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 H, Kohr MJ, Wheeler DG, Ziolo MT. Endothelial nitric oxide synthase decreases ␤-adrenergic responsiveness via inhibition of the L-type Ca 2ϩ current.

The role of sarcolipin (SLN) in cardiac physiology was critically evaluated by generating a transgenic (TG) mouse model in which the SLN to sarco(endoplasmic)reticulum (SR) Ca 2؉ ATPase (SERCA) ratio was increased in the ventricle. Overexpression of SLN decreases SR calcium transport function and results in decreased calcium transient amplitude and rate of relaxation. SLN TG hearts exhibit a significant decrease in rates of contraction and relaxation when assessed by ex vivo work-performing heart preparations. Similar results were also observed with muscle preparations and myocytes from SLN TG ventricles. Interestingly, the inhibitory effect of SLN was partially relieved upon high dose of isoproterenol treatment and stimulation at high frequency. Biochemical analyses show that an increase in SLN level does not affect PLB levels, monomer to pentamer ratio, or its phosphorylation status. No compensatory changes were seen in the expression of other calcium-handling proteins. These studies suggest that the SLN effect on SERCA pump is direct and is not mediated through increased monomerization of PLB or by a change in PLB phosphorylation status. We conclude that SLN is a novel regulator of SERCA pump activity, and its inhibitory effect can be reversed by ␤-adrenergic agonists.The sarco(endo)plasmic reticulum (SR) 2 Ca 2ϩ ATPase (SERCA) plays a dominant role in transporting Ca 2ϩ into the SR during the contraction-relaxation cycle of the heart. The rate and amount of Ca 2ϩ transported into the SR determines both the rate of muscle relaxation and the SR Ca 2ϩ load available for the next cycle of contraction (1-4). It is well established that SERCA function is regulated by phospholamban (PLB), whose inhibitory effect is reversed by phosphorylation by protein kinase A and the calcium/calmodulin-dependent protein kinase (CAMKII) during adrenergic activation (5-7). Recent studies have shown that in addition to PLB, sarcolipin (SLN) could also play an important role in the regulation of SERCA pump activity (8 -12).SLN is a 31-amino acid protein expressed in both cardiac and skeletal muscle (11,(13)(14)(15). We have recently demonstrated that SLN is localized in the cardiac SR membrane, and its distribution pattern is similar to SERCA2a and PLB (11). SLN mRNA is differentially expressed in small as opposed to larger mammals. In rodents, SLN mRNA is abundant in the atria with very low levels in the ventricle and skeletal muscles (11,14,15). In contrast, in larger mammals including humans, SLN mRNA is abundant in fast-twitch skeletal muscle compared with atria and ventricle (13). SLN expression is developmentally regulated (11), and its expression levels are modified under certain pathological conditions of the muscle (16,17). Decreased expression of SLN mRNA has been shown in the atria of patients with atrial fibrillation (16). A recent study also showed that SLN mRNA was up-regulated ϳ50-fold in the hypertrophied ventricles of Nkx2-5-null mice (17). Structural similarities between SLN and PLB indicate that they are homolog...

The peroxynitrite donor SIN-1 decreases beta-adrenergic stimulation by reducing PLB(Ser16) phosphorylation via protein phosphatase activation. This peroxynitrite-induced decrease in PLB phosphorylation may be a key mechanism in the beta-adrenergic dysfunction observed in many cardiomyopathies.

We conclude that SLN may play an important role in regulating the SR calcium ATPase pump, possibly by interacting with phospholamban.