Ellen Dirkx

The hypoxia-inducible microRNA cluster miR-199a∼214 targets myocardial PPARδ and impairs mitochondrial fatty acid oxidation.

Peroxisome proliferator-activated receptor δ (PPARδ) is a critical regulator of energy metabolism in the heart. Here, we propose a mechanism that integrates two deleterious characteristics of heart failure, hypoxia and a metabolic shift toward glycolysis, involving the microRNA cluster miR-199a∼214 and PPARδ. We demonstrate that under hemodynamic stress, cardiac hypoxia activates DNM3os, a noncoding transcript that harbors the microRNA cluster miR-199a∼214, which shares PPARδ as common target. To address the significance of miR-199a∼214 induction and concomitant PPARδ repression, we performed antagomir-based silencing of both microRNAs and subjected mice to biomechanical stress to induce heart failure. Remarkably, antagomir-treated animals displayed improved cardiac function and restored mitochondrial fatty acid oxidation. Taken together, our data suggest a mechanism whereby miR-199a∼214 actively represses cardiac PPARδ expression, facilitating a metabolic shift from predominant reliance on fatty acid utilization in the healthy myocardium toward increased reliance on glucose metabolism at the onset of heart failure.

Regulation of fetal gene expression in heart failure

During the processes leading to adverse cardiac remodeling and heart failure, cardiomyocytes react to neurohumoral stimuli and biomechanical stress by activating pathways that induce pathological hypertrophy. The gene expression patterns and molecular changes observed during cardiac hypertrophic remodeling bare resemblance to those observed during fetal cardiac development. The re-activation of fetal genes in the adult failing heart is a complex biological process that involves transcriptional, posttranscriptional and epigenetic regulation of the cardiac genome. In this review, the mechanistic actions of transcription factors, microRNAs and chromatin remodeling processes in regulating fetal gene expression in heart failure are discussed.

Nfat and miR-25 cooperate to reactivate the transcription factor Hand2 in heart failure

Although aberrant reactivation of embryonic gene programs is intricately linked to pathological heart disease, the transcription factors driving these gene programs remain ill-defined. Here we report that increased calcineurin/Nfat signalling and decreased miR-25 expression integrate to re-express the basic helix-loop-helix (bHLH) transcription factor dHAND (also known as Hand2) in the diseased human and mouse myocardium. In line, mutant mice overexpressing Hand2 in otherwise healthy heart muscle cells developed a phenotype of pathological hypertrophy. Conversely, conditional gene-targeted Hand2 mice demonstrated a marked resistance to pressure-overload-induced hypertrophy, fibrosis, ventricular dysfunction and induction of a fetal gene program. Furthermore, in vivo inhibition of miR-25 by a specific antagomir evoked spontaneous cardiac dysfunction and sensitized the murine myocardium to heart failure in a Hand2-dependent manner. Our results reveal that signalling cascades integrate with microRNAs to induce the expression of the bHLH transcription factor Hand2 in the postnatal mammalian myocardium with impact on embryonic gene programs in heart failure.

Requirement for distinct vesicle-associated membrane proteins in insulin- and AMP-activated protein kinase (AMPK)-induced translocation of GLUT4 and CD36 in cultured cardiomyocytes

Aims/hypothesisUpon stimulation of insulin signalling or contraction-induced AMP-activated protein kinase (AMPK) activation, the glucose transporter GLUT4 and the long-chain fatty acid (LCFA) transporter CD36 similarly translocate from intracellular compartments to the plasma membrane of cardiomyocytes to increase uptake of glucose and LCFA, respectively. This similarity in regulation of GLUT4 traffic and CD36 traffic suggests that the same families of trafficking proteins, including vesicle-associated membrane proteins (VAMPs), are involved in both processes. While several VAMPs have been implicated in GLUT4 traffic, nothing is known about the putative function of VAMPs in CD36 traffic. Therefore, we compared the involvement of the myocardially produced VAMP isoforms in insulin- or contraction-induced GLUT4 and CD36 translocation.MethodsFive VAMP isoforms were silenced in HL-1 cardiomyocytes. The cells were treated with insulin or the contraction-like AMPK activator oligomycin or were electrically stimulated to contract. Subsequently, GLUT4 and CD36 translocation as well as substrate uptake were measured.ResultsThree VAMPs were demonstrated to be necessary for both GLUT4 and CD36 translocation, either specifically in insulin-treated cells (VAMP2, VAMP5) or in oligomycin/contraction-treated cells (VAMP3). In addition, there are VAMPs specifically involved in either GLUT4 traffic (VAMP7 mediates basal GLUT4 retention) or CD36 traffic (VAMP4 mediates insulin- and oligomycin/contraction-induced CD36 translocation).Conclusions/interpretationThe involvement of distinct VAMP isoforms in both GLUT4 and CD36 translocation indicates that CD36 translocation, just like GLUT4 translocation, is a vesicle-mediated process dependent on soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex formation. The ability of other VAMPs to discriminate between GLUT4 and CD36 translocation allows the notion that myocardial substrate preference can be modulated by these VAMPs.

High fat diet induced diabetic cardiomyopathy

In response to a chronic high plasma concentration of long-chain fatty acids (FAs), the heart is forced to increase the uptake of FA at the cost of glucose. This switch in metabolic substrate uptake is accompanied by an increased presence of the FA transporter CD36 at the cardiac plasma membrane and over time results in the development of cardiac insulin resistance and ultimately diabetic cardiomyopathy. FA can interact with peroxisome proliferator-activated receptors (PPARs), which induce upregulation of the expression of enzymes necessary for their disposal through mitochondrial β-oxidation, but also stimulate FA uptake. This then leads to a further increase in FA concentration in the cytoplasm of cardiomyocytes. These metabolic changes are supposed to play an important role in the development of cardiomyopathy. Although the onset of this pathology is an increased FA utilization by the heart, the subsequent lipid overload results in an increased production of reactive oxygen species (ROS) and accumulation of lipid intermediates such as diacylglycerols (DAG) and ceramide. These compounds have a profound impact on signaling pathways, in particular insulin signaling. Over time the metabolic changes will introduce structural changes that affect cardiac contractile characteristics. The present mini-review will focus on the lipid-induced changes that link metabolic perturbation, characteristic for type 2 diabetes, with cardiac remodeling and dysfunction.

Cardiovascular extracellular microRNAs: emerging diagnostic markers and mechanisms of cell-to-cell RNA communication

Cardiovascular diseases are a leading cause of morbidity and mortality in Western societies. It is now well established that microRNAs (miRNAs) are determinant regulators in various medical conditions including cardiovascular diseases. The recent discovery that miRNAs, while associated with different carriers, can be exported out of the cell, has triggered a renewed interest to analyze the potential to use extracellular miRNAs as tools for diagnostic and therapeutic studies. Circulating miRNAs in biological fluids present a technological advantage compared to current diagnostic tools by virtue of their remarkable stability and relative ease of detection rendering them ideal tools for non-invasive and rapid diagnosis. Extracellular miRNAs also represent a novel form of inter-cellular communication by transferring genetic information from a donor cell to a recipient cell. This review briefly summarizes recent insights in the origin, function and diagnostic potential of extracellular miRNAs by focusing on a select number of cardiovascular diseases.

Protein Kinase D1 Is Essential for Contraction-induced Glucose Uptake but Is Not Involved in Fatty Acid Uptake into Cardiomyocytes

Background: Contraction of cardiomyocytes up-regulates glucose and fatty acid uptake by GLUT4 and CD36 translocation to the sarcolemma. Results: Silencing of protein kinase D1 abolishes contraction-induced GLUT4 but not CD36 translocation. Conclusion: Protein kinase D1 signaling mediates cardiac glucose but not fatty acid uptake. Significance: Selective stimulation of glucose uptake is beneficial for diabetic hearts characterized by elevated fatty acid uptake. Increased contraction enhances substrate uptake into cardiomyocytes via translocation of the glucose transporter GLUT4 and the long chain fatty acid (LCFA) transporter CD36 from intracellular stores to the sarcolemma. Additionally, contraction activates the signaling enzymes AMP-activated protein kinase (AMPK) and protein kinase D1 (PKD1). Although AMPK has been implicated in contraction-induced GLUT4 and CD36 translocation in cardiomyocytes, the precise role of PKD1 in these processes is not known. To study this, we triggered contractions in cardiomyocytes by electric field stimulation (EFS). First, the role of PKD1 in GLUT4 and CD36 translocation was defined. In PKD1 siRNA-treated cardiomyocytes as well as cardiomyocytes from PKD1 knock-out mice, EFS-induced translocation of GLUT4, but not CD36, was abolished. In AMPK siRNA-treated cardiomyocytes and cardiomyocytes from AMPKα2 knock-out mice, both GLUT4 and CD36 translocation were abrogated. Hence, unlike AMPK, PKD1 is selectively involved in glucose uptake. Second, we analyzed upstream factors in PKD1 activation. Cardiomyocyte contractions enhanced reactive oxygen species (ROS) production. Using ROS scavengers, we found that PKD1 signaling and glucose uptake are more sensitive to changes in intracellular ROS than AMPK signaling or LCFA uptake. Furthermore, silencing of death-activated protein kinase (DAPK) abrogated EFS-induced GLUT4 but not CD36 translocation. Finally, possible links between PKD1 and AMPK signaling were investigated. PKD1 silencing did not affect AMPK activation. Reciprocally, AMPK silencing did not alter PKD1 activation. In conclusion, we present a novel contraction-induced ROS-DAPK-PKD1 pathway in cardiomyocytes. This pathway is activated separately from AMPK and mediates GLUT4 translocation/glucose uptake, but not CD36 translocation/LCFA uptake.

Overexpression of Vesicle-associated Membrane Protein (VAMP) 3, but Not VAMP2, Protects Glucose Transporter (GLUT) 4 Protein Translocation in an in Vitro Model of Cardiac Insulin Resistance

Background: GLUT4 translocation in cardiomyocytes is impaired during insulin resistance leading to insufficient glucose supply and eventually heart failure. Results: Cardiomyocytes overexpressing VAMP3 maintain full insulin-stimulated GLUT4 translocation and do not accumulate intramyocellular lipids. Conclusion: Overexpression of VAMP3 protects cardiac glucose metabolism under conditions of impaired insulin sensitivity. Significance: These data indicate a mechanism how contraction signaling improves insulin-dependent GLUT4 translocation. Cardiac glucose utilization is regulated by reversible translocation of the glucose transporter GLUT4 from intracellular stores to the plasma membrane. During the onset of diet-induced insulin resistance, elevated lipid levels in the circulation interfere with insulin-stimulated GLUT4 translocation, leading to impaired glucose utilization. Recently, we identified vesicle-associated membrane protein (VAMP) 2 and 3 to be required for insulin- and contraction-stimulated GLUT4 translocation, respectively, in cardiomyocytes. Here, we investigated whether overexpression of VAMP2 and/or VAMP3 could protect insulin-stimulated GLUT4 translocation under conditions of insulin resistance. HL-1 atrial cardiomyocytes transiently overexpressing either VAMP2 or VAMP3 were cultured for 16 h with elevated concentrations of palmitate and insulin. Upon subsequent acute stimulation with insulin, we measured GLUT4 translocation, plasmalemmal presence of the fatty acid transporter CD36, and myocellular lipid accumulation. Overexpression of VAMP3, but not VAMP2, completely prevented lipid-induced inhibition of insulin-stimulated GLUT4 translocation. Furthermore, the plasmalemmal presence of CD36 and intracellular lipid levels remained normal in cells overexpressing VAMP3. However, insulin signaling was not retained, indicating an effect of VAMP3 overexpression downstream of PKB/Akt. Furthermore, we revealed that endogenous VAMP3 is bound by the contraction-activated protein kinase D (PKD), and contraction and VAMP3 overexpression protect insulin-stimulated GLUT4 translocation via a common mechanism. These observations indicate that PKD activates GLUT4 translocation via a VAMP3-dependent trafficking step, which pathway might be valuable to rescue constrained glucose utilization in the insulin-resistant heart.

Non-coding RNA in control of gene regulatory programs in cardiac development and disease.

Organogenesis of the vertebrate heart is a highly specialized process involving progressive specification and differentiation of distinct embryonic cardiac progenitor cell populations driven by specialized gene programming events. Likewise, the onset of pathologies in the adult heart, including cardiac hypertrophy, involves the reactivation of embryonic gene programs. In both cases, these intricate genomic events are temporally and spatially regulated by complex signaling networks and gene regulatory networks. Apart from well-established transcriptional mechanisms, increasing evidence indicates that gene programming in both the developing and the diseased myocardium are under epigenetic control by non-coding RNAs (ncRNAs). MicroRNAs regulate gene expression at the post-transcriptional level, and numerous studies have now established critical roles for this species of tiny RNAs in a broad range of aspects from cardiogenesis towards adult heart failure. Recent reports now also implicate the larger family of long non-coding RNAs (lncRNAs) in these processes as well. Here we discuss the involvement of these two ncRNA classes in proper cardiac development and hypertrophic disease processes of the adult myocardium. This article is part of a Special Issue entitled: Non-coding RNAs.

Protein kinase-D1 overexpression prevents lipid-induced cardiac insulin resistance.

In the insulin resistant heart, energy fuel selection shifts away from glucose utilization towards almost complete dependence on long-chain fatty acids (LCFA). This shift results in excessive cardiac lipid accumulation and eventually heart failure. Lipid-induced cardiomyopathy may be averted by strategies that increase glucose uptake without elevating LCFA uptake. Protein kinase-D1 (PKD1) is involved in contraction-induced glucose, but not LCFA, uptake allowing to hypothesize that this kinase is an attractive target to treat lipid-induced cardiomyopathy. For this, cardiospecific constitutively active PKD1 overexpression (caPKD1)-mice were subjected to an insulin resistance-inducing high fat-diet for 20-weeks. Substrate utilization was assessed by microPET and cardiac function by echocardiography. Cardiomyocytes were isolated for measurement of substrate uptake, lipid accumulation and insulin sensitivity. Wild-type mice on a high fat-diet displayed increased basal myocellular LCFA uptake, increased lipid deposition, greatly impaired insulin signaling, and loss of insulin-stimulated glucose and LCFA uptake, which was associated with concentric hypertrophic remodeling. The caPKD1 mice on high-fat diet showed none of these characteristics, whereas on low-fat diet a shift towards cardiac glucose utilization in combination with hypertrophy and ventricular dilation was observed. In conclusion, these data suggest that PKD pathway activation may be an attractive therapeutic strategy to mitigate lipid accumulation, insulin resistance and maladaptive remodeling in the lipid-overloaded heart, but this requires further investigation.