Standardized Classification of Lung Adenocarcinoma Subtypes and Improvement of Grading Assessment Through Deep Learning.

The histopathologic distinction of lung adenocarcinoma (LADC) subtypes is subject to high interobserver variability, which can compromise the optimal assessment of patient prognosis. Therefore, this study developed convolutional neural networks capable of distinguishing LADC subtypes and predicting disease-specific survival, according to the recently established LADC tumor grades. Consensus LADC histopathologic images were obtained from 17 expert pulmonary pathologists and one pathologist in training. Two deep learning models (AI-1 and AI-2) were trained to predict eight different LADC classes. Furthermore, the trained models were tested on an independent cohort of 133 patients. The models achieved high precision, recall, and F1 scores exceeding 0.90 for most of the LADC classes. Clear stratification of the three LADC grades was reached in predicting the disease-specific survival by the two models, with both Kaplan-Meier curves showing significance (P = 0.0017 and 0.0003). Moreover, both trained models showed high stability in the segmentation of each pair of predicted grades with low variation in the hazard ratio across 200 bootstrapped samples. These findings indicate that the trained convolutional neural networks improve the diagnostic accuracy of the pathologist and refine LADC grade assessment. Thus, the trained models are promising tools that may assist in the routine evaluation of LADC subtypes and grades in clinical practice.

The Long-Term Natural History of Fibrous Dysplasia.

Fibrous dysplasia (FD), a developmental, nonfamilial, benign anomaly of bone development, is characterized by the replacement of normal bone by proliferating fibro-osseous tissue. Marked craniofacial deformities, functional disturbances, and emotional stress are major indications for treatment, and various surgical procedures have been performed; however, excision and regrowth issues have also been reported. While several treatment options are available, no studies have reported the natural history of untreated FD. Here, we report 2 patients, aged 73 and 50 years, respectively, who had not received treatment. Both patients presented to the hospital complaining of noise when moving their heads. Computed tomography scans showed niveau with honeycomb cavities in both patients, indicating abscess formation, and resection was performed. Relatively large cranial FD leads to the development of central necrosis over time. In such cases, surgical intervention should be performed at an early disease stage.

Allyl isothiocyanate and 6-(methylsulfinyl) hexyl isothiocyanate contents vary among wild and cultivated wasabi (Eutrema japonium).

Wasabi (Japanese horseradish, Eutrema japonicum) is the only cultivated species in the genus Eutrema with functional components that provide a strong pungent flavor. To evaluate genetic resources for wasabi breeding, we surveyed variations in the two most abundant isothiocyanate (ITC) components in wasabi, allyl isothiocyanate (AITC) and 6-methylsulfinyl (hexyl) isothiocyanate (6-MSITC, hexaraphane). We also examined the phylogenetic relationships among 36 accessions of wild and cultivated wasabi in Japan using chloroplast DNA analysis. Our results showed that (i) the 6-MSITC content in currently cultivated wasabi accessions was significantly higher than in escaped cultivars, whereas the AITC content was not significantly different. (ii) Additionally, the 6-MSITC content in cultivated wasabi was significantly lower in the spring than during other seasons. This result suggested that the 6-MSITC content responds to environmental conditions. (iii) The phylogenetic position and the 6-MSITC content of accessions from Rebun, Hokkaido Prefecture had different profiles compared with those from southern Honshu, Japan, indicating heterogeneity of the Rebun populations from other Japanese wasabi accessions. (iv) The total content of AITC and 6-MSITC in cultivated wasabi was significantly higher than that of wild wasabi. In conclusion, old cultivars or landraces of wasabi, "zairai", are the most suitable candidates for immediate use as genetic resources.

Presence of heat shock protein 47-positive fibroblasts in cancer stroma is associated with increased risk of postoperative recurrence in patients with lung cancer.

BACKGROUND: Heat shock protein 47 (HSP47), a collagen-binding protein, has a specific role in the intracellular processing of procollagen production. HSP47 expression is associated with cancer growth and metastasis in several types of cancers. However, none of the studies have assessed whether HSP47 expression is associated with the risk of postoperative recurrence of lung cancer until now. Therefore, we aimed to assess this association. METHODS: The study population consisted of a cohort of consecutive patients who underwent surgery for lung cancer at Nagasaki University Hospital, Nagasaki, Japan, from January 2009 to December 2010. Patient characteristics, survival and disease-free survival (DFS), and laboratory findings were compared between patients who tested positive and negative for HSP47 expression in lung cancer cells and between those who showed high and low numbers of HSP47-positive fibroblasts in cancer stroma. RESULTS: A total of 133 patients underwent surgery for lung cancer. Sixty-seven patients (50.4%) had HSP47-positive cancer cells, and 91 patients (68.4%) had a higher number of HSP47-positive fibroblasts. The patients with a high number of HSP47-positive fibroblasts had a shorter DFS than those with a low number of HSP47-positive fibroblasts. Multivariate analysis identified only the presence of a high number of HSP47-positive fibroblasts as an independent risk factor for recurrence of lung cancer after surgery (odds ratio, 4.371; 95% confidence interval, 1.054-29.83; P = 0.042). CONCLUSION: The present study demonstrated that the presence of a high number of HSP47-positive fibroblasts in the cancer stroma was a risk factor for recurrence of lung cancer after surgery.

Simple, Frequent Indicator for Personal Identification-Postmortem and Antemortem Abdominal Computed Tomography Findings of a Charred Body.

Postmortem personal identification in forensic science is performed using various methods. However, severely burnt bodies are hard to identify using odontological or skeletal features because of carbonization, and sometimes DNA profiling is impracticable because of the unavailability of the relatives. We present a case of a burn victim found after a house fire. Personal identification was attempted, but the body was heavily charred to the bones and the use of physical appearance was impracticable. There were no known relatives or personal belongings of the deceased for comparison of DNA typing. We obtained a series of abdominal computed tomography (CT) scans taken antemortem and found bilateral multiple renal cysts, left renal artery calcification, and a big right inguinal hernia, which matched the deceased's postmortem CT findings and autopsy findings. To date, studies of identification by CT have acted for a rise in precision, but they require complicated calculation or high graphical methods. Calcification of the arteries or renal cysts seen in our case are very common lesions present in many adults with abundant variation; thus, they may be helpful as simple indicators for identification.

5-HT2A Receptor Agonist-Induced Hyperthermia Is Induced via Vasoconstriction by Peripheral 5-HT2A Receptors and Brown Adipose Tissue Thermogenesis by Peripheral Serotonin Loss at a High Ambient Temperature.

Recreational drugs such as 3,4-methylenedioxymethamphetamine and cocaine induce hyperthermia, which is affected by ambient temperature. 2-(4-Bromo-2,5-dimethoxyphenyl)-N-(2-methoxybenzyl)ethanamine (25B-NBOMe), a selective agonist of 5-HT2A receptor used as a recreational drug, reportedly induces hyperthermia. This study aimed to verify whether 25B-NBOMe induces ambient temperature-dependent hyperthermia and to clarify its mechanism. Eight-week-old male Sprague-Dawley rats were administered intraperitoneal injection of 25B-NBOMe at an ambient temperature of 23°C or 29°C. 25B-NBOMe administration at 23°C did not change the core body temperature of the rats, whereas administration at 29°C induced significant hyperthermia 30-120 minutes postadministration. Tail surface temperature temporarily decreased 30 minutes postadministration, indicating heat storage by peripheral vasoconstriction despite a high ambient temperature. Because 25B-NBOMe-induced-hyperthermia was suppressed by sarpogrelate, but not by destruction of central noradrenaline or serotonin neurons, peripheral 5-HT2A receptors were considered contributors to the development of hyperthermia at a high ambient temperature, independently from central neurons. The temperature of brown adipose tissue (BAT) increased 60-120 minutes postadministration of 25B-NBOMe at 29°C, indicating thermogenesis. Previous studies have reported that peripheral serotonin contributes to the inhibition of BAT thermogenesis. Decreased plasma serotonin levels were observed at 29°C, and serotonin administration partially suppressed 25B-NBOMe-induced hyperthermia at a high ambient temperature, suggesting that decreased levels of peripheral serotonin induced BAT thermogenesis. Our findings indicate that 25B-NBOMe induces hyperthermia at a high ambient temperature via vasoconstriction regulated by 5-HT2A receptors and BAT thermogenesis mediated by decreased levels of plasma serotonin. Thus, peripheral serotonin plays a partial but important role in thermoregulation.

Still without Sales Restriction? Fatal Caffeine Toxicity A case report.

Caffeine-containing drinks are popular daily beverages worldwide and highly concentrated caffeine in the form of tablets is easily obtainable in local chemists. It is common to detect caffeine in toxicological screens in autopsy cases, but we always have to take in account that caffeine itself possesses toxicity which may sometimes lead to death The case was a 44-year-old woman who was found dead in her room one day in June. Empty packages of 'Estaron Mocha 12®, accounting for 14 tablets, were found at the scene. The autopsy showed nothing remarkable suggesting external forces, apart from white granules in her stomach. Toxicological analysis revealed 127 mg/L of caffeine concentration in the deceased's blood, which was considered to be fatal. Caffeine intoxication cases have been reported from many countries. Some are caused by accidental overtake of energy drinks, and many others are caused by intentional intake of caffeine tablets. We think that some restrictions will be effective to prevent this kind of death.

Pulmonary arterial hypertension in rats due to age-related arginase activation in intermittent hypoxia.

Pulmonary arterial hypertension (PAH) is prevalent in patients with obstructive sleep apnea syndrome (OSAS). Aging induces arginase activation and reduces nitric oxide (NO) production in the arteries. Intermittent hypoxia (IH), conferred by cycles of brief hypoxia and normoxia, contributes to OSAS pathogenesis. Here, we studied the role of arginase and aging in the pathogenesis of PAH in adult (9-mo-old) and young (2-mo-old) male Sprague-Dawley rats subjected to IH or normoxia for 4 weeks and analyzed them with a pressure-volume catheter inserted into the right ventricle (RV) and by pulsed Doppler echocardiography. Western blot analysis was conducted on arginase, NO synthase isoforms, and nitrotyrosine. IH induced PAH, as shown by increased RV systolic pressure and RV hypertrophy, in adult rats but not in young rats. IH increased expression levels of arginase I and II proteins in the adult rats. IH also increased arginase I expression in the pulmonary artery endothelium and arginase II in the pulmonary artery adventitia. Furthermore, IH reduced pulmonary levels of nitrate and nitrite but increased nitrotyrosine levels in adult rats. An arginase inhibitor (N(ω)-hydroxy-nor-1-arginine) prevented IH-induced PAH and normalized nitrite and nitrate levels in adult rats. IH induced arginase up-regulation and PAH in adult rats, but not in young rats, through reduced NO production. Our findings suggest that arginase inhibition prevents or reverses PAH.

MDMA induces cardiac contractile dysfunction through autophagy upregulation and lysosome destabilization in rats.

The underlying mechanisms of cardiotoxicity of 3,4-methylenedioxymethylamphetamine (MDMA, "ecstasy") abuse are unclear. Autophagy exerts either adaptive or maladaptive effects on cardiac function in various pathological settings, but nothing is known on the role of autophagy in the MDMA cardiotoxicity. Here, we investigated the mechanism through which autophagy may be involved in MDMA-induced cardiac contractile dysfunction. Rats were injected intraperitoneally with MDMA (20mg/kg) or saline. Left ventricular (LV) echocardiography and LV pressure measurement demonstrated reduction of LV systolic contractility 24h after MDMA administration. Western blot analysis showed a time-dependent increase in the levels of microtubule-associated protein light chain 3-II (LC3-II) and cathepsin-D after MDMA administration. Electron microscopy showed the presence of autophagic vacuoles in cardiomyocytes. MDMA upregulated phosphorylation of adenosine monophosphate-activated protein kinase (AMPK) at Thr172, mammalian target of rapamycin (mTOR) at Thr2446, Raptor at Ser792, and Unc51-like kinase (ULK1) at Ser555, suggesting activation of autophagy through the AMPK-mTOR pathway. The effects of autophagic inhibitors 3-methyladenine (3-MA) and chloroquine (CQ) on LC3-II levels indicated that MDMA enhanced autophagosome formation, but attenuated autophagosome clearance. MDMA also induced release of cathepsins into cytosol, and western blotting and electron microscopy showed cardiac troponin I (cTnI) degradation and myofibril damage, respectively. 3-MA, CQ, and a lysosomal inhibitor, E64c, inhibited cTnI proteolysis and improved contractile dysfunction after MDMA administration. In conclusion, MDMA causes lysosome destabilization following activation of the autophagy-lysosomal pathway, through which released lysosomal proteases damage myofibrils and induce LV systolic dysfunction in rat heart.

Immobilization Stress With α2-Adrenergic Stimulation Induces Regional and Transient Reduction of Cardiac Contraction Through Gi Coupling in Rats.

Stress cardiomyopathy is characterized by transient apical hypokinesia related to catecholamine overflow. Recently, excessive epinephrine administration was shown to recapitulate stress cardiomyopathy through ß2-adrenoceptor (AR)-inhibitory G protein (Gi) coupling in rats. We aimed to study whether α2-AR and Gi affect cardiac contraction in rats in which emotional stress was evoked using immobilization (IMO). Echocardiography results showed that when male rats were exposed to IMO for 30 minutes and then injected with the α2-AR agonist xylazine (Xy), ejection fraction and the movement of the anterior wall (AW) were suppressed, maximally at 5 minutes post-injection, whereas posterior wall (PW) movement was preserved. At the same time points, the phosphorylation of Ser282 in myosin-binding protein-C (MyBP-C-Ser282) was higher in the PW than in the AW. Pretreatment with the Gi inhibitor pertussis toxin (PTX) reversed the low contractility and MyBP-C-Ser282 phosphorylation in the AW, but induced lethal heart failure in 3 out of 11 rats. Moreover, at 5 minutes after Xy injection following 30 minutes of IMO, serum epinephrine levels were increased. Thus, in rats exposed to psychological stress, α2-AR stimulation triggered transient hypo-contractility and MyBP-C-Ser282 hypo-phosphorylation in the AW, in association with an epinephrine surge. PTX treatment reversed the AW hypo-contractility and MyBP-C hypo-phosphorylation, but induced acute heart failure. These findings suggest α2AR/Gi-dependent signaling attenuates MyBP-C phosphorylation and contractility in the AW through an epinephrine surge in rats subjected to IMO and α2-AR stimulation. This model can recapitulate stress cardiomyopathy and thereby deepen our understanding of regional cardiac hypo-contractility and prosurvival mechanisms.

Intermittent-hypoxia induced autophagy attenuates contractile dysfunction and myocardial injury in rat heart.

Sleep apnea syndrome (SAS) is considered to be associated with heart failure (HF). It is known that autophagy is induced in various heart diseases thereby promotes survival, but its excess may be maladaptive. Intermittent hypoxia (IH) plays pivotal role in the pathogenesis of SAS. We aimed to clarify the relationships among IH, autophagy, and HF. Rats underwent IH at a rate of 20cycles/h (nadir of 4% O2 to peak of 21% O2 with 0% CO2) or normal air breathing (control) for 8h/d for 3weeks. IH increased the cardiac LC3II/LC3I ratio. The IH induced upregulation of LC3II was attenuated by the administration of an inhibitor of autophagosome formation 3-methyladenine (3-MA), but enhanced by an inhibitor of autophagosome-lysosome fusion chloroquine (CQ), showing enhanced autophagic flux in IH hearts. Electron microscopy confirmed an increase in autophagosomes and lysosomes in IH. With 3-MA or CQ, IH induced progressive deterioration of fractional shortening (FS) on echocardiography over 3weeks, although IH, 3-MA, or CQ alone had no effects. With CQ, IH for 4weeks increased serum troponin T levels, reflecting necrosis. Western blotting analyses showed dephosphorylation of Akt and mammalian target of rapamycin (mTOR) at Akt (Ser2448, 2481) sites, suggesting the activation of autophagy via Akt inactivation. Conclusions. IH-mediated autophagy maintains contractile function, whereas when autophagy is inhibited, IH induces systolic dysfunction due to myocyte necrosis. General significance. This study highlighted the potential implications of autophagy in cardio-protection in early SAS patients without comorbidity, reproduced in normal rats by 3~4weeks of IH.

Immunohistochemical study of the autophagy marker microtubule-associated protein 1 light chain 3 in normal and steatotic human livers.

AIM: Autophagy has been implicated in lipid droplet (LD) turnover. Adipose differentiation-related protein (ADRP) and microtubule-associated protein 1 light chain 3 (LC3) monitor LD and autophagosomes, respectively. We examined whether immunohistochemical staining of ADRP and LC3 can monitor LD and autophagy, and if so, whether autophagy is related to LD turnover in post-mortem human livers. METHODS: We performed conventional immunohistochemistry of LC3 in paraffin-embedded human livers with different severities of steatosis, obtained at autopsy. Double immunofluorescence microscopy using anti-LC3 and anti-ADRP antibodies was performed to elucidate the relationship between autophagy and LD turnover. RESULTS: LC3 immunohistochemistry reproducibly delineated puncta in normal human livers, which were preferentially located around the central venal zone. The extent of LC3 immunostaining reduced with progressing steatosis. Double immunofluorescence for ADRP and LC3 demonstrated an inverse relationship between ADRP positive areas and LC3 positive areas, as well as the co-localization of ADRP and LC3 on a part of small LD but not large LD. CONCLUSION: These findings suggest that impaired autophagy promotes steatosis and that autophagy may be implicated in LD turnover.

Lethal pulmonary air embolism caused by the removal of a double-lumen hemodialysis catheter.

Pulmonary air embolisms due to the removal of a central venous catheter are rare, but catheter removal is known to be a high risk factor for air embolism. In particular, the removal of a large catheter, such as a double-lumen hemodialysis catheter, can allow a large amount of air to enter into the bloodstream, which often results in sudden death. So, during catheter removal, special care should be taken to prevent air from entering blood vessels, for example, to ensure that the patient's head is tilted downward, that they have inhaled and are holding their breath, and that a covering gauze and inert ointment have been applied to the exit site. We report a lethal case of pulmonary air embolism caused by the removal of a double-lumen catheter from the right internal jugular vein of a patient who was sitting up and had not been instructed to hold their breath.

Alveolar adenoma with a single cyst: A case report.

Alveolar adenoma is a rare and benign pulmonary tumor, which originates from type II pneumocytes and is often incidentally identified on radiographic images. Alveolar adenoma presents as a peripleural, solitary and cystic nodule in the lung and may mimic other types of lung tumors, thus rendering its differential diagnosis difficult. Alveolar adenoma is diagnosed based on histopathological and immunohistochemical analyses. The present study describes the case of a 50-year-old male patient with alveolar adenoma. He visited a local doctor ~3 years prior due to left chest pain. A chest computed tomography scan revealed a cystic lesion in segment 8 of the left lung. A nodular shadow appeared in the cyst and gradually increased in size; the patient was thus referred to the authors' hospital. The nodule was well-defined, solitary and solid; thus, lung cancer or aspergilloma were suspected. Thoracoscopic wedge resection was performed as diagnostic therapy. The frozen sections were non-diagnostic, and a pathological examination revealed an alveolar adenoma with no evidence of malignancy and a negative culture. The patient had a good post-operative course, with no sign of recurrence at the follow-up evaluation 46 months later. On the whole, alveolar adenoma is a rare, benign pulmonary tumor that is difficult to diagnose pre-operatively.

Connexin-43 hemichannels contribute to the propagation of µ-calpain-mediated neuronal death in a cortical ablation injury model.

We investigated the role of the astrocytic and neuronal hemichannels (HCs) in the spread of cortical neuronal death in a rat cortical injury model. Over time (by 6 h), propidium iodide (PI)-positive cells with labeling either with anti-neuron specific enolase or anti-parvalbumin (indicating GABAnergic interneurons) antibody spread in the deep cortical layers adjacent to the injury and co-localized with activated µ-calpain. Connexin (Cx)-43, glial fibrillary acidic protein (GFAP), activated µ-calpain and α-fodrin breakdown product (FBP) increased post-injury, peaking at 1 h, in the injury and adjacent areas. GFAP-Cx43-positive reactivated astrocytes exhibited similar distribution to the dead neurons. Cx43 and Cx36 primarily comprise HCs in the astrocyte and neuron, respectively. Ethidium bromide (EtBr) uptake was enhanced post-injury, and confirmed in the Cx43- and Cx36-positive cells. A Cx43-HC inhibitor Gap26 prevented the opening of the Cx43-HC and Cx36-HC, µ-calpain activation, α-fodrin proteolysis and death in the deep cortical neurons. Collectively, opening of the astrocytic Cx43-HC and neuronal Cx36-HC would induce the regional spread of cortical neuronal death through µ-calpain activation in the rat brain injury model.

Decrease in the expression of muscle-specific miRNAs, miR-133a and miR-1, in myoblasts with replicative senescence.

Muscles that are injured or atrophied by aging undergo myogenic regeneration. Although myoblasts play a pivotal role in myogenic regeneration, their function is impaired with aging. MicroRNAs (miRNAs) are also involved in myogenic regeneration. MiRNA (miR)-1 and miR-133a are muscle-specific miRNAs that control the proliferation and differentiation of myoblasts. In this study, we determined whether miR-1 and miR-133a expression in myoblasts is altered with cellular senescence and involved in senescence-impaired myogenic differentiation. C2C12 murine skeletal myoblasts were converted to a replicative senescent state by culturing to a high passage number. Although miR-1 and miR-133a expression was largely induced during myogenic differentiation, expression was suppressed in cells at high passage numbers (passage 10 and/or passage 20). Although the senescent myoblasts exhibited a deterioration of myogenic differentiation, transfection of miR-1 or miR-133a into myoblasts ameliorated cell fusion. Treatment with the glutaminase 1 inhibitor, BPTES, removed senescent cells from C2C12 myoblasts with a high passage number, whereas myotube formation and miR-133a expression was increased. In addition, primary cultured myoblasts prepared from aged C57BL/6J male mice (20 months old) exhibited a decrease in miR-1 and miR-133a levels compared with younger mice (3 months old). The results suggest that replicative senescence suppresses muscle-specific miRNA expression in myoblasts, which contributes to the senescence-related dysfunction of myogenic regeneration.

Application of Teeth in Toxicological Analysis of Decomposed Cadavers Using a Carbamazepine-Administered Rat Model.

In a regular autopsy, blood and organs are used to quantify drug and toxicant concentrations; however, specimens such as blood cannot be collected from highly decomposed corpses, making the quantification of drug and toxicants impossible. This study aimed to estimate the blood carbamazepine (CBZ) concentration from teeth, a part of the human body that is best preserved after death. We sampled teeth and blood of rats administered CBZ. The correlation between the tooth and serum CBZ concentrations was analyzed. Rats were euthanized after CBZ administration and kept at 22 °C for 0 to 15 days before sampling the teeth and measuring the CBZ concentration. Undecalcified, fresh, frozen sections of rat teeth were prepared, and CBZ localization was evaluated. CBZ concentrations in both teeth and cardiac blood peaked at 60 min after administration and increased in a dose-dependent manner. CBZ concentration in teeth did not substantially change after death, with high CBZ distribution being observed in the pulp cavity. The tooth and serum CBZ concentrations were highly correlated, suggesting that the measurement of toxicant concentration in sampled teeth would allow for the estimation of blood toxicant concentration in highly decomposed corpses.

Overcoming the Interobserver Variability in Lung Adenocarcinoma Subtyping: A Clustering Approach to Establish a Ground Truth for Downstream Applications.

CONTEXT.­: The accurate identification of different lung adenocarcinoma histologic subtypes is important for determining prognosis but can be challenging because of overlaps in the diagnostic features, leading to considerable interobserver variability. OBJECTIVE.­: To provide an overview of the diagnostic agreement for lung adenocarcinoma subtypes among pathologists and to create a ground truth using the clustering approach for downstream computational applications. DESIGN.­: Three sets of lung adenocarcinoma histologic images with different evaluation levels (small patches, areas with relatively uniform histology, and whole slide images) were reviewed by 17 international expert lung pathologists and 1 pathologist in training. Each image was classified into one or several lung adenocarcinoma subtypes. RESULTS.­: Among the 4702 patches of the first set, 1742 (37%) had an overall consensus among all pathologists. The overall Fleiss κ score for the agreement of all subtypes was 0.58. Using cluster analysis, pathologists were hierarchically grouped into 2 clusters, with κ scores of 0.588 and 0.563 in clusters 1 and 2, respectively. Similar results were obtained for the second and third sets, with fair-to-moderate agreements. Patches from the first 2 sets that obtained the consensus of the 18 pathologists were retrieved to form consensus patches and were regarded as the ground truth of lung adenocarcinoma subtypes. CONCLUSIONS.­: Our observations highlight discrepancies among experts when assessing lung adenocarcinoma subtypes. However, a subsequent number of consensus patches could be retrieved from each cluster, which can be used as ground truth for the downstream computational pathology applications, with minimal influence from interobserver variability.

Ischemia-reperfusion induces myocardial infarction through mitochondrial Ca²âº overload.

Both mitochondria and the sarcoplasmic reticulum (SR) are essential for myocardial homeostasis and control of cardiac function. Uptake of Ca(2+) from the cytosol into SR is mediated by the Ca(2+)-dependent ATPase SERCA2a, which is reversibly inhibited by phospholamban (PLN). We previously showed that removal of PLN inhibition of SERCA2a with an antibody to (anti-) PLN reduces cytosolic Ca(2+) overload, thereby attenuating the spread of contraction bands and fodrin proteolysis, during reperfusion after cardiac ischemia. We have now examined the effects of anti-PLN injection into the heart on the development of myocardial infarction (MI) after ischemia-reperfusion in rats. Whereas anti-PLN injection attenuated cytosolic Ca(2+) overload, it did not affect MI size 6h after the onset of reperfusion and actually increased it at 30 min. The antibody also increased the release of apoptosis-inducing factor (AIF) from mitochondria into the cytosol, indicative of enhanced opening of the mitochondrial permeability transition pore (mPTP). Administration of an mPTP blocker at the time of reperfusion or of a blocker of the mitochondrial Ca(2+) uniporter significantly suppressed the release of AIF and the development of MI. These results indicate that the enhancement of SR Ca(2+) loading by anti-PLN injection facilitated Ca(2+) uniporter-dependent mitochondrial Ca(2+) uptake and thereby induced mPTP opening and MI development during early reperfusion. The enhancement of SR Ca(2+) loading thus aggravates MI in a manner independent of cytosolic Ca(2+) overload. Given that cytosolic Ca(2+) overload induces contraction bands, our findings are inconsistent with a causal relation between contraction bands and MI.

Ischemia induces phospholamban dephosphorylation via activation of calcineurin, PKC-α, and protein phosphatase 1, thereby inducing calcium overload in reperfusion.

Cardiac sarcoplasmic reticulum (SR) Ca(2+) ATPase (SERCA2a) promotes Ca(2+) uptake in the SR. Dephosphorylated phospholamban (PLB) inhibits SERCA2a activity. We found a distinct dephosphorylation of PLB at Thr(17) and Ser(16) after 20-30min of ischemia produced by coronary artery occlusion in rats. The aim of the study was to investigate how PLB is dephosphorylated in ischemia and to determine whether PLB dephosphorylation causes myocardial hypercontraction and calpain activation through Ca(2+) overload in reperfusion. Protein inhibitor-1 (I-1) specifically inhibits protein phosphatase 1 (PP1), the predominant PLB phosphatase in heart. A Ca(2+)-dependent phosphatase calcineurin may also induce PLB dephosphorylation. Ischemia for 30min induced PKC-α translocation, resulting in inactivation of I-1 through PKC-α-dependent phosphorylation at Ser(67). The PP1 activation following I-1 inactivation was thought to induce PLB dephosphorylation in ischemia. Ischemia for 30min activated calcineurin, and pre-treatment with a calcineurin inhibitor, cyclosporine A (CsA), inhibited PKC-α translocation, I-1 phosphorylation at Ser(67), and PLB dephosphorylation in ischemia. Reperfusion for 5min following 30min of ischemia induced spreading of contraction bands (CBs) and proteolysis of fodrin by calpain. Both CsA and an anti-PLB antibody that inhibits binding of PLB to SERCA2a reduced the CB area and fodrin breakdown after reperfusion. These results reveal a novel pathway via which ischemia induces calcineurin-dependent activation of PKC-α, inactivation of I-1 through PKC-α-dependent phosphorylation at Ser(67), and PP1-dependent PLB dephosphorylation. The pathway contributes to the spreading of CBs and calpain activation through Ca(2+) overload in early reperfusion.