MIRO1 controls energy production and proliferation of vascular smooth muscle cells

Reviewer #1 (Public review):

Summary:

In this paper, the authors investigate the effects of Miro1 on VSMC biology after injury. Using conditional knockout animals, they provide the important observation that Miro1 is required for neointima formation. They also confirm that Miro1 is expressed in human coronary arteries. Specifically, in conditions of coronary diseases, it is localized in both media and neointima, and, in atherosclerotic plaque, Miro1 is expressed in proliferating cells.

However, the role of Miro1 in VSMC in CV diseases is poorly studied, and the data available are limited; therefore, the authors decided to deepen this aspect. The evidence that Miro-/- VSMCs show impaired proliferation and an arrest in S phase is solid and further sustained by restoring Miro1 to control levels, normalizing proliferation. Miro1 also affects mitochondrial distribution, which is strikingly changed after Miro1 deletion. Both effects are associated with impaired energy metabolism due to the ability of Miro1 to participate in MICOS/MIB complex assembly, influencing mitochondrial cristae folding. Interestingly, the authors also show the interaction of Miro1 with NDUFA9, globally affecting super complex 2 assembly and complex I activity.

Finally, these important findings also apply to human cells and can be partially replicated using a pharmacological approach, proposing Miro1 as a target for vasoproliferative diseases.

Strengths:

The discovery of Miro1 relevance in neointima information is compelling, as well as the evidence in VSMC that MIRO1 loss impairs mitochondrial cristae formation, expanding observations previously obtained in embryonic fibroblasts.

The identification of MIRO1 interaction with NDUFA9 is novel and adds value to this paper. Similarly, the findings that VSMC proliferation requires mitochondrial ATP support the new idea that these cells do not rely mostly on glycolysis.

Weaknesses:

(1) Figure 3:

I appreciate the system used to assess mitochondrial distribution; however, I believe that time-lapse microscopy to evaluate mitochondrial movements in real time should be mandatory. The experimental timing is compatible with time-lapse imaging, and these experiments will provide a quantitative estimation of the distance travelled by mitochondria and the fraction of mitochondria that change position over time. I also suggest evaluating mitochondrial shape in control and MIRO1-/- VSMC to assess whether MIRO1 absence could impact mitochondrial morphology, altering fission/fusion machinery, since mitochondrial shape could differently influence the mobility.

(2) Figure 6:

The evidence of MIRO1 ablation on cristae remodeling is solid; however, considering that the mechanism proposed to explain the finding is the modulation of MICOS/MIB complex, as shown in Figure 6D, I suggest performing EM analysis in each condition. In my mind, Miro1 KK and Miro1 TM should lead to different cristae phenotypes according to the different impact on MICOS/MIB complex assembly. Especially, Miro1 TM should mimic Miro1 -/- condition, while Miro1 KK should drive a less severe phenotype. This would supply a good correlation between Miro1, MICOS/MIB complex formation and cristae folding.

I also suggest performing supercomplex assembly and complex I activity with each plasmid to correlate MICOS/MIB complex assembly with the respiratory chain efficiency.

(3) I noticed that none of the in vitro findings have been validated in an in vivo model. I believe this represents a significant gap that would be valuable to address. In your animal model, it should not be too complex to analyze mitochondria by electron microscopy to assess cristae morphology. Additionally, supercomplex assembly and complex I activity could be evaluated in tissue homogenates to corroborate the in vitro observations.

(4) I find the results presented in Figure S7 somewhat unclear. The authors employ a pharmacological strategy to reduce Miro1 and validate the findings previously obtained with the genetic knockout model. They report increased mitophagy and a reduction in mitochondrial mass. However, in my opinion, these changes alone could significantly impact cellular metabolism. A lower number of mitochondria would naturally result in decreased ATP production and reduced mitochondrial respiration. This, in turn, weakens the proposed direct link between Miro1 deletion and impaired metabolic function or altered electron transport chain (ETC) activity. I believe this section would benefit from additional experiments and a more in-depth discussion.

Reviewer #2 (Public review):

Summary:

This study identifies the outer‑mitochondrial GTPase MIRO1 as a central regulator of vascular smooth muscle cell (VSMC) proliferation and neointima formation after carotid injury in vivo and PDGF-stimulation ex vivo. Using smooth muscle-specific knockout male mice, complementary in vitro murine and human VSMC cell models, and analyses of mitochondrial positioning, cristae architecture, and respirometry, the authors provide solid evidence that MIRO1 couples mitochondrial motility with ATP production to meet the energetic demands of the G1/S cell cycle transition. However, a component of the metabolic analyses is suboptimal and would benefit from more robust methodologies. The work is valuable because it links mitochondrial dynamics to vascular remodelling and suggests MIRO1 as a therapeutic target for vasoproliferative diseases, although whether pharmacological targeting of MIRO1 in vivo can effectively reduce neointima after carotid injury has not been explored. This paper will be of interest to those working on VSMCs and mitochondrial biology.

Strengths:

The strength of the study lies in its comprehensive approach, assessing the role of MIRO1 in VSMC proliferation in vivo, ex vivo, and importantly in human cells. The subject provides mechanistic links between MIRO1-mediated regulation of mitochondrial mobility and optimal respiratory chain function to cell cycle progression and proliferation. Finally, the findings are potentially clinically relevant given the presence of MIRO1 in human atherosclerotic plaques and the available small molecule MIRO1.

Weaknesses:

(1) There is a consistent lack of reporting across figure legends, including group sizes, n numbers, how many independent experiments were performed, or whether the data is mean +/- SD or SEM, etc. This needs to be corrected.

(2) The in vivo carotid injury experiments are in male mice fed a high-fat diet; this should be explicitly stated in the abstract, as it's unclear if there are any sex- or diet-dependent differences. Is VSMC proliferation/neointima formation different in chow-fed mice after carotid injury?

(3) The main body of the methods section is thin, and it's unclear why the majority of the methods are in the supplemental file. The authors should consider moving these to the main article, especially in an online-only journal.

(4) Certain metabolic analyses are suboptimal, including ATP concentration and Complex I activity measurements. The measurement of ATP/ADP and ATP/AMP ratios for energy charge status (luminometer or mass spectrometry), while high-resolution respirometry (Oroboros) to determine mitochondrial complex I activity in permeabilized VSMCs would be more informative.

(5) The statement that 'mitochondrial mobility is not required for optimal ATP production' is poorly supported. XF Seahorse analysis should be performed with nocodazole and also following MIRO1 reconstitution +/- EF hands.

(6) The authors should consider moving MIRO1 small molecule data into the main figures. A lot of value would be added to the study if the authors could demonstrate that therapeutic targeting of MIRO1 could prevent neointima formation in vivo.

Reviewer #3 (Public review):

Summary:

This study addresses the role of MIRO1 in vascular smooth muscle cell proliferation, proposing a link between MIRO1 loss and altered growth due to disrupted mitochondrial dynamics and function. While the findings are potentially useful for understanding the importance of mitochondrial positioning and function in this specific cell type within health and disease contexts, the evidence presented appears incomplete, with key bioenergetic and mechanistic claims lacking adequate support.

Strengths:

(1) The study focuses on an important regulatory protein, MIRO1, and its role in vascular smooth muscle cell (VSMC) proliferation, a relatively underexplored context.

(2) It explores the link between smooth muscle cell growth, mitochondrial dynamics, and bioenergetics, which is a potentially significant area for both basic and translational biology.

(3) The use of both in vivo and in vitro systems provides a potentially useful experimental framework to interrogate MIRO1 function in this context.

Weaknesses:

(1) The central claim that MIRO1 loss impairs mitochondrial bioenergetics is not convincingly demonstrated, with only modest changes in respiratory parameters and no direct evidence of functional respiratory chain deficiency.

(2) The proposed link between MIRO1 and respiratory supercomplex assembly or function is speculative, lacking mechanistic detail and supported by incomplete or inconsistent biochemical data.

(3) Key mitochondrial assays are either insufficiently controlled or poorly interpreted, undermining the strength of the conclusions regarding oxidative phosphorylation.

(4) The study does not adequately assess mitochondrial content or biogenesis, which could confound interpretations of changes in respiratory activity.

(5) Overall, the evidence for a direct impact of MIRO1 on mitochondrial respiratory function in the experimental setting is weak, and the conclusions overreach the data.