Mitochondrial aldehyde dehydrogenase (ALDH2) rescues cardiac contractile dysfunction in an APP/PS1 murine model of Alzheimer's disease via inhibition of ACSL4-dependent ferroptosis

Abstract

Alzheimer's disease (AD) is associated with high incidence of cardiovascular events but the mechanism remains elusive. Our previous study reveals a tight correlation between cardiac dysfunction and low mitochondrial aldehyde dehydrogenase (ALDH2) activity in elderly AD patients. In the present study we investigated the effect of ALDH2 overexpression on cardiac function in APP/PS1 mouse model of AD. Global ALDH2 transgenic mice were crossed with APP/PS1 mutant mice to generate the ALDH2-APP/PS1 mutant mice. Cognitive function, cardiac contractile, and morphological properties were assessed. We showed that APP/PS1 mice displayed significant cognitive deficit in Morris water maze test, myocardial ultrastructural, geometric (cardiac atrophy, interstitial fibrosis) and functional (reduced fractional shortening and cardiomyocyte contraction) anomalies along with oxidative stress, apoptosis, and inflammation in myocardium. ALDH2 transgene significantly attenuated or mitigated these anomalies. We also noted the markedly elevated levels of lipid peroxidation, the essential lipid peroxidation enzyme acyl-CoA synthetase long-chain family member 4 (ACSL4), the transcriptional regulator for ACLS4 special protein 1 (SP1) and ferroptosis, evidenced by elevated NCOA4, decreased GPx4, and SLC7A11 in myocardium of APP/PS1 mutant mice; these effects were nullified by ALDH2 transgene. In cardiomyocytes isolated from WT mice and in H9C2 myoblasts in vitro, application of Aβ (20 μM) decreased cell survival, compromised cardiomyocyte contractile function, and induced lipid peroxidation; ALDH2 transgene or activator Alda-1 rescued Aβ-induced deteriorating effects. ALDH2-induced protection against Aβ-induced lipid peroxidation was mimicked by the SP1 inhibitor tolfenamic acid (TA) or the ACSL4 inhibitor triacsin C (TC), and mitigated by the lipid peroxidation inducer 5-hydroxyeicosatetraenoic acid (5-HETE) or the ferroptosis inducer erastin. These results demonstrate an essential role for ALDH2 in AD-induced cardiac anomalies through regulation of lipid peroxidation and ferroptosis.

Keywords: ALDH2; Alda-1; Alzheimer’s disease; cardiac function; ferroptosis; landscape perceptions; lipid peroxidation; tolfenamic acid; triacsin C; 5-HETE; erastin.

Fig. 1. The effect of age on myocardial ALDH2 expression and activity and the effect of ALDH2 overexpression on cognitive function, body and organ weight, and cardiac morphology in the APP/PS1 mouse model of Alzheimer’s disease.

a Myocardial ALDH2 levels at 2, 5, and 10 months of age; inset: representative gel bands depicting ALDH2 and GAPDH (loading control). b Myocardial ALDH2 activity at 2, 5, and 10 months of age; (c) Escape latency; (d) Time in target quadrant (%); (e) Body weight; (f) Heart weight; (g) Heart-to-body weight ratio; (h) Liver weight; (i) Kidney weight. j Representative images of FITC-lectin (×200) and Masson’s trichrome staining (×100) showing myocardial morphology. k Quantitative analysis of FITC-lectin cardiomyocyte cross-sectional area. l Quantitative analysis of fibrotic area (Masson’s trichrome-stained area in light blue color normalized to total cardiac area). Mean ± SEM, n = 7–8 mice per group, *P < 0.05 between the indicated groups.

Fig. 2. Effect of ALDH2 overexpression on echocardiographic properties in the APP/PS1 mouse model of Alzheimer’s disease.

a Left ventricular (LV) wall thickness; (b) Septal wall thickness; (c) LV end systolic volume (LVESD); (d) LV end diastolic volume (LVEDD); (e) Fractional shortening; (f) Ejection fraction; (g) LV mass; (h) LV mass-to-body weight ratio; (i) Heart rate. Mean ± SEM, n = 10–11 mice per group, *P < 0.05 between the indicated groups.

Fig. 3. Effect of ALDH2 overexpression on cardiomyocyte mechanical and intracellular Ca²⁺ properties in the APP/PS1 mouse model of Alzheimer’s disease.

a Resting cell length; (b) Peak shortening; (c) Maximal velocity of cell shortening (+dL/dt); (d) Maximal velocity of cell relengthening (−dL/dt); (e) Time-to-peak shortening (TPS); (f) Time-to-90% relengthening (TR₉₀); (g) Baseline Fura-2 fluorescence intensity (FFI); (h) Electrically stimulated rise in FFI (ΔFFI); (i) Intracellular Ca²⁺ decay rate. Mean ± SEM, n = 71 (panels a–f) or 40 (panels g–i) cells from 5–6 mice per group, *P < 0.05 between the indicated groups.

Fig. 4. Mitochondrial membrane potential, mitochondrial permeability transition pore (mPTP) opening, myocardial ultrastructure, and reactive oxygen species (ROS) production in hearts from the APP/PS1 mouse model of Alzheimer’s disease with or without the ALDH2 transgene.

a Representative images of JC-1 fluorescence in cardiomyocytes from various groups (b) Pooled data depicting the ratio of aggregate/monomeric JC-1; (c) Representative transmission electron microscopy (TEM) ultrastructural images (×15,000); (d) Representative DCF fluorescent images depicting ROS production; (e) Mitochondrial area per cardiac area; (f) Percentage of damaged mitochondria; (g) Pooled data from DCF staining depicting ROS levels; (h) mPTP opening evaluated using NAD⁺ levels; (i) Levels of PGC1α; (j) Levels of UCP2. Insets: representative gel blots showing the levels of the mitochondrial proteins PGC1α and UCP2 using specific antibodies (α-tubulin was used as the loading control). Mean ± SEM, n = 7–9 images or mice per group (n = 5–6 images for panels e, f, and h). *P < 0.05 between the indicated groups.

Fig. 5. Levels of ALDH2, Aβ, and markers of apoptosis and inflammation in the myocardium of the APP/PS1 mouse model of Alzheimer’s disease with or without ALDH2 overexpression.

a Representative gel bands depicting ALDH2, Aβ, and apoptosis and inflammation markers using specific antibodies (GAPDH was used as the loading control). b Levels of ALDH2; (c) Levels of Aβ; (d) Levels of Bax; (e) Levels of Bcl-2; (f) Levels of TNFα; (g) Levels of IL-6, and (h) GSH-GSSG ratio. Mean ± SEM, n = 6–9 mice per group, *P < 0.05 between the indicated groups.

Fig. 6. Levels of protein carbonyl damage, lipid peroxidation, the lipid peroxidation regulatory signals SP1 and ACSL4, and ferroptosis in myocardium from the APP/PS1 mouse model of Alzheimer’s disease with or without ALDH2 overexpression.

a Levels of MDA; (b) Levels of protein carbonyl; (c) Representative gel bands depicting SP1, ACSL4, and ferroptosis protein markers using specific antibodies (GAPDH was used as the loading control); (d) Levels of SP1; (e) Levels of ACSL4; (f) Levels of GPx4; (g) Levels of SLC7A11; (h) Levels of NCOA4. Mean ± SEM, n = 6–9 mice per group, *P < 0.05 between the indicated groups.

Fig. 7. Effects of Aβ challenge, inhibition of SP1 and ACSL4 or induction of lipid peroxidation and ferroptosis on cardiomyocyte survival and contractile responses in isolated cardiomyocytes from WT and ALDH2 transgenic mice.

Cardiomyocytes from WT and ALDH2 transgenic mice were treated with Aβ (20 μΜ) for 6 h in the absence or presence of the SP1 inhibitor tolfenamic acid (TA, 50 μM), the ACSL4 inhibitor triacsin C (TC, 10 μM), the ferroptosis inducer erastin (20 μM) or the lipid peroxidation inducer 5-hydroxyeicosatetraenoic acid (5-HETE, 1 μM) prior to assessment of cardiomyocyte mechanical function. a Cell survival evaluated using MTT assay; (b) Peak shortening (normalized to cell length); (c) Maximal velocity of shortening (+dL/dt); (d) Maximal velocity of relengthening (-dL/dt); (e) Time-to-peak shortening (TPS); (f) Time-to-90% relengthening (TR₉₀). Mean ± SEM, n = 5 isolations (panel a) or 31 cells (panels b–f) per group, *P < 0.05 between the indicated groups.

Fig. 8. Effect of Aβ challenge, ALDH2 activation, inhibition of SP1 and ACSL4 or induction of lipid peroxidation and ferroptosis on lipid peroxidation in H9C2 myoblasts.

H9C2 cells were incubated with Aβ (20 μΜ) for 24 h in the absence or presence of the ALDH2 activator Alda-1 (20 μM), the SP1 inhibitor tolfenamic acid (TA, 50 μM), the ACSL4 inhibitor triacsin C (TC, 10 μM), the ferroptosis inducer erastin (20 μM) or the lipid peroxidation inducer 5-HETE (10 μM) prior to assessment of lipid peroxidation using BODIPY1 C11 imaging. a Representative image depicting cells from the various treatment groups; (b) Pooled BODIPY1 C11 fluorescence intensity data; and (c) Schematic diagram depicting our working model of the protective effects of ALDH2 against APP/PS1 mutation-associated pathological changes in cardiac remodeling and function. APP/PS1 mutation evokes the accumulation of Aβ and the induction of SP1-ACSL4-mediated lipid peroxidation and ferroptosis, ultimately evoking mitochondrial injury and cardiac damage. ALDH2 disrupts the Aβ-induced increase in SP1/ACSL4 to suppress lipid peroxidation and ferroptosis, thus preserving cardiac homeostasis under APP/PS1 mutation. Mean ± SEM, n = 7 independent cell cultures per group, *P < 0.05 between the indicated groups.