Hypertrophy, Pathology, and Molecular Markers of Cardiac Pathogenesis

From the Department of Molecular, Cellular, and Developmental Biology (K.L.V., L.A.L.), University of Colorado, Boulder; the Department of Medicine (T.B.), University of Colorado Health Science Center, Denver; and the Department of Pathology (S.M.F.), Albert Einstein College of Medicine, Bronx, NY.

Correspondence to Leslie A. Leinwand, PhD, Professor and Chair, Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Campus Box 347, Boulder, CO 80309-0347. E-mail leinwand{at}stripe.colorado.edu

	Abstract

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Abstract—Increased ventricular expression of several genes, including atrial natriuretic factor (ANF), has been documented in experimental models of cardiac hypertrophy. It remains to be clarified whether altered expression of these genes is a consistent marker of the hypertrophy itself or a marker of some parallel pathogenetic process. Using a transgenic mouse model of hypertrophic cardiomyopathy as a tool, we assessed the relationship between the amount of ventricular ANF gene expression and the degree of hypertrophy as well as the relationship between the cells expressing ANF and tissue pathology. We determined that hypertrophy is not always associated with increased ventricular expression of ANF and that cells expressing ANF are found in regions of tissue pathology. We propose that alteration in the ventricular expression of this gene is a sensitive indicator of cardiac pathogenesis and may result from a number of different stimuli that include, among others, abnormal tissue architecture and hemodynamic load.

Key Words: hypertrophy • gene expression • pathogenesis • atrial natriuretic factor

	Introduction

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Hypertrophy is a compensatory response that allows the heart to cope with the pathogenic stimuli found with many cardiovascular diseases, including the primary myocardial dysfunction associated with cardiomyopathies. The normal and hypertrophied heart exhibit qualitative as well as quantitative differences in gene expression. This observation has spurred efforts over the past decade to understand the molecular mechanisms underlying the hypertrophic process, and a number of genes have been identified that exhibit altered expression patterns in cardiac hypertrophy. Among others, these include transient expression of proto-oncogenes^{1 2 3 4} and increased expression of -skeletal actin^{5 6 7} and ß-myosin heavy chain.^{4 7 8} It is thought that these changes may reflect a shift toward an embryonic program of gene expression (for a review see Reference 9⁹ ).

Changes in the expression of ANF in patient populations and in experimental models of cardiac hypertrophy have been well documented.^{4 10 11 12 13 14 15 16} ANF, a peptide hormone with diuretic, natriuretic, and vasorelaxant properties (for reviews see References 17 and 18^{17 18} ), was first identified in extracts of rat atria.¹⁹ Under normal conditions in the mammalian heart, atrial levels of ANF are 100-fold greater than ventricular levels,²⁰ whereas ventricular expression of ANF increases with conditions of increased hemodynamic load, often concomitant with increases in ventricular mass (for examples see References 4, 10, 11, and 12^{4 10 11 12} ).

However, it remains to be clarified whether altered expression of ANF is a marker of the hypertrophic process or whether it is a marker of a parallel process during cardiac pathogenesis. For example, is there a quantitative relationship between the amount of gene expression and the degree of hypertrophy, such that a threshold level of gene expression is indicative of hypertrophy? Through analysis of a transgenic mouse model of HCM, we show that hypertrophy can occur in the absence of increased ventricular levels of ANF message and that increased levels of this mRNA can also occur in the absence of detectable cardiac hypertrophy. In this genetic model, increases in ventricular levels of the ANF gene product reflect local changes in gene expression that correlate with areas of tissue pathology, prompting the conclusion that to some extent increased ventricular expression of ANF reflects part of a cellular pathological response.

	Materials and Methods

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Animal Husbandry
A previously described transgenic mouse model for HCM²¹ was used for these studies. These animals express a mutant -myosin heavy chain with expression driven by a rat -myosin heavy chain promoter. HCM transgenic mice, line 140,²¹ were maintained under specific pathogen-free conditions with free access to water and food. Heterozygously transgenic lines were maintained with backcrossing to C57/Bl6 mice obtained from the Institute for Behavioral Genetics, University of Colorado at Boulder. At the time of these studies, the transgenic line had been backcrossed to C57/016 for >14 generations. All animal protocols were approved by the Institutional Animal Use and Care Committee at the University of Colorado at Boulder.

Slot Blot Analysis
Total RNA was purified from LV and RV tissue using the guanidinium–acid phenol method.²² Total RNA (3 µg) in denaturing buffer (50% formamide, 6% formaldehyde, 20 mmol/L MOPS, pH 7.0, 50 mmol/L sodium acetate, and 1.0 mmol/L EDTA) was heated to 65°C for 15 minutes and then rapidly cooled on ice. Two volumes of 20x SSC (3 mol/L NaCl and 0.3 mol/L) was added, and the mixture was applied to positively charged nylon membrane using a vacuum slot-blot apparatus. Random-primed ³²P-labeled probes were generated with the Ready-to-Go DNA labeling kit (Pharmacia Biotech) using a PstI fragment from a rat ANF cDNA,²³ a PstI fragment from a rat GAPDH cDNA,²⁴ or a SacI-HindIII fragment corresponding to the 5' untranslated region of murine -skeletal actin mRNA²⁵ as templates. Membranes were hybridized overnight at 37°C in 50% formamide, 5x SSPE (0.9 mol/L NaCl, 0.05 mol/L sodium phosphate, and 5 mmol/L EDTA, pH 7.5), 1x Denhardt's solution (0.02% Ficoll, 0.02% polyvinylpyrrolidone, and 0.02% BSA), 0.2% SDS, 0.2 mg/mL sheared denatured salmon sperm DNA, and 1x10⁶ cpm/mL of ³²P-labeled probe. Afterward, the membranes were washed for 10 minutes at 65°C in each of the following buffers: 2x SSC and 0.2% SDS; 1x SSC, 0.2% SDS, 0.5x SSC, and 0.2% SDS; and 0.1x SSC and 0.2% SDS; the membranes were then exposed to PhosphorImager plates (Molecular Dynamics). Images were acquired using a STORM PhosphorImager (Molecular Dynamics, and the intensity of the signals was measured using ImageQuant software (Molecular Dynamics). Hybridization to yeast tRNA was used to determine background levels.

In Situ Hybridization
A 580-bp PstI fragment from the rat ANF cDNA²³ was subcloned into pBluescriptKS (Stratagene) and used as a template to generate sense and anti-sense ³⁵S-labeled RNA probes. A 364-bp region of the SV40 large T antigen that corresponds to the 3' end of the HCM transgene²¹ was subcloned into pTZ19R (United States Biochemical) and used to generate a transgene-specific anti-sense RNA probe. All RNA probes were synthesized by using the Promega Riboprobe kit and following the manufacturer's instructions.

Hearts were removed from 12-week-old female HCM mice and fixed overnight at 4°C in PBS containing 4% formaldehyde. After they were dehydrated through a graded ethanol series and cleared in cedarwood oil, tissues were embedded in paraffin. Sections (7 µm) were cut on a microtome and adhered to SectionLock slides (Polysciences Inc). Subsequent processing of the specimens followed the procedure described by Sassoon and Rosenthal²⁶ with the addition of an N-ethylmaleimide blocking step.²⁷ After hybridization and washing, slides were dried and then coated with Kodak NTB2 autoradiography emulsion (Eastman Kodak). Slides were developed using D-19 developer (Kodak) and photographed with bright-field optics.

Electron Microscopy
Hearts were removed, rinsed in saline, fixed with a mixture of formaldehyde and gluteraldehyde,²⁸ and then embedded. Abnormal regions within the myocardium were identified in thick sections. Thin sections were then cut from these regions, stained with uranyl acetate and lead citrate, and examined on the electron microscope.

Statistical Analysis
Data are expressed as the mean±SD. Differences between groups were assessed using an unpaired Student t test, and the correlation between various parameters was determined by linear regression analysis. In all cases, differences were considered significant at P<.05.

	Results

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Increased Ventricular Expression of ANF mRNA Does Not Correlate With Hypertrophy in a Murine Model of HCM
To determine the relationship between global hypertrophy, tissue pathology, and the induction of ventricular expression of ANF, we examined the ventricular levels of this message in a transgenic mouse model of HCM.²¹ Previous characterization of this transgenic mouse had shown that 12-week-old animals exhibit significant LV and RV hypertrophy.²¹ When Northern blot analysis was used to assess the mRNA expression of ANF, a disproportionate increase in ANF mRNA levels was detected in the LV samples (Figure 1A), although transgene expression is similar in both the LV and RV. LV and RV RNA from 12-week-old female transgenic animals, with nontransgenic littermates serving as controls, was also subjected to slot blot analysis using probes for ANF and -skeletal actin.^{23 25} GAPDH mRNA levels were also determined for the same blots and used to correct for small variations in sample loading. Under the hybridization conditions used, these probes detect single bands on Northern blots of mouse cardiac RNA (as shown in Figure 1A for ANF). As also seen by Northern blot, ANF mRNA was more abundant in the LVs of the HCM mice than in control LV samples (2.7-fold increase, P<.005). However, the abundance of ANF mRNA in the RV samples was indistinguishable from control values (Figure 1). This finding was surprising, since at this age these animals exhibit significant hypertrophy in both the RV and LV (14% and 12%, respectively²¹ ), and increased expression of -skeletal actin mRNA was detected in these same samples (Figure 1C). When the relative levels of ANF gene expression were plotted as a function of ventricular mass, it was clear that increased ANF mRNA levels are dissociated from LV or RV hypertrophy in the transgenic animals (Figure 2) with no correlation between ANF mRNA levels and LV or RV mass indicated by linear regression analysis (R=0.09 and R=0.11 for LV and RV comparisons, respectively). Similar results were seen in male HCM mice at this age as well (data not shown).

View larger version (21K): [in this window] [in a new window]   Figure 1. A, Northern analysis of transgene and ANF levels in transgenic and nontransgenic littermates, as indicated. Ethidium bromide–stained gel is included to demonstrate loading. B and C, Ventricular ANF (B) and -skeletal actin (C) mRNA levels, normalized to GAPDH mRNA levels, were determined for transgenic animals (solid bars) and nontransgenic littermate controls (open bars). Although both chambers exhibited increased mass, a significant increase in the abundance of ANF message was seen only in the LVs of the transgenic animals (>2.5-fold increases, P<.005). In contrast, increased levels of -skeletal actin transcript were detected in both the RV and LV (C). Data represent the mean±SD from seven animals. *P<.005 versus controls.

View larger version (15K): [in this window] [in a new window]   Figure 2. The relative level of ANF mRNA in LV and RV RNA samples from 12-week-old female transgenic mice, with nontransgenic sex-matched littermates serving as controls, was determined by slot blot analysis, normalized to GAPDH mRNA levels, and plotted against an index of chamber mass (LV or RV weight normalized to tibial length). The increase in ANF mRNA seen in transgenic LV, but not RV, samples did not correlate with the extent of LV hypertrophy (R=0.09; P>.05). indicates control mice; , transgenic mice. The mean values for controls are indicated by dashed lines.

Ventricular Expression of ANF mRNA Does Not Result From Focal Transgene Expression
The discordance between hypertrophy and ANF mRNA levels in the RV suggests that some trigger other than hypertrophy may induce the increased ANF mRNA levels in the LVs of the HCM mice. We had previously noted that tissue pathology in these mice is focal and is more extensive in the LV than the RV,²¹ implying that the greater incidence of abnormal tissue architecture in the LV might be responsible for the increased LV ANF mRNA levels. However, it has been noted that transgene expression under the -myosin heavy chain promoter sometimes results in patchy expression in the heart. Patchy expression of the mutant myosin mRNA in these HCM mice could also contribute to differential gene expression between the RV and LV. To determine the relationship between transgene expression and ANF expression, we performed in situ hybridization analysis with probes recognizing either ANF or transgene mRNAs. In HCM mouse hearts, transgene expression was found uniformly throughout the LV and RV, with lower expression seen in the atria (Figure 3A and 3C). As expected, ANF gene expression was very strong in the atria (Figure 3B). Intensely positive cells were also found in foci throughout the LV (Figure 3B and 3D). A small number of ANF mRNA–positive cells were also seen in control hearts, especially near the endomyocardial surface and at the fibrous base of the cardiac valves (not shown). However, in control animals, the number of ANF-positive cells in the ventricular myocardium was much less than in the HCM hearts, and they were found in scattered groups containing one to three positive cells. These staining patterns do not result from nonspecific hybridization, since the transgene-specific probe was negative in control mouse hearts and a sense-ANF probe was negative in both control and HCM hearts (data not shown).

View larger version (132K): [in this window] [in a new window]   Figure 3. The distribution of transgene (A and C) and ANF (B and D) mRNAs in 12-week-old female HCM mice was determined by in situ hybridization. Transgene expression was found uniformly throughout the ventricular myocardium. The atria were strongly positive for ANF message, as were foci of cells scattered throughout the ventricle. The region indicated by arrows in panel B is shown at higher magnification in panel D. Bar=10 µm (applies to panels C and D).

Foci of ANF-Positive Ventricular Myocytes Are Found in Regions With Tissue Pathology and Fibrosis
The pattern of ANF mRNA–positive cells in the HCM mouse hearts was suggestive of the pattern of tissue pathology seen in the hearts of these mice. To determine whether ANF mRNA expression occurred in regions of tissue pathology, serial sections were obtained from 12-week-old female HCM mice and processed for in situ hybridization with the anti-sense ANF probe or stained with Masson's trichrome to visualize areas of fibrosis (Figure 4). Regions of the ventricular myocardium with foci of intensely positive cells were identified by in situ hybridization (Figure 4A), and the corresponding region in the adjacent section was located (Figure 4B). Foci of ANF-expressing ventricular myocytes were found in regions of the heart with tissue pathology, especially in regions with fibrosis but also including severe disarray. Interestingly, many of these foci of ANF expression and tissue pathology were found adjacent to small intramural coronary vessels.

View larger version (122K): [in this window] [in a new window]   Figure 4. Serial sections from 12-week-old female HCM mice were processed for in situ hybridization with the ANF probe (A) or stained with Masson's trichrome (B). Foci of ANF-positive ventricular myocytes (for examples, see arrows in panel A) were found in regions of the ventricle that in the adjacent section (B) exhibited significant tissue pathology and fibrosis. Bar=250 µm.

ANF Secretory Granules Are Found in Areas of Abnormal Tissue Architecture
The colocalization of foci of ANF expression and regions of tissue pathology described above is limited by the distance between adjacent paraffin sections (7 µm) and by one's ability to match landmarks in two adjacent sections. Therefore, we wished to confirm our findings by using a higher resolution. In atrial myocytes, ANF is stored in distinctive secretory granules that are often called atrial particles.¹⁹ Although these particles are scarce in the normal rodent ventricle,²⁹ they are found more readily in pathological states (for example see Reference 30³⁰ ). We predicted that if areas containing tissue pathology were identified in HCM mouse hearts and then examined by electron microscopy, we would find ventricular myocytes containing atrial particles. Regions of ventricular myocardium with significant cellular disarray or fibrosis were identified in thick sections of embedded tissue, and thin sections of these regions were examined by electron microscopy. Areas with abnormal ultrastructure were readily apparent in these sections, and cells with prominent secretory granules similar in appearance to atrial particles were identified (Figure 5). These cells typically were surrounded by matrix accumulations with prominent collagen fibrils (Figure 5, see asterisks). Although more extensive characterization would be required to conclude that these secretory granules are in fact ANF particles, these morphological data are consistent with our observation that increased ANF gene expression in this mouse model of cardiomyopathy is strongly associated with the presence of tissue pathology.

View larger version (164K): [in this window] [in a new window]   Figure 5. Granules resembling ANF particles were found in ventricular myocytes bordering regions of collagen accumulation. AP, indicates ANP granules; asterisk, collagen fibrils. Bar=0.1 µm.

	Discussion

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The development of cardiac hypertrophy is accompanied by changes in cardiac gene expression that are thought to provide the heart with a means to compensate for increased hemodynamic load.³¹ During cardiac hypertrophy in rodents, increased ventricular expression of genes, such as ANF, -skeletal actin, and ß-myosin heavy chain, normally expressed during development may reflect the reinduction of an embryonic pattern of gene expression.^{9 32} Increases in ventricular expression of ANF have been documented in numerous experimental models of cardiac hypertrophy and failure^{4 10 11 12 14 16 33 34} as well as in human heart failure.^{13 15} In these pathological states, increased ANF levels may serve to reduce preload through the natriuretic and vasodilatory properties of the secreted peptide (for a review see Reference 17¹⁷ ), leading to the hypothesis that increased ventricular levels of ANF may be a molecular marker of cardiac hypertrophy.³⁵

It is clear that increased ventricular expression of ANF can occur in response to a number of different stimuli. Investigators have shown significant correlation between the extent of LV hypertrophy and LV ANF mRNA content in experimental models of cardiac hypertrophy resulting from volume or pressure overload^{4 12} or isoproterenol infusion.³⁶ It has been suggested that changes in gene expression accompanying cardiac hypertrophy are the result of a multifactorial process.¹¹ In the case of ventricular expression of ANF, this is clearly the case. Careful monitoring of the induction of ANF message after the imposition of hemodynamic overload¹¹ or isoproterenol infusion³⁶ demonstrates a biphasic pattern of induction with the greatest increases in ventricular ANF levels occurring within 3 to 4 days after the imposition of the stimulus. Both load-dependent and load-independent mechanisms^{37 38} and the renin-angiotensin system^{10 39} have been implicated in this process.

We have presented data suggesting that cardiac hypertrophy and increased ventricular expression of ANF are not necessarily correlated. Through analysis of a transgenic mouse model of HCM we have shown that hypertrophy can occur in the absence of increased ventricular levels of ANF message and that increased levels of this mRNA can also occur in the absence of detectable cardiac hypertrophy. In this genetic model, increases in ventricular levels of the ANF gene product reflect local changes in gene expression that appear to correlate with areas of tissue pathology. Foci of cells positive for ANF expression were often found near small intramural vessels in the cardiomyopathic hearts (Figure 4). In normal rat heart, solitary myocytes immunopositive for ANF have been detected, albeit rarely, in the vicinity of small intramural vessels.²⁰ In the HCM mice, increased incidence of ANF-positive myocytes near small vessels may reflect a regional pathogenetic process. We had previously reported the presence of abnormal small vessels in the hearts of these transgenic mice,²¹ a feature that is also seen in most patients with HCM.⁴⁰ Local changes in ANF gene expression may reflect the response of the surrounding myocardium to alterations in vessel structure and/or function or may occur concomitant with increasing fibrosis in the vessel vicinity.

Regional changes in cardiac gene expression have been noted in other model systems. Induction of ventricular ANF mRNA levels is greater in the septum of young cardiomyopathic Syrian hamsters than in the RV or LV free wall.⁴¹ During the early phases of cardiac hypertrophy in a rat model of pressure overload, ß-myosin heavy chain gene expression exhibits transmural differences as well as increases around large coronary arteries.^{8 32} In addition, in endomyocardial biopsies taken from HCM patients, increased ANF levels are seen in specimens that also exhibit significant fibrosis and cellular disarray.⁴²

In our experimental model, we detected relatively small changes in ANF gene expression. It should be noted that much greater increases in ANF gene expression have been reported in experimental models of acute pressure or volume overload (10- to 20-fold increases), which may represent a different pathogenetic response. The relatively small increases in ventricular ANF mRNA that we have seen in young HCM mice may reflect an early phase in the pathogenesis of this murine cardiomyopathy. In any case, it is clear that increased ventricular expression of ANF is not always associated with cardiac hypertrophy and thus should not be considered a stable marker of cardiac hypertrophy. We propose that alteration in the ventricular expression of this gene is a sensitive indicator of cardiac pathogenesis and may result from a number of different stimuli that include, among others, abnormal tissue architecture and hemodynamic load.

	Selected Abbreviations and Acronyms

 

ANF = atrial natriuretic factor HCM = hypertrophic cardiomyopathy LV = left ventricle RV = right ventricle

	Acknowledgments

 
This study was supported by a grant to Dr Leinwand from the Colorado/Wyoming Affiliate of the American Heart Association. The authors would like to thank Amy Whitledge for cloning the ANF riboprobe template during an Undergraduate Research Opportunity fellowship supported by the Undergraduate Research Initiative of the Howard Hughes Medical Institute at the University of Colorado, Boulder.

	Footnotes

 
This manuscript was sent to Eugene Braunwald, Consulting Editor, for review by expert referees, editorial decision, and final disposition.

Received September 2, 1997; accepted February 2, 1998.

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