Neuronal nitric oxide synthase signaling within cardiac myocytes targets phospholamban

Abstract

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.

Inotropic Response of Cardiac Ventricular Myocytes to β-Adrenergic Stimulation With Isoproterenol Exhibits Diurnal Variation

“…Although, unlike many gene deletion studies since, these authors did not find any difference in basal Ca 2ϩ transients and sarcomere shortening. Wang et al, 32 also found an increased response to ISO in eNOS Ϫ/Ϫ myocytes, although in this study a high concentration of 1 mmol/L ISO was used and the specific eNOS inhibitor L-N 5 -(1-iminoethyl)-ornithine had little effect on the ISO response of wild-type myocytes. In contrast, Martin et al, 33 describe enhancement of the contractile response to 100 nmol/L ISO in nNOS Ϫ/Ϫ and no significant difference in eNOS Ϫ/Ϫ mouse myocytes.…”

Cardiomyocyte GTP Cyclohydrolase 1 and Tetrahydrobiopterin Increase NOS1 Activity and Accelerate Myocardial Relaxation

“…Increased NOS1 Activity on LV Contraction: Role of I Ca and SR Ca 2؉ Release NOS1 gene deletion has been reported to slow the rate of myocardial relaxation and SR Ca 2ϩ uptake 7,16 -18 and to have variable effects on I Ca , myocardial inotropy 14,15,17 and the diastolic leak of Ca 2ϩ through the RyR2. 23,24 In mGCH1-Tg mice, increased NOS1 activity did not affect LV systolic function (Online Figure II) or cardiomyocyte basal and ␤-adrenergic stimulated contraction ( Figure 4A and Online Figure III) Figure 4B) did not differ between genotypes, whereas the SR Ca 2ϩ content tended to be lower in mGCH1-Tg ( Figure 4C).…”

“…Myocardial overexpression of GCH1 hastens relaxation by increasing Ser 16 PLB phosphorylation. A, The PLB Ser 16 phosphorylated fraction (nϭ20 hearts per genotype) and the SERCA2A/total PLB ratio (nϭ4 hearts per genotype) were significantly increased in mGCH1-Tg mice (***PϽ0.0001 and *PϽ0.05 versus WT, respectively), whereas the PLB Thr 17 …”

“…NOS1 inhibition was achieved by pre-incubating LV myocytes with S-methyl-Lthiocitrulline (1 µmol/L SMTC, Sigma) for 1 hour. Similarly, xanthine oxidoreductase (XOR) or protein kinase A (PKA) inhibition was achieved by incubating LV myocytes with oxypurinol (100 µmol/L, Sigma) or myristoylated PKI (14)(15)(16)(17)(18)(19)(20)(21)(22) The L-type calcium current (I Ca ) was measured as described in 2 . Briefly, LV myocytes were placed in an experimental bath set on the stage of an inverted microscope and perfused with Tyrode solution containing in mmol/L: 140 TEACl, 5.4 CsCl, 1.2 MgCl 2 , 5 HEPES, 11 glucose, 1.4 CaCl 2 , pH 7.4 with CsOH.…”

Abnormal Ca2+Cycling in Failing Ventricular Myocytes: Role of NOS1-Mediated Nitroso-Redox Balance

“…4) (20,85). Our and others' work has shown that NOS1 is a key protein involved in these pathways (10,91,167,170). That is, in NOS1 knockout (NOS1 -/ -) mice, there is a blunted FFR and functional response to b-AR stimulation, which is similar to failing myocytes (i.e., decreased contractile reserve).…”