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BioRxiv : the Preprint Server For... Jun 2024-Related Dilated Cardiomyopathy (DCM) is an autosomal-dominant genetic condition with cardiomyocyte and conduction system dysfunction often resulting in heart failure...
-Related Dilated Cardiomyopathy: Single-Cell Transcriptomics during Patient-derived iPSC Differentiation Support Cell type and Lineage-specific Dysregulation of Gene Expression and Development for Cardiomyocytes and Epicardium-Derived Cells with Lamin A/C Haploinsufficiency.
-Related Dilated Cardiomyopathy (DCM) is an autosomal-dominant genetic condition with cardiomyocyte and conduction system dysfunction often resulting in heart failure or sudden death. The condition is caused by mutation in the Lamin A/C ( ) gene encoding Type-A nuclear lamin proteins involved in nuclear integrity, epigenetic regulation of gene expression, and differentiation. Molecular mechanisms of disease are not completely understood, and there are no definitive treatments to reverse progression or prevent mortality. We investigated possible mechanisms of -Related DCM using induced pluripotent stem cells derived from a family with a heterozygous splice-site mutation. We differentiated one mutant iPSC line derived from an affected female (Patient) and two non-mutant iPSC lines derived from her unaffected sister (Control) and conducted single-cell RNA sequencing for 12 samples (4 Patient and 8 Control) across seven time points: Day 0, 2, 4, 9, 16, 19, and 30. Our bioinformatics workflow identified 125,554 cells in raw data and 110,521 (88%) high-quality cells in sequentially processed data. Unsupervised clustering, cell annotation, and trajectory inference found complex heterogeneity: ten main cell types; many possible subtypes; and lineage bifurcation for Cardiac Progenitors to Cardiomyocytes (CM) and Epicardium-Derived Cells (EPDC). Data integration and comparative analyses of Patient and Control cells found cell type and lineage differentially expressed genes (DEG) with enrichment to support pathway dysregulation. Top DEG and enriched pathways included: 10 genes and RNA polymerase II transcription in Pluripotent cells (PP); and TGF Beta/BMP signaling, sarcomere gene subsets and cardiogenesis, and EMT in CM; and epigenetic regulation and and mTORC1 signaling in EPDC. Top DEG also included: and other X-linked genes, six imprinted genes: , , , , , , and enriched gene sets in metabolism, proliferation, and homeostasis. We confirmed Lamin A/C haploinsufficiency by allelic expression and Western blot. Our complex Patient-derived iPSC model for Lamin A/C haploinsufficiency in PP, CM, and EPDC provided support for dysregulation of genes and pathways, many previously associated with Lamin A/C defects, such as epigenetic gene expression, signaling, and differentiation. Our findings support disruption of epigenomic developmental programs as proposed in other disease models. We recognized other factors influencing epigenetics and differentiation; thus, our approach needs improvement to further investigate this mechanism in an iPSC-derived model.
PubMed: 38915555
DOI: 10.1101/2024.06.12.598335 -
Frontiers in Cardiovascular Medicine 2024Little is known about left ventricular (LV) sequences of contraction and electrical activation in hypertrophic cardiomyopathy (HCM). A better understanding of the...
BACKGROUND
Little is known about left ventricular (LV) sequences of contraction and electrical activation in hypertrophic cardiomyopathy (HCM). A better understanding of the underlying relation between mechanical and electrical activation may allow the identification of predictive response criteria to right ventricular DDD pacing in obstructive patients.
OBJECTIVE
To describe LV mechanical and electrical activation sequences in HCM patients compared to controls.
MATERIALS AND METHODS
We prospectively studied, in 40 HCM patients (20 obstructive and 20 non-obstructive) and 20 healthy controls: (1) mechanical activation using echocardiography at rest and cardiac magnetic resonance imaging, (2) electrical activation using 3-dimensional electrocardiographic mapping (ECM).
RESULTS
In echocardiography, healthy controls had a physiological apex-to-base delay (ABD) during contraction (23.8 ± 16.2 ms). Among the 40 HCM patients, 18 HCM patients presented a loss of this ABD (<10 ms, defining hypersynchrony) more frequently than controls (45% vs. 5%, = 0.017). These patients had a lower LV end-diastolic volume (71.4 ± 9.7 ml/m vs. 82.4 ± 14.8 ml/m, = 0.01), lower native T1 values (988 ± 32 ms vs. 1,028 ± 39 ms, = 0.001) and tended to have lower LV mass (80.7 ± 23.7 g/m vs. 94.5 ± 25.3 g/m, = 0.08) compared with HCM patients that had a physiological contraction sequence. There was no significant relation between ABD and LV outflow tract obstruction. While HCM patients with a physiological contraction sequence presented an ECM close to those encountered in controls, patients with a loss of ABD presented a particular pattern of ECM with the first potential more frequently occurring in the postero-basal region.
CONCLUSION
The LV contraction sequence can be modified in HCM patients, with a loss of the physiological ABD, and is associated with smaller LV dimensions and a particular pattern of ECM. Further research is needed to determine whether this pattern is related to an electrical substrate or is the consequence of the hypertrophied heart's specific geometry.
CLINICAL TRIAL REGISTRATION
ClinicalTrial.gov: NCT02559726.
PubMed: 38911519
DOI: 10.3389/fcvm.2024.1359657 -
PLoS Genetics Jun 2024Filamins are mechanosensitive actin crosslinking proteins that organize the actin cytoskeleton in a variety of shapes and tissues. In muscles, filamin crosslinks actin...
Filamins are mechanosensitive actin crosslinking proteins that organize the actin cytoskeleton in a variety of shapes and tissues. In muscles, filamin crosslinks actin filaments from opposing sarcomeres, the smallest contractile units of muscles. This happens at the Z-disc, the actin-organizing center of sarcomeres. In flies and vertebrates, filamin mutations lead to fragile muscles that appear ruptured, suggesting filamin helps counteract muscle rupturing during muscle contractions by providing elastic support and/or through signaling. An elastic region at the C-terminus of filamin is called the mechanosensitive region and has been proposed to sense and counteract contractile damage. Here we use molecularly defined mutants and microscopy analysis of the Drosophila indirect flight muscles to investigate the molecular details by which filamin provides cohesion to the Z-disc. We made novel filamin mutations affecting the C-terminal region to interrogate the mechanosensitive region and detected three Z-disc phenotypes: dissociation of actin filaments, Z-disc rupture, and Z-disc enlargement. We tested a constitutively closed filamin mutant, which prevents the elastic changes in the mechanosensitive region and results in ruptured Z-discs, and a constitutively open mutant which has the opposite elastic effect on the mechanosensitive region and gives rise to enlarged Z-discs. Finally, we show that muscle contraction is required for Z-disc rupture. We propose that filamin senses myofibril damage by elastic changes in its mechanosensory region, stabilizes the Z-disc, and counteracts contractile damage at the Z-disc.
PubMed: 38905299
DOI: 10.1371/journal.pgen.1011101 -
Journal of the American Heart... Jun 2024ELMSAN1 (ELM2-SANT domain-containing scaffolding protein 1) is a newly identified scaffolding protein of the MiDAC (mitotic deacetylase complex), playing a pivotal role...
BACKGROUND
ELMSAN1 (ELM2-SANT domain-containing scaffolding protein 1) is a newly identified scaffolding protein of the MiDAC (mitotic deacetylase complex), playing a pivotal role in early embryonic development. Studies on knockout mice showed that its absence results in embryo lethality and heart malformation. However, the precise function of ELMSAN1 in heart development and formation remains elusive. To study its potential role in cardiac lineage, we employed human-induced pluripotent stem cells (hiPSCs) to model early cardiogenesis and investigated the function of ELMSAN1.
METHODS AND RESULTS
We generated -deficient hiPSCs through knockdown and knockout techniques. During cardiac differentiation, depletion inhibited pluripotency deactivation, decreased the expression of cardiac-specific markers, and reduced differentiation efficiency. The impaired expression of genes associated with contractile sarcomere structure, calcium handling, and ion channels was also noted in -deficient cardiomyocytes derived from hiPSCs. Additionally, through a series of structural and functional assessments, we found that -null hiPSC cardiomyocytes are immature, exhibiting incomplete sarcomere Z-line structure, decreased calcium handling, and impaired electrophysiological properties. Of note, we found that the cardiac-specific role of ELMSAN1 is likely associated with histone H3K27 acetylation level. The transcriptome analysis provided additional insights, indicating maturation reduction with the energy metabolism switch and restored cell proliferation in knockout cardiomyocytes.
CONCLUSIONS
In this study, we address the significance of the direct involvement of ELMSAN1 in the differentiation and maturation of hiPSC cardiomyocytes. We first report the impact of ELMSAN1 on multiple aspects of hiPSC cardiomyocyte generation, including cardiac differentiation, sarcomere formation, calcium handling, electrophysiological maturation, and proliferation.
PubMed: 38904247
DOI: 10.1161/JAHA.124.034816 -
BioRxiv : the Preprint Server For... Jun 2024The contraction of striated muscle is driven by cycling myosin motor proteins embedded within the thick filaments of sarcomeres. In addition to cross-bridge cycling with...
The contraction of striated muscle is driven by cycling myosin motor proteins embedded within the thick filaments of sarcomeres. In addition to cross-bridge cycling with actin, these myosin proteins can enter an inactive, sequestered state in which the globular S1 heads rest along the thick filament surface and are unable to perform motor activities. Structurally, this state is called the interacting heads motif (IHM) and is a critical conformational state of myosin that regulates muscle contractility and energy expenditure. Structural perturbation of the sequestered state via missense mutations can pathologically disrupt the mechanical performance of muscle tissue. Thus, the IHM state has become a target for therapeutic intervention. An ATP analogue called 2'-deoxy-ATP (dATP) is a potent myosin activator which destabilizes the IHM. Here we use molecular dynamics simulations to study the molecular mechanisms by which dATP modifies the structure and dynamics of myosin in a sequestered state. Simulations with IHM containing ADP.Pi in both nucleotide binding pockets revealed residual dynamics in an otherwise 'inactive' and 'sequestered' state of a motor protein. Replacement of ADP.Pi by dADP.Pi triggered a series of structural changes that modify the protein-protein interface that stabilizes the sequestered state, and changes to this interface were accompanied by allosteric changes in remote regions of the protein complex. A comparative analysis of these dynamics predicted new structural sites that may affect IHM stability.
PubMed: 38895221
DOI: 10.1101/2024.06.06.597809 -
International Journal of Molecular... Jun 2024Levosimendan's calcium sensitizing effects in heart muscle cells are well established; yet, its potential impact on skeletal muscle cells has not been evidently...
Levosimendan's calcium sensitizing effects in heart muscle cells are well established; yet, its potential impact on skeletal muscle cells has not been evidently determined. Despite controversial results, levosimendan is still expected to interact with skeletal muscle through off-target sites (further than troponin C). Adding to this debate, we investigated levosimendan's acute impact on fast-twitch skeletal muscle biomechanics in a length-dependent activation study by submersing single muscle fibres in a levosimendan-supplemented solution. We employed our technology to investigate the calcium sensitivity of skinned single muscle fibres alongside their stress-strain response in the presence or absence of levosimendan (100 µM). While control data are in agreement with the theory of length-dependent activation, levosimendan appears to shift the onset of the 'descending limb' of active force generation to longer sarcomere lengths without notably improving myofibrillar calcium sensitivity. Passive stretches in the presence of levosimendan yielded over twice the amount of enlarged restoration stress and Young's modulus in comparison to control single fibres. Both effects have not been described before and may point towards potential off-target sites of levosimendan.
Topics: Simendan; Animals; Mice; Calcium; Muscle Fibers, Fast-Twitch; Muscle Contraction; Sarcomeres; Male; Myofibrils
PubMed: 38892380
DOI: 10.3390/ijms25116191 -
Biology Open Jun 2024Regular spatial patterns are ubiquitous forms of organization in nature. In animals, regular patterns can be found from the cellular scale to the tissue scale, and from...
Regular spatial patterns are ubiquitous forms of organization in nature. In animals, regular patterns can be found from the cellular scale to the tissue scale, and from early stages of development to adulthood. To understand the formation of these patterns, how they assemble and mature, and how they are affected by perturbations, a precise quantitative description of the patterns is essential. However, accessible tools that offer in-depth analysis without the need for computational skills are lacking for biologists. Here, we present PatternJ, a novel toolset to analyze regular one-dimensional patterns precisely and automatically. This toolset, to be used with the popular imaging processing program ImageJ/Fiji, facilitates the extraction of key geometric features within and between pattern repeats in static images and time-lapse series. We validate PatternJ with simulated data and test it on images of sarcomeres from insect muscles and contracting cardiomyocytes, actin rings in neurons, and somites from zebrafish embryos obtained using confocal fluorescence microscopy, STORM, electron microscopy, and brightfield imaging. We show that the toolset delivers subpixel feature extraction reliably even with images of low signal-to-noise ratio. PatternJ's straightforward use and functionalities make it valuable for various scientific fields requiring quantitative one-dimensional pattern analysis, including the sarcomere biology of muscles or the patterning of mammalian axons, speeding up discoveries with the bonus of high reproducibility.
Topics: Animals; Axons; Image Processing, Computer-Assisted; Zebrafish; Sarcomeres; Somites; Software; Algorithms
PubMed: 38887972
DOI: 10.1242/bio.060548 -
Frontiers in Cardiovascular Medicine 2024Hypertrophic cardiomyopathy (HCM) is a very prevalent inherited disease with a wide global distribution and a prevalence rate of approximately 0.2% in the general... (Review)
Review
Hypertrophic cardiomyopathy (HCM) is a very prevalent inherited disease with a wide global distribution and a prevalence rate of approximately 0.2% in the general population. Left ventricular hypertrophy (LVH) caused by sarcomere mutation is the primary reason of HCM. The histopathology feature is that cardiomyocyte hypertrophy, myocyte disorder and myocardial fibrosis lead to diminished diastolic function, left ventricular outflow tract obstruction (LVOTO) and arrhythmia, all of which result in serious cardiac complications. Previously, HCM was considered a malignant disease that was almost untreatable. With the improvement of medical standards and increasing awareness of HCM, it has become a highly treatable disease in contemporary times, with a significant decrease in mortality rates. However, there are still significant unmet requirements in the therapy of HCM. This paper draws on more than 100 references from the past four decades and summarizes current advances in the treatment of HCM. The article will review the pathogenesis and types, recent development in pharmacotherapy, invasive treatments and gene therapies, as well as dilemma and future development of HCM.
PubMed: 38887447
DOI: 10.3389/fcvm.2024.1387596 -
PNAS Nexus Jun 2024Cardiomyocytes meet their high ATP demand almost exclusively by oxidative phosphorylation (OXPHOS). Adequate oxygen supply is an essential prerequisite to keep OXPHOS...
Cardiomyocytes meet their high ATP demand almost exclusively by oxidative phosphorylation (OXPHOS). Adequate oxygen supply is an essential prerequisite to keep OXPHOS operational. At least two spatially distinct mitochondrial subpopulations facilitate OXPHOS in cardiomyocytes, i.e. subsarcolemmal (SSM) and interfibrillar mitochondria (IFM). Their intracellular localization below the sarcolemma or buried deep between the sarcomeres suggests different oxygen availability. Here, we studied SSM and IFM isolated from piglet hearts and found significantly lower activities of electron transport chain enzymes and FF-ATP synthase in IFM, indicative for compromised energy metabolism. To test the contribution of oxygen availability to this outcome, we ventilated piglets under hyperbaric hyperoxic (HBO) conditions for 240 min. HBO treatment raised OXPHOS enzyme activities in IFM to the level of SSM. Complexome profiling analysis revealed that a high proportion of the FF-ATP synthase in the IFM was in a disassembled state prior to the HBO treatment. Upon increased oxygen availability, the enzyme was found to be largely assembled, which may account for the observed increase in OXPHOS complex activities. Although HBO also induced transcription of genes involved in mitochondrial biogenesis, a full proteome analysis revealed only minimal alterations, meaning that HBO-mediated tissue remodeling is an unlikely cause for the observed differences in OXPHOS. We conclude that a previously unrecognized oxygen-regulated mechanism endows cardiac OXPHOS with spatiotemporal plasticity that may underlie the enormous metabolic and contractile adaptability of the heart.
PubMed: 38881840
DOI: 10.1093/pnasnexus/pgae210 -
Scientific Reports Jun 2024Connectin (also known as titin) is a giant striated muscle protein that functions as a molecular spring by providing elasticity to the sarcomere. Novex-3 is a short...
Connectin (also known as titin) is a giant striated muscle protein that functions as a molecular spring by providing elasticity to the sarcomere. Novex-3 is a short splice variant of connectin whose physiological function remains unknown. We have recently demonstrated using in vitro analyses that in addition to sarcomere expression, novex-3 was also expressed in cardiomyocyte nuclei exclusively during fetal life, where it provides elasticity/compliance to cardiomyocyte nuclei and promotes cardiomyocyte proliferation in the fetus, suggesting a non-sarcomeric function. Here, we analyzed novex-3 knockout mice to assess the involvement of this function in cardiac pathophysiology in vivo. Deficiency of novex-3 compromised fetal cardiomyocyte proliferation and induced the enlargement of individual cardiomyocytes in neonates. In adults, novex-3 deficiency resulted in chamber dilation and systolic dysfunction, associated with Ca dysregulation, resulting in a reduced life span. Mechanistic analyses revealed a possible association between impaired proliferation and abnormal nuclear mechanics, including stiffer nuclei positioned peripherally with stabilized circumnuclear microtubules in knockout cardiomyocytes. Although the underlying causal relationships were not fully elucidated, these data show that novex-3 has a vital non-sarcomeric function in cardiac pathophysiology and serves as an early contributor to cardiomyocyte proliferation.
Topics: Animals; Myocytes, Cardiac; Cell Proliferation; Mice, Knockout; Mice; Cell Nucleus; Connectin; Sarcomeres; Muscle Proteins; Calcium
PubMed: 38877142
DOI: 10.1038/s41598-024-64608-1