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.

Focal adhesion kinase mediates atrial fibrosis via the AKT/S6K signaling pathway in chronic atrial fibrillation patients with rheumatic mitral valve disease.

BACKGROUND: Atrial fibrosis, as a hallmark of atrial structural remodeling, plays a critical role in the maintenance of chronic atrial fibrillation (AF), but the mechanisms responsible for atrial fibrosis are still uncertain. Fibrogenesis represents a complex process in which focal adhesion kinase (FAK) plays an important role. Therefore, we investigated the role of FAK-mediated signaling in atrial fibrosis in patients with chronic AF related to rheumatic mitral valve disease (RMVD). METHODS: Atrial appendages were excised from 45 patients with RMVD and either chronic AF (n=25, AF >6 months) or sinus rhythm (n=20). Fibrosis was assessed by histology, and FAK and its two downstream pathways (AKT/S6K and ERK1/2) were evaluated by western blotting. We further evaluated the role of FAK in fibrogenesis by culturing neonatal rat cardiac fibroblasts to determine the importance of FAK-regulated signaling in cardiac myofibroblast differentiation induced by transforming growth factor-ß1 (TGFß1). RESULTS: Our study revealed that FAK can regulate its downstream signaling to cause fibrosis in atrial tissue and activate isolated fibroblasts. Histology revealed a significant increase in atrial fibrosis in AF patients. The phosphorylation of FAK and its downstream AKT/S6K signaling was increased secondary to TGFß1-induced high expression of α-SMA, a marker of myofibroblast activity. FAK and AKT inhibitors suppressed α-SMA expression in TGFß1-induced fibroblasts. However, ERK1/2 signaling seemed to be unrelated to the fibrotic process in AF patients. CONCLUSION: The FAK-mediated AKT/S6K signaling pathway participated in atrial fibrogenesis and this finding may contribute to the prevention of atrial fibrosis associated with chronic AF in patients with underlying cardiac disease.

Increased transcript level of poly(ADP-ribose) polymerase (PARP-1) in human tricuspid compared with bicuspid aortic valves correlates with the stenosis severity.

Oxidative stress may contribute to the hemodynamic progression of aortic valve stenosis, and is associated with activation of the nuclear enzyme poly(ADP-ribose) polymerase (PARP) 1. The aim of the present study was to assess the transcriptional profile and the topological distribution of PARP-1 in human aortic valves, and its relation to the stenosis severity. Human stenotic aortic valves were obtained from 46 patients undergoing aortic valve replacement surgery and used for mRNA extraction followed by quantitative real-time PCR to correlate the PARP-1 expression levels with the non invasive hemodynamic parameters quantifying the stenosis severity. Primary isolated valvular interstitial cells (VICs) were used to explore the effects of cytokines and leukotriene C(4) (LTC(4)) on valvular PARP-1 expression. The thickened areas of stenotic valves with tricuspid morphology expressed significantly higher levels of PARP-1 mRNA compared with the corresponding part of bicuspid valves (0.501 vs 0.243, P=0.01). Furthermore, the quantitative gene expression levels of PARP-1 were inversely correlated with the aortic valve area (AVA) (r=-0.46, P=0.0469) and AVA indexed for body surface area (BSA) (r=-0.498; P=0.0298) only in tricuspid aortic valves. LTC(4) (1nM) significantly elevated the mRNA levels of PARP-1 by 2.38-fold in VICs. Taken together, these data suggest that valvular DNA-damage pathways may be associated with inflammation and the stenosis severity in tricuspid aortic valves.

Mitral valvular interstitial cell responses to substrate stiffness depend on age and anatomic region.

The material properties of heart valves depend on the subject's age, the state of the disease and the complex valvular microarchitecture. Furthermore, valvular interstitial cells (VICs) are mechanosensitive, and their synthesis of extracellular matrix not only determines the valve's material properties but also provides an adhesive substrate for VICs. However, the interrelationship between substrate stiffness and VIC phenotype and synthetic properties is poorly understood. Given that the local mechanical environment (substrate stiffness) surrounding VICs differs among different age groups and different anatomic regions of the valve, it was hypothesized that there may be an age- and valve-region-specific response of VICs to substrate stiffness. Therefore, 6-week-, 6-month- and 6-year-old porcine VICs from the center of the mitral valve anterior leaflet (MVAC) and posterior leaflet (PML) were seeded onto poly(ethylene) glycol hydrogels of different stiffnesses and stained for markers of VIC activation (smooth muscle alpha-actin (SMaA)) and collagen synthesis (heat shock protein-47 (HSP47), prolyl 4-hydroxylase (P4H)). Six-week-old MVAC demonstrated decreased SMaA, P4H and HSP47 on stiffer gels, while 6-week-old PML only demonstrated decreased HSP47. Six-month-old MVAC demonstrated no difference between substrates, while 6-month-old PML demonstrated decreased SMaA, P4H and HSP47. Six-year-old MVAC demonstrated decreased P4H and HSP47, while 6-year-old PML demonstrated decreased P4H and increased HSP47. In conclusion, the age-specific and valve-region-specific responses of VICs to substrate stiffness link VIC phenotype to the leaflet regional matrix in which the VICs reside. These data provide further rationale for investigating the role of substrate stiffness in VIC remodeling within diseased and tissue engineered valves.

Mitral valve surgery with surgical embolectomy for mitral valve endocarditis complicated by septic coronary embolism.

Acute myocardial infarction (AMI) complicated by septic coronary embolism due to active infective endocarditis is rare but usually fatal. We report a case of successful mitral valve surgery with surgical embolectomy in a 27-year-old man with an AMI complicated by septic coronary embolism due to mitral valve endocarditis. A chest radiograph revealed cardiomegaly and marked pulmonary edema. A transthoracic echocardiogram disclosed severe mitral regurgitation with highly mobile vegetations and hypokinesia of the left ventricular apex. The electrocardiographic findings of ST segment elevation in leads V2-4 and elevated cardiac enzyme levels were strongly suggestive of an acute anterolateral AMI. Nevertheless, emergent cardiac surgery was needed without selective coronary angiography because of intractable heart failure and life-threatening ventricular tachyarrhythmia requiring cardiopulmonary resuscitation. A total occlusion of the distal left anterior descending artery caused by embolic vegetation and thrombus, which was incidentally detected intraoperatively, was successfully recanalized by surgical embolectomy and thrombectomy using a direct coronary incision. The mitral valve endocarditis was managed with wide debridement and mechanical valve replacement. Three years after the surgery a follow-up echocardiogram showed no abnormalities of the regional wall, motion in the left ventricle and the patient is living an active life without any complications.

Matrix metalloproteinase expression in the ascending aorta and aortic valve.

Extracellular matrix degradation and increased proteolytic enzyme (matrix metalloproteinase (MMP)) activity characterise abdominal aortic aneurysm formation. Post-stenotic dilatation of ascending aorta is associated with aortic stenosis and regurgitation, haemodynamically normal bicuspid aortic valve (BAV) and following AV replacement. We aimed to determine an association between ascending aortic pathology and abnormal AV, with particular reference to MMPs, and ascertain differences between BAV and tricuspid (TAV) AV. Subset of the study population (n=19) with a preoperative ascending aorta of >4 cm was analysed. Samples of ascending aorta and AV were obtained from 82 patients (TAV, n=54, BAV, n=28) undergoing surgery. Gene expression of MMP-1, -2, -9 and tissue inhibitor of metalloproteinase (TIMP)-1 and -2 was quantified by real-time RT-PCR. No significant difference was seen in gene expression level of MMPs, TIMPs and ratio of MMPs/TIMPs in ascending aorta and AV between patients with BAV and TAV. MMP-2/TIMP-1 in ascending aorta was greater in BAV, in the subset of patients with preoperative aortic dilatation (P<0.05). No difference exists in gene expression of MMPs in ascending aorta and AV between patients with BAV and TAV. However, patients with larger aortic diameters have increased MMP-2/TIMP-1. Modifying MMP expression may have a role in development of aneurysms.

Endothelial nitric oxide synthase in bicuspid aortic valve disease.

BACKGROUND: The pathogenesis of ascending aortic dilatation in the presence of a bicuspid valve is discussed controversially. Recent experimental evidence suggests that the expression of endothelial nitric oxide synthase (eNOS) may have an influence on aortic valve anatomy and aneurysmal dilatation of the aorta. We investigated the relationship among eNOS expression, valve anatomy, and aortic dilatation in the human aortic wall. METHODS: Aortic wall specimens from 39 patients with aortic valve disease (bicuspid, n = 17; tricuspid, n = 22) were studied. The functional aortic valve pathology was regurgitation (n = 22), stenosis (n = 10), and combined aortic valve disease (n = 7). The specimens were obtained intraoperatively from the aortic wall above the noncoronary sinus. The eNOS protein expression was quantified by western blot analysis after immunoprecipitation from tissue lysates. The eNOS levels were analyzed for correlation with valve anatomy and ascending aortic diameters. RESULTS: The eNOS protein expression of aortic endothelial cells was significantly lower in patients with bicuspid as compared with tricuspid aortic valves (4,615 +/- 489 vs 6,275 +/- 442; p = 0.017). In bicuspid aortic valves there was a significant correlation between eNOS expression and maximum aortic diameter (r = -0.530; p = 0.029) or sinotubular diameter (r = -0.520; p = 0.033). In patients with tricuspid aortic valves, no significant correlation between aortic size and eNOS expression was found. CONCLUSIONS: Our results show an association between eNOS levels and aortic valve anatomy as well as aneurysm formation in patients with bicuspid aortic valves.

Tissue microarray detection of matrix metalloproteinases, in diseased tricuspid and bicuspid aortic valves with or without pathology of the ascending aorta.

OBJECTIVE: The degeneration of bicuspid aortic valve and its frequent association with ascending aortic pathology, point to a still unidentified genetic tissue defect with unknown mediators. Metalloproteinases (MMPs) are lytic enzymes that have been strongly implicated in aneurysm formation. The purpose of this study was to detect the presence of these enzymes in aortic valvular tissue in healthy and diseased aortic valves with or without the presence of synchronous ascending aortic pathology. METHODS: Aortic valve specimens from 26 aortic valve replacement patients as well as 4 healthy control tricuspid aortic valves were included. 10 patients had bicuspid aortic valves, and 16 had tricuspid aortic valves. Half of our patient population had a concomitant aortic procedure for aortic pathology. The study detected MMPs 1,2 and 9 as well as their Tissue inhibitors (TIMPs) 1 and 2. MMP and TIMP detection was accomplished with the construction of a tissue micro array and immunohistochemistry. CONCLUSIONS: MMP-9 expression was significantly higher in bicuspid aortic valves compared to normal valves (P<0.05). When compared to the tricuspid valve group, MMP-9 mean value was significantly higher in bicuspid valves (P<0.05). When the entire rest of the valve group (n=4+16, i.e. control and tricuspid valve groups) was compared to the bicuspid valve group, bicuspid valves had significantly higher MMP-2, and MMP-9 (P<0.01) expression. TIMP expression also changed in diseased valves, among different patient groups. This increased proteolytic presence in bicuspid aortic valves may attribute to the observed decreased elastin and collagen content, and their resultant functional failure.

Is the mitral valve passive flap theory overstated? An active valve is hypothesized.

The concept that the mitral valve of the heart is a passive flap that opens and closes like a barn door has been emphasized for decades by medical and biology professors to their students. But experimental findings, which are outlined in this report, support the theory of an active valve. We hypothesize that the two leaflets of the mitral valve are actively contractile; that physical forces generated in the valve itself may stabilize and add precision to the sum of forces that regulate valve movement. This precision could be of critical significance both in the moments preceding, and during, valve opening and closing. Evidence supporting our active valve hypothesis includes the profuse innervation of motor and sensory nerves that are present in the mitral valves of all animals studied. In addition, multiple contractile cell types have been found in the mitral valve, including cardiac muscle cells, smooth muscle cells, and cardiac valvular interstitial cells. In vitro work in our laboratories using the rat mitral valve shows that not only are the valves capable of contraction and relaxation, but that the contractions and relaxations are nerve-mediated. We theorize that the rich innervation and contractile cells in the mitral valve work together to modulate fine-tuning of valve movements and tone, thereby ensuring the integrity of the valve seal. Other investigators have reported that the mitral valve demonstrates contractile activity and that denervation localized to the mitral valve affects valve competence. The evidence for an active mitral valve presented by these and other experimental studies warrant a reexamination of the validity of the passive valve concept. An accurate and full understanding of the precise movements of the valve leaflets and the mechanisms that regulate these movements is likely to provide the information needed to understand and develop treatments for many different cardiac valve problems, including mitral valve diseases such as prolapse and myxomatous degeneration. In view of the available experimental evidence, the concept that the mitral valve functions only as a passive structure is challenged by numerous anomalies. A reinterpretation of the concept of valve function that incorporates active as well as passive roles for the valve leaflets and other components of the valve apparatus would have significant implications both for the directions taken in research involving the cardiac valves and for the approaches to treatment.

Increased NADPH-diaphorase activity in canine myxomatous mitral valve leaflets.

Comparable pathological changes in the mitral valve have been described in dogs, pigs and human patients with myxomatous mitral valve disease (MMVD), i.e., primary mitral valve prolapse. The progressive myxomatous changes are probably a response to repeated impact on the leaflets, and endothelial stress or damage probably plays a central role in the pathogenesis. Little, however, is known about the vasoactive substances that mediate the subendothelial changes. The aim of this study was to investigate the expression of nitric oxide synthase (NOS) in canine mitral valve leaflets and to relate the findings to MMVD changes. The mitral valve was taken post mortem from 12 dogs (six males and six females) and a whole valve NADPH (the reduced form of nicotinamide-adenine dinucleotide phosphate) diaphorase (NADPH-d) reaction was performed. Macroscopical (semiquantitative) and microscopical (computer image analysis) evaluations of the staining due to NADPH-d activity were performed at four specific areas of the valve and related to microscopical signs of MMVD and gross signs of thickening or prolapse, or both. Macroscopically, the NADPH-d colour grade was correlated with the degree of MMVD (P=0.01). In addition, endothelial NADPH-d staining intensity was correlated with macroscopical signs of disease (P=0.004) as well as with collagen degeneration (P=0.008) and deposition of mucopolysaccharides (P=0.02). Age, gender and specific area of the valve did not seem to influence the NADPH-d activity. In conclusion, increased NADPH-d activity, suggesting increased NOS expression, was found in areas of the mitral valve with myxomatous changes. This indicates that nitric oxide (NO) may play a role in the pathogenesis of MMVD in dogs.

Biochemical pathways of tissue degeneration in bioprosthetic cardiac valves. The role of matrix metalloproteinases.

Degeneration processes that affect bioprosthetic heart valves made from glutaraldehyde treated bovine pericardium are poorly understood. The present study undertook the identification and characterization of matrix metalloproteinases (MMPs) in extracts obtained from 28 pericardial derived bioprosthetic heart valves explanted at surgery. A lysosomal marker was used to assess the incidence of infiltrating extracellular matrix degrading cells. The major biochemical features that were associated with tissue degeneration and bioprosthetic heart valve failure were increased levels of MMP 9, high levels of beta-glucuronidase, and constant levels of active collagenase and MMP 2. The MMPs extracted from ruptured bioprostheses were inhibited by calcium chelators and zinc binding compounds. These data suggest that tissue failure, in addition to known mechanical and calcification related factors, may be contributed to by the intervention of proteolytic enzymes. A schematic working model was proposed that described the major biochemical pathways underlying tissue degeneration, starting from bioprostheses preparation and ending with clinical failure.

An enzyme histochemical study of human sinus node, coronary sinus, and mitral valve muscle.

An enzyme histochemical study of the sinoatrial node, the coronary sinus, and the atrial muscle extending into the anterior mitral valve was performed on human hearts. Investigation of the activity and localization of the structurally bound enzymes was performed by conventional histochemical techniques. Determination of the activity of nonstructurally or weakly structurally bound enzymes was performed by histochemical techniques in which leakage of enzymes during the incubation period was reduced by the application of semipermeable membranes. The sinoatrial node is characterized by a high degree of anaerobic enzyme capacity and a relatively low degree of aerobic enzyme capacity. The discriminatory nature of these reactions allows examination of the structure of the sinoatrial node and its approaches. The presence of transitional cells was confirmed; isolated clusters of nodal cells were found in the atrial myocardium around the sinoatrial node, but no evidence of specialized tissue forming the beginning of an internodal pathway was found by this technique. The specific histochemical reactions that characterize the sinoatrial node also occur in the atrial muscle, extending into the anterior mitral valve, the anterior wall of the coronary sinus, and the atrial tissue near the orifice of the coronary sinus. These observations seem to corroborate the hypothesis that arrhythmic ectopic foci can arise in these regions.