Faculty Bibliography

Rationale: The cardiac sodium channel NaV1.5 has a fundamental role in excitability and conduction. Previous studies have shown that sodium channels cluster together in specific cellular subdomains. Their association with intracellular organelles in defined regions of the myocytes, and the functional consequences of that association, remain to be defined. Objective: To characterize a subcellular domain formed by sodium channel clusters in the crest region of the myocytes, and the subjacent subsarcolemmal mitochondria (SSM).Methods and Results: Through a combination of imaging approaches including super-resolution microscopy and electron microscopy we identified, in adult cardiac myocytes, a NaV1.5 subpopulation in close proximity to SSM; we further found that SSM preferentially host the mitochondrial Na+/Ca2+ exchanger (NCLX). This anatomical proximity led us to investigate functional changes in mitochondria resulting from sodium channel activity. Upon TTX exposure, mitochondria near NaV1.5 channels accumulated more Ca2+ and showed increased ROS production when compared to interfibrillar mitochondria. Finally, crosstalk between NaV1.5 channels and mitochondria was analyzed at a transcriptional level. We found that SCN5A and SLC8B1 (which encode NaV1.5 and NCLX, respectively) are negatively correlated both in a human transcriptome dataset (GTEx) and in human-induced pluripotent stem cell-derived cardiac myocytes deficient in SCN5A. Conclusions: We describe an anatomical hub (a couplon) formed by sodium channel clusters and SSM. Preferential localization of NCLX to this domain allows for functional coupling where the extrusion of Ca2+ from the mitochondria is powered, at least in part, by the entry of sodium through NaV1.5 channels. These results provide a novel entry-point into a mechanistic understanding of the intersection between electrical and structural functions of the heart.

A farnesylated and methylated form of prelamin A called progerin causes Hutchinson-Gilford progeria syndrome (HGPS). Inhibiting progerin methylation by inactivating the isoprenylcysteine carboxylmethyltransferase (ICMT) gene stimulates proliferation of HGPS cells and improves survival of Zmpste24-deficient mice. However, we don't know whether Icmt inactivation improves phenotypes in an authentic HGPS mouse model. Moreover, it is unknown whether pharmacologic targeting of ICMT would be tolerated by cells and produce similar cellular effects as genetic inactivation. Here, we show that knockout of Icmt improves survival of HGPS mice and restores vascular smooth muscle cell numbers in the aorta. We also synthesized a potent ICMT inhibitor called C75 and found that it delays senescence and stimulates proliferation of late-passage HGPS cells and Zmpste24-deficient mouse fibroblasts. Importantly, C75 did not influence proliferation of wild-type human cells or Zmpste24-deficient mouse cells lacking Icmt, indicating drug specificity. These results raise hopes that ICMT inhibitors could be useful for treating children with HGPS.

miR-33 is an intronic microRNA within the gene encoding the SREBP2 transcription factor. Like its host gene, miR-33 has been shown to be an important regulator of lipid metabolism. Inhibition of miR-33 has been shown to promote cholesterol efflux in macrophages by targeting the cholesterol transporter ABCA1, thus reducing atherosclerotic plaque burden. Inhibition of miR-33 has also been shown to improve high-density lipoprotein (HDL) biogenesis in the liver and increase circulating HDL-C levels in both rodents and nonhuman primates. However, evaluating the extent to which these changes in HDL metabolism contribute to atherogenesis has been hindered by the obesity and metabolic dysfunction observed in whole-body miR-33-knockout mice. To determine the impact of hepatic miR-33 deficiency on obesity, metabolic function, and atherosclerosis, we have generated a conditional knockout mouse model that lacks miR-33 only in the liver. Characterization of this model demonstrates that loss of miR-33 in the liver does not lead to increased body weight or adiposity. Hepatic miR-33 deficiency actually improves regulation of glucose homeostasis and impedes the development of fibrosis and inflammation. We further demonstrate that hepatic miR-33 deficiency increases circulating HDL-C levels and reverse cholesterol transport capacity in mice fed a chow diet, but these changes are not sufficient to reduce atherosclerotic plaque size under hyperlipidemic conditions. By elucidating the role of miR-33 in the liver and the impact of hepatic miR-33 deficiency on obesity and atherosclerosis, this work will help inform ongoing efforts to develop novel targeted therapies against cardiometabolic diseases.

Importance/UNASSIGNED:To date, no study has characterized the mucocutaneous features seen in hospitalized children with multisystem inflammatory syndrome in children (MIS-C) or the temporal association of these findings with the onset of systemic symptoms. Objective/UNASSIGNED:To describe the mucocutaneous findings seen in children with MIS-C during the height of the coronavirus disease 2019 (COVID-19) pandemic in New York City in 2020. Design, Setting, and Participants/UNASSIGNED:A retrospective case series was conducted of 35 children admitted to 2 hospitals in New York City between April 1 and July 14, 2020, who met Centers for Disease Control and Prevention and/or epidemiologic criteria for MIS-C. Main Outcomes and Measures/UNASSIGNED:Laboratory and clinical characteristics, with emphasis on mucocutaneous findings, of children who met criteria for MIS-C. The characterization of mucocutaneous features was verified by 2 board-certified pediatric dermatologists. Results/UNASSIGNED:Twenty-five children (11 girls [44%]; median age, 3 years [range, 0.7-17 years]) were identified who met definitional criteria for MIS-C; an additional 10 children (5 girls [50%]; median age, 1.7 years [range, 0.2-15 years]) were included as probable MIS-C cases (patients met all criteria with the exception of laboratory test evidence of severe acute respiratory syndrome coronavirus 2 [SARS-CoV-2] infection or known exposure). The results of polymerase chain reaction tests for SARS-CoV-2 were positive for 10 patients (29%), and the results of SARS-CoV-2 immunoglobulin G tests were positive for 19 patients (54%). Of the 35 patients, 29 (83%) exhibited mucocutaneous changes, with conjunctival injection (n = 21), palmoplantar erythema (n = 18), lip hyperemia (n = 17), periorbital erythema and edema (n = 7), strawberry tongue (n = 8), and malar erythema (n = 6) being the most common findings. Recognition of mucocutaneous findings occurred a mean of 2.7 days (range, 1-7 days) after the onset of fever. The duration of mucocutaneous findings varied from hours to days (median duration, 5 days [range, 0-11 days]). Neither the presence nor absence of mucocutaneous findings was significantly associated with overall disease severity. Conclusions and Relevance/UNASSIGNED:In this case series of hospitalized children with suspected MIS-C during the COVID-19 pandemic, a wide spectrum of mucocutaneous findings was identified. Despite their protean and transient nature, these mucocutaneous features serve as important clues in the recognition of MIS-C.

Purpose/UNASSIGNED:Besides regulating paracellular diffusion, claudin-19 modulates the expression of proteins essential for the retinal pigment epithelium (RPE). This study asks how RPE responds when the expression of claudin-19 is reduced. Methods/UNASSIGNED:In stem cell-derived RPE, claudin-19 and sequestosome-1/p62 (SQSTM1) were knocked down with siRNAs. Expression was monitored by quantitative RT-PCR and western blotting. Morphology and function were monitored by immunocytochemistry and transepithelial electrical resistance (TER). Phagocytosis of photoreceptor outer segments (POSs) was followed by fluorescence-activated cell sorting and western blotting. Pharmacology was used to assess the effects of AMP-activated protein kinase (AMPK) and SQSTM1 on phagocytosis. Enzymatic activity was measured using commercial assay kits. Results/UNASSIGNED:Knockdown of claudin-19 reduced the TER without affecting the integrity of the apical junctional complex, as assessed by the distribution of zonula occludens-1 and filamentous actin. AMPK was activated without apparent effect on autophagy. Activation of AMPK alone had little effect on phagocytosis. Without affecting ingestion, knockdown reduced the rate of POS degradation and increased the steady-state levels of LC3B and SQSTM1. Proteasome inhibitors also retarded degradation, as did knockdown of SQSTM1. The expression of metallothioneins and the activity of superoxide dismutase increased. Conclusions/UNASSIGNED:Knockdown of claudin-19 slowed the degradation of internalized POSs. The study questions the role of activated AMPK in phagocytosis and suggests a role for SQSTM1. Further, knockdown was associated with a partial oxidative stress response. The study opens new avenues of experimentation to explore these essential RPE functions.

INTRODUCTION/BACKGROUND:Transforming growth factor β (TGFβ) promotes cell survival by endorsing DNA damage repair and mediates an immunosuppressive tumor microenvironment. Thus, TGFβ activation in response to radiation therapy is potentially targetable because it opposes therapeutic control. Strategies to assess this potential in the clinic are needed. METHODS:Zr -fresolimumab, a humanized TGFβ neutralizing monoclonal antibody, as means to detect TGFβ activation in intracranial tumor models. TGFβ pathway activity was validated by immunodetection of phosphorylated SMAD2 and TGFβ target, tenascin. The contribution of TGFβ to radiation response was assessed by Kaplan Meier survival analysis of mice bearing intracranial murine tumor models, GL261 and SB28 glioblastoma and brain-adapted 4T1 breast cancer (4T1-BrA) treated with TGFβ neutralizing monoclonal antibody, 1D11, and/or focal radiation (10 Gy). RESULTS:Zr-fresolimumab PET imaging detected engineered, physiological and radiation-induced TGFβ activation, which was confirmed by immunostaining of biological markers. GL261 glioblastoma tumors had more PET signal compared to similar sized SB28 glioblastoma tumors, whereas widespread PET signal of 4T1-BrA intracranial tumors is consistent with its highly dispersed histological distribution. Survival of mice bearing intracranial tumors treated with 1D11 neutralizing antibody alone was similar to that of mice treated with control antibody whereas 1D11 improved survival when given in combination with focal radiation. The extent of survival benefit of combination of radiation and 1D11 was associated with the degree of TGFβ activity detected by PET. CONCLUSIONS:Zr -fresolimumab PET imaging detects radiation-induced TGFβ activation in tumors. Functional imaging indicated a range of TGFβ activity in intracranial tumors, but TGFβ blockade provided survival benefit only in the context of radiation treatment. These studies are further evidence that radiation-induced TGFβ activity opposes therapeutic response to radiation.

GENERAL PURPOSE/UNASSIGNED:To review the various mechanical forces that affect fibroblasts, keratinocytes, endothelial cells, and adipocytes at the cellular and molecular level as well as scar-reducing mechanical devices currently in clinical use. TARGET AUDIENCE/BACKGROUND:This continuing education activity is intended for physicians, physician assistants, nurse practitioners, and nurses with an interest in skin and wound care. LEARNING OBJECTIVES/OUTCOMES/UNASSIGNED:After participating in this educational activity, the participant will:1. Compare and contrast the responses of various types of cells to mechanical forces.2. Identify the mechanical devices and techniques that can help restore skin integrity.

Barth Syndrome (BTHS) is a rare X-linked genetic disorder caused by mutations in the gene encoding the transacylase tafazzin and characterized by loss of cardiolipin and severe cardiomyopathy. Mitochondrial oxidants have been implicated in the cardiomyopathy in BTHS. Eleven mitochondrial sites produce superoxide/H₂ O₂ at significant rates. Which of these sites generate oxidants at excessive rates in BTHS is unknown. Here, we measured the maximum capacity of superoxide/H₂ O₂ production from each site and the ex vivo rate of superoxide/H₂ O₂ production in the heart and skeletal muscle mitochondria of the tafazzin knockdown mice (tazkd) from 3 to 12 months of age. Despite reduced oxidative capacity, superoxide/H₂ O₂ production is indistinguishable between tazkd mice and wildtype littermates. These observations raise questions about the involvement of mitochondrial oxidants in BTHS pathology.

In lung cancer, enrichment of the lower airway microbiota with oral commensals commonly occurs and ex vivo models support that some of these bacteria can trigger host transcriptomic signatures associated with carcinogenesis. Here, we show that this lower airway dysbiotic signature was more prevalent in group IIIB-IV TNM stage lung cancer and is associated with poor prognosis, as shown by decreased survival among subjects with early stage disease (I-IIIA) and worse tumor progression as measured by RECIST scores among subjects with IIIB-IV stage disease. In addition, this lower airway microbiota signature was associated with upregulation of IL-17, PI3K, MAPK and ERK pathways in airway transcriptome, and we identified Veillonella parvula as the most abundant taxon driving this association. In a KP lung cancer model, lower airway dysbiosis with V. parvula led to decreased survival, increased tumor burden, IL-17 inflammatory phenotype and activation of checkpoint inhibitor markers.

BACKGROUND:Platelets are increasingly recognized as immune cells. As such, they are commonly seen to induce and perpetuate inflammation, however, anti-inflammatory activities are increasingly attributed to them. Atherosclerosis is a chronic inflammatory condition. Similar to other inflammatory conditions, the resolution of atherosclerosis requires a shift in macrophages to an M2 phenotype, enhancing their efferocytosis and cholesterol efflux capabilities. OBJECTIVES/OBJECTIVE:To assess the effect of platelets on macrophage phenotype. METHODS:In several in vitro models employing murine (RAW264.7 and bone marrow derived macrophages) and human (THP-1 and monocyte-derived macrophages) cells, we exposed macrophages to media in which non-agonized human platelets were cultured for 60 minutes (platelet conditioned media; PCM) and assessed the impact on macrophage phenotype and function. RESULTS:). CONCLUSIONS:PCM induces an anti-inflammatory, pro-resolving phenotype in macrophages. Our findings suggest that therapies targeting hemostatic properties of platelets, while not influencing pro-resolving, immune-related activities, could be beneficial for the treatment of atherothrombotic disease.