Epicardial deletion of Sox9 leads to myxomatous valve degeneration and identifies Cd109 as a novel gene associated with valve development.

Epicardial-derived cells (EPDCs) are involved in the regulation of myocardial growth and coronary vascularization and are critically important for proper development of the atrioventricular (AV) valves. SOX9 is a transcription factor expressed in a variety of epithelial and mesenchymal cells in the developing heart, including EPDCs. To determine the role of SOX9 in epicardial development, an epicardial-specific Sox9 knockout mouse model was generated. Deleting Sox9 from the epicardial cell lineage impairs the ability of EPDCs to invade both the ventricular myocardium and the developing AV valves. After birth, the mitral valves of these mice become myxomatous with associated abnormalities in extracellular matrix organization. This phenotype is reminiscent of that seen in humans with myxomatous mitral valve disease (MVD). An RNA-seq analysis was conducted in an effort to identify genes associated with this myxomatous degeneration. From this experiment, Cd109 was identified as a gene associated with myxomatous valve pathogenesis in this model. Cd109 has never been described in the context of heart development or valve disease. This study highlights the importance of SOX9 in the regulation of epicardial cell invasion-emphasizing the importance of EPDCs in regulating AV valve development and homeostasis-and reports a novel expression profile of Cd109, a gene with previously unknown relevance in heart development.

The prevalent I686T human variant and loss-of-function mutations in the cardiomyocyte-specific kinase gene TNNI3K cause adverse contractility and concentric remodeling in mice.

TNNI3K expression worsens disease progression in several mouse heart pathology models. TNNI3K expression also reduces the number of diploid cardiomyocytes, which may be detrimental to adult heart regeneration. However, the gene is evolutionarily conserved, suggesting a beneficial function that has remained obscure. Here, we show that C57BL/6J-inbred Tnni3k mutant mice develop concentric remodeling, characterized by ventricular wall thickening and substantial reduction of cardiomyocyte aspect ratio. This pathology occurs in mice carrying a Tnni3k null allele, a K489R point mutation rendering the protein kinase-dead, or an allele corresponding to human I686T, the most common human non-synonymous TNNI3K variant, which is hypomorphic for kinase activity. Mutant mice develop these conditions in the absence of fibrosis or hypertension, implying a primary cardiomyocyte etiology. In culture, mutant cardiomyocytes were impaired in contractility and calcium dynamics and in protein kinase A signaling in response to isoproterenol, indicating diminished contractile reserve. These results demonstrate a beneficial function of TNNI3K in the adult heart that might explain its evolutionary conservation and imply that human TNNI3K variants, in particular the widespread I686T allele, may convey elevated risk for altered heart geometry and hypertrophy.

A novel transgenic Cre allele to label mouse cardiac conduction system.

The cardiac conduction system is a network of heterogeneous cell population that initiates and propagates electric excitations in the myocardium. Purkinje fibers, a network of specialized myocardial cells, comprise the distal end of the conduction system in the ventricles. The developmental origins of Purkinje fibers and their roles during cardiac physiology and arrhythmia have been reported. However, it is not clear if they play a role during ischemic injury and heart regeneration. Here we introduce a novel tamoxifen-inducible Cre allele that specifically labels a broad range of components in the cardiac conduction system while excludes other cardiac cell types and vital organs. Using this new allele, we investigated the cellular and molecular response of Purkinje fibers to myocardial injury. In a neonatal mouse myocardial infarction model, we observed significant increase in Purkinje cell number in regenerating myocardium. RNA-Seq analysis using laser-captured Purkinje fibers showed a unique transcriptomic response to myocardial infarction. Our finds suggest a novel role of cardiac Purkinje fibers in heart injury.

Pitx2 promotes heart repair by activating the antioxidant response after cardiac injury.

Myocardial infarction results in compromised myocardial function and heart failure owing to insufficient cardiomyocyte self-renewal. Unlike many vertebrates, mammalian hearts have only a transient neonatal renewal capacity. Reactivating primitive reparative ability in the mature mammalian heart requires knowledge of the mechanisms that promote early heart repair. By testing an established Hippo-deficient heart regeneration mouse model for factors that promote renewal, here we show that the expression of Pitx2 is induced in injured, Hippo-deficient ventricles. Pitx2-deficient neonatal mouse hearts failed to repair after apex resection, whereas adult mouse cardiomyocytes with Pitx2 gain-of-function efficiently regenerated after myocardial infarction. Genomic analyses indicated that Pitx2 activated genes encoding electron transport chain components and reactive oxygen species scavengers. A subset of Pitx2 target genes was cooperatively regulated with the Hippo pathway effector Yap. Furthermore, Nrf2, a regulator of the antioxidant response, directly regulated the expression and subcellular localization of Pitx2. Pitx2 mutant myocardium had increased levels of reactive oxygen species, while antioxidant supplementation suppressed the Pitx2 loss-of-function phenotype. These findings reveal a genetic pathway activated by tissue damage that is essential for cardiac repair.

Tnni3k alleles influence ventricular mononuclear diploid cardiomyocyte frequency.

Recent evidence implicates mononuclear diploid cardiomyocytes as a proliferative and regenerative subpopulation of the postnatal heart. The number of these cardiomyocytes is a complex trait showing substantial natural variation among inbred mouse strains based on the combined influences of multiple polymorphic genes. One gene confirmed to influence this parameter is the cardiomyocyte-specific kinase Tnni3k. Here, we have studied Tnni3k alleles across a number of species. Using a newly-generated kinase-dead allele in mice, we show that Tnni3k function is dependent on its kinase activity. In an in vitro kinase assay, we show that several common human TNNI3K kinase domain variants substantially compromise kinase activity, suggesting that TNNI3K may influence human heart regenerative capacity and potentially also other aspects of human heart disease. We show that two kinase domain frameshift mutations in mice cause loss-of-function consequences by nonsense-mediated decay. We further show that the Tnni3k gene in two species of mole-rat has independently devolved into a pseudogene, presumably associated with the transition of these species to a low metabolism and hypoxic subterranean life. This may be explained by the observation that Tnni3k function in mice converges with oxidative stress to regulate mononuclear diploid cardiomyocyte frequency. Unlike other studied rodents, naked mole-rats have a surprisingly high (30%) mononuclear cardiomyocyte level but most of their mononuclear cardiomyocytes are polyploid; their mononuclear diploid cardiomyocyte level (7%) is within the known range (2-10%) of inbred mouse strains. Naked mole-rats provide further insight on a recent proposal that cardiomyocyte polyploidy is associated with evolutionary acquisition of endothermy.

Pitx2 maintains mitochondrial function during regeneration to prevent myocardial fat deposition.

Loss of the paired-like homeodomain transcription factor 2 (Pitx2) in cardiomyocytes predisposes mice to atrial fibrillation and compromises neonatal regenerative capacity. In addition, Pitx2 gain-of-function protects mature cardiomyocytes from ischemic injury and promotes heart repair. Here, we characterized the long-term myocardial phenotype following myocardial infarction (MI) in Pitx2 conditional-knockout (Pitx2 CKO) mice. We found adipose-like tissue in Pitx2 CKO hearts 60 days after MI induced surgically at postnatal day 2 but not at day 8. Molecular and cellular analyses showed the onset of adipogenic signaling in mutant hearts after MI. Lineage tracing experiments showed a non-cardiomyocyte origin of the de novo adipose-like tissue. Interestingly, we found that Pitx2 promotes mitochondrial function through its gene regulatory network, and that the knockdown of a key mitochondrial Pitx2 target gene, Cox7c, also leads to the accumulation of myocardial fat tissue. Single-nuclei RNA-seq revealed that Pitx2-deficient hearts were oxidatively stressed. Our findings reveal a role for Pitx2 in maintaining proper cardiac cellular composition during heart regeneration via the maintenance of proper mitochondrial structure and function.

Divergent Requirements for EZH1 in Heart Development Versus Regeneration.

RATIONALE: Polycomb repressive complex 2 is a major epigenetic repressor that deposits methylation on histone H3 on lysine 27 (H3K27me) and controls differentiation and function of many cells, including cardiac myocytes. EZH1 and EZH2 are 2 alternative catalytic subunits with partial functional redundancy. The relative roles of EZH1 and EZH2 in heart development and regeneration are unknown. OBJECTIVE: We compared the roles of EZH1 versus EZH2 in heart development and neonatal heart regeneration. METHODS AND RESULTS: Heart development was normal in Ezh1-/- (Ezh1 knockout) and Ezh2f/f::cTNT-Cre (Ezh2 knockout) embryos. Ablation of both genes in Ezh1-/-::Ezh2f/f::cTNT-Cre embryos caused lethal heart malformations, including hypertrabeculation, compact myocardial hypoplasia, and ventricular septal defect. Epigenome and transcriptome profiling showed that derepressed genes were upregulated in a manner consistent with total EZH dose. In neonatal heart regeneration, Ezh1 was required, but Ezh2 was dispensable. This finding was further supported by rescue experiments: cardiac myocyte-restricted re-expression of EZH1 but not EZH2 restored neonatal heart regeneration in Ezh1 knockout. In myocardial infarction performed outside of the neonatal regenerative window, EZH1 but not EZH2 likewise improved heart function and stimulated cardiac myocyte proliferation. Mechanistically, EZH1 occupied and activated genes related to cardiac growth. CONCLUSIONS: Our work unravels divergent mechanisms of EZH1 in heart development and regeneration, which will empower efforts to overcome epigenetic barriers to heart regeneration.

Hippo signaling impedes adult heart regeneration.

Heart failure due to cardiomyocyte loss after ischemic heart disease is the leading cause of death in the United States in large part because heart muscle regenerates poorly. The endogenous mechanisms preventing mammalian cardiomyocyte regeneration are poorly understood. Hippo signaling, an ancient organ size control pathway, is a kinase cascade that inhibits developing cardiomyocyte proliferation but it has not been studied postnatally or in fully mature adult cardiomyocytes. Here, we investigated Hippo signaling in adult cardiomyocyte renewal and regeneration. We found that unstressed Hippo-deficient adult mouse cardiomyocytes re-enter the cell cycle and undergo cytokinesis. Moreover, Hippo deficiency enhances cardiomyocyte regeneration with functional recovery after adult myocardial infarction as well as after postnatal day eight (P8) cardiac apex resection and P8 myocardial infarction. In damaged hearts, Hippo mutant cardiomyocytes also have elevated proliferation. Our findings reveal that Hippo signaling is an endogenous repressor of adult cardiomyocyte renewal and regeneration. Targeting the Hippo pathway in human disease might be beneficial for the treatment of heart disease.

Pleural plaques and lung function in the Marysville worker cohort: a re-analysis.

BACKGROUND: In the 2014 Integrated Risk Information System (IRIS) assessment for Libby amphibole asbestos (LAA), US EPA calculated a reference concentration (RfC) based on the prevalence of pleural plaques in a group of vermiculite workers in Marysville, Ohio. This RfC is based on the assumption that pleural plaques are associated with adverse lung function. In this study, we evaluated the association between pleural plaques and lung function in the Marysville worker cohort to determine whether they are associated with adverse effects or, rather, are more likely a biomarker of cumulative exposure to LAA. METHODS: We obtained the dataset on the Marysville worker cohort from University of Cincinnati, which included information on demographics, occupational exposures and results of chest high-resolution computed tomography (HRCT)/computed tomography (CT) scans and pulmonary function tests (PFTs). We used multivariate linear regression to estimate mean differences in several lung function parameters, and logistic regression to evaluate the odds of abnormal ventilatory patterns, among men with different pulmonary findings on HRCT/CT scans. RESULTS: No statistically significant differences in FEV1, FVC, FEV1/FVC, TLC, RV or DLCO were observed between workers with normal scans and those with pleural plaques but no other abnormalities. In contrast, workers with other abnormal findings had statistically significant lower FEV1, FVC, TLC and DLCO, compared with those with normal scans. CONCLUSIONS: This study does not indicate that pleural plaques have a significant effect on lung function when past asbestos exposure is accounted for.

Systematic review of pleural plaques and lung function.

CONTEXT: US EPA proposed a Reference Concentration for Libby amphibole asbestos based on the premise that pleural plaques are adverse and cause lung function deficits. OBJECTIVE: We conducted a systematic review to evaluate whether there is an association between pleural plaques and lung function and ascertain whether results were dependent on the method used to identify plaques. METHODS: Using the PubMed database, we identified studies that evaluated pleural plaques and lung function. We assessed each study for quality, then integrated evidence and assessed associations based on the Bradford Hill guidelines. We also compared the results of HRCT studies to those of X-ray studies. RESULTS: We identified 16 HRCT and 36 X-ray studies. We rated six HRCT and 16 X-ray studies as higher quality based on a risk-of-bias analysis. Half of the higher quality studies reported small but statistically significant mean lung function decrements associated with plaques. None of the differences were clinically significant. Many studies had limitations, such as inappropriate controls and/or insufficient adjustment for confounders. There was little consistency in the direction of effect for the most commonly reported measurements. X-ray results were more variable than HRCT results. Pleural plaques were not associated with changes in lung function over time in longitudinal studies. CONCLUSION: The weight of evidence indicates that pleural plaques do not impact lung function. Observed associations are most likely due to unidentified abnormalities or other factors.

Snai1 is important for avian epicardial cell transformation and motility.

BACKGROUND: Formation of the epicardium requires several cellular processes including migration, transformation, invasion, and differentiation in order to give rise to fibroblast, smooth muscle, coronary endothelial and myocyte cell lineages within the developing myocardium. Snai1 is a zinc finger transcription factor that plays an important role in regulating cell survival and fate during embryonic development and under pathological conditions. However, its role in avian epicardial development has not been examined. RESULTS: Here we show that Snai1 is highly expressed in epicardial cells from as early as the proepicardial cell stage and its expression is maintained as proepicardial cells migrate and spread over the surface of the myocardium and undergo epicardial-to-mesenchymal transformation in the generation of epicardial-derived cells. Using multiple in vitro assays, we show that Snai1 overexpression in chick explants enhances proepicardial cell migration at Hamburger Hamilton Stage (HH St.) 16, and epicardial-to-mesenchymal transformation, cell migration, and invasion at HH St. 24. Further, we demonstrate that Snai1-mediated cell migration requires matrix metalloproteinase activity, and MMP15 is sufficient for this process. CONCLUSIONS: Together our data provide new insights into the multiple roles that Snai1 has in regulating avian epicardial development.

A natural loss-of-function deletion of the cytohesin 1 (Cyth1) gene in BALB/cByJ mice does not impact cardiomyocyte polyploidy.

Mammalian cardiomyocytes (CMs) mostly become polyploid shortly after birth. Because this feature may relate to several aspects of heart biology, including regeneration after injury, the mechanisms that cause polyploidy are of interest. BALB/cJ and BALB/cByJ mice are highly related sister strains that diverge substantially in CM ploidy. We identified a large deletion in the Cyth1 gene that arose uniquely in BALB/cByJ mice that creates a null allele. The deletion also results in ectopic transcription of the downstream gene Dnah17, although this transcript is unlikely to encode a protein. By evaluating the natural null allele from BALB/cByJ and an engineered knockout allele in the C57BL/6J background, we determined that absence of Cyth1 does not by itself influence CM ploidy. The ready availability of BALB/cByJ mice may be helpful to other investigations of Cyth1 in other biological processes.

Anti-Ferroptotic Treatment Deteriorates Myocardial Infarction by Inhibiting Angiogenesis and Altering Immune Response.

Mammalian cardiomyocytes have limited regenerative ability. Cardiac disease, such as congenital heart disease and myocardial infarction, causes an initial loss of cardiomyocytes through regulated cell death (RCD). Understanding the mechanisms that govern RCD in the injured myocardium is crucial for developing therapeutics to promote heart regeneration. We previously reported that ferroptosis, a non-apoptotic and iron-dependent form of RCD, is the main contributor to cardiomyocyte death in the injured heart. To investigate the mechanisms underlying the preference for ferroptosis in cardiomyocytes, we examined the effects of anti-ferroptotic reagents in infarcted mouse hearts. The results revealed that the anti-ferroptotic reagent did not improve neonatal heart regeneration, and further compromised the cardiac function of juvenile hearts. On the other hand, ferroptotic cardiomyocytes played a supportive role during wound healing by releasing pro-angiogenic factors. The inhibition of ferroptosis in the regenerating mouse heart altered the immune and angiogenic responses. Our study provides insights into the preference for ferroptosis over other types of RCD in stressed cardiomyocytes, and guidance for designing anti-cell-death therapies for treating heart disease.

Cardiomyocyte-fibroblast interaction regulates ferroptosis and fibrosis after myocardial injury.

Neonatal mouse hearts have transient renewal capacity, which is lost in juvenile and adult stages. In neonatal mouse hearts, myocardial infarction (MI) causes an initial loss of cardiomyocytes. However, it is unclear which type of regulated cell death (RCD) occurs in stressed cardiomyocytes. In the current studies, we induced MI in neonatal and juvenile mouse hearts and showed that ischemic cardiomyocytes primarily undergo ferroptosis, a non-apoptotic and iron-dependent form of RCD. We demonstrated that cardiac fibroblasts (CFs) protect cardiomyocytes from ferroptosis through paracrine effects and direct cell-cell interaction. CFs show strong resistance to ferroptosis due to high ferritin expression. The fibrogenic activity of CFs, typically considered detrimental to heart function, is negatively regulated by paired-like homeodomain 2 (Pitx2) signaling from cardiomyocytes. In addition, Pitx2 prevents ferroptosis in cardiomyocytes by regulating ferroptotic genes. Understanding the regulatory mechanisms of cardiomyocyte survival and death can identify potentially translatable therapeutic strategies for MI.

Pyrolysis mechanism and pyrolysis kinetics of yellow wine lees.

Yellow wine lees, a by-product produced while brewing yellow wine, can be a helpful biomass resource through pyrolysis. However, there have been very few studies on the pyrolysis of yellow wine lees. The kinetics and mechanism of pyrolysis in yellow wine lees were explored through an extensive study of their chemical and elemental composition. The pyrolysis mechanism of yellow wine lees was further studied using thermogravimetric analysis (TGA) from 30 °C to 900 °C. The TG/DTG analysis showed that yellow wine lees thermally decomposed mainly between 121 °C and 500 °C. The maximum decomposition was observed between 218 °C and 326 °C, with a clear peak at 298 °C. Upon analyzing the 3D-FTIR results, the gas phase products at this stage primarily included inorganic molecules like CO2, H2O, and CH4, along with organic compounds such as esters, alcohols, phenols, amines, ethers, aldehydes, ketones, and acids. The Maillard reaction and ketosis decarboxylation primarily occurred in proteins (amino acids) and carbohydrates. The pyrolysis kinetics of yellow wine lees were analyzed utilizing the distributed activation energy model (DAEM). The results of DAEM were simultaneously verified using the Flynn-Wall-Ozawa (FWO) method. The findings indicated that the pyrolysis of yellow wine lees conforms to the assumptions of infinite parallel reactions and activation energy distribution. As the conversion rate increased during pyrolysis, the activation energy of yellow wine lees initially increased to 210-220 kJ mol-1, then stabilized at 190-200 kJ mol-1 and rapidly decreased to approximately 100 kJ mol-1. This study offers a theoretical basis for the application of yellow wine lees using pyrolysis.

Endothelial nitric oxide signaling regulates Notch1 in aortic valve disease.

The mature aortic valve is composed of a structured trilaminar extracellular matrix that is interspersed with aortic valve interstitial cells (AVICs) and covered by endothelium. Dysfunction of the valvular endothelium initiates calcification of neighboring AVICs leading to calcific aortic valve disease (CAVD). The molecular mechanism by which endothelial cells communicate with AVICs and cause disease is not well understood. Using a co-culture assay, we show that endothelial cells secrete a signal to inhibit calcification of AVICs. Gain or loss of nitric oxide (NO) prevents or accelerates calcification of AVICs, respectively, suggesting that the endothelial cell-derived signal is NO. Overexpression of Notch1, which is genetically linked to human CAVD, retards the calcification of AVICs that occurs with NO inhibition. In AVICs, NO regulates the expression of Hey1, a downstream target of Notch1, and alters nuclear localization of Notch1 intracellular domain. Finally, Notch1 and NOS3 (endothelial NO synthase) display an in vivo genetic interaction critical for proper valve morphogenesis and the development of aortic valve disease. Our data suggests that endothelial cell-derived NO is a regulator of Notch1 signaling in AVICs in the development of the aortic valve and adult aortic valve disease.

Perish in the Attempt: Regulated Cell Death in Regenerative and Nonregenerative Tissue.

Significance: A cell plays its roles throughout its life span, even during its demise. Regulated cell death (RCD) is one of the key topics in modern biomedical studies. It is considered the main approach for removing stressed and/or damaged cells. Research during the past two decades revealed more roles of RCD, such as coordinating tissue development and driving compensatory proliferation during tissue repair. Recent Advances: Compensatory proliferation, initially identified in primitive organisms during the regeneration of lost tissue, is an evolutionarily conserved process that also functions in mammals. Among various types of RCD, apoptosis is considered the top candidate to induce compensatory proliferation in damaged tissue. Critical Issues: The roles of apoptosis in the recovery of nonregenerative tissue are still vague. The roles of other types of RCD, such as necroptosis and ferroptosis, have not been well characterized in the context of tissue regeneration. Future Directions: In this review article, we attempt to summarize the recent insights on the role of RCD in tissue repair. We focus on apoptosis, with expansion to ferroptosis and necroptosis, in primitive organisms with significant regenerative capacity as well as common mammalian research models. After gathering hints from regenerative tissue, in the second half of the review, we take a notoriously nonregenerative tissue, the myocardium, as an example to discuss the role of RCD in terminally differentiated quiescent cells. Antioxid. Redox Signal. 39, 1053-1069.

Cardiomyocyte-fibroblast interaction regulates ferroptosis and fibrosis after myocardial injury.

Neonatal mouse hearts have transient renewal capacity which is lost in juvenile and adult hearts. After myocardial infarction (MI) in neonatal hearts, an initial loss of cardiomyocytes occurs but it is unclear through which type of regulated cell death (RCD). In the current studies, we induced MI in neonatal and juvenile mouse hearts, and show that ischemic cardiomyocytes primarily undergo ferroptosis, a non-apoptotic and iron-dependent form of RCD. We demonstrate that cardiac fibroblasts (CFs) protect cardiomyocytes from ferroptosis through paracrine factors and direct cell-cell interaction. CFs show strong resistance to ferroptosis due to high ferritin expression. Meanwhile, the fibrogenic role of CFs, typically considered detrimental to heart function, is negatively regulated by paired-like homeodomain 2 (Pitx2) signaling from cardiomyocytes. In addition, Pitx2 prevents ferroptosis in cardiomyocytes by regulating ferroptotic genes. Understanding the regulatory mechanisms of cardiomyocyte survival and death can identify potentially translatable therapeutic strategies for MI.

Purkinje Cardiomyocytes of the Adult Ventricular Conduction System Are Highly Diploid but Not Uniquely Regenerative.

Adult hearts are characterized by inefficient regeneration after injury, thus, the features that support or prevent cardiomyocyte (CM) proliferation are important to clarify. Diploid CMs are a candidate cell type that may have unique proliferative and regenerative competence, but no molecular markers are yet known that selectively identify all or subpopulations of diploid CMs. Here, using the conduction system expression marker Cntn2-GFP and the conduction system lineage marker Etv1CreERT2, we demonstrate that Purkinje CMs that comprise the adult ventricular conduction system are disproportionately diploid (33%, vs. 4% of bulk ventricular CMs). These, however, represent only a small proportion (3%) of the total diploid CM population. Using EdU incorporation during the first postnatal week, we demonstrate that bulk diploid CMs found in the later heart enter and complete the cell cycle during the neonatal period. In contrast, a significant fraction of conduction CMs persist as diploid cells from fetal life and avoid neonatal cell cycle activity. Despite their high degree of diploidy, the Purkinje lineage had no enhanced competence to support regeneration after adult heart infarction.

Remdesivir increases mtDNA copy number causing mild alterations to oxidative phosphorylation.

SARS-CoV-2 causes the severe respiratory disease COVID-19. Remdesivir (RDV) was the first fast-tracked FDA approved treatment drug for COVID-19. RDV acts as an antiviral ribonucleoside (adenosine) analogue that becomes active once it accumulates intracellularly. It then diffuses into the host cell and terminates viral RNA transcription. Previous studies have shown that certain nucleoside analogues unintentionally inhibit mitochondrial RNA or DNA polymerases or cause mutational changes to mitochondrial DNA (mtDNA). These past findings on the mitochondrial toxicity of ribonucleoside analogues motivated us to investigate what effects RDV may have on mitochondrial function. Using in vitro and in vivo rodent models treated with RDV, we observed increases in mtDNA copy number in Mv1Lu cells (35.26% increase ± 11.33%) and liver (100.27% increase ± 32.73%) upon treatment. However, these increases only resulted in mild changes to mitochondrial function. Surprisingly, skeletal muscle and heart were extremely resistant to RDV treatment, tissues that have preferentially been affected by other nucleoside analogues. Although our data suggest that RDV does not greatly impact mitochondrial function, these data are insightful for the treatment of RDV for individuals with mitochondrial disease.