Published online before print March 22, 2004, doi: 10.1161/01.CIR.0000124231.98250.A8
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(Circulation. 2004;109:1886-1891.)
© 2004 American Heart Association, Inc.
Basic Science Reports |
Myocyte Nitric Oxide Synthase 2 Contributes to Blunted ß-Adrenergic Response in Failing Human Hearts by Decreasing Ca2+ Transients
From the Department of Physiology, Loyola University Medical Center, Maywood, Ill (M.T.Z., L.S.M., J.B., D.M.B.), and the Cardiovascular Research Group, Department of Physiology, Temple University School of Medicine, Philadelphia, Pa (V.P., S.R.H.). Dr Maier is now at the Department of Cardiology, Georg-August-University, Goettingen, Germany.
Correspondence to Donald M. Bers, Department of Physiology, Loyola University Medical Center, 2160 S First Ave, Maywood, IL 60153. E-mail dbers{at}lumc.edu
Received April 9, 2002; de novo received October 8, 2003; revision received December 30, 2003; accepted January 7, 2004.
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Abstract |
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Background— Human heart failure (HF) usually exhibits blunted response to ß-adrenergic receptor (AR) stimulation. Here, we examined whether expression of nitric oxide synthase-2 (NOS2, or inducible NOS) contributes to this loss of inotropic reserve in human HF.
Methods and Results— Failing human hearts were obtained at transplantation. Contraction and [Ca2+]i measurements were performed in isolated cardiac myocytes and trabeculae. In HF myocytes and muscle, isoproterenol (ISO), a ß-AR agonist, led to small inotropic and lusitropic responses. Specific inhibition of NOS2 by aminoguanidine (AG) or L-NIL dramatically increased the ISO-induced inotropy and lusitropy, such that the ISO+AG response in HF approached that seen with ISO alone in nonfailing human myocytes or muscles. Ca2+ transient data directly paralleled these results, indicating that altered cellular Ca2+ handling is responsible. In nonfailing human hearts, NOS2 inhibition had no effects. In addition, NOS2 inhibition also had no effect in 30% of failing hearts, but in these myocytes and muscles, the ISO response alone was similar to that of nonfailing hearts. In line with these functional findings, NOS2 protein expression measured by Western blotting was induced in HF when AG/L-NIL had a functional effect but not when AG/L-NIL had no effect on contractility and Ca2+ transients.
Conclusions— NOS2 expression strongly limited ISO-induced increases in contraction, twitch 
[Ca2+]i, and lusitropy in trabeculae and isolated myocytes from failing human hearts. Thus, the ß-AR hyporesponsiveness in human HF is mediated in large part by NO (or related congeners) produced within cardiac myocytes via NOS2.
Key Words: myocytes • receptors, adrenergic, beta • calcium • heart failure • nitric oxide synthase
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Introduction |
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Cardiac contractility is regulated by excitation-contraction coupling (ECC).1 A well-known modulator of ECC is stimulation of the ß-adrenergic receptor (AR) pathway, which leads to positive inotropic and lusitropic effects because of cAMP-dependent protein kinase (PKA) phosphorylation of proteins in the ECC cascade.2 Nitric oxide (NO) can also regulate cardiac contractility, affecting the same proteins as the ß-AR pathway.3,4
In human heart failure (HF), there is a negative force-frequency relationship but also usually a reduced response to ß-AR stimulation. This reduced ß-AR response has been shown to be partially caused by downregulation of ß1-AR receptor, increased G
i protein, and altered expression of Ca2+-handling proteins.1,5
Human HF also induces the expression of nitric oxide synthase-2 (NOS2, or inducible NOS),6–13 which may also blunt the ß-AR response in human HF.10,14 However, neither the specific role of NOS2, the origin of nitric oxide (NO) production (myocyte versus other cells), nor the mechanism of NOS2-mediated dysfunction has been investigated in human HF.
Here we tested whether (1) expression of NOS2 causes ß-AR hyporesponsiveness in human HF, (2) NOS2 expressed in ventricular myocytes is sufficient to cause the ß-AR hyporesponsiveness, and (3) alterations in myocyte Ca2+ handling mediate these effects.
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Methods |
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Failing Hearts
Experiments were performed on 13 end-stage failing hearts obtained at the time of transplantation and 1 nonfailing heart (not implanted for technical reasons). Individual premedication or pathogenesis of the cardiomyopathy did not affect the results of this study. All tissue was procured according to an Institutional Review Board–approved protocol by Loyola University and pathological specimen handling procedures accepted by Temple University.
Muscle Strips
Muscle strips were prepared as described previously.15 During muscle isolation and mounting, only 30 mmol/L 2,3-butanedione monoxime (BDM) was included in the superfusate used, a modified Krebs-Henseleit buffer (KHB; in mmol/L, NaCl 118, KCl 4.7, NaHCO3 25, KH2PO4 1.2, MgSO4 1.2, CaCl2 2.5, glucose 11, and insulin 10 IU) bubbled with 95% O2, 5% CO2 (pH 7.4). Trabeculae (average diameter, 257±17 µm) were stimulated (1 Hz, 37°C) in KHB, and isometric force was recorded. Some muscles were perfused with KHB that contained L-arginine (1 mmol/L), and this did not affect the NOS2-mediated dysfunction or the aminoguanidine (AG) response.
Human Myocyte Isolation
Myocytes were isolated as described previously.16 A section of left ventricle was excised, and a vessel was cannulated and perfused with solution containing (in mmol/L) NaCl 130, KCl 5.4, NaHCO3 25, NaH2PO4 1.2, MgSO4 1.2, glucose 12.5, lactic acid 1, Na-pyruvate 2, and taurine 20; pH 7.4). Then collagenase (180 U/mL, Worthington type II), BDM (20 mmol/L), and CaCl2 (50 µmol/L) were added to the perfusate. The isolated myocytes were resuspended in Tyrode’s solution with 200 µmol/L Ca2+ without BDM and used within 12 hours.
Myocytes (loaded with fluo 3-AM, 10 µmol/L, Molecular Probes) were field-stimulated (1 Hz) and superfused with Tyrode’s solution containing (in mmol/L) 140 NaCl, 4 KCl, 1 MgCl2, 2 CaCl2, 10 glucose, and 5 HEPES (pH 7.4) at 37°C. Some myocytes were perfused with Tyrode’s solution that contained L-arginine (1 mmol/L), and this did not affect the NOS2-mediated dysfunction or the AG response. All chemicals were purchased from Sigma except L-N6-(1-iminoethyl)lysine (L-NIL) (Calbiochem).
Measurement of NOS2 Expression
Microsomes were prepared from frozen human tissue17 using a protease inhibitor cocktail (set V, Calbiochem) instead of PMSF and subjected to electrophoresis and Western blotting according to instructions from the anti-NOS2 antibody supplier (Affinity Bioreagents) and developed with enhanced chemiluminescence (Amersham).
Statistical Analysis
Results were expressed as mean±SEM. Statistical significance (P<0.05) was determined by repeated-measures ANOVA (followed by Newman-Keuls test).
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Results |
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Effects of NOS2 Inhibition on ß-Adrenergic Stimulated Muscle Strips
Figure 1A shows the effects of 1 µmol/L isoproterenol (ISO), a ß-AR agonist, and 1 mmol/L AG, a specific NOS2 inhibitor, on force development. ISO increased force modestly and also shortened time to peak (TTP) and relaxation time (see inset of figure and Table). When the ISO response reached steady state, superfusion of AG caused a dramatic further increase in force as well as accelerating TTP and relaxation time (see inset and Table). Figure 1B summarizes data on the effects of ISO and ISO+AG on force. ISO alone increased force production by 39±13% of control (P<0.05 versus control). ISO+AG led to a further increase in force production by 118±22% of control (P<0.05 versus control and ISO). Kinetic data are summarized in the Table. Superfusion with AG+ISO further decreased TTP and relaxation time (measured as RT50%). These data show that specific NOS2 inhibition increases myocardial ß-AR responsiveness in HF.
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Effects of NOS2 Inhibition on ß-Adrenergic Stimulated Ventricular Myocytes
Next, we examined whether NO produced via NOS2 within cardiac myocytes (isolated from failing human hearts) was sufficient for the ß-AR hyporesponsiveness observed in human HF. Figure 2A (top traces) shows that ISO produced an increase in shortening (albeit small) and modest decreases in TTP and relaxation time (Table). When the ISO response reached steady state, superfusion of AG caused a marked further increase in shortening amplitude as well as decreasing TTP and relaxation time. Figure 2B (left) shows that on average, ISO increased shortening (by 61±30% of control, P<0.05 versus control). ISO+AG led to a further increase in shortening (by 196±34% of control, P<0.05 versus control and ISO). Kinetic data (Table) show that AG superfusion decreased TTP and relaxation time. These data suggest that NOS2 expression within cardiac myocytes contributes directly to ß-AR hyporesponsiveness in human HF.
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Figure 2A (bottom) shows that myocyte Ca2+ transients were changed by ISO and ISO+AG in a manner that parallels the shortening data. That is, ISO slightly increased Ca2+ transient amplitude (by 41±15% of control, P<0.05 versus control), but with NOS2 inhibition (ISO+AG), Ca2+ transients were more than doubled in amplitude (by 126±27% of control, P<0.05 versus control and ISO, Figure 2B). Similarly, ISO alone produced only a small lusitropic effect and acceleration of [Ca]i decline, but these were dramatically enhanced by ISO+AG (Table). A similar effect was seen with L-NIL (a different specific NOS2 inhibitor; data not shown). In addition, AG alone (without previous ISO treatment) had no significant effect on either contractions or Ca transients (by –8±6% and 1±6%, respectively, Figure 2C). Our results suggest that NOS2 within HF cardiac myocytes causes ß-AR hyporesponsiveness by altering Ca2+ handling.
No Functional Effects of NOS2 Inhibition and ß-Adrenergic Responsiveness
In 2 of the HF trabeculae, addition of AG had no additional effect on the ISO response (by –3% of ISO response; eg, Figure 3A). However, in this case, the muscle already exhibited a large ISO response (by 277% of control versus 39% for all other HF muscles). This is comparable to the large inotropic and lusitropic effects observed in nonfailing muscles (Figure 3B), in which addition of AG (or L-NIL) produced no further effects.
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This lack of AG effect was also observed in a minority of HF cells (Figure 4A). As for the trabeculae, these myocytes already had a large response to ISO alone (Figure 4B; increases as percentage of control were 354±72% for shortening and 163±8% for [Ca2+]i versus 61% and 41% for all other myocytes). Again, the large inotropic effect of ISO in these myocytes (and complete lack of AG effect) was similar to that observed in myocytes from nonfailing human heart (Figure 4D, 312±53% for shortening and 123±27% for [Ca]i). We infer that the hearts that these trabeculae and myocytes were isolated from (4 of 13 HF hearts) might not express functional NOS2 at substantial levels. This would explain both the lack of AG effect and the much greater stimulation by ISO alone.
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Expression of NOS2 in Failing and Nonfailing Human Hearts
Next, we examined NOS2 expression in the failing hearts in which we measured contraction and [Ca]i. Figure 5 shows that NOS2 was not expressed in nonfailing heart but was expressed in failing hearts, in which NOS2 inhibitors had functional effects (F+). In addition, in a failing heart in which NOS2 inhibition had no effect (F–), there was no apparent expression of NOS2 (as in the nonfailing heart). This supports our inference that NOS2 expression is centrally involved in the blunted response to ß-AR stimulation.
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135-kDa band) of various intensity is detected in failing hearts, which showed a functional effect of NOS2 inhibition (F+), whereas no band is detected in failing hearts that had no functional effect of NOS2 inhibition (F–) and in a nonfailing heart (NF).
Relationship Between ß-AR Response and NOS2 Inhibition
Figure 6 shows the inverse relationship between the responses to ISO and to NOS2 inhibition (AG or L-NIL), measured as force production in trabeculae and shortening in myocytes. Thus, when there was a large response to ISO, NOS2 inhibition had little effect on trabeculae force production (top) or myocyte shortening (bottom). Conversely, when the ISO response was small, NOS2 inhibition had a large effect on force production and myocyte shortening. These data are consistent with NOS2 expression and NO production within failing cardiac myocytes, severely limiting the responsiveness to ß-AR in HF.
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Discussion |
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Human HF is associated with altered ß-AR signaling. Myocytes isolated from failing human hearts have a decreased contractile response to ß-AR stimulation and also to forskolin,18 which would bypass both the downregulation of ß-AR receptors and the increased G
i protein expression seen in HF. This has been explained in part by altered expression of Ca2+ handling proteins.1,5 It has also been proposed that NO plays a role in the ß-AR hyporesponsiveness observed in HF.19 This is the first report that specific NOS2 inhibition significantly increased ß-AR inotropic effects in muscle strips and isolated myocytes from failing human hearts.
Effects of NOS2 Inhibition on ß-AR Response in Human HF
Hare and coworkers14 showed in an in vivo study that patients with HF had a larger response to NOS inhibition on potentiating ß-AR responsiveness compared with normal subjects. However, this study used a nonspecific NOS inhibitor, NG-monomethyl-L-arginine (L-NMMA), and the specific role of NOS2 cannot be inferred from this study. Another study10 showed a significant correlation between ß-AR hyporesponsiveness and NOS2 mRNA levels. Unfortunately, this study also used a nonspecific NOS inhibitor (L-NMMA) to further increase the ISO response in isolated muscle strips, again leaving the specific role of NOS2 somewhat equivocal. This is a key issue with regard to NO signaling in HF, because NOS1 and NOS3 expression may be altered20,21 and/or NOS3 activity enhanced because of ß3-AR activation.22 In addition, it has been found that nonspecific NOS inhibition had no effect on the ISO response in myocytes isolated from failing human hearts.23
We show here that specific NOS2 inhibition greatly enhanced the response to ß-AR stimulation in trabeculae from failing human hearts (Figure 1). We also show that specific NOS2 inhibition in isolated cardiac myocytes from failing human hearts restored the ß-AR response (Figure 2). We specifically inhibited NOS2 with AG, which is a specific inhibitor of the NOS2 isoform in cardiac myocytes.24–26 Similar effects were observed when using either AG or L-NIL27 as NOS2 inhibitors. In addition, there was no effect of AG or L-NIL on ISO-treated myocytes or trabeculae from nonfailing human hearts, which do not express NOS2 (present study and Stein et al20 and Thoenes et al28). In the absence of ISO, AG also had no effect on Ca transients or contractions (Figure 2C). Thus, AG and L-NIL (as used here) do not appear to have nonspecific side effects. Therefore, our data suggest that expression of NOS2 within the cardiac myocytes of failing human hearts leads to a dramatic decrease in response to ß-AR stimulation. The lack of effect of AG on basal Ca2+ transients or contractions in HF also suggests that NO produced by NOS2 is exerting its effect primarily by modulating ß-AR signaling.
Mechanism of NOS2-Mediated ß-Adrenergic Hyporesponsiveness in Human HF
We investigated the possible mechanism of how NOS2 expression leads to a reduced response to ß-AR stimulation in isolated cardiac myocytes from failing human hearts. Along with further enhancing myocyte shortening in response to ISO superfusion, we also found that NOS2 inhibition enhanced the Ca2+ transient response to ISO (Figure 2) in failing human hearts. These data suggest that NOS2 expression depresses the ß-AR stimulation of Ca2+ transient amplitude and kinetics.
Altered Sarcoplasmic Reticulum Ca2+ Transport?
There is a decreased level of phospholamban (PLB) phosphorylation with ß-AR stimulation in human HF.29–31 We have recently shown that reduced functional ISO responsiveness in failing human myocardium was associated with less ISO-induced increase in the sarcoplasmic reticulum (SR) Ca2+ load in failing versus nonfailing human myocardium.15 This defect may be partly a result of alterations in PLB phosphorylation. Indeed, we show here that the lusitropic effect of ISO was also severely limited by NOS2 (Table). We have also shown directly that NO can reduce PLB phosphorylation.32 Thus, expression of NOS2 (and increased NO• production, or a related congener) could limit ß-AR–induced PLB phosphorylation, which would decrease SR Ca2+ load, slow relaxation, and reduce ß-AR responsiveness, as is seen in human HF.
Recently, we also found that NOS2 inhibition increases resting Ca2+ spark frequency (in the presence of ß-adrenergic stimulation) in cardiac myocytes isolated from failing human hearts.25 A main determinant of spark frequency is SR Ca2+ load33; hence, this result also supports the above hypothesis. In addition, NOS2 expression can also directly affect ryanodine receptor activity25 and L-type Ca2+ channel amplitude,24 which could also play a role in the ß-AR hyporesponsiveness seen here.
NOS2 and HF in Human Patients
Our study indicates that not all but a majority of failing hearts express NOS2 (Figure 5). This agrees with previous studies, which found NOS2 expression,6–13 did not detect NOS2 expression,20,28 and a study9 with results similar to ours (ie, a majority of failing hearts but not all express NOS2). Thus, most but not all HF patients express NOS2 (and this is independent of pathogenesis or drug therapy).11 In the present study, we further show that the failing hearts that do exhibit NOS2 expression also have a blunted response to ß-AR stimulation.
Another point of controversy with regard to NOS2 signaling in human HF is the source of NO production. Immunohistochemistry studies performed on tissue from human HF have shown NOS2 expression not only in cardiac myocytes but also in endothelial cells (from endocardial and vascular endothelium), vascular smooth muscle cells, infiltrating macrophages, fibroblasts, and circulating monocytes.8,9,11–13 Thus, there is controversy34,35 as to which cell type expresses NOS2 that results in ß-AR hyporesponsiveness.9,11–13 Our present results are the first to show that NOS2 expression within cardiac myocytes is sufficient to cause ß-AR hyporesponsiveness.
No Expression of NOS2 and ß-Adrenergic Responsiveness in Human HF
There is a significant inverse relationship between ß-AR responsiveness and NOS2 inhibition (Figure 6). That is, when there was no effect of NOS2 inhibition on the ISO response, a large response to ß-AR stimulation was already seen in the trabeculae and isolated cardiac myocytes (failing and nonfailing human hearts). In the minority of HF cases in which NOS2 is not expressed, the ISO response was similar to what we observed in trabeculae and myocytes isolated from nonfailing human heart (Figures 3 and 4
and similar to previous studies in nonfailing trabeculae and myocytes15,18,23). Thus, we conclude that NOS2 expression is a key element in the ß-AR hyporesponsiveness associated with human HF.
In conclusion, our experiments are the first to show that NOS2 expression in the failing human heart leads to a reduction in the responsiveness of the heart to ß-AR stimulation. This effect was also observed in isolated cardiac myocytes from failing human hearts. This suggests that expression of NOS2 within the cardiac myocytes is sufficient to cause the ß-AR hyporesponsiveness in human HF. This autocrine effect of NO produced by the myocytes themselves causes alterations in Ca2+ handling that may be sufficient to explain the ß-AR hyporesponsiveness. We suggest that inhibition of NOS2 in failing cardiac myocytes will improve contractile reserve and could be therapeutically beneficial (in failing hearts that express NOS2).
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Acknowledgments |
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This study was supported by grants from the National Institutes of Health (NIH), HL-10122, and the American Heart Association (AHA), 0335385Z (Dr Ziolo); the Deutsche Forschungsgemeinschaft MA 1982/1-2 (Dr Maier); AHA 0010103U (Dr Piacentino); NIH HL-61495 (Dr Houser); and NIH HL-64098 and HL-64724 (Dr Bers). The authors thank the Loyola Department of Thoracic/Cardiovascular Surgery and the Temple Hospital Cardiac Transplant team for providing the human failing hearts and Ken Marguiles, MD, for helpful discussions.
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References |
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