There are 3 natriuretic peptides known to have major effects on the cardiovascular system: atrial natriuretic (ANP), B-type (BNP), and C-type (CNP) natriuretic peptides. ANP and BNP act through a natriuretic peptide receptor (NPR1), and CNP acts through another receptor (NPR2).1,2 By binding to these receptors, the peptides activate a particulate guanylyl cyclase to produce the second messenger cyclic GMP.1,2,3 Increased levels of cyclic GMP exert negative metabolic and functional effects in cardiac myocytes.4,5,6 Cyclic GMP exerts most of these negative effects on cardiac myocytes via a cyclic GMP-dependent protein kinase.5,7,8
Cardiac hypertrophy develops as a basic response of the heart to chronic increases in cardiac work and elevated O2 consumption. If this increased workload continues, the heart hypertrophies; when the work load is reduced, the hypertrophy regresses.9-11 Thyroxine (T4)-induced hypertrophy of rabbit hearts is a high metabolic state-induced cardiac hypertrophy. Excess levels of T4 are associated with hypertension, increased cardiac output, increased oxygen consumption, and cardiac hypertrophy.11-13 The hypertrophic effects of T4 on cardiac myocytes may be indirect and nongenomic through effects on the regulation of alpha and beta adrenergic receptors, as well as direct via binding to nuclear receptors and altering protein synthesis.14-16 This form of cardiac hypertrophy leads to blunting of the negative effects of the nitric oxide-cyclic GMP signal transduction system.7,17 The effects of natriuretic peptides are blunted in pressure-overload hypertrophy.18,19 However, these effects of natriuretic peptides have not been studied in T4-induced cardiac hypertrophy.
We tested the hypothesis that the functional effects of BNP and CNP would be reduced in T4-induced hypertrophic cardiac myocytes. We also examined whether these effects would be altered by inhibition of the cyclic GMP phosphodiesterase (PDE5) with a specific PDE5 inhibitor, zaprinast. Additionally, we measured both particulate and soluble guanylyl cyclase activity and the level of cyclic GMP in order to isolate the defect or alteration in hypertrophic cardiac myocytes. These experiments were conducted in control and T4-treated ventricular myocytes from rabbit.
MATERIALS AND METHODS
These experiments were conducted in accordance with the Guide for the Care of Laboratory Animals (DHHS Publication 85-23, revised 1996) and were approved by our Institutional Animal Care and Use Committee.
New Zealand white rabbits (n = 7) were injected subcutaneously with 0.5 mg/kg T4 daily for 16 days and sacrificed on the seventeenth day. Experimental rabbits were weighed on days 1, 8, and 15 before injection. Additionally, the experimental rabbits were weighed on day 16 before sacrifice. New Zealand white rabbits designated as controls (n = 8) did not receive injections and were also weighed before sacrifice.
The rabbits were anesthetized with 35 mg/kg sodium pentobarbital injected intravenously into the circumflex ear vein. After anesthesia, 1000 units of heparin were injected intravenously into the rabbit's circumflex ear vein. An additional dose of 60 mg/kg pentobarbital was then administered.
Freshly isolated ventricular myocytes from the hearts of New Zealand white rabbits (control, n = 8; experimental, n = 7) were prepared by a standard method described previously.6,20 The heart was rapidly removed immediately and flushed through the aorta with minimum essential medium (MEM; Sigma, St. Louis, MO) supplemented with 10 mM taurine, 2 mM L-glutamic acid, and 20 mM HEPES, pH 7.2. The heart was then weighed. Next, retrograde perfusion of the heart through the aorta was carried out for 5 min with MEM. The heart was then perfused with MEM containing 0.1% type II collagenase (Worthington) for 13 to 20 minutes. All perfusion solutions were maintained at 37oC and equilibrated with water saturated oxygen.
After perfusion with collagenase, the atria were discarded and the ventricles were cut into small pieces. The pieces were placed in MEM containing 0.1% type II collagenase and 0.5% bovine serum albumin (BSA, fraction V; Sigma). The tissue suspension was gently swirled at 2 cycles per sec for 5 min in a 37°C water bath. A slurry containing isolated ventricular myocytes was then decanted and washed 3 times with MEM containing 0.5% BSA. After each wash, the solution was centrifuged at low speed (34 × g) to remove residual collagenase and subcellular debris. Rod-shaped morphology confirmed myocyte viability, and yields were 50% to 70%. Some of the cardiac myocytes were resuspended in MEM containing 0.5% BSA and used for the cell shortening measurements. The remaining cells were used to assay for particulate guanylyl cyclase, soluble guanylyl cyclase, and cyclic GMP levels.
Two millimolar Ca2+ MEM was added to an open chamber (37°C) on the stage of an inverted microscope (Zeiss Axovert 125; Carl Zeiss, Thornwood, NY). A few drops of ventricular myocytes were added to the chamber. The ventricular myocytes were paced with an electrical field stimulation (1 Hz, 5 msec duration, voltage at 10% above threshold, and the polarity alternated with each pulse) via 2 platinum wires that were placed on either side of the chamber. The cells were allowed to equilibrate for at least 5 minutes. After equilibration, 10 consecutive contractions were averaged to determine the mean functional shortening at baseline and during experimental observations. Three cells per experimental group per rabbit heart were studied. In total, there were 4 experimental groups with 12 individual cells per heart (see below). A video edge detector with a video camera (Myotrack system; Crystal Biotech, Data Sciences International, St Paul, MN) measured unloaded cell shortening by detecting changes in the position of the edges of the cell. The outputs of the camera and video edge detector were sent to a television and desktop computer. Percent shortening was calculated as (end diastolic length - end systolic length)/end diastolic length. We also determined maximal rate of shortening and maximal rate of relaxation.
Cyclic GMP Measurement
Frozen myocytes (-80°C) from the study described below were warmed to 0°C and homogenized in ethanol in an ice bath using a Brinkmann Polytron (Westbury, NY). The homogenate was centrifuged at 30,000 × g for 15 min in a Sorvall RC-5B centrifuge (Dupont, Wilmington, DE). The supernatant was saved, and the pellet was reconstituted in 1 mL of a 2:1 ratio of ethanol to water. Centrifugation at 30,000 × g for 15 min was repeated. The supernatants were combined. The combined supernatants were evaporated to dryness by placing them in a 60°C bath under a stream of nitrogen. The residue was reconstituted in 1.5 mL of assay buffer (0.05 mol/L sodium acetate, pH 5.8, containing sodium azide). All samples were analyzed at the same time. Cyclic GMP levels were measured via radioimmunoassay (Amersham Pharmacia). The assay measures the competitive binding of 125I labeled cyclic GMP to a cyclic GMP-specific antibody. After formulation of a standard curve, the experimental results were determined and reported as picomoles of cyclic GMP/105 myocytes.
Guanylyl Cyclase Activity Measurement
The soluble guanylyl cyclase activity of the ventricular myocytes was assayed using a modified protocol from Su et al.18 The assay mixture consisted of 0.2 mL of a reaction solution containing 50 mM Tris HCl (pH 7.6), 10 mM theophylline, and a GTP regeneration system. The GTP regeneration system consisted of 10 mM creatine phosphate, 10 mg of 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). This dose of SNAP was capable of stimulating maximal activity.18 Reactions were initiated by the addition of the reaction solution to ventricular cell extracts and were maintained for 10 min at 37°C. Sodium acetate (0.8 mL, 50 mM, pH 4.0) was then added. The reaction was terminated by placing the tube in 90°C for 3 min.
Particulate guanylyl cyclase activity in ventricular myocytes was assayed using a modified protocol from Agullo et al.21 The addition of 0.1% Triton X-100 to the particulate guanylyl cyclase reaction solution was used to stimulate particulate guanylyl cyclase activity.18 The reaction was maintained for 10 minutes at 37°C and later terminated by the addition of 1 mL of cold ethanol. The production of cyclic GMP from the guanylyl cyclase reactions were determined by radioimmunoassay.
The cells were allowed to equilibrate for at least 5 min until cell shortening measurements were stabilized. Afterwards, 10 consecutive contractions were used as baseline data for the cell. In the first experimental group, BNP (10−7 M) was added to the reaction chamber. The cell was allowed to contract for approximately 5 min and 10 consecutive contractions were measured. After 5 minutes, BNP (10−6M) was added to the reaction chamber, and 10 consecutive contractions were used. Approximately 5 minutes were allowed before data collection (10 consecutive contractions) after the administration of each reagent. In the second group, baseline data collection was followed by addition of CNP (10−7, 10−6 M). The third group consisted of baseline data, then zaprinast [a cyclic GMP phosphodiesterase (PDE5) inhibitor, 10−6 M]. followed by BNP (10−7, 10−6 M). In the fourth group, zaprinast was added after baseline data were gathered. This was followed by CNP (10−7, 10−6 M). Three cells were examined for each group in each animal. In preliminary studies, baseline functional parameters of control and hypertrophic cardiac myocytes were not significantly altered when they were allowed to contract over this time period.
A similar protocol was used for the cyclic GMP measurements. There was a baseline group and an experimental group, which was exposed to the high dose of BNP (10−6 M). The groups were treated similarly to the functional measurement groups. They were electrically paced for 5 minutes. The solution was then centrifuged at a low speed, and the pellets were frozen via liquid nitrogen and stored at -80oC.
Results are expressed as mean ± standard error. A repeated measures analysis of variance (ANOVA) was used to compare experimental and control variables. Duncan post hoc test was used to compare significant differences between baseline and treatment observations. A value of P < 0.05 was considered statistically significant.
The heart weight to body weight ratio of the T4-induced cardiac hypertrophied rabbits (3.9 ± 0.2 g/kg) was significantly greater than the control rabbits (2.3 ± 0.1). Cell length was also significantly greater in the T4 rabbits (153 ± 3 vs. 138 ± 4 μm).
Percent Shortening Data
There were no significant differences in baseline percent shortening between control and T4 cardiac myocytes (Fig. 1). In control myocytes, BNP (10−6 M) significantly reduced percent shortening by 25%. CNP (10−6 M) also significantly reduced percent shortening by 26%. There were no significant effects of BNP or CNP on percent shortening in the T4-treated cardiac myocytes. Exposure to the cyclic GMP phosphodiesterase inhibitor zaprinast (10−6 M) reduced percent shortening in control myocytes. Both BNP and CNP further significantly lowered percent shortening in these control myocytes. However, zaprinast, BNP, and CNP had no significant effect on percent shortening in the T4 myocytes.
Maximal Rate of Shortening Data
There were no significant differences in baseline maximum rate of shortening between control and T4 cardiac myocytes (Fig. 2). In control ventricular myocytes, BNP (10−6 M) significantly reduced maximal rate of shortening by 25%. CNP (10−6 M) also significantly reduced maximal shortening by 23%. In the T4-treated cardiac myocytes, there were no significant effects of BNP or CNP on maximal rate of shortening. Zaprinast (10−6 M) had no significant effects on maximal rate of shortening in control or T4-treated myocytes. Both BNP and CNP further significantly lowered maximal rate of shortening in the control myocytes. However, zaprinast, BNP, and CNP had no significant effect on maximal rate of shortening in the T4 myocytes.
Maximal Rate of Relaxation Data
Baseline maximum rate of relaxation was not different in comparisons between control and T4 cardiac myocytes (Table 1). In control ventricular myocytes, BNP (10−6 M) significantly reduced maximal rate of relaxation by 22%. CNP (10−6 M) also significantly reduced maximal rate of relaxation by 22%. In the T4-treated cardiac myocytes, there were no significant effects of BNP or CNP on maximal rate of relaxation. Zaprinast (10−6 M) had no significant effects on maximal rate of relaxation in control or T4 treated myocytes. Both BNP and CNP further significantly lowered maximal rate of relaxation in the control myocytes. However, zaprinast, BNP, and CNP had no significant effect on maximal rate of relaxation in the T4 myocytes.
Guanylyl Cyclase Data
No significant differences in baseline particulate guanylyl cyclase activity were noted between control and T4 ventricular myocytes (Fig. 3). However, particulate guanylyl cyclase activity post-stimulation was significantly blunted by 43% in the T4 myocytes relative to control myocytes. Triton X-100 increased particulate guanylyl cyclase activity by more than sixteen-fold in the control group. There was no significant difference in soluble guanylyl cyclase activity between control and experimental myocytes at baseline. Stimulation with a nitric oxide donor significantly and similarly increased soluble guanylyl cyclase activity in control and T4 ventricular myocytes.
Cyclic GMP Data
There were no significant differences in the level of cyclic GMP at baseline between the control and T4 myocytes (0.00115 ± 0.0003 and 0.00112 ± 0.0004 femtomoles/cell). However, exposure to 10−6 M BNP caused a significant increase in cyclic GMP levels from baseline by 75% to 0.00202 ± 0.0004 in the control myocytes. However, in the T4-induced hypertrophied myocytes, no significant increase was seen (0.00167 ± 0.0004) after BNP administration.
In control myocytes, the natriuretic peptides (BNP and CNP) reduced myocyte function, including percent shortening, maximal rate of shortening, and maximal rate of relaxation, before and after cyclic GMP phosphodiesterase inhibition with zaprinast. One major finding of this study was that exposure of T4-induced hypertrophied rabbit cardiac myocytes to zaprinast, BNP, or CNP did not have a significant effect on functional parameters. Baseline functional values for the control and hypertrophic myocytes were not significantly different. In addition, basal guanylyl cyclase activity and cyclic GMP levels were similar between control and T4 ventricular myocytes. The second major finding of this study was that stimulation of the particulate guanylyl cyclase in the hypertrophic cells had a significantly reduced effect compared with control cells. In support of the decreased particulate guanylyl cyclase activity in the hypertrophic myocytes, we found that BNP did not significantly increase cyclic GMP levels in T4 myocytes, while it did significantly increased these levels in control myocytes.
There are 3 types of natriuretic peptide receptors. ANP and BNP bind to NPR1 receptors. CNP binds to NPR2 receptors. NPR3 receptors are referred to as clearance receptors and may be responsible for the internalization and degradation of natriuretic peptides.3,22-24 Activation of the NPR1 or NPR2 particulate guanylyl cyclase domain leads to increases in the level of cyclic GMP.6 We observed an increase in cyclic GMP in control myocytes exposed to BNP. The negative functional effects of cyclic GMP are mainly mediated through the cyclic GMP-dependent protein kinase, and this can reduce intracellular Ca2+ by activation of the sarcoplasmic reticulum Ca2+-ATPase.4,5,22,25 The cyclic GMP signaling pathway may also be mediated by cyclic GMP protein kinase-independent interaction with other molecules in the cell, such as cyclic GMP-gated cation channels and certain phosphodiesterases.5,22
The natriuretic peptide associated increases in cyclic GMP can lead to reduced cardiac myocyte function in several species.6,18 We observed this decrease in myocyte function with natriuretic peptides whether or not the cyclic GMP phosphodiesterase was inhibited with zaprinast. This dose of zaprinast decreased percent shortening in controls and was shown previously to decrease myocyte oxygen consumption.20 Zaprinast alone did not significantly alter the effects of BNP (−25% BNP vs. −30% BNP + zaprinast) or CNP (−26% CNP vs. −29% CNP + zaprinast) in control myocytes.
In the present study, we used a 16-day administration of T4 to induce hypertrophic changes in cardiac myocytes. In animals with T4-induced cardiac hypertrophy, we saw increased heart weight/body weight ratios (+70%) and ventricular myocyte length (+11%), indicating that significant cardiac hypertrophy did occur. The hypertrophic changes could be related to a number of causes, including thyroid hormone exerting indirect effects on the regulation of alpha and beta adrenergic receptors, direct effects via binding to nuclear receptors and altering protein synthesis,14,26 and nongenomic effects by activating different cytosolic signaling cascades.14-16 Thyroid hormone is known to increase oxygen consumption in many tissues, including both direct and indirect effects on heart.11 There are usually increases in cardiac metabolism, blood flow, and function in vivo with T411-13; whereas cardiac myocytes may or may not have increased function.7,17,27
In the current study, we observed no functional effects of either BNP or CNP on cardiac myocytes from rabbits that had been chronically treated with T4. This lack of effect was observed whether or not the cyclic GMP phosphodiesterase was inhibited with zaprinast. This was similar to previous reports of reduced functional effects of nitric oxide and cyclic GMP after T4.7,17 These changes were reported to be related to reduced activity of the cyclic GMP-dependent protein kinase.7,17 Previous in vivo studies had also suggested that nitric oxide did not significantly increase myocardial cyclic GMP levels to the same extent as in control hearts after T4.11 There is some variability in baseline levels of cyclic GMP with higher, lower, and unchanged values during hypertrophy.7,10,11,17 Our data reporting a lack of change in basal cyclic GMP are similar to previous in vivo data with T4.11 In the current study, BNP failed to increase cyclic GMP levels after T4. This suggests that there may be multiple defects to the natriuretic peptide-cyclic GMP signaling pathway after T4.
Most of the negative metabolic and functional effects of cyclic GMP are exerted through the cyclic GMP-dependent protein kinase.5,6,28 Two cyclic GMP-dependent protein kinase genes exist. As reported previously, the cyclic GMP-dependent protein kinase in the T4 hypertrophic cells appears to be defective.7,17 In this study, we report another defect in the cyclic GMP signaling pathway after chronic thryroxine administration, which is upstream of the effects of the cyclic GMP-dependent protein kinase.
After chronic T4 administration, we found reduced particulate guanylyl cyclase activity but normal soluble guanylyl activity. We also found a reduced ability of BNP to increase the level of cyclic GMP after T4. The reasons for these reduced effects are not clear. The natriuretic peptide receptors are maximally active in the phosphorylated state and become desensitized with dephosphorylation.24 Such change could occur with T4 exposure. These natriuretic peptide receptors can be desensitized via homologous or heterologous mechanisms.3,24 Homologous desensitization occurs with repeated exposure of the natriuretic peptide to its receptor, the molecular mechanism of which is not completely known.3 Heterologous desensitization can occur with exposure of growth factors and hormones.3 For NPR2, increased intracellular Ca2+ levels via the phospholipase C-inositol triphosphate pathway may also play a role.3,24,29 Perhaps the injected T4 may have caused increased intracellular Ca2+ resulting in desensitization of the receptors. There may have been an upregulation of protein kinase C in the hypertrophic cells. With T4 administration, there may have been release of natriuretic peptides, which could have also desensitized the receptors via homologous desensitization. Alterations in natriuretic peptide levels after T4 may affect downstream components of this signaling system. It is also possible that there were changes in the natriuretic peptide clearance receptor (NPR3) after T4. Neutral endopeptidase (NEP), one enzyme responsible for the degradation of natriuretic peptides, has broad distribution and can be found in the brain, heart, and kidney.30 NEP is expressed on the plasma membrane and can degrade ANP, BNP, and CNP.3 It is also possible that NEP was upregulated in T4-induced cardiac myocytes, causing rapid degradation of the natriuretic peptides.
In summary, stimulation of T4-induced hypertrophied cardiac myocytes with BNP, CNP, or zaprinast did not cause any significant changes in functional parameters (percent shortening, maximal rate of relaxation, and maximal rate of shortening), whereas these substances did cause significant functional decrements in control myocytes. T4-induced hypertrophic cardiac myocytes appear to have 2 regulatory defects in the cyclic GMP signaling pathway. It has been reported that the importance of the cyclic GMP-dependent kinase in regulating the functional effects of the hypertrophic myocytes was diminished with chronic T4 relative to the control myocytes.7,17 Here we report that the particulate guanylyl cyclase is also defective. Unlike control cells, cyclic GMP levels did not significantly increase in the T4 myocytes after exposure to BNP. Further functional parameters were not significantly affected by BNP and CNP in the T4 myocytes.
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