Publications by authors named "Pasi Tavi"

66 Publications

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

Expert Opin Ther Targets 2021 Oct 12. Epub 2021 Oct 12.

Department of Ophthalmology, Institute of Clinical Medicine, University of Eastern Finland, Kuopio, Finland.

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.
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http://dx.doi.org/10.1080/14728222.2021.1991913DOI Listing
October 2021

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

Cardiovasc Res 2021 Jun 4. Epub 2021 Jun 4.

A.I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, Kuopio, Finland.

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 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 heart failure 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.

Translational Perspective: Biological sex is an important variable in clinical medicine, cardiac physiology, and pathogenesis. However, sex-specific clinical practices or therapies are emerging slowly in the absence of deeper understanding of the specific mechanism behind sex dimorphism in cardiac disease progression. Here, we show that energy metabolism has a central role in sex dimorphism of heart failure progression and that a signalling cascade involving PGC-1α might have a role in it. We provide insights into sex specific mechanisms of heart failure development which are necessary to identify sex specific treatment practices for cardiovascular diseases.
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http://dx.doi.org/10.1093/cvr/cvab188DOI Listing
June 2021

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

Biomolecules 2021 03 17;11(3). Epub 2021 Mar 17.

A.I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, 70211 Kuopio, Finland.

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.
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http://dx.doi.org/10.3390/biom11030452DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8002705PMC
March 2021

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

JBMR Plus 2020 Nov 26;4(11):e10397. Epub 2020 Aug 26.

Research Unit of Biomedicine, Pharmacology and Toxicology University of Oulu Oulu Finland.

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. published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.
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http://dx.doi.org/10.1002/jbm4.10397DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7657391PMC
November 2020

Metabolic alterations in Parkinson's disease astrocytes.

Sci Rep 2020 09 2;10(1):14474. Epub 2020 Sep 2.

A.I.Virtanen Institute for Molecular Sciences, University of Eastern Finland, Neulaniementie 2, 70211, Kuopio, Finland.

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 Ca 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.
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http://dx.doi.org/10.1038/s41598-020-71329-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7468111PMC
September 2020

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

Cardiovasc Res 2021 04;117(5):1339-1357

A. I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, 70211 Kuopio, Finland.

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.
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http://dx.doi.org/10.1093/cvr/cvaa219DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8064437PMC
April 2021

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

Physiol Rep 2020 07;8(13):e14474

A.I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, Kuopio, Finland.

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.
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http://dx.doi.org/10.14814/phy2.14474DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7343666PMC
July 2020

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

Oncogene 2019 12 23;38(50):7473-7490. Epub 2019 Aug 23.

Department of Biotechnology and Molecular Medicine, A.I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, P.O. Box 1627, FIN-70211, Kuopio, Finland.

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.
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http://dx.doi.org/10.1038/s41388-019-0956-6DOI Listing
December 2019

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

JCI Insight 2019 03 28;5. Epub 2019 Mar 28.

Department of Physiology and Pharmacology, Molecular Muscle Physiology and Pathophysiology, Karolinska Institutet, Stockholm, Sweden.

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.
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http://dx.doi.org/10.1172/jci.insight.126347DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6538353PMC
March 2019

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

Diabetes 2019 05 4;68(5):918-931. Epub 2019 Mar 4.

Research Unit of Biomedicine, Pharmacology and Toxicology, University of Oulu, Oulu, Finland

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.
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http://dx.doi.org/10.2337/db18-1050DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6477896PMC
May 2019

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

Redox Biol 2019 01 14;20:1-12. Epub 2018 Sep 14.

Arnold and Mabel Beckman Macular Research Center, Doheny Eye Institute, Los Angeles, CA, USA.

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.
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http://dx.doi.org/10.1016/j.redox.2018.09.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6156745PMC
January 2019

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

Sci Rep 2018 08 29;8(1):13036. Epub 2018 Aug 29.

Institute of Public Health and Clinical Nutrition, University of Eastern Finland, P.O. Box 1627, FI-70211, Kuopio, Finland.

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 β-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 β-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.
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http://dx.doi.org/10.1038/s41598-018-31484-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6115339PMC
August 2018

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

Cardiovasc Res 2019 01;115(1):107-118

A.I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, Neulaniementie 2, Kuopio, Finland.

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.
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http://dx.doi.org/10.1093/cvr/cvy155DOI Listing
January 2019

Complex role of peroxisome proliferator activator receptors (PPARs) in nociception.

Scand J Pain 2015 Oct 1;9(1):70-71. Epub 2015 Oct 1.

A.I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, Neulaniementie 2, Kuopio 70211, Kuopio, Finland.

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http://dx.doi.org/10.1016/j.sjpain.2015.08.006DOI Listing
October 2015

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

Front Physiol 2018 7;9:80. Epub 2018 Feb 7.

A.I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, Kuopio, Finland.

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. We have developed a novel 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 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. 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.
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http://dx.doi.org/10.3389/fphys.2018.00080DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5808345PMC
February 2018

Sarcoplasmic reticulum Ca -induced Ca release regulates class IIa HDAC localization in mouse embryonic cardiomyocytes.

Physiol Rep 2018 01;6(2)

Department of Biotechnology and Molecular Medicine, A.I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, Kuopio, Finland.

In embryonic cardiomyocytes, sarcoplasmic reticulum (SR)-derived Ca release is required to induce Ca oscillations for contraction and to control cardiac development through Ca -activated pathways. Here, our aim was to study how SR Ca release regulates cytosolic and nuclear Ca distribution and the subsequent effects on the Ca -dependent localization of class IIa histone deacetylases (HDAC) and cardiac-specific gene expression in embryonic cardiomyocytes. Confocal microscopy was used to study changes in Ca -distribution and localization of immunolabeled HDAC4 and HDAC5 upon changes in SR Ca release in mouse embryonic cardiomyocytes. Dynamics of translocation were also observed with a confocal microscope, using HDAC5-green fluorescent protein transfected myocytes. Expression of class IIa HDACs in differentiating myocytes and changes in cardiac-specific gene expression were studied using real-time quantitative PCR. Inhibition of SR Ca release caused a significant decrease in intranuclear Ca concentration, a rapid nuclear import of HDAC5 and subnuclear redistribution of HDAC4. Endogenous localization of HDAC5 and HDAC4 was mostly cytosolic and at the nuclear periphery, respectively. Downregulated expression of cardiac-specific genes was also observed upon SR Ca release inhibition. Electrical stimulation of sarcolemmal Ca influx was not sufficient to rescue either the HDAC localization or the gene expression changes. SR Ca release controls subcellular Ca distribution and regulates localization of HDAC4 and HDAC5 in embryonic cardiomyocytes. Changes in SR Ca release also caused changes in expression of the developmental phase-specific genes, which may be due to the changes in HDAC-localization.
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http://dx.doi.org/10.14814/phy2.13522DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5789715PMC
January 2018

NRF2 regulates endothelial glycolysis and proliferation with miR-93 and mediates the effects of oxidized phospholipids on endothelial activation.

Nucleic Acids Res 2018 02;46(3):1124-1138

A.I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, 70211 Kuopio, Finland.

Phospholipids, such as 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (PAPC), are the major components of cell membranes. Their exposure to reactive oxygen species creates oxidized phospholipids, which predispose to the development of chronic inflammatory diseases and metabolic disorders through endothelial activation and dysfunction. Although the effects of oxidized PAPC (oxPAPC) on endothelial cells have been previously studied, the underlying molecular mechanisms evoking biological responses remain largely unknown. Here, we investigated the molecular mechanisms of oxPAPC function with a special emphasis on NRF2-regulated microRNAs (miRNAs) in human umbilical vein endothelial cells (HUVECs) utilizing miRNA profiling, global run-on sequencing (GRO-seq), genome-wide NRF2 binding model, and RNA sequencing (RNA-seq) with miRNA overexpression and silencing. We report that the central regulators of endothelial activity, KLF2 for quiescence, PFKFB3 for glycolysis, and VEGFA, FOXO1 and MYC for growth and proliferation, are regulated by transcription factor NRF2 and the NRF2-regulated miR-106b∼25 cluster member, miR-93, in HUVECs. Mechanistically, oxPAPC was found to induce glycolysis and proliferation NRF2-dependently, and oxPAPC-dependent induction of the miR-106b∼25 cluster was mediated by NRF2. Additionally, several regulatory loops were established between NRF2, miR-93 and the essential regulators of healthy endothelium, collectively implying that NRF2 controls the switch between the quiescent and the proliferative endothelial states together with miR-93.
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http://dx.doi.org/10.1093/nar/gkx1155DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5815049PMC
February 2018

PSEN1 Mutant iPSC-Derived Model Reveals Severe Astrocyte Pathology in Alzheimer's Disease.

Stem Cell Reports 2017 12 16;9(6):1885-1897. Epub 2017 Nov 16.

A.I.Virtanen Institute for Molecular Sciences, University of Eastern Finland, 70210 Kuopio, Finland. Electronic address:

Alzheimer's disease (AD) is a common neurodegenerative disorder and the leading cause of cognitive impairment. Due to insufficient understanding of the disease mechanisms, there are no efficient therapies for AD. Most studies have focused on neuronal cells, but astrocytes have also been suggested to contribute to AD pathology. We describe here the generation of functional astrocytes from induced pluripotent stem cells (iPSCs) derived from AD patients with PSEN1 ΔE9 mutation, as well as healthy and gene-corrected isogenic controls. AD astrocytes manifest hallmarks of disease pathology, including increased β-amyloid production, altered cytokine release, and dysregulated Ca homeostasis. Furthermore, due to altered metabolism, AD astrocytes show increased oxidative stress and reduced lactate secretion, as well as compromised neuronal supportive function, as evidenced by altering Ca transients in healthy neurons. Our results reveal an important role for astrocytes in AD pathology and highlight the strength of iPSC-derived models for brain diseases.
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http://dx.doi.org/10.1016/j.stemcr.2017.10.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5785689PMC
December 2017

Aggravated Postinfarct Heart Failure in Type 2 Diabetes Is Associated with Impaired Mitophagy and Exaggerated Inflammasome Activation.

Am J Pathol 2017 Dec 19;187(12):2659-2673. Epub 2017 Sep 19.

Department of Biotechnology and Molecular Medicine, A.I. Virtanen Institute, Kuopio University Hospital, Kuopio, Finland; Heart Center and Gene Therapy Unit, Kuopio University Hospital, Kuopio, Finland. Electronic address:

Type 2 diabetes mellitus (T2DM) is a major risk factor for heart disease. Mortality rates after myocardial infarction (MI) are significantly increased in T2DM patients because of dysfunctional left ventricle (LV). However, molecular pathways underlying accelerated heart failure (HF) after MI in T2DM remain unclear. We investigated the underlying mechanisms by inducing MI in a well-established model of T2DM and control mice. Cardiac imaging revealed a significantly decreased global left ventricular ejection fraction in parallel with increased mortality after MI in T2DM mice compared with control mice. Genome-wide mRNA sequencing, immunoblot, electron microscopy, together with immunofluorescence staining for LC3 and p62 indicated an impaired mitophagy in peri-infarct regions of LV in T2DM mice compared with control mice. Furthermore, defective mitophagy was associated with an increased release of mitochondrial DNA, resulting in Aim2 and NLRC4 inflammasome and caspase-I hyperactivation in cardiomyocytes and cardiac macrophages in peri-infarct regions of LV in T2DM mice. Consistent with inflammasome and caspase-I hyperactivation, cardiomyocyte death and IL-18 secretion were increased in T2DM mice. Our results indicate that T2DM aggravates HF after MI through defective mitophagy, associated exaggerated inflammasome activation, cell death, and IL-18 secretion, suggesting that restoring mitophagy and inhibiting inflammasome activation may serve as novel targets for the prevention and treatment of HF in T2DM.
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http://dx.doi.org/10.1016/j.ajpath.2017.08.023DOI Listing
December 2017

Loss of causes altered neurogenesis in a mouse model of a childhood neurodegenerative disorder.

Dis Model Mech 2017 09 21;10(9):1089-1100. Epub 2017 Jul 21.

A.I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, 70211 Kuopio, Finland

Neural stem/progenitor cells (NPCs) generate new neurons in the brain throughout an individual's lifetime in an intricate process called neurogenesis. Neurogenic alterations are a common feature of several adult-onset neurodegenerative diseases. The neuronal ceroid lipofuscinoses (NCLs) are the most common group of inherited neurodegenerative diseases that mainly affect children. Pathological features of the NCLs include accumulation of lysosomal storage material, neuroinflammation and neuronal degeneration, yet the exact cause of this group of diseases remains poorly understood. The function of the CLN5 protein, causative of the CLN5 disease form of NCL, is unknown. In the present study, we sought to examine neurogenesis in the neurodegenerative disorder caused by loss of Our findings demonstrate a newly identified crucial role for CLN5 in neurogenesis. We report for the first time that neurogenesis is increased in -deficient mice, which model the childhood neurodegenerative disorder caused by loss of Our results demonstrate that, in deficiency, proliferation of NPCs is increased, NPC migration is reduced and NPC differentiation towards the neuronal lineage is increased concomitantly with functional alterations in the NPCs. Moreover, the observed impairment in neurogenesis is correlated with increased expression of the pro-inflammatory cytokine IL-1β. A full understanding of the pathological mechanisms that lead to disease and the function of the NCL proteins are critical for designing effective therapeutic approaches for this devastating neurodegenerative disorder.
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http://dx.doi.org/10.1242/dmm.029165DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5611964PMC
September 2017

Vascular Endothelial Growth Factor-B Induces a Distinct Electrophysiological Phenotype in Mouse Heart.

Front Physiol 2017 31;8:373. Epub 2017 May 31.

A.I. Virtanen Institute for Molecular Sciences, University of Eastern FinlandKuopio, Finland.

Vascular endothelial growth factor B (VEGF-B) is a potent mediator of vascular, metabolic, growth, and stress responses in the heart, but the effects on cardiac muscle and cardiomyocyte function are not known. The purpose of this study was to assess the effects of VEGF-B on the energy metabolism, contractile, and electrophysiological properties of mouse cardiac muscle and cardiac muscle cells. and analysis of cardiac-specific VEGF-B TG mice indicated that the contractile function of the TG hearts was normal. Neither the oxidative metabolism of isolated TG cardiomyocytes nor their energy substrate preference showed any difference to WT cardiomyocytes. Similarly, myocyte Ca signaling showed only minor changes compared to WT myocytes. However, VEGF-B overexpression induced a distinct electrophysiological phenotype characterized by ECG changes such as an increase in QRSp time and decreases in S and R amplitudes. At the level of isolated TG cardiomyocytes, these changes were accompanied with decreased action potential upstroke velocity and increased duration (APD). These changes were partly caused by downregulation of sodium current (I) due to reduced expression of Na1.5. Furthermore, TG myocytes had alterations in voltage-gated K currents, namely decreased density of transient outward current (I) and total K current (I). At the level of transcription, these were accompanied by downregulation of K channel-interacting protein 2 (), a known modulatory subunit for K4.2/3 channel. Cardiac VEGF-B overexpression induces a distinct electrophysiological phenotype including remodeling of cardiomyocyte ion currents, which in turn induce changes in action potential waveform and ECG.
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http://dx.doi.org/10.3389/fphys.2017.00373DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5450225PMC
May 2017

Genome-Wide Dynamics of Nascent Noncoding RNA Transcription in Porcine Heart After Myocardial Infarction.

Circ Cardiovasc Genet 2017 Jun;10(3)

From the Department of Biotechnology and Molecular Medicine, A.I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, Kuopio (M.U.K., P.H., O.H.-F.L., T.T., J.P., P.M., I.S., T.T., E.A., P.T., S.Y.-H.); and Heart Center and Gene Therapy Unit, Kuopio University Hospital, Finland (S.Y.-H.).

Background: Microarrays and RNA sequencing are widely used to profile transcriptome remodeling during myocardial ischemia. However, the steady-state RNA analysis lacks in sensitivity to detect all noncoding RNA species and does not provide separation between transcriptional and post-transcriptional regulations. Here, we provide the first comprehensive analysis of nascent RNA profiles of mRNAs, primary micro-RNAs, long noncoding RNAs, and enhancer RNAs in a large animal model of acute infarction.

Methods And Results: Acute infarction was induced by cardiac catheterization of domestic swine. Nuclei isolated from healthy, border zone, and ischemic regions of the affected heart were subjected to global run-on sequencing. Global run-on sequencing analysis indicated that half of affected genes are regulated at the level of transcriptional pausing. A gradient of induction of inflammatory mediators and repression of peroxisome proliferator-activated receptor signaling and oxidative phosphorylation was detected when moving from healthy toward infarcted area. In addition, we interrogated the transcriptional regulation of primary micro-RNAs and provide evidence that several arrhythmia-related target genes exhibit repression at post-transcriptional level. We identified 450 long noncoding RNAs differently regulated by ischemia, including novel conserved long noncoding RNAs expressed in antisense orientation to myocardial transcription factors GATA-binding protein 4, GATA-binding protein 6, and Krüppel-like factor 6. Finally, characterization of enhancers exhibiting differential expression of enhancer RNAs pointed a central role for Krüppel-like factor, MEF2C, ETS, NFY, ATF, E2F2, and NRF1 transcription factors in determining transcriptional responses to ischemia.

Conclusions: Global run-on sequencing allowed us to follow the gradient of gene expression occurring in the ischemic heart and identify novel noncoding RNAs regulated by oxygen deprivation. These findings highlight potential new targets for diagnosis and treatment of myocardial ischemia.
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http://dx.doi.org/10.1161/CIRCGENETICS.117.001702DOI Listing
June 2017

The role of cardiac energy metabolism in cardiac hypertrophy and failure.

Exp Cell Res 2017 11 24;360(1):12-18. Epub 2017 Mar 24.

A.I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, Kuopio, Finland. Electronic address:

In mammalian heart, incessant production of cellular energy is vital for maintaining continuous mechanical pumping function providing the body for oxygen and nutrients. To ensure this essential function, cardiac muscle adapt to increased energy demand or compromised energy supply by reprogramming the network of genes whose products are necessary to match the production of energy to consumption. Failure in this regulation leads to severe cardiac dysfunction and has been associated with cardiac pathogenesis including cardiac hypertrophy, failure and diabetes. Metabolic adaptations are induced by network of transcriptional pathways that are activated by a variety of factors such as hormones, nutrients, second messengers and oxygen. The metabolic phenotype of the heart is maintained by pathways controlling transcriptional regulators, which include peroxisome proliferator-activated receptors, estrogen-related receptors and nuclear respiratory factors, as well as their common coactivator protein peroxisome proliferator-activated receptor γ coactivator 1. These central regulators of gene expression are complemented with factors such as hypoxia inducible factor 1, which is activated in insufficient oxygenation of the tissue. Here, we discuss how these pathways relate to the cardiac metabolism and how they interact with pathways controlling the contractile phenotype of the heart.
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http://dx.doi.org/10.1016/j.yexcr.2017.03.052DOI Listing
November 2017

Refractoriness in human atria: Time and voltage dependence of sodium channel availability.

J Mol Cell Cardiol 2016 Dec 20;101:26-34. Epub 2016 Oct 20.

Center for Cardiological Innovation and Center for Biomedical Computing, Simula Research Laboratory, Oslo, Norway; Department of Biotechnology and Molecular Medicine, A.I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, Kuopio, Finland. Electronic address:

Background: Refractoriness of cardiac cells limits maximum frequency of electrical activity and protects the heart from tonic contractions. Short refractory periods support major arrhythmogenic substrates and augmentation of refractoriness is therefore seen as a main mechanism of antiarrhythmic drugs. Cardiomyocyte excitability depends on availability of sodium channels, which involves both time- and voltage-dependent recovery from inactivation. This study therefore aims to characterise how sodium channel inactivation affects refractoriness in human atria.

Methods And Results: Steady-state activation and inactivation parameters of sodium channels measured in vitro in isolated human atrial cardiomyocytes were used to parameterise a mathematical human atrial cell model. Action potential data were acquired from human atrial trabeculae of patients in either sinus rhythm or chronic atrial fibrillation. The ex vivo measurements of action potential duration, effective refractory period and resting membrane potential were well-replicated in simulations using this new in silico model. Notably, the voltage threshold potential at which refractoriness was observed was not different between sinus rhythm and chronic atrial fibrillation tissues and was neither affected by changes in frequency (1 vs. 3Hz).

Conclusions: Our results suggest a preferentially voltage-dependent, rather than time-dependent, effect with respect to refractoriness at physiologically relevant rates in human atria. However, as the resting membrane potential is hyperpolarized in chronic atrial fibrillation, the voltage-dependence of excitability dominates, profoundly increasing the risk for arrhythmia re-initiation and maintenance in fibrillating atria. Our results thereby highlight resting membrane potential as a potential target in pharmacological management of chronic atrial fibrillation.
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http://dx.doi.org/10.1016/j.yjmcc.2016.10.009DOI Listing
December 2016

Peroxisome proliferator-activated receptor-γ coactivator 1 α1 induces a cardiac excitation-contraction coupling phenotype without metabolic remodelling.

J Physiol 2016 12;594(23):7049-7071

Department of Biotechnology and Molecular Medicine, A.I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, Kuopio, Finland.

Key Points: Transcriptional co-activator PGC-1α1 has been shown to regulate energy metabolism and to mediate metabolic adaptations in pathological and physiological cardiac hypertrophy but other functional implications of PGC-1α1 expression are not known. Transgenic PGC-1α1 overexpression within the physiological range in mouse heart induces purposive changes in contractile properties, electrophysiology and calcium signalling but does not induce substantial metabolic remodelling. The phenotype of the PGC-1α1 transgenic mouse heart recapitulates most of the functional modifications usually associated with the exercise-induced heart phenotype, but does not protect the heart against load-induced pathological hypertrophy. Transcriptional effects of PGC-1α1 show clear dose-dependence with diverse changes in genes in circadian clock, heat shock, excitability, calcium signalling and contraction pathways at low overexpression levels, while metabolic genes are recruited at much higher PGC-1α1 expression levels. These results imply that the physiological role of PGC-1α1 is to promote a beneficial excitation-contraction coupling phenotype in the heart.

Abstract: The transcriptional coactivator PGC-1α1 has been identified as a central factor mediating metabolic adaptations of the heart. However, to what extent physiological changes in PGC-1α1 expression levels actually contribute to the functional adaptation of the heart is still mostly unresolved. The aim of this study was to characterize the transcriptional and functional effects of physiologically relevant, moderate PGC-1α1 expression in the heart. In vivo and ex vivo physiological analysis shows that expression of PGC-1α1 within a physiological range in mouse heart does not induce the expected metabolic alterations, but instead induces a unique excitation-contraction (EC) coupling phenotype recapitulating features typically seen in physiological hypertrophy. Transcriptional screening of PGC-1α1 overexpressing mouse heart and myocyte cultures with higher, acute adenovirus-induced PGC-1α1 expression, highlights PGC-1α1 as a transcriptional coactivator with a number of binding partners in various pathways (such as heat shock factors and the circadian clock) through which it acts as a pleiotropic transcriptional regulator in the heart, to both augment and repress the expression of its target genes in a dose-dependent fashion. At low levels of overexpression PGC-1α1 elicits a diverse transcriptional response altering the expression state of circadian clock, heat shock, excitability, calcium signalling and contraction pathways, while metabolic targets of PGC-1α1 are recruited at higher PGC-1α1 expression levels. Together these findings demonstrate that PGC-1α1 elicits a dual effect on cardiac transcription and phenotype. Further, our results imply that the physiological role of PGC-1α1 is to promote a beneficial EC coupling phenotype in the heart.
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http://dx.doi.org/10.1113/JP272847DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5134381PMC
December 2016

Potassium Channel Interacting Protein 2 (KChIP2) is not a transcriptional regulator of cardiac electrical remodeling.

Sci Rep 2016 06 28;6:28760. Epub 2016 Jun 28.

Department of Biomedical Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.

The heart-failure relevant Potassium Channel Interacting Protein 2 (KChIP2) augments CaV1.2 and KV4.3. KChIP3 represses CaV1.2 transcription in cardiomyocytes via interaction with regulatory DNA elements. Hence, we tested nuclear presence of KChIP2 and if KChIP2 translocates into the nucleus in a Ca(2+) dependent manner. Cardiac biopsies from human heart-failure patients and healthy donor controls showed that nuclear KChIP2 abundance was significantly increased in heart failure; however, this was secondary to a large variation of total KChIP2 content. Administration of ouabain did not increase KChIP2 content in nuclear protein fractions in anesthetized mice. KChIP2 was expressed in cell lines, and Ca(2+) ionophores were applied in a concentration- and time-dependent manner. The cell lines had KChIP2-immunoreactive protein in the nucleus in the absence of treatments to modulate intracellular Ca(2+) concentration. Neither increasing nor decreasing intracellular Ca(2+) concentrations caused translocation of KChIP2. Microarray analysis did not identify relief of transcriptional repression in murine KChIP2(-/-) heart samples. We conclude that although there is a baseline presence of KChIP2 in the nucleus both in vivo and in vitro, KChIP2 does not directly regulate transcriptional activity. Moreover, the nuclear transport of KChIP2 is not dependent on Ca(2+). Thus, KChIP2 does not function as a conventional transcription factor in the heart.
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http://dx.doi.org/10.1038/srep28760DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4923891PMC
June 2016

Generation of Functional Neuromuscular Junctions from Human Pluripotent Stem Cell Lines.

Front Cell Neurosci 2015 8;9:473. Epub 2015 Dec 8.

Department of Neurobiology, A.I. Virtanen Institute for Molecular Sciences, University of Eastern Finland Kuopio, Finland.

Several neuromuscular diseases involve dysfunction of neuromuscular junctions (NMJs), yet there are no patient-specific human models for electrophysiological characterization of NMJ. We seeded cells of neurally-induced embryoid body-like spheres derived from induced pluripotent stem cell (iPSC) or embryonic stem cell (ESC) lines as monolayers without basic fibroblast factor (bFGF) and observed differentiation of neuronal as well as spontaneously contracting, multinucleated skeletal myotubes. The myotubes showed striation, immunoreactivity for myosin heavy chain, actin bundles typical for myo-oriented cells, and generated spontaneous and evoked action potentials (APs). The myogenic differentiation was associated with expression of MyoD1, myogenin and type I ryanodine receptor. Neurons formed end plate like structures with strong binding of α-bungarotoxin, a marker of nicotinic acetylcholine receptors highly expressed in the postsynaptic membrane of NMJs, and expressed SMI-32, a motoneuron marker, as well as SV2, a marker for synapses. Pharmacological stimulation of cholinergic receptors resulted in strong depolarization of myotube membrane and raised Ca(2+) concentration in sarcoplasm, while electrical stimulation evoked Ca(2+) transients in myotubes. Stimulation of motoneurons with N-Methyl-D-aspartate resulted in reproducible APs in myotubes and end plates displayed typical mEPPs and tonic activity depolarizing myotubes of about 10 mV. We conclude that simultaneous differentiation of neurons and myotubes from patient-specific iPSCs or ESCs results also in the development of functional NMJs. Our human model of NMJ may serve as an important tool to investigate normal development, mechanisms of diseases and novel drug targets involving NMJ dysfunction and degeneration.
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http://dx.doi.org/10.3389/fncel.2015.00473DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4672046PMC
December 2015

Endothelial Bmx tyrosine kinase activity is essential for myocardial hypertrophy and remodeling.

Proc Natl Acad Sci U S A 2015 Oct 1;112(42):13063-8. Epub 2015 Oct 1.

Wihuri Research Institute and Translational Cancer Biology Program, Biomedicum Helsinki, University of Helsinki, FI-00290 Helsinki, Finland;

Cardiac hypertrophy accompanies many forms of heart disease, including ischemic disease, hypertension, heart failure, and valvular disease, and it is a strong predictor of increased cardiovascular morbidity and mortality. Deletion of bone marrow kinase in chromosome X (Bmx), an arterial nonreceptor tyrosine kinase, has been shown to inhibit cardiac hypertrophy in mice. This finding raised the possibility of therapeutic use of Bmx tyrosine kinase inhibitors, which we have addressed here by analyzing cardiac hypertrophy in gene-targeted mice deficient in Bmx tyrosine kinase activity. We found that angiotensin II (Ang II)-induced cardiac hypertrophy is significantly reduced in mice deficient in Bmx and in mice with inactivated Bmx tyrosine kinase compared with WT mice. Genome-wide transcriptomic profiling showed that Bmx inactivation suppresses myocardial expression of genes related to Ang II-induced inflammatory and extracellular matrix responses whereas expression of RNAs encoding mitochondrial proteins after Ang II administration was maintained in Bmx-inactivated hearts. Very little or no Bmx mRNA was expressed in human cardiomyocytes whereas human cardiac endothelial cells expressed abundant amounts. Ang II stimulation of endothelial cells increased Bmx phosphorylation, and Bmx gene silencing inhibited downstream STAT3 signaling, which has been implicated in cardiac hypertrophy. Furthermore, activation of the mechanistic target of rapamycin complex 1 pathway by Ang II treatment was decreased in the Bmx-deficient hearts. Our results demonstrate that inhibition of the cross-talk between endothelial cells and cardiomyocytes by Bmx inactivation suppresses Ang II-induced signals for cardiac hypertrophy. These results suggest that the endothelial Bmx tyrosine kinase could provide a target to attenuate the development of cardiac hypertrophy.
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http://dx.doi.org/10.1073/pnas.1517810112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4620883PMC
October 2015

WDR12, a Member of Nucleolar PeBoW-Complex, Is Up-Regulated in Failing Hearts and Causes Deterioration of Cardiac Function.

PLoS One 2015 27;10(4):e0124907. Epub 2015 Apr 27.

The Institute of Biomedicine, Department of Pharmacology and Toxicology, University of Oulu, Oulu, Finland; Division of Pharmacology and Pharmacotherapy, University of Helsinki, Helsinki, Finland.

Aims: In a recent genome-wide association study, WD-repeat domain 12 (WDR12) was associated with early-onset myocardial infarction (MI). However, the function of WDR12 in the heart is unknown.

Methods And Results: We characterized cardiac expression of WDR12, used adenovirus-mediated WDR12 gene delivery to examine effects of WDR12 on left ventricular (LV) remodeling, and analyzed relationship between MI associated WDR12 allele and cardiac function in human subjects. LV WDR12 protein levels were increased in patients with dilated cardiomyopathy and rats post-infarction. In normal adult rat hearts, WDR12 gene delivery into the anterior wall of the LV decreased interventricular septum diastolic and systolic thickness and increased the diastolic and systolic diameters of the LV. Moreover, LV ejection fraction (9.1%, P<0.05) and fractional shortening (12.2%, P<0.05) were declined. The adverse effects of WDR12 gene delivery on cardiac function were associated with decreased cellular proliferation, activation of p38 mitogen-activated protein kinase (MAPK)/heat shock protein (HSP) 27 pathway, and increased protein levels of Block of proliferation 1 (BOP1), essential for ribosome biogenesis. Post-infarction WDR12 gene delivery decreased E/A ratio (32%, P<0.05) suggesting worsening of diastolic function. In human subjects, MI associated WDR12 allele was associated significantly with diastolic dysfunction and left atrial size.

Conclusions: WDR12 triggers distinct deterioration of cardiac function in adult rat heart and the MI associated WDR12 variant is associated with diastolic dysfunction in human subjects.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0124907PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4411154PMC
January 2016

MicroRNA profiling of pericardial fluid samples from patients with heart failure.

PLoS One 2015 12;10(3):e0119646. Epub 2015 Mar 12.

Department of Biotechnology and Molecular Medicine, A. I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, Kuopio, Finland.

Aims: Multicellular organisms maintain vital functions through intercellular communication. Release of extracellular vesicles that carry signals to even distant target organs is one way of accomplishing this communication. MicroRNAs can also be secreted from the cells in exosomes and act as paracrine signalling molecules. In addition, microRNAs have been implicated in the pathogenesis of a large number of diseases, including cardiovascular diseases, and are considered as promising candidate biomarkers due to their relative stability and easy quantification from clinical samples. Pericardial fluid contains hormones secreted by the heart and is known to reflect the cardiac function. In this study, we sought to investigate whether pericardial fluid contains microRNAs and if so, whether they could be used to distinguish between different cardiovascular pathologies and disease stages.

Methods And Results: Pericardial fluid was collected from heart failure patients during open-heart surgery. MicroRNA profiles of altogether 51 patients were measured by quantitative real-time PCR (qPCR) using Exiqon human panels I and II. On the average, 256 microRNAs were detected per sample, and 70 microRNAs out of 742 profiled microRNAs were detected in every sample. The five most abundant microRNAs in pericardial fluid were miR-21-5p, miR-451a, miR-125b-5p, let-7b-5p and miR-16-5p. No specific signatures for cardiovascular pathologies or clinically assessed heart failure stages could be detected from the profiles and, overall, microRNA profiles of the samples were found to be very similar despite the heterogeneity in the study population.

Conclusion: Measured microRNA profiles did not separate the samples according to the clinical features of the patients. However, several previously identified heart failure marker microRNAs were detected. The pericardial fluid microRNA profile appeared to be a result of an active and selective secretory process indicating that microRNAs may act as paracrine signalling factors by mediating the local crosstalk between cardiac cells.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0119646PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4357463PMC
November 2015
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