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(Circulation Research. 2002;91:741.)
© 2002 American Heart Association, Inc.

Integrative Physiology

Ischemic Protection and Myofibrillar Cardiomyopathy

Dose-Dependent Effects of In Vivo PKC Inhibition

Harvey S. Hahn, Martin G. Yussman, Tsuyoshi Toyokawa, Yehia Marreez, Thomas J. Barrett, K. Chad Hilty, Hanna Osinska, Jeffrey Robbins, Gerald W. Dorn, II

From the Department of Internal Medicine, Division of Cardiology (H.S.H., M.G.Y., T.T., Y.M., T.J.B., G.W.D.), University of Cincinnati Medical Center, Cincinnati, Ohio; and the Division of Cardiovascular Molecular Biology (H.O., J.R.), the Children’s Hospital Research Foundation, Cincinnati, Ohio.

Correspondence to G.W. Dorn II, Division of Cardiology, University of Cincinnati Medical Center, 231 Albert B. Sabin Way, Cincinnati, Ohio 45267-0542. E-mail dorngw{at}ucmail.uc.edu

	Abstract

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To delineate the in vivo cardiac functions requiring normal protein kinase C (PKC) activity, we pursued loss-of-function through transgenic expression of a PKC-specific translocation inhibitor protein fragment, V1, in mouse hearts. Initial results using the mouse -myosin heavy chain (MHC) promoter resulted in a lethal heart failure phenotype. Viable V1 mice were therefore obtained using novel attenuated mutant MHC promoters lacking one or the other thyroid response element (TRE-1 and -2). In transgenic mouse hearts, V1 decorated cytoskeletal elements and inhibited ischemia-induced PKC translocation. At high levels, V1 expression was uniformly lethal, with depressed cardiac contractile function, increased expression of fetal cardiac genes, and formation of intracardiomyocyte protein aggregates. Ultrastructural and immunoconfocal analyses of these aggregates revealed focal cytoskeletal disruptions and localized concentrations of desmin and B-crystallin. In individual cardiomyocytes, cytoskeletal abnormalities correlated with impaired contractile function. Whereas desmin and B-crystallin protein were increased 4-fold in V1 hearts, combined overexpression of these proteins at these levels was not sufficient to cause any detectable cardiac pathology. At low levels, V1 expression conferred striking resistance to postischemic dysfunction, with no measurable effects on basal cardiac structure, function, or gene expression. Intermediate expression of V1 conferred modest basal contractile depression with less ischemic protection, associated with abnormal cardiac gene expression, and a histological picture of infrequent cardiomyocyte cytoskeletal deformities. These results validate an approach of PKC inhibition to protect against myocardial ischemia, but indicate that there is a threshold level of PKC activation that is necessary to maintain normal cardiomyocyte cytoskeletal integrity.

Key Words: ischemia/reperfusion • protein kinase C • myofibrillar cardiomyopathy • congestive heart failure

	Introduction

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The and isoforms of protein kinase C (PKC) are implicated in heart failure, myocardial hypertrophy, and ischemic preconditioning, based largely on patterns of PKC isoform translocation in human heart disease and experimental animal models.^1–5 Accordingly, delineating the in vivo consequences of PKC isoform activation in myocardial tissue is a scientific priority. Studying these signaling molecules by modulating their expression can potentially be confounded by promiscuous interactions between overexpressed signaling proteins and their downstream effectors or substrates, or by opportunistic compensation of related signaling proteins in gene ablation models. These concerns are especially relevant to PKC, a group of ubiquitous enzymes composed of at least 11 different, but related, isoforms, many of which are coexpressed in the same tissue or cell type.^6–8 To avoid alterations in enzyme-substrate stoichiometry that are inherent with PKC isoform overexpression or mutational ablation, we have utilized complementary gain- and loss-of function, achieved by modulating PKC isoform interactions with their respective membrane anchor proteins, or RACKs.^9–12 In this manner, in vivo and PKC activation revealed opposing effects on myocardial ischemic protection, but identical cardiotrophic influences,¹² which suggested a novel therapeutic approach for myocardial ischemic salvage by PKC activation and PKC inhibition, alone or in combination. However, growth-modifying effects of chronically activated and PKC suggest caution in manipulating their myocardial activity. Indeed, chronic in vivo inhibition of myocardial PKC has caused a dilated cardiomyopathy,^10,11 and initial attempts to generate a viable mouse model of chronic PKC inhibition were unsuccessful due to early development of a lethal cardiomyopathy.

In the present study, we report the results of chronic myocardial PKC inhibition through transgenic expression of V1, which encodes the putative PKC RACK binding domain.¹² Viable V1-expressing mice were obtained using mutant -myosin heavy chain (MHC) promoters lacking one or the other thyroid response element (TRE). Mice expressing V1 at the lowest levels exhibited marked resistance to myocardial ischemia but were otherwise normal in terms of myocardial structure, function, and gene expression. In contrast, when expressed at higher levels, V1 transgenic mice developed, in a transgene dose-dependent fashion, a cardiomyopathy characterized histologically and ultrastructurally by cytoskeletal disorganization and intracellular proteinaceous aggregates resembling those in human myofibrillar cardiomyopathies.^13,14 In the context of V1 localization to cytoskeletal elements within cardiac myocytes, these results indicate a role for PKC not only in the myocardial response to ischemia, but also in maintaining normal myofibrillar integrity.

	Materials and Methods

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Generation of Transgenic V1 Mice
Analogous to the previously described PKC selective inhibitory protein fragment, V1,^9,15 V1 was modeled after the first variable region of rat PKC (amino acids 2 to 144), which encodes the putative PKC RACK binding domain.¹² For transgenic expression, the cDNA for the V1 peptide, preceded by a FLAG epitope, was inserted into the SalI-HindIII site of the full-length MHC promoter¹⁶ followed by the human growth hormone polyadenylation sequence (MHC-V1). For attenuated V1 expression, a Bgl-II cassette of the MHC promoter, with either the first or second thyroid responsive element individually ablated,¹⁷ was inserted into the analogous portion of the MHC-V1 construct, generating TRE-1 MHC-V1 and TRE-2 MHC-V1 (Figure 1). All transgene constructs were confirmed by double-stranded sequencing, microinjected into male pronuclei of fertilized mouse oocytes (FVB/N), and implanted into pseudopregnant dames by the University of Cincinnati Transgenic Core (directed by John Neumann). Founders were identified by genomic Southern analysis of tail clip DNA. Studies were performed in accordance with protocols approved by the University of Cincinnati Institutional Animal Care and Use Committee, and animals were supplied by the UC Transgenic Mouse Core facility.

View larger version (24K): [in this window] [in a new window]   Figure 1. Schematic depiction of the three V1-MHC promoter constructs. TRE indicates thyroid responsive element.

SDS-PAGE and Immunoblot Analysis
Western blotting was performed as described.⁹ Briefly, myocardial homogenates were fractionated by differential centrifugation into cytosolic (100 000g supernatant) and Triton X-100–extracted particulate (100 000g pellet) fractions, size-separated on 10% SDS-PAGE gels, transferred to polyvinylidene membranes, blocked with 5% dry milk, and incubated with primary antibody and secondary antibodies. Blots were developed with enhanced chemifluorescence and quantitated using a Storm PhosphorImager. To accurately quantitate cytosolic PKC, it was necessary to load 75 µg of protein, compared with 25 µg in the membrane fraction. Antibodies were as follows: anti–B-crystallin (Calbiochem), anti–FLAG-M2 and anti-desmin (Sigma), anti– and PKC, and anti–heat shock protein 60 (Santa Cruz).

Dot-Blot Analysis
mRNA levels were quantified by dot blotting as described.^18,19 Total ventricular RNA was applied (2 µg/dot) to nylon membranes, hybridized with ³²P-labeled antisense oligonucleotides specific for atrial natriuretic factor (ANF), MHC, ßMHC, sarcoplasmic reticular ATPase (SERCA), skeletal actin, or glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and quantified using a Storm PhosphorImager (Molecular Dynamics).

Ischemic Injury and Recovery
Isolated hearts from 12- to 20-week-old mice were Langendorff-perfused for 20 minutes for equilibration, followed by 40 minutes of no-flow ischemia and a 20-minute reperfusion period.¹² Contractile function was measured as left ventricular developed pressure (LVP) and the peak rate of increase of pressure (+dP/dt). Creatine kinase (CK), a marker of myocyte injury, was assayed in the perfusate using a Sigma kit.

Invasive Hemodynamics
Polyethylene catheters were introduced into the femoral artery and vein and a 1.4F Millar catheter placed into the left ventricle through the right carotid artery to monitor real-time heart rate, arterial and left ventricular pressures, and +dP/dt and -dP/dt. Data were archived on a G4 computer (Apple Computers) using MacLab software and interface.¹⁹

Myocyte Mechanics
Isolated adult ventricular myocytes were prepared²⁰ and analyzed according to genotype and presence of intracellular aggregates determined by phase contrast microscopy. Myocyte +dL/dt (peak rate of unloaded shortening), -dL/dt (peak rate of relengthening), and percent shortening were recorded during field stimulation at 0.25 Hz.

Microscopy
Routine histological examination was performed on formalin-fixed Masson’s trichrome-stained sections. Samples were preserved in glutaraldehyde for ultrastructural analysis. Immunoconfocal studies used desmin, B-crystallin, vimentin, and PKC antibodies (the latter a gift from Dr Daria Mochly-Rosen, Stanford University, Stanford, Calif), all at a 1:200 dilution, with Texas red- or fluorescein-conjugated secondary antibodies (1:200). Imaging was performed with a dual laser Nikon PCM2000 system and a Nikon Eclipse E800 microscope using emission wavelengths of 515±30 nm (fluorescein) and 605±32 nm (Texas Red).

Statistical Analysis
Results are presented as mean±SEM. Experimental groups were compared using Student’s t test or 1-way ANOVA, as appropriate. The Bonferroni test was applied to all significant ANOVA results using SigmaStat software. A value of P<0.05 was considered significant.

	Results

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Premature Lethality and Myofibrillar Cardiomyopathy of MHC-V1 Transgenic Mice
Our prior studies of transgenic mice expressing a PKC activating peptide revealed exaggerated postischemic injury.¹² We therefore considered whether inhibition of PKC could be cardioprotective. To test this possibility, transgenic mice were created expressing the PKC inhibitory protein fragment, V1, in the heart. However, the response of these PKC-inhibited hearts to ischemia could not be assessed due to early lethality. Of three original MHC-V1 founders, two died before breeding, whereas the third sired only 9 first generation V1 transgenic mice (out of 245 pups; 3.7%) before he also died. No MHC-V1 first generation mice survived long enough to breed; all were dead by the age of 41 days (mean 27±3 days). Before death, MHC-V1 transgenic mice appeared lethargic, with rapid respirations and tissue edema, suggesting heart failure. Indeed, although only one MHC-V1 mouse survived to an age and size sufficient for invasive hemodynamic evaluation, this mouse exhibited a 64% reduction in peak dobutamine-stimulated +dP/dt compared with its control (NTG=19 877 versus MHC-V1=6720 mm Hg/s), and pathological examination of all 9 MHC-V1 mice revealed pleural effusions, ascites, and enlarged, thick-walled hearts with intramural thrombi (Figure 2A). Compared with NTG siblings, whole-heart weights of MHC-V1 mice were increased by 89% (93±38 mg NTG versus 176±13 mg MHC-V1; P<0.001), and lung weights were increased, indicating pulmonary congestion (158±20 mg NTG versus 215±21 mg MHC-V1; P=0.024). The thick-walled V1 phenotype contrasts with dilated cardiomyopathy in the analogous PKC inhibitor (V1) transgenic mice (Figure 2A) (which is, however, equally lethal^10,11). Immunoconfocal microscopy of V1 using the incorporated FLAG-epitope (Figure 2G) revealed decoration of cytoskeletal elements, similar to anti-PKC (Figure 2F).²¹ This pattern of subcellular localization contrasts with localization of PKC-derived V1 to cross-striated elements.¹⁰

View larger version (90K): [in this window] [in a new window]   Figure 2. V1 expression induces myofibrillar cardiomyopathy. A, Representative 30-day-old NTG, V1, and V1 hearts. B, Histological section (Masson’s trichrome) from a V1 heart demonstrating protein aggregates (*). C, Electron micrograph of V1 heart demonstrating amorphous regions (*) and sarcomere streaming. D, Aggregate labeled with desmin. E, Aggregate stained for B-crystallin. F, Anti-PKC and (G) anti-FLAG-V1 demonstrating an identical cytoskeletal pattern, but not staining within the aggregates (*).

A unique feature of the V1 phenotype, and one that could explain the development of lethal heart failure without ventricular dilation, was frequent intramyocyte blue-staining areas on Masson’s trichrome-stained myocardial sections that did not specifically label for mucin, collagen, glycogen, or mucopolysaccharides (Figure 2B). On ultrastructural examination these areas corresponded to electron-dense regions of amorphous protein within focal subcellular cytoskeletal distortions (Figure 2C). Mitochondria, other cellular organelles, and streaming fragments of sarcomeres were visualized within these areas (see online Figure in the online data supplement available at http://www.circresaha.org).

The distortions of cardiomyocyte subcellular architecture in MHC-V1 hearts closely resemble those seen in human myofibrillar myopathies^13,14,22 and their genetically modified mouse cardiomyopathy analogs.^23,24 In these myopathies, blue-staining intramyocyte protein aggregates contain high levels of desmin and B-crystallin, due to mutations affecting one or the other protein.^13,14 We therefore examined V1-expressing cardiomyocytes for similar immunohistological features. As shown in Figures 2D and 2E, the intracellular aggregates in V1 myocardium stained positively for both desmin and B-crystallin, the latter of which, as in human myofibrillar myopathies,^14,22 was preferentially localized to the periphery (Figure 2E). In contrast, V1 staining was not more prominent within the aggregates (Figure 2G), and there was no qualitative change in desmin or B-crystallin in normal-appearing V1 cardiomyocytes (not shown). Not surprisingly, given the abnormal accumulation of these proteins into intramyocyte aggregates, immunoblot analysis showed that expression of both desmin and B-crystallin was increased approximately 4-fold in MHC-V1 myocardium, compared with nontransgenic (Figure 3A). In contrast, expression of a related chaperone, HSP60, was not altered (Figure 3A). A direct phosphorylation effect of PKC on desmin and B-crystallin was excluded by 2-dimensional immunoblotting, which showed no differences in phosphorylation states between hearts from MHC-V1, nontransgenics, and the recently described PKC activator mice¹² (not shown).

View larger version (71K): [in this window] [in a new window]   Figure 3. Increased expression of desmin and B-crystallin is not sufficient to cause cardiomyopathy. A, Representative Western blots demonstrating increased desmin and B-crystallin in V1 mice. B, Representative Western blots for desmin and B-crystallin in desmin (Des), B-crystallin (B), NTG, and dual transgenic (Des/B) hearts. C, Normal in vivo contractile function at baseline and with peak dobutamine. D, Masson’s trichrome section demonstrating absence of myocyte aggregates.

Given the previously established roles of mutant desmin and B-crystallin in human myofibrillar myopathies, their upregulation and localization within protein aggregates in cardiomyopathic V1 hearts raised the possibility that increased expression of these two proteins in combination might contribute to the cardiomyopathy. To test this notion, we crossbred recently developed mouse models of desmin and B-crystallin overexpression, neither of which are reported to exhibit myocardial pathology.^23,24 Immunoblot analysis demonstrated that the double transgenics maintained the 4-fold levels of desmin and B-crystallin overexpression of the parent lines (Figure 3B). Confirming the previous characterization of these mice, single transgenic animals were normal in terms of myocardial pathology and cardiac contractile function (Figure 3C and data not shown). The desmin/B-crystallin double transgenic mice also were normal in all respects, including heart weight, ventricular contractility at baseline and in response to dobutamine (Figure 3C and data not shown), and myocardial histological appearance (Figure 3D). From this, we conclude that simple upregulation of myocardial desmin and B-crystallin is not sufficient to cause the V1 myofibrillar cardiomyopathy phenotype, and infer that PKC activation and targeting to the cardiomyocyte cytoskeleton is necessary for normal cardiomyocyte structure and contractile function.

To confirm that the observed cardiomyocyte cytoskeletal abnormalities impaired MHC-V1 myocardial performance, contractile function of isolated ventricular myocytes was assessed as a function of the presence or absence of cytoskeletal distortions. Using phase contrast optics, cardiomyocytes with normal cellular architecture were easily distinguished from those with intracellular aggregates. Accordingly, compared with normal-appearing MHC-V1 cells, the unloaded fractional shortening of abnormal MHC-V1 cardiomyocytes was depressed by 63%, and the peak rate of unloaded shortening was decreased by 59% (Table 1). Normal-appearing MHC-V1 myocytes had unloaded fractional shortening similar to NTG (Table 1).

View this table: [in this window] [in a new window]   Table 1. Isolated Myocyte Mechanics

Phenotypic V1 Dose-Response With Attenuated MHC Promoters and Cardiac Protection With PKC Inhibition
The unexpected phenotype of myofibrillar cardiomyopathy and lethality in the MHC-V1 mice precluded an analysis of the effects of PKC inhibition on the cardiac response to ischemia/reperfusion injury. To obtain viable animals suitable for testing our original hypothesis, two approaches were undertaken. First, the original MHC-V1 transgene construct was reinjected at lower concentrations of DNA (2 versus 6 ng/µL) to obtain additional founders with fewer integrated copies of the transgene. Second, the MHC promoter was modified in order to attenuate its expression while retaining its characteristic cardiomyocyte-specificity. Toward this end, we mutated the V1 construct to individually ablate the two thyroid responsive elements (TRE), which have previously been reported to be necessary for full activity of this promoter.^16,17 Herein, the mutant promoters are designated TRE-1 when the first thyroid response element is ablated, and TRE-2 when the second site is ablated (see Figure 1).

Multiple founders were identified for each transgene construct, MHC-V1, TRE-1, and TRE-2. Because results were similar between lines for each promoter, the data have been combined. Expression of V1, measured by immunoblot blot analysis of the incorporated amino terminal FLAG epitope, corresponded with the anticipated activities of the promoters; TRE-1 mice expressed 72% and TRE-2 mice 40% of the level of the reinjected, lower copy number MHC-V1 mouse (Figure 4A). At levels of V1 expression achieved in the MHC-V1 and TRE-1 mice, heart weight increased in proportion with V1 expression, whereas TRE-2 hearts were normal in size (Table 2). Likewise, the increase in fetal cardiac genes that characterizes cardiomyopathic syndromes²⁵ also showed a relationship to V1 expression level (Figure 4A). Compared with NTG (and TRE-2) mice, MHC-V1 mouse ventricles had a 3-fold increase in ßMHC and a 15-fold increase in ANF mRNA expression, whereas TRE-1 mice similarly demonstrated a 3-fold increase in ßMHC, but an 8-fold increase in ANF mRNA. In addition, MHC-V1 mice had a 50% reduction in SERCA expression, compared with a 30% reduction for TRE-1; again, TRE-2 mice were similar to NTG. TRE-1 and TRE-2 mice exhibited no increase in mortality up to 12 months of age. However, like those of the original V1 line, first generation mice from the reinjected MHC-V1 founder died prematurely from apparent heart failure, with a mean survival time of 18±2 weeks (n=44).

View larger version (25K): [in this window] [in a new window]   Figure 4. Variable V1 expression and phenotype achieved with mutant TRE MHC promoters. A, Top, Western blot analysis of FLAG-V1 expression. Bottom, mRNA expression. B, Selective inhibition of ischemia/reperfusion-induced PKC translocation in V1 expressing hearts. Top, PKC partitioning in NTG at baseline, and NTG, TRE-1, and TRE-2 hearts after ischemia/reperfusion. Bottom, Parallel studies of PKC.

View this table: [in this window] [in a new window]   Table 2. Morphometric Analysis of V1 Mice

The effects of different levels of V1 expression on myocardial PKC activation, assessed as translocation to subcellular membrane particulates, were assessed in isolated perfused mouse hearts. As shown in Figure 4B, a 40-minute period of global ischemia followed by 20 minutes of reperfusion stimulated a marked redistribution of PKC from the cytosolic to membrane subcellular fraction (compare control NTG to ischemia/reperfused NTG). In contrast, translocation was significantly inhibited in TRE-2 hearts (76±2.3% of NTG), and TRE-1 hearts (42±4.4% of NTG; P0.05 versus NTG and TRE-2). No effect of V1 expression was seen on subcellular partitioning of related PKC (Figure 4B). Thus, variable myocardial expression of V1 has specific, dose-related inhibitory effects on ischemia/reperfusion-stimulated PKC translocation.

Because a hallmark feature of the original V1 cardiomyopathy was the presence of intracellular desmin/crystallin-containing aggregates, we performed histological examination to establish that the phenotypic continuum achieved with variable V1 expression extended to these characteristic structural abnormalities. Although (as hoped) none of the new V1 transgenic lines developed a phenotype as severe as the original, the proportion of cardiomyocytes (percent of total) with aggregates that stained blue with Masson’s Trichrome correlated with V1 expression (7.7±0.6% MHC-V1, 1.1±0.4% TRE-1, 0% TRE-2) (Figure 5). Furthermore, cardiac functional abnormalities paralleled the alterations in cardiomyocyte/myocardial structure. Invasive hemodynamic analysis revealed that both the MHC-V1 and TRE-1 mice had significantly depressed basal and stimulated +dP/dt. At the maximal dobutamine dose, MHC-V1 and TRE-1 mice exhibited 43% and 28% reductions in +dP/dt, respectively (12265±740 mm Hg/s MHC-V1, 15418±69 TRE-1, and 21217±720 NTG; P0.05 versus NTG). As with histological appearance, TRE-2 mice were functionally normal (peak +dP/dt 18802±2102 mm Hg/s; P=NS).

View larger version (171K): [in this window] [in a new window]   Figure 5. Pathological aggregate formation parallels V1 transgene expression. Top left, MHC-V1 (Masson’s trichrome). Top right, TRE-1. Bottom left, TRE-2. Bottom right, NTG. Arrows denote representative aggregates.

The varying "dose" of V1 expression afforded by the mutant promoters permitted a correlative analysis of biochemical PKC inhibition and functional response to ischemia/reperfusion. TRE-2 mouse hearts, with only modest PKC inhibition and no detectable abnormalities in structure, function, or gene expression were nevertheless markedly resistant to global ischemia, with rapid recovery of contractile function (Figures 6A and 6B) and strikingly less myocyte damage (assessed as CK release) compared with NTG (Figure 6C). TRE-1 mouse hearts, with significantly decreased PKC translocation inhibition (see Figure 4B), appeared at first glance to have increased ischemic injury (Figure 6A). However, the basal function of TRE-1 hearts was depressed due to the modest myofibrillar cardiomyopathy (Figures 5 and 6A), and when the postischemic data are corrected for the baseline functional abnormality, there is no difference in post-ischemic cardiac functional recovery compared with NTG controls (Figure 6B). However, as with TRE-2, a striking cytoprotective effect was evident (Figure 6C). It was not possible to assess the effects of ischemia/reperfusion on the MHC-V1 mice, due to the severity of the cardiomyopathy.

View larger version (13K): [in this window] [in a new window]   Figure 6. Cardiac protection from ischemia/reperfusion injury. A, Enhanced recovery of left ventricular dP/dt after 40 minutes of ischemia in TRE-2 mice. B, Postischemic function normalized to baseline values (error bars omitted for clarity). C, Attenuated postischemic CK release from TRE-1 and TRE-2 hearts. n=6 to 10.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
These studies establish dose-dependent effects of a PKC translocation inhibitor in the in vivo mouse heart. Lower levels of V1 expression, which inhibited ischemia-stimulated PKC translocation by 24%, protected hearts from ischemia/reperfusion injury. Importantly, these benefits occurred in the absence of any detectable structural or functional alterations, including highly sensitive assays of myocardial gene expression. With progressively more PKC inhibition however, cardiomyopathies developed exhibiting pathological and histological characteristics of myofibrillar myopathies. Cytoskeletal abnormalities in these V1 cardiomyopathies, together with targeting of V1 (and hence activated PKC) to the cardiomyocyte cytoskeleton, identify a critical function for PKC in maintaining myocyte cytoskeletal integrity.

An unexpected finding of these studies was the degree of cardiomyocyte protection (as assessed by CK release) afforded by V1 expression in both the TRE-1 and TRE-2 lines (Figure 6C). We considered that the presence of protein aggregates and depressed basal contractile function of TRE-1 mice would compromise functional recovery after ischemia/reperfusion. Indeed, absolute recovery of contractile function was severely depressed in TRE-1 mice, although when normalized to basal function, TRE-1 mice had exactly the same degree of functional recovery as NTG controls (55.5±8.6% NTG versus 55.5±15.3% TRE-1). Nevertheless, a dramatic cytoprotective effect was observed, exceeding even that of the functionally superior TRE-2 mice. A possible explanation relates to increased expression of B-crystallin in the TRE-1 mice, which was not seen in TRE-2. B-crystallin, aka, heat shock protein 22, is a chaperone for the intermediate filament desmin and is known to exert a cardioprotective effect in ischemic preconditioning.²⁶ Thus, V1-mediated upregulation of B-crystallin could contribute to cytoprotection in these hearts. It is therefore interesting that activation of PKC, which also protects from postischemic cardiac dysfunction, is rather less cytoprotective than PKC inhibition, and is not associated with regulated expression of B-crystallin or desmin⁹ (unpublished results). Another difference between cardiac protection afforded by transgenically expressed RACK versus V1 was delayed development of mild, albeit normally functioning ventricular hypertrophy, with induction of ßMHC gene expression in RACK mice.^9,10 Absence of any such physical or molecular modifications by the chronically expressed PKC inhibitor (at low expression levels) may be advantageous in terms of a potential therapeutic approach.

When V1 was expressed at incrementally higher levels in the heart, a cardiomyopathy developed with severity in proportion to V1 expression level. The most surprising aspect of higher-level V1 expression was that this cardiomyopathy had the histopathological and functional characteristics of specific human genetic disorders, the myofibrillar myopathies. These skeletal and cardiac myopathies are a heterogeneous group of diseases that share a common histopathological picture of intramyocyte proteinaceous aggregates with sarcomere disorganization.^13,14,22 The best characterized of this group are the desmin-related myopathies caused by mutant forms of desmin or B-crystallin. Missense mutations in the carboxy-terminal part of the desmin rod domain cause myopathies by interfering with desmin assembly.¹³ The B-crystallin R120G mutation leads to myopathy by impairing its ability to act as a molecular chaperone for desmin, and interfering with proper desmin integration into intermediate filaments.^14,22 Thus, pathological mutations of either of these proteins result in abnormal accumulation of protein into characteristic intracellular aggregates, accompanied by cytoskeletal and sarcomere disruption. Interestingly, a similar phenotype has been observed in the desmin knockout mouse model,^27,28 which implies that the pathology is associated with loss of desmin function, not increased desmin expression as observed in the V1 mouse model and other hypertrophy conditions.²⁹ Indeed, our present studies demonstrate that neither overexpression of normal desmin alone, nor its overexpression in combination with B-crystallin, causes any measurable functional or structural myocardial abnormalities. Rather, the observation that activated PKC in cardiac myocyte translocates to the cytoskeleton establishes a mechanistic link between PKC function and myofibrils. Studies in cultured rat neonatal cardiomyocytes and in V1 mice (data not shown) demonstrate that activated PKC colocalizes with cytoskeletal vimentin,^30,31 and that vimentin and PKC coimmunoprecipitate.³⁰ Thus, PKC substrates exist at the cytoskeleton, and our results indicate that attenuated PKC activation causes loss of cytoskeletal organization. The identities of critical cytoskeletal PKC substrates, and of the cytoskeletal PKC binding protein, ie, PKC RACK, are currently unknown.

The V1 transgenic approach interrogates PKC function in the in vivo mouse heart through targeted expression of a protein fragment designed to competitively inhibit PKC binding with its membrane anchor protein, or RACK.^12,32 We have previously used the approach of translocation inhibition to characterize in vivo functions of myocardial PKC.^9–11 In those studies, PKC inhibition by transgenic expression of its first variable region, V1, had little effect at lower expression levels, but at the highest expression level caused a lethal dilated cardiomyopathy.¹⁰ In both the current V1 model, and the previous V1 model, expression of PKC V1 peptides at lower levels was well tolerated, but higher expression levels resulted in development of lethal cardiomyopathies. However, the cardiomyopathies themselves are strikingly different. Whereas V1 expressors developed profound ventricular wall thinning and chamber dilation, V1 expressors had thick ventricular walls and normal chamber dimensions, associated with the aforementioned histological and ultrastructural abnormalities. Likewise, the antithetic experiments, ie, facilitated translocation of PKC and PKC through transgenic expression of their respective RACK peptides, resulted in similar hypertrophic phenotypes, but opposite effects on myocardial recovery from ischemia.^10,12 Taken together, these observations indicate that PKC and PKC have distinct roles in myocardial cytoskeletal maintenance and the response to ischemic injury, but an overlapping function in myocardial growth. This latter functional redundancy may explain why mutational ablation of the PKC gene has no overt cardiac phenotype,³³ because PKC may opportunistically compensate for the absent PKC.

In the present studies, a great deal of effort went into generating mouse models with varying levels of transgene expression. The need was to generate V1-expressing animals that survived long enough to study, and to assess the dose-dependent effects of the transgene while retaining the favorable characteristics of MHC promoter, ie, robust, cardiomyocyte-specific ventricular transgene expression that largely begins after birth. Thus, we chose to mutationally attenuate the MHC promoter. Attenuated versions of the MHC promoter were obtained by individually mutating the thyroid response elements, as originally conceived by Rindt and Robbins,^16,17 and the resulting TRE-1 and TRE-2 V1 phenotypes represent form frustes of the original MHC-V1 phenotype, permitting a detailed analysis. Importantly, multiple independent lines of TRE-2 mice did not develop a cardiomyopathy when followed up to one year, indicating that a threshold expression level exists for pathological effects of PKC inhibition. We have observed similar threshold effects when overexpressing either intact signaling molecules, such as Gq,¹⁹ or catalytically inactive peptide fragments, such as the PKC inhibitor, V1.¹⁰ Gq overexpression at twice endogenous levels had no effect on cardiac structure, function, or gene expression, whereas 4- and 5-fold Gq overexpressors developed the characteristic hypertrophy, contractile dysfunction, and abnormalities in gene expression.¹⁹ Likewise, in the lowest expressing line (8 copies) cardiac-specific expression of the PKC inhibitory peptide, V1, had no detectable basal phenotype (although a phenotype was revealed by superimposed expression with Gq¹¹), whereas the highest expressing line (>200 copies) died prematurely of dilated cardiomyopathy, and an intermediate expressing line (40 copies) exhibited modest but significant alterations in contractile function and increased fetal cardiac gene expression.¹⁰ In the context of the present and previous studies where transgenesis has been utilized as a drug delivery system for catalytically inactive peptides (Gq-mini gene,³⁴ ßARK-ct³⁵), delineation of dose-dependent effects should be an essential component of the phenotypic characterization, and the TRE mutant MHC promoter constructs have demonstrated utility for this purpose.

In summary, we have delineated essential roles for PKC in the in vivo myocardial response to ischemic injury and for normal cardiomyocyte cytoskeletal integrity. A critical function for PKC in maintenance of normal cardiomyocyte cytoskeletal structure was indicated not only by the histopathological/ultrastructural findings and associated contractile dysfunction in mice expressing V1, but also by decoration of intermediate filaments by V1, thus identifying the cardiomyocyte cytoskeleton as a subcellular target for activated PKC. Despite the deleterious effects of high-level V1 expression, the potential therapeutic utility of PKC inhibition for postischemic myocardial protection is supported by identification of a therapeutic window, where low-level V1 expression is protective, without adverse consequences.

	Acknowledgments

 
This work is supported by P50 HL52318 and R01 HL58010. We wish to thank Gary Lin for his technical contributions to this project.

Received July 1, 2002; revision received August 28, 2002; accepted September 3, 2002.

	References

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