Angiotensin II type1a receptor gene expression in the heart: AP-1 and GATA-4 participate in the response to pressure overload

Hypertrophy of mammalian cardiac muscle is mediated, in part, by angiotensin II through an angiotensin II type1a receptor (AT1aR)-dependent mechanism. To understand how the level of AT1aRs is altered in this pathological state, we studied the expression of an injected AT1aR promoter-luciferase reporter gene in adult rat hearts subjected to an acute pressure overload by aortic coarctation. This model was validated by demonstrating that coarctation increased expression of the α-skeletal actin promoter 1.7-fold whereas the α-myosin heavy chain promoter was unaffected. Pressure overload increased expression from the AT1aR promoter by 1.6-fold compared with controls. Mutations introduced into consensus binding sites for AP-1 or GATA transcription factors abolished the pressure overload response but had no effect on AT1aR promoter activity in control animals. In extracts from coarcted hearts, but not from control hearts, a Fos-JunB-JunD complex and GATA-4 were detected in association with the AP-1 and GATA sites, respectively. These results establish that the AT1aR promoter is active in cardiac muscle and its expression is induced by pressure overload, and suggest that this response is mediated, in part, by a functional interaction between AP-1 and GATA-4 transcription factors.

Transient cardiac expression of constitutively active Gαq leads to hypertrophy and dilated cardiomyopathy by calcineurin-dependent and independent pathways

Cardiac hypertrophy and dilatation can result from stimulation of signal transduction pathways mediated by heterotrimeric G proteins, especially Gq, whose α subunit activates phospholipase Cβ (PLCβ). We now report that transient, modest expression of a hemagglutinin (HA) epitope-tagged, constitutively active mutant of the Gq α subunit (HAα*q) in hearts of transgenic mice is sufficient to induce cardiac hypertrophy and dilatation that continue to progress after the initiating stimulus becomes undetectable. At 2 weeks, HAα*q protein is expressed at less than 50% of endogenous αq/11, and the transgenic hearts are essentially normal morphologically. Although HAα*q protein declines at 4 weeks and is undetectable by 10 weeks, the animals develop cardiac hypertrophy and dilatation and die between 8 and 30 weeks in heart failure. As the pathology develops, endogenous αq/11 rises (2.9-fold in atria; 1.8-fold in ventricles). At 2 weeks, basal PLC activity is increased 9- to 10-fold in atria but not ventricles. By 10 weeks, it is elevated in both, presumably because of the rise in endogenous αq/11. We conclude that the pathological changes initiated by early, transient HAα*q expression are maintained in part by compensatory changes in signal transduction and other pathways. Cyclosporin A (CsA) prevents hypertrophy caused by activation of calcineurin [Molkentin, J. D., Lu, J.-R., Antos, C. L., Markham, B., Richardson, J., Robbins, J., Grant, S. R. & Olson, E. N. (1998) Cell 93, 215–228]. Because HAα*q acts upstream of calcineurin, we hypothesized that HAα*q might initiate additional pathways leading to hypertrophy and dilatation. Treating HAα*q mice with CsA diminished some, but not all, aspects of the hypertrophic phenotype, suggesting that multiple pathways are involved.

Nonmuscle myosin II-B is required for normal development of the mouse heart

We used targeted gene disruption in mice to ablate nonmuscle myosin heavy chain B (NMHC-B), one of the two isoforms of nonmuscle myosin II present in all vertebrate cells. Approximately 65% of the NMHC-B−/− embryos died prior to birth, and those that were born suffered from congestive heart failure and died during the first day. No abnormalities were detected in NMHC-B+/− mice. The absence of NMHC-B resulted in a significant increase in the transverse diameters of the cardiac myocytes from 7.8 ± 1.8 μm (right ventricle) and 7.8 ± 1.3 μm (left ventricle) in NMHC-B+/+ and B+/− mice to 14.7 ± 1.1 μm and 13.8 ± 2.3 μm, respectively, in NMHC-B−/− mice (in both cases, P < 0.001). The increase in size of the cardiac myocytes was seen as early as embryonic day 12.5 (4.5 ± 0.2 μm for NMHC-B+/+ and B+/− vs. 7.2 ± 0.6 μm for NMHC-B−/− mice (P < 0.01)). Six of seven NMHC-B−/− newborn mice analyzed by serial sectioning also showed structural cardiac defects, including a ventricular septal defect, an aortic root that either straddled the defect or originated from the right ventricle, and muscular obstruction to right ventricular outflow. Some of the hearts of NMHC-B−/− mice showed evidence for up-regulation of NMHC-A protein. These studies suggest that nonmuscle myosin II-B is required for normal cardiac myocyte development and that its absence results in structural defects resembling, in part, two common human congenital heart diseases, tetralogy of Fallot and double outlet right ventricle.

Hypertension, cardiac hypertrophy, and sudden death in mice lacking natriuretic peptide receptor A

Natriuretic peptides, produced in the heart, bind to the natriuretic peptide receptor A (NPRA) and cause vasodilation and natriuresis important in the regulation of blood pressure. We here report that mice lacking a functional Npr1 gene coding for NPRA have elevated blood pressures and hearts exhibiting marked hypertrophy with interstitial fibrosis resembling that seen in human hypertensive heart disease. Echocardiographic evaluation of the mice demonstrated a compensated state of systemic hypertension in which cardiac hypertrophy and dilatation are evident but with no reduction in ventricular performance. Nevertheless, sudden death, with morphologic evidence indicative in some animals of congestive heart failure and in others of aortic dissection, occurred in all 15 male mice lacking Npr1 before 6 months of age, and in one of 16 females in our study. Thus complete absence of NPRA causes hypertension in mice and leads to cardiac hypertrophy and, particularly in males, lethal vascular events similar to those seen in untreated human hypertensive patients.

Signal transducer and activator of transcription 3 in the heart transduces not only a hypertrophic signal but a protective signal against doxorubicin-induced cardiomyopathy

The signal transducer and activator of transcription (STAT) 3, a transcriptional factor downstream of several cytokines, is activated by Janus kinase families and plays a pivotal role in cardiac hypertrophy through gp130. To determine the physiological significance of STAT3 in vivo, transgenic mice with cardiac-specific overexpression of the Stat3 gene (STAT3-TG) were generated. STAT3-TG manifested myocardial hypertrophy at 12 wk of age with increased expression of the atrial natriuretic factor (ANF), β-myosin heavy chain (MHC), and cardiotrophin (CT)-1 genes. The animals were injected i.p. with 15 mg/kg doxorubicin (Dox), an antineoplastic drug with restricted use because of its cardiotoxicity. The survival rates after 10 days were 25% (5/20) for control littermates (WT), but 80% (16/20) for STAT3-TG (P < 0.01). WT showed increased expression of β-MHC and ANF mRNAs in the hearts 1 day after Dox treatment; this expression peaked at 3 days, suggesting that the WT suffered from congestive heart failure. Although the expression of these mRNAs was elevated in STAT3-TG hearts before Dox treatment, no additional increase was observed after the treatment. Dox administration significantly reduced the expression of the cardiac α-actin and Stat3 genes in WT hearts but not in STAT3-TG. These results provide direct evidence that STAT3 transduces not only a hypertrophic signal but also a protective signal against Dox-induced cardiomyopathy by inhibiting reduction of cardiac contractile genes and inducing cardiac protective factors.

Low- and high-level transgenic expression of β2-adrenergic receptors differentially affect cardiac hypertrophy and function in Gαq-overexpressing mice

Transgenic overexpression of Gαq in the heart triggers events leading to a phenotype of eccentric hypertrophy, depressed ventricular function, marked expression of hypertrophy-associated genes, and depressed β-adrenergic receptor (βAR) function. The role of βAR dysfunction in the development of this failure phenotype was delineated by transgenic coexpression of the carboxyl terminus of the βAR kinase (βARK), which acts to inhibit the kinase, or concomitant overexpression of the β2AR at low (≈30-fold, Gαq/β2ARL), moderate (≈140-fold, Gαq/β2ARM), and high (≈1,000-fold, Gαq/β2ARH) levels above background βAR density. Expression of the βARK inhibitor had no effect on the phenotype, consistent with the lack of increased βARK levels in Gαq mice. In marked contrast, Gαq/β2ARL mice displayed rescue of hypertrophy and resting ventricular function and decreased cardiac expression of atrial natriuretic factor and α-skeletal actin mRNA. These effects occurred in the absence of any improvement in basal or agonist-stimulated adenylyl cyclase (AC) activities in crude cardiac membranes, although restoration of a compartmentalized β2AR/AC signal cannot be excluded. Higher expression of receptors in Gαq/β2ARM mice resulted in salvage of AC activity, but hypertrophy, ventricular function, and expression of fetal genes were unaffected or worsened. With ≈1,000-fold overexpression, the majority of Gαq/β2ARH mice died with cardiomegaly at 5 weeks. Thus, although it appears that excessive, uncontrolled, or generalized augmentation of βAR signaling is deleterious in heart failure, selective enhancement by overexpressing the β2AR subtype to limited levels restores not only ventricular function but also reverses cardiac hypertrophy.

A genetic model provides evidence that the receptor for atrial natriuretic peptide (guanylyl cyclase-A) inhibits cardiac ventricular myocyte hypertrophy

Guanylyl cyclase-A (NPR-A; GC-A) is the major and possibly the only receptor for atrial natriuretic peptide (ANP) or B-type natriuretic peptide. Although mice deficient in GC-A display an elevated blood pressure, the resultant cardiac hypertrophy is much greater than in other mouse models of hypertension. Here we overproduce GC-A in the cardiac myocytes of wild-type or GC-A null animals. Introduction of the GC-A transgene did not alter blood pressure or heart rate as a function of genotype. Cardiac myocyte size was larger (approximately 20%) in GC-A null than in wild-type animals. However, introduction of the GC-A transgene reduced cardiac myocyte size in both wild-type and null mice. Coincident with the reduction in myocyte size, both ANP mRNA and ANP content were significantly reduced by overexpression of GC-A, and this reduction was independent of genotype. This genetic model, therefore, separates a regulation of cardiac myocyte size by blood pressure from local regulation by a GC-mediated pathway.

Targeted inhibition of calcineurin attenuates cardiac hypertrophy in vivo

The Ca2+-calmodulin-activated Ser/Thr protein phosphatase calcineurin and the downstream transcriptional effectors of calcineurin, nuclear factor of activated T cells, have been implicated in the hypertrophic response of the myocardium. Recently, the calcineurin inhibitory agents cyclosporine A and FK506 have been extensively used to evaluate the importance of this signaling pathway in rodent models of cardiac hypertrophy. However, pharmacologic approaches have rendered equivocal results necessitating more specific or genetic-based inhibitory strategies. In this regard, we have generated Tg mice expressing the calcineurin inhibitory domains of Cain/Cabin-1 and A-kinase anchoring protein 79 specifically in the heart. ΔCain and ΔA-kinase-anchoring protein Tg mice demonstrated reduced cardiac calcineurin activity and reduced hypertrophy in response to catecholamine infusion or pressure overload. In a second approach, adenoviral-mediated gene transfer of ΔCain was performed in the adult rat myocardium to evaluate the effectiveness of an acute intervention and any potential species dependency. ΔCain adenoviral gene transfer inhibited cardiac calcineurin activity and reduced hypertrophy in response to pressure overload without reducing aortic pressure. These results provide genetic evidence implicating calcineurin as an important mediator of the cardiac hypertrophic response in vivo.

Myocyte-enriched calcineurin-interacting protein, MCIP1, inhibits cardiac hypertrophy in vivo

Signaling events controlled by calcineurin promote cardiac hypertrophy, but the degree to which such pathways are required to transduce the effects of various hypertrophic stimuli remains uncertain. In particular, the administration of immunosuppressive drugs that inhibit calcineurin has inconsistent effects in blocking cardiac hypertrophy in various animal models. As an alternative approach to inhibiting calcineurin in the hearts of intact animals, transgenic mice were engineered to overexpress a human cDNA encoding the calcineurin-binding protein, myocyte-enriched calcineurin-interacting protein-1 (hMCIP1) under control of the cardiac-specific, α-myosin heavy chain promoter (α-MHC). In unstressed mice, forced expression of hMCIP1 resulted in a 5–10% decline in cardiac mass relative to wild-type littermates, but otherwise produced no apparent structural or functional abnormalities. However, cardiac-specific expression of hMCIP1 inhibited cardiac hypertrophy, reinduction of fetal gene expression, and progression to dilated cardiomyopathy that otherwise result from expression of a constitutively active form of calcineurin. Expression of the hMCIP1 transgene also inhibited hypertrophic responses to β-adrenergic receptor stimulation or exercise training. These results demonstrate that levels of hMCIP1 producing no apparent deleterious effects in cells of the normal heart are sufficient to inhibit several forms of cardiac hypertrophy, and suggest an important role for calcineurin signaling in diverse forms of cardiac hypertrophy. The future development of measures to increase expression or activity of MCIP proteins selectively within the heart may have clinical value for prevention of heart failure.

Telomerase expression and activity are coupled with myocyte proliferation and preservation of telomeric length in the failing heart

The role and even the existence of myocyte proliferation in the adult heart remain controversial. Documentation of cell cycle regulators, DNA synthesis, and mitotic images has not modified the view that myocardial growth can only occur from hypertrophy of an irreplaceable population of differentiated myocytes. To improve understanding the biology of the heart and obtain supportive evidence of myocyte replication, three indices of cell proliferation were analyzed in dogs affected by a progressive deterioration of cardiac performance and dilated cardiomyopathy. The magnitude of cycling myocytes was evaluated by the expression of Ki67 in nuclei. Ki67 labeling of left ventricular myocytes increased 5-fold, 12-fold, and 17-fold with the onset of moderate and severe ventricular dysfunction and overt failure, respectively. Telomerase activity in vivo is present only in multiplying cells; this enzyme increased 2.4-fold and 3.1-fold in the decompensated heart, preserving telomeric length in myocytes. The contribution of cycling myocytes to telomerase activity was determined by the colocalization of Ki67 and telomerase in myocyte nuclei. More than 50% of Ki67-positive cells expressed telomerase in the overloaded myocardium, suggesting that these myocytes were the morphological counterpart of the biochemical assay of enzyme activity. Moreover, we report that 20–30% of canine myocytes were telomerase competent, and this value was not changed by cardiac failure. In conclusion, the enhanced expression of Ki67 and telomerase activity, in combination with Ki67-telomerase labeling of myocyte nuclei, support the notion that myocyte proliferation contributes to cardiac hypertrophy of the diseased heart.

Cystic fibrosis gene encodes a cAMP-dependent chloride channel in heart.

cAMP-dependent chloride channels in heart contribute to autonomic regulation of action potential duration and membrane potential and have been inferred to be due to cardiac expression of the epithelial cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel. In this report, a cDNA from rabbit ventricle was isolated and sequenced, which encodes an exon 5 splice variant (exon 5-) of CFTR, with >90% identity to human CFTR cDNA present in epithelial cells. Expression of this cDNA in Xenopus oocytes gave rise to robust cAMP-activated chloride currents that were absent in control water-injected oocytes. Antisense oligodeoxynucleotides directed against CFTR significantly reduced the density of cAMP-dependent chloride currents in acutely cultured myocytes, thereby establishing a direct functional link between cardiac expression of CFTR protein and an endogenous chloride channel in native cardiac myocytes.

Characterization of a voltage-gated K+ channel beta subunit expressed in human heart.

Voltage-gated K+ channels are important modulators of the cardiac action potential. However, the correlation of endogenous myocyte currents with K+ channels cloned from human heart is complicated by the possibility that heterotetrameric alpha-subunit combinations and function-altering beta subunits exist in native tissue. Therefore, a variety of subunit interactions may generate cardiac K+ channel diversity. We report here the cloning of a voltage-gated K+ channel beta subunit, hKv beta 3, from adult human left ventricle that shows 84% and 74% amino acid sequence identity with the previously cloned rat Kv beta 1 and Kv beta 2 subunits, respectively. Together these three Kv beta subunits share > 82% identity in the carboxyl-terminal 329 aa and show low identity in the amino-terminal 79 aa. RNA analysis indicated that hKv beta 3 message is 2-fold more abundant in human ventricle than in atrium and is expressed in both healthy and diseased human hearts. Coinjection of hKv beta 3 with a human cardiac delayed rectifier, hKv1.5, in Xenopus oocytes increased inactivation, induced an 18-mV hyperpolarizing shift in the activation curve, and slowed deactivation (tau = 8.0 msec vs. 35.4 msec at -50 mV). hKv beta 3 was localized to human chromosome 3 by using a human/rodent cell hybrid mapping panel. These data confirm the presence of functionally important K+ channel beta subunits in human heart and indicate that beta-subunit composition must be accounted for when comparing cloned channels with endogenous cardiac currents.

Continuous activation of gp130, a signal-transducing receptor component for interleukin 6-related cytokines, causes myocardial hypertrophy in mice.

To investigate the physiological roles of gp130 in detail and to determine the pathological consequence of abnormal activation of gp130, transgenic mice having continuously activated gp130 were created. This was carried out by mating mice from interleukin 6 (IL-6) and IL-6 receptor (IL-6R) transgenic lines. Offspring overexpressing both IL-6 and IL-6R showed constitutive tyrosine phosphorylation of gp130 and a downstream signaling molecule, acute phase response factor/signal transducer and activator of transcription 3. Surprisingly, the distinguishing feature of such offspring was hypertrophy of ventricular myocardium and consequent thickened ventricular walls of the heart, where gp130 is also expressed, in adulthood. Transgenic mice overexpressing either IL-6 or IL-6R alone did not show detectable myocardial abnormalities. Neonatal heart muscle cells from normal mice, when cultured in vitro, enlarged in response to a combination of IL-6 and a soluble form of IL-6R. The results suggest that activation of the gp130 signaling pathways leads to cardiac hypertrophy and that these signals might be involved in physiological regulation of myocardium.