PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Hypertension. Author manuscript; available in PMC Apr 1, 2011.
Published in final edited form as:
PMCID: PMC2843393
NIHMSID: NIHMS181085
Accelerated Development of Pressure Overload-Induced Cardiac Hypertrophy and Dysfunction in a RyR2-R176Q Knockin Mouse Model
Ralph J. van Oort,a Jonathan L. Respress,a Na Li,a Corey Reynolds,a Angela C. De Almeida,a Darlene G. Skapura,a Leon J. De Windt,b and Xander H.T. Wehrensab1
aFrom the Dept. of Molecular Physiology and Biophysics, and Medicine (Cardiology), Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, U.S.A.
bDepartment of Medical Physiology, University Medical Center Utrecht, Yalelaan 50, 3584 CM Utrecht, The Netherlands
1Address correspondence to: Xander H.T. Wehrens, MD PhD, Baylor College of Medicine, One Baylor Plaza, BCM 335, Houston, TX 77030, U.S.A. Tel: 713-798-4261, Fax: 713-798-3475, wehrens/at/bcm.edu
In response to chronic hypertension, the heart compensates by hypertrophic growth, which frequently progresses to heart failure. Although Ca2+ has a central role in hypertrophic signaling pathways, the Ca2+ source for activating these pathways remains elusive. We hypothesized that pathological sarcoplasmic reticulum Ca2+ leak through defective cardiac intracellular Ca2+ release channels/ ryanodine receptors (RyR2) accelerates heart failure development by stimulating Ca2+-dependent hypertrophic signaling. Mice heterozygous for the gain-of-function mutation R176Q/+ in RyR2 and wildtype (WT) mice were subjected to transverse aortic constriction (TAC). Cardiac function was significantly lower, and cardiac dimensions were larger at 8 weeks after TAC in R176Q/+ compared with WT mice. R176Q/+ mice displayed an enhanced hypertrophic response compared to WT mice as assessed by heart weight to body weight ratios and cardiomyocyte cross sectional areas after TAC. Quantitative PCR revealed increased transcriptional activation of cardiac stress genes in R176Q/+ mice after TAC. Moreover, pressure overload resulted in an increased SR Ca2+ leak, associated with higher expression levels of the exon 4 splice form of regulator of calcineurin-1 (RCAN1-4), and a decrease in nuclear factor of activated T-cells (NFAT) phosphorylation in R176Q/+ mice compared to WT. Taken together, our results suggest that RyR2-dependent SR Ca2+ leak activates the pro-hypertrophic calcineurin/NFAT pathway under conditions of pressure overload.
Keywords: Calcium, heart failure, hypertrophy, ryanodine receptor calcium release channel, sarcoplasmic reticulum
Cardiac hypertrophy is an adaptive physiological mechanism that develops in response to hypertension-associated functional overload of the heart to preserve cardiac output and tissue perfusion.1 Despite these initial benefits, cardiac hypertrophy is also an important independent risk factor for cardiovascular disease and, if left untreated, it often deteriorates into decompensation and heart failure.2 It is now well established that several major signaling pathways involved in the hypertrophic response are activated by increases in intracellular calcium (Ca2+) concentrations.3
Sustained elevations in cytosolic Ca2+ levels activate the Ca2+/calmodulin-dependent serine-threonine phosphatase calcineurin.4 Once activated, calcineurin dephosphorylates members of the Nuclear Factor of Activated T-cells (NFAT) transcription factor family, resulting in translocation of NFAT into the nucleus and the induction of hypertrophic gene expression.2 Another important Ca2+-activated cardiac hypertrophy signaling pathway involves Ca2+/calmodulin-dependent protein kinase (CaMKII) mediated phosphorylation of class II histone deacetylases (HDACs). Upon phosphorylation, HDACs are exported out of the nucleus thereby derepressing activity of the Myocyte Enhancer Factor-2 (MEF2) family of transcription factors.2, 5
Despite the central role of Ca2+-dependent hypertrophic signaling cascades, the subcellular origin of Ca2+ signals that activate these signaling pathways in cardiac myocytes remain mostly unknown.2, 5 The question whether altered patterns of Ca2+ release and reuptake associated with excitation-contraction (EC) coupling may affect hypertrophic signaling pathways remains very controversial.6
In cardiomyocytes, EC coupling is initiated by the influx of extracellular Ca2+ through L-type Ca2+ channels increases Ca2+ levels in the junctional space between transverse (T)-tubules and sarcoplasmic reticulum (SR).7 This increase in local Ca2+ levels activates ryanodine receptor (RyR2)/ intracellular Ca2+ release channels, which release a much greater amount of Ca2+ from the SR known as the contractile Ca2+ transient.6, 8 Myocyte relaxation occurs when cytosolic Ca2+ levels are subsequently reduced to baseline levels through the actions of Ca2+ recycling proteins, such as SR Ca2+-ATPase (SERCA2a) and the Na+/Ca2+-exchanger (NCX1).7 It has been proposed that enhanced Ca2+ leak from the SR during diastole, caused by ‘leaky’ RyR2 Ca2+ release channels, may underlie contractile dysfunction.812 However, it is controversial at present whether this diastolic SR Ca2+ leak might also activate Ca2+-dependent hypertrophic signaling pathways under pathological conditions.
Here, we tested the hypothesis that enhanced SR Ca2+ leak through RyR2 accelerates the development of cardiac hypertrophy through the activation of Ca2+ dependent signaling pathways. To study the specific effects of increased RyR2-mediated Ca2+ release, we studied knock-in mice heterozygous for mutation R176Q in RyR2 (R176Q/+). This mutation was previously shown to increase SR Ca2+ release in atrial myocytes and in ventricular myocytes after catecholaminergic stimulation.13, 14
An expanded Methods section is available in the online Data Supplement at http://hyper.ahajournals.org.
Transverse aortic constriction (TAC) in mice
R176Q/+ knock-in mice were generated as previously described.13 TAC was performed as previously described in detail.15
Transthoracic echocardiography
Cardiac function was assessed by echocardiography using a VisualSonics VeVo 770 Imaging System (VisualSonics, Toronto, Canada), as described.16
Invasive hemodynamic measurements
Pressure-volume relationships were generated by cardiac catheterization of a 1.4F catheter (Millar Instruments, Houston, TX) and analyzed using the IOX data acquisition system (Emka Technologies, Falls Church, VA).
Histology
Paraffin sections of the hearts were stained with hematoxylin-eosin (H&E) for cell morphology. Myocyte cross-sectional areas were measured in sections stained with fluorescein-conjugated wheat germ agglutinin (WGA-FITC).
Quantitative RT-PCR
Real-time PCR was performed using a Mastercycler ep realplex (Eppendorf, Hamburg, Germany), as described before.17
Calcium imaging in isolated ventricular myocytes
Single ventricular myocytes isolation and Ca2+ imaging was performed as described in detail before.14, 16
Immunoprecipitation and Western blot analysis
CaMKII autophosphorylation status was determined by Western blot on mouse heart homogenates using anti-CaMKIIδ (1:1,000) and anti-pThr287-CaMKII (1:1,000; Cayman Chemicals, Ann Arbor, MI) antibodies. To determine NFAT phosphorylation status, 500 µg of protein extract was immunoprecipitated with anti-NFATc3 (Santa Cruz Biotechnology, Santa Cruz, CA) and blotted with anti-phosphoserine (Invitrogen, Carlsbad, CA).
Statistical analysis
Results are expressed as mean ± SEM. A value of P<0.05 was considered statistically significant.
Accelerated Cardiac Failure in R176Q/+ Knockin Mice
R176Q/+ knockin mice (n=28) and WT littermates (n=32) with an average weight of 24.2±1.1 g and 23.1±1.0 g, respectively, were used for this study. At baseline, left ventricular (LV) contractility and dimensions measured by echocardiography were similar in both groups (see Table S1 in the online supplement), consistent with previous studies showing the absence of cardiac hypertrophy in R176Q/+ mice.13
After baseline echocardiography measurements, pressure overload was induced surgically by transverse aorta constriction (TAC) in R176Q/+ (n=19) and WT (n=22) mice. Additionally, 9 R176Q/+ and 10 WT mice were subjected to a sham procedure. The presence of a pressure gradient was evaluated using Doppler ultrasound measurements 7 days after surgery, and was similar in R176Q/+ TAC and WT TAC mice. To evaluate the effects of the R176Q/+ mutation in RyR2 on the development of cardiac failure following pressure overload, LV function was evaluated using serial echocardiography 2, 4, and 8 weeks following TAC. Measurements were made at the same level of anesthesia in all mice, and body temperature was carefully maintained between 36.0 and 37.0 °C. There were no significant differences in heart rates comparing the experimental groups of mice (heart rate at 8 weeks after TAC: WT, 496 ± 10 bpm; R176Q/+ 500 ± 8 bpm; P=N.S.). R176Q/+ TAC mice exhibited an accelerated development of contractile failure compared with WT TAC mice (Figure 1). At each time point, ejection fractions (EF) were significantly lower in R176Q/+ TAC mice compared with WT TAC (Figure 1A). Moreover, R176Q/+ mice developed more pronounced cardiac dilatation following pressure overload (Figure 1B). The increase in end-diastolic LV diameters (EDD) 8 weeks after TAC was significantly larger in R176Q/+ mice (32.5±3.0%) compared with WT mice (26.5±2.9%; P<0.05) (Figure 1B). There were no significant changes in EF or EDD in either R176Q/+ or WT mice following the sham procedure (data not shown).
Figure 1
Figure 1
Accelerated cardiac contractile dysfunction in R176Q/+ mice after pressure overload
At the conclusion of the experiment 8 weeks following TAC, pressure-volume loops were obtained using LV catheterization. These hemodynamic measurements revealed a significantly lower first derivative of LV pressure over time (dP/dtmax) in R176Q/+ TAC mice (n=12) compared with WT TAC mice (n=14, P<0.05), suggesting that the R176Q mutation in RyR2 accelerates the depression in systolic function in R176Q/+ mice following pressure overload (Figure 1C). Furthermore, quantification of the peak negative dP/dt index revealed a strong trend towards more severely impaired diastolic function in R176Q/+ TAC mice (−4268.9±330.7 mmHg/s) compared with WT TAC mice (−5033.2±374.9 mm Hg/s; P=0.07) after pressure overload, although this did not reach statistical significance (Figure 1D).
R176Q Mutation Augments Cardiac Hypertrophy Following Pressure Overload
Eight weeks after surgery, heart and cardiomyocyte size were indistinguishable between sham-operated R176Q/+ and WT mice (Figure 2A). The hypertrophic response observed after aortic constriction, however, was exacerbated in R176Q/+ mice compared to WT mice. Indeed, heart weight-to-body weight ratio was larger in R176Q/+ mice (13.3±1.2 mg/g) compared with WT mice (10.3±0.7 mg/g) after induction of pressure overload (P<0.05) (Figure 2B). Consistently, heart weight-to-tibia length ratio was also larger in R176Q/+ mice (16.2±1.0 mg/mm) compared with WT mice (13.2±0.7 mg/mm) 8 weeks after TAC (P<0.05) (Figure 2C). In contrast, HW/BW and HW/TL ratios were similar in sham operated R176Q/+ and WT mice.
Figure 2
Figure 2
Augmented cardiac enlargement and myocyte hypertrophy in R176Q/+ mice following TAC
To further test whether the R176Q mutation in RyR2 affected the hypertrophic response following pressure overload, we measured LV wall thickness using echocardiography. Figure 2D shows that R176Q/+ mice exhibited a more pronounced hypertrophic response following TAC, evidenced by a significantly larger LV wall thickness (1.1±0.02 mm) compared with WT TAC mice (1.0±0.01 mm; P<0.01).
As a quantitative evaluation of individual myofiber hypertrophy, we measured individual myofibril cross-sectional areas from wheat germ agglutinin (WGA) stained sections (Figure 2A and E). Sham-operated R176Q/+ and WT mice had similar myofiber cross-sectional areas, whereas R176Q/+ mice subjected to TAC exhibited a significantly increased myofiber size (420.8±8.3 µm2) compared to WT TAC mice (352.6±6.5 µm2; P<0.001) (Figure 2E). Enlargement of myofiber diameters occurred throughout the ventricular wall and was not limited to specific areas or layers within the wall.
A hallmark of the stress-associated cardiac remodeling process induced by pressure overload is reactivation of fetal cardiac genes. Therefore, the expression levels of a number of fetal genes were determined using quantitative PCR (Figure 2F and G). Transcripts levels for acta1 (alpha skeletal muscle actin, Acta1, Figure 2F), and nppb (brain natriuretic peptide, BNP, Figure 2G), were higher in R176Q/+ mice compared with WT mice 8 weeks following TAC. In sham-operated animals, there were no differences between any of these transcript levels comparing WT and R176Q/+ mice. Overall, these data suggest that the gain-of-function mutation R176Q in RyR2 augments the myofiber hypertrophy response following biomechanical stress.
Elevated SR Ca2+ Leak and Activation of NFAT-dependent Hypertrophic Signaling in Cardiomyocytes from R176Q/+ Mice
To determine whether pressure overload resulted in an increased SR Ca2+ leakiness in R176Q/+ mice, isolated cardiomyocytes were loaded with a Ca2+ sensitive dye and SR Ca2+ leak was measured using the protocol described by Shannon et al.18 (Figure 3A). There was a non-significant trend towards an increased Ca2+ leak in WT TAC (7.6±0.7 a.u.) compared to WT sham animals (5.8±0.7, P=0.07). The magnitude of SR Ca2+ leak was significantly larger in R176Q/+ TAC mice (10.2±1.0 a.u.) compared to R176Q/+ sham (6.8±0.4 a.u., P<0.01) and WT TAC (7.6±0.7 a.u., P<0.05) animals (Figure 3B).
Figure 3
Figure 3
Increased SR Ca2+ leak in cardiomyocytes from R176Q/+ mice after TAC
Since an increased level of cytosolic Ca2+ might activate Ca2+-dependent hypertrophic signaling pathways, we determined if there is an increased activation of either the CaMKII/HDAC or calcineurin (CaN)/NFAT pathway in R176Q/+ TAC mice (Figure 4). Activation of the CaMKII-HDAC hypertrophic signaling pathway was assessed by Western blotting using antibodies against auto-phosphorylated CaMKII (Figure 4A). There was a trend towards increased levels of CaMKII auto-phosphorylation of WT and R176Q/+ mice after TAC (WT TAC vs sham: P=0.17; R176Q/+ TAC vs sham: P=0.10). However, there was no significant difference in CaMKII auto-phosphorylation comparing R176Q/+ and WT mice 8 weeks after TAC (Figure 4B), suggesting similar CaMKII activity levels in these mice. No differences in total CaMKII levels were detected between groups when normalized for GAPDH protein level (data not shown).
Figure 4
Figure 4
Increased calcineurin/NFAT activity in R176Q/+ mice after TAC
To probe activity of the CaN/NFAT pathway, we determined the phosphorylation level of NFAT (Figure 4C). NFAT was found to be dephosphorylated in R176Q/+ mice at 8 weeks after TAC (Figure 4D), indicating an enhanced activity of the phosphatase calcineurin in these mice. A Western blot for GAPDH on 10% of the input samples was used to verify equal protein levels in each group (data not shown). Next, we assessed transcript levels of the NFAT-regulated exon 4 splice isoform of rcan1 (regulator of calcineurin-1) (Figure 4E).19 Pressure overload induced an increase in RCAN1-4 expression level in WT mice (3.1±1.4 for WT TAC versus 1.0±0.3 for WT sham: P<0.05). RCAN1-4 expression level was more upregulated in R176Q/+ mice (RQ TAC = 7.3±1.0) than WT mice (WT TAC = 3.1±1.4) after 8 weeks of pressure overload (RQ TAC vs WT TAC: P<0.05), reflecting an increase in total NFAT activity downstream of CaN signaling in the heart.20 Thus, these results indicate that the R176Q gain-of-function mutation in RyR2 led to an augmentation of calcineurin-induced structural alterations in the myocardium. Together, these data suggest that the accelerated development of heart failure in R176Q/+ mice might be predominantly mediated by excessive activation of the Ca2+/calcineurin/NFAT signaling pathway as a result of enhanced SR Ca2+ leak via RyR2.
It has been well recognized that Ca2+-dependent signaling pathways play a key role in the development of pathological cardiac hypertrophy.2 Although these intracellular signaling events have been well described during the past decade, the exact nature of the subcellular Ca2+ pool that initiates hypertrophic signaling has remained quite controversial.6 The source of this ‘hypertrophy-associated’ Ca2+ pool might be influx of extracellular Ca2+ via voltage-gated Ca2+ channel 2123 or store-operated Ca2+ channels 2426, release from the sarcoplasmic reticulum via RyR2 12, or release from the nucleus via IP3 receptors.27 Our results suggest that pathological ‘leak’ of Ca2+ from the sarcoplasmic reticulum (SR) through mutant RyR2 accelerates the development of cardiac hypertrophy and heart failure following pressure overload. These data are also consistent with previous reports suggesting that increased diastolic RyR2 Ca2+ leak impairs cardiac contractility due to a secondary decrease in SR Ca2+ loading.2830 In addition, our results provide the first in vivo evidence that increased RyR2-mediated Ca2+ release from the SR leads to activation of the Ca2+-dependent calcineurin/NFAT hypertrophic signaling pathway.
Defects in RyR2 Ca2+ channels regulation are believed to play a central role in the development of contractile dysfunction in heart failure.31 A variety of alterations in the subunit composition of the RyR2 macromolecular complex have been demonstrated in patients with congestive heart failure, including decreased levels of FK506-binding protein 12.6 (FKBP12.6, or calstabin2), protein phosphatase 1 (PP1A), protein phosphatase 2A (PP2A), and phosphodiesterase 4D3 (PDE4D3).3234 In addition, changes in RyR2 posttranslational modifications such as oxidation, S-nitrosylation, and phosphorylation have been shown in patients and animal models with heart failure.3540 The combination of these alterations of RyR2 leads to a decreased ability of the channel to remain closed during diastole, resulting in diastolic Ca2+ leak from the SR.41, 42 Whereas it has been well accepted that increased diastolic SR Ca2+ releases contribute to reduced cardiac contractility in patients with decompensated hypertrophy or heart failure, significant controversy exists about the source of Ca2+ involved in the activation of Ca2+-dependent prohypertrophic signaling cascades.6
Our data suggest that pathological Ca2+ leak from the SR through ‘leaky’ RyR2 may lead to enhanced activation of the calcineurin/NFAT-dependent hypertrophic signaling pathway (Figure 5). Our data are consistent with those obtained in mice in which the calmodulin (CaM) binding site on RyR2 has been disrupted with three mutations (RyR2-W3587A/ L3591D/ F3603A). Homozygous RyR2-ADA knock-in mice exhibit spontaneous SR Ca2+ release episodes, neonatal cardiac hypertrophy, and activation of HDAC4/MEF2 signaling.12 Moreover, MCIP1 (RCAN1-4) levels were elevated suggesting that the calcineurin pathway was activated in RyR2-ADA knock-in mice.12 In contrast, heterozygous RyR2-ADA mice did not exhibit signs of cardiac hypertrophy or transcriptional activation of genes associated with hypertrophy. Together, these data suggest that a minor defect in RyR2 (RyR2-ADA heterozygous, RyR2-R176Q/+ heterozygous mice) might not be sufficient to induce cardiac hypertrophy, whereas a more pronounced defect (RyR2-ADA homozygous), or a minor defect plus an exogenous stressor (chronic hypertension) may induce activation of hypertrophic signaling pathways.
Figure 5
Figure 5
Calcium spillover from the calcium release unit triggers hypertrophic signaling
Additional evidence has emerged from recent clinical studies showing that genetic defects in the RyR2 gene may lead to a predisposition towards the development of hypertrophic cardiomyopathy and cardiac failure in patients.4345 Two common single nucleotide polymorphisms (SNPs) in the human RyR2 gene are associated with arrhythmogenic right ventricular cardiomyopathy, often associated with mild cardiac failure.45 The presence of both SNPs in RyR2 results in channels with a higher open probability, suggesting that these RyR2 lead to aberrant SR Ca2+ release under diastolic conditions. In addition, Fujino et al. identified missense mutations Thr1107Met 46 and Gly2367Arg 47 patients with left ventricular and asymmetrical septal hypertrophy.
Whereas it might seem counterintuitive that Ca2+-sensitive signaling pathways could be regulated by large contractile Ca2+ transients, recent studies suggest otherwise. Colella et al. 3 demonstrated in cultured neonatal cardiomyocytes that an increased frequency of Ca2+ transients, if prolonged for hours, is sufficient to activate the calcineurin/NFAT signaling pathway. These results are similar to those showing that an increased beating frequency of cultured neonatal myocytes or atrial tissue preparations induces NFAT translocation into the nucleus.48, 49 Thus, changes in excitation-contraction coupling-associated Ca2+ fluxes might indeed mediate calcineurin activation and NFAT translocation, although the mechanisms underlying this phenomenon are still unknown.
PERSPECTIVES
Our findings demonstrate that defective Ca2+ release from the SR via mutant RyR2 adversely affects cardiac remodeling as seen during a sustained increase in blood pressure. Our study indicates that Ca2+ leaking from the SR enhances hypertrophic signaling, preferentially by activating the calcineurin/NFAT signaling pathway. This reveals a new role for SR Ca2+ in heart disease, but additional studies are required to test whether modification of its release might improve clinical outcome in cases of human cardiac disease.
Supplementary Material
Supp1
ACKNOWLEDGMENTS
The authors would like to thank Dr. Susan Hamilton for providing the R176Q/+ knockin mice, and Alejandro Garbino for graphical assistance.
SOURCES OF FUNDING
X.H.T.W. is a W.M. Keck Foundation Distinguished Young Scholar in Medical Research, and is also supported by NIH/NHLBI grants R01-HL089598 and R01-R01HL091947, and Muscular Dystrophy Association grant #69238. R.J.v.O. is the recipient of the 2008-2010 American Physiological Society Postdoctoral Fellowship in Physiological Genomics. This work was also supported in part by the Fondation Leducq Alliance for CaMKII Signalling in Heart (X.H.T.W.), a VIDI award 917-863-72 from the Netherlands Organization for Health Research and Development (ZonMW) and the Fondation Leducq Transatlantic Network of Excellence program 08-CVD-03 (to L.J.D.W.).
Footnotes
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
DISCLOSURES
None
1. Chien KR, Olson EN. Converging pathways and principles in heart development and disease: CV@CSH. Cell. 2002;110:153–162. [PubMed]
2. Heineke J, Molkentin JD. Regulation of cardiac hypertrophy by intracellular signalling pathways. Nat Rev Mol Cell Biol. 2006;7:589–600. [PubMed]
3. Colella M, Grisan F, Robert V, Turner JD, Thomas AP, Pozzan T. Ca2+ oscillation frequency decoding in cardiac cell hypertrophy: role of calcineurin/NFAT as Ca2+ signal integrators. Proc Natl Acad Sci U S A. 2008;105:2859–2864. [PubMed]
4. Bueno OF, van Rooij E, Molkentin JD, Doevendans PA, De Windt LJ. Calcineurin and hypertrophic heart disease: novel insights and remaining questions. Cardiovasc Res. 2002;53:806–821. [PubMed]
5. Frey N, Katus HA, Olson EN, Hill JA. Hypertrophy of the heart: a new therapeutic target? Circulation. 2004;109:1580–1589. [PubMed]
6. Houser SR, Molkentin JD. Does contractile Ca2+ control calcineurin-NFAT signaling and pathological hypertrophy in cardiac myocytes? Sci Signal. 2008;1:pe31. [PMC free article] [PubMed]
7. Bers DM. Cardiac excitation-contraction coupling. Nature. 2002;415:198–205. [PubMed]
8. Wehrens XH, Lehnart SE, Marks AR. Intracellular calcium release and cardiac disease. Annu Rev Physiol. 2005;67:69–98. [PubMed]
9. Lehnart SE, Wehrens XH, Marks AR. Defective ryanodine receptor interdomain interactions may contribute to intracellular Ca2+ leak: a novel therapeutic target in heart failure. Circulation. 2005;111:3342–3346. [PubMed]
10. Wehrens XH, Lehnart SE, Reiken S, Vest JA, Wronska A, Marks AR. Ryanodine receptor/calcium release channel PKA phosphorylation: a critical mediator of heart failure progression. Proc Natl Acad Sci U S A. 2006;103:511–518. [PubMed]
11. Xu M, Zhou P, Xu SM, Liu Y, Feng X, Bai SH, Bai Y, Hao XM, Han Q, Zhang Y, Wang SQ. Intermolecular failure of L-type Ca2+ channel and ryanodine receptor signaling in hypertrophy. PLoS Biol. 2007;5:e21. [PMC free article] [PubMed]
12. Yamaguchi N, Takahashi N, Xu L, Smithies O, Meissner G. Early cardiac hypertrophy in mice with impaired calmodulin regulation of cardiac muscle Ca release channel. J Clin Invest. 2007;117:1344–1353. [PMC free article] [PubMed]
13. Kannankeril PJ, Mitchell BM, Goonasekera SA, Chelu MG, Zhang W, Sood S, Kearney DL, Danila CI, De Biasi M, Wehrens XH, Pautler RG, Roden DM, Taffet GE, Dirksen RT, Anderson ME, Hamilton SL. Mice with the R176Q cardiac ryanodine receptor mutation exhibit catecholamine-induced ventricular tachycardia and cardiomyopathy. Proc Natl Acad Sci U S A. 2006;103:12179–12184. [PubMed]
14. Chelu MG, Sarma S, Sood S, Wang S, van Oort RJ, Skapura DG, Li N, Santonastasi M, Muller FU, Schmitz W, Schotten U, Anderson ME, Valderrabano M, Dobrev D, Wehrens XH. Calmodulin kinase II-mediated sarcoplasmic reticulum Ca2+ leak promotes atrial fibrillation in mice. J Clin Invest. 2009;119:1940–1951. [PMC free article] [PubMed]
15. Rockman HA, Ross RS, Harris AN, Knowlton KU, Steinhelper ME, Field LJ, Ross J, Jr, Chien KR. Segregation of atrial-specific and inducible expression of an atrial natriuretic factor transgene in an in vivo murine model of cardiac hypertrophy. Proc Natl Acad Sci U S A. 1991;88:8277–8281. [PubMed]
16. Sood S, Chelu MG, van Oort RJ, Skapura D, Santonastasi M, Dobrev D, Wehrens XH. Intracellular calcium leak due to FKBP12.6 deficiency in mice facilitates the inducibility of atrial fibrillation. Heart Rhythm. 2008;5:1047–1054. [PMC free article] [PubMed]
17. van Oort RJ, van Rooij E, Bourajjaj M, Schimmel J, Jansen MA, van der Nagel R, Doevendans PA, Schneider MD, van Echteld CJ, De Windt LJ. MEF2 activates a genetic program promoting chamber dilation and contractile dysfunction in calcineurin-induced heart failure. Circulation. 2006;114:298–308. [PubMed]
18. Shannon TR, Ginsburg KS, Bers DM. Quantitative assessment of the SR Ca2+ leak-load relationship. Circ Res. 2002;91:594–600. [PubMed]
19. Yang J, Rothermel B, Vega RB, Frey N, McKinsey TA, Olson EN, Bassel-Duby R, Williams RS. Independent signals control expression of the calcineurin inhibitory proteins MCIP1 and MCIP2 in striated muscles. Circ Res. 2000;87:E61–E68. [PubMed]
20. Rothermel BA, Vega RB, Williams RS. The role of modulatory calcineurin-interacting proteins in calcineurin signaling. Trends Cardiovasc Med. 2003;13:15–21. [PubMed]
21. Chiang CS, Huang CH, Chieng H, Chang YT, Chang D, Chen JJ, Chen YC, Chen YH, Shin HS, Campbell KP, Chen CC. The CaV3.2 T-Type Ca2+ Channel Is Required for Pressure Overload-Induced Cardiac Hypertrophy in Mice. Circ Res. 2009;104:522–530. [PubMed]
22. Jaleel N, Nakayama H, Chen X, Kubo H, MacDonnell S, Zhang H, Berretta R, Robbins J, Cribbs L, Molkentin JD, Houser SR. Ca2+ influx through T- and L-type Ca2+ channels have different effects on myocyte contractility and induce unique cardiac phenotypes. Circ Res. 2008;103:1109–1119. [PMC free article] [PubMed]
23. Nakayama H, Chen X, Baines CP, Klevitsky R, Zhang X, Zhang H, Jaleel N, Chua BH, Hewett TE, Robbins J, Houser SR, Molkentin JD. Ca2+- and mitochondrial-dependent cardiomyocyte necrosis as a primary mediator of heart failure. J Clin Invest. 2007;117:2431–2444. [PMC free article] [PubMed]
24. Nakayama H, Wilkin BJ, Bodi I, Molkentin JD. Calcineurin-dependent cardiomyopathy is activated by TRPC in the adult mouse heart. FASEB J. 2006;20:1660–1670. [PMC free article] [PubMed]
25. Ohba T, Watanabe H, Murakami M, Takahashi Y, Iino K, Kuromitsu S, Mori Y, Ono K, Iijima T, Ito H. Upregulation of TRPC1 in the development of cardiac hypertrophy. J Mol Cell Cardiol. 2007;42:498–507. [PubMed]
26. Onohara N, Nishida M, Inoue R, Kobayashi H, Sumimoto H, Sato Y, Mori Y, Nagao T, Kurose H. TRPC3 and TRPC6 are essential for angiotensin II-induced cardiac hypertrophy. EMBO J. 2006;25:5305–5316. [PubMed]
27. Wu X, Zhang T, Bossuyt J, Li X, McKinsey TA, Dedman JR, Olson EN, Chen J, Brown JH, Bers DM. Local InsP3-dependent perinuclear Ca2+ signaling in cardiac myocyte excitation-transcription coupling. J Clin Invest. 2006;116:675–682. [PMC free article] [PubMed]
28. Ai X, Curran JW, Shannon TR, Bers DM, Pogwizd SM. Ca2+/calmodulin-dependent protein kinase modulates cardiac ryanodine receptor phosphorylation and sarcoplasmic reticulum Ca2+ leak in heart failure. Circ Res. 2005;97:1314–1322. [PubMed]
29. Bers DM, Eisner DA, Valdivia HH. Sarcoplasmic reticulum Ca2+ and heart failure: roles of diastolic leak and Ca2+ transport. Circ Res. 2003;93:487–490. [PubMed]
30. Yano M, Yamamoto T, Ikemoto N, Matsuzaki M. Abnormal ryanodine receptor function in heart failure. Pharmacol Ther. 2005;107:377–391. [PubMed]
31. Yano M, Yamamoto T, Ikeda Y, Matsuzaki M. Mechanisms of Disease: ryanodine receptor defects in heart failure and fatal arrhythmia. Nat Clin Pract Cardiovasc Med. 2006;3:43–52. [PubMed]
32. Marx SO, Gaburjakova J, Gaburjakova M, Henrikson C, Ondrias K, Marks AR. Coupled gating between cardiac calcium release channels (ryanodine receptors) Circ Res. 2001;88:1151–1158. [PubMed]
33. Reiken S, Gaburjakova M, Guatimosim S, Gomez AM, D'Armiento J, Burkhoff D, Wang J, Vassort G, Lederer WJ, Marks AR. Protein kinase A phosphorylation of the cardiac calcium release channel (ryanodine receptor) in normal and failing hearts. Role of phosphatases and response to isoproterenol. J Biol Chem. 2003;278:444–453. [PubMed]
34. Lehnart SE, Wehrens XH, Reiken S, Warrier S, Belevych AE, Harvey RD, Richter W, Jin SL, Conti M, Marks AR. Phosphodiesterase 4D deficiency in the ryanodine-receptor complex promotes heart failure and arrhythmias. Cell. 2005;123:25–35. [PMC free article] [PubMed]
35. Marx SO, Reiken S, Hisamatsu Y, Jayaraman T, Burkhoff D, Rosemblit N, Marks AR. PKA phosphorylation dissociates FKBP12.6 from the calcium release channel (ryanodine receptor): defective regulation in failing hearts. Cell. 2000;101:365–376. [PubMed]
36. Zhang T, Maier LS, Dalton ND, Miyamoto S, Ross J, Jr, Bers DM, Brown JH. The deltaC isoform of CaMKII is activated in cardiac hypertrophy and induces dilated cardiomyopathy and heart failure. Circ Res. 2003;92:912–919. [PubMed]
37. Damy T, Ratajczak P, Shah AM, Camors E, Marty I, Hasenfuss G, Marotte F, Samuel JL, Heymes C. Increased neuronal nitric oxide synthase-derived NO production in the failing human heart. Lancet. 2004;363:1365–1367. [PubMed]
38. Mochizuki M, Yano M, Oda T, Tateishi H, Kobayashi S, Yamamoto T, Ikeda Y, Ohkusa T, Ikemoto N, Matsuzaki M. Scavenging free radicals by low-dose carvedilol prevents redox-dependent Ca2+ leak via stabilization of ryanodine receptor in heart failure. J Am Coll Cardiol. 2007;49:1722–1732. [PubMed]
39. Terentyev D, Gyorke I, Belevych AE, Terentyeva R, Sridhar A, Nishijima Y, Carcache de Blanco E, Khanna S, Sen CK, Cardounel AJ, Carnes CA, Gyorke S. Redox modification of ryanodine receptors contributes to sarcoplasmic reticulum Ca2+ leak in chronic heart failure. Circ Res. 2008;103:1466–1472. [PMC free article] [PubMed]
40. Reiken S, Lacampagne A, Zhou H, Kherani A, Lehnart SE, Ward C, Huang F, Gaburjakova M, Gaburjakova J, Rosemblit N, Warren MS, He KL, Yi GH, Wang J, Burkhoff D, Vassort G, Marks AR. PKA phosphorylation activates the calcium release channel (ryanodine receptor) in skeletal muscle: defective regulation in heart failure. J Cell Biol. 2003;160:919–928. [PMC free article] [PubMed]
41. Reiken S, Wehrens XH, Vest JA, Barbone A, Klotz S, Mancini D, Burkhoff D, Marks AR. Beta-blockers restore calcium release channel function and improve cardiac muscle performance in human heart failure. Circulation. 2003;107:2459–2466. [PubMed]
42. Hasenfuss G, Pieske B. Calcium cycling in congestive heart failure. J Mol Cell Cardiol. 2002;34:951–969. [PubMed]
43. Bhuiyan ZA, Hamdan MA, Shamsi ET, Postma AV, Mannens MM, Wilde AA, Al-Gazali L. A novel early onset lethal form of catecholaminergic polymorphic ventricular tachycardia maps to chromosome 7p14-p22. J Cardiovasc Electrophysiol. 2007;18:1060–1066. [PubMed]
44. Chiu C, Tebo M, Ingles J, Yeates L, Arthur JW, Lind JM, Semsarian C. Genetic screening of calcium regulation genes in familial hypertrophic cardiomyopathy. J Mol Cell Cardiol. 2007;43:337–343. [PubMed]
45. Milting H, Lukas N, Klauke B, Korfer R, Perrot A, Osterziel KJ, Vogt J, Peters S, Thieleczek R, Varsanyi M. Composite polymorphisms in the ryanodine receptor 2 gene associated with arrhythmogenic right ventricular cardiomyopathy. Cardiovasc Res. 2006;71:496–505. [PubMed]
46. Fujino N, Ino H, Hayashi K, Uchiyama K, Nagata M, Konno T, Katoh H, Sakamoto Y, Tsubokawa T, Ohsato K, Mizuno S, Yamagishi M. A Novel Missense Mutation in Cardiac Ryanodine Receptor Gene as a Possible Cause of Hypertrophic Cardiomyopathy: Evidence From Familial Analysis. Circulation. 2006;114:II-165.
47. Fujino N, Shimizu M, Ino H, Yamaguchi M, Terai H, Sakato K, Kaneda T, Seidman CE, Seidman J. Missense Mutations in Cardiac Ryanodine Receptor Gene Cause Hypertrophic Cardiomyopathy Associated with Ventricular Arrhythmia of Massive Hypertrophy. Circ J. 2004;68:354.
48. Tavi P, Pikkarainen S, Ronkainen J, Niemela P, Ilves M, Weckstrom M, Vuolteenaho O, Bruton J, Westerblad H, Ruskoaho H. Pacing-induced calcineurin activation controls cardiac Ca2+ signalling and gene expression. J Physiol. 2004;554:309–320. [PubMed]
49. Xia Y, McMillin JB, Lewis A, Moore M, Zhu WG, Williams RS, Kellems RE. Electrical stimulation of neonatal cardiac myocytes activates the NFAT3 and GATA4 pathways and up-regulates the adenylosuccinate synthetase 1 gene. J Biol Chem. 2000;275:1855–1863. [PubMed]