ALS/FTD-causing mutation in cyclin F causes the dysregulation of SFPQ.

Previously, we identified missense mutations in CCNF that are causative of familial and sporadic amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Hallmark features of these diseases include the build-up of insoluble protein aggregates as well as the mislocalization of proteins such as transactive response DNA binding protein 43 kDa (TDP-43). In recent years, the dysregulation of SFPQ (splicing factor proline and glutamine rich) has also emerged as a pathological hallmark of ALS/FTD. CCNF encodes for the protein cyclin F, a substrate recognition component of an E3 ubiquitin ligase. We have previously shown that ALS/FTD-linked mutations in CCNF cause disruptions to overall protein homeostasis that leads to a build-up of K48-linked ubiquitylated proteins as well as defects in autophagic machinery. To investigate further processes that may be affected by cyclin F, we used a protein-proximity ligation method, known as Biotin Identification (BioID), standard immunoprecipitations and mass spectrometry to identify novel interaction partners of cyclin F and infer further process that may be affected by the ALS/FTD-causing mutation. Results demonstrate that cyclin F closely associates with proteins involved with RNA metabolism as well as a number of RNA-binding proteins previously linked to ALS/FTD, including SFPQ. Notably, the overexpression of cyclin F(S621G) led to the aggregation and altered subcellular distribution of SFPQ in human embryonic kidney (HEK293) cells, while leading to altered degradation in primary neurons. Overall, our data links ALS/FTD-causing mutations in CCNF to converging pathological features of ALS/FTD and provides a link between defective protein degradation systems and the pathological accumulation of a protein involved in RNA processing and metabolism.

Decoupling the effects of hydrophilic and hydrophobic moieties at the neuron-nanofibre interface.

Peptide-based nanofibres are a versatile class of tunable materials with applications in optoelectronics, sensing and tissue engineering. However, the understanding of the nanofibre surface at the molecular level is limited. Here, a series of homologous dilysine-diphenylalnine tetrapeptides were synthesised and shown to self-assemble into water-soluble nanofibres. Despite the peptide nanofibres displaying similar morphologies, as evaluated through atomic force microscopy and neutron scattering, significant differences were observed in their ability to support sensitive primary neurons. Contact angle and labelling experiments revealed that differential presentation of lysine moieties at the fibre surface did not affect neuronal viability; however the mobility of phenylalanine residues at the nanofibre surface, elucidated through solid- and gel-state NMR studies and confirmed through tethered bilayer lipid membrane experiments, was found to be the determining factor in governing the suitability of a given peptide as a scaffold for primary neurons. This work offers new insights into characterising and controlling the nanofibre surface at the molecular level.

Peptide Nanofiber Substrates for Long-Term Culturing of Primary Neurons.

The culturing of primary neurons represents a central pillar of neuroscience research. Primary neurons are derived directly from brain tissue and recapitulate key aspects of neuronal development in an in vitro setting. Unlike neural stem cells, primary neurons do not divide; thus, initial attachment of cells to a suitable substrate is critical. Commonly used polylysine substrates can suffer from batch variability owing to their polymeric nature. Herein, we report the use of chemically well-defined, self-assembling tetrapeptides as substrates for primary neuronal culture. These water-soluble peptides assemble into fibers which facilitate adhesion and development of primary neurons, their long-term survival (>40 days), synaptic maturation, and electrical activity. Furthermore, these substrates are permissive toward neuronal transfection and transduction which, coupled with their uniformity and reproducible nature, make them suitable for a wide variety of applications in neuroscience.

Integrins protect cardiomyocytes from ischemia/reperfusion injury.

Ischemic damage is recognized to cause cardiomyocyte (CM) death and myocardial dysfunction, but the role of cell-matrix interactions and integrins in this process has not been extensively studied. Expression of α7ß1D integrin, the dominant integrin in normal adult CMs, increases during ischemia/reperfusion (I/R), while deficiency of ß1 integrins increases ischemic damage. We hypothesized that the forced overexpression of integrins on the CM would offer protection from I/R injury. Tg mice with CM-specific overexpression of integrin α7ß1D exposed to I/R had a substantial reduction in infarct size compared with that of α5ß1D-overexpressing mice and WT littermate controls. Using isolated CMs, we found that α7ß1D preserved mitochondrial membrane potential during hypoxia/reoxygenation (H/R) injury via inhibition of mitochondrial Ca2+ overload but did not alter H/R effects on oxidative stress. Therefore, we assessed Ca2+ handling proteins in the CM and found that ß1D integrin colocalized with ryanodine receptor 2 (RyR2) in CM T-tubules, complexed with RyR2 in human and rat heart, and specifically bound to RyR2 amino acids 165-175. Integrins stabilized the RyR2 interdomain interaction, and this stabilization required integrin receptor binding to its ECM ligand. These data suggest that α7ß1D integrin modifies Ca2+ regulatory pathways and offers a means to protect the myocardium from ischemic injury.

Increased connective tissue growth factor associated with cardiac fibrosis in the mdx mouse model of dystrophic cardiomyopathy.

Cardiomyopathy contributes to morbidity and mortality in Duchenne muscular dystrophy (DMD), a progressive muscle-wasting disorder. A major feature of the hearts of DMD patients and the mdx mouse model of the disease is cardiac fibrosis. Connective tissue growth factor (CTGF) is involved in the fibrotic process in many organs. This study utilized the mdx mouse model to assess the role of CTGF and other extracellular matrix components during the development of fibrosis in the dystrophic heart. Left ventricular function of mdx and control mice at 6, 29 and 43 weeks was measured by echocardiography. Young (6 weeks old) mdx hearts had normal function and histology. At 29 weeks of age, mdx mice developed cardiac fibrosis and increased collagen expression. The onset of fibrosis was associated with increased CTGF transcript and protein expression. Increased intensity of CTGF immunostaining was localized to fibrotic areas in mdx hearts. The upregulation of CTGF was also concurrent with increased expression of tissue inhibitor of matrix metalloproteinases (TIMP-1). These changes persisted in 43 week old mdx hearts and were combined with impaired cardiac function and increased gene expression of transforming growth factor (TGF)-ß1 and matrix metalloproteinases (MMP-2, MMP-9). In summary, an association was observed between cardiac fibrosis and increased CTGF expression in the mdx mouse heart. CTGF may be a key mediator of early and persistent fibrosis in dystrophic cardiomyopathy.

Dysfunction induced by ischemia versus edema: does edema matter?

OBJECTIVES: Recovery from pediatric cardiac surgery is affected by ischemia-reperfusion injury, cardiac edema, and in some cases a low cardiac output syndrome. Although association has been made between the development of edema and dysfunction, modeling is confounded by intercurrent injurious stimuli that also cause cardiac edema and dysfunction. We tested whether a true causal relationship exists between edema and cardiac dysfunction. METHODS: We induced either ischemia or edema alone in isolated cardiomyocytes and whole Langendorff-perfused hearts. Function was measured as shortening dynamics and developed pressure, respectively. RESULTS: Ischemic injury impaired function in both cardiomyocytes and whole hearts. Isolated cells showed significant reduction in peak shortening and departure and relaxation velocities. Whole hearts displayed severely reduced developed pressures. Hyposmotic solution forced cardiomyocytes to swell to 7% greater than their normal size. No significant effect on shortening was seen. Similarly, Langendorff-perfused hearts were induced to take on 3% more water than control-perfused hearts and 9% more water than nonperfused hearts. This additional water was associated with mild dysfunction. CONCLUSIONS: We demonstrate the capacity of the heart to tolerate edema greater than that seen in clinical settings without residual effect. Ischemia results in ongoing contractile dysfunction of both isolated cardiomyocytes and whole hearts. We conclude that dysfunction resulting from edema in ex vivo cardiac models is mild and suggest review of the importance given to edema-mediated dysfunction after cardiac surgery.

Myocardial membrane injury in pediatric cardiac surgery: An animal model.

OBJECTIVE: Reduced myocardial performance invariably follows pediatric cardiac surgery and is manifested by a low cardiac output state in its severest form. The role of myocardial membrane proteins in this setting is unknown. Dystrophin and dysferlin are involved in membrane integrity, whereas aquaporins selectively transport water. These proteins were examined in a model of pediatric cardiac surgery, together with a trial of poloxamer 188, which may reduce membrane injury. METHODS: Eight lambs were randomized to saline with or without poloxamer 188. Lambs underwent 2 hours of cardiopulmonary bypass and aortic crossclamping. After a further 9 hours of monitoring, the hearts were assessed for water content, capillary leak, and protein expression. RESULTS: Dystrophin expression was unaffected by ischemia/reperfusion, but dysferlin expression was reduced. Aquaporin 1 protein increased after ischemia/reperfusion. Poloxamer 188 administration was associated with supranormal levels of dystrophin, preservation of dysferlin expression, and normalization of aquaporin 1 expression. Poloxamer 188 was associated with less capillary leak, maintained colloid osmotic pressure, and less hemodilution. Poloxamer 188 was associated with an improved hemodynamic profile (higher blood pressure, higher venous saturation, and lower lactate), although the heart rate tended to be higher. CONCLUSIONS: Changes in protein expression within the myocardial membrane were found in a clinically relevant model of pediatric cardiac surgery. Indicators of reduced performance, such as lower blood pressure and lower oxygen delivery, were lessened in association with the administration of the membrane protecting poloxamer 188. Poloxamer 188 was also associated with potentially beneficial changes in membrane protein expression, reduced capillary leakage, and less hemodilution.

Myocardial ischemia is more important than the effects of cardiopulmonary bypass on myocardial water handling and postoperative dysfunction: a pediatric animal model.

OBJECTIVES: Low cardiac output state is the principal cause of morbidity after surgical intervention for congenital heart disease. Myocardial ischemia-reperfusion injury, apoptosis, capillary leak syndrome, and myocardial edema are associated factors. We established a clinically relevant model to examine relationships between myocardial ischemia, edema, and cardiac dysfunction and to assess the role of the water transport proteins aquaporins. METHODS: Sixteen lambs were studied. Seven were control animals not undergoing cardiopulmonary bypass, and 9 underwent bypass. Six had 90 minutes of aortic crossclamping with blood cardioplegia and moderate hypothermia. The remaining 3 underwent cardiopulmonary bypass without aortic crossclamping. Hemodynamic and biochemical data were recorded, and myocardial edema, apoptotic markers, and aquaporin expression were determined after death. RESULTS: The group undergoing cardiopulmonary bypass with aortic crossclamping had a low cardiac output state, with early postoperative tachycardia, hypotension, increased serum lactate levels, and impaired tissue oxygen delivery (P < .05) compared with the group undergoing cardiopulmonary bypass without aortic crossclamping. The lambs undergoing cardiopulmonary bypass with aortic crossclamping had increased myocardial water (P < .05) compared with those not undergoing cardiopulmonary bypass and a 2-fold increase in aquaporin 1 mRNA expression (P < .05) compared with those not undergoing cardiopulmonary bypass and those undergoing cardiopulmonary bypass without aortic crossclamping. CONCLUSIONS: A temporal association between hemodynamic dysfunction, myocardial edema, and increased aquaporin 1 expression was demonstrated. Cardiopulmonary bypass without ischemia was associated with minimal edema, negligible myocardial dysfunction, and static aquaporin expression. Ischemic reperfusion injury is the main cause of myocardial edema and myocardial dysfunction, but a causal relationship between edema and dysfunction remains to be proved.

Changes in skeletal muscle expression of AQP1 and AQP4 in dystrophinopathy and dysferlinopathy patients.

Transmembrane water transport is mediated by aquaporins (AQPs), of which AQP1 and AQP4 are expressed in skeletal muscle. AQP4 expression is reduced in Duchenne muscular dystrophy (DMD) patients, and is reported to correlate with decreased alpha1-syntrophin and altered osmotic permeability. In this study, we assessed the relationship between AQP1, AQP4, dystrophin and alpha1-syntrophin in dystrophinopathy and dysferlinopathy patients. Muscle biopsies of patients with DMD (n = 8) and limb-girdle muscular dystrophy type 2B (LGMD2B; n = 5) were screened for AQP1 and AQP4 expression by real-time quantitative RT-PCR or Western blot and immunohistochemistry. AQP expression was further analyzed in primary myotubes derived from DMD and LGMD2B patients by cell culture and immunohistochemistry. AQP1 transcript and protein expression was significantly elevated in DMD biopsies, and was localized to the sarcolemma of muscle fibers and endothelia of muscle capillaries. AQP4 was significantly reduced despite normal dystrophin and alpha1-syntrophin in dysferlinopathy patients, while expression of AQP1 was variably upregulated. Expression of AQP1 and AQP4 was normal in patient-derived primary myotubes, suggesting that altered AQPs observed in biopsies are likely secondary to the dystrophic process. Our study shows that AQP4 downregulation can occur in muscular dystrophies with either normal or disrupted expression of dystrophin-associated proteins, and that this might be associated with upregulation of AQP1.

Myocardial water handling and the role of aquaporins.

Cardiac surgery is performed in approximately 770,000 adults and 30,000 children in the United States of America annually. In this review we outline the mechanistic links between post-operative myocardial stunning and the development of myocardial edema. These interrelated processes cause a decline in myocardial performance that account for significant morbidity and mortality after cardiac surgery. Factors leading to myocardial edema include hemodilution, ischemia and reperfusion as well as osmotic gradients arising from pathological change. Several members of the aquaporin family of water transport proteins have been described in the myocardium although their role in the pathogenesis and resolution of cardiac edema is not established. This review examines evidence for the involvement of aquaporins in myocardial water handling during normal and pathological conditions.

Cardiac aquaporin expression in humans, rats, and mice.

Water accumulation in the heart is important in ischemia-reperfusion injury and operations performed by using cardiopulmonary bypass, with cardiac dysfunction associated with myocardial edema being the principal determinant of clinical outcome. As an initial step in determining the role of aquaporin (AQP) water channels in myocardial edema, we have assessed the myocardial expression of AQPs in humans, rats, and mice. RT-PCR revealed expression of AQP-1, -4, -6, -7, -8, and -11 transcripts in the mouse heart. AQP-1, -6, -7, and -11 mRNAs were found in the rat heart as well as low levels of AQP-4 and -9. Human hearts contained AQP-1, -3, -4, -5, -7, -9, -10, and -11 mRNAs. AQP-1 protein expression was confirmed by Western blot analysis in all three species. AQP-4 protein was detected in the mouse heart but not in the rat or human heart. To determine the potential functional consequences of myocardial AQP expression, water permeability was measured in plasma membrane vesicles from myocardial cells of wild-type versus various AQP knockout mice. Water permeability was reduced by AQP-1 knockout but not by AQP-4 or AQP-8 knockout. With the use of a model of isolated rat heart perfusion, it was found that osmotic and ischemic stresses are not associated with changes in AQP-1 or AQP-4 expression. These studies support a possible functional role of AQP-1 in myocardium but indicate that early adaptations to osmotic and ischemic stress do not involve transcriptional or posttranslational AQP-1 regulation.

Expression of aquaporin 1 in human cardiac and skeletal muscle.

Aquaporins (AQPs) are a family of water channel proteins that assist in maintenance of the cellular osmotic environment and whole body fluid balance. Specialized organ-specific AQPs are important in physiologic and pathologic processes but little is known about AQPs in the human heart. AQP1 has been identified in rodent heart. We investigated the presence and localization of AQP1 in human heart and skeletal muscle using immunohistochemistry and confocal microscopy, western blot and reverse transcriptase-polymerase chain reaction. There was abundant AQP1 present in both cardiac and skeletal muscle. Immunohistochemistry revealed co-localization of AQP1 with vinculin, a t-tubule marker, and caveolin-3. No novel sequences bearing an NPA box motif common to other AQPs were identified in human heart using degenerative PCR analysis. We conclude that AQP1 is present in the human heart. AQP1 co-localizes with t-tubular and caveolar proteins. Cardiac AQPs may have a role during osmotic stresses including ischemia/reperfusion and cardiopulmonary bypass.