Transcriptomic and spatial dissection of human ex vivo right atrial tissue reveals proinflammatory microvascular changes in ischemic heart disease.

Cardiovascular disease plays a central role in the electrical and structural remodeling of the right atrium, predisposing to arrhythmias, heart failure, and sudden death. Here, we dissect with single-nuclei RNA sequencing (snRNA-seq) and spatial transcriptomics the gene expression changes in the human ex vivo right atrial tissue and pericardial fluid in ischemic heart disease, myocardial infarction, and ischemic and non-ischemic heart failure using asymptomatic patients with valvular disease who undergo preventive surgery as the control group. We reveal substantial differences in disease-associated gene expression in all cell types, collectively suggesting inflammatory microvascular dysfunction and changes in the right atrial tissue composition as the valvular and vascular diseases progress into heart failure. The data collectively suggest that investigation of human cardiovascular disease should expand to all functionally important parts of the heart, which may help us to identify mechanisms promoting more severe types of the disease.

BIN1, Myotubularin, and Dynamin-2 Coordinate T-Tubule Growth in Cardiomyocytes.

BACKGROUND: Transverse tubules (t-tubules) form gradually in the developing heart, critically enabling maturation of cardiomyocyte Ca2+ homeostasis. The membrane bending and scaffolding protein BIN1 (bridging integrator 1) has been implicated in this process. However, it is unclear which of the various reported BIN1 isoforms are involved, and whether BIN1 function is regulated by its putative binding partners MTM1 (myotubularin), a phosphoinositide 3'-phosphatase, and DNM2 (dynamin-2), a GTPase believed to mediate membrane fission. METHODS: We investigated the roles of BIN1, MTM1, and DNM2 in t-tubule formation in developing mouse cardiomyocytes, and in gene-modified HL-1 and human-induced pluripotent stem cell-derived cardiomyocytes. T-tubules and proteins of interest were imaged by confocal and Airyscan microscopy, and expression patterns were examined by RT-qPCR and Western blotting. Ca2+ release was recorded using Fluo-4. RESULTS: We observed that in the postnatal mouse heart, BIN1 localizes along Z-lines from early developmental stages, consistent with roles in initial budding and scaffolding of t-tubules. T-tubule proliferation and organization were linked to a progressive and parallel increase in 4 detected BIN1 isoforms. All isoforms were observed to induce tubulation in cardiomyocytes but produced t-tubules with differing geometries. BIN1-induced tubulations contained the L-type Ca2+ channel, were colocalized with caveolin-3 and the ryanodine receptor, and effectively triggered Ca2+ release. BIN1 upregulation during development was paralleled by increasing expression of MTM1. Despite no direct binding between MTM1 and murine cardiac BIN1 isoforms, which lack exon 11, high MTM1 levels were necessary for BIN1-induced tubulation, indicating a central role of phosphoinositide homeostasis. In contrast, the developing heart exhibited declining levels of DNM2. Indeed, we observed that high levels of DNM2 are inhibitory for t-tubule formation, although this protein colocalizes with BIN1 along Z-lines, and binds all 4 isoforms. CONCLUSIONS: These findings indicate that BIN1, MTM1, and DNM2 have balanced and collaborative roles in controlling t-tubule growth in cardiomyocytes.

PGC-1α4 Interacts with REST to Upregulate Neuronal Genes and Augment Energy Consumption in Developing Cardiomyocytes.

Transcriptional coactivator PGC-1α is a main regulator of cardiac energy metabolism. In addition to canonical PGC-1α1, other PGC-1α isoforms have been found to exert specific biological functions in a variety of tissues. We investigated the expression patterns and the biological effects of the non-canonical isoforms in the heart. We used RNA sequencing data to identify the expression patterns of PGC-1α isoforms in the heart. To evaluate the biological effects of the alternative isoform expression, we generated a transgenic mouse with cardiac-specific overexpression of PGC-1α4 and analysed the cardiac phenotype with a wide spectrum of physiological and biophysical tools. Our results show that non-canonical isoforms are expressed in the heart, and that the main variant PGC-1α4 is induced by ß-adrenergic signalling in adult cardiomyocytes. Cardiomyocyte specific PGC-1α4 overexpression in mice relieves the RE1-Silencing Transcription factor (REST)-mediated suppression of neuronal genes during foetal heart development. The resulting de-repression of REST target genes induces a cardiac phenotype with increased cellular energy consumption, resulting in postnatal dilated cardiomyopathy. These results propose a new concept for actions of the PGC-1α protein family where activation of the Pgc-1α gene, through its isoforms, induces a phenotype with concurrent supply and demand for cellular energy. These data highlight the biological roles of the different PGC-1α isoforms, which should be considered when future therapies are developed.

Varied Responses to a High m.3243A>G Mutation Load and Respiratory Chain Dysfunction in Patient-Derived Cardiomyocytes.

The m.3243A>G mutation in mitochondrial tRNA-Leu(UUR) is one of the most common pathogenic mitochondrial DNA mutations in humans. The clinical manifestations are highly heterogenous and the causes for the drastic clinical variability are unknown. Approximately one third of patients suffer from cardiac disease, which often increases mortality. Why only some patients develop cardiomyopathy is unknown. Here, we studied the molecular effects of a high m.3243A>G mutation load on cardiomyocyte functionality, using cells derived from induced pluripotent stem cells (iPSC-CM) of two different m.3243A>G patients, only one of them suffering from severe cardiomyopathy. While high mutation load impaired mitochondrial respiration in both patients' iPSC-CMs, the downstream consequences varied. mtDNA mutant cells from a patient with no clinical heart disease showed increased glucose metabolism and retained cellular ATP levels, whereas cells from the cardiac disease patient showed reduced ATP levels. In this patient, the mutations also affected intracellular calcium signaling, while this was not true in the other patient's cells. Our results reflect the clinical variability in mitochondrial disease patients and show that iPSC-CMs retain tissue specific features seen in patients.

Testing the Role of Glutamate NMDA Receptors in Peripheral Trigeminal Nociception Implicated in Migraine Pain.

The pro-nociceptive role of glutamate in the CNS in migraine pathophysiology is well established. Glutamate, released from trigeminal afferents, activates second order nociceptive neurons in the brainstem. However, the function of peripheral glutamate receptors in the trigeminovascular system suggested as the origin site for migraine pain, is less known. In the current project, we used calcium imaging and patch clamp recordings from trigeminal ganglion (TG) neurons, immunolabelling, CGRP assay and direct electrophysiological recordings from rat meningeal afferents to investigate the role of glutamate in trigeminal nociception. Glutamate, aspartate, and, to a lesser extent, NMDA under free-magnesium conditions, evoked calcium transients in a fraction of isolated TG neurons, indicating functional expression of NMDA receptors. The fraction of NMDA sensitive neurons was increased by the migraine mediator CGRP. NMDA also activated slowly desensitizing currents in 37% of TG neurons. However, neither glutamate nor NMDA changed the level of extracellular CGRP. TG neurons expressed both GluN2A and GluN2B subunits of NMDA receptors. In addition, after removal of magnesium, NMDA activated persistent spiking activity in a fraction of trigeminal nerve fibers in meninges. Thus, glutamate activates NMDA receptors in somas of TG neurons and their meningeal nerve terminals in magnesium-dependent manner. These findings suggest that peripherally released glutamate can promote excitation of meningeal afferents implicated in generation of migraine pain in conditions of inherited or acquired reduced magnesium blockage of NMDA channels and support the usage of magnesium supplements in migraine.

PGC-1α deficiency reveals sex-specific links between cardiac energy metabolism and EC-coupling during development of heart failure in mice.

AIMS: Biological sex has fundamental effects on mammalian heart physiology and pathogenesis. While it has been established that female sex is a protective factor against most cardiovascular diseases (CVDs), this beneficial effect may involve pathways associated with cardiac energy metabolism. Our aim was to elucidate the role of transcriptional coactivator PGC-1α in sex dimorphism of heart failure (HF) development. METHODS AND RESULTS: Here, we show that mice deficient in cardiac expression of the peroxisome proliferator-activated receptor gamma (PPAR-γ) coactivator-1α (PGC-1α) develop dilated HF associated with changes in aerobic and anaerobic metabolism, calcium handling, cell structure, electrophysiology, as well as gene expression. These cardiac changes occur in both sexes, but female mice develop an earlier and more severe structural and functional phenotype associated with dyssynchronous local calcium release resulting from disruption of t-tubular structures of the cardiomyocytes. CONCLUSIONS: These data reveal that the integrity of the subcellular Ca2+ release and uptake machinery is dependent on energy metabolism and that female hearts are more prone to suffer from contractile dysfunction in conditions with compromised production of cellular energy. Furthermore, these findings suggest that PGC-1α is a central mediator of sex-specific differences in heart function and CVD susceptibility.

Mitochondrial NDUFA4L2 is a novel regulator of skeletal muscle mass and force.

The hypoxia-inducible nuclear-encoded mitochondrial protein NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 4-like 2 (NDUFA4L2) has been demonstrated to decrease oxidative phosphorylation and production of reactive oxygen species in neonatal cardiomyocytes, brain tissue and hypoxic domains of cancer cells. Prolonged local hypoxia can negatively affect skeletal muscle size and tissue oxidative capacity. Although skeletal muscle is a mitochondrial rich, oxygen sensitive tissue, the role of NDUFA4L2 in skeletal muscle has not previously been investigated. Here we ectopically expressed NDUFA4L2 in mouse skeletal muscles using adenovirus-mediated expression and in vivo electroporation. Moreover, femoral artery ligation (FAL) was used as a model of peripheral vascular disease to induce hind limb ischemia and muscle damage. Ectopic NDUFA4L2 expression resulted in reduced mitochondrial respiration and reactive oxygen species followed by lowered AMP, ADP, ATP, and NAD+ levels without affecting the overall protein content of the mitochondrial electron transport chain. Furthermore, ectopically expressed NDUFA4L2 caused a ~20% reduction in muscle mass that resulted in weaker muscles. The loss of muscle mass was associated with increased gene expression of atrogenes MurF1 and Mul1, and apoptotic genes caspase 3 and Bax. Finally, we showed that NDUFA4L2 was induced by FAL and that the Ndufa4l2 mRNA expression correlated with the reduced capacity of the muscle to generate force after the ischemic insult. These results show, for the first time, that mitochondrial NDUFA4L2 is a novel regulator of skeletal muscle mass and force. Specifically, induced NDUFA4L2 reduces mitochondrial activity leading to lower levels of important intramuscular metabolites, including adenine nucleotides and NAD+ , which are hallmarks of mitochondrial dysfunction and hence shows that dysfunctional mitochondrial activity may drive muscle wasting.

Therapeutic potential of PGC-1α in age-related macular degeneration (AMD) - the involvement of mitochondrial quality control, autophagy, and antioxidant response.

INTRODUCTION: Age-related macular degeneration (AMD) is the leading, cause of sight loss in the elderly in the Western world. Most patients remain still without any treatment options. The targeting of Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), a transcription co-factor, is a putative therapy against AMD. AREAS COVERED: The characteristics of AMD and their possible connection with PGC-1α as well as the transcriptional and post-transcriptional control of PGC-1α are discussed. The PGC-1α-driven control of mitochondrial functions, and its involvement in autophagy and antioxidant responses are also examined. Therapeutic possibilities via drugs and epigenetic approaches to enhance PGC-1α expression are discussed. Authors conducted a search of literature mainly from the recent decade from the PubMed database. EXPERT OPINION: Therapy options in AMD could include PGC-1α activation or stabilization. This could be achieved by a direct elevation of PGC-1α activity, a stabilization or modification of its upstream activators and inhibitors by chemical compounds, like 5-Aminoimidazole-4-carboxamide riboside, metformin, and resveratrol. Furthermore, manipulations with epigenetic modifiers of PGC-1α expression, including miRNAs, e.g. miR-204, are considered. A therapy aimed at PGC-1α up-regulation may be possible in other disorders besides AMD, if they are associated with disturbances in the mitochondria-antioxidant response-autophagy axis.

The Ablation of VEGFR-1 Signaling Promotes Pressure Overload-Induced Cardiac Dysfunction and Sudden Death.

Molecular mechanisms involved in cardiac remodelling are not fully understood. To study the role of vascular endothelial growth factor receptor 1 (VEGFR-1) signaling in left ventricular hypertrophy (LVH) and heart failure, we used a mouse model lacking the intracellular VEGFR-1 tyrosine kinase domain (VEGFR-1 TK-/-) and induced pressure overload with angiotensin II infusion. Using echocardiography (ECG) and immunohistochemistry, we evaluated pathological changes in the heart during pressure overload and measured the corresponding alterations in expression level and phosphorylation of interesting targets by deep RNA sequencing and Western blot, respectively. By day 6 of pressure overload, control mice developed significant LVH whereas VEGFR-1 TK-/- mice displayed a complete absence of LVH, which correlated with significantly increased mortality. At a later time point, the cardiac dysfunction led to increased ANP and BNP levels, atrial dilatation and prolongation of the QRSp duration as well as increased cardiomyocyte area. Immunohistochemical analyses showed no alterations in fibrosis or angiogenesis in VEGFR-1 TK-/- mice. Mechanistically, the ablation of VEGFR-1 signaling led to significantly upregulated mTOR and downregulated PKCα phosphorylation in the myocardium. Our results show that VEGFR-1 signaling regulates the early cardiac remodelling during the compensatory phase of pressure overload and increases the risk of sudden death.

NRF2 is a key regulator of endothelial microRNA expression under proatherogenic stimuli.

AIMS: Oxidized phospholipids and microRNAs (miRNAs) are increasingly recognized to play a role in endothelial dysfunction driving atherosclerosis. NRF2 transcription factor is one of the key mediators of the effects of oxidized phospholipids, but the gene regulatory mechanisms underlying the process remain obscure. Here, we investigated the genome-wide effects of oxidized phospholipids on transcriptional gene regulation in human umbilical vein endothelial cells and aortic endothelial cells with a special focus on miRNAs. METHODS AND RESULTS: We integrated data from HiC, ChIP-seq, ATAC-seq, GRO-seq, miRNA-seq, and RNA-seq to provide deeper understanding of the transcriptional mechanisms driven by NRF2 in response to oxidized phospholipids. We demonstrate that presence of NRF2 motif and its binding is more prominent in the vicinity of up-regulated transcripts and transcriptional initiation represents the most likely mechanism of action. We further identified NRF2 as a novel regulator of over 100 endothelial pri-miRNAs. Among these, we characterize two hub miRNAs miR-21-5p and miR-100-5p and demonstrate their opposing roles on mTOR, VEGFA, HIF1A, and MYC expressions. Finally, we provide evidence that the levels of miR-21-5p and miR-100-5p in exosomes are increased upon senescence and exhibit a trend to correlate with the severity of coronary artery disease. CONCLUSION: Altogether, our analysis provides an integrative view into the regulation of transcription and miRNA function that could mediate the proatherogenic effects of oxidized phospholipids in endothelial cells.

Obesity Represses CYP2R1, the Vitamin D 25-Hydroxylase, in the Liver and Extrahepatic Tissues.

Low plasma level of 25-hydroxyvitamin D (25-OH-D), namely vitamin D deficiency, is associated with obesity and weight loss improves 25-OH-D status. However, the mechanism behind obesity-induced vitamin D deficiency remains unclear. Here, we report that obesity suppresses the expression of cytochrome P450 (CYP) 2R1, the main vitamin D 25-hydroxylase, in both mice and humans. In humans, weight loss induced by gastric bypass surgery increased the expression of CYP2R1 in the s.c. adipose tissue suggesting recovery after the obesity-induced suppression. At the same time, CYP27B1, the vitamin D 1α-hydroxylase, was repressed by the weight loss. In a mouse (C57BL/6N) model of diet-induced obesity, the plasma 25-OH-D was decreased. In accordance, the CYP2R1 expression was strongly repressed in the liver. Moreover, obesity repressed the expression of CYP2R1 in several extrahepatic tissues, the kidney, brown adipose tissue, and testis, but not in the white adipose tissue. Obesity had a similar effect in both male and female mice. In mice, obesity repressed expression of the vitamin D receptor in brown adipose tissue. Obesity also upregulated the expression of the vitamin D receptor and CYP24A1 in the s.c. adipose tissue of a subset of mice; however, no effect was observed in the human s.c. adipose tissue. In summary, we show that obesity affects CYP2R1 expression both in the mouse and human tissues. We suggest that in mouse the CYP2R1 repression in the liver plays an important role in obesity-induced vitamin D deficiency. Currently, it is unclear whether the CYP2R1 downregulation in extrahepatic tissues could contribute to the obesity-induced low plasma 25-OH-D, however, this phenomenon may affect at least the local 25-OH-D concentrations. © 2020 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Metabolic alterations in Parkinson's disease astrocytes.

In Parkinson`s disease (PD), the loss of dopaminergic (DA) neurons in the substantia nigra pars compacta is associated with Lewy bodies arising from the accumulation of alpha-synuclein protein which leads ultimately to movement impairment. While PD has been considered a disease of the DA neurons, a glial contribution, in particular that of astrocytes, in PD pathogenesis is starting to be uncovered. Here, we report findings from astrocytes derived from induced pluripotent stem cells of LRRK2 G2019S mutant patients, with one patient also carrying a GBA N370S mutation, as well as healthy individuals. The PD patient astrocytes manifest the hallmarks of the disease pathology including increased expression of alpha-synuclein. This has detrimental consequences, resulting in altered metabolism, disturbed Ca2+ homeostasis and increased release of cytokines upon inflammatory stimulation. Furthermore, PD astroglial cells manifest increased levels of polyamines and polyamine precursors while lysophosphatidylethanolamine levels are decreased, both of these changes have been reported also in PD brain. Collectively, these data reveal an important role for astrocytes in PD pathology and highlight the potential of iPSC-derived cells in disease modeling and drug discovery.

Short high-fat diet interferes with the physiological maturation of the late adolescent mouse heart.

Dietary fats are essential for cardiac function. The metabolites of fats known as fatty acids provide most of the energy for cardiac tissue, serve as building blocks for membranes and regulate important signaling cascades. Despite their importance, excess fat intake can cause cardiac dysfunction. The detrimental effects of high-fat diet (HFD) on cardiac health are widely investigated in long-term studies but the short-term effects of fats have not been thoroughly studied. To elucidate the near-term effects of a HFD on the growth and maturation of late adolescent heart we subjected 11-week-old mice to an 8-week long HFD (42% of calories from fat, 42% from carbohydrate, n = 8) or chow diet (12% of calories from fat, 66% from carbohydrate, n = 7) and assessed their effects on the heart in vivo and in vitro. Our results showed that excessive fat feeding interferes with normal maturation of the heart indicated by the lack of increase in dimensions, volume, and stroke volume of the left ventricles of mice on high fat diet that were evident in mice on chow diet. In addition, differences in regional strain during the contraction cycle between mice on HFD and chow diet were seen. These changes were associated with reduced activity of the growth promoting PI3K-Akt1 signaling cascade and moderate changes in glucose metabolism without changes in calcium signaling. This study suggests that even a short period of HFD during late adolescence hinders cardiac maturation and causes physiological changes that may have an impact on the cardiac health in adulthood.

Nrf2 and SQSTM1/p62 jointly contribute to mesenchymal transition and invasion in glioblastoma.

Accumulating evidence suggests that constitutively active Nrf2 has a pivotal role in cancer as it induces pro-survival genes that promote cancer cell proliferation and chemoresistance. The mechanisms of Nrf2 dysregulation and functions in cancer have not been fully characterized. Here, we jointly analyzed the Broad-Novartis Cancer Cell Line Encyclopedia (CCLE) and the Cancer Genome Atlas (TCGA) multi-omics data in order to identify cancer types where Nrf2 activation is present. We found that Nrf2 is hyperactivated in a subset of glioblastoma (GBM) patients, whose tumors display a mesenchymal subtype, and uncover several different mechanisms contributing to increased Nrf2 activity. Importantly, we identified a positive feedback loop between SQSTM1/p62 and Nrf2 as a mechanism for activation of the Nrf2 pathway. We also show that autophagy and serine/threonine signaling regulates p62 mediated Keap1 degradation. Our results in glioma cell lines indicate that both Nrf2 and p62 promote proliferation, invasion and mesenchymal transition. Finally, Nrf2 activity was associated with decreased progression free survival in TCGA GBM patient samples, suggesting that treatments have limited efficacy if this transcription factor is overactivated. Overall, our findings place Nrf2 and p62 as the key components of the mesenchymal subtype network, with implications to tumorigenesis and treatment resistance. Thus, Nrf2 activation could be used as a surrogate prognostic marker in mesenchymal subtype GBMs. Furthermore, strategies aiming at either inhibiting Nrf2 or exploiting Nrf2 hyperactivity for targeted gene therapy may provide novel treatment options for this subset of GBM.

Oxidative hotspots on actin promote skeletal muscle weakness in rheumatoid arthritis.

Skeletal muscle weakness in patients suffering from rheumatoid arthritis (RA) adds to their impaired working abilities and reduced quality of life. However, little molecular insight is available on muscle weakness associated with RA. Oxidative stress has been implicated in the disease pathogenesis of RA. Here we show that oxidative post-translational modifications of the contractile machinery targeted to actin result in impaired actin polymerization and reduced force production. Using mass spectrometry, we identified the actin residues targeted by oxidative 3-nitrotyrosine (3-NT) or malondialdehyde adduct (MDA) modifications in weakened skeletal muscle from mice with arthritis and patients afflicted by RA. The residues were primarily located to three distinct regions positioned at matching surface areas of the skeletal muscle actin molecule from arthritis mice and RA patients. Moreover, molecular dynamic simulations revealed that these areas, here coined "hotspots", are important for the stability of the actin molecule and its capacity to generate filaments and interact with myosin. Together, these data demonstrate how oxidative modifications on actin promote muscle weakness in RA patients and provide novel leads for targeted therapeutic treatment to improve muscle function.

Fasting-Induced Transcription Factors Repress Vitamin D Bioactivation, a Mechanism for Vitamin D Deficiency in Diabetes.

Low 25-hydroxyvitamin D levels correlate with the prevalence of diabetes; however, the mechanisms remain uncertain. Here, we show that nutritional deprivation-responsive mechanisms regulate vitamin D metabolism. Both fasting and diabetes suppressed hepatic cytochrome P450 (CYP) 2R1, the main vitamin D 25-hydroxylase responsible for the first bioactivation step. Overexpression of coactivator peroxisome proliferator-activated receptor γ coactivator 1-α (PGC-1α), induced physiologically by fasting and pathologically in diabetes, resulted in dramatic downregulation of CYP2R1 in mouse hepatocytes in an estrogen-related receptor α (ERRα)-dependent manner. However, PGC-1α knockout did not prevent fasting-induced suppression of CYP2R1 in the liver, indicating that additional factors contribute to the CYP2R1 repression. Furthermore, glucocorticoid receptor (GR) activation repressed the liver CYP2R1, suggesting GR involvement in the regulation of CYP2R1. GR antagonist mifepristone partially prevented CYP2R1 repression during fasting, suggesting that glucocorticoids and GR contribute to the CYP2R1 repression during fasting. Moreover, fasting upregulated the vitamin D catabolizing CYP24A1 in the kidney through the PGC-1α-ERRα pathway. Our study uncovers a molecular mechanism for vitamin D deficiency in diabetes and reveals a novel negative feedback mechanism that controls crosstalk between energy homeostasis and the vitamin D pathway.

Loss of NRF-2 and PGC-1α genes leads to retinal pigment epithelium damage resembling dry age-related macular degeneration.

Age-related macular degeneration (AMD) is a multi-factorial disease that is the leading cause of irreversible and severe vision loss in the developed countries. It has been suggested that the pathogenesis of dry AMD involves impaired protein degradation in retinal pigment epithelial cells (RPE). RPE cells are constantly exposed to oxidative stress that may lead to the accumulation of damaged cellular proteins, DNA and lipids and evoke tissue deterioration during the aging process. The ubiquitin-proteasome pathway and the lysosomal/autophagosomal pathway are the two major proteolytic systems in eukaryotic cells. NRF-2 (nuclear factor-erythroid 2-related factor-2) and PGC-1α (peroxisome proliferator-activated receptor gamma coactivator-1 alpha) are master transcription factors in the regulation of cellular detoxification. We investigated the role of NRF-2 and PGC-1α in the regulation of RPE cell structure and function by using global double knockout (dKO) mice. The NRF-2/PGC-1α dKO mice exhibited significant age-dependent RPE degeneration, accumulation of the oxidative stress marker, 4-HNE (4-hydroxynonenal), the endoplasmic reticulum stress markers GRP78 (glucose-regulated protein 78) and ATF4 (activating transcription factor 4), and damaged mitochondria. Moreover, levels of protein ubiquitination and autophagy markers p62/SQSTM1 (sequestosome 1), Beclin-1 and LC3B (microtubule associated protein 1 light chain 3 beta) were significantly increased together with the Iba-1 (ionized calcium binding adaptor molecule 1) mononuclear phagocyte marker and an enlargement of RPE size. These histopathological changes of RPE were accompanied by photoreceptor dysmorphology and vision loss as revealed by electroretinography. Consequently, these novel findings suggest that the NRF-2/PGC-1α dKO mouse is a valuable model for investigating the role of proteasomal and autophagy clearance in the RPE and in the development of dry AMD.

Heart specific PGC-1α deletion identifies metabolome of cardiac restricted metabolic heart failure.

Aims: Heart failure (HF) is associated with drastic changes in metabolism leading to a cardiac energy deficiency well as maladaptive changes in multiple other tissues. It is still unclear which of these changes originates from cardiomyocyte metabolic remodelling or whether they are induced secondarily by systemic factors. Our aim here was to induce cardiac restricted metabolic changes mimicking those seen in HF and to characterize the associated metabolite changes in the heart, circulation, and peripheral tissues. Methods and results: We generated a cardiac specific PGC-1α knockout mice (KO) to specifically induce metabolic dysregulation typically accompanied by HF and performed a non-targeted LC-MS metabolite profiling analysis of heart, plasma, liver, and skeletal muscle to identify metabolites associated with cardiac specific metabolic remodelling. The KO animals developed a progressive cardiomyopathy with cardiac dilatation leading to fatal HF. At 17 weeks of age, when significant remodelling had occurred but before the onset of HF, isolated PGC-1α deficient cardiomyocytes had suppressed glucose and fatty acid oxidation as well as blunted anaerobic metabolism. KO hearts displayed a distinctive metabolite profile with 92 significantly altered molecular features including metabolite changes in energy metabolism, phospholipid metabolism, amino acids, and oxidative stress signalling. Some of the metabolite changes correlated with the specific parameters of cardiac function. We did not observe any significant alterations in the metabolomes of the other measured tissues or in plasma. Conclusions: Heart specific PGC-1α KO induces metabolic, functional, and structural abnormalities leading to dilating cardiomyopathy and HF. The metabolic changes were limited to the cardiac tissue indicating that cardiomyocyte metabolic remodelling is not sufficient to evoke the body wide metabolic alterations usually associated with HF.

Whole grain intake associated molecule 5-aminovaleric acid betaine decreases ß-oxidation of fatty acids in mouse cardiomyocytes.

Despite epidemiological evidence showing that diets rich in whole grains reduce the risk of chronic life-style related diseases, biological mechanisms for these positive effects are mostly unknown. Increased 5-aminovaleric acid betaine (5-AVAB) levels in plasma and metabolically active tissues such as heart have been associated with consumption of diets rich in whole grains. However, biological effects of 5-AVAB are poorly understood. We evaluated 5-AVAB concentrations in human and mouse heart tissue (3-22 µM and 38-78 µM, respectively) using mass spectrometry. We show that 5-AVAB, at physiological concentration range, dose-dependently inhibits oxygen consumption due to ß-oxidation of fatty acids, but does not otherwise compromise mitochondrial respiration, as measured with oxygen consumption rate in cultured mouse primary cardiomyocytes. We also demonstrate that this effect is caused by 5-AVAB induced reduction of cellular L-carnitine. Reduced L-carnitine levels are at least partly mediated by the inhibition of cell membrane carnitine transporter (OCTN2) as evaluated by in silico docking, and by siRNA mediated silencing of OCTN2 in cultured cardiomyocytes. 5-AVAB caused inhibition of ß-oxidation of fatty acids is a novel mechanism on how diets rich in whole grains may regulate energy metabolism in the body. Elucidating potentially beneficial effects of 5-AVAB e.g. on cardiac physiology will require further in vivo investigations.

Structural Immaturity of Human iPSC-Derived Cardiomyocytes: In Silico Investigation of Effects on Function and Disease Modeling.

Background: Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have emerged as a promising experimental tool for translational heart research and drug development. However, their usability as a human adult cardiomyocyte model is limited by their functional immaturity. Our aim is to analyse quantitatively those characteristics and how they differ from adult CMs. Methods and Results: We have developed a novel in silico model with all essential functional electrophysiology and calcium handling features of hiPSC-CMs. Importantly, the virtual cell recapitulates the immature intracellular ion dynamics that are characteristic for hiPSC-CMs, as quantified based our in vitro imaging data. The strong "calcium clock" is a source for a dual function of excitation-contraction coupling in hiPSC-CMs: action potential and calcium transient morphology vary substantially depending on the activation sequence of underlying ionic currents and fluxes that is altered in spontaneous vs. paced mode. Furthermore, parallel simulations with hiPSC-CM and adult cardiomyocyte models demonstrate the central differences. Results indicate that hiPSC-CMs translate poorly the disease specific phenotypes of Brugada syndrome, long QT Syndrome and catecholaminergic polymorphic ventricular tachycardia, showing less robustness and greater tendency for arrhythmic events than adult CMs. Based on a comparative sensitivity analysis, hiPSC-CMs share some features with adult CMs, but are still functionally closer to prenatal CMs than adult CMs. A database analysis of 3000 hiPSC-CM model variants suggests that hiPSC-CMs recapitulate poorly fundamental physiological properties of adult CMs. Single modifications do not appear to solve this problem, which is mostly contributed by the immaturity of intracellular calcium handling. Conclusion: Our data indicates that translation of findings from hiPSC-CMs to human disease should be made with great caution. Furthermore, we established a mathematical platform that can be used to improve the translation from hiPSC-CMs to human, and to quantitatively evaluate hiPSC-CMs development toward more general and valuable model for human cardiac diseases.