Disruptions in the balance of mitochondrial fission and fusion are implicated in a host of diseases including cardiovascular, metabolic, and neurodegenerative, as well as cancer. Leinheiser et al. proposed a mechanistic model for mitochondrial fission which relies on the oligomerization of Dynamin-related protein 1 (Drp1). In this work, we propose an alternative state-dependent delay-differential equation (sdDDE) framework for mitochondrial fission, which reveals that the intrinsic delay dynamics in Drp1 oligomerization can drive oscillations in the rate of mitochondrial fission. To develop this sdDDE model, we generate a simplified model which disallows oligomer disassembly on the mitochondrial membrane. Following homogenization, the simplified model approaches a steady state dominated by oligomers too small to reach the threshold for fission. Therefore, the fission rate approaches zero when initial conditions reside within the basin of attraction of this fission-free equilibrium. We therefore reincorporate oligomer disassembly on the mitochondrial membrane. However, the attracting, fission-free equilibrium persists. To eliminate this fission-free equilibrium, we incorporate an atomization term into the oligomerization mechanism, highlighting the importance of oligomer disassembly in sustaining mitochondrial fission. Using homogenization techniques, we derive an advection PDE with nonlocal interactions and obtain a reduced sdDDE system governing oligomer partial moments. Analysis of this reduced system reveals an analogous Hopf bifurcation to the Leinheiser et al. fission model, demonstrating that intrinsic delays in Drp1 oligomerization are sufficient to generate oscillatory mitochondrial fission dynamics.

Background:Previous studies have linked mutations in the Mediator complex, specifically Mediator 13 (Med13) and Mediator 13-like (Med13L), with both congenital heart defects and cardiovascular diseases. Med13 and Med13L are mutually exclusive paralogs within the kinase submodule of the Mediator complex that have been shown to have partially redundant functions in embryonic development and transcription, but their combined roles have not been investigated in the adult heart. We investigated the critical yet redundant roles of Med13 and Med13L in adult murine cardiomyocytes for basal cardiac function. Methods:We generated an inducible Med13 and Med13L cardiomyocyte-specific knockout mouse model to investigate Med13 and Med13L regulation of cardiac function and transcription. We performed RNAseq on mice four weeks after the start of tamoxifen to identify changes in gene expression. Differentially expressed genes were compared across cardiac knockouts of Med13/13L, Med13, Med12, Med1, and Med30 elucidating similar mechanisms of cardiac dysfunction. Results:Med13/13L knockout resulted in decreased cardiac function leading to lethal heart failure in a median timeframe of 6 weeks from the start of tamoxifen. There is significant gene dysregulation after Med13/13L knockout with similar gene dysregulation of fibrotic pathways and calcium handling across Mediator cardiac knockouts. Conclusions:Med13 and Med13L function partially redundantly within the heart to maintain basal cardiac function and transcription, as well as redundancies within cardiac phenotypes related to mediator complex disruptions.

Transcription directs the heart's development and adaptation to stress signals, and transcriptional dysregulation contributes to developmental disorders, pathological remodeling and heart failure (HF). Stereotypic changes at the mRNA level in the failing heart can be powerful diagnostics, as dysregulation can precede pathological outcomes such as decreased ejection fraction and increased heart size. The Mediator Complex is a general regulator of transcription in all eukaryotic cells; however, unknown subunit- and tissue-specific functions complicate our understanding of Mediator's influence on the cell. Here, we investigated the subunit-specific responses of Mediator throughout cardiac development, aging, and disease at the single cell- and whole ventricle-resolution using single cell RNA-sequencing, bulk RNA-sequencing, qPCR, and assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) datasets from humans and mice. In the developing heart, we discovered that key stages of growth such as ventricle formation were marked with elevated Mediator component expression, which declined during postnatal maturation, but increased again in the aging heart. Heart failure, a heterogenous disease, presented with a global increase in Mediator expression in human and mouse cardiomyocytes. This increased expression was mirrored by increased chromatin accessibility at the promoters of Mediator genes. Collectively, this study reveals the dynamic expression of Mediator subunits throughout the stages of the cardiomyocyte lifecycle and uncovers potential mechanisms by which Mediator is modulated in response to various pathological stimuli.

Background Gene transcription is crucial for embryo and postnatal development and is regulated by the Mediator complex. Mediator is comprised of four submodules, including the kinase submodule (CKM). The CKM consists of MED13, MED12, CDK8, and CCNC. In mammals, there are paralogs for CKM components, including MED13L, MED12L, and CDK19. Neurological disorders have been associated with mutations in CKM genes including MED13L syndrome. MED13L syndrome is generally characterized as a haploinsufficiency of MED13L with a broad phenotypic response due in part to a wide range of de novo mutations.Results We developed a Med13l heterozygous (HET) mouse model with an exon 11 deletion to evaluate whether Med13l HET mice are a viable research tool to study human phenotypes. We characterized our mouse model using growth, cardiovascular, and skeletal readouts. We observed Med13l HET mice are smaller than wildtype (WT) littermates, and over 60% of them exhibited one of two craniofacial anomalies: a pug snout with midface hypoplasia or a crooked snout. We also observed discontinuous squamosal sutures in a subset of our Med13l HETs.Conclusions Med13l HET mice recapitulate MED13L syndrome phenotypes including a developmental growth delay and craniofacial anomalies. Med13l HET mice represent a novel research tool for MED13L syndrome.

The lysosome integrates anabolic signaling and nutrient sensing to regulate intracellular growth pathways. The leucine-rich repeat-containing 8 (LRRC8) channel complex forms a lysosomal anion channel and regulates PI3K-AKT-mTOR signaling, skeletal muscle differentiation, growth, and systemic glucose metabolism. Here, we define the endogenous LRRC8 subunits localized to a subset of lysosomes in differentiated myotubes. We show that LRRC8A affects leucine-stimulated mTOR; lysosome size; number; pH; expression of lysosomal proteins LAMP2, P62, and LC3B; and lysosomal function. Mutating an LRRC8A lysosomal targeting dileucine motif sequence (LRRC8A-L706A;L707A) in myotubes recapitulates the abnormal AKT signaling and altered lysosomal morphology and pH observed in LRRC8A knockout cells. In vivo, LRRC8A-L706A;L707A knock-in mice exhibit increased adiposity, impaired glucose tolerance and insulin resistance associated with reduced skeletal muscle PI3K-AKT-mTOR signaling, glucose uptake, and impaired incorporation of glucose into glycogen. These data reveal a lysosomal LRRC8-mediated metabolic signaling function regulating lysosomal function, systemic glucose homeostasis, and insulin sensitivity.

BACKGROUND:Calcium (Ca2+) homeostasis in cardiac fibroblasts (CFs) plays a critical role in myocardial repair and remodeling after injury. JPH2 (junctophilin-2; human JPH2 or mouse Jph2) is a structural protein known to regulate intracellular Ca2+ signaling and excitation-contraction coupling in cardiomyocytes. However, the role of JPH2 in CF biology remains unexplored. METHODS:Junctophilin expression was assayed in human and mouse CFs using reverse transcription quantitative polymerase chain reaction, Western blotting, and immunofluorescence. To investigate the functional role of Jph2 in CFs, we assessed Ca2+ handling with live-cell confocal imaging, conducted RNA sequencing analysis, and assayed TGFβ (transforming growth factor β) responses after adenovirus-mediated gene silencing of Jph2 and fibroblast-specific Jph2 knockout. Jph2 interactions in CFs were identified using immunoprecipitation and proximity biotin ligation assays and confirmed by coimmunoprecipitation and rescue experiments. The in vivo role of Jph2 in CFs was investigated in fibroblast-specific Jph2 knockout mice (Col1a2CreERT/Jph2flox/flox, Jph2fKO) at baseline and after myocardial infarction and evaluated for changes in cardiac function, fibrosis, angiogenesis, CF activation, differentiation, and proliferation. RESULTS:Jph2 was identified as the only junctophilin expressed in CFs and acutely upregulated in response to myocardial infarction. Cellular and RNA sequencing analyses revealed that isolated Jph2-deficient CFs exhibited impaired fibroblast activation, reduced extracellular matrix production, and diminished expression of VEGFA (vascular endothelial growth factor A), VEGFB, and VEGFC after TGFβ treatment. In vivo, Jph2fKO mice displayed exacerbated adverse cardiac remodeling after myocardial infarction, characterized by decreased CF activation/extracellular matrix production, enhanced CF proliferation, worsened systolic dysfunction, increased left ventricular dilation, impaired scar maturation, and decreased angiogenesis. Mechanistically, biochemical assays demonstrated that Jph2 interacts directly with the coiled-coil 2 domain of Stim1 (stromal interaction molecule 1) via its joining region. Jph2 knockdown led to Stim1 protein destabilization, defective store-operated Ca2+entry, and a reduction in both canonical and noncanonical TGFβ signaling pathways. Stim1 overexpression partially rescued the sensitivity of Jph2-deficient CFs to TGFβ, as evidenced by increased expression of periostin and fibronectin-1, as well as enhanced Smad3 phosphorylation. CONCLUSIONS:Our results demonstrate that Jph2 is required in CFs to orchestrate Ca2+ homeostasis, CF activation, and extracellular matrix production and promotes angiogenesis in the infarcted heart, positioning it as a central regulator of cardiac repair after injury.

A strategic research plan (SRP) serves as a compass for the patient advocacy organizations driving the therapeutic options for their rare disorder. The MED13L Foundation commissioned the SRP in 2022 through COMBINEDBrain, a consortium of patient advocacy organizations of rare neurodevelopmental disorders, working toward clinical trial readiness. The MED13L Foundation SRP is an objective evaluation of MED13L literature including clinical and basic science knowledge interwoven with an assessment of preclinical trial readiness tools necessary for achieving therapeutic interventions. Clinical evaluation is conducted through a review of the literature documenting symptoms and variant information for each individual with MED13L syndrome. Data is collated and presented as a summary, providing any unique genotype–phenotype, as applicable. Scientific literature is reviewed in the same manner, identifying areas of opportunity to expand knowledge of MED13L syndrome. Researchers and clinicians responsible for growing the understanding of MED13L syndrome are interviewed and information is shared to create an open and collaborative network. Preclinical trial readiness tools are largely framed through Food and Drug Administration guidelines for the development of therapeutics from bench to bedside. Finally, the Foundation infrastructure and community engagement are assessed providing areas of strengths and opportunities to elevate the bond formed to drive patient-centered research forward. Completed, this SRP becomes a living resource for the MED13L Foundation to set priorities, share with researchers and clinicians, and provide direction to reach their organizational goals, including therapies for their community affected by MED13L syndrome.

Mitochondria and endoplasmic reticulum (ER) contact sites (MERCs) are protein- and lipid-enriched hubs that mediate interorganellar communication by contributing to the dynamic transfer of Ca2+, lipid, and other metabolites between these organelles. Defective MERCs are associated with cellular oxidative stress, neurodegenerative disease, and cardiac and skeletal muscle pathology via mechanisms that are poorly understood. We previously demonstrated that skeletal muscle-specific knockdown (KD) of the mitochondrial fusion mediator optic atrophy 1 (OPA1) induced ER stress and correlated with an induction of Mitofusin-2, a known MERC protein. In the present study, we tested the hypothesis that Opa1 downregulation in skeletal muscle cells alters MERC formation by evaluating multiple myocyte systems, including from mice and Drosophila, and in primary myotubes. Our results revealed that OPA1 deficiency induced tighter and more frequent MERCs in concert with a greater abundance of MERC proteins involved in calcium exchange. Additionally, loss of OPA1 increased the expression of activating transcription factor 4 (ATF4), an integrated stress response (ISR) pathway effector. Reducing Atf4 expression prevented the OPA1-loss-induced tightening of MERC structures. OPA1 reduction was associated with decreased mitochondrial and sarcoplasmic reticulum, a specialized form of ER, calcium, which was reversed following ATF4 repression. These data suggest that mitochondrial stress, induced by OPA1 deficiency, regulates skeletal muscle MERC formation in an ATF4-dependent manner.

Doberman Pinschers are known for their increased susceptibility to dilated cardiomyopathy (DCM) relative to other domestic dogs. This makes the Doberman Pinscher a key model for gene-disease investigations. We conducted a genome-wide association study (GWAS) leveraging a database of genetic profiles obtained through collaboration with the Doberman Diversity Project (DDP). We worked in parallel to increase the depth and power of the DDP database. We exchanged direct correspondences with listed breeders and owners to establish health updates for participant dogs. In total, our study included data on 216, 184 single nucleotide polymorphisms (SNPs) in 46 cases and 3226 population control Doberman Pinschers. Using a generalized linear mixed model and saddlepoint approximation to correct for unbalanced group sizes, we identified a cluster of SNPs associated with DCM on chromosome 16.

Mitochondrial hyperfission in response to cellular insult is associated with reduced energy production and programmed cell death. Thus, there is a critical need to understand the molecular mechanisms coordinating and regulating the complex process of mitochondrial fission. We develop a nonlinear dynamical systems model of dynamin related protein one (Drp1)-dependent mitochondrial fission and use it to identify parameters which can regulate the total fission rate (TFR) as a function of time. The TFR defined from a nondimensionalization of the model undergoes a Hopf bifurcation with bifurcation parameter [Formula: see text] where [Formula: see text] is the total concentration of mitochondrial fission factor (Mff) and k+ and k- are the association and dissociation rate constants between oligomers on the outer mitochondrial membrane. The variable μ can be thought of as the maximum build rate over the disassembling rate of oligomers. Though the nondimensionalization of the system results in four dimensionless parameters, we found the TFR and the cumulative total fission (TF) depend strongly on only one, μ. Interestingly, the cumulative TF does not monotonically increase as μ increases. Instead it increases with μ to a certain point and then begins to decrease as μ continues to increase. This non-monotone dependence on μ suggests interventions targeting k+, k-, or [Formula: see text] may have a non-intuitive impact on the fission mechanism. Thus understanding the impact of regulatory parameters, such as μ, may assist future therapeutic target selection.

The COVID-19 disease, caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), emerged in late 2019 and rapidly spread worldwide, becoming a pandemic that infected millions of people and caused significant deaths. COVID-19 continues to be a major threat, and there is a need to deepen our understanding of the virus and its mechanisms of infection. To study the cellular responses to SARS-CoV-2 infection, we performed an RNA sequencing of infected vs. uninfected Calu-3 cells. Total RNA was extracted from infected (0.5 MOI) and control Calu-3 cells and converted to cDNA. Sequencing was performed, and the obtained reads were quality-analyzed and pre-processed. Differential expression was assessed with the EdgeR package, and functional enrichment was performed in EnrichR for Gene Ontology, KEGG pathways, and WikiPathways. A total of 1040 differentially expressed genes were found in infected vs. uninfected Calu-3 cells, of which 695 were up-regulated and 345 were down-regulated. Functional enrichment analyses revealed the predominant up-regulation of genes related to innate immune response, response to virus, inflammation, cell proliferation, and apoptosis. These transcriptional changes following SARS-CoV-2 infection may reflect a cellular response to the infection and help to elucidate COVID-19 pathogenesis, in addition to revealing potential biomarkers and drug targets.

Progesterone prevents development of endometrial cancers through its receptor (PR) although the molecular mechanisms have yet to be fully characterized. In this study, we performed a global analysis of gene regulation by progesterone using human endometrial cancer cells that expressed PR endogenously or exogenously. We found progesterone strongly inhibits multiple components of the platelet derived growth factor receptor (PDGFR), Janus kinase (JAK), signal transducer and activator of transcription (STAT) pathway through PR. The PDGFR/JAK/STAT pathway signals to control numerous downstream targets including AP-1 transcription factors Fos and Jun. Treatment with inhibitors of the PDGFR/JAK/STAT pathway significantly blocked proliferation in multiple novel patient-derived organoid models of endometrial cancer, and activation of this pathway was found to be a poor prognostic signal for the survival of patients with endometrial cancer from The Cancer Genome Atlas. Our study identifies this pathway as central to the growth-limiting effects of progesterone in endometrial cancer and suggests that inhibitors of PDGFR/JAK/STAT should be considered for future therapeutic interventions.

Aging and many illnesses and injuries impair skeletal muscle mass and function, but the molecular mechanisms are not well understood. To better understand the mechanisms, we generated and studied transgenic mice with skeletal muscle–specific expression of growth arrest and DNA damage inducible α (GADD45A), a signaling protein whose expression in skeletal muscle rises during aging and a wide range of illnesses and injuries. We found that GADD45A induced several cellular changes that are characteristic of skeletal muscle atrophy, including a reduction in skeletal muscle mitochondria and oxidative capacity, selective atrophy of glycolytic muscle fibers, and paradoxical expression of oxidative myosin heavy chains despite mitochondrial loss. These cellular changes were at least partly mediated by MAP kinase kinase kinase 4, a protein kinase that is directly activated by GADD45A. By inducing these changes, GADD45A decreased the mass of muscles that are enriched in glycolytic fibers, and it impaired strength, specific force, and endurance exercise capacity. Furthermore, as predicted by data from mouse models, we found that GADD45A expression in skeletal muscle was associated with muscle weakness in humans. Collectively, these findings identify GADD45A as a mediator of mitochondrial loss, atrophy, and weakness in mouse skeletal muscle and a potential target for muscle weakness in humans.

AIMS The study investigates the role and mechanisms of clinically translatable exercise heart rate (HR) envelope effects, without dyssynchrony, on myocardial ischaemia tolerance compared to standard preconditioning methods. Since the magnitude and duration of exercise HR acceleration are tightly correlated with beneficial cardiac outcomes, it is hypothesized that a paced exercise-similar HR envelope, delivered in a maximally physiologic way that avoids the toxic effects of chamber dyssynchrony, may be more than simply a readout, but rather also a significant trigger of myocardial conditioning and stress resistance. METHODS AND RESULTS For 8 days over 2 weeks, sedated mice were atrial-paced once daily via an oesophageal electrode to deliver an exercise-similar HR pattern with preserved atrioventricular and interventricular synchrony. Effects on cardiac calcium handling, protein expression/modification, and tolerance to ischaemia-reperfusion (IR) injury were assessed and compared to those in sham-paced mice and to the effects of exercise and ischaemic preconditioning (IPC). The paced cohort displayed improved myocardial IR injury tolerance vs. sham controls with an effect size similar to that afforded by treadmill exercise or IPC. Hearts from paced mice displayed changes in Ca2+ handling, coupled with changes in phosphorylation of calcium/calmodulin protein kinase II, phospholamban and ryanodine receptor channel, and transcriptional remodelling associated with a cardioprotective paradigm. CONCLUSIONS The HR pattern of exercise, delivered by atrial pacing that preserves intracardiac synchrony, induces cardiac conditioning and enhances ischaemic stress resistance. This identifies the HR pattern as a signal for conditioning and suggests the potential to repurpose atrial pacing for cardioprotection.

Pathological cardiac hypertrophy represents a major risk factor for heart failure (HF). Cardiac hypertrophic responses are coordinated in part through altered transcriptional dynamics involving chromatin structure and accessibility, transcription factor (TF) activation, and enhancer utilization. Mediator is a multiprotein complex that integrates signal-dependent TFs with basal transcriptional machinery to regulate downstream gene expression. Cyclin-dependent kinase 8 (Cdk8) is a Mediator kinase demonstrated to have a complex role in transcription regulation, driving both transcription activation and inhibition in context-specific ways. Previous studies have demonstrated that Cdk8 protein expression is increased in human and mouse HF. Cardiomyocyte-specific Cdk8 overexpression leads to eccentric cardiac hypertrophy consistent with HF, suggesting that Cdk8 activity may play a significant role in the progression of HF. To investigate the therapeutic potential in regulating Cdk8 activity, we used Cdk8 selective inhibitors SenexinA and CCT251545 in an in vitro neonatal rat cardiomyocyte hypertrophy model, resulting in altered regulation of hypertrophic transcriptional programs and blunted cardiomyocyte hypertrophy. To further assess Cdk8 kinase activity in regulating hypertrophic responses, Cdk8 inhibitor CCT251545 was used in an in vivo angiotensin II-induced hypertensive mouse model. It was hypothesized that Cdk8 inhibition by CCT251545 in vivo would lead to reduced hypertrophic responses. Results indicate that CCT251545 is effective at inhibiting Cdk8 activity, leading to blunted hypertrophic responses with altered gene expression. These studies demonstrate a role for Cdk8 activity in transcriptional regulation of genes associated with pathological cardiac hypertrophy, which can ultimately lead to heart failure.

The Mediator co-activation complex is an important component in RNA Pol II-dependent gene expression that functions to integrate intracellular signals, allowing for optimized cellular response to (patho)physiological states. The Mediator kinase submodule functions to fine-tune these molecular transitions. The kinase submodule consists of four proteins, including Med13, Med12, Cdk8, and Cyclin C. Also involved is the Med13 paralog, Med13-like (Med13L), which is both mutually exclusive in the submodule and partially redundant. Approximately 22% of reported human mutations in MED13 or MED13L have congenital heart defects often resulting in dysregulated cardiac metabolism and aberrant cardiovascular physiology. Therefore, we hypothesize that the acute loss of Med13 and Med13L in cardiomyocytes results in disrupted Mediator kinase submodule formation leading to altered gene expression and subsequent pathogenesis of heart failure. To investigate this, we created a Tamoxifen-inducible cardiomyocyte-specific Med13/Med13L double knockout mouse model and treated mice for two weeks with Tamoxifen beginning at eight-weeks-old. Following treatment, mice had significant progressive decreased ejection fraction consistent with severe heart failure. Survival studies demonstrate rapid mortality-50% of Med13/Med13L cardiac knockout mice die within four weeks. Post-mortem analysis showed increased heart weight and heart-weight-to-body-weight ratio as well as gross histology consistent with dilated hypertrophy. To begin elucidating the mechanistic processes underlying these findings, mRNA sequencing was performed prior to observed declines in cardiac function and showed that cardiac stress response pathways were differentially expressed. Specifically, the Wnt-β-catenin pathway was activated, while basal transcription was unchanged. Taken together, Med13 and Med13L are critical for normal Mediator-regulated RNA Pol II-dependent transcription and subsequent cardiac physiology. Therefore, disruptions cause significant cardiac dysfunction and death as sequelae of heart failure.

Background: Transcriptional remodeling is known to contribute to heart failure (HF). Targeting stress-dependent gene expression mechanisms may represent a clinically relevant gene therapy option. We recently uncovered a salutary mechanism in the heart whereby JP2 (junctophilin-2), an essential component of the excitation-contraction coupling apparatus, is site-specifically cleaved and releases an N-terminal fragment (JP2NT [N-terminal fragment of JP2]) that translocates into the nucleus and functions as a transcriptional repressor of HF-related genes. This study aims to determine whether JP2NT can be leveraged by gene therapy techniques for attenuating HF progression in a preclinical pressure overload model. Methods: We intraventricularly injected adeno-associated virus (AAV) (2/9) vectors expressing eGFP (enhanced green fluorescent protein), JP2NT, or DNA-binding deficient JP2NT (JP2NT ΔbNLS/ARR ) into neonatal mice and induced cardiac stress by transaortic constriction (TAC) 9 weeks later. We also treated mice with established moderate HF from TAC stress with either AAV-JP2NT or AAV-eGFP. RNA-sequencing analysis was used to reveal changes in hypertrophic and HF-related gene transcription by JP2NT gene therapy after TAC. Echocardiography, confocal imaging, and histology were performed to evaluate heart function and pathological myocardial remodeling following stress. Results: Mice preinjected with AAV-JP2NT exhibited ameliorated cardiac remodeling following TAC. The JP2NT DNA-binding domain is required for cardioprotection as its deletion within the AAV-JP2NT vector prevented improvement in TAC-induced cardiac dysfunction. Functional and histological data suggest that JP2NT gene therapy after the onset of cardiac dysfunction is effective at slowing the progression of HF. RNA-sequencing analysis further revealed a broad reversal of hypertrophic and HF-related gene transcription by JP2NT overexpression after TAC. Conclusions: Our prevention- and intervention-based approaches here demonstrated that AAV-mediated delivery of JP2NT into the myocardium can attenuate stress-induced transcriptional remodeling and the development of HF when administered either before or after cardiac stress initiation. Our data indicate that JP2NT gene therapy holds great potential as a novel therapeutic for treating hypertrophy and HF.

Impairments in macroautophagy/autophagy, which degrades dysfunctional organelles as well as long-lived and aggregate proteins, are associated with several cardiomyopathies; however, the regulation of cardiac autophagy remains insufficiently understood. In this regard, ULK1 and ULK2 are thought to play primarily redundant roles in autophagy initiation, but whether their function is developmentally determined, potentially having an impact on cardiac integrity and function remains unknown. Here, we demonstrate that perinatal loss of ULK1 or ULK2 in cardiomyocytes (cU1-KO and cU2-KO mice, respectively) enhances basal autophagy without altering autophagy machinery content while preserving cardiac function. This increased basal autophagy is dependent on the remaining ULK protein given that perinatal loss of both ULK1 and ULK2 in cU1/2-DKO mice impaired autophagy causing age-related cardiomyopathy and reduced survival. Conversely, adult loss of cardiac ULK1, but not of ULK2 (i.e., icU1-KO and icU2-KO mice, respectively), led to a rapidly developing cardiomyopathy, heart failure and early death. icU1-KO mice had impaired autophagy with robust deficits in mitochondrial respiration and ATP synthesis. Trehalose ameliorated autophagy impairments in icU1-KO hearts but did not delay cardiac dysfunction suggesting that ULK1 plays other critical, autophagy-independent, functions in the adult heart. Collectively, these results indicate that cardiac ULK1 and ULK2 are functionally redundant in the developing heart, while ULK1 assumes a more unique, prominent role in the adult heart.Abbreviations: ATG4: autophagy related 4, cysteine peptidase; ATG5: autophagy related 5; ATG7: autophagy related 7; ATG9: autophagy related 9; ATG13: autophagy related 13; CYCS: Cytochrome C; DNM1L, dynamin 1-like; MAP1LC3A: microtubule-associated protein 1 light chain 3 alpha; MAP1LC3B: microtubule-associated protein 1 light chain 3 beta; MFN1: mitofusin 1; MFN2: mitofusin 2; MT-CO1: mitochondrially encoded cytochrome c oxidase I; MYH: myosin, heavy polypeptide; NBR1: NBR1 autophagy cargo receptor; NDUFA9: NADH:ubiquinone oxidoreductase subunit A9; OPA1: OPA1, mitochondrial dynamin like GTPase; PPARGC1A, peroxisome proliferator activated receptor, gamma, coactivator 1 alpha; SDHA: succinate dehydrogenase complex, subunit A, flavoprotein (Fp); SQSTM1: sequestosome 1; ULK1: unc-51 like kinase 1; ULK2: unc-51 like kinase 2; UQCRC1: ubiquinol-cytochrome c reductase core protein 1

HomeJournal of the American Heart AssociationVol. 10, No. 9Mining Metabolomics in Mitral Valve Disease Open AccessEditorialPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citations ShareShare onFacebookTwitterLinked InMendeleyRedditDiggEmail Jump toOpen AccessEditorialPDF/EPUBMining Metabolomics in Mitral Valve Disease Evangeline L. Scheibe and Chad E. Grueter, PhD Evangeline L. ScheibeEvangeline L. Scheibe https://orcid.org/0000-0003-0111-5435 Division of Cardiovascular Medicine, , Department of Internal Medicine, , Francois M. Abboud Cardiovascular Research Center, , Fraternal Order of Eagles Diabetes Research Center, , University of Iowa, , Iowa City, , IA and Chad E. GrueterChad E. Grueter * Correspondence to: Chad E. Grueter, PhD, 169 Newton Rd, 4332 Papajohn Biomedical Discovery Building, Department of Internal Medicine, University of Iowa, Iowa City, IA 52242. E‐mail: E-mail Address: [email protected] https://orcid.org/0000-0001-8950-742X Division of Cardiovascular Medicine, , Department of Internal Medicine, , Francois M. Abboud Cardiovascular Research Center, , Fraternal Order of Eagles Diabetes Research Center, , University of Iowa, , Iowa City, , IA Originally published23 Apr 2021https://doi.org/10.1161/JAHA.121.020726Journal of the American Heart Association. 2021;10:e020726This article is a commentary on the followingMetabolomics Analysis Reveals Deranged Energy Metabolism and Amino Acid Metabolic Reprogramming in Dogs With Myxomatous Mitral Valve DiseaseAs the most common naturally acquired cardiovascular disease in dogs, myxomatous mitral valve disease, or MMVD, affects roughly 9% of the canine population,1 with greater incidence in smaller breeds and geriatric dogs. Initially known as Barlow disease2 in humans, it affects an estimated 2% to 3% of the global population, with ≈15% of those individuals requiring valve surgery3; age is a risk factor in developing the disease, as is the case with dogs, and prevalence in humans is expected to increase as the mean age of the population increases.2The disease is typified by ballooning of a section of a mitral leaflet and its chordae, thereby causing mitral regurgitation, which may progress to congestive heart failure. MMVD in dogs is most often detected by the presence of a heart murmur, and ancillary examinations confirm the diagnosis as well as offer prognostic insight.4 However, there are few known molecular or metabolic diagnostic indicators, although it stands to reason that they would effectively supplement the currently used physical and echocardiographic parameters.5In this issue of the Journal of the American Heart Association (JAHA), Li et al identified numerous biochemical markers in an untargeted metabolomic study that compared and analyzed the profiles of each stage of MMVD in dogs.6 The authors generated extensive profiles of 84 client owned dogs, 27 of which were healthy, staff‐owned dogs. The 57 remaining subjects were in various stages of MMVD, as defined by the American College of Veterinary Internal Medicine. In addition to the serum metabolomic assay, echocardiographic studies generated structural information for each subject that indicated the subject's disease severity. Although dogs with severe, systemic diseases or complicating prior conditions were not included in the study, there were no exclusions on the basis of size, age, breed, or sex.Because the development of congestive heart failure can introduce metabolic changes independent of MMVD, the authors were particularly interested in significant differences between healthy dogs and a preclinical stage of MMVD marked by the presence of an mitral regurgitation‐induced heart murmur but the absence of clinical signs of congestive heart failure. The authors identified 173 known metabolites with significant differences in concentration between all groups, of which 94 were between the healthy and the preclinical stages. The identified metabolites were associated with several key pathways, >30% of which were linked to energy production.Ketone bodies were increased in all disease stages, an occurrence that previous studies have linked to congestive heart failure progression in humans.7 Twenty‐two acylcarnitines were positively associated with MMVD severity, as was carnitine concentration, together indicating a disruption in long‐chain fatty acid transport and oxidation. Reduced nicotinamide and increased quinolinate concentrations were observed in all disease groups, which the authors suggest could indicate a compromised nicotinamide adenine dinucleotide (NAD+) salvage pathway and subsequent activation of de novo NAD+ synthesis. Reprogramming of arginine and proline metabolism as well as renal insufficiency were evidenced by changes in numerous uremic toxins, including argininate, 2‐oxoarginine, 4‐guanidinobutanoate, trimethylamine N‐oxide, urea, and uric acid.The study by Li and colleagues offers a wealth of potential biomarkers and insight on the intricate balance of the heart‐kidney‐gut axis.6 Their findings, however, cannot be further explored without also acknowledging the limitations of their work. Their control group was made up of dogs that were significantly younger and larger than the other groups, both of which are factors that lower their risk of developing MMVD.1, 2, 4, 6, 8 It was a sample of convenience, as the authors themselves mention, and in such a preliminary study they certainly cannot be faulted for it. It should not be ignored, however, that such a small sample size, particularly one vulnerable to so many confounding factors, inevitably brings with it the possibility of inaccuracy. The authors did attempt to mitigate this effect with a bootstrap resampling method, and although it was effective in that it greatly increased the accuracy and trustworthiness of their findings, no amount of statistical analysis can completely fix a bad sample or avoid the risk of overfitting. In a similar vein, components, such as diet and breed, were not explored or corrected for as potential confounding effects, even though breed has been suggested to have an effect on MMVD development and progression.8 To their credit, the authors readily admit the shortcomings of their work and suggest many future directions that could eliminate these issues.This study addressed the hypothesis that "serum metabolomics changes reflect adaptations of energy substrates and interruptions of energy metabolic machinery during MMVD progression." By doing so, the authors provide a platform for future hypotheses to test clinical and mechanistic studies of the metabolites identified and intertissue communication in forms of progressive heart disease. The study was undeniably successful in doing what it set out to do: direct further inquiry. From novel therapeutic targets to potential prognostic and diagnostic factors to insight on the subtle interplay between metabolic pathways in disease, the authors brought forth a wealth of knowledge that should both fuel and direct considerable future investigation.DisclosuresNone.Footnotes* Correspondence to: Chad E. Grueter, PhD, 169 Newton Rd, 4332 Papajohn Biomedical Discovery Building, Department of Internal Medicine, University of Iowa, Iowa City, IA 52242. E‐mail: [email protected]eduThe opinions expressed in this article are not necessarily those of the editors or of the American Heart Association.For Disclosures, see page 2.See Article by Li et al.References1 Li Q, Freeman LM, Rush JE, Laflamme DP. Expression profiling of circulating microRNAs in canine myxomatous mitral valve disease. Int J Mol Sci. 2015; 16:14098–14108. DOI: 10.3390/ijms160614098.CrossrefMedlineGoogle Scholar2 Markby GR, Summers KM, MacRae VE, Corcoran BM. Comparative transcriptomic profiling and gene expression for myxomatous mitral valve disease in the dog and human. Vet Sci. 2017; 4:34.–10.3390/vetsci4030034.CrossrefGoogle Scholar3 Neto FL, Marques LC, Aiello VD. Myxomatous degeneration of the mitral valve. Autops Case Rep. 2018; 8:e2018058. DOI: 10.4322/acr.2018.058.MedlineGoogle Scholar4 Mattin MJ, Boswood A, Church DB, Brodbelt DC. 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Published on behalf of the American Heart Association, Inc., by Wiley BlackwellThis is an open access article under the terms of the Creative Commons Attribution‐NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.https://doi.org/10.1161/JAHA.121.020726PMID: 33890481 Originally publishedApril 23, 2021 Keywordsvalvular heart diseasemetabolismbasic scienceheart failureEditorialsbiomarkerPDF download