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.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo, Sao Paulo - SP (FAPESP) [2009/18546-4, 2010/00028-4, 2012/05765-2]Fundacao de Amparo a Pesquisa do Estado de Sao Paulo, Sao Paulo SP (FAPESP)Conselho Nacional de Pesquisa e Desenvolvimento - Brasil (CNPq) [479407/2010-0]Conselho Nacional de Pesquisa e Desenvolvimento Brasil (CNPq)Instituto Nacional de Ciencia e TecnologiaInstituto Nacional de Ciencia e TecnologiaNucleo de Apoio a Pesquisa de Processos Redox em BiomedicinaNucleo de Apoio a Pesquisa de Processos Redox em BiomedicinaFundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP) [2009/12349-2

The “Goldilocks Zoneâ€? from a redox perspectiveâ€â€�Adaptive vs. deleterious responses to oxidative stress in striated muscle

Consequences of oxidative stress may be beneficial or detrimental in physiological systems. An organ system's position on the “hormetic curve� is governed by the source and temporality of reactive oxygen species (ROS) production, proximity of ROS to moieties most susceptible to damage, and the capacity of the endogenous cellular ROS scavenging mechanisms. Most importantly, the resilience of the tissue (the capacity to recover from damage) is a decisive factor, and this is reflected in the disparate response to ROS in cardiac and skeletal muscle. In myocytes, a high oxidative capacity invariably results in a significant ROS burden which in homeostasis, is rapidly neutralized by the robust antioxidant network. The up-regulation of key pathways in the antioxidant network is a central component of the hormetic response to ROS. Despite such adaptations, persistent oxidative stress over an extended time-frame (e.g., months to years) inevitably leads to cumulative damages, maladaptation and ultimately the pathogenesis of chronic diseases. Indeed, persistent oxidative stress in heart and skeletal muscle has been repeatedly demonstrated to have causal roles in the etiology of heart disease and insulin resistance, respectively. Deciphering the mechanisms that underlie the divergence between adaptive and maladaptive responses to oxidative stress remains an active area of research for basic scientists and clinicians alike, as this would undoubtedly lead to novel therapeutic approaches. Here, we provide an overview of major types of ROS in striated muscle and the divergent adaptations that occur in response to them. Emphasis is placed on highlighting newly uncovered areas of research on this topic, with particular focus on the mitochondria, and the diverging roles that ROS play in muscle health (e.g., exercise or preconditioning) and disease (e.g., cardiomyopathy, ischemia, metabolic syndrome)

Bicarbonate/CO2 increase damage in ischemia-reperfusion injury: from observation to molecular characterization

Bicarbonato é uma importante espécie química para os seres vivos, sendo o principal tampão celular, alem de apresentar uma negligenciada atividade redox. Isquemia é um evento no qual existe inibição do aporte de nutrientes e oxigênio, sendo a reperfusão o retorno do fluxo de nutrientes e oxigênio, que é acompanhada por alta produção de radicais livres e morte celular. Nessa tese estudamos o efeito da presença de bicarbonato durante a isquemia-reperfusão. Em nosso modelo nós mantivemos o pH constante e modulamos a quantidade de bicarbonato enquanto células, órgãos e animais foram submetidos a isquemia-reperfusão. Utilizamos condições sem a presença de bicarbonato, a concentração basal sanguínea e uma concentração mais alta simulando o acúmulo de bicarbonato em condições isquêmicas. Nesses diversos modelos mostramos que a presença de bicarbonato aumenta o dano provocado por isquemia-reperfusão e provoca um aumento do acúmulo de proteínas oxidadas. A presença do bicarbonato não modifica a respiração, produção de espécies reativas de oxigênio, ou a morfologia mitocondrial, também não detectamos mudança na atividade do proteassoma e nos indicadores de autofagia geral. Entretanto detectamos um acúmulo de marcadores autofágicos na fração mitocondrial indicando inibição da mitofagia. Essa inibição foi confirmada ao detectarmos o acúmulo de uma proteína degradada especificamente por mitofagia enquanto não houve mudança em outra degradada pelo proteassoma. Além disso, ao inibirmos farmacologicamente a autofagia, reproduzimos o fenótipo causado pelo bicarbonato mesmo na sua ausência. Em conclusão, a presença de bicarbonato é deletéria em condições de isquemia/reperfusão devido a inibição da mitofagiaBicarbonate is an important molecule in all living being, acting as the main cellular buffer. However, its biological and redox activity has been mostly neglected to date. Ischemia is an event in which an inhibition of nutrient availablity and oxygen flow occurs, while reperfusion is the return of nutrients and oxygen, accompanied of a burst of reactive oxygen species production and cell death. Here, we studied the effects of bicarbonate during cardiac ischemia-reperfusion. In our model, we kept the pH stable and changed the concentration of the bicarbonate. We then subjected cells, organs and animals to ischemia-reperfusion under conditions where there was no presence, basal blood concentration or a higher concentration of bicarbonate. In these diverse models, we found that the presence of bicarbonate increased damage after a ischemia-reperfusion, and promoted the accumulation of oxidized proteins. Bicarbonate did not change respiration, production of reactive oxygen species or the morphology of the mitochondria. There were also no changes in proteasome activity and in global autophagy markers, although there was an accumulation of mitophagy markers. We also found that mitophagy was responsible for the increased damage observed, since pharmacological inhibiting of autophagy abolished the increased damage caused by the presence of bicarbonate. In conclusion the presence of bicarbonate is deleterious in ischemia-reperfusion due mitophagy inhibitio

Mitochondrial ion transport pathways: Role in metabolic diseases

Mitochondria are the central coordinators of energy metabolism and alterations in their function and number have long been associated with metabolic disorders such as obesity, diabetes and hyperlipidemias. Since oxidative phosphorylation requires an electrochemical gradient across the inner mitochondrial membrane, ion channels in this membrane certainly must play an important role in the regulation of energy metabolism. However, in many experimental settings, the relationship between the activity of mitochondrial ion transport and metabolic disorders is still poorly understood. This review briefly summarizes some aspects of mitochondrial H(+) transport (promoted by uncoupling proteins, UCPs). Ca(2+) and K(+) uniporters which may be determinant in metabolic disorders. (C) 2009 Elsevier B.V. All rights reserved

Bicarbonate Increases Ischemia-Reperfusion Damage by Inhibiting Mitophagy

<div><p>During an ischemic event, bicarbonate and CO₂ concentration increase as a consequence of O₂ consumption and lack of blood flow. This event is important as bicarbonate/CO₂ is determinant for several redox and enzymatic reactions, in addition to pH regulation. Until now, most work done on the role of bicarbonate in ischemia-reperfusion injury focused on pH changes; although reperfusion solutions have a fixed pH, cardiac resuscitation protocols commonly employ bicarbonate to correct the profound acidosis associated with respiratory arrest. However, we previously showed that bicarbonate can increase tissue damage and protein oxidative damage independent of pH. Here we show the molecular basis of bicarbonate-induced reperfusion damage: the presence of bicarbonate selectively impairs mitophagy, with no detectable effect on autophagy, proteasome activity, reactive oxygen species production or protein oxidation. We also show that inhibition of autophagy reproduces the effects of bicarbonate in reperfusion injury, providing additional evidence in support of this mechanism. This phenomenon is especially important because bicarbonate is widely used in resuscitation protocols after cardiac arrest, and while effective as a buffer, may also contribute to myocardial injury.</p></div

Autophagy markers accumulate in the mitochondrial fraction.

<p>HL-1 cells were subjected to 150 min ischemia followed by 5 min reperfusion, and cell extracts were obtained at the indicated times and probed for p62 by western blot (A). Isolated rat hearts were subjected to 30 min ischemia followed by 15 min reperfusion, and the heart homogenates were fractionated by differential centrifugation to yield cytosol (B, D, F) and mitochondria (C, E, G). The resulting fractions were probed for p62 (B and C), Beclin 1 (D and E) and Drp1 (F and G) by western blot.</p

H₂O₂ production from isolated mitochondria is not increased by bicarbonate.

<p>Mitochondria isolated from rat hearts were subjected to 0 or 10% bicarbonate; hydrogen peroxide production as a function of oxygen consumption was measured in the presence of ADP (A), ADP + Oligomycin (B) or ADP + Oligomycin + CCCP (C). Respiratory control (RC) (D) was measured as described in Materials and Methods. To examine protein oxidative modifications, mitochondria were incubated in media with Succinate (E and G), or Succinate + Antimycin (F and H) for 15 min at 37°C; the mitochondria were then pelleted and protein carbonyl (E and F) and methionine sulfoxide (G and H) content was measured by western blot.</p

Bicarbonate exacerbates ischemia/reperfusion injury.

<p>HL-1 cells were subjected to 150 min ischemia followed by 5 min of reperfusion (A); CK release to the supernatant was measured at the indicated times (B); protein carbonylation content was measured by western blot in the cell lysate before and after the 5 min reperfusion (C). Isolated rat hearts were subjected to ischemia for 30 min and 15 min reperfusion (D); CK release into the perfusate was measured during reperfusion (E); protein carbonylation content was measured by western blot at the end of reperfusion (15 min) (F).</p

Scheme representing the events that lead to increased damage in I/R and sI/R in the presence of bicarbonate.

<p>I/R causes mitochondrial oxidant production with resulting oxidative damage to macromolecules (orange glow). Outer mitochondrial membrane proteins such as TOM70 are degraded by the proteasome, and the damaged mitochondrion is separated from the network by Drp1 and marked for autophagic removal by p62. Bicarbonate interferes with mitophagy, resulting in the accumulation of oxidized proteins, functional impairment, and cell death. Bicarbonate does not affect mitochondrial oxidant production, morphology, proteasome activity, or general autophagy.</p