Cardioprotection by remote ischemic preconditioning of the rat heart is mediated by extracellular vesicles

Remote ischemic preconditioning (RIPC) of the heart is exerted by brief ischemic insults affected on a remote organ or a remote area of the heart before a sustained cardiac ischemia. To date, little is known about the inter-organ transfer mechanisms of cardioprotection by RIPC. Exosomes and microvesicles/microparticles are vesicles of 30-100nm and 100-1000nm in diameter, respectively (collectively termed extracellular vesicles [EVs]). Their content of proteins, mRNAs and microRNAs, render EVs ideal conveyors of inter-organ communication. However, whether EVs are involved in RIPC, is unknown. Therefore, here we investigated whether (1) IPC induces release of EVs from the heart, and (2) EVs are necessary for cardioprotection by RIPC. Hearts of male Wistar rats were isolated and perfused in Langendorff mode. A group of donor hearts was exposed to 3x5-5min global ischemia and reperfusion (IPC) or 30min aerobic perfusion, while coronary perfusates were collected. Coronary perfusates of these hearts were given to another set of recipient isolated hearts. A group of recipient hearts received IPC effluent depleted of EVs by differential ultracentrifugation. Infarct size was determined after 30min global ischemia and 120min reperfusion. The presence or absence of EVs in perfusates was confirmed by dynamic light scattering, the EV marker HSP60 Western blot, and electron microscopy. IPC markedly increased EV release from the heart as assessed by HSP60. Administration of coronary perfusate from IPC donor hearts attenuated infarct size in non-preconditioned recipient hearts (12.9+/-1,6% vs. 25.0+/-2.7%), similarly to cardioprotection afforded by IPC (7.3+/-2.7% vs. 22.1+/-2.9%) on the donor hearts. Perfusates of IPC hearts depleted of EVs failed to exert cardioprotection in recipient hearts (22.0+/-2.3%). This is the first demonstration that EVs released from the heart after IPC are necessary for cardioprotection by RIPC, evidencing the importance of vesicular transfer mechanisms in remote cardioprotection

NOXA contributes to the sensitivity of PERK-deficient cells to ER stress.

PKR-like ER kinase (PERK) deficient mouse embryonic fibroblasts (MEFs) are hypersensitive to ER stress-induced apoptosis. However, the molecular determinants of increased sensitivity of PERK(-/-) MEFs are not clearly understood. Here we show that induction of several Unfolded Protein Response (UPR) target genes is attenuated in PERK(-/-) MEFs. We also report elevated expression of the BH3-only protein, NOXA in PERK(-/-) MEFs. Further, shRNA-mediated knockdown of NOXA rescued the hypersensitivity of PERK(-/-) MEFs to ER stress-induced apoptosis. Taken together our results suggest that compromised induction of UPR and increased NOXA expression contributes to hypersensitivity of PERK(-/-) MEFs to ER stress-induced apoptosis

A szívizom sztressz-adaptációja: a peroxinitrit, a mátrix metalloproteinázok, és a hiperlipidémia szerepe = Stress adaptation of the myocardium: role of peroxynitrite, matrix metalloproteinases, and hyperlipidemia

A hiperlipidémia talaján kialakuló iszkémiás szívbetegség a leggyakoribb halálokok közé tartozik. A 4 éves project során a szívizom iszkémiának és az iszkémiás stressz adaptációs képességének (iszkémiás pre- és posztkondíció) celluláris mechanizmusait vizsgáltuk állatkísérletekben, különösképpen a peroxinitrit és celluláris targetjének, az MMP2-nek a szerepét. Új eredményeink közül néhányat emelünk ki. Kimutattuk, hogy hiperlipidemiában a szívben a peroxitrit képződés és ezáltal az MMP-2 aktivitása fokozódik, ami különösen hiperlipidmémiában jelentős, és ezt a folyamatot a prékondíció gátolja. DNA-chip vizsgálattal feltérképeztük hiperlipidémia hatására a génkifejeződés változásait a szívizomban. Kimutattuk, hogy az alacsony mértékű peroxinitrit képződés a stessz adaptáció kiváltásában igen fontos szerepet tölt be, hiszen olyan mechanizmusokat aktivál, melyek az iszkémiás stressz során túlzott mértékű peroxinitrit-MMP aktivitást csökkenti. Leírtuk, hogy nemspecifikus MMP gátlókkal az infarktus területe csökkenthető még hiperlipidémiás állatban is. Humán ApoB100 transzgenetikus eger modelleken megfigyeltük, hogy az oxidatív/nitrozatív stressz oka a hiperkoleszterinémia, és nem a hipertrigliceridémia. Kimutattuk továbbá, hogy a fiziológiás peroxinitrit szint, melyet a szívizom kapszaicin-érzékeny neuronjai szabályoznak, a normális szívizom relaxációt tartja fent. A project futamideje alatt az adott témában összesen 16 nemzetközi cikket (impakt faktor >70) közöltünk. | Ischemic heart disease developing due to hyperlipidemia is the number one killer in civilized societies. The present 4-year project was aiming at exploration of cellular mechanisms underlying stress adaptation of the myocardium, i.e. pre- and postconditioning, focusing on the role of peroxynitrite and its cellular target matrix metalloproteinase-2 (MMP2). Here we emphasize only some of the most important results of the project. We have shown that in hyperlipidemia, myocardial peroxynitrite formation and thereby MMP2 activity is increased, which is attenuated by preconditioning. We have mapped the changes in gene expression due to hyperlipidemia by the use of DNA-microarray assay. We have observed that moderate peroxynitrite formation is necessary to trigger the stress adaptation mechanisms, which in turn will decrease the pathological activation of the peroxynitrite-MMP2 signaling. We have shown that nonspecific MMP inhibitors are able to reduce infarct size even in the presence of hyperlipidemia. In human ApoB-100 transgene mice, we have observed that oxidative/nitrosative stress is due to hypercholesterolemia and not hypertriglyceridemia. Furthermore, we have shown that baseline physiological peroxynitrite formation, which is regulated by myocardial capsaicin-sensitive sensory nerves, plays an important role in the maintenance of normal relaxation of the myocardium. The present project yielded altogether 16 peer-reviewed papers (impact factor >70)

Cardiac miRNA Expression and their mRNA Targets in a Rat Model of Prediabetes

Little is known about the mechanism of prediabetes-induced cardiac dysfunction. Therefore, we aimed to explore key molecular changes with transcriptomic and bioinformatics approaches in a prediabetes model showing heart failure with preserved ejection fraction phenotype. To induce prediabetes, Long-Evans rats were fed a high-fat diet for 21 weeks and treated with a single low-dose streptozotocin at week 4. Small RNA-sequencing, in silico microRNA (miRNA)-mRNA target prediction, Gene Ontology analysis, and target validation with qRT-PCR were performed in left ventricle samples. From the miRBase-annotated 752 mature miRNA sequences expression of 356 miRNAs was detectable. We identified two upregulated and three downregulated miRNAs in the prediabetic group. We predicted 445 mRNA targets of the five differentially expressed miRNAs and selected 11 mRNAs targeted by three differentially expressed miRNAs, out of which five mRNAs were selected for validation. Out of these five targets, downregulation of three mRNAs i.e., Juxtaposed with another zinc finger protein 1 (Jazf1); RAP2C, member of RAS oncogene family (Rap2c); and Zinc finger with KRAB and SCAN domains 1 (Zkscan1) were validated. This is the first demonstration that prediabetes alters cardiac miRNA expression profile. Predicted targets of differentially expressed miRNAs include Jazf1, Zkscan1, and Rap2c mRNAs. These transcriptomic changes may contribute to the diastolic dysfunction and may serve as drug targets

Hypercholesterolemia downregulates autophagy in the rat heart

Background: We have previously shown that efficiency of ischemic conditioning is diminished in hypercholesterolemia and that autophagy is necessary for cardioprotection. However, it is unknown whether isolated hypercholesterolemia disturbs autophagy or the mammalian target of rapamycin (mTOR) pathways. Therefore, we investigated whether isolated hypercholesterolemia modulates cardiac autophagy-related pathways or programmed cell death mechanisms such as apoptosis and necroptosis in rat heart. Methods: Male Wistar rats were fed either normal chow (NORM; n=9) or with 2% cholesterol and 0.25% cholic acid-enriched diet (CHOL; n=9) for 12 weeks. CHOL rats exhibited a 41% increase in plasma total cholesterol level over that of NORM rats (4.09mmol/L vs. 2.89mmol/L) at the end of diet period. Animals were sacrificed, hearts were excised and briefly washed out. Left ventricles were snap-frozen for determination of markers of autophagy, mTOR pathway, apoptosis, and necroptosis by Western blot. Results: Isolated hypercholesterolemia was associated with a significant reduction in expression of cardiac autophagy markers such as LC3-II, Beclin-1, Rubicon and RAB7 as compared to controls. Phosphorylation of ribosomal S6, a surrogate marker for mTOR activity, was increased in CHOL samples. Cleaved caspase-3, a marker of apoptosis, increased in CHOL hearts, while no difference in the expression of necroptotic marker RIP1, RIP3 and MLKL was detected between treatments. Conclusions: This is the first comprehensive analysis of autophagy and programmed cell death pathways of apoptosis and necroptosis in hearts of hypercholesterolemic rats. Our data show that isolated hypercholesterolemia suppresses basal cardiac autophagy and that the decrease in autophagy may be a result of an activated mTOR pathway. Reduced autophagy was accompanied by increased apoptosis, while cardiac necroptosis was not modulated by isolated hypercholesterolemia. Decreased basal autophagy and elevated apoptosis may be responsible for the loss of cardioprotection reported in hypercholesterolemic animals

Anti-PD-1 Therapy Does Not Influence Hearing Ability in the Most Sensitive Frequency Range, but Mitigates Outer Hair Cell Loss in the Basal Cochlear Region

The administration of immune checkpoint inhibitors (ICIs) often leads to immune-related adverse events. However, their effect on auditory function is largely unexplored. Thorough preclinical studies have not been published yet, only sporadic cases and pharmacovigilance reports suggest their significance. Here we investigated the effect of anti-PD-1 antibody treatment (4 weeks, intraperitoneally, 200 μg/mouse, 3 times/week) on hearing function and cochlear morphology in C57BL/6J mice. ICI treatment did not influence the hearing thresholds in click or tone burst stimuli at 4-32 kHz frequencies measured by auditory brainstem response. The number and morphology of spiral ganglion neurons were unaltered in all cochlear turns. The apical-middle turns (32 kHz). The number of Iba1-positive macrophages has also increased moderately in this high frequency region. We conclude that a 4-week long ICI treatment does not affect functional and morphological integrity of the inner ear in the most relevant hearing range (4-32 kHz; apical-middle turns), but a noticeable preservation of OHCs and an increase in macrophage activity appeared in the >32 kHz basal part of the cochlea