Biology subjects

Boudreau, R. L.

Publications and source records attributed to Boudreau, R. L..

9 recordsLinked to original sources

bioRxiv2026

Overexpression of miR-424(322)/-503 induces severe dilated cardiomyopathy by regulating the fatty acid oxidation gene expression program

Fatty acid oxidation (FAO) is a major energy source in the adult heart, and disruption of cardiac metabolism is closely associated with heart failure. Here, we investigated the effects of cardiac-specific overexpression of the H19X-encoded miR-424(322)/-503 cluster using an inducible transgenic mouse model. Sustained miR-424(322)/-503 overexpression caused rapid metabolic and functional deterioration, with early impairment of fatty acid oxidation. Short-term induction reduced FAO activity and downregulated genes involved in lipid metabolism, while glycolytic enzyme activity remained largely unchanged. Continued miR-424(322)/-503 expression subsequently led to severe dilated cardiomyopathy characterized by ventricular dilation, wall thinning, fibrosis, reduced contractility, and high mortality. Importantly, disease progression was dependent on the level and duration of miR-424(322)/-503 expression, as intermittent or lower-dose induction delayed cardiac dysfunction and prolonged survival. Withdrawal of miR-424(322)/-503 expression after the onset of dysfunction promoted substantial functional recovery. Together, these findings identify miR-424(322)/-503 as a potent regulator of cardiac metabolic reprogramming that disrupts fatty acid metabolism and drives progressive heart failure.

molecular biology↗

bioRxiv2025

Non-canonical Sodium Channel Isoforms Underlie Chamber Specific Cardiac Excitability

Voltage-gated sodium (NaV) channels drive cardiac excitability. While NaV1.5 is the primary cardiac isoform, the composition and functional contributions of non-NaV1.5 isoforms in the heart remain unclear. Here, we developed a chemical-genetic mouse model (NaV1.5-GX) in which NaV1.5 can be selectively and reversibly inhibited by acyl- and aryl-sulfonamide compounds (GX drugs). NaV1.5-GX mice exhibited normal cardiac function at baseline, but acute GX drug administration caused profound conduction defects and arrhythmias. Whole-heart optical mapping revealed dose-dependent chamber-specific sensitivity to NaV1.5 inhibition, with the right ventricle (RV) being the most sensitive, followed by the left ventricle (LV), left atrium (LA), and right atrium (RA). Patch-clamp recordings of isolated cardiomyocytes with application of NaV isoform-selective inhibitors showed that NaV1.5 contributed 93% of sodium current in the LV, 81% in the RV and 78% in the LA. Non-NaV1.5 isoforms were differentially enriched across chambers: NaV1.8 in the LV, NaV1.1/1.3 in the RV, and NaV1.2/1.6/1.7 in the atria. These results reveal a surprising chamber-specific isoform landscape of cardiac sodium currents which may underlie the right ventricular predominant phenotype of Brugada syndrome and highlight non-NaV1.5 isoforms as potential mediators of chamber-specific cardiac pathologies and as pharmacological targets.

physiology↗

bioRxiv2024

MIRO1 controls energy production and proliferation of smooth muscle cells

BackgroundThe outer mitochondrial Rho GTPase 1, MIRO1, mediates mitochondrial motility within cells, but implications for vascular smooth muscle cell (VSMC) physiology and its roles in vascular diseases, such as neointima formation following vascular injury are widely unknown. MethodsCarotid ligation was performed in an in vivo model of selective Miro1 deletion in smooth muscle cells. VSMC proliferation during the cell cycle and molecular mechanisms of smooth muscle cell proliferation were explored in cultured aortic VSMCs by imaging mitochondrial positioning and cristae structure and assessing the effects on ATP production, metabolic function, and interactions with components of the electron transport chain (ETC). MIRO1 expression was also analyzed in human coronary arteries, and its function was assessed via knockdown in human coronary artery VSMCs. ResultsMIRO1 was highly expressed in VSMCs within human atherosclerotic plaques. MIRO1 facilitated VSMC proliferation and neointima formation by regulating mitochondrial positioning and PDGF-stimulated ATP production and respiration, critical for cell-cycle progression at G1/S. Deletion of Miro1 disrupted mitochondrial cristae structure, diminished ETC complex I activity, and impaired supercomplex formation. Notably, restoring MIRO1 function with expression of wild type MIRO1 recovered proliferation and ATP production and respiration, whereas a mutant lacking EF hands, which are essential for mitochondrial motility, only partially rescued these effects. MIRO1 knockdown in human coronary artery VSMCs confirmed its pivotal role in mitochondrial function and VSMC proliferation. ConclusionsThis study highlights two key mechanisms by which MIRO1 regulates VSMC proliferation. First, it maintains ATP synthesis by preserving mitochondrial cristae integrity. Second, its Ca2+-dependent EF hands enable ATP-dependent mitochondrial positioning. By linking mitochondrial motility and energy production to VSMC physiology, these findings position MIRO1 as a critical regulator of vascular remodeling and a potential target for therapeutic interventions.

cell biology↗

bioRxiv2024

microRNA-1 Regulates Metabolic Flexibility in Skeletal Muscle via Pyruvate Metabolism

MicroRNA-1 (miR-1) is the most abundant miRNA in adult skeletal muscle. To determine the function of miR-1 in adult skeletal muscle, we generated an inducible, skeletal muscle-specific miR-1 knockout (KO) mouse. Integration of RNA-sequencing (RNA-seq) data from miR-1 KO muscle with Argonaute 2 enhanced crosslinking and immunoprecipitation sequencing (AGO2 eCLIP-seq) from human skeletal muscle identified miR-1 target genes involved with glycolysis and pyruvate metabolism. The loss of miR-1 in skeletal muscle induced cancer-like metabolic reprogramming, as shown by higher pyruvate kinase muscle isozyme M2 (PKM2) protein levels, which promoted glycolysis. Comprehensive bioenergetic and metabolic phenotyping combined with skeletal muscle proteomics and metabolomics further demonstrated that miR-1 KO induced metabolic inflexibility as a result of pyruvate oxidation resistance. While the genetic loss of miR-1 reduced endurance exercise performance in mice and in C. elegans, the physiological down-regulation of miR-1 expression in response to a hypertrophic stimulus in both humans and mice causes a similar metabolic reprogramming that supports muscle cell growth. Taken together, these data identify a novel post-translational mechanism of adult skeletal muscle metabolism regulation mediated by miR-1.

physiology↗

bioRxiv2024

Upstream alternative polyadenylation in SCN5A produces a short transcript isoform encoding a mitochondria-localized NaV1.5 N-terminal fragment that influences cardiomyocyte respiration

SCN5A encodes the cardiac voltage-gated Na+ channel, NaV1.5, that initiates action potentials. SCN5A gene variants cause arrhythmias and increased heart failure risk. Mechanisms controlling NaV1.5 expression and activity are not fully understood. We recently found a well-conserved alternative polyadenylation (APA) signal downstream of the first SCN5A coding exon. This yields a SCN5A-short transcript isoform expressed in several species (e.g. human, pig, and cat), though rodents lack this upstream APA. Reanalysis of transcriptome-wide cardiac APA-seq and mRNA-seq data shows reductions in both upstream APA usage and short/full-length SCN5A mRNA ratios in failing hearts. Knock-in of the human SCN5A APA sequence into mice is sufficient to enable expression of SCN5A-short transcript, while significantly decreasing expression of full-length SCN5A mRNA. Notably, SCN5A-short transcript encodes a novel protein (NaV1.5-NT), composed of an N-terminus identical to NaV1.5 and a unique C-terminus derived from intronic sequence. AAV9 constructs were able to achieve stable NaV1.5-NT expression in mouse hearts, and western blot of human heart tissues showed bands co-migrating with NaV1.5-NT transgene-derived bands. NaV1.5-NT is predicted to contain a mitochondrial targeting sequence and localizes to mitochondria in cultured cardiomyocytes and in mouse hearts. NaV1.5-NT expression in cardiomyocytes led to elevations in basal oxygen consumption rate, ATP production, and mitochondrial ROS, while depleting NADH supply. Native PAGE analyses of mitochondria lysates revealed that NaV1.5-NT expression resulted in increased levels of disassembled complex V subunits and accumulation of complex I-containing supercomplexes. Overall, we discovered that APA-mediated regulation of SCN5A produces a short transcript encoding NaV1.5-NT. Our data support that NaV1.5-NT plays a multifaceted role in influencing mitochondrial physiology: 1) by increasing basal respiration likely through promoting complex V conformations that enhance proton leak, and 2) by increasing overall respiratory efficiency and NADH consumption by enhancing formation and/or stability of complex I-containing respiratory supercomplexes, though the specific molecular mechanisms underlying each of these remain unresolved.

physiology↗

bioRxiv2024

Mitoregulin self-associates to form likely homo-oligomeric pore-like structures

We and others previously found that a misannotated long noncoding RNA encodes for a conserved mitochondrial transmembrane microprotein named Mitoregulin (Mtln). Beyond an established role for Mtln in lipid metabolism, Mtln has also been shown to more broadly influence mitochondria, boosting respiratory efficiency and Ca2+ retention capacity, while lowering ROS, yet the underlying mechanisms remain unresolved. Prior studies have identified possible Mtln protein interaction partners; however, a lack of consensus persists, and no claims have been made about Mtlns structure. We previously noted two key published observations that seemingly remained overlooked: 1) endogenous Mtln co-immunoprecipitates with epitope-tagged Mtln at high efficiency, and 2) Mtln primarily exists in a [~]66 kDa complex. To investigate if Mtln may self-oligomerize into higher-order complexes, we performed co-immunoprecipitation, protein modeling simulations, and native gel assessments of Mtln-containing complexes in cells and tissues, as well as tested whether synthetic Mtln protein itself forms oligomeric complexes. Our combined results provide strong support that Mtln self-associates and likely forms a hexameric pore-like structure.

molecular biology↗

bioRxiv2024

Mitoregulin supports mitochondrial membrane integrity and protects against cardiac ischemia-reperfusion injury

We and others discovered a highly-conserved mitochondrial transmembrane microprotein, named Mitoregulin (Mtln), that supports lipid metabolism. We reported that Mtln strongly binds cardiolipin (CL), increases mitochondrial respiration and Ca2+ retention capacities, and reduces reactive oxygen species (ROS). Here we extend our observation of Mtln-CL binding and examine Mtln influence on cristae structure and mitochondrial membrane integrity during stress. We demonstrate that mitochondria from constitutive- and inducible Mtln-knockout (KO) mice are susceptible to membrane freeze-damage and that this can be rescued by acute Mtln re-expression. In mitochondrial-simulated lipid monolayers, we show that synthetic Mtln decreases lipid packing and monolayer elasticity. Lipidomics revealed that Mtln-KO heart tissues show broad decreases in 22:6-containing lipids and increased cardiolipin damage/remodeling. Lastly, we demonstrate that Mtln-KO mice suffer worse myocardial ischemia-reperfusion injury, hinting at a translationally-relevant role for Mtln in cardioprotection. Our work supports a model in which Mtln binds cardiolipin and stabilizes mitochondrial membranes to broadly influence diverse mitochondrial functions, including lipid metabolism, while also protecting against stress.

cell biology↗

bioRxiv2024

A Specialized Centrosome-Proteasome Axis Mediates Proteostasis and Influences Cardiac Stress through Txlnb

BackgroundCentrosomes localize to perinuclear foci where they serve multifunctional roles, arranging the microtubule organizing center (MTOC) and anchoring ubiquitin-proteasome system (UPS) machinery. In mature cardiomyocytes, centrosomal proteins redistribute into a specialized perinuclear cage-like structure, and a potential centrosome-UPS interface has not been studied. Taxilin-beta (Txlnb), a cardiomyocyte-enriched protein, belongs to a family of centrosome adapter proteins implicated in protein quality control. We hypothesize that Txlnb plays a key role in centrosomal-proteasomal crosstalk in cardiomyocytes. MethodsIntegrative bioinformatics assessed centrosomal gene dysregulation in failing hearts. Txlnb gain/loss-of-function studies were conducted in cultured cardiomyocytes and mice. Txlnbs role in cardiac proteotoxicity and hypertrophy was examined using CryAB-R120G mice and transverse aortic constriction (TAC), respectively. Molecular modeling investigated Txlnb structure/function. ResultsHuman failing hearts show consistent dysregulation of many centrosome-associated genes, alongside UPS-related genes. Txlnb emerged as a candidate regulator of cardiomyocyte proteostasis that localizes to the perinuclear centrosomal compartment. Txlnbs interactome strongly supports its involvement in cytoskeletal, microtubule, and UPS processes, particularly centrosome-related functions. Overexpressing Txlnb in cardiomyocytes reduced ubiquitinated protein accumulation and enhanced proteasome activity during hypertrophy. Txlnb-knockout (KO) mouse hearts exhibit proteasomal insufficiency and altered cardiac growth, evidenced by ubiquitinated protein accumulation, decreased 26S{beta}5 proteasome activity, and lower mass with age. In Cryab-R120G mice, Txlnb loss worsened heart failure, causing lower ejection fractions. After TAC, Txlnb-KO mice also showed reduced ejection fraction, increased heart mass, and elevated ubiquitinated protein accumulation. Investigations into the molecular mechanisms revealed that Txlnb-KO did not affect proteasomal subunit expression but led to the upregulation of Txlna and several centrosomal proteins (Cep63, Ofd1, and Tubg) suggesting altered centrosomal dynamics. Structural predictions support Txlnbs role as a specialized centrosomal-adapter protein bridging centrosomes with proteasomes, confirmed by microtubule-dependent perinuclear localization. ConclusionsTogether, these data provide initial evidence connecting Txlnb to cardiac proteostasis, hinting at the potential importance of functional bridging between specialized centrosomes and UPS in cardiomyocytes.

physiology↗

bioRxiv2020

Modulation of the Cardiac Sodium Channel Nav1.5 Peak and Late Currents by NAD+ Precursors

RationaleThe cardiac sodium channel NaV1.5, encoded by SCN5A, produces the rapidly inactivating depolarizing current INa that is responsible for the initiation and propagation of the cardiac action potential. Acquired and inherited dysfunction of NaV1.5 results in either decreased peak INa or increased residual late INa (INa,L), leading to tachy/bradyarrhythmias and sudden cardiac death. Previous studies have shown that increased cellular NAD+ and NAD+/NADH ratio increase INa through suppression of mitochondrial reactive oxygen species and PKC-mediated NaV1.5 phosphorylation. In addition, NAD+-dependent deacetylation of NaV1.5 at K1479 by Sirtuin 1 increases NaV1.5 membrane trafficking and INa. The role of NAD+ precursors in modulating INa remains unknown. ObjectiveTo determine whether and by which mechanisms the NAD+ precursors nicotinamide riboside (NR) and nicotinamide (NAM) affect peak INa and INa,L in vitro and cardiac electrophysiology in vivo. Methods and ResultsThe effects of NAD+ precursors on the NAD+ metabolome and electrophysiology were studied using HEK293 cells expressing wild-type and mutant NaV1.5, rat neonatal cardiomyocytes (RNCMs), and mice. NR increased INa in HEK293 cells expressing NaV1.5 (500 M: 51 {+/-} 18%, p=0.02, 5 mM: 59 {+/-} 22%, p=0.03) and RNCMs (500 {micro}M: 60 {+/-} 26%, p=0.02, 5 mM: 75 {+/-} 39%, p=0.03) while reducing INa,L at the higher concentration (RNCMs, 5 mM: -45 {+/-} 11%, p=0.04). NR (5 mM) decreased NaV1.5 K1479 acetylation but increased INa in HEK293 cells expressing a mutant form of NaV1.5 with disruption of the acetylation site (NaV1.5-K1479A). Disruption of the PKC phosphorylation site abolished the effect of NR on INa. Furthermore, NAM (5 mM) had no effect on INa in RNCMs or in HEK293 cells expressing wild-type NaV1.5, but increased INa in HEK293 cells expressing NaV1.5-K1479A. Dietary supplementation with NR for 10-12 weeks decreased QTc in C57BL/6J mice (0.35% NR: -4.9 {+/-} 2.0%, p=0.26; 1.0% NR: -9.5 {+/-} 2.8%, p=0.01). ConclusionsNAD+ precursors differentially regulate NaV1.5 via multiple mechanisms. NR increases INa, decreases INa,L, and warrants further investigation as a potential therapy for arrhythmic disorders caused by NaV1.5 deficiency and/or dysfunction.

physiology↗