Biology subjects

Lederer, W. J.

Publications and source records attributed to Lederer, W. J..

6 recordsLinked to original sources

bioRxiv2025

Pericytes in heart

Pericytes are cells associated primarily with capillaries and are thought to play an important role in the regulation of blood flow. They are often referred to as "mural" cells because they are so frequently found on the exterior walls of small vessels - particularly the capillaries. In heart, high-resolution real-time observations and measurements of pericyte function under physiological conditions are challenging to obtain because of vascular motion, tissue depth and vigorous functional movement. For these reasons, the heart may be one of the most difficult tissues in which to examine pericyte function. Recently, we introduced a perfused papillary muscle preparation (the Z-Prep) that allows us to observe coronary arteries, arterioles, venules, capillaries and myocytes in real time at physiological temperature and pressure while also imaging pericytes 1, 2. Here we present an initial study intended to visualize and characterize quantitatively cardiac pericytes in heart at physiological pressure and temperature conditions. Vascular anatomy was imaged using a z-stack protocol with a rapidly spinning disk confocal microscope. Here the anatomical organization of the pericytes is shown at high resolution with respect to the microcirculation components and cardiac myocytes. The surprising findings include the high abundance of pericytes in native tissue, the extent of their spread on the capillaries themselves, and the existence of major pericyte extensions that travel intimately along the surface of neighboring ventricular myocytes and attach to capillaries on the distant side. These extensions arise from a capillary-based pericyte location and normally end on another capillary endothelial surface and we have named them "bridging" pericytes. Taken together this anatomical organization suggests that the pericytes provide signaling, communication and contractile services to important cellular components of the heart. There is also a provocative suggestion that pericytes in heart are unusually fragile since they suffer an extremely high degree of loss during cellular isolation procedures. However, our investigation of the organization argues against this fragility because of the durability of the dynamic pericyte organization and function despite the stress and brutality of the contracting heart. The work presented here lays the foundation for critical functional studies of pericytes in heart in both health and disease.

physiology↗

bioRxiv2023

Na+/K+ATPase - Cav1.2 nanodomain differentially regulates intracellular and local adrenergic signaling in cardiac myocytes

BackgroundThe intracellular Na+ concentration ([Na+]i) is a crucial but understudied regulator of cardiac myocyte function. The Na+/K+ ATPase (NKA) controls the steady-state [Na+]i and thereby determines the set-point for intracellular Ca2+. Here, we investigate the nanoscopic organization and local adrenergic regulation of the NKA macromolecular complex and how it differentially regulates the intracellular Na+ and Ca2+ homeostases in atrial and ventricular myocytes. MethodsMulticolor STORM super-resolution microscopy, Western Blot analyses, and in vivo examination of adrenergic regulation are employed to examine the organization and function of Na+ nanodomains in cardiac myocytes. Quantitative fluorescence microscopy at high spatiotemporal resolution is used in conjunction with cellular electrophysiology to investigate intracellular Na+ homeostasis in atrial and ventricular myocytes. ResultsThe NKA1 (NKA1) and the L-type Ca2+-channel (Cav1.2) form a nanodomain with a center-to center distance of [~]65 nm in both ventricular and atrial myocytes. NKA1 protein expression levels are [~]3 fold higher in atria compared to ventricle. 100% higher atrial INKA, produced by large NKA "superclusters", underlies the substantially lower Na+concentration in atrial myocytes compared to the benchmark values set in ventricular myocytes. The NKAs regulatory protein phospholemman (PLM) has similar expression levels across atria and ventricle resulting in a much lower PLM/NKA1 ratio for atrial compared to ventricular tissue. In addition, a huge PLM phosphorylation reserve in atrial tissue produces a high {beta}-adrenergic sensitivity of INKA in atrial myocytes. {beta}-adrenergic regulation of INKA is locally mediated in the NKA1-Cav1.2 nanodomain via A-kinase anchoring proteins. ConclusionsNKA1, Cav1.2 and their accessory proteins form a structural and regulatory nanodomain at the cardiac dyad. The tissue-specific composition and local adrenergic regulation of this "signaling cloud" is a main regulator of the distinct global intracellular Na+ and Ca2+ concentrations in atrial and ventricular myocytes.

physiology↗

bioRxiv2023

Arrhythmogenic Ca2+ Signaling in a Metabolic Model of HFpEF

Heart Failure with preserved ejection fraction (HFpEF) is the most prevalent form of heart failure worldwide and its significant mortality is associated with a high rate of sudden cardiac death (SCD; 30% - 40%). Chronic metabolic stress is an important driver of HFpEF, and clinical data show metabolic stress as a significant risk factor for ventricular arrhythmias in HFpEF patients. The mechanisms of SCD and ventricular arrhythmia in HFpEF remain critically understudied and empirical treatment is ineffective. To address this important knowledge gap, we developed a novel preclinical model of metabolic-stress induced HFpEF using Western diet (High fructose and fat) and hypertension induced by nitric oxide synthase inhibition (with L-NAME) in wildtype C57BL6/J mice. After 5 months, mice display all clinical characteristics of HFpEF and present with stress-induced sustained ventricular tachycardia (VT). Mechanistically, we found a novel pattern of arrhythmogenic intracellular Ca2+ handling that is distinct from the well-characterized changes pathognomonic for heart failure with reduced ejection fraction. In addition, we show that the transverse tubular system remains intact in HFpEF and that arrhythmogenic, intracellular Ca2+ mobilization becomes hyper-sensitive to {beta}- adrenergic activation. Finally, in proof-of-concept experiments we show in vivo that the clinically used intracellular calcium stabilizer dantrolene, which acts on the Ca2+ release channels of the sarcoplasmic reticulum (SR), the ryanodine receptors, acutely prevents stress-induced VT in HFpEF mice. Therapeutic control of SR Ca2+ leak may present a novel mechanistic treatment approach in metabolic HFpEF.

physiology↗

bioRxiv2023

Metastatic breast cancer cells have reduced calcium and actin response after ATP-P2Y2 signaling

The tumor microenvironment and wound healing after injury both contain extremely high concentrations of the extracellular signaling molecule, adenosine triphosphate (ATP) compared to normal tissue. P2Y2 receptor, an ATP-activated purinergic receptor, is typically associated with pulmonary, endothelial, and neurological cell signaling. Here we report its role and importance in breast epithelial cell signaling and how it is altered in metastatic breast cancer. In response to ATP activation, P2Y2 receptor signaling causes an increase of intracellular Ca2+ in non-tumorigenic breast epithelial cells, while their tumorigenic and metastatic counterparts have significantly reduced Ca2+ responses. The non-tumorigenic cells respond to increased Ca2+ with actin polymerization and localization to cell edges, while the metastatic cells remained unaffected. The increase in intracellular Ca2+ after ATP stimulation was blunted using a P2Y2 antagonist, which also prevented actin mobilization and caused cell dissemination from spheroids in non-tumorigenic breast epithelial cells. Furthermore, the lack of Ca2+ concentration changes and actin mobilization in the metastatic breast cancer cells could be due to reduced P2Y2 expression, which correlates with poorer overall survival in breast cancer patients. This study elucidates rapid changes that occur after elevated intracellular Ca2+ in breast epithelial cells and how metastatic cancer cells have adapted to evade this cellular response.

cancer biology↗

bioRxiv2022

Calcium and Bicarbonate Signaling Pathways have Pivotal, Resonating Roles in Matching ATP Production to Demand

Mitochondrial ATP production in cardiac ventricular myocytes must be continually adjusted to rapidly replenish the ATP consumed by the working heart. Two systems are known to be critical in this regulation: mitochondrial matrix Ca2+ ([Ca2+]m) and blood flow that is tuned by local ventricular myocyte metabolic signaling. However, these two regulatory systems do not account for the large physiological range of ATP consumption observed. We report here on the identity, location, and signaling cascade of a controversial third regulatory system -- CO2/bicarbonate. CO2 is generated in the mitochondrial matrix as a metabolic waste product produced by oxidation of nutrients which power the production of ATP. It is a lipid soluble gas that equilibrates with bicarbonate (HCO3-) in aqueous solutions. The bicarbonate level is tracked by a bicarbonate-activated adenylyl cyclase, soluble adenylyl cyclase (sAC). Using structural Airyscan super-resolution imaging and functional measurements we find that sAC is primarily inside the mitochondria of ventricular myocytes where it generates cAMP when activated by HCO3-. This cAMP signaling cascade is shown to operate inside the mitochondrial inter-membrane space (IMS) by activating local EPAC1 (Exchange Protein directly Activated by cAMP) which turns on Rap1 (Ras-related protein 1). Thus, mitochondrial ATP production is shown to be increased by bicarbonate-triggered sAC signaling through Rap1. Additional evidence is presented indicating that the cAMP signaling itself does not occur directly in the matrix. We also show that this third signaling process involving bicarbonate and sAC activates the cardiac mitochondrial ATP production machinery by working independently of, yet in conjunction with, [Ca2+]m-dependent ATP production to meet the energy needs of cellular activity in both health and disease. Thus, the resonant, or complementary effects of bicarbonate and [Ca2+]m signaling arms tune mitochondrial ATP production to match the full scale of energy consumption in cardiac ventricular myocytes.

physiology↗

bioRxiv2020

Tubulin acetylation increases cytoskeletal stiffness to regulate mechanotransduction in striated muscle

Microtubules tune cytoskeletal stiffness to regulate the mechanics and mechanotransduction of striated muscle. While recent evidence suggests that microtubules enriched in detyrosinated α-tubulin are responsible for these effects in healthy muscle, and for their excess in disease, the possible contribution from several other α-tubulin modifications has not been investigated. Here we used genetic or pharmacologic strategies in isolated cardiomyocytes or skeletal myofibers to increase the level of acetylated α-tubulin without altering the level of detyrosinated α-tubulin. We show that microtubules enriched in acetylated α-tubulin contribute to the cytoskeletal stiffness and viscoelastic resistance, showing slowed rates of contraction and relaxation during unloaded contraction, and increased activation of NADPH Oxidase 2 (Nox2) by mechanotransduction. Together these findings add to growing evidence that microtubules contribute to the mechanobiology of striated muscle in health and disease.Competing Interest StatementThe authors have declared no competing interest.View Full Text

physiology↗