Cardiomyocyte Nuclei Research Articles

FORCE platform overcomes barriers of oligonucleotide delivery to muscle and corrects myotonic dystrophy features in preclinical models

BackgroundWe developed the FORCETM platform to overcome limitations of oligonucleotide delivery to muscle and enable their applicability to neuromuscular disorders. The platform consists of an antigen-binding fragment, highly specific for the human transferrin receptor 1 (TfR1), conjugated to an oligonucleotide via a cleavable valine-citrulline linker. Myotonic dystrophy type 1 (DM1) is a neuromuscular disorder caused by expanded CUG triplets in the DMPK RNA, which sequester splicing proteins in the nucleus, lead to spliceopathy, and drive disease progression.MethodsMultiple surrogate conjugates were generated to characterize the FORCE platform. DYNE-101 is the conjugate designed to target DMPK and correct spliceopathy for the treatment of DM1. HSALR and TfR1hu/mu;DMSXLTg/Tg mice were used as models of myotonic dystrophy, the latter expresses human TfR1 and a human DMPK RNA with >1,000 CUG repeats. Cynomolgus monkeys were used to determine translatability of DYNE-101 pharmacology to higher species.ResultsIn HSALR mice, a surrogate FORCE conjugate achieves durable correction of spliceopathy and improves myotonia to a greater extent than unconjugated ASO. In patient-derived myoblasts, DYNE-101 reduces DMPK RNA and nuclear foci, consequently improving spliceopathy. In TfR1hu/mu;DMSXLTg/Tg mice, DYNE-101 reduces mutant DMPK RNA in muscle, thereby correcting splicing. Reduction of DMPK foci in cardiomyocyte nuclei accompanies these effects. Low monthly dosing of DYNE-101 in TfR1hu/mu;DMSXLWT/Tg mice or cynomolgus monkeys leads to a profound reduction of DMPK expression in muscle.ConclusionsThese data validate FORCE as a drug delivery platform and support the notion that DM1 may be treatable with low and infrequent dosing of DYNE-101.

Differential cardiac impacts of hematological malignancies and solid tumors: a histopathological and biomarker study

BackgroundCancer patients are known to be associated with increased risk of cardiovascular disease. However, no studies have examined the differential impact of hematologic malignancies (HMs) and solid tumors (STs) on cardiac morphology at the tissue level.ObjectiveWe aimed to examine histopathological features alongside cardiovascular biomarkers in patients with HMs and STs who underwent post-mortem evaluation.MethodsWe analyzed cardiac changes in 198 patients with HMs and 164 patients with solid tumors STs. We compared demographics, echocardiogram data, exposure to various antineoplastic agents, and post-mortem findings. Additionally, cardiac histological validation was conducted on post-mortem cardiac specimens to examine cardiac tissue morphology, focusing on cardiomyocyte nuclear density, collagen content, and collagen fiber orientation.ResultsHM patients displayed significantly disordered collagen fiber alignment (0.71 vs 0.83, P = 0.027), and reduced cardiomyocyte nuclear density (56 vs 72, P = 0.002) compared to ST patients. Similarly, hemoglobin level was decreased (6.71 vs 8.06, P < 0.001) in HM patients compared to ST patients. HM patients also showed elevated B-type natriuretic peptide levels (2,275 vs 867, P < 0.001), without significant differences in creatine-kinase MB and cardiac troponin levels. Multivariate analysis identified increased right ventricular thickness, low diastolic blood pressure, and high cardiac troponin levels as risk factors for cardiac death in HM patients.ConclusionsThis study demonstrates that HM patients have fewer cardiomyocyte nuclei and poorly aligned collagen, with serum biomarker evidence of increased cardiac dysfunction. This supports the necessity for specialized cardiac care for these patients.

Cardiomyocyte proliferation and heart regeneration in adult Xenopus tropicalis evidenced by a transgenic reporter line

Cardiomyocyte proliferation in adult Xenopus tropicalis during heart regeneration has remained largely contentious due to the absence of genetic evidence. Here, we generated a transgenic reporter line Tg(mlc2:H2C) expressing mCherry specifically in cardiomyocyte nuclei driven by the promoter of myosin light chain 2 (mlc2). Using the reporter line, we found that traditional whole-cell staining is not a rigorous way to identify cardiomyocytes in adult Xenopus tropicalis when using a cryosection with common thickness (5 μm) which leading to a high error, but this deviation could be reduced by increasing section thickness. In addition, the reporter line confirmed that apex resection injury greatly increased the proliferation of mlc2+ cardiomyocytes at 3-30 days post-resection (dpr), thereby regenerating the lost cardiac muscle by 30 dpr in adult Xenopus tropicalis. Our findings from the reporter line have rigorously defined cardiomyocyte proliferation in adult heart upon injury, thereby contributing heart regeneration in adult Xenopus tropicalis.

Morphological Changes Of The Heart In Sudden Death Psychiatric Patients

Although sudden cardiac death is often reported in epileptic or psychiatric patients, the etiologyis not fully understood. The aim of this study was to examine whether morphological changes of the heart contribute to sudden cardiac death in epileptic or psychiatric patients, compared with control decedents.Cases of sudden cardiac deathsdue to fatal arrhythmia in patients with epilepsy or psychiatric disease were selected from forensic autopsies performed at the Shiga Prefecture from 2014 to 2021.As the control group, autopsied healthy decedents were selected. The heart weight to normal heart weight ratio, ventricular wall thickness, cardiomyocyte width, nuclear diameter and nuclear density of the left and right ventricle and interventricular septum were examined. The left and right ventricular wall thickness and cardiomyocyte to be smaller in epileptic patients, however, no significant differences were found. The nuclear diameter/body surface area of the left ventricle was significantly smaller in epileptic and psychiatric patients than in control patients.The smaller nuclei of cardiomyocytes in epileptic and psychiatric patients may relate to the etiology of sudden cardiac death.

Microtubule forces drive nuclear damage in LMNA cardiomyopathy.

Nuclear homeostasis requires a balance of forces between the cytoskeleton and nucleus. Mutations in the LMNA gene, which encodes the nuclear envelope proteins lamin A/C, disrupt this balance by weakening the nuclear lamina. This results in nuclear damage in contractile tissues and ultimately muscle disease. Intriguingly, disrupting the LINC complex that connects the cytoskeleton to the nucleus has emerged as a promising strategy to ameliorate LMNA-associated cardiomyopathy. Yet how LINC complex disruption protects the cardiomyocyte nucleus remains unclear. To address this, we developed an assay to quantify the coupling of cardiomyocyte contraction to nuclear deformation and interrogated its dependence on the nuclear lamina and LINC complex. We found that, surprisingly, the LINC complex was mostly dispensable for transferring contractile strain to the nucleus, and that increased nuclear strain in lamin A/C-deficient cardiomyocytes was not rescued by LINC complex disruption. Instead, LINC complex disruption eliminated the cage of microtubules encircling the nucleus. Disrupting microtubules was sufficient to prevent nuclear damage and rescue cardiac function induced by lamin A/C deficiency. We computationally simulated the stress fields surrounding cardiomyocyte nuclei and show how microtubule forces generate local vulnerabilities that damage lamin A/C-deficient nuclei. Our work pinpoints localized, microtubule-dependent force transmission through the LINC complex as a pathological driver and therapeutic target for LMNA-cardiomyopathy.

Ultrastructural Features of Left Ventricle Cardiomyocytes in Preterm Newborn Rats.

The structure of left ventricular cardiomyocytes of 1 day preterm newborn rats was studied using transmission electron microscopy. It was shown that the relative area of the nucleus in cardiomyocytes of preterm rats is lower, and the relative area of the cytoplasm is higher than in full-term rats, while the relative areas of myofibrils and mitochondria do not differ. In cardiomyocytes of preterm rats damaged mitochondria, subsegmental myofibrillar contracture, and cytoplasmic swelling were found on the first postnatal day. Preterm birth in rats, in contrast to birth at term, is accompanied by the development of a number of ultrastructural damages in cardiomyocytes.

Murine neonatal cardiac regeneration depends on Insulin-like growth factor 1 receptor signaling.

Unlike adult mammals, the hearts of neonatal mice possess the ability to completely regenerate from myocardial infarction (MI). This observation has sparked vast interest in deciphering the potentially lifesaving and morbidity-reducing mechanisms involved in neonatal cardiac regeneration. In mice, the regenerative potential is lost within the first week of life and coincides with a reduction of Insulin-like growth factor 1 receptor (Igf1r) expression in the heart. Igf1r is a well-known regulator of cardiomyocyte maturation and proliferation in neonatal mice. To test the role of Igf1r as a pivotal factor in cardiac regeneration, we knocked down (KD) Igf1r specifically in cardiomyocytes using recombinant adeno-associated virus (rAAV) delivery and troponin T promotor driven shRNAmirs. Cardiomyocyte specific Igf1r KD versus control mice were subjected to experimental MI by permanent ligation of the left anterior descending artery (LAD). Cardiac functional and morphological data were analyzed over a 21-day period. Neonatal Igf1r KD mice showed reduced systolic cardiac function and increased fibrotic cardiac remodeling 21days post injury. This cardiac phenotype was associated with reduced cardiomyocyte nuclei mitosis and decreased AKT and ERK phosphorylation in Igf1r KD, compared to control neonatal mouse hearts. Our in vivo murine data show that Igf1r KD shifts neonatal cardiac regeneration to a more adult-like scarring phenotype, identifying cardiomyocyte-specific Igf1r signaling as a crucial component of neonatal cardiac regeneration.

LncRNA DCRT Protects Against Dilated Cardiomyopathy by Preventing NDUFS2 Alternative Splicing by Binding to PTBP1.

Dilated cardiomyopathy is characterized by left ventricular dilation and continuous systolic dysfunction. Mitochondrial impairment is critical in dilated cardiomyopathy; however, the underlying mechanisms remain unclear. Here, we explored the cardioprotective role of a heart-enriched long noncoding RNA, the dilated cardiomyopathy repressive transcript (DCRT), in maintaining mitochondrial function. The DCRT knockout (DCRT-/-) mice and DCRT knockout cells were developed using CRISPR-Cas9 technology. Cardiac-specific DCRT transgenic mice were generated using α-myosin heavy chain promoter. Chromatin coimmunoprecipitation, RNA immunoprecipitation, Western blot, and isoform sequencing were performed to investigate the underlying mechanisms. We found that the long noncoding RNA DCRT was highly enriched in the normal heart tissues and that its expression was significantly downregulated in the myocardium of patients with dilated cardiomyopathy. DCRT-/- mice spontaneously developed cardiac dysfunction and enlargement with mitochondrial impairment. DCRT transgene or overexpression with the recombinant adeno-associated virus system in mice attenuated cardiac dysfunction induced by transverse aortic constriction treatment. Mechanistically, DCRT inhibited the third exon skipping of NDUFS2 (NADH dehydrogenase ubiquinone iron-sulfur protein 2) by directly binding to PTBP1 (polypyrimidine tract binding protein 1) in the nucleus of cardiomyocytes. Skipping of the third exon of NDUFS2 induced mitochondrial dysfunction by competitively inhibiting mitochondrial complex I activity and binding to PRDX5 (peroxiredoxin 5) and suppressing its antioxidant activity. Furthermore, coenzyme Q10 partially alleviated mitochondrial dysfunction in cardiomyocytes caused by DCRT reduction. Our study revealed that the loss of DCRT contributed to PTBP1-mediated exon skipping of NDUFS2, thereby inducing cardiac mitochondrial dysfunction during dilated cardiomyopathy development, which could be partially treated with coenzyme Q10 supplementation.

Abstract Tu089: p21 Regulates Hypertrophic Cardiomyopathy Remodeling By Inhibition of Cardiomyocyte Endoreplication

Background: We previously discovered that a murine model of hypertrophic cardiomyopathy had increased cardiomyocyte endoreplication and replication stress leading to increased DNA damage. Activation of DNA damage response pathways was associated with increased cardiomyocyte expression of the cyclin-dependent kinase inhibitor p21. However, the role of p21 on pathologic myocardial remodeling in hypertrophic cardiomyopathy is poorly understood. Methods: We utilized a murine model of hypertrophic cardiomyopathy that is deficient in cardiac myosin binding protein 3 (Mybpc3 -/- ). We used murine models deficient in p21 (Cdkn1a -/- ) or overexpressed p21 (Cdkn1a SUPER ) to determine the role of p21 on hypertrophic remodeling. Heart structure and function were evaluated by echocardiography. Flow cytometry and immunohistochemistry were used to measure cardiomyocyte DNA content and ploidy. Proteomics and biochemical assays were utilized to identify p21 target proteins. Results: We observed that there was an increased p21 expression in Mybpc3 -/- cardiomyocytes during the postnatal day 7 to 25 time interval. Elimination of p21 expression in Mybpc3 -/- animals (Cdkn1a -/- /Mybpc3 -/- ) caused increased myocardial hypertrophy (Table 1). Conversely, selective overexpression of p21 in cardiomyocytes (Cdkn1a SUPER /Mybpc3 -/- /Myh6Cre) led to reduced myocardial hypertrophy (Table 1). We discovered that isolated cardiomyocyte nuclei from Mybpc3 -/- mice with reduced p21 had increased polyploidy whereas overexpression of cardiomyocyte p21 led to reduced cardiomyocyte polyploidy (Table 1). Using both proteomics and targeted assays we discovered that cardiomyocyte p21 was binding to key cycle regulatory proteins to inhibit cardiomyocyte endoreplication and hypertrophic growth. Conclusions: Collectively, our results show that induction of p21 in hypertrophic cardiomyopathy reduces cardiomyocyte growth by inhibiting endoreplication.

Loss of connectin novex-3 leads to heart dysfunction associated with impaired cardiomyocyte proliferation and abnormal nuclear mechanics

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 Ca2+ 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.

Microtubule-induced restriction of nuclear deformability in hypertrophic cardiomyopathy

Abstract Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): Leducq foundation Background Hypertrophic cardiomyopathy (HCM) is characterized by abnormal thickening of the left ventricular (LV) wall in conjunction with diastolic dysfunction with a prevalence of ~1:200. HCM pathophysiology is incompletely understood. We hypothesized that misregulated mechanotransduction plays an important role in the disease process. Extensive cytoskeletal remodeling has been observed in HCM patients, with a robust increase in tubulin and desmin levels, with a concomitant increase in stabilizing post-translational modifications, which we previously showed contributes to diastolic dysfunction. Microtubules are connected to the nucleus by the LINC complex. Little is known about the interaction of the non-sarcomeric cytoskeleton with the nucleus and the role this may have in the development of HCM. Purpose Relate cytoskeleton changes to nuclear and chromatin morphology in HCM patient myocardium and assess nuclear deformability in contracting cardiomyocytes with and without microtubule destabilizing drugs. Methods Nuclear morphology and chromatin organization was assessed using electron microscopy in cardiac septal tissue from 19 patients with obstructive HCM and compared to samples from 4 non-failing donors (NF). Further nuclear changes were studied using wild type (WT) and homozygous Mybpc3 c.2373InsG mice (Mybpc3c.2373InsG, KI), which have a HCM phenotype. Nuclear morphology was assessed in slices of 6 WT and 6 Mybpc3c.2373InsG paraffin-embedded mouse hearts. To study nuclear deformability during contractions, adult cardiomyocytes were isolated, stained with live cell dyes and imaged live while electrically paced. Lastly, DNA damaged was assessed in 6 WT and 6 Mybpc3c.2373InsG paraffin-embedded mouse hearts. Results Cardiomyocyte nuclei of HCM patients had a significantly higher number of invaginations (0.32±0.09 invaginations/µm in HCM versus 0.18±0.03 invaginations/µm in NF hearts), were more irregular of shape and had altered chromatin organization compared to those from NF hearts. Nuclei of Mybpc3c.2373InsG mice were considerably larger (322±75 µm³ versus 221±24 µm³). While sarcomere shortening is similar (7.0±1.4% in Mybpc3c.2373InsG versus 6.5±1.7% in WT), nuclei from Mybpc3c.2373InsG mice are less deformable than WT nuclei (figure). Addition of microtubule destabilizer nocodazole or detyrosination inhibitor EPO-Y restored nuclear deformability to WT levels, indicating a role of detyrosinated microtubules in nuclear deformability. In addition, nuclei from Mybpc3c.2373InsG have more extensive DNA damage (19.7±3.4 foci in Mybpc3c.2373InsG versus 9.1±3.2 foci in WT). Conclusion Extensive nuclear changes are observed in HCM patients and a HCM mouse model. Cytoskeletal remodeling results in less deformable nuclei that might contribute to chromatin alterations and increased DNA damage. Nuclear deformability can be restored by microtubule modifying drugs, specifically those that decrease microtubule detyrosination.

#1515 Myocardial remodeling is associated with redistribution of phosphate, PIT1/ERK1/2 up-regulation and signaling pathways alterations in mild CKD model

Abstract Background and Aims Patients with CKD have significantly increased cardiovascular risk, and heart failure is a leading cause of morbidity and mortality in these patients. Myocardial hypertrophy is a hallmark of cardiac remodeling and dysfunction in CKD and is known to be mediated by angiotensin II, catecholamines, hyperphosphatemia, hyperparathyroidism, increase in FGF23, decrease in Klotho and alterations in various signaling pathways including calcineurin, mitogen-activated protein kinase ERK1/2, calmodulin-dependent protein kinase II etc. Less well understood are the mechanisms of fibrosis and intramyocardial arterial remodeling in CKD, which may be related to hyperphosphatemia and decreased renal and systemic Klotho. The aim of this study was to evaluate the molecular and cellular features of myocardial remodeling in experimental mild CKD. Method We induced CKD by 3/4 nephrectomy (Nx) in adult male spontaneously hypertensive rats (SHR) with a follow-up period of 4 months. Animals were fed standard chow containing 0.6% phosphate. Sham-operated Wistar rats and SHR served as controls. We measured serum creatinine, renal interstitial fibrosis area, systolic blood pressure (BP), myocardial mass index (MMI), histological indices of myocardial hypertrophy, interstitial fibrosis and intramyocardial artery (IA) remodeling (H&amp;E, Masson's trichrome, von Kossa stain), myocardial phosphorus content, serum levels of inorganic phosphate (Pi), PTH and FGF23. Myocardial expression of phosphate transporters PiT1, ERK1/2, WNT, BMP, Notch, Hedgehog signaling pathways and LRG4 molecules was evaluated by PCR-RT, IHC and IF (Zeiss LSM 710; study performed at the Centre for Collective Use “Confocal Microscopy” of the Pavlov Institute of Physiology, Russian Academy of Sciences). Results Chronic kidney injury and phosphate regulatory factors did not differ, whereas BP and MMI were higher in sham SHR vs. Wistar rats (Fig. 1). Nx rats showed features of mild chronic kidney injury before the elevation of serum PTH, FGF23 levels (Fig. 1). Myocardial P content and serum Pi were higher in Nx and in the combined SHR group and directly correlated with indices of IA remodeling. Cardiomyocyte diameter, myocardial interstitial and perivascular fibrosis were higher in Nx (Figs 1, 2a) and accompanied with upregulation in Ppp3ca mRNA (calcineurin A, Fig. 2b), Mapk1 mRNA (ERK2, Fig. 2b), phospho-ERK1/2 (Figs 1, 3), PiT1 (Figs 1, 2c), Lgr4 (Fig. 2c) and downregulation of Hes1 mRNA (Fig. 2b). PiT1 and pospho-ERK1/2 were co-expressed in IA media from SHR (Fig. 2c). In Nx, nestin and LGR4 were expressed by IA adventitia and media cells, myocardial interstitial cells, and occasionally co-localized (Fig. 2c). Hes1 expression was detected in IA and capillary endothelial cells and co-localised with Gli1 in IA media; Gli1 was also detected in cardiomyocyte nuclei (Fig. 2c). In SHR, beta-catenin expression in cardiomyocytes was more pronounced in Nx (Fig. 2c). Conclusion Mild CKD is associated with histological and molecular features of cardiomyocyte hypertrophy, myocardial fibrosis and intramyocardial arterial remodeling, which are probably modulated by phosphate redistribution and its tissue retention involving upregulation of PiT1 and ERK1/2, as well as alterations in signals related to progenitor regulation and myocyte hypertrophy.

Catecholamine treatment induces reversible heart injury and cardiomyocyte gene expression

BackgroundCatecholamines are commonly used as therapeutic drugs in intensive care medicine to maintain sufficient organ perfusion during shock. However, excessive or sustained adrenergic activation drives detrimental cardiac remodeling and may lead to heart failure. Whether catecholamine treatment in absence of heart failure causes persistent cardiac injury, is uncertain. In this experimental study, we assessed the course of cardiac remodeling and recovery during and after prolonged catecholamine treatment and investigated the molecular mechanisms involved.ResultsC57BL/6N wild-type mice were assigned to 14 days catecholamine treatment with isoprenaline and phenylephrine (IsoPE), treatment with IsoPE and subsequent recovery, or healthy control groups. IsoPE improved left ventricular contractility but caused substantial cardiac fibrosis and hypertrophy. However, after discontinuation of catecholamine treatment, these alterations were largely reversible. To uncover the molecular mechanisms involved, we performed RNA sequencing from isolated cardiomyocyte nuclei. IsoPE treatment resulted in a transient upregulation of genes related to extracellular matrix formation and transforming growth factor signaling. While components of adrenergic receptor signaling were downregulated during catecholamine treatment, we observed an upregulation of endothelin-1 and its receptors in cardiomyocytes, indicating crosstalk between both signaling pathways. To follow this finding, we treated mice with endothelin-1. Compared to IsoPE, treatment with endothelin-1 induced minor but longer lasting changes in cardiomyocyte gene expression. DNA methylation-guided analysis of enhancer regions identified immediate early transcription factors such as AP-1 family members Jun and Fos as key drivers of pathological gene expression following catecholamine treatment.ConclusionsThe results from this study show that prolonged catecholamine exposure induces adverse cardiac remodeling and gene expression before the onset of left ventricular dysfunction which has implications for clinical practice. The observed changes depend on the type of stimulus and are largely reversible after discontinuation of catecholamine treatment. Crosstalk with endothelin signaling and the downstream transcription factors identified in this study provide new opportunities for more targeted therapeutic approaches that may help to separate desired from undesired effects of catecholamine treatment.

The effect of physical activity of varying intensity on cardiomyocyte hypertrophy and myocardial polyploidy in rats

Regular exercise improves cognitive function, reduces the risk of premature death from cardiovascular disease, stroke, diabetes and improves quality of life.Aim: To study the morphological parameters of cardiomyocytes of the left ventricle of the heart and the proliferative activity of rat cardiomyocytes during physical activity of different intensity.Methodology and Research Methods. Outbred male rats (n = 30) were taken as experimental animals and divided into three series. The first series of rats were given light physical activity – the animals swam in the bath for 15 minutes. The animals of the second series were in the bath for 30 minutes (moderate severity), the third series were in the bath until they began to lose strength and sink (in 55–59 minutes). Animals were taken out of the experiment after 10 sessions of water loading. Some rats (5 animals per series) were slaughtered 30 days after the end of the experiment. After extraction, the heart was stained with hematoxylin and eosin, the amount of DNA in the nuclei of cardiomyocytes was determined, binuclear and Ki-67 positive cells were counted, and the diameter of cardiomyocytes was measured. Statistical analysis was carried out in the program “Statistica 10.0”.Results. Histological examination revealed changes only in the myocardium of the left ventricle of rats of the third series: pronounced dystrophic changes in cardiomyocytes. There were cells with necrobiosis, focal necrosis of groups of cardiomyocytes. The highest level of cardiomyocyte polyploidy occurred in the second and third series (the ratio of diploid and tetraploid cardiomyocytes in the control corresponded to 91.6 ± 7.4%; 8.2±6.3%). In these series, there was also a change in the number of binuclear cells (in the control – 12.7 ± 1.9‰). In all series of Ki-67 positive nuclei were not detected.Conclusion. Severe physical activity leads to structural disorders of the myocardium, persistent hypertrophy of cardiomyocytes and is accompanied by a decrease in the proliferative activity of cardiomyocytes.

Nuclear AGO2 promotes myocardial remodeling by activating ANKRD1 transcription in failing hearts

Heart failure (HF) is manifested by transcriptional and post-transcriptional reprogramming of critical genes. Multiple studies have revealed that microRNAs could translocate into subcellular organelles such as nucleus to modify gene expression. However, the functional property of subcellular Argonaute2 (AGO2), the core member of the microRNA machinery, has remained elusive in HF. AGO2 was found to be localized in both cytoplasm and nucleus of cardiomyocytes, and robustly increased in failing hearts of patients and animal models. We demonstrated nuclear AGO2 rather than cytosolic AGO2 overexpression by recombinant adeno-associated virus (serotype 9) with cardiomyocyte-specific troponin T promoter exacerbated the cardiac dysfunction in transverse aortic constriction (TAC)-operated mice. Mechanistically, nuclear AGO2 activates the transcription of ANKRD1, encoding Ankyrin repeat domain-containing protein 1 (ANKRD1), which also has a dual function in the cytoplasm as part of I-band of the sarcomere and in the nucleus as a transcriptional cofactor. Overexpression of nuclear ANKRD1 recaptured some key features of cardiac remodeling by inducing pathological MYH7 activation, whereas cytosolic ANKRD1 seemed cardio-protective. For clinical practice, we found ivermectin, an anti-parasite drug, and ANPep, a ANKRD1 nuclear location signal mimetic peptide, were able to prevent ANKRD1 nuclear import, resulting in the improvement of cardiac performance in TAC-induced HF.

Oxidative Stress in Sepsis: A Focus on Cardiac Pathology.

This study aims to analyze post-mortem human cardiac specimens, to verify and evaluate the existence or extent of oxidative stress in subjects whose cause of death has been traced to sepsis, through immunohistological oxidative/nitrosative stress markers. Indeed, in the present study, i-NOS, NOX2, and nitrotyrosine markers were higher expressed in the septic death group when compared to the control group, associated with also a significant increase in 8-OHdG, highlighting the pivotal role of oxidative stress in septic etiopathogenesis. In particular, 70% of cardiomyocyte nuclei from septic death specimens showed positivity for 8-OHdG. Furthermore, intense and massive NOX2-positive myocyte immunoreaction was noticed in the septic group, as nitrotyrosine immunostaining intense reaction was found in the cardiac cells. These results demonstrated a correlation between oxidative and nitrosative stress imbalance and the pathophysiology of cardiac dysfunction documented in cases of sepsis. Therefore, subsequent studies will focus on the expression of oxidative stress markers in other organs and tissues, as well as on the involvement of the intracellular pattern of apoptosis, to better clarify the complex pathogenesis of multi-organ failure, leading to support the rationale for including therapies targeting redox abnormalities in the management of septic patients.

Anatomical and morphological features of the heart of a domestic dog (Canis Lupus Familiaris L., 1758)

Cardiovascular organopathology in dogs occurs quite often, but is not fully diagnosed. The occurrence of cardiovascular pathology is due to inadequate physical activity, infectious diseases, injuries, blood loss, diseases of the lungs and other systems, hereditary factors, etc. It is known that prevention, diagnosis, surgical intervention and treatment of these pathologies are impossible without knowledge of the morpho-functional parameters of anatomy, histology and physiology. The cardiovascular system in mammals performs vital functions, ensuring the vital activity of the animal and human body. The cardiovascular system is very plastic in relation to the morpho-functional relationship and has not only pronounced hereditary individual traits, but also the ability to quickly adapt to the changing conditions of the organism's existence. It is one of the first to respond to various natural and anthropogenic factors of the environment and physical stress. The study of the cardiovascular system is an urgent issue today. The aim of the study was to study the morphological assessment of macro- and microstructures of the heart of a sexually mature domestic dog (n = 5), class – Mammalia, species – wolf (Canis lupus), subspecies – domestic dog (Canis familiaris). The completed work is a fragment of the scientific topic of the scientific research work of the Department of Normal and Pathological Morphology, Hygiene and Expertise of the Faculty of Veterinary Medicine of the Polish National University on the topic: “Development, morphology and histochemistry of animal organs in normal and pathological conditions”, according to state registration number No. 0113V000900 and scientific topics: “Features of the morphology of the heart of domestic mammals”, state registration No. 0121U108884. For the work, complex morphological research methods were used: anatomical, histological, organo-, histo- and cytometric, statistical, thanks to which new data on the peculiarities of the macro-, histo- and cytomorphometric characteristics of the morphological structures of the heart in the studied animals were presented. The dog's heart has a rounded shape, its absolute weight is 167.58 ± 9.46 g (without epicardial fat – 154.22 ± 8.04 g), relative weight is 0.72 ± 0.005 %. It was established that the microscopic structure of the ventricles and atria of the dog's heart differ by cytometric indicators, depending on their morphofunctional load. Thus, cardiomyocytes of the left ventricle have the largest volume – 2941.76 ± 127.44 μm3, the right one has a smaller volume – 2237.24 ± 103.02 μm3, and the cardiomyocytes of the atria have the smallest volume – 1496.92±98.02 μm3. The volume of the nuclei of cardiomyocytes of the left ventricle is 64.58 ± 5.09 μm3, of the right ventricle – 59.97 ± 5.83 μm3, the volume of cardiomyocytes of the atria is correspondingly smaller – 53.06 ± 6.02 μm3. At the same time, the nuclear-cytoplasmic ratio of cardiomyocytes of the left ventricle is equal to 0.0224±0.0076, cardiomyocytes of the right ventricle are more important – 0.0275 ± 0.0081, and cardiomyocytes of the atria are the most important – 0.0367 ± 0.0105. We associate such ambiguous cytometric parameters of cardiomyocytes with the morphofunctional activity of the ventricles and the functional features of this myocardial tissue, which is capable of spontaneous rhythmic contractions, as a result of which blood moves through a closed vascular system. Information on the morphology of the heart of a domestic dog, including the results of the study of the macro- and microscopic structure of the studied organs, which are presented in the publication, are of great importance for histology and comparative anatomy, and also make a significant contribution to clinical veterinary medicine.

Forensic significance of intracardiac expressions of Nrf2 in acute myocardial ischemia

When exposed to oxidative and electrophilic stress, a protective antioxidant response is initiated by nuclear factor erythroid 2-related factor 2 (Nrf2). However, the extent of its importance in the forensic diagnosis of acute ischemic heart diseases (AIHD), such as myocardial infarction (MI), remains uncertain. On the other hand, immunohistochemical analyses of fibronectin (FN) and the terminal complement complex (C5b-9) prove valuable in identifying myocardial ischemia that precedes necrosis during the postmortem diagnosis of sudden cardiac death (SCD). In this study, we investigated the immunohistochemical levels of Nrf2, FN, and C5b-9 in human cardiac samples to explore their forensic relevance for the identification of acute cardiac ischemia. Heart samples were obtained from 25 AIHD cases and 39 non-AIHD cases as controls. Nrf2 was localized in the nuclei of cardiomyocytes, while FN and C5b-9 were detected in the myocardial cytoplasm. The number of intranuclear Nrf2 positive signals in cardiomyocytes increased in AIHD cases compared to control cases. Additionally, the grading of positive portions of cardiac FN and C5b-9 in the myocardium was also significantly enhanced in AIHD, compared to controls. Collectively, these results indicate that the immunohistochemical investigation of Nrf2 combined with FN, and/or C5b-9 holds the potential for identifying early-stage myocardial ischemic lesions in cases of SCD.

Ineffectiveness of mitoTEMPO on Cardiomyocyte S-phase Activity in TNNI3K-expressing Mice

Background and Hypothesis: The limited regenerative capacity of the mammalian adult myocardium is a significant roadblock for therapeutic approaches in cardiovascular disease. Cell cycle arrest following S-phase is widely considered a primary contributor to the reduced proliferative capacity of adult cardiomyocytes. Recently, expression of troponin I-interacting kinase (Tnni3k) was shown to increase cardiomyocyte S-phase activity in mice. Tnni3k was previously shown to enhance ROS formation and adverse cardiac remodeling following injury. Our primary hypothesis was that TNNI3K-induced cardiomyocyte DNA synthesis resulted from enhanced ROS signaling. To test this, cardiomyocyte S-phase activity in TNNI3K-expressing mice was compared between those treated with the ROS scavenging agent mitoTEMPO and untreated mice. Project Methods: Transgenic mice expressing TNNI3K were subjected to 14 days infusion with mitoTEMPO (experimental group) or vehicle (control group). The mice were also subjected to 14 days infusion with bromodeoxyuridine (BrdU) to identify DNA synthesis during S-phase (all mice carried a cardiomyocyte-restricted nuclear-localized transgenic reporter to aid in cardiomyocyte nuclei identification). The proportion of cardiomyocytes in S-phase was determined and mean S-phase activity was compared between treatment groups. Ploidy analysis was also conducted to determine if cardiomyocytes completing S-phase progressed through karyokinesis. Results: The percentage of cardiomyocytes in S-phase in the control and mitoTEMPO treated group were 0.819% ± 0.163% and 0.855% ± 0.138%, respectively (mean ± SEM, p=0.873). Ploidy analysis revealed no overt difference in DNA content in S-phase-positive cardiomyocyte nuclei between the groups. Hence, we have shown that there is no appreciable difference in cell cycle induction or progression in cardiomyocytes from control vs. mitoTEMPO treated mice expressing TNNI3K. Conclusion and Potential Impact: These data suggest that (a) TNNI3K-induced cardiomyocyte S-phase activity is not secondary to elevated ROS activity, and (b) reduction of ROS activity does not relax the cell cycle block between S-phase and karyokinesis in adultcardiomyocytes.

Long non-coding RNA SNHG18 epigenetic regulates ferroptosis in acute myocardial infarction

Abstract Background Loss of terminally differentiated cardiomyocytes is a key pathogenic factor for the occurrence of acute myocardial infarction (AMI) and the development of lethal heart failure. LncRNAs have been confirmed to be critical in regulating AMI, besides, recently published studies disclosed that ferroptosis is a new form of programmed cell death that contributes to AMI. However, whether LncRNAs are involved in the regulation of ferroptosis and correlated underlying mechanisms in AMI remains unknown. Purpose Our present study is to investigate LncRNA that regulates cardiomyocyte ferroptosis in AMI and uncover the potential mechanisms. Method RNA-sequencing and bioinformatical analyses were conducted to identify the candidate LncRNAs for AMI. Adenovirus for LncRNA knockout and overexpression were constructed to alter the SNHG18 expression of cardiomyocytes in vitro. Cardiomyocyte-specific knockout and overexpression mice were also constructed to alter SNHG18 expression in vivo. Ferroptosis characteristic measurement, including glutathione peroxidase 4 (GPX4), Ferritin heavy chain (FTH-1), ptgs2, MDA, ROS, Fe2+, glutathione, and mitochondrial morphology, was performed to verify the ability of SNHG18 to regulate the ferroptosis of cardiomyocytes. Cardiac morphological, structural, functional, and hemodynamic changes as well as the viability of cardiomyocytes were also measured to assess the effect of SNHG18 on AMI. RNA-pulldown+Mass spectrometry, RNA immunoprecipitation were performed to identify the specific binding factors of SNHG18. Results RNA-sequencing results revealed the expression of SNHG18 was significantly and persistently elevated from 3 to 7 days after AMI. SNHG18 was mainly enriched in the nucleus of cardiomyocytes. Besides, the down-regulation of SNHG18 could specifically alleviate the cell viability loss caused by ferroptosis inducers. Thereafter, gain- and loss-of-function experiments were performed both in vitro and in vivo. Functionally, SNHG18 knockout dramatically enhanced cardiomyocyte vitality, reduced infarct size, improved cardiac performance and alleviated cardiac fibrosis by mitigating cardiomyocyte ferroptosis. However, enforced SNHG18 expression resulted in the opposite effects. Mechanistically, the subunit RBBP4 that forms the PRC2 (Polycomb Repressive Complex 2) complex was a direct target of SNHG18, which was also confirmed by RIP assays. In addition, Chip-PCR analysis also demonstrated that PRC2 could directly bind to the promoter region of the GPX4 and FTH1 gene, thus altering the level of H3K27me3 and inhibiting their transcription. Moreover, subsequent rescue experiments further verified that SNHG18 regulated the expression of GPX4 and FTH1 to ameliorate AMI-induced cardiomyocyte ferroptosis in a PRC2-dependent manner. Conclusion This study demonstrates that SNHG18-PRC2-GPX4/FTH1 pathway is a promising therapeutic target for the treatment of AMI by ameliorating cardiomyocyte ferroptosis.