β
-Spectrin is a membrane cytoskeletal protein with specialized roles in the nervous system and heart. Recent evidence also indicates a fundamental role for β
-spectrin in angiogenesis as its ...endothelial-specific gene deletion in mice enhances embryonic lethality due to hypervascularization and hemorrhagic defects. During early vascular sprouting, β
-spectrin is believed to inhibit tip cell sprouting in favor of the stalk cell phenotype by mediating VEGFR2 internalization and degradation. Despite these essential roles, mechanisms governing β
-spectrin expression remain unknown. Here we identify bone morphogenetic protein 9 (BMP9) as a major inducer of β
-spectrin gene expression in the vascular system. We show that BMP9 signals through the ALK1/Smad1 pathway to induce β
-spectrin expression, which then recruits CaMKII to the cell membrane to induce phosphorylation-dependent VEGFR2 turnover. Although BMP9 signaling promotes stalk cell behavior through activation of hallmark stalk cell genes ID-1/3 and Hes-1 and Notch signaling cross-talk, we find that β
-spectrin acts upstream of these pathways as loss of β
-spectrin in neonate mice leads to retinal hypervascularization due to excessive VEGFR2 levels, increased tip cell populations, and strong Notch inhibition irrespective of BMP9 treatment. These findings demonstrate β
-spectrin as a BMP9 gene target critical for tip/stalk cell selection during nascent vessel sprouting.
Heart failure (HF) is characterized by compromised cardiac structure and function. Previous work has identified a link between upregulation of pro-inflammatory cytokines and HF. Tumor necrosis factor ...(TNF)-like weak inducer of apoptosis (TWEAK) is a pro-inflammatory cytokine, which binds to fibroblast growth factor inducible 14 (Fn14), a ubiquitously expressed cell-surface receptor. The objective of this study was to investigate the role of TWEAK/Fn14 pathway in promoting cardiac inflammation under non ischemic stress conditions.
Wild type (WT) and Fn14 knock out (Fn14−/−) mice were subjected to pressure overload transaortic constriction (TAC) for 1 or 6 weeks. A subset of WT TAC animals were treated with the Fn14 antagonist L524-0366. Cardiac function was measured by echocardiography. Cardiac fibrosis and macrophage infiltration were quantified using immunohistochemistry and flow cytometry, respectively. Cardiac fibroblasts were isolated for quantifying TWEAK-induced chemokine release.
Fn14−/− mice displayed improved cardiac function, reduced fibrosis and lower macrophage infiltration in heart compared to WT following TAC. L524-0366 mitigated maladaptive remodeling with TAC. TWEAK induced secretion of the pro-inflammatory chemokine, monocyte chemoattractant protein 1 from WT but not Fn14−/− fibroblasts in vitro, in part through activation of non-canonical NF-κB signaling. Finally, Fn14 expression was increased in mouse following TAC and in human failing hearts.
Our findings support an important role for the TWEAK/Fn14 promoting macrophage infiltration and fibrosis in heart under non-ischemic stress, with potential for therapeutic intervention to improve cardiac function in the setting of HF.
•Genetic deletion of the TNF receptor family member Fn14 (Fn14−/−) reduces cardiac fibrosis and improves cardiac function in response to chronic pressure overload compared to WT.•Fn14−/− mice show a reduction in resident and infiltrating macrophages in myocardium in response to chronic pressure overload compared to WT•TWEAK-Fn14 signaling activates downstream non-canonical NF-kB pathway to promote MCP-1 secretion in cardiac fibroblasts•TWEAK-Fn14 pathway mediates pressure overload cardiac dysfunction and serves as a potential therapeutic target in non-ischemic HF
Clustered ion channels at nodes of Ranvier are critical for fast action potential propagation in myelinated axons. Axon-glia interactions converge on ankyrin and spectrin cytoskeletal proteins to ...cluster nodal Na
channels during development. However, how nodal ion channel clusters are maintained is poorly understood. Here, we generated mice lacking nodal spectrins in peripheral sensory neurons to uncouple their nodal functions from their axon initial segment functions. We demonstrate a hierarchy of nodal spectrins, where β4 spectrin is the primary spectrin and β1 spectrin can substitute; each is sufficient for proper node organization. Remarkably, mice lacking nodal β spectrins have normal nodal Na
channel clustering during development, but progressively lose Na
channels with increasing age. Loss of nodal spectrins is accompanied by an axon injury response and axon deformation. Thus, nodal spectrins are required to maintain nodal Na
channel clusters and the structural integrity of axons.
Kinase/phosphatase balance governs cardiac excitability in health and disease. Although detailed mechanisms for cardiac kinase regulation are established, far less is known regarding cardiac protein ...phosphatase 2A (PP2A) regulation. This is largely due to the complexity of the PP2A holoenzyme structure (combinatorial assembly of three subunit enzyme from >17 subunit genes) and the inability to segregate “global” PP2A function from the activities of multiple “local” holoenzyme populations. Here we report that PP2A catalytic, regulatory, and scaffolding subunits are tightly regulated at transcriptional, translational, and post-translational levels to tune myocyte function at base line and in disease. We show that past global read-outs of cellular PP2A activity more appropriately represent the collective activity of numerous individual PP2A holoenzymes, each displaying a specific subcellular localization (dictated by select PP2A regulatory subunits) as well as local specific post-translational catalytic subunit methylation and phosphorylation events that regulate local and rapid holoenzyme assembly/disassembly (via leucine carboxymethyltransferase 1/phosphatase methylesterase 1 (LCMT-1/PME-1). We report that PP2A subunits are selectively regulated between human and animal models, across cardiac chambers, and even within specific cardiac cell types. Moreover, this regulation can be rapidly tuned in response to cellular activation. Finally, we report that global PP2A is altered in human and experimental models of heart disease, yet each pathology displays its own distinct molecular signature though specific PP2A subunit modulatory events. These new data provide an initial view into the signaling pathways that govern PP2A function in heart but also establish the first step in defining specific PP2A regulatory targets in health and disease.
Background: PP2A regulates cardiac excitability and physiology.
Results: PP2A regulation in heart occurs through integrative transcriptional, translational, and post-translational control of three classes of subunits (17 genes) to control holoenzyme synthesis, localization, and maintenance; pathways are mechanistically altered in heart disease.
Conclusion: Multiple mechanisms are present for acute and chronic regulation of specific PP2A populations.
Significance: Results provide molecular insight into cardiac PP2A regulation.
RATIONALE:Nav1.5 (SCN5A) is the primary cardiac voltage-gated Nav channel. Nav1.5 is critical for cardiac excitability and conduction, and human SCN5A mutations cause sinus node dysfunction, atrial ...fibrillation, conductional abnormalities, and ventricular arrhythmias. Further, defects in Nav1.5 regulation are linked with malignant arrhythmias associated with human heart failure. Consequently, therapies to target select Nav1.5 properties have remained at the forefront of cardiovascular medicine. However, despite years of investigation, the fundamental pathways governing Nav1.5 membrane targeting, assembly, and regulation are still largely undefined.
OBJECTIVE:Define the in vivo mechanisms underlying Nav1.5 membrane regulation.
METHODS AND RESULTS:Here, we define the molecular basis of an Nav channel regulatory platform in heart. Using new cardiac-selective ankyrin-G mice (conditional knock-out mouse), we report that ankyrin-G targets Nav1.5 and its regulatory protein calcium/calmodulin–dependent kinase II to the intercalated disc. Mechanistically, βIV-spectrin is requisite for ankyrin-dependent targeting of calcium/calmodulin–dependent kinase II-δ; however, βIV-spectrin is not essential for ankyrin-G expression. Ankyrin-G conditional knock-out mouse myocytes display decreased Nav1.5 expression/membrane localization and reduced INa associated with pronounced bradycardia, conduction abnormalities, and ventricular arrhythmia in response to Nav channel antagonists. Moreover, we report that ankyrin-G links Nav channels with broader intercalated disc signaling/structural nodes, as ankyrin-G loss results in reorganization of plakophilin-2 and lethal arrhythmias in response to β-adrenergic stimulation.
CONCLUSIONS:Our findings provide the first in vivo data for the molecular pathway required for intercalated disc Nav1.5 targeting/regulation in heart. Further, these new data identify the basis of an in vivo cellular platform critical for membrane recruitment and regulation of Nav1.5.
Cardiac arrhythmias are a major cause of morbidity and mortality worldwide. Although recent advances in cell-based models, including human-induced pluripotent stem cell-derived cardiomyocytes ...(iPSC-CM), are contributing to our understanding of electrophysiology and arrhythmia mechanisms, preclinical animal studies of cardiovascular disease remain a mainstay. Over the past several decades, animal models of cardiovascular disease have advanced our understanding of pathological remodeling, arrhythmia mechanisms, and drug effects and have led to major improvements in pacing and defibrillation therapies. There exist a variety of methodological approaches for the assessment of cardiac electrophysiology and a plethora of parameters may be assessed with each approach. This guidelines article will provide an overview of the strengths and limitations of several common techniques used to assess electrophysiology and arrhythmia mechanisms at the whole animal, whole heart, and tissue level with a focus on small animal models. We also define key electrophysiological parameters that should be assessed, along with their physiological underpinnings, and the best methods with which to assess these parameters.
Interestingly, patient populations with arterial hypertension, congestive heart failure, and myocardial infarction are particularly vulnerable to the development of hypokalemia 3. ...drugs that ...increase plasma potassium levels help reduce mortality and morbidity rates in certain patient populations 4. ...in more predictable fashion, decreased K+o increases the slow delayed rectifier K+ current, IKs, due to an increase in driving force. ...these simulations effectively underscore the utility of a mathematical model in unraveling mechanism associated with the highly nonlinear cardiac AP.
Ion channel function is fundamental to the existence of life. In metazoans, the coordinate activities of voltage-gated Na(+) channels underlie cellular excitability and control neuronal ...communication, cardiac excitation-contraction coupling, and skeletal muscle function. However, despite decades of research and linkage of Na(+) channel dysfunction with arrhythmia, epilepsy, and myotonia, little progress has been made toward understanding the fundamental processes that regulate this family of proteins. Here, we have identified β(IV)-spectrin as a multifunctional regulatory platform for Na(+) channels in mice. We found that β(IV)-spectrin targeted critical structural and regulatory proteins to excitable membranes in the heart and brain. Animal models harboring mutant β(IV)-spectrin alleles displayed aberrant cellular excitability and whole animal physiology. Moreover, we identified a regulatory mechanism for Na(+) channels, via direct phosphorylation by β(IV)-spectrin-targeted calcium/calmodulin-dependent kinase II (CaMKII). Collectively, our data define an unexpected but indispensable molecular platform that determines membrane excitability in the mouse heart and brain.
Mathematical models are invaluable tools for understanding the relationships between components of a complex system. In the biological context, mathematical models help us understand the complex web ...of interrelations between various components (DNA, proteins, enzymes, signaling molecules etc.) in a biological system, gain better understanding of the system as a whole, and in turn predict its behavior in an altered state (e.g. disease). Mathematical modeling has enhanced our understanding of multiple complex biological processes like enzyme kinetics, metabolic networks, signal transduction pathways, gene regulatory networks, and electrophysiology. With recent advances in high throughput data generation methods, computational techniques and mathematical modeling have become even more central to the study of biological systems. In this review, we provide a brief history and highlight some of the important applications of modeling in biological systems with an emphasis on the study of excitable cells. We conclude with a discussion about opportunities and challenges for mathematical modeling going forward. In a larger sense, the review is designed to help answer a simple but important question that theoreticians frequently face from interested but skeptical colleagues on the experimental side: “What is the value of a model?”
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