The contribution of N-terminal truncated cMyBPC to in vivo cardiac function

Figures

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  Figure 1.. Vector design and construct maps. (a) Schematic of AAV9 vectors containing cMyBPC-FL and –C3C10 with C-terminal c-Myc tag under the control of a truncated chicken cardiac troponin T promoter. (b) Schematics of full-length (FL) cMyBPC and truncated cMyBPC consisting of domains C3 through C10 (C3C10), including the C-terminal c-Myc tag used to identify exogenously expressed cMyBPC.

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  Figure 2.. Expression of AAV9-delivered constructs. (a) Expression of AAV9-FL and –C3C10 as determined by immunoblotting (left) against the c-Myc epitope, normalized to total protein loading (right). (b) Expression of endogenously produced cMyBPC (WT) versus AAV9-FL as determined by immunoblotting (left) against the N-terminus of cMyBPC, normalized to total protein loading (right). (c) Plot of mean ± SEM c-Myc expression normalized to the average c-Myc expression of the FL group, for n = 8 hearts per group. (d) Plot of mean ± SEM cMyBPC expression normalized to the average cMyBPC expression of the WT group. n = 12 hearts per group. Comparisons between C3C10 and FL, and WT and FL, made by one-tailed unpaired t test.

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  Figure 3.. Incorporation of constructs into sarcomeres. Representative IHC of c-Myc- and α-actinin-stained myofibrils from respective hearts showing incorporation of AAV9-derived constructs in doublets between z-lines.

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  Figure 4.. Morphology normalized by C3C10. (a) Representative images of hearts from mice injected with saline or AAV9-FL or –C3C10. (b) Plots of mean ± SEM LVM/BW and LVPW:d. Group comparison made by one-way ANOVA with post-hoc Tukey’s multiple comparisons test.

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  Figure 5.. Contractile function improved by C3C10. (a) Plots of mean ± SEM EF (left) and GLS (right). Group comparison made by one-way ANOVA with post-hoc Tukey’s multiple comparisons test. (b) Representative PSLAX B-mode images (top) with vectors indicating longitudinal strain along the endocardial border. Representative SAX M-mode images (bottom) indicating end diastolic diameter (EDD) and end systolic diameter (ESD).

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  Figure 6.. Diastolic impairment in the absence of NTDs. (a) Representative para-sternal long axis (PSLAX) B-mode images with vectors indicating longitudinal strain along the endocardial border. (b) Plot of mean ± SEM rLSR. Group comparison made by one-way ANOVA with post-hoc Tukey’s multiple comparisons test.

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  Figure 7.. Truncated ejection in the absence of NTDs. (a) Representative pulsed wave Doppler traces depicting the velocity of blood flow through a region of interest positioned at the area of peak inflow during diastole. Velocities above the baseline represent ventricular inflow, and below baseline represent ventricular outflow. (b) AET is indicated, and plotted as mean ± SEM. Group comparison made by one-way ANOVA with post-hoc Tukey’s multiple comparisons test.

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  Figure 8.. Effect of FL and C3C10 cMyBPC on myofilament pCa50 and cooperativity of force development (nH), and effect of FL and C3C10 cMyBPC on dynamic stretch activation parameters. (a) Force–pCa relationship of FL, C3C10, and saline-treated myocardium analyzed by plotting normalized forces generated at a range of pCa. No significant differences were observed between the groups (b). Representative force trace in response to a sudden 2% stretch in ML in an isometrically contracting FL myocardium preparation at an SL of 2.1 µm. The green line represents the rate of cross-bridge detachment (krel) and the orange line represents the rate of cross-bridge recruitment (kdf). (c) The rate of cross-bridge detachment, krel. (d) The rate of cross-bridge recruitment, kdf. Values are expressed as mean ± SEM. Data from n = 4 hearts per group with three fiber preparations per heart was analyzed in a hierarchical, nested manner using one-way repeated measures ANOVA followed by Tukey’s multiple comparisons test.