The electrical properties of the heart are primarily determined by the activity of ion channels and the activity of these molecules is permanently modulated and adjusted to the physiological needs by adrenergic signaling. miRNAs are known to control the expression of many proteins and to fulfill distinct functions in the mammalian heart, though the in vivo effects of miRNAs on the electrical activity of the heart are poorly characterized. The miRNAs miR-1 and miR-133a are the most abundant miRNAs of the heart and are expressed from two miR-1/133a genomic clusters. Genetic modulation of miR-1/133a cluster expression without concomitant severe disturbance of general cardiomyocyte physiology revealed that these miRNA clusters govern cardiac muscle repolarization. Reduction of miR-1/133a dosage induced a longQT phenotype in mice especially at low heart rates. Longer action potentials in cardiomyocytes are caused by modulation of the impact of β-adrenergic signaling on the activity of the depolarizing L-type calcium channel. Pharmacological intervention to attenuate β-adrenergic signaling or L-type calcium channel activity in vivo abrogated the longQT phenotype that is caused by modulation of miR-1/133a activity. Thus, we identify the miR-1/133a miRNA clusters to be important to prevent a longQT-phenotype in the mammalian heart.

Pulmonary arterial hypertension (PAH) is a progressive disorder characterized by remodeling of the pulmonary vasculature and a rise in right ventricular (RV) afterload. The increased RV afterload leads to right ventricular failure (RVF) which is the reason for the high morbidity and mortality in PAH patients. The objective was to evaluate the therapeutic efficacy and antiremodeling potential of the phosphodiesterase type 5 (PDE5) inhibitor sildenafil and the soluble guanylate cyclase stimulator riociguat in a model of pressure overload RV hypertrophy induced by pulmonary artery banding (PAB). Mice subjected to PAB, one week after surgery, were treated with either sildenafil (100 mg/kg/d, n = 5), riociguat (30 mg/kg/d, n = 5), or vehicle (n = 5) for 14 days. RV function and remodeling were assessed by right heart catheterization, magnetic resonance imaging (MRI), and histomorphometry. Both sildenafil and riociguat prevented the deterioration of RV function, as determined by a decrease in RV dilation and restoration of the RV ejection fraction (EF). Although both compounds did not decrease right heart mass and cellular hypertrophy, riociguat prevented RV fibrosis induced by PAB. Both compounds diminished TGF-beta1 induced collagen synthesis of RV cardiac fibroblasts in vitro. Treatment with either riociguat or sildenafil prevented the progression of pressure overload-induced RVF, representing a novel therapeutic approach.

Aldosterone is a mineralocorticoid hormone critically involved in arterial blood pressure regulation. Although pharmacological aldosterone antagonism reduces mortality and morbidity among patients with severe left-sided heart failure, the contribution of aldosterone to the pathobiology of pulmonary arterial hypertension (PAH) and right ventricular (RV) heart failure is not fully understood.

Control of energy homeostasis and metabolism is achieved by integrating numerous pathways, and miRNAs are involved in this process by regulating expression of multiple target genes. However, relatively little is known about the posttranscriptional processing of miRNAs and a potential role for the precursors they derive from. Here, we demonstrate that mature miRNA-22 is more abundant in muscle from male mice relative to females and that this enables sex-specific regulation of muscular lipid metabolism and body weight by repressing estrogen receptor alpha (ERα) expression. We found that the ERα adjusts its own activity by preventing processing of miR-22 via direct binding to a conserved ERα-binding element within the primary miR-22 precursor. Mutation of the ERα binding site within the pri-miR-22 in vivo eliminates sex-specific differences in miR-22 expression. We reason that the resulting tissue selective negative feedback regulation is essential to establish sex-specific differences in muscle metabolism and body weight development.

The right ventricle (RV) differs in several aspects from the left ventricle (LV) including its embryonic origin, physiological role and anatomical design. In contrast to LV hypertrophy, little is known about the molecular circuits, which are activated upon RV hypertrophy (RVH). We established a highly reproducible model of RVH in mice using pulmonary artery clipping (PAC), which avoids detrimental RV pressure overload and thus allows long-term survival of operated mice. Magnetic resonance imaging revealed pathognomonic changes with striking similarities to human congenital heart disease- or pulmonary arterial hypertension-patients. Comparative, microarray based transcriptome analysis of right- and left-ventricular remodeling identified distinct transcriptional responses to pressure-induced hypertrophy of either ventricle, which were mainly characterized by stronger transcriptional responses of the RV compared to the LV myocardium. Hierarchic cluster analysis revealed a RV- and LV-specific pattern of gene activity after induction of hypertrophy, however, we did not find evidence for qualitatively distinct regulatory pathways in RV compared to LV. Data mining of nearly three thousand RV-enriched genes under PAC disclosed novel potential (co)-regulators of long-term RV remodeling and hypertrophy. We reason that specific inhibitory mechanisms in RV restrict excessive myocardial hypertrophy and thereby contribute to its vulnerability to pressure overload.

Long non-coding RNAs (lncRNAs) contribute to cardiac (patho)physiology. Aging is the major risk factor for cardiovascular disease with cardiomyocyte apoptosis as one underlying cause. Here, we report the identification of the aging-regulated lncRNA Sarrah (ENSMUST00000140003) that is anti-apoptotic in cardiomyocytes. Importantly, loss of SARRAH (OXCT1-AS1) in human engineered heart tissue results in impaired contractile force development. SARRAH directly binds to the promoters of genes downregulated after SARRAH silencing via RNA-DNA triple helix formation and cardiomyocytes lacking the triple helix forming domain of Sarrah show an increase in apoptosis. One of the direct SARRAH targets is NRF2, and restoration of NRF2 levels after SARRAH silencing partially rescues the reduction in cell viability. Overexpression of Sarrah in mice shows better recovery of cardiac contractile function after AMI compared to control mice. In summary, we identified the anti-apoptotic evolutionary conserved lncRNA Sarrah, which is downregulated by aging, as a regulator of cardiomyocyte survival.

The leading cause of death in Pulmonary Arterial Hypertension (PAH) is right ventricular (RV) failure. The tumor suppressor p53 has been associated with left ventricular hypertrophy (LVH) and remodeling but its role in RV hypertrophy (RVH) is unclear. The purpose of this study was to determine whether pharmacological activation of p53 by Quinacrine affects RV remodeling and function in the pulmonary artery banding (PAB) model of compensated RVH in mice. The effects of p53 activation on cellular functions were studied in isolated cardiomyocytes, cardiac fibroblasts and endothelial cells (ECs). The expression of p53 was examined both on human RV tissues from patients with compensated and decompensated RVH and in mouse RV tissues early and late after the PAB. As compared to control human RVs, there was no change in p53 expression in compensated RVH, while a marked upregulation was found in decompensated RVH. Similarly, in comparison to SHAM-operated mice, unaltered RV p53 expression 7 days after PAB, was markedly induced 21 days after the PAB. Quinacrine induced p53 accumulation did not further deteriorate RV function at day 7 after PAB. Quinacrine administration did not increase EC death, neither diminished EC number and capillary density in RV tissues. No major impact on the expression of markers of sarcomere organization, fatty acid and mitochondrial metabolism and respiration was noted in Quinacrine-treated PAB mice. p53 accumulation modulated the expression of Heme Oxygenase 1 (HO-1) and Glucose Transporter (Glut1) in mouse RVs and in adult cardiomyocytes. We conclude that early p53 activation in PAB-induced RVH does not cause substantial detrimental effects on right ventricular remodeling and function.

Postnatal maturation of cardiomyocytes is characterized by a metabolic switch from glycolysis to fatty acid oxidation, chromatin reconfiguration and exit from the cell cycle, instating a barrier for adult heart regeneration1,2. Here, to explore whether metabolic reprogramming can overcome this barrier and enable heart regeneration, we abrogate fatty acid oxidation in cardiomyocytes by inactivation of Cpt1b. We find that disablement of fatty acid oxidation in cardiomyocytes improves resistance to hypoxia and stimulates cardiomyocyte proliferation, allowing heart regeneration after ischaemia-reperfusion injury. Metabolic studies reveal profound changes in energy metabolism and accumulation of α-ketoglutarate in Cpt1b-mutant cardiomyocytes, leading to activation of the α-ketoglutarate-dependent lysine demethylase KDM5 (ref. 3). Activated KDM5 demethylates broad H3K4me3 domains in genes that drive cardiomyocyte maturation, lowering their transcription levels and shifting cardiomyocytes into a less mature state, thereby promoting proliferation. We conclude that metabolic maturation shapes the epigenetic landscape of cardiomyocytes, creating a roadblock for further cell divisions. Reversal of this process allows repair of damaged hearts.

Cardiac healing after myocardial ischemia depends on the recruitment and local expansion of myeloid cells, particularly macrophages. Here we identify Reg3β as an essential regulator of macrophage trafficking to the damaged heart. Using mass spectrometry-based secretome analysis, we found that dedifferentiating cardiomyocytes release Reg3β in response to the cytokine OSM, which signals through Jak1 and Stat3. Loss of Reg3β led to a large decrease in the number of macrophages in the ischemic heart, accompanied by increased ventricular dilatation and insufficient removal of neutrophils. This defect in neutrophil removal in turn caused enhanced matrix degradation, delayed collagen deposition and increased susceptibility to cardiac rupture. Our data indicate that OSM, acting through distinct intracellular pathways, regulates both cardiomyocyte dedifferentiation and cardiomyocyte-dependent regulation of macrophage trafficking. Release of OSM from infiltrating neutrophils and macrophages initiates a positive feedback loop in which OSM-induced production of Reg3β in cardiomyocytes attracts additional OSM-secreting macrophages. The activity of the feedback loop controls the degree of macrophage accumulation in the heart, which is instrumental in myocardial healing.

The extracellular matrix is crucial for organogenesis. It is a complex and dynamic component that regulates cell behavior by modulating the activity, bioavailability and presentation of growth factors to cell surface receptors. Here, we determined the role of the extracellular matrix protein Nephronectin (Npnt) in heart development using the zebrafish model system. The vertebrate heart is formed as a linear tube in which myocardium and endocardium are separated by a layer of extracellular matrix termed the cardiac jelly. During heart development, the cardiac jelly swells at the atrioventricular (AV) canal, which precedes valve formation. Here, we show that Npnt expression correlates with this process. Morpholino-mediated knockdown of Npnt prevents proper valve leaflet formation and trabeculation and results in greater than 85% lethality at 7 days post-fertilization. The earliest observed phenotype is an extended tube-like structure at the AV boundary. In addition, the expression of myocardial genes involved in cardiac valve formation (cspg2, fibulin 1, tbx2b, bmp4) is expanded and endocardial cells along the extended tube-like structure exhibit characteristics of AV cells (has2, notch1b and Alcam expression, cuboidal cell shape). Inhibition of has2 in npnt morphants rescues the endocardial, but not the myocardial, expansion. By contrast, reduction of BMP signaling in npnt morphants reduces the ectopic expression of myocardial and endocardial AV markers. Taken together, our results identify Npnt as a novel upstream regulator of Bmp4-Has2 signaling that plays a crucial role in AV canal differentiation.

Mouse models of myocardial infarction (MI) are commonly used to explore the pathophysiological role of the monocytic response in myocardial injury and to develop translational strategies. However, no study thus far has examined the potential impact of inter-individual variability and sham surgical procedures on monocyte subset kinetics after experimental MI in mice. Our goal was to investigate determinants of systemic myeloid cell subset shifts in C57BL/6 mice following MI by developing a protocol for sequential extensive flow cytometry (FCM).

Myogenic vasoconstriction is an autoregulatory function of small arteries. Recently, G-protein-coupled receptors have been involved in myogenic vasoconstriction, but the downstream signalling mechanisms and the in-vivo-function of this myogenic autoregulation are poorly understood. Here, we show that small arteries from mice with smooth muscle-specific loss of G12/G13 or the Rho guanine nucleotide exchange factor ARHGEF12 have lost myogenic vasoconstriction. This defect was accompanied by loss of RhoA activation, while vessels showed normal increases in intracellular [Ca2+]. In the absence of myogenic vasoconstriction, perfusion of peripheral organs was increased, systemic vascular resistance was reduced and cardiac output and left ventricular mass were increased. In addition, animals with defective myogenic vasoconstriction showed aggravated hypotension in response to endotoxin. We conclude that G12/G13- and Rho-mediated signaling plays a key role in myogenic vasoconstriction and that myogenic tone is required to maintain local and systemic vascular resistance under physiological and pathological condition.

Oncostatin M (OSM) and leukemia inhibitory factor (LIF) signaling protects the heart after myocardial infarction (MI). In mice, oncostatin M receptor (OSMR) and leukemia inhibitory factor receptor (LIFR) are selectively activated by the respective cognate ligands while OSM activates both the OSMR and LIFR in humans, which prevents efficient translation of mouse data into potential clinical applications. We used an engineered human-like OSM (hlOSM) protein, capable to signal via both OSMR and LIFR, to evaluate beneficial effects on cardiomyocytes and hearts after MI in comparison to selective stimulation of either LIFR or OSMR. Cell viability assays, transcriptome and immunoblot analysis revealed increased survival of hypoxic cardiomyocytes by mLIF, mOSM and hlOSM stimulation, associated with increased activation of STAT3. Kinetic expression profiling of infarcted hearts further specified a transient increase of OSM and LIF during the early inflammatory phase of cardiac remodeling. A post-infarction delivery of hlOSM but not mOSM or mLIF within this time period combined with cardiac magnetic resonance imaging-based strain analysis uncovered a global cardioprotective effect on infarcted hearts. Our data conclusively suggest that a simultaneous and rapid activation of OSMR and LIFR after MI offers a therapeutic opportunity to preserve functional and structural integrity of the infarcted heart.

Pulmonary hypertension (PH) is characterized by a progressive elevation of mean arterial pressure followed by right ventricular failure and death. Previous studies have indicated that numerous inhibitors of receptor tyrosine kinase signaling could be either beneficial or detrimental for the treatment of PH. Here we investigated the therapeutic potential of the multi-kinase inhibitor regorafenib (BAY 73-4506) for the treatment of PH. A peptide-based kinase activity assay was performed using the PamStation®12 platform. The 5-bromo-2'-deoxyuridine proliferation and transwell migration assays were utilized in pulmonary arterial smooth muscle cells (PASMCs). Regorafenib was administered to monocrotaline- and hypoxia-induced PH in rats and mice, respectively. Functional parameters were analyzed by hemodynamic and echocardiographic measurements. The kinase activity assay revealed upregulation of twenty-nine kinases in PASMCs from patients with idiopathic PAH (IPAH), of which fifteen were established as potential targets of regorafenib. Regorafenib showed strong anti-proliferative and anti-migratory effects in IPAH-PASMCs compared to the control PASMCs. Both experimental models indicated improved cardiac function and reduced pulmonary vascular remodeling upon regorafenib treatment. In lungs from monocrotaline (MCT) rats, regorafenib reduced the phosphorylation of c-Jun N-terminal kinase and extracellular signal-regulated kinase 1/2. Overall, our data indicated that regorafenib plays a beneficial role in experimental PH.

Mammalian models have been instrumental in investigating adult heart function and human disease. However, electrophysiological differences with human hearts and high costs motivate the need for non-mammalian models. The zebrafish is a well-established genetic model to study cardiovascular development and function; however, analysis of cardiovascular phenotypes in adult specimens is particularly challenging as they are opaque.

The serotonin (5-HT) pathway was shown to play a role in pulmonary hypertension (PH), but its functions in right ventricular failure (RVF) remain poorly understood. The aim of the current study was to investigate the effects of Terguride (5-HT2A and 2B receptor antagonist) or SB204741 (5-HT2B receptor antagonist) on right heart function and structure upon pulmonary artery banding (PAB) in mice.

Chromatin modifying enzymes play a critical role in cardiac differentiation. Previously, it has been shown that the targeted deletion of the histone methyltransferase, Smyd1, the founding member of the SET and MYND domain containing (Smyd) family, interferes with cardiomyocyte maturation and proper formation of the right heart ventricle. The highly related paralogue, Smyd2 is a histone 3 lysine 4- and lysine 36-specific methyltransferase expressed in heart and brain. Here, we report that Smyd2 is differentially expressed during cardiac development with highest expression in the neonatal heart. To elucidate the functional role of Smyd2 in the heart, we generated conditional knockout (cKO) mice harboring a cardiomyocyte-specific deletion of Smyd2 and performed histological, functional and molecular analyses. Unexpectedly, cardiac deletion of Smyd2 was dispensable for proper morphological and functional development of the murine heart and had no effect on global histone 3 lysine 4 or 36 methylation. However, we provide evidence for a potential role of Smyd2 in the transcriptional regulation of genes associated with translation and reveal that Smyd2, similar to Smyd3, interacts with RNA Polymerase II as well as to the RNA helicase, HELZ.

Recent studies indicate that tumor-associated macrophages (MΦ) with an M2 phenotype can influence cancer progression and metastasis, but the regulatory pathways remain poorly characterized.

Right ventricular (RV) remodeling represents a complex set of functional and structural adaptations in response to chronic pressure or volume overload due to various inborn defects or acquired diseases and is an important determinant of patient outcome. However, the underlying molecular mechanisms remain elusive. We investigated the time course of structural and functional changes in the RV in the murine model of pressure overload-induced RV hypertrophy in C57Bl/6J mice. Using magnetic resonance imaging, we assessed the changes of RV structure and function at different time points for a period of 21 days. Pressure overload led to significant dilatation, cellular and chamber hypertrophy, myocardial fibrosis, and functional impairment of the RV Progressive remodeling of the RV after pulmonary artery banding (PAB) in mice was associated with upregulation of myocardial gene markers of hypertrophy and fibrosis. Furthermore, remodeling of the RV was associated with accumulation and activation of mast cells in the RV tissue of PAB mice. Our data suggest possible involvement of mast cells in the RV remodeling process in response to pressure overload. Mast cells may thus represent an interesting target for the development of new therapeutic approaches directed specifically at the RV.

GPR116 (ADGRF5) and ELTD1 (ADGRL4) belong to different subfamilies of the adhesion G-protein-coupled receptor group but are both expressed in endothelial cells. We therefore analyzed their functions in mice lacking these receptors. While loss of GPR116 or ELTD1 alone had no obvious effect on cardiovascular or kidney function, mice lacking both, GPR116 and ELTD1, showed malformations of the aortic arch arteries and the cardiac outflow tract leading to perinatal lethality in about 50% of the mutants. In addition to cardiovascular malformations, surviving mice developed renal thrombotic microangiopathy as well as hemolysis and splenomegaly, and their lifespan was significantly reduced. Loss of GPR116 and ELTD1 specifically in endothelial cells or neural crest-derived cells did not recapitulate any of the phenotypes observed in GPR116-ELTD1 double deficient mice, indicating that loss of GPR116 and ELTD1 expressed by other cells accounts for the observed cardiovascular and renal defects.