RATIONALE:Loss-of-function studies in cardiac myocytes (CMs) are currently limited by the need for appropriate conditional knockout alleles. The factors that regulate CM maturation are poorly ...understood. Previous studies on CM maturation have been confounded by heart dysfunction caused by whole organ gene inactivation.
OBJECTIVE:To develop a new technical platform to rapidly characterize cell-autonomous gene function in postnatal murine CMs and apply it to identify genes that regulate transverse tubules (T-tubules), a hallmark of mature CMs.
METHODS AND RESULTS:We developed CRISPR/Cas9/AAV9-based somatic mutagenesis, a platform in which AAV9 delivers tandem guide RNAs targeting a gene of interest and cardiac troponin-T promoter–driven Cre to Rosa neonatal mice. When directed against junctophilin-2 (Jph2), a gene previously implicated in T-tubule maturation, we achieved efficient, rapid, and CM-specific JPH2 depletion. High-dose AAV9 ablated JPH2 in 64% CMs and caused lethal heart failure, whereas low-dose AAV9 ablated JPH2 in 22% CMs and preserved normal heart function. In the context of preserved heart function, CMs lacking JPH2 developed T-tubules that were nearly morphologically normal, indicating that JPH2 does not have a major, cell-autonomous role in T-tubule maturation. However, in hearts with severe dysfunction, both adeno-associated virus–transduced and nontransduced CMs exhibited T-tubule disruption, which was more severe in the transduced subset. These data indicate that cardiac dysfunction disrupts T-tubule structure and that JPH2 protects T-tubules in this context. We then used CRISPR/Cas9/AAV9-based somatic mutagenesis to screen 8 additional genes for required, cell-autonomous roles in T-tubule formation. We identified RYR2 (Ryanodine Receptor-2) as a novel, cell-autonomously required T-tubule maturation factor.
CONCLUSIONS:CRISPR/Cas9/AAV9-based somatic mutagenesis is a powerful tool to study cell-autonomous gene functions. Genetic mosaics are invaluable to accurately define cell-autonomous gene function. JPH2 has a minor role in normal T-tubule maturation but is required to stabilize T-tubules in the failing heart. RYR2 is a novel T-tubule maturation factor.
BACKGROUND:Catecholaminergic polymorphic ventricular tachycardia (CPVT), an inherited cardiac arrhythmia characterized by adrenergically triggered arrhythmias, is inadequately treated by current ...standard of care. Ca/calmodulin-dependent protein kinase II (CaMKII), an adrenergically activated kinase that contributes to arrhythmogenesis in heart disease models, is a candidate therapeutic target in CPVT. However, translation of CaMKII inhibition has been limited by the need for selective CaMKII inhibition in cardiomyocytes. Here, we tested the hypothesis that CaMKII inhibition with a cardiomyocyte-targeted gene therapy strategy would suppress arrhythmia in CPVT mouse models.
METHODS:We developed AAV9-GFP-AIP, an adeno-associated viral vector in which a potent CaMKII inhibitory peptide, autocamtide-2–related inhibitory peptide AIP, is fused to green fluorescent protein (GFP) and expressed from a cardiomyocyte selective promoter. The vector was delivered systemically. Arrhythmia burden was evaluated with invasive electrophysiology testing in adult mice. AIP was also tested on induced pluripotent stem cells derived from patients with CPVT with different disease-causing mutations to determine the effectiveness of our proposed therapy on human induced pluripotent stem cell–derived cardiomyocytes and different pathogenic genotypes.
RESULTS:AAV9-GFP-AIP was robustly expressed in the heart without significant expression in extracardiac tissues, including the brain. Administration of AAV9-GFP-AIP to neonatal mice with a known CPVT mutation (RYR2) effectively suppressed ventricular arrhythmias induced by either β-adrenergic stimulation or programmed ventricular pacing, without significant proarrhythmic effect. Intravascular delivery of AAV9-GFP-AIP to adolescent mice transduced ≈50% of cardiomyocytes and was effective in suppressing arrhythmia in CPVT mice. Induced pluripotent stem cell–derived cardiomyocytes derived from 2 different patients with CPVT with different pathogenic mutations demonstrated increased frequency of abnormal calcium release events, which was suppressed by a cell-permeable form of AIP.
CONCLUSIONS:This proof-of-concept study showed that AAV-mediated delivery of a CaMKII peptide inhibitor to the heart was effective in suppressing arrhythmias in a murine model of CPVT. CaMKII inhibition also reversed the arrhythmia phenotype in human CPVT induced pluripotent stem cell–derived cardiomyocyte models with different pathogenic mutations.
After birth, cardiomyocytes (CM) acquire numerous adaptations in order to efficiently pump blood throughout an animal's lifespan. How this maturation process is regulated and coordinated is poorly ...understood. Here, we perform a CRISPR/Cas9 screen in mice and identify serum response factor (SRF) as a key regulator of CM maturation. Mosaic SRF depletion in neonatal CMs disrupts many aspects of their maturation, including sarcomere expansion, mitochondrial biogenesis, transverse-tubule formation, and cellular hypertrophy. Maintenance of maturity in adult CMs is less dependent on SRF. This stage-specific activity is associated with developmentally regulated SRF chromatin occupancy and transcriptional regulation. SRF directly activates genes that regulate sarcomere assembly and mitochondrial dynamics. Perturbation of sarcomere assembly but not mitochondrial dynamics recapitulates SRF knockout phenotypes. SRF overexpression also perturbs CM maturation. Together, these data indicate that carefully balanced SRF activity is essential to promote CM maturation through a hierarchy of cellular processes orchestrated by sarcomere assembly.
Peripheral glucocorticoid receptors (GRs) play a significant role in the anti-inflammatory effects of glucocorticoids; however, the role of central GRs in nociceptive behaviors after peripheral nerve ...injury (neuropathic pain behaviors) remains unknown. Here we show that the development of neuropathic pain behaviors (thermal hyperalgesia and mechanical allodynia) induced by chronic constriction nerve injury (CCI) in rats was attenuated by either the GR antagonist RU38486 (4 = 2 > 1 = 0.5 microg) or a GR antisense oligonucleotide administered intrathecally twice daily for postoperative days 1-6. The development of thermal hyperalgesia and mechanical allodynia after CCI also was prevented in adrenalectomized rats, whereas the GR agonist dexamethasone (100 microg/kg) given subcutaneously twice daily for postoperative day 1-6 restored CCI-induced neuropathic pain behaviors in the adrenalectomized rats. Mechanistically, CCI induced a time-dependent and region-specific expression of neuronal GRs primarily within the spinal cord dorsal horn ipsilateral to nerve injury, which showed a time course parallel to that of the development of neuropathic pain behaviors. Moreover, the expression of neuronal GR after CCI was mediated in part through an elevated spinal level of interleukin-6 (IL-6) and protein kinase Cgamma (PKCgamma), because intrathecal treatment with an IL-6 antiserum, a PKC inhibitor (cheryrithrine), or PKCgamma knock-out substantially reduced the expression of neuronal GRs as well as neuropathic pain behaviors after CCI. These findings indicate a central role of neuronal GRs in the mechanisms of neuropathic pain behaviors in rats and suggest a potential role for GR antagonists in clinical management of neuropathic pain.
Catecholaminergic polymorphic ventricular tachycardia (CPVT), an inherited cardiac arrhythmia characterized by adrenergically triggered arrhythmias, is inadequately treated by current standard of ...care. Ca
/calmodulin-dependent protein kinase II (CaMKII), an adrenergically activated kinase that contributes to arrhythmogenesis in heart disease models, is a candidate therapeutic target in CPVT. However, translation of CaMKII inhibition has been limited by the need for selective CaMKII inhibition in cardiomyocytes. Here, we tested the hypothesis that CaMKII inhibition with a cardiomyocyte-targeted gene therapy strategy would suppress arrhythmia in CPVT mouse models.
We developed AAV9-GFP-AIP, an adeno-associated viral vector in which a potent CaMKII inhibitory peptide, autocamtide-2-related inhibitory peptide AIP, is fused to green fluorescent protein (GFP) and expressed from a cardiomyocyte selective promoter. The vector was delivered systemically. Arrhythmia burden was evaluated with invasive electrophysiology testing in adult mice. AIP was also tested on induced pluripotent stem cells derived from patients with CPVT with different disease-causing mutations to determine the effectiveness of our proposed therapy on human induced pluripotent stem cell-derived cardiomyocytes and different pathogenic genotypes.
AAV9-GFP-AIP was robustly expressed in the heart without significant expression in extracardiac tissues, including the brain. Administration of AAV9-GFP-AIP to neonatal mice with a known CPVT mutation (RYR2
) effectively suppressed ventricular arrhythmias induced by either β-adrenergic stimulation or programmed ventricular pacing, without significant proarrhythmic effect. Intravascular delivery of AAV9-GFP-AIP to adolescent mice transduced ≈50% of cardiomyocytes and was effective in suppressing arrhythmia in CPVT mice. Induced pluripotent stem cell-derived cardiomyocytes derived from 2 different patients with CPVT with different pathogenic mutations demonstrated increased frequency of abnormal calcium release events, which was suppressed by a cell-permeable form of AIP.
This proof-of-concept study showed that AAV-mediated delivery of a CaMKII peptide inhibitor to the heart was effective in suppressing arrhythmias in a murine model of CPVT. CaMKII inhibition also reversed the arrhythmia phenotype in human CPVT induced pluripotent stem cell-derived cardiomyocyte models with different pathogenic mutations.
Highlights • CTCs reveal the spatial and temporal heterogeneity of tumors. • High concordance was observed in molecular features between CTCs and tumors. • CTCs do not fully represent the full ...mutation spectrum of tumors. • Using CTCs to profile tumors is useful in clinical practice.
Nitrogen-doped graphene aerogels with three-dimensional network structures are fabricated using hydrothermal method which includes the reduction of graphene oxide by organic amine and self-assembly ...of reduced graphene oxide. The effect of amine-containing compounds including aniline, 2-aminoethanol, ethylenediamine, melamine and chitosan on the assembly of nitrogen-doped graphene aerogel is investigated. The microstructure and chemical composition of nitrogen-doped graphene aerogels are characterized. The results reveal that nitrogen-doped graphene aerogel prepared using aniline as nitrogen source possesses a large specific surface area, high nitrogen content, good mechanical strength and excellent electrical conductivity. Based on these features, the as-prepared nitrogen-doped graphene aerogel shows high performance in electrochemical detection of dopamine in the presence of uric acid and ascorbic acid. Given the facile and scalable processability of aerogels, the proposed nitrogen-doped graphene aerogels are expected to have potential applications in sensors and other related devices.
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•Nitrogen-doped graphene aerogels are fabricated.•The effect of amine-containing compounds on the aerogel assembly is investigated.•Aniline is beneficial for the fabrication of aerogel with better performance.•It shows high performance in electrochemical detection of dopamine.
Abstract In August 2022, Pakistan experienced an unprecedented precipitation event that caused significant damage. Analysis of the observations reveals that this extreme rainfall is primarily driven ...by anomalous atmospheric zonal advection, resulting in an anomalous water vapor concentration in Pakistan. The climatological meridional advection also contributes to this flooding. Anomalous easterly winds and low-level vertical convection combine to be critical factors contributing to the moisture concentration over the country. Further investigation identifies the air-sea interaction over the tropical Indian Ocean and abnormal warming over the Arabian Sea are crucial factors influencing this extreme flooding event. The concurrent occurrence of a negative Indian Ocean Dipole event and the warming sea surface temperature anomalies in the northern Arabian Sea intensifies the easterly winds over Pakistan, helping to transfer the anomalous water vapor from the remote region into Pakistan, ultimately contributing to the extreme flooding in 2022.
Background Previous studies have revealed that Galectin-9 (Gal-9) acts as an apoptosis modulator in autoimmunity and rheumatic inflammation. In the present study, we investigated the potential role ...of Gal-9 as a biomarker in patients with rheumatoid arthritis (RA), especially as an indicator of functional limitations and radiographic joint damage. Methods A total of 146 patients with RA and 52 age- and sex-matched healthy controls were included in this study. Clinical data including disease activity, physical function, and radiographic joint damage were assessed. Functional limitation was defined as the Stanford Health Assessment Questionnaire (HAQ) disability index >1. Subjects with joint erosion >0 or joint space narrowing >0 were considered to have radiographic joint damage. Serum Gal-9 levels were detected by an enzyme-linked immunosorbent assay. Univariate and multivariate logistic regression analysis were used to evaluate the association between Gal-9 and high disease activity and functional limitations, and a prediction model was established to construct predictive nomograms. Results Serum levels of Gal-9 were significantly increased in patients with RA compared to those in healthy controls (median 13.1 ng/mL vs. 7.6 ng/mL). Patients with RA who were older (>65 years), had a longer disease duration (>5 years), longer morning stiffness (>60mins), elevated serum erythrocyte sedimentation rate and C-reactive protein, and difficult-to-treat RA had significantly higher Gal-9 levels than those in the corresponding control subgroups (all p <0.05). Patients with RA were divided into two subgroups according to the cut-off value of Gal-9 of 11.6 ng/mL. Patients with RA with Gal-9 >11.6 ng/mL had a significantly higher core clinical disease activity index, HAQ scores, Sharp/van der Heijde modified Sharp scores, as well as a higher percentage of advanced joint damage (all p<0.05) than patients with Gal-9 ≤11.6 ng/mL. Accordingly, patients with RA presenting either functional limitations or radiographic joint damage had significantly higher serum Gal-9 levels than those without (both p <0.05). Furthermore, multivariate logistic regression analysis showed that a serum level of Gal-9 >11.6 ng/mL was an independent risk factor for high disease activity (OR=3.138, 95% CI 1.150–8.567, p=0.026) and presence of functional limitations (OR=2.455, 95% CI 1.017–5.926, p=0.046), respectively. Conclusion Gal-9 could be considered as a potential indicator in patients with RA, especially with respect to functional limitations and joint damage.
Employing low‐dimensional perovskite has been proven to be a promising approach to enhance the efficiency and stability of perovskite solar cells. Here, thiopheniformamidine hydrochloride is ...introduced into CH3NH3PbI3‐based printable mesoscopic perovskite solar cells, to form 1D iodide lead thiophenamidine (TFPbI3) in situ. This judiciously designed low‐dimensional perovskite can effectively passivate the defect of perovskite and induce the perovskite crystals to grow in a direction perpendicular to the substrate. Thus, the obtained 1D@3D perovskite could suppress the charge recombination and promote the charge transfer significantly. Benefiting from its dual effect and robustness, a significantly improved power conversion efficiency of 17.42% is yielded. The authors also develop high‐performance printable mesoscopic perovskite solar cells with a champion efficiency approaching 13% for aperture area about 11.8 cm2, as well as outstanding operational stability, retaining 90% of the original power conversion efficiency after 1000 hours of continuous illumination at the maximum power point in air.
The formation of 1D iodide lead thiophenamidine (TFPbI3) in situ in perovskite films not only passivates the defect, but also leads to oriented growth of perovskite crystal. The highly ordered 1D@3D perovskite films exhibit superior intrinsic stability and photophysical properties. Consequently, 1D@3D‐based printable mesoscopic perovskite mini‐modules demonstrate an efficiency approaching 13% with impressive long‐term operational stability.
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