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(Circulation Research. 2004;95:424.)
© 2004 American Heart Association, Inc.

Integrative Physiology

Protein Kinase C Overexpression Alters Myofilament Properties and Composition During the Progression of Heart Failure

Paul H. Goldspink, David E. Montgomery, Lori A. Walker, Dalia Urboniene, Ronald D. McKinney, David L. Geenen, R. John Solaro, Peter M. Buttrick

From the Program in Cardiovascular Sciences, Departments of Medicine (P.H.G., L.A.W., R.D.M., D.L.G., P.M.B.) and Physiology & Biophysics (D.E.M., D.U., R.J.S.), University of Illinois at Chicago.

Correspondence to Paul Goldspink, PhD, Section of Cardiology, University of Illinois at Chicago, 840 S Wood St, M/C 715, Chicago, IL 60612. E-mail pgolds{at}uic.edu

	Abstract

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We report characterization of a transgenic mouse that overexpresses constitutively active protein kinase C in the heart and slowly develops a dilated cardiomyopathy with failure. The hemodynamic, mechanical, and biochemical properties of these hearts demonstrate a series of temporal events that mark the progression of the disease. In the 3-month transgenic (TG) animals, contractile properties and gene expression measurements are normal, but an increase in myofibrillar Ca²⁺ sensitivity and thin filament protein phosphorylation is noted. At 6 months, there is a decrease in the myofibrillar Ca²⁺ sensitivity, a significant increase in ß-myosin heavy chain mRNA and protein, normal cardiac function, but a blunted response to an inotropic challenge. The transition at 9 months is especially interesting because age-related changes appear to contribute to the decline in function seen in the TG heart. At this point, there is a decline in baseline function and maximum tension produced by the myofibrils, which is coincident with the onset of atrial myosin light chain isoform re-expression in the ventricles. In the 12-month TG mice, there is clear hemodynamic and geometric evidence of failure. Alterations in the composition of the myofibrils persist but the phosphorylation of myosin light chain 2v is dramatically different at this age compared with all others. We interpret these data to implicate the disruption of the myofibrillar proteins and their interactions in the propagation of dilated cardiac disease.

Key Words: contractile proteins • heart failure • protein kinase C

	Introduction

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Heart disease is the most frequent cause of death in the general population, with a dramatically increasing incidence in the elderly.¹ As with the aging heart, many pathologic features of heart failure are related to structural and functional alterations in cardiac muscle cells. However, the molecular mechanisms underlying the progression of heart failure at the level of cardiac muscle function are largely unknown.

A number of diverse lines of evidence have suggested that activation of protein kinase C (PKC) plays a central role in the physiologic and pathophysiologic adaptation of the heart. Studies in vitro and in vivo have shown that PKC phosphorylates a number of important cardiac proteins, including myofilament proteins,² as well as proteins involved in Ca²⁺ homeostasis.³ Clearly, one hypothesis concerning PKC activation is that increased myofilament phosphorylation results in contractile dysfunction, which diminishes cardiac output, which results in a compensatory enlargement of the heart. This is supported by evidence showing that PKC-mediated phosphorylation of the myofilaments is associated with depressed myofilament activity in reconstituted systems.⁴ Multiple isoforms of PKC are expressed in the heart during development, with the predominant isoforms in the adult being the Ca²⁺-dependent () and the Ca²⁺-independent (, ) isoforms.⁵ Protein kinase C, an isoform that translocates to the myofilaments on activation and is linked to the negative inotropic effects of phorbol esters and ₁-adrenergic receptor agonists in vitro, is therefore a likely candidate to regulate myofilament activity.⁶

Our current knowledge of the molecular alterations brought about by PKC isoform activation is derived largely from gain-of-function studies in animal models. Data from transgenic mouse models support the notion that PKC activation is sufficient to induce a hypertrophic response. Hearts of mice that overexpress the Ca²⁺-dependent PKC isoforms demonstrate abnormalities in cellular Ca²⁺ handling and eventually hypertrophy.^7,8 Transgenic mice that overexpress either the constitutively active PKC enzyme or an activator peptide (RACK) develop a hypertrophic phenotype with contractile dysfunction.^9,10 However, the majority of data presented from these murine models focus on single time points of disease progression. To completely explore the hypothesis concerning PKC activation, important issues related to the temporal progression of the maladaptive process also need to be studied.

Here we report the use of a transgenic model that overexpresses a constitutively active PKC in the myocardium and progresses over time to a dilated cardiomyopathic state with signs of failure. This model suggests that the gradual alterations of the myofibrillar proteins and their interactions form the basis of the functional transitions that take place during the progression of heart failure.

	Materials and Methods

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The experiments were conducted in accordance with the institutional Animal Care and Use Committee and National Institutes of Health guidelines. A detailed description of the mouse model and methodology can be found in the online data supplement available at http://www.circresaha.org.

Pressure–Volume Loops
Mice were anesthetized with 0.75% to 1.25% isoflurane delivered through a vaporizer with 100% oxygen, connected to a rodent ventilator with the stroke volume set at 0.2 to 0.4 mL and a respiration rate of 125 breaths per minute.

A 1.4-French pressure-conductance catheter (SPR-839; Millar Instruments, Houston, Tex) was inserted retrograde into the left ventricle and baseline pressure–volume loops were recorded (ARIA Pressure Volume Conductance System; Millar Instruments).

Steady-State Tension Measurements
Steady-state tension was measured in detergent-extracted fiber bundles dissected from left ventricular papillary muscle.¹¹

Back-Phosphorylation
Frozen myocardium was homogenized in isolation buffer (in mmol/L: 100 KCl, 10 imidazole, 1 MgCl₂, 2 EGTA, 4 Na₂ATP, pH 7.2) plus 1% Triton X-100. A 25-µL reaction (containing 5 µL protein homogenate, 15 µCi [-³²P] ATP [Amersham 5000Ci mmol/L], 0.5 µL PKC [human recombinant, CALBIOCHEM], 16.75 µL standard relaxing buffer, 1.75 µL Micelle mix {23 µL 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-L-serine], 12 µL 1,2-dioleoyl-sn-glycerol and 0.5 µL Triton X-100]}) was incubated for 30 minutes at 30°C, followed by SDS-PAGE analysis.

Western Blotting
Ventricular samples were frozen in liquid nitrogen and 5 to 6 animals per group were pooled. Samples were homogenized in buffer (in mmol/L: 20 Tris-HCl [pH 7.4], 2 EDTA, 10 EGTA, 320 sucrose, 0.3 PMSF, 20 µg/mL leupeptin and 10 ß-mercaptoethanol) on ice. Ultracentrifugation was used to fractionate the cytosolic and particulate fractions before separation with SDS-PAGE and transfer to nitrocellulose. Antibodies for PKC isoforms and myosin light chain (MLC)-2 (FL-172) were purchased from Santa Cruz Biotechnology. The antibodies for cTnI and cTnT were purchased from Research Diagnostics Inc and Sigma Chemical Co. The antibody specific for the MLC-2a was from the Center of Biomedical Inventions, University of Texas Southwestern Medical School. The antibody that recognizes phosphorylated MLC-2 was a kind gift from Dr. N. Epstein (National Institutes of Health). Gels were performed on average 4 times and the images shown are representative.

Quantitative Reverse-Transcriptase Polymerase Chain Reaction
Analysis of gene expression was studied using quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) with SYBR Green detection in the LightCycler thermocycler (Roche Diagnostics) as previously described.¹²

Statistics
Data are expressed as means±SE. The data from the normalized pCa–tension relations were fitted to the Hill equation using nonlinear least-square regression to obtain pCa₅₀. Comparisons among groups were determined by ANOVA, followed by Newman–Keuls post-hoc analysis. P<0.05 was taken to indicate statistical significance.

	Results

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To characterize expression of the constitutively active PKC transgene, Western blot analysis was performed on total and fractionated ventricular homogenates. An increase in PKC isoform expression was seen in the total and particulate fraction of TG hearts compared with the age-matched nontransgenic controls (NTG). Also, no change in translocation of the other PKC isoforms expressed in the heart was noted.

A full description of these experiments can be found in the online data supplement available at http://circres.ahajournals.org (see online Figure I).

Morphometric and Hemodynamic Measurements
Table 1 shows no difference in the left ventricular weight-to-body weight (LV/BW) ratios of TG animals. However, when the whole heart-to-body weight ratio was calculated (HW/BW), the 12-month TG mice were significant compared with all other groups (HW/BW ratio; 12mNTG=6.0±0.2 versus 12mTG=8.9±0.9, n=6; P<0.05) because of extensive calcification of the left atria.

View this table: [in this window] [in a new window]   Table 1. Heart Weight and Hemodynamic Measurements Recorded in TG and NTG Mice at Baseline and After Injection With Dobutamine

Hemodynamic measurements taken from 3- and 6-month TG mice show no differences under baseline conditions, whereas at 9 and 12 months, there were significant decreases in systolic pressures and indices of contractility (+dp/dt and –dp/dt) compared with NTG controls (Table 1). To determine whether any contractile abnormalities could be unmasked in the 3- and 6-month TG mice, a cohort was given an inotropic challenge by administrating dobutamine. Both age groups of NTG mice responded with increases in both +dp/dt and –dp/dt (Table 1). A similar increase was also seen in the 3-month TG mice, whereas the 6-month TG demonstrated a blunted response. Peak systolic pressure was also significantly decreased in the 6-month TG mice (6mNTG=123.9±1.9 versus 6mTG=94.8±1.3 mm Hg, n=6; P<0.001), with no change in heart rate.

Echocardiography was performed to characterize the myopathy seen in the 12-month TG mice. Left ventricular end-diastolic and systolic dimensions were significantly increased (LVDd: 12mNTG=0.24±0.03 versus 12mTG=0.30±0.01, and LVSd: 12mNTG=0.13±0.01 versus 12mTG=0.19±0.01 cm, n=4, P<0.05), with no increase in either the interventricular septum or posterior wall thickness, indicating these TG mice have a dilated nonhypertrophic cardiomyopathy. In addition, indices of cardiac function were also significantly depressed in the 12-month TG mice compared with NTG mice (see online Table I).

The decreased cardiac function is evident in the pressure–volume loops that were derived from 12-month mice (Figure 1). The end systolic pressure–volume relationship (ESPVR) shows a decreased slope, and systolic pressure was 20% lower in the TG ventricle compared with the NTG mice (96.8±0.5 versus 77.7±3.0 mm Hg, n=5; P<0.05). The relative ejection fraction was markedly decreased (NTG=45.5 versus TG=26.3%), with no significant differences in heart rate observed between groups. The slope of the end-systolic pressure–volume relationship (ESPVR), a preload-independent measure of contractility, measured after inferior vena cava occlusion was reduced (NTG=11.4 versus TG=6.1) and right-shifted in the TG animals.

View larger version (38K): [in this window] [in a new window]   Figure 1. Pressure–volume loop analysis of 12-month mice. Typical pressure–volume loops generated after IVC occlusion in 12-month NTG and TG mice. The end-systolic pressure–volume relations (ESPVR) are shown as dotted lines.

Myofibrillar Ca²⁺ Sensitivity and Tension Measurements
Ca²⁺ sensitivity and steady-state tension development of the myofilaments were determined. We observed no difference in Ca²⁺ sensitivity in the NTG mice with age, so these measurements were grouped and shown as a single curve (Figure 2A). There was a left shift in the force–pCa curve and an increased pCa₅₀ in the 3-month TG mice (pCa₅₀, NTG=5.65±0.01 versus 3mTG=5.79±0.004, n=8; P<0.05), whereas all other TG mice were no different from the NTG controls. When force is normalized to the cross-sectional area of the fibers, the maximum tension shows a trend toward depression in fibers from 3- and 6-month old TG hearts, which became significant in older TG mice compared with NTG controls (Figure 2B). This temporal pattern of decreased tension correlates with the decreased in situ hemodynamic measurements taken in the 9- and 12-month TG animals.

View larger version (24K): [in this window] [in a new window]   Figure 2. pCa2+–Force relationships in NTG and TG mice. A, No difference in Ca2+ sensitivity in the NTG mice was observed, so these measurements are shown as a single curve. B, Maximum tension generated by the myofibrils in aged-matched NTG and TG mice after being normalized to the cross-sectional area. The data show means±S.E, n=8, *P<0.05.

Myofibrillar Phosphorylation
To correlate phosphorylation of myofibrillar proteins with the changes in Ca²⁺ sensitivity and decreased maximum tension, we assayed for the presence of the constitutively active PKC in the myofibrils. We found that the constitutively active PKC copurified with myofibrils extracted from TG hearts at all ages (Figure 3A). Incubation of the purified myofibrils with [-³²P] ATP resulted in selective labeling of proteins in the TG extracts compared with the myofibrils extracted from NTG hearts, in the absence of exogenous enzyme (Figure 3B). Addition of activated recombinant PKC to NTG myofibrillar extracts showed enhanced labeling of the same bands as seen in TG myofibrillar extracts, suggesting there is specific kinase activity associated with the constitutively active PKC transgene. To determine the extent of myofibrillar protein phosphorylation at the different ages, back-phosphorylation was used. This technique is based on the addition of exogenous enzyme (recombinant PKC), which phosphorylates unoccupied phosphorylation sites. Back-phosphorylation demonstrated differences in 2 myofibrillar proteins between NTG and TG hearts that were identified as cTnT and cTnI based on comigration and antibody detection (Figure 3C). Consistent differences in both cTnI and cTnT phosphorylation could be detected in extracts from older hearts (Figure 3D). The decrease in ³²P incorporation indicates increased phosphorylation of cTnI and cTnT in the TG hearts with age (Table 2).

View larger version (58K): [in this window] [in a new window]   Figure 3. Myofibrillar protein phosphorylation. A, Representative Western blot of PKC expressed in the purified myofibrils from NTG and TG hearts. The lower half of the gel stained with Coomassie Blue. B, Autoradiogram of myofibrillar proteins purified from TG and NTG hearts and incubated with [-32P] for 1 hour with and without exogenous PKC. Similar proteins are phosphorylated in the TG myofibrillar extract (arrows) compared with NTG treated with activated recombinant PKC. Myofibrils purified from NTG hearts show nonspecific incorporation. A representative gel from 4 different samples, performed 6 times. C, Autoradiogram of back-phosphorylation of myofibrils extracted from 3-month NTG and TG hearts. To identify the phosphorylated myofibrillar proteins, identical amounts of labeled (via the back-phosphorylation) and unlabeled protein were performed on the same gel. Half of the gel was exposed to x-ray film and half used for blotting with antibodies. C, Back-phosphorylation of myofibrillar extracts from 3- and 6-month NTG and TG hearts.

View this table: [in this window] [in a new window]   Table 2. Semiquantitative Analysis of Myofibrillar Phosphorylation in TG and NTG Mice

Gene Expression
Quantitative real-time RT-PCR was performed to determine changes in gene expression. A switch in myosin heavy chain (MHC) isoform expression (-MHC and ß-MHC) was evident in the TG mice, with a significant increase in the ß-MHC isoform mRNA detectable at 6 months and older (Figure 4A). Expression of other embryonic/hypertrophic marker genes, ANF and skeletal a-actin, were significantly increased in all the TG mice, including the 3-month mice (Figure 4B and 4C). To explore the extensive calcification in the atria of 12-month TG mice, we examined osteopontin gene expression. A significant increase in osteopontin expression was seen in both the ventricular and atrial tissue of the 12-month TG mice only (Figure 4D and 4E). The expression in the atria was approximately 50-fold greater compared with all other ages and correlates with the appearance of atrial calcification (Figure 4E).

View larger version (32K): [in this window] [in a new window]   Figure 4. Quantitative hypertrophic marker gene expression. A, Expression of the myosin heavy chain isoforms (/ß MHC) in NTG and TG mice. B, Atrial natriuretic factor expression. C, Skeletal -actin expression. D, Osteopontin expression in the ventricles. E, Osteopontin expression in the atria. Expression was normalized to glyceraldehyde-3-phosphate dehydrogenase and the data are reported as means±SE, n=5, *P<0.05 vs age-matched NTG.

Both conventional and quantitative RT-PCR detected changes in MLC isoform expression with age, independent of transgene expression (Figure 5A). Analysis of the atrial MLC isoforms showed higher-than-expected molecular weight bands (MLC-1a, 265 bp; and MLC-2a, 624 bp) in RNA extracted from the ventricles of 9-month NTG and TG mice. The appearance of the higher molecular weight bands was not sensitive to DNAse treatment but was sensitive to RNAse treatment. The absence of any band corresponding to the atrial isoforms in the 3- and 6-month samples was as predicted, and re-expression in the 12-month ventricles yielded the correct size bands based on the primer design. Quantitative analysis clearly shows that expression of the ventricular isoforms is significantly decreased by 12 months in both the NTG and TG ventricles. Conversely, the atrial isoforms not normally expressed in the adult ventricular compartment show significantly increased expression at 9 and 12 months in both NTG and TG mice (Figure 5B). Thus, it appears that re-expression of the atrial MLC isoforms in the ventricle is an age-dependent phenomenon, which may initially occur via gene splicing.

View larger version (53K): [in this window] [in a new window]   Figure 5. Qualitative and quantitative myosin light chain (MCL) isoform gene expression. A, Conventional RT-PCR with primers for the MLC isoforms on RNA extracted from the ventricles. B, Quantitative analysis of MLC isoform expression in ventricular RNA. Expression was normalized to glyceraldehyde-3-phosphate dehydrogenase and the standards used to quantify the isoforms were cloned from the bands in the 12-month samples. The data are reported as means±SE, n=5, #P<0.001 for 9- and 12-month vs 3- and 6-month NTG and TG mice, whereas *P<0.05 is for 12-month vs 9-month NTG and TG mice.

Further analysis of other myofibrillar proteins showed no change in the -Tm/ß-Tm mRNA expression ratio or changes in the appearance of TnT splice variants (TnT₁, TnT₂, TnT₃, and TnT₄), in the TG mice, or with age (data not shown). Analysis of mRNA expression of proteins involved in regulating Ca²⁺ fluxes (SERCA2, phospholamban, ryanodine receptor, and sodium/calcium exchanger) also showed no change in the TG mice or with age (see online Figure II).

Protein Expression
Because both MHC and MLC demonstrated substantial changes in gene expression, we established corresponding changes in protein. Analysis of the MHC protein isoforms showed ß-MHC protein in the 6-month TG hearts, corresponding to the increase in the mRNA (Figure 6A). To determine whether MLC protein isoform switching was occurring, we investigated the presence of MLC-2a in ventricular homogenates using an MLC-2a–specific antibody. The presence of MLC-2a protein was detected in both 12-month NTG and TG ventricular extracts, with slight expression observed at 9 months. The antibody clearly identified the MLC-2a isoform in atrial samples, with an increased abundance in the 12-month TG atria that undergoes calcification (Figure 6B). Probing the same immunoblot with an MLC-2v antibody demonstrates the presence of the MLC-2v isoform in all ventricular homogenates but not the 3-month atria. Interestingly, the presence of the MLC-2v isoform can also be detected in the 12-month TG atria (Figure 6B).

View larger version (62K): [in this window] [in a new window]   Figure 6. Changes in MHC and MLC isoform composition and phosphorylation. A, Expression of /ß MHC isoforms in purified myofibrillar proteins resolved on a 6.5% gel stained with Coomassie blue. B, Representative Western blot of the MLC-2 isoforms expressed in ventricular homogenates from NTG and TG mice separated on a 10% to 20% gradient gel. Top panel, Increased expression of the MLC2a isoform in the ventricles and atria of 12-month mice, probed with an MLC2a-specific antibody. Middle panel, The same blot reprobed with an MLC-2v antibody demonstrating expression in the ventricles but not the 3-month atria. Bottom panel, Top half of the gel stained with Coomassie blue. The first lane (3mNTG) is unloaded as demonstrated by the diminished antibody binding and Coomassie stain. C, Expression of the phosphorylated MLC-2v isoform in purified myofibrillar proteins from NTG and TG ventricles. An apparent loss of the phosphorylated form can be seen in the 12-month NTG and TG samples. D, Prolonged exposure demonstrates that MLC-2v is still phosphorylated but to a much lesser extent. E, Incubation of the same samples with the MLC-2v antibody shows that the decreased MLC-2v phosphorylation is not caused by an absence of the protein. The gels and immunoblots were performed 4 times and the images are representative.

To determine whether a change in MLC-2 phosphorylation was occurring as a result of atrial isoform re-expression, we performed immunoblotting with an antibody specific for phosphorylation at serine 19 on MLC-2. This demonstrated a significant loss of MLC-2 phosphorylation in both the 12-month NTG and TG mice (Figure 6C and 6D). Probing with a pan MLC-2 antibody showed that the loss of MLC-2 phosphorylation was not caused by absence of the protein (Figure 6E).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
PKC signaling has been implicated in a variety of cardiomyopathies by our laboratory and others; however, work to date has not clearly explicated the cellular mechanisms that are responsible for the temporal progression from a compensated to a decompensated phenotype. This is the rationale for the present study, in which a transgenic model expressing constitutively active PKC was analyzed over an extended period of time. This allowed us to report changes in the molecular, biochemical, and functional responses of the heart, which correlate with compensatory and decompensatory cardiac adaptation that occurs against a background of aging. The data presented here strongly implicate the disruption of the myofibrillar proteins and their interactions in the propagation of dilated cardiac disease.

Our data suggest that the initial net effect of PKC overexpression is an increase in the Ca²⁺ sensitivity of the myofilaments, with a tendency toward decreased tension development but no overt effect on cardiac function. The phosphorylation profiles noted in the 3-month TG mice demonstrate that a number of myofibrillar proteins were phosphorylated. It seems likely that the increased Ca²⁺ sensitivity of the myofilaments is caused by increased MLC phosphorylation (at a site distinct from S19), and that the trend toward decreased tension development is caused by cTnI and cTnT phosphorylation.

PKC phosphorylation of MLC has been shown to increase the Ca²⁺-stimulated actomyosin MgATPase activity in skinned fibers.¹³ Moreover, PKC activation with phorbol esters, endothelin-1, and arachidonic acid all increased MLC-2 phosphorylation in isolated cardiac myocytes.¹⁴ In skinned fiber bundles derived from transgenic mice expressing a nonphosphorylatable MLC-2, the force/Ca²⁺ relationship was unchanged after treatment with kinase (MLCK), whereas controls demonstrated a left-shift in the force/pCa²⁺ curve.¹⁵

Although PKC phosphorylation of MLC might contribute to enhanced inotropy, phosphorylation of cTnI by PKC would appear to exert an opposing effect. In vitro, phosphorylation of cTnI by the novel PKC isoforms takes place at serine 43/45 and threonine 144, and it is postulated that negative charges in these positions induce conformational changes, altering interactions among the thin filament proteins.¹⁶ This is supported by studies using reconstituted myofibrils with mutations that mimic the effects of phosphorylation (S43/45E or S43/45D) and result in desensitization to Ca²⁺ and decreased maximum tension development.¹⁷ Studies involving transgenic mice, in which serines 43 and 45 were rendered nonphosphorylatable, show an attenuated effect on the decrease in maximum tension seen in response to -adrenergic and endothelin-1 stimulation, suggesting a role for these sites in vivo.^18,19 Although cTnI phosphorylation has been studied extensively, less is known about cTnT. Transgenic mice that overexpress fast skeletal TnT lack 2 phosphorylation sites (Thr 195, 285) in the amino terminus that are normally found in cardiac TnT. In these mice, the decrease in tension development normally seen in response to PKC activation was substantially blunted.¹¹ Also, recent evidence from reconstituted fiber experiments implicates PKC phosphorylation of cTnT at Thr 206 in decreased myofilament Ca²⁺ sensitivity.²⁰

Prolonged exposure to active PKC for 6 months brings about changes in the composition of thick filament proteins, with an increase in ß-MHC mRNA and protein. There is a decrease in the Ca²⁺ sensitivity of the myofilaments compared with the 3-month TG normal cardiac function under baseline conditions but a blunted inotropic response. The decrease in Ca²⁺ sensitivity correlates with an increase in ß-MHC expression as well as increased cTnI/cTnT phosphorylation. The ß-MHC effect on the force–pCa curve has been demonstrated in myocytes isolated from hypothyroid rats, which express the ß-MHC isoform, and has been suggested in human tissue.²¹

The events at 9 months herald the onset of failure in the TG animals. Most striking is the expression of the atrial MLC isoforms in the ventricles of both NTG and TG animals. This coupled with the expression of ß-MHC in the TG animals changes the relationship between the heavy chains and light chains, a phenomenon described in end-stage human heart failure.²² The ventricular expression of MLC-1a and the ventricular light chains in the atria has been modeled using transgenesis to determine their impact on contractile function.^23,24 Overall, significant changes in cross-bridge kinetics were observed in the absence of overt pathology, suggesting that MLC isoforms are capable of regulating the actomyosin cross-bridge cycling rate and that the different MLC isoforms partially underlie the different contractile properties of the atrium and ventricle. In contrast, transgenic expression of a nonphosphorylatable MLC-2 revealed severe structural and contractile abnormalities, suggesting an important role for MLC phosphorylation in maintaining normal cardiac function.¹⁵ Comparisons between the atrial and ventricular isoforms show there is difference at the N-terminus of the protein, implying that incorporation of the atrial isoforms could alter myofibrillar protein interactions.

Transgenic hearts at 12 months clearly demonstrate the phenotypic changes associated with failure. At this stage, cTnI and cTnT phosphorylation remain elevated whereas MLC phosphorylation is diminished. This change in the balance of myofibrillar protein phosphorylation has been described in the failing human heart and is postulated to underlie the impaired activational properties of the myofibrils associated with failure.²⁵ Also, samples from failing human myocardium demonstrate increased activation of multiple PKC isoforms and phosphorylation of cellular targets such as cTnI.²⁶

Beyond this, the change in the ventricular geometry seen at 12 months might be predicted from this biochemical analysis. Altered myocyte morphology has been noted in cultured neonatal myocytes after infection with an adenovirus expressing constitutively active PKC.²⁷ It is possible that expression of the embryonic isoforms of MHC (ß-MHC) and MLC (atrial MLC) produce a similar composition of myofibrillar proteins, which when phosphorylated by PKC promotes the remodeling process in vitro. Remodeling of the ventricles reflects the need to normalize wall stress in response to an imposed load or loss of myocytes. The mechanisms involved are multiple, but recently it has been shown that cardiac contraction is not only dependent on the gradient of tension because of the orientation of myofibers but also on an associated gradient of MLC-2 phosphorylation.²⁸ This would suggest that phosphorylation of the light chain on a beat-to-beat basis is directly responsible for the gradient of tension development across the wall of the heart. Given this, the replacement of the ventricular with the atrial light chain isoforms could disrupt the gradient of tension development across the wall of the heart and lead to dilation.

The contrast between this model of PKC overexpression and others in which disrupted Ca²⁺ homeostasis is prominent is worth comment. Analysis of the major Ca²⁺ handling proteins mRNA showed no difference between TG and NTG. However, we have seen changes in expression of SERCA and L-type Ca²⁺ channel component (Ca_v1.2 subunit) mRNA coupled to abnormalities in Ca²⁺ current conductance in a transgenic mouse that overexpressed PKCß_II.^12,29 In contrast to the mice described in this article, mice expressing the PKCß_II developed a hypertrophic myopathy with impaired relaxation, suggesting that the triggers to the divergent phenotypic adaptations of hypertrophy and dilation are separable and distinct. This is further supported by proteomic analysis of the signal transduction complexes formed in the PKC overexpression mice. This has revealed associations of PKC with a number of myofibrillar, metabolic, and transcriptional proteins, but not intracellular Ca²⁺ handling proteins.³⁰ However, PKC has been shown to form multiprotein signaling complexes with nonreceptor tyrosine kinases (Lck, PYK2, Src, FAK) in the heart and neonatal cultures, implicating the possible involvement of other kinases in the development of the dilated myopathy in these mice.^31,32

Of final note is the increase in osteopontin gene expression seen in the atria and ventricles of the 12-month TG hearts. Osteopontin is an extracellular matrix protein initially identified in bone but synthesized in many tissues, where it regulates physiological and pathophysiological mineralization. Increased ventricular expression is associated with the transition from decompensated hypertrophy to failure.³³ In our model, it appears to play a similar role. In the TG atria, the 50-fold induction of gene expression was coincident with extensive ectopic calcification, which did impact the total heart weight and presumably atrial mechanics.

We believe the data presented in this study detail a number of molecular, biochemical, and functional events that take place during the progression of heart failure. This model describes the gradual disruption of the myofibrillar proteins and their interactions in response to overexpression of a constitutively active kinase and provides a molecular roadmap for the transition from a compensatory adaptation to a decompensated maladaptation. This is superimposed on the inherent genetic modifications associated with aging. Identification of these transitional moments leading to cardiac maladaptation may help establish stage-specific therapeutic interventions.

	Acknowledgments

 
This work was supported by National Institutes of Health P01 HL62426 (R.J.S. and P.M.B.), R01 HL 64035 (R.J.S.), R01 HL/DK63704 (P.M.B.), and an American Heart Association Scientist Development Grant (P.H.G.).

	Footnotes

 
Original received November 4, 2003; revision received June 25, 2004; accepted June 25, 2004.

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