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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-β in the early viremic phase. TRIF−/− myocytes displayed a TLR4-dependent suppression of IFN-β, and pharmacological treatment of CVB3-infected TRIF−/− mice with murine IFN-β 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.

Pharmacological renin inhibition with aliskiren is an effective antihypertensive drug treatment, but it is currently unknown whether aliskiren is able to attenuate cardiac failure independent of its blood pressure-lowering effects. We investigated the effect of aliskiren on cardiac remodeling, apoptosis, and left ventricular (LV) function after experimental myocardial infarction (MI). C57J/b16 mice were subjected to coronary artery ligation and were treated for 10 days with vehicle or aliskiren (50 mg/kg per day via an SC osmopump), whereas sham-operated animals served as controls. This dose of aliskiren, which did not affect systemic blood pressure, improved systolic and diastolic LV function, as measured by the assessment of pressure-volume loops after MI. Furthermore, after MI LV dilatation, cardiac hypertrophy and lung weights were decreased in mice treated with aliskiren compared with placebo-treated mice after MI. This was associated with a normalization of the mitogen-activated protein kinase P38 and extracellular signal-regulated kinases 1/2, AKT, and the apoptotic markers bax and bcl-2 (all measured by Western blots), as well as the number of TUNEL-positive cells in histology. LV dilatation, as well as the associated upregulation of gene expression (mRNA abundance) and activity (by zymography) of the cardiac metalloproteinase 9 in the placebo group after MI, was also attenuated in the aliskiren-treated group. Aliskiren improved LV dysfunction after MI in a dose that did not affect blood pressure. This was associated with the amelioration of cardiac remodelling, hypertrophy, and apoptosis. (Hypertension. 2008; 52: 1068-1075.)

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-α. In addition, TLR9 deficiency led to an activation of the antiviral cytokine interferon-β in the heart as a result from viral infection. In conclusion, the MyD88/TNF-α axis due to TLR9 activation in the heart contributes the development of acute myocarditis but not of chronic myocarditis.

Aims: One of the hemodynamic mechanisms underlying heart failure with normal EF (HFNEF) is increased LV stiffness. In order to clarify the pathological changes leading to increased stiffness we investigated the left ventricular (LV) function using pressure-volume loops under basal conditions and during atrial pacing and investigated endomyocardial biopsies of patients with HFNEF. Methods: In 36 patients with HFNEF and 8 controls pressure volume loops were measured under basal conditions and during atrial pacing. Furthermore, endomyocardial biopsies were analyzed for changes in the extracellular matrix regulation, cardiac inflammation and changes in the titin isoforms. Results: Patients with HFNEF had an increased LV diastolic stiffness (+350%) compared to controls. During atrial pacing, stroke volume decreased (−35%) due to a leftward shift of the PV loops. This was associated with increased collagen content in cardiac biopsies (+380%), which blunted their cardiac output response to enhanced cardiac demand. This may explain the exercise intolerance in HFNEF patients. The total collagen correlated with cardiac stiffness in the HFNEF group (r2=0.69). This was associated with increased mRNA levels of the pro fibrotic TGF-b. Furthermore, the collagenase matrix metalloproteinases (MMP) 1 was not changed while TIMP-1 was increased (+150%), which supposes a decrease in collagen degradation in HFNEF. Nevertheless, MMP 2 was increased (+160%). This MMP is also associated with inflammation, and coherently, we investigated cardiac inflammation. We documented increased cardiac inflammation by an increment of adhesion molecules (ICAM +55%) and invading cells (CD11b cells +250%) in the cardiac tissue of HFNEF patients compared to controls. Interestingly, there was no change in titin isoforms between both groups. Conclusion: Patients with HFNEF have increased diastolic stiffness leading to an impairment of the Frank-Starling mechanism during atrial pacing. Since increased stiffness correlated with the cardiac collagen content, changes in the ECM regulation might be important for the development of HFNEF and invading cells triggering cardiac inflammation might further participate in this pathophysiology.

We thank Dr Csont and Dr Ferdinandy for their interest in our work.1 In principle, we agree that it is possible and even likely that more that one factor controls the complex events that accompany myocardial infarction (MI). Nevertheless, we have clearly shown that mortality, frequency of ventricular ruptures, and hemodynamic dysfunction were significantly increased 3 weeks post MI in bgn −/ 0 mice.1 These findings prove that absence of biglycan …

Clinical use of the anthracycline doxorubicin (DOX) is limited by its cardiotoxic effects, which are attributed to the induction of apoptosis. To elucidate the possible role of the kinin B1 receptor (B1R) during the development of DOX cardiomyopathy, we studied B1R knockout mice (B1R-/-) by investigating cardiac inflammation and apoptosis after induction of DOX-induced cardiomyopathy. DOX control mice showed cardiac dysfunction measured by pressure-volume loops in vivo. This was associated with a reduced activation state of AKT, as well as an increased bax/bcl2 ratio in Western blots, indicating cardiac apoptosis. Furthermore, mRNA levels of the proinflammatory cytokine interleukin 6 were increased in the cardiac tissue. In DOX B1R-/- mice, cardiac dysfunction was improved compared to DOX control mice, which was associated with normalization of the bax/bcl-2 ratio and interleukin 6, as well as AKT activation state. These findings suggest that B1R is detrimental in DOX cardiomyopathy in that it mediates the inflammatory response and apoptosis. These insights might have useful implications for future studies utilizing B1R antagonists for treatment of human DOX cardiomyopathy.

Background Due to the limited RNA amounts from endomyocardial biopsies (EMBs) and low expression levels of certain genes, gene expression analyses by conventional real-time RT-PCR are restrained in EMBs. We applied two preamplification techniques, the TaqMan® PreAmp Master Mix (T-PreAmp) and a multiplex preamplification following a sequence specific reverse transcription (SSRT-PreAmp). Results T-PreAmp encompassing 92 gene assays with 14 cycles resulted in a mean improvement of 7.24 ± 0.33 Ct values. The coefficients for inter- (1.89 ± 0.48%) and intra-assay variation (0.85 ± 0.45%) were low for all gene assays tested (® gene assays. Only two of the tested Taqman® ABI inventoried gene assays (HPRT-ABI and CD56) did not maintain PreAmp uniformity levels between -1.5 and +1.5. In comparison, the SSRT-PreAmp tested on 8 self-designed gene assays yielded higher Ct improvement (9.76 ± 2.45), however was not as robust regarding the maintenance of PreAmp uniformity related to HPRT-CCM (-3.29 ± 2.40; p < 0.0001), and demonstrated comparable intra-assay CVs (1.47 ± 0.74), albeit higher inter-assay CVs (5.38 ± 2.06; p = 0.01). Comparing EMBs from each 10 patients with dilated cardiomyopathy (DCM) and inflammatory cardiomyopathy (DCMi), T-PreAmp real-time RT-PCR analyses revealed differential regulation regarding 27 (30%) of the investigated 90 genes related to both HPRT-CCM and CDKN1B. Ct values of HPRT and CDKN1B did not differ in equal RNA amounts from explanted DCM and donor hearts. Conclusion In comparison to the SSRT-PreAmp, T-PreAmp enables a relatively simple workflow, and results in a robust PreAmp of multiple target genes (at least 92 gene assays as tested here) by a mean Ct improvement around 7 cycles, and in a lower inter-assay variance in RNA derived from EMBs. Preliminary analyses comparing EMBs from DCM and DCMi patients, revealing differential regulation regarding 30% of the investigated genes, confirm that T-PreAmp is a suitable tool to perform gene expression analyses in EMBs, expanding gene expression investigations with the limited RNA/cDNA amounts derived from EMBs. CDKN1B, in addition to its function as a reference gene for the calculation of PreAmp uniformity, might serve as a suitable housekeeping gene for real-time RT-PCR analyses of myocardial tissues.

Introduction: Inflammation and/or viral infection are both relevant for extracellular matrix remodeling. Therefore the effects of myocardial inflammation on ECM remodeling are analyzed in this study. # Methods and results: Endomyocardial biopsies were obtained from 20 patients with established DCM (2.11±0.44 CD3 cells/mm 2 ) and 29 DCMi patients (18.91±6.41 CD3 cells/mm 2 ) comparable in age (51.45±3.15 vs. 48.48±2.39 years), LVEDD (72.7±2.2 vs. 69.76±1.06mm) and EF (26±2.99 vs. 25.86±2.0%). Total collagen content was quantified by picosiriusredstaining while the MMP2, MMP3 and TIMP1 expression was evaluated by immunohistochemistry. The MMP1, MMP3 and TIMP1 and ICTP expression in the serum was quantified by Elisa. and levels of IL6 and IL8 were measured by BioPlexAssays. Furthermore various cytokines and proteases were evaluated by real-time-PCR. In DCMi patients both CD3-lymphocyte infiltration (8.95fold, pP P P P P Conclusion: Myocardial inflammation in patients with DCMi leads to an imbalance in the MMP/TIMP-system, which attributes to the development of myocardial fibrosis.

Introduction: The biopsy based analyse, with histological, immunohistological and molecular biological analysis of myocardial tissues, represents the only possible tool to investigate the basement of inflammatory cardiomyopathy. Since development of this disease assume involvement of extracellular matrix remodelling, the analyze of this process were aimed in this article. Methods and results: Endomyocardial biopsies from patient with inflammatory cardiomyopathy (n=170) were analysed by RT-PCR, Furthermore, histological, immunohistological and biochemical methods (ELISA) were used to estimate the matrix proteins amount in myocardial tissues (n=36). All results were obtained by comparison of patient groups regarding to left ventricular ejection fraction (LVEF), EF>60 versus EF60 group. The escalating number of active CD3 cells correlated positively with BNP (ρ=0.624, p Conclusion: Myocardial inflammation lead to an imbalance in the MMP/TIMP system with development of myocardial fibrosis with significant correlation to LV-dysfunction. Extracellular matrix remodeling with an imbalance in the MMP/TIMP system plays an important role in the development of left ventricular dysfunction in inflammatory heart disease