Circulating tumor cells (CTCs) are the principal vehicle for the spread of non-hematologic cancer disease from a primary tumor, involving extravasation of CTCs across blood vessel walls, to form secondary tumors in remote organs. Herein, a polydimethylsiloxane-based microfluidic system is developed and characterized for in vitro systematic studies of organ-specific extravasation of CTCs. The system recapitulates the two major aspects of the in vivo extravasation microenvironment: local signaling chemokine gradients in a vessel with an endothelial monolayer. The parameters controlling the locally stable chemokine gradients, flow rate, and initial chemokine concentration are investigated experimentally and numerically. The microchannel surface treatment effect on the confluency and adhesion of the endothelial monolayer under applied shear flow has also been characterized experimentally. Further, the conditions for driving a suspension of CTCs through the microfluidic system are discussed while simultaneously maintaining both the local chemokine gradients and the confluent endothelial monolayer. Finally, the microfluidic system is utilized to demonstrate extravasation of MDA-MB-231 cancer cells in the presence of CXCL12 chemokine gradients. Consistent with the hypothesis of organ-specific extravasation, control experiments are presented to substantiate the observation that the MDA-MB-231 cell migration is attributed to chemotaxis rather than a random process.

Mutations in MYBPC3, the gene encoding cardiac myosin binding protein C (cMyBP-C), are a major cause of hypertrophic cardiomyopathy (HCM). While most mutations encode premature stop codons, missense mutations causing single amino acid substitutions are also common. Here we investigated effects of a single proline for alanine substitution at amino acid 31 (A31P) in the C0 domain of cMyBP-C, which was identified as a natural cause of HCM in cats. Results using recombinant proteins showed that the mutation disrupted C0 structure, altered sensitivity to trypsin digestion, and reduced recognition by an antibody that preferentially recognizes N-terminal domains of cMyBP-C. Western blots detecting A31P cMyBP-C in myocardium of cats heterozygous for the mutation showed a reduced amount of A31P mutant protein relative to wild-type cMyBP-C, but the total amount of cMyBP-C was not different in myocardium from cats with or without the A31P mutation indicating altered rates of synthesis/degradation of A31P cMyBP-C. Also, the mutant A31P cMyBP-C was properly localized in cardiac sarcomeres. These results indicate that reduced protein expression (haploinsufficiency) cannot account for effects of the A31P cMyBP-C mutation and instead suggest that the A31P mutation causes HCM through a poison polypeptide mechanism that disrupts cMyBP-C or myocyte function.

A microfluidic-based platform is developed for in vitro investigation of signaling within the blood-stream leading to preferential homing of specific cells such as white blood cells (WBCs) selectively to injured host organs. A microchannel has been coated with a monolayer of endothelial cells to mimic the microenvironment in blood vessels, while stable chemokine gradients have been generated locally at selected locations along the microchannel sidewalls. We demonstrate that both the chemokine gradients and the endothelial cells are simultaneously maintained during steady flow of cells as in the blood stream.

Introduction: Cardiac myosin binding protein-C (cMyBP-C) is an essential regulator of heart muscle function that is necessary for both normal contraction and for increased contractility in response to inotropic signals. Effects of cMyBP-C on contraction are complex and attributable to dynamic interactions with both actin and myosin. For instance, cMyBP-C binding to actin can exert either activating or inhibitory effects on contraction by shifting tropomyosin on the thin filament or by blocking myosin heads, respectively. Recently, calmodulin (Ca 2+ /CaM) was also shown to bind cMyBP-C at a unique sequence within the regulatory M-domain of cMyBP-C (Lu et al, JBC 287:31596, 2012). Importantly, the binding site for CaM overlaps with a conserved actin binding site that we found was important for mediating the activating and inhibitory effects of cMyBP-C on the thin filament (Bezold et al. JBC 288: 21496, 2013). Hypothesis: Here we hypothesized that CaM competes with actin for cMyBP-C binding and thus influences the functional effects of cMyBP-C that are mediated through the thin filament. Methods: Recombinant N’-terminal domains of cMyBP-C were used in co-sedimentation binding assays, pull-down assays, and actomyosin ATPase assays to determine effects of CaM binding on cMyBP-C function. Results: We found that CaM binds to N’-terminal domains of cMyBP-C in the presence of Ca 2+ (Ca 2+ /CaM) with an affinity comparable to cMyBP-C binding to actin (3-10 UM). We further show that Ca 2+ /CaM reduces apparent binding affinity of cMyBP-C for actin, consistent with Ca 2+ /CaM competing with actin for binding to cMyBP-C. Also consistent with reduced binding of cMyBP-C to actin, Ca 2+ /CaM reversed the inhibitory effects of cMyBP-C N’-terminal domains on actin activated myosin ATPase rates. However, apo-CaM showed reduced binding affinity for cMyBP-C, did not compete with actin, and did not reverse the activating effects of cMyBP-C on the thin filament at low Ca 2+ . Conclusions: These data suggest that Ca 2+ /CaM is a novel modulator of cMyBP-C function that can temporally tune the complex effects of cMyBP-C on contraction, potentially as Ca 2+ rises and falls during a single heartbeat.

No abstract