Personalized Medicine Approach in a DCM Patient with LMNA Mutation Reveals Dysregulation of mTOR Signaling

Figures

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  Figure 1. Patient pedigree. The proband is a 47-year-old male (depicted by an arrow). Square boxes denote males while the circles denote females. Squares or circles filled with dark areas denote individuals with the LMNA mutation (NM_170707.3: c. 481 G > A: p. E161K). Crossed squares or circles denote that individuals have already passed away. Y = years, DCM = dilated cardiomyopathy, HTX = heart transplantation, * year of DCM diagnosis.

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  Figure 2. CRISPR-Cas9 mediated patient-specific knock in mES cell line generation and differentiation into cardiomyocytes. (a) Surveyor nuclease assay to validate two different sgRNAs (1 and 2) for use in mES cells. Arrows indicate the successful cleavage of PCR products by sgRNAs. (b) Hind III restriction digest of PCR product of the target region to identify the insertion of donor DNA and presence of a specific mutation in single mES cell clones. Arrow indicates successful cleavage of PCR product. (c) Confirmation of positive KI clone via Sanger sequencing. Arrows indicate the insertion of the desired mutation in the mES cell genome. (d) WT mES cells are grown on MEF feeder cells. (e) Hanging drop culture to initiate cardiomyocyte differentiation from mES cells. (f) Differentiated cardiomyocyte from WT mES cells as seen on Day 12 in brightfield microscope. (g) Actinin (green) and DAPI (blue) staining in WT cardiomyocytes as seen on Day 12. sgRNA = single guide RNA. Scale bar: 10 μm.

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  Figure 3. Expression of proteins of the mTOR signaling pathway with and without isoprenaline stimulation. WT and LMNA-KI mES cells were differentiated into cardiomyocytes and stimulated on day 12 with either isoprenaline (I) or isoprenaline together with rapamycin (R). (a) Western blot images of total and phosphorylated protein levels of mTOR, P70S6K, and 4EBP1. β-actin is used as a loading control. (b–d) Densitometric quantification of phosphorylated protein relative to total protein, measured by ImageJ program. One-way ANOVA and Tukey’s post hoc analysis were used. Data are shown as mean ± SEM (n = 2–3). **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05. I = isoprenaline, R = rapamycin.

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  Figure 4. Lamin staining of cardiomyocytes differentiated from WT and LMNA-KI mES cells. WT mES cells and LMNA-KI mES cells were differentiated into cardiomyocytes and on day 12 stained with lamin antibody and DAPI (panels a,d,f). Additionally, nuclear lamin organization (b) and nuclear blebs (c) were quantified by counting the cells per field of the 40× microscopic view. Figures are representative of fully (e) or partially (g) organized nuclear lamin. Scale bar: 10 μm. An unpaired t-test was used. Data are shown as mean ± SEM (n = 80). **** p < 0.0001, ** p < 0.01.

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  Figure 5. Functional characterization of cardiomyocytes differentiated from WT and LMNA-KI mES cells. (a) Electrophysiological recordings of spontaneous APs of WT.CMs, WT.CMs pre-incubated with isoprenaline, LMNA-KI.CMs, and LMNA-KI.CMs pre-incubated with either isoprenaline alone or with isoprenaline and rapamycin; red arrows indicate delayed afterdepolarization-like arrhythmias. (b–d) Frequency of APs, contractions, and delayed afterdepolarizations in WT.CM and LMNA-KI.CM under different conditions. (e) WT.CM (black line) and LMNA-KI.CM (red line) phase plot (dV/dt versus V) of a single AP; inset shows the respective AP traces (dotted line indicates 0 mV). Arrow indicates the delayed afterdepolarization-like arrhythmias in LMNA-KI.CM. One-way ANOVA and Tukey’s post hoc analysis were used. Data are shown as mean ± SEM (n = 6–19). **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05. I = isoprenaline, R = rapamycin, dV/dt = rate of action potential.

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  Figure 6. Patient biopsy data and treatment follow-up. Pathologic examination of patient biopsy from left ventricle was done with (a) Masson’s trichrome staining, (b) immunohistochemical staining using anti-phospho-mTOR Ab and (c) HLA-DR staining, illustrating mostly macrophages. Corresponding Masson’s trichrome (d), anti-phospho-mTOR (e) and HLA-DR (f) stainings from a healthy, 61-year-old male revealing normal stainings in the myocardium. The LMNA-affected DCM patient was treated off-label with the mTOR-inhibitor rapamycin at a serum concentration of 8 ng/mL for 6 months as an off-label treatment. (g) NT-BNP serum concentration dropped from 8120 ng/L to 2210 ng/L. Measurements of NT-BNP levels at an interval of 1 week are shown. (h) Echocardiographic information before and 6 months after the rapamycin treatment. NT-BNP = N-terminal brain natriuretic peptide.

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  Figure 7. Schematic diagram of a putative signaling pathway in cardiomyocytes with LMNA mutation. Factors like age or stimulants affect the gene expression of LMNA mutant cells. Dysregulation of gene expression from mutant alleles causes higher activation of the mTOR signaling pathway. This dysregulation causes protein translation imbalance, lamin disorganization, and nuclear blebbing and adds to the disease phenotype. mTOR-inhibitor rapamycin is effective in checking the cellular effects of LMNA mutation.