Autophagy: in: Cell Death

Autophagy is a conserved proteolytic mechanism that degrades cytoplasmic material including cell organelles. Although the importance of autophagy for cell homeostasis and survival has long been appreciated, our understanding of how autophagy is regulated at a molecular level just recently evolved. The importance of autophagy for the quality control of proteins is underscored by the fact that many neurodegenerative and myodegenerative diseases are characterized by an increased but still insufficient autophagic activity. Similarly, if the cellular stress, leading to deoxyribonucleic acid (DNA) damage, mitochondrial damage and/or damaged proteins, does not result in sufficient autophagic repair mechanisms, cells seem to be prone to transform into tumour cells. Therefore, autophagy has multiple roles to play in the causation and prevention of human diseases.

Establishment of a novel intervertebral disc/endplate culture model: analysis of an ex vivo in vitro whole-organ rabbit culture system

STUDY DESIGN: Ex vivo in vitro study evaluating a novel intervertebral disc/endplate culture system. OBJECTIVES: To establish a whole-organ intervertebral disc culture model for the study of disc degeneration in vitro, including the characterization of basic cell and organ function. SUMMARY OF BACKGROUND DATA: With current in vivo models for the study of disc and endplate degeneration, it remains difficult to investigate the complex disc metabolism and signaling cascades. In contrast, more controlled but simplified in vitro systems using isolated cells or disc fragments are difficult to culture due to the unconstrained conditions, with often-observed cell death or cell dedifferentiation. Therefore, there is a demand for a controlled culture model with preserved cell function that offers the possibility to investigate disc and endplate pathologies in a structurally intact organ. METHODS: Naturally constrained intervertebral disc/endplate units from rabbits were cultured in multi-well plates. Cell viability, metabolic activity, matrix composition, and matrix gene expression profile were monitored using the Live/Dead cell viability test (Invitrogen, Basel, Switzerland), tetrazolium salt reduction (WST-8), proteoglycan and deoxyribonucleic acid quantification assays, and quantitative polymerase chain reaction. RESULTS: Viability and organ integrity were preserved for at least 4 weeks, while proteoglycan and deoxyribonucleic acid content decreased slightly, and matrix genes exhibited a degenerative profile with up-regulation of type I collagen and suppression of collagen type II and aggrecan genes. Additionally, cell metabolic activity was reduced to one third of the initial value. CONCLUSIONS: Naturally constrained intervertebral rabbit discs could be cultured for several weeks without losing cell viability. Structural integrity and matrix composition were retained. However, the organ responded to the artificial environment with a degenerative gene expression pattern and decreased metabolic rate. Therefore, the described system serves as a promising in vitro model to study disc degeneration in a whole organ.

Mechanical chondroplasty: early metabolic consequences in vitro

PURPOSE: The purpose of this study was to determine the depth of penetration from mechanical chondroplasty and metabolic consequences of this procedure on the remaining articular cartilage. METHODS: Mechanical chondroplasty was performed in vitro on a portion of fresh grade I or II articular cartilage from 8 human knee arthroplasty specimens. Treated and control (untreated) explants (approximately 30 mg) were cut from the cartilage. The explants were divided into 2 groups, day 1 and day 4, placed separately in a 48-well plate containing media, and incubated at 37 degrees C for 24 hours. After the 24-hour incubation, the explants were weighed on day 1 and day 4, and explant media were removed and tested for total proteoglycan synthesis and aggrecan synthesis. At time 0, 2 sets (2.6 mm each) of treated and control cartilage slices were cut with a precision saw. One set was stained for confocal laser microscopy via a cytotoxicity stain to determine cell viability. The second set was stained with H;E to determine depth of penetration. RESULTS: The mean depth of penetration was 252.8 +/- 78 microm. There was no significant difference (P > .25) between total proteoglycan synthesis for control versus treatment groups on day 1 or 4. Aggrecan synthesis was significantly reduced on day 1 when normalized for tissue weight (P = .019) and double-stranded deoxyribonucleic acid (P = .004). On day 4, no significant difference was detected. Confocal laser microscopy did not show cell death below the zone of treatment. CONCLUSIONS: There was no significant metabolic consequence caused by chondroplasty to the remaining articular cartilage, and the zone of injury was limited to the treatment area. CLINICAL RELEVANCE: Mechanical chondroplasty causes no significant metabolic consequences to articular cartilage under these conditions.

The RYR2-encoded ryanodine receptor/calcium release channel in patients diagnosed previously with either catecholaminergic polymorphic ventricular tachycardia or genotype negative, exercise-induced long QT syndrome: a comprehensive open reading frame mutational analysis.

OBJECTIVES This study was undertaken to determine the spectrum and prevalence of mutations in the RYR2-encoded cardiac ryanodine receptor in cases with exertional syncope and normal corrected QT interval (QTc). BACKGROUND Mutations in RYR2 cause type 1 catecholaminergic polymorphic ventricular tachycardia (CPVT1), a cardiac channelopathy with increased propensity for lethal ventricular dysrhythmias. Most RYR2 mutational analyses target 3 canonical domains encoded by <40% of the translated exons. The extent of CPVT1-associated mutations localizing outside of these domains remains unknown as RYR2 has not been examined comprehensively in most patient cohorts. METHODS Mutational analysis of all RYR2 exons was performed using polymerase chain reaction, high-performance liquid chromatography, and deoxyribonucleic acid sequencing on 155 unrelated patients (49% females, 96% Caucasian, age at diagnosis 20 +/- 15 years, mean QTc 428 +/- 29 ms), with either clinical diagnosis of CPVT (n = 110) or an initial diagnosis of exercise-induced long QT syndrome but with QTc <480 ms and a subsequent negative long QT syndrome genetic test (n = 45). RESULTS Sixty-three (34 novel) possible CPVT1-associated mutations, absent in 400 reference alleles, were detected in 73 unrelated patients (47%). Thirteen new mutation-containing exons were identified. Two-thirds of the CPVT1-positive patients had mutations that localized to 1 of 16 exons. CONCLUSIONS Possible CPVT1 mutations in RYR2 were identified in nearly one-half of this cohort; 45 of the 105 translated exons are now known to host possible mutations. Considering that approximately 65% of CPVT1-positive cases would be discovered by selective analysis of 16 exons, a tiered targeting strategy for CPVT genetic testing should be considered.