Biology subjects

Pena, A.

Publications and source records attributed to Pena, A..

At least 19 recordsLinked to original sources

bioRxiv2026

PDGFD-induced pericyte contractility drives pathological neoangiogenesis

Neovascular tufts (NVTs) are a defining feature of proliferative retinopathies such as diabetic retinopathy and retinopathy of prematurity, in which they compromise retinal integrity and vision. Excessive endothelial cell (EC) sprouting underlies their formation, yet the cellular and mechanical mechanisms that shape NVTs, and the contribution of pericytes (PCs) to this process, remain unclear. In this work, we investigated the role of actomyosin contractility in ECs and PCs during pathological angiogenesis and NVT formation. We identified non-muscle myosin IIA (NMIIA) and phosphorylated myosin light chains (MLCs) as a key signature of PCs and ECs in retinopathy and NVTs. Using inducible deletion of Myh9 in the oxygen-induced retinopathy mouse model, we uncover distinct and opposing roles for NMIIA in the two cell types. Endothelial NMIIA promotes neovascularisation and prevents enlargement of NVTs, whilst PC NMIIA drives neovascular tuft formation. Mechanistically, we identify platelet-derived growth factor D (PDGFD), but not PDGFB, as a driver of pathological PC actomyosin contractility through Rho-associated coiled-coil kinase (ROCK). Genetic deletion of Pdgfd partially phenocopies the loss of NMIIA in PCs, reducing NVT formation and lowering NMIIA and phosphorylated MLC levels within NVTs. Our findings establish cell-type-specific actomyosin contractility as a determinant of NVT pathogenesis and identify PDGFD-ROCK-NMIIA signalling in PCs as a therapeutic target in proliferative retinopathies.

cell biology↗

bioRxiv2026

Distinct senescent β-cell senotypes differentially drive islet aging and dysfunction

Biological aging greatly impacts the bodys ability to handle glucose, and represents a major risk factor the development and progression of type 2 diabetes (T2D). Nonetheless, despite advances in cellular senescence research and the development of new senolytic therapies, the heterogeneity of cellular senescence in the human endocrine pancreas, as well as its roles in normal aging, remains to be elucidated at the single-cell level. Here, we performed single-cell-resolved spatial proteomics and transcriptomics on intact pancreas from 26 donors (ages 20-80) and multiplexed single-cell RNA sequencing and functional assays on dispersed islets from 14 donors (ages 34-69). We identify two discrete SnC subpopulations distinguished by relative expression of CDKN1A and CDKN2A. CDKN1A senescent cells (SnCs) exhibit loss of {beta}-cell identity, impaired insulin secretion, and a proinflammatory SASP associated with increased islet immune infiltration. In contrast, CDKN2A SnCs retain transcriptional identity and functional competence, with lower inflammatory signaling. Together, these findings identify heterogeneous and functionally divergent senotypes in the human pancreas, distinguishing an adaptive (CDKN2A) from a maladaptive (CDKN1A) senescence program, thus providing a mechanism-guided framework for senescence-targeted therapies in T2D.

cell biology↗

bioRxiv2026

Effects of Chitosan as a Permeabilizing Agent in Different Yeast Species. Studying Enzymes in situ.

Chitosan is an oligosaccharide derived from chitin that is protonated at acidic pH to form a polycation. Its positive charge promotes the interaction with negatively charged components of the yeast cell surface, which has been associated with increased cell permeability and growth inhibition. In this study, we investigated the interaction of chitosan with the cell surface and its permeabilizing capacity in three yeast species displaying distinct susceptibility profiles, Saccharomyces cerevisiae, Candida albicans and Debaryomyces hansenii. We evaluated the correlation between differential susceptibility and chitosan association at the cell surface, as well as cell permeabilization, by integrating growth analyses with surface-binding assays, including FITC-conjugated chitosan to monitor surface association and cellular integration over time, and ultrastructural examination by transmission electron microscopy (TEM). Our results showed that chitosan exhibited varying effects on the growth and permeability of each yeast strain, with D. hansenii being the most susceptible. Furthermore, we observed the incorporation of chitosan onto the cell surface and confirmed its role as a permeabilizing agent. Finally, we used chitosan-induced permeabilization as a method to measure the activity of selected enzymes in situ, demonstrating its potential for studying metabolic functions in permeabilized yeast cells. Overall, our findings establish chitosan as a strain-dependent antifungal agent and a useful tool for functional biochemical analyses in yeast.

microbiology↗

bioRxiv2026

Immune checkpoint inhibitors amplify type 2 immune mediated repair bypro-regenerative scaffolds

Extracellular matrix (ECM) scaffolds induce type 2 immunity to promote repair. Here, we show that immune cells recruited to ECM-treated murine muscle injuries and clinical soft tissue defects express immune checkpoints. Specifically, TH2 cells and regulatory T cells (Tregs) increase LAG3 expression, while macrophages express PDL2. TCR analysis and a triple-reporter strain for interleukin (IL)-13 and Treg fate-mapping suggest that Tregs in ECM-treated wounds transition into TH2-like exTregs that express LAG3. Immune checkpoint inhibition (ICI) significantly stimulated type 2 immunity in ECM-treated wounds, including increased TH2 cells, Treg transition to TH2-like exTregs, and pro-regenerative macrophages. Moreover, ICI enhanced muscle repair and reduced fibrosis in ECM-treated wounds. Collectively, these findings show Treg/TH2 plasticity in wound healing and introduce a novel ICI application to enhance immune-mediated regeneration.

bioengineering↗

bioRxiv2025

Comprehensive analysis of proteins associated with light responses and stress tolerance in the gametophyte of the fern Dryopteris affinis ssp. affinis

The fern gametophyte, despite its crucial role as the pioneer stage ensuring the successful establishment of the fern, has remained largely neglected as a subject of research, being particularly evident at the molecular level. In this study, based on an RNA-sequenced gametophyte transcriptome from the apogamous fern Dryopteris affinis ssp. affinis, 1,160 proteins functionally associated with light responses, transport processes, and stress mechanisms were identified. Light-responsive proteins are linked to photosynthesis, photorespiration, xanthophyll metabolism, and photomorphogenesis. Among them are several HIGH CHLOROPHYLL FLUORESCENCE (HCF 136, 173 and 244), which participate in the formation of photosystem II, and ENHANCER OF VARIEGATION 3 (EVR3), which is involved in chloroplast biogenesis. Regarding photomorphogenesis, the protein list includes the photoreceptors PHYTOCHROME B (PHYB), CRYPTOCHROMES 1 and 2 (CRY1 and 2), PHOTOTROPIN 2 (PHOT2), and UV RESISTANCE LOCUS 2, 3 and 8 (UVR2, 3 and 8). Likewise, related to transport, 659 proteins were found either in membranes and cytoplasm, moving a wide range of molecules, such as PERMEASE 2 and 3 (AAP2 and 3), SUCROSE TRANSPORTER 4 (SUC4), AMMONIUM TRANSPORTER 1 (AMT1), MECHANOSENSITIVE ION CHANNEL PROTEIN 1 (MSL1), among others. Furthermore, our results comprised an extensive arsenal of proteins to battle against both biotic and abiotic stress, or involved in plant immunity, such as BRASSINOSTEROID-SIGNALLING KINASE 1 (BSK1), NON-RESPONDING TO OXYLIPINS 7 (NOXY7), and CALLOSE SYNTHASE 12 (CALS12); whereas -MANNOSIDASE 2 (GMII), PALMITOYL-MONOGALACTOSYLDIACYLGLYCEROL {Delta}-7 DESATURASE (ADS3), and FORGETTER 1 (FGT1) are linked to salt, cold or heat stresses, respectively. Analysis of protein-protein interactions revealed MODIFIED TRANSPORT TO THE VACUOLE 17 (MTV17) as the most connected protein, with 19 interactions, mainly supported by text-mining and database evidence. These findings contribute to expanding the limited knowledge at the molecular level of ferns overall, paving the way for future experimental investigations.

plant biology↗

bioRxiv2025

Missense variants in the myosin binding domains of MYBPC3 and MYBPHL impair sarcomere incorporation

Approximately 40% of genetic hypertrophic cardiomyopathy cases involve mutations in MYBPC3, which encodes cardiac myosin binding protein-C (cMyBP-C), a key regulator of sarcomere contractility. The atrial-specific paralog, myosin binding protein-H like (MyBP-HL), has been associated with dilated cardiomyopathy in humans and mice. Both proteins bind to the same binding sites in the thick filament C-zone. In the atria, cMyBP-C and MyBP-HL are found at [~]1:1 ratios, while ventricles only express cMyBP-C, which is found at twice the atrial level, indicating a stoichiometric relationship. In the atria, we hypothesize that missense variants in either gene may cause alterations in thick filament binding affinity and changes in the normal [~]1:1 ratio. Notably, MyBP-HL deletion in atrial myofibrils accelerates relaxation kinetics, suggesting that altered stoichiometry impacts biophysical parameters. We hypothesized that deletion, overexpression, or missense variants in either gene would alter the abundance of the other protein in atrial sarcomeres, affecting sarcomere localization and function. To test this, we engineered two constructs: a mini-C construct comprising thick filament-binding domains of cMyBP-C and a MyBP-HL construct. Selected MYBPC3 and MYBPHL missense variants were introduced and expressed in neonatal rat ventricular cardiomyocytes (NRVMs). Sarcomere localization was assessed by co-localization with endogenous cMyBP-C. MYBPC3 variants were selected across a range of pathogenicity, while MYBPHL variants were based on evolutionary conservation of residues. MYBPHL variants Gly275Ser, Arg285His, and Ala342Thr induced significant sarcomere mislocalization, and MYBPC3 variants Pro1181Ala and Asn1257Lys showed variable effects on sarcomere mislocalization. To assess stoichiometric effects, we developed a T2A/P2A polycistronic construct to co-express mini-C, Td-Tomato, and MyBP-HL. Immunoblotting and mass spectrometry confirmed consistent and reproducible expression. We identified several MYBPC3 and MYBPHL variants that reduced the affinity of their protein for myofilament incorporation. These results suggest that this 2A construct is a useful tool for measuring the effect of myosin binding protein missense variants on sarcomere affinity, with implications for assessing pathogenicity of these variants.

biochemistry↗

bioRxiv2025

Social observation influences the trajectory of performance monitoring across trials: evidence from single-trial estimates of the ERN and CRN

The error-related negativity (ERN) and correct-related negativity (CRN) are event-related potentials (ERPs) that reflect performance monitoring following error and correct responses, respectively. Prior work demonstrates the ERN is sensitive to the motivational significance of errors, which increases under social observation. However, most studies testing how social observation impacts performance monitoring rely on trial-averaged ERPs, potentially obscuring meaningful fluctuations in ERN/CRN over time. Here, we had participants complete a Flanker task twice (social observation vs. alone) and employed mixed-effects modeling of single-trial ERPs to test if social observation impacts ERN/CRN trajectories over short (within blocks) or long (between blocks) timescales. We found that social observation selectively influenced ERN/CRN trajectories over short timescales: for blocks performed under social observation (but not alone), ERN magnitudes increased across trials and CRN magnitudes decreased. At longer timescales, ERN/CRN significantly decreased across all blocks, regardless of social observation and consistent with a vigilance decrement. To our knowledge, this is the first demonstration that social observation influences performance monitoring trajectories over short timescales. Results highlight the importance of analyzing ERN/CRN trajectories over relatively short timescales to fully characterize the impact of social observation on performance monitoring dynamics. These findings lay the groundwork for future investigation into whether social observation interacts with individual differences in motivation/affect to differentially impact performance monitoring dynamics.

neuroscience↗

bioRxiv2025

Benzo(a)pyrene degradation induces coordinated antioxidant and detoxification responses in the marine yeast Debaryomyces hansenii

Polycyclic aromatic hydrocarbons (PAHs), such as benzo(a)pyrene (BaP), are persistent environmental pollutants recognized for their high toxicity and resistance to microbial degradation. In the search for efficient and promising organisms in mycoremediation, the marine yeast Debaryomyces hansenii has emerged as a compelling candidate, due to its ability to thrive under a variety of stressful conditions. This study presents the first integrated molecular and biochemical characterization of BaP degradation in a marine yeast, providing mechanistic insights into the detoxification and antioxidant responses involved. In this study, we explored D. hanseniis capacity to degrade BaP and characterized the associated molecular and biochemical responses induced by this compound. Our findings demonstrate that D. hansenii can tolerate BaP concentrations up to 100 ppm without compromising cell viability and is capable of degrading nearly 70% of the compound within six days. The degradation process appears to be enzymatically mediated, primarily involving cytochrome P450 (CYP), epoxide hydrolase (EH), and glutathione S-transferase (GST), enzymes typically associated with xenobiotic metabolism, and reactive oxygen species (ROS) generation. BaP exposure resulted in pronounced oxidative stress, evidenced by elevated ROS levels, lipid peroxidation, and protein carbonylation. However, D. hansenii activated a robust antioxidant defense, including superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and regulation of the glutathione redox system. Altogether, our data unveil a tightly coordinated cellular strategy that combines oxidation, conjugation, and detoxification pathways to counteract BaP-induced toxicity and sustain redox homeostasis. These insights position D. hansenii as a promising and metabolically adaptive organism for bioremediation of PAH-contaminated environments.

microbiology↗

bioRxiv2025

Transcriptomic profiling of Debaryomyces hansenii reveals detoxification and stress responses to benzo(a)pyrene exposure

The environmental accumulation of polycyclic aromatic hydrocarbons (PAHs), such as benzo(a)pyrene (BaP), poses significant threats to ecosystems and public health due to their persistent nature, mutagenic potential, and well-documented carcinogenicity. In this study, we investigated the ability of the extremophilic yeast Debaryomyces hansenii to activate specialized detoxification mechanisms for BaP degradation, even under nutrient-deprived conditions. When exposed to 100 ppm BaP, D. hansenii eliminated over 70% of the contaminant within three days while maintaining normal growth dynamics. RNA-Seq analysis revealed widespread transcriptional remodeling, with 1179 genes upregulated and 1031 downregulated under BaP-only conditions, and 1067 upregulated and 977 downregulated genes during co-metabolic exposure (2% glucose + 100 ppm BaP), from a total of 6506 annotated genes. Gene Ontology (GO) and KEGG enrichment analyses highlighted the activation of xenobiotic degradation pathways, notably involving cytochrome P450 monooxygenases (CYPs), epoxide hydrolases (EHs), and glutathione S-transferases (GSTs), alongside an enhanced antioxidant response and finely tuned glutathione homeostasis. This work provides the first comprehensive transcriptomic profile of BaP detoxification in D. hansenii, revealing an intricate and highly adaptive stress response. Collectively, these findings position D. hansenii as a promising eukaryotic platform for bioremediation in saline and contaminated environments, especially where conventional microbial candidates fall short due to environmental extremes or nutrient scarcity. ImportancePolycyclic aromatic hydrocarbons (PAHs), such as benzo(a)pyrene (BaP), are long-lasting environmental pollutants with serious health and ecological implications. Although microbial degradation offers a promising strategy for remediation, most efforts have focused on bacterial and filamentous fungal systems, leaving other microbial groups comparatively unexplored. In contrast, extremotolerant yeasts remain largely overlooked despite their inherent resilience. Here, we investigated the marine yeast Debaryomyces hansenii and discovered that it not only tolerates BaP under nutrient-limited conditions but also actively degrades it. This response relies on a combination of detoxifying enzymes and antioxidant defenses, reflecting a well-orchestrated metabolic adaptation to chemical stress. Our findings highlight the untapped and promising potential of D. hansenii as a robust eukaryotic chassis for bioremediation, particularly in environments where conventional microbes may fail to survive.

biochemistry↗

bioRxiv2025

A human and mouse subpopulation of senescent β-cells induces pathologic dysfunction through targetable paracrine signaling

Cellular senescence is a stress response mechanism marked by irreversible growth arrest, upregulation of antiapoptotic pathways, loss of cellular function, and remodelling of the cellular secretory profile. In both humans and mice, pancreatic {beta}-cells undergo senescence with age and insulin resistance. Targeted removal of senescent cells in mouse models of diabetes improves glucose homeostasis, demonstrating the role {beta}-cell senescence in diabetes progression. In contrast, {beta}-cell senescence also promotes immune surveillance, promoting {beta}-cell survival and function. Thus, a better understanding of senescent cells phenotypic and functional heterogeneity is needed to develop effective therapeutic strategies. Herein, we show that subpopulations of senescent {beta}-cells in mice and humans, which were identified through the expression of Cdkn1a (encoding p21Cip1) and Cdkn2a (encoding p16Ink4a) by single-cell RNA sequencing (scRNA-seq), flow cytometry, spatial transcriptomics, and spatial proteomics, exhibit distinct transcriptional and functional identities. The predominant senescent {beta}-cell subpopulation expressed Cdkn1a and was characterized by a lack of glucose responsiveness, high basal insulin secretion, and transcription of canonical SASP factors. The SASP of Cdkn1a-expressing {beta}-cells had non-cell autonomous effects on neighbouring cells. A subset of four SASP factors from Cdkn1a+ cells was sufficient to induce secondary senescence and {beta}-cell dysfunction in vitro. JAK inhibitors (JAK1/2 and JAK1/3) counteracted secondary senescence induction and restored {beta}-cell function in high-fat diet-fed mice and human islets from donors with or without type 2 diabetes. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=196 SRC="FIGDIR/small/648438v1_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@9b8addorg.highwire.dtl.DTLVardef@1b9c4eborg.highwire.dtl.DTLVardef@12f2964org.highwire.dtl.DTLVardef@1468297_HPS_FORMAT_FIGEXP M_FIG C_FIG

developmental biology↗

bioRxiv2025

Non-canonical HIPPO-MST1/2 promotes hyper-proliferation of pulmonary vascular cells through CDC20

HIPPO components mammalian Ste20-like protein kinases 1 and 2 (MST1/2) are well described growth suppressors. However, in pulmonary arterial hypertension (PAH), MST1/2 switch their roles and become pro-proliferative and pro-survival molecules, supporting hyper-proliferation of pulmonary artery (PA) smooth muscle cells (PASMCs) and adventitial fibroblasts (PAAFs), remodeling of small PAs, and pulmonary hypertension. Here, we report that MST1/2 promotes hyper-proliferation and apoptosis resistance of human PAH PASMCs and PAAFs by up-regulating cell division cycle protein 20 (CDC20), establishing novel link between HIPPO-MST1/2 and cell cycle regulation in PAH. Authors Contributionsconception and design of the work (EAG, SSP, TVK); acquisition, analysis, and interpretation of data (TD, IZ, LJ, SOO, AP, TA, DL, DG, JRG, PJW, HD, TK); drafting and editing the manuscript (EAG, SSP, TVK, JRG, PJW).

cell biology↗

bioRxiv2024

A complete approach for circRNA therapeutics from purification to lyophilized delivery using novel ionizable lipids

Circular RNA (circRNA) has gained significant attention as a potential therapeutic tool due to its remarkable stability and resistance to degradation by exonucleases. However, scalable and efficient methods for purification and delivery remain critical challenges that must be addressed. In this study, we developed and evaluated an optimized affinity chromatography method using Oligo(dT) columns for the purification of circRNA, achieving high yield and purity comparing with high-performance liquid chromatography. Additionally, we investigated the in vivo efficacy of circRNA-Oligo(dT) encapsulated in lipid nanoparticles (LNPs) formulated with emerging STAAR ionizable lipids, including CP-LC-0867 and CP-LC-0729. Our results showed that LNPs formulated with CP-LC-0867 consistently produced higher protein expression compared to SM-102, with sustained luciferase activity observed over a 14-day period. Furthermore, we assessed the lyophilization potential of LNP-circRNA-Oligo(dT) using CP-LC-0729 to extend shelf life and eliminate the need for ultra-low temperature storage. Remarkably, the lyophilized LNPs exhibited no significant differences in protein expression compared to their non-lyophilized counterparts, demonstrating that lyophilization is a viable strategy for extending the storage and transport of circRNA therapies. These findings underscore the potential of optimized new ionizable lipids, improved purification strategies, and lyophilization techniques to enhance the scalability, stability, and practical application of circRNA therapies. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=91 SRC="FIGDIR/small/620632v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@8bb7eeorg.highwire.dtl.DTLVardef@d8a1eeorg.highwire.dtl.DTLVardef@6f359forg.highwire.dtl.DTLVardef@1295d05_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2024

The effects of conditional loss of myosin binding protein H-like on cardiac function.

Mutations in the myosin-binding protein H-like (MyBP-HL) gene, MYBPHL, are linked to hereditary dilated cardiomyopathy (DCM), atrial fibrillation, and atrioventricular arrhythmias. MyBP-HL is a sarcomeric protein that is highly expressed in the atria with only scarce, distinct clusters of MyBP-HL positive cells within and surrounding the ventricular conduction system. Constitutive knock-out of MyBP-HL in mice causes atrial dilation, arrhythmia, and DCM. Whether MyBP-HL plays a developmental role, or if knock-down in adulthood will recapitulate a similar phenotype has yet to be examined. Moreover, the significance of the MyBP-HL expressing ventricular cells, or the functional need for differential thick filament regulation is currently unknown. We used a conditional floxed Mybphl mouse to further elucidate the role of MyBP-HL. We crossed this mouse with a ROSA26-Cre(ERT2) LoxP mouse to conditionally knock-down Mybphl after tamoxifen treatment. We also crossed the Mybphl flox mouse with a Contactin-2-Cre mouse that deletes Mybphl solely in the cardiac conduction system from birth. Echocardiography was used to measure contractile function, and conscious telemetry allowed for monitoring of heart rhythm and electrical signal conduction changes. We demonstrate that mice with conditional decrease of MyBP-HL in adulthood develop a hypertrophic phenotype with atrial contractile changes, increased total heart weight to body weight, and increased heart rate variability. Deletion of Mybphl solely within the cardiac conduction system trends toward mild hypercontractility, lower heart rates, and interventricular septal thickening. These data show that MyBP-HL is essential for proper cardiac function, and even minor alteration in protein levels cause a diseased cardiac phenotype.

physiology↗

bioRxiv2024

Multicomponent Thiolactone-Based Ionizable Lipid Screening Platform for an Efficient and Tunable mRNA Delivery to the Lungs

Ionizable lipids are an essential component of lipid nanoparticles (LNPs) for an efficient mRNA delivery. However, optimizing their chemical structures for high protein expression, efficient endosomal escape, and selective organ targeting remains challenging due to complex structure-activity relationships and multistep synthesis. In this study, we introduce a rapid, high-throughput platform for screening ionizable lipids using a two-step, scalable synthesis involving a one-pot 3-component click-like reaction. This method, herein known as the STAAR approach, standing for Sequential Thiolactone Amine Acrylate Reaction, allowed for the combinatorial synthesis and in vivo screening of 91 novel lipids, followed by a structure-activity study. This led to the development of CP-LC-0729, an ionizable lipid that significantly surpasses the benchmark in protein expression while showing no in vivo toxicity. Additionally, the STAAR lipid platform was further validated by incorporating a one-step strategy to yield a permanently cationic lipid which was tested following a fifth-lipid formulation strategy. The in vivo results showed a highly selective lung delivery with a 32-fold increase in protein expression, outperforming current endogenous targeting strategies. All these findings underscore the potential of lipid CP-LC-0729 and the STAAR lipid platform in advancing the efficiency and specificity of mRNA delivery systems, while also advancing the development of new ionizable lipids.

biochemistry↗

bioRxiv2024

MYBPHL nonsense mutations have poor sarcomere binding, are degraded, and cause abnormal contraction

Heart function depends on the cardiomyocyte contractile apparatus and proper sarcomere protein expression. Mutations in sarcomere genes cause inherited forms of cardiomyopathy and arrhythmias, including atrial fibrillation (AF). Recently, a novel sarcomere component, myosin binding protein-H like (MyBP-HL) was identified. MyBP-HL is mainly expressed in cardiac atria and shares homology to the last three C-terminal domains of cardiac myosin binding protein-C (cMyBP-C). The MYBPHL R255X mutation has been linked to atrial enlargement, dilated cardiomyopathy, and atrial and ventricular arrhythmias. Similar nonsense mutations in MYBPC3 result in no myofilament incorporation and a rapid degradation of the truncated protein and are highly associated with development of hypertrophic cardiomyopathy. However, the MYBPHL R255X mutation occurs too frequently in the human population to be highly pathogenic. We sought to determine whether all MYBPHL nonsense mutations lead to impaired MyBP-HL sarcomere integration and degradation of the mutant protein, or if the MYBPHL R255X mutation has a different consequence. We mimicked human MYBPHL nonsense mutations in the mouse Mybphl cDNA sequence and tested their sarcomere incorporation in neonatal rat cardiomyocytes. We demonstrated that wild type MyBP-HL overexpression showed the expected C-zone sarcomere incorporation, like cMyBP-C. Nonsense mutations showed defective sarcomere incorporation. We demonstrated that wild type MyBP-HL and MyBP-HL nonsense mutations were degraded by both proteasome and calpain mechanisms. Additionally, we observed changes in contraction kinetics and calcium transients in cells transfected with MyBP-HL nonsense mutations compared to MyBP-HL full length. Together, these data support the hypothesis that MYBPHL nonsense mutations are largely similar. Short summaryPremature stop mutations in myosin binding protein H-like prevent sarcomere incorporation of the translated protein. Overexpression of truncating mutants causes contractile defects in neonatal rat cardiomyocytes. These effects occur regardless of the location of the premature stop along the protein.

physiology↗

bioRxiv2023

Proteolytic degradation of atrial sarcomere proteins underlies contractile defects in atrial fibrillation

(ii) ABSTRACTO_ST_ABSAimsC_ST_ABSAtrial fibrillation (AFib) is the most common cardiac rhythm disturbance. Treatment of AFib involves restoration of the atrial electrical rhythm. Following rhythm restoration, a period of depressed mechanical function known as atrial stunning occurs that involves decreased blood flow velocity and reduced atrial contractility. This suggests that defects in contractility occur in AFib and are revealed upon restoration of rhythm. The aim of this project is to define the contractile remodeling that occurs in AFib Methods and ResultsTo assess contractile function, we used a canine atrial tachypacing model of induced AFib. Mass spectrometry analysis showed dysregulation of contractile proteins in samples from AFib compared to sinus rhythm atria. Atrial cardiomyocytes showed reduced force of contraction in skinned single cardiomyocyte calcium-force studies. There were no significant differences in myosin heavy chain isoform expression. Resting tension is decreased in the AFib samples correlating with reduced full-length titin in the sarcomere. We measured degradation of other myofilament proteins including cMyBP-C, actinin, and cTnI, showing significant degradation in the AFib samples compared to sinus rhythm atria. Many of the protein degradation products appeared as discrete cleavage products that are generated by calpain proteolysis. We assessed calpain activity and found it to be significantly increased. Skinned cardiomyocytes from AFib atria showed decreased troponin I phosphorylation, consistent with the increased calcium sensitivity that was found within these cardiomyocytes. ConclusionsWith these results it can be concluded that AFib causes alterations in contraction that can be explained by both molecular changes occurring in myofilament proteins and overall myofilament protein degradation. These results provide an understanding of the contractile remodeling that occurs in AFib and provides insight into the molecular explanation for atrial stunning and the increased risk of atrial thrombus and stroke in AFib.

cell biology↗

bioRxiv2023

A non-genetic model of vascular shunts informs on the cellular mechanisms of formation and resolution of arteriovenous malformations

Arteriovenous malformations (AVMs), a disorder characterized by direct shunts between arteries and veins, are associated with genetic mutations. However, the mechanisms leading to the transformation of a capillary into a shunt remain unclear and how shunts can be reverted into capillaries is poorly understood. Here, we report that oxygen-induced retinopathy (OIR) protocol leads to the consistent and stereotypical formation of AV shunts in non-genetically altered mice. OIR-induced AV shunts show all the canonical markers of AVMs. Genetic and pharmacological interventions demonstrated that changes in endothelial cell (EC) volume of venous origin (hypertrophic venous cells) are the initiating step promoting AV shunt formation, whilst EC proliferation or migration played minor roles. Inhibition of mTOR pathway prevents pathological increases in EC volume and significantly reduces the formation of AV shunts. Importantly, we demonstrate that ALK1 signaling cell-autonomously regulates EC volume, demonstrating that our discoveries link with hereditary hemorrhagic telangiectasia (HHT)-related AVMs. Finally, we demonstrate that a combination of EC volume control and EC migration is associated with the regression of AV shunts. We demonstrate that an increase in the EC volume is the key mechanism driving the initial stages of AV shunt formation, leading to asymmetric capillary diameters. Based on our results, we propose a coherent and unifying timeline leading to the fast conversion of a capillary vessel into an AV shunt. Our data advocates for further investigation into the mechanisms regulating EC volume in health and disease as a way to identify therapeutic approaches to prevent and revert AVMs.

cell biology↗

bioRxiv2023

Terpenes from Cannabis sativa Induce Antinociception in Mouse Chronic Neuropathic Pain via Activation of Spinal Cord Adenosine A2A Receptors

Terpenes are small hydrocarbon compounds that impart aroma and taste to many plants, including Cannabis sativa. A number of studies have shown that terpenes can produce pain relief in various pain states in both humans and animals. However, these studies were methodologically limited and few established mechanisms of action. In our previous work, we showed that the terpenes geraniol, linalool, {beta}-pinene, - humulene, and {beta}-caryophyllene produced cannabimimetic behavioral effects via multiple receptor targets. We thus expanded this work to explore the efficacy and mechanism of these Cannabis terpenes in relieving chronic pain. We first tested for antinociceptive efficacy by injecting terpenes (200 mg/kg, IP) into male and female CD- 1 mice with chemotherapy-induced peripheral neuropathy (CIPN) or lipopolysaccharide-induced inflammatory pain, finding that the terpenes produced roughly equal efficacy to 10 mg/kg morphine or 3.2 mg/kg WIN55,212. We further found that none of the terpenes produced reward as measured by conditioned place preference, while low doses of terpene (100 mg/kg) combined with morphine (3.2 mg/kg) produced enhanced antinociception vs. either alone. We then used the adenosine A2A receptor (A2AR) selective antagonist istradefylline (3.2 mg/kg, IP) and spinal cord-specific CRISPR knockdown of the A2AR to identify this receptor as the mechanism for terpene antinociception in CIPN. In vitro cAMP and binding studies and in silico modeling studies further suggested that the terpenes act as A2AR agonists. Together these studies identify Cannabis terpenes as potential therapeutics for chronic neuropathic pain, and identify a receptor mechanism in the spinal cord for this activity.

neuroscience↗