Fig 1.
Schematic of the basic structure of cMyBP-C.
A Diagram showing the domain structure of cMyBP-C. Ovals represent Ig-like domains and diamonds represent FnIII-like domains. P/A is the pro/ala region and M the phosphorylatable domain (* represents phosphorylatable serine sites within this domain). Regions of cMyBP-C that interact with other sarcomeric proteins are shown below this. B cMyBP-C missense and truncation samples used in this study are shown above and below the schematic, respectively.
Table 1.
Patient data of samples used in this study.
Fig 2.
Representative fluorescence decay chase traces from donor, HCMsmn and MYBPC3mut samples.
Fibres were incubated in mATP and chased with ATP, and fluorescence intensity was monitored at 5 second intervals. Decay traces show an initial rapid phase, followed by a slow phase. The slow phase indicates the presence of myosin in the SRX. The decay in fluorescence intensity is nearly identical in donors (•) and HCMsmn (°). The fluorescence intensity from the MYBPC3mut (☐) sample decays much more rapidly and exhibits a smaller slow phase. This represents a destabilised SRX. See S1 Text for the fit to these traces.
Fig 3.
Summary of the variables derived from the two-state exponential in each of the studied groups.
A The P1 is increased in MYBPC3mut compared to the other groups, while the P2 is significantly reduced in MYBPC3mut compared to donor and HCMsmn groups (B). C The lifetime of the rapid phase (T1) was not altered between groups. The lifetime of ATP turnover (T2) was also significantly decreased (D). Together these data show that mutations to MYBPC3 significantly disturb the SRX. Data are expressed as mean ± SEM (see Table 2 for number of samples, technical repeats and statistical significance).
Table 2.
Comparison of the parameters of SRX experiments derived from age matched donor, MYBPC3mut and HCMsmn tissue.
The number of biological samples used in the study is represented by N. while the total number of technical repeats (n) is shown in parenthesis. P2 is an indicator of the number of myosin heads in the SRX. T2 is the lifetime of ATP turnover of these SRX myosin heads. Data are means ± SEMs.
Fig 4.
Reduction of the SRX is a common feature of MYBPC3 mutations.
The type of MYBPC3 mutation (missense vs. truncations) does not alter the level of SRX destabilisation. (A) The proportion of myosin heads in the SRX (P2) and (B) the lifetime of ATP turnover of these SRX myosin are significantly decreased in both missense and truncating mutations compared to age matched donors. Data are expressed as mean ± SEM (n = 8 for donor and 4 for each mutation type; ** represents p < 0.01, **** represents p < 0.0001).
Fig 5.
Expression of cMyBP-C in age matched donor (D), HCMsmn and MYBPC3mut samples.
Representative western blot (A) and corresponding stain-free blot (B) showing protein loadings. To account for changes in loading, cMyBP-C was normalised to α-actinin. (C) shows a significant decrease in the expression of cMyBP-C in MYBPC3mut compared to age matched donors. HCMsmn did not achieve statistical significance, probably due to the small sample population. Each sample was run in duplicate. Data are expressed as mean ± SEM (number of patients = 4 for donors; = 2 for HCMsmn; = 8 for MYBPC3mut; each sample was run in duplicate; * represents p < 0.05).
Fig 6.
cMyBP-C expression plotted against P2.
The western blot- determined expression level of each sample (normalised to α-actinin) was plotted against its respective P2 value, revealing a positive correlation between these two values. This suggests that the cMyBP-C tethers myosin heads to the thick filament. The absence of cMyBP-C destabilises the SRX. Interestingly the theoretical Y-intercept is similar to the P2 observed in mice lacking cMyBP-C. ADU represents “arbitrary density units”, as determined from normalization of cMyBP-C to α-actinin.
Fig 7.
Phosphorylation of key sarcomeric proteins were measured by ProQ diamond staining, A. Panel A also shows the location of the sarcomeric proteins measured in this study. Panel B shows the same gel stained with Sypro Ruby, with the molecular weights of the marker shown to the left. The relative phosphorylation of each protein is shown in panel C, normalised to the donor group. Data are expressed as mean ± SEM (n = 4, 2 and 8 for donor, HCMsmn and MYBPC3mut, each sample was run in duplicate and data pooled respectively; *, **, *** and **** represent p < 0.05, 0.01, 0.001 and 0.0001 compared to donor; # represents p < 0.05 compared to HCMsmn). The molecular weight marker used is the Peppermint Stick Marker, supplied with ProQ Diamond kit.
Fig 8.
Plot of T2 (s) vs. P2 (%) for each sample used in this study.
Donors (•) and HCMsmn (°) cluster towards the top right of the graph. However, the MYBPC3mut (☐) samples have a wide spread of values, with none in the upper right quadrant. This shows destabilisation of the SRX in all MYBPC3mut samples, irrespective of the type of mutation or expression level of cMyBP-C.