Stefan E. Hardt

High-Mobility Group Box-1 in Ischemia-Reperfusion Injury of the Heart

Background— High-mobility group box-1 (HMGB1) is a nuclear factor released by necrotic cells and by activated immune cells. HMGB1 signals via members of the toll-like receptor family and the receptor for advanced glycation end products (RAGE). Although HMGB1 has been implicated in ischemia/reperfusion (I/R) injury of the liver and lung, its role in I/R injury of the heart remains unclear. Methods and Results— Here, we demonstrate that HMGB1 acts as an early mediator of inflammation and organ damage in I/R injury of the heart. HMGB1 levels were already elevated 30 minutes after hypoxia in vitro and in ischemic injury of the heart in vivo. Treatment of mice with recombinant HMGB1 worsened I/R injury, whereas treatment with HMGB1 box A significantly reduced infarct size and markers of tissue damage. In addition, HMGB1 inhibition with recombinant HMGB1 box A suggested an involvement of the mitogen-activated protein kinases jun N-terminal kinase and extracellular signal-regulated kinase 1/2, as well as the nuclear transcription factor nuclear factor-&kgr;B in I/R injury. Interestingly, infarct size and markers of tissue damage were not affected by administration of recombinant HMGB1 or HMGB1 antagonists in RAGE−/− mice, which demonstrated significantly reduced damage in reperfused hearts compared with wild-type mice. Coincubation studies using recombinant HMGB1 in vitro induced an inflammatory response in isolated macrophages from wild-type mice but not in macrophages from RAGE−/− mice. Conclusions— HMGB1 plays a major role in the early event of I/R injury by binding to RAGE, resulting in the activation of proinflammatory pathways and enhanced myocardial injury. Therefore, blockage of HMGB1 might represent a novel therapeutic strategy in I/R injury.

Targeted Mutation Reveals Essential Functions of the Homeodomain Transcription Factor Shox2 in Sinoatrial and Pacemaking Development

Background— Identifying molecular pathways regulating the development of pacemaking and coordinated heartbeat is crucial for a comprehensive mechanistic understanding of arrhythmia-related diseases. Elucidation of these pathways has been complicated mainly by an insufficient definition of the developmental structures involved in these processes and the unavailability of animal models specifically targeting the relevant tissues. Here, we report on a highly restricted expression pattern of the homeodomain transcription factor Shox2 in the sinus venosus myocardium, including the sinoatrial nodal region and the venous valves. Methods and Results— To investigate its function in vivo, we have generated mouse lines carrying a targeted mutation of the Shox2 gene. Although heterozygous animals did not exhibit obvious defects, homozygosity of the targeted allele led to embryonic lethality at 11.5 to 13.5 dpc. Shox2−/− embryos exhibited severe hypoplasia of the sinus venosus myocardium in the posterior heart field, including the sinoatrial nodal region and venous valves. We furthermore demonstrate aberrant expression of connexin 40 and connexin 43 and the transcription factor Nkx2.5 in vivo specifically within the sinoatrial nodal region and show that Shox2 deficiency interferes with pacemaking function in zebrafish embryos. Conclusions— From these results, we postulate a critical function of Shox2 in the recruitment of sinus venosus myocardium comprising the sinoatrial nodal region.

Longitudinal left ventricular function for prediction of survival in systemic light-chain amyloidosis: incremental value compared with clinical and biochemical markers.

OBJECTIVES The aim of the study was to determine whether longitudinal left ventricular (LV) function provides prognostic information in a large cohort of patients with systemic light-chain (AL) amyloidosis. BACKGROUND AL amyloidosis is associated with a high incidence of cardiovascular events. Reduced myocardial longitudinal function is one of the hallmarks of myocardial involvement in this rare disease. METHODS Two hundred six consecutive patients with biopsy-proven AL amyloidosis were investigated in this prospective observational study. Echocardiographic imaging parameters, mean tissue Doppler-derived longitudinal strain (LS), and two-dimensional global longitudinal strain (2D-GLS) of the LV, cardiac serological biomarkers, and comprehensive clinical disease characteristics were assessed. The primary endpoint was all-cause mortality or heart transplantation. RESULTS After a median follow-up of 1207 days, LS and 2D-GLS were significant predictors of survival in AL amyloidosis. The cutoff values discriminating survivors from nonsurvivors were -10.65% for LS and -11.78% for 2D-GLS. In a multivariable echocardiographic Cox model, only diastolic dysfunction and 2D-GLS remained as independent predictors of survival. In comprehensive clinical models, 2D-GLS (p < 0.0001), diastolic dysfunction (p < 0.01), the pathologic free light chains (p < 0.05), cardiac troponin-T (cTnT) (p < 0.01), and the Karnofsky index (p < 0.001) remained as independent predictors. 2D-GLS delineated a superior prognostic value compared with that derived from pathologic free light chains or cTnT in patients evaluated before firstline chemotherapy (n = 113; p < 0.0001), and remained the only independent predictor besides the Karnofsky index in subjects with preserved LV ejection fraction (≥50%; n = 127; p < 0.01). LS and 2D-GLS both offered significant incremental information (p < 0.001) for the assessment of outcome compared with clinical variables (age, Karnofsky index, and New York Heart Association functional class) and serological biomarkers. CONCLUSIONS In the largest serial investigation reported so far, reduced LV longitudinal function served as an independent predictor of survival in AL amyloidosis and offered incremental information beyond standard clinical and serological parameters.

Alterations in cardiac DNA methylation in human dilated cardiomyopathy.

Dilated cardiomyopathies (DCM) show remarkable variability in their age of onset, phenotypic presentation, and clinical course. Hence, disease mechanisms must exist that modify the occurrence and progression of DCM, either by genetic or epigenetic factors that may interact with environmental stimuli. In the present study, we examined genome‐wide cardiac DNA methylation in patients with idiopathic DCM and controls. We detected methylation differences in pathways related to heart disease, but also in genes with yet unknown function in DCM or heart failure, namely Lymphocyte antigen 75 (LY75), Tyrosine kinase‐type cell surface receptor HER3 (ERBB3), Homeobox B13 (HOXB13) and Adenosine receptor A2A (ADORA2A). Mass‐spectrometric analysis and bisulphite‐sequencing enabled confirmation of the observed DNA methylation changes in independent cohorts. Aberrant DNA methylation in DCM patients was associated with significant changes in LY75 and ADORA2A mRNA expression, but not in ERBB3 and HOXB13. In vivo studies of orthologous ly75 and adora2a in zebrafish demonstrate a functional role of these genes in adaptive or maladaptive pathways in heart failure.

Assessment of disease severity and outcome in patients with systemic light-chain amyloidosis by the high-sensitivity troponin T assay

Cardiac biomarkers provide prognostic information in light-chain amyloidosis (AL). Thus, a novel high-sensitivity cardiac troponin T (hs-TnT) assay may improve risk stratification. hs-TnT was assessed in 163 patients. Blood levels were higher with cardiac than renal or other organ involvement and were related to the severity of cardiac involvement. Increased sensitivity was not associated with survival benefit. Forty-seven patients died during follow-up (22.3 ± 1.0 months). Nonsurvivors had higher hs-TnT than survivors. Outcome was worse if hs-TnT more than or equal to 50 ng/L and best less than 3 ng/L. Survival of patients with hs-TnT 3 to 14 ng/L did not differ from patients with moderately increased hs-TnT (14-50 ng/L), but was worse if interventricular septum was more than or equal to 15 mm. Discrimination according to the Mayo staging system was only achieved by the use of the hs-TnT assay, but not by the fourth-generation troponin T assay. Multivariate analysis revealed hs-TnT, NT-proBNP, and left ventricular impairment as independent risk factors for survival. hs-TnT and NT-proBNP predicted survival, even after exclusion of patients with impaired renal function. Plasma levels of the hs-TnT assay are associated with the clinical, morphologic, and functional severity of cardiac AL amyloidosis and could provide useful information for clinicians on cardiac involvement and outcome.

Wnt Signaling Is Critical for Maladaptive Cardiac Hypertrophy and Accelerates Myocardial Remodeling

The evolutionary conserved Wnt signaling pathway regulates cardiogenesis. However, members of the Wnt pathway are also expressed in the adult heart. Although Wnt-signaling is quiescent under normal conditions, we noticed activation on pathological stress of the heart, such as chronic afterload increase. To examine the role of Wnt signaling on the postnatal heart, we modified the expression and function of the Wnt regulator dishevelled 1 (Dvl-1) both in transgenic mice with cardiac-specific overexpression of Dvl-1 (Dvl-1-Tg) and in cultured cardiac myocytes. Dvl-1-Tg mice (3 months) had severe cardiac hypertrophy (heart weight:body weight ratio: 5.2±0.3 mg/g wild-type [WT] versus 6.4±0.7 mg/g Dvl-1-Tg; P<0.01), an increase in cardiomyocyte size (86% increase in Dvl-1-Tg compared with WT; P<0.01) and marked raise of atrial natriuretic factor expression (12-fold increase versus WT; P<0.01). Hypertrophy was associated with left ventricular dilatation in Dvl-1-Tg and a reduction of ejection fraction (4.4±0.1 mm versus 5.5±0.2 mm, 80±2% and 43±4% in WT versus Dvl-1-Tg, respectively; P<0.01). Transgenic animals died prematurely before 6 months of age. Both canonical as well as noncanonical Wnt signaling branches were activated in the Dvl-1-Tg animals. Small interfering RNA–mediated depletion of Dvl-1 was used to further characterize the role of Dvl-1 in cardiac myocytes. Whereas baseline parameters were unaltered, &bgr;-adrenergic hypertrophic response was abrogated in Dvl-1 knockdown cardiac myocytes, indicating a mandatory role in &bgr;-adrenergic stimulation. Therefore, activation of Wnt signaling is sufficient and critical for the induction of myocardial hypertrophy and cardiomyopathy.

Phosphorylation of Eukaryotic Translation Initiation Factor 2Bε by Glycogen Synthase Kinase-3β Regulates β-Adrenergic Cardiac Myocyte Hypertrophy

Abstract— Glycogen synthase kinase 3β (GSK-3β) negatively regulates cardiac hypertrophy. A potential target mediating the antihypertrophic effect of GSK-3β is eukaryotic translation initiation factor 2B3ε (eIF2Bε). Overexpression of GSK-3β increased the cellular kinase activity toward GST-eIF2Bε in neonatal rat cardiac myocytes, whereas LiCl (10 mmol/L) or isoproterenol (ISO) (10 μmol/L), a treatment known to inhibit GSK-3β, decreased it. Immunoblot analyses using anti-S535 phosphospecific eIF2Bε antibody showed that S535 phosphorylation of endogenous eIF2Bε was decreased by LiCl or ISO, suggesting that GSK-3β is the predominant kinase regulating phosphorylation of eIF2Bε-S535 in cardiac myocytes. Decreases in eIF2Bε-S535 phosphorylation were also observed in a rat model of cardiac hypertrophy in vivo. Overexpression of wild-type eIF2Bε alone moderately increased cell size (+31±11%; P< 0.05 versus control), whereas treatment of eIF2Bε-transduced myocytes with LiCl (+73±22% versus eIF2Bε only; P< 0.05) or ISO (+84±33% versus eIF2Bε only; P< 0.05) enhanced the effect of eIF2Bε. Overexpression of eIF2Bε-S535A, which is not phosphorylated by GSK-3β, increased cell size (+107±35%) as strongly as ISO (+95±25%), and abolished antihypertrophic effects of GSK-3β, indicating that S535 phosphorylation of eIF2Bε critically mediates antihypertrophic effects of GSK-3β. Furthermore, expression of eIF2Bε-F259L, a dominant-negative mutant, inhibited ISO-induced hypertrophy, indicating that eIF2Bε is required for β-adrenergic hypertrophy. Interestingly, expression of eIF2Bε-S535A partially increased cytoskeletal reorganization, whereas it did not increase expression of atrial natriuretic factor gene. These results suggest that GSK-3β is the predominant kinase mediating phosphorylation of eIF2Bε-S535 in cardiac myocytes, which in turn plays an important role in regulating cardiac hypertrophy primarily through protein synthesis.

Multivariate miRNA signatures as biomarkers for non-ischaemic systolic heart failure.

AIMS Non-ischaemic heart failure is one of the todays most prevalent cardiovascular disorders. Since modern pharmacotherapy has proved to be very effective in delaying disease progression and preventing death, imaging modalities and molecular biomarkers play an important role in early identification and clinical management as well as risk assessment of patients. The present study evaluated for the first time whole peripheral blood miRNAs as novel biomarker candidates for non-ischaemic heart failure with reduced ejection fraction (HF-REF). METHODS AND RESULTS We assessed genome-wide miRNA expression profiles in 53 HF-REF patients and 39 controls. We could identify and validate several miRNAs that show altered expression levels in non-ischaemic HF-REF, discriminating cases from controls both as single markers or when combined in a multivariate signature. In addition, we demonstrate that the miRNAs of this signature significantly correlate with disease severity as indicated by left ventricular ejection fraction. CONCLUSION Our data further denote that miRNAs are potential biomarkers for systolic heart failure. Since their detection levels in whole blood are also related to the degree of left ventricular dysfunction, they may serve as objective molecular tools to assess disease severity and prognosis.

Myocardial left ventricular dysfunction in patients with systemic lupus erythematosus: new insights from tissue Doppler and strain imaging.

Objective. Systemic lupus erythematosus (SLE) is associated with high cardiovascular morbidity and mortality. Cardiovascular involvement is frequently underestimated by routine imaging techniques. Our aim was to determine if new echocardiographic imaging modalities like tissue Doppler (TDI), strain rate (SRR), and strain (SRI) imaging detect abnormalities in left ventricular (LV) function in asymptomatic patients with SLE. Methods. Sixty-seven young patients with SLE (mean age 42 ± 10 yrs) without typical symptoms or signs of heart failure or angina, and a matched healthy control group (n = 40), underwent standard transthoracic echocardiography, TDI, SRR, and SRI imaging of the LV as well as assessment of disease characteristics. Results. Despite findings within the normal range on routine standard 2-dimensional echocardiography, SLE was associated with significantly impaired systolic and diastolic myocardial velocities of the LV measured by TDI [mean global TDI: systolic (s): 2.9 ± 0.9 vs 3.9 ± 0.7 cm/s, p < 0.05; early (e): 4.3 ± 1.5 vs 6.3 ± 1.3 cm/s, p < 0.05; late (a): 2.9 ± 0.8 vs 3.4 ± 0.8 cm/s, p < 0.05; values ± SD); SRR (s: −0.8 ± 0.1 vs −1.1 ± 0.1 s−1; e: 1.1 ± 0.2 vs 1.6 ± 0.3 s−1; a: 0.7 ± 0.1 vs 1.0 ± 0.2 s−1; all p < 0.05); and SR (−15.11 ± 2.2% vs −19.7 ± 1.9%; p < 0.05) compared to the control group. Further, elevated disease activity, measured with the ECLAM and the SLEDAI score, resulted in significantly lower values for LV longitudinal function measured by SRR and SR, but not by TDI. Conclusion. SLE is associated with a significant impairment of systolic and diastolic LV longitudinal function in patients without cardiac symptoms. New imaging modalities provide earlier insight into cardiovascular involvement in SLE and seem to be superior to standard echocardiography to detect subclinical myocardial disease.

Left atrial appendage occlusion in atrial fibrillation after intracranial hemorrhage

Objective: To evaluate the safety and feasibility of percutaneous left atrial appendage occlusion (LAAO) in patients with atrial fibrillation (AF) and previous intracranial hemorrhage (ICH). Methods: In an explorative, prospective, single-center, observational study, LAAO was performed in patients with previous ICH and AF using the Amplatzer Cardiac Plug device. Risks of ischemic strokes and hemorrhagic complications were estimated using the CHA2DS2Vasc score and the HAS-BLED score. Before and 1, 6, 12, and 24 months after the procedure, clinical status and complications were recorded. Major complications were predefined as periprocedural stroke, death, pericardial effusion, and device embolism. Results: LAAO was performed in 20 patients. Based on CHA2DS2Vasc score (mean 4.5 ± 1.4) and HAS-BLED score (mean 4.7 ± 1.0), annual risks of stroke and hemorrhagic complications were 4.0%–6.7% and 8.7%–12.5%, respectively. No patient had a procedure-related complication. Minor postprocedural complications were observed in 4/20 patients (2 inguinal hematoma, 1 self-limiting asystole, and 1 thrombus formation on device). No ischemic or hemorrhagic stroke occurred during a mean follow-up of 13.6 ± 8.2 months. Conclusions: In this first study of LAAO in patients with previous ICH, LAAO appears feasible and safe. A larger, controlled trial is needed to assess the efficacy and safety of the procedure compared to other preventive measures. Classification of evidence: This study provides Class III evidence that in patients with a history of previous ICH and AF, percutaneous LAAO is safe and feasible.