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Introduction Pulmonary hypertension (PH) is a chronic disease characterized by persistently increases pulmonary arterial pressure, leading on the long term to right ventricular overload and ultimately right heart failure. Until now, therapy focuses on alleviating symptoms and hence the development of a novel strategy for the treatment of this disease remains highly relevant. Purpose The importance of pro-inflammatory cytokines in progression of PH has already been established. Interestingly, patients with increased plasma levels of inflammatory markers present with more severe disease. Regnase-1 is a newly identified endoribonuclease cleaving the mRNA of pro-inflammatory cytokines therefore diminishing their levels of expression. Hence, we hypothesize that regnase-1 overexpression in lung tissue can be translated into a novel therapeutic approach for PH. Methods In vitro experiments were performed in human lung endothelial cells (HUVECs). For our in vivo study, mice were subjected to hypoxic conditions (10% O2) for 3 weeks. Regnase-1 overexpression was achieved in lung endothelial cells by transduction with targeted adeno-associated virus serotype 2 (AAV2-ESGHGYF), administered systemically by tail vein injection 3 days after exposure to hypoxia (early therapy approach). AAV2-EGFP treated mice served as controls. Right ventricular function was monitored by echocardiography and right ventricular systolic pressure was measured by right heart catheterization. The degree of pro-inflammatory cell infiltration was monitored by immunohistochemistry. Induction of fetal gene programme in the right ventricle was measured by real time qPCR. Fibrosis deposition in lung tissue was determined by Sirius Red staining of frozen sections. Results Regnase-1 overexpression led to a significantly decreased level of hypoxia-induced pro-inflammatory cytokines in HUVECs. Importantly, we could show decreased regnase-1 levels in lung endothelial cells of mice subjected to hypoxia for 3 weeks. On the other hand, regnase-1 reintroduction led to a significant improvement in right ventricular function and decreased pulmonary pressure in mice placed under hypoxic conditions. Moreover, the described gene therapy approach induced normalization of fetal gene programme in the cardiac tissue and reduced pro-inflammatory cell infiltration in lungs. Additionally, we could determine mitigation of pulmonary extracellular matrix deposition and fibrosis development in mice receiving AAV2-regnase1 as compared to controls. Conclusion AAV-mediated regnase-1 overexpression in lung endothelial cells results in amelioration of pathological events leading to PH in mice subjected to hypoxia, when the therapeutic AAV is delivered 3 days after disease induction. Further experiments will determine whether this method can be successful used for reversing already established PH. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): DZHK

Background Imbalances in autophagy are known to have either protective or detrimental effects on cardiac function. Although there are significant insights into the early steps of the autophagy pathway, the mechanisms of autophagosome-lysosome transport and fusion in cardiomyocytes are less understood. Fyco1 is highly expressed in the heart and facilitates transport of autophagosomes in cardiomyocytes, enhancing global autophagic flux. Purpose The aim of our study was to investigate the effects of FYCO1 overexpression post myocardial infarction. Our hypothesis is that FYCO1 overexpression leads to enhanced autophagic flux and hence blunted cardiac remodeling and improved heart function. Methods FYCO1-Tg mice were crossbred with RFP-EGFP-LC3-Tg reporter mice (FYCO1-TGxRGF) to enable the analysis of autophagic flux using confocal microscopy. Myocardial ischemia was induced by permanent ligation of left anterior descending artery (LAD). Mice were analyzed 3 days and 30 days post LAD ligation. Cardiac function (ejection fraction, EF) and left ventricular (LV) mass were measured by echocardiography. Myocardial fibrosis, Midline Infarct size (%) and LV circumference were assessed in Masson's Trichrome stainings. Autophagy activaty was analyzed by Western blot for LC3 II and LC3 II/LC3 I. Furthermore, Evans blue/TTC staining was performed to assess the area at risk and to define perfused, ischemic and necrotic area post LAD. Results In FYCO1-TGxRGFP mice 3 days post LAD we observed an overall increased autophagic flux demonstrated by significantly increased LC3 II protein levels (WT sham: 1±0,3; WT LAD: 1,3±0,2; TG sham: 10,8±1,2; TG LAD: 8,8±0,9) and LC3 II/LC3 I ratios (WT sham: 1±0,3; WT LAD: 0,99±0,1; TG sham: 10,5±1,2; TG LAD: 5,6±1) post LAD. Moreover, using confocal microscopy revealed a shift in autophagosomal transport towards plus-end-directed transport, shown by peripheral accumulation of autophagosomes (WT: 1,4±0,8; TG: 46,6±5,8) and increased levels of autolysosomes (WT: 79,2±19,2; TG: 181,1±36,7). In Evans blue/TTC staining, FYCO1-TGxRGFP mice demonstrated comparable ischemic but markedly decreased necrotic area (ischemic: 1,9±1,8%; necrotic: 37,1±11,5%) within the LV as compared to WT (ischemic: 2,1±1,9%; necrotic: 53,4±31,4%). Fibrosis in LV (WT: 19,6±1,8%; TG: 10,6±1,6%) and infarct size (WT: 34,5±3,9%; TG: 5,5±2%) were found significantly decreased in FYCO1-TGxRGFP-mice. FYCO1-TGxRGFP mice presented with increased LV mass (WT: 106,2±25,5 mg; TG: 114,2±38,3mg) but comparable LV circumference/tibia length (WT: 11,1±0,9; TG: 11,4±1,5). Importantly, global heart function of FYCO1-TGxRGFP-mice was significantly improved post LAD compared to WT (EF WT: 36,7±6,3%; TG: 46,3±18,3%). Conclusion Our study demonstrates that the activation of autophagy via FYCO1 overexpression protects against adverse cardiac remodeling and improves cardiac function post myocardial ischemia. These findings may thus offer translational potential in the treatment of myocardial infarction. Funding Acknowledgement Type of funding sources: None.

Background We have found L-2-hydroxyglutarate dehydrogenase (L2HGDH) to be downregulated in the myocardium of mice subjected to transverse aortic constriction (TAC). L2HGDH is an important regulator of mitochondrial bioenergetics by catalyzing the conversion of L-2-hydroxyglutarate (L2-HG) to α-ketoglutarate. However, the connection between L2-HG accumulation and heart failure is not yet understood. Purpose Purpose of our study was to investigate the role of increased L2-HG levels in heart failure and the potential role of L2HGDH overexpression as therapeutic strategy. Methods For in vitro studies, primary rat neonatal cardiomyocytes (NRVCMs) were incubated with L2-HG. L2HGDH was overexpressed using adeno-associated virus (AAV) 6 vectors. Mitochondrial membrane potential was measured using TMRE (tetramethylrhodamine ethyl ester) dye. Mitochondrial reactive oxygen species production was monitored using MitoSOX. We further determined activation of fetal gene program by real time qPCR and macrophage migration using RAW 264.7 cells and transwell inserts. mTOR activation was analyzed by Western blot with antibodies against phosphorylated mTOR and ribosomal protein S6. AAV9 expressing L2HGDH or luciferase was injected in C57BL/6N mice two weeks prior to TAC and heart function was monitored by echocardiography for 6 weeks. Results L2-HG acts as a pro-hypertrophic stimulus in NRVCMs as shown by upregulation of a fetal gene expression pattern and an increase in cardiomyocyte cross-sectional area upon L2-HG treatment. Furthermore, mRNA levels of macrophage chemoattractant protein 1 were increased in L2-HG treated cells, which correlated with enhanced macrophage migration towards supernatant of L2-HG treated NRVCMs. Furthermore, we could confirm that L2-HG augmented mTOR signaling by affecting the phosphorylation status of ribosomal protein S6. AAV-mediated L2HGDH overexpression in NRVCMs led to a significant 2.1-fold decrease in the accumulation of ROS production. Moreover, we found an inhibition of endothelin-1 induced mitochondrial membrane depolarization in AAV6-L2HGDH transduced cells. Pretreatment of mice with AAV9-L2HGDH prior to TAC resulted in significantly reduced heart weight to tibia length ratios (HW/TL) and cardiomyocyte area. Importantly, heart function was notably improved in mice receiving gene therapy (ejection fraction, EF: 36.18±6.63%, fractional shortening, FS: 16.72±4.01%) whereas control animals showed marked decline in myocardial contractility (EF: 20.14±8.24%, FS: 12.66±6.66%). Conclusion L2-HG causes cardiomyocyte dysfunction by activating mTOR signaling pathway, a well-characterized critical inducer of myocyte hypertrophy, and enhancing macrophage migration, leading to establishment of a pro-inflammatory environment in the myocardium. Moreover, our results point out towards a novel preventive approach for cardiac hypertrophy and heart failure by cardiomyocyte-specific L2HGDH overexpression. Acknowledgement/Funding DZHK (Deutsches Zentrum für Herz-Kreislaufforschung)

Objective: In this study, using a Yeast two-hybrid screen, we identified the HECT domain-containing E3 ubiquitin ligase 3 (HECTD3) as one of the novel cardiac binding partners of SUMO2. Present study thus focusses on deciphering the cardiac function of HECTD3. Methods and Results: HECTD3-SUMO2 interaction was further validated by co-immunoprecipitation analysis. Interesting, adenovirus-mediated overexpression of HECTD3 in neonatal rat ventricular cardiomyocytes (NRVCMs) resulted in downregulation of endogenous as well as overexpressed SUMO2. Proteasome inhibitor MG132 treatment however inhibited HECTD3-mediated SUMO2 downregulation, suggesting SUMO2 as a likely substrate of HECTD3 in ubiquitin-proteasome system dependent protein degradation. To get further insights into cardiac substrates of HECTD3, we performed mass-spectrometry analysis of NRVCM protein lysate. Interestingly, several proteins from interferon signaling, including signal transducer and activator of transcription-1 (STAT1), were significantly downregulated by HECTD3 overexpression. Functional dissection of HECTD3-SUMO2-STAT1 interaction revealed attenuation of cellular hypertrophy and LPS mediated activation of inflammatory cascade in NRVCMs. Importantly, in vitro findings were consistent in vivo , where, AAV-9 mediated overexpression of HECTD3 substantially reduced cardiac hypertrophy and fibrosis, inhibited activation of STAT1-mediated downstream signaling, and attenuated infiltration of inflammatory cells to the heart after transverse-aortic constriction or Angiotensin II treatment. Conclusions: In conclusion, we describe here a novel cardioprotective mechanism involving the ubiquitin ligase HECTD3, which exerts anti-hypertrophic and anti-inflammatory effects via dual regulation of SUMO2 and its sumoylation target STAT1. This “dual pathway” inhibition may be exploited for the prevention and/or therapy of cardiac hypertrophy and heart failure.

Application of molecular genetic tools to inherited cardiovascular disorders has provided important insights into the molecular mechanisms underlying cardiomyopathies, arrhythmias, blood pressure regulation, and atherosclerosis. In addition, alteration of gene expression has been observed under common cardiovascular conditions such as cardiac hypertrophy and heart failure. Recent advances in transgenic and gene-targeting approaches allow a sophisticated manipulation of the mouse genome by gene addition, gene deletion, or gene modifications. These transgenic models enable the dissection of in vivo pathways responsible for these complex disease phenotypes. This review describes tissue-specific promoters suitable for targeting candidate genes to the cardiovascular system as well as a number of valuable transgenic animal models of blood pressure regulation, atherogenesis, defects in the coagulation system, cardiac hypertrophy, myocarditis, cardiomyopathies, and heart failure. Limitations and difficulties associated with these transgenic approaches are discussed. Animal models which may provide a basis for future gene therapy of cardiovascular diseases are introduced. Finally, methods are described to regulate the spatial and temporal expression level of a transgene, to inactivate a target gene in a tissue-specific manner, and to introduce specific mutations into the genome. These recent advances in transgenic technology are expected to have a considerable impact on cardiovascular research in the near future.

Background: Desmin is a type III intermediate filament which plays a central role in maintaining the structural integrity of cardiac and skeletal muscle cells. Mutations in the desmin coding gene are associated with a heterogeneous phenotype involving cardiomyopathies, cardiac conduction disease and myopathy. Desminopathies are most commonly diagnosed in adults in their mid 30s and show a progressive course, with cardiac involvement as the most important factor limiting prognosis. As of today, only symptomatic treatment is available for patients. Similar to patients with distinct defects in the desmin gene, knockout mice develop dilative cardiomyopathy compared to wildtype littermates. Aim of our study was to determine whether an adeno-associated-viral vector (AAV) mediated gene transfer of the intact desmin cDNA can either prevent or slow-down the development of cardiomyopathy in desmin-deficient (des-KO) mice. Methods: murine desmin cDNA under the control of the TNNT2 promoter was packaged into AAV serotype 9 capsids and were intravenously injected into adult des-KO mice. AAV9-luciferase vectors were used as controls. Left ventricular function was monitored using echocardiography before vector application and 3 months, 6 months and 9 months after treatment. After left heart catheterisation to assess pressure-volume (PV)-loops mice were sacrificed after 10 months for histological and molecular analyses. Results: AAV9-mediated transfer of the desmin cDNA resulted in an efficient reconstitution of desmin with intact intercalated discs. Echocardiographic analyzes revealed a fractional shortening of 38 + 4% (n=10) in desmin deficient mice treated with desmin cDNA compared to 28 + 4% (n=9) in AAV9-luciferase control-treated mice (1-way ANOVA, p < 0.05). PV-loops showed significant improvement in cardiac contractility with increased dp/dtmax in AAV9-desmin-treated mice 7742 + 539 mmHg/sec (n=7) compared to luciferase-treated mice 5901 + 466 mmHg/sec (n=9) suggesting an amelioration of the cardiomyopathy phenotype in AAV9-desmin-treated mice. Conclusion: Our study establishes an approach to specifically treat desmin-related cardiomyopathy by targeting gene expression into the myocardium through systemic application of AAV vectors.

Background: Smooth muscle cells (SMC) are a preferential target for gene therapeutic approaches in atherosclerosis and restenosis. However, the undesirable expression of putative therapeutic genes in tissues other than the vascular wall is a considerable safety limitation for clinical trials, thus requiring the identification of a smooth-muscle-specific promoter sequence. Since the 2.3 kb rabbit Smooth Muscle Myosin Heavy Chain (SMHC) promoter was shown to be transcriptionally active in primary vascular but not visceral or other non-SMC in vitro, this fragment was chosen for in vivo analysis. Methods and Results: Transgenic mice and rabbits were established expressing a luciferase reporter gene under control of the 2.3 kb rabbit SMHC promoter. In contrast to the endogenous expression pattern of the SMHC gene both species revealed light emission predominantly in the arterial system including coronary arteries. Low activities were measured in large veins and the gastrointestinal system. In situ hybridization of murine embryos using a luciferase riboprobe confirmed reporter gene expression in large arteries with no detectable mRNA in the viscera. Unlike adult animals, ectopic luciferase activities were found in ventricular myocardium during murine development ceasing 1 week post partum. Conclusions: In two animal species, the 2.3 kb SMHC promoter appeared to be effective in discriminating between the pathways regulating vascular and visceral smooth muscle gene expression. The vascular-specific expression profile of the 2.3 kb SMHC promoter suggests that the 2.3 kb SMHC promoter contains the regulatory elements necessary for selective gene targeting into vascular SMC of large arteries including coronary arteries in vivo.