Cardiac contractility is regulated through the activity of various key Ca(2+)-handling proteins. The sarco(endo)plasmic reticulum (SR) Ca(2+) transport ATPase (SERCA2a) and its inhibitor phospholamban (PLN) control the uptake of Ca(2+) by SR membranes during relaxation. Recently, the antiapoptotic HS-1-associated protein X-1 (HAX-1) was identified as a binding partner of PLN, and this interaction was postulated to regulate cell apoptosis. In the current study, we determined that HAX-1 can also bind to SERCA2. Deletion mapping analysis demonstrated that amino acid residues 575-594 of SERCA2's nucleotide binding domain are required for its interaction with the C-terminal domain of HAX-1, containing amino acids 203-245. In transiently cotransfected human embryonic kidney 293 cells, recombinant SERCA2 was specifically targeted to the ER, whereas HAX-1 selectively concentrated at mitochondria. On triple transfections with PLN, however, HAX-1 massively translocated to the ER membranes, where it codistributed with PLN and SERCA2. Overexpression of SERCA2 abrogated the protective effects of HAX-1 on cell survival, after hypoxia/reoxygenation or thapsigargin treatment. Importantly, HAX-1 overexpression was associated with down-regulation of SERCA2 expression levels, resulting in significant reduction of apparent ER Ca(2+) levels. These findings suggest that HAX-1 may promote cell survival through modulation of SERCA2 protein levels and thus ER Ca(2+) stores.

The muscle LIM protein (MLP) and cofilin 2 (CFL2) are important regulators of striated myocyte function. Mutations in the corresponding genes have been directly associated with severe human cardiac and skeletal myopathies, and aberrant expression patterns have often been observed in affected muscles. Herein, we have investigated whether MLP and CFL2 are involved in common molecular mechanisms, which would promote our understanding of disease pathogenesis. We have shown for the first time, using a range of biochemical and immunohistochemical methods, that MLP binds directly to CFL2 in human cardiac and skeletal muscles. The interaction involves the inter-LIM domain, amino acids 94 to 105, of MLP and the amino-terminal domain, amino acids 1 to 105, of CFL2, which includes part of the actin depolymerization domain. The MLP/CFL2 complex is stronger in moderately acidic (pH 6.8) environments and upon CFL2 phosphorylation, while it is independent of Ca 2؉ levels. This interaction has direct implications in actin cytoskeleton dynamics in regulating CFL2-dependent F-actin depolymerization, with maximal depolymerization enhancement at an MLP/ CFL2 molecular ratio of 2:1. Deregulation of this interaction by intracellular pH variations, CFL2 phosphorylation, MLP or CFL2 gene mutations, or expression changes, as observed in a range of cardiac and skeletal myopathies, could impair F-actin depolymerization, leading to sarcomere dysfunction and disease.The muscle LIM protein (MLP) has emerged as a critical player in striated muscle physiology and pathophysiology over recent years. MLP, encoded by the CSRP3 (or CRP3) gene, is a member of the conserved LIM-only protein family since it contains two LIM functional zinc finger domains, and it is expressed exclusively in muscle cells. Specifically, in differentiating striated muscle cells, MLP localizes in the nucleus, promoting myogenic differentiation (8), while in adult muscle, it translocates to the cytoplasm and assumes an essential role in myocyte cytoarchitecture. Through its interactions with structural proteins, such as alpha-actinin, telethonin (T-cap), ␤I-spectrin, and N-RAP, MLP has been suggested to act as a scaffold protein for the basic contractile unit, the sarcomere, and the actin-based cytoskeleton (7,9,25,27,46,73).Mutations in CSRP3 have been directly associated with dilated (DCM) and hypertrophic (HCM) cardiomyopathies (9,31,32,46). HCM is the most common genetic myocardial disease, with a prevalence of 0.2% in adults, and the most frequent cause of sudden cardiac death in young individuals, while DCM is the third most common cause of heart failure (54,55,76). Genetic aberrations in MLP have been shown to lead to marked actin cytoskeleton disorganization and disrupted cardiac myofibrillar cytoarchitecture (9). Similar observations have been described for skeletal muscles, and HCM-CSRP3 mutations have been associated with mild skeletal myopathy (9, 31). Various hypotheses, including an MLP role in mechanical stretch sensing or the mechanical stress response, have...

AimsTo investigate whether genetic variants of the histidine-rich calcium (HRC)-binding protein are associated with idiopathic dilated cardiomyopathy (DCM) and its progression.Methods and resultsWe screened 123 idiopathic DCM patients and 96 healthy individuals by single-strand conformation polymorphism analysis and direct sequencing for genetic variants in HRC. Six polymorphisms were detected: Leu35Leu (A/G), Ser43Asn (G/A), Ser96Ala (T/G), Glu202_Glu203insGlu (−/GAG), Asp261del (GAT/−), and an in-frame insertion of 51 amino acids at His321. The analysis of their frequencies did not reveal any significant correlation with DCM development. However, the Ser96Ala polymorphism exhibited a statistically significant correlation with the occurrence of life-threatening ventricular arrhythmias. During a follow-up of 4.02 ± 2.4 years, the risk for ventricular arrhythmias was higher (HR, 9.620; 95% CI, 2.183–42.394; P = 0.003) in the Ala/Ala patients, compared with Ser/Ser homozygous patients. On multivariable Cox regression analysis, the Ser96Ala polymorphism was the only significant genetic arrythmogenesis predictor in DCM patients (HR, 4.191; 95% CI, 0.838–20.967; P = 0.018).ConclusionThe Ser96Ala genetic variant of HRC is associated with life-threatening ventricular arrhythmias in idiopathic DCM and may serve as an independent predictor of susceptibility to arrhythmogenesis in the setting of DCM.

Hyperpolarized 13 C-magnetic resonance imaging (MRI) is an emerging tool for probing tissue metabolism by measuring 13 C-label exchange between intravenously injected hyperpolarized [1-13 C]pyruvate and endogenous tissue lactate. Here we demonstrate that hyperpolarized 13 C-MRI can be used to detect early response to neoadjuvant therapy in breast cancer. Seven patients underwent multiparametric 1 H-MRI and hyperpolarized 13 C-MRI before and 7-11 days after commencing treatment. An increase in the lactate-topyruvate ratio of ~20% identified three patients who, following 5-6 cycles of treatment, showed pathological complete response. This ratio correlated with gene expression of the pyruvate transporter MCT1, and lactate dehydrogenase A (LDHA), the enzyme catalyzing label exchange between pyruvate and lactate. Analysis of ~2000 breast tumors showed that overexpression of LDHA and the hypoxia marker CAIX were associated with reduced relapse-free and overall survival. Hyperpolarized 13 C-MRI represents a promising method for monitoring very early treatment response in breast cancer and has demonstrated prognostic potential. SignificanceHyperpolarized carbon-13 MRI allows response assessment in breast cancer patients after 7-11 days of neoadjuvant chemotherapy and outperformed state-of-the-art and research quantitative proton MRI techniques.