Research ArticleCardiology

Pacemaker-induced transient asynchrony suppresses heart failure progression

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Science Translational Medicine  23 Dec 2015:
Vol. 7, Issue 319, pp. 319ra207
DOI: 10.1126/scitranslmed.aad2899
  • Fig. 1. PITA improves in vivo cardiac function.

    (A) Example end-diastolic echocardiographic images at baseline (BL) and after 6 weeks (end of study) of dog hearts in the HF and PITA-treated groups. The left ventricle (LV) is outlined in yellow. (B and C) Left ventricular ESV (B) and EF (C) assessed by echocardiography at baseline (n = 10), after 2 weeks of atrial pacing (n = 18), at the end of the 6-week atrial pacing protocol (HF, n = 8), and at the end of the 6-week PITA protocol (n = 9). (D) Left ventricular EDP (LVEDP) from hemodynamic studies (baseline, n = 8; 2 weeks, n = 12; HF, n = 13; PITA, n = 10 dogs). EDP data were non-normal and are displayed as box plots. Data were log10-transformed before one-way analysis of variance (ANOVA). Direct comparison to the 2-week group was made by t test. (E) Example left ventricular pressure waveforms in HF and PITA hearts at baseline and with dobutamine (15 μg kg−1 min−1). (F) Dobutamine dose effect on left ventricular contractility (dP/dtmax/IP) (control, n = 6 dogs; HF, n = 9; PITA, n = 8 dogs; some doses missing for individual dogs). Data points indicate individual animals at each dose; symbols are means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 versus control; P < 0.05, P < 0.001 versus HF by one-way ANOVA and Holm-Sidak post hoc test.

  • Fig. 2. Myocyte function is depressed in HF after βAR stimulation but near-normal with PITA.

    (A) Top: Example tracings of sarcomere shortening and intracellular calcium transients from left ventricular lateral wall myocytes isolated from healthy control (Con), HF, and PITA dogs at baseline and after isoproterenol stimulation (0.1 μM) or after norepinephrine (NE; 0.1 μM) and prazosin (Prz; 1 μM) stimulation. Bottom: Sarcomere shortening and intracellular calcium are quantified as means ± SEM [n = 4 to 7 dogs in each group; number of cells per dog, 5.8 ± 0.4 (mean ± SEM)]. For all three groups, isoproterenol and NE + Prz sarcomere shortening and peak Ca2+ transient data are P < 0.05 versus respective baseline; ***P < 0.001 versus control by two-way ANOVA and Holm-Sidak post hoc test. (B) cAMP activity after isoproterenol or forskolin stimulation. Data are individual dogs; means ± SEM (n = 8). (C) Plasma membrane βAR, β1AR, and β2AR density. Data are individual dogs; means ± SEM (n = 8 per group). In (B) and (C), *P < 0.05, **P < 0.01, ***P < 0.001 versus control by one-way ANOVA and Holm-Sidak post hoc test.

  • Fig. 3. Myofilament function is depressed in HF and recovered with PITA.

    (A) Mean force as a function of calcium concentration (± SEM) and fitted curves for skinned myocytes from the left ventricular lateral wall for control, HF, and PITA. Inset shows force data normalized to Fmax. Summary results for Fmax and EC50 from these curve fits are shown as individual myocytes and means ± SEM [control, n = 15 from 6 dogs; HF, n = 26 myocytes from 8 dogs; PITA, n = 14 myocytes from 4 dogs; number of cells per dog, 3 ± 0.2 (mean ± SEM)]. (B) Representative images (×40) of control, HF, and PITA skinned myocytes stretched to a sarcomere length of 2.1 μm. (C) Myocyte cross-sectional area (CSA) of all myocytes examined in (A). Data are means ± SEM. (D) Mean skinned myocyte width ± SEM (n = 150 cells per group). (E) Fmax from (A) without normalization to CSA. Data are individual myocytes (means ± SEM). (F) Expression of phospho-TnI S22/S23 normalized to total TnI. Data are individual dogs and means ± SEM (n = 4 per group). (G) Expression of phospho–GSK-3β S9 normalized to total GSK-3β (n = 4 per group). For all panels, **P < 0.01, ***P < 0.001 versus control, unless otherwise indicated, by one-way ANOVA with Holm-Sidak post hoc test.

  • Fig. 4. Myofilament structure and function is disrupted in HF but restored in PITA.

    (A) Longitudinal sections of skinned myocytes imaged by EM. Normal structures were observed in control and PITA, but HF sarcomeres showed myofilament disarray. Scale bar, 1 μm. (B) Higher magnification highlighting the disrupted myofilament structures: weak and wavy Z-band and M-band and bent/curved filaments with irregular spaces in between that were observed in some HF myofibrils, as indicated by arrows. Scale bar, 200 nm. (C) Transverse sections of myofilaments showing smaller-diameter myofibrils with increased spaces in between and loss of regular filament lattice structure in HF compared with control and PITA-treated animals. Inset: FFTs of boxed areas confirmed loss of normal lattice structure in the HF group. Scale bar, 200 nm. (D) Percentage of normal and disarrayed sarcomeres identified by EM (n = 3727 sarcomeres examined from n = 3 HF dogs). (E) Disarrayed sarcomeres cluster together in HF. The percent of EM fields containing normal, disarrayed, or a mixture was quantified in the HF group (n = 28 fields). (F) Isolated myofibril force. A subpopulation of fibrils generating extremely low force is indicated by the dashed line. Data are individual myofibrils and shown as box plots because HF data were non-normally distributed (control, n = 15; HF, n = 18; PITA, n = 18 myofibrils). P values determined by Mann-Whitney rank sum nonparametric test. (G) Fmax compared after removing the weak myofibrils from (F) (n removed from the analysis: control, 0; HF, 7; PITA, 1 myofibril). n.s., not significant by one-way ANOVA.

  • Fig. 5.

    Proteomic analysis of myofilament-enriched samples revealed changes in sarcomere assembly chaperones. (A) Ratio of the core myofilament proteins in both HF and PITA normalized to control samples (n = 4). Proteins that lay along the identity line (dashed line, slope = 1) were similarly expressed in HF and PITA. The two red dots (BAG3 and NRAP) were significantly different between HF and PITA: P < 0.0017 (FDR, 0.05) by one-way ANOVA. Tm, tropomyosin; MyBPC, myosin binding protein C; TnC, troponin C; TnT, troponin T. (B) SWATH-MS analysis of BAG3 and NRAP expression. Data are individual animals; means ± SEM (n = 4). **P < 0.01, ***P < 0.001 versus control, unless otherwise indicated, by one-way ANOVA and Holm-Sidak post hoc test. βMHC, β–myosin heavy chain.

  • Fig. 6.

    Randomly distributed right ventricular pacing (dyssynchrony) does not confer beneficial effects compared to HF. (A) Changes in ESV (ΔESV) and EF (ΔEF) assessed by serial echocardiography at 6 weeks versus baseline. (B) Absolute left ventricular EDP in random right ventricular pacing in HF dogs. (C) Influence of dobutamine infusion on contraction (dP/dtmax/IP). In (A) to (C), data are means ± SEM (Rand, n = 6; HF, n = 5 dogs). (D) Peak sarcomere shortening and peak calcium transient at baseline and with isoproterenol stimulation in HF and Rand. Data are means ± SEM (baseline HF, n = 13 cells from 4 dogs; baseline Rand, n = 34 cells from 7 dogs; isoproterenol HF, n = 30 cells from 4 dogs; isoproterenol Rand, n = 71 cells from 7 dogs). Black dashed line, baseline control mean; gray line, isoproterenol-stimulated control mean. #P < 0.05 versus respective baseline by two-way ANOVA and Holm-Sidak post hoc test. (E) Mean force as a function of calcium concentration (±SEM) and fitted curves for myocytes from the left ventricular lateral wall for HF and Rand6 (6 weeks of 6 hours/day randomly distributed right ventricular pacing). Summary data for Fmax and calcium sensitivity (EC50) are shown as means ± SEM (HF, n = 26 myocytes from 8 dogs; Rand6, n = 10 myocytes from 3 dogs). (F) EM structural imaging of sarcomeres. Image is representative of n = 3 sarcomeres. Scale bar, 500 nm. (G) Transverse EM image section, showing filament lattice structure. Scale bar, 200 nm. Inset: FFTs confirmed loss of normal lattice structure.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/7/319/319ra207/DC1

    Methods

    Fig. S1. Holter monitoring confirmed atrial pacing during the day and ventricular pacing at night.

    Fig. S2. β-Adrenergic response in HF and PITA.

    Fig. S3. β2AR stimulation revealed little improvement in PITA.

    Fig. S4. Western blots of βAR signaling.

    Fig. S5. Changes in βAR density were not due to mRNA expression of GRK signaling.

    Fig. S6. Holter monitoring confirmed randomly distributed 2-min periods of right ventricular pacing.

    Table S1. SWATH MS protein areas for each sample, normalized to βMHC.

  • Supplementary Material for:

    Pacemaker-induced transient asynchrony suppresses heart failure progression

    Jonathan A. Kirk, Khalid Chakir, Kyoung Hwan Lee, Edward Karst, Ronald J. Holewinski, Gianluigi Pironti, Richard S. Tunin, Iraklis Pozios, Theodore P. Abraham, Pieter de Tombe, Howard A. Rockman, Jennifer E. Van Eyk, Roger Craig, Taraneh G. Farazi, David A. Kass*

    *Corresponding author. E-mail: dkass{at}jhmi.edu

    Published 23 December 2015, Sci. Transl. Med. 7, 319ra207 (2015)
    DOI: 10.1126/scitranslmed.aad2899

    This PDF file includes:

    • Methods
    • Fig. S1. Holter monitoring confirmed atrial pacing during the day and ventricular pacing at night.
    • Fig. S2. β-Adrenergic response in HF and PITA.
    • Fig. S3. β2AR stimulation revealed little improvement in PITA.
    • Fig. S4. Western blots of βAR signaling.
    • Fig. S5. Changes in βAR density were not due to mRNA expression of GRK signaling.
    • Fig. S6. Holter monitoring confirmed randomly distributed 2-min periods of right ventricular pacing.

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S1 (Microsoft Excel format). SWATH MS protein areas for each sample, normalized to βMHC

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