Myocardial Contractile Function and Heart Rate in Mice With Myocyte-Specific Overexpression of Endothelial Nitric Oxide Synthase
Background— The major source of nitric oxide (NO) in the heart is the constitutive form of NO synthases (eNOS, NOS III) that is expressed in vascular endothelium and cardiac myocytes. NO mediates endothelium-dependent vasodilation and may modulate cardiac function. We examined the role of NO in hearts from transgenic (TG) mice overexpressing eNOS exclusively in cardiac myocytes.
Methods and Results— Three independent TG lines with varying levels of NOS activity were selected, and the hearts were isolated and retrogradely perfused at constant flow. We found that NO is positively inotropic in spontaneously beating hearts from wild-type (WT) mice, whereas hearts overexpressing eNOS had reduced basal contractility that was partially reversed by NOS blockade. Heart rate was not altered. Acetylcholine (10 to 1000 nmol/L) increased contractility in unstimulated hearts and decreased contractility after β-adrenergic stimulation with norepinephrine, and these responses were identical in WT and TG hearts. Finally, resting systolic intracellular calcium (Ca2+i) tended to be lower in TG than in WT hearts, and the beat-to-beat responsiveness to Ca2+i was reduced in hearts with eNOS overexpression.
Conclusions— High levels of endogenous myocyte-derived NO blunt myofilament Ca2+ sensitivity. The similar effects of acetylcholine on contractility and heart rate, as well as the identical basal intrinsic heart rate in WT and TG hearts, provide a solid argument against NO as an important modulator of neurohormonal control of myocardial function.
Received October 17, 2001; revision received November 2, 2001; accepted November 2, 2001.
Nitric oxide (NO) is one of the most important regulatory factors of the cardiovascular system. NO is synthesized from l-arginine by NO synthases (NOS), a family of isoenzymes with distinct functional and regulatory properties.1 NO mediates endothelium-dependent vasodilation and may modulate cardiac function.2 In the heart, the major form of NOS is the endothelial constitutive form (eNOS, NOS III) that is expressed in vascular endothelium and cardiac myocytes.3,4 However, the evidence for a role of NO in the modulation of myocardial function remains inconclusive.5 Exogenous NO, mostly delivered from pharmacological NO donors, slows cardiac rhythm6 and exerts concentration-dependent biphasic effects on myocardial contractility.7,8 The role of endogenous NO produced inside cardiac myocytes remains controversial because both blocking the NO/cGMP pathway with NOS inhibitors9–12 and using mice deficient in eNOS13,14 have yielded contradictory results. We chose a novel approach and examined the role of NO in isolated perfused hearts overexpressing eNOS exclusively in cardiac myocytes. Left ventricular function and the corresponding intracellular Ca2+ concentration ([Ca2+]i) were recorded on a beat-to-beat basis using the aequorin bioluminescence method originally developed for the rat heart.15
Generation of TG Mice
The full-length cDNA for human eNOS (kindly provided by Dr J. Liao, Brigham and Women’s Hospital, Boston, Mass) and the PCR-cloned 3′UTR were subcloned into a vector (clone 26; kindly provided by Dr J. Robbins, Children’s Hospital Medical Center, Cincinnati, Ohio) containing the murine cardiac α-myosin heavy chain (MHC) gene promoter. All cloning junctions were subsequently sequenced. After digestion with Not I and extensive purification, the 10.6-kb minigene (containing the 5.5-kb α-MHC gene promoter, the first 3 exons and 2 introns of the α-MHC gene, the human eNOS cDNA from bp −19 to the end of the 3′ UTR, and an additional 579 bp of the 3′ flanking region) was microinjected at the transgenic facility of the Rockefeller University into oocytes of pseudopregnant mice (F1 of CBA x C57BL/6).
Identification of TG Mice
A total of 33 potential founder mice were obtained, 8 of which were identified as positive by polymerase chain reaction (PCR) analysis of genomic DNA isolated from tail-tip biopsies. The PCR analysis was confirmed by Southern blot analysis of genomic DNA. Genomic DNA (10 μg) was digested with Xmn I to yield a 440-bp fragment that is specific for the transgene and a 1000-bp fragment that is specific for the endogenous mouse eNOS gene. The digested DNA was electrophoresed, blotted, and hybridized to a 32P-labeled Xmn I fragment of human eNOS cDNA, which also hybridizes to murine eNOS. Transgenic mice were backcrossed with C57BL/6 mice for at least 3 generations. All animal experiments were conducted according to the Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, revised 1985).
Mouse tissues were homogenized in 50 mmol/L triethanolamine/HCl buffer (pH 7.4) containing 128 mmol/L β-mercaptoethanol. For preparation of total homogenate protein, 10 mmol/L CHAPS and 0.5 mmol/L EDTA were present during homogenization, and the homogenate was centrifuged at 10 000g for 5 minutes to remove insoluble material. For separation of particulate and supernatant protein fractions, the homogenate was centrifuged in the absence of CHAPS and EDTA, the pellet was resuspended in homogenization buffer supplemented with 10 mmol/L CHAPS and 0.5 mmol/L EDTA, and insoluble material was removed by repeated freezing and thawing, followed by centrifugation. Protein concentration was determined using the Bradford assay.
Western Analysis and eNOS Activity
Total homogenate protein, particulate fraction, or supernatant (50 μg) supplemented with CHAPS (10 mmol/L final concentration) were separated using SDS-PAGE (8% acrylamide), transferred to nitrocellulose, and probed with eNOS antibody prepared in our laboratory. To determine NOS activity, 50 to 200 μg protein was incubated at 37°C for 10 minutes with 10 μmol/L 3H-l-arginine (≈50 000 cpm), 10 μg/mL calmodulin, 10 μmol/L tetrahydrobiopterin, 5 μmol/L flavin adenine dinucleotide, 5 μmol/L flavin mononucleotide, 200 μmol/L nicotinamide adenine dinucleotide phosphate, and 10 mmol/L CHAPS, followed by isolation and counting of 3H-citrulline as has been described previously.16
Isolated Heart Perfusions and Ca2+i Measurements
Mouse hearts were Langendorff-perfused at 2 mL/min with bicarbonate buffer (pH 7.4, 37°C).17 Left ventricular pressure was recorded with a fluid-filled balloon made of polyethylene film; left ventricular end-diastolic pressure was set at 5 mm Hg. The heart rate was obtained from the pressure signal. Perfusion pressure, an index of coronary function, was recorded at the aortic root. Test compounds were added via a sideline in the following order: norepinephrine (NE) (3 to 300 nmol/L), followed by washout; Acetylcholine (Ach) (10 to 1000 nmol/L), followed by washout; and ACh in presence of 3 nmol/L NE. Intracellular Ca2+ transients were measured on a beat-to-beat basis.17 Briefly, hearts were equilibrated for 10 minutes at ambient temperature at spontaneous beating rate before they were arrested by reducing Ca2+ and Mg2+ (0.5 and 0.6 mmol/L), and aequorin (1 to 3 μL; Friday Harbor Photoproteins) was slowly macroinjected into the interstitium of the epicardium at the apex of the heart. Perfusate Ca2+ and Mg2+ were gradually increased to 1.2 mmol/L, the temperature was raised to 37°C, the right auricle was removed, and pacing was initiated at 6.7 Hz (400 min−1). Myocardial parameters were recorded and aequorin light signals were analyzed as previously described.18 Light signals were converted to quantitative Ca2+ concentrations by normalizing the aequorin light after each phase of the protocol by the amount of active aequorin at that time (L/Lmax method), followed by conversion of the normalized light signal to a Ca2+i concentration value using an in vitro (37°C) calibration curve.15 As to the sensitivity of the aequorin method, even the diastolic light signals were always several-fold above the photomultiplier shot noise of ≈700 pA (background).
A minigene was constructed containing the human eNOS cDNA under the control of the murine cardiac α-MHC gene promoter, which has been used previously to direct expression of transgenes exclusively in heart muscle.19 The construct is assembled from the following: the α-MHC gene promoter; 3 exons and 2 introns of the α-MHC gene; the complete cDNA of human eNOS, including the 3′ untranslated region and the polyadenylation signal; and 579 bp of the 3′ flanking region of the eNOS gene. Introns were included in the construct because this has been reported to increase the transcriptional efficiency of transgene expression.20 Transgenic founder mice were initially identified by Southern blot analysis of genomic DNA isolated from tail-tip biopsies (Figure 1a). After backcrossing founder mice with C57BL/6 mice, the offspring were examined for NOS activity in heart tissue. Three independent TG lines were selected: line 11, line 8, and line 23 (26.5±5.7, 45.8±10.8, and 61.8±11.8 pmol 3H-l-citrulline · mg−1 · min−1, respectively). NOS activity in hearts from C57BL/6 (wild-type, WT) mice was 0.7±0.3 pmol · mg−1 · min−1 (n=3 hearts). Western blot analysis of heart homogenates from these 3 TG mouse lines revealed high levels of expression of eNOS (Figure 1b). Low levels of endogenous eNOS were detectable in WT hearts after long exposure, as well as in other organs (Figure 1c). However, eNOS expression did not vary between WT and TG animals in any tissue except the heart, showing that transgene expression is completely restricted to heart tissue.
For the experiments described below, we studied male WT (n=25) and TG mice of line 11 (n=10) and line 23 (n=25) matched for age and body weight. There was no significant difference in mean arterial blood pressure measured in conscious animals using the tail-cuff method (WT, 116±4; TG line 11, 113±2; and TG line 23, 108±3 mm Hg; P=0.61 and 0.16, respectively, versus WT) or heart rate (WT, 473±44; TG line 11, 444±37; and TG line 23, 469±28 beats per minute (bpm); P=0.64 and 0.94, respectively, versus WT) (n=5) between WT and TG mice. Body weights were not different between groups (WT, 30.5±0.4 g; TG line 11, 29.0±0.8 g; TG line 23, 29.5±0.6 g; P=0.17 and 0.14, respectively, versus WT), but the hearts from TG animals of line 23 were slightly lighter than those from WT animals (“wet weights”: WT, 138±2.1 mg; TG line 23, 128±1.6 mg; P=0.003, n=20). The mean wet weight of hearts from TG line 11 was 130±2.7 mg (P=0.07 versus WT hearts; n=5).
Subcellular Distribution and Activity of Overexpressed eNOS
Because the subcellular localization is important for the regulation and activity of eNOS,21 the distribution of the transgenic eNOS between the particulate and supernatant fractions of heart homogenates was examined (Figure 2a). In TG line 11, eNOS was localized to the particulate fraction. In lines 8 and 23, which express higher levels of eNOS, some protein became apparent in the soluble fraction, the proportion of which increased with increasing transgene expression. Reflecting protein expression, eNOS activities were much higher in TG lines 11 and 23 than in WT hearts, with most of the activity (94% to 97%) localized to the particulate fraction and ≈5% to the supernatant (P<0.001, n=5) (Figure 2b). Enzyme activity was reduced to blank level in all preparations in the presence of 0.2 mmol/L NG-nitro-l-arginine (n=3; not shown). Hence, overexpressed eNOS enzyme was functional and inhibitable, and its localization was mostly to cellular membranes, similar to that in WT hearts.
Basal Heart Function and Effect of eNOS Blockade
We assessed basal heart function in isolated spontaneously beating hearts from WT and TG animals. After equilibration, pressure development of the left ventricle (LVDevP) was progressively reduced with increasing transgene expression: WT, 88±3 mm Hg (n=8); TG line 11, 69±2 mm Hg (−22%; n=3, P=0.008; not shown); TG line 23, 59±1 mm Hg (−33%; n=8, P<0.001) (Figure 3a). Basal heart rates were not significantly different between groups (409±8, 405±14, and 412±10 bpm, respectively; P=0.82) (Figure 3b). Coronary perfusion pressure, a measure of coronary resistance in these hearts perfused at constant flow, was 66±5, 67±3 and 65±3 mm Hg, respectively (P=0.88; not shown). The role of endogenous NO was further analyzed in hearts from animals that had been treated with a daily dose of ≈50 mg/kg body weight of the nonselective NOS inhibitor, NG-nitro-l-arginine methyl ester (L-NAME), which was added to the drinking water for 2 days (n=5). Importantly, this treatment led to no increase in blood pressure (WT, 113±3 mm Hg; TG, 119±6 mm Hg; P=NS), but it significantly reversed the depressed basal contractility in hearts with the highest NOS overexpression (TG line 23, +24%; P<0.001), and markedly reduced LVDevP in WT hearts (−16%, P=0.006). In line with these effects, LVDevP rose slightly after L-NAME treatment in hearts with modest overexpression (TG line 11), but the difference (+5%) was not statistically significant (P=0.11; not shown).
We tested whether the reduced contractile function in TG hearts may be a reflection of chronic myocyte damage. However, sections from paraffin-embedded, hematoxylin-eosin stained hearts showed no histological differences between ventricles from WT (Figure 4a and 4c) or TG (Figure 4b and 4d) animals. Specifically, no myocyte disarray, myocyte atrophy, basophilic degeneration, or interstitial or replacement fibrosis were present. We also tested whether ventricles from TG mice produced more superoxide because of “uncoupling” of the NOS enzyme.1 However, superoxide levels, determined with a sensitive standard method,22 were similar in both groups under basal conditions (Figure 4e).
Effects of β-Adrenergic and Muscarinic Stimulation
We assessed the responses to NE and ACh, the 2 cardiac autonomic transmitters, on pump function and heart rate in WT and TG mice with the highest eNOS overexpression (line 23, n=5). NE (3 to 300 nmol/L) increased LVDevP from 87±4 to 188±10 mm Hg (2.2× basal LVDevP) in WT mice and from 60±6 to 156±13 mm Hg (2.6× basal LVDevP) in TG mice. The LVDevP was significantly greater in WT than TG hearts at all NE concentrations (P<0.05 for each NE concentration) (Figure 5a), but the calculated half-maximal effect values (EC50) were similar (47.6±15.6 and 36.3±16.6 nmol/L NE in WT and TG hearts, respectively; P=0.63). These data indicate a similar potency, but possibly lower efficacy of NE in the group with the eNOS transgene. ACh (10 to 1000 nmol/L) raised LVDevP by 11±3 mm Hg in WT and 10±3 mm Hg in TG animals (1.2× basal LVDevP in both cases) (Figure 5b). These increases were significantly different from basal (P=0.02). The lower basal contractility in TG hearts compared with WT hearts (P=0.001) was not affected by ACh. Spontaneous heart rate was similarly influenced in WT and TG hearts by both agonists: NE accelerated heart rate (WT, 619±19 and TG, 620±13 bpm [1.5× basal heart rate in both cases]; P<0.001), whereas ACh decreased heart rate (WT, 394±11 [93% of basal]; P=0.047; TG, 379±7 bpm [92% of basal]; P=0.011). As expected, both agents relaxed the coronary vasculature, but there were no differences between WT and TG hearts (n=5, data not shown).
We studied the interaction between NO and the β-adrenergic pathway in hearts prestimulated with 3 nmol/L NE. Ach, which stimulates NO production by activating endothelial muscarinic receptors,23 reduced LVDevP from 105±7 to 82±2 mm Hg (77% of basal) in WT hearts and from 79±2 to 60±4 mm Hg in TG hearts (76% of basal) (maximal ACh concentration, 1000 nmol/L). The decreases were significantly different from basal at 100 and 1000 nmol/L ACh (P≤0.01, n=5; Figure 5c). On the other hand, muscarinic activation had no significant effect on NE-stimulated heart rate: At 1000 nmol/L, ACh reduced the beating frequency in WT hearts from 493±22 to 470±27 bpm (95% of basal; P=0.53) and in TG hearts from 506±11 to 458±21 bpm (91% of basal; P=0.08). After NOS blockade in vivo as described above, NE (3 nmol/L) was as potent in WT as in TG (line 23) hearts (110±6 and 113±8 mm Hg; see baseline in Figure 5c), and the negative inotropic effect of ACh was similarly reduced in both groups.
[Ca2+]i and Myofilament Responsiveness to Ca2+i
We tested whether the reduced contractility in eNOS-overexpressing hearts may be related to a defect in the handling of intracellular calcium (Ca2+i). In parallel measurements of Ca2+i and LVDevP, ventricles from TG hearts were significantly less responsive to extracellular Ca2+ (Figure 6a) or β-receptor stimulation with NE (3 to 300 nmol/L; n=5; not shown). The resting (basal) systolic levels of Ca2+i were significantly lower in TG than WT hearts (mean difference, 0.04±0.005 μmol/L; n=5, P<0.01; Figure 6b), whereas diastolic Ca2+i levels were not different (0.30±0.001 μmol/L in both groups independent of perfusate [Ca2+]). The relationship between peak systolic pressure and [Ca2+]i is shown in Figure 6c. Although in this analysis myofilament sensitivity was not assessed at steady-state, it is clear that hearts from TG animals generated significantly less pressure at all levels of Ca2+i than did WT hearts (eg, ≈19% less at 0.64 μmol/L; P<0.05). A reduction in myofilament sensitivity also was observed previously in isolated myocytes in response to a high concentration of exogenous NO.24
Myocardial Contraction and Relaxation Kinetics
To assess the effects of NO overproduction on the time course of isometric contraction and the aequorin light signal, we compared the time to peak systolic light (TPL) and peak systolic pressure (TPP), and time to 90% decline from peak systolic light (T90L) and peak systolic pressure (T90P). NO overproduction in hearts from TG animals (line 23) was associated with an abbreviated rise to peak pressure (Figure 7a) and accelerated decline from peak pressure (Figure 7b) (P<0.01 for each variable) which, together, result in the reduced basal contractility of TG hearts described above (Figure 6a). The corresponding time course for the aequorin light transient, T90L, was also accelerated in TG hearts (Figure 7d, P<0.01), whereas TPL was not significantly different between groups (Figure 7c). After in vivo NOS inhibition, contraction and relaxation kinetics were partially normalized in TG hearts but were still significantly different from WT hearts (n=3, not shown).
This study demonstrates that stable myocardial overexpression of eNOS in mice greatly stimulated cardiac NO formation and reduced left ventricular basal contractility, whereas in WT hearts endogenous NO exerted positive inotropic effects. The results strengthen the view of NO as a dual endogenous regulator of myocardial contractility that tonically supports pump function at physiological concentrations, but unloads the heart when generated in great amounts by lowering the calcium sensitivity of the contractile elements. This may be important in humans with cardiac diseases, such as congestive heart failure or inflammatory heart disease, that are associated with pathological NO generation through increased NOS expression in cardiac myocytes.25,26 In addition, the cardio-adaptive effects of high NO levels may also relate to the inhibition of mitochondrial oxygen consumption and glucose uptake in cardiac myocytes.27 The similar positive inotropic effect of NE, as well as the similar antiadrenergic effect of ACh, in WT and TG hearts suggests that receptor-effector coupling was unaffected by NO and that the cellular effects of NO mainly relate to the desensitization of the myofilaments. Furthermore, the unaltered response to ACh in WT and TG hearts with and without NOS blockade (Figure 5) supports the growing evidence for a lack of role for NO in cholinergic modulation of myocardial contractility.28 Finally, the basal beating rate was not altered in response to eNOS overexpression or NOS blockade, and muscarinic stimulation slowed heart rate similarly in hearts from WT and TG animals. This supports more recent findings that generally have cast doubt on the involvement of NO in the muscarinic regulation of heart rate,14,29 in contrast to initial reports.6,10
Conclusions and Clinical Implications
Thus, from the present results obtained in spontaneously beating mouse hearts, we conclude that high concentrations of endogenous NO can produce a negative inotropic effect that is primarily due to a reduction in myofilament responsiveness to Ca2+. Ach increased contractility in unstimulated hearts and decreased contractility after β-adrenergic stimulation, and these responses were identical in WT and TG hearts, further weakening the notion that eNOS is essential for the normal autonomic control of cardiac muscle function. The cardiac-specific differential overexpression of eNOS (NOS III) in mouse hearts provides a promising new model for investigations into the role of NO in human heart diseases associated with increased expression of this enzyme.
This work was supported by grants of the Fonds zur Förderung der Wissenschaftlichen Forschung in Austria (P. 13759 to Dr Brunner, 13013 and 13586 to Dr Mayer, and SFB-F713 to Dr Zechner). We are grateful to Dr J.K. Liao (Brigham and Women’s Hospital, Boston, Mass) for providing the human eNOS cDNA, Dr J. Robbins (Children’s Hospital Medical Center, Cincinnati, Ohio) for the α-MHC promoter/human growth hormone construct, Dr G. Höfler (Department of Pathology, Karl-Franzens-Universität Graz, Graz, Austria) for histological advice, and B. Oberer and M. Rehn for excellent technical assistance.
This article originally appeared Online on November 19, 2001 (Circulation. 2001;104:r34–r39).
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