ß2-Adrenergic receptor signaling in the cardiac myocyte is modulated by interactions with CXCR4.

Chemokines are small secreted proteins with chemoattractant properties that play a key role in inflammation, metastasis, and embryonic development. We previously demonstrated a nonchemotactic role for one such chemokine pair, stromal cell-derived factor-1α and its G-protein coupled receptor, CXCR4. Stromal cell-derived factor-1/CXCR4 are expressed on cardiac myocytes and have direct consequences on cardiac myocyte physiology by inhibiting contractility in response to the nonselective ß-adrenergic receptor (ßAR) agonist, isoproterenol. As a result of the importance of ß-adrenergic signaling in heart failure pathophysiology, we investigated the underlying mechanism involved in CXCR4 modulation of ßAR signaling. Our studies demonstrate activation of CXCR4 by stromal cell-derived factor-1 leads to a decrease in ßAR-induced PKA activity as assessed by cAMP accumulation and PKA-dependent phosphorylation of phospholamban, an inhibitor of SERCA2a. We determined CXCR4 regulation of ßAR downstream targets is ß2AR-dependent. We demonstrated a physical interaction between CXCR4 and ß2AR as determined by coimmunoprecipitation, confocal microscopy, and BRET techniques. The CXCR4-ß2AR interaction leads to G-protein signal modulation and suggests the interaction is a novel mechanism for regulating cardiac myocyte contractility. Chemokines are physiologically and developmentally relevant to myocardial biology and represent a novel receptor class of cardiac modulators. The CXCR4-ß2AR complex could represent a hitherto unknown target for therapeutic intervention.

Quantitation of CXCR4 expression in myocardial infarction using 99mTc-labeled SDF-1alpha.

UNLABELLED: The chemokine stromal-derived factor-1alpha (SDF-1alpha, CXCL12) and its receptor CXCR4 are implicated as key mediators of hematopoietic stem cell retention, cancer metastasis, and HIV infection. Their role in myocardial infarction (MI) is not as well defined. The noninvasive in vivo quantitation of CXCR4 expression is central to understanding its importance in these diverse processes as well in the cardiac response to injury. METHODS: Recombinant SDF-1alpha was radiolabeled under aprotic conditions and purified by gel-filtration chromatography (GFC) using high-specific-activity 99mTc-S-acetylmercaptoacetyltriserine-N-hydroxysuccinimide ([99mTc-MAS3]-NHS) prepared by solid-phase preloading. Radiotracer stability and transmetallation under harsh conditions were quantified by GFC. Affinity, specificity, and maximum number of binding sites (Bmax) were quantified, with adenoviral-expressed CXCR4 on nonexpressing cells and endogenous receptor on rat neonatal cardiomyocytes, using a high-throughput live-cell-binding assay. Blood half-life, biodistribution, and clearance of intravenously injected [99mTc-MAS3]-SDF-1alpha were quantified in Sprague-Dawley rats before and after experimentally induced MI. RESULTS: [99mTc-MAS3]-SDF-1alpha could be prepared in 2 h total with a specific activity of 8.0 x 10(7) MBq/mmol (2,166 Ci/mmol) and a radiochemical purity greater than 98%. Degradation of the radiotracer after boiling for 5 min, with and without 1 mM dithiothreitol, and transmetallation in 100% serum at 37 degrees C for 4 h were negligible. [99mTc-MAS3]-SDF-1alpha exhibits high specificity for CXCR4 on the surface of living rat neonatal cardiomyocytes, with an affinity of 2.7 +/- 0.9 nM and a Bmax of 4.8 x 10(4) binding sites per cell. After intravenous injection, 99mTc-labeled SDF-1alpha displays a blood half-life of 25.8 +/- 4.6 min, rapid renal clearance with only 26.2 +/- 6.1 percentage injected dose remaining in the carcass at 2 h, consistently low uptake in most organs (<0.1 percentage injected dose per gram), and no evidence of blood-brain barrier penetration. After MI was induced, CXCR4 expression levels in the myocardium increased more than 5-fold, as quantified using [99mTc-MAS3]-SDF-1alpha and confirmed using confocal immunofluorescence. CONCLUSION: We describe a 99mTc-labeled SDF-1alpha radiotracer that can be used as a sensitive and specific probe for CXCR4 expression in vivo and demonstrate that this radiotracer is able to quantify changes in CXCR4 expression under different physiologic and pathologic states. Taken together, CXCR4 levels should now be quantifiable in vivo in a variety of animal model systems of human diseases.

Reduced sialylation status in UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase (GNE)-deficient mice.

Sialic acids are widely expressed as terminal carbohydrates on glycoconjugates of eukaryotic cells. They are involved in a variety of cellular functions, such as cell adhesion or signal recognition. The key enzyme of sialic acid biosynthesis is the bifunctional UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase (GNE), which catalyzes the first two steps of sialic acid biosynthesis in the cytosol. Previously, we have shown that inactivation of the GNE by gene targeting causes early embryonic lethality in mice, whereas heterozygous GNE-deficient mice are vital. In this study we compared the amount of membrane-bound sialic acids of wildtype mice with those of heterozygous GNE-deficient mice. For that we quantified membrane-bound sialic acid concentration in various organs of wildtype- and heterozygous GNE-deficient mice. We found an organ-specific reduction of membrane-bound sialic acids in heterozygous GNE-deficient mice. The overall reduction was 25%. Additionally, we analyzed transferrin and polysialylated neural cell adhesion molecule (NCAM) by one- or two-dimensional gel electrophoresis. Transferrin-expression was unchanged in heterozygous GNE-deficient mice; however the isoelectric point of transferrin was shifted towards basic pH, indicating a reduced sialylation. Furthermore, the expression of polysialic acids on NCAM was reduced in GNE-deficient mice.

Muscle cachexia is regulated by a p53-PW1/Peg3-dependent pathway.

Muscle wasting (cachexia) is an incurable complication associated with chronic infection and cancers that leads to an overall poor prognosis for recovery. Tumor necrosis factor-alpha (TNFalpha) is a key inflammatory cytokine associated with cachexia. TNFalpha inhibits myogenic differentiation and skeletal muscle regeneration through downstream effectors of the p53 cell death pathway including PW1/Peg3, bax, and caspases. We report that p53 is required for the TNFalpha-mediated inhibition of myogenesis in vitro and contributes to muscle wasting in response to tumor load in vivo. We further demonstrate that PW1 and p53 participate in a positive feedback regulatory loop in vitro. Consistent with this observation, we find that the number of PW1-expressing stem cells in skeletal muscle declines significantly in p53 nullizygous mice. Furthermore, gene transfer of a dominant-negative form of PW1 into muscle tissue in vivo blocks myofiber atrophy in response to tumor load. Taken together, these results show a novel role for p53 in mediating muscle stem cell behavior and muscle atrophy, and point to new targets for the therapeutic treatment of muscle wasting.

Sialylation is essential for early development in mice.

Sialic acids are widely expressed as terminal carbohydrates on glycoconjugates of eukaryotic cells. Sialylation is crucial for a variety of cellular functions, such as cell adhesion or signal recognition, and regulates the biological stability of glycoproteins. The key enzyme of sialic acid biosynthesis is the bifunctional UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase (UDP-GlcNAc 2-epimerase), which catalyzes the first two steps of sialic acid biosynthesis in the cytosol. We report that inactivation of the UDP-GlcNAc 2-epimerase by gene targeting causes early embryonic lethality in mice, thereby emphasizing the fundamental role of this bifunctional enzyme and sialylation during development. The need of UDP-GlcNAc 2-epimerase for a defined sialylation process is exemplified with the polysialylation of the neural cell adhesion molecule in embryonic stem cells.