Tachycardiomyopathy entails a dysfunctional pattern of interrelated mitochondrial functions

Abstract

Tachycardiomyopathy is characterised by reversible left ventricular dysfunction, provoked by rapid ventricular rate. While the knowledge of mitochondria advanced in most cardiomyopathies, mitochondrial functions await elucidation in tachycardiomyopathy. Pacemakers were implanted in 61 rabbits. Tachypacing was performed with 330 bpm for 10 days (n = 11, early left ventricular dysfunction) or with up to 380 bpm over 30 days (n = 24, tachycardiomyopathy, TCM). In n = 26, pacemakers remained inactive (SHAM). Left ventricular tissue was subjected to respirometry, metabolomics and acetylomics. Results were assessed for translational relevance using a human-based model: induced pluripotent stem cell derived cardiomyocytes underwent field stimulation for 7 days (TACH-iPSC-CM). TCM animals showed systolic dysfunction compared to SHAM (fractional shortening 37.8 ± 1.0% vs. 21.9 ± 1.2%, SHAM vs. TCM, p < 0.0001). Histology revealed cardiomyocyte hypertrophy (cross-sectional area 393.2 ± 14.5 µm² vs. 538.9 ± 23.8 µm², p < 0.001) without fibrosis. Mitochondria were shifted to the intercalated discs and enlarged. Mitochondrial membrane potential remained stable in TCM. The metabolite profiles of ELVD and TCM were characterised by profound depletion of tricarboxylic acid cycle intermediates. Redox balance was shifted towards a more oxidised state (ratio of reduced to oxidised nicotinamide adenine dinucleotide 10.5 ± 2.1 vs. 4.0 ± 0.8, p < 0.01). The mitochondrial acetylome remained largely unchanged. Neither TCM nor TACH-iPSC-CM showed relevantly increased levels of reactive oxygen species. Oxidative phosphorylation capacity of TCM decreased modestly in skinned fibres (168.9 ± 11.2 vs. 124.6 ± 11.45 pmol·O₂·s^-1·mg^-1 tissue, p < 0.05), but it did not in isolated mitochondria. The pattern of mitochondrial dysfunctions detected in two models of tachycardiomyopathy diverges from previously published characteristic signs of other heart failure aetiologies.

Keywords: Acetylome; Mitochondria; Redox; Tachycardiomyopathy.

Fig. 1

Tachypacing induces left ventricular non-fibrotic eccentric hypertrophy and severe heart failure syndrome. After 10 days of rapid ventricular pacing by a permanent pacemaker (A), animals developed progressive LV dilatation (B), systolic dysfunction (C) and left atrial enlargement (D). TCM animals showed severe systolic heart failure as evident in increased natriuretic peptide expression (E), reduced tissue cGMP levels (F), as well as fluid retention with pericardial effusion (G, asterisk) and ascites (H, arrow). Cardiomyocyte hypertrophy was confined to advanced disease with increased cell cross-sectional area and stable nuclear density (I–L). Apoptosis increased in TCM (M–O), resulting in increased high-sensitivity serum troponin T levels (P). Masson’s trichrome stain (Q–S) and measurement of hydroxyproline content (T) did not reveal any increase in fibrosis. Data are shown as mean ± SEM. *p < 0.05, ***p < 0.001 for ANOVA with Tukey post-hoc test (B–D, I, P) and t test (F, M). A fluoroscopy after placement of the right ventricular pacemaker lead. J, K wheat germ agglutinin staining. N, O staining with DAPI (blue) and TUNEL (green). R, S Masson’s trichrome staining. cGMP cyclic guanosine monophosphate, CSA cross-sectional area, LV left ventricular

Fig. 2

Tachycardiomyopathy entails disturbed mitochondrial distribution and morphology. In LV tissue of animals with advanced disease, the mitochondrial network was partially shifted from the sarcomeres to the intercalated discs (A–E). TCM animals showed giant mitochondria in TCM (F, G): LV tissue of TCM animals featured extensive perimitochondrial lipid droplets (H). Protein expression of key regulators of mitochondrial fusion, fission and mitophagy remained unchanged (I–P). Isolated mitochondria of TCM showed an earlier mitochondrial calcium release upon repetitive 10 µmol/l calcium pulses. Inhibition of the mitochondrial permeability transition pore by cyclosporine A (CsA) abolished the increased mitochondrial calcium release in TCM (Q, R). Data are shown as mean ± SEM. R p value for two-way ANOVA. A–C confocal microscopy with staining for HSP60 (green) and N-cadherin (red). D–H transmission electron microscopy. Q calcium retention capacity of isolated mitochondria exposed to repetitive calcium-pulses. Extramitochondrial calcium was monitored. Calcium extrusion was post-hoc calculated (R) from original tracings (Q). CsA cyclosporine A, LI lipid droplet, MF myofibril, MI mitochondrion

Fig. 3

Tachycardiomyopathy is characterised by severe depletion of tricarboxylic acid cycle intermediates and emptying of the NADPH and NADH pool with a more pronounced effect in early disease than in heart failure state. Principal component analysis of metabolomics data of LV tissue clearly separated SHAM, ELVD and TCM (A). TCA intermediates were depleted even more in ELVD than in TCM (B). Acetylomics of isolated LV mitochondria based on SWATH–MS analysis remained without differences in the acetylation status of 25 identified peptides (C), indicating unchanged mitochondrial acetylome in tachycardiomyopathy. ELVD was characterised by diminished amino acids and increased fatty acids (D), being partially recovered in TCM (E). NADH and NADPH were decreased in disease, with a more pronounced loss in ELVD than in TCM (F). **p < 0.01, ***p < 0.001 for ANOVA. B fold change ELVD/SHAM (blue) and TCM/SHAM (red) of identified intermediates in metabolome analysis. LV left ventricular, NAD(H) nicotinamide adenine dinucleotide, NADP(H) nicotinamide adenine dinucleotide phosphate, SWATH–MS sequential window acquisition of all theoretical mass spectra, TCA tricarboxylic acid cycle

Fig. 4

Tachycardiomyopathy implies mild respiratory dysfunction and stable ROS emission of mitochondria. Oxygen consumption of isolated mitochondria was similar for SHAM and TCM in the presence of pyruvate/malate/glutamate/succinate (A carbohydrate metabolism) and upon supplementation with fatty acids (B beta-oxidation). The expression levels of ETC complexes were equal in SHAM and TCM (C). In skinned fibres, OXPHOS declined (D). Simultaneous to oxygen consumption, mitochondrial membrane potential was measured and found to be similar for SHAM and TCM (E, F). Depletion of TCA metabolites was not explained by mitochondrial activities of aconitase, isocitrate dehydrogenase and malate dehydrogenase (G–I). Mitochondrial redox balance was shifted towards a more oxidised state (J). Neither pyruvate/malate/glutamate/succinate- nor fatty acid-respiration were accompanied by altered mitochondrial H₂O₂ emission in TCM (K, L). In LV tissue, mitochondrial aconitase activity and malondialdehyde content were similar for SHAM, ELVD, TCM (M, N). The ratio of the glutathione redox couple did not change (O). Data are shown as mean ± SEM. *p < 0.05, **p < 0.01 for t test. ETS electron transfer system capacity, FA fatty acid, GSH glutathione, GSSG glutathione disulphide, IDH isocitrate dehydrogenase, MDH malate dehydrogenase, NAD nicotinamide adenine dinucleotide, OXPHOS oxidative phosphorylation, PMGS pyruvate–malate–glutamate–succinate

Fig. 5

Mitochondrial function in iPSC–CM tachypaced for 1 and 7 days. Seven days of tachypacing lead to mitochondrial enlargement and pleomorphy. Furthermore, an increased number of lipid droplets was found compared to the control group (A–C). Apoptosis rate did not increase (D). Oxygen consumption remained stable after 24 h, but mildly decreased after 7 days (E). Mitochondrial content was unchanged after 24 h and 7 days (F). Mitochondrial ROS emission (MitoSOX) rose after 24 h, with inhibition of contraction by blebbistatin cancelling most of the effect (G). Data are shown as mean ± SEM. *p < 0.05, **p < 0.01 for paired t test CTRL vs. TACH and t test TACH vs. TACH + BLEB. D n = 7 differentiations of 3 donors. E n = 6 differentiations of 4 donors. F n = 5 differentiations of 2 donors. G n = 9 differentiations of 2 donors for CTRL and TACH, n = 4 differentiations of 1 donor for TACH + BLEB. A–C transmission electron microscopy. BLEB Blebbistatin, CSA citrate synthase activity, ETS electron transfer system capacity, iPSC–CM induced pluripotent stem cell cardiomyocyte, LI lipid droplet, MI mitochondrion, OXPHOS oxidative phosphorylation capacity, PMGS pyruvate–malate–glutamate–succinate

Fig. 6

Tachypacing does not primarily induce right ventricular remodelling. After 30 days of tachypacing, echocardiography revealed a trend towards right ventricular dilatation (A) and decreased right ventricular stroke volume (B). Mitochondrial distribution in right ventricular cardiac myocytes was only subtly altered (C, D). Histology of right ventricular tissue showed a trend towards cardiomyocyte hypertrophy (E, F) and increased apoptosis (G) without fibrosis (H). Aconitase activity and malondialdehyde content remained unchanged (I, J). Thus, no evidence of oxidative stress was found. Data are shown as mean ± SEM. *p < 0.05 for t test. C, D confocal microscopy with staining for HSP60 (green) and N-cadherin (red). CSA cross-sectional area, RV right ventricular

Fig. 7

Mitochondrial functions in tachycardiomyopathy and other heart failure aetiologies. The two columns on the left side summarise our results on mitochondrial functions in tachycardiomyopathy. The three columns on the right side compile the current knowledge on main mitochondrial functions in dilated cardiomyopathy (DCM) as well as in heart failure due to coronary artery disease (CAD) or pressure-overload [, , , , –, –, , , , –, –, , , –64, 69, 70, 72, 74, 76, 81, 85]. EMID enrichment of mitochondria at intercalated discs, Redox balance ratio of reduced to oxidised nicotinamide adenine dinucleotide, ROS reactive oxygen species, TCA intermediates of the tricarboxylic acid cycle