Myeloid differentiation factor-2 activates monocytes in patients with dilated cardiomyopathy.

Plasma levels of myeloid differentiation factor-2 (MD-2), a co-receptor of toll-like-receptor 4 (TLR4), independently predict mortality in patients with dilated cardiomyopathy (DCM). We tested whether monocyte activation by MD-2 contributes to immune activation and inflammatory status in DCM patients. We found increased MD-2 plasma levels in 25 patients with recent-onset DCM (1250 ± 80.7 ng/ml) compared to 25 age- and gender-matched healthy controls (793.4 ± 52.0 ng/ml; p < 0.001). Monocytes isolated from DCM patients showed a higher expression (141.7 ± 12.4%; p = 0.006 vs. controls) of the MD-2 encoding gene, LY96 and an increased NF-κB-activation. Further, the TLR4-activator lipopolysaccharide (LPS) caused a higher increase in interleukin (IL)-6 in monocytes from DCM patients compared to controls (mean fluorescence intensity: 938.7 ± 151.0 vs. 466.9 ± 51.1; p = 0.005). MD-2 increased IL-6 secretion in a TLR4/NF-κB-dependent manner in monocyte-like THP-1-cells as demonstrated by TLR4-siRNA and NF-κB-inhibition. Since endothelial cells (ECs) are responsible for recruiting monocytes to the site of inflammation, ECs were treated with MD-2 leading to an activation of Akt and increased secretion of monocyte-chemoattractant-protein-1 (MCP-1). Activation of ECs by MD-2 was accompanied by an increased expression of the adhesion molecules CD54, CD106 and CD62E, resulting in an increased monocyte recruitment, which was attenuated by CD54 inhibition. In addition, in murine WT but not LY96-KO bone marrow-derived macrophages LPS increased the amount of CD54 and CD49d/CD29. MD-2 facilitates a pro-inflammatory status of monocytes and EC-mediated monocyte recruitment via TLR4/NF-κB. Elevated MD-2 plasma levels are possibly involved in monocyte-related inflammation-promoting disease progression in DCM. Our results suggest that MD-2 contributes to increasing monocytic inflammatory activity and triggers the recruitment of monocytes to ECs in DCM.

Stiffness of left ventricular cardiac fibroblasts is associated with ventricular dilation in patients with recent-onset nonischemic and nonvalvular cardiomyopathy.

BACKGROUND: Ventricular dilation is known as a pivotal predictor in recent-onset cardiomyopathy (ROCM), but its pathophysiology is not fully understood. In the present study we investigated whether single-cell stiffness of right and left ventricular-derived fibroblasts has an effect on cardiac phenotype in patients with ROCM. METHODS AND RESULTS: Patients with endomyocardial biopsy-proven ROCM were included (n=10). Primary cardiac fibroblasts (CFBs) were cultured from left and right ventricular endomyocardial biopsies and their single-cell stiffness was analyzed by quantification of Young's modulus using colloidal probe atomic force microscopy. Cardiac fibrosis was analyzed by Masson's trichrome staining. CFBs from the left ventricle showed significantly decreased stiffness when compared with CFBs from the right ventricle, indexed by decreased stiffness (Young's modulus 3,374±389 vs. 4,837±690 Pa; P<0.05). Young's modulus of CFBs derived from the left ventricle correlated negatively with the left ventricular end-diastolic dimension derived from 2-dimensional echocardiography (R(2)=0.77; P<0.01). Neither left nor right ventricular fibrosis correlated with the respective ventricular dimensions. CONCLUSIONS: Our data suggest that a decrease in single-cell stiffness of left ventricular fibroblasts could trigger left ventricular dilation in patients with ROCM. This implies a new potential mechanism for the ventricular dilation with this disease.

Variants of Toll-like receptor 4 predict cardiac recovery in patients with dilated cardiomyopathy.

The clinical course of patients with dilated cardiomyopathy (DCM) varies from cardiac recovery to end stage heart failure. The etiology of this variability is largely unknown. In this study, we investigated the impact of coding polymorphisms of the innate immune protein Toll-like receptor 4 (TLR4) on left ventricular performance in patients with DCM. Two variants of TLR4 (rs4986790, TLR4 c.1187A→G, p.299D→G and rs4986791,TLR4 c.1487C→T, p.T399I) were investigated in 158 patients with DCM. Other reasons for heart failure were excluded by coronary angiography, myocardial biopsy, and echocardiography. Risk factors, age, gender, or treatment did not differ among the groups. At the follow-up evaluation (median 4.0-5.4 months), patients carrying the TLR4 wild type gene displayed cardiac recovery under intense medical heart failure therapy indexed by reduced left ventricular dilation, improved left ventricular ejection fraction, and reduced NT-probrain natriuretic peptide blood level when compared with the initial evaluation. In contrast, patients carrying both the rs4986790 and the rs4986791 variant showed significantly reduced improvement of left ventricular ejection fraction (p = 0.006) and left ventricular dilation (p = 0.015) at the follow-up evaluation when compared with carriers of the wild type gene under the same treatment conditions. In addition, NT-probrain natriuretic peptide level in carriers of both TLR4 variants did not change significantly at the follow up when compared with the first evaluation. Among patients with DCM, the presence of the TLR4 variants rs4986790 and rs4986791 predicts impaired cardiac recovery independently of medical treatment or cardiac risk factors.

TRIF is a critical survival factor in viral cardiomyopathy.

TRIF is a member of the innate immune system known to be involved in viral recognition and type I IFN activation. Because IFNs are thought to play an important role in viral myocarditis, we investigated the role of TRIF in induced myocarditis in mice. Whereas C57BL/6 (wild-type) mice showed only mild myocarditis, including normal survival postinfection with coxsackievirus group B serotype 3 (CVB3), infection of TRIF(-/-) mice led to the induction of cardiac remodeling, severe heart failure, and 100% mortality (p < 0.0001). These mice showed markedly reduced virus control in cardiac tissues and cardiomyocytes. This was accompained with dynamic cardiac cytokine activation in the heart, including a suppression of the antiviral cytokine IFN-ß in the early viremic phase. TRIF(-/-) myocytes displayed a TLR4-dependent suppression of IFN-ß, and pharmacological treatment of CVB3-infected TRIF(-/-) mice with murine IFN-ß led to improved virus control and reduced cardiac inflammation. Additionally, this treatment within the viremic phase of myocarditis showed a significant long-term outcome indexed by reduced mortality (20 versus 100%; p < 0.001). TRIF is essential toward a cardioprotection against CVB3 infection.

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.

High-Mobility Group Box Protein 1 Is an Independent Prognostic Marker for All-Cause Mortality in Patients With Dilated Cardiomyopathy.

High-mobility group box protein 1 (HMGB1) is released during tissue damage and activates the innate immune system through toll-like receptor 4. Because mortality in dilated cardiomyopathy (DCM) is associated with activation of the innate immune system, we hypothesized that HMGB1 possesses a prognostic value in estimating mortality in patients with DCM. We determined HMGB1 and N-terminal B-type natriuretic peptide (NT-proBNP) levels in 67 patients with DCM (12 women, mean age 53.6 ± 1.5 years). Kaplan-Meier analyzes revealed that higher levels of HMGB1 and NT-proBNP are related to increased all-cause mortality. Multivariable Cox regression confirmed HMGB1 as a risk factor for mortality in patients with DCM, independent of NT-proBNP, age, and gender (hazard ratio per 1 SD 1.920, 95% confidence interval 1.401 to 2.631, p <0.001). HMGB1 is a promising candidate to estimate the prognosis of patients with DCM.

Myeloid differentiation factor-88 contributes to TLR9-mediated modulation of acute coxsackievirus B3-induced myocarditis in vivo.

Toll-like receptor 9 (TLR9) is a member of the innate immune system and has been shown to influence myocardial function, but its role in myocarditis is hitherto unknown. We therefore investigated whether or not TLR9 plays a role in this disease in coxsackievirus B3 (CVB3)-induced myocarditis in mice. Left ventricular (LV) function, cardiac immune cell infiltration, virus mRNA, and components of the TLR9 downstream pathway were investigated in TLR9-deficient [knockout (KO)] and wild-type (WT) mice after infection with CVB3. Murine cardiac TLR9 expression was significantly increased in WT mice with acute CVB3 infection but not in WT mice with chronic myocarditis. Furthermore, in the acute phase of CVB3-induced myocarditis, CVB3-infected KO mice displayed improved LV function associated with reduced cardiac inflammation indexed by reduced amounts of immune cells compared with CVB3-infected WT mice. In contrast, in the chronic phase, LV function and inflammation were not seen to differ among the infected groups. The cardioprotective effects due to TLR9 deficiency were associated with suppression of the TLR9 downstream pathway as indexed by reduced cardiac levels of the adapter protein myeloid differentiation factor (MyD)-88 and the proinflammatory cytokine TNF-alpha. In addition, TLR9 deficiency led to an activation of the antiviral cytokine interferon-beta in the heart as a result from viral infection. In conclusion, the MyD88/TNF-alpha axis due to TLR9 activation in the heart contributes the development of acute myocarditis but not of chronic myocarditis.

Role of left ventricular stiffness in heart failure with normal ejection fraction.

BACKGROUND: Increased left ventricular stiffness is a distinct finding in patients who have heart failure with normal ejection fraction (HFNEF). To elucidate how diastolic dysfunction contributes to heart failure symptomatology during exercise, we conducted a study using an invasive pressure-volume loop approach and measured cardiac function at rest and during atrial pacing and handgrip exercise. METHODS AND RESULTS: Patients with HFNEF (n=70) and patients without heart failure symptoms (n=20) were enrolled. Pressure-volume loops were measured with a conductance catheter during basal conditions, handgrip exercise, and atrial pacing with 120 bpm to analyze diastolic and systolic left ventricular function. During transient preload reduction, the diastolic stiffness constant was measured directly. Diastolic function with increased stiffness was significantly impaired in patients with HFNEF during basal conditions. This was associated with increased end-diastolic pressures during handgrip exercise and with decreased stroke volume and a leftward shift of pressure-volume loops during atrial pacing. CONCLUSIONS: Increased left ventricular stiffness contributed to increased end-diastolic pressure during handgrip exercise and decreased stroke volume during atrial pacing in patients with HFNEF. These data suggest that left ventricular stiffness modulates cardiac function in HFNEF patients and suggests that diastolic dysfunction with increased stiffness is a target for treating HFNEF.

Human apolipoprotein A-I gene transfer reduces the development of experimental diabetic cardiomyopathy.

BACKGROUND: The hallmarks of diabetic cardiomyopathy are cardiac oxidative stress, intramyocardial inflammation, cardiac fibrosis, and cardiac apoptosis. Given the antioxidative, antiinflammatory, and antiapoptotic potential of high-density lipoprotein (HDL), we evaluated the hypothesis that increased HDL via gene transfer (GT) with human apolipoprotein (apo) A-I, the principal apolipoprotein of HDL, may reduce the development of diabetic cardiomyopathy. METHODS AND RESULTS: Intravenous GT with 3x10(12) particles/kg of the E1E3E4-deleted vector Ad.hapoA-I, expressing human apoA-I, or Ad.Null, containing no expression cassette, was performed 5 days after streptozotocin (STZ) injection. Six weeks after apoA-I GT, HDL cholesterol levels were increased by 1.6-fold (P<0.001) compared with diabetic controls injected with the Ad.Null vector (STZ-Ad.Null). ApoA-I GT and HDL improved LV contractility in vivo and cardiomyocyte contractility ex vivo, respectively. Moreover, apoA-I GT was associated with decreased cardiac oxidative stress and reduced intramyocardial inflammation. In addition, compared with STZ-Ad.Null rats, cardiac fibrosis and glycogen accumulation were reduced by 1.7-fold and 3.1-fold, respectively (P<0.05). Caspase 3/7 activity was decreased 1.2-fold (P<0.05), and the ratio of Bcl-2 to Bax was upregulated 1.9-fold (P<0.005), translating to 2.1-fold (P<0.05) reduced total number of cardiomyocytes with apoptotic characteristics and 3.0-fold (P<0.005) reduced damaged endothelial cells compared with STZ-Ad.Null rats. HDL supplementation ex vivo reduced hyperglycemia-induced cardiomyocyte apoptosis by 3.4-fold (P<0.005). The apoA-I GT-mediated protection was associated with a 1.6-, 1.6-, and 2.4-fold induction of diabetes-downregulated phospho to Akt, endothelial nitric oxide synthase, and glycogen synthase kinase ratio, respectively (P<0.005). CONCLUSIONS: ApoA-I GT reduced the development of streptozotocin-induced diabetic cardiomyopathy.

Enhancement of the endothelial NO synthase attenuates experimental diastolic heart failure.

BACKGROUND: Diastolic heart failure is a rising problem with a high incidence and similar mortality and morbidity compared to patients with systolic heart failure. Nevertheless, the underlying pathophysiology is still debated. AIM: We investigated the effect of pharmacological enhancement of endothelial nitric oxide synthase (eNOS) on experimental diastolic heart failure (DHF). METHODS: DHF was induced in 60 DAHL salt-sensitive rats by salt diet in 8-week-old animals. 30 were treated with the eNOS enhancer AVE3085 (DHFeNOS) and 30 with placebo (DHF). Rats with normal salt intake served as controls. RESULTS AND CONCLUSION: Diastolic dysfunction with increased diastolic stiffness constant and increased left ventricular (LV) pressure was analyzed by invasive pressure-volume loop measurements in the DHF group compared to controls. Cardiac hypertrophy as indicated by LV mass measurements by echocardiography, and increased cardiac collagen content as measured by immunohistochemistry were associated with an increased activation state of calcineurin, AKT, ERK(1/2), but not JNK and p38 kinases. Titin isoforms were not altered in this model of DHF. Treatment with AVE3085 significantly increased eNOS mRNA and protein levels in the cardiac tissue and decreases NAD(P)H oxidase subunits p22phox and gp91phox. Diastolic dysfunction was attenuated and cardiac hypertrophy and fibrosis were improved in comparison with untreated DHF animals. This was associated with a normalized activation state of calcineurin, AKT and ERK(1/2). Therefore, we suggest that targeting the NO system might yield a future therapeutic aim for the treatment of DHF.

Viral myocarditis and coagulopathy: increased tissue factor expression and plasma thrombogenicity.

We investigated the effects of viral infection on Tissue Factor (TF) expression and activity in mice within the myocardium to understand increased thrombosis during myocarditis. Mice were infected with coxsackie virus B3 (CVB3) and the hearts were collected at day 4, 8 and 28 post infection (p.i.). Myocardial TF expression and cellular activity as well as plasma activity were analyzed from CVB3 infected mice by Western blot, chromogenic Factor Xa generation assay, in situ staining for active TF and immunohistochemistry. In addition to TF expression, hemodynamic parameters were measured during the time course of infection. Furthermore, we analyzed myocardial tissues from patients with suspected inflammatory cardiomyopathy. TF protein expression was maximally 5-fold elevated 8 days p.i. in mice and remained increased on day 28 p.i. (P<0.001 vs. non-infected controls). Alterations in TF expression were associated with fibrin deposits within the myocardium. The TF pathway inhibitor protein expression in the myocardium was not altered during myocarditis. Active cellular TF co-localized with CD3 positive cells and VCAM-1 positive endothelial cells in the myocardium. The TF expression was positively correlated with the amount of infiltrating CD3 and Mac3 positive cells (Spearman-Rho rho=0.749 P<0.0001 for CD3(+) and rho=0.775 P<0.0001 for Mac3(+); N=35). Increased myocardial TF expression was associated with a 2-fold elevated plasma activity (P<0.05 vs. non-infected controls). In the human hearts, the TF expression correlated positively with an endothelial cell activation marker (rho=0.523 P<0.0001 for CD62E; N=54). Viral myocarditis is a hypercoagulative state which is associated with increased myocardial TF expression and activity. Upregulation of TF contributes to a systemic activation of the coagulation cascade.

Accelerated mitochondrial adenosine diphosphate/adenosine triphosphate transport improves hypertension-induced heart disease.

BACKGROUND: Strong evidence suggests that mitochondrial malfunction, which leads to disturbed energy metabolism and stimulated apoptosis, is a linchpin in the induction and manifestation of cardiac failure. An adequate exchange of ATP and ADP over the inner mitochondrial membrane by the adenine nucleotide translocase (ANT) is thereby essential to guarantee the cellular energy supply. METHODS AND RESULTS: To explore the effect of an ameliorated mitochondrial ATP/ADP transportation on cardiac dysfunction, we generated transgenic rats overexpressing ANT1 in the heart (ANT rats) and crossed them with renin-overexpressing rats (REN rats) suffering from hypertension-induced cardiac insufficiency. Cardiac-specific ANT1 overexpression resulted in a higher ATP/ADP transportation and elevated activities of respiratory chain complexes. Increased ANT activity in double-transgenic (ANT/REN) animals did not influence excessive hypertension seen in REN rats. Hypertension-induced cardiac hypertrophy in the REN rats was prevented by parallel ANT1 overexpression, however, and left ventricular function remarkably improved. The ANT1 overexpression led to a reduction in fibrosis and an improvement in cardiac tissue architecture. Consequently, the survival rate of ANT/REN rats was enhanced. Further investigations into the cardioprotective mechanism of ANT1 overexpression revealed improved mitochondrial structure and function and significantly reduced apoptosis in ANT/REN rats, shown by lowered cytosolic/mitochondrial cytochrome c ratio, reduced caspase 3 level, and prevented DNA degradation. CONCLUSIONS: Myocardial ANT1 overexpression protects against hypertension-induced cardiac pathology. Thus, the improvement in mitochondrial function may be a basic principle for new strategies in treating heart disease.

Contributions of inflammation and cardiac matrix metalloproteinase activity to cardiac failure in diabetic cardiomyopathy: the role of angiotensin type 1 receptor antagonism.

We investigated the effect of the angiotensin type 1 (AT-1) receptor antagonist, irbesartan, on matrix metalloproteinase (MMP) activity and cardiac cytokines in an animal model of diabetic cardiomyopathy. Diabetes was induced in 20 C57/bl6 mice by injection of streptozotocin (STZ). These animals were treated with irbesartan or placebo and were compared with nondiabetic controls. Left ventricular (LV) function was measured by pressure-volume loops with parameters for systolic function (end systolic elastance [Ees]) and diastolic function (cardiac stiffness) 8 weeks after STZ treatment. The cardiac protein content of interleukin (IL)1beta and transforming growth factor (TGF)beta1 were measured by enzyme-linked immunosorbent assay. The total cardiac collagen content and collagen type 1 and 3 were measured by histochemistry, and MMP-2 activity was measured by gelatin zymography. LV dysfunction was documented by impaired Ees and diastolic stiffness in STZ mice compared with controls. This was accompanied by increased TGFbeta, IL1beta, and fibrosis and decreased MMP-2 activity. Treatment with irbesartan attenuated LV dysfunction, IL1beta, TGFbeta, and cardiac fibrosis compared with untreated diabetic animals and normalized MMP activity. These findings present evidence that AT-1 receptor antagonists attenuate cardiac failure by decreasing cardiac inflammation and normalizing MMP activity, leading to normalized cardiac fibrosis in STZ-induced diabetic cardiomyopathy.

Up-regulation of PPARgamma in myocardial infarction.

BACKGROUND: Peroxisome proliferator activated receptors (PPARs) are key regulators for cardiac energy metabolism after myocardial injury. We hypothesized, that PPARs are regulated in myocardial infarction (MI) and their activity is modulated by angiotensin receptor blockers (ARBs). METHODS: Following induction of MI, male rats were treated with placebo or the ARB irbesartan for three weeks. PPARalpha, beta/delta and gamma protein expression and gene expression of PPAR target genes and glucose transporters were measured. PPARgamma-protein expression was analyzed by immunofluorescence. RESULTS: MI decreased LVP and dp/dtmax and increased LVEDP, this effect was counteracted by irbesartan. PPARalpha and PPARbeta/delta protein expression was not altered in MI and was not affected by irbesartan. PPARgamma protein content was increased in the infarcted area and localized to cardiac myocytes and fibroblasts. In parallel, expression of CTGF was increased 10-fold in the infarcted zone. PPAR target genes (CD36, MCAD, ACO and GLUT4) were significantly decreased in infarcted tissue, and this was unaffected by irbesartan. However, CD36 and ACO in the non-infarcted areas were up-regulated by irbesartan. CONCLUSION: Endogenous up-regulation of PPARgamma in MI is insufficient to counteract the decrease in metabolic genes, but parallels an increase in the profibrotic mediator CTGF. Irbesartan increases fatty acid oxidating enzymes after MI independent of PPARgamma regulation.

Toll-like receptor-4 deficiency attenuates doxorubicin-induced cardiomyopathy in mice.

BACKGROUND: Cardiac inflammation and generation of oxidative stress are known to contribute to doxorubicin (Dox)-induced cardiomyopathy. Toll-like receptors (TLRs) are a part of the innate immune system and are involved in cardiac stress reactions. Since TLR4 might play a relevant role in cardiac inflammatory signalling, we investigated whether or not TLR4 is involved in Dox-induced cardiotoxicity. METHODS AND RESULTS: Five days after a single injection of Dox (20 mg/kg; i.p.), left ventricular pressure-volume loops were measured in wild-type and TLR4-deficient mice (TLR4-/-) Dox-treated and control mice. Analyses of possible pathophysiological mechanisms were performed in left ventricular tissue and isolated myocytes, respectively. Dox injection resulted in an impairment of left ventricular function and neurohumoral activation, indexed by increased ET-1 expression. This was further associated with an increase in cardiac oxidative stress, inflammation and apoptosis, as indicated by an up-regulation of cardiac lipid peroxidation, TNF-alpha expression and enhanced content of TUNEL-positive cells. In contrast, TLR4-/- Dox mice showed improved left ventricular function with reduced oxidative and inflammatory stress response including reduced cardiac apoptosis. These results were found to be associated with an increase of GATA-4 expression. CONCLUSIONS: TLR4 deficiency improves left ventricular function and attenuates pathophysiological key mechanisms in Dox-induced cardiomyopathy.

MD-2 is a new predictive biomarker in dilated cardiomyopathy and exerts direct effects in isolated cardiomyocytes.

BACKGROUND: Myeloid differentiation factor-2 (MD-2) has been shown to be an important modulator of the innate immune system, but its role in cardiac diseases is unknown. We investigated whether MD-2 plays a role as risk predictor and contributor in dilated cardiomyopathy (DCM). METHODS AND RESULTS: We included 174 patients with reduced left ventricular (LV) ejection fraction (LVEF <45%) due to DCM. Coronary artery disease and severe valvular diseases were excluded in all patients by angiography or echocardiography. Cardiac inflammation, viral infection and MD-2 expression were analyzed from right ventricular endomyocardial biopsies. MD-2 was quantified by ELISA in serum upon first hospital admission. Myocyte contractility and inflammatory response after stimulation with recombinant MD-2 protein were analyzed in isolated rat cardiomyocytes. Median follow-up of the patients was 3.51 years (2.73; 4.48) with 34 deaths. Absolute mortality risk increases in patients displaying a MD-2 serum concentration greater than the median (302 ng/ml) was 23% (P < 0.0001). Age- and sex-adjusted Cox regression analyses demonstrated that mortality risk was highly related to MD-2 concentrations (P < 0.001), but not to age or sex. An increase of 100 ng/ml in the MD-2 level was associated with an absolute mortality risk increase of 50.4%. Receiver operating characteristic (ROC) analyses showed no difference between MD-2 and nterminal-pro brain natriuretic peptide (NT-pro-BNP), while the combination of both MD-2 and NT-pro-BNP resulted in a significantly increased capability of risk prediction when compared to NT-pro-BNP alone (P = 0.014). In-vitro, recombinant MD-2 decreases cell shortening and modulates cytokine activation in isolated cardiomyocytes. CONCLUSION: MD-2 predicts long-term outcome in DCM patients and improves mortality risk prediction capability compared to NT-pro-BNP alone. In addition, MD-2 exerts direct negative inotropic effects on isolated cardiomyocytes in-vitro. Further randomized trials should confirm MD-2 as a diagnostic and therapeutic target.

Tumor necrosis factor-alpha antagonism protects from myocardial inflammation and fibrosis in experimental diabetic cardiomyopathy.

To investigate the effect of anti-cytokine-based therapy in the course of diabetic cardiomyopathy, we performed a study using an anti-TNF-alpha monoclonal antibody treatment (mab) in Sprague male Dawley (SD) rats with streptozotocin-induced diabetic cardiomyopathy. Five days after streptozotocin injection, rats were treated with the anti-TNF-alpha mAb C432A for 6 weeks.At the end of the study, left ventricular (LV) function was determined by a pressure-catheter. Intercellular adhesion molecule (ICAM)-1, vascular adhesion molecule (VCAM)-1, beta2-lymphocyte-integrins(+) (CD18(+), CD11a(+), CD11b(+)), ED1/CD68(+) and cytokine (TNF-alpha, interleukin (IL)-1beta)- expressing infiltrates, total collagen content and stainings of collagen I and III were quantified by digital image analysis. LV phosphorylated and total ERK protein levels were determined by Western Blot. TNFalpha-antagonism reduced ICAM-1- and VCAM-1 expression and leukocyte infiltration to levels of non-diabetics and decreased macrophage residence by 3.3-fold compared with untreated diabetics. In addition, anti-TNF-alpha mAb-treatment decreased diabetes-induced cardiac TNF-alpha and IL-1beta expression by 2.0-fold and 1.8- fold, respectively, and reduced the ratio of phosphorylated to total ERK by 2.7-fold. The reduction in intramyocardial inflammation was associated with a 5.4-fold and 3.6-fold reduction in cardiac collagen I and III content, respectively. This was reflected by a normalization of cardiac total collagen content to levels of non-diabetics and associated with an improved LV function. TNFalpha-antagonism attenuates the development of experimental diabetic cardiomyopathy associated with a reduction of intramyocardial inflammation and cardiac fibrosis.

Low-dose treatment with atorvastatin leads to anti-oxidative and anti-inflammatory effects in diabetes mellitus.

We investigated whether atorvastatin, given in a dose to low to influence the lipid profile, has any effect on oxidative stress, inflammation and endothelial function under streptozotocin-induced diabetic conditions. Diabetes mellitus was induced in male Sprague Dawley rats by a single injection of streptozotocin. Rats were treated chronically with atorvastatin (50 mg/kg/day; p.o.) or vehicle until day 48 and compared with controls. NAD(P)H activity, protein expression nuclear factor-kappaBp65 (NF-kappaBp65) and phosphorylation of the extracellular signal-regulated kinase (ERK1/2) were assessed in the quadriceps muscle. Protein and mRNA levels of intracellular and vascular adhesion molecules (ICAM-1, VCAM-1) and cytokines were measured by Taqman or immunohistochemistry staining, respectively. Endothelial function was investigated in vivo using the autoperfused hind limb model. Diabetic groups displayed similar severe hyperglycemia. Untreated diabetic rats showed enhanced NAD(P)H activity, activation of the ERK1/2/NF-kappaBp65-pathway, enhanced expression of cytokines and cellular adhesion molecules and impaired vascular function. Low-dose therapy by atorvastatin did not alter the lipid profile but led to a reduction of NAD(P)H activity (-28%, P<0.05) associated with reduced protein expression of NF-kappaBp65 (-53%, P<0.05) and phosphorylation of its regulator mitogen-activated protein kinase (MAPK) ERK1/2 in diabetic rats. Also inflammatory markers were reduced after atorvastatin treatment indexed by reduced mRNA expression of VCAM-1 (-24%), tumor necrosis factor alpha (-59%) and interleukin 1beta (-50%) and reduced ICAM-1 (-81%) and VCAM-1 (-74%) positive staining. These beneficial effects were associated with improved endothelium-dependent vasodilatation (maximal vasodilatation: +101%; P<0.05). Lipid-independent anti-oxidative and anti-inflammatory effects of low-dose atorvastatin involving the ERK1/2/NF-kappaB-pathway are sufficient to improve endothelial function under experimental diabetic conditions.

Chronic inhibition of p38MAPK improves cardiac and endothelial function in experimental diabetes mellitus.

To investigate the influence of p38 mitogen activated kinase (p38MAPK) on the development of diabetic cardiac and endothelial dysfunction, we assessed left ventricular and vascular function as well as inflammatory markers in diabetic rats after chronic pharmacological inhibition of p38MAPK. Diabetes mellitus was induced in rats by a single injection of streptozotocin. Rats were treated with the p38MAPK inhibitor SB 239063 (40 mg/kg/day, p.o.) or vehicle. 48 days after diabetes mellitus-induction, left ventricular function and vascular function were assessed in vivo by TIP-catheter and the autoperfused hindlimb, respectively. Cell adhesion molecules staining was quantified immunohistochemically in the heart and quadriceps muscle, respectively, as well as cardiac phosphorylation of p38MAPK by Western blot analysis. Treated and untreated diabetic groups displayed similar severe hyperglycemia. Left ventricular and endothelial function were impaired in the untreated diabetic group compared to controls (dp/dtmax: -40%, dp/dtmin: +49%, maximal vasodilatation: -57%; P < 0.05) associated with significantly increased cardiac (3-fold) and peripheral cell adhesion molecules staining, respectively. Treatment of diabetic rats with SB 239063 led to a significant reduction of diabetes-induced enhancement of p38MAPK phosphorylation associated with improved left ventricular function (dp/dtmax: +39%, dp/dtmin: +47%; P < 0.05) and peripheral endothelial function (maximal vasodilatation: +71%; P < 0.05) under diabetic conditions. This was associated with reduced cardiac and peripheral inflammation indexed by reduced adhesion molecules content. Pharmacological inhibition of p38MAPK is sufficient to mitigate the development of diabetic cardiac and endothelial dysfunction despite of hyperglycemia. Our data suggest that the anti-inflammatory properties due to p38MAPK inhibition contribute to these beneficial cardiovascular effects.

Diltiazem treatment prevents diastolic heart failure in mice with familial hypertrophic cardiomyopathy.

BACKGROUND: The cardiac troponin T I79N mutation, linked to familial hypertrophic cardiomyopathy, carries a high risk of sudden cardiac death even in the absence of significant cardiac hypertrophy. The pathology underlying this mechanism has not yet been identified. AIMS: To study the underlying mechanism of this phenomenon we characterized the left ventricular (LV) performance of transgenic mice carrying the human troponin T mutation I79N under basal and isoproterenol-induced stress conditions. METHODS AND RESULTS: LV function was analyzed by recording pressure-volume loops using a microconductance catheter. Despite a hypercontractile systolic function under basal conditions TnT-I79N mice showed a diastolic dysfunction indicated by an increase in end-diastolic pressure-volume relationship (EDPVR), a load-independent factor of LV stiffness (0.06+/-0.01 vs. 0.02+/-0.01; P<0.05), when compared to mice expressing human wild-type troponin T (TnT-WT). TnT-I79N mutants developed severe diastolic heart failure and cardiac sudden death under isoproterenol stress. This was prevented after pretreatment with the L-type Ca2+ channel inhibitor diltiazem. CONCLUSIONS: Diastolic dysfunction due to increased LV stiffness in TnT-I79N mice leads to severe primary diastolic heart failure and finally to cardiac sudden death, which can be prevented by diltiazem.