|Home | About | Journals | Submit | Contact Us | Français|
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.
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.8–12 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
Cardiac function was assessed by echocardiography using a VisualSonics VeVo 770 Imaging System (VisualSonics, Toronto, Canada), as described.16
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).
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).
Real-time PCR was performed using a Mastercycler ep realplex (Eppendorf, Hamburg, Germany), as described before.17
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).
Results are expressed as mean ± SEM. A value of P<0.05 was considered statistically significant.
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).
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).
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.
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.
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).
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).
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 21–23 or store-operated Ca2+ channels 24–26, 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.28–30 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).32–34 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.35–40 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.
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.43–45 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.
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.
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.).
Publisher's Disclaimer: 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.