| dc.description.abstracteng | L-type Ca2+ channels (LTCC) are known to be involved in the dysregulation of intracellular Ca2+ handling in heart failure (HF) contributing to contractile dysfunction and arrhythmias. Beside alteration in subcellular localization, reduced LTCC function may be a consequence of altered posttranslational modification. It is known that cAMP-dependent protein kinase A (PKA) can phosphorylate LTCC resulting in increased Ca2+ current (ICa). PKA-dependent LTCC phosphorylation has been shown to occur upon acute pressure overload partly compensating for reduced Ca2+ release from sarcoplasmic reticulum (SR). However, the mechanisms of PKA-dependent LTCC activation upon pressure overload are inadequately understood. Recent data suggest that PKA can be activated upon direct oxidation of its regulatory subunit (RI) without binding to its physiologic agonist, cAMP. In this thesis, I have investigated the pathophysiological relevance of this oxidative PKA activation for intracellular Ca2+ homeostasis, contractile function, and arrhythmogenesis upon pressure overload. Here, a novel PKA redox-dead knock-in mouse line was used harboring a RI point mutation that results in the exchange of cysteine 17 with serine (KI). Oxidation of cysteine 17 with consequent intersubunit disulfide bridge, RI-RI dimer, formation is inhibited. The later is a prerequisite for catalytic subunit release and phosphorylation of target proteins, i.e. oxidative activation of the kinase. At baseline, no alterations in Ca2+ handling (FURA-2-loaded isolated cardiomyocytes) or contractile function (echocardiography) are observed in these mice. However, NAPDH oxidase 2 (NOX2)-dependent oxidants stimulated by angiotensin II (AngII, 1µmol/L) could not result in RI dimer (Western blotting) formation in KI. In accordance, ICa and Ca2+ transient amplitude were significantly smaller in KI mice in the presence of AngII. Similarly, pressure overload by transverse aortic constriction did not result in RI dimer formation in KI. Moreover, compared to WT mice KI mice displayed significantly reduced ICa and Ca2+ transient amplitude with a significantly impaired ejection fraction (echocardiography) upon pressure overload. Western blot analyses revealed that AngII and pressure overload-dependent LTCC phosphorylation at the PKA site were inhibited. Beside disturbed contractile function, KI mice displayed severe QTc prolongation and increased the propensity for ventricular arrhythmias (ECG and programmed ventricular stimulation), which results in significantly reduced survival rates upon pressure overload. In summary, oxidative activation of PKA appears to be important for adaptation of the heart during increased afterload. Thus, stimulation of oxidative PKA activation could be a potential therapeutic option for patients with cardiac diseases, like heart failure. | de |