Research ArticleCardiology

HDAC inhibition improves cardiopulmonary function in a feline model of diastolic dysfunction

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Science Translational Medicine  08 Jan 2020:
Vol. 12, Issue 525, eaay7205
DOI: 10.1126/scitranslmed.aay7205
  • Fig. 1 Experimental study design and remodeling effects of SAHA.

    (A) Baseline measurements were collected, and felines then underwent the aortic banding or sham procedure. After 2 months, banded cats were assigned to either daily subcutaneous injection of vehicle (veh) or SAHA (10 mg/kg) for 2 months. At 4 months after banding, invasive hemodynamic studies were performed. Conventional echocardiography data showing (B) left ventricular wall thickness, (C and D) LA size, (E) LA function, (F) LV diastolic function, (G) LV EF, and (H) LVEDD in sham, vehicle-treated, and SAHA-treated animals. (I) Speckle-tracking–based strain analysis was quantified from sham, vehicle-treated, and SAHA-treated animals. (J) Parasternal long-axis images showing septal wall thickness and LA size. (K) Parasternal short-axis images with superimposed regional radial strain values. Data shown are means ± SEM. Statistical significance was determined by linear mixed-effects models. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 between b + vehicle and sham. §P < 0.05, §§P < 0.01, §§§P < 0.001, §§§§P < 0.0001 between b + SAHA and sham. #P < 0.05, ##P < 0.01, ###P < 0.001, ####P < 0.0001 between b + SAHA and b + vehicle. NS, not significant. Ao, aorta; BGA, blood gas analysis; BL, baseline; E, peak early diastolic transmitral velocity; e′, spectral tissue Doppler-derived peak early diastolic velocity; Echo, echocardiography; EF, ejection fraction; HFpEF, heart failure with preserved ejection fraction; LA, left atrium; LA EF, left atrial ejection fraction; LAVES, left atrial end-systolic volume; LV, left ventricle; LVEDD, left ventricular end-diastolic diameter; PFT, pulmonary function testing; SAHA, suberoylanilide hydroxamic acid. The number of animals included in each parameter is reported in table S7.

  • Fig. 2 SAHA improves diastolic function.

    (A) Aortic band pressure gradient measured in banded animals treated with SAHA or vehicle. Invasive hemodynamics showing (B) CO, (C) heart rate (HR), (D) dP/dtmax, (E) dP/dtmin, (F) t-dia/τ, (G) LVEDP, and (H) mPAP (three banded cats added to b + veh) before and after dobutamine infusion. Data shown are means ± SEM. Statistical significance was determined by linear mixed-effects models. *P < 0.05, **P < 0.01, ***P < 0.001 between b + veh and sham. §P < 0.05, §§P < 0.01 between b + SAHA and sham. #P < 0.05, ##P < 0.01 between b + SAHA and b + veh. Dob−/+, before and after dobutamine; dP/dtmax, maximum rate of pressure rise; dP/dtmin, minimum rate of pressure decay; LVEDP, left ventricular end-diastolic pressure; mPAP, mean arterial pulmonary pressure; τ, isovolumic relaxation constant; t-dia, diastolic time interval. The number of animals included in each parameter is reported in table S7.

  • Fig. 3 SAHA improves myofibril relaxation and exerts antihypertrophic effects.

    Ex vivo myofibril mechanical studies on LV tissue collected at 4 months after banding showing (A) myofibril maximal tension and (B) linear relaxation duration. Correlations of ex vivo linear relaxation duration with in vivo (C) t-dia/τ, (D) LVEDP, and (E) LV wall thickness. LV hypertrophy was assessed by (F) HW/BW ratio and (G) LV cardiomyocyte cross-sectional area. (H) Confocal micrographs of LV tissue section stained for WGA (wheat germ agglutinin) (green) and DAPI (4′,6-diamidino-2-phenylindole; blue) show cardiomyocyte size. Scale bars, 50 μm. Assessment of (I) LV fibrosis in subendocardial and subepicardial layers. Quantified plasma concentrations of (J) NT-proBNP. Data shown are means ± SEM. Statistical significance was determined by (A, B, F, G, and I) Kruskal-Wallis tests with multiple comparisons using two-stage linear step-up procedure, (C to E) Pearson correlation, and (J) linear mixed-effects models. *P < 0.05, **P < 0.01, ***P < 0.001 between b + veh and sham. §P < 0.05 between b + SAHA and sham. #P < 0.05 between b + SAHA and b + veh. †P < 0.05 versus subendo b + veh. @P < 0.05 versus subendo b + SAHA. BW, body weight; CM, cardiomyocyte; CSA, cross-sectional area; HW, heart weight; NT-proBNP, N-terminal probrain natriuretic peptide; τ, isovolumic relaxation constant; t-dia, diastolic time interval. The number of animals included in each parameter is reported in table S7.

  • Fig. 4 SAHA improves lung mechanics, gas exchange, and attenuates lung morphological changes.

    Lung function was assessed by measuring (A) lung compliance, (B) PaO2/FIO2 ratio (FIO2 = 1), (C) A-aDO2, and (D) intrapulmonary shunt fraction. Correlation of lung compliance with (E) LV wall thickness, (F) LA size, and (G) PaO2/FIO2. Quantification of (H) alveolar-capillary wall thickness, (I) alveolar area, (J) expansion index (the ratio of volume of gas exchange to parenchymal space), and (K) ratio of cuff/vessel area of extra-alveolar vessels. (L) Bright-field micrographs of H&E-stained lung sections. Scale bars, 250 μm. Data shown are means ± SEM. Statistical significance was determined by (A) linear mixed-effects models, (B to D and H to K) Kruskal-Wallis tests with multiple comparisons using two-stage linear step-up procedure, and (E to G) Pearson correlation. *P < 0.05, **P < 0.01, ***P < 0.001 between b + veh and sham groups. §P < 0.05 between b + SAHA and sham. #P < 0.05, ##P < 0.01 between b + SAHA and b + veh. Open symbols in (E) and (F) indicate baseline data points. A-aDO2, alveolar-arterial oxygen difference; LA/Ao, left atria to aortic root ratio; PaO2, partial pressure of oxygen in arterial blood; FIO2, fraction of inspired oxygen. *, bronchioles; †, extra-alveolar vessel; arrow, fluid cuff around extra-alveolar vessels. The number of animals included in each parameter is reported in table S7.

  • Fig. 5 SAHA induces switch in skeletal muscle fiber composition and improves mitochondrial function.

    In vivo measurements of (A) oxygen consumption. (B) mRNA expression of genes related to mitochondrial biogenesis (Pgc-1α, Pgc-1β, and Tfam), glucose metabolism (Glut4), and TCA cycle (Idh3α) in skeletal muscle. (C) Skeletal muscle fiber composition. Assessment of (D) mitochondrial membrane potential, (E) mitochondrial structure, and (F) mitochondrial calcium uptake before and after histamine administration in HeLa cells. Scale bars, 3 μm. (G) Oxygen consumption rate and extracellular acidification rate were measured using Seahorse Technology: basal (1) and maximal (3) mitochondria respiration, uncoupling (4), and proton leak (2) in HeLa cells. Data shown are means ± SEM. Statistical significance was determined by (A to C) Kruskal-Wallis tests with multiple comparisons using two-stage linear step-up procedure and (D, F, and G) analysis of variance (ANOVA) followed by Bonferroni post hoc testing to compare mean values between groups. *P < 0.05, **P < 0.01 between b + veh and sham groups. §§P < 0.01 between b + SAHA and sham. #P < 0.05, ##P < 0.01 between b + SAHA and b + veh. @P < 0.05 between Control and SAHA. The number of animals included in each parameter is reported in table S7.

  • Fig. 6 SAHA mediates mitochondrial metabolism via posttranslational modifications.

    Assessment of (A) acetyl-CoA concentrations and (B to F) protein acetylation patterns measured using mass spectrometry of LV samples from sham and sham + SAHA animals. Acetylation patterns on (B) histone 3, proteins of the (C) electron transport chain (ETC), proteins of the (D) malate-aspartate shuttle, proteins of the (E) TCA cycle, and proteins involved in (F) mitochondrial fatty acid oxidation. Data shown are means ± SEM. Statistical significance was determined by two-tailed Student’s t test with subsequent multiple testing correction by permutation-based false discovery rate method (P < 0.05). The number of animals included in each parameter is reported in table S7.

Supplementary Materials

  • stm.sciencemag.org/cgi/content/full/12/525/eaay7205/DC1

    Materials and Methods

    Fig. S1. SAHA treatment in a feline model of diastolic dysfunction.

    Table S1. Assessment of blood count.

    Table S2. Arterial BGA.

    Table S3. Venous BGA.

    Table S4. Protein acetylation (separate file).

    Table S5. Quantitative proteomics (separate file).

    Table S6. Findings from the current study versus those from previously published papers.

    Table S7. Animal numbers.

    Table S8. Primers for PCR analysis.

    Data file S1. Individual subject-level data (separate file).

    References (5683)

  • The PDF file includes:

    • Materials and Methods
    • Fig. S1. SAHA treatment in a feline model of diastolic dysfunction.
    • Table S1. Assessment of blood count.
    • Table S2. Arterial BGA.
    • Table S3. Venous BGA.
    • Legends for tables S4 and S5
    • Table S6. Findings from the current study versus those from previously published papers.
    • Table S7. Animal numbers.
    • Table S8. Primers for PCR analysis.
    • Legend for data file S1
    • References (5683)

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S4 (Microsoft Excel format). Protein acetylation (separate file).
    • Table S5 (Microsoft Excel format). Quantitative proteomics (separate file).
    • Data file S1 (Microsoft Excel format). Individual subject-level data (separate file).

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