Histidine dipeptides are key regulators of excitation-contraction coupling in cardiac muscle: Evidence from a novel CARNS1 knockout rat model.

Histidine-containing dipeptides (HCDs) are abundantly expressed in striated muscles. Although important properties have been ascribed to HCDs, including H+ buffering, regulation of Ca2+ transients and protection against oxidative stress, it remains unknown whether they play relevant functions in vivo. To investigate the in vivo roles of HCDs, we developed the first carnosine synthase knockout (CARNS1-/-) rat strain to investigate the impact of an absence of HCDs on skeletal and cardiac muscle function. Male wild-type (WT) and knockout rats (4 months-old) were used. Skeletal muscle function was assessed by an exercise tolerance test, contractile function in situ and muscle buffering capacity in vitro. Cardiac function was assessed in vivo by echocardiography and cardiac electrical activity by electrocardiography. Cardiomyocyte contractile function was assessed in isolated cardiomyocytes by measuring sarcomere contractility, along with the determination of Ca2+ transient. Markers of oxidative stress, mitochondrial function and expression of proteins were also evaluated in cardiac muscle. Animals were supplemented with carnosine (1.8% in drinking water for 12 weeks) in an attempt to rescue tissue HCDs levels and function. CARNS1-/- resulted in the complete absence of carnosine and anserine, but it did not affect exercise capacity, skeletal muscle force production, fatigability or buffering capacity in vitro, indicating that these are not essential for pH regulation and function in skeletal muscle. In cardiac muscle, however, CARNS1-/- resulted in a significant impairment of contractile function, which was confirmed both in vivo and ex vivo in isolated sarcomeres. Impaired systolic and diastolic dysfunction were accompanied by reduced intracellular Ca2+ peaks and slowed Ca2+ removal, but not by increased markers of oxidative stress or impaired mitochondrial respiration. No relevant increases in muscle carnosine content were observed after carnosine supplementation. Results show that a primary function of HCDs in cardiac muscle is the regulation of Ca2+ handling and excitation-contraction coupling.

Lactate up-regulates the expression of lactate oxidation complex-related genes in left ventricular cardiac tissue of rats.

BACKGROUND: Besides its role as a fuel source in intermediary metabolism, lactate has been considered a signaling molecule modulating lactate-sensitive genes involved in the regulation of skeletal muscle metabolism. Even though the flux of lactate is significantly high in the heart, its role on regulation of cardiac genes regulating lactate oxidation has not been clarified yet. We tested the hypothesis that lactate would increase cardiac levels of reactive oxygen species and up-regulate the expression of genes related to lactate oxidation complex. METHODS/PRINCIPAL FINDINGS: Isolated hearts from male adult Wistar rats were perfused with control, lactate or acetate (20mM) added Krebs-Henseleit solution during 120 min in modified Langendorff apparatus. Reactive oxygen species (O2●-/H2O2) levels, and NADH and NADPH oxidase activities (in enriched microsomal or plasmatic membranes, respectively) were evaluated by fluorimetry while SOD and catalase activities were evaluated by spectrophotometry. mRNA levels of lactate oxidation complex and energetic enzymes MCT1, MCT4, HK, LDH, PDH, CS, PGC1α and COXIV were quantified by real time RT-PCR. Mitochondrial DNA levels were also evaluated. Hemodynamic parameters were acquired during the experiment. The key findings of this work were that lactate elevated cardiac NADH oxidase activity but not NADPH activity. This response was associated with increased cardiac O2●-/H2O2 levels and up-regulation of MCT1, MCT4, LDH and PGC1α with no changes in HK, PDH, CS, COXIV mRNA levels and mitochondrial DNA levels. Lactate increased NRF-2 nuclear expression and SOD activity probably as counter-regulatory responses to increased O2●-/H2O2. CONCLUSIONS: Our results provide evidence for lactate-induced up-regulation of lactate oxidation complex associated with increased NADH oxidase activity and cardiac O2●-/H2O2 driving to an anti-oxidant response. These results unveil lactate as an important signaling molecule regulating components of the lactate oxidation complex in cardiac muscle.

Increased clearance of reactive aldehydes and damaged proteins in hypertension-induced compensated cardiac hypertrophy: impact of exercise training.

BACKGROUND: We previously reported that exercise training (ET) facilitates the clearance of damaged proteins in heart failure. Here, we characterized the impact of ET on cardiac protein quality control during compensated ventricular hypertrophy in spontaneously hypertensive rats (SHR). METHODS AND RESULTS: SHR were randomly assigned into sedentary and swimming-trained groups. Sedentary SHR displayed cardiac hypertrophy with preserved ventricular function compared to normotensive rats, characterizing a compensated cardiac hypertrophy. Hypertensive rats presented signs of cardiac oxidative stress, depicted by increased lipid peroxidation. However, these changes were not followed by accumulation of lipid peroxidation-generated reactive aldehydes and damaged proteins. This scenario was explained, at least in part, by the increased catalytic activity of both aldehyde dehydrogenase 2 (ALDH2) and proteasome. Of interest, ET exacerbated cardiac hypertrophy, improved ventricular function, induced resting bradycardia, and decreased blood pressure in SHR. These changes were accompanied by reduced cardiac oxidative stress and a consequent decrease in ALDH2 and proteasome activities, without affecting small chaperones levels and apoptosis in SHR. CONCLUSION: Increased cardiac ALDH2 and proteasomal activities counteract the deleterious effect of excessive oxidative stress in hypertension-induced compensated cardiac hypertrophy in rats. ET has a positive effect in reducing cardiac oxidative stress without affecting protein quality control.

Efeito da suplementação de creatina, associada ou não ao treinamento de força, sobre a peroxidação lipídica em mulheres idosas / Effects of creatine supplementation associated or not with strength training upon lipid peroxidation in elderly women

O objetivo desse trabalho foi avaliar o efeito da suplementação de creatina associada ou não ao treinamento de força sobre a peroxidação lipídica em mulheres idosas. Foi conduzido um estudo clínico, randomizado, duplo-cego e controlado por placebo, no qual mulheres idosas foram randomizadas para compor quatro grupos: 1) suplementação com placebo (PL; n = 10); 2) suplementação com creatina (CR; n = 10); 3) suplementação com placebo associado ao treinamento de força (PL+TR; n = 6); e 4) suplementação com creatina associado ao treinamento de força (CR+TR; n = 8). Antes (PRE) e após 24 semanas (POS) de intervenção, foram coletadas amostras de sangue para posterior análise das concentrações plasmáticas de hidroperóxidos lipídicos por espectrofotometria. Nenhuma diferença estatística foi observada na concentração de hidroperóxidos lipídicos entre os grupos (PL: PRE = 48,7 ± 36,9; POS = 29,3 ± 18,8; delta = -13,0 ± 26,8; CR: PRE = 51,0 ± 46,0; POS = 54,2 ± 51,6; delta = -8,6 ± 30,2; PL+TR: PRE = 33,0 ± 11,2; POS = 47,3 ± 31,6; Δ = 14,3 ± 39,2; CR+TR: PRE = 18,5 ± 10,1; POS = 28,1 ± 17,9; delta = 9,7 ± 16,4 pmol.mg-1 de proteína total; p = 0,17). A suplementação de creatina associada ou não ao treinamento de força não afetou a peroxidação lipídica, um importante marcador de estresse oxidativo no plasma, em mulheres idosas.

The aim of this study was to evaluate the effects of creatine supplementation associated or not with strength training upon lipid peroxidation in older women. This was a clinical, randomized, double-blind, placebo-controlled trial. Older women were randomly allocated into four groups: 1) placebo supplementation (PL, n = 10), 2) creatine supplementation (CR; n = 10), 3) placebo supplementation associated with strength training (PL + RT, n = 6) and 4) creatine supplementation associated with strength training (CR + RT, n = 8). Before (PRE) and after 24 weeks (POST), blood samples were collected to measure lipid hydroperoxides concentration by spectrophotometry. No statistical difference was observed on the lipid hydroperoxides concentration between groups (PL: PRE = 48.7 ± 36.9; POST = 29.3 ± 18.8; Δ = -13.0 ± 26.8; CR : PRE = 51.0 ± 46.0; POST = 54.2 ± 51.6; Δ = -8.6 ± 30.2; + PL TR: PRE = 33.0 ± 11.2; POST = 47.3 ± 31.6, Δ = 14.3 ± 39.2; CR + TR: PRE = 18.5 ± 10.1; POST = 28.1 ± 17.9, Δ = 9.7 ± 16.4 pmol.mg-1 of total protein, p = 0.17). Creatine supplementation associated or not with strength training did not affect the lipid peroxidation, an important plasmatic marker of oxidative stress, in elderly women.

Efeitos dos treinamentos físicos aeróbicos e resistido sobre a atrofia muscular esquelética na insuficiência cardíaca / Effects of aerobic and resistance exercise training on skeletal muscle atrophy induced by heart failure

A insuficiência cardíaca é uma síndrome que envolve múltiplos sistemas e mecanismos compensatórios neuro-hormonais, acompanhada de altos índices de morbidade e mortalidade, caracterizadas por sintomas clínicos como fadiga, dispnéia e intolerância aos esforços físicos. Além do comprometimento cardíaco, observam-se importantes alterações periféricas, como a atrofia muscular esquelética, a qual contribui para o mau prognóstico e para o aumento da mortalidade dos pacientes. Estudos têm demonstrado que essa atrofia muscular pode ser causada por um desbalanço entre vias de síntese e degradação protéica, pricipalmente pelo aumento da ativação do sistema proteolítico ubiquitina-proteassoma e pela diminuição da ativação da via IGF-1/Akt. O treinamento físico, estratégia não farmacológica no tratamento da insuficiência cardíaca, tem se mostrado capaz de atenuar ou reverter o quadro de miopatia esquelética encontrado nessa síndrome. Portanto, foi objetivo da presente revisão discutir em maiores detalhes a atrofia muscular esquelética observada na insuficiência cardíaca e os efeitos dos treinamentos físicos aeróbicos e resistido sobre esta condição. Embora o treinamento físico aeróbico, de moderada ou alta intensidade, não seja conhecido por promover aumento da massa muscular esquelética, alguns estudos demonstram que este tipo de treinamento promove adaptações importantes na regulação da massa muscular na insuficiência cardíaca, prevenindo a atrofia e, consequentemente, contribuindo para a melhora da tolerância ao esforço físico. Por outro lado, o treinamento físico resistido parece ser mais eficaz em atenuar ou reverter a atrofia muscular esquelética presente na insuficiência cardíaca.

Heart failure is a complex syndrome involving multiple systems and neurohumoral compensatory mechanisms accompanied by high morbidity and mortality, and it is characterized by clinical signs such as fatigue, dyspneia, and exercise intolerance. Although heart failure is a syndrome of cardiac origin, it promotes changes in other tissues, such as skeletal muscle, where modifications of muscle phenotype and the loss of skeletal muscle mass observed in heart failure contribute to poor prognosis and increased mortality of patients. Studies have show that skeletal muscle atrophy is caused by an imbalance between the process of protein synthesis and degradation, mainly due to increased activation of ubiquitin-proteasome proteolytic system and decreased activation of IGF-1/Akt pathway. Exercise training, a non-pharmacological strategy to treat heart failure, has been shown to attenuate or reverse the skeletal myopathy in this syndrome. this review aimed to discuss in details the muscular atrophy observed in heart failure and the effects of aerobic and resistance exercise training on skeletal muscle atrophy induced by this conditions. Although aerobic exercise training, at moderate or high intensity, is not known for promoting skeletal muscle hypertrophy, some studies show that this type of exercise training promotes major changes in skeletal muscle mass in heart failure, preventing atrophy and consequently contributing to improved exercise tolerance. On the other hand, resistance exercise training appears to be more effective in attenuating or reversing the skeletal muscle atrophy in heart failure. However, the benefits promoted by aerobic exercise training in many aspects of heart failure are more established in the literature and should be considered in the treatment of this syndrome in association with resistance exercise training, in order to obtain better results.

Does Branched-Chain Amino Acids Supplementation Modulate Skeletal Muscle Remodeling through Inflammation Modulation? Possible Mechanisms of Action.

Skeletal muscle protein turnover is modulated by intracellular signaling pathways involved in protein synthesis, degradation, and inflammation. The proinflammatory status of muscle cells, observed in pathological conditions such as cancer, aging, and sepsis, can directly modulate protein translation initiation and muscle proteolysis, contributing to negative protein turnover. In this context, branched-chain amino acids (BCAAs), especially leucine, have been described as a strong nutritional stimulus able to enhance protein translation initiation and attenuate proteolysis. Furthermore, under inflammatory conditions, BCAA can be transaminated to glutamate in order to increase glutamine synthesis, which is a substrate highly consumed by inflammatory cells such as macrophages. The present paper describes the role of inflammation on muscle remodeling and the possible metabolic and cellular effects of BCAA supplementation in the modulation of inflammatory status of skeletal muscle and the consequences on protein synthesis and degradation.

Pivotal role of Toll-like receptors 2 and 4, its adaptor molecule MyD88, and inflammasome complex in experimental tubule-interstitial nephritis.

Tubule-interstitial nephritis (TIN) results in decreased renal function and interstitial inflammation, which ultimately leads to fibrosis. Excessive adenine intake can cause TIN because xanthine dehydrogenase (XDH) can convert this purine into an insoluble compound, which precipitates in the tubuli. Innate immune sensors, such as Toll-like receptors (TLR) and inflammasome complex, play a crucial role in the initiation of inflammation. The aim of this study was to evaluate the roles of TLR-2 and -4, Myd88 and inflammasome complex in an experimental model of TIN. Here, we show that wild-type (WT) mice fed adenine-enriched food exhibited significant renal dysfunction and enhanced cellular infiltration accompanied by collagen deposition. They also presented higher gene and protein expression of pro-inflammatory cytokines. In contrast, TLR-2, -4, MyD88, ASC and Caspase-1 KO mice showed renoprotection associated with expression of inflammatory molecules at levels comparable to controls. Furthermore, treatment of WT animals with allopurinol, an XDH inhibitor, led to reduced levels of uric acid, oxidative stress, collagen deposition and a downregulation of the NF-kB signaling pathway. We concluded that MyD88 signaling and inflammasome participate in the development of TIN. Furthermore, inhibition of XDH seems to be a promising way to therapeutically target the developing inflammatory process.

A single bout of moderate-intensity exercise increases vascular NO bioavailability and attenuates adrenergic receptor-dependent and -independent vasoconstrictor response in rat aorta.

The present study investigated the effect of one bout of moderate-intensity exercise on the adrenergic receptor-dependent and -independent vasoconstrictor response in rat aortas, and the role of nitric oxide (NO) bioavailability on these vasomotor responses. One group of rats was submitted to a 60 min of exercise at approximately 60% of maximal exercise capacity on a treadmill (exercise group) and the other one was placed in the treadmill without running (control group). Immediately after this period, both groups were euthanized and the thoracic aorta was removed to evaluate the vasoconstrictor response to norepinephrine and potassium chloride, and to evaluate the vascular nitrite and nitrate concentration. One bout of exercise attenuated the maximal contractile response to both norepinephrine and potassium chloride compared to control group. These differences on vascular reactivity were not observed in endothelium-denuded aortic rings and aortic rings pre-incubated with a nitric oxide synthesis inhibitor. Additionally, exercise group increased NO bioavailability (nitrite and nitrate concentration) as compared to control group. These results demonstrate that one bout of moderate-intensity exercise is able to attenuate adrenergic receptor-dependent and -independent vasoconstrictor response in rat aorta, mainly by increasing vascular NO bioavailability.

Moderate exercise training decreases aortic superoxide production in myocardial infarcted rats.

Myocardial infarction (MI) has been associated with increases in reactive oxygen species (ROS). Exercise training (ET) has been shown to exert positive modulations on vascular function and the purpose of the present study was to investigate the effect of moderate ET on the aortic superoxide production index, NAD(P)H oxidase activity, superoxide dismutase activity and vasomotor response in MI rats. Aerobic ET was performed during 11 weeks. Myocardial infarction significantly diminished maximal exercise capacity, and increased vasoconstrictory response to norepinephrine, which was related to the increased activity of NAD(P)H oxidase and basal superoxide production. On the other hand, ET normalized the superoxide production mostly due to decreased NAD(P)H oxidase activity, although a minor SOD effect may also be present. These adaptations were paralleled by normalization in the vasoconstrictory response to norepinephrine. Thus, diminished ROS production seems to be an important mechanism by which ET mediates its beneficial vascular effects in the MI condition.

Exercício físico e disfunção endotelial / Exercise and endothelial dysfunction

A disfunção endotelial apresenta papel de destaque no desenvolvimento e na progressão da aterosclerose por estar diretamente relacionada a alterações na homeostase da parede vascular e no controle da circulação. Estudos clínicos e experimentais têm evidenciado efeitos benéficos do treinamento físico...

Efeitos do treinamento físico aeróbio sobre a bioatividade do óxido nítrico e a vasodilatação aórtica / Effects of aerobic exercise training opon nitric oxide bioactivity and aorta vasodilatation

O treinamento físico aeróbio (TF) é um importante meio para melhorar a função endotelial. Entretanto, como os vasos se adaptam ao TF ainda não está completamente esclarecido. Desta forma, o presente estudo teve por objetivo investigar, em ratos normotensos, os efeitos do TF sobre a via de produção de óxido nítrico (NO) e a defesa antioxidante vascular, e suas conseqüências sobre a resposta vasodilatadora em aorta isolada. Os ratos foram submetidos a um protocolo de TF aeróbio (esteira rolante, ~55%Veloc. Máx; cinco sessões/sem., 60 min/sessão, período de 11 semanas). Após o TF, foi avaliada a função vasomotora “in vitro” pela curva de concentração-efeito à acetilcolina (ACh) e ao nitroprussiato de sódio (NPS), e realizadas medidas bioquímicas na aorta. O programa de TF aumentou significativamente (P < 0,05) em 62% a expressão da enzima óxido nítrico sintase endotelial (eNOS). Entretanto, o TF não modificou significativamente a expressão e atividade da enzima antioxidante superóxido dismutase.Além disso, o TF não modificou o relaxamento individual e a sensibilidade à ACh. Por outro lado, o TF diminuiu significativamente a sensibilidade ao NPS (-8,26 ± 0,081 vs. -7,79 ± 0,099 Log [M], S vs T, respectivamente, P < 0,001). Os resultados apresentados demonstram que o TF aeróbio foi capaz de alterar um dos mecanismos envolvidos na bioatividade do NO, marcadamente o aumento da expressãoda eNOS. Entretanto, esta modificação não levou à melhora da responsividade vasodilatadora aórtica estimulada pela acetilcolina e provocou menor sensibilidade ao NPS.

Aerobic training (AT) is an important way to improve endothelial function. However, it is not completelyunderstood how the blood vessels adapt themselves to the AT. Therefore, the aim of this study was to investigate exercise training-induced adaptations on the nitric oxide (NO) production, antioxidant defense and aorta vasodilatation in normotensive rats. The rats were subjected to an AT protocol (treadmill,~55% Max Veloc., 5 bouts/week, 60 min/bout, 11 wks). After the AT, it was examined in vitro vasomotor function to acetylcholine (ACh) and sodium nitroprusside (SNP), and biochemical analysis in the aorta. Aerobic training significantly increased (P < 0.05) by 62% the expression of the endothelial nitric oxide synthase (eNOS). However, ET did not modify the relaxation response and sensitivity to ACh. In contrast, AT significantly reduced aortic sensitivity to SNP (-8.26 ± 0.081 vs. -7.79 ± 0.099 Log [M], Sed vs. AT,respectively, P < 0.001). These results demonstrate that aerobic AT was able to modify one important mechanism related to the NO bioactivity, which is the increase of eNOS expression. However, this response did not contribute to improve of the aortic vasodilatation response to acetylcholine and decreased the sensitivity to SNP.