This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Hoch, B. Articles by Karczewski, P. Search for Related Content PubMed PubMed Citation Articles by Hoch, B. Articles by Karczewski, P. Related Collections Genomics Heart failure - basic studies Genetics of cardiovascular disease

(Circulation Research. 1999;84:713-721.)
© 1999 American Heart Association, Inc.

Original Contribution

Identification and Expression of -Isoforms of the Multifunctional Ca²⁺/Calmodulin-Dependent Protein Kinase in Failing and Nonfailing Human Myocardium

Brigitte Hoch, Rudolf Meyer, Roland Hetzer, Ernst-Georg Krause, Peter Karczewski

From the Max Delbrück Center for Molecular Medicine (B.H., E.-G.K., P.K.), Berlin-Buch, and the German Heart Institute (R.M., R.H.), Berlin, Germany.

Correspondence to Dr Peter Karczewski, Max Delbrück Center for Molecular Medicine, Robert-Rössle-Str 10, 13122 Berlin-Buch, Germany. E-mail pkarcze{at}mdc-berlin.de

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Abstract—Despite its importance for the regulation of heart function, little is known about the isoform expression of the multifunctional Ca²⁺/calmodulin-dependent protein kinase (CaMKII) in human myocardium. In this study, we investigated the spectrum of CaMKII isoforms ₂, ₃, ₄, ₈, and ₉ in human striated muscle tissue. Isoform ₃ is characteristically expressed in cardiac muscle. In skeletal muscle, specific expression of a new isoform termed ₁₁ is demonstrated. Complete sequencing of human ₂ cDNA, representing all common features of the investigated CaMKII subclass, revealed its high homology to the corresponding rat cDNA. Comparative semiquantitative reverse transcription–polymerase chain reaction analyses from left ventricular tissues of normal hearts and from patients suffering from dilated cardiomyopathy showed a significant increase in transcript levels of isoform ₃ relative to the expression of glyceraldehyde-3-phosphate dehydrogenase in diseased hearts (101.6±11.0% versus 64.9±9.9% in the nonfailing group; P<0.05, n=6). Transcript levels of the other investigated cardiac CaMKII isoforms remained unchanged. At the protein level, by using a subclass-specific antibody, we observed a similar increase of a -CaMKII–specific signal (7.2±1.0 versus 3.8±0.7 optical density units in the nonfailing group; P<0.05, n=4 through 6). The diseased state of the failing hearts was confirmed by a significant increase in transcript levels for atrial natriuretic peptide (292.9±76.4% versus 40.1±3.2% in the nonfailing group; P<0.05, n=3 through 6). Our data characterize for the first time the -CaMKII isoform expression pattern in human hearts and demonstrate changes in this expression pattern in heart failure.

Key Words: Ca²⁺/calmodulin-dependent protein kinase II • -CaMKII • human cardiac and skeletal muscle • dilated cardiomyopathy

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
The Ca²⁺/calmodulin-dependent protein kinase class II (CaMKII) is a multifunctional enzyme¹ involved in the regulation of gene expression,² cell cycle control,³ and differentiation.^{4 5} Alternative splicing of the primary transcripts encoded by 4 genes (, ß, , and ) results in the generation of different CaMKII variants.^{6 7} Members of the - and ß-classes are expressed mainly in neuronal tissue, whereas - and -CaMKII isoforms are abundantly expressed in all tissues.^{6 8 9} Multifunctionality of the homomultimeric or heteromultimeric holoenzyme composed of 8 through 12 subunits¹⁰ seems to counteract its specific cellular functions. This apparent antagonism is compensated for by controlling the localization of the holoenzyme whereby the isoform composition and its phosphorylation state regulate translocation to different cellular compartments and control the accessibility to substrates.^{11 12 13}

As a major regulator of Ca²⁺ homeostasis, CaMKII is essential for heart function. The importance of this enzyme class for the myocardium is underlined through established cardiac CaMKII targets.^{14 15 16 17} Immunoprecipitation studies have indicated that in the mammalian heart, heteromultimer CaMKII holoenzymes are formed, composed of abundant -isoforms and -subunits, the latter expressed in much lower amounts.¹⁸ Members of the CaMKII -class represent the best characterized cardiac CaMKII isoforms.^{8 9 19 20} At the beginning of this study, from the known -isoforms (termed ₁ through ₁₀ according to Mayer et al⁹ ), the presence of proteins for isoforms ₂, ₃, and probably ₉ has been demonstrated in cardiac tissue.^{19 20 21} Isoform ₃, which contains a functional nuclear localization signal in its variable domain I, may be involved in the regulation of atrial natriuretic factor (ANF) gene expression¹³ and appears to be a dominant isoform in the adult heart.^{20 21} Until now, none of these supposedly highly expressed -CaMKII isoforms has been identified in human myocardium. From studies with noncardiac cells, partial sequences for human isoforms ₂ and ₉ were obtained.²²

Herein we report the identification and expression of individual -CaMKII isoforms in human striated muscle tissues. We demonstrate the presence of large amounts of transcripts for isoforms ₂, ₃, ₈, and ₉ in human cardiac tissue, and we have been able to detect a new isoform, termed ₁₁, specifically expressed in human skeletal muscle tissue. Sequencing of the complete ₂-CaMKII cDNA demonstrates a high degree of homology to the corresponding rat isoform.

In most cases of heart failure, systolic and diastolic dysfunction is caused by abnormalities in Ca²⁺ handling systems.^{23 24 25} To prove whether this mechanism also holds true for the CaMKII system, we compared the -CaMKII isoform pattern of normal left ventricular tissues with corresponding tissue from patients suffering from dilated cardiomyopathy (DCM). As a marker for the diseased state,^{26 27} we determined the transcript levels for ANF in parallel. We demonstrate in this article the increased expression of isoform ₃ at the transcriptional level and of the subclass of -CaMKII isoforms, to which isoform ₃ belongs, at the protein level in the failing compared with nonfailing myocardium and suggest that these changes are linked to heart failure.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Tissue Samples and Histomorphology
Human cardiac tissue samples were obtained from the German Heart Institute, Berlin, Germany, and a sample of human thigh skeletal muscle was obtained from the Robert-Rössle Clinic, Berlin-Buch, Germany. Cardiac tissue samples were taken from the apical part of the left ventricular free wall from hearts not accepted for transplantation (nonfailing hearts) and from explanted DCM hearts from age-matched groups (mean±SEM age in years in the nonfailing group, 48.3±3.1; in the DCM group, 50.7±3.3; see also Table 2). Immediately after preparation, the tissue samples for isolation of RNA or protein were frozen in LN₂ and stored at -80°C. For preparation of histological slides, tissue samples were embedded in paraffin and stained with hematoxylin/eosin and Domagk for the demonstration of connective tissue. The size of heart muscle cells, textural organization of heart muscle cells, grading of hypertrophy, and distribution and volume of connective tissue were determined in a standardized protocol.²⁸ In nonfailing hearts, the sizes of heart muscle cells were in the normal range (diameter of cardiomyocytes <14.9 µm) or only slightly enlarged (<16.9 µm). In the DCM group, corresponding values ranged above normal levels (>17 µm, with some values >20 µm). Permission for the procurement of human tissue samples was granted by the German law of transplantation of December 1997 and/or the personal decision of the organ donor.

View this table: [in this window] [in a new window]   Table 2. Relative Transcript Levels for The Various Transcripts From Individual Hearts

Extraction of RNA
Total RNA from human muscle tissue was extracted according to Kingston et al.²⁹ After isolation, the remaining DNA contaminants were digested in 40 mmol/L Tris-HCl (pH 7.5), 6 mmol/L MgCl₂, and 10 µL of RNase-free DNaseI (Pharmacia) for 10 minutes at 37°C in a total volume of 100 µL. RNA was reextracted with the RNA clean-up procedure of the RNeasy system (Qiagen). Total digestion of genomic DNA was assessed by polymerase chain reaction (PCR) without the reverse transcription (RT) step.

Reverse Transcription–Coupled Polymerase Chain Reaction
For RT, the RNA solution was boiled and immediately chilled on ice. Random-primed cDNA synthesis for PCR was carried out in a total volume of 60 µL containing up to 6 µg of total RNA in the presence of 6 µL of 10x PCR buffer (Eurogentec), 4.8 µL of 25 mmol/L MgCl₂, 16 µL of dNTP (2.5 mmol/L of each nucleotide; US Biochemical), 1.5 µL of RNase inhibitor (RNAguard, Pharmacia), 1.5 µL of 10x hexanucleotide mix (Boehringer), and 3 µL of Superscript II RNaseH^- reverse transcriptase (Gibco BRL). After incubation for 10 minutes at room temperature, RT was performed for 1 hour at 42°C. For each PCR, an aliquot of the RT reaction mixture equivalent to that originally containing 500 ng of total RNA was used. The volume of this aliquot was increased to 100 µL and then to a final amount of 10 µL of 10x PCR buffer, 8 µL of 25 mmol/L MgCl₂, 4 µL of dNTP, 50 pmol of each specific primer (Table 1), and, in the case of the standard protocol, 0.2 µL of thermostable DNA polymerase (GoldStar red DNA polymerase, Eurogentec). Standard PCR was performed in a TRIO thermocycler (Biometra) as follows: 3 minutes at 94°C for the denaturation step; 19 cycles for GAPDH-specific amplification in the linear range, 24 cycles for -CaMKII–specific amplifications in the linear range, or 36 cycles of amplification (30 seconds at 94°C, 1 minute at 55°C, and 1 minute at 72°C); and a final 7-minute elongation step at 72°C. For amplification of ANF, a hot-start PCR protocol was established, starting with a 5-minute denaturation step at 95°C; a 4-minute rest at 80°C (during this time, 0.2 µL of thermostable DNA polymerase was added to the amplification reactions) followed by 19 cycles of amplification (30 seconds at 95°C, 1 minute at 58°C, and 1 minute 30 seconds at 72°C); and a final 14-minute elongation step at 72°C. After PCR, 8 µL of the 100-µL reaction mixture was loaded onto 2% agarose gels containing ethidium bromide. As the size marker for gel electrophoresis, a ready-load 100-bp DNA standard (Gibco BRL) was used. After agarose gel electrophoresis, stained DNA bands were visualized under UV light. Background-subtracted optical density (OD) units from peak areas were obtained with the PCBas system (raytest Isotopenmeßgeräte GmbH). For comparison of different reactions, GAPDH-specific PCRs were carried out in parallel.

View this table: [in this window] [in a new window]   Table 1. Primers Used for PCR

Extraction of PCR Fragments From Agarose Gels and Sequencing of PCR Products
For sequencing, amplification products were loaded onto 2% ethidium bromide–containing agarose gels, and the band of interest was cut out. Extraction of DNA from the gel slice was done with the Qiaquick gel-extraction kit (Qiagen) according to the manufacturer's instructions. Standard cycle sequencing reactions were performed commercially by InViTek (Berlin-Buch, Germany).

Preparation of Total Homogenates and Western Blotting
Approximately 30 mg of tissue was homogenized in a 10-fold volume of homogenization buffer consisting of (in mmol/L) HEPES 10 (pH 7.5), PMSF 0.2, and DTT 0.1, and 1 mg/L leupeptin with an Ultra Turrax T5 FU homogenizer (Janke & Kunkel) for 3x 10 seconds at 50 000 rpm. Homogenate protein was solubilized in the same volume of 2x concentrated SDS sample buffer and electrophoresed through SDS polyacrylamide gels.³⁰ Separated proteins were electrotransferred onto polyvinylidenedifluoride membranes. Processing for immunoblotting was performed as described in Towbin et al.³¹ A -subclass–specific antibody recognizing -CaMKII isoforms that contained variable domain II was used as described in Hoch et al.²⁰ For immunoblotting, the antibody was diluted to 1 µg/mL. A specific antibody detecting sarcoplasmic reticulum Ca²⁺-ATPase (BioMol) was prepared to a final dilution of 1:1000. For detection, the secondary antibodies were anti-rabbit IgG (-CaMKII) and anti-mouse IgG (sarcoplasmic reticulum Ca²⁺-ATPase) (both obtained from Sigma) conjugated with peroxidase. The immunoreaction was visualized using the enhanced chemoluminescence kit (Amersham) and autoradiography on x-ray film. Densitometric analyses of autoradiograms were performed with the PDI imaging system (PDI). Data for protein bands were expressed relative to the myosin heavy-chain band (205 kDa) obtained by densitometrically scanning the polyvinylidenedifluoride membranes stained with Ponceau S (Sigma) to correct for differences in muscle protein content between heart tissue homogenates.

Statistical Evaluation
Results are given as mean±SEM. Mean values were compared using the unpaired t test when the check for a normal distribution was positive and the Mann-Whitney U test otherwise. A value of P<0.05 was assumed to be statistically significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
-CaMKII Isoforms in Human Striated Muscle Tissue
To identify human cardiac -CaMKII isoforms, we performed RT-PCR studies with isoform-specific primer combinations (Table 1). -CaMKII contains 2 variable regions. Variable domains I are identical between isoforms ₂/₆, ₃/₇, ₄/₈, and ₉/₁₀.⁹ Variable domain II is a C-terminal extension present in isoforms ₂, ₃, ₄, and ₉ but lacking in isoforms ₆, ₇, ₈, and ₁₀. Isoforms ₁ and ₅, exclusively neuronally expressed in the rat,⁹ are not considered in this study. Using primer combinations specific for isoforms containing both variable regions, we obtained signals from human heart in the sizes expected for isoforms ₂, ₃, and ₉ (Figure 1A, left). As a positive control for isoform ₄, a skeletal muscle–specific isoform in rat, and as a negative control for isoform ₃, an isoform undetectable in rat skeletal muscle tissue,⁸ we amplified cDNAs derived from human skeletal muscle tissue (Figure 1A, right). Because we were unable to obtain an amplification product for isoform ₄ from human skeletal muscle tissue, we used the same forward primer (specific for the identical variable domain I of isoforms ₄ and ₈) but a different reverse primer (P33), which does not detect variable region II, to obtain signals for isoform ₈. This primer combination led to the generation of amplification products for isoform ₈ in cardiac as well as skeletal muscle tissue (Figure 1A).

View larger version (74K): [in this window] [in a new window]   Figure 1. -CaMKII isoform pattern in human striated muscle tissues. A, RT-PCR signals after 36 cycles of amplification derived from human cardiac (left) and skeletal muscle (right) tissues obtained with various primer combinations specific for -CaMKII isoforms and GAPDH (G; see Table 1). Specificity of the single amplification products was also confirmed by sequencing. Skeletal muscle–derived signal obtained with the 3-specific primer combination is set in brackets; sequencing revealed its identity as isoform 11. (The nucleotide sequence reported in this article has been submitted to the GenBank/EMBL Data Bank with accession number AF071569.) Positions of signals derived from the marker (M) and a lane containing buffer control (C; PCR without nucleic acid) are included. B, Positions and orientations of primers generated for specific amplification of isoform 11 (upper part). Amino acid sequences are given in the single-letter code, and asterisk denotes position of the stop codon. RT-PCR of cardiac (c) and skeletal muscle (s) tissue with this primer combination after 36 cycles of amplification (lower part) is displayed. For comparison of RT-PCR reactions, GAPDH (G)-specific amplification products are given. M denote marker lanes. The following controls were included: 1, RT-PCR of cardiac RNA in the absence of reverse transcriptase; 2, RT-PCR of skeletal muscle RNA in the absence of reverse transcriptase; 3, buffer control of PCR of cardiac tissue–derived cDNA; and 4, buffer control of PCR of skeletal muscle–derived cDNA.

To our surprise, we obtained a skeletal muscle–derived signal for isoform ₃ when we used a forward primer that specifically detects variable domain I of this isoform. This amplification product is set in brackets in Figure 1A, because it seemed to be slightly larger than that derived from cardiac muscle (ie, compare the vertical distances in the corresponding lanes between isoforms ₂ and ₃ in Figure 1A). Sequencing of this skeletal muscle–derived amplification product revealed an insertion of 42 bp between the variable region I specific for isoform ₃ and the conserved 3' part of -CaMKII. This 42-bp insert codes for 14 amino acid residues characteristic of variable region I of isoform ₉. The newly identified -CaMKII isoform was named ₁₁ (Figure 1B, top). We generated a primer specific for detection of this yet-undescribed -CaMKII isoform. Whereas isoform ₁₁ is near the detection limit when cardiac tissue is used, sufficient amplification products were obtained with skeletal muscle tissue (Figure 1B, bottom).

Complete cDNA Sequence of Human ₂-CaMKII
Tombes and Krystal²² identified 329 nucleotides of human ₂-CaMKII–specific sequences (Figure 2, underlined). Our amplification products for isoforms ₂, ₃, ₉, and ₁₁ (Figure 1) include the conserved C-terminal part of the kinase, including variable region II, to the stop codon (Figure 2, dotted line). To obtain amplification products for the missing N-terminal part of the kinase, we used forward primer P16, which is derived from amino acid residues 1 to 8 of the corresponding cDNA from the rat.⁶ This primer was used in combination with reverse primer P44 (Figure 2, arrows). RT-PCR of human cardiac RNA resulted in amplification of a 984-bp product. The sequence of this product is set in boldface letters in Figure 2. Thus, we obtained the complete cDNA sequence of human ₂-CaMKII. Alignment with the corresponding cDNA sequence from the rat showed a 92.6% identity with respect to nucleotides and a 98.9% identity at the protein level.

View larger version (57K): [in this window] [in a new window]   Figure 2. Complete coding sequence of human 2-CaMKII and alignment with homologous sequence from rat.6 Differences between both sequences resulting in changes at the protein level are boxed. Sequence fragment as obtained from Tombes and Krystal22 for human -CaMKII isoform 2 from noncardiac cells is underlined. The dotted line marks the sequence of the 3'-terminal region of the kinase derived from sequencing of amplification products shown in Figure 1. The N-terminal part of human 2-CaMKII is set in boldface type and was obtained by sequencing the amplification product obtained with primer combination P16/P44 (positions and orientations marked by arrows) from human left ventricular tissue. A peptide with the amino acid sequence KENFSGGTSLWQNI derived from the C-terminal end of the kinase was used for generating a subclass-specific antibody.20

Comparison of -CaMKII Isoform Pattern in Failing and Nonfailing Human Myocardium
To compare the amounts of transcripts of individual -CaMKII isoforms from failing and nonfailing hearts in a semiquantitative assay, we used GAPDH transcripts as the standard for normalization. As an additional marker of the diseased state, we determined ANF transcript levels in parallel. The RT reactions for analysis of the CaMKII isoforms, ANF, or GAPDH from 1 tissue sample were carried out in the same reaction tube. For PCR, identical aliquots of cDNA were used (see Materials and Methods). For comparison of amplification products, the signals should be in the linear range of the amplification reaction. We therefore determined the cycle dependence for amplification of GAPDH and ₉-CaMKII from nonfailing tissue samples and for ANF from DCM hearts, because in these tissues high levels of the corresponding signals are expected. Isoform ₉ was chosen because, together with isoforms ₂ and ₃, it appeared to be 1 of the strongest signals obtained (Figure 1A). The upper part of Figure 3 shows the result of amplification of GAPDH, ANF, and ₉-CaMKII from cycles 15 to 40 for 3 independent reactions. Data from these experiments are shown in the lower part of Figure 3. After examining these data, we therefore used 19 cycles for amplification of GAPDH- and ANF-specific products and 24 cycles for amplification of -CaMKII–specific products because they appeared to be within the linear ranges of their respective reactions for semiquantitatively assaying transcript levels of -CaMKII and ANF in human heart samples. In Figure 4A, representative RT-PCRs for isoform ₃, ANF, and GAPDH from 3 nonfailing and 6 diseased hearts are presented. Figure 4B shows the statistical evaluation of similar experiments as shown in Figure 4A. Comparison of the isoform pattern normalized to GAPDH expression (Figure 4B) revealed a significant increase in the amount of transcript of isoform ₃ in the diseased hearts (101.6±11.0% versus 64.9±9.9% in the nonfailing group; P<0.05, n=6 individual hearts). As expected from Northern blotting data published by Arai et al,²⁷ the amount of transcript for ANF was significantly increased in the DCM group (292.9±76.4% versus 40.1±3.2% in the nonfailing group; P<0.05, n=3 through 6), confirming the nonfailing and diseased states of the investigated groups. Transcript levels of the other analyzed cardiac CaMKII isoforms remained unchanged. Table 2 summarizes the calculated values for the various transcripts obtained from individual hearts.

View larger version (45K): [in this window] [in a new window]   Figure 3. Evaluation of linear ranges of amplification reactions for -CaMKII isoforms, ANF, and GAPDH. RT-PCR signals for 9, ANF, and GAPDH after 15, 20, 25, 30, 35, and 40 cycles of amplification (upper part) are displayed. Products from 3 independent RT-PCRs with RNA isolated from 2 nonfailing hearts (9- and GAPDH-specific products) or 3 diseased hearts (ANF-derived products) are shown. Specificity of ANF-specific amplification product was also confirmed by sequencing. Statistical evaluation of the gels is shown in the lower part and reveals cycle numbers (24 for 9 and 19 for GAPDH, ANF; marked by asterisks) that were used for amplification of the corresponding products in the linear range for the subsequent experiments.

View larger version (61K): [in this window] [in a new window]   Figure 4. Amount of transcripts of 3-CaMKII and ANF in failing and nonfailing human hearts. A, Representative RT-PCR from 3 individual nonfailing (NF) and 6 failing (DCM) hearts for CaMKII isoform 3, ANF, and GAPDH after 24 (3) and 19 (ANF, GAPDH) cycles of amplification. Marker lanes are denoted as M. B, Statistical evaluation of similar gels as shown in A. Data from 3 through 6 individual hearts were used for evaluation (see Table 2). Amounts of transcripts for isoform 3 and ANF were expressed relative to GAPDH. Asterisks mark values in the DCM group that were significantly different from those in nonfailing hearts (P<0.05). Calculated values for individual hearts are given in Table 2.

To analyze -CaMKII expression at the protein level in failing and nonfailing human myocardium, an antibody was used that recognizes -CaMKII isoforms from rat that contain variable domain II.²⁰ By reason of the identity between rat and human -CaMKII in this region (Figure 2), the antibody detects signals derived from the corresponding -isoforms in human hearts (Figure 5, upper left). On immunoblots, similar to what has been observed in rat hearts,²⁰ there is a faint band below the major signal that corresponds in size to isoform ₂. The major signal corresponds in size to the molecular masses of isoform ₃ (57.7 kDa)²¹ and ₉, which differs from ₃ by only 3 amino acids.⁹ Statistical evaluation of data from 4 nonfailing and 6 failing hearts revealed a significant increase in this main signal in tissue samples from DCM hearts (7.2±1.0 versus 3.8±0.7 OD units in nonfailing hearts, P<0.05) (Figure 5, lower left). Because the amount of transcript for isoform ₉ was found to be unchanged (Table 2), we propose that the increase in the -CaMKII protein signal results from an increase in the expression of isoform ₃.

View larger version (23K): [in this window] [in a new window]   Figure 5. -CaMKII and sarcoplasmic reticulum Ca2+-ATPase expression in failing and nonfailing human myocardium. Representative immunoblots of tissue homogenates from nonfailing (NF) human hearts and from patients suffering from DCM are shown in the upper part. For detection, a -CaMKII subclass–specific antibody (left) and a sarcoplasmic reticulum Ca2+- ATPase–specific antibody (Serca 2; right) were used. Statistical evaluation of data from 4 nonfailing and 6 failing hearts is shown in the lower part. Amount of homogenate protein was 10 µg per lane. Data for -CaMKII and sarcoplasmic reticulum Ca2+-ATPase were normalized to equal expression of myosin heavy-chain band (see Materials and Methods). Asterisks denote statistically significant differences (P<0.05) vs preparations from nonfailing hearts.

In the same tissue samples, we analyzed the expression of sarcoplasmic reticulum Ca²⁺-ATPase. A representative immunoblot is shown in the upper right portion of Figure 5. A statistical evaluation of 4 through 6 independent experiments is shown in the lower portion of Figure 5 (right), and this revealed a significant decrease in sarcoplasmic reticulum Ca²⁺-ATPase in the DCM group (1.5±0.5 versus 3.6±0.6 OD units in nonfailing hearts, P<0.05).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
This is the first report on the identification of the -CaMKII isoform pattern at the transcriptional level in the adult human heart. By comparison with the -CaMKII isoform patterns in human skeletal muscle, we identified the new isoform ₁₁. This isoform is specifically expressed in skeletal muscle tissue and combines the variable regions I from isoforms ₃ and ₉. Identification of the complete coding sequence of human cardiac ₂-CaMKII revealed high homology of this kinase between rats and humans.

DCM is a severe disease of the myocardium that generally leads to an enlarged, dilated left ventricle, increased heart rate, and a dramatically reduced ejection fraction. The pathogenesis of the disease seems to be multifactorial, with various causes resulting in the common terminal state of the failing heart.³² On the molecular level, changes in systems regulating Ca²⁺ homeostasis as well as in contractile proteins were observed in diseased hearts.^{23 25 33 34} CaMKII is critically involved in the regulation of cardiac Ca²⁺ homeostasis. We investigated in this study the transcript pattern of CaMKII variants of the -class, a CaMKII class that has been presumed to be abundantly expressed in the mammalian myocardium.¹⁸ In addition to the histopathological and clinical characterization of tissue samples obtained from failing and nonfailing hearts, we analyzed ANF transcript level and the protein level of sarcoplasmic reticulum Ca²⁺-ATPase. An increase in the amount of ANF mRNA is a commonly accepted marker for heart failure in human and animal models.^{27 35} To our knowledge, however, this is the first attempt to analyze human ANF mRNA levels in nonfailing and DCM hearts by using a semiquantitative RT-PCR technique. Our data confirm the elevation of ANF transcript level in the failing human heart. In contrast to the well-accepted correlation between human heart failure and increased ANF mRNA levels, the reduction in sarcoplasmic reticulum Ca²⁺-ATPase in the diseased myocardium has been more controversial.³⁶ In the present work, we were able to demonstrate a significantly reduced sarcoplasmic reticulum Ca²⁺-ATPase level in the DCM group, as has also been described by other investigators.^{37 38} CaMKII is known to be a regulator of gene expression in cardiac myocytes¹³ and other cell types.^{2 4 39 40} We demonstrated a significant, 1.5-fold increase in the amount of transcript of CaMKII isoform ₃ in failing compared with nonfailing hearts. The enhancement in ₃ transcript level was accompanied by a nearly 2-fold increase in a subclass of -CaMKII protein, to which ₃ belongs. This clearly indicates significant changes in -CaMKII expression in DCM, most likely due to increased levels of isoform ₃. Ramirez et al¹³ demonstrated a positive correlation between expression of CaMKII isoform ₃, resulting in targeting of the CaMKII holoenzyme to the nucleus, and ANF gene expression in neonatal cardiomyocytes from the rat. The increase in ANF mRNA levels in human heart failure (vide infra and References 26 and 27^{26 27} ) and the specific increase of ₃-CaMKII in DCM as demonstrated here suggest that this mechanism may also be functional in human heart failure. The changes in CaMKII isoform expression at both the mRNA and protein level in DCM may reflect a significant role of this enzyme class for alterations in the gene expression program occurring in human heart failure.

	Acknowledgments

 
This work was supported by a grant from the Deutsche Forschungsgemeinschaft to Peter Karczewski (KA939/5-1). We thank Dorothea Riege and Ingrid Ameln for expert technical assistance.

Received July 1, 1998; accepted January 13, 1999.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 
1. Braun AP, Schulman H. The multifunctional calcium/calmodulin-dependent protein kinase: from form to function. Annu Rev Physiol. 1995;57:417–445.[Medline] [Order article via Infotrieve]

2. Nghiem P, Ollick T, Gardner P, Schulman H. Interleukin-2 transcriptional block by multifunctional Ca²⁺/calmodulin kinase. Nature. 1994;371:347–350.[Medline] [Order article via Infotrieve]

3. Planas-Silva MD, Means AR. Expression of a constitutive form of calcium/calmodulin dependent protein kinase II leads to arrest of the cell cycle in G₂. EMBO J. 1992;11:507–517.[Medline] [Order article via Infotrieve]

4. Wang Y, Simonson MS. Voltage-insensitive Ca²⁺ channels and Ca²⁺/calmodulin-dependent protein kinases propagate signals from endothelin-1 receptors to the c-fos promoter. Mol Cell Biol. 1996;16:5915–5923.[Abstract/Free Full Text]

5. Masse T, Kelly PT. Overexpression of Ca²⁺/calmodulin dependent protein kinase II in PC12 cells alters cell growth, morphology, and nerve growth factor induced differentiation. J Neurosci. 1997;17:924–931.[Abstract/Free Full Text]

6. Tobimatsu T, Fujisawa H. Tissue-specific expression of four types of rat calmodulin-dependent protein kinase II mRNAs. J Biol Chem. 1989;264:17907–17912.[Abstract/Free Full Text]

7. Brocke L, Srinivasan M, Schulman H. Developmental and regional expression of multifunctional Ca²⁺/calmodulin-dependent protein kinase isoforms in rat brain. J Neurosci. 1995;15:6797–6808.[Abstract/Free Full Text]

8. Schworer CM, Rothblum LI, Thekkumkara TJ, Singer HA. Identification of novel isoforms of the subunit of Ca²⁺/calmodulin-dependent protein kinase II. J Biol Chem. 1993;268:14443–14449.[Abstract/Free Full Text]

9. Mayer P, Möhlig M, Idlibe D, Pfeiffer A. Novel and uncommon isoforms of the calcium sensing enzyme calcium/calmodulin dependent protein kinase II in heart tissue. Basic Res Cardiol. 1995;90:372–379.[Medline] [Order article via Infotrieve]

10. Kabasaki T, Ikeuchi Y, Sugiura H, Yamauchi T. Structural features of Ca²⁺/calmodulin-dependent protein kinase II revealed by electron microscopy. J Cell Biol. 1991;115:1049–1060.[Abstract/Free Full Text]

11. Srinivasan M, Edman CF, Schulman, H. Alternative splicing introduces a nuclear localization signal that targets multifunctional CaM kinase to the nucleus. J Cell Biol. 1994;126:839–852.[Abstract/Free Full Text]

12. Heist EK, Srinivasan M, Schulman H. Phosphorylation at the nuclear localization signal of Ca²⁺/calmodulin-dependent protein kinase II blocks its nuclear targeting. J Biol Chem. 1998;273:19763–19771.[Abstract/Free Full Text]

13. Ramirez MT, Zhao X-L, Schulman H, Brown JH. The nuclear _B isoform of Ca²⁺/calmodulin-dependent protein kinase II regulates atrial natriuretic factor gene expression in ventricular myocytes. J Biol Chem. 1997;272:31203–31208.[Abstract/Free Full Text]

14. Anderson ME, Braun AP, Schulman H, Premack BA. Multifunctional Ca²⁺/calmodulin-dependent protein kinase mediates Ca²⁺-induced enhancement of the L-type Ca²⁺ current in rabbit ventricular myocytes. Circ Res. 1994;75:854–861.[Abstract/Free Full Text]

15. Hohenegger M, Suko J. Phosphorylation of the purified cardiac ryanodine receptor by exogenous and endogenous protein kinases. Biochem J. 1993;296:303–308.

16. Hawkins C, Xu A, Narayanan N. Sarcoplasmic reticulum calcium pump in cardiac and slow twitch skeletal muscle but not fast twitch skeletal muscle undergoes phosphorylation by endogenous and exogenous Ca²⁺/calmodulin-dependent protein kinase. J Biol Chem. 1994;269:31198–31206.[Abstract/Free Full Text]

17. Simmerman HKB, Collins JH, Theibert JL, Wegener AD, Jones LR. Sequence analysis of phospholamban: identification of phosphorylation sites and two major structural domains. J Biol Chem. 1986;261:13333–13341.[Abstract/Free Full Text]

18. Singer HA, Benscoter HA, Schworer CM. Novel Ca²⁺/calmodulin-dependent protein kinase II -subunit variants expressed in vascular smooth muscle, brain, and cardiomyocytes. J Biol Chem. 1997;272:9393–9400.[Abstract/Free Full Text]

19. Baltas LG, Karczewski P, Krause EG. The cardiac sarcoplasmic reticulum phospholamban kinase is a distinct -CaM kinase isozyme. FEBS Lett. 1995;373:71–75.[Medline] [Order article via Infotrieve]

20. Hoch B, Haase H, Schulze W, Hagemann D, Morano I, Krause E-G, Karczewski P. Differentiation-dependent expression of cardiac -CaMKII isoforms. J Cell Biochem. 1998;68:259–268.[Medline] [Order article via Infotrieve]

21. Edman CF, Schulman H. Identification and characterization of _B-CaM kinase and _C-CaM kinase from rat heart, two new multifunctional Ca²⁺/calmodulin-dependent protein kinase isoforms. Biochim Biophys Acta. 1994;1221:89–101.[Medline] [Order article via Infotrieve]

22. Tombes RM, Krystal GW. Identification of novel human tumor cell-specific CaMK-II variants. Biochim Biophys Acta. 1997;1355:281–292.[Medline] [Order article via Infotrieve]

23. Morgan JP. Abnormal intracellular modulation of calcium as a major cause of cardiac contractile dysfunction. N Engl J Med. 1991;325:625–632.[Medline] [Order article via Infotrieve]

24. Beuckelmann DJ, Näbauer M, Erdmann E. Intracellular calcium handling in isolated ventricular myocytes from patients with terminal heart failure. Circulation. 1992;85:1046–1055.[Abstract/Free Full Text]

25. de Tombe PP. Altered contractile function in heart failure. Cardiovasc Res. 1998;37:367–380.[Abstract/Free Full Text]

26. Takahashi T, Allen PD, Izumo S. Expression of A-, B-, and C-type natriuretic peptide genes in failing and developing human ventricles: correlation with expression of the Ca²⁺-ATPase gene. Circ Res. 1992;71:9–17.[Abstract/Free Full Text]

27. Arai M, Alpert NR, MacLennan DH, Barton P, Periasamy M. Alterations in sarcoplasmic reticulum gene expression in human heart failure: a possible mechanism for alterations in systolic and diastolic properties of the failing myocardium. Circ Res. 1993;72:463–469.[Abstract/Free Full Text]

28. Kunkel B, Lapp H, Kober G, Kaltenbach M. Light-microscopic evaluation of myocardial biopsy. In: Kaltenbach M, Loogen F, Olsen EGJ eds. Cardiomyopathy and Myocardial Biopsy. Berlin/Heidelberg/New York: Springer-Verlag; 1978;62–70.

29. Kingston RE, Chomczynski P, Sacchi N. Guanidinium methods for total RNA preparation. In: Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Struhl K, eds. Current Protocols in Molecular Biology. New York NY: John Wiley & Sons; 1994;I:4.2.1–4.2.9.

30. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970;227:680–685.[Medline] [Order article via Infotrieve]

31. Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and applications. Proc Natl Acad Sci U S A. 1979;76:4350–4354.[Abstract/Free Full Text]

32. Goldman JH, McKenna WJ. Immunopathogenesis of dilated cardiomyopathies. Curr Opin Cardiol. 1995;10:306–311.[Medline] [Order article via Infotrieve]

33. Anderson PA, Greig A, Mark TM, Malouf NN, Oakeley AE, Ungerleider RM, Allen PD, Kay BK. Molecular basis of human cardiac troponin T isoforms expressed in the developing, adult, and failing heart. Circ Res. 1995;76:681–686.[Abstract/Free Full Text]

34. Arai M, Matsui H, Periasamy M. Sarcoplasmic reticulum gene expression in cardiac hypertrophy and heart failure. Circ Res. 1994;74:555–564.[Free Full Text]

35. Franch HA, Dixon RAF, Blaine EH, Siegl PKS. Ventricular atrial natriuretic factor in the cardiomyopathic hamster model of congestive heart failure. Circ Res. 1988;62:31–36.[Abstract/Free Full Text]

36. Movsesian MA, Schwinger RHG. Calcium sequestration by the sarcoplasmic reticulum in heart failure. Cardiovasc Res. 1998;37:352–359.[Free Full Text]

37. Hasenfuss G, Reinecke H, Studer R, Meyer M, Pieske B, Holtz J, Holubarsch C, Posival H, Just H, Drexler H. Relation between myocardial function and expression of sarcoplasmic reticulum Ca²⁺-ATPase in failing and nonfailing human myocardium. Circ Res. 1994;75:434–442.[Abstract/Free Full Text]

38. Meyer M, Schillinger W, Pieske B, Holubarsch C, Heilmann C, Posival H, Kuwajima G, Mikoshiba K, Just H, Hasenfuss G. Alterations of sarcoplasmic reticulum proteins in failing human dilated cardiomyopathy. Circulation. 1995;92:778–784.[Abstract/Free Full Text]

39. Higuchi H, Nakano K, Kim C-H, Li B-S, Kuo C-H, Taira E, Miki N. Ca²⁺/calmodulin-dependent transcriptional activation of neuropeptide Y gene induced by membrane depolarization: determination of Ca²⁺- and cyclic AMP/phorbol 12-myristate 13-acetate-responsive elements. J Neurochem. 1996;66:1802–1809.[Medline] [Order article via Infotrieve]

40. Yoshida K, Imaki J, Matsuda H, Hagiwara M. Light-induced CREB phosphorylation and gene expression in rat retinal cells. J Neurochem. 1995;65:1499–1504.[Medline] [Order article via Infotrieve]

This article has been cited by other articles:

				T. Zhang, T. Guo, S. Mishra, N. D. Dalton, E. G. Kranias, K. L. Peterson, D. M. Bers, and J. H. Brown Phospholamban Ablation Rescues Sarcoplasmic Reticulum Ca2+ Handling but Exacerbates Cardiac Dysfunction in CaMKII{delta}C Transgenic Mice Circ. Res., February 5, 2010; 106(2): 354 - 362. [Abstract] [Full Text] [PDF]

				W. Peng, Y. Zhang, M. Zheng, H. Cheng, W. Zhu, C.-M. Cao, and R.-P. Xiao Cardioprotection by CaMKII-{delta}B Is Mediated by Phosphorylation of Heat Shock Factor 1 and Subsequent Expression of Inducible Heat Shock Protein 70 Circ. Res., January 8, 2010; 106(1): 102 - 110. [Abstract] [Full Text] [PDF]

				C. M. Sag, D. P. Wadsack, S. Khabbazzadeh, M. Abesser, C. Grefe, K. Neumann, M.-K. Opiela, J. Backs, E. N. Olson, J. H. Brown, et al. Calcium/Calmodulin-Dependent Protein Kinase II Contributes to Cardiac Arrhythmogenesis in Heart Failure Circ Heart Fail, November 1, 2009; 2(6): 664 - 675. [Abstract] [Full Text] [PDF]

				S. M. MacDonnell, J. Weisser-Thomas, H. Kubo, M. Hanscome, Q. Liu, N. Jaleel, R. Berretta, X. Chen, J. H. Brown, A.-K. Sabri, et al. CaMKII Negatively Regulates Calcineurin-NFAT Signaling in Cardiac Myocytes Circ. Res., August 14, 2009; 105(4): 316 - 325. [Abstract] [Full Text] [PDF]

				S. Wagner, E. Hacker, E. Grandi, S. L. Weber, N. Dybkova, S. Sossalla, T. Sowa, L. Fabritz, P. Kirchhof, D. M. Bers, et al. Ca/Calmodulin Kinase II Differentially Modulates Potassium Currents Circ Arrhythm Electrophysiol, June 1, 2009; 2(3): 285 - 294. [Abstract] [Full Text] [PDF]

				J. Backs, T. Backs, S. Neef, M. M. Kreusser, L. H. Lehmann, D. M. Patrick, C. E. Grueter, X. Qi, J. A. Richardson, J. A. Hill, et al. The {delta} isoform of CaM kinase II is required for pathological cardiac hypertrophy and remodeling after pressure overload PNAS, February 17, 2009; 106(7): 2342 - 2347. [Abstract] [Full Text] [PDF]

				D. von Lewinski, J. Kockskamper, D. Zhu, H. Post, A. Elgner, and B. Pieske Reduced Stretch-Induced Force Response in Failing Human Myocardium Caused by Impaired Na+-Contraction Coupling Circ Heart Fail, January 1, 2009; 2(1): 47 - 55. [Abstract] [Full Text] [PDF]

				B. London Understanding Cardiac Calcium Channelopathies Circulation, November 25, 2008; 118(22): 2221 - 2222. [Full Text] [PDF]

				Y. Wang, S. Tandan, J. Cheng, C. Yang, L. Nguyen, J. Sugianto, J. L. Johnstone, Y. Sun, and J. A. Hill Ca2+/Calmodulin-dependent Protein Kinase II-dependent Remodeling of Ca2+ Current in Pressure Overload Heart Failure J. Biol. Chem., September 12, 2008; 283(37): 25524 - 25532. [Abstract] [Full Text] [PDF]

				L. F. Couchonnal and M. E. Anderson The Role of Calmodulin Kinase II in Myocardial Physiology and Disease Physiology, June 1, 2008; 23(3): 151 - 159. [Abstract] [Full Text] [PDF]

				J. Backs, T. Backs, S. Bezprozvannaya, T. A. McKinsey, and E. N. Olson Histone Deacetylase 5 Acquires Calcium/Calmodulin-Dependent Kinase II Responsiveness by Oligomerization with Histone Deacetylase 4 Mol. Cell. Biol., May 15, 2008; 28(10): 3437 - 3445. [Abstract] [Full Text] [PDF]

				J. Pang, C. Yan, K. Natarajan, M. E. Cavet, M. P. Massett, G. Yin, and B. C. Berk GIT1 Mediates HDAC5 Activation by Angiotensin II in Vascular Smooth Muscle Cells Arterioscler Thromb Vasc Biol, May 1, 2008; 28(5): 892 - 898. [Abstract] [Full Text] [PDF]

				X. Chen, X. Zhang, D. M. Harris, V. Piacentino III, R. M. Berretta, K. B. Margulies, and S. R. Houser Reduced effects of BAY K 8644 on L-type Ca2+ current in failing human cardiac myocytes are related to abnormal adrenergic regulation Am J Physiol Heart Circ Physiol, May 1, 2008; 294(5): H2257 - H2267. [Abstract] [Full Text] [PDF]

				J. Bossuyt, K. Helmstadter, X. Wu, H. Clements-Jewery, R. S. Haworth, M. Avkiran, J. L. Martin, S. M. Pogwizd, and D. M. Bers Ca2+/Calmodulin-Dependent Protein Kinase II{delta} and Protein Kinase D Overexpression Reinforce the Histone Deacetylase 5 Redistribution in Heart Failure Circ. Res., March 28, 2008; 102(6): 695 - 702. [Abstract] [Full Text] [PDF]

				Y. Ikeda, M. Hoshijima, and K. R. Chien Toward Biologically Targeted Therapy of Calcium Cycling Defects in Heart Failure Physiology, February 1, 2008; 23(1): 6 - 16. [Abstract] [Full Text] [PDF]

				T. Guo, X. Ai, T. R. Shannon, S. M. Pogwizd, and D. M. Bers Intra Sarcoplasmic Reticulum Free [Ca2+] and Buffering in Arrhythmogenic Failing Rabbit Heart Circ. Res., October 12, 2007; 101(8): 802 - 810. [Abstract] [Full Text] [PDF]

				W. Zhu, A. Y.-H. Woo, D. Yang, H. Cheng, M. T. Crow, and R.-P. Xiao Activation of CaMKII{delta}C Is a Common Intermediate of Diverse Death Stimuli-induced Heart Muscle Cell Apoptosis J. Biol. Chem., April 6, 2007; 282(14): 10833 - 10839. [Abstract] [Full Text] [PDF]

				L. S. Maier and D. M. Bers Role of Ca2+/calmodulin-dependent protein kinase (CaMK) in excitation-contraction coupling in the heart Cardiovasc Res, March 1, 2007; 73(4): 631 - 640. [Abstract] [Full Text] [PDF]

				M. E. Anderson Multiple downstream proarrhythmic targets for calmodulin kinase II: Moving beyond an ion channel-centric focus Cardiovasc Res, March 1, 2007; 73(4): 657 - 666. [Abstract] [Full Text] [PDF]

				Y. Yang, W.-Z. Zhu, M.-l. Joiner, R. Zhang, C. V. Oddis, Y. Hou, J. Yang, E. E. Price, L. Gleaves, M. Eren, et al. Calmodulin kinase II inhibition protects against myocardial cell apoptosis in vivo Am J Physiol Heart Circ Physiol, December 1, 2006; 291(6): H3065 - H3075. [Abstract] [Full Text] [PDF]

				B. London CaM Kinase Inhibition: Apply Directly to the Heart? Circ. Res., November 10, 2006; 99(10): 1027 - 1028. [Full Text] [PDF]

				M. S.C. Khoo, J. Li, M. V. Singh, Y. Yang, P. Kannankeril, Y. Wu, C. E. Grueter, X. Guan, C. V. Oddis, R. Zhang, et al. Death, Cardiac Dysfunction, and Arrhythmias Are Increased by Calmodulin Kinase II in Calcineurin Cardiomyopathy Circulation, September 26, 2006; 114(13): 1352 - 1359. [Abstract] [Full Text] [PDF]

				T. Guo, T. Zhang, R. Mestril, and D. M. Bers Ca2+/Calmodulin-Dependent Protein Kinase II Phosphorylation of Ryanodine Receptor Does Affect Calcium Sparks in Mouse Ventricular Myocytes Circ. Res., August 18, 2006; 99(4): 398 - 406. [Abstract] [Full Text] [PDF]

				M. Kohlhaas, T. Zhang, T. Seidler, D. Zibrova, N. Dybkova, A. Steen, S. Wagner, L. Chen, J. Heller Brown, D. M. Bers, et al. Increased Sarcoplasmic Reticulum Calcium Leak but Unaltered Contractility by Acute CaMKII Overexpression in Isolated Rabbit Cardiac Myocytes Circ. Res., February 3, 2006; 98(2): 235 - 244. [Abstract] [Full Text] [PDF]

				J. Backs and E. N. Olson Control of Cardiac Growth by Histone Acetylation/Deacetylation Circ. Res., January 6, 2006; 98(1): 15 - 24. [Abstract] [Full Text] [PDF]

				X. Ai, J. W. Curran, T. R. Shannon, D. M. Bers, and S. M. Pogwizd Ca2+/Calmodulin-Dependent Protein Kinase Modulates Cardiac Ryanodine Receptor Phosphorylation and Sarcoplasmic Reticulum Ca2+ Leak in Heart Failure Circ. Res., December 9, 2005; 97(12): 1314 - 1322. [Abstract] [Full Text] [PDF]

				A. Mattiazzi, C. Mundina-Weilenmann, C. Guoxiang, L. Vittone, and E. Kranias Role of phospholamban phosphorylation on Thr17 in cardiac physiological and pathological conditions Cardiovasc Res, December 1, 2005; 68(3): 366 - 375. [Abstract] [Full Text] [PDF]

				C. Woischwill, P. Karczewski, H. Bartsch, H.-P. Luther, M. Kott, H. Haase, and I. Morano Regulation of the human atrial myosin light chain 1 promoter by Ca2+-calmodulin-dependent signaling pathways FASEB J, April 1, 2005; 19(6): 503 - 511. [Abstract] [Full Text] [PDF]

				M. Zayzafoon, K. Fulzele, and J. M. McDonald Calmodulin and Calmodulin-dependent Kinase II{alpha} Regulate Osteoblast Differentiation by Controlling c-fos Expression J. Biol. Chem., February 25, 2005; 280(8): 7049 - 7059. [Abstract] [Full Text] [PDF]

				W. Wang, W. Zhu, S. Wang, D. Yang, M. T. Crow, R.-P. Xiao, and H. Cheng Sustained {beta}1-Adrenergic Stimulation Modulates Cardiac Contractility by Ca2+/Calmodulin Kinase Signaling Pathway Circ. Res., October 15, 2004; 95(8): 798 - 806. [Abstract] [Full Text] [PDF]

				T. Zhang and J. H. Brown Role of Ca2+/calmodulin-dependent protein kinase II in cardiac hypertrophy and heart failure Cardiovasc Res, August 15, 2004; 63(3): 476 - 486. [Abstract] [Full Text] [PDF]

				X. H.T. Wehrens, S. E. Lehnart, S. R. Reiken, and A. R. Marks Ca2+/Calmodulin-Dependent Protein Kinase II Phosphorylation Regulates the Cardiac Ryanodine Receptor Circ. Res., April 2, 2004; 94(6): e61 - e70. [Abstract] [Full Text] [PDF]

				T. Zhang, S. Miyamoto, and J. H. Brown Cardiomyocyte Calcium and Calcium/Calmodulin-dependent Protein Kinase II: Friends or Foes? Recent Prog. Horm. Res., January 1, 2004; 59(1): 141 - 168. [Abstract] [Full Text]

				J. BORLAK and T. THUM Hallmarks of ion channel gene expression in end-stage heart failure FASEB J, September 1, 2003; 17(12): 1592 - 1608. [Abstract] [Full Text] [PDF]

				Y. Ji, B. Li, T. D. Reed, J. N. Lorenz, M. A. Kaetzel, and J. R. Dedman Targeted Inhibition of Ca2+/Calmodulin-dependent Protein Kinase II in Cardiac Longitudinal Sarcoplasmic Reticulum Results in Decreased Phospholamban Phosphorylation at Threonine 17 J. Biol. Chem., June 27, 2003; 278(27): 25063 - 25071. [Abstract] [Full Text] [PDF]

				L. S. Maier, T. Zhang, L. Chen, J. DeSantiago, J. H. Brown, and D. M. Bers Transgenic CaMKII{delta}C Overexpression Uniquely Alters Cardiac Myocyte Ca2+ Handling: Reduced SR Ca2+ Load and Activated SR Ca2+ Release Circ. Res., May 2, 2003; 92(8): 904 - 911. [Abstract] [Full Text] [PDF]

				T. Zhang, L. S. Maier, N. D. Dalton, S. Miyamoto, J. Ross Jr, D. M. Bers, and J. H. Brown The {delta}C Isoform of CaMKII Is Activated in Cardiac Hypertrophy and Induces Dilated Cardiomyopathy and Heart Failure Circ. Res., May 2, 2003; 92(8): 912 - 919. [Abstract] [Full Text] [PDF]

				S. Mishra, H. N. Sabbah, J. C. Jain, and R. C. Gupta Reduced Ca2+-calmodulin-dependent protein kinase activity and expression in LV myocardium of dogs with heart failure Am J Physiol Heart Circ Physiol, March 1, 2003; 284(3): H876 - H883. [Abstract] [Full Text] [PDF]

				J. M. Colomer, L. Mao, H. A. Rockman, and A. R. Means Pressure Overload Selectively Up-Regulates Ca2+/Calmodulin-Dependent Protein Kinase II in Vivo Mol. Endocrinol., February 1, 2003; 17(2): 183 - 192. [Abstract] [Full Text] [PDF]

				J. M. Lorenz, M. H. Riddervold, E. A. H. Beckett, S. A. Baker, and B. A. Perrino Differential autophosphorylation of CaM kinase II from phasic and tonic smooth muscle tissues Am J Physiol Cell Physiol, November 1, 2002; 283(5): C1399 - C1413. [Abstract] [Full Text] [PDF]

				M. R. Rosen Blunderbuss to Mickey Mouse: The Evolution of Antiarrhythmic Targets Circulation, September 3, 2002; 106(10): 1180 - 1182. [Full Text] [PDF]

				Y. Wu, J. Temple, R. Zhang, I. Dzhura, W. Zhang, R. Trimble, D. M. Roden, R. Passier, E. N. Olson, R. J. Colbran, et al. Calmodulin Kinase II and Arrhythmias in a Mouse Model of Cardiac Hypertrophy Circulation, September 3, 2002; 106(10): 1288 - 1293. [Abstract] [Full Text] [PDF]

				S. Bartel, B. Hoch, D. Vetter, and E.-G. Krause Expression of Human Angiotensinogen-Renin in Rat: Effects on Transcription and Heart Function Hypertension, February 1, 2002; 39(2): 219 - 223. [Abstract] [Full Text] [PDF]

				P. Boknik, I. Heinroth-Hoffmann, U. Kirchhefer, J. Knapp, B. Linck, H. Luss, T. Muller, W. Schmitz, O.-E. Brodde, and J. Neumann Enhanced protein phosphorylation in hypertensive hypertrophy Cardiovasc Res, September 1, 2001; 51(4): 717 - 728. [Abstract] [Full Text] [PDF]

				Y. Wu, R. J. Colbran, and M. E. Anderson Calmodulin kinase is a molecular switch for cardiac excitation -contraction coupling PNAS, February 27, 2001; 98(5): 2877 - 2881. [Abstract] [Full Text] [PDF]

				J. M. Colomer and A. R. Means Chronic Elevation of Calmodulin in the Ventricles of Transgenic Mice Increases the Autonomous Activity of Calmodulin-Dependent Protein Kinase II, Which Regulates Atrial Natriuretic Factor Gene Expression Mol. Endocrinol., August 1, 2000; 14(8): 1125 - 1136. [Abstract] [Full Text]

				M. E. Anderson Connections Count : Excitation-Contraction Meets Excitation-Transcription Coupling Circ. Res., April 14, 2000; 86(7): 717 - 719. [Full Text] [PDF]

				T. Netticadan, R. M. Temsah, K. Kawabata, and N. S. Dhalla Sarcoplasmic Reticulum Ca2+/Calmodulin-Dependent Protein Kinase Is Altered in Heart Failure Circ. Res., March 17, 2000; 86(5): 596 - 605. [Abstract] [Full Text] [PDF]

				Q. He and M. C. LaPointe Interleukin-1{beta} Regulates the Human Brain Natriuretic Peptide Promoter via Ca2+-Dependent Protein Kinase Pathways Hypertension, January 1, 2000; 35(1): 292 - 296. [Abstract] [Full Text] [PDF]

				A. Krasko, H. C. Schroder, S. Perovic, R. Steffen, M. Kruse, W. Reichert, I. M. Muller, and W. E. G. Muller Ethylene Modulates Gene Expression in Cells of the Marine Sponge Suberites domuncula and Reduces the Degree of Apoptosis J. Biol. Chem., October 29, 1999; 274(44): 31524 - 31530. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Hoch, B. Articles by Karczewski, P. Search for Related Content PubMed PubMed Citation Articles by Hoch, B. Articles by Karczewski, P. Related Collections Genomics Heart failure - basic studies Genetics of cardiovascular disease