Nitroxyl enhances myocyte Ca²⁺ transients by exclusively targeting SR Ca²⁺-cycling

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 (I_Ca) provides the trigger for the release of additional Ca²⁺ from the sarcoplasmic reticulum (SR) via the ryanodine receptors (RyR), thus inducing myocyte contraction (1). I_Ca 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 I_Ca. 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. MATERIALS AND METHODS

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 37°C. 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 22°C 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}Ratio_405/485. 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 (22°C) 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 Ratio_405/485 and {delta}Ratio_405/485. All measurements were recorded at room temperature (22°C).

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 (22°C).

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 I_Ca by inactivation of the Na⁺ current with the pre-pulse; replacement of K⁺ with Cs⁺ eliminates the K⁺ current. I_Ca inactivation (tau, time constant for I_Ca decline) was determined using a single exponential fitted to the decay phase of the current. All measurements were recorded at room temperature (22°C).

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 (22°C).

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 (37°C) (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.

Figure 1.

AS enhances systolic Ca²⁺ transients and cell shortening in cardiomyocytes. A) Individual, steady-state cell shortening (top) and systolic Ca²⁺ transient (bottom) traces representing the effect of control (normal Tyrode) and 500 {micro}mol/L AS in isolated murine cardiomyocytes. NOTE: the timescale found in the upper panel of Figure 1A applies to both the upper and lower panels of Figure 1A. B) Pooled data (mean±S.E.M.) demonstrating the effect of control (normal Tyrode) and 500 {micro}mol/L AS on cell shortening (top) and systolic Ca²⁺ transients (bottom) in cardiomyocytes (n = 14 myocytes/5 hearts). *p<0.05 vs. Control. C) Pooled data (mean±S.E.M.) demonstrating the effect of 500 {micro}mol/L AS on cell shortening (top) and systolic Ca²⁺ transients (bottom) in cardiomyocytes displayed as a % of control at room temperature (22°C) and physiological temperature (37°C).

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 Ratio_405/485, 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).

Figure 2.

Hydroxyl radical exposure decreases cardiomyocyte contraction. Pooled data (mean±S.E.M.) demonstrating the effect of control (normal Tyrode) and hydroxyl radical exposure (2 {micro}mol/L ^·OH) on diastolic Ca²⁺ (left), diastolic cell length (center), and shortening amplitude (right) in rat cardiomyocytes (n = 13 cardiomyocytes/5 hearts). *p<0.05 vs. Control.

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}Ratio_405/485, 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.

Figure 3.

Nitrite does not alter cardiomyocyte contraction. Pooled data (mean±S.E.M.) demonstrating the effect of control (normal Tyrode) and 125 {micro}mol/L NaNO₂ on shortening (top) and systolic Ca²⁺ transients (bottom) in murine cardiomyocytes (n = 13 myocytes/2 hearts).

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).

Figure 4.

AS induces a slight change in AP waveform. A) Individual, steady-state AP traces representing the effect of control (normal Tyrode) and 500 {micro}mol/L AS in isolated murine cardiomyocytes. B) Pooled data (mean±S.E.M.) demonstrating the effect of control (normal Tyrode) and 500 {micro}mol/L AS on resting membrane potential (RMP). C) Pooled data (mean±S.E.M.) demonstrating the effect of control (normal Tyrode) and 500 {micro}mol/L AS on AP duration to 20% repolarization (APD₂₀, left) and to 90% repolarization (APD₉₀, right) (n = 8 cardiomyocytes/3 hearts). *p<0.05 vs. Control.

4.4. AS does not alter I_Ca

We subsequently investigated the effect of AS/HNO on murine myocyte I_Ca. Surprisingly, we observed no change in basal I_Ca 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 I_Ca (Figure 5D). However, AS/HNO did induce significantly faster inactivation of I_Ca, measured as the time constant for I_Ca decline (Control: 42±7 vs. HNO: 36±6 ms, p<0.05 vs. Control). This effect can be seen in the normalized I_Ca traces (Figure 5E) and in the summary data (Figure 5F).

Figure 5.

AS does not alter basal I_Ca, but does hasten the rate of I_Ca inactivation. A) Individual, steady-state I_Ca traces representing the effect of control (normal Tyrode) and 500 {micro}mol/L AS in isolated murine cardiomyocytes. B) Representative I_Ca time plot demonstrating the effect of control (normal Tyrode) and 500 {micro}mol/L AS. C) Pooled data (mean±S.E.M.) demonstrating the effect of control (normal Tyrode) and AS (100 & 500 {micro}mol/L) on cardiomyocyte I_Ca. D) Pooled data (mean±S.E.M.) demonstrating the effect of control (normal Tyrode) and 500 {micro}mol/L AS on the current-voltage relationship for I_Ca. E) Normalized I_Ca traces representing the effect of control (normal Tyrode) and 500 mmol/L AS on I_Ca inactivation in isolated murine cardiomyocytes. F) Pooled data (mean±S.E.M.) demonstrating the effect of control (normal Tyrode) and AS (500 {micro}mol/L) on cardiomyocyte I_Ca inactivation (tau, time constant for I_Ca decline) (n = 8–11 myocytes/4 hearts). *p<0.05 vs. Control.

AS/HNO also failed to elicit a change in I_Ca 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).

Figure 6.

AS does not alter {beta}-AR-stimulated I_Ca. A) Individual, steady-state I_Ca traces representing the effect of control (normal Tyrode), 0.01 {micro}mol/L ISO and 500 {micro}mol/L AS in isolated murine cardiomyocytes. B) Pooled data (mean±S.E.M.) demonstrating the effect of control (normal Tyrode), ISO (0.01 {micro}mol/L) and AS (500 {micro}mol/L) on cardiomyocyte I_Ca (n = 6 cardiomyocytes/3 hearts). *p<0.05 vs. Control.

4.5. AS has no effect during SR inhibition

Since we observed no change in I_Ca, 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 I_Ca. 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⁺.

Figure 7.

SR inhibition attenuates effect of AS. A) Individual, steady-state systolic Ca²⁺ transient (bottom) traces representing the effect of control (normal Tyrode) and 500 {micro}mol/L AS (left) or 0.01 {micro}mol/L ISO (right) during SR inhibition in isolated murine cardiomyocytes. NOTE: the black traces prior to the addition of AS (left) and ISO (right), represent basal systolic Ca²⁺ transients with SR inhibition under control conditions (normal Tyrode). B) Pooled data (mean±S.E.M.) demonstrating the effect of control (normal Tyrode), AS (500 {micro}mol/L), and ISO (0.01 {micro}mol/L) on cardiomyocyte systolic Ca²⁺ transient amplitude during SR inhibition (n = 8–9 cardiomyocytes/3 hearts). *p<0.05 vs. Control and AS. C) Pooled data (mean±S.E.M.) demonstrating the effect of AS on cardiomyocyte systolic Ca²⁺ transient amplitude with and without SR function. *p<0.05 vs. without SR Function.

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 I_Ca, 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 (37°C), compared to the effects observed at room temperature (22°C) (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 I_Ca (15, 20).

5.5. HNO does not alter I_Ca

Despite the HNO-induced increase in the APD₉₀, HNO had no effect on basal I_Ca (Figure 5). This lack of effect on I_Ca 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 I_Ca (Figure 5). However, faster inactivation of I_Ca 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 I_Ca (21, 22). Since peak I_Ca was not altered by HNO, the prolongation of the action potential duration by HNO likely results from the targeting of repolarizing K⁺ channels, namely I_K,slow1, I_K,slow2, and I_ss (23, 24), and warrants further study.

Additionally, HNO was without effect on {beta}-AR-stimulated I_Ca (Figure 6). Thus, the effects of HNO on I_Ca appear to be very different from the effects of exogenous and endogenous NO signaling, which has been shown to decrease {beta}-AR-stimulated I_Ca (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 I_Ca 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 I_Ca (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 I_Ca 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 I_Ca 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 I_Ca 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 I_Ca. Prolonged activation of I_Ca 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, I_Ca 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 I_Ca 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 I_Ca.

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 I_Ca. 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.

Abbreviations

AP

action potential

AS

Angeli’s salt

{beta}-AR

{beta}-adrenergic receptor

FS

fractional shortening

HNO

nitroxyl

ISO

isoproterenol

I_Ca

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