CLINICAL RESEARCH: VALVULAR HEART DISEASE
Increased Oxidative Stress and Cardiomyocyte Myofibrillar Degeneration in Patients With Chronic Isolated Mitral Regurgitation and Ejection Fraction >60%
Mustafa I. Ahmed, MD*,
James D. Gladden, BS*,
Silvio H. Litovsky, MD*,
Steven G. Lloyd, MD, PhD*,
Himanshu Gupta, MD*,
Seidu Inusah, MS*,
Thomas Denney, Jr, PhD ,
Pamela Powell, MS*,
David C. McGiffin, MD* and
Louis J. Dell'Italia, MD*, ,*
* Center for Heart Failure Research, Departments of Medicine, Cardiovascular Surgery, Pathology, and Biostatistics, University of Alabama at Birmingham, Birmingham, Alabama
Birmingham Veteran Affairs Medical Center, Birmingham, Alabama
Auburn University, Samuel Ginn College of Engineering, Auburn, Alabama
Manuscript received December 10, 2008;
revised manuscript received August 20, 2009,
accepted August 31, 2009.
* Reprint requests and correspondence: Dr. Louis J. Dell'Italia, UAB Center for Heart Failure Research, Division of Cardiology, 434 BMR2, 901 19th Street South, Birmingham, Alabama 35294-2180 (Email: loudell{at}uab.edu).
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Abstract
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Objectives: This study assessed myocardial damage in patients with chronic isolated mitral regurgitation (MR) and left ventricular ejection fraction (LVEF) >60%.
Background: Typically, MR patients have decreased LVEF after mitral valve (MV) repair despite normal pre-operative LVEF.
Methods: Twenty-seven patients with isolated MR had left ventricular (LV) biopsies taken at time of MV repair. Magnetic resonance imaging with tissue tagging was performed in 40 normal subjects and in MR patients before and 6 months after MV repair.
Results: LVEF (66 ± 5% to 54 ± 9%, p < 0.0001) and LV end-diastolic volume index (108 ± 28 ml/m2 to 78 ± 24 ml/m2, p < 0.0001) decreased, whereas left ventricular end-systolic (LVES) volume index was 60% above normal pre- and post-MV repair (p < 0.05). The LV circumferential and longitudinal strain rates decreased below normal following MV repair (6.38 ± 1.38 vs. 5.11 ± 1.28, p = 0.0009, and 7.51 ± 2.58 vs. 5.31 ± 1.61, percentage of R to R interval, p < 0.0001), as LVES stress/LVES volume index ratio was depressed at baseline and following MV repair versus normal subjects (0.25 ± 0.10 and 0.28 ± 0.05 vs. 0.33 ± 0.12, p < 0.01). LV biopsies demonstrated cardiomyocyte myofibrillar degeneration versus normal subjects (p = 0.035). Immunostaining and immunoblotting demonstrated increased xanthine oxidase in MR versus normal subjects (p < 0.05). Lipofuscin deposition was increased in cardiomyocytes of MR versus normal subjects (0.62 ± 0.20 vs. 0.33 ± 0.11, percentage of area: p < 0.01).
Conclusions: Decreased LV strain rates and LVES wall stress/LVES volume index following MV repair indicate contractile dysfunction, despite pre-surgical LVEF >60%. Increased oxidative stress could cause myofibrillar degeneration and lipofuscin accumulation resulting in LV contractile dysfunction in MR.
Key Words: mitral regurgitation • oxidative stress • magnetic resonance imaging • tissue tagging
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Abbreviations and Acronyms
| | EF = ejection fraction | | LV = left ventricular | | LVED = left ventricular end-diastolic | | LVES = left ventricular end-systolic | | MR = mitral regurgitation | | MRI = magnetic resonance imaging | | MV = mitral valve | | XDH = xanthine dehydrogenase | | XO = xanthine oxidase |
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Chronic left ventricular (LV) volume overload from isolated mitral regurgitation (MR) results in increased LV diastolic wall stress and eccentric hypertrophy, fostering an initial adaptive LV chamber enlargement (1). A combination of increased pre-load and ejection into the low pressure left atrium fosters favorable loading conditions that can falsely elevate LV ejection fraction (EF) despite underlying cardiomyocyte contractile impairment (2). Therefore, to preserve LV systolic function and improve survival, corrective mitral valve (MV) surgery is recommended if LVEF falls below 60% (3).
There is currently no effective medical therapy to attenuate the progressive LV remodeling resulting from the chronic volume overload stress in isolated MR, and mechanisms of LV myocardial remodeling are poorly understood (3). It is of interest that echocardiographic studies of patients with isolated MR have reported that left ventricular end-diastolic (LVED) dimension decreases as left ventricular end-systolic (LVES) dimension remains unchanged resulting in a decrease in LVEF in patients with isolated MR after MV repair, even when LVEF is >55% (4–7). Thus, it is well accepted that LVEF may belie the degree of dysfunction in the setting of MR.
Indeed, some studies have reported myocardial dysfunction from LV muscle strips, derangement of calcium-handling proteins, and increased cytokines in patients with isolated MR despite LVEF >55% (8–10)—all of which can be associated with and/or attributed to increased oxidative stress. Xanthine oxidase (XO) is well characterized as a major contributor to free radical generation and oxidative stress in the cardiovascular system (11). Furthermore, increased XO has been implicated in cardiovascular disease states including heart failure (12). Recent studies have demonstrated that excessive mechanical stretch in the lung increases XO activity, which plays a prominent role in acute lung injury (13). Whether XO plays a role in adverse LV remodeling and functional impairment resulting from myocardial stretch in isolated MR has not previously been investigated. Accordingly, we obtained LV biopsies at the time of surgery to evaluate the level of myocardial oxidative stress and cardiomyocyte damage in patients who were within conventional guidelines for valve repair (LVEF >60%). In addition, we used magnetic resonance imaging (MRI) with tissue tagging and 3-dimensional analysis to define LV function at the myocardial level before and after MV repair.
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Methods
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Study subjects.
The study protocol was approved by the University of Alabama at Birmingham Institutional Review Board and informed consent was obtained from all participants. The study group consisted of 27 patients (mean age 53 ± 12 years, median age 54 years, age range 34 to 71 years) with severe isolated MR secondary to degenerative MV disease who were referred for corrective MV surgery and all had estimated LVEF >60%. Severe MR was documented on echocardiogram/Doppler studies and cine-MRI in all cases. All patients had cardiac catheterization before surgery and patients with obstructive coronary artery disease (>50% stenosis), aortic valve disease, or concomitant mitral stenosis were excluded from the study. Patients underwent MRI with tissue tagging before surgery and 6 months after MV repair. At the time of surgery, LV myocardial tissue was taken from the lateral endocardial wall of the LV at the level of the tips of the papillary muscles in all patients. MRI with tissue tagging was also performed in control volunteers (age 40 ± 11 years, median age 38 years, age range 21 to 62 years) who had no prior history of cardiovascular disease and were not taking any cardiovascular medications. Of note, 6 control subjects were between the ages of 45 and 50 years, and 9 were over 50 years of age.
Magnetic resonance imaging.
Magnetic resonance imaging was performed on a 1.5-T MRI scanner (Signa GE, Milwaukee, Wisconsin) optimized for cardiac application. Electrocardiographically gated breath-hold steady-state free precision technique was used to obtain standard (2-, 3-, and 4-chamber short-axis) views using the following parameters: slice thickness of the imaging planes 8 mm, field of view 44 x 44, scan matrix 256 x 128, flip angle 45°, repetition/echo times 3.8/1.6 ms.
Three-dimensional LV geometric parameters were measured from endocardial and epicardial contours manually traced on cine-MR images acquired near end diastole and end systole. The contours were traced to exclude the papillary muscles. Cubic B-spline surfaces were fit to the endocardial and epicardial contours for each time frame.
Tagged magnetic resonance images were acquired on the same scanner with repetition/echo times 8/44 ms, and tag spacing 7 mm. Three-dimensional LV strain was measured from tagged images at end systole, which was defined by visual inspection of the image data as the time frame with maximum contraction. Strain computations were conducted using an in-house software package (14,15). Two-dimensional strain rates were measured using harmonic phase analysis (16–18). Harmonic phase analysis measures the local, 2-dimensional strain of the myocardium based on the local spatial frequency of the tag lines. During myocardial contraction, the tag lines become closer to each other and the tag frequency increases in proportion to that contraction. Strain rates were computed at midwall segments as defined by Cerqueira et al. (19).
Calculations.
Three-dimensional wall thickness was computed at the same segments by measuring the distance from a point on the endocardial surface to the closest point on the endocardial surface along a line perpendicular to the epicardial surface. The radius to wall thickness ratio was computed as the reciprocal of the product of the endocardial circumferential curvature ( ) and wall thickness (T). End-systolic wall stress was computed according to the formula:
where P is mean arterial LV blood pressure measured by a cuff measurement at the time of the MR scan. Mean arterial pressure (20) was calculated as: (systolic blood pressure + 2[diastolic pressure])/3.
Surgical methods.
All patients underwent MV repair. The operation was performed through a median sternotomy and employed standard hypothermic cardiopulmonary bypass and cold blood cardioplegia. A variety of methods were used to repair the MV including leaflet resection, chordal replacement, or a combination of each, and all patients had implantation of a flexible annuloplasty ring. The adequacy of repair was assessed by intraoperative transesophageal echocardiography.
Histopathological analysis.
Control LV myocardial specimens for histology were obtained at time of autopsy in patients of comparable age with no evidence of cardiac disease on autopsy (age 24, 28, 31, 34, 45, 49, and 49 years). Biopsies from MR patients were immersion-fixed in 10% neutral-buffered formalin and embedded in paraffin. Hematoxylin and eosin stain was used on 5-µm sections for examination by light microscopy, using high power (40x objective, 1,500x total magnification). A semiquantitative method, with a grading scale of 1 to 4, was used to evaluate 10 randomly selected fields for presence of myofibrillar degeneration. A score of 1 represented 0% to <25% degeneration; 2 represented 25% to <50% degeneration; 3 represented 50% to <75% degeneration; 4 represented 75% to 100% degeneration. A mean grade for each biopsy was used to express myofibrillar degeneration observed in the LV tissue with all measurements performed in a blinded manner.
Immunohistochemistry.
Xanthine Oxidase
Five-micrometer sections were mounted on slides, deparaffinized in xylene and rehydrated in graded solutions of ethanol. After microwave antigen retrieval with 10 mM citrate buffer and blocking with 5% normal serum, sections were incubated with XO antibody (1:50; NeoMarkers, Fremont, California) for 1 h at room temperature, followed with Alexa Fluor 488-conjugated secondary antibody incubation (1:150, Molecular Probes, Eugene, Oregon) for 1 h at room temperature. Slides were mounted with Vectashield Mounting Medium with 4,6-diamino-2-phenylindole for nuclear staining (Vector Laboratories, Burlingame, California). Image acquisition and intensity measurements were performed on a Leica DM6000 epifluorescence microscope with SimplePCI Imaging software (Compix, Inc., Cranberry Township, Pennsylvania). Primary antibody absorbed with purified enzyme served as a negative control for each biopsy section to measure background fluorescence.
Nitrotyrosine
Slides were prepared as above for XO. After blocking with 5% normal serum, sections were incubated with nitrotyrosine antibody (1:100, Upstate, Lake Placid, New York) overnight at 4°C, followed with Alexa Fluor 594-conjugated secondary antibody incubation (1:200, Molecular Probes) for 1 h at room temperature. Primary antibody absorbed with 10 mmol/l of nitrotyrosine served as a negative control for each biopsy section to measure background fluorescence.
Lipofuscin
Five-micrometer sections were stained with lipofuscin stain (AFIP method, Laboratory Methods in Histotechnology, Armed Forces Institute of Pathology, 1994) for imaging. Quantitative analysis was accomplished by light microscopy at medium power (20x objective, 700x total magnification), using a 540-nm (green) filter to provide grayscale contrast for lipofuscin granules. Using images collected by the digital camera, we determined the percent lipofuscin of 30 to 40 randomly selected fields in each section, and the mean value was calculated. All measurements were performed in a blinded manner.
Western blots.
Left ventricular biopsies were obtained from 5 patients undergoing valve repair (4 men, 1 woman, mean age 52 ± 7 years). Normal LV tissue (4 men, 1 woman, mean age 46 ± 4 years) was obtained from Imgenex Corporation (San Diego, California). Samples were homogenized in radioimmunoprecipitation assay buffer containing protease inhibitors. Cell debris and fragments were then removed by centrifugation and the Bio-Rad Bradford assay was performed to determine protein concentration. Fifteen micrograms total protein was separated on a Tris-HCl gel and then transferred to a nitrocellulose membrane. Membranes were then probed with a goat polyclonal antibody directed against human xanthine oxidoreductase (Santa Cruz Biotechnology, Santa Cruz, California). Bound primary antibodies were detected with horseradish peroxidase conjugated secondary antibodies rabbit antigoat immunoglobulin G, followed by a chemiluminescent system. Densitometry analysis was conducted using ImageJ software (National Institutes of Health, Bethesda, Maryland). Membranes were stripped and reprobed with a rabbit polyclonal antibody directed toward calsequestrin (Abcam, Cambridge, Massachusetts). All reported densitometry values are normalized to the loading control calsequestrin.
Statistical analysis.
Values are presented as mean ± SD. The Fisher exact test was used to compare the sex whereas McNemar test was used to compare the other binary characteristics in the pre- and post-surgery groups. Continuous patient characteristics and MRI functional data were analyzed with repeated measures analysis of variance models. Using the MIXED procedure in SAS software (SAS Institute, Cary, North Carolina), these models allowed for comparison of the 3 groups (normal subjects, baseline MR, and 6-month post-MV repair) while adjusting for the within-patient correlation. To avoid inflating the probability of a type I error for the MRI variables, the Bonferroni-Holm step-down test procedure was used to adjust the significance level accordingly. This method is used to adjust the level of significance 0.05, by the number of tests. When comparing biopsy myofibrillar degeneration grades between MR patients and control subjects, Wilcoxon rank sum test, a nonparametric analog of the t test, was used to compare the differences in the mean ranks of the measurements between the MR and control groups. Simple linear regression was used to test associations between biopsy findings (i.e., XO, lipofuscin) and patient characteristics including age and MRI parameters. p < 0.05 was considered significant. All statistical analysis was performed using SAS version 9.1.3.
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Results
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Patient characteristics.
There were no significant differences in body surface areas, heart rates, or blood pressures among control and pre- and post-MV repair groups (Table 1). Mean age of control subjects was 40 ± 11 years and MR patients was 53 ± 12 years. All MR patients were New York Heart Association functional class I (45%) or had only very mild symptoms (class II, 55%) before surgery. No MR patients had atrial fibrillation. All patients had pre-operative LVEF >60%. Nine patients (33%) had pre-surgical LVES dimension  40 mm at time of surgery. Seven patients (26%) had both LVES dimension <40 mm and were asymptomatic at time of surgery.
LV Geometry and Function by MRI with Tissue Tagging
The LVED volume index was increased by >60% in pre-MV repair patients versus control subjects (108 ± 28 ml/m2vs. 66 ± 11 ml/m2, p < 0.05), decreased following MV repair (78 ± 24 ml/m2, p < 0.0001) and did not differ from controls. Pre-MV repair LVES volume index was increased above control subjects (37 ± 14 ml/m2 vs. 23 ± 6 ml/m2, p = 0.0018) and did not change 6 months after MV repair. The LVED and LVES dimensions changed in similar directions as LV volumes (Table 2).
Following MV repair, LVEF decreased from 66 ± 5% to 54 ± 9% (p < 0.001). The LV systolic circumferential and longitudinal strain rates did not differ in control versus MR patients (Fig. 1A). The LV strain rates not only decreased 6 months after MV repair but were also reduced below control subjects (6.38 ± 1.38 vs. 5.11 ± 1.28, p = 0.0009, and 7.51 ± 2.58 vs. 5.31 ± 1.61 percentage of R to R interval, p < 0.0001, respectively) (Fig. 1A). The LVES 3-dimensional radius/wall thickness ratios (Fig. 1B) did not differ significantly between groups (p = 0.192). In contrast, LV mass/volume ratio was decreased (0.76 ± 0.17 vs. 0.67 ± 0.15, p < 0.01) and LV 3-dimensional radius/wall thickness was increased (3.72 ± 0.91 vs. 4.32 ± 1.11, p < 0.01) before MV repair and returned to normal after MV repair, consistent with a reversal of eccentric LV remodeling. Furthermore, when compared with normal subjects, the LV contracted to a higher LVES volume at a relatively small increase in mean arterial pressure that did not achieve statistical significance (91 ± 10 mm Hg vs. 97 ± 10 mm Hg, p = 0.15) but did increase following MV repair (100 ± 10 mm Hg, p < 0.01) (Fig. 2). The LVES wall stress was increased before (p = 0.053) but did increase after MV repair (p < 0.01) compared with normal subjects. However, LVES stress/LVES volume index ratio was decreased before and after MV repair versus normal subjects (0.25 ± 0.10 and 0.28 ± 0.05 vs. 0.33 ± 0.12, p < 0.01).
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Figure 1 LV Systolic Strain Rates and Remodeling Before and 6 Months After MV Repair
The left ventricular (LV) circumferential and longitudinal systolic strain rates (A) are significantly decreased following mitral valve (MV) repair versus before surgery and versus normal control subjects. Three-dimensional left ventricular end-systolic (LVES) (B) radius/wall thickness ratios did not differ between normal and post-surgery groups. Graphs displaying LV mass/volume (C) and left ventricular end-diastolic (LVED) radius/wall thickness (D) demonstrate reversal of eccentric remodeling following MV repair. *p < 0.05 versus control group; p < 0.05 versus before MV repair. RR = R to R interval.
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Figure 2 Indexes of LV Afterload and Function Before and 6 Months After MV Repair
Graphs displaying mean arterial pressures (A), LVES wall stress (B), LVES volume index (C), and LVES stress/volume index (D) in normal subjects and pre- and post-surgical mitral regurgitation patients. Abbreviations as in Figure 1.
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Myocardial biopsy histology findings.
Mitral regurgitation hearts demonstrated marked myofibrillar degeneration (2.32 ± 0.82 vs. 1.25 ± 0.23, p = 0.035 [mean degeneration grade 1 to 4]), as demonstrated in Figures 3A to 3C. Immunostaining of biopsies for XO/xanthine dehydrogenase (XDH) (Figs. 3D to 3F) demonstrated mean intensity increase in MR hearts versus control subjects (88 ± 38% vs. 33 ± 11%, p < 0.01). In normal patients, XO was diffusely distributed and clearly associated with myofibrillar Z bands. In MR patients, however, XO showed a punctate, perinuclear pattern, with distribution in close proximity to areas of myofibrillar degeneration. Immunostaining for lipofuscin (Figs. 3G to 3I) demonstrated marked deposition in MR hearts compared with control subjects (0.59 ± 0.21 vs. 0.33 ± 0.13 volume percentage, p < 0.005). Of note, no significant association was noted between age and lipofuscin deposition in MR patients. Nitrotyrosine staining was prominent in areas of lipofuscin accumulation and myofibrillar degeneration (Fig. 3). Transmission electron microscopy in a representative patient with MR demonstrated the electron-dense bodies that are consistent with lipofuscin located in a perinuclear distribution (Fig. 4). There were no significant associations noted on linear regression analyses between biopsy measures of oxidative stress (XO, lipofuscin) and various MRI measures of LV function in MR patients.
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Figure 3 Myofibrillar Loss and Oxidative Stress in the LV of Patients With Isolated MR
Myocardial biopsy findings in control subjects (n = 10) (A, D, G) and mitral regurgitation (MR) patients (n = 27) demonstrating myofibrillar degeneration (B and C), increased xanthine oxidase (E and F), and increased lipofuscin (H and I). Nitrotyrosine staining in the left ventricle (LV) of an MR patient, demonstrating increased staining in areas of lipofuscin accumulation (J) and a corresponding image (K) with immunoabsorbed antibody with no uptake of antibody and only autofluorescence of lipofuscin. *p < 0.05 versus normal control group. Scale bar = 20 µm.
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Figure 4 Lipofuscin in LV Electron Micrographs of Patients With Isolated MR
Transmission electron microscopy of endomyocardial biopsy samples demonstrating marked lipofuscin deposition (arrowheads) in the hearts of MR patients. A: bar = 2 µm. B: bar = 0.5 µm. Abbreviations as in Figure 3.
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Western blot analysis for XO.
XO is produced by either a transient or permanent modification of XDH. On Western blot analysis (Fig. 5), XDH and transiently modified XO (145-kD band) were increased >5-fold in density in MR patients versus control subjects (p = 0.0125). Permanently modified XO is represented by a 125-kD and an 85-kD band. The 125-kD band demonstrated a >5 fold increase in MR versus normal control subjects (p = 0.00098), whereas the 85-kD band did not differ significantly between groups (p = 0.128). Overall, total XDH/XO expression (145 kD + 125 kD +85 kD) was increased 2.6-fold (p = 0.032) in MR patients versus control subjects.
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Figure 5 Protein Quantification of XO in LV of Patients With Isolated MR
Western blot analysis for xanthine oxidoreductase (XOR) (A) depicts a band at 145 kD, which represents both xanthine dehydrogenase and transiently modified xanthine oxidase (XO), and bands at 125 and 85 kD, which represent permanently modified XO. Densitometry depicting average band intensity normalized to respective calsequestrin loading control (B). *p < 0.05 versus normal control group. Abbreviations as in Figure 3.
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Discussion
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In this study, we demonstrate LV systolic dysfunction 6 months after MV repair in patients with isolated degenerative MR despite pre-surgical LVEF >60%. Decreased LV strain rates in the absence of increases in indexes of LVES wall stress, along with a decreased LVES stress/volume ratio, strongly suggests underlying myocyte dysfunction following MV repair. This contention is further supported by the presence of marked myocyte myofibrillar degeneration and lipofuscin accumulation in hearts of isolated MR patients before surgery.
The decrease in LVEF following MV repair has been attributed to a decrease in pre-load and a relative increase in afterload due to the correction of regurgitation into the low pressure left atrium (2,21). However, other studies report a stable LVEF with chordal preservation (22). In the current study, in which all patients had MV repair, MRI-derived LVED volume index and 3-dimensional radius/wall thickness ratio decreased as LV mass/volume ratio returned to normal, all of which indicate a reversal of eccentric cardiac hypertrophy. However, the LVES stress/volume index ratio, which has been shown to predict adverse outcomes in patients with isolated MR (23), was decreased both before- and after MV repair, suggesting a decreased LV contractility. The LVES wall stress was increased at baseline and at 6 months after repair. Further, when compared with normal subjects, the LV contracted to a higher LVES volume index in the presence of a relatively small increase in mean arterial pressure, which also suggests decreased LV contractility at baseline and at 6 months following MV repair.
Previous studies in patients with isolated MR and LVEF >55% have demonstrated decreases in the force-frequency effect in isolated LV muscle strips (9) and in LV calcium handling proteins (8), indicative of mechanical and biochemical markers of heart failure. In another study, LV tumor necrosis factor alpha was increased in patients with isolated MR and well-preserved LVEF (10). In our patients, there was marked deposition of lipofuscin, a nondegradable material primarily composed of oxidatively modified protein and lipid degradation residues (24). Lipofuscin accumulation is usually seen in the senile heart and is considered to be an irreversible end product of excessive oxidative stress that overwhelms protective mechanisms (25). Lipofuscin accumulation has been shown to have deleterious effects on cellular function including triggering of mitochondrial pro-apoptotic pathways in cardiomyocytes and fibroblasts (26,27), and its accumulation in the heart is irreversible (24). Thus, it is tempting to speculate that the cumulative effects of prolonged oxidative stress and lipofuscin accumulation in the volume overloaded heart could account for LV contractile dysfunction at baseline and at 6 months after MV repair. It must be noted, however, that LV function in patients with aortic regurgitation and aortic stenosis may continue to improve for years following surgery, thus the definitive impact of our pre-operative findings may be unknown at this time (28,29).
Another marker of heart failure was cardiomyocyte vacuolization with extensive myofibrillar degeneration. This has also been reported in the clinically relevant model of MR in the dog (30). Myofibrillar degeneration can occur as a result of increased oxidative stress, and indeed degraded myofibrils may also constitute a part of lipofuscin. We also found increased levels of xanthine oxidoreductase, which when transformed from its parent enzyme XDH into its oxidase form XO, generates superoxide and hydrogen peroxide upon conversion of xanthine to hypoxanthine and hypoxanthine to uric acid, respectively (11). The XO activity was up-regulated in the LV of humans with dilated cardiomyopathy and intracoronary infusion of allopurinol improved LV contractile performance without increasing myocardial oxygen consumption (12). In vitro studies demonstrated that XO depresses myofilament sensitivity to calcium and that it colocalized with nitric oxide synthase-1 in the sarcoplasmic reticulum in the mouse cardiomyocyte, which could regulate excitation-contraction coupling as well as myofilament oxidative damage (31,32). Interestingly, staining of nitrotyrosine—a marker of oxidative and nitrosative damage—was increased in areas of myofibrillar degeneration and lipofuscin accumulation in the MR hearts. Xanthine oxidase is a major source of superoxide and its combination with NO produces peroxynitrite. Thus, the proximity of XO to these markers of excessive nitration in the myocyte suggested a pathophysiological role for XO in the cardiomyocyte myofibrillar degeneration.
The current study cannot directly determine cause and effect of XO-mediated oxidative damage and the decrease in LV function following MV repair. Nevertheless, the marked lipofuscin accumulation and myofibrillar degeneration in LV endomyocardial biopsies are indeed markers of heart failure that were present in patients with otherwise normal LV strain rates and LVEF before surgery. This underscores the unreliability of ejection phase indexes in reflecting underlying LV myocardial damage in our and other studies (8–10). These findings may be of use in defining the optimal timing of MV surgery. Future studies are required to determine whether XO and persistent oxidative stress are causative in maladaptive LV remodeling and offer potential therapeutic targets in ameliorating LV damage in patients with isolated MR.
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Footnotes
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Supported by Specialized Centers of Clinically Oriented Research grant (P50HL077100) in cardiac dysfunction.
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References
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