In vivo measurements of the contributions of protein synthesis and protein degradation in regulating cardiac pressure overload hypertrophy in the mouse

Abstract

Cardiac hypertrophy is generated in response to hemodynamic overload by altering steady-state protein metabolism such that the rate of protein synthesis exceeds the rate of protein degradation. To determine the relative contributions of protein synthesis and degradation in regulating cardiac hypertrophy in mice, a continuous infusion strategy was developed to measure myocardial protein synthesis rates in vivo. Osmotic mini-pumps were implanted in the abdominal cavity to infuse radiolabeled leucine in mice that are conscious and ambulatory. Protein synthesis rates were calculated by measuring incorporation of leucine into myocardial protein over 24 h prior to each time point and dividing by the specific radioactivity of plasma leucine. Compared to sham-operated controls, fractional rates of protein synthesis (K_s) increased significantly at days 1 and 3 of TAC, but was lower on day 7 and returned to control values by day 14. These changes coincided with the curvilinear increase in LV mass that characterizes the hypertrophic response. Fractional rates of protein degradation (K_d) were calculated by subtracting the rate of myocardial growth from the corresponding K_s value. K_d fell at days 1 and 3 of TAC, increased at day 7 and returned to control on day 14. Thus, the increase in LV mass generated in response to pressure overload is caused by acceleration of K_s and suppression of K_d. As the growth rate slows, a new steady-state is achieved once the hypertrophic response is completed.

Introduction

Cardiac hypertrophy is generated in response to hemodynamic overload by altering the balance in steady-state protein metabolism such that the rate of protein synthesis is faster than the rate of protein degradation [1]. Previous studies in canine models of hemodynamic overload have demonstrated that an increase in wall stresses produced by either pressure or volume overloading of the LV in vivo activate distinct mechanisms for shifting the myocardium into an anabolic state with respect to protein metabolism [2–4]. For example, in a pressure overload model of aortic stenosis, the increase in systolic stress accelerates the rate of myocardial protein synthesis [2, 4]. The cardiomyocytes enlarge by concentric hypertrophy as sarcomeres are added in parallel to the existing myofibrils. In contrast, in a volume overload model of mitral regurgitation, an increase in diastolic strain has minimal effects on the rate of myocardial protein synthesis [2, 3]. The rate of myocardial protein degradation is slower during volume overload, and thus, cardiomyocytes are lengthened to produce eccentric hypertrophy as sarcomeres are added in series to extend the myofibrils.

Experimental models of LV pressure overload develop a consistent hypertrophic response in which myocardial growth occurs as a curvilinear function over a time period of ~14 days [5–7]. As a result, a new steady-state is achieved with respect to cardiac mass. In the canine model of LV pressure overload, direct measurements of myosin heavy chain synthesis in vivo revealed that two complementary mechanisms shift the cardiomyocyte into an anabolic state with respect to myocardial protein metabolism. During the linear phase of cardiac hypertrophy, the increase in mass is generated by accelerating the fractional rate of protein synthesis, and by reducing simultaneously the fractional rate of protein degradation [2, 4]. Subsequently, fractional rates of protein synthesis and degradation return to steady-state values as hypertrophic growth is completed. These findings underscore that pressure overload hypertrophy is regulated by mechanisms that coordinate time-dependent changes in protein synthesis and protein degradation. In experimental models of volume overload such as mitral regurgitation and aortic regurgitation, suppression in the rate of protein degradation is the primary mechanism for generating hypertrophic growth [3, 8]. However, in large animal models such as the dog, measurements of cardiac protein synthesis and degradation are limited because a large amount radiolabeled amino acid must be infused, under anesthesia, for a relatively long-time period in order to quantify the specific radioactivity of amino acid incorporated into myocardial protein.

Transgenic mice have become the predominant animal model to investigate mechanisms of cardiac hypertrophy, remodeling, and decompensated heart failure. In particular, the transverse aortic constriction (TAC) model of pressure overload is utilized routinely to determine how gain or loss of gene function impacts cardiac growth and adverse remodeling. Despite the large array of genetic models that have been developed, the main endpoint for hypertrophic growth in mice is the LV mass/body weight ratio. However, depending on the transgenic model and the experimental time points at which LV mass is measured, changes in the hypertrophic response could reflect alterations in the rate of protein synthesis, rate of protein degradation, or combinatorial effects on both mechanisms. To determine the relative contributions of these mechanisms in regulating LV mass, the purpose of this study was to develop a method to measure in vivo rates of myocardial protein synthesis in the mouse. Specifically, osmotic mini-pumps were implanted into the abdominal cavity so that a radiolabeled amino acid precursor for protein synthesis was infused continuously in mice that were conscious and ambulatory. This approach was validated by measuring time-dependent changes in the rate of myocardial protein synthesis during pressure overload hypertrophy produced in response to TAC. Furthermore, this study demonstrates that the rate of myocardial protein degradation can be calculated indirectly by subtracting the rate of myocardial growth from the rate of myocardial protein synthesis.

Materials and methods

TAC model of LV pressure overload

The procedures involving animals were approved by the Institutional Animal Care and Use Committee, and all animals were treated and cared for in accordance with institutional guidelines. TAC was created using the C57bl/6 strain of mice as described before [9]. The mouse was anesthetized with 2 % isoflurane provided through a modified mask assembly, and connected to a rodent ventilator (MiniVent 845, Hugo-Sachs, 250 breaths per minute, tidal volume 250 μl, with 2 % isoflurane). Buprenorphine (0.05 mg/kg sc) was injected for analgesia. Electrodes for surface ECG were inserted subcutaneously and the signal monitored (PONEMAH, LDS Gould). Pedal reflex and heart rate were monitored continuously to assess the depth of anesthesia. Using aseptic techniques, a 5 mm skin incision was made in the neck region and the trachea visualized. The mouse was orotracheally intubated and the correct placement of the endotracheal tube confirmed visually. A horizontal incision of 5 mm was made at the level of the suprasternal notch. Blunt dissection was done using a surgical microscope to expose the transverse aorta. Transverse arch banding was performed by placing a 7–0 braided silk suture over a 27-gauge needle, causing complete occlusion of the aorta. The needle was then removed, restoring the aortic lumen, but leaving a severely stenotic aortic orifice. A drainage tube was placed in the incision, air was evacuated via the drainage tube, and the drainage tube was then removed. The incision was closed, the mouse extubated, and the animal was allowed to recover on a warming blanket in a container supplemented with oxygen. The procedure created severe stenosis of the aorta as indicated by a pressure gradient of 92 ± 1 mmHg (mean ± SEM) between the LV and the thoracic aorta. Sham-operated mice were subjected to a thoracotomy and a suture was passed around the transverse aorta and then removed. LV mass and body weight were measured at the time of dissection [10].

Echocardiography

Echocardiographic measurements were made using a 15-MHz transducer and a Sonos 5500 echocardiograph (Agilent Technologies, Andover, MA). Three to six beats were averaged for each measurement. LV dimension and wall thickness were made at end-diastole and end-systole using the American Society of Echocardiography criteria (10). Mean wall thickness was calculated as the average of the interventricular septal wall thickness (IVS) and left ventricular posterior wall thickness (LVPW). Relative wall thickness (RWTH) was calculated as the mean LV wall thickness divided by the LV internal dimension at end-diastole (LVIDd). LV mass was calculated using the following formula:

$$\begin{gathered} \text{LV}\text{mass}=0.8\cdot (1.04) \\ \times [\text{IVSd}+\text{LVPWd}+{\text{LVIDd}}^3-(\text{LVID}{)}^3] \end{gathered}$$

LV mass was normalized to body weight (LV/BW) ratio. LV end-diastolic volume (LVEDV) and end-systolic volume (LVESV) were determined using the Simpson’s method of disks. Ejection fraction (EF) was calculated by the following formula:

$$\mathrm{E}\mathrm{F}(\mathrm{\%})=100·(\mathrm{L}\mathrm{V}\mathrm{E}\mathrm{D}\mathrm{V}-\mathrm{L}\mathrm{V}\mathrm{E}\mathrm{S}\mathrm{V})/\mathrm{L}\mathrm{V}\mathrm{E}\mathrm{D}\mathrm{V}$$

The aortic pressure gradient created by the TAC was measured using the modified Bernoulli equation:

$$\mathrm{P}\mathrm{r}\mathrm{e}\mathrm{s}\mathrm{s}\mathrm{u}\mathrm{r}\mathrm{e}\mathrm{g}\mathrm{r}\mathrm{a}\mathrm{d}\mathrm{i}\mathrm{e}\mathrm{n}\mathrm{t}=4{\left(V_{\mathrm{p}\mathrm{e}\mathrm{a}\mathrm{k}}\right)}^2$$

where $V_{\mathrm{p}\mathrm{e}\mathrm{a}\mathrm{k}}$ is the peak Doppler velocity across the TAC site.

Calculations of LV systolic wall stress

The effect of pressure overload on ambient in vivo hemodynamic load of the LV myocardium was determined by calculating the LV systolic wall stress using the formula:

$$\begin{gathered} \text{LV}\text{systolic}\text{wall}\text{stress}=1.36\times \text{LVSP}\times \text{ESD}/ \\ \times [4\times \text{PWThs}\times (1+\text{PWThs}/ \\ \text{LVESD})] \end{gathered}$$

where LVSP is LV systolic pressure, LVESD is LV end-systolic dimension, and PWThs is LV end-systolic posterior wall thickness. LVSP was calculated as the sum of the pressure gradient across the TAC and the mean aortic pressure (MAP) distal to the TAC. MAP was determined by using tail cuff pressure measurements made with a CODA system (Kent Scientific Corp, Torrington, CT) in restrained, but nonanesthetized mice using the formula:

$$\mathrm{M}\mathrm{A}\mathrm{P}=(2\times \mathrm{D}\mathrm{B}\mathrm{P}+\mathrm{S}\mathrm{B}\mathrm{P})/3$$

where DBP is diastolic blood pressure and SBP is systolic blood pressure.

Measurements of cardiac protein synthesis rates in vivo

Fractional rates ($K_{\mathrm{s}}$) of total protein synthesis and MHC synthesis were calculated in vivo by the formula:

$$K_{\mathrm{s}}=\frac{P^{\mathrm{*}}}{F^{\mathrm{*}}}$$

where $P^{\mathrm{*}}$ is the specific radioactivity of the amino acid incorporated into protein product (DPM/pmol) × t⁻¹, and $F^{\mathrm{*}}$ is the specific radioactivity of the amino acid precursor pool (DPM/pmol) as measured in the plasma [11].

Two radiolabeling protocols were used to measure the rate of protein synthesis in this study (Fig. 1). In Protocol 1, an Alzet^® osmotic mini-pump (Model 2001) was implanted into the abdominal cavity of either TAC or sham mice at the time of surgery (day 0). Each pump was primed to deliver [³H]-LEU (1 mM, 17.5 mCi/μl) at a rate of ~1 μl per h. Mice were sacrificed after 1, 3, or 7 days of continuous infusion. In Protocol 2, the Alzet mini-pumps were used to deliver [³H]-LEU by implantation 24 h prior to the time of sacrifice at 1, 3, 7, or 14 days of TAC. Hearts were removed and rinsed immediately in saline, which allowed for perfusion of the coronary circulation through spontaneous contractions. In both protocols, the LV was dissected, weighed and homogenized immediately in a buffer containing 0.1 M KCl, 20 mM Tris–HCl pH 7.5, 0.2 % NaN₃. An aliquot of the homogenate was used to precipitate total cardiac protein by adding ethanol to a final concentration of 80 %. The protein pellet was washed in 80 % ethanol and resuspended in 0.3 N NaOH. The specific radioactivity of total cardiac protein (DPM/μg) was determined by liquid scintillation counting and measuring the corresponding protein concentration by the BCA method. $P^{\mathrm{*}}$ was calculated by the following formula using a value of 693.6 pmol of LEU per μg total cardiac protein [12].

Fig. 1.

Experimental protocols for measuring in vivo rates of myocardial protein synthesis in mice by infusion of [³H]-LEU via osmotic mini-pumps

$$\begin{gathered} P^{\mathrm{*}}= \\ \times \left[\frac{\text{Specific}\text{radioactivity}\text{of}\text{cardiac}\text{protein}\left(\frac{\text{DPM}}{\text{mg}\text{protein}}\right)}{\text{LEU}\text{composition}\text{of}\text{cardiac}\text{protein}\left(\frac{693.6\text{p}\text{moles}\text{LEU}}{\text{mg}\text{protein}}\right)}\right] \\ \times {\text{day}}^{-1} \end{gathered}$$

To measure $P^{\mathrm{*}}$ for MHC, cardiac myosin was purified by a centrifugation-based extraction method as follows with modifications [13]. The homogenate was centrifuged at 1500×g and the pellet resuspended in high salt extraction buffer [0.45 M KCl, 1 mM DTT, 0.2 M Mg(CH₃COO)₂, 1 mM EGTA, 20 mM Tris-maleate, pH 6.8, 10 mM ATP]. The extract was centrifuged at 12,000×g and the supernatant diluted with 1 mM HCO₃ to reduce the KCl concentration to 0.125 M. The diluted samples were centrifuged at 12,000×g and the pellet resuspended in high salt buffer-containing 0.5 M KCl, 1 mM DTT, 20 mM Tris–HCl pH 7.5, and protease inhibitor cocktail (Sigma). The KCl concentration was reduced again to 0.125 M by adding HCO₃. After an overnight incubation, the diluted samples were centrifuged to recover the enriched fraction of myosin in the pellet. This fraction was used to purify MHC on 8 % SDS-PAGE gels. The gels were fixed and stained using SYPRO^® Ruby Red Stain. The amount of MHC was quantified by digital image analysis of the stained band by extrapolation using a standard curve of MHC. The MHC band was excised from the gel and eluted for liquid scintillation counting by overnight incubation in Solvable^® at 55 °C. The specific radioactivity of MHC (DPM/μg) was calculated and used to determine $P^{\mathrm{*}}$ for MHC by the formula above assuming a value of 792.7 pmol LEU/μg MHC, which is derived from the amino acid sequence of the murine isoform of cardiac α-MHC.

The specific radioactivity of plasma LEU ($F^{\mathrm{*}}$) was measured as described before with modifications [2]. Serum was isolated from whole blood collected at the time of sacrifice, and serum protein was precipitated by adding HClO₄ to a final concentration of 0.5 N. The sample was centrifuged, and the supernatant was neutralized by adding KHCO₃ to a final concentration of 0.5 M and incubation on ice for 10 min. Following centrifugation, the supernatant was dried in a vacuum centrifuge and resuspended in Na₂CO₃/NaHCO₃ buffer to pH 9.5. Aliquots were reacted with an equal volume of 3 mM [methyl-¹⁴C]-Dansyl chloride (1.5–3 DPM/pmol) and the individual Dansylated amino acids resolved by 2-D thin-layer chromatography on micropolyamide plates. The ³H/¹⁴C ratio of Dansyl-LEU was divided by the specific radioactivity of [¹⁴C]-Dansyl chloride to calculate $F^{\mathrm{*}}$ (DPM/pmol LEU).

Results

In Table 1, measurements of LV mass and ejection fraction of TAC mice are compared to sham-operated controls. The LV mass/body weight ratio was increased significantly after 3 days of TAC, which demonstrates the onset of pressure overload hypertrophy. Hypertrophic growth progressed further as demonstrated by significant increases in LV mass/body weight ratios at 7 and 14 days of TAC with no change in LV chamber dimension. Heart rate and ejection fraction in the TAC mice remained constant, and these values did not differ from sham-operated controls.

Table 1.

Effects of TAC

	Sham n = 6	Time after TAC
		Day 1 n = 10	Day 3 n = 17	Day 7 n = 13	Day 14 n = 4
LV/BW (mg/g)	3.7 ± 0.2	3.8 ± 0.2	4.3 ± 1.0*	5.3 ± 1.6*#	6.2 ± 0.3*#
LV mass (mg)	97.3 ± 7.5	97.2 ± 5.3	104.6 ± 28.8*	127.2 ± 31.0*#	142.5 ± 9.3*#
Pressure gradient (mmHg)	0.0 ± 0.0	90.4 ± 2.1*	99.5 ± 2.2*	98.9 ± 2.4*	99.6 ± 4.1*
Heart rate (beats per min)	430 ± 40	438 ± 32	441 ± 25	438 ± 28	431 ± 35
LVIDd (mm)a	3.44 ± 0.08	3.47 ± 0.03	3.52 ± 0.06	3.49 ± 0.06	3.51 ± 0.08
Ejection fraction (%)	72.5 ± 4.0	70.2 ± 3.7	68.6 ± 2.5	69.1 ± 3.0	70.1 ± 5.2

^*P < 0.05 versus sham-operated controls,

^#P < 0.05 versus day 3

^aLV internal dimension in diastole

The initial series of experiments were designed to test whether osmotic mini-pumps have utility for measuring in vivo rates of cardiac protein synthesis. Following implantation of the mini-pumps, incorporation of radiolabeled LEU into total cardiac protein and into MHC were measured in the LV derived from the same hearts. Figure 2 compares rates of LV protein synthesis in TAC mice versus sham-operated controls as measured after 1, 3, and 7 days of continuous infusion using Protocol 1. The rate of total protein synthesis on 1 day was increased significantly in TAC mice as compared to sham-operated controls (Fig. 2a). Over 7 days of continuous infusion, the incorporation of radiolabeled LEU into protein was not linear. As a result, the rate of total protein synthesis decreased in both sham and TAC mice below the baseline values measured on day 1. These data indicate that turnover of radiolabeled protein becomes substantial during continuous infusion of 3 days or longer.

Fig. 2.

In vivo rates of myocardial protein synthesis in mice as measured using Protocol 1. a Rates of total protein synthesis. b Rates of MHC (myosin heavy chain) synthesis in the same hearts. Values are the mean ± SEM; Sham, n = 6 mice per time point; TAC, n = 12, n = 15, n = 10 mice at day 1, 3, and 7, respectively. *P < 0.05 versus sham-operated controls on day 1 as determined by ANOVA followed by a Student–Newman–Keuls test; ^†P < 0.05 versus TAC on day 1

Figure 2b shows that measurements of MHC synthesis changed in parallel with total protein synthesis. Accordingly, the rate of MHC synthesis increased significantly after 1 day of TAC as compared to the sham-operated controls, and subsequently decreased after 3 and 7 days of continuous infusion. Comparison with Fig. 2a further shows that the rate of total protein synthesis as measured on day 1 was 87 and 40 % higher than the rate of MHC synthesis in TAC and sham-operated groups, respectively. Given the heterogeneity of the LV protein pool, the higher rate of total protein synthesis reflects the contribution of myocardial proteins that turnover at a faster rate than myofibrillar proteins [11]. The apparent decrease in MHC synthesis as measured on days 3 and 7 of continuous infusion occurred because the rate of LEU incorporation into MHC did not remain linear. Thus, both synthesis and degradation contributed to changes in the specific radioactivity of MHC over infusion times of 3 days or longer. These results are consistent with the half-life of cardiac MHC in vivo, which is 5.5 days in the rat [14].

The specific radioactivity of plasma LEU fluctuated during continuous infusion using Protocol 1. In the sham-operated mice, plasma LEU specific radioactivity averaged 2.5 ± 0.3 DPM/pmol (mean ± SEM) on 1 day of infusion, 4.9 ± 0.6 DPM/pmol on day 3, and 3.6 ± 0.3 DPM/pmol on day 7. Thus, the decrease in protein synthesis rates as shown in Fig. 2 were not caused by a corresponding reduction in the specific radioactivity of the plasma LEU pool. Similar results were obtained in the TAC mice as the plasma LEU-specific radioactivity averaged 0.9 ± 0.1 DPM/pmol on day 1, 1.9 ± 0.3 DPM/pmol on day 3, and 3.1 ± 0.6 on day 7.

Protocol 1 has two major limitations for measuring myocardial protein synthesis over prolonged infusion times: (1) significant degradation of newly synthesized proteins occurs after 3 days of infusion, and (2) specific radioactivity of the plasma LEU pool fluctuates as measured after 3 and 7 days. Therefore, Protocol 2 was implemented to measure rates of myocardial protein synthesis during pressure overload hypertrophy by implantation of the mini-pumps 24 h prior to sacrifice at day 1, 3, 7, or 14 in the TAC mice. Figure 3 shows that specific radioactivities of plasma LEU were not significantly different using Protocol 2.

Fig. 3.

Specific radioactivity of the plasma LEU pool in TAC mice as measured using Protocol 2. Values are the mean ± SEM; the number of mice at each time point is indicated in parenthesis

In Fig. 4a, rates of myocardial protein synthesis in TAC mice as measured using Protocol 2 are compared to sham-operated controls. K_s for total protein increased significantly after 1 day of TAC, and the increase was maintained on day 3. Subsequently, a decrease in K_s occurred between days 7 and 14 of TAC that corresponds with a slower rate of growth as measured by the LV/BW ratio (see Table 1). Similar results were obtained for MHC synthesis except that a significant increase in K_s was prolonged until day 7 of TAC.

Fig. 4.

In vivo rates of myocardial protein synthesis in mice as measured by Protocol 2. a Values for K_s (fractional rate of protein synthesis) are the mean ± SEM; the number of mice at each time point is indicated in parenthesis. *P < 0.05 versus sham-operated controls for total protein synthesis; ^†P < 0.05 versus sham-operated controls for MHC synthesis as determined by Student’s t test. b LV systolic wall stress was calculated from echocardiographic data. Values are the mean ± SEM. Statistical comparisons between the sham-operated controls and TAC groups were made using a one-way ANOVA; pair wise comparisons were made using the Bonferroni test

Figure 4b shows that LV systolic wall stress increased significantly in TAC mice as compared to sham-operated controls. Systolic wall stress was highest after 1 day of TAC and then gradually decreased over 14 days. Comparison with Fig. 4a indicates that the decrease in K_s correlated with a reduction in LV systolic wall stress that occurred as a result of compensatory hypertrophic growth. Nonetheless, as compared to sham-operated controls, LV systolic wall stress remained significantly higher after 14 days in the TAC mice. In Fig. 5, K_s values for both total protein and MHC were plotted as a function of LV systolic wall stress. These plots demonstrate a direct correlation between increased rates of myocardial protein synthesis and LV systolic wall stress in the TAC mice.

Fig. 5.

Relationship between rates of myocardial protein synthesis and LV systolic wall stress. Values are the mean ± SEM. Correlations between LV systolic wall stress and K_s were determined using a Pearson’s correlation coefficient

In Fig. 6, LV mass/body weight ratios were used to derive the best-fit nonlinear equation for the rate of hypertrophic growth. The curvilinear increase in LV mass occurred continuously over 14 days of TAC despite the fact that K_s values for total protein synthesis decreased by day 7. To assess the relative contributions of protein synthesis and degradation in regulating hypertrophic growth, the fractional rate of protein degradation (K_d) was calculated at each time point after TAC by subtracting the fractional rate of growth (K_g) from the corresponding K_s value. The K_g values in the lower part of Fig. 6 were calculated by dividing the first derivative of this equation at 3, 7, and 14 days of TAC by the corresponding LV mass/body weight ratios. K_g is considered to be 0 in the sham group because these hearts have attained a steady-state with respect to protein metabolism (K_s = K_d). The K_d values indicate that protein degradation was suppressed between days 3 and 7 of TAC, the period that coincides with accelerated K_s and rapid hypertrophic growth. Subsequently, the rate of growth slowed between days 7 and 14 of TAC by reducing K_s and increasing K_d. Growth still occurred because K_s exceeds K_d during this time period.

Fig. 6.

Rates of protein synthesis and degradation during LV pressure overload hypertrophy. The graph shows the best-fit nonlinear equation for the rate of hypertrophic growth as derived from LV mass/body weight ratios of the TAC mice. Values for K_g were calculated by dividing the first derivative of this equation at each time point after TAC by the corresponding LV mass/body weight ratio. K_d was calculated by subtracting K_g at each time point from the corresponding value for K_s (see Fig. 4a)

Discussion

The relative contributions of protein synthesis and degradation to changes in LV mass are time-dependent and determined by the type of hemodynamic overload, for example pressure versus volume overload [15]. In experimental pressure overload, the fractional rate of protein synthesis is accelerated during the initial phase of hypertrophic growth as measured in mixed protein pools (total protein and myofibrillar fractions), and in specific protein pools such as MHC [4, 16, 17]. The fractional rate of protein degradation is suppressed simultaneously. Consistent with other mammalian species, the continuous infusion method developed here shows that TAC-induced hypertrophy in mice is produced by mechanisms that accelerate the rate of protein synthesis and suppress the rate of protein degradation.

The two most common methods to measure K_s involve using a radiolabeled amino acid precursor that is delivered either by injection of high concentrations (flooding dose) of the amino acid precursor or by continuous i.v. infusion of the amino acid precursor over a longer time period [14, 17–19]. In the flooding dose method, the rate of protein synthesis is measured by a short pulse-labeling time of ~10 min. The resulting specific radioactivity of myocardial protein is low because the amino acid isotope is diluted upon incorporation into the large pool of myofibrillar protein. The specific radioactivity of myofibrillar proteins such as MHC is especially difficult to measure because these proteins are expressed in high abundance and have a relatively slow rate of turnover (~5.5 days for MHC in the rat) [14]. The flooding dose method has been adapted in mice to measure in vivo synthesis rates of a mixed pool of myofibrillar proteins derived from the gastrocnemius muscle by incorporation of a stable isotope tracer of PHE (²H₅-PHE) [20]. The drawback of this method is that several steps are required in order to measure the incorporation of ²H₅-PHE into newly synthesized protein of the myofibrillar fraction. These include hydrolysis and derivatization of the myofibrillar protein fraction, separation of amino acid derivatives by gas chromatography, and detection of ²H₅-PHE by mass spectrometry.

In the continuous infusion method, the rate of protein synthesis typically is measured by incorporation of a radiolabeled amino acid precursor over a longer time period (hours). Previous studies have demonstrated that the specific radioactivity of amino acid in the plasma rises rapidly and can be maintained over several hours of continuous infusion in both rat and rabbit [14, 19]. Similarly, in canine models of hemodynamic overload, the specific radioactivity of LEU can be maintained at a constant value over 6 h of continuous infusion [2]. These studies have shown that the specific radioactivity of myofibrillar proteins can be measured, but a relatively large volume of amino acid precursor must be infused intravenously under a plane of anesthesia. Given that blood volume in rodents is ~7 % of total body weight, hypervolemia is a significant problem because the total volume that can be infused safely and reproducibly into a mouse that weighs ~25 g is limited to about 10 μl/h. Other hemodynamic variables are introduced by surgical anesthesia.

To measure in vivo rates of protein synthesis in mice, the continuous infusion method was modified by using osmotic mini-pumps rate to deliver radiolabeled LEU. Although a brief period of anesthesia is required for implantation of the pump, the mice are conscious and ambulatory over the duration of the labeling period. The calculation of protein synthesis rates assumes that the specific radioactivity of the precursor pool in the plasma is constant and equilibrates with the leucyl-tRNA pool. Although the specific radioactivity of leucyl-tRNA was not measured directly in this study, previous results obtained in several mammalian species have established that the free amino acid pools equilibrate in the myocardium during continuous infusion [2, 14, 18]. Thus, it is unlikely that the LEU pool derived from protein degradation is compartmentalized and is used to charge tRNA before equilibration with the free LEU pool.

The calculated values for K_d indicate that protein degradation is suppressed during the initial growth phase of TAC-induced hypertrophy, and thereby contributes to the increase in LV mass. Although these findings are consistent with other models of experimental pressure overload, the role of protein degradation in regulating cardiac hypertrophy has not been fully defined. For example, it has been reported that pressure overload hypertrophy in the same TAC model of pressure overload hypertrophy in mice causes a twofold increase in proteasome activity, and that development of LV hypertrophy is prevented in this model by administration of a proteasome inhibitor [21]. However, another study in the murine TAC model showed that ubiquitination of myocardial proteins was unchanged after 7 days, but did increase eventually between 7 and 14 days as hypertrophic growth slows down and approaches a new steady-state [22]. Several possibilities could account for the differences in these findings. First, protein ubiquitination may function as a more selective process by regulating the expression of a subset of proteins required to initiate pressure overload hypertrophy. A transient increase in ubiquitination of cytoskeletal-bound proteins occurs during the initial 48 h of RV pressure overload hypertrophy in the cat that returns to near baseline levels by 7 days [23]. Second, increased or decreased protein ubiquitination does not correspond to the rate of myocardial protein degradation, a value that reflects turnover a mixed protein pool [11]. Third, recent studies indicate that myocardial protein degradation may involve coordinated activities of the ubiquitin–proteasome system and calcium-activated proteases, or calpains [24]. Sarcomeric proteins are dissembled by activation of calpain prior to degradation of myofibrillar proteins via the ubiquitin proteasome system. Perhaps the rate of myocardial protein degradation is suppressed during the initial growth phase of pressure overload hypertrophy until enzymes such as calpains break down the large pool of sarcomeric proteins that subsequently becomes a substrate for ubiquitination.

In conclusion, a continuous infusion method has been developed to measure in vivo rates of myocardial protein synthesis in the mouse. Following implantation of an osmotic mini-pump, radiolabeled LEU can be infused continuously in mice that are conscious and ambulatory. The utility of this approach is demonstrated using the TAC model of pressure overload hypertrophy. Similar to other mammalian models of LV pressure overload, the initial increase in LV mass is generated by an accelerated rate of protein synthesis and suppression in the rate of protein degradation. As the rate of hypertrophic growth slows, a new steady-state with respect to protein metabolism is attained. The method is particularly relevant to transgenic models because it can be used to determine time-dependent effects on protein synthesis and degradation during hypertrophic growth and adverse remodeling in the mouse.