Cardioprotection by HO-4038, a novel verapamil derivative, targeted against ischemia and reperfusion-mediated acute myocardial infarction

Abstract

Many cardiac interventional procedures, such as coronary angioplasty, stenting, and thrombolysis, attempt to reintroduce blood flow (reperfusion) to an ischemic region of myocardium. However, the reperfusion is accompanied by a complex cascade of cellular and molecular events resulting in oxidative damage, termed myocardial ischemia-reperfusion (I/R) injury. In this study, we evaluated the ability of HO-4038, an N-hydroxypiperidine derivative of verapamil, on the modulation of myocardial tissue oxygenation (Po₂), I/R injury, and key signaling molecules involved in cardioprotection in an in vivo rat model of acute myocardial infarction (MI). MI was created in rats by ligating the left anterior descending coronary artery (LAD) for 30 min followed by 24 h of reperfusion. Verapamil or HO-4038 was infused through the jugular vein 10 min before the induction of ischemia. Myocardial Po₂ and the free-radical scavenging ability of HO-4038 were measured using electron paramagnetic resonance spectroscopy. HO-4038 showed a significantly better scavenging ability of reactive oxygen radicals compared with verapamil. The cardiac contractile functions in the I/R hearts were significantly higher recovery in HO-4038 compared with the verapamil group. A significant decrease in the plasma levels of creatine kinase and lactate dehydrogenase was observed in the HO-4038 group compared with the verapamil or untreated I/R groups. The left ventricular infarct size was significantly less in the HO-4038 (23 ± 2%) compared with the untreated I/R (36 ± 4%) group. HO-4038 significantly attenuated the hyperoxygenation (36 ± 1 mmHg) during reperfusion compared with the untreated I/R group (44 ± 2 mmHg). The HO-4038-treated group also markedly attenuated superoxide production, increased nitric oxide generation, and enhanced Akt and Bcl-2 levels in the reperfused myocardium. Overall, the results demonstrated that HO-4038 significantly protected hearts against I/R-induced cardiac dysfunction and damage through the combined beneficial actions of calcium-channel blocking, antioxidant, and prosurvival signaling activities.

cardiovascular disease is the leading cause of death worldwide. Approximately six million people die of cardiovascular disease every year. Ischemic heart disease, which is caused by insufficient blood supply to regions of the myocardium, is a major form of cardiovascular disease. Although restoration of blood supply to the ischemic region is essential for salvage of myocardium, the accompanying reperfusion during the first few minutes can worsen rather than improve myocardial dysfunction. This phenomenon, termed myocardial ischemia-reperfusion (I/R) injury, can paradoxically reduce the beneficial effects of myocardial reperfusion.

Ischemia in a healthy myocardium produces a complex cascade of events including decrease in ATP content, impaired function of a number of ion channels, increase in acidosis, and elevation of cytosolic calcium ion concentration (Ca²⁺). Accumulation of intracellular calcium can lead to the activation of phospholipase A₂ (13) and phosphatidyl choline. This lysophosphoglyceride has detergent properties and can alter electrophysiological properties and induce arrhythmias (5), contracture (3), delayed afterdepolarizations (39), and inhibit Na-K APTase activity (15). Elevation of calcium levels can also result in an increase in intracellular oxygen free radicals and mitochondrial damage (28) and impair sarcoplasmic reticulum Ca²⁺ transport (43) and abnormal contractile function after recovery from ischemia (41).

Verapamil, a calcium channel blocker (calcium antagonist), is used for the treatment of hypertension, angina pectoris, migraines, and some types of arrhythmia. Verapamil works by relaxing blood vessels, increasing blood flow and oxygen to the heart, and normalizing heart rhythms. Its primary mechanism of action is to block L-type calcium channel (I_Ca,L) currents and prevent calcium influx into cells of vascular smooth muscle, specialized sinoatrial and atrioventricular nodes (negative chronotropic effects), and myocardium (negative inotropic effects). The blockage of calcium influx into the smooth muscle cells of the coronary vasculature prevents excessive vasoconstriction after I/R injury and allows adequate blood supply to the myocardium and thus improves overall collateral blood flow (8). Verapamil protects the heart from I/R injury by preventing excessive accumulation of intracellular calcium, thus preserving mitochondrial function. Pretreatment of hearts with verapamil has also been shown to reduce ischemia-induced conduction delay (35), attenuate postperfusion myocardial damage (40), and protect lung I/R injury (54).

Reactive oxygen species (ROS) such as superoxide anion (O₂^•−), hydroxyl radical (^•OH), and hydrogen peroxide (H₂O₂) react with various biological tissues including myocardium and contribute to the pathophysiology of ischemic injury. Although reperfusion of ischemic myocardium is the initial step in preventing cardiac damage, reoxygenation itself can induce the formation of various deleterious oxidants, e.g., O₂^•−, ^•OH, H₂O₂, ROOH, and Fe⁴⁺, apart from other oxidants (32, 56). During reperfusion, oxygen interacts with the damaged mitochondrial respiratory chain, xanthine oxidase, NADPH oxidase, and arachidonic pathway to contribute to the generation of ROS (10, 24). ROS produce deleterious effects on myocardial cellular membrane proteins, cellular DNA, and mitochondria of cardiac cells.

Oxidative stress during myocardial reperfusion also decreases the bioavailability of the intracellular signaling molecule nitric oxide (NO), thereby inactivating its cardioprotective properties. Akt, the serine-threonine kinase, activation leads to the survival of many cell types including cardiomyocytes. Various studies have demonstrated that Akt has antiapoptotic effects (2, 31). Several pharmacological agents have been shown to be cardioprotective by modulating the Akt activity (2, 19, 55). The antiapoptotic Bcl-2 gene codes for a 25-kDa protein maintain organelle integrity and prevent the release of cytochrome c from mitochondria. During I/R injury, apoptosis is mediated by different signaling cascades that involve the mitochondria-initiated pathway and are mediated by free radicals and oxidative stress, resulting in the release of cytochrome c from mitochondria, activation of caspase-9, and downregulation of Bcl-2 (9). Hearts from transgenic mice overexpressing Bcl-2 prevent cytosolic acidification during ischemia and reduce I/R injury (14).

A variety of free-radical scavengers, antioxidant molecules, and enzymes including low molecular weight antioxidants (LMWA), metal chelators, superoxide dismutase (SOD), and catalase have been shown to protect hearts from I/R injury (1, 11, 52). The LMWA are usually reducing agents that act stoichiometrically and are depleted rapidly when exposed to persistent oxidative stress. The antioxidant enzymes SOD and catalase are restricted to the extracellular compartments. Nitroxides are a class of LMWA that protect against oxidative damage in various pathological processes. Nitroxides are stable radicals that rapidly cross cell membrane, preempt free-radical formation by oxidizing redox-active metal ions, and function as both intra- and extracellular SOD mimics (22, 44). The reduced form of the nitroxide, namely hydroxylamine, also has antioxidant activity (20, 37).

We hypothesized that the addition of an antioxidant moiety to the verapamil molecule would provide an additional level of protection by scavenging ROS generated during the early minutes of reperfusion. We modified the verapamil molecule with a heterocyclic nitroxide-precursor 1-hydroxy-2,2,6,6-tetramethyl-1,2,3,6-tetrahydropyridine group (Fig. 1), which transforms into piperidine nitroxide in cells and tissues. Thus the new verapamil derivative with its nitroxide precursor, hereafter referred to as HO-4038 (Fig. 1), was hypothesized to exhibit the combined beneficial actions of verapamil and nitroxide antioxidant in the prevention of I/R-induced myocardial injury. We studied the cardioprotective efficacy of HO-4038 compared with verapamil in a rat model of acute myocardial infarction (MI). The results revealed that HO-4038 significantly attenuated the I/R-induced cardiac injury and dysfunction through its antiarrhythmic, antioxidant, and prosurvival activities.

Fig. 1.

Molecular structure and characterization of verapamil and HO-4038. A: molecular structure of verapamil and its piperidine derivative HO-4038. The N-hydroxypiperidine group in HO-4038 can undergo conversion to the corresponding N-oxy-piperidine (nitroxide) in cells and tissues. B: representative electron paramagnetic resonance (EPR) spectrum showing the conversion of HO-4038 into paramagnetic nitroxide in aerated PBS. C: effect of verapamil and HO-4038 on L-type calcium current (I_Ca,L) in canine ventricular myocytes. C, left: representative peak I_Ca,L recorded in response to a depolarizing step to 0 mV from a holding potential of −50 mV. The arrow to the left of the raw traces indicates the zero current line. C, right: data peak I_Ca,L. To avoid potential difference due to cell size, all data were normalized to cell size and measured as cell capacitance. The results are expressed as means ± SD (n = 11, baseline; n = 5, verapamil; n = 6, HO-4038). *P < 0.05 vs. baseline. The results show that HO-4038 is as effective as verapamil in inhibiting I_Ca,L in freshly isolated canine myocytes.

MATERIALS AND METHODS

Chemicals.

Verapamil and HO-4038 were synthesized as previously described (29). The compounds were freshly prepared in DMSO and diluted with PBS before administration. Dihydroethidium (DHE), xanthine, xanthine oxidase, 2,2-azobis-2-amidonopropane dihydrochloride, diethylenetriaminepentaacetate, ferrous ammonium sulfate, and triphenyltetrazolium chloride (TTC) were obtained from Sigma Chemicals (St. Louis, MO). 4-Amino-5-methylamino-2,7-difluorofluorescein (DAF-FM) was obtained from Invitrogen (Molecular Probes). 5-(Diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide (DEPMPO) was obtained from Radical Vision (Jerome, Marseille, France). Nitrite/nitrate (NOx) colorimetric assay kit was obtained from Cayman Chemicals (Ann Arbor, MI). All other reagents were analytical grade or higher and purchased from Sigma-Aldrich, unless otherwise mentioned.

Measurement of superoxide, hydroxyl, and alkylperoxyl radicals by electron paramagnetic resonance spectroscopy.

The superoxide, hydroxyl, and alkylperoxyl radical-scavenging ability of HO-4038 in vitro was determined by using electron paramagnetic resonance (EPR) spectroscopy (18). A mixture of xanthine (0.2 mM) and xanthine oxidase (0.02 U/ml) in PBS (pH 7.4) was used to generate superoxide radicals. Hydroxyl radicals were generated by reacting ferrous ammonium sulfate (0.1 mM) with hydrogen peroxide (0.1 mM) in PBS. Alkylperoxyl radicals were generated through the thermolytic fission of 2,2-azobis-2-amidonopropane dihydrochloride (25 mM) in aerobic PBS solution at 37°C. All EPR measurements were performed in PBS (pH 7.4) containing DEPMPO (1 mM) and diethylenediaminepentaacetate (0.1 mM) in the presence or absence of different concentrations of HO-4038. The superoxide, hydroxyl, and peroxyl radicals were detected as DEPMPO-OOH, DEPMPO-OH, and DEPMPO-OOR adducts, respectively. The attenuation of DEPMPO adduct generation was quantified by double integration and expressed as a percentage of untreated (without HO-4038) levels.

Patch-clamp study of calcium currents in isolated ventricular cardiomyocytes.

Left ventricular midmyocardial myocytes were isolated from healthy canine hearts as described previously (23). Normal cardiac structure and function were confirmed by electrocardiogram and echocardiogram. All canine protocols were approved by the Institutional Animal Care and Use Committee of Ohio State University. After isolation, the myocytes were incubated at room temperature in a standard incubation buffer containing (in mM) 118 NaCl, 4.8 KCl, 1.2 KH₂PO₄, 0.68 glutamine, 10 glucose, 5 pyruvate, and 1 CaCl₂ along with insulin (1 μM) and bovine serum albumin (1%) until use. The myocytes were placed in a laminin-precoated cell chamber (Cell Microcontrols, Norfolk, VA) and superfused with bath solution containing (in mM) 135 NaCl, 5 MgCl₂, 5 KCl, 10 glucose, 1 CaCl₂, 5 HEPES, and 2 4-aminopyridine (to block transient outward K⁺ and ultrarapid-delayed K⁺ rectifier currents) at pH 7.4 and temperature 36 ± 0.5°C. Pipette solution contained (in mM) 20 tetraethylammonium chloride, 125 CsCl, 5 MgATP, 10 EGTA, 3.6 creatine phosphate, and 10 HEPES (pH 7.2). Conventional whole cell patch-clamp techniques were used. All data acquisition was performed with pClamp software (version 8+; Axon Instruments, Union City, CA) and an Axopatch (200A) patch-clamp amplifier (Axon Instruments). A holding potential of −50 mV was used to inactivate the sodium current. A series of 80-ms test steps was used to elicit I_Ca,L at voltages from −40 to +50 mV in 10-mV increments. I_Ca,L recordings began 3 min after patch rupture, and recordings with verapamil or HO-4038 (5 μM) were obtained after 7 min of superfusion, based on the time to reach steady state inhibition in initial pilot studies.

Isolated heart preparation.

The experimental protocol used in the present study was approved by the Institutional Animal Care and Use Committee of Ohio State University and conformed to the Institute of Laboratory Animal Resources (1996). All isolated hearts were perfused and tested using a modified Langendorff apparatus for isolated heart. Male Sprague-Dawley rats (300–350 g) were anesthetized intraperitoneally with 60 mg/kg sodium pentobarbital (Nembutal) and heparinized with 500 IU/kg heparin. Access to the heart was gained surgically via bilateral midaxial thoracotomy, and hearts were removed placed into ice-cold Krebs-Henseleit buffer to residual contractions. The aorta was immediately cannulated to the perfusion apparatus. Hearts were then retrogradely perfused with a modified Krebs-Henseleit buffer containing (in mM) 120 NaCl, 25 NaHCO₃, 1.2 MgSO₄, 1.2 KH₂PO₄, 1.2 CaCl₂, and 11 glucose. The perfusate buffer was saturated with a 95% O₂-5% CO₂ gas mixture and maintained at 37°C. A latex balloon was inserted in the left ventricle via the left atrium and inflated with water to produce an end-diastolic pressure of 8–12 mmHg. The hemodynamic and contractile functions of the heart were continuously recorded with a computerized data acquisition system (PC PowerLab with Chart 5 software; ADI Instruments, Colorado Springs, CO). Coronary flow (CF), left ventricular systolic pressure, left ventricular end-diastolic pressure (LVDP), and heart rate were monitored continuously. The LVDP was calculated as the difference between left ventricular systolic pressure and left ventricular diastolic pressure. The rate-pressure product (RPP), an index of myocardial workload, was calculated as LVDP × heart rate. The CF rate was measured using a flow meter with an inline probe (Transonic Systems, Ithaca, NY).

I/R protocol.

Isolated hearts were perfused for 15 min to stabilize the hemodynamic functions and were then subjected to 30 min of no-flow global ischemia, followed by 45 min of reperfusion at 37°C. Test drugs, verapamil (10 μM) and HO-4038 (50 μM), were administered through a side-arm infusion for 1 min at a controlled infusion rate of 1 ml/min using an infusion apparatus (Harvard Apparatus, Holliston, MA). Immediately after drug infusion, a global no-flow ischemia was induced using an overhead shut-off valve and was continued for 30 min. Aerobic perfusion was then subsequently reintroduced, and hemodynamic data were obtained for 45 min into reperfusion. The hemodynamic measurements, biochemical assays, and infarct size measurements were done on the same hearts (n = 6).

Creatine kinase and lactate dehydrogenase assays.

The extent of myocardial damage was assessed by determining the amount of creatine kinase (CK) and lactate dehydrogenase (LDH) in the coronary effluents collected both before ischemia and during reperfusion. The level of CK and LDH release in the coronary effluents was determined by using commercially available kits obtained from Sigma Diagnostics (St. Louis, MO) (LDH) and Catachem (Bridgeport, CT) (CK). The enzyme activity was determined by measuring the rate of change in absorbance at 340 nm for 5 min using a Varian Cary 50 spectrophotometer (Varian, Palo Alto, CA).

Induction of myocardial I/R in vivo.

Male Sprague Dawley rats (300–350 g) were used for in vivo acute model of MI. Rats were randomly divided into the following three groups of six animals each: 1) I/R (vehicle/PBS treated), 2) verapamil (50 μM, 0.5 ml), and 3) HO-4038 (50 μM, 0.5 ml). Rats were anesthetized with ketamine (50 mg/kg ip) and xylazine (5 mg/kg ip) followed by isoflurane (1.5–2.0%) with air. Animals were infused with the drugs through the jugular vein 10 min before the induction of ischemia. MI was created by ligating the left anterior descending coronary artery (LAD), as described (42). An oblique 12-mm incision was made 8 mm away from the left sternal border toward the left armpit. The chest cavity was opened with scissors by a small incision (10 mm in length) at the level of the fourth or fifth intercostal space, 3 to 4 mm from the left sternal border. The LAD was visualized as a pulsating bright red spike, running through the midst of the heart wall from underneath the left atrium toward the apex. The LAD artery was ligated 1 to 2 mm below the tip of the left auricle using a tapered needle and a 6-0 polypropylene ligature. The ligature was passed underneath the LAD, and a double knot was made to occlude the artery. Occlusion was confirmed by the sudden change in color (pale) of the anterior wall of the left ventricle. The chest cavity was closed by bringing together the fourth and fifth ribs with one 4-0 silk suture. The layers of muscle and skin were also closed with a 4-0 polypropylene suture. After LAD ligation, successful infarction was confirmed by an ST elevation on electrocardiograms that were recognized in all surgical groups of animals. After 30 min, the ligation was released, and the chest was closed. After reinstallation of spontaneous respiration, animals were extubated and allowed to recover from the anesthesia.

Measurement of myocardial Po₂ using in vivo EPR oximetry.

To understand the dynamics of tissue oxygenation during the I/R event and to study the effect of HO-4038 on myocardial tissue oxygenation, we used in vivo EPR oximetry for continuous monitoring of tissue oxygenation (Po₂) in the ischemic site. The principle of EPR oximetry is based on molecular oxygen-induced line-width changes in the EPR spectrum of a paramagnetic probe. The probe is a microcrystal of stable, safe, and nontoxic paramagnetic material that can be permanently implanted in the tissue region of interest using a 25-gauge needle. Once implanted, the probe responds to Po₂ in the immediate surrounding, mostly at the crystal-tissue interface, enabling magnetic resonance-based noninvasive and repeated measurements of Po₂ for a prolonged period from the site of implantation. The technology has been well validated for measurements of Po₂ from single cells to whole organs (16, 26, 38).

The myocardial Po₂ measurements in the present study were performed using an in vivo EPR spectrometer (Magnettech, Berlin, Germany) equipped with automatic coupling and tuning controls for measurements in beating hearts. Microcrystals of lithium octa-n-butoxy-naphthalocyanine (LiNc-BuO) were used as a probe for EPR oximetry. Rats, under inhalation anesthesia (air containing 1.5–2.0% isoflurane), were implanted with the oxygen sensing probe in the left ventricular midmyocardium. The animal was placed in a right lateral position with the chest open to the loop of a surface-coil resonator. EPR spectra were acquired as single 30-s duration scans. The instrument settings were: microwave frequency, 1.2 GHz (L-band), incident microwave power, 4 mW; modulation amplitude, 180 mG, modulation frequency 100 kHz; and receiver time constant, 0.2 s. The peak-to-peak width of the EPR spectrum was used to calculate Po₂ using a standard calibration curve (38). Similarly, myocardial Po₂ was measured after 24 h of reperfusion.

Estimation of plasma CK, LDH, and NOx concentrations.

Plasma concentrations of CK, LDH, and NOx were determined in rats by subjecting them to 30 min of ischemia and 1 h of reperfusion. About 0.5 ml of blood samples were collected by tail nick after 1 h of reperfusion. The blood samples were centrifuged, and the plasma was stored at −80°C until the analyses were performed. Plasma levels of CK and LDH in the circulation were determined using commercially available kits obtained from Catachem and Sigma Diagnostics, respectively, according to the manufacturer's instructions. The quantitative measurement of total NO in plasma is based on the enzymatic conversion of nitrate to nitrite by nitrate reductase, followed by the spectrophotometric quantitation (λ = 550 nm) of nitrite levels using a commercially available Griess reagent kit (Cayman Chemicals) using a Beckman AD 340 ELISA plate reader (Beckman Coulter, Fullerton, CA). The concentration of nitrite (indicative of NOx in the original plasma samples) was calculated from a standard curve (1 to 35 μM) and used for the determination of total NOx concentrations.

Measurement of myocardial infarct size.

After 24 h of reperfusion, the animals from all the groups were euthanized, and their hearts were then cut into four transverse slices. The slices were then incubated at 37°C for 10 min with 1.5% TTC to determine the infarct area and the area at risk. Photographs were taken under a dissecting microscope. Left ventricular area, area at risk, and infarct area were determined by computerized planimetry using MetaVue image analysis software (Molecular Devices, Sunnyvale, CA). The area of myocardial tissue showing white color was defined as infarct, and the region in red was defined as the area at risk. Infarct size was expressed as percentage of area at risk.

Measurement of superoxide in the infarct region of rat heart.

All pretreated rats with vehicle only (I/R), verapamil, and HO-4038 were subjected to 30 min of LAD ligation followed by 10 min of reperfusion. Rats were euthanized at 10 min of reperfusion, and hearts were placed immediately in ice-cold PBS and then embedded in optimal cutting temperature (OCT) for cryosectioning. Frozen heart sections were thawed, and superoxide generation in the heart tissue was determined using DHE fluorescence (34). The cell-permeable DHE is oxidized to fluorescent hydroxyethidium (HE) by superoxide, which is then intercalated into DNA. Since it is known that there is a burst of oxygen-free radical generation in the early minutes of reperfusion in hearts subjected to I/R, we measured the DHE fluorescence at 10 min of reperfusion. The frozen segments from the heart tissue were cut into sections 6 μm thick, which were then placed on glass slides. DHE (10 μM) was topically applied to each tissue section. The slides were incubated in a light-protected chamber at 37°C for 30 min. The slides were then washed several times with PBS to remove nonspecific DHE staining and coverslipped with aqueous mounting media. The images of the tissue sections were obtained using a fluorescence microscope (Nikon, Tokyo, Japan) with a rhodamine filter (green excitation, 550 nm; and red emission, 573 nm). Fluorescence intensity, which positively correlates with the amount of superoxide generation, was determined in the myocardial tissue using MetaMorph (Molecular Devices) image analysis software.

Measurement of myocardial NO production.

The NO production in rat hearts subjected to 30 min ischemia (LAD ligation), followed by 10 min of reperfusion, was measured by using DAF-FM diacetate. DAF-FM diacetate is an important reagent for quantifying low levels of NO production. DAF-FM is essentially nonfluorescent until it reacts with NO to form a fluorescent benzotriazole. DAF-FM diacetate is cell permeable and passively diffuses across cellular membranes. Once inside the cells, it is deacetylated by intracellular esterases to become DAF-FM. Hearts, after 30 min of ischemia or 10 min of reperfusion, were placed in an ice-cold PBS buffer and embedded in OCT for cryosectioning. The frozen segments were cut into 6-μm thick sections and incubated with 10 μM of DAF-FM diacetate for 30 min in the dark at 37°C. The images of the tissue sections were obtained using a Nikon fluorescence microscope with a fluorescein isothiocyanate filter (blue excitation, 495 nm; and green emission, 510 nm). The increase in fluorescence intensity, which correlates with the amount of NO production, was quantitatively determined using MetaMorph (Molecular Devices) image analysis software.

Western blot analysis.

For determination of Akt, phospho-Akt, and Bcl-2, Western blots were performed with the tissue homogenates prepared from the anterior wall of the left ventricles of rats from pre-I/R, I/R, verapamil, and HO-4038 groups. After the treatment, rats were anesthetized and euthanized at 24 h of reperfusion. The hearts were rapidly explanted, rinsed in ice-cold PBS (pH 7.4), containing 500 U/ml heparin to remove red blood cells and clots, frozen in liquid nitrogen, and stored at −80°C until analysis. Heart tissues were homogenized in TN1 lysis buffer containing 50 mM Tris (pH 8.0), 10 mM EDTA, 10 mM NaF, 1% Triton X-100, 125 mM NaCl, 1 mM Na₃VO₄, and 1% protease inhibitor (Sigma). The tissue homogenate was incubated for 60 min on ice, followed by microcentrifuging the cell homogenate at 10,000 g for 15 min at 4°C. Aliquots of 75 μg of protein from each sample were boiled in Laemmli buffer (Bio-Rad) containing 1% 2-mercaptoethanol for 5 min. The protein was separated by SDS-PAGE, transferred to polyvinylidene difluoride membrane, and probed with primary antibodies for Bcl-2, Akt, and phospho-Akt (Ser-473; Cell Signaling, Beverly, MA). The primary antibodies were exposed to overnight at 4°C followed by horseradish peroxidase-conjugated secondary antibodies (Amersham Biosciences) for 1 h. The membranes were then developed by an enhanced chemiluminescence detection system (ECL Advanced kit). The same membranes were reprobed for β-actin. The protein intensities were quantified by an image-scanning densitometer (Scion). To quantify the phosphospecific signal in activated proteins, we first subtracted the background and then normalized the signal to the amount of β-actin or total target protein in the tissue homogenate (47). Data were expressed as percentages of the expression in the control group.

Data analysis.

The statistical significance of the results was evaluated using one-way ANOVA followed by a Student's t-test. The values were expressed as means ± SD. A P value of <0.05 was considered significant.

RESULTS

Metabolic conversion of HO-4038 to antioxidant piperidine nitroxide metabolite.

HO-4038 has been designed to confer antioxidant activity by the introduction of the N-hydroxypiperidine group to the parent verapamil compound (Fig. 1A). The piperidine nitroxide metabolite is paramagnetic and can be measured easily by EPR spectroscopy. HO-4038 also undergoes redox cycling in solutions to maintain a steady-state concentration of nitroxide.

HO-4038 blocks calcium currents as effectively as parent verapamil.

To confirm whether the calcium channel-blocking property of verapamil was retained by the structural modification introduced in HO-4038, we studied the effect of HO-4038 on the inhibition of I_Ca,L in freshly isolated canine left ventricular myocytes using the whole cell patch-clamp technique. HO-4038, at 5 μM concentration, significantly (P < 0.01) inhibited the calcium current in the canine ventricular myocytes (Fig. 1C). Furthermore, the calcium channel-blocking efficacy of HO-4038 was similar to that of verapamil at the same concentration. The results clearly revealed that the calcium channel-blocking property of verapamil was not altered by the structural modification introduced in HO-4038.

Free-radical scavenging abilities of HO-4038.

There is a substantial body of evidence showing that nitroxides are scavengers of free radicals. Hence we studied the superoxide, hydroxyl, and peroxyl radical scavenging ability of HO-4038 in vitro using spin-trapping EPR spectroscopy. DEPMPO spin trap (1 mM) was used for direct detection of superoxide, hydroxyl, and peroxyl radicals as DEPMPO-OOH, DEPMPO-OH, and DEPMPO-OOR adducts, respectively. Figure 2 shows the scavenging effect of HO-4038 against superoxide, hydroxyl, and peroxyl radicals. HO-4038 (100 μM), used against 1 mM DEPMPO, decreased the intensity of the DEPMPO-OOH spectrum by more than 40% (Fig. 2A). Challenging of 1 mM DEPMPO with 1 mM HO-4038 inhibited the superoxide intensity by more than 90%. Likewise, the hydroxyl radical adduct (DEPMPO-OH), measured using 1 mM DEPMPO, was inhibited by more than 40% with 100 μM and 90% with 1 mM HO-4038 (Fig. 2B). Similarly, 100 μM HO-4038 decreased the peroxyl radicals by more than 80% (Fig. 2C) and 500 μM HO-4038 completely abolished the peroxyl radical adduct formation. Overall, the EPR study clearly established that the HO-4038 was capable of scavenging reactive oxygen radicals in a dose-dependent fashion.

Fig. 2.

Free-radical scavenging effect of HO-4038. The superoxide (O₂^•−), hydroxyl (^•OH), and alkylperoxyl (ROO·) radicals were determined in presence of HO-4038 by EPR spectroscopy using 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide (1 mM) as a spin trapping agent. Quantification (left) and EPR spectra (right) show a dose-dependent loss of EPR signal intensity corresponding to superoxide (A), hydroxyl (B), and alkylperoxyl (C) radicals. The bar graphs represent means ± SD for 3 independent experiments.

HO-4038 improves hemodynamic and contractile functions.

Isolated hearts were subjected to 30 min of global ischemia followed by 45 min of reperfusion. CF, LVDP, and RPP were continuously monitored before the induction of global ischemia and during the reperfusion. Verapamil or HO-4038 was administered to the heart via a side arm infusion for 1 min before the onset of ischemia. The concentrations of verapamil and HO-4038 that could be administered without loss of functional recovery were 10 and 50 μM, respectively. Hemodynamic data were collected for 45 min into reperfusion and expressed as percentages of their preischemic baseline values: CF, 16 ± 2 ml/min; LVDP, 111 ± 13 mmHg; and heart rate, 295 ± 8 beats/min. The untreated I/R hearts subjected to 30 min of global ischemia followed by 45 min of reperfusion showed a significant decrease in CF (in percentages; 44 ± 5); LVDP (18 ± 3); and RPP (19 ± 5) compared with preischemic baseline values (Fig. 3, A–C). Hearts pretreated with the verapamil showed a significant (P < 0.05) improvement in the recovery of contractile functions compared with the I/R hearts. Furthermore, HO-4038 showed a significantly better recovery compared with I/R (P < 0.01) as well as verapamil-treated hearts.

Fig. 3.

Effect of HO-4038 on the recovery of hemodynamic and contractile functions. The plot shows the recovery of coronary flow (CF; A), left ventricular (LV) developed pressure (LVDP; B), and rate pressure product (RPP; C) at the end of 45 min of reperfusion. Hearts were infused with verapamil (Vera; 10 μM) or HO-4038 (50 μM) for 1 min before 30 min of global ischemia followed by 45 min of reperfusion at 37°C. The release of creatine kinase (CK; D) and lactate dehydrogenase (LDH; E) in the coronary effluents collected at 15 min of reperfusion are also shown. F: infarct size expressed a percentage of area at risk (AAR). The treatment protocol was the same except that the reperfusion time was 120 min. Myocardial infarction was done by triphenyltetrazolium chloride (TTC) staining. Representative images of TTC-stained infarct (white) and noninfarct (red) region slices are displayed on the respective bar graph. The results are expressed as percentages (means ± SD; n = 5) of preischemic baseline values. *P < 0.05 vs. ischemia-reperfusion (I/R); #P < 0.05 vs. verapamil.

HO-4038 attenuates CK, LDH release, and myocardial infarct size in isolated hearts.

The I/R-induced myocardial damage is associated with cessation of contractile activity, alteration of membrane integrity, and leakage of key enzymes such as CK and LDH in the coronary effluents. Untreated (I/R) hearts showed marked increase in CK activity in coronary effluents collected after 15 min into reperfusion (Fig. 3D). The CK release was significantly decreased in both verapamil- and HO-4038-treated hearts compared with I/R hearts. Similarly, the effluents from hearts treated with verapamil and HO-4038 showed significantly less LDH activity compared with the untreated I/R hearts (Fig. 3E).

The measurement of myocardial infarct size is also an important parameter to assess I/R-induced myocardial damage. Myocardial infarct size (in percentages) was estimated by using TTC staining. I/R control hearts subjected to 30 min of ischemia followed by 120 min of reperfusion showed an infarction of 45.0 ± 4.0 of risk area (Fig. 3F). On the other hand, the infarct size was significantly decreased in hearts treated with verapamil (24 ± 7) and HO-4038 (15.0 ± 0.3). Furthermore, HO-4038 showed a marked reduction in infarct size, which was beyond that of the verapamil.

HO-4038 decreases myocardial oxygen demand in the reperfused tissue in vivo.

We implanted an oxygen-sensing microcrystalline probe, LiNc-BuO, in the left ventricular midmyocardium, the expected site of I/R following LAD ligation and reperfusion. We then performed continuous Po₂ measurements in the beating heart. Figure 4 shows the myocardial tissue Po₂ changes during the 30 min ischemia followed by 60 min reperfusion in rats pretreated with verapamil or HO-4038. The basal (pre-I/R) levels of myocardial Po₂ were in the range of 18–20 mmHg, and there were no significant changes among the different groups. Immediately after the induction of ischemia, the Po₂ dropped sharply and maintained around 2 mmHg during the entire 30 min of ischemia in the untreated I/R group. In verapamil- and HO-4038-treated hearts, the Po₂ dropped slowly and maintained above 2 mmHg during the first minutes of the 30-min ischemic period. Upon restoration of blood flow (reperfusion), a rapid increase in Po₂ leading to marked hyperoxygenation was observed in all groups. The hyperoxygenation continued for 60 min and beyond. At 60 min of reperfusion, the hyperoxygenation in untreated (I/R) hearts was significantly higher compared with pre-I/R hearts (39 ± 3 vs. 19 ± 2 mmHg; P < 0.01). However, verapamil- and HO-4038-treated hearts showed significant (P < 0.05) attenuation of hyperoxygenation compared with untreated I/R hearts. The hyperoxygenation was further attenuated in HO-4038-treated hearts compared with verapamil, suggesting that HO-4038 provided additional protection in attenuating I/R-induced hyperoxygenation beyond that of verapamil.

Fig. 4.

Changes in myocardial tissue Po₂ during I/R, as measured in vivo by EPR oximetry. The rat, under isoflurane inhalation anesthesia, was placed in a right lateral position to the loop of a surface coil resonator. Oxygen-sensing microcrystals were implanted in the LV midmyocardium. Changes in myocardial Po₂ during 30 min of ischemia followed by 60 min of reperfusion in rats preinfused with vehicle (I/R), verapamil (Vera), and HO-4038 were monitored. The peak-to-peak width of the EPR spectrum was used to calculate Po₂ using a standard calibration curve. Data represent means ± SD, obtained from 6 rats/group. *P < 0.05 vs. I/R group at 1 h reperfusion. The results show a significant oxygen overshoot (hyperoxygenation) during reperfusion. LAD, left anterior descending coronary artery.

HO-4038 decreases CK and LDH release and enhances NOx production in plasma in rats subjected to acute MI.

It has been demonstrated that plasma CK and LDH are sensitive and specific biomarkers of cardiac injury. When compared with the sham-operated rats, plasma from the I/R group subjected to 30 min of occlusion of LAD followed by 1 h of reperfusion resulted in a significant (P < 0.05) increase in the release of CK and LDH in the systemic circulation (Fig. 5, A and B). On the other hand, plasma levels of CK and LDH in rats that received HO-4038 before the onset of ischemia were significantly (P < 0.05) decreased compared with untreated I/R hearts. Furthermore, both CK and LDH release in plasma treated with HO-4038 were significantly (P < 0.05) reduced beyond that of verapamil, suggesting that HO-4038 provided an additional protection against I/R-mediated cardiac injury better than that of verapamil.

Fig. 5.

Markers of myocardial injury and infarction. Effect of HO-4038 on the plasma levels of CK (A), LDH (B), and nitrite/nitrate (C) in hearts subjected to I/R. Rats were treated with HO-4038 through jugular vein before 10 min of experimentation. Ischemia was induced by temporarily ligating the LAD for 30 min followed by reperfusion by releasing the ligation. Blood samples were collected from I/R, Vera, and HO-4038 groups at 1 h after reperfusion. *P < 0.05 vs. control (non-I/R); **P < 0.05 vs. I/R group. D: effect of HO-4038 on myocardial infarct size. Representative images show TTC sections of rats pretreated with Vera or HO-4038 and subjected to 30-min ischemia followed by 24 h reperfusion. Infarct results showing AAR (E) and infarct size (F) are shown. Data represent means ± SD (n = 6). *P < 0.05 vs. I/R group. The infarct size was significantly decreased in rats pretreated with HO-4038 compared with I/R and Vera groups.

Verapamil is a potent smooth muscle relaxant with vasodilatory properties. To study the effect HO-4038 on NO levels, the plasma levels of stable metabolites of NO, NOx, were measured. Results showed a significant increase (P < 0.05) in the NOx levels in I/R compared with preischemic (pre-I/R) hearts (Fig. 5C). On the other hand, verapamil- and HO-4038-pretreated hearts showed a further increase in NOx levels at 1 h of reperfusion compared with I/R hearts. HO-4038 induced more NOx production in I/R hearts than verapamil, but the increase was not statistically significant.

Pretreatment with HO-4038 attenuates myocardial infarct size in vivo.

Myocardial infarct size was measured in rats subjected to 30 min of LAD ligation followed by 24 h of reperfusion to allow improved accuracy and optimal contrast between the necrotic tissue area and the area at risk. Myocardial infarct size, expressed as area at risk × 100%, was significantly decreased (23 ± 2%; P < 0.05) in rats pretreated with HO-4038 compared with the untreated I/R group (36 ± 4%; Fig. 5, D–F). There was no significant difference in the size of infarct in the verapamil group (32 ± 3%) compared with the I/R group of hearts.

HO-4038 attenuates superoxide levels in reperfused hearts.

To determine the role of HO-4038 on the superoxide-scavenging ability during I/R, myocardial tissue levels of superoxide in hearts subjected to 30 min of LAD occlusion followed by 10 min of reperfusion were measured by HE florescence. The HE fluorescence intensity was significantly (P < 0.05) increased in the untreated I/R hearts (Fig. 6B). Verapamil-treated hearts showed no significant difference compared with I/R hearts. However, hearts pretreated with HO-4038 showed a marked (P < 0.05) reduction in HE fluorescence intensity compared with untreated I/R hearts. The results clearly demonstrated that HO-4038 suppressed the generation of superoxide during the early minutes of reperfusion.

Fig. 6.

Effect of HO-4038 on the production of superoxide and nitric oxide in the myocardial tissue during I/R in vivo. Superoxide and nitric oxide levels in the excised heart tissue were determined at 10 min into reperfusion, as described in materials and methods. A: representative images (×100) of superoxide and nitric oxide production. B: mean fluorescence intensity from triplicate experiments. Values represent means ± SD. *P < 0.05 vs. I/R; #P < 0.05 vs. Vera. HO-4038 significantly decreased superoxide and increased nitric oxide production in the reperfused hearts.

Pretreatment with HO-4038 enhances NO generation in reperfused hearts.

Since NO plays an important role in cardioprotection against I/R injury, we studied the effect of HO-4038 on myocardial NO production in reperfused hearts. The NO generation in rat hearts subjected to 30 min of ischemia followed by 10 min of reperfusion was measured by DAF-FM fluorescence. The fluorescence intensity, which corresponds to the magnitude of NO in the reperfused heart, was significantly (P < 0.05) higher in verapamil- and HO-4038-treated hearts compared with I/R hearts (Fig. 6, A–B). The fluorescence intensity in the HO-4038 group showed significant (P < 0.05) increase compared with the verapamil group. The results suggested that HO-4038 increased NO production not only in systemic circulation but also in reperfused hearts.

HO-4038 enhances expression of prosurvival signaling proteins in reperfused hearts.

We next wanted to determine the underlying molecular mechanism of signaling pathways involved in the mitigation of postischemic reperfusion damage in the hearts pretreated with HO-4038. Western blot analysis was performed to determine the expression of antiapoptotic proteins Akt and Bcl-2. HO-4038 significantly enhanced the expression of Bcl-2 and phosphorylation of Akt in reperfused hearts compared with untreated I/R hearts (Fig. 7). There was no significant difference in the levels of total Akt among the groups. Furthermore, the induction of Bcl-2 expression was significantly increased in HO-4038-treated hearts (P < 0.05) compared with the verapamil group. The Western blot analyses indicated that HO-4038 treatment enhanced the activation of Akt and Bcl-2, thereby conferring to I/R-mediated cardioprotection in the postischemic hearts at 24 h of reperfusion.

Fig. 7.

Effect of HO-4038 on the I/R-induced phosphorylation of key signaling proteins. The expressions of phosphorylated (p)Akt and Bcl-2 were measured in hearts subjected to 30 min of ischemia followed by 24 h reperfusion. A: representative Western blots of total and pAkt and Bcl-2. B–D: quantitative analysis of pAkt and Bcl-2. Results are expressed as means ± SD of 3 hearts from each group. *P < 0.05 vs. I/R; #P < 0.05 vs. Vera. The Western blot analyses indicated that HO-4038 treatment enhanced the activation of pAkt and Bcl-2 in the reperfused myocardium. au, Arbitrary units.

DISCUSSION

The findings of the present study demonstrated that the administration of HO-4038 significantly improved the recovery of cardiac functions, increased CF, and decreased infarct size and superoxide generation in hearts subjected to I/R injury. Furthermore, in vivo studies showed a significant decrease in myocardial oxygen demand at 1 h of reperfusion.

Verapamil is known to increase myocardial oxygen supply by reducing coronary tone and decreasing myocardial contractility and heart rate. Verapamil also inhibits platelet aggregation and thrombus formation, which may contribute to its anti-ischemic effects. Verapamil has been reported to inhibit human low-density lipoprotein oxidation mediated by oxygen radicals (36). Villari et al. (53) administered gallopamil (a calcium channel antagonist similar to verapamil in structure) with SOD in rabbit hearts subjected to I/R injury and observed a reduction in infarct size. The reduction in infarct was attributed primarily to the inhibition of oxygen-radical generation under gallopamil-mediated decreasing myocardial oxygen demand during ischemia and not due to antioxidant activity of gallopamil. In the present study, we evaluated the cardioprotective properties of a verapamil derivative, HO-4038, containing a pronitroxyl group to the parent verapamil compound. Thus HO-4038, having both calcium antagonistic activity and free-radical scavenging abilities, could afford a multiple approach to prevent I/R injury. The advantage of having the antioxidant moiety directly attached to verapamil may enable HO-4038 to deliver a site-specific antioxidant action in the I/R heart. Therefore, HO-4038 may offer a site-specific combinatorial approach that is more efficacious than the administration of either verapamil or free-radical scavenger nitroxide individually. Structure-activity relationship studies of verapamil derivatives have demonstrated that the aromatic rings, nitrile group, and tertiary amines are important to its calcium antagonistic activity, but not the isopropyl group or substituents of aminoethyl aromatic ring (30). The N-hydroxypiperidines and their one-electron oxidized metabolites (nitroxides), and reduced metabolites (hydroxylamine), can act as proton-donating and multifunctional antioxidants (21, 46).

A whole cell patch-clamp study showed a significant inhibition of calcium currents in canine ventricular myocytes treated with HO-4038. Furthermore, the inhibition exhibited by HO-4038 was similar to that of verapamil at the same concentration. This study clearly demonstrated that the calcium channel-blocking property of verapamil was preserved by the structural modification introduced in HO-4038. The EPR spectroscopic studies shown in Fig. 2 provided direct evidence for the oxygen-derived free-radical scavenging ability of HO-4038. HO-4038 was capable of scavenging superoxide, hydroxyl, and peroxyl radicals in a dose-dependent manner. The observed O₂^•−, ^•OH, and ROO· scavenging of HO-4038 is attributed to both the oxidized and reduced forms of the piperidine group.

Alicyclic amines such as piperidine, pyrroline, and pyrrolidine-based drugs have been recognized as antiarrhythmic agents in animal models (12). Nitroxides have been shown to exhibit potential therapeutic benefits in a variety of disease states including myocardial I/R injury (7, 33, 45) and postischemic reperfusion injury (27, 49).

Infusion of HO-4038 in isolated hearts significantly improved the cardiac contractility and left ventricular function, as evidenced by the increase in CF and LVDP. The improved cardiac functions could be due to the availability of HO-4038 in the myocardium during I/R injury and scavenging free radicals generated during early minutes of reperfusion. We had previously reported the cardioprotective properties of several antiarrhythmic compounds with pyrroline modifications against I/R-induced damage and contractile dysfunction without compromising their individual properties (25, 29, 49). HO-4038-administered hearts also showed a significant decrease in infarct size compared with untreated or verapamil-treated hearts. The decrease in infarct size is reflected in the improved recovery of contractile, hemodynamic, and biochemical alterations upon prolonged reperfusion. This is further supported by the decreased cardiac markers such as CK and LDH in the coronary effluents as indicating less tissue injury occurred in the HO-4038-treated myocardium compared with I/R and verapamil-treated hearts. Overall, the ex vivo results clearly showed that HO-4038 was more effective than parent verapamil in restoring cardiac functions against I/R-induced damage.

Optimal myocardial tissue Po₂ is essential for the survival of cardiomyocytes. I/R is associated with rapid changes in myocardial tissue Po₂. The restoration of blood flow to an ischemic myocardium is known to cause acute hyperoxygenation (17), which may have important implications in the early events triggering oxygen-derived free-radical generation leading to myocardial injury and dysfunction.

A significant work of this study is the in vivo monitoring of myocardial oxygen content using EPR oximetry (Fig. 4). The infusion of both verapamil and HO-4038 caused a significant increase (28–30 mmHg) from baseline (18–20 mmHg). The increase in myocardial Po₂ may be attributed to a reduction in oxygen demand due to the negative chronotropic effect of these drugs. When the hearts were subjected to ischemia (LAD ligation for 30 min), the Po₂ drop in the drug-treated group was not as sharp and deep compared with the untreated control (I/R group). This may again suggest a decreased oxygen demand by cardiomyocytes during ischemia in verapamil and HO-4038 groups. During reperfusion, there was a substantial hyperoxygenation in I/R group. The reperfusion-induced hyperoxygenation was significantly attenuated in the verapamil or HO-4038 compared with the untreated I/R group. The reduction in hyperoxygenation observed in the treated groups may be attributed to increase in NO levels (Fig. 6) as well as oxygen demand due to the improved recovery of cardiac function, as much as a threefold increase in RPP in the HO-4038 group compared with control (Fig. 3).

Since NO is a coronary vasodilator and known to reduce myocardial oxygen consumption (48) and under conditions of limited myocardial oxygen supply, NO is a vital factor in maintaining oxygen demand-supply balance in the myocardium. The decrease in myocardial Po₂ is also a reflection of increased NO production, and decreased oxygen demand, by efficiently utilizing oxygen in the HO-4038-treated group, made the myocardium less susceptible to attack from oxygen-derived free radicals by utilizing less oxygen during the early minutes of sudden oxygenated reperfusion. This is further supported by decreased leak of cardiac markers CK and LDH in plasma and left ventricular infarct size in HO-4038-treated group. In addition, this study also provides evidence for decreased superoxide generation in hearts treated with HO-4038. This could be due to the increased nitroxide in the HO-4038-treated group that could scavenge superoxide in the myocardium during early minutes of reperfusion. These results are in agreement with a recent study from our laboratory of decreased myocardial superoxide generation in isolated working rat hearts pretreated with N-hydroxy-pyrroline modification of the verapamil analog H-3010 (29). Overall, although verapamil showed protection against I/R-induced injury ex vivo and in vivo, the magnitude of protection offered by HO-4038 was significantly higher than that of verapamil. This suggests that the observed cardioprotection is due to the antiarrhythmic and anti-ischemic protection by parent verapamil compound and the scavenging of ROS by the antioxidant moiety added to the parent verapamil compound (both ischemia- and reperfusion-mediated protection) during the first minutes of reperfusion that otherwise lead to myocardial tissue damage and dysfunction during reperfusion.

Several studies have shown that that the activation of Akt and Bcl-2 are cardioprotective (14, 50). Several pharmacological agents have also been shown to be cardioprotective by modulating the expression of Bcl-2 (4, 6). In the present study, we have observed that HO-4038 activated Akt and Bcl-2 expression, and this might lead to a decrease in myocyte apoptosis and thereby protecting the myocardium from I/R-induced injury. Some earlier studies have shown that H-2693, a compound containing a secondary amine nitroxide precursor, and HO-3070, a free-radical scavenging compound, enhanced the I/R-induced activation of Akt and protected the hearts from I/R injury (50, 51). Our observations are also consistent with a recent study from our laboratory that demonstrated the protective effect of nitroxide-precursor derivatives of trimetazidine against I/R-induced damage through the involvement of Akt activity (25). The results of the present study showed that HO-4038 significantly enhanced the expression of prosurvival Akt and Bcl-2, thereby conferring myocardial protection. However, further studies are warranted to uncover the exact molecular mechanisms involved in the I/R-induced cardioprotection by HO-4038.

The modified verapamil design that we have used in this work can provide a pharmacological approach targeting multiple mechanisms of I/R injury and cardiac dysfunction. The new drugs can be used not only to improve ventricular function following an ischemic episode but also to attenuate reperfusion damage caused by reactive free radicals produced during reperfusion. This is a promising approach that can be of great clinical significance to overcome the limitations of verapamil.

In conclusion, our study demonstrated that the new verapamil analog HO-4038 protected the myocardium from I/R-induced injury by scavenging of ROS and decreasing myocardial infarct size and cardiac contractile dysfunction. HO-4038 also protected the myocardium by increasing the bioavailability of NO and decreasing myocardial oxygen demand by efficiently utilizing oxygen in the myocardium during I/R. The protective effect could be attributed to the combined benefits of verapamil including the added pronitroxide antioxidant moiety and prosurvial Akt and Bcl-2 activities. These observations may offer a novel approach for reducing the incidence of I/R injury during coronary revascularization procedures.

GRANTS

This work was supported by National Institutes of Health Grant EB-006153 and Hungarian National Research Fund OTKA T048334. I. K. Mohan was on sabbatical from the Nizam Institute of Medical Sciences (Hyderabad, India).