J Mol





Cyclic AMP in Myocytes Isolated

ik-om Hypea-trqhied


Rat Hearts*


Department of Heart and Hypertension Research and Division of Anesthesiology, Cleveland Clinic Foundation, Cleveland, Ohio, USA and Department of Biology, Cleveland State University, Cleveland, Ohio, USA (Received 29 June 1989, accepted in revisedfom 4 February 1991) R. HEAL-DANDAN AND P. A. KHAIRALLAH. Cyclic AMP in Myocytes Isolated from Hypertrophied Rat Hearts. Journal of Molccakrr and Cellular Cardiology (1991) 23, 705-716. Impaired inotropic responsiveness to isoproterenol stimulation has been reported in the hypertrophied hearts of spontaneously hypertensive rats and renal hypertensive rats. This study was carried out in order to investigate the possibility that a defect in cyclic AMP production by cardiac myocytes is responsible for the impaired inotropic responsiveness of these hearts. Basal and isoproterenol stimulated cyclic AMP levels were measured in ventricular myocytes isolated from hypertrophied rat hearts. Cyclic AMP accumulation was also measured in the presence of isobutyl-methylxanthine, a phosphodiesterase inhibitor, and the results were compared to the appropriate controls. In the spontaneously hypertensive rat, no changes were detected in the basal or isoproterenol stimulated cyclic AMP formation. This suggests that the biochemical alterations leading to a diminished inotropic response in this model of cardiac hypertrophy involve abnormalities in mechanisms other than cyclic AMP production. In the renal hypertensive rat, basal and isoproterenol stimulated cyclic AMP levels were significantly depressed as compared to controls. This suggests that abnormalities in the signal transduction mechanism and formation of cyclic AMP are, at least in part, responsible for the impaired inotropic responsiveness seen in this model. These results confirm that cardiac hypertrophy is a heterogeneous process. Reduced inotropic responsiveness to isoproterenol stimulation in the hypertrophied hearts of the SHR and the RHR, both models of pressure overload hypertrophy, involve different biochemical alterations. Results of this study suggest that the physiologic response of cardiac hypertrophy may not be as important as the underlying cause of hypertrophic stimuli in determining the pathophysiological consequences. KEY WORDS: Isoproterenol.





Introduction Impaired inotropic responsiveness to stimulation of @-adrenergic receptors has been reported in the hypertrophied hearts of spontaneously hypertensive rats (SHR) and two kidney-one clip renal hypertensive rats (RHR), both models of pressure overload hypertrophy (Saragoca et al., 1981a,b). This depressed response is important because the sympathetic nervous system is a major source of cardiac reserve under stress conditions, providing inotropic and chronotropic support to the failing heart (Tarazi, 1983). Thus, diminished inotropic responsiveness to sympathetic stimulation in the hypertrophied myocardium may contribute to the heart failure which has




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been shown to follow hypertrophy (Tarazi, 1983; Bristow et al., 1985). In response to hormonal stimulation of the cardiac fi-adrenergic receptors, the stimulatory subunit of the guanine nucleotide regulatory protein (G,) is activated. G, activates the catalytic subunit (C) of adenylate cyclase (AC), which catalyzes the formation of cyclic adenosine monophosphate (CAMP) from ATP (Evans, 1986). Cyclic AMP acts as a second messenger, mediating the intracellular events in cardiac excitation-contraction coupling. The effects of CAMP are mediated through activation of the CAMP-dependent protein kinases. These kinases phosphorylate Ca* + channels within the sarcolemma and phospholamban within the membrane of the sarcoplas-

Please address all correspondence to: Randa Hilaf-Dandan, University of California Pharmacology, 0636, La Jolla, CA 92093, USA. *Supported in part by a Cleveland State University Graduate Student Research Support tCurrent address: University of California at San Diego, Department of Pharmacology, USA.



at San Award. 0636,



La Jolla,

0 1991 Academic







R. HihI-Dandan

mic reticulum, increasing Ca” + influx and release from intracellular stores and therefore enhancing the inotropic response. Phosphorylation of phospholamban, along with phosphorylation of troponin-I, a component of the contractile proteins, enhances the rate of relaxation (Evans, 1986; Opie, 1986). Several studies have been undertaken to investigate molecular defects in the fl-adrenoceptor pathway which may contribute to the reduced inotropic response of the hypertrophied myocardium. Alterations in the density of the /3adrenoceptors and the activity of AC have been reported in both the SHR and the RHR (Kumano et al., 1985). Results of such studies have varied with different laboratories. The density of the /3-adrenergic receptors in the SHR heart has been reported as decreased (Kunano et al. , 1983) or unchanged (Murakami et al., 1987). The activity of AC has also been shown to be depressed (Bhalla et al., 1978) or unchanged (Kumano et al., 1983). Similar discrepancies have been reported in the RHR heart. The density of @-adrenoceptors has been reported as increased (Kumano et al., 1983), decreased (Ayobe et al., 1983), and unchanged (Gende et al., 1985). AC activity in the RHR heart has been shown to be decreased (Sharma et al., 1982; Kumano et al., 1985). Since the dose response relationship between isoproterenol stimulation and CAMP production has so far not been reported in myocytes from the SHR or RHR, it is difficult to determine whether defective production of the second messenger is responsible for a decrease in the inotropic responsiveness. This study was carried out in order to determine whether the impaired inotropic responsiveness to isoproterenol in hypertrophied hearts from the SHR and RHR is due to abnormalities in CAMP formation. Isolated ventricular myocytes were used for this study because they provide a homogeneous population of cells, whose external conditions The use of may be easily manipulated. myocytes also allows multiple samplings and more rapid determinations of incubation conditions than intact tissue preparations, which may be critical in cyclic nucleotide measurements (Hearse, 1983). Basal and isoproterenol stimulated CAMP levels, as well as CAMP levels in the presence of a phosphodiesterase inhibitor, were measured in myocytes isolated from SHR and RHR hearts.

and P.A. Khairallah The results were compared to the respective cAMP normotensivr controls. Different profiles were found in these two models of cardiac hypertrophy, suggesting that thr biochemical alterations leading to an impaired inotropic response are not the same in these two models and that cardiac hypertrophy is not a general phenomenon leading to decreased inotropic responsiveness, but that the decreased response is due to different underlying mechanisms in each case.






Spontaneously hypertensive ruts Twelve-week-old male SHR and Wistar Kyoto rats (WKY) were obtained from Taconic Farms (Germantown, NY, USA). The rats were maintained in the animal facilities of the Cleveland Clinic Foundation until they were sacrificed at 18 weeks of age. Renal hypertensive rats Seven-week-old male Sprague-Dawley rats weighing 150-170g were obtained from Hilltop Laboratories (Scottsdale, NJ, USA). The rats were kept for 1 week of acclimatization before surgery. Two-kidney, one-clip renal hypertension was induced under ether anesthesia by placing a 0.23 mm silver clip on the left renal artery while leaving the other kidney untouched. Sham operated control rats same surgical (Sham) underwent the procedure without application of the artery clip. The animals were sacrificed 10 weeks after surgery, at 18 weeks of age. Rats were considered hypertensive if they developed a systolic blood pressure of 160 mmHg or more 3 weeks following surgery. All animals were housed and fed under identical conditions. The animals had free access to food and water. Blood pressures were monitored weekly in unanesthetized animals, using a tail cuff method (Bunag, 1984).

Isolation The myocyte modification of al. (1983), Bihler al. (1986). The

of ventricular myocytes isolation procedure was methods described by Eckel et al. (1985) and Engelmann procedure was performed

a et et at



in Hypertrophied

37“C under continuous gassing with 95% 0215% co,. Animals were sacrificed by decapitation according to procedures approved by the Animal Care and Use Committee of the Cleveland Clinic Foundation. Hearts were rapidly excised, placed in ice cold oxygenated Joklik buffer, and weighed. The hearts were cannulated via the aorta and perfused by a peristaltic pump at a flow rate of 8ml/min. Each heart was initially perfused for 5min in Ca* + -free modified Joklik minimum essential medium (MEM) (Gibco) to which the following supplements were added: KHCO,, 30 mM; pyruvate, 5 mM; MgCl,.GH,O, 3.4mM; sucrose, 30mM; taurine, 30mM; glucose, 20 mM; carnitine, 2 mM. Perfusion was continued for 45min with an enzyme digestive medium that consisted of the modified Joklik (MEM) further supplemented with 0.1% Collagenase II (Worthington Biochemicals), 2 units/ml trypsin (Worthington Biochemicals), 0.1% crystalline bovine serum albumin (BSA) fraction V (Sigma), and 25~~ CaCl,. At the end of the perfusion period, the heart was removed from the perfusion apparatus, the atria were discarded, and the ventricles were cut into 6-8 small pieces. Dispersion of the ventricular cells was achieved by gentle agitation of the cardiac tissue through a serologic pipette. This process was continued for 30 min at 37%) until disaggregation was complete. The dispersed cells were filtered through a 400 pm nylon mesh and washed twice at 50 g for 1 min. The cells were incubated with 2 units/ml of trypsin and 2 % BSA for 30 min. This step increased the yield and viability of rod shaped, Ca2 + -tolerant myocytes. The trypsin treatment was terminated by adding soy bean trypsin inhibitor, and the cells were filtered through a 400pm nylon mesh and washed twice at 50 g for 1 min. The freshly isolated cells were then suspended in medium-199 (Gibco), the cell count was determined and viability was assessed, using the criteria of rod shaped morphology and trypan blue exclusion.

Isolation of Ca’ +-tolerant myocytesfrom the SHR Several modifications of the isolation procedure were necessary in order to obtain Ca*+ -tolerant myocytes from the SHR. Initially,



during enzymatic digestion, the Ca2+ levels were started at 5/.4M and gradually increased every 5min throughout perfusion to a final concentration of 50pM. The BSA was also extensively dialyzed against distilled water containing EDTA to avoid hypercontracture of the SHR myocytes due to cationic impurities in the BSA (Wittenberg et al., 1981). The freshly isolated cells were suspended in Ca’+ free Joklik (MEM) and medium-199 was gradually added to the cells in order to prevent hypercontracture and shock in response to high concentrations of Ca*+ . Myocytes isolated from the WKY were prepared in the same way as SHR myocytes. Cyclic AMP


Aliquots of isolated myocytes containing 105cells/ml were used for the CAMP assays. Basal and isoproterenol stimulated CAMP levels were measured, as well as the isoproterenol response of the cells in the presence of the /3-adrenergic receptor antagonist propranolol. For the dose-response curves to isoproterenol stimulation, the cells were preincubated for 5min with 0.5mM of isobutyl-methyl-xanthine (MIBX) (Sigma), and the cells were challenged with varying concentrations of isoproterenol-HCl (lo- 8M to lo-*M) (Sigma) for a total of 3 min each. The incubations were terminated by adding 6% ice-cold trichloroacetic acid (TCA) and freezing the samples in liquid nitrogen. The samples were stored at - 80% until use. Cyclic AMP levels were determined by radioimmunoassay (Steiner et al., 1972) using RIANEN cAMP[‘~~I] RIA kit obtained from DuPont (North Billerica, MA, USA). The samples were extracted 4 times with 5 x volume of water-saturated ether, and evaporated to dryness in a Speed Vat concentrator The residues were then resuspended in sodium acetate assay buffer (pH 6.2) supplied with the kit, and used directly in the radioimmunoassay. The percentage recovery of CAMP was determined by adding 3H-cAMP (4000 cpm; equivalent to 0.1 pmols that were subtracted from calculated values) to the samples prior to extraction. More than 90% of CAMP was recovered from the samples.



DNA and protein assays values were determined

by the


R. I-El&Dandan

diphenylamine method described by Leyva et al. (1974). Samples containing l-2 million cells/ml were homogenized and precipitated on ice for 10min with cold perchloric acid (PCA). The pellets were then incubated with 0.3 ml of 0.5% PCA at 75’C for 1 h, followed by the addition of the diphenylamine reagent and incubation at 37OC overnight. Calf thymus DNA was used as a standard. The DNA standards ranged from 10 to 1OOpg and were processed under identical conditions (Engelmann et al., 1986). The protein content of the cells was determined using the Biorad protein dye binding assay originally described by Bradford (1976) with crystalline BSA as the standard.

Light and electron microscopy Freshly isolated myocytes were fixed with 2% gluteraldehyde in 0.2 M phosphate buffer, pH 7.4, and processed for light and electron micrography. For the scanning electron micrographs, the cells were fixed for l-3 h at 4’C and placed in 0.14 M phosphate buffer containing 7.5% sucrose overnight. The cell pellet was stained with osmium tetroxide (1%) for 2 h and dehydrated through 100 % ethanol. Dehydrated cells were placed in a Polaron critical point apparatus, where the ethanol was replaced with carbon dioxide at 10°C, repeated 6-10 times. The cells were sprinkled on double sided sticky tape and placed in a Polar:n Sputter Coater. Gold and paladium (100 A) were slowly distributed under argon at high vacuum. The specimens were examined with an ETEC SEM at 10KV to determine cellular surface topology and retention of rod shaped morphology.



Using Bioquant morphometry software coupled to an IBM XT for statistical analysis, the myocytes fixed in 2 % Gluteraldehyde were traced on a digitizing pad, and the average area (i.e., the area within the cell margins when cells on a slide are viewed from above) and length of the cells were determined for each group and its control. Measurements were performed on 500 myocytes from each heart, using 10 rats in each group.

and P.A. Khairallab Statistical analysis The data were analyzed using the PROPHET Computer system sponsored by the Biotechnology Resources Program, Division of Research Resources, NIH. Data were tested for normality by the WilkShapiro test. Unpaired, two tailed t-tests were used for testing the differences in CAMP formation between each group and its control. Dose response curves were analyzed using the analysis of Variance (ANOVA) and Covariance including repeated measures (Wallenstein et al. , 1980). The ANOVA for repeated measures takes into consideration the fact that cells isolated from the same heart were used for repeated doses of isoproterenol, so that observations are not independent of one another. The results are considered significant at P

Cyclic AMP in myocytes isolated from hypertrophied rat hearts.

Impaired inotropic responsiveness to isoproterenol stimulation has been reported in the hypertrophied hearts of spontaneously hypertensive rats and re...
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