Biomedical subjects

Donald M Bers

Publications and source records attributed to Donald M Bers.

At least 37 records · Page 2Linked to original sources

PubMed2006

Dynamic changes in free Ca-calmodulin levels in adult cardiac myocytes.

Despite its multifunctional role in cardiac myocyte function, little is known about dynamic changes in activation state of calmodulin (CaM). Thus, the purpose of this study was to develop a tool to measure Ca bound CaM (Ca-CaM) levels in intact cardiac myocytes. For dynamic measurements of Ca-CaM, we generated an adenoviral vector which expresses a cyan and a yellow fluorescent protein linked by a modified version of the Ca-CaM binding domain of avian smooth muscle myosin light chain kinase. Adult rabbit cardiac myocytes were infected with the Ca-CaM sensing probe or simultaneously infected with viruses containing CaM and the Ca-CaM sensing probe for 24-48 h. Myocytes were then field stimulated (1 Hz) and excited at 440 nm with emitted fluorescence measured at 485 and 535 nm. Changes in [Ca-CaM] are expressed as the ratio of 485 nm/535 nm. Small beat-to-beat changes of [Ca-CaM] were detected, but only when CaM was co-expressed with the sensor. However, upon beta-adrenergic stimulation with isoproterenol, there was an increase in the amplitude of the signals during each beat (parallel to the shortening, which is an indirect measure of [Ca]i) and also a rise in the diastolic [Ca-CaM]. Total [CaM] measured by both competitive ELISA and semi-quantitative Western blots was 5-6 microM in isolated adult ventricular myocytes. Our results indicate that there are dynamic changes in free Ca-CaM levels (a phasic component tracking [Ca]i) as well as system memory that integrates the [Ca]i signals (a tonic component).

Adult↗

PubMed2006

Altered cardiac myocyte Ca regulation in heart failure.

Ca is critical in both the electrical and mechanical properties of cardiac myocytes, and much is known about ionic currents and the normal excitation-contraction coupling process. In heart failure, there are significant alterations in how myocyte Ca is regulated, and these alterations are critical in dictating both contractile dysfunction and certain cardiac arrhythmias that are characteristic of heart failure.

Animals↗

PubMed2006

Local InsP3-dependent perinuclear Ca2+ signaling in cardiac myocyte excitation-transcription coupling.

Previous work showed that calmodulin (CaM) and Ca2+-CaM-dependent protein kinase II (CaMKII) are somehow involved in cardiac hypertrophic signaling, that inositol 1,4,5-trisphosphate receptors (InsP3Rs) in ventricular myocytes are mainly in the nuclear envelope, where they associate with CaMKII, and that class II histone deacetylases (e.g., HDAC5) suppress hypertrophic gene transcription. Furthermore, HDAC phosphorylation in response to neurohumoral stimuli that induce hypertrophy, such as endothelin-1 (ET-1), activates HDAC nuclear export, thereby regulating cardiac myocyte transcription. Here we demonstrate a detailed mechanistic convergence of these 3 issues in adult ventricular myocytes. We show that ET-1, which activates plasmalemmal G protein-coupled receptors and InsP3 production, elicits local nuclear envelope Ca2+ release via InsP3R. This local Ca2+ release activates nuclear CaMKII, which triggers HDAC5 phosphorylation and nuclear export (derepressing transcription). Remarkably, this Ca2+-dependent pathway cannot be activated by the global Ca2+ transients that cause contraction at each heartbeat. This novel local Ca2+ signaling in excitation-transcription coupling is analogous to but separate (and insulated) from that involved in excitation-contraction coupling. Thus, myocytes can distinguish simultaneous local and global Ca2+ signals involved in contractile activation from those targeting gene expression.

Active Transport, Cell Nucleus↗

PubMed2006

Regulation of Ca2+ and Na+ in normal and failing cardiac myocytes.

Ca(2+) in cardiac myocytes regulates contractility and relaxation, and Ca(2+) and Na (+)regulation are linked via Na(+)/Ca(2+) exchange (NCX). Heart failure (HF) is accompanied by contractile dysfunction and arrhythmias, both of which may be due to altered cellular Ca(2+) handling. Smaller Ca(2+) transient and sarcoplasmic reticulum (SR) Ca(2+) content cause systolic dysfunction in HF. The reduced SR Ca(2+) content is due to: (a) reduced SR Ca(2+)-ATPase function (which also contributes to diastolic dysfunction), (b) increased expression and function of NCX (which competes with SR Ca(2+)-ATPase during relaxation, but preserves diastolic function), and (c) enhanced diastolic SR Ca(2+) leak. Relative contributions of these may vary with HF etiology and stage. Triggered arrhythmias (e.g., delayed afterdepolarizations [DADs]) are prominent in HF. DADs are due to spontaneous SR Ca(2+) release and consequent activation of transient inward NCX current, which in HF allows DADs to more readily trigger arrhythmogenic action potentials. Thus NCX and Na(+) are critical in systolic and diastolic function and arrhythmias. [Na(+)](i) is elevated in HF, which may limit SR unloading and provide some Ca(2+) influx during the HF action potential, thus limiting the depression of systolic function. High [Na(+)](i) in HF is due to enhanced Na(+) influx. Cellular Na(+)/K(+)-ATPase (NKA) function appears unaltered, despite reduced NKA expression. This dichotomy led us to test NKA regulation by phospholemman (PLM). We find that PLM regulates NKA in a manner analogous to phospholamban regulation of SR Ca(2+)-ATPase (i.e., inhibition that is relieved by PLM phosphorylation). We measured intermolecular FRET between PLM and NKA, which is reduced upon PLM phosphorylation. The lower expression level of more phosphorylated PLM in HF may explain the above dichotomy. Thus, altered Ca(2+) and Na(+) handling contributes to altered contractile function and arrhythmogenesis in HF.

Calcium↗

PubMed2005

Increased sarcoplasmic reticulum calcium leak but unaltered contractility by acute CaMKII overexpression in isolated rabbit cardiac myocytes.

The predominant cardiac Ca2+/calmodulin-dependent protein kinase (CaMK) is CaMKIIdelta. Here we acutely overexpress CaMKIIdeltaC using adenovirus-mediated gene transfer in adult rabbit ventricular myocytes. This circumvents confounding adaptive effects in CaMKIIdeltaC transgenic mice. CaMKIIdeltaC protein expression and activation state (autophosphorylation) were increased 5- to 6-fold. Basal twitch contraction amplitude and kinetics (1 Hz) were not changed in CaMKIIdeltaC versus LacZ expressing myocytes. However, the contraction-frequency relationship was more negative, frequency-dependent acceleration of relaxation was enhanced (tau(0.5Hz)/tau(3Hz)=2.14+/-0.10 versus 1.87+/-0.10), and peak Ca2+ current (ICa) was increased by 31% (-7.1+/-0.5 versus -5.4+/-0.5 pA/pF, P<0.05). Ca2+ transient amplitude was not significantly reduced (-27%, P=0.22), despite dramatically reduced sarcoplasmic reticulum (SR) Ca2+ content (41%; P<0.05). Thus fractional SR Ca2+ release was increased by 60% (P<0.05). Diastolic SR Ca2+ leak assessed by Ca2+ spark frequency (normalized to SR Ca2+ load) was increased by 88% in CaMKIIdeltaC versus LacZ myocytes (P<0.05; in an multiplicity-of-infection-dependent manner), an effect blocked by CaMKII inhibitors KN-93 and autocamtide-2-related inhibitory peptide. This enhanced SR Ca2+ leak may explain reduced SR Ca2+ content, despite measured levels of SR Ca2+-ATPase and Na+/Ca2+ exchange expression and function being unaltered. Ryanodine receptor (RyR) phosphorylation in CaMKIIdeltaC myocytes was increased at both Ser2809 and Ser2815, but FKBP12.6 coimmunoprecipitation with RyR was unaltered. This shows for the first time that acute CaMKIIdeltaC overexpression alters RyR function, leading to enhanced SR Ca2+ leak and reduced SR Ca2+ content but without reducing twitch contraction and Ca2+ transients. We conclude that this is attributable to concomitant enhancement of fractional SR Ca2+ release in CaMKIIdeltaC myocytes (ie, CaMKII-dependent enhancement of RyR Ca2+ sensitivity during diastole and systole) and increased ICa.

Animals↗

PubMed2005

Ca2+/calmodulin-dependent protein kinase modulates cardiac ryanodine receptor phosphorylation and sarcoplasmic reticulum Ca2+ leak in heart failure.

Abnormal release of Ca from sarcoplasmic reticulum (SR) via the cardiac ryanodine receptor (RyR2) may contribute to contractile dysfunction and arrhythmogenesis in heart failure (HF). We previously demonstrated decreased Ca transient amplitude and SR Ca load associated with increased Na/Ca exchanger expression and enhanced diastolic SR Ca leak in an arrhythmogenic rabbit model of nonischemic HF. Here we assessed expression and phosphorylation status of key Ca handling proteins and measured SR Ca leak in control and HF rabbit myocytes. With HF, expression of RyR2 and FK-506 binding protein 12.6 (FKBP12.6) were reduced, whereas inositol trisphosphate receptor (type 2) and Ca/calmodulin-dependent protein kinase II (CaMKII) expression were increased 50% to 100%. The RyR2 complex included more CaMKII (which was more activated) but less calmodulin, FKBP12.6, and phosphatases 1 and 2A. The RyR2 was more highly phosphorylated by both protein kinase A (PKA) and CaMKII. Total phospholamban phosphorylation was unaltered, although it was reduced at the PKA site and increased at the CaMKII site. SR Ca leak in intact HF myocytes (which is higher than in control) was reduced by inhibition of CaMKII but was unaltered by PKA inhibition. CaMKII inhibition also increased SR Ca content in HF myocytes. Our results suggest that CaMKII-dependent phosphorylation of RyR2 is involved in enhanced SR diastolic Ca leak and reduced SR Ca load in HF, and may thus contribute to arrhythmias and contractile dysfunction in HF.

Animals↗

PubMed2005

Biosensors to measure inositol 1,4,5-trisphosphate concentration in living cells with spatiotemporal resolution.

Phosphoinositides participate in many signaling cascades via phospholipase C stimulation, which hydrolyzes phosphatidylinositol 4,5-bisphosphate, producing second messengers diacylglycerol and inositol 1,4,5-trisphosphate (InsP3). Destructive chemical approaches required to measure [InsP3] limit spatiotemporal understanding of subcellular InsP3 signaling. We constructed novel fluorescence resonance energy transfer-based InsP3 biosensors called FIRE (fluorescent InsP3-responsive element) by fusing plasmids encoding the InsP3-binding domain of InsP3 receptors (types 1-3) between cyan fluorescent protein and yellow fluorescent protein sequences. FIRE was expressed and characterized in COS-1 cells, cultured neonatal cardiac myocytes, and incorporated into an adenoviral vector for expression in adult cardiac ventricular myocytes. FIRE-1 exhibits an approximately 11% increase in the fluorescence ratio (F530/F480) at saturating [InsP3] (apparent K(d) = 31.3 +/- 6.7 nm InsP3). In COS-1 cells, neonatal rat cardiac myocytes and adult cat ventricular myocytes FIRE-1 exhibited comparable dynamic range and a 10% increase in donor (cyan fluorescent protein) fluorescence upon bleach of yellow fluorescent protein, indicative of fluorescence resonance energy transfer. In FIRE-1 expressing ventricular myocytes endothelin-1, phenylephrine, and angiotensin II all produced rapid and spatially resolved increases in [InsP3] using confocal microscopy (with free [InsP3] rising to approximately 30 nm). Local entry of intracellular InsP3 via membrane rupture by a patch pipette (containing InsP3)in myocytes expressing FIRE-1 allowed detailed spatiotemporal monitoring of intracellular InsP3 diffusion. Both endothelin-1-induced and direct InsP3 application (via pipette rupture) revealed that InsP3 diffusion into the nucleus occurs with a delay and blunted rise of [InsP3] versus cytosolic [InsP3]. These new biosensors allow studying InsP3 dynamics at high temporal and spatial resolution that will be powerful in under-standing InsP3 signaling in intact cells.

Age Factors↗

PubMed2005

Isoproterenol does not enhance Ca-dependent Na/Ca exchange current in intact rabbit ventricular myocytes.

Cardiac Na/Ca exchange (NCX, NCX1.1) is critical in cardiac myocyte Ca regulation, and its altered function contributes to inotropic state, systolic dysfunction in heart failure and arrhythmogenesis. Regulation of NCX is multifaceted, but protein kinase A (PKA) effects on NCX function are controversial. Here, we use three different and complementary approaches to compare NCX function +/-1 microM isoproterenol (ISO) in intact rabbit cardiac myocytes (in paired comparisons). First, in field-stimulated intact cells we inferred the cytosolic [Ca] ([Ca](i)) dependence of NCX function from the decay rate of caffeine-induced [Ca](i) transients. Second, we measured caffeine-induced [Ca](i) and inward I(NCX) simultaneously (perforated patch voltage clamp), to measure directly the [Ca](i) dependence of NCX rate. Third, using whole cell ruptured patch with [Ca](i) heavily buffered to 100 nM, [Na](i)=10 mM, and I(Ca), SR Ca release and Na/K pump all blocked, we recorded I(NCX) ramps at 37 degrees C. We find that NCX function is not altered by PKA activation under any of these three protocols, where intracellular conditions ranged from near-physiological to highly controlled. This does not rule out NCX modulation by PKA under all conditions, or in species other than rabbit. However, such effects are likely to be either minor (vs. other PKA actions on myocyte Ca handling) or indirect, such as secondary effects dependent on altered local [Ca](i) and [Na](i).

Animals↗

PubMed2005

Spatiotemporal characteristics of SR Ca(2+) uptake and release in detubulated rat ventricular myocytes.

In cardiac ventricular myocytes, sarcoplasmic reticulum (SR) Ca(2+) load is a key determinant of SR Ca(2+) release. This release normally occurs predominantly from SR junctions at sarcolemmal invaginations (t-tubules), ensuring synchronous SR Ca(2+) release throughout the cell. However under conditions of Ca(2+) overload, spontaneous SR Ca(2+) release and propagating Ca(2+) waves can occur, which are pro-arrhythmic. We used detubulated rat ventricular myocytes to determine the dependence of Ca(2+) wave propagation on SR Ca(2+) load, and the role of t-tubules in SR Ca(2+) uptake and spontaneous release. After SR Ca(2+) depletion, recovery of Ca(2+) transient amplitude (and SR Ca(2+) load) was slower in detubulated than control myocytes (half-maximal recovery: 9.9+/-1.4 vs. 5.5+/-0.7 beats). In detubulated myocytes the extent and velocity of Ca(2+) propagation from the cell periphery increased with each beat and depended steeply on SR Ca(2+) load. Isoproterenol (ISO) accelerated recovery, increased maximal propagation velocity and reduced the threshold SR Ca(2+) load for propagation. Ca(2+) spark frequency was uniform across control cell width and was similar at the periphery of detubulated cells. However, internal Ca(2+) spark frequency in detubulated cells was 75% lower (despite comparable local SR Ca(2+) load); this transverse spark frequency profile was similar to that in atrial myocytes. We conclude that: (1) t-tubule Ca(2+) fluxes normally control SR Ca(2+) refilling; (2) Ca(2+) wave propagation depends steeply on SR Ca(2+) content (3) SR-t-tubule junctions are important in initiating SR Ca(2+) release and (4) ISO enhances propagation of SR Ca release, but not the initiation of SR Ca release events (for given SR Ca(2+) loads).

Animals↗

PubMed2005

Regulation of cardiac sarcoplasmic reticulum Ca release by luminal [Ca] and altered gating assessed with a mathematical model.

Cardiac excitation-contraction coupling is initialized by the release of Ca from the sarcoplasmic reticulum (SR) in response to a sudden increase in local cytosolic [Ca] ([Ca]i) within the junctional cleft. We have tested the hypothesis that functional ryanodine receptor (RyR) regulation plays a major role in the regulation of myocyte Ca. A mathematical model with unique characteristics was used to simulate Ca homeostasis. Specifically, the model was designed to accurately represent the SR [Ca]-dependence of release from a variety of experimentally produced data sets. The simulated data for altered RyR Ca sensitivity demonstrated a regulatory feedback loop that resulted in the same release at lower [Ca]SR. This suggests that the primary role of myocyte RyR regulation may be to decrease SR [Ca] without decreasing the size of the [Ca]i transient. The model results suggest that this action moderates the increased SR [Ca] observed with adrenergic stimulation and may keep the [Ca]SR below the threshold for delayed afterdepolarizations and arrhythmia. However, increased Ca affinity of the RyR increased the probability of delayed afterdepolarizations when heart failure was simulated. We conclude that RyR regulation may play a role in preventing arrhythmias in healthy myocytes but that the same regulation may have the opposite effect in chronic heart failure.

Animals↗

PubMed2005

Targeted inhibition of sarcoplasmic reticulum CaMKII activity results in alterations of Ca2+ homeostasis and cardiac contractility.

Transgenic (TG) mice expressing a Ca2+/calmodulin-dependent protein kinase II (CaMKII) inhibitory peptide targeted to the cardiac myocyte longitudinal sarcoplasmic reticulum (LSR) display reduced phospholamban phosphorylation at Thr17 and develop dilated myopathy when stressed by gestation and parturition (Ji Y, Li B, Reed TD, Lorenz JN, Kaetzel MA, and Dedman JR. J Biol Chem 278: 25063-25071, 2003). In the present study, these animals (TG) are evaluated for the effect of inhibition of sarcoplasmic reticulum (SR) CaMKII activity on the contractile characteristics and Ca2+ cycling of myocytes. Analysis of isolated work-performing hearts demonstrated moderate decreases in the maximal rates of contraction and relaxation (+/-dP/dt) in TG mice. The response of the TG hearts to increases in load is reduced. The TG hearts respond to isoproterenol (Iso) in a dose-dependent manner; the contractile properties were reduced in parallel to wild-type hearts. Assessment of isolated cardiomyocytes from TG mice revealed 40-47% decrease in the maximal rates of myocyte shortening and relengthening under both basal and Iso-stimulated conditions. Although twitch Ca2+ transient amplitudes were not significantly altered, the rate of twitch intracellular Ca2+ concentration decline was reduced by approximately 47% in TG myocytes, indicating decreased SR Ca2+ uptake function. Caffeine-induced Ca2+ transients indicated unaltered SR Ca2+ content and Na+/Ca2+ exchange function. Phosphorylation assays revealed an approximately 30% decrease in the phosphorylation of ryanodine receptor Ser2809. Iso stimulation increased the phosphorylation of both phospholamban Ser16 and the ryanodine receptor Ser2809 but not phospholamban Thr17 in TG mice. This study demonstrates that inhibition of SR CaMKII activity at the LSR results in alterations in cardiac contractility and Ca2+ handling in TG hearts.

Animals↗

PubMed2005

Expression and phosphorylation of the na-pump regulatory subunit phospholemman in heart failure.

Intracellular [Na] is approximately 3 mmol/L higher in heart failure (HF; in our arrhythmogenic rabbit model), and this can profoundly affect cardiac Ca and contractile function via Na/Ca exchange and Na/H exchange. Na/K-ATPase is the primary mechanism of Na extrusion. We examine here in HF rabbits (and human hearts) expression of Na/K-ATPase isoforms and phospholemman (PLM), a putative Na/K-ATPase regulatory subunit that inhibits pump function and is a major cardiac phosphorylation target. Na/K-ATPase alpha1- and alpha2-isoforms were reduced in HF in rabbit ventricular homogenates (by 24%) and isolated myocytes (by 30% and 17%), whereas alpha3 was increased (50%) in homogenates and decreased (52%) in myocytes (P<0.05). Homogenate Na/K-ATPase activity in left ventricle was also decreased in HF. However, we showed previously that Na/K-ATPase characteristics in intact ventricular myocytes were unaltered in HF. To reconcile these findings, we assessed PLM expression, phosphorylation, and association with Na/K-ATPase. PLM coimmunoprecipitated with Na/K-ATPase alpha1 and alpha2 in control and HF rabbit myocytes. PLM expression was reduced in HF by 42% in isolated rabbit left ventricular (LV) myocytes, by 48% in rabbit LV homogenates, and by 24% in human LV homogenate. The fraction of PLM phosphorylated at Ser-68 was increased dramatically in HF. Our results are consistent with a role for PLM analogous to that of phospholamban for SR Ca-ATPase (SERCA): inhibition of Na/K-ATPase function that is relieved on PLM phosphorylation. So reduced Na/K-ATPase expression in HF may be functionally offset by lower inhibition by PLM (because of reduced PLM expression and higher PLM phosphorylation).

Animals↗

PubMed2005

Phospholemman-phosphorylation mediates the beta-adrenergic effects on Na/K pump function in cardiac myocytes.

Cardiac sympathetic stimulation activates beta-adrenergic (beta-AR) receptors and protein kinase A (PKA) phosphorylation of proteins involved in myocyte Ca regulation. The Na/K-ATPase (NKA) is essential in regulating intracellular [Na] ([Na]i), which in turn affects [Ca]i via Na/Ca exchange. However, how PKA modifies NKA function is unknown. Phospholemman (PLM), a member of the FXYD family of proteins that interact with NKA in various tissues, is a major PKA substrate in heart. Here we tested the hypothesis that PLM phosphorylation is responsible for the PKA effects on cardiac NKA function using wild-type (WT) and PLM knockout (PLM-KO) mice. We measured NKA-mediated [Na]i decline and current (IPump) to assess beta-AR effects on NKA function in isolated myocytes. In WT myocytes, 1 micromol/L isoproterenol (ISO) increased PLM phosphorylation and stimulated NKA activity mainly by increasing its affinity for internal Na (Km decreased from 18.8+/-1.4 to 13.6+/-1.5 mmol/L), with no significant effect on the maximum pump rate. This led to a significant decrease in resting [Na]i (from 12.5+/-1.8 to 10.5+/-1.4 mmol/L). In PLM-KO mice under control conditions Km (14.2+/-1.5 mmol/L) was lower than in WT, but comparable to that for WT in the presence of ISO. Furthermore, ISO had no significant effect on NKA function in PLM-KO mice. ATPase activity in sarcolemmal vesicles also showed a lower Km(Na) in PLM-KO versus WT (12.9+/-0.9 versus 16.2+/-1.5). Thus, PLM inhibits NKA activity by decreasing its [Na]i affinity, and this inhibitory effect is relieved by PKA activation. We conclude that PLM modulates the NKA function in a manner similar to the way phospholamban affects the related SR Ca-ATPase (inhibition of transport substrate affinity, that is relieved by phosphorylation).

Animals↗

PubMed2005

Transgenic rabbit model for human troponin I-based hypertrophic cardiomyopathy.

BACKGROUND: Transgenic and gene-targeted models have focused on the mouse. Fundamental differences between the mouse and human exist in Ca2+ handling during contraction/relaxation and in alterations in Ca2+ flux during heart failure, with the rabbit more accurately reflecting the human system. METHODS AND RESULTS: Cardiac troponin I (cTnI) mutations can cause familial hypertrophic cardiomyopathy. An inhibitory domain mutation, arginine146-->glycine (cTnI(146Gly)), was modeled with the use of transgenic expression in the rabbit ventricle. cTnI(146Gly) levels >40% of total cTnI were perinatally lethal, whereas replacement levels of 15% to 25% were well tolerated. cTnI(146Gly) expression led to a leftward shift in the force-pCa2+ curves with cardiomyocyte disarray, fibrosis, and altered connexin43 organization. In isolated cTnI(146Gly) myocytes, twitch relaxation amplitudes were smaller than in normal cells, but [Ca]i transients and sarcoplasmic reticulum Ca2+ load were not different. Detrended fluctuation analysis of the QT(max) intervals was used to evaluate the cardiac repolarization phase and showed a significantly higher scaling exponent in the transgenic animals. CONCLUSIONS: Expression of modest amounts of cTnI(146Gly) led to subtle defects without severely affecting cardiac function. Aberrant connexin organization, subtle morphological deficits, and an altered fractal pattern of the repolarization phase of transgenic rabbits, in the absence of entropy or other ECG abnormalities, may indicate an early developing pathology before the onset of more obvious repolarization abnormalities or major alterations in cardiac mechanics.

Animals↗

PubMed2005

Adenoviral gene transfer of mutant phospholamban rescues contractile dysfunction in failing rabbit myocytes with relatively preserved SERCA function.

In heart failure (HF) a main factor in reduced contractility is reduced SR Ca2+ content and reversed force-frequency response (FFR), ie, from positive to negative. Our arrhythmogenic rabbit HF model exhibits decreased contractility mainly due to an increase in Na/Ca exchange (NCX) activity (with only modest decrease in SR Ca2+-ATPase (SERCA) function), similar to many end-stage HF patients. Here we test whether phospholamban (PLB) inhibition using a dominant-negative mutant PLB adenovirus (K3E/R14E, AdPLB-dn, with beta-galactosidase adenovirus as control) could enhance SERCA function and restore Ca2+ transients and positive FFR in ventricular myocytes from these HF rabbits. HF myocytes infected with AdPLB-dn (versus control) had enhanced Ca2+ transient amplitude (2.0+/-0.1 versus 1.6+/-0.05 F/Fo at 0.5 Hz, P<0.05) and had a positive FFR, whereas acutely isolated HF myocytes or those infected with Adbetagal had negative FFR. Ca2+ transients declined faster in AdPLB-dn versus Adbetagal myocytes (RT50%: 317+/-29 versus 551+/-90 ms at 0.5 Hz, P<0.05) and had an increased SR Ca2+ load (3.5+/-0.3 versus 2.6+/-0.2 F/Fo at 0.5 Hz, P<0.05), indicative of increased SERCA function. Furthermore, this restoration of function was not due to changes in NCX or SERCA expression. Thus, increasing SERCA activity in failing myocytes by AdPLB-dn gene transfer reversed the contractile dysfunction (and restored positive FFR) by increasing SR Ca2+ load. This approach could enhance contractile function in failing hearts of various etiologies, even here where reduced SERCA activity is not the main dysfunction.

Adenoviridae↗

PubMed2005

Cardiac type 2 inositol 1,4,5-trisphosphate receptor: interaction and modulation by calcium/calmodulin-dependent protein kinase II.

The type 2 inositol 1,4,5-trisphosphate receptor (InsP(3)R2) was identified previously as the predominant isoform in cardiac ventricular myocytes. Here we reported the subcellular localization of InsP(3)R2 to the cardiomyocyte nuclear envelope (NE). The other major known endo/sarcoplasmic reticulum calcium-release channel (ryanodine receptor) was not localized to the NE, indicating functional segregation of these channels and possibly a unique role for InsP(3)R2 in regulating nuclear calcium dynamics. Immunoprecipitation experiments revealed that the NE InsP(3)R2 associates with Ca(2+)/calmodulin-dependent protein kinase IIdelta (CaMKIIdelta), the major isoform expressed in cardiac myocytes. Recombinant InsP(3)R2 and CaMKIIdelta(B) also co-immunoprecipitated after co-expression in COS-1 cells. Additionally, the amino-terminal 1078 amino acids of the InsP(3)R2 were sufficient for interaction with CaMKIIdelta(B) and associated upon mixing following separate expression. CaMKII can also phosphorylate InsP(3)R2, as demonstrated by (32)P labeling. Incorporation of CaMKII-treated InsP(3)R2 into planar lipid bilayers revealed that InsP(3)-mediated channel open probability is significantly reduced ( approximately 11 times) by phosphorylation via CaMKII. We concluded that the InsP(3)R2 and CaMKIIdelta likely represent two central components of a multiprotein signaling complex, and this raises the possibility that calcium release via InsP(3)R2 in the myocyte NE may activate local CaMKII signaling, which may feedback on InsP(3)R2 function.

Animals↗

PubMed2005

Beyond beta blockers.

Explore the source record for details and available documents.

Adrenergic beta-Antagonists↗

PubMed2005

Calcium signaling in cardiac ventricular myocytes.

Calcium (Ca) is a multifunctional regulator of diverse cellular functions. In cardiac muscle Ca is a direct central mediator of electrical activation, ion channel gating, and excitation-contraction (E-C) coupling that all occur on the millisecond time scale. The key amplification step in E-C coupling is under tight control of very local [Ca]. Ca also directly activates signaling via kinases and phosphatases (e.g., Ca-calmodulin-dependent protein kinase [CaMKII] and calcineurin) that occur over a longer time scale (seconds to minutes), and the co-localization of these Ca-dependent modulators to their targets and to Ca is also critical in distinct signaling pathways. Finally, Ca-dependent signaling is also involved in long-term (minutes to hours/days) alterations in gene expression (or excitation-transcription coupling). These pathways are involved in hypertrophy and heart failure, and they can alter the expression of some of the key Ca regulatory proteins involved in E-C coupling and their regulation by kinases and phosphatases. There may again be physical microenvironments involved in this nuclear transcription, such that they sense a discrete Ca signal that is distinct from that involved in E-C coupling. In this way cells can use Ca signaling in multiple ways that function in spatially and temporally distinct manners.

Animals↗