Cardiomyocyte PANX1 Controls Glycolysis and Neutrophil Recruitment in Hypertrophy.

BACKGROUND: PANX1 (pannexin 1), a ubiquitously expressed ATP release membrane channel, has been shown to play a role in inflammation, blood pressure regulation, and myocardial infarction. However, the possible role of PANX1 in cardiomyocytes in the progression of heart failure has not yet been investigated. METHOD: We generated a novel mouse line with constitutive deletion of PANX1 in cardiomyocytes (Panx1MyHC6). RESULTS: PANX1 deletion in cardiomyocytes had no effect on unstressed heart function but increased the glycolytic metabolism and resulting glycolytic ATP production, with a concurrent decrease in oxidative phosphorylation, both in vivo and in vitro. In vitro, treatment of H9c2 (H9c2 rat myoblast cell line) cardiomyocytes with isoproterenol led to PANX1-dependent release of ATP and Yo-Pro-1 uptake, as assessed by pharmacological blockade with spironolactone and siRNA-mediated knockdown of PANX1. To investigate nonischemic heart failure and the preceding cardiac hypertrophy, we administered isoproterenol, and we demonstrated that Panx1MyHC6 mice were protected from systolic and diastolic left ventricle volume increases as a result of cardiomyocyte hypertrophy. Moreover, we found that Panx1MyHC6 mice showed decreased isoproterenol-induced recruitment of immune cells (CD45+), particularly neutrophils (CD11b+ [integrin subunit alpha M], Ly6g+ [lymphocyte antigen 6 family member G]), to the myocardium. CONCLUSIONS: Together, these data demonstrate that PANX1 deficiency in cardiomyocytes increases glycolytic metabolism and protects against cardiac hypertrophy in nonischemic heart failure at least in part by reducing immune cell recruitment. Our study implies PANX1 channel inhibition as a therapeutic approach to ameliorate cardiac dysfunction in patients with heart failure.

A Single Model for the Thermodynamics and Kinetics of Metal Exsolution from Perovskite Oxides.

Exsolution has emerged as an outstanding route for producing oxide-supported metal nanoparticles. For ABO3-perovskite oxides, various late transition-metal cations can be substituted into the lattice under oxidizing conditions and exsolved as metal nanoparticles after reduction. A consistent and comprehensive description of the point-defect thermodynamics and kinetics of this phenomenon is lacking, however. Herein, supported by hybrid density-functional-theory calculations, we propose a single model that explains diverse experimental observations, such as why substituent transition-metal cations (but not host cations) exsolve from perovskite oxides upon reduction; why different substituent transition-metal cations exsolve under different conditions; why the metal nanoparticles are embedded in the surface; why exsolution occurs surprisingly rapidly at relatively low temperatures; and why the reincorporation of exsolved species involves far longer times and much higher temperatures. Our model's foundation is that the substituent transition-metal cations are reduced to neutral species within the perovskite lattice as the Fermi level is shifted upward within the bandgap upon sample reduction. The calculations also indicate unconventional influences of oxygen vacancies and A-site vacancies. Our model thus provides a fundamental basis for improving existing, and creating new, exsolution-generated catalysts.

Mechanisms of whole body, respiratory, acid-base buffering: a first computer-model test of three physicochemical, acid-base theories.

Acid-base disorders are currently analyzed and treated using a bicarbonate-centered approach derived from blood studies prior to the advent of digital computers, which could solve computer models capable of quantifying the complex physicochemical nature governing distribution of water and ions between fluid compartments. An alternative is the Stewart approach, which can predict the pH of a simple mixture of ions and electrically charged proteins; hence, the role of extravascular fluids has been largely ignored. The present study uses a new, comprehensive computer model of four major fluid compartments, based on a recent blood model, which included ion binding to proteins, electroneutrality constraints, and other essential physicochemical laws. The present model predicts quantitative respiratory acid-base buffering behavior in the whole body, as well as determining roles of each compartment and their species, particularly compartmental electrically charged proteins, largely responsible for buffering. The model tested an early theory that H+ was conserved in the body fluids; hence, when changing Pco2 states, intracellular buffering could be predicted by net changes in bicarbonate and protein electrical charge in the remaining fluids. Even though H+ is not conserved in the model, the theory held in simulated respiratory disorders. Model results also agreed with a second part of the theory, that ion movements between cells and interstitial fluid were linked with H+ buffering, but by electroneutrality constraints, not necessarily by some membrane-related mechanisms, and that the strong ion difference (SID), an amalgamation of ionic electrical charges, was approximately conserved when going between equilibrium states caused by Pco2 changes in the body-fluid system.NEW & NOTEWORTHY For the first time, a physicochemically based, whole body, four-compartment, computer model was used to study respiratory whole body acid-base buffering. An improved approach to quantify acid-base buffering, previously used by this author, was able to determine contributions of the various compartmental fluids to whole body buffering. The model was used to test, for the first time, three fundamental theories of whole body acid-base homeostasis, namely, H+-conservation, its linkage to ion transport, and strong ion difference conservation.

Extracellular Matrix Scaffold-Assisted Tumor Vaccines Induce Tumor Regression and Long-Term Immune Memory.

Injectable scaffold delivery is a strategy to enhance the efficacy of cancer vaccine immunotherapy. The choice of scaffold biomaterial is crucial, impacting both vaccine release kinetics and immune stimulation via the host response. Extracellular matrix (ECM) scaffolds prepared from decellularized tissues facilitate a pro-healing inflammatory response that promotes local cancer immune surveillance. Here, an ECM scaffold-assisted therapeutic cancer vaccine that maintains an immune microenvironment consistent with tissue reconstruction is engineered. Several immune-stimulating adjuvants are screened to develop a cancer vaccine formulated with decellularized small intestinal submucosa (SIS) ECM scaffold co-delivery. It is found that the STING pathway agonist cyclic di-AMP most effectively induces cytotoxic immunity in an ECM scaffold vaccine, without compromising key interleukin 4 (IL-4) mediated immune pathways associated with healing. ECM scaffold delivery enhances therapeutic vaccine efficacy, curing 50-75% of established E.G-7OVA lymphoma tumors in mice, while none are cured with soluble vaccine. SIS-ECM scaffold-assisted vaccination prolonged antigen exposure is dependent on CD8+ cytotoxic T cells and generates long-term antigen-specific immune memory for at least 10 months post-vaccination. This study shows that an ECM scaffold is a promising delivery vehicle to enhance cancer vaccine efficacy while being orthogonal to characteristics of pro-healing immune hallmarks.

Characterization of immunomodulating agents from Staphylococcus aureus for priming immunotherapy in triple-negative breast cancers.

Immunotherapy, specifically immune checkpoint blockade (ICB), has revolutionized the treatment paradigm of triple-negative breast cancers (TNBCs). However, a subset of TNBCs devoid of tumor-infiltrating T cells (TILs) or PD-L1 expression generally has a poor response to immunotherapy. In this study, we aimed to sensitize TNBCs to ICB by harnessing the immunomodulating potential of S. aureus, a breast-resident bacterium. We show that intratumoral injection of spent culture media from S. aureus recruits TILs and suppresses tumor growth in a preclinical TNBC model. We further demonstrate that α-hemolysin (HLA), an S. aureus-produced molecule, increases the levels of CD8+ T cells and PD-L1 expression in tumors, delays tumor growth, and triggers tumor necrosis. Mechanistically, while tumor cells treated with HLA display Gasdermin E (GSDME) cleavage and a cellular phenotype resembling pyroptosis, splenic T cells incubated with HLA lead to selective expansion of CD8+ T cells. Notably, intratumoral HLA injection prior to ICB augments the therapeutic efficacy compared to ICB alone. This study uncovers novel immunomodulatory properties of HLA and suggests that intratumoral administration of HLA could be a potential priming strategy to expand the population of TNBC patients who may respond to ICB.

Cell cycle specific, differentially tagged ribosomal proteins to measure phase specific transcriptomes from asynchronously cycling cells.

Asynchronously cycling cells pose a challenge to the accurate characterization of phase-specific gene expression. Current strategies, including RNAseq, survey the steady state gene expression across the cell cycle and are inherently limited by their inability to resolve dynamic gene regulatory networks. Single cell RNAseq (scRNAseq) can identify different cell cycle transcriptomes if enough cycling cells are present, however some cells are not amenable to scRNAseq. Therefore, we merged two powerful strategies, the CDT1 and GMNN degrons used in Fluorescent Ubiquitination-based Cell Cycle Indicator (FUCCI) cell cycle sensors and the ribosomal protein epitope tagging used in RiboTrap/Tag technologies to isolate cell cycle phase-specific mRNA for sequencing. The resulting cell cycle dependent, tagged ribosomal proteins (ccTaggedRP) were differentially expressed during the cell cycle, had similar subcellular locations as endogenous ribosomal proteins, incorporated into ribosomes and polysomes, and facilitated the recovery of cell cycle phase-specific RNA for sequencing. ccTaggedRP has broad applications to investigate phase-specific gene expression in complex cell populations.

Engineering Tumor Stroma Morphogenesis Using Dynamic Cell-Matrix Spheroid Assembly.

The tumor microenvironment consists of resident tumor cells organized within a compositionally diverse, three-dimensional (3D) extracellular matrix (ECM) network that cannot be replicated in vitro using bottom-up synthesis. We report a new self-assembly system to engineer ECM-rich 3D MatriSpheres wherein tumor cells actively organize and concentrate microgram quantities of decellularized ECM dispersions which modulate cell phenotype. 3D colorectal cancer (CRC) MatriSpheres were created using decellularized small intestine submucosa (SIS) as an orthotopic ECM source that had greater proteomic homology to CRC tumor ECM than traditional ECM formulations such as Matrigel. SIS ECM was rapidly concentrated from its environment and assembled into ECM-rich 3D stroma-like regions by mouse and human CRC cell lines within 4-5 days via a mechanism that was rheologically distinct from bulk hydrogel formation. Both ECM organization and transcriptional regulation by 3D ECM cues affected programs of malignancy, lipid metabolism, and immunoregulation that corresponded with an in vivo MC38 tumor cell subpopulation identified via single cell RNA sequencing. This 3D modeling approach stimulates tumor specific tissue morphogenesis that incorporates the complexities of both cancer cell and ECM compartments in a scalable, spontaneous assembly process that may further facilitate precision medicine.

Pannexin 1 Channels Control Cardiomyocyte Metabolism and Neutrophil Recruitment During Non-Ischemic Heart Failure.

Pannexin 1 (PANX1), a ubiquitously expressed ATP release membrane channel, has been shown to play a role in inflammation, blood pressure regulation, and myocardial infarction. However, a possible role of PANX1 in cardiomyocytes in the progression of heart failure has not yet been investigated. We generated a novel mouse line with constitutive deletion of PANX1 in cardiomyocytes (Panx1 MyHC6 ). PANX1 deletion in cardiomyocytes had no effect on unstressed heart function but increased the glycolytic metabolism both in vivo and in vitro . In vitro , treatment of H9c2 cardiomyocytes with isoproterenol led to PANX1-dependent release of ATP and Yo-Pro-1 uptake, as assessed by pharmacological blockade with spironolactone and siRNA-mediated knock-down of PANX1. To investigate non-ischemic heart failure and the preceding cardiac hypertrophy we administered isoproterenol, and we demonstrate that Panx1 MyHC6 mice were protected from systolic and diastolic left ventricle volume increases and cardiomyocyte hypertrophy. Moreover, we found that Panx1 MyHC6 mice showed decreased isoproterenol-induced recruitment of immune cells (CD45 + ), particularly neutrophils (CD11b + , Ly6g + ), to the myocardium. Together these data demonstrate that PANX1 deficiency in cardiomyocytes impacts glycolytic metabolism and protects against cardiac hypertrophy in non-ischemic heart failure at least in part by reducing immune cell recruitment. Our study implies PANX1 channel inhibition as a therapeutic approach to ameliorate cardiac dysfunction in heart failure patients.

Multi-omic analysis reveals VEGFR2, PI3K, and JNK mediate the small molecule induction of human iPSC-derived cardiomyocyte proliferation.

Mammalian hearts lose their regenerative potential shortly after birth. Stimulating the proliferation of preexisting cardiomyocytes is a potential therapeutic strategy for cardiac damage. In a previous study, we identified 30 compounds that induced the bona-fide proliferation of human iPSC-derived cardiomyocytes (hiPSC-CM). Here, we selected five active compounds with diverse targets, including ALK5 and CB1R, and performed multi-omic analyses to identify common mechanisms mediating the cell cycle progression of hiPSC-CM. Transcriptome profiling revealed the top enriched pathways for all compounds including cell cycle, DNA repair, and kinesin pathways. Functional proteomic arrays found that the compounds collectively activated multiple receptor tyrosine kinases including ErbB2, IGF1R, and VEGFR2. Network analysis integrating common transcriptomic and proteomic signatures predicted that MAPK/PI3K pathways mediated compound responses. Furthermore, VEGFR2 negatively regulated endoreplication, enabling the completion of cell division. Thus, in this study, we applied high-content imaging and molecular profiling to establish mechanisms linking pro-proliferative agents to mechanisms of cardiomyocyte cell cycling.