Human induced pluripotent stem cell (iPSC)-derived cardiomyocytes (CMs) (iPSC-CMs) are a promising cell source for myocardial regeneration, disease modeling and drug assessment. However, iPSC-CMs exhibit immature fetal CM-like characteristics that are different from adult CMs in several aspects, including cellular structure and metabolism. As an example, glycolysis is a major energy source for immature CMs. As CMs mature, the mitochondrial oxidative capacity increases, with fatty acid β-oxidation becoming a key energy source to meet the heart’s high energy demand. The immaturity of iPSC-CMs thereby limits their applications. The aim of this study was to investigate whether the energy substrate fatty acid-treated iPSC-CMs exhibit adult CM-like metabolic properties. After 20 days of differentiation from human iPSCs, iPSC-CMs were sequentially cultured with CM purification medium (lactate+/glucose-) for 7 days and maturation medium (fatty acids+/glucose-) for 3–7 days by mimicking the adult CM’s preference of utilizing fatty acids as a major metabolic substrate. The purity and maturity of iPSC-CMs were characterized via the analysis of: (1) Expression of CM-specific markers (e.g., troponin T, and sodium and potassium channels) using RT-qPCR, Western blot or immunofluorescence staining and electron microscopy imaging; and (2) cell energy metabolic profiles using the XF96 Extracellular Flux Analyzer. iPSCs-CMs (98% purity) cultured in maturation medium exhibited enhanced elongation, increased mitochondrial numbers with more aligned Z-lines, and increased expression of matured CM-related genes, suggesting that fatty acid-contained medium promotes iPSC-CMs to undergo maturation. In addition, the oxygen consumption rate (OCR) linked to basal respiration, ATP production, and maximal respiration and spare respiratory capacity (representing mitochondrial function) was increased in matured iPSC-CMs. Mature iPSC-CMs also displayed a larger change in basal and maximum respirations due to the utilization of exogenous fatty acids (palmitate) compared with non-matured control iPSC-CMs. Etomoxir (a carnitine palmitoyltransferase 1 inhibitor) but not 2-deoxyglucose (an inhibitor of glycolysis) abolished the palmitate pretreatment-mediated OCR increases in mature iPSC-CMs. Collectively, our data demonstrate for the first time that fatty acid treatment promotes metabolic maturation of iPSC-CMs (as evidenced by enhanced mitochondrial oxidative function and strong capacity of utilizing fatty acids as energy source). These matured iPSC-CMs might be a promising human CM source for broad biomedical application.

Cytochrome P450 genes catalyze formation of epoxyeicosatrienoic acids (EETs) from arachidonic acid. The effects of 5,6-EET, 8,9-EET, 11,12-EET, and 14,15-EET microinjected into the ventrolateral periaqueductal gray (vlPAG) on the thermally produced tail-flick response were studied in male Sprague-Dawley rats. 14,15-EET microinjected into vlPAG (3-156 pmol) dosedependently inhibited the tail-flick response (ED 50 ϭ 32.5 pmol). In contrast, 5,6-EET, 8,9-EET, and 11,12-EET at a dose of 156 pmol were not active when injected into the vlPAG. 14,15-EET failed to displace the radiobinding of [

Despite considerable advances in medicine, cardiovascular disease is still rising, with ischemic heart disease being the leading cause of death and disability worldwide. Thus extensive efforts are continuing to establish effective therapeutic modalities that would improve both quality of life and survival in this patient population. Novel therapies are being investigated not only to protect the myocardium against ischemia-reperfusion injury but also to regenerate the heart. Stem cell therapy, such as potential use of human mesenchymal stem cells and induced pluripotent stem cells and their exosomes, will make it possible not only to address molecular mechanisms of cardiac conditioning, but also to develop new therapies for ischemic heart disease. Despite the studies and progress made over the last 15 years on the use of stem cell therapy for cardiovascular disease, the efforts are still in their infancy. Even though the expectations have been high, the findings indicate that most of the clinical trials generally have been small and the results inconclusive. Because of many negative findings, there is certain pessimism that cardiac cell therapy is likely to yield any meaningful results over the next decade or so. Similar to other new technologies, early failures are not unusual and they may be followed by impressive success. Nevertheless, there has been considerable attention to safety by the clinical investigators because the adverse events of stem cell therapy have been impressively rare. In summary, although regenerative biology might not help the cardiovascular patient in the near term, it is destined to do so over the next several decades.

We have previously shown that the naturally occurring levomorphine at a subanalgesic picomolar dose pretreated i.t. induces antianalgesia against levo-morphine-produced antinociception. We now report that the synthetic stereo-enantiomer dextro-morphine, even at an extremely low femtomolar dose, induces antianalgesia against levo-morphine-produced antinociception using the tail-flick (TF) test in male CD-1 mice. Intrathecal pretreatment with dextro-morphine (33 fmol) timedependently attenuated the i.t. levo-morphine-produced TF inhibition for 4 h and returned to the preinjection control level at 24 h. Intrathecal pretreatment with dextro-morphine (0.3-33 fmol), which injected alone did not affect the baseline TF latency, dose-dependently attenuated the TF inhibition produced by i.t.-administered levo-morphine (3.0 nmol). The ED 50 value for dextro-morphine to induce antianalgesia was estimated to be 1.07 fmol, which is 71,000-fold more potent than the ED 50 value of levo-morphine, indicating the high stereoselective action of dextro-morphine over levo-morphine for the induction of antianalgesia. Like levo-morphine, the dextro-morphineinduced antianalgesia against levo-morphine-produced TF inhibition was dose-dependently blocked by the nonopioid dextro-naloxone and its stereo-enantiomer levo-naloxone, a nonselective -opioid receptor antagonist. The antianalgesia induced by levo-morphine and dextro-morphine is reversed by the pretreatment with the glial inhibitor propentofylline (3.3-65 nmol), indicating that the antianalgesia is mediated by glial stimulation. The findings strongly indicate that the antianalgesia induced by levo-morphine and dextro-morphine is mediated by the stimulation of a novel nonopioid receptor on glial cells.Naturally occurring levo-morphine, which is isolated from the juice of the opium poppy, Papaver somniferum, is stereochemically identified as a levorotatory isoform of morphine. levo-Morphine produces potent analgesic and other major pharmacological effects, which are mainly mediated by the stimulation of -opioid receptors. The synthetic dextro-enantiomer of levo-morphine has minimal activity in the -opioid receptor binding assay, the electrically stimulated guinea pig ileum assay, and the inhibition of adenylate cyclase activity in the neuroblastoma ϫ glioma hybrid cell homogenates, indicating that it does not interact with -opioid receptors (Jacquet et al., 1977). Unlike levo-morphine, which produces potent levo-naloxone reversible analgesia, dextro-morphine microinjected into the periaqueductal gray in rats produces minimal analgesia (Jacquet et al., 1977). In the present study, i.t. pretreatment with dextro-morphine, which injected alone does not affect baseline nociceptive latency, attenuates the antinociception produced by i.t.-administered levo-morphine. The phenomenon of the attenuation of levo-morphineproduced analgesia by dextro-morphine has been defined as antianalgesia (Wu et al., 2004b).Nonselective -opioid receptor antagonist levo-naloxone and nonopioid receptor ant...

Amyloid-β (Aβ) has long been implicated as a causative protein in Alzheimer's disease. Cellular Aβ accumulation is toxic and causes mitochondrial dysfunction, which precedes clinical symptoms of Alzheimer's disease pathology. In the present study, we explored the possible use of epoxyeicosatrienoic acids (EETs), epoxide metabolites of arachidonic acid, as therapeutic target against Aβ-induced mitochondrial impairment using cultured neonatal hippocampal astrocytes. Inhibition of endogenous EET production by a selective epoxygenase inhibitor, MS-PPOH, caused a greater reduction in mitochondrial membrane potential in the presence of Aβ (1, 10 μM) exposure versus absence of Aβ. MS-PPOH preincubation also aggravated Aβ-induced mitochondrial fragmentation. Preincubation of the cells with either 14,15- or 11,12-EET prevented this mitochondrial depolarization and fragmentation. EET pretreatment also further improved the reduction observed in mitochondrial oxygen consumption in the presence of Aβ. Preincubation of the cells with EETs significantly improved cellular respiration under basal condition and in the presence of the protonophore, carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP). The uncoupling of ATP synthase from the electron transfer chain that occurred in Aβ-treated cells was also prevented by preincubation with EETs. Lastly, cellular reactive oxygen species production, a hallmark of Aβ toxicity, also showed significant reduction in the presence of EETs. We have previously shown that Aβ reduces EET synthesis in rat brain homogenates and cultured hippocampal astrocytes and neurons (Sarkar P, Narayanan J, Harder DR. Differential effect of amyloid beta on the cytochrome P450 epoxygenase activity in rat brain. Neuroscience 194: 241-249, 2011). We conclude that reduction of endogenous EETs may be one of the mechanisms through which Aβ inflicts toxicity and thus supplementing the cells with exogenous EETs improves mitochondrial dynamics and prevents metabolic impairment.