Mechanism for autoinhibition and activation of the MORC3 ATPase.

Microrchidia 3 (MORC3) is a human protein linked to autoimmune disorders, Down syndrome, and cancer. It is a member of a newly identified family of human ATPases with an uncharacterized mechanism of action. Here, we elucidate the molecular basis for inhibition and activation of MORC3. The crystal structure of the MORC3 region encompassing the ATPase and CW domains in complex with a nonhydrolyzable ATP analog demonstrates that the two domains are directly coupled. The extensive ATPase:CW interface stabilizes the protein fold but inhibits the catalytic activity of MORC3. Enzymatic, NMR, mutational, and biochemical analyses show that in the autoinhibited, off state, the CW domain sterically impedes binding of the ATPase domain to DNA, which in turn is required for the catalytic activity. MORC3 autoinhibition is released by disrupting the intramolecular ATPase:CW coupling through the competitive interaction of CW with histone H3 tail or by mutating the interfacial residues. Binding of CW to H3 leads to a marked rearrangement in the ATPase-CW cassette, which frees the DNA-binding site in active MORC3 (on state). We show that ATP-induced dimerization of the ATPase domain is strictly required for the catalytic activity and that the dimeric form of ATPase-CW might cooperatively bind to dsDNA. Together, our findings uncovered a mechanism underlying the fine-tuned regulation of the catalytic domain of MORC3 by the epigenetic reader, CW.

The nucleosome position-encoding WW/SS sequence pattern is depleted in mammalian genes relative to other eukaryotes.

Nucleosomal DNA sequences generally follow a well-known pattern with ∼10-bp periodic WW (where W is A or T) dinucleotides that oscillate in phase with each other and out of phase with SS (where S is G or C) dinucleotides. However, nucleosomes with other DNA patterns have not been systematically analyzed. Here, we focus on an opposite pattern, namely anti-WW/SS pattern, in which WW dinucleotides preferentially occur at DNA sites that bend into major grooves and SS (where S is G or C) dinucleotides are often found at sites that bend into minor grooves. Nucleosomes with the anti-WW/SS pattern are widespread and exhibit a species- and context-specific distribution in eukaryotic genomes. Unlike non-mammals (yeast, nematode and fly), there is a positive correlation between the enrichment of anti-WW/SS nucleosomes and RNA Pol II transcriptional levels in mammals (mouse and human). Interestingly, such enrichment is not due to underlying DNA sequence. In addition, chromatin remodeling complexes have an impact on the abundance but not on the distribution of anti-WW/SS nucleosomes in yeast. Our data reveal distinct roles of cis- and trans-acting factors in the rotational positioning of nucleosomes between non-mammals and mammals. Implications of the anti-WW/SS sequence pattern for RNA Pol II transcription are discussed.

Persistence of SARS-CoV-2 nasopharyngeal swab PCR positivity in COVID-19 convalescent plasma donors.

BACKGROUND: Nucleic acid persists after symptom resolution and infectivity for many viral infections via delayed clearance of nucleic acid fragments, non-infectious particles, or transmissible virus. For Coronavirus Disease 2019 (COVID-19), the relationship between nasopharyngeal (NP) swab positivity, the development of antibodies against COVID-19, and clinical history are unclear. STUDY DESIGN AND METHODS: Individuals who recovered from COVID-19 and volunteered to donate convalescent plasma (CP) were screened by NP swab PCR, responded to a questionnaire, and were tested for anti-COVID-19 antibodies. RESULTS: A proportion of 11.8% of individuals tested positive for SARS-CoV-2 by NP swab PCR greater than 14 days after the resolution of symptoms of active disease, including one donor who had asymptomatic disease and tested positive by NP swab 41 days after her initial diagnosis. Clinical history did not show a significant correlation with persistence of NP swab positivity. Also, NP swab positivity >14 days from symptom resolution did not correlate with anti-COVID-19 serology results. IgG anti-SARS-CoV-2 spike antibody strength correlated with hospitalization for COVID-19 using two different assays. Total anti-SARS-CoV-2 nucleocapsid antibody strength correlated with time from symptom resolution to sample collection and symptom duration. CONCLUSIONS: SARS-CoV-2 nucleic acid is detectable long after the resolution of symptoms in a significant percentage of previously diagnosed individuals, which is important to consider when interpreting PCR swab results. Persistence of PCR positivity does not correlate with antibody strength or symptoms of COVID-19. If anti-spike antibody is used to assess CP potency, individuals who suffered severe COVID-19 disease symptoms may represent better donors.

Expansion of hospital-based blood collections in the face of COVID-19 associated national blood shortage.

BACKGROUND: When the coronavirus pandemic caused widespread school and business closures in March 2020, blood drives were canceled and the supply of blood decreased suddenly in the United States (US). In response, hospital-based transfusion medicine physicians instituted policies to conserve blood and decrease blood product usage. These efforts were aided by the US Surgeon General recommendation to cancel all elective procedures. Nevertheless, the duration, severity, and impact of the pandemic on the national blood supply was uncertain. Hospitals with in-house donor programs had the opportunity not only to control demand, but also increase supply. STUDY DESIGN AND METHODS: A hospital-based blood donor center was rapidly mobilized to increase the supply of in-house collected blood, in order to counteract a sudden but potentially long-term depletion of the national blood supply during a pandemic. RESULTS: Collections increased approximately five-fold above baseline for whole blood units, while apheresis platelet units were maintained at the historical average for the blood donor center. Cancellation of elective procedures showed a modest, but not yet statistically significant decrease in average blood product usage per day, nevertheless the in-house collection rate was sufficient to meet demand. CONCLUSION: A hospital-based blood donor center can quickly increase collection volumes and capacity in the face of a national emergency or pandemic. The desire to collect units should be balanced with safety concerns, need for sustainability, and blood product demand.

Aldehyde reduction in a novel pericardial tissue reduces calcification using rabbit intramuscular model.

Calcification is a major factor that limits the durability of bioprosthetic valve. A novel bovine pericardial tissue treated with aldehyde capping chemistry and glycerolization was evaluated for its resistance to calcification in comparison with porcine tissues treated with amino oleic acid and bovine pericardial tissue with ethanol rinsing in a rabbit intramuscular model. Tissue discs from the test and control tissues were implanted in rabbits for 60 days. The explanted discs were subject to X-ray imaging, calcium quantification and histology analysis. The test tissue showed 95 and 96 % reduction in calcification in comparison with amino oleic acid treatment and ethanol rinsing treatment, respectively. In addition, the test tissue showed the least inflammatory response as evidenced by a reduced amount of macrophages and giant cells in histology analysis. Furthermore, the aldehyde analysis of the pre-implanted samples showed associated reduction in free aldehyde levels with the test tissue. The reduction in calcification is consistent with previously reported results and is hypothesized to be attributed to the capping of free aldehydes in the test tissue.

Locator retention: does variation in number and configuration matter?

This pilot study was designed to collect initial data on overdenture attachment retention in varying configurations of attachment location in an implant-retained mandibular overdenture. A clear acrylic model of a mandible with 6 numbered implants and Locator resilient abutments was used to simulate implant placement in a patient. A clear acrylic denture was fabricated with 6 Locator housings to match the implants in the model. Attachments were tested in 4 different configurations: 2 implants, 2 and 5 (T25); 4 implants, 2-5 (T2345); 4 implants, 1, 3, 4, and 6 (T1346); and 6 implants, 1-6 (T1-6). Clear nylon male inserts were used for each test. The mean overall retentive strength across all 20 pulls was 576.0 N for configuration T1-6, 354.9 N for configuration T1346, 350.7 N for configuration T2345, and 189.9 N for configuration T25. Mean retentive strength also stabilized after the 7th pull for all 4 configurations, resulting in nonsignificant declines in retentive strength within each specific configuration after 7 pulls. Configuration T1-6 exhibited the greatest retentive strength relative to all other configurations both initially and after repeated application of force. Configurations T1346 and T2345 had similar retentive strengths, and both had greater retentive strength than T25. However, despite these differences, all 4 configurations exhibited similar losses in retentive strength from the repeated application of force during the first 7 pulls until stabilization occurred shortly thereafter.

Comparison and evaluation of biomechanical, electrical, and biological methods for assessment of damage to tissue collagen.

In regard to evaluating tissue banking methods used to preserve or otherwise treat (process) soft allograft tissue, current tests may not be sufficiently sensitive to detect potential damage inflicted before, during, and after processing. Using controlled parameters, we aim to examine the sensitivity of specific biomechanical, electrical, and biological tests in detecting mild damage to collagen. Fresh porcine pulmonary heart valves were treated with an enzyme, collagenase, and incubated using various times. Controls received no incubation. All valves were cryopreserved and stored at -135 °C until being rewarmed for evaluation using biomechanical, permeability, and cell viability tests. Statistically significant time dependent changes in leaflet ultimate stress, (p = 0.006), permeability (p = 0.01), and viability (p ≤ 0.02, four different days of culture) were found between heart valves subjected to 0-15 min of collagenase treatment (ANOVA). However, no statistical significance was found between the tensile modulus of treated and untreated valves (p = 0.07). Furthermore, the trends of decreasing and increasing ultimate stress and viability, respectively, were somewhat inconsistent across treatment times. These results suggest that permeability tests may offer a sensitive, quantitative assay to complement traditional biomechanical and viability tests in evaluating processing methods used for soft tissue allografts, or when making changes to current validated methods. Multiple test evaluation may also offer insight into the mechanism of potential tissue damage such as, as is the case here, reduced collagen content and increased tissue porosity.

Advanced Integrity Preservation Technology Reduces Bioprosthesis Calcification While Preserving Performance and Safety.

BACKGROUND AND AIM OF THE STUDY: Structural valve deterioration (SVD) is the leading failure mode of bioprosthetic heart valves. The Edwards Integrity-Preservation (EIP™) technology was developed to permanently block tissue calcium-binding sites and allow for non-aqueous valve storage. The study aim was to evaluate the efficacy of tissue anti-calcification, valve performance, durability, and safety. METHODS: Bovine pericardial tissue with EIP technology was compared to industry-standard Carpentier-Edwards ThermaFix® and Xenologix® treatments. Anti-calcification efficacy was evaluated in the rabbit model at 60 days, and tissue calcium contents were quantified using atomic absorption spectrophotometry. Valve performance was assessed using an in-vivo 20-week chronic ovine model, and in-vitro hydrodynamic testing (effective orifice area and regurgitation). Valve durability was evaluated by accelerated wear testing at 200 million cycles (equivalent to five years). Valve safety was characterized by biocompatibility testing as per ISO requirements. RESULTS: Calcification results showed that the control and EIP technology tissues had a mean Ca content of 104.95 ± 102.69 and 21.20 ± 38.46 µg/mg dry tissue, respectively; the median Ca contents were 81.15 and 0.43 µg/mg dry tissue, respectively (p < 0.0001). The overall valve performance in the sheep was comparable between control and test. In-vitro hydrodynamics and durability were similar between groups, across all sizes, and met ISO requirements. EIP technology was shown to be biocompatible for use as an implantable device. CONCLUSION: Preclinical in-vitro and in-vivo evaluations showed that EIP technology significantly reduced tissue calcification and preserved valve performance and safety compared to current standards of care. Future studies will determine whether these findings can be replicated in humans.

Impact of storage solution formulation during refrigerated storage upon chondrocyte viability and cartilage matrix.

Various preservation solutions have been evaluated for longer hypothermic cartilage storage for tissue transplantation; however, the results are mixed. This research was carried out to determine whether phosphate-buffered saline (PBS) or organ preservation solutions would preserve both the extracellular matrix and chondrocytes of articular cartilage better than culture medium during refrigerated storage in the time frame that cartilage is stored for clinical use. Porcine cartilage plugs were stored, without the underlying bone, in culture medium with and without fetal bovine serum (FBS), PBS, Belzer's and Unisol solutions for 1 month at 4°C. Metabolic activity was tested using a resazurin reduction method, and matrix permeability was evaluated by measuring electrical conductivity. Storage in culture medium with 10% FBS was shown to provide good cartilage metabolic function for 7 days, decreasing to about 36% after 1 month of storage. There was no significant difference between samples stored in culture medium with and without FBS after 1 month of storage (p = 0.5005). Refrigerated storage of cartilage in PBS and two different solutions (Belzer's and Unisol) designed for optimal refrigerated tissue and organ storage results in loss of chondrocyte function and retention of matrix permeability. In contrast, the opposite, namely significantly better retention of chondrocyte function and loss of matrix permeability, was observed with culture medium. Future research should be focused on combining retention of chondrocyte function and matrix permeability by storage solution formulation.

PKCδ Regulates Chromatin Remodeling and DNA Repair through SIRT6.

Irradiation (IR) is a highly effective cancer therapy; however, IR damage to tumor-adjacent healthy tissues can result in significant comorbidities and potentially limit the course of therapy. We have previously shown that protein kinase C delta (PKCδ) is required for IR-induced apoptosis and that inhibition of PKCδ activity provides radioprotection in vivo. Here we show that PKCδ regulates histone modification, chromatin accessibility, and double-stranded break (DSB) repair through a mechanism that requires Sirtuin 6 (SIRT6). Overexpression of PKCδ promotes genomic instability and increases DNA damage and apoptosis. Conversely, depletion of PKCδ increases DNA repair via nonhomologous end joining (NHEJ) and homologous recombination (HR) as evidenced by increased formation of DNA damage foci, increased expression of DNA repair proteins, and increased repair of NHEJ and HR fluorescent reporter constructs. Nuclease sensitivity indicates that PKCδ depletion is associated with more open chromatin, while overexpression of PKCδ reduces chromatin accessibility. Epiproteome analysis reveals increased chromatin associated H3K36me2 in PKCδ-depleted cells which is accompanied by chromatin disassociation of KDM2A. We identify SIRT6 as a downstream mediator of PKCδ. PKCδ-depleted cells have increased SIRT6 expression, and depletion of SIRT6 reverses changes in chromatin accessibility, histone modification and DSB repair in PKCδ-depleted cells. Furthermore, depletion of SIRT6 reverses radioprotection in PKCδ-depleted cells. Our studies describe a novel pathway whereby PKCδ orchestrates SIRT6-dependent changes in chromatin accessibility to regulate DNA repair, and define a mechanism for regulation of radiation-induced apoptosis by PKCδ. IMPLICATIONS: PKCδ controls sensitivity to irradiation by regulating DNA repair.

Genomic Redistribution of Metal-Response Transcription Factor-1 (MTF-1) in Cadmium Resistant Cells.

(1) Background: Metal homeostasis is an important part of cellular programs and is disrupted when cells are exposed to carcinogenic heavy metals. Metal response is mediated by the metal response element transcription factor MTF-1. However, where MTF-1 binds and how that binding changes in response to heavy metals, such as cadmium, remains unknown. (2) Methods: To investigate the effects of prolonged cadmium exposure on the genomic distribution of MTF-1, we performed MTF-1 CUT&RUN, RNA-seq and ATAC-seq on control and cadmium-resistant cells. (3) Results: Changes in MTF-1 binding primarily occur distal to the transcription start sight. Newly occupied MTF-1 sites are enriched for FOS/JUN DNA binding motifs, while regions that lose MTF-1 binding in cadmium are enriched for the FOX transcription factor family member DNA binding sites. (4) Conclusions: Relocalization of MTF-1 to new genomic loci does not alter the accessibility of these locations. Our results support a model whereby MTF-1 is relocalized to accessible FOS/JUN-bound genomic locations in response to cadmium.

Transition from Transient DNA Rereplication to Inherited Gene Amplification Following Prolonged Environmental Stress.

Cells require the ability to adapt to changing environmental conditions, however, it is unclear how these changes elicit stable permanent changes in genomes. We demonstrate that, in response to environmental metal exposure, the metallothionein (MT) locus undergoes DNA rereplication generating transient site-specific gene amplifications (TSSGs). Chronic metal exposure allows transition from MT TSSG to inherited MT gene amplification through homologous recombination within and outside of the MT locus. DNA rereplication of the MT locus is suppressed by H3K27me3 and EZH2. Long-term ablation of EZH2 activity eventually leads to integration and inheritance of MT gene amplifications without the selective pressure of metal exposure. The rereplication and inheritance of MT gene amplification is an evolutionarily conserved response to environmental metal from yeast to human. Our results describe a new paradigm for adaptation to environmental stress where targeted, transient DNA rereplication precedes stable inherited gene amplification.

PKCδ regulates chromatin remodeling and DNA repair through SIRT6.

Protein kinase C delta (PKCδ) is a ubiquitous kinase whose function is defined in part by localization to specific cellular compartments. Nuclear PKCδ is both necessary and sufficient for IR-induced apoptosis, while inhibition of PKCδ activity provides radioprotection in vivo. How nuclear PKCδ regulates DNA-damage induced cell death is poorly understood. Here we show that PKCδ regulates histone modification, chromatin accessibility, and double stranded break (DSB) repair through a mechanism that requires SIRT6. Overexpression of PKCδ promotes genomic instability and increases DNA damage and apoptosis. Conversely, depletion of PKCδ increases DNA repair via non-homologous end joining (NHEJ) and homologous recombination (HR) as evidenced by more rapid formation of NHEJ (DNA-PK) and HR (Rad51) DNA damage foci, increased expression of repair proteins, and increased repair of NHEJ and HR fluorescent reporter constructs. Nuclease sensitivity indicates that PKCδ depletion is associated with more open chromatin, while overexpression of PKCδ reduces chromatin accessibility. Epiproteome analysis revealed that PKCδ depletion increases chromatin associated H3K36me2, and reduces ribosylation of KDM2A and chromatin bound KDM2A. We identify SIRT6 as a downstream mediator of PKCδ. PKCδ-depleted cells have increased expression of SIRT6, and depletion of SIRT6 reverses the changes in chromatin accessibility, histone modification and NHEJ and HR DNA repair seen with PKCδ-depletion. Furthermore, depletion of SIRT6 reverses radioprotection in PKCδ-depleted cells. Our studies describe a novel pathway whereby PKCδ orchestrates SIRT6-dependent changes in chromatin accessibility to increase DNA repair, and define a mechanism for regulation of radiation-induced apoptosis by PKCδ.

Impact of Hypothermia upon Chondrocyte Viability and Cartilage Matrix Permeability after 1 Month of Refrigerated Storage.

BACKGROUND: The purpose of this research was to assess the extracellular matrix and chondrocytes of articular cartilage during refrigerated storage and to determine whether changes could be detected in the time frame that cartilage is stored for clinical use. MATHODS: Porcine cartilage was stored as either bisected femoral heads with bone attached or plugs without the underlying bone in culture medium with fetal bovine serum for 1 month at 4 °C. Metabolic activity was tested using a resazurin reduction method on intact tissue and viable cell recovery after enzymatic tissue digestion at each time point. Cartilage plug permeability was evaluated by measuring electrical conductivity. RESULTS: Storage in culture medium provided good cartilage viability and metabolic function for 7 days; however, significant changes were observed in femoral heads (p < 0.05). All mean chondrocyte assessment values were <30% of fresh controls at 28 days. Cartilage plugs tended to perform better after 7 days of storage than the femoral heads and retained significantly higher metabolic activity (mean = 94.5% vs. 70.5%; p < 0.05). Cartilage plugs demonstrated consistent changes in electrical conductivity after 28 days of storage (p < 0.05). CONCLUSION: Refrigerated storage of cartilage results in both loss of chondrocyte viability and matrix permeability.

Influence of Tissue Technology on Pannus Formation on Bioprosthetic Heart Valves.

PURPOSE: Bioprosthetic heart valves have several modes of failure. Tissue degeneration and calcification are the major modes of failure with the highest focus of attention, however pannus formation can also be problematic. We studied the effect of a new tissue technology with the absence of any glutaraldehyde-based storage solution and a stable aldehyde capping process on pannus formation. METHODS: Using a juvenile sheep model of mitral valve replacement, valves with the new tissue technology were compared to control valves with contemporary bovine pericardial tissue, regarding pannus formation. Valves were implanted for either a 5- or 8-month period. Explanted valves were examined macroscopically and histologically. Histological observations were made by an independent pathologist, blinded to group identity. RESULTS: Pannus area measured macroscopically on the test valves was significantly lower than the pannus on the control tissue. This was confirmed on the histological samples, where the total pannus overgrowth was significantly lower in the test group compared to the control. CONCLUSION: The new tissue technology leads to less pannus formation. This may beneficially influence both short- and long-term valve behavior of bioprosthetic valves.

Rapidly Establishing a Hospital-Based Convalescent Plasma Collection Center With the Alyx Apheresis Collection Device.

The effort to collect convalescent plasma from individuals who recovered from COVID-19 began in earnest during the spring of 2020. Either whole blood or apheresis donations were obtained, the latter yielding higher numbers of units per donor per collection and more frequent collections. The NorthShore University HealthSystem blood donor center purchased 2 Alyx (Fresenius Kabi) apheresis plasma collection devices and quickly implemented them in order to collect COVID-19 convalescent plasma. Apheresis-experienced and inexperienced phlebotomists operated the instruments. Donors were collected >14 days from symptom resolution and all donors were negative by SARS-CoV-2 nasopharyngeal swab. Both internal metrics of performance as well as a post donation survey were used to evaluate the feasibility implementing this collection program. During the first 100 days of the collection program, 650 plasma units were collected. In particular, during the first week of the program, 38 units were collected and distributed to hospitals under the emergency investigational new drug and expanded access program. Fifty-one donors (15%) were deferred due to vital signs out of range or donor screening questions. Thirty-one donors (10%) were deferred due to positive nasopharyngeal swab. Lower than target yield occurred in 16.6% of collections due to donor reactions or flow errors. Donors rated the overall program lower, but not the staff, when they reported symptoms related to collection. In conclusion, a hospital-based apheresis convalescent plasma collection program can be rapidly implemented. Donor reaction rates and vein infiltration rates should be carefully monitored for each phlebotomist.

Inscribed matter as an energy-efficient means of communication with an extraterrestrial civilization.

It is well known that electromagnetic radiation-radio waves-can in principle be used to communicate over interstellar distances. By contrast, sending physical artefacts has seemed extravagantly wasteful of energy, and imagining human travel between the stars even more so. The key consideration in earlier work, however, was the perceived need for haste. If extraterrestrial civilizations existed within a few tens of light years, radio could be used for two-way communication on timescales comparable to human lifetimes (or at least the longevities of human institutions). Here we show that if haste is unimportant, sending messages inscribed on some material can be strikingly more energy efficient than communicating by electromagnetic waves. Because messages require protection from cosmic radiation and small messages could be difficult to find among the material clutter near a recipient, 'inscribed matter' is most effective for long archival messages (as opposed to potentially short "we exist" announcements). The results suggest that our initial contact with extraterrestrial civilizations may be more likely to occur through physical artefacts-essentially messages in a bottle-than via electromagnetic communication.

Molecular mechanism of the MORC4 ATPase activation.

Human Microrchidia 4 (MORC4) is associated with acute and chronic pancreatitis, inflammatory disorders and cancer but it remains largely uncharacterized. Here, we describe the structure-function relationship of MORC4 and define the molecular mechanism for MORC4 activation. Enzymatic and binding assays reveal that MORC4 has ATPase activity, which is dependent on DNA-binding functions of both the ATPase domain and CW domain of MORC4. The crystal structure of the ATPaseCW cassette of MORC4 and mutagenesis studies show that the DNA-binding site and the histone/ATPase binding site of CW are located on the opposite sides of the domain. The ATPase and CW domains cooperate in binding of MORC4 to the nucleosome core particle (NCP), enhancing the DNA wrapping around the histone core and impeding binding of DNA-associated proteins, such as transcription factors, to the NCP. In cells, MORC4 mediates formation of nuclear bodies in the nucleus and has a role in the progression of S-phase of the cell cycle, and both these functions require CW and catalytic activity of MORC4. Our findings highlight the mechanism for MORC4 activation, which is distinctly different from the mechanisms of action observed in other MORC family members.

Assessing anticalcification treatments in bioprosthetic tissue by using the New Zealand rabbit intramuscular model.

The objective of this work was to demonstrate that the New Zealand White (NZW) rabbit intramuscular model can be used for detecting calcification in bioprosthetic tissue and to compare the calcification in the rabbit to that of native human valves. The rabbit model was compared with the commonly used Sprague-Dawley rat subcutaneous model. Eighteen rabbits and 18 rats were used to assess calcification in bioprosthetic tissue over time (7, 14, 30, and 90 d). The explanted rabbit and rat tissue discs were measured for calcium by using atomic absorption and Raman spectroscopy. Calcium deposits on the human valve explants were assessed by using Raman spectroscopy. The results showed that the NZW rabbit model is robust for detecting calcification in a shorter duration (14 d), with less infection complications, more space to implant tissue groups (thereby reducing animal use numbers), and a more metabolically and mechanically dynamic environment than the rat subcutaneous model . The human explanted valves and rabbit explanted tissue both showed Raman peaks at 960 cm(-1) which is representative of hydroxyapatite. Hydroxyapatite is the final calcium and phosphate species in the calcification of bioprosthetic heart valves and rabbit intramuscular implants. The NZW rabbit intramuscular model is an effective model for assessing calcification in bioprosthetic tissue.

Clinical outcome and phenotypic expression in LAMP2 cardiomyopathy.

CONTEXT: Mutations in X-linked lysosome-associated membrane protein gene (LAMP2; Danon disease) produce a cardiomyopathy in young patients that clinically mimics severe hypertrophic cardiomyopathy (HCM) due to sarcomere protein mutations. However, the natural history and phenotypic expression of this newly recognized disease is incompletely resolved and its identification may have important clinical implications. OBJECTIVES: To determine the clinical consequences, outcome, and phenotypic expression of LAMP2 cardiomyopathy associated with diagnostic and management strategies. DESIGN, SETTING, AND PATIENTS: Clinical course and outcome were assessed prospectively in 7 young patients (6 boys) with defined LAMP2 mutations from the time of diagnosis (age 7-17 years; median, 14 years) to October 2008. Phenotypic expression of this disease was assessed both clinically and at autopsy. MAIN OUTCOME MEASURES: Progressive heart failure, cardiac death, and transplant. RESULTS: Over a mean (SD) follow-up of 8.6 (2.6) years, and by age 14 to 24 years, the study patients developed left ventricular systolic dysfunction (mean [SD] ejection fraction, 25% [7%]) and cavity enlargement, as well as particularly adverse clinical consequences, including progressive refractory heart failure and death (n = 4), sudden death (n = 1), aborted cardiac arrest (n = 1), or heart transplantation (n = 1). Left ventricular hypertrophy was particularly marked (maximum thickness, 29-65 mm; mean [SD], 44 [15] mm), including 2 patients with massive ventricular septal thickness of 60 mm and 65 mm at ages 23 and 14 years, respectively. In 6 patients, a ventricular pre-excitation pattern at study entry was associated with markedly increased voltages of R-wave or S-wave (15-145 mm; mean [SD], 69 [39] mm), and deeply inverted T-waves. Autopsy findings included a combination of histopathologic features that were consistent with a lysosomal storage disease (ie, clusters of vacuolated myocytes) but also typical of HCM due to sarcomere protein mutations (ie, myocyte disarray, small vessel disease, myocardial scarring). CONCLUSIONS: LAMP2 cardiomyopathy is a profound disease process characterized by progressive clinical deterioration leading rapidly to cardiac death in young patients (<25 years). These observations underscore the importance of timely molecular diagnosis for predicting prognosis and early consideration of heart transplantation.