Post-translational modification of the thin filament leads to progressive pump dysfunction

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ABSTRACT Heart disease remains the leading cause of death in the United States. Patients and animal models of heart failure consistently develop elevated protein kinase C activity and downstream phosphorylation of the myofilament molecular switch protein, cardiac troponin I (cTnI) at Ser43/45 (S43/45). Previously, we showed contractile dysfunction is linked to chronic phosphorylation of this cTnI cluster and it impairs both contraction and relaxation in isolated myocytes. The working hypothesis guiding this application is that cTnI p-S43/45 is a master brake for short-term modulation of steady state function, but chronically it causes cardiac dysfunction, and serves a non-traditional role as a sarcomere stress signal. This type of sarcomere stress communicates with mitochondria to activate reactive oxygen species production and initiate mitochondrial remodeling prior to significant contractile dysfunction. This communication lays the foundation for the progressive downward spiral of cardiac dysfunction and remodeling that leads to heart failure The objectives are to demonstrate cTnIS43/45 acts as a rapid in vivo master brake, causes dose-dependent contractile dysfunction under chronic conditions and also communicates sarcomere stress by causing early mitochondrial ROS production, altered energetics and remodeling. For the approach, transgenic mice with a range of phospho-mimetic cTnIS43/45D or a novel phospho-null cTnIS43/45N were generated to achieve dose-dependent replacement of endogenous cTnI. In Aim 1, in vivo and cellular cardiac structure and contractile function are integrated with analysis of myofilament and Ca2+ signaling to gain insight into the role played by this cluster. This approach will show that dose-dependent cTnIS43/45D replacement produces in vivo cardiac and myocyte dysfunction that leads to later remodeling, progressively impaired function and heart failure. Studies in this aim also will prove that prior the significant remodeling, this cluster acts as a brake on the positive inotropic and lusitropic response to β-adrenergic receptor stimulation, and in vivo replacement with cTnIS43/45N is a functionally conservative substitution. Preliminary studies show oxidative stress and downstream mitochondrial alterations develop in adult cTnIS43/45D mice prior to detection of significant dysfunction. Thus, Aim 2 examines the novel idea that cTnIS43/45D plays a non-canonical role to stimulate downstream mitochondrial reactive oxygen species (ROS) production, followed by altered energetics and remodeling as an early route for triggering progressively impaired cardiac performance. Studies in each aim also include proof-of-concept experiments to show that early targeting of cTnI and/or downstream mitochondria prevents and/or attenuates the downward spiral of dysfunction and remodeling to heart failure.