Chaetocin | Pi3k Signal

ALDH2 protects against high fat diet-induced obesity cardiomyopathy and defective autophagy: role of CaM kinase II, histone H3K9 methyltransferase SUV39H, Sirt1, and PGC-1α deacetylation

Shuyi Wang1,2 Cong Wang1 Subat Turdi2 Kacy L. Richmond2 Yingmei Zhang1,2 Jun Ren1,2

Received: 5 October 2017 / Revised: 17 November 2017 / Accepted: 8 December 2017
© Macmillan Publishers Limited, part of Springer Nature 2018

Abstract
Background and aims Uncorrected obesity contributes to cardiac remodeling and contractile dysfunction although the underlying mechanism remains poorly understood. Mitochondrial aldehyde dehydrogenase (ALDH2) is a mitochondrial enzyme with some promises in a number of cardiovascular diseases. This study was designed to evaluate the impact of ALDH2 on cardiac remodeling and contractile property in high fat diet-induced obesity.
Methods Wild-type (WT) and ALDH2 transgenic mice were fed low (10% calorie from fat) or high (45% calorie from fat) fat diet for 5 months prior to the assessment of cardiac geometry and function using echocardiography, IonOptix system, Lectin, and Masson Trichrome staining. Western blot analysis was employed to evaluate autophagy, CaM kinase II, PGC- 1α, histone H3K9 methyltransferase SUV39H, and Sirt-1.
Results Our data revealed that high fat diet intake promoted weight gain, cardiac remodeling (hypertrophy and interstitial
ﬁbrosis, p < 0.0001) and contractile dysfunction (reduced fractional shortening (p < 0.0001), cardiomyocyte function (p < 0.0001), and intracellular Ca2+ handling (p = 0.0346)), mitochondrial injury (elevated O2− levels, suppressed PGC-1α, and enhanced PGC-1α acetylation, p < 0.0001), elevated SUV39H, suppressed Sirt1, autophagy and phosphorylation of AMPK and CaM kinase II, the effects of which were negated by ALDH2 (p ≤ 0.0162). In vitro incubation of the ALDH2 activator
Alda-1 rescued against palmitic acid-induced changes in cardiomyocyte function, the effect of which was nulliﬁed by the Sirt-1 inhibitor nicotinamide and the CaM kinase II inhibitor KN-93 (p < 0.0001). The SUV39H inhibitor chaetocin mimicked Alda-1-induced protection again palmitic acid (p < 0.0001). Examination in overweight human revealed an inverse correlation between diastolic cardiac function and ALDH2 gene mutation (p < 0.05).
Conclusions Taken together, these data suggest that ALDH2 serves as an indispensable factor against cardiac anomalies in diet-induced obesity through a mechanism related to autophagy regulation and facilitation of the SUV39H-Sirt1-dependent PGC-1α deacetylation.

These authors contributed equally: Shuyi Wang and Cong Wang.

Portion of this work was presented in the 2013 American Diabetes Association Scientiﬁc Session in Chicago, IL, USA.

Introduction

Uncorrected obesity imposes a major independent risk for myocardial dysfunction and remodeling, leading to the increased cardiovascular morbidity and mortality [1–3].

Both clinical and experimental evidence suggests a key role

Electronic supplementary material The online version of this article (https://doi.org/10.1038/s41366-018-0030-4) contains supplementary material, which is available to authorized users.

Yingmei Zhang [email protected]
Jun Ren
[email protected]

of excessive fat caloric intake in the onset of obesity and associated cardiac remodeling, myocardial dysfunction, and

1 Shanghai Institute of Cardiovascular Diseases, Zhongshan Hospital, Fudan University, 200032 Shanghai, China
2 Center for Cardiovascular Research and Alternative Medicine, University of Wyoming College of Health Sciences, Laramie, WY 82071, USA

mitochondrial injury [2, 4, 5,]. Several mechanisms have been put forward for obesity-related cardiomyopathy including lipotoxicity, inﬂammation, apoptosis, oxidative
stress, inﬂammation, endoplasmic reticulum (ER) stress, and dysregulated autophagy [2, 6–9]. Recent ﬁnding has also depicted a pivotal role for Ca2+/calmodulin-dependent
protein kinase II (CaM kinase II) in obesity-associated impairment of insulin signaling possibly due to dysregu- lated AMPK signaling [10, 11,]. However, little is known for the ultimate culprit pathological factors or therapeutic intervention for cardiac anomalies in obesity.
Mitochondrial aldehyde dehydrogenase (ALDH2) is a mitochondrial enzyme with a high propensity of genetic polymorphism (rs671) in approximately 40% Eastern Asian populations. Genetic mutation of ALDH2 is linked to the
pathogenesis of cardiovascular diseases including ischemic injury [12–14], hypertension [15, 16,], atherosclerosis [17], heart failure [18–20], dilated and alcoholic cardiomyopathy [21–23], diabetic cardiomyopathy [24, 25,], and aging [26, 27,]. The beneﬁcial role of ALDH2 is mainly attributed to
detoxiﬁcation of 4-hydroxy-2-nonenal (4-HNE) and pre- servation of autophagy homeostasis [12, 14,]. Recent ﬁnd- ings have also indicated an important role for ALDH2 in the regulation of longevity and Sirt1 activity [26–28]. Sirt1, a
sirtuin protein from the class III histone deacetylase
(HDAC) family, functions as a mitochondrial stress sensor for the regulation of mitochondrial proteins governing car- diac homeostasis [29]. Recent evidence suggested that Sirt1 may be controlled by the histone H3K9 methyltransferase SUV39H in myocardial infarction [30]. Given the essential role for ALDH2 in cardiovascular health and the risk of cardiac anomalies in obesity [12, 24, 31,], this study was designed to evaluate the impact of ALDH2 on cardiac function in a high fat diet-induced model of obesity. High fat and/or high caloric diet-induced obesity has been widely used in the study of obesity and obesity complications, recapitulating the challenge of overweight and obesity as a result of the rising issue of high caloric food intake [32, 33,]. The high fat diet varies with regards to the source, complexity and ratio of dietary fat, carbohydrate, and pro- tein [34]. In an effort to examine the possible mechanism of action involved in ALDH2-offered response against obesity cardiomyopathy, if any, wild-type (WT) and ALDH2 transgenic mice were fed high fat diet (Western diet) with 45% of dietary energy from fat for 5 months before cardiac geometry, contractile function, superoxide (O −) produc- tion, autophagy, CaM kinase II, histone H3K9 methyl- transferase SUV39H and its downstream regulatory signal
Sirt1, and the mitochondrial biogenesis cofactor peroxisome proliferator-activated receptor-γ co-activator-1α (PGC-1α) were examined. The transcriptional co-activator PGC-1α and the NAD+-dependent deacetylase Sirt1 are perceived as
crucial inducers of mitochondrial biogenesis via regulation

of nucleus-encoded mitochondrial genes [35]. Emerging evidence from our lab and others has depicted a pivotal role for post-translational modiﬁcation such as acetylation
in the regulation of PGC-1α function in various cardio- vascular disease settings [36–38]. To this end, acetylation of PGC-1α, levels of Sirt1 and its upstream signaling molecules SUV39H [30] and AMPK [39] were also
monitored diet-induced obesity or lipotoxicity both in vivo and in vitro.
Materials and methods

Additional material and method can be found in supplement

Experimental animals and high fat diet feeding

The experimental procedures used here were approved by the Zhongshan Hospital Fudan University (Shanghai, China) and the University of Wyoming (Laramie, WY) Institutional Animal Use and Care Committees and was in compliance with the NIH Guide for the Care and Use of Laboratory Animals. In brief, 4 month-old-male Friend virus B (FVB) wild-type (WT) and ALDH2 transgenic mice were randomly assigned to a low fat (10% of calorie from fat, product D12450B, Research Diets Inc., New Bruns- wick, NJ) or a high fat (45% of total calorie from fat, product D12451) diet for 5 months (see Table S1 for composition ingredients). Production of ALDH2 transgenic
mice in FVB mouse background using the chicken β-actin promoter was described previously [14]. Blood glucose and
plasma insulin levels were measured using glucose meter and ELISA commercial kits. ALDH2 activity was measured using the spectrophotometric absorbance (340 nm) of NAD
+ reduction to NADH and was expressed as nanomoles
NADH per min per mg of protein [24].

Echocardiographic assessment

Cardiac geometry and function were evaluated in anesthe- tized (ketamine 80 mg/kg and xylazine 12 mg/kg, i.p.) mice using a 2-dimensional (2-D) guided M-mode echocardio-
graphy (Phillips Sonos 5500) equipped with a 15–6 MHz linear transducer (Phillips Medical Systems, Andover, MD).
Adequate depth of anesthesia was monitored using toe reﬂex. The heart was imaged in the 2-D mode in the parasternal long-axis view with a depth setting of 2 cm. The M-mode cursor was positioned perpendicular to interventricular sep- tum and posterior wall of left ventricle (LV) at the level of papillary muscles from the 2-D mode. Sweep speed was at 100 mm/s for the M-mode. Diastolic wall thickness, left ventricular (LV) end diastolic dimension (EDD), and LV end

systolic dimension (ESD) were measured. LV fractional shortening was calculated as ((EDD − ESD)/EDD) × 100. Heart rate was averaged over 10 cardiac cycles [24].

Cardiomyocyte isolation and cell mechanics

Hearts were removed rapidly from mice sedated with ketamine (80 mg/kg, ip) and xylazine (12 mg/kg, ip) and perfused with Krebs–Henseleit bicarbonate solution consisting of (in mM) 118 NaCl, 4.7 KCl, 1.2 MgSO4, 1.2 KH2PO4, 25 NaHCO3, 10
HEPES, and 11.1 glucose. Hearts were digested with Liberase BlendzymeTH for 15 min. After removal and mincing of left ventricles, Ca2+ was added back to a concentration of 1.25 mM. Cardiomyocytes with no spontaneous contractions and clear edges were used for shortening and Ca2+ cycling experiments. The IonOptix soft-edge system (IonOptix, Mil- ton, MA) was employed to assess the mechanical properties of isolated myocytes. Myocytes were mounted on the stage of an Olympus IX-70 microscope in contractile buffer containing (in mM) 131 NaCl, 4 KCl, 1CaCl2,1 MgCl2, 10 glucose, and 10 HEPES. Myocytes were stimulated at 0.5 Hz with cell short- ening and relengthening evaluated using the following indices: peak shortening (PS), time-to-peak shortening (TPS), time to 90% relengthening (TR90), and maximal velocities of short- ening/relengthening (±dL/dt) [40]. To evaluate the role of CaM kinase II, SUV39H, AMPK, and Sirt1 on high fat diet intake-induced and ALDH2-induced changes in cardiac function, autophagy, and cell signaling molecules, a cohort of WT was treated palmitic acid (0.5 mM) with the ALDH2
activator Alda-1 (20 μM) [36] or the SUV39H inhibitor
chaetocin (50 nM) [30] in the absence or presence of the CaM kinase II inhibitor KN-93 (0.5 μM) [41], the AMPK activator 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR, 500 μM) [42], the Sirt1 activator SRT1720 (1 μM) [26], or inhibitor nicotinamide (NAM, 20 mM) [43] for 4 h
prior to the assessment of cardiomyocyte mechanical and protein properties.

Histological examination

After anesthesia, hearts were excised and immediately placed in 10% neutral-buffered formalin at room tempera-
ture for 24 h after a brief rinse with PBS. The specimens were embedded in parafﬁn, cut into 5-μm sections and stained with ﬂuorescein isothiocyanate (FITC)-conjugated
wheat germ agglutinin. Cardiomyocyte cross-sectional areas were calculated on a digital microscope (×400) using the Image J (version1.34S) software. The Masson’s trichrome staining was used to detect ﬁbrosis. Percentage of ﬁbrosis
was calculated using the histogram function of the photo- shop software. Brieﬂy, seven random ﬁelds (6 mm2) at ×200 magniﬁcation from each section were assessed for ﬁbrosis. The fraction of the light blue stained area normalized to the

total area was used as an indicator of myocardial ﬁbrosis while omitting ﬁbrosis of the perivascular, epicardial, and endocardial areas from the study [24].

Western blot analysis

After killing, heart tissues were homogenized and sonicated in a lysis buffer containing 20 mM Tris (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton, 0.1% sodium dodecyl sulfate, and a protease inhibitor cocktail. Myo- cardial protein samples were incubated with the anti-cas-
pase-3, anti-PGC-1α, anti-Sirt1, anti-LC3B, anti-Atg7, anti- p62, anti-AMPK, anti-phosphorylated AMPK (pAMPK,
Thr172), anti-mTOR, anti-phosphorylated mTOR (pmTOR, Ser2448), anti-CaMKII, anti-phosphorylated CaMKII (pCaMKII, Thr286), anti-glyceraldehyde-3-phosphate dehy-
drogenase (GAPDH; loading control), anti-α-tubulin (loading control) (1:1000, Cell Signaling Technology,
Danvers, MA), anti-ALDH2 (1:1000, Santa Cruz, CA) as well as the anti-SUV39H1 and SUV39H2 (1:1000, Abcam, Cambridge, MA) antibodies. Horseradish peroxidase- coupled secondary antibodies were used for membrane incubation. After immunoblotting, the ﬁlms were scanned and detected with a Bio-Rad calibrated densitometer and the intensity of immunoblot bands was normalized with corre- sponding band intensity of GAPDH [14].

Immunoprecipitation

Co-IP assay was conducted using a commercial Co-IP kit (Pierce). In brief, 50 μg puriﬁed PGC-1α antibodies were immobilized with coupling resin. Protein extracts (500 μg) were incubated with antibody-coupled resin gently end-over-
end mixing for 2 h at room temperature. The resin was washed, and the protein complexes bound to the antibody were eluted with 50 μl of elution buffer. The eluted protein was boiled and separated by 10% SDS-PAGE, transferred to
a nitrocellulose membrane, and incubated with anti-acetylated lysine antibody. Antibody binding was detected using the enhanced chemiluminescence. The ﬁlm was scanned and the intensity of immunoblotting bands was detected with a Bio- Rad Calibrated Densitometer (model GS-800) [32].

ALDH2 polymorphism in overweight human subjects

Cardiac geometry and function were assessed in a com- munity cohort from Shanghai, China, where 142 overweight individuals with body mass index >25 were enrolled. All subjects were provided informed consent for participation and underwent medical data collection. The protocol was approved by the Fudan University Zhongshan Hospital Ethics Committee. Detection of ALDH2 genetic poly- morphism was described previously [26].

Statistical analysis

Data were mean ± SEM. Statistical comparison was per- formed by a 1-way ANOVA followed by the Tukey post hoc test. Independent student t-test was used for ALDH2 genetic polymorphism evaluation. Signiﬁcance was set at p < 0.05.

Results

Echocardiographic properties of WT and ALDH2 mice challenged with fat diet

Chronic high fat diet feeding signiﬁcantly increased body weight, as well as heart, liver and kidney weights (and organ size when normalized to tibial length) along with elevated plasma levels of insulin and triglycerides. ALDH2 over- expression did not exert any effect on body and organ weight or size (p > 0.05) although it attenuated high fat diet intake- induced rise in heart and liver weights or size (normalized to tibial length) without affecting fat diet-induced changes in body and kidney weights (or size) as well as plasma levels of insulin and triglycerides (p < 0.0001). Tibial length, blood glucose levels, heart rate, LV posterior wall thickness, and LV end diastolic diameter (LV EDD) were unaffected by high fat diet feeding, ALDH2 transgene, or both (p > 0.05). High fat diet intake enhanced LV ESD (p = 0.0013), sup- pressed fractional shortening, and cardiac ALDH2 activity (p
< 0.0001) while displaying a trend of increased LV mass (p
= 0.24), the effects of which were obliterated or masked by ALDH2 transgene. ALDH2 transgene failed to elicit any echocardiographic effect by itself although it enhanced ALDH2 activity as expected (p < 0.0001) (Table S2). It is noteworthy that ALDH2 protein expression was unaffected by high fat diet intake (Fig. 1a). These data suggest that ALDH2 transgenic overexpression is capable of alleviating high fat diet-induced myocardial dysfunction.

Effect of high fat diet intake and ALDH2 on cardiomyocyte contractile function, interstitial ﬁbrosis and cardiac hypertrophy, intracellular Ca2+ handling, and O − production

Neither high fat diet feeding nor ALDH2 overtly affected phenotype of cardiomyocytes or resting cell length in cardi- omyocytes (data not shown). High fat diet intake led to a signiﬁcant decrease in peak shortening (PS, p < 0.0001) and maximal velocities of shortening/relengthening (±dL/dt, p = 0.0125) as well as prolonged TR90 (p < 0.0001) without affecting TPS. Although ALDH2 transgene itself did not
affect these mechanical indices, it signiﬁcantly attenuated or abrogated fat diet-induced mechanical changes (Fig. 1b–f). Data from Masson trichrome staining suggested that chronic

high fat diet intake triggered interstitial ﬁbrosis (p < 0.0001), the effect of which was unaffected by ALDH2. Lectin staining revealed that high fat diet intake facilitated enlar- gement of cardiomyocyte cross-sectional area, the effect of which was nulliﬁed by ALDH2 (p < 0.0001). ALDH2 itself did not elicit any effect of interstitial ﬁbrosis or cardiomyo-
cyte cross-sectional area (Fig. 2a–d). Data presented in Fig. 2e and f displayed that fat diet intake signiﬁcantly
suppressed the rise in fura-2 ﬂuorescence intensity (ΔFFI) and prolonged intracellular Ca2+ decay. Although ALDH2 did not affect these intracellular Ca2+ parameters, it restored high fat diet-induced changes in ΔFFI (p = 0.0346) and intracellular Ca2+ decay rate (p < 0.0001). Data from DHE
staining revealed that high fat intake overtly promoted accumulation of intracellular O −, the effect of which was ablated by ALDH2 (p < 0.0001) with little effect by itself (Fig. 2g), suggesting a potential role for superoxide pro- duction in high fat diet intake-induced and ALDH2 transgene-induced cardiac mechanical responses.

Effect of fat diet and ALDH2 on apoptosis and MMP

Our results indicated that fat diet intake signiﬁcantly pro- moted apoptosis and reduced MMP as evidenced by TUNEL (p = 0.0001) and JC-1 staining (p = 0.0179), the effects of which were attenuated or ablated by ALDH2 (Fig. S1A–E).

Effect of high fat diet intake and ALDH2 on myocardial PGC-1α, Sirt1, and autophagy protein markers

High fat diet intake upregulated the pro-apoptotic protein caspase-3, acetylation of PGC-1α, and SUV39H (H1 and H2) while downregulated levels of PGC-1α and Sirt1. ALDH2 transgene itself failed to alter the levels of these
apoptotic or mitochondrial proteins although it signiﬁcantly attenuated or abrogated fat diet-induced changes in these protein markers (p = 0.0162) (Fig. 3). Our result suggested that fat diet intake downregulated levels of autophagy as evidenced by decreased levels of Atg7, LC3BII, and LC3BII-to-LC3BI ratio, as well as promoted accumulation of the autophagy adaptor protein p62. ALDH2 transgene itself failed to alter the levels of these autophagy protein markers although it obliterated high fat diet-induced chan- ges in autophagy protein markers (p = 0.0089) (Fig. 4).

Effect of high fat diet intake and ALDH2 on AMPK, mTOR, and CaM kinase II

To examine the potential cell signaling mechanisms underneath high fat diet-induced or ALDH2-induced cardiac response, levels of pan and phosphorylated

Fig. 1 Expression of ALDH2 and cardiomyocyte contractile properties in cardiomyocytes from WT and ALDH2 transgenic mice fed low (LF) or high fat (HF) diet for 5 months. a ALDH2 expression. b Peak shortening amplitude. c Maximal velocity of shortening (+dL/dt). d

Maximal velocity of relengthening (−dL/dt). e Time-to-PS (TPS); and f time-to-90% relengthening (TR90). Mean ± SEM, n = 90–110 cells from three mice per group, *p < 0.05 vs. WT-LF group, #p < 0.05 vs. WT-HF group

AMPK, mTOR and CaM kinase II were evaluated. Our result revealed that fat diet intake suppressed phosphor- ylation of AMPK and CaM kinase II while promoting that of mTOR (both absolute and normalized values, p = 0.009), without affecting pan protein expression of

AMPK, CaM kinase II, and mTOR. ALDH2 itself failed to alter the pan protein or phosphorylated levels of these proteins although it obliterated fat diet-induced changes in the phosphorylation of AMPK, CaM kinase II, and mTOR (Fig. S2).

Fig. 2 Cardiac ﬁbrosis, hypertrophy, intracellular Ca2+ properties, and superoxide levels in cardiomyocytes from WT and ALDH2 mice fed low (LF) or high fat (HF) diet for 5 months. a Representative photo- graphs of Masson trichrome staining of WT and ALDH2 mice fed LF or HF diet. b Quantitative analysis of interstitial ﬁbrosis using mea- surements of ~100 cardiomyocytes from ﬁve mice per group. c Representative photographs of Lectin staining of transverse sections of left ventricular myocardium (×400) from WT and ALDH2 mice fed

LF or HF diet. d Quantitative analysis of cardiomyocyte cross- sectional (transverse) area using measurements of ~100 cardiomyo- cytes from ﬁve mice per group. e Electrically stimulated rise in FFI
(ΔFFI). f Intracellular Ca2+ decay rate; and g superoxide levels using DHE ﬂuorescence intensity. Mean ± SEM, n = 8–11 mice per group for Masson trichrome staining; n = 12–16 mice for lectin staining and n = 60–75 cells from three mice per group for fura-2. *p < 0.05 vs. WT-LF group, #p < 0.05 vs. WT-HF group

Fig. 3 Expression of caspase-3, sirt1, PGC-1α, acetyl-PGC-1α, SUV39H1, and SUV39H2 in myocardium from WT and ALDH2 mice fed LF or HF diet. a Representative gel blots depicting levels of ALDH2, caspase-3, sirt1, PGC-1α, and acetyl-PGC-1α; b ALDH2; c

Caspase-3; d Sirt1; e PGC-1α; and f acetyl-PGC-1α. Mean ± SEM, n
= 6–8 mice per group, *p < 0.05 vs. WT-LF group, #p < 0.05 vs. WT- HF group

Fig. 4 Expression of autophagy markers in myocardium from WT and ALDH2 mice fed LF or HF diet. a Representative gel blots depicting levels of LC3B, Atg7, p62, and GAPDH (loading control), b LC3BI, c

LC3BII, d LC3BII-to-I ratio, e Atg7; and f p62. Mean ± SEM, n = 7 mice per group, *p < 0.05 vs. WT-LF group, #p < 0.05 vs. WT-HF group

Role of CaM kinase and Sirt1 in palmitic acid- induced and Alda-1-induced cardiac responses

To discern a role for CaM kinase II and Sirt1 in ALDH2- offered protective effect against fat diet-induced

cardiomyocyte anomalies, isolated murine cardiomyocytes from WT mice were pretreated with 500 μM palmitic acid for 4 h in the absence of the ALDH2 activator Alda-1 (20 μM) or the SUV39H inhibitor chaetocin (50 nM) with or without co-incubation of the CaM kinase II inhibitor KN-93
Fig. 5 Contractile properties of cardiomyocytes of adult male WT mice treated with palmitic acid (PA, 0.5 mM for 4 h) in the presence or absence of the ALDH2 activator Alda-1 (20 μΜ), the SUV39H inhi-
bitor chaetocin (50 nM), the CaMKII inhibitor KN-93 (0.5 μΜ) or the
Sirt1 inhibitor nicotinamide (NAM, 20 mM). a Resting cell length. b

Peak shortening. c Maximal velocity of shortening (+dL/dt). d Max- imal velocity of relengthening (−dL/dt). e Time-to-peak shortening (TPS) and f time-to-90% relengthening (TR90). Mean ± SEM, n = 50–70 cells from three mice per group, *p < 0.05 vs. control group, #p
< 0.05 vs. PA group, †p < 0.05 vs. PA-Alda-1 group

(0.5 μΜ) or the Sirt1 inhibitor nicotinamide (NAM, 20 mM). Assessment of cardiomyocyte function revealed that pal- mitic acid overtly suppressed cardiomyocyte contractile
function as manifested by reduced PS, ±dL/dt and prolonged TR90, in a manner reminiscent to in vivo fat diet-induced obesity. Consistent with the in vivo observation, co- incubation of the ALDH2 activator Alda-1 rescued against palmitic acid-induced cardiomyocyte anomalies, the effect of which was negated by inhibition of either CaM kinase II or Sirt1 (p < 0.0001). Neither KN-93 nor nicotinamide eli- cited any notable effect on cardiomyocyte mechanics by itself. Likewise, the SUV39H inhibitor chaetocin mimicked Alda-1-induced protection against palmitic acid-elicited cardiomyocyte dysfunction without any effect by itself (Fig. 5). Further analysis of cell signaling mechanism revealed that palmitic acid exposure signiﬁcantly suppressed CaM kinase II phosphorylation, Sirt1 expression, and
LC3BII-to-LC3BI ratio while promoting PGC-1α acetyla- tion, the effect of which was negated or signiﬁcantly atte-
nuated by Alda-1 (p < 0.0001), with little effect by Alda-1 itself. Interestingly, inhibition of CaM kinase II or Sirt1 using KN-93 or nicotinamide, respectively, nulliﬁed Alda-1- elicited beneﬁcial effects on PGC-1α acetylation and
autophagy, suggesting a permissive role for CaM kinase II
and Sirt1 in Alda-1 offered cardioprotection. In addition, activation of AMPK and Sirt1 failed to mick Alda-1 offered protection against palmitic acid-induced CaM kinase II dephosphorylation although AMPK activation restored pal- mitic acid-induced downregulation of Sirt1 (p < 0.0001) (Fig. 6). To discern the upstream and downstream relation- ship between CaM kinase II/AMPK and SUV39H, murine
cardiomyocytes from WT mice were pretreated with 500 μM palmitic acid for 4 h in the absence of Alda-1 (20 μM) with or without co-incubation of the CaM kinase II inhibitor KN- 93 (0.5 μΜ) or the AMPK inhibitor compound C (5 μΜ). Our results revealed that Alda-1 reversed palmitic acid-
induced upregulation of SUV39H, the effect of which was mitigated by inhibition of CaM kinase II or AMPK (p < 0.0001) (Fig. S3).

Effects of ALDH2 genetic polymorphism on cardiac function in aging people

Given that 50% of Asian decedents carry mutant alleles of ALDH2 [ALDH2*2/1 (G/A) and ALDH2*2/2 (A/A)]
resulted from a single point mutation of the active ALDH2*1 (G/G) gene [44–46], a total of 142 overweight subjects (BMI >25) were examined with a frequency of GG
genotype of 65.73%, and GA/AA genotype of 34.27%. There was no difference of age, gender and BMI between the two groups. Echocardiography examination showed that LV wall thickness, chamber size and LV systolic function were similar between the two groups (p > 0.05). However,

LV diastolic function as evidenced by A and EE′ was worsened in overweight subjects with GA and AA geno- types compared with those with GG genotype (p-values of
0.046 and 0.013 for A and EE′ indices, respectively, Table S3). Our data revealed that ALDH2 mutation is
associated with cardiac diastolic dysfunction in overweight people.
Discussion

Findings of our study indicated that ALDH2 exerts a pro- tective effect against cardiac remodeling and contractile anomalies in obesity through preservation of autophagy,
CaM kinase II phosphorylation, and PGC-1α function. Our
ﬁndings indicated that high fat diet intake-induced changes
in cardiac remodeling, apoptosis, myocardial mitochondrial, contractile, intracellular Ca2+, and oxidative stress proper- ties are associated with dampened autophagy, altered SUV39H histone H3K9 methyltransferase, its downstream target Sirt1 levels and PGC-1 acetylation. Our data suggest that ALDH2 offers beneﬁt against fat diet-induced obesity cardiomyopathy through reversing high fat diet-induced changes in CaM kinase II, autophagy, SUV39H-Sirt1, and
PGC-1α acetylation. This is supported by Alda-1/chaetocin- elicited action against palmitic acid in autophagy and con-
tractile function, favoring the therapeutic potential of ALDH2 in obesity. Interestingly, Alda-1-elicited beneﬁcial effects against palmitic acid were negated by inhibitors of CaM kinase II and Sirt1, supporting a role for Sirt1 dea- cetylation and CaM kinase in high fat diet- and/or ALDH2- induced regulation of cardiac function. Evidence from overweight human subjects supported that notion that ALDH2 mutation is linked with worsened cardiac function. Unfavorable changes in myocardial geometry and func- tion have been shown in high fat diet-induced obesity including cardiac hypertrophy, interstitial ﬁbrosis, com- promised cardiac contractility, prolonged diastolic duration, and intracellular Ca2+ mishandling [2, 32,]. In our hands, high fat diet-induced obesity triggered cardiac remodeling and myocardial contractile defects including ﬁbrosis, car- diac hypertrophy, enlarged LVESD, reduced fractional shortening, peak shortening, ±dL/dt and prolonged TR90. These ﬁndings are consistent with previous reports for obesity-associated myopathic changes [2, 32, 47,]. Our data revealed interrupted intracellular Ca2+ homeostasis, apop- tosis, loss of MMP and O2− production following fat diet intake manifested as depressed electrically stimulated rise in intracellular Ca2+, prolonged intracellular Ca2+ decay, increased DHE and TUNEL staining as well as loss of JC-1 ﬂuorescence, denoting a possible role for intracellular Ca2+ dysregulation, apoptosis, mitochondrial injury and oxidative stress in obesity cardiomyopathy. Perhaps the most
Fig. 6 Effect of palmitic acid exposure on expression of phosphory- lated CaMKII, Sirt1, acetyl-PGC-1α, and LC3B in myocardium from FVB mice. A cohort of cardiomyocytes from FVB mice were chal- lenge with palmitic acid (PA, 0.5 mM) in vitro for 4 h in the absence or presence of the ALDH2 activator Alda-1 (20 μM),the CaMKII inhi-
bitor KN-93 (0.5 μΜ), the Sirt1 activator SRT17200, or the Sirt1

inhibitor nicotinamide (NAM, 20 mM). a pCaMKII-to-CaMKII ratio; b Sirt1; c acetyl-PGC-1α; d LC3BII-to-I ratio. Mean ± SEM, n = 8–12 mice or isolations per group (a), n = 6–9 mice or isolations per group (b), n = 8–10 mice or isolations per group (c), and n = 6–9 mice or isolations per group (d). *p < 0.05 vs. control group, #p < 0.05 vs. PA group, †p < 0.05 vs. PA-Alda-1 group
intriguing observation from our study is that ALDH2 alle- viated fat diet-induced myocardial remodeling (except ﬁbrosis), contractile anomalies and oxidative stress. Our ﬁnding suggested decreased ALDH2 enzymatic activity following 5-month high fat diet intake, denoting a role for ALDH2 in the disturbed cardiac homeostasis in diet- induced obesity. ALDH2 counteracts high fat diet-induced obesity cardiomyopathy (remodeling, contractile and intra- cellular Ca2+ anomalies) possibly related to regulation of
CaM kinase II, autophagy, SUV39H histone H3K9 methyltransferase, Sirt1, and PGC-1α. In accordance with cardiac dysfunction, decreased Sirt1 and PGC-1α levels were noted following high fat diet intake, the effect of
which was ablated by ALDH2. This was supported by the ﬁndings from in vitro studies where the Sirt1 inhibitor nicotinamide mitigated Alda-1-offered beneﬁcial mechan- ical effect. SUV39H is believed to silence gene expression in the heterochromatin region to trigger Sirt1 repression [30]. Recent evidence has revealed a pivotal role for SUV39H in the regulation of cardiac function in patholo- gical conditions such as myocardial infarction [30]. Our results revealed that Alda-1 reversed palmitic acid-induced upregulation of SUV39H, the effect of which may be mitigated by inhibition of CaM kinase II or AMPK, sug- gesting a permissive role for CaM kinase II and AMPK in the regulation of SUV39H (depicted in Fig. S4). Sirt1 is the
most important deacetylase for the deacetylation of PGC-1α [48]. Our immunoprecipitation data further revealed that
high fat diet intake promoted SUV39H, and PGC-1 acet- ylation, the effect of which was reversed by ALDH2 pos- sibly via Sirt1-dependent deacetylation. These data revealed that cardiac dysfunction under diet-induced obesity is associated with downregulated nuclear genes encoding
mitochondrial proteins as a result of PGC-1α acetylation [49–52]. PGC-1α is essential to cardiac energy metabolism and mitochondrial function. Downregulated or acetylated PGC-1α has been shown in obese or failing hearts in numerous rodent models [36, 50–52]. ALDH2 is reported to lower ROS levels [31] although the mitochondrial enzyme
may not directly function as an antioxidant. Given the recognized role of Sirt1 as a critical inhibitor to ROS [29], it may mediate the ALDH2 effect on O2− levels in our current experimental setting.
Our current data revealed suppressed CaM kinase II phosphorylation in conjunction with dampened AMPK phosphorylation, elevated mTOR phosphorylation, reduced autophagy, and O2− accumulation in obese hearts. CaM kinase II has been reported to regulate insulin sen- sitivity in pathological settings such as obesity [10] through regulation of the CaM kinase II downstream sig- naling molecules including AMPK [10, 11, 53,]. In our study, inhibition of AMPK phosphorylation and subse- quently elevated mTOR phosphorylation may be

responsible for dampened autophagy and upregulated SUV39H methyltransferase levels in obese hearts. Derangement of autophagy, a conservative mechanism for quality control through disposal and recycling of aged or damaged cellular components, is consolidated in obesity complications including cardiomyopathy [33, 54, 55,]. Autophagy has an indispensable role in cardiac home- ostasis under both physiological and pathophysiological conditions [55]. ALDH2 helps to preserve cardiac home- ostasis via autophagy-mediated ridding toxic proteins, lipids, and organelles [14, 28, 56, 57,]. Here in our hands, ALDH2 exerts its beneﬁcial effect through restoring the activity of CaM kinase II en route to restored AMPK and mTOR-dependent autophagy. This is supported by the fact that CaM kinase II inhibition nulliﬁed Alda-1-elicited beneﬁcial response against palmitic acid. Furthermore, inhibition of CaM kinase II or Sirt1 nulliﬁed Alda-1-
elicited beneﬁcial effects on PGC-1α acetylation and autophagy, suggesting a permissive role for CaM kinase II
and Sirt1 in Alda-1 offered protection. In our hands, activation of AMPK and Sirt1 failed to mimic Alda-1 offered protection against palmitic acid-induced CaM kinase II dephosphorylation although AMPK activation restored palmitic acid-induced Sirt1 downregulation. These ﬁndings favor a CaM kinase-AMPK-Sirt mediated signaling mechanism in fat diet intake- and ALDH2
offered regulation on PGC-1α acetylation, autophagy, and cardiac function responses. It is noteworthy that AMPK
activation may directly activate Sirt1 to preserve cardiac homeostasis [58]. The CaM kinase II-dependent AMPK activation axis is also reported in elsewhere such as the cardioprotective effect of adiponectin [53], a cytokine inversely correlated with obesity. Our earlier data sug- gested the ALDH2 accentuated aging-induced decline in Sirt1 [26] although it reconciled diet-induced loss of Sirt1 here in this study. The discrepancy may be related to difference in the manner of Sirt1 activation (NAD+ or AMPK) and age of mice.

Experimental limitations

Although our data suggest that ALDH2 rescues against high fat diet intake-induced cardiac anomalies, several experimental limitations must be considered. First, expression of ALDH2 transgene is non-physiological and may elicit off-target effects such as serving as an anti- oxidant. Second, change in the TUNEL apoptosis is merely associated with alterations in autophagy and car- diac function, further study is warranted to discern the interplay between autophagy and apoptosis in obesity. Last but not least, our data revealed decreased LC3B and elevated p62 in the face of fat diet intake. In the absence of assessment of autophagy ﬂux, caution has to be taken
when interpreting the authentic state of autophagy activity following fat diet intake.
In summary, ﬁndings from our present study have pro- vided evidence for the ﬁrst time that ALDH2 rescues against cardiac anomalies in high fat diet-induced obesity including cardiac remodeling, mechanical defect, and intracellular Ca2+ dysregulation likely through a CaM kinase II/AMPK-dependent regulation of SUV39H/Sirt1
and subsequently, ROS, autophagy, and PGC-1α deacety- lation (depicted in Fig. S4). Observation from overweight
human subjects supported that notion that ALDH2 genetic mutation is linked with worsened cardiac function. These ﬁndings favored the notion that ALDH2 may serve as a target for drug development and clinical management of heart dysfunction in patients with obesity. Further study is warranted to unveil the precise mechanism behind ALDH2- mediated CaM kinase II activation and SUV39H inactiva- tion in a more clinically relevant setting of obesity heart anomalies.

Acknowledgements SW was supported by the University of Wyom- ing Biomedical Science PhD program.

Funding American Diabetes Association (7-13-BS-142-BR), NSFC 81370195, NSFC81570225, NSFC81521001, and NSFC 81522004.

Authors contributions SW, CW, ST, KLR, YZ, data collection; YZ and JR: study design, funding, and manuscript writing.

Compliance with ethical standards
Conﬂict of interest The authors declare that they have no conﬂict of interest.

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