Patricia S. Pardo

MicroRNA-149 inhibits PARP-2 and promotes mitochondrial biogenesis via SIRT-1/PGC-1α network in skeletal muscle.

High-fat diet (HFD) plays a central role in the initiation of mitochondrial dysfunction that significantly contributes to skeletal muscle metabolic disorders in obesity. However, the mechanism by which HFD weakens skeletal muscle metabolism by altering mitochondrial function and biogenesis is unknown. Given the emerging roles of microRNAs (miRNAs) in the regulation of skeletal muscle metabolism, we sought to determine whether activation of a specific miRNA pathway would rescue the HFD-induced mitochondrial dysfunction via the sirtuin-1 (SIRT-1)/ peroxisome proliferator–activated receptor γ coactivator-1α (PGC-1α) pathway, a pathway that governs genes necessary for mitochondrial function. We here report that miR-149 strongly controls SIRT-1 expression and activity. Interestingly, miR-149 inhibits poly(ADP-ribose) polymerase-2 (PARP-2) and so increased cellular NAD+ levels and SIRT-1 activity that subsequently increases mitochondrial function and biogenesis via PGC-1α activation. In addition, skeletal muscles from HFD-fed obese mice exhibit low levels of miR-149 and high levels of PARP-2, and they show reduced mitochondrial function and biogenesis due to a decreased activation of the SIRT-1/PGC-1α pathway, suggesting that mitochondrial dysfunction in the skeletal muscle of obese mice may be because of, at least in part, miR-149 dysregulation. Overall, miR-149 may be therapeutically useful for treating HFD-induced skeletal muscle metabolic disorders in such pathophysiological conditions as obesity and type 2 diabetes.

Induction of Sirt1 by Mechanical Stretch of Skeletal Muscle through the Early Response Factor EGR1 Triggers an Antioxidative Response

Mechanical loading of muscles by intrinsic muscle activity or passive stretch leads to an increase in the production of reactive oxygen species (1, 2). The NAD-dependent protein deacetylase SIRT1 is involved in the protection against oxidative stress by enhancing FOXO-driven Sod2 transcription (3–5). In this report, we unravel a mechanism triggered by mechanical stretch of skeletal muscle cells that leads to an EGR1-dependent transcriptional activation of the Sirt1 gene. The resulting transient increase in SIRT1 expression generates an antioxidative response that contributes to reactive oxygen species scavenging.

FOXO transcription factors are mechanosensitive and their regulation is altered with aging in the respiratory pump

The mechanical regulation of the forkhead box O (FOXO) subclass of transcription factors in the respiratory pump and its implication in aging are completely unknown. We investigated the effects of diaphragm stretch on three FOXO isoforms, Foxo1, Foxo3a, and Foxo4, in normal mice at different ages. We tested the hypotheses that 1) FOXO activities are regulated in response to diaphragm stretch and 2) mechanical properties of aging diaphragm are altered, leading to altered regulation of FOXO with aging. Our results showed that stretch downregulated FOXO DNA-binding activity by a mechanism that required Akt and IKK activation in young mice but that these pathways lost their mechanosensitivity with age. This aberrant regulation of FOXO with aging was associated with altered viscoelasticity, compliance, and extensibility of the aged diaphragm. Curiously, the dramatic decrease of the nuclear content of Foxo1 and Foxo3a, the two isoforms associated with muscle atrophy, with aging correlated with higher basal activation of Akt and IKK signaling in diaphragms of old mice. In contrast, the stability of Foxo4 in the nucleus became dependent on JNK, which is strongly activated in aged diaphragm. This finding suggests that Foxo4 was responsible for the FOXO-dependent transcriptional activity in aging diaphragm. Our data support the hypothesis that aging alters the mechanical properties of the respiratory pump, leading to altered mechanical regulation of the stretch-induced signaling pathways controlling FOXO activities. Our study supports a mechanosensitive signaling mechanism that is responsible for regulation of the FOXO transcription factors by aging.

Neurons in Alzheimer disease emerge from senescence.

A number of cell cycle markers are associated with the selective neuronal pathology found in Alzheimer disease. However, the significance of such cell cycle markers is clouded by duplicity of function in that many such proteins are also involved in apoptosis and/or DNA repair following oxidative damage. To clarify whether or not neurons in Alzheimer disease do in fact emerge from a quiescent status, with subsequent entry into the G1 phase of the cell cycle, in this study we focused on a family of MORF4-related proteins that are associated with emergence from senescence. Our results show that many neurons in vulnerable regions of Alzheimer disease brain, but not in control brain, have increased MORF4-related proteins indicating re-entry into the cell cycle. Immunoblot analysis showed a specific disease-related increase in a 52 kDa protein that is likely the human homologue of the MORF4-related transcription factor. The novel localization of such a transcriptional activating protein to selectively vulnerable neurons in Alzheimer disease provides compelling evidence for mitotic re-entry as part of the pathogenesis of neuronal dysfunction and death in Alzheimer disease.

Early mechanical dysfunction of the diaphragm in the muscular dystrophy with myositis (Ttnmdm) model.

A complex rearrangement mutation in the mouse titin gene leads to an in-frame 83-amino acid deletion in the N2A region of titin. Autosomal recessive inheritance of the titin muscular dystrophy with myositis (Ttn(mdm/mdm)) mutation leads to a severe early-onset muscular dystrophy and premature death. We hypothesized that the N2A deletion would negatively impact the force-generating capacity and passive mechanical properties of the mdm diaphragm. We measured in vitro active isometric contractile and passive length-tension properties to assess muscle function at 2 and 6 wk of age. Micro-CT, myosin heavy chain Western blotting, and histology were used to assess diaphragm structure. Marked chest wall distortions began at 2 wk and progressively worsened until 5 wk. The percentage of myofibers with centrally located nuclei in mdm mice was significantly (P < 0.01) increased at 2 and 6 wk by 4% and 17%, respectively, compared with controls. At 6 wk, mdm diaphragm twitch stress was significantly (P < 0.01) reduced by 71%, time to peak twitch was significantly (P < 0.05) reduced by 52%, and half-relaxation time was significantly (P < 0.05) reduced by 57%. Isometric tetanic stress was significantly (P < 0.05) depressed in 2- and 6-wk mdm diaphragms by as much as 64%. Length-tension relationships of the 2- and 6-wk mdm diaphragms showed significantly (P < 0.05) decreased extensibility and increased stiffness. Slow myosin heavy chain expression was aberrantly favored in the mdm diaphragm at 6 wk. Our data strongly support early contractile and passive mechanical aberrations of the respiratory pump in mdm mice.

Activation of p53 Transcriptional Activity by SMRT: a Histone Deacetylase 3-Independent Function of a Transcriptional Corepressor

ABSTRACT The silencing mediator of retinoic acid and thyroid hormone receptors (SMRT) is an established histone deacetylase 3 (HDAC3)-dependent transcriptional corepressor. Microarray analyses of MCF-7 cells transfected with control or SMRT small interfering RNA revealed SMRT regulation of genes involved in DNA damage responses, and the levels of the DNA damage marker γH2AX as well as poly(ADP-ribose) polymerase cleavage were elevated in SMRT-depleted cells treated with doxorubicin. A number of these genes are established p53 targets. SMRT knockdown decreased the activity of two p53-dependent reporter genes as well as the expression of p53 target genes, such as CDKN1A (which encodes p21). SMRT bound directly to p53 and was recruited to p53 binding sites within the p21 promoter. Depletion of GPS2 and TBL1, components of the SMRT corepressor complex, but not histone deacetylase 3 (HDAC3) decreased p21-luciferase activity. p53 bound to the SMRT deacetylase activation domain (DAD), which mediates HDAC3 binding and activation, and HDAC3 could attenuate p53 binding to the DAD region of SMRT. Moreover, an HDAC3 binding-deficient SMRT DAD mutant coactivated p53 transcriptional activity. Collectively, these data highlight a biological role for SMRT in mediating DNA damage responses and suggest a model where p53 binding to the DAD limits HDAC3 interaction with this coregulator, thereby facilitating SMRT coactivation of p53-dependent gene expression.

MicroRNA-434-3p regulates age-related apoptosis through eIF5A1 in the skeletal muscle.

Increased activation of catabolic pathways, including apoptosis causes sarcopenia. However, the precise molecular mechanism that initiates apoptosis during aging is not well understood. Here, we report that aging alters miRNA expression profile in mouse skeletal muscle as evidenced by miRNA microarray and real-time PCR. We identified miR-434-3p as a highly downregulated miRNA in the skeletal muscle of aging mice. Myocytes transfected with miR-434-3p mimic prevents apoptosis induced by various apoptotic stimuli, and co-transfection of miR-434-3p antagomir abolishes the inhibitory role of miR-434-3p. We found that miR-434-3p inhibits apoptosis by targeting the eukaryotic translation initiation factor 5A1 (eIF5A1). Overexpression of miR-434-3p in myocytes reduces the loss of mitochondrial transmembrane potential, and activation of caspases-3, −8 and −9 by suppressing eIF5A1 in response to various apoptotic stimuli whereas inhibition of miR-434-3p reversed this scenario. Skeletal muscles from aging mice exhibit low levels of miR-434-3p and high levels of eIF5A1, suggesting a possible role for miR-434-3p in the initiation of apoptosis in aging muscle. Overall, our data identified for the first time that miR-434-3p is an anti-apoptotic miRNA that may be therapeutically useful for treating muscle atrophy in various pathophysiological conditions, including sarcopenia.

Anisotropic mechanosensitive pathways in the diaphragm and their implications in muscular dystrophies

The diaphragm is the “respiratory pump;” the muscle that generates pressure to allow ventilation. Diaphragm muscles play a vital function and thus are subjected to continuous mechanical loading. One of its peculiarities is the ability to generate distinct mechanical and biochemical responses depending on the direction through which the mechanical forces applied to it. Contractile forces originated from its contractile components are transmitted to other structural components of its muscle fibers and the surrounding connective tissue. The anisotropic mechanical properties of the diaphragm are translated into biochemical signals that are directionally mechanosensitive by mechanisms that appear to be unique to this muscle. Here, we reviewed the current state of knowledge on the biochemical pathways regulated by mechanical signals emphasizing their anisotropic behavior in the normal diaphragm and analyzed how they are affected in muscular dystrophies.

The Role of the MRG Gene Family in Replicative Senescence and Immortalization

INTRODUCTION. Replicative senescence, the terminal loss of proliferation that normal cells undergo in vitro, is considered a model for aging at the cellular level. It is also considered a mechanism of tumor suppression because fusion of normal with immortal, tumor derived cell lines yields hybrids that regain growth control and cease division. Additional genetic analysis has revealed that a large number of immortal human cell lines assign to only four complementation groups for indefinite division (A-D) and microcell fusion studies have identified human chromosomes 1,4, and 7 as carrying the cell senescence gene loci for groups C, B and D respectively. We have cloned a gene on chromosome 4, MORF 4, that following transfection induces senescence only in immortal cell lines assigned to group B. MORF 4 is a member of a gene family MORF related genes (MRG) and the three expressed family members share interesting common motifs, including a leucine zipper region, a helix-loop-helix domain and nuclear localization signal. MRG 15 has a unique chromodomain at the amino terminus, MORF 4 has lost this region and MRG X has another unique amino terminal sequence with no known homology. We here present an update on our studies with these genes. METHODS. To identify proteins that interact with the MORF 4, MRG 15 and MRG X proteins we used the techniques of yeast two-hybrid, GST-pull down/Western, and immunoprecipitation (IP)/western analyses. Fractionation of nucleoprotein complexes was done on sucrose gradients. Transfection was performed using lipofectamine plus according to manufacturers instructions. RESULTS. We have identified a novel protein PAM (protein associated with MRG) that interacts with the MRG proteins. This identification was initially done by yeast two-hybrid analysis and confirmed by GST- pull-down and IP/Western experiments. We have also determined that MRG 15 and MRG X are in multiple nucleoprotein complexes. They cosediment on sucrose gradients with PAM and are present in the same immunoprecipitates. We are currently determining whether they are present in the same complex(es) or whether they interact with PAM in an independent complex(es), since yeast two-hybrid studies indicate they do not interact directly with each other. Both MRG X and MRG 15 can activate a promoterreporter construct of the b-myb gene and we are characterizing the region(s) of the proteins that is involved by the use of deletion mutants. We are also attempting to characterize the other components of the complex(es). DISCUSSION. Our studies with MRG X and MRG 15 indicate these proteins will be present in complexes that activate genes, and most likely be important in cell cycle progression. Since