Cell Biology

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

Hananeh Fonoudi author has email address
Ali Negahi Shirazi
Hui-Hsuan Kuo
Carlos G Vanoye
Xiaozhi Gao
Susan Doody
Mariam Jouni
Brian Lenny
Achal Neupane
Janavi Kotamarthi
Yadav Sapkota
Jane E Wilcox
Alfred L George Jr.
Paul W Burridge author has email address

Department of Pharmacology, Northwestern University Feinberg School of Medicine, Chicago, United States
Center for Pharmacogenomics, Northwestern University Feinberg School of Medicine, Chicago, United States
Department of Epidemiology and Cancer Control, St. Jude Children’s Research Hospital, Memphis, United States
Division of Cardiology, Department of Medicine, Northwestern University Feinberg School of Medicine, Chicago, United States

https://doi.org/10.7554/eLife.111946.1

Open access
Copyright information

Figures and data

Family pedigrees and schematic diagram of LMNA gene and protein.
A) Family pedigrees of all the individuals in the study. Circle and square indicate female and male individuals respectively. B) Summary of the LMNA variants and their corresponding gene and protein location. The code for each individual (LMNA1-6) is shown in the diagram based on the location and codon changes of the variant present in each individual. Below the gene panel the corresponding protein change for each variant is shown.

Assessment of nuclear membrane and transcriptome analysis of LMNA hiPSC-CMs.
A) Representative images of control and laminopathy hiPSC-CMs stained with TNNT2 and LMNA/C. Scale bar is 200 µm. B) Quantification of the nuclear membrane deformation in controls and LMNA hiPSC-CMs using ImageJ software. Each dot represents the number of assessed fields of view. Control N = 30, LMNA1 N = 5, LMNA2 N = 9, LMNA3 N = 10, LMNA4 N = 10, LMNA5 N = 8, LMNA6 N = 9. The dotted line indicates the value for the average of five control hiPSC-CMs. Data is analysed using one way ANOVA. **** indicate P-value < 0.0001. C) Unsupervised hierarchical clustering of control and laminopathy hiPSC-CMs. Heat maps indicating the gene expression of D) cardiac markers, E) intermediate filaments, F) direct targets of LMNA and G) ion channels, from each laminopathy patient compared to controls. All gene expression data is normalized to the average of controls.

Manual and automated patch clamp analysis of LMNA hiPSC-CMs.
Representative traces of spontaneous (A) and induced (B) action potential recordings of control, LMNA3 and LMNA4 hiPSC-CMs. Quantifications of C) V_max and D) action potential durations (Control N = 12, LMNA3 N = 9, LMNA4 N =8). Quantification of E) V₅₀ and F) slope of Na⁺ channel activation measured using SyncroPatch (Control N = 142, LMNA1 N = 25, LMNA2 N = 22, LMNA3 N = 23, LMNA4 N = 34, LMNA5 N = 17, LMNA6 N = 14). G) Representative Na⁺ channel voltage dependance of activation curves. Quantification of H) V₅₀ and I) slope of Na⁺ channel inactivation (Control N = 196, LMNA1 N = 32, LMNA2 N = 44, LMNA3 N = 29, LMNA4 N = 71, LMNA5 N = 55, LMNA6 N = 30). J) Representative Na⁺ channel voltage dependance of inactivation curves. Quantification of K) V₅₀ and L) slope of Ca²⁺ channel activation (Control N = 119, LMNA1 N = 28, LMNA2 N = 24, LMNA3 N = 48, LMNA4 N = 16, LMNA5 N = 27, LMNA6 N = 20). M) Representative Ca²⁺channel voltage dependance of activation curves. Quantification of N) V₅₀ and O) slope of Ca²⁺ channel activation with external Ba²⁺ (Control N = 138, LMNA1 N = 34, LMNA2 N = 23, LMNA3 N = 60, LMNA4 N = 23, LMNA5 N = 34, LMNA6 N = 21). P) Representative Ca²⁺channel voltage dependance of activation curves with external Ba²⁺. N represent the number of assessed cells. *, **, *** and **** indicate P-value < 0.05, 0.01, 0.001 and 0.0001 respectively.

Calcium transient and contractility of LMNA hiPSC-CMs.
A) Representative traces of calcium transients of LMNA hiPSC-CMs organised based on the location of their variants. B) Quantification of intracellular calcium traces (Control N = 53, LMNA1 N = 39, LMNA2 N = 35, LMNA3 N = 29, LMNA4 N = 26, LMNA5 N = 39, LMNA6 N = 32). C) Representative traces of impedance analysis of LMNA hiPSC-CMs organised based on the location of their variants. D) Quantification of impedance traces (Control N = 63, LMNA1 N = 37, LMNA2 N = 33, LMNA3 N = 20, LMNA4 N = 45, LMNA5 N = 50, LMNA6 N = 48). The dotted line indicates the average value for five control hiPSC-CMs. CTD₇₅: calcium transient duration 75%, FWHM: full width at half maximum, T_75-25: decay time from 75% to 25%. Data presented here are from at least three independent differentiations and the N number for calcium imaging indicates the number of assessed wells and for contractility analysis indicates the number of assessed sweeps. Data analysed using one way ANOVA. *, **, *** and **** indicate P-value < 0.05, 0.01, 0.001 and 0.0001 respectively.

CRISPR/Cas9 correction of LMNA variants.
Sanger sequencing of A) LMNA1, B) LMNA3, and C) LMNA5 before and after correction. D) Calcium transient (LMNA1 N = 37, LMNA1-Corr N = 24), E) impedance (LMNA1 N = 20, LMNA1-Corr N = 13), and F) nuclear membrane deformation (LMNA1 N = 10, LMNA1-Corr N = 10) analysis of LMNA1-Corrected hiPSC-CMs compared to LMNA1. G) Calcium transient (LMNA3 N = 64, LMNA3-Corr N = 94), H) impedance (LMNA3 N = 52, LMNA3-Corr N = 23), and I) nuclear membrane deformation (LMNA3 N = 10, LMNA3-Corr N = 9) analysis of LMNA3-Corrected hiPSC-CMs compared to LMNA3. J) Calcium transient (LMNA5 N = 150, LMNA5-Corr N = 195), K) impedance (LMNA5 N = 84, LMNA5-Corr N = 87), and L) nuclear membrane deformation (LMNA5 N = 10, LMNA5-Corr N = 11) analysis of LMNA5-Corrected hiPSC-CMs compared to LMNA5. CTD₇₅: calcium transient duration 75%, FWHM: full width at half maximum, T_75-25: decay time from 75% to 25%. Data presented here are from at least three independent differentiations and the N number for calcium imaging indicates the number of assessed wells, for contractility analysis indicates the number of assessed sweeps, and for blebbing the number of assessed fields of view. Data analysed using one way ANOVA. *, **, *** and **** indicate P-value < 0.05, 0.01, 0.001 and 0.0001, respectively.

Calcium transient analysis of LMNA hiPSC-CMs post sirolimus treatment.
Calcium transient analysis of A) LMNA1 (Control N = 53, LMNA1 N = 39, 10nM N = 6, 100nM N = 6, 1uM N = 6, 10uM N = 6, 100uM N = 6), B) LMNA3 (Control N = 53, LMNA3 N = 29, 10nM N = 12, 100nM N = 12, 1uM N = 12, 10uM N = 12, 100uM N = 12), and C) LMNA5 (Control N = 53, LMNA5 N = 39, 10nM N = 5, 100nM N = 5, 1uM N = 5, 10uM N = 5, 100uM N = 3) post treatment with different doses of sirolimus. The dotted line indicates the average value for five control hiPSC-CMs. CTD₇₅: calcium transient duration 75%, FWHM: full width at half maximum, T_75-25: decay time from 75% to 25%. Data presented here are from at least three independent differentiations and the N number for calcium imaging indicates the number of assessed wells and for contractility analysis indicates the number of assessed sweeps. Data analysed using one way ANOVA. *, **, *** and **** indicate P-value < 0.05, 0.01, 0.001 and 0.0001 respectively.

Unbiased high throughput drug screening and cyproheptadine response of LMNA hiPSC-CMs.
A) Calcium transient duration 75% of LMNA hiPSC-CMs post treatment with Prestwich drug library. The two red dotted lines indicate the average of control CTD₇₅ ± standard deviation. Each dot is the average of three different sets of experiments. B) Venn diagram presenting the overlap of the compounds from Prestwick drug library correcting calcium transient parameters of CTD₇₅, FWHM and T_75-25 for each LMNA hiPSC-CMs. C) Venn diagram presenting the overlap of the compounds alleviating impaired calcium transient phenotype for each LMNA hiPSC-CMs. D) The number of drugs that corrected all the impaired aspects of calcium transients that were shared between different number of laminopathy patients. Calcium transient analysis of E) LMNA1 (Control N = 53, LMNA1 N = 39, 1nM N = 18, 10nM N = 18, 100nM N = 18, 1uM N = 18, 10uM N = 18, 100uM N = 18), F) LMNA3 (Control N = 53, LMNA3 N = 95, 1nM N = 29, 10nM N = 29, 100nM N = 29, 1uM N = 29, 10uM N = 29, 100uM N = 29) and G) LMNA5 (Control N = 53, LMNA5 N = 77, 1nM N = 19, 10nM N = 19, 100nM N = 19, 1uM N = 19, 10uM N = 19, 100uM N = 19) post treatment with different doses of cyproheptadine. The N number indicates the number of assessed wells. H) Impedance analysis of LMNA1 (Control N = 63, LMNA1 N = 37, 1nM N = 12, 10nM N = 12, 100nM N = 12), LMNA3 (Control N = 63, LMNA3 N = 31, 1nM N = 12, 10nM N = 12, 100nM N = 12), and LMNA5 (Control N = 63, LMNA5 N = 60, 1nM N = 10, 10nM N = 10, 100nM N = 10) based on pulse width 50% post treatment with different doses of cyproheptadine. Data presented here are from at least three independent differentiations and the N number for calcium imaging indicates the number of assessed wells and for contractility analysis indicates the number of assessed sweeps. The dotted line indicates the average value for five control hiPSC-CMs. CTD₇₅: calcium transient duration 75%, FWHM: full width at half maximum, T_75-25: decay time from 75% to 25%. *, **, *** and **** indicate P-value < 0.05, 0.01, 0.001 and 0.0001 respectively.

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