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Aims Heart failure is a major complication in cancer treatment due to the cardiotoxic effects of anticancer drugs, especially from the anthracyclines such as doxorubicin (DXR). DXR enhances oxidative stress and stimulates matrix metalloproteinase-2 (MMP-2) in cardiomyocytes. We investigated whether MMP inhibitors protect against DXR cardiotoxicity given the role of MMP-2 in proteolyzing sarcomeric proteins in the heart and remodelling the extracellular matrix. Methods and results Eight-week-old male C57BL/6J mice were treated with DXR weekly with or without MMP inhibitors doxycycline or ONO-4817 by daily oral gavage for 4 weeks. Echocardiography was used to determine cardiac function and left ventricular remodelling before and after treatment. MMP inhibitors ameliorated DXR-induced systolic and diastolic dysfunction by reducing the loss in left ventricular ejection fraction, fractional shortening, and E′/A′. MMP inhibitors attenuated adverse left ventricular remodelling, reduced cardiomyocyte dropout, and prevented myocardial fibrosis. DXR increased myocardial MMP-2 activity in part also by upregulating N-terminal truncated MMP-2. Immunogold transmission electron microscopy showed that DXR elevated MMP-2 levels within the sarcomere and mitochondria which were associated with myofilament lysis, mitochondrial degeneration, and T-tubule distention. DXR-induced myofilament lysis was associated with increased titin proteolysis in the heart which was prevented by ONO-4817. DXR also increased the level and activity of MMP-2 in human embryonic stem cell-derived cardiomyocytes, which was reduced by ONO-4817. Conclusions MMP-2 activation is an early event in DXR cardiotoxicity and contributes to myofilament lysis by proteolyzing cardiac titin. Two orally available MMP inhibitors ameliorated DXR cardiotoxicity by attenuating intracellular and extracellular matrix remodelling, suggesting their use may be a potential prophylactic strategy to prevent heart injury during chemotherapy.

Titin is a large filamentous protein that forms a sarcomeric myofilament with a molecular spring region that develops force in stretched sarcomeres. The molecular spring has a complex make-up that includes the N2A element. This element largely consists of a 104residue unique sequence (N2A-Us) flanked by immunoglobulin domains (I80 and I81). The N2A element is of interest because it assembles a signalosome with CARP (Cardiac Ankyrin Repeat Protein) as an important component; CARP both interacts with the N2A-Us and I81 and is highly upregulated in response to mechanical stress. The mechanical properties of the N2A element were studied using single-molecule force spectroscopy, including how these properties are affected by CARP and phosphorylation. Three protein constructs were made that consisted of 0, 1, or 2 N2A-Us elements with flanking I80 and I81 domains and with specific handles at their ends for study by atomic force microscopy (AFM). The N2A-Us behaved as an entropic spring with a persistence length (Lp) of ∼0.35 nm and contour length (Lc) of ∼39 nm. CARP increased the Lp of the N2A-Us and the unfolding force of the Ig domains; force clamp experiments showed that CARP reduced the Ig domain unfolding kinetics. These findings suggest that CARP might function as a molecular chaperone that protects I81 from unfolding when mechanical stress is high. The N2A-Us was found to be a PKA substrate, and phosphorylation was blocked by CARP. Mass spectrometry revealed a PKA phosphosite (Ser-9895 in NP_001254479.2) located at the border between the N2A-Us and I81. AFM studies showed that phosphorylation affected neither the Lp of the N2A-Us nor the Ig domain unfolding force (F unfold). Simulating the force-sarcomere length relation of a single titin molecule containing all spring elements showed that the compliance of the N2A-Us only slightly reduces passive force (1.4%) with an additional small reduction by CARP (0.3%). Thus, it is improbable that the compliance of the N2A element has a mechanical function per se. Instead, it is likely that this compliance has local effects on binding of signaling molecules and that it contributes thereby to strain-and phosphorylation-dependent mechano-signaling.

BackgroundThe cancer stem cell (CSC) hypothesis posits that deregulated neural stem cells (NSCs) form the basis of brain tumors such as glioblastoma multiforme (GBM). GBM, however, usually forms in the cerebral white matter while normal NSCs reside in subventricular and hippocampal regions. We attempted to characterize CSCs from a rare form of glioblastoma multiforme involving the neurogenic ventricular wall.MethodsWe described isolating CSCs from a GBM involving the lateral ventricles and characterized these cells with in vitro molecular biomarker profiling, cellular behavior, ex vivo and in vivo techniques.ResultsThe patient’s MRI revealed a heterogeneous mass with associated edema, involving the left subventricular zone. Histological examination of the tumor established it as being a high-grade glial neoplasm, characterized by polygonal and fusiform cells with marked nuclear atypia, amphophilic cytoplasm, prominent nucleoli, frequent mitotic figures, irregular zones of necrosis and vascular hyperplasia. Recurrence of the tumor occurred shortly after the surgical resection. CD133-positive cells, isolated from the tumor, expressed stem cell markers including nestin, CD133, Ki67, Sox2, EFNB1, EFNB2, EFNB3, Cav-1, Musashi, Nucleostemin, Notch 2, Notch 4, and Pax6. Biomarkers expressed in differentiated cells included Cathepsin L, Cathepsin B, Mucin18, Mucin24, c-Myc, NSE, and TIMP1. Expression of unique cancer-related transcripts in these CD133-positive cells, such as caveolin-1 and −2, do not appear to have been previously reported in the literature. Ex vivo organotypic brain slice co-culture showed that the CD133+ cells behaved like tumor cells. The CD133-positive cells also induced tumor formation when they were stereotactically transplanted into the brains of the immune-deficient NOD/SCID mice.ConclusionsThis brain tumor involving the neurogenic lateral ventricular wall was comprised of tumor-forming, CD133-positive cancer stem cells, which are likely the driving force for the rapid recurrence of the tumor in the patient.