J. Francisco Nistal

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.

The CarboMedics Valve: Experience With 1,049 Implants

BACKGROUND The lack of valve rotatability, the structural deterioration, and the rate of valve-related complications with the standard mechanical bileaflet prosthesis led to the development of a new second-generation bileaflet valve in 1986. METHODS Between January 1989 and March 1994, 1,049 CarboMedics valves were implanted in 859 patients. The rotatability was used in 109 mitral prostheses (21.5%) and in 61 aortic prostheses (11.6%). Follow-up was 97.1% complete, with 3,049 patient-years. RESULTS The hospital mortality was 6.9% for the mitral group, 3.4% for the aortic group, and 10.7% for the double-valve group (p < 0.005). The actuarial survival curve at 5 years was 77.3% +/- 3.6%, 90.1% +/- 2.5%, and 79.2% +/- 3.7% (p = 0.0003), freedom from thromboembolism was 89.1% +/- 3.6%, 87.1% +/- 3.8%, and 68.8% +/- 8.2%, freedom from reoperation was 95.9% +/- 1.4%, 98.9% +/- 0.6%, and 94.9% +/- 2.4%, and freedom from valve-related complications was 68.8% +/- 4.1%, 79.5% +/- 3.5%, and 55.3% +/- 5.9% after mitral, aortic, and mitral and aortic valve replacement, respectively. There were five episodes of valve thrombosis, but no structural deterioration occurred. CONCLUSIONS The clinical performance of the CarboMedics valve is quite satisfactory, with a low incidence of valve-related mortality and morbidity. The rotatability feature was useful when the native valve was preserved or for repeat valve replacement.

Plasma Levels of Transforming Growth Factor-β1 Reflect Left Ventricular Remodeling in Aortic Stenosis

Background TGF-β1 is involved in cardiac remodeling through an auto/paracrine mechanism. The contribution of TGF-β1 from plasmatic source to pressure overload myocardial remodeling has not been analyzed. We investigated, in patients with valvular aortic stenosis (AS), and in mice subjected to transverse aortic arch constriction (TAC), whether plasma TGF-β1 relates with myocardial remodeling, reflected by LV transcriptional adaptations of genes linked to myocardial hypertrophy and fibrosis, and by heart morphology and function. Methodology/Principal Findings The subjects of the study were: 39 patients operated of AS; 27 healthy volunteers; 12 mice subjected to TAC; and 6 mice sham-operated. Myocardial samples were subjected to quantitative PCR. Plasma TGF-β1 was determined by ELISA. Under pressure overload, TGF-β1 plasma levels were significantly increased both in AS patients and TAC mice. In AS patients, plasma TGF-β1 correlated directly with aortic transvalvular gradients and LV mass surrogate variables, both preoperatively and 1 year after surgery. Plasma TGF-β1 correlated positively with the myocardial expression of genes encoding extracellular matrix (collagens I and III, fibronectin) and sarcomeric (myosin light chain-2, β-myosin heavy chain) remodelling targets of TGF-β1, in TAC mice and in AS patients. Conclusions/Significance A circulating TGF-β1-mediated mechanism is involved, in both mice and humans, in the excessive deposition of ECM elements and hypertrophic growth of cardiomyocytes under pressure overload. The possible value of plasma TGF-β1 as a marker reflecting preoperative myocardial remodeling status in AS patients deserves further analysis in larger patient cohorts.

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.

Nitric oxide mediates aortic disease in mice deficient in the metalloprotease Adamts1 and in a mouse model of Marfan syndrome

Heritable thoracic aortic aneurysms and dissections (TAAD), including Marfan syndrome (MFS), currently lack a cure, and causative mutations have been identified for only a fraction of affected families. Here we identify the metalloproteinase ADAMTS1 and inducible nitric oxide synthase (NOS2) as therapeutic targets in individuals with TAAD. We show that Adamts1 is a major mediator of vascular homeostasis, given that genetic haploinsufficiency of Adamts1 in mice causes TAAD similar to MFS. Aortic nitric oxide and Nos2 levels were higher in Adamts1-deficient mice and in a mouse model of MFS (hereafter referred to as MFS mice), and Nos2 inactivation protected both types of mice from aortic pathology. Pharmacological inhibition of Nos2 rapidly reversed aortic dilation and medial degeneration in young Adamts1-deficient mice and in young or old MFS mice. Patients with MFS showed elevated NOS2 and decreased ADAMTS1 protein levels in the aorta. These findings uncover a possible causative role for the ADAMTS1–NOS2 axis in human TAAD and warrant evaluation of NOS2 inhibitors for therapy.

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.

Isolated non-compaction of the left ventricular myocardium in an adult treated with heart transplantation

Isolated left ventricular non‐compaction is a rare unclassified cardiomyopathy characterized by arrest of normal embryogenesis that results in the persistence of intertrabecular recesses and the development of a spongy myocardium. The pathological diagnosis is often first established at autopsy. Described herein is the case of a 57‐year‐old man with isolated non‐compaction of the left ventricle who had a 17 year history of worsening heart failure and was successfully treated with heart transplantation. To the authors’ knowledge only seven adult patients with non‐compaction cardiomyopathy have been reported to have undergone heart transplantation. Including the present case the mean age of the adult patients at transplantation was 39.5 years (range, 18–60 years). The male : female ratio was 3:1. Both ventricles were involved in 37.5% of cases. The mean follow up was 6.3 years (range, 2 months−17 years). One patient died because of a malignant tumor 9 years after transplantation. The morphological pattern of the isolated ventricular non‐compaction represents a pathological entity discernible from other cardiomyopathies and should be classified as a specific cardiomyopathy. It is likely that surgical pathologists will encounter this entity more frequently due to involvement in transplantation teams.