Gail D. Thomas

Dystrophins carrying spectrin-like repeats 16 and 17 anchor nNOS to the sarcolemma and enhance exercise performance in a mouse model of muscular dystrophy

Sarcolemma-associated neuronal NOS (nNOS) plays a critical role in normal muscle physiology. In Duchenne muscular dystrophy (DMD), the loss of sarcolemmal nNOS leads to functional ischemia and muscle damage; however, the mechanism of nNOS subcellular localization remains incompletely understood. According to the prevailing model, nNOS is recruited to the sarcolemma by syntrophin, and in DMD this localization is altered. Intriguingly, the presence of syntrophin on the membrane does not always restore sarcolemmal nNOS. Thus, we wished to determine whether dystrophin functions in subcellular localization of nNOS and which regions may be necessary. Using in vivo transfection of dystrophin deletion constructs, we show that sarcolemmal targeting of nNOS was dependent on the spectrin-like repeats 16 and 17 (R16/17) within the rod domain. Treatment of mdx mice (a DMD model) with R16/17-containing synthetic dystrophin genes effectively ameliorated histological muscle pathology and improved muscle strength as well as exercise performance. Furthermore, sarcolemma-targeted nNOS attenuated alpha-adrenergic vasoconstriction in contracting muscle and improved muscle perfusion during exercise as measured by Doppler and microsphere circulation. In summary, we have identified the dystrophin spectrin-like repeats 16 and 17 as a novel scaffold for nNOS sarcolemmal targeting. These data suggest that muscular dystrophy gene therapies based on R16/17-containing dystrophins may yield better clinical outcomes than the current therapies.

Nitric oxide mediates contraction‐induced attenuation of sympathetic vasoconstriction in rat skeletal muscle

1 Sympathetic vasoconstriction is attenuated by metabolic events in contracting rat skeletal muscle, in part by activation of ATP‐sensitive potassium (KATP) channels. However, the specific metabolites in contracting muscle that open KATP channels are not known. We therefore asked if contraction‐induced attenuation of sympathetic vasoconstriction is mediated by the endogenous vasodilators nitric oxide (NO), adenosine, or prostaglandins PGI2 or PGE2, all of which are putative KATP channel openers. 2 In anaesthetized rats, hindlimb contraction alone significantly attenuated the vasoconstrictor responses to lumbar sympathetic nerve stimulation. Inhibition of NO synthase with N‐nitro‐L‐arginine methyl ester (L‐NAME, 5 mg kg−1, i.v.) partially reversed this effect of contraction, resulting in enhanced sympathetic vasoconstriction in contracting hindlimb. Subsequent treatment with the KATP channel blocker glibenclamide (20 mg kg−1, i.v.) had no further effect on sympathetic vasoconstriction in contracting hindlimb. 3 This effect of L‐NAME to partially reverse contraction‐induced attenuation of sympathetic vasoconstriction was not replicated by D‐NAME (5 mg kg−1, i.v.) or angiotensin II (12.5 ng kg−1 min−1, i.v.), the latter used as a hypertensive control. 4 Adenosine receptor blockade with 8‐(p‐sulphophenyl)theophylline (10 mg kg−1, i.v.) or cyclooxygenase inhibition with indomethacin (5 mg kg−1, i.v.) had no effect on contraction‐induced attenuation of sympathetic vasoconstriction. 5 These results suggest that NO plays an important role in the precise regulation of blood flow in exercising skeletal muscles by opposing sympathetic vasoconstriction. Although the underlying mechanism is not known, it may involve NO‐induced activation of vascular KATP channels.

Non-nuclear estrogen receptor α signaling promotes cardiovascular protection but not uterine or breast cancer growth in mice

Steroid hormone receptors function classically in the nucleus as transcription factors. However, recent data indicate that there are also non-nuclear subpopulations of steroid hormone receptors, including estrogen receptors (ERs), that mediate membrane-initiated signaling of unclear basis and significance. Here we have shown that an estrogen-dendrimer conjugate (EDC) that is excluded from the nucleus stimulates endothelial cell proliferation and migration via ERalpha, direct ERalpha-Galphai interaction, and endothelial NOS (eNOS) activation. Analysis of mice carrying an estrogen response element luciferase reporter, ER-regulated genes in the mouse uterus, and eNOS enzyme activation further indicated that EDC specifically targets non-nuclear processes in vivo. In mice, estradiol and EDC equally stimulated carotid artery reendothelialization in an ERalpha- and G protein-dependent manner, and both agents attenuated the development of neointimal hyperplasia following endothelial injury. In contrast, endometrial carcinoma cell growth in vitro and uterine enlargement and MCF-7 cell breast cancer xenograft growth in vivo were stimulated by estradiol but not EDC. Thus, EDC is a non-nuclear selective ER modulator (SERM) in vivo, and in mice, non-nuclear ER signaling promotes cardiovascular protection. These processes potentially could be harnessed to provide vascular benefit without increasing the risk of uterine or breast cancer.

Magnetic Resonance Imaging and Invasive Evaluation of Development of Heart Failure in Transgenic Mice With Myocardial Expression of Tumor Necrosis Factor-α

Background—Transgenic mice expressing tumor necrosis factor-α (TNF-α) in cardiac myocytes develop dilated cardiomyopathy, but the temporal progression to cardiac dysfunction is not well characterized. We asked (1) Does magnetic resonance imaging (MRI) provide a reproducible assessment of cardiac output in mice that correlates with invasive measurements obtained with thermodilution? (2) What is the time course of left ventricular (LV) remodeling in transgenic mice with myocardial expression of TNF-α? Methods and Results—Transgenic mice from 2 different lineages with differing amounts of myocardial TNF-α expression [lineage 1 (L1) and lineage 2 (L2)] and littermate controls (LC) were studied. In protocol 1, cardiac output (CO) and stroke volume (SV) were measured by MRI and thermodilution (TD) in 15 mice (3 L1, 4 L2, 8 LC). In protocol 2, 23 mice (7 L1, 8 L2, 8 LC) were scanned at 1 month of life and every 4 weeks thereafter. In both protocols, cine-MRI was performed with the use of a 1.5-T clinical system ...

Differential sympathetic neural control of oxygenation in resting and exercising human skeletal muscle.

Metabolic products of skeletal muscle contraction activate metaboreceptor muscle afferents that reflexively increase sympathetic nerve activity (SNA) targeted to both resting and exercising skeletal muscle. To determine effects of the increased sympathetic vasoconstrictor drive on muscle oxygenation, we measured changes in tissue oxygen stores and mitochondrial cytochrome a,a3 redox state in rhythmically contracting human forearm muscles with near infrared spectroscopy while simultaneously measuring muscle SNA with microelectrodes. The major new finding is that the ability of reflex-sympathetic activation to decrease muscle oxygenation is abolished when the muscle is exercised at an intensity > 10% of maximal voluntary contraction (MVC). During high intensity handgrip, (45% MVC), contraction-induced decreases in muscle oxygenation remained stable despite progressive metaboreceptor-mediated reflex increases in SNA. During mild to moderate handgrips (20-33% MVC) that do not evoke reflex-sympathetic activation, experimentally induced increases in muscle SNA had no effect on oxygenation in exercising muscles but produced robust decreases in oxygenation in resting muscles. The latter decreases were evident even during maximal metabolic vasodilation accompanying reactive hyperemia. We conclude that in humans sympathetic neural control of skeletal muscle oxygenation is sensitive to modulation by metabolic events in the contracting muscles. These events are different from those involved in either metaboreceptor muscle afferent activation or reactive hyperemia.

Vasomodulation by Skeletal Muscle–Derived Nitric Oxide Requires α-Syntrophin–Mediated Sarcolemmal Localization of Neuronal Nitric Oxide Synthase

Abstract— Neuronal nitric oxide synthase (nNOS) is abundantly expressed in skeletal muscle where it associates with the dystrophin complex at the sarcolemma by binding to the PDZ domain of &agr;-syntrophin. Nitric oxide (NO) produced by skeletal muscle nNOS is proposed to regulate blood flow in exercising muscle by diffusing from the skeletal muscle fibers to the nearby microvessels where it attenuates &agr;-adrenergic vasoconstriction. In the present study, we hypothesized that sarcolemmal localization of nNOS is a critical determinant of the vasoregulatory effect of skeletal muscle–derived NO. To test this hypothesis, we performed experiments in &agr;-syntrophin null mice and in transgenic mice expressing a mutated &agr;-syntrophin lacking the PDZ domain (&Dgr;PDZ), both of which are characterized by reduced sarcolemmal nNOS. We found that modulation of &agr;-adrenergic vasoconstriction was greatly impaired in the contracting muscles of the &agr;-syntrophin null mice and transgenic &Dgr;PDZ mice compared with wild-type mice and transgenic mice expressing full-length &agr;-syntrophin. These in vivo mouse studies highlight the functional importance of appropriate membrane targeting of nNOS by the dystrophin-associated protein &agr;-syntrophin and may have implications for the development of potential gene therapy strategies to treat muscular dystrophy or other muscle-related diseases.

Antiphospholipid antibodies promote leukocyte–endothelial cell adhesion and thrombosis in mice by antagonizing eNOS via β2GPI and apoER2

In antiphospholipid syndrome (APS), antiphospholipid antibodies (aPL) binding to β2 glycoprotein I (β2GPI) induce endothelial cell-leukocyte adhesion and thrombus formation via unknown mechanisms. Here we show that in mice both of these processes are caused by the inhibition of eNOS. In studies of cultured human, bovine, and mouse endothelial cells, the promotion of monocyte adhesion by aPL entailed decreased bioavailable NO, and aPL fully antagonized eNOS activation by diverse agonists. Similarly, NO-dependent, acetylcholine-induced increases in carotid vascular conductance were impaired in aPL-treated mice. The inhibition of eNOS was caused by antibody recognition of domain I of β2GPI and β2GPI dimerization, and it was due to attenuated eNOS S1179 phosphorylation mediated by protein phosphatase 2A (PP2A). Furthermore, LDL receptor family member antagonism with receptor-associated protein (RAP) prevented aPL inhibition of eNOS in cell culture, and ApoER2-/- mice were protected from aPL inhibition of eNOS in vivo. Moreover, both aPL-induced increases in leukocyte-endothelial cell adhesion and thrombus formation were absent in eNOS-/- and in ApoER2-/- mice. Thus, aPL-induced leukocyte-endothelial cell adhesion and thrombosis are caused by eNOS antagonism, which is due to impaired S1179 phosphorylation mediated by β2GPI, apoER2, and PP2A. Our results suggest that novel therapies for APS can now be developed targeting these mechanisms.

Nitric oxide‐dependent modulation of sympathetic neural control of oxygenation in exercising human skeletal muscle

Nitric oxide (NO) attenuates α‐adrenergic vasoconstriction in contracting rodent skeletal muscle, but it is unclear if NO plays a similar role in human muscle. We therefore hypothesized that in humans, NO produced in exercising skeletal muscle blunts the vasoconstrictor response to sympathetic activation. We assessed vasoconstrictor responses in the microcirculation of human forearm muscle using near‐infrared spectroscopy to measure decreases in muscle oxygenation during reflex sympathetic activation evoked by lower body negative pressure (LBNP). Experiments were performed before and after NO synthase inhibition produced by systemic infusion of NG‐nitro‐l‐arginine methyl ester (l‐NAME). Before l‐NAME, LBNP at −20 mmHg decreased muscle oxygenation by 20 ± 2 % in resting forearm and by 2 ± 3 % in exercising forearm (n= 20), demonstrating metabolic modulation of sympathetic vasoconstriction. As expected, l‐NAME increased mean arterial pressure by 17 ± 3 mmHg, leading to baroreflex‐mediated supression of baseline muscle sympathetic nerve activity (SNA). The increment in muscle SNA in response to LBNP at −20 mmHg also was attenuated after l‐NAME (before, +14 ± 2; after, +8 ± 1 bursts min−1; n= 6), but this effect of l‐NAME was counteracted by increasing LBNP to −40 mmHg (+19 ± 2 bursts min−1). After l‐NAME, LBNP at −20 mmHg decreased muscle oxygenation similarly in resting (−11 ± 3 %) and exercising (−10 ± 2 %) forearm (n= 12). Likewise, LBNP at −40 mmHg decreased muscle oxygenation both in resting (−19 ± 4 %) and exercising (−21 ± 5 %) forearm (n= 8). These data advance the hypothesis that NO plays an important role in modulating sympathetic vasoconstriction in the microcirculation of exercising muscle, because such modulation is abrogated by NO synthase inhibition with l‐NAME.

FcγRIIB Mediates C-Reactive Protein Inhibition of Endothelial NO Synthase

C-reactive protein (CRP) is an acute-phase reactant that is positively correlated with cardiovascular disease risk and endothelial dysfunction. Whether CRP has direct actions on endothelium and the mechanisms underlying such actions are unknown. Here we show in cultured endothelium that CRP prevents endothelial NO synthase (eNOS) activation by diverse agonists, resulting in the promotion of monocyte adhesion. CRP antagonism of eNOS occurs nongenomically and is attributable to blunted eNOS phosphorylation at Ser1179. Okadaic acid or knockdown of PP2A by short-interference RNA reverses CRP antagonism of eNOS, indicating a key role for the phosphatase. Aggregated IgG, the known ligand for Fc&ggr; receptors, causes parallel okadaic acid–sensitive loss of eNOS function, Fc&ggr;RIIB expression is demonstrable in endothelium, and heterologous expression studies reveal that CRP antagonism of eNOS requires Fc&ggr;RIIB. In Fc&ggr;RIIB+/+ mice, CRP blunts acetylcholine-induced increases in carotid artery vascular conductance; in contrast, CRP enhances acetylcholine responses in Fc&ggr;RIIB−/− mice. Thus Fc&ggr;RIIB mediates CRP inhibition of eNOS via PP2A, providing a mechanistic link between CRP and endothelial dysfunction.

ATP-sensitive potassium channels mediate contraction-induced attenuation of sympathetic vasoconstriction in rat skeletal muscle.

Sympathetic vasoconstriction is sensitive to inhibition by metabolic events in contracting rat and human skeletal muscle, but the underlying cellular mechanisms are unknown. In rats, this inhibition involves mainly alpha2-adrenergic vasoconstriction, which relies heavily on Ca2+ influx through voltage-dependent Ca2+ channels. We therefore hypothesized that contraction-induced inhibition of sympathetic vasoconstriction is mediated by ATP-sensitive potassium (KATP) channels, a hyperpolarizing vasodilator mechanism that could be activated by some metabolic product(s) of skeletal muscle contraction. We tested this hypothesis in anesthetized rats by measuring femoral artery blood flow responses to lumbar sympathetic nerve stimulation or intraarterial hindlimb infusion of the specific alpha2-adrenergic agonist UK 14,304 during KATP channel activation with diazoxide in resting hindlimb and during KATP channel block with glibenclamide in contracting hindlimb. The major new findings are twofold. First, like muscle contraction, pharmacologic activation of KATP channels with diazoxide in resting hindlimb dose dependently attenuated the vasoconstrictor responses to either sympathetic nerve stimulation or intraarterial UK 14,304. Second, the large contraction-induced attenuation in sympathetic vasoconstriction elicited by nerve stimulation or UK 14,304 was partially reversed when the physiologic activation of KATP channels produced by muscle contraction was prevented with glibenclamide. We conclude that contraction-induced activation of KATP channels is a major mechanism underlying metabolic inhibition of sympathetic vasoconstriction in exercising skeletal muscle.