Overcoming Insulin Insufficiency by Forced Follistatin Expression in ß-cells of db/db Mice.

Diabetes poses a substantial burden to society as it can lead to serious complications and premature death. The number of cases continues to increase worldwide. Two major causes of diabetes are insulin resistance and insulin insufficiency. Currently, there are few antidiabetic drugs available that can preserve or protect ß-cell function to overcome insulin insufficiency in diabetes. We describe a therapeutic strategy to preserve ß-cell function by overexpression of follistatin (FST) using an AAV vector (AAV8-Ins-FST) in diabetic mouse model. Overexpression of FST in the pancreas of db/db mouse increased ß-cell islet mass, decreased fasting glucose level, alleviated diabetic symptoms, and essentially doubled lifespan of the treated mice. The observed islet enlargement was attributed to ß-cell proliferation as a result of bioneutralization of myostatin and activin by FST. Overall, our study indicates overexpression of FST in the diabetic pancreas preserves ß-cell function by promoting ß-cell proliferation, opening up a new therapeutic avenue for the treatment of diabetes.

Enhancing muscle membrane repair by gene delivery of MG53 ameliorates muscular dystrophy and heart failure in δ-Sarcoglycan-deficient hamsters.

Muscular dystrophies (MDs) are caused by genetic mutations in over 30 different genes, many of which encode for proteins essential for the integrity of muscle cell structure and membrane. Their deficiencies cause the muscle vulnerable to mechanical and biochemical damages, leading to membrane leakage, dystrophic pathology, and eventual loss of muscle cells. Recent studies report that MG53, a muscle-specific TRIM-family protein, plays an essential role in sarcolemmal membrane repair. Here, we show that systemic delivery and muscle-specific overexpression of human MG53 gene by recombinant adeno-associated virus (AAV) vectors enhanced membrane repair, ameliorated pathology, and improved muscle and heart functions in δ-sarcoglycan (δ-SG)-deficient TO-2 hamsters, an animal model of MD and congestive heart failure. In addition, MG53 overexpression increased dysferlin level and facilitated its trafficking to muscle membrane through participation of caveolin-3. MG53 also protected muscle cells by activating cell survival kinases, such as Akt, extracellular signal-regulated kinases (ERK1/2), and glycogen synthase kinase-3ß (GSK-3ß) and inhibiting proapoptotic protein Bax. Our results suggest that enhancing the muscle membrane repair machinery could be a novel therapeutic approach for MD and cardiomyopathy, as demonstrated here in the limb girdle MD (LGMD) 2F model.

Post-Natal knockdown of fukutin-related protein expression in muscle by long-termRNA interference induces dystrophic pathology [corrected].

Limb-girdle muscular dystrophy 2I (LGMD2I) is caused by mutations in the fukutin-related protein (FKRP) gene. Unlike its severe allelic forms, LGMD2I usually involves slower onset and milder course without defects in the central nervous system. The lack of viable animal models that closely recapitulate LGMD2I clinical phenotypes led us to use RNA interference technology to knock down FKRP expression via postnatal gene delivery so as to circumvent embryonic lethality. Specifically, an adeno-associated viral vector was used to deliver short hairpin (shRNA) genes to healthy ICR mice. Adeno-associated viral vectors expressing a single shRNA or two different shRNAs were injected one time into the hind limb muscles. We showed that FKRP expression at 10 months postinjection was reduced by about 50% with a single shRNA and by 75% with the dual shRNA cassette. Dual-cassette injection also reduced a-dystroglycan glycosylation and its affinity to laminin by up to 70% and induced α-dystrophic pathology, including fibrosis and central nucleation, in more than 50% of the myofibers at 10 months after injection. These results suggest that the reduction of approximately or more than 75% of the normal level of FKRP expression induces chronic dystrophic phenotypes in skeletal muscles. Furthermore, the restoration of about 25% of the normal FKRP level could be sufficient for LGMD2I therapy to correct the genetic deficiency effectively and prevent dystrophic pathology.

Recovery from decompensated heart failure is associated with a distinct, phase-dependent gene expression profile.

BACKGROUND: Clinical and experimental studies have traditionally focused on understanding the mechanisms for why a heart fails. We hypothesize that the pathways involved with myocardial recovery are not simply the reverse of those that cause heart failure. However, determining when and how a decompensated heart can recover remains unknown. METHODS: Male C57BL/6 mice underwent minimally invasive aortic banding for 3, 4, or 6 wk with or without subsequent band removal for 1 wk (debanding). Physiologic and genomic characterization was performed with intracardiac pressure-volume recordings, rt-PCR, and microarray analysis. RESULTS: Heart weight/body weight ratios and PV loops demonstrated a transition from compensated left ventricular hypertrophy to decompensated heart failure between 3 and 4 wk. Pressure-relief afforded by debanding allowed functional recovery and normalization of LVH after both 3 and 4, but not 6 wk of banding. Whole genome microarrays demonstrated 397 genes differentially expressed in recovered hearts, 250 genes differentially expressed in the nonrecoverable (6 wk) hearts, and only 10 genes shared by both processes. In particular, altered expression patterns of apoptotic and metalloproteinase genes correlated with the heart's ability to functionally recover. CONCLUSIONS: This clinically-relevant model (1) allows us to temporally and mechanistically characterize the failing heart, (2) demonstrates a unique genomic signature that may predict when a failing heart can recover following pressure relief, and (3) will prove useful as a template for testing therapeutic strategies aimed at recovery of the failing heart.

Loss of microRNAs in neural crest leads to cardiovascular syndromes resembling human congenital heart defects.

OBJECTIVE: Congenital heart defects represent the most common human birth defects. Even though the genetic cause of these syndromes has been linked to candidate genes, the underlying molecular mechanisms are still largely unknown. Disturbance of neural crest cell (NCC) migration into the derivatives of the pharyngeal arches and pouches can account for many of the developmental defects. The goal of this study was to investigate the function of microRNA (miRNA) in NCCs and the cardiovascular system. METHODS AND RESULTS: We deleted Dicer from the NCC lineage and showed that Dicer conditional mutants exhibit severe defects in multiple craniofacial and cardiovascular structures, many of which are observed in human neuro-craniofacial-cardiac syndrome patients. We found that cranial NCCs require Dicer for their survival and that deletion of Dicer led to massive cell death and complete loss of NCC-derived craniofacial structures. In contrast, Dicer and miRNAs were not essential for the survival of cardiac NCCs. However, the migration and patterning of these cells were impaired in Dicer knockout mice, resulting in a spectrum of cardiovascular abnormalities, including type B interrupted aortic arch, double-outlet right ventricle, and ventricular septal defect. We showed that Dicer loss of function was, at least in part, mediated by miRNA-21 (miR-21) and miRNA-181a (miR-181a), which in turn repressed the protein level of Sprouty 2, an inhibitor of Erk1/2 signaling. CONCLUSIONS: Our results uncovered a central role for Dicer and miRNAs in NCC survival, migration, and patterning in craniofacial and cardiovascular development which, when mutated, lead to congenital neuro-craniofacial-cardiac defects.

Myocardin inhibits cellular proliferation by inhibiting NF-kappaB(p65)-dependent cell cycle progression.

We previously reported the importance of the serum response factor (SRF) cofactor myocardin in controlling muscle gene expression as well as the fundamental role for the inflammatory transcription factor NF-kappaB in governing cellular fate. Inactivation of myocardin has been implicated in malignant tumor growth. However, the underlying mechanism of myocardin regulation of cellular growth remains unclear. Here we show that NF-kappaB(p65) represses myocardin activation of cardiac and smooth muscle genes in a CArG-box-dependent manner. Consistent with their functional interaction, p65 directly interacts with myocardin and inhibits the formation of the myocardin/SRF/CArG ternary complex in vitro and in vivo. Conversely, myocardin decreases p65-mediated target gene activation by interfering with p65 DNA binding and abrogates LPS-induced TNF-alpha expression. Importantly, myocardin inhibits cellular proliferation by interfering with NF-kappaB-dependent cell-cycle regulation. Cumulatively, these findings identify a function for myocardin as an SRF-independent transcriptional repressor and cell-cycle regulator and provide a molecular mechanism by which interaction between NF-kappaB and myocardin plays a central role in modulating cellular proliferation and differentiation.

The histone methyltransferase Set7/9 promotes myoblast differentiation and myofibril assembly.

The molecular events that modulate chromatin structure during skeletal muscle differentiation are still poorly understood. We report in this paper that expression of the H3-K4 histone methyltransferase Set7 is increased when myoblasts differentiate into myotubes and is required for skeletal muscle development, expression of muscle contractile proteins, and myofibril assembly. Knockdown of Set7 or expression of a dominant-negative Set7 mutant impairs skeletal muscle differentiation, accompanied by a decrease in levels of histone monomethylation (H3-K4me1). Set7 directly interacts with MyoD to enhance expression of muscle differentiation genes. Expression of myocyte enhancer factor 2 and genes encoding contractile proteins is decreased in Set7 knockdown myocytes. Furthermore, we demonstrate that Set7 also activates muscle gene expression by precluding Suv39h1-mediated H3-K9 methylation on the promoters of myogenic differentiation genes. Together, our experiments define a biological function for Set7 in muscle differentiation and provide a molecular mechanism by which Set7 modulates myogenic transcription factors during muscle differentiation.

Periostin is a novel factor in cardiac remodeling after experimental and clinical unloading of the failing heart.

BACKGROUND: Maladaptive left ventricular hypertrophy (LVH) remains a prevalent and highly morbid condition associated with end-stage heart disease. Originally evaluated in the context of bone development, periostin is important in endocardial cushion formation and has recently been implicated in heart failure. Because of its potential role in cardiovascular development, we sought to establish the role of periostin after relief of pressure overload in animal and human models. METHODS: Pressure overload induction of LVH was performed by minimally invasive aortic arch banding of C57Bl6 mice. Bands were removed 1 month later to allow regression. Cardiac tissue was procured in paired samples of patients receiving LV assist devices (LVAD), with subsequent reanalysis at the time of explant for transplantation. RESULTS: One week after debanding, heart weight/body weight ratios and echocardiography confirmed decreased LV mass relative to hypertrophied animals. Gene and protein expression of periostin was measured by real-time polymerase chain reaction and Western blot, and was similarly decreased compared with LVH mice. Immunohistochemical localization of periostin showed it was exclusively in the extracellular matrix of the myocardium. The decrease in periostin with pressure relief paralleled changes in interstitial fibrosis observed by picrosirius red staining. Corroborating the murine data, periostin expression was significantly reduced after LVAD-afforded pressure relief in patients. CONCLUSIONS: Periostin is closely associated with pressure overload-induced LVH and LVH regression in both animal and human models. The magnitude of expression changes and the consistent nature of these changes indicate that periostin may be a mediator of cardiac remodeling.

Muscle ring finger 1 mediates cardiac atrophy in vivo.

Pathological cardiac hypertrophy, induced by various etiologies such as high blood pressure and aortic stenosis, develops in response to increased afterload and represents a common intermediary in the development of heart failure. Understandably then, the reversal of pathological cardiac hypertrophy is associated with a significant reduction in cardiovascular event risk and represents an important, yet underdeveloped, target of therapeutic research. Recently, we determined that muscle ring finger-1 (MuRF1), a muscle-specific protein, inhibits the development of experimentally induced pathological; cardiac hypertrophy. We now demonstrate that therapeutic cardiac atrophy induced in patients after left ventricular assist device placement is associated with an increase in cardiac MuRF1 expression. This prompted us to investigate the role of MuRF1 in two independent mouse models of cardiac atrophy: 1) cardiac hypertrophy regression after reversal of transaortic constriction (TAC) reversal and 2) dexamethasone-induced atrophy. Using echocardiographic, histological, and gene expression analyses, we found that upon TAC release, cardiac mass and cardiomyocyte cross-sectional areas in MuRF1(-/-) mice decreased approximately 70% less than in wild type mice in the 4 wk after release. This was in striking contrast to wild-type mice, who returned to baseline cardiac mass and cardiomyocyte size within 4 days of TAC release. Despite these differences in atrophic remodeling, the transcriptional activation of cardiac hypertrophy measured by beta-myosin heavy chain, smooth muscle actin, and brain natriuretic peptide was attenuated similarly in both MuRF1(-/-) and wild-type hearts after TAC release. In the second model, MuRF1(-/-) mice also displayed resistance to dexamethasone-induced cardiac atrophy, as determined by echocardiographic analysis. This study demonstrates, for the first time, that MuRF1 is essential for cardiac atrophy in vivo, both in the setting of therapeutic regression of cardiac hypertrophy and dexamethasone-induced atrophy.

Regression of pressure-induced left ventricular hypertrophy is characterized by a distinct gene expression profile.

OBJECTIVE: Left ventricular hypertrophy is a highly prevalent and robust predictor of cardiovascular morbidity and mortality. Existing studies have finely detailed mechanisms involved with its development, yet clinical translation of these findings remains unsatisfactory. We propose an alternative strategy focusing on mechanisms of left ventricular hypertrophy regression rather than its progression and hypothesize that left ventricular hypertrophy regression is associated with a distinct genomic profile. METHODS: Minimally invasive transverse arch banding and debanding (or their respective sham procedures) were performed in C57Bl6 male mice. Left ventricular hypertrophy was assessed physiologically by means of transthoracic echocardiographic analysis, structurally by means of histology, and molecularly by means of real-time polymerase chain reaction. Mouse hearts were genomically analyzed with Agilent (Santa Clara, Calif) mouse 44k developmental gene chips. RESULTS: Compared with control animals, animals banded for 28 days had a robust hypertrophic response, as determined by means of heart weight/body weight ratio, histologic analysis, echocardiographic analysis, and fetal gene expression. These parameters were reversed within 1 week of debanding. Whole-genome arrays on left ventricular tissue revealed 288 genes differentially expressed during progression, 265 genes differentially expressed with regression, and only 23 genes shared by both processes. Signaling-related expression patterns were more prevalent with regression rather than the structure-related patterns associated with left ventricular hypertrophy progression. In addition, regressed hearts showed comparatively more changes in energy metabolism and protein production. CONCLUSIONS: This study demonstrates an effective model for characterizing left ventricular hypertrophy and reveals that regression is genomically distinct from its development. Further examination of these expression profiles will broaden our understanding of left ventricular hypertrophy and provide a novel therapeutic paradigm focused on promoting regression of left ventricular hypertrophy and not just halting its progression.

MicroRNA-208a is a regulator of cardiac hypertrophy and conduction in mice.

MicroRNAs (miRNAs) are a class of small noncoding RNAs that have gained status as important regulators of gene expression. Here, we investigated the function and molecular mechanisms of the miR-208 family of miRNAs in adult mouse heart physiology. We found that miR-208a, which is encoded within an intron of alpha-cardiac muscle myosin heavy chain gene (Myh6), was actually a member of a miRNA family that also included miR-208b, which was determined to be encoded within an intron of beta-cardiac muscle myosin heavy chain gene (Myh7). These miRNAs were differentially expressed in the mouse heart, paralleling the expression of their host genes. Transgenic overexpression of miR-208a in the heart was sufficient to induce hypertrophic growth in mice, which resulted in pronounced repression of the miR-208 regulatory targets thyroid hormone-associated protein 1 and myostatin, 2 negative regulators of muscle growth and hypertrophy. Studies of the miR-208a Tg mice indicated that miR-208a expression was sufficient to induce arrhythmias. Furthermore, analysis of mice lacking miR-208a indicated that miR-208a was required for proper cardiac conduction and expression of the cardiac transcription factors homeodomain-only protein and GATA4 and the gap junction protein connexin 40. Together, our studies uncover what we believe are novel miRNA-dependent mechanisms that modulate cardiac hypertrophy and electrical conduction.

Inhibitory kappa B kinase-beta is a target for specific nuclear factor kappa B-mediated delayed cardioprotection.

OBJECTIVE: Myocardial ischemia/reperfusion injury remains a vexing problem. Translating experimental strategies that deliver protective agents before the ischemic insult limits clinical applicability. We targeted 2 proteins in the nuclear factor-kappaB pathway, inhibitory kappa B kinase-beta, and 26S cardiac proteasome to determine their cardioprotective effects when delivered during reperfusion. METHODS: C57BL/6 mice underwent left anterior descending artery occlusion for 30 minutes. An inhibitory kappa B kinase-beta inhibitor (Compound A), a proteasome inhibitor (PS-519), or vehicle was administered at left anterior descending artery release or 2 hours afterward. Infarct size was analyzed 24 hours later. Pressure-volume loops were performed at 72 hours. Serum and left ventricular tissue were collected 1 hour after injury to examine protein expression by enzyme-linked immunosorbent assay and Western blot. RESULTS: Inhibitory kappa B kinase-beta and proteasome inhibition significantly attenuated infarct size and preserved ejection fraction compared with the vehicle groups. When delivered even 2 hours after reperfusion, Compound A, but not PS-519, still decreased infarct size in mice. Finally, when delivered at reperfusion, successful inhibition of phosphorylated-p65 and decreased interleukin-6 and tumor necrosis factor-alpha levels occurred in mice given the inhibitory kappa B kinase-beta inhibitor, but not in mice with proteasome inhibition. CONCLUSION: Although inhibitory kappa B kinase-beta and proteasome inhibition at reperfusion attenuated infarct size after acute ischemia/reperfusion, only inhibitory kappa B kinase-beta inhibition provided cardioprotection through specific suppression of nuclear factor-kappaB signaling. This feature of highly targeted nuclear factor-kappaB inhibition might account for its delayed protective effects, providing a clinically relevant option for treating myocardial ischemia/reperfusion associated with unknown periods of ischemia and reperfusion as seen in cardiac surgery and acute coronary syndromes.

Proteasome inhibition promotes regression of left ventricular hypertrophy.

Current research in left ventricular hypertrophy (LVH) has largely focused on its progression and therapeutic mechanisms to prevent or slow its development. Few studies have centered on the regression or treatment of existing LVH. Nuclear factor-kappaB (NF-kappaB) is an inflammatory transcription factor that has been shown to be involved in LVH development. We hypothesized that proteasome-mediated NF-kappaB inhibition would prevent the development of LVH and promote its regression. A murine model of reversible hypertrophy was employed by administering isoproterenol (Iso) subcutaneously for 7-14 days. The proteasome inhibitor, PS-519, was delivered both concurrently and after Iso treatment. LVH was quantified by heart weight-to-body weight ratios, histology, transthoracic echocardiography, and hypertrophic gene expression. After 7 days of Iso treatment, all measures indicated successful development of LVH. Another group was treated for 7 days and then observed for an additional 7 days. This group experienced normalization of Iso-induced cell size, wall thickness, and beta-myosin heavy chain expression. When administered concurrently, PS-519 prevented Iso-induced LVH at 7 days. Furthermore, when PS-519 was given to animals during the second week of continued Iso treatment, these animals also experienced regression of hypertrophy by several measures. The success of proteasome inhibition in preventing LVH development and in promoting LVH regression, even in the face of continued hypertrophic stimulation, demonstrates its potential use as a clinically accessible strategy for treating patients with a variety of LVH-associated cardiomyopathies.

IKKbeta inhibition attenuates myocardial injury and dysfunction following acute ischemia-reperfusion injury.

Despite years of experimental and clinical research, myocardial ischemia-reperfusion (IR) remains an important cause of cardiac morbidity and mortality. The transcription factor nuclear factor-kappaB (NF-kappaB) has been implicated as a key mediator of reperfusion injury. Activation of NF-kappaB is dependent upon the phosphorylation of its inhibitor, IkappaBalpha, by the specific inhibitory kappaB kinase (IKK) subunit, IKKbeta. We hypothesized that specific antagonism of the NF-kappaB inflammatory pathway through IKKbeta inhibition reduces acute myocardial damage following IR injury. C57BL/6 mice underwent left anterior descending (LAD) artery ligation and release in an experimental model of acute IR. Bay 65-1942, an ATP-competitive inhibitor that selectively targets IKKbeta kinase activity, was administered intraperitoneally either prior to ischemia, at reperfusion, or 2 h after reperfusion. Compared with untreated animals, mice treated with IKKbeta inhibition had significant reduction in left ventricular infarct size. Cardiac function was also preserved following pretreatment with IKKbeta inhibition. These findings were further associated with decreased expression of phosphorylated IkappaBalpha and phosphorylated p65 in myocardial tissue. In addition, IKKbeta inhibition decreased serum levels of TNF-alpha and IL-6, two prototypical downstream effectors of NF-kappaB activity. These results demonstrate that specific IKKbeta inhibition can provide both acute and delayed cardioprotection and offers a clinically accessible target for preventing cardiac injury following IR.

Coupling diurnal cytosolic Ca2+ oscillations to the CAS-IP3 pathway in Arabidopsis.

Various signaling pathways rely on changes in cytosolic calcium ion concentration ([Ca2+]i). In plants, resting [Ca2+]i oscillates diurnally. We show that in Arabidopsis thaliana, [Ca2+]i oscillations are synchronized to extracellular Ca2+ concentration ([Ca2+]o) oscillations largely through the Ca2+-sensing receptor CAS. CAS regulates concentrations of inositol 1,4,5-trisphosphate (IP3), which in turn directs release of Ca2+ from internal stores. The oscillating amplitudes of [Ca2+]o and [Ca2+]i are controlled by soil Ca2+ concentrations and transpiration rates. The phase and period of oscillations are likely determined by stomatal conductance. Thus, the internal concentration of Ca2+ in plant cells is constantly being actively revised.

Nitric oxide represses the Arabidopsis floral transition.

The correct timing of flowering is essential for plants to maximize reproductive success and is controlled by environmental and endogenous signals. We report that nitric oxide (NO) repressed the floral transition in Arabidopsis thaliana. Plants treated with NO, as well as a mutant overproducing NO (nox1), flowered late, whereas a mutant producing less NO (nos1) flowered early. NO suppressed CONSTANS and GIGANTEA gene expression and enhanced FLOWERING LOCUS C expression, which indicated that NO regulates the photoperiod and autonomous pathways. Because NO is induced by environmental stimuli and constitutively produced, it may integrate both external and internal cues into the floral decision.