Myosin light chain 2-based selection of human iPSC-derived early ventricular cardiac myocytes.

Applications of human induced pluripotent stem cell derived-cardiac myocytes (hiPSC-CMs) would be strengthened by the ability to generate specific cardiac myocyte (CM) lineages. However, purification of lineage-specific hiPSC-CMs is limited by the lack of cell marking techniques. Here, we have developed an iPSC-CM marking system using recombinant adenoviral reporter constructs with atrial- or ventricular-specific myosin light chain-2 (MLC-2) promoters. MLC-2a and MLC-2v selected hiPSC-CMs were purified by fluorescence-activated cell sorting and their biochemical and electrophysiological phenotypes analyzed. We demonstrate that the phenotype of both populations remained stable in culture and they expressed the expected sarcomeric proteins, gap junction proteins and chamber-specific transcription factors. Compared to MLC-2a cells, MLC-2v selected CMs had larger action potential amplitudes and durations. In addition, by immunofluorescence, we showed that MLC-2 isoform expression can be used to enrich hiPSC-CM consistent with early atrial and ventricular myocyte lineages. However, only the ventricular myosin light chain-2 promoter was able to purify a highly homogeneous population of iPSC-CMs. Using this approach, it is now possible to develop ventricular-specific disease models using iPSC-CMs while atrial-specific iPSC-CM cultures may require additional chamber-specific markers.

Factor VLeiden inhibits fibrinolysis in vivo.

BACKGROUND: Factor V(Leiden) (fV(Leiden)) predisposes to thrombosis by enhancing thrombin formation. This study tested the hypothesis that fV(Leiden) inhibits fibrinolysis in vivo. METHODS AND RESULTS: Radiolabeled clots were injected into the jugular veins of wild-type mice and mice heterozygous (fV(+/Q)) or homozygous (fV(Q/Q)) for fV(Leiden). Mean percent clot lysis 5 hours later was significantly reduced in fV(Q/Q) mice (14.3+/-3.6%, n=13) compared with wild-type mice (40.2+/-7.0%, n=17; P<0.01) and intermediate in fV(+/Q) mice (29.4+/-8.7%, n=9; P<0.03 versus fV(Q/Q), P=0.36 versus wild type). The rate of in vitro lysis of plasma clots prepared from fV(+/Q) or fV(Q/Q) mice was significantly slower than that of wild-type plasma clots, whereas in vitro clot lysis did not differ significantly between groups after inhibiting thrombin-activatable fibrinolysis inhibitor. CONCLUSIONS: fV(Leiden) inhibits fibrinolysis in vivo, suggesting an additional pathway by which this mutation promotes thrombosis.

Chronic iron administration increases vascular oxidative stress and accelerates arterial thrombosis.

BACKGROUND: Iron overload has been implicated in the pathogenesis of ischemic cardiovascular events. However, the effects of iron excess on vascular function and the thrombotic response to vascular injury are not well understood. METHODS AND RESULTS: We examined the effects of chronic iron dextran administration (15 mg over 6 weeks) on thrombosis, systemic and vascular oxidative stress, and endothelium-dependent vascular reactivity in mice. Thrombus generation after photochemical carotid artery injury was accelerated in iron-loaded mice (mean time to occlusive thrombosis, 20.4+/-8.5 minutes; n=10) compared with control mice (54.5+/-35.5 minutes, n=10, P=0.009). Iron loading had no effect on plasma clotting, vessel wall tissue factor activity, or ADP-induced platelet aggregation. Acute administration of dl-cysteine, a reactive oxygen species scavenger, completely abrogated the effects of iron loading on thrombus formation, suggesting that iron accelerated thrombosis through a pro-oxidant mechanism. Iron loading enhanced both systemic and vascular reactive oxygen species production. Endothelium-dependent vasorelaxation was impaired in iron-loaded mice, indicating reduced NO bioavailability. CONCLUSIONS: Moderate iron loading markedly accelerates thrombus formation after arterial injury, increases vascular oxidative stress, and impairs vasoreactivity. Iron-induced vascular dysfunction may contribute to the increased incidence of ischemic cardiovascular events that have been associated with chronic iron overload.

Structure-function analysis of the streptokinase amino terminus (residues 1-59).

Streptokinase (SK) binds to plasminogen (Pg) to form a complex that converts substrate Pg to plasmin. Residues 1-59 of SK regulate its capacity to induce an active site in bound Pg by a nonproteolytic mechanism and to activate substrate Pg in a fibrin-independent manner. We analyzed 24 SK mutants to better define the functional properties of SK-(1-59). Mutations within the alphabeta1 strand (residues 17-26) of SK completely prevented nonproteolytic active site induction in bound Pg and rendered SK incapable of protecting plasmin from inhibition by alpha2-antiplasmin. However, when fibrin-bound, the activities of alphabeta1 strand mutants were similar to that of wild-type (WT) SK and resistant to alpha2-antiplasmin. Mutation of Ile1 of SK also prevented nonproteolytic active site induction in bound Pg. However, unlike alphabeta1 strand mutants, the functional defect of Ile1 mutants was not relieved by fibrin, and complexes of Ile1 mutants and plasmin were resistant to alpha2-antiplasmin. Plasmin enhanced the activities of alphabeta1 strand and Ile1 mutants, suggesting that SK-plasmin complexes activated mutant SK.Pg complexes by hydrolyzing the Pg Arg561-Val562 bond. Mutational analysis of Glu39 of SK suggested that a salt bridge between Glu39 and Arg719 of Pg is important, but not essential, for nonproteolytic active site induction in Pg. Deleting residues 1-59 rendered SK dependent on plasmin and fibrin to generate plasminogen activator (PA) activity. However, the PA activity of SK-(60-414) in the presence of fibrin was markedly reduced compared with WT SK. Despite its reduced PA activity, the fibrinolytic potency of SK-(60-414) was greater than that of WT SK at higher (but not lower) SK concentrations due to its capacity to deplete plasma Pg. These studies define mechanisms by which the SK alpha domain regulates rapid active site induction in bound Pg, contributes to the resistance of the SK-plasmin complex to alpha2-antiplasmin, and controls fibrin-independent Pg activation.