Heart Myosin Research Articles

Generation of a homozygous MYH7 gene knockout human embryonic stem cell line (WAe009-A-69) using an episomal vector-based CRISPR/Cas9 system

Myosin heavy chain 7 (MYH7) encodes the human heart myosin heavy chain subunit, which plays an important role in myocardial contraction. MYH7 is the main pathogenic gene that causes Hypertrophic cardiomyopathy (HCM) and Dilated cardiomyopathy (DCM). In this experiment, a MYH7 homozygous knockout human embryonic stem cell (hESC) line, WAe009-A-69, was generated using an episomal vector-based CRISPR/Cas9 system. It can be an ideal tool to further study the function of MYH7. The cell line was confirmed with pluripotency, normal karyotype and trilineage differentiation potential.

Influence of Chronic Lead Intoxication on Functional Characteristics and Isoform Composition of Left Ventricular Myosin in the Rat Heart

Influence of Chronic Lead Intoxication on Functional Characteristics and Isoform Composition of Left Ventricular Myosin in the Rat Heart

Autoimmunity against Cardiac Self-Antigen Drives Immune Checkpoint Inhibitor-Associated Myocarditis Development

Abstract Immune checkpoint inhibitor (ICI) blocking PD-1 has been approved by the Food and Drug Administration for treatment of several types of cancers. However, fulminant myocarditis has occurred in the ICI-treated cancer patients, with low incidence but with 50 percent mortality. In this study, we investigated if PD-1 blockade can induce myocarditis in the mouse mimicking ICI-associated myocarditis in the human. We found that anti-PD-1 mAb treatment caused myocarditis development in A/J mice exhibiting enlarged heart and increased cardiac infiltration. In the cardiac infiltration, CD8+ T cells, CD4+ T cells, monocytes and macrophages were increased in the PD-1 blockade group compared to isotype control. Cardiac damage was confirmed by elevated serum level of cardiac troponin I and anti-myosin IgG. In addition, PD-1 blockade caused arrhythmia, right bundle branch block, while controls showed a normal ECG trace. The A/J mice with PD-1 blockade also developed systemic autoimmune responses such as colitis, sialadenitis, pneumonitis and myositis like immune-related adverse events in the human, supporting the immunotoxicity of PD-1 blockade. Next, using myosin-MHC tetramer staining, we found the increase of autoreactive T cells specific for cardiac myosin in the mouse heart with ICI-associated myocarditis compared to control. In the naïve heart, we were able to detect myosin-specific T cells, which number was very few but expressed a high level of PD-1. Collectively, we have developed the mouse model for human ICI-associated myocarditis and showed that the pathogenesis of this disease is an autoimmune reaction against heart-specific antigens.

Improvement in cardiac function of ovariectomized rats by antioxidant tempol

A rise in heart disease incidence in women after menopause has led to investigations into the role of female sex hormones on cardiac function. Although various adverse changes in cardiac contractile function following loss of female sex hormones have been reported, a clear mechanism of action has never been characterized. In order to examine whether an elevation in oxidative stress is a major cause of cardiac contractile dysfunction after female sex hormone deprivation, cardiac functions of ovariectomized rats with and without supplementation of superoxide scavenger tempol were compared to those of sham-operated controls. Chronic deprivation of female sex hormones reduced total oxidative capacity and increased plasma carbonyl protein content. Tempol supplementation of ovariectomized rats significantly ameliorated plasma oxidative stress status. Echocardiography demonstrated a significant decrease in left ventricular ejection fraction in ovariectomized rats, which was completely prevented by tempol supplementation. Decreased myocardial contractility occurs with reduced maximum myofilament force of contraction and amplitude of transient intracellular Ca2+ concentration, both phenomena completely attenuated by tempol supplementation. However, tempol only partially prevented shift of heart myosin heavy chain from dominant α-to β-isoform of ovariectomized rats. Immunoblot analysis of protein carbonylation indicated that tempol supplementation significantly reduced the level of cardiac myofibrillar proteins oxidation increased in ovariectomized rat heart. Taken together, the results indicate changes of cardiac contractile machinery following loss of female sex hormones were, in part, due to an increase in oxidative stress, and antioxidant supplementation could be considered another potential prevention measure in postmenopausal women.

Allele-Selective Knockdown of MYH7 Using Antisense Oligonucleotides

Hundreds of dominant-negative myosin mutations have been identified that lead to hypertrophic cardiomyopathy, and the biomechanical link between mutation and disease is heterogeneous across this patient population. To increase the therapeutic feasibility of treating this diverse genetic population, we investigated the ability of locked nucleic acid (LNA)-modified antisense oligonucleotides (ASOs) to selectively knock down mutant myosin transcripts by targeting single-nucleotide polymorphisms (SNPs) that were found to be common in the myosin heavy chain 7 (MYH7) gene. We identified three SNPs in MYH7 and designed ASO libraries to selectively target either the reference or alternate MYH7 sequence. We identified ASOs that selectively knocked down either the reference or alternate allele at all three SNP regions. We also show allele-selective knockdown in a mouse model that was humanized on one allele. These results suggest that SNP-targeting ASOs are a promising therapeutic modality for treating cardiac pathology.

O-Myo! An O-Shaped Myosin Gliding Assay for Characterizing Long-Term Actin-Myosin Behaviors

Nature has evolved molecular machines that are critical in cellular processes occurring over broad timescales, ranging from seconds to days. As an example, myosin in the human heart take ∼10 steps per second and are collectively responsible for ∼10⁵ heart beats per day. Analogous to the engineering of macro-scale machines, the evolution of molecular machines is likely to be constrained by the requirements of high short-term performance and a long lifetime. Here, we developed a circular gliding assay (O-Myo) that utilizes engineered micron-scale DNA nanotube rings with defined geometrical arrangements of dimeric myosin VI, a model system for myosin motor.

Pharmacotherapy of dilated cardiomyopathy.

The pharmacological treatment of dilated cardiomyopathy overlaps with the treatment of heart failure. The primary objective of this treatment is to slow the progression of disease and improve quality and length of life. All patients, including those with asymptomatic dysfunction of the left ventricle, ought to receive angiotensin converting enzyme inhibitors, (in the case of intolerance, angiotensin receptor blockers), and beta blockers. The results of studies involving aliskiren have been, so far, disappointing. In symptomatic heart failure NYHA II-IV diuretics and mineralcorticoid receptor antagonists should be added to treatment. Digoxin is recommended in the event of atrial fibrillation, and otherwise only in the event of NYHA III and IV. Ivabradine is recommended for patients with sinus rhythm and pulse rate of > 70/min. In decompensation of heart failure, dobutamine, phosphodiesterase inhibitors or levosimendan are administered over the short-term. Of the recent treatment options, the vasopressin blocker and adenosine A1 receptor antagonist (rolofylline) were disappointing. One treatment with potential for the future is omecamtiv mecarbil, a heart myosin activator.

Synthesis and 18F-labeling of the analogues of Omecamtiv Mecarbil as a potential cardiac myosin imaging agent with PET

Cardiac myosin is a potential molecular target for heart failure imaging since its changes can be used to assess the function of heart. In this study, two analogues of Omecamtiv Mecarbil, which is the first selective activator of cardiac myosin, were synthesized and radio-labeled with (18)F. Then the radio-compounds were evaluated as potential cardiac myosin imaging agent. The labeling precursor and the nonradioactive compounds were synthesized and characterized by IR, (1)H NMR, (13)C NMR and MS analysis. By substituting bromo of precursors with (18)F, the radiolabeled compounds [(18)F]8 and [(18)F]10 were prepared and further evaluated for their in vitro physicochemical properties, stabilities, protein binding assay and ex vivo biodistribution. Starting with [(18)F]F(-) Kryptofix 2.2.2./K2CO3 solution, the total reaction time for [(18)F]8 and [(18)F]10 was about 40 min respectively, with final high-performance liquid chromatography purification included. Typical decay-corrected radiochemical yield stayed at 12.47% ± 3.30% (n=8), the radiochemical purity, 98% or more. Their specific activity was estimated as 50 GBq/μmol. Both [(18)F]8 and [(18)F]10 could be stable after incubation in water at room temperature and in serum or binding buffer at 37 °C for 3h. Biodistribution in normal mice showed that both [(18)F]8 and [(18)F]10 have good heart uptake at 2 min post-injection time. Compound [(18)F]10 has better heart retention and higher heart to background ratios than those of [(18)F]8. In vitro protein binding assay demonstrates that [(18)F]10 may have high affinity with myosin from bovine heart. [(18)F]8 and [(18)F]10 were synthesized with good radiochemical yield and high radiochemical purity (>98%). One of the compounds ([(18)F]10) has higher bovine heart myosin binding affinity and better heart/liver ratio. It will be further evaluated as a potent cardiac myosin imaging agent in normal and systolic heart failure model with positron emission tomography in the future.

Detecting a Broken Heart

Iron oxide nanoparticles allow MRI visualization of heart inflammation, facilitating noninvasive diagnosis of myocarditis.

GM-CSF mediates autoimmunity by enhancing IL-6–dependent Th17 cell development and survival

Granulocyte macrophage–colony stimulating factor (GM-CSF) is critically involved in development of organ-related autoimmune inflammatory diseases including experimental allergic encephalitis and collagen-induced arthritis. Roles of GM-CSF in the initiation and in the effector phase of the autoimmune response have been proposed. Our study was designed to investigate the mechanisms of GM-CSF in autoimmunity using a model of autoimmune heart inflammatory disease (myocarditis). The pathological sequel after immunization with heart myosin has been shown previously to depend on IL-1, IL-6, IL-23, and IL-17. We found that innate GM-CSF was critical for IL-6 and IL-23 responses by dendritic cells and generation of pathological Th17 cells in vivo. Moreover, GM-CSF promoted autoimmunity by enhancing IL-6–dependent survival of antigen specific CD4+ T cells. These results suggest a novel role for GM-CSF in promoting generation and maintenance of Th17 cells by regulation of IL-6 and IL-23 in vivo.

Persistence of virus and viral genome in myocardium after coxsackievirus B3-induced murine myocarditis.

Following infection with Coxsackievirus B3 (CVB3), A-strain mice develop ongoing myocarditis that persists after the virus ceases to be cultivatable from heart tissue. We studied the natural history of this virus-induced but apparently autoimmune inflammation by means of in situ hybridization (ISH) and by polymerase chain reaction (PCR). Both ISH and culture allowed detection of virus up to 2 weeks post-infection in virtually all heart tissues. In contrast, PCR revealed the presence of viral genome for a substantially longer period of time, i.e. at least 34 days after CVB3 infection. Similarly, the majority of mice showed myocardial inflammation at this time point. However, the persistence of virus did not correlate with ongoing myocarditis, and vice versa. Most mice with ongoing myocarditis produced heart myosin autoantibodies, most probably as a result of tissue damage. The lack of correlation between presence of ongoing inflammation and persistence of virus supports our previous view that the late phase of CVB3-induced myocarditis is mediated by autoimmunological mechanisms.

The molecular phenotype of human cardiac myosin associated with hypertrophic obstructive cardiomyopathy

AimThe aim of the study was to compare the functional and structural properties of the motor protein, myosin, and isolated myocyte contractility in heart muscle excised from hypertrophic cardiomyopathy patients by surgical myectomy with explanted failing heart and non-failing donor heart muscle.MethodsMyosin was isolated and studied using an in vitro motility assay. The distribution of myosin light chain-1 isoforms was measured by two-dimensional electrophoresis. Myosin light chain-2 phosphorylation was measured by sodium dodecyl sulphate–polyacrylamide gel electrophoresis using Pro-Q Diamond phosphoprotein stain.ResultsThe fraction of actin filaments moving when powered by myectomy myosin was 21% less than with donor myosin (P = 0.006), whereas the sliding speed was not different (0.310 ± 0.034 for myectomy myosin vs. 0.305 ± 0.019 µm/s for donor myosin in six paired experiments). Failing heart myosin showed 18% reduced motility. One myectomy myosin sample produced a consistently higher sliding speed than donor heart myosin and was identified with a disease-causing heavy chain mutation (V606M). In myectomy myosin, the level of atrial light chain-1 relative to ventricular light chain-1 was 20 ± 5% compared with 11 ± 5% in donor heart myosin and the level of myosin light chain-2 phosphorylation was decreased by 30–45%. Isolated cardiomyocytes showed reduced contraction amplitude (1.61 ± 0.25 vs. 3.58 ± 0.40%) and reduced relaxation rates compared with donor myocytes (TT50% = 0.32 ± 0.09 vs. 0.17 ± 0.02 s).ConclusionContractility in myectomy samples resembles the hypocontractile phenotype found in end-stage failing heart muscle irrespective of the primary stimulus, and this phenotype is not a direct effect of the hypertrophy-inducing mutation. The presence of a myosin heavy chain mutation causing hypertrophic cardiomyopathy can be predicted from a simple functional assay.

Production of monoclonal antibodies against Streptococcus mutans antigens

Several studies have been conducted in the last decades aiming to obtain an anti-caries vaccine, however some studies have demonstrated cross reactivity between Streptococcus mutans surface antigens and the human cardiac tissue. In this work, the reactivity of five anti-Streptococcus mutans monoclonal antibodies (MoAb) (24A, 56G, C8, E8 and F6) was tested against oral streptococci, cardiac antigens and skeletal and cardiac myosins, aiming to evaluate the specificity of these MoAb. The hybrid producers of immunoglobulins of the IgG2b class were cloned by limit dilution and expanded in vivo. MoAb were tested by ELISA. The hybrid 24A reacted with S. mutans CCT 1910, S. salivarius CCT 0365 and S. pyogenes T23. No reactivity difference was observed among the tested species. Cross reactivity with heart and cardiac myosin was not confirmed and only reaction with myosin of skeletal muscle was observed (p = 0.0381). The hybrid 56G reacted with all the tested microorganisms and there was statistically significant difference between S. mutans and S. pyogenes T23 (p < 0.001). This hybrid also reacted with myosin of skeletal muscle (p = 0.0095). C8, E8 and F6 presented low reactivity against oral streptococci strains and no reactivity against cardiac antigens. The data of this study showed that the 24A and 56G anti-S. mutans MoAb presented reactivity with S. pyogenes and S. salivarius, reinforcing the occurrence of common antigens between these species. The tested MoAb presented low cross-reactivity with myosin of skeletal muscle, but anti-heart activity could not be confirmed.

Neutralization of IL‐17 by active vaccination inhibits IL‐23‐dependent autoimmune myocarditis

The most common reason for heart failure in young adults is dilated cardiomyopathy often resulting from myocarditis. Clinical studies and animal models provide evidence that an autoimmune response against heart myosin is the underlying reason for the disease. IL-12 has been suggested to play a key role in development of experimental autoimmune myocarditis (EAM), as IL-12p40 and IL-12Rbeta1 knockouts are protected from disease. In this study, we have compared IL-12p40-/- mice, IL-12p35-/- mice and mice treated with a neutralizing IL-23 antibody in EAM and found that in fact IL-23, not IL-12, is responsible for inflammatory heart disease. However, these cytokines appear to have redundant activity for priming and expansion of autoreactive CD4 T cells, as specific T cell proliferation was only defective in the absence of both cytokines. IL-23 has been suggested to promote a pathogenic IL-17-producing T cell population. We targeted IL-17 by capitalizing on an active vaccination approach that effectively breaks B cell tolerance. Neutralization of IL-17 reduced myocarditis and heart autoantibody responses, suggesting that IL-17 is the critical effector cytokine responsible for EAM. Thus, targeting of IL-23 and IL-17 by passive and active vaccination strategies holds promise as a therapeutic approach to treat patients at risk for development of dilated cardiomyopathy.

Reinnervation-induced alterations in rat skeletal muscle

Denervation-induced myofiber atrophy can be reversed by reinnervation. Growing reinnervated myofibers upregulate numerous molecules, many of which determine the muscle fiber type. In the present study we aimed at identifying factors that might contribute specifically to myofiber growth after reinnervation. The common peroneal nerve of 15 male Wistar rats was cut and resutured without delay (9 animals) or with a delay of 4 weeks (6 animals). We studied the transcriptional repertoire of intact reinnervated tibialis anterior muscle by microarray gene analysis. We assessed SC activation by immunolabeling using anti-MyoD and -myogenin antibodies. The percentage of SC expressing MyoD reached up to 50% of M-cadherin+ cells whereas the percentage of SC expressing myogenin was normal (<10%) in all muscles examined. The values of ipsi- and contralateral muscles did not differ significantly from one another between right and left leg (p<0.05). Thirteen known genes were differentially regulated after reinnervation compared with contralateral muscles. Five of them determine the slow-twitch fiber type (four and a half LIM domains 3, cardiac beta-myosin heavy chain, calsequestrin 2, troponin C (slow), and heart myosin light chain), and three of them are neurally regulated (thrombospondin 4, transferrin receptor, cardiac ankyrin repeat protein). The results strengthen the notion that reinnervaton affects the molecular repertoire of the myofibers directly, leading to fiber type transformation and partial reversal of the denervation phenotype. By contrast, SC do not appear to be affected by reinnervation directly. They can be activated both in reinnervated and contralateral muscles, and they do not fully differentiate. This makes them unlikely to contribute to myofiber growth.

Immune response to a major Trypanosoma cruzi antigen, cruzipain, is differentially modulated in C57BL/6 and BALB/c mice

BALB/c mice immunized with cruzipain, a major Trypanosoma cruzi antigen, produce specific and autoreactive immune responses against heart myosin, associated with cardiac functional and structural abnormalities. Preferential activation of the Th2 phenotype and an increase in cell populations expressing CD19 +, Mac-1 + and Gr-1 + markers were found in the spleens of these mice. The aim of the present study was to investigate whether cardiac autoimmunity could be induced by cruzipain immunization of C57BL/6 mice and to compare the immune response elicited with that of BALB/c mice. We demonstrate that immune C57BL/6 splenocytes, re-stimulated in vitro with cruzipain, produced high levels of IFNγ and low levels of IL-4 compatible with a Th1 profile. In contrast to BALB/c mice, spleens from cruzipain immune C57BL/6 mice revealed no significant changes in the number of cells presenting CD19 +, Mac-1 + and Gr-1 + markers. An increased secretion of TGFβ and a greater number of CD4 + TGFβ + cells were found in immune C57BL/6 but not in BALB/c mice. These findings were associated with the lack of autoreactive response against heart myosin and a myosin- or cruzipain-derived peptide. Thus, the differential immune response elicited in C57BL/6 and BALB/c mice upon cruzipain immunization is implicated in the resistance or pathogenesis of experimental Chagas’ disease.

Cardiac myosin phosphatase could modulate LV function

Phosphorylation of myosin in heart seems to play physiological roles including Ca 2+ sensitization of contraction, torsion and hypertrophy. MYPT2, a cardiac specific target subunit of myosin phosphatase (MP), complexes with type 1 phosphatase catalytic subunit (PP1c) and possibly with small regulatory subunit M21, thereby forms cardiac MP holoenzyme. To clarify functional roles of MYPT2 in heart, we generated transgenic mice (Tg) overexpressing MYPT2 using α-MHC promoter. Western blot analyses of Tg heart demonstrated an increased expression of MYPT2 with a concomitant increase of the endogenous PP1cδ-isoform. However, the expressions of other PP1c isoforms and M21 were not significantly affected. These results suggest the specific in vivo interaction of MYPT2 with PP1cδ, which was stabilized by MYPT2 overexpression. Survival rate, body weight, heart rate and blood pressure were not significantly different between Tg and wild type (WT). Heart weight/body weight ratio of Tg was about 1.4 fold higher than that of WT (0.61 ± 0.08 vs 0.49 ± 0.05 %, p < 0.01). Echocardiography demonstrated the dilation of LV dimension (LVDd; 3.1 ± 0.5 vs 2.6 ± 0.3 mm, LVDs; 1.91 ± 0.18 vs 1.34 ± 0.05 mm, p < 0.05) and the moderate impairment of LV function (FS; 0.39 ± 0.10 vs 0.48 ± 0.05, p < 0.05) in Tg as compared with WT. These results suggest that the overexpression of MP decreased LV contractility, resulting in mild LV dilatation. Taken together, MP seems to play a role in cardiac function in vivo.

Tuning the human heart molecular motors by myosin light chains.

Cardiac contraction is triggered by the cyclic interaction of the "molecular motor" protein myosin with the actin filament, consuming ATP as the energy source to produce tension or shortening. The myosin heavy chain (MHC) contains the actin- and ATP-binding sites and represents the molecular motor of muscle contraction. This review describes the various subunits of human heart myosin in health and disease and discusses their functions. Two different MHC genes (alpha and beta) with distinct biochemical features are expressed in the human heart. Alpha-MHC confers a higher ATPase activity and higher shortening velocity to the heart than beta-MHC. Motor function is regulated by myosin light chain (MLC) isoforms. Expression of the atrial MLC-1 isoform in the hypertrophied human ventricle increases cross-bridge cycling and contractility. It is suggested that MLC-1 acts as a MHC/actin tether. Weakening of this tether increases myosin function. MLC-2 slows the rate of tension development of myosin. This relative inhibition is relieved upon phosphorylation of the MLC-2 perhaps caused by "swing-out" of cross-bridges from the myosin filament. Mutations in all ventricular myosin subunits have been found in patients with hypertrophic cardiomyopathy.

The Fas/Fas Ligand System Is Involved in the Pathogenesis of Autoimmune Myocarditis in Rats

The mechanisms responsible for myocardial injury and cell death in myocarditis are still unclear. We examined whether myocardial cell death occurs via apoptosis in myosin-induced autoimmune myocarditis in rats and whether the Fas/Fas ligand (FasL) system plays a role in this apoptosis. On days 14, 17, 21, and 35 after immunization with porcine heart myosin, some cardiomyocytes and infiltrating lymphocytes were found to be apoptotic on in situ terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end-labeling (TUNEL) assay, but none was on day 60 and in control rats. Apoptotic indices peaked at day 17, and laddering of genomic DNA from the affected myocardium was observed on days 17 and 21 on agarose gel electrophoresis. The expression of Fas mRNA and protein was detected on days 17 and 21 in some cardiomyocytes and infiltrating lymphocytes by Northern blot analysis and immunohistochemistry, respectively. In addition, FasL was detected in some infiltrating lymphocytes on days 14, 17, and 21 by both in situ hybridization and immunostaining, and FasL-positive lymphocytes were mainly CD4+ cells. Some rats were injected with anti-Fas Ab (0.1 mg/kg) or anti-FasL Ab (0.1 mg/kg), and subsequently, inflammatory lesions exhibited less severe than did untreated rats with myocarditis. These findings suggest that cell death via apoptosis of cardiomyocytes and lymphocytes is one of the mechanisms of myocardial injury in autoimmune myocarditis, and that the Fas/FasL system might play a role in the induction of this apoptosis.

Expression of the Aryl Hydrocarbon Receptor (AhR) and AhR Nuclear Translocator during Chick Cardiogenesis Is Consistent with 2,3,7,8-Tetrachlorodibenzo-p-dioxin-Induced Heart Defects

We examined cardiotoxicity induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in the chick embryo and the cardiac expression of transcription factors, the aryl hydrocarbon receptor (AhR) which binds TCDD, and its dimer partner, the AhR nuclear translocator (Arnt). Chicken eggs were injected with control (triolein) or 1.0 pmol TCDD/g egg prior to incubation and collected on Day 10 when cardiomorphogenesis is complete. Relative to controls, TCDD increased heart wet weight (27.2 ± 0.5 versus 36.6 ± 1.3 mg,p< 0.001) and dry weight (2.7 ± 0.1 versus 3.1 ± 0.1 mg,p< 0.01), and tended to increase heart myosin content (3.5 ± 0.6 versus 6.3 ± 2.5 μg,p< 0.07), suggesting an increase in cardiac muscle mass and edema. Histologic and morphometric analyses revealed that 10/13 TCDD-exposed hearts exhibited enlarged right and left ventricles, thickened ventricular septum, and a thinner left ventricular wall with increased trabeculation, and 4/13 exhibited ventricular septal defects compared to controls (0/23). To evaluate AhR and Arnt expression, untreated chick embryos were collected on Days 2.2, 3, 4, 5, and 8 of incubation, preserved in Bouin's fixative, sectioned, and stained with AhR and Arnt antibodies. The AhR was expressed ubiquitously in cardiac myocytes, while Arnt expression was restricted to myocytes overlying developing septa: atrioventricular canal, outflow tract, and atrial and ventricular septa. Both proteins were absent from endocardium and endocardial-derived mesenchyme. In addition, cardiac expression of an AhR/Arnt target, cytochrome P4501A1, was restricted to myocardium coexpressing AhR and Arnt. Thus, the spatial and temporal expression of AhR and Arnt suggests that the developing myocardium and cardiac septa are potential targets of TCDD-induced teratogenicity, and such targets are also consistent with cardiac hypertrophy and septal defects observed following TCDD exposure.