Intrinsic Cardiac Catecholamines Help Maintain Beating Activity in Neonatal Rat Cardiomyocyte Cultures

0031-3998/04/5603-0411 PEDIATRIC RESEARCH Vol. 56, No. 3, 2004 Copyright © 2004 International Pediatric Research Foundation, Inc. Printed in U.S.A. Intrinsic Cardiac Catecholamines Help Maintain Beating Activity in Neonatal Rat Cardiomyocyte Cultures ARUNA R. NATARAJAN, QI RONG, ALEXANDER N. KATCHMAN, AND STEVEN N. EBERT Department of Pediatrics [A.R.N.], Department of Pharmacology [Q.R., A.N.K., S.N.E.], Georgetown University Medical Center, Washington, DC 20057, U.S.A. ABSTRACT In the present study, we identify intrinsic cardiac adrenergic indicate that intrinsic cardiac catecholamines help to maintain (ICA) cells in the neonatal rat heart using immunofluorescent beating rates in neonatal rat cardiomyocyte cultures via stimula- ␣ ␤ histochemical staining techniques with antibodies that specifi- tion of 1- and -adrenergic receptors. This information should cally recognize the major enzymes in the catecholamine biosyn- help to increase our understanding of the physiologic mecha- thetic pathway. ICA cells are most concentrated near the endo- nisms governing cardiovascular function in neonates. (Pediatr cardial surface of ventricular myocardium, but are also found Res 56: 411–417, 2004) sporadically throughout the heart. In primary cultures of neonatal rat cardiomyocytes, ICA cells are closely associated with clusters Abbreviations of cardiomyocytes. To investigate a potential role for intrinsi- ICA, intrinsic cardiac adrenergic cally produced catecholamines, we recorded beating rates in the TH, tyrosine hydroxylase presence and absence of the catecholamine-depleting agent re- DBH, dopamine ␤-hydroxylase serpine or the adrenergic receptor blockers prazosin and timolol PNMT, phenylethanolamine N-methyltransferase using videomicroscopy and photodiode sensors. Our results TRITC, tetramethylrhodamine isothiocyanate show that beating rates slow significantly when endogenous DOPS, dihydroxyphenylserine catecholamines are depleted or when their action is blocked with DMEM, Dulbecco’s modified Eagle medium ␤ ␣ either a -oran 1-adrenergic receptor antagonist. These data HBSS, Hank’s balanced salt solution The catecholamines epinephrine and norepinephrine are sympathetic innervation of the heart is incomplete (12), and neurotransmitter stress hormones that influence cardiac func- little is known about the specific distribution of ICA cells in the tion profoundly. We and others have shown that the heart itself heart (13–15). Further, the functional significance of these ICA produces these catecholamines in specialized cells known as cells in the neonatal (or adult) heart is unknown. ICA cells (1–3). We have also shown that these cells are The neonatal period is characterized by dramatic cardiovas- transiently and progressively associated with different regions cular adaptation to extrauterine life. Although peripheral cat- of the embryonic rat heart that are important sites for pace- echolamines are synthesized by the adrenal medulla (16–18) making and conduction tissue development (e.g. sinoatrial and organ of Zuckerkandl (19, 20) in the perinatal period, node, atrioventricular canal, bundle of His, and ventricular functional sympathetic innervation of the heart is not com- septum) (3). ICA cells appear to persist in the heart postnatally pleted until the second week after birth in rats (15, 21). The as they have been found in neonatal (1, 4–6) and adult (1, neonatal rat heart, nevertheless, expresses fully functional ␣- 7–11) hearts. In adult hearts, ICA cells are often found clus- and ␤-adrenergic receptors, allowing for chronotropic re- tered around sites of innervation (6, 7, 10). In the neonate, ␣ ␤ sponses to either 1-or -adrenergic agonists (22–24). Once functional sympathetic innervation of the heart is achieved, ␣ Received August 1, 2003; accepted April 26, 2004. 1-agonists lose their ability to stimulate heart rate (22), which Correspondence: Steven N. Ebert, Ph.D., Department of Pharmacology, Georgetown comes increasingly under ␤-adrenergic control, as seen in the University Medical Center, 3900 Reservoir Rd., NW, Washington, DC 20057, U.S.A.; e-mail: [email protected] adult (25, 26). Well before such innervation-dependent Supported in part by a grant to S.N.E. from the National Institutes of Health changes happen, however, chronotropic responses to exog- (R01HL58743). A.N. was supported by the Mentored Clinical Research Scholar Program Award, Grant RR17613, from the National Center for Research Resources, National enously administered catecholamines occur (27–31). The Institutes of Health, Department of Health and Human Services. source(s) of endogenous catecholamines that regulate heart rate DOI: 10.1203/01.PDR.0000136279.80897.4C during perinatal development remain undefined. 411 412 NATARAJAN ET AL. In the present study, we used immunofluorescence histo- ditions and placed in ice-cold sterile HBSS (no Caϩϩ or chemical and cytochemical staining techniques to demonstrate Mgϩϩ). Enzymatic and mechanical dissociation of cardiomy- the presence of ICA cells in neonatal rat hearts and primary ocytes was then performed using the Neonatal Cardiomyocyte cultures of cardiomyocytes isolated from neonatal rat hearts by Isolation System supplied by Worthington Biochemicals (Free- their ability to express catecholamine biosynthetic enzymes. hold, NJ, U.S.A.). The hearts were minced on ice and digested We also used pharmacological interventions to demonstrate overnight at 4°C with purified trypsin (10 ␮g/mL) in HBSS. possible roles for these locally produced catecholamines during On the following morning, the digested tissue was transferred cardiac development by evaluating beating rate responses in to a 50-mL conical tube and purified soybean trypsin inhibitor primary cultures of neonatal rat cardiomyocytes using video- (40 ␮g/mL) was added to terminate trypsinization. The tissue microscopy, photodiode sensors, and customized data acquisi- was oxygenated and warmed to 37°C. Purified collagenase (10 tion/analysis software. U/mL) was added and digestion proceeded for 45 min at 37°C with intermittent gentle swirling. Mild trituration was then METHODS used to mechanically dissociate the digested tissue, and single- cell suspensions were obtained by filtering this digested mate- Materials. All drugs and chemicals used for this study were rial through 70 ␮m sterile mesh filters. The cells were collected purchased from Sigma Chemical (St. Louis, MO, U.S.A.). by low-speed centrifugation. The supernatant was discarded Drugs were prepared as concentrated stock solutions and fro- and the cell pellet resuspended in DMEM (high glucose, zen in small aliquots at Ϫ80°C. The stock aliquots were Biofluids, Rockville, MD, U.S.A.) culture medium containing thawed immediately before use and were not refrozen. Rabbit 10% defined iron-supplemented bovine calf serum (Hyclone anti-PNMT and anti-DBH antisera have been previously de- Laboratories, Logan, UT, U.S.A.), 2 mM glutamine, and gen- scribed (3). Mouse monoclonal anti-TH and anti-sarcomeric tamicin sulfate (50 ␮g/mL). In some experiments, the serum ␣-actinin antibodies were purchased from Sigma Chemical. was pretreated with charcoal before adding it to the media FITC-conjugated donkey anti-rabbit and TRITC-conjugated (10% volume as above) to remove possible trace amounts of donkey anti-mouse secondary antibodies were obtained from catecholamines that may have been present. The cells were Jackson Immunoresearch (West Grove, PA, U.S.A.). then “preplated” to remove fibroblasts as described previously Animals. Timed-pregnant Sprague-Dawley rats were ob- (7), and the remaining cells counted and seeded onto multi- tained from Taconic Farms (Germantown, NY, U.S.A.). Neo- well culture plates at a density of 75,000 cells per cm2. The natal pups were used within the first 2 d after birth. All media was changed every 2–3 d beginning the day after experiments were conducted in strict concordance with the seeding. Bromodeoxyuridine (0.1 mM) was added to the cul- guidelines provided by the Georgetown University Animal ture media for the first3dtofurther minimize contamination Care and Use Committee and the National Institutes of Health. from fibroblasts (32). Immunofluorescent histochemical and cytochemical stain- Beating rate measurements. Beating activity of cultured ing. Double immunofluorescent staining of neonatal rat hearts cardiomyocytes was measured by edge-detection using photo- and cardiomyocyte cultures was performed essentially as de- diode sensors attached to a monitor screen depicting beating scribed previously (3). For immunofluorescent cytochemical cells viewed by videomicroscopy. The entire microscope was staining of cultured cardiomyocytes, the myocytes were seeded enclosed in a temperature-controlled (37°C), custom-designed onto collagen-coated coverslips (12 mm round) at the time of incubation chamber (Part numbers: 8400–005, 8506–050, and isolation, and allowed to grow in culture for 7–9 d before 8537–025; Coy Laboratory Products, Inc., Grass Lake, MI, fixation. To fix the cells, the culture medium was removed and U.S.A.) fed with 5% CO2/95% air to maintain constant pH. the cells were rinsed twice with PBS. The cells were then The photodiode sensor signal was filtered with an 8-pole exposed to 4% paraformaldehyde in PBS for 15 min at room Bessel low-pass filter (Model 900CT, Frequency Devices, Inc., temperature. At the end of this fixation period, the fixative was Havervill, MA, U.S.A.) with a cutoff frequency of 1000 Hz, removed and the cells were washed twice with PBS. The cells and relayed to a PC equipped with a

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