Are myocardial eNOS and nNOS involved in the beta-adrenergic and muscarinic regulation of inotropy? A systematic investigation.
OBJECTIVE
The role of constitutive nitric oxide (NO) production in the regulation of beta-adrenergic and muscarinic responses remains controversial. Conflicting data in left ventricular (LV) myocytes from eNOS knockout mice (eNOS-/-) have been ascribed to inconsistent experimental conditions (i.e., differences in the choice of controls, age of the mice, myocytes' stimulation frequency, and in the level of beta-adrenergic stimulation); however, the recent identification of a neuronal-like NO synthase (nNOS) in the LV myocardium has raised the possibility that this isoform may be involved in the modulation of beta-adrenergic and muscarinic responses.


METHODS
To address these issues we recorded sarcomere shortening at 35 degrees C under basal conditions, in the presence of isoproterenol (ISO, 10-100 nmol/L) and of ISO plus carbamylcholine (CCh, 1 micromol/L) in LV myocytes isolated from eNOS-/- and nNOS-/- mice, their wild type littermates (eNOS+/+ and nNOS+/+) or C57BL/6J mice. eNOS-/- and control myocytes were studied at 1 and 3 Hz, in the presence of 10 and 100 nmol/L ISO, and responses were compared between young (3 months) and old (> or =12 months) mice.


RESULTS
Contraction did not differ between young eNOS-/- and eNOS+/+ mice at all stages of the experimental protocol, either at 1 or 3 Hz or in response to 10 or 100 nmol/L ISO. However, myocytes from old eNOS-/- mice showed a reduced inotropic response to ISO compared with age-matched eNOS+/+ mice (P = 0.02). Similarly, there was a significant difference in the ISO response between eNOS+/+ and C57BL/6J myocytes (P < 0.01), suggesting that experimental variables such as age and the choice of control animals may have contributed to the inconsistency in the results reported in the literature. In contrast, nNOS-/- myocytes showed greater contraction and slower relaxation at all stages of the experimental protocol (P = 0.0003 and P = 0.01 vs. nNOS+/+ myocytes).


CONCLUSIONS
Constitutive eNOS expression in murine LV myocytes is not essential for the muscarinic-mediated inhibition of beta-adrenergic signalling and does not appear to play a significant role in the regulation of basal and beta-adrenergic myocardial contraction. Our data suggest that nNOS is the myocardial constitutive isoform responsible for the NO-mediated autocrine regulation of myocardial inotropy and relaxation.
Martin
Sarah R
SR
Department of Cardiovascular Medicine, University of Oxford, John Radcliffe Hospital, Oxford OX3 9DU, UK.
Emanuel
Krzysztof
K
Sears
Claire E
CE
Zhang
Yin-Hua
YH
Casadei
Barbara
B
D000318:Adrenergic beta-Agonists / Q000494:pharmacology*; D000375:Aging / Q000502:physiology; D000818:Animals; D002217:Carbachol / Q000494:pharmacology*; D048429:Cell Size / Q000187:drug effects; D018679:Cholinergic Agonists / Q000494:pharmacology*; D004558:Electric Stimulation; D007545:Isoproterenol / Q000494:pharmacology*; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D032383:Myocytes, Cardiac / Q000201:enzymology*; D052248:Nitric Oxide Synthase Type I / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III / Q000235:genetics / Q000378:metabolism*; D013268:Stimulation, Chemical
D000318:Adrenergic beta-Agonists; D018679:Cholinergic Agonists; D002217:Carbachol; D052248:Nitric Oxide Synthase Type I; D052250:Nitric Oxide Synthase Type III; D007545:Isoproterenol