The etiology of rhabdomyolysis: an interaction between genetic susceptibility and external triggers.

BACKGROUND AND PURPOSE: Rhabdomyolysis is a medical emergency characterized by acute skeletal muscle breakdown with a sudden rise and subsequent fall of serum creatine kinase (CK) levels. Rhabdomyolysis events are provoked by exposure to external triggers, possibly in combination with an increased genetic susceptibility. We aimed to describe comprehensively the external triggers and potentially pathogenic genetic variants possibly implicated in increased rhabdomyolysis susceptibility. METHODS: We performed a retrospective single-center study, including a total of 1302 patients with an acute CK level exceeding 2000 IU/l. RESULTS: Anoxia was the most frequently reported trigger (40%). A subset of 193 patients were clinically suspected of an underlying genetic disorder (recurrent episodes, a positive family history, very high or persistently increased CK levels). In 72 of these patients, an unequivocal genetic defect was identified. A total of 22 genes with pathogenic variants were identified, including 52 different variants. Of those, 11 genes have been previously associated with rhabdomyolysis (ACADVL, ANO5, CPT2, DMD, DYSF, FKRP, HADHA, PGM1, LPIN1, PYGM, RYR1). Eleven genes are probably implicated in increased susceptibility (including AGL, CAPN3, CNBP, DMPK, MAGT1, ACADM, SCN4A, SGCA, SGCG, SMPD1, TANGO2). CONCLUSION: These findings suggest that the spectrum of genetic susceptibility for rhabdomyolysis has not yet been completely clarified. With the increasing availability of next-generation sequencing in a diagnostic setting, we expect that in more cases a genetic defect will be identified.

Exertional Heat Stroke and Rhabdomyolysis: A Medical Record Review and Patient Perspective on Management and Long-Term Symptoms.

INTRODUCTION: Exertional heat stroke (EHS) is a medical emergency, occurring when the body generates more heat than it can dissipate, and frequently associated with exertional rhabdomyolysis (ERM). In the present study we aimed to (I) identify clinical features and risk factors, (II) describe current prehospital management, (III) investigate long-term outcomes including the impact on mental health, and review the guidance received during restarting activities. We hope that our approach will improve individual and organizational heat illness preparedness, and improve follow-up care. METHODS: We performed a prospective online survey and retrospective medical record review among athletes and military personnel with an episode of EHS/ERM in the Netherlands between 2010 and 2020. We evaluated prehospital management, risk factors, clinical features and long-term outcomes at 6 and 12 months after the event, including mental health symptoms. Furthermore, we investigated what guidance participants received during follow-up, and assessed the patients' perspective on these outcomes. RESULTS: Sixty participants were included, 42 male (70%) and 18 female (30%), of which 47 presented with EHS (78%) and 13 with ERM (22%). Prehospital management was inconsistent and in the majority of participants not conducted according to available guidelines. Self-reported risk factors included not feeling well-acclimatized to environmental heat (55%) and peer pressure (28%). Self-reported long-term symptoms included muscle symptoms at rest (26%) or during exercise (28%), and neurological sequelae (11%). Validated questionnaires (CIS, HADS and SF-36) were indicative of severe fatigue (30%) or mood/anxiety disorders (11%). Moreover, 90% expressed a lack of follow-up care and that a more frequent and intensive follow-up would have been beneficial for their recovery process. CONCLUSION: Our findings indicate major inconsistencies in the management of patients with EHS/ERM, emphasizing the compelling need for implementing standardized protocols. Based on the results of long-term outcome measures, we recommend to counsel and evaluate every patient not only immediately after the event, but also in the long-term.

Perioperative Care for Patients with Neuromuscular Disorders in the Netherlands - A Questionnaire Study Among Anaesthesiologists, Neurologists and Clinical Geneticists.

BACKGROUND: Patients with neuromuscular disorders are at increased risk of suffering perioperative complications. Current knowledge concerning this topic is based on small retrospective studies and expert opinion. Therefore, an individualized multidisciplinary approach to perioperative anaesthesia planning is invaluable to anticipate difficulties and to optimize outcomes. OBJECTIVE: To evaluate current practice regarding preoperative counselling and perioperative care of neuromuscular patients, with the aim to facilitate standardization and improvement of perioperative care for neuromuscular patients. METHODS: A questionnaire-based cross-sectional, observational study was conducted between July, 1st 2020 and December, 31st, 2020 in Dutch anaesthesia, neurology and clinical genetics departments. Main outcome measures were 1.) frequency of consultation requests for neuromuscular patients prior to surgery, 2.) current practice, educational activities and departmental approach to this topic and 3.) preoperative counselling of neuromuscular patients. RESULTS: A total of 83 departments participated. Consultations for a neuromuscular patient scheduled for anaesthesia were requested from anaesthesia and neurology department only infrequently. Local guidelines concerning perioperative care of neuromuscular patients were available in 36.4% of the participating departments. Quality of specific training for residents and staff anaesthetists/neurologists covering perioperative care of neuromuscular patients was rated as 'very good' or 'good' by 42.9%. Neuromuscular patients scheduled for surgery were 'always' or 'often' discussed in multidisciplinary meetings involving anaesthesiologists and neurologists in 20.8% of the participating departments. CONCLUSION: Perioperative care for neuromuscular patients in the Netherlands is highly variable and might benefit from guidelines, education of health care professionals and multidisciplinary meetings between anaesthesiologists and neurologists on a regular basis.

HyperCKemia and rhabdomyolysis in the neuroleptic malignant and serotonin syndromes: A literature review.

Neuroleptic malignant syndrome and serotonin syndrome are two syndromes whose molecular bases remain poorly understood. The phenotypes of both syndromes overlap with other syndromes that have a clear genetic background, in particular RYR1-related malignant hyperthermia. Through a literature review, performed according to the PRISMA guidelines, we aimed to report the clinical features of both syndromes, and the results of genetic testing performed. 10 case series and 99 case reports were included, comprising 134 patients. A male predominance of 58% was found. The median age was 35 (range 4-84) years. Eight patients experienced recurrent episodes of rhabdomyolysis. Genetic analysis was performed in eleven patients (8%), revealing four RYR1 variants, three likely benign (p.Asp849Asn, p.Arg4645Gln, p.Arg4645Gln) and one variant of uncertain significance (p.Ala612Thr). This review underlines that a subset of patients with neuroleptic malignant syndrome and serotonin syndrome develop recurrent episodes of rhabdomyolysis. This recurrent pattern suggests a possible underlying (genetic) susceptibility. However, the genetic background of neuroleptic malignant syndrome and serotonin syndrome has only been investigated to a very limited degree so far. The increasing availability of next generation sequencing offers an opportunity to identify potentially associated genetic backgrounds, especially in patients with recurrent episodes or a positive family history.

Suicide after inhaling a pyrethrins containing insecticide spray.

Pesticide self-poisoning is rare in developed countries. We report a suicide case after inhalation of a pyrethrins containing insecticide spray. The patient presented at the emergency department with respiratory failure. Despite mechanical ventilation, he developed severe pulmonary inflammation with a systemic inflammatory response syndrome and died 5 days later. Studies reporting on acute pyrethrins or pyrethroids insecticide poisoning in both occupational and non-occupational cases usually describe mild and self-limiting respiratory symptoms as the predominant symptom. Severe or fatal cases of pyrethrins or pyrethroids poisoning are very rare. Patients with asthma or allergies are apparently more at risk for severe symptoms. In these cases, early and aggressive treatment with bronchodilatators, steroids, antihistamines and epinephrine should be considered.

Cationization of catalase, peroxidase, and superoxide dismutase. Effect of improved intraarticular retention on experimental arthritis in mice.

Several enzymes and other proteins were made cationic either by coupling to polylysine or by shielding of anionic sites. These cationic proteins, all having an isoelectric point greater than 8.5 exhibited excellent retention in articular structures when injected in mouse knee joints. Autoradiography and histochemistry showed that cationic forms of catalase, superoxide dismutase, and horseradish peroxidase were firmly retained by synovial and cartilaginous tissues. The half-life of these enzymes in the joint is thus significantly extended compared with native enzymes. The native enzymes and their cationic derivatives were tested for antiinflammatory properties in mice, using antigen-induced arthritis and zymosan-induced arthritis. It was found that injection of cationic catalase or peroxidase induced a marked suppression of some parameters of the inflammatory response in both types of arthritis, as measured by 99m technetium pertechnetate uptake and leakage of 125I-labeled albumin. Native catalase and peroxidase were less, or not at all effective. Cationic superoxide dismutase or cationic nonenzyme proteins did not suppress inflammation. The observed suppression of two different types of inflammation (an immune and a nonimmune arthritis) by catalase and peroxidase suggests that elimination of peroxides contributes to the suppression of an inflammatory response. We would hypothesize that cationic enzymes offer the possibility for investigating the mechanisms of inflammation and, in addition, might be interesting from a therapeutical point of view.

IL-4 gene therapy for collagen arthritis suppresses synovial IL-17 and osteoprotegerin ligand and prevents bone erosion.

Bone destruction is the most difficult target in the treatment of rheumatoid arthritis (RA). Here, we report that local overexpression of IL-4, introduced by a recombinant human type 5 adenovirus vector (Ad5E1mIL-4) prevents joint damage and bone erosion in the knees of mice with collagen arthritis (CIA). No difference was noted in the course of CIA in the injected knee joints between Ad5E1mIL-4 and the control vector, but radiographic analysis revealed impressive reduction of joint erosion and more compact bone structure in the Ad5E1mIL-4 group. Although severe inflammation persisted in treated mice, Ad5E1mIL-4 prevented bone erosion and diminished tartrate-resistant acid phosphatase (TRAP) activity, indicating that local IL-4 inhibits the formation of osteoclast-like cells. Messenger RNA levels of IL-17, IL-12, and cathepsin K in the synovial tissue were suppressed, as were IL-6 and IL-12 protein production. Osteoprotegerin ligand (OPGL) expression was markedly suppressed by local IL-4, but no loss of OPG expression was noted with Ad5E1mIL-4 treatment. Finally, in in vitro studies, bone samples of patients with arthritis revealed consistent suppression by IL-4 of type I collagen breakdown. IL-4 also enhanced synthesis of type I procollagen, suggesting that it promoted tissue repair. These findings may have significant implications for the prevention of bone erosion in arthritis.

Chondrocyte nonresponsiveness of arthritic articular cartilage caused by short term immobilization.

We studied the effect of short term immobilization on chondrocyte synthetic function. Arthritis from intraarticular injection of either glucose oxidase or interleukin 1 (IL-1) led to suppression of proteoglycan synthesis. Extension casting of arthritic knees prevented inhibition of proteoglycan synthesis, whereas later casting restored synthesis to normal. During chronic antigen induced arthritis casting protected chondrocyte synthesis for a period of 5 days. The chondrocyte nonresponsiveness was not from altered penetration of suppressing substances. Radiolabelled biologically active IL-1 showed no difference in retention or localization in articular cartilage of either mobile or immobile knees. Flexion casting did not induce unresponsiveness of chondrocytes and suppression of synthesis was similar to that in mobile arthritic knees, indicating the importance of loading. The nonresponsiveness occurred only during immobilization and rapidly disappeared after removal of the cast, provided that suppressing agents were still present. Thus, during short term immobilization chondrocytes are protected from inhibition of proteoglycan synthesis by suppressing agents. This protection may benefit repair of the depleted cartilage matrix with waning inflammation.

Electrical charge of a protein determines penetration and localization in hyaline articular cartilage. Quantitative and autoradiographic studies on cartilage of different species, including man.

Sufficient antigen retention in joint structures is a prerequisite for sustained antigen-induced arthritis. In this in vitro study we investigated the retention of native and charge-modified bovine serum albumin (BSA) with patellar cartilage of different species (mouse, rat, rabbit, and man). Association of BSA with cartilage due to charge of the protein with that due to immune complex formation was compared. Using radiolabeled proteins we showed quantitatively that the retention of highly positively charged BSA is considerably higher (200-500 times) than retention of the anionic BSA in all cartilage species examined. Mouse, rat, and human cartilage bind more protein per mg dry weight, compared to rabbit cartilage. No clear-cut relation was found with the glycosaminoglycans (GAG) contents. Retention of native BSA by anti-BSA antibodies was low in the dense hyaline patellar cartilage in all species. Enhanced immune-complex formation was found in marginal regions of rabbit patellar cartilage, consisting of fibrous-like cartilage. Localization studies by autoradiography showed that cationic BSA penetrates deeply into the cartilage matrix. Even in a thick cartilage specimen, such as rabbit cartilage, very deep penetration into the calcified zone was demonstrated. This study indicates that binding and deep penetration of cationized protein in cartilage is not restricted to mouse specimen, as had been found previously, but is a general phenomenon.

Circulating immune complexes and rheumatoid arthritis: the induction of immune complex formation between rheumatoid factor and IgG by polyethylene glycol.

Circulating immune complexes (CIC) as detected by the C1q binding assay (C1qBA) in sera from patients with rheumatoid arthritis (RA) were not demonstrable in these sera with the indirect granulocyte phagocytosis test (IGPT). This discrepancy could be explained by the finding that polyethylene glycol (PEG), used in the C1qBA, enhanced the binding of rheumatoid factor IgM (RFIgM) and IgG resulting in immune complex (IC) formation. The addition of PEG to RA sera and subsequent testing of these sera in the IGPT revealed increased uptake of IC by these cells, dependent on the PEG dose. Addition of purified RFIgM to a normal human serum generated a positive IGPT in a dose dependent way. We conclude that in RA sera PEG induces IC between RFIgM and IgG. Therefore, assays devised to measure CIC in RA sera which are based on PEG (like the C1qBA) overestimate the amounts of IC present in the circulation in vivo.

Immune complex formation between IgM rheumatoid factor and IgG generated by hyaluronic acid.

Immunoglobulin inclusions are regularly detected in granulocytes obtained from the synovial fluid of rheumatoid arthritis (RA) patients. In contrast, granulocytes isolated from the blood of the same patients usually contain no immunoglobulin inclusions. Using the indirect granulocyte phagocytosis test (IGPT), we obtained evidence that this discrepancy can be explained by the finding that hyaluronic acid (HA), a component of synovial fluid, increases the avidity of rheumatoid factor (RF), resulting in the formation of IgG-IgM-RF complexes. The addition of HA to RA sera and subsequent testing by IGPT revealed an increased uptake of the induced immune complexes by these cells, which was dependent on the HA dose. Furthermore, supplementation of normal human serum with purified IgM-RF generated a positive IGPT result in a dose-dependent manner in the presence of HA. We conclude that HA, a component of synovial fluid, might facilitate immune complex formation in the joint cavity, resulting in the inflammatory reaction in the joints of RA patients.

Flare-up of antigen-induced arthritis in mice after challenge with oral antigen.

Mice with unilateral chronic mBSA-induced arthritis were orally challenged with mBSA. Three hours after antigen challenge clear flare-up of the chronic arthritis was demonstrable as detected by an increase in the 99mTc uptake of the knee joint and the reaction continued for at least 2 days. The contralateral non-arthritic knee joint was not affected. The dose of mBSA needed to induce a flare-up in nearly all mice within a group was in the order of 20 mg. After oral challenge with 10 or 5 mg of mBSA the incidence was lower and flare-up reactions were only rarely observed after challenge with 2.5 or 1.25 mg mBSA. Histology of knee joints taken at 24 h after oral challenge of 20 mg mBSA revealed an increase in the number of cells in the infiltrate in the synovial tissue and exudate in the joint space, the most conspicuous sign being the increase of PMN. Passage of macromolecules through the gastrointestinal mucosa may be an important principle in the perpetuation of human chronic arthritis.

Immobilization aggravates cartilage damage during antigen-induced arthritis in mice. Attachment of polymorphonuclear leukocytes to articular cartilage.

The early and late effects of short-term immobilization on arthritic joints have been studied. Knee joints of mice in which an antigen-induced unilateral arthritis was elicited were immobilized in extension for 3, 5, and 7 days. After 5 and 7 days' immobilization, arthritis was significantly more severe. More leukocytes infiltrated the periarticular tissues and more cellular exudate was found in the joint space. A striking observation was that large numbers of polymorphonuclear leukocytes (PMN) attached to the surface of the cartilage, a phenomenon not found in mobile arthritic joints. Electron-microscopy confirmed PMN adhesion and showed severe ruffling of the cartilage surface under immobilized conditions. Further examination of factors determining PMN sticking revealed that attachment is rapid when the cartilage surface is already damaged, and that retained immune complexes and complement play a pivotal role. The late effects of immobilization were studied after a remobilization period of 2 weeks. Enhanced matrix depletion and chondrocyte death persisted in arthritic joints that were previously immobilized for 5 and 7 days, and the latter also showed significantly increased osteophyte formation. Although these results are speculative for the human situation, this study indicates that treatment of arthritic joints by complete rest should be applied with caution.

Fate of antigen after intravenous and intraarticular injection into mice. Role of molecular weight and charge.

The role of the charge of differently sized proteins in their penetration, persistence and localization in murine joint tissues was studied. Radiolabeled proteins of variable molecular weight (14-150 kDa) and charge (isoelectric point (pI) 4.5-10) were administered by intravenous and intraarticular routes, and localization studies in the first 2 and at 24 h were done with autoradiography. Small cationic proteins rapidly penetrated joint tissues after intravenous injection and persisted in larger amounts compared to anionic proteins of corresponding size. Small cationic proteins easily penetrated the matrices of both epiphysial and articular cartilage. In contrast larger cationic proteins (cBSA, cIgG) persisted for the greater part in the tissues just around the vessels and cartilage association was less striking. After intraarticular injection it was found that low molecular weight proteins (14 to 47 kDa) are rapidly cleared from the joint. Small cationic proteins persisted somewhat better than anionic ones, but impressive retention was only found with cationic proteins with higher molecular weight (67 to 150 kDa). Our data help to define prerequisite properties of proteins for penetration and interaction with joint structures after systemic supply or local production.

Allergic arthritis induced by cationic proteins: role of molecular weight.

Previous studies have shown that chronic murine allergic arthritis can only be induced with cationized BSA, related to excellent retention of the cationic antigen in the joint. We now investigate the impact of size of cationic proteins on their potential to induce this form of arthritis. After intra-articular injection, antigen retention is much enhanced with high molecular weight cationized proteins, like albumin or immunoglobulin, compared to small-sized proteins like myoglobulin and lysozyme. Consequently, severe chronic arthritis was only found with the former ones. The role of size is further substantiated with poly-L-lysine-coupled lysozyme. This derivative shows excellent retention in vivo and causes a chronic destructive arthritis in preimmunized mice, in contrast to the poor arthritis seen with native cationic lysozyme. Control experiments made it clear that antigen retention is the most important denominator and that differences in chronicity are not related to gross variations in T-cell reactivity. Retention studies in vitro revealed that the potential to bind to joint structures is similar for the various proteins, suggesting that in vivo conditions determine size-related differences in antigen clearance. Our data indicate that cationicity per se does not make a protein a proper arthritogen.

The impact of protein size and charge on its retention in articular cartilage.

The interaction of several globular proteins with intact murine hyaline articular cartilage was studied in vitro. Proteins with molecular weights from 12 to 440 kDa and isoelectric points (pI) from 4.5 to 10 were tested for the ability to penetrate and persist in cartilage. Native proteins were modified for a range of pI. Using radiolabeled proteins we showed that retention of proteins in cartilage is a function of their pI. At pI 8.5-9 all proteins showed a sharp increase in cartilage when incubated at physiologic pH. The molecular weight of a protein and its charge is a determining factor for penetration of cartilage. By autoradiography highly cationic proteins up to 150 kDa (IgG) readily penetrated cartilage. Immunofluorescence confirmed these findings. Cationic catalase (240 kDa) showed superficial penetration, but penetration of cationic ferritin (440 kDa) was not demonstrated, suggesting that 240 to 440 kDa represents the upper range for penetration. Small anionic proteins (cytochrome-c; pI less than 4.5; 12 kDa) penetrate in small quantities but do not persist, whereas larger anionic proteins (IgG; pI less than 4.5; 150 kDa) cannot penetrate at all. Our data help define the properties of proteins that are able to interact with cartilage matrix and chondrocytes.

Role of phagocytic synovial lining cells in experimental arthritis.

We investigated the in vivo role of phagocytic synovial lining cells (SLC) in the onset of experimental arthritis by depleting phagocytic SLC prior to arthritis induction. Phagocytic SLC were depleted by a single intra-articular injection of liposomes encapsulating the drug dichloromethylene diphosphonate (CL2MDP). Seven days after injection optimal depletion was observed and this time point was chosen for induction of arthritis in SLC depleted joints. Joint swelling was highly reduced after elicitation with either zymosan, immune complexes or antigen, as compared to observations in normal non-depleted joints. In addition cellular infiltration was markedly reduced. Further study in the immune complex mediated arthritis revealed that reduced cell influx in SLC depleted knee joints was correlated to lowered chemotactic activity and IL-1 levels as measured in washouts of joint tissues. This indicates that IL-1 driven chemotactic factors might be involved. Furthermore reduced cell influx was also correlated to significantly diminished loss of 35S-prelabeled PG from the cartilage. Out data indicate that SLC are directly involved in the onset of joint inflammation.

Cationic immune complex arthritis in mice--a new model. Synergistic effect of complement and interleukin-1.

A novel cationic immune-complex-mediated arthritis (ICA) model was developed in mice. The highly cationic protein lysozyme was coupled to poly-L-lysine (PLL) and injected intra-articularly into the knee joint of the mouse, shortly after systemic administration of specific antibodies. A vehement joint inflammation developed, characterized by severe joint swelling and the influx of predominantly polymorphonuclear (PMN) leukocyte. Unique properties were combined in this protein. First, an excellent retention of the antigen in joint structures was found, facilitating sufficient IC formation in the synovial tissue and at the cartilage surface. Secondly, PLL.lysozyme appeared to be a potent inducer of interleukin-1 (IL-1). Similar IL-1 production was measured at 6 hours, in both immune or nonimmune mice. Neutralization with antibodies against either IL-1 alpha or IL-1 beta revealed that IL-1 alpha was the dominant cytokine. Resident cells were responsible for this IL-1 production since a comparable IL-1 signal was measured after intra-articular injection of PLL.lys in neutropenic mice. We further investigated whether IL-1 and complement factors were involved in the onset of this ICA. Neutralizing the IL-1 production with antibodies directed against IL-1 alpha and beta showed a significant decrease in joint swelling. Complement depletion by cobra venom factor also prevented the onset of arthritis for the greater part. Only a minor swelling remained at 6 hours after eliciting arthritis, which was similar to the swelling after injecting the antigen alone and probably reflects IL-1 mediated inflammation. In this study, the authors show a synergistic action of IL-1 and complement in the onset of cationic ICA. Unique properties of the antigen such as excellent retention and its ability to induce IL-1 are combined within one molecule and make this antigen arthritogenic in the presence of antibodies and complement activation.