Thermal and cytokine responses to endotoxin challenge during early life.

Sudden infant death syndrome (SIDS) remains the leading cause of infant mortality beyond the neonatal period. An increase in body temperature as a result of high environmental temperature, overwrapping of infants, and (or) infection are associated with SIDS. Endotoxins such as lipopolysaccharide (LPS) and heat stress may perturb cardiorespiratory function and thermoregulation. Although LPS-mediated body temperature and cytokine responses are well documented in older animals, the capacity of LPS to induce fever and cytokine response in young rats remains unclear. Therefore, we sought to investigate the acute effects of LPS on body temperature and cytokine concentrations in rat pups. Postnatal day 7 rat pups were divided into 3 groups: Group 1, rats were administered LPS intraperitoneally (200 µg/kg); Group 2, rats received saline at volume equal to that administered in the LPS group; Group 3, rats received no treatment. Pups were placed in custom-made chambers maintained at ambient temperature of 33 °C. Body surface temperature was continuously monitored for 4 h. Thereafter, the rats were euthanized and serum was collected for cytokine analysis. We demonstrate that LPS treatment increased MIP-1α, IL-10, MCP-1, IP-10, fractalkine, and TNF-α with no concurrent rise in body surface temperature. Although neonatal rats produced an array of cytokines in response to LPS, there was no evidence of fever.

Interactive effects of maternal cigarette smoke, heat stress, hypoxia, and lipopolysaccharide on neonatal cardiorespiratory and cytokine responses.

Maternal cigarette smoke (CS) exposure exhibits a strong epidemiological association with Sudden Infant Death Syndrome, but other environmental stressors, including infection, hyperthermia, and hypoxia, have also been postulated as important risk factors. This study examines whether maternal CS exposure causes maladaptations within homeostatic control networks by influencing the response to lipopolysaccharide, heat stress, and/or hypoxia in neonatal rats. Pregnant dams were exposed to CS or parallel sham treatments daily for the length of gestation. Offspring were studied at postnatal days 6-8 at ambient temperatures (Ta) of 33°C or 38°C. Within each group, rats were allocated to control, saline, or LPS (200 µg/kg) treatments. Cardiorespiratory patterns were examined using head-out plethysmography and ECG surface electrodes during normoxia and hypoxia (10% O2). Serum cytokine concentrations were quantified from samples taken at the end of each experiment. Our results suggest maternal CS exposure does not alter minute ventilation (V̇e) or heart rate (HR) response to infection or high temperature, but independently increases apnea frequency. CS also primes the inflammatory system to elicit a stronger cytokine response to bacterial insult. High Ta independently depresses V̇e but augments the hypoxia-induced increase in V̇e Moreover, higher Ta increases HR during normoxia and hypoxia, and in the presence of an immune challenge, increases HR during normoxia, and reduces the increase normally associated with hypoxia. Thus, while most environmental risk factors increase the burden on the cardiorespiratory system in early life, hyperthermia and infection blunt the normal HR response to hypoxia, and gestational CS independently destabilizes breathing by increasing apneas.

Cardiorespiratory control and cytokine profile in response to heat stress, hypoxia, and lipopolysaccharide (LPS) exposure during early neonatal period.

Sudden infant death syndrome (SIDS) is one of the most common causes of postneonatal infant mortality in the developed world. An insufficient cardiorespiratory response to multiple environmental stressors (such as prone sleeping positioning, overwrapping, and infection), during a critical period of development in a vulnerable infant, may result in SIDS. However, the effect of multiple risk factors on cardiorespiratory responses has rarely been tested experimentally. Therefore, this study aimed to quantify the independent and possible interactive effects of infection, hyperthermia, and hypoxia on cardiorespiratory control in rats during the neonatal period. We hypothesized that lipopolysaccharide (LPS) administration will negatively impact cardiorespiratory responses to increased ambient temperature and hypoxia in neonatal rats. Sprague-Dawley neonatal rat pups were studied at postnatal day 6-8. Rats were examined at an ambient temperature of 33°C or 38°C. Within each group, rats were allocated to control, saline, or LPS (200 µg/kg) treatments. Cardiorespiratory and thermal responses were recorded and analyzed before, during, and after a hypoxic exposure (10% O2). Serum samples were taken at the end of each experiment to measure cytokine concentrations. LPS significantly increased cytokine concentrations (such as TNFα, IL-1ß, MCP-1, and IL-10) compared to control. Our results do not support a three-way interaction between experimental factors on cardiorespiratory control. However, independently, heat stress decreased minute ventilation during normoxia and increased the hypoxic ventilatory response. Furthermore, LPS decreased hypoxia-induced tachycardia. Herein, we provide an extensive serum cytokine profile under various experimental conditions and new evidence that neonatal cardiorespiratory responses are adversely affected by dual interactions of environmental stress factors.

Deletion of Galgt2 (B4Galnt2) reduces muscle growth in response to acute injury and increases muscle inflammation and pathology in dystrophin-deficient mice.

Transgenic overexpression of Galgt2 (official name B4Galnt2) in skeletal muscle stimulates the glycosylation of α dystroglycan (αDG) and the up-regulation of laminin α2 and dystrophin surrogates known to inhibit muscle pathology in mouse models of congenital muscular dystrophy 1A and Duchenne muscular dystrophy. Skeletal muscle Galgt2 gene expression is also normally increased in the mdx mouse model of Duchenne muscular dystrophy compared with the wild-type mice. To assess whether this increased endogenous Galgt2 expression could affect disease, we quantified muscular dystrophy measures in mdx mice deleted for Galgt2 (Galgt2(-/-)mdx). Galgt2(-/-) mdx mice had increased heart and skeletal muscle pathology and inflammation, and also worsened cardiac function, relative to age-matched mdx mice. Deletion of Galgt2 in wild-type mice also slowed skeletal muscle growth in response to acute muscle injury. In each instance where Galgt2 expression was elevated (developing muscle, regenerating muscle, and dystrophic muscle), Galgt2-dependent glycosylation of αDG was also increased. Overexpression of Galgt2 failed to inhibit skeletal muscle pathology in dystroglycan-deficient muscles, in contrast to previous studies in dystrophin-deficient mdx muscles. This study demonstrates that Galgt2 gene expression and glycosylation of αDG are dynamically regulated in muscle and that endogenous Galgt2 gene expression can ameliorate the extent of muscle pathology, inflammation, and dysfunction in mdx mice.

A comparative study of N-glycolylneuraminic acid (Neu5Gc) and cytotoxic T cell (CT) carbohydrate expression in normal and dystrophin-deficient dog and human skeletal muscle.

The expression of N-glycolylneuraminic acid (Neu5Gc) and the cytotoxic T cell (CT) carbohydrate can impact the severity of muscular dystrophy arising from the loss of dystrophin in mdx mice. Here, we describe the expression of these two glycans in skeletal muscles of dogs and humans with or without dystrophin-deficiency. Neu5Gc expression was highly reduced (>95%) in muscle from normal golden retriever crosses (GR, n = 3) and from golden retriever with muscular dystrophy (GRMD, n = 5) dogs at multiple ages (3, 6 and 13 months) when compared to mouse muscle, however, overall sialic acid expression in GR and GRMD muscles remained high at all ages. Neu5Gc was expressed on only a minority of GRMD satellite cells, CD8⁺ T lymphocytes and macrophages. Human muscle from normal (no evident disease, n = 3), Becker (BMD, n = 3) and Duchenne (DMD, n = 3) muscular dystrophy individuals had absent to very low Neu5Gc staining, but some punctate intracellular muscle staining was present in BMD and DMD muscles. The CT carbohydrate was localized to the neuromuscular junction in GR muscle, while GRMD muscles had increased expression on a subset of myofibers and macrophages. In humans, the CT carbohydrate was ectopically expressed on the sarcolemmal membrane of some BMD muscles, but not normal human or DMD muscles. These data are consistent with the notion that altered Neu5Gc and CT carbohydrate expression may modify disease severity resulting from dystrophin deficiency in dogs and humans.

Homozygous founder mutation in desmocollin-2 (DSC2) causes arrhythmogenic cardiomyopathy in the Hutterite population.

BACKGROUND: Dominant mutations in cellular junction proteins are the major cause of arrhythmogenic cardiomyopathy, whereas recessive mutations in those proteins cause cardiocutaneous syndromes such as Naxos and Carvajal syndrome. The Hutterites are distinct genetic isolates who settled in North America in 1874. Descended from <100 founders, they trace their origins to 16th-century Europe. METHODS AND RESULTS: We clinically and genetically evaluated 2 large families of the Alberta Hutterite population with a history of sudden death and found several individuals with severe forms of biventricular cardiomyopathy characterized by mainly left-sided localized aneurysms, regions of wall thinning with segmental akinesis, in addition to typical electric and histological features known for arrhythmogenic right ventricular cardiomyopathy. We identified a homozygous truncation mutation, c.1660C>T (p.Q554X) in desmocollin-2 (DSC2), in affected individuals and determined a carrier frequency of this mutation of 9.4% (1 in 10.6) among 1535 Schmiedeleut Hutterites, suggesting a common founder in that subgroup. Immunohistochemistry of endomyocardial biopsy samples revealed altered expression of the truncated DSC2 protein at the intercalated discs but only minor changes in immunoreactivity of other desmosomal proteins. Recombinant expressed mutant DSC2 protein in cells confirmed a stable, partially processed truncated protein with cytoplasmic and membrane localization. CONCLUSIONS: A homozygous truncation mutation in DSC2 leads to a cardiac-restricted phenotype of an early onset biventricular arrhythmogenic cardiomyopathy. The truncated protein remains partially stable and localized at the intercalated discs. These data suggest that the processed DSC2 protein plays a role in maintaining desmosome integrity and function.

N-Glycolylneuraminic acid deficiency worsens cardiac and skeletal muscle pathophysiology in α-sarcoglycan-deficient mice.

Roughly 3 million years ago, an inactivating deletion occurred in CMAH, the human gene encoding CMP-Neu5Ac (cytidine-5'-monophospho-N-acetylneuraminic acid) hydroxylase (Chou HH, Takematsu H, Diaz S, Iber J, Nickerson E, Wright KL, Muchmore EA, Nelson DL, Warren ST, Varki A. 1998. A mutation in human CMP-sialic acid hydroxylase occurred after the Homo-Pan divergence. Proc Natl Acad Sci USA. 95:11751-11756). This inactivating deletion is now homozygous in all humans, causing the loss of N-glycolylneuraminic acid (Neu5Gc) biosynthesis in all human cells and tissues. The CMAH enzyme is active in other mammals, including mice, where Neu5Gc is an abundant form of sialic acid on cellular membranes, including those in cardiac and skeletal muscle. We recently demonstrated that the deletion of mouse Cmah worsened the severity of pathophysiology measures related to muscular dystrophy in mdx mice, a model for Duchenne muscular dystrophy (Chandrasekharan K, Yoon JH, Xu Y, deVries S, Camboni M, Janssen PM, Varki A, Martin PT. 2010. A human-specific deletion in mouse Cmah increases disease severity in the mdx model of Duchenne muscular dystrophy. Sci Transl Med. 2:42-54). Here, we demonstrate similar changes in cardiac and skeletal muscle pathology and physiology resulting from Cmah deletion in α-sarcoglycan-deficient (Sgca(-/-)) mice, a model for limb girdle muscular dystrophy 2D. These experiments demonstrate that loss of mouse Cmah can worsen disease severity in more than one form of muscular dystrophy and suggest that Cmah may be a general genetic modifier of muscle disease.

Genetic defects in muscular dystrophy.

The muscular dystrophies are a group of neuromuscular disorders associated with muscle weakness and wasting, which in many forms can lead to loss of ambulation and premature death. A number of muscular dystrophies are associated with loss of proteins required for the maintenance of muscle membrane integrity, in particular with proteins that comprise the dystrophin-associated glycoprotein (DAG) complex. Proper glycosylation of O-linked mannose chains on alpha-dystroglycan, a DAG member, is required for the binding of the extracellular matrix to dystroglycan and for proper DAG function. A number of congenital disorders of glycosylation have now been described where alpha-dystroglycan glycosylation is altered and where muscular dystrophy is a predominant phenotype. Glycosylation is also increasingly being appreciated as a genetic modifier of disease phenotypes in many forms of muscular dystrophy and as a target for the development of new therapies. Here we will review the mouse models available for the study of this group of diseases and outline the methodologies required to describe disease phenotypes.

A human-specific deletion in mouse Cmah increases disease severity in the mdx model of Duchenne muscular dystrophy.

During the evolution of humans, an inactivating deletion was introduced in the CMAH (cytidine monophosphate-sialic acid hydroxylase) gene, which eliminated biosynthesis of the common mammalian sialic acid N-glycolylneuraminic acid from all human cells. We found that this human-specific change in sialylation capacity contributes to the marked discrepancy in phenotype between the mdx mouse model for Duchenne muscular dystrophy (DMD) and the human disease. When compared to human patients with DMD, mdx mice show reduced severity or slower development of clinically relevant disease phenotypes, despite lacking dystrophin protein in almost all muscle cells. This is especially true for the loss of ambulation, cardiac and respiratory muscle weakness, and decreased life span, all of which are major phenotypes contributing to DMD morbidity and mortality. These phenotypes occur at an earlier age or to a greater degree in mdx mice that also carry a human-like mutation in the mouse Cmah gene, possibly as a result of reduced strength and expression of the dystrophin-associated glycoprotein complex and increased activation of complement. Cmah-deficient mdx mice are a small-animal model for DMD that better approximates the human glycome and its contributions to muscular dystrophy.

The synaptic CT carbohydrate modulates binding and expression of extracellular matrix proteins in skeletal muscle: Partial dependence on utrophin.

The CT carbohydrate, Neu5Ac/Neu5Gcalpha2,3[GalNAcbeta1,4]Galbeta1,4GlcNAcbeta-, is specifically expressed at the neuromuscular junction in skeletal myofibers of adult vertebrates. When Galgt2, the glycosyltransferase that creates the synaptic beta1,4GalNAc portion of this glycan, is overexpressed in extrasynaptic regions of the myofiber membrane, alpha dystroglycan becomes glycosylated with the CT carbohydrate and this coincides with the ectopic expression of synaptic dystroglycan-binding proteins, including laminin alpha4, laminin alpha5, and utrophin. Here we show that both synaptic and extrasynaptic forms of laminin and agrin have increased binding to the CT carbohydrate compared to sialyl-N-acetyllactosamine, its extrasynaptically expressed precursor. Muscle laminins also show increased binding to CT-glycosylated muscle alpha dystroglycan relative to its non-CT-containing glycoforms. Overexpression of Galgt2 in transgenic mouse skeletal muscle increased the mRNA expression of extracellular matrix (ECM) genes, including agrin and laminin alpha5, as well as utrophin, integrin alpha7, and neuregulin. Increased expression of ECM proteins in Galgt2 transgenic skeletal muscles was partially dependent on utrophin, but utrophin was not required for Galgt2-induced changes in muscle growth or neuromuscular development. These experiments demonstrate that overexpression of a synaptic carbohydrate can increase both ECM binding to alpha dystroglycan and ECM expression in skeletal muscle, and they suggest a mechanism by which Galgt2 overexpression may inhibit muscular dystrophy and affect neuromuscular development.

O-fucosylation of muscle agrin determines its ability to cluster acetylcholine receptors.

Protein O-fucosyltransferase 1 (Pofut1) transfers fucose to serine or threonine on proteins, including Notch receptors, that contain EGF repeats with a particular consensus sequence. Here we demonstrate that agrin is O-fucosylated in a Pofut1-dependent manner, and that this glycosylation can regulate agrin function. Fucosylation of recombinant C45 agrin, both active (neural, z8) and inactive (muscle, z0) splice forms, was eliminated when agrin was overexpressed in Pofut1-deficient cells or by mutation of a consensus site for Pofut1 fucosylation (serine 1726 in the EGF4 domain). Loss of O-fucosylation caused a gain of function for muscle agrin such that it stimulated AChR clustering and MuSK phosphorylation in cultured myotubes at levels normally only found with the neural splice form. Deletion of Pofut1 in cultured primary myotubes and in adult skeletal muscle increased AChR aggregation. In addition, Pofut1 gene and protein expression and Pofut1 activity of the EGF4 domain of agrin were modulated during neuromuscular development. These data are consistent with a role for Pofut1 in AChR aggregation during synaptogenesis via the regulation of the synaptogenic activity of muscle agrin.

Overexpression of the cytotoxic T cell (CT) carbohydrate inhibits muscular dystrophy in the dyW mouse model of congenital muscular dystrophy 1A.

A number of recent studies have demonstrated therapeutic effects of transgenes on the development of muscle pathology in the mdx mouse model for Duchenne muscular dystrophy, but none have been shown also to be effective in mouse models for laminin alpha2-deficient congenital muscular dystrophy (MDC1A). Here, we show that overexpression of the cytotoxic T cell (CT) GalNAc transferase (Galgt2) is effective in inhibiting the development of muscle pathology in the dy(W) mouse model of MDC1A, much as we had previously shown in mdx animals. Embryonic overexpression of Galgt2 in skeletal muscles using transgenic mice or postnatal overexpression using adeno-associated virus both reduced the extent of muscle pathology in dy(W)/dy(W) skeletal muscle. As with mdx mice, embryonic overexpression of the Galgt2 transgene in dy(W)/dy(W) myofibers inhibited muscle growth, whereas postnatal overexpression did not. Both embryonic and postnatal overexpression of Galgt2 in dy(W)/dy(W) muscle increased the expression of agrin, a protein that, in recombinant form, has been shown to ameliorate disease, whereas laminin alpha1, another disease modifier, was not expressed. Galgt2 over-expression also stimulated the glycosylation of a gly-colipid with the CT carbohydrate, and glycolipids accounted for most of the CT-reactive material in postnatal overexpression experiments. These experiments demonstrate that Galgt2 overexpression is effective in altering disease progression in skeletal muscles of dy(W) mice and should be considered as a therapeutic target in MDC1A.

Carvedilol improves left ventricular function in murine coxsackievirus-induced acute myocarditis association with reduced myocardial interleukin-1beta and MMP-8 expression and a modulated immune response.

BACKGROUND: Proinflammatory cytokines induce the expression of matrix metalloproteinases that play a crucial role in myocardial remodeling. Beta-adrenergic receptor stimulation influences the production of cytokines heralding the possibility of modulating cytokine production by beta-adrenergic blockers. METHODS AND RESULTS: In a coxsackievirus B3 murine myocarditis model (BALB/c), effects of carvedilol and metoprolol on myocardial cytokine expression, inflammatory cell infiltration and MMP/TIMP profiles were investigated. In carvedilol-treated mice, a significant improvement in left ventricular function was documented 10 days post infection. In infected mice (n=10), IL-1beta, TNF-alpha, TGF-beta(1) and IL-10 myocardial mRNA abundance were increased significantly (240%, 200%, 161%, and 230%) compared to controls (n=10), while IL-15 mRNA was markedly reduced (70%). Infected mice showed significantly increased infiltrations with CD3-, CD4- and CD8-T-lymphocytes (730%, 1110%, 380%). In the infected mice, myocardial MMP/TIMP profiles presented a significant upregulation of membrane type-1 MMP, MMP-9, MMP-8 and MMP-3 (150%, 160%, 340%, and 270%) and a significant decrease in TIMP-4 levels (75%). Carvedilol attenuated over-expression of myocardial TGF-beta(1), IL-1beta and MMP-8 mRNA expression significantly and induced a relevant IL-10 mRNA expression in the infected mice (n=10). By an unchanged infiltration with CD3-T-lymphocytes, carvedilol showed a representative reduction in CD4-T-lymphocytes. CONCLUSION: Carvedilol treatment in experimental myocarditis leads to reduced expression of proinflammatory cytokines and MMPs, which contributes to reduced matrix degradation and ultimately to improved structural integrity of the heart. Besides the antiadrenergic potential, carvedilol is beneficial due to a wide range of biological activities (antiinflammatory, antifibrotic, antioxidative and immunomodulatory).

Viral heart disease: molecular diagnosis, clinical prognosis, and treatment strategies.

Myocarditis is considered as a potent predisposing factor for dilated cardiomyopathy (DCM). Molecular biological proof of viral genome and immunohistochemical evaluation of intramyocardial inflammation are substantial in the identification and diagnosis of this pathological condition. Viruses are generally thought to be the common causative agents that trigger myocarditis and, therefore, several investigations are indispensable for the detection of viral genome in the myocardium in diagnosing viral myocarditis. The era of molecular diagnosis for viral myocarditis began with the establishment of the slot blot hybridization technique for the detection of viral genome in endomyocardial biopsy specimens. Due to inherent technical inadequacies, this method soon was replaced by in situ hybridization and polymerase chain reaction (PCR). Although in situ hybridization combines both morphological and molecular diagnosis, difficulty in standardization, possibility of nonspecific hybridization, and focal viral infection have led PCR to be an ideal molecular diagnostic strategy for the detection of viral myocarditis. Despite controversies over the specificity of this technique, several studies have substantiated the use of PCR in virological diagnosis. The ability to detect the state of viral replicative activity by demonstrating the presence of enteroviral minus-strand RNA has added a new dimension to studies on viral etiology of myocarditis and DCM. Advances in molecular diagnosis have indicated beyond doubt that persistence of viral infection is associated with disease deterioration and poor prognosis. Viral etiology of myocarditis and its contribution to the development of DCM have suggested antiviral therapy for myocarditis and DCM patients with proven viral infection.

Myocardial remodeling in viral heart disease: possible interactions between inflammatory mediators and MMP-TIMP system.

Matrix metalloproteinases (MMP), a family of proteases, are involved in the degradation of extracellular matrix proteins and hence in the determination of interstitial architecture. In the heart, MMPs have been found to play a significant role in the development of myocardial remodeling and congestive heart failure. Tissue inhibitors of matrix metalloproteinases (TIMPs) represent a family of proteins which are known to regulate the expression and activity of MMPs. TIMPs are endogenous physiological inhibitors of MMPs and their concomitant downregulation in heart failure suggests the existence of a critical balance between MMPs and TIMPs in the normal maintenance of myocardial interstitial homeostasis. In addition, cytokines regulate expression of both MMPs and TIMPs besides eliciting a direct effect on myocardial cell function. Therefore, myocardial inflammation may also contribute to the development of cardiac remodeling along with other stimuli like mechanical stress and humoral factors. Viral myocarditis, a predisposing factor for dilated cardiomyopathy, is a condition in which extent of intramyocardial inflammation is thought to determine the progression of disease. Inflammatory events in the heart following viral infection are speculated to be responsible for the transition of myocarditis to dilated cardiomyopathy. In viral myocarditis and other inflammatory heart diseases, the inflammatory cells and their battery of cytokines may also alter the myocardial MMP-TIMP system and eventually lead to dilation of the heart and ventricular dysfunction. The objective of this review is to present an overall picture of the inflammatory phase in viral myocarditis and discuss the possible interactions between inflammation and myocardial MMP profiles which may lead to the evolution of dilated cardiomyopathy.

[Mechanisms of extracellular matrix remodeling in dilated cardiomyopathy]. / Remodeling der extrazellulären Matrix bei dilatativer Kardiomyopathie.

BACKGROUND: The mechanisms underlying myocardial remodeling during heart failure have historically been attributed as the consequence of intrinsic changes in cardiac myocytes. Nevertheless, over the last several years, it has become increasingly evident that disruption of extracellular matrix (ECM) homeostasis is also a deciding factor for the progression of myocardial failure. PATHOGENETIC MECHANISMS: Collagens, the chief components of extracellular matrix, are a tightly regulated family of proteins that determine the structural and functional integrity of heart. Synthesis of collagens is regulated at the cellular level while deposition of these proteins depend on a balance between matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinase (TIMPs). Infiltrating inflammatory cells are major producers of MMPs though myocardial cells are also found to synthesize these proteolytic enzymes. However, immune-mediated regulation of myocardial collagen synthesis and deposition during myocardial inflammation remains poorly understood. It seems likely that a paracrine/autorine effect of a repertoire of cytokines on inflammatory cells and myocardial cells may lead to an imbalance in myocardial MMP/TIMP ratio resulting, eventually, in altered myocardial extracellular matrix architecture and contribute significantly to the development of left ventricular remodeling and dysfunction. CONCLUSION: Attempts to delineate the cross-talk between immune cells, myocardial cells and extracellular matrix are important as chronic myocardial inflammation is documented in about 50% of patients with dilated cardiomyopathy.