Contractile Signaling Research Articles

The Pvc15 ATPase selectively associates effector proteins with the Photorhabdus virulence cassette.

The Photorhabdus virulence cassette (PVC) is an extracellular contractile injection system. In the producing bacterium, N-terminal signal peptides enable effector 'payloads' to be loaded into the PVC's hollow tube-facilitated by the 'ATPases associated with diverse cellular activities' (AAA) ATPase, Pvc15-ready for injection of the toxin or virulence factor into eukaryotic cytosols. Pvc15's function and its interaction with the signal peptide were unclear. This study describes the signal peptide diversity in extracellular contractile injection system clades and interrogates the Pvc15-signal peptide interaction using ATPase assays, cell respiratory assays and western blot quantification of Escherichia coli lysates and co-purifications of PVCs with their payloads. This study found that extracellular contractile injection system signal peptides can be grouped according to sequence alignment, owing to potentially homologous loading mechanisms. Pvc15 contains three domains, including tandem AAA domains D1 and D2. By constructing Pvc15 mutants, we found that while each domain is necessary for PVC-payload loading, domain D2 is the sole bioactive ATPase domain and rescues unstable payloads via the signal peptide. Finally, truncating the signal peptide abolishes Pvc15-dependent PVC loading and has varying effects on payload stability. This study provides crucial insights into extracellular contractile injection system effector loading mechanisms and their ATPase chaperones, and suggests that these devices could be bioengineered for injection of therapeutic proteins into human cells.

Spaceflight-induced contractile and mitochondrial dysfunction in an automated heart-on-a-chip platform

With current plans for manned missions to Mars and beyond, the need to better understand, prevent, and counteract the harmful effects of long-duration spaceflight on the body is becoming increasingly important. In this study, an automated heart-on-a-chip platform was flown to the International Space Station on a 1-mo mission during which contractile cardiac function was monitored in real-time. Upon return to Earth, engineered human heart tissues (EHTs) were further analyzed with ultrastructural imaging and RNA sequencing to investigate the impact of prolonged microgravity on cardiomyocyte function and health. Spaceflight EHTs exhibited significantly reduced twitch forces, increased incidences of arrhythmias, and increased signs of sarcomere disruption and mitochondrial damage. Transcriptomic analyses showed an up-regulation of genes and pathways associated with metabolic disorders, heart failure, oxidative stress, and inflammation, while genes related to contractility and calcium signaling showed significant down-regulation. Finally, in silico modeling revealed a potential link between oxidative stress and mitochondrial dysfunction that corresponded with RNA sequencing results. This represents an in vitro model to faithfully reproduce the adverse effects of spaceflight on three-dimensional (3D)-engineered heart tissue.

Sanglifehrin A mitigates multiorgan fibrosis by targeting the collagen chaperone cyclophilin B.

Pathological deposition and crosslinking of collagen type I by activated myofibroblasts drives progressive tissue fibrosis. Therapies that inhibit collagen synthesis have potential as antifibrotic agents. We identify the collagen chaperone cyclophilin B as a major cellular target of the natural product sanglifehrin A (SfA) using photoaffinity labeling and chemical proteomics. Mechanistically, SfA inhibits and induces the secretion of cyclophilin B from the endoplasmic reticulum (ER) and prevents TGF-β1-activated myofibroblasts from synthesizing and secreting collagen type I in vitro, without inducing ER stress or affecting collagen type I mRNA transcription, myofibroblast migration, contractility, or TGF-β1 signaling. In vivo, SfA induced cyclophilin B secretion in preclinical models of fibrosis, thereby inhibiting collagen synthesis from fibrotic fibroblasts and mitigating the development of lung and skin fibrosis in mice. Ex vivo, SfA induces cyclophilin B secretion and inhibits collagen type I secretion from fibrotic human lung fibroblasts and samples from patients with idiopathic pulmonary fibrosis (IPF). Taken together, we provide chemical, molecular, functional, and translational evidence for demonstrating direct antifibrotic activities of SfA in preclinical and human ex vivo fibrotic models. Our results identify the cellular target of SfA, the collagen chaperone cyclophilin B, as a mechanistic target for the treatment of organ fibrosis.

Large-scale control over collective cell migration using light-controlled epidermal growth factor receptors.

Receptor tyrosine kinases (RTKs) are thought to play key roles in coordinating cell movement at single-cell and tissue scales. The recent development of optogenetic tools for controlling RTKs and their downstream signaling pathways suggested these responses may be amenable to engineering-based control for sculpting tissue shape and function. Here, we report that a light-controlled EGF receptor (OptoEGFR) can be deployed in epithelial cell lines for precise, programmable control of long-range tissue movements. We show that in OptoEGFR-expressing tissues, light can drive millimeter-scale cell rearrangements to densify interior regions or produce rapid outgrowth at tissue edges. Light-controlled tissue movements are driven primarily by PI 3-kinase signaling, rather than diffusible signals, tissue contractility, or ERK kinase signaling as seen in other RTK-driven migration contexts. Our study suggests that synthetic, light-controlled RTKs could serve as a powerful platform for controlling cell positions and densities for diverse applications including wound healing and tissue morphogenesis.

FgF23 and soluble klotho effects on the myocardium: an ex vivo study

Abstract Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): German Center for Cardiovascular research Background Fibroblast Growth Factor 23 (FGF23) and soluble Klotho (sKL) play a critical role in mineral homeostasis in the body by acting on the kidney to regulate phosphate levels. Circulating FGF23 levels rise in patients with kidney disease and dysregulation of the FGF23 –sKL- axis is a common phenomenon in uremic cardiomyopathy. Studies have shown that elevated FGF23 levels induce cardiac hypertrophy by targeting cardiac myocytes via FGF receptor isoform 4 (FGFR4), while sKL mitigates these effects and may also exert an independent protective effect on the heart(1,2). Purpose We aimed to study the effects of FGF23 and sKL on myocardial contractility, calcium handling and molecular signaling pathways in the human heart using an ex vivo model of cultured contracting myocardial slices. Methods We obtained human heart slices from explanted hearts from patients undergoing cardiac transplantation at the LMU Klinikum Munich, Germany. The 300 µm thick slices were cultured under continuous stimulation (1 Hz, 3 ms pulse width) with 1 mN preload, and contractile function were recorded continuously. Slices with vehicle, recombinant FGF23 protein (100ug/ml), recombinant sKL protein (200ug/ml) and both FGF23((100ug/ml)+sKL (200ug/ml) were continuously monitored over 10 days. We checked for mRNA expression using RT PCR. Results Treatment of human left ventricular tissue with recombinant FGF23 protein resulted in enhanced mRNA expression of the FGFR4. sKL treatment alone induced downregulation of the FGFR4 gene and mitigated upregulation of FGFR4 in FGF23+ sKL treated slices. Post pause potentiation (PPP), assessed by pausing the stimulation of the slices for 120s and then measuring the relative height of the first beat after resumption of pacing, is an indirect measure of sarcoplasmic reticulum Ca2+ handling capacity. PPP was reduced in FGF23 treated slices and heightened in the slices with sKL compared to vehicle treated ones, indicating that FGF23 and sKL play a role in intracellular Ca2+ handling. Conclusions sKL and FGF23 have opposite effects on human cardiac muscle with sKL mitigating FGF23-induced upregulation of FGFR4. Furthermore, sKL improves, while FGF23 impairs myocardial calcium handling . As altered calcium handling may also be involved in hypertrophic signaling altered calcium handling may provide a means by which FGF23 promotes and sKL inhibits myocardial hypertrophy. sKL is advocated as a promising candidate in exploring it therapeutic role in FGF23 mediated cardiomyopathy. We intend to advance our understanding of sKL-FGF23signaling mechanisms by combining transcriptomic analysis with functional data in this model of myocardial slice culture.

Ca2+-dependent contractile signaling induced by dsDNA-cGAS ligation mediates type 1 interferon generation and vascular injury

Acute Lung injury (ALI) is an uncontrolled systemic inflammatory response from bacterial or viral infection. The cyclic GMP-AMP synthase (cGAS) detects cytosolic double stranded DNA (dsDNA) release from bacteria, viruses, or apoptotic cells and induces innate immunity by generating the second messenger cGAMP and activation of stimulator of interferon genes (STING). However, the mechanisms regulating cGAS activity upon binding dsDNA remain elusive. Here, we show for the first in-depth analysis of mechanisms inducing cGAS activation in using lung endothelial cells (EC) and establish its translational relevance in causing ALI using a mouse model. We show that within 15 min, dsDNA ligation with cGAS mediates its interaction with a motor protein, myosin light chain 6 (MYL6), leading to phosphorylation of MLC and actin stress fiber formation. cGAS-MYL6-mediated MLC phosphorylation required calcium release and organization of an ER-Ca2+ sensor, Stromal Interaction Molecule 1 (STIM1) as puncta. Activated STIM1 also induced cGAS tyrosine phosphorylation at Y215/242 residues via cSrc. Interfering with MYL6 or cSrc expression impaired cGAS condensation and cGAMP generation. Liposome delivery of phosphodeficient cGAS (Y215/Y242àF215/F242) in EC of cGAS null mice blocked type1-IFN production during bacterial pneumonia. Our finding suggests that STIM1 is crucial in inducing cGAS activity and type1-IFN production. cGAS thus represents a novel target for inhibiting STING activation, thereby suppressing tissue injury and inflammation. NIH. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Novel Role of YAP1 in Regulating Blood Pressure and Vascular Tone

Introduction: Hypertension is a major risk factor for occlusive vascular diseases, myocardial infarction, heart failure, stroke, and chronic kidney disease. In the U.S., ~30% of adults suffer from hypertension. Merely half of the individuals with hypertension have their blood pressure under control, despite the wide range of antihypertensive medications available, indicating an urgent need for a better understanding of the underlying causes of hypertension to identify novel therapeutic strategies for its treatment and prevention. Recent genome-wide association study (GWAS) has identified an unexpected association between a loss-of-function SNP in the YAP1 gene locus and decreased blood pressure (BP). Moreover, de novo analysis of publicly available RNAseq data revealed that the vascular tissues of hypertensive mice treated with Ang-II exhibit increased YAP1 expression. Suggesting a novel role of YAP1 in blood pressure regulation. However, whether Vascular smooth muscle cell (VSMC) expressed YAP1 has a specific role in VSMC contraction and BP regulation remains completely unknown. Therefore, we hypothesized that YAP1 could play a key role in regulating blood pressure. Methodology: We have generated a novel inducible SM-specific- Yap1 Knockout ( YAP1 iKO) mouse model and employed a pharmacological inhibitor of YAP1, Verteporfin, to delineate the functional role of VSMC-expressed YAP1 in hypertension in regulating vascular tone and BP. Vascular reactivity experiments were performed using wire myography. BP was measured in ambulant mice via radiotelemetry. In vitro gain- and loss-of-function studies utilizing human coronary artery SMCs were conducted to examine the impact of YAP1 on contractility signaling components using western blotting. Results: Consistent with GWAS and RNAseq data, we found that YAP1 is upregulated in vascular tissues of spontaneously hypertensive rats (SHRs). Using aortic rings from WT mice, we discovered that Verteporfin significantly reduced vasoconstrictive responses to Phenylephrine or Serotonin but did not alter endothelium-dependent vasorelaxant responses to Acetylcholine, suggesting a specific role of YAP1 in VSMCs. Consistently, YAP1 iKO exhibited attenuated responses to vasoconstrictors in isolated conduit and resistance vessels. Importantly, radiotelemetry studies demonstrated for the first time that YAP1 iKO mice exhibit a hypotensive phenotype. Mechanistically, YAP1 gain- and loss-of-function studies in human VSMCs showed that YAP1 activates multiple signaling pathways that play a key role in VSMC contractility including RhoA/ROCK1/actin polymerization and PKC/ERK signaling pathways. Summary/Conclusion: This is the first study to demonstrate that inhibition of SM-specific-Yap1 expression regulates BP and vascular tone, likely via the regulation of contractile machinery components. Together, our data suggest that YAP1 is a promising novel therapeutic target for ameliorating hypertension. This work is supported by a grant from National Heart, Lung, and Blood Institute (R00HL153896). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Heavy Metal Scavenger Metallothionein Rescues Against Cold Stress-Evoked Myocardial Contractile Anomalies Through Regulation of Mitophagy.

Cold stress prompts an increased prevalence of cardiovascular morbidity yet the underneath machinery remains unclear. Oxidative stress and autophagy appear to contribute to cold stress-induced cardiac anomalies. Our present study evaluated the effect of heavy metal antioxidant metallothionein on cold stress (4°C)-induced in cardiac remodeling and contractile anomalies and cell signaling involved including regulation of autophagy and mitophagy. Cold stress (3weeks) prompted interstitial fibrosis, mitochondrial damage (mitochondrial membrane potential and TEM ultrastructure), oxidative stress (glutathione, reactive oxygen species and superoxide), lipid peroxidation, protein injury, elevated left ventricular (LV) end systolic and diastolic diameters, decreased fractional shortening, ejection fraction, Langendorff heart function, cardiomyocyte shortening, maximal velocities of shortening/relengthening, and electrically stimulated intracellular Ca2+ rise along with elongated relaxation duration and intracellular Ca2+ clearance, the responses of which were overtly attenuated or mitigated by metallothionein. Levels of apoptosis, cell death (Bax and loss of Bcl2, IL-18), and autophagy (LC3BII-to-LC3BI ratio, Atg7 and Beclin-1) were overtly upregulated with comparable p62 under cold stress. Cold stress also evoked elevated mitophagy (decreased TOM20, increased Parkin and FUNDC1 with unaltered BNIP3). Cold stress overtly dampened phosphorylation of autophagy/mitophagy inhibitory molecules Akt and mTOR, stimulated and suppressed phosphorylation of ULK1 and eNOS, respectively, in the absence of altered pan protein levels. Cold stress-evoked responses in cell death, autophagy, mitophagy and their regulatory domains were overtly attenuated or ablated by metallothionein. Suppression of autophagy and mitophagy with 3-methyladenine, bafilomycin A1, cyclosporine A, and liensinine rescued hypothermia-instigated cardiomyocyte LC3B puncta formation and mechanical anomalies. Our findings support a protective nature for metallothionein in deep hypothermia-evoked cardiac abnormalities associated with regulation of autophagy and mitophagy.

In Vivo Dynamic and Static Analysis of Lymphatic Dysfunction in Lymphedema Using Near-Infrared Fluorescence Indocyanine Green Lymphangiography.

Near-infrared fluorescence indocyanine green lymphangiography, a primary modality for detecting lymphedema, which is a disease due to lymphatic obstruction, enables real-time observations of lymphatics and reveals not only the spatial distribution of drainage (static analysis) but also information on the lymphatic contraction (dynamic analysis). We have produced total lymphatic obstruction in the upper limbs of 18 Sprague-Dawley rats through the dissection of proximal (brachial and axillary) lymph nodes and 20-Gy radiation (dissection limbs). After the model formation for 1 week, 9 animal models were observed for 6 weeks using near-infrared fluorescence indocyanine green lymphangiography by injecting 6-μL ICG-BSA solution of 20-μg/mL concentration. The drainage pattern and leakage of lymph fluid were evaluated and time-domain signals of lymphatic contraction were observed in the distal lymphatic vessels. The obtained signals were converted to frequency-domain spectrums using signal processing. The results of both static and dynamic analyses proved to be effective in accurately identifying the extent of lymphatic disruption in the dissection limbs. The static analysis showed abnormal drainage patterns and increased leakage of lymph fluid to the periphery of the vessels compared with the control (normal) limbs. Meanwhile, the waveforms were changed and the contractile signal frequency increased by 58% in the dynamic analysis. Specifically, our findings revealed that regular lymphatic contractions, observed at a frequency range of 0.08 to 0.13 Hz in the control limbs, were absent in the dissection limbs. The contractile regularity was not fully restored for the follow-up period, indicating a persistent lymphatic obstruction. The dynamic analysis could detect the abnormalities of lymphatic circulation by observing the characteristics of signals, and it provided additional evaluation indicators that cannot be provided by the static analysis. Our findings may be useful for the early detection of the circulation problem as a functional evaluation indicator of the lymphatic system.

A computational bridge between traction force microscopy and tissue contraction.

Arterial wall active mechanics are driven by resident smooth muscle cells, which respond to biological, chemical, and mechanical stimuli and activate their cytoskeletal machinery to generate contractile stresses. The cellular mechanoresponse is sensitive to environmental perturbations, often leading to maladaptation and disease progression. When investigated at the single cell scale, however, these perturbations do not consistently result in phenotypes observed at the tissue scale. Here, a multiscale model is introduced that translates microscale contractility signaling into a macroscale, tissue-level response. The microscale framework incorporates a biochemical signaling network along with characterization of fiber networks that govern the anisotropic mechanics of vascular tissue. By incorporating both biochemical and mechanical components, the model is more flexible and more broadly applicable to physiological and pathological conditions. The model can be applied to both cell and tissue scale systems, allowing for the analysis of in vitro, traction force microscopy and ex vivo, isometric contraction experiments in parallel. When applied to aortic explant rings and isolated smooth muscle cells, the model predicts that active contractility is not a function of stretch at intermediate strain. The model also successfully predicts cell-scale and tissue-scale contractility and matches experimentally observed behaviors, including the hypercontractile phenotype caused by chronic hyperglycemia. The connection of the microscale framework to the macroscale through the multiscale model presents a framework that can translate the wealth of information already collected at the cell scale to tissue scale phenotypes, potentially easing the development of smooth muscle cell-targeting therapeutics.

Single fibre cytoarchitecture in ventilator-induced diaphragm dysfunction (VIDD) assessed by quantitative morphometry second harmonic generation imaging: Positive effects of BGP-15 chaperone co-inducer and VBP-15 dissociative corticosteroid treatment.

Ventilator-induced diaphragm dysfunction (VIDD) is a common sequela of intensive care unit (ICU) treatment requiring mechanical ventilation (MV) and neuromuscular blockade (NMBA). It is characterised by diaphragm weakness, prolonged respirator weaning and adverse outcomes. Dissociative glucocorticoids (e.g., vamorolone, VBP-15) and chaperone co-inducers (e.g., BGP-15) previously showed positive effects in an ICU-rat model. In limb muscle critical illness myopathy, preferential myosin loss prevails, while myofibrillar protein post-translational modifications are more dominant in VIDD. It is not known whether the marked decline in specific force (force normalised to cross-sectional area) is a pure consequence of altered contractility signaling or whether diaphragm weakness also has a structural correlate through sterical remodeling of myofibrillar cytoarchitecture, how quickly it develops, and to which extent VBP-15 or BGP-15 may specifically recover myofibrillar geometry. To address these questions, we performed label-free multiphoton Second Harmonic Generation (SHG) imaging followed by quantitative morphometry in single diaphragm muscle fibres from healthy rats subjected to five or 10days of MV + NMBA to simulate ICU treatment without underlying confounding pathology (like sepsis). Rats received daily treatment of either Prednisolone, VBP-15, BGP-15 or none. Myosin-II SHG signal intensities, fibre diameters (FD) as well as the parameters of myofibrillar angular parallelism (cosine angle sum, CAS) and in-register of adjacent myofibrils (Vernier density, VD) were computed from SHG images. ICU treatment caused a decline in FD at day 10 as well as a significant decline in CAS and VD from day 5. Vamorolone effectively recovered FD at day 10, while BGP-15 was more effective at day 5. BGP-15 was more effective than VBP-15 in recovering CAS at day 10 although not to control levels. In-register VD levels were restored at day 10 by both compounds. Our study is the first to provide quantitative insights into VIDD-related myofibrillar remodeling unravelled by SHG imaging, suggesting that both VBP-15 and BGP-15 can effectively ameliorate the structure-related dysfunction in VIDD.

Anillin and the microtubule bundler PRC1 maintain myosin in the contractile ring to ensure completion of cytokinesis.

Cytokinesis is the last step of cell division, when one cell physically divides into two cells. Cytokinesis is driven by an equatorial contractile ring and signals from antiparallel microtubule bundles (the central spindle) that form between the two masses of segregating chromosomes. Bundling of central spindle microtubules is essential for cytokinesis in cultured cells. Using a temperature-sensitive mutant of SPD-1, the homolog of the microtubule bundler PRC1, we demonstrate that SPD-1 is required for robust cytokinesis in the Caenorhabditis elegans early embryo. SPD-1 inhibition results in broadening of the contractile ring, creating an elongated intercellular bridge between sister cells at the last stages of ring constriction that fails to seal. Moreover, depleting anillin/ANI-1 in SPD-1-inhibited cells results in myosin loss from the contractile ring during the second half of furrow ingression, which in turn results in furrow regression and cytokinesis failure. Our results thus reveal a mechanism involving the joint action of anillin and PRC1, which operates during the later stages of furrow ingression to ensure continued functioning of the contractile ring until cytokinesis is complete.

Chemo-mechanical diffusion waves explain collective dynamics of immune cell podosomes

Immune cells, such as macrophages and dendritic cells, can utilize podosomes, mechanosensitive actin-rich protrusions, to generate forces, migrate, and patrol for foreign antigens. Individual podosomes probe their microenvironment through periodic protrusion and retraction cycles (height oscillations), while oscillations of multiple podosomes in a cluster are coordinated in a wave-like fashion. However, the mechanisms governing both the individual oscillations and the collective wave-like dynamics remain unclear. Here, by integrating actin polymerization, myosin contractility, actin diffusion, and mechanosensitive signaling, we develop a chemo-mechanical model for podosome dynamics in clusters. Our model reveals that podosomes show oscillatory growth when actin polymerization-driven protrusion and signaling-associated myosin contraction occur at similar rates, while the diffusion of actin monomers drives wave-like coordination of podosome oscillations. Our theoretical predictions are validated by different pharmacological treatments and the impact of microenvironment stiffness on chemo-mechanical waves. Our proposed framework can shed light on the role of podosomes in immune cell mechanosensing within the context of wound healing and cancer immunotherapy.

Altered contractility, Ca2+ transients, and cell morphology seen in a patient-specific iPSC-CM model of Ebstein's anomaly with left ventricular noncompaction.

Patients with two congenital heart diseases (CHDs), Ebstein's anomaly (EA) and left ventricular noncompaction (LVNC), suffer higher morbidity than either CHD alone. The genetic etiology and pathogenesis of combined EA/LVNC remain largely unknown. We investigated a familial EA/LVNC case associated with a variant (p.R237C) in the gene encoding Kelch-like protein 26 (KLHL26) by differentiating induced pluripotent stem cells (iPSCs) generated from affected and unaffected family members into cardiomyocytes (iPSC-CMs) and assessing iPSC-CM morphology, function, gene expression, and protein abundance. Compared with unaffected iPSC-CMs, CMs containing the KLHL26 (p.R237C) variant exhibited aberrant morphology including distended endo(sarco)plasmic reticulum (ER/SR) and dysmorphic mitochondria and aberrant function that included decreased contractions per minute, altered calcium transients, and increased proliferation. Pathway enrichment analyses based on RNASeq data indicated that the "structural constituent of muscle" pathway was suppressed, whereas the "ER lumen" pathway was activated. Taken together, these findings suggest that iPSC-CMs containing this KLHL26 (p.R237C) variant develop dysregulated ER/SR, calcium signaling, contractility, and proliferation.NEW & NOTEWORTHY We demonstrate here that iPSCs derived from patients with Ebstein's anomaly and left ventricular noncompaction, when differentiated into cardiomyocytes, display significant structural and functional changes that offer insight into disease pathogenesis, including altered ER/SR and mitochondrial morphology, contractility, and calcium signaling.

Quantifying endodermal strains during heart tube formation in the developing chicken embryo

In the early avian embryo, the developing heart forms when bilateral fields of cardiac progenitor cells, which reside in the lateral plate mesoderm, move toward the embryonic midline, and fuse above the anterior intestinal portal (AIP) to form a straight, muscle-wrapped tube. During this process, the precardiac mesoderm remains in close contact with the underlying endoderm. Previous work has shown that the endoderm around the AIP actively contracts to pull the cardiac progenitors toward the midline. The morphogenetic deformations associated with this endodermal convergence, however, remain unclear, as do the signaling pathways that might regulate this process. Here, we fluorescently labeled populations of endodermal cells in early chicken embryos and tracked their motion during heart tube formation to compute time-varying strains along the anterior endoderm. We then determined how the computed endodermal strain distributions are affected by the pharmacological inhibition of either myosin II or fibroblast growth factor (FGF) signaling. Our data indicate that a mediolateral gradient in endodermal shortening is present around the AIP, as well as substantial convergence and extension movements both anterior and lateral to the AIP. These active endodermal deformations are disrupted if either actomyosin contractility or FGF signaling are inhibited pharmacologically. Taken together, these results demonstrate how active deformations along the anterior endoderm contribute to heart tube formation within the developing embryo.

Comparative Analysis of Normoxia- and Hypoxia-Modified Extracellular Vesicle Therapy in Function, Perfusion, and Collateralization in Chronically Ischemic Myocardium.

We have previously shown that normoxia serum-starved extracellular vesicle (EV) therapy improves myocardial function, perfusion, and angiogenesis in a swine model of chronic myocardial ischemia. Hypoxia-modified EVs have increased abundance of anti-oxidant, pro-angiogenic, and pro-survival proteins. The purpose of this study is to investigate the differential effects of normoxia serum-starved EVs and hypoxia-modified EVs on myocardial function, perfusion, and microvascular density in chronically ischemic myocardium. Yorkshire swine underwent placement of an ameroid constrictor to the left circumflex artery to induce chronic myocardial ischemia. Two weeks later, the pigs underwent intramyocardial injection of either normoxia serum-starved EVs (NOR, n = 10) or hypoxia-modified EVs (HYP, n = 7). Five weeks later, pigs were euthanized, and ischemic myocardium was harvested. Hypoxia EV treatment was associated with improved contractility compared to NOR, as well as improved capillary density, without changes in arteriolar density. There were trends towards improved perfusion at rest and during pacing in the HYP group compared to NOR. Ischemic myocardium in the HYP group had increased pro-angiogenic Akt and ERK signaling and decreased expression of anti-angiogenic markers compared to the NOR group. In the setting of chronic myocardial ischemia, hypoxia-modified EVs may enhance contractility, capillary density, and angiogenic signaling pathways compared to normoxia serum-starved EVs.

S-12-2: ENHANCING MAS1:ETBR INTERACTION AS A POTENTIAL VASOPROTECTIVE STRATEGY IN STROKE-PRONE SHR

We previously demonstrated that Ang-(1–7) has anti-inflammatory effects in endothelial cells by inducing interaction between Mas1 and ETBR. Using HTS of > 20,000 druggable compounds, we identified several compounds that enhance Mas1:ETBR interaction as a novel strategy to promote vasoprotection. The objective of this study was to evaluate effects of 2 candidate compounds, termed enhancers 3 and 4 (Enh3 and Enh4), on functional responses in vascular smooth muscle cells (VSMCs) and their therapeutic potential in hypertensive (SHRSP) rats. VSMCs from normotensive (WKY) and SHRSP rats were exposed to Enh3 and Enh4(10–5 M; 5–30 mins). Expression of pro-contractile and mitogenic molecules was assessed by immunoblotting. Ca2+ influx was evaluated using fluorescence microscopy. ROCK2 activity was assessed using a colorimetric immunoassay which detected MYPT1 phosphorylation at Thr696. Male SHRSP rats (18–21weeks) were treated subcutaneously with Enh4 (10 mg/kg/day) for 7 days. Changes in blood pressure and vascular function were assessed via radiotelemetry and wire myography respectively. In WKY VSMCs, Enh3 reduced basal ERK1/2 phosphorylation (26.8 ± 5.9% vs veh,), an effect absent in SHRSP VSMCs, while Enh4 had no effect. Only in SHRSP VSMCs, Enh3, but not Enh4, reduced basal AKT phosphorylation (63.5 ± 8.9% vs veh). Enh4, but not Enh3, reduced basal MLC20 phosphorylation (54.9 ± 7.5% vs veh) in WKY but not in SHRSP VSMCs. ET-1-induced Ca2+ influx in WKY and SHRSP VSMCs was unaffected by Enh3 and Enh4. Assessment of Ca2+ -independent contractile mechanisms in VSMCs showed that ROCK2 activity is increased in SHRSP compared to WKY VSMCs (WKY: 0.27 ± 0.05; SHRSP:4.78 ± 0.34) and Enh4, but not Enh3, reduced ROCK2 activity in SHRSP VSMCs (61.5 ± 0.43% vs veh). Enh4 treatment in SHRSP rats reduced daytime systolic (SBP), diastolic (DBP) and mean arterial pressure (MAP) (SBP:4.26 ± 1.98; DBP:1.48 ± 0.92; MAP:2.07 ± 0.99 vs veh) compared to baseline. Furthermore, Enh4 treatment reduced U46619 induced contraction in SHRSP rat vessels (Emax(Mn): Veh- 13.16 ± 1.81; Enh4- 9.48 ± 1.31). Taken together, enhancing Mas1:ETBR interaction attenuates mitogenic and contractile signaling pathways in WKY and SHRSP VSMCs, and does not increase Ca2+ signaling, important in VSMC contraction. Enhancing Mas1:ETBR interaction reduces blood pressure and improves vascular function in SHRSP rats. Hence, Mas1:ETBR enhancers may be of potential therapeutic use as a new vasoprotective strategy to improve vascular function and to reduce blood pressure in hypertension.

Cardiovascular Involvement in Pediatric FLNC Variants: A Case Series of Fourteen Patients.

Filamin C is a protein specifically expressed in myocytes and cardiomyocytes and is involved in several biological functions, including sarcomere contractile activity, signaling, cellular adhesion, and repair. FLNC variants are associated with different disorders ranging from striated muscle (myofibrillar distal or proximal) myopathy to cardiomyopathies (CMPs) (restrictive, hypertrophic, and dilated), or both. The outcome depends on functional consequences of the detected variants, which result either in FLNC haploinsufficiency or in an aberrant protein, the latter affecting sarcomere structure leading to protein aggregates. Cardiac manifestations of filaminopathies are most often described as adult onset CMPs and limited reports are available in children or on other cardiac spectrums (congenital heart defects—CHDs, or arrhythmias). Here we report on 13 variants in 14 children (2.8%) out of 500 pediatric patients with early-onset different cardiac features ranging from CMP to arrhythmias and CHDs. In one patient, we identified a deletion encompassing FLNC detected by microarray, which was overlooked by next generation sequencing. We established a potential genotype–phenotype correlation of the p.Ala1186Val variant in severe and early-onset restrictive cardiomyopathy (RCM) associated with a limb-girdle defect (two new patients in addition to the five reported in the literature). Moreover, in three patients (21%), we identified a relatively frequent finding of long QT syndrome (LQTS) associated with RCM (n = 2) and a hypertrabeculated left ventricle (n = 1). RCM and LQTS in children might represent a specific red flag for FLNC variants. Further studies are warranted in pediatric cohorts to delineate potential expanding phenotypes related to FLNC.

Disparate biomechanical properties of the aorta in non‐aneurysmal and aneurysmal mice treated with angiotensin II

In vivo angiotensin II (AngII)‐treatment is a widely used experimental model to induce cardiovascular disease and results in a high likelihood of abdominal aorta aneurysm (AAA) formation. This involves progressive and irreversible focal dilation of the abdominal aorta and induces adverse aortic connective tissue remodeling contributing to aortic wall stiffening through inflammation, elastin degradation, and collagen restructuring. Hence, the present study aimed to investigate how AAA formation in AngII‐treated mice affects aortic function and biomechanics. To this end, C57Bl/6J mice were treated with AngII (1000 ng/[kg.min]) or PBS infusion for 28 days. Peripheral blood pressure, echocardiography, and aortic pulse wave velocity were measured in vivo. Thoracic aorta rings were studied ex vivo in organ chambers, while aortic vascular smooth muscle cell (VSMC) phenotype was investigated histologically. We confirmed peripheral hypertension, cardiac hypertrophy, aortic stiffening, and increased VSMC proliferation and migration after AngII‐treatment. Abdominal aorta aneurysm formation was observed in 8/13 AngII‐treated mice. Ex vivo thoracic aortic rings of both aneurysmal and non‐aneurysmal AngII‐treated mice showed high isobaric aortic stiffness, endothelial dysfunction, heightened α1‐adrenergic contractility, and altered VSMC contractile calcium signaling. However, aortic biomechanics were differently affected, with heightened α1‐adrenoreceptor mediated aortic stiffening in non‐aneurysmal mice, whereas contraction‐dependent stiffening was impaired in aneurysmal mice. In conclusion, although aneurysmal and non‐aneurysmal 4‐week AngII‐treated mice displayed similar changes in aortic physiology, aortic biomechanics were dissimilarly affected.

Serotonin and Pulmonary Hypertension; Sex and Drugs and ROCK and Rho.

Serotonin is often referred to as a "happy hormone" as it maintains good mood, well-being, and happiness. It is involved in communication between nerve cells and plays a role in sleeping and digestion. However, too much serotonin can have pathogenic effects and serotonin synthesis is elevated in pulmonary artery endothelial cells from patients with pulmonary arterial hypertension (PAH). PAH is characterized by elevated pulmonary pressures, right ventricular failure, inflammation, and pulmonary vascular remodeling; serotonin has been shown to be associated with these pathologies. The rate-limiting enzyme in the synthesis of serotonin in the periphery of the body is tryptophan hydroxylase 1 (TPH1). TPH1 expression and serotonin synthesis are elevated in pulmonary artery endothelial cells in patients with PAH. The serotonin synthesized in the pulmonary arterial endothelium can act on the adjacent pulmonary arterial smooth muscle cells (PASMCs), adventitial macrophages, and fibroblasts, in a paracrine fashion. In humans, serotonin enters PASMCs cells via the serotonin transporter (SERT) and it can cooperate with the 5-HT1B receptor on the plasma membrane; this activates both contractile and proliferative signaling pathways. The "serotonin hypothesis of pulmonary hypertension" arose when serotonin was associated with PAH induced by diet pills such as fenfluramine, aminorex, and chlorphentermine; these act as indirect serotonergic agonists causing the release of serotonin from platelets and cells through the SERT. Here the role of serotonin in PAH is reviewed. Targeting serotonin synthesis or signaling is a promising novel alternative approach which may lead to novel therapies for PAH. © 2022 American Physiological Society. Compr Physiol 12: 1-16, 2022.