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Aims: Endoplasmic reticulum (ER) stress has recently emerged as an important mechanism involved in the pathogenesis of cardiovascular diseases. However, the molecular mechanisms by which ER stress leads to cardiac dysfunction remain poorly understood., Methods and Results: In this study, we evaluated the early cardiac effects of ER stress induced by tunicamycin (TN) in mice. Echocardiographic analysis indicated that TN-induced ER stress led to a significant impairment of the cardiac function. Electron microscopic observations revealed that ultrastructural changes of cardiomyocytes in response to ER stress manifested extensively at the level of the reticular membrane system. Smooth tubules of sarcoplasmic reticulum in connection with short sections of rough ER were observed. The presence of rough instead of smooth reticulum was increased at the interfibrillar space, at the level of dyads, and in the vicinity of mitochondria. At the transcriptional level, ER stress resulted in a substantial decrease in the expression of the major regulator of mitochondrial biogenesis PGC-1α and of its targets NRF1, Tfam, CS, and COXIV. At the functional level, ER stress also induced an impairment of mitochondrial Ca2+ uptake, an alteration of mitochondrial oxidative phosphorylation, and a metabolic remodelling characterized by a shift from fatty acid to glycolytic substrate consumption., Conclusions: Our findings show that ER stress induces cytoarchitectural and metabolic alterations in cardiomyocytes and provide evidences that ER stress could represent a primary mechanism that contributes to the impairment of energy metabolism reported in most cardiac diseases.

Women and men, female and male animals and cells are biologically different, and acknowledgement of this fact is critical to advancing medicine. However, incorporating concepts of sex-specific analysis in basic research is largely neglected, introducing bias into translational findings, clinical concepts and drug development. Research funding agencies recently approached these issues but implementation of policy changes in the scientific community is still limited, probably due to deficits in concepts, knowledge and proper methodology. This expert review is based on the EUGenMed project (www.eugenmed.eu) developing a roadmap for implementing sex and gender in biomedical and health research. For sake of clarity and conciseness, examples are mainly taken from the cardiovascular field that may serve as a paradigm for others, since a significant amount of knowledge how sex and oestrogen determine the manifestation of many cardiovascular diseases (CVD) has been accumulated. As main concepts for implementation of sex in basic research, the study of primary cell and animals of both sexes, the study of the influence of genetic vs. hormonal factors and the analysis of sex chromosomes and sex specific statistics in genome wide association studies (GWAS) are discussed. The review also discusses methodological issues, and analyses strength, weaknesses, opportunities and threats in implementing sex-sensitive aspects into basic research., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2017. For permissions, please email: journals.permissions@oup.com.)

Aims: Oestrogen receptor alpha (ERα) and beta (ERβ) are involved in the regulation of pathological myocardial hypertrophy (MH). We hypothesize that both ER are also involved in physiological MH. Therefore, we investigated the role of ER in exercise-induced physiological MH in loss-of-function models and studied potential mechanisms of action., Methods and Results: We performed 1 and 8 weeks of voluntary cage wheel running (VCR) with male and female C57BL/6J wild-type (WT), ERα- and ERβ-deleted mice. In line with other studies, female WT mice ran more than males (P ≤ 0.001). After 8 weeks of VCR, both sexes showed an increase in left ventricular mass (females: P ≤ 0.01 and males: P ≤ 0.05) with more pronounced MH in females (P < 0.05). As previously shown, female ERα-deleted mice run less than female WT mice (P ≤ 0.001). ERβ-deleted mice showed similar running performance as WT mice (females vs. male: P ≤ 0.001), but did not develop MH. Only female WT mice showed an increase in phosphorylation of serine/threonine kinase (AKT), ERK1/2, p38-mitogen-activated protein kinase (MAPK), and ribosomal protein s6, as well as an increase in the expression of key regulators of mitochondrial function and mitochondrial respiratory chain proteins (complexes I, III, and V) after VCR. However, ERβ deletion abolished all observed sex differences. Mitochondrial remodelling occurred in female WT-VCR mice, but not in female ERβ-deleted mice., Conclusion: The sex-specific response of the heart to exercise is modulated by ERβ. The greater increase in physiological MH in females is mediated by induction of AKT signalling, MAPK pathways, protein synthesis, and mitochondrial adaptation via ERβ., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2014. For permissions please email: journals.permissions@oup.com.)

Aims: Melusin is a muscle-specific chaperone protein whose expression is required for a compensatory hypertrophy response to pressure overload. Here, we evaluated the consequences of melusin overexpression in the setting of myocardial infarction (MI) using a comprehensive multicentre approach., Methods and Results: Mice overexpressing melusin in the heart (TG) and wild-type controls (WT) were subjected to permanent LAD ligation and both the acute response (Day 3) and subsequent remodelling (2 weeks) were examined. Mortality in wild-type mice was significant between Days 3 and 7, primarily due to cardiac rupture, but melusin's overexpression strongly reduced mortality (43.2% in wild-type vs. 27.3% in melusin-TG, P = 0.005). At Day 3 after MI, a time point preceding the mortality peak, TG hearts had increased heat shock protein 70 expression, increased ERK1/2 signalling, reduced cardiomyocyte hyper-contractility and inflammatory cell infiltrates, and increased matricellular protein expression in the infarcted area. At 2 weeks after MI, melusin overexpression conferred a favourable adaptive remodelling characterized by reduced left ventricle dilatation and better preserved contractility in the presence of a comparable degree of hypertrophy. Adaptive remodelling in melusin TG mice was characterized by reduced apoptosis and fibrosis as well as increased cardiomyocyte contractility., Conclusions: Consistent with its function as a chaperone protein, melusin overexpression exerts a dual protective action following MI reducing an array of maladaptive processes. In the early phase after MI, reduced inflammation and myocyte remodelling protect against cardiac rupture. Chronically, reduced myocyte loss and matrix remodelling, with preserved myocyte contractility, confer adaptive LV remodelling.

Aims: The optic atrophy 1 (OPA1) protein is an essential protein involved in the fusion of the mitochondrial inner membrane. Despite its high level of expression, the role of OPA1 in the heart is largely unknown. We investigated the role of this protein in Opa1(+/-) mice, having a 50% reduction in OPA1 protein expression in cardiac tissue., Methods and Results: In mutant mice, cardiac function assessed by echocardiography was not significantly different from that of the Opa1(+/+). Electron and fluorescence microscopy revealed altered morphology of the Opa1(+/-) mice mitochondrial network; unexpectedly, mitochondria were larger with the presence of clusters of fused mitochondria and altered cristae. In permeabilized mutant ventricular fibres, mitochondrial functional properties were maintained, but direct energy channelling between mitochondria and myofilaments was weakened. Importantly, the mitochondrial permeability transition pore (PTP) opening in isolated permeabilized cardiomyocytes and in isolated mitochondria was significantly less sensitive to mitochondrial calcium accumulation. Finally, 6 weeks after transversal aortic constriction, Opa1(+/-) hearts demonstrated hypertrophy almost two-fold higher (P< 0.01) than in wild-type mice with altered ejection fraction (decrease in 43 vs. 22% in Opa1(+/+) mice, P< 0.05)., Conclusions: These results suggest that, in adult cardiomyocytes, OPA1 plays an important role in mitochondrial morphology and PTP functioning. These properties may be critical for cardiac function under conditions of chronic pressure overload.

Aims: The myofibrillar and nuclear compartments in cardiomyocytes are known to be sensitive to extracellular mechanical stimuli. Recently, we have shown that alterations in the mitochondrial ionic balance in cells in situ are associated with considerably increased mitochondrial volume. Theoretically, this swelling of mitochondria could impose mechanical constraints on the myofibrils and nuclei in their vicinity. Thus, we studied whether modulation of mitochondrial volume in cardiomyocytes in situ has a mechanical effect on the myofibrillar and nuclear compartments., Methods and Results: We used the measurement of passive force developed by saponin-permeabilized mouse ventricular fibres as a sensor for compression of the myofibrils. Osmotic compression induced by dextran caused an increase in passive force. Similarly, mitochondrial swelling induced by drugs that alter ionic homeostasis (alamethicin and propranolol) markedly augmented passive force (confirmed by confocal microscopy). Diazoxide, a mitochondrial ATP-sensitive potassium channel opener known to cause moderate mitochondrial swelling, also increased passive force (by 28 +/- 5% at 10% stretch, P < 0.01). This effect was completely blocked by 5-hydroxydecanoate (5-HD), a putative specific inhibitor of these channels. Mitochondrial swelling induced by alamethicin and propranolol led to significant nuclear deformation, which was visualized by confocal microscopy. Furthermore, diazoxide decreased nuclear volume, calculated using three-dimensional reconstructed images, in a 5-HD-dependent manner by 12 +/- 2% (P < 0.05). This corresponds to an increase in intracellular pressure of 2.1 +/- 0.3 kPa., Conclusion: This study is the first to demonstrate that mitochondria are able to generate internal pressure, which can mechanically affect the morphological and functional properties of intracellular organelles.

Aims: Increased diastolic sarcoplasmic reticulum (SR) Ca(2+) loss could depress contractility in heart failure. Since the failing myocardium has impaired energetics, we investigated whether Ca(2+) loss is linked to changes in energetic pathways., Methods and Results: Leakage from SR in mouse permeabilized preparations was assessed using exogenous ATP, ATP + phosphocreatine (activation of bound creatine kinase, CK), ATP + mitochondrial substrates (mitochondrial activation), or with all of these together (optimal energetic conditions) in Ca(2+)-free solution. In ventricular fibres caffeine-induced tension transients under optimal energetic conditions were used to estimate SR [Ca(2+)]. In cardiomyocytes, intra-SR Ca(2+) was monitored by use of the fluorescent marker Mag-fluo 4. In fibres, SR Ca(2+) content after 5 min incubation strongly depended on energy supply (100%-optimal energetic conditions; 27 +/- 5%-exogenous ATP only, 52 +/- 5%-endogenous CK activation; 88 +/- 8%-mitochondrial activation, P < 0.01 vs. CK system). The significant loss with only exogenous ATP was not inhibited by the ryanodine receptor blockers tetracaine or ruthenium red. However, the SR Ca(2+)-ATPase (SERCA) inhibitors cyclopiazonic acid or 2,5-di(tert-butyl)-1,4-benzohydroquinone significantly decreased Ca(2+) loss. At 100 nM external [Ca(2+)], the SR Ca(2+) loss was also energy dependent and was not significantly inhibited by tetracaine. In cardiomyocytes, the decline in SR [Ca(2+)] at zero external [Ca(2+)] was almost two times slower under optimal energetic conditions than in the presence of exogenous ATP only., Conclusion: At low extra-reticular [Ca(2+)], the main leak pathway is an energy-sensitive backward Ca(2+) pump, and direct mitochondrial-SERCA ATP channelling is more effective in leak prevention than local ATP generation by bound CK.

Although the concept of energy starvation in the failing heart was proposed decades ago, still very little is known about the origin of energetic failure. Recent advances in molecular biology have started to elucidate the transcriptional events governing mitochondrial biogenesis. In particular, a great step was taken with the discovery that peroxisome proliferator-activated receptor gamma co-activator (PGC-1alpha) is the master regulator of mitochondrial biogenesis. The molecular mechanisms underlying the downregulation of PGC-1alpha and the consequent decrease in mitochondrial function in heart failure are, however, still poorly understood. Indeed, the main pathways involved in mitochondrial biogenesis are thought to be up- rather than down-regulated in pathological hypertrophy and heart failure. The current review summarizes recent advances in this field and is restricted to the heart when cardiac data are available.

Objective: Clinical and experimental studies demonstrate that exercise training improves aerobic capacity and cardiac function in heart failure, even in patients on optimal treatment with angiotensin inhibitors and beta-blockers, but the cellular mechanisms are incompletely understood. Since myocardial dysfunction is frequently associated with impaired energy status, the aim of this study was to assess the effects of exercise training and losartan on myocardial systems for energy production and transfer in heart failure., Methods: Maximal oxygen uptake, cardiac function and energy metabolism were assessed in heart failure after a myocardial infarction induced by coronary artery ligation in female Sprague-Dawley rats. Losartan was initiated one week after infarction and exercise training after four weeks, either as single interventions or combined. Animals were sacrificed 12 weeks after surgery., Results: Heart failure, confirmed by left ventricular diastolic pressure >15 mmHg and by >20 mmHg drop in peak systolic pressure, was associated with 40% lower aerobic capacity and significant reductions in enzymes involved in energy metabolism. Combined treatment yielded best improvement of aerobic capacity and ventricular pressure characteristics. Exercise training completely restored aerobic capacity and partly or fully restored creatine and adenylate kinases, whereas losartan alone further reduced these enzymes. In contrast, losartan reduced left ventricle diastolic pressure, whereas exercise training had a neutral effect., Conclusion: Exercise training markedly improves aerobic capacity and cardiac function after myocardial infarction, either alone or in combination with angiotensin inhibition. The two interventions appear to act by complementary mechanisms; whereas exercise training restores cardiac energy metabolism, mainly at the level of energy transfer, losartan unloads the heart by lowering filling pressure and afterload.

As endurance training improves symptoms and quality of life and decreases mortality rate and hospitalization, it is increasingly recognized as a beneficial practice for heart failure (HF) patients. However, the mechanisms involved in the beneficial effects of exercise training are far from being understood and need further evaluation. Independent of hemodynamics effects, exercise training participates in tissue remodeling. While heart failure induces a generalized metabolic myopathy, adaptation to endurance training mainly improves energetic aspects of muscle function. In the present review, after presenting the main characteristics of cardiac and skeletal muscle energy metabolism and the effects of exercise training, we will discuss the evidence for the beneficial effects of endurance training on cardiac and skeletal muscle oxidative metabolism and intracellular energy transfer in HF. These beneficial effects of exercise training seen in heart failure patients are also relevant to other chronic diseases (chronic obstructive pulmonary disease, diabetes, and obesity) and even for highly sedentary or elderly individuals [Booth F.W., Chakravathy M.V., Spangenburg E.E. Exercise and gene expression: physiological regulation of the human genome through physical activity. J Physiol (Lond) 2002;543:399-411]. Physical rehabilitation is thus a major health issue for populations in industrialized countries.

Objective: Mitochondrial function and metabolic profile of slow and fast skeletal muscles and cardiac muscle are altered in chronic heart failure (CHF), suggesting a generalized metabolic myopathy in this disease. The aim of this study was to investigate the potential beneficial effects of voluntary activity on cardiac and skeletal muscle energetics in heart failure., Methods: Heart failure was induced in rats by aortic stenosis. Four months after surgery, part of sham and CHF animals were randomly assigned to activity cages equipped with running wheels for 8 weeks or kept sedentary. Mitochondrial capacity and regulation were measured using saponin skinned fibers in left ventricle, slow and fast skeletal muscles, and metabolic and myosin profiles were established., Results: Despite four times lower performances of CHF rats, alterations in metabolic and myosin parameters (oxidative capacity, mitochondrial enzymes, cytosolic and mitochondrial creatine kinase, myosin heavy chains) observed in all muscles of CHF animals were almost fully restored in soleus muscle though unchanged in heart and fast skeletal muscles., Conclusions: These results show the powerful beneficial effect of physical activity specifically on active slow oxidative skeletal muscle in CHF, without the worsening of cardiac muscle metabolism.

Objective: To elucidate the role of bound creatine kinase in adenine nucleotide compartmentation in myofibrils, the effects of this enzyme's substrates and products on rigor tension were studied in using isolated skinned rat cardiomyocytes rather than fibers, to avoid restrictions due to concentration gradients within the multicellular preparations., Methods: A new experimental set-up was built to allow continuous and stable measurements of force developed by cells. Triton X-100-treated cardiomyocytes were glued between a glass holder and the needle of a galvanometer. A feedback system allowed the precise measurement of force by recording the coil current necessary to prevent movement of the needle., Results: At very low [Ca2+] (pCa 7), as MgATP level decreased, rigor tension appeared. In the absence of phosphocreatine (PCr), this tension started to rise at MgATP concentrations several times higher than in the presence of 12 mM PCr. In the absence of PCr, the pMgATP/tension curves of single cells usually had a complicated relationship which could not be analyzed by a simple Hill equation. In the absence of PCr, 250 microM MgADP strongly potentiated rigor tension development in the 1 mM-3 microM range of [MgATP]; at 100 microM MgATP, in the presence of MgADP, the tension was 4.6 times higher than in the absence of MgADP. Addition of 12 mM PCr immediately eliminated rigor. Finally, in the presence of 100 microM MgATP and 250 microM MgADP, a decrease in PCr resulted in rigor; the half-maximal contracture being recorded at 1 mM PCr., Conclusions: These results indicate a myofibrillar compartmentation of adenine nucleotides influenced by bound creatine kinase, since at equal MgATP concentrations in extramyofibrillar milieu the response of myofibrils strongly depends on the presence of PCr. Local accumulation of ADP in myofibrils due to a fall in cellular PCr and inability of myofibrillar creatine kinase to rephosphorylate ADP produced by myosin ATPase could be an important mechanism of diastolic tension rise in ischaemic conditions.