Hydralazine and Organic Nitrates Restore Impaired Excitation-Contraction Coupling by Reducing Calcium Leak Associated with Nitroso-Redox Imbalance

Background: Hydralazine and organic nitrates have clinical benefits for heart failure, but the underlying mechanism is controversial.

Results: Hydralazine reduced sarcoplasmic reticulum Ca²⁺ leak and improved Ca²⁺ cycling and contractility; nitroglycerin enhanced contractile efficiency; both were impaired by nitroso-redox imbalance.

Conclusion: These agents exert complementary effects on nitroso-redox imbalance.

Significance: New mechanistic insights for redox-targeted treatments of heart failure.

Abstract

Although the combined use of hydralazine and isosorbide dinitrate confers important clinical benefits in patients with heart failure, the underlying mechanism of action is still controversial. We used two models of nitroso-redox imbalance, neuronal NO synthase-deficient (NOS1^−/−) mice and spontaneously hypertensive heart failure rats, to test the hypothesis that hydralazine (HYD) alone or in combination with nitroglycerin (NTG) or isosorbide dinitrate restores Ca²⁺ cycling and contractile performance and controls superoxide production in isolated cardiomyocytes. The response to increased pacing frequency was depressed in NOS1^−/− compared with wild type myocytes. Both sarcomere length shortening and intracellular Ca²⁺ transient (Δ[Ca²⁺]_i) responses in NOS1^−/− cardiomyocytes were augmented by HYD in a dose-dependent manner. NTG alone did not affect myocyte shortening but reduced Δ[Ca²⁺]_i across the range of pacing frequencies and increased myofilament Ca²⁺ sensitivity thereby enhancing contractile efficiency. Similar results were seen in failing myocytes from the heart failure rat model. HYD alone or in combination with NTG reduced sarcoplasmic reticulum (SR) leak, improved SR Ca²⁺ reuptake, and restored SR Ca²⁺ content. HYD and NTG at low concentrations (1 μm), scavenged superoxide in isolated cardiomyocytes, whereas in cardiac homogenates, NTG inhibited xanthine oxidoreductase activity and scavenged NADPH oxidase-dependent superoxide more efficiently than HYD. Together, these results revealed that by reducing SR Ca²⁺ leak, HYD improves Ca²⁺ cycling and contractility impaired by nitroso-redox imbalance, and NTG enhanced contractile efficiency, restoring cardiac excitation-contraction coupling.

Introduction

Heart failure (HF)³ is a common and increasingly prevalent cause of morbidity and mortality. Prior to the African-American heart failure trial, the only pharmacologic strategy that improved survival in HF was neurohormonal blockade therapy. This trial revealed increased survival among African-American patients with advanced HF treated with the combination of isosorbide dinitrate (ISDN) and hydralazine (HYD) (1–3). However, the precise mechanism(s) by which this regimen reduced mortality remains unclear.

HF is characterized by impaired excitation-contraction (E-C) coupling. For instance, increased contractile force upon increasing frequency of stimulation (force-frequency relationship, FFR) is compromised in failing hearts, giving rise to a blunted or negative FFR (4–6). Defects in calcium (Ca²⁺) handling may be responsible for this phenomenon (7, 8). It is increasingly appreciated that altered signaling in both Ca²⁺ reuptake mechanisms and Ca²⁺ leak through the ryanodine receptor (RyR2) contribute to impairment of the Ca²⁺ cycle in the failing heart. In this regard, Ca²⁺ leak is believed to be mediated by post-translational modifications of the RyR2, associated with nitroso-redox (NO/redox) imbalance (9, 10), a linked abnormality in the failing myocardium (11, 12). As a consequence of the sarcoplasmic reticulum (SR) Ca²⁺ leak, an altered E-C coupling occurs by depleting the SR Ca²⁺ stores resulting in possible impaired contractile function of the heart.

Increased xanthine oxidoreductase (XOR)-, and possibly NADPH oxidase (NOX)-mediated reactive oxygen species (ROS) production also occurs in failing hearts (13–15). Importantly, disruption in signaling pathways due to oxidative stress can be intensified by NO deficiency, a situation of NO/redox disequilibrium (16, 17). One possible explanation of the clinical benefits of HYD-ISDN is restoration of the balance between formation of reactive oxygen and reactive nitrogen species (2).

We used two established models of cardiac NO/redox imbalance to test the hypothesis that HYD and organic nitrates restore NO bioavailability and reactivity, ameliorate NO/redox disequilibrium, and improves myocardial contractility as measured by FFR. Accordingly, we examined the effects of each drug, alone and in combination, on sarcomere length (SL) shortening, Ca²⁺ cycling, and SR Ca²⁺ leak in neuronal NO synthase-deficient (NOS1^−/−) mice and spontaneously hypertensive heart failure (SHHF) rats. ROS production by XOR and NOX were determined in vitro in NOS1^−/− hearts.

EXPERIMENTAL PROCEDURES

Animal Models and Myocyte Isolation

All protocols and experimental procedures were approved by the Institutional Animal Care and Use Committee of The University of Miami following the Guide for the Care and Use of Laboratory Animals (NIH Publication 85–234, revised 1996).

Cardiomyocytes (CMs) were isolated from C57BL/6J mice (WT, 3–5 months old, n = 25; Jackson Laboratories, Bar Harbor, ME) and transgenic mice with homozygous deletions for NOS1 (B6;129S4-Nos1<tm1Plh>J, 3–5 months old, n = 41) or male SHHF rats (22–24 months old, n = 4; Charles River Laboratories Inc., Wilmington, MA) and their normotensive controls, Wistar Kyoto rats (WKY, n = 5) hearts as described in detail (18). Briefly, hearts were harvested and retrograde perfused through the aorta in a modified Langendorf system with an isolation solution containing collagenase type 2 (Worthington Biochemical Corp.) and protease type XIV (Sigma).

Protocols

Cells were loaded with Fura-2. The SL and intracellular Ca²⁺ concentration ([Ca²⁺]_i) were measured simultaneously in CMs stimulated at 0.5, 1, 2, 3, and 4 Hz. Experiments were repeated after a 10-min incubation with HYD (0.1, 1, and 10 μm; Sigma), nitroglycerin (NTG, 0.1, 1 and 10 μm; American Regent Laboratories, Inc., Shirley, NY), 10 μm ISDN (Alexis Biochemicals, Enzo Life Sciences, Inc., PA), or HYD plus either NTG or ISDN. Experiments in SHHF rat CMs were carried out similarly, with the exception that the concentration of drugs was as follows: 10 μm HYD, 10 μm NTG, and their combination. All experiments were conducted at 37 °C.

Contractility and Ca²⁺ Measuring

SL shortening (%ΔSL) was recorded with an IonOptix iCCD camera and calculated as: (resting SL − peak SL) × 100/resting SL. Ca²⁺_i was measured using a dual excitation (340/380 nm) spectrofluorometer (IonOptix LLC, Milton, MA). The “in vivo” calibration was performed using solutions containing 10 μm ionomycin (Sigma) as described by Grynkiewicz et al. (19) and the [Ca²⁺]_i was calculated using the following equation,

where K_d′ (apparent dissociation constant for Fura-2) in adult myocytes was 224 nm. R_min and R_max as well as the scaling factors (Sf₂ and Sb₂) were extracted from the calibration curves. Δ[Ca⁺²]_i amplitude was considered as: peak [Ca⁺²]_i − resting [Ca⁺²]_i.

SR Ca²⁺ Leak and SR Ca²⁺ Load Measurement

SR Ca²⁺ leakage was assessed with tetracaine as described by Shannon et al. (20). Briefly, after pacing was stopped, a fast switch to a 0 Na⁺, 0 Ca²⁺ Tyrode solution (Na⁺ was replaced by an equimolar amount of Li⁺) was performed. After 60 s, similar to Bassani et al. (21), a rapid switching to 0 Na⁺, 0 Ca²⁺ solution containing 20 mm caffeine to assess SR Ca²⁺ content was applied. Following recovery of the cell, the same pacing protocol was assessed. After stop pacing, a switch to 0 Na⁺, 0 Ca²⁺ Tyrode solution containing 1 mmol/liter of tetracaine (Sigma) was performed. The observed drop in the Fura-2 ratio compared with the nontetracaine treated condition was considered the Ca²⁺ leak for a particular CM. After assessing the Ca²⁺ leak, tetracaine was washed out by superfusing fresh 0 Na⁺, 0 Ca²⁺ Tyrode solution and then a new caffeine challenge was applied to estimate the SR Ca²⁺ load. SR Ca²⁺ contents were calculated considering that SR represents 3.5% and cytosol 65% of the CM volumes. The following equation from Shannon et al. (20) was used: [Ca²⁺]_SR = [Ca²⁺]_caff + (β_max-SR × [Ca²⁺]_caff)/([Ca²⁺]_caff + K_d-SR). [Ca²⁺]_SR is the SR Ca²⁺ content, [Ca²⁺]_caff is the SR Ca²⁺ released by caffeine, β_max-SR and K_d-SR are the usual Michaelis parameters for SR Ca²⁺ binding. SR leak-SR load pairs were grouped by comparable SR Ca²⁺ loads and expressed as a leak-load relationship.

Detection of Superoxide by DHE Staining

Fresh isolated mouse CMs were incubated at room temperature for 30 min with dihydroethidium (DHE, 3 μm; Molecular Probes). After washing with PBS, cells were fixed with 2% paraformaldehyde for 5 min at 4 °C. Finally, the samples were mounted in Prolong Gold anti-fade (Invitrogen). The images were obtained using a Zeiss LSM-710 confocal microscope. Nuclear fluorescence captured at 562 nm was quantified using the Image-Pro plus software (MediaCybernetics, Silver Spring, MD) and normalized by cytosolic fluorescence.

NOX-dependent Superoxide Production

NOX-dependent superoxide production was measured in heart homogenates from NOS1^−/− mice using lucigenin (5 μm)-enhanced chemiluminescence (β-NADPH 300 μm; room temperature) on a microplate luminometer (Veritas, Turner Biosystems, Sunnyvale, CA). Chemiluminescence readings were expressed as integrated light units. Experiments were performed in the presence of increasing concentrations of HYD and NTG (0.01, 0.1, and 1 mm).

XOR Activity

XOR activity was investigated by measuring uric acid (22) and superoxide production. Heart homogenates from NOS1^−/− mice were passed through a Sephadex G-25 column (GE Healthcare) and XOR-dependent superoxide production was measured using Amplex Red (Molecular Probes, Eugene, OR). The effluent was assessed at 295 nm for uric acid. Both assay plates were incubated at 37 °C for 30 min in the presence or absence of allopurinol, and HYD and NTG at increasing concentrations.

Assessment of Ca²⁺ Myofilament Responsiveness

Myofilament responsiveness to Ca²⁺ was assessed using the steady-state relationship between SL and [Ca²⁺]_i in intact single CMs tetanized by high-frequency (10 Hz) stimulation after exposure to thapsigargin (0.2 μm for 15 min), as described previously (23). With this approach, the Ca²⁺_i was reversibly clamped during the tetanic contracture for 20 s and then rapidly returned to resting levels upon cessation of electrical stimulation via the Na⁺/Ca²⁺ exchanger. The steady-state levels of Ca²⁺ achieved during tetanus exposure were regulated by subjecting CMs to Tyrode solutions containing increasing concentrations of Ca²⁺ (0.1, 1.0, 5.0, 10, and 20 mm CaCl₂).

Western Blot Immunoanalysis

Hearts from 12 NOS1^−/− mice were perfused with Krebs solution (30 min; n = 6), or Krebs plus 10 μm HYD (30 min; n = 4), or 10 μm ISDN (30 min; n = 2). Hearts were homogenized in cold RIPA buffer containing the protease inhibitor mixture and phosphatase inhibitors. Samples were electrophoresed using a NuPAGE 10% Bis-Tris gel (Invitrogen) and transferred to PVDF membranes (Bio-Rad Laboratories). Immunoblot analysis was performed with goat polyclonal antibody for SERCA2 (Santa Cruz Biotechnology, Inc.), mouse monoclonal antibody for phospholamban (PLB; Pierce, Thermo Scientific), rabbit polyclonal for phospho-PLB(Ser16) (Pierce, Thermo Scientific), PLB phospho Thr-17 (Badrilla, Leeds, UK), NCX1 (RDI, Flanders, NJ), and GAPDH (as loading control; Santa Cruz). Phosphorylated PLB were expressed as a ratio of Ser(P)-16/PLB or Thr(P)-17/PLB and SERCA2, NCX1, or total PLB expression compared with GAPDH.

Statistical Analysis

Data are reported as mean ± S.E. Statistical significance was determined by unpaired Student's t test, one-way ANOVA, or two-way ANOVA followed by Student's-Newman-Keuls or Bonferonni's post hoc tests, as appropriate, using the GraphPad Prism version 4.02 (GraphPad Prism Software Corporation, San Diego, CA). The null hypothesis was rejected at p < 0.05.

RESULTS

Hydralazine and Organic Nitrates Improved Contractility in NOS1^−/− CMs

To determine the effect of the drugs on CM contractility, we measured SL shortening in cells from WT and NOS1^−/− mice paced at 0.5, 1, 2, 3, and 4 Hz (Fig. 1, A and B). At baseline, SL shortening was similar in NOS1^−/− and WT CMs (0.5 Hz; Table 1). However, as previously described (12), the increased SL shortening due to augmented pacing frequency was blunted in NOS1^−/− CMs compared with WT (4Hz, Table 1, and Fig. 1, A and B, p < 0.0001 versus WT, two-way ANOVA).

FIGURE 1.

Contractile performance in NOS1^−/− cardiomyocytes. A, representative traces of SL in WT (black) and NOS1^−/− (red) CMs under increasing rates of pacing from 0.5 to 4 Hz. B, CM contractility expressed as % SL shortening versus resting SL, in NOS1^−/− (■; n = 50–54 cells) compared with WT (□; n = 34–41 cells). C, SL shortening at 4 Hz expressed as the percentage of shortening versus resting SL, in WT control or NOS1^−/− CMs in the absence (bl) or presence of HYD (0.1, 1 or 10 μm), NTG (0.1, 1, or 10 μm), ISDN (10 μm), and a combination (HYD + NTG, 0.1, 0.5 or 10 μm each and HYD + ISDN; 10 μm each). **, p < 0.01; ***, p < 0.001 versus WT control, two-way ANOVA.

TABLE 1.

Baseline myocyte performance

	WT		NOS1−/−
	0.5 Hz	4 Hz	0.5 Hz	4 Hz
Cellsa	40–41	32–34	50–54	40–50
SL shortening (%)b	4.75 ± 0.30	9.30 ± 0.50	4.27 ± 0.30	5.90 ± 0.40c
Diastolic [Ca2+]i (nm)	234 ± 16	298 ± 26	284 ± 19d	337 ± 19
Δ[Ca2+]i transient (nm)	160 ± 13	315 ± 40	254 ± 19e	278 ± 21
Δ[Ca2+]i decay (τ s)	0.198 ± 0.009	0.091 ± 0.004	0.231 ± 0.013d	0.101 ± 0.006
SR Ca2+ load (μm)	42.0 ± 7.4	70.6 ± 7.5	37.9 ± 7.2	52.4 ± 4.7d

^a Five to 10 cells per heart were studied.

^b SL shortening is calculated as: (resting SL − peak SL)/resting SL × 100.

^c p < 0.001 versus WT (unpaired Student's t test).

^d p < 0.05 versus WT (unpaired Student's t test).

^e p < 0.01 versus WT (unpaired Student's t test).

HYD augmented the frequency-dependent SL shortening in NOS1^−/− CMs in a concentration-dependent manner (Figs. 1C and 2E, supplemental Table S1). In contrast, NTG alone did not affect the SL shortening in NOS1^−/− (Fig. 1C, supplemental Table S1). To test whether the drug combination would have a synergistic effect, NOS1^−/− myocytes were incubated with HYD plus NTG. Although this treatment restored the FFR in a dose-dependent manner, the response was not greater than hydralazine alone (Figs. 1C and 2E, Supplemental Table 1). In WT CMs, neither HYD nor NTG, alone or in combination, affected frequency-dependent SL shortening (data not shown).

FIGURE 2.

Intracellular Ca²⁺ transient (Δ[Ca²⁺]_i) in NOS1^−/− cardiomyocytes. A, Δ[Ca²⁺]_i (nm) in NOS1^−/− (■; n = 40–50 cells) compared with WT (□; n = 32–40 cells). B, Δ[Ca²⁺]_i in NOS1^−/− (■) and WT (□) CMs, expressed as % increase versus 0.5 Hz. C, Δ[Ca²⁺]_i at 4 Hz in WT control or NOS1^−/− CMs in the absence (bl) or presence of HYD (0.1, 1 or 10 μm), NTG (0.1, 1 and 10 μm), ISDN (10 μm), and a combination (HYD + NTG; 0.1, 0.5, and 10 μm each; and HYD + ISDN; 10 μm each). D, ΔCa²⁺ frequency response in WT CMs (□) under baseline conditions or NOS1^−/− baseline (■) or treated with 10 μm HYD (red circle) or 10 μm HYD + NTG (blue circle). E, FFR in CMs under the same conditions as mentioned above. The combination induces a significant increase in FFR but does not change ΔCa²⁺ significantly compared with NOS1^−/− CMs. However, the contractile reserve is restored. *, p < 0.05; **, p < 0.01; and ***, p < 0.001 versus NOS1^−/− control; †, p < 0.05 versus HYD alone; two-way ANOVA.

We next assessed whether ISDN, the pharmacologically employed nitrate, had similar effects as NTG. Indeed, the combination of HYD and ISDN also induced an increased frequency-dependent SL shortening in NOS1^−/− compared with untreated NOS1^−/− CMs (Fig. 1C and supplemental Table S1; p = 0.004) at an equimolar concentration of 10 μm. Similar to NTG, 10 μm ISDN did not alter contractility in NOS1^−/− (Fig. 1C and supplemental Table S1).

Divergent Effects of Hydralazine and Nitroglycerin on Ca²⁺ Transient in NOS1^−/− CMs

Resting diastolic [Ca²⁺]_i values were slightly elevated in NOS1^−/− compared with WT CMs, over the range of studied frequencies (Table 1; p < 0.01) as previously described (12, 24). When the [Ca²⁺]_i transient (Δ[Ca²⁺]_i) was studied, [Ca²⁺]_i-FFR was clearly depressed in NOS1^−/− CMs compared with WT, as previously described (Fig. 2, A and B, p < 0.0001 versus WT; two-way ANOVA) (18, 24, 25). HYD induced a Δ[Ca²⁺]_i increase in a concentration-dependent manner (Fig. 2C) as well as significantly increased the [Ca²⁺]_i-FFR (Fig. 2D, supplemental Table S2, p < 0.05). Thus, the increase in contractility in response to HYD was mirrored by a concomitant increase in Δ[Ca²⁺]_i (Fig. 2E).

Although NTG did not affect contractility, it induced a concentration-dependent decrease in the Δ[Ca²⁺]_i amplitude compared with control NOS1^−/− myocytes most clearly seen at 4 Hz. This decrease was reproduced using 10 μm ISDN (Fig. 2C and supplemental Table S2). The combination of HYD and NTG (Fig. 2D) or ISDN (supplemental Table S2) on NOS1^−/− CMs restored the increasing [Ca²⁺]_i-FFR (frequency p = 0.0213 or p = 0.0014, respectively; two-way ANOVA) by reducing the Δ[Ca²⁺]_i amplitude toward the levels in WT CMs (p = 0.042 or p = 0.0005 versus HYD alone, respectively). Thus, for any given improvement in cardiac contraction due to HYD, organic nitrates offset the increase in [Ca²⁺]_i, thereby enhancing contractile efficiency (Fig. 2, D and E). Neither HYD nor NTG, alone or in combination (10 μm), altered the [Ca²⁺]_i-FFR in WT CMs (data not shown).

We also examined the peak [Ca²⁺]_i decay time constant (τ), which was significantly higher in NOS1^−/− than WT CMs (p < 0.0001, Table 1, Fig. 3, A and B). All treatments accelerated τ (particularly at 0.5 Hz, Fig. 3B and supplemental Table S3) in a dose-dependent manner (p < 0.0001 HYD alone or in combination with NTG versus NOS1^−/− baseline; p = 0.001 NTG alone versus NOS1^−/− baseline, two-way ANOVA). ISDN was equally effective in reducing τ, either alone or combined with 10 μm HYD (p < 0.001; supplemental Table S3). Reuptake of Ca²⁺ assessed by decay of caffeine-induced Ca²⁺ transients in a Na⁺ and Ca²⁺-free solution, which avoids any Ca²⁺ flux throughout the sarcolemma, was slower in NOS1^−/− (K = 0.1888/s; T₅₀ = 3.65 ± 0.12 s, fitted by an one-phase exponential equation) compared with WT cells (K = 0.2724/s; T₅₀ = 2.53 ± 0.08 s; p = 0.0005). Treatment with either 10 μm HYD or NTG increased the SR Ca²⁺ reuptake rate in NOS1^−/− (K = 0.240/s, T₅₀ = 2.88 ± 0.17 s, p = 0.0003 and K = 0.2392/s, T₅₀ = 2.88 ± 0.11 s, p < 0.0001, respectively). The combination exerted an additive effect (K = 0.2919/s, T₅₀ = 2.36 ± 0.06 s, p < 0.0001) suggesting that the improvement in the regular field-stimulated Δ[Ca²⁺]_i decay was mediated by enhanced SERCA2 activity. This speculation was confirmed by assessing phosphorylation of PLB in cardiac homogenates from NOS1^−/− hearts treated with either 10 μm HYD or ISDN. HYD increased phosphothreonine 17 (Thr(P)-17) for pentameric PLB (p = 0.0393); but not on total PLB (p = 0.0597) or monomeric PLB (Fig. 3, C and D). ISDN increased total Thr(P)-17 (p = 0.011; Fig. 3, E and F). Neither treatment significantly increased phosphoserine 16 (Ser(P)-16; Fig. 3, D–F).

FIGURE 3.

Hydralazine and organic nitrates enhance Ca²⁺ influx to the SR in NOS1^−/− cardiomyocytes. A, Ca²⁺ decay time constant (tau, τ) versus frequency in NOS1^−/− (■) compared with WT (□) CMs. B, Tau values at 0.5 Hz in WT control or NOS1^−/− CMs treated with or without HYD, NTG, or a combination (HYD + NTG) at the indicated concentrations (μm) (n = 5–21 cells) (*, p < 0.05; **, p < 0.01; and ***, p < 0.001 versus NOS1^−/− control; two-way ANOVA). C, PLB phosphorylation at threonine 17 (Thr(P)-17) in baseline or 10 μm HYD-treated NOS1^−/− hearts (total and pentameric Thr(P)-17 PLB). D, representative Western blots for all studied proteins in the absence or presence of 10 μm HYD. E, PLB Thr(P)-17 in baseline or 10 μm ISDN-treated NOS1^−/− hearts (total and monomeric Thr(P)-17 PLB). Phosphorylated PLB at Ser-16 (Ser(P)-16, bottom) was also measured. F, representative Western blots. No significant changes were observed in Ser(P)-16, SERCA2, or NCX1 with either treatment. †, p < 0.05 versus NOS1^−/− control; Student's t test.

Hydralazine Alone and in Combination with Nitroglycerin, Lowers SR Ca²⁺ Leakage Toward Normal

We next assessed SR Ca²⁺ leak and the SR Ca²⁺ leak-load relationship, using the technique of Shannon et al. (20) using tetracaine, a RyR2 blocker (Fig. 4A). SR Ca²⁺ leak was augmented in NOS1^−/− (R² = 0.5225 for WT and R² = 0.8827 for NOS1^−/−, Fig. 4B; p = 0.0002). The decreased SR Ca²⁺ content and increased diastolic Ca²⁺ exhibited by NOS1^−/− compared with WT CMs are consistent with the elevated Ca²⁺ leak. HYD reduced the leak in a concentration-dependent manner in NOS1^−/− myocytes (Fig. 4C; p = 0.0012). Neither 1 nor 10 μm HYD significantly affected the SR Ca²⁺ leak in WT CMs (data not shown).

FIGURE 4.

Assessment of SR diastolic Ca²⁺ leakage in the presence of hydralazine, organic nitrates, or their combination. A, protocol to assess the SR Ca²⁺ leak using tetracaine to block RyR2 and caffeine to estimate the SR Ca²⁺ content. B, SR Ca²⁺ load-leak relationship in WT (□) and NOS1^−/− (■) CMs. C, SR Ca²⁺ load-leak relationship in NOS1^−/− CMs in the absence (red ■) or presence of HYD (0.1 μm, red ▾, 1 μm red ▴ or 10 μm, red ●). D, in the presence of NTG (0.1 μm, green ▾; 1 μm, green ▴; or 10 μm, green ●). E, in the presence HYD plus NTG (0.1 μm, blue ▾; 0.5 μm, blue ▴; or 10 μm, blue ●). NOS1^−/− CMs exhibited an increased SR Ca²⁺ leak and HYD alone or a combination reduced it in a dose-dependent manner. F, dose-response relationship; effect of increasing concentrations of HYD (red ▴), NTG (green ●), and a combination of HYD + NTG (blue ▾) on SR Ca²⁺ leak in NOS1^−/− CMs (**, p < 0.01 versus nontreated NOS1^−/− CMs; one-way ANOVA). G, SR Ca²⁺ load-leak curves in NOS1^−/− (■) CMs in the absence or presence of 10 μm ISDN alone (dark green ●) or in combination with 10 μm HYD (dark blue ●). Load-leak relationship of WT was included as normal control (□). H, SR Ca²⁺ content at 4 Hz. HYD alone and in combination with NTG restored the SR Ca²⁺ content in NOS1^−/− in a concentration-dependent manner toward normal levels as exhibited by WT CMs. NTG alone had no significant effects. *, p < 0.05 and **, p < 0.01 versus NOS1^−/− control; two-way ANOVA.

NTG did not significantly affect the SR Ca²⁺ leak in NOS1^−/− cells (Fig. 4D). In contrast, the combination of NTG plus HYD reduced the leak in a concentration-dependent manner as shown by the leak-load relationship (Fig. 4E; p = 0.0001). Fig. 4F shows the drug concentration-SR Ca²⁺ leak response relationship. The IC₅₀ for HYD was 0.0705 ± 0.0473 μm (at matched SR load of 50 μm). The combination exerted a comparable effect to HYD alone (IC₅₀ = 0.160 ± 0.128 μm).

The combination of 10 μm HYD and 10 μm ISDN reduced the SR Ca²⁺ leak in NOS1^−/− equal to HYD plus NTG (Fig. 4G; p = 0.0186). ISDN alone exhibited differential effects at the low SR Ca²⁺ load (reduced leak) compared with the higher load (no effect) in NOS1^−/− CMs (Fig. 4G). We also assessed the effects of 10 μm HYD plus 10 μm NTG on the SR Ca²⁺ leak in WT CMs but found no significant difference compared with control, whereas 10 μm NTG significantly reduced the degree of the SR Ca²⁺ leak in WT (data not shown).

Cardiomyocyte SR Ca²⁺ Content

Because SR Ca²⁺ storage is the key determinant of FFR, we estimated the Ca²⁺ content by rapidly infusing caffeine after pacing the myocytes. Although increasing the pacing augmented the SR Ca²⁺ content in both NOS1^−/− and WT CMs (p < 0.05, Table 1), the increase was smaller in NOS1^−/− (38.2%) than WT (66.6%), which was consistent with previous studies (25). In the presence of HYD, NOS1^−/− CMs also exhibited a rise in SR Ca²⁺ content in response to increasing frequency (p < 0.05, data not shown). SR Ca²⁺ content evaluated at 4 Hz was increased by HYD in a concentration-dependent manner, reaching a load similar to WT CMs (p < 0.01) (Fig. 4H). We also determined that, similar to HYD alone, the combination of equal concentrations of NTG and HYD increased the SR Ca²⁺ load in a concentration-dependent manner in NOS1^−/− CMs (Fig. 4H). However, 10 μm HYD alone or combined with 10 μm NTG abolished the frequency-induced rise in SR Ca²⁺ content in WT CMs (p = 0.001 and p = 0.005, respectively; data not shown). NTG alone did not affect the SR Ca²⁺ load in NOS1^−/− (Fig. 4H) or WT myocytes (data not shown).

Detection of Superoxide by DHE Staining

To determine the molecular underpinnings for improved E-C coupling in NOS1^−/− myocytes in response to these drugs, we used the superoxide-sensitive dye DHE to assess the scavenging capacity of HYD and NTG alone, or in combination at 0.1, 1, or 10 μm (Fig. 5, A and B). As DHE is oxidized by superoxide and then translocated to the nucleus (Fig. 5B), the ratio between fluorescence in the nucleus and cytoplasm indexes superoxide production. Although the lowest drug concentrations did not affect superoxide detection, increasing concentrations of HYD as well as NTG scavenged superoxide (p < 0.05; Fig. 5A). In combination they had an additive effect (p < 0.01; Fig. 5A). Thus, at concentrations that reduced the SR Ca²⁺ leak, HYD alone or in combination with NTG exhibited an antioxidant effect.

FIGURE 5.

Oxidative stress is attenuated by hydralazine and nitroglycerin. A, superoxide-mediated oxidation of DHE. The ratio of fluorescence in the nucleus/cytoplasm preincubated 30 min with HYD (red ▴), NTG (green ●) and a combination of HYD + NTG (blue ▾) is shown. B, representative pictures of mice ventricular CMs stained with 3 μm DHE (superoxide-sensitive dye) in the absence (control) or presence (0.1, 1, or 10 μm) of the drugs (Bar, 10 μm). Yellow arrows indicate nuclei highly stained and green arrows show nuclei with little or no dye. C, NOX-dependent superoxide production in NOS1^−/− cardiac homogenates. Effects of HYD and NTG on the NOX enzymatic system containing 0.3 mm NADPH as substrate. D, XOR activity in WT and NOS1^−/− mouse hearts in terms of superoxide production (left) or uric acid generation (right). E, effect of allopurinol (♦), HYD, and NTG on XOR-dependent superoxide production in NOS1^−/− cardiac homogenates. F, effects of allopurinol, HYD, and NTG on uric acid production in the XOR enzymatic system containing 0.2 mm xanthine as substrate. *, p < 0.05; **, p < 0.01; and ***, p < 0.001 versus baseline, one-way ANOVA; †, p < 0.05 versus nontreated NOS1^−/−, Student's t test.

NOX-dependent Superoxide Production

We also studied activation of specific oxidases, XOR and NOX, in mouse heart homogenates in the presence of either HYD or NTG. NOX activity of WT (n = 3) and NOS1^−/− (n = 4) hearts was similar (2.29 ± 0.15 versus 2.19 ± 0.13 integrated light units/μg of protein, respectively). HYD (0.1–1 mm, IC₅₀ 0.42 ± 0.23 mm) decreased NOX activity (Fig. 5C, p < 0.01 for both concentrations versus control) in NOS1^−/− cardiac homogenates. NTG (>0.01 mm, IC₅₀ 0.013 ± 0.06 mm) also inhibited NOX-dependent superoxide production (Fig. 5C; p < 0.05 for 0.01 mm, p < 0.01 for 0.1 and 1 mm versus control).

XOR Activity

NOS1^−/− hearts (n = 3) exhibited up-regulated XOR activity compared with WT (n = 3) using the Amplex Red detection of XOR-mediated ROS production assay (Fig. 5D; 2.1 ± 0.18 versus 1.63 ± 0.09 milliunits/μg of protein, respectively; p < 0.05), confirming previous findings in this mouse (25–27). Amplex Red XOR activity was inhibited by allopurinol (>0.01 mm, IC₅₀ 0.19 ± 0.06 mm, Fig. 5D; n = 3) and HYD (IC₅₀ of 0.7 ± 0.3 mm, Fig. 5E; n = 3) in NOS1^−/− cardiac homogenates. Interestingly, NTG (IC₅₀ 1.0 ± 0.4 mm, Fig. 5E, n = 3) also inhibited XOR. Because the Amplex Red assay measures superoxide levels, reduced activity could also reflect increased O₂^˙̄ scavenging. To address this possibility, we measured uric acid conversion as a direct measurement of enzymatic activity. With this approach, NOS1^−/− hearts (n = 3) again exhibited increased XOR activity compared with WT (n = 3) (Fig. 5D; 9.8 ± 0.46 versus 8.5 ± 0.21 milliunits/mg tissue, respectively, p < 0.05). Allopurinol (>0.1 mm) inhibited XOR enzyme activity in NOS1^−/− mice (Fig. 5F, n = 3). Interestingly, HYD did not, but NTG did display XOR-inhibitory activity in NOS1^−/− (Fig. 5F, n = 3).

Organic Nitrates Increased Myofilament Responsiveness to Ca²⁺ in NOS1^−/− Myocytes

Because our FFR data suggested that organic nitrates improve efficiency in myofilament responsiveness to Ca²⁺_i, we assessed the mechanism by which NTG offsets the HYD-induced Δ[Ca²⁺]_i increase toward normal, and directly tested the myofilament responsiveness in NOS1^−/− as well as WT CMs in the presence of 10 μm NTG. Responsiveness of myofilaments to Ca²⁺ (both sensitivity and maximal response) was lower in NOS1^−/− compared with WT CMs (EC_{50,NOS1−/−} = 0.627 ± 0.0185 and EC_50,WT = 0.398 ± 0.018 μm, respectively; p = 0.0009, Fig. 6, A–D). In agreement with previous studies using different NO donors (23, 28), NTG reduced myofilament responsiveness to Ca²⁺_i in WT (not shown). Surprisingly, but consistent with our results, NTG increased responsiveness to Ca²⁺ in NOS1^−/− (Fig. 6, B and D), as shown by a leftward shift (EC_{50,NOS1−/−,NTG} = 0.427 ± 0.0186 μm, p = 0.0016; Fig. 6C). Therefore, NTG restored the impaired myofilament sensitivity in NOS1^−/− toward normal levels observed in WT CMs. Analysis of [Ca²⁺]_i-SL shortening loops confirm that sensitivity to Ca²⁺ is deficient in NOS1^−/− CMs but treatment with 10 μm ISDN improves it toward WT (Fig. 6E).

FIGURE 6.

Myofilament sensitivity to Ca²⁺ in NOS1^−/− cardiomyocytes. A, representative traces of SL shortening (top) in response to similar increases of Ca²⁺_i (bottom) obtained by high frequency-induced tetanus of the CMs in the presence of thapsigargin. Response of NOS1^−/− was compared with WT cells in the absence (left) or presence of 10 μm NTG (right). B, curves of myofilament sensitivity to [Ca²⁺]_i in NOS1^−/− baseline (■) or 10 μm NTG-treated (green ■) CMs compared with WT (□) as normalized to the maximal response in each group (sigmoidal dose-response − variable slope − fitting). C, EC₅₀ values of the curves. D, myofilament sensitivity curves expressed as absolute values; the fitting provides not only higher EC₅₀ values but also a smaller maximal response in the NOS1^−/− control. E, SL shortening − [Ca²⁺]_i loops of WT (□; n = 27 cells) or NOS1^−/− CMs in the absence (■; n = 30 cells) or presence of 10 μm ISDN (dark green ▾; n = 10 cells). The slopes that fit the relaxation phases of the loops correlate with the sensitivity of myofilaments to Ca²⁺. Sensitivity of NOS1^−/− (—) is lower than WT (- - -); however, treatment with ISDN (—) increased the fitted slope toward the values observed in WT CMs. *, p < 0.05 versus NOS1^−/−, Student's t test.

Frequency-stimulated Contractility and Ca²⁺ Transients Are Improved by Hydralazine and Nitroglycerin in CMs from Heart Failure Rats

To study the effects of HYD and NTG in a model of heart failure (dilated cardiomyopathy), we used SHHF rats. Usually, the SL shortening amplitude in rat CMs is flat or negative in response to increasing the pacing rate. We recently showed that SL shortening drops more abruptly in SHHF than WKY control CMs (13). Here, we also observed a significant decrease in contractility in SHHF compared with WKY CMs (p = 0.0002, Fig. 7, A and B). Treatment with 10 μm HYD significantly improved SL shortening in SHHF myocytes (p = 0.0414). Consistent with observations in NOS1^−/−, 10 μm NTG did not affect the SL shortening in SHHF myocytes (p = 0.2553). The combination of HYD and NTG showed a strong trend toward increased contractility in SHHF myocytes (p = 0.0839), which also exhibited an FFR pattern similar to HYD alone (Fig. 7A). Furthermore, there was a significant difference at 4 Hz compared with the SHHF control (p = 0.0359, Fig. 7B). Normalization of SL shortening as a percentage of 0.5 Hz pacing showed that HYD alone as well as in combination with NTG significantly improved FFR in failing CMs (p = 0.035 and p = 0.0013, respectively, data not shown).

FIGURE 7.

SL shortening and Ca²⁺ transient amplitude-frequency response in SHHF cardiomyocytes. A, FFR in SHHF rat CMs in the absence (■) or presence of 10 μm HYD (red ●), 10 μm NTG (green ●), or a combination (HYD + NTG, 10 μm each; blue ●) compared with WKY control CMs (□). B, bar graph showing contractility data at 4 Hz. C, Ca²⁺-FFR in SHHF rat CMs treated with 10 μm HYD or 10 μm NTG or their combination (HYD + NTG, 10 μm each). D, bar graph showing Δ[Ca²⁺]_i data at 4 Hz. In SHHF CMs, similar effects to those observed in NOS1^−/− were obtained with these treatments (n = 5–15 cells). *, p < 0.05; ***, p < 0.001 versus SHHF control; two-way ANOVA. †, p < 0.05; ††, p < 0.01 versus SHHF control, Student's t test.

The ΔCa²⁺ increased in WKY CMs in response to pacing (p = 0.0019, Fig. 7C), compared with the flat pattern in SHHF CMs. In agreement with the results obtained in NOS1^−/− mice, in failing CMs Δ[Ca²⁺]_i amplitude was augmented by 10 μm HYD (p = 0.0124) in comparison to SHHF controls (Fig. 7C). In this setting, the combination of HYD with 10 μm NTG also induced a nonsignificant rise in Δ[Ca²⁺]_i primarily at the highest frequency (p = 0.1756, Fig. 7D). On the other hand, consistent with the effect observed in contractility in this model, Δ[Ca²⁺]_i was not affected by NTG (Fig. 7, C and D). Therefore treatment with HYD and NTG in a genetic model of heart failure enhances E-C coupling in a similar fashion (yet species appropriate) as observed in NOS1^−/− cells, a model of NO/redox imbalance.

DISCUSSION

Our major new finding is that HYD and NTG corrected the increased Ca²⁺ leak from the SR, thus normalizing contractility-frequency responses by modulating Ca²⁺ cycling in models of NO/redox imbalance. Although HYD improved contractile force in parallel with increasing Ca²⁺ transients and total restoration of leak, NTG offset Ca²⁺ cycling without impairing contractility, thereby improving contractile efficiency. Direct measure of myofilament sensitivity confirmed that NTG restores depressed myofilament sensitivity to Ca²⁺ in NOS1^−/− myocytes. Importantly, NTG inhibited two major sources of cellular ROS production, NOX and XOR, and also exhibited ROS scavenging capacity. HYD scavenged ROS but did not inhibit XOR. Although NOX activity appeared to be inhibited by HYD, this effect could be an epiphenomenon due to the superoxide scavenging properties of HYD, because NOX activity was only assessed by NADPH-induced superoxide production. Despite the lack of NOS1-derived NO and the fact that NOS3 is uncoupled under oxidative stress (29), peroxynitrite (ONOO⁻) was measurable in NOS1^−/− homogenates and was reduced by both HYD and NTG, in a dose-dependent manner (data not shown).

Model of NO-Redox Imbalance

NOS1^−/− mice are widely used to investigate the involvement of NO in E-C coupling because CMs exhibit a particularly impaired performance due to deficient NO production and consequent NO/redox imbalance (12, 25). Sears et al. (30) showed that NOS1^−/− CMs have larger ΔCa²⁺, which is consistent with larger I_Ca and augmented SL shortening compared with control animals, at low frequencies. However, we (12, 18) and others (31) have shown that the contractile reserve is impaired in NOS1^−/− as the increase in ΔCa²⁺ and contractility in response to pacing are depressed. Consistently, we have found that the reduced SR Ca²⁺ load was associated with augmented SR Ca²⁺ leak. In contrast, Sears et al. (30) found that the SR Ca²⁺ load was higher in NOS1^−/−, when measured at 1 Hz. In our hands, SR Ca²⁺ loads at low frequencies were not different between strains, but during pacing with rates more physiologic for murine myocytes, were depressed versus WT (25). Similarly, Wang and colleagues (32) demonstrated that the SR Ca²⁺ leak is reduced in NOS1^−/− myocytes, suggesting that NOS1-derived NO induces RyR2 to leak. However, and in contrast to our experiments, this group studied the SR Ca²⁺ leak at room temperature (22 °C), whereas, we assessed the leak at 37 °C. This simple detail may be responsible for the differences. We recently demonstrated that SHHF rats, a representative heart failure model that exhibits oxidative stress as a hallmark (13), share certain specific features with NOS1^−/− mice. Both models exhibit increased superoxide production associated with elevated XOR activity (25) and blunted NOS1 activity, evidenced by hyponitrosylation of RyR2 (12). Moreover, increased SR Ca²⁺ leak, reduced SR Ca²⁺ load, and depressed Ca²⁺ and force-frequency responses are characteristics of these models (13, 18). Unlike SHHF rats, NOS1^−/− mice are not considered a representative model for heart failure. Rather, they develop age-related cardiac hypertrophy (a pre-heart failure stage) and exhibit accelerated mortality (33, 34). Additionally, after myocardial infarction, NOS1^−/− also undergo exaggerated remodeling and higher mortality than WT controls (27). Consequently, NOS1^−/− mice are an excellent model of NO/redox imbalance that share features with a model of heart failure, the SHHF rat.

Therapeutic Use of Hydralazine and Organic Nitrates

Recently, Cole et al. (2) reviewed the use of HYD-ISDN as a treatment for heart failure (1, 3) highlighting the fact that the mechanistic basis for this regimen to reduce all-cause mortality remains to be established. We show for the first time that this regimen restores disrupted NO/redox equilibrium and in so doing restores E-C coupling derangements.

The effects of HYD and NTG resemble, in part, those of XOR inhibitors. Allopurinol improves myocyte contraction without changing Δ[Ca²⁺]_i enhancing contractile efficiency in both NOS1^−/− mice and heart failure models (25, 35, 36). In large animal models, allopurinol restores mechano-energetic uncoupling (15). Interestingly, XOR inhibitors did not have clinical benefits in a broad heart failure population, although it may improve outcomes in patients with high uric acid levels (37) and may benefit patients with hypertension and ischemic heart disease (38–40).

Mechanism of Action and Pharmacology of Hydralazine

HYD is a peripheral vasodilator used for decades to treat essential hypertension and heart failure. The mechanism of its effect on the vasculature remains unknown. It is proposed to inhibit the endoplasmic reticulum Ca²⁺ release in vascular smooth muscle (41, 42), although the molecular mechanism by which HYD regulates the IP₃ receptor (a member of the superfamily of homotetrameric ligand-gated intracellular Ca²⁺ channels, which includes RyR2) has not been elucidated. Because HYD increases contractility and Ca²⁺ transients in myocardial fibers (43), it may positively affect the unregulated RyR2 activity in NOS1^−/−.

We showed that HYD enhanced depressed NOS1^−/− CM contractility and [Ca²⁺]_i responses resulting from NO/redox imbalance. Importantly, HYD restored the increased SR Ca²⁺ leakage both alone or in combination with NTG. This feature suggests a regulatory action of HYD on RyR2 gating, possibly depressing Ca²⁺ release in diastole and enhancing gating in systole.

Accordingly, a more efficient handling of Ca²⁺ would underlie the recovery of the SR Ca²⁺ content observed in CMs treated with HYD or HYD plus NTG. Biochemically, HYD + NTG exhibited an additive superoxide scavenging capacity at concentrations as low as 1 μm, as shown by DHE staining. This effect may be correlated with the additive acceleration in SR Ca²⁺ reuptake in NOS1^−/− induced by combining HYD with NTG. Peroxynitrite links superoxide production to Ca²⁺ reuptake. Although ONOO⁻ generation depends on superoxide availability, PLB phosphorylation is decreased due to ONOO⁻-dependent activation of phosphatases, impairing Ca²⁺ decay and relaxation in NOS1^−/− (31, 44, 45). Furthermore, HYD and NTG scavenged ONOO⁻ in NOS1^−/− heart homogenates (data not shown). Together these findings suggest a role for scavenging of ONOO⁻ on the recovery of the Ca²⁺ reuptake rate, a key process in E-C coupling in mouse CMs (44, 46). Considerable evidence supports the hypothesis that oxidative stress induces cardiac injury by oxidizing cellular constituents, including proteins critical for E-C coupling, and largely by diminishing NO bioactivity (9), thus playing a role in heart failure pathophysiology (47). Therefore, the effect of HYD, which reduced ONOO⁻ in NOS1^−/− hearts (data not shown), and scavenged XOR-mediated superoxide production may impact the redox regulation of RyR2 gating and the pharmacology of the response. Importantly, our results agree with Leiro et al. (48), who suggested antioxidant properties for HYD.

Mechanism of Action and Pharmacology of Organic Nitrates

Janero et al. (49) showed that NTG increases cardiac nitrosylation in vivo, and this effect was potentiated by xanthine oxidase inhibition with allopurinol, highlighting the interactions between NO and XOR-derived superoxide. Importantly, decreased NOS1 in cardiac SR contributes to depressed contractile reserve in response to pacing. It has been hypothesized that NTG may restore ion channel S-nitrosylation in NOS1^−/− myocytes (24). We showed that NTG and ISDN restored the normal amplitude of calcium transients toward WT levels in NOS1^−/− cells treated with HYD. This effect would be consistent with a cGMP-dependent regulation of L-type Ca²⁺ channels and/or restoration of RyR2 S-nitrosylation (33, 50) and occurs without affecting cardiac contractility, which correlates with the observed improvement in myofilament sensitivity induced by NTG in NOS1^−/− CMs. This improvement may also be attributed to the restoration of the S-nitrosylation/normal redox state of redox-sensitive sites on sarcomeric proteins. Thus, this responsiveness is evident across the broad range of pacing frequencies tested, confirming that organic nitrates enhance contractile efficiency. It is reasonable to speculate that the lack of NOS1 and the resulting NO/redox imbalance reduces responsiveness of myofilaments to Ca²⁺ (as confirmed by our results). This reduction was restored by NO donors. These findings suggest that a feasible mechanism for the positive clinical outcomes of organic nitrates (when combined with HYD) may be by regulating intracellular Ca²⁺ handling and restoring myofilament sensitivity impaired by NO/redox imbalance, thereby modulating E-C coupling toward a more efficient performance.

Inhibition of NOX and XOR by NO has been previously demonstrated (26, 51–53), supporting our results. In contrast to the low concentrations required to scavenge superoxide production in isolated myocytes, the effective concentrations of NTG to inhibit the substrate-induced activity of these enzymes in cardiac homogenates exceeded achievable blood levels of this drug (10 ng/ml, 44 nm) (54).

Despite the successful use of nitrates such as NTG or ISDN in the treatment of a variety of cardiovascular diseases, nitrate tolerance during long-term use in heart failure has been described. Co-treatment with HYD has been demonstrated to prevent this effect (55) and several authors have tried to explain the mechanism of this phenomenon (56–58).

The effects of organic nitrates on SR Ca²⁺ leak appear to be uncertain and depend strongly on several variants such as the concentration or nature of the NO donor. As shown in the supplemental material, NTG exerted differential effects along the range of concentrations from 0.1 to 100 μm. On the other hand, ISDN reduced the SR leak in NOS1^−/− at very low SR Ca²⁺ contents but did not affect it at increasing Ca²⁺ loads. We speculate that this differential response may correspond to the delicate equilibrium of the nitrosylation/denitrosylation cycle involved in the regulation of RyR2 activity (50).

Combination of HYD and NTG

The effect of NTG in NOS1^−/− hearts may appear puzzling. At lower concentrations (0.1 and 1 μm), NTG did not significantly affect SL shortening, Ca²⁺ transient, SR Ca²⁺ leak or Ca²⁺ decline. It reduced the Ca²⁺ frequency response with a concomitant acceleration of Ca²⁺ decline at 10 μm. However, despite 10 μm NTG improving Ca²⁺ reuptake, there was a trend toward increasing SR Ca²⁺ leak (or at least effecting no change on SR Ca²⁺ leak; Fig. 3E), which would counteract the improvement in Ca²⁺ influx by the SR, thereby abolishing the SR Ca²⁺ content recovery. This balancing may explain why there was no change in SR Ca²⁺ load, although this was not reflected on the Ca²⁺ amplitude, which was reduced. Because Ca²⁺ influx (I_Ca) was enhanced in NOS1^−/− (30), the reduced RyR2 gating in the presence of 10 μm NTG (or ISDN) may be caused by the down-regulation of the enhanced I_Ca in NOS1^−/− by exogenous NO (as previously shown) (24). Thus, whereas hydralazine alone reduced RyR2 leak, when combined with NTG (or ISDN), down-regulation of the Ca²⁺-induced Ca²⁺ release (via inhibition of I_Ca) would oppose the effect of hydralazine, thereby leading to a reduced Ca²⁺ transient amplitude. Together with the reduced leak and increased reuptake under these conditions, the contractile reserve was recovered in NOS1^−/−. Thus, the evidence of improved EC coupling in this model using the HYD-NTG treatment would be restoration of Ca²⁺ levels toward those exhibited by WT CMs and recovery of the increasing Ca²⁺ frequency response and myofilament responsiveness to Ca²⁺, where NTG played a key role.

Both HYD and NTG scavenged superoxide but were more potent in combination. However, we must consider that the chemical reactions of each molecule with superoxide yield different products. Although HYD can effectively scavenge the radical, the combination of NO and superoxide yields ONOO⁻, a reactive nitrogen species even more harmful than superoxide. In this sense, we speculate that ONOO⁻ may target RyR2 in a similar manner as superoxide. Therefore, the SR Ca²⁺ leak was still elevated in the presence of NTG. Fig. 4F showed that despite 10 μm NTG inducing a trend toward an increased leak in NOS1^−/−, 100 μm NTG restored it to baseline. This observation suggests that higher concentrations of NTG not only scavenge superoxide but also the surplus would counteract the deficiency of NO in NOS1^−/− and is perhaps the mechanism of the combination of NTG and HYD.

Effects in SHHF Rats

Although this study is focused on NOS1^−/− myocytes as a model of NO/redox imbalance, we also tested the effect of these drugs alone or in combination on failing CMs from SHHF rats. The results obtained in SHHF CMs correlate with those in NOS1^−/− mice. HYD alone or in combination with NTG improved the Ca²⁺-frequency response and FFR. NTG alone induced a trend to increase contractility at 4 Hz with no changes in ΔCa²⁺, suggesting an improvement in myofilament sensitivity. These results allow us to speculate that our approach may be extrapolated to heart failure in humans.

Conclusion

Our data show novel effects of HYD and NTG. In combination they quench superoxide in isolated cells, concomitantly with physiologic functional effects, including improved myocardial contractility and Ca²⁺ cycling. At higher in vitro concentrations, this combination scavenges superoxide and peroxynitrite production from two major ROS generating enzymatic systems, NOX and XOR. This improvement in myocyte performance, normalization of SR Ca²⁺ leak, regulation of RyR2 gating, and myofilament sensitivity by NTG may be associated in part with normalizing NO/redox equilibrium. Thus, these drugs in combination exert direct myocardial effects that provide a mechanistic basis for the favorable functional and structural responses in the treatment of congestive heart failure.