Functional High Throughput Drug Screening Reveals Cyproheptadine as a Novel Treatment for LMNA-related Cardiomyopathy

Reviewer #1 (Public review):

Summary:

In this study, the authors use patient-specific induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) from six patients carrying three distinct pathogenic LMNA variants to investigate disease mechanisms underlying LMNA-associated dilated cardiomyopathy (DCM). The authors report shared abnormalities in nuclear morphology, electrophysiology, calcium handling, and contractility across all patient-derived lines and demonstrate that correction of representative LMNA variants by CRISPR/Cas9 rescues several of these phenotypes. They further develop a high-throughput phenotypic drug screening platform using calcium transient measurements and identify cyproheptadine as the only compound among 1,280 FDA-approved drugs that consistently improves calcium transient abnormalities across all patient-derived lines.

The study addresses an important clinical problem and establishes a technically sophisticated patient-derived screening platform. However, while the experimental work is generally well executed, several of the major biological and translational conclusions are not sufficiently supported by the presented data.

Strengths:

The major strength of this study is the development of a patient-derived functional screening platform using multiple LMNA mutations rather than focusing on a single pathogenic variant. The inclusion of six patient-derived iPSC lines representing three distinct mutations increases the generalizability of the observations and allows identification of disease features that appear reproducible across different genetic backgrounds.

The phenotypic characterization is comprehensive and includes nuclear morphology, transcriptomics, manual and automated electrophysiology, calcium imaging, and impedance-based contractility measurements. Importantly, CRISPR-mediated correction of representative LMNA variants provides convincing evidence that the observed cellular abnormalities are directly attributable to the pathogenic variants.

Finally, the implementation of an unbiased high-throughput drug screen using patient-derived cardiomyocytes represents a valuable technical advance that could facilitate therapeutic discovery in inherited cardiomyopathies.

Weaknesses:

The principal weakness of the manuscript is that the central conclusions substantially exceed what is directly demonstrated by the data.

The manuscript repeatedly concludes that dysregulated calcium handling represents a shared pathogenic mechanism underlying LMNA-associated cardiomyopathy. However, the presented experiments establish only that abnormal calcium handling is a shared cellular phenotype across the studied variants. The data do not distinguish whether calcium dysregulation is a primary disease mechanism or whether it is secondary to the numerous upstream abnormalities already known to result from LMNA dysfunction, including altered nuclear architecture, defective mechanotransduction, chromatin remodeling, and transcriptional dysregulation. This distinction is critical because the manuscript repeatedly interprets correction of calcium handling as correction of the underlying disease process without directly demonstrating this relationship.

A related concern is the interpretation of the drug screening results. The primary screen is entirely based on normalization of calcium transient parameters (CTD75, FWHM, and T75-25). Consequently, the screen identifies compounds capable of correcting calcium cycling rather than compounds that necessarily modify disease biology. Although cyproheptadine subsequently improves impedance-derived contractile parameters, it remains unknown whether treatment rescues other defining features of LMNA cardiomyopathy, including abnormal electrophysiology, nuclear defects, transcriptional alterations, or broader cellular stress responses. Thus, the conclusion that cyproheptadine represents a "novel treatment" for LMNA-associated cardiomyopathy is considerably stronger than the evidence presented.

The mechanistic studies are also insufficient to support the proposed mode of action. The manuscript proposes CHRM2 as the likely mediator of cyproheptadine activity primarily because it is the only appreciably expressed known target in the transcriptomic dataset. However, no functional experiments test this hypothesis. Without genetic or pharmacological interrogation of CHRM2, the proposed mechanism remains speculative. Likewise, alternative mechanisms of cyproheptadine action, including serotonergic signaling, histamine receptor antagonism, direct calcium channel modulation, or antioxidant effects, are not investigated.

Another conceptual weakness is that the manuscript promises to distinguish both shared and variant-specific disease mechanisms but ultimately focuses almost exclusively on shared phenotypes. The transcriptomic analyses remain largely descriptive and are not leveraged to identify mutation-specific biological pathways or explain differences among the three LMNA variants. Given the unique cohort assembled in this study, this represents a missed opportunity to generate broader biological insight into LMNA-associated disease.

The transcriptomic analyses themselves would also benefit from more rigorous interpretation. RNA from multiple independent differentiations was pooled prior to sequencing, limiting assessment of biological variability and reducing confidence in statistical inference. Similarly, many functional analyses emphasize the number of wells, recording sweeps, or individual cells while the number of independent biological differentiations is less prominently presented. Greater emphasis on biological replication would strengthen confidence in the robustness of the findings.

Finally, the translational implications of the work should be interpreted more cautiously. All therapeutic studies are performed in relatively immature two-dimensional iPSC-derived cardiomyocytes. No validation is presented in engineered heart tissues, multicellular cardiac organoids, animal models, or human tissue. The current evidence supports the conclusion that cyproheptadine is a promising in vitro phenotypic modifier rather than an established therapeutic candidate for LMNA-associated cardiomyopathy.

Overall, this study establishes a valuable patient-derived platform for investigating LMNA-associated cardiomyopathy and demonstrates the utility of functional high-throughput screening in identifying compounds that improve disease-associated cellular phenotypes. However, the manuscript currently overstates both the mechanistic significance of calcium dysregulation and the therapeutic implications of cyproheptadine. A more restrained interpretation of the findings together with additional mechanistic validation would substantially strengthen the impact of the work.

Reviewer #2 (Public review):

Summary:

The authors conducted a functional high-throughput drug screening using hiPSC-CMs derived from patients with LMNA-DCM. Cyproheptadine emerged as a therapeutic candidate.

Strengths:

The screen appears well designed.

Weaknesses:

The single candidate that emerged from the screen, cyproheptadine, raises issues with potency. In addition, validation studies that are both expected and necessary for a drug proposed as a novel therapeutic for human cardiomyopathy have not yet been performed. Rigor could be improved once the basic mechanistic and validation studies discussed below have been performed.