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Papers by John Dedman

Circulation-arrhythmia and Electrophysiology, Dec 1, 2011

Background-Digitalis-induced Na ϩ accumulation results in an increase in Ca 2ϩ i via the Na ϩ /Ca... more Background-Digitalis-induced Na ϩ accumulation results in an increase in Ca 2ϩ i via the Na ϩ /Ca 2ϩ exchanger, leading to enhanced sarcoplasmic reticulum (SR) Ca 2ϩ load, responsible for the positive inotropic and toxic arrhythmogenic effects of glycosides. A digitalis-induced increase in Ca 2ϩ i could also activate calcium-calmodulin kinase II (CaMKII), which has been shown to have proarrhythmic effects. Here, we investigate whether CaMKII underlies digitalis-induced arrhythmias and the subcellular mechanisms involved. Methods and Results-In paced rat ventricular myocytes (0.5 Hz), 50 mol/L ouabain increased contraction amplitude by 160Ϯ5%. In the absence of electric stimulation, ouabain promoted spontaneous contractile activity and Ca 2ϩ waves. Ouabain activated CaMKII (p-CaMKII), which phosphorylated its downstream targets, phospholamban (PLN) (Thr17) and ryanodine receptor (RyR) (Ser2814). Ouabain-induced spontaneous activity was prevented by inhibiting CaMKII with 2.5 mol/L KN93 but not by 2.5 mol/L of the inactive analog, KN92. Similar results were obtained using the CaMKII inhibitor, autocamtide-2 related inhibitory peptide (AIP) (1 to 2.5 mol/L), and in myocytes from transgenic mice expressing SR-targeted AIP. Consistently, CaMKII overexpression exacerbated ouabain-induced spontaneous contractile activity. Ouabain was associated with an increase in SR Ca 2ϩ content and Ca 2ϩ spark frequency, indicative of enhanced SR Ca 2ϩ leak. KN93 suppressed the ouabain-induced increase in Ca 2ϩ spark frequency without affecting SR Ca 2ϩ content. Similar results were obtained with digoxin. In vivo, ouabain-induced arrhythmias were prevented by KN93 and absent in SR-AIP mice. Conclusions-These results show for the first time that CaMKII mediates ouabain-induced arrhythmic/toxic effects. We suggest that CaMKII-dependent phosphorylation of the RyR, resulting in Ca 2ϩ leak from the SR, is the underlying mechanism involved. (Circ Arrhythm Electrophysiol. 2011;4:947-957.) Key Words: cardiotonic steroids Ⅲ arrhythmias Ⅲ CaMKII Ⅲ heart failure C ardiotonic glycosides selectively bind to and inhibit the sarcolemmal Na ϩ /K ϩ-ATPase and cause an increase in intracellular Na ϩ , which in the heart reduces Ca 2ϩ extrusion and/or increases Ca 2ϩ influx through the Na ϩ /Ca 2ϩ exchanger (NCX). This increase in Ca 2ϩ i leads to an increase in sarcoplasmic reticulum (SR) Ca 2ϩ load and to a positive inotropic effect, which explains, at least in part, their thera

Resultados Los resultados indican que R luego de isquemia induce la aparición de numerosos latido... more Resultados Los resultados indican que R luego de isquemia induce la aparición de numerosos latidos ectópicos (LE), que disminuyeron significativamente por el tratamiento con el inhibidor de CaMKII, KN-93 (1�M)(LE 46±6 Ctrol vs 11±3 KN-93). Disminuir la entrada de Ca2+ a la célula o la liberación Ca2+ desde el retículo sarcoplasmático (RS) por tratamiento con Nifedipina (Nife) o Rianodina (Ry) respectivamente, disminuyó la aparición de arritmias (46±6 Ctrol vs 8±2 Nife y 25±3 Ry, p<0,05). La incidencia de aritmias de R en ratones transgénicos con inhibición selectiva de CaMKI a nivel de las membranas del RS (SR-AIP) también fue menor (3±1 SR-AIP vs 34±10 WT). La PCaMKI aumentó significativamente en R (198±9%) respecto de lo valores preisquémicos, al igual que la fosforilación de sus sustratos PS2815 de RyR2 y PT17 de PLN. El tratamiento de los corazones con apocinina, un atrapador de especies reactivas del O2, no modificó la aparición de aritmias ni la PCaMKI

American Journal of Physiology-heart and Circulatory Physiology, Oct 1, 2008

Returning to normal pH after acidosis, similar to reperfusion after ischemia, is prone to arrhyth... more Returning to normal pH after acidosis, similar to reperfusion after ischemia, is prone to arrhythmias. The type and mechanisms of these arrhythmias have never been explored and were the aim of the present work. Langendorff-perfused rat/mice hearts and rat-isolated myocytes were subjected to respiratory acidosis and then returned to normal pH. Monophasic action potentials and left ventricular developed pressure were recorded. The removal of acidosis provoked ectopic beats that were blunted by 1 mM of the CaMKII inhibitor KN-93, 1 mM thapsigargin, to inhibit sarcoplasmic reticulum (SR) Ca 21 uptake, and 30 nM ryanodine or 45 mM dantrolene, to inhibit SR Ca 21 release and were not observed in a transgenic mouse model with inhibition of CaMKII targeted to the SR. Acidosis increased the phosphorylation of Thr 17 site of phospholamban (PT-PLN) and SR Ca 21 load. Both effects were precluded by KN-93. The return to normal pH was associated with an increase in SR Ca 21 leak, when compared with that of control or with acidosis at the same SR Ca 21 content. Ca 21 leak occurred without changes in the phosphorylation of ryanodine receptors type 2 (RyR2) and was blunted by KN-93. Experiments in planar lipid bilayers confirmed the reversible inhibitory effect of acidosis on RyR2. Ectopic activity was triggered by membrane depolarizations (delayed afterdepolarizations), primarily occurring in epicardium and were prevented by KN-93. The results reveal that arrhythmias after acidosis are dependent on CaMKII activation and are associated with an increase in SR Ca 21 load, which appears to be mainly due to the increase in PT-PLN. sarcoplasmic reticulum; calcium/calmodulin-dependent protein kinase CARDIAC ARRHYTHMIAS are a leading cause of morbidity and mortality. Despite their importance, a clear comprehension of the mechanisms underlying life-threatening ventricular tachyarrhythmias is lacking (6, 23). Different types of evidence indicate that acidosis is able to generate arrhythmias in the heart (27, 37). This is important in the clinical setting since substantial changes in extracellular and/or intracellular pH may occur in several disorders of different origin, like sleep apnea/ hypopnea syndrome, diabetic ketoacidosis, or in patients on dialysis, which affect cardiac function (32). Moreover, a marked acidosis occurs during myocardial ischemia, which

Calcium-Binding Proteins in Health and Disease, 1987

Publisher Summary This chapter reviews about phenothiazines, which are antipsychotic agents commo... more Publisher Summary This chapter reviews about phenothiazines, which are antipsychotic agents commonly used as affinity ligands for calmodulin. Immunoblots and peptide maps have revealed that the 67 and the 35 kDa calcimedins are immunologically related, and share a number of common peptides. Similar experiments with the 30 and the 33 kDa calcimedins have indicated that they are immunologically unrelated to the other calcimedins. Synexin is also related to the calcimedins but does not correspond directly to any of the calcimedins. Calregulins, p35 and pp36, were found to be immunologically unrelated to the calcimedins. The calcimedins and the immunologically related proteins appear to represent a new family of proteins, distinct from calmodulin, which respond to the calcium signal by developing a functional hydrophobic domain. A single mechanism can result in multiple independent paths of regulation using each individual calcimedin. Tisssue or cell specific expression of both the calcimedins and their target proteins would then provide an additional level of physiological control.

Cell Physiology Source Book, 2001

Publisher Summary This chapter discusses mediation by calcium-binding proteins as an intracellula... more Publisher Summary This chapter discusses mediation by calcium-binding proteins as an intracellular second messenger. The chapter discusses several aspects related to Ca2+ , such as determination of Ca2+ involvement in physiological processes, Ca2+ as an intracellular signal, creation of the Ca 2+ signal, mediation of Ca2+ signal, Ca2+-calmodulin dependent protein kinase II, calcium-dependent phospholipid-binding proteins, and protein kinase C. The chapter discusses the current perspectives related to Ca 2+. It is mentioned that an alternative approach to understanding the precise role of proteins associated with the intracellular Ca2+ signal is to design and construct dominant-negative genes that, when expressed, neutralize the function of specific proteins. There have been numerous studies relating Ca2+ and cell functions, including fertilization, development, differentiation, adhesion, growth, division, movement, contraction, and secretion. This evidence demonstrates a primary regulatory role for ionized Ca2+ in biological systems. Ca2+ has also been associated with a number of diseases, particularly those of the muscular and nervous systems, in which this ion plays an important role in contraction and neurotransmitter release. Understanding the mechanism of Ca2+ action has required approaches and expertise from distinct fields. Ca2+ is unique compared with other second messengers, which are formed as metabolic intermediates, such as cyclic nucleotides, inositol phosphates, and diacylglycerol. Ca2+ is a divalent elemental metal and is not converted to any other form as a part of its cellular regulatory properties. The fact that Ca2+ has been associated with a wide variety of cellular functions brings attention to the fact that the blocking of one Ca2+ regulated function could very likely affect interdependent secondary and tertiary functions. Cell stimulation causes the intracellular Ca2+ to increase transiently. This second-messenger signal is then mediated by Ca2+ -binding proteins. There are three primary molecular mechanisms of transmitting the signal—calmodulin, annexins, and protein kinase C. Each of these pathways intersects and can be cross-regulatory. Cellular studies using specific activators and inhibitors of protein kinase C have shown this Ca2+ pathway to be involved in cell growth, differentiation, and development of tumors. Physiological, cellular, and molecular techniques are being used in combination to define the precise cellular roles of Ca2+ -binding proteins.

Cell Physiology Source Book, 2012

Cell Calcium, 1986

The discovery of troponin C and calmodulin set the tenor for understanding the intracellular mech... more The discovery of troponin C and calmodulin set the tenor for understanding the intracellular mechanism of action of calcium. These proteins represent cellular receptors and distinct mediators of calcium. More recently, additional calcium-binding proteins have been identified. Immunological and sequence data suggest that these proteins represent a novel family of calcium mediators. The precise pathways in which these proteins are involved are not known. However, function by inference, the mediation of intracellular calcium, provides new avenues in which to better understand the complex cellular role calcium plays in regulating cell function.

Biochemical and Biophysical Research Communications, 1989

The 30 kDa calcimedin was found to bind directly to phenyl-Sepharose in a calcium dependent manne... more The 30 kDa calcimedin was found to bind directly to phenyl-Sepharose in a calcium dependent manner similar to calmodulin. The 30 kDa calcimedin was also found to bind to and inhibit DNase I. This calcium-dependent binding was exploited to develop a two-step purification scheme for this calcimedin. In addition, affinity-purified antibodies to the 30 kDa calcimedin were used to examine its tissue distribution. The highest levels were found in lung, trachea and diaphragm while the lowest levels of the 30 kDa calcimedin were found in brain and skeletal muscle.

Annals of the New York Academy of Sciences, 1984

A set of four proteins from muscle tissues, termed calcimedins, bind hydrophobic matrices in a ca... more A set of four proteins from muscle tissues, termed calcimedins, bind hydrophobic matrices in a calcium-dependent manner.' Several properties of these proteins have been investigated by biochemical techniques and compared to those exhibited by calmodulin, the ubiquitous Ca*+-binding protein of eukaryotic cells. The calcimedin proteins are similar to calmodulin in that they are acidic (bind DEAE cellulose), are relatively small (MW,,, = 67K, 35K, 33K, and 30K), are apparently monomeric (no large oligomers observed by gel filtration, non-SDS gel electrophoresis or chemical cross-linking) and have a hydrophobic binding site exposed by CaZ+. These proteins are dissimilar from calmodulin, however, in that they do not contain trimethyl lysine nor do they activate uterine myosin light chain kinase nor brain cyclic nucleotide phosphodiesterase.* These calcimedins are not as widely distributed in tissues as calmodulin, but are fairly abundant in muscle tissue homogenates, compared to calmodulin. All calcium action requires specific intracellular receptor proteins to mediate the signal. Thus troponin C interacts with troponin I whereas calmodulin interacts with several cellular proteins to mediate calcium action. This is also true for the calcimedins. Gizzard extracts are prepared in 0.075 M NaCI, 0.04 M Tris-HCI, pH 7.3 containing 1.0% NaN, and 2 m M EDTA. After centrifugation, CaCI, is added to 1-2 m M above the EDTA level and the calcimedin proteins are removed by fluphenazineSepharose chromatography. The unbound extract is then chromatographed on a calcimedin-Sepharose affinity matrix. Several proteins bind the column in a calciumdependent manner. Proteins binding the calcimedin column were compared to those binding an octyl-Sepharose matrix (Sigma Chemical Co.). The results are shown in FIGURE 1 . Proteins of special interest are those in lane 1 (large arrows) that appear to bind specifically to the calcimedin-Sepharose matrix. Affinity columns were then prepared by coupling the 67K calcimedin or calmodulin to Sepharose 4B. Rat heart tissue was homogenized as above, divided into two fractions and chromatographed on either the calcimedin-Sepharose column or the calmodulin-Sepharose column. FIGURE 2 shows that several proteins bound both matrices. The proteins binding the total calcimedin affinity matrix were rechromatographed on the 67K calcimedin-Sepharose column. One of the four major proteins

Journal of Biological Chemistry, 1987

Identification of a 55-kDa High-affinity Calmodulin-binding Protein from ~~e c~~o p~u r a s elec#... more Identification of a 55-kDa High-affinity Calmodulin-binding Protein from ~~e c~~o p~u r a s elec#rieas*

Physiology, 1995

The annexins are a family of Ca2+-dependent membrane binding proteins that have been shown to reg... more The annexins are a family of Ca2+-dependent membrane binding proteins that have been shown to regulate ion conductances, aggregate vesicles and the cytoskeleton, inhibit phospholipase A2, and inhibit blood coagulation. The annexins represent a unique mechanism of cellular regulation by modifying membrane functions in a Ca2+-dependent manner.

American Journal of Physiology-Cell Physiology, 1987

The Ca2+-dependent binding of [125I]calmodulin (CaM) to hepatic proteins separated by sodium dode... more The Ca2+-dependent binding of [125I]calmodulin (CaM) to hepatic proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was utilized to identify CaM binding or “acceptor” proteins or CAPs. Two proteins of apparent molecular weight of 60,000 (CAP-60) and 45,000 (CAP-45) comprised greater than 80% of the Ca2+-dependent CaM binding in rat liver cytosol. CAP-60 and CAP-45 were partially purified by a variety of chromatographic steps, including affinity chromatography on CaM Sepharose. CAP-60 possessed a native molecular size of 400,000, indicating it to be the CaM-binding “subunit” of a larger oligomeric complex. In contrast, CAP-45 was monomeric as judged by gel filtration. Neither CAP-60 nor CAP-45 possessed chromatographic properties consistent with known CaM-dependent enzymes reported in the literature. Two-dimensional peptide mapping provided convincing evidence that CAP-60 and CAP-45 were unrelated to other well-characterized CAPs, namely Ca2+ (C...

It is quite conceivable that every species tends to produce varieties of a limited number and kin... more It is quite conceivable that every species tends to produce varieties of a limited number and kind, and that the effect of natural selection is to favour the development of some of these, while it opposes the development of others along their predetermined lines of modification." Thomas H. Huxley Evolution in Biology. 1878 PROPERTIES OF ALDOLASE CLASSES Class I Class II Animals, plants, green algae, euglena and chlamydomonas (Autotrophic) Schiff-base intermediate ((-amino lysine-DHAP) Functional carboxyterminal tyrosine Broad pH profile MW 120,000-160,000 s 20,» 6.5-8.0 4 subunits FDP/FIP ratio 50,1,10 Specific Activity 8r25 Bacteria, yeast, fungi, bluegreen algae, euglena & 1 chlamydomonas (Heterotrophic) Divalent metal ion requirement K + activation Functional SH groups Sharp pH profile (7»0-7.5

Comparative Biochemistry and Physiology Part B: Comparative Biochemistry, 1974

1.1. The percentage of sequence similarity of homologous proteins can be estimated from a linear ... more 1.1. The percentage of sequence similarity of homologous proteins can be estimated from a linear composition coefficient analysis of the amino acid compositions.

Biochemical Society Transactions, 1990

Comparative Biochemistry and Physiology Part A: Physiology, 1997

Electric tissue of the electric eel, Electrophorus electricus, has been used extensively as a mod... more Electric tissue of the electric eel, Electrophorus electricus, has been used extensively as a model system for the study of excitable membrane biochemistry and electrophysiology. Membrane receptors, ion channels, and ATPases utilized by electrocytes are conserved in mammalian neurons and myocytes. In this study, we show that Ca 2ϩ predominates as the major mediator of electric tissue phosphorylation relative to cyclic AMP and cyclic GMP-induced phosphorylation. Mastoparan, a calmodulin inhibitor peptide, and a peptide corresponding to the pseudosubstrate region of mammalian calmodulin-dependent protein kinase II (CaMKII (281-302)) attenuated Ca 2ϩ-dependent phosphorylation in a dose-dependent manner. These experiments demonstrated that calmodulin-dependent protein kinase II activity predominates in electric tissue. The Electrophorus kinase was purified by a novel affinity chromatography procedure utilizing Ca 2ϩ /calmodulin-dependent binding to the CaMKII (281-302) peptide coupled to Sepharose. The purified 51 kDa calmodulin-dependent protein kinase II demonstrated extensive autophosphorylation and exhibited a 3-to 4-fold increase in Ca 2ϩ-independent activity following autophosphorylation. Immunofluorescent localization experiments demonstrated calmodulin to be abundant in electrocytes, particularly subjacent to the plasma membrane. Calmodulin-dependent protein kinase II had a punctate distribution indicating that it may be compartmentalized by association with vesicles or the cytoskeleton. As the primary mediator of phosphorylation within electric tissue, CaM kinase II may be critical for the regulation of the specialized electrophysiological function of electrocytes. comp biochem physiol

Respiration Physiology, 1997

Oxygen is a strict requirement for cell function. The cellular mechanisms by which organisms dete... more Oxygen is a strict requirement for cell function. The cellular mechanisms by which organisms detect and respond to changes in oxygen tension remain a major unanswered question in pulmonary physiology. Part of the difficulty in addressing this question is due to the limited scope of experiments that can be performed in vivo. In the past few years, several laboratories have begun to make progress in this area, using a variety of cell culture model systems and sophisticated genetic manipulations. Here, we review the current state of knowledge of regulation of gene expression by hypoxia, and describe novel experimental approaches that promise to broaden our understanding of how cells and whole organisms respond to alterations in O 2 tension.

Cell Physiology Source Book, 1995

Calcium (Ca 2+ ) is an abundant element found throughout nature and in all living tissues. It is ... more Calcium (Ca 2+ ) is an abundant element found throughout nature and in all living tissues. It is considered the primordial second messengers since, unlike other second messengers, such as cyclic nucleotides, steroids or nitric oxide, it is not synthesized or degraded. Intracellular Ca 2+ is maintained at levels lower than extracellular by sequestration within membrane compartments and regulation of membrane permeability via membrane channels, pumps and ion exchangers. An increase in intracellular Ca 2+ [Ca 2+ ] i levels can be initiated by factors such as hormones, neurotransmitters, growth factors, cytokines or changes in membrane potential. The transient rise in [Ca 2+ ] i , visualized by fluorescent indicator dyes, may be a localized unitary spark, a wave or may oscillate the frequency and amplitude modulating the response. The biological action of Ca 2+ is mediated by high affinity, specific binding proteins that are differentially located and expressed. Calmodulin is the most ubiquitous Ca 2+ mediator protein. Ca 2+ -bound calmodulin interacts with and activates enzymes by displacing the pseudosubstrate region from the active site. Metabolic sculpturing of CaMKII, for example, can generate a dynamic range in enzyme activity that modulates neurotransmission and cardiac function. Annexins are Ca 2+ -dependent phospholipid binding proteins that are found in animals and higher plants. Although they do not possess enzymatic activity, they form interlocking Ca 2+ -dependent complexes on specific membranes thereby modifying both membrane fluidity and the activity of membrane-associated protein complexes. Members of the protein kinase C family are activated by Ca 2+ and diacylglycerol. Collectively, effective translation of the Ca 2+ signal is conveyed through a diverse array of mediator proteins.

Placenta, 2001

Annexin V is an intracellular protein that lacks a hydrophobic signal peptide. However, there are... more Annexin V is an intracellular protein that lacks a hydrophobic signal peptide. However, there are several studies reporting the extracellular presence of annexin V. In this study, we designed transgenes of annexin V with or without an attached secretory signal peptide and investigated the secretion of the transgene products in COS-7 cells. The signal peptide, targeted annexin V to the endoplasmic reticulum (ER), the Golgi and culture media of transfected cells. In contrast, without the signal peptide, annexin V was present only in the cytoplasm and was not detected in the medium. To confirm our results we also evaluated the presence of extracellular annexin V in two cultured cell lines: BeWo, a choriocarcinoma cell model of placental trophoblasts, and human umbilical vein endothelial cells (HUVEC). Our results showed that annexin V was immunolocalized on the surfaces of both cells but could not be detected in the culture medium of either cell type. Our results suggest that the secretion of annexin V required the recombinant addition of a hydrophobic signal peptide and that the limited quantities of endogenous cell surface annexin V on BeWo and HUVEC cells is most likely derived from adjacent damaged cells.