Strukturelle und funktionale Veränderungen der atrialen Kalzium-Freisetzungseinheit im Herzinsuffizienzmodell durch Junctophilin-2-Knockdown
Structural und functional changes of the atrial calcium release unit in a heart failure model induced by junctophilin 2 knockdown
von Benjamin Eikenbusch
Datum der mündl. Prüfung:2021-03-18
Erschienen:2021-03-05
Betreuer:Prof. Dr. Stephan E. Lehnart
Gutachter:Prof. Dr. Dörthe Katschinski
Gutachter:Prof. Dr. Thomas Meyer
Dateien
Name:Dissertation_FINAL_EDISS.pdf
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Zusammenfassung
Englisch
In this work, the effects of a cardiac-specific tamoxifen-mediated junctophilin-2 (JPH2) knockdown in mice on structure and function of the atria and atrial cardiomyocytes (AM) were investigated. Atrial JPH2 protein expression before knockdown was approximately 20% compared with the ventricle. Adult transgenic mice of the MerCreMer(MCM)-shJPH2 line showed an atrial JPH2 protein expression level of about 42% compared with MCM mice as control after a single i.p. tamoxifen injection of 40 mg/kg body weight. All animals survived the two-week observation period. In Western Blots, the ratio of JPH2 to ryanodine receptor type 2 (RyR2) expression was significantly decreased, as was the expression of protein kinase A (PKA) dependent phosphorylated RyR2-pS2808. RyR2 expression itself was unchanged. Expression of the sarcoendoplasmic calcium pump (SERCA2a) and caveolin-3 (Cav3) were significantly increased. Echocardiographically, JPH2 knockdown resulted in atrial and ventricular loss of function in structurally (histologically) unchanged hearts, namely absence of dilatation, hypertrophy, and fibrosis. Living isolated AM also lacked signs of hypertrophy but live cell imaging (di8-ANEPPS) showed a strong proliferation of the transverse axial tubule (TAT) network, especially the axial tubules (AT). Immunofluorescence studies of fixed AM via STED and confocal imaging revealed a previously unknown differential distribution of JPH2 into large junctional and small nonjunctional clusters. Junctional JPH2 was associated with AT. Cluster morphology of highly phosphorylated junctional RyR2 channels along AT was disrupted after JPH2 knockdown, and TAT-associated voltage-gated calcium channel (CaV1.2) clusters were also reduced. Electrocardiographically, atrioventricular excitatory conduction was slowed after JPH2 knockdown. These results illustrate that JPH2 knockdown resulted in atrial dysfunction and pathological remodeling but without any mortality (up to 4 weeks after tamoxifen treatment), atrial hypertrophy, dilatation, or fibrosis. In summary, our data demonstrate molecular disruption of atrial junctional membrane complexes (JMC) after cardiac-specific JPH2 knockdown.
Keywords: TATS; JPH2; JP2; Knockdown; Junctophilin-2; CRU; JMC; Tamoxifen; RNA interference; Atria; Atrial cardiomyocytes; Heart failure; Pathological remodeling; Atrial dysfunction; RyR2; STED