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. 2012 Nov 8;491(7423):269-73.
doi: 10.1038/nature11444. Epub 2012 Oct 10.

CaMKII determines mitochondrial stress responses in heart

Mei-Ling A Joiner  1 , Olha M Koval, Jingdong Li, B Julie He, Chantal Allamargot, Zhan Gao, Elizabeth D Luczak, Duane D Hall, Brian D Fink, Biyi Chen, Jinying Yang, Steven A Moore, Thomas D Scholz, Stefan Strack, Peter J Mohler, William I Sivitz, Long-Sheng Song, Mark E Anderson
Affiliations

CaMKII determines mitochondrial stress responses in heart

Mei-Ling A Joiner et al. Nature. .

Abstract

Myocardial cell death is initiated by excessive mitochondrial Ca(2+) entry causing Ca(2+) overload, mitochondrial permeability transition pore (mPTP) opening and dissipation of the mitochondrial inner membrane potential (ΔΨm). However, the signalling pathways that control mitochondrial Ca(2+) entry through the inner membrane mitochondrial Ca(2+) uniporter (MCU) are not known. The multifunctional Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) is activated in ischaemia reperfusion, myocardial infarction and neurohumoral injury, common causes of myocardial death and heart failure; these findings suggest that CaMKII could couple disease stress to mitochondrial injury. Here we show that CaMKII promotes mPTP opening and myocardial death by increasing MCU current (I(MCU)). Mitochondrial-targeted CaMKII inhibitory protein or cyclosporin A, an mPTP antagonist with clinical efficacy in ischaemia reperfusion injury, equivalently prevent mPTP opening, ΔΨm deterioration and diminish mitochondrial disruption and programmed cell death in response to ischaemia reperfusion injury. Mice with myocardial and mitochondrial-targeted CaMKII inhibition have reduced I(MCU) and are resistant to ischaemia reperfusion injury, myocardial infarction and neurohumoral injury, suggesting that pathological actions of CaMKII are substantially mediated by increasing I(MCU). Our findings identify CaMKII activity as a central mechanism for mitochondrial Ca(2+) entry in myocardial cell death, and indicate that mitochondrial-targeted CaMKII inhibition could prevent or reduce myocardial death and heart failure in response to common experimental forms of pathophysiological stress.

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Figures

Figure 1
Figure 1
Isolated mitochondria with transgenic, membrane-targeted CaMKII inhibition (CaMKIIN) are resistant to Ca2+ (200 µM Ca2+ free) challenge. a. Mitochondria mPTP Ca2+-dependent (arrow) opening reflected by a decrease in light scattering corresponding to an increase in mitochondrial volume, n = 3/genotype. b. Increasing [Ca2+] promotes loss of ΔΨm more in mitochondria isolated from WT than CaMKIIN mouse hearts (**p < 0.001, ***p < 0.0001, n = 3 hearts/group with duplicate measurements). c. Inner mitochondrial membrane potential (ΔΨm) measurements in isolated mitochondria using a fluorescent reporter, JC-1. Reduced signal from baseline after addition of Ca2+ (200 µM free Ca2+, at arrow) indicates loss of ΔΨm (p < 0.03 for all time points after the Ca2+ challenge) between WT and CaMKIIN treated with Ca2+ alone (indicated with black bracket), red squares versus open red circles, n = 5 hearts/group with duplicate measurements. d. Normalized traces showing rate of mitochondrial Ca2+ uptake in saponin-permeabolized cardiomyocytes after the addition of Ca2+ (arrow, 100 µM free Ca2+). Mitochondrial Ca2+ uptake was monitored in cells by loss of Ca2+ Green-5N fluorescence. The rate of decline in fluorescence reflects the rate of mitochondrial Ca2+ uptake. e. Summary data show the rate of mitochondrial Ca2+ uptake. Nonlinear regression fits for mitochondria Ca2+ uptake between WT and CaMKIIN cardiomyocytes (***p < 0.0001, n = 6/genotype). f. Summary data show reduced Ca2+ uptake in mitochondria, measured using a cameleon mitochondrial-targeted Ca2+ indicator, from HeLa cells expressing mtCaMKIIN compared to myc-expressing controls (**p = 0.003, n = 23 cells/group). Data represent mean ± s.e.m.
Figure 2
Figure 2
CaMKII agonist actions on IMCU require serines 57 and 92. a. IMCU is a Ca2+-dependent conductance. Inset shows IMCU in 0.2, 5 and 100 mM bath [Ca2+] fit with the Hill equation (standard slope). V½ = 23.8 mM, R2= 0.955 and h = 0.057. Red lines show the 95% confidence intervals (runs test p = 0.743). b. Summary data and time course for IMCU recorded with 0.2 mM Ca2+ after obtaining a high resistance seal and mitoplast membrane rupture (time 0) allowing dialysis of CaMKII T/D. Replacing ATP with non-hydrolyzable ATP (non-ATP) analog (both at 0.1 mM) does not allow a CaMKII-dependent increase in IMCU (all mutant CaMKII at 0.5 µM); T/D and ATP; T/D non-ATP; kinase inactive CaMKII (K/M), CaM and ATP, n = 6 (WT), 7 (T/D), 6 (K/M) and 5 (non-ATP), *p < 0.01, **p < 0.001, ***p < 0.0001 at 20 min. c. Summary data for IMCU recorded with 0.2 mM Ca2+ after addition of 100 nM calyculin A (c-A); c-A with T/D CaMKII (0.5 µM), ATP. n = 7 (control), 9 (C-a) and 7 (C-a with T/D), *p < 0.0001. d. Summary data for IMCU from WT, CaMKIIN and mtCaMKIIN mitochondria. Na+ current (150 mM) recorded in the absence of bath (intermembrane space equivalent) Ca2+ or Ru360 (10 nM). **p < 0.001, ***p < 0.0001, n = 7 (cntl groups), 8 (T/D, WT), 9 (T/D, SS/AA) and 6 (Ru360 groups). e. MCU and CaMKII co-immunoprecipitate from mitochondrial lysate. f. Summary data for IMCU recorded from HEK cell mitoplasts with and without transfection of WT or SS/AA MCU mutants. HEK mitoplast Ca+ currents were inhibited by Ru360 (10 nM). For Ca2+ n = 10 (WT and CaMKIIN) and 7 (mtCaMKIIN); Na+ n = 15 (WT), 13 (CaMKIIN) and 12 (mtCaMKIIN) and for Ru360 n = 8 (WT and CaMKIIN), 5 (mtCaMKIIN), ***p < 0.0001. Data represent mean ± s.e.m, except for inset (see a).
Figure 3
Figure 3
mtCaMKIIN hearts are resistant to I/R injury. a. LVDP recovery following I/R as a percentage of the baseline value (*p = 0.026). b. Representative TTC stained heart sections. The dark red staining represents living myocardium and the solid black outlines form boundaries demarcating viable from dead tissue. c. Summary data from TTC stained hearts showing relative area of infarct normalized to WT, measured as in Supplemental Fig. 2e, *p = 0.006. d. Representative TEM images from hearts treated as in panel a. e. Summary mitochondria injury scores for TEM studies by the criteria used in Supplemental Fig. 2h. (*p = 0.003, more than 500 mitochondria from at least 10 random fields were counted/genotype). f. Caspase 9 activity from hearts treated as in panel a (*p = 0.033, n = number of hearts/genotype). Data represent mean ± s.e.m.
Figure 4
Figure 4
mtCaMKIIN hearts are resistant to apoptosis in vivo. a. Representative images of TUNEL-stained nuclei from WT and mtCaMKIIN heart transverse sections 5 h after MI. Dapi stain shows total number of nuclei. Scale bar indicates 50 µm. b. Upper panel - Summary data for the number of TUNEL-stained nuclei from WT and mtCaMKIIN heart sections 5 h after MI (3 hearts/genotype, 10 images/heart). Lower panel - Summary data for the number of TUNEL-stained nuclei from WT and mtCaMKIIN heart sections 30 min after isoproterenol (ISO) treatment (15 mg/kg, 3 hearts/genotype, 10 images/heart, ***p < 0.0001). Data represent mean ± s.e.m.
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Comment in

  • CaMKII does it again: even the mitochondria cannot escape its influence.
    Correll RN, Molkentin JD. Correll RN, et al. Circ Res. 2013 Apr 26;112(9):1208-11. doi: 10.1161/CIRCRESAHA.113.301263. Circ Res. 2013. PMID: 23620234 No abstract available.
  • Mitochondrial Ca2+ uniporter and CaMKII in heart.
    Fieni F, Johnson DE, Hudmon A, Kirichok Y. Fieni F, et al. Nature. 2014 Sep 25;513(7519):E1-2. doi: 10.1038/nature13626. Nature. 2014. PMID: 25254480 Free PMC article.
  • Joiner et al. reply.
    Joiner ML, Koval OM, Li J, He BJ, Allamargot C, Gao Z, Luczak ED, Hall DD, Fink BD, Chen B, Yang J, Moore SA, Scholz TD, Strack S, Mohler PJ, Sivitz WI, Song LS, Anderson ME. Joiner ML, et al. Nature. 2014 Sep 25;513(7519):E3. doi: 10.1038/nature13627. Nature. 2014. PMID: 25254481 No abstract available.

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