Abstract: MicroRNAs (miRs) are short non-coding RNA molecules involved in post-transcriptional gene regulation by binding to the 3' untranslated region of a messenger RNA (mRNA), thereby inhibiting the translation or inducing mRNA destabilization. MiRs are generally considered to act as intracellular mediators essential for normal cardiac function, and their deregulated expression profiles have been associated with cardiovascular diseases. Recent studies have revealed the existence of freely circulating miRs in human peripheral blood, which are present in a stable nature. This has raised the possibility that miRs may be released in the circulation and can serve as novel diagnostic markers for acute or chronic human disorders, including myocardial infarction (MI). This review summarizes the recent findings of miRs that fulfill the criteria of candidate biomarkers for MI.
Abstract: Cardiac hypertrophy is a thickening of the heart muscle that results in enlargement of the ventricles, which is the primary response of the myocardium to stress or mechanical overload. Cardiac pathological and physiological hemodynamic overload causes enhanced protein synthesis, sarcomeric reorganization and density, and increased cardiomyocyte size, all culminating into structural remodeling of the heart. With clinical evidence demonstrating that sustained hypertrophy is a key risk factor in heart failure development, much effort is centered on the identification of signals and pathways leading to pathological hypertrophy for future rational drug design in heart failure therapy. A wide variety of studies indicate that individual microRNAs exhibit altered expression profiles under experimental and clinical conditions of cardiac hypertrophy and heart failure. Here we review the recent literature, illustrating how single microRNAs regulate cardiac hypertrophy by classifying them by their prohypertrophic or antihypertrophic properties and their specific effects on intracellular signaling cascades, ubiquitination processes, sarcomere composition and by promoting inter-cellular communication.
Abstract: MicroRNAs refer to a subfamily of small non-coding RNA species that are designed to influence gene expression in nearly all cell types studied to date. Studies from the past decade have demonstrated that microRNAs are atypically expressed in the cardiovascular system under specific pathological conditions. Gain- and loss-of-function studies using in vitro and in vivo models have revealed distinct roles for specific microRNAs in cardiovascular development, physiological functions, and cardiac pathological conditions. In this review, the current relevant findings on the role of microRNAs in cardiac hypertrophic growth are updated, the target genes of these microRNAs are summarized, and the future of microRNAs as potential therapeutic targets is discussed.
Abstract: The mutation A341V in the S6 transmembrane segment of KCNQ1, the α-subunit of the slowly activating delayed-rectifier K(+) (I(Ks)) channel, predisposes to a severe long-QT1 syndrome with sympathetic-triggered ventricular tachyarrhythmias and sudden cardiac death.
Abstract: Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear receptor family of ligand-activated transcription factors and consist of the three isoforms, PPARα, PPARβ/δ, and PPARγ. Considerable evidence indicates the importance of PPARs in cardiovascular lipid homeostasis and diabetes, yet the isoform-dependent cardiac target genes remain unknown. Here, we constructed murine ventricular clones allowing stable expression of siRNAs to achieve specifically knockdown for each of the PPAR isoforms. By combining gene profiling and computational peroxisome proliferator response element analysis following PPAR isoform activation in normal versus PPAR isoform-deficient cardiomyocyte-like cells, we have, for the first time, determined PPAR isoform-specific endogenous target genes in the heart. Electromobility shift and chromatin immunoprecipitation assays demonstrated the existence of an evolutionary conserved peroxisome proliferator response element consensus-binding site in an insulin-like growth factor-1 (igf-1) enhancer. In line, Wy-14643-mediated PPARα activation in the wild-type mouse heart resulted in up-regulation of igf-1 transcript abundance and provided protection against cardiomyocyte apoptosis following ischemia/reperfusion or biomechanical stress. Taken together, these data confirm igf-1 as an in vivo target of PPARα and the involvement of a PPARα/IGF-1 signaling pathway in the protection of cardiomyocytes under ischemic and hemodynamic loading conditions.
Abstract: Apoptosis Inducing Factor (AIF) is a highly conserved, ubiquitous flavoprotein localized in the mitochondrial intermembrane space. In vivo, AIF provides protection against neuronal and cardiomyocyte apoptosis induced by oxidative stress. Conversely in vitro, AIF has been demonstrated to have a pro-apoptotic role upon induction of the mitochondrial death pathway, once AIF translocates to the nucleus where it facilitates chromatin condensation and large scale DNA fragmentation. Given that the aif hypomorphic harlequin (Hq) mutant mouse model displays severe sarcopenia, we examined skeletal muscle from the aif hypomorphic mice in more detail. Adult AIF-deficient skeletal myofibers display oxidative stress and a severe form of atrophy, associated with a loss of myonuclei and a fast to slow fiber type switch, both in "slow" muscles such as soleus, as well as in "fast" muscles such as extensor digitorum longus, most likely resulting from an increase of MEF2 activity. This fiber type switch was conserved in regenerated soleus and EDL muscles of Hq mice subjected to cardiotoxin injection. In addition, muscle regeneration in soleus and EDL muscles of Hq mice was severely delayed. Freshly cultured myofibers, soleus and EDL muscle sections from Hq mice displayed a decreased satellite cell pool, which could be rescued by pretreating aif hypomorphic mice with the manganese-salen free radical scavenger EUK-8. Satellite cell activation seems to be abnormally long in Hq primary culture compared to controls. However, AIF deficiency did not affect myoblast cell proliferation and differentiation. Thus, AIF protects skeletal muscles against oxidative stress-induced damage probably by protecting satellite cells against oxidative stress and maintaining skeletal muscle stem cell number and activation.
Abstract: Right ventricular (RV) dysfunction is a major determinant of long-term morbidity and mortality in congenital heart disease. The right ventricle (RV) is genetically different from the left ventricle (LV), but it is unknown as to whether this has consequences for the cellular responses to abnormal loading conditions. In the LV, calcineurin-activation is a major determinant of pathological hypertrophy and an important target for therapeutic strategies. We studied the functional and molecular adaptation of the RV in mouse models of pressure and volume load, focusing on calcineurin-activation.
Abstract: The transcription factor MEF2 is a downstream target for several hypertrophic signalling pathways in the heart, suggesting that MEF2 may act as a valuable therapeutic target in the treatment of heart failure.
Abstract: Gender differences in cardiovascular disease have long been recognized and attributed to beneficial cardiovascular actions of estrogen. Class II histone deacetylases (HDACs) act as key modulators of heart disease by repressing the activity of the myocyte enhancer factor (MEF)2 transcription factor, which promotes pathological cardiac remodeling in response to stress. Although it is proposed that HDACs additionally influence nuclear receptor signaling, the effect of class II HDACs on gender differences in cardiovascular disease remains unstudied.
Abstract: In response to chronic hypertension, the heart compensates by hypertrophic growth, which frequently progresses to heart failure. Although intracellular calcium (Ca(2+)) has a central role in hypertrophic signaling pathways, the Ca(2+) source for activating these pathways remains elusive. We hypothesized that pathological sarcoplasmic reticulum Ca(2+) leak through defective cardiac intracellular Ca(2+) release channels/ryanodine receptors (RyR2) accelerates heart failure development by stimulating Ca(2+)-dependent hypertrophic signaling. Mice heterozygous for the gain-of-function mutation R176Q/+ in RyR2 and wild-type mice were subjected to transverse aortic constriction. Cardiac function was significantly lower, and cardiac dimensions were larger at 8 weeks after transverse aortic constriction in R176Q/+ compared with wild-type mice. R176Q/+ mice displayed an enhanced hypertrophic response compared with wild-type mice as assessed by heart weight:body weight ratios and cardiomyocyte cross-sectional areas after transverse aortic constriction. Quantitative PCR revealed increased transcriptional activation of cardiac stress genes in R176Q/+ mice after transverse aortic constriction. Moreover, pressure overload resulted in an increased sarcoplasmic reticulum Ca(2+) leak, associated with higher expression levels of the exon 4 splice form of regulator of calcineurin 1, and a decrease in nuclear factor of activated T-cells phosphorylation in R176Q/+ mice compared with wild-type mice. Taken together, our results suggest that RyR2-dependent sarcoplasmic reticulum Ca(2+) leak activates the prohypertrophic calcineurin/nuclear factor of activated T-cells pathway under conditions of pressure overload.
Abstract: The molecular biology dogma that DNA replicates its genetic information within nucleotide sequences and transcribes it to RNA where it codes for the generation of mRNA, failed to consider a significant part of the genetic code. Although it has been generally assumed that most genetic information is executed by proteins, recent evidence suggests that the majority of the genomes of mammals and other complex organisms is transcribed into non-coding RNA (ncRNA), many of which are alternatively spliced and/or processed into smaller functional RNA molecules. ncRNAs are predominantly involved in processes that require highly specific nucleic acid recognition, revealing a, so far hidden, layer of internal signals that control various levels of gene expression in developmental and (patho)physiological processes. MicroRNAs (miRNAs) are a large class of evolutionary conserved, small ncRNAs, typically 18 to 24 nucleotides in length, that primarily function at the posttranscriptional level by interacting with the 3' untranslated region (UTR) of specific target mRNAs in a sequence-specific manner. Despite the advances in miRNA discovery, the role of miRNAs in physiological and pathological processes is just rising, revealing their cellular functions in proliferation and differentiation, apoptosis, the stress response and tumorgenesis. MiRNA expression profiling and the manipulation of their expression in cardiac tissue has led to the discovery of regulatory roles for these small ncRNAs during cardiac development and disease, implicating them in regulation of cardiac gene expression. Here we review the basic mechanisms by which cardiovascular miRNAs are regulated in the larger context of cardiogenesis and in cardiovascular disease.
Abstract: Pathological forms of left ventricular hypertrophy (LVH) often progress to heart failure. Specific transcription factors have been identified that activate the gene program to induce pathological forms of LVH. It is likely that apart from activating transcriptional inducers of LVH, constitutive transcriptional repressors need to be removed during the development of cardiac hypertrophy. Here, we report that the constitutive presence of Krüppel-like factor 15 (KLF15) is lost in pathological hypertrophy and that this loss precedes progression toward heart failure. We show that transforming growth factor-beta-mediated activation of p38 MAPK is necessary and sufficient to decrease KLF15 expression. We further show that KLF15 robustly inhibits myocardin, a potent transcriptional activator. Loss of KLF15 during pathological LVH relieves the inhibitory effects on myocardin and stimulates the expression of serum response factor target genes, such as atrial natriuretic factor. This uncovers a novel mechanism where activated p38 MAPK decreases KLF15, an important constitutive transcriptional repressor whose removal seems a vital step to allow the induction of pathological LVH.
Abstract: 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.
Abstract: 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).
Abstract: 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.
Abstract: We have previously shown that cardiac-specific inhibition of NF-kappaB attenuates angiotensin II (AngII)-induced left ventricular (LV) hypertrophy in vivo. We now tested whether NF-kappaB inhibition is able to block LV remodelling upon chronic pressure overload and chronic AngII stimulation.
Abstract: We tested the hypothesis that female and male mice differ in terms of cardiac hypertrophy after deoxycorticosterone acetate (DOCA)+salt hypertension (uninephrectomy and 1% saline in drinking water) and focused on calcineurin signaling. We excluded confounding effects of blood pressure elevation or sex-related blood pressure differences by treating DOCA-salt mice with hydralazine (250 mg/L in drinking water). We found that directly measured mean arterial blood pressure was lowered to control values with hydralazine and corroborated this finding in separate mouse groups with radiotelemetry. Male mice were more responsive to DOCA-salt-related effects. They developed more left ventricular hypertrophy and more renal hypertrophy after 6 weeks of DOCA-salt+hydralazine compared with female mice. In hearts, transcripts for calcineurin Abeta and for myocyte-enriched calcineurin interacting protein 1 were upregulated in male but not in female mice. Enhanced activity of calcineurin Abeta, as indicated by diminished phosphorylation of NFATc2 in male mice, accounted for this sex-specific difference. Stretch-related, inflammatory, and profibrotic responses were also accentuated in male mice, as shown by higher transcript levels of atrial natriuretic peptide, monocyte chemoattractant protein-1, and transforming growth factor-beta. Our results support sex-specific regulation of the calcineurin pathway in response to largely blood pressure-independent mineralocorticoid action. We suggest that sex-specific calcineurin activation determines the maladaptive cardiac and renal hypertrophic responses and accompanying organ injury in male mice.
Abstract: Cardiac function depends upon several factors, including adequate cellular mass, intact contractile machinery, and adequate production of ATP. An appropriate homeostasis on all these levels is crucial for the daunting life-long task the myocardium faces. Not surprisingly, many alterations in the above factors have been spotted when the heart fails and hypothesized to play a causal role in the genesis of the failing heart. Indeed, development of cardiac hypertrophy and failure is associated with chamber remodeling as well as with changes of the phenotype at the level of the individual myocyte. Disturbed energy metabolism with impaired fatty acid oxidation and lower expression of proteins involved in ATP synthesis occurs during myocardial hypertrophy and heart failure. The altered expression of proteins from metabolic pathways may reflect mitochondrial dysfunction as a feature of the transition from compensated myocardial hypertrophy with preserved fatty acid metabolism to impaired energy metabolism in heart failure.
Abstract: 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.
Abstract: We analyzed the effect of conditional, alphaMHC-dependent genetic beta-catenin depletion and stabilization on cardiac remodeling following experimental infarct. beta-Catenin depletion significantly improved 4-week survival and left ventricular (LV) function (fractional shortening: CT(Deltaex3-6): 24 +/- 1.9%; beta-cat(Deltaex3-6): 30.2 +/- 1.6%, P < 0.001). beta-Catenin stabilization had opposite effects. No significant changes in adult cardiomyocyte survival or hypertrophy were observed in either transgenic line. Associated with the functional improvement, LV scar cellularity was altered: beta-catenin-depleted mice showed a marked subendocardial and subepicardial layer of small cTnT(pos) cardiomyocytes associated with increased expression of cardiac lineage markers Tbx5 and GATA4. Using a Cre-dependent lacZ reporter gene, we identified a noncardiomyocyte cell population affected by alphaMHC-driven gene recombination localized to these tissue compartments at baseline. These cells were found to be cardiac progenitor cells since they coexpressed markers of proliferation (Ki67) and the cardiomyocyte lineage (alphaMHC, GATA4, Tbx5) but not cardiac Troponin T (cTnT). The cell population overlaps in part with both the previously described c-kit(pos) and stem cell antigen-1 (Sca-1)(pos) precursor cell population but not with the Islet-1(pos) precursor cell pool. An in vitro coculture assay of highly enriched (>95%) Sca-1(pos) cardiac precursor cells from beta-catenin-depleted mice compared to cells isolated from control littermate demonstrated increased differentiation toward alpha-actin(pos) and cTnT(pos) cardiomyocytes after 10 days (CT(Deltaex3-6): 38.0 +/- 1.0% alpha-actin(pos); beta-cat(Deltaex3-6): 49.9 +/- 2.4% alpha-actin(pos), P < 0.001). We conclude that beta-catenin depletion attenuates postinfarct LV remodeling in part through increased differentiation of GATA4(pos)/Sca-1(pos) resident cardiac progenitor cells.
Abstract: One major intracellular signaling pathway involved in heart failure employs the phosphatase calcineurin and its downstream transcriptional effector nuclear factor of activated T-cells (NFAT). In vivo evidence for the involvement of NFAT factors in heart failure development is still ill defined. Here we reveal that nfatc2 transcripts outnumber those from other nfat genes in the unstimulated heart by severalfold. Transgenic mice with activated calcineurin in the postnatal myocardium crossbred with nfatc2-null mice revealed a significant abrogation of calcineurin-provoked cardiac growth, indicating that NFATc2 plays an important role downstream of calcineurin and validates the original hypothesis that calcineurin mediates myocyte hypertrophy through activation of NFAT transcription factors. In the absence of NFATc2, a clear protection against the geometrical, functional, and molecular deterioration of the myocardium following biomechanical stress was also evident. In contrast, physiological cardiac enlargement in response to voluntary exercise training was not affected in nfatc2-null mice. Combined, these results reveal a major role for the NFATc2 transcription factor in pathological cardiac remodeling and heart failure.
Abstract: Calcineurin/NFAT signaling is involved in multiple aspects of skeletal muscle development and disease. The myogenic basic helix-loop-helix transcription factors, MyoD, myogenin, Myf5, and MRF4 specify the myogenic lineage. Here we show that calcineurin/NFAT (nuclear factor of activated T cells) signaling is required for primary myogenesis by transcriptional cooperation with the basic helix-loop-helix transcription factor MyoD. Calcineurin/NFAT signaling is involved in myogenin expression in differentiating myoblasts, where the myogenic regulatory factor MyoD synergistically cooperates with NFATc2/c3 at the myogenin promoter. Using gel shift and chromatin immunoprecipitation assays, we identified two conserved NFAT binding sites in the myogenin promoter that were occupied by NFATc3 upon skeletal muscle differentiation. The transcriptional integration between NFATc3 and MyoD is crucial for primary myogenesis in vivo, as myogenin expression is weak in myod:nfatc3 double null embryos, whereas myogenin expression is unaffected in embryos with null mutations for either factor alone. Thus, the combined findings provide a novel transcriptional paradigm for the first steps of myogenesis, where a calcineurin/NFATc3 pathway regulates myogenin induction in cooperation with MyoD during myogenesis.
Abstract: Alterations in expression levels of Na(v)1.5, Cx43 and Cx40 have been frequently reported in cardiac disease and are associated with the development of arrhythmias, but little is known about the underlying molecular mechanisms. In this study we investigated electrical conduction and expression of Na(v)1.5, Cx43 and Cx40 in hearts of transgenic mice overexpressing a constitutively active form of calcineurin (MHC-CnA). ECG recordings showed that atrial, atrioventricular and ventricular activation were significantly prolonged in MHC-CnA hearts as compared to wildtype (WT) littermates. Epicardial activation and arrhythmia susceptibility analysis revealed increased ventricular activation thresholds and arrhythmia vulnerability. Moreover, epicardial ventricular activation patterns in MHC-CnA mice were highly discontinuous with multiple areas of block. These impaired conduction properties were associated with severe reductions in Na(v)1.5, Cx43 and Cx40 protein expression in MHC-CnA hearts as visualized by immunohistochemistry and immunoblotting. Real-time RT-PCR demonstrated that the decreased protein levels for Na(v)1.5 and Cx40, but not for Cx43, were accompanied by corresponding reductions at the RNA level. Cx43 RNA isoform analysis indicated that the reduction in Cx43 protein expression is caused by a post-transcriptional mechanism rather than by RNA isoform switching. In contrast, RNA isoform analysis for Cx40 and Na(v)1.5 provided additional evidence that in calcineurin-induced hypertrophy the downregulation of these proteins originates at the transcriptional level. These results provide the molecular rationale for Na(v)1.5, Cx43 and Cx40 downregulation in this model of hypertrophy and failure and the development of the pro-arrhythmic substrate.
Abstract: RING-finger proteins commonly function as ubiquitin ligases that mediate protein degradation by the ubiquitin-proteasome pathway. Muscle-specific RING-finger (MuRF) proteins are striated muscle-restricted components of the sarcomere that are thought to possess ubiquitin ligase activity. We show that mice lacking MuRF3 display normal cardiac function but are prone to cardiac rupture after acute myocardial infarction. Cardiac rupture is preceded by left ventricular dilation and a severe decrease in cardiac contractility accompanied by myocyte degeneration. Yeast two-hybrid assays revealed four-and-a-half LIM domain (FHL2) and gamma-filamin proteins as MuRF3 interaction partners, and biochemical analyses showed these proteins to be targets for degradation by MuRF3. Accordingly, FHL2 and gamma-filamin accumulated to abnormal levels in the hearts of mice lacking MuRF3. These findings reveal an important role of MuRF3 in maintaining cardiac integrity and function after acute myocardial infarction and suggest that turnover of FHL2 and gamma-filamin contributes to this cardioprotective function of MuRF3.
Abstract: Observational studies have identified left ventricular hypertrophy (LVH) as a strong, independent risk factor for the development of heart failure (HF), coronary heart disease and stroke. LVH develops in response to hemodynamic overload. Classical conceptualization has it that LVH would start as an adaptive, beneficial response in order to normalize wall stress. With progression of the disease, deterioration to maladaptive hypertrophy, and further on to HF could occur. Recent experiments in animal models of pressure-overload and myocardial infarction now challenge this concept by demonstrating that blunting the hypertrophic response is actually associated with preserved cardiac function, and with improved survival. These findings may have profound therapeutical implications.
Abstract: Previous studies have suggested that the heart may be capable of limited repair and regeneration in response to a focal injury, while other studies indicate that the mammalian heart has no regenerative capacity. To further explore this issue, we performed a series of superficial and transmural myocardial injuries in C57BL/6 and MRL/MpJ adult mice. At defined time intervals following the respective injury (days 3, 14, 30 and 60), we examined cardiac function using echocardiography, morphology, fluorescence-activated cell sorting for 5-bromo-2-deoxyuridine-positive cells and molecular signature using microarray analysis. We observed restoration of myocardial function in the superficial MRL cryoinjured heart and significantly less collagen deposition compared with the injured hearts of C57BL/6 mice. Following a severe transmural myocardial injury, the MRL mouse has increased survival and decreased ventricular remodeling compared with the C57BL/6 mouse but without evidence of complete regeneration. The cytoprotective program observed in the severely injured MRL heart is in part due to increased cellular proliferation, increased vasculogenesis, and decreased apoptosis that limits the extension of the injury. We conclude that MRL injured hearts have evidence of myocardial regeneration, in response to superficial injury, but the stabilized left ventricular function and improved survival observed in the MRL mouse following severe injury is not associated with complete myocardial regeneration.
Abstract: The development of heart failure is invariably associated with extensive fibrosis. Treatment with Peroxisome Proliferator-Activated Receptor (PPAR) ligands has been shown to attenuate cardiac fibrosis, but the molecular mechanism underlying this protective effect has remained largely unknown. In this study the potential of each PPAR isoform (PPARalpha, delta, and gamma) to attenuate cardiac fibroblast proliferation, fibroblast (CF) to myofibroblast (CMF) transdifferentiation, and collagen synthesis was investigated.
Abstract: Gradually the distinction between signalling pathways originally believed to be specific for either hypertrophy, cell cycle control, apoptosis and cell survival are fading. The subtle variations in stimuli to a cell and the microenvironment will determine cell fate. In cardiomyocytes the entrance into the cell cycle is efficiently blocked. Therefore attention has focused on pathways involved in hypertrophy to assess effects in ischaemic models and vice versa. Interventions at different levels have been shown to be cardiomyocyte protective. Various growth factors (including IGF1 and FGF1,2) have shown to prevent or delay cardiomyocyte loss in and ex vivo. Similar results have been reported for downstream interventions in the signalling pathways. Strong effects after MAPK activation have been shown in gene targeted mice. Especially constitutive activation of the ERK proteins prevents ischemic damage of the heart with conservation of left ventricular function. Evidence for a key role of nuclear Akt in preventing apoptosis is accumulating from various genetic and pharmacological sources. Development of techniques to measure the level of cardiomyocyte death depends on further improvements in molecular imaging in mouse and human. In addition to studying cardiomyocyte cell death, it is crucial to measure myocardial function. Whether hypertrophy following ischaemia is adaptive or maladaptive and whether all apoptosis is detrimental will have to be determined by assessment of left ventricular function through invasive and noninvasive methods.
Abstract: The calcium-activated phosphatase calcineurin is regulated by a binding cofactor known as modulatory calcineurin-interacting protein (MCIP) in yeast up through mammals. The physiologic function of MCIP remains an area of ongoing investigation, because both positive and negative calcineurin regulatory effects have been reported. Here we disrupted the mcip1 and mcip2 genes in the mouse and provide multiple lines of evidence that endogenous MCIP functions as a calcineurin facilitator in vivo. Mouse embryonic fibroblasts deficient in both mcip1/2 showed impaired activation of nuclear factor of activated T cells (NFAT), suggesting that MCIP is required for efficient calcineurin-NFAT coupling. Mice deficient in mcip1/2 showed a dramatic impairment in cardiac hypertrophy induced by pressure overload, neuroendocrine stimulation, or exercise, similar to mice lacking calcineurin Abeta. Moreover, simultaneous deletion of calcineurin Abeta in the mcip1/2-null background did not rescue impaired hypertrophic growth after pressure overload. Slow/oxidative fiber-type switching in skeletal muscle after exercise stimulation was also impaired in mcip1/2 mice, similar to calcineurin Abeta-null mice. Moreover, CD4(+) T cells from mcip1/2-null mice showed enhanced apoptosis that was further increased by loss of calcineurin Abeta. Finally, mcip1/2-null mice displayed a neurologic phenotype that was similar to calcineurin Abeta-null mice, such as increased locomotor activity and impaired working memory. Thus, a loss-of-function analysis suggests that MCIPs serve either a permissive or facilitative function for calcineurin-NFAT signaling in vivo.
Abstract: The purpose of this study was to identify apoptosis-inducing factor (AIF) as a cardiac mitochondrial antioxidant and assess the efficacy of EUK-8, a salen-manganese catalytic free radical scavenger, to protect the AIF-deficient myocardium against pressure overload.
Abstract: Hypertrophic growth, a risk factor for mortality in heart disease, is driven by reprogramming of cardiac gene expression. Although the transcription factor myocyte enhancer factor-2 (MEF2) is a common end point for several hypertrophic pathways, its precise cardiac gene targets and function in cardiac remodeling remain to be elucidated.
Abstract: The crucial functions of atrial natriuretic peptide (ANP) and endothelial nitric oxide/NO in the regulation of arterial blood pressure have been emphasized by the hypertensive phenotype of mice with systemic inactivation of either the guanylyl cyclase-A receptor for ANP (GC-A-/-) or endothelial nitric-oxide synthase (eNOS-/-). Intriguingly, similar levels of arterial hypertension are accompanied by marked cardiac hypertrophy in GC-A-/-, but not in eNOS-/-, mice, suggesting that changes in local pathways regulating cardiac growth accelerate cardiac hypertrophy in the former and protect the heart of the latter. Our recent observations in mice with conditional, cardiomyocyte-restricted GC-A deletion demonstrated that ANP locally inhibits cardiomyocyte growth. Abolition of these local, protective effects may enhance the cardiac hypertrophic response of GC-A-/- mice to persistent increases in hemodynamic load. Notably, eNOS-/- mice exhibit markedly increased cardiac ANP levels, suggesting that increased activation of cardiac GC-A can prevent hypertensive heart disease. To test this hypothesis, we generated mice with systemic inactivation of eNOS and cardiomyocyte-restricted deletion of GC-A by crossing eNOS-/- and cardiomyocyte-restricted GC-A-deficient mice. Cardiac deletion of GC-A did not affect arterial hypertension but significantly exacerbated cardiac hypertrophy and fibrosis in eNOS-/- mice. This was accompanied by marked cardiac activation of both the mitogen-activated protein kinase (MAPK) ERK 1/2 and the phosphatase calcineurin. Our observations suggest that local ANP/GC-A/cyclic GMP signaling counter-regulates MAPK/ERK- and calcineurin/nuclear factor of activated T cells-dependent pathways of cardiac myocyte growth in hypertensive eNOS-/- mice.
Abstract: Since atherosclerosis is a chronic inflammatory disease, we tested the hypothesis that the immunosuppressive drug FK506 would attenuate the development of atherosclerosis using a mouse model of collar-induced atherosclerosis. ApoE-/- mice were treated for 4 weeks with the immunosuppressive drug FK506 (0.05 mg/kg/day), yielding sustained blood levels (approximately 0.2 ng/mL) without systemic side effects. Atherosclerotic plaque development of FK506-treated mice was significantly reduced (63%) while plaque cell density was increased (52%) compared to controls. Importantly, FK506 also blocked progression of pre-existing atherosclerotic plaques. Plaque area of pre-existing plaques was 35% reduced by FK506. Cell density (35%) and collagen content (51%) were significantly increased, whereas necrotic core content was decreased (42%), indicating a more stable plaque morphology. Similar results were found during spontaneous atherosclerotic plaque development in ApoE-/- mice (treatment 17-25 weeks of age). Flow-cytometric analysis showed no peripheral effects on blood cell count or T-cell activation after FK506-treatment. In vitro, FK506 decreased vascular smooth muscle cell (VSMC) apoptosis and inhibited nuclear factor of activated T cells (NFAT)-luciferase reporter activity at concentrations in the range of the in vivo concentration. Low-dose FK506 inhibits collar-induced atherosclerotic plaque development and progression and induces more stable plaque phenotypes in ApoE-/- mice without any peripheral side effects.
Abstract: Apoptosis-inducing factor (AIF), or programmed cell death 8 (Pdcd8), is a highly conserved, ubiquitous flavoprotein localized in the mitochondrial intermembrane space. In vivo, AIF provides protection against neuronal apoptosis induced by oxidative stress. Conversely, in vitro, AIF has been demonstrated to have a proapoptotic role when, on induction of the mitochondrial death pathway, AIF translocates to the nucleus where it facilitates chromatin condensation and large scale DNA fragmentation. To determine the role of AIF in myocardial apoptotic processes, we examined cardiomyocytes from an AIF-deficient mouse mutant, Harlequin (Hq). Hq mutant cardiomyocytes demonstrated increased sensitivity to H2O2-induced cell death. Further, Hq hearts subjected to ischemia/reperfusion revealed more cardiac damage and, unlike wild-type mice, the amount of damage increased with the age of the animal. Aortic banding caused enhanced hypertrophy, increased cardiomyocyte apoptotic and necrotic cell death, and accelerated progression toward maladaptive left ventricular remodeling in Hq mutant mice compared with wild-type counterparts. These findings correlated with a reduced capacity of subsarcolemmal mitochondria from Hq mutant hearts to scavenge free radicals. Together, these data demonstrate a critical role for AIF as a cardiac antioxidant in the protection against oxidative stress-induced cell death and development of heart failure induced by pressure overload.
Abstract: Serum response factor (SRF) is a cardiac transcription factor involved in cell growth and differentiation. We have shown, using the Cre/loxP system, that cardiac-specific disruption of SRF gene in the embryonic heart results in lethal cardiac defects. The role of SRF in adult heart is unknown.
Abstract: Abnormalities in intracellular calcium release and reuptake are responsible for decreased contractility in heart failure (HF). We have previously shown that cardiac ryanodine receptors (RyRs) are protein kinase A-hyperphosphorylated and depleted of the regulatory subunit calstabin-2 in HF. Moreover, similar alterations in skeletal muscle RyR have been linked to increased fatigability in HF. To determine whether restoration of calstabin binding to RyR may ameliorate cardiac and skeletal muscle dysfunction in HF, we treated WT and calstabin-2-/- mice subjected to myocardial infarction (MI) with JTV519. JTV519, a 1,4-benzothiazepine, is a member of a class of drugs known as calcium channel stabilizers, previously shown to increase calstabin binding to RyR. Echocardiography at 21 days after MI demonstrated a significant increase in ejection fraction in WT mice treated with JTV519 (45.8 +/- 5.1%) compared with placebo (31.1 +/- 3.1%; P < 0.05). Coimmunoprecipitation experiments revealed increased amounts of calstabin-2 bound to the RyR2 channel in JTV519-treated WT mice. However, JTV519 did not show any of these beneficial effects in calstabin-2-/- mice with MI. Additionally, JTV519 improved skeletal muscle fatigue in WT and calstabin-2-/- mice with HF by increasing the binding of calstabin-1 to RyR1. The observation that treatment with JTV519 improved cardiac function in WT but not calstabin-2-/- mice indicates that calstabin-2 binding to RyR2 is required for the beneficial effects in failing hearts. We conclude that JTV519 may provide a specific way to treat the cardiac and skeletal muscle myopathy in HF by increasing calstabin binding to RyR.
Abstract: Atrial natriuretic peptide (ANP), through its guanylyl cyclase-A (GC-A) receptor, not only is critically involved in the endocrine regulation of arterial blood pressure but also locally moderates cardiomyocyte growth. The mechanisms underlying the antihypertrophic effects of ANP remain largely uncharacterized. We examined the contribution of the Na+/H+ exchanger NHE-1 to cardiac remodeling in GC-A-deficient (GC-A(-/-)) mice.
Abstract: Human heart failure is preceded by a process termed cardiac remodeling in which heart chambers progressively enlarge and contractile function deteriorates. Programmed cell death (apoptosis) of cardiac muscle cells has been identified as an essential process in the progression to heart failure. The execution of the apoptotic program entails complex interactions between and execution of multiple molecular subprograms. Unlike necrosis, apoptosis is an orderly regulated process and, by inference, a logical therapeutic target if intervention occurs at an early stage. To identify potential therapeutic targets, it is imperative to have a full understanding of the apoptotic pathways that are functional in the cardiac muscle. Accordingly, the present review summarizes the apoptotic pathways operative in cardiac muscle and discusses therapeutic options related to apoptosis for the future treatment of human heart failure.
Abstract: Skeletal muscles are a mosaic of slow and fast twitch myofibers. During embryogenesis, patterns of fiber type composition are initiated that change postnatally to meet physiological demand. To examine the role of the protein phosphatase calcineurin in the initiation and maintenance of muscle fiber types, we used a "Flox-ON" approach to obtain muscle-specific overexpression of the modulatory calcineurin-interacting protein 1 (MCIP1/DSCR1), an inhibitor of calcineurin. Myo-Cre transgenic mice with early skeletal muscle-specific expression of Cre recombinase were used to activate the Flox-MCIP1 transgene. Contractile components unique to type 1 slow fibers were absent from skeletal muscle of adult Myo-Cre/Flox-MCIP1 mice, whereas oxidative capacity, myoglobin content, and mitochondrial abundance were unaltered. The soleus muscles of Myo-Cre/Flox-MCIP1 mice fatigued more rapidly than the wild type as a consequence of the replacement of the slow myosin heavy chain MyHC-1 with a fast isoform, MyHC-2A. MyHC-1 expression in Myo-Cre/Flox-MCIP1 embryos and early neonates was normal. These results demonstrate that developmental patterning of slow fibers is independent of calcineurin, while the maintenance of the slow-fiber phenotype in the adult requires calcineurin activity.
Abstract: Significant gender-related differences exist in the development of left ventricular hypertrophy (LVH). In addition, administration of 17beta-estradiol (E2) to ovariectomized female mice attenuates the development of LVH, demonstrating an antagonistic role for E2 in this process, although no molecular mechanism has been proposed for this phenomenon.
Abstract: Pathological remodeling of the left ventricle (LV) after myocardial infarction (MI) is a major cause of heart failure. Although cardiac hypertrophy after increased loading conditions has been recognized as a clinical risk factor for human heart failure, it is unknown whether post-MI hypertrophic remodeling of the myocardium is beneficial for cardiac function over time, nor which regulatory pathways play a crucial role in this process. To address these questions, transgenic (TG) mice engineered to overexpress modulatory calcineurin-interacting protein-1 (MCIP1) in the myocardium were used to achieve cardiac-specific inhibition of calcineurin activation. MCIP1-TG mice and their wild-type (WT) littermates, were subjected to MI and analyzed 4 weeks later. At 4 weeks after MI, calcineurin was activated in the LV of WT mice, which was significantly reduced in MCIP1-TG mice. WT mice displayed a 78% increase in LV mass after MI, which was reduced by 38% in MCIP1-TG mice. Echocardiography indicated marked LV dilation and loss of systolic function in WT-MI mice, whereas TG-MI mice displayed a remarkable preservation of LV geometry and contractility, a pronounced reduction in myofiber hypertrophy, collagen deposition, and beta-MHC expression compared with WT-MI mice. Together, these results reveal a protective role for MCIP1 in the post-MI heart and suggest that calcineurin is a crucial regulator of postinfarction-induced pathological LV remodeling. The improvement in functional, structural, and molecular abnormalities in MCIP1-TG mice challenges the adaptive value of post-MI hypertrophy of the remote myocardium. The full text of this article is available online at http://circres.ahajournals.org.
Abstract: We investigated whether in vivo closed-chest left ventricular pressure-volume measurements would yield similar values for LV hemodynamics compared with open-chest PV measurements under several anesthetics.
Abstract: In response to a variety of extrinsic and intrinsic stimuli that impose increased biomechanical stress the heart responds by enlarging the individual myofibers. Even though myocardial hypertrophy can normalize wall tension, it instigates an unfavorable outcome and threatens affected patients with sudden death or progression to overt heart failure, suggesting that in most instances hypertrophy is a maladaptive process. Increasing evidence suggests that several of the signaling cascades controlling myocyte growth in the adult heart also function to enhance survival of the myocyte population in response to pleiotropic death stimuli. In this review, we summarize recent insights into hypertrophic signaling pathways and their ability to control the balance between myocyte life and death. As modulation of myocardial growth by antagonizing intracellular signaling pathways is increasingly recognized as a potentially auspicious approach to prevent and treat heart failure, the design of such therapies should respect the dichotomous action of pathways that dictate a balance between myocyte hypertrophy, survival and death.
Abstract: 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.
Abstract: The implementation of molecular biological approaches has led to the discovery of single genetic variations that contribute to the development of cardiac failure. In the present review, the characteristics that are invariably associated with the development of failure in experimental animals and clinical studies are discussed, which may provide attractive biological targets in the treatment of human heart failure. Findings from the Framingham studies have provided evidence that the presence of left ventricular hypertrophy is the main risk factor for subsequent development of heart failure in man. Conventional views identify myocardial hypertrophy as a compensatory response to increased workload, prone to evoke disease. Recent findings in genetic models of myocardial hypertrophy and human studies have provided the molecular basis for a novel concept, which favours the existence of either compensatory or maladaptive forms of hypertrophy, of which only the latter leads the way to cardiac failure. Furthermore, the concept that hypertrophy compensates for augmented wall stress is probably outdated. In this article, we provide the molecular pathways that can distinguish beneficial from maladaptive hypertrophy.
Abstract: 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.
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.
Abstract: The calcium-activated phosphatase calcineurin has been implicated as a critical intracellular signal transducer of cardiomyocyte hypertrophy. Although previous data suggested the nuclear factor of activated T-cells (NFAT) as its sole transcriptional effector, the absolute requirement of NFAT as a mediator of calcineurin signaling has not been examined in the heart. We therefore investigated the expression and activation profile of NFAT genes in the heart. Four members (NFATc1-c4) are expressed in cardiomyocytes, elicit nuclear translocation upon calcineurin activation, and are able to drive transactivation of cardiac promoter luciferase constructs. To define the necessary function of NFAT factors as hypertrophic transducers, a dominant negative NFAT construct was created, encompassing part of the N-terminal region of NFATc4 containing a conserved calcineurin-binding motif. Cotransfection of this construct dose-dependently abrogated promoter activation, irrespective of the NFAT isoform used, whereas a control construct with the calcineurin-binding motif mutated displayed no such effects. Adenoviral gene transfer of dominant negative NFAT rendered cardiomyocytes resistant toward all aspects of calcineurin or agonist-induced cardiomyocyte hypertrophy, whereas adenoviral gene transfer of the control construct had no discernable effect on these parameters. These results indicate that multiple NFAT isoforms are expressed in cardiomyocytes where they function as necessary transducers of calcineurin in facilitating cardiomyocyte hypertrophy.
Abstract: 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 PKC alpha, beta II, delta, and epsilon (only wild-type zeta) was performed in cultured neonatal rat cardiomyocytes. Overexpression of wild-type PKC alpha, beta II, delta, and epsilon revealed distinct subcellular localizations upon activation suggesting unique functions of each isozyme in cardiomyocytes. Indeed, overexpression of wild-type PKC alpha, but not betaI I, delta, epsilon, or zeta induced hypertrophic growth of cardiomyocytes characterized by increased cell surface area, increased [(3)H]-leucine incorporation, and increased expression of the hypertrophic marker gene atrial natriuretic factor. In contrast, expression of dominant negative PKC alpha, beta II, delta, and epsilon revealed a necessary role for PKC alpha as a mediator of agonist-induced cardiomyocyte hypertrophy, whereas dominant negative PKC epsilon reduced cellular viability. A mechanism whereby PKC alpha might regulate hypertrophy was suggested by the observations that wild-type PKC alpha induced extracellular signal-regulated kinase1/2 (ERK1/2), that dominant negative PKC alpha inhibited PMA-induced ERK1/2 activation, and that dominant negative MEK1 (up-stream of ERK1/2) inhibited wild-type PKC alpha-induced hypertrophic growth. These results implicate PKC alpha as a necessary mediator of cardiomyocyte hypertrophic growth, in part, through a ERK1/2-dependent signaling pathway.
Abstract: In the past 2 years, an emerging body of research has focused on a novel transcriptional pathway involved in the cardiac hypertrophic response. Ever since its introduction, the significance of the calcineurin-NFAT module has been subject of controversy. The aim of this review is to provide both an update on the current status of knowledge and discuss the remaining issues regarding the involvement of calcineurin in hypertrophic heart disease. To this end, the molecular biology of calcineurin and its direct downstream transcriptional effector NFAT are discussed in the context of the genetic studies that established the existence of this signaling paradigm in the heart. The pharmacological mode-of-action and specificity of the calcineurin inhibitors cyclosporine A (CsA) and FK506 is discussed, as well as their inherent limitations to study the biology of calcineurin. A critical interpretation is given on studies aimed at analyzing the role of calcineurin in cardiac hypertrophy using systemic immunosuppression. To eliminate the controversy surrounding CsA/FK506 usage, recent studies employed genetic inhibitory strategies for calcineurin, which confirm the pivotal role for this signal transduction pathway in the ventricular hypertrophy response. Finally, unresolved issues concerning the role of calcineurin in cardiac pathobiology are discussed based upon the information available, including its controversial role in cardiomyocyte viability, the reciprocal relationship between myocyte Ca(2+) homeostasis and calcineurin activity and the relative importance of calcineurin in relation to other hypertrophic signaling cascades.
Abstract: The Ca(2+)-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. DeltaCain and DeltaA-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 DeltaCain was performed in the adult rat myocardium to evaluate the effectiveness of an acute intervention and any potential species dependency. DeltaCain 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.
Abstract: Mitogen-activated protein kinase (MAPK) signaling pathways are important regulators of cell growth, proliferation, and stress responsiveness. A family of dual-specificity MAP kinase phosphatases (MKPs) act as critical counteracting factors that directly regulate the magnitude and duration of p38, c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinase (ERK) activation. Here we show that constitutive expression of MKP-1 in cultured primary cardiomyocytes using adenovirus-mediated gene transfer blocked the activation of p38, JNK1/2, and ERK1/2 and prevented agonist-induced hypertrophy. Transgenic mice expressing physiological levels of MKP-1 in the heart showed (1) no activation of p38, JNK1/2, or ERK1/2; (2) diminished developmental myocardial growth; and (3) attenuated hypertrophy in response to aortic banding and catecholamine infusion. These results provide further evidence implicating MAPK signaling factors as obligate regulators of cardiac growth and hypertrophy and demonstrate the importance of dual-specificity phosphatases as counterbalancing regulatory factors in the heart.
Abstract: For the murine heart the relationships between ischemia-reperfusion-induced loss of cardiac function, enzyme release, high-energy phosphate (HEP), and membrane phospholipid metabolism are ill-defined. Accordingly, isolated ejecting murine hearts were subjected to varying periods of ischemia, whether or not followed by reperfusion. On reperfusion, hemodynamic function was almost completely restored after 10 min of ischemia [83 +/- 14% recovery of cardiac output (CO)], but was severely depressed after 15 and 20 min of ischemia (40 +/- 24 and 31 +/- 24% recovery of CO, respectively). Reperfusion was associated with partial recovery of HEP stores and enhanced degradation of phospholipids as indicated by the accumulation of fatty acids (FA). Myocardial FA content and enzyme release during reperfusion were correlated (r = 0.70), suggesting that membrane phospholipid degradation and cellular damage are closely related phenomena. To investigate the role of type IIA secretory phospholipase A2 (sPLA2) in this process, hearts from wild-type and sPLA2-deficient mice were subjected to ischemia-reperfusion. Postischemic functional recovery, ATP depletion, enzyme release, and FA accumulation were not significantly different between wild-type and sPLA2- deficient hearts. These findings argue against a prominent role of type IIA sPLA2 in the development of irreversible cell damage in the ischemic-reperfused murine myocardium.
Abstract: The zinc finger-containing transcription factors GATA4 and GATA6 are important regulators of basal and inducible gene expression in cardiac and smooth muscle cell types. Here we demonstrate a direct functional role for GATA4 and GATA6 as regulators of cardiomyocyte hypertrophic growth and gene expression. To model the increase in endogenous GATA4 and GATA6 transcriptional activity that occurs in response to hypertrophic stimulation, each factor was overexpressed in cardiomyocytes using recombinant adenovirus. Overexpression of either GATA4 or GATA6 was sufficient to induce cardiomyocyte hypertrophy characterized by enhanced sarcomeric organization, a greater than 2-fold increase in cell surface area, and a significant increase in total protein accumulation. In vivo, transgenic mice with 2.5-fold overexpression of GATA4 within the adult heart demonstrated a slowly progressing increase in heart to body weight ratio, histological features of cardiomyopathy, and activation of hypertrophy-associated genes, suggesting that GATA factors are sufficient regulators of cardiomyocyte hypertrophy in vitro and in vivo. To evaluate the requirement of GATA factors as downstream transcriptional mediators of hypertrophy, a dominant negative GATA4-engrailed repressor fusion-encoding adenovirus was generated. Expression of GATA4-engrailed blocked GATA4- and GATA6-directed transcriptional responses and agonist-induced cardiomyocyte hypertrophy, demonstrating that cardiac-expressed GATA factors are necessary mediators of this process.
Abstract: Heart disease remains one of the leading causes of morbidity and mortality in the industrialized nations of the world. Intense investigation has centered around identifying and manipulating intracellular signaling pathways that direct hypertrophic and myopathic responses in an attempt to intervene in the progression or reverse certain forms of heart disease. We show here that cyclosporin A-mediated inhibition of the calcium-regulated phosphatase, calcineurin (PP2B), reverses cardiac hypertrophy and myopathic dilation in two transgenic mouse models of cardiomyopathy. Reversal was demonstrated by gravimetric analysis, echocardiography, histological analysis, and molecular analysis of hypertrophy-associated gene expression. In contrast, a third mouse model of hypertrophic cardiomyopathy due to activated NFAT3 cardiac-specific expression was not affected by cyclosporin A. These results suggest that calcineurin may function in the long-term maintenance of cardiac hypertrophy or myopathic disease states.
Abstract: Cardiac hypertrophy is a major predictor of future morbidity and mortality. Recent investigation has centered around identifying the molecular signaling pathways that regulate cardiac myocyte reactivity with the goal of modulating pathologic hypertrophic programs. One potential regulator of cardiomyocyte hypertrophy is the calcium-sensitive phosphatase calcineurin. We show here that calcineurin enzymatic activity, mRNA, and protein levels are increased in cultured neonatal rat cardiomyocytes by hypertrophic agonists such as angiotensin II, phenylephrine, and 1% fetal bovine serum. This induction of calcineurin activity was associated with an increase in calcineurin Abeta (CnAbeta) mRNA and protein, but not in CnAalpha or CnAgamma. Agonist-dependent increases in calcineurin enzymatic activity were specifically inhibited with an adenovirus expressing a noncompetitive peptide inhibitor of calcineurin known as cain [Lai, M. M., Burnett, P. E., Wolosker, H., Blackshaw, S. & Snyder, S. H. (1998) J. Biol. Chem. 273, 18325-18331]. Targeted inhibition of calcineurin with cain or an adenovirus expressing only the calcineurin inhibitory domain of AKAP79 attenuated cardiomyocyte hypertrophy and atrial natriuretic factor expression in response to angiotensin II, phenylephrine, and 1% fetal bovine serum. These data demonstrate that calcineurin is an important regulator of cardiomyocyte hypertrophy in response to certain agonists and suggest that cyclosporin A and FK506 function to attenuate cardiac hypertrophy by specifically inhibiting calcineurin.
Abstract: We have previously shown that the calcium-calmodulin-regulated phosphatase calcineurin (PP2B) is sufficient to induce cardiac hypertrophy that transitions to heart failure in transgenic mice. Given the rapid onset of heart failure in these mice, we hypothesized that calcineurin signaling would stimulate myocardial cell apoptosis. However, utilizing multiple approaches, we determined that calcineurin-mediated hypertrophy protected cardiac myocytes from apoptosis, suggesting a model of heart failure that is independent of apoptosis. Adenovirally mediated gene transfer of a constitutively active calcineurin cDNA (AdCnA) was performed in cultured neonatal rat cardiomyocytes to elucidate the mechanism whereby calcineurin affected myocardial cell viability. AdCnA infection, which induced myocyte hypertrophy and atrial natriuretic factor expression, protected against apoptosis induced by 2-deoxyglucose or staurosporine, as assessed by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) labeling, caspase-3 activation, DNA laddering, and cellular morphology. The level of protection conferred by AdCnA was similar to that of adenoviral Bcl-x(L) gene transfer or hypertrophy induced by phenylephrine. In vivo, failing hearts from calcineurin-transgenic mice did not demonstrate increased TUNEL labeling and, in fact, demonstrated a resistance to ischemia/reperfusion-induced apoptosis. We determined that the mechanism whereby calcineurin afforded protection from apoptosis was partially mediated by nuclear factor of activated T cells (NFAT3) signaling and partially by Akt/protein kinase B (PKB) signaling. Although calcineurin activation protected myocytes from apoptosis, inhibition of calcineurin with cyclosporine was not sufficient to induce TUNEL labeling in Gqalpha-transgenic mice or in cultured cardiomyocytes. Collectively, these data identify a calcineurin-dependent mouse model of dilated heart failure that is independent of apoptosis.
Abstract: Multiple intracellular signaling pathways have been shown to regulate the hypertrophic growth of cardiomyocytes. Both necessary and sufficient roles have been described for the mitogen activated protein kinase(1) (MAPK) signaling pathway, specific protein kinase C (PKC) isoforms, and calcineurin. Here we investigate the interdependence between calcineurin, MAPK, and PKC isoforms in regulating cardiomyocyte hypertrophy using three separate approaches. Hearts from hypertrophic calcineurin transgenic mice were characterized for PKC and MAPK activation. Transgenic hearts demonstrated activation of c-Jun NH(2)-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK1/2), but not p38 MAPK factors. Calcineurin transgenic hearts demonstrated increased activation of PKCalpha, beta(1), and theta, but not of epsilon, beta(2), or lambda. In a second approach, cultured cardiomyocytes were infected with a calcineurin adenovirus to induce hypertrophy and the effects of pharmacologic inhibitors or co-infection with a dominant negative adenovirus were examined. Calcineurin-mediated hypertrophy was prevented with PKC inhibitors, Ca(2+) chelation, and attenuated with a dominant negative SEK-1 (MKK4) adenovirus, but inhibitors of ERK or p38 activation had no effect. In a third approach, we examined the activation of MAPK factors and PKC isoforms during the progression of load-induced hypertrophy in aortic banded rats with or without cyclosporine. We determined that inhibition of calcineurin activity with cyclosporine prevented PKCalpha, theta, and JNK activation, but did not affect PKCepsilon, beta, lambda, ERK1/2, or p38 activation. Collectively, these data indicate that calcineurin hypertrophic signaling is interconnected with PKCalpha, theta, and JNK in the heart, while PKCepsilon, beta, lambda, p38, and ERK1/2 are not involved in calcineurin-mediated hypertrophy.
Abstract: Vascular hypertension resulting in increased cardiac load is associated with left ventricular hypertrophy and is a leading predicator for progressive heart disease. The molecular signaling pathways that respond to increases in cardiac load are poorly understood. One potential regulator of the hypertrophic response is the calcium-sensitive phosphatase calcineurin.
Abstract: Members of the mitogen-activated protein kinase (MAPK) cascade such as extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 are implicated as important regulators of cardiomyocyte hypertrophic growth in culture. However, the role that individual MAPK pathways play in vivo has not been extensively evaluated. Here we generated nine transgenic mouse lines with cardiac-restricted expression of an activated MEK1 cDNA in the heart. MEK1 transgenic mice demonstrated concentric hypertrophy without signs of cardiomyopathy or lethality up to 12 months of age. MEK1 transgenic mice showed a dramatic increase in cardiac function, as measured by echocardiography and isolated working heart preparation, without signs of decompensation over time. MEK1 transgenic mice and MEK1 adenovirus-infected neonatal cardiomyocytes each demonstrated ERK1/2, but not p38 or JNK, activation. MEK1 transgenic mice and MEK1 adenovirus-infected cultured cardiomyocytes were also partially resistant to apoptotic stimuli. The results of the present study indicate that the MEK1-ERK1/2 signaling pathway stimulates a physiologic hypertrophy response associated with augmented cardiac function and partial resistance to apoptotsis.
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 DeltaAKAP79) 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.
Abstract: An improved, isolated, left ventricular-ejecting, murine heart model is described and evaluated. Special attention was paid to the design and impedance characteristics of the artificial aortic outflow tract and perfusate composition, which contained glucose (10 mM plus insulin) and pyruvate (1.5 mM) as substrates. Temperature of the isolated perfused hearts was maintained at 38.5 degrees C. During antegrade perfusion (preload 10 mm Hg, afterload 50 mm Hg, 2.5 mM Ca2+) proper design of the aortic outflow tract provided baseline values for cardiac output (CO), left ventricular developed pressure (LVDP) and the maximum first derivative of left ventricular pressure (LV dP/dtmax) of 11.1+/-1.7 ml min-1, 83+/-5 mm Hg and 6283+/-552 mm Hg s-1, respectively, resembling findings in the intact mouse. During 100 min normoxic antegrade perfusion CO declined non-significantly by less than 10%. Varying pre- and afterloads resulted in typical Frank-Starling relationships with maximal CO values of 18.6+/-1.8 ml min-1 at pre- and afterload pressures of 25 and 50 mm Hg, respectively. Left ventricular function curves were constructed at free [Ca2+] of 1.5 and 2.5 mM in the perfusion medium. Significantly higher values for CO, LVDP and LV dP/dtmax and LV dP/dtmin were obtained at 2.5 mM Ca2+ at all loading conditions investigated. Phosphocreatine and creatine levels remained stable throughout the perfusion period. Despite a small but significant decline in tissue ATP content, the sum of adenine nucleotides did not change during the normoxic perfusion period. The tissue content of glycogen increased significantly.
Abstract: Under pathophysiological conditions, like myocardial ischemia and reperfusion, cardiac phospholipid homeostasis is severely disturbed, resulting in a net degradation of phospholipids and the accumulation of degradation products, such as lysophospholipids and (non-esterified) fatty acids. The derangements in phospholipid metabolism are thought to be involved in the sequence of events leading to irreversible myocardial injury. The net degradation of phospholipids as observed during myocardial ischemia may result from increased hydrolysis and/or reduced resynthesis, while during reperfusion hydrolysis is likely to prevail in this net degradation. Several studies indicate that the activation of phospholipases A2 plays an important role in the hydrolysis of phospholipids. In this review current knowledge regarding the potential role of the different types of phospholipases A2 in ischemia and reperfusion-induced damage is being evaluated. Furthermore, it is indicated how recent advances in molecular biological techniques could be helpful in determining whether disturbances in phospholipid metabolism indeed play a crucial role in the transition from reversible to irreversible myocardial ischemia and reperfusion-induced injury, the knowledge of which could be of great therapeutic relevance.
Abstract: Phospholipase A2 has been considered to play a role in physiological membrane turnover in cardiac tissue and in the degradation of membrane lipids under pathophysiological conditions, such as ischemia and reperfusion. We report the cloning of a cDNA encoding a member of the Ca2+-dependent, low molecular mass phospholipase A2 (PLA2) present in rat heart. The cDNA predicts a mature protein of 146 amino acid residues including a 21 amino acid sequence at the N-terminal end, which has the features characteristic of eukaryotic secretory signal peptides. The deduced amino acid sequence constitutes an enzyme of the group II class of PLA2s, and resembles PLA2s from other mammalian sources. A Northern blot analysis performed to determine the tissue distribution showed that rat ileum contains the largest amount of the PLA2 transcript among the tissues examined, a weaker signal was present in heart, spleen and soleus muscle, and no signal could be detected in EDL muscle, stomach, liver, kidney, brain and lung. Northern blot analysis and reverse transcriptase-polymerase chain reaction (RT-PCR) techniques indicate the presence of this enzyme in neonatal and adult rat cardiomyocytes and in a cultured rat cardiac fibroblast-like cell line, but not in rat cardiac-derived endothelial cell lines. Transcription levels of rat heart group II PLA2 in isolated neonatal rat cardiomyocytes were found to increase after stimulating the cells with tumor necrosis factor-alpha (TNF-alpha) or the alpha1-adrenergic agonist phenylephrine.
