Skeletal Α-actin Research Articles

Therapeutic impact of systemic AAV-mediated RNA interference in a mouse model of myotonic dystrophy.

RNA interference (RNAi) offers a promising therapeutic approach for dominant genetic disorders that involve gain-of-function mechanisms. One candidate disease for RNAi therapy application is myotonic dystrophy type 1 (DM1), which results from toxicity of a mutant mRNA. DM1 is caused by expansion of a CTG repeat in the 3' UTR of the DMPK gene. The expression of DMPK mRNA containing an expanded CUG repeat (CUG(exp)) leads to defects in RNA biogenesis and turnover. We designed miRNA-based RNAi hairpins to target the CUG(exp) mRNA in the human α-skeletal muscle actin long-repeat (HSA(LR)) mouse model of DM1. RNAi expression cassettes were delivered to HSA(LR) mice using recombinant adeno-associated viral (rAAV) vectors injected intravenously as a route to systemic gene therapy. Vector delivery significantly reduced disease pathology in muscles of the HSA(LR) mice, including a reduction in the CUG(exp) mRNA, a reduction in myotonic discharges, a shift toward adult pre-mRNA splicing patterns, reduced myofiber hypertrophy and a decrease in myonuclear foci containing the CUG(exp) mRNA. Significant reversal of hallmarks of DM1 in the rAAV RNAi-treated HSA(LR) mice indicate that defects characteristic of DM1 can be mitigated with a systemic RNAi approach targeting the nuclei of terminally differentiated myofibers. Efficient rAAV-mediated delivery of RNAi has the potential to provide a long-term therapy for DM1 and other dominant muscular dystrophies.

Characterization of porcine skeletal α-actin gene promoter: expression specificity and regulatory elements

Identification of essential fragments in the promoter region of genes to drive tissue-specific expression of exogenous genes in the skeletal muscle is obligatory for animal transgenic study. The skeletal α-actin is a major protein of the thin filaments of skeletal muscle fiber. In this study, the specificity and activity of porcine skeletal α-actin gene promoter were investigated by approaches of cell transfection and assayed with green fluorescent protein (GFP) and dual luciferase reporter activity, respectively. The results revealed that the obtained 3717 bp fragment of porcine skeletal α-actin gene promoter drives GFP to be expressed uniquely in murine C2C12 cells, and two segments of 2.0–2.3 and 0.06–0.37 kb in the promoter region were essential for porcine skeletal α-actin gene expression. The regulatory elements in the 0.06–0.37 kb fragment were further investigated, and when it is deleted or the CArG box within this fragment was mutated, the promoter activity was reduced to 20 or 43 % (P < 0.01), respectively, compared to the 3.04 kb segment, suggesting an important role of CArG box in regulating porcine skeletal α-actin gene transcription. The results may provide reference for creating transgenic pigs to express exogenous genes uniquely in pork.

Creatine kinase B is necessary to limit myoblast fusion during myogenesis.

Myoblast fusion is critical for proper muscle growth and regeneration. During myoblast fusion, the localization of some molecules is spatially restricted; however, the exact reason for such localization is unknown. Creatine kinase B (CKB), which replenishes local ATP pools, localizes near the ends of cultured primary mouse myotubes. To gain insights into the function of CKB, we performed a yeast two-hybrid screen to identify CKB-interacting proteins. We identified molecules with a broad diversity of roles, including actin polymerization, intracellular protein trafficking, and alternative splicing, as well as sarcomeric components. In-depth studies of α-skeletal actin and α-cardiac actin, two predominant muscle actin isoforms, demonstrated their biochemical interaction and partial colocalization with CKB near the ends of myotubes in vitro. In contrast to other cell types, specific knockdown of CKB did not grossly affect actin polymerization in myotubes, suggesting other muscle-specific roles for CKB. Interestingly, knockdown of CKB resulted in significantly increased myoblast fusion and myotube size in vitro, whereas knockdown of creatine kinase M had no effect on these myogenic parameters. Our results suggest that localized CKB plays a key role in myotube formation by limiting myoblast fusion during myogenesis.

FHL2 expression and variants in hypertrophic cardiomyopathy

Based on evidence that FHL2 (four and a half LIM domains protein 2) negatively regulates cardiac hypertrophy we tested whether FHL2 altered expression or variants could be associated with hypertrophic cardiomyopathy (HCM). HCM is a myocardial disease characterized by left ventricular hypertrophy, diastolic dysfunction and increased interstitial fibrosis and is mainly caused by mutations in genes coding for sarcomeric proteins. FHL2 mRNA level, FHL2 protein level and I-band-binding density were lower in HCM patients than control individuals. Screening of 121 HCM patients without mutations in established disease genes identified 2 novel (T171M, V187L) and 4 known (R177Q, N226N, D268D, P273P) FHL2 variants in unrelated HCM families. We assessed the structural and functional consequences of the nonsynonymous substitutions after adeno-associated viral-mediated gene transfer in cardiac myocytes and in 3D-engineered heart tissue (EHT). Overexpression of FHL2 wild type or nonsynonymous substitutions in cardiac myocytes markedly down-regulated α-skeletal actin and partially blunted hypertrophy induced by phenylephrine or endothelin-1. After gene transfer in EHTs, force and velocity of both contraction and relaxation were higher with T171M and V187L FHL2 variants than wild type under basal conditions. Finally, chronic phenylephrine stimulation depressed EHT function in all groups, but to a lower extent in T171M-transduced EHTs. These data suggest that (1) FHL2 is down-regulated in HCM, (2) both FHL2 wild type and variants partially protected phenylephrine- or endothelin-1-induced hypertrophy in cardiac myocytes, and (3) FHL2 T171M and V187L nonsynonymous variants induced altered EHT contractility. These findings provide evidence that the 2 novel FHL2 variants could increase cardiac function in HCM.Electronic supplementary materialThe online version of this article (doi:10.1007/s00395-014-0451-8) contains supplementary material, which is available to authorized users.

P28, a truncated form of TRα1 regulates mitochondrial physiology

We have previously identified in mitochondria two truncated forms of the T3 nuclear receptor TRα1, with molecular weights of 43kDa (p43) and 28kDa (p28) respectively located in the matrix and in the inner membrane. Previously, we have demonstrated that p43 stimulates mitochondrial transcription and protein synthesis in the presence of T3. Here we report that p28 is targeted into the organelle in a T3-dependent manner and displays an affinity for T3 higher than the nuclear receptor. We tried to generate mice overexpressing p28 using the human α-skeletal actin promoter, however we found an early embryonic lethality that was probably linked to a transient expression of p28 in trophoblast giant cells. This could be partly explained by the observation that overexpression of p28 in human fibroblasts induced alterations of mitochondrial physiology.

Sexually dimorphic myofilament function in a mouse model of nemaline myopathy

Nemaline myopathy, the most common congenital myopathy, is characterized by mutations in genes encoding myofilament proteins such as skeletal α-actin. These mutations are thought to ultimately lead to skeletal muscle weakness. Interestingly, some of the mutations appear to be more potent in males than in females. The underlying mechanisms remain obscure but may be related to sex-specific differences in the myofilament function of both limb and respiratory muscles. To verify this, in the present study, we used skeletal muscles (tibialis anterior and diaphragm) from a transgenic mouse model harbouring the His40Tyr amino acid substitution in skeletal α-actin. In this animal model, 60% of males die by 13weeks of age (the underlying causes of death are obscure but probably due to respiratory insufficiency) whereas females have a normal lifespan. By recording and analysing the mechanics of membrane-permeabilized myofibres, we only observed sex-related differences in the tibialis anterior muscles. Indeed, the concomitant deficits in maximal steady-state isometric force and stiffness of myofibres were less exacerbated in transgenic females than in males, potentially explaining the lower potency in limb muscles. However, the absence of sex-difference in the diaphragm muscles was rather unexpected and suggests that myofilament dysfunction does not solely underlie the sexually dimorphic phenotypes.

Recessive ACTA1 variant causes congenital muscular dystrophy with rigid spine.

Variants in ACTA1, which encodes α-skeletal actin, cause several congenital myopathies, most commonly nemaline myopathy. Autosomal recessive variants comprise approximately 10% of ACTA1 myopathy. All recessive variants reported to date have resulted in loss of skeletal α-actin expression from muscle and severe weakness from birth. Targeted next-generation sequencing in two brothers with congenital muscular dystrophy with rigid spine revealed homozygous missense variants in ACTA1. Skeletal α-actin expression was preserved in these patients. This report expands the clinical and histological phenotype of ACTA1 disease to include congenital muscular dystrophy with rigid spine and dystrophic features on muscle biopsy. This represents a new class of recessive ACTA1 variants, which do not abolish protein expression.

Abstract 71: A Dual PI3K-mTOR Inhibitor Induced Hyperglycemia and Increased Insulin Signals in the Heart

We previously reported that a dual PI3K-mTOR inhibitor BEZ235 (BEZ) induced cardiac hypertrophy. Here, we investigated potential mechanisms. Methods: three month old FVB/n female mice were treated with BEZ for five weeks. Cardiac function was monitored by serial echocardiography during the treatment and hemodynamic measurements at the end of the study. Cell signaling was analyzed by RT-PCR, Western blotting and ELISA. Results: BEZ induced a dose-dependent increase of left ventricular (LV) wall thickness and systolic function. These were associated with increased hypertrophic markers ANP, BNP, β-MHC and α-skeletal actin in the heart. In addition, in chronic BEZ-treated mouse hearts, the activations of PI3Ks, mTOR and ERK were increased. We conducted further studies to understand these contradictory results. We found that BEZ induced an increase of hepatic gluconeogenesis gene expression which was associated with increased fasting glucose, increased serum insulin level, a worsened glucose and pyruvate tolerance and increased IGFR/Insulin receptor activation in the heart. Injections of insulin lowered blood glucose, improved glucose and pyruvate tolerance, but further aggravated BEZ-induced cardiac dysfunction. On the other hand, OSI-906 (an IGFR/IR inhibitor) normalized cardiac function in BEZ-treated mice. Conclusions: Chronic BEZ treatment induced cardiac hypertrophy may be caused by increased insulin receptor activation in the heart.

A heterocyclic molecule kartogenin induces collagen synthesis of human dermal fibroblasts by activating the smad4/smad5 pathway

Declined production of collagen by fibroblasts is one of the major causes of aging appearance. However, only few of compounds found in cosmetic products are able to directly increase collagen synthesis. A novel small heterocyclic compound called kartogenin (KGN) was found to stimulate collagen synthesis of mesenchymal stem cells (MSCs). So, we hypothesized and tested that if KGN could be applied to stimulate the collagen synthesis of fibroblasts.Human dermal fibroblasts in vitro were treated with various concentrations of KGN, with dimethyl sulfoxide (DMSO) serving as the negative control. Real-time reverse-transcription polymerase chain reaction, Western blot, and immunofluorescence analyses were performed to examine the expression of collagen and transforming growth factor beta (TGF-β) signaling pathway. The production of collagen was also tested in vivo by Masson’s trichrome stain and immunohistochemistry in the dermis of mice administrated with KGN.Results showed that without obvious influence on fibroblasts’ apoptosis and viability, KGN stimulated type-I collagen synthesis of fibroblasts at the mRNA and protein levels in a time-dependent manner, but KGN did not induce expression of α-skeletal muscle actin (α-sma) or matrix metallopeptidase1 (MMP1), MMP9 in vitro. Smad4/smad5 of the TGF-β signaling pathway was activated by KGN while MAPK signaling pathway remained unchanged. KGN also increased type-I collagen synthesis in the dermis of BALB/C mice.Our results indicated that KGN promoted the type-I collagen synthesis of dermal fibroblasts in vitro and in the dermis of mice through activation of the smad4/smad5 pathway. This molecule could be used in wound healing, tissue engineering of fibroblasts, or aesthetic and reconstructive procedures.

Loss of NOX2 (gp91phox) prevents oxidative stress and progression to advanced heart failure

Oxidative stress plays a key pathogenic role in experimental and human heart failure. However, the source of ROS (reactive oxygen species) is a key determinant of the cardiac adaptation to pathological stressors. In the present study, we have shown that human dilated cardiomyopathy is associated with increased NOX2 (NADPH oxidase 2) levels, increased oxidative stress with adverse myocardial remodelling and activation of MAPKs (mitogen-activated protein kinases). Advanced heart failure in mice was also associated with increased NOX2 levels. Furthermore, we have utilized the pressure-overload model to examine the role of NOX2in advanced heart failure. Increased cardiomyocyte hypertrophy and myocardial fibrosis in response to pressure overload correlated with increased oxidative stress, and loss of NOX2 prevented the increase in oxidative stress, development of cardiomyocyte hypertrophy, myocardial fibrosis and increased myocardial MMP (matrix metalloproteinase) activity in response to pressure overload. Consistent with these findings, expression of disease markers revealed a marked suppression of atrial natriuretic factor, β-myosin heavy chain, B-type natriuretic peptide and α-skeletal actin expression in pressure-overloaded hearts from NOX2-deficient mice. Activation of MAPK signalling, a well-known mediator of pathological remodelling, was lowered in hearts from NOX2-deficient mice in response to pressure overload. Functional assessment using transthoracic echocardiography and invasive pressure-volume loop analysis showed a marked protection in diastolic and systolic dysfunction in pressure-overloaded hearts from NOX2-deficient mice. Loss of NOX2 prevented oxidative stress in heart disease and resulted in sustained protection from the progression to advanced heart failure. Our results support a key pathogenic role of NOX2in murine and human heart failure, and specific therapy antagonizing NOX2 activity may have therapeutic effects in advanced heart failure.

Deficiency of cardiac Acyl-CoA synthetase-1 induces diastolic dysfunction, but pathologic hypertrophy is reversed by rapamycin

In mice with temporally-induced cardiac-specific deficiency of acyl-CoA synthetase-1 (Acsl1H−/−), the heart is unable to oxidize long-chain fatty acids and relies primarily on glucose for energy. These metabolic changes result in the development of both a spontaneous cardiac hypertrophy and increased phosphorylated S6 kinase (S6K), a substrate of the mechanistic target of rapamycin, mTOR. Doppler echocardiography revealed evidence of significant diastolic dysfunction, indicated by a reduced E/A ratio and increased mean performance index, although the deceleration time and the expression of sarco/endoplasmic reticulum calcium ATPase and phospholamban showed no difference between genotypes. To determine the role of mTOR in the development of cardiac hypertrophy, we treated Acsl1H−/− mice with rapamycin. Six to eight week old Acsl1H−/− mice and their littermate controls were given i.p. tamoxifen to eliminate cardiac Acsl1, then concomitantly treated for 10weeks with i.p. rapamycin or vehicle alone. Rapamycin completely blocked the enhanced ventricular S6K phosphorylation and cardiac hypertrophy and attenuated the expression of hypertrophy-associated fetal genes, including α-skeletal actin and B-type natriuretic peptide. mTOR activation of the related Acsl3 gene, usually associated with pathologic hypertrophy, was also attenuated in the Acsl1H−/− hearts, indicating that alternative pathways of fatty acid activation did not compensate for the loss of Acsl1. Compared to controls, Acsl1H−/− hearts exhibited an 8-fold higher uptake of 2-deoxy[1-14C]glucose and a 35% lower uptake of the fatty acid analog 2-bromo[1-14C]palmitate. These data indicate that Acsl1-deficiency causes diastolic dysfunction and that mTOR activation is linked to the development of cardiac hypertrophy in Acsl1H−/− mice.

Consuming a Western diet for two weeks suppresses fetal genes in mouse hearts

Diets high in sugar and saturated fat (Western diet) contribute to obesity and pathophysiology of metabolic syndrome. A common physiological response to obesity is hypertension, which induces cardiac remodeling and hypertrophy. Hypertrophy is regulated at the level of chromatin by repressor element 1-silencing transcription factor (REST), and pathological hypertrophy is associated with reexpression of a fetal cardiac gene program. Reactivation of fetal genes is commonly observed in hypertension-induced hypertrophy; however, this response is blunted in diabetic hearts, partially due to upregulation of the posttranslational modification O-linked-β-N-acetylglucosamine (O-GlcNAc) to proteins by O-GlcNAc transferase (OGT). OGT and O-GlcNAc are found in chromatin-modifying complexes, but it is unknown whether they play a role in Western diet-induced hypertrophic remodeling. Therefore, we investigated the interactions between O-GlcNAc, OGT, and the fetal gene-regulating transcription factor complex REST/mammalian switch-independent 3A/histone deacetylase (HDAC). Five-week-old male C57BL/6 mice were fed a Western (n = 12) or control diet (n = 12) for 2 wk to examine the early hypertrophic response. Western diet-fed mice exhibited fasting hyperglycemia and increased body weight (P < 0.05). As expected for this short duration of feeding, cardiac hypertrophy was not yet evident. We found that REST is O-GlcNAcylated and physically interacts with OGT in mouse hearts. Western blot analysis showed that HDAC protein levels were not different between groups; however, relative to controls, Western diet hearts showed increased REST and decreased ANP and skeletal α-actin. Transcript levels of HDAC2 and cardiac α-actin were decreased in Western diet hearts. These data suggest that REST coordinates regulation of diet-induced hypertrophy at the level of chromatin.

Cardiac FGF23 expression correlates with left ventricular hypertrophy in patients with chronic kidney disease

Left ventricular hypertrophy (LVH) is the most common cardiac abnormality in children with CKD. Experimental and clinical studies demonstrated an association between elevated serum levels of fibroblast growth factor 23 (FGF23) and LVH in CKD. The aim of our study was to investigate i) the endogenous expression of FGF23 in heart tissue of paediatric CKD patients, ii) to establish the relationship between cardiac FGF23 expression and typical molecular mechanisms for LVH, and iii) to evaluate whether cardiac FGF23 expression is associated with LVH and other clinical parameters. We conducted a retrospective case-control-study in 25 deceased paediatric patients (age 11±8y) with CKD stage 5 and 25 age and sex-matched healthy controls. Myocardial autopsy samples of the left ventricle (LV) were evaluated by immunohistochemistry and qPCR with respect to endogenous FGF23 expression, calcineurin-NFAT signalling, genes regulating cardiac remodelling, and brain natriuretic peptide (BNP) as a marker of LVH. Cardiac FGF23 expression increased in CKD patients compared to controls (p<0.01), and correlated with LVH demonstrated by enhanced BNP expression as well as increased cardiomyocyte cross-sectional area (each p<0.01). The calcineurin expression significantly enhanced in cardiac tissue of CKD patients presenting with LVH (p<0.05), and cardiac remodelling was confirmed by the enhanced expression of skeletal α-actin and the significant reduction of cardiac α-actin compared to controls (p<0.05). In the patient cohort, cardiac FGF23 expression negatively correlated with the eGFR (r=-0.526), and positively correlated with time-averaged serum phosphate levels (r=0.503, each p<0.05). Likewise, cardiac FGF23 expression was not increased in transplanted patients compared to controls. For the first time, endogenous FGF23 expression was detected in myocardial tissue. Cardiac FGF23 expression as well as the FGF23 signalling pathway mediating cardiac hypertrophy and hypertrophic gene programs were induced in LVH positive CKD patients. We conclude that, in extension to circulating FGF23 levels, both, endogenous and circulating FGF23 contribute to LVH in CKD.

2013 Late-Breaking Basic Science Abstracts

### 21998 #### Enhanced Myocardial Repair with CardioChimeras Pearl J Quijada, Jonathan D Cubillo, Claudio Staub, Mark A Sussman; San Diego State Univ, San Diego, CA Dual stem cell transplantation of c-kit positive cardiac progenitor cells (CPCs) and mesenchymal stem cells (MSCs) after infarction support modest improvements in cardiac function, however there are currently no reports substantiating a single stem cell type supporting both direct and indirect mechanisms of myocardial repair. Therefore we created a CardioChimera, a stem/progenitor cell formed by fusion between CPCs and MSCs, resulting in a unique progeny superior to either individual precursor. CardioChimeras were purified after cell fusion with Hemagglutinating virus of Japan and expanded clonally based on dual expression of fluorescent proteins mCherry and eGFP from CPCs and MSCs respectively. CardioChimeras are mono-nucleated, have comparable growth kinetics to parental CPCs and MSCs and display increased cellular size unrelated to cell cycle arrest and/or senescence. CardioChimeras have increased expression of cardiomyogenic lineage markers cardiac troponin T (2.5-fold), smooth muscle 22 (9.3-fold), and CD31 (10.7-fold) concomitantly associated with decreased c-kit protein expression (50%) relative to parent CPCs. Lineage commitment of CardioChimeras is bolstered by dexamethasone treatment measured by mRNA levels of cardiogenic genes and increases in active mitochondria (2.2-fold) after labeling with MitoTracker. Induction of apoptosis is blunted in cardiomyocytes co-cultured with CardioChimeras compared to co-culturing with CPCs and MSCs alone, or combination of non-fused parent cells. CardioChimeras enhance cardiomyocyte growth similar to parent MSCs owing to an increased propensity to secrete pro-growth factors. Collectively, CardioChimeras represent an adaptable cell therapy combining the beneficial properties of CPCs to undergo cardiac specific commitment as well as MSCs that foster an improved microenvironment with protective paracrine secretion. Clinically, CardioChimeras merge the application of distinct cell types into a single entity for increased engraftment, mitigation of inflammation and blunting the progression of heart failure by promoting myocardial regeneration. …

Abstract 97: Histone Deacetylase Inhibition Prevents Cardiac Remodeling in Mice Carrying the Disruption of Guanylyl Cyclase/natriuretic Peptide Receptor-A Gene

Regulation of gene expression plays an obligatorily role in the modification of chromatin structure that dynamically attenuates cardiac hypertrophy. In order to culminate the role of epigenetic regulators in the heart tissue, the current study was undertaken to elucidate the effect of histone deacetylase (HDAC) inhibitor, sodium butyrate (SB) in cardiac remodeling process of Npr1 (coding for GC-A/NPRA) gene-targeted mice. Wild type ( Npr1 +/+ , 2-copy), gene-disrupted ( Npr1 +/- , 1-copy), and gene-duplicated ( Npr1 ++/+- , 3-copy) mice were administered intraperitoneally with SB (0.5 mg/kg/day) for 2 weeks (8 mice/group). Mice with gene-disruption ( Npr1 +/- , 1-copy) exhibited the increase in cardiac hypertrophy, heart weight/body weight (HW/BW) ratio (6.9 ± 0.2), and systolic blood pressure (SBP, 121.5 ± 4 mmHg) compared with 2-copy (HW/BW, 5.1 ± 0.2; SBP; 100.9 ± 6 mmHg) and 3-copy (HW/BW, 4.7 ± 0.1; SBP, 89.4 ± 2 mmHg) mice. In addition, an increased activity of HDAC (3-fold, p&lt;0.01) and decreased activity of histone acetyltransferases (HAT) (2.5-fold, p&lt;0.01) were found in untreated 1-copy mice hearts. Whereas, 1-copy mice treated with SB showed reduced HW/BW ratio (5.7 ± 0.3), SBP (SB, 101.2 ± 2), HDAC activity (p&lt;0.01) and improved HAT activity (3-fold, p&lt;0.001). Also, a stimulatory effect on HAT activity was observed in SB treated 2-copy (30%, p&lt;0.01) and 3-copy (50%, p&lt;0.01) mice. Furthermore, Npr1 +/- mice showed a significant increase in the expression of hypertrophic marker genes such as β-myosin heavy chain (β-MHC, 2-fold), α-skeletal actin (α-SK, 2-fold), c-fos (2.5-fold), and c-jun (3-fold) compared to untreated 2-copy and 3-copy mice. A substantial attenuation in the expression of hypertrophic markers (β-MHC, 2.5-fold; α-SK, 2.4-fold) and matrix genes (MMP-2, p&lt;0.01; MMP-9, p&lt;0.01) was found in SB-treated Npr1 +/- mice. The basal expression levels of matrix proteins were also significantly reduced in 2-copy and 3-copy mice hearts. The results show that sodium butyrate-dependent inhibition of HDAC activity attenuates cardiac hypertrophy and fibrosis by improving HAT activity suggesting that chromatin modification can prevent cardiac remodeling process in a Npr1 gene-dose-dependent manner.

Abstract 204: Sodium Sensing Network in Hypertension-Induced Cardiac Hypertrophy

Hypertension is related to alterations of sodium homeostasis that promote abnormal accumulation of water in the intravascular compartment leading to blood pressure elevation. This process can been associated with increased heart mass resulting in cardiac hypertrophy. At cellular level, the concentration and active transport of Na+ is regulated by Na+,K+-ATPase enzyme (NK). In vitro studies have shown a correlation between high blood pressure, NK and SIK activities. Increases in intracellular Na+ are paralleled by elevations in intracellular Ca2+ via NCX1, leading to the activation of SIK1 by a Ca2+/calmodulin-dependent kinase. Activation of SIK1 results in the dephosphorylation of the NK α-subunit and an increase in its catalytic activity. Accordingly, the aim of the present study was to evaluate the cardiac Na+ sensing network changes associated with hypertension in aging hypertensive rats. Systolic and diastolic blood pressures, determined by the tail-cuff method, were significantly higher in 3, 13 and 21-month old SHR than in age-matched WKY. In the SHR, ageing was associated with increases in cardiac mass, gene expression of hypertrophic (α-skeletal muscle actin and β-myosin heavy chain) markers as well as inflammatory cytokine (IL-6). The decreased expression of heart SIK isoforms, SIK1 and SIK3, was associated with downregulation of transcription factors Snail2, Zeb1, MFAT5c and KLF4, in 13 and 21-month old SHR. Cardiac α2-isoform of NK was found to be decreased in 21 month old SHR. Whereas, NKα1, NHE3 and NCX1 showed no differences between age-matched SHR and WKY. Na+-dependent ASCT2 and Na+-independent LAT1 amino acid transporter transcript levels of were significantly higher in 21 month old SHR than in WKY. In the aged SHR, cardiac hypertrophy was associated with dysregulation of several elements of the intracellular Na+ sensing network. Downregulation of SIK1 and transcription factors is correlated with expression of NKα2, suggesting impaired sodium handling. It is proposed that, cardiac hypertrophy is not exclusively the consequence of mechanical stress but also of other factors associated with elevated blood pressure such as abnormal cell sodium homeostasis.Supported by grant PIC/IC/83204/2007.

Development of a new model of cardiac hypertrophy with pro-arrhythmic features to study the role of hypertrophy in arrhythmogenesis

Purpose: Different factors may trigger the occurrence of arrhythmias in diseased hearts including fibrosis, cardiomyocyte hypertrophy and inflammation. This makes it difficult to establish the relative contribution of each of them to the occurrence of arrhythmias. In this study, we developed an in vitro model of pathological cardiac hypertrophy to study its role in the induction of arrhythmias independent of fibrosis and inflammation. Phorbol 12-myristate 13-acetate (PMA) is a diacylglycerol (DAG)-like activator of protein kinase C (PKC) that not only promotes myocyte hypertrophy but also decrease sarcoplasmic reticulum calcium ATPase (SERCA2) gene expression and impairs the calcium transient. Since pressure overload is associated with DAG production and PKC activation in cardiomyocytes, PMA-induced hypertrophy may be a good model to study hypertension-induced myocardial hypertrophy. Methods: Neonatal rat ventricular cardiomyocytes (nrCMCs) were isolated from 2-day-old animals and cultured as confluent monolayers. To inhibit proliferation of cardiac fibroblasts, cultures were treated with mitomycin-C, 1 day after cell isolation. Pathological hypertrophy was induced by 24-hour treatments with 500 nM PMA at day 4 and day 8 after culture initiation. The pathological nature of the hypertrophic response was confirmed by analysis of cell surface area, protein content and natriuretic peptide type A (NPPA), α-skeletal muscle actin (ASMA) and Serca2a protein levels. Electrophysiological properties of the nrCMCs were analyzed by optical mapping at day 9. Results: The percentage of spontaneous arrhythmias was much higher in the PMA-treated cultures than in the control cultures (46.8 vs 3.2% [p<0.01; n=32]). Local 1-Hz stimulation of the nrCMC cultures resulted in 25% arrhythmias (i.e. triggered activity) in the PMA-treated group (n=12) while the control cells showed no arrhythmias (n=12). Following 1-Hz pacing, PMA-treated nrCMCs showed APD prolongation (494±38.5 vs 242±65.5 ms in control cells [p<0.001; n=32]). Conclusion: A new model of hypertrophy-associated arrhythmias has been developed, which is currently being used to identify and validate specific targets for gene therapeutic interventions with viral vectors.

Abstract 182: CTRP9 - A Novel Cardiac Derived Secreted Factor With Anti-hypertrophic Properties

Background: CTRP9 (also called C1qtnf9) is a newly discovered secreted protein and a paralog of adiponectin. The biological functions of CTRP9, however, are still largely unknown. Results: Although previous data from a semi-quantitative real-time PCR had suggested that CTRP9 is mainly secreted by adipose tissue, we found its mRNA to be predominantly expressed in the heart by quantitative real-time PCR. Interestingly, we identified CTRP9 mRNA as significantly upregulated in hypertrophied mouse hearts (after 2 weeks of aortic constriction, TAC) as well as in hypertrophied human hearts (24±4-fold versus healthy human myocardium; p&lt;0.01). LacZ staining in myocardial sections of C1qtnf9 tm1(KOMP)Vlcg mice (knock-out for CTRP9, containing a lacZ cassette to replace exon 1-3 of the gene) revealed exclusive expression of CTRP9 in capillary and venous endothelial cells. Adenoviral overexpression of CTRP9 or recombinant CTRP9 strongly inhibited cardiomyocyte hypertrophy (assessed as cell size, protein/DNA-ratio, expression of skeletal α-actin) after stimulation with phenylephrine (PE). Accordingly, myocardial overexpression of CTRP9 via a cardioselective adeno-associated virus (AAV9-CTRP9) in mice dramatically reduced cardiac hypertrophy after two weeks of pressure overload (heart weight/body weight ratio, HW/BW in mg/g: AAV9-control 6.5±0.2 versus AAV9-CTRP9 5.6±0.2; p&lt;0.01). In turn, downregulation of CTRP9 by a specific siRNA aggravated cardiomyocyte growth in response to PE in vitro and CTRP9 knock-out (KO) mice exerted an enhanced hypertrophic response after two weeks of TAC in vivo (% increase in HW/BW versus sham: wild-type 77±13, KO 106±9; p&lt;0.05). Mechanistically, we found that CTRP9 binds to the adiponectin receptor 1 (AdipoR1) and inhibits prohypertrophic mTOR signalling in cardiac myocytes. SiRNA mediated downregulation of AdipoR1 or mTOR in neonatal rat cardiomyocytes abolished the anti-hypertrophic effect of CTRP9. Conclusion: Endothelial cell derived CTRP9 inhibits cardiac hypertrophy through binding to AdipoR1 and inhibition of the mTOR pathway in cardiomyocytes. Therefore, myocardial application of CTRP9 could be a novel strategy to combat pathological cardiac hypertrophy.

Distinct Functional Interactions between Actin Isoforms and Nonsarcomeric Myosins

Despite their near sequence identity, actin isoforms cannot completely replace each other in vivo and show marked differences in their tissue-specific and subcellular localization. Little is known about isoform-specific differences in their interactions with myosin motors and other actin-binding proteins. Mammalian cytoplasmic β- and γ-actin interact with nonsarcomeric conventional myosins such as the members of the nonmuscle myosin-2 family and myosin-7A. These interactions support a wide range of cellular processes including cytokinesis, maintenance of cell polarity, cell adhesion, migration, and mechano-electrical transduction. To elucidate differences in the ability of isoactins to bind and stimulate the enzymatic activity of individual myosin isoforms, we characterized the interactions of human skeletal muscle α-actin, cytoplasmic β-actin, and cytoplasmic γ-actin with human myosin-7A and nonmuscle myosins-2A, -2B and -2C1. In the case of nonmuscle myosins-2A and -2B, the interaction with either cytoplasmic actin isoform results in 4-fold greater stimulation of myosin ATPase activity than was observed in the presence of α-skeletal muscle actin. Nonmuscle myosin-2C1 is most potently activated by β-actin and myosin-7A by γ-actin. Our results indicate that β- and γ-actin isoforms contribute to the modulation of nonmuscle myosin-2 and myosin-7A activity and thereby to the spatial and temporal regulation of cytoskeletal dynamics. FRET-based analyses show efficient copolymerization abilities for the actin isoforms in vitro. Experiments with hybrid actin filaments show that the extent of actomyosin coupling efficiency can be regulated by the isoform composition of actin filaments.

Skeletal and cardiac α-actin isoforms differently modulate myosin cross-bridge formation and myofibre force production

Multiple congenital myopathies, including nemaline myopathy, can arise due to mutations in the ACTA1 gene encoding skeletal muscle α-actin. The main characteristics of ACTA1 null mutations (absence of skeletal muscle α-actin) are generalized skeletal muscle weakness and premature death. A mouse model (ACTC(Co)/KO) mimicking these conditions has successfully been rescued by transgenic over-expression of cardiac α-actin in skeletal muscles using the ACTC gene. Nevertheless, myofibres from ACTC(Co)/KO animals generate less force than normal myofibres (-20 to 25%). To understand the underlying mechanisms, here we have undertaken a detailed functional study of myofibres from ACTC(Co)/KO rodents. Mechanical and X-ray diffraction pattern analyses of single membrane-permeabilized myofibres showed, upon maximal Ca(2+) activation and under rigor conditions, lower stiffness and disrupted actin-layer line reflections in ACTC(Co)/KO when compared with age-matched wild-types. These results demonstrate that in ACTC(Co)/KO myofibres, the presence of cardiac α-actin instead of skeletal muscle α-actin alters actin conformational changes upon activation. This later finely modulates the strain of individual actomyosin interactions and overall lowers myofibre force production. Taken together, the present findings provide novel primordial information about actin isoforms, their functional differences and have to be considered when designing gene therapies for ACTA1-based congenital myopathies.