The crucial role of protein kinase G in regulating Ca2(+)/calmodulin-dependent protein-kinase II activity and oxidative stress: disastolic function modulation via PKG-dependent CaMKII-phosphorylation and oxidation

• Applicant: Professorin Nazha Hamdani, Ph.D.
• Subject Area: Cardiology, Angiology ; Anatomy and Physiology ; Pharmacology
• Term: since 2016
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 286627390

The development of effective treatments for heart failure with preserved ejection fraction (HFpEF) is currently limited by poor understanding of the underlying pathophysiology. However, it is clear that co-morbid conditions drive myocardial remodeling in HFpEF, promoting a systemic inflammatory state that contributes to endothelial dysfunction, oxidative stress and modulation of diastolic function. Our recent results allowed us to formulate a comprehensive hypothesis describing how inflammation and oxidative stress affect myocardial dysfunction in heart failure (HF). The core of this hypothesis states that Ca2(+)/calmodulin-dependent protein kinase-II activity, mediated by protein kinase-G (PKG) phosphorylation and cyclic guanosine monophosphate (cGMP), largely determines cardiomyocyte stiffness and diastolic function. This central role for PKG in CaMKII regulation is a wholly novel paradigm in HF research and may be key to developing effective treatments for currently untreatable forms of HF. We will show that this finding is closely allied to another important aspect of cardiac function, the control and maintenance of cardiac stiffness. A crucial contributor to diastolic stiffness is the giant myofilament protein titin, and isoform shifts, and posttranslational modifications of cardiac titin are known to be key modulators of cardiomyocyte stiffness. We have shown that phosphorylation of titin by PKG lowers titin-based stiffness and that this mechanical signaling event is disturbed in HF, particularly in HFpEF, thus increasing myocardial stiffness.In this proposal we wish to further explore CaMKII phosphorylation by PKG as a critical modulator of titin phosphorylation and stiffness. We plan to detect and quantify phospho-sites along the entire CaMKII molecule in vivo using mass spectrometry. Selected CaMKII phospho-sites will be verified in vitro and by phospho-antibody-staining. Single skinned and intact cardiomyocytes will be used for mechanical measurements to test for alterations in passive stiffness in HFpEF animal models and HFpEF patients. Furthermore, various treatment regimens will be assigned and tested in an HFpEF model, with the goal of increasing the cGMP pool, improving PKG-mediated CaMKII phosphorylation, reducing CaMKII oxidation and thereby cardiomyocyte stiffness. This work should settle several important issues, including i) identifying the key signaling pathways leading to improved stiffness, and ii) which pathways lead to elevated cGMP, thereby improving CaMKII post-translational modification, increasing titin phosphorylation and reducing passive stiffness of cardiomyocytes. These data will provide important insights regarding the therapeutic potential for improving diastolic function.

• DFG Programme: Research Grants