Myocardial Sections Research Articles

Cardiac rhabdomyoma: an uncommon culprit in sudden infant death

Sudden death in infants remains a common and poorly understood cause of childhood mortality in the USA. Pediatric cardiac tumors, although rare, may underlie some cases of unexplained sudden infant death. Autopsy is a crucial diagnostic step in these cases, as both gross and microscopic examination of the heart may uncover occult cardiac tumors. Rhabdomyomas are the most common cardiac tumors in childhood and may result in arrythmia and sudden death. We present a case of sudden death in a healthy 5-month-old infant which initially appeared “SIDS-like” until thorough histological analysis revealed an underlying cardiac rhabdomyoma. The case is of particular importance in that the gross examination of the heart was considered completely normal, and the tumor only involved certain portions of the heart microscopically. Had a single random section of myocardium been the only heart section examined microscopically, the diagnosis might have been missed. This case emphasizes the importance of thorough microscopic examination in infant cases, especially in cases where the heart appears grossly normal.

In Situ Structural Characterization of Cardiomyocyte Microenvironment by Multimodal STED Microscopy.

Within the myocardium, cardiomyocytes reside in a complex and dynamic extracellular matrix (ECM) consisting of a basement membrane (BM) and interstitial matrix. The interactions between cardiomyocytes and the myocardial ECM play a critical role in maintaining cardiac geometry and function throughout cardiac development and in adult hearts. Understanding how the structural changes of the myocardial ECM affect cardiomyocyte function requires knowledge of pericellular structures. These structures are of a size beyond the resolution of conventional optical microscopy. Here, we demonstrated multi-scale and multi-aspect characterization of the cardiomyocyte microenvironment in myocardial tissue sections using multimodal stimulated emission depletion (STED) microscopy. Second harmonic generation and autofluorescence facilitated multiplexed imaging, enabling the interpretation of protein distribution in 3D. STED imaging modality revealed BM structures of cardiomyocytes and myocardial capillaries at the subdiffractional level. Moreover, meaningful measurements retrieved from acquired images, such as sarcomere length and capillary density, enabled quantitative assessment of myocardial structures.

Morphological assessment of actin and desmin expression at different cold myocardial ischemia times: observational study

Background. Heart transplantation is currently the treatment of choice for patients in terminal stages of chronic heart failure. The critical shortage of donor organs and the growing need for heart transplantation necessitate the expansion of donor selection criteria, including the estimated ischemia time of the donor heart. Despite numerous studies, the issue remains regarding the safe cold ischemia time; no definite limit to the acceptable preservation time is known and no relevant pathomorphological data are available on the state of the donor heart myocardium at different time parameters. Objective. To comparatively assess the features of cardiomyocyte pathomorphology and expression of protein markers (actin and desmin) in the myocardium of a donor heart prior to the main stage of orthotopic heart transplantation. Methods. The work adopted the design of an observational clinical study, which was prospective in nature. The study used intraoperative myocardial biopsy specimens of the left atrial appendage from donors aged up to 60 years, following cold ischemia of the transplant in Bretschneider solution (Dr. Franz Köhler Chemie GmbH, Germany) lasting up to 240 minutes (Group 1, n = 10) and over 240 minutes (Group 2, n = 7). The nature of pathomorphological myocardial transformation in the left atrial appendage of the donor heart was determined at different cold ischemia times. Histological myocardial sections were stained with hematoxylin and eosin according to standard procedures. After that, they were further studied using light and polarization microscopy; the immunohistochemical method was used to analyze the expression of actin and desmin. Morphometry was performed using the ImageJ 1.48v software (USA). In the analysis of actin and desmin amount, the area of DAB(3,3′-diaminobenzidine)-positive products of the immunohistochemical reaction was estimated as a percentage of the image area. The volume density of immunohistochemically detectable actin and desmin was determined using 20 images at a magnification of 40×10. In order to study the intensity of the immune reaction, a semiquantitative method was used, which involved counting the number of cells in 25 randomly selected fields of view. The types of myocardial contracture damage were assessed via polarization microscopy. Results. Patients included in the first and second groups were comparable in terms of mean age and anthropometric indices. The mean age of patients amounted to 50 [44;59] years in Group 1 and 50 [49;50] years in Group 2, р = 0.193. The body mass index was 25 [22;27] in Group1 and 25 [21;31] in Group 2, р = 0.288. Both groups showed male predominance: 8 (80%) in Group 1 and 6 (85.7%) in Group 2, р = 0.256. The comprehensive morphological assessment of ischemic myocardial damage at different cold ischemia times revealed the uniformity and reversibility of changes in cellular structures (in both groups) that take the form of I–II class contractures, lysis changes in individual cardiomyocytes (only in Group 2), preserved immunohistochemical reactions to actin and desmin in both groups at their average intensity and the complete absence of areas showing no reaction to desmin, which gives an idea about the degree of preservation of their macromolecular structure. Conclusion. The obtained study results showed that due to having a balanced elemental composition that determines the metabolic protection of cells and their ionic balance, the Bretschneider solution effectively protects the donor heart during its transportation, with the myocardial cold ischemia lasting up to 240 min and more.

Hematopoietic Prostaglandin D Synthase Is Increased in Mast Cells and Pericytes in Autopsy Myocardial Specimens from Patients with Duchenne Muscular Dystrophy.

The leading cause of death for patients with Duchenne muscular dystrophy (DMD), a progressive muscle disease, is heart failure. Prostaglandin (PG) D2, a physiologically active fatty acid, is synthesized from the precursor PGH2 by hematopoietic prostaglandin D synthase (HPGDS). Using a DMD animal model (mdx mice), we previously found that HPGDS expression is increased not only in injured muscle but also in the heart. Moreover, HPGDS inhibitors can slow the progression of muscle injury and cardiomyopathy. However, the location of HPGDS in the heart is still unknown. Thus, this study investigated HPGDS expression in autopsy myocardial samples from DMD patients. We confirmed the presence of fibrosis, a characteristic phenotype of DMD, in the autopsy myocardial sections. Additionally, HPGDS was expressed in mast cells, pericytes, and myeloid cells of the myocardial specimens but not in the myocardium. Compared with the non-DMD group, the DMD group showed increased HPGDS expression in mast cells and pericytes. Our findings confirm the possibility of using HPGDS inhibitor therapy to suppress PGD2 production to treat skeletal muscle disorders and cardiomyopathy. It thus provides significant insights for developing therapeutic drugs for DMD.

Thermal evaporation as sample preparation for silver-assisted laser desorption/ionization mass spectrometry imaging of cholesterol in amyloid tissues.

Cholesterol plays an important biological role in the body, and its disruption in homeostasis and synthesis has been implicated in several diseases. Mapping the locations of cholesterol is crucial for gaining a better understanding of these conditions. Silver deposition has proven to be an effective method for analyzing cholesterol using mass spectrometry imaging (MSI). We optimized and evaluated thermal evaporation as an alternative deposition technique to sputtering for silver deposition in MSI of cholesterol. A silver layer with a thickness of 6 nm provided an optimal combination of cholesterol signal intensity and mass resolution. The deposition of an ultrathin nanofilm of silver enabled high-resolution MSI with a pixel size of 10 μm. We used this optimized method to visualize the distribution of cholesterol in the senile plaques in the brains of APP/PS1 mice, a model that resembles Alzheimer's disease pathology. We found that cholesterol was evenly distributed across the frontal cortex tissue, with no evidence of plaque-like accumulation. Additionally, we investigated the presence and distribution of cholesterol in myocardial sections of a human heart affected by wild-type ATTR amyloidosis. We identified the presence of cholesterol in areas with amyloid deposition, but complete colocalization was not observed.

Ehrlichia spp. infection worsens cardiac damage in dogs with canine visceral leishmaniasis.

In endemic areas for canine visceral leishmaniasis (CVL), the occurrence of coinfection with other pathogens, such as Ehrlichia spp., has been associated with worsening of the clinical condition. The study aimed to evaluate the occurrence of histological changes in the myocardia of dogs naturally infected with Leishmania chagasi with or without coinfection with Ehrlichia spp.. We evaluated paraffin-embedded myocardial sections from 31 dogs, affected by either L. chagasi alone or coinfected with L. chagasi and Ehrlichia spp., to compare the extent and degree of cardiac damage. The blocks were divided into two groups. G1 (dogs infected only by L. chagasi) and G2 (dogs coinfected with L. chagasi and Ehrlichia spp.). The right atrium free wall, right ventricle free wall, left ventricle, and interventricular septum of all groups were evaluated. Cardiac alterations were observed in 41.93% (52/124) of the fragments evaluated and inflammatory infiltrate was the most common pattern found. The G2 group showed a higher incidence of myocarditis, with 61.53% (32/52), compared to the G1 group, in which 20 out of 72 cases (27.7%) exhibited histopathological changes (p <0.05). These findings confirmed that coinfection can potentiate cardiac damage in dogs.

Preliminary Evaluation of 18F-Labeled Benzylguanidine Analogs as NET Tracers for Myocardial Infarction Diagnosis.

Heart failure (HF) remains a major cause of late morbidity and mortality after myocardial infarction (MI). To date, no clinically established 18F-labeled sympathetic nerve PET tracers for monitoring myocardial infarction are available. Therefore, in this study, we synthesized a series of 18F-labeled benzyl guanidine analogs and evaluated their efficacy as cardiac neuronal norepinephrine transporter (NET) tracers for myocardial imaging. We also investigated the preliminary diagnostic capabilities of these tracers in myocardial infarction animal models, as well as the structure-activity relationship of these tracers. Three benzyl guanidine-NET tracers, including [18F]1, [18F]2, and [18F]3, were synthesized and evaluated in vivo as PET tracers in a myocardial infarction mouse model. [18F]LMI1195 was used as a positive control for the tracers. H&E staining of the isolated myocardial infarction heart tissue sections was performed to verify the efficacy of the selected PET tracer. Our data show that [18F]3 had a moderate decay corrected labeling yield (~10%) and high radiochemical purity (>95%) compared to other tracers. The uptake of [18F]3 in normal mouse hearts was 1.7±0.1%ID/cc at 1 h post-injection (p. i.), while it was 2.4±0.1, 2.6±0.9, and 2.1±0.4%ID/cc in the MI mouse hearts at 1, 2, and 3 days after surgery, respectively. Compared with [18F]LMI1195, [18F]3 had a better myocardial imaging effect in terms of the contrast between normal and MI hearts. The area of myocardial infarction shown by PET imaging corresponded well with the infarcted tissue demonstrated by H&E staining. With an obvious cardiac uptake contrast between normal mice and the myocardial infarction mouse model, [18F]3 appears to be a potential tool in the diagnosis of myocardial infarction. Therefore, it is necessary to conduct further structural modification studies on the chemical structure of [18F]3 to improve its in vivo stability and diagnostic detection ability to achieve reliable and practical imaging effects.

Macrophages of the cardiosplenal axis and their content in patients with myocardial infarction

The spleen is one of the main reservoirs of monocytes, the leading cells of the post-infarction inflammatory response.Aim: To assess features of splenic macrophage infiltration, its dynamics and correlations with myocardial macrophage infiltration and an adverse course of the myocardial infarction (MI)Material and Methods. The macrophage composition of spleen and myocardium sections of patients (n = 30) with fatal MI and persons from the control group without cardiovascular disease (n = 5) was assessed by immunohistochemistry.Results and conclusion. All investigated cells, as CD68+, CD163+, CD206+, and stabilin-1+ were represented in the spleen regardless of the presence of MI. Their number in spleen in patients with MI remained consistently high regardless of the period of MI, and was accompanied by an increased number of such cells in the infarction area of myocardium. CD68+, CD163+ and stabilin-1+ cells predominated in the red pulp in patients with fatal MI, its number many fold exceeded that in the control group and that in the white pulp and in the infarction area of myocardium. In the white pulp of patients with fatal MI, the number of CD68+ cells predominated, in persons from the control group – CD163+. We revealed only one cell types whose content in the spleen in the control group was higher than in individuals with fatal MI – CD206+in the red pulp. Low content of CD206+ cells in the red and white pulp of the spleen characterized patients with a fatal outcome of MI.

Клинико-морфологическое обоснование использования добутамина при ожоговом шоке

Introduction. An effective way to reduce the volume of infusion (restrictive strategy) in hypovolemic shock, in particular in burn shock, is invasive hemodynamic monitoring and choosing the appropriate regimen for the use of inotropic agents, considering the data obtained. However, the use of inotropic agents in hypovolemic shock is still controversial. The most cutting issue is the choice of effective doses of inotropic agents. Aim. Justification of the need to use inotropic agents and assessment of the safety and effectiveness of dobutamine use in burn shock. Materials and methods. The pilot clinical study conducted in 2021–2022 included 9 patients aged 15–70 years who were admitted to the intensive care unit (ICU) of the Burn Center of the Novosibirsk State Regional Clinical Hospital with a total body surface area burned &gt; 40% (II–III degree), in most cases – thermal inhalation injury, who were in the ICU for over 3 days. Intensive care of burn injury in the acute phase was performed according to conventional clinical recommendations. Myocardial samples for morphological and immunohistochemical studies were taken from 34 patients who died from burn shock (27 men and 7 women, aged from 21 to 51 years) from 2015 to 2019. The samples were taken post mortem, and with minimal atherosclerotic changes of the coronary arteries (lesions no more than 20–25%). The control group for morphological and immunohistochemical studies comprised samples which were taken from 25 individuals who died because of sudden circulatory arrest (19 men, 6 women). Results. Studies have shown a decrease in myocardial contractility in patients with burn shock. Morphological examination revealed contraction lesions of individual cardiomyocytes of varying degree, as well as foci of primary clumping of myofibrils’ cytoplasm and myocytolysis. The immunohistochemical study of myocardial sections showed a decrease in the actin expression by 2.4 times (p &lt; 0.05) and desmin – by 2 times (p &lt; 0.05) in comparison with the control group. The results of the clinical study showed that dobutamine at a rate of up to 5.0 μg/kg/min does not cause an arrhythmogenic effect and/or lactic acidosis. Conclusion. The data obtained on structural changes of the myocardium confirm the need for the use of inotropic agent in burn shock. Dobutamine at a rate of up to 5.0 μg/kg/min allows for a restrictive strategy of fluid resuscitation and rapid recovery from burn shock.

216 ARRHYTHMOGENIC CARDIOMYOPATHY: IS THERE EVIDENCE OF AN INFLAMMATORY MYOCARDIAL DISEASE?

Abstract Background and aim Arrhythmogenic cardiomyopathy (ACM) is a heredo-familiar primary heart muscle disease, mainly due to mutations of desmosomal genes and characterized by progressive myocardial atrophy with fibro-fatty replacement, causing electrical instability at risk of sudden cardiac death (SCD). Since the first anatomopathological descriptions, the hypothesis that myocardial inflammation could have an important role in the onset and evolution of ACM has been advanced. With this work we aim to describe the prevalence of inflammatory cells in ACM and their characterization through histological and immunohistochemical (IHC) analysis in endomyocardial biopsy (EMB) and heart specimens coming from either SCD or heart transplantation (HTx). Material and methods We analysed EMB and whole heart specimens (from juvenile SCD registry – below 40 years of age at the time of death – and HTx) with a diagnosis of ACM, focusing on the presence of inflammatory infiltrates. For the whole hearts, in a subgroup of cases the inflammatory infiltrate was evaluated on 9 myocardial sections by applying a semi-quantitative score according to the number of sections involved (0 = none; 1 = 1 section involved; 2 = 2-5 sections; 3 = &amp;gt;5 sections) and an IHC panel for myocarditis on 1 right ventricular and 1 left ventricular section (CD45, CD3, CD8, CD4, CD68, CD20, CD138 and vimentin- as a control for fibroblast). Results Among the 63 analysed EMB (41 M, mean age 45 years, range 18-71 years), 26 cases (41%) showed foci of inflammation with CD3+ &amp;gt; 7/mm2. In all cases viral genome (DNA and RNA virus) was absent. A total of 106 heart specimens with a diagnosis of ACM were enrolled, including 73 juvenile SCD (49 M, mean age 26 years) and 33 HTx (18 M, mean age 45 years). Inflammation was reported in 95% of SCD an d 67% of HTx. The inflammatory infiltrate was focal or multifocal in all, except one case of juvenile SCD with diffuse myocarditis in the postero-lateral wall of the left ventricle. In the 24 reviewed cases, the inflammation was focal with a mean inflammatory score of 1.75 ±0.9 in the HTx group and 1.5±0.9 in the SD group (p = NS). By IHC we showed that: a) there is no significant difference in the inflammatory score between SD and HTx; b) the inflammation is mostly near-scarring (13 cases); c) inflammatory cells consist prevalently of cytotoxic T lymphocytes CD3+CD8+ (with CD4+&amp;lt;CD8+ both in SD and HTx, p &amp;lt; 0.05). Conclusions Our pathological data confirm that ACM is an inflammatory cardiomyopathy with a higher prevalence of inflammation in SCD cases. The inflammatory infiltrate is mainly composed of cytotoxic T lymphocytes with a variable presence of macrophages. These data, combined with the recent experimental studies demonstrating the role of immune mediators in the evolution of the disease, support the potential application of immunosuppressive therapy in ACM.

The flutemetamol analogue cyano-flutemetamol detects myocardial AL and ATTR amyloid deposits: a post-mortem histofluorescence analysis

Background [18F]flutemetamol is a PET radioligand used to image brain amyloid, but its detection of myocardial amyloid is not well-characterized. This histological study characterized binding of fluorescently labeled flutemetamol (cyano-flutemetamol) to amyloid deposits in myocardium. Methods Myocardial tissue was obtained post-mortem from 29 subjects with cardiac amyloidosis including transthyretin wild-type (ATTRwt), hereditary/variant transthyretin (ATTRv) and immunoglobulin light-chain (AL) types, and from 10 cardiac amyloid-free controls. Most subjects had antemortem electrocardiography, echocardiography, SPECT and cardiac MRI. Cyano-flutemetamol labeling patterns and integrated density values were evaluated relative to fluorescent derivatives of Congo red (X-34) and Pittsburgh compound-B (cyano-PiB). Results Cyano-flutemetamol labeling was not detectable in control subjects. In subjects with cardiac amyloidosis, cyano-flutemetamol labeling matched X-34- and cyano-PiB-labeled, and transthyretin- or lambda light chain-immunoreactive, amyloid deposits and was prevented by formic acid pre-treatment of myocardial sections. Cyano-flutemetamol mean fluorescence intensity, when adjusted for X-34 signal, was higher in the ATTRwt than the AL group. Cyano-flutemetamol integrated density correlated strongly with echocardiography measures of ventricular septal thickness and posterior wall thickness, and with heart mass. Conclusion The high selectivity of cyano-flutemetamol binding to myocardial amyloid supports the diagnostic utility of [18F]flutemetamol PET imaging in patients with ATTR and AL types of cardiac amyloidosis.

Assessing the risk factors for myocardial infarction in diet-induced prediabetes: myocardial tissue changes

BackgroundHyperglycaemia is known to result in oxidative stress tissue injury and dysfunction. Interestingly, studies have reported hepatic and renal oxidative stress injury during prediabetes; however, any injury to the myocardium during prediabetes has not been investigated. Hence this study aims to assess changes in the myocardial tissue in an HFHC diet-induced model of prediabetes.MethodsMale Sprague Dawley rats were randomly grouped into non-prediabetes and prediabetes (n = 6 in each group) and consumed a standard rat chow or fed a high-fat-high-carbohydrate diet respectively for a 20-week prediabetes induction period. Post induction, prediabetes was confirmed using the ADA criteria. Aldose reductase, NADH oxidase 1, superoxide dismutase, glutathione peroxide, cardiac troponins were analysed in cardiac tissue homogenate using specific ELISA kits. Lipid peroxidation was estimated by determining the concentration of malondialdehyde in the heart tissue homogenate according to the previously described protocol. Myocardial tissue sections were stained with H&E stain and analysed using Leica microsystem. All data were expressed as means ± SEM. Statistical comparisons were performed with Graph Pad instat Software using the Student's two-sided t-test. Pearson correlation coefficient was calculated to assess the association. Value of p < 0.05 was considered statistically significant.ResultsThe prediabetes group showed a markedly high oxidative stress as indicated by significantly increased NADH oxidase 1 and malondialdehyde while superoxide dismutase and glutathione peroxide were decreased compared to non-prediabetes group. There was no statistical difference between cardiac troponin I and T in the non-prediabetes and prediabetes groups. Cardiac troponins had a weak positive association with glycated haemoglobin.ConclusionThe findings of this study demonstrate that prediabetes is associated with myocardial injury through oxidative stress. Future studies are to investigate cardiac contractile function and include more cardiac biomarkers.

Characteristics of the Cardiosplenic Axis in Patients with Fatal Myocardial Infarction.

Myocardial ischemia triggers neurohumoral activation of the cardiosplenic axis. In rodents, adverse outcomes occur upon prolonged entrance of mononuclear cells from the spleen into myocardial tissue. The purpose of this study is to assess the features of spleen structure in patients with fatal myocardial infarction (MI), the dynamics of macrophage infiltration of the spleen and its relationship with cardiac macrophage infiltration and unfavorable outcomes. Using immunohistochemistry techniques, we analyzed the macrophage infiltration of the spleen and myocardium sections collected from patients (n = 30) with fatal MI. The spleen of the patients was decreased and showed a predominance of red pulp with a high concentration of CD68+ and stabilin-1+ cells. The white pulp contained many medium and small follicles and a lower concentration of CD68+ and stabilin-1+ cells, which was comparable to that in the infarct area of the myocardium. The concentration of CD68+ and stabilin-1+ cells increased in the myocardium in the late period of MI, but did not show any dynamics in the spleen. A high number of CD68+ cells in the red pulp and reduced concentration of stabilin-1+ cells in the white pulp were associated with unfavorable post-infarction outcomes. These fundamental findings could be a basis for the development of new personalized therapeutic and diagnostic approaches for the treatment of MI and its complications.

Isoorientin attenuates doxorubicin-induced cardiac injury via the activation of MAPK, Akt, and Caspase-dependent signaling pathways

BackgroundChemotherapy drugs especially anthracyclines are widely used in the treatment of hematological malignancies and solid tumors. However, their clinical application is limited by dose-dependent and irreversible heart injury, which increases the risk of congestive heart failure and heart-related mortality. PurposeThis study aims to investigate the effect and mechanism of the natural flavonoid isoorientin (ISO) combined with doxorubicin (DOX) on the proliferation of tumor cells and improve the survival rate of DOX-injured cardiomyocytes. Study design/methodsCardiomyocyte H9c2 and a variety of tumor cells were used to evaluate the protective effect of ISO on DOX-induced myocardial injury and enhance the anticancer effects of DOX. DOX chemotherapy-injured mice were used to evaluate the cardioprotective effect of ISO. ResultsThe antiproliferation of DOX on Hela, HepG2, HT-29, and A549 cells could be increased synergistically when cotreated with ISO in vitro. ISO could also improve the survival rate of DOX-injured cardiomyocytes by reducing reactive oxygen species, maintaining mitochondrial function, and inhibiting apoptosis. In mice receiving DOX, a protective effect on myocardial tissue, which was reflected by improved survival state of mice receiving chemotherapy, was observed. The ECG, myocardial zymogram data, HE staining, and TEM observation of myocardial tissue sections showed that ISO had a dose-dependent protective effect on the mouse hearts injured by DOX. Network pharmacology and cardiomyocyte proteomics were used to seek for related target proteins to reveal the protective mechanism of ISO on mouse models, and some potential targets (including caspase-3, EGFR, MAPK1, ESR1, CDC42, STAT1, JAK2, LCK, and CDK2) were generated. Western blotting was further used to verify that ISO upregulated Nrf2 and TGF-β3 by downregulating the phosphorylation levels of JNK and p38 proteins on the MAPK pathway and the Akt and Stat3 expression levels. The downregulation of cleaved caspase-3 and upregulation of Bcl-xl by ISO further confirmed its inhibition on caspase-dependent cardiomyocyte apoptosis. ConclusionISO could be a potential synergistic anticancer agent with a favorable property of reducing the cardiotoxicity for DOX, and the effect mechanism could refer to the inhibition of ISO on MAPK and caspase-dependent apoptosis pathways.

β-cyclodextrin microencapsulation enhanced antioxidant and antihyperlipidemic properties of Tunisian Periploca angustifolia roots condensed tannins in rats.

This study aimed to investigate the microencapsulation of novel condensed tannins isolated from Periploca angustifolia roots, using β-cyclodextrin macrocyclic oligosaccharides, in order to enhance their antioxidant and antihyperlipidemic potentials. Scanning electron microscopy and Fourier transform infrared spectroscopy results revealed that tannin fraction was successfully included into β-cyclodextrin cavities proved with an encapsulation efficacy of 70%. Our in vitro findings highlighted that both pure and encapsulated tannins have efficient inhibition capacities of pancreatic lipase activity. However, the inclusion complex has the greatest, in vivo, antioxidant, and antihyperlipidemic effects. In fact, results showed that complexed tannins had markedly restored serum lipid biomarkers, lipid peroxidation, protein carbonyl oxidation, and antioxidant enzyme defense. These findings were additionally confirmed by aortic and myocardial muscle sections of histological examination. Consequently, β-cyclodextrin microencapsulation may be considered as an effective and promising technique for tannin delivery with improved antioxidant and antihyperlipidemic activities.

DCM and HCM mutations to TPM1 differentially alter contractile behavior and gene expression profiles in engineered heart tissues

Hypertrophic (HCM) or dilated cardiomyopathy (DCM) can be caused by missense mutations in alpha-tropomyosin (TPM1). In this study, we focus on the TPM1 mutations E62Q (HCM) and E54K (DCM), which were introduced homozygously into separate induced pluripotent stem cell (iPSC) lines using CRISPR/Cas9. iPSC-derived cardiomyocytes were seeded into decellularized myocardial sections to form human engineered heart tissues (EHT) that are suitable for biomechanical evaluation of muscle phenotypes. After two weeks in culture, E62Q EHTs displayed a nearly 3-fold increase in peak isometric twitch force relative to isogenic control with no change in relative slope of length dependent activation.

PANoptosis is a prominent feature of desmoplakin cardiomyopathy.

Arrhythmogenic cardiomyopathy (ACM) is hereditary cardiomyopathy caused by pathogenic variants (mutations) in genes encoding the intercalated disc (ID), particularly desmosome proteins. ACM caused by mutations in the DSP gene encoding desmoplakin (DSP) is characterized by the prominence of cell death, myocardial fibrosis, and inflammation, and is referred to as desmoplakin cardiomyopathy. The aim of this article was to gain insight into the pathogenesis of DSP cardiomyopathy. The Dsp gene was exclusively deleted in cardiac myocytes using tamoxifen-inducible MerCreMer (Myh6-Mcm Tam) and floxed Dsp (Dsp F/F) mice (Myh6-Mcm Tam:Dsp F/F). Recombination was induced upon subcutaneous injection of tamoxifen (30 mg/kg/d) for 5 days starting post-natal day 14. Survival was analyzed by Kaplan-Meier plots, cardiac function by echocardiography, arrhythmias by rhythm monitoring, and gene expression by RNA-Seq, immunoblotting, and immunofluorescence techniques. Cell death was analyzed by the TUNEL assay and the expression levels of specific markers were by RT-PCR and immunoblotting. Myocardial fibrosis was assessed by picrosirius red staining of the myocardial sections, RT-PCR, and immunoblotting. The Myh6-Mcm Tam: Dsp F/F mice showed extensive molecular remodeling of the IDs and the differential expression of ~10,000 genes, which predicted activation of KDM5A, IRFs, and NFκB and suppression of PPARGC1A and RB1, among others in the DSP-deficient myocytes. Gene set enrichment analysis predicted activation of the TNFα/NFκB pathway, inflammation, cell death programs, and fibrosis. Analysis of cell death markers indicated PANoptosis, comprised of apoptosis (increased CASP3, CASP8, BAD and reduced BCL2), necroptosis (increased RIPK1, RIPK3, and MLKL), and pyroptosis (increased GSDMD and ASC or PYCARD) in the DSP-deficient myocytes. Transcript levels of the pro-inflammatory and pro-fibrotic genes were increased and myocardial fibrosis comprised ~25% of the myocardium in the DSP-deficient hearts. The Myh6-Mcm Tam:Dsp F/F mice showed severe cardiac systolic dysfunction and ventricular arrhythmias, and died prematurely with a median survival rate of ~2 months. The findings identify PANoptosis as a prominent phenotypic feature of DSP cardiomyopathy and set the stage for delineating the specific molecular mechanisms involved in its pathogenesis. The model also provides the opportunity to test the effects of pharmacological and genetic interventions on myocardial fibrosis and cell death.

Telomere shortening in heart failure due to excess neurohormonal activation is associated with loss of nuclear TRF-2 and increased telomeric DNA damage and predicts cardiac recovery

Abstract The mechanisms and clinical significance of telomere shortening in heart failure remain elusive. Mammalian cardiomyocyte (CM) regeneration is limited and CM cell division cannot account for telomere shortening. Whether telomere shortening in turn affects cardiac recovery remains unexplored. We induced heart failure by excess neurohormonal activation (NHA), a universal dysregulation in the failing heart. B6 mice were subjected to AngII-infusion, uninephrectomy and high-salt (AngII++) to induce excess NHA for 5 weeks. Cardiac function was assessed by ultrasound. Human iPSC-derived CMs, mouse CMs and rat ventricular CMs (H9C2) were stimulated with AngII. Human cardiac biopsies from patients with heart failure with reduced ejection fraction (HFrEF) were grouped depending on cardiac recovery. Telomere length was quantified by Q-FISH. ROS-induced DNA/RNA-damage was evaluated after staining for 8-oxo-7,8-dihydro-2'-deoxyguanosine, -guanine and – guanosine-residues (8-oxo-dG). H9C2-cells were co-stained for telomeres, telomere repeat binding factor 2 (TRF-2) and 8-oxo-dG, telomere-specific DNA-damage was quantified by automated colocalization analysis. Superoxide (O2-) was quantified by 2-hydroxyethidium (2-HE) using HPLC analysis. Fibrosis was quantified by Sirius red staining. AngII++-mice exhibited left ventricular (LV) hypertrophy (p&amp;lt;0.0001 vs B6) along with reduced LV ejection fraction (p=0.03 vs B6), reduced global longitudinal strain (p&amp;lt;0.001 vs B6) along with significant telomere shortening in CMs isolated from AngII++-mice (p=0.0082 vs B6). Telomere length showed a significant correlation with LV systolic function (r2=0.64, p=0.006). While 8-oxo-dG-staining revealed significantly increased ROS-induced DNA/RNA damage in AngII++-myocardial sections overall (p&amp;lt;0.0001 vs B6), DNA/RNA-damage was highest in areas of fibrotic adverse remodeling congruent with significant telomere shortening (p&amp;lt;0.0122 fibrotic vs non-fibrotic AngII++-myocardium, p&amp;lt;0.0001 vs B6). In all cells tested, NHA resulted in significant telomere shortening. As a potential explanation, CMs stimulated with AngII revealed significantly increased, dose-dependent O2–production (AngII 10 nM p=0.03, AngII 50 nm p=0.0004 vs H9C2) along with significant loss of nucelar TRF2 (AngII nM p=0.0049, Ang II 50 nM p&amp;lt;0.0001 vs H9C2), while colocalization analysis revealed a significantly increased ROS-induced damage to the telomeric DNA (AngII 10/50 nm p&amp;lt;0.05 vs H9C2). HFrEF-patients that had recovered cardiac function (ΔLVEF +5 – 30%) showed significantly longer myocardial telomeres than patients with no recovery/worsening (ΔLVEF −10 – 0%) (p=0.008), overall the telomere signal correlated significantly with ΔLVEF (r2=0.41, p=0.03). Our data provides insides into how excess NHA via increased ROS, loss of nuclear TRF-2 and increased telomeric DNA-damage causes myocardial telomere shortening in heart failure and further that the extent of telomere shortening determines cardiac recovery. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): BMBF 01EO1003

Mechanism of lncRNA-ANRIL/miR-181b in autophagy of cardiomyocytes in mice with uremia by targeting ATG5.

ObjectivesThis study is to investigate whether the cardiac microvascular endothelial cells (CMECs) can regulate the autophagy of cardiomyocytes (CMs) by secreting lncRNA-ANRIL/miR-181b exosomes, thus participating in the occurrence of uremic cardiovascular disease (CVD).MethodsA 5/6 nephrectomy uremia model was established, with the mice injected with ANRIL-shRNA lentivirus vector, miR-181b agomir, and related control reagents, containing the serum creatinine and urea nitrogen measured. The renal tissue sections of mice were stained with Periodic Acid-Schiff (PAS), TUNEL, and Hematoxylin-Eosin (HE) performed on myocardial tissue sections of mice. ANRIL-shRNA, miR-181b mimics, and related control reagents were transfected into CMECs, in which the exosomes were extracted and co-cultured with CMs. The expressions of ANRIL, miR-181b and ATG5 were detected by qRT-PCR, and the expressions of autophagy related proteins by Western blot, as well as the binding of ANRIL and miR-181b by the double luciferase reporter gene experiment.ResultsANRIL down-regulation or miR-181b up-regulation can increase the weight of mice with uremia, as well as the expressions of p62 and miR-181b, and reduce the content of serum creatinine and urea nitrogen, the damage of kidney and myocardial tissues, the number of apoptotic cells in myocardial tissues, as well as the expressions of ANRIL, ATG5, Beclin1, and LC3. CMs can absorb the exosomes of CMECs. Compared with IS+ CMEC-Exo group, the expressions of ANRIL and ATG5 in CMs of IS+ CMEC-Exo + sh lncRNA ANRIL and IS+CMEC-Exo+miR-181b mimics groups was down-regulated, as well as the expressions of ATG5, Beclin1, and LC3, while miR-181b expression was up-regulated as well as P62 expression.ConclusionsCMECs can regulate autophagy of CMs by releasing exosomes containing ANRIL and miR-181b.

Evidence for synergy between sarcomeres and fibroblasts in an in vitro model of myocardial reverse remodeling

We have created a novel in-vitro platform to study reverse remodeling of engineered heart tissue (EHT) after mechanical unloading. EHTs were created by seeding decellularized porcine myocardial sections with a mixture of primary neonatal rat ventricular myocytes and cardiac fibroblasts. Each end of the ribbon-like constructs was fixed to a plastic clip, allowing the tissues to be statically stretched or slackened. Inelastic deformation was introduced by stretching tissues by 20% of their original length. EHTs were subsequently unloaded by returning tissues to their original, shorter length. Mechanical characterization of EHTs immediately after unloading and at subsequent time points confirmed the presence of a reverse-remodeling process, through which stress-free tissue length was increased after chronic stretch but gradually decreased back to its original value within 9 days. When a cardiac myosin inhibitor was applied to tissues after unloading, EHTs failed to completely recover their passive and active mechanical properties, suggesting a role for actomyosin contraction in reverse remodeling. Selectively inhibiting cardiomyocyte contraction or fibroblast activity after mechanical unloading showed that contractile activity of both cell types was required to achieve full remodeling. Similar tests with EHTs formed from human induced pluripotent stem cell-derived cardiomyocytes also showed reverse remodeling that was enhanced when treated with omecamtiv mecarbil, a myosin activator. These experiments suggest essential roles for active sarcomeric contraction and fibroblast activity in reverse remodeling of myocardium after mechanical unloading. Our findings provide a mechanistic rationale for designing potential therapies to encourage reverse remodeling in patient hearts.