AJP - Heart Add DOIs to your references at manuscript stage!
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Heart Circ Physiol 295: H1122-H1131, 2008. First published July 11, 2008; doi:10.1152/ajpheart.00386.2008
0363-6135/08 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
295/3/H1122    most recent
00386.2008v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via Web of Science (2)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Somers, J. R.
Right arrow Articles by Duff, H. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Somers, J. R.
Right arrow Articles by Duff, H. J.

iNOS in cardiac myocytes plays a critical role in death in a murine model of hypertrophy induced by calcineurin

Julie R. Somers,1,* Paul L. Beck,2,* James P. Lees-Miller,1 Daniel Roach,1 Yan Li,2 J. Guo,1 Steven Loken,1 Shan Zhan,1 Lisa Semeniuk,1 and Henry J. Duff1

1Libin Cardiovascular Institute and 2Division of Gastroenterology, University of Calgary, Calgary, Alberta, Canada

Submitted 11 April 2008 ; accepted in final form 9 July 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Transgenic overexpression of calcineurin (CN/Tg) in mouse cardiac myocytes results in hypertrophy followed by dilation, dysfunction, and sudden death. Nitric oxide (NO) produced via inducible NO synthase (iNOS) has been implicated in cardiac injury. Since calcineurin regulates iNOS expression, and since phenotypes of mice overexpressing iNOS are similar to CN/Tg, we hypothesized that iNOS is pathogenically involved in cardiac phenotypes of CN/Tg mice. CN/Tg mice had increased serum and cardiac iNOS levels. When CN/Tg-iNOS–/– and CN/Tg mice were compared, some phenotypes were similar: extent of hypertrophy and fibrosis. However, CN/Tg-iNOS–/– mice had improved systolic performance (P < 0.001) and less heart block (P < 0.0001); larger sodium current density and lower serum TNF-{alpha} levels (P < 0.03); and less apoptosis (P < 0.01) resulting in improved survival (P < 0.0003). To define tissue origins of iNOS production, chimeric lines were generated. Bone marrow (BM) from wild-type or iNOS–/– mice was transplanted into CN/Tg mice. iNOS deficiency restricted to BM-derived cells was not protective. Calcineurin activates the local production of NO by iNOS in cardiac myocytes, which significantly contributes to sudden death, heart block, left ventricular dilation, and impaired systolic performance in this murine model of cardiac hypertrophy induced by the overexpression of calcineurin.

heart block; sodium current; sudden death; inducible nitric oxide synthase; transgenic overexpression of calcineurin

TRANSGENIC OVEREXPRESSION of constitutively active calcineurin (CN/Tg) in cardiac myocytes results in profound concentric hypertrophy followed by ventricular dilatation, interstitial fibrosis, heart failure, and eventually sudden cardiac death (13, 26, 32, 33, 40). The sudden deaths relate to ion channelopathies, which are downstream consequences of abnormalities of calcium homeostasis (17) and an inflammatory process (6, 10, 27, 36).

Previous studies indicate that inducible nitric oxide (NO) synthase (iNOS) is a downstream target of calcineurin (12, 19, 36, 44, 45). The regulation of the iNOS gene promoter by calcineurin has been reported (36). LPS-induced iNOS expression in the heart was abrogated by the pharmacological inhibition of calcineurin (36). Similarly, LPS induced the expression of iNOS in wild-type (WT) hearts but not in the calcineurin Aβ knockout hearts (36). Reciprocally, the overexpression of constitutively active calcineurin in isolated cardiomyocytes caused the dephosphorylation and nuclear accumulation of the transcription factor family originally defined as nuclear factor of activated T-cells (NFAT)c1 and induced strong iNOS expression (36). In addition, chromatin immunoprecipitation confirmed the calcineurin-dependent binding of NFATc1 to the iNOS promoter (36). These data are consistent with the idea that iNOS is a downstream target of calcineurin.

Other data also suggest that the activation of iNOS contributes to the phenotypes seen in mice overexpressing constitutively active calcineurin. Mungrue et al. (34, 35) have reported the phenotype of mice manifesting the cardiac-specific upregulation of iNOS. These mice displayed the following phenotypes: mild inflammatory cell infiltrate, cardiac dilatation, and sudden cardiac death due to heart block. Thus the iNOS overexpressing mouse model displays phenotypes similar to those seen in the transgenic calcineurin mice. These data lead to the hypothesis that the deletion of iNOS in mice overexpressing constitutively active calcineurin will improve cardiac phenotypes.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Generation of mice. This investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH Publication No. 85-23, Revised 1996) and approved by the Animal Care and Use Committee of the University of Calgary.

CN/Tg mice were obtained from one mouse line from Molkentin's original 10 independent founder lines (33). The constitutively active calcineurin gene product is expressed behind the {alpha}-myosin heavy chain promoter, resulting in cardiac myocyte-specific expression (33). Every line demonstrated the same phenotype. WT and CN/Tg mice were separated using PCR analysis. Primer sequences used were as follows: forward, 5'-GTCTGACTAGGTGTCCTTCT-3'; and reverse, 5'-CGTCCTCCTGCTGGTATTAC-3'. iNOS deletion mice (Nos2 tm1Lau) were obtained from Jackson Laboratories (Bar Harbor, ME). The iNOS–/– mice were mated to CN/Tg mice to make transgenic mice that were heterozygous for the iNOS allele. These heterozygous mice were then mated to iNOS–/– mice, and their progeny were genotyped. Thereafter, CN/Tg mice in the iNOS–/– background were mated to iNOS–/– mice. The phenotypes of CN/Tg-iNOS–/– mice were compared with those of the CN/Tg mice. Primer sequences used to identify the iNOS deletion mice were as follows: oIMR1216, 5'-ACATGCAGAATGAGTACCGG-3'; and oIMR1217, 5'-TCAACATCTCCTGGTGGAAC-3'. These primers together amplify a 108 bp DNA fragment from the WT allele. The oMIR1218 sequence is 5'-AAT ATG CGA AGT GGA CCT CG-3'. When oMIR1218 is with oIMR1216, it amplifies a 275-bp fragment from the disrupted allele (2).

Measurements of structure and function. Mice were euthanized under pentobarbital. To assess the extent of fibrosis, Masson Trichrome staining was performed (33). The extent of fibrosis in the different treatment groups was compared by a pathologist unaware of the genotype of the specimen. The extent of fibrosis was quantified using the following scale: none = 0; minimal = 1, scattered interstitial fibrosis; mild = 2, interstitial fibrosis encircling <10%; moderate = 3, myocytes interstitial fibrosis encircling 11–50% myocytes; and severe = 4, interstitial fibrosis encircling >50% myocytes. Similarly, to assess inflammatory cell infiltration, hematoxylin-eosin staining was performed and sections were analyzed in a blinded fashion. To assess the extent of apoptosis, a Roche in situ cell death detection kit was used (54). Paraffin-embedded tissues were dewaxed and placed in citrate buffer. A blocking buffer was applied. The terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling (TUNEL) assay was performed according to the manufacturer's manual. A positive control was also prepared by applying DNAase 1 to additional slides to nick the DNA so that the TUNEL assay would stain the damaged tissue. A negative control was also prepared from additional slides. TNF-{alpha} levels (5) were measured by enzyme-linked immunoabsorbent assay (ELISA) from R&D Systems (Minneapolis, MN) according to the manufacturer's protocol.

Echocardiograms were obtained in conscious mice (40) to assess systolic function using a SONOS 5500 from Agilent Technologies (Andover, MA). Echocardiograms were obtained at 14 wk in the WT (n = 6), iNOS–/– (n = 5), CN/Tg-iNOS–/– (n = 6), and CN/Tg (n = 6) mice. Mice were examined at 14 wk of age because at this time ~50% of the calcineurin mice are usually dead. A previous study described a linear death rate beginning at 10 wk and that all animals are dead by ~28 wk (13). Conscious mice were loosely restrained in syringe case tubing, which had been modified with an opening for the 15-MHz linear transducer ECG probe. This is advantageous because the results are not altered by anesthetics, which are known to suppress cardiac function (40). The intra- and interobserver and repeatability variability measures for our laboratory have been previously reported (40). The results of the echocardiograms were confirmed by measurements of heart weight-to-body weight ratio.

ECGs were acquired using chronically implanted PhysioTel transmitters (EA-F20; Transoma Medical, St. Paul, MN). ECGs were analyzed with ECG prospector, a customized software package initially developed in MATLAB utilizing a continuous wavelet transform to localize the distinct recurring points of the ECG, such as the R waves and P waves. R- and P waves and premature ventricular beats were delineated in all files. One hour of data, at the same time of day, was evaluated for each mouse. The design of the prospector allows the visual validation of beats by a cardiologist (17). This program counted the episodes of heart block and ventricular tachycardia in each file.

Patch-clamp measurements of Na+ currents. The preparation of cardiac myocytes has been previously reported (16). In a previous study, our laboratory reported that Na+ current (INa) density was decreased in CN/Tg myocytes without a shift in steady-state inactivation or recovery from inactivation (16). Voltage-dependent INa were measured in isolated ventricular cardiac myocytes using a conventional patch-clamp method in a voltage-clamp configuration. The extracellular Na+ concentration ([Na+]o) was 10 mM, with the Na+ being replaced by equimolar N-methyl-D-glucamine (NMG). A rapid solution changer was used to expose the myocytes to this low [Na+]o solution just before applying the voltage-clamp pulses. All experiments were performed at room temperature. An Axopatch 200B (Axon) amplifier was used. The access resistance was <5 M{Omega}. The series resistance was routinely compensated to >80% (7). To minimize the effect of potential spontaneous shifts in the inactivation-voltage relationship of INa under the whole cell conditions, the holding potential was held at –120 mV and experiments were completed within 1–5 min after the rupture of the cell membrane. Test pulses of 60 ms in duration were applied from –90 to +50 mV in 5-mV increments with 5 s between pulses.

Solutions. The normal Tyrode's external solution contained (in mM): 145 NaCl, 2 CaCl2, 1.0 MgCl2, 5.0 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), 0.1 CdCl2, and 5.5 glucose (pH 7.4, adjusted with NaOH). The pipette solution for the INa recording contained (in mM): 120 CsCl, 20 tetraethylammonium chloride, 5 ATP-Mg, 0.5 GTP-Na2, 5 EGTA, 10 NaCl, 2 CaCl2, and 10 HEPES (pH 7.2, adjusted with CsOH). The calculated free [Ca2+] in the pipette solution was ~100 nM/l (8). The low [Na+]o extracellular solution contained (in mM): 10 NaCl, 132 NMG, 1 MgCl2, 5 CsCl, 5 HEPES, 5 CoCl2, 1 CaCl2, and 5 glucose (pH 7.4 with HCl).

Assessment of NO synthase activity: measurement of plasma NO metabolites. To assess nitrite/nitrate (NOx) production, plasma nitrate and nitrite levels were assayed via a modified Greiss reaction kit (Bioxytech NO-540; Oxis Research) used according to the manufacturer's guidelines, as described previously (5). Western blotting was used to assess cardiac iNOS expression. Snap-frozen cardiac samples were homogenized in 2.5 mM EDTA, and the protein concentration was determined by Lowry assay. Aliquots were placed in Laemmli's sample buffer (Sigma Chemical, St. Louis, MO). Protein samples were separated by SDS-PAGE and electrotransferred onto polyvinylidene difluoride transfer membranes (Amersham Pharmacia Biotech). The resulting blot was blocked with 5% nonfat dry milk in PBS containing 0.05% Tween 20 and then incubated for 24 h at 4°C in a 1:1,000 dilution of rabbit anti-iNOS (Cat No.: 610332BD; PharMingen). The membrane was then washed and incubated with 1:2,000 HRP-conjugated anti-rabbit IgG for 1 h at room temperature, washed, and detected by enhanced chemiluminescence with ECL Western blotting detection reagents (Amersham). Our immunocytochemical assays have been previously described (40). Unrelated isotype control antibodies and no primary antibody were used as negative controls as previously described (5). All primary monoclonal antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Bone marrow transplant. To assess whether iNOS expressed in circulating immune cells versus iNOS produced by cardiac tissues contributed to the cardiac phenotypes in calcineurin mice, we performed bone marrow (BM) transplant experiments as we have previously described (5). In short, female CN/Tg recipients (6 wk old) were irradiated before receiving BM cells from either WT or iNOS–/– male donors. Confirmation of chimeric status was assessed by flow cytometry and the Y chromosome in situ hybridization as we have described previously (5). In the BM transplanted mice, ECGs were performed in conscious mice at 18 wk of age. Survivorship was evaluated using a Kaplan-Meier curve.

Statistical analysis. Statsview (Abacus Concepts, Berkeley, CA) was used to analyze the data. Data are presented as means ± SE. The log-rank test within the Kaplan-Meier analysis was used to assess survival difference between groups. For multigroup comparisons, one-way ANOVA was used with a Newman-Keuls test. For atrioventricular block frequency measurements and the inflammatory cell counts where the data are not normally distributed, a nonparametric analysis (Friedman test) was performed.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Survival and myocardial performance are improved by iNOS deletion. Figure 1A shows the Kaplan-Meier survival curves comparing CN/Tg versus CN/Tg-iNOS–/– mice. The log-rank test indicates that survival was significantly improved in CN/Tg-iNOS–/– compared with CN/Tg (P < 0.001) mice. Premature deaths are not completely abrogated; however, they are significantly delayed.


Figure 1
View larger version (9K):
[in this window]
[in a new window]

  		Fig. 1. Kaplan-Meier analysis of survival. None of the transgenic overexpression of calcineurin (CN/Tg) mice (n = 19) survived beyond 34 wk, whereas none of the CN-inducible nitric oxide (NO) synthase (iNOS)–/– mice (n = 38) survived beyond 48 wk. Survival was statistically greater in the CN/Tg-iNOS–/– vs. CN/Tg mice (log rank test; P = 0.0003). However, both the CN/Tg-iNOS–/– and the CN/Tg mice die earlier than wild-type (WT) (n = 38) or iNOS–/– (n = 25) mice.

 
Table 1 shows metrics of cardiac hypertrophy and systolic performance in the four groups of mice. Cardiac hypertrophy is not altered by the ablation of iNOS in CN/Tg-iNOS–/– mice. However, left ventricular (LV) end-diastolic dimension and systolic performance were significantly improved in CN/Tg-iNOS–/– versus CN/Tg mice (P < 0.001; Table 1). The hearts were removed, blotted dry, and weighed. The heart weights were (in gm): 0.17 ± 0.02 for WT, 0.16 ± 0.02 for iNOS–/–, 0.38 ± 0.3 for CN/Tg, and 0.36 ± 0.03 for CN/Tg-iNOS–/– mice. The mean body weight measurements (in gm) are: 33.5 ± 2.8 for WT, 26.2 ± 2.2 for iNOS–/–, 23.9 ± 0.9 for CN/Tg, and 24.0 ± 1.1 for CN/Tg-iNOS–/– mice. The mean heart weight-to-body weight ratios are: 0.005 ± 0.001 for WT, 0.006 ± 0.0001 for iNOS–/–, 0.016 ± 0.002 for CN/Tg, and 0.015 ± 0.001 for CN/Tg-iNOS–/– mice.


View this table:
[in this window]
[in a new window]

  		Table 1. Echocardiogram results in conscious mice at 14 wk of age

 
Heart block is suppressed by iNOS ablation in CN/Tg mice. Figure 2A shows representative ECGs in the four groups of mice. CN/Tg mice have frequent episodes of heart block. Previous studies report that this heart block is due to the infra-His downregulation of the INa (17). The cumulative distributions of PR intervals in CN/Tg versus CN/Tg-iNOS–/– mice are compared (Fig. 2B). Note that the PR indicates the time delay to the first R wave, which can be an escape beat in the presence of heart block. The mean number of episodes of heart block per hour is not normally distributed, so it is presented in a box plot (Fig. 2C). The frequency of the episodes of heart block are reduced in CN/Tg-iNOS–/– mice (P < 0.001). Episodes of ventricular tachycardia were so rare, the frequency of this event cannot be rigorously compared.


Figure 2
View larger version (28K):
[in this window]
[in a new window]

  		Fig. 2. Incidence of heart block. A: representative ECG traces in the 4 groups of mice. WT and iNOS–/– mice both manifest normal sinus rhythm. B and C: CN/Tg- iNOS–/– mice have significantly fewer episodes of heart block compared with that of the CN/Tg mice. In C, **P < 0.01 (Friedman test). PR, PR interval of ECG.

 
Current density of INa of CN/Tg versus CN/Tg-iNOS–/– myocytes. Representative examples of the families of INa records elicited by the protocol shown in the inset are shown in Fig. 3A, on the left for CN/Tg-iNOS–/– and on the right for CN/Tg. Figure 3B shows the mean current-voltage relationships comparing CN/Tg (Fig. 3, {circ}) to CN/Tg-iNOS–/– at 15 days (n = 5). Figure 3C shows the mean peak current density at –30 mV was significantly increased in CN/Tg-iNOS–/– myocytes (P < 0.05) compared with CN/Tg myocytes. The mean capacitance values of the CN/Tg myocytes are 103 ± 8 pF versus CN/Tg-iNOS–/– of 102 ± 5 pF [not significant (NS)].


Figure 3
View larger version (14K):
[in this window]
[in a new window]

  		Fig. 3. Density of sodium current (INa) in CN/Tg vs. CN/Tg-iNOS–/– myocytes. A: representative examples of the family of currents elicited by the voltage-clamp pulses shown in the inset. The holding potential is –80 mV. Five seconds before applying the depolarizing pulses, a hyperpolarizing prepulse is introduced. Depolarizing pulses were from –70 to +20 mV in 10-mV increments. B: mean current-voltage relationships with CN/Tg ({circ}) and CN/Tg-iNOS–/– (bullet); CN/iNOS (n = 12) and CN (n = 9) are shown. C: comparison of peak INa density at –30 mV (taken from B) comparing CN/Tg vs. CN/Tg-iNOS–/– myocytes. *P < 0.05.

 
No shift in steady-state inactivation was observed comparing the two groups.

The deletion of iNOS decreases myocardial inflammation and apoptosis in the setting of CN overexpression. As has been previously reported by Molkentin et al. (33), there is an inflammatory infiltrate and an accumulation of interstitial apoptotic cells in CN/Tg hearts (11, 31, 33). We compared the extent of inflammatory infiltration, fibrosis and number of apoptotic cells in CN/Tg versus CN/Tg-iNOS–/– hearts at 14 wk of age. Inflammatory cell counts per high power (40x) field on hematoxylin-eosin slides were compared. Cells counts per high power field were: WT, 89 ± 12; iNOS–/–, 122 ± 14; CN/Tg-iNOS–/–, 245 ± 26; and CN/Tg, 250 ± 21. Thus cell counts were similar comparing histology in CN/Tg versus CN/Tg-iNOS–/– mice (NS). However, the infiltrate was significantly greater in these two types of transgenic mice compared with either WT or iNOS–/– mice (P < 0.001). The extent of fibrosis was evaluated by the Masson Trichrome stain. Mean fibrosis scales were 1.1 ± 0.4 for WT, 1.0 ± 0.1 for iNOS–/– (NS), 3.1 ± 0.6 for CN/Tg-iNOS–/–, and 3.5 ± 0.6 for CN/Tg (NS). Thus extent of fibrosis was similar in CN/Tg versus CN/Tg-iNOS–/– mice.

Since Molkentin's laboratory (10, 33) had previously reported interstitial apoptosis in CN/Tg mice, the number of TUNEL-positive cells were compared (10). The number of TUNEL-positive cells was significantly reduced in CN/Tg-iNOS–/– versus CN/Tg mice (P < 0.001; Fig. 4).


Figure 4
View larger version (7K):
[in this window]
[in a new window]

  		Fig. 4. Myocardial apoptosis. Interstitial apoptosis was significantly greater in CN/Tg compared with CN/Tg-iNOS–/– sample (***P < 0.001). HPF, high power field.

 
Serum TNF-{alpha} levels were markedly increased in CN/Tg mice versus WT mice (Fig. 5). Interestingly, the deletion of iNOS resulted in a significant reduction in serum TNF-{alpha} levels in CN/Tg-iNOS–/– compared with CN/Tg mice, suggesting an altered inflammatory milieu. To further define the inflammatory milieu, NO was assessed in a standard fashion by measuring stable NOx by a modified Greiss reaction (Fig. 6). Clearly, CN/Tg mice had significantly increased serum and cardiac NOx levels compared with those of the WT mice (Figs. 5 and 6). Serum and cardiac NOx levels were reduced in CN/Tg-iNOS–/– mice (compared to CN/Tg mice), suggesting that iNOS was a major contributor to serum and cardiac NOx levels in CN/Tg mice. This was confirmed by Western blotting (Fig. 7), in which cardiac iNOS protein levels were markedly increased in CN/Tg mice.


Figure 5
View larger version (9K):
[in this window]
[in a new window]

  		Fig. 5. Serum and tissue TNF-{alpha} level comparisons. One-way ANOVA analysis with Newman-Keuls test indicates a significant reduction in TNF-{alpha} levels in the CN/Tg-iNOS–/– compared with CN/Tg mice. *P < 0.05; ***P < 0.001.

 

Figure 6
View larger version (9K):
[in this window]
[in a new window]

  		Fig. 6. Serum and cardiac tissue nitrite/nitrate (NOx) levels. NOx in serum and myocardium are compared. NOx are significantly reduced in the CN/Tg-iNOS compared with CN/Tg mice. *P < 0.05; **P < 0.01.

 

Figure 7
View larger version (21K):
[in this window]
[in a new window]

  		Fig. 7. Cardiac tissue NOx levels and Western blots of iNOS expression. NOx in myocardium are compared. NOx are significantly reduced in the CN/Tg-iNOS compared with CN/Tg mice. *P < 0.05; **P < 0.01.

 
To assess the cell distribution of iNOS staining, immunocytochemical staining was performed (Fig. 8). CN/Tg mice showed markedly increased cardiac iNOS staining with the majority of staining being noted in cardiac myocytes (Fig. 8, C and D). There did not appear to be any recognizable pattern of the distribution of iNOS-positive cardiac myocytes in CN/Tg mice. Some iNOS-positive inflammatory cells were noted in CN/Tg mice, primarily localized in areas surrounding vessels. Only faint iNOS staining of cardiac myocytes was noted in WT animals (Fig. 8, A and B). As expected, no iNOS staining was noted in CN/Tg-iNOS–/– mice. The endothelial NOS (eNOS) staining appeared to be restricted to endothelial cells, and there was no significant difference in eNOS staining in CN/Tg compared with WT animals. These data, taken in concert, indicate an improvement in the inflammatory milieu (tissue NOx, TNF-{alpha} levels, and apoptosis) in CN/Tg-iNOS–/– mice.


Figure 8
View larger version (154K):
[in this window]
[in a new window]

  		Fig. 8. Immunohistochemical localization of iNOS in cardiac tissue. Isotype control primary antibody and no primary antibody failed to showed specific staining (A). WT animals had minimal iNOS staining, with only faint staining noted in some cardiac myocytes (B). Marked iNOS staining was noted in the hearts of CN/Tg (C and D), which was primarily localized to cardiac myocytes with some staining noted within inflammatory cells (C). In the CN/Tg mice, faint iNOS staining of cardiac myocytes was noted throughout the myocardium and more intense cardiac myocyte staining was noted in what appeared to be a random distribution (C and D). No staining was noted in CN/Tg-iNOS–/– mice, and the cardiac myocyte staining intensity and pattern in the chimeric lines with either WT-derived bone marrow (BM) or iNOS–/– BM was similar to that seen in the CN/Tg mice.

 
Myocardial iNOS expression plays a critical role in the pathogenesis of the cardiac damage induced by calcineurin overexpression. Since iNOS can be expressed by various cell types, experiments were designed to determine whether iNOS expression by cardiac myocytes was responsible for the cardiac phenotype or whether it was the iNOS expression from the recruited inflammatory cell population. To assess this, chimeric lines were established via BM transplantation of CN/Tg mice with either BM cells from WT or iNOS–/– donors (5). Survival was compared in CN/Tg mice transplanted with donor BM from WT versus iNOS–/– mice. Survival was not significantly different in CN/Tg mice transplanted with iNOS–/– versus WT donor BM (Fig. 9). Furthermore, chimeric CN/Tg lines that received iNOS–/–-derived BM failed to show any improvement in the echocardiographic indexes of dimension, thickness, or systolic performance compared with those lines that received WT-derived BM (Table 2). This is in contrast with our findings when iNOS is completely ablated in CN/Tg-iNOS–/– mice, which did show a significant reduction in the extent of LV dilation (Table 1).


Figure 9
View larger version (10K):
[in this window]
[in a new window]

  		Fig. 9. Kaplan-Meier analysis of survival after BM transplant. Survival was similar in CN/Tg mice transplanted with WT (n = 18) vs. iNOS–/– (n = 22) BM. P = not significant.

 

View this table:
[in this window]
[in a new window]

  		Table 2. Echocardiographic measurements comparing CN/Tg mice transplanted with bone marrow from WT or iNOS–/– mice

 
Immunocytochemical studies confirm the presence of Y chromosome-positive cells in the hearts of female transplanted mice. These data indicate that the loss of iNOS restricted to circulating immune cells/BM-derived cells was not sufficient to improve survival in the setting of CN overexpression. In keeping with this idea, there were no differences in cardiac myocyte iNOS staining in CN/Tg mice that received WT BM versus iNOS–/– BM. Thus it appears that iNOS produced by cardiac myocytes and not by BM-derived cells is responsible for the cardiac phenotype of CN/Tg mice.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The novel information provided by this study includes 1) local NO production by iNOS within CN/Tg cardiac myocytes is associated with apoptosis and increased serum TNF-{alpha} levels; 2) NO production by iNOS within cardiac myocytes is pathogenically involved in cardiac dilation and the deterioration of systolic performance in CN/Tg mice; 3) local NO production by iNOS within cardiac myocytes increases the frequency of episodes of heart block in CN/Tg mice, resulting in sudden death. These features are improved by the ablation of iNOS. 4) the ablation of iNOS in BM-derived cells alone is not sufficient to improve survival or LV dysfunction. In review, calcineurin activates the local production of NO by iNOS in cardiac myocytes, which significantly contributes to sudden death, heart block, LV dilation, and impaired systolic performance. A substantial body of evidence exists (48) that indicates a relationship between calcineurin signaling and the cardiac hypertrophic response with resultant heart failure in a wide range of animal models. Even so, the role of iNOS observed in this genetically modified mouse must be confirmed in other models of hypertrophy and heart failure.

Role of iNOS in reduced systolic performance in CN/Tg mice. Previous studies report that NO regulates the function of L-type calcium channel current (ICa, L) and sarcoplasmic reticulum (SR) function. Interleukin-6 decreased post-rest potentiation in cardiac myocytes, which was associated with a decrease in the phosphorylation of phospholamban and the decreased responsiveness to caffeine. These data suggest the alteration in SR-Ca2+ loading (50). The suppression of post-rest potentiation and the responsiveness to caffeine were abolished by NG-monomethyl-L-arginine and 2-amino-5,6 dihydro-6-methyl-4H-1,3-thiazine (a selective iNOS inhibitor). The effects of NO on calcium entry are more complex and controversial (29, 39). Peroxynitrite donors in guinea pig myocytes have been reported to increase the ventricular calcium current in an AMP- and cyclic GMP-independent manner (29). In contrast NO, signaling via cGMP and the muscarinic receptor decreased β-adrenoreceptor-stimulated ICa, L activity in cardiac myocytes. In the study of Schroder et al. (39), the peak average ICa, L current density, mean open probability, mean availability was not decreased by the NO donor 2-(N,N-diethylamino)-diazenolate-2-oxide (DEA)-NO in unstimulated WT cells. However, DEA-NO decreased ICa, L in mice overexpressing the cGMP-dependent protein kinase type I. In the setting of congestive heart failure, β3 stimulation with agonist BRL decreased ICa, L activity, and this decrease was attenuated by the treatment with a NOS inhibitor or pertussis toxin (PTX) (53). These data indicate that β3 stimulation decreases ICa, L in a PTX-sensitive G protein-mediated and an iNOS-dependent manner. Thus the effects of NO are dependent on the cellular environment including the presence of β1 versus β3 stimulation; cAMP, cGMP and phosphodiesterase activities; and the presence of congestive failure modulate responses. In a previous study (49), the overexpression of calcineurin increased ICa, L activity; however, these studies were done early in the life of calcineurin mice (40). In the present study, the improvement in myocardial performance and dilation in iNOS–/–-CN/Tg myocytes may relate to improved calcium handling.

Role of iNOS in INa and heart block in CN/Tg mice. We had also previously reported that calcineurin downregulates the activity of INa due to the activation of PKC secondary to abnormalities of calcium handling (17). Moreover we presented evidence that the decreased density of INa was likely responsible for the heart block seen in the CN/Tg mice. Our new finding is that density INa in CN/Tg-iNOS–/– mice is significantly greater than that of CN/Tg. NO regulates the function of cardiac INa (1). Previous studies indicate that NO significantly reduces peak whole-cell INa in isolated ventricular myocytes but did not decrease INa when the channel was expressed heterologously (1). The effects of NO to decrease INa could only be blocked by the simultaneous inhibition of both cGMP and cAMP pathways. At least part of the NO effect was mediated through its well-described action to activate the cGMP pathway. In the present study, the ablation of iNOS delayed the development of heart block, possibly by blocking the known effects of NO on the cGMP pathway. More specifically, it appears that iNOS expression within cardiac myocytes plays a critical role since the deletion of iNOS restricted to BM-derived cells had minimal effect. Thus the effect of iNOS on CN/Tg cardiac myocytes appears to be autocrine in nature.

Role of iNOS in apoptosis in CN/Tg mice. Molkentin and colleagues had previously reported an increase in interstial apoptosis in mice overexpressing calcineurin (11, 33). Previous studies indicate that pharmacological inhibition of NOS decreased the extent of apoptosis in an acute rat model of myocardial ischemia-reperfusion (11, 31, 33). Other studies report that both eNOS and iNOS play a critical role in mechanical stretch-induced cardiomyocyte apoptosis (24, 25). In addition, chronic in vivo β1-adrenergic stimulation leads to apoptosis via an iNOS-mediated pathway (24, 25). The pharmacological inhibition of iNOS or targeted deletion of iNOS abrogates β1-adrenergic stimulation-induced apoptosis (24, 25). Thus previous studies are in keeping with the present work that the ablation of iNOS inhibits cardiac apoptosis. Inhibition of apoptosis may contribute to improved LV chamber dimension of the CN/Tg heart and overall contractile performance.

Role of TNF-{alpha} activation of iNOS in CN/Tg mice. The promoter for the TNF-{alpha} gene has four binding sites for NFAT (37, 46), which is downstream of calcineurin. The TNF-{alpha} gene becomes induced in response to abnormalities of intracellular calcium handling (7, 30), as is seen in mice overexpressing calcineurin (8). There is substantial evidence that iNOS is a downstream target of TNF-{alpha} (9, 15, 42, 43). Some ion channelopathies may be mediated by TNF-{alpha}-activated iNOS. For example, TNF-{alpha} treatment can cause the downregulation of the transient outward K+ current activity in cardiac myocytes through iNOS activation and reactive oxygen species generation (14). These data indicate that both calcineurin and TNF-{alpha} pathways converge to activate iNOS.

Ablation of iNOS improves dilation but not cardiac hypertrophy. As assessed by LV septal and posterior wall thickness, the ablation of iNOS did not decrease cardiac hypertrophy; however, it significantly decreased LV chamber dilation. This study is in keeping with studies of Zhang et al. (52), who studied a systolic pressure overload model of hypertrophy followed by congestive heart failure. In that study, the ablation of iNOS decreased dilation but did not change septal or posterior wall thickness. Our data indicate that iNOS is downstream of calcineurin. The literature provides no evidence that iNOS interacts with the cardiac hypertrophic gene-program downstream of NFAT3. This may explain why iNOS ablation does not alter cardiac hypertrophy.

Ablation of iNOS does not improve cardiac fibrosis. During cardiac heart failure, fibroblasts become activated by various growth factors (51). Transforming growth factor (TGF)-β promotes fibroblasts transforming into myofibroblasts (22, 51). There appears to be no direct link between the signal transduction mediators of fibrosis and either calcineurin or iNOS pathways. There is evidence that eNOS (28, 41) can promote fibroblast production. The overexpression of TGF-β is associated with cardiac hypertrophy and fibrosis. (38). However, other studies report that TGF-β negatively regulates iNOS expression (47). Therefore, the iNOS pathway does not appear to act upstream of appropriate signal transduction pathways to impact development of fibrosis.

Role of NO sources from circulating immune cells versus cardiac tissues. In the present study, calcineurin was overexpressed behind an {alpha}-myosin heavy chain promoter, leading to cardiac myocyte-specific expression. Previous studies have suggested that iNOS is a downstream target of calcineurin in cardiac myocytes (36). Even so, the source of NO via iNOS could relate to synthesis in circulating immune cells or in cardiac tissue. To address the role of circulating immune cells/BM-derived cells, we assessed the phenotypic responses to BM transplantation with recipient CN/Tg mice receiving either WT or iNOS–/– BM. No significant difference was observed in these murine lines based on survival or LV performance. These data indicate that the ablation of iNOS exclusively from BM-derived tissues was insufficient to improve structure or function in CN-Tg mice. This study suggests that the critical processes involved in the pathogenesis of the cardiac phenotype in CN/Tg mice may be due to signaling events specific to the cardiac myocyte since the reduced systolic dysfunction, decreased episodes of heart block, and improved survival were only noted in murine lines where the deletion of iNOS involved the cardiac myocyte.

The myocyte-specific deletion of iNOS is necessary for the improvement of the heart block and contractive function phenotype. The serum NOx and myocardial NOx levels are significantly reduced in CN/Tg-iNOS–/– compared with CN/Tg mice but are not different than those seen in WT and only slightly greater than those in iNOS–/– mice. The reason that this modest reduction in NOx levels can explain the major improvements in phenotype is because the NO is created within the cardiac myocytes. During the NO assay, cardiac tissue is snap-frozen and then homogenized. This experimental procedure homogenized both cardiac myocytes and other intramyocardial tissues. Moreover, the homogenization procedure by necessity dilutes the cardiac tissue into homogenization buffer. More importantly, we provide immunocytochemical evidence that the NO is likely being created within myocytes and its main effect is autocrine. The concentrations of NO created within the myocytes and their immediate neighborhood are expected to be much higher than that observed in the chemical assay.

Although no difference in survival of CN/Tg mice transplanted with either WT or iNOS BM donors was observed, the survival of both groups is improved compared with untreated CN/Tg mice. It seems likely that stem/progenitor cells are transplanted along with other circulating BM elements. Our finding is in keeping with other studies showing that the transplantation of BM has been reported to improve survival in a number of rodent models of heart failure (3).

Limitations. The recent Triumph study failed to show improved survivorship by the pharmacological inhibition of iNOS by tilargine in patients with cardiogenic shock, complicating myocardial infarction despite an open infarct artery (36). However, the intervention was introduced late after infarction. Myocyte injury at that time may have been irreversible.

Georgakopoulos and Kass (16) have reported the force-frequency responses at heart rates between 400 and 900 beats/min in in situ mouse hearts. The effects on end-diastolic volume and LV maximal change in pressure over time were modest with heart rate changes from 400 to 500 beats/min. These data were confirmed by Larsen et al. (23) who assessed the effects of frequency on LV end-diastolic pressure. In the article of Larsen et al. (23), the force-frequency relationship between 400 and 500 beats/min is on the relatively flat part of that relation. More substantial changes in heart rate, say from 300 to 600 beats/min, do have a substantial and significant effect. Thus it seems likely that the comparison between CN/Tg (heart rate of 496 beats/min) to CN/Tg-iNOS–/– (heart rate of 388 beats/min) is likely valid. Even so, we cannot exclude a contribution of heart rate changes to measured changes in hemodynamics.

Although iNOS is an important mediator, the premature deaths in CN/Tg mice are not completely abrogated by the deletion of iNOS; this end point is just significantly delayed. Other factors other than iNOS are involved, and further studies are required.

Conclusions

Calcineurin activates the local production of NO by iNOS in cardiac myocytes, which significantly contributes to sudden death, heart block, LV dilation, and impaired systolic performance in this murine model of cardiac hypertrophy induced by the overexpression of calcineurin.

There is an urgent need to develop antiarrhythmic treatments that prolong life. The recent Sudden Cardiac Death in Heart Failure Trial Study shows that the putatively most effective antiarrhythmic drug, amiodarone, is no more effective than placebo in prolonging life (4). The current study presents evidence that ablating iNOS prolongs life and improves heart performance in the setting of progressive structural heart disease.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by the Canadian Institutes of Health Research and the Alberta Heart and Stroke Foundation.


   FOOTNOTES
 

Address for reprint requests and other correspondence: H. J. Duff, Dept. of Cardiac Sciences, Univ. of Calgary, HSC 1632, 3330 Hospital Dr. NW, Calgary, AB, Canada T2N 4N1 (e-mail: hduff{at}ucalgary.ca)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

* J. R. Somers and P. L. Beck contributed equally to this work. Back


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Ahmmed GU, Xu Y, Hong Dong P, Zhang Z, Eiserich J, Chiamvimonvat N. Nitric oxide modulates cardiac Na+ channel via protein kinase A and protein kinase G. Circ Res 89: 1005–1013, 2001.[Abstract/Free Full Text]
  2. Balligand JL, Cannon PJ. Nitric oxide synthases and cardiac muscle. Autocrine and paracrine influences. Arterioscler Thromb Vasc Biol 17: 1846–1858, 1997.[Abstract/Free Full Text]
  3. Balsam LB, Robbins RC. Haematopoietic stem cells and repair of the ischaemic heart. Clin Sci (Lond) 109: 483–492, 2005.[Medline]
  4. Bardy GH, Lee KL, Mark DB, Poole JE, Packer DL, Boineau R, Domanski M, Troutman C, Anderson J, Johnson G, McNulty SE, Clapp-Channing N, Davidson-Ray LD, Fraulo ES, Fishbein DP, Luceri RM, Ip JH, Sudden Cardiac Death in Heart Failure Trial (SCD-HeFT) Investigators. Amiodarone or an implantable cardioverter-defibrillator for congestive heart failure. N Engl J Med 352: 225–237, 2005.[Abstract/Free Full Text]
  5. Beck PL, Li Y, Wong J, Chen CW, Keenan CM, Sharkey KA, McCafferty DM. Inducible nitric oxide synthase from bone marrow-derived cells plays a critical role in regulating colonic inflammation. Gastroenterology 132: 1778–1790, 2007.[CrossRef][Medline]
  6. Bueno OF, Lips DJ, Kaiser RA, Wilkins BJ, Dai YS, Glascock BJ, Klevitsky R, Hewett TE, Kimball TR, Aronow BJ, Doevendans PA, Molkentin JD. Calcineurin Abeta gene targeting predisposes the myocardium to acute ischemia-induced apoptosis and dysfunction. Circ Res 94: 91–99, 2004.[Abstract/Free Full Text]
  7. Canellada A, Cano E, Sanchez-Ruiloba L, Zafra F, Redondo JM. Calcium-dependent expression of TNF-alpha in neural cells is mediated by the calcineurin/NFAT pathway. Mol Cell Neurosci 31: 692–701, 2006.[CrossRef][Web of Science][Medline]
  8. Chu G, Carr AN, Young KB, Lester JW, Yatani A, Sanbe A, Colbert MC, Schwartz SM, Frank KF, Lampe PD, Robbins J, Molkentin JD, Kranias EG. Enhanced myocyte contractility and Ca2+ handling in a calcineurin transgenic model of heart failure. Cardiovasc Res 54: 105–116, 2002.[Abstract/Free Full Text]
  9. Condorelli G, Morisco C, Latronico MV, Claudio PP, Dent P, Tsichlis P, Condorelli G, Frati G, Drusco A, Croce CM, Napoli C. TNF-alpha signal transduction in rat neonatal cardiac myocytes: definition of pathways generating from the TNF-alpha receptor. FASEB J 16: 1732–1737, 2002.[Abstract/Free Full Text]
  10. De Windt LJ, Lim HW, Haq S, Force T, Molkentin JD. Calcineurin promotes protein kinase C and c-Jun NH2-terminal kinase activation in the heart. Cross-talk between cardiac hypertrophic signaling pathways. J Biol Chem 275: 13571–13579, 2000.[Abstract/Free Full Text]
  11. De Windt LJ, Lim HW, Taigen T, Wencker D, Condorelli G, Dorn GW 2nd, Kitsis RN, Molkentin JD. Calcineurin-mediated hypertrophy protects cardiomyocytes from apoptosis in vitro and in vivo: an apoptosis-independent model of dilated heart failure. Circ Res 86: 255–263, 2000.[Abstract/Free Full Text]
  12. Diaz-Ruiz A, Vergara P, Perez-Severiano F, Segovia J, Guizar-Sahagun G, Ibarra A, Rios C. Cyclosporin-A inhibits constitutive nitric oxide synthase activity and neuronal and endothelial nitric oxide synthase expressions after spinal cord injury in rats. Neurochem Res 30: 245–251, 2005.[CrossRef][Web of Science][Medline]
  13. Dong D, Duan Y, Guo J, Roach DE, Swirp SL, Wang L, Lees-Miller JP, Sheldon RS, Molkentin JD, Duff HJ. Overexpression of calcineurin in mouse causes sudden cardiac death associated with decreased density of K+ channels. Cardiovasc Res 57: 320–332, 2003.[Abstract/Free Full Text]
  14. Fernandez-Velasco M, Ruiz-Hurtado G, Hurtado O, Moro MA, Delgado C. TNF-{alpha} downregulates transient outward potassium current in rat ventricular myocytes through iNOS overexpression and oxidant species generation. Am J Physiol Heart Circ Physiol 293: H238–H245, 2007.[Abstract/Free Full Text]
  15. Funakoshi H, Kubota T, Machida Y, Kawamura N, Feldman AM, Tsutsui H, Shimokawa H, Takeshita A. Involvement of inducible nitric oxide synthase in cardiac dysfunction with tumor necrosis factor-{alpha}. Am J Physiol Heart Circ Physiol 282: H2159–H2166, 2002.[Abstract/Free Full Text]
  16. Georgakopoulos D, Kass DA. Minimal force-frequency modulation of inotropy and relaxation of in situ murine heart. J Physiol 534: 535–545, 2001.[Abstract/Free Full Text]
  17. Guo J, Zhan S, Somers J, Westenbroek RE, Catterall WA, Roach DE, Sheldon RS, Lees-Miller JP, Li P, Shimoni Y, Duff HJ. Decrease in density of INa is in the common final pathway to heart block in murine hearts overexpressing calcineurin. Am J Physiol Heart Circ Physiol 291: H2669–H2679, 2006.[Abstract/Free Full Text]
  18. Hamalainen M, Lahti A, Moilanen E. Calcineurin inhibitors, cyclosporin A and tacrolimus inhibit expression of inducible nitric oxide synthase in colon epithelial and macrophage cell lines. Eur J Pharmacol 448: 239–244, 2002.[CrossRef][Web of Science][Medline]
  19. Heger J, Euler G. iNOS—another cardiac target of calcineurin. Cardiovasc Res 71: 612–614, 2006.[Free Full Text]
  20. Kato T, Sano M, Miyoshi S, Sato T, Hakuno D, Ishida H, Kinoshita-Nakazawa H, Fukuda K, Ogawa S. Calmodulin kinases II and IV and calcineurin are involved in leukemia inhibitory factor-induced cardiac hypertrophy in rats. Circ Res 87: 937–945, 2000.[Abstract/Free Full Text]
  21. Kim Y, Moon JS, Lee KS, Park SY, Cheong J, Kang HS, Lee HY, Kim HD. Ca2+/calmodulin-dependent protein phosphatase calcineurin mediates the expression of iNOS through IKK and NF-kappaB activity in LPS-stimulated mouse peritoneal macrophages and RAW 264.7 cells. Biochem Biophys Res Commun 314: 695–703, 2004.[CrossRef][Web of Science][Medline]
  22. Kubota T, Miyagishima M, Frye CS, Alber SM, Bounoutas GS, Kadokami T, Watkins SC, McTiernan CF, Feldman AM. Overexpression of tumor necrosis factor-alpha activates both anti- and pro-apoptotic pathways in the myocardium. J Mol Cell Cardiol 33: 1331–1344, 2001.[CrossRef][Web of Science][Medline]
  23. Larsen TS, Belke DD, Sas R, Giles WR, Severson DL, Lopaschuk GD, Tyberg JV. The isolated working mouse heart: methodological considerations. Pflügers Arch 437: 979–985, 1999.[CrossRef][Web of Science][Medline]
  24. Liao X, Liu JM, Du L, Tang A, Shang Y, Wang SQ, Chen LY, Chen Q. Nitric oxide signaling in stretch-induced apoptosis of neonatal rat cardiomyocytes. FASEB J 20: 1883–1885, 2006.[Abstract/Free Full Text]
  25. Liao X, Wang X, Gu Y, Chen Q, Chen LY. Involvement of death receptor signaling in mechanical stretch-induced cardiomyocyte apoptosis. Life Sci 77: 160–174, 2005.[CrossRef][Web of Science][Medline]
  26. Lim HW, De Windt LJ, Steinberg L, Taigen T, Witt SA, Kimball TR, Molkentin JD. Calcineurin expression, activation, and function in cardiac pressure-overload hypertrophy. Circulation 101: 2431–2437, 2000.[Abstract/Free Full Text]
  27. Lim HW, New L, Han J, Molkentin JD. Calcineurin enhances MAPK phosphatase-1 expression and p38 MAPK inactivation in cardiac myocytes. J Biol Chem 276: 15913–15919, 2001.[Abstract/Free Full Text]
  28. Liu YH, Xu J, Yang XP, Yang F, Shesely E, Carretero OA. Effect of ACE inhibitors and angiotensin II type 1 receptor antagonists on endothelial NO synthase knockout mice with heart failure. Hypertension 39: 375–381, 2002.[Abstract/Free Full Text]
  29. Malan D, Levi RC, Alloatti G, Marcantoni A, Bedendi I, Gallo MP. Cyclic AMP and cyclic GMP independent stimulation of ventricular calcium current by peroxynitrite donors in guinea pig myocytes. J Cell Physiol 197: 284–296, 2003.[CrossRef][Web of Science][Medline]
  30. Mitsuyama H, Kambe F, Murakami R, Cao X, Ishiguro N, Seo H. Calcium signaling pathway involving calcineurin regulates interleukin-8 gene expression through activation of NF-kappaB in human osteoblast-like cells. J Bone Miner Res 19: 671–679, 2004.[CrossRef][Web of Science][Medline]
  31. Molkentin JD. Calcineurin, mitochondrial membrane potential, and cardiomyocyte apoptosis. Circ Res 88: 1220–1222, 2001.[Free Full Text]
  32. Molkentin JD. Calcineurin and beyond: cardiac hypertrophic signaling. Circ Res 87: 731–738, 2000.[Abstract/Free Full Text]
  33. Molkentin JD, Lu JR, Antos CL, Markham B, Richardson J, Robbins J, Grant SR, Olson EN. A calcineurin-dependent transcriptional pathway for cardiac hypertrophy. Cell 93: 215–228, 1998.[CrossRef][Web of Science][Medline]
  34. Mungrue IN, Gros R, You X, Pirani A, Azad A, Csont T, Schulz R, Butany J, Stewart DJ, Husain M. Cardiomyocyte overexpression of iNOS in mice results in peroxynitrite generation, heart block, and sudden death. J Clin Invest 109: 735–743, 2002.[CrossRef][Web of Science][Medline]
  35. Mungrue IN, Husain M, Stewart DJ. The role of NOS in heart failure: lessons from murine genetic models. Heart Fail Rev 7: 407–422, 2002.[CrossRef][Medline]
  36. Obasanjo-Blackshire K, Mesquita R, Jabr RI, Molkentin JD, Hart SL, Marber MS, Xia Y, Heads RJ. Calcineurin regulates NFAT-dependent iNOS expression and protection of cardiomyocytes: co-operation with Src tyrosine kinase. Cardiovasc Res 71: 672–683, 2006.[Abstract/Free Full Text]
  37. Oum JH, Han J, Myung H, Hleb M, Sharma S, Park J. Molecular mechanism of NFAT family proteins for differential regulation of the IL-2 and TNF-alpha promoters. Mol Cell 13: 77–84, 2002.[CrossRef][Web of Science]
  38. Rosenkranz S. TGF-beta1 and angiotensin networking in cardiac remodeling. Cardiovasc Res 63: 423–432, 2004.[Abstract/Free Full Text]
  39. Schroder F, Klein G, Fiedler B, Bastein M, Schnasse N, Hillmer A, Ames S, Gambaryan S, Drexler H, Walter U, Lohmann SM, Wollert KC. Single L-type Ca2+ channel regulation by cGMP-dependent protein kinase type I in adult cardiomyocytes from PKG I transgenic mice. Cardiovasc Res 60: 268–277, 2003.[Abstract/Free Full Text]
  40. Semeniuk LM, Severson DL, Kryski AJ, Swirp SL, Molkentin JD, Duff HJ. Time-dependent systolic and diastolic function in mice overexpressing calcineurin. Am J Physiol Heart Circ Physiol 284: H425–H430, 2003.[Abstract/Free Full Text]
  41. Shimojo N, Jesmin S, Zaedi S, Soma M, Kobayashi T, Maeda S, Yamaguchi I, Goto K, Miyauchi T. EPA effect on NOS gene expression and on NO level in endothelin-1-induced hypertrophied cardiomyocytes. Exp Biol Med (Maywood) 231: 913–918, 2006.[Abstract/Free Full Text]
  42. Shindo T, Ikeda U, Ohkawa F, Kawahara Y, Yokoyama M, Shimada K. Nitric oxide synthesis in cardiac myocytes and fibroblasts by inflammatory cytokines. Cardiovasc Res 29: 813–819, 1995.[Abstract/Free Full Text]
  43. Song W, Lu X, Feng Q. Tumor necrosis factor-alpha induces apoptosis via inducible nitric oxide synthase in neonatal mouse cardiomyocytes. Cardiovasc Res 45: 595–602, 2000.[Abstract/Free Full Text]
  44. Tan JX, Liu CZ, Jie YL, Wang Y, Huang YG. Effect of cyclosporine A on myocardial calcineurin activity of the right ventricle and plasma NO, nitric oxide synthase and endothelin-1 levels in rats with chronic hypoxia. Di Yi Jun Yi Da Xue Xue Bao 24: 656–658, 2004.[Medline]
  45. Trajkovic V, Badovinac V, Jankovic V, Mostarica Stojkovic M. Cyclosporin A inhibits activation of inducible nitric oxide synthase in C6 glioma cell line. Brain Res 816: 92–98, 1999.[CrossRef][Web of Science][Medline]
  46. Tsai EY, Falvo JV, Tsytsykova AV, Barczak AK, Reimold AM, Glimcher LH, Fenton MJ, Gordon DC, Dunn IF, Goldfeld AE. A lipopolysaccharide-specific enhancer complex involving Ets, Elk-1, Sp1, and CREB binding protein and p300 is recruited to the tumor necrosis factor alpha promoter in vivo. Mol Cell Biol 20: 6084–6094, 2000.[Abstract/Free Full Text]
  47. Vodovotz Y, Geiser AG, Chesler L, Letterio JJ, Campbell A, Lucia MS, Sporn MB, Roberts AB. Spontaneously increased production of nitric oxide and aberrant expression of the inducible nitric oxide synthase in vivo in the transforming growth factor beta 1 null mouse. J Exp Med 183: 2337–2342, 1996.[Abstract/Free Full Text]
  48. Wilkins BJ, Molkentin JD. Calcium-calcineurin signaling in the regulation of cardiac hypertrophy. Biochem Biophys Res Commun 322: 1178–1191, 2004.[CrossRef][Web of Science][Medline]
  49. Yatani A, Honda R, Tymitz KM, Lalli MJ, Molkentin JD. Enhanced Ca2+ channel currents in cardiac hypertrophy induced by activation of calcineurin-dependent pathway. J Mol Cell Cardiol 33: 249–259, 2001.[CrossRef][Web of Science][Medline]
  50. Yu XW, Chen Q, Kennedy RH, Liu SJ. Inhibition of sarcoplasmic reticular function by chronic interleukin-6 exposure via iNOS in adult ventricular myocytes. J Physiol 566: 327–340, 2005.[Abstract/Free Full Text]
  51. Zhang HY, Phan SH. Inhibition of myofibroblast apoptosis by transforming growth factor beta1. Am J Respir Cell Mol Biol 21: 658–665, 1999.[Abstract/Free Full Text]
  52. Zhang P, Xu X, Hu X, van Deel ED, Zhu G, Chen Y. Inducible nitric oxide synthase deficiency protects the heart from systolic overload-induced ventricular hypertrophy and congestive heart failure. Circ Res 100: 1089–1098, 2007.[Abstract/Free Full Text]
  53. Zhang ZS, Cheng HJ, Onishi K, Ohte N, Wannenburg T, Cheng CP. Enhanced inhibition of L-type Ca2+ current by beta3-adrenergic stimulation in failing rat heart. J Pharmacol Exp Ther 315: 1203–1211, 2005.[Abstract/Free Full Text]
  54. Zhou F, Ajuebor MN, Beck PL, Le T, Hogaboam CM, Swain MG. CD154-CD40 interactions drive hepatocyte apoptosis in murine fulminant hepatitis. Hepatology 42: 372–380, 2005.[CrossRef][Web of Science][Medline]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
295/3/H1122    most recent
00386.2008v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via Web of Science (2)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Somers, J. R.
Right arrow Articles by Duff, H. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Somers, J. R.
Right arrow Articles by Duff, H. J.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2008 by the American Physiological Society.