Glenn A. Walter, Ph.D. Assistant Professor Phone: (352) 392-0551 Fax: (352) 846-0270 Office: M-548 E-mail: glennw@phys.med.ufl.edu Publications: Search PubMed

Bioengineering of Muscle Structure and Metabolism

Thermodynamic bioengineering with invertebrate genes. We have shown that arginine kinase (AK) can serve as a noninvasive monitoring system for viral mediated gene delivery. The general implication of these results are twofold. First arginine kinase can be used as a noninvasive marker for gene transfer in vertebrate skeletal muscles. Secondly, skeletal muscles that express a combination of creatine kinase and arginine kinase provide an extended thermodynamic buffering range. Thus the coexpression of AK and CK should prove beneficial to skeletal cells under conditions of prolonged ischemia or fatiguing conditions. Following the introduction of AK into the mammalian muscle cytoplasm a large PArg pool is expected to be formed without disturbing the normal levels of ATP or PCr. However, upon depletion of PCr which would occur in ischemia, creatine kinase can no longer buffer changes in ATP. At this point, the PArg pool will continue to buffer changes in ATP levels. In addition, the AK reaction will tend to slow the fall of pH.

Restoration of membrane integrity and sarcolemmal integrity. We use a combination of unsuppressed ¹H-MRS, multi-echo and dynamic, macromolecule contrast enhanced MRI, to investigate the intrinsic properties that lead to changes in muscle T₂ in dystrophy and following damage, ischemia, and exercise. Contraction-induced injury can be characterized using a variety of methods. However, many of the histological methods have the disadvantage they are invasive and require the animal to be sacrificed. MRI/MRS due to its noninvasive nature and short acquisition periods, can be used to follow both primary (0-3 day) and secondary (3-21 day) injury processes within the same mouse providing a tool for longitudinal studies in both control and dystrophic animals. Differences in T₂ provide sensitive markers of muscle damage producing differences in image contrast with a spatial resolution on the order of that of a single muscle fiber. MRI for detection of muscle injury has the added advantage that large muscle volumes can be sampled instead of a small number of muscle fibers or muscles. This is particular important due to monitoring of gene transfer efficacy and expression in clinical settings currently requires invasive techniques. This can be problematic in subjects with extensive muscle damage and necrosis such as children with Duchene muscular dystrophy and LGMD.

Augmenting angiogenesis and perfusion in skeletal muscle. We are using MRI and MRS methods to measure the effectiveness of gene mediated collateral formation as a potential therapy for chronically ischemic muscle. We have found that sufficient signal-to-noise and spatial resolution is achievable at field strengths greater than 4T to examine heterogeneity in muscle perfusion. This work has been extended to study whether the lack of NOS in dystrophic muscle is associated with poor muscle perfusion and exercise ischemia in the murine and human muscular dystrophies. Animal models are used to test different molecule weight contrast agents as to the appropriateness for blood perfusion measures as confirmed by ex vivo microsphere methods.

Monitoring of transgene delivery and cell migration for muscle bioengineering

¹H-NMR detection and MR imaging of gene expression in skeletal muscle: An adenovirus coding for human lactoferrin driven by a truncated muscle specific creatine kinase promoter (rAdMCKLF) was constructed to serve as a MRI/MRS marker gene. Lactoferrin is a 80 kDa single chain metal binding glycoprotein and is a member of the transferrin family. Lactoferrin strongly binds two iron ions concomitantly with two carbonate or bicarbonate ions. We are currently developing methods to transiently express rAdMCKLF in adult skeletal muscle and further investigate its use for the microimaging of gene expression in cardiac and skeletal muscle.

MRI of magnetically tagged stem cells: The highest level of sensitivity can be achieved using magnetite as a contrast agent (~5µm). We are investigating whether magnetodendrimers can be used to successfully tag and noninvasively follow stem cell therapy for skeletal and cardiomyopathies. The most immediate targets for stem cell intervention are the muscular dystrophies. Both direct intramuscular and intravenous injection of normal stem cells can lead to re-expression of dystrophin in dystrophic muscle. We are extending these studies to determine whether a new class of contrast agents, magnetodendrimers, can be used to label C₂C₁₂ cells (immortalized myoblast cell line) and to follow their migration and final incorporation into dystrophic muscle fibers. Due to the extremely high relaxativity of magnetodendrimers it is possible to follow the dynamics of single labeled cells in vivo.