Melanie R. Moody

Cardiac-Specific Overexpression of Tumor Necrosis Factor-α Causes Oxidative Stress and Contractile Dysfunction in Mouse Diaphragm

BackgroundWe have developed a transgenic mouse with cardiac-restricted overexpression of tumor necrosis factor-&agr; (TNF-&agr;). These mice develop a heart failure phenotype characterized by left ventricular dysfunction and remodeling, pulmonary edema, and elevated levels of TNF-&agr; in the peripheral circulation from cardiac spillover. Given that TNF-&agr; causes atrophy and loss of function in respiratory muscle, we asked whether transgenic mice developed diaphragm dysfunction and whether contractile losses were caused by oxidative stress or tissue remodeling. Methods and ResultsMuscles excised from transgenic mice and littermate controls were studied in vitro with direct electrical stimulation. Cytosolic oxidant levels were measured with 2′,7′-dichlorofluorescin diacetate; emissions of the oxidized product were detected by fluorescence microscopy. Force generation by the diaphragm of transgenic animals was 47% less than control (13.2±0.8 [±SEM] versus 25.1±0.6 N/cm2;P <0.001); this weakness was associated with greater intracellular oxidant levels (P <0.025) and was partially reversed by 30-minute incubation with the antioxidant N-acetylcysteine 10 mmol/L (P <0.01). Exogenous TNF-&agr; 500 &mgr;mol/L increased oxidant production in diaphragm of wild-type mice and caused weakness that was inhibited by N-acetylcysteine, suggesting that changes observed in the diaphragm of transgenic animals were mediated by TNF-&agr;. There were no differences in body or diaphragm weights between transgenic and control animals, nor was there evidence of muscle injury or apoptosis. ConclusionsElevated circulating levels of TNF-&agr; provoke contractile dysfunction in the diaphragm through an endocrine mechanism thought to be mediated by oxidative stress.

Cyclic GMP is a second messenger by which nitric oxide inhibits diaphragm contraction.

We have shown that endogenous nitrogen oxides (NOx) modulate excitation-contraction coupling in diaphragm. Because cyclic GMP (cGMP) is a second messenger for nitric oxide (NO) inhibition of smooth muscle contraction, we rested the hypothesis that NO acts via cGMP in diaphragm. Fiber bundles from rat diaphragm were studied in vitro. Immunohistochemical analysis using a cGMP-specific monoclonal antibody confirmed the presence of cGMP in the subsarcolemmal region, near nitric oxide synthase (NOS). cGMP measured by ELISA in control muscle (0.27 pmol/mg +/- 0.01 SE) was significantly increased by the NO donor S-nitroso-N-acetylcysteine 1 mM (0.55+/-0.05; N = 6; P < 0.001). Contractile studies showed that the nitric oxide synthase inhibitor N-nitro-L-arginine (L-NNA) 10 mM increased submaximal (40 Hz) tetanic force (P < 0.0001). L-NNA effects were exaggerated by the guanylate cyclase inhibitor LY83583 5-10 microM; force at 40 Hz was increased (P < 0.001). L-NNA effects were partially reversed by 8-bromo-cGMP 1 mM (8-Br-GMP; a cell-permeable cGMP analogue; P < 0.0001) or dipyridamole 10 microM (DPM; a phosphodiesterase inhibitor; P < 0.0001). 8-Br-GMP and DPM produced more-complete L-NNA reversal in combination (P < 0.0001). We conclude that cGMP functions as a second messenger by which NO inhibits diaphragm contraction.

Lipid-shelled microbubbles for ultrasound-triggered release of xenon

Xenon is a cellular protectant shown to stabilize or reduce neurologic injury in stroke. The goal of this work was to develop lipid-shelled microbubbles for ultrasound-triggered xenon release. Microbubbles loaded with either xenon alone (Xe-MB) or xenon and octafluoropropane (Xe-OFP-MB) were synthesized by high-shear mixing lipids with either 100% xenon, or 90% (v/v) xenon and 10% (v/v) octafluoropropane. The size distribution and the attenuation coefficient of Xe-MB and Xe-OFP-MB were measured using a Coulter counter and a broadband attenuation spectroscopy system, respectively. Gas chromatography/mass spectrometry (GC/MS) was performed to assess the dose and stability of encapsulated xenon. The feasibility of xenon release using 5-MHz pulsed Doppler ultrasound and 220-kHz pulsed ultrasound was tested by ultrasound attenuation spectroscopy. Co-encapsulation of OFP increased the number density, attenuation coefficient, and temporal stability of microbubbles. The GC/MS measurements revealed that 143 ± 20 μL/mg and 131 ± 33 μL/mg of xenon was loaded in Xe-MB and Xe-OFP-MB, respectively. Xe-MB and Xe-OFP-MB retained 54 ± 11% and 66 ± 1% of the xenon payload within 15 min of activation, respectively. Attenuation measurements confirmed ultrasound-triggered xenon release. These results suggest that lipid-shelled microbubbles with OFP could serve as ultrasound-triggered xenon delivery agents to attenuate cellular breakdown. Xenon is a cellular protectant shown to stabilize or reduce neurologic injury in stroke. The goal of this work was to develop lipid-shelled microbubbles for ultrasound-triggered xenon release. Microbubbles loaded with either xenon alone (Xe-MB) or xenon and octafluoropropane (Xe-OFP-MB) were synthesized by high-shear mixing lipids with either 100% xenon, or 90% (v/v) xenon and 10% (v/v) octafluoropropane. The size distribution and the attenuation coefficient of Xe-MB and Xe-OFP-MB were measured using a Coulter counter and a broadband attenuation spectroscopy system, respectively. Gas chromatography/mass spectrometry (GC/MS) was performed to assess the dose and stability of encapsulated xenon. The feasibility of xenon release using 5-MHz pulsed Doppler ultrasound and 220-kHz pulsed ultrasound was tested by ultrasound attenuation spectroscopy. Co-encapsulation of OFP increased the number density, attenuation coefficient, and temporal stability of microbubbles. The GC/MS measurements revealed that 143 ± 20 μ...