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(Circulation. 1997;96:3116-3123.)
© 1997 American Heart Association, Inc.

Articles

Development of Decompensated Dilated Cardiomyopathy Is Associated With Decreased Gene Expression and Activity of the Milrinone-Sensitive cAMP Phosphodiesterase PDE3A

Carolyn J. Smith, PhD; Raymond Huang, BS; Dong Sun, MD, PhD; Sidonnie Ricketts, BA; Carl Hoegler, PhD; Jia-Zhen Ding, MD; Richard A. Moggio, MD; ; Thomas H. Hintze, PhD

From the Departments of Pathology (C.J.S., R.H., S.R.) and Physiology (D.S., C.H., T.H.H.), New York Medical College, and Department of Surgery (J.-Z.D., R.A.M.), Westchester Medical Center, Valhalla, NY; and Department of Biology (C.H.), Marymount College, Tarrytown, NY.

	Abstract

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Background Phosphodiesterase III (PDE3) inhibitors are inotropic agents used to treat congestive heart failure (CHF) and are less effective in patients with severe CHF. Little is known about relative changes in PDE3 activity or gene expression during the evolution of cardiomyopathy.

Methods and Results In the present study, we evaluated temporal changes in PDE3A gene expression before and after pacing-induced CHF in nine mongrel dogs. Three weeks of left ventricular (LV) pacing produced LV end-diastolic pressures of 15±1.7 mm Hg, whereas overt CHF at 4 to 5 weeks was associated with LV end-diastolic pressures of 24±1.7 mm Hg; prepacing values were 6.6±0.6 mm Hg. Total RNA isolated from LV tissues was analyzed on Northern blots; 10 unpaced normal hearts served as tissue controls. Signals for PDE3A mRNAs (7, 8, and 10 kb) or PDE4D (7.6 kb) were normalized against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or ribosomal 18S RNA. Before the onset of CHF, PDE3A/GAPDH ratios were not different between the control and 3-week paced groups. In contrast, all PDE3A/GAPDH ratios were selectively reduced by 52%, and PDE3A/18S was reduced by 70% (P<.05) in CHF; PDE4D/GAPDH (or 18S) was unchanged. LV tissues from four control and four CHF dogs were also processed to isolate cytosolic and microsomal membrane protein for cAMP PDE3 activity assays. CHF was associated with a significant 54% reduction (P<.05) in microsomal but not cytosolic PDE3 activity.

Conclusions Selective downregulation of PDE3A may account in part for the ineffectiveness of milrinone in the treatment of severe CHF.

Key Words: heart failure • inotropic agents • pacing

	Introduction

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Congestive heart failure is characterized by impaired ß-adrenergic receptor activation of contractility in humans and animal models. This contractile dysfunction is associated with alterations in the functional coupling of ß-adrenergic receptors, G proteins, and adenylate cyclase.^{1 2} In view of the reduced synthesis of cAMP in the failing heart, a postreceptor/cyclase mechanism for elevating cAMP, such as PDE inhibition, should increase contractility and be therapeutically useful.^{3 4} However, in the absence of a ß-agonist, the PDE3 inhibitor milrinone has reduced inotropic effects in vitro in failing human^{5 6} and canine⁷ myocardium. This may be due to altered myocardial PDE3 activity, which is decreased⁸ or unchanged^{6 9} in end-stage human CHF. Whether reductions in PDE3 gene expression are linked to changes in PDE activity⁸ during the progression of CHF is unknown.

PDE3 inhibitors were in development as therapeutic agents a decade before cardiac PDE3 was cloned,¹⁰ and there is little information about the genetic regulation of this enzyme in pathophysiological states. PDE3 is a high-affinity cAMP PDE that is competitively inhibited by cGMP and is represented by at least two gene families: the cardiovascular PDE3A (cardiovascular/platelet/placental low-K_m cGMP-inhibited cAMP phosphodiesterase [also known as PDE III])¹⁰ and the "adipocyte" insulin-sensitive PDE3B (PDE3 gene that is insulin sensitive [previously known as the adipocyte cGMP-inhibited PDE or PDE III]).^{11 12} Multiple mRNAs for PDE3A ranging from 4 to 10 kb have been detected in Northern blots of rat heart,¹¹ canine and rabbit heart,¹³ or human placenta,¹⁴ which may account for multiple PDE3 proteins ranging in size from 55 to 135 kD.¹⁵ Both PDE3A and PDE3B are substrates for cAMP-dependent protein kinase,^{16 17 18 19} and phosphorylation of forms of 110 kD is associated with activation of PDE3.^{15 17 18} In myocardial tissue, PDE3 is located in both the cytosol (80 to 116 kD) and sarcoplasmic reticulum (125 to 135 kD),^{14 16 20 21} and the latter enzyme is believed to be responsible for the inotropic effects of PDE3 inhibitors.²²

The reduction in the direct effect of a PDE3 inhibitor on myocardial contractility^{5 6 7} in CHF could reflect depression in the basal synthesis of myocardial cAMP,^{1 2} as mentioned previously, or it could be due to a decrease in the functional activity of the sarcoplasmic reticulum–associated PDE3.^{8 20} To address the latter possibility, the purpose of the present study was to specifically evaluate PDE3A gene expression and PDE3 enzyme activity during the evolution of pacing-induced heart failure in dogs.

	Methods

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Surgical Preparation and Hemodynamic Measurements in Conscious Dogs
Nine adult mongrel dogs (23 to 31 kg body weight) were used in this study. The dogs were sedated with acepromazine (0.3 mg/kg IM, Ayerst) and anesthetized (sodium pentobarbital, 25 mg/kg IV). A thoracotomy was performed in the left fifth intercostal space. A Tygon catheter was placed in the descending aorta, and a second catheter was inserted in the left atrial appendage. A solid-state pressure gauge (P6.5, Konigsberg Instruments) was placed in the apex of the LV. A human screw-type unipolar myocardial pacing lead (model 831, Pacesetter Systems) was placed on the LV. The wires and catheters were run subcutaneously to the intracapsular region. The chest was closed in layers, and the pneumothorax was reduced. The dogs were allowed to fully recover. Antibiotics were given postoperatively. Heart rate and temperature were monitored daily. After 10 days, the dogs were trained to lie quietly on the laboratory table.

The previously implanted catheters were attached to P23ID strain-gauge transducers (Statham Instruments) for the measurement of arterial and atrial pressures. LVP was measured with the solid-state pressure gauge. The data were recorded on a 14-channel tape recorder (model 3700B, Bell and Howell) and played back on a direct-writing oscillograph (model 2800s, Gould). Mean values were derived for pressures using 2-Hz resistance-capacitance filters. Heart rate was measured using a cardiotachometer (model 9857B, Beckman Instruments) from the LVP pulse interval. The first derivative of LVP, LV dP/dt, was derived with an operational amplifier (National Semiconductor 324). Triangular wave signals with known slopes were substituted for the pressure signals to directly calibrate the differentiators. The tape recording system and strip-chart recorder were calibrated periodically during the experiment to eliminate electronic drift.^{23 24} The protocols were approved by the Institutional Animal Care and Use Committee of New York Medical College and conform to the "Guiding Principles for the Use and Care of Laboratory Animals" of the National Institutes of Health and the American Physiological Society.

Production of Heart Failure
Dogs were paced at 210 bpm for 3 weeks, and the pacing was increased to 240 bpm for an additional week using an external pacemaker (model EV4543, Pace Medical) which the dog carried in a vest. Hemodynamic measurements in all nine dogs were made before and at 3 (n=3) or 4 to 5 (n=6) weeks after chronic LV pacing, when the pacer was turned off and with the heart in spontaneous rhythm. In additional studies from this laboratory (n=50 dogs), the onset of heart failure follows a reproducible time course and is evident at 30±1 days of pacing.

Northern Blot Analysis
Total RNA was isolated from grossly normal LV tissue (epicardium and midmyocardium) from the dogs previously described that were subjected to chronic LV pacing for 3 or 4 to 5 weeks (CHF) and from 10 nonpaced historical controls. Frozen tissues were powdered under liquid nitrogen and thawed in guanidine isothiocyanate containing 2% ß-mercaptoethanol and 0.5% N-lauroylsarcosine; tRNA was isolated by centrifugation over a CsCl cushion.²⁵ RNA (15 µg) was denatured by heating (65°C) in 50% (vol/vol) formamide and 4.4 mol/L formaldehyde, electrophoresed through a 1% agarose gel containing 2.2 mol/L formaldehyde, and transferred by capillary blotting to a nylon membrane (BioRad Zeta-Probe). The RNA was cross-linked to the blot by UV irradiation (Stratagene).

cDNA probes were labeled with [³²P]dCTP (1 to 3 x10⁹ cpm/µg) by random priming (Ambion). After a 2-hour prehybridization period, nylon blots were hybridized overnight with radiolabeled cDNA probes under high stringency conditions as indicated below. Hybridizations were carried out at 42°C in 50% formamide, 5x Denhardt's, 28 mmol/L sodium phosphate (pH 7.4), 375 mmol/L NaCl, 1% N-lauroylsarcosine, 0.5 mg/mL heparin, and 0.2 mg/mL salmon sperm DNA. GAPDH, ribosomal 18S, and PDE3A blots were washed at 55°C (GAPDH and ribosomal 18S) or 60°C (PDE3A) for 30 min each in 2x SSC/0.5% SDS, 1x SSC/0.5% SDS, and 0.5x SSC/0.25% SDS. PDE4D (D gene of the rolipram-inhibited low-K_m cAMP phosphodiesterase PDE4 family [also previously known as PDE3 gene of the PDE IV family]) blots were washed at 65°C twice for 30 min in 2x SSC/0.1% SDS and twice for 30 min in 0.1x SSC/0.1% SDS. Blots were dried and exposed to Kodak O-MAT x-ray film in the presence of intensifying screens at -80°C for 24 to 48 hr (GAPDH and PDE3A) or 4 or 6 days (PDE4D) or at room temperature for 30 min (18S). High stringency wash conditions for the PDE3A and PDE4D blots were those used by Movsesian et al¹³ or Swinnen et al,²⁶ respectively. (The 6.8-kb "rat PDE3" mRNA²⁶ is now referred to as PDE4D, and the 4.4-kb "rat PDE4" mRNA is now PDE4B.^{27 28} )

Sufficient LV tissue was processed so that two or three RNA samples from most dogs were run on several blots; the yield of RNA per gram of tissue was comparable among all groups. Five separate Northern blots were prepared. Each blot contained samples from three to five CHF animals (plus one 3-week paced animal ) with three to five controls or three each of 3-week paced and controls. Blots were stripped after probing with one cDNA and then rehybridized with one to three other probes. Optical densities of hybridization signals on several x-ray film exposures were quantified by laser scanning densitometry (LKB Ultrascan) to determine steady state RNA levels normalized to signals with GAPDH or 18S.

Differential Centrifugation of LV Tissue for Isolation of Cytosolic and Microsomal Protein
Cytosolic and sarcoplasmic reticulum-enriched microsomal fractions were prepared from LV myocardium¹⁶ using material from the same animals (four control and four CHF dogs) as in the RNA study (no additional LV material was available after RNA isolations for protein analyses of the 3-week paced group). LV tissues (0.5 g wet weight) were powdered under liquid nitrogen and homogenized (two 10-second bursts at setting 7 on a Brinkmann Instruments Kinematica) in 5 vol of buffer containing 290 mmol/L sucrose; 10 mmol/L 3-(N-morpholino)propanesulfonic acid, pH 7.05 at 4°C; 1 mmol/L EGTA; 3 mmol/L NaN₃; 3 mmol/L benzamidine; 10 µg/mL concentration of pepstatin A, leupeptin, and antipain; 0.8 mmol/L phenylmethylsulfonyl fluoride; and 1 mmol/L dithiothreitol. Two low-speed centrifugations were first used to remove particulate cellular debris (3000 rpm, 10 min, 4°C, and then 8000 rpm, 10 min, in a Sorvall SS-34 rotor), followed by sedimentation of the supernatant at 35 000 rpm (60 min, 4°C, Beckman Instruments 100.4 rotor, TL100 centrifuge). The supernatant from the latter centrifugation was saved as the cytosolic fraction; the 35 000 rpm pellet (microsomal fraction) was washed in sucrose-free buffer containing 0.6 mol/L KCl and resedimented at 50 000 rpm (4°C, 40 min) before storage of membranes at -80°C in sucrose-containing buffer without EGTA or KCl. Recovery of total homogenate protein per gram of LV tissue was comparable between control and CHF groups.

PDE Assay
cAMP PDE activity was assayed in duplicate at 0.1 µmol/L substrate by the two-step method (snake venom conversion of adenine-labeled ³H-5'-AMP to adenosine) as previously described under linear conditions⁹ in the presence and absence of 2 µmol/L OPC 3911, a water-soluble cilostamide derivative and potent PDE3-selective inhibitor synthesized by Otsuka. PDE3 activity was calculated by subtracting cAMP PDE activity measured in the presence of 2 µmol/L OPC 3911 from cAMP PDE activity measured in its absence. This concentration of PDE inhibitor is 20 times higher than the K_i value for OPC 3911 inhibition of human myocardial cAMP PDE activity in the sarcoplasmic reticulum.⁹ Similarly, in other PDE assays, rolipram (10 µmol/L)-dependent inhibition was used to estimate PDE4-specific activity. In human sarcoplasmic reticulum, 10 µmol/L rolipram inhibits low-K_m cAMP PDE activity by <15%,⁹ which is consistent with the <8% inhibition observed in the present study with canine microsomal membranes from control or CHF groups. Membrane-associated PDE4 in canine LV may be more concentrated in the sarcolemma than in the microsomal sarcoplasmic reticulum.²⁹

Proteins were measured according to the Bradford method (BioRad microassay) using bovine serum albumin as standard. The relative recovery of total homogenate protein (100%) between various soluble and particulate fractions was the same in both control and CHF groups. In fractions evaluated for PDE activity, cytosolic protein recoveries were 31±4% and 35±3% and the microsomal membrane protein recoveries were 1.1±0.13% and 1.0±0.13% of homogenate protein for control and CHF, respectively. Total low-K_m (0.1 µmol/L substrate) cAMP PDE activity recoveries were 90±2% and 94±1% in the cytosolic fraction from control and CHF groups, respectively. The recoveries and relative distributions of PDE3 activity between the cytosol (83% to 90%) and microsomal fractions (10±2% CHF and 17±2% control) were similar to our previous studies comparing fractions from normal LV tissues of dog, rabbit and guinea pig, or human (idiopathic dilated cardiomyopathy).¹⁶

Reagents
The 1.5-kb EcoRI fragment of the catalytic domain of the human myocardial PDE3A¹⁰ and the PDE3-selective inhibitor OPC 3911 were kindly provided by Dr Vincent Manganiello (National Heart, Lung, and Blood Institute, National Institutes of Health). A 2-kb EcoRI fragment, which represents a full-length coding region of rodent PDE4D,³⁰ was generously provided by Dr Michael Wigler (Cold Spring Harbor Laboratories) and Dr Graeme Bolger (University of Utah). A 1.1-kb fragment of the cDNA for human GAPDH was purchased from Clontech. A 0.6-kb EcoRI fragment of murine 18S was the gift of Dr Eric Lader (Ambion). RNA molecular weight standards were from Promega. Sources for all other materials are as previously described.^{10 23}

Statistical Analysis
Results are expressed as mean±SEM (n=number of dogs). The Northern and enzyme activity data were analyzed with an unpaired Student's t test; a paired t test was used in dogs to compare hemodynamics in the same animal before and after CHF or 3 weeks of pacing. A value of P<.05 was considered statistically significant.

	Results

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Hemodynamic Function After Chronic Ventricular Pacing
Mongrel dogs were subjected to a ventricular pacing regimen for 3 to 5 weeks, which eventually produced overt CHF as evidenced by ascites, edema, and dyspnea. Hemodynamic characteristics for these animals before and after chronic pacing are summarized in the Table. After 4 weeks of pacing (ie, CHF), LV dP/dt, LV systolic pressure, and mean arterial blood pressure were significantly decreased by 45%, 21%, and 16%, whereas spontaneous heart rate increased by 63% (all P<.02 from prepacing). The greatest change in CHF was a 343% increase in LV end-diastolic pressure. After a shorter period of pacing, LV dysfunction was less marked. At 3 weeks, the pacing-induced increase in LV end-diastolic pressure was 267% and LV dP/dt decreased 36% (both P<.05; Table 1), whereas LV systolic pressure, blood pressure, and spontaneous heart rate were unchanged.^{23 24}

View this table: [in this window] [in a new window]   Table 1. Alterations in Hemodynamics After 3 to 4 Weeks of LV Rapid Pacing

Northern Analyses of Gene Expression in Ventricular Tissue
Three high-molecular-weight PDE3 messages were detected with the catalytic domain of the human PDE3A cDNA^{10 13} : a 7- to 8-kb doublet and a larger single band at 10 kb (Fig 1). This pattern of three messages was obvious in RNA from control, 3-week paced, and CHF dogs (Fig 1). As normalized to GAPDH, both the 7- to 8-kb doublet and the 10-kb PDE3A transcripts were significantly reduced in CHF to average reductions of 50% and 54%, respectively (both P<.05; Fig 2). In contrast, at an earlier time point during ventricular pacing, PDE3A/GAPDH ratios were not different between control and 3-week paced groups: 0.78±0.10 (n=8) and 0.82±0.16 (n=3), respectively (data given for the 7- to 8-kb doublet).

View larger version (80K): [in this window] [in a new window]   Figure 1. Northern blot of PDE3A and GAPDH mRNAs in tRNA isolated from LV myocardium. C indicates control, CHF, pacing-induced CHF (4 weeks); 3 WK, 3-week paced. Autoradiograms of a representative blot containing 15 µg of intact tRNA (ethidium bromide–stained gel under UV illumination [bottom]). The nylon blot was sequentially probed for PDE3A (7- to 8-kb doublet and 10-kb mRNA) and GAPDH (1.5-kb mRNA) using human cDNAs or a murine cDNA for ribosomal 18S (see "Methods"); film exposures were 30 minutes (18S) or 1 (GAPDH) to 5 days (PDE3A). The PDE3A film was deliberately overexposed to illustrate three mRNA signals in CHF for this figure; lighter films were used for scanning densitometry. The quantities of each PDE3A mRNA relative to GAPDH (Fig 2) or relative to 18S (Fig 5) were significantly reduced in CHF versus controls.

View larger version (26K): [in this window] [in a new window]   Figure 2. Densitometric analyses of PDE3A Northern blots. Autoradiograms with mRNA signals for PDE3A and GAPDH mRNA levels in tRNA of canine LV were scanned, and the values (arbitrary absorbance units) were normalized against those obtained for GAPDH to express mRNA levels as ratios. The 7- to 8-kb PDE3A mRNA absorbance largely reflects the lower band of the incompletely resolved doublet (see Fig 1). Results are mean±SEM of four blots prepared with 10 different controls (open bars) and 6 pacing-induced (4 to 5 weeks; filled bars) CHF dogs. *P<.05 versus control.

The possibility that CHF was associated with a specific downregulation of PDE3A mRNA was addressed by probing Northern blots with the cDNA for the rolipram-inhibited, cGMP-insensitive cAMP PDE4D gene (a 7.6-kb mRNA; Fig 3). Unlike PDE3A, PDE4D/GAPDH mRNA levels did not differ significantly (P=.27) between control and CHF groups (Fig 4).

View larger version (78K): [in this window] [in a new window]   Figure 3. Northern blot of PDE4D and GAPDH mRNAs in tRNA isolated from LV myocardium. C indicates control heart. Conditions are as in Fig 1 but illustrated with a different blot. Radiolabeled cDNAs for rodent PDE4D (7.6-kb mRNA), human GAPDH, and murine 18S were used (see "Methods"); a 7-day film exposure is shown for PDE4D. The ratio of PDE4D mRNA relative to GAPDH or 18S was unchanged in CHF compared with controls (Figs 4 and 5).

View larger version (25K): [in this window] [in a new window]   Figure 4. Densitometric analyses of PDE4D Northern blots. Autoradiograms with mRNA signals for PDE4D (7.6 kb) and GAPDH mRNA levels in tRNA of canine LV were scanned, and the values (arbitrary absorbance units) were normalized against those obtained for GAPDH to express mRNA levels as ratios. PDE4D/GAPDH ratios were not significantly different (P=.27) between control and CHF groups. Results are mean±SEM of three blots prepared with 10 different controls (open bars) and 6 pacing-induced (4 to 5 weeks; filled bars) CHF dogs.

In some CHF samples, it appeared that GAPDH was not proportional to tRNA loaded on the gel (Figs 1 and 3; bottom photographs of ethidium bromide–stained gel). The suitability of GAPDH as a normalizing denominator for PDE mRNAs on Northern blots was further evaluated by normalization of various mRNAs versus ribosomal 18S (Figs 3 and 5). The ratio of GAPDH/18S was 37% lower (P<.05; Fig 5) in CHF compared with controls. Despite the CHF-associated reduction in GAPDH, PDE3A/18S ratios were significantly reduced in CHF by 72% (P<.05 for the 7- to 8-kb doublet data shown in Fig 5; similar results were obtained with the 10-kb PDE3A mRNA). These data suggest that the relative CHF-associated reduction in PDE3A was about twice as great as that for GAPDH. In contrast to PDE3A, relative PDE4D mRNA levels were unchanged in CHF compared with controls regardless of mRNA normalization (GAPDH in Fig 4 and 18S in Fig 5).

View larger version (30K): [in this window] [in a new window]   Figure 5. Densitometric analyses of GAPDH and 7- to 8-kb PDE mRNAs relative to ribosomal 18S on Northern blots. Autoradiograms with mRNA signals for GAPDH, the 7- to 8-kb doublet of PDE3A, and PDE4D mRNA levels in tRNA of canine LV were scanned, and the values (arbitrary absorbance units) were normalized against those obtained for 18S to express mRNA levels as ratios. GAPDH/18S and PDE3A/18S ratios were significantly different (*P<.05) between control (open bars) and CHF (filled bars) groups, whereas the PDE4D/ribosomal 18S ratios were not significantly different. A similar reduction (P<.05) was observed for the 10-kb PDE3A/18S ratio in CHF (not shown). Results are mean±SEM from three blots containing RNA from a total of 10 control and 6 CHF dogs.

PDE Activity in Cytosolic and Microsomal Membranes From LV Tissues
High-affinity ("low-K_m") cAMP PDE enzyme activities were evaluated in cytosolic and microsomal membrane fractions prepared from canine LV tissues by carrying out assays at 0.1 µmol/L cAMP substrate. Under these conditions, the apparent specific activity per milligram of protein of the microsomal fraction was two to three times higher than the corresponding value in cytosols from both control and CHF groups (Fig 6). CHF was associated with a significant 50% reduction (P<.05) in low-K_m microsomal PDE activity, which is in contrast to a 33% reduction (P=.17) in cytosolic activity. Because assays at 0.1 µmol/L cAMP could detect several low-K_m PDE activities (eg, PDE3, PDE4, and PDE7), PDE assays were also carried out in the presence of a saturating concentration of a PDE3-selective (OPC 3911) or PDE4-selective (rolipram) inhibitor to estimate PDE3- (Fig 6) or PDE4- (not shown) specific activities. (No selective inhibitors for PDE7 are available to date.³¹ ) PDE3 (ie, OPC 3911 inhibitable) activity represented 39% to 41% of total cytosolic and 65% to 69% of total microsomal activities (Fig 6).¹⁶ In contrast, PDE4 represented an average of 13% (control) to 31% (CHF) of total cytosolic PDE (P=.22 for control versus CHF) or 7% to 8% of total microsomal PDE activities.

View larger version (28K): [in this window] [in a new window]   Figure 6. Low-Km cAMP PDE activities in subcellular fractions prepared from LV tissue. Cytosols and microsomal membranes were isolated as described in "Methods" and assayed at 0.1 µmol/L [3H]cAMP in the absence (Total) and presence of the PDE3 inhibitor OPC3911 (2 µmol/L). The proportion of OPC 3911–inhibited total activity is indicated as PDE3. Results are mean±SEM of four preparations of control (open bars) and 4- to 5-week paced CHF dogs (filled bars), some of which represent the same dogs analyzed for gene expression in Fig 5. *P<.05 versus control values.

PDE3 microsomal specific activity was three to six times higher than that in the cytosol fraction, which is suggestive of a selective compartmentalization of this enzyme in the membrane fraction.^{20 22} In contrast to PDE3, the apparent amount of microsomal PDE4 was minor and was 7-fold lower in specific activity compared with cytosolic PDE4 (PDE4 data not shown). As observed with total low-K_m activity, CHF was associated with a significant 54% reduction (P<.05) in microsomal PDE3-specific activity and a nonsignificant 36% average reduction (P=.30) in cytosolic PDE3-specific activity (Fig 6).

To calculate total recovery of cytosol versus microsomal PDE3 activity, the apparent specific activities per milligram of protein (Fig 6) and yields of homogenate protein were used (see "Methods"). The proportion of total units of PDE3 activity in each group was greater in the cytosol (83±2% control; 90±2% in CHF) than in the microsomal membranes (17±2% control; 10±2% CHF). In a comparison of experimental groups for recovery of PDE3 in each fraction, CHF was associated with a 27% reduction in cytosolic and a 54% reduction in microsomal PDE3 activity units per gram of LV tissue compared with control values. These CHF-associated reductions in recovery of PDE3 activities were quantitatively similar to those based on specific activity per milligram of protein of the individual fractions (Fig 6).

	Discussion

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The most important finding of the present study is that the gene expression for the PDE3A in canine myocardium was specifically reduced after CHF but not after 3 weeks of rapid ventricular pacing. This time-dependent change in myocardial PDE gene expression was selective for a certain PDE family because PDE4D mRNA was not significantly affected after CHF, despite a decrease in GAPDH relative to ribosomal 18S. PDE3 activity in the sarcoplasmic reticulum was significantly reduced after CHF, so reduced gene expression of PDE3A is most likely responsible for reduced PDE3 activity. The decrease in myocardial PDE3A gene expression in CHF also coincides with a depression in mRNA and protein expression for endothelial cell nitric oxide synthase²³ but an increase in GAPDH mRNA in aortic endothelium. These findings raise the possibility that chronic alterations in nitric oxide–stimulated cGMP levels influence expression of the cGMP-inhibitable cAMP PDE3A.

The hemodynamic changes that occurred in our dogs with pacing-induced cardiomyopathy are similar to those in previous studies on endothelial dysfunction.^{23 24} One advantage of our model of CHF is the consistent time course for the development of pacing-induced CHF. Before the onset of CHF, LV dysfunction at 3 weeks was limited to an elevation in LV end-diastolic pressure and a minor reduction in LV dP/dt, without clinical signs of CHF. However, after 4 weeks of pacing, overt CHF was characterized by increases in LV end-diastolic pressure, reductions in myocardial contractile state and mean arterial blood pressure, resting tachycardia, ascites, and edema. The extent of fibrosis has been estimated in this model to represent 3% to 16% by quantitative morphometry^{32 33} and was most evident in the midmyocardium and endocardium. Both hypertrophy and hyperplasia are detected after the development of severe CHF.³³ Because mRNA is a cell component absent from fibrous tissue, the reductions in PDE3A gene expression are independent of this parameter. In addition, the CHF-associated decreases in PDE3 enzyme activities (per milligram of protein) were four to six times greater (ie, 36% to 54%) than the average extent of fibrosis (9%).

It is noteworthy that two distinct states of pacing-induced cardiomyopathy in dogs are associated with reduced contractile effects of milrinone and altered cellular handling of calcium: compensatory hypertrophy³⁴ and CHF without hypertrophy.⁷ In the face of depressed ß-adrenergic receptor function,^{1 2} reduced expression of microsomal PDE3 activity may be a compensatory mechanism to increase cAMP levels (near the sarcoplasmic reticulum) in chronic CHF. Inhibition of membrane-associated PDE3 may favor persistent cAMP-dependent phosphorylation of phospholamban and increased calcium uptake in the sarcoplasmic reticulum.^{3 20 22} Despite the lower recovery of microsomal PDE3 activity (10% to 17%) compared with the cytosolic fraction (83% to 90%), the higher specific activity and subcellular locale of this enzyme with other established enzyme markers of the sarcoplasmic reticulum^{9 20} argue for a functionally important pool of PDE3.²² Furthermore, the relative balance between CHF-dependent reductions in adenylate cyclase and PDE may dictate whether inhibition of PDE3 has an impact on cardiac contractility and relaxation.⁴ Although the direct effects of PDE inhibitors are reduced in CHF,^{5 6 7} PDE3 inhibitors enhance the submaximal activation of adenylate cyclase by ß-agonists^{4 6 35} or forskolin.^{5 7} Pharmacological inhibition of down-regulated PDE3 may not elevate cAMP sufficiently to improve contractile state in late-stage CHF,^{3 7} whereas PDE3 inhibition during an earlier compensatory phase^{3 34} may prove beneficial.

Chronic use of milrinone in late-stage CHF has been associated with increased mortality.³⁶ Vesnarinone, a distinct PDE3 inhibitor that also possesses anti-inflammatory effects,^{37 38} seems to have a more favorable impact on patient survival.^{39 40} If PDE3 is downregulated in late-stage CHF, then the other effects of milrinone to inhibit non-PDE3 isoforms in the myocardium or vasculature³ may predominate. Milrinone is not as PDE3 selective as other PDE3 inhibitors, such as pimobendan,⁶ which also has calcium-sensitizing actions.^{3 4}

Post-translational activation of PDE3, such as phosphorylation of the enzyme, is an important mechanism by which PDE3 activity is rapidly modulated. This may preclude precise estimations of PDE protein mass in the present work and other studies.^{6 8 9} PDE3A (80 to 135 kD) and PDE3B (135 kD) from a variety of tissues are substrates for cAMP-dependent protein kinase.^{16 17 18 19} In addition, cytosolic platelet PDE3A (110 kD) and membrane-associated adipocyte PDE3B (135 kD) are phosphorylated by an insulin-sensitive serine kinase.^{15 17 27 41} Thus, crude enzyme activity⁹ or the apparent V_max of a partially purified PDE^{6 8} could reflect phosphorylation state (at the time of tissue disruption) as well as mass of protein. This may account for equivocal data from humans on altered PDE3 activity in late-stage CHF. To our knowledge, no similar studies in a canine model have been published. In human ventricular tissues, PDE3 K_m or V_max values were unchanged in sarcoplasmic reticulum⁹ or partially purified cytosolic fractions.⁶ On the other hand, another group reported decreased PDE3 V_max values (with no change in K_m or inhibitor sensitivity) in partially purified preparations of both cytosol and microsomal membranes.⁸

Our PDE3 activity data in crude cytosolic and microsomal membrane fractions are quantitatively similar to those of Silver et al⁸ with partially purified human PDE3, except that the reduction in cytosolic PDE3 activity in canine CHF was not statistically significant. In contrast to the specific enrichment of PDE3 in the sarcoplasmic reticulum,^{9 20 22} this isoform does not represent the majority of detectable PDE activity in crude cytosolic fractions. Myocardial tissue contains at least four PDE gene families²⁷ that can hydrolyze cAMP^{20 22} : PDE1 (Ca⁺²/calmodulin sensitive), PDE2 (cGMP stimulated), PDE3 (cGMP inhibited), and PDE4 (cGMP insensitive); low-K_m PDE7 may also be present.³¹ Our data suggest that a combination of PDE3 and PDE4 may account for >74% of total membrane-associated PDE activity^{20 22 29} in the canine heart and 54% to 69% of total cytosolic low-K_m cAMP PDE activity. In consideration of the signal-to-noise for multiple PDE activities in the cytosolic fraction, it may be necessary to separate isoforms by ion-exchange chromatography to clearly discern a selective change in cytosolic PDE3 activity.⁸

Northern blotting was used to identify selective PDE gene regulation for PDE3A¹⁰ and PDE4D.²⁷ Three PDE3A transcripts (7- to 8-kb doublet and 10 kb) were identified with a catalytic domain cDNA for the human PDE3A, which is consistent with the mRNA pattern seen by others in canine and rabbit ventricle.¹³ These messages may account for both cytosolic and sarcoplasmic reticulum–associated enzymes of PDE3.^{14 20 22} Taira et al reported the presence of a 5.1-kb PDE3A (RGIP2) mRNA in rat heart¹¹ (in which PDE3 is predominantly cytosolic²² ), and Kasuya et al¹⁴ identified a 4.4-kb placental cDNA that is translated into a 74-kD cytosolic PDE3A. However, these mRNAs are much smaller than what we observed in Northern blots of canine ventricular tissue. Because we found that only membrane-associated PDE3 activity was significantly reduced in CHF, this may reflect reduced stability and translation of PDE3A mRNAs of >7 kb into proteins of 110 kD.^{15 21} There is some uncertainty in the PDE field¹⁵ regarding the relative importance of differential alternative splicing of the same gene, alternative start sites,¹⁴ and/or proteolysis of membrane-associated PDE3 to generate cytosolic PDE3.^{19 21} Because all three PDE3A messages were similarly reduced in CHF, there does not appear to be differential alternative splicing of high-molecular-weight PDE3A mRNAs. If the cytosol fraction contains PDE3A isoforms of <110 kD^{15 16} which are more active and/or abundant than the 110-kD form,²¹ this may also contribute to the lack of a significant decrease in total cytosolic PDE3 activity in CHF. Alternatively, cytosolic PDE3A activity was recovered in excess of microsomal activity, which may represent some degree of proteolysis (in vivo and/or ex vivo) of the membrane-associated enzyme to truncated and more stable isoforms.²¹

The exact temporal relationship between the onset of decreases in PDE3A mRNA and decreased PDE3A activity/protein remains to be established because insufficient LV tissues were available from the 3-week paced group to characterize both myocardial protein and RNA. Little is known about molecular mechanisms for chronic regulation of PDE3 in cardiovascular tissues. Nevertheless, downregulation of PDE3A mRNA levels in CHF appears to be selective because PDE4D, a similarly large mRNA (7.6 kb), was not reduced. This result also excludes the possibility that large mRNAs were not recovered from CHF tissues. In a variety of endocrine and neural cells, PDE4D is upregulated transcriptionally and translationally by prolonged elevations in cAMP²⁷ ; PDE4 activity is increased in prostacyclin agonist–treated hearts.⁴² However, because PDE4D mRNA and PDE4-like activity were not changed in CHF and adenylate cyclase activity is reduced in CHF,^{1 2} these data argue against cAMP as a direct stimulus for PDE3A downregulation. There are distinct tissue-specific and developmental patterns of PDE3A and PDE3B mRNA expression.^{12 27} The PDE3B gene is associated with the differentiation of adipocytes¹¹ and is upregulated by dexamethasone, insulin,²⁷ and hypothyroidism.^{43 44 45}

In summary, the evolution of canine CHF induced by rapid ventricular pacing is associated with a time-dependent downregulation of several PDE3A mRNAs in myocardial tissue. PDE3A gene expression was not altered at 3 weeks, whereas the selective decrease in CHF was seen for PDE3A but not PDE4D. The reduction in several high-molecular-weight PDE3A mRNAs may account in part for the reduced mass of PDE3 protein because PDE3 activity in the sarcoplasmic reticulum but not cytosol was also reduced in CHF. These data suggest a molecular mechanism to account for reduced inotropic effects of PDE3 inhibitors in late-stage CHF and raise the question of whether inhibition of a downregulated enzyme is a useful therapeutic target.

	Selected Abbreviations and Acronyms

 

CHF = congestive heart failure GAPDH = glyceraldehyde-3-phosphate dehydrogenase LV = left ventricle, ventricular LVP = left ventricular pressure PDE = phosphodiesterase SDS = sodium dodecyl sulfate SSC = standard saline citrate

	Acknowledgments

 
This work was supported by Biomedical Research support grant RR-05398 to New York Medical College from the Division of Research Resources, National Institutes of Health (Dr Smith), American Heart Association, New York State Affiliate, grant GS-94-324 (Dr Smith), and National Institutes of Health, National Heart, Lung, and Blood Institute grants R29-HL-54081 (Dr Smith) and PO-1-HL-43023, HL-50142, and HL-53053 (Dr Hintze). Victor Fried PhD (Cell Biology and Anatomy, New York Medical College) provided access to the Beckman and Sorvall ultracentrifuges, and Alan Springer, PhD (Cell Biology and Anatomy), kindly provided access to Image Analysis software. Gong Zhao, MD, PhD; Xiaobin Xin, MD; and Robert Bernstein, BA (Physiology, New York Medical College), provided surgical assistance and hemodynamic measurements. Jingzhi He, MS, carried out PDE4 enzyme assays, and ShuangDan Sun, MS, prepared cDNA probes.

	Footnotes

 
Reprint requests to Carolyn J. Smith, PhD, Assistant Professor, Department of Pathology, Basic Science Bldg, Room BSB452, New York Medical College, Valhalla, NY 10595.

A preliminary report of these findings was presented at the 1994 Scientific Sessions of the American Heart Association, November 16, 1994, Dallas, Tex.

Received March 24, 1997; revision received May 23, 1997; accepted June 5, 1997.

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