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W J Lederer

Publications and source records attributed to W J Lederer.

At least 19 recordsLinked to original sources
PubMed2006

Probing the outer mitochondrial membrane in cardiac mitochondria with nanoparticles.

The outer mitochondrial membrane (OMM) is the last barrier between the mitochondrion and the cytoplasm. Breaches of OMM integrity result in the release of cytochrome c oxidase, triggering apoptosis. In this study, we used calibrated gold nanoparticles to probe the OMM in rat permeabilized ventricular cells and in isolated cardiac mitochondria under quasi-physiological ionic conditions and during permeability transition. Our experiments showed that under control conditions, the OMM is not permeable to 6-nm particles. However, 3-nm particles could enter the mitochondrial intermembrane space in mitochondria of permeabilized cells and isolated cardiac mitochondria. Known inhibitors of the voltage-dependent anion channel (VDAC), König polyanion, and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid inhibited this entrance. Thus, 3-nm particles must have entered the mitochondrial intermembrane space through the VDAC. The permeation of the isolated cardiac mitochondria OMM for 3-nm particles was approximately 20 times that in permeabilized cells, suggesting low availability of VDAC pores within the cell. Experiments with expressed green fluorescent protein showed the existence of intracellular barriers restricting the VDAC pore availability in vivo. Thus, our data showed that 1), the physical diameter of VDAC pores in cardiac mitochondria is >or=3 nm but <or=6 nm, and 2), permeability transition-related mitochondrial swelling results in breaching and disruption of the OMM.

Animals↗
PubMed2006

Stabilization of cardiac ryanodine receptor prevents intracellular calcium leak and arrhythmias.

Catecholaminergic polymorphic ventricular tachycardia is a form of exercise-induced sudden cardiac death that has been linked to mutations in the cardiac Ca2+ release channel/ryanodine receptor (RyR2) located on the sarcoplasmic reticulum (SR). We have shown that catecholaminergic polymorphic ventricular tachycardia-linked RyR2 mutations significantly decrease the binding affinity for calstabin-2 (FKBP12.6), a subunit that stabilizes the closed state of the channel. We have proposed that RyR2-mediated diastolic SR Ca2+ leak triggers ventricular tachycardia (VT) and sudden cardiac death. In calstabin-2-deficient mice, we have now documented diastolic SR Ca2+ leak, monophasic action potential alternans, and bidirectional VT. Calstabin-deficient cardiomyocytes exhibited SR Ca2+ leak-induced aberrant transient inward currents in diastole consistent with delayed after-depolarizations. The 1,4-benzothiazepine JTV519, which increases the binding affinity of calstabin-2 for RyR2, inhibited the diastolic SR Ca2+ leak, monophasic action potential alternans and triggered arrhythmias. Our data suggest that calstabin-2 deficiency is as a critical mediator of triggers that initiate cardiac arrhythmias.

Animals↗
PubMed2006

Orphaned ryanodine receptors in the failing heart.

Heart muscle is characterized by a regular array of proteins and structures that form a repeating functional unit identified as the sarcomere. This regular structure enables tight coupling between electrical activity and Ca(2+) signaling. In heart failure, multiple cellular defects develop, including reduced contractility, altered Ca(2+) signaling, and arrhythmias; however, the underlying causes of these defects are not well understood. Here, in ventricular myocytes from spontaneously hypertensive rats that develop heart failure, we identify fundamental changes in Ca(2+) signaling that are related to restructuring of the spatial organization of the cells. Myocytes display both a reduced ability to trigger sarcoplasmic reticulum Ca(2+) release and increased spatial dispersion of the transverse tubules (TTs). Remodeled TTs in cells from failing hearts no longer exist in the regularly organized structures found in normal heart cells, instead moving within the sarcomere away from the Z-line structures and leaving behind the sarcoplasmic reticulum Ca(2+) release channels, the ryanodine receptors (RyRs). These orphaned RyRs appear to be responsible for the dyssynchronous Ca(2+) sparks that have been linked to blunted contractility and, probably, Ca(2+)-dependent arrhythmias in diverse models of heart failure. We conclude that the increased spatial dispersion of the TTs and orphaned RyRs lead to the loss of local control and Ca(2+) instability in heart failure.

Animals↗
PubMed2006

Restitution of Ca(2+) release and vulnerability to arrhythmias.

New information has recently been obtained along two essentially parallel lines of research: investigations into the fundamental mechanisms of Ca(2+)-induced Ca(2+) release (CICR) in heart cells, and analyses of the factors that control the development of unstable rhythms such as repolarization alternans. These lines of research are starting to converge such that we can begin to understand unstable and potentially arrhythmogenic cardiac dynamics in terms of the underlying mechanisms governing not only membrane depolarization and repolarization but also the complex bidirectional interactions between electrical and Ca(2+) signaling in heart cells. In this brief review, we discuss the progress that has recently been made in understanding the factors that control the beat-to-beat regulation of cardiac Ca(2+) release and attempt to place these results within a larger context. In particular, we discuss factors that may contribute to unstable Ca(2+) release and speculate about how instability in CICR may contribute to the development of arrhythmias under pathological conditions.

Action Potentials↗
PubMed2005

Aqueous diffusion pathways as a part of the ventricular cell ultrastructure.

The physical organization of the ventricular myocyte includes barriers for the movement of objects of varying dimensions ranging from ions to solid particles. There are two kinds of diffusion in the cell: lateral (in membranes) and aqueous. Here we examine the size constraints of aqueous diffusion pathways and discuss their impact on cellular physiology. Calibrated gold nanoparticles were used to probe the accessibility of the entire transverse-axial tubular system (TATS), the sarcoplasm, and intracellular structures. The TATS tubules, although up to 300 nm in diameter, permitted only particles </=11 nm to enter. When calibrated nanoparticles were added to permeabilized ventricular cells, particles </=11 nm were found in the sarcoplasm. The distribution of nanoparticles in the cells allowed us to conclude that 1), the TATS and the sarcoplasm are accessible only for particles </=11 nm; 2), the gaps between T-tubules and junctional sarcoplasmic reticulum (jSR), jSR and mitochondria, and intermitochondrial contacts are inaccessible for particles with physical size >3 nm; 3), the mitochondrial voltage-dependent anion channel and the nuclear pore complex in ventricular cells could not be penetrated by particles >/=6 nm; and 4), there is a difference in size clearance between transversal and longitudinal sarcoplasmic diffusional pathways.

Animals↗
PubMed2005

The Ca 2+ leak paradox and rogue ryanodine receptors: SR Ca 2+ efflux theory and practice.

Ca(2+) efflux from the sarcoplasmic reticulum (SR) is routed primarily through SR Ca(2+) release channels (ryanodine receptors, RyRs). When clusters of RyRs are activated by trigger Ca(2+) influx through L-type Ca(2+) channels (dihydropyridine receptors, DHPR), Ca(2+) sparks are observed. Close spatial coupling between DHPRs and RyR clusters and the relative insensitivity of RyRs to be triggered by Ca(2+) together ensure the stability of this positive-feedback system of Ca(2+) amplification. Despite evidence from single channel RyR gating experiments that phosphorylation of RyRs by protein kinase A (PKA) or calcium-calmodulin dependent protein kinase II (CAMK II) causes an increase in the sensitivity of the RyR to be triggered by [Ca(2+)](i) there is little clear evidence to date showing an increase in Ca(2+) spark rate. Indeed, there is some evidence that the SR Ca(2+) content may be decreased in hyperadrenergic disease states. The question is whether or not these observations are compatible with each other and with the development of arrhythmogenic extrasystoles that can occur under these conditions. Furthermore, the appearance of an increase in the SR Ca(2+) "leak" under these conditions is perplexing. These and related complexities are analyzed and discussed in this report. Using simple mathematical modeling discussed in the context of recent experimental findings, a possible resolution to this paradox is proposed. The resolution depends upon two features of SR function that have not been confirmed directly but are broadly consistent with several lines of indirect evidence: (1) the existence of unclustered or "rogue" RyRs that may respond differently to local [Ca(2+)](i) in diastole and during the [Ca(2+)](i) transient; and (2) a decrease in cooperative or coupled gating between clustered RyRs in response to physiologic phosphorylation or hyper-phosphorylation of RyRs in disease states such as heart failure. Taken together, these two features may provide a framework that allows for an improved understanding of cardiac Ca(2+) signaling.

Animals↗
PubMed2005

Local recovery of Ca2+ release in rat ventricular myocytes.

Excitation-contraction coupling in the heart depends on the positive feedback process of Ca2+-induced Ca2+ release (CICR). While CICR provides for robust triggering of Ca2+ sparks, the mechanisms underlying their termination remain unknown. At present, it is unclear how a cluster of Ca2+ release channels (ryanodine receptors or RyRs) can be made to turn off when their activity is sustained by the Ca2+ release itself. We use a novel experimental approach to investigate indirectly this issue by exploring restitution of Ca2+ sparks. We exploit the fact that ryanodine can bind, nearly irreversibly, to an RyR subunit (monomer) and increase the open probability of the homotetrameric channel. By applying low concentrations of ryanodine to rat ventricular myocytes, we observe repeated activations of individual Ca2+ spark sites. Examination of these repetitive Ca2+ sparks reveals that spark amplitude recovers with a time constant of 91 ms whereas the sigmoidal recovery of triggering probability lags behind amplitude recovery by approximately 80 ms. We conclude that restitution of Ca2+ sparks depends on local refilling of SR stores after depletion and may also depend on another time-dependent process such as recovery from inactivation or a slow conformational change after rebinding of Ca2+ to SR regulatory proteins.

Animals↗
PubMed2005

Calmodulin kinase II inhibition protects against structural heart disease.

Beta-adrenergic receptor (betaAR) stimulation increases cytosolic Ca(2+) to physiologically augment cardiac contraction, whereas excessive betaAR activation causes adverse cardiac remodeling, including myocardial hypertrophy, dilation and dysfunction, in individuals with myocardial infarction. The Ca(2+)-calmodulin-dependent protein kinase II (CaMKII) is a recently identified downstream element of the betaAR-initiated signaling cascade that is linked to pathological myocardial remodeling and to regulation of key proteins involved in cardiac excitation-contraction coupling. We developed a genetic mouse model of cardiac CaMKII inhibition to test the role of CaMKII in betaAR signaling in vivo. Here we show CaMKII inhibition substantially prevented maladaptive remodeling from excessive betaAR stimulation and myocardial infarction, and induced balanced changes in excitation-contraction coupling that preserved baseline and betaAR-stimulated physiological increases in cardiac function. These findings mark CaMKII as a determinant of clinically important heart disease phenotypes, and suggest CaMKII inhibition can be a highly selective approach for targeting adverse myocardial remodeling linked to betaAR signaling.

Adrenergic beta-Antagonists↗
PubMed2005

Ghost sparks.

Explore the source record for details and available documents.

Animals↗
PubMed2005

Twenty years of calcium imaging: cell physiology to dye for.

The use of fluorescent dyes over the past two decades has led to a revolution in our understanding of calcium signaling. Given the ubiquitous role of Ca(2+) in signal transduction at the most fundamental levels of molecular, cellular, and organismal biology, it has been challenging to understand how the specificity and versatility of Ca(2+) signaling is accomplished. In excitable cells, the coordination of changing Ca(2+) concentrations at global (cellular) and well-defined subcellular spaces through the course of membrane depolarization can now be conceptualized in the context of disease processes such as cardiac arrhythmogenesis. The spatial and temporal dimensions of Ca(2+) signaling are similarly important in non-excitable cells, such as endothelial and epithelial cells, to regulate multiple signaling pathways that participate in organ homeostasis as well as cellular organization and essential secretory processes.

Animals↗
PubMed2005

Calcium biology of the transverse tubules in heart.

Ca(2+) sparks in heart muscle are activated on depolarization by the influx of Ca(2+) through dihydropyridine receptors in the sarcolemmal (SL) and transverse tubule (TT) membranes. The cardiac action potential is thus able to synchronize the [Ca(2+)](i) transient as Ca(2+) release is activated throughout the cell. Increases in the amount of Ca(2+) within the sarcoplasmic reticulum (SR) underlie augmented Ca(2+) release globally and an increase in the sensitivity of the ryanodine receptors (RyRs) to be triggered by the local [Ca(2+)](i). In a similar manner, phosphorylation of the RyRs by protein kinase A (PKA) increases the sensitivity of the RyRs to be activated by local [Ca(2+)](i). Heart failure and other cardiac diseases are associated with changes in SR Ca(2+) content, phosphorylation state of the RyRs, [Ca(2+)](i) signaling defects and arrhythmias. Additional changes in transverse tubules and nearby junctional SR may contribute to alterations in local Ca(2+) signaling. Here we briefly discuss how TT organization can influence Ca(2+) signaling and how changes in SR Ca(2+) release triggering can influence excitation-contraction (EC) coupling. High speed imaging methods are used in combination with single cell patch clamp experiments to investigate how abnormal Ca(2+) signaling may be regulated in health and disease. Three issues are examined in this presentation: (1) normal Ca(2+)-induced Ca(2+) release and Ca(2+) sparks, (2) abnormal SR Ca(2+) release in disease, and (3) the triggering and propagation of waves of elevated [Ca(2+)](i).

Animals↗
PubMed2004

Overexpression of beta2-adrenergic receptors cAMP-dependent protein kinase phosphorylates and modulates slow delayed rectifier potassium channels expressed in murine heart: evidence for receptor/channel co-localization.

The cardiac slow delayed rectifier potassium channel (IKs), comprised of (KCNQ1) and beta (KCNE1) subunits, is regulated by sympathetic nervous stimulation, with activation of beta-adrenergic receptors PKA phosphorylating IKs channels. We examined the effects of 2-adrenergic receptors (beta2-AR) on IKs in cardiac ventricular myocytes from transgenic mice expressing fusion proteins of IKs subunits and hbeta2-ARs. KCNQ1 and beta2-ARs were localized to the same subcellular regions, sharing intimate localization within nanometers of each other. In IKs/B2-AR myocytes, IKs density was increased, and activation shifted in the hyperpolarizing direction; IKs was not further modulated by exposure to isoproterenol, and KCNQ1 was found to be PKA-phosphorylated. Conversely, beta2-AR overexpression did not affect L-type calcium channel current (ICaL) under basal conditions with ICaL remaining responsive to cAMP. These data indicate intimate association of KCNQ1 and beta2-ARs and that beta2-AR signaling can modulate the function of IKs channels under conditions of increased beta2-AR expression, even in the absence of exogenous beta-AR agonist.

Animals↗
PubMed2003

FKBP12.6 deficiency and defective calcium release channel (ryanodine receptor) function linked to exercise-induced sudden cardiac death.

Arrhythmias, a common cause of sudden cardiac death, can occur in structurally normal hearts, although the mechanism is not known. In cardiac muscle, the ryanodine receptor (RyR2) on the sarcoplasmic reticulum releases the calcium required for muscle contraction. The FK506 binding protein (FKBP12.6) stabilizes RyR2, preventing aberrant activation of the channel during the resting phase of the cardiac cycle. We show that during exercise, RyR2 phosphorylation by cAMP-dependent protein kinase A (PKA) partially dissociates FKBP12.6 from the channel, increasing intracellular Ca(2+) release and cardiac contractility. FKBP12.6(-/-) mice consistently exhibited exercise-induced cardiac ventricular arrhythmias that cause sudden cardiac death. Mutations in RyR2 linked to exercise-induced arrhythmias (in patients with catecholaminergic polymorphic ventricular tachycardia [CPVT]) reduced the affinity of FKBP12.6 for RyR2 and increased single-channel activity under conditions that simulate exercise. These data suggest that "leaky" RyR2 channels can trigger fatal cardiac arrhythmias, providing a possible explanation for CPVT.

Animals↗
PubMed2003

Ankyrin-B mutation causes type 4 long-QT cardiac arrhythmia and sudden cardiac death.

Mutations in ion channels involved in the generation and termination of action potentials constitute a family of molecular defects that underlie fatal cardiac arrhythmias in inherited long-QT syndrome. We report here that a loss-of-function (E1425G) mutation in ankyrin-B (also known as ankyrin 2), a member of a family of versatile membrane adapters, causes dominantly inherited type 4 long-QT cardiac arrhythmia in humans. Mice heterozygous for a null mutation in ankyrin-B are haploinsufficient and display arrhythmia similar to humans. Mutation of ankyrin-B results in disruption in the cellular organization of the sodium pump, the sodium/calcium exchanger, and inositol-1,4,5-trisphosphate receptors (all ankyrin-B-binding proteins), which reduces the targeting of these proteins to the transverse tubules as well as reducing overall protein level. Ankyrin-B mutation also leads to altered Ca2+ signalling in adult cardiomyocytes that results in extrasystoles, and provides a rationale for the arrhythmia. Thus, we identify a new mechanism for cardiac arrhythmia due to abnormal coordination of multiple functionally related ion channels and transporters.

Action Potentials↗
PubMed2002

Local Ca(2+) signaling and EC coupling in heart: Ca(2+) sparks and the regulation of the [Ca(2+)](i) transient.

The elementary event of Ca(2+) release in heart is the Ca(2+) spark. It occurs at a low rate during diastole, activated only by the low cytosolic [Ca(2+)](i). Synchronized activation of many sparks is due to the high local [Ca(2+)](i) in the region surrounding the sarcoplasmic reticulum (SR) Ca(2+) release channels and is responsible for the systolic [Ca(2+)](i) transient. The biophysical basis of this calcium signaling is discussed. Attention is placed on the local organization of the ryanodine receptors (SR Ca(2+) release channels, RyRs) and the other proteins that underlie and modulate excitation-contraction (EC) coupling. A brief review of specific elements that regulate SR Ca(2+) release (including SR lumenal Ca(2+) and coupled gating of RyRs) is presented. Finally integrative calcium signaling in heart is presented in the context of normal heart function and heart failure.

Animals↗
PubMed2002

Effects of PP1/PP2A inhibitor calyculin A on the E-C coupling cascade in murine ventricular myocytes.

Calyculin A was used to examine the importance of phosphatases in the modulation of cardiac contractile magnitude in the absence of any neural or humoral stimulation. Protein phosphatase (PP)1 and PP2A activity, twitch contractions, intracellular Ca(2+) concentration ([Ca(2+)](i)) transients, action potentials, membrane currents, and myofilament Ca(2+) sensitivity were measured in isolated mouse ventricular myocytes. Calyculin A (125 nM) inhibited PP1 and PP2A by 50% and 85%, respectively, whereas it doubled the twitch magnitude and increased twitch duration by 50% in field-stimulated cells. Calyculin A-evoked increases in L-type Ca(2+) current (70%) and the resulting [Ca(2+)](i) transient (83%) explain the positive inotropic response. However, increases in twitch and action potential durations did not result from increased myofilament Ca(2+) sensitivity or K(+) current inhibition, respectively. Comparison of the effects of calyculin A and isoproterenol on [Ca(2+)](i) transients and twitch contractions revealed that calyculin A had a much smaller lusitropic effect than the beta-agonist, indicating that calyculin A did not significantly increase sarcoplasmic reticulum Ca(2+) reuptake. Thus while cardiac contractile magnitude is controlled by a steady-state kinase/phosphatase balance, this regulation is not equally operative at all of the steps in the excitation-contraction coupling pathway and may in fact be most important to the regulation of the L-type Ca(2+) channel.

Animals↗
PubMed2001

Heart failure after myocardial infarction: altered excitation-contraction coupling.

BACKGROUND: Heart failure (HF) frequently follows the occurrence of myocardial infarction (MI). Questions about how HF develops and what cellular defects contribute to this dysfunction led to this study. Methods and Results-- MI was induced in rats by coronary artery ligation. Clinical examination of the post-MI (PMI) surviving animals indicated that they were in overt HF by all measures. Cellular examination of the cardiomyocytes by patch-clamp and confocal [Ca(2+)](i) imaging methods indicated that cellular function was significantly compromised. At the single-cell level, [Ca(2+)](i) transient amplitudes were reduced and contractions were decreased and slowed, although Ca(2+) current (I(Ca)) remained unchanged. The excitation-contraction coupling (ECC) gain function measured as Delta[Ca(2+)](i)/I(Ca) was significantly decreased. Ouabain, a cardiotonic steroid that blocks the Na(+),K(+)-ATPase and activates Ca(2+) entry via cardiac Na(+) channels, largely alleviated this defect. CONCLUSIONS: After MI, I(Ca) becomes less able to trigger release of Ca(2+) from the sarcoplasmic reticulum. This failure of ECC is a major factor contributing to the development of contractile dysfunction and HF in PMI animals. The improved ECC gain, enhanced Ca(2+) entry, and augmented Ca(2+) signaling due to cardiotonic steroids contribute to the beneficial effects of these agents.

Animals↗