Medizinische Universität Graz Austria/Österreich - Forschungsportal - Medical University of Graz
Gewählte Publikation:
Schönleitner, P.
Expression and function of Na+ handling proteins in young ß1-adrenergic receptor transgenic mice at an early stage of cardiac remodeling
[ Diplomarbeit ] Medical University of Graz; 2013. pp. 49
[OPEN ACCESS]
FullText
- Autor*innen der Med Uni Graz:
- Betreuer*innen:
- Heinzel Frank
- Wakula-Heinzel Paulina
- Altmetrics:
- Abstract:
- Chronic stimulation of the ß1-adrenergic pathway leads to cardiac hypertrophy and heart failure. In mice overexpressing the ß1-adrenergic receptor (ß1-OE), changes in Ca2+ handling at an early stage of remodeling (8-12 weeks) precede the development of structural hypertrophy. The relaxation of cardiomyocytes is impaired due to slower Ca2+ removal via the Na+/Ca2+ exchanger (NCX) and higher cytosolic Na+ levels. The aim of the thesis is to elucidate the underlying mechanism ultimately leading to the deterioration of cardiac function which inevitably occurs under the influence of disturbed ion handling. Therefore, the expression of major Ca2+ and Na+ handling proteins as well as phosphorylation of regulatory subunits were assessed. Furthermore the main Na+ efflux pathway (Na+/K+-ATPase - NKA) and the influx of Na+ via the late component of the Na+ current (I[late Na+]) were investigated. Single ventricular myocytes were isolated from 8-12 weeks old male ß1 mice (ß1-OE, N=4) and wild-type littermates (WT, N=6) for whole-cell patch-clamp recordings. NKA current (I[pump]) was measured as the difference in outward current, at 0 mV, after rapid solution switch to K+-free solution to inhibit I[pump]. The late component of the Na+ current was measured as current sensitive to 30 µm tetrodotoxin (TTX). Currents were normalized to cell capacitance. Protein expression levels of NKA ¿ 1 and ¿ 2, NHE1, SERCA2a, TRPC1,3 and 6, PLB, P-Thr17 PLB, PLM, P-Ser68, RyR, P-Ser2814 RyR, P-Ser2808 RyR, were determined using Western blotting in whole ventricle homogenates (ß1-OE, N=4-6 vs. WT, N=4-6). Protein densities were normalized against GAPDH. In young ß1-OE mice protein expression of NKA ¿1 was unchanged, whereas NKA ¿2 was significantly decreased. The NKA regulatory subunit PLM was significantly reduced in ß1-OE mice Furthermore, PLM phosphorylation at serine 68, which is the downstream target for ß1-adrenoreceptor stimulation, was increased in ß1-OE mice. The protein levels of NHE1, RyR and SERCA were unchanged. Expression of TRPC3, TRPC1 and TRPC6, non-selective cation channels, were unchanged in lysates of isolated cardiac myocytes of ß1-OE mice. The expression of phospholamban (PLB), the regulatory subunit of SERCA, was unchanged but phosphorylation of threonine 17 was significantly increased. Phosphorylation of RyR Ser2808 was unchanged whereas the RyR Ser2814 showed an increased phosphorylation. With [Na+]pip=10 mM, I[pump] density was significantly increased in ß1 myocytes. Maximal Ipump density was unchanged. I[late Na+] amplitude at the end of pulse as well as the integrated current from 250-750ms were significantly increased in ß1-OE mice. In conclusion, at an early stage of cardiac remodeling, NKA ¿1, NHE1, TRPC3, TRPC6, TRPC1 protein expression were unchanged in young ß1 mice. In ß1-OE mice, increased I[late Na+] may contributes to elevated [Na+]i during chronic sympathetic activation. Higher Na+ affinity of NKA due to less inhibition by phospholemman only partially compensates increased Na+ influx at this early stage of heart failure development. Increased phosphorylation of PLB at threonine 17 and RyR2 at serine 2814 indicates activation of CaMKII in chronic ß1-adrenergic stimulation which might augment I[late Na+].