Impaired cardiac and skeletal muscle bioenergetics in children, adolescents, and young adults with Barth syndrome

Barth syndrome (BTHS) is an X‐linked condition characterized by altered cardiolipin metabolism and cardioskeletal myopathy. We sought to compare cardiac and skeletal muscle bioenergetics in children, adolescents, and young adults with BTHS and unaffected controls and examine their relationships with cardiac function and exercise capacity. Children/adolescents and young adults with BTHS (n = 20) and children/adolescent and young adult control participants (n = 23, total n = 43) underwent (31)P magnetic resonance spectroscopy ((31)P‐MRS) of the lower extremity (calf) and heart for estimation of skeletal muscle and cardiac bioenergetics. Peak exercise testing (VO (2peak)) and resting echocardiography were also performed on all participants. Cardiac PCr/ATP ratio was significantly lower in children/adolescents (BTHS: 1.5 ± 0.2 vs. Control: 2.0 ± 0.3, P < 0.01) and adults (BTHS: 1.9 ± 0.2 vs. Control: 2.3 ± 0.2, P < 0.01) with BTHS compared to Control groups. Adults (BTHS: 76.4 ± 31.6 vs. Control: 35.0 ± 7.4 sec, P < 0.01) and children/adolescents (BTHS: 71.5 ± 21.3 vs. Control: 31.4 ± 7.4 sec, P < 0.01) with BTHS had significantly longer calf PCr recovery (τ PCr) postexercise compared to controls. Maximal calf ATP production through oxidative phosphorylation (Qmax‐lin) was significantly lower in children/adolescents (BTHS: 0.5 ± 0.1 vs. Control: 1.1 ± 0.3 mmol/L per sec, P < 0.01) and adults (BTHS: 0.5 ± 0.2 vs. Control: 1.0 ± 0.2 mmol/L sec, P < 0.01) with BTHS compared to controls. Blunted cardiac and skeletal muscle bioenergetics were associated with lower VO(2peak) but not resting cardiac function. Cardiac and skeletal muscle bioenergetics are impaired and appear to contribute to exercise intolerance in BTHS

At the heart of the matter:imaging cardiac metabolism in insulin resistance

Overweight increases the risk of cancer, fatty liver disease, cardiovascular disease, and the development of diabetes. It is known that diabetes can lead to heart failure (diabetic cardiomyopathy). In prediabetes, a pre-stage of diabetes, the metabolism of the heart and therefore heart function may already be hampered. Using imaging techniques, changes in cardiac metabolism, energy status and cardiac function can be analyzed in patients with prediabetes. Although the energy status (PCr/ATP ratio) measured by a 31P-MRS scan did not appear to reflect mitochondrial function, energy status was related to cardiac function in prediabetes. Thereby, energy status was found to be already reduced in prediabetes, which proves that the heart in patients with prediabetes is already vulnerable to develop heart failure. It was also shown in this thesis that medication can influence the metabolism of the heart in prediabetes, without adverse effects on heart function or insulin sensitivity

Investigating the protective role of the natural hormone Melatonin, in reducing drug-induced cardiotoxicity in the therapy of chronic diseases

Heart failure (HF) is a highly complex disorder and a major end-point of cardiovascular diseases (CVD). The pathogenesis of HF is mostly unresolved but involves interplay between cardiac structural and electrical remodelling, metabolic alterations, cell death and altered gene expression. Mitochondrial dysfunction and HF are common complications of chronic treatment from diverse groups of drugs, in particular anticancer drugs such as doxorubicin (DOX). Treatment of animals and cardiomyocytes with cardiotoxic chemicals such as β-adrenergic receptor agonists (such as isoproterenol) induces cardiac dysfunction and HF. Previous work done by the group have identified the pineal hormone melatonin was protective against stress-induced cardiac arrhythmias and simulated heart failure in cardiomyocytes in vitro. Melatonin synthesis is also dramatically decreased with age and in patients with CVD. The aim of the present project was to better understand the pathogenesis of druginduced cardiac dysfunction and delineate the role of melatonin in cardioprotection in H9c2, a model rat cell line in vitro. Using the Seahorse XF analyser method, it was demonstrated that commonly used medication for chronic diseases such as amiodarone, amitriptyline, and statins all caused altered mitochondrial dysfunction. In addition, cardiotoxic chemicals (isoproterenol, hydrogen peroxide, DOX) altered oxidative phosphorylation and glycolysis in living cardiomyocyte-derived H9c2 cells; these deleterious metabolic changes were ameliorated by melatonin. Flowcytometry and Alamar Blue staining methods demonstrated that DOX robustly induced apoptosis in H9c2 cells (~30%) which was reversed by melatonin. Doxorubicin-induced stress in H9c2 cells dramatically altered gene expression in several key signalling pathways integral in cardiac function and disease. These included mitochondrial metabolism (UCP2, PPARɣ, Drp1, Mfn1, Parp 1, Parp2, Sirt3 and Cav3), apoptosis (Bcl2 and Bcl-xL), cardiac electrophysiology and arrhythmia (Scn5a, SERCA2a), calcium handling (SERCA2a) and cardiac remodelling (Myh7, ms1). Melatonin pre-treatment attenuated or completely blocked this DOX-induced alteration in gene expression in cardiomyocytes. In conclusion, the present result demonstrated for the first time that melatonin is cardioprotective against drug-induced cardiotoxicity and apoptosis via modifying diverse heart failure-related signalling pathways. This provides novel insight on the possible use of melatonin as an adjunct intervention in several therapies including anti-cancer

Investigating the cardiac health of poultry suffering from wooden breast myopathy

Årsak og molekylære mekanismer til wooden breast (WB) i kyllinger er fortsatt ikke godt forstått, men studier viser at det ofte utvikler seg til fibrose i skjelettmuskelen. Hos mennesker fører muskeldystrofier som Duchenne muskeldystrofi (DMD) ofte til kardiomyopati og muskel fibrose. Om WB affekterte kyllinger utvikler kardiomyopati er fortsatt uklart. Hovedmålet med denne studien var å undersøke om hjertemuskelen til WB affekterte Ross 308 kyllinger har samme molekylære fingeravtrykk for fibrose som skjelettmuskelen. Prøver av hjerter til Ross 308 hannkyllinger med mild og alvorlig WB ble tatt ut. Hjerteprøvene ble så kuttet og ventrikkel området ble brukt til analyser av myokardfibrose, da fibrose ofte akkumulerer i dette området ved hjertesykdom. En del av hjerteventrikkelen ble brukt til RNA og proteinisolering, og den andre delen til spektroskopiske analyser. Kvantitativ real-time PCR (qPCR), RNA-Sekvensering (RNA-Seq) og western blotting ble brukt til å analysere genuttrykk og proteiner. NIR og Raman ble brukt til å analysere spektroskopisk profil (fysisk fingeravtrykk) av vann, fett og protein (kollagen) avsetning i prøvene. Ingen tegn til endring i genekspresjon av fibrose markører som ekstracellulære matriksproteiner som kollagen, små-leucine rike proteoglykaner (SLRPs) og kollagen kryssbindingsenzym lysyl-oksidase (LOX) ble observert på mRNA nivå i alvorlig WB sammenlignet med mild WB. Raman-analysen detekterte ikke en endring i spektral topper av kollagen, som støtter genekspresjonen av kollagen. Genekspresjonen av vevsinhibitor av metalloproteinase 2 (TIMP2) var signifikant nedregulert i alvorlige WB-prøver, mens derimot på protein nivå ved western blot analyser ble denne endringen ikke observert. Videre, var ble ingen forskjeller i genekspresjon av syndekaner som er kjente regulatorer av myokardfibrose, observert mellom mild og alvorlig WB affekterte kyllinger. Ingen forskjeller i genekspresjon av kardiomyocytt markører eller cytoskjelett markører ble funnet. Gensett ontologi berikelsesanalyse (GSEA) av biologiske prosesser påviste at mitokondrielt genuttrykk, oksidative fosforyleringen og ATP-elektrontransportkjeden var betydelig nedregulert i alvorlige WB-prøver. Protein uttrykket av cytokrom c (Cyt c) ble så analysert ved western og ble funnet signifikant økt i alvorlige WB prøver. En mindre komparativ analyse av kyllinger ble utført mellom to kyllingeraser i tillegg: Hubbard JA787, en saktevoksende rase og Ross 308, en rasktvoksende rase, da rask vekst har blitt koblet til WB myopati i kylling. Når vi sammenlignet hjertene av milde WB affekterte kyllinger av Ross 308 med hjertene av milde WB affekterte til Hubbard JA787, kunne vi se klare forskjeller kjemisk og fysisk sammensetning fra NIR og Raman, og annerledes protein mønster fra SDS-gel elektroforese og Coomassie farging. MMP2 protein ekspresjonen var høyere i Ross 308, mens Cyt c og kardialt Troponin T (cTnT) var like mellom mild WB gruppene av de to hybridene. I tillegg, fra visuell observasjon, Hubbard JA787 hjertene viste fett infiltrasjon i deres høyre og venstre ventrikkel. På en annen, side ble ingen fibrose karakteristikker eller mitokondriell dysfunksjon funnet i Hubbard gruppene av mild og alvorlig WB individer basert på NIR, Raman og western blot analyser. I konklusjon, resultatene indikerte ikke progresjon til fibrose i Ross 308 WB affekterte kylling hjerter. Derimot, tegn til mitokondriell dysfunksjon ble observert, som mulig kan lede til progresjon av hjerte feil. Fett infiltrasjon i Hubbard JA787 kan indikere hjerteproblemer

Reduced Coupling of Oxidative Phosphorylation In Vivo Precedes Electron Transport Chain Defects Due to Mild Oxidative Stress in Mice

Oxidative stress and mitochondrial function are at the core of many degenerative conditions. However, the interaction between oxidative stress and in vivo mitochondrial function is unclear. We used both pharmacological (2 week paraquat (PQ) treatment of wild type mice) and transgenic (mice lacking Cu, Zn-superoxide dismutase (SOD1−/−)) models to test the effect of oxidative stress on in vivo mitochondrial function in skeletal muscle. Magnetic resonance and optical spectroscopy were used to measure mitochondrial ATP and oxygen fluxes and cell energetic state. In both models of oxidative stress, coupling of oxidative phosphorylation was significantly lower (lower P/O) at rest in vivo in skeletal muscle and was dose-dependent in the PQ model. Despite this reduction in efficiency, in vivo mitochondrial phosphorylation capacity (ATPmax) was maintained in both models, and ex vivo mitochondrial respiration in permeabilized muscle fibers was unchanged following PQ treatment. In association with the reduced P/O, PQ treatment led to a dose-dependent reduction in PCr/ATP ratio and increased phosphorylation of AMPK. These results indicate that oxidative stress uncouples oxidative phosphorylation in vivo and results in energetic stress in the absence of defects in the mitochondrial electron transport chain

Lipotoxicity in type 2 diabetic cardiomyopathy

As obesity and type 2 diabetes are becoming an epidemic in westernized countries, the incidence and prevalence of obesity- and diabetes-related co-morbidities are increasing. In type 2 diabetes ectopic lipid accumulation in the heart has been associated with cardiac dysfunction and apoptosis, a process termed lipotoxicity. Since cardiovascular diseases are the main cause of death in diabetic patients, diagnosis and treatment become increasingly important. Although ischaemic heart disease is a major problem in diabetes, non-ischaemic heart disease (better known as diabetic cardiomyopathy) becomes increasingly important with respect to the impairment of cardiac function and mortality in type 2 diabetes. The underlying aetiology of diabetic cardiomyopathy is incompletely understood but is beginning to be elucidated. Various mechanisms have been proposed that may lead to lipotoxicity. Therefore, this review will focus on the mechanisms of cardiac lipid accumulation and its relation to the development of cardiomyopathy

Age-related transcriptional changes in gene expression in different organs of mice support the metabolic stability theory of aging

Individual differences in the rate of aging are determined by the efficiency with which an organism transforms resources into metabolic energy thus maintaining the homeostatic condition of its cells and tissues. This observation has been integrated with analytical studies of the metabolic process to derive the following principle: The metabolic stability of regulatory networks, that is the ability of cells to maintain stable concentrations of reactive oxygen species (ROS) and other critical metabolites is the prime determinant of life span. The metabolic stability of a regulatory network is determined by the diversity of the metabolic pathways or the degree of connectivity of genes in the network. These properties can be empirically evaluated in terms of transcriptional changes in gene expression. We use microarrays to investigate the age-dependence of transcriptional changes of genes in the insulin signaling, oxidative phosphorylation and glutathione metabolism pathways in mice. Our studies delineate age and tissue specific patterns of transcriptional changes which are consistent with the metabolic stability–longevity principle. This study, in addition, rejects the free radical hypothesis which postulates that the production rate of ROS, and not its stability, determines life span

Biochemical adaptations in cardiac hypertrophy

Cardiac hypertrophy is the adaptive response of the heart to chronic overload. The metabolic adaptations that occur during hypertrophy are initially beneficial, but can ultimately deteriorate into heart failure. The mechanisms underlying this are unknown. Evidence of impaired energy reserve, which may be caused by changes in the profile of substrate use, has been implicated in the transition of compensatory hypertrophy to heart failure. The work of this thesis characterises the alterations in substrate utilisation that occur in the heart, secondary to pressure-overload induced cardiac hypertrophy, the their implications on heart function.Pressure-overload hypertrophy was induced surgically in male Sprague- Dawley rats by inter-renal ligation. 13C-NMR spectroscopy was performed on extracts from hypertrophied and control hearts perfused with 13C-labelled substrate mixtures to determine the profile of substrate utilisation. Nine weeks pressure- overload achieved a moderate hypertrophy, evidenced by a 10-15% increase in heart mass to tibia length. The hypertrophied hearts showed an increased reliance on glucose and endogenous substrate contribution to TCA cycle oxidation for the production of ATP (15.0% versus 11.0%) compared to control hearts.Prolonged fifteen weeks pressure-overload resulted in further metabolic changes including impaired long-chain fatty acid oxidation and the accumulation of long-chain acylcarnitines. Alteration in substrate utilisation preceded any change in heart function and is strong evidence to suggest that impaired substrate delivery at the level of the mitochondria in cardiac hypertrophy plays an important role in the development of heart failure and is not a secondary phenomenon. At high workloads both hypertrophied and control hearts, showed similar profiles of substrate use, with glucose being the predominant substrate utilised for TCA cycle oxidation. At high workloads, hypertrophied hearts initially exhibited significantly higher mechanical function, but was not sustained, suggesting that physiological changes were becoming detrimental. This study highlights that sequential metabolic adaptations occur during the development of hypertrophy and precede any functional abnormality, providing potential prognostic markers

The Reproducibility of 31-Phosphorus MRS Measures of Muscle Energetics at 3 Tesla in Trained Men

Magnetic resonance spectroscopy (MRS) provides an exceptional opportunity for the study of in vivo metabolism. MRS is widely used to measure phosphorus metabolites in trained muscle, although there are no published data regarding its reproducibility in this specialized cohort. Thus, the aim of this study was to assess the reproducibility of (31)P-MRS in trained skeletal muscle

Evaluating changes in reversible cysteine oxidation of cardiac proteins as metabolic syndrome develops into cardiovascular disease

Oxidative stress is commonly associated with diet-induced metabolic syndrome (MetS) and left ventricular cardiac remodeling, but much remains unknown about the role of redox signaling, sensors, and switches in mediating the effects of high fat and sugar intake. In this work, I describe and apply an optimized method for quantifying changes in reversible protein-cysteine oxidation in the heart. This method uses isobaric tagging of cysteine thiols and mass spectrometry in a modified biotin switch on whole tissue lysate. Analyzing the resulting data with systems biology approaches helped delineate redox pathways playing a role in disease development, while cysteine-specificity provided exact targets for mutation-based mechanistic studies. Initial findings in a mouse model for MetS, wherein C57Bl6J mice were fed a high fat/high sucrose diet, identified energy pathways as the primary target of changing reversible cysteine oxidation. In follow-up studies, our collaborators helped validate the pathophysiological role of two particular cysteines in complex II; their early reversible oxidation and later irreversible oxidation contributed to decreased ATP output from cardiac mitochondria. A subsequent, more robust study revealed a weakness in our original method. While investigating the role of hydrogen peroxide-induced oxidative post-translational modifications (OPTMs) in the development of MetS sequelae, analysis of four mouse groups, each with an n=5, revealed that measurements of reversibly oxidized cysteine thiols were highly variable compared to those of all available thiols. Thus, I developed a strategy to address the source of variability and, in the process, improved many additional steps in the switch protocol. Finally, in an effort to clarify the role of the most stable reversible OPTM, glutathionylation (RSSG), we characterized the HFHS diet response in mice engineered to have more or less RSSG via genetic manipulation of glutaredoxin-1 expression. Those with more RSSG suffered worsened cardiac function, making them an ideal model for future studies with the methods optimized in this work. Studying the progression from poor diet to cardiac involvement in these and other mouse models using the methods described herein will aid in the design of diagnostics and targeted therapies against the growing burden of metabolic CVD