Cardiac structure, composition, and function each respond rapidly and reversibly to increases and decreases in hemodynamic loads. Our central hypothesis is that changing load itself is the primary variable responsible for regulating these properties of the cardiac muscle cell, or cardiocyte. This proposal will utilize a unique experimental preparation, the terminally differentiated, quiescent adult feline cardiocyte maintained in long-term cell culture, to evaluate the role of loading conditions as the central regulator of cardiocyte growth. There are two specific aims of this proposal: first, to establish the relative importance of load regulation versus adrenoceptor activation both in maintaining differentiated cellular properties and in initiating growth of isolated adult cardiocytes; second, to identify any new proteins which are unique to early phases of cardiac hypertrophy and thus may initiate growth, either as a depressor or as an inductor, in stimulating expression of structural protein genes. The first specific aim will be accomplished by maintaining quiescent adult feline cardiocytes in cell culture under one of three mechanical conditions: (1) """"""""normally loaded"""""""", where the cells will be attached at their diastolic rest length to a substrate to which they are adherent, (2) """"""""unloaded"""""""", where the cells are maintained in suspension culture and do not adhere to a substrate, and (3) """"""""overloaded"""""""", where substrate adherent cardiocytes are stretched along with the substrate to a defined length greater than their diastolic rest length. The load responses of these cardiocytes will be assessed by the following markers of cardiocyte differentiation and growth: (1) ultrastructural organization characteristic of normal or growing as opposed to atrophic or fetal cardiocytes, (2) cellular function, characterized by the mechanical response to electrical stimulation, (3) cardiocyte protein content, especially for the myofibrillar proteins, (4) non-specific RNA and protein synthesis and synthesis of sarcomere-specific proteins, and (5) degradation rates of cardiocyte proteins, with the emphasis again on the myofibrillar proteins. Adherent cells cultured at rest length and exposed to adrenergic agonists will be evaluated for growth in the same manner. The second specific aim will be accomplished by using the technique of subractive hybridization to compare mRNAs from normal and hypertrophying myocardium. Any probes which appear to be unique to hypertrophy will be validated in """"""""overloaded"""""""" cardiocytes by the use of Northern blots for quantification and in situ hybridization for localization. The long-term objective of this research is to define the mechanisms by which cardiocyte load changes are linked to cellular growth.
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