Biomedical subjects
Publications and source records attributed to E Marban.
We have investigated the regulation of intracellular free calcium in heart muscle using the free acid form of the Ca2+ indicator fura-2 iontophoretically microinjected into rat cardiac trabeculae or ferret papillary muscles. This method shows great promise in elucidating a number of crucial questions in cardiac excitation-contraction coupling.
OBJECTIVES: This study was conducted to elucidate the role of sodium/calcium (Na+/Ca2+) exchange in the mechanism of myocardial stunning. BACKGROUND: Cellular Ca2+ overload mediated by Na+/Ca2+ exchange during reperfusion has been proposed as a mechanism for myocardial stunning. Because no specific pharmacologic inhibitors of the exchanger are available, we increased extracellular sodium concentration ([Na]o) during the early phase of reperfusion to decrease the driving force for Ca2+ influx through the pathway. METHODS: Isovolumetric left ventricular pressure and phosphorus-31 nuclear magnetic resonance spectra were measured in isolated perfused ferret hearts. Hearts were reperfused with different solutions after 15 min of total global ischemia at 37 degrees C. RESULTS: Hearts reperfused with standard solution ([Na]o = 140 mmol/liter; the stunned hearts, n = 8) showed only 69 +/- 3% (mean +/- SEM) recovery of developed pressure relative to preischemic control developed pressure. In contrast, hearts reperfused with a high [Na]o solution ([Na]o = 268 mmol/liter) during the initial 5 min, followed by a gradual decrease of [Na]o to the standard level over 25 min (the high [Na]o group, n = 8) showed significantly better recovery of developed pressure (85 +/- 2%, p < 0.05 vs. the stunned hearts). In contrast, reperfusion with solutions in which the additional Na was substituted either by 256 mmol/liter sucrose or 128 mmol/liter choline chloride did not improve functional recovery, indicating that the beneficial effects of high [Na]o reperfusion are not due to either high ionic strength or high osmolarity. Phosphorus-31 nuclear magnetic resonance spectra showed no correlation between functional recovery and intramyocardial contents of phosphorus compounds or pH. CONCLUSIONS: High [Na]o reperfusion protects against stunning, supporting the concept that Na+/Ca2+ exchange plays an important role in the mechanism of stunned myocardium.
During acute respiratory alkalosis, myocardial contractility initially increases but then declines toward control levels. To elucidate the mechanism of this response, two parallel strategies were adopted: isovolumic left ventricular developed pressure (DP) and intracellular pH (pHi) were measured in isolated ferret hearts using 31P-nuclear magnetic resonance spectroscopy, and isometric developed tension (DT) and intracellular Ca2+ concentration ([Ca2+]i) were measured in ferret papillary muscles using microinjected fura 2 salt. When hypocapnia was induced by sudden introduction of perfusate equilibrated with 2% CO2 (from 5% CO2 in control), DP increased to a maximum of 120 +/- 3% (SE; n = 7) of control within 40 s. Afterward, DP decreased toward control levels, reaching a new steady state in 2-3 min. In contrast, pHi increased from control (7.11 +/- 0.01) only after 30 s of hypocapnia and reached a peak of 7.25 +/- 0.02 between 80 and 100 s. Thus pHi lagged behind contractility. In contrast to pHi, [Ca2+]i changed in parallel with DT: when DT reached a maximum (251 +/- 63% of control; n = 5) during hypocapnia, the amplitude of [Ca2+]i transients also peaked (190 +/- 22% of control; n = 5). A simulation of contractile force based on our measurements of pHi and [Ca2+]i, along with published Ca(2+)-tension relations, described adequately the changes in developed force during hypocapnia. These results indicate that the biphasic changes in [Ca2+]i, coupled with an out-of-phase change in pHi, underlie the biphasic response of myocardial contractility to hypocapnia.
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Background outward K+ currents in guinea pig ventricular myocytes were characterized over a broad range of membrane potentials, including those corresponding to the plateau of the action potential. The background current that is blocked by 1 mM Ba2+ (IK,p) activates within 5 msec at positive potentials, does not inactivate, and deactivates very rapidly on repolarization. IK,p is insensitive to Cl- channel blockers, internal or external [Cl-], dihydropyridines, and sulfonylureas. In contrast, the delayed rectifier K+ current (IK) was not completely blocked even by 30 mM Ba2+. Ba(2+)-sensitive current density increased progressively from 0.16 +/- 0.04 pA/pF at 0 mV to 0.52 +/- 0.21 pA/pF at +80 mV (n = 13, mean +/- SEM). The background current remains present when [K+]o is reduced to 0 mM, which suppresses the inward rectifier K+ current (IK1). These and other features suggest that IK,p is generated by K+ channels that are distinct from IK1 or IK. The kinetics and voltage dependence of IK,p render it capable of modulating both the height and duration of the cardiac action potential.
Free magnesium ions and magnesium-nucleotide complexes can exert opposite effects on many fundamental cellular processes. Although increases in the intracellular concentration of magnesium ions inhibit the L-type calcium current in heart cells, magnesium-adenosine triphosphate complexes (MgATP) would be expected to increase the current by promoting channel phosphorylation. Rapid increases in the intracellular concentration of MgATP induced by flash photolysis of caged magnesium or caged ATP resulted in enhanced calcium current. The increase in calcium current was not prevented by blocking phosphorylation, revealing a previously unrecognized direct regulatory action of the magnesium-nucleotide complex.
Sodium channels are the major proteins that underlie excitability in nerve, heart, and skeletal muscle. Chemical reaction rate theory was used to analyze the blockage of single wild-type and mutant sodium channels by cadmium ions. The affinity of cadmium for the native tetrodotoxin (TTX)-resistant cardiac channel was much higher than its affinity for the TTX-sensitive skeletal muscle isoform of the channel (microliters). Mutation of Tyr401 to Cys, the corresponding residue in the cardiac sequence, rendered microliters highly susceptible to cadmium blockage but resistant to TTX. The binding site was localized approximately 20% of the distance down the electrical field, thus defining the position of a critical residue within the sodium channel pore.
During ischemia or metabolic inhibition, intracellular Na+ concentration ([Na+]i) increases considerably. Elevation of [Na+]i figures critically in the mechanism of cellular injury by promoting Ca2+ influx via the Na+-Ca2+ exchanger, but the exact mechanism of this intracellular Na+ accumulation remains unknown. To test directly the hypothesis that voltage-dependent Na+ channels are involved, we measured Na+ currents (INa) in isolated guinea-pig ventricular myocytes using the patch-clamp technique. The cell-attached configuration was used in order to avoid disturbing the intracellular milieu. Metabolic inhibition was induced by exposing the cells to either iodoacetate (IAA, 1 mM) to inhibit glycolysis or 2,4-dinitrophenol (DNP, 0.2 mM) to uncouple oxidative phosphorylation. The amplitude of INa was measured in multichannel patches before and during exposure to IAA or DNP, by depolarizing the cell to different membrane potentials from a holding potential of -135 mV. Analysis of current-voltage relations before and during metabolic inhibition revealed a modest but significant reduction of peak INa at test potentials positive to -40 mV with DNP; no change was observed with IAA. The voltage dependence of steady-state parameters of inactivation was not altered by either intervention; specifically, no steady-state ("window") current was induced. Although we cannot exclude the possibility that other factors not explored here might lead to different conclusions during genuine ischemia, metabolic inhibition alone does not up-regulate the function of Na+ channels. Thus, we conclude that other mechanisms underlie the accumulation of intracellular Na+ observed during metabolic inhibition.
1. We sought to distinguish two types of Ca2+ channel in guinea-pig ventricular cells (T-type and L-type) and to characterize their respective gating and permeation properties when Ca2+ (1-10 mM) is the charge carrier, as is the case physiologically. 2. Na+ was removed from both the external and internal solutions to eliminate currents through Na+ channels and Na(+)-Ca2+ exchange. Major differences in the voltage dependence of steady-state inactivation were exploited to separate the two Ca2+ current components. 3. From a holding potential of -50 mV, only L-type channels were available to open with depolarization. When holding at -90 mV, T-type channels contributed an additional rapidly inactivating component superimposed upon the L-type current. Only the L-type channels thus identified were sensitive to the dihydropyridine Ca2+ channel blocker nitrendipine. 4. T-type currents, measured by taking the difference between the currents elicited from a holding potential of -90 mV and those elicited from -50 mV, peaked within 10 ms and decayed completely within 50-100 ms. 5. Macroscopic T-type currents were largest during depolarizing pulses between -40 and -30 mV (peak current density of 0.62 +/- 0.21 nA nF-1) and decreased at more positive potentials, becoming unmeasurably small above 0 mV. 6. Unitary currents recorded with similar ionic conditions and voltage protocols exhibited a single-channel conductance of 4-5 pS in 10 mM Ca2+. Ensemble average currents through a single channel reproduced accurately the time course of whole-cell T-type current. Permeation properties could not explain the absence of macroscopic T-type currents at positive test potentials, which must therefore be attributable to gating. 7. Convolution analysis was employed to clarify the single-channel basis of the rapidly decaying current waveform of T-type channels. The latencies to first opening and reopening, which reflect activation and deactivation, influenced the waveform most strikingly. Open times were sufficiently brief that they contributed little to shaping the average current. Thus, macroscopic inactivation largely reflects rate-limiting activation events. 8. The unitary current amplitudes and peak open probabilities measured for single T-type channels, when compared to the average macroscopic T-type current density, predict 10.6 functional channels per picofarad, or approximately 1700 T-type channels per typical ventricular myocyte.
1. We investigated the currents through L-type Ca2+ channels when Ca2+ (1-10 mM) was the charge carrier, as is the case physiologically. 2. Na+ was removed from both the external and internal solutions to eliminate currents through Na+ channels and Na(+)-Ca2+ exchange. 3. From a holding potential of -50 mV only L-type channels were available to open with depolarization. Macroscopic L-type currents were maximal during depolarizing pulses to +10 mV (peak current density of 4.7 +/- 0.3 nA nF-1). 4. During depolarizing steps as long as 180 ms, the decay of current through L-type channels was incomplete, in contrast to that of T-type current. 5. Unitary currents recorded with comparable ionic conditions and voltage protocols exhibited a single-channel conductance of 6.9 pS in 10 mM Ca2+. Ensemble average currents reproduced accurately the features of whole-cell L-type current, including the maintained component. 6. Convolution analysis was employed to clarify the single-channel basis of the complex current waveform of L-type channels. First openings underlie the peak, while the maintained pedestal is generated by multiple re-openings. As with T-type channels, single openings are brief and contribute little to the time course of the average current. 7. The prominent maintained component of macroscopic and ensemble average L-type current cannot be explained by simple Markov models in which current decay reflects the progressive entry of channels into an absorbing inactivated state. 8. We considered the possibility that the maintained component of current arises from the existence of multiple distinct gating patterns, one of which lacks inactivation. Individual sweeps were sorted among three patterns of gating (no openings, active-early and active-late). Patterns of activity are not randomly distributed; instead, they tend to cluster over time. 9. Most of the maintained current is attributable to the 'active-late' pattern of gating. Considered separately, this pattern can be well described by a simple Markov chain lacking an inactivated state. The 'active-early' gating pattern accounts entirely for the initial current transient, and for about one-third of the maintained component; thus, inactivation, even when present, must be reversible rather than absorbing. 10. The unitary current amplitudes and peak open probabilities measured for single L-type channels, when compared to the average macroscopic L-type current density, predict 170 functional channels per picofarad, or 28,000 L-type channels per typical ventricular myocyte.(ABSTRACT TRUNCATED AT 400 WORDS)
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This study examined the hypothesis that glycolysis is required for functional recovery of the myocardium during reperfusion by facilitating restoration of calcium homeostasis. [Ca2+]i was measured in isolated perfused rabbit hearts by using the Ca2+ indicator 1,2-bis(2-amino-5-fluorophenoxy)ethane-N,N,N',N'-tetraacetic acid (5F-BAPTA) and 19F nuclear magnetic resonance spectroscopy. In nonischemic control hearts, inhibition of glycolysis with iodoacetate did not alter [Ca2+]i. In hearts subjected to 20 minutes of global zero-flow ischemia, [Ca2+]i increased from 260 +/- 80 nM before ischemia to 556 +/- 44 nM after 15 minutes of ischemia (p less than 0.05). After reperfusion with 5 mM pyruvate as a carbon substrate, [Ca2+]i increased further in hearts with intact glycolysis to 851 +/- 134 nM (p less than 0.05 versus ischemia) during the first 10 minutes of reperfusion, before returning to preischemic levels. In contrast, inhibition of glycolysis during the reperfusion period resulted in persistent severe calcium overload ([Ca2+]i, 1,380 +/- 260 nM after 15 minutes of reperfusion, p less than 0.02 versus intact glycolysis group). Furthermore, despite the presence of pyruvate and oxygen, inhibition of glycolysis during early reperfusion resulted in greater impairment of functional recovery (rate/pressure product, 3,722 +/- 738 mm Hg/min) than did reperfusion with pyruvate and intact glycolysis (rate/pressure product, 9,851 +/- 590 mm Hg/min, p less than 0.01). Inhibition of glycolysis during early reperfusion was also associated with a marked increase in left ventricular end-diastolic pressure during reperfusion (41 +/- 5 mm Hg) compared with hearts with intact glycolysis (16 +/- 2 mm Hg, p less than 0.01). The detrimental effects of glycolytic inhibition during early reperfusion were, however, prevented by initial reperfusion with a low calcium solution ([Ca]o, 0.63 mM for 30 minutes, then 2.50 mM for 30 minutes). In these hearts, the rate/pressure product after 60 minutes of reperfusion was 12,492 +/- 1,561 mm Hg/min (p less than 0.01 versus initial reflow with [Ca]o of 2.50 mM). These findings indicate that the functional impairment observed in postischemic myocardium is related to cellular Ca2+ overload. Glycolysis appears to play an important role in restoration of Ca2+ homeostasis and recovery of function of postischemic myocardium.
Ventricular fibrillation (VF) is known to produce alterations in myocardial energetics, but the mechanism of these changes remains unclear. To investigate energy metabolism during VF, phosphorus nuclear magnetic resonance spectroscopy and magnetization transfer were applied to isolated perfused ferret hearts. VF was induced either by perfusion with digitalis (strophanthidin, 30 microM) or by high-frequency electrical stimulation. We measured the flux in two critical reactions: from inorganic phosphate (Pi) to ATP (ATP synthesis rate) and from phosphocreatine (PCr) to ATP (energy transfer capacity). During digitalis-induced VF, energy-related phosphates showed changes similar to those during hypoxia: myocardial [Pi] increased and [PCr] decreased. Concomitantly, the ATP synthesis rate increased to levels about threefold higher than control, whereas oxygen consumption increased by only 16%. The ATP synthesis rate exhibited a strong negative correlation with left ventricular pressure during VF (r = -0.95, n = 5, p < 0.02), whereas oxygen consumption did not (r = 0.19, p > 0.05). On the other hand, energy transfer capacity catalyzed by creatine kinase was significantly smaller during VF than in the control condition but still higher than the simultaneous ATP synthesis rate. In contrast to the marked energetic deterioration during VF induced by digitalis, electrically induced VF led to only a small increase in [Pi] and a small decrease in [PCr], and there were no significant changes in the ATP synthesis rate, energy transfer capacity, or O2 consumption. These results indicate that the rundown in energy metabolism during VF induced by digitalis was mainly attributable to a limitation of energy production through oxidative phosphorylation as well as to a marked increase in energy consumption. In contrast, myocardial energy generation remained unimpaired during VF induced by electrical stimulation. Intracellular calcium overload is more severe during VF induced by digitalis than during electrically induced VF (Circ Res 1991;68:1378-1389); severe calcium overload would be expected to compromise the capacity for energy generation by mitochondria. Thus, we propose that known differences in cellular calcium loading underlie the discrepant energetic patterns of the two types of VF.
Cellular calcium homeostasis is disturbed during brief periods of ischemia, with free cytosolic Ca ([Ca2+]i) rising severalfold within 10-15 minutes (or even sooner). Nevertheless, the myocardial cells' ability to regulate Ca recovers quickly after reperfusion, unlike the lingering depression of contractile function known as stunning. This brief commentary considers the hypothesis that the rise in cell calcium during ischemia and during early reperfusion leaves behind stunning as an unwelcome legacy. This idea by no means excludes the involvement of other factors, such as free radicals; instead, it suggests a possible common pathway for cell injury by a variety of specific agents. A provocative feature of the formulation is the idea that the crucial lesion in stunning occurs at the level of the contractile proteins, rather than more proximally.
Changes in cytoplasmic magnesium concentration in the millimolar range are known to evoke directionally opposite changes in the amplitude of cardiac L-type calcium current, but the effects of magnesium changes in the micromolar range are not known. This concentration range is particularly interesting for two reasons: open-channel block of some ion channels (eg, the inward rectifier) occurs with a micromolar Kd; and magnesium at such low levels may limit the availability of magnesium adenosine triphosphate (MgATP) and magnesium guanidine triphosphate (MgGTP) for a variety of enzymatic processes, including calcium channel phosphorylation. To investigate the effects of critical magnesium depletion and its sudden reversal, we used flash photolysis to liberate magnesium from intracellular DM-nitrophen in guinea pig heart cells. Under our conditions, intracellular [Mg2+] changed from 0.25 mumol/L before the flash to less than or equal to 200 mumols/L afterwards. Calcium channel currents carried by either calcium or barium consistently increased after flash, a directional change opposite to the reported effects of elevated [Mg2+] in the millimolar range. Our data do not yet enable us to understand the basis of the magnesium effect, but we are particularly interested in the possibility that the sudden appearance of magnesium enables calcium channel phosphorylation.
1. Unitary Na+ channel currents were recorded from isolated guinea-pig ventricular myocytes using the cell-attached patch-clamp technique with high [Na+] in the pipette to enhance the signal-to-noise ratio. 2. The probability that the channel enters the inactivated state (I) directly from resting states (C) was investigated over a wide range of membrane potentials. 3. At membrane potentials of -60 mV or more positive, Markov chain theory was used to estimate the probability of C----I from histograms of the number of channel openings per depolarizing period. Holding potentials at least as negative as -136 were required to ensure that all channels resided in C prior to depolarization. 4. At membrane potentials negative to -60 mV, a two-pulse protocol was employed to determine the probability of C----I from the fraction of blank sweeps during the pre-pulse with correction for missed events. 5. The probability of C----I was found to be steeply voltage dependent at negative potentials, falling from 0.87 +/- 0.03 (mean +/- S.D.) at -91 mV to 0.42 +/- 0.01 at -76 mV. At potentials positive to -60 mV, this probability was less steeply voltage dependent and decayed to near zero at 0 mV. 6. Under physiological conditions, C----I transitions may produce appreciable Na+ channel inactivation at diastolic potentials. At potentials above the action potential threshold, inactivation is much more likely to occur from the open state.
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The basis of early ischemic contractile failure was investigated in perfused ferret hearts at 27 degrees C. Isovolumic left ventricular developed pressure fell by more than 50% within 30 seconds of the onset of total global ischemia and reached zero by 5 minutes. Monophasic action potential recordings revealed no decrease in excitability during this period. Phosphorus nuclear magnetic resonance spectra obtained at 30-second resolution showed no significant changes in inorganic phosphate or phosphocreatine during the first 30 seconds of ischemia. Intracellular pH (pHi) and ATP changed even more slowly; therefore, none of these metabolites could account for the rapid fall in force. To gauge the contribution of intravascular pressure, we compared ordinary aortic flow occlusion with tissue-level ischemia induced by massive coronary microembolization at the level of the precapillary arterioles. Functional depression developed significantly more slowly in the microembolized hearts, despite accumulation of inorganic phosphate and protons comparable with that in ordinary ischemia. After microembolization, the time course of functional depression reflected much more closely the concomitant inorganic phosphate and pHi changes. Thus, our results provide novel evidence supporting the importance of vascular collapse in the mechanism of early ischemic contractile failure.