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.

The transcriptional regulator JDP2 (Jun dimerization protein 2) has been identified as a prognostic marker for patients to develop heart failure after myocardial infarction. We now performed in vivo studies on JDP2-overexpressing mice, to clarify the impact of JDP2 on heart failure progression. Therefore, during birth up to the age of 4 weeks cardiac-specific JDP2 overexpression was prevented by doxycycline feeding in transgenic mice. Then, JDP2 overexpression was started. Already after 1 week, cardiac function, determined by echocardiography, decreased which was also resembled on the cardiomyocyte level. After 5 weeks blood pressure declined, ejection fraction and cardiac output was reduced and left ventricular dilatation developed. Heart weight/body weight, and mRNA expression of ANP, inflammatory marker genes, collagen and fibronectin increased. Collagen 1 protein expression increased, and fibrosis developed. As an additional sign of elevated extracellular matrix remodeling, matrix metalloproteinase 2 activity increased in JDP2 mice. Thus, JDP2 overexpression is deleterious to heart function in vivo. It can be concluded that JDP2 overexpression provokes cardiac dysfunction in adult mice that is accompanied by hypertrophy and fibrosis. Thus, induction of JDP2 is a maladaptive response contributing to heart failure development.

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.

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.

Mitochondrial biogenesis and adequate energy production in various organs of mammals are necessary for postnatal adaptation to extrauterine life in an environment with high oxygen content. Even though transgenic mice are frequently used as experimental models, to date, no combined detailed molecular and morphological analysis on the mitochondrial compartment in different lung cell types has been performed during postnatal mouse lung development. In our study, we revealed a significant upregulation of most mitochondrial respiratory complexes at protein and mRNA levels in the lungs of P15 and adult animals in comparison to newborns. The majority of adult animal samples showed the strongest increase, except for succinate dehydrogenase protein (SDHD). Likewise, an increase in mRNA expression for mtDNA transcription machinery genes (Polrmt, Tfam, Tfb1m, and Tfb2m), mitochondrially encoded RNA (mt-Rnr1 and mt-Rnr2), and the nuclear-encoded mitochondrial DNA polymerase (POLG) was observed. The biochemical and molecular results were corroborated by a parallel increase of mitochondrial number, size, cristae number, and complexity, exhibiting heterogeneous patterns in distinct bronchiolar and alveolar epithelial cells. Taken together, our results suggest a specific adaptation and differential maturation of the mitochondrial compartment according to the metabolic needs of individual cell types during postnatal development of the mouse lung.

The cytoskeletal signaling protein four and-a-half LIM domains 1 (FHL-1) has recently been identified as a novel key player in pulmonary hypertension as well as in left heart diseases. In this regard, FHL-1 has been implicated in dysregulated hypertrophic signaling in pulmonary arterial smooth muscle cells leading to pulmonary hypertension. In mice, FHL-1-deficiency (FHL-1-/-) led to an attenuated hypertrophic signaling associated with a blunted hypertrophic response of the pressure-overloaded left ventricle (LV). However, the role of FHL-1 in right heart hypertrophy has not yet been addressed.

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.

Among patients meeting diagnostic criteria for idiopathic pulmonary arterial hypertension (IPAH), there is an emerging lung phenotype characterised by a low diffusion capacity for carbon monoxide (DLCO) and a smoking history. The present study aimed at a detailed characterisation of these patients.

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.

Influenza A virus (IAV) infection mobilizes bone marrow-derived macrophages (BMDM) that gradually undergo transition to tissue-resident alveolar macrophages (TR-AM) in the inflamed lung. Combining high-dimensional single-cell transcriptomics with complex lung organoid modeling, in vivo adoptive cell transfer, and BMDM-specific gene targeting, we found that transitioning ("regenerative") BMDM and TR-AM highly express Placenta-expressed transcript 1 (Plet1). We reveal that Plet1 is released from alveolar macrophages, and acts as important mediator of macrophage-epithelial cross-talk during lung repair by inducing proliferation of alveolar epithelial cells and re-sealing of the epithelial barrier. Intratracheal administration of recombinant Plet1 early in the disease course attenuated viral lung injury and rescued mice from otherwise fatal disease, highlighting its therapeutic potential.

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).

Background: Circulating apoptotic signals (CASs) have been described in the pathologies associated with dysregulated apoptosis, such as cancer, heart diseases, and pulmonary hypertension (PH). However, nothing is known about the expression profiles of these markers in the circulation of humans exposed to acute and chronic effects of high altitude (HA). Methods: Gene expression levels of different apoptotic signals (ASs) were analyzed in human pulmonary artery smooth muscle cells (PASMCs) upon hypoxia incubation. In addition, we measured the plasma values of relevant CAS in Kyrgyz volunteers during acute and chronic exposure to HA. Finally, we analyzed the effects of pro-apoptotic mediator Fas ligand (FasL) on apoptosis and proliferation of human PASMCs. Results: Several cellular AS were increased in PASMCs exposed to hypoxia, in comparison to normoxia condition. Among analyzed CAS, there was a prominent reduction of FasL in lowlanders exposed to HA environment. Furthermore, decreased circulatory levels of FasL were found in highlanders with HA-induced PH (HAPH), as compared to the lowland controls. Furthermore, FasL concentration in plasma negatively correlated with tricuspid regurgitant gradient values. Finally, FasL exerted pro-apoptotic and anti-proliferative effects on PASMCs. Conclusion: Our data demonstrated that circulating levels of FasL are reduced during acute and chronic exposure to HA environment. In addition, dysregulated FasL may play a role in the context of HAPH due to its relevant functions on apoptosis and proliferation of PASMCs.

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.

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.

Pulmonary hypertension (PH) is defined by elevated mean pulmonary artery pressure following the pathological remodelling of small pulmonary arteries. An increase in right ventricular (RV) afterload results in RV hypertrophy and RV failure. The pathophysiology of PH, and RV remodelling in particular, is not well understood, thus explaining, at least in part, why current PH therapies have a limited effect. Existing therapies mostly target the pulmonary circulation. Because the remodelled RV fails to support normal cardiac function, patients eventually succumb from RV failure. Developing novel therapies that directly target the function of the RV may therefore benefit patients with PH. In the past decade, several promising studies have investigated novel cardioprotective strategies in experimental models of PH. This review aims to comprehensively discuss and highlight these novel experimental approaches to confer, in the long-term, greater health benefit in patients with PH.

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.

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.

Although the response of the right ventricle (RV) to the increased afterload is an important determinant of the patient outcome, very little is known about the underlying mechanisms. Mast cells have been implicated in the pathogenesis of left ventricular maladaptive remodeling and failure. However, the role of mast cells in RV remodeling remains unexplored. We subjected mast cell-deficient WBB6F1-KitW/W-v (KitW/KitW-v) mice and their mast cell-sufficient littermate controls (MC+/+) to pulmonary artery banding (PAB). PAB led to RV dilatation, extensive myocardial fibrosis, and RV dysfunction in MC+/+ mice. In PAB KitW/KitW-v mice, RV remodeling was characterized by minimal RV chamber dilatation and preserved RV function. We further administered to C57Bl/6J mice either placebo or cromolyn treatment starting from day 1 or 7 days after PAB surgery to test whether mast cells stabilizing drugs can prevent or reverse maladaptive RV remodeling. Both preventive and therapeutic cromolyn applications significantly attenuated RV dilatation and improved RV function. Our study establishes a previously undescribed role of mast cells in pressure overload-induced adverse RV remodeling. Mast cells may thus represent an interesting target for the development of a new therapeutic approach directed specifically at the heart.

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.