Desmoplakin loss leads to PKC-dependent insertion of series sarcomeres and contractile dysfunction in cardiomyocytes

Fig. 1:. Engineered heart tissue model of mutant desmoplakin (DSP-R451G) exhibits hypocontractile phenotype. A. Domain diagram of desmoplakin showing location of R451G mutation within SH3 domain of plakin head. B. Western blot of engineered heart tissue (EHT) lysate showing total nearly loss of DSP protein in DSPR451G EHTs. Dotted lines separate non-consecutive lanes from the same membrane. C. Silver-stained myosin gel showing exclusive expression of β-myosin heavy chain (MHC) in WT and DSPR451G EHTs. Mouse ventricular and porcine left-ventricular lysates were used as a standard for α-MHC and β-MHC, respectively. D. Representative twitches from isometric mechanical testing of WT and DSPR451G EHTs. Constructs were field-stimulated at 1.5 Hz. E. Isometric twitch force generated by EHTs with 1.5 Hz field stimulation (WT N = 15, DSPR451G N = 15, two differentiation batches). F. Systolic kinetics of twitch contraction (TTP = time to peak force from stimulation time). G. Diastolic kinetics of twitch relaxation (RT50 = time from peak force to 50% relaxation). H. Representative Fura-2 calcium transients of whole tissues measured at culture length with 1.5 Hz pacing. Tissues were excited with 340…

Fig. 2:. Application of chronic mechanical stress results in EHT maturation, rather than exacerbating hypocontractile ACM phenotype A. Diagram of experimental design, consisting of 2 weeks of culture after tissue seeding, baseline characterization, and 1 week of culture with 20% biomechanical stretch. B. Schematic of tissue cassette that enables tissue stretch. A 20% stretch stimulus is applied at once after baseline mechanical testing. C. Fold-change in isometric twitch force before and after 20% mechanical stretch. WT and DSPR451G EHTs responded equally to this hypertrophic stimulus. WT, pre-stretch N = 17, post-stretch N = 12; DSPR451G, pre-stretch N = 18, post-stretch N = 12, three differentiations per genotype. Statistical design used two-way ANOVA with repeated measures to account for paired comparison. D. Systolic kinetics (time from stimulation to peak force, TTP) were not affected by mechanical stretch in a genotype-specific manner. E. Diastolic kinetics (time from peak force to 50% relaxation) were not affected by mechanical stretch in a genotype-specific manner. F. Western blot showing equal protein expression of myosin heavy chain (MHC) in WT and DSPR451G EHTs after stretch…

Fig. 3:. DSPR451G EHTs exhibit enhanced length-dependent activation, secondary to shorter diastolic sarcomere length. A. Representative isometric twitches at 1.5 Hz pacing showing lower baseline force production in DSPR451G EHTs, with a greater fold-change in force production over a length sweep from 0 to 15% stretch (3% intervals). B. Whole-mount confocal micrographs of fixed, phalloidin-stained EHTs, showing aligned sarcomeres. Scale bar = 20 μm. C. Length-dependent activation curve demonstrating that both WT and DSPR451G tissues display Frank-Starling behavior. Two-way repeated-measures ANOVA used for statistical analysis (WT and DSPR451G N = 15) D. Normalization of Frank-Starling curve in Panel C suggests that DSPR451G tissues display enhanced length-dependent activation. Peak force values were normalized to culture length (i.e. 0% stretch). E. Sarcomere length difference between WT (N = 3) and DSPR451G (N = 4) EHTs. From each blinded confocal z-stack, 5–10 myofibrils were traced as line scans and peak-to-peak distance calculated in Matlab. Total number of sarcomeres analyzed: WT n = 180, DSPR451G n = 161. Each biological replicate was from a separate differentiation.

Figure 4:. PKC inhibition rescues sarcomere length deficit in 2D iPSC-derived cardiomyocytes A. Representative micrographs of cell clusters stained with phalloidin and Hoechst. Cells were treated with 0.05% vehicle control or 5 μM pan-PKC inhibitor bisindolylmaleimide VIII acetate for 4 h prior to fixation and labeling. B. Drug screen of PKC-activating and inhibiting compounds. Stauro = staurosporine; Bis = bisindolylmaleimide VIII; PMA = phorbol myristate acetate. Cells were treated with indicated compound for 4 h prior to fixation and immunofluorescence. Numbers below each violin are average sarcomere length for each condition. Statistics used two-way ANOVA. Numbers above each violin are p value for multiple comparisons between drug treatment and vehicle control of same genotype. Interaction p-value for drug treatment is <0.0001. Each measurement consists of at least seven fields of view containing at least 25 cardiomyocytes, from which line scans of 5–10 sarcomeres were generated to calculate sarcomere length.

Fig. 5:. Ruboxistaurin rescues sarcomere length in 3D EHTs, with genotype-specific effects on contractile function. A. Ruboxistaurin rescues sarcomere length in DSPR451G EHTs (two-way ANOVA drug-genotype interaction p = 0.0162, N = 3 EHTs per group, with n = 88–136 sarcomeres per condition). B. Representative twitches showing how ruboxistaurin decreases tension-time integral in WT tissues but has a neutral effect in DSPR451G EHTs. C. Isometric twitch force at 1 Hz pacing of vehicle- and ruboxistaurin-treated EHTs. WT vehicle, N = 5; WT ruboxistaurin, N = 4; DSPR451G vehicle, N = 5; DSPR451G ruboxistaurin, N = 6; two-way ANOVA used for statistical comparison. D. Twitch force normalized to each genotype’s vehicle. E. Tension-time integral (area under twitch curve) of WT and DSPR451G EHTs, showing a genotype-specific effect of ruboxistaurin, driven mostly by relaxation kinetics. F. Diastolic kinetics (RT50, time from peak force to 50% relaxation) in DSPR451G and WT tissues.

Fig. 6:. DSPR451G EHTs have enhanced PKC phosphorylation and a loss of stretch-sensitive PKC activation, while DSPR451G myocytes exhibit increased mechanical loading at cell-cell junctions A. Western blot of EHT lysate probed for active (phosphorylated) pan-PKC. βII-S660 site corresponds to the hydrophobic tail site of activated PKC. B. and C. Quantification of phospho-PKC normalized to total protein (Li-Cor Revert). EHTs in stretched and non-stretched cassettes were from different experiments so were not analyzed together. Statistical comparison made using Student’s t-tests. Pre-stretch samples came from 3 (WT) and 4 (DSP) differentiations, post-stretch samples came from 3 differentiations (WT and DSP). D. Widefield immunofluorescence staining of 6-week-old, expanded 2D iPSC-derived cardiomyocytes. Extended-conformation α18 corresponds to an epitope of α-catenin unmasked by mechanical stress. N-cadherin staining used as fiduciary adherens junction marker. Images were processed using watershed algorithm to identify junctions. Scale bar, 20 μm. E. Histogram of α18-catenin to N-cadherin fluorescence ratio at segmented junctions. F. Quantification of data in E., with Student’s t-test for…

Fig. 7:. Working model of force redistribution between desmosomes and adherens junctions resulting in PKC-mediated insertion of series sarcomeres in the setting of DSP haploinsufficiency.