Neuronal nitric oxide synthase signaling within cardiac myocytes targets phospholamban.
Studies have shown that neuronal nitric oxide synthase (nNOS, NOS1) knockout mice (NOS1-/-) have increased or decreased contractility, but consistently have found a slowed rate of intracellular Ca2+ ([Ca2+]i) decline and relengthening. Contraction and [Ca2+]i decline are determined by many factors, one of which is phospholamban (PLB). The purpose of this study is to determine the involvement of PLB in the NOS1-mediated effects. Force-frequency experiments were performed in trabeculae isolated from NOS1-/- and wild-type (WT) mice. We also simultaneously measured Ca2+ transients (Fluo-4) and cell shortening (edge detection) in myocytes isolated from WT, NOS1-/-, and PLB-/- mice. NOS1-/- trabeculae had a blunted force-frequency response and prolonged relaxation. We observed similar effects in myocytes with NOS1 knockout or specific NOS1 inhibition with S-methyl-l-thiocitrulline (SMLT) in WT myocytes (i.e., decreased Ca2+ transient and cell shortening amplitudes and prolonged decline of [Ca2+]i). Alternatively, NOS1 inhibition with SMLT in PLB-/- myocytes had no effect. Acute inhibition of NOS1 with SMLT in WT myocytes also decreased basal PLB serine16 phosphorylation. Furthermore, there was a decreased SR Ca2+ load with NOS1 knockout or inhibition, which is consistent with the negative contractile effects. Perfusion with FeTPPS (peroxynitrite decomposition catalyst) mimicked the effects of NOS1 knockout or inhibition. beta-Adrenergic stimulation restored the slowed [Ca2+]i decline in NOS1-/- myocytes, but a blunted contraction remained, suggesting additional protein target(s). In summary, NOS1 inhibition or knockout leads to decreased contraction and slowed [Ca2+]i decline, and this effect is absent in PLB-/- myocytes. Thus NOS1 signaling modulates PLB serine16 phosphorylation, in part, via peroxynitrite.
Wang
Honglan
H
Department of Physiology and Cell Biology, Davis Heart and Lung Research Institute, The Ohio State University, Columbus, OH 43210, USA.
Kohr
Mark J
MJ
Traynham
Christopher J
CJ
Wheeler
Debra G
DG
Janssen
Paul M L
PM
Ziolo
Mark T
MT
D000318:Adrenergic beta-Agonists / Q000494:pharmacology; D000818:Animals; D020013:Calcium Signaling / Q000187:drug effects; D002135:Calcium-Binding Proteins / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D002956:Citrulline / Q000031:analogs & derivatives / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007545:Isoproterenol / Q000494:pharmacology; D008665:Metalloporphyrins / Q000494:pharmacology; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D009200:Myocardial Contraction / Q000187:drug effects; D032383:Myocytes, Cardiac / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D052248:Nitric Oxide Synthase Type I / Q000037:antagonists & inhibitors / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D030421:Peroxynitrous Acid / Q000378:metabolism; D010766:Phosphorylation; D012519:Sarcoplasmic Reticulum / Q000378:metabolism; D013890:Thiourea / Q000031:analogs & derivatives / Q000494:pharmacology; D013997:Time Factors; D000099211:Phospholamban
D000318:Adrenergic beta-Agonists; D002135:Calcium-Binding Proteins; D002956:Citrulline; D004791:Enzyme Inhibitors; D007545:Isoproterenol; D008665:Metalloporphyrins; D052248:Nitric Oxide Synthase Type I; D030421:Peroxynitrous Acid; D013890:Thiourea; D000099211:Phospholamban; C466420:5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrinato iron(III) chloride; C496321:Nos1 protein, mouse; C086717:S-methylthiocitrulline