The first cardiac transplant experience in a patient with mucopolysaccharidosis

Hunter syndrome (MPSII) is a rare X-linked lysosomal storage disorder that can affect multiple systems but primarily affects the heart. We report the case of a previously asymptomatic 23-year-old patient who had an attenuated form of MPSII and presented with refractory heart failure that required a heart transplant. The diagnosis was confirmed by detection of an increase in urinary excretion of glycosaminoglycans, a deficiency in enzymatic activity, and molecular analysis. A myocardial biopsy revealed hypertrophic cardiomyocytes, mild fibrosis, and lysosomal storage in interstitial cells. Molecular analysis identified a novel mutation in the iduronate-2-sulfatase gene. Although the clinical outcome was not favorable, we believe that this approach may be valid in end-stage heart failure. (C) 2012 Elsevier Inc. All rights reserved.

Focal adhesion kinase governs cardiac concentric hypertrophic growth by activating the AKT and mTOR pathways

The heart responds to sustained overload by hypertrophic growth in which the myocytes distinctly thicken or elongate on increases in systolic or diastolic stress. Though potentially adaptive, hypertrophy itself may predispose to cardiac dysfunction in pathological settings. The mechanisms underlying the diverse morphology and outcomes of hypertrophy are uncertain. Here we used a focal adhesion kinase (FAK) cardiac-specific transgenic mice model (FAK-Tg) to explore the function of this non-receptor tyrosine kinase on the regulation of myocyte growth. FAK-Tg mice displayed a phenocopy of concentric cardiac hypertrophy, reflecting the relative thickening of the individual myocytes. Moreover, FAK-Tg mice showed structural, functional and molecular features of a compensated hypertrophic growth, and preserved responses to chronic pressure overload. Mechanistically, FAK overexpression resulted in enhanced myocardial FAK activity, which was proven by treatment with a selective FAK inhibitor to be required for the cardiac hypertrophy in this model. Our results indicate that upregulation of FAK does not affect the activity of Src/ERK1/2 pathway, but stimulated signaling by a cascade that encompasses PI3K, AKT, mTOR, S6K and rpS6. Moreover, inhibition of the mTOR complex by rapamycin extinguished the cardiac hypertrophy of the transgenic FAK mice. These findings uncover a unique role for FAK in regulating the signaling mechanisms that governs the selective myocyte growth in width, likely controlling the activity of PI3K/AKT/mTOR pathway, and suggest that FAK activation could be important for the adaptive response to increases in cardiac afterload. This article is part of a Special Issue entitled "Local Signaling in Myocytes". (C) 2011 Elsevier Ltd. All rights reserved.

CARNITINE SUPPLEMENTATION EFFECTS ON NONENZYMATIC ANTIOXIDANTS IN YOUNG RATS SUBMITTED TO EXHAUSTIVE EXERCISE STRESS

Bucioli, SA, de Abreu, LC, Valenti, VE, and Vannucchi, H. Carnitine supplementation effects on nonenzymatic antioxidants in young rats submitted to exhaustive exercise stress. J Strength Cond Res 26(6): 1695-1700, 2012-Previous studies have demonstrated that exercise stress increases oxidative stress in rats. However, antioxidant supplement therapy effects on reactive oxygen substances are conflicting. We evaluated the effects of carnitine on renal nonenzymatic antioxidants in young rats submitted to exhaustive exercise stress. Wistar rats were divided into 3 groups: (a) control group (not submitted to exercise stress), (b) exercise stress group, and (c) exercise stress and carnitine group. The rats from group 3 were treated with gavage administration of 1 ml of carnitine (5 mg.kg(-1)) for 7 consecutive days. The animals from groups 2 and 3 were submitted to a bout of swimming exhaustive exercise stress. Kidney samples were analyzed for reactive substances to thiobarbituric acid by malondialdehyde (MDA), reduced glutathione (GSH), and vitamin-E levels. Carnitine treatment attenuated MDA increase caused by exercise stress (1:0.16 +/- 0.02 vs. 2:0.34 +/- 0.07 vs. 3:0.1 +/- 0.01 mmmol per milligram of protein; p < 0.0001). It also increased the renal levels of GSH (1:23 +/- 4 vs. 2:23 +/- 2 vs. 3:58 +/- 9 mu mol per gram of protein; p, 0.0001); however, it did not change renal vitamin E (1:24 +/- 5 vs. 2:27 +/- 1 vs. 3:28 +/- 5 mu M per gram of tissue; p < 0.001). In conclusion, carnitine improved oxidative stress and partially improved the nonenzymatic antioxidant activity in young rats submitted to exhaustive exercise stress.

Early dystrophin disruption in the pathogenesis of experimental chronic Chagas cardiomyopathy

Chronic Chagas cardiomyopathy evolves over a long period of time after initial infection by Trypanosoma cruzi. Similarly, a cardiomyopathy appears later in life in muscular dystrophies. This study tested the hypothesis that dystrophin levels are decreased in the early stage of T cruzi-infected mice that precedes the later development of a cardiomyopathy. CD1 mice were infected with T cruzi (Brazil strain), killed at 30 and 100 days post infection (dpi), and the intensity of inflammation, percentage of interstitial fibrosis, and dystrophin levels evaluated. Echocardiography and magnetic resonance imaging data were evaluated from 15 to 100 dpi. At 30 dpi an intense acute myocarditis with ruptured or intact intracellular parasite nests was observed. At 100 dpi a mild chronic fibrosing myocarditis was detected without parasites in the myocardium. Dystrophin was focally reduced or completely lost in cardiomyocytes at 30 dpi, with the reduction maintained up to 100 dpi. Concurrently, ejection fraction was reduced and the right ventricle was dilated. These findings support the hypothesis that the initial parasitic infection-induced myocardial dystrophin reduction/loss, maintained over time, might be essential to the late development of a cardiomyopathy in mice. (C) 2011 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Vitamin E and Carnitine suplementation effects on blood glucose levels in young rats submitted to exhaustive exercise stress

Background: In this study we evaluated the effects of carnitine and vitamin E supplementation on blood glucose levels in young rats submitted to exhaustive exercise stress. Methods: Wistar rats were divided into four groups: 1) control group; 2) exercise stress group; 3) exercise stress + Vitamin E and; 4) exercise stress + carnitine group. Rats from the group 3 and 4 were treated with gavage administration of 1 mL of Vitamin E (5mg/kg) and carnitine (5mg/kg) for seven consecutive days. Animals from groups 2, 3 and 4 were submitted to a bout of swimming exhaustive exercise stress. We analyzed blood glucose levels after exercise stress. Results: Blood glucose levels after exercise stress were significantly increased in the groups treated with Vitamine E and carnitine (control group: 98.7 +/- 9mg/dL vs. stress group: 84.2 +/- 11 mg/dL vs. carnitine + stress group: 147.4 +/- 15 mg/dL vs. vintamin E + stress: 158.3 +/- 7 mg/dL; p<0.0001). Conclusion: Vitamin E and carnitine supplementation attenuate the hypoglycemia induced by exercise in young rats submitted to exhaustive exercise stress.

Protein quality control disruption by PKC beta II in heart failure: rescue by the selective PKC beta II inhibitor, beta IIV5-3

Myocardial remodeling and heart failure (HF) are common sequelae of many forms of cardiovascular disease and a leading cause of mortality worldwide. Accumulation of damaged cardiac proteins in heart failure has been described. However, how protein quality control (PQC) is regulated and its contribution to HF development are not known. Here, we describe a novel role for activated protein kinase C isoform beta II (PKC beta II) in disrupting PQC. We show that active PKC beta II directly phosphorylated the proteasome and inhibited proteasomal activity in vitro and in cultured neonatal cardiomyocytes. Importantly, inhibition of PKC beta II, using a selective PKC beta II peptide inhibitor (beta IIV5-3), improved proteasomal activity and conferred protection in cultured neonatal cardiomyocytes. We also show that sustained inhibition of PKC beta II increased proteasomal activity, decreased accumulation of damaged and misfolded proteins and increased animal survival in two rat models of HF. Interestingly, beta IIV5-3-mediated protection was blunted by sustained proteasomal inhibition in HF. Finally, increased cardiac PKC beta II activity and accumulation of misfolded proteins associated with decreased proteasomal function were found also in remodeled and failing human hearts, indicating a potential clinical relevance of our findings. Together, our data highlights PKC beta II as a novel inhibitor of proteasomal function. PQC disruption by increased PKC beta II activity in vivo appears to contribute to the pathophysiology of heart failure, suggesting that PKC beta II inhibition may benefit patients with heart failure. (218 words)

FERM domain interaction with myosin negatively regulates FAK in cardiomyocyte hypertrophy

Focal adhesion kinase (FAK) regulates cellular processes that affect several aspects of development and disease. The FAK N-terminal FERM (4.1 protein-ezrin-radixin-moesin homology) domain, a compact clover-leaf structure, binds partner proteins and mediates intramolecular regulatory interactions. Combined chemical cross-linking coupled to MS, small-angle X-ray scattering, computational docking and mutational analyses showed that the FAK FERM domain has a molecular cleft (similar to 998 angstrom(2)) that interacts with sarcomeric myosin, resulting in FAK inhibition. Accordingly, mutations in a unique short amino acid sequence of the FERM myosin cleft, FP-1, impaired the interaction with myosin and enhanced FAK activity in cardiomyocytes. An FP-1 decoy peptide selectively inhibited myosin interaction and increased FAK activity, promoting cardiomyocyte hypertrophy through activation of the AKT-mammalian target of rapamycin pathway. Our findings uncover an inhibitory interaction between the FAK FERM domain and sarcomeric myosin that presents potential opportunities to modulate the cardiac hypertrophic response through changes in FAK activity.

Molecular mapping of the regenerative niche in a murine model of myocardial infarction

Adult stem cells are distributed through the whole organism, and present a great potential for the therapy of different types of disease. For the design of efficient therapeutic strategies, it is important to have a more detailed understanding of their basic biological characteristics, as well as of the signals produced by damaged tissues and to which they respond. Myocardial infarction (MI), a disease caused by a lack of blood flow supply in the heart, represents the most common cause of morbidity and mortality in the Western world. Stem cell therapy arises as a promising alternative to conventional treatments, which are often ineffective in preventing loss of cardiomyocytes and fibrosis. Cell therapy protocols must take into account the molecular events that occur in the regenerative niche of MI. In the present study, we investigated the expression profile of ten genes coding for chemokines or cytokines in a murine model of MI, aiming at the characterization of the regenerative niche. MI was induced in adult C57BL/6 mice and heart samples were collected after 24 h and 30 days, as well as from control animals, for quantitative RT-PCR. Expression of the chemokine genes CCL2, CCL3, CCL4, CCL7, CXCL2 and CXCL10 was significantly increased 24 h after infarction, returning to baseline levels on day 30. Expression of the CCL8 gene significantly increased only on day 30, whereas gene expression of CXCL12 and CX3CL1 were not significantly increased in either ischemic period. Finally, expression of the IL-6 gene increased 24 h after infarction and was maintained at a significantly higher level than control samples 30 days later. These results contribute to the better knowledge of the regenerative niche in MI, allowing a more efficient selection or genetic manipulation of cells in therapeutic protocols.

New insight into the mechanisms associated with the rapid effect of T-3 on AT1R expression

The angiotensin II type 1 receptor (AT1R) is involved in the development of cardiac hypertrophy promoted by thyroid hormone. Recently, we demonstrated that triiodothyronine (T-3) rapidly increases AT1R mRNA and protein levels in cardiomyocyte cultures. However, the molecular mechanisms responsible for these rapid events are not yet known. In this study, we investigated the T-3 effect on AT1R mRNA polyadenylation in cultured cardiomyocytes as well as on the expression of microRNA-350 (miR-350), which targets AT1R mRNA. The transcriptional and translational actions mediated by T-3 on AT1R levels were also assessed. The total content of ubiquitinated proteins in cardiomyocytes treated with T-3 was investigated. Our data confirmed that T-3 rapidly raised AT1R mRNA and protein levels, as assessed by real-time PCR and western blotting respectively. The use of inhibitors of mRNA and protein synthesis prevented the rapid increase in AT1R protein levels mediated by T-3. In addition, T-3 rapidly increased the poly-A tail length of the AT1R mRNA, as determined by rapid amplification of cDNA ends poly-A test, and decreased the content of ubiquitinated proteins in cardiomyocytes. On the other hand, T-3 treatment increased miR-350 expression. In parallel with its transcriptional and translational effects on the AT1R, T-3 exerted a rapid posttranscriptional action on AT1R mRNA polyadenylation, which might be contributing to increase transcript stability, as well as on translational efficiency, resulting to the rapid increase in AT1R mRNA expression and protein levels. Finally, these results show, for the first time, that T-3 rapidly triggers distinct mechanisms, which might contribute to the regulation of AT1R levels in cardiomyocytes. Journal of Molecular Endocrinology (2012) 49, 11-20

Parathyroid hormone and phosphorus overload in uremia: impact on cardiovascular system

Background. Cardiac remodeling in uremia is characterized by left ventricular hypertrophy, interstitial fibrosis and microvascular disease. Cardiovascular disease is the leading cause of death in uremic patients, but coronary events alone are not the prevalent cause, sudden death and heart failure are. We studied the cardiac remodeling in experimental uremia, evaluating the isolated effect of parathyroid hormone (PTH) and phosphorus. Methods. Wistar rats were submitted to parathyroidectomy (PTx) and 5/6 nephrectomy (Nx); they also received vehicle (V) and PTH at normal (nPTH) or high (hPTH) doses. They were fed with a poor-phosphorus (pP) or rich-phosphorus (rP) diet and were divided into the following groups: 'Sham': G1 (V + normal-phosphorus diet (np)) and 'Nx + PTx': G2 (nPTH + pP), G3 (nPTH + rP), G4 (hPTH + pP) and G5 (hPTH + rP). After 8 weeks, biochemical analysis, myocardium morphometry and arteriolar morphological analysis were performed. In addition, using immunohistochemical analysis, we evaluated angiotensin II, alpha-actin, transforming growth factor-beta (TGF-beta) and nitrotyrosine, as well as fibroblast growth factor-23 (FGF-23), fibroblast growth factor receptor-1 (FGFR-1) and runt-related transcription factor-2 (Runx-2) expression. Results. Nx animals presented higher serum creatinine levels as well as arterial hypertension. Higher PTH levels were associated with myocardial hypertrophy and fibrosis as well as a higher coronary lesion score. High PTH animals also presented a higher myocardial expression of TGF-beta, angiotensin II, FGF-23 and nitrotyrosine and a lower expression of alpha-actin. Phosphorus overload was associated with higher serum FGF-23 levels and Runx-2, as well as myocardial hypertrophy. FGFR-1 was positive in the cardiomyocytes of all groups as well as in calcified coronaries of G4 and G5 whereas Runx-2 was positive in G3, G4 and G5. Conclusion. In uremia, PTH and phosphorus overload are both independently associated with major changes related to the cardiac remodeling process, emphasizing the need for a better control of these factors in chronic kidney disease.

Exercise Training Restores Cardiac Protein Quality Control in Heart Failure

Exercise training is a well-known coadjuvant in heart failure treatment; however, the molecular mechanisms underlying its beneficial effects remain elusive. Despite the primary cause, heart failure is often preceded by two distinct phenomena: mitochondria dysfunction and cytosolic protein quality control disruption. The objective of the study was to determine the contribution of exercise training in regulating cardiac mitochondria metabolism and cytosolic protein quality control in a post-myocardial infarction-induced heart failure (MI-HF) animal model. Our data demonstrated that isolated cardiac mitochondria from MI-HF rats displayed decreased oxygen consumption, reduced maximum calcium uptake and elevated H2O2 release. These changes were accompanied by exacerbated cardiac oxidative stress and proteasomal insufficiency. Declined proteasomal activity contributes to cardiac protein quality control disruption in our MI-HF model. Using cultured neonatal cardiomyocytes, we showed that either antimycin A or H2O2 resulted in inactivation of proteasomal peptidase activity, accumulation of oxidized proteins and cell death, recapitulating our in vivo model. Of interest, eight weeks of exercise training improved cardiac function, peak oxygen uptake and exercise tolerance in MI-HF rats. Moreover, exercise training restored mitochondrial oxygen consumption, increased Ca2+-induced permeability transition and reduced H2O2 release in MI-HF rats. These changes were followed by reduced oxidative stress and better cardiac protein quality control. Taken together, our findings uncover the potential contribution of mitochondrial dysfunction and cytosolic protein quality control disruption to heart failure and highlight the positive effects of exercise training in re-establishing cardiac mitochondrial physiology and protein quality control, reinforcing the importance of this intervention as a nonpharmacological tool for heart failure therapy.

Trypanosoma cruzi invades host cells through the activation of endothelin and bradykinin receptors: a converging pathway leading to chagasic vasculopathy

BACKGROUND AND PURPOSE Independent studies in experimental models of Trypanosoma cruzi appointed different roles for endothelin-1 (ET-1) and bradykinin (BK) in the immunopathogenesis of Chagas disease. Here, we addressed the hypothesis that pathogenic outcome is influenced by functional interplay between endothelin receptors (ETAR and ETBR) and bradykinin B2 receptors (B2R). EXPERIMENTAL APPROACH Intravital microscopy was used to determine whether ETR/B2R drives the accumulation of rhodamine-labelled leucocytes in the hamster cheek pouch (HCP). Inflammatory oedema was measured in the infected BALB/c paw of mice. Parasite invasion was assessed in CHO over-expressing ETRs, mouse cardiomyocytes, endothelium (human umbilical vein endothelial cells) or smooth muscle cells (HSMCs), in the presence/absence of antagonists of B2R (HOE-140), ETAR (BQ-123) and ETBR (BQ-788), specific IgG antibodies to each GPCRs; cholesterol or calcium-depleting drugs. RNA interference (ETAR or ETBR genes) in parasite infectivity was investigated in HSMCs. KEY RESULTS BQ-123, BQ-788 and HOE-140 reduced leucocyte accumulation in HCP topically exposed to trypomastigotes and blocked inflammatory oedema in infected mice. Acting synergistically, ETAR and ETBR antagonists reduced parasite invasion of HSMCs to the same extent as HOE-140. Exogenous ET-1 potentiated T. cruzi uptake by HSMCs via ETRs/B2R, whereas RNA interference of ETAR and ETBR genes conversely reduced parasite internalization. ETRs/B2R-driven infection in HSMCs was reduced in HSMC pretreated with methyl-beta-cyclodextrin, a cholesterol-depleting drug, or in thapsigargin-or verapamil-treated target cells. CONCLUSIONS AND IMPLICATIONS Our findings suggest that plasma leakage, a neutrophil-driven inflammatory response evoked by trypomastigotes via the kinin/endothelin pathways, may offer a window of opportunity for enhanced parasite invasion of cardiovascular cells.

Myocardial performance in conscious streptozotocin diabetic rats

Abstract Background In spite of a large amount of studies in anesthetized animals, isolated hearts, and in vitro cardiomyocytes, to our knowledge, myocardial function was never studied in conscious diabetic rats. Myocardial performance and the response to stress caused by dobutamine were examined in conscious rats, fifteen days after the onset of diabetes caused by streptozotocin (STZ). The protective effect of insulin was also investigated in STZ-diabetic rats. Methods Cardiac contractility and relaxation were evaluated by means of maximum positive (+dP/dtmax) and negative (-dP/dtmax) values of first derivative of left ventricular pressure over time. In addition, it was examined the myocardial response to stress caused by two dosages (1 and 15 μg/kg) of dobutamine. One-way analysis of variance (ANOVA) was used to compare differences among groups, and two-way ANOVA for repeated measure, followed by Tukey post hoc test, to compare the responses to dobutamine. Differences were considered significant if P < 0.05. Results Basal mean arterial pressure, heart rate, +dP/dtmax and -dP/dtmax were found decreased in STZ-diabetic rats, but unaltered in control rats treated with vehicle and STZ-diabetic rats treated with insulin. Therefore, insulin prevented the hemodynamic and myocardial function alterations observed in STZ-diabetic rats. Lower dosage of dobutamine increased heart rate, +dP/dtmax and -dP/dtmax only in STZ-diabetic rats, while the higher dosage promoted greater, but similar, responses in the three groups. In conclusion, the results indicate that myocardial function was remarkably attenuated in conscious STZ-diabetic rats. In addition, the lower dosage of dobutamine uncovered a greater responsiveness of the myocardium of STZ-diabetic rats. Insulin preserved myocardial function and the integrity of the response to dobutamine of STZ-diabetic rats. Conclusion The present study provides new data from conscious rats showing that the cardiomyopathy of this pathophysiological condition was expressed by low indices of contractility and relaxation. In addition, it was also demonstrated that these pathophysiological features were prevented by the treatment with insulin.

Morfologia dos cardiomiócitos e quantificação do colágeno no miocárdio de ratas tratadas com isoflavonas ou estrogênios

OBJETIVOS: Avaliar a morfologia dos cardiomiócitos e quantificar o colágeno presente no miocárdio de ratas tratadas com extrato concentrado de soja ou 17β-estradiol (E2). MÉTODOS: Vinte e oito ratas foram divididas em quatro grupos: GCtrl - fase de estro; GOvx - ovariectomizadas (Ovx); GIso - Ovx tratadas com extrato de soja (150 mg/kg, por dia); GE2 - Ovx tratadas com E2 (10 µg/kg, por dia). As drogas e o veículo (0,2 mL de propilenoglicol) foram administrados após 30 dias da realização da ovariectomia, por 30 dias consecutivos. No último dia os animais foram anestesiados, o coração retirado, mergulhado em formaldeído a 10%, e fragmentos dos ventrículos submetidos a processamento histológico, sendo os cortes corados pela hematoxilina e eosina ou pelo picrosirius-red. As análises histomorfométricas (contagem, volume nuclear e quantificação do colágeno) foram realizadas em microscópio de luz e software AxioVision Rel. 4.2, sendo o colágeno determinado pelo programa Imagelab 2000. Os dados foram submetidos ao teste de ANOVA complementado pelo teste de Tukey (p<0,05). RESULTADOS: Notamos maior quantidade de núcleos de cardiomiócitos nos animais dos grupos Ovx e Iso do que no GE2 e GCtrl (GOvx=121,7±20,2=GIso=92,8±15,4>GE2=70,5±14,8=GCtrl=66,3±9,6; p<0,05), sendo o volume nuclear maior nos animais do grupo Ctrl e E2 (GE2=35,7±4,8=GCtrl=29,9±3,6>GIso=26,5±4,5=GOvx=22,4±2,9; p<0,05). Com relação ao colágeno notamos maior concentração no grupo Ovx (GOvx=5,4±0,1>GCtrl=4,0±0,1=GIso=4,4±0,08=GE2=4,3±0,5; p<0,05). CONCLUSÕES: Os estrogênios previnem a diminuição do volume nuclear dos cardiomiócitos e a deposição de colágeno entre as fibras musculares cardíacas. Já a administração de isoflavonas previne somente a deposição de colágeno, o que pode preservar as propriedades mecânicas das fibras cardíacas.

AMPK signaling pathway is rapidly activated by T3 and regulates the cardiomyocyte growth

Previous studies have indicated that AMP-activated protein kinase (AMPK) plays a critical role in the control of cardiac hypertrophy mediated by different stimuli such as thyroid hormone (TH). Although the classical effects of TH mediating cardiac hypertrophy occur by transcriptional mechanisms, recent studies have identified other responses to TH, which are more rapid and take place in seconds or minutes evidencing that TH rapidly modulates distinct signaling pathway, which might contribute to the regulation of cardiomyocyte growth. Here, we evaluated the rapid effects of TH on AMPK signaling pathway in cultured cardiomyocytes and determined the involvement of AMPK in T3-induced cardiomyocyte growth. We found for the first time that T3 rapidly activated AMPK signaling pathway. The use of small interfering RNA against AMPK resulted in increased cardiomyocyte hypertrophy while the pharmacological stimulation of AMPK attenuated this process, demonstrating that AMPK contributes to regulation of T3-induced cardiomyocyte growth.