S100A1's single cysteine is an indispensable redox switch for the protection against diastolic calcium waves in cardiomyocytes.

The EF-hand calcium (Ca2+) sensor protein S100A1 combines inotropic with antiarrhythmic potency in cardiomyocytes (CMs). Oxidative posttranslational modification (ox-PTM) of S100A1's conserved, single-cysteine residue (C85) via reactive nitrogen species (i.e., S-nitrosylation or S-glutathionylation) has been proposed to modulate conformational flexibility of intrinsically disordered sequence fragments and to increase the molecule's affinity toward Ca2+. Considering the unknown biological functional consequence, we aimed to determine the impact of the C85 moiety of S100A1 as a potential redox switch. We first uncovered that S100A1 is endogenously glutathionylated in the adult heart in vivo. To prevent glutathionylation of S100A1, we generated S100A1 variants that were unresponsive to ox-PTMs. Overexpression of wild-type (WT) and C85-deficient S100A1 protein variants in isolated CM demonstrated equal inotropic potency, as shown by equally augmented Ca2+ transient amplitudes under basal conditions and ß-adrenergic receptor (ßAR) stimulation. However, in contrast, ox-PTM defective S100A1 variants failed to protect against arrhythmogenic diastolic sarcoplasmic reticulum (SR) Ca2+ waves and ryanodine receptor 2 (RyR2) hypernitrosylation during ßAR stimulation. Despite diastolic performance failure, C85-deficient S100A1 protein variants exerted similar Ca2+-dependent interaction with the RyR2 than WT-S100A1. Dissecting S100A1's molecular structure-function relationship, our data indicate for the first time that the conserved C85 residue potentially acts as a redox switch that is indispensable for S100A1's antiarrhythmic but not its inotropic potency in CMs. We, therefore, propose a model where C85's ox-PTM determines S100A1's ability to beneficially control diastolic but not systolic RyR2 activity.NEW & NOTEWORTHY S100A1 is an emerging candidate for future gene-therapy treatment of human chronic heart failure. We aimed to study the significance of the conserved single-cysteine 85 (C85) residue in cardiomyocytes. We show that S100A1 is endogenously glutathionylated in the heart and demonstrate that this is dispensable to increase systolic Ca2+ transients, but indispensable for mediating S100A1's protection against sarcoplasmic reticulum (SR) Ca2+ waves, which was dependent on the ryanodine receptor 2 (RyR2) nitrosylation status.

Inhibition of soluble epoxide hydrolase prevents diabetic retinopathy.

Diabetic retinopathy is an important cause of blindness in adults, and is characterized by progressive loss of vascular cells and slow dissolution of inter-vascular junctions, which result in vascular leakage and retinal oedema. Later stages of the disease are characterized by inflammatory cell infiltration, tissue destruction and neovascularization. Here we identify soluble epoxide hydrolase (sEH) as a key enzyme that initiates pericyte loss and breakdown of endothelial barrier function by generating the diol 19,20-dihydroxydocosapentaenoic acid, derived from docosahexaenoic acid. The expression of sEH and the accumulation of 19,20-dihydroxydocosapentaenoic acid were increased in diabetic mouse retinas and in the retinas and vitreous humour of patients with diabetes. Mechanistically, the diol targeted the cell membrane to alter the localization of cholesterol-binding proteins, and prevented the association of presenilin 1 with N-cadherin and VE-cadherin, thereby compromising pericyte-endothelial cell interactions and inter-endothelial cell junctions. Treating diabetic mice with a specific sEH inhibitor prevented the pericyte loss and vascular permeability that are characteristic of non-proliferative diabetic retinopathy. Conversely, overexpression of sEH in the retinal Müller glial cells of non-diabetic mice resulted in similar vessel abnormalities to those seen in diabetic mice with retinopathy. Thus, increased expression of sEH is a key determinant in the pathogenesis of diabetic retinopathy, and inhibition of sEH can prevent progression of the disease.

RGS3L allows for an M2 muscarinic receptor-mediated RhoA-dependent inotropy in cardiomyocytes.

The role and outcome of the muscarinic M2 acetylcholine receptor (M2R) signaling in healthy and diseased cardiomyocytes is still a matter of debate. Here, we report that the long isoform of the regulator of G protein signaling 3 (RGS3L) functions as a switch in the muscarinic signaling, most likely of the M2R, in primary cardiomyocytes. High levels of RGS3L, as found in heart failure, redirect the Gi-mediated Rac1 activation into a Gi-mediated RhoA/ROCK activation. Functionally, this switch resulted in a reduced production of reactive oxygen species (- 50%) in cardiomyocytes and an inotropic response (+ 18%) in transduced engineered heart tissues. Importantly, we could show that an adeno-associated virus 9-mediated overexpression of RGS3L in rats in vivo, increased the contractility of ventricular strips by maximally about twofold. Mechanistically, we demonstrate that this switch is mediated by a complex formation of RGS3L with the GTPase-activating protein p190RhoGAP, which balances the activity of RhoA and Rac1 by altering its substrate preference in cardiomyocytes. Enhancement of this complex formation could open new possibilities in the regulation of the contractility of the diseased heart.

Secretome Analysis of Cardiomyocytes Identifies PCSK6 (Proprotein Convertase Subtilisin/Kexin Type 6) as a Novel Player in Cardiac Remodeling After Myocardial Infarction.

BACKGROUND: Acute occlusion of a coronary artery results in swift tissue necrosis. Bordering areas of the infarcted myocardium can also experience impaired blood supply and reduced oxygen delivery, leading to altered metabolic and mechanical processes. Although transcriptional changes in hypoxic cardiomyocytes are well studied, little is known about the proteins that are actively secreted from these cells. METHODS: We established a novel secretome analysis of cardiomyocytes by combining stable isotope labeling and click chemistry with subsequent mass spectrometry analysis. Further functional validation experiments included ELISA measurement of human samples, murine left anterior descending coronary artery ligation, and adeno-associated virus 9-mediated in vivo overexpression in mice. RESULTS: The presented approach is feasible for analysis of the secretome of primary cardiomyocytes without serum starvation. A total of 1026 proteins were identified to be secreted within 24 hours, indicating a 5-fold increase in detection compared with former approaches. Among them, a variety of proteins have not yet been explored in the context of cardiovascular pathologies. One of the secreted factors most strongly upregulated upon hypoxia was PCSK6 (proprotein convertase subtilisin/kexin type 6). Validation experiments revealed an increase of PCSK6 on mRNA and protein level in hypoxic cardiomyocytes. PCSK6 expression was elevated in hearts of mice after 3 days of ligation of the left anterior descending artery, a finding confirmed by immunohistochemistry. ELISA measurements in human serum also indicate distinct kinetics for PCSK6 in patients with acute myocardial infarction, with a peak on postinfarction day 3. Transfer of PCSK6-depleted cardiomyocyte secretome resulted in decreased expression of collagen I and III in fibroblasts compared with control treated cells, and small interfering RNA-mediated knockdown of PCSK6 in cardiomyocytes impacted transforming growth factor-ß activation and SMAD3 (mothers against decapentaplegic homolog 3) translocation in fibroblasts. An adeno-associated virus 9-mediated, cardiomyocyte-specific overexpression of PCSK6 in mice resulted in increased collagen expression and cardiac fibrosis, as well as decreased left ventricular function, after myocardial infarction. CONCLUSIONS: A novel mass spectrometry-based approach allows investigation of the secretome of primary cardiomyocytes. Analysis of hypoxia-induced secretion led to the identification of PCSK6 as being crucially involved in cardiac remodeling after acute myocardial infarction.

Development of an AAV9-RNAi-mediated silencing strategy to abrogate TRPM4 expression in the adult heart.

The cation channel transient receptor potential melastatin 4 (TRPM4) is a calcium-activated non-selective cation channel and acts in cardiomyocytes as a negative modulator of the L-type Ca2+ influx. Global deletion of TRPM4 in the mouse led to increased cardiac contractility under ß-adrenergic stimulation. Consequently, cardiomyocyte-specific inactivation of the TRPM4 function appears to be a promising strategy to improve cardiac contractility in heart failure patients. The aim of this study was to develop a gene therapy approach in mice that specifically silences the expression of TRPM4 in cardiomyocytes. First, short hairpin RNAmiR30 (shRNAmiR30) sequences against the TRPM4 mRNA were screened in vitro using lentiviral transduction for a stable expression of the shRNA cassettes. Western blot analysis identified three efficient shRNAmiR30 sequences out of six, which reduced the endogenous TRPM4 protein level by up to 90 ± 6%. Subsequently, the most efficient shRNAmiR30 sequences were delivered into cardiomyocytes of adult mice using adeno-associated virus serotype 9 (AAV9)-mediated gene transfer. Initially, the AAV9 vector particles were administered via the lateral tail vein, which resulted in a downregulation of TRPM4 by 46 ± 2%. Next, various optimization steps were carried out to improve knockdown efficiency in vivo. First, the design of the expression cassette was streamlined for integration in a self-complementary AAV vector backbone for a faster expression. Compared to the application via the lateral tail vein, intravenous application via the retro-orbital sinus has the advantage that the vector solution reaches the heart directly and in a high concentration, and eventually a TRPM4 knockdown efficiency of 90 ± 7% in the heart was accomplished by this approach. By optimization of the shRNAmiR30 constructs and expression cassette as well as the route of AAV9 vector application, a 90% reduction of TRPM4 expression was achieved in the adult mouse heart. In the future, AAV9-RNAi-mediated inactivation of TRPM4 could be a promising strategy to increase cardiac contractility in preclinical animal models of acute and chronic forms of cardiac contractile failure.

AAV-mediated expression of NFAT decoy oligonucleotides protects from cardiac hypertrophy and heart failure.

Previous studies have underlined the substantial role of nuclear factor of activated T cells (NFAT) in hypertension-induced myocardial hypertrophy ultimately leading to heart failure. Here, we aimed at neutralizing four members of the NFAT family of transcription factors as a therapeutic strategy for myocardial hypertrophy transiting to heart failure through AAV-mediated cardiac expression of a RNA-based decoy oligonucleotide (dON) targeting NFATc1-c4. AAV-mediated dON expression markedly decreased endothelin-1 induced cardiomyocyte hypertrophy in vitro and resulted in efficient expression of these dONs in the heart of adult mice as evidenced by fluorescent in situ hybridization. Cardiomyocyte-specific dON expression both before and after induction of transverse aortic constriction protected mice from development of cardiac hypertrophy, cardiac remodeling, and heart failure. Singular systemic administration of AAVs enabling a cell-specific expression of dONs for selective neutralization of a given transcription factor may thus represent a novel and powerful therapeutic approach.

Critical role of RAGE and HMGB1 in inflammatory heart disease.

Autoimmune response to cardiac troponin I (TnI) induces inflammation and fibrosis in the myocardium. High-mobility group box 1 (HMGB1) is a multifunctional protein that exerts proinflammatory activity by mainly binding to receptor for advanced glycation end products (RAGE). The involvement of the HMGB1-RAGE axis in the pathogenesis of inflammatory cardiomyopathy is yet not fully understood. Using the well-established model of TnI-induced experimental autoimmune myocarditis (EAM), we demonstrated that both local and systemic HMGB1 protein expression was elevated in wild-type (wt) mice after TnI immunization. Additionally, pharmacological inhibition of HMGB1 using glycyrrhizin or anti-HMGB1 antibody reduced inflammation in hearts of TnI-immunized wt mice. Furthermore, RAGE knockout (RAGE-ko) mice immunized with TnI showed no structural or physiological signs of cardiac impairment. Moreover, cardiac overexpression of HMGB1 using adeno-associated virus (AAV) vectors induced inflammation in the hearts of both wt and RAGE-ko mice. Finally, patients with myocarditis displayed increased local and systemic HMGB1 and soluble RAGE (sRAGE) expression. Together, our study highlights that HMGB1 and its main receptor, RAGE, appear to be crucial factors in the pathogenesis of TnI-induced EAM, because inhibition of HMGB1 and ablation of RAGE suppressed inflammation in the heart. Moreover, the proinflammatory effect of HMGB1 is not necessarily dependent on RAGE only. Other receptors of HMGB1 such as Toll-like receptors (TLRs) may also be involved in disease pathogenesis. These findings could be confirmed by the clinical relevance of HMGB1 and sRAGE. Therefore, blockage of one of these molecules might represent a novel therapeutic strategy in the treatment of autoimmune myocarditis and inflammatory cardiomyopathy.

AMP-activated protein kinase α1-sensitive activation of AP-1 in cardiomyocytes.

AMP-activated protein kinase (Ampk) regulates myocardial energy metabolism and plays a crucial role in the response to cell stress. In the failing heart, an isoform shift of the predominant Ampkα2 to the Ampkα1 was observed. The present study explored possible isoform specific effects of Ampkα1 in cardiomyocytes. To this end, experiments were performed in HL-1 cardiomyocytes, as well as in Ampkα1-deficient and corresponding wild-type mice and mice following AAV9-mediated cardiac overexpression of constitutively active Ampkα1. As a result, in HL-1 cardiomyocytes, overexpression of constitutively active Ampkα1 increased the phosphorylation of Pkcζ. Constitutively active Ampkα1 further increased AP-1-dependent transcriptional activity and mRNA expression of the AP-1 target genes c-Fos, Il6 and Ncx1, effects blunted by Pkcζ silencing. In HL-1 cardiomyocytes, angiotensin-II activated AP-1, an effect blunted by silencing of Ampkα1 and Pkcζ, but not of Ampkα2. In wild-type mice, angiotensin-II infusion increased cardiac Ampkα1 and cardiac Pkcζ protein levels, as well as c-Fos, Il6 and Ncx1 mRNA expression, effects blunted in Ampkα1-deficient mice. Pressure overload by transverse aortic constriction (TAC) similarly increased cardiac Ampkα1 and Pkcζ abundance as well as c-Fos, Il6 and Ncx1 mRNA expression, effects again blunted in Ampkα1-deficient mice. AAV9-mediated cardiac overexpression of constitutively active Ampkα1 increased Pkcζ protein abundance and the mRNA expression of c-Fos, Il6 and Ncx1 in cardiac tissue. In conclusion, Ampkα1 promotes myocardial AP-1 activation in a Pkcζ-dependent manner and thus contributes to cardiac stress signaling.

Inducible cardiomyocyte-specific deletion of CaM kinase II protects from pressure overload-induced heart failure.

CaM kinase II (CaMKII) has been suggested to drive pathological cardiac remodeling and heart failure. However, the evidence provided so far is based on inhibitory strategies using chemical compounds and peptides that also exert off-target effects and followed exclusively preventive strategies. Therefore, the aim of this study was to investigate whether specific CaMKII inhibition after the onset of cardiac stress delays or reverses maladaptive cardiac remodeling and dysfunction. Combined genetic deletion of the two redundant CaMKII genes δ and γ was induced after the onset of overt heart failure as the result of pathological pressure overload induced by transverse aortic constriction (TAC). We used two different strategies to engineer an inducible cardiomyocyte-specific CaMKIIδ/CaMKIIγ double knockout mouse model (DKO): one model bases on tamoxifen-inducible mER/Cre/mER expression under control of the cardiac-specific αMHC promoter; the other strategy bases on overexpression of Cre recombinase via cardiac-specific gene transfer through adeno-associated virus (AAV9) under control of the cardiac-specific myosin light chain promoter. Both models led to a substantial deletion of CaMKII in failing hearts. To approximate the clinical situation, CaMKII deletion was induced 3 weeks after TAC surgery. In both models of DKO, the progression of cardiac dysfunction and interstitial fibrosis could be slowed down as compared to control animals. Taken together, we show for the first time that "therapeutic" CaMKII deletion after cardiac damage is sufficient to attenuate maladaptive cardiac remodeling and to reverse signs of heart failure. These data suggest that CaMKII inhibition is a promising therapeutic approach to combat heart failure.

Osteopontin is indispensible for AP1-mediated angiotensin II-related miR-21 transcription during cardiac fibrosis.

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.

Tyrosine nitration limits stretch-induced CD40 expression and disconnects CD40 signaling in human endothelial cells.

Hemodynamic forces are important effectors of endothelial cell phenotype and function. Because CD40-CD154 interactions between endothelial cells and mononuclear leukocytes or activated platelets play an important role in vascular dysfunction, we investigated the effects of cyclic stretch on CD40 expression in human cultured endothelial cells. Short-term stretch transiently up-regulated CD40 expression while long-term stretch resulted in a distinct decline in CD40 protein which was prevented by inhibition of the 20S proteasome or scavenging of peroxynitrite. Tyrosine nitration of CD40 also occurred under static conditions on addition of authentic peroxynitrite, and according to mass spectrometry analysis Tyr-82 but not Tyr-31 was its target in the native protein. Immunofluorescence analysis of endothelial cells transduced with a control or Tyr-82 to Ala mutated AAV9-CD40-eGFP expression construct confirmed a peroxynitrite-dependent redistribution of the protein from the cell membrane to the cytoplasm, which was prevented by methyl-ß-cyclodextrin. Moreover, CD154-stimulated IL-12p40 and E-selectin expression markedly decreased after exposure to authentic peroxynitrite or cyclic stretch, respectively. Coimmunoprecipitation demonstrated a decreased binding of TRAF2 and TRAF6 to the CD40 protein after tyrosine nitration. Through this posttranslational oxidative modification of an important costimulatory molecule, endothelial cells are able to quickly adapt to unfavorable hemodynamics and maintain their anti-inflammatory phenotype.

AAV-mediated AP-1 decoy oligonucleotide expression inhibits aortic elastolysis in a mouse model of Marfan syndrome.

AIMS: Marfan syndrome is one of the most common inherited disorders of connective tissue caused by fibrillin-1 mutations, characterized by enhanced transcription factor AP-1 DNA binding activity and subsequently abnormally increased expression and activity of matrix-metalloproteinases (MMPs). We aimed to establish a novel adeno-associated virus (AAV)-based strategy for long-term expression of an AP-1 neutralizing RNA hairpin (hp) decoy oligonucleotide (dON) in the aorta to prevent aortic elastolysis in a murine model of Marfan syndrome. METHODS AND RESULTS: Using fibrillin-1 hypomorphic mice (mgR/mgR), aortic grafts from young (9 weeks old) donor mgR/mgR mice were transduced ex vivo with AAV vectors and implanted as infrarenal aortic interposition grafts in mgR/mgR mice. Grafts were explanted after 30 days. For in vitro studies, isolated primary aortic smooth muscle cells (SMCs) from mgR/mgR mice were used. Elastica-van-Giesson staining visualized elastolysis, reactive oxygen species (ROS) production was assessed using dihydroethidine staining. RNA F.I.S.H. verified AP-1 hp dON generation in the ex vivo transduced aortic tissue. MMP expression and activity were assessed by western blotting and immunoprecipitation combined with zymography.Transduction resulted in stable therapeutic dON expression in endothelial and SMCs. MMP expression and activity, ROS formation as well as expression of monocyte chemoattractant protein-1 were significantly reduced. Monocyte graft infiltration declined and the integrity of the elastin architecture was maintained. RNAseq analysis confirmed the beneficial effect of AP-1 neutralization on the pro-inflammatory environment in SMCs. CONCLUSION: This novel approach protects from deterioration of aortic stability by sustained delivery of nucleic acids-based therapeutics and further elucidated how to interfere with the mechanism of elastolysis.

Preclinical evidence for the therapeutic value of TBX5 normalization in arrhythmia control.

AIMS: Arrhythmias and sudden cardiac death (SCD) occur commonly in patients with heart failure. We found T-box 5 (TBX5) dysregulated in ventricular myocardium from heart failure patients and thus we hypothesized that TBX5 reduction contributes to arrhythmia development in these patients. To understand the underlying mechanisms, we aimed to reveal the ventricular TBX5-dependent transcriptional network and further test the therapeutic potential of TBX5 level normalization in mice with documented arrhythmias. METHODS AND RESULTS: We used a mouse model of TBX5 conditional deletion in ventricular cardiomyocytes. Ventricular (v) TBX5 loss in mice resulted in mild cardiac dysfunction and arrhythmias and was associated with a high mortality rate (60%) due to SCD. Upon angiotensin stimulation, vTbx5KO mice showed exacerbated cardiac remodelling and dysfunction suggesting a cardioprotective role of TBX5. RNA-sequencing of a ventricular-specific TBX5KO mouse and TBX5 chromatin immunoprecipitation was used to dissect TBX5 transcriptional network in cardiac ventricular tissue. Overall, we identified 47 transcripts expressed under the control of TBX5, which may have contributed to the fatal arrhythmias in vTbx5KO mice. These included transcripts encoding for proteins implicated in cardiac conduction and contraction (Gja1, Kcnj5, Kcng2, Cacna1g, Chrm2), in cytoskeleton organization (Fstl4, Pdlim4, Emilin2, Cmya5), and cardiac protection upon stress (Fhl2, Gpr22, Fgf16). Interestingly, after TBX5 loss and arrhythmia development in vTbx5KO mice, TBX5 protein-level normalization by systemic adeno-associated-virus (AAV) 9 application, re-established TBX5-dependent transcriptome. Consequently, cardiac dysfunction was ameliorated and the propensity of arrhythmia occurrence was reduced. CONCLUSIONS: This study uncovers a novel cardioprotective role of TBX5 in the adult heart and provides preclinical evidence for the therapeutic value of TBX5 protein normalization in the control of arrhythmia.

Liver alanine catabolism promotes skeletal muscle atrophy and hyperglycaemia in type 2 diabetes.

Both obesity and sarcopenia are frequently associated in ageing, and together may promote the progression of related conditions such as diabetes and frailty. However, little is known about the pathophysiological mechanisms underpinning this association. Here we show that systemic alanine metabolism is linked to glycaemic control. We find that expression of alanine aminotransferases is increased in the liver in mice with obesity and diabetes, as well as in humans with type 2 diabetes. Hepatocyte-selective silencing of both alanine aminotransferase enzymes in mice with obesity and diabetes retards hyperglycaemia and reverses skeletal muscle atrophy through restoration of skeletal muscle protein synthesis. Mechanistically, liver alanine catabolism driven by chronic glucocorticoid and glucagon signalling promotes hyperglycaemia and skeletal muscle wasting. We further provide evidence for amino acid-induced metabolic cross-talk between the liver and skeletal muscle in ex vivo experiments. Taken together, we reveal a metabolic inter-tissue cross-talk that links skeletal muscle atrophy and hyperglycaemia in type 2 diabetes.

The E3 ubiquitin ligase HectD3 attenuates cardiac hypertrophy and inflammation in mice.

Myocardial inflammation has recently been recognized as a distinct feature of cardiac hypertrophy and heart failure. HectD3, a HECT domain containing E3 ubiquitin ligase has previously been investigated in the host defense against infections as well as neuroinflammation; its cardiac function however is still unknown. Here we show that HectD3 simultaneously attenuates Calcineurin-NFAT driven cardiomyocyte hypertrophy and the pro-inflammatory actions of LPS/interferon-γ via its cardiac substrates SUMO2 and Stat1, respectively. AAV9-mediated overexpression of HectD3 in mice in vivo not only reduced cardiac SUMO2/Stat1 levels and pathological hypertrophy but also largely abolished macrophage infiltration and fibrosis induced by pressure overload. Taken together, we describe a novel cardioprotective mechanism involving the ubiquitin ligase HectD3, which links anti-hypertrophic and anti-inflammatory effects via dual regulation of SUMO2 and Stat1. In a broader perspective, these findings support the notion that cardiomyocyte growth and inflammation are more intertwined than previously anticipated.

Cardiac transcriptional and metabolic changes following thoracotomy.

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.

Restriction of essential amino acids dictates the systemic metabolic response to dietary protein dilution.

Dietary protein dilution (DPD) promotes metabolic-remodelling and -health but the precise nutritional components driving this response remain elusive. Here, by mimicking amino acid (AA) supply from a casein-based diet, we demonstrate that restriction of dietary essential AA (EAA), but not non-EAA, drives the systemic metabolic response to total AA deprivation; independent from dietary carbohydrate supply. Furthermore, systemic deprivation of threonine and tryptophan, independent of total AA supply, are both adequate and necessary to confer the systemic metabolic response to both diet, and genetic AA-transport loss, driven AA restriction. Dietary threonine restriction (DTR) retards the development of obesity-associated metabolic dysfunction. Liver-derived fibroblast growth factor 21 is required for the metabolic remodelling with DTR. Strikingly, hepatocyte-selective establishment of threonine biosynthetic capacity reverses the systemic metabolic response to DTR. Taken together, our studies of mice demonstrate that the restriction of EAA are sufficient and necessary to confer the systemic metabolic effects of DPD.

AAV-Mediated Expression of AP-1-Neutralizing RNA Decoy Oligonucleotides Attenuates Transplant Vasculopathy in Mouse Aortic Allografts.

Transplant vasculopathy (TV), characterized by obstructive lesions in affected vessels, represents one of the long-term complications of cardiac transplantation. Activation of the transcription factor activator protein-1 (AP-1) is implicated in smooth muscle cell (SMC) phenotypic switch from contractile to synthetic function, increasing the migration and proliferation rate of these cells. We hypothesize that adeno-associated virus (AAV)-mediated delivery of an RNA hairpin AP-1 decoy oligonucleotide (dON) might effectively ameliorate TV severity in a mouse aortic allograft model. Aortic allografts from DBA/2 mice ex vivo transduced with modified AAV9-SLR carrying a targeting peptide within the capsid surface were transplanted into the infrarenal aorta of C57BL/6 mice. Cyclosporine A (10 mg/kg BW) was administered daily. AP-1 dONs were intracellularly expressed in the graft tissue as small hairpin RNA proved by fluorescent in situ hybridization. Explantation after 30 days and histomorphometric evaluation revealed that AP-1 dON treatment significantly reduced intima-to-media ratio by 41.5% (p < 0.05) in the grafts. In addition, expression of adhesion molecules, cytokines, as well as numbers of proliferative SMCs, matrix metalloproteinase-9-positive cells, and inflammatory cell infiltration were significantly decreased in treated aortic grafts. Our findings demonstrate the feasibility, efficacy, and specificity of the anti-AP-1 RNA dON approach for the treatment of allograft vasculopathy in an animal model. Moreover, the AAV-based approach in general provides the possibility to achieve a prolonged delivery of nucleic-acids-based therapeutics in to the blood vessel wall.

CaMKII activation participates in doxorubicin cardiotoxicity and is attenuated by moderate GRP78 overexpression.

The clinical use of the chemotherapeutic doxorubicin (Dox) is limited by cardiotoxic side-effects. One of the early Dox effects is induction of a sarcoplasmic reticulum (SR) Ca2+ leak. The chaperone Glucose regulated protein 78 (GRP78) is important for Ca2+ homeostasis in the endoplasmic reticulum (ER)-the organelle corresponding to the SR in non-cardiomyocytes-and has been shown to convey resistance to Dox in certain tumors. Our aim was to investigate the effect of cardiac GRP78 gene transfer on Ca2+ dependent signaling, cell death, cardiac function and survival in clinically relevant in vitro and in vivo models for Dox cardiotoxicity.By using neonatal cardiomyocytes we could demonstrate that Dox induced Ca2+ dependent Ca2+ /calmodulin-dependent protein kinase II (CaMKII) activation is one of the factors involved in Dox cardiotoxicity by promoting apoptosis. Furthermore, we found that adeno-associated virus (AAV) mediated GRP78 overexpression partly protects neonatal cardiomyocytes from Dox induced cell death by modulating Ca2+ dependent pathways like the activation of CaMKII, phospholamban (PLN) and p53 accumulation. Most importantly, cardiac GRP78 gene therapy in mice treated with Dox revealed improved diastolic function (dP/dtmin) and survival after Dox treatment. In conclusion, our results demonstrate for the first time that Ca2+ dependent CaMKII activation fosters Dox cardiomyopathy and provide additional insight into possible mechanisms by which GRP78 overexpression protects cardiomyocytes from Doxorubicin toxicity.

Various effects of AAV9-mediated ßARKct gene therapy on the heart in dystrophin-deficient (mdx) mice and δ-sarcoglycan-deficient (Sgcd-/-) mice.

So far effective strategies to treat cardiomyopathy in patients with muscular dystrophies are still not clearly defined. Previously, treatment with ß-blockers showed beneficial effects on the development of cardiomyopathy in dystrophin-deficient (mdx) mice, but not in δ-sarcoglycan-deficient (Sgcd-/-) mice. We therefore aimed to study a more specific approach to target maladaptive ß-adrenergic signalling in these mice. It has been shown that lowering cardiac G-protein-coupled-receptor-kinase-2 (GRK2) activity with ßARKct expression, a peptide inhibitor of protein-coupled-receptor-kinase-2 (GRK2), results in improvement of heart failure in several different animal models. We therefore investigated whether adeno-associated virus type 9 (AAV9)-mediated gene delivery of ßARKct, could ameliorate cardiac pathology in mdx and Sgcd-/- mice. We found that long-term treatment with AAV9- ßARKct-cDNA with a cardiac-specific promoter significantly improves left ventricular systolic function and reduces myocardial hypertrophy in mdx mice, whereas only mild beneficial effects on cardiac function is observed in Sgcd-/- mice. Interestingly, in contrast to mdx mice neither GRK2 nor nuclear-factor-kappaB (NFκB) were upregulated in Sgcd-/- mice. Taken together, effectiveness of AAV-mediated ßARKct therapy may vary between different genetic mutations and presumably depend on the state of adrenergic dysregulation mediated through the upregulation of GRK2.