Overexpression of Transforming Growth Factor-β1 and Insulin-Like Growth Factor-I in Patients With Idiopathic Hypertrophic Cardiomyopathy
Background Idiopathic hypertrophic cardiomyopathy (HCM) is characterized by regional myocardial hypertrophy. To investigate involvement of growth factors on myocardial hypertrophy in HCM patients, we evaluated gene expression and cellular localization of transforming growth factor-β1 (TGF-β1), insulin-like growth factors (IGF-I and IGF-II), and platelet-derived growth factor-B (PDGF-B) in ventricular biopsies obtained from patients with HCM (n=8), aortic stenosis (AS) (n=8), or stable angina (SA) (n=8) and from explanted hearts with ischemic cardiomyopathy (TM) (n=7).
Methods and Results Levels of TGF-β1, IGF-I, IGF-II, and PDGF-B transcripts were quantified with the use of multiplex RT-PCR. Glyceraldehyde 3-phosphate dehydrogenase was used as an internal standard. Antibodies against TGF-β and IGF-I were used to localize their peptides within the myocardium. Antisense and sense (control) cRNA probes of TGF-β1 and IGF-I, labeled with digoxigenin, were used to localize the growth factor transcripts by in situ hybridization. mRNA levels (densitometric ratio of growth factor/glyceraldehyde-3-phosphate dehydrogenase) of TGF-β1 and IGF-I in HCM (0.75±0.05 and 0.85±0.15, respectively; mean±1 SEM) were significantly (P<.01 for all groups) elevated in comparison with non-HCM myocardium (AS: 0.38±0.07, 0.29±0.06; SA: 0.32±0.04, 0.18±0.05; TM: 0.25±0.03, 0.15±0.03). mRNA levels of TGF-β1 and IGF-I in the hypertrophic AS myocardium were greater (P=.02, P=.05) than those in the explanted myocardium (TM). Immunohistochemical and in situ hybridization studies showed increased expression of TGF-β1 and IGF-I in the HCM cardiomyocytes.
Conclusions Gene expression of TGF-β1 and IGF-I was enhanced in idiopathic hypertrophic cardiomyopathy and may be associated with its development.
Idiopathic HCM is a primary cardiac abnormality characterized by regional asymmetrical myocardial hypertrophy. The hypertrophic myocardium can result in obstruction of left ventricular ejection as well as systolic and diastolic dysfunction and myocardial ischemia. Symptoms unresponsive to medical therapy can necessitate surgery.
HCM is described for the most part as a heterogeneous disease of the sarcomeres. At least 34 missense mutations have been described in the β-myosin heavy chain gene, 7 mutations in the cardiac troponin-T gene, and 2 mutations in α-tropomyosin, and other mutation candidate loci also exist.1 2 3 4 However, family studies suggest that the autosomal dominant trait accounts for only 50% of HCM patients.5 The remaining HCM patients show no familial transmission, and the disease occurs sporadically. Myocardial calcium kinetics and sympathetic stimulation have been studied because of diastolic functional abnormalities.6 7 However, none of these findings explain the regional myocardial hypertrophy (cardiomyocyte hypertrophy and oversynthesis of extracellular matrix proteins) observed in most HCM patients. The etiology of this disease remains unknown.
Growth factors may play an important role in cardiomyocyte proliferation, cell hypertrophy, and the overproduction of extracellular matrix. Kardami8 showed that bFGF and IGF-I stimulate cultured neonatal rat and embryonic chicken cardiomyocyte DNA synthesis and cell proliferation. PDGF increased DNA synthesis in cultured adult newt cardiomyocytes.9 Although TGF-β1 inhibits DNA synthesis in cultured cardiomyocytes,8 elevated TGF-β1 levels increase contractile protein synthesis in cultured adult rat cardiomyocytes and extracellular matrix production in cultured myocardial fibroblasts.10 Animal studies suggest that hypertrophic stimuli (pressure overload or norepinephrine) increase TGF-β1, bFGF, and IGF-I gene expression in hypertrophic myocardium.11 12 13 14
Because growth factors have been shown to stimulate cardiomyocyte proliferation and contractile protein synthesis and fibroblast extracellular matrix production in both in vitro and in vivo studies, localized overexpression of growth factors could contribute to the regional asymmetrical ventricular hypertrophy seen in HCM patients. We evaluated TGF-β1, IGF-I, IGF-II, and PDGF-B levels in freshly resected ventricular tissue obtained from patients with HCM, AS, SA, and ischemic cardiomyopathy. We found that TGF-β1 and IGF-I levels were dramatically increased in HCM patients. Immunocytochemistry and in situ hybridization showed that the growth factors were localized to the cardiomyocytes.
Myocardial ventricular biopsies were collected from eight male patients with HCM (44 to 58 years of age) undergoing surgery, eight male patients with acquired left ventricular hypertrophy secondary to AS (56 to 72 years of age) undergoing aortic valve replacement, eight male patients with SA (50 to 61 years of age) undergoing coronary artery bypass graft surgery, and seven explanted hearts from male patients undergoing TM (31 to 63 years of age). Permission was obtained from The Toronto Hospital’s Human Experimentation Committee and from the patients. Half of each biopsy was immediately frozen in liquid nitrogen for quantification of growth factor mRNA, and the other half was fixed in 4% paraformalin for immunohistochemistry and in situ hybridization.
Quantification of Growth Factor mRNA Levels in the Myocardium
Multiplex RT-PCR was used to determine growth factor mRNA levels in HCM and non-HCM ventricular tissue.
Frozen human myocardial tissue (10 to 20 mg) was powdered and then homogenized in 1 mL TRlzoL reagent (BRL-Life Technologies). Total RNA was isolated as outlined by the manufacturer. The integrity of each RNA preparation was determined by electrophoretic fractionation through an agarose/formaldehyde gel. A visual inspection of the 28S and 18S rRNA bands was performed to ensure that the bands were sharp and not degraded.
First-Strand cDNA Synthesis
Total cellular myocardial RNA was reverse transcribed using oligo dT(20),15 and the cDNA was resuspended in 100 μL H2O. Briefly, RNA (10 μg) was added to a mixture containing 1 μg oligo dT20, 4 μL 5× RT-reaction buffer, 1 μL dNTPs (25 mmol/L concentration of each; Pharmacia Biotech), 2 μL of 0.1 mol/L dithiothreitol, 1 μL RNase inhibitor, and 20 U Moloney murine leukemia virus RT. Water was added to bring the total volume to 20 μL, and the sample was incubated at 37°C for 1 hour. The reaction was stopped, and the RNA was denatured by the addition of 30 μL of 0.7 N NaOH/45 mmol/L EDTA and incubated at 65°C for 10 minutes. The first-strand cDNA products were precipitated with 5 μL of 3 mol/L sodium acetate and 110 μL of 100% ethanol. After centrifugation, the pellet was resuspended in 100 μL H2O.
PCR primers corresponding to each respective growth factor sequence were purchased from Clontech. Each primer was designed to discriminate between genomic and cDNA sequences to eliminate possible interference by contaminating genomic DNA. The primer sequences and their sizes are summarized in the Table⇓. For quantitative analysis of growth factor mRNAs, human G3PDH gene served as the internal control in calculation of the densitometric results. The G3PDH and target growth factor mRNAs were coamplified by the multiplex PCR. The PCR solution (total volume, 100 μL) contained 10 μL first-strand cDNA, 1 μL of 25 mmol/L dNTP, 10 μL of 10× PCR buffer, 3 μL of 50 mmol/L MgCl2, 2.5 U Taq DNA polymerase (Perkin-Elmer Cetus), 2 μL of 0.2 μmol/L G3PDH primers (Clontech), 2 μL of 0.2 μmol/L concentration of each growth factor primer, and 67.5 μL H2O. Mineral oil was layered on top of the PCR samples to prevent evaporation. The samples were transferred directly from ice into a thermocycler (Perkin-Elmer Cetus) set to 94°C, and the DNA was denatured for 10 minutes. Subsequently, 33 reaction cycles were performed: denaturation (94°C, 1 minute), annealing (55°C, 1 minute), and primer extension (72°C, 2 minutes). PCR products were separated using agarose gel electrophoresis and visualized by ethidium bromide staining. The densities of G3PDH and the growth factor bands were analyzed using a computer image densitometer (BioRad gel Doc 1000). The ratio of the growth factor to G3PDH was determined.
Localization of Growth Factor mRNA in the Myocardium
In situ hybridization was used to identify sources of TGF-β1 and IGF-I in the HCM and non-HCM myocardium.
The plasmids pST64 and pKT218, containing human TGF-β1 and IGF-I genes (American Type Culture Collection), were isolated and purified using a QIAGEN-tip 100 (Qiagen). The pSP64 plasmid was digested with EcoR1, and the pKT218 plasmid was digested with Pst I. The TGF-β1 (1.05 kb, 17 ng) and IGF-I (0.659 kb, 17 ng) were inserted into the EcoR1 site of pGEM 7Zf+ (100 ng) or the Pst I site of pSL301 (100 ng), respectively, and ligated. The reconstructed phagemid DNA was transfected into JM109 competent cells. The cells were amplified, and the plasmids were isolated and purified using a QIAGEN-tip 100. The size and orientation (sense or antisense) of each respective insert were determined with enzymes restricting analysis and DNA sequencing.
cRNA Probe Synthesis and Labeling
cRNA probes for TGF-β1 and IGF-I were synthesized in vitro and labeled with DIG. The pGEM 7Zf+ DNA containing TGF-β1 was digested with Sma I and Xho I. The TGF-β1 cRNA probes were synthesized at 37°C for 2 hours in a mixture composed of 1 μg template DNA; 1 μL of ATP, CTP, GTP, and UTP mixture solution (25 μmol/L concentration of each); 2 μL transcription buffer; and 20 U RNase inhibitor with 30 U SP6 polymerase for the antisense strand or 40 U T7 RNA polymerase for sense strand. The pSL301 DNA containing the IGF-I fragment was digested with Hpa I and Xho I. The IGF-I probes were synthesized as described above using IGF-I DNA as a template and 40 U T3 polymerase for the antisense and 40 U T7 RNA polymerase for the sense. The reactions were stopped by heating at 65°C for 15 minutes, and plasmid DNA was removed by incubating the solution with 20 U DNase 1 (RNase-free) for 15 minutes at 37°C.
The cRNA probes were labeled with the use of a DIG 3′-end labeling kit (Boehringer-Mannheim Canada). The DIG-labeled cRNA was dissolved in 20 μL sterile distilled water and stored at −80°C.
In Situ Hybridization
In situ hybridization was performed as described by Zerbini et al.16 Briefly, the myocardium was fixed in 4% paraformalin, embedded in paraffin, and sectioned (6 μm thickness). Myocardial sections from HCM, AS, SA, and TM groups were processed concurrently. After deparaffination of the tissue section with xylene and rehydration in a graded series of ethanol solutions (100%, 95%, 90%, 85%, 80%, and 70% in distilled water) for 5 minutes at each step, the section was washed with PBS and then treated with 0.3% Triton X-100 for 15 minutes and digested with proteinase K (1 μg/mL) at 37°C for 20 minutes. The sample was then washed with PBS (three times for 5 minutes each) and prehybridized in 25 mL of a buffer containing 50% formanide, 5× SSC, 50 mmol/L sodium phosphate (pH 7.2), 2% blocking reagent, 2% SDS, and 0.1% N-lauroylsarcosine at 42°C for 1 hour. The slides were transferred into hybridization buffer containing either antisense (30 ng/mL) or sense (30 ng/mL) DIG-labeled probes, separately. The samples were hybridized at 42°C for 16 hours and washed with 2× SSC/0.1% SDS at room temperature (twice for 5 minutes each) and 0.1× SSC/0.1% SDS at 68°C (twice for 15 minutes each). After washing in 0.3% Tween 20 in buffer A (0.1 mol/L maleic acid, 0.15 mol/L NaCl, pH 7.5) for 1 minute and incubation for 30 minutes in buffer B (1% blocking reagent in buffer A), the samples were incubated at room temperature with anti-DIG antibody conjugated with alkaline phosphatase (1:3000 dilution) for 60 minutes (Boehringer-Mannheim Canada). After washing with buffer A (twice for 10 minutes each), the samples were incubated with a color substrate solution (nitroblue tetrazolium and X-phosphate) for 16 hours at room temperature as described by the manufacturer. The color reaction was stopped by washing the samples with buffer C (10 mmol/L Tris-HCl and 1 mmol/L EDTA, pH 8.0), and the slides were photographed.
Antibodies against IGF-I and TGF-β were used to localize the growth factors in the myocardium by the avidin-biotin-peroxidase complex technique as described by Hsu et al.17 Myocardial tissue was fixed with 4% paraformalin in 10 mmol/L PBS for 2 hours. The sample was dehydrated in a graded series of ethanol solutions (70%, 80%, 85%, 90%, 95%, and 100% in distilled water) for 5 to 15 minutes at each step, permeated in 100% chloroform for 30 minutes at room temperature, embedded in paraffin, and cut into serial sections (6 μm thick).
Immunohistochemistry was performed concurrently on the tissue sections of HCM, AS, SA, and TM groups. After the samples were deparaffined and rehydrated as described above and incubated in 10 mmol/L PBS, they were incubated with a solution of 3% H2O2 in 70% methanol for 30 minutes to inhibit endogenous myocardial peroxidase. Triton X-100 (0.2%) was used to treat samples for 10 minutes to enhance cell permeability. After blocking nonspecific protein binding with 2% normal goat serum in 0.05 mol/L Tris buffer (pH 7.4) for 15 minutes, primary antibodies against human TGF-β (1:1000) or IGF-I (1:1000) (Cedarlane Laboratories) were added, and the samples were incubated at 37°C for 30 minutes followed by an overnight incubation at 4°C. Both antibodies were monoclonal and growth factor specific. The antibody against TGF-β did not distinguish among β1, β2, and β3. Negative control samples were incubated in PBS (without the primary antibodies) under the same conditions. After samples were washed with PBS (three times for 5 minutes each), a biotin-labeled secondary antibody (1:250, Vector Laboratories) was added to the specimens and incubated at room temperature for 1 hour. The samples were rinsed three times for 5 minutes each in fresh PBS and reacted with an avidin-biotin complex conjugated with peroxidase at room temperature for 45 minutes. Visualization was performed with a diaminobenzidine solution (0.25 mg/mL in 0.05 Tris-HCl buffer containing 0.02% H2O2) for 10 minutes. The cellular nuclei were counterstained with hematoxylin for 1 minute. The samples were covered with crystal mounts and photographed.
Data are expressed as mean±1 SEM. Comparison among growth factor mRNA levels in HCM, AS, SA, and TM was performed with one-way ANOVA. Differences between the groups were analyzed with Student’s t test.
Ventricular myocardium expressed TGF-β1, IGF-I, IGF-II, PDGF-B, and G3PDH genes (Figs 1⇓ and 3⇓). The pattern of TGF-β1 levels in HCM, AS, SA, and TM was similar to that of IGF-I (Fig 2⇓). RT-multiplex-PCR data (densitometric ratio of growth factor/G3PDH) showed that the TGF-β1 in HCM myocardium (0.75±0.05) was significantly higher than that in the AS (0.38±0.07, P=.003), SA (0.32±0.04, P=.0001), and TM (0.25±0.03, P=.000006) patients (Figs 1⇓ and 2⇓). The AS TGF-β1 mRNA level was greater (P=.02) than that in TM. The HCM IGF-I mRNA level (0.85±0.15) was significantly greater than that in the AS (0.29±0.06, P=.003), SA (0.18±0.05, P=.0008), and TM (0.15±0.03, P=.002) (Figs 1⇓ and 2⇓). IGF-I mRNA level in the AS myocardium was greater (P=.05) than the TM myocardium but not that of the SA myocardium. There were no significant differences among HCM, AS, SA, and TM in IGF-II and PDGF-B mRNA levels (Figs 3⇓ and 4⇓).
The difference of growth factor levels between the patient groups was not due to patient sex or age. All myocardial biopsies were obtained from male patients. Patients in the AS group were older (67.0±5.0 years, P<.05) than those in the HCM (51±6.0 years), SA (57±5.0 years), or TM (51±11 years) group. However, the correlation between growth factor mRNA levels and patient age was not significant (correlation coefficients = 0.21304 and 0.2785 for TGF-β1 and IGF-I, respectively).
The overexpression of IGF-I and TGF-β1 in HCM myocardium found in RT-PCR studies was confirmed by in situ hybridization. mRNA expression of IGF-I and TGF-β1 in HCM myocardium was greater than that in non-HCM myocardium (Figs 5⇓ and 6⇓). The growth factor mRNAs were localized within the cardiomyocytes. Staining with the use of sense probes for both growth factors was negative (Figs 5J⇓ and 6J⇓).
Immunohistochemical studies demonstrated that the HCM cardiomyocytes contained more IGF-I and TGF-β proteins than did non-HCM myocardial tissue because HCM cardiomyocytes stained more strongly for IGF-I and TGF-β than did the SA, AS, and TM cardiomyocytes (Figs 5⇑ and 6⇑). The hypertrophic cardiomyocytes in the AS myocardium also stained more strongly than nonhypertrophic TM cardiomyocytes. The connective tissue and negative controls (Figs 5I⇑ and 6I⇑) did not stain.
HCM is a common cause of sudden death and affects ≈1 of 5000 people at any age.18 19 The natural history of the patients can be benign to severely symptomatic, requiring surgery.20 21 It is a primary cardiac abnormality characterized by a hypertrophied myocardium that is unrelated to pressure or volume overload.22 Histological findings in HCM are distinctive, with striking cardiomyocyte hypertrophy and significant increases in extracellular matrix connective tissue.23 24
To better understand the growth factor results, an understanding of the study limitations is important. Patients with AS were significantly older than those in the other study groups; age may have affected growth factor levels in the AS patients. However, statistical analysis did not reveal an effect of age on growth factor levels within any patient group or overall in patients enrolled in the study. All tissue samples were obtained from the left ventricle, although the tissue was obtained from different parts of the ventricle in each group. Tissue obtained from the HCM and AS patients was resected from the septum, whereas tissue from the SA and TM groups was taken from the left ventricular free wall. A comparison of the growth factor levels between hypertrophic and nonhypertrophic myocardium from the same HCM patient would have been invaluable. However, it was impractical due to ethical concerns. The TGF-β antibodies used in the immunochemistry were not specific for the β1 isoform and probably would cross-react with β2 and β3. We attribute the increase in TGF-β to TGF-β1 because of the specificity of the PCR primers and the cRNA probe to the β1 isoform. It is also important to note that the anti–TGF-β antibodies detect both latent and active TGF-β. Likewise, the technique used to measure TGF-β1 mRNA does not distinguish between active and latent forms.
This study examined growth factor levels in hypertrophic myocardium (HCM and AS) and nonhypertrophic myocardium (SA and TM). IGF-I and TGF-β1 gene expressions at the mRNA and protein levels were significantly higher in the HCM myocardium than in the AS, SA, and TM myocardium. Although the IGF-I and TGF-β1 mRNA and protein levels in the AS myocardium were higher than those in the TM myocardium, the AS levels were similar to those in the SA myocardium. The difference in mRNA and protein levels of IGF-I and TGF-β1 between the AS and TM myocardium could be explained by a decrease in these growth factors in the severely damaged explanted myocardium. The IGF-II and PDGF-B levels were similar among HCM, AS, SA, and TM myocardium.
The significance of the present study is that IGF-I and TGF-β1 mRNA levels in HCM patients were 2.9 and 2.0 times higher than those in the hypertrophic myocardium secondary to AS, 4.7 and 2.3 times higher compared with their levels in the nonhypertrophic myocardium affected by SA, and 5.7 and 3.0 times greater than those in the explanted dilated ischemic cardiomyopathic myocardium. In agreement with the PCR results, in situ hybridization for mRNA and immunohistochemistry for growth factor peptides demonstrated that these growth factors were overexpressed in the cardiomyocytes of the HCM myocardium. Significant elevation of IGF-I and TGF-β1 in hypertrophic myocardium of HCM patients suggested that these growth factors may play an important role in the regional hypertrophy of HCM patient hearts. It is important to note that this study does not show whether the marked increases in IGF-I and TGF-β1 mRNA and protein levels in the HCM myocardium are primary or secondary causes of the hypertrophy. Localized increases in myocardial IGF-I and TGF-β1 resulting in regional myocardial hypertrophy in patients with HCM need to be shown; this is difficult because of safety considerations involved in taking tissue biopsies from hypertrophied and nonhypertrophied myocardium from the same HCM patient.
The AS results are in agreement with results from animal studies. In a rat model of cardiac hypertrophy induced by pressure overload, Villarreal and Dillman14 demonstrated that TGF-β1 mRNA levels increased 1.7-fold over sham-operated animals at 12 hours after surgery and were returned to control levels by 14 days. In a similar pressure-overload rat model of cardiac hypertrophy, Hanson et al11 showed that IGF-I mRNA levels paralleled the development of the hypertrophy. The animal results are consistent with the tendency for increased myocardial IGF-I and TGF-β1 mRNA and protein levels in the AS myocardium.
IGF-I is highly correlated with myocardial hypertrophy. Decker et al28 showed that IGF-I increased protein accumulation in cultured adult rabbit cardiac myocytes. The addition of the growth factor to culture medium stimulated cardiomyocytes to synthesize contractile proteins.29 30 31 Clinical studies have reported a high correlation between circulating IGF-I levels and left ventricular hypertrophy in hypertensive patients.32 Increased IGF-I levels in growth hormone–deficient patients resulted in increased left ventricular mass.33 The present study demonstrates that IGF-I is overexpressed in the HCM myocardium. The elevated IGF-I levels could evoke autocrine and paracrine mechanisms to stimulate cardiomyocytes to overproduce contractile proteins, resulting in cell hypertrophy. The overexpressed IGF-I could be synergistic with TGF-β1 in the HCM myocardium,34 which could accelerate myocardial hypertrophy. IGF-I receptor increase was also found in human hypertrophic myocardium compared with nonhypertrophic myocardium.27
Although TGF-β1 inhibits cardiac myocyte DNA synthesis,8 TGF-β1 is a strong trophic factor. Elevated TGF-β1 was found to stimulate cultured rat cardiomyocytes to synthesize contractile proteins and myocardial fibroblasts to overproduce connective tissue.10 Similar results were observed in cultured adult rabbit cardiomyocytes.28 TGF-β1 is also involved in myocardial extracellular matrix production by stimulating fibroblasts to synthesize and secrete collagen.34 Sakata35 showed a significant increase in TGF-β1 mRNA levels in the hypertrophic left ventricles of cardiomyopathic Syrian hamsters. The elevated TGF-β1 levels in the HCM myocardium could stimulate (1) contractile protein synthesis in cardiomyocytes and (2) overproduction of extracellular matrix proteins in myocardial fibroblasts, resulting in extensive cardiomyocyte hypertrophy and myocardial fibrosis in HCM patients. In addition, overexpressed TGF-β1 in HCM myocardium may stimulate myocardial fibroblasts to synthesize contractile proteins, which may convert fibroblasts into cardiomyocyte-like cells.36
Although the etiology of HCM has been studied extensively, the present study demonstrates for the first time increased myocardial levels of IGF-I and TGF-β1, which could account for the myocardial hypertrophy in idiopathic HCM patients. Whether there exists a causal relationship among increased growth factor levels and idiopathic HCM and contractile gene mutations requires further study.
Selected Abbreviations and Acronyms
|bFGF||=||basic fibroblast growth factor|
|IGF||=||insulin-like growth factor|
|PCR||=||polymerase chain reaction|
|PDGF-B||=||platelet-derived growth factor-B|
|SSC||=||standard saline citrate|
|TGF-β1||=||transforming growth factor-β1|
|TM||=||explanted heart with ischemic cardiomyopathy|
This study was supported by the Heart and Stroke Foundation of Ontario and Medical Research Council of Canada (MT-13665 and MT-10392). Dr Li is a Research Scholar of the Heart and Stroke Foundation of Canada. Dr Weisel is a Career Investigator of the Heart and Stroke Foundation of Ontario. Drs Rao and Merante are Research Fellows of Heart and Stroke Foundation of Ontario. We are indebted to Drs C. Feindel, C. Peniston, R. Cusimano, and V. DeSouza for the myocardial tissue.
- Received December 9, 1996.
- Revision received February 4, 1997.
- Accepted February 16, 1997.
- Copyright © 1997 by American Heart Association
Elstein E, Leiw C-C, Sole MJ. The genetic basis of hypertrophic cardiomyopathy. J Mol Cell Cardiol. 1992;24:1471-1477.
Hengstenberg C, Schwartz K. Molecular genetics of familial hypertrophic cardiomyopathy. J Mol Cell Cardiol. 1994;26:3-10.
Thierfelder L, Watkins H, MacRae C, Lamas R, McKenna W, Vosberg HP, Seidman JG, Seidman CE. Alpha-tropomyosin and cardiac troponin T mutations cause familial hypertrophic cardiomyopathy: a disease of the sarcomere. Cell. 1994;75:701-712.
Watkins H, Seidman CE, MacRae C, Seidman JG, McKenna W. Progress in familial hypertrophic cardiomyopathy: molecular genetic analysis in the original family studies by Teare. Br Heart J. 1992;67:34-38.
Gilligan DM, Cleland JGF, Oakley CM. The genetics of hypertrophic cardiomyopathy. Br Heart J. 1991;66:193-195.
Golf S, Myhre E, Abdelnoor M, Andersen D, Hansson V. Hypertrophic cardiomyopathy characterized by beta-andrenoceptor density, relative amount of beta-adrenoceptor subtypes and adenylate cyclase activity. Cardiovasc Res. 1985;19:693-699.
Wagner JA, Sax FL, Weisman HF, Porterfied J. Calcium-antagonist receptors in the atrial tissue of patients with hypertrophic cardiomyopathy. N Engl J Med. 1989;320:755-761.
Kardami E. Stimulation and inhibition of cardiac myocyte proliferation in vitro. Mol Cell Biochem. 1990;92:129-135.
Soonpaa MH, Oberpriller JO, Oberpriller JC. Stimulation of DNA synthesis by PDGF in the newt cardiac myocyte. J Mol Cell Cardiol. 1992;24:1039-1046.
Villarrel FJ, Lee AA, Dillmann WH, Giordano FJ. Adenovirus-mediated overexpression of human transforming growth factor-β1 in rat cardiac fibroblasts, myocytes and smooth muscle cells. J Mol Cell Cardiol. 1996;28:735-742.
Hanson MC, Fath KA, Alexander RW, Delafontaine P. Induction of cardiac insulin-like growth factor I gene expression in pressure overload hypertrophy. Am J Med Sci. 1993;306:69-74.
Padua R, Kardami E. Increased basic fibroblast growth factor accumulation and distinct patterns of localization in isoproterenol-induced cardiomyocyte injury. Growth Factor. 1993;8:291-306.
Takahashi N, Calderone A, Izzo NJ Jr, Maki TM, Marsh JD, Colucci WS. Hypertrophic stimuli induce transforming growth factor-beta 1 expression in rat ventricular cardiomyocytes. J Clin Invest. 1994;94:1470-1476.
Villarrel FJ, Dillmann WH. Cardiac hypertrophy-induced changes in mRNA levels for TGF-beta 1, fibronectin, and collagen. Am J Physiol. 1992;262:H1861-H1866.
Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press; 1989:14.5-14.20.
Zerbini M, Musiani M, Gibellini D, Gentilomi G, Venturoli S, Gallinella G, La Placa M. Evaluation of strand-specific RNA probes visualized by colorimetric and chemiluminescent reactions for the detection of B19 parvovirus DNA. J Virol Meth. 1993;45:169-178.
Hsu SM, Raine L, Fanger H. The use of antiavidin antibody and avidin-biotin-peroxidase complex in immunoperoxidase technics. Am J Clin Pathol. 1981;75:816-821.
Bjarnason I, Jonsson S, Hardarson T. Mode of inheritance of hypertrophic cardiomyopathy in Iceland: echocardiographic study. Br Heart J. 1982;47:122-129.
Codd MB, Sugrue DD, Gersh BJ, Melton CJ. Epidemiology of idiopathic dilated and hypertrophic cardiomyopathy: a population-study of Olmsted county, Minnesota, 1975-1984. Circulation. 1989;80:564-572.
Spirito P, Chiarella F, Carratino L, Berisso MZ, Bellotti P, Vecchio C. Clinical course and prognosis of hypertrophic cardiomyopathy in an outpatient population. N Engl J Med. 1989;320:749-755.
Wigle ED, Rakowski H, Kimball BP, Williams WG. Hypertrophic cardiomyopathy: clinical spectrum and treatment. Circulation. 1995;92:1680-1692.
Maron BJ, Epstein SE. Hypertrophic cardiomyopathy: a discussion of nomenclature. Am J Cardiol. 1979;43:1242-1244.
Maron BJ, Roberts WC. Hypertrophic cardiomyopathy. In: Schlant RC, Alexander RW, eds. The Heart: Arteries and Veins. 8th ed. New York, NY: McGraw-Hill Health Professions Division; 1994:1621-1635.
Factor SM, Butany J, Sole MJ, Wigle D, Williams WC, Rojkind M. Pathologic fibrosis and matrix connective tissue in the subaortic myocardium of patients with hypertrophic cardiomyopathy. J Am Coll Cardiol. 1991;17:1343-1351.
Engelman GL, Boehm KD, Haskell JF, Khairallah PA, Ilan J. Insulin-like growth factors and neonatal cardiomyocyte development: ventricular gene expression and membrane receptor variations in hormotensive and hypertensive rats. Mol Cell Endocrinol. 1989;63:1-14.
Reiss K, Kajstura J, Capasso JM, Marino TM, Anversa P. Impairment of myocyte contractility following coronary artery narrowing is associated with activation of the myocyte IGF-1 autocrine system, enhanced expression of late growth related genes, DNA synthesis, and myocyte nuclear mitotic division in rats. Exp Cell Res. 1994;207:348-360.
Toyozaki T, Hiroe M, Hasumi M, Hosoda S, Tsushima T, Sekiguchi M. Insulin-like growth factor I receptors in human cardiac myocytes and their relation to myocardial hypertrophy. Jpn Circ J. 1993;57:1120-1127.
Decker RS, Cook MG, Behnke-Barclay M, Decker ML. Some growth factors stimulate cultured adult rabbit ventricular myocyte hypertrophy in the absence of mechanical loading. Circ Res. 1995;77:544-555.
Adachi S, Ito H, Akimoto H, Tanaka M, Fujisaki H, Marumo F, Hiroe M. Insulin-like growth factor-II induces hypertrophy with increased expression of muscle specific genes in cultured rat cardiomyocytes. J Mol Cell Cardiol. 1994;26:789-795.
Fuller SJ, Mynett JR, Sugden PH. Stimulation of cardiac protein synthesis by insulin-like growth factors. Biochem J. 1992;282:85-90.
Ito H, Hiroe M, Hirara Y, Tsujino M, Adachi S, Shichiri M, Koike A, Nogami A, Marumo F. Insulin-like growth factor-I induces hypertrophy with enhanced expression of muscle specific genes in cultured rat cardiomyocytes. Circulation. 1993;87:1715-1721.
Diez J, Laviades C. Insulin-like growth factor-1 and cardiac mass in essential hypertension: comparative effects of captopril, lisinopril and quinapril. J Hypertens. 1994;12:s31-s36.
Amato G, Carella C, Fazio S, Montagna GL, Cittadini A, Sabatini D, Mariano-Mone C, Sacca L, Bellastella A. Body composition, bone metabolism, and heart structure and function in growth hormone (GH)-deficient adults before and after GH replacement therapy at low dose. J Clin Endocrinol Metab. 1993;77:1671-1676.
Pricci F, Pugliese G, Romano G, Romeo G, Locuratolo N, Pugliese F, Mene P, Galli G, Casini A, Rotella CM, Mario UD. Insulin-like growth factor I and II stimulate extracellular matrix production in human glomerular mesangial cells: comparison with transforming growth factor-beta. Endocrinology. 1996;137:879-885.
Sakata Y. Tissue factors contributing to cardiac hypertrophy in cardiomyopathic hamsters (BIO14.6): involvement of transforming growth factor-beta 1 and tissue. Hokkaido Igaku Zasshi-Hokkaido J Med Sci. 1993;68:18-28.
Eghbali M, Tomek R, Woods C, Bhambi B. Cardiac fibroblasts are predisposed to convert into myocyte phenotype: specific effect of transforming growth factor β. Proc Natl Acad Sci U S A. 1991;88:795-799.