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Background: Mutations in the human desmin gene cause myopathies and cardiomyopathies. This study aimed to elucidate molecular mechanisms initiated by the heterozygous R406W-desmin mutation in the development of a severe and early-onset cardiac phenotype., Methods: We report an adolescent patient who underwent cardiac transplantation as a result of restrictive cardiomyopathy caused by a heterozygous R406W-desmin mutation. Sections of the explanted heart were analyzed with antibodies specific to 406W-desmin and to intercalated disc proteins. Effects of the R406W mutation on the molecular properties of desmin were addressed by cell transfection and in vitro assembly experiments. To prove the genuine deleterious effect of the mutation on heart tissue, we further generated and analyzed R405W-desmin knock-in mice harboring the orthologous form of the human R406W-desmin., Results: Microscopic analysis of the explanted heart revealed desmin aggregates and the absence of desmin filaments at intercalated discs. Structural changes within intercalated discs were revealed by the abnormal organization of desmoplakin, plectin, N-cadherin, and connexin-43. Next-generation sequencing confirmed the DES variant c.1216C>T (p.R406W) as the sole disease-causing mutation. Cell transfection studies disclosed a dual behavior of R406W-desmin with both its integration into the endogenous intermediate filament system and segregation into protein aggregates. In vitro, R406W-desmin formed unusually thick filaments that organized into complex filament aggregates and fibrillar sheets. In contrast, assembly of equimolar mixtures of mutant and wild-type desmin generated chimeric filaments of seemingly normal morphology but with occasional prominent irregularities. Heterozygous and homozygous R405W-desmin knock-in mice develop both a myopathy and a cardiomyopathy. In particular, the main histopathologic results from the patient are recapitulated in the hearts from R405W-desmin knock-in mice of both genotypes. Moreover, whereas heterozygous knock-in mice have a normal life span, homozygous animals die at 3 months of age because of a smooth muscle-related gastrointestinal phenotype., Conclusions: We demonstrate that R406W-desmin provokes its severe cardiotoxic potential by a novel pathomechanism, where the concurrent dual functional states of mutant desmin assembly complexes underlie the uncoupling of desmin filaments from intercalated discs and their structural disorganization.

Background: FAP (fibroblast activation protein) plays an important role in cardiac wound healing and remodeling. Although initially developed as a theranostic ligand for metastasized cancer, FAPI (FAP inhibitor) tracers have recently been used to study cardiac remodeling following myocardial infarction in small-animal models. The aim of the study was to evaluate the activity of FAP via FAPI-positron emission tomography-computed tomography scans in human hearts., Methods: FAPI-positron emission tomography-computed tomography scans of 229 patients of 2 consecutive cohorts (modeling cohort: n=185; confirmatory cohort: n=44) suffering from metastasized cancer were analyzed applying the American Heart Association 17-segment model of the left ventricle. Logistic regression models were created using data from the modeling cohort. Multivariate regression models were established using Akaike information criterion in a step-down approach., Results: Fourteen percent of patients had preexisting coronary artery disease (n=31), 33% arterial hypertension (n=75), and 12% diabetes mellitus type II (n=28). Forty-three percent had been treated with platin derivatives (n=100), 14% with anthracyclines (n=32), and 10% had a history of prior radiation to the chest (n=23). High left ventricular FAPI signals correlated with the presence of cardiovascular risk factors (odds ratio [OR], 4.3, P =0.0029), a focal myocardial signal pattern (OR, 3.9, P =0.0068), diabetes mellitus type II (OR, 4.1, P =0.046), and beta-blocker use (OR, 3.8, P =0.049) in univariate regression models. In a multivariate analysis, increased signal intensity was significantly higher in patients with cardiovascular risk factors (overweight [OR, 2.6, P =0.023], diabetes mellitus type II [OR, 2.9, P =0.041], certain chemotherapies [platinum derivatives; OR, 3.0, P =0.034], and a history of radiation to the chest [OR, 3.5, P =0.024]). A focal enrichment pattern was more frequently observed in patients with known cardiovascular risk factors ( P <0.0001)., Conclusions: FAPI-positron emission tomography-computed tomography scans represent a new imaging modality to investigate cardiac FAP. High signal intensities correlate with cardiovascular risk factors and metabolic disease.

Non-cardiac surgery is associated with significant cardiovascular complications. Reported mortality rate ranges from 1.9% to 4% in unselected patients. A postoperative surge in pro-inflammatory cytokines is a well-known feature and putative contributor to these complications. Despite much clinical research, little is known about the biomolecular changes in cardiac tissue following non-cardiac surgery. In order to increase our understanding, we analyzed whole-transcriptional and metabolic profiling data sets from hearts of mice harvested two, four, and six weeks following isolated thoracotomy. Hearts from healthy litter-mates served as controls. Functional network enrichment analyses showed a distinct impact on cardiac transcription two weeks after surgery characterized by a downregulation of mitochondrial pathways in the absence of significant metabolic alterations. Transcriptional changes were not detectable four and six weeks following surgery. Our study shows distinct and reversible transcriptional changes within the first two weeks following isolated thoracotomy. This coincides with a time period, in which most cardiovascular events happen.

Background: Cardiac amyloidosis, caused by deposition of immunoglobulin light chains (AL) or transthyretin (ATTR), carries a poor prognosis. Established risk scores for amyloidosis may not predict outcomes in those patients who develop advanced heart failure and who are potential candidates for heart transplantation. Here, we aimed to identify predictive parameters for patients with severe heart failure due to amyloidosis., Methods: Out of > 1000 patients with cardiac amyloidosis (AL or ATTR) admitted to our centre between September 1998 and January 2016, a cohort of 120 patients with a complete cardiac assessment at diagnosis, including right heart catheterization, echocardiography and biomarkers, was analysed retrospectively in this study. Primary endpoint was all-cause mortality. We performed univariate and multivariate Cox regression analysis, generated risk scores to predict outcomes in AL and ATTR amyloidosis and compared those to established risk models for amyloidosis., Results: In the Cox multivariate model, high-sensitivity troponin T (hsTnT; hazard ratio (HR) 1.003; confidence interval (CI) 1.001-1.005; p = 0.009) and mean pulmonary artery pressure (HR 1.061; CI 1.024-1.100; p = 0.001) were found to significantly and independently predict outcomes for AL amyloidosis, whereas QRS duration (HR 1.021; CI 1.004-1.039; p = 0.013), hsTnT (HR 1.021; CI 1.006-1.036; p = 0.006) and N-terminal pro-brain natriuretic peptide (HR 1.0003; CI 1.0001-1.0004; p = 0.002) were the best predictors for ATTR amyloidosis. A simple risk score ("HeiRisk") including these parameters for AL and ATTR allowed a more precise risk stratification in our patient population compared to established risk models., Conclusions: Risk stratification for cardiac amyloidosis with the newly developed "HeiRisk" score may be superior to other staging systems for patients with advanced heart failure due to amyloid cardiomyopathy.

Aims: Our aim was to investigate the glucose uptake in cancer patients suffering from different entities, using 18 F-FDG positron emission tomography-computed tomography scans. We further aimed at identifying potential variables altering cardiac and skeletal muscle glucose metabolism., Methods and Results: In a retrospective cohort study, we analysed cardiac and skeletal muscle 18 F-FDG uptake in onco-positron emission tomography-computed tomography scans in adult patients suffering from Hodgkin's lymphoma, non-Hodgkin's lymphoma, and non-lymphatic cancer including patients suffering from thyroid cancer, bronchial carcinoma, and malignant melanoma. Univariate logistic regression models were created for increased cardiac and skeletal muscle 18 F-FDG uptake using cancer entity, sex, age, previous radiation, previous chemotherapy, diabetes, obesity, serum glucose levels, renal function, and thyroid function as parameters. Multivariate models were created by selecting variables according to Akaike's information criterion in a step-down approach. Between 2014 and 2018, a total of 337 consecutive patients suffering from Hodgkin's lymphoma (n = 52), non-Hodgkin's lymphoma (n = 57), and non-lymphatic cancer (n = 228) were included in the analysis. Univariate logistic regression models showed high serum glucose levels to be associated with lower absorption rates in both cardiac and skeletal muscle (odds ratio [OR] 0.38 [0.23, 0.60, 95% confidence interval-CI], P < 0.0001, and 0.52 [0.33, 0.82, 95% CI], P < 0.005, respectively). Hodgkin's lymphoma was associated with an increase in cardiac uptake (OR 2.4 [1.3, 4.5, 95% CI], P < 0.005). Decreased skeletal muscle 18 F-FDG uptake was noted in elderly and obese patients. In our multivariate analysis, Hodgkin's lymphoma patients showed higher cardiac 18 F-FDG uptake, while non-Hodgkin's lymphoma patients did not differ significantly from non-lymphatic cancer patients (OR 1.6 [0.7, 3.3, 95% CI], P = 0.24). High serum glucose levels and prior chemotherapy were both associated with a significantly decreased cardiac 18 F-FDG uptake (OR 0.40 [0.24, 0.65, 95% CI], P < 0.0005, and 0.50 [0.27, 0.90, 95% CI], P < 0.05, respectively). Notably, prior chemotherapy did not influence FDG uptake in skeletal muscle to the same extent. Obesity and older age were both significantly associated with decreased gluteal 18 F-FDG uptake (OR 0.49 [0.27, 0.89, 95% CI], P < 0.05, and 0.47 [0.25, 0.87, 95% CI], P < 0.05)., Conclusions: Our data provide evidence for metabolic alterations in patients with Hodgkin's lymphoma related to cardiac glucose uptake in humans. This effect was independent from skeletal muscle metabolism., (© 2019 The Authors. ESC Heart Failure published by John Wiley & Sons Ltd on behalf of the European Society of Cardiology.)

Cardiac two-pore-domain potassium (K 2P ) channels have been proposed as novel antiarrhythmic targets. K 2P 13.1 (THIK-1) channels are expressed in the human heart, and atrial K 2P 13.1 levels are reduced in patients with atrial fibrillation (AF) or heart failure. The first objective of this study was to investigate acute effects of antiarrhythmic drugs on human K 2P 13.1 currents. Second, we assessed atrial K 2P 13.1 remodeling in AF pigs to validate the porcine model for future translational evaluation of K 2P 13.1-based antiarrhythmic concepts. K 2P 13.1 protein expression was studied in domestic pigs during AF induced by atrial burst pacing. AF was associated with 66% reduction of K 2P 13.1 levels in the right atrium at 21-day follow-up. Voltage clamp electrophysiology was employed to elucidate human K 2P 13.1 channel pharmacology in Xenopus oocytes. Propafenone (-26%; 100 μM), mexiletine (-75%; 1.5 mM), propranolol (-38%; 200 μM), and lidocaine (-59%; 100 μM) significantly inhibited K 2P 13.1 currents. By contrast, K 2P 13.1 channels were not markedly affected by quinidine, carvedilol, metoprolol, amiodarone and verapamil. Concentration-dependent K 2P 13.1 blockade by mexiletine occurred rapidly with membrane depolarization and was frequency-independent. Mexiletine reduced K 2P 13.1 open rectification properties and shifted current-voltage relationships towards more negative potentials. In conclusion, atrial expression and AF-associated downregulation of K 2P 13.1 channels in a porcine model resemble human findings and support a general role for K 2P 13.1 in AF pathophysiology. K 2P 13.1 current sensitivity to antiarrhythmic drugs provides a starting point for further development of an emerging antiarrhythmic paradigm., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Aims: Heart failure is characterized by structural and metabolic cardiac remodelling. The aim of the present study is to expand our understanding of the complex metabolic alterations in the transition from pathological hypertrophy to heart failure and exploit the results from a translational perspective., Methods and Results: Mice were subjected to transverse aortic constriction (TAC) or sham surgery and sacrificed 2 weeks, 4 weeks, or 6 weeks after the procedure. Samples from plasma, liver, skeletal muscle, and heart were collected and analysed using metabolomics. Cardiac samples were also analysed by transcriptional profiling. Progressive alterations of key cardiac metabolic pathways and gene expression patterns indicated impaired mitochondrial function and a metabolic switch during transition to heart failure. Similar to the heart, liver, and skeletal muscle revealed significant metabolic alterations such as depletion of essential fatty acids and glycerolipids in late stages of heart failure. Circulating metabolites, particularly fatty acids, reflected cardiac metabolic defects, and deteriorating heart function. For example, inverse correlation was found between plasma and the heart levels of triacylglycerol (C18:1, C18:2, C18:3), and sphingomyelin (d18:1, C23:0) already at an early stage of heart failure. Interestingly, combining metabolic and transcriptional data from cardiac tissue revealed that decreased carnitine shuttling and transportation preceded mitochondrial dysfunction. We, thus, studied the therapeutic potential of OCTN2 (Organic Cation/Carnitine Transporter 2), an important factor for carnitine transportation. Cardiac overexpression of OCTN2 using an adeno-associated viral vector significantly improved ejection fraction and reduced interstitial fibrosis in mice subjected to TAC., Conclusion: Comprehensive plasma and tissue profiling reveals systemic metabolic alterations in heart failure, which can be used for identification of novel biomarkers and potential therapeutic targets., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2018. For permissions, please email: journals.permissions@oup.com.)

Background: The diagnostic performance of adenosine stress cardiovascular magnetic resonance (CMR) for the detection of significant stenosis in infarct-related arteries is widely unknown. Two different types of perfusion defects can be observed: (a) larger than or (b) equal size as scar.We hypothesized that: (a) defect>scar predicts significant coronary stenosis, and (b) defect=scar predicts an unobstructed infarct-related artery, and (c) angina symptoms might be of additional value in stratification., Patients and Methods: Patients with previous myocardial infarction referred for work-up of myocardial ischemia undergoing adenosine stress CMR were included if they had coronary angiography within 4 weeks of CMR., Results: Two hundred patients with a mean age of 66±11 years, ischemic scars (subendocardial/transmural), and a mean left ventricular ejection fraction of 53% were included. In patients with defect>scar, the positive predictive value was excellent (88%) and typical angina was reported only in the stenosis group (P=0.002). However, patients with defect=scar (with 50% showing subendocardial scar) had a prevalence of 37% for stenosis, yielding a low negative predictive value of 63%. In this group, symptoms of typical angina were independent of stenosis (P=1.0)., Conclusion: A perfusion defect larger than scar is highly predictive for significant stenosis in infarct-related arteries. However, more than a third of the patients with perfusion defect of equal size as scar also showed significant coronary stenosis. As half of these patients showed still viable (subendocardial) scars, there is a high-risk of reinfarction. The addition of angina symptoms seems to increase diagnostic accuracy only in patients with perfusion defects larger than scar.

Background: Stroke can lead to cardiac dysfunction in patients, but the mechanisms underlying the interaction between the injured brain and the heart are poorly understood. The objective of the study is to investigate the effects of experimental murine stroke on cardiac function and molecular signalling in the heart., Methods and Results: Mice were subjected to filament-induced left middle cerebral artery occlusion for 30 or 60 min or sham surgery and underwent repetitive micro-echocardiography. Left ventricular contractility was reduced early (24-72 h) but not late (2 months) after brain ischaemia. Cardiac dysfunction was accompanied by a release of high-sensitive cardiac troponin (hsTNT (ng/ml): d1: 7.0 ± 1.0 vs. 25.0 ± 3.2*; d3: 7.3 ± 1.1 vs. 52.2 ± 16.7*; d14: 5.7 ± 0.8 vs. 5.2 ± 0.3; sham vs. 60 min. MCAO; mean ± SEM; *p < 0.05); reduced heart weight (heart weight/tibia length ratio: d1: 6.9 ± 0.2 vs. 6.4 ± 0.1*; d3: 6.7 ± 0.2 vs. 5.8 ± 0.1*; d14: 6.7 ± 0.2 vs. 6.4 ± 03; sham vs. 60 min. MCAO; mean ± SEM; *p < 0.05); resulting from cardiomyocyte atrophy (cardiomyocyte size: d1: 12.8% ± 0.002**; d3: 13.5% ± 0.002**; 14d: 6.3% ± 0.003*; 60 min. MCAO vs. sham; mean ± SEM; **p < 0.01; *p < 0.05), accompanied by increased atrogin-1 and the E3 ubiquitin ligase murf-1. Net norepinephrine but not synthesis was increased, suggesting a reduced norepinephrine release or an increase of norepinephrine re-uptake, resulting in a functional denervation. Transcriptome analysis in cardiac tissue identified the transcription factor peroxisome proliferator-activated receptor gamma as a potential mediator of stroke-induced transcriptional dysregulation involved in cardiac atrophy., Conclusions: Stroke induces a complex molecular response in the heart muscle with immediate but transient cardiac atrophy and dysfunction., (© 2018 The Authors. Journal of Cachexia, Sarcopenia and Muscle published by John Wiley & Sons Ltd on behalf of the Society on Sarcopenia, Cachexia and Wasting Disorders.)

Two-pore-domain potassium (K 2P ) channels modulate cellular excitability. The significance of stretch-activated cardiac K 2P channels (K 2P 2.1, TREK-1, KCNK2; K 2P 4.1, TRAAK, KCNK4; K 2P 10.1, TREK-2, KCNK10) in heart disease has not been elucidated in detail. The aim of this work was to assess expression and remodeling of mechanosensitive K 2P channels in atrial fibrillation (AF) and heart failure (HF) patients in comparison to murine models. Cardiac K 2P channel levels were quantified in atrial (A) and ventricular (V) tissue obtained from patients undergoing open heart surgery. In addition, control mice and mouse models of AF (cAMP-response element modulator (CREM)-IbΔC-X transgenic animals) or HF (cardiac dysfunction induced by transverse aortic constriction, TAC) were employed. Human and murine KCNK2 displayed highest mRNA abundance among mechanosensitive members of the K 2P channel family (V > A). Disease-associated K 2P 2.1 remodeling was studied in detail. In patients with impaired left ventricular function, atrial KCNK2 (K 2P 2.1) mRNA and protein expression was significantly reduced. In AF subjects, downregulation of atrial and ventricular KCNK2 (K 2P 2.1) mRNA and protein levels was observed. AF-associated suppression of atrial Kcnk2 (K 2P 2.1) mRNA and protein was recapitulated in CREM-transgenic mice. Ventricular Kcnk2 expression was not significantly altered in mouse models of disease. In conclusion, mechanosensitive K 2P 2.1 and K 2P 10.1 K + channels are expressed throughout the heart. HF- and AF-associated downregulation of KCNK2 (K 2P 2.1) mRNA and protein levels suggest a mechanistic contribution to cardiac arrhythmogenesis., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Background: Genetic variation is an important determinant of RNA transcription and splicing, which in turn contributes to variation in human traits, including cardiovascular diseases., Results: Here we report the first in-depth survey of heart transcriptome variation using RNA-sequencing in 97 patients with dilated cardiomyopathy and 108 non-diseased controls. We reveal extensive differences of gene expression and splicing between dilated cardiomyopathy patients and controls, affecting known as well as novel dilated cardiomyopathy genes. Moreover, we show a widespread effect of genetic variation on the regulation of transcription, isoform usage, and allele-specific expression. Systematic annotation of genome-wide association SNPs identifies 60 functional candidate genes for heart phenotypes, representing 20% of all published heart genome-wide association loci. Focusing on the dilated cardiomyopathy phenotype we found that eQTL variants are also enriched for dilated cardiomyopathy genome-wide association signals in two independent cohorts., Conclusions: RNA transcription, splicing, and allele-specific expression are each important determinants of the dilated cardiomyopathy phenotype and are controlled by genetic factors. Our results represent a powerful resource for the field of cardiovascular genetics.

A disturbed inflammatory response following myocardial infarction (MI) is associated with poor prognosis and increased tissue damage. Monocytes are key players in healing after MI, but little is known about the role of the cardiac niche in monocyte activation. This study investigated microenvironment-dependent changes in inflammatory monocytes after MI RNA sequencing analysis of murine Ly6C high monocytes on day 3 after MI revealed differential regulation depending on location. Notably, the local environment strongly impacted components of the WNT signaling cascade. Analysis of WNT modulators revealed a strong upregulation of WNT Inhibitory Factor 1 (WIF1) in cardiomyocytes-but not fibroblasts or endothelial cells-upon hypoxia. Compared to wild-type (WT) littermates, WIF1 knockout mice showed severe adverse remodeling marked by increased scar size and reduced ejection fraction 4 weeks after MI While FACS analysis on day 1 after MI revealed no differences in neutrophil numbers, the hearts of WIF1 knockouts contained significantly more inflammatory monocytes than hearts from WT animals. Next, we induced AAV-mediated cardiomyocyte-specific WIF1 overexpression, which attenuated the monocyte response and improved cardiac function after MI, as compared to control-AAV-treated animals. Finally, WIF1 overexpression in isolated cardiomyocytes limited the activation of non-canonical WNT signaling and led to reduced IL-1β and IL-6 expression in monocytes/macrophages. Taken together, we investigated the cardiac microenvironment's interaction with recruited monocytes after MI and identified a novel mechanism of monocyte activation. The local initiation of non-canonical WNT signaling shifts the accumulating myeloid cells toward a pro-inflammatory state and impacts healing after myocardial infarction., (© 2017 The Authors. Published under the terms of the CC BY 4.0 license.)

In heart failure (HF), a disturbed cardiac norepinephrine (NE) homeostasis is characterized by depleted cardiac NE stores, impairment of the cardiac NE re-uptake by the neuronal norepinephrine transporter (NET) and enhanced cardiac NE net release. Reduced cardiac NE content appears to be caused by enhanced cardiac NE net release from sympathetic neurons in HF, triggered by neurohumoral activation. However, it remains unclear whether reduced NE itself has an impact on cardiac NE re-uptake, independent of neurohumoral activation. Here, we evaluated whether depletion of cardiac NE stores alone can regulate cardiac NE re-uptake. Treatment of Wistar rats with reserpine (5 mg/kg/d) for one (1d) or five days (5d) resulted in markedly reduced cardiac NE content, comparable to NE stores in experimental HF due to pressure overload. In order to assess cardiac NE re-uptake, the specific cardiac [3H]-NE uptake via the NET in a Langendorff preparation was measured. Reserpine treatment led to decreased NE re-uptake at 1d and 5d compared to saline treatment. Expression of tyrosine hydroxylase (TH), the rate-limiting enzyme of the NE synthesis, was elevated in left stellate ganglia after reserpine. Mechanistically, measurement of NET mRNA expression in left stellate ganglia and myocardial NET density revealed a post-transcriptional downregulation of the NET by reserpine. In summary, present data demonstrate that depletion of cardiac NE stores alone is sufficient to impair cardiac NE re-uptake via downregulation of the NET, independent of systemic neurohumoral activation. Knowledge about the regulation of the cardiac NE homeostasis may offer novel therapeutic strategies in HF.

Background: Cardiac amyloid load has not been analyzed for its effect on mortality in patients with amyloid light-chain (AL) cardiac amyloidosis., Objectives: This study retrospectively compared histological amyloid load with common clinical predictors of mortality., Methods: This study assessed 216 patients with histologically confirmed cardiac amyloidosis at a single center with electrocardiography, echocardiography, and laboratory testing., Results: AL amyloid deposits were usually distributed in a reticular/pericellular pattern, whereas transthyretin amyloid (ATTR) more commonly showed patchy deposits. Median amyloid load was 30.5%; no amyloid load was above 70%. During follow-up (median 19.1 months), 112 patients died. Chemotherapy had a significant effect on overall survival in AL amyloidosis (16.2 months vs. 1.4 months; p = 0.003). Patients with <20% AL amyloid load who responded to chemotherapy showed significantly better survival than nonresponders. According to univariate analysis, predictors of survival in AL amyloidosis included sex, Karnofsky index, New York Heart Association (NYHA) functional class, diastolic blood pressure, estimated glomerular filtration rate, N-terminal pro-B-type natriuretic peptide, mineralocorticoid receptor antagonists, low voltage, ineligibility for chemotherapy, response to chemotherapy, and amyloid load. Independent predictors of mortality by multivariate analysis included NYHA functional class (III vs. II), estimated glomerular filtration rate, responders to chemotherapy, and amyloid load. In ATTR amyloidosis, survival correlated with NYHA functional class, diastolic blood pressure, and use of diuretic agents. Following Cox regression analysis, NYHA functional class (III vs. II; p < 0.05) remained the only independent predictor of patient survival in ATTR amyloidosis., Conclusions: Early identification of subjects with AL amyloid is essential given that in late-stage disease with extensive amyloid load, our data suggested that outcomes are not affected by administration of chemotherapy., (Copyright © 2016 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.)

Aims: Regulatory proteins of the sarcomere are pivotal for normal heart function and when affected by mutations are frequently causing cardiomyopathy. The exact function of these regulatory proteins and how mutations in these translate into distinct cardiomyopathy phenotypes remains poorly understood. Mutations in the essential myosin light chain (ELC) are linked to human cardiomyopathy characterized by a marked variability in disease phenotypes and high incidences of sudden death. Here we studied the role of the highly conserved S195 phosphorylation site of ELC using heterozygous adult zebrafish lazy susan (laz(m647)) in regulating contractile function in normal physiology and disease., Methods and Results: Echocardiography revealed signs of systolic dysfunction in otherwise phenotypically unremarkable heterozygote mutants. However, after physical stress, heart function of laz heterozygous zebrafish severely deteriorated causing heart failure and sudden death. Mechanistically, we show that upon physical stress, ELCs become phosphorylated and lack of S195 dominant-negatively impairs ELC phosphorylation. In vitro motility analysis with native myosin from adult heterozygous hearts demonstrates that S195 loss, specifically following physical stress, results in altered acto-myosin sliding velocities and myosin binding cooperativity, causing reduced force generation and organ dysfunction., Conclusion: Using adult heterozygous zebrafish, we show that ELC S195 phosphorylation is pivotal for adaptation of cardiac function to augmented physical stress and we provide novel mechanistic insights into the pathogenesis of ELC-linked cardiomyopathy., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2016. For permissions please email: journals.permissions@oup.com.)

Cardiac gene therapy is a promising approach for treating heart diseases. Although clinical studies are ongoing, effective and targeted transgene delivery is still a major obstacle. We sought to improve and direct transgene expression in myocardium by ultrasound-targeted microbubble destruction (UTMD). In pigs, adeno-associated virus-derived (AAV) vectors harboring the luciferase reporter gene were delivered via retroinfusion into the anterior interventricular coronary vein (AIV). AAV vectors were either loaded to lipid microbubbles before injection or injected unmodified. Upon injection of AAV/microbubble solution, UTMD was performed. After 4 weeks, reporter gene expression levels in the anterior wall (target area), in the posterior wall (control area), and in noncardiac organs were analyzed. Retroinfusion of AAV-luciferase vectors loaded to lipid microbubbles led to a significant increase in transgene expression, with an increase in UTMD targeted areas of the anterior wall. Moreover, off-target expression was reduced in comparison to control animals, receiving AAV-luciferase without microbubbles. Besides an increase in overall target area transgene expression, UTMD alters the spatial expression of the luciferase transgene, focusing expression to ultrasound-targeted left ventricular wall. These data suggest UTMD as a promising approach for directing AAV to specific cardiac segments.

Aims: Inhibition of β-adrenergic signalling plays a key role in treatment of heart failure. Gsα is essential for β-adrenergic signal transduction. In order to reduce side-effects of beta-adrenergic inhibition diminishing β-adrenergic signalling in the heart at the level of Gsα is a promising option., Methods and Results: We analyzed the influence of Gsα on regulation of myocardial function and development of cardiac hypertrophy, using a transgenic mouse model (C57BL6/J mice) overexpressing a dominant negative Gsα-mutant under control of the α-MHC-promotor. Cardiac phenotype was characterized in vivo and in vitro and under acute and chronic β-adrenergic stimulation. At rest, Gsα-DN-mice showed bradycardia (602 ± 13 vs. 660 ± 17 bpm, p<0.05) and decreased dp/dtmax (5037 ± 546- vs. 6835 ± 505 mmHg/s, p = 0.02). No significant differences were found regarding ejection fraction, heart weight and cardiomyocyte size. β-blockade by propranolol revealed no baseline differences of hemodynamic parameters between wildtype and Gsα-DN-mice. Acute adrenergic stimulation resulted in decreased β-adrenergic responsiveness in Gsα-DN-mice. Under chronic adrenergic stimulation, wildtype mice developed myocardial hypertrophy associated with increase of LV/BW-ratio by 23% (4.4 ± 0.2 vs. 3.5 ± 0.1 mg/g, p<0.01) and cardiac myocyte size by 24% (14927 ± 442 px vs. 12013 ± 583 px, p<0.001). In contrast, both parameters were unchanged in Gsα-DN-mice after chronic isoproterenol stimulation., Conclusion: Overexpression of a dominant negative mutant of Gsα leads to decreased β-adrenergic responsiveness and is protective against isoproterenol-induced hypertrophy. Thus, Gsα-DN-mice provide novel insights into β-adrenergic signal transduction and its modulation in myocardial overload and failure.

Aims: Osteopontin (OPN) is a multifunctional cytokine critically involved in cardiac fibrosis. However, the underlying mechanisms are unresolved. Non-coding RNAs are powerful regulators of gene expression and thus might mediate this process., Methods and Results: OPN and miR-21 were significantly increased in cardiac biopsies of patients with myocardial fibrosis. Ang II infusion via osmotic minipumps led to specific miRNA regulations with miR-21 being strongly induced in wild-type (WT) but not OPN knockout (KO) mice. This was associated with enhanced cardiac collagen content, myofibroblast activation, ERK-MAP kinase as well as AKT signalling pathway activation and a reduced expression of Phosphatase and Tensin Homologue (PTEN) as well as SMAD7 in WT but not OPN KO mice. In contrast, cardiotropic AAV9-mediated overexpression of OPN in vivo further enhanced cardiac fibrosis. In vitro, Ang II induced expression of miR-21 in WT cardiac fibroblasts, while miR-21 levels were unchanged in OPN KO fibroblasts. As pri-miR-21 was also increased by Ang II, we studied potential involved upstream regulators; Electrophoretic Mobility Shift and Chromatin Immunoprecipitation analyses confirmed activation of the miR-21 upstream-transcription factor AP-1 by Ang II. Recombinant OPN directly activated miR-21, enhanced fibrosis, and activated the phosphoinositide 3-kinase pathway. Locked nucleic acid-mediated miR-21 silencing ameliorated cardiac fibrosis development in vivo., Conclusion: In cardiac fibrosis related to Ang II, miR-21 is transcriptionally activated and targets PTEN/SMAD7 resulting in increased fibroblast survival. OPN KO animals are protected from miR-21 increase and fibrosis development due to impaired AP-1 activation and fibroblast activation., (© The Author 2015. Published by Oxford University Press on behalf of the European Society of Cardiology.)

Objectives: T1 mapping by cardiac magnetic resonance imaging (CMR) is able to determine the extracellular volume fraction. Wild-type transthyretin amyloidosis (WT-ATTR) is characterized by extracellular amyloid deposition in the heart. Recent reports indicated a reduction of left ventricular (LV) myocardial mass in WT-ATTR after consumption of epigallocatechin-3-gallate, the main catechin in green tea. It remained unclear, whether reduction of LV myocardial mass reflects decrease of amyloid load or progressive atrophy of cardiomyocytes., Methods: This study included 7 male patients with CMR repetitively performed before and 12 months after daily consumption of green tea extract (600 mg epigallocatechin-3-gallate). Short axis slices as well as 2-, 3-, and 4-chamber views were acquired using SSFP sequences. T1 mapping was created out of 11 mid-ventricular short axis views with increasing inversion times using a single breath-hold modified look-locker inversion recovery sequence before and 15 min after Gadolinium contrast administration., Results: After 12 months, a significant decrease of LV myocardial mass [198 (160; 212) vs. 180 (142; 204) g; p < 0.05] was observed. Moreover, a significant decrease of native [T1 1110 (1072; 1150) ms vs. 1080 (970; 1101), p < 0.05 or p = 0.03] was noticed. The calculated extracellular volume decreased in 5 patients (62.5%) by 7% and increased in 2 patients (37.5%) by 9.5%, in trend resulting in a (not significant) decrease of median ECV by 2.4%. Left ventricular ejection fraction (LVEF) [57 (48; 65) vs. 55 (47; 64) %; p = 0.3] remained unchanged., Conclusions: This study provided further evidence of LV myocardial mass reduction in patients with WT-ATTR daily consuming green tea extract. Additionally, this study gave first insights into the histomorphological correlate of LV mass reduction using T1 mapping. LV mass reduction appeared to be rather due to a decrease of amyloid load than atrophy of cardiomyocytes.

Background: Since the differentiation between physiological and pathological trabeculation is challenging, we assessed its distribution in a reference population of selected healthy volunteers., Methods: We studied 117 subjects (58 males) stratified into age tertiles and by gender. Cardiovascular magnetic resonance images were acquired using a standard SSFP-sequence. Left and right ventricular (LV/RV) end-diastolic (EDV), end-systolic (ESV) and trabeculated volumes indexed to the body surface area as well as ejection fraction (EF) were quantified in short-axis views. The maximum non-compacted-to-compacted (NC/C) ratio was measured in long-axis views., Results: The trabeculated volumes were significantly larger in men than in women and decreased with age. The correlation between both was moderate (r=0.46; p<0.001). LV trabeculated volume was positively associated with EDV and ESV (r=0.74; r=0.59; both p<0.001) and negatively with EF (r=-0.27; p<0.005). It was no independent predictor for EF. The maximum NC/C ratio was >2.3 in 46.2% and >2.5 in 37.6% of the subjects, which is regarded as abnormal in current literature. The fraction of subjects with a maximum NC/C ratio >2.3 and the mean maximum NC/C ratio differed significantly between gender but not between age groups. An increasing NC/C ratio was associated with a significant decrease in EF (r=-0.21; p<0.05)., Conclusion: A considerable amount of healthy volunteers fulfils the current diagnostic criteria of LV noncompaction with female subjects showing a higher fraction of false-positive results than males. LV trabeculated volume is more pronounced in young subjects and declines with age. The use of age- and gender-specific reference values as provided in this study may facilitate the delineation of physiological and pathological findings., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Aims: To investigate the prognostic impact of left-ventricular (LV) cardiac magnetic resonance (CMR) deformation imaging in patients with non-ischaemic dilated cardiomyopathy (DCM) compared with late-gadolinium enhancement (LGE) quantification and LV ejection fraction (EF)., Methods and Results: A total of 210 subjects with DCM were examined prospectively with standard CMR including measurement of LGE for quantification of myocardial fibrosis and feature tracking strain imaging for assessment of LV deformation. The predefined primary endpoint, a combination of cardiac death, heart transplantation, and aborted sudden cardiac death, occurred in 26 subjects during the median follow-up period of 5.3 years. LV radial, circumferential, and longitudinal strains were significantly associated with outcome. Using separate multivariate analysis models, global longitudinal strain (average of peak negative strain values) and mean longitudinal strain (negative peak of the mean curve of all segments) were independent prognostic parameters surpassing the value of global and mean LV radial and circumferential strain, as well as NT-proBNP, EF, and LGE mass. A global longitudinal strain greater than -12.5% predicted outcome even in patients with EF < 35% (P < 0.01) and in those with presence of LGE (P < 0.001). Mean longitudinal strain was further investigated using a clinical model with predefined cut-offs (EF < 35%, presence of LGE, NYHA class, mean longitudinal strain greater than -10%). Mean longitudinal strain exhibited an independent prognostic value surpassing that provided by NYHA, EF, and LGE (HR = 5.4, P < 0.01)., Conclusion: LV longitudinal strain assessed with CMR is an independent predictor of survival in DCM and offers incremental information for risk stratification beyond clinical parameters, biomarker, and standard CMR., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2014. For permissions please email: journals.permissions@oup.com.)

Mutations of the human desmin gene on chromosome 2q35 cause autosomal dominant, autosomal recessive and sporadic forms of protein aggregation myopathies and cardiomyopathies. We generated R349P desmin knock-in mice, which harbor the ortholog of the most frequently occurring human desmin missense mutation R350P. These mice develop age-dependent desmin-positive protein aggregation pathology, skeletal muscle weakness, dilated cardiomyopathy, as well as cardiac arrhythmias and conduction defects. For the first time, we report the expression level and subcellular distribution of mutant versus wild-type desmin in our mouse model as well as in skeletal muscle specimens derived from human R350P desminopathies. Furthermore, we demonstrate that the missense-mutant desmin inflicts changes of the subcellular localization and turnover of desmin itself and of direct desmin-binding partners. Our findings unveil a novel principle of pathogenesis, in which not the presence of protein aggregates, but disruption of the extrasarcomeric intermediate filament network leads to increased mechanical vulnerability of muscle fibers. These structural defects elicited at the myofiber level finally impact the entire organ and subsequently cause myopathy and cardiomyopathy.

Impairment of the cardiac norepinephrine (NE) reuptake by the neuronal NE transporter contributes to enhanced cardiac NE net release in congestive heart failure. Elevated plasma levels of aldosterone (AL) promote sympathetic overstimulation in failing hearts by unclear mechanisms. Our aim was to evaluate if elevated AL and/or alterations in Na(+) intake regulate cardiac NE reuptake. To test the effects of AL and Na(+) on cardiac NE reuptake, Wistar rats were fed a normal-salt (NS) diet (0.2% NaCl), a low-salt (LS) diet (0.015% NaCl), or a high-salt (HS) diet (8% NaCl). Another group of animals received AL infusion alone (0.75 μg/h) or AL infusion plus HS diet. Specific cardiac [(3)H]NE uptake via the NE transporter in a Langendorff preparation and AL plasma levels were measured at different time points between 5 and 42 days of treatment. To compare these findings from healthy animals with a disease model, Dahl salt-sensitive rats were investigated as a model of congestive heart failure with endogenously elevated AL. In summary, neither exogenous nor endogenous elevations of AL alone were sufficient to reduce cardiac NE reuptake. Only the HS diet induced a reduction of NE reuptake by 26%; additional infusion of AL augmented this effect to a further reduction of NE reuptake by 36%. In concordance, Dahl salt-sensitive rats treated with a HS diet displayed elevated AL and a marked reduction of NE reuptake. We conclude that exogenous or endogenous AL elevations alone do not reduce cardiac NE reuptake, but AL serves as an additional factor that negatively regulates cardiac NE reuptake in concert with HS intake., (Copyright © 2014 the American Physiological Society.)

Background: Ca(2+)-dependent signaling through CaM Kinase II (CaMKII) and calcineurin was suggested to contribute to adverse cardiac remodeling. However, the relative importance of CaMKII versus calcineurin for adverse cardiac remodeling remained unclear., Methods and Results: We generated double-knockout mice (DKO) lacking the 2 cardiac CaMKII genes δ and γ specifically in cardiomyocytes. We show that both CaMKII isoforms contribute redundantly to phosphorylation not only of phospholamban, ryanodine receptor 2, and histone deacetylase 4, but also calcineurin. Under baseline conditions, DKO mice are viable and display neither abnormal Ca(2+) handling nor functional and structural changes. On pathological pressure overload and β-adrenergic stimulation, DKO mice are protected against cardiac dysfunction and interstitial fibrosis. But surprisingly and paradoxically, DKO mice develop cardiac hypertrophy driven by excessive activation of endogenous calcineurin, which is associated with a lack of phosphorylation at the auto-inhibitory calcineurin A site Ser411. Likewise, calcineurin inhibition prevents cardiac hypertrophy in DKO. On exercise performance, DKO mice show an exaggeration of cardiac hypertrophy with increased expression of the calcineurin target gene RCAN1-4 but no signs of adverse cardiac remodeling., Conclusions: We established a mouse model in which CaMKII's activity is specifically and completely abolished. By the use of this model we show that CaMKII induces maladaptive cardiac remodeling while it inhibits calcineurin-dependent hypertrophy. These data suggest inhibition of CaMKII but not calcineurin as a promising approach to attenuate the progression of heart failure., (© 2014 American Heart Association, Inc.)

Effective and safe pharmacological management of cardiac arrhythmia still constitutes a major clinical challenge. Outward potassium currents mediated by two-pore-domain potassium (K2P) channels promote repolarization of excitable cells. In the heart, inhibition or genetic inactivation of K2P currents results in action potential prolongation. Human K2P3.1 (TASK-1) channels are predominantly expressed in the atria and represent targets for the treatment of atrial fibrillation. In addition, stretch-sensitive K2P2.1 (TREK-1) channels are implicated in mechanoelectrical feedback and arrhythmogenesis in atrial and ventricular tissue. K2P current inhibition by clinically used antiarrhythmic drugs indicates a role of the channels as potential drug targets. This work summarizes the current knowledge on function, pharmacology, and significance of cardiac K2P channels. Therapeutic implications with emphasis on atrial fibrillation are highlighted., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Members of the S100 protein family have been reported to function as endogenous danger signals (alarmins) playing an active role in tissue inflammation and repair when released from necrotic cells. Here, we investigated the role of S100A1, the S100 isoform with highest abundance in cardiomyocytes, when released from damaged cardiomyocytes during myocardial infarction (MI). Patients with acute MI showed significantly increased S100A1 serum levels. Experimental MI in mice induced comparable S100A1 release. S100A1 internalization was observed in cardiac fibroblasts (CFs) adjacent to damaged cardiomyocytes. In vitro analyses revealed exclusive S100A1 endocytosis by CFs, followed by Toll-like receptor 4 (TLR4)-dependent activation of MAP kinases and NF-κB. CFs exposed to S100A1 assumed an immunomodulatory and anti-fibrotic phenotype characterized i.e. by enhanced intercellular adhesion molecule-1 (ICAM1) and decreased collagen levels. In mice, intracardiac S100A1 injection recapitulated these transcriptional changes. Moreover, antibody-mediated neutralization of S100A1 enlarged infarct size and worsened left ventricular functional performance post-MI. Our study demonstrates alarmin properties for S100A1 from necrotic cardiomyocytes. However, the potentially beneficial role of extracellular S100A1 in MI-related inflammation and repair warrants further investigation., (© 2014 The Authors. Published under the terms of the CC BY 4.0 license.)

The ultrarapidly activating delayed rectifier current, I(Kur), is a main determinant of atrial repolarization in humans. I(Kur) and the underlying ion channel complex Kv1.5/Kvβ1.2 are negatively regulated by protein kinase C. However, the exact mode of action is only incompletely understood. We therefore analyzed isoenzyme-specific regulation of the Kv1.5/Kvβ1.2 ion channel complex by PKC. Cloned ion channel subunits were heterologously expressed in Xenopus oocytes, and measurements were performed using the double-electrode voltage-clamp technique. Activation of PKC with phorbol 12-myristate 13-acetate (PMA) resulted in a strong reduction of Kv1.5/Kvβ1.2 current. This effect could be prevented using the PKC inhibitor staurosporine. Using the bisindolylmaleimide Ro-31-8220 as an inhibitor and ingenol as an activator of the conventional PKC isoforms, we were able to show that the Kv1.5/Kvβ1.2 ion channel complex is mainly regulated by conventional isoforms. Whereas pharmacological inhibition of PKCα with HBDDE did not attenuate the PMA-induced effect, current reduction could be prevented using inhibitors of PKCβ. Here, we show the isoform βII plays a central role in the PKC-dependent regulation of Kv1.5/Kvβ1.2 channels. These results add to the current understanding of isoenzyme-selective regulation of cardiac ion channels by protein kinases.

Stimulation of myocardial β(1)-adrenoceptors (AR) is a major mechanism that increases cardiac function. We investigated the functional consequences of genetic β(1)-AR knockdown in three-dimensional engineered heart tissue (EHT). For β(1)-AR knockdown, short interfering RNA (siRNA) sequences targeting specifically the β(1)-AR (shB1) and a scrambled control (shCTR) were subcloned into a recombinant adeno-associated virus (AAV)-short hairpin RNA (shRNA) expression system. Transduction efficiency was ∼100%, and radioligand binding revealed 70% lower β(1)-AR density in AAV6-shB1-transduced EHTs. Force measurements, performed over the culture period of 14 days, showed paradoxically higher force generation in AAV6-shB1 compared with shCTR under basal (0.19 ± 0.01 versus 0.13 ± 0.01 mN) and after β-AR-stimulated conditions with isoprenaline (Δfractional shortening: 72 ± 5% versus 34 ± 4%). Large scale gene expression analysis revealed that AAV6-shCTR compared with nontransduced EHTs showed only few differentially regulated genes (<20), whereas AAV6-shB1 induced marked changes in gene expression (>250 genes), indicating that β(1)-AR knockdown itself determines the outcome. None of the regulated genes pointed to obvious off-target effects to explain higher force generation. Moreover, compensational regulation of β(2)-AR signaling or changes in prominent β(1)-AR downstream targets could be ruled out. In summary, we show paradoxically higher force generation and isoprenaline responses after efficient β(1)-AR knockdown in EHTs. Our findings 1) reveal an unexpected layer of complexity in gene regulation after specific β(1)-AR knockdown rather than unspecific dysregulations through transcriptional interference, 2) challenge classic assumptions on the role of cardiac β(1)-AR, and 3) may open up new avenues for β-AR loss-of-function research in vivo.

Class IC antiarrhythmic drugs are commonly used for rhythm control in atrial fibrillation. In addition, class I drugs are administered to suppress ventricular tachyarrhythmia in selected cases. The multichannel blocking profile of class I compounds includes reduction of cardiac potassium currents in addition to their primary mechanism of action, sodium channel inhibition. Blockade of two-pore-domain potassium (K2P) channels in the heart causes action potential prolongation and may provide antiarrhythmic action in atrial fibrillation. This study was designed to elucidate inhibitory effects of class I antiarrhythmic drugs on K2P channels. Human K2P2.1 (TREK1) and hK2P3.1 (TASK1) channels were systematically tested for their sensitivity to clinically relevant class IA (ajmaline), class IB (mexiletine), and class IC (propafenone) antiarrhythmic compounds using whole-cell patch clamp and two-electrode voltage clamp electrophysiology in Chinese hamster ovary cells and in Xenopus oocytes. Mexiletine and propafenone inhibited hK2P2.1 (IC50,mexiletine=173µM; IC50,propafenone=7.6µM) and hK2P3.1 channels (IC50,mexiletine=97.3µM; IC50,propafenone=5.1µM) in mammalian cells. Ajmaline did not significantly reduce current amplitudes. K2P channels were blocked in open and closed states, resulting in resting membrane potential depolarization. Open rectification properties of the channels were not affected by class I drugs. In summary, class I antiarrhythmic drugs target cardiac K2P K(+) channels. Blockade of hK2P2.1 and hK2P3.1 potassium currents provides mechanistic evidence to establish cardiac K2P channels as antiarrhythmic drug targets., (© 2013 Elsevier B.V. All rights reserved.)

Over the past 2 decades, cardiac troponins (cTn) have emerged as the preferred biomarkers for the noninvasive detection of myocardial injury. In conjunction with typical clinical findings of ischaemia, elevated cTn levels in blood confirm a diagnosis of myocardial infarction. However, neither cTnT or cTnI are exclusively released as a result of ischaemic myocardial cell necrosis, but also with numerous nonischaemic acute and chronic cardiac conditions, such as myopericarditis, toxic injury, or severe cardiac overload. With the advent of high-sensitivity assays, causes of cTn elevation not related to an acute coronary syndrome (ACS) have become common findings in patients with chest pain and in those with acute or chronic systemic disorders. Elevated cTn levels in blood are associated with increased rates of cardiac events and mortality, independently of the underlying disease. However, the clinical conditions leading to cTn release in patients who do not have ACS, and the appropriate diagnostic and therapeutic strategies for these individuals, are largely unknown. Here, we provide an overview of the many causes and the prognostic importance of the release of cTn not related to ACS. We also recommend strategies to discriminate between ischaemic and nonischaemic cTn elevation, and describe the clinical evaluation of these patients.

Summary: The detailed molecular mechanisms following activation of apoptosis in ischemia-reperfusion injury are unknown. This study using different transgenic mouse models provided first evidence that apoptosis in myocardial ischemia-reperfusion injury is rather linked to the mitochondrial pathway than to death receptor pathway., Introduction: There is a wealth of evidence for activation of apoptosis in ischemia-reperfusion injury. However, the understanding of detailed molecular mechanism is lacking., Methods: The extent of myocardial infarction after ligation of the left anterior descending artery in mice carrying different transgenes for inhibition of either the intrinsic or the extrinsic or a combination of both apoptotic cascades was evaluated. The extent of myocardial damage was assessed by echocardiographic determination of left ventricular (LV) ejection fraction, LV hemodynamics, troponin T, and histology. The rate of apoptosis was analyzed by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and caspase-3 staining., Results: Highest perioperative rate of death was observed in the dominant-negative form of a truncated Fas-associated death domain (FADD-DN) group. Infarction size by 2,3,5-triphenyltetrazolium chloride (TTC) staining was smaller in the Bcl-2, but not in the other groups as compared to wild-type mice. This was accompanied by lower troponin T values in Bcl-2 transgenic mice as compared to the all other groups. Troponin T correlated well with macroscopic extent of myocardial infarction by TTC staining. A lower decline of LV ejection fraction was seen in the Bcl-2 as compared to wild-type or FADD-DN mice. A smaller number of TUNEL- and caspase-3-positive myocyte nuclei were observed in the Bcl-2 and FADD-DN group as compared to wild-type mice., Conclusions: We provide first evidence for protective effects on the myocardium in a transgenic mouse model of myocardial ischemia-reperfusion due to inhibition of the Bcl-2, but not the FADD pathway despite that reduced apoptotic cells were observed in both groups as compared to wild-type mice., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Ageing is the predominant risk factor for cardiovascular diseases and contributes to a significantly worse outcome in patients with acute myocardial infarction. MicroRNAs (miRNAs) have emerged as crucial regulators of cardiovascular function and some miRNAs have key roles in ageing. We propose that altered expression of miRNAs in the heart during ageing contributes to the age-dependent decline in cardiac function. Here we show that miR-34a is induced in the ageing heart and that in vivo silencing or genetic deletion of miR-34a reduces age-associated cardiomyocyte cell death. Moreover, miR-34a inhibition reduces cell death and fibrosis following acute myocardial infarction and improves recovery of myocardial function. Mechanistically, we identified PNUTS (also known as PPP1R10) as a novel direct miR-34a target, which reduces telomere shortening, DNA damage responses and cardiomyocyte apoptosis, and improves functional recovery after acute myocardial infarction. Together, these results identify age-induced expression of miR-34a and inhibition of its target PNUTS as a key mechanism that regulates cardiac contractile function during ageing and after acute myocardial infarction, by inducing DNA damage responses and telomere attrition.

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., (Copyright © 2013 The Authors. Published by John Wiley and Sons, Ltd on behalf of EMBO.)

Cardiac troponins (cTns) T and I are exclusively expressed at high concentrations in cardiac muscle and have emerged as the preferred biomarker in the universal definition of myocardial infarction (MI). With the recent introduction of high-sensitivity (hs) assays, diagnostic sensitivity for earlier detection of MI has substantially improved. However, lowering the diagnostic cut-off has increased the detection of myocardial injuries in various non-acute coronary syndrome (ACS) conditions, which are not related to myocardial ischemia, leading to rising difficulties in diagnosing MI in clinical situations. Several approaches, such as serial sampling and incorporation of relative or absolute δ-changes, have been proposed to overcome the limitation of decreased sensitivity for MI diagnosis with hs-cTn assays. Current consensus for rapid rule-in proposes a 20% increase within 3 or 6h when baseline cTn levels are elevated. In the case of negative baseline values, relative increases ≥50% above the 99(th) percentile were found to be adequate to improve accuracy of MI diagnosis. Besides improved diagnostic accuracy for myocardial injury, even minor cTn elevations provide important prognostic information, and increased levels of cTn are associated with adverse outcomes in both the ACS and non-ACS condition, irrespective of whether the underlying cause is an acute or chronic illness. Thus, it is highly likely that lowering the diagnostic cut-off with even more sensitive assays might improve risk stratification in both conditions.

Dronedarone is currently used for the treatment of paroxysmal and persistent atrial fibrillation (AF). Pharmacological inhibition of cardiac two-pore-domain potassium (K(2P)) channels results in action potential prolongation and has recently been proposed as novel antiarrhythmic strategy. We hypothesized that blockade of human K(2P) channels contributes to the electrophysiological efficacy of dronedarone in AF. Two-electrode voltage clamp and whole-cell patch clamp electrophysiology was used to record K(2P) currents from Xenopus oocytes and Chinese hamster ovary cells. All functional human K(2P) channels were screened for dronedarone sensitivity, revealing significant and concentration-dependent inhibition of cardiac K(2P)2.1 (TREK1; IC(50) = 26.7 μM) and K(2P)3.1 channels (TASK1; IC(50) = 18.7 μM) with maximum current reduction of 60.3 and 65.5 % in oocytes. IC(50) values obtained from mammalian cells yielded 6.1 μM (K(2P)2.1) and 5.2 μM (K(2P)3.1). The molecular mechanism of action was studied in detail. Dronedarone block affected open and closed channels. K(2P)3.1 currents were reduced in frequency-dependent fashion in contrast to K(2P)2.1. Mutagenesis studies revealed that amino acid residues implicated in K(2P)3.1 drug interactions were not required for dronedarone blockade. The class III antiarrhythmic drug dronedarone targets multiple human cardiac two-pore-domain potassium channels, including atrial-selective K(2P)3.1 currents. K(2P) current inhibition by dronedarone represents a previously unrecognized mechanism of action that extends the multichannel blocking profile of the drug.

Background: Growth differentiation factor 15 (GDF15) is a stress-responsive cytokine and biomarker that is produced after myocardial infarction and that is related to prognosis in acute coronary syndrome (ACS). We hypothesized that secreted proteins that activate GDF15 production may represent new ACS biomarkers., Methods: We expressed clones from an infarcted mouse heart cDNA library in COS1 cells and assayed for activation of a luciferase reporter gene controlled by a 642-bp fragment of the mouse growth differentiation factor 15 (GDF15) gene promoter. We measured the circulating concentrations of follistatin-like 1 (FSTL1) and GDF15 in 1369 patients with ACS., Results: One cDNA clone that activated the GDF15 promoter-luciferase reporter encoded the secreted protein FSTL1. Treatment with FSTL1 activated GDF15 production in cultured cardiomyocytes. Transgenic production of FSTL1 stimulated GDF15 production in the murine heart, whereas cardiomyocyte-selective deletion of FSTL1 decreased production of GDF15 in cardiomyocytes, indicating that FSTL1 is sufficient and required for GDF15 production. In ACS, FSTL1 emerged as the strongest independent correlate of GDF15 (partial R(2) = 0.26). A total of 106 patients died of a cardiovascular cause during a median follow-up of 252 days. Patients with an FSTL1 concentration in the top quartile had a 3.7-fold higher risk of cardiovascular death compared with patients in the first 3 quartiles (P < 0.001). FSTL1 remained associated with cardiovascular death after adjustment for clinical, angiographic, and biochemical variables., Conclusions: Our study is the first to use expression cloning for biomarker discovery upstream of a gene of interest and to identify FSTL1 as an independent prognostic biomarker in ACS.

Dystrophin plays an important role in muscle contraction, linking the intracellular cytoskeleton to the extracellular matrix. Mutations of the dystrophin gene leading to a complete loss of the protein cause Duchenne muscular dystrophy (DMD), frequently associated with severe cardiomyopathy. Early clinical trials in DMD using gene transfer to skeletal muscle are underway, but gene transfer to dystrophic cardiac muscle has not yet been tested in humans. The aim of this study was to develop an optimized protocol for cardiac gene therapy in the mouse model of dystrophin deficiency (mdx), using a cardiac promoter for expression of a microdystrophin (μDys) transgene packaged into an adeno-associated virus serotype 9 vector (AAV9). In this study adult mdx mice were intravenously injected with 1×10(12) genomic particles of AAV9 vectors carrying a cDNA encoding μDys under the control of either a ubiquitously active cytomegalovirus (CMV) promoter or a cardiac-specific CMV-enhanced myosin light chain (MLC0.26) promoter. After 10 months, both AAV9 vectors led to sustained μDys expression in cardiac muscle, but the MLC promoter conferred about 4-fold higher protein levels. AAV9-CMV-MLC0.26-μDys resulted in significant protection of cardiac morphology and function as assessed by histopathology, echocardiography, and left ventricular catheterization. In conclusion, we established an AAV9-mediated gene transfer approach for efficient and specific long-term μDys expression in the hearts of mdx mice, resulting in a sustained therapeutic effect. Thus, this approach might be a basis for further translation into a treatment strategy for DMD-associated cardiomyopathy.

Muscle LIM protein (MLP) has been proposed to be a central player in the pathogenesis of heart muscle disease. In line with this notion, the homozygous loss of MLP results in cardiac hypertrophy and dilated cardiomyopathy. Moreover, MLP is induced in several models of cardiac hypertrophy such as aortic banding and myocardial infarction. We thus hypothesized that overexpression of MLP might change the hypertrophic response to cardiac stress. In order to answer the question whether MLP modulates cardiac hypertrophy in vivo, we generated a novel transgenic mouse model with cardiac-specific overexpression of MLP. Three independent transgenic lines did not show a pathological phenotype under baseline conditions. Specifically, contractile function and heart weight to body weight ratios at different ages were normal. Next, the transgenic animals were challenged with pressure overload due to aortic constriction. Surprisingly, transgenic mice developed cardiac hypertrophy to the same extent as their wild-type littermates. Moreover, neither contractile dysfunction nor pathological gene expression in response to pressure overload were differentially affected by MLP overexpression. Finally, in a milder in vivo model of hypertrophy induced by chronic infusion of angiotensin-II, cardiac mass and hypertrophic gene expression were again identical in MLP transgenic mice and controls. Taken together, we provide evidence that cardiac overexpression of MLP does not modulate the heart's response to various forms of pathological stress.

Purpose: To study myocardial perfusion reserve and myocellular metabolic alterations indicated by triglyceride content as possible causes of diastolic dysfunction in patients with type 2 diabetes mellitus, preserved systolic function, and without clinically evident coronary artery disease., Materials and Methods: Patients with type 2 diabetes mellitus (n = 42) underwent cardiac magnetic resonance (CMR) for quantification of 1) myocardial contractility by strain-encoded MR (SENC); 2) myocardial triglyceride content by proton magnetic resonance spectroscopy ((1) H-MRS); and 3) myocardial perfusion reserve during pharmacologic hyperemia. Age-matched healthy volunteers (n = 16) also underwent CMR to acquire normal values for myocardial strain and perfusion reserve., Results: Stress CMR procedures were successfully performed in all subjects, and no regional inducible perfusion defects were observed in type 2 diabetes mellitus patients. Diastolic strain rate and myocardial perfusion reserve were significantly impaired in patients with type 2 diabetes mellitus compared to control subjects (P < 0.001 for both). Interestingly, impaired diastolic function in type 2 diabetes mellitus was not associated with impaired myocardial perfusion reserve (r = 0.12, P = NS). Conversely a significant association was observed between diastolic dysfunction and myocardial triglyceride content (r = -0.71, P < 0.001), which proved to be independent of age, gender, diabetes duration, blood pressure, and fasting blood glucose., Conclusion: Myocardial steatosis may represent an early marker of diabetic heart disease, triggering subclinical myocardial dysfunction irrespective of myocardial perfusion reserve., (Copyright © 2011 Wiley Periodicals, Inc.)

Human heart failure is a disease with multifactorial causes, considerable morbidity, and high mortality. Several circulating autoantibodies, some of them being heart-specific, play a crucial role in the progression and induction of heart failure. However the precise mechanisms on how these autoantibodies perpetuate or even induce an organ specific autoimmune response are not yet fully understood. Also it is being a matter of current research to elucidate a potential pathophysiological role of the innate immune system in generating auto-reactive antibodies. In this review we will summarize the current available literature on circulating autoantibodies which are related to human heart failure. We will present clinical and animal studies that demonstrate the occurrence and pathophysiological relevance of several autoantibodies in heart failure, as well as point out biological mechanisms on molecular and cellular level. Finally the beneficial therapeutic effects of numerous clinical studies that target the humoral arm of the immune system by using either intravenous immunoglobulins and/or immunoadsorption will be critically discussed.

Assembly, maintenance and renewal of sarcomeres require highly organized and balanced folding, transport, modification and degradation of sarcomeric proteins. However, the molecules that mediate these processes are largely unknown. Here, we isolated the zebrafish mutant flatline (fla), which shows disturbed sarcomere assembly exclusively in heart and fast-twitch skeletal muscle. By positional cloning we identified a nonsense mutation within the SET- and MYND-domain-containing protein 1 gene (smyd1) to be responsible for the fla phenotype. We found SMYD1 expression to be restricted to the heart and fast-twitch skeletal muscle cells. Within these cell types, SMYD1 localizes to both the sarcomeric M-line, where it physically associates with myosin, and the nucleus, where it supposedly represses transcription through its SET and MYND domains. However, although we found transcript levels of thick filament chaperones, such as Hsp90a1 and UNC-45b, to be severely upregulated in fla, its histone methyltransferase activity - mainly responsible for the nuclear function of SMYD1 - is dispensable for sarcomerogenesis. Accordingly, sarcomere assembly in fla mutant embryos can be reconstituted by ectopically expressing histone methyltransferase-deficient SMYD1. By contrast, ectopic expression of myosin-binding-deficient SMYD1 does not rescue fla mutants, implicating an essential role for the SMYD1-myosin interaction in cardiac and fast-twitch skeletal muscle thick filament assembly.

As a prerequisite for clinical application, we determined the long-term therapeutic effectiveness and safety of adeno-associated virus (AAV)-S100A1 gene therapy in a preclinical large animal model of heart failure. S100A1, a positive inotropic regulator of myocardial contractility, becomes depleted in failing cardiomyocytes in humans and animals, and myocardial-targeted S100A1 gene transfer rescues cardiac contractile function by restoring sarcoplasmic reticulum calcium (Ca(2+)) handling in acutely and chronically failing hearts in small animal models. We induced heart failure in domestic pigs by balloon occlusion of the left circumflex coronary artery, resulting in myocardial infarction. After 2 weeks, when the pigs displayed significant left ventricular contractile dysfunction, we administered, by retrograde coronary venous delivery, AAV serotype 9 (AAV9)-S100A1 to the left ventricular, non-infarcted myocardium. AAV9-luciferase and saline treatment served as control. At 14 weeks, both control groups showed significantly decreased myocardial S100A1 protein expression along with progressive deterioration of cardiac performance and left ventricular remodeling. AAV9-S100A1 treatment prevented and reversed these functional and structural changes by restoring cardiac S100A1 protein levels. S100A1 treatment normalized cardiomyocyte Ca(2+) cycling, sarcoplasmic reticulum calcium handling, and energy homeostasis. Transgene expression was restricted to cardiac tissue, and extracardiac organ function was uncompromised. This translational study shows the preclinical feasibility of long-term therapeutic effectiveness of and a favorable safety profile for cardiac AAV9-S100A1 gene therapy in a preclinical model of heart failure. Our results present a strong rationale for a clinical trial of S100A1 gene therapy for human heart failure that could potentially complement current strategies to treat end-stage heart failure.

Aims: Overexpression of therapeutic genes with potential disease-limiting effects, specifically at the site of inflammation, remains a major clinical challenge. In this study, we investigate the potential of adeno-associated virus (AAV)-9-mediated cardiac expression of the anti-inflammatory mediators interleukin (IL)-10 and a dominant-negative inhibitor of monocyte chemoattractant protein-1 (MCP1-7ND) on prevention of autoimmune myocarditis., Methods and Results: Autoimmune myocarditis was induced by immunizing A/J mice with subcutaneous injection of 120 µg cardiac troponin I (cTnI) on Days 0, 7, and 14. Two weeks prior to initial immunization, each mouse received a single systemic dose of 10(12) AAV9 vectors carrying the coding sequence of IL-10 or MCP1-7ND transcriptionally targeted to the heart. Mice were sacrificed 28 days after initial immunization for further analysis. Only expression of IL-10 resulted in a highly significant decrease in myocardial inflammation and fibrosis, as well as an increased ejection fraction compared with controls. Further analyses of cytokine profiles of cTnI-stimulated splenocytes from IL-10 and MCP1-7ND-treated mice revealed significant alterations compared with controls. In addition, transcript levels of chemokine receptor CCR4 and T-cell activation gene were significantly reduced in hearts of IL-10-treated mice as determined by quantitative real-time PCR., Conclusion: Our study suggests that cardiac expression of IL-10 with AAV9 vectors is a promising therapeutic approach for autoimmune myocarditis.

Background: We sought to determine the performance of the new high sensitivity cardiac troponin T assay (TnThs) for early diagnosis of myocardial infarction in patients with suspected acute coronary syndrome (ACS) and compare it with the fourth generation cTnT assay, myoglobin and heart-type fatty acid binding protein (h-FABP)., Methods: Ninety-four patients with diagnosis of suspected ACS without ST-segment elevation admitted to our chest pain unit were included. Patients were divided according to time from onset of symptoms to presentation into an early presenter group (<4 h) and a late presenter group (≥4 h). A median of six samples (range 2-8) were available per patient. The diagnostic performance of TnThs was assessed using ROC analysis. Areas under the curve (AUC) of baseline and follow-up results of TnThs, cTnT, myoglobin, and h-FABP were compared using c statistics., Results: The TnThs assay allows an excellent prediction of non-ST-segment elevation myocardial infarction (non-STEMI) at presentation, particularly among late presenters. A follow-up sample improves diagnostic performance in a time-dependent manner. The AUC of TnThs was superior to cTnT at all time points. The performance of TnThs was at least as good as myoglobin and h-FABP at presentation and during follow-up., Conclusions: A baseline sample of TnThs allows an earlier prediction of non-STEMI than the less sensitive and precise fourth generation cTnT assay. Probably, this excellent performance of TnThs at baseline and follow-up could obviate the need for other early markers of necrosis in future.