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Aim: To evaluate the frequency of Mycoplasma pneumoniae in nasopharyngeal specimens from children with respiratory tract infections (RTIs) and to detail clinical characteristics and management., Methods: The study was designed as a retrospective cohort study. All children with RTI and nucleic acid amplification testing from nasopharyngeal specimens were analysed. Clinical data were extracted from electronic health records for all M. pneumoniae-positive cases. Stored samples of cases and a random selection of matched controls were retested using a M. pneumoniae-specific nucleic acid amplification test., Results: Of 4460 children, 70 (1.6%) were positive for M. pneumoniae with a median age of 6.4 (IQR: 2.7-9.7). M. pneumoniae was the only organism identified in 50/64 (78%) cases. Macrolide treatment was prescribed in 52/65 (80%); prescription was empirical in 29/52 (56%) and targeted in 23/52 (44%) with no difference regarding patient age, oxygen requirement or duration of hospitalisation., Conclusion: The prevalence of M. pneumoniae in nasopharyngeal specimens of children with RTI was low. The detection of M. pneumoniae influenced antibiotic prescriptions, but the benefit of early empirical versus targeted treatment remains unclear., (©2019 Foundation Acta Paediatrica. Published by John Wiley & Sons Ltd.)

Objectives: Standard inflammatory markers and chest radiography lack the ability to discriminate bacterial from non-bacterial lower respiratory tract infection (LRTI). Cytokine profiles may serve as biomarkers for LRTI, but their applicability to identify aetiology, severity of disease and need for antibiotic prescription in children remains poorly defined. Objectives were to determine the cytokine kinetic profiles over 5 days in paediatric patients with LRTI, to investigate the relationship between cytokine patterns, and clinical and laboratory variables., Methods: We included patients aged 1 month to 18 years, with febrile LRTI and three consecutive cytokines measurements on days 1, 3 and 5 of a randomized controlled trial (ProPAED study). We evaluated differences in cytokine concentrations between days and associations with clinical and laboratory variables., Results: A total of 181 patients (median age 4.1 years) were included; 72/181 (40%) received antibiotics. Serum concentrations of interferon (IFN)-γ, interleukin (IL)-1ra, IL-6, IL-10, IFN-γ-inducible protein (IP)-10 and tumor necrosis factor-α were elevated on day 1 and decreased subsequently, with the greatest decline between day 1 and 3 (by -8 to >-94%). Procalcitonin (PCT) and C-reactive protein (CRP) values showed a protracted decrease with the most prominent reduction in concentrations between days 3 and 5. Significantly elevated IL-6 concentrations were associated with hospital admission, antibiotic treatment, and prolonged antibiotic treatment. Bacteraemic LRTI patients had higher concentrations of IL-1ra (p <0.0055) and IL-6 (p <0.0055) on day 1., Conclusions: We observed an earlier decrease of elevated cytokines compared to PCT or CRP. Both pro- and anti-inflammatory cytokines may serve as markers for severity of LRTI., (Copyright © 2018 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Cytokine-based diagnostic assays are increasingly used in research and clinical practice. Assays developed for adults such as the interferon-gamma release assay for tuberculosis show inferior performance in children. Limited evidence suggests that release of cytokines is influenced by age but normal ranges of cytokines in children are lacking. Whole blood of healthy children (0-12 years) undergoing elective/diagnostic procedures was stimulated with SEB, PHA, Candida albicans for 24 hours or left unstimulated. Concentrations of eight cytokines were measured by multiplex bead-based immunoassays and associations with age and other factors quantified by regression analysis. 271 children (median age 5.2 years) were included. In unstimulated samples IL-1ra, IP-10 and TNF-α concentrations decreased by up to -60% with age. Following antigen stimulation, an age-associated increase (ranging from +90% to +500%) was observed for all cytokines except IL-1ra (significant for IL-4, IFN-γ and TNF-α). Inter-individual variability in cytokine concentrations was large with a coefficient of variation ranging from 42% to 1412%. Despite inter-individual variation age was identified as a strong influencing factor of cytokine concentrations. Age-specific normal values need to be considered for cytokine-based diagnostic purposes. These results are relevant for development of novel cytokine-based diagnostic assays and for optimal dosing of therapeutic agents targeting cytokines.

Cytokines have attracted much attention as diagnostic biomarkers for infectious and inflammatory diseases in recent years. However, understanding of maturation and normal age-associated values is limited. This review summarizes evidence on the influence of age and other factors on expression profiles of soluble and intracellular cytokines in healthy children. IFN-γ, IL-6, IL-10, and TNF-α are the most frequently investigated cytokines, of which an age-associated increase was shown consistently for IFN-γ and TNF-α. An age-associated decrease of IL-13 was seen in resource-limited settings. For other cytokines, including IL-1RA, IL-2, and IL-10, uni- or bimodal curves have been described, and results were influenced by study setting. To conclude, despite limited current understanding of the development of cytokine expression, age clearly influences expression profiles in healthy children. Dynamics of cytokine expression in childhood need to be considered when these are measured in diagnostic assays or as biomarkers. In addition, cytokine-targeting agents may require adjustment for normal values when used in children.

Background: Clinical studies in children are necessary yet conducting multiple visits at study centers remains challenging. The success of "care-at-home" initiatives and remote clinical trials suggests their potential to facilitate conduct of pediatric studies. This pilot aimed to study the feasibility of remotely collecting valid (i.e. complete and correct) saliva samples and clinical data utilizing mobile technology., Methods: Single-center, prospective pilot study in children undergoing elective tonsillectomy at the University of Basel Children's Hospital. Data on pain scores and concomitant medication and saliva samples were collected by caregivers on two to four inpatient study days and on three consecutive study days at home. A tailored mobile application developed for this study supported data collection. The primary endpoint was the proportion of complete and correct caregiver-collected data (pain scale) and saliva samples in the at-home setting. Secondary endpoints included the proportion of complete and correct saliva samples in the inpatient setting, subjective feasibility for caregivers, and study cost., Results: A total number of 23 children were included in the study of which 17 children, median age 6.0 years (IQR 5.0, 7.4), completed the study. During the at-home phase, 71.9% [CI = 64.4, 78.6] of all caregiver-collected pain assessments and 53.9% [CI = 44.2, 63.4] of all saliva samples were complete and correct. Overall, 64.7% [CI = 58.7, 70.4] of all data collected by caregivers at home was complete and correct. The predominant reason for incorrectness of data was adherence to the timing of predefined patient actions. Participating caregivers reported high levels of satisfaction and willingness to participate in similar trials in the future. Study costs for a potential sample size of 100 patients were calculated to be 20% lower for the at-home than for a traditional in-patient study setting., Conclusions: Mobile device supported studies conducted at home may provide a cost-effective approach to facilitate conduct of clinical studies in children. Given findings in this pilot study, data collection at home may focus on electronic data capture rather than biological sampling.

Background: Microscopic polyangiitis (MPA) is an ANCA-associated vasculitis (AAV; ANCA denotes antineutrophil cytoplasmic antibody) that causes necrotizing inflammation of small blood vessels. Renal and pulmonary manifestations are common whereas central nervous system (CNS) involvement, and in particular spinal disease, is rare., Methods: We reviewed a case of MPA presenting with spinal intradural hemorrhage and intracerebral hemorrhage. We also summarized all reported cases of AAV with spinal cord involvement in the literature (database search included MEDLINE, Embase, Scopus, and Proquest with no date or language restriction)., Results: We reviewed 20 cases of AAV with spinal cord involvement (12 granulomatosis with polyangiitis [GPA], 4 eosinophilic granulomatosis with polyangiitis, 2 MPA, and 2 cases diagnosed as AAV only) and reported demographic information, clinical features, methods of diagnosis, treatment, and patient outcome. Although CNS involvement has been associated with a poor prognosis, 14 of 18 cases that reported outcome data achieved remission during follow-up. Death occurred in 3 patients diagnosed with GPA and in 1 patient with MPA. Our patient with MPA deteriorated rapidly despite use of prednisone and died., Conclusions: AAV can present with brain and spinal cord involvement, even in the absence of systemic disease. CNS disease may be responsive to immunosuppressive therapy (e.g., steroids and cyclophosphamide) in several of the cases reviewed., (Copyright © 2016 National Stroke Association. Published by Elsevier Inc. All rights reserved.)

Cardiac myosin binding protein C (cMyBP-C) is a myofibrillar protein important for normal myocardial contractility and stability. In mutated form it can cause cardiomyopathy and heart failure. cMyBP-C appears to have separate regions for different functions. Three phosphorylation sites near the N terminus modulate contractility by their effect on both the kinetics of contraction and the binding site of the N-terminus. The C terminal region binds to myosin rods and stabilizes thick filament structure. The aim of the study reported here was to test whether cMyBPC is important in producing the structural and functional changes that result from ischemia/reperfusion. In this study the sequential changes in cMyBP-C, contractility, and thick filament structure following dephosphorylation of cMyBP-C associated with ischemia and reperfusion have been studied in biopsied specimens from chronically instrumented dogs. One and two dimensional electrophoresis, electron microscopy and immunocytochemistry with multiple antibodies generated against different domains in cMyBP-C have been used to follow structural changes in cMyBP-C. Ischemia produced dephosphorylation of cMyBP-C. Subsequent reperfusion released the dephosphorylated cMyBP-C from myofibrils and activated proteolysis of the cytoplasmic cMyBP-C. This in turn leads to increased vulnerability of cMyBP-C to proteolysis and increased degradation of thick filaments. The state of cMyBP-C appears to be closely related to phosphorylation and dephosphorylation of serine 282. In the absence of the stabilizing action of cMyBP-C either as a consequence of genetic mutation or dephosphorylation, premature degradation of thick filaments occurs and is accompanied by persistent contractile dysfunction., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Hutchinson-Gilford Progeria Syndrome (HGPS) is a rare premature aging disorder caused by mutations in the gene LMNA, which encodes the nuclear matrix protein lamin A. Previous research has shown that the average telomere length in fibroblasts from HGPS patients is shorter than in age-matched controls. How mutations in lamin A lead to shortened telomere lengths is not known nor is the contribution of individual chromosome ends to the low average length understood. To measure the telomere length of individual chromosomes, we used quantitative fluorescence in situ hybridization (Q-FISH). In agreement with previous studies, we found that the average telomere length in HPGS fibroblasts is greatly reduced; however, the telomere length at chromosome ends was variable. In contrast, the telomere length in hematopoietic cells which typically do not express lamin A, was within the normal range for three out of four HGPS patient samples. Our results suggest that mutant lamin A decreases telomere length via a direct effect and that expression of mutant LMNA is necessary for telomere loss in HGPS.

Objectives: To assess the proportion of children being stunted and underweight-for-age at 3, 9 and 15 months in Lambaréné, Gabon, using the WHO child growth standards released in 2006 as compared with the Centers for Disease Control and Prevention (CDC) 2000 and the National Center for Health Statistics (NCHS) 1978 child growth charts/references., Design and Setting: Prospective birth cohort in Lambaréné, Gabon., Subjects: Two hundred and eighty-nine children from birth to 15 months of age., Methods: Weight and length were recorded at 3, 9 and 15 months. Corresponding Z scores for stunting and underweight-for-age were calculated for the three different standards/references. Children with a height-for-age or weight-for-age below -2 SD of the corresponding reference median (Z score < or = -2) were classified as stunted or underweight-for-age, respectively., Results: With the new WHO 2006 standards a higher proportion (4.0%) of 3-month-old infants were underweight compared with the CDC (1.0%) or the NCHS (0.7%) child growth charts/references. In contrast to the NCHS references or the CDC charts, this proportion did not increase from 3 to 9 months or from 9 to 15 months. The proportion of children being stunted was highest (above 20%) with the WHO 2006 standards at all three ages. Again, in contrast to the old standards, this proportion did not increase from 3 to 9 months or from 9 to 15 months., Conclusions: The present results show considerably different growth faltering patterns for Gabonese children depending on the growth charts used to assess the prevalence of stunting and underweight. Shifting to the new WHO child growth standards may have important implications for child health programmes.

Background: Insecticide-treated bed nets (ITNs) range among the most effective measures of malaria prophylaxis, yet their implementation level in sub-Saharan Africa is still low. The goal of this study was to investigate the influence of socio-economic factors on the use of bed nets by mothers in Gabon., Methods: A cross-sectional study was conducted completing pre-tested, interviewer-administered questionnaires exploring socioeconomic proxy measures with 397 mothers or guardians of young children. Respondents were grouped according to their socio-economic situation, using scores. The condition of the bed nets was evaluated during a home visit., Results: Socio-economic factors of wellbeing were negatively associated with bed net use, such as living in a stone house (OR 0.26, 95% CI 0.14-0.48), running water in the house (OR 0.44, 95% CI 0.21-0.92), shower/flush toilet in the house (OR 0.39/0.34, 95% CI 0.21-0.75/0.16-0.73), ownership of a freezer (OR 0.50, 95% CI 0.26-0.96) and belonging to the highest group in the economic score (OR 0.32, 95% CI 0.15-0.67). In contrast, similar factors were positively associated with a good maintenance condition of the bed nets: higher monthly income (OR 5.64, 95% CI 2.41-13.19) and belonging to the highest group in the economic score (OR 2.55, 95% CI 1.19 - 5.45)., Conclusion: Among the poorest families in Lambaréné the coverage with untreated nets (UTNs) is the highest, but the condition of these UTNs is the worst. To achieve a broad implementation of ITNs in Lambaréné, there is an urgent need for educational programmes as well as need-tailored marketing strategies for ITNs.

Generalized anxiety disorder (GAD) has been conceptualized as being characterized by the experience of intense emotion, limited understanding of the emotional experience, poor ability to adaptively modulate emotions, and an aversive response to emotions [Mennin, D. S., Turk, C. L., Heimberg, R. G., & Carmin, C. N. (2004). Focusing on the regulation of emotion: a new direction for conceptualizing and treating generalized anxiety disorder. In: M. A. Reinecke, & D. A. Clark (Eds.), Cognitive therapy over the lifespan: theory, research and practice (pp. 60-89). New York: Wiley]. In order to test aspects of this model, participants completed daily diaries and a questionnaire measuring emotion regulation strategies. As hypothesized, relative to controls, individuals classified as having GAD reported more intense daily emotional experiences. Contrary to predictions and previous research, those with GAD did not show poor emotion differentiation and used several emotion regulation strategies more often than control participants. The implications for both the emotion dysregulation model and treatment of GAD are discussed.

Background: Contractile dysfunction develops in the chronically instrumented canine myocardium after bouts of low-flow ischemia and persists after reperfusion. The objective of this study is to identify whether changes in the phosphorylation state of myosin-binding protein C (MyBP-C) are a potential cause of dysfunction., Methods and Results: During low-flow ischemia, MyBP-C is dephosphorylated, and the number of actomyosin cross-bridges in the central core of the sarcomere decreases as thick filaments dissemble from the periphery of the myofibril. During reperfusion, MyBP-C remains dephosphorylated, and its degradation is accelerated., Conclusions: Dephosphorylation of MyBP-C may initiate changes in myofibril thick filament structure that decrease the interaction of myosin heads with actin thin filaments. Limiting the formation of actomyosin cross-bridges may contribute to the contractile dysfunction that is apparent after low-flow ischemia. Breakdown of MyBP-C during reperfusion may prolong myocardial stunning.

Canine myocardium was exposed to bouts of low-flow ischemia to identify the interactions that develop between the microtubule-based cytoskeleton and the heat shock protein 70 (HSP70) family of heat shock proteins in viable cardiomyocytes. "Moderate" or "severe" low-flow ischemia was produced in chronically instrumented dogs by reducing circumflex coronary flow by 50% for 2 h or by 75% for 5 h followed by reperfusion for 2 and 24 h, respectively. Electron and immunofluorescence microscopy demonstrated either partial or nearly complete depolymerization of the intermyofibrillar microtubules in areas of myofibril disruption and partial dissolution of the perinuclear microtubule girdle. In contrast, centrosomal tubulin arrays appeared to remain intact following low-flow ischemia. In cardiomyocytes displaying myofibril disruption, constitutively expressed HSP73 (HSC73) colocalized with intact but not disrupted microtubules and with perinuclear and centrosomal tubulin following moderate ischemia. Microtubule depolymerization and high molecular weight tubulin-HSC73 complexes were present in more severely ischemic tissue. These results suggest that HSC73 directly interacts with tubulin and may protect selected elements of the microtubule network and limit myofibril disruption during reversible low-flow ischemia.

Chronically instrumented dogs underwent 2- or 5-h regional reductions in coronary flow that were followed, respectively, by balanced reductions in myocardial contraction and O(2) consumption ("hibernation") and persistently reduced contraction despite normal myocardial O(2) consumption ("stunning"). Previously unidentified myofibrillar disruption developed during flow reduction in both experimental models and persisted throughout the duration of reperfusion (2-24 h). Aberrant perinuclear aggregates that resembled thick filaments and stained positively with a monoclonal myosin antibody were present in 34 +/- 3.8% (SE) and 68 +/- 5.9% of "hibernating" and "stunned" subendocardial myocytes in areas subjected to flow reduction and in 16 +/- 2.5% and 44 +/- 7.4% of subendocardial myocytes in remote areas of the same ventricles. Areas of myofibrillar disruption also showed glycogen accretion and unusual heterochromatin clumping adjacent to the inner nuclear envelope. The degrees of flow reduction employed were sufficient to reduce regional myofibrillar creatine kinase activity by 25-35%, but troponin I degradation was not evident. The observed changes may reflect an early, possibly reversible, phase of the myofibrillar loss characteristic of hypocontractile myocardium in patients undergoing revascularization.

Cardiac myocytes maintained in cell culture develop hypertrophy both in response to mechanical loading as well as to receptor-mediated signaling mechanisms. However, it has been shown that the hypertrophic response to these stimuli may be modulated through effects of intercellular contact achieved by maintaining cells at different plating densities. In this study, we show that the myocyte plating density affects not only the hypertrophic response and features of the differentiated phenotype of isolated adult myocytes, but also plays a significant role influencing myocyte survival in vitro. The native rod-shaped phenotype of freshly isolated adult myocytes persists in an environment which minimizes myocyte attachment and spreading on the substratum. However, these conditions are not optimal for long-term maintenance of cultured adult cardiac myocytes. Conditions which promote myocyte attachment and spreading on the substratum, on the other hand, also promote the re-establishment of new intercellular contacts between myocytes. These contacts appear to play a significant role in the development of spontaneous activity, which enhances the redevelopment of highly differentiated contractile, junctional, and sarcoplasmic reticulum structures in the cultured adult cardiomyocyte. Although it has previously been shown that adult cardiac myocytes are typically quiescent in culture, the addition of beta-adrenergic agonists stimulates beating and myocyte hypertrophy, and thereby serves to increase the level of intercellular contact as well. However, in densely-plated cultures with intrinsically high levels of intercellular contact, spontaneous contractile activity develops without the addition of beta-adrenergic agonists. In this study, we compare the function, morphology, and natural history of adult feline cardiomyocytes which have been maintained in cultures with different levels of intercellular contact, with and without the addition of beta-adrenergic agonists. Intercellular contact, communication, and transmission of contractile forces between myocytes appears to play a primary role in remodeling the 2-dimensional cell layer into a parallel alignment of elongated myocytes with highly developed intercalated disk-like junctions. This highly differentiated state is very stable, and cultures which achieve this state exhibit significantly greater longevity than more sparsely plated myocytes. These myocytes typically continue beating, and survive from 6 to more than 12 weeks in culture. When this level of contact and differentiation are not achieved, even among beta-adrenergic stimulated myocytes, contractile activity is not sustained, myofibrils atrophy, there is little or no development of junctional complexes, and the period of myocyte viability is typically no more than 5 weeks in vitro.

Fluctuations in hemodynamic load have been documented to modulate contractile protein turnover and myofibrillar structure in the heart; however, the relative importance of active and passive loading in regulating adult cardiocyte growth remains unresolved. To address this issue at the cellular level, adult feline cardiocytes were cultured either on Silastic membranes or plastic surfaces. Cardiocyte-laden membranes were stretched 10% of their rest length to enhance passive loading, whereas heart cells cultured on plastic or Silastic were field stimulated at 1 Hz to mimic active loading. Turnover of contractile proteins and structural integrity of the contractile-cytoskeletal apparatus were monitored for periods ranging from 4 to 72 h. Active and passive loading elevated contractile protein synthesis nearly equally (approximately 50%) and promoted the attachment of remodeled myofibrils to vinculin-positive focal contacts and/or costameres during the first 24 h of loading. Thereafter, rates of contractile protein synthesis returned to control values in passively stretched heart cells but remained elevated in field-stimulated cultures. The fractional rate of growth was increased significantly (approximately 8%/day) in electrically paced cells, whereas in passively stretched cardiocytes the growth rate rose only modestly (approximately 2%/day). Changes in the rate of myocyte growth appeared more closely correlated with the development of focal contacts and myofibril remodeling than with changes in myofibrillar protein turnover per se. 2,3-Butanedione monoxime, nifedipine, and, to a lesser extent, ryanodine blocked field-stimulated contractile protein synthesis and myofibrillar remodeling but had no impact on protein turnover or myofibril reassembly in passively loaded cardiocytes. The results of these experiments imply that both active and passive loading stimulate contractile protein turnover and myofibril remodeling, but the generation of active tension accelerates cardiocyte growth to a greater extent than passive loading. Furthermore, pharmacological interventions suggest that unique pathways may mediate these cellular events in actively and passively loaded adult cardiocytes.

Cultured adult rabbit cardiac myocytes treated with recombinant growth factors display enhanced rates of protein accumulation (ie, growth) in response to insulin and insulin-like growth factors (IGFs), but epidermal growth factor, acidic or basic fibroblast growth factor, and platelet-derived growth factor failed to increase contractile protein synthesis or growth of the heart cells. Insulin and IGF-1 increased growth rates by stimulating anabolic while simultaneously inhibiting catabolic pathways, whereas IGF-2 elevated growth modestly by apparently inhibiting lysosomal proteolysis. Neutralizing antibodies directed against either IGF-1 or IGF-2 or IGF binding protein 3 blocked protein accumulation. A monoclonal antibody directed against the IGF-1 receptor also inhibited changes in protein turnover provoked by recombinant human IGF-1 but not IGF-2. Of the other growth factors tested, only transforming growth factor-beta 1 increased the fractional rate of myosin heavy chain (MHC) synthesis, with beta-MHC synthesis being elevated and alpha-MHC synthesis being suppressed. However, the other growth factors were able to modestly stimulate the rate of DNA synthesis in this preparation. Bromodeoxyuridine labeling revealed that these growth factors increased DNA synthesis in myocytes and nonmyocytes alike, but the heart cells displayed neither karyokinesis or cytokinesis. In contrast, cocultures of cardiac myocytes and nonmyocytes and nonmyocyte-conditioned culture medium failed to enhance the rate of cardiac MHC synthesis or its accumulation, implying that quiescent heart cells do not respond to "conditioning" by cardiac nonmyocytes. These findings demonstrated that insulin and the IGFs promote passively loaded cultured adult rabbit heart cells to hypertrophy but suggest that other growth factors tested may be limited in this regard.

Adult feline ventricular myocytes cultured on a laminin-coated substratum reestablish intercellular junctions, yet disassemble their myofibrils. Immunofluorescence microscopy reveals that these non-beating heart cells lack vinculin-positive focal adhesions; moreover, intercellular junctions are also devoid of vinculin. When these quiescent myocytes are stimulated to contract with the beta-adrenergic agonist, isoproterenol, extensive vinculin-positive focal adhesions and intercellular junctions emerge. If solitary myocytes are stimulated to beat, an elaborate series of vinculin-positive focal adhesions develop which appear to parallel the reassembly of myofibrils. In cultures where neighboring myocytes reestablish cell-cell contact, myofibrils appear to reassemble from the fascia adherens rather than focal contacts. Activation of beating is accompanied by a significant reduction in the rate of total and cytoskeletal protein synthesis; in fact, myofibrillar reassembly, redevelopment of focal adhesions and fascia adherens junctions require no protein synthesis for at least 24 h, implying the existence of an assembly competent pool of cytoskeletal proteins. Maturation of the fasciae adherens and the appearance of vinculin within Z-line/costameres, does require de novo synthesis of new cytoskeletal proteins. Changes in cytoskeletal protein turnover appear dependent on beta agonist-induced cAMP production, but myofibrillar reassembly is a cAMP-independent event. Such observations suggest that mechanical forces, in the guise of contractile activity, regulate vinculin distribution and myofibrillar order in cultured adult feline heart cells.

When rabbit ventricular myocytes were cultured for 1 wk and then exposed to alpha- and/or beta-adrenergic agonists, such nonbeating heart cell preparations disclosed increased protein-to-DNA ratios and elevated RNA content, indicative of cellular hypertrophy. Norepinephrine, isoproterenol, and phenylephrine provoked hypertrophy with norepinephrine eliciting a greater response than isoproterenol or phenylephrine. Specific alpha- and beta-antagonists blocked growth by inhibiting catecholamine-induced changes in protein turnover. Each catecholamine enhanced the fractional rate of protein synthesis within 48 h; however, changes in growth rates appeared to be modulated, in part, by alterations in protein degradation. Even though rates of total protein and actin synthesis resembled values measured in vivo, myosin heavy chain fractional rate of synthesis was only 22% of in vivo levels. Double label immunofluorescence microscopy further illustrated that catecholamine treatment accelerated myofibrillar disruption in these quiescent heart cells. These observations suggested that in the absence of beating, neurohumoral modulation of contractile protein turnover was not associated with the maintenance of myofibrillar integrity even though catecholamines induced cellular hypertrophy.

The isolation and culture of adult cardiac myocytes has proved to be an ideal model system to explore myocardial biology at the cellular level. A major criticism of this model, however, has been that organ-specific characteristics such as cell shape and subcellular structural organization cannot be retained in vitro for prolonged periods of time. Encasing freshly isolated myocytes in a matrix of calcium alginate enables one to maintain the rod-like, three-dimensional (3D) shape of the cultured myocyte. Such preparations more closely resemble their in vivo counterparts with respect to the organization of the contractile apparatus, the transverse tubular system and the sarcoplasmic reticulum than do heart cells cultured on a two-dimensional (2D) plastic surface. Stereologic measurements reveal that myofibrillar volume density (VvMYF) decreases in both non-beating preparations over a 2-week interval, but VvMYF is conserved in cells cultured in an alginate matrix when compared to those myocytes maintained on a laminin-coated substratum. The present observations suggest that in the absence of contractile function myofibrillar atrophy appears responsible for the decline in VvMYF in alginate (3D) preparations, whereas atrophy and subcellular remodelling probably mediate the myofibrillar loss and reorganization that develops when adult heart cells are cultured on a 2D surface.

Rabbit cardiac myocytes remain quiescent for more than 1 month when cultured at low density. During this period, myofibrillar volume density declines sixfold as myofibrils are disassembled or degraded and are replaced by actin and alpha-actinin-positive, myosin-negative structures that resemble myofibrils but lack thick filaments. Such structures are termed minute myofibrils. The length of the sarcomeres in these altered myofibrils is significantly less than length values obtained from freshly isolated heart cells or from contracting myocytes. A number of high density cultures develop spontaneous, synchronous contraction during the second week of culture. Myofibrillar volume density is stabilized when beating begins, and no further decline is observed in the succeeding weeks of culture. Such contracting myocytes display myofibrils typical of normal heart with no visible evidence of minute myofibrils. The volume density of the transverse tubular system also declines significantly in both beating and nonbeating myocytes, and its reduction appears more closely correlated with cell spreading than with beating per se. No quantitative changes in volume density of mitochondria or sarcoplasmic reticulum could be documented, but the structural organization of the sarcoplasmic reticulum seems to be greatly influenced by the physiological state of the heart cell. The present observations document the importance of mechanical factors in regulating the integrity of the contractile apparatus in cardiac myocytes and emphasize the utility of the cultured heart cell to directly investigate structure-function relations in individual myocytes.

Isolated rabbit ventricular cardiac myocytes adapt readily to primary culture. As the myocytes spread and flatten over the culture substratum, the myofibrillar apparatus retains a "rod-like" orientation. Development of contractile activity is crucial in the maintenance of the integrity of the myofibrillar apparatus during prolonged culture. Myocytes that fail to beat display morphological indications of atrophy; conversely, myocytes that commence beating show no such morphological signs of myofibrillar disorganization. The subcellular organization of other elements of the contractile apparatus, including the transverse tubular system and the sarcoplasmic reticulum, retain their structural relationship with the myofibrils in beating myocytes but not in quiescent cells. Cultured adult myocytes represent an important model to investigate the influence of mechanical factors on the organization and maintenance of the adult cardiac phenotype.

The present observations demonstrate that quiescent calcium-tolerant adult rabbit cardiac myocytes can be isolated by collagenase-hyaluronidase perfusion and maintained in primary culture for at least 2 wk. Culturing large numbers of myocytes requires that the freshly isolated cells be attached to a suitable substratum such as laminin, type IV collagen, or fetal bovine serum. The cultured myocytes retain their rod-like morphology for approximately 7 days before gradually spreading into a flattened conformation by 14 days. During the 1st wk of culture, contaminating interstitial cells rapidly proliferate, making cultures unsuitable for long-term study. Pure myocyte populations can be established and maintained if freshly isolated cells are cultured in the presence of cytosine arabinoside (Ara-C, 10 microM). This antimetabolite does not appear to adversely affect high-energy phosphates, since ATP and creatine phosphate (CrP) content of the myocytes is maintained at levels normally found in biopsy samples of rabbit myocardium. These results illustrate that an energetically stable population of adult cardiac myocytes can be maintained in primary culture in sufficient numbers to make them useful for future investigations of myocyte function.

Lysosomal cathepsin D has been localized with the electron microscope employing an indirect immunohistochemical method using peroxidase labeled, monospecific antibody Fab' subunits. The acid proteinase has been demonstrated within secondary lysosomes of cardiac myocytes and interstitial cells, but not in components of the Golgi apparatus or endoplasmic reticulum. Incubations with a variety of peroxidatic inhibitors suggests that the staining that is observed in secondary lysosomes is attributable to the peroxidase-labeled antibody and not to endogenous oxidation of DAB. The protocol outlined here provides a reproducible method to localize the major lysosomal acid proteinase of the heart at the subcellular level.

By use of a combined morphologic, immunocytochemical, and biochemical approach, this study demonstrates the changes in the lysosomal vacuolar apparatus that accompany thyroxine-induced cardiac hypertrophy. During the 1st wk of thyroxine administration, immunocytochemical studies revealed a decline in cathepsin D within many myocytes, but an increase within interstitial cells. These events transpired with only a modest rise in cathepsin D activity. During the 2nd wk, cathepsin D reappeared within most myocytes and continued to increase within the interstitial population and was associated with a general rise in lysosomal enzyme activity. These data demonstrate the importance of employing both immunocytochemical and biochemical approaches to evaluate the status of the lysosomal vacuolar apparatus, and suggests that the lysosomal vacuolar apparatus may be involved in the apparent remodeling that attends rapid cardiac growth, which may represent one component of the enhanced rates of proteolysis documented in this model of cardiac hypertrophy.

Cardiac myocytes whose lysosomes had been pre-labelled with acridine orange were exposed to either L-amino acid methyl esters (L-leucine or methionine methyl ester) or to 'lysosomotropic' weak bases (chloroquine, methylamine, and NH4Cl) for 1 h. Both types of interventions dilated lysosomes equally and inhibited proteolysis to varying degrees. The weak bases produced no apparent alterations in the acridine orange staining, whereas the methylated amino acids induced a marked redistribution of the fluorescent dye from lysosomes into the myoplasm, suggesting that they may have provoked a change in lysosomal membrane permeability. A brief exposure to weak bases failed to enhance acid proteinase secretion into the culture medium but apparently inactivated cellular cathepsin B activity. In contrast, methylated amino acids induced no alterations in acid proteinase activity or the cellular distribution of the two proteolytic enzymes. Lastly, weak bases markedly elevated intralysosomal pH as measured with fluorescein dextran, while only modest rises were observed after amino acid methyl ester treatment. The present observations imply that amino acid methyl esters represent a new class of reagents with actions distinctly different from those of chloroquine and NH4Cl, and they may provide a unique and valuable means of studying secondary lysosomal function in cell culture.