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Michael D Stern

Publications and source records attributed to Michael D Stern.

At least 19 recordsLinked to original sources
PubMed2006

Membrane potential fluctuations resulting from submembrane Ca2+ releases in rabbit sinoatrial nodal cells impart an exponential phase to the late diastolic depolarization that controls their chronotropic state.

Stochastic but roughly periodic LCRs (Local subsarcolemmal ryanodine receptor-mediated Ca(2+) Releases) during the late phase of diastolic depolarization (DD) in rabbit sinoatrial nodal pacemaker cells (SANCs) generate an inward current (I(NCX)) via the Na(+)/Ca(2+) exchanger. Although LCR characteristics have been correlated with spontaneous beating, the specific link between LCR characteristics and SANC spontaneous beating rate, ie, impact of LCRs on the fine structure of the DD, have not been explicitly defined. Here we determined how LCRs and resultant I(NCX) impact on the DD fine structure to control the spontaneous SANC firing rate. Membrane potential (V(m)) recordings combined with confocal Ca(2+) measurements showed that LCRs impart a nonlinear, exponentially rising phase to the DD later part, which exhibited beat-to-beat V(m) fluctuations with an amplitude of approximately 2 mV. Maneuvers that altered LCR timing or amplitude of the nonlinear DD (ryanodine, BAPTA, nifedipine or isoproterenol) produced corresponding changes in V(m) fluctuations during the nonlinear DD component, and the V(m) fluctuation response evoked by these maneuvers was tightly correlated with the concurrent changes in spontaneous beating rate induced by these perturbations. Numerical modeling, using measured LCR characteristics under these perturbations, predicted a family of local I(NCX) that reproduced V(m) fluctuations measured experimentally and determined the onset and amplitude of the nonlinear DD component and the beating rate. Thus, beat-to-beat V(m) fluctuations during late DD phase reflect the underlying LCR/I(NCX) events, and the ensemble of these events forms the nonlinear DD component that ultimately controls the SANC chronotropic state in tight cooperation with surface membrane ion channels.

Action Potentials↗
PubMed2006

High basal protein kinase A-dependent phosphorylation drives rhythmic internal Ca2+ store oscillations and spontaneous beating of cardiac pacemaker cells.

Local, rhythmic, subsarcolemmal Ca2+ releases (LCRs) from the sarcoplasmic reticulum (SR) during diastolic depolarization in sinoatrial nodal cells (SANC) occur even in the basal state and activate an inward Na(+)-Ca2+ exchanger current that affects spontaneous beating. Why SANC can generate spontaneous LCRs under basal conditions, whereas ventricular cells cannot, has not previously been explained. Here we show that a high basal cAMP level of isolated rabbit SANC and its attendant increase in protein kinase A (PKA)-dependent phosphorylation are obligatory for the occurrence of spontaneous, basal LCRs and for spontaneous beating. Gradations in basal PKA activity, indexed by gradations in phospholamban phosphorylation effected by a specific PKA inhibitory peptide were highly correlated with concomitant gradations in LCR spatiotemporal synchronization and phase, as well as beating rate. Higher levels of basal PKA inhibition abolish LCRs and spontaneous beating ceases. Stimulation of beta-adrenergic receptors extends the range of PKA-dependent control of LCRs and beating rate beyond that in the basal state. The link between SR Ca2+ cycling and beating rate is also present in vivo, as the regulation of beating rate by local beta-adrenergic receptor stimulation of the sinoatrial node in intact dogs is markedly blunted when SR Ca2+ cycling is disrupted by ryanodine. Thus, PKA-dependent phosphorylation of proteins that regulate cell Ca2+ balance and spontaneous SR Ca2+ cycling, ie, phospholamban and L-type Ca2+ channels (and likely others not measured in this study), controls the phase and size of LCRs and the resultant Na(+)-Ca2+ exchanger current and is crucial for both basal and reserve cardiac pacemaker function.

Action Potentials↗
PubMed2005

HIF-1alpha-targeted pathways are activated by heat acclimation and contribute to acclimation-ischemic cross-tolerance in the heart.

Hypoxia-inducible factor-1 (HIF-1) is a key regulator of the cellular hypoxic response. We previously showed that HIF-1 activation is essential for heat acclimation (AC) in Caenorhabditis elegans. Metabolic changes in AC rat hearts indicate HIF-1alpha activation in mammals as well. Here we characterize the HIF-1alpha profile and the transcriptional activation of its target genes following AC and following heat stress (HS) in hearts from nonacclimated (C; 24 degrees C) and AC (34 degrees C, 1 mo) rats. We used Western blot and immunohistochemistry to measure HIF-1alpha levels and EMSA and RT-PCR/quantitative RT-PCR to detect expression of the HIF-1alpha-targeted genes, including vascular endothelial growth factor (Vegf), heme oxygenase-1 (HO1), erythropoietin (Epo), and Epo receptor (EpoR). EpoR and Epo mRNA levels were measured to determine systemic effects in the kidneys and cross-tolerance effects in C and AC ischemic hearts (Langendorff, 75% ischemia, 40 min). The results demonstrated that 1) after AC, HIF-1alpha protein levels were increased, 2) HS alone induced transient HIF-1alpha upregulation, and 3) VEGF and HO1 mRNA levels increased after HS, with greater magnitude in the AC hearts. Epo mRNA in AC kidneys and EpoR mRNA in AC hearts were also elevated. In AC hearts, EpoR expression was markedly higher after HS or ischemia. Hearts from AC rats were dramatically protected against infarction after ischemia-perfusion. We conclude that HIF-1 contributes to the acclimation-ischemia cross-tolerance mechanism in the heart by induction of both chronic and inducible adaptive components.

Animals↗
PubMed2005

Concerted vs. sequential. Two activation patterns of vast arrays of intracellular Ca2+ channels in muscle.

To signal cell responses, Ca(2+) is released from storage through intracellular Ca(2+) channels. Unlike most plasmalemmal channels, these are clustered in quasi-crystalline arrays, which should endow them with unique properties. Two distinct patterns of local activation of Ca(2+) release were revealed in images of Ca(2+) sparks in permeabilized cells of amphibian muscle. In the presence of sulfate, an anion that enters the SR and precipitates Ca(2+), sparks became wider than in the conventional, glutamate-based solution. Some of these were "protoplatykurtic" (had a flat top from early on), suggesting an extensive array of channels that activate simultaneously. Under these conditions the rate of production of signal mass was roughly constant during the rise time of the spark and could be as high as 5 microm(3) ms(-1), consistent with a release current >50 pA since the beginning of the event. This pattern, called "concerted activation," was observed also in rat muscle fibers. When sulfate was combined with a reduced cytosolic [Ca(2+)] (50 nM) these sparks coexisted (and interfered) with a sequential progression of channel opening, probably mediated by Ca(2+)-induced Ca(2+) release (CICR). Sequential propagation, observed only in frogs, may require parajunctional channels, of RyR isoform beta, which are absent in the rat. Concerted opening instead appears to be a property of RyR alpha in the amphibian and the homologous isoform 1 in the mammal.

Animals↗
PubMed2004

The quantal nature of Ca2+ sparks and in situ operation of the ryanodine receptor array in cardiac cells.

Intracellular Ca(2+) release in many types of cells is mediated by ryanodine receptor Ca(2+) release channels (RyRCs) that are assembled into two-dimensional paracrystalline arrays in the endoplasmic/sarcoplasmic reticulum. However, the in situ operating mechanism of the RyRC array is unknown. Here, we found that the elementary Ca(2+) release events, Ca(2+) sparks from individual RyRC arrays in rat ventricular myocytes, exhibit quantized Ca(2+) release flux. Analysis of the quantal property of Ca(2+) sparks provided a view of unitary Ca(2+) current and gating kinetics of the RyRC in intact cells and revealed that spark activation involves dynamic recruitment of small, variable cohorts of RyRCs. Intriguingly, interplay of RyRCs in multichannel sparks renders an unusual, thermodynamically irreversible mode of channel gating that is unshared by an RyRC acting solo, nor by RyRCs in vitro. Furthermore, an array-based inhibitory feedback, overriding the regenerative Ca(2+)-induced Ca(2+) release of RyRCs, provides a supramolecular mechanism for the microscopic stability of intracellular Ca(2+) signaling.

Animals↗
PubMed2004

Putting out the fire: what terminates calcium-induced calcium release in cardiac muscle?

The majority of contractile calcium in cardiac muscle is released from stores in the sarcoplasmic reticulum (SR), by a process of calcium-induced calcium release (CICR) through ryanodine receptors. Because CICR is intrinsically self-reinforcing, the stability of and graded regulation of cardiac EC coupling appear paradoxical. It is now well established that this gradation results from the stochastic recruitment of varying numbers of elementary local release events, which may themselves be regenerative, and which can be directly observed as calcium sparks. Ryanodine receptors (RyRs) are clustered in dense lattices, and most calcium sparks are now believed to involve activation of multiple RyRs. This implies that local CICR is regenerative, requiring a mechanism to terminate it. It was initially assumed that this mechanism was inactivation of the RyR, but during the decade since the discovery of sparks, no sufficiently strong inactivation mechanism has been demonstrated in vitro and all empirically determined gating schemes for the RyR give unstable EC coupling in Monte Carlo simulations. We consider here possible release termination mechanisms. Stochastic attrition is the spontaneous decay of active clusters due to random channel closure; calculations show that it is much too slow unless assisted by another process. Calcium-dependent RyR inactivation involving third-party proteins remains a viable but speculative mechanism; current candidates include calmodulin and sorcin. Local depletion of SR release terminal calcium could terminate release, however calculations and measurements leave it uncertain whether a sufficient diffusion resistance exists within the SR to sustain such depletion. Depletion could be assisted by dependence of RyR activity on SR lumenal [Ca(2+)]. There is substantial evidence for such lumenal activation, but it is not clear if it is a strong enough effect to account for the robust termination of sparks. The existence of direct interactions among clustered RyRs might account for the discrepancy between the inactivation properties of isolated RyRs and intact clusters. Such coupled gating remains controversial. Determining the mechanism of release termination is the outstanding unsolved problem of cardiac EC coupling, and will probably require extensive genetic manipulation of the EC coupling apparatus in its native environment to unravel the solution.

Animals↗
PubMed2004

Diastolic calcium release controls the beating rate of rabbit sinoatrial node cells: numerical modeling of the coupling process.

Recent studies employing Ca2+ indicators and confocal microscopy demonstrate substantial local Ca2+ release beneath the cell plasma membrane (subspace) of sinoatrial node cells (SANCs) occurring during diastolic depolarization. Pharmacological and biophysical experiments have suggested that the released Ca2+ interacts with the plasma membrane via the ion current (INaCa) produced by the Na+/Ca2+ exchanger and constitutes an important determinant of the pacemaker rate. This study provides a numerical validation of the functional importance of diastolic Ca2+ release for rate control. The subspace Ca2+ signals in rabbit SANCs were measured by laser confocal microscopy, averaged, and calibrated. The time course of the subspace [Ca2+] displayed both diastolic and systolic components. The diastolic component was mainly due to the local Ca2+ releases; it was numerically approximated and incorporated into a SANC cellular electrophysiology model. The model predicts that the diastolic Ca2+ release strongly interacts with plasma membrane via INaCa and thus controls the phase of the action potential upstroke and ultimately the final action potential rate.

Animals↗
PubMed2003

Can transcriptome size be estimated from SAGE catalogs?

MOTIVATION: SAGE (Serial Analysis of Gene Expression) can be used to estimate the number of unique transcripts in a transcriptome. A simple estimator that corrects for sequencing and sampling errors was applied to a SAGE library (137 832 tags) obtained from mouse embryonic stem cells, and also to Monte Carlo simulated libraries generated using assumed distributions of 'true' expression levels consistent with the data. RESULTS: When the corrected data themselves were taken as the underlying model of 'ground truth', the estimator converged to the 'true' value (53 535) only after counting 300 000 simulated tags, more than twice the number in the experiment. The SAGE data could also be well fit by a Monte Carlo model based on a truncated inverse-square distribution of expression levels, with 130 000 'true' transcripts and 10(6) samples needed for convergence. We conclude that the size of a transcriptome is ill-determined from SAGE libraries of even moderately large size. In order to obtain a valid estimate, one must sample a number of tags inversely proportional to the lowest abundance level, which is not known a priori. This constrains the design of SAGE experiments intended to determine biological complexity. AVAILABILITY: The 'homemade' software used for this analysis was not designed for general or 'production' use, but the authors will be happy to share Fortran sourcecode with interested parties. CONTACT: sternm@grc.nia.nih.gov

Algorithms↗
PubMed2003

Cyclic variation of intracellular calcium: a critical factor for cardiac pacemaker cell dominance.

While a diversity of cell types and distribution within the sinoatrial node and cell-cell interactions add complexity to a complete elucidation of the heart's pacemaker function, it has become clear that cyclic variation of submembrane [Ca2+] and activation of the Na+-Ca2+ exchanger during diastolic depolarization (DD) act in concert with ion channels to confer on sinoatrial node cells (SANCs) their status of dominance with respect to pacemaker function. Studies using confocal microscopy indicate that subsarcolemmal Ca2+ release via ryanodine receptors occurs not only in response to the action potential (AP) upstroke, but also during the DD, and this is augmented by beta-adrenergic receptor (beta-AR) stimulation. Spontaneous APs simulated by mathematical SANC models beat at a faster rate when this subsarcolemmal Ca2+ waveform measured under beta-AR stimulation is introduced into the modeling scheme. Thus, in future investigation of pacemaker functioning in health, disease, and disease therapies the "bar ought to be raised" to embrace the impact of cyclic variation in submembrane [Ca2+] on pacemaker function. The full text of this article is available at http://www.circresaha.org.

Adrenergic beta-Agonists↗
PubMed2003

The new role of SAGE in gene discovery.

The sequencing of the human genome has led to in silico predictions of far fewer genes than anticipated. Recent studies using serial analysis of gene expression have cast doubt on this finding. One study predicts the presence of many unidentified low abundance transcripts, whereas two others have mapped unique tags to previously unpredicted exons. Genome and transcriptome complexity is thus greater than predicted and many of the missing genes are probably expressed in low copy numbers or only in early embryonic tissues.

Animals↗
PubMed2002

The ryanodine receptor modulates the spontaneous beating rate of cardiomyocytes during development.

In adult myocardium, the heartbeat originates from the sequential activation of ionic currents in pacemaker cells of the sinoatrial node. Ca(2+) release via the ryanodine receptor (RyR) modulates the rate at which these cells beat. In contrast, the mechanisms that regulate heart rate during early cardiac development are poorly understood. Embryonic stem (ES) cells can differentiate into spontaneously contracting myocytes whose beating rate increases with differentiation time. These cells thus offer an opportunity to determine the mechanisms that regulate heart rate during development. Here we show that the increase in heart rate with differentiation is markedly depressed in ES cell-derived cardiomyocytes with a functional knockout (KO) of the cardiac ryanodine receptor (RyR2). KO myocytes show a slowing of the rate of spontaneous diastolic depolarization and an absence of calcium sparks. The depressed rate of pacemaker potential can be mimicked in wild-type myocytes by ryanodine, and rescued in KO myocytes with herpes simplex virus (HSV)-1 amplicons containing full-length RyR2. We conclude that a functional RyR2 is crucial to the progressive increase in heart rate during differentiation of ES cell-derived cardiomyocytes, consistent with a mechanism that couples Ca(2+) release via RyR before an action potential with activation of an inward current that accelerates membrane depolarization.

Action Potentials↗
PubMed2002

SAGE identification of gene transcripts with profiles unique to pluripotent mouse R1 embryonic stem cells.

The identification of signals that regulate pluripotentiality and self-renewal is fundamental to the understanding of stem cell biology. To quantify the functionally active genome of pluripotent R1 embryonic stem (ES) cells, we used the method of serial analysis of gene expression (SAGE) to sequence a total of 140,313 SAGE tags. Of 44,569 unique transcripts, 9% matched known genes in the nonredundant GenBank database, whereas >35% of the unique tags did not match any known mouse sequence. Comparisons of relatively abundant (> or = 20) tags in the ES cell SAGE catalog with publicly available SAGE data sets identified 16 transcripts with an abundance profile unique to pluripotent R1 ES cells. We confirmed 12 by RT-PCR including those encoding KLF2, a transcription factor; galanin, a hypothalamic neurohormone; BAX, a proapoptotic signaling factor; and CDK4 and PAL31, cell cycle progression associated proteins. The data from this study provide a starting point for detailed transcriptome analyses of stem cells.

Animals↗
PubMed2002

A quantitative and validated SAGE transcriptome reference for adult mouse heart.

Transcriptome comparisons facilitate the identification of developmental, aging, and disease-related genes. To quantify the functionally active genome of adult C57BL/6 mouse heart (AMH), we used serial analysis of gene expression (SAGE) to sequence a total of 88,860 tags or 23,941 unique tags. Over 66% of the unique tags matched either known genes or ESTs. Mitochondrial transcripts accounted for 18.7% of the total transcripts, whereas sarcomeric proteins accounted for 3.2% of all tags. After comparison of AMH expression profiles obtained by SAGE and cDNA arrays, we observed numerous quantitative discrepancies (for example: arrays, mt-Co1 > mt-Co2 > mt-Co3; SAGE, mt-Co1 >> mt-Co3 >or= mt-Co2). We carried out quantitative PCR analyses as an independent test of transcript abundance and determined that SAGE yielded quantitatively reliable data. These SAGE results thus represent the first quantitative expression profile of AMH and serve as a reliable transcriptome reference to identify dynamic changes in cardiac gene expression.

Animals↗
PubMed2002

Alterations in properties of L-type Ca channels in aging rat heart.

Previous studies of whole-cell L-type Ca currents in aging heart have demonstrated an increase in the peak Ca current magnitude in proportion to the increase in membrane area, and a slowing of the time course for inactivation. However, the single-channel mechanisms underlying this upregulation, and for the slowed inactivation are not known. We have therefore compared the properties of single L-type Ca channel currents recorded from ventricular myocytes obtained from young adult (3 month), adult (6-8 month) and aging (24 month) Wistar rats, using 5 m m Ba ions as the permeant ion. We report that the peak ensemble-averaged single Ca channel currents from aging heart (-280+/-57 fA) were enhanced compared to those from young adult (-137+/-16 fA), or adult hearts (-144+/-38 fA). This surprising result was related, in part, to an apparent increase in the number of active Ca channels per patch in aging (1.90+/-0.23) v young adult (1.33+/-0.19) or adult heart (1.50+/-0.2). Moreover, there was an increase in the time constant for inactivation of the ensemble-averaged Ca currents of aging (471+/-169 ms), compared with young adult (198+/-43 ms), or adult heart (196+/-32 ms). The aging-related changes were also traced to alterations in single Ca channel gating, including an increase in the average probability of being open, and an increase in the availability of single Ca currents in aging heart. In contrast, the unitary Ca current amplitude was unchanged with aging. These novel findings suggest that the compensatory increase in the L-type Ca currents during aging is a consequence of an apparent increase in both the number, and the activity of individual L-type Ca channels.

Aging↗
PubMed2002

Thermodynamically irreversible gating of ryanodine receptors in situ revealed by stereotyped duration of release in Ca(2+) sparks.

For a single or a group of Markov channels gating reversibly, distributions of open and closed times should be the sum of positively weighted decaying exponentials. Violation of this microscopic reversibility has been demonstrated previously on a number of occasions at the single channel level, and has been attributed to possible channel coupling to external sources of free energy. Here we show that distribution of durations of Ca(2+) release underlying Ca(2+) sparks in intact cardiac myocytes exhibits a prominent mode at approximately 8 ms. Analysis of the cycle time for repetitive sparks at hyperactive sites revealed no intervals briefer than approximately 35 ms and a mode at approximately 90 ms. These results indicate that, regardless of whether Ca(2+) sparks are single-channel or multi-channel in origin, they are generated by thermodynamically irreversible stochastic processes. In contrast, data from planar lipid bilayer experiments were consistent with reversible gating of RyR under asymmetric cis (4 microM) and trans Ca(2+) (10 mM), suggesting that the irreversibility for Ca(2+) spark genesis may reside at a supramolecular level. Modeling suggests that Ca(2+)-induced Ca(2+) release among adjacent RyRs may couple the external energy derived from Ca(2+) gradients across the SR to RyR gating in situ, and drive the irreversible generation of Ca(2+) sparks.

Animals↗
PubMed2002

Intracellular Ca(2+) release as irreversible Markov process.

In striated muscles, intracellular Ca(2+) release is tightly controlled by the membrane voltage sensor. Ca(2+) ions are necessary mediators of this control in cardiac but not in skeletal muscle, where their role is ill-understood. An intrinsic gating oscillation of Ca(2+) release-not involving the voltage sensor-is demonstrated in frog skeletal muscle fibers under voltage clamp. A Markov model of the Ca(2+) release units is shown to reproduce the oscillations, and it is demonstrated that for Markov processes to have oscillatory transients, its transition rates must violate thermodynamic reversibility. Such irreversibility results in permanent cycling of the units through a ring of states, which requires a source of free energy. Inhibition of the oscillation by 20 to 40 mM EGTA or partial depletion of Ca(2+) in the sarcoplasmic reticulum (SR) identifies the SR [Ca(2+)] gradient as the energy source, and indicates a location of the critical Ca(2+)-sensing site at distances greater than 35 nm from the open channel. These results, which are consistent with a recent demonstration of irreversibility in gating of cardiac Ca(2+) sparks, (Wang, S.-Q., L.-S. Song, L. Xu, G. Meissner, E. G. Lakatta, E. Ríos, M. D. Stern, and H. Cheng. 2002. Biophys. J. 83:242-251) exemplify a cell-wide oscillation caused by coupling between ion permeation and channel gating.

Algorithms↗
PubMed2002

Modulation of the gating of unitary cardiac L-type Ca(2+) channels by conditioning voltage and divalent ions.

Although a considerable number of studies have characterized inactivation and facilitation of macroscopic L-type Ca(2+) channel currents, the single channel properties underlying these important regulatory processes have only rarely been examined using Ca(2+) ions. We have compared unitary L-type Ca(2+) channel currents recorded with a low concentration of Ca(2+) ions with those recorded with Ba(2+) ions to elucidate the ionic dependence of the mechanisms responsible for the prepulse-dependent modulation of Ca(2+) channel gating kinetics. Conditioning prepulses were applied across a wide range of voltages to examine their effects on the subsequent Ca(2+) channel activity, recorded at a constant test potential. All recordings were made in the absence of any Ca(2+) channel agonists. Moderate-depolarizing prepulses resulted in a decrease in the probability of opening of the Ca(2+) channels during subsequent test voltage steps (inactivation), the extent of which was more dramatic with Ca(2+) ions than Ba(2+) ions. Facilitation, or increase of the average probability of opening with strong predepolarization, was due to long-duration mode 2 openings with Ca(2+) ions and Ba(2+) ions, despite a decrease in Ca(2+) channel availability (inactivation) under these conditions. The degree of both prepulse-induced inactivation and facilitation decreased with increasing Ba(2+) ion concentration. The time constants (and their proportions) describing the distributions of Ca(2+) channel open times (which reflect mode switching) were also prepulse-, and ion-dependent. These results support the hypothesis that both prior depolarization and the nature and concentration of permeant ions modulate the gating properties of cardiac L-type Ca(2+) channels.

Animals↗