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Cardiovascular Research 2002 53(2):294-303; doi:10.1016/S0008-6363(01)00451-5
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Copyright © 2001, European Society of Cardiology

Old and new tools to dissect calcineurin's role in pressure-overload cardiac hypertrophy

Weiguo Zhang*

Department of Internal Medicine/Hypertension Division, The University of Texas Southwestern Medical Center at Dallas, Dallas, TX 75390-8586, USA

wzhang{at}mednet.swmed.edu

* Tel.: +1-214-648-7944; fax: +1-214-648-7902

Received 13 July 2001; accepted 24 August 2001


   Abstract
Top
 Abstract
1. Introduction
 2. Calcineurin hypothesis of...
 3. Calcineurin inhibitors and...
 4. Calcineurin inhibitors and...
 5. New genetic approaches
 6. Conclusions
 References
 
In the last several years, a number of experiments have implicated a pivotal role of the calcium/calmodulin-calcineurin dependent pathway as a final common signaling mechanism by which diverse hypertrophic stimuli converge to mediate hypertrophic responses in cardiomyocytes. Calcineurin inhibitors, i.e. cyclosporine A (CsA) and FK506, can interrupt the pathway, thereby preventing cardiac hypertrophy. The data that convincingly support this novel hypothesis were derived either from in vitro studies in cultured cardiomyocytes or from in vivo studies in transgenic mice. However, when the hypothesis was tested in clinically relevant animal models of cardiac hypertrophy, controversial results and conclusions emerged. In conventional models of cardiac hypertrophy, two questions remain to be answered: (1) whether calcineurin is activated in hypertrophied cardiac muscle, and (2) whether calcineurin inhibitors prevent cardiac hypertrophy. In addition, clinical observations have revealed that calcineurin inhibitors appear to exert pro-hypertrophic effects in organ transplant recipients. The controversies suggest that current calcineurin inhibitors are blunt tools for testing the hypothesis in pressure-overload hypertrophy in vivo, because there are so many confounding effects that are associated with systemic administration of the drugs. As such, new genetic approaches may overcome some of the problems associated with pharmacological inhibitors. This invited review will focus on the controversies surrounding the ability of calcineurin inhibition to prevent conventional (pressure-overload) cardiac hypertrophy and the new genetic approaches to address the question.

KEYWORDS ACE inhibitors; Gene expression; Hypertension; Hypertrophy


   1. Introduction
Top
 Abstract
 1. Introduction
2. Calcineurin hypothesis of...
 3. Calcineurin inhibitors and...
 4. Calcineurin inhibitors and...
 5. New genetic approaches
 6. Conclusions
 References
 
Cardiac hypertrophy has been firmly established as one of the most powerful clinical predictors for cardiovascular morbidity and mortality [1,2]. Although many of the extrinsic and intrinsic factors that determine cardiomyocyte hypertrophy have been identified, a unifying hypothesis that explains how multiple diverse stimuli are transduced into the final hypertrophic response has remained elusive. A major breakthrough occurred recently when a series of elegantly designed and carefully performed experiments in cultured cardiomyocytes and transgenic mice implicated a pivotal role for calcineurin, the calcium-calmodulin dependent phosphatase (PP2B), in the signal transduction pathway that culminates in cardiomyocyte hypertrophy. Calcineurin inhibitors such as cyclosporine A (CsA) and FK506 were found to block cardiomyocyte hypertrophy in vitro, and transgenic overexpression of cardiac calcineurin in mice was found to produce profound cardiac hypertrophy leading to heart failure in vivo. This basic research led numerous teams of investigators to ask whether calcineurin plays an important role in mediating the hypertrophic response to the common forms of hypertrophy seen in patients. The most important cardiac hypertrophy is pressure-overload mediated, such as that resulting from long-standing hypertension or aortic valve stenosis. The answer to this question has far reaching clinical implications, because calcineurin constitutes a potential novel target to intervene in the hypertrophic process.

Although CsA effectively blocks cardiomyocyte hypertrophy in cultured cells and in certain transgenic mouse models, cardiac hypertrophy is prevalent in patients with CsA-induced hypertension. In clinically relevant conventional rodent models, the ability of CsA to prevent cardiac hypertrophy has been controversial. The purpose of this invited article is to review clinical and experimental animal studies, the latter utilizing either conventional pharmacological approaches or new targeted genetic approaches.


   2. Calcineurin hypothesis of cardiomyocyte hypertrophy
Top
 Abstract
 1. Introduction
 2. Calcineurin hypothesis of...
3. Calcineurin inhibitors and...
 4. Calcineurin inhibitors and...
 5. New genetic approaches
 6. Conclusions
 References
 
Calcineurin, or phosphatase 2B, is a serine/threonine protein phosphatase. It is most abundant in mammalian brains, accounting for 10% of total brain protein, and it also is widely distributed in peripheral tissues. In the early 1990s, calcineurin was shown to play a pivotal role in the activation of T lymphocytes and to be the common cellular target for the immunosuppressive drugs CsA and FK506. In T-cells, the activation of the membrane receptors by antigens promotes an increase in intracellular calcium, which after coupling with its binding protein calmodulin (calcium "receptor"), results in calcineurin activation. The activated calcineurin dephosphorylates a transcriptional factor NFAT (nuclear factor of activated T-cell). Dephosphorylated NFAT translocates to the nucleus, where it interacts with the interleukin II gene, resulting in the expression of cytokines involved in the immune response. Potent calcineurin inhibitors cyclosporine A (CsA), which forms immunophilin complexes with cyclophilin, and FK506, which forms immunophilin complexes with FK binding protein 12, can interrupt the pathway, thereby blocking cytokine-induced immune responses (for a comprehensive review, see Ref. [3]).

One fascinating aspect of this new area of cytosolic signaling is that some of the same components of the calcineurin pathway found in T-cells also are present in cardiomyocytes [4,5]. A series of in vitro and in vivo experiments were performed to determine if a similar signaling pathway connects those components and induces the hypertrophic responses in cardiomyocytes [6]. The pathway is depicted schematically in Fig. 1. Either extrinsic hypertrophic stimuli such as pressure-overload or intrinsic stimuli such as angiotensin II can produce sustained increases in cytosolic calcium that constitute a potent physiological and/or pathological stimulus to calcineurin. Activated calcineurin was shown to dephosphorylate NFAT, which then enters the nucleus to cause transcriptional activation of hypertrophic fetal genes leading to cardiomyocyte hypertrophy.


Figure 1
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  		Fig. 1 Schematic illustration of calcineurin's role in cardiac hypertrophy. Either extrinsic hypertrophic stimuli such as pressure-overload or intrinsic stimuli such as angiotensin II produce sustained increases in cytosolic calcium that constitute a potent physiological and/or pathological stimulus to activate calcineurin. The activated calcineurin dephosphorylates nuclear factor of activated T-cell (NFAT, a transcriptional factor). Dephosphorylated NFAT enters the nucleus where it interacts with another transcriptional factor (GATA4) and causes transcriptional activation of hypertrophic fetal genes leading to cardiomyocyte hypertrophy.

 
The initial physiological confirmation of the functional importance of this signaling pathway was as follows. In cultured neonatal cardiomyocytes, the hypertrophic response to angiotensin II or an {alpha}-adrenergic agonist was abrogated by pretreatment with either CsA or FK506. In transgenic mice, expression of constitutively active calcineurin in the heart under transcriptional control of {alpha}-myosin heavy chain ({alpha}-MHC) promoter induced profound cardiac hypertrophy leading to dilated cardiomyopathy, heart failure, and sudden death. This dramatic cardiac hypertrophy/heart failure phenotype was rescued by systemic CsA, which confirmed calcineurin mediation [7]. Even though the hypothesis requires additional tests (for example, to determine if knockout mice lacking calcineurin or NFAT3 in the heart are protected from hypertrophic stimuli), numerous studies in cultured cardiomyocytes and in transgenic animal models [7–12] have provided experimental support, although a couple of studies [8,13,14] have led to contradictory findings. Further, cardiac hypertrophy was also produced in transgenic mice with cardiac-specific expression of constitutively active NFAT3. Because NFAT3 is downstream in the calcium/calmodulin-calcineurin signaling pathway, the CsA sensitive step, the hypertrophy in NFAT-transgenic mice, was unaffected by CsA [6]. The importance of calcineurin and related pathways in cardiac hypertrophy has been comprehensively reviewed in recent years [15–22].

In contrast to the robust anti-hypertrophic effect of systemic calcineurin in these transgenic animal models, the ability of systemic CsA or FK506 to modulate pressure-overload hypertrophy in a clinical setting and in conventional rodent models has been more difficult to determine.


   3. Calcineurin inhibitors and LV hypertrophy in organ transplant recipients
Top
 Abstract
 1. Introduction
 2. Calcineurin hypothesis of...
 3. Calcineurin inhibitors and...
4. Calcineurin inhibitors and...
 5. New genetic approaches
 6. Conclusions
 References
 
Since the clinical introduction of CsA in the early 1980s, the systemic administration of CsA and (now) FK506 has emerged as an important cause of secondary hypertension. In heart transplant recipients, for example, the prevalence of hypertension has increased from 20% in the pre-CsA era to more than 90% currently (for a comprehensive review, see Ref. [23]). This hypertension typically is moderate to severe and may not be optimally controlled even with multiple antihypertensive medications. A characteristic feature of this hypertension is that the normal nocturnal dip in blood pressure is blunted [24–27a], so that the overall time-integral burden of blood pressure on the left ventricle (LV) is accentuated. Therefore, it is not surprising that cardiac hypertrophy has been shown to be prevalent in organ-transplant recipients with CsA- or FK506-induced hypertension (Table 1) [24,27a–37].


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  		Table 1 State of the effect of calcineurin inhibitors on cardiac mass in patients by the end of April 2001

 
Although most of the clinical studies were longitudinal or retrospective, and therefore lacking adequate controls, an echocardiographic study by Ventura et al. [30] was well designed to eliminate potential confounding factors. In this study, 25 cardiac transplant recipients on CsA treatment were matched by mean arterial pressure, gender, height and weight to 25 patients with established essential hypertension, and 25 normotensive subjects matched by age, gender and body habitus were used as controls. Patients with essential hypertension and hypertensive cardiac transplant recipients had greater posterior wall thickness, left ventricular mass index and left ventricular mass/height ratio than normotensives. More importantly, hypertensive cardiac transplant recipients had a greater left ventricular mass than patients with established essential hypertension (245±7 vs. 223±8 g, P<0.05) (Fig. 2). The explanation for the greater hypertrophic response to CsA-induced hypertension than to essential hypertension is unknown, but may be related to a greater time-integral BP burden and/or to subclinical allograft rejection in the CsA group.


Figure 2
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  		Fig. 2 An echocardiographic study showing that the cardiac transplant recipients who were on CsA developed hypertension and left ventricular (LV) hypertrophy. In this study, 25 cardiac transplant recipients were matched by mean arterial pressure (MAP), gender, height and weight to 25 patients with established essential hypertension, and 25 normotensive subjects matched by age, gender, body weight and height were used as controls. Essential hypertension patients and hypertensive cardiac transplant recipients had greater LV mass, LV mass index and LV mass/height than normotensives and, more importantly, hypertensive cardiac transplant recipients had a greater left ventricular mass than patients with established essential hypertension (modified, with permission, from Ref. [30]).

 
Regardless of the precise explanation, in this clinical setting the hypertensive effects of calcineurin inhibitors appear to outweigh any putative protective effects on cardiac hypertrophy.


   4. Calcineurin inhibitors and pressure-overload hypertrophy in rodents
Top
 Abstract
 1. Introduction
 2. Calcineurin hypothesis of...
 3. Calcineurin inhibitors and...
 4. Calcineurin inhibitors and...
5. New genetic approaches
 6. Conclusions
 References
 
4.1 Studies from the author's laboratory
To bridge the gap between the basic laboratory research and clinical observations, we conducted a series of experiments to determine whether systemic administration of CsA or FK506 attenuates left ventricular pressure-overload hypertrophy in two well-established rat models: spontaneously hypertensive rats (SHR) and aortic banding in normotensive Sprague–Dawley rats. The single most important feature of these experiments compared to the previously published studies in this field was the detailed measurement of the hemodynamic stimulus to hypertrophy. We studied the hypertrophic response in banded rats treated with CsA (10 or 20 mg/kg/day), FK506 (0.3 mg/kg/day), or vehicle for 2–4 weeks. The results were all statistically negative (Fig. 3). The only protocol in which there was a trend for the LV/BW ratio to be lower with CsA/FK506 than vehicle was when rats were treated with CsA for 4 weeks. However, this tendency may represent a nonspecific effect related to an attenuated body weight gain that occurred with both CsA and FK506 treatment. Although the mechanism is unknown, this nonspecific effect of CsA and FK506 has been well described [38–46], and it is a major problem in the use of these agents to test the calcineurin hypothesis in the intact animal. We found that the aortic pressure gradient across the stenosis was less severe in rats treated with CsA or FK506 compared with vehicle-treated rats. When young rats undergo aortic banding, the magnitude of the pressure gradient normally increases over time as the aorta grows around a fixed extrinsic stenosis. In banded rats receiving CsA or FK506, the attenuated weight gain could lead to a proportionate attenuation in growth of the aorta, creating a smaller pressure gradient across the stenosis. Another potential explanation for these negative results is that, in the initial experiments, CsA was administered after the banding. We therefore performed an additional series of experiments in which we reduced the duration of the experiment to 2 weeks and began CsA 2 days before the banding, i.e. well before the initiation of the hypertrophic stimulus. Under these conditions, the pressure gradients were comparable in banded rats treated with CsA or vehicle, and the LV/BW ratios were indistinguishable, as were the LVEDPs, a measure of diastolic function, and ventricular ANP mRNA, a marker of cardiac fetal hypertrophic gene expression. It should be pointed out that these doses of CsA produced trough blood levels that were 10 times higher than those achieved with clinical doses in patients. As explained in greater detail later, these doses of CsA were sufficient to inhibit 90% of the maximal stimulated cardiac calcineurin activity in extracts from the hypertrophied ventricles.


Figure 3
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  		Fig. 3 (A) Aortic-banded rats were treated with CsA at two doses (10 and 20 mg/kg/day), FK506 (0.3 mg/kg/day), and restricted food to match the caloric intake of the CsA 10 mg/kg/day (PairFed). After 4 weeks of treatment the LVW/BW ratio and pressure gradient across the stenosis were determined. Calcineurin inhibitors did not affect the contribution of each mmHg increase in pressure gradient to the LVW/BW ratio. (B) Aortic-banded rats were pretreated with CsA (10 mg/kg/day) for 2 days (three injections) before the banding surgery so that they were exposed to calcineurin inhibition before the initiation of the hypertrophic stimulus. The treatment was maintained for 2 weeks. Despite the fact that calcineurin phosphatase activity in LV was inhibited by 90%, CsA did not inhibit hypertrophic gene expression, improve left ventricular function and attenuate LVW/BW or LVW/BW/gradient (modified, with permission, from Ref. [40]).

 
Aortic banding represents a short-term model of pressure-overload and we wondered whether an effect of CsA or FK506 might be more evident with the more gradual hypertrophic response that accompanies the progressive hypertension seen in SHR. The time course of this response more closely mimics the clinical situation. In this experimental protocol, SHR were treated with a more clinically relevant dose of CsA, 5 mg/kg/day for 6 weeks, and the LV/BW in CsA-treated SHR was not affected. This was consistent with that from a previous study [39] and was confirmed by recent studies [42,46]. However, CsA slightly exaggerated the hypertension in SHR. So, from this negative finding, we could not exclude the possibility that CsA caused a relative attenuation in the hypertrophic response, given the slightly higher conscious blood pressures.

In an additional series of SHR experiments, we increased the dose of CsA four-fold and performed a head-to-head comparison of CsA vs. ramipril, an angiotensin converting enzyme inhibitor (ACEI) that is firmly established to prevent left ventricular hypertrophy (LVH) in this model. Although the dose of ramipril used in our experiments (0.2 mg/kg/day subcutaneously) did not cause a significant lowering of blood pressure, it abrogated the hypertrophic response in the left ventricle as expected. By comparison with this potent internal control, CsA had no effect (Fig. 4, unpublished data).


Figure 4
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  		Fig. 4 Spontaneously hypertensive rats (SHR) and their normotensive control Wistar–Kyoto rats (WKY) (5 weeks old, n=10–11 each) were treated with vehicle or CsA (20 mg/kg/day, subcutaneous injection) for 6 weeks. SHR were also treated with ramipril (0.2 mg/kg/day), an angiotensin converting enzyme inhibitor (ACEI). This four-fold higher dose of CsA did not alter the left ventricular weight/body weight (LVW/BW) ratio at all. Unlike CsA, ramipril abrogated left ventricular hypertrophy in SHR. *P<0.05 vs. WKY on vehicle; §P<0.05 vs. SHR on vehicle (from unpublished data).

 
While the results of each of our multiple protocols could be open to alternative interpretations, taken together the data led us to conclude that these two conventional rat models of pressure-overload hypertrophy are basically insensitive to systemic calcineurin inhibitors.

4.2 Studies from other laboratories
At the time of writing this review, 18 publications have reported the effects of systemic CsA or FK506 on cardiac hypertrophy using in vivo rodent models of pressure-overload LVH (summarized in Table 2) [7,39–42,45–57]. In prevention experiments, eight studies/eleven protocols were interpreted as being overwhelmingly negative, six studies/six protocols were interpreted as being overwhelming positive, and the remaining four studies/four protocols were interpreted as indicating a partial attenuation of LVH by the calcineurin inhibitors. In regression experiments, cardiac hypertrophy was not reversed in one study and was partially reversed in two studies.


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  		Table 2 State of calcineurin hypothesis in conventional models of cardiac (pressure-overload) hypertrophy by the end of April 2001 (in chronological order). The author's new data (Fig. 3) are not included

 
What is evident from Table 2 is that these 18 studies using similar experimental designs have not produced consistent results regarding the effects of systemic CsA or FK506 on pressure-overload cardiac hypertrophy, i.e. in some experiments the drugs prevented hypertrophy and in others they failed. One interpretation is that the problem is not with the underlying hypothesis but rather with the methods used to test it in vivo. Systemic CsA and FK506 do not only inhibit calcineurin, but also produce a diverse spectrum of adverse effects, including cardiotoxicity, nephrotoxicity and neurotoxicity [23,58–61]. A number of other problems (as detailed below) are concomitant with the use of current calcineurin inhibitors in rodents, making it difficult to probe calcineurin's role in pressure-overload hypertrophy.

4.3 Some major problems related to the systemic use of calcineurin inhibitors
4.3.1 Nonspecific effects of systemic CsA and FK506 on body weight
When administered chronically, CsA and FK506 reduce weight gain in young rats and cause weight loss in older rats [38–46]. They also decrease locomotor activity and induce a generally ill appearance in rats, though the mechanism for these effects is unknown. Decreased body weight represents a major problem when testing the effects of these drugs on LVH, because the LV weight typically is normalized for body weight as an index for cardiac hypertrophy. Some investigators have tried to circumvent this problem by normalizing LV weight to tibial length. If the rats are post-pubescent at the time of study, tibial length will be relatively constant during the protocol and thus will not correct for the nonspecific changes in body mass. Because body weight is one of the most powerful determinants of LV weight, a reduced LV weight-to-tibial length ratio could be due to a specific effect of calcineurin inhibition in the heart or to a nonspecific effect of weight loss. Such results cannot discriminate between these alternative interpretations.

For example, the data of Øie et al. (No. 15 in Table 2) [54] were interpreted as showing that CsA attenuated cardiac hypertrophy in rats post myocardial infarction. CsA attenuated right ventricular hypertrophy (RVW/TL) by 38%, cardiac hypertrophy (HW/TL) by 20% and left ventricular hypertrophy (LVW/TL) by 15%, respectively. Although tibial length was unaffected by CsA treatment, body weight was 17% lower in the CsA group. If one recalculates the same data using body weight to normalize HW, LVW and RVW, CsA attenuated right ventricular hypertrophy (RVW/BW) by only 26%, cardiac hypertrophy (HW/BW) by only 4.5%, and did not attenuate left ventricular hypertrophy (LVW/BW), but instead increased it by 1.6%. In the same study, the expressions of four hypertrophic genes were measured as molecular markers of the hypertrophy. Except for a slight decrease in the mRNA level of skeletal {alpha}-actin, there were no changes in mRNA levels of ANP, BNP and TGF-β in CsA compared to vehicle-treated hearts. Similarly, the data from Shimoyama et al. (No. 16 in Table 2) [55] were interpreted to suggest that FK506 attenuates the development and induces the regression of cardiac hypertrophy in rats with Dahl salt-sensitive hypertension. However, the conclusion was based mainly on the LVW/TL ratio. When the same data are expressed as LVW/BW, FK506 had no effect on hypertensive LVH. Furthermore, of the four hypertrophic cardiac fetal genes measured in this study, mRNA levels of ANP and skeletal {alpha}-actin were up-regulated in the hypertrophic hearts, but unaffected by FK506.

Thus, even carefully designed and well performed "positive" studies are not definitively positive due to the anorexic effects of CsA and FK506.

4.3.2 Systemic hemodynamic effects of CsA and FK506 in animal experiments in vivo
Of the 18 papers cited in Table 2, some performed no assessment of the hemodynamic stimulus to pressure-overload hypertrophy [7,45,47,48]. Of those studies in which invasive hemodynamics was measured, most were done under general anesthesia, and thus the results may not be reflective of the chronic hemodynamic burden.

The importance of hemodynamic measurement arises because both acute and chronic administration of CsA or FK506 is usually accompanied by an increase in blood pressure in many species including mice and rats [39,42,44,46,62–70]. Thus there is a possibility that a minor attenuation of the hypertrophy was actually achieved in those previously mentioned negative studies, but masked by a slight increase in blood pressure as seen in the author's first SHR protocol [40], in which the conscious blood pressure was 14 mmHg higher in CsA-treated than vehicle-treated SHR. In addition, the acute hypertensive response to calcineurin inhibitors has been shown to be associated with sympathetic nerve activation in rodents [63,65,69–74] (whether the chronic hypertension is associated with sympathetic activation has not yet been documented), which also contributes to the development of cardiac hypertrophy [75,76].

Surprisingly, in some studies CsA or FK506 actually appeared to lower blood pressure [50,54], which itself could account for reduced hypertrophy and suggests that either the pressure-overload was not adequately developed or that these drugs made the animals sick. Anesthesia also decreases blood pressure. If anesthesia caused profound hypotension, then echocardiography performed under such conditions to document cardiac function and structure (ejection fraction, wall stress, wall thickness, etc.) may not be very valid.

Thus, without precise measurement of blood pressure, the effects of calcineurin inhibitors on LVH could be overlooked or overestimated.

4.3.3 Measurement of cardiac calcineurin activity
Because the disparate conclusions in Table 2 are based on the systemic administration of CsA or FK-506, a critical issue is whether the drug regimens produced nearly complete inhibition of cardiac calcineurin activity. The doses of CsA used in these rodent experiments produce enormous trough CsA blood levels that are 10-fold higher than the therapeutic blood levels needed to inhibit calcineurin signaling in T-cells [77]. Although half of the studies cited in Table 2 made no attempt to measure cardiac calcineurin activity, similar dosing regimens have been shown to inhibit calcineurin-mediated transcriptional activation in rat hind limb muscle [78]. The remaining studies attempted to measure the extent to which systemic CsA or FK506 inhibited cardiac calcineurin activity. Using the only available assay, these investigators measured calcineurin phosphatase activity in vitro from left ventricular samples extracted from control animals or animals exposed to a hypertrophic stimulus (i.e., hypertension or aortic banding) alone or in combination with CsA or FK506.

The details of this assay are as follows. Total phosphatase activity of the cell-lysed tissue extracts is evaluated by measuring the rate of dephosphorylation of a synthetic [32P]-ATP-labeled phosphopeptide substrate (R-II peptide) in the presence of excess exogenous CaCl2 and calmodulin to maximally activate calcineurin. First, the activity of phosphatase 1 and 2a present in these samples is blocked with a low dose of okadaic acid which is relatively specific for these Ca2+-independent phosphatases. Then, the fraction of remaining phosphatase activity due to activated calcineurin is measured by blocking calcineurin activity with an immunosuppressive drug complex (1.0 µM each of CsA and recombinant human cyclophilin B) [40,79].

The results of this assay were used to answer two questions: (1) In the absence of CsA or FK506, is pressure-overload hypertrophy accompanied by activation of calcineurin in the heart? (2) Are the systemic doses of CsA or FK506 sufficient to inhibit this putative activation of cardiac calcineurin? It needs to be emphasized that this is a cell-lysed assay that measures maximal calcineurin activity in the presence of exogenous Ca2+ and calmodulin [40]. Thus, regardless of whether hypertrophy is accompanied by increased, decreased or unchanged maximal cardiac calcineurin activity (Table 2), these in vitro data provide limited information about the extent to which the phosphatase was activated by increased cytosolic Ca2+ levels in vivo. Every study that measured cardiac calcineurin activity after CsA or FK506 found that the maximal stimulated activity in the tissue extracts was markedly inhibited by the various dosing regimens. From these data, it is reasonable to argue that the very high systemic doses of CsA or FK506 used in these different rodent studies likely were more than sufficient to inhibit whatever in vivo activation of cardiac calcineurin was produced by pressure-overload.

4.3.4 Adequate controls
From the standpoint of in vivo organ/system physiology, at least two types of control groups would seem critically important in the interpretation of the studies summarized in Table 2. First, given the various outcome measures used to estimate LVH, any putative new strategy to ameliorate LVH should be compared against the benchmark of conventional antihypertrophic therapy [80–86]. With the exception of our new data presented above, there is only one study in Table 2 in which a head-to-head comparison between CsA and an ACEI was performed [53]. Such a control with ACEI may also help to dissect the reason for the high mortality seen in animals receiving CsA or FK506 [7,40,47,50,55], which was attributed to the lack of an adequate hypertrophic response to the pressure-overload by some authors, though many investigations have demonstrated that preventing cardiac hypertrophy by current anti-hypertrophic agents, e.g. ACEI, can increase survival rate [83,86,87]. The second type of control group is needed to address the specificity of a positive response to CsA or FK506. Rapamycin would constitute a good control for some effects that is concomitant with systemic calcineurin inhibition [88–90], because this immunophilin ligand inhibits T-cell function and causes immunosuppression via an independent signaling pathway that does not involve calcineurin [91].

For the reasons discussed above, it is obvious that the current calcineurin inhibitors, when administered systemically to intact rodents, are blunt tools to probe calcineurin's role in pressure-overload hypertrophy.


   5. New genetic approaches
Top
 Abstract
 1. Introduction
 2. Calcineurin hypothesis of...
 3. Calcineurin inhibitors and...
 4. Calcineurin inhibitors and...
 5. New genetic approaches
6. Conclusions
 References
 
To overcome the limitations inherent in these pharmacologic approaches, two research teams recently developed targeted genetic approaches to specifically inhibit cardiac calcineurin activity in vivo [92,93]. Published as back-to-back papers in the March 13, 2001 issue of the Proceedings of the National Academy of Sciences of the United States of America, these fascinating studies demonstrate that forced cardiac overexpression of three different calcineurin-inhibitory peptides effectively blocks the hypertrophic response of the LV to four different hypertrophic stimuli: cardiac expression of constitutively active calcineurin, exercise (physiological hypertrophy), β-adrenergic agonist (systemic isoproterenol), or pressure-overload (aortic banding).

These investigators discovered that calcineurin's phosphatase activity is sensitive to inhibition not only by the immunophilin ligands CsA and FK506 (fungal products), but also by three different types of mammalian proteins: AKAP79 (A-kinase anchoring protein 79), cain/cabin-1, and MCIP-1 (myocyte-enriched calcineurin-interacting protein-1). All three proteins can inhibit calcineurin activity and hypertrophic responses in cardiac myocytes. Of these, MCIP-1 is most likely to constitute an endogenous calcineurin inhibitor because it is the only one of the three peptides that is expressed in high levels in both cardiac and skeletal muscle. Furthermore, MCIP-1 expression is negatively regulated by calcineurin, suggesting an endogenous negative feedback mechanism.

In the study by Rothermel et al. [93] transgenic mice were engineered to over-express a human cDNA encoding a MCIP-1 under the control of cardiac-specific, {alpha}-MHC promoter. The cardiac-specific expression of hMCIP1 inhibited the pharmacological hypertrophy from β-adrenergic receptor stimulation with systemic isoproterenol, the physiological hypertrophy from exercise training, and the genetic hypertrophy from expression of constitutively active cardiac calcineurin. In the study by De Windt et al. [92] transgenic mice expressing the calcineurin inhibitory domains of cain/cabin-1 or AKAP79 specifically in the heart, partially inhibited hypertrophic responses to β-adrenergic receptor stimulation and to aortic banding-induced pressure-overload. In rats, {Delta}Cain adenoviral gene transfer also inhibited cardiac calcineurin activity and reduced hypertrophic response to aortic banding-induced pressure-overload, suggesting the potential for future clinical applications.

Clearly, these genetic approaches have overcome several of the key disadvantages of the pharmacological approaches and their advantages include: (1) the absence of nonspecific abnormal phenotypes (e.g., weight loss) in the unstressed transgenic mice; (2) preserved T-cell function; and (3) no evidence of congestive heart failure despite prevention of cardiac hypertrophy.

The challenge now is to translate this exciting basic science from bench to clinical bedside.


   6. Conclusions
Top
 Abstract
 1. Introduction
 2. Calcineurin hypothesis of...
 3. Calcineurin inhibitors and...
 4. Calcineurin inhibitors and...
 5. New genetic approaches
 6. Conclusions
References
 
The current calcineurin inhibitors, when administered systemically to intact rodents, are blunt tools to probe calcineurin's role in pressure-overload hypertrophy. Genetic approaches to inhibit calcineurin have overcome some of the disadvantages of the pharmacological approach. The controversies surrounding the ability of calcineurin inhibition to prevent pressure-overload cardiac hypertrophy could be settled when potent and cardiac-specific calcineurin inhibitors become available, which go straight to the "heart" of the matter, and when cardiac calcineurin or NFAT knockout animals are made, in which the heart does not respond to hypertrophic stimuli.

Time for primary review 27 days.


   Acknowledgements
 
The author appreciates feedback from Drs. Ronald G. Victor, Gail D. Thomas, Robert Augustyniak, Eric N. Olson and Mr. Qiong Zhang during the preparation of this review. The studies cited from the author's lab were supported by NIH (HL44010).


   References
Top
 Abstract
 1. Introduction
 2. Calcineurin hypothesis of...
 3. Calcineurin inhibitors and...
 4. Calcineurin inhibitors and...
 5. New genetic approaches
 6. Conclusions
 References
 

  1. Kannel W.B, Dawber T.R, Kagan A, Revotskie V, Stokes J. Factors of risk in the development of coronary heart disease — six year follow-up experience. The Framingham Heart Study. Ann Intern Med (1961) 55:44–50.
  2. Kannel W.B, Gordon T, Offutt D. Left ventricular hypertrophy by electrocardiogram. Prevalence, incidence and the mortality in the Framingham Study. Ann Intern Med (1969) 71:89–105.[ISI][Medline]
  3. Rusnak F, Mertz P. Calcineurin: form and function. Physiol Rev (2000) 80:1483–1521.[Abstract/Free Full Text]
  4. Herzig T.C, Jobe S.M, Aoki H, et al. Angiotensin II type 1a receptor gene expression in the heart: AP-1 and GATA-4 participate in the response to pressure overload. Proc Natl Acad Sci USA (1997) 94:7543–7548.[Abstract/Free Full Text]
  5. Hasegawa K, Lee S.J, Jobe S.M, Markham B.E, Kitsis R.N. cis-Acting sequences that mediate induction of beta-myosin heavy chain gene expression during left ventricular hypertrophy due to aortic constriction. Circulation (1997) 96:3943–3953.[Abstract/Free Full Text]
  6. Molkentin J.D, Lu J.R, Antos C.L, et al. A calcineurin-dependent transcriptional pathway for cardiac hypertrophy. Cell (1998) 93:215–228.[CrossRef][ISI][Medline]
  7. Sussman M.A, Lim H.W, Gude N, et al. Prevention of cardiac hypertrophy in mice by calcineurin inhibition. Science (1998) 281:1690–1693.[Abstract/Free Full Text]
  8. Mende U, Kagen A, Cohen A, et al. Transient cardiac expression of constitutively active Galphaq leads to hypertrophy and dilated cardiomyopathy by calcineurin-dependent and independent pathways. Proc Natl Acad Sci USA (1998) 95:13893–13898.[Abstract/Free Full Text]
  9. Mervaala E, Muller D.N, Park J.K, et al. Cyclosporin A protects against angiotensin II-induced end-organ damage in double transgenic rats harboring human renin and angiotensinogen genes. Hypertension (2000) 35:360–366.[Abstract/Free Full Text]
  10. Lim H.W, De Windt L.J, Mante J, et al. Reversal of cardiac hypertrophy in transgenic disease models by calcineurin inhibition. J Mol Cell Cardiol (2000) 32:697–709.[CrossRef][ISI][Medline]
  11. Fu M, Xu S, Zhang J, et al. Involvement of calcineurin in angiotensin II-induced cardiomyocyte hypertrophy and cardiac fibroblast hyperplasia of rats. Heart Vessels (1999) 14:283–288.[ISI][Medline]
  12. Taigen T, De Windt L.J, Lim H.W, Molkentin J.D. Targeted inhibition of calcineurin prevents agonist-induced cardiomyocyte hypertrophy. Proc Natl Acad Sci USA (2000) 97:1196–1201.[Abstract/Free Full Text]
  13. He Q, LaPointe M.C. Interleukin-1beta regulates the human brain natriuretic peptide promoter via Ca(2+)-dependent protein kinase pathways. Hypertension (2000) 35:292–296.[Abstract/Free Full Text]
  14. Fatkin D, McConnell B.K, Mudd J.O, et al. An abnormal Ca(2+) response in mutant sarcomere protein-mediated familial hypertrophic cardiomyopathy. J Clin Invest (2000) 106:1351–1359.[ISI][Medline]
  15. Homcy C.J. Signaling hypertrophy: how many switches, how many wires. Circulation (1998) 97:1890–1892.[Free Full Text]
  16. Walsh R.A. Calcineurin inhibition as therapy for cardiac hypertrophy and heart failure: requiescat in pace? Circ Res (1999) 84:741–743.[Free Full Text]
  17. Hunter J.J, Chien K.R. Signaling pathways for cardiac hypertrophy and failure. New Engl J Med (1999) 341:1276–1283.[Free Full Text]
  18. Olson E.N, Molkentin J.D. Prevention of cardiac hypertrophy by calcineurin inhibition: hope or hype? Circ Res (1999) 84:623–632.[Free Full Text]
  19. McKinsey T.A, Olson E.N. Cardiac hypertrophy: sorting out the circuitry. Curr Opin Genet Dev (1999) 9:267–274.[CrossRef][ISI][Medline]
  20. Sugden P.H. Signaling in myocardial hypertrophy: life after calcineurin? Circ Res (1999) 84:633–646.[Free Full Text]
  21. Olson E.N, Williams R.S. Calcineurin signaling and muscle remodeling. Cell (2000) 101:689–692.[CrossRef][ISI][Medline]
  22. Chien K.R. Meeting Koch's postulates for calcium signaling in cardiac hypertrophy. J Clin Invest (2000) 105:1339–1342.[ISI][Medline]
  23. Sander M, Lyson T, Thomas G.D, Victor R.G. Sympathetic neural mechanisms of cyclosporine-induced hypertension. Am J Hypertens (1996) 9:121S–138S.[CrossRef][Medline]
  24. Lipkin G.W, Tucker B, Giles M, Raine A.E. Ambulatory blood pressure and left ventricular mass in cyclosporin- and non-cyclosporin-treated renal transplant recipients. J Hypertens (1993) 11:439–442.[CrossRef][ISI][Medline]
  25. van den Dorpel M.A, van den Meiracker A.H, Lameris T.W, et al. Cyclosporin A impairs the nocturnal blood pressure fall in renal transplant recipients. Hypertension (1996) 28:304–307.[Abstract/Free Full Text]
  26. Keitel E, Fuchs F.D, Costa C.A, et al. Loss of blood pressure circadian rhythm in renal transplant recipients taking cyclosporine. Transplant Proc (1997) 29:222.[CrossRef][ISI][Medline]
  27. Galiatsou E, Morris S.T.W, Jardine A.G, et al. Cardiac and vascular abnormalities in renal transplant patients: differential effects of cyclosporin and azathioprine. J Nephrol (2000) 13:185–192.[ISI][Medline]
  28. McKoy R.C, Uretsky B.F, Kormos R, et al. Left ventricular hypertrophy in cyclosporine-induced systemic hypertension after cardiac transplantation. Am J Cardiol (1988) 62:1140–1142.[CrossRef][ISI][Medline]
  29. Farge D, Julien J, Amrein C, et al. Effect of systemic hypertension on renal function and left ventricular hypertrophy in heart transplant recipients. J Am Coll Cardiol (1990) 15:1095–1101.[Abstract]
  30. Dart A.M, Yeoh J.K, Jennings G.L, Cameron J.D, Esmore D.S. Circadian rhythms of heart rate and blood pressure after heart transplantation. J Heart Lung Transplant (1992) 11:784–792.[ISI][Medline]
  31. Ventura H.O, Lavie C.J, Messerli F.H, et al. Cardiovascular adaptation to cyclosporine-induced hypertension. J Hum Hypertens (1994) 8:233–237.[ISI][Medline]
  32. Gill E.A, Borrego C, Bray B.E, et al. Left ventricular mass increases during cardiac allograft vascular rejection. J Am Coll Cardiol (1995) 25:922–926.[Abstract]
  33. Husain A.N, Siddiqui M.T, Reddy V.B, et al. Postmortem findings in lung transplant recipients. Mod Pathol (1996) 9:752–761.[ISI][Medline]
  34. Globits S, De Marco T, Schwitter J, et al. Assessment of early left ventricular remodeling in orthotopic heart transplant recipients with cine magnetic resonance imaging: potential mechanisms. J Heart Lung Transplant (1997) 16:504–510.[ISI][Medline]
  35. Schwitter J, De Marco T, Globits S, et al. Influence of felodipine on left ventricular hypertrophy and systolic function in orthotopic heart transplant recipients: possible interaction with cyclosporine medication. J Heart Lung Transplant (1999) 18:1003–1013.[CrossRef][ISI][Medline]
  36. Atkison P, Joubert G, Barron A, et al. Hypertrophic cardiomyopathy associated with tacrolimus in paediatric transplant patients. Lancet (1995) 345:894–896.[CrossRef][ISI][Medline]
  37. Chang R.K, Alzona M, Alejos J, Jue K, McDiarmid S.V. Marked left ventricular hypertrophy in children on tacrolimus (FK506) after orthotopic liver transplantation. Am J Cardiol (1998) 81:1277–1280.[CrossRef][ISI][Medline]
  38. Nakata Y, Yoshibayashi M, Yonemura T, et al. Tacrolimus and myocardial hypertrophy. Transplantation (2000) 69:1960–1962.[CrossRef][ISI][Medline]
  39. Burdmann E.A, Andoh T.F, Nast C.C, et al. Prevention of experimental cyclosporin-induced interstitial fibrosis by losartan and enalapril. Am J Physiol (1995) 269:F491–F499.[ISI][Medline]
  40. Mervaala E.M, Pere A.K, Lindgren L, et al. Effects of dietary sodium and magnesium on cyclosporin A-induced hypertension and nephrotoxicity in spontaneously hypertensive rats. Hypertension (1997) 29:822–827.[Abstract/Free Full Text]
  41. Zhang W, Kowal R.C, Rusnak F, et al. Failure of calcineurin inhibitors to prevent pressure-overload left ventricular hypertrophy in rats. Circ Res (1999) 84:722–728.[Abstract/Free Full Text]
  42. Shimoyama M, Hayashi D, Takimoto E, et al. Calcineurin plays a critical role in pressure overload-induced cardiac hypertrophy. Circulation (1999) 100:2449–2454.[Abstract/Free Full Text]
  43. Mervaala E, Lassila M, Vaskonen T, et al. Effects of ACE inhibition on cyclosporine A-induced hypertension and nephrotoxicity in spontaneously hypertensive rats on a high-sodium diet. Blood Pressure (1999) 8:49–56.[CrossRef][ISI][Medline]
  44. Takada S, Takahara S, Nishimura K, et al. Effect of hepatocyte growth factor on tacrolimus-induced nephrotoxicity in spontaneously hypertensive rats. Transpl Int (1999) 12:27–32.[CrossRef][ISI][Medline]
  45. Takeda Y, Miyamori I, Furukawa K, Inaba S, Mabuchi H. Mechanisms of FK 506-induced hypertension in the rat. Hypertension (1999) 33:130–136.[Abstract/Free Full Text]
  46. Lim H.W, De Windt L.J, Steinberg L, et al. Calcineurin expression, activation, and function in cardiac pressure-overload hypertrophy. Circulation (2000) 101:2431–2437.[Abstract/Free Full Text]
  47. Lassila M, Finckenberg P, Pere A.K, et al. Comparison of enalapril and valsartan in cyclosporine A-induced hypertension and nephrotoxicity in spontaneously hypertensive rats on high-sodium diet. Br J Pharmacol (2000) 130:1339–1347.[CrossRef][ISI][Medline]
  48. Luo Z, Shyu K.G, Gualberto A, Walsh K. Calcineurin inhibitors and cardiac hypertrophy. Nat Med (1998) 4:1092–1093.[CrossRef][ISI][Medline]
  49. Muller J.T, Nemoto S, Laser M, Carabello B.A, Menick D.R. Calcineurin inhibition and cardiac hypertrophy. Science (1998) 282:1007a.[Free Full Text]
  50. Ding B, Price R.L, Borg T.K, et al. Pressure overload induces severe hypertrophy in mice treated with cyclosporine, an inhibitor of calcineurin. Circ Res (1999) 84:729–734.[Abstract/Free Full Text]
  51. Meguro T, Hong C, Asai K, et al. Cyclosporine attenuates pressure-overload hypertrophy in mice while enhancing susceptibility to decompensation and heart failure. Circ Res (1999) 84:735–740.[Abstract/Free Full Text]
  52. Eto Y, Yonekura K, Sonoda M, et al. Calcineurin is activated in rat hearts with physiological left ventricular hypertrophy induced by voluntary exercise training. Circulation (2000) 101:2134–2137.[Abstract/Free Full Text]
  53. Hill J.A, Karimi M, Kutschke W, et al. Cardiac hypertrophy is not a required compensatory response to short-term pressure overload. Circulation (2000) 101:2863–2869.[Abstract/Free Full Text]
  54. Hayashida W, Kihara Y, Yasaka A, Sasayama S. Cardiac calcineurin during transition from hypertrophy to heart failure in rats. Biochem Biophys Res Commun (2000) 273:347–351.[CrossRef][ISI][Medline]
  55. Øie E, Bjornerheim R, Clausen O.P, Attramadal H. Cyclosporin A inhibits cardiac hypertrophy and enhances cardiac dysfunction during postinfarction failure in rats. Am J Physiol Heart Circ Physiol (2000) 278:H2115–2123.[Abstract/Free Full Text]
  56. Shimoyama M, Hayashi D, Zou Y, et al. Calcineurin inhibitor attenuates the development and induces the regression of cardiac hypertrophy in rats with salt-sensitive hypertension. Circulation (2000) 102:1996–2004.[Abstract/Free Full Text]
  57. Sakata Y, Masuyama T, Yamamoto K, et al. Calcineurin inhibitor attenuates left ventricular hypertrophy, leading to prevention of heart failure in hypertensive rats. Circulation (2000) 102:2269–2275.[Abstract/Free Full Text]
  58. Murat A, Pellieux C, Brunner H.R, Pedrazzini T. Calcineurin blockade prevents cardiac mitogen-activated protein kinase activation and hypertrophy in renovascular hypertension. J Biol Chem (2000) 275:40867–40873.[Abstract/Free Full Text]
  59. Spencer C.M, Goa K.L, Gillis J.C. Tacrolimus. An update of its pharmacology and clinical efficacy in the management of organ transplantation. Drugs (1997) 54:925–975.[ISI][Medline]
  60. Taler S.J, Textor S.C, Canzanello V.J, Schwartz L. Cyclosporin-induced hypertension: incidence, pathogenesis and management. Drug Saf (1999) 20:437–449.[CrossRef][ISI][Medline]
  61. Olyaei A.J, de Mattos A.M, Bennett W.M. Immunosuppressant-induced nephropathy: pathophysiology, incidence and management. Drug Saf (1999) 21:471–488.[CrossRef][ISI][Medline]
  62. Plosker G.L, Foster R.H. Tacrolimus: a further update of its pharmacology and therapeutic use in the management of organ transplantation. Drugs (2000) 59:323–389.[CrossRef][ISI][Medline]
  63. Sitsen J.M, de Jong W. Development of high blood pressure in spontaneously hypertensive rats is delayed by treatment with cyclosporin at an early age. Experientia (1987) 43:403–405.[CrossRef][ISI][Medline]
  64. Morgan B.J, Lyson T, Scherrer U, Victor R.G. Cyclosporine causes sympathetically mediated elevations in arterial pressure in rats. Hypertension (1991) 18:458–466.[Abstract/Free Full Text]
  65. Textor S.C, Canzanello V.J, Taler S.J, et al. Cyclosporine-induced hypertension after transplantation. Mayo Clin Proc (1994) 69:1182–1193.[ISI][Medline]
  66. Lyson T, McMullan D.M, Ermel L.D, Morgan B.J, Victor R.G. Mechanism of cyclosporine-induced sympathetic activation and acute hypertension in rats. Hypertension (1994) 23:667–675.[Abstract/Free Full Text]
  67. Hohage H, Bruckner D, Arlt M, et al. Influence of cyclosporine A and FK506 on 24 h blood pressure monitoring in kidney transplant recipients. Clin Nephrol (1996) 45:342–344.[ISI][Medline]
  68. Oriji G.K, Keiser H.R. Role of nitric oxide in cyclosporine A-induced hypertension. Hypertension (1998) 32:849–855.[Abstract/Free Full Text]
  69. Morris S.T, McMurray J.J, Rodger R.S, Farmer R, Jardine A.G. Endothelial dysfunction in renal transplant recipients maintained on cyclosporine. Kidney Int (2000) 57:1100–1106.[CrossRef][ISI][Medline]
  70. Zhang W, Victor R.G. Calcineurin inhibitors cause renal afferent activation in rats: a novel mechanism of cyclosporine-induced hypertension. Am J Hypertens (2000) 13:999–1004.[CrossRef][ISI][Medline]
  71. Zhang W, Li J.L, Hosaka M, et al. Cyclosporine A-induced hypertension involves synapsin in renal sensory nerve endings. Proc Natl Acad Sci USA (2000) 97:9765–9770.[Abstract/Free Full Text]
  72. Duruibe V.A, Okonmah A, Panton L, Blyden G.T. Effect of cyclosporin A on rat kidney catecholamines. Life Sci (1990) 47:255–261.[CrossRef][ISI][Medline]
  73. Chiu P.J, Vemulapalli S, Sabin C, et al. Sympathoadrenal stimulation, not endothelin, plays a role in acute pressor response to cyclosporine in anesthetized rats. J Pharmacol Exp Ther (1992) 261:994–999.[Abstract/Free Full Text]
  74. Grobecker H.F, Riebel K, Wellenhofer T. Cyclosporine A-induced hypertension in SHR and WKY: role of the sympatho-adrenal system. Clin Exp Pharmacol Physiol (1995) 22(Suppl_1):S94–S95.[CrossRef]
  75. Reis F, Tavares P, Teixeira F. The distribution of catecholamines between plasma and platelets in cyclosporin A-induced hypertensive rats. Pharmacol Res (2000) 41:129–135.[CrossRef][ISI][Medline]
  76. Cohn J.N. Sympathetic nervous system activity and the heart. Am J Hypertens (1989) 2:353S–356S.[Medline]
  77. Julius S. Effect of sympathetic overactivity on cardiovascular prognosis in hypertension. Eur Heart J (1998) 19(Suppl F):F14–F18.[ISI][Medline]
  78. el Gamel A, Keevil B, Rahman A, et al. Cardiac allograft rejection: do trough cyclosporine levels correlate with the grade of histologic rejection? J Heart Lung Transplant (1997) 16:268–274.[ISI][Medline]
  79. Chin E.R, Olson E.N, Richardson J.A, et al. A calcineurin-dependent transcriptional pathway controls skeletal muscle fiber type. Genes Dev (1998) 12:2499–2509.[Abstract/Free Full Text]
  80. Rusnak F, Beressi A.H, Haddy A, Tefferi A. Calcineurin protein phosphatase activity in peripheral blood lymphocytes. Bone Marrow Transplant (1996) 17:309–313.[ISI][Medline]
  81. Linz W, Scholkens B.A, Ganten D. Converting enzyme inhibition specifically prevents the development and induces regression of cardiac hypertrophy in rats. Clin Exp Hypertens [A] (1989) 11:1325–1350.[ISI][Medline]
  82. Linz W, Schaper J, Wiemer G, Albus U, Scholkens B.A. Ramipril prevents left ventricular hypertrophy with myocardial fibrosis without blood pressure reduction: a one year study in rats. Br J Pharmacol (1992) 107:970–975.[ISI][Medline]
  83. Pfeffer J.M, Pfeffer M.A, Mirsky I, Braunwald E. Regression of left ventricular hypertrophy and prevention of left ventricular dysfunction by captopril in the spontaneously hypertensive rat. Proc Natl Acad Sci USA (1982) 79:3310–3314.[Abstract/Free Full Text]
  84. Pfeffer J.M, Pfeffer M.A, Braunwald E. Influence of chronic captopril therapy on the infarcted left ventricle of the rat. Circ Res (1985) 57:84–95.[Abstract/Free Full Text]
  85. Pfeffer J.M, Pfeffer M.A. Angiotensin converting enzyme inhibition and ventricular remodeling in heart failure. Am J Med (1988) 84:37–44.[ISI][Medline]
  86. Rockman H.A, Wachhorst S.P, Mao L, Ross J Jr. ANG II receptor blockade prevents ventricular hypertrophy and ANF gene expression with pressure overload in mice. Am J Physiol (1994) 266:H2468–H2475.[ISI][Medline]
  87. Bruckschlegel G, Holmer S.R, Jandeleit K, et al. Blockade of the renin–angiotensin system in cardiac pressure-overload hypertrophy in rats. Hypertension (1995) 25:250–259.[Abstract/Free Full Text]
  88. Kim S, Yoshiyama M, Izumi Y, et al. Effects of combination of ACE inhibitor and angiotensin receptor blocker on cardiac remodeling, cardiac function, and survival in rat heart failure. Circulation (2001) 103:148–154.[Abstract/Free Full Text]
  89. Ryffel B, Weber E, Mihatsch M.J. Nephrotoxicity of immunosuppressants in rats: comparison of macrolides with cyclosporin. Exp Nephrol (1994) 2:324–333.[ISI][Medline]
  90. Sadoshima J, Izumo S. Rapamycin selectively inhibits angiotensin II-induced increase in protein synthesis in cardiac myocytes in vitro. Potential role of 70-kD S6 kinase in angiotensin II-induced cardiac hypertrophy. Circ Res (1995) 77:1040–1052.[Abstract/Free Full Text]
  91. Boluyt M.O, Zheng J.S, Younes A, et al. Rapamycin inhibits alpha 1-adrenergic receptor-stimulated cardiac myocyte hypertrophy but not activation of hypertrophy-associated genes. Evidence for involvement of p70 S6 kinase. Circ Res (1997) 81:176–186.[Abstract/Free Full Text]
  92. Perico N, Remuzzi G. Prevention of transplant rejection: current treatment guidelines and future developments. Drugs (1997) 54:533–570.[ISI][Medline]
  93. De Windt L.J, Lim H.W, Bueno O.F, et al. Targeted inhibition of calcineurin attenuates cardiac hypertrophy in vivo. Proc Natl Acad Sci USA (2001) 98:3322–3327.[Abstract/Free Full Text]
  94. Rothermel B.A, McKinsey T.A, Vega R.B, et al. Myocyte-enriched calcineurin-interacting protein, MCIP1, inhibits cardiac hypertrophy in vivo. Proc Natl Acad Sci USA (2001) 98:3328–3333.[Abstract/Free Full Text]

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