The role of ferroptosis in cell-to-cell propagation of cell death initiated from focal injury in cardiomyocytes.

AIMS: Ferroptosis has grown in importance as a key factor in ischemia-reperfusion (I/R) injury. This study explores the mechanism underlying fibrotic scarring extending along myofibers in cardiac ischemic injury and demonstrates the integral role of ferroptosis in causing a unique cell death pattern linked to I/R injury. MAIN METHODS: Cadaveric hearts from individuals who had ischemic injury were examined by histological assays. We created a novel model of inducing cell death in H9c2 cells, and used it to demonstrate ferroptotic cell death extending in a cell-to-cell manner. Ex vivo Langendorff-perfused hearts were used alongside the model to replicate cell death extension along myofibers while also demonstrating protective effects of a ferroptosis inhibitor, ferrostatin-1 (Fer-1). KEY FINDINGS: Human hearts from individuals who had I/R injury demonstrated scarring along myofibers that was consistent with mouse models, suggesting that cell death extended from cell-to-cell. Treatment with Ras-selective lethal 3 (RSL3), a ferroptosis inducer, and exposure to excess iron exacerbated cell death propagation in in vitro models, and inhibition of ferroptosis by Fer-1 blunted this effect in both settings. In ex vivo models, Fer-1 was sufficient to reduce cell death along the myofibers caused by external injury. SIGNIFICANCE: The unique I/R injury-induced pattern of cell death along myofibers requires novel injury models that mimic this phenomenon, thus we established new methods to replicate it. Ferroptosis is important in propagating injury between cells and better understanding this mechanism may lead to therapeutic responses that limit I/R injury.

mTOR-mediated calcium transients affect cardiac function in ex vivo ischemia-reperfusion injury.

The mechanistic target of rapamycin (mTOR) is a key mediator of energy metabolism, cell growth, and survival. While previous studies using transgenic mice with cardiac-specific overexpression of mTOR (mTOR-Tg) demonstrated the protective effects of cardiac mTOR against ischemia-reperfusion (I/R) injury in both ex vivo and in vivo models, the mechanisms underlying the role of cardiac mTOR in cardiac function following I/R injury are not well-understood. Torin1, a pharmacological inhibitor of mTOR complex (mTORC) 1 and mTORC2, significantly decreased functional recovery of LV developed pressure in ex vivo I/R models (p < 0.05). To confirm the role of mTOR complexes in I/R injury, we generated cardiac-specific mTOR-knockout (CKO) mice. In contrast to the effects of Torin1, CKO hearts recovered better after I/R injury than control hearts (p < 0.05). Interestingly, the CKO hearts had exhibited irregular contractions during the reperfusion phase. Calcium is a major factor in Excitation-Contraction (EC) coupling via Sarcoplasmic Reticulum (SR) calcium release. Calcium is also key in opening the mitochondrial permeability transition pore (mPTP) and cell death following I/R injury. Caffeine-induced SR calcium release in isolated CMs showed that total SR calcium content was lower in CKO than in control CMs. Western blotting showed that a significant amount of mTOR localizes to the SR/mitochondria and that GSK3-ß phosphorylation, a key factor in SR calcium mobilization, was decreased. These findings suggest that cardiac mTOR located to the SR/mitochondria plays a vital role in EC coupling and cell survival in I/R injury.

The effects of Tel2 on cardiomyocyte survival.

AIMS: Overexpression of the mechanistic target of rapamycin (mTOR), a member of the PIKK (phosphoinositide kinase-related kinase) family, protects cardiomyocytes from cell death induced by pathological stimuli such as ischemia. We previously reported that posttranslational modification of mTOR plays an important role in regulating cardiac mTOR expression. The aim of this study was to see if Tel2 (telomere maintenance 2), a protein that regulates the abundance of PIKKs, confers similar cardioprotective effects as mTOR. Tel2 is not well-characterized in cardiomyocytes, therefore we examined the effects of Tel2 on cardiomyocyte viability under ischemic stress conditions. MATERIALS AND METHODS: We overexpressed Tel2 or silenced Tel2 with siRNA in the HL-1 cardiomyocyte cell line to survey the effects of Tel2 overexpression and downregulation on cell survival during hypoxia. Adult mouse cardiomyocytes transfected with Tel2 adenoviruses were used to test whether Tel2 sufficiently prevented cardiomyocyte cell death against hydrogen peroxide (H2O2). KEY FINDINGS: Overexpressing Tel2 increased mTOR expression with a concomitant increase in mTOR Complex 1 (mTORC1) and mTORC2 activity in HL-1 cells. Tel2 deletion decreased mTOR expression, and mTORC1 and mTORC2 activity accordingly. In both HL-1 cells and adult mouse cardiomyocytes, Tel2 overexpression protected cardiomyocytes under ischemic stress. These effects were mTOR-dependent, as mTOR inhibitors blunted the effects of Tel2. While gene silencing of Tel2 did not affect cell survival under normoxia, Tel2 silencing made cardiomyocytes more vulnerable to cell death under hypoxia. SIGNIFICANCE: Upregulating Tel2 expression increases mTOR-mediated cardiomyocyte survival and targeting Tel2 could be another therapeutic strategy against ischemic heart disease.

Cardiac mTOR rescues the detrimental effects of diet-induced obesity in the heart after ischemia-reperfusion.

Diet-induced obesity deteriorates the recovery of cardiac function after ischemia-reperfusion (I/R) injury. While mechanistic target of rapamycin (mTOR) is a key mediator of energy metabolism, the effects of cardiac mTOR in ischemic injury under metabolic syndrome remains undefined. Using cardiac-specific transgenic mice overexpressing mTOR (mTOR-Tg mice), we studied the effect of mTOR on cardiac function in both ex vivo and in vivo models of I/R injury in high-fat diet (HFD)-induced obese mice. mTOR-Tg and wild-type (WT) mice were fed a HFD (60% fat by calories) for 12 wk. Glucose intolerance and insulin resistance induced by the HFD were comparable between WT HFD-fed and mTOR-Tg HFD-fed mice. Functional recovery after I/R in the ex vivo Langendorff perfusion model was significantly lower in HFD-fed mice than normal chow diet-fed mice. mTOR-Tg mice demonstrated better cardiac function recovery and had less of the necrotic markers creatine kinase and lactate dehydrogenase in both feeding conditions. Additionally, mTOR overexpression suppressed expression of proinflammatory cytokines, including IL-6 and TNF-α, in both feeding conditions after I/R injury. In vivo I/R models showed that at 1 wk after I/R, HFD-fed mice exhibited worse cardiac function and larger myocardial scarring along myofibers compared with normal chow diet-fed mice. In both feeding conditions, mTOR overexpression preserved cardiac function and prevented myocardial scarring. These findings suggest that cardiac mTOR overexpression is sufficient to prevent the detrimental effects of diet-induced obesity on the heart after I/R, by reducing cardiac dysfunction and myocardial scarring.