SECOND HARMONIC GENERATION IMAGING (SHG) DETECTS AUTOPHAGIC LESIONS IN MUSCLE FIBERS FROM THE POMPE DISEASE MOUSE MODEL AND FROM HUMAN SUBJECTS

SHG's sensitivity to the orientation and alignment of myofibrils in unstained muscle fibers (1) give this imaging mode a tremendous potential in the detection of muscle pathologies. We study Pompe Disease, a lysosomal storage disorder resulting from a deficiency in acid-α-glucosidase (GAA, also called acid maltase). Glycogen accumulates inside lysosomes of various tissues but, clinically, the most severely affected are cardiac and skeletal muscles. Immunofluorescence of single muscle fibers of GAA knockout (KO) mice has recently shown a fiber type-dependent accumulation of autophagic debris in large discrete regions of the myofibrillar core (2). However, staining of single fibers is time-consuming and the in situ relative positioning of the fibers is lost. We reasoned that in SHG imaging the autophagic regions should appear as holes, while autofluorescence of these regions may be increased. Bundles of fibers, 200-400 mm thick, were cut from fixed unstained hindlimb muscles from GAA-KO mice and mounted in 90% glycerol between a coverslip and a glass slide separated by a silicone gasket. SHG was excited at 820 nm on a 2-photon Leica SP2 system using a 63x N.A. 1.4 oil immersion objective and collected in the forward direction using a transmitted light detector. A 405/ 20 nm bandpass filter was inserted in the emission path to exclude fluorescence. Autofluorescence was recorded simultaneously in back-scattered mode. Fibers from fast muscles of GAA-KO mice had the striated SHG pattern found in control animals, but the SHG image was interrupted by large holes up to several hundred ∝m in length and 5-15 ∝m in diameter, occasionally split. These holes contained autofluorescent structures of up to 5 ∝m in diameter. Fibers of slow muscles presented a very different distribution of autofluorescence and no large holes in their core. All GAA-KO fibers also showed smaller lesions likely to represent enlarged lysosomes. Preliminary examination of biopsies from human Pompe patients showed a mosaic pattern: nearly normal fibers next to fibers with SHG holes, with or without increased autofluorescence. The degree of damage varied between subjects. We conclude that SHG, alone and in combination with autofluorescence, will be helpful in monitoring and understanding the progression of Pompe Disease in mice and in human subjects.