Differential Cardiac Remodeling in Preload Versus Afterload
Background— Hemodynamic load regulates myocardial function and gene expression. We tested the hypothesis that afterload and preload, despite similar average load, result in different phenotypes.
Methods and Results— Afterload and preload were compared in mice with transverse aortic constriction (TAC) and aortocaval shunt (shunt). Compared with sham mice, 6 hours after surgery, systolic wall stress (afterload) was increased in TAC mice (+40%; P<0.05), diastolic wall stress (preload) was increased in shunt (+277%; P<0.05) and TAC mice (+74%; P<0.05), and mean total wall stress was similarly increased in TAC (69%) and shunt mice (67%) (P=NS, TAC versus shunt; each P<0.05 versus sham). At 1 week, left ventricular weight/tibia length was significantly increased by 22% in TAC and 29% in shunt mice (P=NS, TAC versus shunt). After 24 hours and 1 week, calcium/calmodulin-dependent protein kinase II signaling was increased in TAC. This resulted in altered calcium cycling, including increased L-type calcium current, calcium transients, fractional sarcoplasmic reticulum calcium release, and calcium spark frequency. In shunt mice, Akt phosphorylation was increased. TAC was associated with inflammation, fibrosis, and cardiomyocyte apoptosis. The latter was significantly reduced in calcium/calmodulin-dependent protein kinase IIδ-knockout TAC mice. A total of 157 mRNAs and 13 microRNAs were differentially regulated in TAC versus shunt mice. After 8 weeks, fractional shortening was lower and mortality was higher in TAC versus shunt mice.
Conclusions— Afterload results in maladaptive fibrotic hypertrophy with calcium/calmodulin-dependent protein kinase II–dependent altered calcium cycling and apoptosis. Preload is associated with Akt activation without fibrosis, little apoptosis, better function, and lower mortality. This indicates that different loads result in distinct phenotype differences that may require specific pharmacological interventions.
Received June 5, 2009; accepted June 24, 2010.
In the heart, hemodynamic load is a critical regulator of myocardial function, gene expression, and phenotype appearance.1 Specific structures involved in perception of hemodynamic load have been identified, and influencing or deleting these structures has been associated with cardiac dysfunction and disease.2 Two types of load can be differentiated. Preload builds up during diastolic filling and stretches cardiomyocytes. This results in immediate recruitment of contractile units and increased cardiac performance through the Frank-Starling mechanism. In addition, proteins such as titin and associated molecules are stretched with subsequent effects on myocardial elasticity and gene expression.3 Systolic force matching afterload is generated by each cardiomyocyte to produce cardiac stroke work against vascular resistance. This is accomplished by the contractile protein complex. During ejection, preload declines and titin is unloaded. Both preload and afterload influence load-dependent ion channels and intracellular ion concentrations,4 which in turn may also influence cardiac function and gene expression. From a hemodynamic point of view, afterload-mediated concentric hypertrophy was considered beneficial because of stress compensation through increased wall thickness according to the law of Laplace.5 In contrast, preload-mediated eccentric hypertrophy was considered maladaptive because of uncompensated wall stress. However, cardiac geometry and macroscopic phenotype are only one aspect. Myocardial hypertrophic phenotype (ie, the protein composition of the myocardium) is another, and there is good evidence that the latter may be more relevant to the transition to heart failure.6
Clinical Perspective on p 1003
In previous studies in isolated muscle preparations, we showed that preload and afterload differentially regulate expression of fetal genes.7,8 The present study was performed to compare differences in phenotypes, signaling, and gene expression of preload- and afterload-induced hypertrophy in vivo. Therefore, we used the aorta–vena cava mouse fistula model (shunt), which generates volume overload and predominantly increased preload, and the transverse aortic constriction (TAC) model, which generates pressure overload and reflects increased afterload. Shunt and TAC mice were graded to match average load, measured as average left ventricular wall stress. Our data show that eccentric hypertrophy in shunt mice is more beneficial than concentric hypertrophy in TAC mice with increased inflammation, fibrosis, and cardiomyocyte apoptosis. The shunt model is associated with Akt activation, whereas the TAC model is associated with altered calcium cycling and calcium/calmodulin-dependent protein kinase II (CaMKII)9 activation.
Only a short description of Methods is given here. An expanded version can be found in the online-only Data Supplement.
Animal Experiments and In Vivo Characterization
The investigation conforms to the Guide for the Care and Use of Laboratory Animals (National Institutes of Health publication No. 85–23, revised 1996). In 12-week-old female mice, volume overload was induced by the creation of a shunt between the aorta and vena cava inferior. Pressure overload was induced by TAC. Female mice were used because of high mortality in male mice. Echocardiography, in vivo hemodynamic measurements, cardiomyocyte isolation, cardiomyocyte shortening, calcium measurements, and patch-clamp experiments were performed with standard protocols.
Protein and gene expression were measured with standard protocols of Western immunoblots and quantitative real-time polymerase chain reaction (Biorad iQ-Cycler). Fibrosis, cardiomyocyte apoptosis, and inflammation were quantified in histological sections. For measurement of the cell cycle rate, 3H-thymidine autoradiography was measured. The 11.0 miRCURY LNA microRNA array (Exiqon, Denmark) was used for microRNA, and the Affymetrix mouse 430 2.0 GeneChip array was used for gene expression analysis. Gene expression microarray data have been deposited in the ArrayExpress database (accession No. E-MEXP-2498).
Calculation and Statistical Analysis
Data are presented as mean±SEM. P<0.05 was considered statistically significant. Gene- and protein-expression and electrophysiological experiments were analyzed statistically with the use of unpaired Student t test, 1-way ANOVA on ranks (Dunn’s method), or 1-way ANOVA followed by the Tukey post hoc test, where appropriate. Survival was analyzed by Kaplan-Meier and Fisher tests. Wall stress was calculated according to the law of Laplace. Raw microarray data were imported into R version 2.9.1 and analyzed with Bioconductor packages. Pathway analysis was performed by the gene set enrichment analysis approach with the use of the Category and GSEABase Bioconductor packages, querying the Kyoto Encyclopedia of Genes and Genomes pathway database (for references, see the statistical section of the online-only Data Supplement). The numbers of animals or cells are shown in the figure legends in the following order: n=sham (TAC)/TAC/sham (shunt)/shunt.
Hemodynamic Function and Wall Stress
Conductance catheter pressure volume analysis 6 hours after respective surgical procedures (Figure 1A) showed that left ventricular systolic pressure was increased in the TAC group (+41%; P<0.05), and left ventricular end-diastolic pressure and volume were increased in the shunt group (+97%; P<0.05; Figure I in the online-only Data Supplement). Wall stress was calculated at 4 time points during the cardiac cycle (midsystolic, end-systolic, mid-diastolic, end-diastolic; Figure IIA in the online-only Data Supplement). In the TAC group, midsystolic wall stress was increased by 40% (P<0.05; Figure IIB in the online-only Data Supplement), and end-diastolic wall stress was increased by 277% in the shunt group (P<0.05) and by 74% in the TAC group (each P<0.05; Figure IIE in the online-only Data Supplement). Mean total wall stress during 1 cardiac contraction-relaxation cycle yielded similar values for TAC and shunt groups (TAC, 69%; shunt, 67%; each P<0.05 versus sham; Figure 1B), which indicates similar average load elevation immediately after surgery in both models. Echocardiographic measurements 24 hours after intervention confirmed left ventricular dilatation in the shunt group by increased left ventricular end-diastolic diameter (P<0.05), whereas fractional shortening was not changed at this time point (Figure III in the online-only Data Supplement).
Remodeling and Left Ventricular Hypertrophy
After 1 week, left ventricular hypertrophy as indicated by left ventricular weight per tibia length was increased similarly in both models, being concentric in the TAC group and eccentric in the shunt group (TAC, +22%; shunt, +29%; each P<0.01; TAC versus shunt, P=NS; Figure 1C). Cardiomyocyte minimal fiber diameter was increased in both models (Figures 1D and 1E), whereas cardiomyocyte length was increased only in the shunt group (Figures 1D and 1F). Echocardiographic characterization is shown in the online-only Data Supplement (Figure IV in the online-only Data Supplement). At this time point, myocardial function was not reduced in either model (Figure IVD in the online-only Data Supplement).
Hypertrophy in the TAC group was associated with a significant increase in myocardial fibrosis (perivascular fibrosis +490%; P<0.01; Figure 2A and 2B). Cell cycle rate showed a clear increase in TAC in the noncardiomyocytes but not in the shunt group (Figure 2A; for discrimination of cardiomyocytes and noncardiomyocytes, nuclear-ßGAL transgenic mice were used). This suggests that increased fibrosis in the TAC group resulted in part from fibroblast proliferation. In addition, inflammation was increased in TAC by 114% (P<0.001; Figure 2A and 2C). In the shunt group, fibrosis and inflammation were not elevated (Figure 2A through 2C). Cardiomyocyte apoptosis was elevated in both the TAC and shunt groups, but it was significantly higher in the TAC group (Figure 2D through 2F).
Long-Term Myocardial Function and Mortality
Eight weeks of increased load were associated with a moderate increase in septum thickness in the shunt group (+7%; P<0.05) and a large increase in TAC (+39%; P<0.01; Figure 3A). End-diastolic diameter was increased in the TAC group by 14% (P<0.05) and in the shunt group by 38% (P<0.01; Figure 3B). Fractional shortening was considerably reduced in the TAC group (−38%; P<0.01), whereas it was only slightly reduced in the shunt group (−18%; P<0.01 versus sham, P<0.01 versus TAC; Figure 3D). After 8 weeks, the percentage of surviving animals was significantly lower in the TAC group (4/13) than in the shunt group (7/10; P<0.05). In addition, Kaplan-Meier analysis yielded significantly higher mortality in the TAC versus the shunt group (P<0.05; Figure 3E).
Load-Dependent Regulation of Gene Expression and Signal Transduction Pathways
Left ventricular gene expression and protein phosphorylation were analyzed 24 hours and 7 days after intervention to show persistent activation (Tables 1 and 2⇓). Brain natriuretic peptide (BNP) was upregulated at both time points only in the TAC group (24 hours, +507%, P<0.05; 7 days, +384%, P<0.05; Table 1). An isoform shift from α-myosin heavy chain (MHC) to β-MHC at these time points occurred in the TAC but not in the shunt model (Table 1). Upregulation of ß-MHC gene expression in the TAC model already occurred after 24 hours. The expression of SERCA and the other calcium-regulated proteins was not changed at 24 hours and 7 days after intervention in both models, with the exception of a transient upregulation of NCX in TAC (Table 1).
CaMKII, histone deacetylase, Akt, GSK3ß, and mitogen-activated protein kinase expression and activation were measured with specific antibodies as well as with phosphospecific antibodies (Figures V and VI in the online-only Data Supplement). If protein expression was not altered, the ratio of phosphorylated protein to total protein is shown. When total protein expression was altered, phosphorylated protein normalized to GAPDH as well as the ratio of phosphorylated protein to total protein and total protein expression to GAPDH is shown. An absolute increase of phosphorylated protein is considered to reflect increased biological activity.
Increased biological activity at 24 hours and 7 days was seen only for CaMKII and Akt. In TAC, the biological CaMKII activity was increased after 24 hours and 7 days by 78% and 82%, respectively (Figure 4A and 4B and Table 2). None of these changes were observed in the shunt group (Figure 4A and 4B and Table 2), whereas Akt phosphorylation was increased in the shunt group exclusively (24 hours, +70%, P<0.05; 7 days, 41%, P<0.05; Table 2).
The other kinases studied were not consistently activated at 24 hours and 7 days: p38 phosphorylation but not total biological activity was increased at both time points in the TAC group (Table 2). Changes in ERK phosphorylation also occurred only in the TAC group (24 hours, +74%, P<0.05; 7 days, −61%, P<0.05; Table 2). In addition, histone deacetylase phosphorylation was increased only in the TAC group (Table 2). GSK3β phosphorylation was increased in the shunt group after 24 hours (+26%, P<0.05; Table 2). mRNA expression of myocyte-enriched calcineurin-interacting protein as an indicator of calcineurin activity was selectively upregulated after 7 days in TAC (myocyte-enriched calcineurin-interacting protein, +454%, P<0.05; Table 2).
Analysis of left ventricular human heart samples from patients with aortic stenosis exhibiting myocardial hypertrophy but still preserved cardiac function (see Methods in the online-only Data Supplement) showed an increase in CaMKIIδ expression compared with samples from control hearts that were not hypertrophied (+24.7±9.7%; Figure 4C and 4D).
Gene Array Analysis
To further define the molecular signature of the 2 load models, we performed genome-wide expression profiling. Applying a moderated linear model and the false discovery rate method for multiple testing with a threshold of 5% resulted in 1399 (1954 probes sets) upregulated and 1513 (1896 probes sets) downregulated annotated genes in the TAC compared with the sham group. In the shunt group compared with sham, 315 (384 probes sets) upregulated and 704 (853 probes sets) downregulated annotated genes were identified (Figure 5A and 5B and Tables I and II in the online-only Data Supplement). Comparing the differences between TAC and shunt groups directly (up or down) resulted in 157 differentially expressed genes (187 probes sets; Table III in the online-only Data Supplement). Unsupervised hierarchical clustering of these 157 genes (187 probes sets) resulted in accurate identification of the cardiac gene expression profiles of individual animals in the appropriate groups (Figure VII in the online-only Data Supplement). Of the candidate genes identified by the aforementioned protein analysis, an increased expression of CaMKIIδ was selectively found in the TAC but not in the shunt group. Of the 157 differentially expressed genes, 122 were regulated only in TAC, 21 were regulated only in shunt, and 14 were significantly regulated in both models. Of the 14 genes, 6 were regulated in parallel but at a different amount and 8 in an opposite direction (brain-derived neurotrophic factor; protein phosphatase 1B, magnesium dependent, beta isoform; c-myc binding protein; RIKEN complementary DNA 1190002N15 gene; phosphodiesterase 4D interacting protein [myomegalin]; serine/threonine kinase 38 like; amine oxidase, copper-containing 3; B and T lymphocyte associated).
Furthermore, pathway analysis revealed 90 pathways selectively regulated in the TAC group, 10 pathways similarly regulated in the TAC and shunt groups, and 4 pathways selectively regulated in the shunt group (Tables IV and V in the online-only Data Supplement). We confirmed cardiomyocyte apoptosis, inflammation, increased cell cycle activity, and fibrosis in the pathway analysis confined to the TAC model. Among the selectively regulated pathways in the shunt model, activation of the Wnt signaling is of special interest (Table 3).
Load-Dependent Regulation of MicroRNAs
The expression of microRNAs was evaluated in both models. At 24 hours, none of the microRNAs assayed by microarray exhibited differential expression between the experimental groups (data not shown). After 7 days of subjection to load, we identified 13 microRNAs differentially regulated between TAC and shunt groups. Of these, 9 were selectively regulated in the TAC model, 3 were selectively regulated in the shunt model, and 1 was regulated in parallel but at significantly different amounts in both models (Figure 5C through 5E).
Single Cell Function
Seven days after intervention, increased cardiomyocyte fractional shortening (+24%; P<0.05) and intracellular calcium transients (+13%; P<0.05) were seen at a 1-Hz stimulation rate in TAC mice (Figure 6A, 6B, 6E, and 6F). Sarcoplasmic reticulum (SR) calcium load was unchanged, whereas fractional SR calcium release was significantly increased in TAC mice by 27% (Figure 6G and 6H). In contrast, in cardiomyocytes from shunt mice, fractional shortening and calcium cycling were not different from those of sham mice (Figure 6C through 6H).
Mechanisms underlying the observed alteration in calcium cycling in TAC mice were studied in detail. The increase in SR calcium fractional release was clearly CaMKII dependent because KN-93, a CaMKII inhibitor, normalizes SR calcium fractional release (Figure 7A). The enhancement of fractional SR calcium release in TAC mice could be due to increased L-type calcium current or increased ryanodine receptor sensitivity, both regulated by CaMKII.9 Calcium spark frequency as an estimate of ryanodine receptor sensitivity was largely increased in TAC mice (+206%, P<0.001; Figure 7B and 7C). Measurement of action potentials as an indicator of calcium influx showed a prolongation of the action potential duration (APD90) in TAC compared with sham mice (P<0.001; Figure 7D and 7E). This prolongation could be abolished by CaMKII inhibition with autocamtide-2 related inhibitory peptide (P<0.001) or L-type calcium channel inhibition with nifedipine (P<0.001; Figure 7D and 7E). Furthermore, direct measurement of L-type calcium current showed a significant increase in TAC versus sham mice (P<0.05), which could be normalized by addition of the CaMKII inhibitor autocamtide-2 related inhibitory peptide (P<0.05; Figure 7F and 7G). This indicates that increased L-type calcium current is the primary mechanism of the APD prolongation and suggests that increased fractional release results from both increased L-type current and increased ryanodine receptor sensitivity.
Analysis of CaMKIIδ-Knockout Mice
To better understand the role of CaMKII activation in pressure-overload hypertrophy, we used the CaMKIIδ-knockout mouse model.10 One week after TAC, the amount of perivascular fibrosis was similarly increased in wild-type and knockout mice (Figure 8A and 8B). However, the amount of cardiomyocyte apoptosis after TAC was significantly lower in the knockout compared with the wild-type hearts (P<0.01; Figure 8C).
The present study shows that with a comparable elevation of wall stress myocardial structure, largely different molecular phenotypes develop with preload versus afterload, although the extent of cardiac hypertrophy is similar. (1) Increased afterload with TAC resulted in increased BNP expression, which was not seen by increased preload in the shunt model during the first 7 days. (2) TAC resulted in concentric hypertrophy with increased fibrosis, inflammation, and cardiomyocyte apoptosis, whereas eccentric hypertrophy in the shunt model occurred without increased fibrosis, without inflammation, and with less cardiomyocyte apoptosis. (3) TAC hypertrophy resulted in development of severe left ventricular dysfunction and higher mortality compared with shunt hypertrophy. (4) Signaling analysis showed that the hypertrophic phenotype in TAC was associated with persistent activation of CaMKII and disturbed intracellular calcium cycling. None of these changes happened in the shunt model, in which Akt was persistently activated. (5) CaMKIIδ-knockout mice and pharmacological CaMKII inhibition led to normalization of disturbed calcium cycling and a reduced rate of cardiomyocyte apoptosis. We conclude that afterload results in disturbed calcium cycling and CaMKII activation and maladaptive hypertrophy, whereas preload results in a more favorable type of hypertrophy through stretch-mediated activation of Akt.
Load and neurohormones regulate the function and phenotypic characteristics of the myocardium. Afterload results in a concentric hypertrophic phenotype that was often viewed as compensatory in nature because increased wall thickness reduces wall stress according to the law of Laplace. Preload results in eccentric hypertrophy. Although earlier studies suggested that volume overload with uncompensated wall stress would be disadvantageous, more recent studies suggested that volume overload was associated with a more favorable remodeling compared with pressure overload.11,12 The present study shows that with identical wall stress myocardial structure, cardiac function and mortality are more favorable with the shunt model than with the TAC model. This suggests that elevation of wall stress by itself may not be a critical issue. Indeed, genetically modified mice with improved cardiac function and prognosis after TAC exhibited modified protein expression but absence of hypertrophic response.13 Thus, the composition of the myocardium in response to increased load by compensation on a molecular level may be more relevant than the degree of hypertrophy (ie, wall thickness).
We studied signaling at 24 hours, assuming that at this early time point load-mediated activation of signaling cascades and gene expression dominates over secondary effects such as neurohumoral activation, and we studied signaling at 7 days to identify those signals being consistently activated by load. The maladaptive phenotype in TAC can be partially attributed to CaMKII signaling, which is functionally associated with defective calcium cycling. Wang et al14,15 suggested previously that afterload activates L-type calcium current. The present study indicates that increased L-type calcium current predominantly results from CaMKII activation because it can be reversed with CaMKII inhibition. Furthermore, CaMKIIδ activation induces cardiomyocyte apoptosis in TAC, as indicated by the reduced rate of cardiomyocyte apoptosis in the CaMKIIδ-knockout animals. This confirms in vivo the recent suggestions in an in vitro study showing that CaMKII inhibition prevented cardiomyocyte apoptosis after angiotensin II exposure of rat and mouse cardiomyocytes.16 Furthermore, the mechanistic relevance of CaMKII-dependent cardiomyocyte apoptosis for the development of the maladaptive hypertrophic phenotype is supported by previous data showing that in CaMKIIδ-knockout mice, progression to heart failure is reduced after TAC.17 Interestingly, in a previous study we showed reduced interstitial fibrosis in TAC in CaMKIIδ-knockout mice at 3 weeks.10 This previous work also showed that CaMKIIδ-knockout does not result in upregulation of other CaMK isoforms. This suggests that increased cardiomyocyte apoptosis may be a major component of CaMKII-mediated maladaptive hypertrophy during increased afterload. Interstitial fibrosis at later stages may reflect replacement fibrosis after cardiomyocyte apoptosis. However, these data also suggest that early perivascular fibrosis (1 week) occurs independently from CaMKII activity and is possibly related to inflammation.
In shunt-induced preload elevation, calcium cycling is normal, and none of the afterload-related signals are activated. In contrast, Akt is activated, which is generally believed to promote a more adaptive hypertrophic phenotype.18 One may speculate that Akt could be activated by a preload-mediated stretch of titin or related proteins involved in the sensing of preload.
Interestingly, ventricular BNP expression occurs only with increased afterload and not with elevated preload. This supports for the first time in vivo the suggestions from previous in vitro studies.7,8 BNP may prevent more pronounced hypertrophy in TAC.
Gene array analysis shows a largely different gene expression pattern between TAC and shunt models. Interestingly, 8 genes were significantly regulated in the opposite direction in both models. Among these, the phosphodiesterase 4D interacting protein (myomegalin) is upregulated in the TAC and downregulated in the shunt model. Myomegalin is localized in the Z-disc,19 interacts with the phosphodiesterase 4D, and is also involved in regulation of stress-dependent nuclear trafficking of myopodin.20 Furthermore, gene array pathway analysis confirms an activation of inflammation, fibrosis, and cardiomyocyte apoptosis pathways in the TAC model (Table 3), whereas in the shunt model, selective activation of the Wnt pathway is an interesting finding. The Wnt pathway may influence eccentric hypertrophy via Akt, which was shown here to be consistently activated by phospho-protein analysis.21
MicroRNAs control expression of gene clusters. MicroRNA array analysis also identifies significant differences between both load models. These include absence of regulation of microRNAs 133, 30, and 208 in the shunt model, which are regulated in TAC and associated with hypertrophy and fibrosis.22–24 Furthermore, microRNA 208 expression is necessary for ß-MHC upregulation after TAC.23 Here microRNA 208b upregulation is paralleled by ß-MHC upregulation. MicroRNA 140*, 320, and 455 are regulated in shunt but not in TAC models. MicroRNA 140* and 320 have been described as upregulated in TAC22 or human heart failure,25 and upregulation of microRNA 320 is associated with apoptosis.26 MicroRNA 455 has not been described previously to be regulated in the heart. Interestingly, microRNA regulation was not observed after 24 hours following surgery. This may suggest that microRNAs regulate later changes in gene expression during overload. Differentially regulated genes in TAC and shunt models support qualitative differences in both load forms and eliminate concerns that preload is just a milder stress than afterload. The mechanistic relevance of newly identified genes and pathways in load-dependent cardiac hypertrophy is hypotheses generating and warrants further studies with differential consideration of preload and afterload.
Pathophysiological and Therapeutic Implications
TAC is associated with inflammation, fibrosis, and cardiomyocyte apoptosis, which may impair myocardial function directly. Moreover, fibrosis may critically increase the diffusion distance of oxygen to myocytes and thereby influence cardiac energetics and function.27 In the shunt group, no inflammation and fibrosis were observed. A small level of apoptosis may be the mechanism underlying late decline of cardiac function.28 The present data also suggest that increased load may require differential pharmacological interventions depending on the contribution of preload and afterload. With increased afterload such as occurs in arterial hypertension or aortic stenosis, strategies to inhibit CaMKII signaling may be beneficial by reducing cardiomyocyte apoptosis. In diseases associated with increased preload predominantly, such as mitral regurgitation or aortic regurgitation, CaMKII inhibition would not be a rational approach to treat this type of hypertrophy.
The present findings may also be relevant for the design of experimental studies. Many studies use the TAC model in genetically modified mice to study the influence of genes on increased load. One should now consider that the shunt model may provide additional information.29,30 Furthermore, load experiments in isolated cardiomyocytes are frequently performed by flex membrane stretching of cells. Under those circumstances, it should be taken into account that stretch of cardiomyocytes may have completely different effects depending on its occurrence in diastole, where it elevates preload, or in systole, where it elevates afterload.
We are grateful to Brigitte Korff and Thomas Sowa for excellent technical assistance.
Sources of Funding
This work was supported by the Deutsche Forschungsgemeinschaft (grant Klinische Forschergruppe 155: TP1,3,4,5,7,8 [KO 1872/2–1 to Dr Hasenfuss; SE 1117/1–1 to Dr Seidler; LE 1313/2–1 to Dr Lehnart; MA 1982/2–2 to Dr Maier; EN 84/21–1 to Dr Schäfer; LI 690/7–2 to Dr Linke]), National Institutes of Health grant HL085098 (to Dr Field), Emmy-Noether Program (BA2258–2-1 to Dr Backs), and EUGeneHeart (project No. LSHM-CT-2005–018833). Dr Maier is also funded by a DFG-Heisenberg grant (MA 1982/4–1) and the Fondation Leducq Award to the Alliance for Calmodulin-Kinase Signaling in Heart Disease.
Linke WA. Sense and stretchability: the role of titin and titin-associated proteins in myocardial stress-sensing and mechanical dysfunction. Cardiovasc Res. 2008; 77: 637–648.
von Lewinski D, Stumme B, Fialka F, Luers C, Pieske B. Functional relevance of the stretch-dependent slow force response in failing human myocardium. Circ Res. 2004; 94: 1392–1398.
Norton JM. Toward consistent definitions for preload and afterload. Adv Physiol Educ. 2001; 25: 53–61.
Kögler H, Schott P, Toischer K, Milting H, Nguyen van P, Kohlhaas M, Grebe C, Kassner A, Domeier E, Teucher N, Seidler T, Knöll R, Maier LS, El-Banayosy A, Körfer R, Hasenfuss G. Relevance of brain natriuretic peptide in preload-dependent regulation of cardiac sarcoplasmic reticulum Ca2+ ATPase expression. Circulation. 2006; 113: 2724–2732.
Toischer K, Kögler H, Tenderich G, Grebe C, Seidler T, Nguyen Van P, Jung K, Knöll R, Körfer R, Hasenfuss G. Elevated afterload, neuroendocrine stimulation, and human heart failure increase BNP levels and inhibit preload-dependent SERCA upregulation. Circ Heart Fail. 2008; 1: 265–271.
Maier LS, Bers DM. Role of Ca2+/calmodulin-dependent protein kinase (CaMK) in excitation-contraction coupling in the heart. Cardiovasc Res. 2007; 73: 631–640.
Backs J, Backs T, Neef S, Kreusser MM, Lehmann LH, Patrick DM, Grueter CE, Qi X, Richardson JA, Hill JA, Katus HA, Bassel-Duby R, Maier LS, Olson EN. The delta isoform of CaM kinase II is required for pathological cardiac hypertrophy and remodeling after pressure overload. Proc Natl Acad Sci U S A. 2009; 106: 2342–2347.
Hill JA, Karimi M, Kutschke W, Davisson RL, Zimmerman K, Wang Z, Kerber RE, Weiss RM. Cardiac hypertrophy is not a required compensatory response to short-term pressure overload. Circulation. 2000; 101: 2863–2869.
Wang Y, Tandan S, Cheng J, Yang C, Nguyen L, Sugianto J, Johnstone JL, Sun Y, Hill JA. Ca2+/calmodulin-dependent protein kinase II-dependent remodeling of Ca2+ current in pressure overload heart failure. J Biol Chem. 2008; 283: 25524–25532.
Wang Z, Kutschke W, Richardson KE, Karimi M, Hill JA. Electrical remodeling in pressure-overload cardiac hypertrophy: role of calcineurin. Circulation. 2001; 104: 1657–1663.
Palomeque J, Rueda OV, Sapia L, Valverde CA, Salas M, Petroff MV, Mattiazzi A. Angiotensin II–induced oxidative stress resets the Ca2+ dependence of Ca2+-calmodulin protein kinase II and promotes a death pathway conserved across different species. Circ Res. 2009; 105: 1204–1212.
Ling H, Zhang T, Pereira L, Means CK, Cheng H, Gu Y, Dalton ND, Peterson KL, Chen J, Bers D, Heller Brown J. Requirement for Ca2+/calmodulin-dependent kinase II in the transition from pressure overload-induced cardiac hypertrophy to heart failure in mice. J Clin Invest. 2009; 119: 1082–1085.
Shiojima I, Walsh K. Regulation of cardiac growth and coronary angiogenesis by the Akt/PKB signaling pathway. Genes Dev. 2006; 20: 3347–3365.
Verde I, Pahlke G, Salanova M, Zhang G, Wang S, Coletti D, Onuffer J, Jin SL, Conti M. Myomegalin is a novel protein of the golgi/centrosome that interacts with a cyclic nucleotide phosphodiesterase. J Biol Chem. 2001; 276: 11189–11198.
Faul C, Dhume A, Schecter AD, Mundel P. Protein kinase A, Ca2+/calmodulin-dependent kinase II, and calcineurin regulate the intracellular trafficking of myopodin between the Z-disc and the nucleus of cardiac myocytes. Mol Cell Biol. 2007; 27: 8215–8227.
Sayed D, Hong C, Chen IY, Lypowy J, Abdellatif M. MicroRNAs play an essential role in the development of cardiac hypertrophy. Circ Res. 2007; 100: 416–424.
van Rooij E, Sutherland LB, Qi X, Richardson JA, Hill J, Olson EN. Control of stress-dependent cardiac growth and gene expression by a microRNA. Science. 2007; 316: 575–579.
Carè A, Catalucci D, Felicetti F, Bonci D, Addario A, Gallo P, Bang ML, Segnalini P, Gu Y, Dalton ND, Elia L, Latronico MV, Høydal M, Autore C, Russo MA, Dorn GW II, Ellingsen O, Ruiz-Lozano P, Peterson KL, Croce CM, Peschle C, Condorelli G. MicroRNA-133 controls cardiac hypertrophy. Nat Med. 2007; 13: 613–618.
Thum T, Galuppo P, Wolf C, Fiedler J, Kneitz S, van Laake LW, Doevendans PA, Mummery CL, Borlak J, Haverich A, Gross C, Engelhardt S, Ertl G, Bauersachs J. MicroRNAs in the human heart: a clue to fetal gene reprogramming in heart failure. Circulation. 2007; 116: 258–267.
Ren XP, Wu J, Wang X, Sartor MA, Qian J, Jones K, Nicolaou P, Pritchard TJ, Fan GC. MicroRNA-320 is involved in the regulation of cardiac ischemia/reperfusion injury by targeting heat-shock protein 20. Circulation. 2009; 119: 2357–2366.
Chaturvedi RR, Herron T, Simmons R, Shore D, Kumar P, Sethia B, Chua F, Vassiliadis E, Kentish JC. Passive stiffness of myocardium from congenital heart disease and implications for diastole. Circulation. 2010; 121: 979–988.
Zheng J, Chen Y, Pat B, Dell'italia LA, Tillson M, Dillon AR, Powell PC, Shi K, Shah N, Denney T, Husain A, Dell'Italia LJ. Microarray identifies extensive downregulation of noncollagen extracellular matrix and profibrotic growth factor genes in chronic isolated mitral regurgitation in the dog. Circulation. 2009; 119: 2086–2095.
Hemodynamic load regulates myocardial function and gene expression. Increased load triggers molecular, structural, and functional remodeling and eventually heart failure. Increased left ventricular load acts either as preload because of left to right shunt or aortic or mitral regurgitation or as increased afterload because of aortic stenosis or arterial hypertension. In the present study, different cardiac gene expression, signaling, and remodeling with preload or afterload were studied in mice with aortocaval shunt (preload) or transverse aortic constriction (afterload) with matched mean total wall stresses. Here we show that increased afterload results in maladaptive hypertrophy with increased fibrosis, inflammation, cardiomyocyte apoptosis, development of heart failure, and increased mortality. Increased preload results in a more favorable type of hypertrophy without increased fibrosis and inflammation and with less apoptosis and better survival. Ventricular brain natriuretic peptide expression is increased only with afterload but not with preload. Gene expression and signaling pathways differ considerably with preload and afterload. Calcium/calmodulin-dependent protein kinase IIδ–mediated signaling may be the dominant maladaptive pathway in pressure overload, whereas activation of the Akt pathway may be dominant in volume overload. The data indicate that differential therapeutic strategies should be developed to address the specific signaling pathways activated with preload versus afterload. This may prevent or delay the development of heart failure in patients with increased preload or afterload, respectively.
↵*Drs Toischer and Rokita contributed equally to this study, and Drs Maier and Hasenfuss contributed equally to this study.
The online-only Data Supplement is available with this article at http://circ.ahajournals.org/cgi/content/full/CIRCULATIONAHA.110.943431/DC1.