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BioRxiv : the Preprint Server For... Jun 2024Vascular stabilization is a mechanosensitive process, in part driven by blood flow. Here, we demonstrate the involvement of the mechanosensitive ion channel, Piezo1, in...
Vascular stabilization is a mechanosensitive process, in part driven by blood flow. Here, we demonstrate the involvement of the mechanosensitive ion channel, Piezo1, in promoting arterial accumulation of vascular smooth muscle cells (vSMCs) during zebrafish development. Using a series of small molecule antagonists or agonists to temporally regulate Piezo1 activity, we identified a role for the Piezo1 channel in regulating levels and altered targeting of vSMCs between arteries and veins. Increasing Piezo1 activity suppressed and increased vSMC association with the cardinal vein, while inhibition of Piezo1 activity increased levels and decreased vSMC association with arteries. We supported the small molecule data with genetic suppression of and in zebrafish, resulting in loss of vSMCs on the dorsal aorta. Further, endothelial cell (EC)-specific knockout in mice was sufficient to decrease vSMC accumulation along the descending dorsal aorta during development, thus phenocopying our zebrafish data, and supporting functional conservation of Piezo1 in mammals. To determine mechanism, we used modeling assays to demonstrate that differential sensing of pulsatile versus laminar flow forces across endothelial cells changes the expression of mural cell differentiation genes. Together, our findings suggest a crucial role for EC Piezo1 in sensing force within large arteries to mediate mural cell differentiation and stabilization of the arterial vasculature.
PubMed: 38915529
DOI: 10.1101/2024.06.11.598539 -
Circulation Jun 2024Alterations in the buffering of intracellular Ca, for which myofilament proteins play a key role, have been shown to promote cardiac arrhythmia. It is interesting that...
BACKGROUND
Alterations in the buffering of intracellular Ca, for which myofilament proteins play a key role, have been shown to promote cardiac arrhythmia. It is interesting that although studies report atrial myofibrillar degradation in patients with persistent atrial fibrillation (persAF), the intracellular Ca buffering profile in persAF remains obscure. Therefore, we aim to investigate the intracellular buffering of calcium and its potential arrhythmogenic role in persAF.
METHODS
Simultaneous transmembrane fluxes (patch-clamp) and intracellular Ca signaling (fluo-3-acetoxymethyl ester) were recorded in myocytes from right atrial biopsies of sinus rhythm (control) and patients with persAF, alongside human atrial subtype induced pluripotent stem cell-derived cardiac myocytes (iPSC-CMs). Protein levels were quantified by immunoblotting of human atrial tissue and induced pluripotent stem cell-derived cardiac myocytes. Mouse whole heart and atrial electrophysiology was measured on a Langendorff system.
RESULTS
Cytosolic Ca buffering was decreased in atrial myocytes of patients with persAF because of a depleted amount of Ca buffers. In agreement, protein levels of selected Ca binding myofilament proteins, including cTnC (cardiac troponin C), a major cytosolic Ca buffer, were significantly lower in patients with persAF. Small interfering RNA (siRNA)-mediated knockdown of cTnC in induced pluripotent stem cell-derived cardiac myocytes (si-cTnC) phenocopied the reduced cytosolic Ca buffering observed in persAF. Si-cTnC induced pluripotent stem cell-derived cardiac myocytes exhibited a higher predisposition to spontaneous Ca release events and developed action potential alternans at low stimulation frequencies. Last, indirect reduction of cytosolic Ca buffering using blebbistatin in an ex vivo mouse whole heart model increased vulnerability to tachypacing-induced atrial arrhythmia, validating the direct mechanistic link between impaired cytosolic Ca buffering and atrial arrhythmogenesis.
CONCLUSIONS
Our findings suggest that loss of myofilament proteins, particularly reduced cTnC protein levels, causes diminished cytosolic Ca buffering in persAF, thereby potentiating the occurrence of spontaneous Ca release events and AF susceptibility. Strategies targeting intracellular buffering may represent a promising therapeutic lead in AF management.
PubMed: 38910563
DOI: 10.1161/CIRCULATIONAHA.123.066577 -
Journal of Bone and Mineral Research :... Jun 2024In previous studies, we have demonstrated that stress response-induced high glucocorticoid levels could be the underlying cause of traumatic heterotopic ossification...
In previous studies, we have demonstrated that stress response-induced high glucocorticoid levels could be the underlying cause of traumatic heterotopic ossification (HO), and we have developed a glucocorticoid-induced ectopic mineralization (EM) mouse model by systemic administration of a high dose of dexamethasone (DEX) to animals with muscle injury induced by cardiotoxin injection. In this model, dystrophic calcification (DC) developed into HO in a cell autonomous manner. However, it is not clear how DC is formed after DEX treatment. Therefore, in this study, we aimed to explore how glucocorticoids initiate muscle EM at a cellular and molecular level. We showed that DEX treatment inhibited inflammatory cell infiltration into injured muscle but inflammatory cytokine production in the muscle was significantly increased, suggesting that other non-inflammatory muscle cell types may regulate the inflammatory response and the muscle repair process. Accompanying this phenotype, transforming growth factor β1 (TGF-β1) expression in fibro-adipogenic progenitors (FAPs) was greatly down-regulated. Since TGF-β1 is a strong immune suppressor and FAP's regulatory role has a large impact on muscle repair, we hypothesized that down-regulation of TGF-β1 in FAPs after DEX treatment resulted in this hyperinflammatory state and subsequent failed muscle repair and EM formation. To test our hypothesis, we utilized a transgenic mouse model to specifically knock out Tgfb1 gene in PDGFRα positive FAPs to investigate if the transgenic mice could recapitulate the phenotype that was induced by DEX treatment. Our results showed that the transgenic mice completely phenocopied this hyperinflammatory state and spontaneously developed EM following muscle injury. On the contrary, therapeutics that enhanced TGF-β1 signaling in FAPs inhibited the inflammatory response and attenuated muscle EM. In summary, these results indicate that FAPs-derived TGF-β1 is a key molecule in regulating muscle inflammatory response and subsequent EM, and that glucocorticoids exert their effect via down-regulating TGF-β1 in FAPs.
PubMed: 38896028
DOI: 10.1093/jbmr/zjae097 -
BioRxiv : the Preprint Server For... Jun 2024Our sense of hearing is critically dependent on the spiral ganglion neurons (SGNs) that connect the sound receptors in the organ of Corti (OC) to the cochlear nuclei of...
Our sense of hearing is critically dependent on the spiral ganglion neurons (SGNs) that connect the sound receptors in the organ of Corti (OC) to the cochlear nuclei of the hindbrain. Type I SGNs innervate inner hair cells (IHCs) to transmit sound signals, while type II SGNs (SGNIIs) innervate outer hair cells (OHCs) to detect moderate-to-intense sound. During development, SGNII afferents make a characteristic 90-degree turn toward the base of the cochlea and innervate multiple OHCs. It has been shown that the Planar Cell Polarity (PCP) pathway acts non-autonomously to mediate environmental cues in the cochlear epithelium for SGNII afferent turning towards the base. However, the underlying mechanisms are unknown. Here, we present evidence that PCP signaling regulates multiple downstream effectors to influence cell adhesion and the cytoskeleton in cochlear supporting cells (SCs), which serve as intermediate targets of SGNII afferents. We show that the core PCP gene Vangl2 regulates the localization of the small GTPase Rac1 and the cell adhesion molecule Nectin3 at SC-SC junctions through which SGNII afferents travel. Through genetic analysis, we also show that loss of Rac1 or Nectin3 partially phenocopied SGNII peripheral afferent turning defects in mutants, and that Rac1 plays a non-autonomous role in this process in part by regulating PCP protein localization at the SC-SC junctions. Additionally, epistasis analysis indicates that Nectin3 and Rac1 likely act in the same genetic pathway to control SGNII afferent turning. Together, these experiments identify Nectin3 and Rac1 as novel regulators of PCP-directed SGNII axon guidance in the cochlea.
PubMed: 38895287
DOI: 10.1101/2024.06.05.597585 -
BioRxiv : the Preprint Server For... Jun 2024Paclitaxel is a standard of care neoadjuvant therapy for patients with triple negative breast cancer (TNBC); however, it shows limited benefit for locally advanced or...
Paclitaxel is a standard of care neoadjuvant therapy for patients with triple negative breast cancer (TNBC); however, it shows limited benefit for locally advanced or metastatic disease. Here we used a coordinated experimental-computational approach to explore the influence of paclitaxel on the cellular and molecular responses of TNBC cells. We found that escalating doses of paclitaxel resulted in multinucleation, promotion of senescence, and initiation of DNA damage induced apoptosis. Single-cell RNA sequencing (scRNA-seq) of TNBC cells after paclitaxel treatment revealed upregulation of innate immune programs canonically associated with interferon response and downregulation of cell cycle progression programs. Systematic exploration of transcriptional responses to paclitaxel and cancer-associated microenvironmental factors revealed common gene programs induced by paclitaxel, IFNB, and IFNG. Transcription factor (TF) enrichment analysis identified 13 TFs that were both enriched based on activity of downstream targets and also significantly upregulated after paclitaxel treatment. Functional assessment with siRNA knockdown confirmed that the TFs FOSL1, NFE2L2 and ELF3 mediate cellular proliferation and also regulate nuclear structure. We further explored the influence of these TFs on paclitaxel-induced cell cycle behavior via live cell imaging, which revealed altered progression rates through G1, S/G2 and M phases. We found that ELF3 knockdown synergized with paclitaxel treatment to lock cells in a G1 state and prevent cell cycle progression. Analysis of publicly available breast cancer patient data showed that high ELF3 expression was associated with poor prognosis and enrichment programs associated with cell cycle progression. Together these analyses disentangle the diverse aspects of paclitaxel response and identify ELF3 upregulation as a putative biomarker of paclitaxel resistance in TNBC.
PubMed: 38895265
DOI: 10.1101/2024.06.04.596911 -
Diet-microbiome interactions promote enteric nervous system resilience following spinal cord injury.BioRxiv : the Preprint Server For... Jun 2024Spinal cord injury (SCI) results in a plethora of physiological dysfunctions across all body systems, including intestinal dysmotility and atrophy of the enteric nervous...
Spinal cord injury (SCI) results in a plethora of physiological dysfunctions across all body systems, including intestinal dysmotility and atrophy of the enteric nervous system (ENS). Typically, the ENS has capacity to recover from perturbation, so it is unclear why intestinal pathophysiologies persist after traumatic spinal injury. With emerging evidence demonstrating SCI-induced alterations to the gut microbiome composition, we hypothesized that modulation of the gut microbiome could contribute to enteric nervous system recovery after injury. Here, we show that intervention with the dietary fiber, inulin prevents ENS atrophy and limits SCI-induced intestinal dysmotility in mice. However, SCI-associated microbiomes and exposure to specific SCI-sensitive gut microbes are not sufficient to modulate injury-induced intestinal dysmotility. Intervention with microbially-derived short-chain fatty acid (SCFA) metabolites prevents ENS dysfunctions and phenocopies inulin treatment in injured mice, implicating these microbiome metabolites in protection of the ENS. Notably, inulin-mediated resilience is dependent on signaling by the cytokine IL-10, highlighting a critical diet-microbiome-immune axis that promotes ENS resilience following SCI. Overall, we demonstrate that diet and microbially-derived signals distinctly impact recovery of the ENS after traumatic spinal injury. This protective diet-microbiome-immune axis may represent a foundation to uncover etiological mechanisms and future therapeutics for SCI-induced neurogenic bowel.
PubMed: 38895207
DOI: 10.1101/2024.06.06.597793 -
Cell Death & Disease Jun 2024As the second most common malignant tumor in the urinary system, renal cell carcinoma (RCC) is imperative to explore its early diagnostic markers and therapeutic...
As the second most common malignant tumor in the urinary system, renal cell carcinoma (RCC) is imperative to explore its early diagnostic markers and therapeutic targets. Numerous studies have shown that AURKB promotes tumor development by phosphorylating downstream substrates. However, the functional effects and regulatory mechanisms of AURKB on clear cell renal cell carcinoma (ccRCC) progression remain largely unknown. In the current study, we identified AURKB as a novel key gene in ccRCC progression based on bioinformatics analysis. Meanwhile, we observed that AURKB was highly expressed in ccRCC tissue and cell lines and knockdown AURKB in ccRCC cells inhibit cell proliferation and migration in vitro and in vivo. Identified CDC37 as a kinase molecular chaperone for AURKB, which phenocopy AURKB in ccRCC. AURKB/CDC37 complex mediate the stabilization of MYC protein by directly phosphorylating MYC at S67 and S373 to promote ccRCC development. At the same time, we demonstrated that the AURKB/CDC37 complex activates MYC to transcribe CCND1, enhances Rb phosphorylation, and promotes E2F1 release, which in turn activates AURKB transcription and forms a positive feedforward loop in ccRCC. Collectively, our study identified AURKB as a novel marker of ccRCC, revealed a new mechanism by which the AURKB/CDC37 complex promotes ccRCC by directly phosphorylating MYC to enhance its stability, and first proposed AURKB/E2F1-positive feedforward loop, highlighting AURKB may be a promising therapeutic target for ccRCC.
Topics: Humans; Carcinoma, Renal Cell; E2F1 Transcription Factor; Kidney Neoplasms; Cell Cycle Proteins; Cell Line, Tumor; Disease Progression; Phosphorylation; Proto-Oncogene Proteins c-myc; Aurora Kinase B; Cell Proliferation; Animals; Gene Expression Regulation, Neoplastic; Mice, Nude; Mice; Cell Movement; Chaperonins
PubMed: 38890303
DOI: 10.1038/s41419-024-06827-y -
Advanced Science (Weinheim,... Jun 2024Dysferlin is a multi-functional protein that regulates membrane resealing, calcium homeostasis, and lipid metabolism in skeletal muscle. Genetic loss of dysferlin...
Dysferlin is a multi-functional protein that regulates membrane resealing, calcium homeostasis, and lipid metabolism in skeletal muscle. Genetic loss of dysferlin results in limb girdle muscular dystrophy 2B/2R (LGMD2B/2R) and other dysferlinopathies - rare untreatable muscle diseases that lead to permanent loss of ambulation in humans. The mild disease severity in dysferlin-deficient mice and diverse genotype-phenotype relationships in LGMD2B patients have prompted the development of new in vitro models for personalized studies of dysferlinopathy. Here the first 3-D tissue-engineered hiPSC-derived skeletal muscle ("myobundle") model of LGMD2B is described that exhibits compromised contractile function, calcium-handling, and membrane repair, and transcriptomic changes indicative of impaired oxidative metabolism and mitochondrial dysfunction. In response to the fatty acid (FA) challenge, LGMD2B myobundles display mitochondrial deficits and intracellular lipid droplet (LD) accumulation. Treatment with the ryanodine receptor (RyR) inhibitor dantrolene or the dissociative glucocorticoid vamorolone restores LGMD2B contractility, improves membrane repair, and reduces LD accumulation. Lastly, it is demonstrated that chemically induced chronic RyR leak in healthy myobundles phenocopies LGMD2B contractile and metabolic deficit, but not the loss of membrane repair capacity. Together, these results implicate intramyocellular Ca leak as a critical driver of dysferlinopathic phenotype and validate the myobundle system as a platform to study LGMD2B pathogenesis.
PubMed: 38887849
DOI: 10.1002/advs.202400188 -
Circulation Jun 2024HIF (hypoxia inducible factor) regulates many aspects of cardiac function. We and others previously showed that chronic HIF activation in the heart in mouse models...
BACKGROUND
HIF (hypoxia inducible factor) regulates many aspects of cardiac function. We and others previously showed that chronic HIF activation in the heart in mouse models phenocopies multiple features of ischemic cardiomyopathy in humans, including mitochondrial loss, lipid accumulation, and systolic cardiac dysfunction. In some settings, HIF also causes the loss of peroxisomes. How, mechanistically, HIF promotes cardiac dysfunction is an open question.
METHODS
We used mice lacking cardiac pVHL (von Hippel-Lindau protein) to investigate how chronic HIF activation causes multiple features of ischemic cardiomyopathy, such as autophagy induction and lipid accumulation. We performed immunoblot assays, RNA sequencing, mitochondrial and peroxisomal autophagy flux measurements, and live cell imaging on hearts and isolated cardiomyocytes. We used CRISPR-Cas9 gene editing in mice to validate a novel mediator of cardiac dysfunction in the setting of chronic HIF activation.
RESULTS
We identify a previously unknown pathway by which cardiac HIF activation promotes the loss of mitochondria and peroxisomes. We found that DEPP1 (decidual protein induced by progesterone 1) is induced under hypoxia in a HIF-dependent manner and localizes inside mitochondria. DEPP1 is both necessary and sufficient for hypoxia-induced autophagy and triglyceride accumulation in cardiomyocytes ex vivo. DEPP1 loss increases cardiomyocyte survival in the setting of chronic HIF activation ex vivo, and whole-body Depp1 loss decreases cardiac dysfunction in hearts with chronic HIF activation caused by loss in vivo.
CONCLUSIONS
Our findings identify DEPP1 as a key component in the cardiac remodeling that occurs with chronic ischemia.
PubMed: 38881449
DOI: 10.1161/CIRCULATIONAHA.123.066628 -
The Journal of the Association of... May 2024Brugada phenocopies are conditions that have an electrocardiography (ECG) pattern that mimics typical patterns seen in Brugada syndrome (BS). We report a rare case of a...
Brugada phenocopies are conditions that have an electrocardiography (ECG) pattern that mimics typical patterns seen in Brugada syndrome (BS). We report a rare case of a patient who had a Brugada-like ECG pattern caused by ischemia due to strangulation of the septal artery. The patient was treated with thrombolytic therapy after a probable diagnosis of ST-elevation myocardial infarction (STEMI), which resulted in hematologic complications.
Topics: Humans; Brugada Syndrome; Electrocardiography; Male; Middle Aged; ST Elevation Myocardial Infarction
PubMed: 38881121
DOI: 10.59556/japi.72.0507