Cardiac Troponin Mutations and Restrictive Cardiomyopathy

Mutations in sarcomeric proteins have recently been established as heritable causes of Restrictive Cardiomyopathy (RCM). RCM is clinically characterized as a defect in cardiac diastolic function, such as, impaired ventricular relaxation, reduced diastolic volume and increased end-diastolic pressure. To date, mutations have been identified in the cardiac genes for desmin, α-actin, troponin I and troponin T. Functional studies in skinned muscle fibers reconstituted with troponin mutants have established phenotypes consistent with the clinical findings which include an increase in myofilament Ca2+ sensitivity and basal force. Moreover, when RCM mutants are incorporated into reconstituted myofilaments, the ability to inhibit the ATPase activity is reduced. A majority of the mutations cluster in specific regions of cardiac troponin and appear to be mutational “hot spots”. This paper highlights the functional and clinical characteristics of RCM linked mutations within the troponin complex

Sarcospan Regulates Cardiac Isoproterenol Response and Prevents Duchenne Muscular Dystrophy-Associated Cardiomyopathy.

BackgroundDuchenne muscular dystrophy is a fatal cardiac and skeletal muscle disease resulting from mutations in the dystrophin gene. We have previously demonstrated that a dystrophin-associated protein, sarcospan (SSPN), ameliorated Duchenne muscular dystrophy skeletal muscle degeneration by activating compensatory pathways that regulate muscle cell adhesion (laminin-binding) to the extracellular matrix. Conversely, loss of SSPN destabilized skeletal muscle adhesion, hampered muscle regeneration, and reduced force properties. Given the importance of SSPN to skeletal muscle, we investigated the consequences of SSPN ablation in cardiac muscle and determined whether overexpression of SSPN into mdx mice ameliorates cardiac disease symptoms associated with Duchenne muscular dystrophy cardiomyopathy.Methods and resultsSSPN-null mice exhibited cardiac enlargement, exacerbated cardiomyocyte hypertrophy, and increased fibrosis in response to β-adrenergic challenge (isoproterenol; 0.8 mg/day per 2 weeks). Biochemical analysis of SSPN-null cardiac muscle revealed reduced sarcolemma localization of many proteins with a known role in cardiomyopathy pathogenesis: dystrophin, the sarcoglycans (α-, δ-, and γ-subunits), and β1D integrin. Transgenic overexpression of SSPN in Duchenne muscular dystrophy mice (mdx(TG)) improved cardiomyofiber cell adhesion, sarcolemma integrity, cardiac functional parameters, as well as increased expression of compensatory transmembrane proteins that mediate attachment to the extracellular matrix.ConclusionsSSPN regulates sarcolemmal expression of laminin-binding complexes that are critical to cardiac muscle function and protects against transient and chronic injury, including inherited cardiomyopathy

TBK1 Kinase Addiction in Lung Cancer Cells Is Mediated via Autophagy of Tax1bp1/Ndp52 and Non-Canonical NF-kappa B Signalling

K-Ras dependent non-small cell lung cancer (NSCLC) cells are 'addicted' to basal autophagy that reprograms cellular metabolism in a lysosomal-sensitive manner. Here we demonstrate that the xenophagy-associated kinase TBK1 drives basal autophagy, consistent with its known requirement in K-Ras-dependent NSCLC proliferation. Furthermore, basal autophagy in this context is characterised by sequestration of the xenophagy cargo receptor Ndp52 and its paralogue Tax1bp1, which we demonstrate here to be a bona fide cargo receptor. Autophagy of these cargo receptors promotes non-canonical NF-κB signalling. We propose that this TBK1-dependent mechanism for NF-κB signalling contributes to autophagy addiction in K-Ras driven NSCLC

Predicting Cardiomyopathic Phenotypes by Altering the Calcium Affinity of Cardiac Troponin C

Cardiac diseases associated with mutations in Tn subunits include hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM) and restrictive cardiomyopathy (RCM). Altered calcium handling in these diseases is evidenced by changes in the Ca2+ sensitivity of contraction. Mutations were generated to increase/ decrease the Ca2+ sensitivity of skinned fibers, and create the classified effects of DCM, HCM and RCM. This study mimicked the changes in Ca2+ sensitivity and relaxation properties of the muscle to determine if this was sufficient to recreate the disease. Four mutants (A23Q, S37G, V44Q, L48Q) were identified with RCM-like properties; a large increase in Ca2+ sensitivity, increased basal force and loss of ATPase inhibition. Two mutations were identified (E40A, I61Q) with DCM properties; decreased Ca2+ sensitivity in skinned fibers, decreased force recovery (%), and decreased activation of the ATPase at high Ca2+ levels (pCa 6-4). Also, the functional effects of four newly identified cTnC mutations associated with HCM were reported. Three of these HCM mutations A8V, C84Y, and D145E displayed HCM characteristics, increased Ca2+ sensitivity in skinned fibers and ATPase and A8V and D145E increased the force recovery. Only, D145E significantly increased the ATPase activation of the reconstituted thin filament. Also, Ca2+ affinity measurements using IAANS fluorescence were performed. No significant changes were found for E134D. The C84Y IAANS fluorescence measurements revealed that cTnC Ca2+ affinity of the cTn complex was unaltered. The Ca2+ affinity increased for D145E in isolated cTnC and the cTn complex, however in the regulated thin filament (RTF) with myosin subfragment-1 (S1) and rigor crossbridges the Ca2+ affinity values were similar to the fiber Ca2+ sensitivity. For A8V, the RTF significantly increased the Ca2+ affinity, and addition of S1 and rigor crossbridges caused the values to parallel the Ca2+ sensitivity values. In conclusion, direct and indirect protein-protein interactions contribute to the enhanced Ca2+ sensitivity of the HCM mutants. The cTnC mutant screen allowed selection of mutations that mimic the disease states: S37G (RCM) and, E40A (DCM); A8V (HCM) from the patient study for analysis in knock-in mice for futures studies to determine if these disease states can be recapitulated in vivo