Parkin is transcriptionally regulated by ATF4: evidence for an interconnection between mitochondrial stress and ER stress.

Loss of parkin function is responsible for the majority of autosomal recessive parkinsonism. Here, we show that parkin is not only a stress-protective, but also a stress-inducible protein. Both mitochondrial and endoplasmic reticulum (ER) stress induce an increase in parkin-specific mRNA and protein levels. The stress-induced upregulation of parkin is mediated by ATF4, a transcription factor of the unfolded protein response (UPR) that binds to a specific CREB/ATF site within the parkin promoter. Interestingly, c-Jun can bind to the same site, but acts as a transcriptional repressor of parkin gene expression. We also present evidence that mitochondrial damage can induce ER stress, leading to the activation of the UPR, and thereby to an upregulation of parkin expression. Vice versa, ER stress results in mitochondrial damage, which can be prevented by parkin. Notably, the activity of parkin to protect cells from stress-induced cell death is independent of the proteasome, indicating that proteasomal degradation of parkin substrates cannot explain the cytoprotective activity of parkin. Our study supports the notion that parkin has a role in the interorganellar crosstalk between the ER and mitochondria to promote cell survival under stress, suggesting that both ER and mitochondrial stress can contribute to the pathogenesis of Parkinson's disease.

Electrophysiological features of the mouse sinoatrial node in relation to connexin distribution.

OBJECTIVE: The sinoatrial (SA) node consists of a relatively small number of poorly coupled cells. It is not well understood how these pacemaker cells drive the surrounding atrium and at the same time are protected from its hyperpolarizing influence. To explore this issue on a small tissue scale we studied the activation pattern of the mouse SA node region and correlated this pattern with the distribution of different gap junction proteins, connexin (Cx)37, Cx40, Cx43 and Cx45. METHODS AND RESULTS: The mouse SA node was electrophysiologically mapped using a conventional microelectrode technique. The primary pacemaker area was located in the corner between the lateral and medial limb of the crista terminalis. Unifocal pacemaking occurred in a group of pacemaking fibers consisting of 450 cells. In the nodal area transitions of nodal and atrial waveform were observed over small distances ( approximately 100 microm). Correlation between the activation pattern and connexin distribution revealed extensive labeling by anti-Cx45 in the primary and secondary pacemaker area. Within these nodal areas no gradient in Cx45 labeling was found. A sharp transition was found between Cx40- and Cx43-expressing myocytes of the crista terminalis and the Cx45-expressing myocytes of the node. In addition, strands of myocytes labeled for Cx43 and Cx40 protrude into the nodal area. Cx37 labeling was only present between endothelial cells. Furthermore, a band of connective tissue largely separates the nodal from the atrial tissue. CONCLUSIONS: Our results demonstrate strands of Cx43 and Cx40 positive atrial cells protruding into the Cx45 positive nodal area and a band of connective tissue largely separating the nodal and atrial tissue. This organization of the mouse SA node provides a structural substrate that both shields the nodal area from the hyperpolarizing influence of the atrium and allows fast action potential conduction from the nodal area into the surrounding atrium.

Calcium-activated Cl(-) current contributes to delayed afterdepolarizations in single Purkinje and ventricular myocytes.

BACKGROUND: The ionic mechanism underlying the transient inward current (I(ti)), the current responsible for delayed afterdepolarizations (DADs), appears to be different in ventricular myocytes and Purkinje fibers. In ventricular myocytes, I(ti) was ascribed to a Na(+)-Ca(2+) exchange current, whereas in Purkinje fibers, it was additionally ascribed to a Cl(-) current and a nonselective cation current. If Cl(-) current contributes to I(ti) and thus to DADs, Cl(-) current blockade may be potentially antiarrhythmogenic. In this study, we investigated the ionic nature of I(ti) in single sheep Purkinje and ventricular myocytes and the effects of Cl(-) current blockade on DADs. METHODS AND RESULTS: In whole-cell patch-clamp experiments, I(ti) was induced by repetitive depolarizations from -93 to +37 mV in the presence of 1 micromol/L norepinephrine. In both Purkinje and ventricular myocytes, I(ti) was inward at negative potentials and outward at positive potentials. The anion blocker 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) blocked outward I(ti) completely but inward I(ti) only slightly. The DIDS-sensitive component of I(ti) was outwardly rectifying, with a reversal close to the reversal potential of Cl(-) currents. Blockade of Na(+)-Ca(2+) exchange by substitution of extracellular Na(+) by equimolar Li(+) abolished the DIDS-insensitive component of I(ti). DIDS reduced both DAD amplitude and triggered activity based on DADs. Conclusions-In both Purkinje and ventricular myocytes, I(ti) consists of 2 ionic mechanisms: a Cl(-) current and a Na(+)-Ca(2+) exchange current. Blockade of the Cl(-) current may be potentially antiarrhythmogenic by lowering DAD amplitude and triggered activity based on DADs.

Two types of action potential configuration in single cardiac Purkinje cells of sheep.

Membrane potentials and currents of isolated sheep Purkinje and ventricular cells were compared using patch-clamp and microelectrode techniques. In approximately 50% of Purkinje cells, we observed action potentials that showed a prominent phase 1 repolarization and relatively negative plateau (LP cells). Action potential configuration of the remaining Purkinje cells was characterized by little phase 1 repolarization and relatively positive plateau (HP cells). Microelectrode impalement of Purkinje strands also revealed these two types of action potential configuration. In LP cells, the density of L-type Ca(2+) current (I(Ca,L)) was lower, whereas the density of transient outward K(+) current was higher, than in HP cells. Action potentials of HP cells strongly resembled those of ventricular cells. Densities of inward rectifier current and I(Ca,L) were significantly higher in ventricular cells compared with densities in both LP and HP Purkinje cells. Differences in current densities explain the striking differences in action potential configuration and the stimulus frequency dependency thereof that we observed in LP, HP, and ventricular cells. We conclude that LP Purkinje cells, HP Purkinje cells, and ventricular cells of sheep each have a unique action potential configuration.

Contribution of L-type Ca2+ current to electrical activity in sinoatrial nodal myocytes of rabbits.

The role of L-type calcium current (ICa,L) in impulse generation was studied in single sinoatrial nodal myocytes of the rabbit, with the use of the amphotericin-perforated patch-clamp technique. Nifedipine, at a concentration of 5 microM, was used to block ICa,L. At this concentration, nifedipine selectively blocked ICa,L for 81% without affecting the T-type calcium current (ICa,T), the fast sodium current, the delayed rectifier current (IK), and the hyperpolarization-activated inward current. Furthermore, we did not observe the sustained inward current. The selective action of nifedipine on ICa,L enabled us to determine the activation threshold of ICa,L, which was around -60 mV. As nifedipine (5 microM) abolished spontaneous activity, we used a combined voltage- and current-clamp protocol to study the effects of ICa,L blockade on repolarization and diastolic depolarization. This protocol mimics the action potential such that the repolarization and subsequent diastolic depolarization are studied in current-clamp conditions. Nifedipine significantly decreased action potential duration at 50% repolarization and reduced diastolic depolarization rate over the entire diastole. Evidence was found that recovery from inactivation of ICa,L occurs during repolarization, which makes ICa,L available already early in diastole. We conclude that ICa,L contributes significantly to the net inward current during diastole and can modulate the entire diastolic depolarization.

Effects of ischemia on discontinuous action potential conduction in hybrid pairs of ventricular cells.

BACKGROUND: Acute ischemia often occurs in cardiac tissue that has prior injury, resulting in spatially inhomogeneous distributions of membrane properties and intercellular coupling. Changes in action potential conduction with ischemia, which can be associated with release of catecholamines, may be particularly important in tissue that has discontinuous conduction resulting from prior infarction, hypertrophy, or myopathy. METHODS AND RESULTS: Isolated guinea pig ventricular myocytes were electrically coupled by a coupling-clamp circuit to a comprehensive computer model of a guinea pig ventricular myocyte to assess alterations in the critical value of coupling conductance required for action potential conduction from the real cell to the model cell when the real cell was exposed to a solution that included hypoxia, acidosis, and an elevated extracellular potassium concentration to simulate acute ischemia. The "ischemic" solution increased critical coupling conductance from 6.2+/-0.1 to 7.4+/-0.2 nS and decreased the associated maximum conduction delay from 31+/-1 to 23+/-1 ms (mean+/-SEM, n=11). The ischemic solution plus 1 micromol/L norepinephrine decreased critical coupling conductance from 5.9+/-0.2 to 5.0+/-0.1 nS and increased maximum conduction delay from 31+/-2 to 54+/-4 ms (mean+/-SEM, n=8). CONCLUSIONS: The release of catecholamines with ischemia, in a setting of partially uncoupled cells, may play a major role in producing long conduction delays, which may allow reentrant pathways.

The graduate training in medical information sciences in the Academic Medical Centre at the University of Amsterdam.

Since its inception in 1987, the 4-year Medical Information Sciences (MIS) curriculum at the Academic Medical Centre (AMC), Amsterdam has gone through several major changes. The present curriculum started in 1994. The course takes 4 years, the first 3 years are programmed in integrated modules of 7 weeks in duration each. In these modules much attention is given to interactive teaching, problem based learning and private study. Typical for the Amsterdam curriculum is a strong emphasis on the role and significance of data and information in health care and its management. The authors see information technology per se as auxiliary to this orientation. Presently, about 150 students follow the courses.

Electrical interactions between a rabbit atrial cell and a nodal cell model.

Atrial activation involves interactions between cells with automaticity and slow-response action potentials with cells that are intrinsically quiescent with fast-response action potentials. Understanding normal and abnormal atrial activity requires an understanding of this process. We studied interactions of a cell with spontaneous activity, represented by a "real-time" simulation of a model of the rabbit sinoatrial (SA) node cell, simultaneously being electrically coupled via our "coupling clamp" circuit to a real, isolated atrial myocyte with variations in coupling conductance (Gc) or stimulus frequency. The atrial cells were able to be driven at a regular rate by a single SA node model (SAN model) cell. Critical Gc for entrainment of the SAN model cell to a nonstimulated atrial cell was 0.55 +/- 0.05 nS (n = 7), and the critical Gc that allowed entrainment when the atrial cell was directly paced at a basic cycle length of 300 ms was 0.32 +/- 0.01 nS (n = 7). For each atrial cell we found periodic phenomena of synchronization other than 1:1 entrainment when Gc was between 0.1 and 0.3 nS, below the value required for frequency entrainment, when the atrial cell was directly driven at a basic cycle length of either 300 or 600 ms. In conclusion, the high input resistance of the atrial cells allows successful entrainment of nodal and atrial cells at low values of Gc, but further uncoupling produces arrhythmic interactions.

Distribution of atrial and nodal cells within the rabbit sinoatrial node: models of sinoatrial transition.

BACKGROUND: In the sinoatrial node (SAN) the course of the action potential gradually changes from the primary pacemaker region toward the atrium. It is not known whether this gradient results from different intrinsic characteristics of the nodal cells, from an increasing electrotonic interaction with the atrium, or from both. Therefore we have characterized the immunohistochemical, morphological, and electrophysiological correlates of this functional gradient. METHODS AND RESULTS: The distribution of rabbit nodal myocytes in the SAN has been studied by immunohistochemistry. After cell isolation, the electrophysiological characteristics of different nodal cell types were measured. (1) The staining pattern of a neurofilament protein coincides with the electrophysiologically mapped pacemaker region in the SAN. (2) Enzymatic digestion of the SAN reveals three morphologically different nodal cell types and one atrial type. Of each nodal cell type, neurofilament-positive as well as neurofilament-negative myocytes are found. Atrial cells are all neurofilament-negative. (3) In contrast to previous findings, we observed atrial cells in the very center of the SAN. The relative number of atrial cells gradually increases from the central pacemaker area toward the atrium. (4) Differences in electrophysiological characteristics between individual nodal cells are not associated with differences in cell type. CONCLUSIONS: (1) The expression of neurofilaments can be used to delineate the nodal area in the intact SAN but is not sufficiently sensitive for characterizing all individual isolated nodal cells. (2) A fundamentally different organization of the SAN is presented: The gradual increase in density of atrial cells from the dominant area toward the crista terminalis in the SAN causes a gradual increase of atrial electrotonic influence that may be an important cause of the gradual transition of the nodal to the atrial type of action potential.

Pacemaker synchronization of electrically coupled rabbit sinoatrial node cells.

The effects of intercellular coupling conductance on the activity of two electrically coupled isolated rabbit sinoatrial nodal cells were investigated. A computer-controlled version of the "coupling clamp" technique was used in which isolated sinoatrial nodal cells, not physically in contact with each other, were electrically coupled at various values of ohmic coupling conductance, mimicking the effects of mutual interaction by electrical coupling through gap junctional channels. We demonstrate the existence of four types of electrical behavior of coupled spontaneously active cells. As the coupling conductance is progressively increased, the cells exhibit: (a) independent pacemaking at low coupling conductances, (b) complex dynamics of activity with mutual interactions, (c) entrainment of action potential frequency at a 1:1 ratio with different action potential waveforms, and (d) entrainment of action potentials at the same frequency of activation and virtually identical action potential waveforms. The critical value of coupling conductance required for 1:1 frequency entrainment was <0.5 nS in each of the five cell pairs studied. The common interbeat interval at a relatively high coupling conductance (10 nS), which is sufficient to produce entrainment of frequency and also identical action potential waveforms, is determined most by the intrinsically faster pacemaker cell and it can be predicted from the diastolic depolarization times of both cells. Evidence is provided that, at low coupling conductances, mutual pacemaker synchronization results mainly from the phase-resetting effects of the action potential of one cell on the depolarization phase of the other. At high coupling conductances, the tonic, diastolic interactions become more important.

[Medical education of tomorrow: a glance into the (near) future]. / Medisch onderwijs van morgen: een blik in de (naaste) toekomst.

Educational reform is a topical subject in Dutch medical schools. Public visitation reports were issued in 1992 and 1997. In 1994 an 'outline plan' was presented, setting forth communal training requirements (final results). An important consequence will be reorganization of (assistant) housemanships. Options are to start already in the 3rd- and 4th-year with a few (assistant) housemanships, to reduce the number but increase the duration of the (assistant) housemanships and to introduce a more comprehensive training (including elements from several disciplines). Other areas for special attention are the insufficient professionalization of teachers in medicine, the bureaucracy weighing down the teaching and the continuing need of educational research. Regarding the future doctors' image of their profession, emphasis should be placed on the international trend toward more attention for prevention and cost control, apart from the physician's traditional curative task. With respects to curriculum reorganization, six items should be considered: the need of a basic philosophy (per faculty), the priority to be given to the process of medical problem solving over that of acquiring knowledge and information, the desirability of increasing orientation to practice, the necessity of an integrated approach of medical teaching (teaching elements to be deduced from the final terms, instead of vice versa), development of a professional attitude and the realization of a master-apprentice relationship in medical education.

Biphasic response of action potential duration to metabolic inhibition in rabbit and human ventricular myocytes: role of transient outward current and ATP-regulated potassium current.

Inhibition of cell metabolism is associated with significant changes in action potential duration. The aim of this study was to investigate the time course of the changes in action potential duration during metabolic inhibition and to determine what changes in membrane currents are responsible. The amphotericin perforated patch clamp technique was used to study membrane currents and voltage in single rabbit and human ventricular myocytes. In all myocytes inhibition of cell metabolism, induced by hypoxia (PO2 < 5 mmHg) or by addition of 100 microM 2,4-dinitrophenol (DNP), resulted in action potential shortening, which was accompanied by an increase in outward current, likely to be carried by ATP-regulated potassium channels. In about 65% of the rabbit and 50% of the human ventricular myocytes, however, action potential shortening was preceded by an initial prolongation. During this action potential prolongation, the L-type calcium current and the steady-state outward current remained unchanged. The transient outward current (Ito), however, was almost completely inhibited, suggesting that the action potential prolongation is caused by a decreased Ito. This interpretation was further supported by the observations that: (1) Action potential prolongation was found in all subepicardial myocytes, as was Ito, but only in a minority of the subendocardial myocytes. (2) Addition of DNP failed to cause action potential prolongation in subepicardial myocytes in the presence of 4-aminopyridine, a blocker of Ito. In conclusion, these data suggest that the phenomenon of action potential prolongation preceding action potential shortening during metabolic inhibition is mainly restricted to myocytes from subepicardial origin, and is due to a decrease in Ito.

Delayed rectifier channels in human ventricular myocytes.

BACKGROUND: Previous studies have shown that in heart there are two kinetically distinct components of delayed rectifier current: a rapidly activating component (IKr) and a more slowly activating component (IKs). The presence of IKr and/or IKs appears to be species dependent. We studied the nature of the delayed rectifier current in human ventricle in whole-cell and single-channel experiments. METHODS AND RESULTS: Ventricular myocytes were obtained from hearts of patients with ischemic or dilated cardiomyopathy. Single-channel currents and whole-cell tail currents were recorded at negative potentials directly after return from a depolarizing step. Single-channel currents were measured in the cell-attached patch configuration with 140 mmol/L K+ in the pipette. In the present study, we identified a voltage-dependent channel with a single-channel conductance of 12.9 +/- 0.8 pS (mean +/- SEM, n = 5) and a reversal potential near to the K+ equilibrium potential, suggesting that the channel is selective to K+ ions. Channel activity was observed only after a depolarizing step and increased with the duration and amplitude of the depolarization, indicating time- and voltage-dependent activation. Activation at +30 mV was complete within 300 milliseconds, and the time constant of activation, determined in the whole-cell configuration, was 101 +/- 25 milliseconds (mean +/- SEM, n = 4). The voltage dependence of activation could be described by a Boltzmann equation with a half-activation potential of -29.9 mV and a slope factor of 9.5 mV. The addition of the class III antiarrhythmic drug E-4031 completely blocked channel activity in one patch. No indications for the presence of IKs were found in these experiments. CONCLUSIONS: The conformity between the properties of IKr and those of the K+ channel in the present study strongly suggests that IKr is present in human ventricle.

Age-related changes in structure and relative collagen content of the human and feline sinoatrial node. A comparative study.

Age-related changes in the structure and size of the human and cat sinoatrial nodes were studied by light microscopy, with emphasis on changes in relative collagen volume. Sinoatrial nodes from 41 humans (aged 0-94 years) and 21 cats (aged 6 weeks-18 years) were used. It was found that there were no changes in the dimensions of the sinoatrial node during adult life in either species. In Sirius Red F3 Ba stained sections, the relative volume of collagen was measured using an interactive image analysis system. The relative volume of collagen in the human sinoatrial node increases from 38% during childhood to 70% during adulthood. Once adulthood is reached, there are no further changes in the relative volume of collagen. In the cat sinoatrial node the relative volume of collagen is only 27% and does not change with age. The organisation of collagen in the sinoatrial node, however, demonstrates an age-dependent change in both humans and cats. From coarse strands between clusters of nodal cells it gradually changes into a fine network of isolated collagen fibres which surround individual nodal cells. This process is more pronounced in humans. It is concluded that age-related changes in sinoatrial node function are not related to an increase in collagen content in the sinoatrial node.

Effects of delayed rectifier current blockade by E-4031 on impulse generation in single sinoatrial nodal myocytes of the rabbit.

The role of the delayed rectifier current (IK) in impulse generation was studied in single sinoatrial nodal myocytes of the rabbit. We used the class III antiarrhythmic drug E-4031, which blocks IK in rabbit ventricular myocytes. In single sinoatrial nodal cells, E-4031 (0.1 mumol/L) significantly prolonged cycle length and action potential duration, depolarized maximum diastolic potential, and reduced both the upstroke velocity of the action potential and the diastolic depolarization rate. Half of the cells were arrested completely. At higher concentrations (1 and 10 mumol/L), spontaneous activity ceased in all cells. Three ionic currents fundamental for pacemaking, ie, IK, the long-lasting inward calcium current (ICa,L), and the hyperpolarization-activated current (I(f)), were studied by using the whole-cell and amphotericin-perforated patch technique. E-4031 blocked part of the outward current during depolarizing steps as well as the tail current upon subsequent repolarization (ITD) in a dose-dependent manner. E-4031 (10 mumol/L) depressed ITD (88 +/- 4%) (n = 6), reduced peak ICa,L at 0 mV (29 +/- 15%) (n = 4), but did not affect I(f). Lower concentrations did not affect ICa,L. Additional use of 5 mumol/L nifedipine demonstrated that ITD is carried in part by a calcium-sensitive current. Interestingly, complete blockade of IK and ICa,L unmasked the presence of a background current component with a reversal potential of -32 +/- 5.4 mV (n = 8) and a conductance of 39.5 +/- 5.6 pS/pF, which therefore can contribute both to the initial part of repolarization and to full diastolic depolarization.(ABSTRACT TRUNCATED AT 250 WORDS)

Electrophysiology of the ageing rabbit and cat sinoatrial node--a comparative study.

Intrinsic properties of the human sinoatrial (SA) node have been shown to decline with age. In the present study we aimed at investigating the underlying mechanisms of age-dependent changes in intrinsic cycle length and sinoatrial conduction time. To this end, the cycle length and transmembrane potentials of the SA nodes of rabbits (2 days-5.6 years) and cats (6 weeks-18 years) were recorded and nodal conduction was reconstructed. The size of the SA nodes was measured in Sirius Red stained sections. Cycle length increases with age in both the rabbit and cat SA node, and in both species cycle length is dependent on diastolic depolarization rate and action potential duration. Nodal action potential duration increases with age in both rabbit and cat, whereas diastolic depolarization rate decreases in the cat only. The location of the primary pacemaker is not related to age. With age, sinoatrial conduction time increases in both species as a result of an enlargement of the area with low phase 0 upstroke velocities. The size of the SA node of adult animals does not increase with age.

Single delayed rectifier channels in the membrane of rabbit ventricular myocytes.

In rabbit ventricular cells, the delayed rectifier current (IK) has not been extensively studied, and properties of single IK channels still need to be determined. In this study, we present data on a voltage-dependent channel in rabbit ventricular cells; the properties indicate that it is an IK channel. Patch-clamp experiments were carried out on cell-attached and inside-out patches of rabbit ventricular cells. Single-channel currents were recorded at negative potentials as inward currents with 150 mM K+ in the pipette. Voltage-dependent channel activity was only present after the return from a depolarizing test pulse, indicating activation on depolarization. Single-channel conductance calculated from the current-voltage relation was 13.1 pS (pooled data, n = 8). The shift in reversal potential of the unitary currents, determined at 150 and 300 mM K+ at the intracellular side of the membrane, showed that the channels were highly permeable to potassium ions. Increase of the duration or the amplitude of the depolarizing test pulse increased channel activity. The time constant for activation at +30 mV was 187 msec (pooled data, n = 4). Half-activation potential was -4.9 +/- 3.8 mV (mean +/- SD), and the slope factor was 7.2 +/- 3.7 mV (mean +/- SD). Current tails, reconstructed from averaged single-channel currents, revealed that the time course of deactivation decreased from 694 +/- 73 msec at -80 mV to 136 +/- 39 msec at -110 mV. Additional evidence that the channel was indeed an IK channel was provided by the observation that the channel was blocked by 10(-7) M E-4031, a class III antiarrhythmic agent that has been shown to block a component of the macroscopic IK in guinea pig heart.