Brandon J. Biesiadecki, Ph.D. - US grants

DESCRIPTION (provided by applicant): The experiments proposed in this study investigate the hypothesis that tropomyosin phosphorylation (Tm-P) in the heart is altered by cardiac stress and this charge modification of Tm alters it's interactions within the thin filament to affect contraction. To date the modification of Tm resulting from cardiac stress and its effect on contraction are unknown. We propose three aims: I) To evaluate Tm-P in the stressed heart. Tm-P will be characterized in isolated perfused hearts treated with ischemia/reperfusion by phosphoprotein specific staining of 2-D gels and mass spectrometry. II) To determine the effect of Tm-P on binding to actin, troponin and to actin in the presence of troponin. Tm binding will be carried out by co-sedimentation and quantified on 2-D gels with densitometry. Ill) To determine the effect of Tm-P on the development of force in cardiac muscle. Calcium activated tension will be measured in skinned papillary bundles treated with gelsolin to selectively remove the thin filament and reconstituted with actin, troponin and non-phosphorylated or phosphorylated Tm. The knowledge gained from these experiments will provide novel insight into the regulatory mechanism of Tm-P and its effect on cardiac muscle contraction in health and disease.

DESCRIPTION (provided by applicant): Tropomyosin (Tm) is modified by both posphorylation and nitration post-translational modifications. To date the functional significance of Tm phosphorylation and nitration are not well understood. It is the goal of this proposal to identify the effect of Tm phosphorylation and nitration on its interactions with the sarcomere proteins and on activation of the sarcomere thin filament. Recent data has suggested the post-translational modification of Tm may be regulated, therefore we further propose to investigate the effect of cardiac stress on the level of Tm post-translational modifications as a novel signaling mechanism to alter cardiac sarcomeric contraction. This application contains three specific aims to investigate these questions. 1) Does cardiac stress alter the post-tranlational modification of Tm to affect contractile function? 2) Does the posttranslational modification of Tm by phsophorylation or nitration alter its interactions within the thin filament protein network? 3) Do post-translational modifications of Tm alter Ca2+ activation of the thin filament? These aims will be carried out by investigating the effect of Tm that has been modified by either phosphorylation or nitration on Tm binding affinity to the other sarcomeric thin filament proteins, the binding of Ca2+ to reconstituted thin filaments and the ATPase activity of reconstituted thin filaments. We also propose to investigate the effect of cardiac stress on Tm post-translational modifications by treating cardiac myocytes with ischemic reperfusion followed by identification of Tm phosphorylation and nitration levels. These studies will provide a much needed molecular understanding of how the post-translaitonal modification of Tm functions to affect cardiac contraction at the sarcomeric level and its role as a novel signaling mechanism to affect cardiac contraction in ischemic reperfusion. The findings from these studies are directly relevant to understanding the molecular basis of cardiac dysfunction in human myocardial infarction. These studies will also provide new insight into potential pharmacotheriputic treatments to improve heart function in both myocardial infarction and disease.

Tropomyosin (Tm) is modified by both posphorylation and nitration post-translational modifications. To date tiie functional significance of Tm pfiospliorylation and nitration are not well understood. It is the goal of this proposal to identify the effect of Tm phosphorylation and nitration on its interactions with the sarcomeric proteins and on activation of the sarcomeric thin filament. Recent data has suggested the post-transiational modification of Tm may be regulated, therefore we further propose to investigate the effect of cardiac stress on the level of Tm post-translational modifications as a novel signaling mechanism to alter cardiac sarcomeric contraction. This application contains three specific aims to investigate these questions. 1) Does cardiac stress alter the post-tranlational modification of Tm to affect contractile function? 2) Does the posttranslational modification of Tm by phsophorylation or nitration alter its interactions within the thin filament protein network? 3) Do post-transiational modifications of Tm alter Ca2+ activation of the thin filament? These aims wiil be carried out by investigating the effect of Tm that has been modified by either phosphorylation or nitration on Tm binding affinity to the other sarcomeric thin filament proteins, the binding of Ca2+ and the ATPase activity of reconstituted thin filaments. We also propose to investigate the effect of cardiac stress on Tm post-translational modifications by treating cardiac myocytes with ischemic reperfusion followed by identification of Tm phosphorylation and nitration levels. We will investigate the significance of Tm phosphorylation by investigating calcium regulated force development in cardiac fibers from transgenic mice overexpressing pseudo-phosphorylated Tm. These studies will provide a much needed molecular understanding of how the post-translaitonal modification of Tm functions to affect cardiac contraction at the sarcomeric level and its role as a novel signaling mechanism to affect cardiac contraction in ischemic reperfusion. The findings from these studies are direclty relevant to understanding the molecular basis of cardiac dysfunction in human myocardial infarction. These studies wiil also provide new insight into potential pharmacotheriputic treatments to improve heart function in both myocardial infarction and disease.

DESCRIPTION (provided by applicant): Heart disease resulting from cardiac contractile dysfunction is the number one killer of people in the US today. The proteins that regulate cardiac contractile are under the tight post-translational modification (PTM) regulation of kinase phosphorylation and phosphatase de-phosphorylation. Central to the regulation of cardiac muscle contraction is the troponin (Tn) protein complex. In response to cardiac stress the Tn complex is subjected to a number of PTMs. Phosphorylation represents the primary mechanism of muscle PTM and it's tight balance is critical to normal heart function. Other non-phosphorylation Tn PTMs also occur. To date most Tn protein PTMs have been studied as isolated events, however the combined function of multiple Tn PTMs cannot be determined from function of the isolated events. This proposal investigates the primary troponin contractile regulation phosphorylation and other novel PTM events as combined events to better understand the physiological regulatory function of Tn PTMs as they occur in the heart. To this end, we will employ model troponin proteins and transgenic animal models containing single or multiple PTMs to evaluate biochemical and animal level function. The goal of these studies is to develop a more physiological understanding of the events that regulate heart function in order to identify novel drug targets to treat cardiac dysfunction.