During development, during hypertrophic growth and in response to injury, the heart undergoes profound remodeling at both the tissue and cellular level. Defects in the assembly and integration of new myofibrils can lead to myofibril disarray, myocyte death, and congestive heart failure. Obscurin is a novel giant sarcomeric protein links the sarcomere to the sarcoplasmic membrane through interactions with titin and ankyrin. As such, it may have vital roles in myofibril assembly and in proper positioning of ankyrin-associated ion channels relative to the contractile apparatus.
The specific aims of this study will therefore be to 1) examine the effects of antisense morpholino-mediated obscurin inhibition on in vivo myofibril assembly and cardiac and skeletal muscle development in zebrafish embryos using immunohistochemical analysis and videographic assessment of cardiac function 2) characterize physical interactions between obscurin and other sarcomeric proteins using yeast two hybrid and in vitro binding studies, and determine their physiologic significance through competitve inhibition of the interaction in beating cardiac myocytes in culture 3) determine if the ankyrin-obscurin interaction is disrupted by pathologic processes such as chronic arrhythmia (using a canine model of atrial fibrillation) or hypoxia (in cultured cardiac myocytes) and examine the potential role of ankyrin and/or obscurin phosphorylation in regulating the interaction 4) using conditional gene targeting to inhibit the obscurin Rho guanine nucleotide exchange factor (RhoGEF) signaling in a tissue- and stage-specific manner in mice, determine its specific contributions to myofibril assembly during adaptive cardiac hypertrophy (secondary to aortic constriction) and skeletal muscle repair. Characterizing obscurin's roles in new myofibril assembly and in sarcomere-sarcoplasma organization may identify new strategies to treat congestive heart failure and prevent myofibril damage during ischemia.
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