Cardiomyocyte Surface Area Research Articles

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.

Asiatic acid inhibits cardiac hypertrophy by blocking interleukin-1β-activated nuclear factor-κB signaling in vitro and in vivo.

Activated interleukin (IL)-1β signaling pathway is closely associated with pathological cardiac hypertrophy. This study investigated whether asiatic acid (AA) could inhibit IL-1β-related hypertrophic signaling, and thus suppressing the development of cardiac hypertrophy. Transverse aortic constriction (TAC) induced cardiac hypertrophy in C57BL/6 mice and cultured neonatal cardiac myocytes stimulated with IL-1β were used to evaluate the role of AA in cardiac hypertrophy. The expression of atrial natriuretic peptide (ANP) was evaluated by quantitative polymerase chain reaction (qPCR) and the nuclear factor (NF)-κB binding activity was measured by electrophoretic mobility shift assays (EMSA). AA pretreatment significantly attenuated the IL-1β-induced hypertrophic response of cardiomyocytes as reflected by reduction in the cardiomyocyte surface area and the inhibition of ANP mRNA expression. The protective effect of AA on IL-1β-stimulated cardiomyocytes was associated with the reduction of NF-κB binding activity. In addition, AA prevented TAC-induced cardiac hypertrophy in vivo. It was found that AA markedly reduced the excessive expression of IL-1β and ANP, and inhibited the activation of NF-κB in the hypertrophic myocardium. Our data suggest that AA may be a novel therapeutic agent for cardiac hypertrophy. The inhibition of IL-1β-activated NF-κB signaling may be the mechanism through which AA prevents cardiac hypertrophy.

Abstract P215: Angiotensin II-Mediated Cardiac Remodeling in cChAT Mice

It has been demonstrated that the cardiac non-neuronal cholinergic system (NNCS) plays a role in regulating cardiac homeostasis under physiological conditions. To examine a possible role played by NNCS upon cardiac remodeling, we submitted mice with genetic deletion of cardiomyocyte-specific choline acetyltransferase (cChAT) to chronic subcutaneous infusion of angiotensin II (Ang II). Male mice aged six months were assigned into four experimental groups: wild-type (WT) + Saline; WT + Ang II; cChAT + Saline and cChAT + Ang II. Ang II did not change the heart weight in WT mice (10.4±0.3 vs. 8.5±0.2 mg/mm in WT+Saline) but determined an increase in cChAT mice (12.8±1 vs. 8.8±0.6 mg/mm in cChAT+Saline). In WT mice, Ang II decreased left ventricular (LV) fractional shortening (23±2 vs. 35±2 % in WT+Saline) and LV ejection fraction (54±4 vs. 73±2 % in WT+Saline). However, cChAT+Ang II mice exhibited greater decrease in both LV fractional shortening (12±1.8 vs. 23±2 % in WT+Ang II) and ejection fraction (31±4 vs. 54±4 % in WT+Ang II). cChAT mice displayed cardiomyocyte hypertrophy (2002±116 vs. 1377±48 μm2 in WT+Saline) even when receiving saline. Ang II increased the cardiomyocyte surface area in both WT and cChAT mice; however, the cChAT mice exhibited greater myocyte hypertrophy (3035±181 vs. 2603±122 μm2 in WT+Ang II). Hematoxylin and eosin staining revealed that Ang II promoted greater disruption of myocardial structure in cChAT mice. Additionally, Trichrome C staining revealed that WT+Ang II mice presented increased collagen deposition (2.86±0.2 vs. 0.45±0.07 % in WT+Saline); nevertheless, the fibrotic response in cChAT+Ang II mice was greater than that observed in WT animals (6.16±1.1 vs. 2.86±0.2 % in WT+Ang II). Therefore, mice with deficiency for ChAT displayed exacerbated ventricular dysfunction induced by chronic Ang II administration, providing support for a role of the NNCS in the progression of cardiac remodeling.

NF‐қB Activation is Required for Thyroid Hormone Induced Cardiomyocyte Hypertrophy

The nuclear factor NF‐қB promotes inflammatory response and mediates cardiac growth by different hypertrophic stimuli. We have previous results obtained by microarray experiments indicating that thyroid hormone (triiodotyronine, T3) up‐regulates pro‐inflammatory genes. Considering that, we investigated the influence of T3 on NF‐қB signaling and the possible participation of this nuclear factor in cardiomyocyte hypertrophy induced by T3. Primary cultures of neonatal rat ventricular cardiomyocytes were treated with T3 for 10, 30 minutes and 24 hours and the NF‐қB signaling was analyzed by western blot. Also, cardiomyocytes were treated with an inhibitor of NF‐қB (JSH‐23) followed by treatment with T3 for evaluation of mRNA expression of atrial natriuretic factor (ANF) and cell surface area analysis. Data were presented as mean ± SD and were analyzed using one‐way ANOVA and Tukey multiple comparisons test. Values of p<0.05 were considered statistically significant. T3 rapidly increased the nuclear protein expression of NF‐қB without alterations on IKK and IқB expression. The inhibition of NF‐қB reduced both ANF mRNA levels and the surface area of cardiomyocytes compared to T3 treated cells. These results confirm that NF‐қB is required for development of cardiomyocyte hypertrophy by T3. The possible interactions of NF‐қB with genes associated with cardiac growth are being investigated. Financial support: Fapesp (2013/22480‐4 and 2011/23352‐4).

Abstract 311: Early Losartan Treatment Prevents the Developmental Programming of Cardiac Dysfunction Caused by Neonatal High Oxygen Exposure in Rats

Neonatal oxidative stress is a major postnatal deleterious factor predisposing preterm born infants to classical complications of prematurity (retinopathy, bronchopulmonary dysplasia) which are characterized by impaired vascular development. Our group has previously shown that rats transiently exposed to high O2 as newborns (mimicking human preterms oxidative stress conditions) develop high blood pressure (BP), cardiac remodeling and dysfunction later in life, in part mediated by the renin angiotensin system (RAS). Cardiac RAS activation is characterized by AT1/AT2 receptors imbalance, with increased AT1R at adult age. In order to study the role of RAS at early stages of the developmental programming of cardiac dysfunction caused by high O2 exposure, we assessed whether an early and short-term treatment with AT1R blocker Losartan, prevents cardiac alterations at young male 4 wks-old rats (prior to the elevation of BP in this model). Sprague-Dawley newborns rats were kept with their mother in 80% O2 (O2 group, n=9) or room air (Ctrl, n=9) from days 3-10 of life (P3-P10). Losartan (LOS, n=10, 20 mg/Kg) or water was administered by gavage in O2 rats from P8-P10 (last 2 days of O2 to avoid impact on nephrogenesis). At 4 wks, echocardiography reveals that O2 rats have decreased fraction of shortening compared to Ctrl (FS: 37±2 vs 42±2 %), suggesting impaired systolic function in O2. Cardiac hypertrophy evaluated by heart/body weight and cardiomyocyte surface area (CSA) is also increased in O2 vs Ctrl (141±13 vs 118±4 μm2). LOS treatment prevented the impairment of systolic function in O2 by ameliorating FS (43±2 %) and reducing CSA (121±11 μm2). LOS treatment also modulated RAS genes expression (RT-PCR): LOS restored AT1/AT2 balance in O2 hearts by decreasing AT1b subunit (0.8±0.2 O2 vs 1.3±0.3 O2+LOS vs 0.9±0.2 Ctrl) as well as increasing ACE2 (1.5±0.4 O2 vs 0.8±0.1 O2+LOS vs 1.1±0.4 Ctrl) expressions. In conclusion, a short-term treatment with LOS during neonatal O2 exposure prevents the impairment of cardiac systolic function and hypertrophy at young age. This data reinforces the key role of RAS in the developmental programming of cardiac dysfunction and reveals LOS as an effective strategy to prevent early cardiac alterations caused by neonatal high O2 exposure.

Dapper-1 is essential for Wnt5a induced cardiomyocyte hypertrophy by regulating the Wnt/PCP pathway.

The Wnt signaling pathway was identified as crucial mediator of cardiomyocyte hypertrophy. In this study we found that activation of non-canonical Wnt signaling by Wnt5a stimulates protein synthesis and enlargement of cardiomyocyte surface area. These hypertrophic features were inhibited in Dapper-1 (Dpr1) depleted cells. On the molecular level, we observed inhibition of the non-canonical Wnt/planar-cell-polarity (PCP) pathway denoted by reduction of c-jun-n-terminal-kinase (JNK) phosphorylation. Upstream of JNK, increased protein levels of the Wnt/PCP trans-membrane receptor van-Gogh-like-2 (Vangl2) were observed along with an enrichment of Vangl2 in perinuclear located vesicles. The findings suggest that Dpr1 is essential for execution of the Wnt/PCP pathway and regulation of the Vangl2/JNK axis. Depletion of Dpr1 inhibits non-canonical Wnt signaling induced cardiomyocyte hypertrophy by blocking Wnt/PCP signaling.

Asiatic acid attenuates cardiac hypertrophy by blocking transforming growth factor-β1-mediated hypertrophic signaling in vitro and in vivo.

Cardiac hypertrophy is a major cause of morbidity and mortality worldwide. Transforming growth factor-β1 (TGF-β1) signaling has been considered as a trigger causally contributing to pathological cardiac hypertrophy. Asiatic acid (AA) is a triterpenoid compound extracted from Centella asiatica and exhibits a variety of pharmacological effects. In this study, we investigated the anti-hypertrophic effects and mechanisms of action of AA in a TGF-β1-stimulated hypertrophic response using cultured neonatal cardiomyocytes in vitro and in a mouse model of cardiac hypertrophy induced by pressure overload in vivo. Treatment with AA markedly attenuated the TGF-β1-induced hypertrophic responses of cardiomyocytes as reflected by reduction in the cardiomyocyte surface area and the inhibition of atrial natriuretic peptide (ANP) mRNA expression. The protective effects of AA on hypertrophic cardiomyocytes were associated with the blocking of p38 and extracellular signal‑regulated kinase (ERK)1/2 phosphorylation and the reduction of nuclear factor-κB (NF-κB) binding activity. In vivo experiments indicated that the administration of AA prevented cardiac hypertrophy and dysfunction induced by pressure overload. It was found that AA markedly reduced the excessive production of TGF-β1 in the hypertrophic myocardium, blocked the phosphorylation of p38 and ERK1/2 and inhibited the activation of NF-κB. Our data suggest that AA may be a novel therapeutic agent for cardiac hypertrophy. The inhibition of TGF-β1‑mediated hypertrophic signaling may be the mechanism through which AA prevents cardiac hypertrophy.

SIRT1 functions as an important regulator of estrogen-mediated cardiomyocyte protection in angiotensin II-induced heart hypertrophy.

Background. Sirtuin 1 (SIRT1) is a member of the sirtuin family, which could activate cell survival machinery and has been shown to be protective in regulation of heart function. Here, we determined the mechanism by which SIRT1 regulates Angiotensin II- (AngII-) induced cardiac hypertrophy and injury in vivo and in vitro. Methods. We analyzed SIRT1 expression in the hearts of control and AngII-induced mouse hypertrophy. Female C57BL/6 mice were ovariectomized and pretreated with 17β-estradiol to measure SIRT1 expression. Protein synthesis, cardiomyocyte surface area analysis, qRT-PCR, TUNEL staining, and Western blot were performed on AngII-induced mouse heart hypertrophy samples and cultured neonatal rat ventricular myocytes (NRVMs) to investigate the function of SIRT1. Results. SIRT1 expression was slightly upregulated in AngII-induced mouse heart hypertrophy in vivo and in vitro, accompanied by elevated cardiomyocyte apoptosis. SIRT1 overexpression relieves AngII-induced cardiomyocyte hypertrophy and apoptosis. 17β-Estradiol was able to protect cardiomyocytes from AngII-induced injury with a profound upregulation of SIRT1 and activation of AMPK. Moreover, estrogen receptor inhibitor ICI 182,780 and SIRT1 inhibitor niacinamide could block SIRT1's protective effect. Conclusions. These results indicate that SIRT1 functions as an important regulator of estrogen-mediated cardiomyocyte protection during AngII-induced heart hypertrophy and injury.

Transient Neonatal High Oxygen Exposure Leads to Early Adult Cardiac Dysfunction, Remodeling, and Activation of the Renin–Angiotensin System

Perinatal conditions (such as preterm birth) can affect adult health and disease, particularly the cardiovascular system. Transient neonatal high O(2) exposure in rat in adulthood (a model of preterm birth-related complications) leads to elevated blood pressure, vascular rigidity, and dysfunction with renin-angiotensin system activation. We postulate that neonatal hyperoxic stress also affects myocardial structure, function, and expression of renin-angiotensin system components. Sprague-Dawley pups were kept with their mother in 80% O(2) or in room air (control) from days 3 to 10 of life. Left ventricular function was assessed in 4-, 7-, 12-week-old (echocardiography) and in 16-week-old (intraventricular catheterization) male O(2)-exposed versus control rats. At 16 weeks, hearts from O(2)-exposed rats showed cardiomyocyte hypertrophy, enhanced fibrosis, and increased expression of transforming growth factor-β1, senescence-associated proteins p53 and Rb, upregulation of angiotensin II type 1 (AT1) receptor expression (protein and AT1a/b mRNA), and downregulation of AT2 receptors. At 4 weeks (before blood pressure increase), the expression of cardiomyocyte surface area, fibrosis, p53, and AT1b was significantly increased and AT2 decreased in O(2)-exposed animals. After 4 weeks of continuous angiotensin II infusion (starting at 12 weeks), O(2)-exposed rats developed severe heart failure, with impaired myocardial mechanical properties compared with saline-infused rats. Transient neonatal O(2) exposure in rats leads to left ventricular hypertrophy, fibrosis and dysfunction, and increased susceptibility to heart failure under pressure overload. These results are relevant to the growing population of individuals born preterm who may be at higher risk of cardiac dysfunction when faced with increased peripheral resistance associated with hypertension, vascular diseases, and aging.

Abstract 185: Transient Neonatal High Oxygen Exposure Leads to Cardiac Dysfunction and Renin Angiotensin System Activation in Rats

Preterm infants are exposed to high oxygen (O2) pressure (relative to intrauterine levels) leading to systemic oxidative stress and impaired vascular development; as children and adults, preterm born subjects have higher blood pressure. In rats, high O2 exposure induces vascular dysfunction, in part mediated by the renin angiotensin system (RAS). However, impact of neonatal high O2 exposure on heart development and whether RAS activation prevails in the heart is unknown. We aimed to assess early heart alterations and activation of RAS after neonatal high O2 exposure. METHODS AND RESULTS: Sprague-Dawley pups were kept with their mother in 80% O2 (O2, n=8) or room air (Ctrl, n=8) from days 3-10 of life. Hearts were extracted at day 3 (pre O2-exposure, P3), day 5 (during, P5), 10 (after, P10) and 4 wks to assess myocardium hypertrophy (HE), fibrosis (Masson’s trichrome) and RAS components gene expressions by RT-PCR. Echocardiography was performed at 4 weeks to assess heart function. RAS components mRNA expression in O2 vs Ctrls shows up-regulation of AT1a and ACE2 genes at P5 (AT1a: 152±28 vs 58±16/ACE2: 140±20 vs 77±6% of P3) and P10 (AT1a: 374±35 vs 250±30 /ACE2: 326±70 vs 208±18% of P3) relative to values at P3. With age, AT1b and AT2 expressions decrease in Ctrls, whereas are maintained in O2-exposed rats to values similar to P3 (Ctrl vs O2, P5: AT1b: 18±5 vs 71±12/AT2: 48±20 vs 120±16; P10: AT1b: 33±9 vs 105±26/AT2: 27±8 vs 77±7% of P3). At P10, cardiomyocyte surface area is increased in O2 vs Ctrls (4.9±0.2 vs 2.9±0.1 μm2). At 4 wks, O2 group show increased fibrosis (49±4 vs 29±2 % pixels), left ventricular (LV) cavity diameter (3.6±0.2 vs 3.0±0.1 mm) and systolic dysfunction by decreased fraction of shortening (39±2 vs 47±2 %). AT1b gene expression (2.6±1.7 vs 0.3±0.2) is increased and AT2 receptor (7.5±0.9 vs 6.0±0.6) is decreased in O2 group vs Ctrl. CONCLUSION: Neonatal O2 exposure activates RAS in hearts. AT1 receptor is up-regulated at all ages studied in O2-exposed rat hearts while AT2 is up-regulated at P5 and P10. At 4 wks, AT1/AT2 imbalance prevails in O2 group hearts along with LV dysfunction and enhanced fibrosis and hypertrophy. RAS activation by neonatal O2-exposure might significantly impact in heart development and programming of cardiac dysfunction in rats.

Abstract 190: A Mechanotransduction Study In Embryonic Zebrafish Heart

Background: While proper heart contraction requires an intact sarcomere structure, contraction reciprocally regulates sarcomere assembly and cardiomyocyte shape during heart morphogenesis and heart remodeling. However, how contraction modulates heart morphogenesis and the underlying molecular mechanism remains unclear. Here we address this question by manipulating cardiac contractility in a developing zebrafish embryo. Results: Cessation of heart contraction was achieved by treating zebrafish embryos with blebbistatin, a myosin II specific inhibitor, or Niferdipine, a Ca 2+ channel inhibitor. Loss of cardiac contractility reduces ventricular chamber size in 1 dpf embryo, but significantly increases ventricular chamber size in 2 dpf and 3 dpf embryos. In both circumstances, cessation of contraction consistently prevents or disrupts the lateral fusion of myofibril. Cessation of contraction-induced heart enlargement is reversible and also dependent on the time of drug treatment. This heart-remodeling event in 2dpf embryo, which manifests as increased cardiomyocyte surface area accompanied by reduced thickness, can be attenuated by reducing the transmural pressure towards the ventricle wall. This mechanotransductive process occurs in the myocardium without need of endocardium. At the molecular level, by screening several compounds, we found that PI3K signaling confers this contraction-regulated cardiomyocyte shape remodeling process without affecting the lateral fusion of sarcomere. Conclusion: Our data in an in vivo animal model distinguish two different and relatively independent functions of contraction in regulating lateral fusion of sarcomere and cardiomyocytes shape remodeling. The contraction-regulated heart shape remodeling is conveyed by PI3k signaling mediated mechanotransduction independent of endocardium.

Rheb (Ras Homologue Enriched in Brain)-dependent Mammalian Target of Rapamycin Complex 1 (mTORC1) Activation Becomes Indispensable for Cardiac Hypertrophic Growth after Early Postnatal Period

Cardiomyocytes proliferate during fetal life but lose their ability to proliferate soon after birth and further increases in cardiac mass are achieved through an increase in cell size or hypertrophy. Mammalian target of rapamycin complex 1 (mTORC1) is critical for cell growth and proliferation. Rheb (Ras homologue enriched in brain) is one of the most important upstream regulators of mTORC1. Here, we attempted to clarify the role of Rheb in the heart using cardiac-specific Rheb-deficient mice (Rheb(-/-)). Rheb(-/-) mice died from postnatal day 8 to 10. The heart-to-body weight ratio, an index of cardiomyocyte hypertrophy, in Rheb(-/-) was lower than that in the control (Rheb(+/+)) at postnatal day 8. The cell surface area of cardiomyocytes isolated from the mouse hearts increased from postnatal days 5 to 8 in Rheb(+/+) mice but not in Rheb(-/-) mice. Ultrastructural analysis indicated that sarcomere maturation was impaired in Rheb(-/-) hearts during the neonatal period. Rheb(-/-) hearts exhibited no difference in the phosphorylation level of S6 or 4E-BP1, downstream of mTORC1 at postnatal day 3 but showed attenuation at postnatal day 5 or 8 compared with the control. Polysome analysis revealed that the mRNA translation activity decreased in Rheb(-/-) hearts at postnatal day 8. Furthermore, ablation of eukaryotic initiation factor 4E-binding protein 1 in Rheb(-/-) mice improved mRNA translation, cardiac hypertrophic growth, sarcomere maturation, and survival. Thus, Rheb-dependent mTORC1 activation becomes essential for cardiomyocyte hypertrophic growth after early postnatal period.

Trypanosoma cruzi infection induces morphological reorganization of the myocardium parenchyma and stroma, and modifies the mechanical properties of atrial and ventricular cardiomyocytes in rats

This study investigates morphofunctional adaptations of the heart stroma and parenchyma in rats that are chronically infected with Trypanosoma cruzi. Four-month-old male Wistar rats were randomized into control (n=14) and infected (n=14) groups. Infected animals were inoculated with T. cruzi Y strain. After 9 weeks, the animals were euthanized, and the right atrium (RA) and left ventricle (LV) were removed for biochemical, stereological, and cardiomyocyte mechanical analyses. Infected animals presented cardiomyocyte atrophy and myocardial fibrosis. For these animals, the total volume, length, surface area, and cross-sectional area of cardiomyocytes were significantly reduced, and the total interstitial and collagen volumes were significantly increased in the RA and LV compared to the controls. The total volume and length of blood vessels were significantly increased in the LV, and the total blood vessel surface area was significantly higher in the RA of infected animals. RA and LV cardiomyocytes from infected animals exhibited a significant reduction in cell shortening (43.02% and 24.98%, respectively), prolongation of the time to the peak of contraction (17.09%) and the time to half relaxation (23.68%) compared to non-infected animals. Lipid hydroperoxides, but not mineral concentrations, were significantly increased in the RA and LV from infected animals, showing an inverse correlation with cell shortening. T. cruzi infection induces global structural remodeling of the RA and LV in rats. This remodeling coexists with cardiomyocyte contractility dysfunction, which is possibly related to the abnormal organization of the myocardial stroma and increased cellular lipid peroxidation.

MiR-30-Regulated Autophagy Mediates Angiotensin II-Induced Myocardial Hypertrophy

Dysregulated autophagy may lead to the development of disease. Role of autophagy and the diagnostic potential of microRNAs that regulate the autophagy in cardiac hypertrophy have not been evaluated. A rat model of cardiac hypertrophy was established using transverse abdominal aortic constriction (operation group). Cardiomyocyte autophagy was enhanced in rats from the operation group, compared with those in the sham operation group. Moreover, the operation group showed up-regulation of beclin-1 (an autophagy-related gene), and down-regulation of miR-30 in cardiac tissue. The effects of inhibition and over-expression of the beclin-1 gene on the expression of hypertrophy-related genes and on autophagy were assessed. Angiotensin II-induced myocardial hypertrophy was found to be mediated by over-expression of the beclin-1 gene. A dual luciferase reporter assay confirmed that beclin-1 was a target gene of miR-30a. miR-30a induced alterations in beclin-1 gene expression and autophagy in cardiomyocytes. Treatment of cardiomyocytes with miR-30a mimic attenuated the Angiotensin II-induced up-regulation of hypertrophy-related genes and decreased in the cardiomyocyte surface area. Conversely, treatment with miR-30a inhibitor enhanced the up-regulation of hypertrophy-related genes and increased the surface area of cardiomyocytes induced by Angiotensin II. In addition, circulating miR-30 was elevated in patients with left ventricular hypertrophy, and circulating miR-30 was positively associated with left ventricular wall thickness. Collectively, these above-mentioned results suggest that Angiotensin II induces down-regulation of miR-30 in cardiomyocytes, which in turn promotes myocardial hypertrophy through excessive autophagy. Circulating miR-30 may be an important marker for the diagnosis of left ventricular hypertrophy.

Effect of distinct sources of Ca2+ on cardiac hypertrophy in cardiomyocytes

It is believed that intracellular calcium (Ca(2+)) overload can cause the cardiac hypertrophy, but it is possible that the Ca(2+) entering the cytoplasm through distinct pathways will induce various effects on cardiomyocytes. The aim of the present study is to explore the effect of different sources of Ca(2+) on cardiomyocyte hypertrophy. The cardiomyocytes isolated from neonatal Sprague-Dawley rats were treated with three agents (ionomycin, caffeine and angiotensin II [Ang II]) that increased the intracellular Ca(2+) concentration via different pathways. Treatments with ionomycin, caffeine and Ang II for 24 h caused a significant increase in resting [Ca(2+)](i) by 108.0 ± 7.8%, 102.0 ± 6.9% and 59.8 ± 3.3%, respectively. Caffeine and Ang II increased the cell surface area of cardiomyocytes and the mRNA level of atrial natriuretic peptide, brain natriuretic peptide and β-myosin heavy chain, but ionomycin did not. Moreover, sarcoplasmic reticulum Ca(2+)-ATPase (SERCA2a) activity and the amplitudes of the twitch [Ca(2+)](i) transients were reduced in the caffeine-treated group and Ang II-treated group. Furthermore, cardiomyocyte hypertrophy induced by caffeine was inhibited by cyclosporin A (CsA) and KN93, whereas cardiomyocyte hypertrophy induced by Ang II was inhibited by KN93, but not CsA. Our results show that cardiomyocyte hypertrophy is associated with SERCA2a activity, contractile performance and signaling pathways of CaMKII and/or calcineurin, whereas the Ca(2+) overload is not sufficient to cause the cardiomyocyte hypertrophy.

Abstract P208: Depletion of Dapper-1 Acts Antihypertrophic on Cardiomyocytes and Inhibits Wnt3a- and Wnt5a-Mediated Activation of the Wnt Signaling Pathway

Background: Cardiac development is regulated by the evolutionary conserved Wnt signaling pathway. While Wnt activity is silenced in the adult heart under normal conditions this pathway is activated during myocardial remodeling following pathological injury such as myocardial infarction. Among other members of this pathway Dapper-1 was suggested to antagonize Wnt activation but its role in Wnt signaling is poorly understood. In a murine myocardial infarction model as well as in a cardiomyocyte hypoxia model we found a robust over-expression of Dapper-1. To elucidate its function in cardiomyocytes we performed Dapper-1 specific siRNA mediated knockdown experiments. Results: Depletion of Dapper-1 leads to reduction of cardiomyocyte surface area (-10% ± 4%, p<0.001 vs. control), attenuation of global protein content (-20% ± 3%, p<0.02 vs. control) and diminishes global protein synthesis (-27% ± 10%, p<0.02 vs. control). Augmentation of protein synthesis by β-adrenic Isoproterenol stimulation is widely inhibited. Wnt3a induced stimulation of protein synthesis and enlargement of cardiomyocyte surface area is blocked in Dapper-1 knockdown cardiomyocytes. Furthermore, we observed reduced total- and active-β-catenin protein levels. Moreover, Dapper-1 knockdown inhibits β-catenin from translocation to the nucleus following Wnt3a stimulation. TCF/LEF reporter activity was reduced in Dapper-1-diminished -and elevated in Dapper-1-overexpressed cardiomyocytes after Wnt3a treatment. Robust Ser9 -and weak Tyr216 phosphorylation of GSK3β suggested a GSK3β independent inhibition of canonical Wnt signaling. In addition, activation of non-canonical Wnt/JNK pathway by Wnt5a conditioned medium was completely inhibited in Dapper-1 knockdown cells. Summary: Dapper1 is an essential modulator for global protein synthesis. Furthermore, Dapper-1 is required to activate canonical and noncanonical Wnt signaling pathways in cardiomyocytes.

Left ventricular remodeling with exercise in hypertension

We investigated how exercise training superimposed on chronic hypertension impacted left ventricular remodeling. Cardiomyocyte hypertrophy, apoptosis, and proliferation in hearts from female spontaneously hypertensive rats (SHRs) were examined. Four-month-old SHR animals were placed into a sedentary group (SHR-SED; n = 18) or a treadmill running group (SHR-TRD, 20 m/min, 1 h/day, 5 days/wk, 12 wk; n = 18). Age-matched, sedentary Wistar Kyoto (WKY) rats were controls (n = 18). Heart weight was greater in SHR-TRD vs. both WKY (P < 0.01) and SHR-SED (P < 0.05). Morphometric-derived left ventricular anterior, posterior, and septal wall thickness were increased in SHR-SED relative to WKY and augmented in SHR-TRD. Cardiomyocyte surface area, length, and width were increased in SHR-SED relative to WKY and further increased in SHR-TRD. Calcineurin abundance was increased in SHR-SED vs. WKY (P < 0.001) and attenuated in SHR-TRD relative to SHR-SED (P < 0.05). Protein abundance and mRNA of Akt was not different among groups. The rate of apoptosis was increased in SHR-SED relative to WKY and mitigated in SHR-TRD. The abundance of Ki-67(+) cells across groups was not statistically different across groups. The abundance of cardiac progenitor cells (c-Kit(+) cells) was increased in SHR-TRD relative to WKY. These data suggest that exercise training superimposed on hypertension augmented cardiomyocyte hypertrophy, despite attenuating calcineurin abundance. Exercise training also mitigated apoptosis in hypertension and showed a tendency to enhance the abundance of cardiac progenitor cells, resulting in a more favorable cardiomyocyte number in the exercise-trained hypertensive heart.

Angiotensin II Induces Cardiomyocyte Hypertrophy Probably through Histone Deacetylases

Angiotensin II (Ang II) plays a pathophysiological role in the genesis of cardiac hypertrophy as a hypertrophic stimulus. But little is known about the terminal steps, in which Ang II reprograms cardiac gene expression. Histone deacetyltransferases (HDACs) are considered as the integrators of divergent stress-response pathways during heart remodeling. However, the exact role of HDACs in the hypertrophic process is not clear yet. Therefore, we studied the expression of HDAC2, one of Class I HDACs, and the effect of valproic acid (VPA), a nonspecific HDAC inhibitor, in the Ang II-induced cardiomyocyte hypertrophy. Primary cultures of neonatal rat cardiomyocytes were prepared from 1-day-old Wistar rats and treated with Ang II. The mRNA levels of HDAC2 and beta-myosin heavy chain (beta-MHC), a hypertrophic marker gene, were determined by reverse transcription-polymerase chain reaction (RT-PCR). The protein expression of HDAC2 and c-fos, an immediate early response gene, was evaluated by immunohistochemistry, and the surface areas of cardiomyocytes were measured using Motic Images software. The expression levels of HDAC2 mRNA and protein were increased in a time-dependent manner during the hypertrophic process, accompanied with the increment of beta-MHC and c-fos proteins. Ang II also increased the surface area of cardiomyocytes by more than twofold. VPA significantly reversed these changes. These results suggest that Ang II may induce cardiomyocyte hypertrophy through HDACs in combination with c-fos and that VPA has the protective effect on cardiomyocyte hypertrophy. Thus, HDAC inhibition is a feasible therapeutic strategy that holds promise in the treatment of cardiac hypertrophy.

Effects of dl-praeruptorin a on cultured neonatal rat ventricular cardiomyocytes with hypertrophy induced by endothelin-1

The present study investigated whether dl-praeruptorin (Pd-Ia) prevents endothelin-1 (ET-1)-induced cardiomyocyte hypertrophy and the potential pathways that underlie such an effect. We assessed cardiomyocyte surface area, protein synthesis, the expression of Bax/Bcl2 and Jun genes, the expression of atrial natriuretic factor (ANF) and Ca2+/calmodulin-dependent kinase II (CaMK-II) activity in cultured neonatal rat ventricular cardiomyocytes with ET-1-induced hypertrophy. It was found that Pd-Ia decreased the surface area and protein synthesis rate in cardiomyocytes exposed to ET-1. Additionally, the expression of Bcl2 and Bax was increased in both the ET-1-exposed and Pd-Ia+ET- 1-treated groups compared with the control group, although this was not significant. In cardiomyocytes incubated with ET-1, the expression of ANF (Nppa) significantly increased relative to the control and Pd-Ia groups. The expression of Jun significantly increased in cardiomyocytes incubated with ET-1, but not in the Pd-Ia group, where Jun levels were similar to those found for the control group. Moreover, it was found that Pd-Ia inhibited the ET-1-induced increase in intracellular Ca(2+) concentration. The results showed that Pd-Ia could conceivably be an effective therapeutic drug for treating the contractile defects associated with cardiac hypertrophy and failure. This activity may be associated with its Ca2+-antagonist effect and modulation of the expression of immediate-early genes that play important roles in the mitogen-activated protein (MAP) kinase pathway.

246 INVITED Targeting Her: Can resistance to EGFR inhibitors be overcome?

The Forkhead box-containing protein, O subfamily 3 (FoxO3) transcription factor negatively regulates myocardial hypertrophy, and its transcriptional activity is finely conditioned by diverse posttranslational modifications, such as phosphorylation, acetylation, ubiquitination, methylation and glycosylation. Here, we introduce a novel modification of the FoxO3 protein in cardiomyocytes: poly(ADP-ribosyl)ation (PARylation) mediated by poly(ADP-ribose) polymerase-1 (PARP1). This process catalyzes the NAD+-dependent synthesis of polymers of ADP-ribose (PAR) and their subsequent attachment to target proteins by PARPs. Primary-cultured neonatal rat cardiomyocytes were incubated with isoproterenol (ISO) to induce hypertrophy, or were infected with recombinant adenovirus vectors harboring PARP1 cDNA (Ad-PARP1). Sprague-Dawley (SD) rats were treated with ISO to induce cardiac hypertrophy, or were injected with Ad-PARP1 into the anterior and posterior left ventricular walls. Cardiomyocyte surface area, the mRNA expression of hypertrophic biomarkers, echocardiography, morphometry of the hearts were measured. The PARP1 activity was tested by cellular PAR levels. Interactions of PARP1 and FoxO3 were investigated by co-immunoprecipitation and immunofluorescence technique. PARylation of FoxO3 mediated by PARP1 facilitated its phosphorylation at the T32, S252 and S314 sites, triggered its nucleus export and suppressed its transcriptional activity and target genes expression, ultimately inducing cardiac hypertrophy. Additionally, PARP1 silencing or specific inhibition by 3-Aminobenzamide (3AB) and veliparib (ABT-888) alleviated the inhibition of FoxO3 activity by ISO, thus suppressing ISO-induced cardiac hypertrophy. Our data provide the first evidence that PARP1 exacerbates cardiac hypertrophy by PARylation of FoxO3.