casein-kinase-ii and Heart-Failure

Recent studies have shown that the sensitivity of apamin-sensitive K(+) current (I KAS, mediated by apamin-sensitive small conductance calcium-activated potassium channels subunits) to intracellular Ca(2+) is increased in heart failure (HF), leading to I KAS upregulation, action potential duration shortening, early after depolarization, and recurrent spontaneous ventricular fibrillation. We hypothesized that casein kinase 2 (CK2) interacted with small conductance calcium-activated potassium channels (SK) is decreased in HF, and protein phosphatase 2A (PP2A) is increased on the opposite, upregulating the sensitivity of I KAS to intracellular Ca(2+) in HF. Rat model of volume-overload HF was established by an abdominal arteriovenous fistula procedure. The expression of SK channels, PP2A and CK2 was detected by Western blot analysis. Interaction and colocalization of CK2 with SK channel were detected by co-immunoprecipitation analysis and double immunofluorescence staining. In HF rat left ventricle, SK3 was increased by 100 % (P < 0.05), and SK2 was not significantly changed. PP2A protein was increased by 94.7 % in HF rats (P < 0.05), whereas the level of CK2 was almost unchanged. We found that CK2 colocalized with SK2 and SK3 in rat left ventricle. With anti-CK2α antibody, SK2 and SK3 were immunoprecipitated, the level of precipitated SK2 decreased by half, whereas precipitated SK3 was almost unchanged. In conclusion, the increased expression of total PP2A and decreased interaction of CK2 with SK2 may underlie enhanced sensitivity of I KAS to intracellular Ca(2+) in volume-overload HF rat.

Topics: Action Potentials; Animals; Apamin; Casein Kinase II; Disease Models, Animal; Echocardiography; Germinal Center Kinases; Heart Failure; Male; Myocytes, Cardiac; Potassium; Protein Binding; Protein Phosphatase 2; Protein Serine-Threonine Kinases; Protein Transport; Rats; Small-Conductance Calcium-Activated Potassium Channels; Up-Regulation

L-type calcium (Ca(2+)) currents conducted by voltage-gated Ca(2+) channel CaV1.2 initiate excitation-contraction coupling in cardiomyocytes. Upon activation of β-adrenergic receptors, phosphorylation of CaV1.2 channels by cAMP-dependent protein kinase (PKA) increases channel activity, thereby allowing more Ca(2+) entry into the cell, which leads to more forceful contraction. In vitro reconstitution studies and in vivo proteomics analysis have revealed that Ser-1700 is a key site of phosphorylation mediating this effect, but the functional role of this amino acid residue in regulation in vivo has remained uncertain. Here we have studied the regulation of calcium current and cell contraction of cardiomyocytes in vitro and cardiac function and homeostasis in vivo in a mouse line expressing the mutation Ser-1700-Ala in the CaV1.2 channel. We found that preventing phosphorylation at this site decreased the basal L-type CaV1.2 current in both neonatal and adult cardiomyocytes. In addition, the incremental increase elicited by isoproterenol was abolished in neonatal cardiomyocytes and was substantially reduced in young adult myocytes. In contrast, cellular contractility was only moderately reduced compared with wild type, suggesting a greater reserve of contractile function and/or recruitment of compensatory mechanisms. Mutant mice develop cardiac hypertrophy by the age of 3-4 mo, and maximal stress-induced exercise tolerance is reduced, indicating impaired physiological regulation in the fight-or-flight response. Our results demonstrate that phosphorylation at Ser-1700 alone is essential to maintain basal Ca(2+) current and regulation by β-adrenergic activation. As a consequence, blocking PKA phosphorylation at this site impairs cardiovascular physiology in vivo, leading to reduced exercise capacity in the fight-or-flight response and development of cardiac hypertrophy.

Topics: Adaptation, Physiological; Adrenergic beta-Agonists; Amino Acid Substitution; Animals; Arrhythmias, Cardiac; Barium; Calcium; Calcium Channels, L-Type; Cardiomyopathy, Hypertrophic; Casein Kinase II; Dihydropyridines; Exercise Tolerance; Heart Failure; Ion Transport; Isoproterenol; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Models, Molecular; Mutation, Missense; Myocardial Contraction; Myocytes, Cardiac; Phosphorylation; Phosphoserine; Point Mutation; Protein Conformation; Protein Processing, Post-Translational; Receptors, Adrenergic, beta; Signal Transduction; Transfection

This study examined the hypothesis that 26S proteasome dysfunction in human end-stage heart failure is associated with decreased docking of the 19S regulatory particle to the 20S proteasome. Previous studies have demonstrated that 26S proteasome activity is diminished in human end-stage heart failure associated with oxidation of the 19S regulatory particle Rpt5 subunit. Docking of the 19S regulatory particle to the 20S proteasome requires functional Rpt subunit ATPase activity and phosphorylation of the α-type subunits.. An enriched proteasome fraction was prepared from 7 human nonfailing and 10 failing heart explants. Native gel electrophoresis assessed docking of 19S to 20S proteasome revealing 3 proteasome populations (20S, 26S, and 30S proteasomes). In failing hearts, 30S proteasomes were significantly lower (P=0.048) by 37% suggesting diminished docking. Mass spectrometry-based phosphopeptide analysis demonstrated that the relative ratio of phosphorylated:non phosphorylated α7 subunit (serine250) of the 20S proteasome was significantly less (P=0.011) by almost 80% in failing hearts. Rpt ATPase activity was determined in the enriched fraction and after immunoprecipitation with an Rpt6 antibody. ATPase activity (ρmol PO4/μg protein per hour) of the total fraction was lowered from 291±97 to 194±27 and in the immunoprecipitated fraction from 42±12 to 3±2 (P=0.005) in failing hearts.. These studies suggest that diminished 26S activity in failing human hearts may be related to impaired docking of the 19S to the 20S as a result of decreased Rpt subunit ATPase activity and α7 subunit phosphorylation.

Topics: Casein Kinase II; Female; Heart Failure; Humans; Male; Middle Aged; Myocardium; Native Polyacrylamide Gel Electrophoresis; Phosphorylation; Proteasome Endopeptidase Complex; Protein Processing, Post-Translational