Purpose: To determine the efficacy of the superoxide dismutase mimetic, manganese(III) tetrakis(1-methyl-4-pyridyl) porphyrin (Mn-TM-2-PyP), in vitro in human corneal epithelial (HCE-T) cells and in vivo in a preclinical mouse model for dry-eye disease (DED). Methods: In vitro, HCE-T cultures were exposed either to tertbutylhydroperoxide (tBHP) to generate oxidative stress or to hyperosmolar conditions modeling cellular stress during DED. Cells were pre-treated with Mn-TM-2-PyP or vehicle. Mn-TM-2-PyP permeability across stratified HCE-T cells was assayed, In vivo, Mn-TM-2-PyP (0.1% w/v in saline) was delivered topically as eye drops in a desiccating stress / scopolamine model for DED. Preclinical efficacy was compared to untreated, vehicle-and ophthalmic cyclosporine emulsion-treated mice. Results: Mn-TM-2-PYP protected HCE-T cells in a dose-dependent manner against tBHPinduced oxidative stress as determined by calculating the IC 50 for tBHP in the resazurin, MTT and lactate dehydrogenase release cell viability assays. Mn-TM-2-PyP did not protect HCE-T cells

Timely reperfusion is still the most effective approach to limit infarct size in humans. Yet, despite advances in care and reduction in door-to-balloon times, nearly 25% of patients develop heart failure postmyocardial infarction, with its attendant morbidity and mortality. We previously showed that cardioprotection results from a skin incision through the umbilicus in a murine model of myocardial infarction. In the present study, we show that an electrical stimulus or topical capsaicin applied to the skin in the same region induces significantly reduced infarct size in a murine model. We define this class of phenomena as nociceptor-induced conditioning (NIC) based on the peripheral nerve mechanism of initiation. We show that NIC is effective both as a preconditioning and postconditioning remote stimulus, reducing infarct size by 86% and 80%, respectively. NIC is induced via activation of skin C-fiber nerves. Interestingly, the skin region that activates NIC is limited to the anterior of the T9−T10 vertebral region of the abdomen. Cardioprotection after NIC requires the integrity of the spinal cord from the region of stimulation to the thoracic vertebral region of the origin of the cardiac nerves but does not require that the cord be intact in the cervical region. Thus, we show that NIC is a reflex and not a central nervous system-mediated effect. The mechanism involves bradykinin 2 receptor activity and activation of PKC, specifically, PKC-α. The similarity of the neuroanatomy and conservation of the effectors of cardioprotection supports that NIC may be translatable to humans as a nontraumatic and practical adjunct therapy against ischemic disease. NEW & NOTEWORTHY This study shows that an electrical stimulus to skin sensory nerves elicits a very powerful cardioprotection against myocardial infarction. This stimulus works by a neurogenic mechanism similar to that previously elucidated for remote cardioprotection of trauma. Nociceptor-induced conditioning is equally potent when applied before ischemia or at reperfusion and has great potential clinically.

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ΔAbs, the difference between the two absorbances; ACE, angiotensin converting enzyme; B 2 : bradykinin B 2 receptor; (a number [95.0%CI number, number]): effect size, 95.0% confidence interval, width lower bound, upper bound]); CCA, common carotid arteries; DTNB, 5,5'-dithio-bis-(2-nitrobenzoic acid); GMP, Good Manufacturing Practices; IC 50, the concentration of an inhibitor that inhibits the enzyme activity by 50% relative to control activity; K i , the dissociation equilibrium constant of an enzyme-inhibitor complex; K m , Michaelis constant (the concentration of the substrate at which the enzyme is at half maximal velocity); LAD, left anterior descending coronary artery; MCA, middle cerebral artery; eNOS, endothelial nitric oxide synthase; RRR, Relative Risk Reduction; tMCAO, transient middle cerebral artery occlusion; ST-115, [(S)-2-mercapto-4-methylpentanoyl]-4(S)-fluoro-Pro-Pro-3(R)-beta-Pro; tPA, tissue plasminogen activator; TTC, triphenyltetrazolium chloride.

IntroductionCardiovascular disease is the leading cause of death in the US with coronary heart disease (ischemia/reperfusion; I/R injury) accounting for approximately half of these deaths. Recent data from our lab suggests that alternative polyadenylation (APA) plays a role in cardiac gene expression following I/R via modulation of mRNA 3′UTR length, leading to the inclusion or exclusion of regulatory sequence elements. The goal of this work is to identify specific RNA binding proteins (RBPs) that are likely to mediate APA in the myocardium.MethodsThe 3′UTR of the heat shock protein 70.3 (Hsp70.3) mRNA was randomly biotinylated throughout and incubated with cardiac protein extract associated with differential APA of the Hsp70.3 3′UTR. Biotinylated RNA and RBPs were co‐precipitated and bound RBPs were identified using mass spectrometry.ResultsWe identified a total of 45 known RBPs interacting with the Hsp70.3 3′UTR. Of these, 9 unique RBPs were found to bind only under conditions associated with a long 3′UTR, whereas 10 unique RBPs were found to bind only in association with APA truncation of the 3′UTR. Our results identified RBPs previously indicated to play a role in APA and/or mRNA degradation as well as RBPs whose function remains unknown. Work is ongoing to determine the functional role of these RBPs in alternative polyadenylation and cardiac I/R injury.This work was partially funded by a UC CCTST Grant (MT).

Ischemic preconditioning (IPC) is a well-characterized cardioprotective phenomenon involving multiple brief, non-lethal ischemic episodes to the heart. Remote ischemic preconditioning (RIPC) elicits cardioprotection via repetitive ischemia and reperfusion to a distant vascular bed, organ, or limb. A non-ischemic, nociceptive stimulus in distant tissues can result in the most powerful cardioprotection reported - an 80% decrease in infarct size - called remote nociceptor-induced cardioprotection (NIC). Electrically induced cardioprotection via electrical stimulation with electroacupuncture needles (EA) or skin patches (ES) on the abdomen may be a clinically feasible way to induce NIC. A 5 volt, 15 minute electrical stimulus applied as either preconditioning or postconditioning reduces infarct size in a mouse surgical model of myocardial infarction. This study’s objective is to determine the molecular mechanism of electrically-induced NIC. Our previous work shows that IPC requires upregulation and post-transcriptional regulation of heat shock proteins (HSPs), which act in unity to assist in protein folding, repair, and degradation following myocardial injury. However, Western blotting and PCR revealed non-significant changes in HSP mRNA and protein relative to sham, which were inconsistent with the large, significant increases in HSPs observed after IPC. Studies using mice with a genetic deletion of HSP70.1 revealed that this heat shock protein is not required for protection. The transcription factor NF-κB regulates many cardioprotective genes in the heart after IPC, including, but not limited to, heat shock proteins. Based on our work in IPC, we hypothesize that electrical stimulation of the skin is cardioprotective via a unique induction of NF-κB-dependent genes. Pharmacological and genetic inhibition of NF-κB were utilized to evaluate the role of this transcription factor in electrically-induced cardioprotection. Deep sequencing studies are in progress to delineate the presence and quantity of additional transcripts that may be involved in electrically-induced cardioprotection. Future investigations will address the neurobiology of electrically-induced cardioprotection via skin stimulation.

IntroductionHeat shock protein 70.3 (Hsp70.3) expression is increased in response to cellular stress and plays a cytoprotective role, including a cardioprotective role in the heart against ischemia/reperfusion (I/R) injury. Recent work from our lab shows that Hsp70.3 expression following I/R is controlled through alternative polyadenylation (APA). Truncation of the 3′UTR removes potential regulatory sequences and is observed concomitant with upregulation of Hsp70.3 expression. Herein, we investigated the hypothesis that APA truncation of the 3′UTR enhances polysome loading of the Hsp70.3 transcript.MethodsSucrose gradient ultracentrifugation was used to isolate polysome bound mRNA from HL‐1 cardiac myocyte cells subjected to control (long 3′UTR) or a 1 hr heat shock (HS; short 3′UTR).ResultsCells subjected to HS for one hour showed a decrease in the total amount of polysome loaded mRNA. The Hsp70.3 transcript found in the polysome dense fractions existed as a higher ratio of the APA truncated short 3′UTR, thus supporting our hypothesis that APA truncation of the Hsp70.3 3′UTR increases polysome loading.Our results provide an enhanced understanding of APA on the regulation of Hsp70.3 and will have a significant impact in understating the regulation of cardiac gene regulation after I/R injury.This work was partially funded by a University of Cincinnati Provost Pilot Research Project grant (MT).

A non‐ischemic, nociceptive stimulus such as electroacupunture (EA) in distant tissues can result in the most powerful cardioprotection reported – an 80% decrease in infarct size – called remote preconditioning of trauma (RPCT). It is known that the mechanisms underlying ischemic preconditioning, remote ischemic preconditioning, and RPCT are similar. Whether the downstream genes implicated in other cardioprotective stimuli are also involved in RPCT remains to be determined and is the objective of this study. Specifically, this study will address the hypothesis that electroacupuncture (EA) as a remote nociceptive stimulus is cardioprotective and increases NF‐κB dependent expression of heat shock proteins in the heart. Activation of heat shock proteins (HSPs) by the transcription factor NF‐κB is necessary for late IPC. Preliminary data supports that NF‐κB is required for protection against myocardial infarction 24h after RPCT. qRT‐PCR and Western Blotting were used to assess the changes in gene and protein levels following EA in a mouse model. Previous studies indicate that EA reduces infarct size after occlusion of the left coronary artery. We show that EA of skin results in significant increases in levels of cardiac mRNA levels of HSP90 and DNAJs 3 hours post‐stimulus. Protein accumulation and kinetics are under investigation and confirmatory studies with knockout mice are planned. Future studies will further address the molecular mechanisms of cardioprotection associated with EA therapy. Understanding the mechanism of RPTC via nociception would facilitate the development of non‐invasive therapies, which might be used to precondition patients against ischemia/reperfusion injury by mimicking remote cardioprotection.