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Am J Physiol Heart Circ Physiol 293: H3122-H3129, 2007. First published September 7, 2007; doi:10.1152/ajpheart.00687.2007
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Calcineurin inhibition normalizes beta-adrenergic responsiveness in the spontaneously hypertensive rat

Scott M. MacDonnell,1 Hajime Kubo,1 David M. Harris,5 Xiongwen Chen,1 Remus Berretta,1 Mary F. Barbe,3 Stephen Kolwicz,2 Patricia O. Reger,2 Andrea Eckhart,5 Brian F. Renna,2 Walter J. Koch,5 Steven R. Houser,1,4 and Joseph R. Libonati1,2,4

1Cardiovascular Research Center and the Departments of 2Kinesiology, 3Physical Therapy, and 4Physiology, Temple University, Philadelphia; and 5Center for Translational Medicine, Thomas Jefferson University, Philadelphia, Pennsylvania

Submitted 13 June 2007 ; accepted in final form 6 September 2007


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Calcineurin, a Ca2+-regulated protein phosphatase, links myocardial Ca2+ signaling with hypertrophic gene transcription. Calcineurin abundance increases in pressure-overload hypertrophy and may reduce agonist-mediated phospholamban (PLB) phosphorylation to underlie blunted beta-adrenergic receptor (beta-AR) responsiveness in hypertension. This hypothesis was tested by measuring the effects of calcineurin inhibition on changes in cardiac contractility caused by beta-adrenergic stimulation in spontaneously hypertensive rats (SHR). Female SHR (age: 7 mo) and age-matched female Wistar-Kyoto rats (WKY) were studied. Heart weight-to-body weight ratio (P < 0.01) and systolic blood pressure (P < 0.01) were greater in SHR compared with WKY and were associated with increased myocardial calcineurin mRNA (CnAbeta) and activity (P < 0.05). beta-AR stimulation with isoproterenol (Iso) increased calcineurin activity (P < 0.05) in both WKY and SHR hearts, and this activity was suppressed with cyclosporin A (CsA) treatment. In SHR, CsA improved left ventricular whole heart and isolated myocyte beta-AR responsiveness by normalizing PLB phosphorylation at Ser16 and Thr17 (P < 0.05). These CsA-induced, PLB-mediated effects were associated with an augmentation in cardiomyocyte peak Ca2+ and a reduced rate (time constant of isovolumic pressure relaxation, tau) and magnitude of diastolic Ca2+ during beta-AR stimulation. In conclusion, CsA normalized the blunted beta-AR responsiveness associated with hypertension, in part, by mitigating calcineurin activity while improving PLB phosphorylation and subsequent sarcoplasmic reticulum Ca2+ regulation.

myocardium; hypertrophy; phosphatase; protein kinase A; calcium calmodulin kinase II

HYPERTENSION is a significant health concern, as it has increased 30% over the last decade and afflicts nearly 65 million Americans (8). Chronic hypertension induces pathological hypertrophy, which is associated with cardiomyocyte structural and functional abnormalities and the recapitulation of a fetal cardiac phenotype (4). Early in the hypertrophic cascade, significant alterations in Ca2+ signaling pathways have been observed (20). One such specific Ca2+-dependent signaling pathway involves calcineurin, the type 2B Ca2+ calmodulin-dependent phosphatase (9, 21, 34, 37). Calcineurin induces myocardial hypertrophy by activating hypertrophic target genes via dephosphorylation of transcription factors associated with the nuclear factor of activated T cells (NFAT) family and is well established in the pathogenesis of pressure-overload hypertrophy (9, 21, 34, 37).

Functionally, reduced beta-adrenergic receptor (beta-AR) responsiveness is a hallmark of pressure-overload cardiac hypertrophy (5, 7, 14, 18, 19, 29). Alterations in beta-AR responsiveness contribute to impaired Ca2+ handling and subsequent disturbances in myocardial inotropy and lusitropy, particularly during acute situations of hemodynamic stress. Mice overexpressing constitutively active calcineurin, a model of enhanced calcineurin activity independent of pressure overload, develop myocardial hypertrophy (25), hypercontractile myocytes (6, 25), and a reduced beta-AR inotropic response to both isoproterenol (Iso) and forskolin (25). These data indicate that calcineurin can impact beta-AR signaling downstream of the beta-ARs, possibly at the effector protein level since lowering extracellular Ca2+ concentration, and hence calcineurin activity, restored beta-AR responsiveness (25). beta-AR signaling appears to be compromised in a genetic model of enhanced calcineurin activity; however, this relationship has not been established in the experimental model that best mimics essential hypertension, the spontaneously hypertensive rat (SHR) (10, 36).

Calcineurin is colocalized to the t-tubules (38) and has previously been shown in normal myocardium to impair sarcoplasmic reticulum (SR) Ca2+ signaling (33) by opposing protein kinase A (PKA)-mediated phospholamban (PLB) phosphorylation (28, 31). However, to our knowledge, no study to date has interrogated the therapeutic potential of calcineurin inhibition to normalize beta-AR desensitization in pressure-overload hypertrophy. Thus, on the basis of previous work in normal myocardium, we hypothesized that beta-AR desensitization in SHR (19) would be improved by calcineurin inhibition with cyclosporin A (CsA). CsA antagonizes calcineurin by binding to the cytoplasmic receptor cyclophilin, forming immunophilin complexes, which are potent calcineurin inhibitors (17, 27, 35). Thus the purpose of the present study was to determine if calcineurin negatively regulates cardiomyocyte beta-AR responsiveness in hypertension through competitive dephosphorylation of specific Ca2+ regulatory proteins.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals

Female Wistar-Kyoto rats (WKY, n = 28) and female SHR (n = 27) were obtained from Charles River Laboratories (St.-Constant, Quebec, Canada) at 16 wk of age. All rats were housed three per cage, maintained on a 12:12-h light-dark cycle, and fed ad libitum (Harlan Teklad Global Diets, 18% Protein Diet, Madison, WI). Resting systolic blood pressures (mean of 25 cardiac cycles) were collected utilizing a tail-cuff apparatus before the rats were killed (Kent Scientific, Torrington, CT; XBP1000). At 28 wk of age, all animals were killed, and the studies were performed. All animal protocols were approved by the Animal Care and Use Committee of Temple University. All animals received humane care in compliance with Temple University standards and "Principles of Laboratory Animal Care" formulated by the National Society for Medical Research and the Guide for the Care and Use of Laboratory Animals prepared by the Institute of Laboratory Animal Resources and published by the National Institutes of Health (NIH Publication No. 85–23, Revised 1996).

RT-PCR mRNA Quantitation

Analysis of mRNA expression levels from untreated (basal) tissue (WKY, n = 6; SHR, n = 6) for calcineurin A protein (CnAbeta) and atrial natriuretic peptide (ANP) were performed with primers designed to detect rat gene products. CnAbeta primers were 5'-CCACAGGGATGTTGCCTAGTG (forward) and 5'-GTCCCGTGGTTCTCAGTGGTA (reverse) (32); ANP primers were 5'-TGCGGTAGAAGATGAGGTC (forward) and 5'-TGCTTTTCAAGAGGGCAGAT (reverse). RT-PCR reactions were performed with 1 µg of cDNA created from RNA using iScript (Bio-Rad) followed by 22 cycles of PCR amplification (annealing temperature 62.5°C) using a Bio-Rad iCycler. Appropriate melt curves were also run to ensure specificity of the product. The expression levels were compared to and normalized to levels of 28S: 5'-TTGAAAATCCGGGGGAGAG (forward) and 5'-ACATTGTTCCAACATGCCAG (reverse).

Immunoprecipitation/Western Blot Analysis

Following the completed isolated heart experiments and in a subset of untreated (basal) animals, an apical section of the left ventricle (LV) was frozen in liquid nitrogen. Basal hearts were perfused before collection of the apical section. These tissue sections were utilized for both immunoprecipitation and Western blot analysis. To demonstrate activated calcineurin, protein extracts were made from the LV apical sections in immunoprecipitation buffer (20 mmol/l Tris, 250 mmol/l NaCl, 1 mmol/l Ca2+, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 5 µg/ml pepstatin, 200 µg/ml benzamidine). For immunoprecipitation, 500 µg of protein extract was incubated with 5 µg of calmodulin rabbit polyclonal antibody (Zymed) in 100 µl at 4°C with gentle rocking for 12 h followed by the addition of 50 µl of protein A/G agarose (Santa Cruz) and another hour of incubation at 4°C. The samples were washed three times with 200 µl of immunoprecipitation buffer and subjected to SDS-PAGE. Immunodetection was then performed with a calcineurin antibody (Transduction Laboratories) followed by a calmodulin-specific antibody (Sigma).

Tissue protein abundance and phosphorylation levels in isolated protein were analyzed using Western blot analysis as previously described (19). Target antigens were probed with the non-phosphorylation-specific monoclonal antibodies, phospholamban total (PLBt) (Upstate Biotechnology) and sarcomeric actin (Sigma). Phosphorylation-specific polyclonal antibodies Ser16-PLB (Ser16) and Thr17-PLB (Thr17) (Badrilla) were also probed.

Films were scanned (UMAX PowerLook 1100), and band intensities were quantified with densitometric analysis using the Scion Image program (Alpha 4.0.3.2 [EC] ). To normalize blot-to-blot differences in protein loading or transfer efficiency, a common sample was included.

Isolated Rat Heart Preparation/Experimental Design

Langendorff isolated heart performance was determined as previously described (19). Briefly, rats were anesthetized with pentobarbital sodium (60 mg/kg ip) and heparinized intravenously (500 U iv). The heart was excised, trimmed of excess tissue, and rapidly immersed in cold (4°C) Ca2+-free Krebs-Henseleit buffer (KHB). Hearts were placed on a Langendorff perfusion apparatus (ML785B2, ADI Instruments, Colorado Springs, CO) and perfused at 16 ml/min (STH pump controller ML175, ADI Instruments) with a modified KHB (2 mmol/l Ca2+) solution (19). LV pressure (LVP), LV end-diastolic pressure (LVEDP), the maximum rate of positive and negative change in LVP (±dP/dt), and coronary perfusion pressure were continuously recorded by means of a data-acquisition system (Powerlab/8SP, ADInstruments, Colorado Springs, CO). LV developed pressure (LV Dev P) was calculated by subtracting the LVEDP from the LV systolic pressure. The time constant of isovolumic pressure relaxation (tau) was calculated using LVP and dP/dt, from peak –dP/dt to 5 mmHg above LVEDP, allowing a shift of the exponential baseline. A minimum of five pressure waves were analyzed to determine each data point.

Isoproterenol + CsA administration. As previously described, pre-agonist baseline data were recorded following equilibration. Five-minute infusions of isoproterenol (Iso, DMSO = 0.02%) were then initiated at concentrations ranging from 1 x 10–10 mol/l to 1 x 10–7 mol/l (WKY, n = 9; SHR, n = 8). Hearts were paced at 8.5 Hz for 20 s at baseline and at each Iso concentration. We examined LV performance during pacing to create conditions that should cause calcineurin activation (33). A second group of hearts underwent an identical Iso protocol (described above) concomitant with CsA infusion (CsA, 0.2 µmol/l, Santa Cruz Biotechnology). This dose of CsA in isolated rat hearts has been previously shown to protect against dopamine-induced apoptosis and ischemia-induced damage where FK506 was ineffective (11, 23). Briefly, after baseline with KHB, a 5-min infusion of CsA diluted in DMSO (final concentration of DMSO = 0.02%) was administered. Following CsA treatment, hearts were subjected to 5-min infusions of Iso + CsA (WKY, n = 8; SHR, n = 9).

Isolated Cell Preparation/Experimental Design

Myocyte isolation. LV myocytes were isolated as described previously (15, 16, 30). In brief, hearts were quickly explanted, weighed, and cannulated on a constant-flow Langendorff apparatus. The hearts (WKY, n = 5; SHR, n = 4) were perfused (16 ml/min) with nonrecirculating Ca2+-free KHB followed by 15–20 min of recirculated KHB supplemented with 180 U/ml collagenase. The hearts were removed from the cannula, and the atria, right ventricle, and septum were separated from the LV. The remaining LV tissue was gently minced and filtered. Initial yields from the cell isolation were consistently 70–80% rod-shaped (of total cell count). Cells were maintained at room temperature with a 5% CO2-95% O2 overlay. Throughout the isolation procedure, all solutions were equilibrated with 5% CO2-95% O2 and warmed to 37°C. All cells were studied within 6 h of isolation.

Physiological measurement. All experiments were performed on single rod-shaped myocytes with clear sarcomeric cross striations. In brief, isolated ventricular myocytes were loaded with fluo-3 AM and perfused with normal Tyrode solution (in mmol/l: 150 NaCl, 5 HEPES, 2 Na-pyruvate, 5.4 KCl, 1.2 magnesium chloride, pH 7.4) with 1 mmol/l Ca2+. Contractions were recorded by video edge detection (VED-104, Crescent Electronics, Sandy, UT). Initially, myocytes were paced at 1.0 Hz during a 10-min stabilization period. Myocytes were then paced at 3.0 Hz for 30 s, and pre-agonist baseline data were recorded (vehicle). Subsequently, a 5-min Iso infusion was initiated at 1 x 10–8 mol/l with and without pretreatment (5 min) with CsA (100 nmol/l, Santa Cruz Biotechnology) (28) diluted in DMSO. pCLAMP 8.2 software (Axon Instruments) was used for data acquisition and analysis.

Data Analysis and Interpretation

Animal characteristics, RT-PCR, immunoprecipitation, protein abundance, and baseline data were compared with one-way ANOVA followed by a Tukey post hoc analysis. Iso + CsA dose-response relationships in whole hearts and isolated cells were compared with ANOVA for repeated measures followed by one-way ANOVA and Tukey post hoc at each drug concentration. All analyses were performed on SPSS (SPSS, Chicago, IL, release 15.0.). Significance was set at an {alpha}-level of P < 0.05. Data are reported as means ± SE.


   RESULTS
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 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Calcineurin Mediates Pressure-Overload Hypertrophy and is Activated During beta-Adrenergic Stimulation

As displayed in Table 1, body weight was greater in WKY vs. SHR (P < 0.01). While absolute heart weight was similar between groups, a 13% increase in heart weight-to-body weight ratio was observed in SHR (P < 0.01). Systolic blood pressure was elevated in SHR (WKY 140 ± 2 vs. SHR 176 ± 3 mmHg, P < 0.001) consistent with our previously published work in this model (14, 19).


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  		Table 1. Animal characteristics

 
There are three known genes that encode catalytic calcineurin A protein: CnA{alpha}, CnAbeta, and CnA{gamma}. Previous work has shown that only CnAbeta was increased by stimulation with hypertrophic agonists (32). CnAbeta mRNA levels from LV basal tissue extracts were increased in SHR compared with control (Fig. 1A). We also assayed for ANP as a marker of the hypertensive phenotype. As expected, ANP mRNA levels were greater in SHR compared with control (Fig. 1A).


Figure 1
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  		Fig. 1. RT-PCR and immunoprecipitation. Following the completed isolated heart experiments and in a subset of untreated (basal) animals, RT-PCR and immunoprecipitation were performed. A: both calcineurin mRNA (CnAbeta; left) and atrial natriuretic peptide (ANP; right) levels relative to 28S molecules were increased in basal tissue from spontaneously hypertensive rats (SHR) vs. Wistar-Kyoto rats (WKY). B: levels of immunoprecipitated (IP) calcineurin (CnA) were increased with isoproterenol (Iso) and reduced with cyclosporin A (CsA; 0.2 µmol/l) treatment, suggesting that calcineurin was activated during beta-adrenergic receptor (beta-AR) stimulation and was sensitive to CsA. CaM, calmodulin. C: representative immunoprecipitated calcineurin and calmodulin blots are presented. Basal represents untreated tissue. WKY: n = 6 basal, n = 6 Iso, n = 6 Iso + CsA; SHR: n = 6 basal, n = 6 Iso, n = 6 Iso + CsA. Data are presented as means ± SE. *P < 0.05 vs. WKY basal, {dagger}P < 0.05 vs. Iso, {ddagger}P < 0.05 vs. SHR basal.

 
Calcineurin activity was elevated in LV protein extracts from basal SHR and Iso-treated hearts as demonstrated by calcineurin-specific Western blot after calmodulin immunoprecipitation (Fig. 1, B and C). Levels of immunoprecipitated calcineurin were normalized to levels of calmodulin coprecipitated. An increase in calcineurin protein abundance was observed in both WKY and SHR (P < 0.05) after Iso treatment. The increase in calcineurin abundance observed was attenuated (P < 0.05) in hearts treated with the calcineurin inhibitor CsA before Iso administration (Fig. 1, B and C).

PLB Phosphorylation is Regulated by Calcineurin

Total PLB abundance and serine16 (Ser16) and threonine17 (Thr17) phosphorylation were measured in basal tissues and from isolated hearts after treatment with Iso or Iso + CsA (Fig. 2). The total PLB (PLBt) protein levels were greater (30%) in SHR hearts compared with WKY (P < 0.05). No difference in basal phosphorylation state (Ser16, Thr17) was observed between WKY and SHR. Interestingly, Iso failed to increase PLB phosphorylation at both Ser16 and Thr17 in SHR, but a significant Iso response was noted in WKY (P < 0.05). Consistent with our previously published data, Iso-mediated PLB phosphorylation at both Ser16 and Thr17 was lower in SHR compared with WKY (19). However, beta-AR-mediated PLB phosphorylations (Ser16 and Thr17) were identical in SHR and WKY hearts treated with CsA (Fig. 2). These data suggest that in SHR, beta-AR-mediated activation of calcineurin negatively regulates the increase in the phosphorylation state of PLB caused by catecholamines.


Figure 2
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  		Fig. 2. Representative Western blots for total phospholamban (PLBt) abundance and phosphorylated phospholamban (PLB-Ser16 and PLB-Thr17) are presented. PLBt abundance was increased in SHR compared with WKY. Iso failed to increase PLB phosphorylation at both Ser16 and Thr17 in SHR, but a significant Iso response was noted in WKY. beta-AR-mediated (Iso) PLB phosphorylations at both Ser16 and Thr17 were lower in SHR compared with WKY; however, beta-AR-mediated PLB phosphorylations (Ser16 and Thr17) were normalized in SHR hearts treated with CsA. Basal represents untreated tissue. WKY: n = 6 basal, n = 7 Iso, n = 8 Iso + CsA; SHR: n = 6 basal, n = 7 Iso, n = 8 Iso + CsA. Data are presented as means ± SE. *P < 0.05 vs. WKY, {dagger}P < 0.05 vs. WKY basal, {ddagger}P < 0.05 vs. WKY Iso.

 
Calcineurin Dynamically Mediates beta-Adrenergic Responsiveness in the Hypertensive Myocardium

Langendorff baseline performance. Table 2 illustrates the baseline in vitro isolated heart performance in WKY and SHR without pacing (intrinsic heart rate) or with pacing (8.5 Hz). In the absence of pacing, LV end-diastolic volume was set to yield a LVEDP of 10 mmHg. At intrinsic hearts rates, SHR generated greater pressures than WKY (P < 0.01), similar to that observed in mice with cardiac-specific expression of constitutively active calcineurin (6, 25). Similar to our previously published data (19), a reduction in LV Dev P (P < 0.001) and an increase in both LVEDP (P < 0.001) and tau (P < 0.05) were observed during pacing, demonstrating impaired relaxation kinetics. Perfusion pressure was greater in SHR during both intrinsic and paced conditions (P < 0.01).


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  		Table 2. Left ventricular baseline performance

 
Langendorff beta-adrenergic responsiveness. beta-AR responsiveness (Iso) during pacing in both the presence and absence of CsA is depicted in Fig. 3. Blunted beta-AR responsiveness was evident in SHR (Fig. 3, A and B). In paced WKY hearts, Iso increased LV Dev P (Fig. 3A), while in SHR, LV Dev P fell to below baseline values with the administration of Iso (Fig. 3B). In contrast, Iso caused a positive inotropic response in CsA-treated SHR hearts (P < 0.05 vs. Iso) that was similar to the Iso response observed in WKY. The ±dP/dt responses to Iso both in the presence and absence of CsA were similar in WKY (Fig. 3, C and E). However, the ±dP/dt response was significantly improved with CsA in SHR at 10–9 mol/l Iso (Fig. 3, D and F).


Figure 3
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  		Fig. 3. Langendorff performance was established during an Iso dose response (1 x 10–10 to 1 x 10–7 mol/l) and pacing stress (8.5 Hz) in both the presence (Iso + CsA, filled symbols) and absence (Iso, open symbols) of the calcineurin inhibitor CsA (0.2 µmol/l). Administration of Iso (WKY, n = 9; SHR (B), n = 8) increased left ventricular developed pressure (LV Dev P) beyond baseline values in WKY (A) but failed to produce a significant inotropic effect in SHR (B). The combination of Iso + CsA (WKY, n = 8; SHR, n = 9) significantly improved LV Dev P in SHR compared with Iso alone (B). Data represent delta values from pacing baseline. Data are presented as means ± SE. Delta +dP/dt, delta maximum rate of positive change in LV pressure from pacing baseline in WKY (C) and SHR (D). Delta –dP/dt, delta maximum rate of negative change in LV pressure from pacing baseline in WKY (E) and SHR (F).*P < 0.05 vs. Iso + CsA.

 
Isolated myocyte function. In an effort to further define the mechanistic role of calcineurin in the hypertensive myocardium, myocyte contractions and Ca2+ transients were measured in myocytes isolated from both WKY and SHR myocardium. As displayed in Fig. 4, A and B, the hypercontractile phenotype observed in our isolated hearts was also present in SHR myocytes, which displayed greater maximal velocities of shortening/relengthening (±dL/dts) during baseline perfusion with normal Tyrode (vehicle). As presented in Fig. 4, A and B, Iso had significant inotropic and lusitropic effects (±dL/dt) in WKY but failed to elicit a response in SHR. However, consistent with the increased beta-AR responsiveness observed in CsA-treated Langendorff-perfused SHR hearts, CsA-treated (100 nmol/l) isolated myocytes (Iso + CsA) exhibited greater ±dL/dt (P < 0.05) in response to beta-AR with Iso vs. Iso treatment only (Fig. 4, A and B). Functionally, these data support the hypotheses that calcineurin acts as a negative regulator of contractile performance during beta-AR stimulation. As a control, CsA infusion in the absence of Iso failed to significantly alter myocyte function in WKY and SHR (data not shown).


Figure 4
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  		Fig. 4. A and B: average isolated myocyte maximal velocity of shortening/relengthening (±dL/dt) are presented. Compared with vehicle, Iso had significant inotropic and lusitropic effects (±dL/dt) in WKY but failed to elicit a response in SHR. However, in SHR, CsA-treated (100 nmol/l) myocytes (Iso + CsA) exhibited greater ±dL/dt values (P < 0.05) in response to Iso (Iso: 1 x 10–8 mol/l) (A and B). C–E: transient data. C: Iso (1 x 10–8 mol/l) increased peak Ca2+ in WKY but not SHR. Iso + CsA increased peak Ca2+ in both WKY and SHR (Iso + CsA). D: Iso (1 x 10–8 mol/l) reduced diastolic Ca2+ in WKY with no effect in SHR. Iso + CsA treatment significantly reduced diastolic Ca2+ in SHR. F/F0, fractional fluorescence change. E: time constant of Ca2+ transient decay was reduced with Iso in WKY; however, CsA was necessary to elicit a similar effect in SHR. Data from an average of 18 myocytes were recorded in each condition. Data are presented as means ± SE. Treatment comparisons are identified by lines at the following {alpha}-levels: *P < 0.05, {dagger}P < 0.01. {ddagger}P < 0.01 indicates WKY vehicle vs. SHR vehicle.

 
Isolated myocyte Ca2+ handling. As illustrated in Fig. 4C, administration of Iso (1 x 10–8 mol/l) significantly increased peak systolic Ca2+ in WKY but not in SHR. Consistent with the isolated myocyte functional studies, Iso + CsA increased peak systolic Ca2+ in SHR (Fig. 4C). Pacing (1–3 Hz) caused an increase in diastolic Ca2+ in both WKY [1.0 Hz, 0.90 ± 0.06 vs. 3.0 Hz, 1.44 ± 0.13 fractional fluorescence change (F/F0), P < 0.001] and SHR (1.0 Hz, 0.93 ± 0.05 vs. 3.0 Hz, 1.20 ± 0.07 F/F0, P < 0.001). Relative to vehicle, Iso treatment attenuated diastolic Ca2+ in WKY (Fig. 4D) but not in SHR (Fig. 4D). Iso + CsA diminished diastolic Ca2+ in SHR (P < 0.05) but not significantly in WKY (Fig. 4D). Iso treatment reduced tau in WKY but had no effect in SHR (Fig. 4E), while SHR myocytes pretreated with CsA exhibited a reduction in tau vs. Iso only (Fig. 4E). These data are in accordance with the Langendorff-isolated heart data in SHR where Iso treatment caused a reduction in LV Dev P possibly by causing an acute increase in intracellular Ca2+ concentration ([Ca2+]i) and the subsequent activation of calcineurin.


   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The novel finding of this study is that calcineurin antagonism increased beta-AR responsiveness in hypertension. These effects were seen in an animal model with persistent hypertension, hypertrophy, and increased calcineurin mRNA levels. Treatment with CsA in SHR improved cardiomyocyte Ca2+ regulation by increasing Ser16 and Thr17 PLB phosphorylation, which potentially augmented SR Ca2+ uptake and SR Ca2+ loading. These data suggest that in hypertension, activation of calcineurin is centrally involved in disrupted beta-AR signaling, in part via its actions on Ca2+ regulation.

To our knowledge, no data exist with respect to the functional role of calcineurin in the hypertensive heart. However, in other experimental models, calcineurin has been shown to oppose both PKA-mediated (31) and CamKII-mediated (22) phosphorylation of PLB and thereby alter SR Ca2+ loading (28). Thus we hypothesized that the depressed contractile responsiveness to beta-AR stimulation observed in hypertension (19) may be, in part, due to calcineurin's antagonistic action on PLB phosphorylation (22, 31).

It is well established that an increase in the [Ca2+]i transient is associated with the initiation of cardiac hypertrophy (21). Both elevated heart rates (33), as observed in vivo in SHR (19), and extrinsic hypertrophic stimuli, such as hypertension (1), can produce a sustained increase in myocardial [Ca2+]i and thereby activate calcineurin through interaction with Ca2+-calmodulin (21, 32). In the present investigation, we examined LV performance during pacing to create conditions that should cause calcineurin activation (33). Our results show that CsA treatment normalized beta-AR-mediated PLB phosphorylation in SHR and improved beta-AR-induced increases in contractility in whole hearts and isolated cardiomyocytes. The reduced diastolic Ca2+ and faster rate of decay of the Ca2+ transient in CsA-treated SHR cardiomyocytes suggest enhanced Ca2+ uptake by the SR consistent with the normalization of PLB phosphorylation. Our peak Ca2+ data also support that increased SR Ca2+ resequestration may have more optimally loaded the SR for subsequent Ca2+ release. However, we cannot rule out direct interaction of calcineurin on the ryranodine receptor or the L-type Ca2+ channel. Calcineurin may likewise control the phosphorylation status of these Ca2+ release mechanisms during hypertension (2).

It is clear that beta-AR-mediated Ca2+ regulation in cardiac myocytes is governed by numerous overlapping mechanisms. Work by Santana et al. (28) in normotensive mice demonstrates the functional coupling of PKA and calcineurin in ventricular myocytes. They observed increased myocyte Ca2+ transients, currents, and a threefold increase in Ca2+ sparks in CsA (100 nmol/l)-treated myocytes. Interestingly, caffeine-induced Ca2+ release was greater in CsA-treated myocytes, suggesting that inhibition of calcineurin increases whole cell [Ca2+]i transients, at least in part, through elevation of the SR Ca2+ content. Additionally, calcineurin inhibition in smooth muscle has been reported to increase Ca2+ influx by evoking new persistent Ca2+ sparklet sites and by increasing the activity of active sites (24). Depressed Na+/Ca2+ exchanger activity observed in phenylephrine-treated neonatal myocytes has also been shown to be reversed by acute inhibition of calcineurin with little effect on myocyte hypertrophic phenotype (13). Taken together, these data suggest that calcineurin acts not only as a critical mediator of myocardial hypertrophy, but also appears to be a multipotent phosphatase with an important functional role in myocardial E-C coupling.

Recent data in mice expressing constitutively active calcineurin demonstrate myocardial hypertrophy (25) and a hypercontractile phenotype (6, 25) with impaired beta-AR signaling (25), results of which are similar to our model of spontaneous hypertension. However, in contrast to our molecular profile, Chu et al. (6) showed reduced total PLB and increased phosphorylated PLB (Ser16) with enhanced SR Ca2+ handling in calcineurin transgenic mice. Similarly, an enhanced force-frequency relationship was observed in calcineurin transgenic mice, which suggests an increased resequestration rate of Ca2+ by the SR (25). These data are in contrast to the pacing-induced decrease in LV Dev P observed in the present investigation and may be due to differences in the animal models utilized. In myocardial hypertrophy induced by progressive pressure overload, alterations in metabolic, structural, and hormonal regulation occur as adaptational responses to the increased hemodynamic pressures. In our model, increased calcineurin mRNA and activity are secondary to elevated circulating catecholamines (12) and increased intracellular Ca2+ (1). These differences may be responsible for the disparity observed between these studies.

The SHR model was selected for our study because it resembles essential hypertension in humans. We chose to study these animals at 7 mo of age because cardiac function is well maintained and fibrosis is not yet accelerated (3, 26). However, there are several limitations that may affect the interpretability of the present study. First, the intracellular actions of CsA are not limited to calcineurin inhibition. CsA also increases SR Ca2+ leak and L-type Ca2+ channel activity (34). Second, the results of our study are limited to acute CsA treatment such that chronic CsA treatment may confer a differential response. Additionally, to remain consistent with our previous work, we utilized an elevated bath Ca2+ (2 mmol/l), which may have impacted our ability to detect a significant beta-AR effect. Last, our data are limited to the female SHR of which the estrus cycle was not controlled.

In summary, our study suggests that calcineurin is activated in persistent hypertension. Activated calcineurin is then responsible for many cellular effects, including induction of LV hypertrophy. The new finding of this study is that calcineurin antagonism increased beta-AR responsiveness in hypertension. Our data suggest that acute inhibition of calcineurin increases LV beta-AR responsiveness in SHR by normalizing phosphorylation of the Ca2+ regulatory protein PLB. These data suggest that in hypertension, calcineurin acts as a critical mediator of reduced inotropic reserve in addition to its role as a hypertrophic signaling molecule. The identification of calcineurin as a multipotent molecular modulator of hypertrophy and contractility makes it an attractive therapeutic target in hypertrophic heart disease.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by a postdoctoral fellowship grant from the American Heart Association, Pennsylvania Delaware Affiliate (0625509U), Beginning-Grant-in-Aide from the American Heart Association, Mid-Atlantic Affiliate (J. R. Libonati), and National Heart, Lung, and Blood Institute Grants HL-33921 (S. R. Houser), HL-61690 (W. J. Koch), and HL-56205 (W. J. Koch).


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. R. Libonati, Temple Univ., 122 Pearson Hall, 1800 N. Broad St., Philadelphia, PA 19122 (e-mail: jlibonat{at}temple.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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