Aims: The histidine-rich calcium-binding protein (HRC) Ser96Ala variant has previously been identified as a potential biomarker for ventricular arrhythmias and sudden cardiac death in patients with idiopathic dilated cardiomyopathy. Herein, the role of this variant in cardiac pathophysiology is delineated through a novel mouse model, carrying the human mutation in the homologous mouse position. Methods and results: The mouse HRC serine 81, homologous to human HRC serine 96, was mutated to alanine, using knock-in gene targeting. The HRC-Ser81Ala mice presented increased mortality in the absence of structural or histological abnormalities, indicating that early death may be arrhythmia-related. Indeed, under stress-but not baseline-conditions, the HRC-Ser81Ala mice developed ventricular arrhythmias, whilst at the cardiomyocyte level they exhibited increased occurrence of triggered activity. Cardiac contraction was decreased in vivo, ex vivo, and in vitro. Additionally, Ca2+ transients and SR Ca2+ load were both reduced suggesting that cytosolic Ca2+ overload is not the underlying proarrhythmic mechanism. Interestingly, total SR Ca2+ leak was increased in HRC-Ser81Ala cardiomyocytes, without an increase in Ca2+ spark and wave frequency. However, Ca2+ wave propagation was significantly slower and the duration of the associated Na/Ca exchange current was increased. Moreover, action potential duration was also increased. Notably, Ca2+/Calmodulin kinase II (CaMKII) phosphorylation of the ryanodine receptor was increased, whilst KN-93, an inhibitor of CaMKII, reduced the occurrence of arrhythmias. Conclusions: The homologous mutation Ser81Ala in HRC in mice, corresponding to Ser96Ala in humans, is associated with sudden death and depressed cardiac function. Ventricular arrhythmias are related to abnormal Ca2+ cycling across the SR. The data further support a role for CaMKII with the perspective to treat arrhythmias through CaMKII inhibition.

Neurodegenerative disease are frequently characterized by microglia activation and/or leukocyte infiltration in the parenchyma of the central nervous system and at the molecular level by increased oxidative modifications of proteins, lipids and nucleic acids. NADPH oxidases (NOX) emerged as a novel promising class of pharmacological targets for the treatment of neurodegeneration due to their role in oxidant generation and presumably in regulating microglia activation. The unique function of NOX is the generation of superoxide anion (O2(*-)) and hydrogen peroxide (H2O2). However in the context of neuroinflammation, they present paradoxical features since O2(*-)/H2O2 generated by NOX and/or secondary reactive oxygen species (ROS) derived from O2(*-)/H2O2 can either lead to neuronal oxidative damage or resolution of inflammation. The role of NOX enzymes has been investigated in many models of neurodegenerative diseases by using either genetic or pharmacological approaches. In the present review we provide a critical assessment of recent findings related to the role of NOX in the CNS as well as how the field has advanced over the last 5 years. In particular, we focus on the data derived from the work of a consortium (Neurinox) funded by the European Commission's Programme 7 (FP7). We discuss the evidence gathered from animal models and human samples linking NOX expression/activity with neuroinflammation in neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and Creutzfeldt-Jakob disease as well as autoimmune demyelinating diseases like multiple sclerosis (MS) and chronic inflammatory demyelinating polyneuropathy (CIDP). We address the possibility to use measurement of the activity of the NOX2 isoform in blood samples as biomarker of disease severity and treatment efficacy in neurodegenerative disease. Finally we clarify key controversial aspects in the field of NOX, such as NOX cellular expression in the brain, measurement of NOX activity, impact of genetic deletion of NOX in animal models of neurodegeneration and specificity of NOX inhibitors.

Precise Ca cycling through the sarcoplasmic reticulum (SR), a Ca storage organelle, is critical for proper cardiac muscle function. This cycling initially involves SR release of Ca via the ryanodine receptor, which is regulated by its interacting proteins junctin and triadin. The sarco/endoplasmic reticulum Ca ATPase (SERCA) pump then refills SR Ca stores. Histidine-rich Ca-binding protein (HRC) resides in the lumen of the SR, where it contributes to the regulation of Ca cycling by protecting stressed or failing hearts. The common Ser96Ala human genetic variant of HRC strongly correlates with life-threatening ventricular arrhythmias in patients with idiopathic dilated cardiomyopathy. However, the underlying molecular pathways of this disease remain undefined. Here, we demonstrate that family with sequence similarity 20C (Fam20C), a recently characterized protein kinase in the secretory pathway, phosphorylates HRC on Ser96. HRC Ser96 phosphorylation was confirmed in cells and human hearts. Furthermore, a Ser96Asp HRC variant, which mimics constitutive phosphorylation of Ser96, diminished delayed aftercontractions in HRC null cardiac myocytes. This HRC phosphomimetic variant was also able to rescue the aftercontractions elicited by the Ser96Ala variant, demonstrating that phosphorylation of Ser96 is critical for the cardioprotective function of HRC. Phosphorylation of HRC on Ser96 regulated the interactions of HRC with both triadin and SERCA2a, suggesting a unique mechanism for regulation of SR Ca homeostasis. This demonstration of the role of Fam20C-dependent phosphorylation in heart disease will open new avenues for potential therapeutic approaches against arrhythmias.

Alzheimer's and Parkinson's diseases are the most prevalent neurodegenerative diseases that generate important health-related direct and indirect socio-economic costs. They are characterized by severe neuronal losses in several disease-specific brain regions associated with deposits of aggregated proteins. In Alzheimer's disease, beta-amyloid peptide-containing plaques and intraneuronal neurofibrillary tangles composed of hyperphosphorylated microtubule-associated protein tau are the two main neuropathological lesions, while Parkinson's disease is defined by the presence of Lewy Bodies that are intraneuronal proteinaceous cytoplasmic inclusions. alpha-Synuclein has been identified as a major protein component of Lewy Bodies and heavily implicated in the pathogenesis of Parkinson's disease. In the past few years, evidence has emerged to explain how these aggregate-prone proteins can undergo spontaneous self-aggregation, propagate from cell to cell, and mediate neurotoxicity. Current research now indicates that oligomeric forms are probably the toxic species. This article discusses recent progress in the understanding of the pathogenesis of these diseases, with a focus on the underlying mechanisms of protein aggregation, and emphasizes the pathophysiological molecular mechanisms leading to cellular toxicity. Finally, we present the putative direct link between beta-amyloid peptide and tau in causing toxicity in Alzheimer's disease as well as alpha-synuclein in Parkinson's disease, along with some of the most promising therapeutic strategies currently in development for those incurable neurodegenerative disorders.

Muscle Lim Protein (MLP) is a protein with multiple functional roles in striated muscle physiology and pathophysiology. Herein, we demonstrate that MLP directly binds to slow, fast, and cardiac myosin-binding protein C (MyBP-C) during myogenesis, as shown by yeast two-hybrid and a range of protein-protein interaction assays. The minimal interacting domains involve MLP inter-LIM and MyBP-C [C4]. The interaction is sensitive to cytosolic Ca(2+) concentrations changes and to MyBP-C phosphorylation by PKA or CaMKII. Confocal microscopy of differentiating myoblasts showed MLP and MyBP-C colocalization during myoblast differentiation. Suppression of the complex formation with recombinant MyBP-C [C4] peptide overexpression, inhibited myoblast differentiation by 65%. Suppression of both MLP and MyBP-C expression in myoblasts by siRNA revealed negative synergistic effects on differentiation. The MLP/MyBP-C complex modulates the actin activated myosin II ATPase activity in vitro, which could interfere with sarcomerogenesis and myofilaments assembly during differentiation. Our data demonstrate a critical role of the MLP/MyBP-C complex during early myoblast differentiation. Its absence in muscles with mutations or aberrant expression of MLP or MyBP-C could be directly implicated in the development of cardiac and skeletal myopathies.