A novel EPO receptor agonist improves glucose tolerance via glucose uptake in skeletal muscle in a mouse model of diabetes.

Patients treated with recombinant human Epo demonstrate an improvement in insulin sensitivity. We aimed to investigate whether CNTO 530, a novel Epo receptor agonist, could affect glucose tolerance and insulin sensitivity. A single administration of CNTO 530 significantly and dose-dependently reduced the area under the curve in a glucose tolerance test in diet-induced obese and diabetic mice after 14, 21, and 28 days. HOMA analysis suggested an improvement in insulin sensitivity, and this effect was confirmed by a hyperinsulinemic-euglycemic clamp. Uptake of (14)C-2-deoxy-D-glucose indicated that animals dosed with CNTO 530 transported more glucose into skeletal muscle and heart relative to control animals. In conclusion, CNTO530 has a profound effect on glucose tolerance in insulin-resistant rodents likely because of improving peripheral insulin sensitivity. This effect was observed with epoetin-α and darbepoetin-α, suggesting this is a class effect, but the effect with these compounds relative to CNTO530 was decreased in duration and magnitude.

Protein phosphatase 1 and an opposing protein kinase regulate steady-state L-type Ca2+ current in mouse cardiac myocytes.

Studies have suggested that integration of kinase and phosphatase activities maintains the steady-state L-type Ca(2+) current in ventricular myocytes, a balance disrupted in failing hearts. As we have recently reported that the PP1/PP2A inhibitor calyculin A evokes pronounced increases in L-type I(Ca), the goal of this study was to identify the counteracting kinase and phosphatase that determine 'basal'I(Ca) in isolated mouse ventricular myocytes. Whole-cell voltage-clamp studies, with filling solutions containing 10 mm EGTA, revealed that calyculin A (100 nm) increased I(Ca) at test potentials between -42 and +49 mV (44% at 0 mV) from a holding potential of -80 mV. It also shifted the V(0.5) (membrane potential at half-maximal) of both activation (from -17 to -25 mV) and steady-state inactivation (from -32 to -37 mV) in the hyperpolarizing direction. The broad-spectrum protein kinase inhibitor, staurosporine (300 nm), was without effect on I(Ca) when added after calyculin A. However, by itself, staurosporine decreased I(Ca) throughout the voltage range examined (50% at 0 mV) and blocked the response to calyculin A, indicating that the phosphatase inhibitor's effects depend upon an opposing kinase activity. The PKA inhibitors Rp-cAMPs (100 microm in the pipette) and H89 (1 microm) failed to reduce basal I(Ca) or to block the calyculin A-evoked increase in I(Ca). Likewise, calyculin A was still active with 10 mm intracellular BAPTA or when Ba(2+) was used as the charge carrier. These data eliminate roles for protein kinase A (PKA) and calmodulin-dependent protein kinase II (CaMKII) as counteracting kinases. However, the protein kinase C (PKC) inhibitors Ro 31-8220 (1 microm) and Gö 6976 (200 nm) decreased steady-state I(Ca) and blunted the effect of calyculin A. PP2A is not involved in this regulation as intracellular applications of 10-100 nm okadaic acid or 500 nm fostriecin failed to increase I(Ca). However, PP1 is important, as dialysis with 2 microm okadaic acid or 500 nm inhibitor-2 mimicked the increases in I(Ca) seen with calyculin A. These in situ studies identify constitutive activity of PP1 and the counteracting activity of certain isoforms of PKC, in pathways distinct from receptor-mediated signalling cascades, as regulatory components that determine the steady-state level of cardiac L-type I(Ca).

Ankyrin-B mutation causes type 4 long-QT cardiac arrhythmia and sudden cardiac death.

Mutations in ion channels involved in the generation and termination of action potentials constitute a family of molecular defects that underlie fatal cardiac arrhythmias in inherited long-QT syndrome. We report here that a loss-of-function (E1425G) mutation in ankyrin-B (also known as ankyrin 2), a member of a family of versatile membrane adapters, causes dominantly inherited type 4 long-QT cardiac arrhythmia in humans. Mice heterozygous for a null mutation in ankyrin-B are haploinsufficient and display arrhythmia similar to humans. Mutation of ankyrin-B results in disruption in the cellular organization of the sodium pump, the sodium/calcium exchanger, and inositol-1,4,5-trisphosphate receptors (all ankyrin-B-binding proteins), which reduces the targeting of these proteins to the transverse tubules as well as reducing overall protein level. Ankyrin-B mutation also leads to altered Ca2+ signalling in adult cardiomyocytes that results in extrasystoles, and provides a rationale for the arrhythmia. Thus, we identify a new mechanism for cardiac arrhythmia due to abnormal coordination of multiple functionally related ion channels and transporters.

Effects of PP1/PP2A inhibitor calyculin A on the E-C coupling cascade in murine ventricular myocytes.

Calyculin A was used to examine the importance of phosphatases in the modulation of cardiac contractile magnitude in the absence of any neural or humoral stimulation. Protein phosphatase (PP)1 and PP2A activity, twitch contractions, intracellular Ca(2+) concentration ([Ca(2+)](i)) transients, action potentials, membrane currents, and myofilament Ca(2+) sensitivity were measured in isolated mouse ventricular myocytes. Calyculin A (125 nM) inhibited PP1 and PP2A by 50% and 85%, respectively, whereas it doubled the twitch magnitude and increased twitch duration by 50% in field-stimulated cells. Calyculin A-evoked increases in L-type Ca(2+) current (70%) and the resulting [Ca(2+)](i) transient (83%) explain the positive inotropic response. However, increases in twitch and action potential durations did not result from increased myofilament Ca(2+) sensitivity or K(+) current inhibition, respectively. Comparison of the effects of calyculin A and isoproterenol on [Ca(2+)](i) transients and twitch contractions revealed that calyculin A had a much smaller lusitropic effect than the beta-agonist, indicating that calyculin A did not significantly increase sarcoplasmic reticulum Ca(2+) reuptake. Thus while cardiac contractile magnitude is controlled by a steady-state kinase/phosphatase balance, this regulation is not equally operative at all of the steps in the excitation-contraction coupling pathway and may in fact be most important to the regulation of the L-type Ca(2+) channel.