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.

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.

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.

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.

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.

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.

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.

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.

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.

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

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.

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

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.

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

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.

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

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.

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

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.

AAV9-mediated VEGF-B gene transfer improves systolic function in progressive left ventricular hypertrophy.

Mechanisms of the transition from compensatory hypertrophy to heart failure are poorly understood and the roles of vascular endothelial growth factors (VEGFs) in this process have not been fully clarified. We determined the expression profile of VEGFs and relevant receptors during the progression of left ventricular hypertrophy (LVH). C57BL mice were exposed to transversal aortic constriction (TAC) and the outcome was studied at different time points (1 day, 2, 4, and 10 weeks). A clear compensatory phase (2 weeks after TAC) was seen with following heart failure (4 weeks after TAC). Interestingly, VEGF-C and VEGF-D as well as VEGF receptor-3 (VEGFR-3) were upregulated in the compensatory hypertrophy and VEGF-B was downregulated in the heart failure. After treatment with adeno-associated virus serotype 9 (AAV9)-VEGF-B(186) gene therapy in the compensatory phase for 4 weeks the function of the heart was preserved due to angiogenesis, inhibition of apoptosis, and promotion of cardiomyocyte proliferation. Also, the genetic programming towards fetal gene expression, a known phenomenon in heart failure, was partly reversed in AAV9-VEGF-B(186)-treated mice. We conclude that VEGF-C and VEGF-D are associated with the compensatory LVH and that AAV9-VEGF-B(186) gene transfer can rescue the function of the failing heart and postpone the transition towards heart failure.

Ca2+-calmodulin-dependent protein kinase II represses cardiac transcription of the L-type calcium channel alpha(1C)-subunit gene (Cacna1c) by DREAM translocation.

Recent studies have demonstrated that changes in the activity of calcium-calmodulin-dependent protein kinase II (CaMKII) induce a unique cardiomyocyte phenotype through the regulation of specific genes involved in excitation-contraction (E-C)-coupling. To explain the transcriptional effects of CaMKII we identified a novel CaMKII-dependent pathway for controlling the expression of the pore-forming α-subunit (Cav1.2) of the L-type calcium channel (LTCC) in cardiac myocytes. We show that overexpression of either cytosolic (δC) or nuclear (δB) CaMKII isoforms selectively downregulate the expression of the Cav1.2. Pharmacological inhibition of CaMKII activity induced measurable changes in LTCC current density and subsequent changes in cardiomyocyte calcium signalling in less than 24 h. The effect of CaMKII on the α1C-subunit gene (Cacna1c) promoter was abolished by deletion of the downstream regulatory element (DRE), which binds transcriptional repressor DREAM/calsenilin/KChIP3. Imaging DREAM-GFP (green fluorescent protein)-expressing cardiomyocytes showed that CaMKII potentiates the calcium-induced nuclear translocation of DREAM. Thereby CaMKII increases DREAM binding to the DRE consensus sequence of the endogenous Cacna1c gene. By mathematical modelling we demonstrate that the LTCC downregulation through the Ca2+-CaMKII-DREAM cascade constitutes a physiological feedback mechanism enabling cardiomyocytes to adjust the calcium intrusion through LTCCs to the amount of intracellular calcium detected by CaMKII.

Mitochondrial uncoupling downregulates calsequestrin expression and reduces SR Ca2+ stores in cardiomyocytes.

AIMS: Mitochondrial cardiomyopathy is associated with deleterious remodelling of cardiomyocyte Ca(2+) signalling that is partly due to the suppressed expression of the sarcoplasmic reticulum (SR) Ca(2+) buffer calsequestrin (CASQ2). This study was aimed at determining whether CASQ2 downregulation is directly caused by impaired mitochondrial function. METHODS AND RESULTS: Mitochondrial stress was induced in cultured neonatal rat cardiomyocytes by means of the mitochondrial uncoupler carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP). Ca(2+) transients and reactive oxygen species (ROS) were measured by confocal microscopy using the indicators fluo-4 and MitoSOX red, respectively. Mitochondrial stress led to concentration-dependent downregulation of calsequestrin (CASQ2) and changes in the Ca(2+) signals of the cardiomyocytes that were accompanied by reduction in SR Ca(2+) content and amplitude and duration of Ca(2+) sparks. Caspase 3, p38, and p53 inhibitors had no effect on FCCP-induced CASQ2 downregulation; however, it was attenuated by the ROS scavenger N-acetylcysteine (NAC). Importantly, NAC not only decreased FCCP-induced ROS production, but it also restored the Ca(2+) signals, SR Ca(2+) content, and Ca(2+) spark properties to control levels. CONCLUSION: Mitochondrial uncoupling results in fast transcriptional changes in CASQ2 expression that manifest as compromised Ca(2+) signalling, and these changes can be prevented by ROS scavengers. As impaired mitochondrial function has been implicated in several cardiac pathologies as well as in normal ageing, the mechanisms described here might be involved in a wide spectrum of cardiac conditions.

Impaired Ca2+ handling and contraction in cardiomyocytes from mice with a dominant negative thyroid hormone receptor alpha1.

The profound effects of thyroid hormone (TH) on heart development and function are mediated by the thyroid hormone receptors (TR) alpha(1) and beta(1). While numerous patients with TRbeta(1) mutations have been identified, patients with similar mutations in TRalpha(1) are yet to be discovered. Recently generated heterozygous mice with a dominant negative mutation in TRalpha(1) (TRalpha(1)+/m mice) have normal TH levels, which may have hampered the discovery of patients with such mutations. We now measure intracellular Ca(2+) and contraction in cardiomyocytes isolated from TRalpha(1)+/m mice and wildtype littermates (WT). TRalpha(1)+/m cardiomyocytes showed a phenotype similar to that in hypothyroidism with significant slowing of voltage-activated Ca(2+) transients and contractions. Increased stimulation frequency (from 0.5 to 3 Hz) or beta-adrenergic stimulation reduced the differences between TRalpha(1)+/m and WT cardiomyocytes. However, in TRalpha(1)+/m cells stimulation at 3 Hz gave a marked increase in diastolic Ca(2+) and beta-adrenergic stimulation triggered spontaneous Ca(2+) release events during relaxation. Both TRalpha(1)+/m and WT cardiomyocytes responded to TH treatment by displaying a "hyperthyroid" phenotype with faster and larger Ca(2+) transients and contractions. Excised TRalpha(1)+/m hearts showed an increased expression of phospholamban (PLB). In conclusion, isolated TRalpha(1)+/m cardiomyocytes display major dysfunctions with marked slowing of the Ca(2+) transients and contractions.