Transcriptional bursts and heterogeneity among cardiomyocytes in hypertrophic cardiomyopathy

Figure 1. Independent, stochastic transcription of TNNI3- and MYBPC3-alleles. (A) Exonic and intronic mRNA of either TNNI3 or MYBPC3 were hybridized with fluorescently labeled DNA probes to visualize active transcription in cardiomyocyte nuclei in cryosections from left ventricular heart tissue. Representative cardiomyocyte nucleus with one active transcription site (aTS) for MYBPC3 from the cMyBP-Ctrunc patient. Left panel (green) non-specific fluorescence, middle panels intronic RNA (red) and exonic RNA (orange) and right panel merge. Intronic and exonic signals are shown merged with DAPI (blue) to stain the nucleus. One layer of the z-stack is shown. White arrow indicates aTS with co-localization of Quasar 570 and Quasar 670 fluorescence. The other fluorescent spots in the nucleus are only stained by exonic probes and presumably represent spliced mRNA. (B) Distribution of the numbers of aTS per nucleus in cardiomyocytes for TNNI3 and MYBPC3. Percentage of nuclei with 0, 1, 2, 3, 4, and >4 aTS was plotted for different individuals. (C) TNNI3- or MYBPC3-mRNA copy number per cell plotted against the cell cross-sectional area. Linear correlation was tested using the Pearson correlation…

Figure 2. Allelic imbalance of TNNI3 in cTnIR145W patient and donor cardiomyocytes. Individual cTnIR145W cardiomyocytes and donor cardiomyocytes were isolated by laser-microdissection from cryosections and reverse transcribed. cDNA was split in two technical replicates and subjected to nested PCR. (A) RT-PCR-products from single laser-microdissected cTnIR145W cardiomyocytes were treated with MwoI to generate allele-specific restriction fragments. Shown are three different cells in technical replicates. Expected fragment sizes are 202 bp for TNNI3c.433C>T-allele, 160 bp for WT-allele and 115 bp for both WT- and TNNI3c.433C>T-allele corresponding to 100% of the PCR-product. L indicates the DNA standard. (B) RT-PCR-products from donor cardiomyocytes with heterozygous SNP rs3729841 were treated with BbsI to generate allele-specific restriction fragments. Shown are three different cells in technical replicates. Expected fragment sizes are 317 bp for WT-allele and 273 bp for SNP-allele. L indicates the DNA standard. (C) Fractions of mutant TNNI3-allele from single cTnIR145W cardiomyocytes (two patients) and of SNP-allele from donor cardiomyocytes. Each dot represents the mean of two replicates…

Figure 3. Unequal expression of cMyBP-C in cardiomyocytes of the cMyBP-Ctrunc patient. (A) Western blot of one donor sample and three cMyBP-Ctrunc patients H84 (MYBPC3c.927−2A>G), H45 (MYBPC3c.1458−6G>A) and H36 (MYBPC3c.2864_2865delCT) was incubated with an N-terminal cMyBP-C antibody. Detection of full-length WT-cMyBP-C (~140 kDa) and possible truncated cMyBP-C was performed with 5 s exposure time. (B) Relative protein amount of cMyBP-C was calculated from IOD of the cMyBP-C band divided by IOD of α-actinin for normalization. IOD of α-actinin band at 103 kDa was quantified as loading control. IOD ratio (cMyBP-C/α-actinin) from one donor sample (blue dots, n = 12 lanes) and compared to data from cMyBP-Ctrunc patients (red dots, n = 6 lanes each). For comparison of patient results (H84, H45, H36) with donor (H108) reference one-way analysis of variance (ANOVA) and Dunnett's post-hoc test were performed. ANOVA yielded significant variation among groups [F(3, 26) = 10.17, p = 0.00013]. Mean±SD and p-values from Dunnett's test are indicated in the figure. (C) Cryosections (5 or 10 μm) from cMyBP-Ctrunc patients and donor myocardium were stained with an N-terminus-specific antibody for cMyBP-C…

Figure 4. Contractile imbalance among cardiomyocytes from cMyBP-Ctrunc and cTnIR145W. Individual cardiomyocytes from cTnIR145W patient, cMyBP-Ctrunc (c.927-2G>A) patient and respective donors were permeabilized and subjected to different calcium concentrations to measure calcium-dependent force generation. (A) Maximum force generation (pCa 4.18) and (B) pCa50 values of individual cTnIR145W cardiomyocytes (n = 20), of cMyBP-Ctrunc (n = 28) and of the corresponding donor cardiomyocytes (n = 20 and n = 29). pCa50 was derived from Hill-fits of normalized force-pCa relations. p-values of U test are indicated in the figure. (C) Force-pCa relations were plotted for individual cTnIR145W cardiomyocytes, for cMyBP-Ctrunc cardiomyocytes and the respective donor cardiomyocytes. Patient cardiomyocytes, red symbols and solid lines; donor cardiomyocytes, blue symbols and dashed lines. Force values in black boxes are plotted in (D). (D) Normalized forces of individual cardiomyocytes are shown for cTnIR145W vs. the respective donor at pCa 5.24, and for cMyBP-Ctrunc versus the respective donor at pCa 5.74. Each symbol represents an individual cardiomyocyte. Mean±SD and p-values for U-test and Levene's test…

Figure 5. Experimental results from donor and from patients with TNNI3-mutations in comparison to simulation results from the model. (A) TNNI3-aTS distribution from three donors (H89, H108, and H113) compared to aTS distribution simulated by model. Percentage of nuclei with 0, 1, 2, 3, 4, and >4 aTS was plotted. (B) Percentage of TNNI3-SNP-allele transcripts from a donor and of TNNI3-mutant-allele transcripts from two cTnIR145W patients (densitometric analysis of allele-specific restriction fragments), compared to the simulated distribution. Each data point represents the mean of replicates from one cell. Violin plots represent probability density of experimental data (blue and red) and probability density simulated by model (violet). Horizontal bar, median; white rectangle, interquartile range. (C) Total TNNI3-mRNA counts in donor cardiomyocytes. Violin plot probability density of the data (blue) compared to outcome of model simulation (violet). (D) Force values of individual cardiomyocytes for donor and for TNNI3c.433C>T patient at pCa 5.24, normalized to respective maximum force, compared to simulation outcome. Dots represent individual cardiomyocytes. Violin plots, probability density…