Die Bedeutung der δ-Isoform der Kalzium-Calmodulin-abhängigen Proteinkinase II (CaMKII) für die Azidose bei isolierten Kardiomyozyten

The effect of Ca²⁺/calmodulin-dependent protein kinase II isoform δ (CaMKIIδ) on acidosis in isolated cardiomyocytes

by Henrik Bäumer
Doctoral thesis
Date of Examination:2013-10-29
Date of issue:2013-10-23
Advisor:Prof. Dr. Lars S. Maier
Referee:Prof. Dr. Lars Klinge
Referee:Prof. Dr. Martin Oppermann
Persistent Address: http://dx.doi.org/10.53846/goediss-4080

 

 

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Abstract

English

Objective: CaMKII-dependent Thr-17 phosphorylation of PLB leading to higher SERCA activity has been suggested to maintain or even partially recover fractional shortening (FS) and intracellular Ca transient amplitude (ΔF/F0) during a late phase of acidosis in isolated cardiac myocytes. In accordance, CaMKII-inhibition using KN-93, which unselectively blocks all CaMKII isoforms, was reported to completely inhibit recovery from acidosis for FS as well as for intracellular Ca transients. However, the individual contribution of each CaMKII isoform is so far unknown. As CaMKIIδ is the dominant isoform of CaMKII in the heart, we investigated its relevance for maintenance/recovery of FS and Ca transients during late phase acidosis in a murine knockout model (CaMKIIδ-KO). Methods: Ventricular cardiac myocytes were isolated from WT and CaMKIIδ-KO mouse littermates. Acidosis was induced by lowering pH0 from 7.4 (control) to pH0 6.75 (acidosis). Fractional shortening (% vs. resting cell length, % RCL) and intracellular Ca transients (Fluo-3 AM, ΔF/F0, 10 µM) were elicited using 1 Hz stimulation frequency. Data are presented as means±S.E.M. Two-way analysis of variance (ANOVA) for repeated measurement (RM) tests combined with Fisher-LSD post hoc test were performed where appropriate and values of P<0.05 were considered as statistically significant. Results: In WT mouse myocytes (n=49) FS clearly decreased from 3.85±0.24 % RCL during control to 1.54±0.15 % RCL during early phase of acidosis (P<0.05) and could be maintained during late acidosis (1.56±0.19 % RCL). Similarly, Ca transient amplitude initially decreased from 1.13±0.05 a.U.F. in control to 0.82±0.05 a.U.F. in early acidosis (P<0.05) and remained relatively constant during late acidosis (0.83±0.06 a.U.F.). In parallel, RT90% (90% relaxation of shortening) reaccelerated from 283±23 ms in early acidosis to 253±24 ms in late acidosis (P<0.05), and Ca transient decline (τCa) slightly reaccelerated from 139±9 ms 133±8 ms. In addition, SR Ca content measured by caffeine contractures (10 mM), increased from 2.31±0.21 a.U.F. in early acidosis to 2.66±0.33 a.U.F. after 10 min of acidosis (P<0.05). In contrast, in myocytes from CaMKIIδ-KO mice (n=41) FS decreased from 3.33±0.24 % RCL in control to 1.64±0.17 % (P<0.05) in early acidosis and further decreased to 1.09±0.17 % RCL in late acidosis (P<0.05). Similarly, Ca transients also showed a significant further decrease from early to late acidosis (1.02±0.06 a.U.F. in control, 0.80±0.05 a.U.F. in early acidosis, 0.65±0.06 a.U.F. in late acidosis; P<0.05). Furthermore, there was no reacceleration of any relaxation parameter in CaMKIIδ-KO cardiomyocytes (P<0.05 vs. WT using ANOVA). In addition, SR Ca content declined from 2.25±0.15 a.U.F. in early acidosis to 1.49±0.15 a.U.F. after 10 min of acidosis (P<0.05 vs. early acidosis/WT). Conclusion: Knockout of the δ isoform of CaMKII significantly impairs maintenance/recovery of FS and Ca transients during late phase of acidosis, pointing to an essential role of this particular CaMKII isoform in handling acidosis. This impairment most likely results from an inability to increase SR Ca content sufficiently during late phase acidosis. As CaMKII-inhibition could be of future therapeutical relevance, possible negative effects of a pharmacologically-induced lower CaMKIIδ-activity during acidosis should be carefully considered.
Keywords: CaMKIIδ; acidosis; CaMKIIδ-KO
Schlagwörter: CaMKIIδ; Azidose
 
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