A recent study demonstrated that approximately half of critically ill patients with a normal ejection fraction (EF) and no previous history of heart failure (HF) exhibited evidence of left ventricular (LV) diastolic dysfunction (1). Several studies have demonstrated that immunoinflammatory activation has a pivotal role in the development and progression of HF in critically burned patients (2, 3). Specifically, serum levels of tumor necrosis factor α (TNF-α) and interleukin 6 (IL-6) are positively correlated with deteriorating heart function and is predictive of mortality (2–4).
Previous animal model experiments found that proinflammatory processes were associated with progression of cardiac diastolic dysfunction (5). Therefore, one hypothesis for how inflammation affects the heart of critically ill patients is that inflammatory cytokines, such as TNF-α or IL-6, directly induce cardiac diastolic dysfunction (6). Recently, we showed that TNF-α and IL-6 influence cardiac function. There was a significant correlation between the immunoinflammatory system and the development of diastolic HF (DHF) (7, 8). We also demonstrated that these inflammatory cytokines downregulated the sarcoplasmic reticulum Ca2+-ATPase 2 (SERCA2) protein, one of the most essential calcium-handling proteins, subsequently leading to LV diastolic dysfunction.
Diffuse thermal injury in severe burns causes loss of the normal skin barrier and impacts several organs beyond skin. The patient often presents with severe inflammation. The postinjury phase of major burns is characterized by severe hypermetabolic reaction, immune compromise, and significant cytokine release (9). Traditionally, mortality of major burn injuries is related to patient age, the total body surface area burned (TBSA), and the presence or absence of inhalation injury (10). It is possible that acute cardiac dysfunction due to severe burn injury may also contribute to adverse clinical prognosis. Only a few studies have focused on acute cardiac dysfunction in severely burned patients, and the underlying cellular and molecular mechanisms are poorly understood (11–13).
In this study, we compared the levels of serum levels of TNF-α and IL-6 and clinical outcomes in severely burned patients. We assessed the association of plasma levels of IL-6 and TNF-α and LV diastolic dysfunction indices in this specific group of patients. We explored the direct effect of the sera from burn patients on the mRNA levels SERCA2 in cardiomyocytes. We also investigated the prognostic influence of LV diastolic function after adjusting for other possible factors.
MATERIALS AND METHODS
Between January 2007 and June 2010; we consecutively enrolled 56 burn patients who had been admitted to the burn unit of the intensive care unit (ICU) at the National Taiwan University Hospital. Exclusion criteria included children younger than 12 years, burns involving less than 10% TBSA, admissions for conditions other than burns (e.g., purpura vulgaris, toxic shock syndrome, Stevens-Johnson syndrome), and patients for whom a decision to treat with palliative care was made within the first 24 h of admission. Patients with significant hepatic disease, significant coronary artery disease, secondary hypertension, pericardial disease, severe valvular heart disease, cancer, chronic atrial fibrillation, or death within the first 48 h of being admitted to the ICU were excluded. A control population consisted of risk factor–matched controls with no symptoms of HF failure and no objective evidence of diastolic dysfunction was selected from the clinic of the same hospital. The control population was age, sex-matched nonburned patients. They had no acute or chronic inflammatory disease, and none of them received statin therapy. The study was approved by the local institutional review board, and all subjects provided their informed consent.
Date of admission and discharge, age, sex, admission weight, height, body mass index, TBSA, presence or absence of inhalation injury, length of hospital stay, and in-hospital mortality were prospectively recorded for all patients. Percentage of burns (TBSA) was assessed using the Lund and Browder chart, and depth was determined by clinical observation. All patients received echocardiographic examinations. Blood samples were obtained for measuring the plasma levels of TNF-α and IL-6. To compare the change of diastolic function and cytokine levels upon improved health, TNF-α and IL-6 were measured while the patient was in the ICU and after being transferred to the general ward. The echocardiographic parameters of diastolic dysfunction were selected according to the recent consensus statement of the European Society of Cardiology and our previous articles (14–17). The burn patients were classified as having LV diastolic dysfunction or not, according to the echocardiographic findings.
Measurements of plasma TNF-α and IL-6
In all patients, blood samples were collected from the antecubital vein between 8:00 and 10:00 AM, in supine position, after 12 h of fasting. Serum IL-6 and TNF-α were measured with high-sensitivity enzyme-linked immunosorbent assays. Further detailed methods are provided in Methods, Supplemental Digital Contents 1, at http://links.lww.com/SHK/A128.
Left atrial diameter, LV end-diastolic diameter and systolic diameter, interventricular septum thickness, LV posterior wall thickness, mitral inflow early rapid filling wave (E), peak velocity of the late filling wave due to atrial contraction (A), E/A ratio, E wave deceleration time (DT), and mitral annular early diastolic velocity (Em) were measured according to the American Society of Echocardiography guidelines with the use of Sonos 7500 echocardiography (Philips, Andover, Mass) with a 1- to 3-MHz transducer attached (18). Left ventricular mass index was calculated from the LV end-diastolic diameter and systolic diameter, interventricular septum thickness, and LV posterior wall thickness according to the method of Devereux et al. (19). Doppler and color Doppler studies were performed to detect valvular heart disease. Significant valvular heart disease was defined as at least moderate aortic or mitral stenosis/regurgitation.
Measurements of TBSA
Percentage of burns (TBSA) was assessed using the Lund and Browder chart, and depth was determined by clinical observation. Total burned surface area was carefully calculated according to documented charts with appropriate modification for obese patients. In brief, for obese patients, their height, length, and circumference of body segments were measured and converted to TBSA with the linear method. The surface area was determined by multiplying the circumference of a body segment by its length and a factor correcting for shape (20, 21).
HL-1 cardiomyocytes were cultured in Claycomb medium (JRH Bioscience, Lenexa, Kan) supplemented with 10% fetal bovine serum and maintained in a humid 10% CO2 incubator at 37°C, as previously described (8).
RNA extraction, quantitative real-time reverse transcription polymerase chain reaction
The cells were harvested 24 h after treatment with sera from burn patients for RNA extraction and quantification. Total mRNA was isolated and reverse transcribed. The single-stranded cDNA was amplified (ABI-Prism 7900; Applied Biosystems, Foster City, Calif), using SYBR green dye. For mouse SERCA2, the primers were designed according to the published gene sequences (8), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA levels were used as an internal control. The primer sequences and other detailed methods are provided in Methods, Supplemental Digital Contents 1, at http://links.lww.com/SHK/A128.
Data were analyzed using SPSS 15.0 software (SPSS Inc, Chicago, Ill). Continuous variables are presented as mean (SD), whereas categorical variables are presented as frequencies. Associations between categorical variables were tested by Pearson χ2 test. To test whether the data were normally distributed, the Kolmogorov-Smirnov test was applied. The comparisons between the data showing normal distribution were performed using the Student t test, or otherwise by the Mann-Whitney U test. The associations between cytokines and burn surface area or Doppler parameters were studied with the use of Pearson correlation coefficient if the data met the criteria for normal distribution, or otherwise by Spearman correlation test. Echocardiographic and serum cytokines levels of the burn patients were compared by paired t test for data before and after treatments. To evaluate the survival influence of LV diastolic dysfunction, a multiple Cox regression model in which the traditional prognostic factors after burns including age, major comorbidities, TBSA, and inhalation injury were added on top of LV diastolic dysfunction was applied. P < 0.05 was considered statistically significant.
The demographic data of burn patients with or without LV diastolic dysfunction are shown in Table 1. More than half of major burn patients had echocardiographic finding of LV diastolic dysfunction. Although the baseline characteristics and even LV EF of the two groups are comparable, patients with LV diastolic dysfunction had much significantly higher percentages of TBSA and higher plasma inflammatory cytokines levels: for IL-6: 319.2 (SD, 182.1) pg/ml versus 626.1 (SD, 253.6) pg/ml, P < 0.001; and for TNF-α: 39.2 (SD, 25.1) pg/ml versus 73.4 (SD, 24.8) pg/ml, P < 0.001 (Table 1). We also compared the differences between burn patients and the control subjects of general population (see Table, Supplemental Digital Contents 2, at http://links.lww.com/SHK/A129); the LV diastolic dysfunction parameters were significantly different in the two groups along with much significantly higher inflammatory cytokines levels in burn patients.
Cytokine levels and diastolic dysfunction parameter changes in burn ICU patients
Total body surface area was associated with both TNF-α (r2 = 0.77, P < 0.001) and IL-6 (r2 = 0.81, P < 0.001) (Fig. 1). Diastolic parameters were also associated with TNF-α and IL-6 (Fig. 1, C–H). Nevertheless, there was no association between inflammatory cytokines and diastolic parameter in the controls (see Figure, Supplemental Digital Contents, at http://links.lww.com/SHK/A130).
Interleukin 6 and TNF-α serum levels decreased after patients were moved from the ICU to the general ward (Table 2). Echocardiographic parameters for diastolic dysfunction (including decelerating time, E/Em, and mitral inflow E/A) improved significantly after the patient’s general condition improved (Table 2).
LV diastolic dysfunction was associated with burn patients’ survival
There were 7 patients (22%) with LV diastolic dysfunction, and only 1 patient (7%) without LV diastolic dysfunction died. All patients died because of sepsis and finally progressed to multiorgan failure. Kaplan-Meier analysis indicated that there was a significant difference between the two groups (Fig. 2). To evaluate the independent effect of LV diastolic function, we adjusted the traditional prognostic factors of burns including age, diabetes, hypertension, TBSA, and inhalation injury. We found that LV diastolic dysfunction appeared to increase 4-fold the chance of dying in the burn patients (hazard ratio, 3.92; 95% confidence interval, 1.04–14.69; P = 0.034) (Table 3).
Serum from burn patients attenuated SERCA2 expression in cardiomyocytes
Earlier studies from our group found that TNF-α and IL-6 decreased SERCA2 expression in cardiomyocytes (7). Therefore, we investigated if high serum levels of TNF-α and IL-6 from burn patients were associated with changes in SERCA2 expression. The SERCA2 mRNA level decreased significantly in cardiomyocytes after treatment with sera from burn patients (Fig. 3). The sera from patients with greater TBSA further decreased cardiomyocyte SERCA mRNA levels (Fig. 3).
In this study, we showed that the levels of the proinflammatory cytokines IL-6 and TNF-α correlated to LV diastolic dysfunction parameters in burn patients and found that the sera of these patients inhibited SERCA2 mRNA levels. Moreover, improvement in LV diastolic dysfunction was correlated with a decrease in TNF-α and IL-6 serum levels and also was associated with the clinical outcomes of burn patients. Because of the strong correlation between LV diastolic function and SERCA2 mRNA expression, we hypothesize that plasma levels of these proinflammatory cytokines influence LV diastolic dysfunction in these burn patients by, at least in part, downregulating SERCA2 expression, which in turn influences patient outcomes.
There are only few prior studies that investigated LV diastolic dysfunction during burn injury, and most were animal model and correlation studies. To our best knowledge, we are the first to investigate the molecular mechanism that underlies LV diastolic dysfunction failure in burn patients. In our study, LV DHF, defined by the consensus statement of the European Society of Cardiology, was found to be an independent risk factor for clinical outcome in burned patients. Aggressive intravenous fluid supplement, which can overdistend the ventricles, is mandatory treatment for burn injuries during resuscitation and may increase the risk of LV diastolic dysfunction. The failure of the LV to completely diastole may lead to inadequate cardiac preload and decreased cardiac output, inducing organ dysfunction and even death. To further clarify change in LV diastolic function, it is important to carefully investigate the hemodynamic tracing during acute care in the burn ICU. A better understanding of this pathophysiology may help guide clinical treatment and improve the prognosis of burn patients.
Earlier studies from our group found that TNF-α and IL-6 decreased SERCA2 expression in cardiomyocytes (7). Therefore, we investigated whether high serum levels of TNF-α and IL-6 from burn patients were associated with changes in SERCA2 expression. According to a recent article regarding burn size and inflammatory response, the inflammatory response significantly rose in those with mean TBSA of greater than 50% (22). In our current study, the sera from patients with TBSA of greater than 50% attenuated SERCA2 levels than the control and patients with less TBSA and may lead to a higher probability for the development of LV diastolic dysfunction clinically. Burn injury is well known for its vigorous inflammatory reaction and cytokine release, a condition of systemic inflammatory response syndrome. In previous studies, C-reactive protein is upregulated in response to burn injury and related to the severity of burn trauma and to the clinical outcome (23). Proinflammatory cytokines, TNF α and IL-6, are also secreted by monocytes and macrophage during acute phase of burn injury (24). These cytokines produce the hemodynamic, metabolic, immunological, and pathological manifestations of the disease, which in turn lead to tissue damage, shock, and organ failure (23, 25).
In our study, the serum levels of TNF-α and IL-6 differed between patients who survived and those who died because of burn injuries, suggesting their clinical relevance. However, the use of anti-inflammation therapy is controversial in the treatment of these patients (26, 27) and has not been validated in clinical practice. Numerous factors may regulate this process of inflammatory cytokine release and adverse prognosis. To improve clinical outcome in burn patients, it is necessary to further investigate and better understand the complex underlying molecular regulatory mechanisms. In this study, we found that DHF may be a valuable burn-related outcome to study for understanding the role of burn-induced inflammation and clinical outcomes; however, more studies are required to further understand these medical issues.
Sarcoplasmic reticulum Ca2+-ATPase 2 is one of the most extensively studied calcium-handling proteins with respect to LV diastolic dysfunction. In cardiomyocytes, free calcium increases during systole and induces mechanical contraction by binding to the troponin complex. Subsequently, calcium is removed from the cytosol primarily by the action of SERCA2 during diastolic relaxation (28). Decreased activity of SERCA2 slows the removal of calcium from the cytosol, which impairs the diastolic relaxation of contractile proteins (29). Inflammatory response has numerous effects on cardiomyocytes that might influence LV function and remodeling (30, 31). For example, TNF-α may modulate the β-adrenergic receptor function and thus result in contractile dysfunction (32). Proinflammatory cytokines may inhibit contractility of papillary muscles directly through mediation of myocardial nitric oxide synthase (33). Cytokines can also impact myocardial function via their effects on the extracellular matrix (6). However, one possible rapid influence of inflammatory responses over LV diastolic dysfunction may be through calcium dynamics, as reported in the current study. Burns induce serious inflammatory response that may be even stronger than severe sepsis. Left ventricular diastolic dysfunction might subsequently develop by impairment of calcium homeostasis (34). The resulting fluid accumulation, tissue edema accompanied with LV diastolic dysfunction, could lead to poor wound recovery and clinical outcomes for burn patients.
Left ventricular contractile dysfunction after burn injury has been reported in some earlier studies (35, 36). Horton (37) concluded that postburn rise in serum TNF-α provided a measure of immune response. Proinflammatory cytokines after burn injury also provided a better prognostic indicator of patient susceptibility to sepsis. These cytokines could lead to upregulation of p38 mitogen-activated protein kinase and are associated with burn-related myocardial contractile defects (38). Other studies have also shown that burn trauma could activate α1-adrenergic pathway, which in turn initiates RhoA/Rho-kinase activation (39). Activation of Rho-kinase pathway plays a pivotal role in injury-related myofibrillar remodeling and is associated with myocardial contractile function. Horton (37) also proposed that heat shock proteins, matrix metalloproteinase from burn wounds, and adverse effects for mesenteric lymph from the gastrointestinal tract were possible sources of myocardial depressant factors. However, most of the above studies focused on LV systolic dysfunction after burn injury, rarely investigating the change of LV diastolic dysfunction. Our research mainly analyzed parameters of LV diastolic dysfunction and proved the prognostic value of cardiac diastolic function. We also hypothesized that inflammation cytokines might exert influence over cardiomyocytes through calcium-handling protein.
Our study had limitations. In our current study, whether TNF-α and IL-6 in patients sera cause SERCA2 suppression still requires verification. It would be most advantageous to prove the mechanism by performing blocking studies. To delineate the actual mechanism for how and whether TNF-α and IL-6 cause SERCA2 suppression, further detailed analysis is required.
This study found a significant correlation between the hyperactivating immunoinflammatory system after burn injuries and the development of LV diastolic dysfunction, which improved as inflammation declined. We also demonstrated that the sera from burn patients reduced SERCA2, one of the most essential calcium-handling proteins; mRNA levels and this reduction may be pertinent to diastolic dysfunction at the cellular level. More importantly, the development of LV diastolic dysfunction had a strong correlation with mortality in this specific group of patients. Although there is little information regarding the medical management of burn patients, a better understanding of the nature of inflammatory activation in burn patients with LV diastolic dysfunction may lead to improvement of outcomes by applying novel therapies aimed at limiting inflammatory reactions.
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