Endurance Exercise Ameliorates Aging-Related Bradyarrhythmia in Drosophila Resulting from miR-283 Knockdown in LN_vs

Reviewer #1 (Public review):

In this study, Li et al et al. investigated the role of miR-283 in regulating cardiac aging and its potential contribution to age-related bradyarrhythmia. Using Drosophila as a model, the authors demonstrated that systemic overexpression or knockdown of miR-283 induced age-associated bradycardia. Notably, the study found that miR-283 knockdown in ventral-lateral neurons (LNvs), rather than in the heart, was sufficient to induce bradyarrhythmia, an effect the authors linked to the upregulation of miR-283 expression in both the brain and heart. The study also explored the beneficial impact of exercise on cardiac aging, showing that endurance training mitigated bradyarrhythmia, correlating with reduced miR-283 accumulation in the brain and myocardium.

The conclusions of this paper are mostly well supported by data; however, some concerns arise from the unexpected finding that bradyarrhythmia was triggered by miR-283 knockdown in LNvs rather than in the heart, suggesting a non-cell-autonomous mechanism. A more precise mechanistic explanation linking miR-283 dysregulation in LNvs to cardiac dysfunction would strengthen the study's conclusions. While the authors propose cwo as a potential target of miR-283, no functional experiments were conducted to confirm its role in mediating miR-283's effects. Additionally, it remains unclear whether reduced miR-283 levels in LNvs lead to accelerated aging rather than a cardiac-specific effect. Likewise, the potential influence of miR-283 on the circadian clock and its broader impact on aging warrant further investigation.

Major Comments:

(1) A significant concern arises from the unexpected outcome observed in miR-283 knockdown in LNvs, which suggests a non-cell-autonomous mechanism. Elucidating the mechanisms by which miR-283 deficiency leads to the observed phenotypes would provide a more comprehensive understanding of the study's implications.

(2) The authors propose cwo as a potential target of miR-283; however, no functional experiments were conducted to confirm its role in mediating miR-283's effects. Similarly, direct evidence demonstrating that cwo is a bona fide target of miR-283 in LNvs should be provided.

(3) It remains unclear whether miR-283 knockdown in LNvs results in accelerated aging rather than a cardiac-specific effect. This hypothesis is supported by observations that pdf>miR-283SP animals exhibit systemic premature senescence (elevated SA-β-gal activity in both the heart and brain), cardiac dysfunction, impaired climbing ability, and reduced lifespan.

(4) The finding that reduced miR-283 levels in LNvs lead to accelerated aging raises an important, yet unexplored, question: does miR-283 influence the circadian clock, thereby broadly affecting aging?

Two aspects of this question should be addressed:
(a) Is the circadian rhythm disrupted in miR-283 knockdown experiments?
(b) Do circadian rhythm defects impact aging?

(5) The authors state that miR-283 knockdown in LNvs led to bradyarrhythmia, which was mainly caused by miR-283 upregulation in the whole brain and heart. However, it is unclear which experiments support this conclusion. Could the authors clarify this point?

(6) Given that miR-283 expression varies with age, could the upregulation of miR-283 in both the brain and heart be a consequence of accelerated aging rather than a specific effect of miR-283 knockdown in LNvs?

(7) While the beneficial effects of exercise on cardiac function appear clear, the claim that this effect is mediated through miR-283 function in LNvs seems premature. The data suggest that exercise-induced improvement occurs in both wild-type and miR-283-SP animals, raising the possibility that exercise acts through a miR-283-independent mechanism.

Reviewer #2 (Public review):

Summary:

The manuscript presents findings that indicate a role in controlling Drosophila heart rate for a conserved miRNA (miR-238 in flies). Further, the manuscript localizes the relevant tissue for the function of this miRNA to a subset of neurons that are heavily involved in circadian regulation, thus presenting an interesting mechanistic link between the circadian system and heart rate. Either ubiquitous knockout or ubiquitous overexpression negatively impacts several aspects of heart performance, with a pronounced effect on heart rate. Interestingly, knockdowns in the heart itself are innocuous, but knockdown in LNvS neurons recapitulates the effect on heart rate. Authors use bioinformatics to identify the clockwork orange (cwo) gene as a potential target and validate that cwo expression is reduced when miR-238 is knocked down in LNvS neurons in vivo and also validate that cwo is regulated by miR-238 in cell culture luciferase assays. Exercise shows a modest ability to restore normal cwo expression and a trend toward an effect on survival, but shows a much stronger rescue of the heart rate phenotype.

Strengths:

Evidence is strong for the effect of miR-238 in pdf-positive neurons on the control of heart rate and for cwo as a downstream effector of miR-238.

Work to identify specific targets of miR-283 is well-done and successfully identified a key downstream regulator in cwo.

The potential mechanism using miR-238 to link circadian neurons to heart rate regulation is novel and exciting.

Weaknesses:

The evidence that this is related to normal aging is rather weak, and the effect of exercise on the observed parameters is small and not necessarily working through the miR-238/cwo mechanism.

The authors seem to be conflating two hypotheses in their interpretations. Is miR-283 working through circadian mechanisms or age-related mechanisms? While it is true that aging tends to reduce heart rate, I don't think that means that any intervention that reduces heart rate is causing "senescence". Similarly, reduced survival in miR-283 knockdown flies does not prove that miR-283 promotes healthy aging per se, just that miR-283 is required for health regardless of age.

Survival reduction is quite modest which does not necessarily support the idea that the bradycardia is causing major health issues or premature senescence for the flies. The interpretation of the longevity experiments throughout the manuscript seems overstated.

The study would benefit greatly from a direct test of the author's proposed pathway for exercise to improve bradycardia.

The statement in the discussion "inducing endurance exercise of anti gravity climbing in flies with miR-283 knockdown in LNvs can improve bradyarrhythmic features by decreasing brain miR-283 expression" is not fully supported by data in the paper. There is an association there, but it cannot be said to be the full cause (or even required) without doing more experiments

The summary figure includes both data-supported mechanistic relationships and mechanisms that are inferred or assumed.