Biology subjects

Margulies, K. B.

Publications and source records attributed to Margulies, K. B..

At least 19 recordsLinked to original sources

bioRxiv2026

SureQuant IS-PRM Enables Cross-Species Targeted Quantification of Retinoid Metabolism and Signaling Proteins in the Heart

Retinoic acid signaling is critical for cardiac development and homeostasis. Dysregulation of all-trans retinoic acid metabolism contributes to vascular atherogenesis, restenosis, calcification, and heart failure. Therefore, assessment of proteins involved in retinoid metabolism and signaling has gained interest for identifying potential biomarkers and therapeutic targets in cardiovascular disease. However, quantifying these proteins remains challenging due to limitations of antibody-based methods. We developed a targeted proteomics approach using SureQuant internal standard-triggered parallel reaction monitoring mass spectrometry to profile these proteins. We designed a panel of 80 stable isotope-labeled (heavy) peptides representing proteins involved in retinoid signaling and metabolism, with sequences applicable to human samples and conserved across multiple species. Survey experiments using directed data-dependent acquisition on the Orbitrap Fusion Lumos mass spectrometer determined precursor and product ion masses for each heavy peptide, which were programmed into the SureQuant method for continuous monitoring. Upon detection of these heavy internal standards, the instrument transitions to a targeted PRM acquisition mode in which repeated high-resolution MS/MS spectra of both endogenous (light) and heavy peptides are acquired. Using this method, retinoid pathway-associated proteins were quantified to as low as 10 attomoles for selected targets across multiple tissues and developmental stages. Distinct tissue-specific retinoid metabolic networks were identified across lung, liver, retinal cell lines and cardiac tissues. Developmental profiling of mouse and rat hearts revealed remodeling of retinoid pathway proteins from embryonic to postnatal and adult stages, suggesting a functional transition from retinoid-driven cardiac development toward maintenance of retinoid homeostasis in the mature heart.

biochemistry↗

bioRxiv2026

Label free multimodal optical imaging of metabolic heterogeneity in aging by integrating SRS, MPF, FLIM, and SHG

Cellular metabolism is governed by the coordinated organization of macromolecules, including lipids and proteins, together with redox-active cofactors such as NADH and FAD. However, resolving these biochemical features quantitatively and spatially at subcellular resolution remains challenging because no single imaging modality can capture molecular composition, redox state, and tissue architecture simultaneously without labeling. Here, we present MANIFEST (Multimodal Analysis of Nonlinear Imaging for Functional Endogenous Spatial correlaTion), a label-free imaging platform that integrates stimulated Raman scattering (SRS), second harmonic generation (SHG), multiphoton fluorescence (MPF), and fluorescence lifetime imaging microscopy (FLIM) with their spatial correlation. MANIFEST combines chemical imaging of lipids with autofluorescence- and lifetime-based quantification of NADH and FAD metabolism, enabling spatially resolved analysis of metabolic heterogeneity at organelle and tissue-compartment levels. We apply this framework to four distinct aging or disease models: amyloid-beta-treated tri-cultured brain cells, high-fat diet mouse liver, human non-ischemic cardiomyopathy tissue, and aging mouse retina. Across these systems, MANIFEST reveals disease-associated lipid remodeling, redox imbalance, disrupted metabolic zonation, collagen reorganization, and layer-specific metabolic changes. By integrating complementary nonlinear optical modalities into a single label-free platform, MANIFEST provides an extensible, proof-of-concept platform for high-resolution metabolic phenotyping in complex biological systems and offers new opportunities for studying disease mechanisms, aging biology, and metabolism-driven tissue pathology.

bioengineering↗

bioRxiv2025

Effects of Sodium Glucose Co-Transporter Inhibitors on Work in Human Hypertrophic Cardiomyopathy Living Myocardial Slices

BackgroundDisease modifying therapies for hypertrophic cardiomyopathy (HCM) remain a prevailing unmet need. Human-based experimental platforms capable of controlled manipulation of preload and afterload can distinguish direct myocardial and systemic effects and facilitate development of targeted cardiac therapeutics. Sodium glucose cotransporter inhibitors (SGLTi) may directly affect cardiac contractility, potentially related to increased ketone availability. These effects have not been adequately studied in human HCM myocardium under defined loading conditions. AimsWe sought to establish human living myocardial slices (LMS) as a platform to interrogate load-dependent myocardial mechanics in HCM and to quantify the acute effects of metabolic and pharmacologic interventions--including SGLTi--on myocardial work under physiologic loading conditions. MethodsHuman myocardial tissue was procured from non-failing donor hearts or individuals with HCM undergoing septal myectomy. Freshly prepared human LMS were mechanically tested to generate biomimetic work loops across a range of physiologic preloads and afterloads in either glucose-only fuel or glucose supplemented with ketone. Following baseline measurements, slices were loaded with drug (isoproterenol, mavacamten, sotagliflozin, or empagliflozin) or vehicle (DMSO) and work loop analysis was repeated, allowing each slice to serve as its own control. Mixed effects linear regression models incorporating random effects for heart and slice and fixed effects for clinical characteristics evaluated determinants of myocardial work and drug response across loading conditions. ResultsA total of 120 LMS from 32 individuals (16 non-failing and 16 HCM) were analyzed. At baseline, myocardial work was positively associated with younger age, hypertension, and ejection fraction. Ketone supplementation augmented work and work-strain slope particularly in HCM LMS at high afterloads. We validated our drug testing methodology by demonstrating increased work with known positive inotrope isoproterenol, decreased work with negative inotrope mavacamtem most pronounced in HCM LMS, and a null effect of DMSO. Acute exposure to SGLTi sotagliflozin and empagliflozin directly reduced myocardial work, with increased potency of sotagliflozin at high afterloads. ConclusionsOur LMS platform enables assessment of myocardial mechanics across controlled loading conditions and is an ideal platform to rigorously phenotype human myocardial tissue and interrogate direct effects of pharmacologic intervention. We demonstrate that SGLTi and ketones have distinct and discordant effects on human myocardial contractility.

physiology↗

bioRxiv2025

Telomere 3'-overhang attrition and persistent telomeric DNA damage response in failing human hearts

BackgroundTelomere homeostasis is critical for normal cellular and organ function, and its dysregulation is implicated in aging and chronic diseases. Although telomere length (TL) is critical for normal telomere function, its functional status can also be altered by many other factors. The organization and protective status of telomere in failing human heart (FHH) remains incompletely understood. MethodsUsing left ventricular tissues from patients with idiopathic dilated cardiomyopathy (IDC), ischemic heart disease (IHD), and non-failing heart (NFH), we performed a comprehensive analysis that included measurements of overall TL and 3 overhang length in left ventricles and cardiomyocytes (CM), assessment of telomere-binding protein associations, and transcriptomic profiling through RNA sequencing. ResultsAlthough TL varied among individuals, reduced median TL was observed only in IDC, while both IDC and IHD showed an increased frequency of very short telomeres, falling below the 5th percentile, in CMs. Strikingly, significant telomere 3-overhang attrition was detected in both disease groups and strongly correlated with elevated H2AX phosphorylated on serine 139 ({gamma}H2AX) in heart tissues, indicating DNA damage. This was accompanied by persistent activation of ataxia telangiectasia mutated (ATM) protein-mediated DNA damage responses and the formation of telomere dysfunction-induced foci (TIFs) in CMs from FHH. Concomitantly, the association of telomeres with the single-stranded telomere-binding protein, Protection of Telomeres 1 (POT1), and the double-stranded telomere-interacting protein, telomere repeat-binding factor 2 (TERF2), was markedly reduced, accompanied by an increase in association of {gamma}H2AX and ssDNA binding protein, RPA with telomeres in IDC and IHD relative to NFH, signifying telomere de-protection. ConclusionsCM telomere dysfunction, characterized by 3 overhang attrition and de-protection, is a common feature in FHH, leading to persistent DNA damage response in telomeres. Better understanding of telomere biology throughout the progression of different heart diseases to heart failure will provide more effective prevention and treatment strategies. GRAPHICAL ABSTRACTHuman telomeres safeguard chromosome ends through a higher-order telomere-loop (t-loop) structure and the shelterin complex, preventing their recognition as DNA double-strand breaks (DSBs). In the failing human heart (FHH) with idiopathic dilated cardiomyopathy (IDC) and ischemic heart disease (IHD), cardiomyocyte telomeres lose protection due to disruption of the shelterin complex, driven by oxidative DNA damage from reactive oxygen species (ROS) targeting G-rich telomeric sequences. This leads to t-loop unfolding, DDR cascade activation, 3 overhang excision, and persistent DNA damage responses with Telomere Dysfunction-Induced Foci (TIFs) formation at the telomeric regions of cardiomyocytes in FHH. The ATM-mediated DDR, along with engagement of both classical non-homologous end joining (cNHEJ) and homologous recombination (HDR) repair pathways, is integral to FHH progression. This study underscores the significance of telomere 3 overhang attrition in the pathogenesis of FHH with IDC and IHD. Unlike changes in overall telomere length, 3 overhang shortening directly correlates with increased DNA damage in human myocardial tissue. Preventing telomere 3 overhang attrition and oxidative damage offers a promising avenue for attenuating heart disease progression toward heart failure. In particular, selective ATM inhibition, as opposed to ATR inhibition, presents a potential therapeutic strategy for patients with IDC and IHD. Future efforts to pinpoint deficiencies in cNHEJ and HDR pathways in failing human hearts may reveal novel targets for heart failure prevention and treatment, providing new strategies in the management of this patient population. CMs, cardiomyocytes; DDR, DNA double-strand break repair; FHH, failing human heart; ROS, reactive oxygen species; t-loop, telomere-loop. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=120 SRC="FIGDIR/small/669993v1_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@1d21937org.highwire.dtl.DTLVardef@757f54org.highwire.dtl.DTLVardef@1fa7472org.highwire.dtl.DTLVardef@1f2e961_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2025

Matrix Stiffening Induces Mechanical Memory and Nuclear Fragility in Cardiomyocytes via Microtubule-Lamin Coupling

BackgroundMechanical memory (MM) describes the persistent phenotypic remodeling following exposure to a transient extrinsic biomechanical cue. Short-term biomechanical stress is a feature of several etiologies of cardiomyopathy, including dysfunction of the viable myocardium following a large myocardial infarction. However, the nuclear mechanisms linking stiffness to persistent cellular remodeling remain poorly understood. MethodsWe cultured human iPSC-cardiomyocytes on a magnetorheological elastomer (MRE) with tunable stiffness (9-56 kPa) to mimic physiological and pathological myocardium. This allowed us to assess how transient increases in stiffness influence cellular responses such as nuclear structure, and DNA damage in hiPSC-cardiomyocytes. Using a combination of Immunofluorescence imaging, Western Blot and pharmacological interventions, we examined the role of microtubule detyrosination and the LINC (Linker of Nucleoskeleton and Cytoskeleton) complex in transducing mechanical signals from the cytoskeleton to the nucleus with a focus on MM induction. ResultsShort-term (6 h) stiff priming induced reversible phenotypic changes upon resoftening. However, 48 h of stiff priming triggered persistent MM, characterized by nuclear rupture, increased lamin A/C expression, DNA damage, and cytoplasmic leakage of DNA repair factors like KU80. Disruption of either a-tubulin detyrosination or the LINC complex prevented MM and nuclear damage, indicating that these elements are essential for nuclear mechanotransduction. In contrast, depletion of lamin A/C or DNA repair components accelerated stiffness-induced phenotypes and promoted MM onset within 6 hours. Finally, inhibition of a-tubulin detyrosination using ADV-TTL reversed both MM and DNA damage. ConclusionsAcceleration of MM induction by lamin knockdown suggests that hereditary laminopathies may be associated with increased cardiomyocyte vulnerability to transient mechanical stress inducing DNA damage and senescence. Conversely, the protective effects of limiting stiffness-induced -tubulin detyrosination, or nuclear mechano-transduction suggest potential cardioprotective strategies in the setting of laminopathies and/or sustained increases in extracellular matrix stiffness.

cell biology↗

bioRxiv2025

Cardiac fibroblasts counterbalance cardiomyocytes in LMNA cardiomyopathy pathogenesis

Genetic cardiomyopathies arising from mutations in the LMNA gene, encoding nuclear intermediate filaments lamin A/C, display variable age of onset, severity, and fibrosis development. This variability suggests a fundamental element in disease pathogenesis that has yet to be elucidated. Given the central role cardiac fibroblasts play in fibrosis, we explored the relevance of lamin A/C in cardiac fibroblast function, as very little is known in this regard. Using primary cardiac fibroblasts and in vivo mouse models, we show that Lmna mutations impact various aspects of cardiac fibroblast function in response to myocyte damage. We show that both lamin A/C depletion and point-mutant variant expression impair cardiac fibroblast proliferation and contraction whereas other functions such as cell migration appears to be mutation dependent. In vivo depletion of lamin A/C simultaneously in cardiomyocytes and cardiac fibroblasts significantly delayed disease progression, improved cardiac function, and prolonged survival, indicating that lamin A/C mediate an opposing balance between cardiomyocytes and cardiac fibroblasts in driving disease pathogenesis. Our results elucidate previously unexplored roles of lamin A/C in cardiac fibroblasts and suggest that interactions between cardiac fibroblasts and cardiomyocytes are important determinants of the rate of progression and the severity of LMNA cardiomyopathy.

cell biology↗

bioRxiv2025

Desmoplakin Haploinsufficiency Underlies Cell-Cell Adhesion Failure in DSP Cardiomyopathy and is Rescued by Transcriptional Activation

BackgroundTruncating variants in desmoplakin (DSPtv), are a leading cause of arrhythmogenic cardiomyopathy (ACM), often presenting with early fibrosis and arrhythmias disproportionate to systolic dysfunction. DSP is critical for cardiac mechanical integrity, linking desmosomes to the cytoskeleton to withstand contractile forces. While loss-of-function is implicated, direct evidence, both for DSP haploinsufficiency in human hearts and for the impact of mechanical stress on cardiomyocyte adhesion, has been limited, leaving the pathogenic mechanism unclear. MethodsWe analyzed explanted human heart tissue from patients with DSPtv (N=3), titin truncating variants (TTNtv, N=5), and controls (N=5) using RNA-sequencing and mass spectrometry. We generated human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) harboring patient-derived or CRISPR-Cas9 engineered DSPtv to model a range of DSP expression levels. Using a 2D cardiac muscle bundle (CMB) platform enabling live visualization of cell junctions, we developed an assay to assess cell-cell adhesion upon heightened contractile stress in response to the contractile agonist endothelin-1. CRISPR-interference (CRISPRi) was used to confirm the role of DSP loss, and CRISPR-activation (CRISPRa) was tested for therapeutic rescue. ResultsCompared to both control and TTNtv hearts, DSPtv human hearts exhibited reduced DSP at both the mRNA and protein level, as well as broadly disrupted desmosomal stoichiometry. Transcriptomic and proteomic analyses implicated cell adhesion, extracellular matrix, and inflammatory pathways. iPSC-CM models recapitulated DSP haploinsufficiency and desmosomal disruption. DSPtv CMBs showed normal baseline contractile function. However, they displayed marked cell-cell adhesion failure with contractile stress (75% failure vs. 8% in controls, p<0.001). Adhesion failure was prevented by the myosin inhibitor, mavacamten. CRISPRi-mediated DSP knockdown replicated this susceptibility to adhesion failure. Conversely, CRISPRa robustly increased DSP expression and rescued cell-cell adhesion failure in DSPtv CMBs (9% failure post-CRISPRa, p<0.001 vs. un-treated). Rescue occurred even when only the DSPII isoform was upregulated in a model with biallelic DSP transcript 1 loss of function. ConclusionsDSP haploinsufficiency is the major cause of DSP cardiomyopathy with a primary consequence of conferring vulnerability to cardiomyocyte cell-cell adhesion failure under heightened contractile stress. Transcriptional activation of DSP reverses this defect in preclinical models, establishing proof-of-concept for a potential therapeutic strategy in DSP cardiomyopathy.

genetics↗

bioRxiv2025

Microtubule detyrosination alters nuclear mechanotransduction and leads to pro-hypertrophic signaling in hypertrophic cardiomyopathy

BackgroundHypertrophic cardiomyopathy (HCM) is characterized by left ventricular hypertrophy and diastolic dysfunction and is accompanied by extensive cytoskeletal remodeling, including increased protein levels of desmin, tubulin and detyrosinated tubulin. We have previously demonstrated that tubulin detyrosination contributes to diastolic dysfunction. Microtubules are connected to the nucleus by the LINC complex, yet the role of cytoskeleton-nucleus interactions in HCM remain poorly understood. ObjectivesWe investigated whether cytoskeletal remodeling in HCM alters nuclear morphology and mechanics, and modulates hypertrophy-associated signaling pathways. MethodsNuclear morphology was assessed by electron microscopy and immunofluorescence in cardiac septal tissue from obstructive HCM patients (N=19) and non-failing donors (NF, N=8), as well as in Wild-Type (WT, N=18) and homozygous HCM-associated Mybpc3 c.2373InsG mice (Mybpc3c.2373InsG, N=19). A novel live-cell imaging approach was used to study nuclear deformation during cardiomyocyte contraction. YAP1 nuclear translocation was measured to evaluate downstream mechanosensitive signaling and detyrosination inhibitor epoY was used to test causality. ResultsNuclei were highly invaginated and enlarged in HCM in both patients and mice. Nuclear deformation during contraction was restricted in HCM cardiomyocytes, indicating altered mechanotransduction. These changes were associated with increased YAP1 nuclear localization and induction of YAP1 target and hypertrophic genes. Inhibition of microtubule detyrosination reduced nuclear invaginations, restored nuclear deformation and decreased YAP1 nuclear translocation. ConclusionCytoskeletal remodeling in HCM is associated with altered nuclear morphology and mechanotransduction, accompanied by YAP1 translocation which may contribute to hypertrophic remodeling. Targeting microtubule detyrosination rescues this phenotype, identifying nuclear mechanotransduction as a potential therapeutic target for HCM. Unstructured abstractHypertrophic cardiomyopathy (HCM) is characterized by left ventricular hypertrophy and diastolic dysfunction and is accompanied by extensive cytoskeletal remodeling, including increased desmin, tubulin, and detyrosinated tubulin levels, which we previously showed contribute to impaired relaxation. Since microtubules connect to the nucleus via the LINC complex, we investigated whether cytoskeletal remodeling alters nuclear morphology, mechanics, and hypertrophy-associated signaling. Using electron microscopy and immunofluorescence in cardiac septal tissue from obstructive HCM patients and non-failing donors, and immunofluorescence and live-cell imaging in septal tissue from Wild-Type and homozygous HCM-associated Mybpc3 c.2373InsG mice, we found that nuclei were enlarged and highly invaginated, with restricted nuclear deformation during contraction, indicating altered mechanotransduction. These changes were associated with increased YAP1 nuclear localization and increased expression of YAP1 target and hypertrophic genes. Inhibiting microtubule detyrosination reduced nuclear abnormalities, restored nuclear deformation, and decreased YAP1 nuclear translocation, identifying nuclear mechanotransduction as a potential therapeutic target in HCM. Visual abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=122 SRC="FIGDIR/small/646061v2_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@7f915forg.highwire.dtl.DTLVardef@12131c6org.highwire.dtl.DTLVardef@4aff5eorg.highwire.dtl.DTLVardef@1c3a3a8_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LICytoskeletal remodeling contributes to nuclear abnormalities and impaired mechanotransduction in hypertrophic cardiomyopathy. C_LIO_LIMicrotubule detyrosination promotes nuclear invaginations and increased YAP1 signaling in hypertrophic cardiomyopathy. C_LIO_LIPharmacological reduction of detyrosinated tubulin normalizes nuclear structure and mechanosignaling. C_LIO_LITargeting nuclear-cytoskeletal coupling may provide new therapeutic strategies for hypertrophic cardiomyopathy. C_LI

physiology↗

bioRxiv2025

Single cell transcriptomic analyses of human heart failure with preserved ejection fraction

BackgroundHeart failure with preserved ejection fraction (HFpEF) is a poorly understood, multi-system disease with high morbidity and mortality. To improve our understanding of its underlying biology, we used single-nucleus RNA sequencing (snRNA-seq) to characterize cell-specific gene expression patterns in human HFpEF myocardium. MethodsSeptal myocardial biopsies (2-3 mg) from 30 HFpEF patients and 29 non-failing donor controls were analyzed using the 10X Genomics platform, with nuclei isolated from combined samples (6 patients/pool). Genotype-based demultiplexing was performed with souporcell, and gene expression quantified with CellRanger and CellBender. After quality control, nuclei were clustered and annotated by cell types based on specific marker genes. Differential expression (DE) by cell-type in HFpEF vs controls was performed using limma-voom and functional analysis performed using Gene Set Enrichment Analysis. Data were compared to dilated cardiomyopathy (DCM) using prior snRNA-seq in DCM vs respective controls. ResultsWe successfully demultiplexed pooled myocardial biopsies, assigning >75% of droplets to individual patients. From eight pooled samples (19 HFpEF, 24 controls), we recovered 48,886 nuclei and identified 14 cell types. Cardiomyocytes (5159 differentially expressed [DE] genes, 36%) and fibroblasts (5905 DE genes, 49%) showed the most DE genes, while endothelial cells (2143), pericytes (1812), and macrophages (1405) had fewer. Enriched pathways common to multiple cell types included transcription/translation, immune activation, metabolism, and protein quality control. Of 7848 DE genes identified via pseudo-bulk snRNA-seq, 51% were DE in fibroblasts and 47% in cardiomyocytes, compared to <20% in other cell types. Unlike dilated cardiomyopathy (DCM), sub-clustering fibroblasts did not reveal an activated fibroblast population in HFpEF. Comparative analysis between HFpEF and DCM identified transcriptional differences primarily in cardiomyocytes. ConclusionsThis study demonstrates the power of genotype-based demultiplexing for single-cell transcriptomic analyses of small endomyocardial biopsies and identifies cardiomyocytes as the principal cell type with distinct transcriptional changes in HFpEF versus DCM. These findings, coupled with differential gene expression and functional pathway analyses, illuminate HFpEF pathways and may nominate compelling targets for future mechanistic studies and therapeutic efforts for HFpEF. Clinical PerspectiveWhat is new? We successfully used genotype-based demultiplexing to perform single nucleus RNA-seq from myocardial biopsies. The snRNA-seq analysis revealed distinct enrichment of pathways related to transcription/translation, immune activation, metabolism, and protein quality control across multiple cell types in HFpEF. In contrast to DCM, HFpEF is distinguished by the absence of an activated fibroblast population and a predominance of transcriptional differences within cardiomyocytes, highlighting a distinct disease mechanism. What are the clinical implications? The cell-selective HFpEF myocardial transcriptional landscape highlights altered metabolism, protein translation and quality control, immune activation and growth/matrix organization. These pathways provide compelling new targets for developing effective treatments.

bioinformatics↗

bioRxiv2025

Ultrastructure analysis of mitochondria, lipid droplet and sarcoplasmic reticulum apposition in human heart failure

BackgroundCardiomyocyte structural remodeling is reported as a causal contributor to heart failure (HF) development and progression. Growing evidence highlights the role of organelle apposition in cardiomyocyte function and homeostasis. Disruptions in organelle crosstalk, such as that between the sarcoplasmic reticulum (SR) and mitochondria, are thought to impact numerous cellular processes such as calcium handling and cellular bioenergetics; two processes that are disrupted and implicated in cardiac pathophysiology. While the physical distance between organelles is thought to be essential for homeostatic cardiomyocyte function, whether the interactions and coupling of organelles are altered in human heart failure remains unclear. MethodsHere, we utilized transmission electron microscopy and careful quantification of ultrastructure to characterize the changes in organelle apposition in cardiomyocytes isolated from the hearts of patients diagnosed with various types of HF. Subsequently we employed molecular approaches to examine the expression of proposed organelle tethers. ResultsWe demonstrate that cardiomyocytes isolated from dilated cardiomyopathy, hypertrophic cardiomyopathy and ischemic cardiomyopathy hearts display smaller, more rounded mitochondria, as compared to nonfailing controls. Failing cardiomyocytes also exhibited disrupted SR-mitochondria juxtaposition and changes in the expression of proposed molecular tethers. Further analysis revealed alterations in lipid droplet dynamics including decreased lipid droplet content and less lipid droplets in association with mitochondria in failing cardiomyocytes. ConclusionHere we observed changes in organelle dynamics in cardiomyocytes isolated from heart failure patients diagnosed with differing etiologies. Our results suggest that organelle structure and apposition may be a ubiquitous contributor to human HF progression. RESEARCH PERSPECTIVEWhat is New? O_LIWe provide a detailed analysis of organelle apposition in human heart failure, which has been understudied, and report that that failing human cardiomyocytes display an increase in distance between mitochondria and both the sarcoplasmic reticulum and lipid droplets. C_LIO_LIStructural changes in organelles are correlated with the expression of proposed organelle tethers. C_LIO_LIResource of ultrastructural changes in organelle apposition in human heart failure resulting from various etiologies. C_LI What Questions Should be Addressed next? O_LIThe results from this study provide rationale for causal experimentation to elucidate the contribution of organelle apposition to the progression of heart failure. Future studies examining mechanisms of mitochondrial tethering to the SR or lipid droplet will evaluate specific targets for therapeutic application. C_LI

cell biology↗

bioRxiv2024

Increased CPT1a expression is a critical cardioprotective response to pathological stress that suppresses gene programs for remodeling and enables rescue by gene transfer

Text AbstractO_ST_ABSBackgroundC_ST_ABSCarnitine palmitoyl transferase 1 (CPT1) is a rate-limiting enzyme for long chain fatty acid oxidation (FAO) in cardiac mitochondria. In adult hearts, CPT1b predominates, while CPT1a is co-expressed at lower levels. Pathological stress on the heart induces greater CPT1a expression, and this coincides with a reduction in FAO, yet the role of CPT1a in pathological cardiac remodeling is unknown. MethodsCPT1 isoform expression was assayed in myocardium of human heart failure (HF) patients with nonischemic cardiomyopathy (NICM) and a preclinical mouse model of heart failure. To explore the role of CPT1a upregulation in response to pathological stress, mice were subjected to afterload stress via transverse aortic constriction (TAC) or sham surgery (sham) with cardiac-specific CPT1a knockdown or cardiac-specific, AAV9-mediated CPT1a overexpression (AAV9.cTNTN.Cpt1a), versus empty virus or PBS infusions as controls. MiR370, known to suppress hepatic CPT1a, was assayed and overexpressed to determine if miR370 regulates cardiac CPT1a expression. ResultsCPT1a protein was elevated and miR370 reduced in myocardium of male and female NICM patients (204% vs. non-failing unused donor hearts), as well as in failing mouse hearts. AAV mediated miR370 overexpression in mouse hearts suppressed CPT1a expression and attenuated the response of CPT1a to TAC. Preventing CPT1a upregulation in response to TAC in cardiac specific CPT1a knockout mice (csCPT1a ko) exacerbated adverse remodeling, causing severe dysfunction and increased mortality. In contrast, CPT1a overexpression (2.8 fold), attenuated impaired ejection fraction (EF, by 54%) and fractional shortening (FS, 65%) vs. PBS-infused TAC hearts (p<0.05). Delivery of AAV9.cTnT.Cpt1a 4 wks after TAC surgery, led to significant rescue of EF and FS vs. animals receiving empty virus and mitigated the exacerbated dysfunction of csCPT1a ko hearts at 4 wks TAC. RNA-seq and reverse transcription-quantitative PCR revealed a novel function of CPT1a in suppressing hypertrophic, profibrotic and cell death gene programs in both sham and TAC hearts, irrespective of changes in FAO. ConclusionsThe effects of CPT1a in the heart extend beyond FAO and include a non-canonical regulation of cardiac gene programs. In addition to an animal model of HF, CPT1a upregulation occurs in NICM, and is a critical cardioprotective adaptation to pathological stress.

physiology↗

bioRxiv2023

Right Ventricular Sarcomere Contractile Depression and the Role of Thick Filament Activation in Human Heart Failure with Pulmonary Hypertension

RationaleRight ventricular (RV) contractile dysfunction commonly occurs and worsens outcomes in heart failure patients with reduced ejection fraction and pulmonary hypertension (HFrEF-PH). However, such dysfunction often goes undetected by standard clinical RV indices, raising concerns that they may not reflect aspects of underlying myocyte dysfunction. ObjectiveTo determine components of myocyte contractile depression in HFrEF-PH, identify those reflected by clinical RV indices, and elucidate their underlying biophysical mechanisms. Methods and ResultsResting, calcium- and load-dependent mechanics were measured in permeabilized RV cardiomyocytes isolated from explanted hearts from 23 HFrEF-PH patients undergoing cardiac transplantation and 9 organ-donor controls. Unsupervised machine learning using myocyte mechanical data with the highest variance yielded two HFrEF-PH subgroups that in turn mapped to patients with depressed (RVd) or compensated (RVc) clinical RV function. This correspondence was driven by reduced calcium-activated isometric tension in RVd, while surprisingly, many other major myocyte contractile measures including peak power, maximum unloaded shortening velocity, and myocyte active stiffness were similarly depressed in both groups. Similar results were obtained when subgroups were first defined by clinical indices, and then myocyte mechanical properties in each group compared. To test the role of thick-filament defects, myofibrillar structure was assessed by X-ray diffraction of muscle fibers. This revealed more myosin heads associated with the thick filament backbone in RVd but not RVc, as compared to controls. This corresponded to reduced myosin ATP turnover in RVd myocytes, indicating less myosin in a cross-bridge ready disordered-relaxed (DRX) state. Altering DRX proportion (%DRX) affected peak calcium-activated tension in the patient groups differently, depending on their basal %DRX, highlighting potential roles for precision-guided therapeutics. Lastly, increasing myocyte preload (sarcomere length) increased %DRX 1.5-fold in controls but only 1.2-fold in both HFrEF-PH groups, revealing a novel mechanism for reduced myocyte active stiffness and by extension Frank-Starling reserve in human HF. ConclusionsWhile there are multiple RV myocyte contractile deficits In HFrEF-PH, clinical indices primarily detect reduced isometric calcium-stimulated force related to deficits in basal and recruitable %DRX myosin. Our results support use of therapies to increase %DRX and enhance length-dependent recruitment of DRX myosin heads in such patients.

biophysics↗

bioRxiv2023

Perinuclear damage from nuclear envelope deterioration elicits stress responses that contribute to LMNA cardiomyopathy

Mutations in the LMNA gene encoding nuclear lamins A/C cause a diverse array of tissue-selective diseases, with the heart being the most commonly affected organ. Despite progress in understanding the molecular perturbations emanating from LMNA mutations, an integrative understanding of the pathogenesis leading to cardiac dysfunction remains elusive. Using a novel cell-type specific Lmna deletion mouse model capable of translatome profiling, we found that cardiomyocyte-specific Lmna deletion in adult mice led to rapid cardiomyopathy with pathological remodeling. Prior to the onset of cardiac dysfunction, lamin A/C-depleted cardiomyocytes displayed nuclear envelope deterioration, golgi dilation/fragmentation, and CREB3-mediated golgi stress activation. Translatome profiling identified upregulation of Med25, a transcriptional co-factor that can selectively dampen UPR axes. Autophagy is disrupted in the hearts of these mice, which can be recapitulated by disrupting the golgi or inducing nuclear damage by increased matrix stiffness. Systemic administration of pharmacological modulators of autophagy or ER stress significantly improved the cardiac function. These studies support a hypothesis wherein stress responses emanating from the perinuclear space contribute to the development of LMNA cardiomyopathy. TeaserInterplay of stress responses underlying the development of LMNA cardiomyopathy

cell biology↗

bioRxiv2022

Tissue- and sex-specific DNA damage tracks aging in rodents and humans

DNA damage causes genomic instability underlying many human diseases. Traditional approaches to DNA damage analysis provide minimal insights into the spectrum of disease-driving DNA lesions and the mechanisms causing imbalances in damage formation and repair. Here we used untargeted mass spectrometry-based adductomics1 to discover 114 putative DNA lesions and modifications consistently detected in humans and two independent analyses in rats, showing species-, tissue-, age-, and sex-biases. As evidence of methodologic rigor, 10 selected adductomic signals were structurally validated as epigenetic marks: 5-MdC, 5-HMdC, 5-FdC; DNA damage products: N2-CMdG, 1,N6 {varepsilon}-dA, 3,N4-{varepsilon}dC, M1dG, O6/N2-MdG, and 8-Oxo-dG; and established analytical artifacts: cyclobutane dimers of 2-deoxycytosine. With steady-state levels of putative DNA adducts integrating multiple cell types in each tissue, there was strong age-dependent variation for many putative adducts, including N2-CMdG, 5-HMdC, and 8-Oxo-dG in rats and 1,N6 {varepsilon}-dA in human heart, as well as sex biases for 67 putative adducts in rat tissues. These results demonstrate the potential of untargeted adductomic analysis for defining DNA adducts as disease determinants, assigning substrates to DNA repair pathways, discovering new metabolically-driven DNA lesions, and quantifying inter-individual variation in DNA damage and repair across populations.

biochemistry↗

bioRxiv2021

TRAF2, an innate immune sensor, reciprocally regulates mitophagy and inflammation to maintain cardiac myocyte homeostasis.

Mitochondrial damage triggers cell death signaling with catastrophic consequences in long-lived and irreplaceable cells, such as cardiac myocytes. Sensing of leaked mitochondrial DNA upon mitochondrial damage is also a potent trigger of inflammation. Whether the innate immune response pathways monitor mitochondrial damage in mitochondria-rich cardiac myocytes to prevent inflammation and cell death, remains unknown. TRAF2, an adaptor protein downstream of innate immune receptors, localizes to the mitochondria in the unstressed heart, with increased mitochondrial targeting in cardiomyopathic human hearts and after cardiac ischemia-reperfusion injury in mice. Inducible cardiomyocyte-specific deletion of TRAF2 in young adult mice impairs mitophagy with rapid decline in mitochondrial quality, upregulates TLR9 expression in cardiac myocytes, and results in inflammation and cell death manifesting as a fulminant cardiomyopathy. Preventing TLR9-mediated mitochondrial DNA sensing and resultant inflammation provides a short-term reprieve from cardiomyopathy, but persistence of damaged mitochondria results in long-term recrudescence. Restoration of wild-type TRAF2, but not the E3 ubiquitin ligase deficient mutant, improves mitochondrial quality and rescues cardiomyopathy to restore homeostasis. Thus, the innate immune response acts via TRAF2 as the first line of defense against mitochondrial damage by orchestrating homeostatic mitophagy to dampen myocardial inflammation and prevent cell death.

cell biology↗

bioRxiv2021

Apolipoprotein M attenuates doxorubicin cardiotoxicity by regulating transcription factor EB

ObjectivesDetermine the role of apolipoprotein M (ApoM) in anthracycline (Dox) cardiotoxicity. BackgroundApoM binds the cardioprotective sphingolipid sphingosine-1-phosphate (S1P). Circulating ApoM is inversely associated with mortality in human heart failure (HF). MethodsIn the Penn HF Study (PHFS), we tested the relationship between ApoM and mortality in a subset with anthracycline-induced cardiomyopathy. We measured ApoM in humans and mice treated with Dox and utilized hepatic ApoM transgenic (ApomTG), ApoM knockout (ApomKO), ApoM knock-in mice with impaired S1P binding, and S1P receptor 3 (S1PR3) knockout mice in Dox cardiotoxicity. We assayed autophagy in left ventricular tissue from anthracycline-induced HF patients versus donor controls. ResultsApoM was inversely associated with mortality in PHFS, and Dox reduced circulating ApoM in mice and breast cancer patients. ApomTG mice were protected from Dox-induced cardiac dysfunction and loss of left ventricular mass. ApomTG attenuated Dox-induced impairment in autophagic flux in vivo and accumulation of insoluble p62, which was also observed in the myocardium of patients with anthracycline-induced HF. In vehicle-treated mice, ApoM negatively regulated transcription factor EB (TFEB), a master regulator of autophagy and lysosomal biogenesis. The effect of ApoM on TFEB required both S1P binding and S1PR3. In the presence of Dox, ApoM preserved TFEB and cardiomyocyte lysosomal abundance assessed as lysosomal associated membrane protein 1 positive structures in vivo, while S1P mimetic pretreatment of cardiomyocytes prevented Dox-induced changes in lysosomal pH. ConclusionsApoM attenuates Dox cardiotoxicity via the autophagy-lysosome pathway. The association between ApoM and reduced mortality may be explained by its role in sustaining autophagy. HighlightsO_LICirculating ApoM is inversely associated with survival in human anthracycline-induced cardiomyopathy C_LIO_LIAnthracycline treatment reduces circulating ApoM in humans and mice C_LIO_LIIncreasing ApoM attenuates doxorubicin cardiotoxicity, lysosomal injury and preserves myocardial autophagic flux, but does not impact doxorubicin anti-neoplastic efficacy C_LIO_LIAutophagic impairment is characteristic of human anthracycline cardiomyopathy C_LI

pharmacology and toxicology↗

bioRxiv2020

Transcriptional and Cellular Diversity of the Human Heart

IntroductionThe human heart requires a complex ensemble of specialized cell types to perform its essential function. A greater knowledge of the intricate cellular milieu of the heart is critical to increase our understanding of cardiac homeostasis and pathology. As recent advances in low input RNA-sequencing have allowed definitions of cellular transcriptomes at single cell resolution at scale, here we have applied these approaches to assess the cellular and transcriptional diversity of the non-failing human heart. MethodsMicrofluidic encapsulation and barcoding was used to perform single nuclear RNA sequencing with samples from seven human donors, selected for their absence of overt cardiac disease. Individual nuclear transcriptomes were then clustered based upon transcriptional profiles of highly variable genes. These clusters were used as the basis for between-chamber and between-sex differential gene expression analyses and intersection with genetic and pharmacologic data ResultsWe sequenced the transcriptomes of 287,269 single cardiac nuclei, revealing a total of 9 major cell types and 20 subclusters of cell types within the human heart. Cellular subclasses include two distinct groups of resident macrophages, four endothelial subtypes, and two fibroblasts subsets. Comparisons of cellular transcriptomes by cardiac chamber or sex reveal diversity not only in cardiomyocyte transcriptional programs, but also in subtypes involved in extracellular matrix remodeling and vascularization. Using genetic association data, we identified strong enrichment for the role of cell subtypes in cardiac traits and diseases. Finally, intersection of our dataset with genes on cardiac clinical testing panels and the druggable genome reveals striking patterns of cellular specificity. ConclusionsUsing large-scale single nuclei RNA sequencing, we have defined the transcriptional and cellular diversity in the normal human heart. Our identification of discrete cell subtypes and differentially expressed genes within the heart will ultimately facilitate the development of new therapeutics for cardiovascular diseases.

genomics↗

bioRxiv2019

Detailed Regulatory Interaction Map of the Human Heart Facilitates Gene Discovery for Cardiovascular Disease

Most disease-associated variants identified by population based genetic studies are non-coding, which compromises finding causative genes and mechanisms. Presumably they interact through looping with nearby genes to modulate transcription. Hi-C provides the most complete and unbiased method for genome-wide identification of potential regulatory interactions, but finding chromatin loops in Hi-C data remains difficult and tissue specific data are limited. We have generated Hi-C data from primary cardiac tissue and developed a method, peakHiC, for sensitive and quantitative loop calling to uncover the human heart regulatory interactome. We identify complex CTCF-dependent and -independent contact networks, with loops between coding and non-coding gene promoters, shared enhancers and repressive sites. Across the genome, enhancer interaction strength correlates with gene transcriptional output and loop dynamics follows CTCF, cohesin and H3K27Ac occupancy levels. Finally, we demonstrate that intersection of the human heart regulatory interactome with cardiovascular disease variants facilitates prioritizing disease-causative genes.

genomics↗