Sean M. Garvey

The deacetylase HDAC4 controls myocyte enhancing factor-2-dependent structural gene expression in response to neural activity.

Histone deacetylase 4 (HDAC4) binds and inhibits activation of the critical muscle transcription factor myocyte enhancer factor‐2 (MEF2). However, the physiological significance of the HDAC4‐MEF2 complex in skeletal muscle has not been established. Here we show that in skeletal muscle, HDAC4 is a critical modulator of MEF2‐dependent structural and contractile gene expression in response to neural activity. We present evidence that loss of neural input leads to concomitant nuclear accumulation of HDAC4 and transcriptional reduction of MEF2‐regulated gene expression. Cell‐based assays show that HDAC4 represses structural gene expression via direct binding to AT‐rich MEF2 response elements. Notably, using both surgical denervation and the neuromuscular disease amyotrophic lateral sclerosis (ALS) model, we found that elevated levels of HDAC4 are required for efficient repression of MEF2‐dependent structural gene expression, indicating a link between the patho‐logical induction of HDAC4 and subsequent MEF2 target gene suppression. Supporting this supposition, we show that ectopic expression of HDAC4 in muscle fibers is sufficient to induce muscle damage in mice. Our study identifies HDAC4 as an activity‐dependent regulator of MEF2 function and suggests that activation of HDAC4 in response to chronically reduced neural activity suppresses MEF2‐dependent gene expression and contributes to progressive muscle dysfunction observed in neuromuscular diseases.—Cohen, T. J., Barrientos, T., Hartman, Z. C., Garvey, S. M., Cox, G. A., Yao, T.‐P. The deacetylase HDAC4 controls myocyte enhancing factor‐2 dependent structural gene expression in response to neural activity. FASEB J. 23, 99‐106 (2009)

Cyclosporine Up-Regulates Krüppel-Like Factor-4 (KLF4) in Vascular Smooth Muscle Cells and Drives Phenotypic Modulation In Vivo

Cyclosporine A (CSA, calcineurin inhibitor) has been shown to block both vascular smooth muscle cell (VSMC) proliferation in cell culture and vessel neointimal formation following injury in vivo. The purpose of this study was to determine molecular and pathological effects of CSA on VSMCs. Using real-time reverse transcription-polymerase chain reaction, Western blot analysis, and immunofluorescence microscopy, we show that CSA up-regulated the expression of Krüppel-like factor-4 (KLF4) in VSMCs. KLF4 plays a key role in regulating VSMC phenotypic modulation. KLF4 antagonizes proliferation, facilitates migration, and down-regulates VSMC differentiation marker gene expression. We show that the VSMC differentiation marker genes smooth muscle α-actin (ACTA2), transgelin (TAGLN), smoothelin (SMTN), and myocardin (MYOCD) are all down-regulated by CSA in VSMC monoculture, whereas cyclin-dependent kinase inhibitor-1A (CDKN1A) and matrix metalloproteinase-3 (MMP3) are up-regulated. CSA did not affect the abundance of the VSMC microRNA (MIR) markers MIR143 and MIR145. Administration of CSA to rat carotid artery in vivo resulted in acute and transient suppression of ACTA2, TAGLN, SMTN, MYOCD, and smooth muscle myosin heavy chain (MYH11) mRNA levels. The tumor suppressor genes KLF4, p53, and CDKN1A, however, were up-regulated, as well as MMP3, MMP9, and collagen-VIII. CSA-treated arteries showed remarkable remodeling, including breakdown of the internal elastic lamina and reorientation of VSMCs, as well as increased KLF4 immunostaining in VSMCs and endothelial cells. Altogether, these data show that cyclosporin up-regulates KLF4 expression and promotes phenotypic modulation of VSMCs.

Integrative genomics identifies DSCR1 (RCAN1) as a novel NFAT-dependent mediator of phenotypic modulation in vascular smooth muscle cells

Vascular smooth muscle cells (SMCs) display remarkable phenotypic plasticity in response to environmental cues. The nuclear factor of activated T-cells (NFAT) family of transcription factors plays a critical role in vascular pathology. However, known functional NFAT gene targets in vascular SMCs are currently limited. Publicly available whole-genome expression array data sets were analyzed to identify differentially expressed genes in human, mouse and rat SMCs. Comparison between vehicle and phenotypic modulatory stimuli identified 63 species-conserved, upregulated genes. Integration of the 63 upregulated genes with an in silico NFAT-ome (a species-conserved list of gene promoters containing at least one NFAT binding site) identified 18 putative NFAT-dependent genes. Further intersection of these 18 potential NFAT target genes with a mouse in vivo vascular injury microarray identified four putative NFAT-dependent, injury-responsive genes. In vitro validations substantiated the NFAT-dependent role of Cyclooxygenase 2 (COX2/PTGS2) in SMC phenotypic modulation and uncovered Down Syndrome Candidate Region 1 (DSCR1/RCAN1) as a novel NFAT target gene in SMCs. We show that induction of DSCR1 inhibits calcineurin/NFAT signaling through a negative feedback mechanism; DSCR1 overexpression attenuates NFAT transcriptional activity and COX2 protein expression, whereas knockdown of endogenous DSCR1 enhances NFAT transcriptional activity. Our integrative genomics approach illustrates how the combination of publicly available gene expression arrays, computational databases and empirical research methods can answer specific questions in any cell type for a transcriptional network of interest. Herein, we report DSCR1 as a novel NFAT-dependent, injury-inducible, early gene that may serve to negatively regulate SMC phenotypic switching.

Molecular and metabolomic effects of voluntary running wheel activity on skeletal muscle in late middle‐aged rats

We examined the molecular and metabolomic effects of voluntary running wheel activity in late middle‐aged male Sprague Dawley rats (16–17 months). Rats were assigned either continuous voluntary running wheel access for 8 weeks (RW+) or cage‐matched without running wheel access (RW−). The 9 RW+ rats averaged 83 m/day (range: 8–163 m), yet exhibited both 84% reduced individual body weight gain (4.3 g vs. 26.3 g, P = 0.02) and 6.5% reduced individual average daily food intake (20.6 g vs. 22.0 g, P = 0.09) over the 8 weeks. Hindlimb muscles were harvested following an overnight fast. Muscle weights and myofiber cross‐sectional area showed no difference between groups. Western blots of gastrocnemius muscle lysates with a panel of antibodies suggest that running wheel activity improved oxidative metabolism (53% increase in PGC1α, P = 0.03), increased autophagy (36% increase in LC3B‐II/‐I ratio, P = 0.03), and modulated growth signaling (26% increase in myostatin, P = 0.04). RW+ muscle also showed 43% increased glycogen phosphorylase expression (P = 0.04) and 45% increased glycogen content (P = 0.04). Metabolomic profiling of plantaris and soleus muscles indicated that even low‐volume voluntary running wheel activity is associated with decreases in many long‐chain fatty acids (e.g., palmitoleate, myristoleate, and eicosatrienoate) relative to RW− rats. Relative increases in acylcarnitines and acyl glycerophospholipids were also observed in RW+ plantaris. These data establish that even modest amounts of physical activity during late middle‐age promote extensive metabolic remodeling of skeletal muscle.

Cellular and Physiological Effects of Dietary Supplementation with β-Hydroxy-β-Methylbutyrate (HMB) and β-Alanine in Late Middle-Aged Mice.

There is growing evidence that severe decline of skeletal muscle mass and function with age may be mitigated by exercise and dietary supplementation with protein and amino acid ingredient technologies. The purposes of this study were to examine the effects of the leucine catabolite, beta-hydroxy-beta-methylbutyrate (HMB), in C2C12 myoblasts and myotubes, and to investigate the effects of dietary supplementation with HMB, the amino acid β-alanine and the combination thereof, on muscle contractility in a preclinical model of pre-sarcopenia. In C2C12 myotubes, HMB enhanced sarcoplasmic reticulum (SR) calcium release beyond vehicle control in the presence of all SR agonists tested (KCl, P<0.01; caffeine, P = 0.03; ionomycin, P = 0.03). HMB also improved C2C12 myoblast viability (25 μM HMB, P = 0.03) and increased proliferation (25 μM HMB, P = 0.04; 125 μM HMB, P<0.01). Furthermore, an ex vivo muscle contractility study was performed on EDL and soleus muscle from 19 month old, male C57BL/6nTac mice. For 8 weeks, mice were fed control AIN-93M diet, diet with HMB, diet with β-alanine, or diet with HMB and β-alanine. In β-alanine fed mice, EDL muscle showed a 7% increase in maximum absolute force compared to the control diet (202 ± 3vs. 188± 5 mN, P = 0.02). At submaximal frequency of stimulation (20 Hz), EDL from mice fed HMB plus β-alanine showed an 11% increase in absolute force (88.6 ± 2.2 vs. 79.8 ± 2.4 mN, P = 0.025) and a 13% increase in specific force (12.2 ± 0.4 vs. 10.8 ± 0.4 N/cm2, P = 0.021). Also in EDL muscle, β-alanine increased the rate of force development at all frequencies tested (P<0.025), while HMB reduced the time to reach peak contractile force (TTP), with a significant effect at 80 Hz (P = 0.0156). In soleus muscle, all experimental diets were associated with a decrease in TTP, compared to control diet. Our findings highlight beneficial effects of HMB and β-alanine supplementation on skeletal muscle function in aging mice.

Dietary HMB and β-alanine co-supplementation does not improve in situ muscle function in sedentary, aged male rats

This study evaluated the effects of dietary β-hydroxy-β-methylbutyrate (HMB) combined with β-alanine (β-Ala) in sedentary, aged male rats. It has been suggested that dietary HMB or β-Ala supplementation may mitigate age-related declines in muscle strength and fatigue resistance. A total of 20 aged Sprague-Dawley rats were studied. At age 20 months, 10 rats were administered a control, purified diet and 10 rats were administered a purified diet supplemented with both HMB and β-Ala (HMB+β-Ala) for 8 weeks (approximately equivalent to 3 and 2.4 g per day human dose). We measured medial gastrocnemius (MG) size, force, fatigability, and myosin composition. We also evaluated an array of protein markers related to muscle mitochondria, protein synthesis and breakdown, and autophagy. HMB+β-Ala had no significant effects on body weight, MG mass, force or fatigability, myosin composition, or muscle quality. Compared with control rats, those fed HMB+β-Ala exhibited a reduced (41%, P = 0.039) expression of muscle RING-finger protein 1 (MURF1), a common marker of protein degradation. Muscle from rats fed HMB+β-Ala also exhibited a 45% reduction (P = 0.023) in p70s6K phosphorylation following fatiguing stimulation. These data suggest that HMB+β-Ala at the dose studied may reduce muscle protein breakdown by reducing MURF1 expression, but has minimal effects on muscle function in this model of uncomplicated aging. They do not, however, rule out potential benefits of HMB+β-Ala co-supplementation at other doses or durations of supplementation in combination with exercise or in situations where extreme muscle protein breakdown and loss of mass occur (e.g., bedrest, cachexia, failure-to-thrive).

Genome-Wide Microarray Analyses Identify the Protein C Receptor as a Novel Calcineurin/Nuclear Factor of Activated T Cells–Dependent Gene in Vascular Smooth Muscle Cell Phenotypic Modulation

Objective—Calcineurin (Cn) and the nuclear factor of activated T cells (NFAT) family of transcription factors are critical in vascular smooth muscle cell (SMC) development and pathology. Here, we used a genomics approach to identify and validate NFAT gene targets activated during platelet-derived growth factor-BB (PDGF-BB)–induced SMC phenotypic modulation. Methods and Results—Genome-wide expression arrays were used to identify genes both (1) differentially activated in response to PDGF-BB and (2) whose differential expression was reduced by both the Cn inhibitor cyclosporin A and the NFAT inhibitor A-285222. The 20 most pharmacologically sensitive genes were validated by quantitative reverse transcription–polymerase chain reaction analysis of PDGF-BB-stimulated SMCs in the presence of Cn/NFAT inhibitors, including the VIVIT peptide. In all experiments, protein C receptor (PROCR) gene activation was reduced. We showed that PROCR expression was virtually absent in untreated, quiescent SMCs. PDGF-BB stimulation, however, induced significant PROCR promoter activation and downstream protein expression in a Cn/NFAT-dependent manner. Mutation of a species-conserved, NFAT binding motif significantly attenuated PDGF-BB-induced PROCR promoter activity, thereby distinguishing NFAT as the first PROCR transcriptional activator to date. Moreover, SMC PROCR expression was upregulated in the neointima as early as 7 days following acute vascular injury in rat carotid arteries. Conclusion—We hereby report PROCR as a novel, NFAT-dependent gene that may be implicated in vascular restenosis and consequent inward remodeling.

Effects of running wheel activity and dietary HMB and B—alanine co-supplementation on muscle quality in aged male rats

ObjectiveLoss of skeletal muscle function is linked to increased risk for loss of health and independence in older adults. Dietary interventions that can enhance aging muscle function, alone or in combination with exercise, may offer an effective way to reduce these risks. The goal of this study was to evaluate the muscular effects of beta-hydroxy-beta-methylbutyrate (HMB) and beta-alanine (β-Ala) co-supplementation in aged Sprague-Dawley rats with voluntary access to running wheels (RW).MethodsAged (20 months) rats were housed with ad libitum access to RW while on a purified diet for 4 weeks, then balanced for RW activity and assigned to either a control or an experimental diet (control + HMB and β-Ala) for the next 4 weeks (n = 10/group). At the end of the study, we assessed muscle size, in situ force and fatigability in the medial gastrocnemius muscles, as well as an array of protein markers related to various age- and activity-responsive signaling pathways.ResultsDietary HMB+β-Ala did not improve muscle force or fatigue resistance, but a trend for increased muscle cross-sectional area (CSA) was observed (P = 0.077). As a result, rats on the experimental diet exhibited reduced muscle quality (force/CSA; P = 0.032). Dietary HMB+β-Ala reduced both the abundance of PGC1-α (P = 0.050) and the ratio of the lipidated to non-lipidated forms of microtubule-associated protein 1 light chain 3 beta (P = 0.004), markers of mitochondrial biogenesis and autophagy, respectively. Some alterations in myostatin signaling also occurred in the dietary HMB+β-Ala group. There was an unexpected difference (P = 0.046) in RW activity, which increased throughout the study in the animals on the control diet, but not in animals on the experimental diet.ConclusionsThese data suggest that the short-term addition of dietary HMB+β-Ala to modest physical activity provided little enhancement of muscle function in this model of uncomplicated aging.

Effect of acute muscle contusion injury, with and without dietary fish oil, on adult and aged male rats: contractile and biochemical responses

Aim Contusion injury in aging muscle has not been studied in detail, but older adults are at risk for such injuries due to increased risk of falls. As falls in older populations are unlikely to be eliminated, interventions to minimize the negative impact of falls, including contusion injury should be pursued. Dietary fish oil (FO) is a common often supplement in older adults, which is associated with factors that might reduce or worsen the negative impact of contusion. Methods Here, we investigate whether 8 weeks of FO can blunt the impact of contusion injury in adult (n = 14) and aged (n = 12) rats. We assessed contractility and several biochemical markers in adult and aged gastrocnemius muscles 48 h post‐contusion injury, using the uninjured muscles as controls. Results Injury reduced force production ˜40% (P < 0.001), sarcoplasmic reticulum calcium release by ˜20% (P = 0.003) and significantly increased several markers of muscle damage (i.e., protein carbonyls, Grp78 abundance (P = 0.022, 0.006, respectively)), and these injury‐related changes were not affected by aging. The effects of FO were limited. A main effect (P = 0.018) for FO to increase the myogenic factor Myf5 was observed. In addition FO reduced the injury‐associated decline in the mitophagy factor DRP1 (P = 0.027). Conclusion Although age‐related differences in certain protein markers differed, aged muscles exhibited no greater acute functional deficits following injury. Similarly, while FO did not reduce functional deficits, it did not worsen them. However, changes in Myf5 and DRP1 with dietary FO suggest the potential to improve recovery from contusion injury, which should be investigated in future studies. HighlightsContusion injury functionally impairs aged muscles no more than adult muscles.Aging increases and reduces muscle cell stress and autophagy markers, respectivelyFish oil (FO) does not reduce or worsen the acute negative impact of contusionFO affects markers satellite cell commitment and mitochondrial remodelingThese finding suggest a potential role for FO during recovery from contusion

Integrative Genomics Identifies DSCR1 (RCAN1) as a Novel NFAT-Dependent Mediator of Phenotypic Modulation in Vascular Smooth Muscle Cells

Meeting Abstract from 10th Annual Conference on Arteriosclerosis, Thrombosis and Vascular Biology held in Washington, DC, on April 29 - May 01, 2009. American Heart Association Council Arteriosclerosis, Thrombosis & Vascular Biology.Meeting Abstract from the 10th Annual Conference on Arteriosclerosis, Thrombosis and Vascular Biology held in Washington, DC, on April 29 - May 01, 2009.Objective: Chronic infusion of angiotensin II (AngII) augments the development of atherosclerosis and induces abdominal aortic aneurysms (AAAs) in hypercholesterolemic mice. Smooth muscle cells (SMCs) play a pivotal role in the development of these two pathologies. AngII-induced AAA is associated with the downregulation of peroxisome proliferator-activated receptor gamma (PPARgamma). In addition, activation of PPARgamma by ligands attenuates atherosclerosis in male mice. The purpose of the study was to define whether deficiency of SMC-specific PPARgamma influences AngII-induced vascular pathologies. Methods and Results: Male and female LDL receptor -/- mice were bred with homozygous floxed PPARgamma alleles (PPARgammaf/f). Parental males were also developed that were hemizygous for Cre under the control of the SM22 promoter. Littermates were generated that did not express Cre (Cre0/0, n=20) or were hemizygous for Cre(Cre1/0, n=16) and fed a saturated fat-enriched diet (21% wt/wt milk fat; 0.15% wt/wt cholesterol). Mice were infused with AngII (1,000 ng/kg/min) by osmotic minipump for 4 weeks. AngII increased systolic blood pressure equivalently in both groups. SMC-specific deficiency of PPARgamma had no effect on serum cholesterol concentrations (Cre0/0, 1544±112; Cre1/0, 1548±87 mg/dL) or lipoprotein-cholesterol distributions. AngII infusion led to a similar incidence of AAA formation (84% of male Cre0/0 versus 80% of male Cre1/0 mice). Ex vivo measurement of maximal diameter of abdominal aorta showed a comparable dilation in Cre0/0 and Cre1/0 mice (2.16±0.16 versus 2.17±0.31 mm, P=NS). Male Cre1/0 mice had significantly larger atherosclerotic lesion areas in both arch and thoracic aortic regions by en face measurement compared to male f/fCre0/0 mice (Arch area intimal lesions; Cre0/0 = 4.74±1.13%; Cre1/0 = 10.24±1.67%, Thoracic area intimal lesions; Cre0/0 = 4.12±0.69%; Cre1/0 = 8.99±2.28%, P<0.05, n=12–18 per group). SMC specific PPARgamma deficiency in female LDL receptor -/- mice did not influence either AngII-induced atherosclerosis or AAAs. Conclusion: SMC specific PPARgamma deficiency augments atherosclerosis but does not contribute to the formation of AAAs induced by AngII in male hyperlipidemic mice.Arteriosclerosis, Thrombosis, and Vascular Biology Annual Conference 2009 April 29–May 1, 2009 Omni Shoreham Hotel Washington, DC Sponsored by the American Heart Association Council on Arteriosclerosis, Thrombosis, and Vascular Biology. Conference Chair: Alan Daugherty, PhD, DSc, FAHA Conference Co-Chairs: Robert A. Hegele, MD, FAHA; Murray W. Huff, PhD, FAHA Conference Program Committee Timothy Hla, PhD; Larry Kraiss, MD, FAHA; Steven R. Lentz, MD, PhD, FAHA; Joseph M. Miano, PhD; Kathryn J. Moore, PhD, FAHA; Susan S. Smyth, MD, PhD, FAHA; Mary Sorci-Thomas, PhD, FAHA; Hartmut Weiler, PhD; Cuihua Zhang, MD, PhD, FAHA Conference Program Committee Liaisons Jeanine D’Armiento, MD, PhD; William P. Fay, MD, FAHA; Kathy Griendling, PhD, FAHA; John S. Parks, PhD, FAHA; Muredach P. Reilly, MB, FAHA; Lawrence L. Rudel, PhD, FAHA D ow nladed from http://journals.org by on N ovem er 5, 2019