Apoptotic cell death of cardiomyocytes is a characteristic hallmark of ischemia–reperfusion (I/R) injury. The master hypoxamiR, microRNA-210 (miR-210), is considered the primary driver of the cellular response to hypoxic stress. However, to date, no consensus has emerged with regards to the polarity of the miR-210-elicited cellular response, as miR-210 has been shown to exacerbate as well as attenuate hypoxia-driven apoptotic cell death. Herein, in AC-16 cardiomyocytes subjected to hypoxia-reoxygenation (H-R) stress, we unravel novel facets of miR-210 biology and resolve the biological response mediated by miR-210 into the hypoxia and reoxygenation temporal components. Using transient overexpression and decoy/inhibition vectors to modulate miR-210 expression, we elucidated a Janus role miR-210 in the cellular response to H-R stress, wherein miR-210 mitigated the hypoxia-induced apoptotic cell death but exacerbated apoptotic cell death during cellular reoxygenation. We further delineated the underlying cellular mechanisms that confer this diametrically opposite effect of miR-210 on apoptotic cell death. Our exhaustive biochemical assays cogently demonstrate that miR-210 attenuates the hypoxia-driven intrinsic apoptosis pathway, while significantly augmenting the reoxygenation-induced caspase-8-mediated extrinsic apoptosis pathway. Our study is the first to unveil this Janus role of miR-210 and to substantiate the cellular mechanisms that underlie this functional duality.

Apoptotic cell death is a deleterious consequence of hypoxia-induced cellular stress. The master hypoxamiR, microRNA-210 (miR-210), is considered the primary driver of the cellular response to hypoxia stress. We have recently demonstrated that miR-210 attenuates hypoxia-induced apoptotic cell death. In this paper, we unveil that the miR-210-induced inhibition of the serine/threonine kinase Glycogen Synthase Kinase 3 beta (GSK3β) in AC-16 cardiomyocytes subjected to hypoxia stress underlies the salutary protective response of miR-210 in mitigating the hypoxia-induced apoptotic cell death. Using transient overexpression vectors to augment miR-210 expression concomitant with the ectopic expression of the constitutive active GSK3β S9A mutant (ca-GSK3β S9A), we exhaustively performed biochemical and molecular assays to determine the status of the hypoxia-induced intrinsic apoptosis cascade. Caspase-3 activity analysis coupled with DNA fragmentation assays cogently demonstrate that the inhibition of GSK3β kinase activity underlies the miR-210-induced attenuation in the hypoxia-driven apoptotic cell death. Further elucidation and delineation of the upstream cellular events unveiled an indispensable role of the inhibition of GSK3β kinase activity in mediating the miR-210-induced mitigation of the hypoxia-driven BAX and BAK insertion into the outer mitochondria membrane (OMM) and the ensuing Cytochrome C release into the cytosol. Our study is the first to unveil that the inhibition of GSK3β kinase activity is indispensable in mediating the miR-210-orchestrated protective cellular response to hypoxia-induced apoptotic cell death.

Following myocardial infarction, reperfusion injury (RI) is commonly observed due to the excessive formation of, e.g., reactive oxygen species (ROS). Doxorubicin (DOX), a widely used anti-cancer drug, is also known to cause cardiotoxicity due to excessive ROS production. Exercise training has been shown to protect the heart against both RI- and DOX-induced cardiotoxicity, but the exact mechanism is still unknown. Neuron-derived orphan receptor 1 (NOR-1) is an important exercise-responsive protein in the skeletal muscle which has also been reported to facilitate cellular survival during hypoxia. Therefore, we hypothesized that NOR-1 could protect cardiomyocytes (CMs) against cellular stress induced by DOX. We also hypothesized that NOR-1 is involved in preparing the CMs against a stress situation during nonstimulated conditions by increasing cell viability. To determine the protective effect of NOR-1 in CMs stressed with DOX challenge, we overexpressed NOR-1 in AC16 human CMs treated with 5 µM DOX for 12 h or the respective vehicle control, followed by performing Lactate dehydrogenase (LDH) activity, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and caspase-3 activity assays to measure cell death, cell viability, and apoptosis, respectively. In addition, Western blotting analysis was performed to determine the expression of key proteins involved in cardioprotection. We demonstrated that NOR-1 overexpression decreased cell death (p < 0.105) and apoptosis (p < 0.01) while increasing cell viability (p < 0.05) in DOX-treated CMs. We also observed that NOR-1 overexpression increased phosphorylation of extracellular signal-regulated kinase (ERK) (p < 0.01) and protein expression levels of B cell lymphoma extra-large (Bcl-xL) (p < 0.01). We did not detect any significant changes in phosphorylation of protein kinase B (Akt), glycogen synthase kinase-3β (GSK-3β) and signal transducer and activator of transcription 3 (STAT3) or expression levels of superoxide dismutase 2 (SOD2) and cyclin D1. Furthermore, we demonstrated that NOR-1 overexpression increased the cell viability (p < 0.0001) of CMs during nonstimulated conditions without affecting cell death or apoptosis. Our findings indicate that NOR-1 could serve as a potential cardioprotective protein in response to Doxorubicin-induced cellular stress.

Experimental evidence, both in vitro and in vivo, has indicated cardioprotective effects of extracellular vesicles (EVs) derived from various cell types, including induced pluripotent stem cell-derived cardiomyocytes. The biological effects of EV secretion, particularly in the context of ischemia and cardiac electrophysiology, remain to be fully explored. Therefore, the goal of this study was to unveil the effects of exosome (EXO)-mediated cell–cell signaling during hypoxia by employing a simulated preconditioning approach on human-induced pluripotent stem cell-derived cardiomyocytes (hIPSC-CMs). Electrophysiological activity of hIPSC-CMs was measured using a multielectrode array (MEA) system. A total of 16 h of hypoxic stress drastically increased the beat period. Moreover, hIPSC-CMs preconditioned with EXOs displayed significantly longer beat periods compared with non-treated cells after 16 h of hypoxia (+15.7%, p < 0.05). Furthermore, preconditioning with hypoxic EXOs resulted in faster excitation–contraction (EC) coupling compared with non-treated hIPSC-CMs after 16 h of hypoxia (−25.3%, p < 0.05). Additionally, microRNA (miR) sequencing and gene target prediction analysis of the non-treated and pre-conditioned hIPSC-CMs identified 10 differentially regulated miRs and 44 gene targets. These results shed light on the intricate involvement of miRs, emphasizing gene targets associated with cell survival, contraction, apoptosis, reactive oxygen species (ROS) regulation, and ion channel modulation. Overall, this study demonstrates that EXOs secreted by hIPSC-CM during hypoxia beneficially alter electrophysiological properties in recipient cells exposed to hypoxic stress, which could play a crucial role in the development of targeted interventions to improve outcomes in ischemic heart conditions.

Ischemia–reperfusion (I-R) injury is a cardinal pathophysiological hallmark of ischemic heart disease (IHD). Despite significant advances in the understanding of what causes I-R injury and hypoxia–reoxygenation (H-R) stress, viable molecular strategies that could be targeted for the treatment of the deleterious biochemical pathways activated during I-R remain elusive. The master hypoxamiR, microRNA-210 (miR-210), is a major determinant of protective cellular adaptation to hypoxia stress but exacerbates apoptotic cell death during cellular reoxygenation. While the hypoxia-induced transcriptional up-regulation of miR-210 is well delineated, the cellular mechanisms and molecular entities that regulate the transcriptional induction of miR-210 during the cellular reoxygenation phase have not been elucidated yet. Herein, in immortalized AC-16 cardiomyocytes, we delineated the indispensable role of the ubiquitously expressed transcription factor, NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) in H-R-induced miR-210 expression during cellular reoxygenation. Using dominant negative and dominant active expression vectors encoding kinases to competitively inhibit NF-κB activation, we elucidated NF-κB activation as a significant mediator of H-R-induced miR-210 expression. Ensuing molecular assays revealed a direct NF-κB-mediated transcriptional up-regulation of miR-210 expression in response to the H-R challenge that is characterized by the NF-κB-mediated reorchestration of the entire repertoire of histone modification changes that are a signatory of a permissive actively transcribed miR-210 promoter. Our study confers a novel insight identifying NF-κB as a potential novel molecular target to combat H-R-elicited miR-210 expression that fosters augmented cardiomyocyte cell death.

Small extracellular vesicles (sEVs) are increasingly recognized as crucial mediators of cell-cell communication. This study aims to determine the influence of sEV mediation on cardiomyocytes (CMs) under hypoxic conditions and identify the molecular modifications induced by hypoxia-derived sEVs (H-sEVs). Using a preconditional approach, we administered hypoxic AC16 CM-derived sEVs to recipient AC16 CMs before hypoxic stimulation. Molecular and biochemical analyses were performed to evaluate the biological effects of H-sEVs, e.g., cell viability assay, caspase assays, Western blotting, and quantitative sandwich ELISA. Our results showed a significant decrease in CM viability following hypoxic stimulation, as well as elevated caspase-3 and caspase-8 activity, and increased Bcl-2-associated X protein (BAX) translocation to the mitochondria followed by an increased release of Cytochrome C from mitochondria to cytosol. Preconditioning with H-sEVs further exacerbated caspase-3 and caspase-8 activation, which was explained by aggravated translocation of BAX to the mitochondria with consequences of cytochrome C release and increased apoptotic signaling. Our research indicates that sEVs secreted by hypoxic AC16 CMs negatively impact the biological properties of recipient CMs exposed to hypoxic stress. Thus, these findings widen our current understanding of sEV-mediated cellular communication during hypoxic events and provide insights into potential therapeutic targets for cardiac ischemic injury.