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Myocardial oxidative stress in patients with active infective endocarditis
IJCA-14349; No of Pages 7 International Journal of Cardiology xxx (2012) xxx–xxx
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International Journal of Cardiology journal homepage: www.elsevier.com/locate/ijcard
Myocardial oxidative stress in patients with active infective endocarditis Stanislaw Ostrowski a, Marek Kasielski b, Jacek Kordiak c, Anna Zwolinska d, Anna Wlodarczyk d, Dariusz Nowak d,⁎ a
Department of Cardiosurgery, Medical University of Lodz, Poland Bases of Clinical Medicine Teaching Center, Medical University of Lodz, Poland 90-153 Lodz, Kopcinskiego St. 20 Department of Chest Surgery, Oncologic and General Surgery, University Hospital No. 2, Medical University of Lodz, Poland d Department of Clinical Physiology, Medical University of Lodz, Mazowiecka St. 6/8, 92-215 Lodz, Poland b c
a r t i c l e
i n f o
Article history: Received 11 February 2011 Received in revised form 13 June 2011 Accepted 24 December 2011 Available online xxxx Keywords: Infective endocarditis Valvular heart disease Oxidative stress Hydrogen peroxide Thiobarbituric acid reactive substances
a b s t r a c t Background: Infective endocarditis (IE) induces the rise of pro-inflammatory cytokines. Some of them can stimulate oxidants production in myocardium with subsequent peroxidative damage to various biomolecules. We compared indices of oxidative stress: H2O2, thiobarbituric acid-reactive substances (TBARs), thiols in myocardium specimens between patients with active IE and those with valvular heart disease (VHD) of rheumatic etiology who underwent surgical valve replacement. Methods: 17 left ventricle papillary muscle specimens and 28 specimens of auricle of the right heart were collected from 45 IE patients, and 16 papillary muscle and 12 auricle specimens from 28 VHD patients, respectively. Patients groups had similar NYHA functional class and majority of echocardiographic indices of heart morphology. H2O2 and TBARs were determined fluorometrically in myocardium homogenates whereas thiols with photometric method. Between and within groups comparisons and mutual correlations between variables were analyzed. Results: H2O2 generation from all myocardium specimens and auricles was 2.14- and 2.59- times higher (p b 0.001) in IE patients than in VHD group. Auricles had the highest H2O2 levels within IE group. TBARs were 10-times higher (p b 0.05) in IE when compared to VHD group in auricles and papillary muscles. Thiols did not differ between groups. H2O2 positively correlated with TBARs and negatively with thiols in all IE myocardium specimens (r = 0.31 and r = − 0.46, p b 0.05) and auricles (r = 0.58 and r = − 0.67, p b 0.05), respectively. No such associations were noted in VHD specimens. Conclusions: Active IE induces enhanced myocardial production of H2O2 and formation of TBARs which proves occurrence of oxidative stress in the heart. © 2011 Published by Elsevier Ireland Ltd.
1. Introduction Oxidative stress is implicated in the development of congestive heart failure in humans [1,2]. Evidences for this are coming from ex vivo analysis of myocardium specimens from patients with end-stage heart failure [3–6] and are consistent with the results from experiments on animals with pacing- ,aortic banding-, and pharmacologicallyinduced heart insufficiency [7–10]. Increased activity of NADPH oxidase seems to be the main source of ROS overproduction in the failing heart [3,11]. Angiotensin II, endothelin-1, TNF-α, and also mechanical forces belong to activators of cardiomyocyte NADPH oxidase [1]. This enzyme generates excessive amounts of superoxide radicals that subsequently dismutate into hydrogen peroxide (H2O2). Both, superoxide radicals and H2O2 together with tissue iron drive hydroxyl
⁎ Corresponding author. Tel.: + 48 426781800; fax: + 48 436782661. E-mail address: [email protected] (D. Nowak).
radicals formation in the failing myocardium [12]. Hydroxyl radicals are extremely reactive and can induce peroxidative damage to membrane lipids, proteins, carbohydrates, and nucleic acids resulting in the rise of myocardial levels of thiobarbituric acid-reactive substances (TBARS) in patients with heart failure [3,6]. Enhanced myocardial ROS generation (e.g. hydroxyl radicals) correlated with contractile dysfunction of the left ventricle [12], and was accompanied by decreased concentrations of reduced glutathione a main donor of sulfhydryl groups in myocardium homogenates [7]. However, little is known about the effect of infective processes in the heart on oxidants-antioxidants balance in the myocardium. Infective endocarditis (IE) represents an infection that most commonly involves heart valves and results frequently in the refractory congestive heart failure due to new or worsening valve dysfunction [13]. IE is accompanied by local [14] and strong systemic inflammatory response with elevated circulating IL-6, IL-2R and IL-1β concentrations [15–17]. These cytokines together with a variety of bacteria-derived products (e.g. formylated chemotactic peptides, lipopolysaccharides)
0167-5273/$ – see front matter © 2011 Published by Elsevier Ireland Ltd. doi:10.1016/j.ijcard.2011.12.102
Please cite this article as: Ostrowski S, et al, Myocardial oxidative stress in patients with active infective endocarditis, Int J Cardiol (2012), doi:10.1016/j.ijcard.2011.12.102
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can enhance (via activation of NADPH oxidase) ROS production by circulating polymorphonuclear leukocytes and monocytes [18]. Although plasma TNF-α levels were not elevated in the course of IE [15], this cytokine immunoreactivity was found in right atrium specimens and heart valves of patient suffering from this disease [14,19]. It is likely that some of these mediators may enhance myocardial ROS generation in the course of cardiac failure related to IE-induced valve damage. Therefore, in this study we compared H2O2 generation in homogenates of myocardium specimens obtained during surgical valve replacement in patients with active IE and with acquired valvular heart disease (VHD). Furthermore, the concentrations of low molecular weight thiols (R-SH, e.g. cysteine, reduced glutathione) and TBARs as a marker of peroxidative damage to myocardium were also determined. In the end, the mutual associations between these variables and their relationships with selected clinical parameters were evaluated.
Table 2 Patients preoperative condition. Parameter
Infective endocarditis
Acquired valvular heart disease
ESR (mm/h) RBC (x106/μl)
50 ± 37 (43 ;2–145) * 4.07 ± 0.78 (3.90; 2.82–6.08) 8.9 ± 3.6 (8.4; 6.5–20.0) * 37 ± 7 (35; 25–55) 248 ± 102 (228 ;68–493) 1.14 ± 0.76 (0.90; 0.60–4.60) * 43 ± 27 (38 ;10–154) 0.97 ± 0.67 (0.73; 0.20–3.05) 27 ± 15 (25; 10–78) 32 ± 23 (25; 7–124) 6.58 ± 0.73 (6.78; 4.61–7.95) * 1025 ± 341 (929; 516–1867) 25.4 ± 13.3 (22.7; 3.1–66.7) *
20 ± 11 (17 ;5–53) 4.52 ± 0.55 (4.53;3.47–5.44) 5.9 ± 1.8 (5.5 ;3.1–9.3) 42 ± 4 (42; 32–53) 201 ± 66 (189;75–345) 0.97 ± 0.26 (0.90; 0.49–1.59) 42 ± 13 (44; 22–71) 1.43 ± 1.20 (1.17; 0.32–2.82) 28 ± 11 (27; 11–44) 28 ± 15 (24; 8–72) 6.97 ± 0.63 (6.98; 5.69–8.76) 978 ± 316 (867; 337–1460) 15.4 ± 6.9 (14.9; 6.1–27.7)
WBC (x103/μl) HCT (%) PLT (x103/ μl) Creatinine (mg/dl) Urea (mg/dl) Bilirubin (mg/dl) AST (IU/L) ALT (IU/L) Total protein (g/dl) FRAP [μmol/L]
2. Materials and methods DPPH decomposition [%]
2.1. Study population and study protocol The study was conducted from March 2007 to January 2010 and involved 45 patients with active infective endocarditis (IE) and 28 patients with acquired valvular heart disease (VHD) of rheumatic etiology that underwent surgical treatment (valve replacement) using standard cardiopulmonary bypass (CPB) with crystalloid cardioplegia (Tables 1 and 2). Patients with VHD were without any signs of infection. The clinical diagnosis of IE was established in all patients according to the modified Duke criteria [20]. Native valve IE was present in 42 patients and prosthetic valve IE in 3. The inclusion criteria were: presence of active IE or acquired VHD of rheumatic etiology, age between 40 and 80 years, and a written informed consent. The exclusion criteria included the presence of any other active infectious or inflammatory process that could affect cellular ROS production, any history of autoimmune-disorders (apart from past rheumatic fever), acquired and inherited immunological defects, diabetes, pregnancy, alcohol and illicit drug abuse, chronic renal and liver insufficiency, current cigarette smoking, and usage of any vitamins and food supplements with antioxidant activities within 3 weeks preceding the cardiac surgery. Previous risk factors in patients with native valve IE included: preexisting defective valve (n = 17), prior endocarditis (n = 1), untreated dental caries (n= 7), prior respiratory tract infection (n= 7), prior urinary tract infection (n= 2), implanted pacemaker (n= 1), unknown (n = 10). Five ml of venous blood were drawn into EDTA-K3 vacuette-tubes (Greiner bio-one GmbH, Kremsmunster, Austria) at approximately 8:30 a.m. the day before cardiac
Table 1 Patients’ characteristics. Variable
Number Sex F/M Age (years) NYHA functional class EF (%) LA (cm) LVEDD (cm) LVESD (cm) LVPWDD (cm) LVPWSD (cm) RA (cm) RVEDD (cm) IVSDD (cm) IVSSD (cm) Embolism (CNS/peripheral) Atrial fibrillation
Patients that underwent cardiac valve surgery Infective endocarditis
Acquired valvular heart disease
45 15/30 52 ± 12 (53; 43–77) * I- 0, II- 8, III-28, IV-9 57 ± 8 (58; 38–80) 4.9 ± 0.8 (4.9; 2.8–7.0) 5.8 ± 0.7 (5.7; 4.6–8.1) * 4.0 ± 0.8 (4.0; 2.4–5.8) * 1.1 ± 0.1 (1.1; 0.8–1.5) 1.4 ± 0.2 (1.5; 1.1–1.8) 5.3 ± 0.7 (5.4; 3.2–6.7) 2.6 ± 0.4 (2.5; 1.6–3.5) 1.2 ± 0.2 (1.2; 0.8–1.6) 1.4 ± 0.2 (1.4; 1.0–2.2) 8 (3/5) 9
28 20/8 62 ± 10 (62 ;41–89) I-0, II-7, III-15, IV-6 56 ± 8 (57; 40–75) 5.1 ± 0.8 (4.9; 3.3–6.7) 5.1 ± 0.7 (4.9; 4.1–6.7) 3.4 ± 0.6 (3.3; 2.5–5.4) 1.1 ± 0.2 (1.0; 0.8–1.4) 1.4 ± 0.2 (1.3; 1.1–1.8) 5.4 ± 0.9 (5.2; 4.0–7.3) 2.6 ± 0.3 (2.5; 2.2–3.5) 1.1 ± 0.1 (1.1; 0.9–1.5) 1.4 ± 0.2 (1.4; 1.1–1.9) 6 (4/2) 11
EF – ejection fraction of the left ventricle; LA - left atrial diameter; LVEDD – left ventricular end-diastolic diameter; LVESD – left ventricular end-systolic diameter, LVPWDD – left ventricular posterior wall diastolic diameter; LVPWSD – left ventricular posterior wall systolic diameter; RA – right atrial diameter; RVEDD – right ventricular end-diastolic diameter; IVSDD – interventricular septum diastolic diameter; IVSSD – interventricular septum systolic diameter; CNS – central nervous system. Echocardiographic indices of heart morphology were determined within 7 days before surgery. * different vs. acquired valvular heart disease group; p b 0.05. In parentheses, median with minimal and maximal value.
ESR – erythrocyte sedimentation rate; RBC – red blood cells; WBC – white blood cells; HCT – hematocrit; PLT – platelets; AST – aspartate aminotransferase; ALT – alanine aminotransferase; FRAP - ferric reducing ability of plasma, DPPH decomposition serum DPPH-radical scavenging activity. * different vs. acquired valvular heart disease group; p b 0.05. In parentheses, median with minimal and maximal value.
surgery for the determination of blood cell count, chemistry and blood antioxidant activity. Samples of myocardium (slice of papillary muscle of the left heart or auricle of the right heart, 50 to 70 mg of wet tissue) were collected during heart surgery for ex vivo assessment of selected parameters of oxidative stress. The study, conformed to the Declaration of Helsinki, was approved by The Medical University of Lodz Ethics Committee (approval No RNN/139/KE). All participants provided a written, informed consent prior to their inclusion in the study.
2.2. Preoperative patient management Antibiotics that were shown to be effective through blood culture tests were used for 3 to 4 weeks prior to surgery in patients with active IE. When blood cultures were negative, vancomycin alone or in combination with gentamicin was used empirically. Patients with VHD did not receive any antibiotics before surgery. Both patient groups received diuretics, digitalis, and beta-blockers if required. No patient received systemic steroids or non-steroidal anti-inflammatory drugs within the three weeks preceding the heart operation.
2.3. Collection of myocardium samples Patients premedication, anesthesia, ventilation mode, CPB with crystalloid cardioplegia were the same in both groups and were described previously [18]. Slice of auricle of the right heart was taken just before placing a cannula of CPB in the right atrium mostly in patients with infected or insufficient aortic valve. Sample of papillary muscle was collected during excision of involved mitral valve in patients on CPB after infusion of cardioplegic solution and aorta clamping. One sample of myocardium was taken from one patient. Thus 40 auricle samples (28 from IE patients and 12 from VHD patients) and 33 left ventricle papillary muscle samples (17 from IE patients and 16 from VHD patients) were collected for further analysis. All patients underwent surgical valve replacement with mechanical valve prostheses (St Jude medical or Medtronic Hall). The duration of aortic clamping (cross-clamp time) and CPB as well as the volume of cardioplegic solution used during surgery were similar in both groups (Table 3). Intra-operative valve-tissue cultures were positive in 29 patients with IE (Staphylococcus sp. = 13, Streptococcus sp. = 8, Enterococcus sp. = 7, Corynebacterium sp. = 1). These cultures performed in patients with VHD were always negative.
2.4. Storage of myocardium samples and preparation of tissue homogenate Specimens of myocardium (slices of papillary muscle and auricle of the right heart) were washed in ice-cold sterile 0.9% NaCl, desiccated with lignin and stored at − 80 °C for not longer than 2 weeks. Samples of myocardium (25 mg) were homogenized in 1 ml of 1.15 mol/l KCl (glass homogenizer, on ice), centrifuged (5 min, 1500 ×g, 4 °C) and supernatant immediately used for measurement of TBARs, protein concentration, and spontaneous H2O2 release. Part of tissue specimens (25 mg) were homogenized in 0.5 ml of ice-cold 6% trichloroacetic acid, then centrifuged (10,000 ×g, 20 min, 4 °C), and supernatant assayed for concentration of R-SH.
Please cite this article as: Ostrowski S, et al, Myocardial oxidative stress in patients with active infective endocarditis, Int J Cardiol (2012), doi:10.1016/j.ijcard.2011.12.102
S. Ostrowski et al. / International Journal of Cardiology xxx (2012) xxx–xxx Table 3 Position of infected and or insufficient valve and operative data in the studied groups. Parameter
Involved valve Aortic Mitral Aortic + Mitral CPB time (min) Cross-clamp time (min) Cardioplegic solution (ml) Normothermia Hypothermia (32 °C)
Patients that underwent cardiac valve surgery* Infective endocarditis
Acquired valvular heart disease
16 21 8 143 ± 51 (150; 65–240) 80 ± 24 (77; 45–132)
8 14 6 176 ± 28 (180; 80–185) 74 ± 21 (73; 40–125)
1250 ± 310 (1200; 800–2600) 32 13
1320 ± 300 (1300; 900–1800) 20 8
CPB – cardiopulmonary bypass. * replacement with mechanical valve prostheses. In parentheses, median with minimal and maximal value.
3. Reagents Albumin from bovine serum, catalase from Aspergillus niger (6600 U/mg protein), 2,2-diphenyl-1-picryl-hydrazyl radical (DPPH), 5-5’-dithio-bis-(2-nitrobenzoic acid) (DTNB), ethylenediaminetetraacetic acid (EDTA), homovanillic acid (HVA), peroxidase from horseradish type II (HRP, 291 U/mg protein), reduced glutathione (GSH), thiobarbituric acid (TBA), and 2,4,6-tripirydyl-s-triazyne (TPTZ) were purchased from Sigma (St Louis, MO, USA). Sterile 0.9% NaCl and phosphate buffered saline (PBS, pH 7.4) were acquired from Biomed (Lublin, Poland). All other reagents were obtained from POCH (Gliwice, Poland) and were of analytical grade. Sterile, deionized, pyrogen-free water for HPLC (resistance > 18 Ωcm, Water Purification System USF ELGA, Buckinghamshire, UK) was used throughout the study. 3.1. Spontaneous hydrogen peroxide release from myocardium homogenates Hydrogen peroxide generation was determined according to the Ruch method [21] with some modifications [22]. A 10 μl aliquot of myocardium homogenate was mixed with 90 μl of PBS and 100 μl of HRP (1 U/ml) containing 400 μmol/l HVA and incubated for 60 min at 37 °C. Then, the sample was mixed with 300 μl of PBS and 125 μl of 0.1 mol/l glycine-NaOH buffer (pH 12.0) with the addition of 25 mmol/l EDTA. The HVA-derived oxidation product, as a measure of the amount of H2O2, was determined fluorometrically (Perkin Elmer Luminescence Spectrometer LS-50-B, Norwalk, CT, U.S.A.). Excitation length was set at 312 nm and emission was measured at 420 nm. Control samples received: (A) myocardium homogenate and HRP but without HVA, and (B) 10 μl of PBS instead of organ homogenate. Readings were converted into μmoles of H2O2 per liter (μmol/l) using calibration curve obtained from 3 series of calibration experiments with increasing standard concentrations of H2O2 (10 nmol/l to 10 μmol/l, 19 concentration-points). The lower limit of H2O2 detection was 0.08 μmol/l. Twenty units of catalase added to samples of myocardium homogenate (n = 5) before mixing with further substrates completely abolished HVA oxidation. 3.2. Measurement of TBARs in myocardium homogenate The concentration of TBARs was estimated as previously described [22]. Briefly, 100 μl of myocardium homogenate was mixed for 30 s with 1 ml of 0.05 mol/l sulphuric acid and 0.5 ml of 1.23 mol/l trichloroacetic acid and then allowed to stand for 5 min. After centrifugation (1500 ×g, 10 min, 4 °C), the supernatant was discarded and the pellet resuspended in 2 ml of TBA solution (0.67 g dissolved in
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100 ml distilled water, then diluted 1:1 with glacial acetic acid) with addition of 10 μl of 0.01% butylated hydroxytoluene. The solution was boiled for 30 min, allowed to cool at room temperature and the chromogen formed in the reaction was extracted into 2.5 ml of butanol by vigorous shaking for 30 s. Following centrifugation (1500 ×g, 10 min, 20 °C), TBA-reactive substances in the butanol layer were measured fluorometrically (excitation 515 nm, emission 546 nm). Control samples received 100 μl of 1.15 mol/l KCl instead of myocardium homogenate. Readings were converted into μmoles per liter using calibration curve obtained from 3 series of calibration experiments with increasing standard concentrations of tetramethoxy-propane (0.01 to 10 μmol/l, 10 concentration-points). The lower limit of TBARs detection was 0.07 μmol/l. 3.3. Measurement of thiols concentration in myocardium homogenates Thiols (R-SH) concentration in myocardium homogenates was measured with DTNB assay as previously described [23] with some modifications. Briefly: 250 μl of myocardium homogenate was mixed with 250 μl of 0.3 mol/l Na2HPO4 buffer (pH = 7.5) and 250 μl of 0.04% DTNB in 10% sodium citrate and after 5 min incubation at 37 °C the absorbance was measured at 412 nm (Ultrospec III, Pharmacia LKB spectrophotometer). Control samples received 250 μl of 6% trichloroacetic acid solution instead of myocardium homogenate. Results were expressed in μmoles per liter using the calibration curve executed with the concentration of GSH increasing from 0.1 to 200 μmol/l (20 concentration-points). The method sensitivity was 0.25 μmol/l of GSH. 3.4. Other techniques Ferric reducing ability of plasma (FRAP) and serum DPPH-radical scavenging activity were measured as previously described [24,25] and expressed in μmol/l of reduced Fe 3+ over 3 min incubation and % of decomposed DPPH radical over 30 min incubation, respectively. Protein concentration in samples of myocardium homogenate was determined using the Lowry method [26] with bovine serum albumin as a standard. 3.5. Statistical analysis All data were expressed as the mean and standard deviation (SD) as well as the Median (Me) with minimal (Min) and maximal (Max) value. Shapiro-Wilk W test was used for normality testing. The differences between groups were assessed using the t-student test for normal data and with U Mann-Whitney test for data with abnormal distribution. Correlations between measured variables were determined using Pearson's r (normal data) and Spearman's ρ (abnormal data distribution). A p value of b0.05 was considered significant. 4. Results 4.1. Preoperative conditions -comparison between IE and VHD patients Tables 1 and 2 show the comparison of IE group and VHD patients. Majority of echocardiographic indices of the heart morphology and function did not differ between these groups. Only the left ventricular end-diastolic diameter and left ventricular end-systolic diameter were higher in IE patients than in VHD subjects (Table 1). IE group had higher pre-surgery erythrocyte sedimentation rate, white blood cell count, and lower total plasma protein suggesting persistent strong inflammatory response in this group (Table 2). Moreover, blood antioxidant activity, especially serum DPPH-radical scavenging activity, was higher in IE patients than in VHD group, probably as a compensatory response to increased ROS production by circulating phagocytes [18]. Mean plasma creatinine was higher in IE group.
Please cite this article as: Ostrowski S, et al, Myocardial oxidative stress in patients with active infective endocarditis, Int J Cardiol (2012), doi:10.1016/j.ijcard.2011.12.102
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S. Ostrowski et al. / International Journal of Cardiology xxx (2012) xxx–xxx
4.2. Hydrogen peroxide release from myocardium homogenates Spontaneous H2O2 release from all myocardium specimens (right auricle plus papillary muscle) was 2.14 times higher in patients with IE than in VHD group (Fig. 1). This difference was even bigger when only auricle specimens were compared. In this case the H2O2 release was 2.59-times higher (123.7 ± 43.8 μmol/L vs. 47.7 ± 21.9 μmol/L) in specimens obtained from patients with IE than from those with VHD. Although homogenates of left ventricle papillary muscle slices from IE group generated 1.52- times more H2O2 than those from VHD patients, this difference did not reach statistical significance (p = 0.28), probably due to the high inter-individual variability of H2O2 readings especially in the IE patients (Fig. 1). There was a significant difference (p b 0.001) between H2O2 generation in auricle (n = 28) and papillary muscle (n = 17) homogenates within IE group. No such difference was noted within group of VHD patients. There were no significant differences in H2O2 release (and other measured markers of oxidative stress) between myocardium specimens from IE patients with (n = 29) and without (n = 16) positive culture of intra-operative valve-tissue (data not shown). Similarly, there was no difference between female and male patients within IE group and VHD group, respectively. 4.3. Thiobarbituric homogenates
acid-reactive
substances
in
myocardium
Both, auricle and papillary muscle homogenates from IE group revealed means TBARs concentrations are about 10-times higher (p b 0.05) than corresponding samples from VHD patients (Fig. 2). No significant differences were noted between TBARs content in papillary muscle and auricle slices homogenates within the IE and the VHD groups, respectively. Corresponding specimens from male and female patients revealed similar TBARs levels within both groups (data not shown). 4.4. Protein and thiols concentration in myocardium homogenates There were no significant differences (p > 0.05) between mean protein concentrations in auricle (4.6 ± 1.9 mg/ml vs. 5.4 ± 1.1 mg/ ml), papillary muscle (4.3 ± 1.6 mg/ml vs. 5.6 ± 2.5 mg/ml), and all myocardium (4.5 ± 1.9 mg/ml vs. 5.5 ± 2.0 mg/ml) specimens homogenates between IE and VHD patients, respectively. Similarly,
there was no difference between corresponding specimens within patient groups. Concentration of R-SH did not differ (p > 0.05) between corresponding specimens and was as follows: IE group – papillary muscle 9.2 ± 9.7 (Me11.0; Min 0.1– Max 24.1) μmol/l , auricle 14.5 ± 7.4 (12.6; 0.5–33.9) μmol/l, all myocardium specimens 12.8 ± 8.4 (12.1; 0.1–33.9) μmol/l: VHD patients – papillary muscle 11.0 ± 14.6 (11.6; 0.2–57.4) μmol/l, auricle 10.7 ± 6.1 (10.7; 0.4–20.6) μmol/l, and all myocardium specimens 10.9 ± 11.3 (11.3; 0.2–57.4) μmol/l. There was no significant difference between corresponding myocardium specimens between and within patient groups and also between female and male patients.
4.5. Correlations between measured variables There was a positive moderate correlation between H2O2 activity and TBARs levels both in auricle homogenates and all myocardium specimens obtained from patients with IE (Table 4). These specimens revealed also significant negative correlation between TBARs levels and concentrations of R-SH. Negative correlation between H2O2 activity and R-SH was noted in homogenized samples of auricles from IE group (Table 4). No significant associations between measured variables (H2O2 with TBARs, H2O2 with R-SH, TBARs with R–SH) were noted in papillary muscle homogenates either in IE group or in VHD group. Negative correlation between H2O2 activity and the concentration of RSH in all myocardium specimen homogenates was the only significant association observed in patients with VHD (Table 4). Majority of data shown in Tables 1 and 2 (FRAP and DPPH test, measures of plasma antioxidant activity, patients age) did not correlate with oxidative stress parameters determined in myocardium specimens in both patient groups. No positive association between H2O2 activity as well as TBARs concentration and echocardiographic indices of heart morphology was noted in patients with IE and VHD. In particular H2O2 activity did not correlate (p > 0.05) with left ventricular end-diastolic diameter (r = − 0.01), left ventricular endsystolic diameter (r = 0.06), left ventricular posterior wall diastolic diameter (r = − 0.23) and left ventricular posterior wall systolic diameter (r = − 0.10) in all myocardium specimens from IE patients. Similarly no significant correlations were found between myocardial TBARs levels and left ventricular end-diastolic diameter (r = −0.11) and left ventricular end-systolic diameter (r = − 0.03), respectively.
Fig. 1. Spontaneous H2O2 generation in homogenates of myocardium specimens (auricle of the right heart; 28 – infective endocarditis, 12 – valvular heart disease, and papillary muscle of the left heart; 17- infective endocarditis, 16- valvular heart disease) collected during valve replacement surgery from 45 patients with active infective endocarditis (closed bars) and 28 patients with acquired valvular heart disease of rheumatic etiology (open bars). * p b 0.001 vs. valvular heart disease group. In parentheses, median with minimal and maximal value.
Please cite this article as: Ostrowski S, et al, Myocardial oxidative stress in patients with active infective endocarditis, Int J Cardiol (2012), doi:10.1016/j.ijcard.2011.12.102
S. Ostrowski et al. / International Journal of Cardiology xxx (2012) xxx–xxx
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Fig. 2. Concentration of thiobarbituric acid-reactive substances (TBARs) in homogenates of myocardium specimens (auricle of the right heart and papillary muscle of the left heart) collected during valve replacement surgery from 45 patients with active infective endocarditis (closed bars) and 28 patients with acquired valvular heart disease of rheumatic etiology (open bars). Other details as for Fig. 1. # p b 0.05, * p b 0.001, ** p b 0.01 vs. valvular heart disease group. In parentheses, median with minimal and maximal value.
Surprisingly, a negative moderate association between TBARs concentrations in all myocardium specimens and interventricular septum diastolic diameter (r = −0.32, p = 0.05 and left ventricular posterior wall diastolic diameter (r = −0.33, p = 0.04) was found in the IE group. Also in this group a negative correlation between left ventricular end-diastolic diameter and total protein concentration in all myocardium specimens was noted (r = −38, p = 0.02). However, these associations were not significant in papillary muscle specimens (r = −0.20, p = 0.46; r = −0.17, p = 0.54; and r = −0.10, p = 0.72). 5. Discussion The most important finding emerging from the current study is that myocardial tissue specimens (auricle of right atrium, left ventricle papillary muscles) of IE patients revealed increased H2O2 and TBARs levels in comparison to specimens from VHD patients without any signs of infection. Moreover, there was a positive association between myocardial H2O2 and TBARs concentrations suggesting the occurrence of oxidative stress in this tissue. To our knowledge this is the first study showing such abnormalities in myocardium in the course of active IE. Activated NADPH oxidase and mitochondria are recognized as the main potential sources of ROS in myocardium hypertrophy of varied etiology [1,2]. Although, we did not measure myocardial NADPH oxidase activity and ROS generation form mitochondria, results of other Table 4 Mutual correlation coefficients between markers of oxidative stress in homogenates of myocardium specimens obtained during surgical valve replacement from patients with active infective endocarditis and patients with acquired valvular heart disease. Correlated variables Patients with infective endocarditis Patients with acquired valvular heart disease H2O2 with TBARs H2O2 with R-SH TBARs with R-SH H2O2 with TBARs H2O2 with R-SH TBARs with R-SH H2O2 with TBARs H2O2 with R-SH TBARs with R-SH
All myocardium specimens 0.31# − 0.11 − 0.46# Auricle specimens 0.58* − 0.60* − 0.67* Papillary muscle specimens 0.34 0.21 − 0.39
− 0.05 − 0.42# 0.21 − 0.33 − 0.18 0.46 0.11 − 0.48 0.08
TBARs- thiobarbituric reactive substances, H2O2 – hydrogen peroxide, R-SH - low molecular weight thiols (e.g. cysteine, reduced glutathione). * p b 0.01, # p b 0.05.
related clinical and experimental studies allow us to speculate possible mechanism leading to enhanced ROS generation and oxidative stress in the heart of patients with IE. Positive blood cultures belong to the major criteria for IE diagnosis according to the Duke criteria [13]. This suggests at least transient presence of bacteria and their biologically active products in the circulating blood of patients with active IE. Bacteria and their toxins stimulated the synthesis of pro-inflammatory cytokines (IL-1, Il-6, TNF-alpha, interferon-gamma) in the whole blood system and in cultures of human blood mononuclear cells in vitro [27–29]. Combination of LPS and INF-gamma induced the rise of NADPH oxidase activity in cultivated microvascular endothelial cells [30,31]. Taking these into consideration, one can assume that the rise of circulating pro-inflammatory cytokines induced by infection could be a mechanism leading to the rise of NADPH oxidase activity in the myocardium with subsequent increased H2O2 production. This can be supported by the following observations: (A)- patients with IE had elevated plasma concentrations of IL-1, IL-6 [15–17]; (B)- proinflammatory cytokines (IL-6, IL- 8, monocyte chemoattractant protein) were detected in human valve interstitial cells from autopsy specimens of subjects died due to IE but not in cells from normal control specimens [32]; (C)- IL-8 and TNF-alpha-containing cells were found in heart valve stromas and vegetations in the course of active IE [14]; (D)– cardiomyocytes exposed to TNF-alpha produced more ROS and this was blocked by apocynin, a NADPH oxidase inhibitor [33]. Thus myocardial NADPH oxidase could be activated by inflammatory signals generated locally in the close neighborhood of an infected valve or by systemic signals generated in response to bacteriemia via coronary circulation and lymphatic vessels of the heart. Another important source of ROS are mitochondria. During normal mitochondrial function up to 3% of consumed oxygen is partially reduced to superoxide radicals due to interaction of a single electron with molecular oxygen at complex I and III of the respiratory chain [34]. Mitochondria from failing myocardium produced more superoxide radicals than normal mitochondria in medium containing NADH [35]. In a rat model of right ventricular failure-induced by pulmonary hypertension, isolated mitochondria had increased ROS production associated with the rise in complex II activity [8]. Mitochondria from specimens of left ventricular papillary muscles obtained from patients who underwent mitral valve replacement had decreased oxygen consumption, total cytochrome content, and ATPase activity [36]. Similar results were found with mitochondria isolated from
Please cite this article as: Ostrowski S, et al, Myocardial oxidative stress in patients with active infective endocarditis, Int J Cardiol (2012), doi:10.1016/j.ijcard.2011.12.102
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specimens of right ventricular outflow tract muscle of patients with tetralogy of Fallot [37]. These observations suggest that valvular defects can induce mitochondrial dysfunction that might lead to increased leakage of superoxide radicals that are the immediate precursors of H2O2. Since our patients had insufficient valve/valves (including mitral valve), this mitochondrial dysfunction could occur in myocardium specimens from both studied groups and not be a culprit of observed differences in myocardial H2O2 generation. On the other hand, infection was reported to increase mitochondrial ROS generation as was documented in animal model of sepsis [38] and meningitis [39], as well as in healthy volunteers after challenge with endotoxin [40]. In addition, TNF-alpha increased mitochondrial ROS generation in left ventricle myocardium in rats [41]. Therefore, mitochondria derived ROS as a consequence of inflammatory response could contribute to increased H2O2 generation from myocardial specimens in patients with active IE. However, further studies are necessary to confirm this hypothesis. Mechanical stretch of cardiac myocytes was reported to activate NADPH oxidase and induce generation of ROS [42,43]. Both patients with IE and VHD presented heart insufficiency (NYHA functional class ranging from II to IV) suggesting a previous and actual exposition of myocardium to prolonged mechanical stretch. They underwent mitral or aortic valve or both valves replacement. Although mean left ventricular end-diastolic and left ventricular end-systolic diameters were higher in the IE group, these differences seem to be not the contributing factor responsible for increased H2O2 generation in myocardium specimens in these patients. There are at least two reasons supporting this: first, the highest difference in H2O2 generation was observed for comparison of auricle specimens collected from right atria that had similar size in both studied groups; second, H2O2 activity did not correlate with echocardiographic indices of heart morphology especially with left ventricular end-diastolic and left ventricular end-systolic diameter in IE group. Moreover, no significant positive correlations between myocardial TBARs and left ventricle dimensions were found. Some studies suggested that left ventricular remodeling proportionately affects papillary muscles and they are subjected to mechanical stretch conditions in a similar manner to adjacent left ventricular wall [44,45]. However, patients involved in the mentioned studies had lower ejection fraction and did not suffer from VHD as those in our study. Moreover, our results showed that papillary muscles generated less H2O2 than auricle specimens within IE group. Therefore, mechanical stretch and its supposed differences between studied groups cannot be responsible for the higher myocardial H2O2 activity in IE patients. Myocardial specimens from IE patients revealed a tremendous rise of TBARs in comparison to those from VHD patients. Since TBARs represent a group of compounds (e.g. malodialdehyde and hydroxynoneal) generated during peroxidative damage to lipids, proteins, DNA and carbohydrates [46], one may conclude that active IE is accompanied by oxidative stress in myocardium. There was a positive correlation between TBARs and H2O2 in myocardial specimens and also a negative correlation between R-SH level and TBARs. These suggest that enhanced H2O2 generation induces peroxidative damage to a variety of biomolecules of the heart and R-SH extent can protect myocardial structures from this peroxidative injury. Myocardial tissue contains iron that can be liberated from inactive complexes by superoxide radicals and subsequently can react with H2O2 to form extremely reactive hydroxyl radicals [12]. They can induce peroxidative damage to cardiomyocytes that is reflected by increased TBARs formation. Lack of above mentioned correlations in myocardial homogenates from VHD group suggests that low H2O2 release in these patients is not able to induce excessive hydroxyl radicals’ formation and antioxidant defense sufficiently prevents myocardium from peroxidative injury.
Although this is the first study showing myocardial oxidative stress in the course of IE, one may understand the helpfulness of antioxidants in preventing cardiomyocytes damage. However, we do not know the possible effect of this oxidative stress on treatment outcomes in patients with IE. Therefore, further prospective studies are necessary to establish whether the determination of the intensity of myocardial oxidative stress can have any predictive value for heart performance in patients with IE. Acknowledgement The authors of this manuscript have certified that they comply with the Principles of Ethical Publishing in the International Journal of Cardiology [47]. References [1] Murdoch CE, Grieve DJ, Cave AC, Looi YH, Shah AM. NADPH oxidase and heart failure. Curr Opin Pharmacol 2006;6:148–53. [2] Murdoch CE, Zhang M, Cave AC, Shah AM. NADPH oxidase-dependent redox signalling in cardiac hypertrophy, remodelling and failure. Cardiovasc Res 2006;71: 208–15. [3] Borchi E, Bargelli V, Stillitano F, et al. Enhanced ROS production by NADPH oxidase is correlated to changes in antioxidant enzyme activity in human heart failure. Biochim Biophys Acta 2010;1802:331–8. [4] Sam F, Kerstetter DL, Pimental DR, et al. Increased reactive oxygen species production and functional alterations in antioxidant enzymes in human failing myocardium. J Card Fail 2005;11:473–80. [5] Gupte RS, Vijay V, Marks B, et al. Upregulation of glucose-6-phosphate dehydrogenase and NAD(P)H oxidase activity increases oxidative stress in failing human heart. J Card Fail 2007;13:497–506. [6] Nediani C, Borchi E, Giordano C, et al. NADPH oxidase-dependent redox signaling in human heart failure: relationship between the left and right ventricle. J Mol Cell Cardiol 2007;42:826–34. [7] Gupte SA, Levine RJ, Gupte RS, et al. Glucose-6-phosphate dehydrogenase-derived NADPH fuels superoxide production in the failing heart. J Mol Cell Cardiol 2006;41:340–9. [8] Redout EM, Wagner MJ, Zuidwijk MJ, et al. Right-ventricular failure is associated with increased mitochondrial complex II activity and production of reactive oxygen species. Cardiovasc Res 2007;75:770–81. [9] Dhalla AK, Hill MF, Singal PK. Role of oxidative stress in transition of hypertrophy to heart failure. J Am Coll Cardiol 1996;28:506–14. [10] Lionetti V, Linke A, Chandler MP, et al. Carnitine palmitoyl transferase-I inhibition prevents ventricular remodeling and delays decompensation in pacing-induced heart failure. Cardiovasc Res 2005;66:454–61. [11] Kuroda J, Ago T, Matsushima S, Zhai P, Schneider MD, Sadoshima J. NADPH oxidase 4 (Nox4) is a major source of oxidative stress in the failing heart. Proc Natl Acad Sci U S A 2010;107:15565–70. [12] Ide T, Tsutsui H, Kinugawa S, et al. Direct evidence for increased hydroxyl radicals originating from superoxide in the failing myocardium. Circ Res 2000;86:152–7. [13] Moreillon P, Que YA. Infective endocarditis. Lancet 2004;363:139–49. [14] Ekdahl C, Broqvist M, Franzén S, Ljunghusen O, Maller R, Sander B. IL-8 and tumor necrosis factor alpha in heart valves from patients with infective endocarditis. Scand J Infect Dis 2002;34:759–62. [15] Watkin RW, Harper LV, Vernallis AB, et al. Pro-inflammatory cytokines IL6, TNFalpha, IL1beta, procalcitonin, lipopolysaccharide binding protein and C-reactive protein in infective endocarditis. J Infect 2007;55:220–5. [16] Rawczynska-Englert I, Hryniewiecki T, Dzierzanowska D. Evaluation of serum cytokine concentrations in patients with infective endocarditis. J Heart Valve Dis 2000;9:705–9. [17] Alter P, Hoeschen J, Ritter M, Maisch B. Usefulness of cytokines interleukin-6 and interleukin-2R concentrations in diagnosing active infective endocarditis involving native valves. Am J Cardiol 2002;89:1400–4. [18] Ostrowski S, Kasielski M, Kordiak J, Nowak D. Elevated resting and agonistinduced whole blood chemiluminescence in patients with active infective endocarditis. Interact Cardiovasc Thorac Surg 2009;8:12–6. [19] Doyama K, Fujiwara H, Fukumoto M, et al. Tumour necrosis factor is expressed in cardiac tissues of patients with heart failure. Int J Cardiol 1996;54:217–25. [20] Li JS, Sexton DJ, Mick N, et al. Proposed modifications to the Duke criteria for the diagnosis of infective endocarditis. Clin Infect Dis 2000;30:633–8. [21] Ruch W, Cooper PH, Baggiolini M. Assay for H2O2 production by macrophages and neutrophols with homovanilic acid and horse-radish peroxidase. J Immunol Methods 1983;63:347–57. [22] Zieba M, Suwalski M, Kwiatkowska S, et al. Comparison of hydrogen peroxide generation and the content of lipid peroxidation products in lung cancer tissue and pulmonary parenchyma. Respir Med 2000;94:800–5. [23] Szkudlarek U, Zdziechowski A, Witkowski K, et al. Effect of inhaled Nacetylcysteine on hydrogen peroxide exhalation in healthy subjects. Pulm Pharmacol Ther 2004;17:155–62.
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International Journal of Cardiology journal homepage: www.elsevier.com/locate/ijcard
Myocardial oxidative stress in patients with active infective endocarditis Stanislaw Ostrowski a, Marek Kasielski b, Jacek Kordiak c, Anna Zwolinska d, Anna Wlodarczyk d, Dariusz Nowak d,⁎ a
Department of Cardiosurgery, Medical University of Lodz, Poland Bases of Clinical Medicine Teaching Center, Medical University of Lodz, Poland 90-153 Lodz, Kopcinskiego St. 20 Department of Chest Surgery, Oncologic and General Surgery, University Hospital No. 2, Medical University of Lodz, Poland d Department of Clinical Physiology, Medical University of Lodz, Mazowiecka St. 6/8, 92-215 Lodz, Poland b c
a r t i c l e
i n f o
Article history: Received 11 February 2011 Received in revised form 13 June 2011 Accepted 24 December 2011 Available online xxxx Keywords: Infective endocarditis Valvular heart disease Oxidative stress Hydrogen peroxide Thiobarbituric acid reactive substances
a b s t r a c t Background: Infective endocarditis (IE) induces the rise of pro-inflammatory cytokines. Some of them can stimulate oxidants production in myocardium with subsequent peroxidative damage to various biomolecules. We compared indices of oxidative stress: H2O2, thiobarbituric acid-reactive substances (TBARs), thiols in myocardium specimens between patients with active IE and those with valvular heart disease (VHD) of rheumatic etiology who underwent surgical valve replacement. Methods: 17 left ventricle papillary muscle specimens and 28 specimens of auricle of the right heart were collected from 45 IE patients, and 16 papillary muscle and 12 auricle specimens from 28 VHD patients, respectively. Patients groups had similar NYHA functional class and majority of echocardiographic indices of heart morphology. H2O2 and TBARs were determined fluorometrically in myocardium homogenates whereas thiols with photometric method. Between and within groups comparisons and mutual correlations between variables were analyzed. Results: H2O2 generation from all myocardium specimens and auricles was 2.14- and 2.59- times higher (p b 0.001) in IE patients than in VHD group. Auricles had the highest H2O2 levels within IE group. TBARs were 10-times higher (p b 0.05) in IE when compared to VHD group in auricles and papillary muscles. Thiols did not differ between groups. H2O2 positively correlated with TBARs and negatively with thiols in all IE myocardium specimens (r = 0.31 and r = − 0.46, p b 0.05) and auricles (r = 0.58 and r = − 0.67, p b 0.05), respectively. No such associations were noted in VHD specimens. Conclusions: Active IE induces enhanced myocardial production of H2O2 and formation of TBARs which proves occurrence of oxidative stress in the heart. © 2011 Published by Elsevier Ireland Ltd.
1. Introduction Oxidative stress is implicated in the development of congestive heart failure in humans [1,2]. Evidences for this are coming from ex vivo analysis of myocardium specimens from patients with end-stage heart failure [3–6] and are consistent with the results from experiments on animals with pacing- ,aortic banding-, and pharmacologicallyinduced heart insufficiency [7–10]. Increased activity of NADPH oxidase seems to be the main source of ROS overproduction in the failing heart [3,11]. Angiotensin II, endothelin-1, TNF-α, and also mechanical forces belong to activators of cardiomyocyte NADPH oxidase [1]. This enzyme generates excessive amounts of superoxide radicals that subsequently dismutate into hydrogen peroxide (H2O2). Both, superoxide radicals and H2O2 together with tissue iron drive hydroxyl
⁎ Corresponding author. Tel.: + 48 426781800; fax: + 48 436782661. E-mail address: [email protected] (D. Nowak).
radicals formation in the failing myocardium [12]. Hydroxyl radicals are extremely reactive and can induce peroxidative damage to membrane lipids, proteins, carbohydrates, and nucleic acids resulting in the rise of myocardial levels of thiobarbituric acid-reactive substances (TBARS) in patients with heart failure [3,6]. Enhanced myocardial ROS generation (e.g. hydroxyl radicals) correlated with contractile dysfunction of the left ventricle [12], and was accompanied by decreased concentrations of reduced glutathione a main donor of sulfhydryl groups in myocardium homogenates [7]. However, little is known about the effect of infective processes in the heart on oxidants-antioxidants balance in the myocardium. Infective endocarditis (IE) represents an infection that most commonly involves heart valves and results frequently in the refractory congestive heart failure due to new or worsening valve dysfunction [13]. IE is accompanied by local [14] and strong systemic inflammatory response with elevated circulating IL-6, IL-2R and IL-1β concentrations [15–17]. These cytokines together with a variety of bacteria-derived products (e.g. formylated chemotactic peptides, lipopolysaccharides)
0167-5273/$ – see front matter © 2011 Published by Elsevier Ireland Ltd. doi:10.1016/j.ijcard.2011.12.102
Please cite this article as: Ostrowski S, et al, Myocardial oxidative stress in patients with active infective endocarditis, Int J Cardiol (2012), doi:10.1016/j.ijcard.2011.12.102
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can enhance (via activation of NADPH oxidase) ROS production by circulating polymorphonuclear leukocytes and monocytes [18]. Although plasma TNF-α levels were not elevated in the course of IE [15], this cytokine immunoreactivity was found in right atrium specimens and heart valves of patient suffering from this disease [14,19]. It is likely that some of these mediators may enhance myocardial ROS generation in the course of cardiac failure related to IE-induced valve damage. Therefore, in this study we compared H2O2 generation in homogenates of myocardium specimens obtained during surgical valve replacement in patients with active IE and with acquired valvular heart disease (VHD). Furthermore, the concentrations of low molecular weight thiols (R-SH, e.g. cysteine, reduced glutathione) and TBARs as a marker of peroxidative damage to myocardium were also determined. In the end, the mutual associations between these variables and their relationships with selected clinical parameters were evaluated.
Table 2 Patients preoperative condition. Parameter
Infective endocarditis
Acquired valvular heart disease
ESR (mm/h) RBC (x106/μl)
50 ± 37 (43 ;2–145) * 4.07 ± 0.78 (3.90; 2.82–6.08) 8.9 ± 3.6 (8.4; 6.5–20.0) * 37 ± 7 (35; 25–55) 248 ± 102 (228 ;68–493) 1.14 ± 0.76 (0.90; 0.60–4.60) * 43 ± 27 (38 ;10–154) 0.97 ± 0.67 (0.73; 0.20–3.05) 27 ± 15 (25; 10–78) 32 ± 23 (25; 7–124) 6.58 ± 0.73 (6.78; 4.61–7.95) * 1025 ± 341 (929; 516–1867) 25.4 ± 13.3 (22.7; 3.1–66.7) *
20 ± 11 (17 ;5–53) 4.52 ± 0.55 (4.53;3.47–5.44) 5.9 ± 1.8 (5.5 ;3.1–9.3) 42 ± 4 (42; 32–53) 201 ± 66 (189;75–345) 0.97 ± 0.26 (0.90; 0.49–1.59) 42 ± 13 (44; 22–71) 1.43 ± 1.20 (1.17; 0.32–2.82) 28 ± 11 (27; 11–44) 28 ± 15 (24; 8–72) 6.97 ± 0.63 (6.98; 5.69–8.76) 978 ± 316 (867; 337–1460) 15.4 ± 6.9 (14.9; 6.1–27.7)
WBC (x103/μl) HCT (%) PLT (x103/ μl) Creatinine (mg/dl) Urea (mg/dl) Bilirubin (mg/dl) AST (IU/L) ALT (IU/L) Total protein (g/dl) FRAP [μmol/L]
2. Materials and methods DPPH decomposition [%]
2.1. Study population and study protocol The study was conducted from March 2007 to January 2010 and involved 45 patients with active infective endocarditis (IE) and 28 patients with acquired valvular heart disease (VHD) of rheumatic etiology that underwent surgical treatment (valve replacement) using standard cardiopulmonary bypass (CPB) with crystalloid cardioplegia (Tables 1 and 2). Patients with VHD were without any signs of infection. The clinical diagnosis of IE was established in all patients according to the modified Duke criteria [20]. Native valve IE was present in 42 patients and prosthetic valve IE in 3. The inclusion criteria were: presence of active IE or acquired VHD of rheumatic etiology, age between 40 and 80 years, and a written informed consent. The exclusion criteria included the presence of any other active infectious or inflammatory process that could affect cellular ROS production, any history of autoimmune-disorders (apart from past rheumatic fever), acquired and inherited immunological defects, diabetes, pregnancy, alcohol and illicit drug abuse, chronic renal and liver insufficiency, current cigarette smoking, and usage of any vitamins and food supplements with antioxidant activities within 3 weeks preceding the cardiac surgery. Previous risk factors in patients with native valve IE included: preexisting defective valve (n = 17), prior endocarditis (n = 1), untreated dental caries (n= 7), prior respiratory tract infection (n= 7), prior urinary tract infection (n= 2), implanted pacemaker (n= 1), unknown (n = 10). Five ml of venous blood were drawn into EDTA-K3 vacuette-tubes (Greiner bio-one GmbH, Kremsmunster, Austria) at approximately 8:30 a.m. the day before cardiac
Table 1 Patients’ characteristics. Variable
Number Sex F/M Age (years) NYHA functional class EF (%) LA (cm) LVEDD (cm) LVESD (cm) LVPWDD (cm) LVPWSD (cm) RA (cm) RVEDD (cm) IVSDD (cm) IVSSD (cm) Embolism (CNS/peripheral) Atrial fibrillation
Patients that underwent cardiac valve surgery Infective endocarditis
Acquired valvular heart disease
45 15/30 52 ± 12 (53; 43–77) * I- 0, II- 8, III-28, IV-9 57 ± 8 (58; 38–80) 4.9 ± 0.8 (4.9; 2.8–7.0) 5.8 ± 0.7 (5.7; 4.6–8.1) * 4.0 ± 0.8 (4.0; 2.4–5.8) * 1.1 ± 0.1 (1.1; 0.8–1.5) 1.4 ± 0.2 (1.5; 1.1–1.8) 5.3 ± 0.7 (5.4; 3.2–6.7) 2.6 ± 0.4 (2.5; 1.6–3.5) 1.2 ± 0.2 (1.2; 0.8–1.6) 1.4 ± 0.2 (1.4; 1.0–2.2) 8 (3/5) 9
28 20/8 62 ± 10 (62 ;41–89) I-0, II-7, III-15, IV-6 56 ± 8 (57; 40–75) 5.1 ± 0.8 (4.9; 3.3–6.7) 5.1 ± 0.7 (4.9; 4.1–6.7) 3.4 ± 0.6 (3.3; 2.5–5.4) 1.1 ± 0.2 (1.0; 0.8–1.4) 1.4 ± 0.2 (1.3; 1.1–1.8) 5.4 ± 0.9 (5.2; 4.0–7.3) 2.6 ± 0.3 (2.5; 2.2–3.5) 1.1 ± 0.1 (1.1; 0.9–1.5) 1.4 ± 0.2 (1.4; 1.1–1.9) 6 (4/2) 11
EF – ejection fraction of the left ventricle; LA - left atrial diameter; LVEDD – left ventricular end-diastolic diameter; LVESD – left ventricular end-systolic diameter, LVPWDD – left ventricular posterior wall diastolic diameter; LVPWSD – left ventricular posterior wall systolic diameter; RA – right atrial diameter; RVEDD – right ventricular end-diastolic diameter; IVSDD – interventricular septum diastolic diameter; IVSSD – interventricular septum systolic diameter; CNS – central nervous system. Echocardiographic indices of heart morphology were determined within 7 days before surgery. * different vs. acquired valvular heart disease group; p b 0.05. In parentheses, median with minimal and maximal value.
ESR – erythrocyte sedimentation rate; RBC – red blood cells; WBC – white blood cells; HCT – hematocrit; PLT – platelets; AST – aspartate aminotransferase; ALT – alanine aminotransferase; FRAP - ferric reducing ability of plasma, DPPH decomposition serum DPPH-radical scavenging activity. * different vs. acquired valvular heart disease group; p b 0.05. In parentheses, median with minimal and maximal value.
surgery for the determination of blood cell count, chemistry and blood antioxidant activity. Samples of myocardium (slice of papillary muscle of the left heart or auricle of the right heart, 50 to 70 mg of wet tissue) were collected during heart surgery for ex vivo assessment of selected parameters of oxidative stress. The study, conformed to the Declaration of Helsinki, was approved by The Medical University of Lodz Ethics Committee (approval No RNN/139/KE). All participants provided a written, informed consent prior to their inclusion in the study.
2.2. Preoperative patient management Antibiotics that were shown to be effective through blood culture tests were used for 3 to 4 weeks prior to surgery in patients with active IE. When blood cultures were negative, vancomycin alone or in combination with gentamicin was used empirically. Patients with VHD did not receive any antibiotics before surgery. Both patient groups received diuretics, digitalis, and beta-blockers if required. No patient received systemic steroids or non-steroidal anti-inflammatory drugs within the three weeks preceding the heart operation.
2.3. Collection of myocardium samples Patients premedication, anesthesia, ventilation mode, CPB with crystalloid cardioplegia were the same in both groups and were described previously [18]. Slice of auricle of the right heart was taken just before placing a cannula of CPB in the right atrium mostly in patients with infected or insufficient aortic valve. Sample of papillary muscle was collected during excision of involved mitral valve in patients on CPB after infusion of cardioplegic solution and aorta clamping. One sample of myocardium was taken from one patient. Thus 40 auricle samples (28 from IE patients and 12 from VHD patients) and 33 left ventricle papillary muscle samples (17 from IE patients and 16 from VHD patients) were collected for further analysis. All patients underwent surgical valve replacement with mechanical valve prostheses (St Jude medical or Medtronic Hall). The duration of aortic clamping (cross-clamp time) and CPB as well as the volume of cardioplegic solution used during surgery were similar in both groups (Table 3). Intra-operative valve-tissue cultures were positive in 29 patients with IE (Staphylococcus sp. = 13, Streptococcus sp. = 8, Enterococcus sp. = 7, Corynebacterium sp. = 1). These cultures performed in patients with VHD were always negative.
2.4. Storage of myocardium samples and preparation of tissue homogenate Specimens of myocardium (slices of papillary muscle and auricle of the right heart) were washed in ice-cold sterile 0.9% NaCl, desiccated with lignin and stored at − 80 °C for not longer than 2 weeks. Samples of myocardium (25 mg) were homogenized in 1 ml of 1.15 mol/l KCl (glass homogenizer, on ice), centrifuged (5 min, 1500 ×g, 4 °C) and supernatant immediately used for measurement of TBARs, protein concentration, and spontaneous H2O2 release. Part of tissue specimens (25 mg) were homogenized in 0.5 ml of ice-cold 6% trichloroacetic acid, then centrifuged (10,000 ×g, 20 min, 4 °C), and supernatant assayed for concentration of R-SH.
Please cite this article as: Ostrowski S, et al, Myocardial oxidative stress in patients with active infective endocarditis, Int J Cardiol (2012), doi:10.1016/j.ijcard.2011.12.102
S. Ostrowski et al. / International Journal of Cardiology xxx (2012) xxx–xxx Table 3 Position of infected and or insufficient valve and operative data in the studied groups. Parameter
Involved valve Aortic Mitral Aortic + Mitral CPB time (min) Cross-clamp time (min) Cardioplegic solution (ml) Normothermia Hypothermia (32 °C)
Patients that underwent cardiac valve surgery* Infective endocarditis
Acquired valvular heart disease
16 21 8 143 ± 51 (150; 65–240) 80 ± 24 (77; 45–132)
8 14 6 176 ± 28 (180; 80–185) 74 ± 21 (73; 40–125)
1250 ± 310 (1200; 800–2600) 32 13
1320 ± 300 (1300; 900–1800) 20 8
CPB – cardiopulmonary bypass. * replacement with mechanical valve prostheses. In parentheses, median with minimal and maximal value.
3. Reagents Albumin from bovine serum, catalase from Aspergillus niger (6600 U/mg protein), 2,2-diphenyl-1-picryl-hydrazyl radical (DPPH), 5-5’-dithio-bis-(2-nitrobenzoic acid) (DTNB), ethylenediaminetetraacetic acid (EDTA), homovanillic acid (HVA), peroxidase from horseradish type II (HRP, 291 U/mg protein), reduced glutathione (GSH), thiobarbituric acid (TBA), and 2,4,6-tripirydyl-s-triazyne (TPTZ) were purchased from Sigma (St Louis, MO, USA). Sterile 0.9% NaCl and phosphate buffered saline (PBS, pH 7.4) were acquired from Biomed (Lublin, Poland). All other reagents were obtained from POCH (Gliwice, Poland) and were of analytical grade. Sterile, deionized, pyrogen-free water for HPLC (resistance > 18 Ωcm, Water Purification System USF ELGA, Buckinghamshire, UK) was used throughout the study. 3.1. Spontaneous hydrogen peroxide release from myocardium homogenates Hydrogen peroxide generation was determined according to the Ruch method [21] with some modifications [22]. A 10 μl aliquot of myocardium homogenate was mixed with 90 μl of PBS and 100 μl of HRP (1 U/ml) containing 400 μmol/l HVA and incubated for 60 min at 37 °C. Then, the sample was mixed with 300 μl of PBS and 125 μl of 0.1 mol/l glycine-NaOH buffer (pH 12.0) with the addition of 25 mmol/l EDTA. The HVA-derived oxidation product, as a measure of the amount of H2O2, was determined fluorometrically (Perkin Elmer Luminescence Spectrometer LS-50-B, Norwalk, CT, U.S.A.). Excitation length was set at 312 nm and emission was measured at 420 nm. Control samples received: (A) myocardium homogenate and HRP but without HVA, and (B) 10 μl of PBS instead of organ homogenate. Readings were converted into μmoles of H2O2 per liter (μmol/l) using calibration curve obtained from 3 series of calibration experiments with increasing standard concentrations of H2O2 (10 nmol/l to 10 μmol/l, 19 concentration-points). The lower limit of H2O2 detection was 0.08 μmol/l. Twenty units of catalase added to samples of myocardium homogenate (n = 5) before mixing with further substrates completely abolished HVA oxidation. 3.2. Measurement of TBARs in myocardium homogenate The concentration of TBARs was estimated as previously described [22]. Briefly, 100 μl of myocardium homogenate was mixed for 30 s with 1 ml of 0.05 mol/l sulphuric acid and 0.5 ml of 1.23 mol/l trichloroacetic acid and then allowed to stand for 5 min. After centrifugation (1500 ×g, 10 min, 4 °C), the supernatant was discarded and the pellet resuspended in 2 ml of TBA solution (0.67 g dissolved in
3
100 ml distilled water, then diluted 1:1 with glacial acetic acid) with addition of 10 μl of 0.01% butylated hydroxytoluene. The solution was boiled for 30 min, allowed to cool at room temperature and the chromogen formed in the reaction was extracted into 2.5 ml of butanol by vigorous shaking for 30 s. Following centrifugation (1500 ×g, 10 min, 20 °C), TBA-reactive substances in the butanol layer were measured fluorometrically (excitation 515 nm, emission 546 nm). Control samples received 100 μl of 1.15 mol/l KCl instead of myocardium homogenate. Readings were converted into μmoles per liter using calibration curve obtained from 3 series of calibration experiments with increasing standard concentrations of tetramethoxy-propane (0.01 to 10 μmol/l, 10 concentration-points). The lower limit of TBARs detection was 0.07 μmol/l. 3.3. Measurement of thiols concentration in myocardium homogenates Thiols (R-SH) concentration in myocardium homogenates was measured with DTNB assay as previously described [23] with some modifications. Briefly: 250 μl of myocardium homogenate was mixed with 250 μl of 0.3 mol/l Na2HPO4 buffer (pH = 7.5) and 250 μl of 0.04% DTNB in 10% sodium citrate and after 5 min incubation at 37 °C the absorbance was measured at 412 nm (Ultrospec III, Pharmacia LKB spectrophotometer). Control samples received 250 μl of 6% trichloroacetic acid solution instead of myocardium homogenate. Results were expressed in μmoles per liter using the calibration curve executed with the concentration of GSH increasing from 0.1 to 200 μmol/l (20 concentration-points). The method sensitivity was 0.25 μmol/l of GSH. 3.4. Other techniques Ferric reducing ability of plasma (FRAP) and serum DPPH-radical scavenging activity were measured as previously described [24,25] and expressed in μmol/l of reduced Fe 3+ over 3 min incubation and % of decomposed DPPH radical over 30 min incubation, respectively. Protein concentration in samples of myocardium homogenate was determined using the Lowry method [26] with bovine serum albumin as a standard. 3.5. Statistical analysis All data were expressed as the mean and standard deviation (SD) as well as the Median (Me) with minimal (Min) and maximal (Max) value. Shapiro-Wilk W test was used for normality testing. The differences between groups were assessed using the t-student test for normal data and with U Mann-Whitney test for data with abnormal distribution. Correlations between measured variables were determined using Pearson's r (normal data) and Spearman's ρ (abnormal data distribution). A p value of b0.05 was considered significant. 4. Results 4.1. Preoperative conditions -comparison between IE and VHD patients Tables 1 and 2 show the comparison of IE group and VHD patients. Majority of echocardiographic indices of the heart morphology and function did not differ between these groups. Only the left ventricular end-diastolic diameter and left ventricular end-systolic diameter were higher in IE patients than in VHD subjects (Table 1). IE group had higher pre-surgery erythrocyte sedimentation rate, white blood cell count, and lower total plasma protein suggesting persistent strong inflammatory response in this group (Table 2). Moreover, blood antioxidant activity, especially serum DPPH-radical scavenging activity, was higher in IE patients than in VHD group, probably as a compensatory response to increased ROS production by circulating phagocytes [18]. Mean plasma creatinine was higher in IE group.
Please cite this article as: Ostrowski S, et al, Myocardial oxidative stress in patients with active infective endocarditis, Int J Cardiol (2012), doi:10.1016/j.ijcard.2011.12.102
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4.2. Hydrogen peroxide release from myocardium homogenates Spontaneous H2O2 release from all myocardium specimens (right auricle plus papillary muscle) was 2.14 times higher in patients with IE than in VHD group (Fig. 1). This difference was even bigger when only auricle specimens were compared. In this case the H2O2 release was 2.59-times higher (123.7 ± 43.8 μmol/L vs. 47.7 ± 21.9 μmol/L) in specimens obtained from patients with IE than from those with VHD. Although homogenates of left ventricle papillary muscle slices from IE group generated 1.52- times more H2O2 than those from VHD patients, this difference did not reach statistical significance (p = 0.28), probably due to the high inter-individual variability of H2O2 readings especially in the IE patients (Fig. 1). There was a significant difference (p b 0.001) between H2O2 generation in auricle (n = 28) and papillary muscle (n = 17) homogenates within IE group. No such difference was noted within group of VHD patients. There were no significant differences in H2O2 release (and other measured markers of oxidative stress) between myocardium specimens from IE patients with (n = 29) and without (n = 16) positive culture of intra-operative valve-tissue (data not shown). Similarly, there was no difference between female and male patients within IE group and VHD group, respectively. 4.3. Thiobarbituric homogenates
acid-reactive
substances
in
myocardium
Both, auricle and papillary muscle homogenates from IE group revealed means TBARs concentrations are about 10-times higher (p b 0.05) than corresponding samples from VHD patients (Fig. 2). No significant differences were noted between TBARs content in papillary muscle and auricle slices homogenates within the IE and the VHD groups, respectively. Corresponding specimens from male and female patients revealed similar TBARs levels within both groups (data not shown). 4.4. Protein and thiols concentration in myocardium homogenates There were no significant differences (p > 0.05) between mean protein concentrations in auricle (4.6 ± 1.9 mg/ml vs. 5.4 ± 1.1 mg/ ml), papillary muscle (4.3 ± 1.6 mg/ml vs. 5.6 ± 2.5 mg/ml), and all myocardium (4.5 ± 1.9 mg/ml vs. 5.5 ± 2.0 mg/ml) specimens homogenates between IE and VHD patients, respectively. Similarly,
there was no difference between corresponding specimens within patient groups. Concentration of R-SH did not differ (p > 0.05) between corresponding specimens and was as follows: IE group – papillary muscle 9.2 ± 9.7 (Me11.0; Min 0.1– Max 24.1) μmol/l , auricle 14.5 ± 7.4 (12.6; 0.5–33.9) μmol/l, all myocardium specimens 12.8 ± 8.4 (12.1; 0.1–33.9) μmol/l: VHD patients – papillary muscle 11.0 ± 14.6 (11.6; 0.2–57.4) μmol/l, auricle 10.7 ± 6.1 (10.7; 0.4–20.6) μmol/l, and all myocardium specimens 10.9 ± 11.3 (11.3; 0.2–57.4) μmol/l. There was no significant difference between corresponding myocardium specimens between and within patient groups and also between female and male patients.
4.5. Correlations between measured variables There was a positive moderate correlation between H2O2 activity and TBARs levels both in auricle homogenates and all myocardium specimens obtained from patients with IE (Table 4). These specimens revealed also significant negative correlation between TBARs levels and concentrations of R-SH. Negative correlation between H2O2 activity and R-SH was noted in homogenized samples of auricles from IE group (Table 4). No significant associations between measured variables (H2O2 with TBARs, H2O2 with R-SH, TBARs with R–SH) were noted in papillary muscle homogenates either in IE group or in VHD group. Negative correlation between H2O2 activity and the concentration of RSH in all myocardium specimen homogenates was the only significant association observed in patients with VHD (Table 4). Majority of data shown in Tables 1 and 2 (FRAP and DPPH test, measures of plasma antioxidant activity, patients age) did not correlate with oxidative stress parameters determined in myocardium specimens in both patient groups. No positive association between H2O2 activity as well as TBARs concentration and echocardiographic indices of heart morphology was noted in patients with IE and VHD. In particular H2O2 activity did not correlate (p > 0.05) with left ventricular end-diastolic diameter (r = − 0.01), left ventricular endsystolic diameter (r = 0.06), left ventricular posterior wall diastolic diameter (r = − 0.23) and left ventricular posterior wall systolic diameter (r = − 0.10) in all myocardium specimens from IE patients. Similarly no significant correlations were found between myocardial TBARs levels and left ventricular end-diastolic diameter (r = −0.11) and left ventricular end-systolic diameter (r = − 0.03), respectively.
Fig. 1. Spontaneous H2O2 generation in homogenates of myocardium specimens (auricle of the right heart; 28 – infective endocarditis, 12 – valvular heart disease, and papillary muscle of the left heart; 17- infective endocarditis, 16- valvular heart disease) collected during valve replacement surgery from 45 patients with active infective endocarditis (closed bars) and 28 patients with acquired valvular heart disease of rheumatic etiology (open bars). * p b 0.001 vs. valvular heart disease group. In parentheses, median with minimal and maximal value.
Please cite this article as: Ostrowski S, et al, Myocardial oxidative stress in patients with active infective endocarditis, Int J Cardiol (2012), doi:10.1016/j.ijcard.2011.12.102
S. Ostrowski et al. / International Journal of Cardiology xxx (2012) xxx–xxx
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Fig. 2. Concentration of thiobarbituric acid-reactive substances (TBARs) in homogenates of myocardium specimens (auricle of the right heart and papillary muscle of the left heart) collected during valve replacement surgery from 45 patients with active infective endocarditis (closed bars) and 28 patients with acquired valvular heart disease of rheumatic etiology (open bars). Other details as for Fig. 1. # p b 0.05, * p b 0.001, ** p b 0.01 vs. valvular heart disease group. In parentheses, median with minimal and maximal value.
Surprisingly, a negative moderate association between TBARs concentrations in all myocardium specimens and interventricular septum diastolic diameter (r = −0.32, p = 0.05 and left ventricular posterior wall diastolic diameter (r = −0.33, p = 0.04) was found in the IE group. Also in this group a negative correlation between left ventricular end-diastolic diameter and total protein concentration in all myocardium specimens was noted (r = −38, p = 0.02). However, these associations were not significant in papillary muscle specimens (r = −0.20, p = 0.46; r = −0.17, p = 0.54; and r = −0.10, p = 0.72). 5. Discussion The most important finding emerging from the current study is that myocardial tissue specimens (auricle of right atrium, left ventricle papillary muscles) of IE patients revealed increased H2O2 and TBARs levels in comparison to specimens from VHD patients without any signs of infection. Moreover, there was a positive association between myocardial H2O2 and TBARs concentrations suggesting the occurrence of oxidative stress in this tissue. To our knowledge this is the first study showing such abnormalities in myocardium in the course of active IE. Activated NADPH oxidase and mitochondria are recognized as the main potential sources of ROS in myocardium hypertrophy of varied etiology [1,2]. Although, we did not measure myocardial NADPH oxidase activity and ROS generation form mitochondria, results of other Table 4 Mutual correlation coefficients between markers of oxidative stress in homogenates of myocardium specimens obtained during surgical valve replacement from patients with active infective endocarditis and patients with acquired valvular heart disease. Correlated variables Patients with infective endocarditis Patients with acquired valvular heart disease H2O2 with TBARs H2O2 with R-SH TBARs with R-SH H2O2 with TBARs H2O2 with R-SH TBARs with R-SH H2O2 with TBARs H2O2 with R-SH TBARs with R-SH
All myocardium specimens 0.31# − 0.11 − 0.46# Auricle specimens 0.58* − 0.60* − 0.67* Papillary muscle specimens 0.34 0.21 − 0.39
− 0.05 − 0.42# 0.21 − 0.33 − 0.18 0.46 0.11 − 0.48 0.08
TBARs- thiobarbituric reactive substances, H2O2 – hydrogen peroxide, R-SH - low molecular weight thiols (e.g. cysteine, reduced glutathione). * p b 0.01, # p b 0.05.
related clinical and experimental studies allow us to speculate possible mechanism leading to enhanced ROS generation and oxidative stress in the heart of patients with IE. Positive blood cultures belong to the major criteria for IE diagnosis according to the Duke criteria [13]. This suggests at least transient presence of bacteria and their biologically active products in the circulating blood of patients with active IE. Bacteria and their toxins stimulated the synthesis of pro-inflammatory cytokines (IL-1, Il-6, TNF-alpha, interferon-gamma) in the whole blood system and in cultures of human blood mononuclear cells in vitro [27–29]. Combination of LPS and INF-gamma induced the rise of NADPH oxidase activity in cultivated microvascular endothelial cells [30,31]. Taking these into consideration, one can assume that the rise of circulating pro-inflammatory cytokines induced by infection could be a mechanism leading to the rise of NADPH oxidase activity in the myocardium with subsequent increased H2O2 production. This can be supported by the following observations: (A)- patients with IE had elevated plasma concentrations of IL-1, IL-6 [15–17]; (B)- proinflammatory cytokines (IL-6, IL- 8, monocyte chemoattractant protein) were detected in human valve interstitial cells from autopsy specimens of subjects died due to IE but not in cells from normal control specimens [32]; (C)- IL-8 and TNF-alpha-containing cells were found in heart valve stromas and vegetations in the course of active IE [14]; (D)– cardiomyocytes exposed to TNF-alpha produced more ROS and this was blocked by apocynin, a NADPH oxidase inhibitor [33]. Thus myocardial NADPH oxidase could be activated by inflammatory signals generated locally in the close neighborhood of an infected valve or by systemic signals generated in response to bacteriemia via coronary circulation and lymphatic vessels of the heart. Another important source of ROS are mitochondria. During normal mitochondrial function up to 3% of consumed oxygen is partially reduced to superoxide radicals due to interaction of a single electron with molecular oxygen at complex I and III of the respiratory chain [34]. Mitochondria from failing myocardium produced more superoxide radicals than normal mitochondria in medium containing NADH [35]. In a rat model of right ventricular failure-induced by pulmonary hypertension, isolated mitochondria had increased ROS production associated with the rise in complex II activity [8]. Mitochondria from specimens of left ventricular papillary muscles obtained from patients who underwent mitral valve replacement had decreased oxygen consumption, total cytochrome content, and ATPase activity [36]. Similar results were found with mitochondria isolated from
Please cite this article as: Ostrowski S, et al, Myocardial oxidative stress in patients with active infective endocarditis, Int J Cardiol (2012), doi:10.1016/j.ijcard.2011.12.102
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specimens of right ventricular outflow tract muscle of patients with tetralogy of Fallot [37]. These observations suggest that valvular defects can induce mitochondrial dysfunction that might lead to increased leakage of superoxide radicals that are the immediate precursors of H2O2. Since our patients had insufficient valve/valves (including mitral valve), this mitochondrial dysfunction could occur in myocardium specimens from both studied groups and not be a culprit of observed differences in myocardial H2O2 generation. On the other hand, infection was reported to increase mitochondrial ROS generation as was documented in animal model of sepsis [38] and meningitis [39], as well as in healthy volunteers after challenge with endotoxin [40]. In addition, TNF-alpha increased mitochondrial ROS generation in left ventricle myocardium in rats [41]. Therefore, mitochondria derived ROS as a consequence of inflammatory response could contribute to increased H2O2 generation from myocardial specimens in patients with active IE. However, further studies are necessary to confirm this hypothesis. Mechanical stretch of cardiac myocytes was reported to activate NADPH oxidase and induce generation of ROS [42,43]. Both patients with IE and VHD presented heart insufficiency (NYHA functional class ranging from II to IV) suggesting a previous and actual exposition of myocardium to prolonged mechanical stretch. They underwent mitral or aortic valve or both valves replacement. Although mean left ventricular end-diastolic and left ventricular end-systolic diameters were higher in the IE group, these differences seem to be not the contributing factor responsible for increased H2O2 generation in myocardium specimens in these patients. There are at least two reasons supporting this: first, the highest difference in H2O2 generation was observed for comparison of auricle specimens collected from right atria that had similar size in both studied groups; second, H2O2 activity did not correlate with echocardiographic indices of heart morphology especially with left ventricular end-diastolic and left ventricular end-systolic diameter in IE group. Moreover, no significant positive correlations between myocardial TBARs and left ventricle dimensions were found. Some studies suggested that left ventricular remodeling proportionately affects papillary muscles and they are subjected to mechanical stretch conditions in a similar manner to adjacent left ventricular wall [44,45]. However, patients involved in the mentioned studies had lower ejection fraction and did not suffer from VHD as those in our study. Moreover, our results showed that papillary muscles generated less H2O2 than auricle specimens within IE group. Therefore, mechanical stretch and its supposed differences between studied groups cannot be responsible for the higher myocardial H2O2 activity in IE patients. Myocardial specimens from IE patients revealed a tremendous rise of TBARs in comparison to those from VHD patients. Since TBARs represent a group of compounds (e.g. malodialdehyde and hydroxynoneal) generated during peroxidative damage to lipids, proteins, DNA and carbohydrates [46], one may conclude that active IE is accompanied by oxidative stress in myocardium. There was a positive correlation between TBARs and H2O2 in myocardial specimens and also a negative correlation between R-SH level and TBARs. These suggest that enhanced H2O2 generation induces peroxidative damage to a variety of biomolecules of the heart and R-SH extent can protect myocardial structures from this peroxidative injury. Myocardial tissue contains iron that can be liberated from inactive complexes by superoxide radicals and subsequently can react with H2O2 to form extremely reactive hydroxyl radicals [12]. They can induce peroxidative damage to cardiomyocytes that is reflected by increased TBARs formation. Lack of above mentioned correlations in myocardial homogenates from VHD group suggests that low H2O2 release in these patients is not able to induce excessive hydroxyl radicals’ formation and antioxidant defense sufficiently prevents myocardium from peroxidative injury.
Although this is the first study showing myocardial oxidative stress in the course of IE, one may understand the helpfulness of antioxidants in preventing cardiomyocytes damage. However, we do not know the possible effect of this oxidative stress on treatment outcomes in patients with IE. Therefore, further prospective studies are necessary to establish whether the determination of the intensity of myocardial oxidative stress can have any predictive value for heart performance in patients with IE. Acknowledgement The authors of this manuscript have certified that they comply with the Principles of Ethical Publishing in the International Journal of Cardiology [47]. References [1] Murdoch CE, Grieve DJ, Cave AC, Looi YH, Shah AM. NADPH oxidase and heart failure. Curr Opin Pharmacol 2006;6:148–53. [2] Murdoch CE, Zhang M, Cave AC, Shah AM. NADPH oxidase-dependent redox signalling in cardiac hypertrophy, remodelling and failure. Cardiovasc Res 2006;71: 208–15. [3] Borchi E, Bargelli V, Stillitano F, et al. Enhanced ROS production by NADPH oxidase is correlated to changes in antioxidant enzyme activity in human heart failure. Biochim Biophys Acta 2010;1802:331–8. [4] Sam F, Kerstetter DL, Pimental DR, et al. Increased reactive oxygen species production and functional alterations in antioxidant enzymes in human failing myocardium. J Card Fail 2005;11:473–80. [5] Gupte RS, Vijay V, Marks B, et al. Upregulation of glucose-6-phosphate dehydrogenase and NAD(P)H oxidase activity increases oxidative stress in failing human heart. J Card Fail 2007;13:497–506. [6] Nediani C, Borchi E, Giordano C, et al. NADPH oxidase-dependent redox signaling in human heart failure: relationship between the left and right ventricle. J Mol Cell Cardiol 2007;42:826–34. [7] Gupte SA, Levine RJ, Gupte RS, et al. Glucose-6-phosphate dehydrogenase-derived NADPH fuels superoxide production in the failing heart. J Mol Cell Cardiol 2006;41:340–9. [8] Redout EM, Wagner MJ, Zuidwijk MJ, et al. Right-ventricular failure is associated with increased mitochondrial complex II activity and production of reactive oxygen species. Cardiovasc Res 2007;75:770–81. [9] Dhalla AK, Hill MF, Singal PK. Role of oxidative stress in transition of hypertrophy to heart failure. J Am Coll Cardiol 1996;28:506–14. [10] Lionetti V, Linke A, Chandler MP, et al. Carnitine palmitoyl transferase-I inhibition prevents ventricular remodeling and delays decompensation in pacing-induced heart failure. Cardiovasc Res 2005;66:454–61. [11] Kuroda J, Ago T, Matsushima S, Zhai P, Schneider MD, Sadoshima J. NADPH oxidase 4 (Nox4) is a major source of oxidative stress in the failing heart. Proc Natl Acad Sci U S A 2010;107:15565–70. [12] Ide T, Tsutsui H, Kinugawa S, et al. Direct evidence for increased hydroxyl radicals originating from superoxide in the failing myocardium. Circ Res 2000;86:152–7. [13] Moreillon P, Que YA. Infective endocarditis. Lancet 2004;363:139–49. [14] Ekdahl C, Broqvist M, Franzén S, Ljunghusen O, Maller R, Sander B. IL-8 and tumor necrosis factor alpha in heart valves from patients with infective endocarditis. Scand J Infect Dis 2002;34:759–62. [15] Watkin RW, Harper LV, Vernallis AB, et al. Pro-inflammatory cytokines IL6, TNFalpha, IL1beta, procalcitonin, lipopolysaccharide binding protein and C-reactive protein in infective endocarditis. J Infect 2007;55:220–5. [16] Rawczynska-Englert I, Hryniewiecki T, Dzierzanowska D. Evaluation of serum cytokine concentrations in patients with infective endocarditis. J Heart Valve Dis 2000;9:705–9. [17] Alter P, Hoeschen J, Ritter M, Maisch B. Usefulness of cytokines interleukin-6 and interleukin-2R concentrations in diagnosing active infective endocarditis involving native valves. Am J Cardiol 2002;89:1400–4. [18] Ostrowski S, Kasielski M, Kordiak J, Nowak D. Elevated resting and agonistinduced whole blood chemiluminescence in patients with active infective endocarditis. Interact Cardiovasc Thorac Surg 2009;8:12–6. [19] Doyama K, Fujiwara H, Fukumoto M, et al. Tumour necrosis factor is expressed in cardiac tissues of patients with heart failure. Int J Cardiol 1996;54:217–25. [20] Li JS, Sexton DJ, Mick N, et al. Proposed modifications to the Duke criteria for the diagnosis of infective endocarditis. Clin Infect Dis 2000;30:633–8. [21] Ruch W, Cooper PH, Baggiolini M. Assay for H2O2 production by macrophages and neutrophols with homovanilic acid and horse-radish peroxidase. J Immunol Methods 1983;63:347–57. [22] Zieba M, Suwalski M, Kwiatkowska S, et al. Comparison of hydrogen peroxide generation and the content of lipid peroxidation products in lung cancer tissue and pulmonary parenchyma. Respir Med 2000;94:800–5. [23] Szkudlarek U, Zdziechowski A, Witkowski K, et al. Effect of inhaled Nacetylcysteine on hydrogen peroxide exhalation in healthy subjects. Pulm Pharmacol Ther 2004;17:155–62.
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Please cite this article as: Ostrowski S, et al, Myocardial oxidative stress in patients with active infective endocarditis, Int J Cardiol (2012), doi:10.1016/j.ijcard.2011.12.102