Heavy Chain mRNA Expression Research Articles

Puerarin attenuates isoproterenol‑induced myocardial hypertrophy via inhibition of the Wnt/β‑catenin signaling pathway.

Myocardial hypertrophy (MH) is an independent risk factor for cardiovascular disease, which in turn lead to arrhythmia or heart failure. Therefore, attention must be paid to formulation of therapeutic strategies for MH. Puerarin is a key bioactive ingredient isolated from Pueraria genera of plants that is beneficial for the treatment of MH. However, its molecular mechanism of action has not been fully determined. In the present study, 40 µM puerarin was demonstrated to be a safe dose for human AC16 cells using Cell Counting Kit-8 assay. The protective effects of puerarin against MH were demonstrated in AC16 cells stimulated with isoproterenol (ISO). These effects were characterized by a significant decrease in surface area of cells (assessed using fluorescence staining) and mRNA and protein expression levels of MH-associated biomarkers, including atrial and brain natriuretic peptide, assessed using reverse transcription-quantitative PCR and western blotting, as well as β-myosin heavy chain mRNA expression levels. Mechanistically, western blotting demonstrated that puerarin inhibited activation of the Wnt signaling pathway. Puerarin also significantly decreased phosphorylation of p65; this was mediated via crosstalk between the Wnt and NF-κB signaling pathways. An inhibitor (Dickkopf-1) and activator (IM-12) of the Wnt signaling pathway were used to demonstrate that puerarin-mediated effects alleviated ISO-induced MH via the Wnt signaling pathway. The results of the present study demonstrated that puerarin pre-treatment may be a potential therapeutic strategy for preventing ISO-induced MH and managing MH in the future.

Preliminary Investigation of In vitro, Bidirectional Vocal Fold Muscle-Mucosa Interactions.

Oversimplified clinical dogma suggests that laryngeal diseases fall into two broad, mutually exclusive diagnostic categories-mucosal injury or neuromuscular/functional disorders. Extensive investigation in the lower airway as well as other organ systems suggest complex interactions between tissue types underlying both tissue health and pathological states. To date, no such relationship has been described in the vocal folds, likely the most bioactive organ in the body. We hypothesize interactions between the vocal fold muscle and mucosa likely contribute to aberrant phonatory physiology and warrant further investigation to ultimately develop novel therapeutic strategies. Primary culture of myoblasts from rat thyroarytenoid muscle and fibroblasts from the vocal fold mucosa were established. Co-culture and conditioned media experiments were performed to established bidirectional interactions between cell types. Transforming Growth Factor (TGF)-β was employed to stimulate a fibrotic phenotype in culture. In addition to quantitative PCR, standard migration and proliferation assays were performed as well as immunocytochemistry. Bidirectional cell-cell interactions were observed. Without TGF-β stimulation, myoblast conditioned media inhibited fibroblast migration, but enhanced proliferation. Conversely, fibroblast conditioned media increased both myoblast proliferation and migration. Myoblast conditioned media decreased TGF-β-mediated gene expression and of particular interest, ACTA2 mRNA expression. In both co-culture and in response to fibroblast conditioned media, myosin heavy chain (Myh2) mRNA expression decreased in myoblasts. These data are the first to describe interactions between cell types within the vocal fold. The implications for these interactions in vivo warrant further investigation to develop and refine optimal treatment strategies.

Decreased vascular smooth muscle contractility in Hutchinson\u2013Gilford Progeria Syndrome linked to defective smooth muscle myosin heavy chain expression

Children with Hutchinson–Gilford Progeria Syndrome (HGPS) suffer from multiple cardiovascular pathologies due to the expression of progerin, a mutant form of the nuclear envelope protein Lamin A. Progerin expression has a dramatic effect on arterial smooth muscle cells (SMCs) and results in decreased viability and increased arterial stiffness. However, very little is known about how progerin affects SMC contractility. Here, we studied the LaminAG609G/G609G mouse model of HGPS and found reduced arterial contractility at an early age that correlates with a decrease in smooth muscle myosin heavy chain (SM-MHC) mRNA and protein expression. Traction force microscopy on isolated SMCs from these mice revealed reduced force generation compared to wild-type controls; this effect was phenocopied by depletion of SM-MHC in WT SMCs and overcome by ectopic expression of SM-MHC in HGPS SMCs. Arterial SM-MHC levels are also reduced with age in wild-type mice and humans, suggesting a common defect in arterial contractility in HGPS and normal aging.

Allele-Selective Knockdown of MYH7 Using Antisense Oligonucleotides

Hundreds of dominant-negative myosin mutations have been identified that lead to hypertrophic cardiomyopathy, and the biomechanical link between mutation and disease is heterogeneous across this patient population. To increase the therapeutic feasibility of treating this diverse genetic population, we investigated the ability of locked nucleic acid (LNA)-modified antisense oligonucleotides (ASOs) to selectively knock down mutant myosin transcripts by targeting single-nucleotide polymorphisms (SNPs) that were found to be common in the myosin heavy chain 7 (MYH7) gene. We identified three SNPs in MYH7 and designed ASO libraries to selectively target either the reference or alternate MYH7 sequence. We identified ASOs that selectively knocked down either the reference or alternate allele at all three SNP regions. We also show allele-selective knockdown in a mouse model that was humanized on one allele. These results suggest that SNP-targeting ASOs are a promising therapeutic modality for treating cardiac pathology.

Si-Miao-Yong-An Decoction Protects Against Cardiac Hypertrophy and Dysfunction by Inhibiting Platelet Aggregation and Activation.

Objective: The aim of this study was to determine whether Si-Miao-Yong-An decoction (SMYAD) could ameliorate pressure overload-induced heart hypertrophy and its mechanisms.Methods: C57BL/6 mice were subjected to either sham or transverse aortic constriction (TAC) surgery to induce heart hypertrophy. SMYAD (14.85 g/kg/day, ig) or captopril (16.5 mg/kg/day, ig) was administered to the mice for 4 weeks. Cardiac function was evaluated based on echocardiography. Heart hypertrophy was detected using hematoxylin and eosin or wheat germ agglutinin staining. Protein expression of CD41, CD61, and P-selectin were measured with Western blot and immunohistochemistry. The expression levels of atrial natriuretic peptide, brain natriuretic peptide, β-myosin heavy chain, β-thromboglobulin, and von Willebrand factor were evaluated by quantitative polymerase chain reaction.Results: Four weeks after TAC, mice developed exaggerated cardiac hypertrophy and demonstrated a strong decrease in left ventricular ejection fraction compared with sham (29.9 ± 9.3% versus 66.0 ± 9.9%; P < 0.001). Conversely, SMYAD improved cardiac dysfunction with preserved left ventricular ejection fraction (66.5 ± 17.2%; P < 0.001). Shortening fraction was increased by SMYAD, while the left ventricular internal diameter and left ventricular volume were decreased in SMYAD group. SMYAD treatment significantly attenuated cardiac hypertrophy as reflected by the inhibition of atrial natriuretic peptide, brain natriuretic peptide, β-myosin heavy chain mRNA expression, and by the decreasing of cardiac myocyte cross-sectional area. Furthermore, Western blot and immunohistochemistry indicated that the protein expression of platelet aggregation markers (CD41 and CD61) and platelet activation marker (P-selectin) were significantly higher in model mice compared with control. These pathological alterations in TAC-induced mice were significantly ameliorated or blocked by SMYAD administration.Conclusions: Our results suggested that SMYAD exerted its effect by inhibiting platelet aggregation and activation as revealed by CD41/CD61/P-selectin downregulation. Inhibition the activation of the platelets might contribute to the therapeutic effect of SMYAD in failing heart.

Calcimimetic R568 improved cardiac remodeling by classic and novel renin-angiotensin system in spontaneously hypertensive rats.

One major cause of cardiac mortality is heart disease caused by hypertension. The formation of cyclic adenosine monophosphate (cAMP) is inhibited by calcium-sensitive receptor (CaSR) activation which increases intracellular Ca2+ concentrations and suppresses renin release. As we know, renin-angiotensin system (RAS) is closely related to development of essential hypertension (EH). Therefore, we focused on exploring the roles of NPSR568 (R568)-activated CaSR in cardiac remodeling of spontaneously hypertensive rats (SHRs), as well as the activity of classic and novel RAS. Wistar-Kyoto rats (WKYs) and SHRs were treated by R568 for four and eight weeks, respectively, and their blood pressure (BP), echocardiographic values, heart-to-body weight ratio (HW/BW%), and left ventricle-to-body weight ratio (LVW/BW%) were evaluated. Then Masson’s trichrome staining and hematoxylin and eosin staining as well as RT-qPCR analysis of β-isoform of myosin heavy chain and brain natriuretic peptide mRNA expression were performed. A Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay and analysis of apoptosis marker proteins were used to assess the extent of myocardial apoptosis. The CaSR expression and the activity of classic and novel RAS were examined by immunohistochemistry, western blotting, and enzyme-linked immunosorbent assay. The present study revealed that the development of hypertension was accompanied by increased BP, apoptosis, hypertrophy, and fibrosis, along with decreased expression of CaSR, decreased novel RAS, and increased classic RAS in myocardial tissues. R568 administration for four and eight weeks reduced BP and myocardial remodeling and reversed the low expression of CaSR; moreover, classic RAS was suppressed and novel RAS was activated in the myocardium. Taken together, these data indicate that R568 may effectively inhibit EH myocardial remodeling by inhibiting classic RAS and activating novel RAS in SHRs. Impact statement Our study reveals that low calcium-sensitive receptor (CaSR) expression is associated with the occurrence and development of essential hypertension-mediated myocardial remodeling. The activation of CaSR can reverse adverse myocardia remodeling by inhibiting local classical renin-angiotensin system (RAS) and activating novel RAS in cardiac tissues. CaSR is closely related to many cardiovascular diseases, but its specific mechanism remains not to be elucidated. To date, CaSR has not been investigated with regard to cardiovascular treatment; however, given the important relationship between CaSR and cardiovascular disease, CaSR regulators can be potential drugs for the treatment of cardiovascular disease.

Sibjotang attenuates Doxorubicin‐Induced Cardiac Hypertrophy: Involvement of necroptosis

Doxorubicin is one for the most widely used anti‐neoplastic agents, however severely limited due to its cumulative cardiotoxicity, which develops over time into congestive heart failure. Sibjotang (Shizaotang), traditional herbal medicine formula, has been reported regulates the body fluid blood pressure homeostasis. The present study was aimed at determining the effect and regulatory mechanism of Sibjotang in doxorubicin‐induced cardiotoxicity in H9c2 cells. Immunofluorescence staining analysis showed that Sibjotang decreased doxorubicin‐induced increase in H9c2 cardiomyocytes size in does dependent manner. Pre‐treatment with Sibjotang significantly inhibited doxorubicin‐induced cardiac hypertrophic markers such as ANP (atrial natriuretic peptide), BNP (brain natriuretic peptide) and β‐MHC (β‐myosin heavy chain) mRNA expression levels. Sibjotang reduced hypertrophy‐associated transcription factor GATA 4 (GATA binding factor 4) expression. Pre‐treatment with Sibjotang significantly inhibited doxorubicin‐induced necroptosis‐associated kinase such as receptor‐interacting serine/threonine‐protein kinase RIPK1 and RIPK3 without affecting cell viability. In conclusion, doxorubicin‐induced cardiac hypertrophy and necroptosis were attenuated by Sibjotang. Therefore, Sibjotang may be potential herbal therapies targeting cardiotoxicity resulting from anti‐cancer agents.Support or Funding InformationThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Leucine regulates slow-twitch muscle fibers expression and mitochondrial function by Sirt1/AMPK signaling in porcine skeletal muscle satellite cells.

A previous study demonstrated that leucine upregulates the slow myosin heavy chain mRNA expression in C2C12 cells. However, the role of leucine in slow-twitch muscle fibers expression and mitochondrial function of porcine skeletal muscle satellite cells as well as its mechanism remain unclear. In this study, porcine skeletal muscle satellite cells cultured in differentiation medium were treated with 2 mM leucine for 3days. Sirt1 inhibitor EX527, AMPK inhibitor compound C, and AMPKα1 siRNA were used to examine its underlying mechanism. Here we showed that leucine increased slow-twitch muscle fibers and mitochondrial function-related gene expression, as well as increased succinic dehydrogenase (SDH) and malate dehydrogenase (MDH) activities. Moreover, leucine increased the protein levels of Sirt1 and phospho-AMPK. We also found that AMPKα1 siRNA, AMPK inhibitor compound C, or Sirt1 inhibitor EX527 attenuated the positive effect of leucine on slow-twitch muscle fibers and mitochondrial function-related gene expression. Finally, we showed that Sirt1 was required for leucine-induced AMPK activation. Our results provide, for the first time, evidence that leucine induces slow-twitch muscle fibers expression and improves mitochondrial function through Sirt1/AMPK signaling pathway in porcine skeletal muscle satellite cells.

Intracellular response to process optimization and impact on productivity and product aggregates for a high-titer CHO cell process.

A key goal in process development for antibodies is to increase productivity while maintaining or improving product quality. During process development of an antibody, titers were increased from 4 to 10 g/L while simultaneously decreasing aggregates. Process development involved optimization of media and feed formulations, feed strategy, and process parameters including pH and temperature. To better understand how CHO cells respond to process changes, the changes were implemented in a stepwise manner. The first change was an optimization of the feed formulation, the second was an optimization of the medium, and the third was an optimization of process parameters. Multiple process outputs were evaluated including cell growth, osmolality, lactate production, ammonium concentration, antibody production, and aggregate levels. Additionally, detailed assessment of oxygen uptake, nutrient and amino acid consumption, extracellular and intracellular redox environment, oxidative stress, activation of the unfolded protein response (UPR) pathway, protein disulfide isomerase (PDI) expression, and heavy and light chain mRNA expression provided an in-depth understanding of the cellular response to process changes. The results demonstrate that mRNA expression and UPR activation were unaffected by process changes, and that increased PDI expression and optimized nutrient supplementation are required for higher productivity processes. Furthermore, our findings demonstrate the role of extra- and intracellular redox environment on productivity and antibody aggregation. Processes using the optimized medium, with increased concentrations of redox modifying agents, had the highest overall specific productivity, reduced aggregate levels, and helped cells better withstand the high levels of oxidative stress associated with increased productivity. Specific productivities of different processes positively correlated to average intracellular values of total glutathione. Additionally, processes with the optimized media maintained an oxidizing intracellular environment, important for correct disulfide bond pairing, which likely contributed to reduced aggregate formation. These findings shed important understanding into how cells respond to process changes and can be useful to guide future development efforts to enhance productivity and improve product quality.

Deletion of Kvβ2 Alters Murine Skeletal Muscle Morphology and Phenotype

IntroductionMammalian skeletal muscle expresses various potassium channels that regulate myogenesis. Recent evidence points to the role of Kv1.5 in cell cycle progression in myoblasts as pharmacological inhibition of this channel in L6E9 cells inhibited G0/G1 transition. Additionally, skeletal muscle also expresses voltage‐gated potassium channel beta subunits 1–3 (Kvβ, 2, 3) of aldo‐keto reductases (AK6) family, although their role in skeletal myogenesis is not yet known. Given that Kvβ subunits binds to and regulate members of Kv1 family including Kv1.5, it is plausible that Kvβ subunits play a significant role in skeletal myogenesis.Hypothesiswe hypothesized that Kvβ2 modulates skeletal myogenesis.MethodsAge matched, 8–12 week old, wild type (Wt) and Kvβ2 knockout mice (KO) mice were used in this study. Transcript expression analysis of key transcription factors, muscle growth regulators and contractile genes was conducted utilizing real time quantitative PCR (qPCR). Myofiber size was estimated in biceps femoris muscle cross‐sections using Hematoxylin and eosin staining. Protein expression was assessed by Western blots in lysates isolated from Biceps femoris muscle.ResultsBody weight of KO mice recorded at 6, 11 or 16 weeks of age, was significantly (p &lt; 0.05) less than that of age matched Wt mice. Body weight normalized to tibia length in 16 week old KO mice was nearly 30% less than that of age matched Wt mice. Biceps femoris muscle cross‐sections in KO mice revealed significantly smaller myofiber area when compared to that of Wt mice (p &lt; 0.05). Myosin heavy chain mRNA expression profiles were also significantly different between the KO and Wt mice. when compared with that of Wt mice, gastrocnemius and biceps femoris of KO mice showed marginal (p &lt; 0.1) to significantly (p &lt; 0.05) higher expression of myosin heavy chain IIA and IIX isoforms, while myosin heavy chain I mRNA expression was not different between the groups. Soleus muscle of KO mice showed significantly higher myosin heavy chain I expression, while the expression of myosin heavy chain 2B was profoundly (p &lt; 0.05) decreased. Interestingly, the expression of major myogenic regulatory factors was unchanged between the two genotypes, with the exception that MyoD expression showed a consistently decreasing trend in all three muscles of KO mice when compared with that of Wt. In C2C12 cells, Kvβ2 mRNA expression declined steadily during the course of differentiation reaching to approximately 10% of that observed before differentiation (p &lt; 0.05).ConclusionOur data suggest that Kvβ2 subunit is essential for skeletal myogenesis. Kvβ2 appear to effect muscle fitness by altering myosin heavy chain composition, suggesting a significant role of Kvβ subunits in skeletal muscle size and phenotype determination.

Exogenous apelin changes alpha and beta myosin heavy chain mRNA expression and improves cardiac function in PTU-induced hypothyroid rats

The most important conditions associated with hypothyroidism is the cardiac dysfunction. Apelin is an endogenous ligand, involved in energy storage and metabolism which improves cardiac contractility. This study was done to evaluate the effects of apelin, l-Thyroxin (T4) or a combination of both, on cardiac function and mRNA expression of two contractile proteins, α and β myosin heavy chain (α-MHC and β-MHC), in 6-propyl-2-thiouracil (PTU)-induced hypothyroid rats. Forty male Wistar rats were randomly assigned into five groups: Ctrl (Control), and 4 hypothyroid groups (H, HA, HT, and HAT). The Hypothyroid (H) group received 0.05% PTU in the drinking water for six weeks; the next 3 groups, along with PTU, received apelin (HA, 200μg/kg/day, ip), T4 (HT, 20μg/kg/day, gavage), or a combination of both drugs (HAT) for the last 2weeks (weeks 5 and 6). TSH and T4 were measured using ELISA kit. Isolated hearts of animals were perfused in Langendorff apparatus and left ventricular developed pressure, cardiac contractility, heart rate, rate pressure product and perfusion pressure were assessed using PowerLab ADInstruments. In addition α-MHC and β-MHC mRNA expression were evaluated by RT-PCR method in heart tissue. Apelin alone or accompanied by T4 significantly increased cardiac contractility and performance as compared to hypothyroid group. Apelin also significantly increased the alpha-MHC mRNA expression and in the presence of T4 significantly decreased beta-MHC mRNA expression.It seems that apelin alone may improve cardiac function in hypothyroid rats via genomic pathways.

Neurofilament heavy chain expression and neuroplasticity in rat auditory cortex after unilateral and bilateral deafness

Deafness induces many plastic changes in the auditory neural system. For instance, dendritic changes cause synaptic changes in neural cells. SMI-32, a monoclonal antibody reveals auditory areas and recognizes non-phosphorylated epitopes on medium- and high-molecular-weight subunits of neurofilament proteins in cortical pyramidal neuron dendrites. We investigated SMI-32-immunoreactive (-ir) protein levels in the auditory cortices of rats with induced unilateral and bilateral deafness. Adult male Sprague–Dawley rats were divided into unilateral deafness (UD), bilateral deafness (BD), and control groups. Deafness was induced by cochlear ablation. All rats were sacrificed, and the auditory cortices were harvested for real-time quantitative polymerase chain reaction (RT-qPCR) and western blot analyses at 2, 4, 6, and 12 weeks after deafness was induced. Immunohistochemical staining was performed to evaluate the location of SMI-32-ir neurons. Neurofilament heavy chain (NEFH) mRNA expression and SMI-32-ir protein levels were increased in the BD group. In particular, SMI-32-ir protein levels increased significantly 6 and 12 weeks after deafness was induced. In contrast, no significant changes in protein level were detected in the right or left auditory cortices at any time point in the UD group. NEFH mRNA level decreased at 4 weeks after deafness was induced in the UD group, but recovered thereafter. Taken together, BD induced plastic changes in the auditory cortex, whereas UD did not affect the auditory neural system sufficiently to show plastic changes, as measured by neurofilament protein level.

Klotho inhibits angiotensin II-induced cardiomyocyte hypertrophy through suppression of the AT1R/beta catenin pathway

Myocardial hypertrophy is an independent risk factor for cardiac morbidity and mortality. The antiaging protein klotho reportedly possesses a protective role in cardiac diseases. However, the precise mechanisms underlying the cardioprotective effects of klotho remain unknown. This study was aimed to determine the effects of klotho on angiotensin II (Ang II)-induced hypertrophy in neonatal rat cardiomyocytes and the possible mechanism of actions. We found that klotho significantly inhibited Ang II-induced hypertrophic growth of neonatal cardiomyocytes, as evidenced by decreased [3H]-Leucine incorporation, cardiomyocyte surface area and β-myosin heavy chain (β-MHC) mRNA expression. Meanwhile, klotho inhibited Ang II–stimulated activation of the Wnt/β-catenin pathway in cardiomyocytes, as evidenced by decreased protein expression of active β-catenin, downregulated protein and mRNA expression of the β-catenin target genes c-myc and cyclin D1, and increased β-catenin phosphorylation. Inhibition of the Wnt/β-catenin pathway by the specific inhibitor XAV939 markedly attenuated Ang II-induced cardiomyocyte hypertrophy. The further study revealed that klotho treatment significantly downregulated protein expression of Ang II receptor type I (AT1R) but not type II (AT2R). The AT1R antagonist losartan inhibited Ang II–stimulated activation of the Wnt/β-catenin pathway and cardiomyocyte hypertrophy. Our findings suggest that klotho inhibits Ang II-induced cardiomyocyte hypertrophy through suppression of the AT1R/β-catenin signaling pathway, which may provide new insights into the mechanism underlying the protective effects of klotho in heart diseases, and raise the possibility that klotho may act as an endogenous antihypertrophic factor by inhibiting the Ang II signaling pathway.

Augmented endothelial l-arginine transport ameliorates pressure-overload-induced cardiac hypertrophy.

What is the central question of this study? What is the potential role of endothelial NO production via overexpression of the l-arginine transporter, CAT1, as a mitigator of cardiac hypertrophy? What is the main finding and its importance? Augmentation of endothelium-specific l-arginine transport via CAT1 can attenuate pressure-overload-dependent cardiac hypertrophy and fibrosis. Our findings support the conclusion that interventions that improve endothelial l-arginine transport may provide therapeutic utility in the setting of myocardial hypertrophy. Such modifications may be introduced by exercise training or locally delivered gene therapy, but further experimental and clinical studies are required. Endothelial dysfunction has been postulated to play a central role in the development of cardiac hypertrophy, probably as a result of reduced NO bioavailability. We tested the hypothesis that increased endothelial NO production, mediated by increased l-arginine transport, could attenuate pressure-overload-induced cardiac hypertrophy. Echocardiography and blood pressure measurements were performed 15 weeks after transverse aortic constriction (TAC) in wild-type (WT) mice (n = 12) and in mice with endothelium-specific overexpression of the l-arginine transporter, CAT1 (CAT+; n = 12). Transverse aortic constriction induced greater increases in heart weight to body weight ratio in WT (by 47%) than CAT+ mice (by 25%) compared with the respective controls (P ≤ 0.05). Likewise, the increase in left ventricular wall thickness induced by TAC was significantly attenuated in CAT+ mice (P = 0.05). Cardiac collagen type I mRNA expression was greater in WT mice with TAC (by 22%; P = 0.03), but not in CAT+ mice with TAC, compared with the respective controls. Transverse aortic constriction also induced lesser increases in β-myosin heavy chain mRNA expression in CAT+ mice compared with WT (P ≤ 0.05). Left ventricular systolic pressure after TAC was 36 and 39% greater in WT and CAT+ mice, respectively, compared with the respective controls (P ≤ 0.001). Transverse aortic constriction had little effect on left ventricular end-diastolic pressure in both genotypes. Taken together, these data indicate that augmenting endothelial function by overexpression of l-arginine transport can attenuate pressure-overload-induced cardiac hypertrophy.

Reliable determination of training-induced alterations in muscle fiber composition in human skeletal muscle using quantitative polymerase chain reaction.

Determination of muscle fiber composition in human skeletal muscle biopsies is often performed using immunohistochemistry, a method that tends to be both time consuming, technically challenging, and complicated by limited availability of tissue. Here, we introduce quantitative reverse transcriptase polymerase chain reaction (qRT-PCR)-based Gene-family profiling (GeneFam) of myosin heavy chain (MyHC) mRNA expression as a high-throughput, sensitive, and reliable alternative. We show that GeneFam and immunohistochemistry result in similar disclosures of alterations in muscle fiber composition in biopsies from musculus vastus lateralis and musculus biceps brachii of previously untrained young women after 12 weeks of progressive strength training. The adaptations were evident as (a) consistent increases in MyHC2A abundance; (b) consistent decreases in MyHC2X abundance; and (c) consistently stable MyHC1 abundance, and were not found using traditional reference gene-based qRT-PCR analyses. Furthermore, muscle fiber composition found using each of the two approaches was correlated with each other (r = 0.50, 0.74, and 0.78 for MyHC1, A, and X, respectively), suggesting that GeneFam may be suitable for ranking of individual muscle phenotype, particularly for MyHC2 fibers. In summary, GeneFam of MyHC mRNA resulted in reliable assessment of alterations in muscle fiber composition in skeletal muscle of previously untrained women after 12 weeks of strength training.

Abstract 199: Distinctive Roles of Linker Histone H1 Variants in the Hypertrophic Response of Cardiomyocytes

Heart failure results when cardiac output is insufficient to meet physiological requirements and is often preceded by development of cardiomyocyte hypertrophy. As cardiac myocytes respond to hypertrophic stresses they re-express developmentally important genes, normally senescent in the adult. The chromatin structural events underlying this “fetal gene program” are unknown. We previously showed by proteomics that histones, components of the chromatin protein functional unit, the nucleosome, are altered during hypertrophic and failing phases of pressure overload in mouse: linker histone variants H1.2 and H1.5 decreased in hypertrophied myocardium while H1.0 increased during the transition to failure. The linker histone H1 family influences higher order chromatin structure and gene expression, although the role of this family in the heart is unknown. To assess the role of linker histones in hypertrophy, neonatal rat ventricular cardiomyocytes (NRVMs) were transfected with siRNAs individually targeting six H1 variants. Loss of H1.3 and H1.4 respectively induced a significant 26.1% (76 of 90) and 13.5% (80 of 94) increase in cell size area (µm2). A role of H1 in the hypertrophic response is evidenced by its influence on myosin heavy chain (MHC) mRNA expression. Knock-down of individual H1 variants significantly altered the MHC isoform ratio: loss of H1.3 increased α-MHC levels 1.5 fold and decreased β-MHC 1.6 fold while H1.5 depletion decreased α-MHC 2.5 fold. Both H1.3 and H1.4 knock-down increased atrial natriuretic factor (ANF) 1.3 fold while H1.5 loss decreased ANF 6.2 fold shown by qRT-PCR. Treatment with hypertrophy-inducing agents Isoproterenol (1μM), Endothelin (2nM) or Phenylephrine (10μM), reduced H1 mRNA levels however with subtle effects on protein abundance. To evaluate whether H1 loss shifted NRVM nuclei from a predominantly heterochromatic toward euchromatic state favoring gene accessibility we examined distinct histone markers of chromatin states. Histone H1.5 knock-down significantly decreased H3K9Me3 levels, a silencing mark associated with heterochromatin, 1.7 fold. Therefore we conclude that variants package distinctive regions of the genome and that H1.3 and H1.4 controls genes involved in the hypertrophic response.

GW24-e0068 The effect of intermedin on angiotensin II and endothelin-1 induced ventricular myocyte hypertrophy in neonatal rat

Intermedin (IMD) is a novel peptide related to calcitonin gene-related peptide (CGRP) and adrenomedullin (ADM), which may have localised actions as a modulator of cardiac function. The aim of the...

Tetramethylpyrazine inhibits angiotensin II-induced cardiomyocyte hypertrophy and tumor necrosis factor-α secretion through an NF-κB-dependent mechanism

Tetramethylpyrazine (TMP), a bioactive compound isolated from the Chinese herb, Ligusticum wallichii Franchat, has been reported to play a protective role in cardiac diseases. However, the cellular and molecular mechanisms behind the protective effects of TMP on the heart remain to be elucidated. In this study, we aimed to determine the effects of TMP on angiotensin II (Ang II)-induced hypertrophy in neonatal rat cardiomyocytes and its possible mechanisms of action. In addition, we investigated whether TMP regulates tumor necrosis factor-α (TNF-α) secretion and expression. We found that TMP significantly inhibited the Ang II-induced hypertrophic growth of neonatal cardiomyocytes, as evidenced by the decrease in [3H]leucine incorporation and β-myosin heavy chain (β-MHC) mRNA expression. TMP inhibited Ang II-stimulated TNF-α protein secretion and mRNA expression in the cardiomyocytes. Further experiments revealed that Ang II increased the level of the phosphorylated form of the transcription factor, nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB), as well as NF-κB-DNA binding activity in the cardiomyocytes; treatment with TMP significantly inhibited the Ang II-induced activation of NF-κB. Furthermore, the inhibition of NF-κB by the specific inhibitor, pyrrolidine dithiocarbamate (PDTC), markedly attenuated the Ang II-induced increase in [3H]leucine incorporation, β-MHC mRNA expression and TNF-α protein secretion. Our findings suggest that TMP inhibits Ang II-induced cardiomyocyte hypertrophy and TNF-α production through the suppression of the NF-κB pathway, which may provide new insight into the mechanisms underlying the protective effects of TMP in heart diseases.

Developmental changes in diaphragm muscle function in the preterm and postnatal lamb

The preterm diaphragm is structurally and functionally immature, potentially contributing to an increased risk of respiratory distress and failure. We investigated developmental changes in contractile function and susceptibility to fatigue of the costal diaphragm in the fetal lamb to understand factors contributing to the risk of developing diaphragm dysfunction and respiratory disorders. We hypothesized that the functional capacity of the diaphragm will vary with maturational stage as will its susceptibility to fatigue. Lambs were studied at 75, 100, 125, 145, 154, 168, and 200 days postconceptional age (term = 147 days). Lambs were euthanized (sodium pentobarbitone, 100 mg/kg) either at delivery or immediately prior to post-mortem for postnatal lambs. Contractile function was assessed on longitudinal strips of intact muscle fibers and the remaining tissue frozen in liquid nitrogen for analysis of myosin heavy chain (MHC) mRNA expression and protein content. Fetal development of diaphragm function was characterized by a significant increase in maximum specific force, increased susceptibility to fatigue, reduced twitch contraction times, and a progressive increase in MHCI and MHCII protein content. Postnatally, there was a progressive decrease in the susceptibility to fatigue that coincided with an increase in the MHC I:II protein ratio. These data indicate that the functional capacity of the diaphragm varies with maturational age and may be an important determinant of the susceptibility to preterm respiratory failure.

The effect of Intermedin on Angiotensin II and Endothelin-1 Induced Ventricular Myocyte Hypertrophy in Neonatal Rat

Intermedin (IMD), a novel peptide related to calcitonin gene-related peptide (CGRP) and adrenomedullin (ADM), may have localized actions as a modulator of cardiac function. The aim of the study is to explore the effect of IMD on angiotensin II (Ang II) and endothelin-1 (ET-1) induced hypertrophy in ventricular myocytes of neonatal rat and to try to elucidate the possible mechanism. Neonatal rat cardiomyocytes were cultured in serum-free medium with and without AngII (1 micromol/L) or ET-1 (60 micromol/L) in the presence and absence of IMD (1 micromol/L). Hypertrophic responses (including cell surface area, alpha-actin, and beta-myosin heavy chain mRNA expression) and cardiomyocyte expression of NADPH oxidase gp91phox were determined. Ang II induced increases in cardiomyocyte size to 305 +/- 32 microm2 (n = 198, p < 0.05, at 48 hours), alpha-actin expression to 4 +/- 2.8-fold (n = 6, p < 0.05, at 48 hours) and beta-myosin heavy chain expression to 11 +/- 4.8-fold (n = 6, p < 0.05, at 48 hours), and expression of the gp91phox subunit of NADPH oxidase to 29.4 +/- 12.7-fold (n = 6, p < 0.05, at 48 hours). These effects were all significantly inhibited by IMD; cardiomyocyte size, alpha-actin expression, beta-myosin heavy chain expression, and gp91phox expression were reduced to 265 +/- 32 microm2 (n = 374, p < 0.05), 3.0 +/- 1.7-fold (n = 6, p < 0.05), 8.7 +/- 4.9-fold (n = 6, p < 0.05), 3.9 +/- 3-fold (n = 6, p < 0.05), respectively. IMD also significantly inhibited ET1-induced increases in cardiomyocyte size and superoxide generation. IMD exerts an antihypertrophic effect on neonatal cardiomyocytes by reduced levels of superoxide, suggesting that an antioxidant action contributes to the antihypertrophic actions of IMD.