Sleep disordered breathing (SDB) is a widespread disease and is associated with contractile dysfunction. However, current therapeutic options are limited, and new approaches are warranted, demanding detailed mechanistic insights. Interestingly, increased activity of Ca/calmodulin-dependent protein kinase (CaMKII), a key regulator of cardiac Ca homeostasis, can lead to contractile dysfunction and arrhythmias.
Purpose:
We tested whether mice with SDB exhibit a CaMKII-dependent dysregulation of cellular Ca homeostasis.
Methods:
SDB was induced by injecting polytetrafluorethylene (PTFE, 100 µl) into the tongue of wildtype (WT) and CaMKII knock-out (CKO) mice. After two weeks, apneas and inspiratory flow limitations (IFL) were measured during murine sleep phases for 8 h using whole-body plethysmography. Eight weeks after the injection, isolated ventricular cardiomyocytes were incubated with the Ca-sensitive dye FURA-2 for 15 minutes. Stimulated Ca transients, as well as cardiomyocyte shortening (fig. A), were measured using epifluorescence microscopy (1 Hz, 20 V for 4 ms). Sarcoplasmic reticulum (SR) Ca content was estimated by rapid application of 10 µM caffeine and measurement of the induced Ca transient (fig. D).
Results:
Sonographic measurements revealed a significant increase in mean tongue diameter from (in mm) 3.7±0.1 to 5.1±0.1 after PTFE injection (n=23, p<0.0001), which correlated significantly with the frequency of apneas in PTFE mice (p=0.046, r2=0.19). Compared to untreated littermates, there was a significant decrease in Ca transient amplitude in WT PTFE mice (FURA ratio 340/380 nm) from 0.41±0.14 to 0.30±0.08 (n=13 vs. 15, p=0.048, fig. B), while CKO mice were protected (0.41±0.12, n=13, p=0.38). The PTFE-dependent decrease in Ca transient amplitude was accompanied by a significant decrease in fractional shortening from 0.07±0.03 to 0.05±0.02 in WT (p=0.048, fig. C), but not in CKO mice (0.08±0.02, p=0.21). In accordance, PTFE treatment also significantly reduced caffeine-induced Ca transient amplitude from 0.86±0.25 to 0.55±0.17 in PTFE-treated WT mice (n=7 vs. 7, p=0.037, fig. E), indicating a decreased SR Ca content. This resulted in a significant negative correlation with the IFL frequency (p=0.0076, r2= 0.43, fig. F). Importantly, CKO mice showed a normal caffeine-induced Ca transient amplitude upon PTFE treatment (0.83±0.14, n=8) and the correlation with the IFL frequency was completely absent (p=0.98, r2<0.01).
Conclusion:
Cardiac Ca homeostasis was dysregulated in a mouse model of obstructive sleep apnoea. Interestingly, mice with a genetic knock-out of CaMKII were protected, which may have therapeutic implications.
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