Friedreich's Ataxia Cardiomyopathy Research Articles

Comparative multi-omic analyses of cardiac mitochondrial stress in three mouse models of frataxin deficiency.

Cardiomyopathy is often fatal in Friedreich ataxia (FA). However, FA hearts maintain adequate function until advanced disease stages, suggesting initial adaptation to the loss of frataxin (FXN). Conditional cardiac knockout mouse models of FXN show transcriptional and metabolic profiles of the mitochondrial integrated stress response (ISRmt), which could play an adaptive role. However, the ISRmt has not been investigated in models with disease-relevant, partial decrease in FXN. We characterized the heart transcriptomes and metabolomes of three mouse models with varying degrees of FXN depletion: YG8-800, KIKO-700 and FXNG127V. Few metabolites were changed in YG8-800 mice, which did not provide a signature of cardiomyopathy or ISRmt; several metabolites were altered in FXNG127V and KIKO-700 hearts. Transcriptional changes were found in all models, but differentially expressed genes consistent with cardiomyopathy and ISRmt were only identified in FXNG127V hearts. However, these changes were surprisingly mild even at advanced age (18 months), despite a severe decrease in FXN levels to 1% of those of wild type. These findings indicate that the mouse heart has low reliance on FXN, highlighting the difficulty in modeling genetically relevant FA cardiomyopathy.

PO-01-126 SEXUAL DIMORPHISM AND CARDIAC ARRHYTHMIAS IN A MOUSE MODEL OF FRIEDREICH'S ATAXIA: WHAT DO WE KNOW?

Friedreich ataxia (FA) is a monogenic recessive disorder caused by the reduction of the mitochondrial protein, frataxin (FXN). Despite neurological deficits place patients with FA in the wheelchairs, most patients die from lethal cardiomyopathy in their 40s. FA patients develop progressive cardiac disease with characteristics similar to primary hypertrophic cardiomyopathy but with more progressive cardiac fibrosis, both diastolic and systolic dysfunction, and arrhythmias. However, the mechanisms leading to cardiomyopathy and arrhythmias in FA patients are poorly understood. To characterize electrical signal propagation and cardiac function in a mouse model which imitates the late-stage cardiomyopathy in FA patients. Cardiac-specific McK-Cre FXN knockout (FXN cKO) mice were used as a model of severe cardiomyopathy which is characteristic for the late-stage of FA. Due to premature death, animals of either sex at 8-weeks of age were used to assess cardiac function in-vivo by echocardiography and surface electrocardiogram (ECG). We found that FXN cKO mice developed idiosyncratic arrhythmias with varying time durations for beat-to-beat (RR) intervals and heart rate variability (HRV) parameters such as root mean squared successive differences (RMSSD, p<0.0358), revealing increased vagal activity. Mice presented extended P-wave (p<0.0113) and QTc intervals (>150 ms, p<0.0015), suggesting impaired atrial and left-ventricular function, resembling human patients. HRV frequency parameters showed increased low-frequency (0.023), high-frequency (p<0.264) and total (p<0.0326) power. Non-linear parameters showed varying short-term variation (SD1, p<0.019), increased long-term variation (SD2, p<0.0017) and ellipse area values (S, p<0.0074). Poincare plots of FXN cKO males included multiple clustering of RR and RR+1 identities while females generally sustained a single clustered ellipse (area, p<0.028). Similarly, QTc intervals were increased mostly in FXN cKO males. Contractile dysfunction was also more severe in FXN cKO males as evident by reduction in left ventricular fractional shortening, ejection fraction, stroke volume and cardiac output. Our study revealed sexual dimorphism and significant impairment in electrical and contractile function in FXN cKO males compared to females. A detailed characterization of impaired electrical signals in FA hearts will build a platform for drug testing to treat lethal cardiomyopathy in FA.

Gene Therapy Hits Its Stride in the Clinic

Gene Therapy Hits Its Stride in the Clinic

Cellular pathophysiology of Friedreich's ataxia cardiomyopathy

Friedreich's ataxia (FRDA) is a hereditary neuromuscular disorder. Cardiomyopathy is the leading cause of premature death in FRDA. FRDA cardiomyopathy is a complex and progressive disease with no cure or treatment to slow its progression. At the cellular level, cardiomyocyte hypertrophy, apoptosis and fibrosis contribute to the cardiac pathology. However, the heart is composed of multiple cell types and several clinical studies have reported the involvement of cardiac non-myocytes such as vascular cells, autonomic neurons, and inflammatory cells in the pathogenesis of FRDA cardiomyopathy. In fact, several of the cardiac pathologies associated with FRDA including cardiomyocyte necrosis, fibrosis, and arrhythmia, could be contributed to by a diseased vasculature and autonomic dysfunction. Here, we review available evidence regarding the current understanding of cellular mechanisms for, and the involvement of, cardiac non-myocytes in the pathogenesis of FRDA cardiomyopathy.

Mechanisms of impaired mitochondrial homeostasis and NAD+ metabolism in a model of mitochondrial heart disease exhibiting redox active iron accumulation

Due to the high redox activity of the mitochondrion, this organelle can suffer oxidative stress. To manage energy demands while minimizing redox stress, mitochondrial homeostasis is maintained by the dynamic processes of mitochondrial biogenesis, mitochondrial network dynamics (fusion/fission), and mitochondrial clearance by mitophagy. Friedreich's ataxia (FA) is a mitochondrial disease resulting in a fatal hypertrophic cardiomyopathy due to the deficiency of the mitochondrial protein, frataxin. Our previous studies identified defective mitochondrial iron metabolism and oxidative stress potentiating cardiac pathology in FA. However, how these factors alter mitochondrial homeostasis remains uncharacterized in FA cardiomyopathy. This investigation examined the muscle creatine kinase conditional frataxin knockout mouse, which closely mimics FA cardiomyopathy, to dissect the mechanisms of dysfunctional mitochondrial homeostasis. Dysfunction of key mitochondrial homeostatic mechanisms were elucidated in the knockout hearts relative to wild-type littermates, namely: (1) mitochondrial proliferation with condensed cristae; (2) impaired NAD+ metabolism due to perturbations in Sirt1 activity and NAD+ salvage; (3) increased mitochondrial biogenesis, fusion and fission; and (4) mitochondrial accumulation of Pink1/Parkin with increased autophagic/mitophagic flux. Immunohistochemistry of FA patients' heart confirmed significantly enhanced expression of markers of mitochondrial biogenesis, fusion/fission and autophagy. These novel findings demonstrate cardiac frataxin-deficiency results in significant changes to metabolic mechanisms critical for mitochondrial homeostasis. This mechanistic dissection provides critical insight, offering the potential for maintaining mitochondrial homeostasis in FA and potentially other cardio-degenerative diseases by implementing innovative treatments targeting mitochondrial homeostasis and NAD+ metabolism.

An unusual combination of large Eustachian valve in a young patient with Friedreich's ataxia cardiomyopathy.

An unusual combination of large Eustachian valve in a young patient with Friedreich's ataxia cardiomyopathy.

Stress-Induced Mouse Model of the Cardiac Manifestations of Friedreich's Ataxia Corrected by AAV-mediated Gene Therapy.

Friedreich's ataxia (FA), an autosomal recessive disorder caused by a deficiency in the expression of frataxin (FXN), is characterized by progressive ataxia and hypertrophic cardiomyopathy. Although cardiac dysfunction is the most common cause of mortality in FA, the cardiac disease remains subclinical for most of the clinical course because the neurologic disease limits muscle oxygen demands. Previous FXN knockout mouse models exhibit fatal cardiomyopathy similar to human FA, but in contrast to the human condition, untreated mice become moribund by 2 months of age, unlike humans where the cardiac disease often does not manifest until the third decade. The study was designed to create a mouse model for early FA disease relevant to the time for which a gene therapy would likely be most effective. To generate a cardiac-specific mouse model of FA cardiomyopathy similar to the human disease, we used a cardiac promoter (αMyhc) driving CRE recombinase cardiac-specific excision of FXN exon 4 to generate a mild, cardiac-specific FA model that is normal at rest, but exhibits the cardiac phenotype with stress. The hearts of αMyhc mice had decreased levels of FXN and activity of the mitochondrial complex II/complex IV respiratory chain. At rest, αMyhc mice exhibited normal cardiac function as assessed by echocardiographic assessment of ejection fraction and fractional shortening, but when the heart was stressed chemically with dobutamine, αMyhc mice compared with littermate control mice had a 62% reduction in the stress ejection fraction (p < 2 × 10-4) and 71% reduction in stress-related fractional shortening (p < 10-5). When assessing functional cardiac performance using running on an inclined treadmill, αMyhc mice stayed above the midline threefold less than littermate controls (p < 0.02). A one-time intravenous administration of 1011 genome copies of AAVrh.10hFXN, an adeno-associated virus (AAV) serotype rh10 gene transfer vector expressing human FXN, corrected the stress-induced ejection fraction and fractional shortening phenotypes. Treated αMyhc mice exhibited exercise performance on a treadmill indistinguishable from littermate controls (p > 0.07). These αMyhc mice provide an ideal model to study long-term cardiac complications due to FA and AAV-mediated gene therapy correction of stress-induced cardiac phenotypes typical of human FA.

Is left ventricular longitudinal strain a good prognostic factor in Friedreich ataxia?

Friedreich ataxia (FRDA) is a rare genetic ataxia. The causal mutation is an expanded trinucleotide repeat (GAA) in the frataxin gene. Hypertrophic cardiomyopathy in FRDA is the major cause of early death. Patients with progressive decline of the left ventricular ejection fraction (LVEF) have the worst prognosis. The aim of the study was to evaluate the prognostic value of 2D global longitudinal strain (GLS) compared to LVEF in FRDA patients. From 2003 to 2017 consecutive patients with genetically confirmed FRDA were included. GLS was retrospectively performed. News was obtained for all patients until April 2018. The study included 156 patients of 35 ± 12 years (mean ± SD) with an age at disease onset of 17 ± 11 y and GAA repeat on the shorter allele of 590 ± 241 pb. The following echocardiographic parameters were studied: LVEF 64 ± 9%, GLS -19.8 ± 5% ( n = 141), septal wall thickness (SWT) 11.4 ± 2.5 mm, posterior wall thickness (PWT) 10.4 ± 1.8 mm, LV end diastolic diameter (LVEDD) 44.4 ± 6 mm. Correlation between GLS and LVEF was 0.31 ( P = 0.0002). After a mean follow-up of 7.7 ± 4.0 y, 17 (11%) patients died and the outcome (cardiac arrhythmia, heart failure, stroke or death) concerned 28 (18%) patients. In univariate analysis (Cox model), factors associated with mortality were: GLS (HR:1.2;95%CI 1.10–1.32, P = 0.0001), LVEF (HR:0.88;95%CI 0.85–0.92, P < 0.0001), GAA (HR:1.28;95%CI 1.11–1.47, P = 0.0008), age at onset (HR:0.84;95%CI 0.76–0.94, P = 0.002), LVMi (HR:1.02;95%CI 1.01–1.04, P = 0.0078), SWT (HR:1.18%CI 1.01–1.36, P = 0.03) and LVEDD (HR:1.09;95%CI 1.00–1.19, P = 0.04). In multivariate analysis LVEF was the only independent predictor of long-term mortality (HR:0.93;95%CI 0.88;099, P = 0.02). GLS was also an independent predictor of the composite outcome in multivariate analysis. GLS is a predictor of morbimortality but is not superior to LVEF in FRDA patients.

Calcium Deregulation: Novel Insights to Understand Friedreich's Ataxia Pathophysiology.

Friedreich’s Ataxia (FRDA) is a neurodegenerative disorder, characterized by degeneration of dorsal root ganglia, cerebellum and cardiomyopathy. Heart failure is one of the most common causes of death for FRDA patients. Deficiency of frataxin, a small mitochondrial protein, is responsible for all clinical and morphological manifestations of FRDA. The focus of our study was to investigate the unexplored Ca2+ homeostasis in cerebellar granule neurons (CGNs) and in cardiomyocytes of FRDA cellular models to understand the pathogenesis of degeneration. Ca2+ homeostasis in neurons and cardiomyocytes is not only crucial for the cellular wellbeing but more importantly to generate action potential in both neurons and cardiomyocytes. By challenging Ca2+ homeostasis in CGNs, and in adult and neonatal cardiomyocytes of FRDA models, we have assessed the impact of frataxin decrease on both neuronal and cardiac physiopathology. Interestingly, we have found that Ca2+ homeostasis is altered both cell types. CGNs showed a Ca2+ mishandling under depolarizing conditions and this was also reflected in the endoplasmic reticulum (ER) content. In cardiomyocytes we found that the sarcoplasmic reticulum (SR) Ca2+ content was pathologically reduced, and that mitochondrial Ca2+ uptake was impaired. This phenomenon is due to the excess of oxidative stress under FRDA like conditions and the consequent aberrant modulation of key players at the SR/ER and mitochondrial level that usually restore the Ca2+ homeostasis. Our findings demonstrate that in both neurons and cardiomyocytes the decreased Ca2+ level within the stores has a comparable detrimental impact in their physiology. In cardiomyocytes, we found that ryanodine receptors (RyRs) may be leaking and expel more Ca2+ out from the SR. At the same time mitochondrial uptake was altered and we found that Vitamin E can restore this defect. Moreover, Vitamin E protects from cell death induced by hypoxia-reperfusion injury, revealing novel properties of Vitamin E as potential therapeutic tool for FRDA cardiomyopathy.

Nicotinamide mononucleotide requires SIRT3 to improve cardiac function and bioenergetics in a Friedreich's ataxia cardiomyopathy model.

Increasing NAD+ levels by supplementing with the precursor nicotinamide mononucleotide (NMN) improves cardiac function in multiple mouse models of disease. While NMN influences several aspects of mitochondrial metabolism, the molecular mechanisms by which increased NAD+ enhances cardiac function are poorly understood. A putative mechanism of NAD+ therapeutic action exists via activation of the mitochondrial NAD+-dependent protein deacetylase sirtuin 3 (SIRT3). We assessed the therapeutic efficacy of NMN and the role of SIRT3 in the Friedreich's ataxia cardiomyopathy mouse model (FXN-KO). At baseline, the FXN-KO heart has mitochondrial protein hyperacetylation, reduced Sirt3 mRNA expression, and evidence of increased NAD+ salvage. Remarkably, NMN administered to FXN-KO mice restores cardiac function to near-normal levels. To determine whether SIRT3 is required for NMN therapeutic efficacy, we generated SIRT3-KO and SIRT3-KO/FXN-KO (double KO [dKO]) models. The improvement in cardiac function upon NMN treatment in the FXN-KO is lost in the dKO model, demonstrating that the effects of NMN are dependent upon cardiac SIRT3. Coupled with cardio-protection, SIRT3 mediates NMN-induced improvements in both cardiac and extracardiac metabolic function and energy metabolism. Taken together, these results serve as important preclinical data for NMN supplementation or SIRT3 activator therapy in Friedreich's ataxia patients.

Progressive mitochondrial protein lysine acetylation and heart failure in a model of Friedreich's ataxia cardiomyopathy.

IntroductionThe childhood heart disease of Friedreich’s Ataxia (FRDA) is characterized by hypertrophy and failure. It is caused by loss of frataxin (FXN), a mitochondrial protein involved in energy homeostasis. FRDA model hearts have increased mitochondrial protein acetylation and impaired sirtuin 3 (SIRT3) deacetylase activity. Protein acetylation is an important regulator of cardiac metabolism and loss of SIRT3 increases susceptibility of the heart to stress-induced cardiac hypertrophy and ischemic injury. The underlying pathophysiology of heart failure in FRDA is unclear. The purpose of this study was to examine in detail the physiologic and acetylation changes of the heart that occur over time in a model of FRDA heart failure. We predicted that increased mitochondrial protein acetylation would be associated with a decrease in heart function in a model of FRDA.MethodsA conditional mouse model of FRDA cardiomyopathy with ablation of FXN (FXN KO) in the heart was compared to healthy controls at postnatal days 30, 45 and 65. We evaluated hearts using echocardiography, cardiac catheterization, histology, protein acetylation and expression.ResultsAcetylation was temporally progressive and paralleled evolution of heart failure in the FXN KO model. Increased acetylation preceded detectable abnormalities in cardiac function and progressed rapidly with age in the FXN KO mouse. Acetylation was also associated with cardiac fibrosis, mitochondrial damage, impaired fat metabolism, and diastolic and systolic dysfunction leading to heart failure. There was a strong inverse correlation between level of protein acetylation and heart function.ConclusionThese results demonstrate a close relationship between mitochondrial protein acetylation, physiologic dysfunction and metabolic disruption in FRDA hypertrophic cardiomyopathy and suggest that abnormal acetylation contributes to the pathophysiology of heart disease in FRDA. Mitochondrial protein acetylation may represent a therapeutic target for early intervention.

The significance of intercalated discs in the pathogenesis of Friedreich cardiomyopathy

Friedreich ataxia (FRDA) is an autosomal recessive disorder with a complex clinical and neuropathological phenotype, but the most frequent cause of death is cardiomyopathy. The principal autopsy findings in FRDA hearts are concentric hypertrophy, enlargement of cardiomyocytes, myofiber necrosis, inflammatory infiltration, scarring, and random accumulation of iron. In addition, the myocardium shows generalized disorganization of intercalated discs (ICD), the Velcro-like end-to-end connections of heart fibers that provide mechanical cohesion and ionic coupling. The principal components of ICD are fascia adherens junctions (FAJ), desmosomes, and gap junctions. Frataxin deficiency in FRDA may cause improper assembly of ICD early in life, making hearts vulnerable to mechanical stress in childhood and adolescence. We studied the ICD in the myocardium of left ventricular wall (LVW), right ventricular wall, and ventricular septum in 18 genetically confirmed FRDA patients (age of death, 10 to 87years) and 12 normal controls (age of death, 13 to 69years). In cases with juvenile onset, electron microscopy and immunohistochemistry of N-cadherin and vinculin, two abundant FAJ proteins, showed enlargement of ICD, discontinuity, and hyperconvolution. Reaction product of the desmosomal protein desmoglein 2 was similar. The distribution of the gap junction protein connexin 43 at ICD was also irregular and displayed abnormal lateralization to the plasma membranes of cardiomyocytes. Confocal immunofluorescence microscopy of α-actinin, affinity fluorescence microscopy of actin with rhodamine-labeled phalloidin, and electron microscopy, revealed the principal integrity of sarcomeres of the myocardium in FRDA. In two late-onset long-surviving FRDA patients (ages 79 and 87), clinical cardiomyopathy was absent, and ICD were normal. The described observations in patients with a broad range of disease onset and duration allow us to conclude that faulty assembly of ICD interferes with proper end-to-end adhesion of cardiomyocytes of the growing heart and contributes to the pathogenesis of FRDA cardiomyopathy.

54. Prevention of Sensory Ataxia in a Novel Mouse Model of Friedreich Ataxia Using Gene Therapy Approach

Friedreich's ataxia (FRDA), the most common autosomal recessive ataxia, is characterized by a sensory and spinocerebellar ataxia, hypertrophic cardiomyopathy and increase incidence of diabetes. FRDA is caused by reduced levels of frataxin (FXN), an essential mitochondrial protein involved in the biosynthesis of iron-sulfur (Fe-S) clusters. Impaired mitochondrial oxidative phosphorylation, bioenergetics imbalance, deficit of Fe-S cluster enzymes and mitochondrial iron overload occur in individuals with FRDA. Proprioceptive neurons within the dorsal root ganglia (DRG) and cardiomyocytes are the most affected tissues in FRDA patients. To date there are not effective treatment for FRDA. We have previously established the primary proof-of-concept for developing gene therapy of FRDA cardiomyopathy and showed that adeno-associated virus (AAV) rh. 10 vector expressing human FXN injected intravenously not only prevented the onset of the cardiac disease in a faithful FRDA cardiac mouse model, but also, when administered at the time of heart failure, rapidly and completely reversed the cardiac disease. To date, there were unfortunately no adequate neuronal mouse model to address the possibility of gene therapy for the neuronal aspects of FRDA. We therefore recently generated a novel mouse model that recapitulates faithfully the sensory ataxia associated to FRDA using the conditional approach to delete frataxin specifically in the proprioceptive neurons of the DRG. By behavioural analysis, the mice exhibit an ataxic phenotype beginning at 3 weeks of age, which is rapidly progressive. Electrophysiological studies reveal a significant decrease of sensory wave already at 4.5 weeks and almost a complete loss at 8 weeks of age. A significant loss of sensory neurons within dorsal root ganglia is observed at 17.5 weeks of age compare to age matched controls. Ultrastructural analysis of sciatic and saphenous nerves showed abnormalities at early time points. Using this mouse model, we have developed an AAV gene therapy approach based on an intravenous delivery of AAV9-CAG-hFXN-HA vector at an early symptomatic stage of the disease. Mice displayed a complete prevention of the ataxic phenotype and electrophysiological analysis showed maintenance of the sensory wave in the treated animals. Histological studies revealed a complete prevention of neuronal loss in the DRG as well as a normal ultrastructure of saphenous and sciatic nerve. As results are encouraging, we plan to evaluate the potential therapeutical effect at a more advanced stage of the disease.

Abundance and Significance of Iron, Zinc, Copper, and Calcium in the Hearts of Patients With Friedreich Ataxia

Cardiomyopathy is a frequent cause of death in patients with Friedreich ataxia (FA), and a characteristic pathological feature is the focal accumulation of iron (Fe) in cardiomyocytes. This restricted localization of the metal contrasts with the diffuse cardiac Fe overload in hemochromatosis and transfusion siderosis. Nevertheless, heart Fe in FA contributes to cardiomyocyte necrosis, inflammation, and scarring as the disease progresses. A putative mechanism of cardiomyopathy in FA is Fe-mediated oxidative damage. Two other transition metals zinc (Zn) and copper (Cu), are diffusely distributed throughout normal hearts and the hearts of patients with FA. The myocardium in FA is also prone to deposits of calcium in the form of scattered concretions. In this study, heart tissues (left and right ventricular walls and ventricular septum) of 23 patients with genetically confirmed FA and 8 normal controls were obtained at autopsy and analyzed for Fe, Zn, Cu, and calcium. The principal assay methods were inductively coupled plasma optical emission spectrometry and plasma mass spectrometry. Total levels of Fe in bulk extracts were not significantly higher than normal, and the concentrations of Zn also remained in the normal range. Cu levels, however, were significantly lower in FA. In conclusion, the decrease of Cu may be important in consideration of the potential benefit of Cu supplements in FA cardiomyopathy.

0161: Usefulness of plasma high sensitive troponin t and Nt-proBNP in the diagnosis of cardiopathy in Friedreich ataxia

Friedreich ataxia, due to mitochondrial dysfunction, is the most common genetic sensory ataxia. It’s due to lack of frataxin. Hypertrophic cardiomyopathy is associated with Friedreich ataxia and is the major cause of death, (80%). This study concerned the role of plasma biomarkers, high sensitive troponin and Nt-proBNP, in the diagnostic of cardiopathy in Friedreich ataxia. From December 2012 to January 2015, we included 76 genetically confirmed Friedreich’s ataxia patients in Pitié-Salpêtrière Hospital. Clinical examination, ECG, echocardiography and blood samples were obtained. Patients were aged of 38±12 years, (mean±sd), 50% were male. 4 patients had palpitations, 2 dyspnea and no patients had chest pain. 89% had negative T waves on the ECG. 49% had echographic cardiac hypertrophy according to Henry’s nomogram. Patients with hypertrophy were younger: 34±10 years versus 42±14 years, age at onset of the disease was earlier: 15±6 years versus 21±15 years. Interventricular septal wall thickness was 12,9±1,7mm versus 10±1,2mm, and posterior wall thickness was 11,3±1,5mm versus 9,4±1mm. Left ventricle ejection fraction was similar (64%). For patients with hyper-trophy, troponin was higher: 22±21 ng/L versus 10±7 ng/L. Plasma NtproBNP was the same between the 2 groups 104±170 ng/L versus 64±122 ng/ L. 5 patients had plasma Nt-proBNP ≥300 ng/L, they all had had atrial fibrillation or heart failure. Plasma High sensitive troponin is a diagnostic marker of hypertrophic cardiomyopathy in Friedreich ataxia’s patients, whereas plasma Nt-proBNP is associated with cardiac events and could be a prognostic marker in these patients.

The pathogenesis of cardiomyopathy in Friedreich ataxia.

Friedreich ataxia (FA) is an autosomal recessive disease with a complex neurological phenotype, but the most common cause of death is heart failure. This study presents a systematic analysis of 15 fixed and 13 frozen archival autopsy tissues of FA hearts and 10 normal controls (8 frozen) by measurement of cardiomyocyte hypertrophy; tissue frataxin assay; X-ray fluorescence (XRF) of iron (Fe) and zinc (Zn) in polyethylene glycol-embedded samples of left and right ventricular walls (LVW, RVW) and ventricular septum (VS); metal quantification in bulk digests by inductively-coupled plasma optical emission spectrometry (ICP-OES); Fe histochemistry; and immunohistochemistry and immunofluorescence of cytosolic and mitochondrial ferritins and of the inflammatory markers CD68 and hepcidin. FA cardiomyocytes were significantly larger than normal and surrounded by fibrotic endomysium. Frataxin in LVW was reduced to less than 15 ng/g wet weight (normal 235.4±75.1 ng/g). All sections displayed characteristic Fe-reactive inclusions in cardiomyocytes, and XRF confirmed significant regional Fe accumulation in LVW and VS. In contrast, ICP-OES analysis of bulk extracts revealed normal total Fe levels in LVW, RVW, and VS. Cardiac Zn remained normal by XRF and assay of bulk digests. Cytosolic and mitochondrial ferritins exhibited extensive co-localization in cardiomyocytes, representing translational and transcriptional responses to Fe, respectively. Fe accumulation progressed from a few small granules to coarse aggregates in phagocytized cardiomyocytes. All cases met the “Dallas criteria” of myocarditis. Inflammatory cells contained CD68 and cytosolic ferritin, and most also expressed the Fe-regulatory hormone hepcidin. Inflammation is an important factor in the pathogenesis of FA cardiomyopathy but may be more evident in advanced stages of the disease. Hepcidin-induced failure of Fe export from macrophages is a likely contributory cause of damage to the heart in FA. Frataxin replacement and anti-inflammatory agents are potential therapies in FA cardiomyopathy.

Monitoring cardiac function during idebenone therapy in Friedreich's ataxia.

Friedreich's ataxia (FA) is associated with progressive cardiac hypertrophy resulting from a genetic abnormality in the frataxin gene. Cardiac involvement is the most common cause of death (59%) in FA patients. Cardiac related death occurs at a significantly younger age than non-cardiac related death. Idebenone is a short-chain quinone analogue with a potent free-radical scavenger action. This drug has the potential to preserve and even improve mitochondrial function.Studies on Idebenone treatment showed rather conflicting results on FA cardiomyopathy. The present article reviews the clinical features of FA cardiomyopathy, imaging techniques used to diagnose, follow and monitor therapy which aimed to revert FA cardiomyopathy.

Prevention and reversal of severe mitochondrial cardiomyopathy by gene therapy in a mouse model of Friedreich's ataxia

Cardiac failure is the most common cause of mortality in Friedreich's ataxia (FRDA), a mitochondrial disease characterized by neurodegeneration, hypertrophic cardiomyopathy and diabetes. FRDA is caused by reduced levels of frataxin (FXN), an essential mitochondrial protein involved in the biosynthesis of iron-sulfur (Fe-S) clusters. Impaired mitochondrial oxidative phosphorylation, bioenergetics imbalance, deficit of Fe-S cluster enzymes and mitochondrial iron overload occur in the myocardium of individuals with FRDA. No treatment exists as yet for FRDA cardiomyopathy. A conditional mouse model with complete frataxin deletion in cardiac and skeletal muscle (Mck-Cre-Fxn(L3/L-) mice) recapitulates most features of FRDA cardiomyopathy, albeit with a more rapid and severe course. Here we show that adeno-associated virus rh10 vector expressing human FXN injected intravenously in these mice fully prevented the onset of cardiac disease. Moreover, later administration of the frataxin-expressing vector, after the onset of heart failure, was able to completely reverse the cardiomyopathy of these mice at the functional, cellular and molecular levels within a few days. Our results demonstrate that cardiomyocytes with severe energy failure and ultrastructure disorganization can be rapidly rescued and remodeled by gene therapy and establish the preclinical proof of concept for the potential of gene therapy in treating FRDA cardiomyopathy.

0300: Systemic delivery of AAVrh10 expressing frataxin corrects the severe mitochondrial cardiomyopathy of frataxin deficient mice

Cardiac failure is the most common cause of mortality in Friedreich ataxia (FRDA), a mitochondrial disease with neurodegeneration, hypertrophic cardiomyopathy and diabetes. FRDA is caused by reduced levels of frataxin (FXN), an essential mitochondrial protein involved in mitochondrial bioenergetics. To date, no treatment exists for FRDA cardiomyopathy. Here, we show that adenoassociated virus (AAV) rh10 vector expressing human FXN injected intravenously not only prevented the onset of the cardiac disease in a faithful FRDA cardiac mouse model, but also, when administered at the time of heart failure, reversed rapidly and completely cardiac disease at the functional, cellular and molecular level in all treated animals. Our results also demonstrate the capacity of defective cardiomyocytes with severe energy failure and ultrastructure disorganization to be rapidly corrected and remodeled by gene therapy. These results establish the primary proof-of-concept for developing gene therapy of FRDA cardiomyopathy.

Molecular and Functional Alterations in a Mouse Cardiac Model of Friedreich Ataxia: Activation of the Integrated Stress Response, eIF2α Phosphorylation, and the Induction of Downstream Targets

Friedreich ataxia (FA) is a neurodegenerative and cardiodegenerative disease resulting from marked frataxin deficiency. The condition is characterized by ataxia with fatal cardiomyopathy, but the pathogenic mechanisms are unclear. We investigated the association between gene expression and progressive histopathological and functional changes using the muscle creatine kinase conditional frataxin knockout (KO) mouse; this mouse develops a severe cardiac phenotype that resembles that of FA patients. We examined KO mice from 3 weeks of age, when they are asymptomatic, to 10 weeks of age, when they die of the disease. Positive iron staining was identified in KO mice from 5 weeks of age, with markedly reduced cardiac function from 6 weeks. We identified an early and marked up-regulation of a gene cohort responsible for stress-induced amino acid biosynthesis and observed markedly increased phosphorylation of eukaryotic translation initiation factor 2α (p-eIF2α), an activator of the integrated stress response, in KO mice at 3 weeks of age, relative to wild-type mice. Importantly, the eIF2α-mediated integrated stress response has been previously implicated in heart failure via downstream processes such as autophagy and apoptosis. Indeed, expression of a panel of autophagy and apoptosis markers was enhanced in KO mice. Thus, the pathogenesis of cardiomyopathy in FA correlates with the early and persistent eIF2α phosphorylation, which precedes activation of autophagy and apoptosis.