S. N. Prasanna Murthy

Vimentin Organization Modulates the Formation of Lamellipodia

The disassembly and withdrawal of vimentin intermediate filaments (VIF) from the plasma membrane induces membrane ruffling and the formation of a lamellipodium. Conversely, lamellipodium formation is inhibited when VIF are present.

Autonomic Remodeling in the Left Atrium and Pulmonary Veins in Heart Failure – Creation of a Dynamic Substrate for Atrial Fibrillation

Background—Atrial fibrillation (AF) is commonly associated with congestive heart failure (CHF). The autonomic nervous system is involved in the pathogenesis of both AF and CHF. We examined the role of autonomic remodeling in contributing to AF substrate in CHF. Methods and Results—Electrophysiological mapping was performed in the pulmonary veins and left atrium in 38 rapid ventricular–paced dogs (CHF group) and 39 control dogs under the following conditions: vagal stimulation, isoproterenol infusion, &bgr;-adrenergic blockade, acetylcholinesterase (AChE) inhibition (physostigmine), parasympathetic blockade, and double autonomic blockade. Explanted atria were examined for nerve density/distribution, muscarinic receptor and &bgr;-adrenergic receptor densities, and AChE activity. In CHF dogs, there was an increase in nerve bundle size, parasympathetic fibers/bundle, and density of sympathetic fibrils and cardiac ganglia, all preferentially in the posterior left atrium/pulmonary veins. Sympathetic hyperinnervation was accompanied by increases in &bgr;1-adrenergic receptor R density and in sympathetic effect on effective refractory periods and activation direction. &bgr;-Adrenergic blockade slowed AF dominant frequency. Parasympathetic remodeling was more complex, resulting in increased AChE activity, unchanged muscarinic receptor density, unchanged parasympathetic effect on activation direction and decreased effect of vagal stimulation on effective refractory period (restored by AChE inhibition). Parasympathetic blockade markedly decreased AF duration. Conclusions—In this heart failure model, autonomic and electrophysiological remodeling occurs, involving the posterior left atrium and pulmonary veins. Despite synaptic compensation, parasympathetic hyperinnervation contributes significantly to AF maintenance. Parasympathetic and/or sympathetic signaling may be possible therapeutic targets for AF in CHF.Background— Atrial fibrillation (AF) is commonly associated with congestive heart failure (CHF). The autonomic nervous system is involved in the pathogenesis of both AF and CHF. We examined the role of autonomic remodeling in contributing to AF substrate in CHF. Methods and Results— Electrophysiological mapping was performed in the pulmonary veins and left atrium in 38 rapid ventricular–paced dogs (CHF group) and 39 control dogs under the following conditions: vagal stimulation, isoproterenol infusion, β-adrenergic blockade, acetylcholinesterase (AChE) inhibition (physostigmine), parasympathetic blockade, and double autonomic blockade. Explanted atria were examined for nerve density/distribution, muscarinic receptor and β-adrenergic receptor densities, and AChE activity. In CHF dogs, there was an increase in nerve bundle size, parasympathetic fibers/bundle, and density of sympathetic fibrils and cardiac ganglia, all preferentially in the posterior left atrium/pulmonary veins. Sympathetic hyperinnervation was accompanied by increases in β1-adrenergic receptor R density and in sympathetic effect on effective refractory periods and activation direction. β-Adrenergic blockade slowed AF dominant frequency. Parasympathetic remodeling was more complex, resulting in increased AChE activity, unchanged muscarinic receptor density, unchanged parasympathetic effect on activation direction and decreased effect of vagal stimulation on effective refractory period (restored by AChE inhibition). Parasympathetic blockade markedly decreased AF duration. Conclusions— In this heart failure model, autonomic and electrophysiological remodeling occurs, involving the posterior left atrium and pulmonary veins. Despite synaptic compensation, parasympathetic hyperinnervation contributes significantly to AF maintenance. Parasympathetic and/or sympathetic signaling may be possible therapeutic targets for AF in CHF.

Capturing the stem cell paracrine effect using heparin-presenting nanofibres to treat cardiovascular diseases

The mechanism for stem cell‐mediated improvement following acute myocardial infarction has been actively debated. We support hypotheses that the stem cell effect is primarily paracrine factor‐linked. We used a heparin‐presenting injectable nanofibre network to bind and deliver paracrine factors derived from hypoxic conditioned stem cell media to mimic this stem cell paracrine effect. Our self‐assembling peptide nanofibres presenting heparin were capable of binding paracrine factors from a medium phase. When these factor‐loaded materials were injected into the heart following coronary artery ligation in a mouse ischaemia‐reperfusion model of acute myocardial infarction, we found significant preservation of haemodynamic function. Through media manipulation, we were able to determine that crucial factors are primarily < 30 kDa and primarily heparin‐binding. Using recombinant VEGF‐ and bFGF‐loaded nanofibre networks, the effect observed with conditioned media was recapitulated. When evaluated in another disease model, a chronic rat ischaemic hind limb, our factor‐loaded materials contributed to extensive limb revascularization. These experiments demonstrate the potency of the paracrine effect associated with stem cell therapies and the potential of a biomaterial to bind and deliver these factors, pointing to a potential therapy based on synthetic materials and recombinant factors as an acellular therapy. Copyright

Cross‐Linking Sites of the Human Tau Protein, Probed by Reactions with Human Transglutaminase

Abstract: A portion of the neurofibrillary tangles of Alzheimers disease has the characteristics of cross‐linked protein. Because the principal component of these lesions is the microtubule‐associated protein tau, and because a major source of cross‐linking activity within neurons is supplied by tissue transglutaminase (TGase), it has been postulated that isopeptide bond formation is a major posttranslational modification leading to the formation of insoluble neurofibrillary tangles. Here we have mapped the sites on two isoforms of human tau protein (τ23 and τ40) capable of participating in human TGase‐mediated isopeptide bond formation. Using dansyl‐labeled fluorescent probes, it was shown that eight Gln residues can function as amine acceptor residues, with two major sites being Gln351 and Gln424. In addition, 10 Lys residues were identified as amine donors, most of which are clustered adjacent to the microtubule‐binding repeats of tau in regions known to be solvent accessible in filamentous tau. The distribution of amine donors correlated closely with that of Arg residues, suggesting a link between neighboring positive charge and the TGase selectivity for donor sites in the protein substrate. Apart from revealing the sites that can be cross‐linked during the TGase‐catalyzed assembly of tau filaments, the results suggest a topography for the tau monomers so assembled.

Conserved tryptophan in the core domain of transglutaminase is essential for catalytic activity

Transglutaminase 2 (TG2) is a distinctive member of the family of Ca2+-dependent enzymes recognized mostly by their abilities to catalyze the posttranslational crosslinking of proteins. TG2 uniquely binds and hydrolyzes GTP; binding GTP inhibits its crosslinking activity but allows it to function in signal transduction (hence the Gh designation). The core domain of TG2 (residues 139–471, rat) comprises the papain-like catalytic triad and the GTP-binding domain (residues 159–173) and contains almost all of the conserved tryptophans of the protein. Examining point mutations at Trp positions 180, 241, 278, 332, and 337 showed that, upon binding 2′-(or 3′)-O-(N-methylanthraniloyl)GTP (mantGTP), the Phe-332 mutant was the weakest (35% less than wild type) in resonance energy transfer from the protein (λexc, max = 290 nm) to the mant fluorophore (λem = 444 nm) and had a reduced affinity for mantGTP. Trp-332, situated near the catalytic center and the nucleotide-binding area of TG2, may be part of the allosteric relay machinery that transmits negative effector signals from nucleotide binding to the active center of TG2. A most important observation was that, whereas no enzyme activity could be detected when Trp-241 was replaced with Ala or Gln, partial preservation of catalytic activity was seen with substitutions by Tyr > Phe > His. The results indicate that Trp-241 is essential for catalysis, possibly by stabilizing the transition states by H-bonding, quadrupole–ion, or van der Waals interactions. This contrasts with the evolutionarily related papain family of cysteine proteases, which uses Gln-19 (papain) for stabilizing the transition state.

Inroads into the Structure and Function of Intermediate Filament Networks

Although intermediate filaments are one of three major cytoskeletal systems of vertebrate cells, they remain the least understood with respect to their structure and function. This is due in part to the fact that they are encoded by a large gene family which is developmentally regulated in a cell and tissue type specific fashion. This article is in honor of Ueli Aebi. It highlights the studies on IF that have been carried out by our laboratory for more than 40 years. Many of our advances in understanding IF are based on conversations with Ueli which have taken place during adventurous and sometimes dangerous hiking and biking trips throughout the world.

Selectivity in the post-translational, transglutaminase-dependent acylation of lysine residues

Transglutaminases (TGs) are known to exhibit remarkable specificities not only for the Q (or Gln) sites but also for the K (or Lys) sites of proteins with which they react. To gain further insight into K-site specificity, we examined the reactions of dansyl-epsilon-aminocaproyl-GlnGlnIleVal with three chemically and structurally well-characterized proteins (bovine pancreatic ribonuclease A, bovine pancreatic trypsin inhibitor, and chicken egg white lysozyme), as catalyzed by TG2, a biologically important post-translational enzyme. The substrates represent a total of 20 potential surface sites for acylation by the fluorescent Gln probe, yet only two of the lysine side chains reacted with TG2. While the K1 site of ribonuclease and the K15 site of the trypsin inhibitor could be readily acylated by the enzyme, none of the lysines in lysozyme were modified. The findings lead us to suggest that the selection of lysine residues by TG2 is not encoded in the primary amino acid sequence surrounding the target side chain but depends primarily on its being positioned in an accessible segment of the protein structure.

Activation of transglutaminase in μ-calpain null erythrocytes

Intracellular transglutaminases (protein-glutamine: amine gamma-glutamyltransferase, EC 2.3.2.13) are calcium-dependent thiol enzymes that catalyze the covalent cross-linking of proteins, including those in the erythrocyte membrane. Several studies suggest that the activation of some transglutaminases is positively regulated by the calcium-dependent cysteine protease, mu-calpain. Using mu-calpain null (Capn1(-/-)) mouse erythrocytes, we demonstrate that the activation of soluble as well as membrane-bound forms of transglutaminase (TG2) in mouse erythrocytes was independent of mu-calpain. Also, the absence of mu-calpain or any detectable cysteine protease did not affect the transglutaminase activity in the erythrocyte lysate. Our studies also identify physiological substrates of mu-calpain in the erythrocyte membrane and show that their cleavage has no discernible effect on the transglutaminase mediated cross-linking of membrane proteins. Taken together, these data suggest the existence of a calpain-independent mechanism for the activation of transglutaminase 2 by calcium ions in the mouse erythrocytes and presumably also in non-erythroid cells.

Autonomic Remodeling in the Left Atrium and Pulmonary Veins in Heart FailureClinical Perspective

Background—Atrial fibrillation (AF) is commonly associated with congestive heart failure (CHF). The autonomic nervous system is involved in the pathogenesis of both AF and CHF. We examined the role of autonomic remodeling in contributing to AF substrate in CHF. Methods and Results—Electrophysiological mapping was performed in the pulmonary veins and left atrium in 38 rapid ventricular–paced dogs (CHF group) and 39 control dogs under the following conditions: vagal stimulation, isoproterenol infusion, &bgr;-adrenergic blockade, acetylcholinesterase (AChE) inhibition (physostigmine), parasympathetic blockade, and double autonomic blockade. Explanted atria were examined for nerve density/distribution, muscarinic receptor and &bgr;-adrenergic receptor densities, and AChE activity. In CHF dogs, there was an increase in nerve bundle size, parasympathetic fibers/bundle, and density of sympathetic fibrils and cardiac ganglia, all preferentially in the posterior left atrium/pulmonary veins. Sympathetic hyperinnervation was accompanied by increases in &bgr;1-adrenergic receptor R density and in sympathetic effect on effective refractory periods and activation direction. &bgr;-Adrenergic blockade slowed AF dominant frequency. Parasympathetic remodeling was more complex, resulting in increased AChE activity, unchanged muscarinic receptor density, unchanged parasympathetic effect on activation direction and decreased effect of vagal stimulation on effective refractory period (restored by AChE inhibition). Parasympathetic blockade markedly decreased AF duration. Conclusions—In this heart failure model, autonomic and electrophysiological remodeling occurs, involving the posterior left atrium and pulmonary veins. Despite synaptic compensation, parasympathetic hyperinnervation contributes significantly to AF maintenance. Parasympathetic and/or sympathetic signaling may be possible therapeutic targets for AF in CHF.Background— Atrial fibrillation (AF) is commonly associated with congestive heart failure (CHF). The autonomic nervous system is involved in the pathogenesis of both AF and CHF. We examined the role of autonomic remodeling in contributing to AF substrate in CHF. Methods and Results— Electrophysiological mapping was performed in the pulmonary veins and left atrium in 38 rapid ventricular–paced dogs (CHF group) and 39 control dogs under the following conditions: vagal stimulation, isoproterenol infusion, β-adrenergic blockade, acetylcholinesterase (AChE) inhibition (physostigmine), parasympathetic blockade, and double autonomic blockade. Explanted atria were examined for nerve density/distribution, muscarinic receptor and β-adrenergic receptor densities, and AChE activity. In CHF dogs, there was an increase in nerve bundle size, parasympathetic fibers/bundle, and density of sympathetic fibrils and cardiac ganglia, all preferentially in the posterior left atrium/pulmonary veins. Sympathetic hyperinnervation was accompanied by increases in β1-adrenergic receptor R density and in sympathetic effect on effective refractory periods and activation direction. β-Adrenergic blockade slowed AF dominant frequency. Parasympathetic remodeling was more complex, resulting in increased AChE activity, unchanged muscarinic receptor density, unchanged parasympathetic effect on activation direction and decreased effect of vagal stimulation on effective refractory period (restored by AChE inhibition). Parasympathetic blockade markedly decreased AF duration. Conclusions— In this heart failure model, autonomic and electrophysiological remodeling occurs, involving the posterior left atrium and pulmonary veins. Despite synaptic compensation, parasympathetic hyperinnervation contributes significantly to AF maintenance. Parasympathetic and/or sympathetic signaling may be possible therapeutic targets for AF in CHF.

Autonomic Remodeling in the Left Atrium and Pulmonary Veins in Heart FailureClinical Perspective: Creation of a Dynamic Substrate for Atrial Fibrillation

Background—Atrial fibrillation (AF) is commonly associated with congestive heart failure (CHF). The autonomic nervous system is involved in the pathogenesis of both AF and CHF. We examined the role of autonomic remodeling in contributing to AF substrate in CHF. Methods and Results—Electrophysiological mapping was performed in the pulmonary veins and left atrium in 38 rapid ventricular–paced dogs (CHF group) and 39 control dogs under the following conditions: vagal stimulation, isoproterenol infusion, &bgr;-adrenergic blockade, acetylcholinesterase (AChE) inhibition (physostigmine), parasympathetic blockade, and double autonomic blockade. Explanted atria were examined for nerve density/distribution, muscarinic receptor and &bgr;-adrenergic receptor densities, and AChE activity. In CHF dogs, there was an increase in nerve bundle size, parasympathetic fibers/bundle, and density of sympathetic fibrils and cardiac ganglia, all preferentially in the posterior left atrium/pulmonary veins. Sympathetic hyperinnervation was accompanied by increases in &bgr;1-adrenergic receptor R density and in sympathetic effect on effective refractory periods and activation direction. &bgr;-Adrenergic blockade slowed AF dominant frequency. Parasympathetic remodeling was more complex, resulting in increased AChE activity, unchanged muscarinic receptor density, unchanged parasympathetic effect on activation direction and decreased effect of vagal stimulation on effective refractory period (restored by AChE inhibition). Parasympathetic blockade markedly decreased AF duration. Conclusions—In this heart failure model, autonomic and electrophysiological remodeling occurs, involving the posterior left atrium and pulmonary veins. Despite synaptic compensation, parasympathetic hyperinnervation contributes significantly to AF maintenance. Parasympathetic and/or sympathetic signaling may be possible therapeutic targets for AF in CHF.Background— Atrial fibrillation (AF) is commonly associated with congestive heart failure (CHF). The autonomic nervous system is involved in the pathogenesis of both AF and CHF. We examined the role of autonomic remodeling in contributing to AF substrate in CHF. Methods and Results— Electrophysiological mapping was performed in the pulmonary veins and left atrium in 38 rapid ventricular–paced dogs (CHF group) and 39 control dogs under the following conditions: vagal stimulation, isoproterenol infusion, β-adrenergic blockade, acetylcholinesterase (AChE) inhibition (physostigmine), parasympathetic blockade, and double autonomic blockade. Explanted atria were examined for nerve density/distribution, muscarinic receptor and β-adrenergic receptor densities, and AChE activity. In CHF dogs, there was an increase in nerve bundle size, parasympathetic fibers/bundle, and density of sympathetic fibrils and cardiac ganglia, all preferentially in the posterior left atrium/pulmonary veins. Sympathetic hyperinnervation was accompanied by increases in β1-adrenergic receptor R density and in sympathetic effect on effective refractory periods and activation direction. β-Adrenergic blockade slowed AF dominant frequency. Parasympathetic remodeling was more complex, resulting in increased AChE activity, unchanged muscarinic receptor density, unchanged parasympathetic effect on activation direction and decreased effect of vagal stimulation on effective refractory period (restored by AChE inhibition). Parasympathetic blockade markedly decreased AF duration. Conclusions— In this heart failure model, autonomic and electrophysiological remodeling occurs, involving the posterior left atrium and pulmonary veins. Despite synaptic compensation, parasympathetic hyperinnervation contributes significantly to AF maintenance. Parasympathetic and/or sympathetic signaling may be possible therapeutic targets for AF in CHF.