Heart Samples Research Articles

Seroepidemiology of Toxoplasma gondii infection and parasite DNA in free-range chickens in Aguascalientes, Mexico

The objectives of the study were to estimate the prevalence of anti-Toxoplasma gondii antibodies in free-range chickens in Aguascalientes, Mexico, its association with certain risk factors, and the frequency ofparasite DNA in the heart. Eighty-one small rural family farms were included, and blood and heartsamples were taken from 150 clinically healthy, adult, free-range chickens. Serum samples were processed by indirect immunofluorescence antibody test considering a dilution of 1:16 as a positive reaction, while the DNA detection was done by PCR. The correlation between the presence of antibodies and the potential risk factors was estimated with logistic regression. The overall seroprevalence in the examined populations was 67%, with a range from 33% to 100% among municipalities, and 78% of the farms having at least one seropositive bird. The PCR test identified the presence of parasite DNA for one case only. Among the variables, the presence of cats and the presence of other animal species on the farm were the ones that had the highest values, while the presence of feral cats and the presence of noxious fauna were also identified as potential risk factors. The results indicate high soil contamination by T. gondii oocysts, which implies the active role of cats living on the farms.

Australian Donation and Transplantation Biobank: A Research Biobank Integrated Within a Deceased Organ and Tissue Donation Program.

Feasibility of a research biobank integrated within the deceased organ and tissue donation program was assessed. DonateLife Victoria sought consent for ADTB donation after consent was received for organ donation for transplantation from the donor's senior available next of kin. ADTB samples were collected during donation surgery and distributed fresh to researchers or stored for future research. The main outcome measures were ADTB donation rates, ADTB sample collection, ADTB sample use, and to identify ethical considerations. Over 2 y, samples were collected for the ADTB from 69 donors (28% of 249 donors). Samples were obtained from the spleen (n = 59, 86%), colon (n = 57, 83%), ileum (n = 56, 82%), duodenum (n = 55, 80%), blood (n = 55, 80%), bone marrow (n = 55, 80%), skin (n = 54, 78%), mesenteric lymph nodes (n = 56, 81%), liver (n = 21, 30%), lung (n = 29, 42%), and lung-draining lymph node (n = 29, 42%). Heart (n = 20), breast (n = 1), and lower urinary tract (n = 1) samples were obtained in the second year. Five hundred fifty-six samples were used in 19 ethics-approved research projects spanning the fields of immunology, microbiology, oncology, anatomy, physiology, and surgery. The integration of routine deceased donation and transplantation activities with a coordinated system for retrieval and allocation of donor samples for use in a range of research projects is feasible and valuable.

Microscopic and Molecular Detection of Sarcocystis cruzi (Apicomplexa: Sarcocystidae) in the Heart Muscle of Cattle.

Sarcocystis is a genus of coccidian protozoa that at least seven species of it can parasitize cattle. The global prevalence of Sarcocystis is close to 100% in adult cattle. The main aim of this study was to identify the infection rate of Sarcocystis spp. in heart of cattle in Tehran, Iran by microscopy and PCR-RFLP methods. Totally, 100 bovine heart samples were collected from the main slaughterhouse of Shahriar, Meysam slaughterhouse, west of Tehran in 2016. At first, heart samples were completely examined for the presence of sarcocystic macrocysts. Then, for microscopic examination, 50 g of each heart was digested in sterile condition using pepsin acid digestion method. Then, the species of the parasite were detected by PCR-RFLP technique and sequencing. Overall, 97 of 100 of the heart muscle samples were infected with Sarcocystis. All the samples were detected as S. cruzi through similar patterns in PCR-RFLP. S. cruzi is the most common species in the heart of cattle slaughtered in Shahriar.

Effects of Dietary Supplementation With Soybean And Canola Oil on Lipid Profiles and Heart in Swiss Albino Mice

Soybean and canola oil are two of the most widely used cooking oils on the planet. The experiment was conducted to study the dose dependent toxicity of dietary supplementation of soybean and canola oil on heart of Swiss albino mice. In the study, 30 male Swiss albino mice aged 6 weeks were randomly assigned to one of five equivalent groups: Group A was considered as control by feeding rat pellet only, group B1 and B2 were supplemented with 25 and 35 ml soybean oil/kg pellet and group C1 and C2 were provided with 25 and 35 ml canola oil/kg pellet, respectively for 60 days, adopting established research procedure. After conducting the experimental period, blood and organ (heart) samples were collected from control and oil supplemented mice through deep anesthesia. The biochemical study of blood revealed that maximum total cholesterol, triglycerides and high density lipoprotein values (153.85 ± 0.44, 88.82 ± 0.37 and 105.50 ± 0.33 mg/dl) were found statistically significant (P<0.05) in soybean oil supplemented group (B2) as compared to other groups (B1, C2, C1 and A). Gross study revealed that highest mean weight of heart (0.41 ± 0.01 g) was recorded in soybean oil supplemented group (B2) significantly (P<0.05) in comparison to other experimental groups B1, C2, C1 and A. Histopathological study revealed that marked lymphocytic infiltration was found in the ventricular wall of the heart of high dose supplemented groups. So, it may conclude that dietary supplementation of soybean and canola oil have adverse effects on blood lipid profiles and heart of mice.Ann. Bangladesh Agric. (2021) 25 (2) : 87-95

Morphometric Analysis And Anatomical Variations Of Right Coronary Artery In “Human Cadaveric Hearts” In South Indian Population

Aim And Objective: The study objective is to know the anatomical variations of “right coronary artery” through dissection method and to know its clinical significance. Materials And Methods: In this study, 45 heart samples were implied and approx 30 hearts were calmed from Anatomy Department of “SRM Medical College”. 15 hearts are calmed from the anatomy department of “Sri Ramachandra medical college”. “The right coronary arteries” were observed as well as dissected for its normal anatomy and for any variations, and observations were noted out. Results: The present study shows that during dissection of “right coronary artery”, the “coronary ostium” was examined in all the sample inside the “anterior aortic sinus”, in that 9 sample presents different ostium for “right conus artery” and 1 specimen presents a several ostium for “SA nodal artery” in the same sinus, right marginal artery was present in all specimen with average length of 7.36cms.And with respect to PIV branch, the coronary dominance in right - 75.56%, left – 13.3% and co dominance - 11.11%. Conclusion: In view of this detailed study, I would like to conclude that the noted variations of this artery would be of effective use to the interventional along with “cardiologist plus radiologist” to destine the abnormalities to provide excellent maintainer about “cardiac diseases” and interventional procedures.

Availability of extractives from various Norway spruce (Picea abies) stumps assortments

Stumps and knotwood of Norway spruce (Picea abies) are valuable sources of wood extractives. Although lignans from knotwood have already been utilized in value-added products, the behavior and valorization of stump-derived extractives are less studied. In this study, the composition of lipophilic and hydrophilic extractives, particularly lignans, from various spruce stump samples (stump bottom, stump heart, and crushed stump samples) stored outside were studied. Lipophilic and hydrophilic extracts were separated with an accelerated solvent extraction (ASE) apparatus using n-hexane and hot water, respectively. The detailed extractives content of samples was then determined by gas chromatography equipped with a flame ionization detector and a mass detector (GC-FID/MS) and high-performance liquid chromatography (HPLC). In stump bottom samples, an apparent decrease in total dissolved solids was observed in all the major extractives groups during storage: lignans, sugars, stilbene–glucosides, organic acids, resin acids, fatty acids, diterpenoids, and sterols. While a definitive decrease in extractives could not be demonstrated due to the moderately high variation of extractives among different samples, a good indication of the accessibility of important extractives in weathered stumps was obtained. Of the identified hydrophilic extractives, 79% were lignans, 53% of them being composed of 7-hydroxymatairesinol (HMR), 16% conidendric acid, and 12% todolactol. After 12 weeks of storage, the total amount of lignans was 15.3 mg/g of dry matter in stump bottom, 17.0 mg/g of dry matter in stump heart samples, and 10.2 mg/g of dry matter in crushed stump samples.

Long term toxicities following developmental exposure to perfluorooctanoic acid: Roles of peroxisome proliferation activated receptor alpha

Perfluorooctanoic acid (PFOA) is a widespread persistent organic pollutant. Fertile chicken eggs were exposed to PFOA and incubated to hatch. At three time points post hatch (0-, 1- and 3-months old), chickens were subjected to electrocardiography and sacrificed. Serum was subjected to LC-MS/MS for PFOA concentration, and organs were subjected to histopathological assessments. Additionally, PPARα-silencing lentivirus was co-applied with PFOA exposure, and the corresponding phenotypes were evaluated. Western blotting was performed to assess expressions of FABPs and pSMAD2 in heart and liver samples. Considerable amount of PFOA were detected in hatchling chicken serum, but not in one-month-old or three-month-old chicken serum. PFOA exposure resulted in developmental cardiotoxicity and hepatotoxicity in hatchling chickens. Meanwhile, one-month-old chickens still exhibited elevated heart rate, but classical cardiac remodeling (thicker right ventricular wall) were observed in exposed animals. Three-month-old chickens exhibited similar results as one-month-old ones. PPARα silencing only had partial protective effects in hatchling chickens, but the protective effects seemed to increase as chickens aged. Western blotting results indicated that L-FABP was involved in PFOA-induced hepatotoxicity, while pSMAD2 was involved in PFOA-induced cardiotoxicity. In summary, developmental exposure to PFOA resulted in persistent cardiotoxicity, but not hepatotoxicity. PPARα participates in both cardiotoxicity and hepatotoxicity.

Ramosin: The First Antibacterial Peptide Identified on Bolitoglossa ramosi Colombian Salamander.

The discovery and improvements of antimicrobial peptides (AMPs) have become an alternative to conventional antibiotics. They are usually small and heat-stable peptides, exhibiting inhibitory activity against Gram-negative and Gram-positive bacteria. In this way, studies on broad-spectrum AMPs found in amphibians with the remarkable capability to regenerate a wide array of tissues are of particular interest in the search for new strategies to treat multidrug-resistant bacterial strains. In this work, the use of bioinformatic approaches such as sequence alignment with Fasta36 and prediction of antimicrobial activity allowed the identification of the Ramosin peptide from the de novo assembled transcriptome of the plethodontid salamander Bolitoglossa ramosi obtained from post-amputation of the upper limb tissue, heart, and intestine samples. BLAST analysis revealed that the Ramosin peptide sequence is unique in Bolitoglossa ramosi. The peptide was chemically synthesized, and physicochemical properties were characterized. Furthermore, the in vitro antimicrobial activity against relevant Gram-positive and Gram-negative human pathogenic bacteria was demonstrated. Finally, no effect against eukaryotic cells or human red blood cells was evidenced. This is the first antibacterial peptide identified from a Colombian endemic salamander with interesting antimicrobial properties and no hemolytic activity.

Exercise Training and Verbena officinalis L. Affect Pre-Clinical and Histological Parameters.

Verbena officinalis L. or vervain is an herbal medicine and dietary supplement used worldwide. It is used for antidepressant and anticonvulsant purposes, as well as to treat inflammatory disorders, skin burns, abrasions, and gastric diseases, among others. Here, we investigated the biochemical, antioxidant, and histopathological effects of vervain against chronic physical stress. Male Wistar rats were submitted to chronic physical training and oral administration of 200 mg/kg of extract for 7 weeks. Control animals were not treated with either stress or vervain. Body weight was monitored during the study. Liver, kidney, spleen, testis, epididymis, heart, skeletal muscle, and brain samples were collected. Blood cholesterol, lactate dehydrogenase (LDH), bilirubin, and creatinine kinase (CREA), among others, were studied. Glutathione peroxidase (GPox) and superoxide dismutase (SOD) antioxidant activity was analyzed in the blood, liver, and kidney. Testosterone measurements were also performed on whole testis extracts. We found significant weight ratios differences in the epididymis, brain, and heart. Animals submitted to training showed hemorrhagic livers. Kidney histology was affected by both stress and vervain. Cell disruption and vacuolization were observed in the testes and epididymis of animals submitted to stress. Hematological and biochemical markers as CREA, LDH, TP, CKI, URCA, γGT, and glucose revealed statistically significantly differences. Additionally, the activity of glutathione peroxide (GPox) and superoxide dismutase (SOD) in the blood was also impacted. Both stress and vervain have significant in vivo effects. Infusions of vervain include phenylpropanoids, iridoids, verbenalin, hastatoside, and flavonoids, amongst others, which interact synergistically to produce the preclinical effects reported here.

Inflammation and vascular remodeling in COVID-19 hearts

A wide range of cardiac symptoms have been observed in COVID-19 patients, often significantly influencing the clinical outcome. While the pathophysiology of pulmonary COVID-19 manifestation has been substantially unraveled, the underlying pathomechanisms of cardiac involvement in COVID-19 are largely unknown. In this multicentre study, we performed a comprehensive analysis of heart samples from 24 autopsies with confirmed SARS-CoV-2 infection and compared them to samples of age-matched Influenza H1N1 A (n = 16), lymphocytic non-influenza myocarditis cases (n = 8), and non-inflamed heart tissue (n = 9). We employed conventional histopathology, multiplexed immunohistochemistry (MPX), microvascular corrosion casting, scanning electron microscopy, X-ray phase-contrast tomography using synchrotron radiation, and direct multiplexed measurements of gene expression, to assess morphological and molecular changes holistically. Based on histopathology, none of the COVID-19 samples fulfilled the established diagnostic criteria of viral myocarditis. However, quantification via MPX showed a significant increase in perivascular CD11b/TIE2 + —macrophages in COVID-19 over time, which was not observed in influenza or non-SARS-CoV-2 viral myocarditis patients. Ultrastructurally, a significant increase in intussusceptive angiogenesis as well as multifocal thrombi, inapparent in conventional morphological analysis, could be demonstrated. In line with this, on a molecular level, COVID-19 hearts displayed a distinct expression pattern of genes primarily coding for factors involved in angiogenesis and epithelial-mesenchymal transition (EMT), changes not seen in any of the other patient groups. We conclude that cardiac involvement in COVID-19 is an angiocentric macrophage-driven inflammatory process, distinct from classical anti-viral inflammatory responses, and substantially underappreciated by conventional histopathologic analysis. For the first time, we have observed intussusceptive angiogenesis in cardiac tissue, which we previously identified as the linchpin of vascular remodeling in COVID-19 pneumonia, as a pathognomic sign in affected hearts. Moreover, we identified CD11b + /TIE2 + macrophages as the drivers of intussusceptive angiogenesis and set forward a putative model for the molecular regulation of vascular alterations.

Betamethasone Toxicity Induced by Topical Maternal Exposure Prior to Fertilization in Heart of Newborn Rabbits

Background: Betamethasone treatment is widely used and recommended worldwide. Although, there are no enough studies to look for the risk or potential alterations due to accumulation of this drug before fertilization. In addition, previous studies payed attention to intramuscular injection during pregnancy neglecting the long-term impact of the use of topical solutions. So, the aim of the present study was to determine this side effect. Methods: We used two doses of betamethasone: 0.02 and 0.2 mg/Kg b.w. were and at the end of the experimental period (after 3 weeks of delivery), six new-born rabbits of both sexes (1 new born rabbit per mother) were dissected under light anesthesia and heart samples were collected. All the measurements were performed in one assay. Result: The results of this study revealed maternal exposure to betamethasone prior to fertilization, induced oxidative stress, alterations in levels of enzymes. Furthermore, these biochemical alterations were supported by histopathological and ultrastructural observations. So, this study suggested that females; desiring to be pregnant, should stop treatment of psoriasis with betamethasone before fertilization to avoid heart toxicity of their offspring.

TPEN/TPGS (T2) combo dramatically reduces Philadelphia chromosome-positive pro-lymphoblastic B leukemia in BALB/c mice

Acute lymphoblastic leukemia (ALL) is hematological neoplasia that affects human beings from early life to adulthood. Although ALL treatment has been effective, an important percentage of ALL patients are resilient to treatment. Therefore, there is an urgent need for testing a new combination of compounds for the treatment of this disease. Recently, combined TPEN and TPGS (T2 combo) have shown selective cytotoxic effects in vitro leukemia cells such as Jurkat, K562, and Ba/F3 cells. In this study, we aimed to test the effect of combined TPEN and TPGS agents (T2 combo) at a fixed dose (TPEN 5 mg/kg: TPGS 100 mg/kg) on leukemic Ba/F3-BCR-ABL P210 BALB-c mice model. We found that 4 successive 2-day apart intravenous injections of T2 combo showed a statistically significant reduction of Ba/F3 BCR-ABL leukemia cells (− 69%) in leukemia BALB/c mice (n = 6) compared to untreated leukemia group (n = 6). Moreover, the T2 combo was innocuous to non-leukemia BALB/c mice (n = 3) compared to untreated non-leukemia mice (control, n = 3). After treatments (day 42), all mice were left to rest until day 50. Outstandingly, the leukemia BALB/c mice treated with the T2 combo showed a lower percentage of Ba/F3-BCR-ABL P210 cells (− 84%) than untreated leukemia BALB/c mice. Furthermore, treatment of leukemia and non-leukemia mice with T2 combo showed no significant tissue alteration/damage according to the histopathological analysis of brain, heart, liver, kidney, and spleen samples; however, T2 combo significantly reduced the number of leukocytes in the bone marrow of treated leukemia mice. We conclude that the T2 combo specifically affects leukemia cells but no other tissue/organs. Therefore, we anticipate that the T2 combo might be a potential pro-oxidant combination for the treatment of leukemia patients.

Abstract 14024: Role of Endothelial CXCR4 on the Development of Aortic Valve Stenosis

Background. CXCL12/CXCR4 signaling is essential in cardiac development and repair, however, its contribution to the Aortic valve stenosis (AVS) remains unclear. In this study, we tested the role of endothelial CXCR4 on the development of AVS. Methods. We generated CXCR4 endothelial cell-specific knockout mice (EC CXCR4 KO) by crossing CXCR4 fl/fl mouse with Tie2-Cre mouse to study the role of endothelial cell CXCR4 in AVS. CXCR4 fl/fl mice with no Tie2-Cre gene present were used as control. Echocardiography was used to assess the aortic valve and cardiac function. Heart samples containing the aortic valve were stained using Alizarin Red for detection of calcification. Masson’s Trichrome staining was used for the detection of fibrosis. Apex samples were stained with wheat germ agglutinin to visualize ventricular hypertrophy. Results. Compared with the control group, the deletion of CXCR4 in endothelial cells led to significantly increased aortic valve peak velocity and aortic valve peak pressure gradient, and decreased aortic valve area and ejection fraction. EC CXCR4 KO mice also developed cardiac hypertrophy evidenced by increased left ventricular mass, diastolic and systolic left ventricle posterior wall (LVPW), cardiac myocyte size, and heart weight (HW) to body weight (BW) ratio. Our data also confirmed increased microcalcifications, interstitial fibrosis, and thickened valvular leaflets of the EC CXCR4 KO mice. Conclusions. We found that the deletion of CXCR4 in endothelial cells leads to aortic valve stenosis and left ventricular hypertrophy. This indicates endothelial cell CXCR4 plays a protective role against AVS, and EC CXCR4 KO mice can be used as a novel model for AVS study.

Abstract 11983: Associations Between Circulating Proteins and Intracardiac Immune Populations in Immune Checkpoint Inhibitor-Associated Myocarditis

Introduction: Myocarditis due to immune checkpoint inhibitors (ICIs), a type of cancer immunotherapy, is associated with high morbidity and mortality. The cellular and molecular pathogenesis of ICI myocarditis remains largely unknown. Identification of circulating factors associated with intracardiac pathology may aid new clinical approaches. Hypothesis: We hypothesized that ICI myocarditis is associated with increased abundance of intracardiac immune cells and upregulated inflammatory genes in the heart and serum. Methods: Heart tissue from 13 patients with ICI myocarditis was acquired by endomyocardial biopsy or autopsy; peripheral blood mononuclear cells (PBMCs) were also collected from ten of these patients and eight additional ICI myocarditis patients. Control heart tissue was derived from six hearts declined for transplantation (non-ICI-exposed) and from the biopsy and autopsy of one ICI-treated patient without myocarditis. The 10x Genomics Chromium system was used to generate single-cell RNA sequencing (scRNAseq) data from heart and PBMC specimens. Serum proteins were measured by core lab assay (for troponin T) or by multiplexed Luminex immunoassay. Results: Unbiased clustering of scRNAseq data from 77,071 cells recovered from heart samples revealed 37 cell subsets across 10 cell lineages. Myocarditis heart tissue demonstrated enrichment of T/NK cells (odds ratio 8.3, p=0.0006), B/plasma cells (OR 5.1, p=0.01), and dendritic cells (DCs) (OR 9.2, p=0.006) relative to controls. Circulating DC abundance was decreased in fatal (n=3) versus nonfatal (n=15) myocarditis cases (p=0.008), while intracardiac DC abundance was directly associated with serum troponin T values (p=0.02). The levels of five immunomodulatory factors - IL-15, CXCL9, CCL3, TNFα, and CCL21 - were found to be significantly upregulated transcriptionally in at least one intracardiac cell subset and at the protein level in the serum of myocarditis cases. Conclusions: Fatality status and troponin level associated with intracardiac DC abundance and multiple immunomodulatory genes were upregulated in both the heart and serum in ICI myocarditis. These observations may guide novel diagnostic and therapeutic strategies.

First Molecular Identification of Sarcocystis Buffalonis in Goats Worldwide in Misan Governorate, Iraq

The current study was conducted in Misan Governorate to detect the parasite Sarcocystis spp. by both conventional and molecular methods and to identify the species of this genus in goats as intermediate hosts for multiple species of this parasite. Regarding to the traditional study, the rates of infection with the macroscopic type of Sarcocystis spp. were low and recorded 2% in esophageal samples only in goats. The sizes of macroscopic parasite cysts ranged between 1-10 mm in length and 2-6 mm in width. Infection with the microscopic type of parasite recorded high rates. When comparing the efficiency of the traditional methods used in our study to diagnose the infection of the microscopic type in all of the examined organs isolated from goats, which included the methods of digestion with pepsin, squeezing, trichinoscopy. It was found that the method of digestion with pepsin was the most efficient traditional method for diagnosing the microscopic type of parasite, followed by the trichinoscopy. While the squeezing method recorded the least efficient traditional method for diagnosing Sarcocystis spp. microscopically. The molecular study of Sarcocystis spp. included molecular diagnosis of the parasite's identity at the species level through gene amplification using polymerase chain reaction (PCR) of the target segment of DNA extracted from the parasite samples of goats for 18S rRNA gene by 120 microscopic samples and 4 macroscopic samples. 87.9% of goat samples, showed positive infection with the 18S rRNA gene; In which 100% of the macroscopic and microscopic samples of the esophagus were recorded, and both animals had a positive infection, while the infection rates for the diaphragm, tongue and heart samples were recorded as 90%, 86.7 and 80%, respectively. The results obtained from sequence analysis of 18S rRNA gene for six samples showed that four microscopic samples of goats (OP474017.1, OP474018.1, OP476699.1 and OP474036.1) were closely related to the Malaysian species; S. capracanis with accession number (KR155191.1) with identity score percentages as 99.50%, 99.01%, 99.76% and 99.13%, respectively. Finally, the two macroscopic samples of goats (OP474061.1 and OP474062.1) were identical with the Egyptian species; S. buffalonis, with accession numbers (KU247913.1) and (KU247913.1) respectively, with a matching percentage of 84.39% and 85.64% respectively. S. buffalonis has identified in goats at the first time globally in present study. Keywords: Sarcocystis buffalonis, Goats, OP474061.1, OP474062.1.

Metal accumulation in juvenile and sub-adult loggerhead and green turtles in northern Cyprus

Sea turtles are considered pollution bioindicators due to their tendency to accumulate high metal levels in their tissues during their long lifespans. In this context, we aimed to analyse the concentrations of 12 elements in liver, kidney, heart and muscle samples from green turtles (Chelonia mydas; n = 41) and loggerhead turtles (Caretta caretta; n = 14) found stranded in Northern Cyprus. The samples were collected between 2019 and 2021, stored in sterile Eppendorf tubes at −20 °C until metal analysis, and analysed with an inductively coupled plasma mass spectrometer. With this study, we contribute to the limited number of studies on metal accumulation in heart tissue and present the first data for Mg accumulation in the heart, liver, muscle and kidney tissues of both species. We found that metal accumulation levels differed among the two study species’ tissues, with some elements in the same tissue (AlKidney, AsHeart, AsLiver, FeMuscle, FeKidney, FeHeart, MnHeart, PbHeart, ZnMuscle and ZnKidney) significantly differing between species. The observed variation likely resulted from their different feeding habits, which cause them to be exposed to different levels of metals. We also found significant associations among elements within tissues, as well as between the same element across different tissues in both species, which may indicate the differential accumulation of elements among organs due to physiological processes in turtle metabolism, bioaccumulation or excretion.

Unpredicted Aberrant Splicing Products Identified in Postmortem Sudden Cardiac Death Samples.

Molecular screening for pathogenic mutations in sudden cardiac death (SCD)-related genes is common practice for SCD cases. However, test results may lead to uncertainty because of the identification of variants of unknown significance (VUS) occurring in up to 70% of total identified variants due to a lack of experimental studies. Genetic variants affecting potential splice site variants are among the most difficult to interpret. The aim of this study was to examine rare intronic variants identified in the exonic flanking sequence to meet two main objectives: first, to validate that canonical intronic variants produce aberrant splicing; second, to determine whether rare intronic variants predicted as VUS may affect the splicing product. To achieve these objectives, 28 heart samples of cases of SCD carrying rare intronic variants were studied. Samples were analyzed using 85 SCD genes in custom panel sequencing. Our results showed that rare intronic variants affecting the most canonical splice sites displayed in 100% of cases that they would affect the splicing product, possibly causing aberrant isoforms. However, 25% of these cases (1/4) showed normal splicing, contradicting the in silico results. On the contrary, in silico results predicted an effect in 0% of cases, and experimental results showed >20% (3/14) unpredicted aberrant splicing. Thus, deep intron variants are likely predicted to not have an effect, which, based on our results, might be an underestimation of their effect and, therefore, of their pathogenicity classification and family members’ follow-up.

Early Doxorubicin Myocardial Injury: Inflammatory, Oxidative Stress, and Apoptotic Role of Galectin-3.

Doxorubicin (DOXO) is an effective drug that is used in the treatment of a large number of cancers. Regardless of its important chemotherapeutic characteristics, its usage is restricted because of its serious side effects; the most obvious is cardiotoxicity, which can manifest acutely or years after completion of treatment, leading to left ventricular dysfunction, dilated cardiomyopathy, and heart failure. Galectin 3 (Gal-3) is a beta galactoside binding lectin that has different roles in normal and pathophysiological conditions. Gal-3 was found to be upregulated in animal models, correlating with heart failure, atherosclerosis, and myocardial infarction. Male C57B6/J and B6.Cg-Lgals3 <tm 1 Poi>/J Gal-3 knockout (KO) mice were used for a mouse model of acute DOXO-induced cardiotoxicity. Mice were given DOXO or vehicle (normal saline), after which the mice again had free access to food and water. Heart and plasma samples were collected 5 days after DOXO administration and were used for tissue processing, staining, electron microscopy, and enzyme-linked immunosorbent assay (ELISA). There was a significant increase in the heart concentration of Gal-3 in Gal-3 wild type DOXO-treated mice when compared with the sham control. There were significantly higher concentrations of heart cleaved caspase-3, plasma troponin I, plasma lactate dehydrogenase, and plasma creatine kinase in Gal-3 KO DOXO-treated mice than in Gal-3 wild type DOXO-treated mice. Moreover, there were significantly higher heart antioxidant proteins and lower oxidative stress in Gal-3 wild type DOXO-treated mice than in Gal-3 KO DOXO-treated mice. In conclusion, Gal-3 can affect the redox pathways and regulate cell survival and death of the myocardium following acute DOXO injury.

Las mutaciones missense en el dominio ROD2 de la filamina C muestran un fenotipo con miocardiopatía restrictiva/hipertrófica y miocardio en dientes de sierra

Introduction and objectivesMissense mutations in the filamin C (FLNC) gene have been reported as cause of inherited cardiomyopathy. Knowledge of the pathogenicity and genotype-phenotype correlation remains scarce. Our aim was to describe a distinctive cardiac phenotype related to rare missense FLNC variants in the ROD2 domain. MethodsWe recruited 21 unrelated families genetically evaluated because of hypertrophic cardiomyopathy (HCM)/restrictive cardiomyopathy (RCM) phenotype carrying rare missense variants in the ROD2 domain of FLNC (FLNC-mRod2). Carriers underwent advanced cardiac imaging and genetic cascade screening. Myocardial tissue from 3 explanted hearts of a missense FLNC carrier was histologically analyzed and compared with an FLNC-truncating variant heart sample and a healthy control. Plasmids independently containing 3 FLNC missense variants were transfected and analyzed using confocal microscopy. ResultsEleven families (52%) with 20 assessed individuals (37 [23.7-52.7]) years showed 15 cases with a cardiac phenotype consisting of an overlap of HCM-RCM and left ventricular hypertrabeculation (saw-tooth appearance). During a median follow-up of 6.49 years, they presented with advanced heart failure: 16 (80%) diastolic dysfunction, 3 heart transplants, 3 heart failure deaths) and absence of cardiac conduction disturbances or skeletal myopathy. A total of 6 families had moderate genotype-phenotype segregation, and the remaining were de novo variants. Differential extracellular matrix remodeling and FLNC distribution among cardiomyocytes were confirmed on histology. HT1080 and H9c2 cells did not reveal cytoplasmic aggregation of mutant FLNC. ConclusionsFLNC-mRod2 variants show a high prevalence of an overlapped phenotype comprising RCM, HCM and deep hypertrabeculation with saw-tooth appearance and distinctive cardiac histopathological remodeling.Full English text available from:www.revespcardiol.org/en

A miRNA derived from the 17/92 cluster is a novel player in programming pulmonary hypertension in response to gestational hypoxia via NADPH oxidases and the mTOR pathway

Abstract Background Gestational hypoxia can lead to intrauterine growth restriction (IUGR) and programming of cardiovascular diseases in adulthood via different, although not completely understood, epigenetic mechanisms. We have previously shown that reactive oxygen species (ROS) derived from NADPH oxidases contribute to hypoxia induced pulmonary hypertension (PH). However, their role in disease programming by gestational hypoxia is not resolved. Purpose We aimed to elucidate the role of NADPH oxidases and redox sensitive miRNAs in the cardiovascular response of adult offspring to transient gestational hypoxia. Methods Wildtype (WT) and nmf333 mice, lacking a functional p22phox dependent NADPH oxidase, were exposed to gestational hypoxia from E10.5 to E11.5. Embryos and adult offspring were analyzed by functional, immunohistochemical and molecular methods including mRNA and miRNA profiling. miRNA mimics or inhibitors were applied in vivo and in vitro using embryoid bodies (EBs). Heart samples from patients with right heart disease associated with congenital heart disease (CHD) were used to validate target miRNA expression. Results Transient gestational hypoxia was sufficient to induce IUGR in embryos and pulmonary hypertension (PH) characterized by right ventricular hypertrophy, increased right ventricular pressure and pulmonary vascular remodeling in adult offspring from WT mice while p22phox-deficient nmf333 mice were protected. Cardiac mRNA and miRNA profiling followed by qPCR validation identified several single miRNAs derived from the 17/92 miRNA cluster as downregulated by gestational hypoxia and NADPH oxidases in embryos and adult offspring hearts. In vitro analyses using EBs validated a single miRNA from this cluster to be sufficient to induce cardiac differentiation in response to hypoxia and p22phox, and target analyses identified the mTOR pathway as dysregulated. Treatment with an inhibitor targeting this miRNA was sufficient to induce PH in adult mice, while treating offspring derived from gestational hypoxia pregnancies with an miRNA mimic prevented the development of PH in the adult and dysregulation of the mTOR pathway. Importantly, expression of this single miRNA from the miRNA 17/92 cluster was negatively correlated to right ventricular pressure in CHD patients. Conclusions In this study, we demonstrate that gestational hypoxia programs PH in adulthood by upregulating NADPH oxidases and downregulating miRNAs derived from the 17/92 cluster subsequently affecting the mTOR pathway. Since dysregulation of a single miRNA from this cluster induced not only PH but was also associated with right ventricular pressure in CHD patients, these findings might open novel options for diagnosis, prevention and treatment of right heart disease. Funding Acknowledgement Type of funding sources: Public Institution(s). Main funding source(s): DZHK (German Centre for Cardiovascular Research)