Hsp22 Deficiency Induces Age-Dependent Cardiac Dilation and Dysfunction by Impairing Autophagy, Metabolism, and Oxidative Response

Round 1

Reviewer 1 Report

Summary: In this study the authors examine the effect of Hspb8/Hsp22 deletion on cardiac function with aging. They find that loss of the protein results in progressive dilated cardiomyopathy that they associate with loss of BAG3 expression, and defects in autophagy and metabolic activity. In addition, aged hearts lacking Hsp22 showed evidence of increased oxidative damage.

Overall, this is a very good study. The experiments are appropriate and the conclusions are logically supported by the data. I have no major comments.

Minor Comments:

1. Please correct sentence fragment on page 2, line 92/93.
2. Please provide a reference to mouse age/human age equivalence on page 5, line 181.

Author Response

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Author Response File: Author Response.pdf

Reviewer 2 Report

Here, the authors characterize the role of HSP22 in cardiac ageing by using KO mice with in vivo and in vitro approaches.  They certainly establish an important role for HSP22 in ageing, but mechanistic data is somewhat lacking.  Overall I found this to be a fairly well designed study and well written paper, which will be of interest to scientists in the field.  However, I do have some concerns with the way certain data are presented, and I believe that a few additional experiments will be necessary. 

Major points:

Figure 1: Since HW/TL ratios are being shown at multiple time points, this leads me to believe that a separate group of mice was used for each study group, since measuring HW:TL length requires killing a mouse.  Is this correct?  If so, all of the graphs in Fig 1 should be changed to show young/middle/old mouse data separately.  Showing the data as connected points between young and old implies it is all data taken from the same mice over time, which is misleading.   

Figure 1:  Please provide representative images showing differences in CSA and fibrosis (aka WGA and Picrosirius red staining) between different groups in old mice.  

Figure 2: Please provide representative M-mode echocardiographic images for each group in old mice. 

Page 7 line 273, referring to Figure 2: “In addition, older KO mice exhibited a remarkable reduction in HR” – this may not demonstrate a phenotype so much as an issue with recording echo data.  Ideally heart rate should be very similar for all groups when recording anesthetized echo data, as significant differences in HR can seriously affect contractile parameters obtained from echo.  Fortunately, the difference in HR here is pretty small, but I certainly wouldn’t claim it to be a phenotype. 

Figure 5-6: While the extensive Western blotting data the authors show is certainly interesting, and likely indicates significant differences in metabolic flux between WT and KO mice, it doesn’t prove that metabolic changes are indeed occurring.  Changes in protein expression do not necessarily cause changes in cellular or organismal behavior.  This data would be far more convincing in combination with one or more assays directly interrogating cardiac metabolism: for example, direct measurement of metabolite levels, metabolic flux assays, measurement of ATP levels, or even enzymatic activity assays of key metabolic enzymes.  Please include such data in the revision.  At a minimum, I would strongly recommend measuring ATP levels. 

Figure 7: The increased levels of oxidative damage markers in older-HSP22 KO mice is interesting, but again is descriptive.  What is this oxidative damage doing to cause loss of cardiac function?  A possible explanation is increased cardiac apoptosis.  It would be helpful to see measurements of apoptosis (such as TUNEL staining on heart sections, or cleaved caspase 3 staining or protein expression in heart lysates) or other more mechanistic studies here.

Minor points:

Figure 5: Why was CPT1 not blotted for in young mice? Since it was in older mice…

Page 2 line 92-93: “Hsp22 KO mice during the aging transition.”  This isn’t a complete sentence; please revise.

Page 5 line 186: “As shown in Fig1A-B, compared to the young mice, WT mice exhib-186 ited progressively increased LV posterior wall thickness at end-systole and end-diastole…”  Saying “Compared to young mice, WT mice showed…” makes it sound like the young mice aren’t WT.  Please rephrase this. 

Page 5 line 212: “However, middle-aged Hsp22 KO mice revealed a significantly dilated cardiomyopathy (DCM)” – cardiac dilation and DCM aren’t the same thing.  I don’t think you can claim these mice have DCM without showing considerably more data to prove this. 

Page 14, line 501: “In addition, our results also 500 showed that Hsp22 is utterly necessary for multiple physiological processes” – this is to me a significant overstatement.  If HSP22 were utterly necessary, these processes wouldn't simply be depressed; they wouldn’t occur at all and HSP22-KO would probably be embryo lethal.

Author Response

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Author Response File: Author Response.pdf

Reviewer 3 Report

The manuscript by Wu et al. analyzes the role of the small heat shock protein 22 (Hsp22; H11 kinase / HspB8 / aC-crystallin), that it  is predominantly expressed in skeletal and cardiac muscle, in heart homeostasis in an age-dependent fashion; this was previously unexplored. The central knockout (KO) model was previously generated and authors demonstrated a critical role of Hsp22 in protecting heart against cardiac stress.

In this study, authors show that Hsp22 deletion, compared with age-matched wild-type (WT) littermates, induces a progressive cardiac dilation paralleled with a functional declined function along the aging transition (evaluated at three-time points: young (_^~17 weeks), middle-aged (_^~33 weeks), and older (_^~53 weeks), which are proposed to be equivalent to humans 26-30, 40-44 and 58-62years old, respectively). The data indicate that, notably, LV chamber size at end-systole (LVESD and LVESV) showed an even earlier increase in middle-aged KO mice vs. young KO mice, which may imply a gradual weakening in contractility starting at this age. The described cardiac dilation is further exacerbated in older KO mice vs. WT mice. In agreement, total heart weight (HW) and left ventricular weight (LVW), normalized to tibial length, showed no age-dependent variations in WT animals but very significant differences in old mice compared to middle age and young animals. In addition, results showed that cardiac myocyte size and cardiac fibrosis were significantly increased in old Hsp22 KO mice.  All these structural alterations precede and provoke a clear progressive functional deficit involving a severe contractile deficit, staring in the middle age that worsens in old animals.

Based on previously knowledge, authors evaluate, mechanistically, that the loss of Hsp22 impaired BCL-2–associated athanogene 3 (BAG3) expression and its associated cardiac roles; autophagy is significantly affected and multiple levels and authors present also a very interesting comparative analysis of cardiac energy metabolism in homeostasis, that demonstrated a significant altered FA oxidation and glucose metabolisms. The metabolic defects evolve with aging, affecting in old KO animals also to the mitochondrial pyruvate decarboxylation. BAG3 is a co-chaperone protein also abundantly expressed in the heart, and whose mutation or deficient expression has been associated with human cardiomyopathy.

Finally, all the structural and functionals alteration are interpreted in the context of a clear incremented oxidative stress using different molecular markers, with a different penetrance at different aging stages. Then, it is concluded that the progressive metabolic remodeling occurs along the anomalous cardiac aging and age-related cardiomyopathy, which is inherently triggered and advanced by a significant alteration of autophagy and  globally incremented oxidative stress.

Therefore, this study clearly show that Hsp22 plays an essential role in non-stressed heart during the early stages of aging, and its deficiency provokes a progressive functional defect. As stated by the authors, this model could be of great interest to understand the onset of some age-related cardiac pathologies with an unquestionable relevance for dilated cardiomyopathy.

The manuscript is interesting, well-developed, clearly written and very good presented. Below I indicate some anecdotic questions that could be interesting to be considered.

Major point

1. Although it can be inferred from western blot presented in Fig. 4, I think it would be interesting to include in a dedicated panel showing the evolution of Hsp22 with respect to aging. This could be complemented with the parallel analysis of BAG3, Hsp70 and other small heat shock proteins that interact with Hsp22.
2. The earlier marker of the incremented oxidative stress appears to be 8-OHdG, already 5-fold increase in young KO animals. It could be interesting to evaluate directly different ROS levels in early stages, to define more precisely the onset events.
3. For further development of the project it would interesting to know which of their dual activities (kinase and chaperone) would be more important for the interesting phenotype described.

Minor points

1. Page 6, line 220. Although indicated in the figure legend, first time is referred in the main text please indicate full description (TL).

Author Response

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Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Most of my concerns from the original submission have been addressed.  Thank you.  No further suggestions.