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Abstract-Microtubules have been shown to alter contraction in cardiac myocytes through changes in cellular stiffness.However, an effect on excitation-contraction coupling has not been examined. Here we analyze the effects of microtubule disruption by 1 mol/L colchicine on calcium currents (I Ca ) and [Ca 2ϩ ] i transients in rat ventricular myocytes. I Ca was studied using the whole-cell patch-clamp technique. Colchicine treatment increased I Ca density (peak values, Ϫ4.6Ϯ0.4 and Ϫ9.1Ϯ1.3 pA/pF in 11 control and 12 colchicine-treated myocytes, respectively; PϽ0.05). I Ca inactivation was well fitted by a biexponential function. The slow component of inactivation was unchanged, whereas the fast component was accelerated after colchicine treatment (at Ϫ10 mV, 11.8Ϯ1.0 versus 6.7Ϯ1.0 ms in control versus colchicine-treated cells; PϽ0.005). [Ca 2ϩ ] i transients were analyzed by fluo-3 epifluorescence simultaneously with I Ca . Peak [Ca 2ϩ ] i transients were significantly increased in cardiac myocytes treated with colchicine. The values of F/F 0 at 0 mV were 1.1Ϯ0.02 in 9 control cells and 1.4Ϯ0.1 in 11 colchicine-treated cells (PϽ0.05). ␤-Adrenergic stimulation with 1 mol/L isoproterenol increased both I Ca and [Ca 2ϩ ] i transient in control cells. However, no significant change was induced by isoproterenol on colchicine-treated cells. Colchicine and isoproterenol effects were similar and not additive. Inhibition of adenylyl cyclase by 200 mol/L 2Ј-deoxyadenosine 3Ј-monophosphate blunted the colchicine effect. We suggest that ␤-adrenergic stimulation and microtubule disruption share a common pathway to enhance The microtubule network is dynamic, composed by the self-association of ␣,␤-tubulin dimers. Thus, by polymerization and depolymerization, the cell can change the amount of microtubules at constant tubulin amount. The presence of microtubules in the cardiac myocytes is well known, but its role in physiology and pathology is thought to be purely mechanical. In this regard, it has been shown that in pressureoverload cardiac hypertrophy, there is an increase in the microtubule network, which would be responsible for the contractile dysfunction in hypertrophied cells. 1 In this elegant work, Tsutsui et al 1 studied right ventricular cardiac myocytes isolated from cats subjected to pulmonary artery constriction. Under these experimental conditions, hypertrophied cells presented an increased number of microtubules and contracted weakly. When treated with the depolymerizing agent colchicine, hypertrophied myocytes contracted normally. Tsutsui et al 1 concluded that the contractile defect of hypertrophied cells is due to an increase in stiffness and viscosity on the cell imposed by the increased microtubule network triggered by the pressure overload. 2 However, it is also possible that microtubule polymerization and depolymerization play other roles in addition to the mechanical one. In this regard, we have recently shown that heart failure after pressure-overload cardiac hypertrophy induces a dysfunction of...

Abstract-We recently showed that phosphoinositide-3-kinase-␥-deficient (PI3K␥ Ϫ/Ϫ ) mice have enhanced cardiac contractility attributable to cAMP-dependent increases in sarcoplasmic reticulum (SR) Ca 2ϩ content and release but not L-type Ca 2ϩ current (I Ca,L ), demonstrating PI3K␥ locally regulates cAMP levels in cardiomyocytes. Because phosphodiesterases (PDEs) can contribute to cAMP compartmentation, we examined whether the PDE activity was altered by PI3K␥ ablation. Selective inhibition of PDE3 or PDE4 in wild-type (WT) cardiomyocytes elevated Ca 2ϩ transients, SR Ca 2ϩ content, and phospholamban phosphorylation (PLN-PO 4 ) by similar amounts to levels observed in untreated PI3K␥

Background-Cardiac hypertrophy underlies arrhythmias and sudden death, for which mineralocorticoid receptor (MR) activity has recently been implicated. We sought to establish the sequence of ionic events that link the initiating insult and MR to hypertrophy development. Methods and Results-Using whole-cell, patch-clamp and quantitative reverse transcription-polymerase chain reaction techniques on right ventricular myocytes of a myocardial infarction (MI) rat model, we examined the cellular response over time. One week after MI, no sign of cellular hypertrophy was found, but action potential duration (APD) was lengthened. Both an increase in Ca 2ϩ current (I Ca ) and a decrease in K ϩ transient outward current (I to ) underlay this effect. Consistently, the relative expression of mRNA coding for the Ca 2ϩ channel ␣1C subunit (Ca v 1.2) increased, and that of the K ϩ channel K v 4.2 subunit decreased. Three weeks after MI, AP prolongation endured, whereas cellular hypertrophy developed. I Ca density, Ca v 1.2, and K v 4.2 mRNA levels regained control levels, but I to density remained reduced. Long-term treatment with RU28318, an MR antagonist, prevented this electrical remodeling. In a different etiologic model of abdominal aortic constriction, we confirmed that APD prolongation and modifications of ionic currents precede cellular hypertrophy. Conclusions-Electrical remodeling, which is triggered at least in part by MR activation, is an initial, early cellular response to hypertrophic insults.

Abstract-Ca2ϩ influx through the L-type Ca 2ϩ channel (I Ca,L ) is a key determinant of cardiac contractility and is modulated by multiple signaling pathways. Because the regulation of I Ca,L by phosphoinositide-3-kinases (PI3Ks) and phosphoinositide-3-phosphatase (PTEN) is unknown, despite their involvement in the regulation of myocardial growth and contractility, I Ca,L was recorded in myocytes isolated from mice overexpressing a dominant-negative p110␣ mutant (DN-p110␣) in the heart, lacking the PI3K␥ gene (PI3K␥ Ϫ/Ϫ ) or with muscle-specific ablation of PTEN (PTEN Ϫ/Ϫ ). Combinations of these genetically altered mice were also examined. Although there were no differences in the expression level of Ca V 1.2 proteins, basal I Ca,L densities were larger (PϽ0.01) in PTEN Ϫ/Ϫ myocytes compared with littermate controls, PI3K␥ Ϫ/Ϫ , or DN-p110␣ myocytes and showed negative shifts in voltage dependence of current activation. The I Ca,L differences seen in PTEN Ϫ/Ϫ mice were eliminated by pharmacological inhibition of either PI3Ks or protein kinase B (PKB) as well as in PTEN Ϫ/Ϫ /DN-p110␣ double mutant mice but not in PTEN Ϫ/Ϫ /PI3K␥ Ϫ/Ϫ mice. On the other hand, application of insulin-like growth factor-1 (IGF-1), an activator of PKB, increased I Ca,L in control and PI3K␥ Ϫ/Ϫ , while having no effects on I Ca,L in DN-p110␣ or PTEN Ϫ/Ϫ mice. The I Ca,L increases induced by IGF-1 were abolished by PKB inhibition. Our results demonstrate that IGF-1 treatment or inactivation of PTEN enhances I Ca,L via PI3K␣-dependent increase in PKB activation. T he phosphoinositide 3-kinases (PI3Ks) are a family of ubiquitously expressed enzymes catalyzing the phosphorylation of membrane phosphoinositides at the inositol 3-OH position. 1 Of special interest is the class I receptorregulated PI3Ks that link many extracellular stimuli to intracellular response. Class I PI3Ks are divided into class 1A (PI3K␣,␤,␦) kinases, which are activated by receptor or nonreceptor tyrosine kinases and class 1B (PI3K␥) kinases, which are activated by G protein-coupled receptors via G ␤␥ . 1 Both class 1A and 1B PI3Ks selectively phosphorylate phosphatidylinositol 4,5-biphosphate (PI(4,5)P 2 ) to generate the phosphatidylinositol 3,4,5-triphosphate (PI(3,4,5)P 3 ), 1,2 a lipid second messenger that recruits many signaling proteins to the membrane including the serine/threonine kinase protein kinase B (PKB) (also named Akt). 2,3 The PI3K-dependent activation of PKB in turn regulates numerous targets involved in the regulation of nutrient metabolism and cell growth, differentiation, and survival. 4 PI3K actions are antagonized by the phosphoinositide 3-phosphatase PTEN (Phosphatase and TENsin homolog deleted on chromosome ten), which catalyzes conversion of PI(3,4,5)P 3 back to PI(4,5)P 2 . 5,6 PI3Ks and PTEN can also exert biological effects via their protein kinase and phosphatase activities 1,5 as well as by direct protein-protein interaction for PI3K␥. 7 Recent evidence suggests an important role for PI3K-PTEN signaling pathways in the regulation of my...

Abstract-Using the whole-cell patch-clamp configuration in rat ventricular myocytes, we recently reported that microtubule disruption increases calcium current (I Ca ) and [Ca 2ϩ ] i transient and accelerates their kinetics by adenylyl cyclase activation. In the present report, we further analyzed the effects of microtubule disruption by 1 mol/L colchicine on Ca 2ϩ signaling in cardiac myocytes with intact sarcolemma. In quiescent intact cells, it is possible to investigate ryanodine receptor (RyR)

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Abstract-We recently showed that phosphoinositide-3-kinase-␥-deficient (PI3K␥ Ϫ/Ϫ ) mice have increased cardiac contractility without changes in heart size compared with control mice (ie, PI3K␥