Right ventricular cardiomyocyte expansion accompanies cardiac regeneration in newborn mice after large left ventricular infarcts.

Cauterization of the root of the left coronary artery (LCA) in the neonatal heart on postnatal day 1 (P1) resulted in large, reproducible lesions of the left ventricle (LV), and an attendant marked adaptive response in the right ventricle (RV). The response of both chambers to LV myocardial infarction involved enhanced cardiomyocyte (CM) division and binucleation, as well as LV revascularization, leading to restored heart function within 7 days post surgery (7 dps). By contrast, infarction of P3 mice resulted in cardiac scarring without a significant regenerative and adaptive response of the LV and the RV, leading to subsequent heart failure and death within 7 dps. The prominent RV myocyte expansion in P1 mice involved an acute increase in pulmonary arterial pressure and a unique gene regulatory response, leading to an increase in RV mass and preserved heart function. Thus, distinct adaptive mechanisms in the RV, such as CM proliferation and RV expansion, enable marked cardiac regeneration of the infarcted LV at P1 and full functional recovery.

Electrophysiological Properties of Tetraploid Cardiomyocytes Derived from Murine Pluripotent Stem Cells Generated by Fusion of Adult Somatic Cells with Embryonic Stem Cells.

Most cardiomyocytes (CMs) in the adult mammalian heart are either binucleated or contain a single polyploid nucleus. Recent studies have shown that polyploidy in CMs plays an important role as an adaptive response to physiological demands and environmental stress and correlates with poor cardiac regenerative ability after injury. However, knowledge about the functional properties of polyploid CMs is limited. In this study, we generated tetraploid pluripotent stem cells (PSCs) by fusion of murine embryonic stem cells (ESCs) and somatic cells isolated from bone marrow or spleen and performed a comparative analysis of the electrophysiological properties of tetraploid fusion-derived PSCs and diploid ESC-derived CMs. Fusion-derived PSCs exhibited characteristics of genuine ESCs and contained a near-tetraploid genome. Ploidy features and marker expression were also retained during the differentiation of fusion-derived cells. Fusion-derived PSCs gave rise to CMs, which were similar to their diploid ESC counterparts in terms of their expression of typical cardiospecific markers, sarcomeric organization, action potential parameters, response to pharmacologic stimulation with various drugs, and expression of functional ion channels. These results suggest that the state of ploidy does not significantly affect the structural and electrophysiological properties of murine PSC-derived CMs. These results extend our knowledge of the functional properties of polyploid CMs and contribute to a better understanding of their biological role in the adult heart.

A PI3Kγ mimetic peptide triggers CFTR gating, bronchodilation, and reduced inflammation in obstructive airway diseases.

Cyclic adenosine 3',5'-monophosphate (cAMP)-elevating agents, such as ß2-adrenergic receptor (ß2-AR) agonists and phosphodiesterase (PDE) inhibitors, remain a mainstay in the treatment of obstructive respiratory diseases, conditions characterized by airway constriction, inflammation, and mucus hypersecretion. However, their clinical use is limited by unwanted side effects because of unrestricted cAMP elevation in the airways and in distant organs. Here, we identified the A-kinase anchoring protein phosphoinositide 3-kinase γ (PI3Kγ) as a critical regulator of a discrete cAMP signaling microdomain activated by ß2-ARs in airway structural and inflammatory cells. Displacement of the PI3Kγ-anchored pool of protein kinase A (PKA) by an inhaled, cell-permeable, PI3Kγ mimetic peptide (PI3Kγ MP) inhibited a pool of subcortical PDE4B and PDE4D and safely increased cAMP in the lungs, leading to airway smooth muscle relaxation and reduced neutrophil infiltration in a murine model of asthma. In human bronchial epithelial cells, PI3Kγ MP induced unexpected cAMP and PKA elevations restricted to the vicinity of the cystic fibrosis transmembrane conductance regulator (CFTR), the ion channel controlling mucus hydration that is mutated in cystic fibrosis (CF). PI3Kγ MP promoted the phosphorylation of wild-type CFTR on serine-737, triggering channel gating, and rescued the function of F508del-CFTR, the most prevalent CF mutant, by enhancing the effects of existing CFTR modulators. These results unveil PI3Kγ as the regulator of a ß2-AR/cAMP microdomain central to smooth muscle contraction, immune cell activation, and epithelial fluid secretion in the airways, suggesting the use of a PI3Kγ MP for compartment-restricted, therapeutic cAMP elevation in chronic obstructive respiratory diseases.

Imaging of Gαq Proteins in Mouse and Human Organs and Tissues.

G protein-coupled receptors (GPCRs) transfer extracellular signals across cell membranes by activating intracellular heterotrimeric G proteins. Several studies suggested G proteins as novel drug targets for the treatment of complex diseases, e.g., asthma and cancer. Recently, we developed specific radiotracers, [³H]PSB-15900-FR and [³H]PSB-16254-YM, for the Gαq family of G proteins by tritiation of the macrocyclic natural products FR900359 (FR) and YM-254890 (YM). In the present study, we utilized these potent radioligands to perform autoradiography studies in tissues of healthy mice, mouse models of disease, and human tissues. Specific binding was high, while non-specific binding was extraordinarily low, giving nearly identical results for both radioligands. High expression levels of Gαq proteins were detected in healthy mouse organs showing the following rank order of potency: kidney > liver > brain > pancreas > lung > spleen, while expression in the heart was low. Organ sub-structures, e.g., of mouse brain and lung, were clearly distinguishable. Whereas an acute asthma model in mice did not result in altered Gαq protein expressions as compared to control animals, a cutaneous melanoma model displayed significantly increased expression in comparison to healthy skin. These results suggest the future development of Gαq-protein-binding radio-tracers as novel diagnostics.

Bone marrow CD73+ mesenchymal stem cells display increased stemness in vitro and promote fracture healing in vivo.

Mesenchymal stem cells (MSCs) are multipotent and considered to be of great potential for regenerative medicine. We could show recently (Breitbach, Kimura et al. 2018) that a subpopulation of MSCs as well as sinusoidal endothelial cells (sECs) in the bone marrow (BM) of CD73-EGFP reporter mice could be labeled in vivo. We took advantage of this model to explore the plasticity and osteogenic potential of CD73-EGFP+ MSCs in vitro and their role in the regenerative response upon bone lesion in vivo. Herein we show that isolated CD73-EGFP+ MSCs displayed more pronounced stemness and stronger in vitro differentiation capacity into the osteogenic lineage compared to CD73-EGFP- MSCs. In a bone fracture model, endogenous BM-resident CD73-EGFP+ MSCs were found to migrate to the fracture site and differentiate into cartilage and bone cells. Our analysis also showed that CD73-EGFP+ sECs contributed to the neovascularization of the fracture site. In addition, grafting of CD73-EGFP+ MSCs into acute bone lesions revealed their capacity to differentiate into chondrocytes and osteocytes in vivo and their contribution to callus formation in the regeneration process of fracture healing. Thus, CD73+ MSCs display enhanced stemness and osteogenic differentiation potential in vitro and in vivo illustrating a prominent role of the CD73+ MSC subpopulation to promote fracture repair.

Macrocyclic Gq Protein Inhibitors FR900359 and/or YM-254890-Fit for Translation?

Guanine nucleotide-binding proteins (G proteins) transduce extracellular signals received by G protein-coupled receptors (GPCRs) to intracellular signaling cascades. While GPCRs represent the largest class of drug targets, G protein inhibition has only recently been recognized as a novel strategy for treating complex diseases such as asthma, inflammation, and cancer. The structurally similar macrocyclic depsipeptides FR900359 (FR) and YM-254890 (YM) are potent selective inhibitors of the Gq subfamily of G proteins. FR and YM differ in two positions, FR being more lipophilic than YM. Both compounds are utilized as pharmacological tools to block Gq proteins in vitro and in vivo. However, no detailed characterization of FR and YM has been performed, which is a prerequisite for the compounds' translation into clinical application. Here, we performed a thorough study of both compounds' physicochemical, pharmacokinetic, and pharmacological properties. Chemical stability was high across a large range of pH values, with FR being somewhat more stable than YM. Oral bioavailability and brain penetration of both depsipeptides were low. FR showed lower plasma protein binding and was metabolized significantly faster than YM by human and mouse liver microsomes. FR accumulated in lung after chronic intratracheal or intraperitoneal application, while YM was more distributed to other organs. Most strikingly, the previously observed longer residence time of FR resulted in a significantly prolonged pharmacologic effect as compared to YM in a methacholine-induced bronchoconstriction mouse model. These results prove that changes within a molecule which seem marginal compared to its structural complexity can lead to crucial pharmacological differences.

Proximity to injury, but neither number of nuclei nor ploidy define pathological adaptation and plasticity in cardiomyocytes.

The adult mammalian heart consists of mononuclear and binuclear cardiomyocytes (CMs) with various ploidies. However, it remains unclear whether a variation in ploidy or number of nuclei is associated with distinct functions and injury responses in CMs, including regeneration. Therefore, we investigated transcriptomes and cellular as well as nuclear features of mononucleated and binucleated CMs in adult mouse hearts with and without injury. To be able to identify the role of ploidy we analyzed control and failing human ventricular CMs because human CMs show a larger and disease-sensitive degree of polyploidization. Using transgenic Myh6-H2BmCh to identify mononucleated and binucleated mouse CMs, we found that cellular volume and RNA content were similar in both. On average nuclei of mononuclear CMs showed a 2-fold higher ploidy, as compared to binuclear CMs indicating that most mononuclear CMs are tetraploid. After myocardial infarction mononucleated and binucleated CMs in the border zone of the lesion responded with hypertrophy and corresponding changes in gene expression, as well as a low level of induction of cell cycle gene expression. Human CMs allowed us to study a wide range of polyploidy spanning from 2n to 16n. Notably, basal as well as pathological gene expression signatures and programs in failing CMs proved to be independent of ploidy. In summary, gene expression profiles were induced in proximity to injury, but independent of number of nuclei or ploidy levels in CMs.

Sensitive LC-MS/MS Method for the Quantification of Macrocyclic Gαq Protein Inhibitors in Biological Samples.

The cyclic depsipeptide FR900359 (FR) isolated from the plant Ardisia crenata and produced by endosymbiotic bacteria acts as a selective Gq protein inhibitor. It is a powerful tool to study G protein-coupled receptor signaling, and has potential as a novel drug for the treatment of pulmonary diseases and cancer. For pharmacokinetic studies, sensitive quantitative measurements of drug levels are required. In the present study we established an LC-MS/MS method to detect nanomolar concentrations of FR and the structurally related natural product YM-254890 (YM) in biological samples. HPLC separation coupled to ESI-QTOF-MS and UV-VIS detection was applied. For identification and quantification, the extract ion chromatogram (EIC) of M+1 was evaluated. Limits of detection (LOD) of 0.53-0.55 nM and limits of quantification (LOQ) of 1.6-1.7 nM were achieved for both FR and YM. This protocol was subsequently applied to determine FR concentrations in mouse organs and tissues after peroral application of the drug. A three-step liquid-liquid extraction protocol was established, which resulted in adequate recovery rates of typically around 50%. The results indicated low peroral absorption of FR. Besides the gut, highest concentrations were determined in eye and kidney. The developed analytical method will be useful for preclinical studies to evaluate these potent Gq protein inhibitors, which may have potential as future drugs for complex diseases.

Adenosine/A2B Receptor Signaling Ameliorates the Effects of Aging and Counteracts Obesity.

The combination of aging populations with the obesity pandemic results in an alarming rise in non-communicable diseases. Here, we show that the enigmatic adenosine A2B receptor (A2B) is abundantly expressed in skeletal muscle (SKM) as well as brown adipose tissue (BAT) and might be targeted to counteract age-related muscle atrophy (sarcopenia) as well as obesity. Mice with SKM-specific deletion of A2B exhibited sarcopenia, diminished muscle strength, and reduced energy expenditure (EE), whereas pharmacological A2B activation counteracted these processes. Adipose tissue-specific ablation of A2B exacerbated age-related processes and reduced BAT EE, whereas A2B stimulation ameliorated obesity. In humans, A2B expression correlated with EE in SKM, BAT activity, and abundance of thermogenic adipocytes in white fat. Moreover, A2B agonist treatment increased EE from human adipocytes, myocytes, and muscle explants. Mechanistically, A2B forms heterodimers required for adenosine signaling. Overall, adenosine/A2B signaling links muscle and BAT and has both anti-aging and anti-obesity potential.

Cell-permeable high-affinity tracers for Gq proteins provide structural insights, reveal distinct binding kinetics and identify small molecule inhibitors.

BACKGROUND AND PURPOSE: G proteins are intracellular switches that transduce and amplify extracellular signals from GPCRs. The Gq protein subtypes, which are coupled to PLC activation, can act as oncogenes, and their expression was reported to be up-regulated in cancer and inflammatory diseases. Gq inhibition may be an efficient therapeutic strategy constituting a new level of intervention. However, diagnostic tools and therapeutic drugs for Gq proteins are lacking. EXPERIMENTAL APPROACH: We have now developed Gq -specific, cell-permeable 3 H-labelled high-affinity probes based on the macrocyclic depsipeptides FR900359 (FR) and YM-254890 (YM). The tracers served to specifically label and quantify Gq proteins in their native conformation in cells and tissues with high accuracy. KEY RESULTS: FR and YM displayed low nanomolar affinity for Gαq , Gα11 and Gα14 expressed in CRISPR/Cas9 Gαq -knockout cells, but not for Gα15 . The two structurally very similar tracers showed strikingly different dissociation kinetics, which is predicted to result in divergent biological effects. Computational studies suggested a "dowel" effect of the pseudoirreversibly binding FR. A high-throughput binding assay led to the discovery of novel Gq inhibitors, which inhibited Gq signalling in recombinant cells and primary murine brown adipocytes, resulting in enhanced differentiation. CONCLUSIONS AND IMPLICATIONS: The Gq protein inhibitors YM and FR are pharmacologically different despite similar structures. The new versatile tools and powerful assays will contribute to the advancement of the rising field of G protein research.

Optogenetic stimulation of Gs-signaling in the heart with high spatio-temporal precision.

The standard technique for investigating adrenergic effects on heart function is perfusion with pharmaceutical agonists, which does not provide high temporal or spatial precision. Herein we demonstrate that the light sensitive Gs-protein coupled receptor JellyOp enables optogenetic stimulation of Gs-signaling in cardiomyocytes and the whole heart. Illumination of transgenic embryonic stem cell-derived cardiomyocytes or of the right atrium of mice expressing JellyOp elevates cAMP levels and instantaneously accelerates spontaneous beating rates similar to pharmacological ß-adrenergic stimulation. Light application to the dorsal left atrium instead leads to supraventricular extrabeats, indicating adverse effects of localized Gs-signaling. In isolated ventricular cardiomyocytes from JellyOp mice, we find increased Ca2+ currents, fractional cell shortening and relaxation rates after illumination enabling the analysis of differential Gs-signaling with high temporal precision. Thus, JellyOp expression allows localized and time-restricted Gs stimulation and will provide mechanistic insights into different effects of site-specific, long-lasting and pulsatile Gs activation.

Treatment with mononuclear cell populations improves post-infarction cardiac function but does not reduce arrhythmia susceptibility.

BACKGROUND: Clinical and experimental data give evidence that transplantation of stem and progenitor cells in myocardial infarction could be beneficial, although the underlying mechanism has remained elusive. Ventricular tachyarrhythmia is the most frequent and potentially lethal complication of myocardial infarction, but the impact of mono nuclear cells on the incidence of ventricular arrhythmia is still not clear. OBJECTIVE: We aimed to characterize the influence of splenic mononuclear cell populations on ventricular arrhythmia after myocardial infarction. METHODS: We assessed electrical vulnerability in vivo in mice with left ventricular cryoinfarction 14 days after injury and intramyocardial injection of specific subpopulations of mononuclear cells (MNCs) (CD11b-positive cells, Sca-1-positive cells, early endothelial progenitor cells (eEPCs)). As positive control group we used embryonic cardiomyocytes (eCMs). Epicardial mapping was performed for analysing conduction velocities in the border zone. Left ventricular function was quantified by echocardiography and left heart catheterization. RESULTS: In vivo pacing protocols induced ventricular tachycardia (VT) in 30% of non-infarcted mice. In contrast, monomorphic or polymorphic VT could be evoked in 94% of infarcted and vehicle-injected mice (p<0.01). Only transplantation of eCMs prevented post-infarction VT and improved conduction velocities in the border zone in accordance to increased expression of connexin 43. Cryoinfarction resulted in a broad aggravation of left ventricular function. All transplanted cell types augmented left ventricular function to a similar extent. CONCLUSIONS: Transplantation of different MNC populations after myocardial infarction improves left ventricular function similar to effects of eCMs. Prevention of inducible ventricular arrhythmia is only seen after transplantation of eCMs.

PECAM/eGFP transgenic mice for monitoring of angiogenesis in health and disease.

For the monitoring of vascular growth as well as adaptive or therapeutic (re)vascularization endothelial-specific reporter mouse models are valuable tools. However, currently available mouse models have limitations, because not all endothelial cells express the reporter in all developmental stages. We have generated PECAM/eGFP embryonic stem (ES) cell and mouse lines where the reporter gene labels PECAM+ endothelial cells and vessels with high specificity. Native eGFP expression and PECAM staining were highly co-localized in vessels of various organs at embryonic stages E9.5, E15.5 and in adult mice. Expression was found in large and small arteries, capillaries and in veins but not in lymphatic vessels. Also in the bone marrow arteries and sinusoidal vessel were labeled, moreover, we could detect eGFP in some CD45+ hematopoietic cells. We also demonstrate that this labeling is very useful to monitor sprouting in an aortic ring assay as well as vascular remodeling in a murine injury model of myocardial infarction. Thus, PECAM/eGFP transgenic ES cells and mice greatly facilitate the monitoring and quantification of endothelial cells ex vivo and in vivo during development and injury.

A Novel iRFP-Incorporated in vivo Murine Atherosclerosis Imaging System.

By using near-infrared fluorescent protein (iRFP)-expressing hematopoietic cells, we established a novel, quantitative, in vivo, noninvasive atherosclerosis imaging system. This murine atherosclerosis imaging approach targets macrophages expressing iRFP in plaques. Low-density lipoprotein receptor-deficient (LDLR-/-) mice transplanted with beta-actin promoter-derived iRFP transgenic (TG) mouse bone marrow (BM) cells (iRFP → LDLR-/-) were used. Atherosclerosis was induced by a nonfluorescent 1.25% cholesterol diet (HCD). Atherosclerosis was compared among the three differently induced mouse groups. iRFP → LDLR-/- mice fed a normal diet (ND) and LDLR-/- mice transplanted with wild-type (WT) BM cells were used as controls. The in vivo imaging system (IVIS) detected an enhanced iRFP signal in the thoracic aorta of HCD-fed iRFP → LDLR-/- mice, whereas iRFP signals were not observed in the control mice. Time-course imaging showed a gradual increase in the signal area, which was correlated with atherosclerotic plaque progression. Oil red O (ORO) staining of aortas and histological analysis of plaques confirmed that the detected signal was strictly emitted from plaque-positive areas of the aorta. Our new murine atherosclerosis imaging system can noninvasively image atherosclerotic plaques in the aorta and generate longitudinal data, validating the ability of the system to monitor lesion progression.

Sonic hedgehog signaling regulates the mammalian cardiac regenerative response.

Certain organisms, including zebrafish, are capable of complete cardiac regeneration in response to injury. This response has also been observed in newborn mice, although in this case, the regenerative capacity is lost at approximately one week of age. The mechanisms regulating this short temporal window of cardiac regeneration in mice are not well understood. Here, we show that sonic hedgehog (Shh) signaling modulates the neonatal mouse regenerative response. In particular, we demonstrate that following apical resection of the heart on postnatal day 1, mice activate Shh ligand expression and downstream signaling. This response is largely absent when surgery is performed on non-regenerative, postnatal day 7 pups. Furthermore, an enhanced cardiac regeneration response was detected in ptch heterozygous mice which have a genetically-based constitutive increase in Shh signaling. We further show that Shh ligand is produced in the myocardium by non-myocytes and appears to regulate cardiomyocyte proliferation, as well as the recruitment of monocytes/macrophages to the regenerating area. Finally, we demonstrate that a small molecule activator of Shh signaling promotes heart regeneration, whereas an inhibitor of Shh signaling impairs the regenerative response. Together, these results implicate Shh signaling as a regulator of mammalian heart regeneration and suggest that modulating this pathway may lead to new potential therapies for cardiovascular diseases.

Intra-Cardiac Release of Extracellular Vesicles Shapes Inflammation Following Myocardial Infarction.

RATIONALE: A rapid and massive influx of inflammatory cells occurs into ischemic area after myocardial infarction (MI), resulting in local release of cytokines and growth factors. Yet, the mechanisms regulating their production are not fully explored. The release of extracellular vesicles (EVs) in the interstitial space curbs important biological functions, including inflammation, and influences the development of cardiovascular diseases. To date, there is no evidence for in situ release of cardiac EVs after MI. OBJECTIVE: The present study tested the hypothesis that local EV generation in the infarcted heart coordinates cardiac inflammation after MI. METHODS AND RESULTS: Coronary artery ligation in mice transiently increases EV levels in the left ventricle when compared with sham animals. EVs from infarcted hearts were characterized as large vesicles (252±18 nm) expressing cardiomyocyte and endothelial markers and small EVs (118±4 nm) harboring exosomal markers, such as CD (cluster of differentiation) 63 and CD9. Cardiac large EVs generated after MI, but not small EVs or sham EVs, increased the release of IL (interleukin)-6, CCL (chemokine ligand) 2, and CCL7 from fluorescence-activated cell-sorted Ly6C+ cardiac monocytes. EVs of similar diameter were also isolated from fragments of interventricular septum obtained from patients undergoing aortic valve replacement, thus supporting the clinical relevance of our findings in mice. CONCLUSIONS: The present study demonstrates that acute MI transiently increases the generation of cardiac EVs characterized as both exosomes and microvesicles, originating mainly from cardiomyocytes and endothelial cells. EVs accumulating in the ischemic myocardium are rapidly taken up by infiltrating monocytes and regulate local inflammatory responses.

In Vivo Labeling by CD73 Marks Multipotent Stromal Cells and Highlights Endothelial Heterogeneity in the Bone Marrow Niche.

Despite much work studying ex vivo multipotent stromal cells (MSCs), the identity and characteristics of MSCs in vivo are not well defined. Here, we generated a CD73-EGFP reporter mouse to address these questions and found EGFP+ MSCs in various organs. In vivo, EGFP+ mesenchymal cells were observed in fetal and adult bones at proliferative ossification sites, while in solid organs EGFP+ cells exhibited a perivascular distribution pattern. EGFP+ cells from the bone compartment could be clonally expanded ex vivo from single cells and displayed trilineage differentiation potential. Moreover, in the central bone marrow CD73-EGFP+ specifically labeled sinusoidal endothelial cells, thought to be a critical component of the hematopoietic stem cell niche. Purification and molecular characterization of this CD73-EGFP+ population revealed an endothelial subtype that also displays a mesenchymal signature, highlighting endothelial cell heterogeneity in the marrow. Thus, the CD73-EGFP mouse is a powerful tool for studying MSCs and sinusoidal endothelium.

Visualization of endothelial cell cycle dynamics in mouse using the Flt-1/eGFP-anillin system.

Endothelial cell proliferation is a key process during vascular growth but its kinetics could only be assessed in vitro or ex vivo so far. To enable the monitoring and quantification of cell cycle kinetics in vivo, we have generated transgenic mice expressing an eGFP-anillin construct under control of the endothelial-specific Flt-1 promoter. This construct labels the nuclei of endothelial cells in late G1, S and G2 phase and changes its localization during the different stages of M phase, thereby enabling the monitoring of EC proliferation and cytokinesis. In Flt-1/eGFP-anillin mice, we found eGFP+ signals specifically in Ki67+/PECAM+ endothelial cells during vascular development. Quantification using this cell cycle reporter in embryos revealed a decline in endothelial cell proliferation between E9.5 to E12.5. By time-lapse microscopy, we determined the length of different cell cycle phases in embryonic endothelial cells in vivo and found a M phase duration of about 80 min with 2/3 covering karyokinesis and 1/3 cytokinesis. Thus, we have generated a versatile transgenic system for the accurate assessment of endothelial cell cycle dynamics in vitro and in vivo.