LAMC2 mitigates ER stress by enhancing ER-mitochondria interaction via binding to MYH9 and MYH10.

Highly proliferative and metastatic tumors are constantly exposed to both intrinsic and extrinsic factors that induce adaptation to stressful conditions. Chronic adaptation to endoplasmic reticulum (ER) ER stress is common to many different types of cancers, and poses a major challenge for acquired drug resistance. Here we report that LAMC2, an extracellular matrix protein upregulated in many types of cancers, is localized in the ER of lung, breast, and liver cancer cells. Under tunicamycin-induced ER stress, protein level of LAMC2 is upregulated. Transfection of cancer cells with LAMC2 resulted in the attenuation of ER stress phenotype, accompanied by elevation in mitochondrial membrane potential as well as reduction in reactive oxygen species (ROS) levels and apoptosis. In addition, LAMC2 forms protein complexes with MYH9 and MYH10 to promote mitochondrial aggregation and increased ER-mitochondria interaction at the perinuclear region. Moreover, overexpression of LAMC2 counteracts the effects of ER stress and promotes tumor growth in vivo. Taken together, our results revealed that in complex with MYH9 and MYH10, LAMC2 is essential for promoting ER-mitochondria interaction to alleviate ER stress and allow cancer cells to adapt and proliferate under stressful conditions. This study provides new insights and highlights the promising potential of LAMC2 as a therapeutic target for cancer treatment.

Fluorescent hiPSC-derived MYH6-mScarlet cardiomyocytes for real-time tracking, imaging, and cardiotoxicity assays.

Human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) hold great potential in the cardiovascular field for human disease modeling, drug development, and regenerative medicine. However, multiple hurdles still exist for the effective utilization of hiPSC-CMs as a human-based experimental platform that can be an alternative to the current animal models. To further expand their potential as a research tool and bridge the translational gap, we have generated a cardiac-specific hiPSC reporter line that differentiates into fluorescent CMs using CRISPR-Cas9 genome editing technology. The CMs illuminated with the mScarlet fluorescence enable their non-invasive continuous tracking and functional cellular phenotyping, offering a real-time 2D/3D imaging platform. Utilizing the reporter CMs, we developed an imaging-based cardiotoxicity screening system that can monitor distinct drug-induced structural toxicity and CM viability in real time. The reporter fluorescence enabled visualization of sarcomeric disarray and displayed a drug dose-dependent decrease in its fluorescence. The study also has demonstrated the reporter CMs as a biomaterial cytocompatibility analysis tool that can monitor dynamic cell behavior and maturity of hiPSC-CMs cultured in various biomaterial scaffolds. This versatile cardiac imaging tool that enables real time tracking and high-resolution imaging of CMs has significant potential in disease modeling, drug screening, and toxicology testing.

Kallikrein-related peptidase-8 (KLK8) aggravated hypoxia-induced right ventricular hypertrophy by targeting P38 MAPK/P53 signaling pathway.

Right ventricular (RV) hypertrophy and further heart failure are major co-morbidities, resulting in the premature death of patients with hypoxic pulmonary hypertension (HPH). The regulatory effects of kallikrein-related peptidase (KLK) family members on cardiac function have been extensively studied. However, to the best of the authors' knowledge, the regulatory effects of KLK8 on RV hypertrophy caused by HPH have yet to be reported. The aim of the present study was to assess KLK8 expression in the RV tissue of HPH-modeled rats, and to further explore the effects and underlying mechanism of KLK8 in regulating the hypertrophy of hypoxia-induced H9c2 cardiomyocytes. In HPH model rats, increases in the right ventricle hypertrophy index, the right ventricular systolic pressure, cardiac output, as well as pulmonary artery wall thickness were observed. Western blot analysis revealed that KLK8 expression and MAPK/p53 signaling activity were enhanced in the RVs of rats in an RV HPH rat model. In hypoxia-induced H9c2 cardiomyocytes, KLK8 overexpression promoted cardiomyocyte hypertrophy, whereas KLK8 silencing showed the opposite results. KLK8 overexpression increased the expression levels of ventricular hypertrophy markers, including atrial natriuretic peptide, brain natriuretic peptide and myosin heavy chain 7, which were blocked upon addition of the p38 MAPK inhibitor, SB202190. Conversely, KLK8 silencing caused a decrease in the expression levels of the ventricular hypertrophy markers, which were further reduced via inhibition of the p38 MAPK/p53 signaling pathway. Taken together, the results of the present study have shown that KLK8 may subtly regulate RV hypertrophy, and therefore KLK8 may be a promising therapeutic target for treating HPH-induced RV hypertrophy.

Periurethral and Intravenous Injections of Adipose-Derived Stem Cells to Promote Local Tissue Recovery in a Rat Model of Stress Urinary Incontinence.

OBJECTIVE: To compare the effects of periurethral and intravenous injection of adipose-derived stem cells (ADSCs) on voiding function and tissue recovery in a stress urinary incontinence (SUI) rat model. METHODS: Sixty-four postpartum rats were randomly allocated to a normal group and the SUI model was established in 48 rats by vagina balloon dilation and bilateral ovariectomy. The SUI rats were randomized into 3 groups and received urethral injection of PBS (SUI group), periurethral injection of ADSCs (PU group), and intravenous injection of ADSCs (IV group) in 10 days after the ovariectomy. After 1, 7, and 14 days, ADSCs were tracked in urethra specimen. The urinary function of the remaining rats was analyzed at day 28, and urethral tissues were harvested for Western blotting and histochemical analyses. RESULTS: Alpha smooth muscle actin, myosin heavy chain, vascular endothelial growth factor, and neurofilament protein expression was increased in the IV and PU groups. Voiding function was also improved, with no significant differences between the IV and PU groups. The cell retention rate in rat urethral tissues was higher in the PU group than that in the IV group. Compared with the IV group, myosin heavy chain, vascular endothelial growth factor, neurofilament and transforming growth factor-ß1 (TGF-ß1)/Smad pathway protein expression levels were significantly higher in the PU group, while alpha smooth muscle actin expression was significantly lower (P < .05). CONCLUSION: Periurethral and intravenous injection of ADSCs induces different degrees of recovery of the urethral sphincter, cytokine secretion levels and cell retention rates in the urethral tissues in SUI rats, however, there was no significant difference in 2 methods.

Mononuclear cell secretome protects from experimental autoimmune myocarditis.

AIMS: Supernatants of serum-free cultured mononuclear cells (MNC) contain a mix of immunomodulating factors (secretome), which have been shown to attenuate detrimental inflammatory responses following myocardial ischaemia. Inflammatory dilated cardiomyopathy (iDCM) is a common cause of heart failure in young patients. Experimental autoimmune myocarditis (EAM) is a CD4+ T cell-dependent model, which mirrors important pathogenic aspects of iDCM. The aim of this study was to determine the influence of MNC secretome on myocardial inflammation in the EAM model. METHODS AND RESULTS: BALB/c mice were immunized twice with an alpha myosin heavy chain peptide together with Complete Freund adjuvant. Supernatants from mouse mononuclear cells were collected, dialysed, and injected i.p. at Day 0, Day 7, or Day 14, respectively. Myocarditis severity, T cell responses, and autoantibody formation were assessed at Day 21. The impact of MNC secretome on CD4+ T cell function and viability was evaluated using in vitro proliferation and cell viability assays. A single high-dose application of MNC secretome, injected at Day 14 after the first immunization, effectively attenuated myocardial inflammation. Mechanistically, MNC secretome induced caspase-8-dependent apoptosis in autoreactive CD4+ T cells. CONCLUSION: MNC secretome abrogated myocardial inflammation in a CD4+ T cell-dependent animal model of autoimmune myocarditis. This anti-inflammatory effect of MNC secretome suggests a novel and simple potential treatment concept for inflammatory heart diseases.

Ex vivo analysis of tumor antigen specific CD8+ T cell responses using MHC/peptide tetramers in cancer patients.

The development of soluble tetrameric MHC/peptide complexes has opened the possibility to directly identify and monitor antigen-specific CD8+ T cells in different clinical situations. This represents a technological breakthrough for the field of cell-mediated immunity. For example, the direct identification and enumeration of tumor-specific CD8+ T cells at the tumor site and in blood has recently provided compelling evidence that strong anti-tumoral responses naturally occur in some cancer patients. Moreover, the use of tetramers plays an essential role in the design of vaccination protocols aimed at inducing a strong and protective CD8+ T cell-mediated anti-tumoral response in cancer patients. The monitoring of antigen-specific T cell responses elicited by various peptide-based vaccines tested in phase I clinical trials clearly indicates that tumor-specific CD8+ T cells can be activated effectively at least in some cancer patients. Thus, multiparameter monitoring of antigen-specific T cell responses that combines ex vivo tetramer staining with various phenotyping and functional assays provides a novel approach to assess the functional potential of tumor-specific T lymphocytes and may also facilitate the optimization of vaccination protocols.

Oncogenic src, raf, and ras stimulate a hypertrophic pattern of gene expression and increase cell size in neonatal rat ventricular myocytes.

In response to hormones and growth factors, cultured neonatal ventricular myocytes increase in profile, exhibit myofibrillogenesis, and re-express genes whose expression is normally restricted to the fetal stage of ventricular development. These include atrial natriuretic factor (ANF), beta-myosin heavy chain (beta-MHC), and skeletal muscle (SkM)-alpha-actin. By using luciferase reporter plasmids, we examined whether oncogenes that activate the extracellular signal-regulated kinase cascade (srcF527, Ha-rasV12, and v-raf) increased expression of "fetal" genes. Transfection of myocytes with srcF527 stimulated expression of ANF, SkM-alpha-actin, and beta-MHC by 62-, 6.7-, and 50-fold, respectively, but did not induce DNA synthesis. Stimulation of ANF expression by srcF527 was greater than by Ha-rasV12, which in turn was greater than by v-raf. General gene expression was also increased but to a lesser extent. The response to srcF527 was inhibited by dominant-negative Ha-rasN17. Myocyte area was increased by srcF527, Ha-rasV12, and v-raf, and although it altered myocyte morphology by causing a pseudopodial appearance, srcF527 did not detectably increase myofibrillogenesis either alone or in combination with Ha-rasV12. A kinase-dead src mutant increased myocyte size to a much lesser extent than srcF527 and also did not inhibit ANF-luciferase expression in response to phenylephrine. We conclude that members of the Src family of tyrosine kinases may be important in mediating the transcriptional changes occurring during cardiac myocyte hypertrophy and that Ras and Raf may be downstream effectors.

A myosin-derived peptide C109 binds to GLUT4-vesicles and inhibits the insulin-induced glucose transport stimulation and GLUT4 recruitment in rat adipocytes.

The yeast-based two-hybrid screening of a human cardiac myocyte cDNA library revealed a peptide, C109 that interacted with the C-terminal cytoplasmic domain of GLUT4 (GLUT4C). cDNA-deduced amino acid sequence of C109 was identical to the human cardiac muscle myosin heavy chain beta isoform sequence 1469-1909. GST-fusion protein of C109 (GST-C109) bound synthetic GLUT4C-peptide in vitro, but not GLUT1C-peptide. GST-C109 avidly bound to the GLUT4-vesicles isolated from basal rat adipocytes but not those isolated from insulin treated adipocytes. Furthermore, the incorporation of C109 into rat adipocytes greatly reduced the plasma membrane GLUT4 level and the 3-O-methyl D glucose flux in host cells without affecting total cellular GLUT4 content. These findings suggest that myosin or a myosin-like protein plays a key role in insulin-regulated movement of GLUT4 to the plasma membrane in rat adipocytes.