Double knockout of Akt2 and AMPK accentuates high fat diet-induced cardiac anomalies through a cGAS-STING-mediated mechanism

High fat diet intake contributes to undesired cardiac geometric and functional changes although the underlying mechanism remains elusive. Akt and AMPK govern to cardiac homeostasis. This study examined the impact of deletion of Akt2 (main cardiac isoform of Akt) and AMPKα2 on high fat diet intake-induced cardiac remodeling and contractile anomalies and mechanisms involved. Cardiac geometry, contractile, and intracellular Ca²⁺ properties were evaluated using echocardiography, IonOptix® edge-detection and fura-2 techniques in wild-type (WT) and Akt2-AMPK double knockout (DKO) mice receiving low fat (LF) or high fat (HF) diet for 4 months. Our results revealed that fat diet intake elicit obesity, cardiac remodeling (hypertrophy, LV mass, LVESD, and cross-sectional area), contractile dysfunction (fractional shortening, peak shortening, maximal velocity of shortening/relengthening, time-to-90% relengthening, and intracellular Ca²⁺ handling), ultrastructural disarray, apoptosis, O₂⁻, inflammation, dampened autophagy and mitophagy. Although DKO did not affect these parameters, it accentuated high fat diet-induced cardiac remodeling and contractile anomalies. High fat intake upregulated levels of cyclic GMP-AMP synthase (cGAS), stimulator of interferon genes (STING), and STING phosphorylation while suppressing phosphorylation of ULK1 (Ser⁷⁵⁷ and Ser⁷⁷⁷), with a more pronounced effect in DKO mice. In vitro data revealed that inhibition of cGAS and STING using PF-06928215 and Astin C negated palmitic acid-induced cardiomyocyte contractile dysfunction. Biological function analysis for all differentially expressed genes (DEGs) depicted that gene ontology terms associated with Akt and AMPK signaling processes were notably changed in high fat-fed hearts. Our data indicate that Akt2-AMPK ablation accentuated high fat diet-induced cardiac anomalies possibly through a cGAS-STING-mechanism.     

Inhibition of CYP2E1 attenuates myocardial dysfunction in a murine model of insulin resistance through NLRP3-mediated regulation of mitophagy

Insulin resistance leads to myocardial contractile dysfunction and deranged autophagy although the underlying mechanism or targeted therapeutic strategy is still lacking. This study was designed to examine the impact of inhibition of the cytochrome P450 2E1 (CYP2E1) enzyme on myocardial function and mitochondrial autophagy (mitophagy) in an Akt2 knockout model of insulin resistance. Adult wild-type (WT) and Akt2^?/? mice were treated with the CYP2E1 inhibitor diallyl sulfide (100?mg/kg/d, i.p.) for 4?weeks. Cardiac geometry and function were assessed using echocardiographic and IonOptix systems. Western blot analysis was used to evaluate autophagy, mitophagy, inducible NOS (iNOS), and the NLRP3 inflammasome, a multi-protein intracellular pattern recognition receptor complex. Akt2 deletion triggered insulin resistance, compromised cardiac contractile and intracellular Ca²⁺ property, mitochondrial ultrastructural damage, elevated O2^– production, as well as suppressed autophagy and mitophagy, accompanied with elevated levels of NLRP3 and iNOS, the effects of which were significantly attenuated or ablated by diallyl sulfide. In vitro studies revealed that the NLRP3 activator nigericin nullified diallyl sulfide-offered benefit against Akt2 knockout on cardiomyocyte mechanical function and mitophagy (using Western blot and colocalization of GFP-LC3 and MitoTracker Red). Moreover, inhibition of iNOS but not mitochondrial ROS production attenuated Akt2 deletion-induced activation of NLRP3, substantiating a role for iNOS-mediated NLRP3 in insulin resistance-induced changes in mitophagy and cardiac dysfunction. In conclusion, these data depict that insulin resistance through CYP2E1 may contribute to the pathogenesis of myopathic changes including myocardial contractile dysfunction, oxidative stress and mitochondrial injury, possibly through activation of iNOS and NLRP3 signaling.     

Pentacyclic triterpene oleanolic acid protects against cardiac aging through regulation of mitophagy and mitochondrial integrity

Advanced aging exhibits altered cardiac geometry and function involving mitochondrial anomaly. Natural compounds display promises in the regulation of cardiac homeostasis via governance of mitochondrial integrity in aging. This study examined the effect of oleanolic acid (OA), a natural pentacyclic triterpenoid with free radical scavenging and P450 cyclooxygenase-regulating properties, on cardiac aging and mechanisms involved with a focus on mitophagy. Young (4–5 month-old) and old (22–24 month-old) mice were treated with OA for 6 weeks prior to assessment of cardiac function, morphology, ultrastructure, mitochondrial integrity, cell death and autophagy. Our data revealed that OA treatment alleviated aging-induced changes in myocardial remodeling (increased heart weight, chamber size, cardiomyocyte area and interstitial fibrosis), contractile function and intracellular Ca²⁺ handling, apoptosis, necroptosis, inflammation, autophagy and mitophagy (LC3B, p62, TOM20 and FUNDC1 but not BNIP3 and Parkin). OA treatment rescued aging-induced anomalies in mitochondrial ultrastructure (loss of myofilament alignment, swollen mitochondria, increased circularity), mitochondrial biogenesis and O₂^? production without any notable effect at young age. Interestingly, OA-offered benefit against cardiomyocyte aging was nullified by deletion of the mitophagy receptor FUNDC1 using FUNDC1 knockout mice, denoting an obligatory role for FUNDC1 in OA-elicited preservation of mitophagy. OA reconciled aging-induced changes in E3 ligase MARCH5 but not FBXL2, and failed to affect aging-induced rises in IP3R3. Taken together, our data indicated a beneficial role for OA in attenuating cardiac remodeling and contractile dysfunction in aging through a FUNDC1-mediated mechanism.     

Mitochondrial ALDH2 protects against lipopolysaccharide-induced myocardial contractile dysfunction by suppression of ER stress and autophagy

Lipopolysaccharide (LPS), an essential component of outer membrane of the Gram-negative bacteria, plays a pivotal role in myocardial anomalies in sepsis. Recent evidence depicted an essential role for mitochondrial aldehyde dehydrogenase (ALDH2) in cardiac homeostasis. This study examined the effect of ALDH2 on endotoxemia-induced cardiac anomalies. Echocardiographic, cardiac contractile and intracellular Ca²⁺ properties were examined. Our results indicated that LPS impaired cardiac contractile function (reduced fractional shortening, LV end systolic diameter, peak shortening, maximal velocity of shortening/relengthening, prolonged relengthening duration, oxidation of SERCA, and intracellular Ca²⁺ mishandling), associated with ER stress, inflammation, O₂⁻ production, increased autophagy, CAMKKβ, phosphorylated AMPK and suppressed phosphorylation of mTOR, the effects of which were significantly attenuated or negated by ALDH2. LPS promoted early endosomal formation (as evidenced by RAB4 and RAB5a), apoptosis and necrosis (MTT and LDH) while decreasing late endosomal formation (RAB7 and RAB 9), the effects were reversed by ALDH2. In vitro study revealed that LPS-induced SERCA oxidation, autophagy and cardiac dysfunction were abrogated by ALDH2 activator Alda-1, the ER chaperone TUDCA, the autophagy inhibitor 3-MA, or the AMPK inhibitor Compound C. The beneficial effect of Alda-1 against LPS was nullified by AMPK activator AICAR or rapamycin. CAMKKβ inhibition failed to rescue LPS-induced ER stress. Tunicamycin–induced cardiomyocyte dysfunction was ameliorated by Alda-1 and autophagy inhibition, the effect of which was abolished by rapamycin. These data suggested that ALDH2 protected against LPS-induced cardiac anomalies via suppression of ER stress, autophagy in a CAMKKβ/AMPK/mTOR-dependent manner.     

AMP-dependent kinase and autophagic flux are involved in aldehyde dehydrogenase-2-induced protection against cardiac toxicity of ethanol

Mitochondrial aldehyde dehydrogenase-2 (ALDH2) alleviates ethanol toxicity although the precise mechanism is unclear. This study was designed to evaluate the effect of ALDH2 on ethanol-induced myocardial damage with a focus on autophagy. Wild-type FVB and transgenic mice overexpressing ALDH2 were challenged with ethanol (3 g/kg/day, ip) for 3 days and cardiac mechanical function was assessed using the echocardiographic and IonOptix systems. Western blot analysis was used to evaluate essential autophagy markers, Akt and AMPK, and the downstream signal mTOR. Ethanol challenge altered cardiac geometry and function as evidenced by enlarged ventricular end systolic and diastolic diameters, decreased cell shortening and intracellular Ca²⁺ rise, prolonged relengthening and intracellular Ca²⁺ decay, as well as reduced SERCA Ca²⁺ uptake, which effects were mitigated by ALDH2. Ethanol challenge facilitated myocardial autophagy as evidenced by enhanced expression of Beclin, ATG7, and LC3B II, as well as mTOR dephosphorylation, which was alleviated by ALDH2. Ethanol challenge-induced cardiac defect and apoptosis were reversed by the ALDH2 agonist Alda-1, the autophagy inhibitor 3-MA, and the AMPK inhibitor compound C, whereas the autophagy inducer rapamycin and the AMPK activator AICAR mimicked or exacerbated ethanol-induced cell injury. Ethanol promoted or suppressed phosphorylation of AMPK and Akt, respectively, in FVB but not ALDH2 murine hearts. Moreover, AICAR nullified Alda-1-induced protection against ethanol-triggered autophagic and functional changes. Ethanol increased GFP-LC3 puncta in H9c2 cells, the effect of which was ablated by Alda-1 and 3-MA. Lysosomal inhibition using bafilomycin A1, E64D, and pepstatin A obliterated Alda-1- but not ethanol-induced responses in GFP-LC3 puncta. Our results suggest that ALDH2 protects against ethanol toxicity through altered Akt and AMPK signaling and regulation of autophagic flux.     

Mitochondrial aldehyde dehydrogenase obliterates endoplasmic reticulum stress-induced cardiac contractile dysfunction via correction of autophagy

ER stress triggers myocardial contractile dysfunction while effective therapeutic regimen is still lacking. Mitochondrial aldehyde dehydrogenase (ALDH2), an essential mitochondrial enzyme governing mitochondrial and cardiac function, displays distinct beneficial effect on the heart. This study was designed to evaluate the effect of ALDH2 on ER stress-induced cardiac anomalies and the underlying mechanism involved with a special focus on autophagy. WT and ALDH2 transgenic mice were subjected to the ER stress inducer thapsigargin (1 mg/kg, i.p., 48 h). Echocardiographic, cardiomyocyte contractile and intracellular Ca^2 + properties as well as myocardial histology, autophagy and autophagy regulatory proteins were evaluated. ER stress led to compromised echocardiographic indices (elevated LVESD, reduced fractional shortening and cardiac output), cardiomyocyte contractile and intracellular Ca^2 + properties and cell survival, associated with upregulated autophagy, dampened phosphorylation of Akt and its downstream signal molecules TSC2 and mTOR, the effects of which were alleviated or mitigated by ALDH2. Thapsigargin promoted ER stress proteins Gadd153 and GRP78 without altering cardiomyocyte size and interstitial fibrosis, the effects of which were unaffected by ALDH2. Treatment with thapsigargin in vitro mimicked in vivo ER stress-induced cardiomyocyte contractile anomalies including depressed peak shortening and maximal velocity of shortening/relengthening as well as prolonged relengthening duration, the effect of which was abrogated by the autophagy inhibitor 3-methyladenine and the ALDH2 activator Alda-1. Interestingly, Alda-1-induced beneficial effect against ER stress was obliterated by autophagy inducer rapamycin, Akt inhibitor AktI and mTOR inhibitor RAD001. These data suggest a beneficial role of ALDH2 against ER stress-induced cardiac anomalies possibly through autophagy reduction.     

Cardiomyocyte-specific knockout of endothelin receptor a attenuates obesity cardiomyopathy

Endothelin (ET)-1 is implicated in the pathophysiology of cardiovascular diseases although its role in obesity anomalies has not been fully elucidated. This study was designed to examine the impact of ET-1 receptor A (ET_A) ablation on obesity-induced changes in cardiac geometry and contractile function, as well as the mechanisms involved with a focus on autophagy. Cardiomyocyte-specific ET_A receptor knockout (ETAKO) and WT mice were fed either low-fat (10% calorie from fat) or high-fat (45% calorie from fat) diet for 24?weeks. Glucose tolerance test was examined to confirm insulin resistance. High-fat diet intake compromised myocardial geometry (enlarged left ventricular diameters in systole and diastole), morphology (cardiac hypertrophy, increased wall thickness and interstitial fibrosis), contractile function (reduced fractional shortening, ejection fraction and cardiomyocyte shortening) and intracellular Ca²⁺ handling, the effect of which was significantly attenuated by ETAKO. TUNEL staining revealed overt apoptosis in high-fat-fed group, the effect was reverted by ETAKO. Western blot analysis noted that high-fat intake downregulated leptin receptor and PPARγ, insulin signaling (elevated basal/dampened insulin-stimulated phosphorylation of Akt and IRS1), phosphorylation of AMPK, ACC, upregulated GATA-4, ANP, NFATc3, PPARα, m-TOR/p70s6k signaling, which were attenuated by ETAKO with the exception of AMPK/ACC. Furthermore, high-fat intake suppressed cardiac autophagy, which was abrogated by ETAKO. In cultured murine cardiomyocytes, palmitic acid challenged mimicked high-fat diet-induced hypertrophic and autophagic responses, the effect of which were abolished by the ET_A receptor antagonist BQ123 or mTOR inhibitor rapamycin. These results suggest that inhibition of ET_A rescues high-fat intake-induced cardiac anomalies possibly through autophagy regulation.     

CCCP induces autophagy in an AMPK-independent manner

AMP-activated protein kinase (AMPK) is an important sensor of cellular energy status, and is involved in cell growth and autophagy through mammalian target of rapamycin complex 1 (mTORC1). Carbonyl cyanide m-chlorophenylhydrazone (CCCP), a mitochondrial uncoupler, leads to AMPK activation and Parkin-dependent mitophagy, respectively. However, the detailed biochemical mechanism of how CCCP induces autophagy or mitophagy has not been investigated yet. Here, we showed that CCCP inhibits mTORC1 independently of AMPK, although CCCP induces AMPK activation. Using wild type (WT) and AMPKα1/α2 double knockout (DKO) MEFs, we observed that CCCP promotes endogenous LC3 lipidation and formation of RFP-LC3 puncta, indicating autophagosome or autolysosome, in an AMPK-independent manner. Finally, we also revealed that the percentage of CCCP-dependent colocalization between mitochondria and RFP-LC3 puncta is similar both in WT and AMPKα1/α2 DKO MEFs. Based on these data, we concluded that AMPK is not essential in regulation of CCCP-induced autopahgy including mitophagy.     

Rapamycin enhances growth inhibition on urothelial carcinoma cells through LKB1 deficiency-mediated mitochondrial dysregulation

Rapamycin, a mammalian target of rapamycin (mTOR) inhibitor, has significant potential for application in the treatment of urothelial carcinoma (URCa) of the bladder. Previous studies have shown that regulation of the AMP-activated serine/threonine protein kinase (AMPK)–mTOR signaling pathway enhances apoptosis by inducing autophagy or mitophagy in bladder cancer. Alteration of liver kinase B1 (LKB1)-AMPK signaling leads to mitochondrial dysfunction and the accumulation of autophagy-related proteins as a result of mitophagy, resulting in enhanced cell sensitivity to drug treatments. Therefore, we hypothesized that LKB1 deficiency in URCa cells could lead to increased sensitivity to rapamycin by inducing mitochondrial defect-mediated mitophagy. To test this, we established stable LKBI-knockdown URCa cells and analyzed the effects of rapamycin on their growth. Rapamycin enhanced growth inhibition and apoptosis in stable LKB1-knockdown URCa cells and in a xenograft mouse model. In spite of the stable downregulation of LKB1 expression, rapamycin induced AMPK activation in URCa cells, causing loss of the mitochondrial membrane potential, ATP depletion, and ROS accumulation, indicating an alteration of mitochondrial biogenesis. Our findings suggest that the absence of LKB1 can be targeted to induce dysregulated mitochondrial biogenesis by rapamycin treatment in the design of novel therapeutic strategies for bladder cancer.     

Tauroursodeoxycholic Acid Mitigates High Fat Diet-Induced Cardiomyocyte Contractile and Intracellular Ca2+ Anomalies

ObjectivesThe endoplasmic reticulum (ER) chaperone tauroursodeoxycholic acid (TUDCA) has exhibited promises in the treatment of obesity, although its impact on obesity-induced cardiac dysfunction is unknown. This study examined the effect of TUDCA on cardiomyocyte function in high-fat diet-induced obesity.MethodsAdult mice were fed low or high fat diet for 5 months prior to treatment of TUDCA (300 mg/kg. i.p., for 15d). Intraperitoneal glucose tolerance test (IPGTT), cardiomyocyte mechanical and intracellular Ca²⁺ property, insulin signaling molecules including IRS-1, Akt, AMPK, ACC, GSK-3β, c-Jun, ERK and c-Jun N terminal kinase (JNK) as well as ER stress and intracellular Ca²⁺ regulatory proteins were examined. Myocardial ultrastructure was evaluated using transmission electron microscopy (TEM).ResultsHigh-fat diet depressed peak shortening (PS) and maximal velocity of shortening/relengthenin as well as prolonged relengthening duration. TUDCA reversed or overtly ameliorated high fat diet-induced cardiomyocyte dysfunction including prolongation in relengthening. TUDCA alleviated high-fat diet-induced decrease in SERCA2a and phosphorylation of phospholamban, increase in ER stress (GRP78/BiP, CHOP, phosphorylation of PERK, IRE1α and eIF2α), ultrastructural changes and mitochondrial permeation pore opening. High-fat diet feeding inhibited phosphorylation of AMPK and promoted phosphorylation of GSK-3β. TUDCA prevented high fat-induced dephosphorylation of AMPK but not GSK-3β. High fat diet promoted phosphorylation of IRS-1 (Ser³⁰⁷), JNK, and ERK without affecting c-Jun phosphorylation, the effect of which with the exception of ERK phosphorylation was attenuated by TUDCA.ConclusionsThese data depict that TUDCA may ameliorate high fat diet feeding-induced cardiomyocyte contractile and intracellular Ca²⁺ defects through mechanisms associated with mitochondrial integrity, AMPK, JNK and IRS-1 serine phosphorylation.     

Autophagosome formation and cargo sequestration in the absence of LC3/GABARAPs

It has been widely assumed that Atg8 family LC3/GABARAP proteins are essential for the formation of autophagosomes during macroautophagy/autophagy, and the sequestration of cargo during selective autophagy. However, there is little direct evidence on the functional contribution of these proteins to autophagosome biogenesis in mammalian cells. To dissect the functions of LC3/GABARAPs during starvation-induced autophagy and PINK1-PARK2/Parkin-dependent mitophagy, we used CRISPR/Cas9 gene editing to generate knockouts of the LC3 and GABARAP subfamilies, and all 6 Atg8 family proteins in HeLa cells. Unexpectedly, the absence of all LC3/GABARAPs did not prevent the formation of sealed autophagosomes, or selective engulfment of mitochondria during PINK1-PARK2-dependent mitophagy. Despite not being essential for autophagosome formation, the loss of LC3/GABARAPs affected both autophagosome size, and the efficiency at which they are formed. However, the critical autophagy defect in cells lacking LC3/GABARAPs was failure to drive autophagosome-lysosome fusion. Relative to the LC3 subfamily, GABARAPs were found to play a prominent role in autophagosome-lysosome fusion and recruitment of the adaptor protein PLEKHM1. Our work clarifies the essential contribution of Atg8 family proteins to autophagy in promoting autolysosome formation, and reveals the GABARAP subfamily as a key driver of starvation-induced autophagy and PINK1-PARK2-dependent mitophagy. Since LC3/GABARAPs are not essential for mitochondrial cargo sequestration, we propose an additional mechanism of selective autophagy. The model highlights the importance of ubiquitin signals and autophagy receptors for PINK-PARK2-mediated selectivity rather than Atg8 family-LIR-mediated interactions.     

Adiponectin knockout accentuates high fat diet-induced obesity and cardiac dysfunction: Role of autophagy

Adiponectin (APN), an adipose-derived adipokine, offers cardioprotective effects although the precise mechanism of action remains unclear. This study was designed to examine the role of APN in high fat diet-induced obesity and cardiac pathology. Adult C57BL/6 wild-type and APN knockout mice were fed a low or high fat diet for 22 weeks. After 40 day feeding, mice were treated with 2 mg/kg rapamycin or vehicle every other day for 42 days on respective fat diet. Cardiomyocyte contractile and Ca^2 + transient properties were evaluated. Myocardial function was evaluated using echocardiography. Dual energy X-ray absorptiometry was used to evaluate adiposity. Energy expenditure, metabolic rate and physical activity were monitored using a metabolic cage. Lipid deposition, serum triglyceride, glucose tolerance, markers of autophagy and fatty acid metabolism including LC3, p62, Beclin-1, AMPK, mTOR, fatty acid synthase (FAS) were evaluated. High fat diet intake induced obesity, systemic glucose intolerance, cardiac hypertrophy, dampened metabolic ability, cardiac and intracellular Ca^2 + derangements, the effects of which were accentuated by APN knockout. Furthermore, APN deficiency augmented high fat diet-induced upregulation in the autophagy adaptor p62 and the decline in AMPK without affecting high fat diet-induced decrease in LC3II and LC3II-to-LC3I ratio. Neither high fat diet nor APN deficiency altered Beclin-1. Interestingly, rapamycin negated high fat diet-induced/APN-deficiency-accentuated obesity, cardiac hypertrophy and contractile dysfunction as well as AMPK dephosphorylation, mTOR phosphorylation and p62 buildup. Our results collectively revealed that APN deficiency may aggravate high fat diet-induced obesity, metabolic derangement, cardiac hypertrophy and contractile dysfunction possibly through decreased myocardial autophagy.     

Mammalian Atg8s: One is simply not enough

Yeast Atg8, a key factor in the autophagic process, is a ubiquitin-like protein that undergoes a unique conjugation to phosphatidylethanolamine (PE). Atg8 plays a dual role in early stages of autophagosome formation: It was implicated in recruitment of cargo proteins such as Atg19 and Atg32 for Cvt and mitophagy, respectively, and in autophagosome biogenesis, serving as an elongation factor by mediating membrane hemi-fusion. Similarly, the mammalian Atg8 proteins, LC3s and GABARAPs, recruit cargo into autophagosomes by binding to adaptor proteins such as p62, NBR1 and Nix. These functions, however, are not essential for bulk autophagic flux. Other studies in which the activity of the mammalian Atg8s was blocked either by knockout of the E2-like enzyme Atg3 or by using a dominant negative mutant of the promiscuous protease Atg4B revealed, in agreement with the yeast Atg8 data, that the mammalian factors are crucial for the formation of normal and mature autophagosomes. While it seems that the single yeast Atg8 and the mammalian Atg8s share similar roles, it is still unclear why the mammalian system employs several homologs. Recent publications demonstrated that the mammalian Atg8s differ in their cargo specificity, as Nix, for example, binds exclusively to GABARAP-L1. This may suggest that these proteins exhibit distinct activity also in autophagosome biogenesis. In our study we divided the mammalian Atg8s into two subfamilies of homologs based on amino acid similarity, the LC3 and GABARAP/GATE-16 subfamilies, and tested their essentiality and role in autophagy. In agreement with previous studies we found that the mammalian Atg8s are essential for autophagy but, more importantly, that each of these subfamilies has a distinct role in the process of autophagosome biogenesis.     

Backbone and side chain resonance assignments for a structured domain within Atg32

Autophagy is a catabolic cellular process that targets cytosolic material, including mitochondria, to the vacuole or lysosomes for degradation. The selective degradation of mitochondria by autophagy is termed mitophagy. Dysfunctional mitophagy, which leads to the accumulation of damaged mitochondria, has been implicated in Parkinson’s disease, cancer, cardiac disease and metabolic disease. In Saccharomyces cerevisiae, mitophagy is initiated by the autophagy receptor Atg32, an outer mitochondrial membrane protein. A lack of structural information for Atg32 has hindered our understanding of the molecular mechanisms of mitophagy initiation. To gain new structural insight into Atg32, we have identified the location of a structured domain within the cytosolic region of Atg32 and completed the backbone and side chain resonance assignments for this domain.     

CD74 knockout attenuates alcohol intake-induced cardiac dysfunction through AMPK-Skp2-mediated regulation of autophagy

CD74, a non-polymorphic type II transmembrane glycoprotein and MHC class II chaperone, is the cell surface receptor for the inflammatory cytokine macrophage migration inhibitory factor (MIF) and participates in inflammatory signaling regulation. This study examined the potential role of CD74 in binge drinking-induced cardiac contractile dysfunction. WT and CD74 knockout mice were exposed to ethanol (3 g/kg/d, i.p., for 3 days). Echocardiography, cardiomyocyte function, histological staining and autophagy signaling including AMPK, mTOR, and AMPK downstream signals Skp2 and Sirt1 were evaluated. Our results revealed that ethanol challenge overtly compromised echocardiographic, cardiomyocyte contractile, intracellular Ca²⁺ and ultrastructural properties along with overt apoptosis, inflammation (elevated MIF, IL-1β and IL-6) and mitochondrial O₂^– production (p < 0.01), the effect of which was reconciled by CD74 ablation (p < 0.01 vs. ethanol group) with the exception of MIF expression. Ethanol challenge upregulated autophagy (p < 0.001), promoted AMPK phosphorylation and Sirt1 levels (p < 0.003) while suppressing mTOR phosphorylation and Skp2 levels (p < 0.02). These effects were reversed by CD74 ablation. In vitro studies demonstrated that short-term ethanol challenge compromised cardiomyocyte contractile function and facilitated GFP-Puncta formation, which were mitigated by CD74 knockout (p < 0.0001). Moreover, the CD74 ablation-offered beneficial effects against ethanol-induced cardiomyocyte dysfunction, and GFP-Puncta formation were nullified by the AMPK activator AICAR, the Skp2 inhibitor C1 or the Sirt1 activator SRT1720 (p < 0.0001). Taken together, our data revealed that CD74 ablation counteracts acute ethanol challenge-induced myocardial dysfunction, inflammation and apoptosis possibly through an AMPK-mTOR-Skp2-mediated regulation of autophagy.     

Cathepsin K knockout alleviates aging‐induced cardiac dysfunction

Aging is a major risk factor for cardiovascular disease. It has previously been shown that protein levels of cathepsin K, a lysosomal cysteine protease, are elevated in the failing heart and that genetic ablation of cathepsin K protects against pressure overload‐induced cardiac hypertrophy and contractile dysfunction. Here we test the hypothesis that cathepsin K knockout alleviates age‐dependent decline in cardiac function. Cardiac geometry, contractile function, intracellular Ca²⁺ properties, and cardiomyocyte apoptosis were evaluated using echocardiography, fura‐2 technique, immunohistochemistry, Western blot and TUNEL staining, respectively. Aged (24‐month‐old) mice exhibited significant cardiac remodeling (enlarged chamber size, wall thickness, myocyte cross‐sectional area, and fibrosis), decreased cardiac contractility, prolonged relengthening along with compromised intracellular Ca²⁺ release compared to young (6‐month‐old) mice, which were attenuated in the cathepsin K knockout mice. Cellular markers of senescence, including cardiac lipofuscin, p21 and p16, were lower in the aged‐cathepsin K knockout mice compared to their wild‐type counterpart. Mechanistically, cathepsin K knockout mice attenuated an age‐induced increase in cardiomyocyte apoptosis and nuclear translocation of mitochondrial apoptosis‐inducing factor (AIF). In cultured H9c2 cells, doxorubicin stimulated premature senescence and apoptosis. Silencing of cathepsin K blocked the doxorubicin‐induced translocation of AIF from the mitochondria to the nuclei. Collectively, these results suggest that cathepsin K knockout attenuates age‐related decline in cardiac function via suppressing caspase‐dependent and caspase‐independent apoptosis.     

AMP‐activated protein kinase deficiency exacerbates aging‐induced myocardial contractile dysfunction

Aging is associated with myocardial dysfunction although the underlying mechanism is unclear. AMPK, a key cellular fuel sensor for energy metabolism, is compromised with aging. This study examined the role of AMPK deficiency in aging‐associated myocardial dysfunction. Young or old wild‐type (WT) and transgenic mice with overexpression of a mutant AMPK α₂ subunit (kinase dead, KD) were used. AMPK α isoform activity, myocardial function and morphology were examined. DCF and JC‐1 fluorescence probes were employed to quantify reactive oxygen species (ROS) and mitochondrial membrane potential (ΔΨm), respectively. KD mice displayed significantly reduced α₂ but not α₁ AMPK isoform activity at both ages with a greater effect at old age. Aging itself decreased α₁ isoform activity. Cardiomyocyte contractile function, intracellular Ca²⁺ handling, and SERCA2a levels were compromised with aging, the effects of which were exacerbated by AMPK deficiency. H&E staining revealed cardiomyocyte hypertrophy with aging, which was more pronounced in KD mice. TEM micrographs displayed severe disruption of mitochondrial ultrastructure characterized by swollen, irregular shape and disrupted cristae in aged KD compared with WT mice. Aging enhanced ROS production and reduced ΔΨm, the effects of which were accentuated by AMPK deficiency. Immunoblotting data depicted unchanged Akt phosphorylation and a significant decrease in mitochondrial biogenesis cofactor PGC‐1α in aged groups. AMPK deficiency but not aging decreased the phosphorylation of ACC and eNOS. Expression of membrane Glut4 and HSP90 was decreased in aged KD mice. Moreover, treatment of the AMPK activator metformin attenuated aging‐induced cardiomyocyte contractile defects. Collectively, our data suggest a role for AMPK deficiency in aging‐induced cardiac dysfunction possibly through disrupted mitochondrial function and ROS production.     

Ablation of FUNDC1-dependent mitophagy renders myocardium resistant to paraquat-induced ferroptosis and contractile dysfunction

Paraquat is a quaternary nitrogen herbicide evoking mitochondrial damage and heart failure with little therapeutic remedies available. Recent reports depicted a role for unchecked autophagy in paraquat-induced cardiotoxicity. This study was designed to examine the role of the mitophagy receptor protein FUNDC1 in paraquat-induced cardiac contractile and mitochondrial injury using a murine model of FUNDC1 knockout (FUNDC1^?/?) mice. WT and FUNDC1^?/? mice were challenged with paraquat (45 mg/kg, single injection, i.p.) for 72 h prior to examination of cardiac contractile and intracellular Ca²⁺ properties, mitochondrial integrity, mitochondrial function, O₂^? production, apoptosis, autosis and ferroptosis. Our results found that paraquat challenge compromised echocardiographic, contractile and intracellular Ca²⁺ properties in conjunction with mitochondrial damage (reduced levels of PGC1α, UCP2, NAD+, and citrate synthase activity along with fragmentation manifested by elevated Drp1 and TEM ultrastructural changes), the effects of which were overtly attenuated or obliterated by FUNDC1 ablation. Paraquat triggered ferroptosis, apoptosis (but not autosis) and unchecked mitophagy as evidenced by downregulation of GPx4, SLC7A11, Bcl2, TOM20 and ferritin as well as upregulated levels of Bax, TNFα, IL6, NCOA4 and FUNDC1, the effects of which were relieved by FUNDC1 ablation. Further study noted dephosphorylation of JNK upon paraquat challenge, the effect of which was obliterated by FUNDC1 knockout. In vitro evaluation of BODIPY ferroptosis and cardiomyocyte function revealed FUNDC1 ablation inhibited paraquat-induced increase in BODIPY lipid peroxidation and cardiomyocyte contractile dysfunction, the effects of which were nullified and mimicked by inhibition of JNK or ferroptosis and activation of JNK, respectively. Taken together, our data suggest an essential role for FUNDC1/JNK-mediated ferroptosis in paraquat exposure-evoked cardiac and mitochondrial injury.     

Inhibition of autophagy in the heart induces age-related cardiomyopathy

Constitutive autophagy is important for control of the quality of proteins and organelles to maintain cell function. Damaged proteins and organelles accumulate in aged organs. We have previously reported that cardiac-specific Atg5 (autophagy-related gene 5)-deficient mice, in which the gene was floxed out early in embryogenesis, were born normally, and showed normal cardiac function and structure up to 10 weeks old. In the present study, to determine the longer-term consequences of Atg5-deficiency in the heart, we monitored cardiac-specific Atg5-deficient mice for further 12 months. First, we examined the age-associated changes of autophagy in the wild-type mouse heart. The level of autophagy, as indicated by decreased LC3-II (microtubule-associated protein 1 light chain 3-II) levels, in the hearts of 6-, 14- or 26-month-old mice was lower than that of 10-week-old mice. Next, we investigated the cardiac function and life-span in cardiac-specific Atg5-deficient mice. The Atg5-deficient mice began to die after the age of 6 months. Atg5-deficient mice exhibited a significant increase in left ventricular dimension and decrease in fractional shortening of the left ventricle at the age of 10 months, compared to control mice, while they showed similar chamber size and contractile function at the age of 3 months. Ultrastructural analysis revealed a disorganized sarcomere structure and collapsed mitochondria in 3- and 10-month-old Atg5-deficient mice, with decreased mitochondrial respiratory functions. These results suggest that continuous constitutive autophagy has a crucial role in maintaining cardiac structure and function.