Published online before print January 29, 2007, doi: 10.1161/CIRCULATIONAHA.106.645499
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(Circulation. 2007;115:743-751.)
© 2007 American Heart Association, Inc.
Molecular Cardiology |
Cardiac Hypertrophy and Reduced Contractility in Hearts Deficient in the Titin Kinase Region
From the Department of Veterinary and Comparative Anatomy, Pharmacology, and Physiology, Washington State University, Pullman (J.P, Y.W., H.G., M.G.); Neuromuscular and Cardiovascular Cell Biology, Max-Delbrück Center for Molecular Medicine, Berlin, Germany (K.R., M.G.); Division of Molecular Cardiovascular Biology, Department of Pediatrics, Children’s Hospital Medical Center, Cincinnati, Ohio (J.M.); and Department of Anesthesiology, Mannheim University, Mannheim, Germany (S.L.).
Correspondence to Michael Gotthardt, VCAPP, Washington State University, Wegner Hall, Room 205, Pullman, WA 99164–6520 (e-mail gotthard{at}vetmed.wsu.edu); or Max-Delbrück-Center for Molecular Medicine Berlin-Buch, Robert Rössle Str 10, 13125 Berlin, Germany (e-mail gotthardt@mdc-berlin.de).
Received June 13, 2006; accepted November 20, 2006.
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Abstract |
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Background— Titin is a giant protein crucial for the assembly and elasticity of the sarcomere. Recently, titin has been linked to signal transduction through its kinase domain, which has been proposed to sense mechanical load. We developed a knockout in which expression of M-line–deficient titin can be induced in adult mice and investigated the role of the titin kinase region in cardiac function.
Methods and Results— Isolated heart experiments revealed that in titin M-line–deficient mice, the contractile response to ß-adrenergic agonists and extracellular calcium is reduced. However, the Ca2+ sensitivity and cooperativity of activation of skinned cardiac muscle were unchanged. In knockout mice, calcium transients showed a reduced rate of calcium uptake, and expression analysis showed reduced levels of calmodulin, phospholamban, and SERCA2. Ultimately, knockout mice developed cardiac hypertrophy and heart failure, which involves protein kinase C signal transduction but not the mitogen-activated protein kinase pathway.
Conclusions— The titin kinase region emerges as a regulator of contractile function through effects on calcium handling and hypertrophy through protein kinase signal transduction. These novel functions of titin might provide a rationale for future therapeutic approaches to attenuate or reverse symptoms of heart failure.
Key Words: cardiac output • cardiomyopathy • cells • genes • heart failure • mechanics • models, animal
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Introduction |
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Heart failure is one of the main causes of mortality in the developed world. It has various underlying causes such as cardiomyopathy, coronary artery disease, hypertension, and diabetes, which lead to reduced contractility (systolic dysfunction) or altered relaxation (diastolic dysfunction).1–3 Among the genetic defects that lead to heart failure are mutations in the sarcomeric protein titin, which cause dilated or hypertrophic cardiomyopathy, depending on the underlying defect.4–6 Titin, a giant sarcomeric protein that contributes to the diastolic properties of the heart through its elastic domains,7–9 is involved in signal transduction through its kinase region.10,11 Although the elastic properties of titin have been studied extensively,12–16 its signaling functions are less well understood. The titin kinase (TK) domain has been proposed to regulate protein expression in striated muscle in a strain-dependent manner via its substrates nbr1 and p62.17 TK is encoded in M-line exon 1, together with binding sites for proteins such as the ubiquitin ligase MuRF-1,18 the adaptor protein FHL2/DRAL, which binds both signaling proteins and metabolic enzymes,19 and calmodulin, which has been shown to regulate TK activity in vitro.20
Clinical Perspective p 751
To study the kinase region in vivo, we have previously used a conditional knockout (KO) approach to selectively delete this region (M-line exon 1 and M-line exon 2) in striated muscle using the MCKcre transgene.21 Although this approach results in live offspring, neonatal mice develop cardiomyopathy and skeletal muscle wasting, resulting in early death. To restrict the phenotype to the adult heart, we used MerCreMer (MCM)-transgenic mice, which express a tamoxifen-inducible Cre fusion protein under control of the 
-myosin heavy chain promoter.22 We show that titin M-line–deficient mice have a significantly attenuated response to adrenergic stimulation and extracellular calcium, have severe cardiac hypertrophy, and ultimately develop congestive heart failure. Thus, titin not only is critical for diastole but also plays an important role in contractile function.
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Methods |
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TK Region KO
The generation of mice carrying the floxed TK region (M-line exon 1/M-line exon 2) has been described.21 To generate the tamoxifen-inducible KOs, we mated these animals with the MCM-transgenic mouse.22 The breeding scheme is shown in Figure IB (see the online Data Supplement). MCM-transgenic animals homozygous for the floxed kinase region (MCM+/–, TK loxP+/+) were injected with tamoxifen or vehicle at 8 weeks of age (10 mg/mL tamoxifen in 10% ethanol and 90% peanut oil for 1 or 2 weeks22). Recombination efficiency was monitored as described in the online Data Supplement. Experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals (National Academy of Sciences, Washington DC, 1996).
Immunoelectron Microscopy and Immunofluorescence Analysis
Left ventricular (LV) muscle strips were stretched, fixed, immunolabeled with antibodies directed against the kinase region (A169/A170) and the N2B unique sequence (Un) of titin, embedded, and processed for immunoelectron microscopy as described.23 Immunofluorescence methods are described in the online Data Supplement.
Isolated Heart Experiments
The developed and passive pressure-volume relationships were determined using the isolated heart preparation. Steady-state analysis of LV function was performed by changing LV filling volume between 17 and 28 µL to generate Frank-Starling curves (details are given in the online Data Supplement). The LV pressure was converted into LV wall stress (
) using a thick-walled spherical model: =P/[(LVw/1.05V+1)2/3–1], where LVw is the weight of the LV wall. The model normalizes for differences in LV size and wall thickness, and obtained values reflect the intrinsic stress generated by the myocardium (see elsewhere for details24).
Muscle Mechanics
To evaluate the Ca2+ sensitivity of myofilaments in kinase-deficient hearts, 2 groups, Vh(30d/2) and T(30d/2), were selected. LV wall muscle strips were dissected and skinned overnight at 4°C in relaxing solution (for composition, see elsewhere25) containing 1% (wt/vol) Triton X-100, followed by washing with relaxing solution. The skinned muscles were mounted to a force transducer and a high-speed motor. The cross-sectional area was measured to convert force to tension.9 Experiments were performed at room temperature (20°C to 22°C). Sarcomere length was measured by laser diffraction,26 and force-pCa relations were measured at a sarcomere length of 2.0 µm. Active tensions at submaximal activations were normalized to those produced at pCa 4.5. The relation between relative tension and pCa was fitted to the following equation: relative tension=[Ca2+]nH/(K+[Ca2+]nH), where nH is the Hill coefficient and PCA50 is –(log K)/nH.
Measurements of Calcium Transients
Isolated LV cardiac myocytes from Vh(30d/2) and T(30d/2) KO mice were incubated with Fura-2 AM in Tyrode’s solution (1 µmol/L, TEFLabs, Austin, Tex) for 20 minutes at room temperature and resuspended in Tyrode’s solution containing 1.8 mmol/L Ca2+. Myocytes were perfused with Tyrode’s solution and field stimulated (1 Hz, square waves). Fluorescence was measured ratiometrically with the IonOptix photometry system (IonOptix Corp, Milton, Calif). Fura-2 was excited alternately at 340 and 380 nm, and emission was recorded at 510 nm. Background fluorescence was subtracted for each excitation wavelength. Measurements were taken from the average of 5 steady-state transients obtained both under baseline conditions and after application of dobutamine (0.3 µmol/L). Calcium uptake was fitted with a monoexponential function to determine the time constant (
). All measurements were carried out at 37±1°C.
Expression Analysis
The SDS-agarose gel system used to separate titin isoforms has been described.27 Western blot analysis for the remaining proteins and 2-dimensional gel electrophoresis are described in the Data Supplement.
Statistical Analysis
Results are reported as mean±SEM. Differences between mean values were evaluated by Student t test or ANOVA, with values of P<0.05 indicating a significant difference. Multiple linear regression analysis was used to analyze and compare the -volume relationship. Data were fitted with the following regression model: Y=b0+bxX+bx2X2+bdD+bdxDxX+bdx2DxX2, where Y is the response variable (wall stress, ), X is the predictor variable (volume), D is a dummy variable to encode control, b0 is the intercept, and the bi symbols are regression coefficients. In all cases, the data could be well fit by the regression model, with R2 
0.90. Also included in the regression model (but not shown for clarity) are dummy variables to encode the different mice so that the repeated observations on each mouse were accounted for in the model. The entire regression curves were compared by the F test, and 2 curves are considered significantly different if P<0.05.
The authors had full access to and take responsibility for the integrity of the data. All authors have read and agree to the manuscript as written.
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Results |
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Inducible Deletion of the TK Region in the Adult Mouse Heart: Development of Cardiac Hypertrophy and Failure
To obtain adult TK-deficient animals, homozygous floxed mice carrying the MCM transgene (MCM+/–, TK loxP+/+) were injected with tamoxifen to activate the Cre recombinase and to excise the kinase region in cardiac muscle (online Data Supplement, Figure IA through IC). Recombination was monitored at the protein level with SDS-agarose gel electrophoresis. Tamoxifen-injected animals showed an additional titin band of higher mobility (Figure ID). Western blot analysis confirmed that this high-mobility band represents mutant titin that lacks the TK region (Figure IE).
We adjusted the level of mutant titin by varying the tamoxifen injection schedule (1 or 2 bouts of injections, each lasting 1 week; see Methods) and the time after injection (5, 10, 20, 30, or 80 days). Treatment groups are labeled by injected substance (Vh for vehicle and T for tamoxifen), number of days after injection, and number of injection bouts. The ratio of truncated titin to wild-type titin varied from 28% [T(5d/1)] to 52% [(T(80d/2; see Figure 1A]. On a cellular level, a fraction (
40%) of the cardiomyocytes showed recombination (Figure 1B), but both M-line–deficient cells and control cells showed normal sarcomere structure 30 days after tamoxifen injection (Figure 1C). This includes both proper incorporation of the I-band region of titin into the sarcomere and proper alignment of the sarcomeres without signs of disassembly. Secondary to loss of the kinase region, progressive structural changes of the sarcomere occurred later in the development of the myopathy (faintly stained sarcomeres and ultimately disassembly). The phenomenon is noticeable 30 days after 1 bout of tamoxifen injections and increases to >50% of the myofibrillar area 80 days after 2 bouts (Figure 2A).
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Titin deficiency induced by 1 bout of tamoxifen injections did not induce an obvious phenotype. However, 2 bouts of tamoxifen injections resulted in an increased ratio of ventricular to body weight 30 days after treatment and heart failure with fibrosis (increased expression of collagen type I and III) and pulmonary edema 80 days after treatment (Table I and Figure 2B through 2E). The hypertrophy phenotype is in contrast to the normal heart size in striated muscle TK KO mice, which are deficient in skeletal titin and cardiac titin.21 This difference might be due to kinetics of Cre-mediated recombination in MCKcre versus MCM animals or relate to the underlying atrophy in striated muscle knockouts.
Cardiac Function Is Impaired in TK Region–Deficient Mice
To determine the functional effect of the TK region, we measured the diastolic stress ()-volume relations in isolated hearts. Under baseline conditions (Figure 3A and 3D), both the developed and diastolic -volume relationships are not significantly different when either 29% or 44% mutant titin is expressed. However, when the level exceeds 50% mutant titin, developed wall stress (dev) is reduced and diastolic increased. This is most prominent for the T(80d/2) group, which has 52% mutant titin. We plotted dev at low and high ventricular volumes, chosen as the equilibrium volume (Veq, volume at which diastolic is 0) and 1.4xVeq, represented by the open bars in Figure IIA and IIC, respectively. The effect of mutant titin on dev is most prominent at high volumes (Figure IIA and IIC). Furthermore, even in the late-stage cardiomyopathic heart [T(80d/2)] with 50% sarcomere disassembly (Figure 2A), there is still enough contractile reserve to produce increased under baseline conditions when the volume is increased from 1.0 to 1.4xVequ (up from 15 mm Hg in Figure IIA to 22 mm Hg Figure IIC [open bars labeled with ++]).
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Next we examined the effect of ß-adrenergic stimulation with dobutamine. Both control and TK-deficient mice showed a robust adrenergic response (increased dev at all volumes tested), which was reversible with propranolol (Figure 3B and 3C). We also plotted the dev difference between baseline and dobutamine-stimulated hearts, both at low volume (Vequ) and high volume (1.4 Vequ). This revealed that the increase in after dobutamine treatment (
dev) is significantly smaller in the T(30d/1), T(30d/2), and T(80d/2) groups. This effect is more pronounced at high volumes (compare Figure IIB and IID). Maximum rate of pressure development (dP/dtmax) and relaxation (–dP/dtmax) (Figure III) essentially mirrored these results.
The reduced response to dobutamine suggests cross-talk between the TK region and the adrenergic signaling pathway, which could occur at the level of G-protein–coupled receptor signaling, calcium homeostasis, or the myofilament. To explore the contribution of G-protein–coupled receptor signaling versus Ca2+ handling, we examined the inotropic effect of extracellular Ca2+. Increasing the extracellular Ca2+ level from 2.0 mmol/L (used in all experiments described so far) to 3.5 mmol/L induced a significant inotropic effect in vehicle control but not in kinase-deficient hearts (Figure 4). Analogous to the dobutamine experiments, both developed wall stress, and speed of contraction/relaxation was increased after high Ca2+ treatment; this effect was absent in the TK-deficient hearts (Figure 4 and Figure IIIE and IIIF).
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To investigate whether the blunted response to inotropic stimulation by dobutamine and calcium in the isolated heart has a source in the myofilaments, we measured the active tension-PCA relation of skinned muscle fibers from the LV wall of control and T(30/2) mice. We found no change in calcium sensitivity (pCa 50) or cooperativity of activation (Hill coefficient) (Figure 5A and 5B). Although the reduction in maximal active tension in the muscles with mutant titin was not significant, the noted trend toward reduced tension might be due to the sarcomere disarray that was present in T(30/2) mice (Figure 2A). We conclude that the calcium sensitivity of myofilaments is not altered in the TK-deficient hearts. Thus, the attenuated response of the TK-deficient hearts to dobutamine and extracellular calcium is upstream of the myofilaments, most likely in the excitation-contraction coupling system.
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To gain further insight into calcium handling, we measured calcium transients and compared results of cardiac myocytes from vehicle control- and tamoxifen-treated animals. The amplitude of the calcium transient in cardiomyocytes from KO hearts is significantly reduced even under baseline condition (Figure 5C and 5D), and this reduction is amplified after dobutamine stimulation. Importantly, the time constant of calcium uptake () is significantly increased in kinase-deficient myocytes both under baseline conditions and in the presence of dobutamine (Figure 5D, bottom). Thus, Ca2+ uptake is severely reduced in titin M-line–deficient mice, and the reduced contractility in KO hearts is likely due to changes in calcium handling.
Molecular Basis of the Contractility and Hypertrophy Phenotype
We used 2-dimensional gel electrophoresis, Western blotting, and real-time polymerase chain reaction to monitor changes in protein and gene expression. KO and control hearts were compared at (30d/2), when both the reduced adrenergic response and the hypertrophic changes became significant (Figure II and Table I).
The most prominent changes were the induction of structural and cytoskeletal proteins, stress-activated proteins, and metabolic enzymes (Table II). Many of these proteins are targets of protein kinase C (PKC) signaling (PKC
and PKC
) and thus are implicated in the hypertrophy response. Of these 2 PKC isoforms, only PKC is upregulated, which is consistent with the hypertrophy of the KO heart [T(30/2)] (Figure 6A and 6D).
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To connect altered PKC signaling to the TK region, we focused on the titin-binding protein MuRF-1 because its binding site is located in the kinase region and it links to both hypertrophy and contractile function (MuRF-1 has been implied in an antihypertrophy signaling pathway in neonatal rat ventricular myocytes28 and has been shown to degrade cardiac troponin I and thus suppress cardiomyocyte contractility29). We used various antibodies directed against MuRF-1 and its target proteins to investigate expression levels in the KO animals, which lack the MuRF-1 binding site (Figure 6). At T(30d/2) with >50% truncated titin, MuRF-1 protein was upregulated, which is inconsistent with a role in hypertrophy. Furthermore, its targets, cardiac troponin I and PKC, are unchanged (Figure 6D). Of the various additional TK region–binding proteins that we investigated, only calmodulin was changed (downregulated in KO; Figure 6C).
To differentiate the contribution of intracellular and extracellular Ca2+ handling to the contractility phenotype, we compared expression levels of proteins involved in Ca2+ handling and found that the L-type Ca2+ channel was unchanged in KO mice but that phospholamban and SERCA2 were both downregulated (Figure 6D). Altered expression of these calcium handling proteins was present from day 5, before changes in cardiac function became apparent (Figure 6E).
At the earliest time point at which the ratio of heart to body weight was significantly increased in KO animals [Table II; T(30/2)], hypertrophy markers were strongly increased (atrial and brain natriuretic peptide by a factor 10 and 4, respectively). The MAP kinase pathway and hypertrophy-related transcription factors Mef2C and GATA4 were unchanged (Figure 6A).
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Discussion |
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Patients with titin mutations develop cardiomyopathy and/or muscular dystrophy,4–6,17,30 highlighting the importance of titin for muscle function. Various animal and tissue culture models have been generated to dissect the structural, mechanical, and potential signaling functions of titin.21,31–34 The inducible KO provides the unique opportunity to study contractile function in adult hearts with kinase-deficient titin incorporated in the sarcomere (Figures 1C and 2
A). We used isolated heart experiments to correlate titin deficiency, adrenergic response, ventricular volume, and wall stress. We found a significant effect on contractility and cross-talk between titin and the adrenergic signaling pathway. The reduced contractile response to dobutamine in animals with >40% kinase-deficient titin could be due to changes at the level of adrenergic receptor, calcium handling, or the myofilament. Our results suggest that the mechanism for reduced contractility is at the level of calcium handling. A mechanism upstream of calcium handling is unlikely because a reduced contractile response is present in response to both ß-adrenergic stimulation and increased extracellular calcium (Figure 4). A downstream effect is unlikely because the developed wall stress of the isolated heart is responsive to increased volume and because the calcium sensitivity of myofilaments in skinned fibers is unaltered in KO mice (Figures II and 5). Supportive evidence for a mechanism at the level of calcium handling is derived from the analysis of calcium transients in KO cardiomyocytes that revealed a decreased calcium uptake (Figure 5) and an early reduction in the calcium handling proteins SERCA2 and phospholamban from day 5 of induction of the KO (Figure 6).
Multiple proteins bind in and near the TK region (MuRF-1, nbr1, calmodulin, FHL2, myomesin), and any of these may play a role in the phenotype of the M-line KO mice. As for the recently reported nbr1-p62-MuRF-2 signaling complex, it is unlikely to be involved because titin mutations that disrupt this pathway do not lead to a cardiac phenotype.17 The potential kinase substrate Tcap and the kinase region–binding protein FHL2 have both been suggested to play a role in cardiac hypertrophy,35,36 but their expression levels were not altered in the KO mice. In contrast, MuRF-1 protein was upregulated (Figure 6C). MuRF-1 binds to the titin repeats A168/A169 adjacent to the kinase domain and has been reported to prevent cardiac hypertrophy by inhibiting the activation of PKC. Although MuRF-1 was upregulated in the KO, there is no change in PKC expression (Figure 6D), indicating that it is unlikely that the MuRF-1-PKC pathway plays a major role in cardiac hypertrophy in the TK KO model. In addition, MuRF-1 functions as a negative regulator of contractility by degrading troponin I. However, troponin I protein levels were unchanged (Figure 6D), suggesting that involvement of the MuRF-1–troponin I pathway in reduced contractility in the KO hearts also is unlikely. This conclusion is consistent with the absence of changes in the skinned fiber contractility (Figure 5A and 5B).
The only additional alteration in titin-binding protein levels was the downregulation of calmodulin in the KO mice (Figure 6). Calmodulin interacts with the regulatory domain of TK and has been proposed to bind and activate TK.20 Hence, calmodulin is a possible link between the kinase region and Ca2+ signaling. Calmodulin also participates in calcium release of the sarcoplasmic reticulum, regulating the activity of SERCA and phospholamban through calmodulin-dependent kinase II.37,38 We speculate that the mechanism underlying the contractility phenotype of the KO mouse involves the TK-binding protein calmodulin.
Significant changes also were found in structural proteins, stress-activated proteins, and metabolic enzymes (Table II), most of which are downstream of PKC. Thus, these changes might reflect increased PKC and PKC activity in titin-deficient animals.39,40 The expression of PKC was not significantly affected in the KO hearts (Figure 6). In contrast, PKC was upregulated in TK knockouts at both the RNA and the protein levels. This upregulation was associated with increased expression of known targets of PKC such as pyruvate kinase 3 and peroxiredoxin 6 (Table II).
Increased PKC activity has been associated with postischemic contractile dysfunction,41 and evidence for a function in hypertrophy is starting to emerge. Sustained activation of PKC in cardiomyocytes leads to hypertrophy and ultimately disruption of the cytoskeleton.42 In the titin M-line KO mice, the upregulation of PKC could be a secondary change or an indication of cross-talk between protein kinase signaling and the TK region. This issue warrants future follow-up work.
In summary, in addition to the well-established role of titin in muscle elasticity, we have demonstrated that deficiency in the TK region directly affects contractile function at the level of intracellular Ca2+ handling. The impaired contractile properties lead to cardiac hypertrophy and reduced ß-adrenergic responsiveness, effects that are likely to involve altered intracellular Ca2+ handling and PKC signal transduction.
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Acknowledgments |
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We are grateful to Dr Bryan Slinker for statistical expertise; Dirk Albrecht and Falko Hochgräfe for protein identification; and Beate Goldbrich, Mark McNabb, and Gemaine Wright for expert technical assistance.
Sources of Funding
This work was supported by the National Institutes of Health (HL 69008 and HL 61497), Alexander von Humboldt Foundation, and American Heart Association (postdoctoral fellowship 0620031Z to Dr Peng).
Disclosures
Dr Molkentin has received a research grant (NIH) and an AHA established investigator award. Drs Labeit, Granzier, and Gotthardt have received research grants from the Deutsche Forschungsgemeinschaft (DFG) and the NIII. Dr Gotthardt also has received an AHA grant-in-aid (0455494Z) and the Kovalevskaya award. The other authors report no conflicts.
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The online-only Data Supplement, consisting of expanded Methods, Tables I and II, and Figures I through V, is available with this article at http://circ.ahajournals.org/cgi/content/full/CIRCULATIONAHA.106.645499/DC1.
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