Search

Vascular corrosion casting is a method used to visualize the three dimensional anatomy and branching pattern of blood vessels, guiding insight into health and cardiovascular disease pathogenesis and progression. A polymer resin is injected in the vascular system and, after curing, the surrounding tissue is removed. This corrosion process often deforms or even fractures the fragile cast, resulting in an overall loss of information. Here, we propose a method that does not require corrosion of the tissue, based on in-situ high-resolution computed tomography (micro-CT) scans. Since there is a lack of CT contrast between the polymer cast and the animals’ surrounding soft tissue, we introduce hafnium oxide nanocrystals (HfO2 NCs) as CT contrast agents into the resin. The NCs dramatically improve the overall CT contrast of the cast and allow for straightforward segmentation in the CT scans. We designed the NC surface chemistry to ensure colloidal stability of the NCs in the casting resin, resulting in a homogeneous dispersion that remains stable during casting and curing. Using only 5 m% of HfO2 NCs, high-quality casts of both zebrafish and mouse models could be segmented using CT imaging software, allowing us to differentiate even μm scale details, without having to alter the resin injection method or affecting the resin’s mechanical properties. Our new method of virtual dissection by visualizing casts in-situ using contrast enhanced CT imaging greatly expands the application potential of the technique.

Introduction Marfan syndrome (MFS) is caused by a defect in fibrillin-1, which binds TGF-beta via interaction with latent TGF-beta binding proteins (LTBPs). The role of TGF-beta in MFS is controversial. Objectives use dedicated mouse models for MFS, with defects interfering with TGF-beta binding and function, to gain insights into the role of TGF-beta signaling in aneurysm formation and dissection. Material & methods Mice lacking the binding site for LTBPs (Fbn1H1Δ/+ and Fbn1H1Δ/H1Δ), mice with a truncated fibrillin-1 (Fbn1GT-8/+), and mice with a combination (Fbn1GT-8/H1Δ) were subjected to cardiac ultrasound and ex vivo synchrotron X-ray imaging. Results Only Fbn1GT-8/H1Δ mice showed increased mortality (aortic rupture) starting at 4–5 months, whereas all other mice had a normal life span. Aortic root dilatation occurred both in Fbn1GT-8/+ and Fbn1GT-8/H1Δ mice at 6 months, but not in Fbn1H1D/+ or Fbn1H1Δ/H1Δ mice. Significant elastin fragmentation was observed in the thoracic aortic wall of Fbn1GT-8/+ mice, and to a larger extent in Fbn1GT-8/H1Δ mice. Surprisingly, localized elastin fragmentation was also found in the ascending aorta of Fbn1H1Δ/+ and Fbn1H1Δ/H1Δ mice, despite a lack of aortic aneurysm formation. Moreover, Fbn1H1Δ/H1Δ mice displayed more severe aortic wall damage. Conclusion Our data suggest that loss of LTBP binding to fibrillin-1 leads to the development of localized aortic microdissections in the absence of aortic aneurysm, and exacerbates the aortic wall morphology abnormalities in mice with truncated fibrillin-1. We therefore hypothesize that local TGF-beta sequestration is required to maintain aortic homeostasis.

Introduction Marfan syndrome (MFS) is the most common type of fibrillinopathy with a high predisposition to develop TAAD. A thorough understanding of the underlying mechanisms is still lacking, indicating a particular need for more flexible in vivo models to address this knowledge gap. Objectives We aimed to generate a relevant zebrafish model to gain insight into the molecular mechanisms relating fibrillin defects to the cardiovascular system. Methods The CRISPR/Cas9 system was used to systematically target the three different fibrillin genes (fbn1, fbn2a and fbn2b) in Tg(kdrl:GFP) reporter zebrafish. Time-lapse fluorescent microscopy was used to evaluate the cardiovascular phenotype. Results zebrafish lacking fbn1 and/or fbn2a do not show any cardiovascular phenotype during early-stage development. On the other hand, approximately 50% of homozygous fbn2b mutant (fbn2b-/-) zebrafish embryo's show a severe phenotype characterized by endocardial detachment, leading to vascular embolism and premature mortality at 7–9 dpf. Interestingly, the remaining fbn2b-/- zebrafish survive until adulthood, but during larval stages already develop a dilation of the bulbus arteriosus. The caudal vein of all fbn2b-/- embryos also develops abnormally as a cavernous structure lacking vessel integrity. This phenotype is resolved in embryos retaining normal blood flow and aggravated upon pharmacological inhibition of blood flow during development. Conclusion These data indicate that fbn2b-/- zebrafish model recapitulates cardiovascular complications, and can be considered as a relevant model to study the mechanisms underlying MFS pathogenesis. Our preliminary data suggest that there is an interplay between fibrillin deficiency and biomechanical signaling in the regulation of cardiovascular development.

Marfan syndrome (MFS) is a heritable connective tissue disorder caused by pathogenic variants in the gene coding for the extracellular matrix protein fibrillin-1. While the disease affects multiple organ systems, the most life-threatening manifestations are aortic aneurysms leading to dissection and rupture. Other cardiovascular complications, including mitral valve prolapse, primary cardiomyopathy, and arrhythmia, also occur more frequently in patients with MFS. The standard medical care relies on cardiovascular imaging at regular intervals, along with pharmacological treatment with β-adrenergic receptor blockers aimed at reducing the aortic growth rate. When aortic dilatation reaches a threshold associated with increased risk of dissection, prophylactic surgical aortic replacement is performed. Although current clinical management has significantly improved the life expectancy of patients with MFS, no cure is available and fatal complications still occur, underscoring the need for new treatment options. In recent years, preclinical studies have identified a number of potentially promising therapeutic targets. Nevertheless, the translation of these results into clinical practice has remained challenging. In this review, we present an overview of the currently available knowledge regarding the underlying pathophysiological processes associated with MFS cardiovascular pathology. We then summarize the treatment options that have been developed based on this knowledge and are currently in different stages of preclinical or clinical development, provide a critical review of the limitations of current studies and highlight potential opportunities for future research.

Cutis laxa (CL) syndromes are a large and heterogeneous group of rare connective tissue disorders that share loose redundant skin as a hallmark clinical feature, which reflects dermal elastic fiber fragmentation. Both acquired and congenital-Mendelian- forms exist. Acquired forms are progressive and often preceded by inflammatory triggers in the skin, but may show systemic elastolysis. Mendelian forms are often pleiotropic in nature and classified upon systemic manifestations and mode of inheritance. Though impaired elastogenesis is a common denominator in all Mendelian forms of CL, the underlying gene defects are diverse and affect structural components of the elastic fiber or impair metabolic pathways interfering with cellular trafficking, proline synthesis, or mitochondrial functioning. In this chapter we provide a detailed overview of the clinical and molecular characteristics of the different cutis laxa types and review the latest insights on elastic fiber assembly and homeostasis from both human and animal studies.

International audience; In eukaryotic cells, the balance between the synthesis and the degradation decides the steady-state levels of messenger RNAs (mRNA). The removal of adenosine residues from the poly(A) tail, called deadenylation, is the first and the most crucial step in the process of mRNA degradation. Poly (A)-specific ribonuclease (PARN) is one such enzyme that catalyses the process of deadenylation. Although PARN has been primarily known as the regulator of the mRNA stability, recent evidence clearly suggests several other functions of PARN, including a role in embryogenesis, oocyte maturation, cell-cycle progression, telomere biology, non-coding RNA maturation and ribosome biogenesis. Also, deregulated PARN activity is shown to be a hallmark of specific disease conditions. Pathogenic variants in the RN gene have been observed in various cancers and inherited bone marrow failure syndromes. The focus in this review is to highlight the emerging functions of PARN, particularly in the context of human diseases.

Objective Facial dysostosis is a group of rare craniofacial congenital disabilities requiring multidisciplinary long-term care. This report presents the phenotypic and genotypic information from South India. Design The study is a case series. Setting This was an international collaborative study involving a tertiary craniofacial clinic and medical genetics unit. Patients, Participants The participants were 9 families with 17 affected individuals of facial dysostosis. Intervention Exome analysis focused on known genes associated with acrofacial and mandibulofacial syndromes. Main Outcome Measure The outcome measure was to report phenotyptic and genetic heterogeneity in affected individuals. Results A Tessier cleft was seen in 7 (41%), lower eyelid coloboma in 12 (65%), ear anomalies in 10 (59%), uniolateral or bilateral aural atresia in 4 (24%), and deafness in 6 (35%). The facial gestalt of Treacher Collins syndrome (TCS) showed extensive phenotypic variations. Pathogenic variants in TCOF1 (Treacher Collins syndrome) were seen in six families, POLR1A (acrofacial dysostosis, Cincinnati type) and EFTUD2 (mandibulofacial dysostosis with microcephaly) in one each. One family (11.1%) had no detectable variation. Five out of six probands with Treacher Collins syndrome had other affected family members (83.3%), including a non-penetrant mother, identified after sequencing. Conclusion Our report illustrates the molecular heterogeneity of mandibulofacial dysostosis in India.

Background Marfan syndrome (MFS) is a rare disease caused by a defect in the fibrillin-1 gene (FBN1), with potentially severe cardiovascular manifestations. MFS patients are particularly susceptible to a progressive aortic dilation leading to potential dissection and wall rupture. No causal treatment for the disease is available and current medical treatment is aimed at slowing aortic disease progression to minimize severe complications. When indicated, surgical repair of the aortic defect is performed. Although these strategies have clearly led to improved survival, some patients still present with fatal complications. Purpose To generate a new flexible zebrafish model of MFS to gain a better understanding of the underlying pathophysiological mechanisms and to find new treatment options. Methods We used the CRISPR/Cas9 method to disrupt the 3 fibrillin genes in zebrafish (fbn1, fbn2a, and fbn2b). The Tg(kdrl:EGFP) reporter was used to visualize cardiovascular structure by fluorescent microscopy up to 8 days post fertilization (dpf). A subset of embryos was treated with the myosin inhibitor 2,3-butanedione monoxime (BDM). Results We found that zebrafish lacking fbn1 and/or fbn2a do not show any detectable phenotype during development. No evidence of induction of genetic compensation was found in these mutant lines. Zebrafish deficient in fbn2b however do show strong phenotypes, including fully penetrant finfold atrophy (Fig. 1A-B and E, arrowhead). On average 50% of homozygous fbn2b mutant (fbn2b−/−) zebrafish embryos show endocardial detachment (Fig. 1C-D; a:atrium, v:ventricle), leading to vascular embolism, pericardial edema (Fig. 1B, arrow), loss of blood flow, and ultimately death at 7–9 dpf. Interestingly, fbn2b−/− without endocardial detachment survive normally, but develop a dilated bulbus arteriosus phenotype during larval stages (Fig. 1F-G, arrow; 1H: diameter during minimal and maximal distension, *: P All fbn2b−/− embryos show abnormal early development of the caudal vein as a cavernous structure lacking vessel integrity (Fig. 2, arrowheads). This phenotype resolves in embryos retaining normal blood flow. We found that fbn2b−/− embryos raised in BDM to inhibit blood flow show a more severe caudal vein phenotype than wild-type (WT) controls (Fig. 2, yellow line: severe vascular dilation). Conclusion Loss of fbn2b, but not the other fibrillin genes, in zebrafish results in cardiovascular manifestations overlapping with MFS. These data indicate that fbn2b−/− zebrafish can be a relevant model to explore the mechanisms leading from fibrillin deficiency to the cardiovascular symptoms observed in MFS. Our preliminary results suggest that there is an interplay between fibrillin deficiency and biomechanical signaling. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): Fund Baillet Latour Grant for Medical Research Figure 1. Phenotype of fbn2b−/− larvaeFigure 2. Effect of blood flow in fbn2b−/−

Summary Latent transforming growth factor β (TGFβ)-binding proteins (LTBPs) are microfibril-associated proteins essential for anchoring TGFβ in the extracellular matrix (ECM) as well as for correct assembly of ECM components. Variants in LTBP2, LTBP3, and LTBP4 have been identified in several autosomal recessive Mendelian disorders with skeletal abnormalities with or without impaired development of elastin-rich tissues. Thus far, the human phenotype associated with LTBP1 deficiency has remained enigmatic. In this study, we report homozygous premature truncating LTBP1 variants in eight affected individuals from four unrelated consanguineous families. Affected individuals present with connective tissue features (cutis laxa and inguinal hernia), craniofacial dysmorphology, variable heart defects, and prominent skeletal features (craniosynostosis, short stature, brachydactyly, and syndactyly). In vitro studies on proband-derived dermal fibroblasts indicate distinct molecular mechanisms depending on the position of the variant in LTBP1. C-terminal variants lead to an altered LTBP1 loosely anchored in the microfibrillar network and cause increased ECM deposition in cultured fibroblasts associated with excessive TGFβ growth factor activation and signaling. In contrast, N-terminal truncation results in a loss of LTBP1 that does not alter TGFβ levels or ECM assembly. In vivo validation with two independent zebrafish lines carrying mutations in ltbp1 induce abnormal collagen fibrillogenesis in skin and intervertebral ligaments and ectopic bone formation on the vertebrae. In addition, one of the mutant zebrafish lines shows voluminous and hypo-mineralized vertebrae. Overall, our findings in humans and zebrafish show that LTBP1 function is crucial for skin and bone ECM assembly and homeostasis.

The inability to maintain a strictly regulated endo(lyso)somal acidic pH through the proton-pumping action of the vacuolar-ATPases (v-ATPases) has been associated with various human diseases including heritable connective tissue disorders. Autosomal recessive (AR) cutis laxa (CL) type 2C syndrome is associated with genetic defects in the ATP6V1E1 gene and is characterized by skin wrinkles or loose redundant skin folds with pleiotropic systemic manifestations. The underlying pathological mechanisms leading to the clinical presentations remain largely unknown. Here, we show that loss of atp6v1e1b in zebrafish leads to early mortality, associated with craniofacial dysmorphisms, vascular anomalies, cardiac dysfunction, N-glycosylation defects, hypotonia, and epidermal structural defects. These features are reminiscent of the phenotypic manifestations in ARCL type 2C patients. Our data demonstrates that loss of atp6v1e1b alters endo(lyso)somal protein levels, and interferes with non-canonical v-ATPase pathways in vivo. In order to gain further insights into the processes affected by loss of atp6v1e1b, we performed an untargeted analysis of the transcriptome, metabolome, and lipidome in early atp6v1e1b-deficient larvae. We report multiple affected pathways including but not limited to oxidative phosphorylation, sphingolipid, fatty acid, and energy metabolism together with profound defects on mitochondrial respiration. Taken together, our results identify complex pathobiological effects due to loss of atp6v1e1b in vivo., Author summary Cutis laxa syndromes are pleiotropic disorders of the connective tissue, characterized by skin redundancy and variable systemic manifestations. Cutis laxa syndromes are caused by pathogenic variants in genes encoding structural and regulatory components of the extracellular matrix or in genes encoding components of cellular trafficking, metabolism, and mitochondrial function. Pathogenic variants in genes coding for vacuolar-ATPases, a multisubunit complex responsible for the acidification of multiple intracellular vesicles, cause type 2 cutis laxa syndromes, a group of cutis laxa subtypes further characterized by neurological, skeletal, and rarely cardiopulmonary manifestations. To investigate the pathomechanisms of vacuolar-ATPase dysfunction, we generated zebrafish models that lack a crucial subunit of the vacuolar-ATPases. The mutant zebrafish models show morphological and functional features reminiscent of the phenotypic manifestations in cutis laxa patients carrying pathogenic variants in ATP6V1E1. In-depth analysis at multiple -omic levels identified biological signatures that indicate impairment of signaling pathways, lipid metabolism, and mitochondrial respiration. We anticipate that these data will contribute to a better understanding of the pathogenesis of cutis laxa syndromes and other disorders involving defective v-ATPase function, which may eventually improve patient treatment and management.

Cutis laxa (CL) syndromes are a large and heterogeneous group of rare connective tissue disorders that share loose redundant skin as a hallmark clinical feature, which reflects dermal elastic fiber fragmentation. Both acquired and congenital-Mendelian- forms exist. Acquired forms are progressive and often preceded by inflammatory triggers in the skin, but may show systemic elastolysis. Mendelian forms are often pleiotropic in nature and classified upon systemic manifestations and mode of inheritance. Though impaired elastogenesis is a common denominator in all Mendelian forms of CL, the underlying gene defects are diverse and affect structural components of the elastic fiber or impair metabolic pathways interfering with cellular trafficking, proline synthesis, or mitochondrial functioning. In this chapter we provide a detailed overview of the clinical and molecular characteristics of the different cutis laxa types and review the latest insights on elastic fiber assembly and homeostasis from both human and animal studies.

The inability to maintain a strictly regulated endo(lyso)somal acidic pH through the proton-pumping action of the vacuolar-ATPases has been associated with various human diseases including heritable connective tissue disorders, neurodegenerative diseases and cancer. Multiple studies have investigated the pleiotropic effects of reduced acidification in vitro, but the mechanisms elicited by impaired endo(lyso)somal acidification in vivo remain poorly understood. Here, we show that loss of atp6v1e1b in zebrafish leads to early mortality, associated with craniofacial dysmorphisms, vascular anomalies, cardiac dysfunction, hypotonia and epidermal structural defects, reminiscent of the phenotypic manifestations in cutis laxa patients carrying a defect in the ATP6V1E1 gene. Mechanistically, we found that in vivo genetic depletion of atp6v1e1b leads to N-glycosylation defects and reduced maturation of endosomal and lysosomal vesicles, but retains the hypoxia-mediated response. In order to gain further insights into the processes affected by aberrant organelle acidification, we performed an untargeted analysis of the transcriptome and metabolome in early atp6v1e1b-deficient larvae. We report multiple affected pathways including but not limited to oxidative phosphorylation, sphingolipid and fatty acid metabolism with profound defects in mitochondrial respiration. Taken together, our results identify new complex biological effects of reduced organelle acidification in vivo, which likely contribute to the multisystemic manifestations observed in disorders caused by v-ATPase deficiency.

Patients with Marfan syndrome (MFS), a connective tissue disorder caused by pathogenic variants in the gene encoding the extracellular matrix protein fibrillin-1, have an increased prevalence of primary cardiomyopathy, arrhythmias, and sudden cardiac death. We have performed an in-depth in vivo and ex vivo study of the cardiac phenotype of Fbn1mgR/mgR mice, an established mouse model of MFS with a severely reduced expression of fibrillin-1. Using ultrasound measurements, we confirmed the presence of aortic dilatation and observed cardiac diastolic dysfunction in male Fbn1mgR/mgR mice. Upon post-mortem examination, we discovered that the mutant mice consistently presented myocardial lesions at the level of the right ventricular free wall, which we characterized as spontaneous pseudoaneurysms. Histological investigation demonstrated a decrease in myocardial compaction in the MFS mouse model. Furthermore, continuous 24 h electrocardiographic analysis showed a decreased heart rate variability and an increased prevalence of extrasystolic arrhythmic events in Fbn1mgR/mgR mice compared to wild-type littermates. Taken together, in this paper we document a previously unreported cardiac phenotype in the Fbn1mgR/mgR MFS mouse model and provide a detailed characterization of the cardiac dysfunction and rhythm disorders which are caused by fibrillin-1 deficiency. These findings highlight the wide spectrum of cardiac manifestations of MFS, which might have implications for patient care.

Zebrafish have come to the forefront as a flexible, relevant animal model to study human disease, including cardiovascular disorders. Zebrafish are optically transparent during early developmental stages, enabling unparalleled imaging modalities to examine cardiovascular structure and function in vivo and ex vivo. At later stages, however, the options for systematic cardiovascular phenotyping are more limited. To visualise the complete vascular tree of adult zebrafish, we have optimised a vascular corrosion casting method. We present several improvements to the technique leading to increased reproducibility and accuracy. We designed a customised support system and used a combination of the commercially available Mercox II methyl methacrylate with the Batson's catalyst for optimal vascular corrosion casting of zebrafish. We also highlight different imaging approaches, with a focus on scanning electron microscopy (SEM) and X-ray microtomography (micro-CT) to obtain highly detailed, faithful three-dimensional reconstructed images of the zebrafish cardiovascular structure. This procedure can be of great value to a wide range of research lines related to cardiovascular biology in small specimens.

Dysregulated nitric oxide (NO) signaling contributes to the pathogenesis of hypertension, a prevalent and often sex-specific risk factor for cardiovascular disease. We previously reported that mice deficient in the α1-subunit of the NO receptor soluble guanylate cyclase (sGCα1−/− mice) display sex- and strain-specific hypertension: male but not female sGCα1−/− mice are hypertensive on an 129S6 (S6) but not a C57BL6/J (B6) background. We aimed to uncover the genetic and molecular basis of the observed sex- and strain-specific blood pressure phenotype. Via linkage analysis, we identified a suggestive quantitative trait locus associated with elevated blood pressure in male sGCα1−/−S6 mice. This locus encompasses Cyp4a12a, encoding the predominant murine synthase of the vasoconstrictor 20-hydroxy-5,8,11,14-eicosatetraenoic acid (20-HETE). Renal expression of Cyp4a12a in mice was associated with genetic background, sex, and testosterone levels. In addition, 20-HETE levels were higher in renal preglomerular microvessels of male sGCα1−/−S6 than of male sGCα1−/−B6 mice. Furthermore, treating male sGCα1−/−S6 mice with the 20-HETE antagonist 20-hydroxyeicosa-6(Z),15(Z)-dienoic acid (20-HEDE) lowered blood pressure. Finally, 20-HEDE rescued the genetic background- and testosterone-dependent impairment of acetylcholine-induced relaxation in renal interlobar arteries associated with sGCα1 deficiency. Elevated Cyp4a12a expression and 20-HETE levels render mice susceptible to hypertension and vascular dysfunction in a setting of sGCα1 deficiency. Our data identify Cyp4a12a as a candidate sex-specific blood pressure-modifying gene in the context of deficient NO-sGC signaling.

The cyclic adenosine monophosphate (AMP) responsive element binding protein 3-like 1 (CREB3L1) gene codes for the endoplasmic reticulum stress transducer old astrocyte specifically induced substance (OASIS), which has an important role in osteoblast differentiation during bone development. Deficiency of OASIS is linked to a severe form of autosomal recessive osteogenesis imperfecta (OI), but only few patients have been reported. We identified the first homozygous pathogenic missense variant (p.(Ala304Val)) in a patient with lethal OI, which is located within the highly conserved basic leucine zipper domain, four amino acids upstream of the DNA binding domain. In vitro structural modeling and luciferase assays demonstrate that this missense variant affects a critical residue in this functional domain, thereby decreasing the type I collagen transcriptional binding ability. In addition, overexpression of the mutant OASIS protein leads to decreased transcription of the SEC23A and SEC24D genes, which code for components of the coat protein complex type II (COPII), and aberrant OASIS signaling also results in decreased protein levels of SEC24D. Our findings therefore provide additional proof of the potential involvement of the COPII secretory complex in the context of bone-associated disease.

Significance Type I collagenopathies are a heterogenous group of connective tissue disorders, caused by genetic defects in type I collagen. Inherent to these disorders is a large clinical variability, of which the underlying molecular basis remains undefined. By systematically analyzing skeletal phenotypes in a large set of type I collagen zebrafish mutants, we show that zebrafish models are able to both genocopy and phenocopy different forms of human type I collagenopathies, arguing for a similar pathogenetic basis. This study illustrates the future potential of zebrafish as a tool to further dissect the molecular basis of phenotypic variability in human type I collagenopathies, to improve diagnostic strategies as well as promote the discovery of new targetable pathways for pharmacological intervention of these disorders., The type I collagenopathies are a group of heterogeneous connective tissue disorders, that are caused by mutations in the genes encoding type I collagen and include specific forms of osteogenesis imperfecta (OI) and the Ehlers–Danlos syndrome (EDS). These disorders present with a broad disease spectrum and large clinical variability of which the underlying genetic basis is still poorly understood. In this study, we systematically analyzed skeletal phenotypes in a large set of zebrafish, with diverse mutations in the genes encoding type I collagen, representing different genetic forms of human OI, and a zebrafish model resembling human EDS, which harbors a number of soft connective tissues defects, typical of EDS. Furthermore, we provide insight into how zebrafish and human type I collagen are compositionally and functionally related, which is relevant in the interpretation of human type I collagen-related disease models. Our studies reveal a high degree of intergenotype variability in phenotypic expressivity that closely correlates with associated OI severity. Furthermore, we demonstrate the potential for select mutations to give rise to phenotypic variability, mirroring the clinical variability associated with human disease pathology. Therefore, our work suggests the future potential for zebrafish to aid in identifying unknown genetic modifiers and mechanisms underlying the phenotypic variability in OI and related disorders. This will improve diagnostic strategies and enable the discovery of new targetable pathways for pharmacological intervention.

Fibrillins are major constituents of microfibrils, which are essential components of the extracellular matrix of connective tissues where they contribute to the tissue homeostasis. Although it is known that microfibrils are abundantly expressed in the left ventricle of the heart, limited data are available about the presence of microfibrils in the other parts of the myocardial tissue and whether there are age or sex-related differences in the spatial arrangement of the microfibrils. This basic knowledge is essential to better understand the impact of fibrillin-1 pathogenic variants on the myocardial tissue as seen in Marfan related cardiomyopathy. We performed histological analyses on wild-type male and female murine myocardial tissue collected at different time-points (1, 3 and 6 months). Fibrillin-1 and -2 immunofluorescence stainings were performed on cross-sections at the level of the apex, the mid-ventricles and the atria. In addition, other myocardial matrix components such as collagen and elastin were also investigated. Fibrillin-1 presented as long fibres in the apex, mid-ventricles and atria. The spatial arrangement differed between the investigated regions, but not between age groups or sexes. Collagen had a similar broad spatial arrangement to that of fibrillin-1, whereas elastic fibres were primarily present in the atria and the vessels. In contrast to fibrillin-1, limited amounts of fibrillin-2 were observed. Fibrillin-rich fibres contribute to the architecture of the myocardial tissue in a region-dependent manner in wild-type murine hearts. This knowledge is helpful for future experimental set-ups of studies evaluating the impact of fibrillin-1 pathogenic variants on the myocardial tissue.

© 2018 Sips et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Cyanide is a potent toxic agent, and the few available antidotes are not amenable to rapid deployment in mass exposures. As a result, there are ongoing efforts to exploit different animal models to identify novel countermeasures. We have created a pipeline that combines high-throughput screening in zebrafish with subsequent validation in two mammalian small animal models as well as a porcine large animal model. We found that zebrafish embryos in the first 3 days post fertilization (dpf) are highly resistant to cyanide, becoming progressively more sensitive thereafter. Unbiased analysis of gene expression in response to several hours of ultimately lethal doses of cyanide in both 1 and 7 dpf zebrafish revealed modest changes in iron-related proteins associated with the age-dependent cyanide resistance. Metabolomics measurements demonstrated significant age-dependent differences in energy metabolism during cyanide exposure which prompted us to test modulators of the tricarboxylic acid cycle and related metabolic processes as potential antidotes. In cyanide-sensitive 7 dpf larvae, we identified several such compounds that offer significant protection against cyanide toxicity. Modulators of the pyruvate dehydrogenase complex, as well as the small molecule sodium glyoxylate, consistently protected against cyanide toxicity in 7 dpf zebrafish larvae. Together, our results indicate that the resistance of zebrafish embryos to cyanide toxicity during early development is related to an altered regulation of cellular metabolism, which we propose may be exploited as a potential target for the development of novel antidotes against cyanide poisoning.

Hypertension is an important modifiable risk factor for coronary heart disease, congestive heart failure, stroke, end-stage renal disease, and peripheral vascular disease, but many of the molecular mechanisms and genetic factors underlying the development of the most common forms of human hypertension remain to be defined. Abundant evidence suggests that nitric oxide (NO) and one of its primary targets, the cyclic guanosine monophosphate (cGMP)-generating enzyme soluble guanylate cyclase (sGC), have a critical role in regulating blood pressure. The availability of murine models of hypertension and the revolution in human genetics research (e.g., genome-wide association studies [GWAS]), resulting in the identification of dozens of genetic loci that affect normal variation in blood pressure and susceptibility to hypertension, provide a unique opportunity to dissect the mechanisms by which NO-cGMP signaling regulates blood pressure and to gain important insights into the pathogenesis of hypertension. In this review, we will give an overview of the current knowledge relating to the role of sGC in the regulation of blood pressure, discussing data obtained from genetically modified mouse models as well as from human genetic studies.

Myocardial depression is an important contributor to morbidity and mortality in septic patients. Nitric oxide (NO) plays an important role in the development of septic cardiomyopathy, but also has protective effects. Recent evidence has indicated that NO exerts many of its downstream effects on the cardiovascular system via protein S-nitrosylation, which is negatively regulated by S-nitrosoglutathione reductase (GSNOR), an enzyme promoting denitrosylation. We tested the hypothesis that reducing cardiomyocyte S-nitrosylation by increasing GSNOR activity can improve myocardial dysfunction during sepsis. Therefore, we generated mice with a cardiomyocyte-specific overexpression of GSNOR (GSNOR-CMTg mice) and subjected them to endotoxic shock. Measurements of cardiac function in vivo and ex vivo showed that GSNOR-CMTg mice had a significantly improved cardiac function after lipopolysaccharide challenge (LPS, 50 mg/kg) compared with wild-type (WT) mice. Cardiomyocytes isolated from septic GSNOR-CMTg mice showed a corresponding improvement in contractility compared with WT cells. However, systolic Ca2+release was similarly depressed in both genotypes after LPS, indicating that GSNOR-CMTg cardiomyocytes have increased Ca2+sensitivity during sepsis. Parameters of inflammation were equally increased in LPS-treated hearts of both genotypes, and no compensatory changes in NO synthase expression levels were found in GSNOR-overexpressing hearts before or after LPS challenge. GSNOR overexpression however significantly reduced total cardiac protein S-nitrosylation during sepsis. Taken together, our results indicate that increasing the denitrosylation capacity of cardiomyocytes protects against sepsis-induced myocardial depression. Our findings suggest that specifically reducing protein S-nitrosylation during sepsis improves cardiac function by increasing cardiac myofilament sensitivity to Ca2+.

Background: Volatile anesthetics increase levels of the neurotransmitter nitric oxide (NO) and the secondary messenger molecule cyclic guanosine monophosphate (cGMP) in the brain. NO activates the enzyme guanylyl cyclase (GC) to produce cGMP. We hypothesized that the NO-GC-cGMP pathway contributes to anesthesia-induced unconsciousness. Methods: Sevoflurane-induced loss and return of righting reflex (LORR and RORR, respectively) were studied in wild-type mice (WT) and in mice congenitally deficient in the GC-1 alpha subunit (GC-1(-/-) mice). Spatial distributions of GC-1 alpha and the GC-2 alpha subunit in the brain were visualized by in situ hybridization. Brain cGMP levels were measured in WT and GC-1(-/-) mice after inhaling oxygen with or without 1.2% sevoflurane for 20 min. Results: Higher concentrations of sevoflurane were required to induce LORR in GC-1(-/-) mice than in WT mice (1. 5 +/- 0.1 vs. 1.1 +/- 0.2%, respectively, n = 14 and 14, P < 0.0001). Similarly, RORR occurred at higher concentrations of sevoflurane in GC-1(-/-) mice than in WT mice (1.0 +/- 0.1 vs. 0.8 +/- 0.1%, respectively, n = 14 and 14, P < 0.0001). Abundant GC-1 alpha and GC-2 alpha mRNA expression was detected in the cerebral cortex, medial habenula, hippocampus, and cerebellum. Inhaling 1.2% sevoflurane for 20 min increased cGMP levels in the brains of WT mice from 2.6 +/- 2.0 to 5.5 +/- 3.7 pmol/mg protein (n = 13 and 10, respectively, P = 0.0355) but not in GC-1(-/-) mice. Conclusion: Congenital deficiency of GC-1 alpha abolished the ability of sevoflurane anesthesia to increase cGMP levels in the whole brain, and increased the concentration of sevoflurane required to induce LORR. Impaired NO-cGMP signaling raises the threshold for producing sevoflurane-induced unconsciousness in mice.

Coronary flow (CF) measured ex vivo is largely determined by capillary density that reflects angiogenic vessel formation in the heart in vivo. Here we exploit this relationship and show that CF in the rat is influenced by a locus on rat chromosome 2 that is also associated with cardiac capillary density. Mitochondrial tryptophanyl-tRNA synthetase (Wars2), encoding an L53F protein variant within the ATP-binding motif, is prioritized as the candidate at the locus by integrating genomic data sets. WARS2(L53F) has low enzyme activity and inhibition of WARS2 in endothelial cells reduces angiogenesis. In the zebrafish, inhibition of wars2 results in trunk vessel deficiencies, disordered endocardial-myocardial contact and impaired heart function. Inhibition of Wars2 in the rat causes cardiac angiogenesis defects and diminished cardiac capillary density. Our data demonstrate a pro-angiogenic function for Wars2 both within and outside the heart that may have translational relevance given the association of WARS2 with common human diseases., Blood supply to the heart is crucial for cardiac function. Here, the authors show that the mitochondrial tryptophanyl-tRNA synthetase, WARS2, drives blood vessel generation in zebrafish and rats and that inhibition of Wars2 diminishes blood vessel growth both within and outside in the heart, suggesting a new target for manipulating angiogenesis.

Objective: Soluble guanylate cyclase (sGC), a heterodimer composed of α and β subunits, synthesizes cGMP in response to nitric oxide (NO). NO modulates vascular tone and structure but the relative contributions of cGMP-dependent versus cGMP-independent mechanisms remain uncertain. We studied the response to vascular injury in male (M) and female (F) mice with targeted deletion of exon 6 of the sGCα1 subunit (sGCα1-/-), resulting in a non-functional heterodimer. Methods: We measured aortic cGMP levels and mRNA transcripts encoding sGC α1, α2, and β1 subunits in wild type (WT) and sGCa1-/- mice. To study the response to vascular injury, BrdU-incorporation and neointima formation (maximum intima to media (I/M) ratio) were determined 5 and 28 days after carotid artery ligation, respectively. Results: Aortic cGMP levels were 4-fold higher in F than in M mice in both genotypes, and, within each gender, 4-fold higher in WT than in sGCa1-/-. In contrast, sGCα1, sGCα2, and sGCβ1 mRNA expression did not differ between groups. 3H-thymidine incorporation in cultured sGCa1-/- smooth muscle cells (SMC) was 27%±12% lower than in WT SMC and BrdU-incorporation in carotid arteries 5 days after ligation was significantly less in sGCa1-/- M than in WT M. Neointima area and I/M 28 days after ligation were 65% and 62% lower in sGCa1-/- M than in WT M mice (p Conclusion: Functional deletion of sGCa1 resulted in reduced cGMP levels in male sGCa1-/- mice and a gender-specific effect on the adaptive response to vascular injury.

Nitric oxide (NO) activates soluble guanylate cyclase (sGC) to produce guanosine-3',5'-cyclic-monophosphate (cGMP). The aim of this study was to investigate the nitrergic regulation of jejunal motility in sGCalpha(1) knockout (KO) mice. Functional responses to nitrergic stimuli and cGMP levels in response to nitrergic stimuli were determined in circular muscle strips. Intestinal transit was determined. Nitrergic relaxations induced by electrical field stimulation and exogenous NO were almost abolished in male KO strips, but only minimally reduced and sensitive to ODQ in female KO strips. Basal cGMP levels were decreased in KO strips, but NO still induced an increase in cGMP levels. Transit was not attenuated in male nor female KO mice. In vitro, sGCalpha(1)beta(1) is the most important isoform in nitrergic relaxation of jejunum, but nitrergic relaxation can also occur via sGCalpha(2)beta(1) activation. The latter mechanism is more pronounced in female than in male KO mice. In vivo, no important implications on intestinal motility were observed in male and female KO mice.

The effects of nitric oxide (NO) in the cardiovascular system are attributed in part to cGMP synthesis by the alpha1beta1 isoform of soluble guanylate cyclase (sGC). Because available sGC inhibitors are neither enzyme- nor isoform-specific, we generated knockout mice for the alpha1 subunit (sGCalpha1(-/-) mice) in order to investigate the function of sGCalpha1beta1 in the regulation of blood pressure and cardiac function.Blood pressure was evaluated, using both non-invasive and invasive haemodynamic techniques, in intact and gonadectomized male and female sGCalpha1(-/-) and wild-type (WT) mice. Cardiac function was assessed with a conductance catheter inserted in the left ventricle of male and female sGCalpha1(-/-) and WT mice. Male sGCalpha1(-/-) mice developed hypertension (147 +/- 2 mmHg), whereas female sGCalpha1(-/-) mice did not (115 +/- 2 mmHg). Orchidectomy and treatment with an androgen receptor antagonist prevented hypertension, while ovariectomy did not influence the phenotype. Chronic testosterone treatment increased blood pressure in ovariectomized sGCalpha1(-/-) mice but not in WT mice. The NO synthase inhibitor Nomega-nitro-L-arginine methyl ester hydrochloride raised blood pressure similarly in male and female WT and sGCalpha1(-/-) mice. The ability of NO donor compounds to reduce blood pressure was slightly attenuated in sGCalpha1(-/-) male and female mice as compared to WT mice. The direct sGC stimulator BAY 41-2272 reduced blood pressure only in WT mice. Increased cardiac contractility and arterial elastance as well as impaired ventricular relaxation were observed in both male and female sGCalpha1(-/-) mice.These findings demonstrate that sGCalpha1beta1-derived cGMP signalling has gender-specific and testosterone-dependent cardiovascular effects and reveal that the effects of NO on systemic blood pressure do not require sGCalpha1beta1.

The principal target of the relaxant neurotransmitter nitric oxide (NO) is soluble guanylate cyclase (sGC). As the α1β1-isoform of sGC is the predominant one in the gastrointestinal tract, the aim of this study was to investigate the role of sGC in nitrergic regulation of gastric motility in male and female sGCα1 knock-out (KO) mice. In circular gastric fundus muscle strips, functional responses and cGMP levels were determined in response to nitrergic and non-nitrergic stimuli. sGC subunit mRNA expression in fundus was measured by real-time RT-PCR; in vivo gastric emptying of a phenol red meal was determined. No changes were observed in sGC subunit mRNA levels between wild-type (WT) and KO tissues. Nitrergic relaxations induced by short trains of electrical field stimulation (EFS) were abolished, while those by long trains of EFS were reduced in KO strips; the latter responses were abolished by 1H[1,2,4,]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ). The relaxations evoked by exogenous NO and the NO-independent sGC activator BAY 41-2272 were reduced in KO strips but still sensitive to ODQ. Relaxations induced by vasoactive intestinal peptide (VIP) and 8-bromo-cGMP were not influenced. Basal cGMP levels were decreased in KO strips but NO, long train EFS and BAY 41-2272 still induced a moderate ODQ-sensitive increase in cGMP levels. Gastric emptying, measured at 15 and 60 min, was increased at 15 min in male KO mice. sGCα1β1 plays an important role in gastric nitrergic relaxation in vitro, but some degree of nitrergic relaxation can occur via sGCα2β1 activation in sGCα1 KO mice, which contributes to the moderate in vivo consequence on gastric emptying.

Soluble guanylyl cyclase (sGC), the predominant receptor for nitric oxide (NO), exists in 2 active isoforms (alpha(2)beta(1) and alpha(1)beta(1)). In vascular tissue sGCalpha(1)beta(1) is believed to be the most important. The aim of our study was to investigate the functional importance of the sGCalpha(1)-subunit in vasorelaxation.Aortic and femoral artery segments from male and/or female sGCalpha(1)(-/-) mice and wild-type littermates were mounted in a small-vessel myograph for isometric tension recording. This was supplemented with biochemical measurements of the cGMP concentration and sGC enzyme activity.The functional importance of sGCalpha(1)beta(1) was demonstrated by the significantly decreased relaxing effects of acetylcholine (ACh), sodium nitroprusside (SNP), S-nitroso-N-acetylpenicillamine (SNAP), NO gas, YC-1, BAY 41-2272 and T-1032 in the sGCalpha(1)(-/-) mice of both genders. Moreover, the basal and SNP-stimulated cGMP levels and basal sGC activity were significantly lower in the sGCalpha(1)(-/-) mice. However, the relaxing effects of NO, BAY 41-2272 and YC-1 seen in blood vessels from sGCalpha(1)(-/-) mice indicate a role for an sGCalpha(1)beta(1)-independent mechanism. The increase in sGC activity after addition of BAY 41-2272 and the inhibition of the ACh-, SNP-, SNAP- and NO gas-induced response by the sGC inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) in the sGCalpha(1)(-/-) mice are observations suggesting that the sGCalpha(2)beta(1) isoform is also functionally active. However, the insignificant increase in cGMP in response to SNP and the non-upregulated sGCalpha(2) expression level in the sGCalpha(1)(-/-) mice suggest rather the involvement of (an) sGC-independent mechanism(s).We conclude that sGCalpha(1)beta(1) is involved in the vasorelaxation induced by NO-dependent and NO-independent sGC activators in both genders. However, the remaining relaxation seen in the sGCalpha(1)(-/-) mice suggests that besides sGCalpha(1)beta(1) also the minor isoform sGCalpha(2)beta(1) and/or (an) sGC-independent mechanism(s) play(s) a substantial role.

Introduction: Mutations in the cardiac myosin-binding protein C ( MYBPC3 ) gene have been implicated in over one third of hypertrophic cardiomyopathy (HCM) cases. These mutations have been reported to be associated with delayed expression of hypertrophy and a relatively good prognosis. However, we previously reported that a patients with a S593Pfs*9 mutation (a single base deletion of a thymidine residue at nucleotide 11645 in exon 18) in this gene deteriorated into the“end-stage” HCM, characterized by left ventricular systolic dysfunction, cavity dilation, and heart failure. This mutation causes a truncated MYBPC3. The precise functional and pathological consequences of this mutation are not fully characterized. The aim of this study was to recapitulate the S593Pfs*9 mutation in the zebrafish to define the pathologic mechanisms. Methods: We used the CRISPR/Cas9 system to generate a genocopy of the S593Pfs*9 with a specific guide RNA injected with Cas9 into one-cell stage embryos. Results: Gross morphological examination of the mutants showed a smaller ventricle and pericardial edema compared with controls. There were significant reductions in heart rate (125 +/- 31 vs 148 +/- 9 /min), end-diastolic volume (EDV) (0.41 +/- 0.16 vs 0.72 +/- 0.18 nl), end-systolic volume (ESV) (0.24 +/- 0.08 vs 0.36 +/- 0.13 nl), stroke volume (SV) (0.16 +/- 0.12 vs 0.36 +/- 0.09 nl) in mutants compared with control embryos at 48 hours post fertilization (hpf). In addition, the calcium transient amplitude (0.17 +/- 0.02 vs 0.23 +/- 0.02 ratio units) was also significantly reduced in the mutant hearts compared with the control hearts at 48 hpf. Conclusions: Our data demonstrate that the S593Pfs*9 mutants exhibit reduced EDV, ESV, and SV and define early abnormalities of Ca 2+ handling that may drive the development of heart failure at later stages. This model will allow the mechanistic dissection of MYBPC-associated heart failure and enable chemical screens for novel drugs.

Oxidative stress, a central mediator of cardiovascular disease, results in loss of the prosthetic haem group of soluble guanylate cyclase (sGC), preventing its activation by nitric oxide (NO). Here we introduce Apo-sGC mice expressing haem-free sGC. Apo-sGC mice are viable and develop hypertension. The haemodynamic effects of NO are abolished, but those of the sGC activator cinaciguat are enhanced in apo-sGC mice, suggesting that the effects of NO on smooth muscle relaxation, blood pressure regulation and inhibition of platelet aggregation require sGC activation by NO. Tumour necrosis factor (TNF)-induced hypotension and mortality are preserved in apo-sGC mice, indicating that pathways other than sGC signalling mediate the cardiovascular collapse in shock. Apo-sGC mice allow for differentiation between sGC-dependent and -independent NO effects and between haem-dependent and -independent sGC effects. Apo-sGC mice represent a unique experimental platform to study the in vivo consequences of sGC oxidation and the therapeutic potential of sGC activators., Haem-free, NO-insensitive soluble guanylate cyclase (apo-sGC) generated during oxidative stress contributes to cardiovascular pathology. By generating and characterizing apo-sGC knock-in mice, Thoonen et al. provide a scientific ground for the therapeutic concept of sGC activators, and dissect the relevance of the NO-sGC axis.

Anaphylactic shock is a sudden, life-threatening allergic reaction associated with severe hypotension. Platelet-activating factor (PAF) is implicated in the cardiovascular dysfunctions occurring in various shock syndromes, including anaphylaxis. Excessive production of the vasodilator NO causes inflammatory hypotension and shock, and it is generally accepted that transcriptionally regulated inducible iNOS is responsible for this. Nevertheless, the contribution of NO to PAF-induced shock or anaphylactic shock is still ambiguous. We studied PAF and anaphylactic shock in conscious mice. Surprisingly, hyperacute PAF shock depended entirely on NO, produced not by inducible iNOS, but by constitutive eNOS, rapidly activated via the PI3K pathway. Soluble guanylate cyclase (sGC) is generally regarded as the principal vasorelaxing mediator of NO. Nevertheless, although methylene blue partially prevented PAF shock, neither 1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one (ODQ) nor sGCα1 deficiency did. Also, in 2 different models of active systemic anaphylaxis, inhibition of NOS, PI3K, or Akt or eNOS deficiency provided complete protection. In contrast to the unsubstantiated paradigm that only excessive iNOS-derived NO underlies cardiovascular collapse in shock, our data strongly support the unexpected concept that eNOS-derived NO is the principal vasodilator in anaphylactic shock and define eNOS and/or PI3K or Akt as new potential targets for treating anaphylaxis.

Cisplatin holds an illustrious position in the history of chemistry most notably for its role in the virtual cure of testicular cancer. Here we describe a role for this small molecule in cyanide detoxification in vivo. Cyanide kills organisms as diverse as insects, fish, and humans within seconds to hours. Current antidotes exhibit limited efficacy and are not amenable to mass distribution requiring the development of new classes of antidotes. The binding affinity of the cyanide anion for the positively charged metal platinum is known to create an extremely stable complex in vitro. We therefore screened a panel of diverse cisplatin analogs and identified compounds that conferred protection from cyanide poisoning in zebrafish, mice, and rabbits. Cumulatively, this discovery pipeline begins to establish the characteristics of platinum ligands that influence their solubility, toxicity, and efficacy, and provides proof of concept that platinum-based complexes are effective antidotes for cyanide poisoning.

Septic shock is the leading cause of death in noncoronary intensive care units and the 10th leading cause of death overall. Several lines of evidence support an important role for the vasodilator NO in hypotension, a hallmark of septic shock. However, NO may also positively or negatively regulate inflammation, apoptosis, and oxidative stress. These dual effects of NO may relate to its isoform specific production but also to differences in cellular and/or temporal expression. Via bone marrow transplantations, we examined the contribution of hematopoietic cells to the dramatically elevated NO levels seen in (septic) shock. Surprisingly, hematopoietic cells are not responsible at all for the production of circulating NO after systemic tumor necrosis factor or lipopolysaccharide challenge and contribute only marginally in a bacteremic (Salmonella) model of septic shock. Immunohistochemistry identified the nonhematopoietic sources of NO as hepatocytes, paneth cells, and intestinal and renal epithelial cells. In contrast, during granulomatous Bacillus Calmette-Guérin inflammation, the hematopoietic cell population represents the sole source of systemic NO. These mouse data demonstrate that, in contrast to the general conjecture, the dramatically elevated levels of NO during (septic) shock are not produced by hematopoietic cells such as monocytes/macrophages but rather by parenchymal cells in liver, kidney and gut.

Nitric oxide (NO)-soluble guanylate cyclase (sGC)-dependent signaling mechanisms have a profound effect on the regulation of blood pressure (BP). In this review, we will discuss recent findings in the field that support the importance of sGC in the development of hypertension.The importance of sGC in BP regulation was highlighted by studies using genetically modified animal models, chemical stimulators/activators and inhibitors of the NO/sGC signaling pathway, and genetic association studies in humans. Many studies further support the role of NO/sGC in vasodilation and vascular dysfunction, which is underscored by the early clinical success of synthetic sGC stimulators for the treatment of pulmonary hypertension. Recent work has uncovered more details about the structural basis of sGC activation, enabling the development of more potent and efficient modulators of sGC activity. Finally, the mechanisms involved in the modulation of sGC by signaling gases other than NO, as well as the influence of redox signaling on sGC, have been the subject of several interesting studies.sGC is fast becoming an interesting therapeutic target for the treatment of vascular dysfunction and hypertension, with novel sGC stimulating/activating compounds as promising clinical treatment options.

The nitric oxide (NO)-cyclic guanosine monophosphate (cGMP) signaling system is a well-characterized modulator of cardiovascular function, in general, and blood pressure, in particular. The availability of mice mutant for key enzymes in the NO-cGMP signaling system facilitated the identification of interactions with other blood pressure modifying pathways (e.g. the renin-angiotensin-aldosterone system) and of gender-specific effects of impaired NO-cGMP signaling. In addition, recent genome-wide association studies identified blood pressure-modifying genetic variants in genes that modulate NO and cGMP levels. Together, these findings have advanced our understanding of how NO-cGMP signaling modulates blood pressure. In this review, we will summarize the results obtained with mice with disrupted NO-cGMP signaling and highlight the relevance of this pathway as a potential therapeutic target for the treatment of hypertension.

Background— Sudden cardiac arrest (CA) is a leading cause of death worldwide. Breathing nitric oxide (NO) reduces ischemia/reperfusion injury in animal models and in patients. The objective of this study was to learn whether inhaled NO improves outcomes after CA and cardiopulmonary resuscitation (CPR). Methods and Results— Adult male mice were subjected to potassium-induced CA for 7.5 minutes whereupon CPR was performed with chest compression and mechanical ventilation. One hour after CPR, mice were extubated and breathed air alone or air supplemented with 40 ppm NO for 23 hours. Mice that were subjected to CA/CPR and breathed air exhibited a poor 10-day survival rate (4 of 13), depressed neurological and left ventricular function, and increased caspase-3 activation and inflammatory cytokine induction in the brain. Magnetic resonance imaging revealed brain regions with marked water diffusion abnormality 24 hours after CA/CPR in mice that breathed air. Breathing air supplemented with NO for 23 hours starting 1 hour after CPR attenuated neurological and left ventricular dysfunction 4 days after CA/CPR and markedly improved 10-day survival rate (11 of 13; P =0.003 versus mice breathing air). The protective effects of inhaled NO on the outcome after CA/CPR were associated with reduced water diffusion abnormality, caspase-3 activation, and cytokine induction in the brain and increased serum nitrate/nitrite levels. Deficiency of the α1 subunit of soluble guanylate cyclase, a primary target of NO, abrogated the ability of inhaled NO to improve outcomes after CA/CPR. Conclusions— These results suggest that NO inhalation after CA and successful CPR improves outcome via soluble guanylate cyclase–dependent mechanisms.

Nitric oxide (NO)-dependent soluble guanylate cyclase (sGC) activation is an important component of cardiac signal transduction pathways, including the cardioprotective signaling cascade induced by ischemic preconditioning (IPC). The sGCα subunit, which binds to the common sGCβ1 subunit, exists in two different isoforms, sGCα1 and sGCα2, but their relative physiological roles remain unknown. In the present study, we studied Langendorff-perfused isolated hearts of genetically engineered mice lacking functional sGCα1 (sGCα1KO mice), which is the predominant isoform in the heart. Our results show that the loss of sGCα1 has a positive inotropic and lusitropic effect on basal cardiac function, indicating an important role for sGCα1 in regulating basal myocardial contractility. Surprisingly, IPC led to a similar 35–40% reduction in infarct size and concomitant protein kinase Ce (PKCe) phosphorylation in both wild-type (WT) and sGCα1KO hearts subjected to 40 min of global ischemia and reperfusion. Inhibition of the activation of all sGC isoforms by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one (ODQ, 10 μmol/L) completely abolished the protection by IPC in WT and sGCα1KO hearts. NO-stimulated cGMP production was severely attenuated in sGCα1KO hearts compared to WT hearts, indicating that the sGCα2 isoform only produces minute amounts of cGMP after NO stimulation. Taken together, our results indicate that although sGCα1 importantly regulates cardiac contractility, it is not required for cardioprotection by IPC. Instead, our results suggest that possibly only minimal sGC activity, which in sGCα1KO hearts is provided by the sGCα2 isoform, is sufficient to transduce the cardioprotective signal induced by IPC via phosphorylation of PKCe.