ABSTRACT
Three-dimensional (3-D) tissue imaging offers substantial benefits to a wide range of biomedical investigations from cardiovascular biology, diabetes, Alzheimer's disease to cancer. Two-photon tissue cytometry is a novel technique based on high-speed multiphoton microscopy coupled with automated histological sectioning, which can quantify tissue morphology and physiology throughout entire organs with subcellular resolution. Furthermore, two-photon tissue cytometry offers all the benefits of fluorescence-based approaches including high specificity and sensitivity and appropriateness for molecular imaging of gene and protein expression. We use two-photon tissue cytometry to image an entire mouse heart at subcellular resolution to quantify the 3-D morphology of cardiac microvasculature and myocyte morphology spanning almost five orders of magnitude in length scales.
Subject(s)
Image Cytometry/instrumentation , Image Enhancement/instrumentation , Image Interpretation, Computer-Assisted/methods , Imaging, Three-Dimensional/instrumentation , Microscopy, Fluorescence, Multiphoton/instrumentation , Myocardium/cytology , Myocytes, Cardiac/ultrastructure , Animals , Equipment Design , Equipment Failure Analysis , Image Cytometry/methods , Image Enhancement/methods , Image Interpretation, Computer-Assisted/instrumentation , Imaging, Three-Dimensional/methods , Mice , Microscopy, Fluorescence, Multiphoton/methods , Reproducibility of Results , Sensitivity and Specificity , Tissue Culture Techniques/instrumentation , Tissue Culture Techniques/methodsABSTRACT
BACKGROUND: Although cellular redox balance plays an important role in mechanically induced cardiac hypertrophy, the mechanisms of regulation are incompletely defined. Because thioredoxin is a major intracellular antioxidant and can also regulate redox-dependent transcription, we explored the role of thioredoxin activity in mechanically overloaded cardiomyocytes in vitro and in vivo. METHODS AND RESULTS: Overexpression of thioredoxin induced protein synthesis in cardiomyocytes (127+/-5% of controls, P<0.01). Overexpression of thioredoxin-interacting protein (Txnip), an endogenous thioredoxin inhibitor, reduced protein synthesis in response to mechanical strain (89+/-5% reduction, P<0.01), phenylephrine (80+/-3% reduction, P<0.01), or angiotensin II (80+/-4% reduction, P<0.01). In vivo, myocardial thioredoxin activity increased 3.5-fold compared with sham controls after transverse aortic constriction (P<0.01). Aortic constriction did not change thioredoxin expression but reduced Txnip expression by 40% (P<0.05). Gene transfer studies showed that cells that overexpress Txnip develop less hypertrophy after aortic constriction than control cells in the same animals (28.1+/-5.2% reduction versus noninfected cells, P<0.01). CONCLUSIONS: Thus, even though thioredoxin is an antioxidant, activation of thioredoxin participates in the development of pressure-overload cardiac hypertrophy, demonstrating the dual function of thioredoxin as both an antioxidant and a signaling protein. These results also support the emerging concept that the thioredoxin inhibitor Txnip is a critical regulator of biomechanical signaling.
