PREreview of p38β/MAPK11 Deficiency Exacerbates Cardiac Structural and Electrophysiological Remodeling and Contributes to Immune Dysregulation in the Aging Heart

The p38 MAPK family has been shown to regulate cardiac remodeling and contribute to multiple cardiac dysfunctions. In myocardial injury models, p38 family members have been reported to have isoform specific roles in both worsening and ameliorating cardiac function. In these models, p38-beta has shown to have a cardio protective effect. In this study, the authors investigate whether cardioprotective effects of p38 extend into aging.

The manuscript claims the authors have identified p38-beta as a critical regulator of structural, electrophysiological, and immune homeostasis in the aging heart and demonstrate that its loss promotes maladaptive remodeling and arrhythmogenic vulnerability. Although the evidence supports p38-beta deletion contributes to cardiac abnormalities, it is unclear what molecular mechanisms mediate these changes. In addition, there are several major concerns regarding the interpretation of findings, the experimental design, the statistical analysis, and the strength of the conclusions. Overall, this study is an important step in characterizing the effects of systemic deletion of p38-beta in the aged heart and provides a foundation to investigate how p38-beta deletion regulates the molecular mechanisms underlying these cardiac changes.

Major Points

1)The authors compare structural, functional, transcriptomic profiles, and immune microenvironments between wild type and p38-beta knockout mice to show cardiac changes in aging. However, by not including a young control for wild type and knock out mice I can’t decipher whether these changes are dependent on age. The authors may consider including additional young cohorts to establish whether the reported changes only appear with age in the young p38-beta knock out mice. Including these controls will strengthen the papers age related conclusions by helping to exclude the possibility that effects are dependent on the p38-beta deletion and not age.

2)Echocardiographic analyses in Supplemental Fig 1 shows a decrease in body weight and the LVpWd only in female KO mice (panel B) and an increase in LV/Body Weight only in male KO mice (panel A). However, to test whether there is a difference between males and females with p38-beta deletion it needs to be determined if there is an interaction between genotype and sex. This can be accomplished using a two-way ANOVA test that accounts for both genotype and sex on the measured parameters. Others have reported sex-differences in p38-beta KO mice with female mice showing an increase in maladaptive remodeling compared to male mice under DOX treatment (DOI: 10.1152/ajpheart.00458.2023). Consider reanalyzing this data with the appropriate statistical test to confirm there are no sex-differences. This is an important comparison while interpreting the data.

3)Figure 4 asks the question whether p38-beta deletion alters calcium handling across different pacing frequencies. The data shows calcium transient parameters are slower in rise time (panel C), CaTD 30 and CaTD 50 (panels D & E). Since the analysis compares changes over different cycle lengths to get the restitution curves, it is unclear whether calcium transient parameters were significant at all pacing frequencies or just certain BCLs. There is large variance in multiple BCLs (Basic Cycle Lengths) across the panels listed above. The authors may consider running additional statistical analysis to determine if there are differences in specific BCLs and not only in a comparison of overall curve between the genotypes. Providing this information will help determine if these differences are dependent on pacing frequency and help evaluate the strength of the interpretation.

4)The manuscript claims p38-beta alters calcium handling in early calcium reuptake. In Figure 4, calcium transients show differences in CaTD at 30 and 50, but these results in combination with slower CaRT doesn’t rule out the possibility that the slower RT is contributing to longer transient duration instead of impaired calcium reuptake. Please provide a description of how CaTD’s were calculated to verify if the slower CaRT contributes. In addition, the authors may consider choosing a different representative trace that reflects the differences in both delayed CaTD’s and RT to provide visual clarity. The current trace doesn’t reflect a change in CaRT (panel B). To further improve clarity, the authors may consider including a different style of representation for the trace that is an averaged trace with highlights that show variance in transients.

5)In Figure 5, the manuscript claims p38-beta deletion increases susceptibility to ventricular arrhythmias under 300 nM isoproterenol and a burst pacing protocol. However, in panels I and J, the bursting protocol differs between genotypes with a pace every 100 ms in wild type mice compared to every 450 ms in the knockout mouse. Different pacing frequencies between genotypes are not an appropriate comparison. Please consider providing a detailed description of the bursting protocol(s) used in this analysis. Providing a detailed description of the bursting protocol in addition to addressing the pace discrepancy in comparing EADs between genotypes would provide more clarity in this experiment.

6)In Figure 6, the authors compare protein levels of cytokines and chemokines between wild type and p38-beta knockout mice by performing a Student’s t-test on each protein. However, performing multiple t-tests increases the chances of a false-positive by not accounting for multiple comparisons and may artificially inflate significance, which may or may not change the interpretation of the data. The authors could consider adapting the statistical strategy described in Figure 7 and provide an adjusted p-value and a test for false discovery rate. This would improve the statistical rigor of the analysis.

7)While the findings suggest p38-beta may be correlated with cardiac protection, using a germline knockout mouse introduces the possibility that the reported findings may be influenced by compensatory mechanisms from other p38 isoforms. Although functional studies would be necessary to evaluate this possibility, the authors could provide a preliminary analysis of gene expression of the remaining p38 MAPK family members from the bulk sequencing already performed in Figure 8 to address this concern as best as possible without running additional experiments. Additionally, acknowledging this as a limitation of the study would be sufficient and benefit the paper by clarifying the scope of the claims that can be drawn from the evidence provided.

Minor Points:

1)There are many occasions where comparisons of animals are separated, but they don’t have the same scale bar on the y-axis. Consistent y-axis labels help improve ease of visual comparison. Please check Supplemental Figures 1, 2, & 3.

2)Figure 1S shows representative images of ventricle cross sectional slices Although the cross-sectional areas were quantified in Figure 1T, including a high mag image of a single cross sectional area from each genotype will help readers visualize the hypertrophic phenotype.

3)Supplementary Tables 2 and 3 show comparisons between genotyped males and females for echocardiography and electrocardiography. Unlike Supplementary Table 1, they don’t report heart rate. Including this parameter in the table will improve consistency in data reporting.

4)In Figure 2, the manuscript claims prolonged QT and QTc interval (panels G and I) in p38-beta deficient mice indicate deficiencies in ventricular repolarization and depolarization. However, since the QRS duration is not statistically significant, this suggests that prolongation primarily reflects changes in the ventricular repolarization. The authors may consider separating the differences in ventricular depolarization and repolarization by acknowledging that in 20 month old female p38-beta deficient mice, prolongation of the QRS interval (Supplemental Figure 3 panel C) in addition to the QT and QTc interval (Supplemental Figure 3 panel D & F) were significantly different from wild type female mice. Distinguishing between these findings will clarify the interpretation of the ECG results.

5)In Figure 3 and 4 (B panels) the y-axis of the representative traces is missing. It is hard to decipher whether the traces are normalized or raw fluorescence data. By including a description of how the traces were normalized in the methods section would improve reader clarity.

6)Please add the cryosection slice thicknesses in the histology staining’s in the figure legends or methods section. Including this information will improve the reproducibility of the staining protocols.

I am one of 2 participants from the same research lab that reviewed this preprint as part of the Transparent and Accountable Peer Review training program. This report was written with the support of my research mentor.

Competing interests

The authors declare that they have no competing interests.

Use of Artificial Intelligence (AI)

The authors declare that they did not use generative AI to come up with new ideas for their review.