Myofibroblast senescence promotes arrhythmogenic remodeling in the aged infarcted rabbit heart
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eLife assessment
This study describes important results and convincing evidence linking myofibroblast senescence in the aged heart with a pro-arrhythmogenic phenotype. This is in turn related to higher mortality after myocardial infarction in the aged rabbit heart. These constitute important empiric as opposed to detailed findings. They nevertheless will be of interest to clinician scientists studying cardiac function and disease.
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Abstract
Progressive tissue remodeling after myocardial infarction (MI) promotes cardiac arrhythmias. This process is well studied in young animals, but little is known about pro-arrhythmic changes in aged animals. Senescent cells accumulate with age and accelerate age-associated diseases. Senescent cells interfere with cardiac function and outcome post-MI with age, but studies have not been performed in larger animals, and the mechanisms are unknown. Specifically, age-associated changes in timecourse of senescence and related changes in inflammation and fibrosis are not well understood. Additionally, the cellular and systemic role of senescence and its inflammatory milieu in influencing arrhythmogenesis with age is not clear, particularly in large animal models with cardiac electrophysiology more similar to humans than previously studied animal models. Here, we investigated the role of senescence in regulating inflammation, fibrosis, and arrhythmogenesis in young and aged infarcted rabbits. Aged rabbits exhibited increased peri-procedural mortality and arrhythmogenic electrophysiological remodeling at the infarct border zone (IBZ) compared to young rabbits. Studies of the aged infarct zone revealed persistent myofibroblast senescence and increased inflammatory signaling over a 12-week timecourse. Senescent IBZ myofibroblasts in aged rabbits appear to be coupled to myocytes, and our computational modeling showed that senescent myofibroblast-cardiomyocyte coupling prolongs action potential duration (APD) and facilitates conduction block permissive of arrhythmias. Aged infarcted human ventricles show levels of senescence consistent with aged rabbits, and senescent myofibroblasts also couple to IBZ myocytes. Our findings suggest that therapeutic interventions targeting senescent cells may mitigate arrhythmias post-MI with age.
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eLife assessment
This study describes important results and convincing evidence linking myofibroblast senescence in the aged heart with a pro-arrhythmogenic phenotype. This is in turn related to higher mortality after myocardial infarction in the aged rabbit heart. These constitute important empiric as opposed to detailed findings. They nevertheless will be of interest to clinician scientists studying cardiac function and disease.
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Reviewer #1 (Public Review):
Baggett C., Murphy K. R., and Sengun E. et al. investigated cell senescence as the basis of pro-arrhythmogenic changes associated with myocardial infarction in the aged heart using the rabbit as a model, with validation of senescence markers on human heart specimens. The study is interesting and addresses a relevant biological and health issue. The authors demonstrate that aged rabbits are prone to arrhythmogenesis associated with higher mortality within 72 h after induction of myocardial infarction. Analysis of scar morphology determined that fibrosis is not sufficient to explain age-associated arrhythmogenesis. Instead, the authors show that senescence, assessed by -galactosidase activity, expression of regulators of the senescence-associated secretory phenotype, and H2AX, is increased in myofibroblasts …
Reviewer #1 (Public Review):
Baggett C., Murphy K. R., and Sengun E. et al. investigated cell senescence as the basis of pro-arrhythmogenic changes associated with myocardial infarction in the aged heart using the rabbit as a model, with validation of senescence markers on human heart specimens. The study is interesting and addresses a relevant biological and health issue. The authors demonstrate that aged rabbits are prone to arrhythmogenesis associated with higher mortality within 72 h after induction of myocardial infarction. Analysis of scar morphology determined that fibrosis is not sufficient to explain age-associated arrhythmogenesis. Instead, the authors show that senescence, assessed by -galactosidase activity, expression of regulators of the senescence-associated secretory phenotype, and H2AX, is increased in myofibroblasts compared to endothelial cells in infarcted aged rabbit hearts. Accordingly, H2AX was detected in αSMA+ cells in human-aged hearts. The authors tested the influence of myofibroblasts on cardiomyocyte electrophysiology by exposing cardiomyocytes in vitro to conditioned media from fibroblasts in which senescence was induced by treatment with etoposide. Such treatment did not affect action potential duration, leading the authors to conclude that senescent fibroblasts are unlikely to influence cardiomyocytes through paracrine signaling. Instead, the authors propose a possible yuxtacrine effect. To test this, they performed immunofluorescence to infer potential myofibroblast-cardiomyocyte coupling by the presence of connexin 43 in the cell-cell interphase and tested the potential electrophysiological effects of coupling using a computational model.
The analysis of peri-procedure mortality, arrhythmogenesis, and senescence in young and aged rabbits subjected to myocardial infarction is valuable, represents a significant amount of work, and the results support the conclusions drawn. Stronger evidence that senescent myofibroblasts couple with cardiomyocytes in the aged heart is needed to support the proposed model.
The authors conclude a propensity of myofibroblast senescence based on the finding that 80% of αSMA+ cells are also positive for H2AX. Showing the immunofluorescence results on hearts 2 weeks after MI would help to more convincingly illustrate the result. From these immunofluorescence experiments, it is also concluded that most of the persistent senescent cells in the scar correspond to myofibroblasts. The results presented show a continued increase in the proportion of H2AX+ cells in aged hearts up to 12 weeks after myocardial infarction. According to results in Figures 4F and G, these cells do not correspond to either myofibroblasts or endothelial cells. Given that H2AX+ cells are significantly increased in the aged heart, could the results presented suggest that a different cell type might be more important for the aged heart's response to MI? Providing some insight into the identity of these cells would be helpful to better understand the results presented. For example, cardiomyocyte senescence could contribute to arrhythmic phenotypes.
The results presented show that treatment of cardiomyocytes with conditioned media from, and co-cultured with, senescent myofibroblasts did not change action potential duration in cardiomyocytes. This led to the conclusion that paracrine signalling is unlikely to contribute to a pro-arrhythmogenic phenotype. It is possible that cardiomyocytes do couple with myofibroblasts in the in vitro system used. In which case, the results presented would not favor the proposed model. Another important possibility to be considered is that myofibroblasts might not have produced senescence-associated secretory phenotype-mediators at concentrations high enough to alter action potential duration in the conditions tested. Experimental evidence of the levels of selected mediators of the senescence-associated secretory phenotype in conditioned media would help assess a potential paracrine effect.
The evidence of coupling, i.e., the presence of connexin-43 in the interphase between αSMA+ and cardiomyocytes needs to be strengthened. Perhaps analyzing Z-stack 3D reconstructions would help to better define adjacent cells and more precisely reveal the localization of connexin-43.
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Reviewer #2 (Public Review):
- A detailed step-by-step approach to validation of some previously known outcomes.
- Useful for more focus to be placed on data from the second half of the paper.
- Some reflection on the media used to study paracrine effects is needed - more experiments here would be beneficial.
- Path clamp experiments - how does bath solution alter the effect of any limited paracrine effect - we are removing cells from the treatment media and putting them in physiological solutions - an opportunity to recover?
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