There is increasing support for the idea that excessive production of reactive oxygen species (ROS) contributes to the pathogenesis of diabetes. In particular, a strong correlation has been made between increased ROS, activation of specific protein kinase C (PKC) isoforms, and many functional consequences of diabetes. This correlation has been strengthened by recent transgenic experiments in the heart where overexpression of the PKCB2 isoform but not PKCB, decreased cardiac function. Because PKCB2 activity is increased in hearts from diabetic animals, this transgenic model represents a new approach to understand some of the pathogenic mechanisms of diabetic cardiomyopathy. We have developed a novel hypothesis based on data from our laboratory that p90 ribosomal S6 kinase (p90RSK) is a physiological substrate of PKCB2, and that Troponin I (TnI) phosphorylation by p90RSK contributes to decreased cardiac function in diabetes. Data in support of our hypothesis include. 1) TnI phosphorylation inhibits Ca2+-stimulated myosin MgATPase activity, indicating an important role for TnI phosphorylation in contractile function. 2) Phosphorylation of TnI is increased in PKCB2, but not in PKCe transgenic mice. 3) p90RSK activity is increased in myocardial samples from patients with end-stage heart failure. 4) p90RSK activity is increased in myocardial samples from PKCB2, but not PKCe transgenic mice. 5) p90RSK phosphorylates TnI with -ImM substrate affinity. To prove that PKCB2-dependent p90RSK mediated phosphorylation of TnI causes to cardiac contractile dysfunction we propose two aims:
Aim 1 : Define the role of p90RSK in TnI phosphorylation and regulation of Ca2+-stimulated myosin MgATPase activity;
Aim 2 : Determine the role of p90RSK and TnI phosphorylation in cardiac contractile function in vivo.
In Aim 1, we will characterize the sites in TnI phosphorylated by p90RSK using two-dimensional tryptic phosphopeptide mapping, and show that p90RSK phosphorylation of TnI inhibits calcium-dependent myosin MgATPase activity. Next we will show that mutating the TnI residues phosphorylated by p90RSK prevents alterations in MgATPase activity.
In Aim 2, we will generate p90RSK wild-type overexpressing transgenic mice which we expect will exhibit cardiac dysfunction. Then, based on TnI mutation results in Aim 1, we will make a double transgenic that we anticipate will reverse the cardiac dysfunction associated with p90RSK. These results of these experiments will provide a molecular mechanism for the cardiac dysfunction associated with increased PKC activity, based on phosphorylation of TnI by p90RSK.
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