Introduction and objective

The absence of insulin and insulin like growth factors (IGF) has been reported to play an important role in impaired contractility seen in DM. Insulin and IGF is required by several cell-types including connective tissue fibroblasts and also the smooth and striated myocytes.  In addition to stimulating growth, insulin and it’s congeners also promote the production of secondary growth factors as fibroblast growth factor (FGF) and connective tissue growth factor (CTGF) peptides.  DM disrupts various growth factors signaling pathways mediated by PKC isoforms. The objective of our study was to interrogate the structural and molecular changes occurring in pelvic floor tissue seen in DM.

Methods

Samples of the levator ani striated muscle and fibroconnective tissue of the control (WT) and transgenic rat model of DM were cryosectioned and digitally imaged with an epifluoresence microscope (Leica).  PKC isoforms Cy-3 (a, b, e, d, h, x) were examined by indirect immunofluoresence against C- terminal antigens. Sections were simultaneously doubly or triply stained to depict surface glycoproteins (Oregon green labeled wheat germ agglutinin), or actin (Texas red phalloidin) and nuclear DNA (bisbenzamide).

Results

The connective tissue sheath covering striated pelvic muscle (epimysium), individual cells, and fiber bundles (endomysium and perimysium, respectively) is severely diminished in DM. Connective tissue covered fascicles that enter the muscle, carrying nerves and vessels are also decreased in number and size in DM.  This is apparent from i. the decreased staining of sialylated and glucuronylated proteins detected by the lectin and wheat germ agglutinin, ii. the thinned basophilic areas in conventional H&E stain, iii. By the attenuated autofluorescence of elastin, and iv. Decreased cell number indicated by nuclear stain.  In muscle from diabetic animals the endomysial connective tissue between cells also appears particularly susceptible to rupture and premature tear. In addition, changes in expression and distribution of PKC isoforms, or mitochondrial Hsp75 suggest phenotypic remodeling of striated muscle, vascular and connective tissues in DM.   The epimysium in diseased muscle is reduced to 15-45 microns compared to 65-150 microns in controls. Similarly the endomysium between muscle cells is reduced from 4.8+0.3 to 2.7+0.4 microns in diseased muscle.

Conclusions

These findings suggest that DM induce fundamental changes in the intracellular structure and signaling pathways of the pelvic floor tissues. These molecular changes may explain the pathological morphology and contractile dysfunction seen in lower urinary tract and pelvic floor tissues in DM.