The goal of this K08 application is to provide the candidate with the skills needed to become an independent translational researcher in the field of ciliary muscle physiology and juvenile myopia. The applicant will gain an understanding of how to induce myopia in the guinea pig, and he will receive advanced training in microscopy and genetic analysis in order to characterize smooth muscle growth and development. The applicant will also learn tissue culture immortalization and cell stretching techniques. The full mechanism of emmetropization, eye growth that produces a clear image on the retina, is unknown. Visually guided eye growth using foveal defocus cues has been the main paradigm, but treatments based on this hypothesis have resulted in little clinical benefit. Primate and human studies suggest that peripheral hyperopic defocus from a relatively prolate ocular shape may be a stronger stimulant to ocular growth than foveal defocus. An alternative hypothesis proposed by this laboratory is that the enlarged eye at risk for myopia places mechanical stretch on the ciliary muscle, which triggers specific biochemical pathways (mechanotransduction) leading to hypertrophy, altered ocular shape, and accelerated axial elongation. Increased expression of ?-smooth muscle actin (?-SMA) along with myosin light chain kinase (MLCK) and increased cell size are known markers of hypertrophy. Experiments in Aim 1 will determine alterations in in vitro ciliary muscle cell structure, gene expression, and cell modulus after mechanical stretch of primary guinea pig and human ciliary muscle cells. Human immortal cells will then be developed and stretched, and results will be compared to the primary cells. Experiments in Aim 2 will determine the ex vivo consequences of experimentally induced myopia and mechanical stretch on guinea pig ciliary muscle. We will induce minus spectacle lens myopia monocularly, and we will biaxially stretch segments of ciliary muscle;results from both experiments will be compared to untreated ciliary muscle from the fellow eye.
In Aims 1 and 2, we will measure cytoskeletal architecture and cell volume with Multi-Photon Confocal, Scanning Electron, and Transmission Electron Microscopy. Using Atomic Force Microscopy, we will evaluate mechanical changes in cells (modulus). We will measure alterations in ?-SMA and MLCK expression with ELISA. ?-SMA and MLCK expression will be confirmed with RNA-Seq. RNA-Seq will also indicate if other pathways are altered in response to ocular growth and/or ciliary muscle stretch. We chose the guinea pig model because the guinea pig can accommodate, and because spectacle lenses can induce a significant amount of myopia. To achieve these Aims, the applicant has recruited a strong, multidisciplinary mentoring team, which comprises a primary mentor, Dr. Donald Mutti, who conducts human juvenile myopia research, and three co-mentors: Dr. Kirk McHugh, a cell and molecular biologist who is an expert in the study of smooth muscle in bladder and obstructive (stretch) nephropathy in the mouse;Dr. Andy Fischer, a neuroscientist who is also an expert in chicken myopia development and molecular biology, and Dr. Sudha Agarwal, an immunologist who is an expert in exercise mechanotransduction. This K08-supported research will form the basis for a future R01 grant application for independent research aimed at creating targeted interventions for treating and preventing ciliary muscle related myopic growth.
Improved understanding of how mechanical forces alter ciliary smooth muscle will lead to more effective treatments and management of ciliary muscle related conditions such as myopia, accommodative dysfunction, presbyopia, and glaucoma.
|Pucker, Andrew D; Jackson, Ashley R; Morris, Hugh J et al. (2015) Ciliary Muscle Cell Changes During Guinea Pig Development. Invest Ophthalmol Vis Sci 56:7691-6|