Cardiomyocyte Compartments Research Articles

REMODELING OF EXTRACELLULARLY RECORDED ACTION POTENTIALS OF RAT HEART SUBEPICARDIAL CARDIOMYOCYTES AFTER ISCHEMIA REPERFUSION INJURY

A number of systemic heart diseases leading to the development of heart failure (aortic stenosis, hypertension, diabetic cardiomyopathy, reperfusion injury etc.) are accompanied by a pronounced reorganization of the T-system of cardiomyocytes, both in humans and animals. However, structural-functional changes within this membrane compartment of cardiomyocytes following ischemia-reperfusion (IR) have not been thoroughly studied. The aim of the work was to study the remodeling of the T-system in the subepicardial cardiomyocytes of the left ventricle of the rat heart after IR injury using confocal microscopy and extracellular recording methods. The study was carried out after 24 hours, two weeks, and four weeks following IR. A remodeling of action potentials, recorded extracellularly in the cardiomyocyte membrane patches devoid of t-tubule entrances (type 1 eAP), was observed. Starting from 24 hours up to 4 weeks after IR, there was an increase in the duration of their decline time (T90) and the formation of eAP after-hyperpolarization phase, reaching maximum values by the fourth week after IR. A decrease in the second peak’s amplitude of eAPs, measured from cardiomyocyte surface locations with t-tubule openings, was also noticed four weeks after IR. In this investigation, no observable changes in the structural organization of the T-system were found. These data suggest that functional modifications of the epicardial cardiomyocyte T-system after IR injury may precede its structural modifications.

УЛЬТРАСТРУКТУРНАЯ ПЕРЕСТРОЙКА МИОКАРДА НА 60-e СУТКИ ПОСЛЕ ДОКСОРУБИЦИН-ИНДУЦИРОВАННОЙ КАРДИОМИОПАТИИ У КРЫС

Background. To assess the cardiotoxic effects of doxorubicin, it is of great importance to elucidate the nature and severity of tissue and cell damage, their ultrastructural changes, as well as to elucidate the features of myocardial remodeling in such conditions. Objective. The aim of the study was a qualitative and quantitative analysis of ultrastructural changes in the myocardium on the 60th day after doxorubicin-induced cardiomyopathy in rats. Material and methods. Cardiomyopathy in rats was induced by doxorubicin given at a cumulative dose of 15 mg/kg for 14 days. The animals were removed from the experiment on the 60th day after last administered dose of the medicine. An electron microscopic research method was used. Ultrastructural analysis was performed using a JEM-100 CX microscope, and morphometric evaluation was done using the ImageJ data processing software. Results. Ultrastructural disorders of the myocardium of rats on the 60th day after doxorubicin-induced cardiomyopathy were characterized by: alterative changes of contractile apparatus and nuclear compartment of cardiomyocytes, activation of autophagic and necrotic cell death and compensatory mitochondria hyperplasia with T-system channels dilation; a variety of reactive changes of capillary endothelium in the form of nuclear hypertrophy and organelle hyperplasia in some endothelial cells along with intracellular edema and destruction of organelles in others, which was accompanied by a significant decrease in cross-sectional area of capillaries; activation of fibroblasts with a formation of interstitial and perivascular fibrosis; activation of autophagy processes in cardiomyocytes, fibroblastic cells and capillary endothelium. Conclusions. The established ultrastructural changes in the rat’s myocardium on the 60th day after the doxorubicin- induced cardiomyopathy indicate the complex effect of metabolic and angiogenic mechanisms of pathology development. Activation of autophagy processes plays an important role in myocardial reorganization in the late stages of doxorubicin myocardial damage.

Genetically targeted fluorescent probes reveal dynamic calcium responses to adrenergic signaling in multiple cardiomyocyte compartments.

Calcium (Ca2+), an important second messenger, regulates many cellular activities and varies spatiotemporally within the cell. Conventional methods to monitor Ca2+ changes, such as synthetic Ca2+ indicators, are not targetable, while genetically encoded Ca2+ indicators (GECI) can be precisely directed to cellular compartments. GECIs are chimeric proteins composed of calmodulin (or other proteins that change conformation on Ca2+ binding) coupled with two fluorescent proteins that come closer together after an increase in [Ca2+], and enhance Förster resonance energy transfer (FRET) that allows for ratiometric [Ca2+] assessment. Here, adult rat ventricular myocytes were transfected with specifically targeted calmodulin-based GECIs and Ca2+ responses to a physiological stimulus, norepinephrine (NE, 10 μM), were observed in a) sarcoplasmic reticulum (SR), b) mitochondria, c) the space between the mitochondria and SR, termed the Mitochondria Associated Membrane space (MAM) and d) cytosol for 10 min after stimulation. In SR and mitochondria, NE increased the [Ca2+] ratio by 17% and by 8%, respectively. In the MAM the [Ca2+] ratio decreased by 16%, while in cytosol [Ca2+] remained unchanged. In conclusion, adrenergic stimulation causes distinct responses in the cardiomyocyte SR, mitochondria and MAM. Additionally, our work provides a toolkit-update for targeted [Ca2+] measurements in multiple cellular compartments.

A heart-enriched antisense long non-coding RNA regulates the balance between cardiac and skeletal muscle triadin

Non-coding RNAs play major roles in cardiac pathophysiology. Recent studies reported that long non-coding RNAs (lncRNAs) are dysregulated in the failing heart, but how they contribute to heart failure development is unclear. In this study, we aimed to identify heart-enriched lncRNAs and investigate their regulation and function in the failing heart. ResultsAnalysis of a RNA-seq dataset of 15 Caucasian tissues allowed the identification of 415 heart-enriched lncRNAs. Fifty-three lncRNAs were located on the genome in close vicinity to protein-coding genes associated with cardiac function and disease. Analysis of a second RNA-seq dataset of 16 failing human hearts highlighted one lncRNA which we arbitrarily named TRDN-AS due to its localisation in the antisense position of the gene encoding triadin (TRDN). Expression of TRDN-AS and cardiac TRDN was up-regulated in biopsies from failing human hearts compared to control hearts. In failing hearts, TRDN-AS was positively correlated with a cardiac isoform of TRDN and negatively correlated with a skeletal muscle isoform of TRDN. A murine homolog of human TRDN-AS was identified and found to be enriched in the heart and localised in the nuclear compartment of cardiomyocytes. Trdn-AS expression as well as the ratio between cardiac and skeletal muscle isoforms were down-regulated after experimental myocardial infarction. In murine cardiomyocytes, activation of Trdn-AS transcription with the CRISPR/dCas9-VPR system enhanced the ratio between cardiac and skeletal isoforms of Trdn. ConclusionThe lncRNA TRDN-AS regulates the balance between cardiac and skeletal isoforms of triadin. This finding may have implications for the treatment of heart failure.

Sub-cellular localization specific SUMOylation in the heart

Although the majority of SUMO substrates are localized in the nucleus, SUMOylation is not limited to nuclear proteins and can be also detected in extra-nuclear proteins. In this review, we will highlight and discuss how SUMOylation in different cellular compartments regulate biological processes. First, we will discuss the key role of SUMOylation of proteins in the extra-nuclear compartment in cardiomyocytes, which is overwhelmingly cardio-protective. On the other hand, SUMOylation of nuclear proteins is generally detrimental to the cardiac function mainly because of the trans-repressive nature of SUMOylation on many transcription factors. We will also discuss the potential role of SUMOylation in epigenetic regulation. In this review, we will propose a new concept that shuttling of SUMO proteases between the nuclear and extra-nuclear compartments without changing their enzymatic activity regulates the extent of SUMOylation in these compartments and determines the response and fate of cardiomyocytes after cardiac insults. Approaches focused specifically to inhibit this shuttling in cardiomyocytes will be necessary to understand the whole picture of SUMOylation and its pathophysiological consequences in the heart, especially after cardiac insults. This article is part of a Special Issue entitled: Genetic and epigenetic control of heart failure - edited by Jun Ren & Megan Yingmei Zhang.

Connecting heart failure with preserved ejection fraction and renal dysfunction: the role of endothelial dysfunction and inflammation

Renal dysfunction in heart failure with preserved ejection fraction (HFpEF) is common and is associated with increased mortality. Impaired renal function is also a risk factor for developing HFpEF. A new paradigm for HFpEF, proposing a sequence of events leading to myocardial remodelling and dysfunction in HFpEF, was recently introduced, involving inflammatory, microvascular, and cardiac components. The kidney might play a key role in this systemic process. Renal impairment causes metabolic and systemic derangements in circulating factors, causing an activated systemic inflammatory state and endothelial dysfunction, which may lead to cardiomyocyte stiffening, hypertrophy, and interstitial fibrosis via cross-talk between the endothelium and cardiomyocyte compartments. Here, we review the role of endothelial dysfunction and inflammation to explain the link between renal dysfunction and HFpEF, which allows for identification of new early risk markers, prognostic factors, and unique targets for intervention.

Cardiogenic Genes Expressed in Cardiac Fibroblasts Contribute to Heart Development and Repair

Cardiac fibroblasts are critical to proper heart function through multiple interactions with the myocardial compartment, but appreciation of their contribution has suffered from incomplete characterization and lack of cell-specific markers. To generate an unbiased comparative gene expression profile of the cardiac fibroblast pool, identify and characterize the role of key genes in cardiac fibroblast function, and determine their contribution to myocardial development and regeneration. High-throughput cell surface and intracellular profiling of cardiac and tail fibroblasts identified canonical mesenchymal stem cell and a surprising number of cardiogenic genes, some expressed at higher levels than in whole heart. While genetically marked fibroblasts contributed heterogeneously to interstitial but not cardiomyocyte compartments in infarcted hearts, fibroblast-restricted depletion of one highly expressed cardiogenic marker, T-box 20, caused marked myocardial dysmorphology and perturbations in scar formation on myocardial infarction. The surprising transcriptional identity of cardiac fibroblasts, the adoption of cardiogenic gene programs, and direct contribution to cardiac development and repair provoke alternative interpretations for studies on more specialized cardiac progenitors, offering a novel perspective for reinterpreting cardiac regenerative therapies.

The transmembrane receptor Uncoordinated5 (Unc5) is essential for heart lumen formation in Drosophila melanogaster

Transport of liquids or gases in biological tubes is fundamental for many physiological processes. Our knowledge on how tubular organs are formed during organogenesis and tissue remodeling has increased dramatically during the last decade. Studies on different animal systems have helped to unravel some of the molecular mechanisms underlying tubulogenesis. Tube architecture varies dramatically in different organs and different species, ranging from tubes formed by several cells constituting the cross section, tubes formed by single cells wrapping an internal luminal space or tubes that are formed within a cell. Some tubes display branching whereas others remain linear without intersections. The modes of shaping, growing and pre-patterning a tube are also different and it is still not known whether these diverse architectures and modes of differentiation are realized by sharing common signaling pathways or regulatory networks. However, several recent investigations provide evidence for the attractive hypothesis that the Drosophila cardiogenesis and heart tube formation shares many similarities with primary angiogenesis in vertebrates. Additionally, another important step to unravel the complex system of lumen formation has been the outcome of recent studies that junctional proteins, matrix components as well as proteins acting as attractant and repellent cues play a role in the formation of the Drosophila heart lumen. In this study we show the requirement for the repulsively active Unc5 transmembrane receptor to facilitate tubulogenesis in the dorsal vessel of Drosophila. Unc5 is localized in the luminal membrane compartment of cardiomyocytes and animals lacking Unc5 fail to form a heart lumen. Our findings support the idea that Unc5 is crucial for lumen formation and thereby represents a repulsive cue acting during Drosophila heart tube formation.

Being there: cellular targeting of voltage-gated sodium channels in the heart

Voltage-gated sodium (Nav) channels in cardiomyocytes are localized in specialized membrane domains that optimize their functions in propagating action potentials across cell junctions and in stimulating voltage-gated calcium channels located in T tubules. Mutation of the ankyrin-binding site of Nav1.5, the principal Nav channel in the heart, was previously known to cause cardiac arrhythmia and the retention of Nav1.5 in an intracellular compartment in cardiomyocytes. Conclusive evidence is now provided that direct interaction between Nav1.5 and ankyrin-G is necessary for the expression of Nav1.5 at the cardiomyocyte cell surface.

Lysosomal, cytoskeletal, and metabolic alterations in cardiomyopathy of cathepsin L knockout mice

Although lysosomal proteases are expressed in the heart at considerable levels, their specific functions in this organ remain elusive. Mice deficient for the lysosomal cysteine protease cathepsin L (CTSL) develop a late onset dilated cardiomyopathy (DCM) that is characterized by cardiac chamber dilation, fibrosis, and impaired cardiac contraction at 12 month of age. Investigation of the pathogenic sequence of DCM in ctsl-/- mice revealed numerous dysmorphic lysosome-like structures in heart muscle as early as 3 days after birth, whereas skeletal muscle was not affected. Labeling of the acidic cell compartment of neonatal cardiomyocytes and detection of lysosomal markers after subcellular fractionation confirmed increased lysosome content in CTSL deficient myocardium; however, specific storage materials were not detected. The myocardium of ctsl+/+ and ctsl-/- mice revealed no differences in incidence of cell death, proliferation, and capillary density during DCM progression. However, an observed increase in mRNA expression of natriuretic peptides in young adult mice indicates the activation of the adaptive "fetal" gene program, while proteome analysis revealed decreased levels of the sarcomere-associated proteins alpha-tropomyosin, desmin, and calsarcin 1, as well as considerable changes of metabolic enzymes. Bioinformatic pathway analysis suggested a switch to anaerobic catabolism and impairment of mitochondrial respiration. This interpretation was supported by a 50% reduction in resting state oxygen consumption and impaired respiration capacity in ctsl-/- myocardial homogenates. In summary, the data indicate an essential role of CTSL in maintaining the structure of the endosomal/lysosomal compartment in cardiomyocytes. Lysosomal impairment in ctsl-/- hearts results in metabolic and sarcomeric alterations that promote DCM development.

Ultrastructure of nuclear compartment in cardiomyocytes during regenerative and plastic insufficiency of the myocardium

Ultrastructure of nuclear compartment in cardiomyocytes during regenerative and plastic insufficiency of the myocardium

Ultrastructure of nuclear compartment in cardiomyocytes during regenerative and plastic insufficiency of the myocardium.

We studied ultrastructure of nuclear compartment in cardiomyocytes during regenerative and plastic insufficiency of the myocardium induced by anthracycline antibiotic daunomycin. A peculiarity of ultrastructural organization of cardiomyocyte nuclei under these conditions is almost complete disappearance of the heterochromatin lumps. The earliest changes in nucleoli under conditions of disturbed DNA-dependent RNA synthesis are segregation of the granular and fibrillar nucleolonema components. Deep alterations in the nucleoli manifested by fragmentation and annulation correlate with pronounced changes in cardiomyocytes ultrastructure, intensive lysis of the myofilaments, reduction of the organelles, and enhanced autophagocytosis.