Calmodulin Kinase II Inhibition Shortens Action Potential Duration by Upregulation of K+ Currents
The multifunctional Ca2+/calmodulin-dependent protein kinase II (CaMKII) is activated by elevated intracellular Ca2+ (Ca2+i), and mice with chronic myocardial CaMKII inhibition (Inh) resulting from transgenic expression of a CaMKII inhibitory peptide (AC3-I) unexpectedly showed action potential duration (APD) shortening. Inh mice exhibit increased L-type Ca2+ current (ICa), because of upregulation of protein kinase A (PKA) activity, and decreased CaMKII-dependent phosphorylation of phospholamban (PLN). We hypothesized that CaMKII is a molecular signal linking Ca2+i to repolarization. Whole cell voltage-clamp recordings revealed that the fast transient outward current (Ito,f) and the inward rectifier current (IK1) were selectively upregulated in Inh, compared with wild-type (WT) and transgenic control, mice. Breeding Inh mice with mice lacking PLN returned Ito,f and IK1 to control levels and equalized the APD and QT intervals in Inh mice to control and WT levels. Dialysis of AC3-I into WT cells did not result in increased Ito,f or IK1, suggesting that enhanced cardiac repolarization in Inh mice is an adaptive response to chronic CaMKII inhibition rather than an acute effect of reduced CaMKII activity. Increasing PKA activity, by cell dialysis with cAMP, or inhibition of PKA did not affect IK1 in WT cells. Dialysis of WT cells with cAMP also reduced Ito,f, suggesting that PKA upregulation does not increase repolarizing K+ currents in Inh mice. These findings provide novel in vivo and cellular evidence that CaMKII links Ca2+i to cardiac repolarization and suggest that PLN may be a critical CaMKII target for feedback regulation of APD in ventricular myocytes.
Cardiac contraction depends on a tightly orchestrated interplay between action potential duration (APD) and intracellular Ca2+ (Ca2+i). Increases in APD cause increases in Ca2+i, leading to strengthened contraction.1 Changes in Ca2+i, however, are also a source of feedback control for ionic currents that ultimately determine APD.2 The mechanisms for feedback regulation of APD by Ca2+i remain to be elucidated, but we hypothesized that Ca2+i-activated signaling molecules may be important for regulating APD.
The multifunctional Ca2+/calmodulin-dependent protein kinase II (CaMKII) is a serine/threonine kinase that is abundant in heart and targets key Ca2+i homeostatic proteins.3 We studied mice with chronic cardiomyocyte-delimited CaMKII inhibition (Inh) by transgenic expression of a specific CaMKII inhibitory peptide (AC3-I). In addition to wild-type littermates (WT), we also used transgenic control mice (Con) with myocardial expression of an inactive, scrambled version of AC3-I (AC3-C). Inh mice have shortened optically recorded APDs, despite an increased inward L-type Ca2+ current (ICa) that results from increased activity of protein kinase A (PKA).4 Because increased inward ICa, unopposed by upregulation of other repolarizing currents, would cause APD lengthening rather than APD shortening, we hypothesized that CaMKII inhibition also increases repolarizing K+ currents.
Most Ca2+i for each heart beat is released from intracellular sarcoplasmic reticulum (SR) Ca2+ stores.5 Overexpression of a SR Ca2+ ATPase (SERCA) results in reductions in repolarizing K+ currents and APD lengthening.2 On the other hand, Inh mice have reduced SR Ca2+ content and APD abbreviation compared with Con or WT mice.4 These 2 findings suggested the hypothesis that SR Ca2+ uptake is important for feedback regulation of APD. To test the potential role of CaMKII in this mechanism, we bred Inh (and Con) mice with mice null for phospholamban (PLN−/−),6 a negative regulatory protein for SERCA and a phosphorylation target for CaMKII.7
Recordings from ventricular myocytes isolated from Inh mice revealed significant upregulation of 2 repolarizing K+ currents: the fast component of the transient outward current (Ito,f) and the inward rectifier current (IK1), whereas other K+ currents were unchanged compared with Con or WT cells. Increased Ito,f and IK1 appear to represent an adaptive response to chronic myocardial CaMKII inhibition and not an acute consequence of CaMKII inhibition, because neither Ito,f nor IK1 was increased in WT cells dialyzed with AC3-I. Furthermore, increased Ito,f and IK1 were not reversed by cell dialysis with a PKA inhibitory peptide in Inh cardiomyocytes, suggesting increased PKA activity is not involved in the feedback regulation of APD during CaMKII inhibition. In addition, quantitative real-time PCR (qRT PCR) and quantitative immunoblotting did not reveal correlative changes in the expression levels of several K+ channel subunit genes/proteins, including subunits previously shown to contribute to Ito,f and IK1,8,9 in the ventricles of Inh mice. PLN ablation eliminated the differences in QT interval, APD, Ito,f and IK1 in Inh, Con and WT mice ex vivo and in vivo. These findings reveal that CaMKII is an important signal element for a previously unrecognized SR-dependent mechanism for feedback control of cardiac repolarization.
Materials and Methods
An expanded Materials and Methods section is available in the online data supplement at http://circres.ahajournals.org.
Mice With Myocardial CaMKII Inhibition
The AC3-I (Inh) and AC3-C (Con) mice were generated by synthesis of a minigene based on the peptide sequence of AC3-I (KKALHRQEAVDCL) or AC3-C (KKALHAQERVDCL).4 This approach avoids the complications inherent in many previous studies using CaMKII inhibitory drugs that are known to also act as CaMKII-independent K+ current antagonists.10 For some experiments, PLN-null mice (PLN−/−) were bred with AC3-I and AC3-C mice for >4 generations.6,11
ECGs were recorded in vivo or ex vivo from Langendorff-perfused hearts.12 Total outward K+ currents, IK1, and Ito,f were recorded from isolated ventricular myocytes using previously described methods.12 Reagents for manipulating CaMKII and PKA activity were included in the pipette solution for some experiments: cAMP (1 mmol/L); protein kinase A inhibitory peptide (PKI) (50 μmol/L); CaMKII inhibitory peptide AC3-I and the inactive AC3-I congener peptide AC3-C (both at 30 μmol/L).4
K+ Channel Subunit Protein Expression and mRNA Measurements
Western blots and qRT PCR were performed on fractionated lysates prepared from the ventricles of 11- to 13-week-old male and female WT, Con, and Inh mice. Identical analyses were performed on hearts from PLN−/− and PLN−/− mice interbred with Con or Inh mice.
Analysis of Cav1.2 Phosphorylation
Immunosignals for Cav1.2 were detected by chemiluminescence and quantified by densitometry as described.13
Data analysis was performed using SigmaStat. Values are means±SEM. Statistical significance was determined using ANOVA or Student’s t test, as appropriate. Post hoc comparisons were assessed using Bonferroni’s correction for parametric data and Dunn’s test on ranks for nonparametric data. The null hypothesis was rejected for P>0.05.
Abbreviated Ventricular Repolarization in Hearts With Genetic CaMKII Inhibition
The QT interval reflects the duration of ventricular repolarization. Inh mice had significantly shorter QT intervals compared with WT and Con mice in vivo and ex vivo (Figure 1). Spontaneous heart rates in isolated, Langendorff-perfused hearts were not different among Inh, WT, or Con hearts (supplemental Figure I), whereas PR intervals were prolonged in Inh hearts (supplementary Figure I), as occurs in vivo.14 The APDs at 90%, 75%, and 50% repolarization were all significantly shorter in Inh than in WT or Con hearts (Figure 1). Thus, in vivo and ex vivo measures are in agreement and show that chronic myocardial inhibition significantly abbreviates repolarization in the ventricular myocardium.
IK1 and Ito,f Are Selectively Increased in Mice With Chronic CaMKII Inhibition
Inh mice have increased ICa,4,6 but an unopposed increase in L-type Ca2+ current (ICa) should prolong the APD; therefore, we hypothesized that the mechanism for APD shortening by CaMKII inhibition was enhancement of repolarizing K+ currents. We measured K+ currents and found that peak outward K+ current densities (at +80 mV) were significantly (P=0.05) higher in Inh (113±7 pA/pF; n=30), compared with WT (97±7 pA/pF; n=26) or Con (91±6 pA/pF; n=24) ventricular myocytes. Densities of IK1 (Figure 2) and Ito,f (Figure 3) were significantly upregulated in Inh, compared with Con and WT, cells. In contrast, other repolarizing K+ currents were similar in Inh, Con, and WT ventricular myocytes (Figure 3). These findings show that with chronic in vivo CaMKII inhibition results in increased outward K+ currents caused by a selective upregulation of 2 critical repolarizing currents and suggest that these currents are important for shortening repolarization in Inh mice.
Increases in IK1 and Ito,f did not appear to be part of an electrical remodeling program related to cardiomyocyte hypertrophy,15 because cell membrane capacitances were similar in WT (145.4±3.5 pF, n=27), Inh (138.6±5.1 pF, n=31), and Con (151.6±3.8 pF, n=27) myocytes, consistent with the similarities in Inh, Con, and WT hearts.4 We used qRT PCR to determine the expression levels of atrial natriuretic factor (ANF) and β-myosin heavy chain (β-MHC) as genetic markers of latent hypertrophy. ANF and β-MHC expression levels were not increased Inh (or Con), compared with WT hearts (supplemental Figure II). Taken together, these data suggest that upregulation of IK1 and Ito,f in Inh mice do not represent coordinate components of a hypertrophic signaling program.
Acute CaMKII Inhibition Does Not Increase IK1 or Ito,f
CaMKII activity is chronically suppressed in adult Inh mice because expression of the CaMKII antagonist peptide is driven by the α-MHC promoter that becomes active near the time of birth.16 However, the previous experiments do not provide insight into whether these changes are a direct result of CaMKII inhibition or, alternatively, reflect an indirect adaptive response to chronic CaMKII inhibition. We dialyzed ventricular myocytes isolated from WT mice with the Inh (AC3-I) peptide under conditions previously shown to prevent CaMKII actions on L-type Ca2+ current.17 Acute (10-minute) exposure of ventricular myocytes to AC3-I failed to increase IK1 or Ito,f compared with WT cells without AC3-I or compared with WT cells dialyzed with the inactive control peptide AC3-C (supplemental Figure III). These findings do not support a connection between acute CaMKII inhibition and increased IK1 or Ito,f and, therefore, suggest that shortened repolarization observed in Inh mice is not directly related to reduced CaMKII-dependent phosphorylation of ion channel proteins.
Increased PKA Activity Directly Affects Cav1.2
Upregulation of ventricular ICa in Inh mice is attributable to enhancement of PKA activity, without a change in expression of the pore-forming α1 Cav1.2 subunit.4 We used a phospho- and site-specific antibody to measure directly the phosphorylation state of α1 at Ser1928,13 a validated site for PKA phosphorylation.18 Western blot analyses revealed that phosphorylation of Cav1.2 at Ser1928 is significantly increased in Inh compared with Con hearts (Figure 4). There are 2 isoforms of α1 1.2: a full-length 240-kDa form and a short form in which the C-terminal ≈300 residues have been cleaved off by the calcium-activated protease calpain.19 Because the short form is predicted to be more active than the long form, we also evaluated the expression levels of the 2 isoforms separately. These experiments revealed no measurable differences in the expression level of either of the 2 Cav1.2 isoforms in Inh, compared with WT or Con, ventricles (Figure 4).
Increased PKA Activity Is Not a General Mechanism for Increasing IK1 and Ito,f
We next asked if enhanced PKA activity was a general compensatory response to chronic CaMKII inhibition and a mechanism for increasing IK1 and Ito,f, similar to the PKA-dependent mechanism for ICa upregulation seen in Inh hearts.4 We dialyzed WT, Inh, and Con cells with the PKA activator cAMP or the PKA inhibitory peptide PKI for 10 minutes. We previously found that cAMP increased peak ICa in WT and Con myocytes to Inh levels, whereas PKI reduced ICa in Inh cells to WT and Con levels.4 In contrast, the increased IK1 in Inh, compared with Con and WT, ventricular myocytes was not consistently affected by dialysis with cAMP or PKI (Figure 5a and 5b), although PKI dialysis for 10 minutes increased peak IK1 recorded at −60 mV more in Inh than in WT or Con cells (Figure 5a). The larger inward component of IK1, recorded at −100 mV, remained greater in Inh compared with WT or Con cells after cAMP (P=0.03) or PKI (P=0.05). These findings are not consistent with the concept that upregulation of PKA activity in Inh cardiomyocytes accounts for increases in IK1 in Inh cardiomyocytes.
Peak Ito,f was reduced significantly (P<0.001) by cAMP in Inh, Con, and WT cells compared with cells dialyzed with PKI (Figure 5c). The pattern of relative increase in peak Ito,f recorded from Inh compared with WT and Con cardiomyocytes was maintained, but the differences among WT, Inh, and Con cells were no longer significantly different after 10 minutes of cAMP (P=0.129) or PKI (P=0.199). These findings suggest that Ito,f is responsive to PKA signaling but are most consistent with a model where PKA signaling reduces, rather than increases, peak Ito,f. Taken together, these findings showed that upregulation of IK1 and Ito,f is not likely caused by increased PKA activity and suggest that PKA upregulation is not a general mechanism for cellular adaptation to chronic CaMKII inhibition.
Phospholamban Is Required for Enhanced Repolarization by CaMKII Inhibition
Inh mice have significantly reduced CaMKII-dependent PLN phosphorylation compared with Con and WT counterparts,4 and recent findings have shown that overexpression of SERCA1a markedly reduces the expression of repolarizing K+ currents in heart.2 To test whether shortened repolarization observed in Inh mice was linked to the SR Ca2+ regulation, we interbred the Inh and Con mice with mice lacking PLN (PLN−/−). PLN is a negative regulator of SR Ca2+ uptake that is phosphorylated by CaMKII. The reduction in SR Ca2+ content seen in Inh ventricular myocytes4 is prevented by PLN ablation, and ventricular myocytes from PLN−/− mice and PLN−/− mice expressing the CaMKII inhibitory peptide AC3-I (Inh×PLN−/−) and expressing the inactive control peptide AC3-C (Con×PLN−/−) have equivalent SR Ca2+ content.6 Thus, if reduced SR Ca2+ content were part of the mechanism for enhanced repolarization in Inh mice, then repolarization should be equivalent in Inh and Con mice after breeding into a PLN−/− background, unless the interbred mice exhibited cardiac hypertrophy. The interbreed mice did not show histological or morphometric evidence of hypertrophy.11 In addition, ANF and β-MHC mRNA were not increased in Inh×PLN−/− compared with WT and Con×PLN−/− hearts (supplemental Figure IV).
CaMKII inhibition–dependent effects on repolarization were prevented by ablation of PLN. Inh×PLN−/− and Con×PLN−/− mice had similar repolarization durations, ex vivo (Figure 6a through 6c) and in vivo (Figure 6d and 6e). In the absence of PLN, chronic CaMKII inhibition did not result in increased IK1 (Figure 6f and 6g) or Ito,f (Figure 6h) compared with PLN−/− or Con×PLN−/− myocytes or compared with WT and Con myocytes (see Figures 2 and 3⇑). PLN ablation, therefore, interrupts the effects of CaMKII inhibition on repolarization, consistent with the concept that the SR is a critical target for the observed remodeling of action potential repolarization by chronic myocardial CaMKII inhibition.
Remodeling of K+ Channel Subunit mRNA and Protein Expression Levels With CaMKII Inhibition and Deletion of PLN
CaMKII activity can regulate transcriptional activity and so a possible mechanism for increased IK1 and Ito,f in Inh mice is upregulation of mRNAs encoding K+ channel proteins. We performed qRT PCR using primer pairs specific for several K+ channel pore-forming subunit genes expressed in adult mouse ventricles (supplemental Table), including KCND2 and KCND3, which encode Kv4.2 and Kv4.3, respectively, and contribute to Ito,f8; and KCNJ2 and KCNJ12, which encode Kir2.1 and Kir2.2, respectively, and contribute to IK1.9 These experiments revealed that the expression levels of KCND2, KCND3, KCNJ2, and KCNJ12, as well as of other K+ channel pore-forming subunit genes, including KCNA5 (Kv1.5) and KCNB1 (Kv2.1), are similar in Inh, Con, and WT ventricles (Figure 7a). Unexpectedly, these experiments also revealed markedly reduced expression of KCNIP2 (which encodes the K+ channel accessory subunit KChIP220) in Inh, compared with WT and Con, ventricles (Figure 7a). Expression levels of another accessory subunit gene, KCNAB1 (which encodes Kvβ1 subunits) were similar in Inh, Con, and WT ventricles. Similar results were obtained in the PLN−/− background (Figure 7b). These findings suggest that the increased IK1 and Ito,f in Inh mice are not attributable to transcriptional upregulation of the genes encoding IK1 and Ito,f channels.
The Kv4.2, Kv4.3, and KChIP2 proteins coassemble to produce Ito,f,8 whereas Kir2.1 is the critical pore-forming subunit for the generation of IK1,9 and increases in these subunits could potentially explain the observed increases in Ito,f and IK1 in Inh myocytes. Western blot analyses, however, revealed that the expression levels of Kv4.2, Kv4.3, and Kir2.1, as well as of other K+ channel subunits present in adult mouse ventricles, including Kv1.5, Kv2.1, Kir2.2, and Kvβ1, were similar in Inh, Con, and WT ventricles (Figure 8a through 8e). PLN ablation did not measurably affect the relative expression levels of these channel subunit proteins (Figure 8f). These experiments also revealed small, but statistically significant (P<0.05) differences in KChIP2 protein expression in Con, compared with WT and Inh, ventricles (Figure 8c and 8e), as well as in the ventricles of Inh and Con compared with WT (P<0.05) mice lacking PLN (Figure 8f).
CaMKII Regulates Myocardial Repolarization
This study identifies Ito,f and IK1 as determinants for APD shortening in Inh mice with CaMKII inhibition. CaMKII is activated by Ca2+i and regulates key Ca2+i homeostatic processes including SR Ca2+ cycling.21 Our findings point to a potential connection between SR Ca2+ handling and CaMKII for sculpting action potential repolarization, because PLN ablation eliminated the effects of CaMKII inhibition on APD, Ito,f, and IK1. CaMKII activity is already known to be increased by the elevation in Ca2+i caused by APD prolongation,10 and the present findings suggest that CaMKII can also have a role as a regulatory feedback signal for connecting changes in Ca2+i to cardiac repolarization.
Model of Chronic In Vivo CaMKII Inhibition
One surprising finding was that upregulation of Ito,f and IK1 did not appear to be caused by the acute effects of CaMKII-dependent phosphorylation, because neither current was increased by cellular dialysis with AC3-I. The finding that increases in Ito,f and IK1 were not recapitulated by AC3-I dialysis suggested that cellular responses to chronic CaMKII inhibition are different from those to acute suppression of CaMKII activity. CaMKII inhibition by chronic inhibitory drug therapy or by cardiomyocyte-delimited AC3-I expression confers resistance to myocardial infarction, catecholamine toxicity,4apoptosis,11 and myocardial dysfunction and arrhythmias in calcineurin cardiomyopathy22; therefore, insight into the mechanisms of cellular compensation to chronic CaMKII inhibition could be important for developing future therapies.
CaMKII can directly increase ICa by increasing the opening probability of L-type Ca2+ channels17; therefore, we initially anticipated that ICa would be less in Inh than in Con or WT mice. However, Inh mice have ICa upregulation by increased PKA activity.4 Here we show direct evidence for enhanced phosphorylation of Cav1.2 at Ser1928, but our experiments using cellular dialysis with cAMP and PKA failed to yield evidence that PKA activity contributes to increased Ito,f or IK1 in Inh mice. PKA is typically associated with reduction in Ito,f,23 and cAMP reduced and PKI increased Ito,f, consistent with these reports. Our findings do not suggest that increased PKA activity is regulating Ito,f in Inh mice, because Ito,f is greater in Inh than in Con or WT animals. On the other hand, IK1 has been reported to increase24 and decrease25 in response to PKA stimulation. Cell dialysis with cAMP or PKI failed to increase IK1 in Inh, Con, or WT cells, suggesting that PKA was not a critical determinant of IK1 under our experimental conditions. Overall, our findings do not support a model in which increased PKA activity is important in the upregulation of Ito,f or IK1 in Inh mice.
SR Ca2+ Feedback Regulation of Repolarizing K+ Currents
It is now established that prolongation of repolarization can elevate Ca2+i26 that contributes to increased cellular contraction1 and arrhythmias.27 In some studies, increased APD results in hypertrophy,28 but hypertrophy is not present in other mouse models with action potential prolongation.29 Recent evidence also supports the concept that “retrograde” signaling from SR Ca2+ to ionic currents is important for regulating K+ channel synthesis and regulating APD.2 Our results clearly reveal that targeting CaMKII activity selectively affects the functional expression of repolarizing cardiac K+ currents by a mechanism that requires PLN, without measurably affecting the expression levels of several subunit proteins that contribute to the formation of these channels.
Transgenic mice with enhanced myocardial SR Ca2+ uptake caused by overexpression of a skeletal muscle isoform of SERCA (SERCA1a) show APD prolongation and reduced expression of proteins underlying Ito,f (KChiP2, Kv4.2, Kv4.3) and IK,slow (Kv2.1 and Kv1.5).2 Inh mice were previously shown to have reduced SR Ca2+ content, equivalent spark frequency, and markedly diminished PLN phosphorylation at Thr17 compared with Con mice.4,6 The increases in Ito,f and IK1 in Inh mice are reversed to baseline by PLN ablation, a maneuver that eliminates CaMKII inhibition mediated reduction in SR Ca2+ content,6 suggesting that PLN and SR Ca2+ are key components connecting CaMKII-dependent alterations in K+ currents to the physiology of myocardial repolarization. Taken together, these findings in Inh and SERCA1a-overexpressing mice point to an important relationship between SR Ca2+i uptake and action potential repolarization.
The mechanisms underlying the alterations in K+ current densities in the Inh and SERCA1a overexpressing models are different. SERCA1a overexpressing mice have increased SR Ca2+ content and QT interval prolongation,2 whereas Inh mice have reduced SR Ca2+4 and show QT and APD shortening caused by increases in Ito,f and IK1. SERCA1a-overexpressing mice show reduced Ito,f and reduced slow (IK,slow) and steady-state (Iss) currents, whereas IK1 was not reported.2 The different IK,slow responses in Inh and SERCA1a-overexpressing mice may indicate that individual repolarizing currents couple to distinct components of SR Ca2+ uptake regulation (eg, SERCA versus PLN). Although the feedback mechanisms linking SR Ca2+ content and cardiac repolarization remain incompletely understood in SERCA1a-overexpressing and Inh mice, neither model exhibited signs of cardiac hypertrophy. Thus, the feedback relationship between SR Ca2+ and cardiac repolarization seems to be operative even in the absence of underlying cardiac disease. There are similarities but also substantial differences in the molecular mechanisms for repolarization between mice and larger mammals, including humans.30 An important future direction will be to determine whether feedback mechanisms among CaMKII, SR Ca2+ reuptake, and myocardial repolarization are similar in mice and in larger mammals.
CaMKII Is a Dual Signal for Cardiac Hypertrophy and Electrical Remodeling
APD prolongation is a stereotyped “electrical remodeling” response to structural heart disease from diverse causes that may contribute to arrhythmias in animal models and in patients with cardiac hypertrophy and heart failure.15 The APD shortening in Inh mice suggests the possibility of a “reverse electrical remodeling” response to chronic CaMKII inhibition. Acute CaMKII inhibition can reduce arrhythmias in the setting of electrical remodeling and cardiac hypertrophy, but without changing APD.31 On the other hand, chronic CaMKII inhibition can improve myocardial function, reduce mortality, and suppress ventricular arrhythmias in mice with calcineurin cardiomyopathy,22 suggesting that distinct mechanisms control antiarrhythmic properties of acute and chronic CaMKII inhibition. Myocardial responses to chronic CaMKII inhibition are complex but are apparently adaptive and the present findings establish that they include a reordering of repolarization that causes APD shortening by a mechanism that requires PLN.
The PLN−/− mice were a generous gift from Dr E. G. Kranias.
Sources of Funding
We acknowledge financial support from the NIH (grants HL070250, HL062494, and HL046681 to M.E.A.; HL034161 and HL066388 to J.M.N.; and NS035563 to J.W.H.) and the American Heart Association. M.E.A. is an Established Investigator of the American Heart Association, and C.M. is the recipient of a Postdoctoral Fellowship from the American Heart Association, Heartland Affiliate.
Original received August 19, 2005; resubmission received August 23, 2006; revised resubmission received September 28, 2006; accepted September 28, 2006.
Bouchard RA, Clark RB, Giles WR. Effects of action potential duration on excitation-contraction coupling in rat ventricular myocytes. Action potential voltage-clamp measurements. Circ Res. 1995; 76: 790–801.
Zhang R, Khoo MS, Wu Y, Yang Y, Grueter CE, Ni G, Price EE, Thiel W, Guatimosim S, Song LS, Madu EC, Shah AN, Vishnivetskaya TA, Atkinson JB, Gurevich VV, Salama G, Lederer WJ, Colbran RJ, Anderson ME. Calmodulin kinase II inhibition protects against structural heart disease. Nat Med. 2005; 11: 409–417.
Guo W, Li H. Aimond F, Johns DC, Rhodes KJ, Trimmer JS, Nerbonne JM. Role of heteromultimers in the generation of myocardial transient outward K+ currents. Circ Res. 2002; 90: 586–593.
Anderson ME, Braun AP, Wu Y, Lu T, Schulman H, Sung RJ. KN-93, an inhibitor of multifunctional Ca++/calmodulin-dependent protein kinase, decreases early afterdepolarizations in rabbit heart. J Pharm Exp Ther. 1998; 287: 996–1006.
Yang Y, Zhu WZ, Joiner ML, Zhang R, Oddis CV, Hou Y, Yang J, Price EE, Gleaves L, Eren M, Ni G, Vaughan DE, Xiao RP, Anderson ME. Calmodulin Kinase II Inhibition Protects Against Myocardial Cell Apoptosis in vivo. Am J Physiol Heart Circ Physiol. In press.
Li J, McLerie M, Lopatin AN. Transgenic upregulation of IK1 in the mouse heart leads to multiple abnormalities of cardiac excitability. Am J Physiol Heart Circ Physiol. 2004; 287: H2790–H2802.
Davare MA, Hell JW. Increased phosphorylation of the neuronal L-type Ca(2+) channel Ca(v) 1.2 during aging. Proc Natl Acad Sci U S A. 2003; 100: 16018–16023.
Tomaselli GF, Zipes DP. What causes sudden death in heart failure? Circ Res. 2004; 95: 754–763.
Subramaniam A, Jones WK, Gulick J, Wert S, Neumann J, Robbins J. Tissue-specific regulation of the alpha-myosin heavy chain gene promoter in transgenic mice. J Biol Chem. 1991; 266: 24613–24620.
De Jongh KS, Murphy BJ, Colvin AA, Hell JW, Takahashi M, Catterall WA. Specific phosphorylation of a site in the full-length form of the alpha 1 subunit of the cardiac L-type calcium channel by adenosine 3′,5′-cyclic monophosphate-dependent protein kinase. Biochemistry. 1996; 35: 10392–10402.
Hell JW, Westenbroek RE, Breeze LJ, Wang KK, Chavkin C, Catterall WA. N-methyl-D-aspartate receptor-induced proteolytic conversion of postsynaptic class C L-type calcium channels in hippocampal neurons. Proc Natl Acad Sci U S A. 1996; 93: 3362–3367.
Khoo MS, Li J, Singh MV, Yang Y, Kannankeril P, Wu Y, Grueter CE, Guan X, Oddis CV, Zhang R, Mendes L, Ni G, Madu EC, Yang J, Bass M, Gomez RJ, Wadzinski BE, Olson EN, Colbran RJ, Anderson ME. Death, cardiac dysfunction, and arrhythmias are increased by calmodulin kinase II in calcineurin cardiomyopathy. Circulation. 2006; 114: 1352–1359.
Gallego M, Setien R, Puebla L, Boyano-Adanez MC, Arilla E, Casis O. alpha1-Adrenoceptors stimulate a Galphas protein and reduce the transient outward K+ current via a cAMP/PKA-mediated pathway in the rat heart. Am J Physiol Cell Physiol. 2005; 288: C577–C585.
Zitron E, Kiesecker C, Luck S, Kathofer S, Thomas D, Kreye VA, Kiehn J, Katus HA, Schoels W, Karle CA. Human cardiac inwardly rectifying current IKir2.2 is upregulated by activation of protein kinase A. Cardiovasc Res. 2004; 63: 520–527.
Wischmeyer E, Karschin A. Receptor stimulation causes slow inhibition of IRK1 inwardly rectifying K+ channels by direct protein kinase A-mediated phosphorylation. Proc Natl Acad Sci U S A. 1996; 93: 5819–5823.
Wu Y, Roden DM, Anderson ME. Calmodulin kinase inhibition prevents development of the arrhythmogenic transient inward current. Circ Res. 1999; 84: 906–912.
Kassiri Z, Zobel C, Nguyen TT, Molkentin JD, Backx PH. Reduction of I(to) causes hypertrophy in neonatal rat ventricular myocytes. Circ Res. 2002; 90: 578–585.
Nerbonne JM, Nichols CG, Schwarz TL, Escande D. Genetic manipulation of cardiac K(+) channel function in mice: what have we learned, and where do we go from here? Circ Res. 2001; 89: 944–956.
Wu Y, Temple J, Zhang R, Dzhura I, Zhang W, Trimble RW, Roden DM, Passier R, Olson EN, Colbran RJ, Anderson ME. Calmodulin kinase II and arrhythmias in a mouse model of cardiac hypertrophy. Circulation. 2002; 106: 1288–1293.