Diana Lindner

Cardiac Inflammation Contributes to Changes in the Extracellular Matrix in Patients With Heart Failure and Normal Ejection Fraction

Background—The pathophysiology of heart failure with normal ejection fraction (HFNEF) is still under discussion. Here we report the influence of cardiac inflammation on extracellular matrix (ECM) remodeling in patients with HFNEF. Methods and Results—We investigated left ventricular systolic and diastolic function in 20 patients with HFNEF and 8 control patients by conductance catheter methods and echocardiography. Endomyocardial biopsy samples were also obtained, and ECM proteins as well as cardiac inflammatory cells were investigated. Primary human cardiac fibroblasts were outgrown from the endomyocardial biopsy samples to investigate the gene expression of ECM proteins after stimulation with transforming growth factor-&bgr;. Diastolic dysfunction was present in the HFNEF patients compared with the control patients. In endomyocardial biopsy samples from HFNEF patients, we found an accumulation of cardiac collagen, which was accompanied by a decrease in the major collagenase system (matrix metalloproteinase-1) in the heart. Moreover, a subset of inflammatory cells, which expressed the profibrotic growth factor transforming growth factor-&bgr;, could be documented in the HFNEF patients. Stimulation of primary human cardiac fibroblasts from HFNEF patients with transforming growth factor-&bgr; resulted in transdifferentiation of fibroblasts to myofibroblasts, which produced more collagen and decreased the amount of matrix metalloproteinase-1, the major collagenase in the human heart. A positive correlation between cardiac collagen, as well as the amount of inflammatory cells, and diastolic dysfunction was evident and suggests a direct influence of inflammation on fibrosis triggering diastolic dysfunction. Conclusions—Cardiac inflammation contributes to diastolic dysfunction in HFNEF by triggering the accumulation of ECM.

Reduced Degradation of the Chemokine MCP-3 by Matrix Metalloproteinase-2 Exacerbates Myocardial Inflammation in Experimental Viral Cardiomyopathy

Background— Myocarditis is an important cause for cardiac failure, especially in younger patients, followed by the development of cardiac dysfunction and death. The present study investigated whether gene deletion of matrix metalloproteinase-2 influences cardiac inflammation and function in murine coxsackievirus B3 (CVB3)–induced myocarditis. Methods and Results— Matrix metalloproteinase-2 knockout mice (MMP-2−/−) and their wild-type controls (WT) were infected with CVB3 to induce myocarditis. Three days after infection, an increased invasion of CD4+-activated T cells into the myocardium was documented, followed by an excess of inflammatory cells 7 days after infection, which was significantly higher in the MMP-2−/−animals compared with the WT animals. Moreover, cardiac apoptosis, remodeling, viral load, and function were deteriorated in MMP-2−/− animals after CVB3 infection. This overwhelming inflammation was followed by 100% mortality after 15 days. This was associated with increased levels of MCP-3 in the cardiac tissue of MMP-2−/− mice. Because MMP-2 cleaves the chemokine MCP-3, the loss of this cleavage lead to an overreaction of the immune system with pronounced myocardial damage mediated by the inflammatory cells. When a neutralizing antibody against MCP-3 was given to MMP-2−/− mice, this exaggerated reaction of the immune system could be normalized to levels similar to WT-CVB3 animals. Conclusions— Loss of MMP-2 increased the inflammatory response after CVB3 infection, which impaired cardiac function and survival during CVB3-induced myocarditis. Matrix metalloproteinase-2–mediated chemokine cleavage has an important role in cardiac inflammation as a negative feedback mechanism.

Role of Heart Rate Reduction in the Prevention of Experimental Heart Failure: Comparison Between If-Channel Blockade and β-Receptor Blockade

To investigate whether heart rate reduction via If-channel blockade and &bgr;-receptor blockade prevents left ventricular (LV) dysfunction, we studied ivabradine and metoprolol in angiotensin II–induced heart failure. Cardiac dysfunction in C57BL/6J mice was induced by implantation of osmotic pumps for continuous subcutaneous dosing of angiotensin II (1.8 mg/kg per day SC) over a period of 3 weeks. Ivabradine (10 mg/kg per day) and metoprolol (90 mg/kg per day), which resulted in similar heart rate reduction, or placebo treatments were simultaneously started with infusion of angiotensin II. After 3 weeks, LV function was estimated by conductance catheter technique, cardiac remodeling assessed by estimation of cardiac hypertrophy, fibrosis, and inflammatory stress response by immunohistochemistry or PCR, respectively. Compared with controls, angiotensin II infusion resulted in hypertension in impaired systolic (LV contractility, stroke volume, end systolic elastance, afterload, index of arterial-ventricular coupling, and cardiac output; P<0.05) and diastolic (LV relaxation, LV end diastolic pressure, &tgr;, and stiffness constant &bgr;; P<0.05) LV function. This was associated with a significant increase in cardiac hypertrophy and fibrosis. Increased cardiac stress was also indicated by an increase in cardiac inflammation and apoptosis. Both ivabradine and metoprolol led to a similar reduction in heart rate. Metoprolol also reduced systolic blood pressure. Ivabradine led to a significant improvement in systolic and diastolic LV function (P<0.05). This was associated with less cardiac hypertrophy, fibrosis, inflammation, and cardiac apoptosis (P<0.05). Metoprolol treatment did not prevent the reduction in cardiac function and adverse remodeling, despite a reduction of the inflammatory stress response. Behind heart rate reduction, additional beneficial cardiac effects contribute to heart failure prevention with If-channel inhibition.

Cardiac fibroblasts support cardiac inflammation in heart failure

Cardiac remodeling and inflammation are hallmarks of cardiac failure and correlate with outcome in patients. However, the basis for the development of both remains unclear. We have previously reported that cardiac inflammation triggers transdifferentiation of fibroblasts to myofibroblasts and therefore increase accumulation of cardiac collagen, one key pathology in cardiac remodeling. Hence, identifying key pathways for inflammation would be beneficial for patients suffering from heart failure also. Besides their well-characterized function in matrix regulation, we here investigate the role of fibroblasts in the inflammatory process. We address for the first time the role of fibroblasts as inflammatory supporter cells in heart failure. Using endomyocardial biopsies from patients with heart failure and dilated cardiomyopathy, we created a primary human cardiac fibroblast cell culture system. We found that mechanical stretch mimicking cardiac dilation in heart failure induces activation of fibroblasts and not only stimulates production of extracellular matrix but more interestingly up-regulates chemokine production and triggers typical inflammatory pathways in vitro. Moreover, the cell culture supernatant of stretched fibroblasts activates inflammatory cells and induces further recruitment of monocytes by allowing transendothelial migration into the cardiac tissue. Our findings reveal that cardiac fibroblasts provide pro-inflammatory mediators and may act as sentinel cells activated by mechanical stress. Those cells are able to recruit inflammatory cells into the cardiac tissue, a process known to aggravate outcome of patients. This might be important in different forms of heart failure and therefore may be one general mechanism specific for fibroblasts.

Interleukin-23 Deficiency Leads to Impaired Wound Healing and Adverse Prognosis after Myocardial Infarction

Background— CD4+ cells are implicated in the healing process after myocardial infarction (MI). We sought to investigate the role of interleukin-23 (IL-23) deficiency, a cytokine important in differentiation of CD4+ cells, in scar formation of the ischemic heart. Methods and Results— MI was performed in wild-type and IL23p19−/− mice. Thirty-day mortality, hemodynamic function 4 days after MI and myocardial inflammation, and remodeling 4 and 30 days after MI were examined. Differentiation of fibroblasts from infarcted and noninfarcted hearts into myofibroblasts was examined under basal conditions and after stimulation with interferon-&ggr;, IL-17&agr; and IL-23. Interleukin-23p19−/− mice showed higher expression of proinflammatory cytokines and immune cell infiltration in the scar early after MI compared with wild-type mice. A stronger interferon-&ggr;/Th1 reaction seemed to be responsible for the increased inflammation under IL-23 deficiency. Expression of &agr;-smooth muscle actin (&agr;-SMA), collagen I and III was significantly higher in the heart tissue and isolated cardiac fibroblasts 4 days after MI in the wild-type mice. Interleukin-23p19−/− mice showed impaired healing compared with wild-type mice, as seen by significantly higher mortality because of ventricular rupture (40% higher after 30 days) and stronger left ventricular dilation early after MI. Stimulation of cardiac fibroblasts with interferon-&ggr;, the main Th1 cytokine, but not with IL-23 or IL-17&agr;, led to a significant downregulation of &agr;-smooth muscle actin, collagen I and III and decreased migration and differentiation to myofibroblasts. Conclusions— IL-23 deficiency leads to increased myocardial inflammation and decreased cardiac fibroblast activation, associated with impaired scar formation and adverse remodeling after MI.

Cardiac Fibroblasts Aggravate Viral Myocarditis: Cell Specific Coxsackievirus B3 Replication

Myocarditis is an inflammatory disease caused by viral infection. Different subpopulations of leukocytes enter the cardiac tissue and lead to severe cardiac inflammation associated with myocyte loss and remodeling. Here, we study possible cell sources for viral replication using three compartments of the heart: fibroblasts, cardiomyocytes, and macrophages. We infected C57BL/6j mice with Coxsackievirus B3 (CVB3) and detected increased gene expression of anti-inflammatory and antiviral cytokines in the heart. Subsequently, we infected cardiac fibroblasts, cardiomyocytes, and macrophages with CVB3. Due to viral infection, the expression of TNF-α, IL-6, MCP-1, and IFN-β was significantly increased in cardiac fibroblasts compared to cardiomyocytes or macrophages. We found that in addition to cardiomyocytes cardiac fibroblasts were infected by CVB3 and displayed a higher virus replication (132-fold increase) compared to cardiomyocytes (14-fold increase) between 6 and 24 hours after infection. At higher virus concentrations, macrophages are able to reduce the viral copy number. At low virus concentration a persistent virus infection was determined. Therefore, we suggest that cardiac fibroblasts play an important role in the pathology of CVB3-induced myocarditis and are another important contributor of virus replication aggravating myocarditis.

Protective Function of STAT3 in CVB3-Induced Myocarditis.

The transcription factor signal transducer and activator of transcription 3 (STAT3) is an important mediator of the inflammatory process. We investigated the role of STAT3 in viral myocarditis and its possible role in the development to dilated cardiomyopathy. We used STAT3-deficent mice with a cardiomyocyte-restricted knockout and induced a viral myocarditis using Coxsackievirus B3 (CVB3) which induced a severe inflammation during the acute phase of the viral myocarditis. A complete virus clearance and an attenuated inflammation were examined in both groups WT and STAT3 KO mice 4 weeks after infection, but the cardiac function in STAT3 KO mice was significantly decreased in contrast to the infected WT mice. Interestingly, an increased expression of collagen I was detected in STAT3 KO mice compared to WT mice 4 weeks after CVB3 infection. Furthermore, the matrix degradation was reduced in STAT3 KO mice which might be an explanation for the observed matrix deposition. Consequently, we here demonstrate the protective function of STAT3 in CVB3-induced myocarditis. Since the cardiomyocyte-restricted knockout leads to an increased fibrosis, it can be assumed that STAT3 signalling in cardiomyocytes protects the heart against increased fibrosis through paracrine effects.

Altered physiological functions and ion currents in atrial fibroblasts from patients with chronic atrial fibrillation

The contribution of human atrial fibroblasts to cardiac physiology and pathophysiology is poorly understood. Fibroblasts may contribute to arrhythmogenesis through fibrosis, or by directly altering electrical activity in cardiomyocytes. The objective of our study was to uncover phenotypic differences between cells from patients in sinus rhythm (SR) and chronic atrial fibrillation (AF), with special emphasis on electrophysiological properties. We isolated fibroblasts from human right atrial tissue for patch‐clamp experiments, proliferation, migration, and differentiation assays, and gene expression profiling. In culture, proliferation and migration of AF fibroblasts were strongly impaired but differentiation into myofibroblasts was increased. This was associated with a higher number of AF fibroblasts expressing functional Nav1.5 channels. Strikingly Na+ currents were considerably larger in AF cells. Blocking Na+ channels in culture with tetrodotoxin did not affect proliferation, migration, or differentiation in neither SR nor AF cells. While freshly isolated fibroblasts showed mostly weak rectifier currents, fibroblasts in culture developed outward rectifier K+ currents of similar amplitude between the SR and AF groups. Adding the K+ channel blockers tetraethylammonium and 4‐aminopyridin in culture reduced current amplitude and inhibited proliferation in the SR group only. Analysis of gene expression revealed significant differences between SR and AF in genes encoding for ion channels, collagen, growth factors, connexins, and cadherins. In conclusion, this study shows that under AF conditions atrial fibroblasts undergo phenotypic changes that are revealed in culture. Future experiments should be performed in situ to understand the nature of those changes and whether they affect cardiac electrical activity.

Diagnosing heart failure with preserved ejection fraction.

INTRODUCTION Heart failure with preserved ejection fraction (HFPEF) is a common syndrome, accounting for about 50% of all patients with heart failure (HF). Morbidity and mortality are similar to patients with HF with reduced ejection fraction (HFREF), yet no effective treatment has been identified in randomized clinical trials. AREAS COVERED This article provides an overview of the available literature regarding diagnosing established HFPEF and potential new therapeutic targets for the early diagnosis of HFPEF. Vascular dysfunction, ventricular-arterial coupling, oxidative stress, extracellular matrix regulation, chronotropic incompetence, pulmonary hypertension, exercise testing and biomarkers were taken into consideration next to conventional measurements of diastolic dysfunction. EXPERT OPINION Measuring diastolic dysfunction in HFPEF is considered important in many patients. Nevertheless, today we know that other causes besides diastolic dysfunction are also involved in the pathophysiology of many HFPEF patients and need to be investigated in order to make a correct diagnosis. Therefore, further research is required to allow better and more specific diagnostic and treatment options to reduce the morbidity and mortality for this ever-expanding HF population.

Cardiac Function Remains Impaired Despite Reversible Cardiac Remodeling after Acute Experimental Viral Myocarditis

Background. Infection with Coxsackievirus B3 induces myocarditis. We aimed to compare the acute and chronic phases of viral myocarditis to identify the immediate effects of cardiac inflammation as well as the long-term effects after resolved inflammation on cardiac fibrosis and consequently on cardiac function. Material and Methods. We infected C57BL/6J mice with Coxsackievirus B3 and determined the hemodynamic function 7 as well as 28 days after infection. Subsequently, we analyzed viral burden and viral replication in the cardiac tissue as well as the expression of cytokines and matrix proteins. Furthermore, cardiac fibroblasts were infected with virus to investigate if viral infection alone induces profibrotic signaling. Results. Severe cardiac inflammation was determined and cardiac fibrosis was consistently colocalized with inflammation during the acute phase of myocarditis. Declined cardiac inflammation but no significantly improved hemodynamic function was observed 28 days after infection. Interestingly, cardiac fibrosis declined to basal levels as well. Both cardiac inflammation and fibrosis were reversible, whereas the hemodynamic function remains impaired after healed viral myocarditis in C57BL/6J mice.