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Concise appraisal

One line — Cardiomorphs are a high-fidelity iPSCderived human heart organoid that recapitulate multicellular myocardium and model KearnsSayre syndrome (KSS); an FDAapproved drug screen identified Betaxolol and Nadolol as mutationagnostic agents that rebalance mitochondria by coupling selective mitophagy with biogenesis and restore contractility in KSS tissues Cardiomorphs model and betablocker rescue.

Paper Review Human heart organoids reveal a regenerative strategy for mitochondrial disease

Structured critical analysis

1) What the paper does (facts with citations)

• Establishes cardiomorphs (CDMs): selforganising, iPSCderived cardiac organoids that include cardiomyocytes, endothelial, mural, neuronal, macrophage and stromal lineages and mature along a developmental timeline comparable to mouse and human stages Cardiomorph derivation and benchmarking.
• Models KearnsSayre syndrome (KSS) in CDMs using patient iPSCs with large mtDNA deletions, reproducing hallmark molecular, structural and functional phenotypes (reduced mtDNA content, loss of deleted transcripts, cristae disruption, reduced OXPHOS, glycolytic shift, contractile dysfunction and hypertrophy) KSS CDM phenotype.
• Performs complexome and pulseSILAC analyses to show destabilisation of MICOSSAMM superassemblies and reduced respiratory subunit turnover in KSS CDMs, supporting a mechanistic deficit in cristae architecture and 'microregeneration' of OXPHOS components Complexome and turnover.
• Carries out a targeted screen of approved drugs affecting oxygen metabolism and identifies Betaxolol (β1selective) and Nadolol (nonselective) as agents that reduce oxygen consumption, increase intracellular oxygenation, induce selective mitophagy of depolarised mitochondria, stimulate mitochondrial biogenesis, reduce mutant mtDNA fraction, and restore contractility in KSS CDMs Drug screen and mechanisms.

2) Strengths

• Multicellular, developmental benchmarking increases physiological relevance versus simple 2D cardiomyocytes; single nucleus RNAseq with comparison to human ventricular biopsies strengthens claims of tissuelike transcriptional composition snRNAseq benchmarking.
• Deep multimodal phenotyping (TEM, complexome, pulsedSILAC, metabolomics, Seahorse, live imaging) provides convergent evidence for mitochondrial structural and functional pathology in KSS CDMs and for drug action — mechanistic depth is unusually high for an organoid study Multimodal mechanistic assays.
• Practical translational tilt: using clinically approved βblockers lowers the barrier for repurposing and early clinical testing, and the authors explicitly discuss dosing/tissue penetration caveats Translational perspective.

3) Limitations and critical caveats

1. In vitro organoid constraints: despite multi-lineage content, CDMs lack full systemic physiology (blood flow, native immune system, endocrine inputs) that shape mitochondrial selection pressures in vivo. Therefore rescue in CDMs does not guarantee in vivo efficacy or safety In vitro versus in vivo.
2. Heteroplasmy complexity and cell specificity: measured reduction in mutant mtDNA fraction by targeted qPCR is promising, but questions remain about celltype specific heteroplasmy shifts (cardiomyocytes vs nonmyocytes), absolute amounts, and the durability of the shift after drug withdrawal Heteroplasmy measurement limits.
3. Mechanistic depth but partial chain of causation: authors show reduced oxygen consumption, increased intracellular oxygenation, enhanced mitophagy and biogenesis; however, the precise signalling cascade linking βadrenergic blockade to mitophagy induction and biogenesis (for example via PGC1α/TFAM modulation, mTOR/AMPK, ROS/HIF axes) requires more direct perturbation evidence (genetic or pharmacologic) to prove necessity and sufficiency Mechanistic chain gaps.
4. Drug class specificity and offtarget concerns: Betaxolol and Nadolol affect adrenergic signalling broadly; systemic use in patients with mitochondrial disease could have cardiovascular and metabolic side effects (bradycardia, hypotension), and the effective tissue concentrations needed for organellelevel effects are unknown; longterm safety in mitochondrial disease patients is untested and not recommended here Translational safety concerns.
5. Sample sizes and iPSC line variability: many organoid studies show interline variability; the paper reports n=3 or 4 replicates for several assays which is common for organoid work but warrants replication across additional patient lines and independent labs to assess generality and reproducibility Sample and line variability.

4) Reproducibility and data availability

Authors deposited raw proteomics/complexome datasets to PRIDE (PXD063313, PXD063322111) and provide extensive methods (LCMS parameters, snRNAseq pipelines, Seahorse protocols) which supports reproducibility; reviewer access credentials are noted in the supplement (good transparency). However, full raw sequencing read access and processed single nucleus matrices would accelerate reanalysis; links to PRIDE given in the manuscript were included but direct downloads should be confirmed because PRIDE entries sometimes require public release steps Data deposition PRIDE.

5) How convincing is the central claim (mutationagnostic organelle selection via oxygen modulation)

The authors present convergent evidence that pharmacological suppression of oxygen consumption (via βblockers) increases intracellular oxygenation and biases mitochondrial population dynamics toward wildtype organelles by increasing mitophagy of depolarised mitochondria while stimulating biogenesis of polarised mitochondria. The claim is mechanistically plausible and supported by multiple assays (oxygen sensors, JC10, mitophagy imaging, mtDNA qPCR, Seahorse), but remains correlative without perturbation of the proposed intermediates (e.g. block mitophagy genetically or inhibit biogenesis to test necessity). Therefore the central claim is promising and wellsupported but not proven to the level of causal sufficiency Evidence strength evaluation.

6) Suggested followup experiments to strengthen claims (concise)

1. Genetic blockade of mitophagy (e.g. PINK1 or Parkin knockdown/KO in CDMs) to test whether Betaxolol/Nadolol require mitophagy to reduce mutant mtDNA fraction and restore contractility.
2. Block mitochondrial biogenesis (e.g. PGC1α or TFAM knockdown) to test necessity of biogenesis for the rescue.
3. Lineageresolved heteroplasmy: singlecell or singlemitochondrion assays (e.g. singlecell mtDNA sequencing or FISH) to map which cell types lose mutant genomes.
4. In vivo translation: test pharmacokinetics, myocardial tissue penetration, heteroplasmy shifts and cardiac function in an animal model with mtDNA deletions or heteroplasmy (if available) before clinical trials.

7) Practical translational assessment

Repurposing Betaxolol/Nadolol is attractive but clinical translation requires: (1) demonstration of efficacy and safety in an appropriate in vivo model, (2) PK/PD to define myocardial exposures required for organellelevel effects, (3) assessments of offtarget cardiovascular effects in patients already prone to conduction disease (KSS patients frequently have conduction defects), and (4) longterm studies to ensure durable heteroplasmy improvements and absence of adverse remodelling Translational roadmap.

8) Blindspots, biases and sources of error to watch

• Publication and positive result bias: organoid studies that produce clean therapeutic effects may be preferentially published; independent replication is essential.
• Financial or translational incentive bias: repurposing approved cardiovascular drugs has commercial appeal — examine COI statements and funding sources (authors declared multiple public grants; COI not flagged) Funding and COI.
• Model generality: KSS is a prototypical deletion syndrome — other mtDNA lesions (point mutations, tRNA defects) may not respond similarly to oxygen modulation.

9) Recreated figure suggestions and interactive data to request

I recommend the authors or subsequent reanalyses publish: (1) processed snRNAseq matrices for independent celltype heteroplasmy and expression analyses; (2) raw Seahorse traces and JC10 plate reader files; (3) singleorganoid timecourses of heteroplasmy during treatment and washout to judge durability.

Summary judgement

This is a technically strong, mechanistically rich paper that advances organoid modelling of cardiac mitochondrial disease and nominates a plausible, mutationagnostic therapeutic strategy. The evidence is convergent and substantial within the organoid model, but key causal links (necessity experiments) and in vivo validation are still required before clinical translation. The conceptual novelty (exploiting organelle competition via oxygen modulation) is high and could be broadly influential if replicated across other models and lesions Conceptual novelty.

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