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Cocaine Activates Calcium/Calmodulin Kinase II and Causes Cardiomyocyte Hypertrophy

Henning, Robert J MD*; Cuevas, Javier PhDwith the assistance of Douglas Ivancsits, BS and Anthony Sanchez, MPH

Journal of Cardiovascular Pharmacology: July 2006 - Volume 48 - Issue 1 - p 802-813
doi: 10.1097/01.fjc.0000211796.45281.46
Original Article
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Cardiac hypertrophy occurs in as many as 47% of normotensive individuals who chronically use cocaine. We investigated the effects of cocaine, in concentrations commonly found in chronic cocaine users, on calcium/calmodulin kinase (CaMK), and whether cocaine can activate CaMK, increase cardiac myocyte protein expression, and cause cardiac hypertrophy in this manner. In series I to III, 0 (control) or cocaine in concentrations of 108 to 105 mol/L was added to cultured adult rat cardiac ventricular myocytes to determine by Western blots and by 32P incorporation the optimal treatment time and the optimal dose for CaMK activation. In series I, cocaine, 106 mol/L, increased myocyte CaMKII translocation from myocyte soluble to particulate fractions by ≥73 ± 9% (P < 0.01) in comparison with controls but did not cause the translocation of CaMKI or CaMKIV. In series II and III, cocaine treatment of myocytes for 15 minutes increased maximal CaMKII activity by 86.5 ± 13.3% (P < 0.001) and a cocaine dose of 5×106 mol/L increased CaMKII activity by 169.5 ± 18.1% (P < 0.001). In series IV we measured by silver staining β-myosin heavy chain protein (β-MHC) expression in myocytes before and after cocaine and also CaMK inhibition with KN-62 (1-[N,O-bis-(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine). In these experiments, cocaine, 5×106 mol/L, increased myocyte protein concentration by 29.2 ± 2.8%, and β-MHC by 93.2 ± 8.8% (P < 0.001). In series V and VI, cocaine effects on calcium currents (ICa) and intracellular Ca2+ ([Ca2+]i) were determined before and after CaMK inhibition with KN-62 in rat myocytes. Cocaine, 10−6 mol/L, enhanced ICa peak amplitude in a voltage-dependent manner (by 173.9 ± 14.9% at −20 mV and by 38.4 ± 6.9% at 0 mV P < 0.01). Cocaine, 106 to 105 mol/L, in series VI promoted Ca2+ transients from myocyte sarcoplasmic reticulum and increased [Ca2+]i to 607 ± 141 × 109 mol/L (P < 0.05). KN-62 decreased cocaine-induced myocyte protein expression by 76.6%, and β-MHC by 66.2% (P < 0.01) and significantly decreased cocaine-induced Ca2+ transients and [Ca2+]i. We conclude that CaMKII activation is an important mechanism whereby cocaine can cause myocyte hypertrophy.

From the *Departments of Medicine and †Molecular Pharmacology and Physiology, University of South Florida College of Medicine and the James A. Haley VA Hospital, Tampa, Florida.

Received for publication November 29, 2005; accepted May 24, 2006.

Reprints: Robert J. Henning, MD, University of South Florida College of Medicine/James A. Haley VA Hospital, 13000 Bruce B. Downs Boulevard, 111, Tampa, FL 33612 (e-mail: rhenning@hsc.usf.edu).

Thirty million Americans have used cocaine at least once, and approximately 5 million Americans use cocaine on a regular basis. Cocaine can cause, in these individuals, acute myocardial ischemia and infarction, cardiomyopathy, cardiac arrhythmias, and sudden death.1 Recently, cocaine has been found to produce cardiac ventricular hypertrophy, which can occur in as many as 47% of normotensive individuals, especially blacks, who chronically use cocaine.2,3

We have previously reported that cocaine directly increases adult cardiomyocyte protein expression and specifically increases the expression of β-myosin heavy chain protein (β-MHC) in cardiomyocytes.4,5 Cocaine can directly cause cardiac hypertrophy in this manner. However, the signaling pathways by which this hypertrophy occurs are not known. Mitogen-activated protein kinases (MAPK) are 1 group of kinases that modify transcription factors and gene transcription and can contribute to cardiac hypertrophy. In this regard, we have examined the effects of cocaine on MAPK including ERK (extracellular signal regulated kinase), SAPK/JNK (stress-activated protein kinase/Jun N-terminal kinase), and p38 and found that cocaine only slightly increases the phosphorylation of ERK.5

Increased intracellular calcium also regulates cardiomyocyte gene expression and growth and can lead to cardiac hypertrophy and heart failure. Studies have shown that prolongations in L-type calcium channel opening and/or calcium release from the sarcoplasmic reticulum ryanodine receptors increase intracellular calcium and can ultimately cause cardiomyocyte hypertrophy.6,7 Activation of calcium/calmodulin kinase (CaMK) I, II, and IV can increase intracellular calcium and may be associated with cardiomyocyte hypertrophy. In this regard, CaMKII predominates in normal hearts.8 CaMKII can regulate intracellular calcium in cardiomyocytes and can cause ventricular hypertrophy by a signaling pathway that is separate from the MAPK pathways. In the cytosol, CaMKII modulates L-type calcium channels and calcium currents (ICa) and therefore can alter intracellular calcium.9-11 CaMKII also phosphorylates the sarcoplasmic reticulum ryanodine receptors thereby increasing their open probability.12-14 This causes an increase in calcium transients and an elevation in intracellular calcium. These CaMKII-induced alterations in cardiomyocyte calcium handling can contribute to cardiac hypertrophy and ultimately to heart failure.

CaMKI can induce hypertrophic responses in cardiomyocytes by increasing α-actin and atrial natriuretic peptide formation.15 However, CaMKI has only 2.5% to 20% of the activity of CaMKII and does not significantly change during pressure overload induced cardiac ventricular hypertrophy in mice.8 In contrast, CaMKIV can produce cardiomyocyte hypertrophy when activated by Leukemia Inhibitor Factor in vitro or when overexpressed in transgenic mice.15,16 On the basis of these CaMK investigations, we determined whether cocaine can activate calmodulin kinase I, II, or IV and increase calcium-mediated signaling events in adult ventricular myocytes, and in this manner increase cardiomyocyte protein expression and hypertrophy.

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MATERIALS AND METHODS

Isolation of Adult Rat Cardiomyocytes

Ventricular cardiomyocytes were isolated from 250 to 300g male, Sprague-Dawley rat hearts (Harlan) by methods that we have previously described.4,5 All experiments conformed to the guidelines established by the National Institutes of Health for animal care and were approved by the animal care and use committee of our medical center. The cardiomyocytes were cultured at a density of 2.0×106 cardiomyocytes/15 mL Dulbecco's modified Eagle's medium with 1% penicillin/streptomycin in suspension cultures by techniques previously described.4,5,17,18 The cardiomyocyte cultures were incubated at 37°C in humidified air with 5% CO2 for 1 to 7 days depending on each series of experiments.

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Series I: Expression of CaMK by Western Blotting

In 5 series I experiments, cocaine, 0 (control) or 106 mol/L, was added for 1, 10, 30, 60, 240, 480, or 1440 minutes to cardiomyocytes previously cultured for 24 hours. We determined the optimal time by Western blot techniques for CaMKI, II, and IV translocation from soluble to particulate cardiomyocyte fractions. In 5 subsequent experiments, cocaine-CaMK dose-response determinations were performed to determine the cocaine dose that produced the largest translocation of CaMK I, II, or IV. The cocaine doses of 105 to 108 mol/L were based on cocaine concentrations commonly found in hospitalized chronic cocaine users.1,4,5 The cardiomyocytes in these experiments were cultured for 24 hours and were then treated with either vehicle or cocaine for 6 hours.

The cardiomyocytes were lysed with lysis buffer [20 mM N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES), 1 mM dithiothreitol, 10 μg/μL leupeptin, 10 μg/μL aprotinin, 50 mM β-glycerophosphate, 1 mM Na3VO4, 10% glycerol with 0.4 mM phenylmethylsulfonyl fluoride, and 300 nM microcystin-RR, pH 7.4].19 The lysate was centrifuged at 40,000 rpm for 90 minutes. The supernatant was saved as the soluble fraction. The pellet was resuspended in lysis buffer plus 1% Triton X-100 and recentrifuged at 40,000 rpm for 90 minutes, and the supernatant was saved as the particulate fraction. Soluble and particulate total protein concentrations were determined by the Bradford method (BioRad). Fifty microgram protein samples were then separated by SDS-PAGE on 10% Tris-HCl Ready-Gels (BioRad) and then transblotted onto polyvinylidene fluoride membranes (BioRad). Each membrane was blocked with 5% nonfat dry milk then incubated with rabbit monoclonal antibodies to CaMKI antibody, 1:300 dilution (Affinity BioReagent) for 12 hours at 4°C or CaMKII antibody, 1:150 dilution (BD Biosciences) for 12 hours at 4°C and then secondary goat antimouse IgG horseradish peroxidase conjugated antibody 1:3000 dilution for CaMKI or 1:3000 dilution for CaMKII for 1 hour. For CaMKIV determinations, the membranes were incubated for 12 hours at 4°C with an anti-CaMKIV antibody, 1:300 dilution (Affinity Bioreagents), or with an anti-CaMKIV antibody, 1:1500 dilution, generously provided by Gary Wayman, PhD (Oregon Health Sciences Center), for 12 hours at 4°C and then with secondary IgG horseradish peroxidase conjugated antibody (1:3000) for 1 hour. The membranes were subjected to an Enhanced Chemiluminescence Detection Kit (Amersham) and exposed on radiographic film (Amersham). Protein bands were then quantified with the use of a densitometer (UVP Gel Documentation).

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Series II to III: Radioisotopic CAMK Activity Determinations

To more precisely quantitate the effects of cocaine on CaMK, time and dose-response determinations were performed using radioisotopic techniques for CaMKII and CaMKIV. CaMKII and CaMKIV activity was determined using cardiomyocyte lysates and lysates selectively immunoprecipitated for CaMKII and IV.16,20 Cardiomyocyte lysate assays were performed to determine whether the data were concordant with the immunoprecipitate assays which are specific for CaMKII and CaMKIV.

Series II examined the time dependence of the effect of cocaine on the CaMK response. Cardiomyocytes were incubated in humidified air with 5% CO2 for 24 hours then treated with cocaine 106 mol/L for 5 minutes (Lysate L=4 experiments, Immunoprecipitate I=4 experiments), 10 minutes (L=4, I=4), 15 minutes (L=5, I=4), 20 minutes (L=2, I=2), 30 minutes (L=8, I=10), or 1080 minutes (L=8, I=6) for CaMKII responses.

Eight separate experiments were also performed with CaMKIV with cardiac myocyte lysate and immunoprecipitates at the same time intervals. Anti-CaMKII or IV antibodies (BD Biosciences) or mouse monoclonal anti-CaMKIV antibodies (Gary Wayman, PhD, Oregon Health Sciences Center) were used for the immunoprecipitation experiments. For the CaMK determinations, the synthetic CaMKII substrate autocamtide-2 (Sigma), and the synthetic CaMKIV peptide-γ substrate (BioMol) were used.16,20,21 In addition, 6 separate experiments were performed with cardiomyocytes treated with phenylephrine, 106 mol/L, for 5 to 30 minutes to activate CaMK as a positive control for comparison with the cocaine experiments.

In 6 series III experiments, cocaine dose-CaMKII response determinations were performed with myocyte lysates and immunoprecipitates. Cardiomyocyte cultures were incubated for 24 hours then treated with cocaine 108, 107, 5×107, 106, 5×106, or 105 mol/L for 15 minutes. The cocaine incubation time was based on our pilot and series II experiments. Separate cardiomyocyte cultures were also treated initially with the CaMKII antagonist KN-62, 106 mol/L, and then with cocaine, 106 mol/L.22,23 The cardiomyocytes were then lysed with the lysis buffer described in series I. The lysate was centrifuged for 30 minutes at 12,000 rpm at 4°C, and the supernatant was collected. The protein concentrations were determined by the Bradford method.

For the immunoprecipitates, 100 μL of Protein A/G beads (Santa Cruz Biotechnology) was added to each sample tube and centrifuged for 5 minutes at 1000 rpm at 4°C. The pellet was resuspended with an equal volume of lysis buffer 3 separate times. Anti-CaMKII, 1.7 μg (BD Biosciences), was added to each sample tube, which was rotated at 4°C for 1 hour and centrifuged for 5 minutes at 1000 rpm at 4°C. The pellet was resuspended in an equal volume of lysis buffer, centrifuged for 5 minutes, and resuspended in lysis buffer. For each immunoprecipitate measurement, 150 μg of protein was added to each sample tube. For cardiomyocyte lysates, 150 μg of protein was added directly to microcentrifuge tubes. The immunoprecipitate and the lysates samples were rotated overnight at 4°C, centrifuged for 5 minutes at 1000 rpm at 4°C, and washed with phosphate-buffered saline 4 separate times. Cell lysate and immunoprecipitated protein samples were then added to separate mixtures containing 40×106 mol/L adenosine triphosphate (ATP) (Sigma), 6.7×106 mol/L calmodulin (Sigma), 15μg CaMKII substrate autocamtide-2 (Sigma) or CaMKIV substrate peptide-γ, 5μCi [γ-32P]ATP (Amersham), and 5× CaMK assay buffer (20×103 mol/L MOPS, 10×103 mol/L MgCl2, 10×103 mol/L CaCl2, and 103 mol/L DTT).16,20,21

In series II and III, the CaMK reactions occurred in separate tubes at 37°C for 1 hour. Fifty microliters of each sample was transferred onto separate 2.5 cm P81 phosphocellulose absorption papers (Whatman Inc), and washed 5 times for 5 minutes each in a solution of 75×103 mol/L phosphoric acid. All papers were then washed in acetone for 5 minutes. The radioactivity of absorbed 32P from phosphorylated CaMKII or CaMKIV was determined in duplicate samples by liquid scintillation counting (Tracor) and expressed in counts per minute.

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Series IV: Determination of Cocaine-induced β-MHC Expression

Cocaine-induced β-MHC expression was determined in series IV experiments by silver staining.24-26 Cardiomyocytes were cultured for 24 hours and the suspension cultures were then treated with cocaine 5×106 mol/L (n=9), 106 mol/L (n=10), or phenylephrine 106 mol/L (n=3) for 6 days on the basis of our previous experiments.4,5 Separate cardiomyocyte cultures (n=8) were treated initially with the CaMK inhibitor KN-62, 106 mol/L, for 60 minutes, and then cocaine 106 mol/L for 6 days. The culture media and the drug(s) in each myocyte culture were changed every 3 days. The myocytes were then harvested and lysed, and the protein concentrations determined.24-26

Forty micrograms of myocyte protein was loaded onto mini-gels [7.5% acrylamide and 5% (vol/vol) glycerol] and subjected to electrophoresis in a Mini-Protean II apparatus (BioRad).24 After electrophoresis, gels were fixed in 30% acetic acid/10% ethanol for 1 hour then fixed in a 1:10 solution of glutaraldehyde (Sigma) overnight at 4°C. The gels were subjected to silver staining with SilverSNAP II kits (Pierce) and the myosin isoform bands were then quantitated with a densitometer (UVP).

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Series V and VI: Determination of Cocaine-induced Ca2+ Currents and Intracellular Ca2+ ([Ca2+]i)

Acutely isolated cardiomyocytes from 2 to 8 cell isolation experiments were examined in the absence and presence of cocaine or cocaine plus inhibitor. An aliquot of myocytes was placed in a 300 μL open-flow chamber mounted on an inverted Zeiss microscope. The myocytes were superfused continuously at a flow rate of ∼1 mL/min with a solution containing (in 103 mol/L): 140 NaCl, 3 KCL, 2.5 CaCl2, 1.2 MgCl2, 7.7 glucose, 10 HEPES, pH adjusted to 7.4 with NaOH at 22 to 23°C. A solenoid valve switching system (Parker Hannifin) was used to change between reservoirs for the addition of control buffer or cocaine to the microscope chamber. The whole-cell perforated patch configuration of the patch clamp technique was used which prevented dialysis of the cell interior and concomitant loss of second messengers and other small molecules that might otherwise decrease myocyte function and cause “run-down” of Ca2+ currents. Micropipettes were used with tip resistances of 1 to 3 MΩ that were back-filled with (in 103 mol/L): 75 Cs2SO4, 55 CsCl, 5 MgSO4, 10 HEPES, N-methyl-D-glucamine to pH 7.4, and 360 mg/mL amphotericin B in 0.6% DMSO.27 To minimize voltage error, series resistance (RS) was compensated by 75% to ≤2 MΩ. Cells with final RS ≥ 5 μΩ or which exhibited currents indicative of voltage escape were excluded from our study. Membrane responses were amplified and filtered (5kHz) using an Axopatch-200A amplifier (Axon), digitized using a Digidata 1200B (20 kHz), and stored and analyzed in a PC using pClamp 8 programs.

In series V, cardiomyocytes were voltage-clamped and depolarization-activated Ca2+ channel currents were evoked using voltage jumps (250 ms) from −60 mV to more positive potentials. Capacitive and leak currents were subtracted using the P/4 protocol with a −60 mV subsweep holding level and the polarity of the pulses opposite the test potentials. This protocol determines the linear relationship for these currents. Such a linear relationship is predicted under the ionic and pharmacological conditions used here (ie, isolated Ca2+ channel currents).27 The extracellular solution for these experiments consisted of (in 103 mol/L): 70 NaCl, 70 tetraethyl ammonium Cl, 3 KCl, 5 BaCl2, 1.2 MgCl2, 7.7 glucose, 10 HEPES, 0.002 tetrodotoxin (TTX), adjusted to pH 7.4 with NaOH. Isolation was achieved by inactivating Na+ channels (−60 mV holding potential) and blocking the remaining Na+ channels with extracellular TTX (2×106 mol/L). Potassium channels were inhibited with intracellular Cs+, and extracellular TEA (70×103 mol/L). Barium (Ba) was used as the charge carrier to further depress K+ channel currents and effectively isolate ICa.

To compare the effects of cocaine on the voltage dependence of calcium channel activation, the whole-cell conductance was determined by dividing the measured peak current at each test potential by the difference between the membrane potential and the observed reversal potential (−43 mV) for Ba. Whole-cell conductance values were normalized by dividing the peak conductance obtained at each voltage by that obtained at +10 mV, and best fit using a single Boltzmann equation: GBa=a/{1 + exp[−(VVh)/k]}, where GBa is the conductance, a is the asymptotic maximum, Vh is the voltage for half-maximal activation, and k is the slope parameter.

In series VI experiments, intracellular calcium ([Ca2+]i) was quantified using the ratiometric Ca2+ indicator fura-2 (Molecular Probes) by techniques we have previously described.28 These experiments were conducted in quiescent cells in the absence of field stimulation to mimic the conditions used in series I to IV. These recordings permitted the study of cocaine's effect on [Ca2+]i and the relationship between [Ca2+]i and ICa. Cardiomyocytes were loaded with fura 2-acetoxymethyl ester (3×106 mol/L) by incubation at 37°C for 30 minutes. After loading with fura-2, the myocytes were washed and examined for fura-2 loading and uniform fluorescence. Excitation light was applied using a DG-4 (Sutter Instruments) ultra-high speed wavelength switcher/xenon arc lamp (175 W), and emitted fluorescence light was collected with a Cooke SensiCam CCD camera mounted on a Zeiss Axiovert (40×Plan-Apochromat) microscope. Time-lapse image capture and processing was conducted using the SlideBook (Intelligent Imaging) software with the fura-2 ratio imaging module. The system was calibrated using a fura-2 calcium imaging calibration kit (Molecular Probes), and autofluorescence values at each wavelength, collected from the unloaded cardiomyocytes, were also input into the SlideBook Program and adjusted for in the calculation of [Ca2+]i.

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Statistical Analysis

All results are expressed as mean ± standard error of the mean (SEM). Differences between more than 2 groups were tested by analyses of variance. The Bonferroni modified t test was used to test the difference between 2 groups for a priori comparisons and the Scheffe procedure was used for post hoc comparisons. A value of P < 0.05 was judged significant.

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RESULTS

In series I, cocaine, 106 mol/L, was added to cardiac myocyte cultures for 1, 10, 30, 60, 240, 480, or 1440 minutes. Cocaine caused translocation of CaMKII from cardiomyocyte soluble to particulate fractions as determined by Western blots. In these experiments, the cardiomyocyte CamKII particulate/soluble ratio increased by 96 ± 12% after 240 minutes of cocaine treatment (P < 0.004). The CamKII particulate/soluble ratio slightly decreased thereafter but remained significantly greater than the control (Fig. 1A). CaMKI and CaMKIV were detected in the control cardiomyocyte soluble fractions; however, cocaine did not cause translocation of CaMKI or CaMKIV in these experiments.

FIGURE 1

FIGURE 1

Cocaine dose (108 to 105 mol/L)-CaMK response determinations were then performed to determine the cocaine dose that produced the largest CaMK translocation from soluble to particulate fractions. In these experiments, cocaine at 106 mol/L caused the maximal translocation of CaMKII from soluble to particulate fractions and increased the CaMKII particulate/soluble ratio by 73 ± 9% in comparison with controls as shown in Figure 1B. Cocaine, 108 to 105 mol/L, did not, however, increase CaMKI or CaMKIV translocation in these experiments. Because cocaine did not affect CaMKI, which was only weakly detected in the soluble cardiomyocyte fraction, series II experiments focused on CaMKII and IV.

In series II, we quantitated the cocaine-induced activation of CaMKII and the phosphorylation of the CaMKII-specific substrate autocamtide-2 with [γ-32P]ATP measurements using myocyte lysates and also lysates selectively immunoprecipitated for CaMKII. In these experiments, separate myocyte cell cultures were treated with cocaine, 106 mol/L, for 5, 10, 15, 20, 30, or 1080 minutes. With the autocamtide technique, cocaine maximally increased CaMKII activity in myocyte lysates at 15 minutes by 86.5 ± 13.3% (P < 0.001) but also increased CaMKII activity at 5, 10, 30, and 1080 minutes (P < 0.05) as shown in Figure 2A. In comparison, phenylephrine, 10−6 mol/L, maximally increased CaMKII activity by 110.4 ± 15.1% (P < 0.001) in cardiomyocytes (range 93.5% to 110.4% with 5 to 30 min of incubation). There was no significant difference between the cocaine-induced increase in CaMKII activity at 15 minutes and the maximal increase in CaMKII activity due to phenylephrine. Cocaine treatment for 5 to 1080 minutes did not, however, increase CaMKIV activation or phosphorylation of the CaMKIV-specific substrate peptide γ.

FIGURE 2

FIGURE 2

Figure 2B shows the effects of cocaine, 106 mol/L, on CaMKII activity on cardiomyocyte lysates after immunoprecipitation of CaMKII. Separate cardiomyocyte cultures were treated with cocaine for 5 to 1080 minutes. Cocaine increased CaMKII activity at all times and maximally increased CaMKII activity by 68.8 ± 9.5% (P < 0.001) at 15 minutes of incubation in comparison with controls. Phenylephrine, 10−6 mol/L, maximally increased CaMKII activity by 45.8 ± 10.9% at 15 minutes (P < 0.001). There was no significant difference between the maximal increase in CaMKII activity induced by cocaine or phenylephrine. Although CaMKIV activity was present in control cardiomyocyte immunoprecipitates, cocaine treatment did not increase the activation of CaMKIV. We therefore focused our subsequent experiments on CaMKII.

In series III, the effects of cocaine in concentrations of 0 (control), 108, 107, 5×107, 106, 5×106, or 105 mol/L were determined on CaMKII activity in myocyte lysates as shown in Figure 3A. On the basis of pilot experiments and series II experiments, cardiomyocyte cultures were treated with cocaine at each dose for 15 minutes. Cocaine at 5×106 mol/L maximally increased CaMKII activity in the myocyte lysates by 169.5 ± 18.1% (P < 0.001). The effect of cocaine on myocyte lysates selectively immunoprecipitated for CaMKII is shown in Figure 3B. Cocaine at 5×106 mol/L maximally increased CaMKII activity in the immunopreciptates by 103.3 ± 11.7% (P < 0.001). When the CaMKII antagonist KN-62 was added to myocytes cultures before cocaine, the effect of cocaine on CaMKII activity in the myocyte lysates and immunoprecipitates was nearly completely inhibited (Figs. 3A, B). However, CaMKII activity was not significantly inhibited by KN-62 when the KN-62 was added to the myocyte cultures after cocaine.

FIGURE 3

FIGURE 3

In series IV, we quantitated the role of CaMKII in cocaine-induced myocyte protein expression, and specifically the expression of β-MHC. In these experiments, separate myocyte cultures were treated with cocaine in doses of 0 (control), 106, or 5×106 mol/L for 1 week based on our current and previous experiments.4,5 Cocaine increased the total cardiomyocyte protein content by 29.2 ± 2.8% at 5×106 mol/L and by 20.5 ± 2.8% at 106 mol/L (both P < 0.001) as shown in Figure 4A. In comparison, phenylephrine, 106 mol/L, increased total myocyte protein by 35.8 ± 3.7% (P < 0.001), which was greater than that of cocaine (P < 0.01). KN-62, 10−6 mol/L, treatment of myocyte cultures before cocaine, 10−6 mol/L, decreased cocaine-induced cardiomyocyte protein expression by 59.1% and phenylephrine-induced cardiomyocyte protein expression by 76.6% (P < 0.01). The cardiomyocyte protein measurements after treatment with KN-62 plus cocaine were not statistically different from the control measurements.

FIGURE 4

FIGURE 4

Cocaine increased β-MHC expression by 93.2 ± 8.8% (P < 0.001) at 5×106 mol/L and by 62.6 ± 8.3% (P < 0.01) at 106 mol/L (Fig. 4B). In comparison, phenylephrine, 106 mol/L, increased β-MHC expression by 134 ± 15.2% (P < 0.001), which was significantly more than the cocaine-induced increase in β-MHC expression (P < 0.05). KN-62, 106 mol/L, treatment of myocyte cultures before cocaine significantly decreased the cocaine-induced protein expression of β-MHC by 66.2% (P < 0.01) to a level that was not statistically different from the controls (Fig. 4B).

In series V experiments, whole-cell patch-clamp experiments were conducted under voltage-clamp mode to determine the effects of cocaine on high-voltage-activated Ca2+ channels in cardiomyocytes. Figure 5A shows representative barium currents through calcium channels (IBa) evoked by step depolarizations to the indicated membrane potentials, and recorded from cardiomyocytes in the absence and presence of cocaine. Cocaine potentiated the peak amplitude of IBa when cardiomyocytes were depolarized to either −20 or 0 mV. This increase in current amplitude showed little rundown over 15 minutes and was fully reversible (data not shown). The current-voltage (I-V) relationship obtained from myocytes in 4 separate experiments showed that cocaine enhanced peak IBa amplitude at all test potentials studied (Fig. 5B). However, for both conditions (± cocaine), IBa activated at approximately −30 mV, the I-V relation was maximal near 0 mV and reversed at approximately −45 mV. These values are consistent with a calcium channel current under the ionic conditions used. The fact that cocaine fails to alter these parameters suggests that cocaine acts specifically on calcium channels and is not altering conductance through a second channel type.

FIGURE 5

FIGURE 5

Although cocaine increased peak current at all potentials tested, the magnitude of this potentiation was voltage dependent (Fig. 5C). Whereas cocaine, 106 mol/L, enhanced peak IBa by 173.9 ± 14.9% at −20 mV, this increase was only 38.4 ± 6.9% at 0mV. To determine whether cocaine shifts the voltage dependence of activation of Ca2+ channels to more positive potentials, the whole-cell conductance-voltage (G-V) relationship for Ca2+ channels was determined by measuring the peak current at that potential divided by the difference between membrane potential and observed reversal potential from myocytes in 4 separate experiments in the absence (control) and presence of 106 mol/L cocaine (Fig. 5D). The data demonstrate a sigmoidal G-V relationship in the absence and presence of cocaine, and are best-fit using single Boltzmann distributions. Table 1 summarizes the results of the fits using this equation. Cocaine significantly shifted the voltage of half-maximal activation and increased the peak relative conductance (P < 0.01) (Fig. 5D). The conductance was increased even at the most positive test potentials used here, which suggests a voltage-independent effect of cocaine.

TABLE 1

TABLE 1

In series VI, we determined whether cocaine alters intracellular free Ca2+ concentrations ([Ca2+]i) in cardiomyocytes. Fura-2 fluorometric measurements of [Ca2+]i were carried out in isolated cardiomyocytes in the absence and presence of cocaine. Application of cocaine evoked transient [Ca2+]i elevations in myocytes that were observed 10 to 15 minute after the initiation of drug treatment (Fig. 6A). The magnitude of the peak [Ca2+]i was directly dependent on the cocaine concentration, and increased more than 6-fold from the control value (Fig. 6B) to a mean peak value of 607 ± 141×109 mol/L in the presence of 10×106 mol/L cocaine (P < 0.05). The cocaine-induced elevations in [Ca2+]i were significant (P < 0.05) at both 106 and 10×106 mol/L cocaine.

FIGURE 6

FIGURE 6

Experiments in isolated cardiomyocytes were then performed to determine whether activation of high-voltage membrane Ca2+ channels contribute to the transient Ca2+ elevations observed. In 6 separate experiments, cocaine, 10×106 mol/L, increased the amplitude of transient elevations in [Ca2+]i observed in myocytes, and this increase was not significantly depressed by coapplication of nifedipine, 10×106 mol/L (Figs. 7A, B). In contrast, cocaine failed to increase [Ca2+]i when cardiomyocytes were pretreated with the same concentration of nifedipine for 5 to 10 minutes and then cocaine (Figs. 7C, D).

FIGURE 7

FIGURE 7

Experiments were then conducted to ascertain whether the cocaine-induced elevations in [Ca2+]i in cardiomyocytes were dependent on Ca2+ mobilization from ryanodine stores. Figure 8A shows a representative trace of [Ca2+]i recorded in the presence of cocaine before and after application of ryanodine, 106 mol/L, which acts as an inhibitor of ryanodine receptors at this concentration.28 Although cocaine, 10×106 mol/L, alone elicited elevations of [Ca2+]i in cardiomyocytes, ryanodine, 10×106 mol/L, blocked the cocaine-evoked Ca2+ transients and the increase in [Ca2+]i (P < 0.05). In 4 separate experiments shown in Figure 8B, ryanodine blocked cocaine-evoked Ca2+ transients in a statistically significant manner (P < 0.05). The time dependence of the cocaine effect on [Ca2+]i was similar to that observed for activation of CaMKII (compare Figs. 6A, 7A, 8A with Figs. 2A, B). Furthermore, the concentration dependence of both of these responses was similar. We therefore determined whether cocaine modulates [Ca2+]i via a CaMKII signal transduction cascade by performing experiments with the CaMKII antagonist KN-62 as shown in Figure 9. Cocaine alone evoked Ca2+ transients in myocytes (Fig. 9A) and also evoked Ca2+ transients when myocytes were subsequently treated with KN-62. However, cocaine failed to increase [Ca2+]i in myocytes that were preincubated with KN-62 for 5 minutes (Fig. 9B). Figure 9C shows that in 4 separate experiments cocaine (10×106 mol/L) alone increased [Ca2+]i nearly 4-fold (P < 0.05). However, preincubation of myocytes with KN-62 (10×106 mol/L) inhibited the cocaine-induced mobilization of Ca2+ such that there was no significant difference in [Ca2+]i when compared with the controls (Fig. 9C).

FIGURE 8

FIGURE 8

FIGURE 9

FIGURE 9

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DISCUSSION

The present experiments demonstrate that cocaine, in concentrations commonly found in chronic cocaine users, increases the activation of CaMKII in cardiomyocytes. The present experiments also demonstrate that cocaine increases intracellular calcium by increasing calcium currents through sarcolemma calcium channels and calcium release from the sarcoplasmic reticulum mediated by ryanodine receptors. The cocaine-induced increase in calcium currents most likely activates CaMKII which in turn facilitates calcium release mediated by the ryanodine receptors. This process is inhibited by nifedipine, ryanodine, and the specific CaMK inhibitor KN-62. Moreover, KN-62 significantly inhibits the cocaine-induced increases in cardiomyocyte total protein expression and specifically inhibits the expression of β-MHC. Taken together, the present experiments indicate that CaMKII is an important component in the development of cocaine-induced cardiomyocyte hypertrophy.

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Cocaine-induced Cardiac Hypertrophy

Cocaine is known to cause acute myocardial infarction, cardiomyopathy, and congestive heart failure but only recently has cocaine been discovered to produce cardiac ventricular hypertrophy in approximately 47% of blacks and 18% of white, normotensive chronic cocaine users.2,3 Cocaine can increase cardiac ventricular weight by ≥30%, the left ventricular mass by 69%, and the left ventricular wall thickness by 47%.2,3 The cardiac hypertrophy is not due to sustained increases in systemic arterial pressure, heart rate, or persistent increases in the plasma concentrations of renin, aldosterone, norepinephrine, or cortisol.29 Moreover, the presence and degree of cocaine-induced ventricular hypertrophy is not correlated with the use of other substances of abuse such as alcohol.30 Although cocaine can cause transient catecholamine surges in the body, such catecholamine surges increase α-myosin rather than β-MHC in the heart.31

We have previously demonstrated that cocaine in a dose-dependent manner can directly increase adult cardiomyocyte protein concentration by as much as 29 ± 2% and β-MHC by 81 ± 10%.4,5 We have also reported that the cocaine-induced increase in cardiomyocyte protein expression is not directly dependent on the ERK, SAPK/JNK, or p38 MAP kinase pathways,5 which can contribute to other forms of cardiac hypertrophy. We therefore investigated in the present experiments the CaMK pathway, which when activated can also cause cardiac hypertrophy.8,15,32,33

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CaMK in the Heart

CaMK is distributed in a wide variety of cells where it is localized in the cytosol, cytoskeleton, membrane, and the nucleus. CaMK can regulate cell cycle control, gene expression, the synthesis and release of neurotransmitters, ion permeation across cell membranes and contractility.34 Only CaMKII, however, predominates in the normal heart.8

Transgenic mice that overexpress CaMKII increase left ventricle to body weight ratios, increase LV diameters, decrease dP/dT, and up-regulate β-MHC expression in the heart.32,33 Constriction of the transverse aorta in research animals increases CaMKII activity in the heart by as much as 39% and causes ventricular hypertrophy.8 In addition, CaMKII activity is increased 2 to 3-fold in cardiomyopathic and failing human hearts and in cardiomyocytes undergoing apoptosis.35-37 In contrast, inhibition of CaMK II with KN-62 prevents the induction of β-MHC and protects cardiomyocytes against apoptosis.19,37,38 However, KN-62, in doses up to 100×106 mol/L, does not significantly affect the activity of protein kinase A, protein kinase C, or myosin light chain kinase.22,23 Moreover, KN-62 does not inhibit CaMKII activity once CaMKII autophosphorylation has occurred.22,23

Transient initial phosphorylation converts CaMKII from a calcium-dependent enzyme to a calcium-independent enzyme capable of autophosphorylation.39 Activated CaMKII can phosphorylate L-type calcium channels,9 ryanodine receptors,14 sarcoplasmic reticular Ca2+-ATPase,8 phospholamban,40 cAMP response element binding protein, and also transcription factor-1 in cardiomyocytes.41,42 In addition, CaMKII mediates the transcription of c-fos and the Fos/Jun heterodimer complex AP-1 that is involved in the expression of fetal protein genes in hypertrophied hearts.19

The present studies demonstrate that cocaine, in a time-dependent and dose-dependent manner, significantly increases the activity of CaMKII by as much as 69% to 103% (series II to III) in comparison with controls. As a consequence, cardiomyocyte total protein concentration increases by 29.2% and β-MHC increases by as much as 93.2% (series IV) which is similar to the phenylephrine-induced increases in total protein and β-MHC content in cardiomyocytes. The fact that the CaMK inhibitor KN-62 substantially inhibits this process indicates that cocaine activation of CaMKII is important in causing cocaine-induced cardiomyocyte and ultimately cardiac hypertrophy. Concentrations of cocaine equal to or greater than 105 M may have direct toxic effects on cardiomyocytes and this may explain the decrease in calmodulin kinase activity with 105 M cocaine in the present experiments.43

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Cocaine-induced Increased Intracellular Calcium

The present experiments also demonstrate that cocaine potentiates calcium channel currents and shifts the voltage dependence of calcium channel activation to more negative potentials. Thus, the modulation of Ca2+ channels in cardiomyocytes by cocaine exhibits both voltage-dependent and voltage-independent components. Although the effects of cocaine on calcium current amplitude in the present investigation are consistent with a previous report,44 our observation concerning the effects of cocaine on calcium channel activation kinetics is novel. By shifting the voltage dependence of calcium channel activation to more negative potentials, cocaine most likely facilitates the opening of these channels at membrane potentials closer to the resting cardiomyocyte membrane potential and enhances calcium entry into myocytes. The potentiation of calcium currents by cocaine shows little rundown or desensitization over time which suggests that subacute or chronic exposure to cocaine can significantly increase calcium currents and contribute to calcium overload in cardiomyocytes and therefore cardiac arrhythmias.

Cocaine, in a dose-dependent manner, also causes the release of calcium from the sarcoplasmic reticulum via the ryanodine receptors as demonstrated in series VI by the cocaine-induced increases in calcium transients and intracellular calcium. Ryanodine, a competitive inhibitor of the sarcoplasmic reticulum ryanodine receptors, and the CaMKII antagonist KN-62 significantly inhibit this process. In this regard, CaMKII, when activated, can phosphorylate the ryanodine receptors, increase the open probability and activity of these receptors and thereby increase intracellular calcium.12,13 The effects of cocaine on ryanodine receptors are dependent on activation of both L-type calcium channels and CaMKII because treatment of cardiomyocytes with nifedipine or KN-62 than cocaine in the present experiments significantly inhibits the increases in calcium signals and intracellular calcium. The fact that neither nifedipine nor KN-62 are able to inhibit the cocaine-induced increases in intracellular calcium once this process has been initiated in cardiomyocytes suggests that L-type Ca2+ channel modulation and increased calcium currents are involved in the initiation of the calcium signal elevations owing to CaMKII activation. Our observations also suggest that CaMKII autophosphorylation promotes calcium signal activity once initiated, because neither nifedipine nor KN-62 inhibits CaMKII activity once CaMKII autophosphorylation has occurred.22,23

Hyperphosphorylation of the ryanodine receptors occurs in subjects with heart failure, which enhances calcium leakage from the sarcoplasmic reticulum during diastole and thereby decreases the calcium pool available in the sarcoplasmic reticulum for release during the contractile cycle.37 Hyperphosphorylation of the ryanodine receptors by CaMKII may explain the results obtained in the present experiments, and suggests that this process may be important in cocaine-induced cardiomyopathy. Moreover, the increases in [Ca2+]i observed in the present experiments frequently elicited contractions of the cardiomyocytes. Consequently, the direct effects of cocaine on cardiomyocytes may promote cardiac arrhythmias. In this regard, inhibition of CaMKII has been shown to significantly decrease cardiac early after depolarizations and lethal cardiac arrhythmias.45 CaMK inhibitors might therefore be useful in suppressing cardiac arrhythmias in subjects with cocaine-induced cardiac hypertrophy and cardiomyopathy.

In summary, the present experiments indicate that cocaine can increase calcium currents across the sarcolemma membrane. The increase in intracellular calcium most likely activates CaMKII. CaMKII subsequently increases calcium release from the sarcoplasmic reticulum and up-regulates protein formation within cardiomyocytes. These processes can be important in cocaine-induced cardiomyocyte hypertrophy and in cocaine-induced cardiac arrhythmias.

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ACKNOWLEDGMENTS

The authors thank Denise Cooper, PhD and Ray Olsson, MD for their assistance. This work was supported, in part, by grants to RJH from the Bugher Foundation, the Florida Affiliate of the American Heart Association, and the Office of Research and Development, Department of Veterans' Affairs. Robert J. Henning, MD and Javier Cuevas, PhD made equally important contributions to these investigations.

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Keywords:

cardiomyopathy; β-myosin heavy chain; calcium; calcium currents; intracellular calcium; cardiovascular pharmacology

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