Facilitated ethanol metabolism promotes cardiomyocyte contractile dysfunction through autophagy in murine hearts

Abstract

Chronic drinking leads to myocardial contractile dysfunction where ethanol metabolism plays an essential role. Acetaldehyde, the main ethanol metabolite, mediates alcohol-induced cell injury although the underlying mechanism is still elusive. This study was designed to examine the mechanism involved in accelerated ethanol metabolism-induced cardiac defect with a focus on autophagy. Wild-type FVB and cardiac-specific overexpression of alcohol dehydrogenase mice were placed on a 4% nutrition-balanced alcohol diet for 8 weeks. Myocardial histology, immunohistochemistry, autophagy markers and signal molecules were examined. Expression of micro RNA miR-30a, a potential target of Beclin 1, was evaluated by real-time PCR. Chronic alcohol intake led to cardiac acetaldehyde accumulation, hypertrophy and overt autophagosome accumulation (LC3-II and Atg7), the effect of which was accentuated by ADH. Signaling molecules governing autophagy initiation including class III PtdIns3K, phosphorylation of mTOR and p70S6K were enhanced and dampened, respectively, following alcohol intake. These alcohol-induced signaling responses were augmented by ADH. ADH accentuated or unmasked alcohol-induced downregulation of Bcl-2, Bcl-xL and MiR-30a. Interestingly, ADH aggravated alcohol-induced p62 accumulation. Autophagy inhibition using 3-MA abolished alcohol-induced cardiomyocyte contractile anomalies. Moreover, acetaldehyde led to cardiomyocyte contractile dysfunction and autophagy induction, which was ablated by 3-MA. Ethanol or acetaldehyde increased GFP-LC3 puncta in H9c2 cells, the effect of which was ablated by 3-MA but unaffected by lysosomal inhibition using bafilomycin A₁, E64D and pepstatin A. In summary, these data suggested that facilitated acetaldehyde production via ADH following alcohol intake triggered cardiac autophagosome formation along with impaired lysosomal degradation, en route to myocardial defect.

Introduction

Chronic alcohol intake leads to deleterious cardiovascular consequence including dilated cardiomyopathy, disrupted myofibrillary architecture and myocardial contractile anomalies.^1,2 A number of scenarios have been put forward for the pathogenesis of alcoholic cardiomyopathy including cardiotoxicity of alcohol, oxidative stress and accumulation of fatty acid ethyl esters^3,4 although the ultimate culprit factor(s) remain poorly understood. Recent evidence indicates a pivotal role of the ethanol metabolite acetaldehyde in the onset and development of alcoholic cardiomyopathy.^3,5 As the major ethanol metabolite, acetaldehyde plays a culprit role in the regulation of myocardial structure and contractile function including interrupted intracellular Ca²⁺ homeostasis, impaired cardiac contractile function, disturbed excitation-contractile coupling, oxidative damage, lipid peroxidation and hypertrophic responses.⁶ In addition, acetaldehyde interrupts myocardial protein synthesis and mitochondrial integrity leading to disrupted cellular metabolism required for cardiac homeostasis.^4,5,7 Recent findings from our lab depicted that alcohol dehydrogenase⁸ exacerbates cardiac geometric and functional changes associated with protein damage, oxidative and endoplasmic reticulum (ER) stress following alcohol intake.^9-11 Nonetheless, the precise mechanism of action behind the ADH enzyme facilitated ethanol metabolism and cardiac injury following chronic alcohol exposure remains poorly understood.

Ample evidence has suggested an essential role of autophagy in the regulation of cardiac structure and function.¹² As a vital dynamic process in the heart, autophagy participates in the lysosomal turnover of dysfunctional cytosolic components and serves as a catabolic energy source during times of starvation. Autophagy is present at basal level to maintain cardiomyocyte function, ventricular mass and cellular homeostasis. Impaired autophagy has been associated with cardiac diseases. For example, poor autophagic removal of mitochondria may serve as a source of lipofuscin, a toxic waste product that builds up during the life span,¹³ while impairment of lysosomal degradation leads to compromised myocardial function.^14,15 A number of studies have demonstrated that autophagy may be upregulated in response to pathological stress such as ER stress, ischemia/reperfusion injury and heart failure.^16-18 The upregulated autophagy process is expected to antagonize ventricular hypertrophy by promoting protein degradation during the transition from hypertrophic cardiomyopathy to heart failure.^17,19 On the other hand, excess of autophagy leads to abnormal removal of intracellular protein aggregates, resulting in oxidative stress, reduced ATP production, collapse of cellular catabolic machinery, loss of cardiomyocytes and cell death.¹² Furthermore, ethanol metabolism may promote autophagosome formation and suppress lysosomal function in hepatic cells to contribute to the facilitated intracellular virus accumulation and steatosis in HCV-infected alcohol-consuming patients.²⁰ More recent findings from our own group also depicted a unique role of autophagy and autophagic flux in ethanol-induced cardiac contractile dysfunction.²¹

To examine the role of autophagy in alcohol-induced and ADH-facilitated myocardial injury, our present study was designed to address the role of autophagy in a facilitated alcoholic cardiomyopathy model using ADH transgenic model. Using the cardiac-specific overexpression of ADH, we were able to mimic an “acetaldehyde overload” model of alcoholic cardiomyopathy.²² Cardiomyocyte contractile properties, myocardial histology, immunohistochemistry, GFP-LC3 puncta, autophagy markers including Atg7, Beclin 1, LC3 and p62 were closely monitored. Expression of autophagy signal molecules including class III phosphatidylinositol-3-kinase (PtdIns3K, which forms a complex with Beclin 1 to play a key role in the autophagy-lysosomal pathway²³), mTOR, p70S6K as well as the Beclin 1-related proteins Bcl-2 and Bcl-xL were examined by immunoblotting. Levels of Ca²⁺-calmodulin-dependent protein kinase (CaMKII), which plays a key role in modulating cardiac hypertrophy,²⁴ were also measured in an effort to delineate hypertrophic signaling mechanism. The fold change of micro RNA miR-30a, a potential target of Beclin 1, was evaluated using real-time PCR.

Results

Effect of chronic alcohol ingestion on myocardial histology in FVB and ADH mice

Neither chronic alcohol intake nor ADH transgene overtly affected body weight, heart rate, systolic and diastolic blood pressures. Levels of blood alcohol and acetaldehyde were significantly increased, in a comparable manner, in both FVB and ADH mice following chronic alcohol intake. Cardiac tissue acetaldehyde levels were significantly elevated following alcohol intake in both mouse groups, with a more pronounced rise in ADH mice. Myocardial ADH enzymatic activity was significantly enhanced with ADH transgenic overexpression in the heart. Chronic alcohol intake exerted little effect on cardiac ADH enzymatic activity (Table 1). Chronic alcohol feeding significantly increased the heart weight, the effect of which was accentuated by ADH. Along the same line, evaluation of myocardial histology revealed that chronic alcohol intake significantly increased cardiomyocyte cross-sectional area with a more pronounced rise in ADH mice. The H&E staining data failed to note any overt change in myocardial histology by ADH transgene itself. Along the same line, phosphorylation of CaMKII, an essential signaling molecule governing cardiac hypertrophy,²⁴ was significantly elevated in both FVB and ADH groups following chronic alcohol intake with a more pronounced rise in ADH mice (absolute and normalized value). ADH transgene did not affect the activation of CaMKII. Expression of pan CaMKII was unaffected by either alcohol intake or ADH transgene (Fig. 1).

Table 1. Biometric parameters of FVB and ADH mice following 8-week alcohol feeding.

Parameter	FVB	FVB-EtOH	ADH	ADH-EtOH
Body Weight (g)	30.6 ± 0.7	29.1 ± 0.4	31.2 ± 2.9	30.1 ± 1.2
Heart Rate (bpm)	474 ± 16	454 ± 15	464 ± 18	466 ± 18
Systolic blood pressure (mmHg)	109.5 ± 3.8	112.0 ± 3.7	106.5 ± 3.8	111.2 ± 4.5
Diastolic blood pressure (mmHg)	85.2 ± 3.0	86.7 ± 4.3	84.0 ± 4.7	86.0 ± 5.7
Blood Alcohol (mg/dl)	Undetectable	72.8 ± 6.5*	Undetectable	76.2 ± 6.4*
Blood Acetaldehyde (μM)	4.1 ± 0.6	52.6 ± 5.0*	4.7 ± 0.8	57.4 ± 6.5*
Cardiac Acetaldehyde (nmol/mg)	6.2 ± 0.5	46.3 ± 4.7*	6.1 ± 0.9	164.1 ± 13.6*,#
Cardiac ADH activity (nmol NADH/min/mg protein)	5.0 ± 0.5	5.4 ± 0.6	24.3 ± 3.2*	27.4 ± 2.7*,#

Mean ± SEM, n = 7–8 mice per group, undetectable: < 2.5 mg/dl, *p < 0.05 vs. FVB group, ^#p < 0.05 vs. FVB-EtOH group.

Figure 1.

Histological, biometric features and hypertrophic protein markers of hearts from FVB and ADH mice with or without ethanol intake (4% ethanol liquid diet for 8 weeks). (A–D) H&E staining micrographs of transverse sections of left ventricular myocardium (× 400) from FVB, FVB-EtOH, ADH and ADH-EtOH groups. (E) Quantitative analysis of cardiomyocyte cross-sectional (transverse) area using measurements of ~150 cardiomyocytes from 3–5 mice per group. (F) Heart weight. (G) Expression of hypertrophic marker CaMKII. (H) Phosphorylated CaMKII (pCaMKII); and (I) (pCaMKII-to-CaMKII ratio). Mean ± SEM, n = 5–6 mice per group (F–I), *p < 0.05 vs. FVB, ^#p < 0.05 vs. FVB-EtOH group.

Effect of chronic alcohol ingestion on autophagy markers in FVB and ADH mice

To examine the potential role of autophagy in alcoholic heart damage, expression of the autophagy markers Atg7, Beclin 1, LC3I/II and the cargo receptor p62 were evaluated in the myocardium from FVB and ADH mice following chronic alcohol intake. Immunoblotting results shown in Figure 2 revealed overtly elevated levels of LC3-II (also LC3-II-to-LC3-I ratio) and Atg7 without changes in LC3-I and Beclin 1 in myocardium from FVB mice following alcohol intake. Although ADH transgene did not affect the expression of these autophagy markers, it significantly promoted chronic alcohol intake-induced the onset of autophagy (expression of LC3-II and Atg7 as well as the LC3-II-to-LC3-I ratio). In addition, ADH transgene unmasked an alcohol intake-induced up- and downregulation of Beclin 1 and LC3-I expression, respectively. Intriguingly, ADH significantly exacerbated the p62 accumulation followed by chronic alcohol intake.

Figure 2.

Expression of autophagic markers in hearts from FVB and ADH mice with or without ethanol intake (4% ethanol liquid diet for 8 weeks). (A) Representative gel blots depicting levels of Atg7, Beclin 1, LC3-I, LC3-II and GAPDH (loading control) using specific antibodies; (B) LC3-I expression; (C) LC3-II expression; (D) LC3-II/LC3-I ratio; (E) Atg7 expression; and (F) Beclin 1 expression. Mean ± SEM, n = 5–6 mice per group, *p < 0.05 vs. FVB group, ^#p < 0.05 vs. FVB-EtOH group.

Effect of chronic alcohol ingestion on LC3 expression (fluorescent immunohistochemistry)

To assess the impact of ADH and chronic alcohol intake on autophagosome formation, LC3B expression was determined using fluorescence immunostaining technique (LC3B-positive cells shown as red fluorescence normalized to the DAPI stained nucleus number). As shown in Figure 3, chronic alcohol intake significantly increased the LC3B-positive cells visualized as red fluorescence, the effect of which was accentuated by the ADH transgene. There was little difference in LC3B expression between FVB and ADH groups in the absence of alcohol intake.

Figure 3.

Fluorescent immunohistochemistry determination on LC3B expression in myocardium from FVB and ADH mice with or without chronic alcohol exposure (4% alcohol for 8 weeks). (A1–A3, B1–B3, C1–C3 and D1–D3) Immunostaining micrographs of left ventricular myocardium sections (× 400) from FVB, FVB-EtOH, ADH and ADH-EtOH groups. (A1–D1) The immunostaining of LC3B in each group; (A2–D2) the nucleus staining using DAPI in each group; (A3–D3) overlays of LC3B immunostaining and DAPI in each group. Arrowheads indicate LC3B expression (small red puncta) in cytoplasm; and (E) percentage of the LC3 positive cells to the number of the nucleus. Mean ± SEM, n = 1500–3000 cells per group from three independent experiments, *p < 0.05 vs. FVB group, ^#p < 0.05 vs. FVB-EtOH group, Scale bar = 50 μm.

Effect of chronic alcohol intake on autophagy initiation signaling in FVB and ADH mice

To better understand the cell signaling mechanisms involved in alcohol intake and ADH-induced autophagic responses, levels of class III PtdIns3K, mTOR and phosphorylation of mTOR (Ser2448) and p70S6K (Thr389) were examined. Chronic alcohol intake significantly elevated the expression of class III PtdIns3K and decreased phosphorylation of mTOR and p70S6K without affecting the expression of mTOR. While ADH transgene itself failed to affect the levels of class III PtdIns3K and phosphorylation of mTOR and p70S6K, it significantly augmented chronic alcohol intake-induced changes in these autophagy signaling molecules (Fig. 4).

Figure 4.

Expression of autophagy signaling proteins in FVB and ADH mice with or without chronic alcohol exposure (4% alcohol for 8 weeks). (A) Representative gel blots depicting expression of PtdIns3K class III, phospho-p70S6K (Thr389), phosphor-mTOR (Ser2448), mTOR and α-tubulin (loading control); (B) class III PtdIns3K; (C) mTOR expression; (D) pmTOR level; (E) pmTOR-to-mTOR ratio; and (F) phospho-p70S6K (Thr389). Mean ± SEM, n = 5–6 per group, *p < 0.05 vs. FVB group, ^#p < 0.05 vs. FVB-EtOH group.

Effect of chronic alcohol intake on levels of Beclin 1 related proteins and miR-30a

To further evaluate the role of Beclin 1 in alcohol intake and ADH-induced autophagy, levels of Bcl-2, Bcl-xL proteins and miR-30a which plays an essential regulatory role in the autophagic response through becn1 were monitored. Our results showed a significant decrease in Bcl-2 expression by chronic alcohol intake, the effect of which was accentuated by ADH. Although chronic alcohol intake did not affect levels of Bcl-xL and miR-30a in FVB mice, ADH transgene significantly unmasked a decrease in both Bcl-xL and miR-30a following alcohol intake. Last but not least, ADH transgene itself did not affect the levels of Bcl-2, Bcl-xL and miR-30a (Fig. 5).

Figure 5.

Expression of the Beclin 1-related proteins Bcl-2 and Bcl-xL as well as miR-30a in hearts from FVB and ADH mice with or without chronic alcohol intake (4% alcohol, 8 weeks). (A) Representative gels depicting levels of Bcl-2, Bcl-xL and GAPDH (loading control); (B) Bcl-2 expression; (C) Bcl-xL expression; and (D) Fold change in miR-30a. Mean ± SEM, n = 5–6 per group, *p < 0.05 vs. FVB group, ^#p < 0.05 vs. FVB-EtOH group.

Effect of ethanol and 3-MA on cardiomyocyte contractile function in FVB and ADH mice

To further assess the role of autophagy in alcohol-induced cardiac contractile response in vivo, FVB and ADH transgenic mice were injected intraperitoneally with ethanol (3 g/kg/d) for three consecutive days with or without daily pretreatment of the autophagy inhibitor 3-MA (10 mg/kg/d). Assessment of cardiomyocyte mechanics revealed that ethanol challenge significantly depressed peak shortening, maximal velocity of shortening/relengthening (± dL/dt) as well as prolonged time-to-90% relengthening (TR₉₀) without affecting resting cell length and time-to-peak shortening (TPS) in cardiomyocytes from FVB mice, the effects of which were significantly exacerbated by ADH transgene. ADH itself failed to alter these mechanical indices although the transgene unmasked a prolonged TPS in response to alcohol challenge. Interestingly, autophagy inhibition using 3-MA effectively ablated alcohol challenge-induced cardiomyocyte mechanical anomalies in both FVB and ADH mice. 3-MA alone failed to elicit any effect on cardiomyocyte mechanics. These data favored a permissive role for autophagy in ethanol- and ADH metabolic product acetaldehyde -induced cardiac contractile dysfunction (Fig. 6).

Figure 6.

Contractile properties of cardiomyocytes from FVB and ADH mice challenged with ethanol (3 g/kg/d, i.p.) in the absence or presence of pretreatment of 3-MA (10 mg/kg/d, i.p.) or saline (vehicle control). (A) Resting cell length; (B) Peak shortening (% of cell length); (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 = 70–75 cells from three mice per group, *p < 0.05 vs. FVB group, ^#p < 0.05 vs. FVB-EtOH group, ^†p < 0.05 vs. ADH-EtOH group.

Effect of acetaldehyde, 3-MA and thapsigargin on cardiomyocyte contractile function

To further examine the role of the ADH metabolic product acetaldehyde and autophagy in alcohol-induced cardiac contractile response, freshly isolated cardiomyocytes from FVB mice were treated with or without thapsigargin prior to the exposure of acetaldehyde in the absence or presence of the autophagy inhibitor 3-MA. Our data shown in Figure 7 revealed that acetaldehyde significantly depressed PS, ± dL/dt and prolonged TR₉₀ without affecting resting cell length and TPS in cardiomyocytes. Although 3-MA itself did not affect cardiomyocyte mechanics, it ablated or significantly attenuated acetaldehyde-induced cardiomyocyte mechanical anomalies (Fig. 7). More interestingly, pretreatment of cardiomyocytes with the SERCA inhibitor thapsigargin prior to 3-MA and acetaldehyde exposure significantly attenuated or ablated 3-MA-exerted protection against acetaldehyde-induced cardiomyocyte dysfunction. Thapsigargin itself did not elicit any significant effect on cardiomyocyte contractile properties at the concentration tested nor did it exacerbate acetaldehyde-induced cardiomyocyte anomalies (depressed PS, ± dL/dt and prolonged TR₉₀). These data favored a likely role of intracellular Ca²⁺ homeostasis; in particular SERCA function in autophagy inhibition offered a beneficial effect against acetaldehyde-induced cardiac contractile dysfunction.

Figure 7.

Contractile properties of cardiomyocytes from FVB mice incubated for 2 h with acetaldehyde (ACA, 200 μM) in the absence or presence of the autophagy inhibitor 3-methyladenine (3-MA, 10 mM). A cohort of cardiomyocytes was also treated with or without the SERCA inhibitor thapsigargin (TG, 10⁻⁸ M) for 1 h prior to incubation of 3-MA (10 mM) and/or acetaldehyde (200 μM). (A) Resting cell length; (B) peak shortening (% of cell length); (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 = 61–83 cells from three mice per group, *p < 0.05 vs. control group, ^#p < 0.05 vs. ACA group, ^†p < 0.05 vs. ACA-3-MA group.

Effect of acetaldehyde on autophagy markers in cardiomyocytes

To access the effect of acetaldehyde on autophagy markers, Beclin 1 and LC3 proteins were determined. Acetaldehyde treatment elicited a significant upregulation of Beclin 1 and LC3-II expression, the effect of which was attenuated by 3-MA. 3-MA itself did not affect the expression of Beclin 1 and LC3-II. As expected, the autophagy inducer rapamycin promoted autophagy (shown as increased levels of Beclin 1 and LC3-II). Interestingly, rapamycin elicited a further increase in LC3-II expression and displayed a trend of increase (although not statistically significant) in Beclin 1 levels in the presence of acetaldehyde compared with rapamycin alone. These data favor a role of acetaldehyde in promoting autophagosome formation and impedance of autophagic flux (Fig. 8A and B).

Figure 8.

Effect of acetaldehyde on autophagy and autophagic flux. (A and B) Level of autophagy markers Beclin 1 and LC3-II in FVB cardiomyocytes incubated with acetaldehyde (ACA, 200 μM) for 2 h in the absence or presence of the autophagy inhibitor 3-methyladenine (3-MA, 10 mM) or rapamycin (Rapa, 5 μM). Mean ± SEM, n = 4–6 mice per group, *p < 0.05 vs. control group, ^#p < 0.05 vs. ACA group. (C–L) Effect of inhibition of lysosomal enzyme and autophagosome formation on ethanol and acetaldehyde-induced autophagy in H9c2 myoblasts. H9c2 cells were transfected with adenovirus for 24 h to express the GFP-LC3 fusion protein. Cells were then exposed to ethanol (240 mg/dl) or acetaldehyde (ACA, 200 μM) for 4 h in the absence or presence of the autophagosome formation inhibitor 3-MA (10 mM) or the mixture of the cell-permeable lysosomal inhibitors bafilomycin A₁ (50 nM), E64D (2.5 μg/ml) and pepstatin A methyl ester (5 μg/ml). The autophagy inducer rapamycin (5 μM) was used as the positive control. DAPI stating was used for identification of nucleus. (C–L) Representative images (GFP, DAPI and Merged) depicting GFP-LC3 puncta in H9c2 cells following ethanol exposure with or without 3-MA or lysosomal inhibitors; (fluorescent images with better resolution and larger size are shown in Fig. S2) and (M) percentage of cells with autophagosomes. Cells with 10 or more punctate spots were scored as positive for autophagosomes. Mean ± SEM, n = 300–400 cells per group from three independent experiments, *p < 0.05 vs. control group, ^#p < 0.05 vs. ethanol group, ^†p < 0.05 vs. acetaldehyde group.

Effect of lysosomal inhibitors and 3-MA on acetaldehyde-induced autophagosome accumulation in H9c2 cells

To determine if ethanol and its metabolic product, acetaldehyde-induced autophagosome accumulation, was due to changes in early or late stages of autophagy process, H9c2 myoblasts were transfected with an adenovirus expressing GFP-LC3 fusion protein for 24 h prior to exposure of ethanol (240 mg/dL, 6 h) and acetaldehyde (200 μM, 2 h) in the absence or presence of mixed lysosomal inhibitors (bafilomycin A₁, E64D and pepstatin A methyl ester) to suppress autophagosome-lysosome fusion, cysteine proteases and cathepsin D.¹⁴ Data shown in Figure 8C–M revealed that lysosomal inhibition did not alter the ethanol or acetaldehyde-induced GFP-LC3 puncta, indicating a role of lysosomal inhibition in ethanol or acetaldehyde-induced autophagic response. The lysosomal inhibitors themselves displayed a tendency to increase GFP-LC3 puncta although it failed to reach statistical significance. The autophagy inhibitor 3-MA blocked ethanol and acetaldehyde-induced rise in GFP-LC3 puncta. 3-MA itself failed to affect GFP-LC3 puncta. The autophagy inducer rapamycin (5 μM) was employed as a positive control for GFP-LC3 puncta formation. These findings suggest that ethanol and its metabolite acetaldehyde-induced autophagosome accumulation may be due to impaired autophagic flux and induction of autophagosome formation. To evaluate if ethanol- and acetaldehyde-exerted cell survival responses were related to their ability to promote cell death, cell viability was measured using MTT assay in both cardiomyocytes and H9c2 myoblasts. Our data presented in Figure S1 revealed that neither ethanol nor acetaldehyde affected cell viability at the concentration and incubation duration used elsewhere in this study.

Discussion

The salient findings from the present study supported the hypothesis that acetaldehyde may serve as a key candidate toxin for the onset of alcoholic cardiomyopathy including compromised cardiac morphology and contractility.^2,3,25 Our earlier findings revealed that overexpression of ADH in the heart led to enhanced cardiac acetaldehyde exposure and augmented alcohol intake-induced cardiomyopathy manifested as cardiac hypertrophy, protein damage, ER stress, oxidative stress and mitochondrial damage.^9,22,26,27 Results from our current study confirmed cardiac acetaldehyde accumulation had a more pronounced effect in ADH mice (despite comparable levels of blood alcohol and acetaldehyde between FVB and ADH mice) following chronic alcohol intake. More importantly, data from our present study provided evidence for the first time that acetaldehyde through ADH metabolism may effectively initiate myocardial autophagy en route to myocardial geometric and functional defects. Autophagy inhibition using 3-MA abolished alcohol challenge-induced cardiomyocyte contractile anomalies. Measurement of GFP-LC3 positive puncta in the absence (steady-state autophagosomes) or presence (cumulative autophagosomes) of lysosomal inhibitors¹⁴ revealed that ethanol/acetaldehyde-induced autophagosome accumulation may be more likely due to an inhibition of the late-stage autophagic flux as opposed to induction of the early-stage autophagosome formation.

Previous findings from our lab revealed development of myocardial contractile dysfunction including enlarged end diastolic and systolic diameters, depressed factional shortening, maximal left ventricular tension development, the first derivative of left ventricular tension development and cardiomyocyte shortening capacity following alcohol administration, the effects of which may be exacerbated by ADH transgene.^5,9,22 These findings favor the notion of an aggravated or accelerated alcoholic cardiomyopathy with higher ADH-mediated ethanol metabolism. Data from our present study revealed that ADH accentuated alcohol intake-induced cardiac hypertrophy and cardiomyocyte hypertrophy, the effect of which may be likely attributed to the ADH metabolic product acetaldehyde. This is consistent with our earlier findings using the same ADH transgenic mice where cardiac hypertrophy (heart weight and LV mass) and upregulated expression of the hypertrophic markers α-skeletal actin and atrial natriuretic factor (ANF) were noted in ADH but not FVB mice following chronic alcohol intake.^22,28 Moreover, CaMKII activation, which was considered as one of the main pathways to promote myocardial hypertrophy,²⁴ was significantly enhanced in both FVB and ADH mice with a further rise in ADH group. Although the precise mechanism behind alcohol and ADH-induced cardiac hypertrophic response is still unclear at this time, it may be speculated that the excess of autophagy may contribute to the development of cardiac hypertrophy to promote heart failure.²⁹ This is supported by the findings that autophagy may serve as an intracellular phenomenon of failing heart.¹⁹ In addition, autophagic degeneration has been noted as a possible mechanism of myocardial cell death caused by dilated cardiomyopathy or myocardial hypertrophy due to hemodynamic overload.^29,30 Last but not least, heart rate and blood pressure were found to be comparable among our mouse groups, thus excluding potential contribution of changes in heart rhythm and afterload to cardiac contractile function in our current experimental setting.

Data from our current study revealed elevated levels of LC3-II (as well as LC3-II-to-LC3-I ratio), and Atg7 in hearts following chronic alcohol exposure with a more pronounced increase in ADH transgenic mice. Moreover, ADH unmasked an alcohol-induced upregulation of Beclin 1. These autophagic responses were supported by the observation that autophagy inhibition using 3-MA mitigated alcohol challenge-induced cardiomyocyte contractile dysfunction in both FVB and ADH mice (in particular the ADH-augmented anomalies in ethanol-induced cardiomyocyte contractile dysfunction). These autophagy data were in line with the in vitro observation where acetaldehyde facilitates autophagy induction, suggesting a role of acetaldehyde in promoting alcohol-induced cardiac autophagy initiation. Our data revealed that acetaldehyde compromised cardiomyocyte contractile properties which was ameliorated by 3-MA. This is supported by our recent finding that acetaldehyde detoxification reduces autophagy induction in conjunction with myocardial contractile anomalies in the heart.²¹ We found that acetaldehyde elicited a further increase (or a trend) in LC3-II and Beclin 1 levels in the presence of rapamycin, favoring the notion that acetaldehyde may impair autophagic flux thus facilitating accumulation of autophagosomes. Under normal conditions, autophagy depends on efficient fusion of autophagosomes with functional lysosomes to maintain cellular homeostasis including removing damaged organelles or proteins.^31,32 However, excessive autophagy impairs functional proteins and organelles, resulting in cardiac dysfunction.¹² Furthermore, impaired autophagic degradation contributes to the pathogenesis of human diseases such as myopathic heart and lysosomal storage diseases.^33,34 The maladaptive autophagy has received some recent attention in the disease progression including heart diseases under stressful situations.^12,35 These findings supported a role of autophagy in acetaldehyde-induced cardiac contractile dysfunction, suggesting that the acetaldehyde-induced initiation of autophagy may be primarily a pro-death rather than a prosurvival mechanism. Nonetheless, our present study did not note any direct effect of ethanol and acetaldehyde (at the experimental conditions used in this study) on cell viability, excluding a direct cytotoxic effect from ethanol or acetaldehyde. Although it is beyond the scope of our current study, results from this study revealed that SERCA inhibition with thapsigargin canceled off the protective effect of autophagy inhibition against acetaldehyde. Given that acetaldehyde has long been known to compromise cardiac contractile function through interrupted intracellular Ca²⁺ homeostasis,^4,25 our data favor the notion that autophagy inhibition benefits cardiac contractile function dampened by ethanol or its metabolite acetaldehyde likely through SERCA-mediated maintenance of intracellular Ca²⁺ homeostasis.

The microtuble-associated protein LC3 is a homolog of the yeast protein Atg8 used as a marker for autophagy. During autophagy, formation of autophagosomes allows LC3 to be targeted to autophagic membranes, resulting the presence of lower migrating LC3-II in autophagosomes (conversion of the 16KD LC3-I to the 14KD LC3-II often used as an indicator of autophagy).³⁶ Meanwhile, detection of LC3 by immunostaining or the GFP-LC3 puncta has become a reliable technique to monitor autophagy and autophagic cell death.³⁷ As shown in our study, the more pronounced increase of LC3B-positive cells in ADH mice following alcohol intake and the high percentage of GFP-LC3 puncta in H9c2 cells following acetaldehyde exposure favored that ADH or acetaldehyde may trigger autophagosome formation.³⁸ Beclin 1, the mammalian homolog of yeast Atg6, possesses a key role in the initiation of autophagy. It was found in a multiprotein complex depended on the activation of class III PtdIns3K required for the formation of the autophagosome.²³ In this study, levels of Beclin 1 and class III PtdIns3K were dramatically increased following alcohol intake in ADH mice. These data were in line with the in vitro findings from acetaldehyde treatment. However, little change was noted in Beclin 1 expression in alcohol-fed FVB mice, suggesting that acetaldehyde but not ethanol itself is more likely to be responsible for autophagy induction. Meanwhile, Bcl-2 and its homolog Bcl-xL, which inhibit the autophagic function of Beclin 1, were decreased (or showed a trend) after alcohol intake in FVB mice, which was accentuated by ADH. These findings suggest that Bcl-2 and Bcl-xL are required for cell survival and may possess important roles in acetaldehyde-induced autophagy.³⁹ Altered levels of the becn1 gene or mRNA were found in several human diseases including cancers⁴⁰ although the nature behind the aberrant expression of this key autophagy-promoting gene remains unknown. Zhu and colleagues reported that becn1 is a potential target for miRNA miR-30a which negatively regulates becn1 expression to suppress autophagic activity.⁴¹ In our study, the fold change in miR-30a was drastically decreased following alcohol challenge in ADH mice, suggesting that a role of this miRNA in targeting becn1 in response to alcoholic insult.

It has been well accepted that static levels of LC3-II renders possible misinterpretation for autophagy as LC3-II levels may increase, decrease or remain unchanged following autophagic induction.⁸ Therefore it is pertinent to measure LC3-II levels in the presence and absence of lysosomal inhibitors to block the degradation of LC3-II through autophagic flux.^8,14 Moreover, our data depicted that acetaldehyde and ethanol-induced GFP-LC3 puncta formation was unaffected by lysosomal inhibitors. Furthermore, we also noted dramatically elevated cargo receptor p62, which interacts with LC3-II,⁴² following chronic alcohol intake (data not shown). These findings suggest that acetaldehyde and ethanol-induced accumulation of autophagosome may be attributed predominantly to stimulation of the late-stage autophagic flux mechanism. Our data revealed that phosphorylation of mTOR and p70S6K was significantly decreased following alcohol intake, the effect of which was further accentuated by ADH. This is in agreement with the notion that inhibition of mTOR promotes autophagy induction.⁴³ The marked reduction of phosphorylation of p70S6K further supported the notion of reduced mTOR activity. Taken together, these findings favored a causal role of mTOR signaling cascade in mediating the ADH-facilitated autophagy in response to alcohol intake.

Experimental limitations

There are a number of pitfalls for our present study. First, the use of acetaldehyde in vitro cannot fully recapitulate the buildup of the toxin following alcohol intake. Thus, caution has to be taken when interpreting these in vitro findings to the setting of in vivo alcohol exposure. Nonetheless, this in vitro approach does provide some advantages such as use of inhibitors, drug specificity issue and toxicity which may not be overcome in the in vivo setting.

In conclusion, our study provided convincing evidence, for the first time, that cardiac overexpression of ADH facilitated chronic alcohol intake-induced autophagy. This is supported by the observation that alcohol/acetaldehyde-induced cardiac contractile defects and autophagy may be inhibited by 3-MA. In addition, the acetaldehyde-induced autophagosome accumulation may likely be due to an inhibition of the late-stage autophagic flux as oppose to induction of the early-stage autophagosome formation. These results collectively supported an essential role of ethanol metabolism and acetaldehyde but not ethanol per se in alcoholic myopathic alteration. Although it is still premature to discern the precise mechanisms involved in autophagy following alcohol intake, our study should shed some light toward a better understanding of the role of ADH in alcohol-induced myocardial and autophagic response. Further scrutiny is still required to unveil the precise mechanism for autophagy induction following alcohol intake such as oxidative stress which was already considered to be an important contributor for autophagy.⁴⁴

Materials and Methods

Experimental animals and chronic alcohol feeding

All animal procedures were approved by the Institutional Animal Care and Use Committee at the University of Wyoming. Production of ADH transgenic mice was described in detail previously.⁴⁵ In brief, the cDNA for murine class I ADH was inserted behind mouse α-myosin heavy chain promoter to achieve the cardiac-specific overexpression in albino friendly virus-B type (FVB) mice. This cDNA was chosen because class I ADH is most efficient in the oxidation of ethanol. A second transgene with a cDNA encoding tyrosinase was co-injected with ADH. This enzyme produces coat color pigmentation in albino mice and was used to conveniently identify transgenic animals. All mice were housed in a temperature-controlled room under a 12hr/12hr-light/dark and with access to water ad libitum. Three-month-old adult female and male FVB and ADH mice were placed on a nutritionally complete liquid diet (Shake & Pour Bioserv Inc., F1258SP) for a one-week acclimation period. The use of a liquid diet is based on the scenario that ethanol self-administration resulted in less nutritional deficiencies and less stress to animals in comparison to forced-feeding regimens, intravenous administration, or aerosolized inhalation. Upon completion of the acclimation period, half of the FVB and ADH mice were maintained on the regular liquid diet (without ethanol), and the remaining half began an 8-week period of isocaloric 4% (vol/vol) ethanol diet feeding. An isocaloric pair-feeding regimen was employed to eliminate the possibility of nutritional deficits. Control mice were offered the same quantity of diet ethanol-consuming mice drank the previous day. Body weight was monitored weekly.⁹ Heart rate was measured at the end of the 8-week feeding period using echocardiography.⁴⁶ Systolic and diastolic blood pressures were examined at the end of the 8-week feeding period using a KODA semi-automated, amplified tail cuff device (Kent Scientific Corporation).⁴⁷ Blood levels of alcohol and acetaldehyde as well as cardiac tissue levels of acetaldehyde were measured using the gas chromatography technique equipped with a flame ionization detector as described previously.⁵

ADH enzymatic activity assay

Freshly dissected myocardial tissues were homogenized and sonicated. The supernatants were centrifuged at 13,000 × g for 20 min at 4°C. ADH enzymatic activity was measured at 50°C in 200 μl of 100 mM glycine buffer (pH 8.8) containing 2.4 mM NAD⁺, 33 mM ethanol and 300 μg of myocardial protein extract. Production of NADH was determined by absorbance intensity at 340 nm obtained every min for 5 min using a SpectraMax 190 Microplate Spectrophotometer equipped with a kinetic software module. Molar absorbance coefficient of NADH was 6220 M/cm and one unit of enzyme activity was defined as the amount of enzyme that catalyzed the generation of 1 μmol NADH/min under the above experimental condition. The final result was normalized to the tissue protein content.⁴⁸

Histological examination and immunostaining

Following anesthesia, hearts were excised and immediately placed in 10% neutral-buffered formalin at room temperature for 24 h after a brief rinse with PBS. Thereafter, heart tissues were dehydrated through serial alcohols and cleared in xylenes. The specimen were embedded in paraffin, cut in 5 µm sections and stained with hematoxylin and eosin (H&E) as described.⁴⁹ A cohort of samples was used for fluorescent immunohistochemistry to detect LC3B antibody expression and distribution. In brief, after the antigen unmasking by the citrate, sections were stained with the rabbit anti-LC3B antibody (1:500, Cell Signaling, 3868) followed by incubation of the goat anti-rabbit IgGsecondary antibody (Alexa Fluor^® 594, Invitrogen, A-11012).⁵⁰ Nuclei were counterstained with DAPI. Cardiac sections were observed and the ratio between LC3B-stained cell and nucleus⁵¹ was then calculated on a digital microscope (× 400) using the Image J (version1.34S) and Photoshop software.^5,46

Isolation of murine cardiomyocytes and drug treatment

FVB and ADH mice were challenged with ethanol (3 g/kg/d, i.p.) for three consecutive days with each ethanol preceded by injection of the autophagy inhibitor 3-MA (10 mg/kg/d, i.p.)⁵² or saline. After ketamine/xylazine sedation, hearts were removed and perfused with Ca²⁺ -free Tyrode’s solution containing (in mM): NaCl 135, KCl 4.0, MgCl₂ 1.0, HEPES 10, NaH₂PO₄ 0.33, glucose 10, butanedione monoxime 10, and the solution was gassed with 5% CO₂/95% O₂. Hearts were digested with Liberase Blendzyme 4 (Hoffmann-La Roche, Inc., 11988476001) for 20 min. Left ventricles were removed and minced before being filtered. Tissue pieces were gently agitated and pellet of cells was resuspended. Extracellular Ca²⁺ was added incrementally back to 1.20 mM over a period of 30 min. Isolated myocytes were used within 8 h of isolation. A yield of 50–60% viable rod-shaped cardiomyocytes with clear sarcomere striations was achieved. Only rod-shaped myocytes with clear edges were selected for mechanical study. To assess the effect of 3-MA and rapamycin on cardiomyocyte contractile function and autophagy level in response to acetaldehyde, an in vitro experiment was performed. In brief, cardiomyocytes from FVB mice were treated with or without the acetaldehyde (200 μM) at 37°C for 2 h⁵³ in the absence or presence of the autophagy inhibitor 3-methyladenine (3-MA, 10 mM)⁵⁴ or the autophagy inducer rapamycin (5 µM)⁵⁵ prior to mechanical and biochemical evaluation. To further understand the mechanism of action behind autophagy regulation-induced cardiomyocyte contractile response following ethanol exposure, FVB murine cardiomyocytes were pretreated with the sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA) inhibitor thapsigargin (10⁻⁸ M) for 1 h^56,57 prior to treatment of 3-MA and acetaldehyde.

Cell shortening/relengthening

Mechanical properties of cardiomyocytes were assessed using a SoftEdge MyoCam system (IonOptix). In brief, cells were placed in a Warner chamber mounted on the stage of an inverted microscope (Olympus IX-70) and superfused (~1 ml/min at 25°C) with a buffer containing (in mM) 131 NaCl, 4 KCl, 1 CaCl2, 1 MgCl2, 10 glucose, and 10 HEPES at pH 7.4. The cells were field stimulated with suprathreshold voltage at a frequency of 0.5 Hz using a pair of platinum wires placed on opposite sides of the chamber connected to a FHC stimulator. The myocyte being studied was displayed on the computer monitor using an IonOptix MyoCam camera. An IonOptix SoftEdge software was used to capture changes in cell length during shortening and relengthening. Cell shortening and relengthening were assessed using the following indices: resting cell length, peak shortening,⁴⁶ time-to-PS (TPS), time-to-90% relengthening (TR₉₀), and maximal velocity of shortening/relengthening (± dL/dt).¹⁰

LC3B-GFP-adenovirus production and infection in H9C2 cells

Given the poor transfection efficacy and relatively short duration of survival for murine cardiomyocytes,⁵⁸ H9c2 cells, a clonal cell line derived from fetal rat hearts, were purchased from American Type Culture Collection (ATCC, CRL-1446^TM) to assess autophagy using GFP fluorescence. In brief, cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (Gibco, 10438-034) and 1% penicillin and streptomycin and maintained in 95% air and 5% CO₂ at 37°C. Cells were grown to 80% confluence prior to usage. Adenovirus containing GFP-LC3 construct (kindly provided by Dr. Cindy Miranti from Van Andel Institute) was propagated using HEK293 cell line. Cells were then transfected with GFP-LC3 adenovirus as described previously in our laboratory.⁵⁹ Upon plaque formation, infected cells were collected, washed with PBS, resuspended in culture medium and lysed by 3 cycles of freeze -thaw (37°C). Cell debris was collected by centrifugation and aliquots of supernatant with viral particles were stored at -80°C. Adenovirus was purified using an Adeno-X Maxi purification kit (Clontech Laboratories, Inc. 631533). H9c2 cells were grown to confluence on Lab-Tek chamber slide. Cells were infected at an MOI of 2 with adenoviruses expressing GFP-LC3 fusion protein. Medium was replaced with fresh DMEM after 6 h. Twenty four hrs later, cells were visualized for autophagy using fluorescence microscopy.⁵⁹

Quantification of the GFP-LC3

H9c2 cells transfected with GFP-LC3 adenovirus were treated with or without ethanol (240 mg/dl, at 37°C, 6 h) or acetaldehyde (200 μM, 37°C, 2 h) in the absence or presence of 3-MA (10 mM).⁵⁴ Rapamycin (5 µM) was used as the positive control.⁵⁵ To evaluate autophagic flux, GFP-LC3 positive cells were evaluated in ethanol or acetaldehyde-treated cells incubated with (cumulative autophagosomes) or without (steady-state autophagosomes) of a mixture of lysosomal inhibitors [bafilomycin A₁ (50 nM), E64D (2.5 μg/ml) and pepstatin A methyl ester (5 μg/ml)].¹⁴ The cells were fixed with 4% paraformaldehyde in PBS for 10 min at room temperature followed by three times washing with PBS. Coverslips were mounted on the slides using Vecta mountTM AQ-aqueous mounting medium (Vector Laboratories, Inc., H-5501). For autophagy assessment, cells were visualized at 40 × magnification using a Olympus BX51fluorescence microscope (Olympus America, Inc.) and percentage of GFP-LC3 positive cells showing numerous GFP-LC3 puncta (> 10 dots/cell) were scored as described previously.⁶⁰ A minimum of 300–400 cells were scored in at least three independent experiments.

MicroRNA extraction and reverse-transcriptional PCR analysis

Expression of miR-30a, a potential target of becn1, was evaluated by real-time PCR according to the mirVana™ qRT-PCR miRNA Isolation and Detection Kit (Ambion, Inc. AM1560, AM1561, AM1558). Briefly, total RNA containing microRNA was extracted from frozen heart tissues according to the isolation protocol, then reverse-transcribed followed by amplifying using mirVanaTM qRT-PCR miRNA detection kit. The has-miR-30a-5p primer set was purchased from AB Applied Biosystems^TM (Rack ID 871251).

Western blot analysis

Total protein was prepared as described.⁴⁵ In brief, tissue samples from ventricles were removed and homogenized in a lysis buffer containing 10% 10 × RIPA, 1% NaF, 1% Na₃VO₄ and 1% protease inhibitor cocktail. Samples were sonicated on ice for 15 sec and centrifuged at 13,000× g for 20 min at 4°C. Murine cardiomyocytes were collected and sonicated in a lysis buffer. The protein concentration of the supernatant was evaluated using a Protein Assay Reagent (Bio-Rad Laboratories, 500-0006). Protein samples were then mixed 1:2 with Laemmli sample buffer with 5% 2-mercaptoethanol and heated at 95°C for 5 min. Equal amounts (50 µg protein/lane) of proteins were separated on 10% or 15% SDS-polyacrylamide gels in a minigel apparatus (Mini-PROTEAN II, Bio-Rad) and transferred electrophoretically to nitrocellulose membranes (0.2 µm pore size, Bio-Rad Laboratories, Inc.). Membranes were incubated for 1 h in a blocking solution containing 5% milk in Tris-buffered saline (TBS). Membranes were washed briefly in TBS and incubated overnight at 4°C with the anti-Atg7 (1:500), anti-LC3B (1:500), anti-Beclin 1 (1:1,000), anti-p62 (1:1000), anti-Bcl-xL (1:1,000), anti-Bcl-2 (1:200), anti-phospho-p70S6K (1:1,000, Thr389), anti-PtdIns3K class III (1:1,000), anti-mTOR (1:1,000), anti-phospho-mTOR (1:1,000, Ser2448), anti-CaMKII (1:1000), anti-phospho-CaMKII (1:1,000, Thr286), anti-GAPDH (loading control, 1:1,000) and anti-α-tubulin (loading control, 1:1000) antibodies. After washing blots to remove excessive primary antibody binding, blots were incubated for 1 h with horseradish peroxidase (HRP)–conjugated secondary antibody (1:2,500). Antigens were detected by the luminescence method. Quantification of band density was determined using Quantity One software (Bio-Rad, version 4.4.0, ChemiDoc XRS) and reported in optical density per square millimeter.

Data analysis

Data were mean ± SEM. Statistical significance (p < 0.05) for each variable was estimated by analysis of variance (ANOVA) followed by a Tukey’s post hoc analysis.

Glossary

Abbreviations:

3-MA

3-methyladenine

± dL/dt

maximal velocity of shortening/relengthening

ADH

alcohol dehydrogenase

ANF

atrial natriuretic factor

CaMKII

Ca²⁺-calmodulin-dependent protein kinase

ER

endoplasmic reticulum

FVB

friendly virus-B type

H&E

hematoxylin and eosin

mTOR

mammalian target of rapamycin

PtdIns3K

phosphatidylinositol-3-kinase

PS

peak shortening

SERCA

sarco(endo)plasmic reticulum Ca²⁺-ATPase

TPS

time-to-90% peak shortening

TR₉₀

time-to-90% relengthening

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.