Molecular Mechanism of the Association Between Atrial Fibrillation and Heart Failure Includes Energy Metabolic Dysregulation Due to Mitochondrial Dysfunction

Abstract

Background: Atrial fibrillation (AF) and heart failure (HF) commonly coexist, yet the molecular mechanisms of this association have not been determined. We hypothesized that an energy deficit due to mitochondrial dysfunction plays a significant role in pathogenic link between AF and HF.

Methods and results: Myocardial energy metabolism and mitochondria were examined in atrial tissue samples from patients and mice (cardiac-specific LKB1 knock-out) with HF and/or AF. There was significant atrial energy (ATP) deficit in patients with HF (11.5±1.3 nmol/mg, n=10; vs without HF 17±3.8 nmol/mg, n=5, P = .032). AF was associated with further energy depletion (ATP 5.4±1.2 nmol/mg, n=9) in HF (P = .001) and metabolic stress (AMP/ATP 1.6±0.1 vs 0.7±0.2 in HF alone; P = .043). The left atrium demonstrated lower ATP than the right (P = .004). Mitochondrial dysfunction and remodeling caused ATP depletion with impaired oxidative phosphorylation complexes (succinate dehydrogenase and cytochrome c oxidase), increased reactive oxygen species, and mtDNA damage in mice and human atria with AF and HF.

Conclusions: Molecular mechanisms of the association between HF and AF include an energy deficit due to mitochondrial dysfunction in atrial myocardium. Mitochondrial functional and structural remodeling in human and mouse atria is associated with energy metabolic dysregulation and oxidative stress that promote AF in HF and vice versa.

Keywords: Atrial fibrillation; energy metabolism; heart failure; mitochondria.

Figure 1:. Atrial Energetics and Mitochondrial Dysfunction in Human Atrial Tissue in Patients with Heart Failure and Atrial Fibrillation.

A) Atrial myocardial energetic was significantly decreased in patients with heart failure (HF) and atrial fibrillation (AF) as shown with low ATP levels. Atrial ATP concentration was lowest in patients with persAF in HF compared to HF in sinus rhythm. Thus, presence of AF severely reduces atrial ATP levels in HF. Inset shows high performance liquid chromatography measurement of nucleotide profile. B) HF with or without AF was associated with significant metabolic stress in atrial myocardium as reflected with increased AMP/ATP ratio. Metabolic stress was more prominent in patients with coexisting persAF and HF. C) Left atrium ATP content was significantly lower than right atrium in advanced HF with or without AF. However, left atrium has the worst energy deficit in AF. D&E) Impaired energetic in atrial myocardium with HF was associated with reduced mitochondrial oxidative phosphorylation protein complexes: succinate dehydogenase (SDH, complex II) and cytochrome c oxidase (COX, complex IV). These two essential mitochondrial electron transport complexes were significantly low in HF with AF. F) Atrial myocardial energy deficit and metabolic stress in AF and HF were associated with oxidative stress as reflected with increased superoxide dismutase (SOD) generation as response to significantly higher reactive oxygen species generation. Insets show western blotting for specific proteins in panels D-F. The immunoblot analysis is corrected with loading control, antibody against GAPDH. (*P<0.05; ANOVA presents comparing all groups for any difference, t-tests were used pairwise comparisons ).

Figure 2:. Atrial Energetics and Mitochondrial Dysfunction in Mouse Model of Atrial Fibrillation with Heart Failure.

A, B & C) As opposed to wild type (WT) mice, a novel mouse model of atrial fibrillation (AF) shows significant electrical and structural remodeling. Electrocardiograms demonstrate sinus rhythm (SR) in wild type (WT) (A) and LKB1 knockout (KO) mice (B). LKB1 KO mice develop spontaneous AF (C) and then heart failure (HF). Cardiac magnetic resonance imaging showed significant right and left atrial enlargement in LKB1 KO mice in AF and HF (lower panel). D) ECG analysis demonstrated heart rate was similar in WT and KO mice in SR while ventricular rate was slower in KO in AF and HF. E). Atrial size significantly increased in AF and HF with progressive right and left atrial enlargement compare to WT mice. F) Left ventricular ejection fraction was reduced in AF. G, H & I) Disrupted atrial energetics and metabolism was associated with development of atrial remodeling, AF and HF. Atrial myocardial content of nucleotides including ATP (G) and ADP (H) was significantly lower in LKB1 KO mice in SR and AF compare to WT mice. As shown by high performance liquid chromatography measurement (G), ATP was significantly depleted in LKB1 KO atrial myocardium starting in SR and further decrease in AF. Metabolic stress, as reflected in AMP/ATP ratio (I), was more significant in LKB1 KO hearts with AF than in WT. Thus, AF is associated with impaired myocardial energetics and metabolism. The immunoblot analysis is corrected with loading control, antibody against GAPDH. (*P<0.05; Comparisons between WT versus KO mice; The Student t-test, ANOVA)

Figure 3:. Mitochondrial Dysfunction in mechanism of Atrial Fibrillation and Heart Failure in Mice Atria.

A-F) Mitochondrial electron transport chain, matrix, inner- and outer membrane proteins were significantly impaired in LKB1 knockout (KO) heart atria compared with wild type (WT) atria, particularly in atrial fibrillation (AF) and heart failure (HF) co-existence. In KO atria with AF and HF, succinate dehydrogenase (complex II) (A), cytochrome c oxidase (complex IV) (B), pyruvate dehydrogenase (C), voltage gated anion channel (D), prohibitins 1 (E) and cytochrome c (F) levels were significantly lower compare to WT atria and KO atria in sinus rhythm (SR) without HF. Functional and structural impairment started in SR and then worsen in persAF and HF. G-I) Mitochondrial dysfunction was associated with oxidative stress and mitochondrial DNA (mtDNA) damage. There was significantly higher reactive oxygen species (ROS) generation in AF and HF compared to WT atrial tissue as shown in elevated level of hydrogen peroxide (H₂O₂) that was measured by using 2,7-dichlorofluorescein (DCF) diacetate fluorescence (G) and superoxide as shown elevated ROS scavenger, superoxide dismutase (SOD) (H). Mitochondrial DNA (mtDNA, long fragment: 8636-bp) was significantly damaged in AF and HF (I, upper panel). However, the short fragment of mtDNA was similarly amplified in both groups (I, lower panel). Thus, impaired mitochondrial complexes and oxidative stress were associated with mtDNA damage in AF and HF. Insets show western blotting for specific proteins in panels A-F and H. The immunoblot analysis is corrected with loading control, antibody against GAPDH. (*P<0.05; Comparisons between WT versus KO mice; The Student t-test and the Mann–Whitney–Wilcoxon test for heterogenous data)

Figure 4.. Mitochondrial Ultrastructural Remodeling in Mechanism of Atrial Fibrillation and Heart Failure.

A-F) Mitochondrial ultrastructure was significantly disrupted in atrial fibrillation (AF) and heart failure (HF). Electron microscopy showed that atrial cardiomyocyte with AF and HF has higher number of mitochondria than in wild type (WT) (A) and larger mitochondrion-volume (B). Mitochondrial matrix edema and disruption of the inner and outer membranes were demonstrated by measurement of crista density that was significantly lower in AF and HF indicating mitochondrial damage (C). Electron microscopy images showed significantly impaired mitochondrial ultrastructure in cardiomyocytes with AF compare to WT in SR (D, E & F). Mitochondrial functional and structural damage were associated with activation of mitochondrial apoptotic cascade (caspase 9) in mice atria (G), and human atria (H) with HF and AF. Mitochondrial apoptotic cascade, was activated in KO atria in AF compare to WT atria. Insets show western blotting for specific proteins in panels G and H. The immunoblot analysis is corrected with loading control, antibody against GAPDH. (*P<0.05; Comparisons between WT versus KO mice from panel A to G; Comparisons between patients with no HF versus patients with HF with/without AF, panel H; The Student t-test and the Mann–Whitney–Wilcoxon test)