Change in expression of heart carnitine palmitoyltransferase I isoforms with electrical stimulation of cultured rat neonatal cardiac myocytes.

Electrical stimulation of neonatal rat cardiac myocytes in culture produces increases in myocyte size (hypertrophy) and organization of actin into myofibrillar arrays. The maturation of the cells is associated with enhanced contractile parameters and cellular calcium content. The numbers and intensity of cellular mitochondrial profiles increase, as measured by scanning laser confocal microscopy. Consistent with the hypertrophic response is increased cellular content of β-myosin heavy chain and cytochrome oxidase subunit Va messages, as well as increases in cytochrome oxidase activity in the stimulated cardiac myocytes. Myocyte contractile capacity is associated with increased expression of the muscle carnitine palmitoyltransferase (CPT-I) isoform as measured by Northern analysis, immunoblotting, and altered sensitivity of CPT-I activity to malonyl-CoA in the stimulated cells. The data suggest that a switch from the liver isoform of CPT-I, prominent in the neonatal rat heart, to the muscle CPT-I which predominates in adult rat heart, takes place in the neonatal cardiac myocytes over the same time period as the hypertrophic-mediated changes in myofibrillar assembly and increased contractile activity.

Electrical stimulation of neonatal rat cardiac myocytes in culture produces increases in myocyte size (hypertrophy) and organization of actin into myofibrillar arrays. The maturation of the cells is associated with enhanced contractile parameters and cellular calcium content. The numbers and intensity of cellular mitochondrial profiles increase, as measured by scanning laser confocal microscopy. Consistent with the hypertrophic response is increased cellular content of ␤-myosin heavy chain and cytochrome oxidase subunit Va messages, as well as increases in cytochrome oxidase activity in the stimulated cardiac myocytes. Myocyte contractile capacity is associated with increased expression of the muscle carnitine palmitoyltransferase (CPT-I) isoform as measured by Northern analysis, immunoblotting, and altered sensitivity of CPT-I activity to malonyl-CoA in the stimulated cells. The data suggest that a switch from the liver isoform of CPT-I, prominent in the neonatal rat heart, to the muscle CPT-I which predominates in adult rat heart, takes place in the neonatal cardiac myocytes over the same time period as the hypertrophic-mediated changes in myofibrillar assembly and increased contractile activity.
Cardiac myocytes adapt to hemodynamic stress by compensatory hypertrophy which increases cell mass, but not cell number. As a result of increased energy demand due either to pathological or physiological sequellae, gene expression was altered, mediating the expression of the adaptive process. The signaling mechanisms by which the cardiac myocytes translate the increase in hemodynamic stress to activate intermediate early gene and contractile protein gene expression are not well understood, but may involve production of growth factors expressed not only by myocytes but also by non-myocytic cells in the intact heart. Demonstration that some of these growth factors, e.g. transforming growth factor-␤, insulin-like growth factor, endothelin, and angiotensin, are present in cardiac myocytes (see Ref. 1 for review) suggests that autocrine pathways may mediate, in part, the hypertrophic response. The ability of mechanical stimuli applied to cultured neonatal cardiac myocytes in the absence of serum to enhance c-fos expression (2,3) and to stimulate myofibrillar growth and organization in vitro (4) suggests that increases in contractile activity of the cardiac myocyte in culture may lead to the hypertrophic response by an autocrine pathway. Although the alterations in contractile pro-tein content and expression and their role in ventricular remodelling have been extensively studied in cultured neonatal rat cardiac myocytes, the role of contractile stimulation of cardiac myocytes in the nuclear transcription of mitochondrialspecific proteins has not been described. It is reasonable to anticipate that alterations in energy supply and demand would require compensatory proliferation of mitochondrial proteins involved in energy transduction.
Following electrical stimulation of primary ventricular myocytes in culture, increased myofibrillar content and organization is accompanied by increased expression of cardiac myosin light chain-2 and atrial natriuretic factor (4,5). To date, this valuable model has not been explored for effects of electrical stimulation on cardiac mitochondrial gene expression. Mitochondrial protein expression is known to increase in skeletal muscle with exercise training and chronic electrical stimulation of intact muscle (see Ref. 6 for review). In multicellular organisms, encoding of nuclear respiratory genes is under both developmental and metabolic control as demonstrated by coordinate expression of several proteins of the electron transport chain (7). Moreover, tissue specific isoforms representing different mechanisms of regulation or catalysis within an organ have been described for the adenine nucleotide translocase (8) as well as for cytochrome c and cytochrome oxidase subunits (9,10). The level of mRNA encoding for these isoforms is likely growth-regulated as shown by stimulation of mRNA levels by the presence of serum and growth factors (11). An enzyme system crucial to the energy metabolism of the heart via oxidation of long-chain fatty acids is the mitochondrial carnitine palmitoyltransferase-I (CPT-I) 1 (12,13). In cardiac muscle, this protein exists in two isoforms recently described as the liver and muscle isoforms (14). These proteins differ from each other in their affinities for both substrate and for the metabolic inhibitor, malonyl-CoA. The liver isoform contributes ϳ25% to total CPT-I activity in the neonatal heart, this level then decreasing to comprise only 2-3% of CPT-I activity in the adult (15). Therefore, the growth related expression of mitochondrial protein and of the heart isoforms of CPT-I was investigated in this model of electrical stimulation where hypertrophic growth and maturation of the cardiac myocyte occur after prolonged increases in mechanical activity.

Cell Culture
Neonatal (1-3 day old) cardiac myocytes were isolated and plated in 12-well dishes in Dulbecco's modified Eagle's medium, supplemented with 10% Hyclone calf serum at a plating density of 4 ϫ 10 5 cells/21 cm, as described previously by this laboratory (16). After 24 h, the serumcontaining medium was removed and the cells were washed and subsequently maintained for 72 h in Dulbecco's modified Eagle's medium in the absence of serum, containing 1% bovine serum albumin (Fraction V). The medium was exchanged for fresh serum-free medium at 2 days.
After four days in culture (24 h ϩ serum, 72 h Ϫ serum), three wells containing myocytes were stimulated for 72 h using the method of Brevet et al. (17) as modified by McDonough and Glembotski (4), using a GRASS stimulator, Ag:AgCl electrodes, and agarose gel cross-bridges that link the wells in parallel. Stimuli are 80 V with a pulse duration of 10 ms at a pacing frequency of 5 Hz. A cyclic polarity reversal, installed in the circuit by Dr. Craig Hartley, Baylor College of Medicine, reverses the output from the stimulator every 20 s. Three additional wells in the dish were maintained in the same medium as the stimulated cells, but in the absence of contractile stimulation. During the experimental period, the medium bathing the control and stimulated cells was changed after 48 h to fresh Dulbecco's modified Eagle's medium ϩ 1% bovine serum albumin.

Microscopic Techniques
Cell Size Measurement-Cells were cultured on laminin-coated (10 g) glass coverslips inserted into the multiwell dishes. After 72 h electrical stimulation (80 V, 5 Hz), the cells were permeabilized with 0.5% Triton X-100, fixed in 3.7% formaldehyde (Tousimis Research Corp., Rockville, MD), and stained for actin with BODIPY FL phallacidin (Molecular Probes, Inc., Eugene, OR). The fluorescent images of the control and stimulated cells were obtained at an excitation wavelength of 488 nm with emission at 510 nm, using a Molecular Dynamics 2001 Scanning Laser Confocal microscope. The size of the cells was measured by planimetry of the two-dimensional images.
Myocyte Contractility-Cells plated on laminin-coated glass coverslips were placed in the wells and cultured for 72 h under control and stimulating conditions. Contractility measurements were performed on the cells on coverslips in a Sykes-Moore chamber filled with 1 ml of medium, inserted into a heated microscope stage. A video edge detector system from Crystal Biotech (Boston, MA) was used to measure changes in contractile parameters. The cells were paced at 50 volts for 5 ms and 90 pulses/min. Raster lines were attached to cell edges (60 ϫ lens) and the analogue motion signal was digitized and analyzed.
Calcium Content-Calcium content was measured on control and stimulated cells on laminin-coated glass coverslips following 72 h, as described above. The calcium-specific dye, Fluo-3 AM (2 ng/ml) (Molecular Probes, Inc.), which fluoresces only upon chelation of the dye with calcium, was used to monitor averaged cell calcium levels using the Molecular Dynamics 2001 Scanning Laser Confocal microscope with an argon ion laser set for excitation at 488 nm and emission at 510 nm.
Mitochondrial Content-Cardiac myocytes were cultured and stimulated as described above. After 72 h, the control and stimulated cells were stained with 1000 nM MitoTracker (Molecular Probes, Inc.) dissolved in dimethyl sulfoxide by incubation for 15 min under serum-free culture conditions. The loading solution was then replaced with fresh medium and cell fluorescence was visualized using the Molecular Dynamics 2001 Scanning Laser Confocal microscope with excitation set at 514 nm and emission at 535 nm. For comparison of relative fluorescent intensities in the cells, a three-dimensional "fishnet plot," generated by the Molecular Dynamics Image Space software was employed.

Enzyme Activity Measurements
Carnitine Palmitoyltransferase-CPT-I and CPT-II activities were measured in the cardiac myocytes as described previously by this laboratory (16). Briefly, 5 M digitonin was used to permeabilize the cells for assay of CPT-I by incubating the cells for 10 min in 0.5 ml of a permeabilizing medium ("Medium J," Ref. 18) containing digitonin, 40 mM Hepes, 140 mM KCl, 20 mM NaCl, 5 mM MgCl 2 , 1 mM EGTA, 0.566 mM CaCl 2 , 5 mM ATP, 6 g/ml oligomycin, and 5 M NaN 3 . Following permeabilization, fresh Medium J (0.5 ml) containing 1% bovine serum albumin (Fraction V) was added for assay of CPT-I. The CPT-I reaction was carried out for 20 min at 37°C in the presence of 30 M palmitoyl-CoA and 0.4 mM [ 14 C] -carnitine, specific activity ϭ 2600 dpm/nmol. The sensitivity of CPT-I to inhibition by malonyl-CoA was determined after 3 min preincubation over a final concentration range of malonyl-CoA from 20 nM to 60 M prior to initiation of the reaction by [ 14 C]carnitine. The reaction was terminated with butanol-saturated 1 N HCl and the radioactive product extracted and washed as described previously (19). CPT-II was measured by addition of Triton X-100 (0.1%) to the cells in Medium J, a condition which fully expresses CPT-II and inactivates CPT-I (16). The assay was then carried out as described for CPT-I except that no digitonin-permeabilization step was required. Cellular protein was measured by the Lowry method (20).
Cytochrome Oxidase-Following permeabilization of the cardiac myocytes with 1% octyl glucoside, cytochrome oxidase activity was measured by following oxidation of reduced cytochrome c at 550 nm (21).

Immunoblots of CPT-I Muscle Isoform
The [ 3 H]etomoxir-labeled protein in mitochondria was reported to represent the malonyl-CoA sensitive catalytic subunit of CPT-I (22), with the ϳ80-kDa protein being characteristic of the muscle isoform present in cardiac and skeletal muscle (14). Polyclonal antibodies prepared to an etomoxir-labeled 79-kDa protein from rat heart was kindly provided by Dr. Loran Bieber, Michigan State University. Cellular protein was suspended in 0.2 ml of lysis buffer (150 mM NaCl, 1% Nonidet P-40, and 2 mM EDTA) in the presence of protease inhibitors (40 g of phenylmethylsulfonyl fluoride, and 5 g each of leupeptin and aprotinin). The supernatant (100 -150 g of protein) was denatured in sample buffer and electrophoresed at 200 V on 10% polyacrylamide gels. After transfer onto a 0.45-m nitrocellulose membrane at 30 V overnight, the membrane-bound protein was incubated in blocking solution containing 5% non-fat dry milk in TBS (20 mM Tris-HCl, pH 7.5, and 250 mM NaCl), rinsed in TBS ϩ 0.1% Tween 20, and then incubated in 1:2500 dilution of CPT-I muscle isoform antibody with 1% non-fat dry milk overnight. After washing, the blot was incubated with goat antirabbit IgG-conjugated peroxidase antibody (1:1500 in 1% non-fat dry milk) for 1 h. The membrane was again washed with TBS and developed using a chemiluminescent detection system (Amersham, ECL blotting kit). Only one band at 79 kDa was observed (23).

Preparation of cDNA Probes
DNA sequences of oligonucleotides used to generate the cDNA probes for this study were as follows.
Carnitine Palmitoyltransferase I (Liver Isoform) (24) Primers were designed from published sequences. DNA and protein homology searches were performed with BLAST, using GenEMBL and/or SWISS-PROT data bases. The probes for CPT-I, ␤-myosin, and cyclophilin were obtained using total RNA from neonatal liver (CPT-I liver isoform, 738 bp), neonatal skeletal muscle (CPT-I muscle isoform, 428 bp), and neonatal rat heart (␤-myosin, 408 bp and cyclophilin, 402 bp) by reverse transcriptase-polymerase chain reaction (PCT-100 TM , Watertown, MA) and the reverse transcriptase-polymerase chain reaction kit from Promega Corp. The cDNA probes were sequenced by the dideoxy-mediated chain termination method and sequence analysis was performed using the Genetics Computer Group sequence analysis package, version 7.2 (University of Wisconsin, Madison, WI). The 650-bp cDNA fragment of cytochrome oxidase subunit Va was purified from the vector pOC 1318/Eco (provided by Dr. E. A. Schon, Columbia University) after digestion with EcoRI. 28 S rRNA cDNA was a gift from Dr. K. Chien. 18 S rRNA cDNA was provided by Dr. Sue-Hwa Lin (M.D. Anderson Cancer Center) and purchased from Ambion, Inc. The cDNA was purified from pT7 RNA 18 S using EcoRI and HindIII resulting in a 150-bp cDNA fragment for use with cyclophilin as internal standards of RNA loading. The cDNA probes were purified by 1% agarose gel electrophoresis where the uv detected bands were cut from the gels and isolated by electrical elution. The cDNA probes were labeled to high specific activity using random primer labeling (Promega Corp.).

RNA Isolation and Northern Blot Analysis
RNA was isolated from the cultured neonatal rat cardiac myocytes and from various neonatal rat tissues by using the Ultraspec TM RNA isolation system (Biotex Laboratories, Houston, TX). Total RNA and RNA purity were quantitated spectrophotometrically using absorbance at 260 and 260/280 nm ratios. For Northern blotting, 10 g of total cellular RNA was electrophoresed in 1% agarose containing 0.6% formaldehyde at 45 V for 3 h. Fractionated RNA was transferred to a Duralon-UV nylon membrane (Stratagene, La Jolla, CA) by capillary action for 24 h. After blotting, the RNA was cross-linked to the filter by uv irradiation. Following prehybridization overnight at 42°C, the blot was hybridized in the same prehybridization solution containing 1% dextran sulfate as well as the labeled cDNA of choice. After washing, the blots were visualized by autoradiography following storage at Ϫ70°C for 24 -72 h.

Statistics
The significance of the changes reported was determined using Student's t test for paired and non-paired variates. Data are presented as the mean Ϯ S.E.

RESULTS
To confirm the hypertrophic effects of electrical stimulation, cardiac myocytes were stained with the immunofluorescent probe, BODIPY phallacidin (data not shown). Dramatic differences between the control and the stimulated myocytes were seen, not only in cell size, as described previously (4), but also in the organization of actin from punctate cores of actin characteristic of the immature cardiac myocyte (29) into striated arrays of well organized myofilaments. The effects of electrical stimulation were assessed using planimetry, for changes in cell area, and for cellular protein and RNA content. Cell size increases almost 4-fold, from 253 Ϯ 45 m 2 to 970 Ϯ 88.5 m 2 (p Ͻ 0.005). The alteration in size was accompanied by significant increases in both protein (124 Ϯ 3.2 to 162 g Ϯ 7.4/4 ϫ 10 5 cells, p Ͻ 0.01) and RNA content (1.57 Ϯ 0.2 to 2.5 g Ϯ 0.8/4 ϫ 10 5 cells, p Ͻ 0.01) in the control versus stimulated cells, respectively. The increases observed were consistent with the magnitude of changes observed in other models of neonatal cardiac myocyte hypertrophy (30).
In serum-free medium and in the absence of pacing, control cells were often quiescent for prolonged periods. With pacing, the contractility parameters of the control cells were significantly depressed when compared to cells which were exposed to 72 h of electrical stimulation. In the stimulated myocytes, time to peak contraction was abbreviated to 32.3 Ϯ 9% of control values and percent shortening was dramatically increased by 7.85-fold from 6.6 Ϯ 1 to 52.2 Ϯ 2.7% (p Ͻ 0.01) in the stimulated cells. The peak calcium fluorescent intensities also increase in parallel with the enhancement in cellular contractility from relative peak fluorescence intensities of 42.6 Ϯ 5.8 to 87.8 Ϯ 9.3 in the stimulated cells.
Subsequent studies were designed to monitor the effect of the hypertrophic response, and accompanying increases in contractile function, on the cellular capacity for oxidative metabolism and energy production. Mitotracker labels mitochondria within living cells where it was oxidized as a fluorescent product (31). A perinuclear distribution of fluorescence was easily discerned in the control myocyte and the relative fluorescent intensity of the dye was displayed in the "fishnet" plot (Fig. 1A). There was a dramatic increase in both mitochondrial content and fluorescent intensity in the cells which were electrically stimulated for 72 h (Fig. 1B).
Consistent with the increased mitochondrial content in the stimulated cells was a 1.7-fold increase in cytochrome oxidase activity (Fig. 2). This increase in cytochrome oxidase activity was accompanied by a 2.38-fold (densitometry units) increase in cytochrome oxidase (subunit Va) mRNA content in the stimulated cells (Fig. 3). The level of ␤-myosin heavy chain mRNA was also significantly elevated after 72 h of stimulation (Fig. 3). The re-expression of embryonic contractile protein isoforms was characteristic of the hypertrophic growth response in neonatal cardiac myocytes (32).
Tissue-specific isoforms of mitochondrial proteins provide regulatory and/or catalytic properties which were uniquely respon- sive to metabolic signaling pathways of the organ. The heart contains two CPT-I isoforms, the liver isoform which decreases and the muscle isoform which increases in content with cardiac development (15). To confirm the presence of message for these two proteins in the neonatal heart cells, Northern blots of the total RNA extracted from liver, heart, and muscle were probed with the cDNA sequences for the liver and skeletal muscle CPT-I isoforms. Liver CPT-I cDNA hybridizes with liver and neonatal heart RNA, but no signal was obtained from muscle total RNA (Fig. 4). Conversely, the cDNA probe for the muscle CPT-I hybridizes with RNA from heart and muscle, but not with liver RNA (Fig. 4). After 72 h of stimulation, the mRNA content of the liver isoform appears to decrease in the cardiac myocytes, whereas the mRNA content of the muscle isoform was increased in the stimulated cells (Fig. 4). The relative intensity of the signals from the control and stimulated cells was quantitated by densitometry. The mRNA content for the CPT-I liver isoform in the stimulated myocytes was approximately half the content that was present in the control cells (Fig. 5). Conversely, the mRNA for the muscle isoform increases 2.5-fold in the stimulated cells when compared to control (Fig. 5).
To determine whether there was increased expression of the muscle CPT-I protein, immunoblotting of the control and stimulated cell lysates was carried out using the antibody against the etomoxir-binding protein from rat heart. A 2.8-fold increase, determined by densitometric scanning, in the amount of immunoreactivity representing the predominant cardiac CPT-I isoform was detected in the stimulated myocytes (Fig. 6). This increase in the lower molecular weight isoform of CPT-I, which has been identified as the muscle isoform (33) was accompanied by a 3-fold increase in CPT-I activity in the stimulated cells (Fig. 6). The CPT-I activity in the control and in the stimulated cells display differing affinities of the enzyme for its inhibitor, malonyl-CoA. The serum-deprived control cells display a lower CPT-I activity and an IC 50 for malonyl-CoA which was in the range observed for the liver isoform of CPT-I, i.e. low M (Fig.  7). The stimulated cells have a prominent high affinity inhibitory response to malonyl-CoA with an I 50 of approximately 90 nM which resembles the sensitivity of the adult cardiac muscle with its predominant muscle CPT-I isoform (14). DISCUSSION The RNA levels of mitochondrial proteins such as the adenine nucleotide translocase increase in response to serum (11), similar to other growth-regulated genes, e.g. c-fos and c-myc (34,35). The adenine nucleotide translocase mRNA appears to increase in direct response to a growth factor signal (11). This response of mitochondrial proteins to serum was consistent with the increased energy demands of proliferating cells (11,36). Nuclear promoter sequences as well as the cognate nuclear factors for these sequences have been characterized for cytochrome c by Evans and Scarpulla (36). The physiological affector molecules which interact with the promoter elements were potentially important to the modulation of respiratory gene expression as energy demands of the cell were changed. This is the first report that documents mitochondrial proliferation in response to contractile stimulation in the absence of exogenous growth factors as evidenced by the higher levels of cytochrome oxidase mRNA and enzyme activity as well as the increased numbers of mitochondria observed microscopically in the stimulated cells. In addition, the effects of contractile stimulation on expression of the musclespecific isoform of carnitine palmitoyltransferase I in the cultured cardiac myocytes is the first model system where a tissue-FIG. 2. Cytochrome oxidase activity in control and stimulated neonatal cardiac myocytes in culture. After stabilization of the cardiac neonatal myocytes in serum-free medium, the control and stimulated cells were maintained in culture for an additional 72 h, after which the myocytes were permeabilized with 1% octyl glucoside. Cytochrome oxidase activity was measured by following oxidation of cytochrome c at 550 nm (21). The results are the mean Ϯ S.E. of four different cell cultures.
FIG. 3. Increased content of ␤-myosin heavy chain and cytochrome oxidase (subunit Va) mRNA in control and stimulated neonatal cardiac myocytes in culture. Following stabilization of the cells in serum-free medium, control and stimulated cells were maintained in culture for an additional 72 h. Northern blot hybridization was carried out using the cDNA probes described under "Experimental Procedures" for ␤-myosin, cytochrome oxidase, subunit Va, and cyclophilin. The hybridization signals for ␤-myosin heavy chain and for cytochrome oxidase are shown in the top two panels of total RNA from control (C) and stimulated (S) neonatal cardiac myocytes. The lower panels are the hybridization signals obtained from cyclophilin, 28 and 18 S RNA, used as internal standards for RNA loading. The Northern blot shown is representative of blots obtained with RNA preparations from four different cell cultures.
FIG. 4. Northern blot of liver and muscle CPT-I isoforms: tissue specificity and abundance of mRNA in control and stimulated neonatal cardiac myocytes. The total RNA is isolated from liver, skeletal muscle, and cardiac tissue of neonatal rats and from control and stimulated neonatal cardiac myocytes after 72 h in serumfree medium in the presence and absence of electrical stimulation. The cDNA probes representing the muscle and liver isoforms of CPT-I were hybridized to the total RNA (10 g/lane) from the different tissues and cells, where the abbreviations, L, H, M, C, and S, stand for liver, heart, skeletal muscle, and control and stimulated neonatal cardiac myocytes. In the top lane, the 4.3-kilobase signal represents the mRNA for the liver CPT-I isoform (ϳ4.7 kilobases, Ref. 24) and the 2.8-kilobase signal, the mRNA for the muscle CPT-I isoform (25). The blot is representative of blots obtained with five different cell cultures. specific production of a mitochondrial protein can been "switched on" in the absence of serum factors.
A change in the expression of CPT-I isoforms has been documented in the developing rat heart where liver CPT-I was reported to contribute ϳ25% to total cardiac CPT-I activity at birth (15). This value declines to 2-3% as the rat pups mature to adulthood (15). Recently, a cDNA clone encoding the muscle isoform of heart CPT-I has been identified as distinct from the liver CPT-I cDNA sequence (25) and expressed (26), thus providing the molecular basis for the biochemical findings. Confirmation that both the liver and the muscle isoforms for CPT-I may be present to varying degrees in the neonatal heart myocyte cultures suggests that both proteins are important to cardiac development and to the energy metabolism of the contracting heart. The physiological importance of early expression of the liver CPT-I may relate to its low K m (30 M) for carnitine, in keeping with the low cardiac carnitine content at birth (15). In the serum-free cell cultures, we have evidence suggesting that the predominant CPT mRNA expressed is the liver isoform, implying that serum withdrawal leads to a less active contractile state in which muscle-specific gene expression is diminished. In response to contractile stimulation and activation of transcription of the muscle CPT-I isoform, an increase in CPT-I activity is observed, as well as an increase in the amount of immunoreactive 79-kDa protein which corresponds to the dominant adult rat cardiac etomoxir-binding protein (muscle CPT-I). Enhanced expression of this protein in the stimulated cells was further supported by a dramatic alteration in the high affinity binding properties for malonyl-CoA, with an approximate 40-fold decrease in the I 50 for malonyl-CoA after 72 h of electrical stimulation. A double reciprocal plot of CPT-I activity versus carnitine concentration was also carried out (data not presented). Whereas the stimu-  6. Increased cellular content of muscle CPT-I isoform and enzyme activity in neonatal heart cells in culture following electrical stimulation. The immunoblot of total cellular protein in the control and stimulated cells was incubated with polyclonal antibodies to a 79-kDa etomoxir-labeled protein from rat heart and subsequently developed using a chemiluminescent detection system (upper panel, 100 g of total protein loaded per lane). CPT-I activity (lower histogram) is defined as malonyl-CoA sensitive activity measured in digitonin permeabilized cells as described previously (16). The activities represent the mean Ϯ S.E. for four different cultures where: *, p Ͻ 0.005 between control and stimulated cells; and Ⅺ, control cells; and f, stimulated cells. lated cells demonstrated a single slope with high activity and a K m which was consistent with cardiac CPT-I activity, the control myocytes exhibit kinetic behavior consistent with the presence of two different isoforms, i.e. two distinct linear regions with differing affinities for carnitine. These results were consistent with the presence of both isoforms of CPT-I in the cardiac myocyte, with the liver isoform being in high enough concentration in the control neonatal cells to contribute significantly to the velocities measured.
Tissue-specific isoforms have been described for other mitochondrial proteins including cytochrome c (9), the ␥ subunit isoform of mitochondrial ATP synthase (37), as well as the adenine nucleotide translocase (38). It might be anticipated that the kinetic properties and metabolic regulation of the isoforms should reflect the physiological demands of that tissue as has been reported for mitochondrial ATP synthase (39). The predominance of the high K m muscle isoform for CPT-I in skeletal muscle and heart probably reflects the higher carnitine concentrations endogenous to these organs (40). Still unresolved, however, is the adaptation required by the heart that results in such a profound difference in the I 50 values for malonyl-CoA. Since measured malonyl-CoA concentrations in heart may exceed the I 50 values for malonyl-CoA in that tissue by as much as 1000-fold, investigators have hypothesized that the majority of the malonyl-CoA in heart was compartmentalized away from the inhibitory site for malonyl-CoA on the outer mitochondrial membrane (40). Alternatively, it has been suggested that the presence of the liver isoform in heart may have a role in facilitating basal rates of fatty acid oxidation when malonyl-CoA was in an inhibitory range for the muscle isoform (15). Unlike the liver CPT-I, the activity and malonyl-CoA sensitivity of the muscle (cardiac) isoform was not influenced by diabetes and starvation, conditions which alter the sensitivity of the liver CPT-I for its inhibitor (41).
The mechanisms which act to regulate the expression of nuclear genes during mitochondrial proliferation with subsequent differentiation to reflect tissue-specific expression include transcription, message stabilization, and activation of translation rates (42). The two mRNAs which encode the muscle and liver CPT-I isoforms may be the product of different genes, as has been suggested for the adenine nucleotide translocase (38). This possibility was strengthened by the observations that the liver and muscle CPT-I proteins were immunologically distinct (43) and that there is tissue specificity exhibited by the two cDNA probes (25). Alternatively, the liver and muscle CPT-I proteins may be products of the same gene by either different transcriptional or post-transcriptional processing. It is possible that the nuclear encoded CPT-I muscle isoform, like the subunit Va of cytochrome oxidase, responds directly to electrical stimulation as a consequence of growth factor activation of cardiac myocyte calcium channels (44) or to other endogenous intracellular signals which promote cell growth in the absence of serum. In this regard, we have reported a potent mitogenic effect of insulin growth factor-1 (10 ng/ml) on CPT-I and CPT-II expression in the neonatal cardiac myocytes in the absence of serum (45). Significantly, transient expression of insulin growth factor-1 occurred and increased insulin growth factor-1 mRNA levels were seen with pressure overload hypertrophy (46). Therefore, the model of electrical stimulation provides an excellent tool to assess the role of mitochondrial proteins and the energy-producing mechanisms of the cell in the development of cardiac myocyte hypertrophy and in the physiological adaptation to increased energy demands as part of the mitogenic response. The role of c-fos oncogene expression in the mediation of this pathway during electrical stimulation is currently under investigation.