Targeting Peroxisome Proliferator Activated Receptor α (PPAR α) for the Prevention of Mitochondrial Impairment and Hypertrophy in Cardiomyocytes

Background/Aims: Morphological and biochemical maladaptation of cardiomyocytes are associated with mitochondrial dysfunction and dysregulation in hypertrophic conditions. Peroxisome proliferator activated receptor α (PPARα), a drug target for dyslipidemia, is known to be downregulated in cardiomyocytes in response to hypertrophic stimuli. The current study was undertaken to investigate the role of PPARα signaling in mitochondrial remodeling and thereby dysregulation of cardiomyocytes due to hypertrophy in vitro. Methods: Rat cardiomyocytes H9c2 (2-1) and neonatal rat ventricular myocytes (NRVMs) were cultured and treated with α1-adrenergic agonist phenylephrine (PE, 100 µM, 24 hours) in the presence or absence of 10 µM fenofibrate or bezafibrate. Cellular hypertrophy was observed by atomic force microscopy and immunofluorescence with F-actin antibody. mRNA levels of hypertrophic marker genes and other genes were examined by quantitative real time PCR. Structural as well as functional remodeling of the mitochondria were evaluated by immunofluorescence (F-actin and COX-I), live cell imaging microscopy (JC-I, mitotracker), mitochondrial complex V activity, MPTP activity and ATP assay. Oxidative stress was measured by using sensitive fluorescent indicator probes. Cellular and mitochondrial calcium were measured by using fluorescent indicator probes Rhod-2 AM and X-rhod-1 AM, respectively. Targetscan prediction analysis was performed to find out miRNAs as putative regulators of VDAC. Luciferase assay was conducted to confirm binding of miR28 with VDAC. Results: Co-treatment of H9c2(2-1) cells with PE and fenofibrate restricted increase in cell size and expression of marker genes such as atrial-natriuretic peptide (ANP), brain-natriuretic peptide (BNP) and β-myosin heavy chain (β-MHC) compared to those with PE alone. Fenofibrate prevented PE-induced down regulation of PPARα-target genes like CPT-I and MCAD. Mitochondrial trans-membrane potential (Δψm) and motility were reduced by PE which were significantly checked by fenofibrate. Increased ROS production and calcium level in PE-treated cells were ameliorated by fenofibrate. Mitochondrial activity and ATP generation were reduced by PE which was rescued by fenofibrate. Fenofibrate also prevented PE-induced down regulation of mitochondrial genes like VDAC-I and COX-IV. Expression of several miRNAs was altered in hypertrophic cardiomyocytes which were restored when co-treated with fenofibrate. miR28 was found to target 3’ untranslated region of VDAC-I. Conclusion: Overall, the results demonstrate that PPARα signaling is critically involved in mitochondrial dysfunction in hypertrophic cardiomyocytes in which miR28 plays a pivotal role

Millisecond radio pulsars with known masses: Parameter values and equation of state models

The recent fast growth of a population of millisecond pulsars with precisely measured mass provides an excellent opportunity to characterize these compact stars at an unprecedented level. This is because the stellar parameter values can be accurately computed for known mass and spin rate and an assumed equation of state (EoS) model. For each of the 16 such pulsars and for a set of EoS models from nucleonic, hyperonic, strange quark matter and hybrid classes, we numerically compute fast spinning stable stellar parameter values considering the full effect of general relativity. This first detailed catalogue of the computed parameter values of observed millisecond pulsars provides a testbed to probe the physics of compact stars, including their formation, evolution and EoS. We estimate uncertainties on these computed values from the uncertainty of the measured mass, which could be useful to quantitatively constrain EoS models. We note that the largest value of the central density ρc in our catalogue is ∼5.8 times the nuclear saturation density ρsat, which is much less than the expected maximum value 13ρsat. We argue that the ρc-values of at most a small fraction of compact stars could be much larger than 5.8ρsat. Besides, we find that the constraints on EoS models from accurate radius measurements could be significantly biased for some of our pulsars, if stellar spinning configurations are not used to compute the theoretical radius values

Probabilistic Quantum Teleportation

We consider a generalized quantum teleportation protocol for an unknown qubit using non-maximally entangled state as a shared resource. Without recourse to local filtering or entanglement concentration, using standard Bell-state measurement and classical communication one cannot teleport the state with unit fidelity and unit probability. We show that using non-maximally entangled measurements one can teleport an unknown state with unit fidelity albeit with reduced probability, hence probabilistic teleportation. We also give a generalized protocol for entanglement swapping using non-maximally entangled states.Comment: Latex file, 11 pages, No figure

Newborn Aides: An Innovative Approach in Sick Newborn Care at a District-level Special Care Unit

A Sick Newborn Care Unit (SNCU), established in a district hospital in India, substantially reduced the neonatal mortality rate in the district; it, however, suffered from a dearth of trained nurses. Local girls with 10-12 years of school education underwent structured and hands-on training for six months, followed by a six-month internship at the SNCU and were assigned to it as stipendiary ‘Newborn Aides’. Based on the results of formal examinations, internal on-the-job assessment and interview of doctors, nurses, and parents and their technical skills and motivation were rated very high. Although the incremental cost of training is small, the cost of sustaining them, i.e. stipend and replacing attrition, needs to be addressed. Trained Newborn Aides may substantially alleviate human-resource constraint for SNCUs and Sick Newborn Stabilization units in smaller peripheral hospitals for care of sick newborns at an affordable cost

Ca2+-Dependent Interaction between FKBP12 and Calcineurin Regulates Activity of the Ca2+ Release Channel in Skeletal Muscle

AbstractCalcineurin is a Ca2+ and calmodulin-dependent protein phosphatase with diverse cellular functions. Here we examined the physical and functional interactions between calcineurin and ryanodine receptor (RyR) in a C2C12 cell line derived from mouse skeletal muscle. Coimmunoprecipitation experiments revealed that the association between RyR and calcineurin exhibits a strong Ca2+ dependence. This association involves a Ca2+ dependent interaction between calcineurin and FK506-binding protein (FKBP12), an accessory subunit of RyR. Pretreatment with cyclosporin A, an inhibitor of calcineurin, enhanced the caffeine-induced Ca2+ release (CICR) in C2C12 cells. This effect was similar to those of FK506 and rapamycin, two drugs known to cause dissociation of FKBP12 from RyR. Overexpression of a constitutively active form of calcineurin in C2C12 cells, ΔCnA(391–521) (deletion of the last 131 amino acids from calcineurin), resulted in a decrease in CICR. This decrease in CICR activity was partially recovered by pretreatment with cyclosporin A. Furthermore, overexpression of an endogenous calcineurin inhibitor (cain) or an inactive form of calcineurin (ΔCnA(H101Q)) in C2C12 cells resulted in up-regulation of CICR. Taken together, our data suggest that a trimeric-interaction among calcineurin, FKBP12, and RyR is important for the regulation of the RyR channel activity and may play an important role in the Ca2+ signaling of muscle contraction and relaxation

Evaluation of the OPTC gene in primary open angle glaucoma: functional significance of a silent change

BACKGROUND: We investigated the molecular basis of primary open-angle glaucoma (POAG) using Opticin (OPTC) as a candidate gene on the basis of its expression in the trabecular meshwork cells involved in the disease pathogenesis. Two hundred POAG patients and 100 controls were enrolled in this study. The coding sequence of OPTC was amplified by PCR from genomic DNA of POAG patients, followed by SSCP, DHPLC and DNA sequencing. Subsequent bioinformatic analysis, site-directed mutagenesis, quantitative RT-PCR and western blot experiments were performed to address the functional significance of a 'silent' change in the OPTC coding region while screening for mutations in POAG patients. RESULTS: We detected two missense (p.Glu66Gly & p.Ile89Thr) and one silent change (p.Phe162Phe; c.602 C>T) that was present in 3 different patients but in none of the 100 controls screened. The mutant (c.602T) mRNA was predicted to have remarkably different secondary structure compared to the wild-type transcript by in silico approaches. Subsequent wet-lab experiments showed lower expression of the gene both at the mRNA and protein levels. CONCLUSION: Our study suggests OPTC as a candidate gene for POAG. Further, it highlights the importance of investigating the 'silent' variations for functional implication that might not be apparent from only in silico analysis

Development and physicochemical evaluation of bilayered floating tablet of diltiazem hydrochloride prepared from Plantago ovata seed husk

The aim of the present study is to prepare bilayered floating tablets of diltiazem with different ratios of polymers like HPMC K4M, carbopol 934 P, sodium alginate, Plantago ovata seed husk (psyllium) and to carry out evaluation of the physicochemical parameters of tablets like hardness, friability, content uniformity, weight variation, in vitro buoyancy and in vitro dissolution profiles. In vivo X-ray study was done in human volunteers to determine the floating characteristics of the placebo tablets for a period of 12 h.Colegio de Farmacéuticos de la Provincia de Buenos Aire

Interaction of annexin A6 with alpha actinin in cardiomyocytes

<p>Abstract</p> <p>Background</p> <p>Annexins are calcium dependent phospholipid binding proteins that are expressed in a wide variety of tissues and implicated in various extra- and intracellular processes. In myocardial tissue, annexins A2, A5 and A6 are particularly abundant, of which the expression levels of annexin A6 has been found to be maximal. Conflicting reports from transgenic mice overexpressing annexin A6 or null mice lacking annexin A6 showed imbalances in intracellular calcium turnover and disturbed cardiac contractility. However, few studies have focussed on the signalling module of annexin A6 in the heart either in normal or in pathological state.</p> <p>Results</p> <p>To identify the putative binding partners of annexin A6 in the heart, ventricular extracts were subjected to glutathione S-transferase (GST)- annexin A6 pull down assay and the GST- annexin A6 bound proteins were identified by mass spectrometry. The pull down fractions of ventricular extracts with GST-full length annexin A6 as well as GST-C terminus deleted annexin A6 when immunoblotted with anti sarcomeric alpha (α)-actinin antibody showed the presence of α-actinin in the immunoblot which was absent when GST-N terminus deleted annexin A6 was used for pull down. Overexpression of green fluorescent protein (GFP) tagged full length annexin A6 showed z-line like appearance in cardiomyocytes whereas GFP-N termimus deleted annexin A6 was mostly localized to the nucleus. Overexpression of GFP-C terminus deleted annexin A6 in cardiomyocytes showed aggregate like appearance in the cytoplasm. Double immunofluorescent staining of cardiomyocytes with anti annexin A6 and anti sarcomeric α-actinin antibodies showed perfect co-localization of these two proteins with annexin A6 appearing like a component of sarcomere. Transient knockdown of annexin A6 in cardiomyocytes by shRNA significantly enhances the contractile functions but does not affect the z-band architecture, as revealed by α-actinin immunostaining in shRNA treated cells.</p> <p>Conclusions</p> <p>In overall, the present study demonstrated for the first time that annexin A6 physically interacts with sarcomeric α-actinin and alters contractility of cardiomyocytes suggesting that it might play important role in excitation and contraction process.</p