Kaori Shintani-Ishida

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.

Ischemia–reperfusion induces myocardial infarction through mitochondrial Ca2+ 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.

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

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.

Determination of amobarbital and phenobarbital in serum by gas chromatography–mass spectrometry with addition of formic acid to the solvent

A rapid and accurate method for quantification of amobarbital and phenobarbital was developed using gas chromatography-mass spectrometry (GC-MS) without derivatization. Though the compounds measured without derivatization showed low sensitivity because of adsorption, addition of 3% formic acid to the solvent improved the sensitivity for the analytes. Taking account of matrix effect, solid-phase and liquid-liquid extraction from serum were examined. The correlation coefficients of the calibration curves were 0.9995 or better, and the accuracy and precision of intraday and interday assays were in line with Food and Drug Administration (FDA) criteria.

Brief exposure to carbon monoxide preconditions cardiomyogenic cells against apoptosis in ischemia-reperfusion

We examined whether and how pretreatment with carbon monoxide (CO) prevents apoptosis of cardioblastic H9c2 cells in ischemia-reperfusion. Reperfusion (6 h) following brief ischemia (10 min) induced cytochrome c release, activation of caspase-9 and caspase-3, and apoptotic nuclear condensation. Brief CO pretreatment (10 min) or a caspase-9 inhibitor (Z-LEHD-FMK) attenuated these apoptotic changes. Ischemia-reperfusion increased phosphorylation of Akt at Ser472/473/474, and this was enhanced by CO pretreatment. A specific Akt inhibitor (API-2) blunted the anti-apoptotic effects of CO in reperfusion. In normoxic cells, CO enhanced O2(-) generation, which was inhibited by a mitochondrial complex III inhibitor (antimycin A) but not by a NADH oxidase inhibitor (apocynin). The CO-enhanced Akt phosphorylation was suppressed by an O2(-) scavenger (Tiron), catalase or a superoxide dismutase (SOD) inhibitor (DETC). These results suggest that CO pretreatment induces mitochondrial generation of O2(-), which is then converted by SOD to H2O2, and subsequent Akt activation by H2O2 attenuates apoptosis in ischemia-reperfusion.

Mitochondrial m-calpain opens the mitochondrial permeability transition pore in ischemia–reperfusion

BACKGROUND/OBJECTIVES Opening of the mitochondrial permeability transition pore (mPTP) is involved in ischemia-reperfusion injury. Isoforms of Ca(2+)-activated cysteine proteases, calpains, are implicated in the development of myocardial infarction in ischemia-reperfusion. Growing evidence has revealed the presence of calpains in the mitochondria. We aimed to characterize mitochondrial calpains in the rat heart and to investigate the roles of calpains in mPTP opening after ischemia-reperfusion. METHODS AND RESULTS Western blotting analysis showed the expression of μ-calpain, m-calpain and calpain 10 in mitochondria isolated from male Sprague-Dawley rats, but casein zymography detected only m-calpain activity. Subcellular fractionation of mitochondria demonstrated the distribution of m-calpain to the matrix fraction. Addition of >500μM of Ca(2+) to isolated mitochondria induced mitochondrial swelling, reflecting mPTP opening, and calpain activation. Ca(2+)-induced mitochondrial swelling was inhibited partially by the calpain inhibitor calpeptin. These results support a partial contribution of calpain in the opening of the mPTP. The addition of Ca(2+) to the mitochondria induced inactivation of complex I of the electron transport chain, and cleavage of the ND6 complex I subunit, which were inhibited by calpeptin. Mitochondria isolated from rat hearts that underwent 30min of coronary occlusion followed by 30min of reperfusion showed activation of mitochondrial calpains, ND6 cleavage, complex I inactivation, and mPTP opening, which were inhibited by pretreatment with calpain inhibitor 1. CONCLUSIONS We demonstrated for the first time the presence of mitochondrial matrix m-calpain, and its contribution to complex I inactivation and mPTP opening after postischemic reperfusion in the rat heart.

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.

A Death Due to Perirenal Hematoma Complicating Extracorporeal Shockwave Lithotripsy

Abstract:  Perirenal hematoma is an occasional complication of extracorporeal shockwave lithotripsy (ESWL) which does not usually require treatment. A 79‐year‐old woman died 23 h after ESWL. Forensic autopsy was performed to determine whether medical treatment contributed to her death. The cause of death was hemorrhagic shock due to massive hematoma from a ruptured small vein in the perirenal adipose capsule. No injury to other organs was found and the patient had neither coagulation abnormality nor venous disease. Perirenal hematoma can easily be diagnosed with abdominal sonography, if pain or symptoms of anemia develop. Doctors must be aware of the possibilities of severe renal hematomas after ESWL.

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 patients 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.

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 6h), 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 1h, in the injury and adjacent areas. GFAP-Cx43-positive reactivatedastrocytes 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.