Cellular mechanism underlying leptin-induced anion secretion of rat epididymal epithelial cells.

BACKGROUND: A large number of studies have shown that leptin plays an important role in the regulation of fertility via the hypothalamus-pituitary-gonad axis. However, its peripheral function in epididymis was still elusive. OBJECTIVE: The purpose of this study was to determine the pro-secretion effect of leptin on the rat epididymal epithelium. MATERIALS AND METHODS: In the present study, real-time quantitative polymerase chain reaction, western blot, and immunohistochemical analysis were employed to detect the expression pattern of leptin receptors in rat epididymis. The pro-secretion effect of leptin on epididymal epithelial cells was measured by short-circuit current, and the prostaglandin E2 and cyclic adenosine monophosphate level was evaluated by enzyme-linked immunosorbent assay. RESULTS: We verified that the leptin receptor was located on the epididymal epithelium, with a relatively high expression level in corpus and cauda epididymis. Ussing chamber experiments showed that leptin stimulated a significant rise of the short-circuit current in rat epididymal epithelial cells, which could be abolished by the specific leptin receptor antagonist peptide Allo-aca, or by removing the ambient Cl- and HCO3 -. Furthermore, the leptin-stimulated short-circuit current response could be abrogated by blocking the apical cystic fibrosis transmembrane regulator or the basolateral Na+-K+-2Cl- cotransporter. Our pharmacological experiments manifested that interfering with the prostaglandin H synthase-2-prostaglandin E2-EP2/EP4-adenylate cyclase pathways could significantly blunt the cystic fibrosis transmembrane regulator-mediated anion secretion induced by leptin. The enzyme-linked immunosorbent assay demonstrated that leptin could induce a substantial increase in prostaglandin E2 release and cyclic adenosine monophosphate synthesis of primary cultured rat cauda epididymal epithelial cells. Our data also suggested that JAK2, ERK, and PI3K-dependent phosphorylation may be involved in the activation of prostaglandin H synthase-2 and the subsequent prostaglandin E2 production. CONCLUSIONS: The present study demonstrated the pro-secretion function of leptin in rat epididymal epithelium via the activation of cystic fibrosis transmembrane regulator and Na+-K+-2Cl- cotransporter, which was dependent on the paracrine/autocrine prostaglandin E2 stimulated EP2/EP4-adenylate cyclase pathways, and thus contributed to the formation of an appropriate microenvironment essential for sperm maturation.

Intraductal papillary mucinous neoplasm originating from a jejunal heterotopic pancreas: A case report.

BACKGROUND: Intraductal papillary mucinous neoplasm (IPMN) is a rare pancreatic tumor and has the potential to become malignant. Surgery is the most effective treatment at present, but there is no consensus on the site of resection. Heterotopic pancreas occurs in the gastrointestinal tract, especially the stomach and duodenum but is asymptomatic and rare. We report a case of ectopic pancreas with IPMN located in the jejunum. CASE SUMMARY: A 56-year-old male patient suffered from severe pain, nausea and vomiting due to a traffic accident and sought emergency treatment at our hospital. Contrast-enhanced computed tomography of the whole abdomen suggested splenic congestion, which was considered to be splenic rupture. Emergency laparotomy was performed, and the ruptured spleen was removed during the operation. Unexpectedly, a cauliflower-like mass of about 2.5 cm × 2.5 cm in size was incidentally found about 80 cm from the ligament of Treitz during the operation. A partial small bowel resection was performed, and postoperative pathology confirmed the small bowel mass as heterotopic pancreas with low-grade IPMN. CONCLUSION: Ectopic pancreas occurs in the jejunum and is pathologically confirmed as IPMN after surgical resection.

Aerobic exercises regulate the epididymal anion homeostasis of high-fat diet-induced obese rats through TRPA1-mediated Cl- and HCO3- secretion†.

Aerobic exercises could improve the sperm motility of obese individuals. However, the underlying mechanism has not been fully elucidated, especially the possible involvement of the epididymis in which sperm acquire their fertilizing capacity. This study aims to investigate the benefit effect of aerobic exercises on the epididymal luminal milieu of obese rats. Sprague-Dawley male rats were fed on a normal or high-fat diet (HFD) for 10 weeks and then subjected to aerobic exercises for 12 weeks. We verified that TRPA1 was located in the epididymal epithelium. Notably, aerobic exercises reversed the downregulated TRPA1 in the epididymis of HFD-induced obese rats, thus improving sperm fertilizing capacity and Cl- concentration in epididymal milieu. Ussing chamber experiments showed that cinnamaldehyd (CIN), agonist of TRPA1, stimulated an increase of the short-circuit current (ISC) in rat cauda epididymal epithelium, which was subsequently abolished by removing the ambient Cl- and HCO3-. In vivo data revealed that aerobic exercises increased the CIN-stimulated Cl- secretion rate of epididymal epithelium in obese rats. Pharmacological experiments revealed that blocking cystic fibrosis transmembrane regulator (CFTR) and Ca2+-activated Cl- channel (CaCC) suppressed the CIN-stimulated anion secretion. Moreover, CIN application in rat cauda epididymal epithelial cells elevated intracellular Ca2+ level, and thus activate CACC. Interfering with the PGHS2-PGE2-EP2/EP4-cAMP pathway suppressed CFTR-mediated anion secretion. This study demonstrates that TRPA1 activation can stimulate anion secretion via CFTR and CaCC, which potentially forming an appropriate microenvironment essential for sperm maturation, and aerobic exercises can reverse the downregulation of TRPA1 in the epididymal epithelium of obese rats.

Discussion on gemcitabine combined with targeted drugs in the treatment of pancreatic cancer.

Pancreatic cancer is a malignant tumor with poor prognosis. The treatment of pancreatic cancer depends on the tumor stage and type, and includes local treatment (surgery, radiotherapy and ablation intervention) and systemic therapy (chemotherapy, targeted therapy and immunotherapy). We read with great interest the review "Effective combinations of anti-cancer and targeted drugs for pancreatic cancer treatment" published on World J Gastroenterol and intended to share some of our perspectives in pancreatic cancer treatment. This review presents the therapeutic effects of the combination of gemcitabine and targeted drugs, which gives us a deeper insight into the combination treatments for pancreatic cancer.

Mechanosensitive Piezo1 channel in rat epididymal epithelial cells promotes transepithelial K+ secretion.

The Piezo1 channel, a mechanosensitive channel that exhibit a preference for Ca2+, play multifarious physiological and pathological roles in the endothelium and epithelium of various tissues. However, the functional expression of Piezo1 channel in the epithelium of the male reproductive tract remains unknown. In the present study, the expression of Piezo1 channel in the rat epididymis was determined by real-time quantitative PCR, western blot and immunohistochemical analysis. Our data revealed that Piezo1 channel was located in the epithelial layer of the rat epididymis, with higher expression levels in the corpus and cauda regions. The pro-secretion function of Piezo1 channel was then investigated using short circuit current (ISC) and intracellular Ca2+ imaging techniques. Application of Yoda1, a selective Piezo1 channel activator, stimulated a remarkable decrease in the ISC of the epididymal epithelium. Pharmacological experiments revealed that the ISC response induced by Piezo1 channel activation was abolished by pretreating epithelial cells with the Yoda1 analogue, Dooku1, the selective mechanosensitive cation channel blocker, GsMTx4, or removal of basolateral K+. Meanwhile, we demonstrated that activation of Piezo1 channel triggered a robust Ca2+ influx in epididymal epithelial cells. The possible involvement of Ca2+- activated K+channels (KCa) in transepithelial K+ secretion was then evaluated. And that big conductance KCa (BK), but not small conductance or intermediate conductance KCa, mediated Piezo1-elicited transepithelial K+ secretion. Moreover, we demonstrated that NKCC and NKA were responsible for supplying substrate K+ during transepithelial K+ secretion. These data demonstrate that the activation of Piezo1 channel promotes BK-mediated transepithelial K+ secretion, and thus may plays an important role in the formation of a high K+ concentration in epididymal intraluminal fluid.

Activation of TRPV4 stimulates transepithelial K+ secretion in rat epididymal epithelium.

The maturation of sperms is dependent on the coordinated interactions between sperm and the unique epididymal luminal milieu, which is characterized by high K+ content. This study investigated the involvement of transient receptor potential vanilloid 4 (TRPV4) in the K+ secretion of epididymal epithelium. The expression level and cellular localization of TRPV4 and Ca2+-activated K+ channels (KCa) were analyzed via RT-PCR, real-time quantitative PCR, western blot and immunofluorescence. The functional role of TRPV4 was investigated using short-circuit current (ISC) and intracellular Ca2+ imaging techniques. We found a predominant expression of TRPV4 in the corpus and cauda epididymal epithelium. Activation of TRPV4 with a selective agonist, GSK1016790A, stimulated a transient decrease in the ISC of the epididymal epithelium. The ISC response was abolished by either the TRPV4 antagonists, HC067047 and RN-1734, or the removal of basolateral K+. Simultaneously, the application of GSK1016790A triggered Ca2+ influx in epididymal epithelial cells. Our data also indicated that the big conductance KCa (BK), small conductance KCa (SK) and intermediate conductance KCa (IK) were all expressed in rat epididymis. Pharmacological studies revealed that BK, but not SK and IK, mediated TRPV4-elicited transepithelial K+ secretion. Finally, we demonstrated that TRPV4 and BK were localized in the epididymal epithelium, which showed an increased expression level from caput to cauda regions of rat epididymis. This study implicates that TRPV4 plays an important role in the formation of high K+ concentration in epididymal intraluminal fluid via promoting transepithelial K+ secretion mediated by BK.

FUT8-mediated aberrant N-glycosylation of B7H3 suppresses the immune response in triple-negative breast cancer.

Most patients with triple negative breast cancer (TNBC) do not respond to anti-PD1/PDL1 immunotherapy, indicating the necessity to explore immune checkpoint targets. B7H3 is a highly glycosylated protein. However, the mechanisms of B7H3 glycosylation regulation and whether the sugar moiety contributes to immunosuppression are unclear. Here, we identify aberrant B7H3 glycosylation and show that N-glycosylation of B7H3 at NXT motif sites is responsible for its protein stability and immunosuppression in TNBC tumors. The fucosyltransferase FUT8 catalyzes B7H3 core fucosylation at N-glycans to maintain its high expression. Knockdown of FUT8 rescues glycosylated B7H3-mediated immunosuppressive function in TNBC cells. Abnormal B7H3 glycosylation mediated by FUT8 overexpression can be physiologically important and clinically relevant in patients with TNBC. Notably, the combination of core fucosylation inhibitor 2F-Fuc and anti-PDL1 results in enhanced therapeutic efficacy in B7H3-positive TNBC tumors. These findings suggest that targeting the FUT8-B7H3 axis might be a promising strategy for improving anti-tumor immune responses in patients with TNBC.

AKT-mediated regulation of chromatin ubiquitylation and tumorigenesis through Mel18 phosphorylation.

Polycomb repressor complex 1 (PRC1) is linked to the regulation of gene expression and histone ubiquitylation conformation, which contributes to carcinogenesis. However, the upstream regulators of PRC1 biogenesis machinery remain obscure. Here, we report that the polycomb group-related mammalian gene Mel18 is a target of the protein kinase AKT. AKT phosphorylates Mel18 at T334 to disrupt the interaction between Mel18 and other PRC1 members, leading to attenuated PRC1-dependent ubiquitylation of histone H2A at Lys119. As such, PRC1 target genes, many of which are known oncogenes, are derepressed upon T334-Mel18 phosphorylation, which promotes malignant behaviours, including cell proliferation, tumour formation, migration and invasion, bone and brain metastatic lesion formation. Notably, a positive correlation between AKT activity and pT334-Mel18 is observed, and prognostic models based on p-AKT and pT334-Mel18 that predicted overall survival and distant metastasis-free survival in breast cancer patients are established. These findings have implications for understanding the role of AKT and its associated proteins in chromatin ubiquitylation, and also indicate the AKT-Mel18-H2AK119ub axis as a novel prognostic biomarker and therapeutic target for cancer patients.

MAPK1/3 kinase-dependent ULK1 degradation attenuates mitophagy and promotes breast cancer bone metastasis.

The function of mitophagy in cancer is controversial. ULK1 is critical for induction of macroautophagy/autophagy and has a more specific role in mitophagy in response to hypoxia. Here, we show that ULK1 deficiency induces an invasive phenotype of breast cancer cells under hypoxia and increases osteolytic bone metastasis. Mechanistically, ULK1 depletion attenuates mitophagy ability during hypoxia. As a result, the accumulation of damaged, ROS-generating mitochondria leads to activation of the NLRP3 inflammasome, which induces abnormal soluble cytokines secretion, then promotes the differentiation and maturation of osteoclasts, and ultimately results in bone metastasis. Notably, phosphorylation of ULK1 by MAPK1/ERK2-MAPK3/ERK1 kinase triggers its interaction with BTRC and subsequent K48-linked ubiquitination and proteasome degradation. Also, a clearly negative correlation between the expression levels of ULK1 and p-MAPK1/3 was observed in human breast cancer tissues. The MAP2K/MEK inhibitor trametinib is sufficient to restore mitophagy function via upregulation of ULK1, leading to inhibition of NLRP3 inflammasome activation, thereby reduces bone metastasis. These results indicate that ULK1 knockout-mediated mitophagy defect promotes breast cancer bone metastasis and provide evidence to explore MAP2K/MEK- MAPK1/3 pathway inhibitors for therapy, especially in cancers displaying low levels of ULK1.Abbreviations: ATG: autophagy-related; Baf A1: bafilomycin A1; BTRC/ß-TrCP: beta-transducin repeat containing E3 ubiquitin protein ligase; CHX: cycloheximide; CM: conditioned media; FBXW7/FBW7: F-box and WD repeat domain containing 7; MAPK1: mitogen-activated protein kinase 1; MTDR: MitoTracker Deep Red; mtROS: mitochondrial reactive oxygen species; microCT: micro-computed tomography; mtROS: mitochondrial reactive oxygen species; OCR: oxygen consumption rate; SQSTM1: sequestosome 1; ACP5/TRAP: acid phosphatase, tartrate resistant; ULK1: unc-51 like autophagy activating kinase 1.

Disruption of super-enhancer-driven tumor suppressor gene RCAN1.4 expression promotes the malignancy of breast carcinoma.

BACKGROUND: Super-enhancers (SEs) play a crucial role in cancer, which is often associate with activated oncogenes. However, little is known about how SEs facilitate tumour suppression. Individuals with Down syndrome exhibit a remarkably reduced incidence of breast cancer (BC), moving the search for tumor suppressor genes on human chromosome 21 (HSA21). In this study, we aim to identify and explore potential mechanisms by which SEs are established for tumor suppressor RCAN1.4 on HSA21 in BC. METHODS: In silico analysis and immunohistochemical staining were used to assess the expression and clinical relevance of RCAN1.4 and RUNX3 in BC. Function experiments were performed to evaluate the effects of RCAN1.4 on the malignancy of breast carcinoma in vitro and in vivo. ChIP-seq data analysis, ChIP-qPCR, double-CRISPR genome editing, and luciferase reporter assay were utilized to confirm RUNX3 was involved in regulating RCAN1.4-associated SE in BC. The clinical value of co-expression of RCAN1.4 and RUNX3 was evaluated in BC patients. RESULTS: Here, we characterized RCAN1.4 as a potential tumour suppressor in BC. RCAN1.4 loss promoted tumour metastasis to bone and brain, and its overexpression inhibited tumour growth by blocking the calcineurin-NFATc1 pathway. Unexpectedly, we found RCAN1.4 expression was driven by a ~ 23 kb-long SE. RCAN1.4-SEdistal was sensitive to BRD4 inhibition, and its deletion decreased RCAN1.4 expression by over 90% and induced the malignant phenotype of BC cells. We also discovered that the binding sites in the SE region of RCAN1.4 were enriched for consensus sequences of transcription factor RUNX3. Knockdown of RUNX3 repressed the luciferase activity and also decreased H3K27ac enrichment binding at the SE region of RCAN1.4. Furthermore, abnormal SE-driven RCAN1.4 expression mediated by RUNX3 loss could be physiologically significant and clinically relevant in BC patients. Notably, we established a prognostic model based on RCAN1.4 and RUNX3 co-expression that effectively predicted the overall survival in BC patients. CONCLUSIONS: These findings reveal an important role of SEs in facilitating tumour suppression in BC. Considering that the combination of low RCAN1.4 and low RUNX3 expression has worse prognosis, RUNX3-RCAN1.4 axis maybe a novel prognostic biomarker and therapeutic target for BC patients.

Autophagy deficiency promotes triple-negative breast cancer resistance to T cell-mediated cytotoxicity by blocking tenascin-C degradation.

Most triple-negative breast cancer (TNBC) patients fail to respond to T cell-mediated immunotherapies. Unfortunately, the molecular determinants are still poorly understood. Breast cancer is the disease genetically linked to a deficiency in autophagy. Here, we show that autophagy defects in TNBC cells inhibit T cell-mediated tumour killing in vitro and in vivo. Mechanistically, we identify Tenascin-C as a candidate for autophagy deficiency-mediated immunosuppression, in which Tenascin-C is Lys63-ubiquitinated by Skp2, particularly at Lys942 and Lys1882, thus promoting its recognition by p62 and leading to its selective autophagic degradation. High Tenascin-C expression is associated with poor prognosis and inversely correlated with LC3B expression and CD8+ T cells in TNBC patients. More importantly, inhibition of Tenascin-C in autophagy-impaired TNBC cells sensitizes T cell-mediated tumour killing and improves antitumour effects of single anti-PD1/PDL1 therapy. Our results provide a potential strategy for targeting TNBC with the combination of Tenascin-C blockade and immune checkpoint inhibitors.

Activation of canonical transient receptor potential channels preserves Ca2+ entry and endothelium-derived hyperpolarizing factor-mediated function in vitro in porcine coronary endothelial cells and coronary arteries under conditions of hyperkalemia.

OBJECTIVES: Although membrane depolarization by hyperkalemia is known to reduce Ca2+ influx in endothelial cells, the mechanism by which endothelial Ca2+ channel is affected by hyperkalemia remains poorly studied. We studied the effect of hyperkalemia on canonical transient receptor potential channels, in particular canonical transient receptor potential channel 3, in modulation of endothelial intracellular Ca2+ concentration. Endothelium-derived hyperpolarizing factor-mediated function is Ca2+ dependent, and hyperkalemic cardioplegia/organ preservation solutions impair endothelium-derived hyperpolarizing factor-mediated function. We explored the role of canonical transient receptor potential channel 3 in endothelium-derived hyperpolarizing factor-mediated function and investigated whether modulation of these channels preserves endothelial Ca2+ influx and endothelium-derived hyperpolarizing factor-mediated function under the condition of hyperkalemic/cardioplegic exposure. METHODS: Intracellular Ca2+ concentration was measured with fluorescent dye in primary cultured porcine coronary endothelial cells exposed to hyperkalemic/cardioplegic solutions containing mild to extreme high K+ concentration. Endothelium-derived hyperpolarizing factor-mediated relaxation under hyperkalemic/cardioplegic exposure was studied in small porcine coronary arteries in a myograph in the presence of cyclooxygenase and nitric oxide synthase inhibitors and nitric oxide scavenger. RESULTS: Canonical transient receptor potential channel 3 blocker inhibited bradykinin-induced Ca2+ influx and attenuated endothelium-derived hyperpolarizing factor-mediated response. Hyperkalemic exposure inhibited canonical transient receptor potential channel 3-mediated Ca2+ influx in a K+ concentration-dependent manner (120>20>10 mmol/L). Ca2+ influx decreased in porcine coronary endothelial cells exposed to histidine-tryptophan-ketoglutarate, St Thomas' Hospital, and University of Wisconsin solutions that contained mild (10 mmol/L), moderate (20 mmol/L), and extreme high (125 mmol/L) K+ concentration, respectively. Canonical transient receptor potential channel activator prevented the reduction of Ca2+ influx in porcine coronary endothelial cells exposed to solutions containing mild to moderate high [K+]o and restored endothelium-derived hyperpolarizing factor-mediated response that was impaired by hyperkalemic exposure. CONCLUSIONS: Canonical transient receptor potential channel 3 is involved in endothelium-derived hyperpolarizing factor-mediated function in coronary arteries. Hyperkalemia inhibited canonical transient receptor potential channel 3-mediated Ca2+ influx in endothelial cells. Canonical transient receptor potential channel activation restores Ca2+ influx suppressed by hyperkalemia and prevents dysfunction of endothelium-derived hyperpolarizing factor.

Use of intermediate/small conductance calcium-activated potassium-channel activator for endothelial protection.

OBJECTIVES: Endothelial dysfunction occurs in hypoxia-related states such as ischemic heart disease or heart surgery. Intermediate- and small-conductance calcium-activated potassium channels (IKCa and SKCa) are closely related to endothelium-dependent hyperpolarizing factor-mediated endothelial function. However, the status of these KCa under hypoxia is unknown. We investigated whether endothelial dysfunction under hypoxic state is related to the alterations of IKCa and SKCa and whether use of IKCa/SKCa activator may protect endothelium from hypoxia-reoxygenation injury. METHODS: Isometric tension measurement, patch-clamp technique, intracellular membrane potential recording, and molecular methods were used to study porcine coronary arteries and endothelial cells. RESULTS: Hypoxia-reoxygenation (60-30 minutes) decreased endothelium-dependent hyperpolarizing factor-mediated relaxation at normothermia in Krebs solution (43.3%±6.3% vs 82.3%±2.9%) and in St Thomas' Hospital cardioplegic solution (28.9%±1.8% vs 78.1%±3.0%) (P<.001) as well as at hypothermia in St Thomas' Hospital solution (43.1%±2.6%, P<.001). Hypoxia-reoxygenation markedly reduced endothelial IKCa (2.8±0.6 vs 6.9±0.6 pA/pF) and SKCa currents (1.5±0.3 vs 4.3±0.4 pA/pF) (P<.05) and downregulated endothelial IKCa expression. IKCa/SKCa activator 1-ethyl-2-benzimidazolinone enhanced K+ current in endothelial cells that was blunted by hypoxia. Further, 1-ethyl-2-benzimidazolinone restored (P<.001) endothelium-dependent hyperpolarizing factor-mediated relaxation with hyperpolarization recovered from 6.0±0.3 to 7.8±0.4 mV (P<.05). CONCLUSIONS: In porcine coronary arteries, hypoxia markedly reduced endothelial K+ currents related to IKCa and SKCa with downregulation of protein expression and endothelium-derived hyperpolarizing factor function. IKCa/SKCa activator may preserve endothelium-dependent hyperpolarizing factor-mediated relaxation with enhancement of K+ current in endothelial cells and cellular membrane potential hyperpolarization in smooth muscle cells and may become a new strategy to protect coronary endothelium in cardiac surgery or transplantation.

New strategy of endothelial protection in cardiac surgery: use of enhancer of endothelial nitric oxide synthase.

BACKGROUND: Endothelial dysfunction related to the loss of nitric oxide (NO) production remains an important issue in cardiac surgery. We examined the hypothesis that AVE3085, a novel compound that enhances eNOS transcription, may protect coronary endothelium against hypoxia-reoxygenation (H-R) injury during cardioplegic arrest and the possible mechanism by which this occurs. METHODS: Porcine coronary small arteries (600-800-microm diameter) were subjected to hypoxia (PO(2) <5 mmHg) in St. Thomas cardioplegic (ST) solution with or without AVE3085 (10 microM) or L-arginine (10 mM) at either 37 or 4 degrees C for 60 min, followed by 30-min reoxygenation. Bradykinin (-10 to -6.5 LogM)-induced, endothelium-dependent relaxation was studied in a myograph in U(46619) precontraction before and after H-R. Protein expressions of eNOS and phosphorylated eNOS at Ser-1177 (p-eNOS(Ser1177)) were also determined. RESULTS: Exposure to ST solution with H-R at both 37 and 4 degrees C markedly reduced bradykinin-induced relaxation in coronary small arteries. Addition of AVE3085 in ST solution at 37 degrees C preserved the vasorelaxant response to bradykinin (95.7 +/- 2.1% vs. 69.2 +/- 6.6%, p < 0.01), with the protective effect comparable to that of L-arginine (96.1 +/- 3.3% vs. 70.6 +/- 8.7%, p < 0.05). eNOS and p-eNOS(Ser1177) expressions in coronary endothelial cells were significantly increased by the addition of AVE3085 in ST solution during hypoxia (p < 0.05). Protection of endothelium-dependent relaxation from H-R by AVE3085 (70.3 +/- 7.2% vs. 90.5 +/- 2.4%, p < 0.05) also reached a level similar to that by L-arginine (69.9 +/- 9.0% vs. 94.7 +/- 3.9%, p < 0.05) at 4 degrees C. CONCLUSIONS: We have demonstrated a new mechanism to protect coronary endothelium from H-R injury by using eNOS enhancers. This may form a new strategy in the future development of cardioplegic/preservation solutions with direct targeting of eNOS expression in coronary vasculature.