Raquel García

Myocardial and circulating levels of microRNA-21 reflect left ventricular fibrosis in aortic stenosis patients

BACKGROUND Various human cardiovascular pathophysiological conditions associate aberrant expression of microRNAs (miRNAs) and circulating miRNAs are emerging as promising biomarkers. In mice, myocardial miR-21 overexpression is related to cardiac fibrosis elicited by pressure overload. This study was designed to determine the role of myocardial and plasmatic miR-21 in the maladaptive remodeling of the extracellular matrix induced by pressure overload in aortic stenosis (AS) patients and the clinical value of miR-21 as a biomarker for pathological myocardial fibrosis. METHODS In left ventricular biopsies from 75 AS patients and 32 surgical controls, we quantified the myocardial transcript levels of miR-21, miR-21-targets and ECM- and TGF-β-signaling-related elements. miR-21 plasma levels were determined in 25 healthy volunteers and in AS patients. In situ hybridization of miR-21 was performed in myocardial sections. RESULTS The myocardial and plasma levels of miR-21 were significantly higher in the AS patients compared with the controls and correlated directly with the echocardiographic mean transvalvular gradients. miR-21 overexpression was confined to interstitial cells and absent in cardiomyocytes. Using bootstrap validated multiple linear regression, the variance in myocardial collagen expression was predicted by myocardial miR-21 (70% of collagen variance) or plasma miR-21 (52% of collagen variance), together with the miR-21 targets RECK and PDCD4, and effectors of TGF-ß signaling. CONCLUSIONS Our results support the role of miR-21 as a regulator of the fibrotic process that occurs in response to pressure overload in AS patients and underscore the value of circulating miR-21 as a biomarker for myocardial fibrosis.

BAMBI (BMP and activin membrane-bound inhibitor) protects the murine heart from pressure-overload biomechanical stress by restraining TGF-β signaling

Left ventricular (LV) pressure overload is a major cause of heart failure. Transforming growth factors-β (TGF-βs) promote LV remodeling under biomechanical stress. BAMBI (BMP and activin membrane-bound inhibitor) is a pseudoreceptor that negatively modulates TGF-β signaling. The present study tests the hypothesis that BAMBI plays a protective role during the adverse LV remodeling under pressure overload. The subjects of the study were BAMBI knockout mice (BAMBI(-/-)) undergoing transverse aortic constriction (TAC) and patients with severe aortic stenosis (AS). We examined LV gene and protein expression of remodeling-related elements, histological fibrosis, and heart morphology and function. LV expression of BAMBI was increased in AS patients and TAC-mice and correlated directly with TGF-β. BAMBI deletion led to a gain of myocardial TGF-β signaling through canonical (Smads) and non-canonical (TAK1-p38 and TAK1-JNK) pathways. As a consequence, the remodeling response to pressure overload in BAMBI(-/-) mice was exacerbated in terms of hypertrophy, chamber dilation, deterioration of long-axis LV systolic function and diastolic dysfunction. Functional remodeling associated transcriptional activation of fibrosis-related TGF-β targets, up-regulation of the profibrotic micro-RNA-21, histological fibrosis and increased metalloproteinase-2 activity. Histological remodeling in BAMBI(-/-) mice involved TGF-βs. BAMBI deletion in primary cardiac fibroblasts exacerbated TGF-β-induced profibrotic responses while BAMBI overexpression in NIH-3T3 fibroblasts attenuated them. Our findings identify BAMBI as a critical negative modulator of myocardial remodeling under pressure overload. We suggest that BAMBI is involved in negative feedback loops that restrain the TGF-β remodeling signals to protect the pressure-overloaded myocardium from uncontrolled extracellular matrix deposition in humans and mice.

Myocardial gene expression of microRNA-133a and myosin heavy and light chains, in conjunction with clinical parameters, predict regression of left ventricular hypertrophy after valve replacement in patients with aortic stenosis

Background Left ventricular (LV) reverse remodelling after valve replacement in aortic stenosis (AS) has been classically linked to the hydraulic performance of the replacement device, but myocardial status at the time of surgery has received little attention. Objective To establish predictors of LV mass (LVM) regression 1 year after valve replacement in a surgical cohort of patients with AS based on preoperative clinical and echocardiographic parameters and the myocardial gene expression profile at surgery. Methods Transcript levels of remodelling-related proteins and regulators were determined in LV intraoperative biopsies from 46 patients with AS by RT-PCR. Using multiple linear regression analysis, an equation was developed (adjusted R2=0.73; p<0.0001) that included positive [preoperative LVM, microRNA-133a, serum response factor (SRF, which is known to be a transactivator of miR-133) and age] and negative [body mass index (BMI), Wolf-Hirschhorn syndrome candidate-2 (WHSC2, which is a target for repression by miR-133a), β-myosin heavy chain, myosin light chain-2, diabetes mellitus, and male gender] independent predictors of LVM reduction. Results Aortic valve area gain or the reduction in transvalvular gradient maintained no significant relationships with the dependent variable. Logistic regression analysis identified microRNA-133a as a significant positive predictor of LVM normalisation, whereas β-myosin heavy chain and BMI constituted negative predictors. Conclusions Hypertrophy regression 1 year after pressure overload release is related to the preoperative myocardial expression of remodelling-related genes, in conjunction with the patients clinical background. In this scenario, miR-133 emerges as a key element of the reverse remodelling process. Postoperative improvement of valve haemodynamics does not predict the degree of hypertrophy regression or LVM normalisation. These results led us to reconsider the current reverse remodelling paradigm and (1) to include criteria of hypertrophy reversibility in the decision algorithm used to decide timing for the operation; and (2) to modify other prevailing factors (overweight, diabetes, etc) known to maintain LV hypertrophy.

Androgens Contribute to Sex Differences in Myocardial Remodeling under Pressure Overload by a Mechanism Involving TGF-β

Background In clinical studies, myocardial remodeling in aortic valve stenosis appears to be more favorable in women than in men, even after menopause. In the present study, we assessed whether circulating androgens contribute to a less favorable myocardial remodeling under pressure overload in males. We examined sex-related differences in one-year-old male and female mice. Whereas male mice at this age exhibited circulating androgen levels within the normal range for young adults, the circulating estrogens in females were reduced. The contribution of gonadal androgens to cardiac remodeling was analyzed in a group of same-age castrated mice. Methodology/Principal Findings Animals were subjected to transverse aortic constriction (TAC). Echocardiography was performed 2 weeks after TAC and myocardial mRNA levels of TGF-βs, Smads 2 and 3, collagens, fibronectin, β-myosin heavy chain and α-myosin heavy chain were determined by q-PCR. Protein detection of p-SMAD2/3 was performed by Western Blot. Histological staining of fibrosis was performed with picrosirius red and Massons trichrome. Compared with females, males developed more severe tissue fibrosis, LV dilation and hemodynamic dysfunction. TAC-males showed higher myocardial expression levels of TGF-βs and the treatment with a neutralizing antibody to TGF-β prevented myocardial fibrosis development. Orchiectomy diminished TAC-induced up-regulation of TGF-βs and TGF-β target genes, and it also reduced fibrosis and hemodynamic dysfunction. The capability of androgens to induce TGF-β expression was confirmed in NIH-3T3 fibroblasts and H9C2 cardiomyocytes exposed to dihydrotestosterone. Conclusions/Significance Our results indicate that circulating androgens are responsible for the detrimental effects in the myocardium of older male mice subjected to pressure overload through a mechanism involving TGF-βs.

Circulating Levels of miR‐133a Predict the Regression Potential of Left Ventricular Hypertrophy After Valve Replacement Surgery in Patients With Aortic Stenosis

Background Myocardial microRNA‐133a (miR‐133a) is directly related to reverse remodeling after pressure overload release in aortic stenosis patients. Herein, we assessed the significance of plasma miR‐133a as an accessible biomarker with prognostic value in predicting the reversibility potential of LV hypertrophy after aortic valve replacement (AVR) in these patients. Methods and Results The expressions of miR‐133a and its targets were measured in LV biopsies from 74 aortic stenosis patients. Circulating miR‐133a was measured in peripheral and coronary sinus blood. LV mass reduction was determined echocardiographically. Myocardial and plasma levels of miR‐133a correlated directly (r=0.46, P<0.001) supporting the myocardium as a relevant source of plasma miR‐133a. Accordingly, a significant gradient of miR‐133a was found between coronary and systemic venous blood. The preoperative plasma level of miR‐133a was higher in the patients who normalized LV mass 1 year after AVR than in those exhibiting residual hypertrophy. Logistic regression analysis identified plasma miR‐133a as a positive predictor of the hypertrophy reversibility after surgery. The discrimination of the model yielded an area under the receiver operator characteristic curve of 0.89 (P<0.001). Multiple linear regression analysis revealed plasma miR‐133a and its myocardial target Wolf‐Hirschhorn syndrome candidate 2/Negative elongation factor A as opposite predictors of the LV mass loss (g) after AVR. Conclusions Preoperative plasma levels of miR‐133a reflect their myocardial expression and predict the regression potential of LV hypertrophy after AVR. The value of this bedside information for the surgical timing, particularly in asymptomatic aortic stenosis patients, deserves confirmation in further clinical studies.

p-SMAD2/3 and DICER promote pre-miR-21 processing during pressure overload-associated myocardial remodeling.

Transforming growth factor-β (TGF-β) induces miR-21 expression which contributes to fibrotic events in the left ventricle (LV) under pressure overload. SMAD effectors of TGF-β signaling interact with DROSHA to promote primary miR-21 processing into precursor miR-21 (pre-miR-21). We hypothesize that p-SMAD-2 and -3 also interact with DICER1 to regulate the processing of pre-miR-21 to mature miR-21 in cardiac fibroblasts under experimental and clinical pressure overload. The subjects of the study were mice undergoing transverse aortic constriction (TAC) and patients with aortic stenosis (AS). In vitro, NIH-3T3 fibroblasts transfected with pre-miR-21 responded to TGF-β1 stimulation by overexpressing miR-21. Overexpression and silencing of SMAD2/3 resulted in higher and lower production of mature miR-21, respectively. DICER1 co-precipitated along with SMAD2/3 and both proteins were up-regulated in the LV from TAC-mice. Pre-miR-21 was isolated bound to the DICER1 maturation complex. Immunofluorescence analysis revealed co-localization of p-SMAD2/3 and DICER1 in NIH-3T3 and mouse cardiac fibroblasts. DICER1-p-SMAD2/3 protein-protein interaction was confirmed by in situ proximity ligation assay. Myocardial up-regulation of DICER1 constituted a response to pressure overload in TAC-mice. DICER mRNA levels correlated directly with those of TGF-β1, SMAD2 and SMAD3. In the LV from AS patients, DICER mRNA was up-regulated and its transcript levels correlated directly with TGF-β1, SMAD2, and SMAD3. Our results support that p-SMAD2/3 interacts with DICER1 to promote pre-miR-21 processing to mature miR-21. This new TGFβ-dependent regulatory mechanism is involved in miR-21 overexpression in cultured fibroblasts, and in the pressure overloaded LV of mice and human patients.

Extracellular heat shock protein 90 binding to TGFβ receptor I participates in TGFβ-mediated collagen production in myocardial fibroblasts

The pathological remodeling heart shows an increase in left ventricular mass and an excess of extracellular matrix deposition that can over time cause heart failure. Transforming growth factor β (TGFβ) is the main cytokine controlling this process. The molecular chaperone heat shock protein 90 (Hsp90) has been shown to play a critical role in TGFβ signaling by stabilizing the TGFβ signaling cascade. We detected extracellular Hsp90 in complex with TGFβ receptor I (TGFβRI) in fibroblasts and determined a close proximity between both proteins suggesting a potential physical interaction between the two at the plasma membrane. This was supported by in silico studies predicting Hsp90 dimers and TGFβRI extracellular domain interaction. Both, Hsp90aa1 and Hsp90ab1 isoforms participate in TGFβRI complex. Extracellular Hsp90 inhibition lessened the yield of collagen production as well as the canonical TGFβ signaling cascade, and collagen protein synthesis was drastically reduced in Hsp90aa1 KO mice. These observations together with the significant increase in activity of Hsp90 at the plasma membrane pointed to a functional cooperative partnership between Hsp90 and TGFβRI in the fibrotic process. We propose that a surface population of Hsp90 extracellularly binds TGFβRI and this complex behaves as an active participant in collagen production in TGFβ-activated fibroblasts. We also offer an in vivo insight into the role of Hsp90 and its isoforms during cardiac remodeling in murine aortic banding model suffering from pathological cardiac remodeling and detect circulating Hsp90 overexpressed in remodeling mice.

BMP-7 attenuates left ventricular remodelling under pressure overload and facilitates reverse remodelling and functional recovery

AIMS TGF-β regulates tissue fibrosis: TGF-β promotes fibrosis, whereas bone morphogenetic protein (BMP)-7 is antifibrotic. To demonstrate that (i) left ventricular (LV) remodelling after pressure overload is associated with disequilibrium in the signalling mediated by these cytokines, and (ii) BMP-7 exerts beneficial effects on LV remodelling and reverse remodelling. METHODS AND RESULTS We studied patients with aortic stenosis (AS) and mice subjected to transverse aortic constriction (TAC) and TAC release (de-TAC). LV morphology and function were assessed by echocardiography. LV biopsies were analysed by qPCR, immunoblotting, and histology. Pressure overload reduced BMP-7 and pSmad1/5/8 and increased TGF-β and pSmad2/3 in AS patients and TAC mice. BMP-7 correlated inversely with collagen, fibronectin, and β-MHC expressions, and with hypertrophy and diastolic dysfunction, and directly with the systolic function. Multiple linear regression disclosed BMP-7 and TGF-β as hypertrophy predictors, negative and positive, respectively. BMP-7 prevented TGF-β-elicited hypertrophic program in cardiomyocytes, and Col1A1 promoter activity in NIH-3T3 fibroblasts. The treatment of TAC mice with rBMP-7 attenuated the development of structural damage and dysfunction, and halted ongoing remodelling. The reverse remodelling after pressure overload release was facilitated by rBMP-7, and hampered by disrupting BMP-7 function using a neutralizing antibody or genetic deletion. CONCLUSION The disequilibrium between BMP-7 and TGF-β signals plays a relevant role in the LV remodelling response to haemodynamic stress in TAC mice and AS patients. Our observations may provide new important insights aimed at developing novel therapies designed to prevent, halt, or reverse LV pathological remodelling in pressure overload cardiomyopathy.

Experimental modelling of cardiac pressure overload hypertrophy: Modified technique for precise, reproducible, safe and easy aortic arch banding-debanding in mice

Pressure overload left ventricular hypertrophy is a known precursor of heart failure with ominous prognosis. The development of experimental models that reproduce this phenomenon is instrumental for the advancement in our understanding of its pathophysiology. The gold standard of these models is the controlled constriction of the mid aortic arch in mice according to Rockman’s technique (RT). We developed a modified technique that allows individualized and fully controlled constriction of the aorta, improves efficiency and generates a reproducible stenosis that is technically easy to perform and release. An algorithm calculates, based on the echocardiographic arch diameter, the intended perimeter at the constriction, and a suture is prepared with two knots separated accordingly. The aorta is encircled twice with the suture and the loop is closed with a microclip under both knots. We performed controlled aortic constriction with Rockman’s and the double loop-clip (DLC) techniques in mice. DLC proved superiority in efficiency (mortality and invalid experiments) and more homogeneity of the results (transcoarctational gradients, LV mass, cardiomyocyte hypertrophy, gene expression) than RT. DLC technique optimizes animal use and generates a consistent and customized aortic constriction with homogeneous LV pressure overload morphofunctional, structural, and molecular features.

MicroRNA-30c-5p modulates neuropathic pain in rodents

miR-30c-5p expression modulates pain in rodent models of nerve injury and is overexpressed in patients with neuropathic pain due to leg ischemia. Targeting a microRNA for neuropathic pain Neuropathic pain is a debilitating condition resulting from nerve damage. Often, patients do not achieve pain relief with currently available therapies. A better understanding of the mechanisms mediating neuropathic pain could help to identify more effective therapies. Now, Tramullas et al. show that neuropathic pain in rodents was associated with increased expression of the microRNA miR-30c-5p in the brain, cerebrospinal fluid, and plasma. In addition, miR-30c-5p was up-regulated in plasma and cerebrospinal fluid from patients with peripheral ischemia–induced pain. Inhibiting this microRNA in the rodent model produced analgesic effects. The results suggest that targeting miR-30c-5p might be an effective strategy for treating neuropathic pain. Neuropathic pain is a debilitating chronic syndrome that is often refractory to currently available analgesics. Aberrant expression of several microRNAs (miRNAs) in nociception-related neural structures is associated with neuropathic pain in rodent models. We have exploited the antiallodynic phenotype of mice lacking the bone morphogenetic protein and activin membrane-bound inhibitor (BAMBI), a transforming growth factor–β (TGF-β) pseudoreceptor. We used these mice to identify new miRNAs that might be useful for diagnosing, treating, or predicting neuropathic pain. We show that, after sciatic nerve injury in rats, miR-30c-5p was up-regulated in the spinal cord, dorsal root ganglia, cerebrospinal fluid (CSF) and plasma and that the expression of miR-30c-5p positively correlated with the severity of allodynia. The administration of a miR-30c-5p inhibitor into the cisterna magna of the brain delayed neuropathic pain development and reversed fully established allodynia in rodents. The mechanism was mediated by TGF-β and involved the endogenous opioid system. In patients with neuropathic pain associated with leg ischemia, the expression of miR-30c-5p was increased in plasma and CSF compared to control patients without pain. Logistic regression analysis in our cohort of patients showed that the expression of miR-30c-5p in plasma and CSF, in combination with other clinical variables, might be useful to help to predict neuropathic pain occurrence in patients with chronic peripheral ischemia.