1 Donald W. Reynolds Cardiovascular Clinical Research Center and 2 Division of Cardiology, Department of Internal Medicine, and 3 Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, Texas; and Departments of 4 Internal Medicine and 5 Surgery, University of Iowa Carver College of Medicine, Iowa City, Iowa
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ABSTRACT |
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signal transduction; myocardium
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INTRODUCTION |
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Hypertrophic growth of the myocardium occurs in most forms of HF and may contribute to the pathogenesis of the failure state (2, 26). Hypertrophic remodeling takes place as a response to chronic stress, such as that occuring in individuals with hypertension. Cardiac hypertrophy with concomitant ventricular dysfunction also develops as a consequence of acute-onset stress, such as after a loss of ventricular mass due to massive infarction. Little is known about the regulatory mechanisms governing the often-coexisting phenotypes of hypertrophy, systolic failure, and diastolic stiffness that characterize clinical disease.
Numerous signaling pathways have been implicated in hypertrophic transformation of the heart (for a review, see Ref. 18). Many studies have examined the association between activation of molecular signaling pathways and human HF, but, as yet, the reported findings have been incomplete and often contradictory. Thus it is not known whether activation of a distinct signaling pathway triggers a specific clinical phenotype or whether disparate degrees of activation of the same pathways are responsible for a clinical spectrum ranging from compensated hypertrophy (Comp Hyp) to decompensated HF.
The development of surgical models of cardiac pressure overload in mice has provided a platform for genetic and mechanistic studies of load-induced failure. An increase in left ventricular (LV) pressure is achieved by constriction of either the ascending, transverse or descending aorta. The magnitude of resulting afterload elevation is dependent on both the location and severity of the constriction. Unfortunately, similar techniques in different hands result in a wide spectrum of experimental outcomes ranging from Comp Hyp to decompensated HF. This has made it difficult to compare results from one group with those of another. It also suggests that relatively small differences in hemodynamic stress can result in vastly different clinical phenotypes.
We hypothesized that intracellular signaling pathways are differentially activated by graded degrees of hemodynamic stress. Furthermore, we postulated that unique profiles of stress-response signaling underlie unique phenotypes: ventricular hypertrophy with preserved systolic function (21) versus hypertrophy with ventricular dysfunction and clinical HF. To test this, we developed models of graded pressure stress in mice and used them to make a direct comparison between Comp Hyp and pressure-overload HF. Surgical interventions were designed to be similar, on either side of a threshold separating compensated from decompensated responses. Our findings revealed two dramatically different hypertrophic phenotypes with modest differences in the activation of relevant intracellular signaling pathways. Furthermore, we uncovered a functional requirement of calcineurin signaling in each model such that calcineurin suppression blunted hypertrophic growth. Remarkably, in each case, suppression of calcineurin signaling was not associated with clinical deterioration or increased mortality.
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EXPERIMENTAL PROCEDURES |
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Myocyte isolation and surface area determinations.
The surface area of a two-dimensional silhouette was estimated by measuring the length and width of 20 randomly selected myocytes. Our reported two-dimensional surface area (length x width) is directly proportional to the surface area of a cylinder (2 radius x length).
RNA dot blots.
Total RNA was isolated from the LV using TRIzol (Invitrogen) reagent. Expression of hypertrophic marker genes was assessed by hybridization of RNA dot blots as previously described (29).
Western blot analysis.
Protein lysates were subjected to polyacrylamide gel electrophoresis, transferred to polyvinylidene difluoride membranes, and subjected to immunoblot analysis. The ratio of antigen to antibody, detergent concentration, and duration and temperature of the reaction was optimized for each antibody. Data were quantified by densitometric analysis and normalized to GAPDH protein levels.
Echocardiography.
Transthoracic echocardiograms were recorded in conscious-sedated mice as described (21, 22). Briefly, views were taken in planes that approximated the parasternal short-axis view (chordal level) and the apical long-axis view in humans. LV internal diameters and wall thicknesses were measured (at least 3 cardiac cycles) at end systole and end diastole. LV mass and volumes were calculated using the area-length method (32).
Reagents.
Antibodies were purchased from Cell Signaling [ERK no. 9102, phosphorylated (p)ERK no. 9106, JNK no. 9252, pJNK no. 9251, p38 no. 9212, pp38 no. 9211, Akt no. 9272, and pAkt no. 9271], Affinity Bioreagents [calsequestrin no. PA1-913, phospholamban (PLB) MA3922], BD Biosciences [calcineuring A (CaN) no. 610259], Upstate (Ser16-pPLB 07-052), or Research Diagnostics (GAPDH no. TRK5G-6C5). Modulatory calcineuring-interacting protein 1 (MCIP1) antiserum (7) was a kind gift of Erik Bush (Myogen).
Statistical methods.
Data are reported as means ± SE. Statistical significance was analyzed using a Student's unpaired t-test or one-way ANOVA followed by Bonferroni's method for post hoc pair-wise multiple comparisons.
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RESULTS |
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Animals subjected to standard TAB were clinically indistinguishable from sham-operated controls. Measurements of heart mass (Fig. 1 and Table 1) revealed significant hypertrophic growth. In contrast, mice subjected to severe TAB gradually developed signs of circulatory failure, including lethargy, impaired mobility, and diminished appetite. Hearts dissected from these mice were markedly enlarged and dilated, and morphometric analysis revealed significant increases in heart mass (Fig. 1).
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Circulating levels of tumor necrosis factor (TNF) and other cytokines are frequently increased in patients with HF, a response that has been implicated in the pathophysiology of the disease (13). Consistent with a clinical diagnosis of HF in severe TAB mice, levels of circulating TNF (17) were markedly increased (P < 0.05) relative to sham-operated controls (Fig. 2 and Table 1). In contrast, TNF levels were not elevated in TAB mice (P = NS vs. sham).
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Development of ventricular dysfunction and clinical HF.
At death, the integrity of aortic banding was confirmed by inspection of the surgical constriction and by visualization of marked differences in caliber of the right and left carotid arteries. Proximal aortic pressures (Fig. 3) were decreased (P < 0.05) in severe TAB mice relative to TAB mice, consistent with decreased cardiac output from diminished pump function (Table 1).
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Thus, despite the similarity in surgical intervention, the clinical phenotypes differed markedly in these models of pressure-induced ventricular remodeling, ranging from Comp Hyp in TAB mice to pressure-overload HF in severe TAB mice. Whereas markers of cardiac size and performance demonstrated hypertrophic growth in each case, echocardiographic and clinical markers of progressive failure were manifest exclusively in HF mice.
Activation of signaling cascades.
Hypertrophic growth of the myocardium is accompanied by a pattern of gene expression mimicking that seen during embryonic development. To investigate reactivation of the "fetal gene program" in load-induced HF, we measured transcript levels of natriuretic peptides after each phenotype had attained steady state (3 wk postoperative). Increases in atrial natriuretic factor (ANF) and B-type natriuretic peptide (BNP) transcript were observed in both Comp Hyp and HF hearts (Fig. 4). Whereas the extent of hypertrophy, measured as heart weight normalized to body weight, was greater in HF hearts compared with Comp Hyp hearts, the increases in ANF and BNP were significantly greater in Comp Hyp hearts than in HF hearts (P < 0.05). In contrast, ß-myosin heavy chain (MHC) transcript abundance was increased to a similar extent in both Comp Hyp and HF tissues. Together, these findings are suggestive of differential activation of transcriptional programs in compensated versus failing hypertrophic phenotypes.
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Alterations in calcium handling.
Depletion of Ca2+ from sarcoplasmic reticulum (SR) stores contributes to the disordered excitation-contraction coupling of systolic HF. Calsequestrin is a high-capacity Ca2+-binding protein that regulates sarco(endo)plasmic reticulum Ca2+ release by the ryanodine receptor (28). In a mouse model of hypertrophic cardiomyopathy, calsequestrin levels are diminished in advance of changes in cardiac histology or morphology (37). Consistent with the notion that dysregulation of Ca2+-responsive signaling is a proximal event in the transition from hypertrophy to failure, we observed a trend toward diminished calsequestrin levels in TAB mice with Comp Hyp (Fig. 6). In failing severe TAB hearts, calsequestrin expression was significantly (P < 0.05) diminished, reflecting graded changes in calcium handling in response to hemodynamic stress.
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Calcineurin signaling.
Calcineurin links cytoplasmic Ca2+ to transcriptional regulation of multiple genes involved in the cardiac hypertrophic program. We observed a slight but reproducible increase in the level of calcineurin catalytic subunit CnA protein levels in Comp Hyp hearts compared with sham-operated controls (P < 0.05; Fig. 6). Interestingly, there was no significant change in CnA levels in severe TAB failing hearts (P = NS) compared with sham-operated controls.
Calcineurin is activated upon the binding of a calcium/calmodulin complex. Thus an increase in calcineurin protein levels per se (Fig. 6) does not necessarily indicate that calcineurin signaling is activated. Rather, the level of calcineurin activity depends both on the abundance of calcineurin enzyme and the availability of intracellular Ca2+ and calmodulin. To assess calcineurin activity, we measured the abundance of modulatory calcineurin-interacting protein-1 (MCIP1). MCIP1 is an endogenous feedback inhibitor of calcineurin, whose expression is under the control of calcineurin (47). Under steady-state conditions, equilibrium is established between MCIP1 protein levels and calcineurin activity, such that the level of MCIP1 protein is proportional to calcineurin activity (41). MCIP1 protein levels were elevated in both Comp Hyp and HF hearts (Fig. 6). MCIP1 protein was more abundant in HF hearts than in Comp Hyp hearts, a pattern consistent with that observed for PLB phosphorylation.
To determine the functional requirement for calcineurin activity in the pathogenesis of compensated and failing hypertrophy, we inhibited calcineurin with systemic administration of cyclosporin A (CsA). Consistent with previous reports (21, 44), CsA abolished the hypertrophic growth response in TAB mice (Fig. 7). Inhibition of calcineurin also blunted hypertrophy in the context of severe TAB failing hearts, measured in terms of heart mass normalized to body mass (decreased 62%, P < 0.01) or tibia length (decreased 58%, P < 0.01). As reported previously, CsA had no detrimental effect on the survival of TAB mice (21). However, CsA decreased the survival of mice subjected to severe TAB (Fig. 8).
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In contrast to CsA, the -MHC-MCIP1 transgene did not cause in increase in mortality (P = NS) relative to wild-type littermate controls subjected to similar stress (Fig. 8). These results suggest that extracardiac effects of CsA may provoke toxicity in the setting of clinical HF. Our results with the
-MHC-MCIP1 transgenic mice, however, suggest that cardiomyocyte-specific suppression of calcineurin-dependent signaling pathwaysand consequent blunting of heart growthcan be well tolerated, despite the persistence of elevated afterload stress.
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DISCUSSION |
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In our hands, working with male C57BL6 mice, surgical stenosis of the thoracic aorta to the degree of a 27-gauge needle induces Comp Hyp without clinical HF or increased mortality [Hill et al. (21) and the present study]. Slightly tighter stenosis (differing by only 1/20th mm) induced ventricular dysfunction and clinical HF despite comparable degrees of cardiomyocyte hypertrophic growth. We found that the phenotypes resulting from these surgical interventions were highly reproducible, allowing us to make direct comparisons between compensated and decompensated forms of hypertrophy.
A wide range of parameters was used to assess the clinical outcome of our surgical procedures. Severe TAB animals manifested numerous features of HF not observed in TAB animals. These included lethargic behavior, increased levels of circulating TNF-, and evidence of both left-sided and right-sided venous engorgement and edema. There was a decrease in systolic function and an increase in end-diastolic volume, consistent with HF. Simultaneous measurements of distal and proximal carotid artery pressures demonstrated that transstenotic pressure gradients were diminished in Severe TAB mice, consistent with impaired pump function of failing hearts. Animals subjected to standard TAB demonstrated none of these clinical features despite the fact that they manifested an increase in cardiac mass. The geometry of hearts with stable Comp Hyp was very different from that of the failing, severe TAB hearts.
Within 24 h after surgery, there were similar rates of mortality among the sham-operated, TAB, and severe TAB mice, presumably due to the initial surgical trauma. However, after this initial 24-h recovery period, TAB was well tolerated, whereas half of the severe TAB animals died over the next 7 days. Three weeks was chosen as an end point to assure sufficient animals for analysis and because we (21) have shown previously that the Comp Hyp resulting from standard TAB has reached steady state at this point. Survival experiments extending up to 18 mo have shown that TAB is well tolerated. In contrast, mortality in the severe TAB model continues with few surviving past 2 mo.
In this model, an incremental increase in the degree of aortic constriction is sufficient to trigger hypertrophic HF as opposed to Comp Hyp. It is important to note that, despite vastly different geometry and function, both the TAB and severe TAB hearts expressed elevated levels of ANF and BNP (molecular markers of HF), suggesting that both undergo pathological hypertrophic remodeling. Physiological hypertrophy, in contrast, occurs during development, in response to exercise, and during pregnancy, without an associated reactivation of the fetal gene program.
Although indicative that the heart is under stress, expression of ANF and BNP has antihypertrophic properties, as mice lacking the gene encoding guanylyl cyclase A, a common receptor for ANF and BNP, have marked hypertrophy and fibrosis (24, 31). Recombinant human BNP (nesiritide) is efficacious in the clinical treatment of decompensated HF (11). Thus the elevated expression of ANF and BNP in the heart may be a protective stress response. It is interesting to note that the severe TAB failing hearts had significantly lower levels of ANF and BNP expression than hearts with stable Comp Hyp. It is impossible to determine from these steady-state, end-point measurements whether this stems from inability of the failing heart to mount an effective stress response or because expression of these protective peptides decreases as HF progresses. Close examination of the temporal dynamics of this response could yield important clues relevant to the role of vasoactive peptides in the pathogenesis and therapeutics of HF.
Profiles of stress-responsive signaling and calcium handling.
Evidence in the literature is conflicting regarding the role of intracellular signaling mechanisms in the pathogenesis of HF. Several studies have reported an association between HF and activation of MAPK (9, 20), protein kinase C (3, 4), Akt (20), or calcineurin (20, 25, 46) pathways. Others find no such association (40). A number of signaling cascades are activated within minutes of the imposition of hemodynamic load, triggering an array of downstream kinases during the acute phase response (16). Some evidence points to a second, later phase of activation, suggesting that certain pathways may have distinct roles at different stages of HF progression (23). By inference, this double peak of activation implicates feedback by negative regulators capable of arresting disease progression under certain circumstances. An attractive approach to the treatment and management of heart disease would be to capitalize on these endogenous negative regulators. It is for this reason that we assessed the activation profile of signaling cascades during the steady-state, maintenance phases of load-induced hypertrophy and failure, with a goal of identifying novel targets of therapy in chronic disease.
MAPK pathways provide a crucial link between extracellular stimuli and transcriptional regulation of numerous genes. Each of the three major MAPK arms has been implicated in hypertrophic signaling, and each has been shown to be activated during the acute response to TAB, although controversy persists regarding their relative importance and their contribution to the maladaptive features of hypertrophy (6, 19). TAB and severe TAB provoke acute-onset mechanical stress on the heart; the resulting, transient activation of ERK, JNK, and p38 kinases, however, likely has little bearing on chronic heart disease. Focusing on load-induced HF under steady-state conditions, we observed selective activation of ERK and JNK. We speculate that persistent activation of these stress-responsive signaling pathways may contribute to progressive myocardial decompensation, a hypothesis that we are currently testing.
Both Comp Hyp and HF hearts manifested changes in calsequestrin and PLB that would result in altered calcium handling. These changes were more pronounced in HF hearts. Decreased calsequestrin will reduce the Ca2+ buffering capacity of the SR, reducing both Ca2+ release and uptake. Protein kinase A-dependent phosphorylation of PLB at Ser16, however, would be expected to release SERCA from tonic inhibition, working to augment Ca2+ uptake (27). Indeed, PLB phosphorylation was highest in failing, severe TAB hearts. Taken as a whole, differential Ca2+ handling provokes changes in contractility and directly impacts the activity of Ca2+/calmodulin kinase and calcineurin, both of which have been linked to pathological remodeling of the heart. Changes in calsequestrin levels and PLB phosphorylation were greatest in HF hearts compared with Comp Hyp hearts, indicative of a larger perturbation in Ca2+ handling.
Calcineurin signaling.
Calcineurin is a Ca2+/calmodulin-regulated cytoplasmic protein phosphatase whose activity is required for hypertrophic transformation of the heart in the setting of many, but not all, forms of stress (for a review, see Ref. 18). The importance of calcineurin signaling in HF has been the subject of debate, with some investigators reporting activation (20, 25) but others detecting none (40). Some evidence from infarction models suggests that calcineurin activation correlates with the severity of the pathological condition (46).
We found evidence of increased calcineurin protein levels in Comp Hyp but not in pressure-overload HF. These data do not necessarily, however, point to increased calcineurin activity, as calcineurin is not active without a rise in cytoplasmic Ca2+ and binding of calmodulin. However, increased MCIP1 protein abundance is an indication that calcineurin is more active in both the hypertrophic and failing hearts compared with sham-operated control hearts. Once again, this is consistent with a greater shift in Ca2+ handling in the failing heart.
The calcineurin inhibitor CsA blunted hypertrophic growth in both the Comp Hyp and HF hearts, demonstrating functional involvement of calcineurin in both. Recent strides in deciphering mechanisms that underlie pathological growth of the heart suggest that calcineurin mediates a maladaptive form of hypertrophy. In fact, suppression of calcineurin-dependent hypertrophy is surprisingly well tolerated in the setting of moderate hemodynamic stress (for a review, see Ref. 18). Thus calcineurin inhibition has been proposed as a therapeutic approach to cardiac hypertrophy and failure. It was therefore of concern that CsA increased mortality in severe TAB hearts. Similarly, CsA has been shown to worsen outcomes in genetic models of hypertrophy (36). CsA can alter expression of the cardiac Na+/Ca2+ exchanger (45) and L-type Ca2+ channel (unpublished observations), perhaps contributing to the increased concentrations of intracellular Ca2+ observed in cells treated with CsA (14). CsA can also affect cellular targets other than calcineurin, such as the mitochondrial permeability transition pore (38, 43). Calcineurin is a ubiquitous protein, and systemic administration of CsA has effects on other, noncardiac organ systems.
Transgenic overexpression of MCIP1 was employed to inhibit calcineurin specifically in cardiac myocytes. MCIP1 overexpression blunts the hypertrophic response to activated calcineurin (35), hormonal or exercise stress (34), and pressure overload (22). Here, we report that MCIP1 blunted the hypertrophic growth response to severe pressure-overload stress, yet blunted hypertrophy was not associated with clinical deterioration or mortality detriment. Together, these findings point to calcineurin as a nodal point in the complex network of hypertrophicand now failuresignaling. Furthermore, they suggest that the increased mortality in severe TAB mice treated with CsA may be a consequence of secondary toxicity.
Targeted calcineurin inhibition by MCIP1 has been shown to blunt hypertrophic remodeling in response to myocardial infarction and improve survival (41). In our model of HF, however, calcineurin inhibition by MCIP1 did not improve survival despite substantial blunting of hypertrophic growth. Certainly, much of the morbidity associated with HF stems from its negative impact on quality of life; in our animal models, however, it is impossible to assess improvement in symptoms. In any event, our results with cardiac-specific MCIP1 overexpression suggest that calcineurin inhibitors that target the myocardium with calcineurin specificity may provide clinical benefit. A major challenge for the future will be the development of such compounds. In addition, further work will be required to understand the compensatory mechanisms that regulate circulatory performance when "compensatory" hypertrophy is eliminated.
Limitations.
Aortic banding induces abrupt elevations in ventricular wall stress, analogous to that seen in the spared regions of the infarcted LV. This model is less applicable to hypertension or aortic stenosis, however, where ventricular pressures typically rise gradually over time and myocyte hypertrophy precedes failure. Furthermore, these studies focus on rodents whose levels of physical exertion are limited by their being maintained in cages. To minimize the effects of gender, we studied only male mice. However, apparent strain differences were seen, as C57BL6 mice manifested a trend (P = NS) toward higher mortality from severe TAB (Fig. 8A) compared with wild-type littermates of the MCIP transgenic line (Fig. 8B). For all these reasons, it is difficult to extrapolate our findings to freely mobile large animals subjected to chronic, low-level afterload stress.
Summary and perspective.
Recent studies have demonstrated that therapeutic targeting of hypertension-induced hypertrophy is a viable strategy that confers long-term mortality benefit (12, 30). The prospect of modulating hypertrophic growth of the myocardium in other contexts, without provoking cardiovascular compromise, requires that we identify commonalities and differences in the signaling systems that promote stable Comp Hyp versus HF. In this study, we found that signaling pathways and Ca2+ handling are differentially altered in pressure-overload hypertrophy and failure, suggesting that targets of therapeutic relevance may differ in these two important disease phenotypes. In both cases, however, calcineurin activation led to pathological remodeling, and suppression of calcineurin signaling was well tolerated.
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GRANTS |
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ACKNOWLEDGMENTS |
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Present address of K.-D. Yoo: St. Vincents Hospital, Catholic University of Korea, Seoul, Korea 442-723.
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FOOTNOTES |
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Address for reprint requests and other correspondence: J. A. Hill, Division of Cardiology, Univ. of Texas Southwestern Medical Center, NB11.200, 5323 Harry Hines Blvd., Dallas, TX 75390-8573 (e-mail: joseph.hill{at}UTsouthwestern.edu).
1 The Supplemental Material for this article (Supplemental Fig. S1) is available online at http://physiolgenomics.physiology.org/cgi/content/full/00061.2005/DC1.
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REFERENCES |
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