Cardiomyopathy in Transgenic myf5 Mice
Abstract To explore the compatibility of skeletal and cardiac programs of gene expression, transgenic mice that express a skeletal muscle myogenic regulator, bmyf5, in the heart were analyzed. These mice develop a severe cardiomyopathy and exhibit a significantly shorter life span than do their nontransgenic littermates. The transgene was expressed from day 7.5 post coitum forward, resulting in activation of skeletal muscle genes not normally seen in the myocardium. Cardiac pathology was not apparent at midgestation but was evident by day 2 of postnatal life, and by 42 days, hearts exhibited multifocal interstitial inflammation, fibrosis, cellular hypertrophy, and occasional myocyte degeneration. All four chambers of the heart were enlarged to varying degrees, with the atria demonstrating the most significant hypertrophy (>100% in 42-day-old mice). The transgene and several skeletal muscle–specific genes were expressed only in patchy areas of the heart in heterozygous mice. However, molecular markers of hypertrophy (such as α-skeletal actin and atrial myosin light chain-1) were expressed with a wider distribution, suggesting that their induction was secondary to the expression of the transgene. In older (28-week-old) mice, lung weights were also significantly increased, consistent with congestive heart failure. The life span of bmyf5 mice was significantly shortened, with an average life span of 109 days, compared with at least a twofold longer life expectancy for nontransgenic littermates. Expression of the transgene was associated with an increase in Ca2+-stimulated myofibrillar ATPase in myofibrils obtained from the left ventricles of 42-day-old bmyf5 mice. Myocardial bmyf5 expression therefore induces a program of skeletal muscle gene expression that results in progressive cardiomyopathy that may be due to incompatibility of heart and skeletal muscle structural proteins.
The MDFs, MyoD, myf5, myogenin, and MRF4/herculin/myf6, direct determination and differentiation of skeletal muscle. They are characterized by a highly homologous 70–amino acid segment that contains a bHLH region. This region also shares sequence homology with a number of other transcriptional activators that are involved with cell-fate specification, such as daughterless and twist.1 MDFs are thought to be responsible for the determination and differentiation of myogenic progenitor cells destined to become skeletal muscle cells. The MDF proteins function as transcriptional activators of skeletal muscle–specific genes through distinct cis-acting elements.2 Additionally, they act in collaboration with other transcription factors, such as MEF2.3 MDFs are expressed at high levels during skeletal muscle development and continue to be expressed at low levels in adult skeletal myofibers. Although their role in adult skeletal muscle is unknown, they appear to be influenced by variations in neuronal activity and hormonal levels.4 5 6 The roles for the different MDFs have been examined by determining the temporal and spatial patterns of expression7 and the effects of targeted disruption of each of these genes.8 In all cases of inactivation of one or more MDFs, mice were born with beating hearts, clearly indicating that targeted disruption of the MDFs had no apparent effect on cardiac formation or development.
The myocardium originates in a region of rostral mesoderm called the cardiac crescent. The primitive heart appears as a simple tube between 7.5 and 8 days PC.9 10 Although a number of transcription factors have been isolated that are expressed in the heart,11 12 none of these has been shown to be cardiac specific. In Drosophila, tinman and cripto are expressed before heart formation and lie in a critical pathway for heart development.13 14 One potential cardiac determination factor has been reported by Litvin et al,15 with homology to the second helix of MyoD. An antibody directed against this helix supershifts a complex consisting of chick heart extract and the E-box of the muscle creatine kinase gene. This implicates a bHLH protein in myocardial gene expression. This finding might be expected, since several cardiac-specific genes contain the E-box motif within their promoters.16 17
The ability of MDFs to activate a skeletal program of gene expression has been demonstrated in a variety of cell types.18 19 In most cases, expression results in conversion of the host cell to that of a myogenic cell. Ectopic expression of MyoD in the hearts of transgenic mice activated some muscle-specific skeletal sarcomeric genes and resulted in embryonic lethality with severe cardiac abnormalities in fetuses heterozygous for the transgene.20 Cardiac and skeletal muscle share some common structural features, and during development, many isoforms of sarcomeric proteins are coexpressed in both types of muscle, such as α-skeletal actin and β-MHC. However, many of the sarcomeric components of skeletal and cardiac muscle are distinct and show restricted patterns of expression. In an attempt to further our understanding of the compatibility of cardiac and skeletal muscle genetic programs, we have studied the effects of bmyf5 expression in the murine myocardium. Heterozygous mice survive to adulthood but have severe pathology and develop heart failure. The results demonstrate that ectopic expression of bmyf5 activates a skeletal muscle gene program that is incompatible with normal cardiac function.
Materials and Methods
Transgenic animals were generously provided by R. Santerre and C. Smith (Eli Lilly Laboratories) and have been previously described.21 Briefly, the coding region and 3′ untranslated regions of the bovine myf5 cDNA were cloned into the unique EcoRI site of pEMSVscribe, which contains the Moloney LTR.22 The line used in the present study (line 25-37) contained more than 30 copies of the transgene. All animals studied were heterozygous and were bred against an FVB/NJ background. Animals were maintained in the animal care facility in accordance with the Guide for Care and Use of Laboratory Animals (Department of Health and Human Services Publication NIH 86-23). To determine presence of the transgene, a portion of the tail was removed from young animals. From this sample, genomic DNA was prepared, and the presence of the transgene was determined as described by Santerre et al.21
Histology and Immunocytochemistry
Hearts from 15.5-day embryos and 2- and 42-day-old mice were removed and placed in 4% formaldehyde/PBS overnight at 4°C. They were infiltrated with paraffin and sectioned on a microtome (MicroM). Sections were stained using hematoxylin and eosin or Masson’s trichrome. For immunocytochemical analysis, sections were fixed in 3.7% formaldehyde/PBS for 5 minutes at room temperature before being permeabilized with 0.5% Triton X-100/PBS for 5 minutes. Nonspecific binding was blocked by incubation with 5% normal goat serum overnight at 4°C. The primary antibody was then added, and the sections were incubated overnight at 4°C. The tissues were washed three times in 0.05% Tween 20/PBS for 10 minutes. A secondary antibody conjugated to horseradish peroxidase was added, and the sections were incubated for 60 minutes at 37°C, followed by washing as described above, before being developed with diaminobenzidine and counterstained with hematoxylin.
For Northern analysis, RNA was prepared from heart or skeletal muscle by the method of Chomczynski and Sacchi.23 Total RNA (10 μg) was separated on a 1% agarose/formaldehyde gel. The quality and relative amounts of RNA were determined from ethidium bromide staining. RNA was transferred to a nylon membrane (BioTrans, ICN Biochemicals) by capillary action. Probes used were isolated from unique cDNA coding regions of embyronic MHC,24 α-skeletal actin,25 ANF,26 α-tubulin,27 and cardiac LC128 and were labeled by random priming with [32P]dCTP. Single-stranded oligonucleotides were also used for α-MHC (5′-GAGGGTCTGCTGGAGAGG-3′) and β-MHC (5′-TGTTGCAAAGGCTCCAGGTCTGAGGGCTTC-3′).29 Blots were hybridized in 6× SSC, 1% SDS, and 100 μg/mL sonicated salmon DNA. Blots probed with cDNA were prehybridized and hybridized at 65°C; blots probed with oligonucleotides were hybridized at 55°C. Washes (2× SSC/2% SDS, 1× SSC/2% SDS, and 0.5× SSC/2% SDS) were performed at 65°C or the calculated hybridization temperature. Quantification of hybridized bands was carried out using a phosphorimager (Molecular Dynamics).
The onset of transgene expression was determined by RT-PCR. Male mice positive for the transgene were used for breeding with control female mice. The presence of a vaginal plug was designated 0.5 day PC. Pregnant females were killed at select times after conception, and the embryos were removed. The embryos were divided and used for preparation of genomic DNA and total RNA, except for the 4.5-day and 7.5-day PC embryos. Because of limiting amounts of tissue, simultaneous preparation of DNA and RNA was achieved by use of TRI reagent (Iowa Biotech Inc). To determine the presence of the transgene, PCR analysis, as indicated above, was performed on genomic DNA. For determination of expression of the transgene, an RT/thermocycling (RT-PCR) reaction was performed. An antisense primer common to the first exon of the bovine and murine myf5 genes was incubated with 1 μg total RNA and AMV-RT (Promega) for 60 minutes at 42°C. After this, species-specific primers unique to the 5′ untranslated regions were used in a thermocycling reaction to amplify the single-strand cDNA. A portion of each reaction was run on a 4% 1× TAE (mmol/L: Tris-Cl 10 [pH 7.4], sodium acetate 10, and EDTA 1) agarose gel and visualized by ethidium bromide staining.
In Situ Hybridization
Embryos were fixed in 4% PBS, dehydrated, and infiltrated with paraffin. The paraffin blocks were cut to give sections of 5 to 7 μm. The hybridization and posthybridization procedures are detailed in Lyons et al.30 The myogenin and MyoD1 probes were used as described by Sassoon et al.31 The murine myf5 probe was used as described by Ott et al.32 The embryonic MHC probe was used as described by Lyons et al.30 The α-skeletal actin probe was used as described by Sassoon et al.25 The ALC1 probe was used as described by Lyons et al.28 The MEF2C probe was used as described by Edmondson et al.33 The MEF2B probe was used as described by Lyons et al.34
Cardiac Contractile Proteins
Cardiac myofibrils were purified using established methods.35 Ca2+-activated ATPase activity of cardiac myofibrils was measured at 30°C in the presence of (mmol/L) Tris-Cl 50 (pH 7.6), CaCl2 10, KCl 300, and ATP 5 using 0.1 to 0.3 mg of protein.36 Myofibrillar ATPase activities were measured in the presence of 3 mmol/L Mg2+ and either with 2.5 mmol/L EDTA or across a range of free Ca2+ concentrations. ATPase data are expressed as micromoles Pi per minute per milligram protein. Myosin isoenzymes were analyzed by nondenaturing electrophoresis as described by D’Albis et al,37 and the relative amounts were estimated by integration of densitometric scans.
Hearts were perfused with a PBS solution before being infused with fixation solution (4% paraformaldehyde, 2.5% glutaraldehyde, and 100 mmol/L calcium cacodylate). They were then fixed in Trump’s EM fixative for 5 days and postfixed in 2% osmium for 1 hour. Hearts were then embedded in acetonitrile/araldite/epoxy resin overnight, flat-embedded in araldite/epoxy resin, and allowed to polymerize for 2 days. Thin sections were stained with uranyl acetate and then by lead citrate. Thin sections were examined using a Siemens No. 102 microscope.
Developmental Profile of Transgene Expression
Transgenic mice were produced to express the bovine myf5 gene under the control of the MSV LTR in their brains and hearts. Mice heterozygous for the transgene exhibited abnormal morphology in both tissues.21 In the brain, this abnormality took the form of striated myofibers, and in the heart, the manifestation was an increase in size. To further characterize these mice and to define their cardiovascular pathology, hearts were analyzed histologically and characterized for their expression of the transgene and for activation of skeletal muscle genes. To determine when, during development, the transgene was expressed, RT-PCR was performed. Embryos were collected and used to determine the presence of the transgene and its expression pattern. In order to understand the pathogenesis, it was important to determine the onset of transgene expression to the developmental events of cardiac morphogenesis. It was observed that the transgene was not expressed in 4.5-day PC embryos but was expressed in 7.5-day PC embryos and thereafter (data not shown). These data indicate that the transgene was expressed before the formation of the heart, although since whole embryos were assayed, it cannot be determined whether this expression occurred in tissues that were destined to become the myocardium.
Life Span of Transgenic Animals
All of the animals studied were heterozygous. Although the transgenic females were fertile, they exhibited a decreased incidence of conception, and most pregnant females went into apparent heart failure, as evidenced by labored breathing and cardiomyopathy. Additionally, transgenic animals were frequently found dead during routine visits. Autopsies were not routinely done, although in animals examined, heart enlargement was in evidence. The survival rates of the bmyf5 transgenic mice were compared with a group of randomly chosen littermate control mice (Fig 1⇓). For the bmyf5 transgenic mice, the mean life age at the time of death was 109±19 days, with no significant gender differences. At the end point of the study (220 days), none of the wild-type littermates had died. Not included were animals that were euthanized because they appeared to be suffering from labored breathing, presumably due to heart failure. Therefore, this estimate of life expectancy may be an underestimate.
Cardiac Hypertrophy in bmyf5 Mice
Heart chamber weights were determined in animals at 6 to 7 weeks and 6 months of age. In animals that were 6 to 7 weeks of age, body weights were not significantly different between the groups (control, 21.4±0.6 g; bmyf5, 21.3±0.9 g), but all four chambers exhibited increases in mass. Both atria were increased >100%, whereas both ventricles were increased >10% (Table⇓). Tissue weights were also obtained from those surviving to 6 months. Greater increases in the ventricles and left atria were observed in the 6-month-old animals compared with the 6-week-old animals (Table⇓). Additionally, in the 6-month-old mice, there was a significant increase in the ratio of lung to body weight, suggesting the presence of congestive heart failure. These data may represent an underestimate of the extent of disease, since not all animals maintained in the colony survived for 6 months. However, the data clearly demonstrate that cardiac enlargement exists in the transgenic animals and that it appears to progress with age.
Histopathology in bmyf5 Transgenic Hearts
A previous report21 suggested fibrosis and some myocyte degeneration in bmyf5 mice. We extended these studies to include a time course as well as electron microscopic analysis. Since bmyf5 expression produced hypertrophy of all four cardiac chambers, evidence of cellular pathology seemed likely. Hearts from 15.5-day embryos demonstrated no apparent pathology (data not shown). However, in both 2-day-old and 6-week-old bmyf5 animals, there were clear indications of pathology (Fig 2⇓). In the 2-day-old animals, myocyte degeneration was observed as well as an increased number of interstitial cells. Multifocal interstitial cellular infiltration and edema were also observed. The presence of these cells in immunologically naive neonates suggests that they do not represent a cellular immune response. In the 6-week-old animals, multifocal interstitial inflammation and fibrosis were observed along with myocyte hypertrophy. Additionally, myocyte degeneration and necrosis were also observed. The left ventricular free wall and base, papillary muscle, and left ventricular subendocardial wall and midwall all exhibited these pathological features. In an attempt to identify the interstitial cells, sections were stained with an anti-desmin antibody, a muscle-specific marker. Whereas the ventricular myocytes showed a characteristic striated pattern, the interstitial cells were desmin negative, suggesting that there were not any cells of myogenic lineage (Fig 3C⇓). There was no obvious evidence of multinucleated skeletal muscle cells in the hearts of these animals, suggesting that expression of bmyf5 did not convert cells in the myocardium to morphologically distinct skeletal muscle cells.
Staining with Masson’s trichrome defines type 1 collagen associated with fibrosis present within the heart. Within control hearts, some collagen (which stains blue) is present (Fig 3A⇑). In the hearts from bmyf5 mice, there were varying degrees of fibrosis present, with some areas displaying remarkable fibrosis interspersed among areas of relatively normal morphology (Fig 3B⇑). Although the degree of fibrosis varied among animals and across a single section, there was clear evidence of significant damage within the bmyf5 hearts.
To examine the impact of this pathology on the ultrastructure of the myocyte and the sarcomere, electron microscopy was carried out. Shown in Fig 4⇓ are representative electron micrographs from the heart of a 6-week-old bmyf5 animal. The photographs indicate that within the same heart, it was evident that adjacent fibers may display differing degrees of organization. There were many regions in which active growth was evident, as indicated by the presence of large numbers of ribosomes and newly formed sarcomeres (Fig 4A⇓). Other cells in the process of forming sarcomeres had disorganized Z lines (Fig 4B⇓). Many regions contained well-organized sarcomeres, and there were no indications of a significant change in their morphology. Also observed were many fibrotic cells and large deposits of collagen (data not shown). These data suggest that the myocardium from 6-week-old bmyf5 mice appeared to be compensating for the extracellular damage by actively forming new sarcomeres, consistent with cellular hypertrophy.
Induction of Skeletal Muscle Gene Expression
It is clear from the above analysis that expression of the bmyf5 transgene is disruptive to the myocardium. The simplest hypothesis is that expression of bmyf5 in the heart has activated a skeletal muscle program of gene expression that is incompatible in some way with that normally found in the heart. Given the considerable homology between many of the structural proteins in cardiac and skeletal muscle, it might be expected that the programs would be compatible. To determine whether transgene expression induced the downstream expression of skeletal muscle genes, analysis of left ventricular RNA was performed by RT-PCR and Northern analysis. Several skeletal muscle genes, including myogenin, herculin, and embryonic MHC, were induced in 6-week-old hearts, as assayed by RT-PCR (data not shown). Northern analysis indicated that ventricular myosin LC1, α-MHC, and α-tubulin were not significantly altered by expression of the bmyf5 transgene (Fig 5⇓). Embryonic MHC was undetected by Northern analysis. Given the observed hypertrophy in 6-week-old animals, RNA was also analyzed for the induction of a number of markers classically associated with compensatory hypertrophy, including β-MHC, α-skeletal actin, ANF, and ALC1a. Expression of these genes was increased in the left ventricles of bmyf5 mice compared with the left ventricles of littermate control mice (Fig 5⇓). These data clearly show that there is an increase in the expression of gene products associated with compensated hypertrophy.
Localization of Skeletal Muscle Transcripts
The dispersed pathology across the hearts of transgenic animals suggested that bmyf5 expression may not be uniform. To determine the distribution of bmyf5 and other skeletal muscle transcripts, sections were cut from the hearts of 2-day-old animals and hybridized to riboprobes for several different skeletal muscle gene products (Fig 6⇓). Two distinct patterns of expression are evident from the sections shown: patchy expression and relatively uniform expression. Expression of bmyf5 was not evenly distributed throughout the heart. This patchy distribution of expression was also observed for the myogenic regulator myogenin (Fig 6B⇓) and for the muscle transcription factors MEF2B (Fig 6C⇓) and MEF2C (data not shown) as well as embryonic skeletal MHC (Fig 6D⇓). Counterstaining the slides with eosin visualized cell boundaries. It was determined that the patchy expression patterns of the MDFs and embryonic MHC were associated with cardiomyocytes and not the fibrotic or desmin-negative regions (data not shown). A second group of genes showed a very different, broader distribution pattern including several structural muscle gene products, such as α-skeletal actin (Fig 6F⇓) and ALC1a (Fig 6E⇓). Additionally, it was observed that α-skeletal actin was expressed in the atria of the transgenic animals but not control animals. The genes whose expression patterns showed a broader distribution have previously been shown to be associated with compensatory hypertrophy. This suggests that they are not directly induced by bmyf5 expression but are likely to represent a secondary response. This hypothesis is also supported by the observation that the expression patterns of ANF and β-MHC were not induced by bmyf5 expression in the hearts of 2-day-old animals but were induced by 6 weeks of age (Fig 5⇑). In the hearts of 2-day-old animals, expression of the bmyf5 transgene did not noticeably alter the expression of several structural proteins and transcription factors, including perinatal MHC, ALC2A, muscle creatine kinase, smooth muscle α-actin, dystrophin, MyoD, sno, MRF4, GATA-4, MEF2D, and Nkx-2.5 (data not shown). These data indicate that the expression of some skeletal muscle gene products was directly dependent upon expression of the transgene, whereas the response patterns of others suggest a compensatory response.
Functional Consequences of bmyf5 Expression
It is unclear how transgene expression and the resulting pathology affect cardiovascular function, but our results suggest compensatory hypertrophy. We have previously observed that the fractional shortening found in 6-month-old bmyf5 mice was slightly depressed compared with that found in littermate control mice.38 These data indicate that there was a functional change in the pumping capacity of the bmyf5 hearts. Several pathological and physiological models have been shown to alter the contractile protein profile of the heart and its functional properties. BALB/c mice that overexpress the α-skeletal actin isoform in their hearts have been shown to have an increase in contractility.39 Pregnant female mice have been shown to have an increase in the β-MHC isoform expression concomitant with a decrease in Ca2+-activated ATPase activity.40
To determine whether ectopic bmyf5 expression caused a shift in the biochemical profile of cardiac myofibrils, Ca2+-activated ATPase activity was determined. Myofibrils from the left ventricle were partially purified, and Ca2+-activated myofibrillar ATPase activity was measured as a function of Ca2+ concentration. Myofibrils from hearts of bmyf5 mice compared with littermate control mice had a significant increase in the Ca2+-activated ATPase activity, which is a measure of the physiologically relevant actin-activated ATPase activity (Fig 7⇓, top). In the presence of high salts and absence of Mg2+, actin and myosin are dissociated; thus, the measured activity reflects inherent myosin ATPase activity.41 42 It was observed that Mg2+-free Ca2+-activated ATPase activity was not significantly different between the hearts of control and bmyf5 mice (Fig 7⇓, bottom). Additionally, electrophoresis under nondenaturing conditions of the same myofibrillar preparations did not indicate a shift in the isoform distribution of the MHCs nor the stoichiometry of the major structural proteins (data not shown). These data suggest that there was not a detectable shift in MHC content. However, the discrete areas of bmyf5 expression may not be sufficient to produce enough proteins to detect a shift in the MHC isoform when myofibrils from the total left ventricle are analyzed. Nevertheless, changes in Ca2+-activated myofibrillar ATPase activity indicate that a functional change at the biochemical level has resulted from transgene expression.
Expression of the bovine myf5 transgene promotes a pattern of skeletal muscle gene expression in the myocardium with ensuing myocardial pathology. The distribution of activated gene expression assumed two forms. One pattern of expression correlated with the expression of bmyf5. A second pattern of skeletal muscle gene expression that included α-skeletal actin and ALC1 extended beyond the region of bmyf5 expression. The activation of these molecular markers of hypertrophy suggests a pattern of primary cellular pathology followed by compensatory hypertrophy. From one point of view, these results are somewhat surprising, because of the high degree of sequence identity among many heart and skeletal contractile proteins. Additionally, since the sarcomeric structure of heart and skeletal muscle are very similar, protein integration ought to be functionally compatible. However, the extraordinarily small changes that appear to be critical for the etiology of familial hypertrophic cardiomyopathy suggest the very fine control under which the sarcomere is constructed and maintained.43
None of the bHLH MDF factors affiliated with skeletal muscle formation have been detected in the developing normal myocardium. Thus, this group of bHLH proteins does not play a role in cardiomyogenesis. However, several cardiac-specific genes contain the E-box motif in their regulatory regions, suggesting that they may be influenced by bHLH proteins and that there may be cardiac equivalents for the MDFs. Although several homeobox proteins have been implicated, to date, no bHLH proteins have been reported that direct the formation of the myocardium.11 12 One promising candidate is the anti-H2 activity described by Litvin et al.15
There was no indication of a morphological transformation of cells in the myocardium into skeletal muscle fibers. This suggests that although the myocardium may be permissive for the MDFs, in terms of muscle-specific gene expression, there is some block to complete transformation into skeletal muscle. Clearly, normal cardiac structure and function are not compatible with expression of the skeletal muscle MDFs. It is also unknown whether the pathology seen in the bmyf5 hearts is the result of an internal sarcomeric organization event or the result of an extracellular event. Both may ultimately be involved, as suggested by the two different patterns of skeletal muscle gene expression: patchy and localized versus diffuse. The EM photographs show that cells containing newly formed sarcomeres are adjacent to cells containing normal sarcomeres. Speculatively, the initial synthesis of skeletal muscle proteins within a myocardial cell could lead to dysfunction and perhaps necrosis. This, in turn, may lead to a compensatory synthesis of other proteins that have been associated with hypertrophy. Alternatively, the induction of the skeletal muscle proteins may not be balanced in the small number of cells expressing the transgene, and the overexpression of specific proteins may cause the formation of disorganized sarcomeres.
Inappropriate expression patterns of structural proteins may be the underlying cause of the cardiopathology observed. The present results are similar to observations made by Miner et al,20 who examined the effects of ectopic expression of MyoD in the hearts of transgenic mice. Embryonic skeletal muscle MHC gene expression was observed in a patchy pattern. However, in that study, the hearts were misshapen, failed to pump effectively, and proved to be lethal during gestation. In the present study, the animals were viable into adulthood. The theoretical reasons for these differences in viability could be gene dosage or the onset of transgene expression. The MyoD transgene was under the control of the MCK promoter, whereas the bmyf5 transgene was under the control of the MSV LTR. In the mouse, the MCK gene is initially expressed at ≈day 13.44 Expression of cardiac-specific proteins is observed much earlier than this, with a beating heart observed by day 9.0 PC.9 In the present study, bmyf5 transgene expression was observed before the onset of cardiac formation. However, the degree of pathology appears to be less than that observed by Miner et al,20 consistent with the hypothesis that the level of expression may play a major role in phenotypic differences. Nonetheless, in both cases, the atria appeared to be preferentially hypertrophied. The present study suggests that the expression of the bmyf5 MDF does not necessarily interfere with early heart development but that activation of skeletal muscle genes induces pathology very early in postnatal life. Expression of mutant contractile proteins has been linked to hypertrophic cardiomyopathy in patients and in transgenic mice (References 43 and 45, and K.L. Vikstrom, S. Factor, and L. Leinwand, unpublished data, 1995). Collectively, these studies suggest that the expression of sarcomeric proteins not normally found in the myocardium is not tolerated.
Cardiac pathology was evident 2 days after birth. Large numbers of infiltrating desmin-negative cells were evident, suggesting that they were not of muscle origin. The cells observed would not have been due to infiltration of macrophages or leukocytes, because the immune system is undeveloped in such young animals. By 6 weeks of age, the number of infiltrating cells was decreased between the two groups, and the degree of fibrosis appeared to be greater. This suggests that events occurring early in life were resolved as the animals matured.
When we examined the functional consequences of bmyf5 expression on cardiac biochemistry, we obtained seemingly paradoxical observations. Given the pathology observed, the increase in myofibrillar ATPase activity was surprising. There are several possible mechanisms responsible for an increase in the ATPase activity including (1) a shift in the phosphorylation state of several contractile proteins, (2) a change in the Ca2+ dynamics, and (3) an increase in the expression of α-skeletal actin. Hewett et al,39 using a BALB/c mouse strain that constitutively expresses α-skeletal actin in the heart, showed a significant positive correlation between the percentage of α-skeletal actin mRNA and +dP/dt, suggesting that the shift in the actin isoform influenced the contractility within those hearts. These changes occurred in the absence of hypertrophy or any remarkable pathology. This suggests that the response is not inherently pathological but represents an adaptive response by the myocardium. Increased α-skeletal actin expression is observed in response to many physiological stimuli, including various forms of overload, adrenergic stimulation, and thyroid hormone.46 Within the bmyf5 hearts, the spatial distribution of α-skeletal actin expression was greater than that of the transgene. Potentially, this represents a compensatory change by the healthy cardiomyocytes in response to the increased stress due to the pathology present. Similarly, the increases in ANF expression and the concomitant production and release of ANF would serve to decrease the work of the heart, a compensatory response to the pathology.
In summary, ectopic cardiac expression of bmyf5 induces the expression of several skeletal muscle–specific gene products. Although the onset of transgene expression preceded the heart’s formation, it did not appear to interfere with embryonic developmental patterns, but pathology was observed soon after birth. These results indicate the incompatibility of the cardiac and skeletal muscle gene programs, demonstrating the need for tight regulation to maintain normal cardiac function.
Selected Abbreviations and Acronyms
|ALC1||=||atrial light chain-1|
|AMV||=||avian myeloblastosis virus|
|ANF||=||atrial natriuretic factor|
|MCK||=||muscle creatine kinase|
|MDF||=||myogenic determination factor|
|MEF||=||muscle enhancer factor|
|MHC||=||myosin heavy chain|
|MSV||=||murine sarcoma virus|
|PCR||=||polymerase chain reaction|
This research was supported by National Institutes of Health grants HL-2R01-GM-29090 (Dr Leinwand) and grants from the Muscular Dystrophy Association (Dr Lyons) and the American Heart Association (92-GB-27). The authors are grateful to R. Santerre and C. Smith for providing us with the myf5 transgenic mice. The authors are grateful for the technical help from J. Rivera, L. Guther, S. Schailis, S. Maithias, and Yvonne Cress.
This manuscript was sent to Robert J. Lefkowitz, Consulting Editor, for review by expert referees, editorial decision, and final disposition.
- Received July 13, 1995.
- Accepted December 4, 1995.
- © 1996 American Heart Association, Inc.
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