Leptin-dependent differential remodeling of visceral and pericardial adipose tissue following chronic exercise and psychosocial stress.

Obesity is driven by an imbalance between caloric intake and energy expenditure, causing excessive storage of triglycerides in adipose tissue at different sites around the body. Increased visceral adipose tissue (VAT) is associated with diabetes, while pericardial adipose tissue (PAT) is associated with cardiac pathology. Adipose tissue can expand either through cellular hypertrophy or hyperplasia, with the former correlating with decreased metabolic health in obesity. The aim of this study was to determine how VAT and PAT remodel in response to obesity, stress, and exercise. Here we have used the male obese Zucker rats, which carries two recessive fa alleles that result in the development of hyperphagia with reduced energy expenditure, resulting in morbid obesity and leptin resistance. At 9 weeks of age, a group of lean (Fa/Fa or Fa/fa) Zucker rats (LZR) and obese (fa/fa) Zucker rats (OZR) were treated with unpredictable chronic mild stress or exercise for 8 weeks. To determine the phenotype for PAT and VAT, tissue cellularity and gene expression were analyzed. Finally, leptin signaling was investigated further using cultured 3T3-derived adipocytes. Tissue cellularity was determined following hematoxylin and eosin (H&E) staining, while qPCR was used to examine gene expression. PAT adipocytes were significantly smaller than those from VAT and had a more beige-like appearance in both LZR and OZR. In the OZR group, VAT adipocyte cell size increased significantly compared with LZR, while PAT showed no difference. Exercise and stress resulted in a significant reduction in VAT cellularity in OZR, while PAT showed no change. This suggests that PAT cellularity does not remodel significantly compared with VAT. These data indicate that the extracellular matrix of PAT is able to remodel more readily than in VAT. In the LZR group, exercise increased insulin receptor substrate 1 (IRS1) in PAT but was decreased in the OZR group. In VAT, exercise decreased IRS1 in LZR, while increasing it in OZR. This suggests that in obesity, VAT is more responsive to exercise and subsequently becomes less insulin resistant compared with PAT. Stress increased PPAR-γ expression in the VAT but decreased it in the PAT in the OZR group. This suggests that in obesity, stress increases adipogenesis more significantly in the VAT compared with PAT. To understand the role of leptin signaling in adipose tissue remodeling mechanistically, JAK2 autophosphorylation was inhibited using 5 µM 1,2,3,4,5,6-hexabromocyclohexane (Hex) in cultured 3T3-derived adipocytes. Palmitate treatment was used to induce cellular hypertrophy. Hex blocked adipocyte hypertrophy in response to palmitate treatment but not the increase in lipid droplet size. These data suggest that leptin signaling is necessary for adipocyte cell remodeling, and its absence induces whitening. Taken together, our data suggest that leptin signaling is necessary for adipocyte remodeling in response to obesity, exercise, and psychosocial stress.

Selective activation of prodrugs in breast cancer using metabolic glycoengineering and the tetrazine ligation bioorthogonal reaction.

Increasing the selectivity of chemotherapies by converting them into prodrugs that can be activated at the tumour site decreases their side effects and allows discrimination between cancerous and non-cancerous cells. Herein, the use of metabolic glycoengineering (MGE) to selectively label MCF-7 breast cancer cells with tetrazine (Tz) activators for subsequent activation of prodrugs containing the trans-cyclooctene (TCO) moiety by a bioorthogonal reaction is demonstrated. Three novel Tz-modified monosaccharides, Ac4ManNTz 7, Ac4GalNTz 8, and Ac4SiaTz 16, were used for expression of the Tz activator within sialic-acid rich breast cancer cells' surface glycans through MGE. Tz expression on breast cancer cells (MCF-7) was evaluated versus the non-cancerous L929 fibroblasts showing a concentration-dependant effect and excellent selectivity with ≥35-fold Tz expression on the MCF-7 cells versus the non-cancerous L929 fibroblasts. Next, a novel TCO-N-mustard prodrug and a TCO-doxorubicin prodrug were analyzed in vitro on the Tz-bioengineered cells to probe our hypothesis that these could be activated via a bioorthogonal reaction. Selective prodrug activation and restoration of cytotoxicity were demonstrated for the MCF-7 breast cancer cells versus the non-cancerous L929 cells. Restoration of the parent drug's cytotoxicity was shown to be dependent on the level of Tz expression where the Ac4ManNTz 7 and Ac4GalNTz 8 derivatives (20 µM) lead to the highest Tz expression and full restoration of the parent drug's cytotoxicity. This work suggests the feasibility of combining MGE and tetrazine ligation for selective prodrug activation in breast cancer.

Halogenated Flavonoid Derivatives Display Antiangiogenic Activity.

Antiangiogenic agents attenuate tumours' growth and metastases and are therefore beneficial as an adjuvant or standalone cancer regimen. Drugs with dual antiproliferative and antiangiogenic activities can achieve anticancer efficacy and overcome acquired resistance. In this study, synthetic flavones (5a,b) with reported anticancer activity, and derivatives (4b and 6a), exhibited significant inhibition of endothelial cell tube formation (40-55%, 12 h) at 1 µM, which is comparable to sunitinib (50% inhibition at 1 µM, 48 h). Flavones (4b, 5a,b and 6a) also showed 25-37% reduction in HUVECs migration at 10 µM. In a Western blotting assay, 5a and 5b subdued VEGFR2 phosphorylation by 37% and 57%, respectively, suggesting that VEGFR2 may be their main antiangiogenic target. 5b displayed the best docking fit with VEGFR2 in an in silico study, followed by 5a, emphasizing the importance of the 7-hydroxyl group accompanied by a 4-C=S for activity. Conversely, derivatives with a 4-carbonyl moiety fitted poorly into the target's binding pocket, suggesting that their antiangiogenic activity depends on a different target. This study provides valuable insight into the Structure Activity Relationships (SAR) and modes of action of halogenated flavones with VEGFR2 and highlights their therapeutic potential as antiangiogenic/anticancer lead compounds.

Role of Perivascular Adipose Tissue and Exercise on Arterial Function with Obesity.

Adipose tissue and arterial dysfunction are common in the obese state. Perivascular adipose tissue (PVAT) plays an important role in mediating arterial health, and with obesity, the PVAT dysfunction negatively affects arterial health. Exercise training exerts direct and beneficial effects on PVAT, providing an additional and novel pathway by which exercise can improve arterial health in diseased populations.

Intact myocardial preparations reveal intrinsic transmural heterogeneity in cardiac mechanics.

Determining transmural mechanical properties in the heart provides a foundation to understand physiological and pathophysiological cardiac mechanics. Although work on mechanical characterisation has begun in isolated cells and permeabilised samples, the mechanical profile of living individual cardiac layers has not been examined. Myocardial slices are 300 µm-thin sections of heart tissue with preserved cellular stoichiometry, extracellular matrix, and structural architecture. This allows for cardiac mechanics assays in the context of an intact in vitro organotypic preparation. In slices obtained from the subendocardium, midmyocardium and subepicardium of rats, a distinct pattern in transmural contractility is found that is different from that observed in other models. Slices from the epicardium and midmyocardium had a higher active tension and passive tension than the endocardium upon stretch. Differences in total myocyte area coverage, and aspect ratio between layers underlined the functional readouts, while no differences were found in total sarcomeric protein and phosphoprotein between layers. Such intrinsic heterogeneity may orchestrate the normal pumping of the heart in the presence of transmural strain and sarcomere length gradients in the in vivo heart.

Nuclear accumulation of myocyte muscle LIM protein is regulated by heme oxygenase 1 and correlates with cardiac function in the transition to failure.

KEY POINTS: The present study investigated the mechanism associated with impaired cardiac mechanosensing that leads to heart failure by examining the factors regulating muscle LIM protein subcellular distribution in myocytes. In myocytes, muscle LIM protein subcellular distribution is regulated by cell contractility rather than passive stretch via heme oxygenase-1 and histone deacetylase signalling. The result of the present study provide new insights into mechanotransduction in cardiac myocytes. Myocyte mechanosensitivity, as indicated by the muscle LIM protein ratio, is also correlated with cardiac function in the transition to failure in a guinea-pig model of disease. This shows that the loss mechanosensitivity plays an important role during the transition to failure in the heart. The present study provides the first indication that mechanosensing could be modified pharmacologically during the transition to heart failure. ABSTRACT: Impaired mechanosensing leads to heart failure and a decreased ratio of cytoplasmic to nuclear CSRP3/muscle LIM protein (MLP ratio) is associated with a loss of mechanosensitivity. In the present study, we tested whether passive or active stress/strain was important in modulating the MLP ratio and determined whether this correlated with heart function during the transition to failure. We exposed cultured neonatal rat myocytes to a 10% cyclic mechanical stretch at 1 Hz, or electrically paced myocytes at 6.8 V (1 Hz) for 48 h. The MLP ratio decreased by 50% (P < 0.05, n = 4) only in response to electrical pacing, suggesting impaired mechanosensitivity. Inhibition of contractility with 10 µm blebbistatin resulted in an ∼3-fold increase in the MLP ratio (n = 8, P < 0.05), indicating that myocyte contractility regulates nuclear MLP. Inhibition of histone deacetylase (HDAC) signalling with trichostatin A increased nuclear MLP following passive stretch, suggesting that HDACs block MLP nuclear accumulation. Inhibition of heme oxygenase1 (HO-1) activity with protoporphyrin IX zinc(II) blocked MLP nuclear accumulation. To examine how mechanosensitivity changes during the transition to heart failure, we studied a guinea-pig model of angiotensin II infusion (400 ng kg(-1)  min(-1) ) over 12 weeks. Using subcellular fractionation, we showed that the MLP ratio increased by 88% (n = 4, P < 0.01) during compensated hypertrophy but decreased significantly during heart failure (P < 0.001, n = 4). The MLP ratio correlated significantly with the E/A ratio (r = 0.71, P < 0.01, n = 12), a clinical measure of diastolic function. These data indicate for the first time that myocyte mechanosensitivity as indicated by the MLP ratio is regulated primarily by myocyte contractility via HO-1 and HDAC signalling.

Disorder Prediction Methods, Their Applicability to Different Protein Targets and Their Usefulness for Guiding Experimental Studies.

The role and function of a given protein is dependent on its structure. In recent years, however, numerous studies have highlighted the importance of unstructured, or disordered regions in governing a protein's function. Disordered proteins have been found to play important roles in pivotal cellular functions, such as DNA binding and signalling cascades. Studying proteins with extended disordered regions is often problematic as they can be challenging to express, purify and crystallise. This means that interpretable experimental data on protein disorder is hard to generate. As a result, predictive computational tools have been developed with the aim of predicting the level and location of disorder within a protein. Currently, over 60 prediction servers exist, utilizing different methods for classifying disorder and different training sets. Here we review several good performing, publicly available prediction methods, comparing their application and discussing how disorder prediction servers can be used to aid the experimental solution of protein structure. The use of disorder prediction methods allows us to adopt a more targeted approach to experimental studies by accurately identifying the boundaries of ordered protein domains so that they may be investigated separately, thereby increasing the likelihood of their successful experimental solution.

Modulation of stretch-induced myocyte remodeling and gene expression by nitric oxide: a novel role for lipoma preferred partner in myofibrillogenesis.

Prolonged hemodynamic load as a result of hypertension eventually leads to maladaptive cardiac adaptation and heart failure. The signaling pathways that underlie these changes are still poorly understood. The adaptive response to mechanical load is mediated by mechanosensors that convert the mechanical stimuli into a biological response. We examined the effect of cyclic mechanical stretch on myocyte adaptation using neonatal rat ventricular myocytes with 10% (adaptive) or 20% (maladaptive) maximum strain at 1 Hz for 48 h to mimic in vivo mechanical stress. Cells were also treated with and without nitro-L-arginine methyl ester (L-NAME), a general nitric oxide synthase (NOS) inhibitor to suppress NO production. Maladaptive 20% mechanical stretch led to a significant loss of intact sarcomeres that were rescued by L-NAME (P < 0.05; n ≥ 5 cultures). We hypothesized that the mechanism was through NO-induced alteration of myocyte gene expression. L-NAME upregulated the mechanosensing proteins muscle LIM protein (MLP; by 100%; P < 0.05; n = 5 cultures) and lipoma preferred partner (LPP), a novel cardiac protein (by 80%; P < 0.05; n = 4 cultures). L-NAME also significantly altered the subcellular localization of LPP and MLP in a manner that favored growth and adaptation. These findings suggest that NO participates in stretch-mediated adaptation. The use of isoform selective NOS inhibitors indicated a complex interaction between inducible NOS and neuronal NOS isoforms regulate gene expression. LPP knockdown by small intefering RNA led to formation of α-actinin aggregates and Z bodies showing that myofibrillogenesis was impaired. There was an upregulation of E3 ubiquitin ligase (MUL1) by 75% (P < 0.05; n = 5 cultures). This indicates that NO contributes to stretch-mediated adaptation via the upregulation of proteins associated with mechansensing and myofibrillogenesis, thereby presenting potential therapeutic targets during the progression of heart failure.

Bioorthogonal activation of prodrugs, for the potential treatment of breast cancer, using the Staudinger reaction.

Selective prodrug activation at a tumor site is crucial to maximise the efficiency of chemotherapy approaches and minimise side effects due to off-site activation. In this paper, a new prodrug activation strategy is reported based on the bioorthogonal Staudinger reaction. The feasibility of this prodrug activation strategy was initially demonstrated using 9-azido sialic acid 4 as a trigger and two novel triphenylphosphine-modified N-mustard-PRO 10 and doxorubicin-PRO 12 prodrugs in an HPLC-monitored release study. Then, the azide reporter group was introduced on cancer cells' surfaces through metabolic glycoengineering of sialic acid-rich surface glycans using azide-modified monosaccharides (9-azido sialic acid 4, tetra-O-acetylated-9-azido sialic acid 5 and tetra-O-acetyl azidomannosamine). Next, the N-mustard-PRO 10 and doxorubicin-PRO 12 prodrugs were employed in vitro with the bioengineered cells, and activation of the prodrugs, which allowed selective release of the cytotoxic moiety at the tumour cell, was assessed. Release of the parent drugs from the prodrugs was shown to be dependent on the level of metabolic labelling, where tetra-O-acetyl azidomannosamine allowed the highest level of azide reporter generation in tumor cells and led to full recovery of the parent cytotoxic drug's potency. The selectivity of azide expression on breast cancer MCF-7 cells versus normal fibroblast L929 cells was also probed, with the 9-azido sialic acid and tetra-O-acetylated-9-azido sialic acid showing ∼17-fold higher azide expression on the former. Taken together, these data demonstrate the feasibility of the Staudinger reaction for selective activation of prodrugs targeted to the MCF-7 breast cancer cells.

Inhibition of activin A receptor signalling attenuates age-related pathological cardiac remodelling.

In the heart, ageing is associated with DNA damage, oxidative stress, fibrosis and activation of the activin signalling pathway, leading to cardiac dysfunction. The cardiac effects of activin signalling blockade in progeria are unknown. This study investigated the cardiac effects of progeria induced by attenuated levels of Ercc1, which is required for DNA excision and repair, and the impact of activin signalling blockade using a soluble activin receptor type IIB (sActRIIB). DNA damage and oxidative stress were significantly increased in Ercc1Δ/- hearts, but were reduced by sActRIIB treatment. sActRIIB treatment improved cardiac systolic function and induced cardiomyocyte hypertrophy in Ercc1Δ/- hearts. RNA-sequencing analysis showed that in Ercc1Δ/- hearts, there was an increase in pro-oxidant and a decrease in antioxidant gene expression, whereas sActRIIB treatment reversed this effect. Ercc1Δ/- hearts also expressed higher levels of anti-hypertrophic genes and decreased levels of pro-hypertrophic ones, which were also reversed by sActRIIB treatment. These results show for the first time that inhibition of activin A receptor signalling attenuates cardiac dysfunction, pathological tissue remodelling and gene expression in Ercc1-deficient mice and presents a potentially novel therapeutic target for heart diseases.

Electrical Stimulation of Adipose-Derived Stem Cells in 3D Nanofibrillar Cellulose Increases Their Osteogenic Potential.

Due to the ageing population, there is a steadily increasing incidence of osteoporosis and osteoporotic fractures. As conventional pharmacological therapy options for osteoporosis are often associated with severe side effects, bone grafts are still considered the clinical gold standard. However, the availability of viable, autologous bone grafts is limited making alternative cell-based strategies a promising therapeutic alternative. Adipose-derived stem cells (ASCs) are a readily available population of mesenchymal stem/stromal cells (MSCs) that can be isolated within minimally invasive surgery. This ease of availability and their ability to undergo osteogenic differentiation makes ASCs promising candidates for cell-based therapies for bone fractures. Recent studies have suggested that both exposure to electrical fields and cultivation in 3D can positively affect osteogenic potential of MSCs. To elucidate the osteoinductive potential of a combination of these biophysical cues on ASCs, cells were embedded within anionic nanofibrillar cellulose (aNFC) hydrogels and exposed to electrical stimulation (ES) for up to 21 days. ES was applied to ASCs in 2D and 3D at a voltage of 0.1 V/cm with a duration of 0.04 ms, and a frequency of 10 Hz for 30 min per day. Exposure of ASCs to ES in 3D resulted in high alkaline phosphatase (ALP) activity and in an increased mineralisation evidenced by Alizarin Red S staining. Moreover, ES in 3D aNFC led to an increased expression of the osteogenic markers osteopontin and osteocalcin and a rearrangement and alignment of the actin cytoskeleton. Taken together, our data suggest that a combination of ES with 3D cell culture can increase the osteogenic potential of ASCs. Thus, exposure of ASCs to these biophysical cues might improve the clinical outcomes of regenerative therapies in treatment of osteoporotic fractures.

Myocyte remodeling in response to hypertrophic stimuli requires nucleocytoplasmic shuttling of muscle LIM protein.

CSRP3 or muscle LIM protein (MLP) is a nucleocytoplasmic shuttling protein and a mechanosensor in cardiac myocytes. MLP regulation and function was studied in cultured neonatal rat myocytes treated with pharmacological or mechanical stimuli. Either verapamil or BDM decreased nuclear MLP while phenylephrine and cyclic strain increased it. These results suggest that myocyte contractility regulates MLP subcellular localization. When RNA polymerase II was inhibited with alpha-amanitin, nuclear MLP was reduced by 30%. However, when both RNA polymerase I and II were inhibited with actinomycin D, there was a 90% decrease in nuclear MLP suggesting that its nuclear translocation is regulated by both nuclear and nucleolar transcriptional activity. Using cell permeable synthetic peptides containing the putative nuclear localization signal (NLS) of MLP, nuclear import of the protein in cultured rat neonatal myocytes was inhibited. The NLS of MLP also localizes to the nucleolus. Inhibition of nuclear translocation prevented the increased protein accumulation in response to phenylephrine. Furthermore, cyclic strain of myocytes after prior NLS treatment to remove nuclear MLP resulted in disarrayed sarcomeres. Increased protein synthesis and brain natriuretic peptide expression were also prevented suggesting that MLP is required for remodeling of the myofilaments and gene expression. These findings suggest that nucleocytoplasmic shuttling MLP plays an important role in the regulation of the myocyte remodeling and hypertrophy and is required for adaptation to hypertrophic stimuli.

Assembly and maintenance of the sarcomere night and day.

The assembly of sarcomeric proteins into the highly organized structure of the sarcomere is an ordered and complex process involving an array of structural and associated proteins. The sarcomere has shown itself to be considerably more complex than ever envisaged and may be considered one of the most complex macromolecular assemblies in biology. Studies over the last decade have helped to put a new face on the sarcomere, and, as such, the sarcomere is being redefined as a dynamic network of proteins capable of generating force and signalling with other cellular compartments and metabolic enzymes capable of controlling many facets of striated myocyte biology.

The exon junction complex senses energetic stress and regulates contractility and cell architecture in cardiac myocytes.

The exon junction complex (EJC) is the main mechanism by which cells select specific mRNAs for translation into protein. We hypothesized that the EJC is involved in the regulation of gene expression during the stress response in cardiac myocytes, with implications for the failing heart. In cultured rat neonatal myocytes, we examined the cellular distribution of two EJC components eukaryotic translation initiation factor 4A isoform 3 (eIF4A3) and mago nashi homologue (Mago) in response to metabolic stress. There was significant relocalization of eIF4A3 and Mago from the nucleus to cytoplasm following 18 h of hypoxia. Treating myocytes with 50 mM NaN3 for 4 h to mimic the metabolic stress induced by hypoxia also resulted in significant relocalization of eIF4A3 and Mago to the cytoplasm. To examine whether the effects of metabolic stress on the EJC proteins were dependent on the metabolic sensor AMP kinase (AMPK), we treated myocytes with 1 µM dorsomorphin (DM) in combination with NaN3 DM augmented the translocation of Mago and eIF4A3 from the nucleus to the cytoplasm. Knockdown of eIF4A3 resulted in cessation of cell contractility 96 h post-treatment and a significant reduction in the number of intact sarcomeres. Cell area was significantly reduced by both hypoxia and eIF4A3 knockdown, whilst eIF4A3 knockdown also significantly reduced nuclear size. The reduction in nuclear size is unlikely to be related to apoptosis as it was reversed in combination with hypoxia. These data suggest for the first time that eIF4A3 and potentially other EJC members play an important role in the myocyte stress response, cell contractility and morphology.

Exploring quercetin and luteolin derivatives as antiangiogenic agents.

The formation of new blood vessels from the pre-existing vasculature (angiogenesis) is a crucial stage in cancer progression and, indeed, angiogenesis inhibitors are now used as anticancer agents, clinically. Here we have explored the potential of flavonoid derivatives as antiangiogenic agents. Specifically, we have synthesised methoxy and 4-thio derivatives of the natural flavones quercetin and luteolin, two of which (4-thio quercetin and 4-thio luteolin) had never been previously reported. Seven of these compounds showed significant (p < 0.05) antiangiogenic activity in an in vitro scratch assay. Their activity ranged from an 86% inhibition of the vascular endothelium growth factor (VEGF)-stimulated migration (observed for methoxyquercetin at 10 µM and for luteolin at 1 µM) to a 36% inhibition (for thiomethoxy quercetin at 10 µM). Western blotting studies showed that most (4 out of 7) compounds inhibited phosphorylation of the VEGF receptor-2 (VEGFR2), suggesting that the antiangiogenic activity was due to an interference with the VEGF/VEGFR2 pathway. Molecular modelling studies looking at the affinity of our compounds towards VEGFR and/or VEGF confirmed this hypothesis, and indeed the compound with the highest antiangiogenic activity (methoxyquercetin) showed the highest affinity towards VEGFR and VEGF. As reports from others have suggested that structurally similar compounds can elicit biological responses via a non-specific, promiscuous membrane perturbation, potential interactions of the active compounds with a model lipid bilayer were assessed via DSC. Luteolin and its derivatives did not perturb the model membrane even at concentrations 10 times higher than the biologically active concentration and only subtle interactions were observed for quercetin and its derivatives. Finally, cytotoxicity assessment of these flavonoid derivatives against MCF-7 breast cancer cells demonstrated also a direct anticancer activity albeit at generally higher concentrations than those required for an antiangiogenic effect (10 fold higher for the methoxy analogues). Taken together these results show promise for flavonoid derivatives as antiangiogenic agents.

Lipoma preferred partner is a mechanosensitive protein regulated by nitric oxide in the heart.

Adaptor proteins play an important role in signaling pathways by providing a platform on which many other proteins can interact. Malfunction or mislocalization of these proteins may play a role in the development of disease. Lipoma preferred partner (LPP) is a nucleocytoplasmic shuttling adaptor protein. Previous work shows that LPP plays a role in the function of smooth muscle cells and in atherosclerosis. In this study we wanted to determine whether LPP has a role in the myocardium. LPP expression increased by 56% in hearts from pressure overload aortic-banded rats (p < 0.05 n = 4), but not after myocardial infarction, suggesting hemodynamic load regulates its expression. In vitro, LPP expression was 87% higher in cardiac fibroblasts than myocytes (p < 0.05 n = 3). LPP expression was downregulated in the absence of the actin cytoskeleton but not when microtubules were disassembled. We mechanically stretched cardiac fibroblasts using the Flexcell 4000 for 48 h (1 Hz, 5% maximum strain), which decreased total LPP total expression and membrane localization in subcellular fractions (p < 0.05, n = 5). However, L-NAME, an inhibitor of nitric oxide synthase (NOS), significantly upregulated LPP expression. These findings suggest that LPP is regulated by a complex interplay between NO and mechanical cues and may play a role in heart failure induced by increased hemodynamic load.

Cardiac dysfunction and heart failure are associated with abnormalities in the subcellular distribution and amounts of oligomeric muscle LIM protein.

Prolonged hemodynamic overload results in cardiac hypertrophy and failure with detrimental changes in myocardial gene expression and morphology. Cysteine-rich protein 3 or muscle LIM protein (MLP) is thought to be a mechanosensor in cardiac myocytes. Therefore, the subcellular location of MLP may have functional implications in health and disease. Our hypothesis is that MLP becomes mislocalized after prolonged overload, resulting in impaired mechanosensing in cardiac myocytes. Using the techniques of biochemical subcellular fractionation and immunocytochemistry, we found MLP exhibits oligomerization in the membrane and cytoskeleton of cultured cardiac rat neonatal myocytes. Nuclear MLP was always monomeric. MLP translocated to the nucleolus in response to 10% cyclic stretch at 1 Hz for 48 h. This was associated with a threefold increase in S6 ribosomal protein (P < 0.01; n = 3 cultures). Adenoviral overexpression of MLP also resulted in a twofold increase in S6 protein, suggesting that MLP can activate ribosomal protein synthesis in the nucleolus. In ventricles from aortic-banded and myocardially infarcted rat hearts, nuclear MLP increased by twofold (P < 0.01; n = 7) along with a significant decrease in the nonnuclear oligomeric fraction. The ratio of nuclear to nonnuclear MLP increased threefold in both groups (P < 0.01; n = 7). In failing human hearts, there was almost a complete loss of oligomeric MLP. Using a flag-tagged adenoviral MLP, we demonstrate that the COOH terminus is required for oligomerization and that this is a precursor to stretch sensing and subsequent nuclear translocation. Therefore, reduced oligomeric MLP in the costamere and cytoskeleton may contribute to impaired mechanosensing in heart failure.

The circadian protein Clock localizes to the sarcomeric Z-disk and is a sensor of myofilament cross-bridge activity in cardiac myocytes.

In the mammalian heart, the circadian protein Clock regulates glucose and fatty acid metabolism. In this study, we determined some of the factors that regulate Clock expression and subcellular distribution in myocytes. Using immunochemistry and biochemical subcellular fractionation, we have shown that Clock localizes to the Z-disk of the myofilaments. Increasing calcium and cross-bridge cycling with 10 microM phenylephrine for 48 h resulted in a threefold increase in Clock and a translocation of the protein to the nucleus. When myofilament cross-bridge cycling was inhibited with 10 microM verapamil or 7.5mM butanedione monoxime for 48 h, both significantly reduced the presence of Clock in the nucleus and cytoskeleton. These results suggest that the expression and subcellular distribution of Clock can be altered by changes in cross-bridge cycling, a major source of energy expenditure in myocytes. We suggest that the circadian Clock protein may help coordinate the sensing of energy expenditure with energy supply.

RGD and YIGSR synthetic peptides facilitate cellular adhesion identical to that of laminin and fibronectin but alter the physiology of neonatal cardiac myocytes.

In the mammalian heart, the extracellular matrix plays an important role in regulating cell behavior and adaptation to mechanical stress. In cell culture, a significant number of cells detach in response to mechanical stimulation, limiting the scope of such studies. We describe a method to adhere the synthetic peptides RGD (fibronectin) and YIGSR (laminin) onto silicone for culturing primary cardiac cells and studying responses to mechanical stimulation. We first examined cardiac cells on stationary surfaces and observed the same degree of cellular adhesion to the synthetic peptides as their respective native proteins. However, the number of striated myocytes on the peptide surfaces was significantly reduced. Focal adhesion kinase (FAK) protein was reduced by 50% in cardiac cells cultured on YIGSR peptide compared with laminin, even though beta(1)-integrin was unchanged. Connexin43 phosphorylation increased in cells adhered to RGD and YIGSR peptides. We then subjected the cardiac cells to cyclic strain at 20% maximum strain (1 Hz) for 48 h. After this period, cell attachment on laminin was reduced to approximately 50% compared with the unstretched condition. However, in cells cultured on the synthetic peptides, there was no significant difference in cell adherence after stretch. On YIGSR peptide, myosin protein was decreased by 50% after mechanical stimulation. However, total myosin was unchanged in cells stretched on laminin. These results suggest that RGD and YIGSR peptides promote the same degree of cellular adhesion as their native proteins; however, they are unable to promote the signaling required for normal FAK expression and complete sarcomere formation in cardiac myocytes.