Ubiquitin Pathway Is Associated with Worsening Left Ventricle Function after Mitral Valve Repair: A Global Gene Expression Study.

The molecular mechanism for worsening left ventricular (LV) function after mitral valve (MV) repair for chronic mitral regurgitation remains unknown. We wished to assess the LV transcriptome and identify determinants associated with worsening LV function post-MV repair. A total of 13 patients who underwent MV repair for chronic primary mitral regurgitation were divided into two groups, preserved LV function (N = 8) and worsening LV function (N = 5), for the study. Specimens of LV from the patients taken during surgery were used for the gene microarray study. Cardiomyocyte cell line HL-1 cells were transfected with gene-containing plasmids and further evaluated for mRNA and protein expression, apoptosis, and contractile protein degradation. Of 67,258 expressed sequence tags, microarrays identified 718 genes to be differentially expressed between preserved-LVF and worsening-LVF, including genes related to the protein ubiquitination pathway, bone morphogenetic protein (BMP) receptors, and regulation of eIF4 and p70S6K signaling. In addition, worsening-LVF was associated with altered expressions of genes pathologically relevant to heart failure, such asdownregulated apelin receptors and upregulated peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PPARGC1A). HL-1 cardiomyocyte cells transfected with ubiquitination-related genes demonstrated activation of the protein ubiquitination pathwaywith an increase in the ubiquitin activating enzyme E1 (UAE-E1). It also led to increased apoptosis, downregulated and ubiquitinated X-linked inhibitor of apoptosis protein (XIAP), and reduced cell viability. Overexpression of ubiquitination-related genes also resulted in degradation and increased ubiquitination of α-smooth muscle actin (SMA). In conclusion, worsening-LVF presented differential gene expression profiles from preserved-LVF after MV repair. Upregulation of protein ubiquitination-related genes associated with worsening-LVF after MV repair may exert adverse effects on LV through increased apoptosis and contractile protein degradation.

Regulation of left atrial fibrosis induced by mitral regurgitation by SIRT1.

SIRT1 (silent information regulator 1) is a histone deacetylase. It can sense the energy level in cells and delay cell senescence, leading to resistance to external stress and improving metabolism. Mitral regurgitation (MR) is a common disease in cardiac surgery. However, there are no previous studies on SIRT1 and left atrial fibrosis caused by MR. In this study, we aimed to explore the regulatory effect of SIRT1 on left atrial fibrosis induced by MR. We used Guizhou miniature pigs to establish an MR model and a sham operation model after anaesthesia induction and respiratory intubation, and these model animals were followed for 30 months after the surgery. The differential distribution and expression of SIRT1 and collagen I in the left atrium was determined by immunofluorescence and Western blotting. Furthermore, we treated NIH3T3 fibroblasts (CFs) with resveratrol and Angiotensin II (Ang II) to analyse the specific mechanism involved in the development of myocardial fibrosis. The results showed that the MR model was successfully constructed. There were 8 pigs in the MR group and 6 pigs in the control group. In both the animal experiments and the cell experiments, the expression of collagen I in the MR group was increased significantly compared to that in the control group, while the expression of SIRT1 was decreased.

Antioxidative enzyme activity and total antioxidant capacity in serum of dogs with degenerative mitral valve disease.

This study was designed to evaluate the antioxidative status of serum by measuring its total antioxidant capacity, as well as the antioxidant enzyme activity (superoxide dismutase, catalase, and glutathione reductase), in dogs with various stages of degenerative mitral valve disease (DMVD) compared to healthy controls. In total, 71 client-owned dogs in different stages of DMVD, which included healthy controls, took part in the study. Following an anamnesis, clinical examination, standard transthoracic echocardiograpic examination, chest X-ray, complete blood (cell) count, and serum biochemistry, dogs were divided into 2 study groups. Blood was drawn from each dog once at the time of presentation and selected antioxidant parameters were measured using commercially available assay kits. The activity of superoxide dismutase gradually decreased in the more advanced stages of DMVD, while the activity of catalase was significantly higher in the group of dogs with asymptomatic DMVD compared to healthy controls and dogs with symptomatic DMVD. No significant changes were noted in total antioxidant capacity and the activity of glutathione reductase. Results suggested that DMVD has a significant impact on the activity of superoxide dismutase and catalase in the serum of the tested dogs. Knowledge of changes in the activity of antioxidative enzymes may warrant further studies, possibly to evaluate the potential role of compounds with antioxidative properties in the clinical outcome of dogs with DMVD.

La présente étude a été conçue afin d'évaluer le statut antioxydant du sérum en mesurant sa capacité antioxydante totale, ainsi que l'activité antioxydante enzymatique (superoxyde dismutase, catalase, et glutathion réductase), chez des chiens avec des degrés divers de maladie dégénérative de la valvule mitrale (DMVD) comparativement à des témoins en santé. Au total, 71 chiens appartenant à des clients à différents stades de DMVD, qui incluaient des témoins en santé, ont pris part à cette étude. À la suite de la prise d'anamnèse, d'un examen clinique, d'un examen échocardiographie transthoracique standard, de radiographie thoracique, d'un comptage cellulaire sanguin complet, et d'analyse biochimique sérique, les chiens étaient séparés en deux groupes d'étude. Du sang fut prélevé de chaque chien une fois au moment de la présentation et les paramètres antioxydants sélectionnés furent mesurés à l'aide d'une trousse disponible commercialement. L'activité de la superoxyde dismutase diminuait graduellement dans les stades plus avancés de DMVD, alors que l'activité de la catalase était significativement plus élevée dans le groupe de chiens avec une DMVD asymptomatique comparativement aux témoins en santé et aux chiens avec une DMVD symptomatique. Aucun changement significatif n'était noté dans la capacité antioxydante totale et dans l'activité de la glutathion réductase. Les résultats suggèrent que la DMVD a un impact significatif sur l'activité de la superoxyde dismutase, et de la catalase dans le sérum des chiens testés. Des connaissances sur les changements dans l'activité des enzymes antioxydantes pourraient justifier des études additionnelles, possiblement pour évaluer le rôle potentiel de produits avec des propriétés antioxydantes dans le devenir clinique de chiens avec DMVD.(Traduit par Docteur Serge Messier).

Correlation between NT-proBNP and lipase levels according to the severity of chronic mitral valve disease in dogs.

Chronic mitral valve disease (CMVD) is the most common cardiovascular disease in dogs, causing decreased cardiac output that results in poor tissue perfusion and tissue damage to kidneys, pancreas, and other organs. The purpose of this study was to evaluate the relationships between heart disease severity and N-terminal pro B-type natriuretic peptide (NT-proBNP) and lipase in dogs with CMVD, as well as to evaluate longitudinal changes in these values. A total of 84 dogs participated in this 2015 to 2017 study. Serum values of NT-proBNP and lipase were analyzed; radiography was used to measure the vertebral heart score and assess various echocardiographic values. NT-proBNP showed a strong positive correlation with increasing stage of heart disease; lipase showed a mild positive correlation with heart disease stage. When the three values (NT-proBNP, lipase and month) were continuously measured at 6-month intervals, all showed a correlation with the increasing length of the disease.

Temporal changes in myocardial collagen, matrix metalloproteinases, and their tissue inhibitors in the left ventricular myocardium in experimental chronic mitral regurgitation in rodents.

Mitral regurgitation (MR) imposes left ventricular volume overload, triggering rapid ventricular dilatation, increased myocardial compliance, and, ultimately, cardiac dysfunction. Breakdown of the extracellular matrix has been hypothesized to drive these rapid changes, partially from an imbalance in the matrix metalloproteinases (MMPs) and their tissue inhibitors [tissue inhibitors of metalloproteinase (TIMPs)]. In the present study, we developed a rat model of severe MR that mimics the human condition and investigated the temporal changes in extracellular matrix-related genes, collagen biosynthesis proteins, and proteolytic enzymes over a 20-wk period. Male Sprague-Dawley rats were anesthetized to a surgical plane with mechanical ventilation, and a thoracotomy was performed to expose the apex. Using transesophageal ultrasound guidance, a needle was inserted into the beating heart to perforate the anterior mitral leaflet and create severe MR. Animals were survived for 20 wk, with some animals terminated at 2, 10, and 20 wk for analysis of left ventricular tissue. A sham group that underwent the same surgery without mitral leaflet perforation and MR were used as controls. At 2 wk post-MR, increased collagen gene expression was measured, but protein levels of collagen did not corroborate this finding. In parallel, MMP-1-to-TIMP-4, MMP-2-to-TIMP-1, and MMP-2-to-TIMP-3 ratios were significantly elevated, indicating a proteolytic milieu in the myocardium, possibly causing collagen degradation. By 20 wk, many of the initial differences seen in the proteolytic ratios were not observed, with an increase in collagen compared with the 2-wk time point. Altogether, this data indicates that an imbalance in the MMP-to-TIMP ratio may occur early and potentially contribute to the early dilatation and compliance observed structurally. NEW & NOTEWORTHY In this rodent model of severe mitral regurgitation that mimics the human condition, eccentric left ventricular dilatation occurred rapidly and persisted over the 20-wk period with parallel changes in myocardial collagen and matrix metalloproteinases that may drive the extracellular matrix breakdown.

Association of altered collagen content and lysyl oxidase expression in degenerative mitral valve disease.

BACKGROUND: Collagen cross-linking is mediated by lysyl oxidase (LOX) enzyme in the extracellular matrix (ECM) of mitral valve leaflets. Alterations in collagen content and LOX protein expression in the ECM of degenerative mitral valve may enhance leaflet expansion and disease severity. METHODS: Twenty posterior degenerative mitral valve leaflets from patients with severe mitral regurgitation were obtained at surgery. Five normal posterior mitral valve leaflets procured during autopsy served as controls. Valvular interstitial cells (VICs) density was quantified by immunohistochemistry, collagen Types I and III by picro-sirius red staining and immunohistochemistry, and proteoglycans by alcian blue staining. Protein expression of LOX and its mediator TGFß1 were quantified by immunofluorescence and gene expression by PCR. RESULTS: VIC density was increased, structural Type I collagen density was reduced, while reparative Type III collagen and proteoglycan densities were increased (P<.0001) with an increase in spongiosa layer thickness in myxomatous valves. These changes were associated with a reduction in LOX (P<.0001) and increase in TGFß1 protein expression (P<.0001). However, no significant change was seen in gene expression. Linear regression analysis identified a correlation between Type I collagen density and LOX grade (R2=0.855; P<.0001). CONCLUSIONS: Reduced Type I collagen density with a simultaneous increase in Type III collagen and proteoglycan densities possibly contributes to spongiosa layer expansion resulting in incompetent mitral valve leaflets. Observed changes in Type I and III collagen densities in Degenerative Mitral Valve Disease may be secondary to alterations in LOX protein expression, contributing to disorganization of ECM and disease severity.

Enhanced expression of ROCK in left atrial myocytes of mitral regurgitation: a potential mechanism of myolysis.

BACKGROUND: Severe mitral regurgitation (MR) may cause myolysis in the left atrial myocytes. Myolysis may contribute to atrial enlargement. However, the relationship between Rho-associated kinase (ROCK) and myolysis in the left atrial myocytes of MR patients remain unclear. METHODS: This study comprised 22 patients with severe MR [12 with atrial fibrillation (AF) and ten in sinus rhythm]. Left atrial appendage tissues were obtained during surgery. Normal left atrial tissues were purchased. Immunofluorescence histochemical and immunoblotting studies were performed. RESULTS: The expression of ROCK2 in the myolytic left atrial myocytes of MR AF patients (p = 0.009) and MR sinus patients (p = 0.011) were significantly higher than that of the normal subjects. Similarly, the expression of ROCK1 in the myolytic left atrial myocytes of MR AF patients was significantly higher than that of the normal subjects (p = 0.010), and the expression of ROCK1 in the myolytic left atrial myocytes of MR sinus patients was higher than that of the normal subjects (p = 0.091). Immunofluorescence study revealed significant co-localization and juxtaposition of ROCK2 and cleaved caspase-3 in the left atrial myocytes both in the MR AF group (Pearson's coefficient = 0.74 ± 0.03) and the MR sinus group (Pearson's coefficient = 0.73 ± 0.02). Similarly, immunofluorescence study revealed significant co-localization and juxtaposition of ROCK1 and cleaved caspase-3 in the left atrial myocytes both in the MR AF group (Pearson's coefficient = 0.65 ± 0.03) and the MR sinus group (Pearson's coefficient = 0.65 ± 0.03). Correlation analysis demonstrated that there was a significant direct relationship between the expression of ROCK2 in the myolytic left atrial myocytes and left atrial diameter in the MR patients (p = 0.041; r = 0.440). Moreover, the ratio of phosphorylated myosin-binding subunit of myosin light chain phosphatase (pMBS)/total MBS of left atrial tissues was significantly higher in the MR AF group (p < 0.04) and the MR sinus group (p < 0.04) compared with the normal control group. CONCLUSIONS: The enhanced expression of ROCKs might be involved in the myolysis of the left atrial myocytes of MR patients.

Decreased paraoxonase 1, arylesterase enzyme activity, and enhanced oxidative stress in patients with mitral and aortic valve insufficiency.

BACKGROUND: Oxidative stress is reportedly associated with several cardiovascular diseases. The antioxidant ability of high density lipoprotein (HDL) is, at least in part, attributable to the pleiotropic serum paraoxonase (PON1). The aim of the study was to investigate the body oxidant/antioxidant balance in patients with mitral regurgitation (MR) and aortic regurgitation (AR) to get new points of view for the underlying oxidative mechanisms. METHODS: Oxidative stress index (OSI), total oxidant status (TOS), and total antioxidant status (TAS) were examined in addition to the PON1 and arylesterase (ARE) enzyme activities in fifty-six patients and thirty-seven healthy control subjects. RESULTS: Serum PON1 and ARE enzyme activities were statistically significantly reduced in heart valve disease (HVD) patients (p = 0.0005 and p < 0.0001, respectively), whereas TOS and OSI levels were found to be significantly higher (p = 0.0021 and p < 0.0001, respectively). CONCLUSIONS: Serum PON1 activity is reduced in patients with HVD, caused by elevated oxidative stress and disturbances of heart valve metabolism. The findings from this novel detailed approach, implicate an inflammatory/oxidative stress process in the pathogenesis of the valve's presentation associated with the HVD. The strength of the significance in differences encourage us to propose that the role of oxidative stress in HVD pathogenesis is very prominent, and oxidative stress markers are potential ancillary tests to evaluate the state of the disease.

Volume overload induces differential spatiotemporal regulation of myocardial soluble guanylyl cyclase in eccentric hypertrophy and heart failure.

Nitric oxide activation of soluble guanylyl cyclase (sGC) blunts the cardiac stress response, including cardiomyocyte hypertrophy. In the concentric hypertrophied heart, oxidation and re-localization of myocardial sGC diminish cyclase activity, thus aggravating depressed nitric oxide-cyclic guanosine monophosphate (NO-cGMP) signaling in the pressure-overloaded failing heart. Here, we hypothesized that volume-overload differentially disrupts myocardial sGC activity during early compensated and late decompensated stages of eccentric hypertrophy. To this end, we studied the expression, redox state, subcellular localization, and activity of sGC in the left ventricle of dogs subjected to chordal rupture-induced mitral regurgitation (MR). Unoperated dogs were used as Controls. Animals were studied at 4weeks and 12months post chordal rupture, corresponding with early (4wkMR) and late stages (12moMR) of eccentric hypertrophy. We found that the sGC heterodimer subunits relocalized away from caveolae-enriched lipid raft microdomains at different stages; sGCß1 at 4wkMR, followed by sGCα1 at 12moMR. Moreover, expression of both sGC subunits fell at 12moMR. Using the heme-dependent NO donor DEA/NO and NO-/heme-independent sGC activator BAY 60-2770, we determined the redox state and inducible activity of sGC in the myocardium, within caveolae and non-lipid raft microdomains. sGC was oxidized in non-lipid raft microdomains at 4wkMR and 12moMR. While overall DEA/NO-responsiveness remained intact in MR hearts, DEA/NO responsiveness of sGC in non-lipid raft microdomains was depressed at 12moMR. Caveolae-localization protected sGC against oxidation. Further studies revealed that these modifications of sGC were also reflected in caveolae-localized cGMP-dependent protein kinase (PKG) and MAPK signaling. In MR hearts, PKG-mediated phosphorylation of vasodilator-stimulated phosphoprotein (VASP) disappeared from caveolae whereas caveolae-localization of phosphorylated ERK5 increased. These findings show that differential oxidation, re-localization, and expression of sGC subunits distinguish eccentric from concentric hypertrophy as well as compensated from decompensated heart failure.

Long-term efficacy of hematopoietic stem cell transplantation on brain involvement in patients with mucopolysaccharidosis type II: a nationwide survey in Japan.

Hematopoietic stem cell transplantation (HSCT) has not been indicated for patients with mucopolysaccharidosis II (MPS II, Hunter syndrome), while it is indicated for mucopolysaccharidosis I (MPS I) patients <2 years of age and an intelligence quotient (IQ) of ≥ 70. Even after the approval of enzyme replacement therapy for both of MPS I and II, HSCT is still indicated for patients with MPS I severe form (Hurler syndrome). To evaluate the efficacy and benefit of HSCT in MPS II patients, we carried out a nationwide retrospective study in Japan. Activities of daily living (ADL), IQ, brain magnetic resonance image (MRI) lesions, cardiac valvular regurgitation, and urinary glycosaminoglycan (GAG) were analyzed at baseline and at the most recent visit. We also performed a questionnaire analysis about ADL for an HSCT-treated cohort and an untreated cohort (natural history). Records of 21 patients were collected from eight hospitals. The follow-up period in the retrospective study was 9.6 ± 3.5 years. ADL was maintained around baseline levels. Cribriform changes and ventricular dilatation on brain MRI were improved in 9/17 and 4/17 patients, respectively. Stabilization of brain atrophy was shown in 11/17 patients. Cardiac valvular regurgitation was diminished in 20/63 valves. Urinary GAG concentration was remarkably lower in HSCT-treated patients than age-matched untreated patients. In the questionnaire analysis, speech deterioration was observed in 12/19 patients in the untreated cohort and 1/7 patient in HSCT-treated cohort. HSCT showed effectiveness towards brain or heart involvement, when performed before signs of brain atrophy or valvular regurgitation appear. We consider HSCT is worthwhile in early stages of the disease for patients with MPS II.

Atrial myocardial nox2 containing NADPH oxidase activity contribution to oxidative stress in mitral regurgitation: potential mechanism for atrial remodeling.

BACKGROUND: Oxidative stress is linked with several cardiovascular diseases. However, the NADPH oxidase activity in severe mitral regurgitation patients with and without atrial fibrillation has not yet been explored. METHODS: This study involved 16 adult patients (eight patients with persistent atrial fibrillation and eight with sinus rhythm) with severe mitral and moderate-to-severe tricuspid regurgitation and five control patients without mitral and tricuspid disease. Atrial tissues of the right and left atrial appendages were obtained during surgery. Superoxide anion production was measured by lucigenin-enhanced chemiluminescence, and the expression of nox2 containing NADPH oxidase mRNA was measured by quantitative real-time RT-PCR. Additionally, immunohistochemical study was performed. RESULTS: NADPH-stimulated superoxide release was significantly higher than basal superoxide production from right [5671.9±3498.7 vs. 232.7±70.0 relative light units per second per milligram of protein (RLU s(-1) mg protein(-1)), P=.008) and left atrial homogenates (6475.1±1890.8 vs. 229.0±79.6 RLU s(-1) mg protein(-1), P=.008) in atrial fibrillation patients. The NADPH-stimulated superoxide release from right atrial homogenates was also significantly higher than basal superoxide production in sinus patients (6809.1±1327.1 vs. 244.2±65.5 RLU s(-1) mg protein(-1), P=.008). Additionally, there was a borderline significant correlation between NADPH-stimulated superoxide production from left atrial homogenates and left atrial sizes (r=0.683, P=.062) in atrial fibrillation patients. Membrane-bound nox2 containing NADPH oxidase mRNA expression was increased and was similar in both the atrial fibrillation patients and sinus patients. The NADPH-stimulated superoxide production in right atrial homogenates in control atrial samples was 1863.7±137.2 RLU s(-1) mg protein(-1). Immunohistochemical study demonstrated increased expression of nox2 in myocytes with moderate-to-severe myolysis and hypertrophy. CONCLUSIONS: Results of this study demonstrate that membrane-bound nox2 containing NADPH oxidase activity and expression in the atrial myocardium is increased in patients with severe mitral regurgitation, possibly contributing to atrial remodeling in this clinical setting.

Selective activation of the "b" splice variant of phospholipase Cbeta1 in chronically dilated human and mouse atria.

Atrial fibrillation (AF) is commonly associated with chronic dilatation of the left atrium, both in human disease and animal models. The immediate signaling enzyme phospholipase C (PLC) is activated by mechanical stretch to generate the Ca2+-releasing messenger inositol(1,4,5)trisphosphate (Ins(1,4,5)P3) and sn-1,2-diacylglycerol (DAG), an activator of protein kinase C subtypes. There is also evidence that heightened activity of PLC, caused by the receptor coupling protein Gq, can contribute to atrial remodelling. We examined PLC activation in right and left atrial appendage from patients with mitral valve disease (VHD) and in a mouse model of dilated cardiomyopathy caused by transgenic overexpression of the stress-activated protein kinase, mammalian sterile 20 like kinase 1 (Mst1) (Mst1-TG). PLC activation was heightened 6- to 10-fold in atria from VHD patients compared with right atrial tissue from patients undergoing coronary artery bypass surgery (CABG) and was also heightened in the dilated atria from Mst1-TG. PLC activation in human left atrial appendage and in mouse left atria correlated with left atrial size, implying a relationship between PLC activation and chronic dilatation. Dilated atria from human and mouse showed heightened expression of PLCbeta1b, but not of other PLC subtypes. PLCbeta1b, but not PLCbeta1a, caused apoptosis when overexpressed in neonatal rat cardiomyocytes, suggesting that PLCbeta1b may contribute to chamber dilatation. The activation of PLCbeta1b is a possible therapeutic target to limit atrial remodelling in VHD patients.

Modulation of the tissue reninangiotensin-aldosterone system in dogs with chronic mild regurgitation through the mitral valve.

OBJECTIVE: To investigate whether the tissue and plasma renin-angiotensin-aldosterone system (RAAS) is activated in dogs with mild regurgitation through the mitral valve and determine the contribution of chymase and angiotensin-converting enzyme (ACE) to the activation of the RAAS and potential production of angiotensin II during the chronic stage of mild mitral valve regurgitation. ANIMALS: 5 Beagles with experimentally induced mild mitral valve regurgitation and 6 clinically normal (control) Beagles. PROCEDURES: Tissue ACE and chymase-like activities and plasma RAAS were measured and the RAAS evaluated approximately 1,000 days after experimental induction of mitral valve regurgitation in the 5 dogs. RESULTS: Dogs with experimentally induced mitral valve regurgitation did not have clinical signs of the condition, although echocardiography revealed substantial eccentric hyper- trophy. On the basis of these findings, dogs with mitral valve regurgitation were classified as International Small Animal Cardiac Health Council class Ib. Plasma activity of renin and plasma concentrations of angiotensin I, angiotensin II, and aldosterone were not significantly different between dogs with mitral valve regurgitation and clinically normal dogs. Tissue ACE activity was significantly increased and chymase-like activity significantly decreased in dogs with mitral valve regurgitation, compared with values in clinically normal dogs. CONCLUSIONS AND CLINICAL RELEVANCE: The tissue RAAS was modulated without changes in the plasma RAAS in dogs with mild mitral valve regurgitation during the chronic stage of the condition. An ACE-dependent pathway may be a major route for production of angiotensin II during this stage of the condition.

Increased expression and phosphorylation of focal adhesion kinase correlates with dysfunction in the volume-overloaded human heart.

FAK (focal adhesion kinase) has been shown to mediate the hypertrophic growth of the left ventricle. Experimental results also suggest that FAK may contribute to the structural and functional deterioration of the chronically overloaded left ventricle. In the present study, we postulated that FAK expression and phosphorylation may be altered in the volume-overloaded heart in humans. FAK expression and phosphorylation at Tyr(397) were detected by Western blotting and immunohistochemistry in samples from endomyocardial biopsies from patients with MR (mitral regurgitation; n=21) and donor subjects (n=4). Hearts from patients with MR had degenerated cardiac myocytes and areas of fibrosis. In this group, the myocardial collagen area was increased (18% in MR hearts compared with 3% in donor hearts respectively) and correlated negatively with left ventricular ejection fraction (r=-0.74; P>0.001). FAK expression and phosphorylation at Tyr(397) (a marker of the enzyme activity) were increased in samples from MR hearts compared with those from donor hearts (3.1- and 4.9-fold respectively). In myocardial samples from donor hearts, anti-FAK staining was almost exclusively restricted to cardiac myocytes; however, in myocardial samples from MR hearts, staining with the anti-FAK antibody was found to occur in myocytes and the interstitium. There was a positive correlation between collagen and the interstitial areas stained with the anti-FAK antibody (r=0.76; P>0.001). Anti-FAK and anti-vimentin staining of the interstitial areas of samples from MR hearts were extensively superimposed, indicating that most of the interstitial FAK was located in fibroblasts. In conclusion, FAK expression and phosphorylation are increased and may contribute to the underlying structural and functional abnormalities in the volume-overloaded heart in humans.

Reduced myocardial expression of calcium handling protein in patients with severe chronic mitral regurgitation.

OBJECTIVE: Left ventricle (LV) function was shown to be a principal determinant of morbidity and mortality in both uncorrected and surgically corrected mitral regurgitation (MR). However, the cellular mechanisms that develop in the LV remodeling secondary to volume overload in chronic severe MR is still not well defined. In single ventricular myocyte, a reduced contraction and slowed relaxation have been mainly attributed to defective intracellular Ca2+ currents. Between several Ca2+ handling proteins, sarcoplasmic reticulum Ca2+-ATPase 2 (SERCA2) expression and activity determines not only the extent and rate of relaxation, but also the rate and amplitude of contraction. The aim of the study was to determine whether modifications of SERCA2 gene expression occurs in LV wall remodeling process secondary to chronic severe MR. METHODS: The LV samples were obtained from 12 patients presented LV wall remodeling (LV: diastolic/systolic diameter-70+/-7 mm vs 46+/-10 mm; diastolic/systolic volume-260+/-65 ml vs 102+/-68 ml) due to chronic, severe MR. Expressions of SERCA2 isoforms-SERCA2a and 2b mRNAs were estimated by semiquantitative RT-PCR and normalized to GAPDH. The protein levels of SERCA2 were determined by Western blot after normalization to actin. Results were compared with samples from non-failing human hearts (NFH). RESULTS: On SERCA2 mRNA levels, important reduction on both SERCA isoforms SERCA2a (-40%) and SERCA2b (-49%) compared to NFH, together with significant correlation between isoforms (r = 0.89; p = 0.01) were observed. SERCA2 protein levels were decreased (-38%) in MR compared to NFH. Also significant correlations between SERCA2a/2b and SERCA2 protein expression (r = 0.83, p = 0.017; r = 0.68, p = 0.05, respectively) were observed. Moreover, a negative correlation between protein levels of SERCA2 (r = -0.64, p = 0.053) and left ventricular diastolic diameter was observed. CONCLUSIONS: In chronic volume overload the down-regulation of SERCA2a and 2b at the mRNA and SERCA2 protein levels exist. Moreover, protein levels of SERCA2 tend to correlate to the grade of left ventricular diastolic dilatation and suggest an important role LV remodeling.

Neurohormonal and circulatory effects of short-term treatment with enalapril and quinapril in dogs with asymptomatic mitral regurgitation.

The aim of the study was to compare the effect of 2 angiotensin-converting enzyme (ACE) inhibitors on neurohormonal and circulatory variables in Cavalier King Charles Spaniels (CKCSs) with asymptomatic mitral regurgitation (MR). Ten CKCSs with mild to severe untreated MR were treated with 2 ACE inhibitors, quinapril and enalapril (each at 0.5 mg/kg PO q24h for 7 days), in a double-blind, crossover study with a washout period of 7 days between treatments. Blood samples were drawn and echocardiography was performed on days 0, 7, 14, and 21. Both treatments reduced ACE activity (P < .001) and increased renin activity (P < .001) and atrial natriuretic peptide concentration (P < .005). The ACE inhibitors had no effect on the concentrations of nitrate and nitrite (NOx) or asymmetric dimethylarginine (ADMA). On day 0, a lower NOx concentration (P = .02) was found in samples taken in the clinic as compared to samples taken in the homes of the dogs. Quinapril caused a significant reduction in more variables that reflect the severity of MR (eg, jet size and left ventricular end diastolic diameter) than did enalapril. However, in terms of specific variables, no significant difference was identified between the effects of the 2 treatments on MR. These results suggest that ACE inhibitors do not affect NOx and ADMA concentrations in asymptomatic dogs, but exercise, stress, or some combination may influence NOx concentrations in these dogs.

Influence of angiotensin-converting enzyme gene insertion/deletion polymorphism on rheumatic valve involvement, valve severity and subsequent valve calcification.

BACKGROUND AND AIM OF THE STUDY: The relationship between the severity of chronic rheumatic heart disease (RHD) and predisposing factors is unknown, and genetic predictors for severe scarring and calcification of the mitral valve are not well defined. A high angiotensin-converting enzyme (ACE) activity has been demonstrated in valve tissue. Thus, a case-control study was conducted to investigate any possible relationship between ACE gene polymorphisms and chronic mitral valve disease severity and calcification. METHODS: This case-control study included 82 patients (24 males, 58 females; mean age 40.3 +/- 14.7 years) with chronic rheumatic mitral valve, and 154 control subjects (53 males, 101 females; mean age 43.4 +/- 13.4 years). ACE gene insertion/deletion (I/D) polymorphisms were identified using polymerase chain reaction methods. RESULTS: Among RHD subjects, 31 (30.6%) were D/D, 25 (32.7%) were I/D, and 26 (18.8%) were I/I. Among controls, 57 (57.4%) were D/D, 69 (61.3%) were I/D, and 28 (35.2%) were I/I. The frequency of ACE I/I genotype was higher in RHD subjects than in controls (chi2 = 7.4, df = 2, p < 0.030; D/D versus I/D versus I/I), or (chi2 = 5.5, df = 1, p < 0.019; DD + ID versus II). Predisposition to RHD was significantly less frequent in the D/D genotype. There was no statistically significant difference in the genetic analysis of RHD with respect to mitral valve score, severity of mitral regurgitation and left atrial diameter. Mitral valve calcification was significantly associated with a higher frequency of I/I genotype and I/D genotype than D/D genotype alone (chi2 = 6.2, df = 2, p = 0.043). The ACE I/I genotype was associated with a predisposition to a greater risk of severe calcific valve disease. CONCLUSION: The ACE I/I genotype is more common in patients with rheumatic valve disease than in the normal population. This suggests that the ACE gene polymorphism may be involved in the pathogenesis of rheumatic heart disease.

Matrix metalloproteinases and atrial remodeling in patients with mitral valve disease and atrial fibrillation.

BACKGROUND: Atrial fibrillation (AF) is associated with extracellular matrix remodeling involving atrial fibrosis and atrial dilatation. Angiotensin II mediated pathways and matrix metalloproteinases (MMPs) have been implicated in these processes. Our aim was to study atrial structural remodeling and the expression of the angiotensin receptor subtypes and MMPs and their inhibitors (TIMPs) in patients with mitral valve disease with and without AF. METHODS AND RESULTS: Biopsies from right and left atrial appendages (RA and LA) were taken from patients undergoing CABG (n=9, all in sinus rhythm (SR)) or mitral valve surgery (MVS; n=19; 9 with permanent AF and 10 in SR). Patients with MVS and AF had significantly larger atria (versus MVS and SR: p=0.02; versus CABG: p<0.01). The MVS patients had significantly more fibrosis than the control CABG group. Fibrosis was increased in both the AF and SR MVS groups in the LA, but only in the MVS-AF group in the RA. These AF patients had significantly more tricuspid regurgitation than SR patients. MMP-1 was down-regulated in LA of MVS patients (p=0.02) independent of the underlying rhythm (SR or AF; p=0.95). In RA biopsies, MMP-1 was down-regulated only in the MVS and AF group. MMP-9 was down-regulated in the MVS patients compared to CABG both in the RA and LA, and without a difference between the SR and AF groups. Protein expression of AT-1, AT-2, MMP-2, TIMP-1, -2 and -4, TNF-alpha, and TNF-alpha-converting enzyme did not differ significantly between the 3 groups. CONCLUSIONS: Concordant changes between MMP-expression and fibrosis during mitral valve disease, both in LA and RA, suggest involvement of MMPs in structural atrial remodeling. AF itself did not contribute to altered fibrosis or MMP-expression in the LA. The association between AF and RA changes may be precipitated by greater hemodynamic load due to tricuspid regurgitation in these patients.

[Study on the total activity of PKC and the quantity of PKC (alpha, beta) in left auricle tissues of the patients with mitral disease and atrial fibrillation].

OBJECTIVE: To investigate the possible relation of atrial fibrillation (AF) with the total activity of protein kinase C (PKC) and the quantity of PKC (alpha, beta) in the left auricle tissues of the patients with mitral disease. METHODS: Thirty-five patients hospitalized for valve replacement surgery were divided into four groups: group A (simple mitral stenosis with AF), group B(simple mitral stenosis with sinus rhythm), group C(simple mitral regurgitation with AF), group D (simple mitral regurgitation with sinus rhythm), The total PKC, activity and the quantity of PKC alpha and PKC beta were measured and compared in an attempt to find out whether significant difference exists between the groups. RESULTS: There was no significant difference in total PKC activity between the four groups (P > 0.05). The quantity of PKC alpha in patients with AF was lower than that in patients with sinus rhythm, the quantity of PKC beta in patients with AF was higher than in patients with sinus rhythm. But these differences were not statistically significant (P > 0.05). CONCLUSION: Under conditions of the same rhythm and the same heart disease, the total PKC activity and the quantity of PKC (alpha, beta) of the four groups showed no significant difference. No obvious relation was seen between the total activity of PKC in left auricle tissue and the occurrence of AF in patients with mitral disease, but more research on the relation between AF and the quantity of PKC isoforms in left auricle tissues would be needed.

Cardiac mast cell- and chymase-mediated matrix metalloproteinase activity and left ventricular remodeling in mitral regurgitation in the dog.

The present study tested the hypothesis that cardiac mast cells and chymase are associated with matrix metalloproteinase (MMP) activation and extracellular matrix (ECM) degradation in the evolution of left ventricular (LV) chamber remodeling secondary to experimental mitral regurgitation (MR) in dogs. LV mast cell density, chymase activity, and angiotensin II (ANG II) levels were significantly increased 2 and 4 weeks post-MR, while an increase in angiotensin-converting enzyme (ACE) activity was not seen prior to the chronic 24 week stage. As early as 2 and 4 weeks, there was a significant decrease in interstitial myocardial collagen content that was associated with an increase in LV end-diastolic diameter (LVEDD) but a normal LVEDD/wall thickness ratio. While mast cell density decreased to normal at 24 weeks, both chymase and MMP-2 activity remained increased throughout the entire 24-week period post-MR. By 24 weeks a transition to an adverse pattern of LV remodeling characterized by a 2-fold increase in the LVEDD/wall thickness ratio had occurred. Thus, this study supports the hypothesis that mast cells and chymase are important modulators of MMP activity and ECM degradation, contributing to adverse LV remodeling in chronic volume overload secondary to MR.