The aim of the present doctoral thesis is to investigate the detailed molecular
mechanism by which proinflammatory cytokines are involved in the pathegenesis of diastolic heart failure (DHF). We realized that the sarcoplasmic reticulum Ca2+ ATPase (SERCA) is essential to the regulation of intracellular calcium levels in cardiac, smooth and skeletal muscles. 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 SERCA during diastolic relaxation. It has also been shown that increasing activity of SERCA2 improves
ventricular diastolic relaxation in animal and human models. However, our pilot study reveal that AngiotensinII does not appear to exert an effect on SERCA2 levels in cardiomyocytes, but pro-inflammatory cytokines such as TNF-α or IL-6 have even greater effect over SERCA2 expression in cardiomyocyte models. Therefore, we explored the effect of IL-6 and TNF-α on the transcriptional regulation and function of SERCA2 in cardiomyocytes in the subsequent study.
Regarding TNF-α or IL-6 and SERCA2 signal transduction, we investigated whether TNF-α or IL-6 modulates SERCA2 calcium current through transcriptional regulation, using a HL-1 cells model. We cloned a 1754 bp promoter fragment of rat SERCA2 gene and amplified by polymerase chain reaction (PCR). We determined that TNF-α or IL-6 decreased SERCA2 mRNA, protein levels and SERCA2 promoter activities, which resulted in an attenuation calcium transient. TNF-α or IL-6 also significantly decreased promoter activity of SERCA2 gene in a concentration- and time-dependent manner. We also treated HL-1 cardiomyocyte with sera from cirtically illness patients and also demonstrated a significant decrease of SERCA2 expression comparing to the controls.In summary, we explored the possible mechanism, and found that inflammatory cytokines
had a direct effect on SERCA2 at the transcription level. Due to the strong correlation between heart diastolic function and SERCA2 expression, we hypothesized that plasma levels of TNF-α and IL-6 are associated with DHF through downregulation of
SERCA2. The above experiment found that TNF-α decrease Sarcoplasmic reticulum Ca2+-ATPase (SERCA2) gene expression, and attenuated diastolic calcium re-uptake in cardiomyocyte.
Accordingly, in the following study, we explored the effect of TNF-α on the transcriptional regulation of SERCA2 cardiomyocytes. We used a murine cell line which continuously divides and maintains a differentiated cardiac phenotype to study the detailed signaling mechanisms by which TNF-α regulates the expression of SERCA2 protein. We first found that TNF-α decreased SERCA2 gene expression and induced left ventricular (LV) diastolic dysfunction through Nuclear Factor-KappaB (NF-κB) Element–Binding Protein–Dependent Pathway. By inhibition of TNF-α or NF-κB to block the pathway, we also discovered an improvement of SERCA2 level and LV diastolic function. We also used an in vivo rat model of hyper-TNF-α to verify the results obtained in the cellular study. In summary, we first demonstrated that
TNF-α decreased the transcription of SERCA2 through a NF-κB binding element in the promoter region of SERCA2 gene both in vivo and in vitro. The upstream NF-κB dependent pathway was critical for TNF-α–induced decreased transcription of SERCA2 gene. The NF-κB blocker (PDTC) and pre-incubation of simvastatin
inhibited TNF-α–induced NF-κB dependent pathway and augmented expression of SERCA2 gene which in turns lead to improvement of LV diastolic function from an in vivo rat model of LPS injection. LV diastolic function has recently been found to play an important role for prognosis in different populations. Therefore, our results may implicate the therapeutic effect of statins or NF-κB inhibitors to prevent TNF-α–
induced NF-κB generation in cardiomyocytes and further ameliorate diastolic function, although the statin concentrations used in the present study may be high compared with clinically relevant concentrations and are known to have other pleiotropic effects.
Though the cell and animal models in our current studies represented acute illness with high inflammation, subclinical inflammatory status induced by adiposity or metabolic syndrome are also known to be potentially associated with the development of LV diastolic dysfunction. Previous study suggested that reduction in myocardial oxidative stress and related protein modifications might be able to prevent the so-called
“metabolic heart disease.” Our current studies may conclude that use of statin might be able to treat LV diastolic dysfunction which resulted from chronic inflammatory status.
Regarding clinical studies, to evaluate the association between cytokines (TNF-αor IL-6) and left ventricular dysfunction indices, first, we sought to assess plasma levels of IL-6 and TNF-α in patients with DHF, including critically ill patients. We included 2 goups of patients including the first group consisted of pure DHF patients admitted to the cardiovascular ward of National Taiwan University Hospital and its affiliated hospital for coronary angiography or a health examination and the second group enrolled 30 consecutive patients with left ventricular diastolic dysfuction that were admitted to the intensive care units (ICUs) of National Taiwan University Hospital. A control population for study group 1 consisted of risk-factor matched controls with no symptoms of HF failure and no objective evidence of diastolic dysfunction. The results showed that patients with DHF had significantly higher plasma levels of TNF-α and IL-6 than the controls. Significant correlations (p < .01 for each) were found for TNF-α and E/Em (r = 0.87) and E/A (r = -0.69), and for IL-6 and E/Em (r = 0.80) and E/A (r = -0.65). Cytokine levels were also correlated with diastolic function in critical ill patients,
and diastolic function improved significantly in association with decrease of cytokines.
We first demonstrated that the levels of pro-inflammatory cytokines IL-6 and TNF-α were higher in non-complicated DHF patients and improvement of cytokines levels along with cardiac diastolic function after treatment of the disease for ICU patients. We may conclude that while there is little information regarding the medical management of DHF, a better understanding of the nature of inflammatory activation in patients with DHF may lead to improvement of outcomes by applying novel therapies aimed at limiting inflammatory reactions, particularly in the setting of patients in intensive care units. We later included 56 critically burned patients who admitted the intensive care unit and performed transthoracic echocardiography to evaluate LV diastolic function.
The total body surfaced area of burned patients was proportional to serum level of interleukin-6 and TNF-α (p < 0.001 for each). Significant correlations were found for tumor necrosis factor-alpha and decelerating time, E/A, and E/Em (r2 = 0.59, 0.45, and 0.52; p <0.001 for each), and for interleukin-6 and decelerating time, E/A, and E/Em (r2
= 0.63, 0.60, and 0.62; p < 0.001 for each). Diastolic function improved significantly in association with decrease of cytokines after burned patients transferred to general ward (p < 0.001). Tumor necrosis factor-alpha, interleukin-6, and sera from critically burned patients downregulated the expression of the SERCA2 gene in HL-1 cardiomyocytes.
There was a significant correlation between LV diastolic dysfunction and in-hospital mortality in critically burned patients (HR = 3.99, p = 0.038) after risk factors adjusted.
With the above experiment, 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.
This study found a significant correlation between the hyper-activating
immunoinflammatory system after burn injuries and the development of LV diastolic dysfunction which improved as inflammation declined. More importantly, the
development of LV diastolic dysfunction had a strong correlation with mortality in this specific group of patients.
Regarding the patients in the general population with higher inflammatory status, we further included 2 other goups of patients to prove the above hypothesis. First we we included 102 otherwise-healthy adults. The participants were classified as having LV diastolic dysfunction by echocardiographic findings. Serum C-reactive protein (CRP) and lipid profile were also measured. The homeostasis model of insulin resistance (HOMA) was calculated. Central obesity was assessed by computerized tomography at the L4 level. In a multivariate regression analysis, the relationship between visceral adipose tissue (VAT) and LV diastolic dysfunction became insignificant when CRP was introduced into the model, although CRP itself was significantly associated with LV diastolic dysfunction (OR: 1.32, 95% CI: 1.01~1.72, p=0.04). A significant correlation was also found between VAT and CRP (r = 0.70; p<0.001). We then performed path analysis as illustrated by the structural equation model. This proved our hypotheses that VAT might affect LV diastolic dysfunction through the effect of CRP (total fat load with inflammation (B = 1.133, p < 0.001) and that inflammation might affect LV diastolic dysfunction (B = 0.373. p < 0.001). In this study, we found a significant association between LV diastolic dysfunction and fat as measured by CT in a sample of the general population. There were strong associations among visceral fat, inflammation, and LV diastolic dysfunction in adjusted logistic regression models. In order to propose a potential biological pathway, we constructed a structural equation model and performed a path analysis. With this analysis, we verified that fat deposition acts mainly through
inflammation to affect LV diastolic dysfunction and less by its own effect (e.g. fat infiltration of LV). The strength of this study is in its precise definitions. We measured central obesity by CT and defined LV diastolic dysfunction by a combination of variable echocardiographic parameters. In conclusion, for the first time, we delineated the
complex relationship among central obesity, inflammation, and LV diastolic dysfunction using SEM. We showed that greater amounts of visceral adipose tissue were associated with low-grade inflammation, which may lead to subclinical LV diastolic dysfunction in non-diabetic, otherwise-healthy subjects. We also tried to establish the association between inflammation and left ventricular (LV) diastolic dysfunction in peritoneal dialysis (PD) and non-PD patients. We also tested the above association and whether inflammation interacts with PD to increase LV diastolic dysfunction risks. 120 subjects with normal creatinine levels and 101 PD patients were recruited. Echocardiographic parameters were assessed in all patients and LV diastolic dysfunction were indentified by echocardiography. Blood was sampled at the baseline for measurement of
inflammation markers, including tissue necrosis factor alpha (TNF-α) and
interleukin-6 (IL-6). Subjects with LV diastolic dysfunction had higher proinflammation cytokines levels in both groups. Inflamed markers correlated significantly with
echocardiography parameters for LV diastolic dysfunction in patients receiving PD. In a multivariate regression analysis adjusting for all the factors associated with LV diastolic dysfunction, inflammation is still significantly associated with left ventricular diastolic dysfunction (TNF-alpha, OR: 2.6, 95% CI: 2.0~3.35, p<0.001; IL-6, OR: 1.26, 95% CI:
1.25~1.26, p=0.01). In addition, the interaction of PD and inflammation significantly contributed to the development of LV diastolic dysfunction (PD*TNF-α: OR: 1.45, 95% CI: 1.13 – 1.79, P=0.004). We find there is a significant correlation between inflammatory cytokines and LV diastolic dysfunction in PD patients and the correlation
was significantly stronger than in subjects with normal creatinine levels. Concurrently, we also notice that there is an interaction between systemic inflammation and CAPD, especially TNF-α and CAPD, which had a synergistic effect for the development of LV diastolic dysfunction in CAPD subjects. From this study, we showed that there is a significant correlation between LV diastolic dysfunction and serum inflammatory cytokines (TNF-alfa, and IL-6) in patients receiving CAPD. An interaction between CAPD and inflammation, especially TNF-α, was also shown to further aggravate LV diastolic dysfunction. Hence it is reasonable that the association between LV diastolic dysfunction and CAPD was at least in part due to the excess of plasma cytokines, which suggests increase of systemic inflammation.
Finally, we investigated an emerging marker for tissue fibrosis, connective tissue growth factor (CTGF) and its association with cardiac diastolic function using cellular and animal models and clinical human data. We recruited a total of 125 patients with a diagnosis of DHF from 1283 patient of the Taiwan Diastolic Heart Failure Registry. The severity of DHF was determined by tissue doppler imaging (E/e’). Cardiac magnetic resonance imaging (CMRI) was used to evaluate myocardial fibrosis. Stretch of cardiomyocytes on flexible membrane base serves as a cellular phenotype of cardiac diastolic dysfunction (DD). A canine model of DD was induced by aortic banding.
Significant correlation was found between plasma CTGF and E/e’ in DHF patients.
Severity of cardiac fibrosis evaluated by CMRI also correlated with CTGF. In the cell model, stretch increased secretion of CTGF from cardiomyocytes. In the canine model, myocardial tissue CTGF expression and fibrosis significantly increased after 2 weeks of aortic banding. Notable, the expression of CTGF paralleled the severity of LV DD (r=
0.40, p<0.001 for E/e’) and hemodynamic changes (r= 0.80, p< 0.001). After adjusting for confounding factors, CTGF levels still correlated with diastolic parameters in both
human and canine models (human plasma CTGF, p<0.001; canine tissue CTGF, p = 0.04). We demonstrated that the level of plasma CTGF was not only an early, sensitive marker for the diagnosis of DHF but also correlated with the severity of LV diastolic dysfunction as well as the severity of cardiac fibrosis. We found that CTGF correlated with echocardiographic parameters for diastolic dysfunction along with plasma NT-proBNP at different stages of human DHF. Accordingly, in the animal model, we also demonstrated that cardiac CTGF increased significantly as early as 2 weeks of pressure overload. Moreover, the expression of CTGF paralleled the severity of LV diastolic dysfunction and the subtle, incremental blood pressure changes. In the cellular model, we also found that CTGF secretion increased after 24 h of stretch stimulation (mimicking pressure overload stimulation). Our study implies that the CTGF level may directly reflect the changes of LV diastolic function and serve as a platform to monitor the effect of treatment.
In conclusions, the present doctoral thesis combined genetic association studies, molecular studies and clinical studies to demonstrate how cytokines (including RAS system, TNF-α, IL-6 and CTGF) are involved in the pathogeneses of cardiac fibrosis, SERCA2 regulation, which are important substrates of DHF. We first showed the
association between RAS genetic variants and the prognosis of DHF. Second, we further investigated the possible molecular mechanisms by which TNF-αis involved in the pathogeneses of SERCA2 regulation. Third, we found that inflammatory status
correlated with clinical diastolic indices closely from patients with severe inflammation to subclinical inflammatory status and LV diasotic dysfunction it self is an independent prognostic marker for severe illness patients. Finally, we proved CTGF to be a useful tool to non-invasively evaluate the status and severity of LV diastolic function using cellular and animal models and clinical human data.