Piezo1 and BKCa channels in human atrial fibroblasts: Interplay and remodelling in atrial fibrillation.

AIMS: Atrial Fibrillation (AF) is an arrhythmia of increasing prevalence in the aging populations of developed countries. One of the important indicators of AF is sustained atrial dilatation, highlighting the importance of mechanical overload in the pathophysiology of AF. The mechanisms by which atrial cells, including fibroblasts, sense and react to changing mechanical forces, are not fully elucidated. Here, we characterise stretch-activated ion channels (SAC) in human atrial fibroblasts and changes in SAC- presence and activity associated with AF. METHODS AND RESULTS: Using primary cultures of human atrial fibroblasts, isolated from patients in sinus rhythm or sustained AF, we combine electrophysiological, molecular and pharmacological tools to identify SAC. Two electrophysiological SAC- signatures were detected, indicative of cation-nonselective and potassium-selective channels. Using siRNA-mediated knockdown, we identified the cation-nonselective SAC as Piezo1. Biophysical properties of the potassium-selective channel, its sensitivity to calcium, paxilline or iberiotoxin (blockers), and NS11021 (activator), indicated presence of calcium-dependent 'big potassium channels' (BKCa). In cells from AF patients, Piezo1 activity and mRNA expression levels were higher than in cells from sinus rhythm patients, while BKCa activity (but not expression) was downregulated. Both Piezo1-knockdown and removal of extracellular calcium from the patch pipette resulted in a significant reduction of BKCa current during stretch. No co-immunoprecipitation of Piezo1 and BKCa was detected. CONCLUSIONS: Human atrial fibroblasts contain at least two types of ion channels that are activated during stretch: Piezo1 and BKCa. While Piezo1 is directly stretch-activated, the increase in BKCa activity during mechanical stimulation appears to be mainly secondary to calcium influx via SAC such as Piezo1. During sustained AF, Piezo1 is increased, while BKCa activity is reduced, highlighting differential regulation of both channels. Our data support the presence and interplay of Piezo1 and BKCa in human atrial fibroblasts in the absence of physical links between the two channel proteins.

Myocardial transcription of inflammatory and remodeling markers in cats with hypertrophic cardiomyopathy and systemic diseases associated with an inflammatory phenotype.

Feline hypertrophic cardiomyopathy (HCM) is characterized by macrophage-driven myocardial remodeling processes in a pro-inflammatory environment. To further investigate the mechanisms behind these processes, the myocardial transcription of cytokines and remodeling enzymes was comparatively assessed in cats with HCM and cats without cardiac diseases. Sixty-seven cats were included, 17 cats with HCM (including 5 with atrial thrombus; AT), and 50 cats without cardiac diseases. The latter comprised 10 control cats (no cardiac or relevant systemic disease), 34 cats with diseases suspected to be associated with a systemic inflammatory state of which 18 suffered from feline infectious peritonitis (FIP), and 6 cats with multicentric lymphoma. Samples from atria, ventricular free walls and interventricular septum were examined using quantitative reverse transcriptase PCR. The overall highest myocardial marker transcriptions were observed in cats with multicentric lymphoma, FIP and HCM, followed by diseases likely associated with a systemic inflammatory state, and control cats. Inflammatory marker transcription predominated in the myocardium of cats with systemic inflammatory diseases, whereas in HCM the transcription of remodeling enzymes prevailed. Sex significantly influenced the myocardial transcription of several remodeling enzymes. These results suggest a versatile myocardial response depending on the disease and illustrates the relevance of sex for the cardiac response to cardiac and systemic disease in cats. A systemic inflammatory state appears to elicit an inflammatory phenotype in the myocardium, whereas in HCM, the myocardium mediates its own remodeling. In HCM, the identified markers might be involved in the ongoing remodeling processes causing structural and functional changes.

The association between impairment of HDL cholesterol efflux capacity and atrial remodeling in atrial fibrillation.

This cross-sectional study enrolled 202 patients with atrial fibrillation (AF) who had undergone catheter ablation and evaluated the association between high-density lipoprotein (HDL) functionality, cholesterol efflux capacity (CEC) of HDL, and the pathophysiology of left atrial structural remodeling. Participants were divided into two groups, based on their left atrial volume index (LAVI) (< 34 mL/m2, n = 60 vs. LAVI ≥ 34 mL/m2, n = 142). We quantified three types of HDL CECs by the presence or absence of cyclic-AMP, as entire, and CEC dependent or not dependent on ATP binding cassette transporter A1 (ABCA1) and termed them Global CEC, ABCA1 CEC, and Non-ABCA1 CEC, respectively. Consequently, Global and Non-ABCA1 CECs were significantly impaired in patients with an enlarged LA (Global CEC: p = 0.039, Non-ABCA1 CEC: p = 0.022). Logistic regression analyses demonstrated that Non-ABCA1 CEC was significantly associated with an enlarged LA after adjusting for the conventional risk factors of AF. Furthermore, the association of higher Non-ABCA1 CEC with an enlarged LA was independent of serum levels of HDL cholesterol and serum myeloperoxidase (Odds ratio of 1 standard deviation higher: 0.64, 95% confidence interval: 0.43-0.95, p = 0.027). The findings of this study indicate the potential contribution of reduced Non-ABCA1 CEC in HDL to the pathophysiology in left atrial structural remodeling of patients with AF.

Regulation of mitochondrial cristae remodelling by acetylcholine alleviates palmitate-induced cardiomyocyte hypertrophy.

Mitochondrial dysfunction is associated with obesity-induced cardiac remodelling. Recent research suggests that the cristae are the true bioenergetic components of cells. Acetylcholine (ACh), the major neurotransmitter of the vagus nerve, exerts cardio-protective effects against ischaemia. This study investigated the role of cristae remodelling in palmitate (PA)-induced neonatal rat cardiomyocyte hypertrophy and explored the beneficial effects of ACh. We found loose, fragmented and even lysed cristae in PA-treated neonatal cardiomyocytes along with declines in mitochondrial network and complex expression and overproduction of mitochondrial reactive oxygen species (ROS); these changes ultimately resulted in increased myocardial size. Overexpression of mitofilin by adenoviral infection partly improved cristae shape, mitochondrial network, and ATP content and attenuated cell hypertrophy. Interestingly, siRNA-mediated AMP-activated protein kinase (AMPK) silencing increased the number of cristae with a balloon-like morphology without disturbing mitofilin expression. Furthermore, AMPK knockdown abolished the effects of mitofilin overexpression on cristae remodelling and inhibited the interaction of mitofilin with sorting and assembly machinery 50 (Sam50) and coiled-coil helix coiled-coil helix domain-containing protein 3 (CHCHD3), two core components of the mitochondrial contact site and cristae organizing system (MICOS) complex. Intriguingly, ACh upregulated mitofilin expression and AMPK phosphorylation via the muscarinic ACh receptor (MAChR). Moreover, ACh enhanced protein-protein interactions between mitofilin and other components of the MICOS complex, thereby preventing PA-induced mitochondrial dysfunction and cardiomyocyte hypertrophy; however, these effects were abolished by AMPK silencing. Taken together, our data suggest that ACh improves cristae remodelling to defend against PA-induced myocardial hypertrophy, presumably by increasing mitofilin expression and activating AMPK to form the MICOS complex through MAChR. These results suggest new and promising therapeutic approaches targeting mitochondria to prevent lipotoxic cardiomyopathy.

Systemic Inflammation Rapidly Induces Reversible Atrial Electrical Remodeling: The Role of Interleukin-6-Mediated Changes in Connexin Expression.

Background Systemic inflammation is a strong predictor of atrial fibrillation. A key role for electrical remodeling is increasingly recognized, and experimental data suggest that inflammatory cytokines can directly affect connexins resulting in gap-junction dysfunction. We hypothesized that systemic inflammation, regardless of its origin, promotes atrial electric remodeling in vivo, as a result of cytokine-mediated changes in connexin expression. Methods and Results Fifty-four patients with different inflammatory diseases and elevated C-reactive protein were prospectively enrolled, and electrocardiographic P-wave dispersion indices, cytokine levels (interleukin-6, tumor necrosis factor-α, interleukin-1, interleukin-10), and connexin expression (connexin 40, connexin 43) were measured during active disease and after reducing C-reactive protein by >75%. Moreover, peripheral blood mononuclear cells and atrial tissue specimens from an additional sample of 12 patients undergoing cardiac surgery were evaluated for atrial and circulating mRNA levels of connexins. Finally, in vitro effects of interleukin-6 on connexin expression were studied in HL-1 mouse atrial myocytes. In patients with active inflammatory diseases, P-wave dispersion indices were increased but rapidly decreased within days when C-reactive protein normalizes and interleukin-6 levels decline. In inflammatory disease patients, both P-wave dispersion indices and interleukin-6 changes were inversely associated with circulating connexin levels, and a positive correlation between connexin expression in peripheral blood mononuclear cells and atrial tissue was demonstrated. Moreover, interleukin-6 significantly reduced connexin expression in HL-1 cells. Conclusions Our data suggest that regardless of specific etiology and organ localization, systemic inflammation, via interleukin-6 elevation, rapidly induces atrial electrical remodeling by down-regulating cardiac connexins. Although transient, these changes may significantly increase the risk for atrial fibrillation and related complications during active inflammatory processes.

A genetic mouse model of severe iron deficiency anemia reveals tissue-specific transcriptional stress responses and cardiac remodeling.

Iron is a micronutrient fundamental for life. Iron homeostasis in mammals requires sustained postnatal intestinal iron absorption that maintains intracellular iron concentrations for central and systemic metabolism as well as for erythropoiesis and oxygen transport. More than 1 billion people worldwide suffer from iron deficiency anemia (IDA), a state of systemic iron insufficiency that limits the production of red blood cells and leads to tissue hypoxia and intracellular iron stress. Despite this tremendous public health concern, very few genetic models of IDA are available to study its progression. Here we developed and characterized a novel genetic mouse model of IDA. We found that tamoxifen-inducible deletion of the mammalian iron exporter ferroportin exclusively in intestinal epithelial cells leads to loss of intestinal iron absorption. Ferroportin ablation yielded a robust phenotype of progressive IDA that develops in as little as 3 months following disruption of intestinal iron absorption. We noted that, at end-stage IDA, tissue-specific transcriptional stress responses occur in which the heart shows little to no hypoxic and iron stress compared with other peripheral organs. However, morphometric and echocardiographic analysis revealed massive cardiac hypertrophy and chamber dilation, albeit with increased cardiac output at very low basal heart rates. We propose that our intestine-specific ferroportin knockout mouse model of end-stage IDA could be used in future studies to investigate IDA progression and cell-specific responses to hypoxic and iron stress.

Tachycardia-induced CD44/NOX4 signaling is involved in the development of atrial remodeling.

Atrial fibrillation (AF) is associated with oxidative stress and Ca2+-handling abnormalities in atrial myocytes. Our prior study has demonstrated the involvement of CD44, a membrane receptor for hyaluronan (HA), in the pathogenesis of AF. This study further evaluated whether CD44 and its related signaling mediate atrial tachycardia-induced oxidative stress and Ca2+-handling abnormalities. Tachypacing in atrium-derived myocytes (HL-1 cell line) induced the activation of CD44-related signaling, including HA and HA synthase (HAS) expression. Blocking HAS/HA/CD44 signaling attenuated tachypacing-induced oxidative stress (NADPH oxidase [NOX] 2/4 expression) and Ca2+-handling abnormalities (oxidized Ca2+/calmodulin-dependent protein kinase II [ox-CaMKII] and phospho-ryanodine receptor type 2 [p-RyR2] expression) in HL-1 myocytes. Furthermore, a direct association between CD44 and NOX4 was documented in tachy-paced HL-1 myocytes and atrial tissues from AF patients. In vitro, Ca2+ spark frequencies in atrial myocytes isolated from CD44-/- mice were lower than those from wild-type mice. Furthermore, administration of an anti-CD44 blocking antibody in atrial myocytes isolated from wild-type mice diminished the frequency of Ca2+ spark. Ex vivo tachypacing models of CD44-/- mice exhibited a lower degree of oxidative stress and expression of ox-CaMKII/p-RyR2 in their atria than those of wild-type mice. In vivo, burst atrial pacing stimulated a less inducibility of AF in CD44-/-mice than in wild-type mice. In conclusion, atrial tachypacing-induced Ca2+-handling abnormalities are mediated via CD44/NOX4 signaling, which provides a possible explanation for the development of AF.

Targeting transcriptional machinery to inhibit enhancer-driven gene expression in heart failure.

Pathological cardiac remodeling is induced through multiple mechanisms that include neurohumoral and biomechanical stress resulting in transcriptional alterations that ultimately become maladaptive and lead to the development of heart failure (HF). Although cardiac transcriptional remodeling is mediated by the activation of numerous signaling pathways that converge on a limited number of transcription factors (TFs) that promote hypertrophy (pro-hypertrophic TFs), the current therapeutic approach to prevent HF utilizes pharmacological inhibitors that largely target specific receptors that are activated in response to pathological stimuli. Thus, there is limited efficacy with the current pharmacological approaches to inhibit transcriptional remodeling associated with the development of HF. Recent evidence suggests that these pro-hypertrophic TFs co-localize at enhancers to cooperatively activate transcription associated with pathological cardiac remodeling. In disease states, including cancer and HF, evidence suggests that the general transcriptional machinery is disproportionately bound at enhancers. Therefore, pharmacological inhibition of transcriptional machinery that integrates pro-hypertrophic TFs may represent a promising alternative therapeutic approach to limit pathological remodeling associated with the development of HF.

The protective effect of high mobility group protein HMGA2 in pressure overload-induced cardiac remodeling.

High mobility group protein AT-hook 2 (HMGA2), an architectural transcription factor, has previously been reported to play an essential role in regulating the expression of many genes through architectural remodeling processes. However, the effects of HMGA2 on cardiovascular disease, especial cardiac remodeling, is unclear. This study was aimed at investigating the functional role of HMGA2 in pressure overload-induced cardiac remodeling. Mice that were subjected to aortic banding (AB) for 8 weeks developed myocardial hypertrophy and cardiac dysfunction, which were associated with altered expression of HMGA2. Cardiac-specific expression of the human HMGA2 gene in mice with an adeno-related virus 9 delivery system ameliorated cardiac remodeling and improve cardiac function in response to pressure overload by activating PPARγ/NRF2 signaling. Knockdown of HMGA2 by AAV9-shHMGA2 accelerated cardiac remodeling after 1 weeks of AB surgery. Additionally, knockdown of heart PPARγ largely abolished HMGA2 overexpression-mediated cardioprotection. HMGA2-mediated cardiomyocyte protection was largely abrogated by knocking down NRF2 and inhibiting PPARγ in cardiomyocytes. PPARγ activation was mediated by C/EBPß, which directly interacted with HMGA2. Knocking down C/EBPß offset the effects of HMGA2 on PPARγ activation and cardioprotection. These findings show that the overexpression of HMGA2 ameliorates the remodeling response to pressure overload, and they also imply that the upregulation of HMGA2 may become a treatment strategy in cardiac pathologies.

Activin Receptor-Like Kinase 4 Haplodeficiency Mitigates Arrhythmogenic Atrial Remodeling and Vulnerability to Atrial Fibrillation in Cardiac Pathological Hypertrophy.

Background Activin receptor-like kinase 4 ( ALK 4) is highly expressed in mammal heart. Atrial fibrillation ( AF ) is closely related to ventricular pressure overload. Because pressure overload increases atrial pressure and leads to atrial remodeling, it would be informative to know whether ALK 4 exerts potential effects on atrial remodeling and AF vulnerability in a pressure-overload model. Methods and Results Wild-type littermates and ALK 4+/- mice were subjected to abdominal aortic constriction or a sham operation. After 4 or 8 weeks, echocardiographic and hemodynamic measurements were performed, and inducibility of AF was tested. The hearts were divided into atria and ventricles and then were fixed in formalin for staining, or they were weighted and snap-frozen for quantitative real-time polymerase chain reaction and Western blot analysis. Compared with wild-type littermates, ALK 4+/- mice demonstrated a similar extent of atrial hypertrophy but significantly suppressed atrial fibrosis at 8 weeks post-abdominal aortic constriction. ALK 4 haplodeficiency partially blocked abdominal aortic constriction-induced upregulation of monocyte chemotactic protein 1 and interleukin-6, and the increased chemotaxin of macrophages. ALK 4 haplodeficiency also blunted a reduction of connexin 40 and redistribution of connexin 43 from the intercalated disk to the lateral membranes, thereby improving localized conduction abnormalities. Meanwhile, ALK 4 haplodeficiency inhibited abdominal aortic constriction-induced decreased INa, ICa-L and IK1 densities as well as the accompanying action potential duration shortening. Mechanistically, ALK 4 haploinsufficiency resulted in the suppression of Smad2/3 activity in this model. Conclusions Our results demonstrate that ALK 4 haplodeficiency ameliorates atrial remodeling and vulnerability to AF in a pressure-overload model through inactivation of the Smad2/3 pathway, suggesting that ALK 4 might be a potential therapeutic target in combating pressure overload-induced AF .

Blockade to pathological remodeling of infarcted heart tissue using a porcupine antagonist.

The secreted Wnt signaling molecules are essential to the coordination of cell-fate decision making in multicellular organisms. In adult animals, the secreted Wnt proteins are critical for tissue regeneration and frequently contribute to cancer. Small molecules that disable the Wnt acyltransferase Porcupine (Porcn) are candidate anticancer agents in clinical testing. Here we have systematically assessed the effects of the Porcn inhibitor (WNT-974) on the regeneration of several tissue types to identify potentially unwanted chemical effects that could limit the therapeutic utility of such agents. An unanticipated observation from these studies is proregenerative responses in heart muscle induced by systemic chemical suppression of Wnt signaling. Using in vitro cultures of several cell types found in the heart, we delineate the Wnt signaling apparatus supporting an antiregenerative transcriptional program that includes a subunit of the nonfibrillar collagen VI. Similar to observations seen in animals exposed to WNT-974, deletion of the collagen VI subunit, COL6A1, has been shown to decrease aberrant remodeling and fibrosis in infarcted heart tissue. We demonstrate that WNT-974 can improve the recovery of heart function after left anterior descending coronary artery ligation by mitigating adverse remodeling of infarcted tissue. Injured heart tissue exposed to WNT-974 exhibits decreased scarring and reduced Col6 production. Our findings support the development of Porcn inhibitors as antifibrotic agents that could be exploited to promote heart repair following injury.

Differential left-to-right atria gene expression ratio in human sinus rhythm and atrial fibrillation: Implications for arrhythmogenesis and thrombogenesis.

BACKGROUND: Atrial fibrillation (AF) causes atrial remodeling, and the left atrium (LA) is the favored substrate for maintaining AF. It remains unclear if AF remodels both atria differently and contributes to LA arrhythmogenesis and thrombogenesis. Therefore, we wished to characterize the transcript profiles in the LA and right atrium (RA) in sinus rhythm (SR) and AF respectively. METHODS: Paired LA and RA appendages acquired from patients receiving cardiac surgery were used for ion-channel- and whole-exome-based transcriptome analysis. The ultrastructure was evaluated by immunohistochemistry. RESULTS: Twenty-two and twenty ion-channels and transporters were differentially expressed between the LA and RA in AF and SR, respectively. Among these, 15 genes were differentially expressed in parallel between AF and SR. AF was associated with increased LA/RA expression ratio in 9 ion channel-related genes, including genes related to calcium handling. In microarray, AF was associated with a differential LA/RA gene expression ratio in 309 genes, and was involved in atherosclerosis-related signaling. AF was associated with the upregulation of thrombogenesis-related genes in the LA appendage, including P2Y12, CD 36 and ApoE. Immunohistochemistry showed higher expressions of collagen-1, oxidative stress and TGF-ß1 in the RA compared to the LA. CONCLUSIONS: AF was associated with differential LA-to-RA gene expression related to specific ion channels and pathways as well as upregulation of thrombogenesis-related genes in the LA appendage. Targeting the molecular mechanisms underlying the LA-to-RA difference and AF-related remodeling in the LA appendage may help provide new therapeutic options in treating AF and preventing thromboembolism in AF.

Regulation of cardiac microRNAs induced by aerobic exercise training during heart failure.

Exercise training (ET) has beneficial effects on the myocardium in heart failure (HF) patients and in animal models of induced cardiac hypertrophy and failure. We hypothesized that if microRNAs (miRNAs) respond to changes following cardiac stress, then myocardial profiling of these miRNAs may reveal cardio-protective mechanisms of aerobic ET in HF. We used ascending aortic stenosis (AS) inducing HF in Wistar rats. Controls were sham-operated animals. At 18 wk after surgery, rats with cardiac dysfunction were randomized to 10 wk of aerobic ET (HF-ET) or to a heart failure sedentary group (HF-S). ET attenuated cardiac remodeling as well as clinical and pathological signs of HF with maintenance of systolic and diastolic function when compared with that of the HF-S. Global miRNA expression profiling of the cardiac tissue revealed 53 miRNAs exclusively dysregulated in animals in the HF-ET, but only 11 miRNAs were exclusively dysregulated in the HF-S. Out of 23 miRNAs that were differentially regulated in both groups, 17 miRNAs exhibited particularly high increases in expression, including miR-598, miR-429, miR-224, miR-425, and miR-221. From the initial set of deregulated miRNAs, 14 miRNAs with validated targets expressed in cardiac tissue that respond robustly to ET in HF were used to construct miRNA-mRNA regulatory networks that revealed a set of 203 miRNA-target genes involved in programmed cell death, TGF-ß signaling, cellular metabolic processes, cytokine signaling, and cell morphogenesis. Our findings reveal that ET attenuates cardiac abnormalities during HF by regulating cardiac miRNAs with a potential role in cardio-protective mechanisms through multiple effects on gene expression.

Feedback mechanisms for cardiac-specific microRNAs and cAMP signaling in electrical remodeling.

BACKGROUND: Loss of transient outward K(+) current (Ito) is well documented in cardiac hypertrophy and failure both in animal models and in humans. Electrical remodeling contributes to prolonged action potential duration and increased incidence of arrhythmias. Furthermore, there is a growing body of evidence linking microRNA (miR) dysregulation to the progression of both conditions. In this study, we examined the mechanistic basis underlying miR dysregulation in electrical remodeling and revealed a novel interaction with the adrenergic signaling pathway. METHODS AND RESULTS: We first used a tissue-specific knockout model of Dicer1 in cardiomyocytes to reveal the overall regulatory effect of miRs on the ionic currents and action potentials. We then validated the inducible cAMP early repressor as a target of miR-1 and took advantage of a clinically relevant model of post myocardial infarction and miR delivery to probe the mechanistic basis of miR dysregulation in electrical remodeling. These experiments revealed the role of inducible cAMP early repressor as a repressor of miR-1 and Ito, leading to prolonged action potential duration post myocardial infarction. In addition, delivery of miR-1 and miR-133a suppressed inducible cAMP early repressor expression and prevented both electrical remodeling and hypertrophy. CONCLUSIONS: Taken together, our results illuminate the mechanistic links between miRs, adrenergic signaling, and electrical remodeling. They also serve as a proof-of-concept for the therapeutic potential of miR delivery post myocardial infarction.

Investigating inherent functional differences between human cardiac fibroblasts cultured from nondiabetic and Type 2 diabetic donors.

INTRODUCTION: Type 2 diabetes mellitus (T2DM) promotes adverse myocardial remodeling and increased risk of heart failure; effects that can occur independently of hypertension or coronary artery disease. As cardiac fibroblasts (CFs) are key effectors of myocardial remodeling, we investigated whether inherent phenotypic differences exist in CF derived from T2DM donors compared with cells from nondiabetic (ND) donors. METHODS: Cell morphology (cell area), proliferation (cell counting over 7-day period), insulin signaling [phospho-Akt and phospho-extracellular signal-regulated kinase (ERK) Western blotting], and mRNA expression of key remodeling genes [real-time reverse transcription-polymerase chain reaction (RT-PCR)] were compared in CF cultured from atrial tissue from 14 ND and 12 T2DM donors undergoing elective coronary artery bypass surgery. RESULTS: The major finding was that Type I collagen (COL1A1) mRNA levels were significantly elevated by twofold in cells derived from T2DM donors compared with those from ND donors; changes reflected at the protein level. T2DM cells had similar proliferation rates but a greater variation in cell size and a trend towards increased cell area compared with ND cells. Insulin-induced Akt and ERK phosphorylation were similar in the two cohorts of cells. CONCLUSION: CF from T2DM individuals possess an inherent profibrotic phenotype that may help to explain the augmented cardiac fibrosis observed in diabetic patients. MINI SUMMARY: We investigated whether inherent phenotypic differences exist between CF cultured from donors with or without Type 2 diabetes. Cell morphology, proliferation, insulin signaling, and gene expression were compared between multiple cell populations. The major finding was that Type I collagen levels were elevated in fibroblasts from diabetic donors, which may help explain the augmented cardiac fibrosis observed with diabetes.

Modulation of microRNAs in hypertension-induced arterial remodeling through the ß1 and ß3-adrenoreceptor pathways.

BACKGROUND: Dysregulation of microRNAs (miRNAs) in arterial dysfunction and hypertension has not been extensively investigated yet. This project determined the effects of two anti-hypertensive ß1 adrenergic selective blockers on miRNA expression in the Dahl Salt Sensitive (DSS) hypertensive rat model. METHODS AND RESULTS: Microarray analysis showed that a set of miRNAs is differently expressed in the aorta of high salt (HS) treated rats with miR-320 increased and miR-26b and -21 decreased. All of these changes were reverted to normal by nebivolol (NEB, a ß1 selective-blocker and ß3 activator). The selective ß3-adrenoceptor antagonist S-(-)-cyanopindolol (Syc) counteracted the effect of NEB on these miRNAs. Atenolol (ATN, a pure ß1-blocker) combined with specific ß3 agonist BRL37344 restored the expression of all three miRNAs, similar to NEB, while ATN alone had only a partial effect on miR-320 expression. Computational analysis found Insulin Growth Factor-1 Receptor (IGF1R) as a putative target of miR-320, and Phosphatase and tensin homolog on chromosome ten (PTEN) as a putative target of miR-26b and -21. The targets were verified by luciferase reporter assays. Inhibition of miR-320 by an antisense inhibitor or NEB increased IGF1R expression, while miR-320 overexpression reversed the effect of NEB. Overexpression of miR-26b or -21 or NEB decreased PTEN levels, while inhibition of miR-26b or -21 attenuated the effect of NEB. HS diet induced downregulation of IGF1R and upregulation of PTEN in the aorta. NEB normalized the aberrant expression of IGF1R and PTEN and also improved the impairment of vascular AKT/eNOS signaling. Moreover, both NEB and ATN showed to have protective effects on salt-induced hypertension, oxidative stress, and vascular remodeling. NEB had a greater effect than ATN. CONCLUSIONS: Our data supports a differential miRNA expression profile in salt-induced hypertension. Manipulation of dysregulated miRNAs by ß-blockers may substantially induce alterations of gene expression and prevent arterial dysfunction and remodeling.