Development of a Model System for Culturing Neonatal Rat Ventricular Myocytes in vitro to Monitor Integrin Activation in Response to Altered Contractile Force
Hypertrophic cardiomyopathy (HCM) affects 1 in 500 individuals and remains the leading cause of sudden cardiac death (SCD) in young adults. Recent evidence links mutations within sarcomeric proteins to the HCM disease state. The interplay between the forces generated by the contractile apparatus and sensed by integrins is central to understanding the pathogenesis of HCM. Integrins are likely the mechanotransducers in cardiomyocytes, converting mechanical stress into chemical signals that stimulate cardiac hypertrophy. Outside-in integrin signaling has been extensively studied, while inside-out signaling in response to changes in contractile force remains undefined. An in vitro culture of cardiomyocytes exhibits disorganization of myofibril structure and intercalated disc formation, making it difficult to determine the physiological relevance. Therefore, we have developed an in vitro tissue model to mimic in vivo physiology. Specifically, we have developed a flexible micropatterned scaffold to better mirror native ECM and allow more meaningful correlation of findings to living systems. The micropatterned laminin matrix allows organized growth and differentiation of neonatal rat ventricular myocytes (NRVMs) into adult phenotype. We have constructed an adenovirus expressing a talin-GFP fusion protein to monitor integrin activation in response to changes in contractile force. We have confirmed talin-GFP colocalization with integrins at cell-ECM contacts using confocal microscopy. Utilizing total internal fluorescence (TIRF) microscopy we have completed preliminary measurements of intensity changes overtime at focal adhesions. Utilizing ImageJ technology, we aim to determine how changes in acto-myosin force affect integrin activation by measuring the changes in focal adhesion intensity, as observed through GFP localization. We hypothesize that mutations and inhibition of sarcomere contractile elements will cause perturbations in traction forces and alter inside-out integrin signaling, initiating a pathological hypertrophic phenotype.
Contractile Force, Integrin Activation, In Vitro Culture of NRVMs
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