Research supported by this grant during the previous twenty-four years has been built around extensive data showing that cardiac structure, composition, and function each respond rapidly and reversibly to changes in hemodynamic load. The first set of studies supported by this grant used isolated cells, or cardiocytes, and intact animals to demonstrate the role of load as a central regulator of cardiocyte growth. The second set of studies supported by this grant, which also used load change as the primary experimental variable, led to our discovery of a dense cardiocyte microtubule network during severe pressure-overload cardiac hypertrophy that contrib- utes to the contractile dysfunction which occurs in this setting. The initial goals for the subsequent studies of this abnormal microtubule network were to determine how it contributes to the contractile dysfunction of hypertrophied myocardium. Major findings have been that 1) it is based both on increased tubulin, and thus microtubules, and on greater microtubule stability, 2) the major car- diac microtubule-stabilizing microtubule-associated protein, MAP4, is greatly upregulated in pressure overload hypertrophy and binds extensively to microtubules, and 3) contractile dysfunction is caused by viscous loading imposed on shortening myofilaments by the dense microtubule network. However, the most important normal role of the microtubules in an interphase cell such as the cardiocyte is not to determine cellular rheological properties but rather to subserve intracellular transport of macromolecules and vesicles via the microtubule-associated kinesin and dynein families of motor proteins. Indeed, this is an absolutely essential role in the extremely diffusion-restricted cytoplasm of the adult cardiocyte. For this reason, and because of the known inhibition of microtubule-dependent intracellular transport by excessive decoration of microtubules with MAPs, we next asked if microtubule-based transport of the activated 2-adrenergic receptor and/or mRNA - ribonucleoprotein complexes was inhibited by MAP4 binding to microtubules in pressure- overload hypertrophy. Such, in fact. was the case. Building on this most recent work, we propose to examine here the potential role of alterations in microtubule network organization and MAP4 binding in causing abnormal transport and localization of connexin43 [Cx43], a gap junction protein known to undergo functionally important alterations in quantity and localization during pathological cardiac hypertrophy. The basic research in the first objective will use isolated cells as well as oper- ated and transgenic mice to determine whether MAP4 decoration of microtubules, and the attendant densifica- tion of the microtubule network, inhibit the normal transport of Cx43 to gap junctions as well as Cx43-depen- dent electrophysiological function. The translational research in the second and third objectives will compare an equal degree & duration of pathological pressure vs. physiological volume overload hypertrophy. We will first extend the findings of the first objective to ask if MAP4 decoration of the dense microtubule network in pathological hypertrophy has a role in the altered Cx43 transport and localization that are important clinically in forming an arrhythmogenic substrate. We will then ask if 2-receptor blockade in pathological hypertrophy, which early data indicates will prevent the abnormal microtubule phenotype, will also prevent the abnormal Cx43 phenotype in this setting. In the first objective we will use murine models, and in the second and third objectives we will use our long- standing feline models of physiological versus pathological hypertrophy. While we recognize that it is prefer- able to use a single species, in this research the initial mechanistic portion can only be done in the mouse, but the later quantitative translational portions require very reproducible animal models that can be reliably and verifiably 2-blocked and have an equivalent degree and duration of physiological vs. pathological hypertrophy, with ex- tensively characterized cytoskeletal properties in each setting.
Pathological cardiac hypertrophy is one of the two most common serious cardiac abnormalities which we encounter in the care of our patients in VA Medical Centers. Our attempts to deal in a definitive as opposed to a palliative way with this problem must be based on a mechanistic understanding of the causes of this entity. Preliminary data for this grant application suggest that specific cytoskeletal changes accompanying severe pres- sure overload cardiac hypertrophy may not only be responsible for contractile dysfunction but may also be responsible, at least in part, for altered transport within the cardiac muscle cell that can lead to the rhythm disturbances that characterize heart failure. Since specific endogenous phosphatases and kinases regulate the molecular events that cause these cytoskeletal changes, this work has the potential to lead to very important therapeutic interventions.
