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Aims Pro-protein convertase subtilisin-kexin type 9 (PCSK9), which is expressed mainly in the liver and at low levels in the heart, regulates cholesterol levels by directing low-density lipoprotein receptors to degradation. Studies to determine the role of PCSK9 in the heart are complicated by the close link between cardiac function and systemic lipid metabolism. Here, we sought to elucidate the function of PCSK9 specifically in the heart by generating and analysing mice with cardiomyocyte-specific Pcsk9 deficiency (CM-Pcsk9−/− mice) and by silencing Pcsk9 acutely in a cell culture model of adult cardiomyocyte-like cells. Methods and results Mice with cardiomyocyte-specific deletion of Pcsk9 had reduced contractile capacity, impaired cardiac function, and left ventricular dilatation at 28 weeks of age and died prematurely. Transcriptomic analyses revealed alterations of signalling pathways linked to cardiomyopathy and energy metabolism in hearts from CM-Pcsk9−/− mice vs. wild-type littermates. In agreement, levels of genes and proteins involved in mitochondrial metabolism were reduced in CM-Pcsk9−/− hearts. By using a Seahorse flux analyser, we showed that mitochondrial but not glycolytic function was impaired in cardiomyocytes from CM-Pcsk9−/− mice. We further showed that assembly and activity of electron transport chain (ETC) complexes were altered in isolated mitochondria from CM-Pcsk9−/− mice. Circulating lipid levels were unchanged in CM-Pcsk9−/− mice, but the lipid composition of mitochondrial membranes was altered. In addition, cardiomyocytes from CM-Pcsk9−/− mice had an increased number of mitochondria–endoplasmic reticulum contacts and alterations in the morphology of cristae, the physical location of the ETC complexes. We also showed that acute Pcsk9 silencing in adult cardiomyocyte-like cells reduced the activity of ETC complexes and impaired mitochondrial metabolism. Conclusion PCSK9, despite its low expression in cardiomyocytes, contributes to cardiac metabolic function, and PCSK9 deficiency in cardiomyocytes is linked to cardiomyopathy, impaired heart function, and compromised energy production.

Aims Cardiac injury and remodelling are associated with the rearrangement of cardiac lipids. Glycosphingolipids are membrane lipids that are important for cellular structure and function, and cardiac dysfunction is a characteristic of rare monogenic diseases with defects in glycosphingolipid synthesis and turnover. However, it is not known how cardiac glycosphingolipids regulate cellular processes in the heart. The aim of this study is to determine the role of cardiac glycosphingolipids in heart function. Methods and results Using human myocardial biopsies, we showed that the glycosphingolipids glucosylceramide and lactosylceramide are present at very low levels in non-ischaemic human heart with normal function and are elevated during remodelling. Similar results were observed in mouse models of cardiac remodelling. We also generated mice with cardiomyocyte-specific deficiency in Ugcg, the gene encoding glucosylceramide synthase (hUgcg–/– mice). In 9- to 10-week-old hUgcg–/– mice, contractile capacity in response to dobutamine stress was reduced. Older hUgcg–/– mice developed severe heart failure and left ventricular dilatation even under baseline conditions and died prematurely. Using RNA-seq and cell culture models, we showed defective endolysosomal retrograde trafficking and autophagy in Ugcg-deficient cardiomyocytes. We also showed that responsiveness to β-adrenergic stimulation was reduced in cardiomyocytes from hUgcg–/– mice and that Ugcg knockdown suppressed the internalization and trafficking of β1-adrenergic receptors. Conclusions Our findings suggest that cardiac glycosphingolipids are required to maintain β-adrenergic signalling and contractile capacity in cardiomyocytes and to preserve normal heart function., Graphical Abstract Cardiac glycosphingolipids are required to maintain β-adrenergic signaling and contractile capacity in cardiomyocytes and to preserve normal heart function.

Cardiovascular disease (CVD) is largely related to complications from atherosclerotic disease such as myocardial infarction (MI) and ischemic stroke and accounts for more than 30% of overall global mortality. Understanding the biochemical changes that occur in cardiac tissue following myocardial infarction is critical for clarifying the mechanisms underlying the impaired heart function seen after a myocardial infarction. Lipids have been shown to accumulate in ischemic cardiac tissue following an infarction. Recent data indicate that this cardiac lipid accumulation induces apoptosis and loss of muscle cells during the post-infarction period, which aggravate the functional impairment in the heart and limit its adaptive capacity for compensatory remodelling. It is therefore important to identify the lipids and molecular mechanisms that induce these destructive responses. In this study, the spatial distribution of lipids in mouse cardiac tissue after surgically induced infarction were identified using ToF-SIMS imaging with a gas cluster ion beam (GCIB). The benefits of frozen hydrated analysis versus freeze dried sample preparation were assessed as was the suitability of different multivariate analysis techniques for identification of localised chemical changes in the tissue. Results show that differences in intensity of the peaks in the mass spectrum corresponding to different lipids can be detected between the infarcted region of the heart and normal tissue region as well as specific accumulation of acyl-carnitine species at the boundary of the damaged region. Different spatial distributions of lipids were detected in both positive and negative ion mode providing insights into the changes in lipid metabolism following infarction. The ToF-SIMS results were compared with conventional lipidomics where although many lipid classes show similar changes between infarcted and non-infarcted hearts the ToF-SIMS data revealed differences due to salt adduct formation and most importantly where the changes in lipid signal are highly localised at the border between the infarcted and non-infarcted regions of the heart.

The pathogenesis of non-alcoholic fatty liver disease (NAFLD) and hepatocellular carcinoma (HCC) has been associated with altered expression of liver-specific genes including pyruvate kinase liver and red blood cell (PKLR), patatin-like phospholipase domain containing 3 (PNPLA3) and proprotein convertase subtilisin/kexin type 9 (PCSK9). Here, we inhibited and overexpressed the expression of these three genes in HepG2 cells, generated RNA-seq data before and after perturbation and revealed the altered global biological functions with the modulation of these genes using integrated network (IN) analysis. We found that modulation of these genes effects the total triglycerides levels within the cells and viability of the cells. Next, we generated IN for HepG2 cells, identified reporter transcription factors based on IN and found that the modulation of these genes affects key metabolic pathways associated with lipid metabolism (steroid biosynthesis, PPAR signalling pathway, fatty acid synthesis and oxidation) and cancer development (DNA replication, cell cycle and p53 signalling) involved in the progression of NAFLD and HCC. Finally, we observed that inhibition of PKLR lead to decreased glucose uptake and decreased mitochondrial activity in HepG2 cells. Hence, our systems level analysis indicated that PKLR can be targeted for development efficient treatment strategy for NAFLD and HCC.

/SummaryMyocardial infarction (MI) promotes a range of systemic effects, many of which are unknown. Here, we investigated the alterations associated with MI progression in heart and other metabolically active tissues (liver, skeletal muscle, and adipose) in a mouse model of MI (induced by ligating the left ascending coronary artery) and sham-operated mice. We performed a genome-wide transcriptomic analysis on tissue samples obtained 6- and 24-hours post MI or sham operation. By generating tissue-specific biological networks, we observed: (1) dysregulation in multiple biological processes (including immune system, mitochondrial dysfunction, fatty-acid beta-oxidation, and RNA and protein processing) across multiple tissues post MI; and (2) tissue-specific dysregulation in biological processes in liver and heart post MI. Finally, we validated our findings in two independent MI cohorts. Overall, our integrative analysis highlighted both common and specific biological responses to MI across a range of metabolically active tissues.

Adaptation to chronic intermittent hypoxia (CIH) is associated with reactive oxygen species (ROS) generation implicated in the improved cardiac tolerance against acute ischemia–reperfusion injury. Phospholipases A2(PLA2s) play an important role in cardiomyocyte phospholipid metabolism influencing membrane homeostasis. Here we aimed to determine the effect of CIH (7000 m, 8 h/day, 5 weeks) on the expression of cytosolic PLA2(cPLA2α), its phosphorylated form (p-cPLA2α), calcium-independent (iPLA2), and secretory (sPLA2IIA) at protein and mRNA levels, as well as fatty acids (FA) profile in left ventricular myocardium of adult male Wistar rats. Chronic administration of antioxidant tempol was used to verify the ROS involvement in CIH effect on PLA2s expression and phospholipid FA remodeling. While CIH did not affect PLA2s mRNA levels, it increased the total cPLA2α protein in cytosol and membranes (by 191% and 38%, respectively) and p-cPLA2α (by 23%) in membranes. On the contrary, both iPLA2and sPLA2IIA were downregulated by CIH. CIH further decreased phospholipid n-6 polyunsaturated FA (PUFA) and increased n-3 PUFA proportion. Tempol treatment prevented only CIH-induced cPLA2α up-regulation and its phosphorylation on Ser505. Our results show that CIH diversely affect myocardial PLA2s and suggest that ROS are responsible for the activation of cPLA2α under these conditions.

The microsomal triglyceride transport protein (MTTP) is critical for assembly and secretion of apolipoprotein B (apoB)-containing lipoproteins and is most abundant in the liver and intestine. Surprisingly, MTTP is also expressed in the heart. Here we tested the functional relevance of cardiac MTTP expression.We combined clinical studies, advanced expression analysis of human heart biopsies and analyses in genetically modified mice lacking cardiac expression of the MTTP-A isoform of MTTP.Our results indicate that lower cardiac MTTP expression in humans is associated with structural and perfusion abnormalities in patients with ischemic heart disease. MTTP-A deficiency in mice heart does not affect total MTTP expression, activity or lipid concentration in the heart. Despite this, MTTP-A deficient mice displayed impaired cardiac function after a myocardial infarction. Expression analysis of MTTP indicates that MTTP expression is linked to cardiac function and responses in the heart.Our results indicate that MTTP may play an important role for the heart function in conjunction to ischemic events.

Myocardial dysfunction is commonly associated with accumulation of cardiac lipid droplets (LDs). Perilipin 2 (Plin2) is a LD protein that is involved in LD formation, stability and trafficking events within the cell. Even though Plin2 is highly expressed in the heart, little is known about its role in myocardial lipid storage. A recent report shows that cardiac overexpression of Plin2 result in massive myocardial steatosis suggesting that Plin2 stabilizes LDs. In this study, we hypothesized that deficiency in Plin2 would result in reduced myocardial lipid storage. In contrast to our hypothesis, we found increased accumulation of triglycerides in hearts, and specifically in cardiomyocytes, from Plin2 −/− mice. Although Plin2 −/− mice had markedly enhanced lipid levels in the heart, they had normal heart function under baseline conditions and under mild stress. However, after an induced myocardial infarction, stroke volume and cardiac output were reduced in Plin2 −/− mice compared with Plin2 +/+ mice. We further demonstrated that the increased triglyceride accumulation in Plin2-deficient hearts was caused by altered lipophagy. Together, our data show that Plin2 is important for proper hydrolysis of LDs.

BackgroundMyocardial ischemia is associated with alterations in cardiac metabolism, resulting in decreased fatty acid oxidation and increased lipid accumulation. Here we investigate how myocardial lipid content and dynamics affect the function of the ischemic heart, and focus on the role of the lipid droplet protein perilipin 5 (Plin5) in the pathophysiology of myocardial ischemia.Methods and resultsWe generated Plin5−/− mice and found that Plin5 deficiency dramatically reduced the triglyceride content in the heart. Under normal conditions, Plin5−/− mice maintained a close to normal heart function by decreasing fatty acid uptake and increasing glucose uptake, thus preserving the energy balance. However, during stress or myocardial ischemia, Plin5 deficiency resulted in myocardial reduced substrate availability, severely reduced heart function and increased mortality. Importantly, analysis of a human cohort with suspected coronary artery disease showed that a common noncoding polymorphism, rs884164, decreases the cardiac expression of PLIN5 and is associated with reduced heart function following myocardial ischemia, indicating a role for Plin5 in cardiac dysfunction.ConclusionOur findings indicate that Plin5 deficiency alters cardiac lipid metabolism and associates with reduced survival following myocardial ischemia, suggesting that Plin5 plays a beneficial role in the heart following ischemia.

Hepatocellular carcinoma (HCC) is one of the most frequent forms of liver cancer, and effective treatment methods are limited due to tumor heterogeneity. There is a great need for comprehensive approaches to stratify HCC patients, gain biological insights into subtypes, and ultimately identify effective therapeutic targets. We stratified HCC patients and characterized each subtype using transcriptomics data, genome-scale metabolic networks and network topology/controllability analysis. This comprehensive systems-level analysis identified three distinct subtypes with substantial differences in metabolic and signaling pathways reflecting at genomic, transcriptomic, and proteomic levels. These subtypes showed large differences in clinical survival associated with altered kynurenine metabolism, WNT/beta-catenin-associated lipid metabolism, and PI3K/AKT/mTOR signaling. Integrative analyses indicated that the three subtypes rely on alternative enzymes (e.g., ACSS1/ACSS2/ACSS3, PKM/PKLR, ALDOB/ALDOA, MTHFD1L/MTHFD2/MTHFD1) to catalyze the same reactions. Based on systems-level analysis, we identified 8 to 28 subtype-specific genes with pivotal roles in controlling the metabolic network and predicted that these genes may be targeted for development of treatment strategies for HCC subtypes by performing in silico analysis. To validate our predictions, we performed experiments using HepG2 cells under normoxic and hypoxic conditions and observed opposite expression patterns between genes expressed in high/moderate/low-survival tumor groups in response to hypoxia, reflecting activated hypoxic behavior in patients with poor survival. In conclusion, our analyses showed that the heterogeneous HCC tumors can be stratified using a metabolic network-driven approach, which may also be applied to other cancer types, and this stratification may have clinical implications to drive the development of precision medicine. QC 20190423

Redox metabolism is often considered for cancer treatment but a systematic examination of redox behavior in hepatocellular carcinoma (HCC) is missing. We employed systems biology approaches to identify key features in redox metabolism in HCC. Several redox genes, including novel prognostic markers, are co-expressed with liver-specific, immune and inflammation genes. HCC also display antagonistic redox behavior, associated with altered fatty acid and amino acid metabolisms, differentiation, proliferation, and antioxidant defense. These responses are observed at the transcriptomic, metabolomic and protein levels, validated in independent cohorts, and experimentally observed using mice transcriptomics. Our integrative approach highlights mechanistic differences associated with HCC subtypes and progression, enabling tumor stratification and identification of survival signatures and potential therapeutic targets in redox metabolism.

Myocardial triglycerides stored in lipid droplets are important in regulating the intracellular delivery of fatty acids for energy generation in mitochondria, for membrane biosynthesis, and as agonists for intracellular signaling. Previously, we showed that deficiency in the lipid droplet protein perilipin 5 (Plin5) markedly reduces triglyceride storage in cardiomyocytes and increases the flux of fatty acids into phospholipids. Here, we investigated whether Plin5 deficiency in cardiomyocytes alters mitochondrial function. We found that Plin5 deficiency reduced mitochondrial oxidative capacity. Furthermore, in mitochondria from Plin5-/- hearts, the fatty acyl composition of phospholipids in mitochondrial membranes was altered and mitochondrial membrane depolarization was markedly compromised. These findings suggest that mitochondria isolated from hearts deficient in Plin5, have specific functional defects.

Dysfunction or abnormalities in the regulation of fatty acid translocase Cd36, a multifunctional membrane protein participating in uptake of long-chain fatty acids, has been linked to the development of heart diseases both in animals and humans. We have previously shown that the Cd36 transgenic spontaneously hypertensive rat (SHR-Cd36), with a wild type Cd36, has higher susceptibility to ischemic ventricular arrhythmias when compared to spontaneously hypertensive rat (SHR) carrying a mutant Cd36 gene, which may have been related to increased β-adrenergic responsiveness of these animals (Neckar et al., 2012 Physiol. Genomics 44:173-182). The present study aimed to determine whether the insertion of the wild type Cd36 into SHR would affect the function of myocardial G protein-regulated adenylyl cyclase (AC) signaling. β-Adrenergic receptors (β-ARs) were characterized by radioligand-binding experiments and the expression of selected G protein subunits, AC, and protein kinase A (PKA) was determined by RT-PCR and Western blot analyses. There was no significant difference in the amount of trimeric G proteins, but the number of β-ARs was higher (by about 35 %) in myocardial preparations from SHR-Cd36 as compared to SHR. Besides that, transgenic rats expressed increased amount (by about 20 %) of the dominant myocardial isoforms AC5/6 and contained higher levels of both nonphosphorylated (by 11 %) and phosphorylated (by 45 %) PKA. Differently stimulated AC activity in SHR-Cd36 significantly exceeded (by about 18-30 %) the enzyme activity in SHR. Changes at the molecular level were reflected by higher contractile responses to stimulation by the adrenergic agonist dobutamine. In summary, it can be concluded that the increased susceptibility to ischemic arrhythmias of SHR-Cd36 is attributable to upregulation of some components of the β-AR signaling pathway, which leads to enhanced sensitization of AC and increased cardiac adrenergic responsiveness.

CD36 fatty acid translocase plays a key role in supplying heart with its major energy substrate, long-chain fatty acids (FA). Previously, we found that the spontaneously hypertensive rat (SHR) harbors a deletion variant of Cd36 gene that results in reduced transport of long-chain FA into cardiomyocytes and predisposes the SHR to cardiac hypertrophy. In the current study, we analyzed the effects of mutant Cd36 on susceptibility to ischemic ventricular arrhythmias and myocardial infarction in adult SHR- Cd36 transgenic rats with wild-type Cd36 compared with age-matched SHR controls. Using an open-chest model of coronary artery occlusion, we found that SHR- Cd36 transgenic rats showed profound arrhythmogenesis resulting in significantly increased duration of tachyarrhythmias (207 ± 48 s vs. 55 ± 21 s, P < 0.05), total number of premature ventricular complexes (2,623 ± 517 vs. 849 ± 250, P < 0.05) and arrhythmia score (3.86 ± 0.18 vs. 3.13 ± 0.13, P < 0.001). On the other hand, transgenic SHR compared with SHR controls showed significantly reduced infarct size (52.6 ± 4.3% vs. 72.4 ± 2.9% of area at risk, P < 0.001). Similar differences were observed in isolated perfused hearts, and the increased susceptibility of transgenic SHR to arrhythmias was abolished by reserpine, suggesting the involvement of catecholamines. To further search for possible molecular mechanisms of altered ischemic tolerance, we compared gene expression profiles in left ventricles dissected from 6-wk-old transgenic SHR vs. age-matched controls using Illumina-based sequencing. Circadian rhythms and oxidative phosphorylation were identified as the top KEGG pathways, while circadian rhythms, VDR/RXR activation, IGF1 signaling, and HMGB1 signaling were the top IPA canonical pathways potentially important for Cd36-mediated effects on ischemic tolerance. It can be concluded that transgenic expression of Cd36 plays an important role in modulating the incidence and severity of ischemic and reperfusion ventricular arrhythmias and myocardial infarct size induced by coronary artery occlusion. The proarrhythmic effect of Cd36 transgene appears to be dependent on adrenergic stimulation.

This study examined the effects of a novel orally active 14,15-epoxyeicosatrienoic acid analog (EET-A) on blood pressure (BP) and myocardial infarct size (IS) in two-kidney, one-clip (2K1C) Goldblatt hypertensive rats during sustained phase of hypertension. Between days 31 and 35 after clip placement the rats were treated with EET-A and BP was monitored by radiotelemetry; sham-operated normotensive rats were used as controls. Tissue concentrations of epoxyeicosatrienoic acids served as a marker of production of epoxygenase metabolites. The rats were subjected to acute myocardial ischemia/reperfusion (I/R) injury and IS was determined. We found that EET-A treatment did not lower BP in 2K1C rats and did not alter availability of biologically active epoxygenase metabolites in 2K1C or in sham-operated rats. The myocardial IS was significantly smaller in untreated 2K1C rats as compared with normotensive controls and EET-A reduced it in controls but not in 2K1C rats. Our findings suggest that during the phase of sustained hypertension 2K1C Goldblatt hypertensive rats exhibit increased cardiac tolerance to I/R injury as compared with normotensive controls, and that in this animal model of human renovascular hypertension short-term treatment with EET-A does not induce any antihypertensive and cardioprotective actions.

Disruption to the sensitive balance of long-chain fatty acids and glucose in the heart could cause cardiovascular diseases. Searching for a possible role of novel protein kinase C (nPKC) in heart with disrupted energy balance, we compared the insulin-resistant spontaneously hypertensive rats (SHR), which carry a nonfunctional variant of the fatty acid transporter FAT/CD36, with the less insulin-resistant congenic strain SHR-4 that is genetically identical except for a segment on chromosome 4 including a wild-type gene for a functional FAT/CD36. We analyzed expression of the nPKC-δ and -e isoforms plus triacylglycerols (TAG) content in the myocardium of both FAT/CD36 strains and after a high sucrose diet (HSD). Two weeks before killing, males of both strains were randomly divided into two groups and fed either a standard laboratory chow or an HSD. PKC was determined by Western blotting in particulate and cytosolic fractions from left ventricles. The SHR-4 rats exhibited lower serum levels of insulin and free fatty acids than did SHR rats and higher amounts of PKC-e in the heart particulate fraction. HSD caused accumulation of heart TAG in SHR but not in SHR-4. HSD increased PKC-δ and decreased PKC-e expression in particulate fraction from left ventricles of SHR-4 while having no effects in SHR. These results demonstrate that reduced insulin resistance in SHR-4 rats with wild-type FAT/CD36 is associated with the insulin signaling pathway involving nPKCs.

Ceramide accumulation is known to accompany acute myocardial ischemia, but its role in the pathogenesis of ischemic heart disease is unclear. In this study, we aimed to determine how ceramides accumulate in the ischemic heart and to determine if cardiac function following ischemia can be improved by reducing ceramide accumulation. To investigate the association between ceramide accumulation and heart function, we analyzed myocardial left ventricle biopsies from subjects with chronic ischemia and found that ceramide levels were higher in biopsies from subjects with reduced heart function. Ceramides are produced by either de novo synthesis or hydrolysis of sphingomyelin catalyzed by acid and/or neutral sphingomyelinase. We used cultured HL-1 cardiomyocytes to investigate these pathways and showed that acid sphingomyelinase activity rather than neutral sphingomyelinase activity or de novo sphingolipid synthesis was important for hypoxia-induced ceramide accumulation. We also used mice with a partial deficiency in acid sphingomyelinase (Smpd1+/− mice) to investigate if limiting ceramide accumulation under ischemic conditions would have a beneficial effect on heart function and survival. Although we showed that cardiac ceramide accumulation was reduced in Smpd1+/− mice 24 h after an induced myocardial infarction, this reduction was not accompanied by an improvement in heart function or survival. Our findings show that accumulation of cardiac ceramides in the post-ischemic heart is mediated by acid sphingomyelinase. However, targeting ceramide accumulation in the ischemic heart may not be a beneficial treatment strategy.