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Am J Physiol Heart Circ Physiol 288: H1367-H1373, 2005. First published November 18, 2004; doi:10.1152/ajpheart.00880.2004
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Functional effects of C-type natriuretic peptide and nitric oxide are attenuated in hypertrophic myocytes from pressure-overloaded mouse hearts

Jun Su,1 Qihang Zhang,1 Jacob Moalem,2 James Tse,3 Peter M. Scholz,2 and Harvey R. Weiss1

Heart and Brain Circulation Laboratory, Departments of 1Physiology and Biophysics, 2Surgery, and 3Anesthesia, University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, Piscataway, New Jersey

Submitted 25 August 2004 ; accepted in final form 11 November 2004


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Increases in the myocardial level of cGMP usually exert negative inotropic effects in the mammalian hearts. We tested the hypothesis that the negative functional effects caused by nitric oxide (NO) or C-type natriuretic peptide (CNP) through cGMP would be blunted in hypertrophied cardiac myocytes. Contractile function, guanylyl cyclase activity, cGMP-dependent protein phosphorylation, and calcium transients were assessed in ventricular myocytes from aortic stenosis-induced hypertrophic and age-matched control mice. Basal percentage shortening was similar in control and hypertrophic myocytes. S-nitroso-N-acetyl-penicillamine (SNAP, an NO donor, 10–6 and 10–5 M) or CNP (10–8 and 10–7 M) reduced percentage shortening in both groups, but their effects were blunted in hypertrophic myocytes. Maximal rates of shortening and relaxation were depressed at the basal level, and both reagents had attenuated effects in hypertrophy. Similar results were also found after treatment with guanylin and carbon monoxide, other stimulators of particulate, and soluble guanylyl cyclase, respectively. Guanylyl cyclase activity was not significantly changed in hypertrophy. Addition of Rp-8-[(4-chlorophenyl)thio]-cGMPS triethylamine (an inhibitor of cGMP-dependent protein kinase, 5 x 10–6 M) blocked SNAP or the effect of CNP in control mice but not in hypertrophy, indicating the cGMP-dependent kinase (PKG) may not mediate the actions of cGMP induced by NO or CNP in the hypertrophic state. Calcium transients after SNAP or CNP were not significantly changed in hypertrophy. These results suggest that in hypertrophied mice, diminished effects of NO or CNP on ventricular myocyte contraction are not due to changes in guanylyl cyclase activity. The data also indicated that PKG-mediated pathways were diminished in hypertrophied myocardium, contributing to blunted effects.

cardiomyocytes; guanosine 3',5'-cyclic monophosphate; guanosine 3',5'-cyclic monophosphate protein kinase; calcium; cardiac hypertrophy

CARDIAC HYPERTROPHY, in response to increased biomechanical stress (5), is currently recognized as a major precursor to heart failure and sudden death. Hypertrophy is characterized by a plethora of altered gene expression, including reexpressed immature fetal cardiac genes, genes modifying intracellular ion homeostasis (such as sarcoplasmic and endoplasmic reticulum calcium ATPases), and some parasympathetic and sympathetic receptor genes (10, 14). Basal nitric oxide (NO) production in hypertrophic hearts is increased in rats (11), and atrial natriuretic peptide expression is elevated (4). Recently, several lines of evidence suggested that the cGMP-dependent signal transduction pathway might be an important pathway for cardiac hypertrophy due to its potential to oppose cardiomyopathy (9, 23). In addition, there is some evidence in hypertrophied myocytes that the cGMP signaling pathway activated by NO stimulation is blunted (12), and the effect of natriuretic peptide through the cGMP signaling pathway is also attenuated (27). However, a parallel study of both pathways in the same type of hypertrophied myocardium is lacking.

cGMP is a ubiquitous intracellular second messenger. Increases in the myocardial level of cGMP usually exert negative metabolic as well as inotropic effects in mammalian hearts (16, 25). The downstream protein cGMP-dependent protein kinase is generally regarded as the major mediator of the action of cGMP in the myocardium. There are also two types of guanylyl cyclases that produce cGMP but occupy different sites in intracellular space: soluble guanylyl cyclase (sGC) in the cytosol and particulate guanylyl cyclase (pGC) right beneath the plasma membrane. They can be stimulated by their specific ligands: pGC is activated by natriuretic peptides, and heat-stable enterotoxin-like peptides such as guanylin and sGC can be activated by NO or carbon monoxide (CO). Recently, it was observed that cGMP from these two different sources had distinct mechanisms and differential effects in various studies (7, 21, 33). It is not known whether this pattern remains during the cardiac reprogramming of hypertrophy.

In this study, we hypothesized that stimulation of both the pGC and sGC pathways would be attenuated in myocytes from hypertrophied hearts. We tested this hypothesis in ventricular myocytes freshly isolated from a mouse hypertrophy model that was established via aortic stenosis. We demonstrated that activation of sGC and pGC by their specific ligands induced similarly reduced effects on ventricular myocyte contraction. This was not dependent on altered guanylyl cyclase activity but was related to a reduced cGMP protein kinase effect. Our results suggested that the downstream cGMP pathways were attenuated in the hypertrophied myocardium.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
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All experiments were conducted in accordance with Guide for the Care of Laboratory Animals (DHHS Publications No. 85-23, Revised 1996) and approved by our Institutional Animal Care and Use Committee. All the chemicals were obtained from Sigma Chemical (St. Louis, MO) unless specified. The animals used in the study were from Jackson Laboratory (Bar Harbor, ME). We used transverse aorta banding to induce hypertrophy in adult mice (strain C57BL/6J, 6–8 wk old, either sex). Specifically, animals were prepared under sterile, anesthetized (Avertin, ip) conditions. The transverse aorta was exposed between the right innominate artery and the left carotid. Aortic constriction was created by tying a 6-0 suture against a 3-mm length of a 27-gauge needle. This produced 65–85% aortic narrowing. The mice were allowed to recover after the operation. After 4 wk, the animals were euthanized, and the body weight and heart weight were measured.

Myocyte isolation. Freshly isolated ventricular myocytes were prepared by a standard protocol as described previously by our laboratory (26) with limited modifications. The animals were anesthetized, and the hearts were rapidly removed after intraperitoneal injection of pentobarbital (50 mg/kg). The aorta was rapidly cannulated, and the heart was subsequently mounted on a Langendorff perfusion apparatus. Aortic retrograde perfusion was initiated at 70 mmHg constant pressure with modified minimal essential medium (MEM) supplemented with (in mM) 10 taurine, 2 L-glutamic acid, and 20 HEPES; pH 7.35. After 2 min, the heart was perfused with MEM containing 0.1% type II collagenase (Worthington, Freehold, NJ) for 10 min. The perfusion buffer was maintained at 37°C and equilibrated with a water-saturated gas mixture (95% O2-5% CO2). After collagenase digestion, the heart was removed from the apparatus, and the ventricles were lacerated into small pieces. The tissue was subsequently pipetted in the 0.1% collagenase solution until all the large pieces were dispersed in cell suspension. The supernatant containing isolated myocytes was then transferred to 15-ml centrifuge tubes. After being washed once in MEM supplemented with 0.5% bovine serum albumin (BSA), the cells finally were subjected to calcium reintroduction to 1 mM in the presence of 10 mM 2,3-butanedione monoxime. After being washed three times, the myocytes were suspended in the media supplemented with 0.5% BSA and 1 mM CaCl2 at room temperature. Cell viability was assessed by the maintenance of rod-shaped morphology and routinely is ~40–60%.

Cell contraction measurements. Details of the analysis of myocyte function have been reported previously (32). Briefly, isolated cardiac myocytes were put into a chamber on the stage of an inverted microscope (Zeiss Axiovert 125) in 2 ml of MEM solution supplemented with 0.5% BSA and 2 mM CaCl2. Two platinum wires placed in the chamber were used to pace the myocytes (1 Hz, 5-ms duration, voltage 10% above threshold, and polarity alternated with each pulse). The chamber was maintained at 37°C throughout the measurement. The contraction of individual myocytes was measured with a Myotrack system containing a camera and a video-edge detector (Crystal Biotech, Patton Biomedical). The contraction data were continuously obtained on a television monitor and a desktop computer. The parameters obtained during a single contraction included the diastolic cell length, percentage shortening, maximal rate of relaxation, maximal rate of shortening, time to peak shortening, time to 50% relaxation, and time to 90% relaxation.

Intracellular Ca2+ transients measurements. The ventricular myocytes were loaded with 2 µM fura-2 acetoxymethyl ester (fura 2-AM, Molecular Probes, Eugene, OR) for 60 min at 37°C in MEM buffer supplemented with 1 mM CaCl2 and 0.5% BSA. The cells were then washed and resuspended in the same buffer. The fluorescence was measured with a dual-excitation fluorescence photomultiplier system (Ionoptix, Milton, MA). Myocytes were placed in a 37°C chamber on the stage of a Nikon inverted microscope (model TS100) and imaged through a fluor x40 objective. The cells were exposed to light excitation at 360 or 380 nm wavelength while being stimulated to contract at 1 Hz, 5-ms duration, voltage 10% above threshold, and polarity alternated with each pulse. Fluorescence emission was detected between 480 and 520 nm by a photomultiplier tube, and qualitative changes in intracellular concentration were determined from the ratio of the fluorescence intensity at 360/380 wavelengths. Each experimental protocol was performed in at least three myocytes from each animal, and nine animals were used for each group.

Guanylyl cyclase activity measurements. The activity of sGC in isolated ventricular myocytes was evaluated with a modified assay used by Sadoff et al. (24). Briefly, a reaction system was established with a total volume of 0.2 ml containing 50 mM Tris·HCl (pH 7.6) and 10 mM theophylline and a GTP regeneration system that contains 10 mM creatine phosphate, 10 µg creatine phosphokinase (200 U/mg protein), 4 mM MgCl2, and 1 mM GTP, in the absence or presence of 0.1 mM S-nitroso-N-acetyl-penicillamine (SNAP). The dose of SNAP was capable of activating the enzyme maximally. Reactions were initiated by the addition of the above enzyme to the cell extract and maintained at 37°C for 10 min. Sodium acetate (0.8 ml, 50 mM, pH 4.0) was added, and the tube was placed in water at 90°C for 3 min to terminate the reaction.

pGC activity was measured using a modified protocol of Agullo et al. (1). The GTP regeneration system was set up in the same way; however, 0.1% Triton X-100 was added into the collected solution containing the particulate fraction to elicit an enhancement of pGC activity. The reaction was maintained at 37°C for 10 min and later terminated by the addition of 1 ml cold ethanol. The cGMP produced by enzyme activity was determined by radioimmunoassay.

Protein phosphorylation assay. Myocytes obtained from the hypertrophic and control mice were subjected to a cell extract preparation and in vitro phosphorylation assay. A suspension of myocytes was centrifuged at 34 g. The pellet was collected and frozen immediately at –80°C. Myocytes were homogenized for 15 s at 20,000 rpm (Polytron homogenizer) in a buffer (0.25 M sucrose, 5 mM Tris, and 1 mM MgCl2, pH 7.4) and centrifuged at 15,500 rpm at 4°C for 20 min. The supernatant was harvested and used as the myocyte extract for the phosphorylation assay. The protein concentration of each sample was determined by the Bradford dye-binding procedure using a spectrometer at 595 nm and adjusted to 5 mg/ml. 8-Bromo-cGMP (8-Br-cGMP, 2.5 x 10–4 M, an activator of cGMP protein kinase) was added to a 10-µl cell extract from control or hypertrophic animals. The reactants were placed at room temperature and allowed for equilibration for 10 min. After that, they were cooled on ice, and 0.5 µl [{gamma}-33P]ATP at 10 µCi/µl (Amersham) was added to initiate the reaction. An equal volume of reducing sample buffer was added after 15 min to terminate the phosphorylation reaction. The samples were then subjected to 95°C for 5 min, and electrophoresis was performed with miniature 12% SDS-polyacrylamide slab gels. Subsequently, the gels were stained with Coomassie brilliant blue, destained overnight, dried with gel drier, and exposed to X-ray film at –20°C for 36 h. The assay was repeated four times. The developed films were used for phosphoprotein analysis. The molecular weights were determined with standards, and intensities of protein bands on the films were calculated with One-Dscan software (Scanalytics, Fairfax, VA).

Experimental protocol. The following protocol was used for the myocyte shortening measurements. After the myocytes were loaded in the measurement system, a stabilization period of 3 min was allowed before the contraction data for the individual ventricular myocyte were recorded. A 3-min interval was allowed between reagent treatments, and 10 consecutive contractions were used for analysis. In the first group, C-type natriuretic peptide (CNP) was added at a concentration of 10–8 M, followed by a higher dose of CNP (10–7 M). In the second group, SNAP, a NO donor, was added at a concentration of 10–6 M, followed by a higher dose of SNAP (10–5 M). In the third group, Rp-8-[(4-chlorophenyl)thio]-cGMPS triethylamine (Rp-8-pCPT-cGMP, a specific inhibitor of the cGMP protein kinase) was administrated to the myocyte at a dose of 5 x 10–6 M, and then CNP or SNAP was added at a concentration of 10–7 or 10–5 M, respectively. In the fourth group, guanylin (a stimulator of particulate guanylyl cyclase) was added with a concentration of 10–6 M. In the fifth group, CO (a stimulator of sGC)-saturated MEM was added with the concentration of CO at about 8 x 10–6 M. The solution was bubbled with pure CO gas at room temperature and 1 atmosphere for at least 20 min before use. Each group was repeated at least two more times for each animal.

Statistical analysis. A repeated measure analysis of variance was used to compare variables measured in the experiment. Duncan's multiple range test was used to compare differences between baseline and the various treatments. A value of P < 0.05 was used as the level of statistical significance. All values are expressed as means ± SE.


   RESULTS
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 METHODS
 RESULTS
DISCUSSION
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The heart weight and heart weight-to-body weight ratio were significantly increased in aortic stenosed mice (237 ± 9, n = 9, vs. control, 174 ± 11 mg, n = 9, P < 0.001; 10.6 ± 0.3, n = 9 vs. control, 7.3 ± 0.2 mg/g, n = 9, P < 0.001), but body weights were not changed markedly (22.5 ± 0.6, n = 9 vs. control, 23.7 ± 1.2 g, n = 9). At baseline, the maximal rates of shortening (48.9 ± 2.9, n = 9 vs. control, 58.1 ± 3.2 µm/s, n = 9, P < 0.05) and relaxation (48.0 ± 2.5, n = 9 vs. control, 68.4 ± 2.1 µm/s, n = 9, P < 0.05) were severely depressed in the hypertrophic myocytes, but there were no significant differences in the other measured parameters such as percentage shortening, time to peak shortening, and time to 90% relaxation.

CNP significantly decreased percentage shortening in a dose-dependent pattern in ventricular myocytes from both groups of animals (Fig. 1A), but at the high dose, the decrements were much less in hypertrophied animals. Specifically, the percentage shortening of the myocytes from control mice was reduced by 32%, whereas it was only by 15% in the myocytes from hypertrophied animals. In a similar way, although CNP markedly reduced the maximal rates of shortening and relaxation in control animals, the effect was diminished in the myocytes from hypertrophied animals (Table 1). As another independent stimulator of cGMP production, SNAP had a similar effect (Fig. 1B). Although it depressed myocyte percentage shortening dose dependently in both hypertrophy and control mice, SNAP decreased percentage shortening by 18% in hypertrophy mice and 37% in control mice. Even though SNAP significantly depressed the maximal rates of shortening and relaxation in both animals, its effect was diminished in the hypertrophied myocytes (Table 1). Specifically, it decreased the maximal rate of shortening by 35% in control but only 17% in hypertrophy mice. It reduced the maximal rate of relaxation by 35% in control but 16% in hypertrophy mice.



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  		Fig. 1. Effects of C-type natriuretic peptide (CNP, A) and S-nitroso-N-acetyl-penicillamine (SNAP, B) on percentage shortening in freshly isolated ventricular myocytes. Data are presented as means ± SE; n = 9 mice hearts, *P < 0.05 vs. baseline; +P < 0.05 vs. control mice.

 

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  		Table 1. Modification of CNP and SNAP effects on maximal rate of shortening and maximal rate of relaxation by Rp-8-pCPT-cGMPS

 
Guanylin and CO can stimulate pGC and sGC, respectively, and promote cGMP production. Similar to CNP and SNAP, guanylin and CO decreased myocyte percentage shortening and maximal rate of shortening in hypertrophy mice (Table 2), but the decrement degree was attenuated in hypertrophied myocytes. Specifically, guanylin decreased percentage shortening in control myocytes by 30%, whereas there was only a 14% decrease in hypertrophic myocytes. CO depressed percentage shortening in control myocytes by 31%, whereas there was only a 17% decrease in hypertrophic myocytes.


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  		Table 2. Guanylin and CO effect on percentage shortening and maximal rate of shortening

 
Enzymatic activities of both pGC and sGC in control and hypertrophied myocytes were studied. However, there were no significant alterations of these enzyme activities at basal level or after stimulation with respective reagents between control and hypertrophied mice (Fig. 2). This strongly suggests that the blunted effects of CNP and NO in hypertrophy were not due to reduced activity of either the particulate or sGC.



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  		Fig. 2. Particulate guanylyl cyclase (pGC) and soluble guanylyl cyclase (sGC) activities in ventricular myocytes under basal condition and after stimulation with Triton X-100 and SNAP, respectively. n = 5 mice hearts. There was no significant difference between control and hypertrophy.

 
To test whether the diminished effects of these reagents in hypertrophied myocytes were related to altered cGMP protein kinase-dependent pathways, a specific inhibitor of cGMP protein kinase Rp-8-pCPT-cGMPS was added before treatment with CNP or SNAP. Rp-8-pCPT-cGMPS blocked the effect of CNP and SNAP on percentage shortening in control mice (Fig. 3, A and C) but not in hypertrophied animals (Fig. 3, B and D). Similar results were also obtained when the maximal rates of shortening or relaxation were examined (Table 1).



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  		Fig. 3. Effects of Rp-8-[(4-chlorophenyl)thio]-cGMPS triethylamine (Rp-8-pCPT-cGMPS) on percentage shortening responses to CNP (A) and SNAP (B) in freshly isolated ventricular myocytes from control and hypertrophied mice. Both SNAP and CNP significantly lowered percent shortening in control and hypertrophic myocytes. Note that Rp-8-pCPT-cGMPS changed the effect of CNP and SNAP only in control myocytes. n = 9 mice hearts. *P < 0.05 vs. baseline value.

 
The pattern of protein phosphorylation caused by the cGMP protein kinase was examined after stimulation with 8-Br-cGMP. In the control myocytes, there was a basal level of protein phosphorylation (Fig. 4, lane 1). The intensities of several were significantly enhanced on addition of 8-Br-cGMP (Fig. 4, lane 2). The overall background phosphorylation pattern from cardiac hypertrophic myocytes was similar to that of control (Fig. 4, lane 3). Proteins also underwent cGMP-dependent phosphorylation (lane 4). However, the degree of phosphorylation of these protein bands induced by cGMP was significantly reduced when the cardiac hypertrophic myocyte pattern was compared with control. This indicated the downstream pathway for cGMP was changed, and the cGMP protein kinase was not the major mediator of the action of cGMP in the hypertrophied mice.



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  		Fig. 4. Effects of cGMP-dependent protein phosphorylation on ventricular myocytes from control (lanes 1 and 2) and hypertrophic cardiac myocytes (lanes 3 and 4) groups of mice. An equal amount of protein (5 µg) was loaded on each lane. Lanes 1 and 3 show the basal activities of protein phosphorylation for the two groups. Lanes 2 and 4 show protein phosphorylation after addition (+) of 8-bromo-cGMP (8-Br-cGMP). Molecular weights standards are shown on left. Note the increased phosphorylation with 8-Br-cGMP and the blunting of this effect in hypertrophic mouse myocytes.

 
To examine whether the ability of these reagents to regulate Ca2+ dynamics was shifted in hypertrophy, the effects of both reagents on intracellular Ca2+ transients were studied in control and hypertrophied myocytes. CNP at 10–7 M significantly decreased the amplitude of Ca2+ transients in both animals, and there was no significant difference between them (Fig. 5A). SNAP had little effect in control mice as well as in hypertrophied mice in changing the Ca2+ transient peaks (Fig. 5B). There was no difference between the resting basal fluorescence intensity ratio (360/380 nm) in control and hypertrophic myocytes. The data suggest that although the reagents used different pathways to depress myocyte contraction, neither the effect of CNP nor SNAP on Ca2+ transients were changed in hypertrophy.



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  		Fig. 5. Effects of CNP (A) and SNAP (B) on percentage peak height of fluorescence intensity in ventricular myocytes. Data are shown as means ± SE; n = 5 mice hearts. *P < 0.05 vs. baseline. There was no difference between control and hypertrophy.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
A major finding of this study was that the contractile responses of cardiac myocytes to elevation in intracellular cGMP caused by stimulation of guanylyl cyclases were significantly diminished in hypertrophied myocardium. We found that in ventricular myocytes from hypertrophied mice, stimulation of both the particulate and sGC with CNP and the NO donor SNAP, respectively, had blunted effects on cell contraction. The diminished actions, however, were not related to decreased guanylyl cyclase activity, because the activities of pGC and sGC were not significantly depressed. In addition, another major finding of the present study was that the downstream cGMP pathway was changed and cGMP protein kinase inhibition did not have significant impact on the effect of either reagent. cGMP-dependent protein phosphorylation was reduced in hypertrophy. Finally, we also observed that calcium transients were not significantly changed in hypertrophy.

Negative myocardial functional effects of both natriuretic peptides and NO have been observed in various studies. The administration of atrial natriuretic peptide has been observed to depress contractility of freshly isolated myocytes (27) and the whole heart (19). Cardiorelaxation effects were also reported with brain natriuretic peptide (BNP) and CNP, the other two members of this family (18). Consistent with these studies, we found that CNP has a concentration-dependent negative inotropic effect in normal mice. CNP was chosen for this study due to it potency in reducing cardiac contractility in mouse hearts (20), and the doses used to treat myocytes were similar to the above studies. Although the doses are substantially higher than the picomolar concentration in the circulating plasma, it has been suggested that natriuretic peptides may act as a local endothelial-derived relaxing factor with autocrine or paracrine effects in the heart (17). Because of the ubiquitous and intimate interaction between myocytes and underlying endothelium (3), the local concentration of natriuretic peptides may be high enough to produce physiological effects. In addition, the negative functional effect of CNP was also observed with guanylin, which stimulates another member of pGC family and also significantly increases the cGMP level in mammalian hearts (6). However, there have also been reports of positive effects of CNP in some studies (8, 18, 20). Presumably one explanation for the discrepancy could be that in our study, we used isolated ventricular myocytes rather than the working whole heart where other cell types or factors present in coronary circulation may modify the effect of CNP on the myocyte.

We also observed that SNAP, the NO donor, inhibited myocyte function in a dose-dependent pattern, similar to a previous study from this laboratory (26). The negative functional effects of NO have been demonstrated in numerous studies across many species using various models, suggesting NO is a common cardiac suppressant agent in normal situations at least at these doses (6, 11, 26). NO is produced by several isoforms of NO synthase found in the heart (3, 7, 16, 25). Both the endothelial and neuronal forms have been reported in cardiac myocytes (25). In the current study, we added exogenous NO, so differential effects of NO synthase would not affect our results. Similarly, the effect of NO was also observed after CO, another stimulator of sGC. Taken together, the data strongly indicated that stimulation of both pGC and sGC have negative inotropic effects in isolated mouse ventricular myocytes.

It has been demonstrated that basal contractility can be depressed in pressure-overloaded cardiac hypertrophy (28). The diastolic properties, in terms of the relaxation velocity, were also reported to be abnormal in hypertrophy. Our data showed that although the percentage shortening was not significantly changed, both the maximum shortening velocity and relaxation velocity were significantly depressed. It has already been shown that the level of sarcoplasmic and endoplasmic reticulum calcium ATPases is reduced in pressure overload (28), and abnormalities in calcium handling by hypertrophic myocytes may play important roles in this process.

The effects of cGMP were reported changed in the hypertrophied myocardium. There are some lines of evidence showing that in various hypertrophic models the sGC-cGMP signaling pathway is blunted (12) and the effects of ANP on contractility are also attenuated (27). Here we report, to the best of our knowledge, on the effects of CNP on the contraction of hypertrophied myocardium for the first time. We found that similar to ANP, the inotropic responses to CNP that were observed in normal myocytes were blunted or abolished in myocytes from hypertrophied hearts. In addition, the effects of NO were also blunted or abolished in a parallel pattern. Because both reagents' performances were repeated by guanylin and CO, stimulators of pGC and sGC, respectively, this strongly suggests that the action of cGMP was generally diminished in hypertrophy regardless of its intracellular sources.

The blunted cGMP effect may not be caused by diminished cGMP production in myocytes, because the current study showed both the activities of pGC and sGC were not markedly changed in pressure-overloaded hypertrophy. Certain types of phosphodiesterase activity can be shifted in response to pressure (31), but it has also been reported to be unchanged in other studies (24). Because homogenized myocardial tissues were used in those studies, currently it is not certain what the activity changes actually were in ventricular myocytes. In addition, although it was reported that the basal level of cGMP was elevated in some hypertrophy models (24), discordant evidence was also found (29). Alterations in the cGMP level may be contingent on the species or stimuli used to induce cardiac hypertrophy. Further investigation is needed to adequately address this issue.

cGMP can reduce cardiac contraction through several pathways. In the current study, we demonstrated in normal hearts that the contractile responses of CNP and NO were via the cGMP-cGMP protein kinase pathway, because blockage of this pathway with a specific inhibitor abolished their negative inotropic effects on cell contraction. It also seemed that the cGMP protein kinase was an important mediator for the action of cGMP regardless of where the cGMP was produced. However, this pathway appears to have a reduced importance in mediating the effect of cGMP in hypertrophy, because treatment of the myocytes with specific inhibitors before addition of CNP or NO did not have any significant impact on the effect of the reagents. Additionally, we demonstrated that the degree of cGMP-stimulated protein phosphorylation was reduced in myocytes from the cardiac hypertrophic mice, indicating that the action of the cGMP protein kinase was diminished in these hypertrophic mouse myocytes. The data were consistent with several previous studies (12, 30) that also suggested that there was a reduced cGMP protein kinase pathway in the hypertrophied myocardium, which might be due to the reduced expression level of cGMP protein kinase (15). This suggests that some other cGMP-dependent pathway must still be active in these hypertrophic myocytes, such as the cGMP-affected cAMP phosphodiesterases (12, 31).

Altered sarcoplasmic reticulum calcium handling can be a major candidate mechanism responsible for the contractile defects in hypertrophied or failing hearts. It has been demonstrated that calcium uptake by the sarcoplasmic reticulum is decreased in hypertrophied hearts (13) and that the impairment in sarcoplasmic reticulum calcium uptake resulted from decreased calcium ATPase activity. However, we did not observe significant changes in calcium dynamics in the basal state. Similar findings were also reported in other hypertrophic models. Because the expression of many genes is altered in hypertrophy (10), some compensatory effects may happen. Additionally, we did not observe that the effects of CNP on calcium transient were altered, indicating that other non-cGMP pathways such as the natriuretic peptide type C receptor-G protein pathway (22) may be involved in hypertrophied myocardium. Because CNP and NO have reduced effects on myocyte contraction, but their effects on calcium transient were unchanged, the data suggest that myofilament calcium sensitivity may be upregulated in hypertrophic mouse hearts. The increased calcium sensitivity may account for the unchanged percentage shortening in hypertrophied myocytes, where maximal rates of shortening and relaxation were depressed at basal level. It has also been demonstrated that myofilament calcium sensitivity was altered in some cardiac hypertrophy and failure models (2). It is largely unknown what happens to the calcium sensitivity in this pressure-overloaded model, and further study is necessary to identify these changes.

In summary, we found that cGMP generated by both the particulate and sGC produced negative inotropic effects in normal ventricular myocytes. This effect was mediated by the cGMP protein kinase. In hypertrophied myocytes from pressure-overloaded hearts, this negative inotropic effect of CNP and NO was blunted. This was not related to altered guanylyl cyclase activity. However, the importance of the cGMP protein kinase in this effect was reduced in hypertrophic myocytes. These changes in hypertrophy were not related to altered calcium transients.


   GRANTS
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 ABSTRACT
 METHODS
 RESULTS
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 GRANTS
REFERENCES
 
The study was partially supported by National Heart, Lung, and Blood Institute Grant HL-4032.


   ACKNOWLEDGMENTS
 
The authors are greatly thankful to Yiqi He for contribution on the biochemical measurement and Elizabeth Katz for help with the mouse surgery.


   FOOTNOTES
 

Address for reprint requests and other correspondence: H. R. Weiss, Dept. of Physiology and Biophysics, UMDNJ-Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway, NJ 08854-5635 (E-mail: hweiss{at}umdnj.edu)

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.


   REFERENCES
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 ABSTRACT
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 DISCUSSION
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