Cardiac resynchronization therapy restores sympathovagal balance in the failing heart by differential remodeling of cholinergic signaling

Abstract

Rationale: Cardiac resynchronization therapy (CRT) is the only heart failure (HF) therapy documented to improve left ventricular function and reduce mortality. The underlying mechanisms are incompletely understood. Although β-adrenergic signaling has been studied extensively, the effect of CRT on cholinergic signaling is unexplored.

Objective: We hypothesized that remodeling of cholinergic signaling plays an important role in the aberrant calcium signaling and depressed contractile and β-adrenergic responsiveness in dyssynchronous HF that are restored by CRT.

Methods and results: Canine tachypaced dyssynchronous HF and CRT models were generated to interrogate responses specific to dyssynchronous versus resynchronized ventricular contraction during hemodynamic decompensation. Echocardiographic, electrocardiographic, and invasive hemodynamic data were collected from normal controls, dyssynchronous HF and CRT models. Left ventricular tissue was used for biochemical analyses and functional measurements (calcium transient, sarcomere shortening) from isolated myocytes (n=42-104 myocytes per model; 6-9 hearts per model). Human left ventricular myocardium was obtained for biochemical analyses from explanted failing (n=18) and nonfailing (n=7) hearts. The M2 subtype of muscarinic acetylcholine receptors was upregulated in human and canine HF compared with nonfailing controls. CRT attenuated the increased M2 subtype of muscarinic acetylcholine receptor expression and Gαi coupling and enhanced M3 subtype of muscarinic acetylcholine receptor expression in association with enhanced calcium cycling, sarcomere shortening, and β-adrenergic responsiveness. Despite model-dependent remodeling, cholinergic stimulation completely abolished isoproterenol-induced triggered activity in both dyssynchronous HF and CRT myocytes.

Conclusions: Remodeling of cholinergic signaling is a critical pathological component of human and canine HF. Differential remodeling of cholinergic signaling represents a novel mechanism for enhancing sympathovagal balance with CRT and may identify new targets for treatment of systolic HF.

Keywords: acetylcholine; autonomic nervous system; cardiac resynchronization therapy; heart failure; receptors, muscarinic; vagal nerve stimulation.

© 2015 American Heart Association, Inc.

Figures

Figure 1. Echocardiographic, hemodynamic and electrophysiological characteristics of normal, DHF and CRT animals before sacrifice at 6 weeks

Invasive pressure measurements revealed LV end-systolic pressures (panel a) were similar between DHF (N=10) and CRT (N=10), but both were reduced compared to normal controls (N=8). Regional LV longitudinal strain (b) derived from echocardiographic speckle tracking analysis revealed similar, simultaneous strains in all regions for normal controls, but in DHF, septal shortening preceded the lateral wall with reciprocal septal stretch when the lateral wall contracted; restoration of synchrony was observed with CRT. Echocardiography-derived ejection fractions (c) and stroke volumes (d) were decreased in DHF and improved by CRT, but both remained lower than normal controls. Invasive pressure measurements revealed higher LV end-diastolic pressures (e) and lower dP/dtmax [normalized to instantaneous developed pressure (IP)] (f) in DHF compared to CRT and normal controls. Despite a similar increase in heart rate (g) in DHF and CRT, the heart rate variability (h) was significantly lower in DHF compared to normal and CRT. In all panels of this figure, *p

Figure 2. Response to cholinergic stimulation in the setting of tonic β-adrenergic stimulation

a. Representative CaT and SS of LV cardiomyocytes from normal control (black tracing), DHF (red), and CRT (blue) hearts are shown for the following solution exchange protocol in sequence (indicated by the horizontal bars at top):

1. β-adrenergic stimulation with isoproterenol [(I), left column];

   cholinergic stimulation with carbamylcholine (CCh) in the continued presence of isoproterenol [(I+C), middle column];

atropine to assess reversal of mAChR-specific effects [(I+C+A), right column]. b. The ratio of the peak responses to CCh added to isoproterenol compared to isoproterenol alone (I+C:I) on CaT (top panel) and SS (bottom) in normal control (empty bar), DHF (red) and CRT (blue) myocytes is plotted on the left column (log2 scale; mean±SEM; n=30–52 myocytes from N=6–9 hearts for each bar). Similarly, the ratio of I+C+A to I+C (I+C+A:I+C) is plotted on the right column. The individual data points are plotted in Online Figure IIa. Cholinergic stimulation reduced the respective peak CaT and SS by 59±4% and 74±4% in DHF; by 41±3% and 55±3% in normal control; and by 23±3% and 36±2% in CRT myocytes. In 15% of DHF myocytes, contraction was arrested despite continued isoproterenol exposure, and subsequent exposure to atropine fully restored CaT and contraction (Supplementary Video 1); these data were not included in this analysis. c. The ratio of the 80% duration of CaT and SS are plotted in a format similar to panel b. The individual data points are plotted in Online Figure IIb. Cholinergic stimulation markedly prolonged the respective CaT and SS by 81±18% and 74±14% in DHF; by 65±6% and 36±7% in normal control; and by 47±8% and 22±7% in CRT myocytes. d. Representative immunohistochemical staining sections of canine LV tissue (top) revealed increased M2-mAChR density in DHF myocytes at intercalated discs and sarcolemma. Quantitative analyses of RT-PCR, Western blot and immunohistochemical fluorescence data in canine LV tissue (bottom) revealed increased mRNA and protein expression of M2-mAChR in DHF but not in CRT myocytes (N=5 hearts/group). Similar results were obtained with Western blot analysis of cardiomyocytes isolated on a perchol gradient (data not shown), suggesting these M2-mAChR protein changes occur within cardiomyocytes. e. Representative immunohistochemistry staining sections of human LV tissue samples from failing and non-failing hearts are shown on top. In failing human LV, M2-mAChR expression was upregulated and redistributed from the intercalated discs to the entire cell border (“lateralization”), similar to that observed in canines. Western blot analysis of tissue lysates from human LV (bottom) revealed 2-fold higher M2-mAChR protein expression in HF (N=18) compared to non-HF (N=7), regardless of ischemic (ICM; N=4) or non-ischemic cardiomyopathy (NICM; N=14). In all panels of this figure, *p

Figure 3. Response to β-adrenergic stimulation in the setting of tonic cholinergic stimulation

a. Representative CaT and SS from normal control, DHF and CRT myocytes are plotted in a format similar to Figure 2a. The myocytes were sequentially exposed to:

1. CCh [(C), left column];

2. isoproterenol [(C+I), middle column];

and atropine [(+Atr; C+I+A), right column]. b. The ratio of the peak responses for C+I:C and C+I+A:C+I for CaT (top panel) and SS (bottom) in normal control (empty bar), DHF (filled) and CRT (striped) myocytes is plotted in a format similar to Figure 2b (n=19–32 myocytes/bar; N=6–9 hearts/bar). The individual data points are plotted in Online Figure IIIa. β-adrenergic stimulation markedly increased the respective peak CaT and SS by 176±26% and 525±57% in normal control; and by 119±20% and 620±111% in CRT myocytes; but by only 65±9% and 165±29% in DHF. Addition of atropine caused an additional increase in respective peak CaT and SS by 93±24% and 228±58% in DHF myocytes whereas normal and CRT myocytes showed little response (22–63%). c. The ratio of the 80% duration of CaT and SS are plotted in a format similar to panel b. The individual data points are plotted in Online Figure IIIb. In the continued presence of CCh, isoproterenol shortened the respective durations of CaT and SS by 15±3% and 21±4% in DHF; by 21±4 and 30±4% in normal controls; and by 29±3% and 33±5% in CRT myocytes. Atropine further shortened the durations by 35±3% and 37±4% in DHF; by 25±5% and 18±4% in normal control; and by 10±3% and 14±5% in CRT myocytes. In all panels of this figure, *p

Figure 4. Response to cholinergic stimulation alone

a. Representative CaT and SS are shown from normal controls, DHF, and CRT myocytes sequentially exposed to standard Tyrode’s extracellular solution [(ECS; E), thin gray line] followed by CCh [(C), thick black line]. b. The ratio of the peak responses for C:E and after the addition of atropine to CCh compared to CCh alone (C+A:C) for CaT (top panel) and SS (bottom) in normal control (empty bar), DHF (filled) and CRT (striped) myocytes is plotted in a format similar to Figure 3b (n=20–30 myocytes/bar; N=6–9 hearts/bar). The individual data points are plotted in Online Figure IVa. Cholinergic stimulation decreased the respective peak CaT and SS by 18±5% and 27±7% in DHF but had little or no effect in normal and CRT myocytes. These effects were reversed by subsequent addition of atropine. c. The ratio of the 80% duration of CaT and SS are plotted in a format similar to panel b. The individual data points are plotted in Online Figure IVb. Cholinergic stimulation prolonged the CaT and SS durations by 14±3% and 5±3% in DHF whereas in normal and CRT myocytes, the CaT and SS durations was either shortened or unchanged. d. The ratio of the peak responses to atropine (A:E) and washout for CaT (top panel) and SS (bottom) in normal control (empty bar), DHF (filled) and CRT (striped) myocytes is plotted in a format similar to panel b (n=8 myocytes/bar; N=3 hearts/bar). Atropine increased the peak CaT (left panel) and SS (right) by 26±5% and 33±11% in DHF, and by 9±4% and 10±3% in normal controls, but had no effect in CRT myocytes. These effects were reversed by washing off atropine. In all panels of this figure, *p

Figure 5. Cholinergic stimulation suppresses after-transients and after-contractions trigged by β-adrenergic stimulation

a. Representative CaT and SS from normal control, DHF and CRT myocytes are plotted (top) in a format similar to Figure 2a. Exposure to isoproterenol (I) induced after-transients and after-contractions that subsequently, were suppressed by CCh (I+C) and recurred with atropine (I+C+A). The bar graph (bottom) shows the percent of normal, DHF and CRT myocytes that demonstrated triggered activity (after-transients and after-contractions) in response to the corresponding solution exposures depicted at the top of the panel (n=42–104 myocytes/group; N=6–9 hearts/group). Myocytes that demonstrated isoproterenol-induced triggered activity were used only for analysis in this section and excluded from the analyses shown in Figures 2–4 and 6. b. Representative CaT and SS from CRT myocytes are plotted (top) in a format similar to Figure 3a. In the presence of CCh (C), addition of isoproterenol (C+I) did not induce after-transients and after-contractions until addition of atropine (C+I+A). The percent of normal, DHF and CRT myocytes demonstrating triggered activity are plotted corresponding to the protocol panel c (n=25–63 myocytes/group; N=6–9 hearts/group). Again, the effects of atropine were not recapitulated by pirenzapine or 4-DAMP (data not shown). Myocytes that demonstrated isoproterenol-induced triggered activity were used only for analysis in this section and excluded from the analyses shown in Figures 2–4 and 6. c. Representative CaT and SS from normal control, DHF and CRT myocytes pretreated with pertussis toxin (PTX) to inhibit Gαi are plotted in a format similar to panel a. In all myocytes from all models, sustained after-transients and after-contractions were noted with isoproterenol regardless of exposure to CCh. In all panels of this figure, *p

Figure 6. Cholinergic stimulation mediates positive and negative inotropic effects via distinct muscarinic receptor subtypes

a. The peak SS responses (mean±SEM) corresponding to the indicated solution exchange protocol are compared in the absence (empty bars) or presence (filled) of PTX for normal control (black), DHF (red bars) and CRT (blue) myocytes (n=30–52 myocytes from N=6–9 hearts for each bar). The individual data points are plotted in Online Figure Va. PTX increased the peak SS response to isoproterenol (left column) in DHF myocytes, but had no effect in normal and CRT myocytes. This is consistent with enhanced baseline Gαi activity in DHF. In the continued presence of isoproterenol, pre-treatment with PTX abolished the negative inotropic effects of cholinergic stimulation in all groups (middle column). The peak SS after addition of atropine was not significantly different with and without PTX for normal (p=0.43), DHF (p=0.13) and CRT (p=0.32) myocytes (right column). These data suggest that in the presence of saturating β-adrenegic stimulation, the negative inotropic effect from cholinergic stimulation is mediated via M2-mAChR-Gαi signaling. b. The ratio of the peak SS responses to CCh alone compared to ECS (C:E) using the same protocol as in Figure 4b are plotted in the absence and presence of PTX and a M3-mAChR-specific inhibitor (M3i) (n=8–30 myocytes/bar; N=3–9 hearts/bar). All myocytes were continuously perfused with pirenzapine to block M1-mAChR-specific effects. The individual data points are plotted in Online Figure Vb–c. Compared to the absence of PTX, cholinergic stimulation in the presence of PTX increased the peak SS by 45±8% in CRT myocytes, but this effect was abolished with M3i. In DHF myocytes, PTX abolished the negative inotropic effect from cholinergic stimulation, but M3i had no significant effect. These data suggest that CRT myocytes are biased towards M3-mAChR-mediated positive inotropic effect whereas normal and DHF myocytes are not. c. Representative immunohistochemical staining sections of canine mid-myocardial tissue from the LV lateral wall (top) revealed increased M3-mAChR density in CRT myocytes at the intercalated discs. Western blots of tissue lysates (5 hearts per group) revealed CRT increased M3-mAChR protein expression without any change in Gαq/11 protein expression. d. Proposed mechanism for autonomic remodeling in DHF and with CRT. Cholinergic stimulation can produce both inhibitory and stimulatory calcium and contractile responses in the heart via well-characterized M2-mAChR-Gαi and M3-mAChR-Gαq coupled signaling, respectively. DHF (red arrows and tracings) is associated with down-regulation of β1-adrenergic receptors (β1AR) and inhibition of adenylate cyclase (AC) from direct interactions with the α subunit of the PTX‐sensitive inhibitory G protein (Gαi) selectively coupled to M2-mAChRs. Coordinated increases in M2-mAChR-Gαi-coupled expression and signaling chronically inhibits basal AC-mediated downstream signaling and markedly impairs the efficiency of β-adrenergic responsiveness, resulting in smaller amplitudes and prolonged relaxation of CaT and SS. CRT (blue arrows and tracings) reverses this phenotype by differentially remodeling cholinergic signaling. By concurrently decreasing M2-mAChR and increasing RGS2 expression, CRT decreases the negative inotropic effects of Gαi signaling. Further, CRT increases M3-mAChR-Gαq-mediated signaling associated with positive inotropic responses and putative cardioprotective effects. In all panels of this figure, *p