Peer review of: Mitochondrial CaMKII causes adverse metabolic reprogramming and dilated (2020)

Reviewers' comments: 

 

Reviewer #1 (Remarks to the Author): 

 

Summary: 

The manuscript titled “Mitochondrial CaMKII causes metabolic reprogramming, energetic 
insufficiency, and dilated cardiomyopathy” investigates the mechanisms linking pathological stress 
to the altered myocardial metabolism observed in the failing heart. Cytosolic CaMKII is a key 
mediator of cardiac hypertrophy and has been implicated in dilated cardiomyopathy, but little is 
known about the specific mechanisms by which CaMKII may contribute to the dilation process. 
Previous work by this group and others demonstrated that CaMKII is present in cardiac 
mitochondria, and that inhibition of CaMKII in the mitochondria protects against mPTP opening, loss 
of delta psi, and cell death in myocardial ischemia reperfusion injury, suggesting a critical role for 
CaMKII in modulating mitochondrial function after an acute pathological insult. Given these data and 
the association between poor mitochondrial function and heart failure/dilation, here the authors 
investigated how mitochondrial CaMKII activity specifically affects chronic changes in myocardial 
energetics and pathological remodeling post-MI. 

 

Using genetic mouse models of mitochondrial CaMKII overexpression or inhibition, the authors 
present data showing that increased mitochondrial CaMKII activity persists 1 week post-MI, when 
hearts are becoming dilated, and that inhibition of mitochondrial CaMKII blocks MI-induced cardiac 
dilation and dysfunction. Furthermore, genetic overexpression of mitochondrial CaMKII alone is 
sufficient to cause spontaneous dilated cardiomyopathy, which is associated with compromised 
myocardial energetics that ultimately impairs cytosolic calcium handling that contributes to 
contractile dysfunction. Finally, the authors demonstrate that replenishment of mitochondrial 
creatine kinase in mice with mitochondrial CaMKII overexpression is sufficient to improve 
myocardial bioenergetics and partially rescue the dilated cardiomyopathy phenotype. 

 

Major Concerns: 

 

1. More data is needed to clearly show that mito-CamKII phosphorylation is increased after injury. A 
cytosolic loading control and also showing cytosolic CamKII phosphorylation would help bolster the 
claim that this is indeed mito-localized. Perhaps the inclusion of sub-fraction assays using protease 
degradation to show this is matrix localized would help this claim. Can CamKII activity be examined 
in the mito localized fraction? 

 

2. The results shown in Figure 1C are astounding. It is virtually impossible to see such a huge 
protective effect only 1 week after permanent LCA ligation where no reperfusion is present. 

This model lends itself to see changes in LV remodeling/infarct expansion, but not so much to acute 
changes in structure or function. A couple mice in the mtCaMKIIN group appear to have almost no 
dysfunction 1wk post MI. Is the ligation/infarct size the same in these groups? The differences at 1 
week are tough to believe. The authors may want to make sure all mice in the study received a 
similar infarct. 

 

3. For all direct measurements of HW/BW used to demonstrate effects on hypertrophy (Fig. 1G, etc), 
please show raw data (HW and BW measurements separately), and/or HW/TL measurements. Given 
the dramatic cardiac phenotype in the mtCaMKII mice, is body weight changing between genotypes? 
Direct histological measurements of cardiomyocyte cross sectional area would also be informative 
here and help to strengthen claims about mtCaMKII’s effect on hypertrophy. 

 

4. Cytoplasmic marker is needed in Fig 2A. 

 

5. The data interpretation in Figure 2 that the phenotype is mitoCamKII dependent is not clear to 
me. The inhibitor AC3-I is almost entirely localized to the cytosol yet it is significantly improving EF 
and ESV in the mtCaMKII mice. It appears that much of the phenotype is due to cytosolic activity 
especially since the inhibitor is probably not 100% effective. 

 

6. The 2nd line of evidence is shown in Figure D but the authors do not show the percent of 
mtCAMKIIN that is in the cytosol vs. the mitochondria. 

 

7. The data showing a difference in ATP and pCRr levels is at a a time with massive cardiac 
pathology. It’s difficult to attribute this as a direct result of mtCaMKII and not secondary to the 
severe phenotype. 

 

8. The data regarding diastolic calcium correction with ATP is nice (Fig 3E,F). Can the authors provide 
the same data with different pacing for amplitude, rise-time and decay? It’s a little difficult to 
understand that if there is insufficient ATP to manage diastolic calcium (likely an effect at SERCA, 
since it is the most ATP dependent) how the individual transient decay is not affected. 

 

9. Fig 4A-D. How well do the CKmito and CK-M antibodies differentiate these two isoforms? The 
blots shown here are from total heart lysates, so it is difficult to assess if CK in the mitochondria 

specifically is truly altered in mtCaMKII hearts, or whether the CKmito and CK-M antibodies might be 
picking up both myofibrillar and mitochondrial isoforms. Showing cytosolic vs. mitochondrial 
fractions would be helpful here or since there are not significant differences these data could be 
disregarded. 

 

10. Is there a significant increase in CK protein in the CK-mito Tg mouse? Did the authors also 
examine mRNA? Is there a tag on the transgene that can be exploited? 

 

11. Fig. 4. Is increased CKmito phosphorylation associated with any alterations in CKmito activity? 
And is loss or inhibition of CKmito alone sufficient to drive diminished mitochondrial energetics and 
contractile dysfunction/dilation seen in mtCaMKII mice? Is CKmito a direct phosphorylation target of 
mtCaMKII? 

 

12. Fig. 4G. How does CKmito expression in mtCaMKII hearts recover both ATP content and PCr 
content, rather than just shifting the balance between the two? How does CKmito increase the total 
pool of high energy phosphate species in this context? 

 

13. Fig 5. Does CKmito expression in the mtCaMKII mice rescue Complex I activity as measured in-gel 
or by OCR, in addition to rescuing complex I expression? What is the link with complex I expression? 
How does the CK-mito rescue have such a big effect on complex I formation? 

 

14. How does the PDH activity increase in mtCaMKII hearts correlate with the increase in phosphor-
PDH reported in the mass-spec data, since phosphor-PDH is inhibited? Is this the inhibitory site? 
Also, there was no change in mitochondrial calcium which is a strong physiological regulator of PDH 
activity. 

 

 

 

 

 

 

Reviewer #2 (Remarks to the Author): 

 

Luczak and colleagues found that mitochondrial CaMKII is activated upon myocardial infarction and 
that targeted inhibition of CaMKII in mitochondria can preserve cardiac function. They then 
generated a model of CaMKII overexpression in mitochondria and show a phenotype of ATP 
depletion and dilated cardiomyopathy which can be rescued by creatine kinase re-expression. This is 
an interesting manuscript describing effects of CaMKII on mitochondrial energy production in 
association with dilatation of the myocardium. Many findings are novel and advance the field. 

 

Major comments that should be addressed: 

1. Fig. 1A: To assess CaMKII activity in MI, additional measures other than pCaMKII should be used 
because pCaMKII as measure has been reported to be limited due to cross reactivity of most of the 
antibodies used in the field. Can the authors measure mitochondrial CaMKII phosphorylation targets 
as they do in Fig. 2G for mtCaMKII, e.g. MCU phsophorylation? 

2. Moreover, looking more closely to the delta C / delta B ratio in Fig. 1A, it looks alike a previous 
finding of Weinreuter et al in 2014 (EMBO Mol Med, Fig. 2A) that the ratio shifts towards delta C. 
Can the authors quantify this in their own experiment again? 

3. Can the authors show that hyperphosphorylation of the MCU upon MI is inhibited by mtCaMKIIN 
to judge whether CaMKII is indeed inhibited in this model? 

4. Fig. 1C: The authors show that mtCaMKIIN mice are protected from cardiac dysfunction one week 
after myocardial infarction. But isn’t that expected in light of the prior paper that these mice have 
less infarct size in the I/R model? Have the authors measured initial infarct size or troponin T levels 
in this experiment? This is important to assess the the relative contribution of different CaMKII 
inhibition-dependent protective effects on cardiac function. 

5. The authors refer to refs 15 and 21 to say that CaMKII is dispensable for cardiac function but in 
these studies only CaMKII delta was deleted and CaMKII activity was not completely abolished. They 
then correctly also refer to Kreusser et al. (ref 76) because in this paper no residual CaMKII activity 
was detectable because the two redundant isoforms CaMKII delta and gamma were knocked out 
simultanously. The same mouse model without any CaMKII activity was also used by Weinreuter et 
al. in an extensive ischemia/reperfusion study. How do the authors discuss their own findings in light 
of the Weinreuter paper? In that paper cardiac function upon I/R was protected only at later stages 
most likely to a reduced pro-inflammatory burden. Can the authors measure also pro-inflammatory 
cytokines and leucocyte infiltration in their model as alternative causes of contractile dysfunction? 

6. Loss of creatine as shown by the Neubauer lab does not cause exacerbation due to MI. How do 
the authors explain this in relation to the own findings? 

 

 

Minor: 

1. MW markers should be shown in the Western blot experiments 

 

In summary, I recommend a major revision. 

 

 

 

 

 

Reviewer #3 (Remarks to the Author): 

 

The paper by Luczak et al. investigates the role of mitochondrial calcium/calmodulin-dependent 
protein kinase II (CaMKII) in cardiac myocytes and its role in regulating mitochondrial function during 
pathology. First, the authors observed that one week after myocardial infarction (MI), mitochondrial 
CaMKII expression and phosphorylation (activity) is increased. Genetic inhibition of mitochondrial 
CaMKII protects from left ventricular (LV) dilation and dysfunction after MI. Mice with 
overexpression of mitochondrial CaMKII in the heart have dilated cardiomyopathy with energetic 
deficit. This can be rescued when mitochondrial creatine kinase (mitCK) is overexpressed in parallel. 
The authors conclude that these insights provide two new strategies how to prevent LV dilation after 
MI, i.e., increasing mitoCK or inhibiting mitochondrial CaMKII. 

 

The study is generally very well conducted and addresses a clinically import topic, i.e., heart failure 
after MI, one of the most frequent causes for cardiovascular and total deaths worldwide. 
Furthermore, gaining insights into mitochondrial dysfunction after ischemia/reperfusion may also be 
of broader interest since similar mechanisms may (or may not) be in place after stroke or ischemia of 
other regions of the body. 

 

The findings of the paper are indeed novel. The manuscript is nicely written, and the Figures are 
clear and well organized. A particular strength of the study is the combination of extensive genetic 
work on in vivo mouse models with functional experiments on isolated mitochondria and cardiac 
myocytes. The findings would have an important impact on the field, since the control of 
mitochondrial function in particular, after MI and in the context of maladaptive remodeling, is not 
fully understood, and CaMKII is a key regulatory hub that receives widespread attention in the 
scientific community. 

 

However, the study has also several shortcomings that limit my enthusiasm about the conclusions 
drawn. These are i) the mere use of transgenic techniques to overexpress and target certain proteins 

in cardiac myocytes (which may be more artificial) rather than using knock-out technology, ii) the 
fact that no clear (molecular) mechanism of how CaMKII enters mitochondria upon stress and 
influences mitochondrial function under physiological or pathological situations is proposed, and iii) 
some of the conclusions drawn are not (yet) fully supported by the experiments. In the following, I 
will address these issues in more detail: 

 

Major: 

1) Figure 1: The localization of CaMKII in mitochondria is not very convincing yet. It is a technical 
challenge to isolate mitochondria without contamination by the ER/SR, based on a close interaction 
and tethering of these two organelles. Can you verify that the CaMKII expression and 
phosphorylation assessed in isolated mitochondria stems from a pure mitochondrial isolation? You 
show the presence of VDAC, verifying mitochondrial protein, but do not show the absence of SR-
related proteins. 

 

2) Another important information, also in view of your results on regulation of mitoCK and Krebs 
cycle enzymes by CaMKII (see below), is the actual localization of CaMKII within mitochondria (inner 
or outer mitochondrial membrane, matrix). In the introduction, you mention matrix localization. For 
this, further biochemical experiments with differential digestion protocols would be required to 
determine the actual localization of native CaMKII. The same applies to the transgenic 
overexpressed and targeted mitoCaMKII (Figure 2A). The immune-fluorescence colocalization of 
CaMKII with Cox IV (Figure 1E) alone hardly answers this, especially since CoxIV is in the inner 
mitochondrial membrane. If CaMKII resides indeed in the matrix, how would it be regulated from 
upstream, and would that be via signals stemming from inside or outside the mitochondria? 

 

3) The transgenic overexpression of CaMKII in mitochondria is rather an artificial condition. This 
should be at least discussed, since in this project, no KO strategy is used, which would provide a 
somewhat cleaner information. This is of particular relevance because in some aspects, your 
previous results on reduction of infarct size by mitochondrial CaMKII inhibition differ from 
observations in CaMKII delta/gamma double KO mice (Weinreuter et al., EMBO Mol Med 2014), in 
which infarct size was unaffected by the KO (although the subsequent remodeling was ameliorated). 

 

4) Figure 2I-J: The observation that overexpression of mitochondrial CaMKII does not affect MCU-
mediated Ca uptake stands at odds with your previously proposed concept in which CaMKII actually 
does control MCU-mediated mitochondrial Ca uptake (Joiner, Nature). In this context, did you also 
re-test whether CaMKII-IN expression affects mitochondrial Ca uptake? And from a technical side, 
you may get a cleaner view on the actual mitochondrial Ca uptake if you perform the Ca retention 
assays on isolated mitochondria in the presence of cyclosporine A, since differential PTP activation 
(potentially controlled by CaMKII?) could blur your view on uptake by affecting Ca release via PTP. 
Therefore, I suggest to perform these experiments in the absence and presence of CsA. 

 

5) Figure 2L: In the methods you mention sequential additions of pyruvate/malate, rotenone and 
antimycin A. Which of these conditions do you show in the figure? Can you show all? Did you also 
measure H2O2 emission in the presence of ADP to simulate more physiological (respiring) 
conditions? Mitochondrial H2O2 emission is controlled also by NADPH and the transhdydrogenase 
(Nnt), which is not expressed in BL/6J mice. Do you know about the expression of the Nnt in your 
various mouse models? This may have an impact on the results of H2O2 emission and, if indeed 
most are in BL/6J background, should be at least critically discussed. 

 

6) Figure 3: Why did you use Cs-based bath and pipette solutions in your experiments determining 
cytosolic Ca transients? Would it not be better to allow EC coupling as during physiological 
conditions? And since the in vivo phenotype is a systolic form of heart failure, I would be interested 
in the amplitudes and kinetics of cytosolic calcium transients as well; from the traces you show one 
could imagine that in the mitoCaMKII, they are somewhat reduced? 

 

7) You mostly argue with reduced ATP, but you may consider that also an accumulation of ADP per 
se has a substantial impact on diastolic function (see Sequeira et al., J Physiol 2015), since ADP can 
induce diastolic dysfunction on the sarcomere level. Also, a change in the ATP/ADP ration and 
thereby, delta G ATP may impact the rate of SERCA, and SERCA function may be reflected by the 
rates of calcium decay in your cell experiments. Therefore, please add the kinetics to these results. 

 

8) Figure 5: Do you have any idea how complex I could be downregulated so strongly by mtCaMKII 
overexpression, and how changes of ATP could upregulate the expression of complex I (since CKmito 
expression partially improves complex I expression)? 

 

9) Figure 6: Your results of NADH/NAD+ in resting cardiac myocytes make sense, since in light of the 
upregulation of TCA cycle enzymes and downregulation of complex I, one indeed expects a more 
reduced redox state of NADH/NAD+. However, I did not understand why NADH becomes more 
oxidized in response to pacing, which is even more a situation of electrons flowing from the Krebs 
cycle into the respiratory chain and thereby, should aggravate any alterations in redox state which 
may be related to the alterations in complex I and Krebs cycle enzymes, respectively. Furthermore, 
one can assume that during pacing, the actual redox state of NADH/NAD+ is similar between both 
groups, since the WT start more oxidized but become more reduced, while the mtCaMKII start more 
reduced but become oxidized. So what is the net steady-state redox state of NADH/NAD during 
pacing? Finally, if you collect also the redox state of FADH2/FAD in these myocytes, you may get a 
good idea how your complex II upregulation affects the redox state of that pool (since FADH2 fuels 
into complex II). 

 

10) Figure 7: The ATP/ADP ratio is rather small, I would expect values of >100? 

 

11) You mention that in mtCaMKII mice, CKmito is hyperphosphorylated. How specific is this result? 
Is there a known phosphorylation site of CK by CaMKII? Is this phosphorylation blunted in CaMKII KO 
mice? And is CKmito downregulated in HF (for instance, in your MI model)?