Cardioprotection by the adiponectin receptor agonist ALY688 in a preclinical mouse model of heart failure with reduced ejection fraction (HFrEF).

AIMS: Adiponectin has been shown to mediate cardioprotective effects and levels are typically reduced in patients with cardiometabolic disease. Hence, there has been intense interest in developing adiponectin-based therapeutics. The aim of this translational research study was to examine the functional significance of targeting adiponectin signaling with the adiponectin receptor agonist ALY688 in a mouse model of heart failure with reduced ejection fraction (HFrEF), and the mechanisms of cardiac remodeling leading to cardioprotection. METHODS AND RESULTS: Wild-type mice were subjected to transverse aortic constriction (TAC) to induce left ventricular pressure overload (PO), or sham surgery, with or without daily subcutaneous ALY688-SR administration. Temporal analysis of cardiac function was conducted via weekly echocardiography for 5 weeks and we observed that ALY688 attenuated the PO-induced dysfunction. ALY688 also reduced cardiac hypertrophic remodeling, assessed via LV mass, heart weight to body weight ratio, cardiomyocyte cross sectional area, ANP and BNP levels. ALY688 also attenuated PO-induced changes in myosin light and heavy chain expression. Collagen content and myofibroblast profile indicated that fibrosis was attenuated by ALY688 with TIMP1 and scleraxis/periostin identified as potential mechanistic contributors. ALY688 reduced PO-induced elevation in circulating cytokines including IL-5, IL-13 and IL-17, and the chemoattractants MCP-1, MIP-1ß, MIP-1alpha and MIP-3α. Assessment of myocardial transcript levels indicated that ALY688 suppressed PO-induced elevations in IL-6, TLR-4 and IL-1ß, collectively indicating anti-inflammatory effects. Targeted metabolomic profiling indicated that ALY688 increased fatty acid mobilization and oxidation, increased betaine and putrescine plus decreased sphingomyelin and lysophospholipids, a profile indicative of improved insulin sensitivity. CONCLUSION: These results indicate that the adiponectin mimetic peptide ALY688 reduced PO-induced fibrosis, hypertrophy, inflammation and metabolic dysfunction and represents a promising therapeutic approach for treating HFrEF in a clinical setting.

Statins Protect Against Early Stages of Doxorubicin-induced Cardiotoxicity Through the Regulation of Akt Signaling and SERCA2.

Background: Doxorubicin-induced cardiomyopathy (DICM) is one of the complications that can limit treatment for a significant number of cancer patients. In animal models, the administration of statins can prevent the development of DICM. Therefore, the use of statins with anthracyclines potentially could enable cancer patients to complete their chemotherapy without added cardiotoxicity. The precise mechanism mediating the cardioprotection is not well understood. The purpose of this study is to determine the molecular mechanism by which rosuvastatin confers cardioprotection in a mouse model of DICM. Methods: Rosuvastatin was intraperitoneally administered into adult male mice at 100 µg/kg daily for 7 days, followed by a single intraperitoneal doxorubicin injection at 10 mg/kg. Animals continued to receive rosuvastatin daily for an additional 14 days. Cardiac function was assessed by echocardiography. Optical calcium mapping was performed on retrograde Langendorff perfused isolated hearts. Ventricular tissue samples were analyzed by immunofluorescence microscopy, Western blotting, and quantitative polymerase chain reaction. Results: Exposure to doxorubicin resulted in significantly reduced fractional shortening (27.4% ± 1.11% vs 40% ± 5.8% in controls; P < 0.001) and re-expression of the fetal gene program. However, we found no evidence of maladaptive cardiac hypertrophy or adverse ventricular remodeling in mice exposed to this dose of doxorubicin. In contrast, rosuvastatin-doxorubicin-treated mice maintained their cardiac function (39% ± 1.26%; P < 0.001). Mechanistically, the effect of rosuvastatin was associated with activation of Akt and phosphorylation of phospholamban with preserved sarcoplasmic/endoplasmic reticulum Ca2+ transporting 2 (SERCA2)-mediated Ca2+ reuptake. These effects occurred independently of perturbations in ryanodine receptor 2 function. Conclusions: Rosuvastatin counteracts the cardiotoxic effects of doxorubicin by directly targeting sarcoplasmic calcium cycling.

Contexte: La cardiomyopathie induite par la doxorubicine (CMID) est l'une des complications pouvant limiter le traitement d'un nombre considérable de patients atteints de cancer. Dans des modèles animaux, l'administration de statines peut prévenir l'apparition d'une CMID. Ainsi, l'utilisation de statines avec les anthracyclines pourrait vraisemblablement permettre aux patients de compléter leur chimiothérapie en évitant une cardiotoxicité supplémentaire. Le mécanisme précis qui sous-tend cet effet cardioprotecteur n'est pas entièrement élucidé. Cette étude a pour objectif de déterminer dans un modèle murin de CMID le mécanisme moléculaire par lequel la rosuvastatine confère une cardioprotection. Méthodologie: La rosuvastatine a été administrée par voie intrapéritonéale à des souris adultes mâles à une dose de 100 µg/kg par jour pendant sept jours, suivie d'une dose unique de doxorubicine de 10 mg/kg administrée par injection intrapéritonéale. Les animaux poursuivaient ensuite le traitement par la rosuvastatine une fois par jour pendant 14 jours supplémentaires. La fonction cardiaque a été mesurée par échocardiographie. Une cartographie optique du calcium a été réalisée sur des cœurs isolés soumis à une perfusion rétrograde selon la méthode de Langendorff. Des échantillons de tissu ventriculaire ont été analysés par microscopie en immunofluorescence, par buvardage de western et par mesure quantitative de l'amplification en chaîne par polymérase. Résultats: L'exposition à la doxorubicine a entraîné une diminution significative de la fraction de raccourcissement (27,4 % ± 1,11 % vs 40 % ± 5,8 % dans le groupe témoin; p < 0,001) et la réexpression du programme génique fœtal. Toutefois, aucune hypertrophie cardiaque inadaptée ni aucun remodelage ventriculaire indésirable n'ont été observés chez les souris ayant été exposées à la dose de doxorubicine étudiée. En revanche, la fonction cardiaque a été préservée chez les souris traitées par l'association rosuvastatine-doxorubicine (39 % ± 1,26 %; p < 0,001). Sur le plan du mode d'action, l'effet de la rosuvastatine a été associé à une activation de l'Akt et à une phosphorylation du phospholambane, avec préservation du recaptage de Ca2+ médié par la pompe SERCA2 (sarcoplasmic/endoplasmic reticulum Ca 2+ transporting 2). Ces effets sont survenus indépendamment des perturbations de la fonction du récepteur RyR2 (ryanodine receptor 2). Conclusions: La rosuvastatine neutralise les effets cardiotoxiques de la doxorubicine en ciblant directement la circulation sarcoplasmique du calcium.

Cardioprotective effects of dantrolene in doxorubicin-induced cardiomyopathy in mice.

BACKGROUND: Doxorubicin (Dox) is a potent chemotherapeutic agent, but its usage is limited by dose-dependent cardiotoxicity. Intracellular calcium dysregulation has been reported to be involved in doxorubicin-induced cardiomyopathy (DICM). The cardioprotective role of RyR stabilizer dantrolene (Dan) on the calcium dynamics of DICM has not yet been explored. OBJECTIVE: To evaluate the effects of dantrolene on intracellular calcium dysregulation and cardiac contractile function in a DICM model. METHODS: Adult male C57BL/6 mice were randomized into 4 groups: (1) Control, (2) Dox Only, (3) Dan Only, and (4) Dan + Dox. Fractional shortening (FS) and left ventricular ejection fraction (LVEF) were assessed by echocardiography. In addition, mice were sacrificed 2 weeks after doxorubicin injection for optical mapping of the heart in a Langendorff setup. RESULTS: Treatment with Dox was associated with a reduction in both FS and LVEF at 2 weeks (P < .0001) and 4 weeks (P < .006). Dox treatment was also associated with prolongation of calcium transient durations CaTD50 (P = .0005) and CaTD80 (P < .0001) and reduction of calcium amplitude alternans ratio (P < .0001). Concomitant treatment with Dan prevented the Dox-induced decline in FS and LVEF (P < .002 at both 2 and 4 weeks). Dan also prevented Dox-induced prolongation of CaTD50 and CaTD80 and improved the CaT alternans ratio (P < .0001). Finally, calcium transient rise time was increased in the doxorubicin-treated group, indicating RyR2 dyssynchrony, and dantrolene prevented this prolongation (P = .02). CONCLUSION: Dantrolene prevents cardiac contractile dysfunction following doxorubicin treatment by mitigating dysregulation of calcium dynamics.

Inhibiting the Pkm2/b-catenin axis drives in vivo replication of adult cardiomyocytes following experimental MI.

Adult mammalian cardiomyocytes (CM) are postmitotic, differentiated cells that cannot re-enter the cell cycle after any appreciable injury. Therefore, understanding the factors required to induce CM proliferation for repair is of great clinical importance. While expression of muscle pyruvate kinase 2 (Pkm2), a cytosolic enzyme catalyzing the final step in glycolysis, is high in end-stage heart failure (HF), the loss of Pkm2 promotes proliferation in some cellular systems, in vivo. We hypothesized that in the adult heart CM proliferation may require low Pkm2 activity. Thus, we investigated the potential for Pkm2 to regulate CM proliferation in a mouse model of myocardial infarction (MI) employing inducible, cardiac-specific Pkm2 gene knockout (Pkm2KOi) mice. We found a lack of cardiac hypertrophy or expression of the fetal gene program in Pkm2KOi mice post MI, as compared to vehicle control animals (P < 0.01), correlating with smaller infarct size, improved mitochondrial (mt) function, enhanced angiogenesis, reduced degree of CM apoptosis, and reduced oxidative stress post MI. There was significantly higher numbers of dividing CM in the infarct zone between 3-9 days post MI (P < 0.001). Mechanistically, we determined that Pkm2 interacts with ß-catenin (Ctnnb1) in the cytoplasm of CM, inhibiting Ctnnb1 phosphorylation at serine 552 and tyrosine 333, by Akt. In the absence of Pkm2, Ctnnb1 translocates to the nucleus leading to transcriptional activation of proliferation-associated target genes. All these effects are abrogated by genetic co-deletion of Pkm2 and Ctnnb1. Collectively, this work supports a novel antiproliferative function for Pkm2 in CM through the sequestration of Ctnnb1 in the cytoplasm of CM whereas loss of Pkm2 is essential for CM proliferation. Reducing cardiac Pkm2 expression may provide a useful strategy for cardiac repair after MI in patients.

Haploinsufficiency of RREB1 causes a Noonan-like RASopathy via epigenetic reprogramming of RAS-MAPK pathway genes.

RAS-MAPK signaling mediates processes critical to normal development including cell proliferation, survival, and differentiation. Germline mutation of RAS-MAPK genes lead to the Noonan-spectrum of syndromes. Here, we present a patient affected by a 6p-interstitial microdeletion with unknown underlying molecular etiology. Examination of 6p-interstitial microdeletion cases reveals shared clinical features consistent with Noonan-spectrum disorders including short stature, facial dysmorphia and cardiovascular abnormalities. We find the RAS-responsive element binding protein-1 (RREB1) is the common deleted gene in multiple 6p-interstitial microdeletion cases. Rreb1 hemizygous mice display orbital hypertelorism and cardiac hypertrophy phenocopying the human syndrome. Rreb1 haploinsufficiency leads to sensitization of MAPK signaling. Rreb1 recruits Sin3a and Kdm1a to control H3K4 methylation at MAPK pathway gene promoters. Haploinsufficiency of SIN3A and mutations in KDM1A cause syndromes similar to RREB1 haploinsufficiency suggesting genetic perturbation of the RREB1-SIN3A-KDM1A complex represents a new category of RASopathy-like syndromes arising through epigenetic reprogramming of MAPK pathway genes.

Anthracycline-induced cardiomyopathy: cellular and molecular mechanisms.

Despite the known risk of cardiotoxicity, anthracyclines are widely prescribed chemotherapeutic agents. They are broadly characterized as being a robust effector of cellular apoptosis in rapidly proliferating cells through its actions in the nucleus and formation of reactive oxygen species (ROS). And, despite the early use of dexrazoxane, no effective treatment strategy has emerged to prevent the development of cardiomyopathy, despite decades of study, suggesting that much more insight into the underlying mechanism of the development of cardiomyopathy is needed. In this review, we detail the specific intracellular activities of anthracyclines, from the cell membrane to the sarcoplasmic reticulum, and highlight potential therapeutic windows that represent the forefront of research into the underlying causes of anthracycline-induced cardiomyopathy.

Tracking adiponectin biodistribution via fluorescence molecular tomography indicates increased vascular permeability after streptozotocin-induced diabetes.

Adiponectin, a highly abundant polypeptide hormone in plasma, plays an important role in the regulation of energy metabolism in a wide variety of tissues, as well as providing important beneficial effects in diabetes, inflammation, and cardiovascular disease. To act on target tissues, adiponectin must move from the circulation to the interstitial space, suggesting that vascular permeability plays an important role in regulating adiponectin action. To test this hypothesis, fluorescently labeled adiponectin was used to monitor its biodistribution in mice with streptozotocin-induced diabetes (STZD). Adiponectin was, indeed, found to have increased sequestration in the highly fenestrated liver and other tissues within 90 min in STZD mice. In addition, increased myocardial adiponectin was detected and confirmed using computed tomography (CT) coregistration. This provided support of adiponectin delivery to affected cardiac tissue as a cardioprotective mechanism. Higher adiponectin content in the STZD heart tissues was further examined by ex vivo fluorescence molecular tomography (FMT) imaging, immunohistochemistry, and Western blot analysis. In vitro mechanistic studies using an endothelial monolayer on inserts and three-dimensional microvascular networks on microfluidic chips further confirmed that adiponectin flux was increased by high glucose. However, in the in vitro model and mouse heart tissue, high glucose levels did not change adiponectin receptor levels. An examination of the tight junction (TJ) complex revealed a decrease in the TJ protein claudin (CLDN)-7 in high glucose-treated endothelial cells, and the functional significance of this change was underscored by increased endothelium permeability upon siRNA-mediated knockdown of CLDN-7. Our data support the idea that glucose-induced effects on permeability of the vascular endothelium contribute to the actions of adiponectin by regulating its transendothelial movement from blood to the interstitial space. These observations are physiologically significant and critical when considering ways to harness the therapeutic potential of adiponectin for diabetes.

An adiponectin-S1P axis protects against lipid induced insulin resistance and cardiomyocyte cell death via reduction of oxidative stress.

BACKGROUND: Adiponectin exerts several beneficial cardiovascular effects, however their specific molecular mechanisms require additional understanding. This study investigated the mechanisms of adiponectin action in the heart during high fat diet (HFD) feeding or in palmitate (PA) treated H9c2 cardiomyoblasts. METHODS: 6-week-old male adiponectin knock out (Ad-KO) mice were fed chow or 60% HFD for 6 weeks then received saline or recombinant adiponectin (3µg/g body weight) for an additional 2 weeks. After acute insulin stimulation (4 U/kg), tissue and serum samples were collected for analysis. H9c2 cardiomyocytes were treated ±0.1 mM PA, the adiponectin receptor agonist AdipoRon, or the antioxidant MnTBAP then assays to analyze reactive oxygen species (ROS) production and cell death were conducted. To specifically determine the mechanistic role of S1P, gain and loss of function studies were conducted with adding S1P to cells or the inhibitors THI and SKI-II, respectively. RESULTS: HFD feeding induced cardiac insulin resistance in Ad-KO mice, which was reversed following replenishment of normal circulating adiponectin levels. In addition, myocardial total triglyceride was elevated by HFD and lipidomic analysis showed increased levels of ceramides and sphingosine-1-phosphate (S1P), with only the latter being corrected by adiponectin administration. Similarly, treatment of H9C2 cardiomyoblasts with PA led to a significant increase of intracellular S1P but not in conditioned media whereas AdipoRon significantly increased S1P production and secretion from cells. AdipoRon or the antioxidant MnTBAP significantly reduced PA-induced cell death. Gain and loss of function studies suggested S1P secretion and autocrine receptor activation mediated the effect of AdipoRon to attenuate PA-induced ROS production and cell death. CONCLUSION: Our data establish adiponectin signaling-mediated increase in S1P secretion as a mechanism via which HFD or PA induced cardiomyocyte lipotoxicity, leading to insulin resistance and cell death, is attenuated.

Molecular mechanisms in cardiomyopathy.

Cardiomyopathies represent a heterogeneous group of diseases that negatively affect heart function. Primary cardiomyopathies specifically target the myocardium, and may arise from genetic [hypertrophic cardiomyopathy (HCM), arrhythmogenic right ventricular cardiomyopathy/dysplasia (ARVC/D), mitochondrial cardiomyopathy] or genetic and acquired [dilated cardiomyopathy (DCM), restrictive cardiomyopathy (RCM)] etiology. Modern genomics has identified mutations that are common in these populations, while in vitro and in vivo experimentation with these mutations have provided invaluable insight into the molecular mechanisms native to these diseases. For example, increased myosin heavy chain (MHC) binding and ATP utilization lead to the hypercontractile sarcomere in HCM, while abnormal protein-protein interaction and impaired Ca2+ flux underlie the relaxed sarcomere of DCM. Furthermore, expanded access to genetic testing has facilitated identification of potential risk factors that appear through inheritance and manifest sometimes only in the advanced stages of the disease. In this review, we discuss the genetic and molecular abnormalities unique to and shared between these primary cardiomyopathies and discuss some of the important advances made using more traditional basic science experimentation.

The E3 ligase Mule protects the heart against oxidative stress and mitochondrial dysfunction through Myc-dependent inactivation of Pgc-1α and Pink1.

Cardiac homeostasis requires proper control of protein turnover. Protein degradation is principally controlled by the Ubiquitin-Proteasome System. Mule is an E3 ubiquitin ligase that regulates cellular growth, DNA repair and apoptosis to maintain normal tissue architecture. However, Mule's function in the heart has yet to be described. In a screen, we found reduced Mule expression in left ventricular samples from end-stage heart failure patients. Consequently, we generated conditional cardiac-specific Mule knockout (Mule fl/fl(y);mcm) mice. Mule ablation in adult Mule fl/fl(y);mcm mice prevented myocardial c-Myc polyubiquitination, leading to c-Myc accumulation and subsequent reduced expression of Pgc-1α, Pink1, and mitochondrial complex proteins. Furthermore, these mice developed spontaneous cardiac hypertrophy, left ventricular dysfunction, and early mortality. Co-deletion of Mule and c-Myc rescued this phenotype. Our data supports an indispensable role for Mule in cardiac homeostasis through the regulation of mitochondrial function via maintenance of Pgc-1α and Pink1 expression and persistent negative regulation of c-Myc.

Cellular, structural and functional cardiac remodelling following pressure overload and unloading.

BACKGROUND: The cardiac remodelling process in advanced heart failure due to pressure overload has not been clearly defined but likely involves mechanisms of cardiac fibrosis and cardiomyocyte hypertrophy. The aim of this study was to examine pressure overload (PO)-induced cardiac remodelling processes and their reversibility after unloading in both humans with heart failure and a mouse model of PO induced by aortic constriction. METHODS & RESULTS: Speckle tracking echocardiography showed PO-induced cardiac dysfunction in mice was reversible after removal of aortic constriction to unload. Masson's Trichrome staining suggested that PO-induced myocardial fibrosis was reversible, however detailed analysis of 3-dimensional collagen architecture by scanning electron microscopy demonstrated that matrix remodelling was not completely normalised as a disorganised network of thin collagen fibres was evident. Analysis of human left ventricular biopsy samples from HF patients revealed increased presence of large collagen fibres which were greatly reduced in paired samples from the same individuals after unloading by left ventricular assist device implantation. Again, an extensive network of small collagen fibres was still clearly seen to closely surround cardiomyocytes after unloading. Other features of PO-induced remodelling including increased myofibroblast content, cardiomyocyte disarray and hypertrophy were largely reversed upon unloading in both humans and mouse model. Previous work in humans demonstrated that receptors for adiponectin, an important mediator of cardiac fibrosis and hypertrophy, decreased in heart failure patients and returned to normal after unloading. Here we provide novel data showing a similar trend for adiponectin receptor adaptor protein APPL1, but not APPL2 isoform. CONCLUSIONS: LV unloading diminishes PO-induced cardiac remodelling and improves function. These findings add new insights into the cardiac remodelling process, and provide novel targets for future pharmacologic therapies.

Adiponectin is required for cardiac MEF2 activation during pressure overload induced hypertrophy.

Cardiomyocyte (CM) hypertrophy and increased heart mass in response to pressure overload are associated with hyper-activation of the myocyte enhancer factor-2 (MEF2) family of transcriptional regulators, and concomitant initiation of the fetal gene program. Adiponectin, an adipokine that is reduced in individuals with obesity and diabetes, has been characterized both as a negative regulator or permissive factor in cardiac hypertrophy. We therefore sought to analyze temporal regulation of MEF2 activity in response to pressure overload (PO) and changes in adiponectin status. To address this we crossed a well characterized transgenic MEF2 "sensor" mouse (MEF2-lacZ) with adiponectin null mice (Ad-KO) to create compound MEF2 lacZ/Ad-KO mice. Initially, we established that transverse aortic banding induced PO in wild-type (WT) mice increased heart mass and CM hypertrophy from 1 to 4weeks following surgery, indicated by increased CM diameter and heart weight/tibia length ratio. This was associated with cardiac dysfunction determined by echocardiography. Hypertrophic changes and dysfunction were observed in Ad-KO mice 4weeks following surgery. MEF2 lacZ activity and endogenous ANF mRNA levels, used as indicators of hypertrophic gene activation, were both robustly increased in WT mice after MTAB but attenuated in the Ad-KO background. Furthermore, activation of the pro-hypertrophic molecule p38 was increased following MTAB surgery in WT mice, but not in Ad-KO animals, and treatment of primary isolated CM with recombinant adiponectin induced p38 phosphorylation in a time dependent manner. Adiponectin also increased MEF2 activation in primary cardiomyocytes, an effect attenuated by p38 MAPK inhibition. In conclusion, our data indicate that robust hypertrophic MEF2 activation in the heart in vivo requires a background of adiponectin signaling and that adiponectin signaling in primary isolated CM directly enhances MEF2 activity through activation of p38 MAPK. We conclude that adiponectin is required for full induction of cardiomyocyte MEF2 activation, thus contributing to the myocardial hypertrophic gene expression program in response to PO.

Pressure Overload-Induced Cardiac Dysfunction in Aged Male Adiponectin Knockout Mice Is Associated With Autophagy Deficiency.

Heart failure is a leading cause of death, especially in the elderly or obese and diabetic populations. Various remodeling events have been characterized, which collectively contribute to the progression of heart failure. Of particular interest, autophagy has recently emerged as an important determinant of cardiac remodeling and function. Here, we used aged, 13-month-old, male adiponectin knockout (Ad-KO) or wild-type (wt) mice subjected to aortic banding to induce pressure overload (PO). Cardiac strain analysis using speckle tracking echocardiography indicated significant dysfunction at an earlier stage in Ad-KO than wt. Analysis of autophagy by Western blotting for Light Chain 3 or microtubule-associated proteins 1B and Sequestosome 1 together with transmission electron microscopy of left ventricular tissue indicated a lack of PO-induced cardiac autophagy in Ad-KO compared with wt mice. Associated with this was mitochondrial degeneration and evidence of enhanced endoplasmic reticulum stress. Western blotting for Light Chain 3 or microtubule-associated proteins 1B, examination of flux using tandem fluoresent tagged-Light Chain 3, and analysis of lysosomal activity in H9c2 cardiac myoblasts treated with adiponectin indicated that adiponectin enhanced autophagy flux. In conclusion, adiponectin directly stimulates autophagic flux and the lack of autophagy in response to PO in aged mice lacking adiponectin may contribute to cellular events which exacerbate the development of cardiac dysfunction.

Temporal and Molecular Analyses of Cardiac Extracellular Matrix Remodeling following Pressure Overload in Adiponectin Deficient Mice.

Adiponectin, circulating levels of which are reduced in obesity and diabetes, mediates cardiac extracellular matrix (ECM) remodeling in response to pressure overload (PO). Here, we performed a detailed temporal analysis of progressive cardiac ECM remodelling in adiponectin knockout (AdKO) and wild-type (WT) mice at 3 days and 1, 2, 3 and 4 weeks following the induction of mild PO via minimally invasive transverse aortic banding. We first observed that myocardial adiponectin gene expression was reduced after 4 weeks of PO, whereas increased adiponectin levels were detected in cardiac homogenates at this time despite decreased circulating levels of adiponectin. Scanning electron microscopy and Masson's trichrome staining showed collagen accumulation increased in response to 2 and 4 weeks of PO in WT mice, while fibrosis in AdKO mice was notably absent after 2 weeks but highly apparent after 4 weeks of PO. Time and intensity of fibroblast appearance after PO was not significantly different between AdKO and WT animals. Gene array analysis indicated that MMP2, TIMP2, collagen 1α1 and collagen 1α3 were induced after 2 weeks of PO in WT but not AdKO mice. After 4 weeks MMP8 was induced in both genotypes, MMP9 only in WT mice and MMP1α only in AdKO mice. Direct stimulation of primary cardiac fibroblasts with adiponectin induced a transient increase in total collagen detected by picrosirius red staining and collagen III levels synthesis, as well as enhanced MMP2 activity detected via gelatin zymography. Adiponectin also enhanced fibroblast migration and attenuated angiotensin-II induced differentiation to a myofibroblast phenotype. In conclusion, these data indicate that increased myocardial bioavailability of adiponectin mediates ECM remodeling following PO and that adiponectin deficiency delays these effects.

Adiponectin mediated APPL1-AMPK signaling induces cell migration, MMP activation, and collagen remodeling in cardiac fibroblasts.

Defects in adiponectin action have been implicated in the development of cardiac dysfunction in obesity and diabetes. Cardiac fibroblasts play an important role in regulating extracellular matrix remodeling yet little is known regarding the direct effects of adiponectin on cardiac fibroblasts. In this study, we first demonstrated temporal relocalization of cellular APPL1 in response to adiponectin in primary cardiac fibroblasts and that siRNA-mediated knockdown of APPL1 attenuated stimulation of AMPK by adiponectin. The cell surface content of MT1-MMP and activation of MMP2 were induced by adiponectin and these responses were dependent on AMPK signaling. Enhanced MMP activity facilitated increased fibroblast migration in response to adiponectin which was also prevented by inhibition of AMPK, with no change in cell proliferation observed. Collagen and elastin immunofluorescence demonstrated reorganization of the extracellular matrix in accordance with increased MMP activity, whereas quantitative mRNA analysis, (3) H-proline incorporation and picrosirius red assays showed no change in intracellular or extracellular total collagen levels in response to adiponectin. In summary, these data are the first to report the adiponectin stimulated APPL1-AMPK signaling axis in cardiac fibroblasts and characterize MT1-MMP translocation, MMP2 activity and cell migration as functional outcomes. These effects may be of significance in heart failure associated with obesity and diabetes.

Adiponectin action: a combination of endocrine and autocrine/paracrine effects.

The widespread physiological actions of adiponectin have now been well characterized as clinical studies and works in animal models have established strong correlations between circulating adiponectin level and various disease-related outcomes. Thus, conventional thinking attributes many of adiponectin's beneficial effects to endocrine actions of adipose-derived adiponectin. However, it is now clear that several tissues can themselves produce adiponectin and there is growing evidence that locally produced adiponectin can mediate functionally important autocrine or paracrine effects. In this review article we discuss regulation of adiponectin production, its mechanism of action via receptor isoforms and signaling pathways, and its principal physiological effects (i.e., metabolic and cardiovascular). The role of endocrine actions of adiponectin and changes in local production of adiponectin or its receptors in whole body physiology is discussed.

Sensitivity of repetitive facial-nerve stimulation in patients with myasthenia gravis.

Repetitive stimulation of the facial nerve is commonly performed in cases of suspected myasthenia gravis (MG) because bulbar weakness is often present, but the most sensitive facial muscle is unknown. We compared the sensitivity of repetitive nerve stimulation (RNS) to the frontalis and nasalis muscles in 244 patients with suspected MG. We found no difference in sensitivity of RNS when recording from these muscles in both ocular and generalized MG. In addition, we confirmed the low sensitivity of RNS for ocular (18%) or generalized (47%) MG. The specificity of facial RNS for both muscles was 100% and, in certain circumstances, may obviate the need for further diagnostic testing.