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The cardiac extracellular matrix plays essential roles in homeostasis and injury responses. Although the role of fibrillar collagens have been thoroughly documented, the functions of non-fibrillar collagen members remain underexplored. These include a distinct group of non-fibrillar collagens, termed, fibril-associated collagens with interrupted triple helices (FACITs). Recent reports of collagen type XIX (encoded by Col19a1) expression in adult heart and evidence of its enhanced expression in cardiac ischemia suggest important functions for this FACIT in cardiac ECM structure and function. Here, we examined the cellular source of collagen XIX in the adult murine heart and evaluated its involvement in ECM structure and ventricular function. Immunodetection of collagen XIX in fractionated cardiovascular cell lineages revealed fibroblasts and smooth muscle cells as the primary sources of collagen XIX in the heart. Based on echocardiographic and histologic analyses, Col19a1 null (Col19a1 N/N) mice exhibited reduced systolic function, thinning of left ventricular walls, and increased cardiomyocyte cross-sectional areas-without gross changes in myocardial collagen content or basement membrane morphology. Col19a1 N/N cardiac fibroblasts had augmented expression of several enzymes involved in the synthesis and stability of fibrillar collagens, including PLOD1 and LOX. Furthermore, second harmonic generation-imaged ECM derived from Col19a1 N/N cardiac fibroblasts, and transmission electron micrographs of decellularized hearts from Col19a1 N/N null animals, showed marked reductions in fibrillar collagen structural organization. Col19a1 N/N mice also displayed enhanced phosphorylation of focal adhesion kinase (FAK), signifying de-repression of the FAK pathway-a critical mediator of cardiomyocyte hypertrophy. Collectively, we show that collagen XIX, which had a heretofore unknown role in the mammalian heart, participates in the regulation of cardiac structure and function-potentially through modulation of ECM fibrillar collagen structural organization. Further, these data suggest that this FACIT may modify ECM superstructure via acting at the level of the fibroblast to regulate their expression of collagen synthetic and stabilization enzymes.

Voltage-gated potassium (Kv) channels control myocardial repolarization. Pore-forming Kvα proteins associate with intracellular Kvβ subunits, which bind pyridine nucleotides with high affinity and differentially regulate channel trafficking, plasmalemmal localization and gating properties. Nevertheless, it is unclear how Kvβ subunits regulate myocardial K(+) currents and repolarization. Here, we tested the hypothesis that Kvβ2 subunits regulate the expression of myocardial Kv channels and confer redox sensitivity to Kv current and cardiac repolarization. Co-immunoprecipitation and in situ proximity ligation showed that in cardiac myocytes, Kvβ2 interacts with Kv1.4, Kv1.5, Kv4.2, and Kv4.3. Cardiac myocytes from mice lacking Kcnab2 (Kvβ2(−/−)) had smaller cross sectional areas, reduced sarcolemmal abundance of Kvα binding partners, reduced I(to), I(K,slow1), and I(K,slow2) densities, and prolonged action potential duration compared with myocytes from wild type mice. These differences in Kvβ2(−/−) mice were associated with greater P wave duration and QT interval in electrocardiograms, and lower ejection fraction, fractional shortening, and left ventricular mass in echocardiographic and morphological assessments. Direct intracellular dialysis with a high NAD(P)H:NAD(P)(+) accelerated Kv inactivation in wild type, but not Kvβ2(−/−) myocytes. Furthermore, elevated extracellular levels of lactate increased [NADH](i) and prolonged action potential duration in wild type cardiac myocytes and perfused wild type, but not Kvβ2(−/−), hearts. Taken together, these results suggest that Kvβ2 regulates myocardial electrical activity by supporting the functional expression of proteins that generate I(to) and I(K,slow), and imparting redox and metabolic sensitivity to Kv channels, thereby coupling cardiac repolarization to myocyte metabolism.

Objectives The purpose of this study was to examine the cellular and molecular mechanisms underlying alcoholic cardiomyopathy. Background The mechanism for alcoholic cardiomyopathy remains largely unknown. Methods The chronic cardiac effects of alcohol were examined in mice feeding with alcohol or isocaloric control diet for 2 months. Signaling pathways of alcohol-induced cardiac cell death were examined in H9c2 cells. Results Compared with controls, hearts from alcohol-fed mice exhibited increased apoptosis, along with significant nitrative damage, demonstrated by 3-nitrotyrosine abundance. Alcohol exposure to H9c2 cells induced apoptosis, accompanied by 3-nitrotyrosine accumulation and nicotinamide adenine dinucleotide phosphate oxidase (NOX) activation. Pre-incubation of H9c2 cells with urate (peroxynitrite scavenger), N G -nitro-l-arginine methyl ester (a nitric oxide synthase inhibitor), manganese(III) tetrakis(1-methyl-4-pyridyl)porphyrin (a superoxide dismutase mimetic), and apocynin (NOX inhibitor) abrogated alcohol-induced apoptosis. Furthermore, alcohol exposure significantly increased the expression of angiotensin II and its type 1 receptor (AT1) . A protein kinase C (PKC)-α/β1 inhibitor or PKC-β1 small interfering RNA and an AT1 blocker prevented alcohol-induced activation of NOX, and the AT1 blocker losartan significantly inhibited the expression of PKC-β1, indicating that alcohol-induced activation of NOX is mediated by PKC-β1 via AT1. To define the role of AT1-mediated PKC/NOX-derived superoxide generation in alcohol-induced cardiotoxicity, mice with knockout of the AT1 gene and wild-type mice were simultaneously treated with alcohol for 2 months. The knockout AT1 gene completely prevented cardiac nitrative damage, cell death, remodeling, and dysfunction. More importantly, pharmacological treatment of alcoholic mice with superoxide dismutase mimetic also significantly prevented cardiac nitrative damage, cell death, and remodeling. Conclusions Alcohol-induced nitrative stress and apoptosis, which are mediated by angiotensin II interaction with AT1 and subsequent activation of a PKC-β1–dependent NOX pathway, are a causal factor in the development of alcoholic cardiomyopathy.

Neuroglobin (Ngb) and Cytoglobin (Cygb) are new members of the globin family and display heterotopic expression patterns. To examine the effect of different hypoxia profiles on expression of Ngb and Cygb in rodent brain, rats were exposed to either sustained hypoxia (SH; 10% O 2 ) or intermittent hypoxia (IH; 10% and 21% O 2 alternating every 90 s) for 1, 3, 7 and 14 days, and mRNA and protein expression of Ngb and Cygb were assessed in brain cortex. SH increased Ngb mRNA and protein expression throughout the exposure, while IH only elicited slight increases in Ngb expression at day 1. Neither SH nor IH elicited increases in Cygb expression. Thus, hypoxic stimulus presentation is a major determinant of the regulation of hypoxic sensitive genes such as Ngb. Furthermore, disparities between Ngb and Cygb responses to hypoxia further suggest that these two globins may play divergent roles in brain.

Background. Intermittent hypoxia during sleep (IH), as occurs in sleep disordered breathing (SDB), induces spatial learning deficits associated with regulation of transcription factors associated with learning and memory in the hippocampal CA1 region in rats. high fat refined carbohydrate diet (HF/RC) can induce similar deficits and associated changes in signaling pathways under normoxic conditions. Methods. Sprague–Dawley adult male rats were fed either with (HF/RC) or low fat/complex carbohydrate diet (LF/CC) starting at post-natal day 30 for 90 days, and were then exposed for 14 days during light phase (12 h/day) to either normoxia (RA) or IH (21% and 10% O 2 alternations every 90 s). Place-training reference memory task deficits were assessed in the Morris water maze. Total and ser-133 phosphorylated CREB were assessed in different brain regions by Western blotting and immunostaining in rats exposed to normoxia or IH and to LF/CC or HF/RC. Results. Substantial decreases in CREB phosphorylation occurred in CA1 but not in motor cortex following either IH, HF/RC, and HF/RC + IH. Place-training reference memory task deficits were observed in rats exposed to IH and to HF/RC, and to a much greater extent in rats exposed to HF/RC + IH. Conclusions. Nutritional factors alter recruitment of transcription factors, possibly via oxidative-related pathways, and modulate the vulnerability of the CA1 region of the hippocampus to the episodic hypoxia that characterizes SDB, thereby enhancing neurocognitive susceptibility in SDB patients.

Background: Recurrent tonsillitis and sleep apnea are the major indications for tonsillectomy in children. We hypothesized that the recurrent vibration in the upper airway of snoring children would promote inflammatory changes in the tonsillar tissue and would lead to the up-regulation of cysteinyl leukotriene (LT) receptors (Rs). Objective: To assess the expression patterns of the human LT-Rs in children undergoing tonsillectomy, and compare those patterns in children having recurrent throat infections (RIs) and children with obstructive sleep apnea syndrome (SA). Methods: Tonsillar tissue from 17 children with SA and 13 with RIs was subjected to quantitative polymerase chain reaction using specific primers for LT1-R and LT2-R, and to immunohistochemistry and Western blotting for protein expression of LT1-R and LT2-R. Results: Messenger RNA encoding for the expression of LT1-R and LT2-R was detected in the tonsils of all children. Immunoblots revealed significantly higher expressions of LT1-R and LT2-R in the tonsils of children with SA. The topographic pattern of both receptors differed among the tonsils of children with SA and RI. Conclusion: LT1-R and LT2-R are expressed in pediatric tonsillar tissue, are more abundant in SA patients, and demonstrate a specific topographic pattern of expression. These findings suggest that an inflammatory process involving LT expression and regulation occurs in children with SA. (CHEST 2004; 126:13–18)

Intermittent hypoxia (IH) during sleep induces temporally defined increases in apoptosis within vulnerable brain regions such as the hippocampal CA1 region in rats. Protein kinase B (AKT) has emerged as major signal transduction protein underlying inhibition of apoptosis and consequent increases in cell survival. Sprague Dawley adult male rats were exposed during sleep to IH or to normoxia (RA) for periods ranging from 0 to 30 days, and expression of total and phosphorylated AKT, of forkhead family members FKHR and FKHRL1, and of glycogen synthase kinase 3β (GSK3β) was assessed. Decreases in phosphorylation occurred as early as 1 h IH exposure, reached a nadir at 6 h–3 days, and then progressively returned to baseline levels at 14–30 days. Phosphorylated AKT and GSK3β were intensely expressed and highly colocalized within neuronal cells (Neu-N positive) in the CA1 region. Thus, IH induces time-dependent biphasic changes in AKT survival pathways within the CA1 region that are temporally correlated with the initial increases and subsequent decreases in neuronal apoptosis.

Intermittent hypoxia (IH) during sleep, a characteristic feature of sleep-disordered breathing (SDB) is associated with time-dependent apoptosis and spatial learning deficits in the adult rat. The mechanisms underlying such neurocognitive deficits remain unclear. Activation of the cAMP-response element binding protein (CREB) transcription factor mediates critical components of neuronal survival and memory consolidation in mammals. CREB phosphorylation and DNA binding, as well as the presence of apoptosis in the CA1 region of the hippocampus were examined in Sprague-Dawley male rats exposed to IH. Spatial reference task learning was assessed with the Morris water maze. IH induced significant decreases in Ser-133 phosphorylated CREB (pCREB) without changes in total CREB, starting as early as 1 h IH, peaking at 6 h-3 days, and returning toward normoxic levels by 14-30 days. Double-labeling immunohistochemistry for pCREB and Neu-N (a neuronal marker) confirmed these findings. The expression of cleaved caspase 3 (cC3) in the CA1, a marker of apoptosis, peaked at 3 days and returned to normoxic values at 14 days. Initial IH-induced impairments in spatial learning were followed by partial functional recovery starting at 14 days of IH exposure. We postulate that IH elicits time-dependent changes in CREB phosphorylation and nuclear binding that may account for decreased neuronal survival and spatial learning deficits in the adult rat. We suggest that CREB changes play an important role in the neurocognitive morbidity of SDB patients.