Reactive oxygen and nitrogen species balance in the determination of thyroid hormones-induced cardiac hypertrophy mediated by renin-angiotensin system

Role of reactive oxygen species (ROS)/nitric oxide (NO) balance and renin-angiotensin system in mediating cardiac hypertrophy in hyperthyroidism was evaluated in an in vivo and in vitro experimental model. Male Wistar rats were divided into four groups: control, thyroid hormone, vitamin E (or Trolox, its hydrosoluble analogue), thyroid hormone + vitamin E. Angiotensin II receptor (AT1/AT2) gene expression, immunocontent of AT1/AT2 receptors, angiotensinogen, NADPH oxidase (Nox2), and nitric oxide synthase isoforms, as well as ROS concentration (hydrogen peroxide and superoxide anion) were quantified in myocardium. Thyroid hormone increased ROS and NO metabolites, iNOS, nNOS and eNOS isoforms and it was accompanied by cardiac hypertrophy. AT1/AT2 expression and the immunocontent of angiotensinogen and Nox2 were enhanced by thyroid hormone. Antioxidants reduced ROS levels, Nox2, AT1/AT2, NOS isoforms and cardiac hypertrophy. In conclusion, ROS/NO balance may play a role in the control of thyroid hormone-induced cardiac hypertrophy mediated by renin-angiotensin system. (C) 2011 Elsevier Ireland Ltd. All rights reserved.

Blockage of Angiotensin II type 2 receptor prevents thyroxine-mediated cardiac hypertrophy by blocking Akt activation

Although most of effects of Angiotensin II (Ang II) related to cardiac remodelling can be attributed to type 1 Ang II receptor (AT(1)R), the type 2 receptor (AT(2)R) has been shown to be involved in the development of some cardiac hypertrophy models. In the present study, we investigated whether the thyroid hormone (TH) action leading to cardiac hypertrophy is also mediated by increased Ang II levels or by change on AT(1)R and AT(2)R expression, which could contribute to this effect. In addition, we also evaluated the possible contribution of AT(2)R in the activation of Akt and in the development of TH-induced cardiac hypertrophy. To address these questions, Wistar rats were treated with thyroxine (T(4), 0.1 mg/kg BW/day, i.p.), with or without AT(2)R blocker (PD123319), for 14 days. Cardiac hypertrophy was identified based on heart/body weight ratio and confirmed by analysis of atrial natriuretic factor mRNA expression. Cardiomyocyte cultures were used to exclude the influence of TH-related hemodynamic effects. Our results demonstrate that the cardiac Ang II levels were significantly increased (80%, P < 0.001) as well as the AT(2)R expression (50%, P < 0.05) in TH-induced cardiac hypertrophy. The critical involvement of AT(2)R to the development of this cardiac hypertrophy in vivo was evidenced after administration of AT(2) blocker, which was able to prevent in 40% (P < 0.01) the cardiac mass gain and the Akt activation induced by TH. The role of AT(2)R to the TH-induced cardiomyocyte hypertrophy was also confirmed after using PD123319 in the in vitro studies. These findings improve understanding of the cardiac hypertrophy observed in hyperthyroidism and provide new insights into the generation of future therapeutic strategies.

Angiotensin type 1 receptor mediates thyroid hormone-induced cardiomyocyte hypertrophy through the Akt/GSK-3 beta/mTOR signaling pathway

Several studies have implicated the renin angiotensin system in the cardiac hypertrophy induced by thyroid hormone. However, whether Angiotensin type 1 receptor (AT(1)R) is critically required to the development of T(3)-induced cardiomyocyte hypertrophy as well as whether the intracellular mechanisms that are triggered by AT(1)R are able to contribute to this hypertrophy model is unknown. To address these questions, we employed a selective small interfering RNA (siRNA, 50 nM) or an AT(1)R blocker (Losartan, 1 mu M) to evaluate the specific role of this receptor in primary cultures of neonatal cardiomyocytes submitted to T(3) (10 nM) treatment. The cardiomyocytes transfected with the AT(1)R siRNA presented reduced mRNA (90%, P < 0.001) and protein (70%, P < 0.001) expression of AT(1)R. The AT(1)R silencing and the AT(1)R blockade totally prevented the T(3)-induced cardiomyocyte hypertrophy, as evidenced by lower mRNA expression of atrial natriuretic factor (66%, P < 0.01) and skeletal alpha-actin (170%, P < 0.01) as well as by reduction in protein synthesis (85%, P < 0.001). The cardiomyocytes treated with T(3) demonstrated a rapid activation of Akt/GSK-3 beta/mTOR signaling pathway, which was completely inhibited by the use of PI3K inhibitors (LY294002, 10 mu M and Wortmannin, 200 nM). In addition, we demonstrated that the AT(1)R mediated the T(3)-induced activation of Akt/GSK-3 beta/mTOR signaling pathway, since the AT(1)R silencing and the AT(1)R blockade attenuated or totally prevented the activation of this signaling pathway. We also reported that local Angiotensin I/II (Ang I/II) levels (120%, P < 0.05) and the AT(1)R expression (180%, P < 0.05) were rapidly increased by T(3) treatment. These data demonstrate for the first time that the AT(1)R is a critical mediator to the T(3)-induced cardiomyocyte hypertrophy as well as to the activation of Akt/GSK-3 beta/mTOR signaling pathway. These results represent a new insight into the mechanism of T(3)-induced cardiomyocyte hypertrophy, indicating that the Ang I/II-AT(1)R-Akt/GSK-3 beta/mTOR pathway corresponds to a potential mediator of the trophic effect exerted by T(3) in cardiomyocytes.

Muscle atrophy and hypertrophy signaling in patients with chronic obstructive pulmonary disease

Rationale: The molecular mechanisms of muscle atrophy in chronic obstructive pulmonary disease (COPD) are poorly understood. In wasted animals, muscle mass is regulated by several AKT-related signaling pathways.
Objectives: To measure the protein expression of AKT, forkhead box class O (FoxO)-1 and -3, atrogin-1, the phosphophrylated form of AKT, p70^S6K glycogen synthase kinase-3ß (GSK-3ß), eukaryotic translation initiation factor 4E binding protein-1 (4E-BP1), and the mRNA expression of atrogin-1, muscle ring finger (MuRF) protein 1, and FoxO-1 and -3 in the quadriceps of 12 patients with COPD with muscle atrophy and 10 healthy control subjects. Five patients with COPD with preserved muscle mass were subsequently recruited and were compared with six patients with low muscle mass.
Methods: Protein contents and mRNA expression were measured by Western blot and quantitative polymerase chain reaction, respectively.
Measurements and Main Results: The levels of atrogin-1 and MuRF1 mRNA, and of phosphorylated AKT and 4E-BP1 and FoxO-1 proteins, were increased in patients with COPD with muscle atrophy compared with healthy control subjects, whereas atrogin-1, p70^S6K, GSK-3ß, and FoxO-3 protein levels were similar. Patients with COPD with muscle atrophy showed an increased expression of p70^S6K, GSK-3ß, and 4E-BP1 compared with patients with COPD with preserved muscle mass.
Conclusions: An increase in atrogin-1 and MuRF1 mRNA and FoxO-1 protein content was observed in the quadriceps of patients with COPD. The transcriptional regulation of atrogin-1 and MuRF1 may occur via FoxO-1, but independently of AKT. The overexpression of the muscle hypertrophic signaling pathways found in patients with COPD with muscle atrophy could represent an attempt to restore muscle mass.

Intrinsic resistance of female hearts to an ischemic insult is abrogated in primary cardiac hypertrophy

Important sex differences in cardiovascular disease outcomes exist, including conditions of hypertrophic cardiomyopathy and cardiac ischemia. Studies of sex differences in the extent to which load-independent (primary) hypertrophy modulates the response to ischemia-reperfusion (I/R) damage have not been characterized. We have previously described a model of primary genetic cardiac hypertrophy, the hypertrophic heart rat (HHR). In this study the sex differences in HHR cardiac function and responses to I/R [compared to control normal heart rat (NHR)] were investigated ex vivo. The ventricular weight index was markedly increased in HHR female (7.82 ± 0.49 vs. 4.80 ± 0.10 mg/g; P < 0.05) and male (5.76 ± 0.22 vs. 4.62 ± 0.07 mg/g; P < 0.05) hearts. Female hearts of both strains exhibited a reduced basal contractility compared with strain-matched males [maximum first derivative of pressure (dP/dtmax): NHR, 4,036 ± 171 vs. 4,258 ± 152 mmHg/s; and HHR, 3,974 ± 160 vs. 4,540 ± 259 mmHg/s; P < 0.05]. HHR hearts were more susceptible to I/R (I = 25 min, and R = 30 min) injury than NHR hearts (decreased functional recovery, and increased lactate dehydrogenase efflux). Female NHR hearts exhibited a significantly greater recovery (dP/dtmax) post-I/R relative to male NHR (95.0 ± 12.2% vs. 60.5 ± 9.4%), a resistance to postischemic dysfunction not evident in female HHR (29.0 ± 5.6% vs. 25.9 ± 6.3%). Ventricular fibrillation was suppressed, and expression levels of Akt and ERK1/2 were selectively elevated in female NHR hearts. Thus the occurrence of load-independent primary cardiac hypertrophy undermines the intrinsic resistance of female hearts to I/R insult, with the observed abrogation of endogenous cardioprotective signaling pathways consistent with a potential mechanistic role in this loss of protection.

Regulation of STARS and its downstream target suggest a novel pathway involved in human skeletal hypertrophy and atrophy

Skeletal muscle atrophy is a severe consequence of ageing, neurological disorders and chronic disease. Identifying the intracellular signalling pathways controlling changes in skeletal muscle size and function is vital for the future development of potential therapeutic interventions. Striated activator of Rho signalling (STARS), an actin-binding protein, has been implicated in rodent cardiac hypertrophy; however its role in human skeletal muscle has not been determined. This study aimed to establish if STARS, as well as its downstream signalling targets, RhoA, myocardin-related transcription factors A and B (MRTF-A/B) and serum response factor (SRF), were increased and decreased respectively, in human quadriceps muscle biopsies taken after 8 weeks of both hypertrophy-stimulating resistance training and atrophy-stimulating de-training. The mRNA levels of the SRF target genes involved in muscle structure, function and growth, such as α-actin, myosin heavy chain IIa (MHCIIa) and insulin-like growth factor-1 (IGF-1), were also measured. Following resistance training, STARS, MRTF-A, MRTF-B, SRF, α-actin, MHCIIa and IGF-1 mRNA, as well as RhoA and nuclear SRF protein levels were all significantly increased by between 1.25- and 3.6-fold. Following the de-training period all measured targets, except for RhoA, which remained elevated, returned to base-line. Our results show that the STARS signalling pathway is responsive to changes in skeletal muscle loading and appears to play a role in both human skeletal muscle hypertrophy and atrophy.

Identification and characterisation of novel genes involved in skeletal muscle hypertrophy

There is mounting evidence in support of the view that skeletal muscle hypertrophy results from the complex and coordinated interaction of numerous signalling pathways. Well characterised components integral to skeletal muscle adaptation include the transcriptional activity of the members of the myogenic regulatory factors, numerous secreted peptide growth factors, and the regenerative potential of satellite cells. Whilst studies investigating isolated components or pathways have enhanced our current understanding of skeletal muscle hypertrophy, our knowledge of how all of these components react in concert to a common stimulus remains limited. The broad aim of this thesis was to identify and characterise novel genes involved in skeletal muscle hypertrophy. We have created a customised human skeletal muscle specific microarray which contains ∼11,000 cDNA clones derived from a normalised human skeletal muscle cDNA library as well as 270 genes with known functional roles in human skeletal muscle. The first aspect of this thesis describes the production of the microarray and evaluates the robustness and reproducibility of this analytical technique. Study one aimed to use this microarray in the identification of genes that are differentially expressed during the forced differentiation of human rhabdomyosarcoma cells, an in vitro model of skeletal muscle development. Firstly using this unique model of aberrant myogenic differentiation we aimed to identify genes with previously unidentified roles in myogenesis. Secondly, the data from this study permitted the examination of the performance of the microarray in detecting differential gene expression in a biological system. We identified several new genes with potential roles in the myogenic arrest of rhabdomyosarcoma and further characterised the expression of muscle specific genes in rhabdomyosarcoma differentiation. In study two, the molecular responses of cell cycle regulators, muscle regulatory factors, and atrophy related genes were mapped in response to a single bout of resistance exercise in human skeletal muscle. We demonstrated an increased expression of MyoD, myogenin and p21, whilst the expression of myostatin was decreased. The results of this study contribute to the existing body of knowledge on the molecular regulation skeletal muscle to a hypertrophic stimulus. In study three, the muscle samples collected in study two were analysed using the human skeletal muscle specific microarray for the identification of novel genes with potential roles in the hypertrophic process. The analysis uncovered four interesting genes (TXNIP, MLP, ASB5, FLJ 38973) that have not previously been examined in human skeletal muscle in response to resistance exercise. The functions of these genes and their potential roles in skeletal muscle are discussed. In study four, the four genes identified in study three were examined in human primary skeletal muscle cell cultures during myogenic differentiation. Human primary skeletal muscle cells were derived from the vastus lateralis muscle of 8 healthy volunteers (6 males and 2 females). Cell cultures were differentiated using serum withdrawal and serum withdrawal combined with IGF-1 supplementation. Markers of the cell proliferation, cell cycle arrest and myogenic differentiation were examined to assess the effectiveness of the differentiation stimulus. Additionally, the expressions of TXNIP, MLP, ASB5 and FLJ 38973 measured in an attempt to characterise further their roles in skeletal muscle. The expression of TXNIP changed markedly in response to both differentiation stimuli, whilst the expression of the remaining genes were not altered. Therefore it was suggested that expression of these genes might be responsive to the mechanical strain or contraction induced by the resistance exercise. In order to examine whether these novel genes responded specifically to resistance type exercise, their expression was examined following a single bout of endurance exercise. The expression of TXNIP, MLP, and FLJ 38973 remained unchanged whilst ASB5 increased 30 min following the cessation of the exercise.

Elevated intracardiac angiotensin II leads to cardiac hypertrophy and mechanical dysfunction in normotensive mice

Introduction
Angiotensin II (Ang II) is known to induce cardiac growth and modulate myocardial contractility. It has been reported that elevated levels of endogenous Ang II contribute to the development of cardiac hypertrophy in hypertensives. However, the long-term functional effects of cardiac exposure to Ang II in normotensives is unclear.

A recently developed transgenic mouse (TG1306/1R), in which cardiac-specific overproduction of Ang II produces primary hypertrophy, provides a new experimental model for investigation of this phenotype. The aim of the present study was to use this model to investigate whether there is a functional deficit in primary hypertrophy that may predispose to cardiac failure and sudden death. We hypothesised that primary cardiac hypertrophy is associated with mechanical dysfunction in the basal state.

Methods
Normotensive heterozygous TG1306/1R mice harbouring multiple copies of a cardiac-specific rat angiotensinogen gene were studied at age 30—40 weeks and compared with age-matched wild-type littermates. Left ventricular function was measured ex vivo in bicarbonate buffer-perfused, Langendorffmounted hearts ( at a perfusion pressure of 80 mmHg, 37°C) using a fluid-filled PVC balloon interfaced to a pressure transducer and digital data acquisition system.

Results
There was no difference in the mean (±SEM) intrinsic heart rate of TG1306/1R and wild-type control mice (357.4±11.8 vs. 367.5±20.9 bpm, n=9 & 7). Under standardised end-diastolic pressure conditions, TG1306/1R hearts exhibited a significant reduction in peak developed pressure (132.2±9.4 vs. 161.5±3.1 mmHg, n=9 & 7, p<0.05) and maximum rate of pressure development (3566.7±323.7 vs. 4486.3±109.4 mmHg, n=9 & 7, p<0.05). TG1306/1R mice show a significant correlation between incidence of arrhythmia and increasing heart size (Spearman's correlation coefficient 0.61).

Conclusion
These data demonstrate that chronic in vivo exposure to elevated levels of intra-cardiac Ang II is associated with significant contractile abnormalities evident in the ex vivo intact heart. Our findings suggest that endogenous overproduction of cardiac Ang II, independent of changes in blood pressure, is sufficient to induce ventricular remodelling that culminates in impaired cardiac function which may precede failure.

Threshold effects of glucose transporter-4 (GLUT4) deficiency on cardiac glucose uptake and development of hypertrophy

The aim of this study was to investigate the metabolic and structural consequences of a decrease in glucose transporter-4 (GLUT4) levels on the heart. The CreLoxP system was utilised to delete GLUT4 in muscle tIssue including heart. The presence of the PGK-neoR cassette in the GLUT4-Lox mice resulted in reduced expression in all tIssues to levels 15-30% of wild-type control mice. In mice expressing Cre recombinase, there was a further reduction of GLUT4 in cardiac tIssue to almost undetectable levels. Cardiac glucose uptake was measured basally and during a uglycaemic/hyperinsulinaemic clamp using 2-deoxy-[1-(14)C]glucose. Insulin-stimulated glucose uptake was normal in hearts expressing 15% of normal GLUT4 levels but markedly reduced in mice with more profound reduction in GLUT4. Cardiac enlargement occurred only when GLUT4 levels were less than 5% of normal values. In heart there is a threshold level of GLUT4 above which insulin-stimulated glucose uptake is maintained. As little as 5% of normal GLUT4 levels expressed in heart is sufficient to prevent the development of cardiac hypertrophy. 2-deoxy-[1-14C]glucose. Insulin-stimulated glucose uptake was normal in hearts expressing 15% of normal GLUT4 levels but markedly reduced in mice with more profound reduction in GLUT4. Cardiac enlargement occurred only when GLUT4 levels were less than 5% of normal values. In heart there is a threshold level of GLUT4 above which insulin-stimulated glucose uptake is maintained. As little as 5% of normal GLUT4 levels expressed in heart is sufficient to prevent the development of cardiac hypertrophy.

Angiotensin II receptor imbalance associated with neonatal cardiac growth restriction is a prelude to adult cardiac hypertrophy

The Hypertrophic Heart Rat (HHR) displays spontaneous cardiomyocyte hypertrophy in association with an apparent reduction in myocyte number in adulthood. This suggests the possibility of reduced hyperplasia or increased apoptosis during early cardiac development. The angiotensin AT1 and AT2 receptor subtypes have been implicated in both cellular growth and apoptosis, but the precise mechanisms are unclear. The aim of this study was to determine the relationship between cardiac AngII receptor expression levels and neonatal cardiomyocyte growth and apoptotic responses in the HHR compared with the Normal Heart Rat (NHR) control strain. Cardiac tissues were freshly harvested from male HHR and NHR at several developmental stages (p2 and 4, 6, 8, 12wks). HHR cardiac weight indices were considerably smaller than NHR at day 2 (4.330.19 vs 5.010.08 mg/g), but ‘caught-up’ to NHR by 4 weeks (5.100.15 vs 5.160.11 mg/g). By 12 weeks, HHR hearts were 27% larger than NHR. Tissue AT1A and AT2 mRNA expression levels were quantified by real-time RT-PCR. Relative to NHR, HHR neonatal hearts exhibited a 4.6-fold higher AT2/AT1 mRNA expression ratio. Cultured neonatal cardiomyocytes were infected with AT1A and/or AT2 receptor-expressing adenoviruses to achieve a physiological level of receptor expression (150 fmol receptor protein/mg total cell protein). In addition, to emulate receptor expression in neonatal HHR hearts, cells were co-infected with AT1A and AT2 receptors at a 4:1 ratio. Apoptosis incidence was studied by morphological analysis after 72 hours exposure to 0.1 M AngII. When infected with the AT1A receptor alone, a higher proportion of HHR myocytes appeared apoptotic than NHR (22.7 4.1% vs 1.1 0.6%, P 0.001). This implies that intrinsic differences predispose HHR cells to accentuated AT1-mediated apoptosis. Interestingly, the bax-1/bcl-2 mRNA expression ratio was significantly higher (50%) in HHR neonatal hearts. When cells were co-infected with AT1A and AT2 receptors, evidence of apoptosis in HHR cells virtually disappeared (0.4 0.1%). These findings suggest a novel capacity of AT2 receptors to counteract accentuated AT1A receptor-induced apoptosis in the HHR in early cardiac growth.

Elevated dietary sodium intake exacerbates myocardial hypertrophy associated with cardiac-specific overproduction of angiotensin II

Introduction/hypothesis
Cardiac hypertrophy is an independent risk factor predictive of cardiovascular disease and is significantly associated with morbidity and mortality. The mechanism by which angiotensin II (Ang II) and dietary sodium exert additive effects on the development of cardiac hypertrophy is unclear. The goal of this study was to evaluate the hypothesis that, where there is a genetic predisposition to Ang II-dependent hypertrophy, there is also an increased susceptibility to sodium-induced hypertrophy mediated by AT₁-receptor expression.

Methods
Diets of low sodium (LS, 0.3% w:w) and high sodium (HS, 4.0% w:w) content were fed to adult (age 25 weeks) control wild-type mice (WT) and to weeks) control wild-type mice (WT) and to transgenic mice exhibiting cardiac specific overexpression of angiotensinogen (TG). At the conclusion of a 40-day dietary treatment period, cardiac tissue weights were compared and the relative expression levels of Ang II receptor subtypes (AT_1A and AT₂) were evaluated using RT-PCR.

Results
WT and TG mice fed HS and LS diets maintained comparable weight gains during the treatment period. The normalised heart weights of TG mice were elevated compared to WT, and the extent of the increase was greater for mice maintained on the HS diet treatments (WT 12% vs. TG 41% increase in cardiac weight index). While a similar pattern of growth was observed for ventricular tissues, the atrial weight parameters demonstrated an additional significant effect of dietary sodium intake on tissue weight, independent of animal genetic type. No differences in the relative (GAPDH normalised) expression levels of AT_1A- and AT₂-receptor mRNA were observed between diet or animal genetic groups.

Conclusion
This study demonstrates that, where there is a pre-existing genetic condition of Ang II-dependent cardiac hypertrophy, the pro-growth effect of elevated dietary sodium intake is selectively augmented. In TG and WT mice, this effect was evident with a relatively short dietary treatment intervention (40 days). Evaluation of the levels of Ang II receptor mRNA further demonstrated that this differential growth response was not associated with an altered relative expression of either AT_1A- or AT₂-receptor subtypes. The cellular mechanistic bases for this specific Ang II-dietary sodium interaction remain to be elucidated.

Normal hypertrophy accompanied by phosphoryation and activation of AMP-activated protein kinase α1 following overload in LKB1 knockout mice

The activation of the AMP-activated protein kinase (AMPK) and inhibition of the mammalian target of rapamycin complex 1 (mTORC1) is hypothesized to underlie the fact that muscle growth following resistance exercise is decreased by concurrent endurance exercise. To directly test this hypothesis, the capacity for muscle growth was determined in mice lacking the primary upstream kinase for AMPK in skeletal muscle, LKB1. Following either 1 or 4 weeks of overload, there was no difference in muscle growth between the wild type (wt) and LKB1−/− mice (1 week: wt, 38.8 ± 7.75%; LKB1−/−, 27.8 ± 12.98%; 4 week: wt, 75.8 ± 15.2%; LKB1−/−, 85.0 ± 22.6%). In spite of the fact that the LKB1 had been knocked out in skeletal muscle, the phosphorylation and activity of the α1 isoform of AMPK were markedly increased in both the wt and the LKB1−/− mice. To identify the upstream kinase(s) responsible, we studied potential upstream kinases other than LKB1. The activity of both Ca2+–calmodulin-dependent protein kinase kinase α(CaMKKα) (5.05 ± 0.86-fold) and CaMKKβ (10.1 ± 2.59-fold) increased in the overloaded muscles, and this correlated with their increased expression. Phosphorylation of TAK-1 also increased 10-fold following overload in both the wt and LKB1 mice. Even though the α1 isoform of AMPK was activated by overload, there were no increases in expression of mitochondrial proteins or GLUT4, indicating that the α1 isoform is not involved in these metabolic adaptations. The phosphorylation of TSC2, an upstream regulator of the TORC1 pathway, at the AMPK site (Ser1345) was increased in response to overload, and this was not affected by LKB1 deficiency. Taken together, these data suggest that the α1 isoform of AMPK is preferentially activated in skeletal muscle following overload in the absence of metabolic adaptations, suggesting that this isoform might be important in the regulation of growth but not metabolism.