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Calcium dynamics in the failing heart: restoration by -adrenergic receptor blockade

Divisions of ¹Molecular Cardiovascular Biology and ²Cardiology, The Children's Hospital and Research Foundation, Cincinnati 45229; ³Department of Physiology and Cell Biology, College of Medicine and Public Health, The Ohio State University, Columbus, Ohio 43210; ⁴Cardiovascular Research Institute and Department of Medicine, New Jersey Medical School, University of Medicine and Dentistry of New Jersey, Newark, New Jersey 07103; ⁵Department of Physiology, University of Wisconsin Medical School, Madison, Wisconsin 53706; and ⁶Research Center, Montreal Heart Institute, Montreal, Quebec, Canada H1T 1C8

Submitted 20 May 2002 ; accepted in final form 11 March 2003

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Changes in calcium (Ca²⁺) regulation contribute to loss of contractile function in dilated cardiomyopathy. Clinical treatment using -adrenergic receptor antagonists (-blockers) slows deterioration of cardiac function in end-stage heart failure patients; however, the effects of -blocker treatment on Ca²⁺ dynamics in the failing heart are unknown. To address this issue, tropomodulin-overexpressing transgenic (TOT) mice, which suffer from dilated cardiomyopathy, were treated with a nonselective -receptor blocker (5 mg · kg^-1 · day^-1 propranolol) for 2 wk. Ca²⁺ dynamics in isolated cardiomyocytes of TOT mice significantly improved after treatment compared with untreated TOT mice. Frequency-dependent diastolic and Ca²⁺ transient amplitudes were returned to normal in propranolol-treated TOT mice and but not in untreated TOT mice. Ca²⁺ kinetic measurements of time to peak and time decay of the caffeine-induced Ca²⁺ transient to 50% relaxation were also normalized. Immunoblot analysis of untreated TOT heart samples showed a 3.6-fold reduction of sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA), whereas Na⁺/Ca²⁺ exchanger (NCX) concentrations were increased 2.6-fold relative to nontransgenic samples. Propranolol treatment of TOT mice reversed the alterations in SERCA and NCX protein levels but not potassium channels. Although restoration of Ca²⁺ dynamics occurred within 2 wk of -blockade treatment, evidence of functional improvement in cardiac contractility assessed by echocardiography took 10 wk to materialize. These results demonstrate that -adrenergic blockade restores Ca²⁺ dynamics and normalizes expression of Ca²⁺-handling proteins, eventually leading to improved hemodynamic function in cardiomyopathic hearts.

cardiomyopathy; -adrenergic receptor antagonists; dilated

Altered Ca²⁺ dynamics associated with heart failure impede normal contractile function, resulting in poor cardiac output. Inefficient myocardial function is compensated for by neuroendocrine release of norepinephrine and epinephrine, which stimulate -adrenergic receptors. Associated G_s-coupled protein activates adenylyl cyclase, causing cAMP production. Accumulation of cAMP increases protein kinase A activity, causing phosphorylation of several downstream substrates that influence Ca²⁺ cycling and contractility. These substrates include the sarcolemmal L-type Ca²⁺ channel and phospholamban in the SR. Phosphorylation of phospholamban increases SERCA activity, thereby promoting SR Ca²⁺ accumulation. Although initially increasing inotropy, chronic -receptor signaling may ultimately be damaging to the failing myocardium by altering Ca²⁺-regulatory protein levels and/or function, leading to impaired Ca²⁺ cycling (1, 9, 13, 30).

Human cardiac dysfunction exhibits altered Ca²⁺-handling protein levels (8, 22). Immunoblot and patchclamp analyses of samples from failing human hearts possess increased NCX (37), reduced SERCA (22), and possibly reduced L-type Ca²⁺ channel density (35, 45). Recorded Ca²⁺ transients of cardiomyocytes from failing hearts show prolonged Ca²⁺ reuptake and frequency-dependent elevation in diastolic Ca²⁺ levels (29, 42, 43). Normalizing these regulatory proteins restores intracellular Ca²⁺ dynamics and reestablishes improved function to the failing myocardium (10, 28, 41).

Once believed to be contraindicated for heart failure, -blockers are now known to be beneficial for patients with mild to moderate dilated and ischemic cardiomyopathy (7, 26, 28). Contractile dysfunction of patients with dilated cardiomyopathy is normalized through chronic (>8 mo) -adrenergic receptor blockade. It is possible that -adrenergic receptor blockade indirectly normalizes Ca²⁺-regulatory proteins, resulting in improved intracellular Ca²⁺ cycling and, in turn, reversing cardiac dysfunction (7, 28). However, the temporal progression of changes in Ca²⁺ dynamics and Ca²⁺-regulatory proteins at the beginning states of -blocker therapy remains unknown.

The tropomodulin-overexpressing transgenic (TOT) mouse is an experimental model for dilated cardiomyopathy, exhibiting poor hemodynamics, sarcomeric remodeling, ventricular wall thinning, and altered Ca²⁺ transients comparable to those in human heart failure (7, 45). Restorative effects of -blockade on Ca²⁺ handling and regulatory proteins in TOT mice after a brief 2-wk treatment with propranolol were assessed by rapid line-scanning confocal microscopy as well as by biochemical and echocardiographic analyses. Results demonstrate that -blockade restores Ca²⁺ dynamics and Ca²⁺-handling protein content relatively quickly, but improvement of cardiac contractile function lags behind, suggesting that long-term remodeling processes are required in addition to normalization of Ca²⁺ handling.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Heart failure model. TOT FVB/N mice 4–6 wk of age were injected intraperitoneally with (N = 25) or without (N = 25) propranolol (Wyeth-Ayerst) totaling 5 mg · kg^-1 · day^-1 for 14 days. Nontransgenic (NTG) mice (N = 15) served as controls. For long-term -blockade experiments, propranolol or metoprolol (both from Sigma Chemical; St. Louis, MO) was administered in tap water (both at 0.5 g/l) that was refreshed every third day. All aspects of animal care and experimentation performed in this study were approved by the Institutional Animal Care and Use Committee of the Children's Hospital Medical Center.

Myocyte isolation and fluo 3-AM loading. Hearts were excised from isoflurane-anesthetized FVB/N normal mice and cannulated onto a modified Langendorff perfusion apparatus. Hearts were perfused in low-Ca²⁺ minimal essential medium (S-MEM, Joklik's modified medium, GIBCO-BRL Life Technologies; Bethesda, MD) for 5 min at 37°C and then switched to S-MEM containing 0.7 mg/ml collagenase D (Boehringer-Mannheim) for 7–15 min. Both solutions were gassed with 95% O₂-5% CO₂ and adjusted to pH 7.4 with 5 N NaOH. Dissociated cells were loaded with fluo 3-AM (Molecular Probes; Eugene, OR) in a final concentration of 2 µmol/l in 2% horse serum-S-MEM. Cells were washed twice and deesterified in S-MEM. For TOT mice, dilated hearts were perfused (5 min) and digested (7–10 min) with 0.7 mg/ml collagenase D in a Ca²⁺-and bicarbonate-free Hanks' buffer with HEPES (CBFHH) solution containing (in mmol/l) 137 NaCl, 5.36 KCl, 0.81 MgSO₄, 20.06 HEPES, 0.44 K₂PO₄, and 0.34 Na₂HPO₄. Both solutions were gassed with 95% O₂-5% CO₂ and adjusted to pH 7.4 with 5 N NaOH. Different methods for isolation of cardiomyocytes from normal and TOT mice were necessary to preserve Ca²⁺-handling function. Isolation of NTG cardiomyocytes using CBFHH solution resulted in unresponsive cells on resuspension in 1 mmol/l Ca²⁺-Tyrode solution. In contrast, TOT cells were unable to regulate Ca²⁺ release in low-Ca²⁺ S-MEM digestion due to their inherent compromised Ca²⁺ regulation and enhanced sparking activity, resulting in irreversible contracture. These two different protocols were used to maintain structural integrity, rod shape morphometry, and stimulation responsiveness of cells in these two types of mice. Dissociated cells were loaded in a final concentration of 2 µmol/l fluo 3-AM in 2% horse serum-CBFHH solution. The myocytes were washed twice and deesterified in CBFHH solution.

Ca²⁺ transient recordings. Fluo 3-AM-loaded myocytes were attached to a laminin-coated glass coverslip in a flow-through chamber and bathed in 1 mmol/l Ca²⁺-modified Tyrode solution (pH 7.4) containing (in mmol/l) 140 NaCl, 2 KCl, 0.5 MgCl₂, 1 CaCl₂, 10 HEPES, 0.25 NaH₂PO₄, 5.6 glucose, 10 Na-pyruvate, and 5 L-carnitine, with 5 mg/l insulin. Ca²⁺ transients of fluo 3-AM-loaded myocytes were measured by rapid line-scan confocal microscopy (Molecular Dynamics CLSM 2010) using a x40/1.40 oil objective. Image analysis was performed using Imagespace software (version 3.2) on an Indy platform (Silicon Graphics; Mountain View, CA). Line scans were performed at 100 Hz. Myocytes were field stimulated using parallel platinum wires by 7-ms pulses 20% above threshold. Ca²⁺ transients were recorded after steady-state pacing at 1 Hz, and line-scan images were acquired at frequencies of 0.5 and 2.0 Hz.

Measurement of intracellular Ca²⁺ concentration transients. Intracellular Ca²⁺ concentrations were determined relative to Ca²⁺ calibration curves generated by confocal microscopy. After acquisition of Ca²⁺ transient data, 100-µl aliquots of fluo 3-AM-loaded myocytes from the same batch of cells used for Ca²⁺ measurements were placed in separate conical vials, each containing a known Ca²⁺ concentration of 0, 300, 500, 700, or 1,000 nmol/l Ca²⁺ using Ca²⁺ Calibration Kit Concentrate (Molecular Probes). The K_d value for Ca-EGTA was adjusted according to pH, ionic strength, and temperature to arrive at the appropriate standard concentrations (6, 21). In addition, each vial contained 10 µmol/l calcimycin (Molecular Probes), 50 mmol/l 2,3-butanedione monoxime, 200 µmol/l dinitrophenol, 140 mmol/l NaCl, 2 mmol/l KCl, 0.5 mmol/l MgCl₂, 10 mmol/l HEPES, 0.25 mmol/l NaH₂PO₄, 5.6 mmol/l glucose, 10 mmol/l Na⁺-pyruvate, 5 mmol/l L-carnitine, and 5 mg/l insulin. Ca²⁺ standardizations were all performed using functionally inactivated cardiomyocytes. The myocytes were allowed to equilibrate in each Ca²⁺ concentration, transferred to slides, and scanned by confocal microscopy. Increasing fluo 3-AM fluorescence corresponded linearly to increasing Ca²⁺ concentration. Fluo 3-AM fluorescence was represented by pixel intensity ranging from 0 to 255 in a pseudocolor format. Images for each Ca²⁺ transient were optimized by photomultiplier adjustment to use the full linear range of pseudocolor pixel intensity. Live cell Ca²⁺ transient recordings were normalized to corresponding calibration curves. The linear regression equation derived from the Ca²⁺ calibration curve converted Ca²⁺ transient pixel intensity to intracellular Ca²⁺ concentration (in nmol/l) for all stimulated cardiomyocytes. The integrated intracellular Ca²⁺ concentration at 0.5 Hz was determined by gravimetric analysis. For SR Ca²⁺ loading, cardiomyocytes were paced at 0.5 or 2.0 Hz until steady state. Cells were rapidly exposed to 10 mmol/l caffeine in Na⁺-free/Ca²⁺-free Tyrode solution using a rapid solution exchanger device (53). The peak of the Ca²⁺ transient induced by caffeine was used as an index of SR load (24).

SERCA function was measured in isolated cardiomyocytes by blocking NCX function and then exposing the cells to a short pulse of 10 mmol/l caffeine as previously described (52). Briefly, cells were paced at 0.5 Hz for 1 min under a stream of 1 mmol/l Ca²⁺-Tyrode solution using a rapid solution exchanger device. The stream was switched to Ca²⁺-free Tyrode solution for 1 min and then captured in a stream of Na⁺-free/Ca²⁺-free solution to inactivate NCX protein, with complete replacement of Na⁺ by 140 mmol/l Li⁺. Cells were abruptly exposed to a short pulse of 10 mmol/l caffeine in the Na⁺-free/Ca²⁺-free solution for 100 ms. SERCA function was determined as the time decay (in ms) of the caffeine-induced Ca²⁺ transient to 50% (t₅₀) of the amplitude.

Protein immunoblot analysis. Hearts were collected from mice at 6–8 wk of age. The hearts were frozen in liquid nitrogen, pulverized, and suspended in lysis buffer (10 mmol/l Tris · HCl, 5 mmol/l EDTA, 50 mmol/l NaCl, 30 mmol/l Na-pyrophosphate, 50 mmol/l NaF, 100 µmol/l Na-orthovanadate, 1% Triton X-100, 1 mmol/l PMSF, 10 nmol/l cypermethrin, 10 nmol/l okadaic acid, 100 µmol/l phenylarsine oxide, 1 mmol/l dithiothreitol, 10 µg/ml pepstatin, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 10 µg/ml N-()-p-tosyl-L-lysine-chloromethyl-ketone, and 10 µg/ml N-tosyl-L-phenylalanyl-chloromethyl-ketone) and sonicated. Fifty micrograms per lane of protein were mixed with an equal volume of sample buffer and boiled for 5 min before being loaded onto a 10% SDS-PAGE gel for electrophoresis. Separated proteins were transferred to Hybond transfer membranes (Amersham Pharmacia Biotech; Piscataway, NJ) using a Bio-Rad Trans-Blot Cell at 145 V for 3 h at 4°C. Transferred proteins were stained with 0.2% Ponceau S (BP103-10, Fisher; Pittsburgh, PA) solution containing 0.3% TCA to detect protein bands. The blot was rinsed in water and washed in Tris-buffered saline-Tween 20 (TBS-T) buffer [50 mmol/l Tris · HCl (pH 7.6), 150 mmol/l NaCl, and 0.1% Tween 20] and blocked in 5% BSA-TBS-T solution for 1 h. Primary antibodies for SERCA and NCX (both from Affinity Bioreagents; Golden, CO) were incubated with blots overnight at 4°C. The next day, membranes were washed three times for 5 min in TBS-T solution, and alkaline-phosphatase secondary antibody (Amersham Pharmacia Biotech) in TBS-T solution was added for 1 h at 25°C. Membranes were washed and analyzed by chemiflourescence using Molecular Dynamics Storm 860. Quantitation of SERCA and NCX protein content was performed using ImageQuant 1.2 software provided with the Storm 860. Protein loading was standardized versus the analysis of actin in a similar fashion to SERCA and NCX using anti-actin antibody (C4 clone, kindly provided by Jim Lessard, Children's Hospital Medical Center). Actin protein expression for standardization was comparable to the expression of the housekeeping enzyme glyceraldehyde-3-phosphate dehydrogenase (RDI Research Diagnostics; Flanders, NJ) in cardiac lysates (data not shown).

Electrophysiological measurements. Left ventricular (LV) myocytes were isolated by previously described methods (32). Whole cell currents were recorded using patch-clamp techniques. Cell capacitance was measured using voltage ramps of 0.8 V/s from a holding potential of -50 mV. Ca²⁺ currents (I_Ca) were recorded using an external solution containing (in mmol/l) 2 CaCl₂, 1 MgCl₂, 135 tetraethylammonium chloride, 5 4-aminopyridine, 10 glucose, and 10 HEPES (pH 7.3). The pipette solution contained (in mmol/l) 100 Cs-aspartate, 20 CsCl, 1 MgCl₂, 2 MgATP, 0.5 GTP, 5 EGTA, and 5 HEPES (pH 7.3). These solutions provided isolation of Ca²⁺ currents from other membrane currents and the NCX exchanger. We (6, 21) have previously shown that Ca²⁺-dependent inactivation properties can be reliably measured under these experimental conditions. The method of recording NCX current was similar to that previously described by Kimura et al. (27). The external solution contained (in mmol/l) 150 NaCl, 2 CsCl, 2 MgCl_2, 1 CaCl₂, 10 glucose, 5 HEPES, and 0.002 nifedipine (pH 7.3 with 1 N CsOH). For the Li⁺ external solution, equimolar LiCl was replaced with NaCl. The pipette solution contained (in mmol/l) 20 NaOH, 10 CsOH, 50 aspartic acid, 1 MgCl₂, 2 MgATP, 42 EGTA, and 5 HEPES (pH 7.4 with 1 N CsOH). The concentration of free internal Ca²⁺ was adjusted to 67 nmol/l by adding 21 mmol/l CaCl₂. For the measurement of K⁺ currents, nifedipine (1 µmol/l) was added to block I_Ca, and the patch pipette solution contained (in mmol/l) 110 K⁺ aspartate, 20 KCl, 2 MgCl₂, 2 ATP, 0.5 GTP, 5 EGTA, and 5 HEPES (pH 7.3 with 1 N KOH).

[³H]ryanodine binding assays. [³H]ryanodine binding was conducted in 200 mmol/l KCl, 20 mmol/l NaHEPES (pH 7.2), 1 mmol/l EGTA, and variable CaCl₂ concentrations to yield the specified free Ca²⁺. Homogenates (50 µg) of control mice, TOT mice, or both groups treated with propranolol were incubated for 90 min at 36°C with the indicated concentrations of [³H]ryanodine. Samples (total volume 100 µl) were filtered through Whatman GF/C filters, washed twice with 5 ml of deionized water, and dried before being counted.

Antibodies. Antibodies used for immunoblots recognized the phosphorylated form of the RyR (P-S2809, Badrilla), SERCA, NCX (Affinity Bioreagents), and -actin (clone C4, a gift from Jim Lessard, Childrens Hospital Research Foundation).

Ribonuclease protection assay. Measurement of mRNA levels of selected K⁺ channel isoforms was performed using a RNase protection assay as previously described (50).

Statistics. Results are expressed as means ± SE. Comparisons between groups were evaluated using ANOVA, with P < 0.05 considered significant.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Ca²⁺ dynamics are impaired in TOT cardiomyocytes but restored by -blockade. Rapid line-scanning confocal microscopy was used to observe Ca²⁺ transients from fluo 3-AM-loaded isolated cardiomyocytes. Representative line scans (Fig. 1A, top) and the corresponding Ca²⁺ transient profile (Fig. 1A, bottom) show that electrically paced TOT cells have prolonged Ca²⁺ transients at 1.0-Hz stimulation (Fig. 1A, middle). Propranolol treatment of cardiomyopathic TOT mice restored Ca²⁺ handling (Fig. 1A, top right) and transients (Fig. 1A, bottom right). Identical propranolol treatment of NTG mice had no effect on Ca²⁺ dynamics (data not shown). Subcellular Ca²⁺ localization was investigated in whole cell transients (Fig. 1B). Overlay comparison of cell center (CC) with subsarcolemmal level (SS) Ca²⁺ transient profiles showed equal Ca²⁺ handling throughout the interior of NTG cardiomyocytes (Fig. 1B, top left and bottom left). In contrast, TOT cells showed a differential in Ca²⁺ handling between CC and SS (Fig. 1B, bottom middle), with the CC exhibiting delayed Ca²⁺ reuptake over the SS at 0.5 Hz stimulation. However, propranolol-treated TOT mice showed normalized subcellular Ca²⁺ gradients at the CC and SS (Fig. 1B, top right and bottom right).

View larger version (46K): [in this window] [in a new window]   Fig. 1. Characterization of Ca2+ transients. Nontransgenic (NTG), tropomodulin-overexpressing transgenic (TOT), and propranolol (Pro)-treated TOT (TOT Pro) cells were electrically paced to record intracellular Ca2+ dynamics using confocal microscopy. A: cardiomyopathic TOT cells show altered Ca2+ handling similar to human heart failure cells (18). Untreated TOT show prolonged Ca2+ reuptake (middle). Short-term Pro treatment restores Ca2+ dynamics in TOT cells (right). Left, NTG cells. B: representative line scans and Ca2+ transient profile at cell center (CC; black) and subsarcolemmal level (SS; red) for NTG (left), TOT (middle), and Pro-treated TOT cells (right). Intracellularly, TOT cells show prolonged Ca2+ reuptake at the CC compared with SS (middle). Two-week Pro treatment normalizes subcellular Ca2+ localization in TOT cells (right) compared with untreated TOT cells (middle).

Normalization of frequency-dependent Ca²⁺ dynamics in TOT cardiomyocytes by -blockade. To understand Ca²⁺ handling at different stimulation rates, we examined frequency-dependent Ca²⁺ dynamics in isolated cardiomyocytes electrically paced at 0.5 and 2.0 Hz. Compared with NTG cells, TOT cells demonstrated elevated diastolic Ca²⁺ levels at 2.0 Hz (Fig. 2A, middle) but not at 0.5 Hz. Two weeks of propranolol treatment normalized Ca²⁺ handling in isolated TOT cardiomyocytes (Fig. 2A, bottom). Prolonged action potential coupled with impaired Ca²⁺ reuptake leads to loss of coordinated Ca²⁺ cycling in TOT cells at stimulation frequencies >2.0 Hz, unlike NTG cardiomyocytes, which could be paced at frequencies as high as 10 Hz while maintaining appropriate Ca²⁺ dynamics (data not shown).

View larger version (42K): [in this window] [in a new window]   Fig. 2. Frequency-dependent Ca2+ dynamics. A: representative Ca2+ transient profiles acquired from line-scan images of cells paced at 0.5 and 2.0 Hz. Untreated TOT cells (middle) show elevated cytosolic Ca2+ levels at 2.0 Hz, whereas Pro-treated TOT cells (bottom) exhibit improved diastolic Ca2+ levels. Top, NTG cells. B: peak systolic Ca2+ levels at 0.5 and 2.0 Hz for NTG, TOT, and Pro-treated TOT cells. C: diastolic Ca2+ levels in NTG, TOT, and Pro-treated TOT cells at 0.5 and 2.0 Hz. Diastolic Ca2+ levels of untreated TOT cells are significantly elevated at 2.0 Hz compared with NTG cells (*P < 0.001). -Blockade decreased frequency-induced Ca2+ elevation at 2.0 Hz in treated TOT mice (**P < 0.00001). Untreated TOT and Pro-treated TOT mice also show a significant reduction of diastolic Ca2+ level relative to NTG controls (#P < 0.01). D: Ca2+ amplitude at 0.5 and 2.0 Hz in NTG, TOT, and Pro-treated TOT cardiomyocytes. Ca2+ transient amplitude was significantly reduced in TOT cardiomyocytes at 2.0 Hz compared with NTG cardiomyocytes (*P < 0.001). Pro-treated TOT cardiomyocytes show normalization of Ca2+ amplitude (**P < 0.001 vs. TOT). NTG: 0.5 Hz, N = 14, and 2.0 Hz, N = 10; TOT: 0.5 Hz, N = 22, and 2.0 Hz, N = 20; Pro-treated TOT: 0.5 Hz, N = 25, and 2.0 Hz, N = 22.

Normalized diastolic Ca²⁺ levels in TOT cardiomyocytes by -blockade. Diastolic and systolic Ca²⁺ concentrations were calculated from field-stimulated cells at 0.5 and 2.0 Hz. Stimulation frequencies at 2.0 Hz showed no differences in peak systolic Ca²⁺ concentration among the NTG, TOT, and propranolol-treated TOT groups (690.7 ± 97.4 nmol/l in the NTG group versus 686.2 ± 78.4 nmol/l in the TOT group or 667.4 ± 77.4 nmol/l in the propranolol-treated TOT group, P > 0.05; Fig. 2B). NTG cardiomyocytes showed comparable diastolic Ca²⁺ concentrations at 0.5 and 2.0 Hz (120.6 ± 41.8 nmol/l, N = 14, and 149.7 ± 36.1 nmol/l, N = 10; respectively; Fig. 2C). Untreated TOT cardiomyocytes showed a significant rise in diastolic Ca²⁺ concentrations concomitant with higher stimulation frequency (266.6 ± 95.8 nmol/l at 2.0 Hz, N = 21, P < 0.001 vs. NTG). Propranolol treatment reduced the diastolic Ca²⁺ level in TOT cardiomyocytes to levels below comparably stimulated NTG cells (116.1 ± 41.3 nmol/l at 2.0 Hz, N = 23). The reduction of diastolic Ca²⁺ by propranolol was significant when untreated TOT cells were compared with treated TOT cells (P < 0.001).

Ca²⁺ transient amplitudes are normalized in TOT cardiomyocytes by -blockade. Ca²⁺ amplitude release at 0.5 and 2.0 Hz was obtained by calculating the difference between systolic and diastolic Ca²⁺ concentrations at 0.5 and 2.0 Hz (Fig. 2D). At 0.5 Hz, there was no significant difference (P > 0.05) in Ca²⁺ amplitudes between NTG, propranolol-treated TOT, and untreated TOT cardiomyocytes (NTG, 591.7 ± 111.3 nmol/l; TOT, 535.0 ± 69.0 nmol/l; propranolol-treated TOT, 561.1 ± 94.8 nmol/l). However, at 2.0 Hz, propranolol-treated TOT cardiomyocytes showed normalized Ca²⁺ amplitude compared with untreated TOT cardiomyocytes (551.3 ± 75.9 vs. 441.7 ± 95.7 nmol/l, respectively, P < 0.00001), demonstrating that Ca²⁺ transients are restored with -blockade treatment. There was no difference (P > 0.05) between NTG and propranolol-treated TOT cardiomyocytes (NTG, 541.0 ± 95.4 nmol/l) at 2.0 Hz.

Normalized caffeine-induced SR Ca²⁺ release. The results of Ca²⁺ transient amplitude calculations showed that Ca²⁺ release from the SR was normalized with propranolol treatment. Caffeine-induced Ca²⁺ release was examined to determine frequency-dependent SR Ca²⁺ loading. At 2.0 Hz, SR loading in cardiomyocytes from untreated TOT mice was significantly reduced compared with NTG cells (653.7 ± 127.2 nmol/l in TOT cardiomyocytes vs. 826.2 ± 98.1 nmol/l in NTG cardiomyocytes, P < 0.0001; Fig. 3). With propranolol treatment, SR loading was restored in TOT cardiomyocytes (856.2 ± 64.2 nmol/l) relative to untreated TOT cardiomyocytes (P < 0.0001). Lower stimulation frequency of cardiomyocytes at 0.5 Hz failed to decrease SR Ca²⁺ loading in TOT cells (NTG, 876.5 ± 69.0 nmol/l; TOT, 864.7 ± 137.2 nmol/l; propranolol-treated TOT, 861.2 ± 71.7 nmol/l; P > 0.05).

View larger version (15K): [in this window] [in a new window]   Fig. 3. Caffeine-induced sarcoplasmic reticulum (SR) Ca2+ release. NTG, TOT, and Pro-treated TOT cells were stimulated at 0.5 and 2.0 Hz and then exposed to 10 mmol/l caffeine. The caffeine-induced Ca2+ transient was used as an index of SR load. Low-frequency (0.5 Hz) stimulation produced no differences among the three groups (NTG, N = 11; TOT, N = 25; Pro-treated TOT, N = 22). However, at a stimulation frequency of 2.0 Hz, untreated TOT cells (N = 27) demonstrate impaired Ca2+ reuptake, as evidenced by decreased caffeine-induced SR Ca2+ release compared with NTG cells (N = 11). -Blockade reverses impairment of Ca2+ reuptake and restores SR loading in Pro-treated TOT cardiomyocytes (N = 22) after 2 wk of therapy. (*P < 0.001 vs. NTG; **P < 0.001 vs. TOT).

Ca²⁺ kinetics are restored in TOT cardiomyocytes by -blockade. Human failing hearts demonstrate altered Ca²⁺ kinetics in isolated cardiomyocytes with delayed time to peak and prolonged Ca²⁺ transient relaxation [t₅₀ (28)]. Similarly, TOT cardiomyocytes showed delayed time to peak and prolonged relaxation (Fig. 4, A and B). The onset of the peak Ca²⁺ transient is delayed in TOT cells compared with nonfailing cells (TOT, 137.5 ± 70.7 ms; NTG, 58.6 ± 22.9 ms; P < 0.001; Fig. 4A). However, cells from propranolol-treated TOT mice showed a significant reduction in time to peak compared with cells from untreated TOT mice, which were comparable to NTG cells (propranolol-treated TOT, 70.2 ± 29 ms, P < 0.0001 vs. TOT; P > 0.05 vs. NTG). Propranolol-treated TOT cells showed significantly improved Ca²⁺ reuptake compared with untreated TOT cells (t₅₀: propranolol-treated TOT, 387.5 ± 74.6 ms; TOT, 647.8 ± 90.7 ms; NTG, 345.1 ± 131.1 ms; P < 0.00001, propranolol-treated TOT vs. untreated TOT; P > 0.05, propranolol-treated vs. NTG; P < 0.000001, NTG vs. untreated TOT; Fig. 4B). Integration of Ca²⁺ transients demonstrated that -blockade reversed chronically elevated cytosolic Ca²⁺ concentrations per second in treated TOT cells (propranolol-treated TOT, 80.0 ± 26.9 nmol · l^-1 · s^-1; TOT, 123.0 ± 26.3 nmol · l^-1 · s^-1; NTG, 87.3 ± 22.4 nmol · l^-1 · s^-1; P < 0.0001, propranolol-treated TOT vs. untreated TOT; P > 0.05, propranolol-treated TOT vs. NTG; P < 0.001, NTG vs. untreated TOT; Fig. 4C). SERCA function was measured using confocal microscopy in isolated cardiomyocytes by inactivating NCX function and then exposing the cells to a short pulse of 10 mmol/l caffeine (Fig. 4D). SERCA-mediated Ca²⁺ reuptake was significantly prolonged in untreated TOT cells (3,050 ± 797 ms) compared with NTG (1,194 ± 495 ms). Similar to results for analysis of Ca²⁺ transients (Fig. 4, A–C), propranolol treatment normalized SERCA function in treated TOTs (1,235 ± 423 ms).

View larger version (19K): [in this window] [in a new window]   Fig. 4. Ca2+ transient kinetics. A: time to peak for NTG, untreated TOT, and Pro-treated TOT cells. TOT cells show prolonged time to peak for Ca2+ transients compared with NTG cells. Delayed time to peak is restored after Pro treatment in TOT cells. B: relaxation time (t50) for Ca2+ transients at 0.5 Hz. Altered Ca2+ resequestration in untreated TOT cells was demonstrated by prolonged relaxation time compared with NTG cells. C: Ca2+ concentration per s at 0.5 Hz. Integration of Ca2+ transient peaks show untreated TOT cells exhibit elevated Ca2+ levels over time. This was normalized with Pro treatment of TOT cells. NTG, N = 14; TOT, N = 22; Pro-treated TOT, N = 25. *P < 0.001 vs. NTG; **P < 0.001 vs. TOT. D: sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) function in isolated cardiomyocytes. Inactivation of the Na+/Ca2+ exchanger (NCX) under a stream of Na+-free/Ca2+-free Tyrode solution was followed by a short pulse (100 ms) of caffeine solution (10 mmol/l) using a rapid solution exchanger device. SERCA function is significantly impaired in TOT cardiomyocytes and is restored by Pro treatment NTG, N = 10; TOT, N = 11; Pro-treated TOT, N = 10. Values are means ± SE. *P < 0.00001 vs. NTG; **P < 0.00001 vs. TOT; not significant, NTG vs. TOT Pro.

Ca²⁺-regulatory proteins are normalized in TOT cardiomyocytes by -blockade. Protein content levels of SERCA and NCX were examined by immunoblot analysis. TOT hearts demonstrated a 2.4-fold increase in NCX protein content compared with NTG hearts (Fig. 5, A and B). Conversely, SERCA protein content was reduced by 3.6-fold in untreated TOT hearts (Fig. 5, C and D). -Blockade treatment of TOT mice restored SERCA (Fig. 5, C and D) and NCX (Fig. 5, A and B) levels to normal levels. Identical propranolol treatment of normal mice had no effect on SERCA or NCX protein levels (data not shown).

View larger version (28K): [in this window] [in a new window]   Fig. 5. SERCA and NCX immunoblots. A and B: NCX immunoblot (A) and bar graph (B). NCX level was 2.4-fold higher in TOT than NTG mice (*P < 0.05) but not statistically different from the level in Pro-treated TOT mice (P > 0.05). C and D: SERCA immunoblot (C) and bar graph (D). SERCA protein levels are reduced 3.6-fold in the TOT heart compared with the NTG heart (*P < 0.05). Pro-treated TOT hearts exhibit normalized SERCA (**P < 0.05 vs. TOT). SERCA and NCX values were normalized to actin to correct for minor variations in sample loading.

L-type Ca²⁺channel function and density are normalized in TOT cardiomyocytes by -blockade. Ca²⁺ entry in isolated cardiomyocytes was investigated using whole cell patch clamp as previously described (40). Figure 6A illustrates representative I_Ca and channel density of cardiomyocytes from NTG, TOT, and propranolol-treated TOT hearts. Cellular remodeling of TOT cells is consistent with increased cell capacitance, which was unaffected by propranolol treatment (Fig. 6B). In contrast, reduced I_Ca density in TOT mice was restored by propranolol treatment (Fig. 6C), as was L-type Ca²⁺ channel inactivation (Fig. 6D). Ryanodine exposure dissolved differences in channel inactivation (half-decay) in NTG, untreated TOT, and propranololtreated TOT cardiomyocytes (Fig. 6E).

View larger version (24K): [in this window] [in a new window]   Fig. 6. L-type Ca2+ channel. A: representative L-type Ca2+ channel currents in NTG, TOT, and Pro-treated TOT ventricular mouse myocytes. Traces show currents elicited from a holding potential of -50 mV to the indicated test potentials at 0.1 Hz. Averaged current-voltage relationships of peak inward Ca2+ currents normalized to the cell capacitance (in pA/pF) is plotted at the bottom. B–E: average cell capacitance (B), Ca2+ current density (C), half-decay time (T1/2) of the current (D), and T1/2 in the presence of ryanodine (10 µmol/l; E), which normalized the current inactivation time (*P < 0.05 vs. NTG, **P < 0.05 vs. TOT). Test potential was +10 mV and holding potential was -50 mV. All patch-clamp experiments were performed at room temperature. The numbers above the bars refer to the number of cells recorded in each sample.

-Blockade reduces NCX current. Extrusion of cytosolic Ca²⁺ is primarily regulated by NCX. Voltageclamped cardiomyocytes were switched from 150 mmol/l Na⁺ to 150 mmol/l Li⁺ to generate an outward exchange current at +40 mV (representative NCX recorded currents, Fig. 7, A and B). NCX current density was calculated by dividing the exchanger current by the cell capacitance (Fig. 7C). Supporting by Western blot analysis, untreated TOT cells demonstrated elevated NCX current density compared with NTG cells. -Blockade reduced NCX current density in dilated TOT hearts (Fig. 7C) but was not significant compared with untreated TOT hearts.

View larger version (20K): [in this window] [in a new window]   Fig. 7. NCX current density. NCX exchange current measured from NTG, TOT, and Pro-treated TOT ventricular mouse myocytes is shown. A and B: examples of the NCX exchange currents recorded in NTG (A) and TOT (B) myocytes. The exchange current was estimated as the outward current when the external solution was changed from 150 mmol/l Na+ to 150 mmol/l Li+ measured at +40 mV. C: current density was calculated by dividing the peak outward current by the capacitance of the cell (in pA/pF). Data are means ± SE. *P < 0.05 vs. NTG.

RyR phosphorylation unchanged by -blockade. Altered density and/or function of RyRs may produce important changes in intra-SR Ca²⁺ and intracellular Ca²⁺ homeostasis. We performed [³H]ryanodine binding assays and Western blot analyses to measure density, Ca²⁺ dependence, and the phosphorylation state of RyRs. The latter two parameters are among the most important indicators of RyR function (5, 38). Western blots probed with a RyR antibody specific for phosphorylated RyR did not demonstrate a difference between propranolol-treated and untreated groups (Fig. 8, A and B). The equilibrium [³H]ryanodine binding curves and Ca²⁺ dependence of [³H]ryanodine binding showed no difference between groups (Fig. 8, C and D, respectively). Thus RyRs appeared to play no significant role in the improvement of Ca²⁺ dynamics of the TOT model induced by -adrenergic blockade.

View larger version (33K): [in this window] [in a new window]   Fig. 8. Ryanodine receptors (RyRs) in TOT mice. A: Western blots of homogenates (50 µg each) from control (NTG), TOT, Pro-treated NTG (NTG Pro), and Pro-treated TOT mice probed with an anti-RyR2 antibody (P-S2809) that recognizes the phosphorylated form of the RyR2 only (Badrilla). B: pooled results from Western blot experiments. RyR band intensity measurements from NTG, TOT, Protreated NTG, and Pro-treated TOT samples (N = 4 for each group) are plotted as bars. Band intensity values from samples were not statistically different. a.u., Arbitrary units. C: equilibrium [3H]ryanodine binding curves from pooled NTG, TOT, Pro-treated NTG (NTGp), and Pro-treated TOT (TOTp) groups (n = 4 each). The maximal number of RyR sites (Bmax) was 0.07 ± 0.008, 0.09 ± 0.011, 0.09 ± 0.012, and 0.07 ± 0.082 pmol/mg protein, respectively. The dissociation constant (Kd) of the [3H]ryanodine-RyR complex was 1.1 ± 0.4, 2.0 ± 0.6, 1.3 ± 0.5, and 1.7 ± 0.5 nmol/l, respectively. Kd and Bmax values were not statistically different among groups. D: Ca2+ dependence of [3H]ryanodine binding curves showed no difference among groups.

K⁺ currents were unchanged in TOT hearts after -blockade. The dramatic restoration of SERCA, NCX, and L-type Ca²⁺ channel led us to investigate whether -blockade affects ion channels outside those involved in Ca²⁺ regulation. TOT hearts demonstrated low expression of outward K⁺ currents and corresponding cardiac K⁺ channel isoforms (Fig. 9A). Outward K⁺ currents were unchanged after propranolol treatment (Fig. 9B).

View larger version (21K): [in this window] [in a new window]   Fig. 9. K+ channel mRNA expression and K+ current density. A, left: bar graphs comparing the relative abundance of Kv1.5 (top) and Kv4.2 (bottom) mRNA transcripts in NTG and TOT mouse ventricular muscle, determined by RNase protection assay. Values were normalized to the -actin signal. Relative abundance was calculated with the value from NTG mice as a reference of 100%. *P < 0.001 vs. NTG. Right, representative autoradiographs of mRNA levels for the K+ channel isoforms measured in the two NTG and two TOT RNA samples (2.5 µg total RNA/sample). Yeast represents the negative control (yeast total RNA). B, top: transient outward K+ currents (Ito) recorded in NTG (left) and TOT (right) myocytes. Representative families of currents elicited by voltage steps from -60 to +60 mV in 20-mV increments from a holding potential of -80 mV are shown. Current-voltage relationships in NTG, TOT, and Pro-treated TOT myocytes are summarized at the bottom. The bottom left graph shows current amplitude at the peak (open symbols) and the bottom right graph shows that of the end of 300-ms pulses (filled symbols). The current amplitudes were normalized to the cell capacitance to give current densities (in pA/pF). Data points are means ± SE; NTG, N = 42; TOT, N = 52; Pro-treated TOT, N = 70. #P < 0.05 vs. TOT and Pro-treated TOT.

Cardiac function improved in TOT mice after 10 wk of -blocker therapy. Echocardiographic analysis demonstrated that TOT cardiac function did not improve after 2 wk of treatment with -blockers as our Ca²⁺ analysis results would suggest (data not shown). Because functional improvements after the initiation of -blockade therapy may take longer than molecular phenotype reversal to manifest, TOT mice were subjected to extended -blockade therapy, and functional improvement was monitored bimonthly by echocardiography. Function improvement as assessed by the shortening fraction became significant (P < 0.02) after 10 wk of metoprolol or propranolol treatment (Fig. 10). Thus restoration of Ca²⁺ dynamics occurs more rapidly, followed later by functional improvement.

View larger version (13K): [in this window] [in a new window]   Fig. 10. Functional improvement in TOT mice treated with -blockade. Percentage values for the shortening fraction derived from echocardiographic analysis of TOT mice before (Pre) and after (Post) treatment with -blockers for 10 wk are shown. Both metoprolol and propranolol treatment improve cardiac contractility significantly, as evidenced by greater fractional shortening (27% and 35% improvement, respectively). NTG mice treated comparably showed no significant change in shortening fraction (data not shown). *Significant differences between pre- and posttreatment values for each group, with actual P values as shown.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The beneficial effects of chronic -adrenergic receptor blockade on myocardial pump function are well documented (2, 3, 7, 12, 15, 17, 20, 25, 36, 39, 49, 52). With regard to Ca²⁺ handling, normal dynamics are restored in cardiomyocytes from heart failure patients chronically treated (>8 mo) with -blockers (28). Our study demonstrates that propranolol-treated TOT mice showed improved Ca²⁺ transient characteristics compared with untreated TOT mice after a brief 2-wk therapy (Fig. 2B) and functional improvement after 10 wk (Fig. 10). Additionally, we also found significantly improved frequency-dependent Ca²⁺ handling in isolated cardiomyocytes from -blockade-treated mice. Specifically, abnormally high diastolic Ca²⁺ levels at higher stimulation rates were normalized in the treated group (Fig. 4C). Perhaps one major mechanistic action of -blockers is to reduce cytosolic Ca²⁺ levels, because in vivo heart rates for a mouse would exceed that used in our experiments by at least fourfold. Maintaining lower diastolic Ca²⁺ levels over time could alleviate activation of maladaptive Ca²⁺-dependent pathways associated with the pathophysiology of heart failure (33). Our study begins to uncover the inhibition of such mechanisms through the action of -blockade.

SERCA, NCX, and L-type Ca²⁺ channel current density levels were normalized by propranolol treatment, explaining the improved Ca²⁺ handling in treated TOT mice. The shared normalization of SERCA, NCX, and L-type Ca²⁺ channel by -blockade in our heart failure model suggests that common regulatory pathways or Ca²⁺-handling proteins may exist downstream of -receptors (14, 16). SERCA and L-type Ca²⁺ channel demonstrated enhanced restoration in protein content compared with NCX, suggesting that SERCA and L-type Ca²⁺ channel regulation may be linked to -adrenergic blockade given the short duration of treatment (16). These findings are in agreement with other studies that report improved SERCA and NCX levels after -blockade in different heart failure models (19, 28, 47).

SERCA is responsible for restoring SR Ca²⁺ load per excitation-contraction cycle. Decreasing SERCA content is associated with reduced SR Ca²⁺ loading and elevated cytoplasmic Ca²⁺ levels (10, 24). Ito et al. (24) showed that stabilizing SERCA levels prevent alterations in SR and cytosolic Ca²⁺ content, attenuating the transition to heart failure in pressure-overloaded mice. The relatively brief 2-wk propranolol treatment normalized SERCA levels from a 3.6-fold deficit (Fig. 5, C and D), improving SR (Fig. 3) and diastolic Ca²⁺ levels (Fig. 2C) in failing TOT hearts. Reducing cytosolic Ca²⁺ over time may dampen or reverse Ca²⁺-dependent signaling cascades that lead to cardiomyopathy (34). Restoration of SERCA levels is likely to be a critical factor in normalization of Ca²⁺ uptake in our line-scan and frequency-dependent experiments. Other salutatory changes could also involve decreased NCX expression and posttranslational modifications such as the phosphorylation state.

An alternative mechanism for reduction of cytosolic Ca²⁺ is extrusion from the cell via the NCX. Upregulation of NCX in heart failure has been proposed as a compensatory reaction for reduced SERCA levels (23, 48). Maladaptive changes in NCX protein elicit frequency-dependent functional abnormalities in muscle strips (22) and cellular preparations (42). -Blockers reverse rising NCX levels in failing human hearts (28), suggesting that normalizing this protein may improve myocardial function. Similarly, prolonged -adrenergic blockade in TOT hearts restored NCX to near control levels and may have influenced the observed improvement of cardiac function at 10 wk (Fig. 10).

L-type Ca²⁺ channel levels are transcriptionally regulated and directly targeted by the -adrenergic pathway (14). Response to -blockade was evident in propranolol-treated TOT cardiomyocytes, which showed normalized channel current density, kinetics (Fig. 6A), and time to peak in Ca²⁺ transient measurements (Fig. 4A). Additionally, normalized channel current density has been associated as a contributing factor for improving Ca²⁺ amplitude (5), supporting our data in treated TOT mice (Fig. 2D). Although -adrenergic transcriptional regulation of SERCA, NCX, and L-type Ca²⁺ channel remains obscure (11, 14, 16), our results indicate a dynamic interplay exists between -blockade and reversal of maladaptive Ca²⁺-handling protein expression, as recently shown to occur in human dilated cardiomyopathy (31). Others (11, 38) report that -blockade stabilizes FK506 binding protein 12.6 binding to the RyR, resulting in reduced Ca²⁺ channel leak and cytosolic Ca²⁺ overload, attenuating heart failure. Increased Ca²⁺ leak due to altered RyR function can also contribute to impaired Ca²⁺ resequestration. Indeed, we observed increased sparking activity in cardiomyocytes from TOT mice compared with NTG cells, reflecting RyR openings. However, alterations of density, Ca²⁺ dependence, or hyperphosphorylation of the RyR did not play a role in this phenomenon (Fig. 8), suggesting that L-type Ca²⁺ channel activity in the failing TOT cell may be a factor in this event (44). Unlike Ca²⁺-regulatory proteins, K⁺-regulatory proteins were unaffected by -blockade (Fig. 9B), indicating that inhibition of -adrenergic signaling does not normalize abnormalities in K⁺-regulatory proteins in this model of heart failure.

The relatively brief 2-wk treatment of TOT mice with ₁- and ₂-receptor blockade improves Ca²⁺ handling but does not lead to markedly improved cardiac function as assessed by echocardiography (data not shown). This finding suggests that either 1) the 2-wk duration of -blockade administration was insufficient to restore function, 2) reversal of preexisting remodeling cannot be accomplished by restoration of Ca²⁺ dynamics, or 3) severity of remodeling in the TOT model renders it incapable of increasing hemodynamic function despite improved Ca²⁺ handling. Of course, improvements in hemodynamic function may be too subtle to be revealed by echocardiographic analyses. Cardiomyocytes from failing TOT mice showed substantial cellular remodeling as shown by cell capacitance measurements (Fig. 6B). Indeed, whole cell patch-clamp analysis failed to demonstrate a reversal in cellular remodeling in propranolol-treated TOT mice, which may partially explain the persistence of impaired hemodynamic function after 2-wk -receptor blockade. Analysis of hemodynamic function in humans suffering from dilated cardiomyopathy indicates that 6 mo of -blockade therapy improves function in 80% of patients (31). This supports our analysis that 10 wk of treatment in TOT mice improved hemodynamic function as assessed by echocardiography (Fig. 10). Furthermore, preliminary results indicate that prophylactic treatment of suckling pups prevents development of dilated cardiomyopathy in TOT litters (unpublished results), indicating that TOT cardiomyopathy is caused in part by -adrenergic overdrive.

In summary, -receptor blockade dramatically restored Ca²⁺ regulation after a brief 2-wk in vivo treatment in our TOT mouse model of heart failure. Our results are the first to show rapid normalization of Ca²⁺-handling protein content and Ca²⁺ regulation in a dilated cardiomyopathic model without a concomitant improvement in cardiac function. -Adrenergic regulation for these proteins requires further studies to elucidate a possible mechanism to explain the restoration from a failing state. The influence of ₁- and ₂-specific receptor blockade on Ca²⁺-regulatory proteins will hopefully provide further insight regarding the relationship of Ca²⁺-handling proteins and Ca²⁺ dynamics to -adrenergic signaling.

	ACKNOWLEDGMENTS

 
C. Fiset thanks Chantale St. Michel for technical assistance with the manuscript.

This work was supported by National Institutes of Health Grants HL-58224, HL-66035, and HL-67245 (to M. A. Sussman), GM-54169 and HL-61476 (to A. Yatani), HL-64140 (to M. Periasamy), PO1 HL-47053 (to H. H. Valdivia), and HL07752 (to D. M. Plank) and by American Heart Association Ohio Valley Affiliate Predoctoral Training Grant 0110127B (to D. M. Plank) National Grant 0040051N (to M. A. Sussman). C. Fiset is a Research Scholar of the Heart and Stroke Foundation of Canada. M. A. Sussman is an Established Investigator of the American Heart Association

	FOOTNOTES

 

Address for reprint requests and other correspondence: M. A. Sussman, Div. of Molecular and Cardiovascular Biology, Rm. 3033, The Children's Hospital and Research Foundation, 3333 Burnet Ave., Cincinnati, OH 45229 (E-mail: sussman{at}heart.chmcc.org).

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 MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

1. Antos CL, Frey N, Marx SO, Reiken S, Gaburjakova M, Richardson JA, Marks AR, and Olson EN. Dilated cardiomyopathy and sudden death resulting from constitutive activation of protein kinase A. Circ Res 89: 997-1004, 2001.
2. Aoyagi T, Yonekura K, Eto Y, Matsumoto A, Yokoyama I, Sugiura S, Momomura S, Hirata Y, Baker DL, and Periasamy M. The sarcoplasmic reticulum Ca²⁺-ATPase (SERCA2) gene promoter is decreased in response to severe left ventricular pressure-overload hypertrophy in rat hearts. J Mol Cell Cardiol 31: 919-926, 1999.
3. Asai K, Yang G, Geng Y, Takagi G, Bishop S, Ishikawa Y, Shannon RP, Wagner TE, Vatner DE, Homcy CJ, and Vatner SF. -Adrenergic receptor blockade arrests myocyte damage and preserves cardiac function in the transgenic G_s mouse. J Clin Invest 104: 551-558, 1999.
4. Barrere-Lamaire S, Piot C, Leclercq F, Nargeot J, and Richard S. Facilitation of L-type calcium currents by diastolic depolarization in cardiac cells: impairment in heart failure. Cardiovasc Res 47: 336-349, 2000.
5. Bers DM. Excitation-Contraction Coupling and Cardiac Contractile Force. Dordrecht, The Netherlands: Kluwer, 1991.
6. Bers DM, Patton CW, and Nuccitelli R. A practical guide to the preparation of Ca²⁺ buffers. In: Methods in Cell Biology, edited by Nuccitelli R. San Diego, CA: Academic, 1994, p. 4-28.
7. Bristow MA. -Adrenergic receptor blockade in chronic heart failure. Circulation 88: 895-902, 2000.
8. Bueckelmann DL, Nabauer M, and Erdmann E. Intracellular calcium handling in isolated ventricular myocytes from patients with terminal heart failure. Circulation 85: 1046-1055, 1992.
9. Dash R, Kadambi FJ, Schmidt AG, Tepe NM, Biniakiewicz D, Gerst MJ, Canning AM, Abraham WT, Hoit BD, Liggett SB, Lorenz JN, Dorn GW, and Kranias EG. Interactions between phospholamban and -adrenergic drive may lead to cardiomyopathy and early mortality. Circulation 103: 889-896, 2001.
10. Del Monte F, Harding SE, Schmidt U, Matsui T, Kang ZB, Dec GW, Gwathmey JK, Rosenzweig A, and Hajjar RJ. Restoration of contractile function in isolated cardiomyocytes from failing hearts by gene transfer of SERCA2a. Circulation 100: 2308-2311, 1999.
11. Doi M, Yano M, Kobayashi S, Kohno M, Tokuhisa T, Okuda S, Suetsugu M, Hisamatsu Y, Ohkusa T, Kohno T, Michihiro M, and Matsuzaki M. Propranolol prevents the development of heart failure by restoring FKBP12.6-mediated stabilization of ryanodine receptor. Circulation 105: 1374-1379, 2002.
12. Eichhorn EJ, Heesch CM, Barnett JH, Alvarez LG, Fass SM, Grayburn PA, Hatfield BA, Marcoux LG, and Malloy CR. Effect of metoprolol on myocardial function and energetics in patients with nonischemic dilated cardiomyopathy: a randomized, double-blind, placebo-controlled study. J Am Coll Cardiol 24: 1310-1320, 1994.
13. Engelhardt S, Hein L, Wiesmann F, and Lohse MJ. Progressive hypertrophy and heart failure in ₁-adrenergic receptor transgenic mice. Proc Natl Acad Sci USA 96: 7059-7064, 1999.
14. Fan QI, Chen B, and Marsh JD. Transcriptional regulation of L-type calcium channel expression in cardiac myocytes. J Mol Cell Cardiol 32: 1841-1849, 2000.
15. Glass MG, Fuleihan F, Liao R, Lincoff AM, Chapados R, Hamlin R, Apstein CS, Allen PD, Ingwall JS, Hajjar RJ, Cory DR, O'Brien PJ, and Gwathmey JK. Differences in cardioprotective efficacy of adrenergic receptor antagonists and Ca²⁺ channel antagonists in animal model of dilated cardiomyopathy. Effects of gross morphology, global cardiac function, and twitch force. Circ Res 73: 1077-1089, 1993.
16. Golden KL, Ren J, O'Connor J, Dean A, Dicarlo SE, and Marsh JD. In vivo regulation of Na/Ca exchanger expression by adrenergic effectors. Am J Physiol Heart Circ Physiol 280: H1376-H1382, 2001.
17. Gwathmey JK. Morphological changes associated with furazolidone-induced cardiomyopathy: effects of digoxin and propranolol. J Comp Pathol 104: 33-45, 1991.
18. Gwathmey JK, Copelas L, MacKinnon R, Schoen FJ, Feldman MD, Grossman W, and Morgan JP. Abnormal intracellular calcium handling in myocardium from patients with end-stage heart failure. Circ Res 61: 70-76, 1987.
19. Gwathmey JK, Kim CS, Hajjar RJ, Khan F, DiSalvo TG, Matsumori A, and Bristow MR. Cellular and molecular remodeling in heart failure model treated with -blocker carteolol. Am J Physiol Heart Circ Physiol 276: H1678-H1690, 1999.
20. Harding VB, Jones LR, Lefkowitz RJ, Koch WJ, and Rockman HA. Cardiac ARK1 inhibition prolongs survival and augments -blocker therapy in a mouse model of severe heart failure. Proc Natl Acad Sci USA 97: 5809-5814, 2001.
21. Harrison SM and Bers DM. Correction of proton and calcium association constants of EGTA for temperature and ionic strength. Am J Physiol Cell Physiol 256: C1250-C1256, 1989.
22. Hasenfuss G, Schillinger W, Lehnart SE, Preuss M, Pieske B, Maier LS, Prestle J, Minami K, and Just H. Relationship between Na⁺-Ca²⁺-exchanger protein levels and diastolic function of failing human myocardium. Circulation 99: 641-648, 1999.
23. Houser SR, Piacentino V, and Weisser J. Abnormalities of calcium cycling in the hypertrophied and failing heart. J Mol Cell Cardiol 32: 1595-1607, 2000.
24. Ito K, Yan X, Feng X, Manning WJ, Dillman WH, and Lorell BH. Transgenic expression of sarcoplasmic reticulum Ca²⁺ ATPase modifies the transition from hypertrophy to early heart failure. Circ Res 89: 422-429, 2001.
25. Jasmin G, Solymoss B, and Proschek L. Therapeutic trials in hamster dystrophy. Ann NY Acad Sci 317: 338-348, 1979.
26. Just H. Pathophysiological targets for -blocker therapy in congestive heart failure. Eur Heart J 17, Suppl B: 2-7, 1996.
27. Kimura J, Miyamae S, and Noma A. Identification of sodiumcalcium exchange current in single ventricular cells of guinea pig. J Physiol 384: 199-222, 1987.
28. Kubo H, Margulies KB, Piacentino V, Gaughan J, and Houser SR. Patients with end-stage congestive heart failure treated with -adrenergic receptor antagonists have improved ventricular myocyte calcium regulatory protein abundance. Circulation 104: 1012-1018, 2001.
29. Lehnart SE, Schillinger W, Pieske B, Prestle J, Just H, and Hasenfuss G. Sarcoplasmic reticulum proteins in heart failure. Ann NY Acad Sci 853: 220-230, 1998.
30. Liggett SB, Tepe NM, Lorenz JN, Canning AM, Jantz TD, Mitarai S, Yatani A, and Dorn GW. Early and delayed consequences of ₂-adrenergic receptor overexpression in mouse hearts. Circulation 101: 1707-1714, 2000.
31. Lowes BD, Gilbert EM, Abraham WT, Minbobe WA, Larrabee P, Ferguson D, Wolfel EE, Lindenfeld J, Tsvetkova T, Robertson AD, Quaife RA, and Bristow MR. Myocardial gene expression in dilated cardiomyopathy treated with betablocking agents. N Engl J Med 346: 1357-1365, 2002.
32. Masaki H, Sato Y, Luo W, Kranias EG, and Yatani A. Phospholamban deficiency alters inactivation kinetics of L-type Ca²⁺ channel in mouse ventricular myocytes. Am J Physiol Heart Circ Physiol 272: H606-H612, 1997.
33. Molkentin JD. Calcineurin and beyond: cardiac hypertrophic signaling. Circ Res 87: 731-738, 2000.
34. Molkentin JD, Lu JR, Antos CL, Markham B, Richardson J, Robbins J, Grant SR, and Olson EN. A calcineurin-dependent transcriptional pathway from cardiac hypertrophy. Cell 93: 215-228, 1998.
35. Ouadid H, Albat B, and Nargeot J. Calcium currents in diseased human cardiac cells. J Cardiovasc Pharmacol 25: 282-291, 1995.
36. Packer M, Bristow MR, Cohn JN, Colucci WS, Fowler MB, Gilbert EM, and Shusterman NH. The effect of carvedilol on morbidity and mortality in patients with chronic heart failure. N Engl J Med 334: 1349-1355, 1996.
37. Pieske B, Maier LS, Bers DM, and Hasenfuss G. Ca²⁺ handling and sarcoplasmic reticulum Ca²⁺ content in isolated failing and nonfailing human myocardium. Circ Res 85: 38-46, 1999.
38. Reiken S, Gaburjakova M, Gaburjakova J, He K, Prieto A, Becker E, Yi G, Wang J, Burkhoff D, and Marks AR. -Adrenergic receptor blockers restore cardiac calcium release channel (ryanodine receptor) structure and function in heart failure. Circulation 104: 2843-2848, 2001.
39. Sabbah HN, Shimoyama H, Kono T, Gupta R, Sharov V, Scicli G, Levine B, and Goldstein S. Effects of long-term monotherapy with enalapril, metoprolol, and digoxin on the progression of left ventricular dysfunction and dilation in dogs with reduced ejection fraction. Circulation 89: 2852-2859, 1994.
40. Sako H, Green S, Kranias EG, and Yatano A. Modulation of Ca²⁺ channel currents by isoproterenol studied in transgenic ventricular myocytes with altered SR Ca²⁺ content. Am J Physiol Cell Physiol 273: C1666-C1672, 1997.
41. Schillinger W, Janssen PML, Emami S, Henderson SA, Ross RS, Teucher J, Zeitz O, Philipson KD, Prestle J, and Hasenfuss G. Impaired contractile performance of cultured rabbit ventricular myocytes after adenoviral gene transfer of Na⁺-Ca²⁺ exchanger. Circ Res 87: 581-587, 2000.
42. Schlotthauer K, Schattmann J, Bers DM, Maier LS, Schütt U, Minami K, Just H, Hasenfuss G, and Pieske B. Frequency-depedent changes in contribution of SR Ca²⁺ to Ca²⁺ transients in failing human myocardium assessed with ryanodine. J Mol Cell Cardiol 30: 1285-1294, 1998.
43. Schmidt U, Hajjar RJ, Helm PA, Kim CS, Doye AA, and Gwathmey JK. Contribution of abnormal sarcoplasmic reticulum ATPase activity to systolic and diastolic dysfunction in human heart failure. J Mol Cell Cardiol 30: 1929-1937, 1998.
44. Schroder F, Handrock R, Beuckelmann DJ, Hirt S, Hullin R, Priebe L, Schwinger RH, Weil J, and Herzig S. Increased availability and open probability of single L-type calcium channel from failing compared with nonfailing human ventricle. Circulation 98: 969-976, 1998.
45. Sussman MA, Welch S, Cambon N, Klevitsky R, Hewett TE, Price R, Witt SA, and Kimball TR. Myofibril degeneration caused by tropomodulin overexpression leads to dilated cardiomyopathy in juvenile mice. J Clin Invest 101: 51-61, 1998.
46. Takahashi T, Allen PD, Lacro RV, Marks AR, Dennis AR, Schoen FJ, Grossman W, Marsh JD, and Izumo S. Expression of dihydropyridine receptor (Ca²⁺ channel) and calsequestrin genes in the myocardium of patients with end-stage heart failure. J Clin Invest 90: 927-935, 1992.
47. Takeo S, Elmoselhi AB, Goel R, Sentex E, Wang J, and Dhalla NS. Attenuation of changes in sarcoplasmic reticular gene expression in cardiac hypertrophy by propranolol and verapamil. Mol Cell Biochem 213: 111-118, 2000.
48. Terracciano CM, Philipson KD, and MacLeod KT. Overexpression of the Na⁺-Ca²⁺ exchanger and inhibition of the sarcoplasmic Ca²⁺-ATPase in ventricular myocytes from transgenic mice. Cardiovasc Res 49: 38-47, 2001.
49. Tominaga M, Matsumori A, Okada I, Yamada T, and Kawai C. -Blocker treatment of dilated cardiomyopathy: beneficial effect of carteolol in mice. Circulation 83: 2021-2028, 1991.
50. Trépanier-Boulay V, St-Michel C, Tremblay A, and Fiset C. Gender-based differences in cardiac repolarization in mouse ventricle. Circ Res 89: 437-444, 2001.
51. Waagstein F, Caidahl K, Wallentin I, Bergh CH, and Hjalmarson A. Long-term -blockage in dilated cardiomyopathy: effects of short- and long-term metoprolol treatment followed by withdrawal and readministration of metoprolol. Circulation 80: 551-563, 1989.
52. Waagstein F, Hjalmarson A, Varnauskas E, and Wallentin I. Effect of chronic beta-adrenergic receptor blockade in congestive cardiomyopathy. Br Heart J 37: 1022-1036, 1975.
53. Yao A, Su Y, Nonaka A, Zubair I, Lu L, Philipson KD, Bridge J, and Barry WH. Effects of overexpression of the Na⁺-Ca²⁺ exchanger on [Ca²⁺]_i transients in murine ventricular myocytes. Circ Res 82: 657-665, 1998.

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