-261. Results of this experiment show that methylation of CpG sites is reduced in the DNA obtained from high salt treated liver of both haplotypes. However, DNA from high salt treated liver of Hap-II animals had least number of methylated CpG sites and therefore open chromatin that leads to higher expression of the hAGT gene in this haplotype.

Human AGT -p.Met268Thr and coronary heart disease risk: a case-control study and meta-analysis

Summary Background. Polymorphisms in genes, which is involved in the renin–angiotensin system, play an important role in the pathogenesis of coronary heart disease (CHD). Polymorphism of c.803T>C in the human angiotensinogen gene results in methionine (M) to threonine (T) substitution at codon 268 (p.Met268Thr), which traditionally has been known as M235T. This polymorphism may contribute to cardiovascular diseases. Objectives. The aim of this study was to investigate the association between p.Met268Thr polymorphism in the angiotensinogen gene and coronary heart disease (CHD) through a case-control study, which is followed by a meta-analysis. Material and methods. In the case-control study, c.803T>C genotyping of 217 subjects (102 CHD cases vs 115 controls) was investigated by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. In the meta-analysis, 31 studies were included, reflecting 12,028 people with CHD and 16,362 healthy controls. Results. The data from the case-control study revealed that MT (OR, 1.875; 95%CI, 1.060–3.316; p = 0.031) and TT (OR, 3.389; 95%CI, 1.251–9.179; p = 0.016) genotypes are significantly associated with CHD. The meta-analysis revealed a significant association in the recessive model (OR, 1.156; 95%CI, 1.011–1.321; p = 0.034). Conclusions. Although the pooled OR of the meta-analysis showed that there is an increased risk of CHD conferred by p.Met268Thr of the AGT gene, this association was weak, which could be attributed to a bias in publications. © Copyright by Wydawnictwo Continuo.

OP 26 Preeclampsia changes cardiac function and structure

Introduction Preeclampsia complicates about 5% of all pregnancies worldwide and is the major cause of maternal, fetal and neonatal morbidity and mortality in the western world. Long-term morbidity has also been observed in former preeclamptic mothers, with a substantial increased risk for coronary heart disease. Recent studies demonstrate that women after preterm PE show echocardiographic signs of AHA classification stage B (asymptomatic heart failure) 2 years postpartum although they were normotensive. Objectives The main objective is to detect where and what kind of changes during preeclamptic pregnancy occur. Materials and methods We established a transgenic rat model for preeclampsia, featuring hypertension, proteinuria and intrauterine growth restriction (IUGR) where a female rat harbouring the human angiotensinogen gene is mated with a male transgenic for the human renin. For evaluation we used echocardiography, immunostaining and mRNA expression levels. Results Echocardiographic data show a decreasing ejection fraction and decreasing stroke volume in c ombination with a reduced left ventricular diameter during diastole stage at the end of pregnancy in the preeclamptic rat. The pathology is visible already at day 18, five days after blood pressure rises. In addition, speckle tracking analysis allows description of myocardial deformation normalized to its original shape (strain) and the speed at which this occurs (strain rate). In all dimensions (longitudinal, circumferential and radial) in long as well as in short axis significant reduced global function of strain and strain rate was observed in the heart of preeclamptic rats compared to controls. In the posterior part of the left ventricle we could detect a regional decrease of strain rate which was not found in septal part. Furthermore, an increased wall thickness and increased perivascular fibrosis was measured in this impaired part of the heart (posterior wall). Conclusion These results demonstrate alterations based on a pathological pregnancy leading to a change in structure and function of the left ventricle heart. Future studies will show how long these changes will remain postpartum and how big the impact on later cardiovascular health is.