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.

Matrix Metalloproteinases (MMPs), Their Genetic Variants and miRNA in Mitral Valve Diseases: Potential Biomarker Tools and Targets for Personalized Treatments.

Mitral valve diseases (MVD)s, comprising congenital and acquired forms, are characterized by a diverse etiology, pathophysiology, prevalence, and incidence. In industrialized countries, the acquired forms represent 2.5% of all cardiovascular diseases, with a marked augmentation after the age of 65 years. In addition, all forms of MVDs (i.e., degenerative forms) have a difficult clinical management. The major challenge is 'the early diagnosis', and echocardiographic analysis has been shown inappropriate for diagnosing MVD in moderate forms. Thus, there is a strong need to identify more appropriate biomarker tools to diagnose MVDs at early clinical stage before complications occur and worsen the prognosis. Innovative biomarker tools may particularly be appropriate for the complex treatment of elderly patients, the clinical management of which is very difficult due to the high risk of surgical interventions and no clear benefits in terms of life expectancy or quality of life compared to younger patients. These biomarker tools may be identified as genetic factors and/or components of cellular and molecular pathways related to the mechanisms of MDV pathophysiology. In this review, emphasis is placed on the possibility of proposing matrix metalloproteinase (MMP) pathways, their genetic variants and microRNA as promising predictive, diagnostic and prognostic biomarkers and targets for personalized treatments. Evidence is also provided of the lack of any consistent evidence which actually hampers their clinical application. Thus, criticisms and concerns are underlined, as well as suggestions to close the existing gaps.

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.

Matrix metalloproteinase-1 mitral expression and -1607 1G/2G gene promoter polymorphism in mitral chordae tendinae rupture.

Understanding the pathogenesis of mitral chordae tendinae rupture (MCTR) is essential for identification of risk factors. Mitral matrix metalloproteinase (MMP) triggers the signal cascade that instigates cardiac fibrosis, which may be a predisposing factor in MCTR. We investigated associations among MMP1 expression, MMP1 -1607 1G/2G polymorphism and mitral chordae tendinae rupture (MCTR). This study enrolled 185 patients (group A) receiving mitral valve replacement. Group A included 65 patients with MCTR and 120 controls without MCTR. MMP1 was assessed on a semiquantitative scale (0-3) by immunohistochemical staining. For genetic association study, another 227 subjects were recruited for group B, including 75 with MCTR and 152 controls. The gene polymorphisms were analyzed by polymerase chain reaction. In group A, MCTR patients had a higher MMP1 expression compared to controls (P < 0.001). Binary regression analysis showed the variation in the MCTR patients was independently explained by MMP1 (P = 0.027). Hypertension and MMP1 staining had a synergistic effect on the MCTR occurrence (P < 0.001). In group B, MMP1 -1607 1G allele was increased in patients with MCTR compared to controls (P = 0.014). The odds ratio for the 1G/1G genotype to the 2G/2G genotype was 3.22 (P = 0.009). Univariate and logistic regression analysis showed an independent association between MCTR and MMP1 -1607 1G/2G polymorphism (P = 0.028 and 0.032, respectively). Since MMP1 mitral expression and -1607 1G/2G polymorphism were associated with MCTR independently of other baseline characteristics, MMP1 may play a role in the individual susceptibility to MCTR.

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.

Distribution of NADPH-diaphorase and AChE activity in the anterior leaflet of rat mitral valve.

The mitral valve, as an active flap, forms the major part of the left ventricular inflow tract and therefore plays an important function in many aspects of left ventricular performance. The anterior leaflet of this valve is the largest and most ventrally placed of two leaflets that come together during ventricular systole to close the left atrioventricular orifice. Various neurotransmitters are responsible for different functions including controlling valve movement, inhibiting or causing the failure of impulse conduction in the valve and the sensation of pain. Nitric oxide acts as a gaseous free radical neurotransmitter, neuromediator and effective cardiovascular modulator. Acetyl-choline is known to function as a typical neurotransmitter. Histochemical methods for detection of nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d), as an indirect nitric oxide-synthase marker, and method for detection of acetylcholinesterase (AChE) were used. Both methods were performed on the same valve sample. A widespread distribution of nerve fibres was observed in the anterior leaflet of the mitral valve. The fine NADPH-d positive (nitrergic) nerve fibres were identified in all zones of valve leaflet. AChE positive (cholinergic) nerve fibres were identified forming dense network and fibres organized in stripes. Endocardial cells and vessels manifested heavy NADPH-d activity. Our observations suggest a different arrangement of nitrergic and cholinergic nerve fibres in the anterior leaflet of the mitral valve. The presence of nitrergic and cholinergic activity confirms the involvement of both neurotransmitters in nerve plexuses and other structures of mitral valve.

Synergistic effect of mitral expression of tissue inhibitor of metalloproteinase-2 with hypertension on the occurrence of mitral chordae tendinae rupture.

OBJECTIVES: We previously found the association between mitral chordae tendinae ruptures (MCTR) and hypertension. Tissue inhibitor of metalloproteinase-2 (TIMP2), which expresses differently under pressure loads, could trigger a signal cascade instigating cardiac fibrosis, possibly predisposing to MCTR. We aimed to elucidate the relationship between the TIMP2 and hypertension and the effect they may have on the occurrence of MCTR. METHODS: Using a cross-sectional study in a tertiary medical center in Taiwan, we enrolled 186 patients who had received mitral valve replacements and classified them into two groups: 64 (34%) with MCTR and 122 (66%) without MCTR. Expression of mitral TIMP2 was assessed on a semiquantitative scale (grade 0-3) by immunohistochemical staining using antibodies against TIMP2. RESULTS: TIMP2 expression was significantly higher in MCTR patients (P < 0.001). Multiple logistic regression analysis showed four independent risk factors: TIMP2 [odds ratio (OR) = 1.82, 95% confidence interval (CI) = 1.18-2.81, P = 0.007], hypertension (OR = 2.40, CI = 1.08-5.34, P = 0.032), rheumatic heart disease (OR = 0.18, CI = 0.05-0.70, P = 0.014), and left ventricular end-diastolic dimension (OR = 1.10, CI = 1.05-1.15, P < 0.001). Among nonhypertensive patients, the higher expression of TIMP2 (grade 2 and 3 vs. 0 and 1) was associated with a 3.27-fold risk. However, hypertensive patients with higher TIMP2 expression had a significantly 10-fold higher risk (P < 0.001 for interaction). CONCLUSION: Mitral TIMP2 expression is higher in patients with MCTR and there is a synergistic effect of mitral TIMP2 staining with hypertension on the occurrence of MCTR.

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.

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.

Differential protein kinase C isoform abundance in ascending aortic aneurysms from patients with bicuspid versus tricuspid aortic valves.

BACKGROUND: It is recognized that different events contribute to the initiation of ascending thoracic aortic aneurysms (ATAAs) in patients with bicuspid aortic valves (BAV) versus patients with tricuspid aortic valves (TAV), but the molecular signaling pathways driving aneurysm formation remain unclear. Protein kinase C (PKC) is a superfamily of kinases which differentially mediate signaling events that lead to altered gene expression and cellular function, and may regulate downstream mediators of vascular remodeling. The present study tested the hypothesis that ATAA development in patients with BAV versus TAV proceeds by independent signaling pathways involving differential PKC signaling. METHODS AND RESULTS: ATAA samples were collected from BAV (n=57) and TAV (n=55) patients and assessed for 10 different PKC isoforms by immunoblotting. Results were expressed as a percent change in abundance (mean+/-SEM) from a nonaneurysmal control group (100%, n=21). Correlation analysis was performed, and relationships between PKC and matrix metalloproteinase abundance were reported. In the BAV group, classic and novel PKC isoforms (PKC-alpha, betaI, gamma, epsilon, theta) were increased, whereas PKC-eta and atypical PKC-zeta were decreased. In the TAV group, classic and novel isoforms were decreased and atypical PKC-zeta was elevated. Positive correlations between PKC and matrix metalloproteinase abundance were identified. CONCLUSIONS: Differential PKC isoform abundance was observed in ATAA samples from patients with BAV versus TAV, suggesting independent molecular signaling pathways may be operative. Induction of independent transcriptional programs may result and may provide a mechanistic foundation for developing selective diagnostic/therapeutic strategies for patients with ATAAs secondary to BAV or TAV.

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.

Direct measurements of nitric oxide release in relation to expression of endothelial nitric oxide synthase in isolated porcine mitral valves.

The aim of this study was to measure the direct release of nitric oxide (NO) from the porcine mitral valve using a NO microelectrode. Furthermore, the expression and localization of endothelial nitric oxide synthase (eNOS) in the mitral valve was studied using immunohistochemistry, Western blotting and RT-PCR. Results show that bradykinin increases NO release from mitral valves (DeltaBradykinin: 33.71 +/- 10.41 nm NO, P < 0.001, n = 10), whereas N-nitro-l-arginine methyl esther (l-NAME) decreases NO release when compared with basal level (Deltal-NAME: 82.69 +/- 15.66 nm NO, P < 0.005, n = 4). Both protein and mRNA expression of eNOS in mitral valves and in isolated valvular endothelial cells suggest that the NO release is mainly associated with the mitral valve endothelium. It is concluded that direct NO release from porcine mitral valves coincides with eNOS expression. This study documents useful techniques for investigations into the role of local NO release in mitral valve diseases.

Mitral valve thrombosis and infective endocarditis in a patient with prothrombin mutation.

This report deals with a 25-year-old female patient, who was admitted to a medical centre with complaints of fatigue and arthralgia. A non-homogeneous mass originating from the posterior papillary muscle and reaching the posterior and anterior cusps of the mitral valve was evident on the patient's echocardiogram. Her intractable fever, despite adequate antibiotic therapy, led us to consult with the cardiac surgeons. A mitral valve replacement was performed on the 8th day of admission. The pathological examination of the mitral valve and the vegetative lesion revealed the presence of organized thrombus and infective endocarditis. The patient had a history of oral contraceptive use for one year and at genetic examination we detected a heterozygous prothrombin mutation (G20210A). The association of infective endocarditis with native mitral valvular thrombosis in a case with prothrombin mutation and history of oral contraceptive use encouraged us to share our experience with our colleagues.

Lactatdehydrogenase (LDH) prior and post implantation of ATS heart valves.

Establishing guidelines towards an assessment of prostheses dysfunction using LDH as a marker is difficult as shown by [M. Suedkamp, A.J. Lercher, F. Mueller-Riemenschneider, K. LaRosee, P. Tossios, U. Mehlhorn, Hemolysis parameters of St Jude Medical hemodynamic valves in aortic position, Int. J. Cardiol (95) (2004) 89-93]. In response to their work we would like to add our data concerning ATS valves (AP) and say a word of caution in interpreting an increase of LDH values.

Nitric oxide promotes in vitro interstitial cell heart valve repair.

BACKGROUND: The cell and molecular biology of heart valve wound repair is not well understood. Valve interstitial cells (IC) are thought to play an important role in valvular wound repair. Because nitric oxide (NO) has been implicated in wound repair, we tested the hypothesis that NO promotes valvular wound repair by examining the presence of the inducible form of nitric oxide synthase (iNOS) in wounded IC monolayers, in vitro. METHODS: Linear denuding wounds were made in confluent monolayers of porcine mitral valve IC plated on glass coverslips. Cultures were fixed at various times (0 to 48 h postwounding), and iNOS was localized in the cells by immunofluorescence microscopy. Cultures were also incubated with iNOS inhibitors L-N(G)-nitroarginine methyl ester (L-NAME) and N-(3-(Aminomethyl)benzyl)acetamidine (1400W), and the extent of wound closure with and without inhibitor was measured at 24, 48 and 72 h postwounding. RESULTS: From 6 to 24 h postwounding, iNOS localization was increased at the wound edge. At 48 h, iNOS was localized beyond the wound edge, into the monolayer, where the intensity of the signal gradually diminished until it was virtually imperceptible. At 24 and 48 h, the inhibition of iNOS with both L-NAME and 1400W resulted in a significant delay in wound closure. CONCLUSION: NO promotes valve wound repair through an effect on IC migration.

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.

Expression of MMP2, MMP9, MT1-MMP, TIMP1, and TIMP2 mRNA in valvular lesions of the heart.

Matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) play an important role in several diseases. This study was undertaken to investigate the mRNA synthesis of MMP2, MMP9, membrane-type 1 (MT1)-MMP, and matrix metalloproteinase inhibitors TIMP1 and TIMP2 by in situ hybridization in a set of heart mitral and aortic valves operatively removed due to degenerative or inflammatory valvular diseases. The material consisted of 21 valves, eight with endocarditis and 13 with a degenerative valvular disease. The samples were studied by in situ hybridization with specific probes for MMP2, MMP9, MT1-MMP, TIMP1, and TIMP2. Synthesis of MMP2 mRNA was found in seven valves, five with endocarditis and two with degenerative valvular disease. Signals for MMP9 mRNA were found in two cases with endocarditis and five cases with degenerative valvular disease. No signal for MT1-MMP mRNA was found in the lesions. TIMP1 mRNA, on the other hand, was found in 17 cases, both endocarditis and degenerative valvular disease. TIMP2 mRNA was found in three cases of endocarditis. The signals for MMP2, MMP9, TIMP1, and TIMP2 mRNA were localized in endothelial cells and in fibroblast-like cells expressing alpha-smooth muscle actin, thus showing myofibroblast-type differentiation. The results show that matrix metalloproteinases MMP2 and MMP9, and matrix metalloproteinase inhibitors TIMP1 and TIMP2 mRNAs are synthesized in diseased valves and suggest that they may contribute to matrix remodelling in valvular disease.