Autophagy and cardiac diseases: Therapeutic potential of natural products.

The global incidence of cardiac diseases is expected to increase in the coming years, imposing a substantial socioeconomic burden on healthcare systems. Autophagy is a tightly regulated lysosomal degradation mechanism important for cell survival, homeostasis, and function. Accumulating pieces of evidence have indicated a major role of autophagy in the regulation of cardiac homeostasis and function. It is well established that dysregulation of autophagy in cardiomyocytes is involved in cardiac hypertrophy, myocardial infarction, diabetic cardiomyopathy, and heart failure. In this sense, autophagy seems to be an attractive therapeutic target for cardiac diseases. Recently, multiple natural products/phytochemicals, such as resveratrol, berberine, and curcumin have been shown to regulate cardiomyocyte autophagy via different pathways. The autophagy-modifying capacity of these compounds should be taken into consideration for designing novel therapeutic agents. This review focuses on the role of autophagy in various cardiac diseases and the pharmacological basis and therapeutic potential of reported natural products in cardiac diseases by modifying autophagic processes.

T-bet transduction enhances anti-tumor efficacy of IFN-producing dendritic cell (IKDC) against hepatocellular carcinoma via apoptosis induction.

Hepatocellular carcinoma (HCC) remains a public health challenge that requires dedication to develop new treatment options due to its high recurrence rate and poor prognosis. Interferon-producing killer dendritic cell (IKDC) is a subset of INF-γ secreting immune cells that modulates acquired immunity and possesses cytolytic ability. We modified IKDC isolated from the murine spleen with T-bet lentiviral transduction to enhance its cytotoxicity against HCC, and acquired IKDC overexpressing T-bet (T-bet-IKDC) for the first time. T-bet-IKDC has increased INF-γ secretion and surface expression of NKG2D and TRAIL. In vitro study by MTS assay and flow cytometry showed enhanced anti-tumor effect against H22 cells via apoptosis induction in a dose- and time-dependent manner. In vivo study on H22-bearing mice confirmed increased INF-γ secretion, reduced tumor size, increased caspase 3 cleavage, and up-regulation of cytotoxic molecules after T-bet-IKDC administration. The study suggested prospective application of T-bet-IKDC in future immunotherapy for HCC treatment.

Urolithin B, a gut microbiota metabolite, protects against myocardial ischemia/reperfusion injury via p62/Keap1/Nrf2 signaling pathway.

Ischemia/reperfusion (IR) induces additional damage during the restoration of blood flow to ischemic myocardium. Urolithin B (UB) is one of the gut metabolites of ellagitannins, a class of antioxidant polyphenols, which was found to be protective against oxidative stress in multiple organs. However, the role of UB in cardiovascular disease remains elusive. Adult Sprague Dawley rats were subjected to left anterior descending artery ligation for 30 min followed by 120 min of reperfusion, with or without UB treatment. In vitro, the H9c2 cardiomyocytes were subjected to hypoxia (94 %N2/5 %CO2/1 %O2) for 3 h, followed by reoxygenation (74 %N2/5 %CO2/21 %O2) for 3 h (HR). UB was found to decrease myocardial infarct size and attenuate the cardiac dysfunction in the rats after IR, and protect against HR injury in H9c2 cardiomyocytes. Mechanistically, UB inhibited autophagy by activating Akt/mTOR/ULK1 pathway and protected against oxidative stress and caspase 3-dependent cell apoptosis. In particular, UB induced accumulation of p62 and its interaction with Keap1, which promoted Nrf2 nuclear translocation during HR insult. Of note, the protection of UB against superoxide production and apoptotic cell death was compromised with Nrf2 gene silencing. Taken together, our findings suggested that UB protected against myocardial IR injury at least partially via the p62/Keap1/Nrf2 signaling pathway, which highlights the potential of UB as a novel therapy for ischemic heart disease.

Nobiletin attenuates adverse cardiac remodeling after acute myocardial infarction in rats via restoring autophagy flux.

BACKGROUND: Our previous study showed that autophagy flux was impaired with sustained heart ischemia, which exacerbated adverse cardiac remodeling after acute myocardial infarction (AMI). Here we investigated whether Nobiletin, a citrus polymethoxylated flavonoids, could restore the autophagy flux and improve cardiac prognosis after AMI. AMI was induced by ligating left anterior descending (LAD) coronary artery in rats. Nobiletin improved the post-infarct cardiac dysfunction significantly and attenuated adverse cardiac remodeling. Meanwhile, Nobiletin protected H9C2 cells against oxygen glucose deprivation (OGD) in vitro. The impaired autophagy flux due to ischemia was ameliorated after Nobiletin treatment by testing the autophagy substrate, LC3BⅡ and P62 protein level both in vivo and in vitro. GFP-mRFP-LC3 adenovirus transfection also supported that Nobiletin restored the impaired autophagy flux. Specifically, the autophagy flux inhibitor, chloroquine, but not 3 MA, alleviated Nobiletin-mediated protection against OGD. Notably, Nobiletin does not affect the activation of classical upstream autophagy signaling pathways. However, Nobiletin increased the lysosome acidation which also supported that Nobiletin accelerated autophagy flux. Taken together, our findings suggested that Nobiletin restored impaired autophagy flux and protected against acute myocardial infarction, suggesting a potential role of autophagy flux in Nobiletin-mediated myocardial protection.

Identification and characterization of coiled-coil motifs across Autographa californica multiple nucleopolyhedrovirus genome.

Coiled coils (CCs) are protein structural motifs universally found in proteins and mediate a plethora of biological interactions, and thus their reliable annotation is crucial for studies of protein structure and function. Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is a large double-stranded DNA (dsDNA) virus and encodes 154 proteins. In this study, genome-wide scans of previously uncharacterized CC motifs throughout AcMNPV was conducted using CC prediction software. In total, 24 CC motifs in 19 CC proteins with high confidence were identified. The characteristic of viral CC motifs were analyzed. The CC proteins could be divided into 12 viral structural proteins and 7 non-structural proteins, including viral membrane fusion proteins, enzymes, and transcription factors. Moreover, CC motifs are conserved in the baculoviral orthologs of 14 of the 19 proteins. It is noted that five CC proteins, including Ac51, Ac66, Exon0, Ac13, and GP16, were previously identified to function in the nuclear egress of nucleocapsids, and Ac66 contains multiple CC motifs, the longest of which comprises 252 amino acids, suggesting a role of CC motifs in this process. Taken together, the CC motifs identified in this study are valuable resource for studying protein function and protein interaction networks during virus replication.

Nobiletin ameliorates cardiac impairment and alleviates cardiac remodeling after acute myocardial infarction in rats via JNK regulation.

Nobiletin was found to protect against acute myocardial infarction (AMI)-induced cardiac function decline and myocardial remodeling, although the dose-effect relationship and underlying pathways remained unclear. In the current research, different doses of Nobiletin (7.5, 15 and 30 mg/kg/day) were administered to AMI rat model for 21 days. Survival rate, echocardiography, and histological analysis were assessed in vivo. In addition, MTT assay, flow cytometry, and Western blotting were conducted to explore Nobiletin's cytotoxicity and antiapoptotic effect on H9C2 cells. Mechanistically, the activation of MAPK effectors and p38 in vivo was studied. The results showed medium- and high-dose Nobiletin could significantly improve survival rate and cardiac function and reduce the area of infarction and cardiac fibrosis. Medium dose showed the best protection on cardiac functions, whereas high dose showed the best protective effect on cellular apoptosis and histological changes. JNK activation was significantly inhibited by Nobiletin in vivo, which could help to explain the partial contribution of autophagy to AMI-induced apoptosis and the discrepancy on dose-effect relationships. Together, our study suggested that JNK inhibition plays an important role in Nobiletin-induced antiapoptotic effect in myocardial infarction, and medium-dose Nobiletin demonstrated the strongest effect in vivo.

Functional ion channels in mouse cardiac c-kit(+) cells.

Cardiac c-kit(+) cells are generally believed to be the major population of stem/progenitor cells in the heart and can be used as a cell source for cardiomyoplasty; however, the cellular electrophysiological properties are not understood in this type of cells. The present study was designed to investigate functional ion channels in undifferentiated mouse cardiac c-kit(+) cells using approaches of whole cell patch voltage clamp, RT-PCR, and cell proliferation assay. It was found that three types of ionic currents were present in mouse cardiac c-kit(+) cells, including a delayed rectifier K(+) current (IK(DR)) inhibited by 4-aminopyridine (4-AP), an inward rectifier K(+) current (I(Kir)) decreased by Ba(2+), and a volume-sensitive chloride current (I(Cl.vol)) inhibited by 5-nitro-1-(3-phenylpropylamino) benzoic acid (NPPB). RT-PCR revealed that the corresponding ion channel genes, Kv1.1, Kv1.2, and Kv1.6 (for IK(DR)), Kir.1.1, Kir2.1, and Kir2.2 (likely responsible for I(Kir)), and Clcn3 (for I(Cl.vol)), were significant in mouse cardiac c-kit(+) cells. The inhibition of I(Cl.vol) with NPPB and niflumic acid, but not IK(DR) with 4-AP and tetraethylammonium, reduced cell proliferation and accumulated the cell progression at G(0)/G(1) phase in mouse cardiac c-kit(+) cells. Our results demonstrate that three types of functional ion channel currents (i.e., IK(DR), I(Kir), and I(Cl.vol)) are present in mouse cardiac c-kit(+) cells, and I(Cl.vol) participates in regulating cell proliferation.

Autophagy is an important action mode for functionalized selenium nanoparticles to exhibit anti-colorectal cancer activity.

Selenium nanoparticles (SeNPs) have attracted much interest as potential anticancer nanodrugs. Our previous studies also demonstrated that SeNPs could be developed as carriers of clinically used anticancer drugs to achieve synergistic efficacy. Here, we describe the synthesis of Pleurotus tuber-regium (PTR)-conjugated SeNPs (PTR-SeNPs) and their application in the treatment of colorectal cancer (CRC), which is one of the principal causes of cancer morbidity and mortality in the world. PTR-SeNPs were absorbed by cancer cells via clathrin-mediated endocytosis into lysosomes and caveolae-mediated endocytosis into the Golgi apparatus. Internalized PTR-SeNPs trigger intracellular dose- and time-dependent G2/M phase arrest and apoptosis. Moreover, as shown by using a pEGFP-LC3 plasmid transfection model, PTR-SeNPs activate autophagy to promote the death of cancer cells via upregulation of beclin 1-related signaling pathways. In summary, this study demonstrates the high efficacy of functionalized SeNPs for therapy of colorectal cancer and reveals the important role of autophagy in promoting apoptosis and cell cycle arrest to induce cell death.

Induction of apoptosis and cell cycle arrest in A549 human lung adenocarcinoma cells by surface-capping selenium nanoparticles: an effect enhanced by polysaccharide-protein complexes from Polyporus rhinocerus.

Surface-capping agents play key roles in cellular uptake and biological activity of functional nanomaterials. In the present study, functionalized selenium nanoparticles (SeNPs) have been successfully synthesized using Polyporus rhinocerus water-soluble polysaccharide-protein complexes (PRW) as the capping agent during the reduction of selenium salts. The acquired monodisperse, spherical PRW-SeNPs presented desirable size distribution and stability in the solution. Moreover, PRW surface decoration significantly enhanced the cellular uptake of SeNPs via endocytosis. Exposure to PRW-SeNPs significantly inhibited the growth of A549 cells through induction of apoptosis and G2/M phase arrest (IC50 = 4.06 ± 0.25 µM) supported by an increase of sub-G1 and G2/M phase cell populations, DNA fragmentation, and chromatin condensation. Caspase-3/8 activation induced by PRW-SeNPs indicated that the activation of death receptors was the main cause of PRW-SeNP-induced apoptosis. Collectively, the results suggest that it is highly efficient to use PRW as a surface decorator of SeNPs to enhance cellular uptake and anticancer efficacy, and the PRW-SeNPs are potential chemopreventive agents for lung cancer therapy.

Ascorbic acid enhances the cardiac differentiation of induced pluripotent stem cells through promoting the proliferation of cardiac progenitor cells.

Generation of induced pluripotent stem cells (iPSCs) has opened new avenues for the investigation of heart diseases, drug screening and potential autologous cardiac regeneration. However, their application is hampered by inefficient cardiac differentiation, high interline variability, and poor maturation of iPSC-derived cardiomyocytes (iPS-CMs). To identify efficient inducers for cardiac differentiation and maturation of iPSCs and elucidate the mechanisms, we systematically screened sixteen cardiomyocyte inducers on various murine (m) iPSCs and found that only ascorbic acid (AA) consistently and robustly enhanced the cardiac differentiation of eleven lines including eight without spontaneous cardiogenic potential. We then optimized the treatment conditions and demonstrated that differentiation day 2-6, a period for the specification of cardiac progenitor cells (CPCs), was a critical time for AA to take effect. This was further confirmed by the fact that AA increased the expression of cardiovascular but not mesodermal markers. Noteworthily, AA treatment led to approximately 7.3-fold (miPSCs) and 30.2-fold (human iPSCs) augment in the yield of iPS-CMs. Such effect was attributed to a specific increase in the proliferation of CPCs via the MEK-ERK1/2 pathway by through promoting collagen synthesis. In addition, AA-induced cardiomyocytes showed better sarcomeric organization and enhanced responses of action potentials and calcium transients to ß-adrenergic and muscarinic stimulations. These findings demonstrate that AA is a suitable cardiomyocyte inducer for iPSCs to improve cardiac differentiation and maturation simply, universally, and efficiently. These findings also highlight the importance of stimulating CPC proliferation by manipulating extracellular microenvironment in guiding cardiac differentiation of the pluripotent stem cells.

In vitro differentiation of rat embryonic stem cells into functional cardiomyocytes.

The recent breakthrough in the generation of rat embryonic stem cells (rESCs) opens the door to application of gene targeting to create models for the study of human diseases. In addition, the in vitro differentiation system from rESCs into derivatives of three germ layers will serve as a powerful tool and resource for the investigation of mammalian development, cell function, tissue repair, and drug discovery. However, these uses have been limited by the difficulty of in vitro differentiation. The aims of this study were to establish an in vitro differentiation system from rESCs and to investigate whether rESCs are capable of forming terminal-differentiated cardiomyocytes. Using newly established rESCs, we found that embryoid body (EB)-based method used in mouse ESC (mESC) differentiation failed to work for the serum-free cultivated rESCs. We then developed a protocol by combination of three chemical inhibitors and feeder-conditioned medium. Under this condition, rESCs formed EBs, propagated and differentiated into three embryonic germ layers. Moreover, rESC-formed EBs could differentiate into spontaneously beating cardiomyocytes after plating. Analyses of molecular, structural, and functional properties revealed that rESC-derived cardiomyocytes were similar to those derived from fetal rat hearts and mESCs. In conclusion, we successfully developed an in vitro differentiation system for rESCs through which functional myocytes were generated and displayed phenotypes of rat fetal cardiomyocytes. This unique cellular system will provide a new approach to study the early development and cardiac function, and serve as an important tool in pharmacological testing and cell therapy.