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Basic Science Aspects

INCREASED SARCOLEMMAL PERMEABILITY AS AN EARLY EVENT IN EXPERIMENTAL SEPTIC CARDIOMYOPATHY

A POTENTIAL ROLE FOR OXIDATIVE DAMAGE TO LIPIDS AND PROTEINS

Celes, Mara R.N.*; Torres-Dueñas, Diego; Prado, Cibele M.*; Campos, Erica C.*; Moreira, Jorge E.*‡; Cunha, Fernando Q.; Rossi, Marcos A.*

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doi: 10.1097/SHK.0b013e3181b38ef6
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Abstract

INTRODUCTION

Clinical studies have shown that myocardial contractility is reduced in severe sepsis and septic shock in the absence of changes of ventricular preload or afterload (1). This cardiac dysfunction has been recognized as a serious manifestation in ≅40% of patients with sepsis, with mortality rate from 70% to 90% in contrast with 20% in septic patients without cardiovascular involvement (2). In recent years, the concept of septic cardiomyopathy has evolved (3-5), which implies alterations of myocardium phenotype in response to a variety of agents acting on heart cells.

Clinically unrecognized myocardial injury as revealed by elevated levels of cardiac troponins I and T has been reported in clinically ill patients (6-8). However, the exact mechanism of the increased troponin levels in patients with sepsis remains unknown. Possible mechanisms are diffuse necrosis, cardiac troponin proteolysis, or leakage of cytoplasmic cardiac troponins with reversible damage to the contractile complex of heart muscle cells. Previous experimental studies have shown either an association between sepsis and structural myocardial changes, such as myocardial edema (9), mitochondrial injury, and myofibrillar loss (10-12), interstitial edema (11, 13, 14), subendocardial hemorrhage (12), and subendocardial necrosis (10), or an impairment of myocardial contractility in the absence of tissue injury (15, 16).

Recently, we reported myocardial structural changes in long-term human severe sepsis classified as an inflammatory cardiomyopathy characterized by increased myocardial macrophage infiltration, intramyocyte accumulation of lipids, scattered foci of partial lack of cross-striations or irregularly disorganized cross-striations within cardiomyocytes, and lysis of myofilaments (4).

Considering that the sarcolemma constitutes the permeability barrier to cardiomyocytes and its disruption is an early event that precedes cellular degenerative changes (17, 18), it is conceivable the occurrence of permeability alterations of sarcolemmal membrane in septic hearts as a possible primary mechanism of cardiac injury in septic patients. We describe herein increased sarcolemmal permeability and myofilamentar damage that occur together with oxidative damage to lipids and proteins in the myocardium in experimental sepsis induced by cecal ligation and puncture (CLP) in mice. These findings make plausible the hypothesis that increased sarcolemmal permeability might be a primary event in myocardial injury in severe sepsis possibly due to oxidative damage to lipids and proteins that could precede phenotypic changes that characterize a septic cardiomyopathy.

MATERIALS AND METHODS

Animals

Male C57BL/6 mice (20-22 g) were acclimated to the laboratory environment for at least 24 h before surgery and received water and food ad libitum. After surgery, the mice were maintained at a constant temperature (22°C ± 2°C) on a 12-h/12-h light-dark cycle. The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH publication 85-23, revised 1996). The animal experiments performed in the present study were conducted according to the guidelines of the Animal Care Committee of the Faculty of Medicine of Ribeirão Preto (process 012/2004).

Sepsis model: CLP

Sepsis was induced through CLP with modifications in relation to the original proposition (19). Briefly, mice were anesthetized with tribromoethanol 250 mg/kg i.p., the abdomens shaved, and an approximately 1-cm midline incision made. The cecum was exposed and ligated with 5-0 silk below the ileocecal valve without causing bowel obstruction. A single puncture was made through the cecum using a 30- or 18-gauge needle to induce moderate septic injury (MSI) or severe septic injury (SSI), respectively. The cecum was then replaced in its original position, and the abdomen sutured. Sham-operated animals (controls) underwent identical laparotomy but without cecum ligation and puncture. All mice received subcutaneous doses of saline (50 mL/kg of body weight) immediately and 12 h after the procedure to prevent dehydration. The survival rate was monitored each 6 h for 72 h after surgery using 10 animals per group (these animals were given subcutaneous doses of saline 36 and 60 h after injury to prevent dehydration).

Number of bacteria in the peritoneal cavity and blood

Six hours after CLP surgery, the animals were killed (n = 10 animals/group); the blood and peritoneal exudates were collected under sterile conditions and assessed as described previously (20). For peritoneal cavity bacterial count, aliquots of serial log dilutions of peritoneal aspirate were plated on Mueller-Hinton agar dishes (Difco Laboratories, Detroit, Mich) and incubated at 37°C for 24 h. Colony-forming units (CFUs) were then counted, and the results were expressed as log of CFUs/peritoneal cavity. For bacteremia, blood was collected, diluted in sterile saline, and plated on Mueller-Hinton agar dishes (Difco). The number of CFUs were counted 24 h after incubation at 37°C, and the results were expressed as log of CFUs/mL of blood.

Cytokine and chemokine measurements

Six hours after CLP, the concentrations of TNF-α and monocyte inflammatory protein 1α (MIP-1α) in the peritoneal exudate and serum (n = 10 animals/group) were determined by using a double-ligand enzyme-linked immunosorbent assay. Briefly, each well of 96-well microtiter plates was coated with antibody anti-TNF-α (2 mg/mL) or MIP-1α (1 mg/mL), diluted in coating buffer, and incubated overnight at 4°C. Plates were washed; samples (nondiluted or diluted 1:2) and standards were loaded onto plates. The plates were thoroughly washed, and the appropriate monoclonal anticytokine antibody was added. After 1 h, the plates were washed; avidin-peroxidase was added (diluted 1:5,000) in each well, and then each plate was thoroughly washed again. After, substrate was added (0.4 mg of o-phenylenediamine and 0.4 mL of H2O2 in 1 mL of substrate buffer), and the reaction was stopped with H2SO4 (1 M), and the optical density was measured on an enzyme-linked immunosorbent assay plate scanner (Spectra Max 250; Molecular Devices, Menlo Park, Calif) at 490 nm. The results were expressed as picograms of TNF-α and MIP-1α per milliliter of supernatant or serum (pg/mL). Recombinant murine TNF-α and MIP-1α standard curve were used to calculate the cytokine concentrations.

Measurement of mean carotid blood pressure

Mean carotid artery blood pressure was measured in anesthetized animals, immediately after cannulation of the carotid artery simulating the time 0 of the experiment and 6, 12, and 24 h after surgery (n = 6 to10 animals/group in each time). Briefly, a polyethylene catheter (PE-10) filled with heparinized saline was introduced and positioned in the right carotid artery and exteriorized in the neck. Immediately after surgical preparation, with stable hemodynamics, mean carotid pressure was recorded connecting the catheter to a pressure transducer PowerLab (AD Instruments, Castle Hill, Australia). The results are expressed as millimeters of mercury.

Histopathology

Hearts from SSI, MSI, and sham mice obtained 12 and 24 h after surgery (n = 6 animals/group) were sectioned into anterior and posterior halves, fixed in phosphate-buffered 10% formalin, and embedded in paraffin; 5-μm-thick sections were stained with hematoxylin-eosin. The other halves were embedded in Historesin (Leica Instruments, Heidelberg, Germany) for high-resolution light microscopy, and 2-μm-thick sections were stained with toluidine blue.

Morphometry

The diameters of individual cardiomyocytes were measured at 12 and 24 h after surgery. The myocardium was divided into five regions intending to cover the entire left ventricle: base of the left ventricle free wall, base of the septum, middle portion of the left ventricle free wall, middle portion of the septum and apex. Fifty measures were collected in the mid myocardium of each region (n = 6 animals/group), totaling 250 measurements for each heart, using a magnification of ×400. Leica QWin software (Leica Imaging Systems, Cambridge, UK) in conjunction with a Leica DMR microscope (Leica Systems, Wetzlar GmbH, Germany) was used for morphometry. The fields, measuring 0.093 mm2 each, were chosen at random. The averages were calculated for each experimental group. The measurements were made by a skilled observed blinded to the treatment groups.

Immunofluorescence

The immunofluorescence labeling was performed in myocardium frozen sections from SSI and sham mice (n = 5 animals/group) obtained 24 h after surgery using primary antibodies to cardiac myosin heavy chain (MHC) (Abcam, Cambridge, Mass) and α-sarcomeric actin (Vector Laboratories, Burlingame, Calif). Fluorescein-conjugated secondary antibody antimouse (Santa Cruz Biotechnology, Santa Cruz, Calif) was used. Albumin immunofluorescent staining was done to evaluate the sarcolemmal integrity of cardiomyocytes using fluoresceinated antibody to albumin-fluorescein isothiocyanate-conjugated (Bethyl Laboratories, Montgomery, Tex). Wheat germ agglutinin (WGA) was used as a selective probe for plasma membrane delineation (Vector Laboratories). To determinate if administration of superoxide dismutase (SOD), a highly selective superoxide scavenger, could prevent the increased sarcolemmal permeability in septic myocardium, an additional experiment was done: sham and SSI mice were treated with SOD (2,000 U/kg of body weight, 100 μL i.p. per animal, immediately after surgery) (Sigma-Aldrich Inc, St Louis, Mo), and sham and SSI mice were treated with saline as placebo (100 μL i.p. per animal, immediately after surgery). To prevent dehydration, all mice received saline subcutaneously (50 mL/kg of body weight) immediately and 12 h after septic injury.

Western blot analysis

To determine the amount of myosin in septic (MSI and SSI) and sham mice (n = 3-5 animals/group), fresh hearts were excised 24 h after septic injury and then washed in cold phosphate-buffered saline; the left and right ventricles were isolated and homogenized in extraction buffer and protease inhibition cocktail (Sigma-Aldrich Inc). Total heart protein (40 μg of protein/well) was resolved on a 7% or 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membrane (Amersham Pharmacia Biotech, Amersham, UK). The membranes were incubated overnight at 4°C with the primary antibodies anti-MHC (Abcam) or anti-α-sarcomeric actin (Vector Laboratories). The blots were washed and incubated with horseradish peroxidase-conjugated secondary antibody (goat anti-mouse IgG; Vector Laboratories) for 1 h at room temperature. The membranes were washed, developed using ECL (Amersham Pharmacia Biotech), and exposed to hyperfilm ECL (Amersham Pharmacia Biotech). The quantification was done using the National Institutes of Health public-domain software ImageJ (http://rbs.info.nih.gov/nih-image/).

Ultrastructure

Small blocks (1 mm3) of myocardial tissue from the left ventricular free wall of mice (n = 5 animals/group) 24 h after septic injury were fixed with 2.5% glutaraldehyde in cacodylate buffer and postfixed in 1% osmium tetroxide. Ultrathin sections were double-stained with uranyl acetate and lead citrate and examined in a Zeiss EM109/900 electron microscope (Carl Zeiss, Oberköchen, Germany) at 80 kV.

Immunohistochemistry for myocardial 4-hydroxy-2-nonenal and nitrotyrosine

The immunohistochemistry labeling was performed in paraffin sections from SSI, MSI, and sham mice (n = 5 animals/group) obtained 24 h after surgery. The avidin-biotin-peroxidase complex method was used to detect 4-hydroxy-2-nonenal (4-HNE) protein adducts (rabbit antiserum to [E] 4-HNE; A. G. Scientific Inc, San Diego, Calif) and NT (rabbit antinitrotyrosine [anti-NT]; Upstate, Lake Placid, NY). For quantification of mean area (%) of 4-HNE immunoreactivity in the base, middle portion, and apex of the left ventricle hearts, 5 to 10 randomly selected images of longitudinally oriented myofibers of 0.0093 mm2 were captured from each group and analyzed with the Leica QWin software (Leica Imaging Systems). The intensity of NT staining was estimated using a semiquantitative score 0 to 4 (0 = no visible staining, 1 = slight staining, 2 = slight to moderate staining, 3 = moderate staining, and 4 = strong staining) examining the same regions of the left ventricles of septic and sham hearts as for the determination on 4-HNE expression. The averages were calculated for each experimental group and presented as an index of expression (arbitrary unit [AU]). All histological evaluation was made by a skilled observed blinded to the groups.

Statistical analysis

All data are presented as mean ± SEM, except the data of carotid blood pressure, bacterial count, and cytokine and chemokine levels, which were expressed as mean ± SD. Multiple comparisons were made using ANOVA followed by the Tukey or Bonferroni posttest. Two groups' comparisons were made using Student t test. The survival rate was expressed as the percentage of live animals. The survival rates were constructed by the Kaplan-Meier method, and differences in mortality were compared using the log-rank test. P < 0.05 was considered statistically significant.

RESULTS

Experimental model

Twenty-four hours after surgery, the animals submitted to SSI and MSI presented reduced activity, piloerection, and exudation around the eyes and nose. In contrast, the sham animals demonstrated full recovery from anesthesia and surgery and had no detectable signs of disease. Sham-operated animals presented a 100% survival rate through the analyzed period of 72 h. The MSI mice showed 90% survival at 12 h after injury declining progressively to 50% survival at 72 h. At 24 h, defined as the period of study, the survival rate of MSI animals was 80%. The SSI mice showed 70% mortality 12 h after injury, increasing to 90% 24 h after cecal puncture, and remaining steady at this rate until 72 h after surgery (Fig. 1A).

Fig. 1
Fig. 1:
A, Mortality of mice submitted to CLP. Curves of actuarial mortality of mice submitted to sham operation and MSI and SSI. The survival rate was determined daily until 72 h after surgery (n = 10 animals/group; the results are representative of three different experiments). B, Mean carotid blood pressure (in mmHg) at 0, 6, 12, and 24 h after CLP or sham operation. The number of animals is 6 to 10 for each group at each time. Data are expressed as mean ± SD. Asterisk (*) indicates difference in comparison with sham control (P < 0.001). C and D, Peritoneal cavity infection and bacteremia. The numbers of CFUs in peritoneal cavity (C) and serum (D) of mice submitted to sham operation and MSI and SSI mice were evaluated 6 h after surgery. Results are expressed as mean number of CFUs/cavity or CFUs/mL of blood (n = 10 animals/group; the results are representative of three different experiments). The mean number of CFUs in the peritoneal cavity of SSI mice was higher than that of MSI and sham control animals. E and F, Peritoneal exudate and serum TNF-α levels. The concentrations of the cytokine were quantified in peritoneal exudate (E) and serum (F) 6 h after surgery in sham-operated, MSI, and SSI mice (n = 10 animals/group; the results are representative of three different experiments). Data are expressed as mean ± SD.

The mean values of carotid blood pressure simulating the time zero of the experiment, immediately after surgery, were similar in the three groups: 120.20 ± 7.23 mmHg, 114.70 ± 7.67 mmHg, and 117.60 ± 6.95 mmHg in sham-operated, MSI, and SSI mice, respectively. The mean blood pressure was significantly lower in SSI and MSI mice undergoing CLP 6 h (54.3% and 28.2% of reduction, respectively) and 12 h (59.7% and 27.3% of reduction, respectively) after injury as compared with sham-operated controls. Twenty-four hours after injury, the values of mean arterial blood pressure of SSI mice remained very low (reduced by 44.8%), whereas the arterial blood pressure of MSI mice returned to values next to those in the sham-operated group (Fig. 1B).

The analyses of bacterial count in the peritoneal cavity and blood cultures 6 h after cecal puncture were significantly positive in SSI mice in contrast to MSI mice that presented extremely low number of CFUs in the peritoneal cavity and blood, not statistically different from sham-operated controls with negative peritoneal cavity and blood cultures (Fig. 1, C and D).

Figure 1, E and F, shows the concentrations of TNF-α in the peritoneal exudate and serum 6 h after surgery in sham-operated, MSI, and SSI mice. The TNF-α levels in peritoneal exudate and serum in MSI mice were not different from those in sham controls. In the SSI mice, the peritoneal cavity and serum TNF-α concentrations were markedly increased in comparison with MSI and sham mice.

Light microscopic analysis and morphometry

No myocardial changes were observed in the three groups 12 h after sham or sepsis operation (data not shown). Twenty-four hours after sepsis induction, only the myocardium from SSI group presented spread foci of myocytolysis and cardiomyocytes tumefaction, contrasting with the myocardium of sham controls (Fig. 2, A and B). No inflammatory infiltrate was detected in the SSI myocardium, similarly to sham hearts. The use of plastic embedding allowed thin sections with adequate resolution of details (Fig. 2, C and D).

Fig. 2
Fig. 2:
The myocardium of mice submitted to severe septic injury (B and D) presents spread foci of myocytolysis (arrows) and tumefaction of cardiac myocells compared with sham-operated myocardium (A and C) (24 h after surgery). A and B, Hematoxylin-eosin. C and D, Plastic-embedded material, toluidine blue. Scale bars: 50 μm (n = 6 animals/group at each time).

To analyze the heterogeneity of the myocytes in SSI, MSI, and sham hearts, the individual minor diameters of myocytes were measured. The box-and-whisker plot of the diameter size of left ventricle myocytes of septic and sham mice is shown in Figure 3, A and C. Twelve hours after surgery (Fig. 3A), the results demonstrated that the mean diameter of cardiomyocytes from SSI was 16.59 ± 1.52 μm, 17% higher than the value 14.13 ± 1.30 μm of sham controls and 10% higher than the value 15.03 ± 1.62 μm of MSI hearts. Significantly, 24 h after CLP (Fig. 3C), the mean diameter of cardiomyocytes from SSI mice was 18.10 ± 1.46 μm, 21% higher than the value 14.94 ± 1.17 μm of MSI and 31% higher than 13.76 ± 1.12 μm of sham myocardium, very likely reflecting intracellular edema. When the percentile frequency distribution of minor diameter of cardiomyocytes in each group was plotted, the shift to the right of the values in the cardiac myocytes of SSI mice can clearly be seen in comparison with the values in MSI and sham-operated animals. Moreover, the occurrence of discrete groups of larger cells corresponding to foci of myocytes tumefaction and/or myocytolysis could be clearly demonstrated when the frequency distributions of minor diameter of myocyte in each group were plotted, more pronounced 24 h after surgery (Fig. 3, B and D).

Fig. 3
Fig. 3:
A and C, Box plots of the minor diameter size of left ventricle myocytes of sham-operated and septic mice 12 h (A) and 24 h (C) after CLP surgery. B and D, Percentile frequency distribution of minor diameter of cardiomyocytes in sham, MSI, and SSI hearts. A shift to the right of the values in MSI and SSI hearts 12 h (B) and 24 h (D) after injury can be seen, more pronounced at 24 h. Moreover, the occurrence of discrete groups of larger cells corresponding to foci of myocytes tumefaction and myocytolysis is clearly depicted, notoriously at 24 h (n = 6 animals/group at each time).

Actin and myosin immunofluorescence

In the sham-operated myocardium, the actin and myosin labeling was regularly stained, showing distinctly the presence of clearly green fluorescent I bands corresponding to actin (appearing as ladder rungs) and A bands corresponding to myosin (appearing as brick piles) within the sarcomeric structures throughout the cardiac muscle cells (Fig. 4, left panels). In contrast, in the SSI myocardium, 24 h after CLP, the localization of actin and myosin was focally disrupted, reflecting disarrangement of the sarcomeric structure; also, spread foci of myosin and actin filaments forming amorphous masses due to collapse of myofilaments were seen (Fig. 4, right panels).

Fig. 4
Fig. 4:
Myosin and actin immunolabeling (green fluorescence) in sham-operated animals shows distinctly the presence of A bands, corresponding to myosin and appearing as brick piles, and I bands, corresponding to actin and appearing as ladder rungs. The localization of actin and myosin was focally disrupted; spread foci of myosin and actin filaments forming amorphous masses due to collapse of myofilaments are seen (arrows) in mice submitted to severe septic injury (SSI) (24 h after surgery). Scale bars: 15 μm.

Sarcolemmal permeability evaluated by albumin staining

In sham controls (with or without saline as placebo), albumin was localized in the interstitial space as a delicate network and in the vascular lumina. In SSI hearts, albumin staining was more intense in the interstitial space, and spread large blocks of cardiomyocytes showed intracytoplasmic albumin labeling. In SSI+SOD hearts, albumin staining was similar to that of sham controls, restricted to the interstitial space, except for a few cardiomyocytes around great intramyocardial vessels showing discrete cytoplasmic albumin staining. Wheat germ agglutinin staining clearly delineated the cardiomyocytes in the three groups. The merge of albumin and WGA fluorescent images clearly depicted the cardiac muscle cells in the three groups, and spread blocks of muscle cells exhibit a uniformly diffuse pattern of green fluorescent cytoplasmic albumin staining in SSI hearts (with or without placebo) and small blocks of just a few myofibers exhibiting mild green fluorescent albumin staining in SSI+SOD hearts (Fig. 5).

Fig. 5
Fig. 5:
Immunolabeling for albumin (green fluorescence), plasma membrane labeling with WGA (red fluorescence), and nuclear staining with DAPI (blue fluorescence) (24 h after surgery). In sham controls (with or without saline as placebo), albumin was localized in the interstitial space as a delicate network and in the vascular lumina. In SSI hearts, albumin staining was more intense in the interstitial space, and spread large blocks of cardiomyocytes showed intracytoplasmic albumin labeling. In SSI+SOD hearts, albumin staining was similar to that of sham controls, restricted to the interstitial space, except for a few cardiomyocytes around great intramyocardial vessels showing discrete cytoplasmic albumin staining. Wheat germ agglutinin staining clearly delineated the cardiomyocytes in the three groups. The merge of albumin and WGA fluorescent images clearly depicted the cardiac muscle cells in the three groups, and spread blocks of muscle cells exhibit a uniformly diffuse pattern of green fluorescent cytoplasmic albumin staining in SSI hearts (with or without placebo) and small blocks of just a few myofibers exhibiting mild green fluorescent albumin staining in SSI+SOD hearts. Scale bars: 50 μm.

MHC and α-sarcomeric actin (actin) expression

The steady-state MHC levels were markedly reduced in SSI mice, 55% lower in comparison with sham hearts and 25% lower in comparison with the MSI hearts. The MHC levels in the MSI hearts was moderately reduced, 27% lower compared with the values in sham hearts. Similarly, the steady-state actin levels were conspicuously reduced in SSI mice, 79% lower in comparison with hearts of sham animals and 53% lower in comparison with MSI hearts. The actin levels in the MSI hearts were remarkably reduced 53% lower compared with the sham hearts (Fig. 6, E and F).

Fig. 6
Fig. 6:
A, Representative electron micrograph of sham-operated myocardium (24 h after surgery). Myofibrils bands alternate with single rows of mitochondria (mi). Scale bar: 3 μm. B and C, Representative electron micrographs of the SSI myocardium. Myofibrillar derangement and fragmentation and dissolution of myofibrils (*), intracellular edema, mitochondrial swelling, contraction bands, and lipofuscin bodies. Scale bars: 5 μm (B) and 3 μm (C). D, Representative electron micrograph of SSI myocardium. Myofibrillar dissolution and fragmentation (*), interstitial edema, and rupture of the sarcolemma (arrow). Scale bar: 2 μm. E and F, Western blot analysis of myosin (E) and actin (F). The amounts of myosin and actin of mice submitted to MSI and SSI and sham operation were measured 24 h after surgery and expressed in arbitrary units. Results (n = 3 to 5 per group) are representative of three different experiments. IS indicates interstitial space; Z, Z line; A, A band; I, I band; ID, intercalated disc; mi, mitochondria; cb, contraction band; sarc, sarcolemma; cap, capillary; lip, lipofuscin body; mf, myofibril.

Ultrastructural changes

The actin and myosin filaments were arranged lengthwise in myofibrils, showing a symmetrical and parallel organization, presenting as arrays of myofibrils separated by rows of mitochondria (Fig. 6A). The myocardium of mice submitted to severe sepsis presented striking degenerative changes characterized by myofibrillar derangement and fragmentation and dissolution of myofibrils, intracellular edema, mitochondrial swelling, contracture bands, lipofuscin bodies, and, occasionally, rupture of the sarcolemma of cardiomyocytes (Fig. 6, B-D). No apoptotic or necrotic cells were detected by electron microscopic observation of cardiac tissue from any of the septic groups.

4-HNE immunoreactivity

4-Hydroxy-2-nonenal was used to detect lipid peroxidation as an indicator of oxidative stress and disruption of plasma membrane lipids. There were minimal cardiomyocytes 4-HNE immunostaining present in the negative controls and sham-operated hearts (Fig. 7, A and B, respectively). However, there was evidence of increased 4-HNE immunoreactivity in cytosol and mainly in the sarcolemma of cardiomyocytes in the myocardium of MSI group (Fig. 7C) and markedly increased in the myocardium of SSI group (Fig. 7D). The mean area of 4-HNE protein adducts was significantly increased in SSI hearts compared with controls (SSI: 9.19% ± 0.40% vs. sham: 1.40% ± 0.15%) and also significantly increased in MSI hearts compared with controls (MSI: 5.34% ± 0.43% vs. sham: 1.40% ± 0.15%). The levels of 4-HNE-modified proteins in SSI hearts were significantly higher than the values in MSI hearts (Fig. 7E).

Fig. 7
Fig. 7:
Immunohistochemical evaluation of the expression of 4-HNE-modified protein in the myocardium of septic and sham mice (24 h after surgery). A, Immunostaining pattern in a septic heart using a secondary antibody alone (negative control). B, Representative image of the minimal 4-HNE immunoreactivity present in all hearts in the sham-operated group. C, Representative image of the increased 4-HNE immunoreactivity in MSI hearts. D, Representative image of the markedly increased 4-HNE immunoreactivity in SSI hearts. Positive immunostaining (brown) for 4-HNE-modified protein is distinct in the cytosol and mainly in the sarcolemma of cardiac myocytes. Scale bars: 50 μm. E, The graph shows the mean expression level of 4-HNE protein adducts as percentage area of microscopic field in septic hearts compared with sham controls. The mean expression level of 4-HNE protein adducts in SSI and MSI hearts were 6.5 and 3.8 times higher than the mean level in sham controls, respectively. Moreover, the mean expression level of 4-HNE protein adducts in SSI hearts was 72% higher compared with the mean level in MSI hearts. Sham indicates sham-operated mice; MSI, hearts of mice submitted to MSI; SSI, hearts of mice submitted to SSI.

NT immunoreactivity

There were minimal cardiomyocytes NT immunostaining present in all of the hearts in sham controls (Fig. 8B). However, there was evidence of increased NT immunoreactivity in the myocardial tissue obtained from MSI (Fig. 8C) and SSI (Fig. 8D) mice. Negative controls showed no positive staining (Fig. 8A). The immunoreactivities of NT in SSI and MSI mice were similar. Positive immunolabeling for NT was distinct in the cytosol, focally spread throughout the left ventricle myocardial tissue. The mean expressions of NT in the myocardium from SSI (2.80 ± 0.34 AUs) and MSI (2.29 ± 0.22 AUs) mice were significantly increased compared with sham controls (1.00 ± 0.23 AU) (Fig. 8E).

Fig. 8
Fig. 8:
Immunohistochemical evaluation of the expression of NT in the myocardium of septic and sham mice (24 h after surgery). Immunostaining pattern in a septic heart using a secondary antibody alone (negative control). A, Immunostaining pattern in a septic heart using a secondary antibody alone (negative control). B, Representative image of the minimal NT immunoreactivity present in all hearts in the sham-operated group. C, Representative image of the increased NT immunoreactivity in MSI hearts. D, Representative image of the markedly increased NT immunoreactivity in SSI hearts. Positive immunostaining (brown) for NT is distinct in the cytosol, focally spread throughout the myocardial tissue. E, The graph shows the mean expression levels of NT (estimated using a semiquantitative score 0-4) in septic hearts compared with sham controls. The immunoreactivity scores of NT in SSI and MSI hearts were 2.8 and 2.3 times higher than the score in sham controls, respectively. The immunoreactivity scores in SSI and MSI were not statistically different.

DISCUSSION

The present study examined the myocardial changes in a mice model of sepsis induced by CLP surgery. The CLP model in rodents has been widely used for the investigation of several features of sepsis and septic shock and is considered the criterion standard for sepsis research (19). The induction of sepsis by CLP in mice is efficient in producing a clinical situation similar to that observed in human sepsis (4). Many of the attributes for an appropriate model were satisfied: it is polymicrobial, has focal infection origin, produced septicemia, induced increased TNF-α levels in peritoneal exudate and serum, and circulatory shock.

There are two major findings in this study: first, sarcolemmal and myofilamentar damage in cardiomyocytes, and second, evidence for the existence of lipid peroxidation and protein nitration as possible mechanisms implicated in these changes.

The heart as an important target in severe sepsis

Previous experimental studies have shown either an association between sepsis and structural myocardial changes, such as myocardial edema (9), mitochondrial injury and myofibrillar loss (10-12), interstitial edema (11, 13, 14), subendocardial hemorrhage (13), and subendocardial necrosis (9), or impaired myocardial contractility in the absence of tissue injury (15, 16). Recently, we reported myocardial structural changes in long-term human severe sepsis classified as an inflammatory cardiomyopathy (4). Diffuse foci of myocytolysis associated with focal disruption of actin/myosin contractile apparatus could be observed in the myocardium of SSI mice 24 h after injury. However, no inflammatory infiltrate in the myocardium of MSI and SSI mice could be seen. The morphological changes were detected only in the late periods of the septic process. All stresses and injurious influences are known to exert their effects first at the molecular or biochemical level, there being a lag between the stress and the morphological changes of cell injury or death, this delay varying according to the evaluation method sensitivity. The detection of intracellular albumin to evaluate plasma membrane permeability provided evidence of severe injury of the sarcolemma in SSI hearts. The ultrastructural study showed spread blocks of a few myocytes with fragmentation and dissolution of myofibrils, intracellular edema, and, occasionally, ruptured cardiomyocytes sarcolemma. The ultrastructural myofibrillar disruption agrees with the markedly reduced levels of MHC and actin in SSI hearts, whereas the MHC levels in MSI hearts were moderately and the actin levels were markedly reduced as compared with sham controls. The increased sarcolemmal permeability resulted in cellular tumefaction, determined morphometrically. Increased sarcolemmal permeability is considered a prominent feature of ischemic myocardial injury (21) and could be the initiating event leading to myocyte damage and the progression of cardiac dysfunction (22). Considering that the sarcolemma, composed of plasma and basement membranes, constitutes the permeability barrier to the cardiac myocytes, and its disruption with increased permeability is an early event that precedes cellular degenerative changes (17, 18), it is conceivable the occurrence of permeability alterations of sarcolemmal membrane in septic hearts. The blocks of cardiomyocytes exhibiting a uniform pattern of albumin staining in the cytoplasm associated with normal WGA staining may reflect a reversible ischemic damage. Moreover, the marked reduction of sarcolemmal permeability with the administration of a superoxide scavenger indicates the involvement of free radicals in its genesis.

The mechanism of cell injury could be ascribed to a dysfunctional microcirculation, hallmark of septic shock that produces regional flow disturbances and abnormal tissue oxygenation, causing relative ischemia in various organs, including the heart (23-25). A heterogeneous distribution of blood flow, with certain microvascular beds underperfused whereas others are normally or hyperperfused, is one of the key features in sepsis (26-28). Moreover, the focal nature of the lesions suggests that the microcirculation could be involved; that is, the primary site of disease could be at a level capable of causing focal myocytolysis in discrete groups. On the other hand, because in sepsis there is inability to consume or use molecular oxygen, a mitochondrial defect has been suggested to underlie sepsis-associated organ dysfunction (29, 30).

Evidence for the existence of lipid peroxidation and peroxynitrite-mediated protein nitration in sepsis

Our study demonstrated, for the first time, increased immunohistochemical detection of 4-HNE proteins adducts in septic hearts. Because 4-HNE can be formed only by the interaction of free radicals with lipids (31), it can be concluded that sepsis promotes the formation of free radicals that causes lipid peroxidation and in turn leads to modifications of cardiac proteins. Because lipid and protein modification of this type causes loss of function (32, 33), this cytotoxic metabolite may represent a factor that contributes to the loss of cellular homeostasis. The focal alterations observed in the hearts of septic mice could be directly related to ischemia-mediated peroxidative degradation of membrane lipids and consequent increased sarcolemmal permeability. Several previous studies have reported that myocardial ischemia is associated with the generation of lipid peroxidation unsaturated aldehyde products such as 4-HNE (34) and covalent adducts between 4-HNE and myocardial proteins (35). The focal actin/myosin disruption and downregulation of actin and myosin in septic hearts can be ascribed to the diffuse focal expression of 4-HNE-modified protein. Elevated levels of 4-HNE-modified protein have been shown in the myocardium of patients with dilated and hypertrophic cardiomyopathy, apparently proportional to left ventricular dilatation and systolic dysfunction (36). The less pronounced presence of 4-HNE in MSI hearts associated with attenuated changes compared with SSI hearts favors the idea that lipid peroxidation may play a pivotal role in the pathogenic process of experimental septic cardiomyopathy.

Excessive production of NO and endogenous formation of peroxynitrite from NO and superoxide have been suggested as key players in the setting of cardiac depression in sepsis (16,37). The increased focal expression of NT in myocardial cells of animals with severe sepsis indicates an increased production of NO and superoxide that interact to produce peroxynitrite, a powerful oxidizing and nitrating agent. Previous study demonstrated that myocardial tyrosine nitration occurs in both sepsis and myocarditis and suggested a role for peroxynitrite derived from NO in the myocardial depression associated with these conditions (38). The most abundant proteins for nitration within cardiac myocyte are myosin and actin. The observation of scattered foci of actin and myosin filament disruption and downregulation of actin and myosin in septic hearts support the idea that tyrosine nitration could potentially decrease myocardial contractility by directly modifying the contractile apparatus; that is, the cardiac myofilaments could be an important target in this setting.

These findings make plausible the hypothesis that increased sarcolemmal permeability might be a primary event in myocardial injury in severe sepsis possibly due to oxidative damage to lipids and proteins that could precede phenotypic changes that characterize a septic cardiomyopathy. These alterations, which seem to be mostly reversible, may be responsible, at least partly, for cardiac depression in sepsis. Although the functional consequences of our findings are not clear at this moment, because we do not know whether the observed changes necessarily establish a direct cause-effect relationship with myocardial dysfunction in SSI hearts, the occurrence of circulatory shock suggests myocardial depression. After further studies, these abnormal variables can emerge as therapeutic targets, and their modulation might provide beneficial effects on future cardiovascular outcomes and mortality in sepsis.

ACKNOWLEDGMENTS

The authors thank Monica A. Abreu, Maria Elena Riul, and Lígia B. Santoro for excellent technical assistance.

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Keywords:

Sepsis; septic cardiomyopathy; sarcolemmal permeability; oxidative damage; 4-hydroxy-2-nonenal (4-HNE); nitrotyrosine (NT)

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