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Cardiac function alters with advanced age reminiscent of ... decay time; FDT: fluorescence signal decay; FFI: ... advanced age and sustained hypertension.
Cellular and Molecular Biology 47, N° 3, OL15-OL22 DOI

ISSN 1165-158X 2001 Cell. mol. Biol.

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LOSS OF CARDIAC CONTRACTILE RESPONSE TO TETRAHYDROPAPAVEROLINE WITH ADVANCED AGE AND HYPERTENSION Jun REN1✍, Mellisa NATAVIO2, LeQuishia JEFFERSON2, Michelle L. PAVLIK2 and Ricardo A. BROWN2 1✍ Department of Pharmacology, Physiology, and Therapeutics, University of North Dakota, School of Medicine and Health Sciences, 501 N. Columbia Road, Grand Forks, ND 58203, USA Fax: +1 701 777 4490; E-mail: [email protected] 2 Department of Physiology, Wayne State University School of Medicine, Detroit, MI, USA

Received May 5, 2000; Accepted September 20, 2000

Abstract - Tetrahydropapaveroline (THP), a condensation product of ethanol-derived acetaldehyde, potentiates cardiac function through β-adrenoceptor. We have recently shown that THP-induced cardiac contractile action is likely due to its action at the single myocyte level, and is markedly diminished during early hypertension. Cardiac function alters with advanced age reminiscent of hypertension. This study was to examine cardiac contractile response to THP with advanced age and hypertension. Left ventricular papillary muscles and myocytes were isolated from normotensive (WKY) or hypertensive (SHR) rats, and stimulated to contract at 0.5 Hz. Mechanical parameters evaluated include: peak tension developed (PTD)/peak shortening (PS), time-to-PTD/PS (TPT/TPS), time-to-90% relaxation/relengthening (RT90/TR90), and maximal velocities of contraction/relaxation (± VT/± dLdt). Intracellular Ca2+ transients were measured as fura-2 fluorescence intensity changes (∆FFI). THP (0.1–100 µM) increased PTD in 10- but not 36-wk-old WKY rat myocardium. THP elicited positive, negative or no response on PS in myocytes from 10-wk WKY, 36-wk WKY, and 36-wk SHR groups, respectively. Interestingly, THP elicited discrepant response on intracellular Ca2+ transient compared with that of myocyte shortening. THP increased ∆FFI in 10-wk WKY and 36-wk SHR myocytes while exhibiting a significant inhibiting action in 36-wk WKY myocytes. Lastly, THP shortened TPT/TPS, RT90/TR90 and increased ±VT in all animal groups. These results indicate that the THP-induced myocardial contractile response is altered in advanced age and hypertension, in a manner similar to early stage of hypertension. It is possible that altered intracellular Ca2+ responsiveness may be involved in THP-induced action. Key words: Tetrahydropapaveroline, age, hypertension, cardiac contraction, intracellular Ca2+

INTRODUCTION Chronic ethanol consumption contributes to impaired myocardial function such as ventricular hypertrophy, depressed contractile performance, electrophysiologic abnormalities and heart failure (3,15). Similarly, advanced age and hypertension are often associated with cardiac hypertrophy and electromechanical dysfunction as well as increased risk for stroke and myocardial infarction Abbreviations: ACA: acetaldehyde; FDT: fluorescence decay time; FDT: fluorescence signal decay; FFI: fluorescence intensity; KHB: Krebs-Henseleit bicarbonate; PS: shortening time; PTD: peak tension developed; SHR: hypertensive; THP: tetrahydropapaveroline; TIQ: tetrahydroisoquinoline; TPS: time-to-PS; TPT: time-toPTD; WKY: normotensive Wistar-Kyoto

(11,12). Although a positive association of advanced age with hypertension and the prevalence of hypertension with chronic ethanol consumption have been described both clinically and experimentally (3,10,12), whether ethanol consumption poses as a risk factor for advanced age and hypertension is still not clear. Ethanol is known to elicit both pressor and depressor actions (1, 16). The depressor action of ethanol is believed associated with the ethanol-induced depression of cardiac myocyte contraction, due to reduced intracellular Ca2+ level and/or myofibril sensitivity (8,16). Acetaldehyde (ACA), the main metabolite of ethanol, has also been considered to play a role in myocardial depression at both the tissue and myocyte levels (4). On the other hand, the pressor action of ethanol is poorly understood although

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sympathetic excitation and the biphasic response of ACA have been speculated (4). Recent evidence has suggested that tetrahydropapaveroline (THP), the ethanol-derived alkaloid from the condensation of dopamine with ACA, enhances cardiac contractility and chronotropicity through an β-adrenoceptor-mediated pathway (18,21). We have further revealed that the THP-induced potentiation of cardiac contraction is diminished during early stage of hypertension (18). Therefore, the purpose of this study was to determine the contractile response to THP at the myocardium and single ventricular myocyte levels in advanced age and sustained hypertension.

MATERIALS AND METHODS Experimental animals Experimental procedure was approved by our institutional Animal Investigation Committees and has been previously described (4). Weight-matched adult male spontaneous hypertensive (SHR) and normotensive Wistar-Kyoto (WKY) rats were obtained at 4 wks of age from Taconic Farms (Germantown, NY) and maintained through 36 wks of age. A cohort of WKY rats was sacrificed at 10 wks of age. The animals were individually housed in a temperature-controlled room under a 12-hrs. light/dark illumination cycle and were allowed to standard rat chow and tap water ad libitum. Systolic blood pressures and body weights were measured on a wkly basis using the tail-cuff method and a standard laboratory balance, respectively. Tension measurement of papillary muscle Tension measurement was conducted with a Grass FT03 force transducer (3). In brief, rats were euthanized under ketamine/xylazine sedation (3: 1, 1.32 mg/kg i.p.) and the hearts were rapidly excised and immersed in oxygenated (95% O2, 5% CO2) Tyrode’s solution (in mM: KCl 5.4, NaCl 136.9, NaHCO3 11.9, MgCl2 0.50, CaCl2 2.70, NaH2PO4 0.45 and glucose 5.6, pH 7.4) at 37oC. Left ventricular papillary muscles were dissected and mounted vertically in temperature-controlled 50 ml organ baths containing Tyrode’s solution. Preparation was allowed to equilibrate for 60 min while being electrically stimulated at a frequency of 0.5 Hz for establishment of baseline isometric peak tension developed (PTD). The signal was output to a chart recorder coupled to an analog-digital board. To reduce the inter-preparation variance, PTD was normalized to respective control value. The following parameters were measured: PTD, time-toPTD (TPT), time-to-90% relaxation (RT90) and the maximal velocities of contraction and relaxation (± VT). Cell isolation procedures Single ventricular myocytes were enzymatically isolated as described (4). Briefly, hearts were rapidly removed and perfused (at 37oC) with Krebs-Henseleit bicarbonate (KHB) buffer containing (in mM): 118 NaCl, 4.7 KCl, 1.25 CaCl2, 1.2 MgSO4, 1.2 KH2PO4, 25 NaHCO3, 10 N-[2-hydro-ethyl]-piperazine-N’-[2-ethanesulfonic acid] (HEPES) and 11.1 glucose. Hearts were perfused with a Ca2+-free KHB buffer containing 176 U/ml collagenase (Worthington Biochem., Freehold, NJ) and 0.1 mg/ml hyaluronidase (Sigma Chem., St. Louis, MO). After perfusion, left ventricle was removed and minced before being further digested with trypsin (Sigma) for 3-5 min. Myocytes were resuspended in a sterile filtered, Ca2+-free Tyrode’s solution supplemented with 2% bovine serum albumin, with a pH of 7.4. Extracellular Ca2+ was added incrementally back (1.25 mM) to avoid

Ca2+ paradox. Cells were maintained for 12-24 hr in a serum-free medium (Medium 199, Sigma) with Earl’s salts containing 25 mM HEPES and NaHCO3. Myocytes with obvious sarcolemmal blebs or spontaneous contractions were not used. Cell shortening/relengthening Ventricular myocyte shortening/relengthening was assessed as described (4). In brief, cells were placed in a chamber mounted on the stage of an Olympus inverted microscope (Olympus IX-70) and superfused (~2 ml/min at 30oC) with a buffer containing (in mM): 131 NaCl, 4 KCl, 1 CaCl2, 1 MgCl2, 10 glucose, 10 HEPES at pH 7.4. The cells were field stimulated at 0.5 Hz. A video imaging edge-detector (IonOptix, Milton, MA) was used to capture and convert changes in cell shortening and relengthening into an analogue voltage signal. Mechanical properties of the myocytes assessed include the following: peak shortening (PS), time-to-PS (TPS), time-to-90% relengthening (TR90) and the maximal velocities of shortening and relengthening (± dLdt). Intracellular Ca2+ fluorescence measurement Myocytes were loaded with fura-2/AM (0.5 µM) for 15 min at 30oC, and fluorescence measurements were recorded with a dualexcitation fluorescence photomultiplier system as described (4). Myocytes were exposed to light emitted by a 75 W lamp and passed through either 360 or 380 nm filter (bandwidths were ± 15 nm). Fluorescence emissions were detected between 480-520 nm by an IonOptix photomultiplier tube after first illuminating cells at 360 nm for 0.5 sec then at 380 nm for the duration of the recording protocol (333 Hz sampling rate). The 360 nm excitation scan was repeated at the end of the protocol and qualitative changes in intracellular Ca2+ concentration were inferred from the ratio of the fluorescence intensity at two wavelengths (360/380). Fluorescent properties of the myocytes assessed include fura-2 fluorescence intensity (FFI) and fluorescence decay time (FDT). Experimental protocol Dose dependent response of THP (Sigma) was constructed by delivering the drug at 5-min intervals. Maximal response was achieved within 3 min and maintained for at least 40 min. Data analysis Data are expressed as mean ± SEM. Differences between means within groups were calculated by repeated measures ANOVA followed by Dunnettes post hoc analysis. Differences between groups were assessed with two-way ANOVA. p