Insulin-Like Growth Factor-1 and PTEN Deletion Enhance Cardiac L-Type Ca2+ Currents via Increased PI3K/PKB Signaling
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Hui Sun, Benoit-Gilles Kerfant, Dongling
参见附件。
the Departments of Physiology and Medicine (H.S., B.G.-K., D.Z., M.G.T., G.Y.O., P.H.B.), Division of Cardiology (M.G.T., G.Y.O., P.H.B.), University Health Network
Heart & Stroke/Richard Lewar Centre of Excellence in Cardiovascular Research (P.H.B.), University of Toronto, Canada
Institute for Molecular Biotechnology (J.M.P.), Austrian Academy of Science, Vienna.
Abstract
Ca2+ influx through the L-type Ca2+ channel (ICa,L) is a key determinant of cardiac contractility and is modulated by multiple signaling pathways. Because the regulation of ICa,L by phosphoinositide-3-kinases (PI3Ks) and phosphoinositide-3-phosphatase (PTEN) is unknown, despite their involvement in the regulation of myocardial growth and contractility, ICa,L was recorded in myocytes isolated from mice overexpressing a dominant-negative p110 mutant (DN-p110) in the heart, lacking the PI3K gene (PI3K–/–) or with muscle-specific ablation of PTEN (PTEN–/–). Combinations of these genetically altered mice were also examined. Although there were no differences in the expression level of CaV1.2 proteins, basal ICa,L densities were larger (P<0.01) in PTEN–/– myocytes compared with littermate controls, PI3K–/–, or DN-p110 myocytes and showed negative shifts in voltage dependence of current activation. The ICa,L differences seen in PTEN–/– mice were eliminated by pharmacological inhibition of either PI3Ks or protein kinase B (PKB) as well as in PTEN–/–/DN-p110 double mutant mice but not in PTEN–/–/PI3K–/– mice. On the other hand, application of insulin-like growth factor-1 (IGF-1), an activator of PKB, increased ICa,L in control and PI3K–/–, while having no effects on ICa,L in DN-p110 or PTEN–/– mice. The ICa,L increases induced by IGF-1 were abolished by PKB inhibition. Our results demonstrate that IGF-1 treatment or inactivation of PTEN enhances ICa,L via PI3K-dependent increase in PKB activation.
Key Words: PI3K isoforms PTEN Akt/PKB L-type Ca2+ channels
Introduction
The 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 receptor-regulated 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)P2) to generate the phosphatidylinositol 3,4,5-triphosphate (PI(3,4,5)P3),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)P3 back to PI(4,5)P2.5,6 PI3Ks and PTEN can also exert biological effects via their protein kinase and phosphatase activities1,5 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 myocardial growth, contractility, and response to -adrenergic stimulation.8 Specifically, PTEN ablation in the heart induces hypertrophy, while impairing myocardial contractility. Simultaneous inactivation of PI3K in PTEN-deficient hearts prevented the hypertrophy without influencing the contractility.9 On the other hand, deletion of PI3K rescued the impaired contractility in PTEN-deficient hearts, while having no effect on the heart size.9 Because L-type Ca2+ current (ICa,L) is critical in regulating myocyte contractility as well as myocardial growth,10,11 we examined ICa,L in mice with altered PI3Ks signaling. Our results establish that ICa,L was unaffected by genetic inhibition of PI3K or PI3K, while being enhanced in PTEN-deficient cardiomyocytes as a result of increased PI3K/PKB activity, an effect that is mediated by PI3K but not PI3K. Furthermore, we found that insulin-like growth factor-1 (IGF-1), an important regulator of myocardial growth and function both during development and in response to pathological stress,12,13 also increases ICa,L via PI3K/PKB signaling pathway.
Materials and Methods
Generation of Mutant Mice
Mice used in our studies were in the C57B6 background and were 10 to 14 weeks of age. Mice lacking PI3K activity in the heart (DN-p110) overexpressed a truncated p110 mutant lacking the catalytic domain under the control of the -myosin heavy chain promoter, as previously described.14 Littermate controls lacked the p110 mutant gene. Mice lacking PI3K (PI3K–/–) were generated by disrupting both alleles of the p110 gene as described previously.15 Littermate controls had either 1 (PI3K+/–) or both (PI3K+/+) of the normal PI3K genes. Skeletal and cardiac muscle-specific inactivation of PTEN gene (PTEN–/–) was achieved by flanking exons 4 and 5 of the PTEN gene with lox P sites and overexpressing Cre-recombinase under the control of the muscle creatine kinase promoter as previously described.9 Littermate controls possessed one copy of the PTEN gene (PTEN+/–). Double mutant mice, DN-p110/PTEN–/– and PI3K–/–/PTEN–/–, were generated by crossing DN-p110 mice or PI3K–/– mice with PTEN–/– mice, respectively. Genotyping was performed by PCR using genomic DNA isolated from the tail tip of mice. The primers for identifying the carriers of different transgene were previously described.9,14,15 The care and use of animals conformed to the standards of the Canadian Council on Animal Care.
Myocyte Isolation
Myocytes were isolated from left ventricular free wall by retrograde perfusion of the heart with a Ca2+-free Tyrode solution followed by the same Tyrode solution containing Type II collagenase (0.4 mg/mL; Boehringer-Mannheim) and Type XIV protease (0.03 mg/mL; Sigma). Following isolation, myocytes were stored in solutions containing (in mmol/L) 120 K-glutamate, 10 KCl, 10 KH2PO4, 2 MgSO4, 10 taurine, 5 creatine, 10 HEPES, 10 glucose, 0.5 EGTA, and 0.1% BSA (pH 7.2). The nominally Ca2+-free solution contained (in mmol/L) 136 NaCl, 5.4 KCl, 1 MgCl2, 0.33 NaH2PO4, 10 HEPES, and 10 D-glucose (pH 7.4).
Electrophysiology
ICa,L was recorded at physiological temperatures (35.5°C to 36.0°C) using whole-cell patch clamp technique (Axopatch 200B, Axon Instruments). The external recording solution contained (in mmol/L) 137 NaCl, 5.4 CsCl, 1 MgCl2, 1.2 CaCl2, 10 HEPES, 10 glucose, 2 4-aminopyridine (4-AP, pH 7.35). The pipette resistance ranged between 1.0 and 1.6 M when filled with a solution containing (in mmol/L) 120 CsCl, 20 TEA-Cl, 1 MgCl2, 5 MgATP, 0.2 Na2GTP, 10 HEPES, and 10 EGTA (pH 7.2). Series resistance (RS) was compensated by 80% to 90%. The average RS before and after compensation was 3.51±0.03 M and 0.70±0.03 M for PTEN–/– myocytes (n=49) and 3.65±0.13 M and 0.73±0.03 M for littermate control myocytes (n=51). Myocytes were held at –80 mV, and Na+ currents were inactivated by applying a prepulse to –40 mV for 300 ms before step depolarizing to voltages ranging from –40 to +60 mV for 300 ms. Steady-state inactivation of ICa,L was studied with 500-ms conditioning pulses ranging from –50 mV to +10 mV followed 10 ms later by a 200-ms test pulse to 0 mV.
Immunoblotting
Proteins (50 μg/lane) from solubilized heart crude membranes were run on a 2-layer (6% and 12%) SDS-PAGE. Following transfer to a polyvinylidene fluoride (PVDF) membrane, antibodies targeting the intracellular loop between II and III domains of the 1C subunit (Alomone), followed by anti-rabbit IgG-coupled horseradish peroxidase (Amersham), were used to detect L-type Ca2+ channel expression. GAPDH was also blotted and used as loading control.
Measurements of Myocyte Contraction
Unloaded myocyte shortening was measured in field-stimulated myocytes (1 Hz) with a video edge detector as previously described.16 The amplitude of cell shortening was expressed as percentage shortening relative to the resting diastolic length.
Chemicals
Wortmannin (WM) (Sigma), LY294002 (Calbiochem), and SH-6 (Alexis) stocks were prepared in DMSO and then diluted to final concentrations in bath solutions before use. IGF-1 (Sigma) was prepared directly in bath solution before use.
Data Analysis and Statistics
Current recordings were analyzed using pClamp software (Clampfit 8.2, Axon, Calif). The ICa,L amplitude was measured as the difference between the peak current and the current level remaining at the end of 300 ms depolarization. The cord conductance (G) was calculated using the equation G=I/(Vm–Erev), where Vm represents the depolarizing voltages and Erev the reversal potential estimated from the current-voltage relation of ICa,L. The maximal conductance (Gmax), the voltage at which 50% activation occurs (V1/2), and the slop factor k were obtained by fitting conductance data to the Boltzmann function: G(Vm)=Gmax/(1+exp[{Vm–V1/2}/k]). The steady-state inactivation curves were obtained by fitting the relative current (I/Imax) obtained during test pulse to the Boltzmann function: I/Imax= 1/(1+exp[{V1/2.inac–Vm}/k]), where Vm refers to the voltage of conditioning pulse, V1/2.inac the voltage at which 50% inactivation occurs and k the slop factor.
Statistical significance of differences between two means was assessed using pair or unpaired Student’s t tests. Comparisons among multiple means were performed using 1-way ANOVA with Bonferroni test. Comparisons of inactivation time constants obtained in control and PTEN–/– groups at different voltages were performed using 2-way ANOVA. A probability value of <0.05 was considered to indicate significance. Group data are expressed as the mean±SEM.
Results
We have previously demonstrated that cardiac PTEN ablation impairs contractile function and induces cardiac hypertrophy in mice.9 To elucidate the underlying mechanisms, we examined the effects of PTEN deficiency on ICa,L, a major determinant of cardiac contractility. Although PTEN–/– myocytes were larger (P<0.01) than littermate controls (ie, PTEN+/–, 235±6 pF, n=49, versus 178±4 pF, n=51), as reported previously,9 the densities and maximal conductance (Gmax) of peak ICa,L were increased (P<0.05) by &20% in conjunction with a negative shift (P<0.05) in the peak ICa,L–voltage relationship and the voltage for half-maximal current activation (V1/2, –19.6±0.4 mV versus –16.8±0.2 mV) (supplemental Table I available at http://circres.ahajournals.org and Figure 1). It is conceivable that the shifts in V1/2 observed in PTEN–/– myocytes were related to incomplete RS compensation because PTEN–/– myocytes had larger ICa,L amplitude. However, correction of applied voltages for the uncompensated RS showed that V1/2 was still negatively shifted (P<0.001) in PTEN–/– myocytes (–17.8±0.3 mV) compared to littermate controls (–15.1±0.2 mV).
The voltage for 50% inactivation was not altered (P=0.2) in PTEN–/– myocytes (–33.0±0.3 mV, n=4) compared with control (–35.8±1.8 mV, n=4). The decay of ICa,L in both groups was best fit with a biexponential function at voltages between –20 and +20 mV. The magnitude of the faster time constant (fast) was smaller (P<0.01), whereas the amplitude of the fast component was larger at –20 and –10 mV in PTEN–/– myocytes compared with littermate control myocytes. The magnitude of the slower time constant (slow) was also smaller at –20 mV in PTEN–/– myocytes. For example, at –20 mV fast was 9.3±0.5 ms (amplitude, 70.1±1.2%) and slow was 60.9±2.0 ms (amplitude, 29.9±1.2%) in PTEN–/– myocytes (n=49), whereas fast was 13.7±0.6 ms (amplitude, 61.3±0.9%) and slow was 68.0±1.5 ms (amplitude, 38.7±0.9%) in PTEN+/– myocytes (n=51). These changes in ICa,L inactivation are consistent with enhanced Ca2+-mediated inactivation (ie, increased fast inactivation) when ICa,L is increased as previously reviewed.17
To determine whether enhanced ICa,L in PTEN–/– mice was related to altered PI3K signaling or loss of protein phosphatase activities of PTEN,1,5 the effects of PI3Ks inhibitors were examined. Incubation of myocytes with 100 nmol/L WM for 90 minutes had no effect (P=0.22) on basal ICa,L in control myocytes (Figure 2A). By contrast, ICa,L recorded in WM-treated PTEN–/– myocytes were indistinguishable (P=0.21 and P=0.26) from ICa,L recorded in littermate control myocytes with or without WM (Figure 2B). Similar observations were obtained using LY294002 (LY), another PI3K inhibitor (Figure 3). These effects of PI3K inhibitions suggest that the PI3K signaling is required for the increased ICa,L density observed in PTEN-deficient cardiomyocytes.
To further confirm that the PI3K activity is necessary for ICa,L enhancement in PTEN-deficient hearts and to identify the PI3K isoforms involved, ICa,L was recorded in mice lacking both PTEN and PI3K activities (PTEN–/–/DN-p110) or lacking both PTEN and PI3K activities (PTEN–/–/PI3K–/–). Inactivation of either PI3K or PI3K alone did not affect ICa,L relative to littermate controls (Figure 4A and 4B). On the other hand, compared with control or PI3K–/– myocytes, ICa,L density and Gmax were elevated (P<0.01), whereas V1/2 was shifted to negative voltages in PTEN–/–/PI3K–/– myocytes, exactly as in the PTEN–/– myocytes (Figure 4B and 4C and supplemental Table I). By contrast, the magnitude and current-voltage relationship of ICa,L in PTEN–/–/DN-p110 were similar to control or DN-p110 myocytes (Figure 4A and 4C and supplemental Table I). These results establish that PI3K, not PI3K, mediates the increase in ICa,L observed in PTEN–/– myocytes.
To determine whether the PI3K-mediated enhancement of ICa,L current density in PTEN-deficient hearts results from alterations in channel expression, the protein levels of the Cav1.2 (ie, the 1C subunit of L-type Ca2+ channels) were measured. Relative to control hearts, the levels of 1C were not different (P=0.74) in PTEN–/– (103.1±4.9%) or DN-p110/PTEN–/– (97.9±6.7%) hearts (n=12 for each group), supporting the conclusion that increased ICa,L in PTEN-deficient myocytes originates from posttranslational modification.
Because previous studies established that PKB is activated in PTEN-deficient mice and that inactivation of PI3K (but not PI3K) reduced this increased PKB activities,9 we postulated that PKB may be responsible for the enhancement of ICa,L in PTEN-deficient hearts. Consistent with our hypothesis, application of a PKB inhibitor, SH-6,18 at 10 μmol/L did not affect ICa,L density or Gmax in control myocytes (Figure 5A), but it did reduce (P<0.01) ICa,L density and Gmax in PTEN–/– myocytes to the same levels observed in littermate controls with or without SH-6 treatment (Figure 5B). Despite reducing ICa,L density to control levels, SH-6 did not affect the V1/2 for activation in PTEN–/– myocytes (supplemental Table I). The lack of effect of PKB inhibitor on V1/2 probably reflects a nonspecific effect of the agent because a negative shift in V1/2 was observed in control myocytes following SH-6 application (Figure 5A and supplemental Table I).
To evaluate the contribution of PTEN deficiency-induced increases in ICa,L to myocyte contractile function, the effects of PKB inhibition on myocyte shortening were examined. Treatment with SH-6 led to 18% reduction (P<0.05) of cell-shortening amplitude in PTEN–/– myocytes (Figure 5C), while having no significant effects on control myocytes (data not shown). These results establish that increases in ICa,L induced by PTEN ablation can have positive inotropic effects.
Our results establish a novel regulation of cardiac ICa,L by PI3K and PKB. Because PI3K and PKB mediate the actions of IGF-1,19,20 a hormone essential for cardiac development, morphology, and function,12,13 we examined the effects of IGF-1 on ICa,L. Superfusion of IGF-1 (0.1 μmol/L) caused an enhancement of ICa,L in littermate control myocytes that required &1 to 2 minutes to fully develop (Figure 6A). After 90 seconds of application, IGF-1 increased (P<0.001) ICa,L by 17% in littermate control myocytes without affecting ICa,L in DN-p110 myocytes (Figure 6B). By contrast, IGF-1 increased ICa,L similarly in PI3K–/– myocytes and littermate control myocytes (Figure 6C). IGF-1 failed to enhance ICa,L in PTEN–/– myocytes although it increased ICa,L in littermate control myocytes (Figure 6D) to similar levels observed in PTEN–/– myocytes. This increase in ICa,L could be prevented by treatment with SH-6, suggesting that PKB activation is required for the IGF-1-induced increase in ICa,L (Figure 6E). These results establish that IGF-1 enhances ICa,L in mouse hearts via activation of PI3K/PKB pathway, and this pathway is fully stimulated in PTEN-deficient hearts.
Discussion
Our study demonstrates that myocytes lacking the PTEN gene have elevated cardiac L-type Ca2+ currents accompanied by negative shifts in voltage-dependent channel activation, which were unrelated to incomplete voltage control arising from uncompensated RS. Our results further establish that the elevations in ICa,L observed in PTEN-deficient hearts occur without increases in channel expression but are eliminated by PI3K or PKB inhibitors and by dominant-negative PI3K suppression. These findings suggest that ICa,L enhancements and voltage shifts of ICa,L activation in PTEN-deficient myocytes result from elevated PI(3,4,5)P3 levels leading to tonic PKB activation and phosphorylation of target proteins involved in L-type Ca2+ channel regulation. These results are consistent with the conclusion that the dominant physiological function of PTEN is the breakdown of the PI3K-generated second messenger PI(3,4,5)P3 to PI(4,5)P2.21 The increases in ICa,L observed in PTEN-deficient myocytes are similar to increases in ICa,L density (without changes in 1C expression) observed in mouse ventricular myocytes overexpressing PKB.22 This conclusion also agrees with previous studies in vascular smooth muscle cells showing that dialysis with anti-PI(3,4,5)P3 antibodies abolished the angiotensin II-induced and PI3K-mediated stimulation of L-type Ca2+ current.23 Increases in ICa,L without change in 1C expression observed in PTEN-deficient hearts contrasts with results showing that, in COS-7 cells and neurons transfected with PI3K, PKB activation increases Cav2.2 membrane expression as a result of 2a subunit phosphorylation by PKB.24
Although cardiac ICa,L was increased in PTEN-deficient mice, it was not different in myocytes with inhibition of cardiac PI3K or with disruption of PI3K compared with control mice. Nor was ICa,L affected by treatment with PI3Ks or PKB inhibitors in control mice. Collectively, these results suggest that basal PKB activity in subcellular compartments containing L-type Ca2+ channels is insufficient to upregulate cardiac ICa,L in myocytes when PTEN phosphatase activity is normal. However, when PTEN phosphatase activity is abolished, the PI3K activity is sufficient to activate PKB and increase ICa,L, consistent with PI3K-dependent myocyte hypertrophy observed in PTEN-deficient mice.9 These observations are similar to those reported previously in vascular smooth muscle cells in which dialysis with antibodies against PI3K or PI3K had no effect on basal ICa,L,25 whereas introduction of various purified class I PI3Ks into myocyte by dialysis led to increased ICa,L.25 The ability of PI3K disruption, but not PI3K disruption, to reverse the elevated ICa,L observed in PTEN-deficient cardiomyocytes suggests that PKB activation occurs in microdomains containing L-type Ca2+ channels and PI3K as well as PTEN. This possibility is consistent with the increases in ICa,L observed in response to stimulation of IGF-1 receptors because previous studies established that receptor tyrosine kinase stimulation activates PKB via PI3K.19,20 By contrast, PI3K does not contribute significantly to PKB activation under basal conditions,9 although it can also activate PKB directly following G-protein-coupled receptor stimulation26 or via Ras activation27 (possibly by indirect transactivation of receptor tyrosine kinases).28
The regulation pattern of ICa,L by PI3K (via PKB activation) versus PI3K parallels the clear separation of myocardial growth regulation by PI3K versus contractility regulation by PI3K.7,9,14,20 Because ICa,L is a key determinant of cardiac contractility, it is somewhat surprising that PKB-dependent increases of ICa,L occur in PTEN-deficient myocytes, which have reduced contractility.9 Although basis for the reduced contractility in PTEN–/– myocytes remains to be determined, it is likely to depend complexly on interactions between increased PI3K- and increased PI3K-mediated signaling. For example, increased contractility in PI3K–/– myocytes has been linked to increased sarcoplasmic reticulum (SR) Ca2+ loads (without alteration in ICa,L) as a result of compartmentalized subcellular elevations of cAMP.29 Therefore, decreased cAMP levels observed in PTEN–/– hearts9 as a result of altered PI3K-dependent signaling may lead to reduced SR Ca2+ loads. However, increased expression of -myosin heavy chain9 combined with decreased myofilament Ca2+ sensitivity, possibly as a consequence of PIP3-dependent protein kinase C (PKC) activation,2,30 could also lead to reduced cardiac tension development in PTEN-deficient mice.31 In addition, nonselective PI3K inhibition enhances the ICa,L increases induced by 1-adrenergic stimulation,32 suggesting that the modulation of ICa,L by PI3K can depend complexly on the experimental conditions and the signaling pathways activated.
Although the cause of reduced contractility in myocytes lacking PTEN is unclear, PKB inhibition with SH-6 reduced ICa,L in PTEN-deficient myocytes to control levels and attenuated the magnitude of cell shortening, suggesting elevated ICa,L in PTEN-deficient myocytes enhance myocyte contractility. Interestingly, enhanced contractility (and pressure relaxation) have been observed in hearts from mice with PKB overexpression,22,33 which was associated with increased SERCA2a expression and increased ICa,L (when constitutively active Akt E40K is overexpressed 25-fold22) or with enhanced phospholamban phosphorylation (when Akt targeted to the nucleus is overexpressed 10-fold33). Thus, it is conceivable that, in addition to increased ICa,L, SERCA2a expression or phospholamban phosphorylation might also be altered in PTEN–/– myocytes where phospho-PKB levels are increased &4-fold.9 However, overexpression of PKB in selected cellular compartments may not accurately reproduce the activation pattern and effects of PKB by PI3K when PTEN is deleted. Consistent with this, we did not observe alterations in the relaxation kinetics of Ca2+ transients in PTEN–/– myocytes or following PKB inhibition (data not shown), as observed previously in mice overexpressing PKB.22,33 These observations are not incompatible with previous studies in PKB-overexpressing mice because the acute effects of PKB on phospholamban phosphorylation or SERCA2a activity were not examined.22,33 Regardless, any changes in SERCA2a function combined with ICa,L increases are insufficient to offset the actions of other factor(s) responsible for reduced contractility in the PTEN-deficient hearts. Future investigations are clearly warranted to examine the contribution of SERCA2a and other molecular mechanisms to altered contractility in PTEN–/– hearts.
Our results also establish that IGF-1, like the loss of PTEN, increases ICa,L in mouse ventricle via activation of PI3K/PKB signaling pathway, consistent with the notion that tyrosine kinase receptors signal via PI3K activation.1 These increases in ICa,L by IGF-1 has important implications because IGF-1 regulates cardiac remodeling and contractility in patients with cardiomyopathy,34 heart failure12,13 as well as in compensatory hypertrophy during pressure-overload.35 Previous studies have established that ICa,L is critical for induction of myocardial hypertrophy11,36–38 and that Ca2+ is an essential cofactor for many hypertrophy signaling pathways including PKC, mitogen-activated protein kinases (MAPKs), and calcineurin.39,40 Thus, because cardiac growth and hypertrophy are strongly regulated by PI3K/PKB signaling pathways,8,20,39 increase in ICa,L may play a role in the hypertrophic effects of IGF-1 or PTEN ablation. Consistent with this possibility, ICa,L inhibition blocks hypertrophy induced by IGF-1 in cultured cardiomyocytes.41 Increased ICa,L may also contribute to the positive inotropic effects of IGF-1 on myocardium,13,42 a suggestion made previously in myocytes isolated from heart failure patients in which inhibition of ICa,L attenuates the positive inotropic response of IGF-1.43 Indeed, when ICa,L was returned to baseline levels by PKB inhibition, contractility was reduced in our PTEN-deficient myocytes. The contribution of increased ICa,L to cardiac growth and enhanced contractility mediated by IGF-1 and the PTEN/PI3K/PKB signaling pathways will require further investigation.
The molecular mechanism for the PKB-dependent increases of ICa,L in PTEN deficient cardiomyocytes and following IGF-1 treatment is unclear. Increased ICa,L observed in PTEN-deficient myocytes and following IGF-1 treatment was accompanied by negative voltage shifts in the steady-state activation curve along with accelerated rates of channel inactivation. This pattern of change in ICa,L resembles that observed following PKA activation,17 suggesting phosphorylation L-type Ca2+ channel. Examination of the amino acid sequence of mammalian cardiac L-type Ca2+ channel subunits revealed 2 absolutely conserved consensus motifs for PKB phosphorylation19 in 1C subunits (located at S863 in the II-III intracellular linker and at S2015 in the C terminus, mouse sequence) and 1 in the C terminus of the 2a subunit (at S624, mouse sequence). Further studies will be required to unravel the molecular mechanism for the regulation of cardiac ICa,L by PKB.
In summary, we have demonstrated that cardiac L-type Ca2+ channels are regulated in a PKB-dependent manner by PI3K–PTEN signaling pathway. This signaling pathway also mediates IGF-1–induced increase in ICa,L in mouse ventricle. This mechanism for ICa,L regulation may play an important role in the regulation of myocardial growth and contractility by PI3K, PTEN, and growth factors in normal and diseased hearts.
Acknowledgments
This work was funded by a Canadian Institute of Health Research (CIHR) operating grant (MOP 43947 to P.H.B.). P.H.B. is a Career Investigator of the Heart & Stroke Foundation of Ontario. H.S. is supported by a Diana Meltzer Abramsky Research Fellowship from the Thyroid Foundation of Canada. B.-G.K. and M.G.T. are supported by fellowships from the Heart & Stroke Foundation of Canada and TACTICS-CIHR). G.Y.O. was supported by fellowships from CIHR and TACTICS-CIHR. J.M.P. is supported by grants from the Austrian National Bank and EuGeneHeart (6th European Commission Framework Programme).
Footnotes
Both authors contributed equally to this study.
Original received January 28, 2005; resubmission received March 9, 2006; revised resubmission received April 5, 2006; accepted April 7, 2006.
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the Departments of Physiology and Medicine (H.S., B.G.-K., D.Z., M.G.T., G.Y.O., P.H.B.), Division of Cardiology (M.G.T., G.Y.O., P.H.B.), University Health Network
Heart & Stroke/Richard Lewar Centre of Excellence in Cardiovascular Research (P.H.B.), University of Toronto, Canada
Institute for Molecular Biotechnology (J.M.P.), Austrian Academy of Science, Vienna.
Abstract
Ca2+ influx through the L-type Ca2+ channel (ICa,L) is a key determinant of cardiac contractility and is modulated by multiple signaling pathways. Because the regulation of ICa,L by phosphoinositide-3-kinases (PI3Ks) and phosphoinositide-3-phosphatase (PTEN) is unknown, despite their involvement in the regulation of myocardial growth and contractility, ICa,L was recorded in myocytes isolated from mice overexpressing a dominant-negative p110 mutant (DN-p110) in the heart, lacking the PI3K gene (PI3K–/–) or with muscle-specific ablation of PTEN (PTEN–/–). Combinations of these genetically altered mice were also examined. Although there were no differences in the expression level of CaV1.2 proteins, basal ICa,L densities were larger (P<0.01) in PTEN–/– myocytes compared with littermate controls, PI3K–/–, or DN-p110 myocytes and showed negative shifts in voltage dependence of current activation. The ICa,L differences seen in PTEN–/– mice were eliminated by pharmacological inhibition of either PI3Ks or protein kinase B (PKB) as well as in PTEN–/–/DN-p110 double mutant mice but not in PTEN–/–/PI3K–/– mice. On the other hand, application of insulin-like growth factor-1 (IGF-1), an activator of PKB, increased ICa,L in control and PI3K–/–, while having no effects on ICa,L in DN-p110 or PTEN–/– mice. The ICa,L increases induced by IGF-1 were abolished by PKB inhibition. Our results demonstrate that IGF-1 treatment or inactivation of PTEN enhances ICa,L via PI3K-dependent increase in PKB activation.
Key Words: PI3K isoforms PTEN Akt/PKB L-type Ca2+ channels
Introduction
The 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 receptor-regulated 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)P2) to generate the phosphatidylinositol 3,4,5-triphosphate (PI(3,4,5)P3),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)P3 back to PI(4,5)P2.5,6 PI3Ks and PTEN can also exert biological effects via their protein kinase and phosphatase activities1,5 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 myocardial growth, contractility, and response to -adrenergic stimulation.8 Specifically, PTEN ablation in the heart induces hypertrophy, while impairing myocardial contractility. Simultaneous inactivation of PI3K in PTEN-deficient hearts prevented the hypertrophy without influencing the contractility.9 On the other hand, deletion of PI3K rescued the impaired contractility in PTEN-deficient hearts, while having no effect on the heart size.9 Because L-type Ca2+ current (ICa,L) is critical in regulating myocyte contractility as well as myocardial growth,10,11 we examined ICa,L in mice with altered PI3Ks signaling. Our results establish that ICa,L was unaffected by genetic inhibition of PI3K or PI3K, while being enhanced in PTEN-deficient cardiomyocytes as a result of increased PI3K/PKB activity, an effect that is mediated by PI3K but not PI3K. Furthermore, we found that insulin-like growth factor-1 (IGF-1), an important regulator of myocardial growth and function both during development and in response to pathological stress,12,13 also increases ICa,L via PI3K/PKB signaling pathway.
Materials and Methods
Generation of Mutant Mice
Mice used in our studies were in the C57B6 background and were 10 to 14 weeks of age. Mice lacking PI3K activity in the heart (DN-p110) overexpressed a truncated p110 mutant lacking the catalytic domain under the control of the -myosin heavy chain promoter, as previously described.14 Littermate controls lacked the p110 mutant gene. Mice lacking PI3K (PI3K–/–) were generated by disrupting both alleles of the p110 gene as described previously.15 Littermate controls had either 1 (PI3K+/–) or both (PI3K+/+) of the normal PI3K genes. Skeletal and cardiac muscle-specific inactivation of PTEN gene (PTEN–/–) was achieved by flanking exons 4 and 5 of the PTEN gene with lox P sites and overexpressing Cre-recombinase under the control of the muscle creatine kinase promoter as previously described.9 Littermate controls possessed one copy of the PTEN gene (PTEN+/–). Double mutant mice, DN-p110/PTEN–/– and PI3K–/–/PTEN–/–, were generated by crossing DN-p110 mice or PI3K–/– mice with PTEN–/– mice, respectively. Genotyping was performed by PCR using genomic DNA isolated from the tail tip of mice. The primers for identifying the carriers of different transgene were previously described.9,14,15 The care and use of animals conformed to the standards of the Canadian Council on Animal Care.
Myocyte Isolation
Myocytes were isolated from left ventricular free wall by retrograde perfusion of the heart with a Ca2+-free Tyrode solution followed by the same Tyrode solution containing Type II collagenase (0.4 mg/mL; Boehringer-Mannheim) and Type XIV protease (0.03 mg/mL; Sigma). Following isolation, myocytes were stored in solutions containing (in mmol/L) 120 K-glutamate, 10 KCl, 10 KH2PO4, 2 MgSO4, 10 taurine, 5 creatine, 10 HEPES, 10 glucose, 0.5 EGTA, and 0.1% BSA (pH 7.2). The nominally Ca2+-free solution contained (in mmol/L) 136 NaCl, 5.4 KCl, 1 MgCl2, 0.33 NaH2PO4, 10 HEPES, and 10 D-glucose (pH 7.4).
Electrophysiology
ICa,L was recorded at physiological temperatures (35.5°C to 36.0°C) using whole-cell patch clamp technique (Axopatch 200B, Axon Instruments). The external recording solution contained (in mmol/L) 137 NaCl, 5.4 CsCl, 1 MgCl2, 1.2 CaCl2, 10 HEPES, 10 glucose, 2 4-aminopyridine (4-AP, pH 7.35). The pipette resistance ranged between 1.0 and 1.6 M when filled with a solution containing (in mmol/L) 120 CsCl, 20 TEA-Cl, 1 MgCl2, 5 MgATP, 0.2 Na2GTP, 10 HEPES, and 10 EGTA (pH 7.2). Series resistance (RS) was compensated by 80% to 90%. The average RS before and after compensation was 3.51±0.03 M and 0.70±0.03 M for PTEN–/– myocytes (n=49) and 3.65±0.13 M and 0.73±0.03 M for littermate control myocytes (n=51). Myocytes were held at –80 mV, and Na+ currents were inactivated by applying a prepulse to –40 mV for 300 ms before step depolarizing to voltages ranging from –40 to +60 mV for 300 ms. Steady-state inactivation of ICa,L was studied with 500-ms conditioning pulses ranging from –50 mV to +10 mV followed 10 ms later by a 200-ms test pulse to 0 mV.
Immunoblotting
Proteins (50 μg/lane) from solubilized heart crude membranes were run on a 2-layer (6% and 12%) SDS-PAGE. Following transfer to a polyvinylidene fluoride (PVDF) membrane, antibodies targeting the intracellular loop between II and III domains of the 1C subunit (Alomone), followed by anti-rabbit IgG-coupled horseradish peroxidase (Amersham), were used to detect L-type Ca2+ channel expression. GAPDH was also blotted and used as loading control.
Measurements of Myocyte Contraction
Unloaded myocyte shortening was measured in field-stimulated myocytes (1 Hz) with a video edge detector as previously described.16 The amplitude of cell shortening was expressed as percentage shortening relative to the resting diastolic length.
Chemicals
Wortmannin (WM) (Sigma), LY294002 (Calbiochem), and SH-6 (Alexis) stocks were prepared in DMSO and then diluted to final concentrations in bath solutions before use. IGF-1 (Sigma) was prepared directly in bath solution before use.
Data Analysis and Statistics
Current recordings were analyzed using pClamp software (Clampfit 8.2, Axon, Calif). The ICa,L amplitude was measured as the difference between the peak current and the current level remaining at the end of 300 ms depolarization. The cord conductance (G) was calculated using the equation G=I/(Vm–Erev), where Vm represents the depolarizing voltages and Erev the reversal potential estimated from the current-voltage relation of ICa,L. The maximal conductance (Gmax), the voltage at which 50% activation occurs (V1/2), and the slop factor k were obtained by fitting conductance data to the Boltzmann function: G(Vm)=Gmax/(1+exp[{Vm–V1/2}/k]). The steady-state inactivation curves were obtained by fitting the relative current (I/Imax) obtained during test pulse to the Boltzmann function: I/Imax= 1/(1+exp[{V1/2.inac–Vm}/k]), where Vm refers to the voltage of conditioning pulse, V1/2.inac the voltage at which 50% inactivation occurs and k the slop factor.
Statistical significance of differences between two means was assessed using pair or unpaired Student’s t tests. Comparisons among multiple means were performed using 1-way ANOVA with Bonferroni test. Comparisons of inactivation time constants obtained in control and PTEN–/– groups at different voltages were performed using 2-way ANOVA. A probability value of <0.05 was considered to indicate significance. Group data are expressed as the mean±SEM.
Results
We have previously demonstrated that cardiac PTEN ablation impairs contractile function and induces cardiac hypertrophy in mice.9 To elucidate the underlying mechanisms, we examined the effects of PTEN deficiency on ICa,L, a major determinant of cardiac contractility. Although PTEN–/– myocytes were larger (P<0.01) than littermate controls (ie, PTEN+/–, 235±6 pF, n=49, versus 178±4 pF, n=51), as reported previously,9 the densities and maximal conductance (Gmax) of peak ICa,L were increased (P<0.05) by &20% in conjunction with a negative shift (P<0.05) in the peak ICa,L–voltage relationship and the voltage for half-maximal current activation (V1/2, –19.6±0.4 mV versus –16.8±0.2 mV) (supplemental Table I available at http://circres.ahajournals.org and Figure 1). It is conceivable that the shifts in V1/2 observed in PTEN–/– myocytes were related to incomplete RS compensation because PTEN–/– myocytes had larger ICa,L amplitude. However, correction of applied voltages for the uncompensated RS showed that V1/2 was still negatively shifted (P<0.001) in PTEN–/– myocytes (–17.8±0.3 mV) compared to littermate controls (–15.1±0.2 mV).
The voltage for 50% inactivation was not altered (P=0.2) in PTEN–/– myocytes (–33.0±0.3 mV, n=4) compared with control (–35.8±1.8 mV, n=4). The decay of ICa,L in both groups was best fit with a biexponential function at voltages between –20 and +20 mV. The magnitude of the faster time constant (fast) was smaller (P<0.01), whereas the amplitude of the fast component was larger at –20 and –10 mV in PTEN–/– myocytes compared with littermate control myocytes. The magnitude of the slower time constant (slow) was also smaller at –20 mV in PTEN–/– myocytes. For example, at –20 mV fast was 9.3±0.5 ms (amplitude, 70.1±1.2%) and slow was 60.9±2.0 ms (amplitude, 29.9±1.2%) in PTEN–/– myocytes (n=49), whereas fast was 13.7±0.6 ms (amplitude, 61.3±0.9%) and slow was 68.0±1.5 ms (amplitude, 38.7±0.9%) in PTEN+/– myocytes (n=51). These changes in ICa,L inactivation are consistent with enhanced Ca2+-mediated inactivation (ie, increased fast inactivation) when ICa,L is increased as previously reviewed.17
To determine whether enhanced ICa,L in PTEN–/– mice was related to altered PI3K signaling or loss of protein phosphatase activities of PTEN,1,5 the effects of PI3Ks inhibitors were examined. Incubation of myocytes with 100 nmol/L WM for 90 minutes had no effect (P=0.22) on basal ICa,L in control myocytes (Figure 2A). By contrast, ICa,L recorded in WM-treated PTEN–/– myocytes were indistinguishable (P=0.21 and P=0.26) from ICa,L recorded in littermate control myocytes with or without WM (Figure 2B). Similar observations were obtained using LY294002 (LY), another PI3K inhibitor (Figure 3). These effects of PI3K inhibitions suggest that the PI3K signaling is required for the increased ICa,L density observed in PTEN-deficient cardiomyocytes.
To further confirm that the PI3K activity is necessary for ICa,L enhancement in PTEN-deficient hearts and to identify the PI3K isoforms involved, ICa,L was recorded in mice lacking both PTEN and PI3K activities (PTEN–/–/DN-p110) or lacking both PTEN and PI3K activities (PTEN–/–/PI3K–/–). Inactivation of either PI3K or PI3K alone did not affect ICa,L relative to littermate controls (Figure 4A and 4B). On the other hand, compared with control or PI3K–/– myocytes, ICa,L density and Gmax were elevated (P<0.01), whereas V1/2 was shifted to negative voltages in PTEN–/–/PI3K–/– myocytes, exactly as in the PTEN–/– myocytes (Figure 4B and 4C and supplemental Table I). By contrast, the magnitude and current-voltage relationship of ICa,L in PTEN–/–/DN-p110 were similar to control or DN-p110 myocytes (Figure 4A and 4C and supplemental Table I). These results establish that PI3K, not PI3K, mediates the increase in ICa,L observed in PTEN–/– myocytes.
To determine whether the PI3K-mediated enhancement of ICa,L current density in PTEN-deficient hearts results from alterations in channel expression, the protein levels of the Cav1.2 (ie, the 1C subunit of L-type Ca2+ channels) were measured. Relative to control hearts, the levels of 1C were not different (P=0.74) in PTEN–/– (103.1±4.9%) or DN-p110/PTEN–/– (97.9±6.7%) hearts (n=12 for each group), supporting the conclusion that increased ICa,L in PTEN-deficient myocytes originates from posttranslational modification.
Because previous studies established that PKB is activated in PTEN-deficient mice and that inactivation of PI3K (but not PI3K) reduced this increased PKB activities,9 we postulated that PKB may be responsible for the enhancement of ICa,L in PTEN-deficient hearts. Consistent with our hypothesis, application of a PKB inhibitor, SH-6,18 at 10 μmol/L did not affect ICa,L density or Gmax in control myocytes (Figure 5A), but it did reduce (P<0.01) ICa,L density and Gmax in PTEN–/– myocytes to the same levels observed in littermate controls with or without SH-6 treatment (Figure 5B). Despite reducing ICa,L density to control levels, SH-6 did not affect the V1/2 for activation in PTEN–/– myocytes (supplemental Table I). The lack of effect of PKB inhibitor on V1/2 probably reflects a nonspecific effect of the agent because a negative shift in V1/2 was observed in control myocytes following SH-6 application (Figure 5A and supplemental Table I).
To evaluate the contribution of PTEN deficiency-induced increases in ICa,L to myocyte contractile function, the effects of PKB inhibition on myocyte shortening were examined. Treatment with SH-6 led to 18% reduction (P<0.05) of cell-shortening amplitude in PTEN–/– myocytes (Figure 5C), while having no significant effects on control myocytes (data not shown). These results establish that increases in ICa,L induced by PTEN ablation can have positive inotropic effects.
Our results establish a novel regulation of cardiac ICa,L by PI3K and PKB. Because PI3K and PKB mediate the actions of IGF-1,19,20 a hormone essential for cardiac development, morphology, and function,12,13 we examined the effects of IGF-1 on ICa,L. Superfusion of IGF-1 (0.1 μmol/L) caused an enhancement of ICa,L in littermate control myocytes that required &1 to 2 minutes to fully develop (Figure 6A). After 90 seconds of application, IGF-1 increased (P<0.001) ICa,L by 17% in littermate control myocytes without affecting ICa,L in DN-p110 myocytes (Figure 6B). By contrast, IGF-1 increased ICa,L similarly in PI3K–/– myocytes and littermate control myocytes (Figure 6C). IGF-1 failed to enhance ICa,L in PTEN–/– myocytes although it increased ICa,L in littermate control myocytes (Figure 6D) to similar levels observed in PTEN–/– myocytes. This increase in ICa,L could be prevented by treatment with SH-6, suggesting that PKB activation is required for the IGF-1-induced increase in ICa,L (Figure 6E). These results establish that IGF-1 enhances ICa,L in mouse hearts via activation of PI3K/PKB pathway, and this pathway is fully stimulated in PTEN-deficient hearts.
Discussion
Our study demonstrates that myocytes lacking the PTEN gene have elevated cardiac L-type Ca2+ currents accompanied by negative shifts in voltage-dependent channel activation, which were unrelated to incomplete voltage control arising from uncompensated RS. Our results further establish that the elevations in ICa,L observed in PTEN-deficient hearts occur without increases in channel expression but are eliminated by PI3K or PKB inhibitors and by dominant-negative PI3K suppression. These findings suggest that ICa,L enhancements and voltage shifts of ICa,L activation in PTEN-deficient myocytes result from elevated PI(3,4,5)P3 levels leading to tonic PKB activation and phosphorylation of target proteins involved in L-type Ca2+ channel regulation. These results are consistent with the conclusion that the dominant physiological function of PTEN is the breakdown of the PI3K-generated second messenger PI(3,4,5)P3 to PI(4,5)P2.21 The increases in ICa,L observed in PTEN-deficient myocytes are similar to increases in ICa,L density (without changes in 1C expression) observed in mouse ventricular myocytes overexpressing PKB.22 This conclusion also agrees with previous studies in vascular smooth muscle cells showing that dialysis with anti-PI(3,4,5)P3 antibodies abolished the angiotensin II-induced and PI3K-mediated stimulation of L-type Ca2+ current.23 Increases in ICa,L without change in 1C expression observed in PTEN-deficient hearts contrasts with results showing that, in COS-7 cells and neurons transfected with PI3K, PKB activation increases Cav2.2 membrane expression as a result of 2a subunit phosphorylation by PKB.24
Although cardiac ICa,L was increased in PTEN-deficient mice, it was not different in myocytes with inhibition of cardiac PI3K or with disruption of PI3K compared with control mice. Nor was ICa,L affected by treatment with PI3Ks or PKB inhibitors in control mice. Collectively, these results suggest that basal PKB activity in subcellular compartments containing L-type Ca2+ channels is insufficient to upregulate cardiac ICa,L in myocytes when PTEN phosphatase activity is normal. However, when PTEN phosphatase activity is abolished, the PI3K activity is sufficient to activate PKB and increase ICa,L, consistent with PI3K-dependent myocyte hypertrophy observed in PTEN-deficient mice.9 These observations are similar to those reported previously in vascular smooth muscle cells in which dialysis with antibodies against PI3K or PI3K had no effect on basal ICa,L,25 whereas introduction of various purified class I PI3Ks into myocyte by dialysis led to increased ICa,L.25 The ability of PI3K disruption, but not PI3K disruption, to reverse the elevated ICa,L observed in PTEN-deficient cardiomyocytes suggests that PKB activation occurs in microdomains containing L-type Ca2+ channels and PI3K as well as PTEN. This possibility is consistent with the increases in ICa,L observed in response to stimulation of IGF-1 receptors because previous studies established that receptor tyrosine kinase stimulation activates PKB via PI3K.19,20 By contrast, PI3K does not contribute significantly to PKB activation under basal conditions,9 although it can also activate PKB directly following G-protein-coupled receptor stimulation26 or via Ras activation27 (possibly by indirect transactivation of receptor tyrosine kinases).28
The regulation pattern of ICa,L by PI3K (via PKB activation) versus PI3K parallels the clear separation of myocardial growth regulation by PI3K versus contractility regulation by PI3K.7,9,14,20 Because ICa,L is a key determinant of cardiac contractility, it is somewhat surprising that PKB-dependent increases of ICa,L occur in PTEN-deficient myocytes, which have reduced contractility.9 Although basis for the reduced contractility in PTEN–/– myocytes remains to be determined, it is likely to depend complexly on interactions between increased PI3K- and increased PI3K-mediated signaling. For example, increased contractility in PI3K–/– myocytes has been linked to increased sarcoplasmic reticulum (SR) Ca2+ loads (without alteration in ICa,L) as a result of compartmentalized subcellular elevations of cAMP.29 Therefore, decreased cAMP levels observed in PTEN–/– hearts9 as a result of altered PI3K-dependent signaling may lead to reduced SR Ca2+ loads. However, increased expression of -myosin heavy chain9 combined with decreased myofilament Ca2+ sensitivity, possibly as a consequence of PIP3-dependent protein kinase C (PKC) activation,2,30 could also lead to reduced cardiac tension development in PTEN-deficient mice.31 In addition, nonselective PI3K inhibition enhances the ICa,L increases induced by 1-adrenergic stimulation,32 suggesting that the modulation of ICa,L by PI3K can depend complexly on the experimental conditions and the signaling pathways activated.
Although the cause of reduced contractility in myocytes lacking PTEN is unclear, PKB inhibition with SH-6 reduced ICa,L in PTEN-deficient myocytes to control levels and attenuated the magnitude of cell shortening, suggesting elevated ICa,L in PTEN-deficient myocytes enhance myocyte contractility. Interestingly, enhanced contractility (and pressure relaxation) have been observed in hearts from mice with PKB overexpression,22,33 which was associated with increased SERCA2a expression and increased ICa,L (when constitutively active Akt E40K is overexpressed 25-fold22) or with enhanced phospholamban phosphorylation (when Akt targeted to the nucleus is overexpressed 10-fold33). Thus, it is conceivable that, in addition to increased ICa,L, SERCA2a expression or phospholamban phosphorylation might also be altered in PTEN–/– myocytes where phospho-PKB levels are increased &4-fold.9 However, overexpression of PKB in selected cellular compartments may not accurately reproduce the activation pattern and effects of PKB by PI3K when PTEN is deleted. Consistent with this, we did not observe alterations in the relaxation kinetics of Ca2+ transients in PTEN–/– myocytes or following PKB inhibition (data not shown), as observed previously in mice overexpressing PKB.22,33 These observations are not incompatible with previous studies in PKB-overexpressing mice because the acute effects of PKB on phospholamban phosphorylation or SERCA2a activity were not examined.22,33 Regardless, any changes in SERCA2a function combined with ICa,L increases are insufficient to offset the actions of other factor(s) responsible for reduced contractility in the PTEN-deficient hearts. Future investigations are clearly warranted to examine the contribution of SERCA2a and other molecular mechanisms to altered contractility in PTEN–/– hearts.
Our results also establish that IGF-1, like the loss of PTEN, increases ICa,L in mouse ventricle via activation of PI3K/PKB signaling pathway, consistent with the notion that tyrosine kinase receptors signal via PI3K activation.1 These increases in ICa,L by IGF-1 has important implications because IGF-1 regulates cardiac remodeling and contractility in patients with cardiomyopathy,34 heart failure12,13 as well as in compensatory hypertrophy during pressure-overload.35 Previous studies have established that ICa,L is critical for induction of myocardial hypertrophy11,36–38 and that Ca2+ is an essential cofactor for many hypertrophy signaling pathways including PKC, mitogen-activated protein kinases (MAPKs), and calcineurin.39,40 Thus, because cardiac growth and hypertrophy are strongly regulated by PI3K/PKB signaling pathways,8,20,39 increase in ICa,L may play a role in the hypertrophic effects of IGF-1 or PTEN ablation. Consistent with this possibility, ICa,L inhibition blocks hypertrophy induced by IGF-1 in cultured cardiomyocytes.41 Increased ICa,L may also contribute to the positive inotropic effects of IGF-1 on myocardium,13,42 a suggestion made previously in myocytes isolated from heart failure patients in which inhibition of ICa,L attenuates the positive inotropic response of IGF-1.43 Indeed, when ICa,L was returned to baseline levels by PKB inhibition, contractility was reduced in our PTEN-deficient myocytes. The contribution of increased ICa,L to cardiac growth and enhanced contractility mediated by IGF-1 and the PTEN/PI3K/PKB signaling pathways will require further investigation.
The molecular mechanism for the PKB-dependent increases of ICa,L in PTEN deficient cardiomyocytes and following IGF-1 treatment is unclear. Increased ICa,L observed in PTEN-deficient myocytes and following IGF-1 treatment was accompanied by negative voltage shifts in the steady-state activation curve along with accelerated rates of channel inactivation. This pattern of change in ICa,L resembles that observed following PKA activation,17 suggesting phosphorylation L-type Ca2+ channel. Examination of the amino acid sequence of mammalian cardiac L-type Ca2+ channel subunits revealed 2 absolutely conserved consensus motifs for PKB phosphorylation19 in 1C subunits (located at S863 in the II-III intracellular linker and at S2015 in the C terminus, mouse sequence) and 1 in the C terminus of the 2a subunit (at S624, mouse sequence). Further studies will be required to unravel the molecular mechanism for the regulation of cardiac ICa,L by PKB.
In summary, we have demonstrated that cardiac L-type Ca2+ channels are regulated in a PKB-dependent manner by PI3K–PTEN signaling pathway. This signaling pathway also mediates IGF-1–induced increase in ICa,L in mouse ventricle. This mechanism for ICa,L regulation may play an important role in the regulation of myocardial growth and contractility by PI3K, PTEN, and growth factors in normal and diseased hearts.
Acknowledgments
This work was funded by a Canadian Institute of Health Research (CIHR) operating grant (MOP 43947 to P.H.B.). P.H.B. is a Career Investigator of the Heart & Stroke Foundation of Ontario. H.S. is supported by a Diana Meltzer Abramsky Research Fellowship from the Thyroid Foundation of Canada. B.-G.K. and M.G.T. are supported by fellowships from the Heart & Stroke Foundation of Canada and TACTICS-CIHR). G.Y.O. was supported by fellowships from CIHR and TACTICS-CIHR. J.M.P. is supported by grants from the Austrian National Bank and EuGeneHeart (6th European Commission Framework Programme).
Footnotes
Both authors contributed equally to this study.
Original received January 28, 2005; resubmission received March 9, 2006; revised resubmission received April 5, 2006; accepted April 7, 2006.
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