Persistently increased CaMKIIδ autophosphorylation mediates pathologic SR Ca loss in a murine model of Doxorubicin-induced cardiomyopathy

Figures

• 

  Fig. 1. CaMKIIδ autophosphorylation reduces systolic Ca transients and SR Ca content upon acute DOX exposure. a-Reduced systolic Ca transient amplitudes upon acute DOX exposure in redox-sensitive CaMKIIδMet281/282 (left) and redox-dead CaMKIIδVal281/282 cardiomyocytes (right) that can be restored by pharmacological CaMKII inhibition using AIP (mean data). b-DOX-dependent impairment of systolic Ca transient amplitudes in CaMKIIδ+/+ cardiomyocytes (left) is absent upon pharmacological and genetic CaMKIIδ inhibition (right. Mean data). c&d-Mean data illustrate DOX-show reduction of SR Ca content in CaMKIIδMet281/282 and CaMKIIδVal281/282 cardiomyocytes (c), but not upon pharmacological CaMKII inhibition using AIP or in CaMKIIδ−/− cardiomyocytes (d). e&f-Original traces of Fura-2 AM loaded cardiomyocytes illustrate reduced SR Ca content in CaMKIIδMet281/282, CaMKIIδVal281/282 (e) and CaMKIIδ+/+ cardiomyocytes. Cardiomyocytes with genetic deletion of CaMKIIδ (CaMKIIδ−/−) are protected from DOX-related SR Ca depletion (f). P-values were calculated using One-Way-ANOVA combined with Holm-Sidak post-hoc test (c) or Kruskal-Wallis test (a,b.d). n = cells/mice. DOX: doxorubicin; SR: sarcoplasmic…

• 

  Fig. 2. CaMKIIδ autophosphorylation mediates SR Ca loss upon acute DOX exposure. a-Mean data illustrate increased diastolic Ca spark frequency (CaSpF) in DOX-exposed CaMKIIδMet281/282 and CaMKIIδVal281/282 cardiomyocytes, which is prevented by pharmacological CaMKII inhibition (AIP). b-Original confocal line scan images of Fluo-4 AM loaded CaMKIIδMet281/282 and CaMKIIδVal281/282 cardiomyocytes. c-Diastolic CaSpF is absent upon genetic deletion of CaMKIIδ (CaMKIIδ−/−). d-Original confocal line scan images of Fluo-4 AM loaded CaMKIIδ+/+ and CaMKIIδ−/− cardiomyocytes. P-values were calculated using Kruskal-Wallis test. n = cells/mice. CaSpF: calcium spark frequency; DOX: doxorubicin; SR: sarcoplasmic reticulum.

• 

  Fig. 3. Increased CaMKIIδ autophosphorylation and CaMKIIδ-dependent RyR2-hyperphosphorylation at pS2814 upon acute DOX exposure. a-c- Original western blots (a) show increased CaMKII autophosphorylation at threonine-287 (pT287) (normalized to total CaMKIIδ expression) in DOX-exposed CaMKIIδMet281/282 and CaMKIIδVal281/282 cardiomyocytes (c), while CaMKII oxidation is prevented in CaMKIIδVal281/282 (b). d-f- Original western blots (d) show increased CaMKII oxidation and autophosphorylation in CaMKIIδ+/+ while CaMKIIδ expression is deleted in CaMKIIδ−/− cardiomyocytes (e&f). Original western blots (g) show CaMKII-specific hyperphosphorylation of pS2814 at the RyR2 in CaMKIIδMet281/282 and CaMKIIδVal281/282 cardiomyocytes (h), which is absent in CaMKIIδ−/− cardiomyocytes (i&j). P-values were calculated using Mann-Whitney-U test (b) or unpaired Student's t-test (c,e,f,h,j). n = mice. DOX: doxorubicin; pS2814: CaMKII-specific RyR2 phosphorylation site at serine-2814; pT287: CaMKII autophosphorylation site at threonine-287; RyR2: ryanodine receptor.

• 

  Fig. 4. CaMKIIδ−/− mice are protected from LV dysfunction in DICM. Original recordings of parasternal short axis at BL (upper panel) and after 12 weeks of DOX treatment (lower panel) in CaMKIIδMet281/282 (a), CaMKIIδVal281/282 (b), CaMKIIδ+/+ (c) and CaMKIIδ−/− mice (d). e-Mean data illustrate that DOX-treated CaMKIIδMet281/282 and CaMKIIδVal281/282 mice develop comparably impaired left ventricular ejection fraction (EF).f-Mean data for heart rate (HR) in CaMKIIδMet281/282 and CaMKIIδVal281/282 mice. g-EF is reduced in DOX-treated CaMKIIδ+/+ and CaMKIIδ−/− mice. However, CaMKIIδ−/− mice are significantly protected upon DOX as compared to CaMKIIδ+/+ littermates. h-Mean data for HR during in CaMKIIδ+/+ and CaMKIIδ−/− mice. P-values were calculated using mixed effect analysis combined with Fisher LSD post hoc test. n = mice. BL: baseline; DOX: doxorubicin; EF: left ventricular ejection fraction; HR: heart rate; 12w: 12 weeks. ⁎indicates significance vs. BL. #indicates significance vs. genotype.

• 

  Fig. 5. CaMKIIδ autophosphorylation cause reduced systolic Ca transients and SR Ca depletion in DICM. a-Reduced systolic Ca transient amplitudes in DICM as evident in CaMKIIδMet281/282 and CaMKIIδVal281/282 myocytes can be restored by pharmacological inhibition of CaMKII (AIP). b-Reduced Ca transient amplitudes in DICM as evident in CaMKIIδ+/+ myocytes are not present upon pharmacological CaMKII inhibition in CaMKIIδ+/+, and following genetic deletion of CaMKIIδ in CaMKIIδ−/−. c-Long-term DOX treatment results in SR Ca depletion in CaMKIIδMet281/282 and CaMKIIδVal281/282 myocytes that can be rescued by pharmacological inhibition of CaMKII. d-Genetic deletion of CaMKIIδ prevents DOX-induced SR Ca depletion. e-Original traces of caffeine-induced Ca transients demonstrate reduced SR Ca content in Fura-2 AM loaded CaMKIIδMet281/282 and CaMKIIδVal281/282 myocytes. f-Original traces of caffeine-induced Ca transients demonstrate maintained SR Ca content in DOX-treated CaMKIIδ−/− myocytes. P-values were calculated using Kruskal-Wallis test (a,b,c) or One-Way-ANOVA combined with Holm-Sidak post-hoc test (d). n = cells/mice. DOX: doxorubicin; SR: sarcoplasmic reticulum.

• 

  Fig. 6. CaMKIIδ autophosphorylation mediates SR Ca loss in DICM. a-Increased diastolic Ca spark frequency (CaSpF) in DOX-treated CaMKIIδMet281/282 and CaMKIIδVal281/282 myocytes, which is abolished by pharmacological CaMKII inhibition (AIP). b-Original confocal line scan images of Fluo-4 AM loaded CaMKIIδMet281/282 and CaMKIIδVal281/282 cardiomyocytes. c-CaMKIIδ+/+ myocytes exhibit increased CaSpF upon DOX, which is not present in CaMKIIδ−/− myocytes or upon pharmacological CaMKII inhibition (AIP). d-Original confocal line scan images of Fluo-4 AM loaded CaMKIIδ+/+ and CaMKIIδ−/− cardiomyocytes. P-values were calculated using Kruskal-Wallis test. n = cells/mice. CaSpF: calcium spark frequency; DOX: doxorubicin;

• 

  Fig. 7. Persistently increased CaMKIIδ autophosphorylation and CaMKIIδ-dependent RyR2-hyperphosphorylation at pS2814 in DICM. a-c- Original western blots (a) show persistently increased CaMKIIδ autophosphorylation at threonine-287 (pT287) (normalized to total CaMKIIδ expression) in CaMKIIδMet281/282 and CaMKIIδVal281/282 cardiomyocytes in DICM (c), while CaMKIIδ oxidation is unaltered (b). Original western blots (d) show increased CaMKIIδ autophosphorylation in CaMKIIδ+/+ while CaMKIIδ expression is deleted in CaMKIIδ−/− cardiomyocytes (f). Original western blots (g) show persistent CaMKII-specific hyperphosphorylation of pS2814 at the RyR2 in CaMKIIδMet281/282 and CaMKIIδVal281/282 cardiomyocytes in DICM (h), which is absent in CaMKIIδ−/− cardiomyocytes (i&j). P-Values were calculated using unpaired Student's t-test (b,c,e,f,j) or Mann-Whitney-U test (h). n = mice. DOX: doxorubicin; pS2814: CaMKII-specific RyR2 phosphorylation site at serine-2814; pT287: CaMKII autophosphorylation site at threonine-287; RyR2: ryanodine receptor.

• 

  Fig. 8. CaMKII mediates SR Ca loss in DOX exposed ventricular human cardiomyocytes. a-DOX exposed ventricular human cardiomyocytes show increased diastolic calcium spark frequency (CaSpF) as compared to vehicle-exposed cardiomyocytes. Pharmacological CaMKII inhibition using AIP prevents DOX-induced CaSpF. b-Original line-scans of Fluo-4 AM loaded human ventricular cardiomyocytes. P-values were calculated using One-Way-ANOVA combined with Holm-Sidak post-hoc test. n = cells/human samples. CaSpF: calcium spark frequency; DOX: doxorubicin.