Author + information
- Received December 6, 1999
- Revision received August 22, 2000
- Accepted October 2, 2000
- Published online February 1, 2001.
- Stephan B Felix, MD∗,* (, )
- Verena Stangl, MD†,
- Peter Pietsch, PhD†,
- Peter Bramlage, MD†,
- Alexander Staudt, MD∗,
- Sabine Bartel, PhD‡,
- Ernst-Georg Krause, MD‡,
- Jörn-Uwe Borschke, MD§,
- Klaus-Dieter Wernecke, PhD∥,
- Gerrit Isenberg, MD§ and
- Gert Baumann, MD†
- ↵*Reprint requests and correspondence: Dr. Stephan Felix, Klinik für Innere Medizin B, Ernst-Moritz-Arndt-Universität Greifswald, Friedrich Loeffler Strasse 23a, 17489 Greifswald, Germany
This study was designed to investigate the effects of cardiodepressant substances released from postischemic myocardial tissue on myocardial calcium-regulating pathways.
We have recently reported that new cardiodepressant substances are released from isolated hearts during reperfusion after myocardial ischemia.
After 10 min of global ischemia, isolated rat hearts were reperfused, and the coronary effluent was collected for 30 s. We tested the effects of the postischemic coronary effluent on cell contraction, Ca2+transients and Ca2+currents of isolated rat cardiomyocytes by applying fluorescence microscopy and the whole-cell, voltage-clamp technique. Changes in intracellular phosphorylation mechanisms were studied by measuring tissue concentrations of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), as well as activities of cAMP-dependent protein kinase (cAMP-dPK) and protein kinase C (PKC).
The postischemic coronary effluent, diluted with experimental buffer, caused a concentration-dependent reduction of cell shortening and Ca2+transient in the field-stimulated isolated cardiomyocytes of rats, as well as a reduction in peak L-type Ca2+current in voltage-clamped cardiomyocytes. The current reduction resulted from reduced maximal conductance—not from changes in voltage- and time-dependent gating of the L-type Ca2+channel. The postischemic coronary effluent modified neither the tissue concentrations of cAMP or cGMP nor the activities of cAMP-dPK and PKC. However, the effluent completely eliminated the activation of glycogen phosphorylase after beta-adrenergic stimulation.
Negative inotropic substances released from isolated postischemic hearts reduce Ca2+transient and cell contraction through cAMP-independent and cGMP-independent blockage of L-type Ca2+channels.
We have recently reported that previously unknown and still unidentified negative inotropic substances (NIS) are released from the postischemic myocardium (1,2). The present study was performed to analyze the mechanism of the negative inotropic effects mediated by NIS. We used isolated rat cardiomyocytes as an in vitro bioassay system in which the effects of postischemic coronary effluent from isolated rat hearts were analyzed. In isolated ventricular myocytes, contractility can be quantified by the extent and rate of unloaded shortening (3). Because contractile activation is controlled by the Ca2+transient (i.e., by changes in cytosolic Ca2+concentration [Ca2+]c), the effects of the postischemic coronary effluent on cell shortening and Ca2+transient of isolated cardiomyocytes were studied together. The Ca2+transient is primarily based on Ca2+release from the sarcoplasmic reticulum triggered by Ca2+influx through sarcolemmal L-type Ca2+channels (3–6). We therefore conducted a separate group of experiments with whole-cell, voltage-clamp experiments to test the effect of NIS on the L-type Ca2+channel current, ICa. As phosphorylation mechanisms are involved in L-type Ca2+channel regulation (7,8), we conducted a further series of experiments to address the question of whether NIS modulate Ca2+channel activity by changes in tissue concentrations of cyclic adenosine monophosphate (cAMP) or cyclic guanosine monophosphate (cGMP).
Collection of coronary effluent from isolated rat hearts after myocardial ischemia
Isolated rat hearts were perfused in the noncirculating mode at a constant flow (10 ml/min) with a modified Krebs-Henseleit buffer (KHB), as described elsewhere (1,2). The hearts were subjected to 10 min of global stop-flow ischemia followed by reperfusion. The postischemic coronary effluent was then collected immediately at the onset of reperfusion, over a period of 30 s. The coronary effluent from 20 postischemic hearts was pooled, and aliquots of the pooled solution were stored at −70°C before testing. Postischemic pooled coronary effluent was diluted with experimental buffer. In addition, the coronary effluent of five control hearts, with no previous ischemia, was collected and pooled by identical techniques.
Isolation of ventricular myocytes
Adult isolated rat hearts were perfused with oxygenated Ca2+-free KHB (37°C, pH 7.4) containing (in mmol/liter) 110 NaCl, 2.6 KCl, 1.2 KH2PO4, 1.2 MgSO4, 25 HEPES and 11 glucose. After 3 min, the hearts were perfused with KHB containing 30 μmol/liter of Ca2+and collagenase type II (355 U/ml). After 30 min of collagenase digestion, the hearts were minced and incubated for another 15 min in the same solution. The following washing steps increased (Ca2+)oincrementally, in steps of 200, 500 and 1000 μmol/liter. The cells were next layered over a 4% bovine serum albumin (BSA) gradient and centrifuged for 1 min at 19 g; the resulting pellet was then resuspended in the experimental buffer (in mmol/liter: 117 NaCl, 2.8 KCl, 0.6 MgCl2, 1.2 KH2PO4, 1.2 CaCl2, 20 glucose and 10 HEPES (pH 7.3). Typically, about 2 × 106cells per rat heart were obtained, 95% of which showed the typical rod-shaped morphology with no blebs or granulation.
The cells were plated on four-well chamber glass slides (Nunc, Naperville, Illinois), which had been coated with 10 μg/ml of laminin. After an attachment period of 30 min, the buffer was exchanged for a staining solution containing 0.1% dimethyl sulfoxide (DMSO), 0.025% Pluronic F-127, 0.2% BSA and 5 μmol/liter of Fluo 3-AM. The cells were incubated at room temperature for 45 min on an orbital shaker, with oscillation of 40 rpm. After incubation, the loading solution was replaced with fresh buffer, and the incubation continued for 30 min.
Measurement of Ca2+transients and cell shortening
The cells were superfused continuously with experimental buffer at a flow rate of 2 ml/min and kept at room temperature to minimize the cell leakage of fluorescent probes (9). They were field-stimulated by a custom-built stimulator through bipolar silver-chloride electrodes at a frequency of 1 Hz for a duration of 5 ms. After a 2-min equilibration period with 1-Hz stimulation, the coronary effluent was superfused.
Cell shortening and Ca2+-dependent changes in Fluo-3 fluorescence of single-ventricular myocytes were simultaneously measured by use of an Odyssey XL confocal laser scan microscope (Noran Instruments, Middleton, Wisconsin), using an argon ion laser with 6 mW of excitation at 488 nm. Emitted light was long-pass–filtered (>515 nm) and collected at a frame rate of 120 images/s. Data were stored on the hard disk of an Indy workstation (Silicon Graphics, Mountain View, California). At an interval of every 8 ms, Ca2+transients and cell length were determined using a custom-written macro function for Object-Image (10). Under control conditions, the rat ventricular myocytes (n = 36) shortened during stimulation by 7.3 ± 0.4% (mean ± SEM). Changes in Ca2+transients are calculated as peak systolic relative fluorescence units (rfu) minus diastolic rfu without the calibration effort, owing to the uncertain subcellular compartmentalization of the probes (9). At baseline, Fluo-3 fluorescence increased from diastolic 23.1 ± 1.9 rfu to peak systolic 59.1 ± 5.4 rfu.
Rat cardiomyocytes were isolated as described previously (11,12)and superfused with saline solution (37°C) containing (in mmol/liter) 150 NaCl, 5.4 KCl, 2 CaCl2, 1.2 MgCl2, 20 glucose and 5 HEPES, with adjustment by NaOH to pH 7.4. For whole-cell, patch-clamp experiments, borosilicate glass capillaries of 2 mm in diameter were pulled to the tips of ∼2 μm in diameter (1.5 to 2 MΩ resistance). Currents were recorded with a RK300 input amplifier (Biologic, Echiroll, France), filtered at 2 kHz and sampled at 5 kHz. Superimposed K+currents were reduced by Cs+electrode solution, as described elsewhere (12). In the whole-cell mode of the voltage clamp, 100- or 140-ms pulses to 0 mV were applied at 1 Hz. The pulses started from a holding potential of −45 or −50 mV to inactivate the Na+channel currents (3,12). Experiments with seal resistance <2 GΩ were discarded. Membrane capacitance was measured by a train of 10 pulses between −45 and −50 mV. The average membrane capacitance was 121 ± 1.4 picofarad (pF) (58 cells). No data correction took place for membrane leakage. The ICawas expressed by current densities (picoampere [pA]/pF after division by cell capacitance) to remove the variability of ICawith cell size.
Effects of the coronary effluent on myocardial tissue concentrations of cAMP and cGMP: activities of cAMP-dPK, PKC and glycogen phosphorylase
Isolated rat hearts were separately perfused at a constant flow (10 ml/min), which was followed by serial perfusion, as described elsewhere (1,2). At the onset of serial perfusion, the coronary effluent of a donor heart (heart no. 1) was reoxygenated and transported to a serially perfused second heart (heart no. 2). Serial perfusion of the two hearts began: 1) after 30 min of separate perfusion at a constant flow or 2) after 20 min of separate perfusion and 10 subsequent min of global ischemia of heart no. 1.
Heart no. 2 was freeze-clamped 30 s after the onset of serial perfusion for biochemical examination. Tissue levels of cAMP were assayed according to Gilman (13), after purification by column chromatography, as described elsewhere (14). Cyclic GMP contents were analyzed by radioimmunoassay, according to the technique of Harper et al. (15). Cardiac cAMP-dPK activity was measured in the particulate tissue fraction according to a modified method of Murray et al. (16). Cyclic AMP-dPK activity was expressed as the activity ratio of malantide phosphorylation in the absence and presence of 2.8 μmol/liter of cAMP. Activity of PKC was determined in the cytosolic and particulate fractions, as described elsewhere (17,18), using the PKC kit (BIOTRAK, Braunschweig, Germany). The activity of glycogen phosphorylase bto atransformation (expressed as percent total activity) was estimated according to the method of England (19). Protein was measured as described elsewhere (20), with use of ovalbumin as the standard agent.
The investigation conforms to the “Position of the American Heart Association on Research Animal Use,” adopted by the Association in November 1984.
The results are expressed as the mean value ± SEM. For comparison of different groups, Kruskal-Wallis analysis of variance was performed, followed by the one-sided Mann-Whitney Utest at p < 0.05. For comparison of different values in the same group, the one-sided Wilcoxon matched pairs test was performed at p < 0.05. Adjustments of the post hoc test for multiple comparisons (more than two groups) were carried out using a sequentially rejective test procedure (Bonferroni-Holm).
Unless otherwise stated, substances were purchased from Sigma Chemicals (Deisenhofen, Germany). Collagenase type II was purchased from Worthington (Freehold, New Jersey); BayK8644 from Bayer Pharmaceutical (Leverkusen, Germany); and (32P)-γ-ATP, (3H)-cAMP, the (125J)-cGMP assay system and the PKC kit BIOTRAK from Amersham-Buchler. BayK8644 and nifedipine were prepared in DMSO, and aliquots were stored at −20°C. Further dilutions were made in experimental buffer.
Reduction of cell shortening and Ca2+transient
Figure 1shows the original fast-time base recording of cell shortening and (Ca2+)cfluorescence of a field-stimulated cardiomyocyte under control conditions and during superfusion of postischemic coronary effluent, diluted to 1:4. The effluent reduced the Ca2+transient and systolic cell shortening by 25% and 38%, respectively. The effect of the effluent was reversible during superfusion with fresh experimental buffer (washout).
Figure 2shows the dependence of systolic cell shortening, Ca2+transient and peak systolic Fluo-3 fluorescence on the concentration of postischemic coronary effluent. At all dilutions tested, a decrease in Ca2+transient and systolic cell shortening reached steady-state within 1 min and was reversible within 1 min on washout. The effects of the effluent were evaluated once stable steady-state conditions were reached. Increasing concentrations of postischemic coronary effluent depressed cell contraction to a greater extent than they did Ca2+transient. Diastolic Fluo-3 fluorescence did not change significantly. Superfusion of undiluted nonischemic coronary effluent modified neither cell contraction nor Ca2+transient (n = 6; data not shown).
Depression of the Ca2+channel current, ICa
Figure 3Ashows the net membrane current of a rat ventricular myocyte produced by clamp-step depolarization from −45 to 0 mV for 100 ms. The K+outward currents were largely blocked by cell dialysis with Cs+ions, and Na+inward current was inactivated by starting the clamp step from the holding potential of −45 mV. The resulting inward current can accordingly be attributed to L-type ICa(11). In Figure 3A, ICapeaks within 3 ms to −12.4 pA/pF and decays along a double-exponential time course (thin-fitting lines in Fig. 3A). The fast exponential demonstrated a time constant of 4 ms; its amplitude contributed to the peak current by 74%. The slow exponential had a time constant of 20 ms and contributed with an amplitude of 26%. Superfusion of pooled postischemic coronary effluent diluted by 1:4 reduced the peak ICafrom −12.4 to −5.3 pA/pF (Fig. 3A). The effect became steady within ∼1 min. No change took place in the time to peak current. It was possible to fit the decay of the effluent-modified ICato the same time constants of 4 and 20 ms and to a very similar ratio of the amplitudes (78% and 22%, respectively). These last results indicate that the kinetics of the decay were insensitive to the effluent. Accordingly, we conclude that the reduction of peak ICaby the postischemic effluent is unlikely to be caused by changes in the activation or inactivation variables. Nonischemic coronary effluent collected from isolated control hearts did not modify the ICaof the isolated cardiomyocytes (data not shown).
The voltage dependence of peak ICa(Fig. 3B)is typical for currents through Ca2+channels of the L-type. The fit of the corresponding current-voltage (iv) curves (according to the Boltzman formula ) yielded the following gating variables for the control currents: −12 mV for the potential of half-maximal activation, −6.2 mV for the slope factor and 48 mV for the reversal potential. Figure 3Bindicates that the effluent did not change these steady-state gating variables. In the presence of the postischemic coronary effluent diluted to 1:4, these variables yielded: −12 mV, −6.8 mV and 51 mV, respectively. However, it reduced the whole-cell Ca2+conductance from 644 to 289 picosiemens [pS]/pF, a decrease of 55%, as evaluated from the slope of the iv curve.
The effect of the postischemic effluent on the Ca2+inward current was reversible on superfusion with experimental buffer. Figure 4shows the time course for the development of this effect on peak ICaduring superfusion and washout of the postischemic effluent. The postischemic coronary effluent diluted by 1:4 suppressed peak ICawithin 1 min to a new steady-state value. During superfusion with fresh experimental buffer (washout), the effect of the postischemic coronary effluent on peak ICawas reversible within 1 min.
On a statistical basis, the pooled postischemic coronary effluent significantly reduced the peak ICain the cells tested. In cells with complete iv curves, the potential of half-maximal activation, the slope factor and the reversal potential were not significantly changed. The reduction of peak ICareached steady-state within 1 min. The time dependence of the decay in ICadid not change significantly (p > 0.05). The paired comparison indicated a reduction of peak ICaby 54%, from −11.8 ± 0.7 to −5.4 ± 0.4 pA/pF (n = 9, p < 0.05) when the postischemic coronary effluent was diluted to 1:4, and by 80%, from −12.1 ± 0.6 to −2.4 ± 0.3 pA/pF (n = 9, p < 0.05) when the dilution was 1:2.
Maximal Ca2+conductance was significantly reduced, by 56 ± 2.7% (p < 0.05), during exposure to postischemic coronary effluent (dilution 1:4). The slope conductance between −60 and 100 mV was not significantly influenced (31 ± 3 pS/pF before and 25 ± 2 pS/pF after addition of postischemic coronary effluent) (p > 0.05). Likewise, the holding current at −50 mV was not significantly changed by the postischemic coronary effluent (Fig. 4). In only five of nine cells, the inward currents between −60 and −100 mV tended to become more negative (as shown in Fig. 3B). On a statistical basis, this effect was not significant. We attributed the minimal reduction of inward currents to a more complete block of K+currents by Cs+when cell dialysis increased the cytosolic Cs+concentration with time.
Reduction of ICaby the postischemic coronary effluent does not depend on prestimulation by isoproterenol or BayK8644
We investigated whether the reduction of peak ICaby the postischemic coronary effluent (dilution 1:4) depended on the status of the Ca2+channel, as it may be influenced by cAMP-dependent phosphorylation or by Ca2+channel openers. Application of 30 nmol/liter of isoproterenol (n = 6) increased peak ICafrom −12.6 ± 1 to −19.5 ± 1.2 pA/pF, and addition of the postischemic coronary effluent reduced prestimulated peak ICawithin 1 min to −10.5 ± 1.2 pA/pF (i.e., by 54%) (p < 0.05). Similarly, addition of 2 μmol/liter of 8-Br-cAMP (n = 4) increased peak ICafrom −11.5 ± 1.1 to −20.8 ± 1.7 pA/pF, and the postischemic coronary effluent reduced ICato −10.3 ± 1.6 pA/pF. Superfusion of 0.5 μmol/liter of BayK8644 (n = 5) increased peak ICafrom −11.8 ± 0.8 to −43.7 ± 1.8 pA/pF, and the postischemic coronary effluent reduced prestimulated peak ICato −14.5 ± 1.1 pA/pF (p < 0.05). When peak ICawas reduced by superfusion of 0.1 μmol/liter of nifedipine to −4 ± 0.6 pA/pF, addition of the postischemic coronary effluent induced a further reduction to −1.7 ± 0.3 pA/pF (n = 3). In summary, our results indicate that the reducing effect of the postischemic coronary effluent on the amplitude of peak ICadoes not depend on the status of the channel, as it is modified by cAMP-dependent phosphorylation or by dihydropyridines.
Changes in tissue levels of cAMP and cGMP and activities of cAMP-dPK and PKC
To establish whether the observed effects of the postischemic coronary effluent are modulated by an intracellular phosphorylation/dephosphorylation mechanism of the L-type Ca2+channel, we conducted serial perfusions of two isolated hearts. We measured the tissue levels of cAMP and cGMP, as well as the activity of cAMP-dPK in serially perfused heart no. 2. As summarized in Table 1, the basal levels of cAMP and cGMP in heart no. 2 were not significantly influenced by the postischemic coronary effluent of heart no. 1, compared with nonischemic effluent. The tissue level of cAMP in heart no. 2 was elevated by isoproterenol stimulation (5 nmol/liter). We then investigated whether the postischemic coronary effluent of heart no. 1 had an antiadrenergic effect in heart no. 2 through cAMP-mediated signaling, compared with nonischemic effluent. As shown in Table 1, there were no significant differences between the two groups in generation of cAMP or in the cAMP-dPK activity ratio. The myocardial tissue level of cGMP did not increase significantly in the presence of isoproterenol in the two groups.
To preclude the possibility that signaling cascades other than beta-adrenoceptor signaling may be involved in the effects of the postischemic coronary effluent, we also measured the distribution of PKC activity in the cytosolic and particulate fractions of the heart. As shown in Table 1, PKC activity in heart no. 2 was not influenced by the postischemic coronary effluent (Table 1).
Activity of glycogen phosphorylase
We studied activation of glycogen phosphorylase because this enzyme is regulated by phosphorylase kinase, which in turn is activated by either cAMP-dPK or an increase in (Ca2+)c(21–23). The glycogen phosphorylase bto atransformation, measured in the presence of AMP, increased in heart no. 2 from 7.5 ± 1.3% (controls, n = 4) to 49.9 ± 3.0% during intracoronary infusion of 5 nmol/liter of isoproterenol (n = 5) (p < 0.05). When serial perfusion was performed after 10 min of global ischemia of the first heart (n = 5), the activation of glycogen phosphorylase during exposure to isoproterenol (5 nmol/liter) was reduced to 9.9 ± 2.5% (p < 0.05 vs. values during infusion of isoproterenol without preceding ischemia of heart no. 1).
Negative inotropic substances released from isolated postischemic hearts
The present study investigated the mechanism by which NIS cause negative inotropic effects in the isolated, field-stimulated ventricular myocytes of rats. Our results indicate that the postischemic coronary effluent contains NIS that decrease contractility (cell shortening) by depression of Ca2+transient, rather than by Ca2+desensitization. Furthermore, our results reveal that the substance(s) reduce Ca2+influx through L-type Ca2+channels. Accordingly, we postulate that this Ca2+channel blockage is the initial event in the signaling cascade.
The chemical structure of NIS has remained unknown until now. As evidenced by data that we have recently reported, the substance(s) involved here are stable, heat-resistant molecules that, most likely, are not proteins (1). We have excluded the possibility that ionic imbalances, lactic acid or pH shifts in the pooled coronary effluent of the postischemic hearts contributed to its effects on contraction and Ca2+transient of the isolated cardiomyocytes. The substances(s) unidentified thus far are small molecules: the pooled effluent was dialyzed against water (1:1000) by means of a diaphragm with a pore size of 0.5 kd. The remaining dialyzed solution—containing molecules >0.5 kd—once lyophilized and dissolved in an appropriate experimental buffer, did not influence cell shortening or Ca2+fluorescence of isolated cardiomyocytes. In contrast, when the postischemic coronary effluent was filtered through a 0.5-kd filter (YCO5 Amicon membrane, Millipore, Eschborn, Germany), its effects on the calcium transients and cell shortening were comparable to those of untreated postischemic coronary effluent (data not shown).
Endogenous negative inotropic factors have been identified by other investigators using different experimental procedures. Splanchnic hypoperfusion induces production of a myocardial depressant factor in the splanchnic region (24). Myocardial depressant factor is a low molecular weight peptide that decreases myocardial contractile force. In addition, a cardiodepressant factor has been isolated by column chromatography from the plasma of dogs after hypovolemic-traumatic shock (25). Further purification yielded a hydrophilic peptide. Finally, several studies have shown that endocardial and coronary vascular endothelium modulate myocardial function (26–28). Endothelial cells have been shown to release unidentified mediators that upregulate or downregulate myocardial contractility (28). A recent study has also disclosed the presence—in the superfusate of cultivated vascular and endocardial endothelial cells—of an unidentified low molecular weight factor that reduces myocardial contraction, predominantly by reducing the myofilament response to calcium (29). In contrast, we have recently provided evidence that the negative inotropic mediators released from postischemic hearts are not derived from endothelial cells (2).
Reduction of the l-type Ca2+current
The voltage-clamp analysis described in the present study indicates that NIS reduce the L-type Ca2+current ICaand intracellular systolic Ca2+concentration, with the consequence of negative inotropy. In addition, a reduced intracellular Ca2+concentration causes metabolic effects, such as a decrease in activation of glycogen phosphorylase. In contrast to our data, Yang et al. (30)recently reported that the coronary effluent of isolated hypoxic rat hearts contains unidentified substances that depress contraction of isolated ventricular myocytes, with only a minor reduction in intracellular Ca2+transient. Furthermore, hypoxic endothelial cells produce diffusible factors that inhibit the myosin cross-bridge function (31). The apparent discrepancies between these reports and our data are most likely due to different experimental protocols. The cited reports describe superfusate of hypoxic endothelial cells or coronary effluent from hypoxic hearts; in our study, the isolated hearts were subjected to 10 min of stop-flow ischemia, followed by reperfusion. We therefore hypothesize that the substances, unidentified until now, that are released during reperfusion and after ischemia are different from those released during hypoxia.
The L-type Ca2+channel activity is known to be upregulated by cAMP and downregulated by cGMP-dependent phosphorylation (7,8). The present results indicate that the suppressing effect of NIS on ICais not modified by prestimulating the Ca2+channel activity through cAMP-dependent phosphorylation (with either isoproterenol or 8-Br-cAMP). Hence, modulation of the cAMP-dPK pathway by NIS seems unlikely. This conclusion is in accordance with our findings that the postischemic coronary effluent did not modify cAMP tissue concentration or cAMP-dPK activity in serially perfused isolated hearts. Similarly, signaling through cGMP or PKC is unlikely, because cGMP tissue levels and PKC activity were not modulated by the effluent. Because the postischemic coronary effluent blocked the ICaindependently of the aforementioned mechanisms that initiate Ca2+channel activation, it appears unlikely that dephosphorylation of subunits of the L-type Ca2+channel is involved. We therefore postulate that NIS interact with the Ca2+channel more directly (i.e., by plugging the pore or by binding to the channel protein) (32). Binding of NIS to the dihydropyridine binding site of the channel protein is unlikely, because suppression of ICaby the effluent was not modified when the cells were pretreated with the dihydropyridines BayK8644 or nifedipine. The mode of interaction between NIS and the Ca2+channel remains to be elucidated by forthcoming single-channel studies.
Potential pathophysiologic role of the negative inotropic substances
In an attempt toward teleologic interpretation of our findings, it may be considered whether the observed reduction in Ca2+influx, Ca2+transient and cell shortening could serve as an endogenous protection mechanism. In terms of cardiac energy balance, the decrease in Ca2+transient and in contractility may reflect a salutary effect that allows enhanced metabolic recovery of the cardiomyocytes before full contractility is restored. Furthermore, the question arises whether the effects of NIS interfere with the phenomenon of “stunning” (33,34). The potential underlying mechanisms of this postischemic contractile abnormality are the release of free radicals, cellular Ca2+overload and decreased sensitivity of the contractile filaments to calcium (reviewed in ). Overload of Ca2+, in concert with oxygen free radicals, may induce activation of Ca2+-dependent protease activity and consequent troponin I proteolysis (36). Reduction in the L-type Ca2+current by NIS may therefore represent a compensatory mechanism of the myocardium, in the form of counteracting Ca2+overload during reperfusion. However, the apparent rapid reversibility of the negative inotropic effect during washout may suggest that NIS play only a minor role in changes of contractility of the postischemic myocardium. In contrast, it may be possible that NIS are continuously released into the microenvironment and retained by the postischemic myocardial tissue. With its use of spillover from the mediators into the coronary effluent, our experimental study may well have failed to accurately ascertain the tissue concentrations of NIS in the postischemic ventricle. These concentrations, indeed, may be much higher (and may decrease over a greater span of time) than those in the coronary effluent.
The present data confirm the release of NIS during reperfusion of isolated hearts after ischemia. Our findings indicate that these substance(s) or these mediator(s) of yet unknown chemical structure reduce the L-type Ca2+current. Presumably, the Ca2+channel blockage is the first step in a chain of cellular events leading to suppressed cytosolic Ca2+transient and, in turn, to reduced contractions. Because the putative L-type Ca2+channel antagonistic effect of NIS was not mediated by changes in cAMP or cGMP levels, nor by changes in cAMP-dPK and PKC activities, we postulate that there is a phosphorylation-independent interaction between NIS and the Ca2+channel.
We thank Gregory H. Joss (Macquarie University, Sydney, Australia) for his excellent assistance in developing the macrofunction for Object-Image. Object-Image is a spin-off of the public domain National Institutes of Health (NIH, Bethesda, Maryland) image program. We also appreciate the technical assistance of Yihua Wang and Angelika Westphal.
☆ This study was supported by the Deutsche Forschungsgemeinschaft (DFG) (Fe 250/3-1, 3-2).
- bovine serum albumin
- cyclic adenosine monophosphate
- cAMP-d PK
- cAMP-dependent protein kinase
- cyclic guanosine monophosphate
- dimethyl sulfoxide
- Krebs-Henseleit buffer
- negative inotropic substances
- protein kinase C
- relative fluorescence units
- sarcoplasmic reticulum
- Received December 6, 1999.
- Revision received August 22, 2000.
- Accepted October 2, 2000.
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