Cardiomyocyte Sarcomere Research Articles

Abstract We039: Adducin Promotes Cardiomyocyte Sarcomere Disassembly

Introduction: Recent interest in understanding the cardiomyocyte cell cycle has been driven by potential therapeutic applications in cardiomyopathy. Despite advancements, the intricacies of cardiomyocyte mitosis, especially the breakdown of sarcomeres to enable daughter cardiomyocytes' division, remain largely unexplored. Hypothesis: This study investigates the role of Adducin in the disassembly of sarcomeres during cardiomyocyte mitosis, hypothesizing that Adducin serves as a crucial regulator in this process. Goals: Our objective is to elucidate the function of Adducin in sarcomere disassembly during the mitosis of mammalian cardiomyocytes and understand how this process is regulated. Methods: We examined the expression patterns of α/γ-Adducins, particularly in neonatal cardiomyocytes undergoing mitosis. Subsequently, we experimented with the overexpression of various α-Adducin splices and phosphorus in vitro and in vivo, with a keen interest in the phosphorylation effects of α-Adducin on sarcomere disassembly. Results: Here, we identify Adducin as a regulator of sarcomere disassembly during mammalian cardiomyocyte mitosis. α/γ-Adducins are selectively expressed in neonatal mitotic cardiomyocytes, and their levels decline precipitously thereafter. Cardiomyocyte-specific overexpression of various splice isoforms and phosphoforms of α-Adducin in vitro and in vivo identified Thr445/Thr480 phosphorylation of a short isoform of α-Adducin as a potent inducer of neonatal cardiomyocyte sarcomere disassembly. Additionally, co-overexpressing this α-Adducin variant with γ-Adducin in adult mice stabilizes the Adducin complex and maintains sarcomere disassembly, facilitated through interactions with α-Actinin. Conclusion: The study highlights Adducin, particularly α-Adducin phosphorylated at Thr445/Thr480, as a pivotal regulator of sarcomere disassembly during neonatal cardiomyocyte mitosis. This mechanism is essential for coordinating the cytoskeletal changes necessary for the mitotic process in cardiomyocytes, offering insights into potential therapeutic targets for cardiomyopathy.

Localized translation and sarcomere maintenance requires ribosomal protein SA in mice.

Cardiomyocyte sarcomeres contain localized ribosomes, but the factors responsible for their localization and the significance of localized translation are unknown. Using proximity labeling, we identified Ribosomal Protein SA (RPSA) as a Z-line protein. In cultured cardiomyocytes, the loss of RPSA led to impaired local protein translation and reduced sarcomere integrity. By employing CAS9 expressing mice along with adeno-associated viruses expressing CRE recombinase and single-guide RNAs targeting Rpsa, we knocked out Rpsa in vivo and observed mis-localization of ribosomes and diminished local translation. These genetic mosaic mice with Rpsa knockout in a subset of cardiomyocytes developed dilated cardiomyopathy, featuring atrophy of RPSA-deficient cardiomyocytes, compensatory hypertrophy of unaffected cardiomyocytes, left ventricular dilation, and impaired contractile function. We demonstrate that RPSA C-terminal domain is sufficient for localization to the Z-lines and that if the microtubule network is disrupted RPSA loses its sarcomeric localization. These findings highlight RPSA as a ribosomal factor essential for ribosome localization to the Z-line, facilitating local translation and sarcomere maintenance.

Adducin Regulates Sarcomere Disassembly During Cardiomyocyte Mitosis.

Recent interest in understanding cardiomyocyte cell cycle has been driven by potential therapeutic applications in cardiomyopathy. However, despite recent advances, cardiomyocyte mitosis remains a poorly understood process. For example, it is unclear how sarcomeres are disassembled during mitosis to allow the abscission of daughter cardiomyocytes. Here, we use a proteomics screen to identify adducin, an actin capping protein previously not studied in cardiomyocytes, as a regulator of sarcomere disassembly. We generated many adeno-associated viruses and cardiomyocyte-specific genetic gain-of-function models to examine the role of adducin in neonatal and adult cardiomyocytes in vitro and in vivo. We identify adducin as a regulator of sarcomere disassembly during mammalian cardiomyocyte mitosis. α/γ-adducins are selectively expressed in neonatal mitotic cardiomyocytes, and their levels decline precipitously thereafter. Cardiomyocyte-specific overexpression of various splice isoforms and phospho-isoforms of α-adducin in vitro and in vivo identified Thr445/Thr480 phosphorylation of a short isoform of α-adducin as a potent inducer of neonatal cardiomyocyte sarcomere disassembly. Concomitant overexpression of this α-adducin variant along with γ-adducin resulted in stabilization of the adducin complex and persistent sarcomere disassembly in adult mice, which is mediated by interaction with α-actinin. These results highlight an important mechanism for coordinating cytoskeletal morphological changes during cardiomyocyte mitosis.

Sarcomere. The structural unit of the myofibrillar complex of typical cardiomyocytes. Isometric aspects of sarcomere organization

Background. The complexity of understanding the structure and function of cardiomyocytes in the myocardium is a fundamental problem in the field of heart morphology. Sarcomeres are the main structural and functional units of myofibrils. Understanding the organization and dynamics of sarcomeres in cardiomyocytes is crucial for establishing the mechanisms responsible for the process of forming the contractile system of cardiomyocytes. Objective. The main purpose of this study is a review of literary sources that complement and expand the essential findings about the construction of myofibrils and their components when studied by effective methods of microscopic techniques. Materials. Literature review databases, systematic search of primary sources related to the research topic. Methods of visual examination of the components of sarcomeres and elements of their organization. Results. Transmission electron microscopy provides high-resolution imaging of sarcomeres, which is necessary for detailed analysis of their ultrastructure, including visualization of sarcomere components such as Z-discs, M-lines, thick and thin filaments. Morphometric methods are crucial for studying the compaction and orientation of myofibrils in cardiomyocytes. Conclusion. Morphometric methods provide valuable information about the structural remodeling of myofibrils in response to physiological stimuli or pathological conditions. These techniques are needed to improve our understanding of the mechanisms underlying muscle contraction and to develop methods to study cardiac remodeling processes in response to the adaptive response.

Mechanisms of vibration-induced structural myocardial remodeling

The review analyzes literature data on structural changes in the heart of patients with vibration disease, as detected by echocardiographic methods. Particularly, it highlights concentric remodeling of the left ventricle chambers and disturbances in diastolic function. The review also discusses a 1.2-fold decrease in heart structure intensity compared to healthy individuals (p 0.05). Furthermore, it examines changes in morphometric and bioenergetic parameters of cardiomyocytes under different experimental vibration modes (7 and 56 sessions at a frequency of 8 Hz), confirming the disruptions in the relationship between the spatial configuration of the heart cavities, contractile ability, and energy supply potential. Loss of cardiac myofibrils represents the transition from myocardial hypertrophy to decompensation, accompanied by an increase in degenerative (dystrophic) signs such as the loss of sarcomeres in cardiomyocytes. Understanding these pathological (morphological) processes requires consideration of various mediators that regulate cell metabolism, proliferation, growth, and survival, including stromal interaction molecule, calcium ATPase of the endo(sarco)plasmic reticulum, inositol-1,4,5-triphosphate receptor, protein that forms CRAC channels, and transient receptor potential canonical. The degradation system of the extracellular matrix, including matrix metalloproteinases and tissue inhibitors, plays a crucial role in structural cardiac remodeling. This system regulates the rate of mRNA synthesis on the DNA matrix by binding to specific DNA regions that control cardiac nutrition and plasticity. The review suggests that these findings can help explain some patterns of cardiac remodeling development in patients with vibration disease and determine the direction of pathogenetically based therapy. This therapy should consider not only the vibration-protective effect of drugs but also their ability to inhibit and regress myocardial remodeling.

Nesprin-2 is a novel scaffold protein for telethonin and FHL-2 in the cardiomyocyte sarcomere

Nesprins comprise a family of multi-isomeric scaffolding proteins, forming the linker of nucleoskeleton-and-cytoskeleton complex with lamin A/C, emerin and SUN1/2 at the nuclear envelope. Mutations in nesprin-1/-2 are associated with Emery-Dreifuss muscular dystrophy (EDMD) with conduction defects and dilated cardiomyopathy (DCM). We have previously observed sarcomeric staining of nesprin-1/-2 in cardiac and skeletal muscle, but nesprin function in this compartment remains unknown. In this study we show that specific nesprin-2 isoforms are highly expressed in cardiac muscle and localise to the Z-disc and I band of the sarcomere. Expression of GFP-tagged nesprin-2 giant spectrin repeats 52-53, localised to the sarcomere of neonatal rat cardiomyocytes. Yeast two-hybrid screening of a cardiac muscle cDNA library identified telethonin and four and half LIM domain (FHL)-2 as potential nesprin-2 binding partners. GST pull-down and immunoprecipitation confirmed the individual interactions between nesprin-2/telethonin and nesprin-2/FHL-2, and showed that nesprin-2 and telethonin binding was dependent on telethonin phosphorylation status. Importantly, the interactions between these binding partners were impaired by mutations in nesprin-2, telethonin and FHL-2 identified in EDMD with DCM and hypertrophic cardiomyopathy patients. These data suggest that nesprin-2 is a novel sarcomeric scaffold protein that may potentially participate in maintenance and/or regulation of sarcomeric organisation and function.

Advances in Multi-Modality Imaging in Hypertrophic Cardiomyopathy

Hypertrophic cardiomyopathy (HCM) is characterized by abnormal growth of the myocardium with myofilament disarray and myocardial hyper-contractility, leading to left ventricular hypertrophy and fibrosis. Where culprit genes are identified, they typically relate to cardiomyocyte sarcomere structure and function. Multi-modality imaging plays a crucial role in the diagnosis, monitoring, and risk stratification of HCM, as well as in screening those at risk. Following the recent publication of the first European Society of Cardiology (ESC) cardiomyopathy guidelines, we build on previous reviews and explore the roles of electrocardiography, echocardiography, cardiac magnetic resonance (CMR), cardiac computed tomography (CT), and nuclear imaging. We examine each modality’s strengths along with their limitations in turn, and discuss how they can be used in isolation, or in combination, to facilitate a personalized approach to patient care, as well as providing key information and robust safety and efficacy evidence within new areas of research.

Decellularized heart extracellular matrix alleviates activation of hiPSC-derived cardiac fibroblasts

Human induced pluripotent stem cell derived cardiac fibroblasts (hiPSC-CFs) play a critical role in modeling human cardiovascular diseases in vitro. However, current culture substrates used for hiPSC-CF differentiation and expansion, such as Matrigel and tissue culture plastic (TCPs), are tissue mismatched and may provide pathogenic cues. Here, we report that hiPSC-CFs differentiated on Matrigel and expanded on tissue culture plastic (M-TCP-iCFs) exhibit transcriptomic hallmarks of activated fibroblasts limiting their translational potential. To alleviate pathogenic activation of hiPSC-CFs, we utilized decellularized extracellular matrix derived from porcine heart extracellular matrix (HEM) to provide a biomimetic substrate for improving hiPSC-CF phenotypes. We show that hiPSC-CFs differentiated and expanded on HEM (HEM-iCFs) exhibited reduced expression of hallmark activated fibroblast markers versus M-TCP-iCFs while retaining their cardiac fibroblast phenotype. HEM-iCFs also maintained a reduction in expression of hallmark genes associated with pathogenic fibroblasts when seeded onto TCPs. Further, HEM-iCFs more homogenously integrated into an hiPSC-derived cardiac organoid model, resulting in improved cardiomyocyte sarcomere development. In conclusion, HEM provides an improved substrate for the differentiation and propagation of hiPSC-CFs for disease modeling.

Early-life exposure to lead changes cardiac development and compromises long-term cardiac function

Lead (Pb) is widely used in industrial and daily-use consumer products. Early-life exposure may increase the risk of lead-related heart problems in childhood. However, the effects of early-life lead exposure on fetal heart development and long-term cardiac outcomes are unknown. In this study, pregnant ICR mice were exposed to lead acetate trihydrate (50 mg/kg/d) via oral gavage from gestation day 1.5 until offspring weaning. Thereafter, the second hit model was established, two groups of offspring (4 weeks old) were either administered sterile saline or Angiotensin II (Ang II) for 4 weeks until euthanasia. We investigated lead-induced offspring heart damage from embryonic period to adulthood by echocardiographic analysis, pathological H&E staining, and ultrastructural examination, as well as mitochondrial function detection. The results showed early-life lead exposure predisposed offspring mice to decreased ejection fraction, increased left ventricular volume, accompanied by hypertrophy and dilation, cardiomyocyte sarcomere dysplasia, abnormal mitochondrial structure, mitochondrial dysfunction, and decreased expression of key sarcomeric and mitochondrial genes, rendering them more susceptible to cardiac hypertrophy, vascular wall thickening, cardiac fibrosis, apoptosis, and heart failure induced by Ang II infusion. This study elucidates early-life low dose lead exposure compromises cardiac development and exacerbates second hit-induced cardiac pathological responses in adulthood, which furnishes crucial scientific evidence pertaining to the cardiac toxicity and risk evaluation associated with early-life exposure to lead.

Assessment of sarcomere shortening and calcium transient in primary human and dog ventricular myocytes

Understanding translation from preclinical observations to clinical findings is important for evaluating the efficacy and safety of novel compounds. Of relevance to cardiac safety is profiling drug effects on cardiomyocyte (CM) sarcomere shortening and intracellular Ca2+ dynamics. Although CM from different animal species have been used to assess such effects, primary human CM isolated from human organ donor heart represent an ideal non-animal alternative approach. We performed a study to evaluate primary human CM and have them compared to freshly isolated dog cardiomyocytes for their basic function and responses to positive inotropes with well-known mechanisms. Our data showed that simultaneous assessment of sarcomere shortening and Ca2+-transient can be performed with both myocytes using the IonOptix system. Amplitude of sarcomere shortening and Ca2+-transient (CaT) were significantly higher in dog compared to human CM in the basic condition (absence of treatment), while longer duration of sarcomere shortening and CaT were observed in human cells. We observed that human and dog CMs have similar pharmacological responses to five inotropes with different mechanisms, including dobutamine and isoproterenol (β-adrenergic stimulation), milrinone (PDE3 inhibition), pimobendan and levosimendan (increase of Ca2+sensitization as well as PDE3 inhibition). In conclusion, our study suggests that myocytes obtained from both human donor hearts and dog hearts can be used to simultaneously assess drug-induced effects on sarcomere shortening and CaT using the IonOptix platform.

Administration of metformin rescues age-related vulnerability to ischemic insults through mitochondrial energy metabolism

Ischemic heart disease (IHD) is the leading cause of death on a global scale. Despite significant advances in the reperfusion treatment of acute myocardial infarction, there is still a significant early mortality rate among the elderly, as angioplasty-achieved reperfusion can exacerbate myocardial damage, leading to severe ischemia/reperfusion (I/R) injury and induce fatal arrhythmias. Mitochondria are a key mediator of ischemic insults; a transient blockade of the electron transport chain (ETC) at complex I during reperfusion can reduce myocardial infarct caused by ischemic insults. The reversible, transient modulation of complex I during early reperfusion is limited by the available of clinically tractable agents. We employed the novel use of acute, high dose metformin to modulate complex I activity during early reperfusion to decrease cardiac injury in the high-risk aged heart. Young (3–6 months) and aged (22–24 months) male and female C57BL/6 J mice were subjected to in vivo regional ischemia for 45 min, followed by metformin (2 mM, i. v.) injection 5 min prior to reperfusion for 24 h. The cardiac functions were measured with echocardiography. A Seahorse XF24 Analyzer was used to ascertain mitochondrial function. Cardiomyocyte sarcomere shortening and calcium transients were measured using the IonOptix Calcium and Contractility System. The results demonstrated that administration of acute, high dose metformin at the onset of reperfusion significantly limited cardiac damage and rescued cardiac dysfunction caused by I/R in both young and aged mice. Importantly, metformin treatment improves contractile functions of isolated cardiomyocytes and maintains mitochondrial integrity under I/R stress conditions. Thus, acute metformin administration at the onset of reperfusion has potential as a mitochondrial-based therapeutic to mitigate reperfusion injury and reduce infarct size in the elderly heart attack patient who remains at greater mortality risk despite reperfusion alone.

High throughput image processing software for analyzing hiPSC-CM sarcomere physiology and cardiac fibroblast activation.

Heart disease is the leading cause of death worldwide and discovering therapies that treat its underlying cause is paramount to improving survivability and patient health. Since heart disease often results in detrimental remodeling of heart tissue, high-throughput screening (HTS) using cardiomyocytes and cardiac fibroblasts is frequently used for drug discovery. A major challenge in analyzing HTS data is ensuring that analysis is both fast and minimally biased. In many forms of heart disease and remodeling, cardiomyocytes will suffer from loss of sarcomeres and sarcomere disarray, while fibroblasts will often express increased α-smooth muscle actin (α-SMA) stress fibers when activated into myofibroblasts. Two custom MATLAB HTS image processing scripts named ‘Tamarack’ and ‘Porcupine’ were developed for quantifying human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) sarcomere morphology and cardiac fibroblast activation, respectively. Both scripts use wavelet transforms to detect subcellular structures that are of interest for analyzing phenotypes at the cellular level. Tamarack enables quantification of sarcomere metrics, including sarcomere count, length, orientation, and α-actinin rich areas. Porcupine enables quantification of fibroblast activation metrics, including α-SMA stress fiber count, length, orientation, nuclei and α-SMA overlap, and F-actin and α-SMA overlap. Both scripts enable analysis of other cellular features, such as nuclei count and stain intensity/protein expression. Tamarack obtained statistically significant results when comparing control hiPSC-CMs with hiPSC-CMs subjected to siRNA knockdowns of genes of interest. Porcupine was validated by analyzing fibroblasts with increasing concentrations of TGF-β which induced increasing activation. By developing technology for analyzing cardiomyocyte and fibroblast imaged-based screens, drug candidates can be efficiently evaluated without bias on cell types that are relevant to human heart disease. This research may help expand drug candidate portfolios and improve the number of treatments available to patients worldwide.

Extracellular matrix and cyclic stretch alter fetal cardiomyocyte proliferation and maturation in a rodent model of heart hypoplasia.

Birth defects, particularly those that affect development of the heart, are a leading cause of morbidity and mortality in infants and young children. Babies born with heart hypoplasia (heart hypoplasia) disorders often have a poor prognosis. It remains unclear whether cardiomyocytes from hypoplastic hearts retain the potential to recover growth, although this knowledge would be beneficial for developing therapies for heart hypoplasia disorders. The objective of this study was to determine the proliferation and maturation potential of cardiomyocytes from hypoplastic hearts and whether these behaviors are influenced by biochemical signaling from the extracellular matrix (ECM) and cyclic mechanical stretch. Congenital diaphragmatic hernia (CDH)-associated heart hypoplasia was induced in rat fetuses by maternal exposure to nitrofen. Hearts were isolated from embryonic day 21 nitrofen-treated fetuses positive for CDH (CDH+) and from fetuses without nitrofen administration during gestation. CDH+ hearts were smaller and had decreased myocardial proliferation, along with evidence of decreased maturity compared to healthy hearts. In culture, CDH+ cardiomyocytes remained immature and demonstrated increased proliferative capacity compared to their healthy counterparts. Culture on ECM derived from CDH+ hearts led to a significant reduction in proliferation for both CDH+ and healthy cardiomyocytes. Healthy cardiomyocytes were dosed with exogenous nitrofen to examine whether nitrofen may have an aberrant effect on the proliferative ability of cardiomyocyte, yet no significant change in proliferation was observed. When subjected to stretch, CDH+ cardiomyocytes underwent lengthening of sarcomeres while healthy cardiomyocyte sarcomeres were unaffected. Taken together, our results suggest that alterations to environmental cues such as ECM and stretch may be important factors in the pathological progression of heart hypoplasia.

Влияние сердечного миозин-связывающего белка С на характеристики одиночного актин-миозинового взаимодействия в миокарде

. Myocardial contraction is the result of the interaction of myosin, which makes up the thick filament, with actin, which forms the basis of the thin filament, and is regulated by calcium through the regulatory proteins troponin and tropomyosin. Recently, it was found that, in addition to regulatory proteins, cardiac myosin-binding protein-C (cMyBP-C) is involved in the regulation of actin-myosin interaction. cMyBP-C is one of the integral proteins of the cardiomyocyte sarcomere, which has binding sites for the main sarcomere proteins, myosin, actin, and tropomyosin. cMyBP-C controls the number of myosin heads interacting with the thin filament and participates in its activation. In this work, the influence of cMyBP-C on the characteristics of a single actin-myosin interaction, myosin step size and interaction duration, was studied using an optical trap method. Cardiac myosin was extracted from rabbit left ventricular myocardium, actin was isolated from rabbit fast skeletal muscle, and cMyBP-C was obtained from chicken ventricles. cMyBP-C was added to cardiac myosin in a physiological ratio of 1:5. In an in vitro motility assay, the addition of cMyBP-C was found to slow actin sliding velocity over myosin by 30%. It was found that cMyBP-C does not affect step size of myosin but increases the duration of its interaction with the actin filament. The results obtained indicate a direct effect of cMyBP-C on a single actin-myosin interaction.

Abstract 10583: A Critical Contribution of Cardiac Myofibroblasts in Rv Failure and the Role of Ucp2 Snps in the Predisposition to Rv Decompensation in Pulmonary Arterial Hypertension

Introduction: The compensatory stage in right ventricle hypertrophy (RVH) in pulmonary arterial hypertension (PAH) is much shorter than in left ventricle hypertrophy (LVH) in systemic hypertension. Between 2 patients with the same degree of PAH one may have a much shorter compensated RVH (cRVH) stage than the other. The cardiac myofibroblasts (CMF) may contribute to these differences. Their ability to contract (stiffening), produce collagen and promote inflammation contributes to heart failure. Decreased mitochondrial calcium (mCa++) promotes CMF differentiation (from cardiac fibroblasts). Methylation of the mCa++ Uptake 1 (MICU1) and lack of UCP2 (uncoupling protein 2, a component of the mCa++ uniporter complex) activate CMF in the LV. Hypothesis: RV CMF are major contributors to RV failure in PAH, and loss of CMF-UCP2 promotes myofibroblast activation and RV decompensation. Methods: In a rat PAH model (Monocrotaline-PAH), we measured the RV pressure from isolated perfused hearts and sarcomere shortening from isolated RV cardiomyocytes. In a cohort (n=25) of patients with pulmonary hypertension that had been treated with the same protocol for years and controlled for age/sex/duration of disease, the correlation between UCP2 loss-of-function SNPs and RV function was measured. Results: In isolated hearts, RV pressure is lower in decompensated RVH (dRVH) compared to normal RV, but in vitro there is no difference in cardiomyocyte sarcomere shortening between the two groups, pointing to potential detrimental role of RV CMF rather than contractile failure of RV cardiomyocytes. dRVH has much more CMF compared to normal RV. CMF from dRVH have increased MICU1 methylation and decreased UCP2 levels compared to normal RV fibroblasts. PAH patients with homozygotes for UCP2 SNPs have worse RV function compared to heterozygote or wild-type, despite similar PA pressures. Conclusions: Our preliminary work suggests that RV CMF rather than RV cardiomyocytes may drive RV decompensation independent of RV afterload in PAH patients and this may be predicted by UCP2 SNPs that are relatively frequent in the population and have been shown to be associated with PAH outcomes (10-year survival, time to transplantation).

Cooperation between myofibril growth and costamere maturation in human cardiomyocytes.

Costameres, as striated muscle-specific cell adhesions, anchor both M-lines and Z-lines of the sarcomeres to the extracellular matrix. Previous studies have demonstrated that costameres intimately participate in the initial assembly of myofibrils. However, how costamere maturation cooperates with myofibril growth is still underexplored. In this work, we analyzed zyxin (costameres), α-actinin (Z-lines) and myomesin (M-lines) to track the behaviors of costameres and myofibrils within the cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs). We quantified the assembly and maturation of costameres associated with the process of myofibril growth within the hiPSC-CMs in a time-dependent manner. We found that asynchrony existed not only between the maturation of myofibrils and costameres, but also between the formation of Z-costameres and M-costameres that associated with different structural components of the sarcomeres. This study helps us gain more understanding of how costameres assemble and incorporate into the cardiomyocyte sarcomeres, which sheds a light on cardiomyocyte mechanobiology.

Effect of Chronic Treatment with Uridine on Cardiac Mitochondrial Dysfunction in the C57BL/6 Mouse Model of High-Fat Diet-Streptozotocin-Induced Diabetes.

Long-term hyperglycemia in diabetes mellitus is associated with complex damage to cardiomyocytes and the development of mitochondrial dysfunction in the myocardium. Uridine, a pyrimidine nucleoside, plays an important role in cellular metabolism and is used to improve cardiac function. Herein, the antidiabetic potential of uridine (30 mg/kg/day for 21 days, i.p.) and its effect on mitochondrial homeostasis in the heart tissue were examined in a high-fat diet–streptozotocin-induced model of diabetes in C57BL/6 mice. We found that chronic administration of uridine to diabetic mice normalized plasma glucose and triglyceride levels and the heart weight/body weight ratio and increased the rate of glucose utilization during the intraperitoneal glucose tolerance test. Analysis of TEM revealed that uridine prevented diabetes-induced ultrastructural abnormalities in mitochondria and sarcomeres in ventricular cardiomyocytes. In diabetic heart tissue, the mRNA level of Ppargc1a decreased and Drp1 and Parkin gene expression increased, suggesting the disturbances of mitochondrial biogenesis, fission, and mitophagy, respectively. Uridine treatment of diabetic mice restored the mRNA level of Ppargc1a and enhanced Pink1 gene expression, which may indicate an increase in the intensity of mitochondrial biogenesis and mitophagy, and as a consequence, mitochondrial turnover. Uridine also reduced oxidative phosphorylation dysfunction and suppressed lipid peroxidation, but it had no significant effect on the impaired calcium retention capacity and potassium transport in the heart mitochondria of diabetic mice. Altogether, these findings suggest that, along with its hypoglycemic effect, uridine has a protective action against diabetes-mediated functional and structural damage to cardiac mitochondria and disruption of mitochondrial quality-control systems in the diabetic heart.

The Cardiac Sarcomere and Cell Cycle.

The lack of adult human cardiomyocyte proliferative capacity impairs cardiac regeneration such as after myocardial injury. The sarcomere, a specialized actin cytoskeletal structure that is essential for twitch contraction in cardiomyocytes, has been considered a critical factor limiting adult human cardiomyocyte proliferation through incompletely understood mechanisms. This review summarizes known and emerging regulatory mechanisms connecting the human cardiomyocyte sarcomere to cell cycle regulation including structural and signaling mechanisms. Cardiac regeneration could be augmented through targeting the inhibitory effects of the sarcomere on cardiomyocyte proliferation.

Sarcomere Disassembly and Transfection Efficiency in Proliferating Human iPSC-Derived Cardiomyocytes.

Contractility of the adult heart relates to the architectural degree of sarcomeres in individual cardiomyocytes (CMs) and appears to be inversely correlated with the ability to regenerate. In this study we utilized multiple imaging techniques to follow the sequence of sarcomere disassembly during mitosis resulting in cellular or nuclear division in a source of proliferating human pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). We observed that both mono- and binuclear hiPSC-CMs give rise to mononuclear daughter cells or binuclear progeny. Within this source of highly proliferative hiPSC-CMs, treated with the CHIR99021 small molecule, we found that Wnt and Hippo signaling was more present when compared to metabolic matured non-proliferative hiPSC-CMs and adult human heart tissue. Furthermore, we found that CHIR99021 increased the efficiency of non-viral vector incorporation in high-proliferative hiPSC-CMs, in which fluorescent transgene expression became present after the chromosomal segregation (M phase). This study provides a tool for gene manipulation studies in hiPSC-CMs and engineered cardiac tissue. Moreover, our data illustrate that there is a complex biology behind the cellular and nuclear division of mono- and binuclear CMs, with a shared-phenomenon of sarcomere disassembly during mitosis.

Sarc-Graph: Automated segmentation, tracking, and analysis of sarcomeres in hiPSC-derived cardiomyocytes.

A better fundamental understanding of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) has the potential to advance applications ranging from drug discovery to cardiac repair. Automated quantitative analysis of beating hiPSC-CMs is an important and fast developing component of the hiPSC-CM research pipeline. Here we introduce "Sarc-Graph," a computational framework to segment, track, and analyze sarcomeres in fluorescently tagged hiPSC-CMs. Our framework includes functions to segment z-discs and sarcomeres, track z-discs and sarcomeres in beating cells, and perform automated spatiotemporal analysis and data visualization. In addition to reporting good performance for sarcomere segmentation and tracking with little to no parameter tuning and a short runtime, we introduce two novel analysis approaches. First, we construct spatial graphs where z-discs correspond to nodes and sarcomeres correspond to edges. This makes measuring the network distance between each sarcomere (i.e., the number of connecting sarcomeres separating each sarcomere pair) straightforward. Second, we treat tracked and segmented components as fiducial markers and use them to compute the approximate deformation gradient of the entire tracked population. This represents a new quantitative descriptor of hiPSC-CM function. We showcase and validate our approach with both synthetic and experimental movies of beating hiPSC-CMs. By publishing Sarc-Graph, we aim to make automated quantitative analysis of hiPSC-CM behavior more accessible to the broader research community.