Mosaic Deficiency in Mitochondrial Oxidative Metabolism Promotes Cardiac Arrhythmia during Aging

ArticleinCell Metabolism 21(5):667-77 · May 2015with 160 Reads 
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Abstract
Aging is a progressive decline of body function, during which many tissues accumulate few cells with high levels of deleted mitochondrial DNA (mtDNA), leading to a defect of mitochondrial functions. Whether this mosaic mitochondrial deficiency contributes to organ dysfunction is unknown. To investigate this, we generated mice with an accelerated accumulation of mtDNA deletions in the myocardium, by expressing a dominant-negative mutant mitochondrial helicase. These animals accumulated few randomly distributed cardiomyocytes with compromised mitochondrial function, which led to spontaneous ventricular premature contractions and AV blocks at 18 months. These symptoms were not caused by a general mitochondrial dysfunction in the entire myocardium, and were not observed in mice at 12 months with significantly lower numbers of dysfunctional cells. Therefore, our results suggest that the disposition to arrhythmia typically found in the aged human heart might be due to the random accumulation of mtDNA deletions and the subsequent mosaic respiratory chain deficiency. Copyright © 2015 Elsevier Inc. All rights reserved.

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  • ... In "mutator" mice with homozygous mutation of mitochondrial polymerase gamma (Polg m/m ), and in mice with disruption of the mitochondrial DNA helicase Twinkle (Twnk), mitochondrial function was compromised, leading to oxidative damage as well as accelerated cardiac aging and cardiac dysfunction. [171][172][173] As early as 8 weeks old, the accumulation rate of mtDNA mutations is 3 to 5 times higher in mutator mice compared with wild-type controls in multiple tissues including the heart. By 6 months, mutator mice develop premature aging phenotypes. ...
    ... [174][175][176] Similarly, in mice overexpressing dominant-negative Twnk helicase in the myocardium, mtDNA deletions accumulated at an accelerated rate in cardiomyocytes, and this was paralleled by mitochondrial deficiency and the development of arrhythmias, a common correlate of aging. 172 In contrast, mice with targeted mutation of the p66 Shc gene involved in mitochondrial ROS production display prolonged lifespan, reduced ROS production and resistance to ROS-mediated apoptosis. 177 Moreover, disruption of p66 Shc protected against angiotensin-II induced cardiac hypertrophy and cardiomyocyte apoptosis as well as reducing oxidative damage in multiple cardiovascular lineages in diabetic mice. ...
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    During aging, deterioration in cardiac structure and function leads to increased susceptibility to heart failure. The need for interventions to combat this age-related cardiac decline is becoming increasingly urgent as the elderly population continues to grow. Our understanding of cardiac aging, and aging in general, is limited. However, recent studies of age-related decline and its prevention through interventions like exercise have revealed novel pathological and cardioprotective pathways. In this review, we summarize recent findings concerning the molecular mechanisms of agerelated heart failure and highlight exercise as a valuable experimental platform for the discovery of much-needed novel therapeutic targets in this chronic disease.
  • ... In "mutator" mice with homozygous mutation of mitochondrial polymerase gamma (Polg m/m ), and in mice with disruption of the mitochondrial DNA helicase Twinkle (Twnk), mitochondrial function was compromised, leading to oxidative damage as well as accelerated cardiac aging and cardiac dysfunction. [171][172][173] As early as 8 weeks old, the accumulation rate of mtDNA mutations is 3 to 5 times higher in mutator mice compared with wild-type controls in multiple tissues including the heart. By 6 months, mutator mice develop premature aging phenotypes. ...
    ... [174][175][176] Similarly, in mice overexpressing dominant-negative Twnk helicase in the myocardium, mtDNA deletions accumulated at an accelerated rate in cardiomyocytes, and this was paralleled by mitochondrial deficiency and the development of arrhythmias, a common correlate of aging. 172 In contrast, mice with targeted mutation of the p66 Shc gene involved in mitochondrial ROS production display prolonged lifespan, reduced ROS production and resistance to ROS-mediated apoptosis. 177 Moreover, disruption of p66 Shc protected against angiotensin-II induced cardiac hypertrophy and cardiomyocyte apoptosis as well as reducing oxidative damage in multiple cardiovascular lineages in diabetic mice. ...
    Article
    During aging, deterioration in cardiac structure and function leads to increased susceptibility to heart failure. The need for interventions to combat this age-related cardiac decline is becoming increasingly urgent as the elderly population continues to grow. Our understanding of cardiac aging, and aging in general, is limited. However, recent studies of age-related decline and its prevention through interventions like exercise have revealed novel pathological and cardioprotective pathways. In this review, we summarize recent findings concerning the molecular mechanisms of age-related heart failure and highlight exercise as a valuable experimental platform for the discovery of much-needed novel therapeutic targets in this chronic disease.
  • ... Just a small number of compromised cells can then lead to a pathology ( Figure 2C). The presence of mitochondrially compromised cells has been shown to cause pathologies including arrhythmias in the heart [74] and damage in muscle fibres [75]. ...
    ... Wider distributions have more probability of crossing mutant load thresholds (dashed lines). (C) Even a small number of high-mutant-load, dysfunctional cells can compromise organ-wide functionality [74,75]. If the cell-to-cell variance of heteroplasmy is low, few cells will cross the threshold and the organ can function as normal. ...
    Article
    Full-text available
    Mitochondrial DNA (mtDNA) encodes vital respiratory machinery. Populations of mtDNA molecules exist in most eukaryotic cells, subject to replication, degradation, mutation, and other population processes. These processes affect the genetic makeup of cellular mtDNA populations, changing cell-to-cell distributions, means, and variances of mutant mtDNA load over time. As mtDNA mutant load has nonlinear effects on cell functionality, and cell functionality has nonlinear effects on tissue performance, these statistics of cellular mtDNA populations play vital roles in health, disease, and inheritance. This mini review will describe some of the better-known ways in which these populations change over time in different organisms, highlighting the importance of quantitatively understanding both mutant load mean and variance. Due to length constraints, we cannot attempt to be comprehensive but hope to provide useful links to some of the many excellent studies on these topics.
  • ... For example, heart rate is influenced not only by the loss of cells in the sinoatrial node (responsible for controlling heart rate) but also by structural changes in the heart, including fibrosis and hypertrophy, which slow propagation of electric impulse (Csepe et al. 2015). A recent work has clearly demonstrated that age-dependent mitochondrial DNA damage is an important substrate underpinning the pathophysiology of cardiac arrhythmias(Baris et al. 2015). In this study, genetically modified mice with accelerated accumulation of mtDNA deletions in the myocardium accumulated few randomly distributed cardiomyocytes with compromised mitochondrial function, which led to spontaneous ventricular premature contractions and atrioventricular blocks at the age of 18 months. ...
    ... These symptoms were not caused by a general mitochondrial dysfunction in the entire myocardium, and were not observed in mice at 12 months with significantly lower numbers of dysfunctional cells. These results suggest that the disposition to arrhythmia typically found in the aged human heart might be due to the random accumulation of mtDNA deletions and the subsequent mosaic respiratory chain deficiency(Baris et al. 2015). Another important pathological feature associated with aging is the calcification of aortic and mitral valves which triggers stenosis/insufficiency resulting in cardiac pressure/volume overload (Freeman et al. 2005). ...
    Article
    Age is one of the major risk factors associated with cardiovascular disease (CVD). About one fifth of the world population will be aged 65 or older by 2030 with an exponential increase in CVD prevalence. It is well established that environmental factors (overnutrition, smoking, pollution, sedentary lifestyles) may lead to premature defects in mitochondrial functionality, insulin signalling, endothelial homeostasis and redox balance fostering early senescent features. Over the last few years, molecular investigations unveiled common signalling networks which may link the aging process with deterioration of cardiovascular homeostasis and metabolic disturbances, namely insulin resistance. These different processes seem to be highly interconnected and their interplay may favour adverse vascular and cardiac phenotypes responsible for myocardial infarction, stroke and heart failure. In the present review, we carefully describe novel molecular cues underpinning aging, metabolism and CVD. In particular, we describe a dynamic interplay between emerging pathways such as FOXOs, AMPK, SIRT1, p66(Shc) , JunD and NF-kB. Such an overview will provide the background for attractive molecular targets to prevent age-driven pathology in the vasculature and the heart. This article is protected by copyright. All rights reserved.
  • ... With age, somatically derived mtDNA deletions clonally accumulate within a subset of individual cells (Wanagat et al., 2001;Ekstrand et al., 2007;McKiernan et al., 2007;Baris et al., 2015). These cells lack subunits of oxidative phosphorylation (OXPHOS) complex IV and cytochrome c oxidase (COX) activity. ...
    ... These cells lack subunits of oxidative phosphorylation (OXPHOS) complex IV and cytochrome c oxidase (COX) activity. COX negative and electron transport chain (ETC) deficient cells have been detected in the brain, heart, kidney, and skeletal muscle of aged mammals (Wanagat et al., 2001;Ekstrand et al., 2007;McKiernan et al., 2007;Baris et al., 2015). To test the hypothesis that 17-month-old Polg mut/mut mice may display ETC deficient cells in SGNs of the cochlea, we used sequential immunohistochemistry to examine mouse cochlea for SGNs possessing mitochondria (Porin-positive) but lacking mtDNA-encoded COX subunit I (COI-negative) (Fig. 8J-M). ...
  • ... With age, somatically derived mtDNA deletions clonally accumulate within a subset of individual cells (Wanagat et al., 2001;Ekstrand et al., 2007;McKiernan et al., 2007;Baris et al., 2015). These cells lack subunits of oxidative phosphorylation (OXPHOS) complex IV and cytochrome c oxidase (COX) activity. ...
    ... These cells lack subunits of oxidative phosphorylation (OXPHOS) complex IV and cytochrome c oxidase (COX) activity. COX negative and electron transport chain (ETC) deficient cells have been detected in the brain, heart, kidney, and skeletal muscle of aged mammals (Wanagat et al., 2001;Ekstrand et al., 2007;McKiernan et al., 2007;Baris et al., 2015). To test the hypothesis that 17-month-old Polg mut/mut mice may display ETC deficient cells in SGNs of the cochlea, we used sequential immunohistochemistry to examine mouse cochlea for SGNs possessing mitochondria (Porin-positive) but lacking mtDNA-encoded COX subunit I (COI-negative) (Fig. 8J-M). ...
  • ... Paradoxically, despite the importance of functional mitochondria for heart development, the loss of OXPHOS activity is insufficient to trigger the death of an adult cardiomyocyte [121]. Cardiomyocytes that have lost OXPHOS can be detected using histochemistry as cytochrome oxidase negative (COX-) and can have a detrimental effect on the overall heart function. ...
    ... Extensive death of cardiomyocytes will result in a large connective tissue scar, whose subsequent remodeling or inflammation can predispose the affected tissue to rupture under pressure. While COXcardiomyocytes are less harmful than fibrotic areas, also they are functionally impaired and could have a pathogenic outcome such as conductance block when reaching a certain threshold [121][122][123]. ...
    Article
    Mitochondria are essential for the development as well as maintenance of the myocardium, the most energy consuming tissue in the human body. Mitochondria are not only a source of ATP energy but also generators of reactive oxygen species (ROS), that cause oxidative damage, but also regulate physiological processes such as the switch from hyperplastic to hypertrophic growth after birth. As excess ROS production and oxidative damage are associated with cardiac pathology, it is not surprising that much of the research focused on the deleterious aspects of free radicals. However, cardiomyocytes are naturally highly adapted against repeating oxidative insults, with evidence suggesting that moderate and acute ROS exposure has beneficial consequences for mitochondrial maintenance and cardiac health. Antioxidant defenses, mitochondrial quality control, mtDNA maintenance mechanisms as well as mitochondrial fusion and fission improve mitochondrial function and cardiomyocyte survival under stress conditions. As these adaptive processes can be induced, promoting mitohormesis or mitochondrial biogenesis using controlled ROS exposure could provide a promising strategy to increase cardiomyocyte survival and prevent pathological remodeling of the myocardium.
  • ... mtDNA maintenance disorders, such as those caused by nuclear gene mutations in POLG, TWNK, or thymidine phosphorylase (TYMP), also caused mtDNA deletions or point mutations and induced FGF21 [57][58][59]. Consistent with human data, mouse models accumulating multiple mtDNA deletions in skeletal muscle [52,60], or those with a single large heteroplasmic mtDNA deletion [61], induced FGF21, clearly linking mtDNA deletions to the cytokine response [56]. ...
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    Mitochondrial oxidative phosphorylation disorders are extremely heterogeneous conditions. Their clinical and genetic variability makes the identification of reliable and specific biomarkers very challenging. Until now, only a few studies have focused on the effect of a defective oxidative phosphorylation functioning on the cell’s secretome, although it could be a promising approach for the identification and pre-selection of potential circulating biomarkers for mitochondrial diseases. Here, we review the insights obtained from secretome studies with regard to oxidative phosphorylation dysfunction, and the biomarkers that appear, so far, to be promising to identify mitochondrial diseases. We propose two new biomarkers to be taken into account in future diagnostic trials.
  • ... The generation of Tfam EKO mice was previously described (Baris et al., 2011). K320E-Twinkle Epi mice were generated by crossing keratin 14-Cre mice (Hafner et al., 2004) with R26-K320E-Twinkle loxP/þ mice ( Baris et al., 2015). Cre recombination leads to the expression of both K320E-TWINKLE and GFP in the epidermis. ...
    Article
    Accumulation of large-scale mitochondrial DNA (mtDNA) deletions and chronic, subclinical inflammation are concomitant during skin aging, thus raising the question of a causal link. To approach this, we generated mice expressing a mutant mitochondrial helicase (K320E-TWINKLE) in the epidermis in order to accelerate the accumulation of mtDNA deletions in this skin compartment. Mice displayed low amounts of large-scale deletions as well as a dramatic depletion of mtDNA in the epidermis and showed macroscopic signs of severe skin inflammation. The mtDNA alterations led to an imbalanced stoichiometry of mitochondrial respiratory chain complexes, inducing a unique combination of cytokine expression, causing a severe inflammatory phenotype, with massive immune cell infiltrates already before birth. Altogether, these data unraveled a previously unknown link between an imbalanced stoichiometry of the mitochondrial respiratory chain complexes and skin inflammation, and suggest that severe respiratory chain dysfunction, as observed in few cells leading to a mosaic in aged tissues, might be involved in the development of chronic sub-clinical inflammation.
  • ... Applying fluorescence and genetic mitochondrial tracking tools to bone marrow transplantation in which mtDNA damage occurs as a consequence of recipient preconditioning, and to embryonic developmental mosaicism, will open up the fields of transplantation medicine and early development to studies of intercellular mitochondrial trafficking. Finally, a mouse model of ageing cardiac muscle is characterized by cellular mosaicism involving pockets of bioenergetically malfunctional cells [49], raising questions about whether regenerative approaches involving mobilization and activation of quiescent stem cells as mitochondrial donors may be applicable not only to cardiac arrhythmias in ageing, but also to degenerative brain diseases like Parkinson's and Alzheimer's. ...
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    Full-text available
    Current dogma holds that genes are the property of individual mammalian cells and partition between daughter cells during cell division. However, and rather unexpectedly, recent research has demonstrated horizontal cell-to-cell transfer of mitochondria and mitochondrial DNA in several mammalian cell culture systems. Furthermore, unequivocal evidence that mitochondrial DNA transfer occurs in vivo has now been published. While these studies show horizontal transfer of mitochondrial DNA in pathological settings, it is also possible that intercellular mitochondrial transfer is a fundamental physiological process with a role in development and tissue homeostasis.
  • ... The effect of endurance exercise seems to be mediated by induction of mitochondrial biogenesis, prevention of mtDNA mutations, increased mitochondrial oxidative capacity and respiratory chain assembly and reduction in apoptosis (Safdar et al. 2011). Very recently, a new animal model has been generated showing accelerated accumulation of mtDNA deletions in the myocardium (Baris et al. 2015). Like PolG animals, these mice accumulate randomly distributed cardiomyocytes with compromised mitochondrial function, which seems to promote premature cardiac arrhythmia. ...
    Article
    As average lifespan of humans increases in western countries, cardiac diseases become the first cause of death. Aging is among the most important risk factors that increase susceptibility for developing cardiovascular diseases. The heart has very aerobic metabolism, and is highly dependent on mitochondrial function, since mitochondria generate more than 90 % of the intracellular ATP consumed by cardiomyocytes. In the last few decades, several investigations have supported the relevance of mitochondria and oxidative stress both in heart aging and in the development of cardiac diseases such as heart failure, cardiac hypertrophy, and diabetic cardiomyopathy. In the current review, we compile different studies corroborating this role. Increased mitochondria DNA instability, impaired bioenergetic efficiency, enhanced apoptosis, and inflammation processes are some of the events related to mitochondria that occur in aging heart, leading to reduced cellular survival and cardiac dysfunction. Knowing the mitochondrial mechanisms involved in the aging process will provide a better understanding of them and allow finding approaches to more efficiently improve this process.
  • ... The physiological impact of mitochondrial mutations is demonstrated by the effect of AZT treatment (Rayapureddi et al., 2010), accelerated aging of mitochondrial DNA polymerase gamma mutator mice (Trifunovic Mitochondrial mutations and cell loss, A. Herbst et al. et al., 2004), and cardiac arrhythmias in Twinkle mitochondrial helicase mutant mice (Baris et al., 2015). Previous experiments demonstrated that a 7 weeks GPA treatment of 27-month-old rats resulted in an increased incidence (3.7-fold) of ETC abnormal fibers, but did not result in measureable fiber loss (Herbst et al., 2013). ...
    Article
    Full-text available
    With age, somatically derived mitochondrial DNA (mtDNA) deletion mutations arise in many tissues and species. In skeletal muscle, deletion mutations clonally accumulate along the length of individual fibers. At high intrafiber abundances, these mutations disrupt individual cell respiration and are linked to the activation of apoptosis, intrafiber atrophy, breakage, and necrosis, contributing to fiber loss. This sequence of molecular and cellular events suggests a putative mechanism for the permanent loss of muscle fibers with age. To test whether mtDNA deletion mutation accumulation is a significant contributor to the fiber loss observed in aging muscle, we pharmacologically induced deletion mutation accumulation. We observed a 1200% increase in mtDNA deletion mutation-containing electron transport chain-deficient muscle fibers, an 18% decrease in muscle fiber number and 22% worsening of muscle mass loss. These data affirm the hypothesized role for mtDNA deletion mutation in the etiology of muscle fiber loss at old age.
  • ... 4,5 Cell-to-cell variances of mtDNA copy number and heteroplasmy are of particular importance, owing to their implications for maternal transmission of dangerous mutations 6 and the manifestation of pathologies dependent on the range of heteroplasmies present in a tissue 7 -even a very small proportion of cells exceeding a heteroplasmy threshold can lead to pathologies. 8 Stochastic behavior underlies much of cell biology and contributes to this cell-to-cell variability; cellular processes including gene expression, [9][10][11] DNA replication, 12 and mitochondrial and mtDNA dynamics [13][14][15][16] are subject to fundamentally stochastic influences. Variability in mitochondria can be a leading contributor to cell physiological behavior, making mitochondria an important target for explanatory stochastic models. ...
    Article
    Populations of physiologically vital mitochondrial DNA (mtDNA) molecules evolve in cells under control from the nucleus. The evolution of populations of mixed mtDNA types is complicated and poorly understood, and variability of these controlled admixtures plays a central role in the inheritance and onset of genetic disease. Here, we develop a mathematical theory describing the evolution of, and variability in, these stochastic populations for any type of cellular control, showing that cell-to-cell variability in mtDNA and mutant load inevitably increases with time, according to rates that we derive and which are notably independent of the mechanistic details of feedback signaling. We show with a set of experimental case studies that this theory explains disparate quantitative results from classical and modern experimental and computational research on heteroplasmy variance in different species. We demonstrate that our general model provides a host of specific insights, including a modification of the often-used but hard-to-interpret Wright formula to correspond directly to biological observables, the ability to quantify selective and mutational pressure in mtDNA populations, and characterization of the pronounced variability inevitably arising from the action of possible mtDNA quality-control mechanisms. Our general theoretical framework, supported by existing experimental results, thus helps us to understand and predict the evolution of stochastic mtDNA populations in cell biology.
  • ... The amount of mtDNA deletions varies between different tissues and even between individual cells of a given tissue due to random drift of the deleted mtDNA molecules (Bua et al., 2006;Campbell et al., 2014;Herbst et al., 2007;Larsson, 2010). Because of this mosaicism, PCR-based methods always identify low levels of mtDNA deletions in tissue homogenates (<10%), whereas single-cell-based approaches can show very high levels in individual cells (Baris et al., 2015;Brierley et al., 1998). In line with this, muscle fiber regions with the highest levels of deletions co-localize within regions of COX deficiency and muscle atrophy in aging individuals (Cao et al., 2001;Wanagat et al., 2001). ...
    Article
    Mitochondria were first postulated to contribute to aging more than 40 years ago. During the following decades, multiple lines of evidence in model organisms and humans showed that impaired mitochondrial function can contribute to age-associated disease phenotypes and aging. However, in contrast to the original theory favoring oxidative damage as a cause for mtDNA mutations, there are now strong data arguing that most mammalian mtDNA mutations originate as replication errors made by the mtDNA polymerase. Currently, a substantial amount of mitochondrial research is focused on finding ways to either remove or counteract the effects of mtDNA mutations with the hope of extending the human health- and lifespan. This review summarizes the current knowledge regarding the formation of mtDNA mutations and their impact on mitochondrial function. We also critically discuss proposed pathways interlinked with mammalian mtDNA mutations and suggest future research strategies to elucidate the role of mtDNA mutations in aging. Impaired mitochondrial function can contribute to age-associated diseases and aging. In this review, Kauppila et al. discuss recent data on the origin of mtDNA mutations as replication errors made by the mtDNA polymerase, mitophagy, and potential therapeutic strategies for improving mitochondrial function.
  • ... mtDNA maintenance disorders, such as those caused by nuclear gene mutations in POLG, C10orf2, or TYMP, also cause mtDNA deletions or point mutations, [22][23][24] and induced FGF21. Consistent with human data, mouse models accumulating multiple mtDNA deletions in skeletal muscle, 25,26 or mice with a single large heteroplasmic mtDNA deletion, 27 induced Fgf21, clearly linking mtDNA deletions to the cytokine response. Mpv17 KO mice did not elicit Fgf21 response, which may be linked with mtDNA depletion or the unknown Mpv17 function. ...
    Article
    Full-text available
    Objective: To validate new mitochondrial myopathy serum biomarkers for diagnostic use. Methods: We analyzed serum FGF21 (S-FGF21) and GDF15 from patients with (1) mitochondrial diseases and (2) nonmitochondrial disorders partially overlapping with mitochondrial disorder phenotypes. We (3) did a meta-analysis of S-FGF21 in mitochondrial disease and (4) analyzed S-Fgf21 and skeletal muscle Fgf21 expression in 6 mouse models with different muscle-manifesting mitochondrial dysfunctions. Results: We report that S-FGF21 consistently increases in primary mitochondrial myopathy, especially in patients with mitochondrial translation defects or mitochondrial DNA (mtDNA) deletions (675 and 347 pg/mL, respectively; controls: 66 pg/mL, p < 0.0001 for both). This is corroborated in mice (mtDNA deletions 1,163 vs 379 pg/mL, p < 0.0001). However, patients and mice with structural respiratory chain subunit or assembly factor defects showed low induction (human 335 pg/mL, p < 0.05; mice 335 pg/mL, not significant). Overall specificities of FGF21 and GDF15 to find patients with mitochondrial myopathy were 89.3% vs 86.4%, and sensitivities 67.3% and 76.0%, respectively. However, GDF15 was increased also in a wide range of nonmitochondrial conditions. Conclusions: S-FGF21 is a specific biomarker for muscle-manifesting defects of mitochondrial translation, including mitochondrial transfer-RNA mutations and primary and secondary mtDNA deletions, the most common causes of mitochondrial disease. However, normal S-FGF21 does not exclude structural respiratory chain complex or assembly factor defects, important to acknowledge in diagnostics. Classification of evidence: This study provides Class III evidence that elevated S-FGF21 accurately distinguishes patients with mitochondrial myopathies from patients with other conditions, and FGF21 and GDF15 mitochondrial myopathy from other myopathies.
  • ... Some of these deletions, distributed between cells in a highly heterogenic manner, cause respiratory deficiency in a small fraction of cells. It has been demonstrated on mouse hearts that, in a complex functional network of cells, even a small number of dysfunctional units can lead to arrhythmia and, as result, to heart failure [20]. In these experiments mutant Twinkle helicase was used to generate aged-dependent accumulation of deletions in the mtDNA of the animals and to mimic the situation in aged humans. ...
    Article
    Full-text available
    Recent deep sequencing data has provided compelling evidence that the spectrum of somatic point mutations in mitochondrial DNA (mtDNA) in aging tissues lacks G > T transversion mutations. This fact cannot, however, be used as an argument for the missing contribution of reactive oxygen species (ROS) to mitochondria-related aging because it is probably caused by the nucleotide selectivity of mitochondrial DNA polymerase γ (POLG). In contrast to point mutations, the age-dependent accumulation of mitochondrial DNA deletions is, in light of recent experimental data, still explainable by the segregation of mutant molecules generated by the direct mutagenic effects of ROS (in particular, of HO· radicals formed from H2O2 by a Fenton reaction). The source of ROS remains controversial, because the mitochondrial contribution to tissue ROS production is probably lower than previously thought. Importantly, in the discussion about the potential role of oxidative stress in mitochondria-dependent aging, ROS generated by inflammation-linked processes and the distribution of free iron also require careful consideration.
  • ... Defects in the aggregation of ion channels as a result of structural perturbance of sarcolemma, cytoskeleton, and sarcomere represent a common pathomechanism for arrhythmogenesis in many cases of DCM [29]. Furthermore, the presence of mitochondrial dysfunction has recently been shown to promote arrhythmias [30]. In this setting, arrhythmias associated with mitochondrial dysfunction have been proposed to result from disturbances in the cardiac calcium metabolism. ...
    Article
    Full-text available
    Background Mutations in the human desmin gene (DES) cause autosomal-dominant and -recessive cardiomyopathies, leading to heart failure, arrhythmias, and AV blocks. We analyzed the effects of vascular pressure overload in a patient-mimicking p.R349P desmin knock-in mouse model that harbors the orthologue of the frequent human DES missense mutation p.R350P. Methods and results Transverse aortic constriction (TAC) was performed on heterozygous (HET) DES-p.R349P mice and wild-type (WT) littermates. Echocardiography demonstrated reduced left ventricular ejection fraction in HET-TAC (WT-sham: 69.5 ± 2.9%, HET-sham: 64.5 ± 4.7%, WT-TAC: 63.5 ± 4.9%, HET-TAC: 55.7 ± 5.4%; p<0.01). Cardiac output was significantly reduced in HET-TAC (WT sham: 13088 ± 2385 μl/min, HET sham: 10391 ± 1349μl/min, WT-TAC: 8097 ± 1903μl/min, HET-TAC: 5793 ± 2517μl/min; p<0.01). Incidence and duration of AV blocks as well as the probability to induce ventricular tachycardias was highest in HET-TAC. We observed reduced mtDNA copy numbers in HET-TAC (WT-sham: 12546 ± 406, HET-sham: 13526 ± 781, WT-TAC: 11155 ± 3315, HET-TAC: 8649 ± 1582; p = 0.025), but no mtDNA deletions. The activity of respiratory chain complexes I and IV showed the greatest reductions in HET-TAC. Conclusion Pressure overload in HET mice aggravated the clinical phenotype of cardiomyopathy and resulted in mitochondrial dysfunction. Preventive avoidance of pressure overload/arterial hypertension in desminopathy patients might represent a crucial therapeutic measure.
  • ... El estudio de Safdar et al. indica que ese efecto del ejercicio físico podría estar mediado al menos en parte por la inducción de la biogénesis mitocondrial, la prevención de las mutaciones de ADNmt y la reducción de procesos apoptóticos 31 . Recientemente se ha desarrollado un nuevo modelo animal que presenta una acumulación aumentada de deleciones de ADNmt en el miocardio 32 . Como los animales polG, estos ratones acumulan cardiomiocitos con la función mitocondrial alterada, que podría estar relacionado con la arritmia cardíaca prematura que desarrollan. ...
    Article
    Resumen De acuerdo con diferentes organizaciones como la Asociación Americana del Corazón o la Organización Mundial de la Salud, las enfermedades cardiovasculares se han convertido en la primera causa de muerte en países occidentales. Aunque la exposición a diferentes factores de riesgo, en particular los relacionados con el estilo de vida, contribuyen de manera significativa a la etiopatogénesis de enfermedades cardíacas, el incremento en la esperanza de vida y el envejecimiento de la población asociado a él se consideran los determinantes principales del inicio y desarrollo de las mismas. Las mitocondrias y el estrés oxidativo se han señalado como factores relevantes tanto en el envejecimiento del corazón como en el desarrollo de enfermedades cardíacas como la insuficiencia cardíaca, la hipertrofia cardíaca y la miocardiopatía diabética. Durante el envejecimiento, diferentes procesos celulares relacionados con la función mitocondrial, como la bioenergética, procesos de apoptosis o de inflamación, se ven alterados, lo que conlleva una reducción en la supervivencia celular, y como consecuencia, disfunción cardíaca. Aumentar nuestro conocimiento sobre los mecanismos mitocondriales relacionados con el proceso de envejecimiento proporcionará nuevas estrategias para mejorar de forma más eficiente este proceso y las diferentes enfermedades relacionadas con él, en particular las cardiovasculares.
  • ... Thus neurons have a higher probability to acquire harmful proportions of mutated mtDNA species which can lead to failure of the mitochondrial ATP generation with all its consequences from altered neuronal excitability to cell death. It was recently demonstrated in the mouse heart that even a small proportion of cells harboring dysfunction-causing mtDNA deletions can lead to disturbance of a complex cellular network [2]. In the brain, parvalbumin-positive inhibitory interneurons are especially rich of mitochondria, and are likely to be severely affected by mitochondrial dysfunction (Fig. 4). ...
    Article
    Full-text available
    Accumulation of mitochondrial DNA (mtDNA) deletions has been proposed to be responsible for the presence of respiratory-deficient neurons in several CNS diseases. Deletions are thought to originate from double-strand breaks due to attack of reactive oxygen species (ROS) of putative inflammatory origin. In epileptogenesis, emerging evidence points to chronic inflammation as an important feature. Here we aimed to analyze the potential association of inflammation and mtDNA deletions in the hippocampal tissue of patients with mesial temporal lobe epilepsy (mTLE) and hippocampal sclerosis (HS). Hippocampal and parahippocampal tissue samples from 74 patients with drug-refractory mTLE served for mtDNA analysis by multiplex PCR as well as long-range PCR, single-molecule PCR and ultra-deep sequencing of mtDNA in selected samples. Patients were sub-classified according to neuropathological findings. Semi-quantitative assessment of neuronal cell loss was performed in the hippocampal regions CA1-CA4. Inflammatory infiltrates were quantified by cell counts in the CA1, CA3 and CA4 regions from well preserved hippocampal samples (n = 33). Samples with HS showed a significantly increased frequency of a 7436-bp mtDNA deletion (p < 0.0001) and a higher proportion of somatic G>T transversions compared to mTLE patients with different histopathology. Interestingly, the number of T-lymphocytes in the hippocampal CA1, CA3 and CA4 regions was, similar to the 7436-bp mtDNA deletion, significantly increased in samples with HS compared to other subgroups. Our findings show a coincidence of HS, increased somatic G>T transversions, the presence of a specific mtDNA deletion, and increased inflammatory infiltrates. These results support the hypothesis that chronic inflammation leads to mitochondrial dysfunction by ROS-mediated mtDNA mutagenesis which promotes epileptogenesis and neuronal cell loss in patients with mTLE and HS.
  • ... Crossing the MnSOD +/− mouse with the systemic Twinkle overexpression mouse abolishes the MnSOD +/− phenotype (Pohjoismaki et al., 2013). Additionally, a transgenic knock-in mouse was created expressing a dominant-negative K320E Twinkle in the heart (Baris et al., 2015). These mice develop ventricular arrhythmia with aging due to dysfunctional ETC enzyme activity. ...
    Article
    Full-text available
    As regulators of bioenergetics in the cell and the primary source of endogenous reactive oxygen species (ROS), dysfunctional mitochondria have been implicated for decades in the process of aging and age-related diseases. Mitochondrial DNA (mtDNA) is replicated and repaired by nuclear-encoded mtDNA polymerase γ (Pol γ) and several other associated proteins, which compose the mtDNA replication machinery. Here, we review evidence that errors caused by this replication machinery and failure to repair these mtDNA errors results in mtDNA mutations. Clonal expansion of mtDNA mutations results in mitochondrial dysfunction, such as decreased electron transport chain (ETC) enzyme activity and impaired cellular respiration. We address the literature that mitochondrial dysfunction, in conjunction with altered mitochondrial dynamics, is a major driving force behind aging and age-related diseases. Additionally, interventions to improve mitochondrial function and attenuate the symptoms of aging are examined.
  • ... However, COX-negative cells, but only very few of them, have been detected in the muscles, heart, liver and brain of aged humans [23,24] , giving rise to tissue mosaics, the physiological relevance of which was not known previously [25] . Recently, we have shown that a proportion as low as 0.5% of COX-negative cardiomyocytes is sufficient to cause age-related ventricular arrhythmias [26] . Interestingly, COX-negative cells have never been reported, even in very old mice -at least to our knowledge -with the exception of colon crypts, which for unknown reasons seem to be a highly mutagenic environment for mtDNA as well [27] . ...
    Article
    Background: Aging is a multifactorial process characterized by organ loss of function and degeneration, but the mechanisms involved remain elusive. We have shown recently that catecholamine metabolism drives the accumulation of mitochondrial DNA (mtDNA) deletions in dopaminergic cells, which likely contribute to their degeneration during aging. Here we investigated whether the well documented degeneration and altered function of adrenals during aging is linked to catecholamine production in the medulla followed by accumulation of mtDNA deletions. Material and methods: We analyzed adrenal medullary and cortical samples from both mouse and human origin covering a wide range of ages for mtDNA deletion content, mtDNA copy number, mitochondrial and cellular integrity as well as aging-related tissue changes such as fibrosis. Results: Indeed, we demonstrate that adrenal medulla accumulates a strikingly high amount of mtDNA deletions with age in mice and humans, causing mitochondrial dysfunction in the adrenal medulla, but also in cortex, accompanied by apoptosis, and, more importantly, by severe inflammation and remarkable fibrosis. Additionally, a concomitant and dramatic loss of medullary and cortical cells is observed in old animals. Conclusion: Thus, our results show that accumulation of mtDNA deletions, and the ensuing mitochondrial dysfunction is a hallmark of adrenal aging, further strengthening the hypothesis that catecholamine metabolism is detrimental for mtDNA integrity, mitochondrial function and cell survival. Moreover, the cell loss potentially induced by mitochondrial dysfunction could explain the decline of adrenal hormonal and steroidal secretion during aging.
  • ... Elucidating the mechanisms by which cells acquire this heterogeneity has emerged as an important theme in cancer research, leading to numerous sub-typing methods to define specific cell populations that contribute to the prognosis of the disease [42]. The development of arrhythmia typically found in the aged human heart has been reported to be caused by random accumulation of mitochondrial DNA deletions and subsequent heterogeneous respiratory chain deficiency [43]. Heterogeneous Myh7 expression has also been reported under stress conditions such as constitutive renin transgene expression and pressure overload, resulting in cardiac fibrosis and hypertrophy [7,8]. ...
    Article
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    Background: The heart responds to hemodynamic overload through cardiac hypertrophy and activation of the fetal gene program. However, these changes have not been thoroughly examined in individual cardiomyocytes, and the relation between cardiomyocyte size and fetal gene expression remains elusive. We established a method of high-throughput single-molecule RNA imaging analysis of in vivo cardiomyocytes and determined spatial and temporal changes during the development of heart failure. Methods and results: We applied three novel single-cell analysis methods, namely, single-cell quantitative PCR (sc-qPCR), single-cell RNA sequencing (scRNA-seq), and single-molecule fluorescence in situ hybridization (smFISH). Isolated cardiomyocytes and cross sections from pressure overloaded murine hearts after transverse aortic constriction (TAC) were analyzed at an early hypertrophy stage (2 weeks, TAC2W) and at a late heart failure stage (8 weeks, TAC8W). Expression of myosin heavy chain β (Myh7), a representative fetal gene, was induced in some cardiomyocytes in TAC2W hearts and in more cardiomyocytes in TAC8W hearts. Expression levels of Myh7 varied considerably among cardiomyocytes. Myh7-expressing cardiomyocytes were significantly more abundant in the middle layer, compared with the inner or outer layers of TAC2W hearts, while such spatial differences were not observed in TAC8W hearts. Expression levels of Myh7 were inversely correlated with cardiomyocyte size and expression levels of mitochondria-related genes. Conclusions: We developed a new image-analysis pipeline to allow automated and unbiased quantification of gene expression at the single-cell level and determined the spatial and temporal regulation of heterogenous Myh7 expression in cardiomyocytes after pressure overload.
  • ... Interestingly, the RC is hardly active in growth plate cartilage in newborns, but growth plate RC activity markedly increases in juvenile mice, when secondary ossification centers are formed and vascular networks are established at the proximal and distal end of the growth plate. We then generated transgenic mice, which have an inactivated RC only in cartilage, using the cartilage-specific expression of an mtDNA helicase Twinkle mutant (Baris et al., 2015;Weiland et al., 2018). Here, we show that these mice, as a consequence of the lack of RC activation after birth, develop postnatal growth retardation and growth plate cartilage degeneration caused by energy deficiency, altered metabolic signaling, destabilization of the hypertrophic ECM, and increased chondrocyte death at the cartilage-bone junction. ...
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    In childhood, skeletal growth is driven by transient expansion of cartilage in the growth plate. The common belief is that energy production in this hypoxic tissue mainly relies on anaerobic glycolysis and not on mitochondrial respiratory chain (RC) activity. However, children with mitochondrial diseases causing RC dysfunction often present with short stature, which indicates that RC activity may be essential for cartilage-mediated skeletal growth. To elucidate the role of the mitochondrial RC in cartilage growth and pathology, we generated mice with impaired RC function in cartilage. These mice develop normally until birth, but their later growth is retarded. A detailed molecular analysis revealed that metabolic signaling and extracellular matrix formation is disturbed and induces cell death at the cartilage–bone junction to cause a chondrodysplasia-like phenotype. Hence, the results demonstrate the overall importance of the metabolic switch from fetal glycolysis to postnatal RC activation in growth plate cartilage and explain why RC dysfunction can cause short stature in children with mitochondrial diseases.
  • ... Accumulation of mtDNA deletions in cells leads to respiratory chain deficiency. 102 Mitochondrial DNA copy number is decreased in aged skeletal muscle and liver compared to adults. 103 However, mitochondrial DNA copy number is not altered in the aged heart, 103,104 including SSM and IFM. ...
    Article
    Altered mitochondrial metabolism is the underlying basis for the increased sensitivity in the aged heart to stress. The aged heart exhibits impaired metabolic flexibility, with a decreased capacity to oxidize fatty acids and enhanced dependence on glucose metabolism. Aging impairs mitochondrial oxidative phosphorylation, with a greater role played by the mitochondria located between the myofibrils, the interfibrillar mitochondria. With aging, there is a decrease in activity of complexes III and IV, which account for the decrease in respiration. Furthermore, aging decreases mitochondrial content among the myofibrils. The end result is that in the interfibrillar area, there is approximate to 50% decrease in mitochondrial function, affecting all substrates. The defective mitochondria persist in the aged heart, leading to enhanced oxidant production and oxidative injury and the activation of oxidant signaling for cell death. Aging defects in mitochondria represent new therapeutic targets, whether by manipulation of the mitochondrial proteome, modulation of electron transport, activation of biogenesis or mitophagy, or the regulation of mitochondrial fission and fusion. These mechanisms provide new ways to attenuate cardiac disease in elders by preemptive treatment of age-related defects, in contrast to the treatment of disease-induced dysfunction.
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    Aging induces structural and functional changes in the heart that are associated with increased risk of cardiovascular disease and impaired functional capacity in the elderly. Exercise is a diagnostic and therapeutic tool, with the potential to provide insights into clinical diagnosis and prognosis, as well as the molecular mechanisms by which aging influences cardiac physiology and function. In this review, we first provide an overview of how aging impacts the cardiac response to exercise, and the implications this has for functional capacity in older adults. We then review the underlying molecular mechanisms by which cardiac aging contributes to exercise intolerance, and conversely how exercise training can potentially modulate aging phenotypes in the heart. Finally, we highlight the potential use of these exercise models to complement models of disease in efforts to uncover new therapeutic targets to prevent or treat heart disease in the aging population.
  • Article
    Background: During aging a mosaic of normal cells and cells with mitochondrial deficiency develops in various tissues including the heart. Whether this contributes to higher susceptibility for arrhythmia following myocardial infarction (MI) is unknown. Methods and results: Myocardial cryoinfarction was performed in 12-month-old transgenic mice with accelerated accumulation of deletions in mitochondrial DNA. Occurrence and pathogenesis of arrhythmia was investigated after two weeks. Holter-ECG recordings revealed higher rates of premature ventricular complexes (incidence > 10/24 h: 100% vs. 20%; p = 0.048) and more severe spontaneous arrhythmia during stress test in mutant mice with MI as compared to control mice with MI. Mice with mitochondrial dysfunction exhibited longer spontaneous AV-blocks (467 ± 26 ms vs. 377 ± 24 ms; p = 0.013), an increased probability for induction of ventricular tachycardia during in vivo electrophysiological investigation (22% vs. 9%; p = 0.044), and a reduced conduction velocity in the infarct borderzone (38.5 ± 0.5 cm/s vs. 55.3 ± 0.9 cm/s; p = 0.001). Furthermore, mutant mice exhibited a significant reduction of the phospho-Cx43/Cx43 ratio in right (0.59 ± 0.04 vs. 0.85 ± 0.01; p = 0.027) and left ventricular myocardium (0.72 ± 0.01 vs. 0.86 ± 0.02; p = 0.023). Conclusions: Aging-related cardiac mosaic respiratory chain dysfunction facilitates the occurrence of spontaneous and inducible cardiac arrhythmia after myocardial infarction and is associated with slowing of electrical impulse propagation in the infarct borderzone.
  • Article
    Age-induced mitochondrial DNA deletion mutations may underlie cell loss and tissue aging. Rapamycin extends mouse lifespan and modulates mitochondrial quality control. We hypothesized that reduced deletion mutation abundance may contribute to rapamycin's life extension effects. To test this hypothesis, genetically heterogeneous male and female mice were treated with rapamycin, compounded in chow at 14 or 42 ppm, from 9 months to 22 months of age. Mice under a 40% dietary restriction were included as a control known to protect mtDNA quality. To determine if chronic rapamycin treatment affects mitochondrial DNA quality, we assayed mtDNA deletion frequency and electron transport chain deficient fiber abundances in mouse quadriceps muscle. At 42 ppm rapamycin, we observed a 57% decrease in deletion frequency, a 2.8-fold decrease in ETC deficient fibers, and a 3.4-fold increase in the number of mice without electron transport chain deficient fibers. We observed a similar trend with the 14 ppm dose. DR significantly decreased ETC deficient fiber abundances with a trend toward lower mtDNA deletion frequency. The effects of rapamycin treatment on mitochondrial DNA quality were greatest in females at the highest dose. Rapamycin treatment at 14 ppm did not affect muscle mass or function. Dietary restriction also reduced deletion frequency and ETC deficient fibers. These data support the concept that the lifespan extending effects of rapamycin treatment result from enhanced mitochondrial DNA quality.
  • Article
    As the elderly segment of the world population increases, it is critical to understand the changes in cardiac structure and function during the normal aging process. In this review, we outline the key molecular pathways and cellular processes that underlie the phenotypic changes in the heart and vasculature that accompany aging. Reduced autophagy, increased mitochondrial oxidative stress, telomere attrition, altered signaling in insulin-like growth factor, growth differentiation factor 11, and 5'- AMP-activated protein kinase pathways are among the key molecular mechanisms underlying cardiac aging. Aging promotes structural and functional changes in the atria, ventricles, valves, myocardium, pericardium, the cardiac conduction system, and the vasculature. We highlight the factors known to accelerate and attenuate the intrinsic aging of the heart and vessels in addition to potential preventive and therapeutic avenues. A greater understanding of the processes involved in cardiac aging may facilitate our ability to mitigate the escalating burden of CVD in older individuals and promote healthy cardiac aging.
  • Article
    Cardiovascular disease is the leading cause of morbidity and mortality worldwide. Advancing age is a major risk factor for developing cardiovascular disease because of the lifelong exposure to cardiovascular risk factors and specific alterations affecting the heart and the vasculature during ageing. Indeed, the ageing heart is characterized by structural and functional changes that are caused by alterations in fundamental cardiomyocyte functions. In particular, the myocardium is heavily dependent on mitochondrial oxidative metabolism and is especially susceptible to mitochondrial dysfunction. Indeed, primary alterations in mitochondrial function, which are subsequently amplified by defective quality control mechanisms, are considered to be major contributing factors to cardiac senescence. In this Review, we discuss the mechanisms linking defective mitochondrial quality control mechanisms (that is, proteostasis, biogenesis, dynamics, and autophagy) to organelle dysfunction in the context of cardiac ageing. We also illustrate relevant molecular pathways that might be exploited for the prevention and treatment of age-related heart dysfunction.
  • Article
    The age-induced, exponential accumulation of mitochondrial DNA (mtDNA) deletion mutations contributes to muscle fiber loss. The causes of these mutations are not known. Systemic inflammation is associated with decreased muscle mass in older adults and is implicated in the formation of sporadic mtDNA deletions. Macrophage migration inhibitory factor knockout (MIF-KO) mice are long-lived with decreased inflammation. We hypothesized that aged MIF-KO mice would have lower mtDNA deletion frequencies and fewer electron transport chain (ETC) deficient fibers. We measured mtDNA copy number and mutation frequency as well as the number and length of ETC deficient fibers in 22-month old MIF-KO and F2 hybrid control mice. We also measured mtDNA copy number and deletion frequency in female UM-HET3 mice, a strain whose lifespan matches the MIF-KO mice. We did not observe a significant effect of MIF ablation on muscle mtDNA deletion frequency. There was a significantly lower mtDNA copy number in the MIF-KO mice and the lifespan-matched UM-HET3 mice compared to the F2 hybrids, suggesting the importance of genetic background in mtDNA copy number control. Our data do not support a definitive role for MIF in age-induced mtDNA deletions.
  • Article
    In this issue, Baris et al. (2015) describe cardiac rhythm abnormalities in a mouse model of mitochondrial dysfunction in widely distributed cells of the aging human heart. How do a few metabolically challenged cells disrupt cardiac rhythm? We suggest that these cells provide “crystallization centers” for latent dysfunctional zones to allow arrhythmia emergence. In this issue, Baris et al. describe cardiac rhythm abnormalities in a mouse model of mitochondrial dysfunction in widely distributed cells of the aging human heart. How do a few metabolically challenged cells disrupt cardiac rhythm? We suggest that these cells provide “crystallization centers” for latent dysfunctional zones to allow arrhythmia emergence.
  • Article
    Transgenic mouse models provided a powerful tool to evaluate the physiological significance of altered quantities or characteristics of specific gene products, such as cardiac ion channels. We have developed a system to record and analyze changes in the electrocardiogram in the mouse using an implantable telemetry system. The R-R and Q-T intervals were measured on individual beats and on signalaveraged complexes derived from 1, 2, or 4 s of contiguous data each hour during a 24-h period in three male and three female FVB mice. Duration of averaging had minimal effect on the measured Q-T. The Q-T interval was shown to be related to the square root of the R-R interval, and an appropriate formula for a rate-corrected Q-T interval (Q-Tc) was derived. Ketamine anesthesia was shown to markedly increase duration and variability in R-R, Q-T, and Q-Tc intervals. In conscious animals, variability in Q-T was low across animals and over time, suggesting that this should be a sensitive model for detection of changes in the Q-T interval in transgenic mice with ion channel defects.
  • Article
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    Heart rate turbulence (HRT) is a prognostic parameter for risk stratification in patients suffering from coronary artery disease. The aims of this study were to demonstrate the feasibility of quantifying HRT in mice, both in long-term electrocardiograms (ECGs) as well as after extrastimulus pacing, and to analyse its characteristics.METHODS AND RESULTS: We performed long-term ECG recordings using implanted telemetric chips and electrophysiological (EP) investigations, using transvenously inserted EP catheters, in healthy mice. Heart rate turbulence was calculated using the established turbulence onset (TO) and slope (TS) algorithm. After spontaneous ventricular premature complexes (VPCs), we found a negative TO (-2.2 ± 7.5%) and positive TS (15.5 ± 18.3 ms/RR interval). Electrophysiological investigations revealed positive values for TO (0.6 ± 1.1%) and TS (6.5 ± 2.9 ms/RR interval) after extrastimulus pacing maneuvers. The shortening of the extrastimuli coupling intervals delivered during EP investigations significantly influenced TO (r = 0.57; P = 0.01): shorter coupling intervals provoked more positive TO values.CONCLUSION: Mice display both spontaneous and induced HRT. In terms of TO, VPCs generated by extrastimulus pacing are significantly dependent on the coupling interval. Determining HRT in mice is feasible and provides insight into basic mechanisms of blood pressure regulation, which is realized by the baroreflex.
  • Article
    -Sudden cardiac death often involves arrhythmias triggered by metabolic stress. Loss of mitochondrial function is thought to contribute to the arrhythmogenic substrate, but how mitochondria contribute to uncoordinated electrical activity is poorly understood. It has been proposed that the formation of "metabolic current sinks", caused by the non-uniform collapse of mitochondrial inner-membrane potential (ΔΨm), contributes to reentrant arrhythmias because ΔΨm depolarization is tightly coupled to the activation of sarcolemmal ATP-sensitive K(+) (KATP) channels, hastening action potential repolarization and shortening the refractory period. -Here we use computational and experimental methods to investigate how ΔΨm instability can induce reentrant arrhythmias. We develop the first tissue level model of cardiac electrical propagation incorporating cellular electrophysiology, excitation-contraction coupling, mitochondrial energetics and reactive oxygen species (ROS) balance. Simulations show that reentry and fibrillation can be initiated by regional ΔΨm loss, due to the disparity of refractory periods inside and outside of the metabolic sink. Computational results are compared with the effects of a metabolic sink generated experimentally by local perfusion of a mitochondrial uncoupler in a monolayer of cardiac myocytes. -The results demonstrate that regional mitochondrial depolarization triggered by oxidative stress activates sarcolemmal KATP currents to form a metabolic sink. Consequent shortening of the action potential inside, but not outside, the sink increases the propensity for reentry. ΔΨm recovery during pacing can lead to novel mechanisms of ectopic activation. The findings highlight the importance of mitochondria as potential therapeutic targets for sudden death associated with cardiovascular disease.
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    Accumulation of mitochondrial DNA deletions is observed especially in dopaminergic neurons of the substantia nigra during ageing and even more in Parkinson's disease. The resulting mitochondrial dysfunction is suspected to play an important role in neurodegeneration. However, the molecular mechanisms involved in the preferential generation of mitochondrial DNA deletions in dopaminergic neurons are still unknown. To study this phenomenon, we developed novel polymerase chain reaction strategies to detect distinct mitochondrial DNA deletions and monitor their accumulation patterns. Applying these approaches in in vitro and in vivo models, we show that catecholamine metabolism drives the generation and accumulation of these mitochondrial DNA mutations. As in humans, age-related accumulation of mitochondrial DNA deletions is most prominent in dopaminergic areas of mouse brain and even higher in the catecholaminergic adrenal medulla. Dopamine treatment of terminally differentiated neuroblastoma cells, as well as stimulation of dopamine turnover in mice over-expressing monoamine oxidase B both induce multiple mitochondrial DNA deletions. Our results thus identify catecholamine metabolism as the driving force behind mitochondrial DNA deletions, probably being an important factor in the ageing-associated degeneration of dopaminergic neurons.
  • Article
    OBJECTIVE: s: Our goal was to provide bedside evidence for the potential link between cardiac mitochondrial dysfunction and arrhythmia as reported in bench studies. BACKGROUND: Atrial fibrillation (AF) is a frequent complication of cardiac surgery. Underlying mechanisms of post-operative atrial fibrillation (POAF) remain largely unknown. Since cardiac mitochondrial dysfunction has been reported in clinical conditions with high risk of POAF, we investigated whether a causal link exists between POAF onset and pre-operative function of cardiac mitochondria. METHODS: Pre-operative mitochondrial respiration and calcium retention capacity, respiratory complex activity and myocardial oxidative stress were quantified in right atrial tissue from 104 consecutive patients with metabolic syndrome, in sinus rhythm, and undergoing CABG surgery. RESULTS: In this high risk population, POAF occurred in 44% of patients. Decreased pre-operative mitochondrial respiration and increased sensitivity to calcium-induced mitochondrial permeability transition pore opening were significantly associated with POAF. ADP-stimulated mitochondrial respiration supported by palmitoyl-L-carnitine was significantly lower in POAF patients and remained independently associated with AF onset after adjustment for age, BMI, heart rate, beta-blockers and statin medication (multivariate logistic regression coefficient per unit = -0.314 ± 0.144; p = 0.028). Gene expression profile analysis identified a general down-regulation of the mitochondria/oxphos gene cluster in pre-operative atrial tissue of patients who developed AF. CONCLUSIONS: Our prospective study identifies an association between pre-operative mitochondrial dysfunction of the atrial myocardium and AF occurrence after cardiac surgery in patients with metabolic disease, providing novel insights into the link between mitochondria and arrhythmias in patients.
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    Known disease mechanisms in mitochondrial DNA (mtDNA) maintenance disorders alter either the mitochondrial replication machinery (POLG, POLG2 and C10orf2) or the biosynthesis pathways of deoxyribonucleoside 5'-triphosphates for mtDNA synthesis. However, in many of these disorders, the underlying genetic defect has yet to be discovered. Here, we identify homozygous nonsense and missense mutations in the orphan gene C20orf72 in three families with a mitochondrial syndrome characterized by external ophthalmoplegia, emaciation and respiratory failure. Muscle biopsies showed mtDNA depletion and multiple mtDNA deletions. C20orf72, hereafter MGME1 (mitochondrial genome maintenance exonuclease 1), encodes a mitochondrial RecB-type exonuclease belonging to the PD-(D/E)XK nuclease superfamily. We show that MGME1 cleaves single-stranded DNA and processes DNA flap substrates. Fibroblasts from affected individuals do not repopulate after chemically induced mtDNA depletion. They also accumulate intermediates of stalled replication and show increased levels of 7S DNA, as do MGME1-depleted cells. Thus, we show that MGME1-mediated mtDNA processing is essential for mitochondrial genome maintenance.
  • Article
    Full-text available
    Mutations of mitochondrial DNA (mtDNA) cause several well-recognized human genetic syndromes with deficient oxidative phosphorylation1, 2, 3, 4 and may also have a role in ageing and acquired diseases of old age5. We report here that hallmarks of mtDNA mutation disorders can be reproduced in the mouse using a conditional mutation strategy to manipulate the expression of the gene encoding mitochondrial transcription factor A (Tfam, previously named mtTFA), which regulates transcription and replication of mtDNA (Refs 6,7). Using a loxP-flanked Tfam allele (TfamloxP; ref. 8 ) in combination with a cre-recombinase transgene under control of the muscle creatinine kinase promoter9,10, we have disrupted Tfam in heart and muscle. Mutant animals develop a mosaic cardiac-specific progressive respiratory chain deficiency, dilated cardiomyopathy, atrioventricular heart conduction blocks and die at 2-4 weeks of age. This animal model reproduces biochemical, morphological and physiological features of the dilated cardiomyopathy of Kearns-Sayre syndrome1, 2, 3, 4. Furthermore, our findings provide genetic evidence that the respiratory chain is critical for normal heart function.
  • Article
    Normal aging is associated with a multitude of changes in the cardiovascular system, including decreased compliance of blood vessels, mild concentric left ventricular hypertrophy, an increased contribution of atrial contraction to left ventricular filling, and a higher incidence of many cardiac arrhythmias, both bradyarrhythmias and tachyarrhythmias. Conduction disorders also become more common with age, and may either be asymptomatic, or cause hemodynamic changes requiring treatment. The epidemiology of common arrhythmias and conduction disorders in the elderly is reviewed.
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    Mitochondrial disease refers to a heterogenous group of genetic disorders that result from dysfunction of the final common pathway of energy metabolism. Mitochondrial DNA mutations affect key components of the respiratory chain and account for the majority of mitochondrial disease in adults. Owing to critical dependence of the heart on oxidative metabolism, cardiac involvement in mitochondrial disease is common and may occur as the principal clinical manifestation or part of multisystem disease. Recent advances in our understanding of the clinical spectrum and genetic aetiology of cardiac involvement in mitochondrial DNA disease have important implications for cardiologists in terms of the investigation and multi-disciplinary management of patients.
  • Article
    The processes of excitation-contraction coupling in cardiac myocytes require enormous amounts of energy in the form of ATP, which is produced by oxidative phosphorylation in mitochondria. Due to the constantly varying workloads of the heart, efficient matching of energy supply to demand is a requisite for proper heart function. Ca(2+) is taken up by mitochondria via the mitochondrial Ca(2+) uniporter (MCU) where it stimulates key dehydrogenases of the Krebs cycle to match regeneration of NADH to its oxidation by the respiratory chain. The kinetics of mitochondrial Ca(2+) uptake, however, remain controversial due to the low Ca(2+) sensitivity of the MCU. Here, we review the evidence for the existence of a "mitochondrial Ca(2+) microdomain", in which the close association of the sarcoplasmic reticulum (SR) to mitochondria provides "hot spots" of very high Ca(2+) concentrations in the vicinity of mitochondria, sufficient to overcome the low Ca(2+) affinity of the MCU. Mitofusins 1 and 2 play redundant roles in regulating mitochondrial dynamics by controlling fusion of mitochondria with each other. Recent work revealed a unique role for mitofusin 2 in tethering mitochondria to the sarco-/endoplasmic reticulum in various cell types, including cardiac myocytes. Disruption of SR-mitochondrial Ca(2+) cross talk in heart failure through spatial and ionic alterations may give rise to energetic deficit and oxidative stress, two factors believed to play causal roles in the progression of the disease. On the other hand, excessive mitochondrial Ca(2+) uptake can trigger programmed necrosis, substantiating the ambiguity of the close interplay between these cousin organelles in health and disease. This article is part of a Special Issue entitled 'Focus on Cardiac Metabolism SI'.
  • Article
    Full-text available
    Respiratory chain (RC) complexes are organized into supercomplexes forming 'respirasomes'. The mechanism underlying the interdependence of individual complexes is still unclear. Here, we show in human patient cells that the presence of a truncated COX1 subunit leads to destabilization of complex IV (CIV) and other RC complexes. Surprisingly, the truncated COX1 protein is integrated into subcomplexes, the holocomplex and even into supercomplexes, which however are all unstable. Depletion of the m-AAA protease AFG3L2 increases stability of the truncated COX1 and other mitochondrially encoded proteins, whereas overexpression of wild-type AFG3L2 decreases their stability. Both full-length and truncated COX1 proteins physically interact with AFG3L2. Expression of a dominant negative AFG3L2 variant also promotes stabilization of CIV proteins as well as the assembled complex and rescues the severe phenotype in heteroplasmic cells. Our data indicate that the mechanism underlying pathogenesis in these patients is the rapid clearance of unstable respiratory complexes by quality control pathways, rather than their impaired assembly.
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    Somatic stem cell (SSC) dysfunction is typical for different progeroid phenotypes in mice with genomic DNA repair defects. MtDNA mutagenesis in mice with defective Polg exonuclease activity also leads to progeroid symptoms, by an unknown mechanism. We found that Polg-Mutator mice had neural (NSC) and hematopoietic progenitor (HPC) dysfunction already from embryogenesis. NSC self-renewal was decreased in vitro, and quiescent NSC amounts were reduced in vivo. HPCs showed abnormal lineage differentiation leading to anemia and lymphopenia. N-acetyl-L-cysteine treatment rescued both NSC and HPC abnormalities, suggesting that subtle ROS/redox changes, induced by mtDNA mutagenesis, modulate SSC function. Our results show that mtDNA mutagenesis affected SSC function early but manifested as respiratory chain deficiency in nondividing tissues in old age. Deletor mice, having mtDNA deletions in postmitotic cells and no progeria, had normal SSCs. We propose that SSC compartment is sensitive to mtDNA mutagenesis, and that mitochondrial dysfunction in SSCs can underlie progeroid manifestations.
  • Article
    Full-text available
    The mitochondrion is the main site of production of ATP that represents the source of energy for a large number of cellular processes. Mitochondrial diseases that result in a deficit in ATP production can affect almost every organ system with a large spectrum of clinical phenotypes. Cardiomyocytes are particularly vulnerable to limited ATP supply because of their large energy requirement. Abnormalities in the mitochondrial function are increasingly recognized in association with dilated and hypertrophic cardiomyopathy, cardiac conduction defects, endothelial dysfunction and coronary artery disease. Cardiologists should, therefore, be alerted to symptoms and signs suggestive of mitochondrial diseases and become familiar with the general issues related to multisystem disease management, genetic counseling and testing.
  • Article
    Aging is an inevitable time-dependent progression associated with a functional decline of the cardiovascular system even in 'healthy' individuals. Age positively correlates with an increasing risk of cardiac problems including arrhythmias. Not only the prevalence but also the severity of arrhythmias escalates with age. The reasons for this are multifactorial but dysregulation of intracellular calcium within the heart is likely to play a key role in initiating and perpetuating these life-threatening events. We now know that several aspects of cardiac calcium regulation significantly change with advancing age - changes that could produce electrical instability. Further development of knowledge of the mechanisms underlying these changes will allow us to reduce what currently is an inevitable increase in the incidence of arrhythmias in the elderly.
  • Article
    Full-text available
    Although mitochondrial mutation abundance has been recognized to increase in an age-dependent manner, the impact of mutation has been more difficult to establish. Using quantitative polymerase chain reaction, we measured the intracellular abundance of mutant and wild-type mitochondrial genomes along the length of individual laser-captured microdissected muscle fibers from aged rat quadriceps. Aged muscle fibers possessed segmental, clonal intracellular expansions of unique somatically derived mitochondrial DNA (mtDNA) deletion mutations. When the mutation abundance surpassed 90% of the total mitochondrial genomes, the fiber lost cytochrome c oxidase activity and exhibited an increase in succinate dehydrogenase activity. In addition to the mitochondrial enzymatic abnormalities, some fibers displayed abnormal morphology such as fiber splitting, atrophy, and breakage. Deletion mutation accumulation was linked to these aberrant morphologies with more severe cellular pathologies resulting from higher deletion mutation abundance. In summary, our measurements indicate that age-induced mtDNA deletion mutations expand within individual muscle fibers, eliciting fiber dysfunction and breakage.
  • Article
    Transgenic mouse models provided a powerful tool to evaluate the physiological significance of altered quantities or characteristics of specific gene products, such as cardiac ion channels. We have developed a system to record and analyze changes in the electrocardiogram in the mouse using an implantable telemetry system. The R-R and Q-T intervals were measured on individual beats and on signal-averaged complexes derived from 1, 2, or 4 s of contiguous data each hour during a 24-h period in three male and three female FVB mice. Duration of averaging had minimal effect on the measured Q-T. The Q-T interval was shown to be related to the square root of the R-R interval, and an appropriate formula for a rate-corrected Q-T interval (Q-Tc) was derived. Ketamine anesthesia was shown to markedly increase duration and variability in R-R, Q-T, and Q-Tc intervals. In conscious animals, variability in Q-T was low across animals and over time, suggesting that this should be a sensitive model for detection of changes in the Q-T interval in transgenic mice with ion channel defects.
  • Article
    Previous studies of cytochrome c oxidase (complex IV of the respiratory chain) in the heart of a 26-year-old man with longstanding Kearns-Sayre syndrome and fatal congestive cardiomyopathy had revealed the presence of randomly distributed enzyme-deficient cardiomyocytes, both in the contractile and the conducting myocardium. In the present study, the conduction system of the heart was screened for the occurrence of the common 4977 base pair deletion (8, 482-13, 459) of mitochondrial DNA (mtDNA) in formalin-fixed, paraffin-embedded tissue and compared with the contractile myocardium. Polymerase chain reaction analysis revealed that in the sinus node, the atrioventricular node, and the bundle branches, 35 to 40% of total mtDNA molecules harbored the common deletion. In contrast, in the contractile myocardium, 10 to 20% of total mtDNA was deleted (P = .05). These results demonstrate that in Kearns-Sayre syndrome, the conduction system of the heart preferentially accumulates the common deletion. This finding might help to explain the high prevalence of cardiac dysrhythmias in this syndrome.
  • Article
    In this review article about fibrosis and arrhythmias, we show that the amount of collagen, a normal element of the heart muscle, increases with age and in heart disease. The relation between fibrosis and electrophysiological parameters such as conduction, fractionation of electrograms, abnormal impulse initiation as well as arrhythmogenicity is discussed. Next to the amount of fibrosis, we offer data suggesting that collagen texture too plays a role in conduction slowing and arrhythmia vulnerability. Data are shown revealing that fibrosis can also be induced by reduced sodium channel and connexin43 expression. Finally contrast-enhanced magnetic resonance to detect fibrosis and ventricular tachycardia vulnerability in a noninvasive way as well as a reduction of fibrosis and arrhythmogenicity by inhibition of the renin-angiotensin-aldosterone system is discussed.
  • Article
    Full-text available
    Despite a high prevalence of sudden cardiac death throughout the world, the mechanisms that lead to ventricular arrhythmias are not fully understood. Over the last 20 years, a growing body of evidence indicates that cardiac mitochondria are involved in the genesis of arrhythmia. In this review, we have attempted to describe the role that mitochondria play in altering the heart's electrical function by introducing heterogeneity into the cardiac action potential. Specifically, we have focused on how the energetic status of the mitochondrial network can alter sarcolemmal potassium fluxes through ATP-sensitive potassium channels, creating a 'metabolic sink' for depolarizing wave-fronts and introducing conditions that favour catastrophic arrhythmia. Mechanisms by which mitochondria depolarize under conditions of oxidative stress are characterized, and the contributions of several mitochondrial ion channels to mitochondrial depolarization are presented. The inner membrane anion channel in particular opens upstream of other inner membrane channels during metabolic stress, and may be an effective target to prevent the metabolic oscillations that create action potential lability. Finally, we discuss therapeutic strategies that prevent arrhythmias by preserving mitochondrial membrane potential in the face of oxidative stress, supporting the notion that treatments aimed at cardiac mitochondria have significant potential in attenuating electrical dysfunction in the heart.
  • Article
    Mutations in the Twinkle (PEO1) gene are a recognized cause of autosomal dominant progressive external ophthalmoplegia (adPEO), resulting in the accumulation of multiple mitochondrial DNA (mtDNA) deletions and cytochrome c oxidase (COX)-deficient fibers in skeletal muscle secondary to a disorder of mtDNA maintenance. Patients typically present with isolated extraocular muscle involvement, with little apparent evidence of the clinical heterogeneity documented in other mtDNA maintenance disorders, in particular POLG-related disease. We reviewed the clinical, histochemical, and molecular genetics analysis of 33 unreported patients from 26 families together with all previous cases described in the literature to define the clinical phenotype associated with PEO1 mutations. Ptosis and ophthalmoparesis were almost universal clinical features among this cohort, with 52% (17/33) reporting fatigue and 33% (11/33) having mild proximal myopathy. Features consistent with CNS involvement were rarely described; however, in 24% (8/33) of the patients, cardiac abnormalities were reported. Mitochondrial histochemical changes observed in muscle showed remarkable variability, as did the secondary mtDNA deletions, which in some patients were only detected by PCR-based assays and not Southern blotting. Moreover, we report 7 novel PEO1 variants. Our data suggest a shared clinical phenotype with variable mild multiorgan involvement, and that the contribution of PEO1 mutations as a cause of adPEO may well be underestimated. Direct sequencing of the PEO1 gene should be considered in adPEO patients prior to muscle biopsy.
  • Article
    Mitochondrial dysfunction is heavily implicated in the multifactorial aging process. Aging humans have increased levels of somatic mtDNA mutations that tend to undergo clonal expansion to cause mosaic respiratory chain deficiency in various tissues, such as heart, brain, skeletal muscle, and gut. Genetic mouse models have shown that somatic mtDNA mutations and cell type-specific respiratory chain dysfunction can cause a variety of phenotypes associated with aging and age-related disease. There is thus strong observational and experimental evidence to implicate somatic mtDNA mutations and mosaic respiratory chain dysfunction in the mammalian aging process. The hypothesis that somatic mtDNA mutations are generated by oxidative damage has not been conclusively proven. Emerging data instead suggest that the inherent error rate of mitochondrial DNA (mtDNA) polymerase gamma (Pol gamma) may be responsible for the majority of somatic mtDNA mutations. The roles for mtDNA damage and replication errors in aging need to be further experimentally addressed.
  • Article
    Full-text available
    Pacemaker and conduction system myocytes play crucial roles in initiating and regulating the contraction of the cardiac chambers. Genetic defects, acquired diseases, and aging cause dysfunction of the pacemaker and conduction tissues, emphasizing the clinical necessity to understand the molecular and cellular mechanisms of their development and homeostasis. Although all cardiac myocytes of the developing heart initially possess pacemaker properties, the majority differentiates into working myocardium. Only small populations of embryonic myocytes will form the sinus node and the atrioventricular node and bundle. Recent efforts have revealed that the development of these nodal regions is achieved by highly localized suppression of working muscle differentiation, and have identified transcriptional repressors that mediate this process. This review will summarize and reflect new experimental findings on the cellular origin and the molecular control of differentiation and morphogenesis of the pacemaker tissues of the heart. It will also shed light on the etiology of inborn and acquired errors of nodal tissues.
  • Article
    To test the feasibility of cardiac MR imaging in mice using a clinical 3 Tesla whole body MR system for structural and functional analysis. Standard protocols for bright blood cine imaging were adapted for murine dimensions. To validate measurements of functional parameters the MR data were compared with high-resolution echocardiographic measurements. Cardiac imaging was carried out in CD 1 wild-type mice (n = 8). MR imaging studies were performed using a clinical 3 Tesla MR system (Achieva, Philips). All mice received 2 MR scans and 1 echocardiographic evaluation. For optimal MR signal detection a dedicated solenoid receive-only coil was used. Electrocardiogram signal was recorded using a dedicated small animal electrocardiogram monitoring unit. For imaging we used a retrospectively triggered TFE sequence with a repetition time of 12 ms and an echo time of 4 ms. A dedicated software patch allowed for triggering of cardiac frequency of up to 600 BPM. Doppler-echocardiography was performed using a VisualSonics Vevo 770 high-resolution imaging system with a 30 MHz scanhead. Axial/lateral resolution was 40 of 100 microm and temporal resolution was 150 to 300 frames/s (B-mode) and 1000 frames/s (M-mode) depending on the setting. MR imaging was successfully carried out in all mice with a sufficient temporal resolution and good signal-to-noise ratio and contrast-to-noise ratio levels allowing for identification of all relevant structures. Accordingly, there was a good scan-rescan reproducibility of MR measurements: Interassay coefficients of variance ranged from 4% for ejection fraction to 12% for endsystolic volume (ESV). Magnetic resonance imaging and echocardiography gave comparable results when using the same geometric model (Teichholz method): EDV: 60.2 +/- 6.1 microL/59.1 +/- 12.3 microL, ESV: 20.0 +/- 2.6 microL/20.7 +/- 7.7 microL, EF: 66.7% +/- 4.0%/65.2% +/- 9.9%, CO 19.5 +/- 3.6 mL/17.9 +/- 2.9 mL. Bland-Altman analysis gave acceptable limits of agreement between both methods: EDV (+28.2/-26.1), ESV (+16.3/-17.7), EF (+19.0/-16.1), CO (10.7/-7.5). When applying the Simpson's method MR volume estimates were significantly higher compared with echocardiography resulting in a lower estimate for the ejection fraction (60% +/- 3.9% vs. 66.7% +/- 4.0%). Cardiac MR imaging of mice using a clinical 3 Tesla MR system for functional analysis is feasible with sufficient spatial and temporal resolution, good repeatability and reliable results when compared with high-resolution echocardiography.
  • Article
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    1. Cardiovascular diseases most commonly occur in the elderly and are a frequent cause of disability or death. However, the effect of age itself on cardiac function is not well understood. 2. Studies in both human and animal hearts indicate that contractile function is unaffected by age while at rest. However, the ability to increase cardiac contractile force during strenuous activities, such as exercise, declines with age. 3. Similar findings have been observed in individual ventricular myocytes isolated from aged hearts. When myocytes are stimulated with β-adrenoceptor agonists or rapid pacing frequencies, aged cells show a much smaller increase in peak contractions and Ca2+ transients than young adult cells. In addition, contractions and Ca2+ transients are prolonged in aged cells compared with younger cells under these conditions. 4. These observations suggest that the age-related decline in cardiac contractile function originates at the cellular level and may reflect modifications in processes involved in excitation–contraction (EC) coupling. 5. Biochemical studies have shown that there are age-related modifications in the expression, regulation and function of a number of proteins essential to EC coupling in the heart. 6. Functional studies indicate that these changes in EC coupling proteins disrupt Ca2+ homeostasis and contribute to decrease in peak contraction and prolongation of contraction duration observed in myocytes from aged hearts. 7. The present review describes modifications in cardiac contractile function that occur in the ageing heart and evaluates underlying alterations in the EC coupling pathway that may be responsible for this decline in contractile function in ageing.
  • Article
    Age is a major risk for cardiovascular diseases. Although mitochondrial reactive oxygen species have been proposed as one of the causes of aging, their role in cardiac aging remains unclear. We have previously shown that overexpression of catalase targeted to mitochondria (mCAT) prolongs murine median lifespan by 17% to 21%. We used echocardiography to study cardiac function in aging cohorts of wild-type and mCAT mice. Changes found in wild-type mice recapitulate human aging: age-dependent increases in left ventricular mass index and left atrial dimension, worsening of the myocardial performance index, and a decline in diastolic function. Cardiac aging in mice is accompanied by accumulation of mitochondrial protein oxidation, increased mitochondrial DNA mutations and deletions and mitochondrial biogenesis, increased ventricular fibrosis, enlarged myocardial fiber size, decreased cardiac SERCA2 protein, and activation of the calcineurin-nuclear factor of activated T-cell pathway. All of these age-related changes were significantly attenuated in mCAT mice. Analysis of survival of 130 mice demonstrated that echocardiographic cardiac aging risk scores were significant predictors of mortality. The estimated attributable risk to mortality for these 2 parameters was 55%. This study shows that cardiac aging in the mouse closely recapitulates human aging and demonstrates the critical role of mitochondrial reactive oxygen species in cardiac aging and the impact of cardiac aging on survival. These findings also support the potential application of mitochondrial antioxidants in reactive oxygen species-related cardiovascular diseases.
  • Article
    Previous studies have revealed cytochrome-c-oxidase-deficient cardiomyocytes and the 4,977 base pair deletion ("common deletion") of mitochondrial DNA (position 8,482-13,459) in the heart of a patient with dilatative cardiomyopathy and Kearns-Sayre syndrome. In the present investigation the co-localization of the enzymatic and genomic defects was studied. In situ hybridization of mitochondrial DNA (mtDNA) revealed different hybridization patterns in the cytochrome-c-oxidase-deficient cells: (1) a selective reduction of the hybridization signal with an mtDNA probe recognizing the common deletion, indicating predominance of the deleted over the nondeleted mtDNA molecules in the cytochrome-c-oxidase-deficient cells; (2) a reduced hybridization signal with different mtDNA probes, indicating depletion of mtDNA; and (3) normal hybridization signals with different probes in single cytochrome-c-oxidase-deficient cardiomyocytes. These results indicate that different mechanisms may co-exist in Kearns-Sayre syndrome and may lead to defective respiratory chain function. The question of the pathogenetic interrelationship is discussed.
  • Article
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    Multiple deletions of mitochondrial DNA (mtDNA) have recently been reported in familial progressive external ophthalmoplegia (PEO), in a case of progressive encephalomyopathy, and in inherited recurrent myoglobinuria. The inheritance of familial PEO has been autosomal dominant, which indicates that a mutation in an unknown nuclear gene results in several mtDNA deletions of different sizes in these patients. We report a patient with autosomal dominant PEO, whose major clinical symptom, however, was severe retarded depression. The morphological analyses of the tissue samples derived from autopsy showed various abnormalities in the mitochondria in all the tissues studied. The activities of the respiratory chain enzymes encoded by mtDNA were remarkably reduced in the skeletal muscle. The mtDNA analyses confirmed that besides myopathy, this patient had a multisystem disorder with widespread distribution of multiple deletions of mtDNA. The highest percentage of mutated mtDNA was found in the brain, skeletal muscle and the heart, the relative quantity of mutated mtDNA correlating to the severity of the clinical symptoms.
  • Article
    Little is known concerning the molecular mechanisms responsible for changes in sarcoplasmic reticulum (SR) function during ontogenic development and aging except that the amount of SR Ca(2+)-ATPase mRNA varies in these conditions. The aim of the present work was to determine whether SR maturation requires expression of specific isoforms and synchronous accumulation of mRNAs encoding proteins located in SR. Thus, we have studied expression of SR Ca(2+)-ATPase and calsequestrin genes in the rat at different developmental stages from 14 fetal days to 24 months of age. Analysis of alternative splicing of the major Ca(2+)-ATPase gene expressed in heart by nuclease S1 mapping led us to conclude that the Ca(2+)-ATPase gene expressed in heart was not differentially spliced during ontogenic development and senescence. A single calsequestrin mRNA isoform was also detected in rat heart whatever the developmental stage. The amount of specific mRNA was then measured by dot blot and normalized to 18S ribosomal RNA or to myosin heavy chain mRNA. The amount of Ca(2+)-ATPase mRNA relative to 18S RNA increases substantially at the end of fetal life and in the early postnatal period (9.5 +/- 0.5% in the 14-15 day fetus versus 99 +/- 7% in the 4-day-old rat). A stable high level is observed during adulthood. In aged rats (24 months), Ca(2+)-ATPase mRNA represents only 44.6% the amount observed in young adults (1-2 months).(ABSTRACT TRUNCATED AT 250 WORDS)
  • Article
    To determine the effects of age on the myocardium, the functional and structural characteristics of the heart were studied in rats at 4, 12, 20, and 29 months of age. Mean arterial pressure, left ventricular pressure and its first derivative (dP/dt), and heart rate were comparable in rat groups up to 20 months. During the interval from 20 to 29 months, elevated left ventricular end-diastolic pressure and decreased dP/dt indicated that a significant impairment of ventricular function occurred with senescence. In the period between 4 and 12 months, a reduction of nearly 19% in the total number of myocytes was measured in both ventricles. In the subsequent ages, similar decreases in myocyte cell number were found in the left ventricle, whereas in the right ventricle, the initial loss was fully reversed by 20 months. Moreover, from 20 to 29 months, a 59% increase in the aggregate number of myocytes occurred in the right ventricular myocardium. In the left ventricle, a 3% increment was also seen, but this small change was not statistically significant. These estimations of myocyte cellular hyperplasia, however, were complicated by the fact that cell loss continued to take place with age. The volume fraction of collagen in the tissue, in fact, progressively increased from 8% and 7% at 4 months to 16% and 22% at 29 months in the left and right ventricles, respectively. In conclusion, myocyte cellular hyperplasia tends to regenerate the ventricular mass being lost with age in the adult mammalian rat heart.
  • Article
    Intracellular Ca2+ release and reuptake are necessary for normal contraction and relaxation of the human heart. Intracellular Ca2+ transients were recorded with aequorin during isometric contraction of myocardium from patients with end-stage heart failure. In contrast to controls, contractions and Ca2+ transients of muscles from failing hearts were markedly prolonged, and the Ca2+ transients exhibited two distinct components. Muscles from the failing hearts showed a diminished capacity to restore a low resting Ca2+ level during diastole. These data obtained in actively contracting human myocardium suggest that intracellular Ca2+ handling is abnormal and might cause both systolic and diastolic dysfunction in heart failure. The inotropic effectiveness of drugs that act to increase intracellular levels of cyclic adenosine monophosphate (AMP), such as beta-adrenergic agonists and phosphodiesterase inhibitors, was markedly reduced in muscles from patients with heart failure. In contrast, the effectiveness of inotropic stimulation with drugs that act by cyclic AMP-independent mechanisms, such as the cardiotonic steroids and DPI 201-106, were preserved. Stimulation of intracellular cyclic AMP production by the adenylate cyclase activator forskolin restored the inotropic response to phosphodiesterase inhibitors. These studies indicate that an abnormality in cyclic AMP production may be a fundamental defect in patients with end-stage heart failure that may markedly diminish the effectiveness of agents that depend on generation of this nucleotide for a positive inotropic effect. Moreover, deficient production of cyclic AMP seems, at least in part, to account for the reversal of the force-frequency relation that characterizes failing myocardium. Of interest, direct measurement of total cellular cyclic AMP content and protein kinase activity did not reveal significant differences between the control and myopathic tissue, suggesting the presence in human ventricular muscle of physiologically distinct compartmentalized pools of cyclic AMP. Finally, changes in the sensitivity of the contractile apparatus to Ca2+ also seem to play an important role in the differential responsiveness to drugs of myopathic versus normal human myocardium.
  • Article
    Cytochrome-c-oxidase, the terminal enzyme of the respiratory chain, was studied in 140 hearts from men obtained at autopsy revealing randomly distributed cardiomyocytes without enzyme activity. The expression of the defect was independent of an underlying heart disease and was observed both in normal hearts and in hearts with hypertrophy and/or coronary arteriosclerosis. In contrast, age was a discriminating factor: The defects occurred sporadically in the second decade, but were regularly present from the sixth decade on. Also, the number of defects/sq cm (defect density) increased with age from 2 to 3 in the second and third decade, to about 50 defects in advanced age. Irrespective of the defect density, the enzyme defect always affected isolated cardiomyocytes and ended abruptly at the intercalated disc of neighboring heart muscle cells, as revealed by ultracytochemistry. The results indicate that cytochrome-c-oxidase deficient heart muscle cells represent a degenerative lesion associated with cellular ageing and may be involved in the reduction of myocardial contractile ability in senescence.
  • Article
    The clinical and postmortem findings in a 26 year old man with Kearns-Sayre syndrome are described. In the last years of his life he suffered from cardiac arrhythmias and a congestive cardiomyopathy, dying of cardiac pump failure. The heart was enlarged, especially the left ventricle which was fibrotic and excessively dilated. Histological and fine structural investigation revealed an excessive loss of myofibrils and an increase of enlarged mitochondria with lamellar and atypically tubular cristae in widespread heart muscle cells. Mitochondrial anomalies were also observed in some cells of the conductive system. This patient thus suffered not only from a mitochondrial myopathy with ragged red fibers but also from a fatal mitochondrial cardiomyopathy. The anomalies observed in the mitochondria of the conductive system cells suggest that the well-known conductive abnormalities in patients with Kearns-Sayre syndrome might be at least partly caused by disturbed function of these mitochondria.
  • Article
    Mutational damage to human mitochondrial DNA (mtDNA) can cause disorders in oxidative phosphorylation; speculation that such damage is involved in degenerative diseases and aging is common. We have detected deletions in mouse mtDNA which resemble those found in elderly humans or patients with certain mtDNA disorders. Five different mtDNA deletions, predicted from the positions of short, direct DNA repeats, were present in aged, but not young, mice. Deleted regions were surrounded by either exact or inexact repeats and occurred in both the major and minor regions of the mtDNA genome. The abundance of a particular deletion was generally related to the thermodynamic stability of the bounding repeat sequence. Deletions in aged mice were present at low levels (less than 0.01% of total mtDNA). However, in contrast to results from aged humans, deletions were more abundant in liver than in brain, heart, or skeletal muscle. These results make it possible to predict the location and relative abundance of deletions in any sequenced mtDNA, including inbred mouse strains differing in inherent natural lifespan. The inbred mouse model will allow a critical examination of the relationship between the presence and abundance of mtDNA deletions and the aging process.
  • Article
    This paper presents a collection of quantitative values for respiratory chain activities in human tissues. These were measured in the most widely used tissues in screening procedures for respiratory chain deficiencies. Investigations were mainly carried out using the different standardized micro-methods previously detailed (Rustin et al., Clin Chim Acta, 1994). The potential effect of the age of the patients on both absolute and relative levels of respiratory chain activities in their skeletal muscle tissue was first considered. No evidence for any significant difference between the various age groups in the studied population (ranging from 0 to above 50 years of age) was observed. Moreover, a quite similar picture of the organization of the respiratory chain was suggested independent of the tissue or the cells investigated. In particular, it was found that roughly identical enzyme activity ratios could be measured in all tissues, which allowed study of the differential involvement of organs and tissues in patients potentially affected by a respiratory chain deficiency. Some tissue-specific features were, however, observed, including varying rates of glycerol-3-phosphate dehydrogenase activities and increased succinate dehydrogenase activity in liver. The technical limitations remaining in the investigations of respiratory chain disorders in man are discussed in the conclusion.
  • Article
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    This paper describes our present strategy for the investigation of respiratory chain disorders in humans. Because very few of the underlying mutations causing mitochondrial disorders in humans are currently known, biochemical studies constitute a major tool in screening procedures for respiratory chain deficiencies. All biochemical and molecular methods described are scaled-down methods, allowing investigation in both adults and young children. Polarographic studies and/or spectrophotometric studies on whole cells (circulating lymphocytes), isolated mitochondria (skeletal muscle) and tissue homogenates are presented. Advantages and limitations of each approach, as well as useful parameters for the characterization of defects and comparison between various tissues are discussed.
  • Article
    The MHC class II gene Aa was disrupted by targeted mutation in embryonic stem (ES) cells derived from C57BL/6 mice to prevent expression of MHC class II molecules. Contrary to previous reports, the effect of the null-mutation on T cell development was investigated in C57BL/6 mice, which provide a defined genetic background. The complete lack of cell surface expression of MHC class II molecules in B6-Aa0/Aa0 homozygous mutant mice was directly demonstrated by cytofiuorometric analysis using anti-Ab and anti-la specific mAbs. Development of CD4+CD8− T cells in the thymus was largely absent except for a small population of thymocytes expressing high levels of CD4 together with low amounts of CD8. The majority of these cells express the TCR at high density. Although mature CD4+CD8− T cells were undetectable in the thymus, some T cells with a CD4+CD8−TCRhigh phenotype were found in lymph nodes and spleen. Peripheral T cells from themutant mice can be polyclonally activated in vitro with the mitogen concanavalin A. However, they could not be stimulated with staphylococcal enterotoxin B in autologous lymphocyte reactions, thereby demonstrating the absence of MHC class II expression in these mice. Peripheral B cells in B6-Aa0/Aa0 mutants were functional and responded to the T cell independent antigen levan by the production of antigenspecific IgM antibodies similar to wild-type cells. The B6-Aa0/Aa0 mutant mice described in this study represent an important tool to investigate the involvement of MHC class II molecules in lymphocyte maturation and the immune response.
  • Article
    To determine the prevalence of palpitations, cardiac arrhythmias and associated cardiovascular risk factors in an ambulatory elderly population, 1454 ambulatory elderly people (219 men and 1235 women, age range 60-94 years) were assessed in a territory-wide health survey including anthropometric measurements, biochemical blood tests, questionnaire interview and resting surface ECG examination. Prevalence of palpitations and ECG abnormalities were determined and correlated with coronary risk factors and biochemical blood tests. Palpitations were present in 121 subjects (8.3%) and cardiac arrhythmias were found in 183 subjects (12.6%). Conduction abnormalities and sinus bradycardia were the commonest findings (9.8%). Premature beats (atrial 2.3%; ventricular 1%) were the next most frequent arrhythmia. Atrial fibrillation was the commonest sustained arrhythmia that was present in 19 subjects (1.3%). Compared with those without arrhythmia on ECG, people with arrhythmias were predominantly males and were older (72 +/- 8 years vs. 70 +/- 6 years, P < 0.05), had a higher prevalence of smoking (12.9% vs. 5%, P < 0.05) and coronary heart disease (30.7% vs. 11.4%, P < 0.05). The prevalence of palpitations between subjects with documented arrhythmias (excluding conduction disturbance) and those without arrhythmias on surface ECG was similar (9% vs. 7.7%, P = N.S.). We conclude that cardiac arrhythmias are common in the elderly and are often asymptomatic. Subjects with ECG documented arrhythmias are more common in males, and are associated with smoking and ischaemic heart disease. Palpitation was a frequent complaint in the ambulatory elderly with no bearing on arrhythmias recorded on resting ECG.
  • Article
    This chapter presents the cytochemical and immunohistochemical methods that, in the experience, appear to be the most reliable for the correct identification of mitochondria on frozen tissue sections, to illustrate their potential using specific examples, and to provide an updated version of the methods. Although the described protocols refer to muscle mitochondria, the methods described can be applied to any cell type. The mitochondria are the primary adenosine triphosphate (ATP)-generating organelles in all mammalian cells and they contain their own DNA (mtDNA), which is maternally inherited. The human mitochondrial genome contains genes encoding for thirteen subunits of different respiratory complexes. These include seven subunits of complex I (NADH dehydrogenase–ubiquinone oxidoreductase), one subunit of complex III (ubiquinone–cytochrome-c oxidoreductase), three subunits of complex IV (cytochrome-c oxidase (COX)), and two subunits of complex V (ATP synthase). Although mitochondria have their transcriptional and translational machinery, most of the proteins located within mitochondria are encoded by the nuclear DNA (nDNA). These nuclear gene products are synthesized on cytoplasmic ribosomes and are subsequently imported into the mitochondria.
  • Article
    Ventricular arrhythmias can be challenging because there are several investigational and therapeutic options. The problem is even more challenging in the elderly because ventricular arrhythmias increase with advancing age, as does the prevalence of structural heart disease, including left ventricular hypertrophy. Furthermore, there may be a reluctance to subject elderly patients to invasive diagnostic studies and device therapy. However, although the elderly are at increased risk for procedure-related morbidity and mortality, they benefit as much as their younger counterparts. Selection of pharmacological therapy for the elderly should take altered drug absorption metabolism and excretion into consideration. Finally, beta-blockers tend not to be used as often in the elderly as in younger postmyocardial infarction patients, and the use of beta-blockers should be encouraged in the absence of contraindications.
  • Article
    Skeletal muscle insulin resistance is among the earliest detectable defects in humans with type 2 diabetes mellitus. To determine the contribution of muscle insulin resistance to the metabolic phenotype of diabetes, we used the Cre-loxP system to disrupt the insulin receptor gene in mouse skeletal muscle. The muscle-specific insulin receptor knockout mice exhibit a muscle-specific > 95% reduction in receptor content and early signaling events. These mice display elevated fat mass, serum triglycerides, and free fatty acids, but blood glucose, serum insulin, and glucose tolerance are normal. Thus, insulin resistance in muscle contributes to the altered fat metabolism associated with type 2 diabetes, but tissues other than muscle appear to be more involved in insulin-regulated glucose disposal than previously recognized.
  • Article
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    Quantitative information on the cell-to-cell distribution of all possible mitochondrial DNA (mtDNA) mutations in young and aged tissues is needed to assess the relevance of these mutations to the aging process. In the present study, we used PCR amplification of full-length mitochondrial genomes from single cells to scan human cardiomyocytes for all possible large deletions in mtDNA. Analysis of more than 350 individual cells that were derived from three middleaged and four centenarian donors demonstrates that while most of the cells contain no deletions, in certain cardiomyocytes a significant portion of the mtDNA molecules carried one particular deletion. Different affected cells contained different deletions. Although similar numbers of cells were screened for each donor, these deletion-rich cells were found only in the hearts of old donors, where they occurred at a frequency of up to one in seven cells. These initial observations demonstrate the efficiency of the method and indicate that mitochondrial mutations have the potential to play an important role in human myocardial aging.
  • Article
    We have examined the transcript levels of a variety of oxidative phosphorylation (OXPHOS) and associated bioenergetic genes in tissues of a patient carrying the myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) A3243G mitochondrial DNA (mtDNA) mutation and the skeletal muscles of 14 patients harboring other pathogenic mtDNA mutations. The patients' tissues, which harbored 88% or more mutant mtDNA, had increased levels of mtDNA transcripts, increased nuclear OXPHOS gene transcripts including the ATP synthase beta subunit and the heart-muscle isoform of the adenine nucleotide translocator, and increased ancillary gene transcripts including muscle mitochondrial creatine phosphokinase, muscle glycogen phosphorylase, hexokinase I, muscle phosphofructokinase, the E1alpha subunit of pyruvate dehydrogenase, and the ubiquinone oxidoreductase. A similar coordinate induction of bioenergetic genes was observed in the muscle biopsies of severe pathologic mtDNA mutations. The more significant coordinated expression was found in muscle from patients with the MELAS, myoclonic epilepsy with ragged red fibers, and chronic progressive external ophthalmoplegia deletion syndromes, with ragged red muscle fibers and mitochondrial paracrystalline inclusions. High levels of mutant mtDNAs were linked to a high induction of the mtDNA and nuclear OXPHOS genes and of several associated bioenergetic genes. These observations suggest that human tissues attempt to compensate for OXPHOS defects associated with mtDNA mutations by stimulating mitochondrial biogenesis, possibly mediated through redox-sensitive transcription factors.
  • Article
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    Sublimons, originally identified in plant mitochondria, are defined as rearranged mtDNA molecules present at very low levels. We have analysed the primary structures of sublimons found in human cells and tissues and estimated their abundance. Each tissue of a given individual contains a wide range of different sublimons and the most abundant species differ between tissues in a substantially systematic manner. Sublimons are undetectable in rho(0) cells, indicating that they are bona fide derivatives of mtDNA. They are most prominent in post-mitotic tissue subject to oxidative stress. Rearrangement break-points, often defined by short direct repeats, are scattered, but hotspot regions are clearly identifiable, notably near the end of the D-loop. The region between the replication origins is therefore frequently eliminated. One other hotspot region is located adjacent to a known site of protein binding, suggesting that recombination may be facilitated by protein-protein interactions. For a given primary rearrangement, both deleted and partially duplicated species can be detected. Although each sublimon is typically present at a low level, at most a few copies per cell, sublimon abundance in a given tissue can vary over three orders of magnitude between healthy individuals. Collectively, therefore, they can represent a non-negligible fraction of total mtDNA. Their structures are very similar to those of the rearranged molecules found in pathological states, such as adPEO and MNGIE; therefore, we propose that, as in plants, human mtDNA sublimons represent a pool of variant molecules that can become amplified under pathological conditions, thus contributing to cellular dysfunction.
  • Article
    The incidence of atrial fibrillation increases with age. We hypothesized that aging-associated changes in the atrial action potential (AP) and conduction velocity provide a substrate for abnormal conduction and arrhythmogenesis. We used microelectrode techniques to record AP from the endocardium of the right atrial wall of dogs aged 1-5 (adult) and >8 years (old). Conduction velocity was measured between two microelectrodes 3-10 mm apart. Histological study was carried out to assess fibrosis. Whereas resting potential, AP amplitude and V(max) did not differ with age, the plateau was more negative and AP duration was longer in old tissue. The L-type calcium current (I(Ca,L)) agonist Bay K8644 (10(-8)-10(-6) mol/l) elevated the plateau and shortened APD more in old than in adult, such that AP contour in old atria approached that of adult. In contrast, the I(Ca,L) blocker nisoldipine (10(-8)-10(-5) mol/l) depressed the plateau in adult and had no effect in old. There was no difference between the two groups in conduction velocity of normal beats, whereas for early premature impulses, reduced conduction velocity and a wider time window manifesting slow conduction were detected in old in comparison to adult tissue. A twofold increase in the amount of fibrous tissue was detected in old atria. Our data show significant differences in contour of AP in adult and old atria. The responses to Bay K8644 and nisoldipine suggest a decreased I(Ca,L) in old atrial tissue. The alterations in AP contour and increased fibrosis may be responsible for slower conduction of early premature beats in old atria. The age-related changes in conduction of premature beats are consistent with those observed in patients with paroxysmal atrial fibrillation and may contribute to the greater propensity to atrial fibrillation in the aged.
  • Article
    Age-related changes in cardiovascular function and structure in healthy adult volunteer community dwelling subjects (from 20 to 85 years) is remarkable for changes in pump function [impaired left ventricular (LV) ejection reserve capacity manifest by a reduced ejection fraction and accompanied by diminished cardioacceleration, LV dilation at end diastole and an altered diastolic filling pattern] and increased vascular afterloading. There is also evidence for a reduction in the number of cardiac myocytes with advancing age. Subcellular changes with aging (best understood in rodents) include certain regulatory factors of excitation-contraction-relaxation coupling (i.e. calcium handling), modulation by adrenergic receptor (AR) stimulation, and changes in the generation and sensitivity to the damaging effects of ROS. Coordinated changes in gene expression and/or protein function with aging result in a prolonged action potential (AP), Ca(i) transient, and contraction. L-type Ca(2+) current (I(Ca)) inactivates more slowly, and outwardly-directed K(+) currents are reduced, and likely contribute to AP-prolongation. The rate of Ca(2+) sequestration by the sarcoplasmic reticulum (SR) decreases in the senescent myocardium, in part underlying the prolonged Ca(i) transient. An age-associated reduction in transcription of the SERCA2 gene, coding for the SR Ca(2+) pump, accounts in part for a decrease in the SR pump site density. The contractile response to both beta(1)-AR and beta(2)-AR stimulation diminishes with aging due to decreased adrenergic augmentation of I(Ca), and thus the Ca(i) transient, in senescent vs. young hearts. The age-associated reduction in the postsynaptic response of myocardial cells to beta(1)-AR stimulation appears to be due to multiple changes in molecular and biochemical receptor coupling and post-receptor mechanisms. An increased basal production of ROS is paralleled by increased ROS-sensitivity, markers of chronic ROS damage and mitochondrial functional decline. Overall, these changes lead to a diminished (but not necessarily exhausted) capacity of the heart to adapt to physiological or pathological stress with advancing age.
  • Article
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    The study of mitochondrial diseases has revealed dramatic variability in the phenotypic presentation of mitochondrial genetic defects. To attempt to understand this variability, different authors have studied energy metabolism in transmitochondrial cell lines carrying different proportions of various pathogenic mutations in their mitochondrial DNA. The same kinds of experiments have been performed on isolated mitochondria and on tissue biopsies taken from patients with mitochondrial diseases. The results have shown that, in most cases, phenotypic manifestation of the genetic defect occurs only when a threshold level is exceeded, and this phenomenon has been named the 'phenotypic threshold effect'. Subsequently, several authors showed that it was possible to inhibit considerably the activity of a respiratory chain complex, up to a critical value, without affecting the rate of mitochondrial respiration or ATP synthesis. This phenomenon was called the 'biochemical threshold effect'. More recently, quantitative analysis of the effects of various mutations in mitochondrial DNA on the rate of mitochondrial protein synthesis has revealed the existence of a 'translational threshold effect'. In this review these different mitochondrial threshold effects are discussed, along with their molecular bases and the roles that they play in the presentation of mitochondrial diseases.
  • Article
    The cardiac troponin T (TnT) I79N mutation has been linked to familial hypertrophic cardiomyopathy and high incidence of sudden death, despite causing little or no cardiac hypertrophy in patients. Transgenic mice expressing mutant human TnT (I79N-Tg) have increased cardiac contractility, but no ventricular hypertrophy or fibrosis. Enhanced cardiac function has been associated with myofilament Ca2+ sensitization, suggesting altered cellular Ca2+ handling. In the present study, we compare cellular Ca2+ transients and electrophysiological parameters of 64 I79N-Tg and 106 control mice in isolated myocytes, isolated perfused hearts, and whole animals. Ventricular action potentials (APs) measured in isolated I79N-Tg hearts and myocytes were significantly shortened only at 70% repolarization. No significant differences were found either in L-type Ca2+ or transient outward K+ currents, but inward rectifier K+ current (IK1) was significantly decreased. More critically, Ca2+ transients of field-stimulated ventricular I79N-Tg myocytes were reduced and had slow decay kinetics, consistent with increased Ca2+ sensitivity of I79N mutant fibers. AP differences were abolished when myocytes were dialyzed with Ca2+ buffers or after the Na+-Ca2+ exchanger was blocked by Li+. At higher pacing rates or in presence of isoproterenol, diastolic Ca2+ became significantly elevated in I79N-Tg compared with control myocytes. Ventricular ectopy could be induced by isoproterenol-challenge in isolated I79N-Tg hearts and anesthetized I79N-Tg mice. Freely moving I79N-Tg mice had a higher incidence of nonsustained ventricular tachycardia (VT) during mental stress (warm air jets). We conclude that the TnT-I79N mutation causes stress-induced VT even in absence of hypertrophy and/or fibrosis, arising possibly from the combination of AP remodeling related to altered Ca2+ transients and suppression of IK1.
  • Article
    The authors describe siblings with progressive external ophthalmoplegia (PEO) due to a novel heterozygous A to G transition at nucleotide 955 of C10Orf2 (Twinkle). The mutation was not identified in parents' blood, hair follicles, buccal mucosa, or urinary epithelium, indicating germ line mosaicism. One sibling presented with sensory ataxic neuropathy, dysarthria, and ophthalmoparesis (SANDO), a phenotype previously associated with the POLG1 gene, highlighting the clinical overlap in autosomal PEO.
  • Article
    Although the incidence of atrial fibrillation (AF) increases with age, the cellular electrophysiological changes that render the atria of aged individuals more susceptible to AF remain poorly understood. We hypothesized that dispersion of atrial repolarization increases with aging, creating a substrate for initiation of AF. Four groups of dogs were studied: adult and old dogs in normal sinus rhythm (SR) and adult and old dogs with chronic AF (CAF) induced by rapid atrial pacing. In each dog, action potentials (AP) were recorded with microelectrodes from isolated endocardial preparations of four regions of right atrium and three regions of left atrium. Two indices of AP duration (APD) heterogeneity were obtained in each dog by calculating standard deviation (SD) and the coefficient of variation (COV=[SD/mean] x 100%). In SR groups, APD averaged across all regions was significantly longer in old than in adult tissues. Both indices of APD heterogeneity were higher in old dogs in comparison to adult. At both ages, CAF was associated with significant APD shortening and a decrease in APD adaptation to rate. While CAF significantly increased both indices of APD heterogeneity in adult dogs, it significantly decreased them in old dogs. The increase of spatial variability in repolarization in old atria may contribute to the initiation of AF in the aged. CAF-induced APD shortening and a decrease in APD adaptation appear to be important for the maintenance of sustained AF in both adult and old atria. The CAF-induced increase in dispersion of repolarization may be important for AF stabilization in adults, while previously reported fibrosis and slowed conduction of premature beats may be important in the old for both AF initiation during SR and subsequent stabilization of AF.
  • Article
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    Defects of mitochondrial DNA (mtDNA) maintenance have recently been associated with inherited neurodegenerative and muscle diseases and the aging process. Twinkle is a nuclear-encoded mtDNA helicase, dominant mutations of which cause adult-onset progressive external ophthalmoplegia (PEO) with multiple mtDNA deletions. We have generated transgenic mice expressing mouse Twinkle with PEO patient mutations. Multiple mtDNA deletions accumulate in the tissues of these mice, resulting in progressive respiratory dysfunction and chronic late-onset mitochondrial disease starting at 1 year of age. The muscles of the mice faithfully replicate all of the key histological, genetic, and biochemical features of PEO patients. Furthermore, the mice have progressive deficiency of cytochrome c oxidase in distinct neuronal populations. These “deletor” mice do not, however, show premature aging, indicating that subtle accumulation of mtDNA deletions and progressive respiratory chain dysfunction are not sufficient to accelerate aging. This model is a valuable tool for therapy development and testing for adult-onset mitochondrial disorders. • mouse model • progressive external ophthalmoplegia • mitochondrial DNA replication