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In vivo left ventricular functional capacity is compromised in cMyBP-C null mice

Department of Physiology, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin

Submitted 21 September 2006 ; accepted in final form 17 November 2006

	ABSTRACT

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Cardiac myosin binding protein-C (cMyBP-C) is a thick filament-associated protein that binds tightly to myosin and has a potential role for modulating myocardial contraction. We tested the hypothesis that cMyBP-C 1) contributes to the enhanced in vivo contractile state following -adrenergic stimulation and 2) is necessary for myocardial adaptation to chronic increases in afterload. In vivo pressure-volume relations demonstrated that left ventricular (LV) systolic and diastolic function were compromised under basal conditions in cMyBP-C^–/– compared with WT mice. Moreover, whereas -adrenergic treatment significantly improved ejection fraction, peak elastance, and the time to peak elastance in WT mice, these functional indexes remained unchanged in cMyBP-C^–/– mice. Morphological and functional changes were measured through echocardiography in anesthetized mice following 5 wk of aortic banding. Adaptation to pressure overload was diminished in cMyBP-C^–/– mice as characterized by a lack of an increase in posterior wall thickness, increased LV diameter, deterioration of fractional shortening, and prolonged isovolumic relaxation time. These results suggest that the absence of cMyBP-C significantly diminishes in vivo LV function and markedly attenuates the increase in LV contractility following -adrenergic stimulation or adaptation to pressure overload.

aortic banding; pressure-volume relations; dobutamine

Numerous studies have investigated the effects of targeted cMyBP-C gene ablation (cMyBP-C^–/–) on cardiac structure and function. Mice lacking cMyBP-C display a profound hypertrophic response with reduced ejection, prolonged relaxation (12), an increased rate of ventricular stiffening during isovolumic contraction, and decreased peak elastance (E_max) (26). These studies suggest that cMyBP-C is important for normal systolic and diastolic function, possibly by modulating the rate of cross-bridge cycling and/or availability of cross bridges to actin (34, 35); that is, cMyBP-C is normally repressive in this regard, but this effect is relieved following ablation of cMyBP-C.

Less is known about the specific effects of -adrenergic stimulation on cMyBP-C. -Adrenergic stimulation of cardiac muscle results in the PKA-mediated phosphorylation of cardiac troponin I (TnI) and cMyBP-C, which in skinned myocardium are associated with decreased Ca²⁺ sensitivity of force and increased rates of cross-bridge cycling (9). In living cardiac muscle, these contractile effects associated with -adrenergic stimulation would be expected to contribute to increased twitch force, decreased twitch duration, and increased rates of relaxation, all of which are observed in vivo. The specific mechanisms of contractile effects due to cMyBP-C phosphorylation may be related to the observation that phosphorylation increases the radial dispersion of cross bridges (38), which in living myocardium would increase the likelihood of cross-bridge binding to actin and thereby increase the cooperative activation of the myocardial thin filament by strong-binding cross bridges. Thus, similar to the accelerated force development noted upon ablation of cMyBP-C (20, 34, 35), phosphorylation of cMyBP-C may act to reduce its repressive effect on cross-bridge function (2, 21).

The observation that ablation of cMyBP-C induces hypertrophy (12) is similar to the response repeatedly demonstrated following pressure overload. A classic hypothesis is that hypertrophy is a compensatory response to normalize wall stress and to preserve systolic pressure (10, 28), although there is recent evidence to indicate that hypertrophy is not required for compensation (14). It is currently unknown how the myocardium would respond to mechanical stress in the absence of cMyBP-C.

In the present study we tested the hypothesis that cMyBP-C contributes to the enhanced myocardial contractile state following -adrenergic stimulation. Second, we sought to probe the capacity of the cMyBP-C null myocardium to respond to pressure overload induced by aortic banding. Here we show that the absence of cMyBP-C significantly diminishes in vivo left ventricular (LV) functional capacity and markedly attenuates the increase in myocardial contractility following -adrenergic stimulation. Additionally, cMyBP-C^–/– mice were unable to compensate for the increased load following aortic banding and experienced a rapid deterioration in systolic and diastolic function. Collectively, these results suggest that the absence of cMyBP-C attenuates the inotropic reserve in LV contractility typically observed as a consequence of -adrenergic stimulation and diminishes compensatory adaptations to pressure overload necessary for the maintenance of ventricular function.

	MATERIALS AND METHODS

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Experimental Animals

Homozygous cMyBP-C knockout mice (cMyBP-C^–/–) were generated as previously described (12) and were maintained on an SV/129 background. Age-matched wild-type (WT) SV/129 mice were obtained from Taconic Farms (Germantown, NY). There is no difference in mortality between cMyBP-C^–/– and WT mice. All experimental protocols were in accordance with the Animal Care and Use Committees of the University of Wisconsin Madison and the Association for Assessment and Accreditation of Laboratory Care International.

Isolated Intact Cardiomyocytes

Enzymatic digestion. Single, living ventricular cardiomyocytes were obtained by enzymatic digestion, as described previously (41), with all solutions at 37°C and bubbled with 100% oxygen. Briefly, the heart was rapidly removed from an anesthetized mouse and placed in ice-cold Ca²⁺-free Tyrode solution supplemented with 1 µg/ml of insulin containing (in mM) 130 NaCl, 0.4 NaH₂PO₄, 5.8 NaHCO₃, 0.5 MgCl₂, 5.4 KCl, 22 HEPES, and 25 glucose (pH 7.4 at 22°C). The aorta was cannulated, and the heart was placed on a temperature-controlled Langendorff coronary perfusion apparatus. The coronary circulation was perfused with Ca²⁺-free Tyrode solution containing BSA (1 mg/ml) for 5 min, at which time collagenases (type I, 0.30 mg/ml; and type II, 1 mg/ml) were added to the perfusate. The digestion was continued for an additional 5–15 min, depending on coronary flow rate. The heart was then removed from the apparatus and placed in a flask containing the enzyme solution with 0.1 mM CaCl₂. The ventricles were cut into small pieces, and the flask was placed in a 37°C water bath for 5–10 min. During this time, the ventricular pieces were gently triturated every 2–3 min to disperse the intact cardiomyocytes. The resulting cellular suspension was filtered through a 0.25-mm nylon mesh and placed in a 15-ml Falcon tube. After the cells were permitted to settle, the supernatant was carefully removed and the cells were resuspended in Ca²⁺-Tyrode solution (1 mM Ca²⁺). After this procedure was repeated once, the cells were stored at room temperature (22–24°C) until used.

Cell shortening and intracellular Ca²⁺ transients. Changes in cardiomyocyte cell length and myoplasmic [Ca²⁺] were recorded simultaneously in intact cardiomyoctes both before and during -adrenergic stimulation using isoproterenol (43). Briefly, intact ventricular myocytes were loaded with 5 µM fluo-3 AM by incubating them in Tyrode solution containing 1.8 mM CaCl₂ for 20 min. Fluo-3-loaded cardiomyocytes were then placed in a perfusion chamber that was equipped with platinum electrodes for field stimulation at 0.5 Hz with inflow and outflow tubes for continuous perfusion of myocytes with Tyrode solution containing 1.8 mM CaCl₂. Ca²⁺ transients were visualized by using the Plan-Apochromat x63 oil-immersion objective Zeiss LSM 510 confocal microscope in line-scan mode at 1.5 ms/line. The 488-nm line of the argon laser was used to illuminate, and emission was collected at >505 nm. The line of scan was selected parallel to the longitudinal cell axis to measure cell shortening expressed as percentage of cell length. Image analysis was performed by homemade routines using IDL software (Research Systems) to measure the peak amplitude of the Ca²⁺ transient (expressed as F/F₀, where F is the fluorescence signal and F₀ is the diastolic fluorescence), and the monoexponential Ca²⁺ transient decay time constant (). -Adrenergic stimulation was achieved by continuous perfusion with Tyrode solution containing 1.8 mM CaCl₂ and 1 µM isoproterenol.

Echocardiography

Transthoracic echocardiography was performed by using an Acuson Sequoia ultrasonograph with a 15-MHz transducer (Agilent Technologies) as described previously (12). Mice were lightly anesthestized with ketamine (100 mg/kg) and maintained on a heated platform. Two-dimensionally guided M-mode images of the LV and Doppler studies were acquired at the tip of the papillary muscles. LV mass-to-body weight ratio (LV/BW), LV dimension in diastole (LVDd), thickness of the anterior and posterior walls in diastole, and isovolumic relaxation time were recorded. Endocardial fractional shortening (EnFS) was calculated as (LVDd-LVDs)/LVDd x 100, where LVDs is LV dimension in systole. The functional index is calculated as EnFS/myocardial performance index, which is a Doppler-based ratio of the isovolumic contraction and relaxation times to the ejection time. All parameters were measured over at least three consecutive cycles.

Hemodynamic Measurements

Mice were anesthetized by inhalation of isoflurane, intubated, and maintained on a heated pad. A ventral midline skin incision was made from the lower mandible posteriorly to the sternum, and the right carotid artery was isolated. Two silk ligatures were passed under the carotid artery and were used for both ligation and retraction. The anterior suture was used to ligate the artery and was placed just caudal to the bifurcation of the interior and exterior carotid arteries. A posterior suture placed loosely 0.5 cm from the anterior tie was used for temporary occlusion of the carotid artery during insertion of a 1.4-Fr high-fidelity Mikro-Tip conductance catheter (Millar Instruments, Houston, TX). The occlusive posterior suture was released, and the pressure transducer was carefully advanced into the LV. The resultant pressure-volume tracings were recorded on commercially available software (Notocord, Croissy Sur Seine, France) to ascertain the proper location for the pressure and volume catheter. Heart rate, peak LV pressure, end-diastolic and end-systolic LV pressures, maximum rate of pressure development (dP/dt_max), maximum rate of pressure decrease (dP/dt_min), and the time constant for diastolic function () were calculated from digitized data that was recorded. The inferior vena cava was isolated and briefly occluded to obtain alterations in venous return for the determination of end-systolic pressure relations. Dobutamine, a -adrenergic agonist, was then infused (10 µg·kg^–1·min^–1) into the right jugular vein. After stabilization, the inferior vena cava was briefly occluded to obtain alterations in venous return for determination of end-systolic pressure relations following -adrenergic stimulation. Anesthesia was maintained under isoflurane, and hearts were rapidly excised and flash frozen in liquid nitrogen.

Aortic Banding

Mice were anesthetized with isoflurane and intubated, and the chest cavity was entered in the second intercostal space at the left upper sternal border through a small incision. Transverse aortic constriction was performed by tying a 7-0 nylon suture ligature against a 0.012-in. stainless steel wire to yield a narrowing 0.4 mm in diameter when the needle was removed (30). Control mice were subjected to a sham operation in which the transverse aorta was visualized but not banded. Mice were given subcutaneous buprenorphine (0.8 mg/kg) for pain relief and recovered in a heated chamber with 100% oxygen. Mice were maintained in housing with food and water ad libitum for 5 wk of postbanding.

Protein Analysis

Total heart proteins were prepared by homogenization in relaxing solution containing (in mmol/l) 100 KCl, 20 imidazole, 2 EGTA, 4 ATP, and 7 MgCl₂ (pH 7.0) using a Polytron homogenizer as previously described (27). The cellular homogenate was centrifuged at 120 g for 2 min, the resulting pellet was washed with fresh relaxing solution, and myofibrils were resuspended in relaxing solution containing 1% Triton X-100 for 30 min. Myofibrils were placed in 10 µl of sample buffer (containing 62.5 mM Tris, 75 mM dithiothreitol, 25% glycerol, 2% SDS, and 0.01% bromophenol blue), heated for 3 min at 100°C, and loaded onto polyacrylamide gels. Myosin heavy chain (MHC) isoform content was determined by using 6% SDS-PAGE cross-linked with N,N'-diallyltartardiamide (37). Gels were run using a Bio-Rad Protean 3 unit at 16 mA for 2 h at 4°C and then were silver stained by using Bio-Rad Silver Stain Plus kit. The relative proportions of - and -MHC isoforms were determined by densitometric analysis of silver-stained gels using LaserPix software (Bio-Rad) and expressed as a percentage of the total of both bands.

Statistical Analysis

Comparisons were made by Student's t-tests. A value of P < 0.01 was considered significant. All values are expressed as means ± SE.

	RESULTS

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Cardiac Morphology and Function in cMyBP-C^–/– Mice

Morphological characteristics presented in Table 1 show overt LV hypertrophy in cMyBP-C^–/– mice. Echocardiographic indexes are similar to those previously described (12), including reduced EnFS and prolonged isovolumic relaxation time (data not shown). LV functional measurements derived from pressure-volume loop (Fig. 1) analysis indicate systolic dysfunction in the cMyBP-C^–/– mice (Table 2) as evidenced by reduced ejection fraction, E_max, and time to E_max (E_t), along with diastolic dysfunction characterized by decreased dP/dt_min.

View larger version (29K): [in this window] [in a new window]   Fig. 1. Pressure-volume relationship in wild-type (WT) and cardiac myosin binding protein-C knockout (cMyBP-C–/–) mice following occlusion of the inferior vena cava.

Cardiac Responsiveness to -Adrenergic Stimulation in cMyBP-C^–/– Mice

Treatment with dobutamine, a -adrenergic agonist, significantly increased systolic and diastolic function in WT mice, whereas LV contractility remained unchanged in cMyBP-C^–/– mice (Table 2). The percent change in WT mice for parameters derived from pressure-volume analysis, dP/dt_max, E_max, and E_t, were 81.71%, 84.4%, and 25.5%, respectively. The percent increases in these same indexes of LV contractility in cMyBP-C^–/– mice following dobutamine treatment were 20.1%, 36.9%, and 5.9%, respectively (Fig. 2).

View larger version (18K): [in this window] [in a new window]   Fig. 2. Effect of dobutamine on left ventricular contractility in WT and cMyBP-C–/– mice. Parameters were derived from pressure-volume analysis and expressed as percent change in the maximum rate of pressure development (dP/dtmax), peak elastance (Emax), and time to Emax (Et). Values are means ± SE. *P < 0.01.

Cell Shortening and Intracellular Ca²⁺ Transients in cMyBP-C^–/– Mice Under Basal and -Adrenergic Simulation Conditions

-Adrenergic stimulation accelerates myocardial contraction in part by PKA-mediated phosphorylation of myofibrillar proteins, including cardiac TnI and cMyBP-C. -Adrenergic stimulation was utilized in both WT and cMyBP-C^–/– intact cardiomyocytes to assess the impact of cMyBP-C on Ca²⁺ handling. The extent of cell shortening, peak amplitude of the Ca²⁺ transient (expressed as F/F₀), and the Ca²⁺ transient decay time constant () are summarized in Table 3. The extent of shortening and the peak amplitude of the Ca²⁺ transients increased, and the time constant of decay decreased in the WT and cMyBP-C^–/– cardiomyocytes in response to isoproterenol. Neither the extent of shortening nor the peak amplitude of the Ca²⁺ transients was different between the WT and cMyBP-C^–/– cardiomyocytes before or during -adrenergic stimulation. T was 19% longer in cMyBP-C^–/– compared with WT cardiomyocytes before -adrenergic stimulation; however, there was no difference from the WT in the presence of isoproterenol. Representative confocal line-scan images of Ca²⁺ transients elicited from WT and cMyBP-C^–/– cardiomyocytes, field stimulated at 0.5 Hz with the associated Ca²⁺ transient and cell shortening profiles before and after perfusion with 1 µM isoproterenol, are depicted in Fig. 3.

View larger version (36K): [in this window] [in a new window]   Fig. 3. Ca2+ transients in WT and cMyBP-c–/– mice, both in the absence and presence of isoproterenol (Iso). Representative confocal line-scan images of Ca2+ transients elicited from cardiomyocytes, field stimulated at 0.5 Hz (middle), with the associated Ca2+ transient (top) and cell shortening (bottom) profiles. Left to right: representative WT cardiomyocyte, WT cardiomyocyte after perfusion with 1 µM Iso, representative cMyBP-C–/– cardiomyocyte, cMyBP-C–/– cardiomyocyte after perfusion with 1 µM Iso. Fluo-3 AM (Molecular Probes) was used as the Ca2+ indicator for all experiments measuring Ca2+ transients. F/F0, peak amplitude of the Ca2+ transient, where F is the fluorescence signal and F0 is the diastolic fluorescence.

Increased expression of -MHC is considered a hallmark of hypertrophy (16, 17). Surprisingly, -MHC content remained virtually unchanged in WT mice following aortic banding (-MHC = 2.0 ± 1.9 of total MHC). Ablation of cMyBP-C resulted in an increased -MHC expression (-MHC = 13 ± 1.5% of total MHC), and expression was further increased in response to aortic banding (-MHC = 24 ± 2.7% of total MHC) (Fig. 4).

View larger version (23K): [in this window] [in a new window]   Fig. 4. Protein analysis for - and -myosin heavy chain (MHC) isoforms. Representative 6% SDS-PAGE silver-stained gel showing MHC isoforms in ventricular myofibrillar preparations. Density of the bands corresponding to the - and -MHC isoforms is expressed as a percentage of the total of both bands. Lane 1, WT control; lane 2, WT banded; lane 3, cMyBP-C–/– banded; lane 4, cMyBP-C–/– control. There is increased expression of -MHC in cMyBP-C–/– vs. WT control mice (P < 0.01), which increased with aortic banding (P < 0.05).

In addition to the overt hypertrophy noted as a result of cMyBP-C ablation, 5 wk of aortic banding induced hypertrophic responses in both the cMyBP-C^–/– and WT mice (Table 1). The thickness of the anterior and posterior walls increased only in the WT mice in response to banding (Table 4). Dilation of the LV noted in cMyBP-C^–/– control mice became more apparent following aortic banding (Fig. 5). Cardiac function as obtained through transthoracic two-dimensionally directed M-mode echocardiography indicates systolic and diastolic dysfunction in cMyBP-C^–/– mice, as previously described (12). In response to 5 wk of aortic banding, the WT mice were able to compensate and maintain LV function as indicated by a relatively unchanged EnFS and twofold improvement in functional index. In contrast, the cMyBP-C^–/– mice exhibited a nearly 30% decline in EnFS and relatively no improvement in functional index.

View larger version (22K): [in this window] [in a new window]   Fig. 5. Morphological and functional changes in WT and cMyBP-C–/– mice following aortic banding assessed through echocardiography. Top, left: posterior wall diameter in diastole (PWd). Top, right: left ventricular diameter in diastole (LVDd). Bottom, left: endocardial fractional shortening (EnFS). Bottom, right: isovolumic relaxation time (IVRT). Values are means ± SE. *P < 0.01, significantly different WT vs. MyBP-C–/– within each experimental group; #P < 0.01, significantly different due to banding.

	DISCUSSION

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Response to -Adrenergic Stimulation in cMyBP-C^–/– Mice

The structural and functional contributions of cMyBP-C have been well studied by using mouse models of gene ablation or truncation and include hypertrophy, LV dilation, myocyte disarray, prolonged relaxation, and reduced ejection (12, 23). Interestingly, despite the compromise in cardiac systolic function, skinned cardiomyocyes from cMyBP-C^–/– mice exhibit an increased rate of force redevelopment, loaded shortening velocity, and power output (20). These observations were reconciled in a recent study that described cMyBP-C as playing an essential role in maintaining force and stiffness during the later phase of systole and a less critical role in early force development (26). Collectively, these results and data presented herein are consistent with the idea that cMyBP-C decreases myocardial contractility; that is, cMyBP-C normally acts to constrain the availability of myosin cross-bridges for binding to actin and subsequently limit steady state for development and the kinetics of cross-bridge interaction (34, 35).

Regulation of myocardial contractility is modulated by -stimulation, which has well-characterized positive inotropic and lusitropic effects, including altered Ca²⁺ handling, increased twitch force, decreased twitch duration, and increased rates of relaxation. There are three -adrenergic-mediated phosphorylation sites on the NH₂-terminal binding domain of cMyBP-C (13, 39). However, the role of cMyBP-C phosphorylation on contractility has been difficult to discern, in part because other myofibrillar proteins such as TnI are targets for PKA. In the present study, we show that the -agonist dobutamine significantly increased the LV functional capacity of WT mice in vivo and that absence of cMyBP-C significantly diminished the ability of the myocardium to respond to -adrenergic stimulation. The absence of cMyBP-C depleted the inotropic reserve, as evidenced by the lack of increase in ejection fraction, dP/dt_max, and E_max with dobutamine, and suggests that the accelerated cross-bridge kinetics previously noted in cMyBP-C^–/– mice (20) cannot be further enhanced with -adrenergic stimulation. Diastolic dysfunction observed in cMyBP-C^–/– mice as measured by decreased dP/dt_min and prolonged was not affected by treatment with dobutamine. This result is consistent with the idea that cMyBP-C phosphorylation accelerates cross-bridge binding to actin (35). In contrast to previous reports (31), dobutamine failed to increase dP/dt_min and had only a modest affect on (17% decrease, P = 0.02) in WT myocardium. Basal phosphorylation levels of cMyBP-C have been reported to be >90% in mouse myocardium (31), which provides one possible explanation for the lack of improvement in relaxation in response to dobutamine.

There are several other possible explanations for the lack of response to -adrenergic stimulation in cMyBP-C^–/– mice. We propose that phosphorylation of other myofibrillar proteins such as TnI are ineffective at increasing ejection fraction, dP/dt_max, and E_max because the acceleration in cross-bridge kinetics due to the absence of cMyBP-C has already been maximized by ablation of cMyBP-C. However, we cannot rule out the possibility that cMyBP-C is permissive for the effects of TnI phosphorylation, which reportedly accelerate myofibrillar relaxation rate and contribute to faster cross-bridge cycling kinetics (18). Downregulation of -adrenergic receptors and abnormalities in Ca²⁺ handling provide other plausible explanations for the lack of response to dobutamine (15, 29). Although we do not have direct evidence that -adrenergic receptors are unaltered by the absence of cMyBP-C, we do show a positive chronotropic effect in response to dobutamine in cMyBP-C^–/– and WT mice (15% and 21% increase in heart rate, respectively). We also investigated abnormalities in Ca²⁺ handling as an underlying mechanism for the lack of increased LV contractility in cMyBP-C^–/– mice. Our evidence indicates that the absence of cMyBP-C does not impact peak Ca²⁺ transients or the extent of shortening, nor does it impair the response of intact myocytes to -adrenergic stimulation. Consistent with the diastolic dysfunction noted on echocardiography, cMyBP-C^–/– myocytes have a prolonged rate constant for Ca²⁺ transient decay compared with WT under basal conditions; however, this is ameliorated upon treatment with isoproterenol.

Response to Pressure Overload in cMyBP-C^–/– Mice

A major aim of the present study was to probe the capacity of the myocardium to respond to pressure overload in the absence of cMyBP-C. It has been well established that chronic pressure overload leads to LV hypertrophy and is associated with diminished -adrenergic responsiveness (29), increased -MHC expression (17), altered contractile protein phosphorylation (31, 36), and abnormalities in Ca²⁺ handling (15) during the progression to failure. Little is known about the ability of the myocardium to adapt to pressure overload in the absence of cMyBP-C.

As expected, aortic banding resulted in LV hypertrophy in both WT and cMyBP-C^–/– mice. Interestingly, the hypertrophic morphology and functional outcome between the two groups were dramatically different following 5 wk of aortic banding. WT mice displayed increased thickness of the posterior and anterior walls during diastole and maintained fractional shortening. In contrast, the ventricular walls in cMyBP-C^–/– mice failed to thicken, the LV dilated out, and fractional shortening and relaxation were impaired. Taken together, these results suggest that cMyBP-C is a crucial sarcomeric protein for myocardial adaptation to pressure overload.

We demonstrate that pressure overload exacerbates the hypertrophic response (41% increase in LV/BW) and expression of -MHC already present in cMyBP-C^–/– mice. WT mice exhibited a similar hypertrophic response (56% increase in LV/BW), suggesting a similar hemodynamic stress was imposed by aortic banding but that WT mice did not show an increase in -MHC expression. Although -MHC is a hallmark of hypertrophy, these changes are not necessarily coordinately regulated (42). Whereas there is no inherent difference in mortality between the two groups, the postbanding mortality rate was nearly twofold higher in the cMyBP-C^–/– mice. Therefore, we conclude that the magnitude of hemodynamic stress required to elicit upregulation of the fetal gene program and decompensation in function is greater in WT compared with cMyBP-C^–/– mice. This underscored the integral role of cMyBP-C in the capacity of the myocardium to compensate for an increase in afterload.

Pressure overload in other transgenic mice, including ₁- and ₂-adrenergic receptor and phospholamban knockouts (19, 25) and -MHC mutation models (32), have failed to evoke differences in the hypertrophic or functional response compared with WT-banded mice. One interpretation is that the ability to compensate for pressure overload is not predicated on these proteins, and thus a major finding of the current investigation is that cMyBP-C is required for the myocardial compensation in response to pressure overload.

We are unable to distinguish the importance of the cMyBP-C phosphorylation in response to pressure overload. Downregulation of -adrenergic receptors and diminished responsiveness to -adrenergic stimulation are well documented during the progression to heart failure (29). Phosphorylation of cMyBP-C was reported to decrease during the progression to heart failure induced by aortic banding or ischemia-reperfusion (31), and the stability of contractile myofilaments has also been shown, at least in part, to be regulated by the phosphorylation state of cMyBP-C (4, 22). From the current study, we are only able to comment on the importance of cMyBP-C in the response of the myocardium to pressure overload.

We conclude that the absence of cMyBP-C attenuates the inotropic reserve in LV contractility typically observed as a consequence of -adrenergic stimulation. Increases in contractility due to phosphorylation of other proteins, such as TnI, are insufficient to significantly increase dP/dt_max, E_max, and E_t in the absence of cMyBP-C. The capacity of the myocardium to respond to mechanical stress induced by aortic banding is also severely compromised in the absence of cMyBP-C. Collectively, these results suggest that cMyBP-C is an essential sarcomeric protein for the contractile response to -adrenergic stimulation and compensatory response to pressure overload.

	GRANTS

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This study was funded by the National Heart, Lung, and Blood Institute Grant P01-HL-47053 (to R. L. Moss) and National Research Service Award T32-HL-07936 (to Univ. of Wisconsin Cardiovascular Research Center for its Training Program in Transitional Cardiovascular Science, which selects trainees for the program. R. L. Moss is a trainer in the program).

	FOOTNOTES

 

Address for reprint requests and other correspondence: S. Brickson, Dept. of Physiology, Univ. of Wisconsin Medical School, 601 Science Dr., Madison, WI 53711 (e-mail: sbrickson{at}physiology.wisc.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.

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