Regulation Of Cardiac Hypertrophy Research Articles

MicroRNA‐101b attenuates cardiomyocyte hypertrophy by inhibiting protein kinase C epsilon signaling

Previously, a surgical regression model identified microRNA-101b (miR-101b) as a potential inhibitor of cardiac hypertrophy. Here, we investigated the antihypertrophic mechanism of miR-101b using neonatal rat ventricular myocytes. miR-101b markedly suppressed agonist-induced cardiac hypertrophy as shown by cell size and fetal gene expression. By systems biology approaches, we identified protein kinase C epsilon (PKCε) as the major target of miR-101b. Our results from qRT-PCR, western blot, and luciferase reporter assays confirm that PKCε is a direct target of miR-101b. In addition, we found that effectors downstream of PKCε (p-AKT, p-ERK1/2, p-NFAT, and p-GSK3β) are also affected by miR-101b. Our study reveals a novel inhibitory mechanism for miR-101b as a negative regulator of cardiac hypertrophy.

Caveolin-1 Deletion Prevents Hypertensive Vascular Remodeling Induced by Angiotensin II.

It has been proposed that membrane microdomains, caveolae, in vascular cells are critical for signal transduction and downstream functions induced by angiotensin II (AngII). We have tested our hypothesis that caveolin-1 (Cav1), a major structural protein of vascular caveolae, plays a critical role in the development of vascular remodeling by AngII via regulation of epidermal growth factor receptor and vascular endothelial adhesion molecule-1. Cav1-/- and control Cav+/+ mice were infused with AngII for 2 weeks to induce vascular remodeling and hypertension. On AngII infusion, histological assessments demonstrated medial hypertrophy and perivascular fibrosis of aorta and coronary and renal arteries in Cav1+/+ mice compared with sham-operated Cav1+/+ mice. AngII-infused Cav1+/+ mice also showed a phenotype of cardiac hypertrophy with increased heart weight to body weight ratio compared with control Cav1+/+ mice. In contrast, Cav1-/- mice infused with AngII showed attenuation of vascular remodeling but not cardiac hypertrophy. Similar levels of AngII-induced hypertension were found in both Cav1+/+ and Cav1-/- mice as assessed by telemetry. In Cav1+/+ mice, AngII enhanced tyrosine-phosphorylated epidermal growth factor receptor staining in the aorta, which was attenuated in Cav1-/- mice infused with AngII. Enhanced Cav1 and vascular endothelial adhesion molecule-1 expression was also observed in aorta from AngII-infused Cav1+/+ mice but not in Cav1-/- aorta. Experiments with vascular cells further provided a potential mechanism for our in vivo findings. These data suggest that Cav1, and presumably caveolae, in vascular smooth muscle and the endothelium plays a critical role in vascular remodeling and inflammation independent of blood pressure or cardiac hypertrophy regulation.

The oxoglutarate receptor 1 (OXGR1) modulates pressure overload-induced cardiac hypertrophy in mice

The G-protein-coupled receptors (GPCRs) family of proteins play essential roles in the heart, including in the regulation of cardiac hypertrophy. One member of this family, the oxoglutarate receptor 1 (OXGR1), may have a crucial role in the heart because it acts as a receptor for α-ketoglutarate, a metabolite that is elevated in heart failure patients. OXGR1 is expressed in the heart but its precise function during cardiac pathophysiological process is unknown. Here we used both in vivo and in vitro approaches to investigate the role of OXGR1 in cardiac hypertrophy.Genetic ablation of Oxgr1 in mice (OXGR1−/−) resulted in a significant increase in hypertrophy following transverse aortic constriction (TAC). This was accompanied by reduction in contractile function as indicated by cardiac fractional shortening and ejection fraction. Conversely, adenoviral mediated overexpression of OXGR1 in neonatal rat cardiomyocytes significantly reduced phenylephrine-induced cardiomyocyte hypertrophy, a result that was consistent with the in vivo data. Using a combination of yeast two hybrid screening and phospho-antibody array analysis we identified novel interacting partner and downstream signalling pathway that might be regulated by the OXGR1. First, we found that OXGR1 forms a molecular complex with the COP9 signalosome complex subunit 5 (CSN5). Secondly, we observed that the STAT3 signalling pathway was upregulated in OXGR1−/− hearts. Since CSN5 interacts with TYK2, a major upstream regulator of STAT3, OXGR1 might regulate the pro-hypertrophic STAT3 pathway via interaction with the CSN5-TYK2 complex.In conclusion, our study has identified OXGR1 as a novel regulator of pathological hypertrophy via the regulation of the STAT3. Identification of molecules that can specifically activate or inhibit this receptor may be very useful in the development of novel therapeutic approach for pathological cardiac hypertrophy.

2,5-Dimethylcelecoxib prevents pressure-induced left ventricular remodeling through GSK-3 activation.

Glycogen synthase kinase-3 (GSK-3) is a crucial regulator of cardiac hypertrophy. We previously reported that 2,5-dimethylcelecoxib (DM-celecoxib), a celecoxib derivative unable to inhibit cyclooxygenase-2, prevented cardiac remodeling by activating GSK-3, resulting in lifespan prolongation in a mouse model of genetic dilated cardiomyopathy. In the present study, we investigated whether DM-celecoxib can also prevent pressure-induced cardiac remodeling and heart failure, elicited by transverse aortic constriction (TAC). Before testing the effects of DM-celecoxib, we compared the effects of TAC on the hearts of wild-type and GSK-3β hetero-deficient (GSK-3β+/-) mice to determine the role of GSK-3 in cardiac remodeling and heart failure. GSK-3β+/- mouse hearts exhibited more severe hypertrophy, which was characterized by accelerated interstitial fibrosis, than wild-type mouse hearts after TAC, suggesting that reduced GSK-3β activity aggravates pressure-induced left ventricular remodeling. We subsequently examined the effects of DM-celecoxib on TAC-induced cardiac remodeling. DM-celecoxib inhibited left ventricular systolic functional deterioration, and prevented left ventricular hypertrophy and fibrosis. It also activated GSK-3α and β by inhibiting Akt, suppressing the activity of β-catenin and nuclear factor of activated T-cells and thereby decreasing the expression of the Wnt/β-catenin target gene products fibronectin and matrix metalloproteinase-2. These results suggest that DM-celecoxib is clinically useful for treating pressure-induced heart diseases.

Abstract P234: CTMP Negatively Regulates Pathological Cardiac Hypertrophy via Akt-dependent Manner

Pathological cardiac hypertrophy which represents a leading cause of morbidity and mortality worldwide is a pathological process related to multifactorial and multiple molecules and regulated by numerous signaling pathways. Deregulation of AKT signaling is important in cardiac hypertrophy and cardiac dysfunction, but the underlying mechanism is not fully understood. In this study, we identified carboxy-terminal modulator protein (CTMP), an endogenous AKT inhibitor, as a key regulator of cardiac hypertrophy in response to pressure overload. Our results demonstrated that CTMP levels were downregulated by about 40% in aortic banding (AB)–induced hypertrophied mice hearts and 50% in failing human hearts compared to their controls respectively. Mice overexpressing CTMP specifically in the heart were resistant to AB-induced cardiac hypertrophy, whereas cardiac-specific conditional CTMP-knockout mice exhibited an aggravated phenotype induced by pressure overload. Additionally, gain-or-loss of function experiments mediated by adenovirus demonstrated that CTMP also prevented an angiotensin II–induced hypertrophic response in isolated cardiomyocytes in vitro . Mechanistically, we discovered that AKT signaling was significantly activated in AB-treated WT hearts, which was blocked by cardiac overexpression of CTMP, whereas being enhanced by loss of CTMP in response to chronic pressure overload and agonist stimulation. Moreover, rescue-experiments revealed that inhibition of AKT activation through LY294002 ameliorated the cardiac abnormalities in CTMP-knockout mice after AB. Taken together, our present study provides both in vitro and in vivo evidences that CTMP functions as a novel negative regulator factor of pathological cardiac hypertrophy. The underlying mechanisms responsible for CTMP-elicited effects are dependent on the inhibition of AKT signaling. The above-mentioned findings also expand our knowledge of the mechanisms of cardiac hypertrophy and provide potential therapeutic targets for pathological cardiac hypertrophy and heart failure.

Regulation of cardiac hypertrophy and remodeling through the dual-specificity MAPK phosphatases (DUSPs)

Mitogen-activated protein kinases (MAPKs) play a critical role in regulating cardiac hypertrophy and remodeling in response to increased workload or pathological insults. The spatiotemporal activities and inactivation of MAPKs are tightly controlled by a family of dual-specificity MAPK phosphatases (DUSPs). Over the past 2 decades, we and others have determined the critical role for selected DUSP family members in controlling MAPK activity in the heart and the ensuing effects on ventricular growth and remodeling. More specifically, studies from mice deficient for individual Dusp genes as well as heart-specific inducible transgene-mediated overexpression have implicated select DUSPs as essential signaling effectors in the heart that function by dynamically regulating the level, subcellular and temporal activities of the extracellular signal-regulated kinases (ERKs), c-Jun N-terminal kinases (JNKs) and p38 MAPKs. This review summarizes recent literature on the physiological and pathological roles of MAPK-specific DUSPs in regulating MAPK signaling in the heart and the effect on cardiac growth and remodeling.

Ubiquitin-specific protease 14 regulates cardiac hypertrophy progression by increasing GSK-3β phosphorylation

Cardiac hypertrophy, a compensatory response to various stimuli in the heart, independently predicts cardiovascular ailments and related deaths. Increasing evidence indicates ubiquitin-proteasome signaling contributes to cardiac hypertrophy regulation. Here, we identified ubiquitin-specific protease 14 (USP14), a 19S proteasome associated deubiquitinase (DUB), as a novel target for cardiac hypertrophy therapy via inhibition of the GSK-3β pathway. Indeed, USP14 expression was increased in an animal model of abdominal aorta constriction. In an angiotensin II (AngII) induced primary neonatal rat cardiomyocyte hypertrophy model, USP14 expression was increased in a time-dependent manner, and reduced USP14 deubiquitinase activity or USP14 knockdown resulted in lower expression levels of the myocardial hypertrophy specific marker β-MHC, and subsequent decreased GSK-3β phosphorylation. In conclusion, USP14 mediates the development of cardiac hypertrophy by promoting GSK-3β phosphorylation, suggesting that USP14 might represent a novel therapeutic target for cardiac hypertrophy treatment.

Abstract 91: Genome-wide Identification and Characterization of Cardiac Hypertrophy-related Long Noncoding RNAs in Mice

Long noncoding RNAs (LncRNAs) are RNA transcripts longer than 200 nucleotides that lack protein-coding potential. Although thousands of lncRNAs have been identified, only a few have been linked to cardiac gene expression and function. In this study, we identified, from genome-scale RNA-seq data, 12 candidate lncRNAs associated with cardiac hypertrophy (CH-lncRNAs). The expression of these lncRNAs was altered in mouse models of cardiac hypertrophy induced by transverse aortic constriction (TAC)- or CnA transgene. To determine the function of these lncRNAs, we developed an adeno-associated virus serotype 9 (AAV9)-based functional screening in postnatal mice. An AAV9:cTNT vector, in which the cardiac troponin T (cTNT) promoter was used to direct cardiac-specific expression of target genes, was utilized to overexpress or knockdown candidate lncRNAs in mouse hearts. Postnatal day 1 wild type or CnA transgenic pups were injected with AAV9 viruses and cardiac function was measured one and two months later. Thus far, we have tested 15 candidate lncRNAs for both gain- and loss-of-function studies. Among them, two lncRNAs were demonstrated regulating hypertrophy growth when knocked down. Finally, we identified the human homologues of CH-lncRNA through analyzing the conservation of the promoter regions of lncRNA genes. We showed that the expression of these human CH-lncRNA was dysregulated in human diseased hearts, suggesting the functional conservation of these lncRNAs in cardiac disease. Our study therefore demonstrated that lncRNAs are important regulator of cardiac hypertrophy and disease.

Abstract 9: Involvement of Caveolin-1 in Vascular Remodeling and Inflammation Induced by Angiotensin II

We have recently reported that caveolin-1 (Cav1) enriched membrane microdomains in vascular smooth muscle cells (VSMC) mediate a metalloprotease ADAM17-dependent EGF receptor (EGFR) transactivation, which is linked to vascular remodeling induced by AngII. We have tested our hypothesis that Cav1, a major structural protein of caveolae, plays a critical role for development of vascular remodeling by AngII via regulation of ADAM17 and EGFR. Here, 8 week old male Cav1-/- and control Cav+/+ wild-type mice (WT) were infused with AngII (1 μg/kg/min) for 2 weeks to induce vascular remodeling and hypertension. Upon AngII infusion, histological assessments demonstrated medial hypertrophy and perivascular fibrosis of coronary and renal arteries in WT mice compared with saline-infused control mice. The AngII-infused WT mice also showed a phenotype of cardiac hypertrophy with increased HW/BW ratio (mg/g: 8.0±0.6 vs 5.7±0.7 p<0.01) compared with WT control. In contrast to AngII-infused WT mice, Cav1-/- mice with AngII showed attenuation of vascular remodeling but not cardiac hypertrophy ; HW/BW ratio (8.6±0.5 vs 6.4±0.2 p<0.05). Similar levels of AngII-induced hypertension were observed in both WT and Cav1-/- mice assessed by telemetry (MAP mmHg: 142±9 vs 154±20). In WT mice, Ang II enhanced ADAM17 expression and phospho-Tyr EGFR staining in heart and kidney vasculature. These events were attenuated in vessels from Cav1-/- mice infused with AngII. In addition, IHC analysis revealed less ER stress in heart and kidney vasculature of AngII-infused Cav1-/- mice compared with WT mice. Enhanced Cav1 and VCAM-1 expression were also observed in the aorta from AngII-infused WT mice but not in Cav1-/- aorta. These data suggest that Cav1 and presumably vascular caveolae play critical roles for vascular remodeling and inflammation via induction of ADAM17 and activation of EGFR independent of blood pressure or cardiac hypertrophy regulation.

The H19 long noncoding RNA is a novel negative regulator of cardiomyocyte hypertrophy.

The H19 lncRNA, a highly abundant and conserved imprinted gene, has been implicated in many essential biological processes and diseases. However, the function of H19 in the heart remains unknown. In this study, we investigated the function and underlying mechanism of H19 in regulating cardiomyocyte hypertrophy. We first detected the expression of H19 and its encoded miR-675 in both normal and diseased hearts and verified their up-regulations in pathological cardiac hypertrophy and heart failure. Adenovirus-mediated expression and a siRNA-mediated silence of H19 showed that H19 overexpression reduced cell size both at baseline and in response to phenylephrine, whereas knock-down of H19 induced cardiomyocyte hypertrophy. Overexpression or knock-down of miR-675 in cardiomyocytes demonstrated that miR-675 also inhibited cardiomyocyte hypertrophy. Moreover, inhibition of miR-675 reversed the reduction of cardiomyocyte size in H19-overexpressing cardiomyocytes, while infection with an adenovirus carrying H19 fragment without pre-miR-675 (H19-Tru) or with mutant sequences of pre-miR-675 (H19-Mut) failed to reduce cardiomyocyte size, indicating that miR-675 mediated the inhibitory effect of H19 on cardiomyocyte hypertrophy. We also identified that CaMKIIδ was a direct target of miR-675 and partially mediated the effect of H19 on cardiomyocyte hypertrophy. Furthermore, in vivo silencing of miR-675 using a specific antagomir in a pressure overload-induced mouse model of heart failure increased cardiac CaMKIIδ expression and exacerbated cardiac hypertrophy. These findings reveal a novel function of H19-miR-675 axis targeting CaMKIIδ as a negative regulator of cardiac hypertrophy, suggesting its potential therapeutic role in cardiac diseases.

Cardiac-Specific EPI64C Blunts Pressure Overload–Induced Cardiac Hypertrophy

The calcium-responsive molecule, calcineurin, has been well characterized to play a causal role in pathological cardiac hypertrophy over the past decade. However, the intrinsic negative regulation of calcineurin signaling during the progression of cardiomyocyte hypertrophy remains enigmatic. Herein, we explored the role of EPI64C, a dual inhibitor of both Ras and calcineurin signaling during T-cell activation, in pressure overload-induced cardiac hypertrophy. We generated a cardiac-specific Epi64c conditional knockout mouse strain and showed that loss of Epi64c remarkably exacerbates pressure overload-induced cardiac hypertrophy. In contrast, EPI64C gain-of-function in cardiomyocyte-specific Epi64c transgenic mice exerts potent protective effects against cardiac hypertrophy. Mechanistically, the cardioprotective effects of EPI64C are largely attributed to the disrupted calcineurin signaling but are independent of its Ras suppressive capability. Molecular studies have indicated that the 406 to 446 C-terminal amino acids in EPI64C directly bind to the 287 to 337 amino acids in the catalytic domain of calcineurin, which is responsible for the EPI64C-mediated suppressive effects. We further extrapolated our studies to cynomolgus monkeys and showed that gene therapy based on lentivirus-mediated EPI64C overexpression in the monkey hearts blunted pressure overload-induced cardiac hypertrophy. Our study thus identified EPI64C as a novel negative regulator in cardiac hypertrophy by targeting calcineurin signaling and demonstrated the potential of gene therapy and drug development for treating cardiac hypertrophy.

Cardiac Stim1 Silencing Impairs Adaptive Hypertrophy and Promotes Heart Failure Through Inactivation of mTORC2/Akt Signaling.

Stromal interaction molecule 1 (STIM1) is a dynamic calcium signal transducer implicated in hypertrophic growth of cardiomyocytes. STIM1 is thought to act as an initiator of cardiac hypertrophic response at the level of the sarcolemma, but the pathways underpinning this effect have not been examined. To determine the mechanistic role of STIM1 in cardiac hypertrophy and during the transition to heart failure, we manipulated STIM1 expression in mice cardiomyocytes by using in vivo gene delivery of specific short hairpin RNAs. In 3 different models, we found that Stim1 silencing prevents the development of pressure overload-induced hypertrophy but also reverses preestablished cardiac hypertrophy. Reduction in STIM1 expression promoted a rapid transition to heart failure. We further showed that Stim1 silencing resulted in enhanced activity of the antihypertrophic and proapoptotic GSK-3β molecule. Pharmacological inhibition of glycogen synthase kinase-3 was sufficient to reverse the cardiac phenotype observed after Stim1 silencing. At the level of ventricular myocytes, Stim1 silencing or inhibition abrogated the capacity for phosphorylation of Akt(S473), a hydrophobic motif of Akt that is directly phosphorylated by mTOR complex 2. We found that Stim1 silencing directly impaired mTOR complex 2 kinase activity, which was supported by a direct interaction between STIM1 and Rictor, a specific component of mTOR complex 2. These data support a model whereby STIM1 is critical to deactivate a key negative regulator of cardiac hypertrophy. In cardiomyocytes, STIM1 acts by tuning Akt kinase activity through activation of mTOR complex 2, which further results in repression of GSK-3β activity.

Long Non-Coding RNA Malat-1 Is Dispensable during Pressure Overload-Induced Cardiac Remodeling and Failure in Mice

BackgroundLong non-coding RNAs (lncRNAs) are a class of RNA molecules with diverse regulatory functions during embryonic development, normal life, and disease in higher organisms. However, research on the role of lncRNAs in cardiovascular diseases and in particular heart failure is still in its infancy. The exceptionally well conserved nuclear lncRNA Metastasis associated in lung adenocarcinoma transcript 1 (Malat-1) is a regulator of mRNA splicing and highly expressed in the heart. Malat-1 modulates hypoxia-induced vessel growth, activates ERK/MAPK signaling, and scavenges the anti-hypertrophic microRNA-133. We therefore hypothesized that Malat-1 may act as regulator of cardiac hypertrophy and failure during cardiac pressure overload induced by thoracic aortic constriction (TAC) in mice.ResultsAbsence of Malat-1 did not affect cardiac hypertrophy upon pressure overload: Heart weight to tibia length ratio significantly increased in WT mice (sham: 5.78±0.55, TAC 9.79±1.82 g/mm; p<0.001) but to a similar extend also in Malat-1 knockout (KO) mice (sham: 6.21±1.12, TAC 8.91±1.74 g/mm; p<0.01) with no significant difference between genotypes. As expected, TAC significantly reduced left ventricular fractional shortening in WT (sham: 38.81±6.53%, TAC: 23.14±11.99%; p<0.01) but to a comparable degree also in KO mice (sham: 37.01±4.19%, TAC: 25.98±9.75%; p<0.05). Histological hallmarks of myocardial remodeling, such as cardiomyocyte hypertrophy, increased interstitial fibrosis, reduced capillary density, and immune cell infiltration, did not differ significantly between WT and KO mice after TAC. In line, the absence of Malat-1 did not significantly affect angiotensin II-induced cardiac hypertrophy, dysfunction, and overall remodeling. Above that, pressure overload by TAC significantly induced mRNA levels of the hypertrophy marker genes Nppa, Nppb and Acta1, to a similar extend in both genotypes. Alternative splicing of Ndrg2 after TAC was apparent in WT (isoform ratio; sham: 2.97±0.26, TAC 1.57±0.40; p<0.0001) and KO mice (sham: 3.64±0.37; TAC: 2.24±0.76; p<0.0001) and interestingly differed between genotypes both at baseline and after pressure overload (p<0.05 each).ConclusionThese findings confirm a role for the lncRNA Malat-1 in mRNA splicing. However, no critical role for Malat-1 was found in pressure overload-induced heart failure in mice, despite its reported role in vascularization, ERK/MAPK signaling, and regulation of miR-133.

Adiponectin Upregulates MiR-133a in Cardiac Hypertrophy through AMPK Activation and Reduced ERK1/2 Phosphorylation.

Adiponectin and miR-133a are key regulators in cardiac hypertrophy. However, whether APN has a potential effect on miR-133a remains unclear. In this study, we aimed to investigate whether APN could regulate miR-133a expression in Angiotensin II (Ang II) induced cardiac hypertrophy in vivo and in vitro. Lentiviral-mediated adiponectin treatment attenuated cardiac hypertrophy induced by Ang II infusion in male wistar rats as determined by reduced cell surface area and mRNA levels of atrial natriuretic peptide (ANF) and brain natriuretic peptide (BNP), also the reduced left ventricular end-diastolic posterior wall thickness (LVPWd) and end-diastolic interventricular septal thickness (IVSd). Meanwhile, APN elevated miR-133a level which was downregulated by Ang II. To further investigate the underlying molecular mechanisms, we treated neonatal rat ventricular myocytes (NRVMs) with recombinant rat APN before Ang II stimulation. Pretreating cells with recombinant APN promoted AMP-activated protein kinase (AMPK) phosphorylation and inhibited ERK activation. By using the inhibitor of AMPK or a lentiviral vector expressing AMPK short hairpin RNA (shRNA) cancelled the positive effect of APN on miR-133a. The ERK inhibitor PD98059 reversed the downregulation of miR-133a induced by Ang II. These results indicated that the AMPK activation and ERK inhibition were responsible for the positive effect of APN on miR-133a. Furthermore, adiponectin receptor 1 (AdipoR1) mRNA expression was inhibited by Ang II stimulation. The positive effects of APN on AMPK activation and miR-133a, and the inhibitory effect on ERK phosphorylation were inhibited in NRVMs transfected with lentiviral AdipoR1shRNA. In addition, APN depressed the elevated expression of connective tissue growth factor (CTGF), a direct target of miR-133a, through the AMPK pathway. Taken together, our data indicated that APN reversed miR-133a levels through AMPK activation, reduced ERK1/2 phosphorylation in cardiomyocytes stimulated with Ang II, revealing a previously undemonstrated and important link between APN and miR-133a.

A circular RNA protects the heart from pathological hypertrophy and heart failure by targeting miR-223.

Sustained cardiac hypertrophy accompanied by maladaptive cardiac remodelling represents an early event in the clinical course leading to heart failure. Maladaptive hypertrophy is considered to be a therapeutic target for heart failure. However, the molecular mechanisms that regulate cardiac hypertrophy are largely unknown. Here we show that a circular RNA (circRNA), which we term heart-related circRNA (HRCR), acts as an endogenous miR-223 sponge to inhibit cardiac hypertrophy and heart failure. miR-223 transgenic mice developed cardiac hypertrophy and heart failure, whereas miR-223-deficient mice were protected from hypertrophic stimuli, indicating that miR-223 acts as a positive regulator of cardiac hypertrophy. We identified ARC as a miR-223 downstream target to mediate the function of miR-223 in cardiac hypertrophy. Apoptosis repressor with CARD domain transgenic mice showed reduced hypertrophic responses. Further, we found that a circRNA HRCR functions as an endogenous miR-223 sponge to sequester and inhibit miR-223 activity, which resulted in the increase of ARC expression. Heart-related circRNA directly bound to miR-223 in cytoplasm and enforced expression of HRCR in cardiomyocytes and in mice both exhibited attenuated hypertrophic responses. These findings disclose a novel regulatory pathway that is composed of HRCR, miR-223, and ARC. Modulation of their levels provides an attractive therapeutic target for the treatment of cardiac hypertrophy and heart failure.

Abstract 14133: Angiotensin II-induced Vascular Fibrosis was Prevented by Caveolin-1 Deficient Mice

Introduction: We have recently reported that caveolin-1 (Cav1) enriched membrane microdomains in vascular smooth muscle cells (VSMC) mediates a metalloprotease ADAM17-dependent EGF receptor (EGFR) transactivation, which is linked to vascular remodeling induced by AngII. Hypothesis: We have tested our hypothesis that Cav1, a major structural protein of caveolae, plays a critical role in AngII-mediated vascular remodeling via regulation of ADAM17 and EGFR. Methods: In vivo: 8 week old male Cav1-/- and control Cav+/+ wild-type mice (WT) were infused with AngII (1 μg/kg/min) for 2 weeks to induce vascular remodeling and hypertension. In vitro: Rat aortic VSMCs were cultured and stimulated with 100 nM AngII. Results: Upon AngII infusion, histological assessments demonstrated medial hypertrophy and perivascular fibrosis of coronary and renal arteries in WT mice compared to sham-operated mice. The AngII-infused WT mice also showed a phenotype of cardiac hypertrophy with increased HW/BW ratio (mg/g: 8.0±0.6 vs 5.7±0.7 p&lt;0.01) compared with sham-operated WT control. In contrast, vascular remodeling but not cardiac hypertrophy were attenuated in Cav1-/- mice with AngII infusion; HW/BW ratio (8.6±0.5 vs 6.4±0.2 p&lt;0.05) compared to sham-operated mice. However, AngII induced similar levels of hypertension in both WT and Cav1-/- mice as assessed by telemetry (MAP mmHg: 142±9 vs 154±20). AngII infusion in WT mice enhanced ADAM17 and phospho-Tyr EGFR staining in heart and kidney vasculature, whereas AngII-infused Cav1-/- mice showed diminished ADAM17 and phospho-Tyr EGFR staining within the vasculature. In addition, IHC analyses revealed reduced vascular ER stress in heart and kidney samples of AngII-infused Cav1-/- mice compared to WT mice. In cultured rat VSMC, Cav1 silencing attenuated induction and activation of ADAM17 by AngII. Conclusions: These data suggest that Cav1, and presumably caveolae microdomains, plays a critical role in vascular fibrosis via vascular induction of ADAM17 and activation of EGFR independent of blood pressure or cardiac hypertrophy regulation.

Cardiac remodeling in Gαq and Gα11 knockout mice

BackgroundAlthough both Gαq- and Gα11-protein signaling are believed to be involved in the regulation of cardiac hypertrophy, their detailed contribution to myocardial function remains elusive. Methods and resultsWe studied remodeling processes in healthy transgenic mice with genetically altered Gαq/Gα11-expression, in particular a global Gα11-knockout and a novel inducible cardiac specific Gαq-knockout, as well as a combined double knockout (dKO) mouse line. Echocardiography and telemetric ECG recordings revealed that compared with wild type mice, hearts of dKO mice showed an increased ejection fraction and a decreased heart rate, irrespective of age resulting in a maintained cardiac output. We attributed these findings to the lack of Gα11, which the absence was associated with a decreased afterload. Histological analysis of the extracellular matrix in the heart depicted a diminished presence of collagen in aging hearts of dKO mice compared to wild-type mice. The results of a transcriptome analysis on isolated ventricular cardiac myocytes revealed alterations of the activity of genes involved in the Gαq/Gα11-dependent regulation of the extracellular matrix, such as the matricellular protein Cyr61. ConclusionsFrom our data we conclude that Gαq/Gα11 signaling pathways play a pivotal role in maintaining gene activity patterns. For the heart we revealed their importance in modulating the properties of the extracellular matrix, a mechanism that might be an important contributor and mechanistic basis for the development of pressure-overload induced cardiac hypertrophy.

Abstract P108: Caveolin-1 is Required for Vascular Remodeling Induced by Angiotensin Ii

We have recently reported that caveolin-1 (Cav1) enriched membrane microdomains in vascular smooth muscle cells (VSMC) mediates a metalloprotease ADAM17-dependent EGF receptor (EGFR) transactivation, which is linked to vascular remodeling induced by AngII. We have tested our hypothesis that Cav1, a major structural protein of caveolae, plays a critical role in AngII-mediated vascular remodeling via regulation of ADAM17 and EGFR. 8 week old male Cav1-/- and control Cav+/+ wild-type mice (WT) were infused with AngII (1 μg/kg/min) for 2 weeks to induce vascular remodeling and hypertension. Upon AngII infusion, histological assessments demonstrated medial hypertrophy and perivascular fibrosis of coronary and renal arteries in WT mice compared to sham-operated mice. The AngII-infused WT mice also showed a phenotype of cardiac hypertrophy with increased HW/BW ratio (mg/g: 8.0±0.6 vs 5.7±0.7 p&lt;0.01) compared with sham-operated WT control. In contrast, vascular remodeling but not cardiac hypertrophy were attenuated in Cav1-/- mice with AngII infusion; HW/BW ratio (8.6±0.5 vs 6.4±0.2 p&lt;0.05) compared to sham-operated mice. However, AngII induced similar levels of hypertension in both WT and Cav1-/- mice as assessed by telemetry (MAP mmHg: 142±9 vs 154±20). AngII infusion in WT mice enhanced ADAM17 and phospho-Tyr EGFR staining in heart and kidney vasculature, whereas AngII-infused Cav1-/- mice showed diminished ADAM17 and phospho-Tyr EGFR staining within the vasculature. In addition, IHC analyses revealed reduced vascular ER stress in heart and kidney samples of AngII-infused Cav1-/- mice compared to WT mice. Expression of Cav1 was predominantly observed within the endothelium and was enhanced upon AngII infusion in WT mice. These data suggest that Cav1, and presumably endothelial caveolae microdomains, plays a critical role in vascular remodeling via vascular induction of ADAM17 and activation of EGFR independent of blood pressure or cardiac hypertrophy regulation.

MicroRNA‐150 Protects Against Pressure Overload‐Induced Cardiac Hypertrophy

Cardiac hypertrophy is the response of the heart to a variety of hypertrophic stimuli; this condition progresses to heart failure and sudden death. MicroRNAs (miRs) are a family of small, non-coding RNAs that mediate posttranscriptional gene silencing. Recent studies have identified miRs as important regulators in cardiac hypertrophy. One specific miR, miR-150 has been reported to be downregulated in hypertrophic murine hearts. However, the role of miR-150 as a regulator of cardiac hypertrophy remains unclear. In the present study, we used gain-of-function and loss-of-function approaches to investigate the functional roles of miR-150 in cardiac hypertrophy induced by aortic banding. The extent of the cardiac hypertrophy was evaluated by echocardiography and by pathological and molecular analyses of heart samples. Our results revealed that transgenic mice that overexpress miR-150 in the heart were resistant to cardiac hypertrophy and fibrosis through down-regulation of serum response factor (SRF). Conversely, the loss of function of miR-150 by genetic knockdown or antagomiR approaches produced the opposite effects. These studies suggest that miR-150 plays an important role in the regulation of cardiac hypertrophy and SRF is involved in miR-150 mediated anti-hypertrophic effect. Thus, miR-150 may be a new therapeutic target for cardiac hypertrophy.

ALK7 protects against pathological cardiac hypertrophy in mice.

Activin receptor-like kinase 7 (ALK7), one of the type I transforming growth factor-β receptors, is expressed in various tissues, including the heart. However, the participation of ALK7 in the regulation of cardiac hypertrophy has not yet been studied. Here, we sought to determine the regulatory role and underlying mechanisms of ALK7 in cardiac hypertrophy. We performed aortic banding (AB) in ALK7-knockout mice, cardiac-specific ALK7-transgenic mice, and the wild-type littermates of these mice. Cardiac hypertrophy was evaluated using pathological analysis, echocardiographic measurement, haemodynamic measurement, and molecular analysis. Our results revealed that ALK7 disruption led to an aggravated cardiac hypertrophic response that was accompanied by increased cardiac fibrosis and reduced contractile function, whereas cardiac-specific ALK7 overexpression exhibited the opposite phenotype in response to pressure overload. Similarly, ALK7 protected against angiotensin II-induced cardiomyocyte hypertrophy in vitro. Mechanistically, we demonstrated that ALK7-dependent cardioprotection was mediated largely through inhibition of the MEK-ERK1/2 signalling pathway. Our data suggest that ALK7 acts as a novel regulator of pathological cardiac hypertrophy via the negative regulation of MEK-ERK1/2 signalling and may serve as a potential therapeutic target for pathological cardiac hypertrophy.