Sreejayan Nair

Akt2 knockout preserves cardiac function in high-fat diet-induced obesity by rescuing cardiac autophagosome maturation

Dear Editor, Accumulating studies have demonstrated that the autophagy–lysosome pathway, a major pathway governing protein and organelle degradation and recycling, is a house keeper in cardiomyocytes under physiological conditions (Mizushima and Klionsky, 2007). However, the role of autophagy in the heart under pathological conditions is still controversial (Nemchenko et al., 2011). In vivo studies depicted that inhibition of mammalian target of rapamycin (mTOR), a primary inhibitory regulator of autophagy, is capable of attenuating pressure overload-induced cardiac dysfunction (McMullen et al., 2004). To the contrary, recent studies have also indicated that suppressing autophagy is beneficial for cardiac hypertrophy (Cao et al., 2011). Along the same line, activated autophagy has been proved detrimental for pressure overload-induced heart failure (Zhu et al., 2007). However, the role of autophagy in the heart in high-fat diet (HFD)-induced obesity is poorly understood. To date, there is little evidence suggesting a role of autophagy in heart anomalies associated with diet-induced obesity, although a number of upstream regulators of autophagy have been identified to play a role in HFD-induced obesity. For example, the primary inhibitor of autophagy mTOR may be hyperactivated by an HFD and contribute to the development of cardiac dysfunction (Birse et al., 2010). As the major activator of mTOR, the Akt family of serine–threonine kinases is also activated by an HFD in the heart. However, the precise role of Akt2, one of the three Akt isoforms predominantly found in the heart, in autophagy regulation in HFD-induced obesity still remains elusive. To this end, the present study was designed to evaluate the role of autophagy and autophagy flux in HFD feeding-induced cardiac geometric and functional changes with a special focus on Akt2 signaling. HFD intake significantly increased body and organ (heart, liver, kidney and adipose tissue) weights compared with low-fat diet (LFD) feeding (Supplementary Table S2). Western blot analysis confirmed the absence of Akt2 in hearts from Akt2 mice (Supplementary Figure S1A and B). Interestingly, HFD feeding upregulated cardiac expression of Akt2 (Supplementary Figure S1A and B) but not that of Akt1 (Supplementary Figure S7A and B) and Akt3 (Supplementary Figure S7A and C). Akt2 knockout did not affect body or organ weight in LFD-fed mice (Supplementary Table S2). However, Akt2 knockout effectively nullified HFD-induced gain in body and organ/tissue weights, in particular the heart (Supplementary Table S2). Accumulating studies have demonstrated that Akt regulates cell growth and lipid biosynthesis through mTORC1. Accordingly, we found that an HFD-activated Akt (Supplementary Figure S6A and B) and mTORC1 (Supplementary Figure S7A and I) in the heart, both of which were mitigated by Akt2 knockout. These data depict a beneficial effect of Akt2 knockout against HFD-induced weight gain possibly through the inhibition of Akt-mTORC1 activation. In addition, HFD feeding significantly increased the level of triglyceride, the effect of which was ablated by Akt2 knockout (Supplementary Figure S1C). Further scrutiny of glucose metabolism using intraperitoneal glucose tolerance test revealed overt glucose intolerance following HFD intake in the wild type (WT) which was partially attenuated in the Akt2 mice (Supplementary Figure S1D and E). HFD feeding significantly compromised myocardial geometry and function as evidenced by overtly increased LV ESD, LV EDD and LV mass, as well as decreased fractional shortening associated with unchanged septum and posterior wall thickness. Interestingly, Akt2 knockout ameliorated HFD feeding-induced cardiac geometric and contractile anomalies (Figure 1A and B and Supplementary Figure S2A–E). Further assessment of cardiomyocyte contractile function revealed consistent findings. HFD feeding dampened cardiomyocyte contractile capacity (decreased peak shortening and maximal velocity of shortening/re-lengthening) associated with unchanged duration of shortening and re-lengthening, which was recovered by Akt2 knockout (Figure 1C and D and Supplementary Figures S2F–I and S3A–C). Besides, Akt2 knockout significantly ameliorated intracellular Ca2+ handling dysfunction induced by an HFD in the WT mice (Supplementary Figure S2J–O). Additionally, HFD feeding induced cardiac hypertrophy (Supplementary Figure S4A–H), interstitial fibrosis (Supplementary Figure S5A and B), and activated cardiac protein synthesis pathway (Supplementary Figures S6A–K and S7A, J, and K), which were obliterated by Akt2 knockout. Taken together, these results supported that Akt2 knockout protected murine hearts against HFD-induced cardiac pathological hypertrophy. Interestingly, our data revealed that the expression of LC3B I (microtubule-associated protein light chain 3 I, type B) was dramatically increased following HFD feeding in both the WT and Akt2 mice, indicating that HFD feeding may trigger the initial autophagy steps (Figure 1E and Supplementary Figure S8A, C–G, J, and L– O). LC3B II integrates onto the autophagosomal membrane, and is widely used as a marker of autophagosomes. Nonetheless, an increase in LC3B II may represent either an increase in autophagosome formation (initiation of autophagy) or a doi:10.1093/jmcb/mjs055 Journal of Molecular Cell Biology (2013), 5, 61–63 | 61 Published online December 19, 2012

Autophagy and cardiovascular aging: lesson learned from rapamycin.

The biological aging process is commonly associated with increased risk of cardiovascular diseases. Several theories have been put forward for aging-associated deterioration in ventricular function, including attenuation of growth hormone (insulin-like growth factors and insulin) signaling, loss of DNA replication and repair, histone acetylation and accumulation of reactive oxygen species. Recent evidence has depicted a rather unique role of autophagy as another important pathway in the regulation of longevity and senescence. Autophagy is a predominant cytoprotective (rather than self-destructive) process. It carries a prominent role in determination of lifespan. Reduced autophagy has been associated with aging, leading to accumulation of dysfunctional or damaged proteins and organelles. To the contrary, measures such as caloric restriction and exercise may promote autophagy to delay aging and associated comorbidities. Stimulation of autophagy using rapamycin may represent a novel strategy to prolong lifespan and combat aging-associated diseases. Rapamycin regulates autophagy through inhibition of the nutrient-sensing molecule mammalian target of rapamycin (mTOR). Inhibition of mTOR through rapamycin and caloric restriction promotes longevity. The purpose of this review is to recapitulate some of the recent advances in an effort to better understand the interplay between rapamycin-induced autophagy and decelerating cardiovascular aging.

Cathepsin K Knockout Mitigates High-Fat Diet–Induced Cardiac Hypertrophy and Contractile Dysfunction

The cysteine protease cathepsin K has been implicated in pathogenesis of cardiovascular disease. We hypothesized that ablation of cathepsin K protects against obesity-associated cardiac dysfunction. Wild-type mice fed a high-fat diet exhibited elevated heart weight, enlarged cardiomyocytes, increased left ventricular wall thickness, and decreased fractional shortening. All these changes were reconciled in cathepsin K knockout mice. Cathepsin K knockout partly reversed the impaired cardiomyocyte contractility and dysregulated calcium handling associated with high-fat diet. Additionally, cathepsin K knockout alleviated whole-body glucose intolerance and improved insulin-stimulated Akt phosphorylation in high-fat diet–fed mice. High-fat feeding increased the expression of cardiac hypertrophic proteins and apoptotic markers, which were inhibited by cathepsin K knockout. Furthermore, high-fat feeding resulted in cathepsin K release from lysosomes into the cytoplasm. In H9c2 myoblasts, silencing of cathepsin K inhibited palmitic acid–induced release of cytochrome c from mitochondria and expression of proapoptotic signaling molecules. Collectively, our data indicate that cathepsin K contributes to the development of obesity-associated cardiac hypertrophy and may represent a potential target for the treatment to obesity-associated cardiac anomalies.

Macrophage Migration Inhibitory Factor Deletion Exacerbates Pressure Overload–Induced Cardiac Hypertrophy Through Mitigating Autophagy

The proinflammatory cytokine macrophage migration inhibitory factor (MIF) has been shown to be cardioprotective under various pathological conditions. However, the underlying mechanisms still remain elusive. In this study, we revealed that MIF deficiency overtly exacerbated abdominal aorta constriction–induced cardiac hypertrophy and contractile anomalies. MIF deficiency interrupted myocardial autophagy in hypertrophied hearts. Rapamycin administration mitigated the exacerbated hypertrophic responses in MIF−/− mice. Using the phenylephrine-induced hypertrophy in vitro model in H9C2 myoblasts, we confirmed that MIF governed the activation of AMP-activated protein kinase–mammalian target of rapamycin–autophagy cascade. Confocal microscopic examination demonstrated that MIF depletion prevented phenylephrine-induced mitophagy in H9C2 myoblasts. Myocardial Parkin, an E3 ubiquitin ligase and a marker for mitophagy, was significantly upregulated after sustained pressure overload, the effect of which was prevented by MIF knockout. Furthermore, our data exhibited that levels of MIF, AMP-activated protein kinase activation, and autophagy were elevated concurrently in human failing hearts. These data indicate that endogenous MIF regulates the mammalian target of rapamycin signaling to activate autophagy to preserve cardiac geometry and protect against hypertrophic responses.

Chronic Akt Activation Attenuated Lipopolysaccharide-Induced Cardiac Dysfunction via Akt/GSK3β-Dependent Inhibition of Apoptosis and ER Stress

Sepsis is characterized by systematic inflammation and contributes to cardiac dysfunction. This study was designed to examine the effect of protein kinase B (Akt) activation on lipopolysaccharide-induced cardiac anomalies and underlying mechanism(s) involved. Mechanical and intracellular Ca²⁺ properties were examined in myocardium from wild-type and transgenic mice with cardiac-specific chronic Akt overexpression following LPS (4 mg/kg, i.p.) challenge. Akt signaling cascade (Akt, phosphatase and tensin homologue deleted on chromosome ten, glycogen synthase kinase 3 beta), stress signal (extracellular-signal-regulated kinases, c-Jun N-terminal kinases, p38), apoptotic markers (Bcl-2 associated X protein, caspase-3/-9), endoplasmic reticulum (ER) stress markers (glucose-regulated protein 78, growth arrest and DNA damage induced gene-153, eukaryotic initiation factor 2α), inflammatory markers (tumor necrosis factor α, interleukin-1β, interleukin-6) and autophagic markers (Beclin-1, light chain 3B, autophagy-related gene 7 and sequestosome 1) were evaluated. Our results revealed that LPS induced marked decrease in ejection fraction, fractional shortening, cardiomyocyte contractile capacity with dampened intracellular Ca²⁺ release and clearance, elevated reactive oxygen species (ROS) generation and decreased glutathione and glutathione disulfide (GSH/GSSG) ratio, increased ERK, JNK, p38, GRP78, Gadd153, eIF2α, BAX, caspase-3 and -9, downregulated B cell lymphoma 2 (Bcl-2), the effects of which were significantly attenuated or obliterated by Akt activation. Akt activation itself did not affect cardiac contractile and intracellular Ca²⁺ properties, ROS production, oxidative stress, apoptosis and ER stress. In addition, LPS upregulated levels of Beclin-1, LC3B and Atg7, while suppressing p62 accumulation. Akt activation did not affect Beclin-1, LC3B, Atg7 and p62 in the presence or absence of LPS. Akt overexpression promoted phosphorylation of Akt and GSK3β. In vitro study using the GSK3β inhibitor SB216763 mimicked the response elicited by chronic Akt activation. Taken together, these data showed that Akt activation ameliorated LPS-induced cardiac contractile and intracellular Ca²⁺ anomalies through inhibition of apoptosis and ER stress, possibly involving an Akt/GSK3β-dependent mechanism.

Apelin administration ameliorates high fat diet-induced cardiac hypertrophy and contractile dysfunction

Apelin has been recognized as an adipokine that plays an important role in regulating energy metabolism and is credited with antiobesity and antidiabetic properties. This study was designed to examine the effect of exogenous apelin on obesity-associated cardiac dysfunction. Oral glucose tolerance test, echocardiography, cardiomyocyte contractile and intracellular Ca(2+) properties were assessed in adult C57BL/6J mice fed - low or a - high-fat diet for 24weeks followed by apelin treatment (100nmol/kg, i.p. for 2weeks). High-fat diet resulted in increased left ventricular diastolic and systolic diameters, and wall thickness, compromised fractional shortening, impaired cardiomyocyte mechanics (peak-shortening, maximal velocity of shortening/relengthening, and duration of shortening and relengthening) and compromised intracellular Ca(2+) handling, all of which were reconciled by apelin. Apelin treatment also reversed high fat diet-induced changes in intracellular Ca(2+) regulatory proteins, ER stress, and autophagy. In addition, microRNAs (miR) -133a, miR-208 and miR-1 which were elevated following high-fat feeding were attenuated by apelin treatment. In cultured cardiomyocytes apelin reconciled palmitic acid-induced cardiomyocyte contractile anomalies. Collectively, these data depict a pivotal role of apelin in obesity-associated cardiac contractile dysfunction, suggesting a therapeutic potential of apelin in the management of cardiac dysfunction associated with obesity.

Proteases in cardiometabolic diseases: Pathophysiology, molecular mechanisms and clinical applications

Cardiovascular disease is the leading cause of death in the U.S. and other developed countries. Metabolic syndrome, including obesity, diabetes/insulin resistance, hypertension and dyslipidemia is a major threat for public health in the modern society. It is well established that metabolic syndrome contributes to the development of cardiovascular disease collective called as cardiometabolic disease. Despite documented studies in the research field of cardiometabolic disease, the underlying mechanisms are far from clear. Proteases are enzymes that break down proteins, many of which have been implicated in various diseases including cardiac disease. Matrix metalloproteinase (MMP), calpain, cathepsin and caspase are among the major proteases involved in cardiac remodeling. Recent studies have also implicated proteases in the pathogenesis of cardiometabolic disease. Elevated expression and activities of proteases in atherosclerosis, coronary heart disease, obesity/insulin-associated heart disease as well as hypertensive heart disease have been documented. Furthermore, transgenic animals that are deficient in or over-express proteases allow scientists to understand the causal relationship between proteases and cardiometabolic disease. Mechanistically, MMPs and cathepsins exert their effect on cardiometabolic diseases mainly through modifying the extracellular matrix. However, MMP and cathepsin are also reported to affect intracellular proteins, by which they contribute to the development of cardiometabolic diseases. On the other hand, activation of calpain and caspases has been shown to influence intracellular signaling cascade including the NF-κB and apoptosis pathways. Clinically, proteases are reported to function as biomarkers of cardiometabolic diseases. More importantly, the inhibitors of proteases are credited with beneficial cardiometabolic profile, although the exact molecular mechanisms underlying these salutary effects are still under investigation. A better understanding of the role of MMPs, cathepsins, calpains and caspases in cardiometabolic diseases process may yield novel therapeutic targets for treating or controlling these diseases. This article is part of a Special Issue entitled: Autophagy and protein quality control in cardiometabolic diseases.

Protein tyrosine phosphatase 1B and insulin resistance: role of endoplasmic reticulum stress/reactive oxygen species/nuclear factor kappa B axis.

Obesity-induced endoplasmic reticulum (ER) stress has been proposed as an important pathway in the development of insulin resistance. Protein-tyrosine phosphatase 1B (PTP1B) is a negative regulator of insulin signaling and is tethered to the ER-membrane. The aim of the study was to determine the mechanisms involved in the crosstalk between ER-stress and PTP1B. PTP1B whole body knockout and C57BL/6J mice were subjected to a high-fat or normal chow-diet for 20 weeks. High-fat diet feeding induced body weight gain, increased adiposity, systemic glucose intolerance, and hepatic steatosis were attenuated by PTP1B deletion. High-fat diet- fed PTP1B knockout mice also exhibited improved glucose uptake measured using [3H]-2-deoxy-glucose incorporation assay and Akt phosphorylation in the skeletal muscle tissue, compared to their wild-type control mice which received similar diet. High-fat diet-induced upregulation of glucose-regulated protein-78, phosphorylation of eukaryotic initiation factor 2α and c-Jun NH2-terminal kinase-2 were significantly attenuated in the PTP1B knockout mice. Mice lacking PTP1B showed decreased expression of the autophagy related protein p62 and the unfolded protein response adaptor protein NCK1 (non-catalytic region of tyrosine kinase). Treatment of C2C12 myotubes with the ER-stressor tunicamycin resulted in the accumulation of reactive oxygen species (ROS), leading to the activation of protein expression of PTP1B. Furthermore, tunicamycin-induced ROS production activated nuclear translocation of NFκB p65 and was required for ER stress-mediated expression of PTP1B. Our data suggest that PTP1B is induced by ER stress via the activation of the ROS-NFκB axis which is causes unfolded protein response and mediates insulin resistance in the skeletal muscle under obese condition.

Cathepsin K Knockout Alleviates Pressure Overload–Induced Cardiac Hypertrophy

Evidence from human and animal studies has documented elevated levels of lysosomal cysteine protease cathepsin K in failing hearts. Here, we hypothesized that ablation of cathepsin K mitigates pressure overload–induced cardiac hypertrophy. Cathepsin K knockout mice and their wild-type littermates were subjected to abdominal aortic constriction, resulting in cardiac remodeling (heart weight, cardiomyocyte size, left ventricular wall thickness, and end diastolic and end systolic dimensions) and decreased fractional shortening, the effects of which were significantly attenuated or ablated by cathepsin K knockout. Pressure overload dampened cardiomyocyte contractile function along with decreased resting Ca2+ levels and delayed Ca2+ clearance, which were partly resolved by cathepsin K knockout. Cardiac mammalian target of rapamycin and extracellular signal-regulated kinases (ERK) signaling cascades were upregulated by pressure overload, the effects of which were attenuated by cathepsin K knockout. In cultured H9c2 myoblast cells, silencing of cathepsin K blunted, whereas cathepsin K transfection mimicked phenylephrine–induced hypertrophic response, along with elevated phosphorylation of mammalian target of rapamycin and ERK. In addition, cathepsin K protein levels were markedly elevated in human hearts of end-stage dilated cardiomyopathy. Collectively, our data suggest that cathepsin K ablation mitigates pressure overload–induced hypertrophy, possibly via inhibition of the mammalian target of rapamycin and ERK pathways.

CARD9 knockout ameliorates myocardial dysfunction associated with high fat diet-induced obesity.

Obesity is associated with chronic inflammation which plays a critical role in the development of cardiovascular dysfunction. Because the adaptor protein caspase recruitment domain-containing protein 9 (CARD9) in macrophages regulates innate immune responses via activation of pro-inflammatory cytokines, we hypothesize that CARD9 mediates the pro-inflammatory signaling associated with obesity en route to myocardial dysfunction. C57BL/6 wild-type (WT) and CARD9(-/-) mice were fed normal diet (ND, 12% fat) or a high fat diet (HFD, 45% fat) for 5months. At the end of 5-month HFD feeding, cardiac function was evaluated using echocardiography. Cardiomyocytes were isolated and contractile properties were measured. Immunofluorescence was performed to detect macrophage infiltration in the heart. Heart tissue homogenates, plasma, and supernatants from isolated macrophages were collected to measure the concentrations of pro-inflammatory cytokines using ELISA kits. Western immunoblotting analyses were performed on heart tissue homogenates and isolated macrophages to explore the underlying signaling mechanism(s). CARD9 knockout alleviated HFD-induced insulin resistance and glucose intolerance, prevented myocardial dysfunction with preserved cardiac fractional shortening and cardiomyocyte contractile properties. CARD9 knockout also significantly decreased the number of infiltrated macrophages in the heart with reduced myocardium-, plasma-, and macrophage-derived cytokines including IL-6, IL-1β and TNFα. Finally, CARD9 knockout abrogated the increase of p38 MAPK phosphorylation, the decrease of LC3BII/LC3BI ratio and the up-regulation of p62 expression in the heart induced by HFD feeding and restored cardiac autophagy signaling. In conclusion, CARD9 knockout ameliorates myocardial dysfunction associated with HFD-induced obesity, potentially through reduction of macrophage infiltration, suppression of p38 MAPK phosphorylation, and preservation of autophagy in the heart.