Leon J. De Windt

MiR423-5p As a Circulating Biomarker for Heart Failure

Rationale: Aberrant expression profiles of circulating microRNAs (miRNAs) have been described in various diseases and provide high sensitivity and specificity. We explored circulating miRNAs as potential biomarkers in patients with heart failure (HF). Objective: The goal of this study was to determine whether miRNAs allow to distinguish clinical HF not only from healthy controls but also from non-HF forms of dyspnea. Methods and Results: A miRNA array was performed on plasma of 12 healthy controls and 12 HF patients. From this array, we selected 16 miRNAs for a second clinical study in 39 healthy controls and in 50 cases with reports of dyspnea, of whom 30 were diagnosed with HF and 20 were diagnosed with dyspnea attributable to non–HF-related causes. This revealed that miR423-5p was specifically enriched in blood of HF cases and receiver-operator-characteristics (ROC) curve analysis showed miR423-5p to be a diagnostic predictor of HF, with an area under the curve of 0.91 (P<0.001). Five other miRNAs were elevated in HF cases but also slightly increased in non-HF dyspnea cases. Conclusion: We identify 6 miRNAs that are elevated in patients with HF, among which miR423-5p is most strongly related to the clinical diagnosis of HF. These 6 circulating miRNAs provide attractive candidates as putative biomarkers for HF.

A Calcineurin-NFATc3-Dependent Pathway Regulates Skeletal Muscle Differentiation and Slow Myosin Heavy-Chain Expression

ABSTRACT The differentiation and maturation of skeletal muscle cells into functional fibers is coordinated largely by inductive signals which act through discrete intracellular signal transduction pathways. Recently, the calcium-activated phosphatase calcineurin (PP2B) and the family of transcription factors known as NFAT have been implicated in the regulation of myocyte hypertrophy and fiber type specificity. Here we present an analysis of the intracellular mechanisms which underlie myocyte differentiation and fiber type specificity due to an insulinlike growth factor 1 (IGF-1)–calcineurin–NFAT signal transduction pathway. We demonstrate that calcineurin enzymatic activity is transiently increased during the initiation of myogenic differentiation in cultured C2C12 cells and that this increase is associated with NFATc3 nuclear translocation. Adenovirus-mediated gene transfer of an activated calcineurin protein (AdCnA) potentiates C2C12 and Sol8 myocyte differentiation, while adenovirus-mediated gene transfer of noncompetitive calcineurin-inhibitory peptides (cain or ΔAKAP79) attenuates differentiation. AdCnA infection was also sufficient to rescue myocyte differentiation in an IGF-depleted myoblast cell line. Using 10T1/2 cells, we demonstrate that MyoD-directed myogenesis is dramatically enhanced by either calcineurin or NFATc3 cotransfection, while a calcineurin inhibitory peptide (cain) blocks differentiation. Enhanced myogenic differentiation directed by calcineurin, but not NFATc3, preferentially specifies slow myosin heavy-chain expression, while enhanced differentiation through mitogen-activated protein kinase kinase 6 (MKK6) promotes fast myosin heavy-chain expression. These data indicate that a signaling pathway involving IGF-calcineurin-NFATc3 enhances myogenic differentiation whereas calcineurin acts through other factors to promote the slow fiber type program.

Conditional Dicer Gene Deletion in the Postnatal Myocardium Provokes Spontaneous Cardiac Remodeling

Background— Dicer, an RNAse III endonuclease critical for processing of pre-microRNAs (miRNAs) into mature 22-nucleotide miRNAs, has proven a useful target to dissect the significance of miRNAs biogenesis in mammalian biology. Methods and Results— To circumvent the embryonic lethality associated with germline null mutations for Dicer, we triggered conditional Dicer loss through the use of a tamoxifen-inducible Cre recombinase in the postnatal murine myocardium. Targeted Dicer deletion in 3-week-old mice provoked premature death within 1 week accompanied by mild ventricular remodeling and dramatic atrial enlargement. In the adult myocardium, loss of Dicer induced rapid and dramatic biventricular enlargement, accompanied by myocyte hypertrophy, myofiber disarray, ventricular fibrosis, and strong induction of fetal gene transcripts. Comparative miRNA profiling revealed a set of miRNAs that imply causality between miRNA depletion and spontaneous cardiac remodeling. Conclusions— Overall, these results indicate that modifications in miRNA biogenesis affect both juvenile and adult myocardial morphology and function.

MicroRNA-199b targets the nuclear kinase Dyrk1a in an auto-amplification loop promoting calcineurin/NFAT signalling

MicroRNAs (miRs) are a class of single-stranded, non-coding RNAs of about 22 nucleotides in length. Increasing evidence implicates miRs in myocardial disease processes. Here we show that miR-199b is a direct calcineurin/NFAT target gene that increases in expression in mouse and human heart failure, and targets the nuclear NFAT kinase dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 1a (Dyrk1a), constituting a pathogenic feed forward mechanism that affects calcineurin-responsive gene expression. Mutant mice overexpressing miR-199b, or haploinsufficient for Dyrk1a, are sensitized to calcineurin/NFAT signalling or pressure overload and show stress-induced cardiomegaly through reduced Dyrk1a expression. In vivo inhibition of miR-199b by a specific antagomir normalized Dyrk1a expression, reduced nuclear NFAT activity and caused marked inhibition and even reversal of hypertrophy and fibrosis in mouse models of heart failure. Our results reveal that microRNAs affect cardiac cellular signalling and gene expression, and implicate miR-199b as a therapeutic target in heart failure.

Targeted Disruption of NFATc3, but Not NFATc4, Reveals an Intrinsic Defect in Calcineurin-Mediated Cardiac Hypertrophic Growth

ABSTRACT A calcineurin-nuclear factor of activated T cells (NFAT) regulatory pathway has been implicated in the control of cardiac hypertrophy, suggesting one mechanism whereby alterations in intracellular calcium handling are linked to the expression of hypertrophy-associated genes. Although recent studies have demonstrated a necessary role for calcineurin as a mediator of cardiac hypertrophy, the potential involvement of NFAT transcription factors as downstream effectors of calcineurin signaling has not been evaluated. Accordingly, mice with targeted disruptions in NFATc3 and NFATc4 genes were characterized. Whereas the loss of NFATc4 did not compromise the ability of the myocardium to undergo hypertrophic growth, NFATc3-null mice demonstrated a significant reduction in calcineurin transgene-induced cardiac hypertrophy at 19 days, 26 days, 6 weeks, 8 weeks, and 10 weeks of age. NFATc3-null mice also demonstrated attenuated pressure overload- and angiotensin II-induced cardiac hypertrophy. These results provide genetic evidence that calcineurin-regulated responses require NFAT effectors in vivo.

Targeted inhibition of calcineurin attenuates cardiac hypertrophy in vivo

The Ca2+-calmodulin-activated Ser/Thr protein phosphatase calcineurin and the downstream transcriptional effectors of calcineurin, nuclear factor of activated T cells, have been implicated in the hypertrophic response of the myocardium. Recently, the calcineurin inhibitory agents cyclosporine A and FK506 have been extensively used to evaluate the importance of this signaling pathway in rodent models of cardiac hypertrophy. However, pharmacologic approaches have rendered equivocal results necessitating more specific or genetic-based inhibitory strategies. In this regard, we have generated Tg mice expressing the calcineurin inhibitory domains of Cain/Cabin-1 and A-kinase anchoring protein 79 specifically in the heart. ΔCain and ΔA-kinase-anchoring protein Tg mice demonstrated reduced cardiac calcineurin activity and reduced hypertrophy in response to catecholamine infusion or pressure overload. In a second approach, adenoviral-mediated gene transfer of ΔCain was performed in the adult rat myocardium to evaluate the effectiveness of an acute intervention and any potential species dependency. ΔCain adenoviral gene transfer inhibited cardiac calcineurin activity and reduced hypertrophy in response to pressure overload without reducing aortic pressure. These results provide genetic evidence implicating calcineurin as an important mediator of the cardiac hypertrophic response in vivo.

Lung ischemia-reperfusion injury: a molecular and clinical view on a complex pathophysiological process

Lung ischemia-reperfusion injury remains one of the major complications after cardiac bypass surgery and lung transplantation. Due to its dual blood supply system and the availability of oxygen from alveolar ventilation, the pathogenetic mechanisms of ischemia-reperfusion injury in the lungs are more complicated than in other organs, where loss of blood flow automatically leads to hypoxia. In this review, an extensive overview is given of the molecular and cellular mechanisms that are involved in the pathogenesis of lung ischemia-reperfusion injury and the possible therapeutic strategies to reduce or prevent it. In addition, the roles of neutrophils, alveolar macrophages, cytokines, and chemokines, as well as the alterations in the cell-death related pathways, are described in detail.

PKCα regulates the hypertrophic growth of cardiomyocytes through extracellular signal–regulated kinase1/2 (ERK1/2)

Members of the protein kinase C (PKC) isozyme family are important signal transducers in virtually every mammalian cell type. Within the heart, PKC isozymes are thought to participate in a signaling network that programs developmental and pathological cardiomyocyte hypertrophic growth. To investigate the function of PKC signaling in regulating cardiomyocyte growth, adenoviral-mediated gene transfer of wild-type and dominant negative mutants of PKCα, βII, δ, and ɛ (only wild-type ζ) was performed in cultured neonatal rat cardiomyocytes. Overexpression of wild-type PKCα, βII, δ, and ɛ revealed distinct subcellular localizations upon activation suggesting unique functions of each isozyme in cardiomyocytes. Indeed, overexpression of wild-type PKCα, but not βII, δ, ɛ, or ζ induced hypertrophic growth of cardiomyocytes characterized by increased cell surface area, increased [3H]-leucine incorporation, and increased expression of the hypertrophic marker gene atrial natriuretic factor. In contrast, expression of dominant negative PKCα, βII, δ, and ɛ revealed a necessary role for PKCα as a mediator of agonist-induced cardiomyocyte hypertrophy, whereas dominant negative PKCɛ reduced cellular viability. A mechanism whereby PKCα might regulate hypertrophy was suggested by the observations that wild-type PKCα induced extracellular signal–regulated kinase1/2 (ERK1/2), that dominant negative PKCα inhibited PMA-induced ERK1/2 activation, and that dominant negative MEK1 (up-stream of ERK1/2) inhibited wild-type PKCα–induced hypertrophic growth. These results implicate PKCα as a necessary mediator of cardiomyocyte hypertrophic growth, in part, through a ERK1/2-dependent signaling pathway.

MEK1-ERK2 Signaling Pathway Protects Myocardium From Ischemic Injury In Vivo

Background—Myocardial infarction causes a rapid and largely irreversible loss of cardiac myocytes that can lead to sudden death, ventricular dilation, and heart failure. Members of the mitogen-activated protein kinase (MAPK) signaling cascade have been implicated as important effectors of cardiac myocyte cell death in response to diverse stimuli, including ischemia-reperfusion injury. Specifically, activation of the extracellular signal–regulated kinases 1/2 (ERK1/2) has been associated with cardioprotection, likely through antagonism of apoptotic regulatory pathways. Methods and Results—To establish a causal relationship between ERK1/2 signaling and cardioprotection, we analyzed Erk1 nullizygous gene-targeted mice, Erk2 heterozygous gene-targeted mice, and transgenic mice with activated MEK1-ERK1/2 signaling in the heart. Although MEK1 transgenic mice were largely resistant to ischemia-reperfusion injury, Erk2+/− gene-targeted mice showed enhanced infarction areas, DNA laddering, and terminal deoxynucleotidyl transferase–mediated dUTP biotin nick-end labeling (TUNEL) compared with littermate controls. In contrast, enhanced MEK1-ERK1/2 signaling protected hearts from DNA laddering, TUNEL, and preserved hemodynamic function assessed by pressure-volume loop recordings after ischemia-reperfusion injury. Conclusions—These data are the first to demonstrate that ERK2 signaling is required to protect the myocardium from ischemia-reperfusion injury in vivo.

Estrogenic hormone action in the heart: regulatory network and function.

Cardiovascular diseases are the leading cause of death in the industrialised countries and display significant gender-based differences. Estrogen plays an important role in the pathogenesis of heart disease and is able to modulate the progression of cardiovascular disease. The focus on the beneficial influence of estrogen is gradually shifting from the vascular system to the myocardium. The presence of functional estrogen receptors in the myocardium has been demonstrated. Estrogen is important for cardiovascular baseline physiology and modulates the myocardial response under pathological conditions. Here we summarise the current knowledge of the regulatory network of estrogenic action in the myocardium and its effects on cardiovascular function.