[Frontiers in Bioscience E2, 614-626, January 1, 2010]
Nitroxyl enhances myocyte Ca2+ transients by exclusively targeting SR Ca2+-cycling
Mark J Kohr1, Nina Kaludercic2, Carlo G Tocchetti2, Wei Dong Gao3, David A Kass2, Paul ML Janssen1, Nazareno Paolocci2,4, Mark T Ziolo1
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TABLE OF CONTENTS
1. ABSTRACT
Nitroxyl (HNO), the 1-electron reduction product of nitric oxide, improves myocardial contraction in normal and failing hearts. Here we test whether the HNO donor Angeli's salt (AS) will change myocyte action potential (AP) waveform by altering the L-type Ca2+ current (ICa) and contrast the contractile effects of HNO with that of the hydroxyl radical (.OH) and nitrite (NO2-), two potential breakdown products of AS. We confirmed the positive effect of AS/HNO on basal cardiomyocyte function, as opposed to the detrimental effect of .OH and the negligible effect of NO2-. Upon examination of the myocyte AP, we observed no change in resting membrane potential or AP duration to 20% repolarization with AS/HNO, whereas AP duration to 90% repolarization was slightly prolonged. However, perfusion with AS/HNO did not elicit a change in basal ICa, but did hasten ICa inactivation. Upon further examination of the SR, the AS/HNO-induced increase in cardiomyocyte Ca2+ transients was abolished with inhibition of SR Ca2+-cycling. Therefore, the HNO-induced increase in Ca2+ transients results exclusively from changes in SR Ca2+-cycling, and not from ICa.
2. INTRODUCTION
The process of excitation-contraction coupling underlies cardiomyocyte contraction. In this process, an influx of Ca2+ through the L-type Ca2+ current (ICa) provides the trigger for the release of additional Ca2+ from the sarcoplasmic reticulum (SR) via the ryanodine receptors (RyR), thus inducing myocyte contraction (1). ICa can also serve to load the SR with Ca2+ and directly activate myofilament contraction. In order for cardiomyocyte relaxation to occur, the Ca2+ available for contraction must either be re-sequestered into the SR via the SR Ca2+ ATPase (SERCA2a) or extruded out of the myocyte through the Na+/Ca2+ exchanger.
We recently reported that nitroxyl (HNO), the one-electron reduction product of nitric oxide (NO), improves myocardial contraction in both normal (2) and failing hearts (3). This functional improvement was elicited upon administration of the HNO donor Angeli's salt (AS), and was demonstrated to occur independent of {beta}-adrenergic receptor ({beta}-AR) stimulation, and distinct from the effects NO and cGMP (3-5). The effects of HNO are due, in part, to the enhancement of SR Ca2+-cycling (4), and likely occur through the targeting of critical thiol groups (6, 7). More specifically, HNO increases ATP-dependent SR Ca2+ uptake (4), and the activity of RyR (4, 8). HNO also works to enhance myocardial contraction by increasing the sensitivity of the myofilaments to Ca2+, thus allowing for increased force development without a concomitant increase in actomyosin ATPase activity (5). The net result of HNO administration is an improvement in myocardial contraction, resulting from an increase in systolic Ca2+ transients, which serve to drive a more Ca2+-sensitive contractile apparatus. However, previous reports have not examined the role of extracellular Ca2+ in the positive inotropic action of HNO. Further, under certain extreme biochemical conditions, the breakdown of AS may lead to the formation the hydroxyl radical (.OH) (9, 10), which is a well-known negative modulator of myocardial function (11-13). Another potential breakdown product of AS includes nitrite (NO2-). Therefore, it is important to establish that the effect of AS on myocardial contraction occurs through the formation of HNO, and not via hydroxyl radical or nitrite production.
Here we investigate the role of extracellular Ca2+ in the positive inotropic action of HNO by examining action potential waveform and ICa. We also contrast the contractile effects of HNO with that of the hydroxyl radical, a possible breakdown product of AS, and nitrite, a reactive nitrogen species co-released during the decomposition of AS. Additionally, we provide further evidence that the effects of HNO are independent from {beta}-AR stimulation and are distinct from those effects typically observed with NO, cGMP, and other reactive oxygen and nitrogen species (14-17).
3. METHODS AND MATERIALS
3.1. Cardiomyocyte Isolation
Ventricular cardiomyocytes were isolated from mouse (C57BL/6, male) and rat (LBN-F1, male) hearts, as previously described (16). Briefly, hearts were excised from animals anesthetized via intraperitoneal injection of sodium pentobarbital (50 mg kg-1). Using a Langendorff apparatus, hearts were perfused with nominally Ca2+-free Joklik Modified MEM (Sigma, St. Louis, MO) for 4 minutes at 37oC. Perfusion was then switched to the same solution, but now containing Liberase Blendzyme 4 (Roche Diagnostics, Indianapolis, IN). Hearts were digested until the drip rate reached one per second. Following digestion, the heart was taken down and the tissue minced, triturated, and filtered. The cell suspension was then rinsed and stored in Joklik Modified MEM containing 200 {micro}mol/L Ca2+. Cells were used within 6 hours of isolation. This investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996) and was approved by the Institutional Laboratory Animal Care and Use Committee at The Ohio State University.
3.2. Simultaneous Measurement of Systolic Ca2+ Transients and Shortening
Systolic Ca2+ transients and shortening were measured in isolated myocytes as previously described (16). Briefly, isolated myocytes were loaded at 22oC with 10 {micro}mol/L Fluo-4 AM (Molecular Probes, Eugene, OR) for 30 minutes. Excess dye was removed by washout with 200 {micro}mol/L Ca2+ normal Tyrode solution. Myocytes were then de-esterfied for an additional 30 minutes. Following loading, cells were stimulated at 1 Hz via platinum electrodes connected to a Grass Telefactor S48 stimulator (West Warwick, RI). Fluo-4 was excited with 480�20 nm light, and the fluorescent emission of a single cell was collected at 530�25 nm using an epifluorescence system (Cairn Research Limited, Faversham, UK). The illumination field was restricted to collect the emission of a single cell. Data were expressed as {delta}F/F0, where F was the fluorescence intensity and F0 was the intensity at rest. For experiments utilizing sodium nitrite, 10 {micro}mol/L Indo-1 AM (Molecular Probes) was utilized. Indo-1 was excited with 365�10 nm light, and the fluorescent emission of a single cell was collected at 405�30 nm and 485�25 nm. Data were expressed as {delta}Ratio405/485. Simultaneous measurement of shortening was performed using an edge detection system (Crescent Electronics, Sandy, UT). Cardiomyocyte shortening amplitude was normalized to resting cell length (%RCL). For experiments utilizing sodium nitrite, sarcomere shortening was measured using the IonOptix MyoCam (Milton, MA). Sarcomere shortening amplitude was expressed as the percent of fractional shortening (%FS). All measurements were recorded at room temperature (22oC) except where noted.
3.3. Hydroxyl Radical Generation
Hydroxyl radicals were generated via Fenton chemistry using the H2O2+Fe2+-nitrilotriaceticacetate (Fe2+-NTA) system, as previously described (13). In this system, the concentration of Fe2+-NTA within the perfusion solution was 10 {micro}mol/L; H2O2 was infused into the perfusion solution through a separate line to a final concentration of 3.75 {micro}mol/L. This allows hydroxyl radical formation to occur as closely to the preparation as possible. With the use of this system, the concentration of hydroxyl radicals generated in the perfusion solution is approximately 2 {micro}mol/L (12, 13). Systolic Ca2+ transients and shortening were simultaneously recorded as described above at a frequency of 1 Hz, with the exception that cells were loaded with 10 {micro}mol/L Indo-1 AM (Molecular Probes) instead of Fluo-4 AM, as hydroxyl radical exposure is known to induce bleaching of the Ca2+ indicator. Therefore, the ratiometric properties of Indo-1 AM will serve to counteract the effect of hydroxyl radical exposure on the Ca2+ indicator. Indo-1 was excited with 365�10 nm light, and the fluorescent emission of a single cell was collected at 405�30 nm and 485�25 nm. Data were expressed as Ratio405/485 and {delta}Ratio405/485. All measurements were recorded at room temperature (22oC).
3.4. Action Potential Measurement
Action potentials were recorded using the whole cell ruptured patch current clamp technique and an Axopatch-200B amplifier with pCLAMP 9.0 software (Axon Instruments), as previously described (15). Electrodes (borosilicate glass tubing) with a resistance of 8-12 M{ohm} were filled with (in mmol/L): K-aspartate (130), KCl (10), NaCl (8), HEPES (5), and MgATP (5); pH 7.2 adjusted with KOH. All measurements were recorded at room temperature (22oC).
3.5. L-Type Ca2+ Current Measurement
L-type Ca2+ current was measured using the whole cell ruptured patch voltage clamp technique and an Axopatch-200B amplifier with pCLAMP 9.0 software (Axon Instruments), as previously described (15). Electrodes (borosilicate glass tubing) with a resistance of 1.5-3 M{ohm}, were filled with (in mmol/L): CsCl (120), MgCl2 (6), EGTA (10), HEPES (10), and MgATP (2); pH 7.2 adjusted with CsOH. The bath solution consisted of (in mmol/L): NaCl (120), CsCl (4), MgCl2 (1), CaCl2 (1), glucose (10), HEPES (5), L-arginine (1); pH 7.4 adjusted with CsOH or HCl. L-type Ca2+ current was elicited by 200 ms pulses to 0 mV from a holding potential of -80 mV (following a pre-pulse to -40 mV) at a frequency of 0.2 Hz. This procedure isolates ICa by inactivation of the Na+ current with the pre-pulse; replacement of K+ with Cs+ eliminates the K+ current. ICa inactivation (tau, time constant for ICa decline) was determined using a single exponential fitted to the decay phase of the current. All measurements were recorded at room temperature (22oC).
3.6. SR Inhibition
For SR inhibition, cardiomyocytes were pretreated with 1 {micro}mol/L thapsigargin for 15 minutes in order to completely block SR function. SR inhibition was verified by the absence of 10 mmol/L caffeine-induced Ca2+ transients. To enhance the Ca2+ influx-induced Ca2+ transients, the (Ca2+) in the perfusion solution was increased to 20 mmol/L (18). Systolic Ca2+ transients were recorded as described above at frequency of 0.5 Hz. All measurements were recorded at room temperature (22oC).
3.7. Solutions and Drugs
Normal Tyrode control solution consisted of (in mmol/L): NaCl (140), KCl (4), MgCl2 (1), CaCl2 (1), Glucose (10), and HEPES (5); pH = 7.4 adjusted with NaOH/HCl. Angeli's salt (AS; Calbiochem, La Jolla, Ca) was dissolved in 10 mmol/L NaOH and used as an HNO donor. Sodium nitrite (NaNO2, Sigma, St. Louis, MO) was used as a source of nitrite (NO2-). Isoproterenol (ISO; Sigma) was used as a non-selective {beta}-AR agonist. Thapsigargin (Sigma) was dissolved in dimethyl sulfoxide (DMSO, Sigma) and used as a specific inhibitor of SERCA activity. All solutions were made fresh on the day of experimentation.
3.8. Statistics
Data are presented as the mean�S.E.M. Statistical significance (p<0.05) was determined between groups using an ANOVA (followed by Newman-Keuls test) for multiple groups or a paired Student's t-test for two groups.
4. RESULTS
4.1. Effect of AS on cardiomyocyte function
We first confirmed the positive effect of Angeli's salt (AS, HNO donor) on basal function in isolated murine cardiomyocytes. AS (500 {micro}mol/L) significantly increased basal systolic Ca2+ transients and myocyte shortening (Systolic Ca2+ Transient: 0.7�0.1 vs. 1.1�0.1 {delta}F/F0, Shortening: 4.0�0.5% vs. 10.5�2.5% RCL, p<0.05 vs. Control). This effect is shown in the representative traces (Figure 1A) and in the summary data (Figure 1B). AS/HNO also increased the time to peak of the systolic Ca2+ transient, while accelerating the decay of the systolic Ca2+ transient (data not shown). Similar effects were observed with AS/HNO upon repetition of the same experimental protocol at physiological temperature (37oC) (Systolic Ca2+ Transient: 47�10% vs. 55�10% change from Control, Shortening: 219�72% vs. 162�47% change from Control, p = NS). This effect is shown in the summary data expressed as a percent of control (Figure 1C). In a previous publication, we demonstrated that AS did not alter diastolic Ca2+ or diastolic cell length (4). Thus, AS/HNO induces positive inotropic effects in isolated cardiomyocytes.
4.2. Effect of hydroxyl radical and nitrite exposure on cardiomyocyte function
The possibility exists for AS to generate the hydroxyl radical (.OH) under certain extreme biochemical conditions (9, 10). Therefore, we examined the effect of acute hydroxyl radical exposure on isolated rat cardiomyocyte function. Acute hydroxyl radical exposure slightly increased systolic Ca2+ transients (data not shown), and induced a significant increase in diastolic Ca2+ that was accompanied by a significant decrease in diastolic cell length and myocyte shortening (Diastolic Ca2+: 2.4�0.6 vs. 2.8�0.7 Ratio405/485, Diastolic cell length: 110�4.9 vs. 105�5.2 {micro}m, Shortening: 7.2�0.9% vs. 5.0�0.6% RCL, p<0.05 vs. Control). This effect is shown in the summary data expressed as a percent of control (Figure 2).
Additionally, nitrite (NO2-) is another breakdown product of AS. In our previous publication, we determined that AS yielded approximately 25% nitrite after 15 minutes of continuous infusion (4). Therefore, we examined the effect of 125 {micro}mol/L NaNO2 on isolated murine cardiomyocyte function. Exposure to nitrite, however, yielded no appreciable effect on basal systolic Ca2+ transients or sarcomere shortening (Systolic Ca2+: 0.7�0.1 vs. 0.6�0.1 {delta}Ratio405/485, Shortening: 2.6�0.3% vs. 2.8�0.4% FS, p = NS). This effect is shown in the summary data (Figure 3). Therefore, the acute effects of hydroxyl radical and nitrite exposure are distinct from those effects observed with AS/HNO.
4.3. AS and action potential waveform
We next examined the effect of AS/HNO on murine myocyte AP waveform. AS (500 {micro}mol/L) did not alter resting membrane potential (RMP: -79�3 vs. -78�4 mV) or the AP duration (APD) to 20% repolarization (APD20: 1.0�0.2 vs. 1.3�0.3 ms), but did induce a slight prolongation of the APD to 90% repolarization (APD90: 59�8 vs. 77�13 ms, p<0.05 vs. Control). This effect is shown in the representative traces (Figure 4A) and in the summary data (Figure 4B-C). Additionally, we did not observe any delayed afterdepolarizations (DADs) with the prolongation of the APD90 (data not shown).
4.4. AS does not alter ICa
We subsequently investigated the effect of AS/HNO on murine myocyte ICa. Surprisingly, we observed no change in basal ICa with either 100 {micro}mol/L or 500 {micro}mol/L AS (100 {micro}mol/L: 2.8�0.5 vs. 2.7�0.5 -pA/pF; 500 {micro}mol/L: 2.5�0.5 vs. 2.5�0.5 -pA/pF). This lack of effect is shown in the representative traces (Figure 5A), the representative time plot (Figure 5B), and in the summary data (Figure 5C). Further, AS/HNO had no effect on the current-voltage relationship for ICa (Figure 5D). However, AS/HNO did induce significantly faster inactivation of ICa, measured as the time constant for ICa decline (Control: 42�7 vs. HNO: 36�6 ms, p<0.05 vs. Control). This effect can be seen in the normalized ICa traces (Figure 5E) and in the summary data (Figure 5F).
AS/HNO also failed to elicit a change in ICa following pre-stimulation with 0.01 {micro}mol/L ISO (Control: 2.0�0.8 vs. ISO: 4.2�0.9* vs. ISO+AS: 3.9�0.5* -pA/pF, *p<0.05 vs. Control). This lack of effect can be seen in the representative traces (Figure 6A) and in the summary data (Figure 6B).
4.5. AS has no effect during SR inhibition
Since we observed no change in ICa, we further investigated the role of SR Ca2+-cycling in the effects of AS/HNO. Upon complete inhibition of SR Ca2+-cycling, systolic Ca2+ transients should be derived entirely from extracellular Ca2+ influx, mainly via ICa. Therefore, to further examine the role of SR Ca2+-cycling, we examined the effect of AS on systolic Ca2+ transients in murine cardiomyocytes during inhibition of SR Ca2+-cycling with thapsigargin. SR inhibition was verified by the absence of 10 mmol/L caffeine-induced systolic Ca2+ transients (data not shown). Further evidence of SR inhibition can be seen in the small size of the systolic Ca2+ transient, the slowed decline of the systolic Ca2+ transient, and the inability of ISO to hasten the systolic Ca2+ transient decline (Figure 7A). To enhance the Ca2+ influx-induced Ca2+ transients, the (Ca2+) in the perfusion solution was increased to 20 mmol/L (18). SR inhibition completely abolished the positive effect of 500 {micro}mol/L AS on systolic Ca2+ transient amplitude (0�4% change from control), as seen in the representative traces (Figure 7A) and in the summary data (Figure 7B). Conversely, 0.01 {micro}mol/L ISO still induced a significant increase in systolic Ca2+ transient amplitude even with SR inhibition (46�16% change from control, p<0.05 vs. Control), as seen in the representative traces (Figure 7A) and in the summary data expressed as a percent of control (Figure 7B). Vehicle treatment alone (DMSO) did not alter basal myocyte contraction or the response to 0.01 {micro}mol/L ISO (data not shown). Thus, HNO works exclusively at the level of the SR to increase systolic Ca2+ transients in isolated cardiomyocytes (Figure 7C), and not from the recruitment of extracellular Ca2+.
5. DISCUSSION
Nitroxyl (HNO) was previously demonstrated to enhance myocardial contraction partly through effects on SR Ca2+-cycling (2-4). However, our previous studies did not examine the role of extracellular Ca2+ in the positive inotropic action of HNO. In our current study, we demonstrate for the first time that HNO induces a slight change in AP waveform, but does not recruit additional extracellular Ca2+, namely ICa, in order to increase systolic Ca2+. Additionally, we did not observe DADs with the HNO-induced prolongation of the APD. Thus, sarcolemmal Ca2+ does not contribute to the HNO-induced increase in systolic Ca2+ transients. However, SR inhibition completely abolished the positive effect of HNO on systolic Ca2+ transients. Moreover, the current study is the first to contrast the positive inotropic action of HNO with that of the hydroxyl radical and nitrite. The generation of the former occurs from the breakdown of AS during extreme biochemical conditions, whereas the latter is normally co-released with HNO by AS. Therefore, the AS-induced increase in cardiomyocyte systolic Ca2+ occurs from the direct enhancement of SR Ca2+-cycling by HNO.
5.1. HNO enhances cardiomyocyte contraction
We confirmed the positive effect of HNO in murine cardiomyocytes, and noted a significant increase in systolic Ca2+ transients and cell shortening (Figure 1). HNO also increased the time to peak of the systolic Ca2+ transient, while accelerating the decay of the systolic Ca2+ transient. These results are consistent with our previous study (4), which also demonstrated that HNO was without effect on diastolic Ca2+ or diastolic cell length. Further, the positive inotropic action of HNO was unaffected by temperature, as we observed similar increases in systolic Ca2+ transients and cell shortening at physiological temperature (37oC), compared to the effects observed at room temperature (22oC) (Figure 1). This result is consistent with a previous publication, where we examined the effect of HNO in vivo and observed a large increase in myocardial contractility, as well as enhanced myocardial relaxation (3).
5.2. Hydroxyl radical exposure is detrimental to cardiomyocyte function
AS is considered to be an HNO donor, but the chemistry of AS is rather complex (19). Studies have demonstrated hydroxyl radical production using very high concentrations of AS (>1 mmol/L) under conditions of very low pH (pH 4-6) (9, 10). Although hydroxyl radical production is minimal under our experimental conditions at pH 7.4, hydroxyl radical production may occur in certain intracellular compartments of low pH (i.e., mitochondria). Therefore, we conducted additional experiments in order to demonstrate that the contractile effects induced by AS were distinct from those observed with hydroxyl radical exposure. Hydroxyl radical exposure proved to be extremely detrimental to cardiomyocyte function by increasing diastolic Ca2+, and decreasing diastolic cell length and myocyte shortening (Figure 2). These results are consistent with previous findings (11-13), and are in contrast to the effects of AS. Treatment with AS caused a large increase in systolic Ca2+ transients and myocyte shortening (Figure 1), without a change in diastolic Ca2+ or diastolic cell length (4). The effects of hydroxyl radical exposure are indicative of Ca2+-overload, as evidenced by the changes in diastolic Ca2+ and length. However, AS does not appear to lead to Ca2+-overload, as we observed no change in diastolic Ca2+ or length. This indicates that the effects of AS are distinct from those observed with the hydroxyl radical and likely other reactive oxygen species, and do not result from a generalized thiol oxidation. Thus, hydroxyl radical generation via AS is not likely to be a confounding factor in our experimental design.
5.3. Nitrite does not alter cardiomyocyte function
Nitrite is co-released with HNO during the decomposition of AS at physiological pH and temperature. However, nitrite had no effect on systolic Ca2+ transients or sarcomere shortening (Figure 3). These effects are consistent with our previous study (4), and are in direct contrast to those effects observed with HNO (Figure 1). Therefore, nitrite production via AS is not likely to underlie the positive inotropic action of AS/HNO.
5.4. HNO alters action potential waveform
Although HNO enhanced cardiomyocyte contraction, HNO did not induce a change in RMP or the APD20, but did slightly prolong the APD90 (Figure 4). Importantly, we did not observe DADs with the HNO-induced prolongation of the APD90. These effects on AP waveform are distinct from the effects of NO signaling, which has been shown to reduce the APD (15). Studies have shown that prolongation of the APD90 can result from changes in ICa (15, 20).
5.5. HNO does not alter ICa
Despite the HNO-induced increase in the APD90, HNO had no effect on basal ICa (Figure 5). This lack of effect on ICa is surprising given that HNO greatly enhanced systolic Ca2+ transients and prolonged the APD90. HNO also had no effect on the current-voltage relationship for ICa (Figure 5). However, faster inactivation of ICa was observed with HNO (Figure 5). These data are consistent with the HNO-induced increase in SR Ca2+-cycling, which may accelerate the Ca2+-dependent inactivation of ICa (21, 22). Since peak ICa was not altered by HNO, the prolongation of the action potential duration by HNO likely results from the targeting of repolarizing K+ channels, namely IK,slow1, IK,slow2, and Iss (23, 24), and warrants further study.
Additionally, HNO was without effect on {beta}-AR-stimulated ICa (Figure 6). Thus, the effects of HNO on ICa appear to be very different from the effects of exogenous and endogenous NO signaling, which has been shown to decrease {beta}-AR-stimulated ICa (15, 25). Further, the effects of {beta}-AR signaling also differ from HNO, as {beta}-AR stimulation has been demonstrated to either increase or decrease ICa depending on which {beta}-AR subtype (i.e., {beta}1-AR, {beta}2-AR, {beta}3-AR) is activated (15, 26, 27).
5.6. SR inhibition abolishes the effects of HNO
Inhibition of SR function completely abolished the positive effects of HNO on systolic Ca2+ transients (Figure 7), and indicates that SR Ca2+-cycling is the sole source for the HNO-induced enhancement of systolic Ca2+ transients. Since {beta}-AR stimulation increases ICa (Figure 6), ISO was used as a positive control in order to verify that myocytes with complete SR inhibition could still exhibit an increase in systolic Ca2+ transients (Figure 7). Additionally, these results provide further verification that ICa and other extracellular Ca2+ influx do not play a role in the effects of HNO. This is particularly important given the pathologic nature of enhanced extracellular Ca2+ influx (28). The enhanced inactivation of ICa with HNO is also consistent with an increase in SR Ca2+-cycling.
5.7. Limitations
A potential limitation of the current study involves the use of rodent cardiomyocytes. More specifically, the rodent AP tends to be more triangular compared to larger mammals (rabbit, human, etc.), and has a very short plateau phase (29). This brief plateau phase can be attributed to the presence of repolarizing currents that are markedly different from larger mammals. Rodent cardiomyocytes are also less reliant upon extracellular Ca2+ influx via ICa during the process of excitation-contraction coupling, but are instead more reliant upon Ca2+ derived from the SR. Therefore, future studies will address the effects of HNO on isolated cardiomyocyte function in larger mammal species.
Another potential limitation of the current study results from indicator loss. Angeli's salt slightly decreased the fluorescent emission of Fluo-4 in cuvette studies conducted over the same time course as our functional experiments (<10%). The possibility also exists for hydroxyl radical exposure to decrease the fluorescent emission of Indo-1. Indeed, a previous study found that hydroxyl radical exposure reduced the fluorescent emission of Indo-1 at both wavelengths (405, 485 nm), without altering the Ca2+ sensitivity of the indicator (11). Further, these changes were not wavelength dependent. Therefore, the ratiometric properties of Indo-1 should overcome the loss in fluorescence intensity due to hydroxyl radical exposure. In addition, our cell shortening measurements during hydroxyl radical exposure were consistent with the changes in Ca2+ observed with Indo-1 (i.e., increased diastolic Ca2+, decreased diastolic cell length). We also observed an increase in diastolic force in trabecular preparations following hydroxyl radical exposure in a prior study (13). This increase in diastolic force is consistent with an increase in diastolic Ca2+.
5.8. Conclusions
In heart failure the process of excitation-contraction coupling becomes dysfunctional due to a reduction in SR Ca2+-cycling (30, 31). Since SR Ca2+-cycling is diminished, cardiomyocyte contraction is also reduced. Classical pharmacological agents used in the treatment of heart failure ({beta}-AR agonists, phosphodiesterase inhibitors, etc.) have been shown to be detrimental over the long term due to adverse remodeling, increased arrhythmogenesis and increased apoptosis (32-35). These effects are likely due, in part, to the detrimental effects of enhanced extracellular Ca2+ influx via ICa. Prolonged activation of ICa results in adverse remodeling and has been shown to lead to pathological cardiac hypertrophy through activation of the calcineurin/NFAT signaling pathway (36, 37). Additionally, ICa can increase the generation of arrhythmias and has been demonstrated to increase the incidence of both early afterdepolarizations (EADs) and DADs (15, 38-40). We did not observe DADs at the myocyte level with AS/HNO, and HNO administration did not trigger arrhythmias in vivo, even with concomitant {beta}-AR stimulation (3). Further, activation of ICa can induce apoptotic cell death in the myocardium (28). However, the pool of Ca2+ that induces cardiomyocyte contraction, such as that enhanced by HNO, appears distinct from the Ca2+ pool that contributes to pathological signaling (41). The pool of Ca2+ which contributes to pathological signaling seems to be composed mainly of Ca2+ derived from enhanced extracellular influx. The differential regulation of contraction and pathological signaling by Ca2+ is likely due to the presence of specialized subcellular Ca2+ signaling domains in the cardiomyocyte. Thus, these data support the potential use of HNO donors as therapeutics for heart failure, as HNO works independent of ICa.
In conclusion, the HNO-induced enhancement of systolic Ca2+ transients in cardiomyocytes is independent and distinct from the non-specific effects of the hydroxyl radical and nitrite, and stems exclusively from an increase in SR Ca2+ release and re-uptake without the recruitment of extracellular Ca2+ via ICa. Thus, the positive inotropic action of HNO results from the enhancement of systolic Ca2+, exclusive to SR Ca2+-cycling, and increased myofilament Ca2+ sensitization. Interestingly, these changes are likely mediated through the targeting of specific cysteine residues of critical excitation-contraction coupling proteins. More specifically, we previously demonstrated that the effects of nitroxyl were due, in part, to the formation of a disulfide bond between two cysteine residues of phospholamban (6). This modification served to alter the confirmation of phospholamban, thus relieving sarcoplasmic reticulum Ca2+-ATPase inhibition. Another study demonstrated that nitroxyl increased ryanodine receptor activity via disulfide bond formation (8), while a third report demonstrated that nitroxyl increased sarcoplasmic reticulum Ca2+-ATPase activity through the direct glutathiolation of cysteine 674 (7). Although it is possible for HNO to increase cardiomyocyte contraction by targeting cysteine residues found in other excitation-contraction coupling proteins, the present study provides definitive evidence that extracellular Ca2+ is not required for the positive inotropic action of HNO.
6. ACKNOLEDGEMENTS
Supported by the American Heart Association (Pre-doctoral Fellowship 0715159B, MJK; Post-doctoral Fellowship 0825491E, NK; Scientist Development Grant 0435154N, NP; Established Investigator Award 0740040N, PMLJ), the Italian Society of Cardiology (CGT) and the National Institutes of Health (K02HL094692, R01HL079283, MTZ; R01HL075265, NP).
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Abbreviations: AP, action potential; AS, Angeli's salt; {beta}-AR, {beta}-adrenergic receptor; FS, fractional shortening; HNO, nitroxyl; ISO, isoproterenol; ICa, L-type Ca2+ current; NO, nitric oxide; NO2-, nitrite; .OH, hydroxyl radical; PLB, phospholamban; RCL, resting cell length; RyR, ryanodine receptor; SERCA, sarco-endoplasmic reticulum Ca2+-ATPase; SR, sarcoplasmic reticulum
Key Words: Excitation-contraction coupling; Cardiomyocyte; Electrophysiology; L-type Ca2+ current; Action potential; Thapsigargin; Heart failure
Send correspondence to: Mark T Ziolo, Department of Physiology and Cell Biology, Davis Heart and Lung Research Institute, The Ohio State University, 304 Hamilton Hall, 1645 Neil Avenue, Columbus, OH 43210, USA, Tel: 614-688-7905, Fax: 614-688-7999, E-mail:ziolo.1@osu.edu