Molecular and structural transition mechanisms in long-term volume overload

Abstract

Aim: We have previously reported that early phase (1 week) of experimental volume overload (VO) has an adaptive phenotype while wall stress-matched pressure overload (PO) is maladaptive. Here we investigate the transition from adaptation to heart failure (HF) in long-term VO.

Methods and results: FVB/N wild-type mice were subjected to VO induced by aortocaval shunt, and were followed by serial echocardiography until in vivo left ventricular ejection fraction was below <50% (135 ± 35 days). Heart failure was evident from increased lung and liver weight and increased mortality compared with sham. Maladaptive remodelling resulted in significantly reduced sarcomeric titin phosphorylation (causing increased sarcomeric stiffness), whereas interstitial fibrosis was not increased. This was paralleled by re-expression of the fetal gene program, activation of calcium/calmodulin-dependent protein kinase II (CaMKII), decreased protein kinase B (Akt) phosphorylation, high oxidative stress, and increased apoptosis. Consistently, development of HF and mortality were significantly aggravated in Akt-deficient mice.

Conclusion: Transition to HF in VO is associated with decreased Akt and increased CaMKII signalling pathways together with increased oxidative stress and apoptosis. Lack of interstitial fibrosis together with sarcomeric titin hypophosphorylation indicates an increased stiffness at the sarcomeric but not matrix level in VO-induced HF (in contrast to PO). Transition to HF may result from myocyte loss and myocyte dysfunction owing to increased stiffness.

Keywords: Akt signalling; Aortocaval shunt; Eccentric hypertrophy; Heart failure; Volume overload.

Figure 1

Cardiac dilatation, dysfunction, increased mortality and circulatory congestion in response to 20 weeks of volume overload. (A) Left ventricle weight‐to‐tibia length (LVW/TL) and right ventricle weight‐to‐tibia length (RVW/TL) ratios. (B) Echocardiographic M‐mode images. (C) Average values for left ventricular end‐diastolic diameter (LVEDD), left ventricular volume at diastole, septum thickness and ejection fraction (EF). (D) Kaplan–Meier curves depicting survival in sham‐ and shunt‐operated mice (n = 10 mice per group). (E) Mean values for lung weight‐to‐tibia length (LuW/TL) and liver weight‐to‐tibia length (LiW/TL) ratios. Data are means ± SEM; ^* P < 0.05; ^** P < 0.01; ^*** P < 0.001 versus sham. Numbers in bars are number of mice.

Figure 2

Pathological cardiac remodelling in long‐term volume overload. (A) Haematoxylins–eosin (H&E)‐stained transverse sections. Scale bar: 1 mm. (B) Representative photomicrographs illustrating ventricular myocyte cross‐sections stained with wheat germ agglutinin (WGA) (left panel). Scale bar: 50 µm. Cross sectional area (CSA) quantification is shown in the right panel. (C) Terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL)‐assay (left panel), arrows indicate apoptotic nuclei. Bar: 50 µm. Quantification is shown in the right panel. (D) Reactive oxygen species production assessed by dihydroethidium (DHE) conversion to red fluorescent ethidium. Bar: 30 µm. (E) Capillary density expressed as the number of capillaries/total cells (left panel). Bar: 30 µm. Quantification of results is shown in the right panel. (F) Masson's trichrome staining (left panel). Bar: 50 µm. Quantification of results is shown in the right panel. Data are means ± SEM; ^* P < 0.05 vs. sham. At least 4 mice/ group were analyzed.

Figure 3

Effect of haemodynamic volume overload on classical hypertrophy signalling pathways. (A) Re‐expression of fetal genes and expression of Rcan1.4 in chronic volume overload. (B) Total protein and phosphorylation levels of classical signalling pathways mediating the development of heart failure. Original immunoblots (left panel), and phosphorylated protein levels normalized to the respective total proteins (right panel). Data are means ± SEM; ^* P < 0.05; ^** P < 0.01; ^*** P < 0.001 vs. sham. Numbers in bars are number of mice; analysis per heart in duplicate.

Figure 4

Cardiac titin phosphorylation in volume overload‐subjected mice. (A,B) Graphs showing all‐titin phosphorylation in chronic (A) and acute (B) shunt normalized to sham. (C) Schematic of extensible I‐band titin region illustrating the epitope positions of the phosphospecific titin antibodies (mouse titin; UniProtKB #A2ASS6). (D,E) Graphs illustrating site‐specific phosphorylation of titin N2B‐unique sequence (N2Bus) and PEVK in chronic (D) and acute (E) shunt normalized to sham. Data are means ± SEM; ^* P < 0.05; ^** P < 0.01; ^*** P < 0.001 vs. sham. Numbers in bars are number of mouse hearts; analysis per heart in duplicate.

Figure 5

Akt^−/− mice exhibit an increased mortality and greater cardiac decompensation upon volume overload than wild‐type (WT) mice. (A) Kaplan–Meier plots showing survival rates in the indicated genotypes (n = 11–12 mice per group). (B–E) Left ventricular weight‐to‐body weight (LVW/BW) (B), septum thickness (C), left ventricular end‐diastolic diameter (LVEDD) (D), and ejection fraction (EF) (E) in WT and Akt^−/− mice at 4 weeks and 20 weeks of volume overload. Data are means ± SEM; ^* P < 0.05 vs. corresponding sham, ^# P < 0.05 vs. WT shunt. Numbers in bars are number of mice.

Figure 6

Schematic diagram for the transition to heart failure in volume overload. Akt, protein kinase B; CaMKIIδ, calcium/calmodulin‐dependent protein kinase IIδ; CN, calcineurin; EC coupling, excitation–contraction coupling. Evidence is provided in this and our previous publication.4 Preserved matrix stiffness owing to unchanged fibrosis; increased sarcomere stiffness owing to titin hypophosphorylation; increased apoptosis indicated by increased Bax/Bcl‐2 and terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL); deteriorated EC‐coupling indicated by decreased Serca2a and increased CaMKIIδ (CaMKIIδ modifies EC‐coupling).29