Pacemaker-induced transient asynchrony suppresses heart failure progression

Abstract

Uncoordinated contraction from electromechanical delay worsens heart failure pathophysiology and prognosis, but restoring coordination with biventricular pacing, known as cardiac resynchronization therapy (CRT), improves both. However, not every patient qualifies for CRT. We show that heart failure with synchronous contraction is improved by inducing dyssynchrony for 6 hours daily by right ventricular pacing using an intracardiac pacing device, in a process we call pacemaker-induced transient asynchrony (PITA). In dogs with heart failure induced by 6 weeks of atrial tachypacing, PITA (starting on week 3) suppressed progressive cardiac dilation as well as chamber and myocyte dysfunction. PITA enhanced β-adrenergic responsiveness in vivo and normalized it in myocytes. Myofilament calcium response declined in dogs with synchronous heart failure, which was accompanied by sarcomere disarray and generation of myofibers with severely reduced function, and these changes were absent in PITA-treated hearts. The benefits of PITA were not replicated when the same number of right ventricular paced beats was randomly distributed throughout the day, indicating that continuity of dyssynchrony exposure is necessary to trigger the beneficial biological response upon resynchronization. These results suggest that PITA could bring the benefits of CRT to the many heart failure patients with synchronous contraction who are not CRT candidates.

Conflict of interest statement

Competing Interests: J.A.K. and D.A.K. are listed as inventors on a pending patent on the use of temporary dyssynchrony to improve cardiac function in failing hearts. H.A.R. is a scientific cofounder for Trevena Inc., a company that is developing GPCR-targeted drugs. E.K. and T.G.F. hold stock in St. Jude Medical, Inc., which developed the pacemaker system and software.

Copyright © 2015, American Association for the Advancement of Science.

Figures

Figure 1. Pacemaker induced transient asynchrony (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 heart failure (HF) and PITA treated group. The left-ventricle (LV) is outlined in yellow. (B and C) LV end-systolic volume (ESV) (B) and ejection fraction (EF) (C) assessed by echocardiography at baseline (BL, n = 10), after two weeks of atrial pacing (n = 18), at the end of the six-week atrial pacing protocol (HF, n = 8), and at the end of six-week PITA protocol (n = 9). (D) LV end-diastolic pressure (LVEDP) from hemodynamic studies (n: Con = 8, 2wks = 12, HF = 13, PITA = 10 dogs). EDP data were non-normal, and are displayed as box plots. Data were log10-transformed before one-way ANOVA. Direct comparison to 2wks group made by t-test). (E) Example LV pressure waveforms in HF and PITA hearts at baseline and with 15 mcg/kg/min dobutamine. (F) Dobutamine dose-effect on LV contractility (dP/dtmax/IP) (n: Control = 6, HF = 9, PITA = 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 vs. Control; †p<0.05, ‡p<0.001 vs. HF by one-way ANOVA and Holm Sidak post-hoc test.

Figure 2. Myocyte function is depressed in HF after β-adrenergic receptor stimulation, but near normal with PITA

(A) Example tracings of sarcomere shortening and intracellular calcium transients from LV lateral wall myocytes isolated from healthy control, HF, and PITA dogs at baseline and after isoproterenol stimulation (0.1 μM) or after norepinephrine and prazosin stimulation (NE, 0.1 μM; Prz, 1 μM). Sarcomere shortening and intracellular calcium are quantified below as means ± SEM (n = 4–7 dogs in each group, cells/dog: 5.8 ± 0.4, mean ± SEM). For all three groups, Iso and NE+Prz sarcomere shortening and peak Ca2+ transient data are p<0.05 versus respective baseline; ***p<0.001 vs. Control by two-way ANOVA and Holm-Sidak post-hoc test. (B) Cyclic AMP activity after isoproterenol or forskolin (FSK) stimulation. Data are individual dogs (n = 8), with means ± S.E.M. (C) Plasma membrane β-AR, β1-AR, and β2-AR density. Data are individual dogs and means ± SEM (n = 8 per group). In B and C, *p<0.05, **p<0.01, ***p<0.001 vs. Control by one-way ANOVA and Holm-Sidak post-hoc test.

Figure 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 LV lateral wall for Con, 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 (n: Con = 15 from 6 dogs, HF = 26 myocytes from 8 dogs, PITA = 14 myocytes from 4 dogs, cells/dog = 3 ± 0.2, mean ± SEM). (B) Representative images (40x) of Con, 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 and means ± SEM. (F) Phospho-serine 22/23 troponin I (TnI) protein expression normalized to total TnI. Data are individual dogs and means ± SEM (n = 4 per group). (G) Phospho-serine 9 GSK-3β normalized to total GSK-3β (n = 4 per group). For all panels, **p<0.01, ***p<0.001 vs. Control, unless otherwise indicated, by one-way ANOVA with Holm-Sidak post-hoc test.

Figure 4. Myofilament structure and function is disrupted in HF, but restored in PITA

(A) Longitudinal sections of skinned myocytes imaged by electron microscopy. 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, bent/curved filaments with irregular spaces between that were observed in some HF myofibrils, as indicated by arrows. Scale bar, 200 nm. (C) Transverse sections of myofilaments, showing smaller diameter of myofibrils with increased space between, and loss of regular filament lattice structure in HF compared with Control and PITA-treated animals. (inset) Fast-Fourier Transforms (FFTs) of boxed areas confirmed loss of normal lattice structure in HF group. Scale bar, 200 nm. (D) Percentage of normal and disarrayed sarcomeres identified by electron microscopy (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 sub-population of fibrils generating extremely low force is indicated by the dashed line. Data are individual myofibrils and box plots, as HF data were non-normally distributed (n: Con = 15, HF = 18, PITA = 18 myofibrils). P values determined by Mann-Whitney Rank Sum non-parametric test. (G) Fmax compared after removing the weak myofibrils from (F) (n removed from the analysis: Con = 0, HF = 7, PITA = 1 myofibril). n.s., not significant by one-way ANOVA.

Figure 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 (Bcl2-associated athanogene 3, BAG3, and nebulin-related anchoring protein, NRAP) were significantly different between HF and PITA: p<0.0017 (false discovery rate = 0.05) by one-way ANOVA. (B) SWATH mass spectrometry analysis for BAG3 and NRAP expression. Data are individual animals (n = 4) and means ± SEM. **p<0.01, ***p<0.001 vs. Control, unless otherwise indicated, by one-way ANOVA and Holm-Sidak post-hoc test.

Figure 6. Randomly distributed RV pacing (dyssynchrony) does not confer beneficial effects compared to HF

(A) Change in end-systolic volume (ΔESV) and ejection fraction (ΔEF) assessed by serial echocardiography at 6 weeks versus baseline. (B) Absolute LVEDP in random RV pacing in HF dogs. (C) The influence of dobutamine infusion on contraction (dP/dtmax/IP). In (A to C), data are means ± SEM (n: Rand = 6, HF = 5 dogs). (D) Peak sarcomere shortening and peak calcium transient at baseline and with isoproterenol stimulation in HF and Rand. Data are means ± SEM (n: Baseline HF: 13 cells from 4 dogs; Baseline Rand: 34 cells from 7 dogs; Iso HF: 30 cells from 4 dogs; Iso Rand: 71 cells from 7 dogs). Black dashed line: baseline control mean, gray line: isoproterenol-stimulated control mean. #p<0.05 vs. 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 LV lateral wall for HF and Rand6 (six weeks of 6 h/day randomly distributed RV pacing). Summary data for Fmax and calcium sensitivity (EC50) are shown as means ± SEM (n: HF = 26 myocytes from 8 dogs, Rand6 = 10 myocytes from 3 dogs). (F) EM structural imaging of sarcomeres. Image is representative of n = 3. 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.