Altered coronary artery function, arteriogenesis and endothelial YAP signaling in postnatal hypertrophic cardiomyopathy

Figures

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  FIGURE 1. Age-dependent myofilament Ca2+ response. (A) Force-Ca2+ relation at P7, (B) at P14, and (C) at P28. (D) Summary of myofilament Ca2+ sensitivity (pCa50), (E) Hill slope, and (F) maximum tension. Data are presented as mean ± SEM. Data were analyzed by 2-way ANOVA followed by Fisher’s LSD test; N = 4–13. NTG, non-transgenic; TG, transgenic.

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  FIGURE 2. Timeline of myofilament isoform switches. Representative images of Western blots and SDS-PAGE summary data of expression of (A) troponin T (TnT) isoforms (N = 2 for both groups), (B) myosin heavy chain (MHC) isoforms, and (C) troponin I (TnI) isoforms in NTG and TG hearts from P2 to P28. For (B,C) N = 4 for both groups. Data are presented as mean ± SEM. NTG, non-transgenic; TG, transgenic. TnT3—adult splicing form 3 of Troponin T; TnT4—adult splicing form 4 of Troponin T; myc-R92Q, myc tag for TnT-R92Q mutation; ssTnI, slow skeletal isoform of TnI; cTnI, cardiac isoform of TnI.

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  FIGURE 3. Morphological, systolic and diastolic changes in NTG and HCM hearts. Summary data represent: (A) heart weight, (B) heart weight to body weight (HW/BW), (C) lung weight to body weight (LW/BW), (D) left atrial diameter (LA), (E) left ventricular internal diastolic diameter (LVIDd), (F) left ventricular internal systolic diameter (LVIDs), (G) left ventricular mass calculated based on echocardiography (LV Mass), (H) isovolumic relaxation time (IVRT), (I) E/A ratio represents peak velocity of early diastolic mitral flow divided by peak velocity of late diastolic mitral inflow, (J) E/e’ ratio represents peak velocity of early diastolic transmitral flow divided by peak velocity of early diastolic mitral annual motion, (K) ejection time (ET), (L) ejection fraction (EF), (M) heart rate (HR), (N) stroke volume (SV), and (O) cardiac output. Data reported as mean ± SEM. N = 4–9 for panels (A–D). N = 6–9 for data presented in panels (E–O). Data were analyzed by 2-way ANOVA followed by Fisher’s LSD test; NTG, non-transgenic; TG, transgenic.

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  FIGURE 4. Time-dependent changes in coronary flow. (A) Representative Doppler echo images of P14 coronary flow charts, with systolic flow highlighted in blue and diastolic in yellow. Summary data represent: (B) mean systolic coronary flow velocity, (C) peak systolic coronary flow velocity, (D) mean diastolic coronary flow velocity, (E) peak diastolic coronary velocity, (F) coronary diastolic acceleration time, and (G) total diastolic flow time (FT) normalized to cardiac cycle length. Data are presented as mean ± SEM, NTG n = 6, TG n = 6 and analyzed by 2-way ANOVA followed by Fisher’s LSD test; NTG, non-transgenic; TG transgenic.

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  FIGURE 5. Time-dependent changes in fibrosis in apical and midventricular regions of NTG and TG hearts. (A) Representative trichrome-stained midpapillary images of NTG and TG hearts at P14 and P28. (B) Quantitation of collagen deposition presented as % of covered area at P7, P14, and P28 at apical, midpapillary and total (apical and midpapillary regions combined). Data are presented as mean ± SEM. Data were analyzed by 2-way ANOVA followed by Fisher’s LSD test; NTG n = 4–6, TG n = 5–6; NTG, non-transgenic; TG, transgenic.

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  FIGURE 6. Time-dependent assessment of localized fibrosis. Representative images of areas with high levels of collagen deposition and quantitation of average collagen deposition in four specific locations: (A) coronary arteries, (B) right ventricular insertion (RVI), (C) interventricular septum (IVS) and (D) lateral free wall (LW). Data presented as mean ± SEM and analyzed using Two-way ANOVA followed by Fisher’s LSD test; NTG n = 5–6, TG n = 5–6. NTG, non-transgenic; TG, transgenic.

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  FIGURE 7. Microvessel density and arteriolar cross-sectional area at midpapillary section. (A) Quantitation of CD31 coverage in NTG and TG hearts. (B) Representative images photomicrographs of CD31 labeled (indicating endothelial cells) immunofluorescent sections taken at midpapillary levels. (C) Representative photomicrographs of midpapillary sections of whole-heart sections with arterioles are highlighted in red at P14. (D) Quantification of arteriolar cross-sectional area (%). N = 5–6. Data presented as mean ± SEM; Two-way ANOVA followed by Fisher’s LSD test.

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  FIGURE 8. Time-dependent expression and localization of YAP. Western blot analysis of YAP abundance (upper panel) and phosphorylation (p-YAP) (Ser-127) (lower panel) at P14 in TG and NTG hearts (A); Representative immunohistochemistry (IHC) images of coronary vessels from NTG and TG hearts stained with fluorescent antibodies against CD31, YAP, α-SMA and DAPI. The merged images represent CD31/YAP/α-SMA with nuclear DAPI counterstaining of the coronary artery region at the basal section of the heart at P14 (B). Inserts below were taken from YAP and merged images at higher magnification; Cytosolic and nuclear YAP fluorescence signal in endothelial (endo) and smooth muscle cells (smc) in the heart sections and a ratio of nuclear/cytosolic YAP expression in the endothelial and smooth muscle cells (smc) in the heart sections at P14 (C), and P28 (D). Data presented as mean ± SEM and analyzed using Student’s t-test (A), NTG N = 6, TG N = 6 and One-way ANOVA followed by Fisher’s LSD test. NTG N = 5–6, TG N = 5–6 (C,D). NTG, non-transgenic; TG, transgenic.

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  FIGURE 9. The effects of R92Q-cTnT on Calcium Signaling—CAMKII and SERCA2a abundance. (A) Western blot analysis of CAMKII abundance and phosphorylation of CAMKII and (B) SERCA2a abundance in NTG and TG hearts. NTG, non-transgenic; TG, transgenic. Data presented as mean ± SEM and analyzed using unpaired Student’s t-test, NTG N = 6, TG N = 5–6.

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  FIGURE 10. The effects of R92Q-cTnT on calcium signaling—phospholamban (PLN) abundance and phosphorylation. Western blot analysis of PLN abundance (A) and phosphorylation of PLN at Ser16 and Thr17 (B) in NTG and TG hearts. NTG, non-transgenic; TG, transgenic. Data presented as mean ± SEM and analyzed using unpaired Student’s t-test, NTG N = 6, TG N = 5–6.

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  FIGURE 11. The Effects of R92Q-cTnT and age on myofilament phosphorylation. (A) PhosTag separation of RLC at P7. Left most panel representative PhosTag Western blot of RLC phosphorylated species (P1-P3) and unmodified RLC (U). The histograms depict the densitometric analysis of relative percent phosphorylation from the left most representative image. (B) PhosTag separation of RLC at P14. Left most panel representative PhosTag Western blot of RLC phosphorylated species (P1-P3) and unmodified RLC (U). The histograms depict the densitometric analysis of relative percent phosphorylation from the left most representative image. (C) Western blot analysis of cardiac troponin I (cTnI) and its phosphorylation abundance at positions 23/24 at P7. The left most panel is an analysis of the phosphorylation abundances while the right most panel is the analysis of total troponin I abundance. Images above the histograms are representative Western blot images. (D) Western blot analysis of cardiac troponin I (cTnI) and its phosphorylation abundance at positions 23/24 at P14. The left most panel is an analysis of the phosphorylation abundances while the right most panel is the analysis of total troponin I…