Metformin increases degradation of phospholamban via autophagy in cardiomyocytes

Phospholamban (PLN) is an effective inhibitor of the sarco(endo)plasmic reticulum Ca²⁺ ATPase (SERCA). Here, we examined PLN stability and degradation in primary cultured mouse neonatal cardiomyocytes (CMNCs) and mouse hearts using immunoblotting, molecular imaging, and [³⁵S]methionine pulse-chase experiments, together with lysosome (chloroquine and bafilomycin A1) and autophagic (3-methyladenine and Atg5 siRNA) antagonists. Inhibiting lysosomal and autophagic activities promoted endogenous PLN accumulation, whereas accelerating autophagy with metformin enhanced PLN degradation in CMNCs. This reduction in PLN levels was functionally correlated with an increased rate of SERCA2a activity, accounting for an inotropic effect of metformin. Metabolic labeling reaffirmed that metformin promoted wild-type and R9C PLN degradation. Immunofluorescence showed that PLN and the autophagy marker, microtubule light chain 3, became increasingly colocalized in response to chloroquine and bafilomycin treatments. Mechanistically, pentameric PLN was polyubiquitinylated at the K3 residue and this modification was required for p62-mediated selective autophagy trafficking. Consistently, attenuated autophagic flux in HECT domain and ankyrin repeat-containing E3 ubiquitin protein ligase 1-null mouse hearts was associated with increased PLN levels determined by immunoblots and immunofluorescence. Our study identifies a biological mechanism that traffics PLN to the lysosomes for degradation in mouse hearts.Phospholamban (PLN) is a 52-amino acid peptide located in the sarcoplasmic reticulum (SR) membrane in cardiac, slow-twitch skeletal, and smooth muscle, where it exists as a monomer or pentamer. Whereas monomeric PLN physically interacts with sarco(endo)plasmic reticulum Ca²⁺ ATPase type 2a (SERCA2a) to antagonize its function, pentameric PLN complexes are thought to be a reservoir of inactive PLN (1–3). The physical interaction between SERCA2a and PLN reduces the apparent affinity of SERCA2a for Ca²⁺, thereby making SERCA2a less active in transporting Ca²⁺ from the cytoplasm to the lumen of the SR at the same concentration of cytoplasmic Ca²⁺. The physical interaction between the two proteins is regulated by phosphorylation of PLN at Ser16 by protein kinase A or at Thr17 by Ca²⁺/calmodulin-dependent protein kinase II (2). Phosphorylation of PLN reduces its affinity for SERCA2a, thereby increasing SERCA2a activity (2). Evidence from transgenic mice also supports the inhibitory function of PLN. Although targeted PLN deletion enhances baseline cardiac performance, cardiac-specific overexpression of superinhibitory forms of PLN leads to decreases in the affinity of SERCA2a for Ca²⁺ (2). These observations underscore the primary role of PLN as a regulator of SERCA2a activity and, therefore, as a crucial regulator of cardiac contractility. PLN inhibition of SERCA2a can be reversed by either external (i.e., activation of β-adrenergic receptors) or internal (i.e., increased intracellular Ca²⁺ concentration) stimuli.Previous studies identified three PLN mutations in families of patients with hereditary dilated cardiomyopathy. These mutations, the substitution of Cys for Arg9 (R9C) (4), Arg14 deletion (RΔ14) (5), and the substitution of TGA for TAA in the Leu39 codon, creating a stop codon (L39stop) (6), also lead to dilated cardiomyopathy in transgenic mice. At the cellular level, ectopically expressed RΔ14 and L39stop PLN mutants localize at the plasma membrane in HEK-293T cells, cultured mouse neonatal cardiomyocytes, and cardiac fibroblasts, whereas wild-type and the R9C mutant reside within the endoplasmic reticulum (ER)/SR (6, 7). These data, together with a recent study by Sharma et al. (8), suggest a highly ordered trafficking of PLN, ultimately ensuring correct localization, and thus function, within the SR. However, PLN trafficking and degradation mechanisms in mammalian cardiomyocytes have not been clearly established.Protein degradation and clearance of damaged organelles are critical for cellular physiology, and failure in proper clearance has been shown to have pathological repercussions (9). Autophagy is a major mechanism that mediates protein and organelle degradation in response to external and internal signals. External stimulation through pharmacological agonists, such as metformin and rapamycin, promotes autophagy via AMP-activated protein kinase (AMPK) and mammalian target of rapamycin signal pathways, whereas amino acid starvation and an increased intracellular AMP/ATP ratio serve as internal signals to promote autophagy via the Ca²⁺/Calmodulin-dependent kinase kinase-β (10). Steps in the autophagy pathway involve nucleation of targeted macromolecules on the ER membrane, trafficking of autophagosomes to lysosomes and, finally, fusion of the autophagosome-lysosome, resulting in targeted protein degradation (11). In the heart, autophagy plays a crucial role in response to insults, in part by relieving ER stress (12) and removing damaged mitochondria (13). Loss of autophagy could result in irreversible apoptosis and reduced cardiac functioning (14).To characterize PLN degradation, we conducted a series of assays in cultured mouse neonatal cardiomyocytes (CMNCs) and the hearts of HECT domain and ankyrin repeat-containing E3 ubiquitin protein ligase 1 (Hace1)-null mice. Our results show that PLN degradation required both polyubiquitinylation and p62-mediated selective autophagy in CMNCs. Loss of HACE1 was associated with increased PLN levels, supporting the notion that selective autophagy modulates PLN degradation in vivo. Metformin promoted wild-type and R9C PLN degradation through autophagic pathways, resulting in metformin-induced inotropic enhancement.     

Prolonged mandibular hypomobility patient with a "square mandible" configuration with coronoid process and angle hyperplasia

The objective of this study was the surgical management of chronic severe mandibular hypomobility patients associated with square mandible morphology with coronoid process and angle hyperplasia, and one-year follow-up data is reported. Ten patients were studied. All patients were female and had a history of gradual severe jaw hypomobility. Clinical findings were similar to those of a "closed lock" patient. However, the facial appearance in these patients showed a characteristic square mandible facial configuration. Coronoid process thickening and overgrowth of the mandibular angle was evident in the radiographic findings. Diagnostic imaging scarcely depicted any disk derangement, but a severely limited jaw opening was noted in spite of acceptable excursive jaw movements. Bilateral coronoidotomy or coronoidectomy was done initially, and then masseter muscle stripping via the intraoral approach. After successful reduction of jaw hypomobility, a selective mandibular anglectomy was completed. Physical therapy began within three to five days after the surgery. Postoperatively, all patients were questioned about their jaw function and their subjective assessment of the treatment. Interincisal jaw opening was recorded with a ruler marked in millimeters. Bilateral coronoidotomy or coronoidectomy and masseter muscle stripping were done for all patients; the mandibular anglectomy was performed in seven of the cases at 13 sites. Simultaneous TMJ surgery was done on three joints for three patients. Most patients reported improvement of jaw function, and the patients' subjective assessment revealed an average satisfaction rate of 74.6%. A preoperative mean jaw opening distance of 25.6 mm increased to 36.6 mm postoperatively at a one-year follow-up (p < 0.05). The conclusion was that surgical intervention is indicated when nonsurgical treatment is unsuccessful. Etiology is unknown, but masseter and temporal muscle contracture associated with mandibular coronoid and angle hyperplasia may be a strong component of the pathophysiology.     

Monophasic epithelial synovial sarcoma arising in the temporomandibular joint

Synovial sarcoma is a mesenchymal spindle-cell tumour that occurs infrequently in the head and neck. It originates from unknown stem cells differentiating into mesenchymal and/or epithelial structures. Most synovial sarcomas are biphasic in character, consisting of epithelial and spindle-cell elements. Here is reported a case of monophasic epithelial synovial sarcoma arising in the temporomandibular joint. The tumour was of a predominantly epithelial pattern, although a minute area of sarcomatous cells was found. The primary mode of treatment was wide en-bloc excision. Two years after surgery, the patient died of hepatocellular carcinoma, but there was no evidence of synovial sarcoma recurrence.     

Efficacy of mandibular manipulation technique for temporomandibular disorders patients with mouth opening limitation: a randomized controlled trial for comparison with improved multimodal therapy

Purpose

Manual therapy has been used for the treatment of patients with temporomandibular disorders (TMD) with mouth-opening limitations. However, the curative effect of manipulation differs among researchers, and its necessity remains controversial. The purpose of this study was to confirm the efficacy of manipulation using a randomized controlled trial (RCT).

Does Nonpayment for Hospital-Acquired Catheter-Associated Urinary Tract Infections Lead to Overtesting and Increased Antimicrobial Prescribing?

Background.?On 1 October 2008, in an effort to stimulate efforts to prevent catheter-associated urinary tract infection (CAUTI), the Centers for Medicare & Medicaid Services (CMS) implemented a policy of not reimbursing hospitals for hospital-acquired CAUTI. Since any urinary tract infection present on admission would not fall under this initiative, concerns have been raised that the policy may encourage more testing for and treatment of asymptomatic bacteriuria. Methods.?We conducted a retrospective multicenter cohort study with time series analysis of all adults admitted to the hospital 16 months before and 16 months after policy implementation among participating Society for Healthcare Epidemiology of America Research Network hospitals. Our outcomes were frequency of urine culture on admission and antimicrobial use. Results.?A total of 39 hospitals from 22 states submitted data on 2?362?742 admissions. In 35 hospitals affected by the CMS policy, the median frequency of urine culture performance did not change after CMS policy implementation (19.2% during the prepolicy period vs 19.3% during the postpolicy period). The rate of change in urine culture performance increased minimally during the prepolicy period (0.5% per month) and decreased slightly during the postpolicy period (-0.25% per month; P?相似文献