Noninvasive measurement of gene expression in skeletal muscle

Abstract

We have developed a noninvasive detection method for expression of viral-mediated gene transfer. A recombinant adenovirus was constructed by using the gene for arginine kinase (AK), which is the invertebrate correlate to the vertebrate ATP-buffering enzyme, creatine kinase. Gene expression was noninvasively monitored using (31)P-magnetic resonance spectroscopy ((31)P-MRS). The product of the AK enzyme, phosphoarginine (PArg), served as an MRS-visible reporter of AK expression. The recombinant adenovirus coding for arginine kinase (rAdCMVAK) was injected into the right hindlimbs of neonatal mice. Two weeks after injection of rAdCMVAK, a unique (31)P-MRS resonance was observed. It was observable in all rAdCMVAK injected hindlimbs and was not present in the contralateral control or the vehicle injected limb. PArg and phosphocreatine (PCr) concentrations were calculated to be 11.6 +/- 0.90 and 13.6 +/- 1.1 mM respectively in rAdCMVAK injected limbs. AK activity was demonstrated in vivo by monitoring the decreases in PArg and ATP resonances during prolonged ischemia. After 1 h of ischemia intracellular pH was 6.73 +/- 0.06, PCr/ATP was decreased by 77 +/- 8%, whereas PArg/ATP was decreased by 50 +/- 15% of basal levels. PArg and PCr returned to basal levels within 5 min of the restoration of blood flow. AK activity persisted for at least 8 mo after injection, indicating that adenoviral-mediated gene transfer can produce stable expression for long periods of time. Therefore, the cDNA encoding AK provides a useful reporter gene that allows noninvasive and repeated monitoring of gene expression after viral mediated gene transfer to muscle.

Figure 1

(A) Schematic diagram for rAdCMVAK gene construct. Expression was driven by nonspecific CMV promoter and was stabilized by an simian virus 40 polyadenylation sequence (SV40pA). From this construct, a ΔE1–ΔE3 adenovirus was prepared by using published methods (17). (B) RT-PCR was used to detect the presence of AK transcripts in rAdCMVAK-injected muscles. Total RNA isolated from frozen tissue was subjected to RT-PCR by using oligonucleotides specific for Drosophila AK and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). AK transcripts were detected in rAdCMVAK-injected muscles (+AdAK) but not in the contralateral control (No Inj). AK and GAPDH primers served as positive controls for the procedure (Pos Ctrl).

Figure 2

³¹P-MRS spectra of a model solution containing expected physiological concentrations of ATP, P_i, PCr, and PArg. The model solution consisted of 30 mM PCr, 10 mM Pi, 30 mM PArg, and 8 mM ATP in 0.4 ml of distilled water. In solution, PArg was well resolved from PCr and γATP with a chemical shift of 0.47 ppm relative to PCr. The model solution was used to determine the appropriate spectral analysis package for the analysis of both phantom and mouse skeletal muscle data with the minimal amount of user bias. The solid line is the best fit to the free induction decay using a Hankel single value decomposition algorithm (18), which avoided peak selection and the use of previous knowledge.

Figure 3

In vivo basal ³¹P spectra from the hindlimbs of a 6-mo-old mouse. ³¹P-MRS spectra from the rAdCMVAK-injected limb (Upper spectrum) reveal a ³¹P resonance at the chemical shift for PArg that is not present in the contralateral control limb (Lower spectrum).

Figure 4

Stack plot of changes in high energy phosphates during prolonged ischemia in the rAdCMVAK-injected limb. During circulatory occlusion, PCr levels are depleted and inorganic Pi levels rise within 15 min of ischemia followed by the depletion of PArg.