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Published online on February 20, 2007, 10.1073/pnas.0608543104
PNAS | February 27, 2007 | vol. 104 | no. 9 | 3444-3449


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BIOLOGICAL SCIENCES / MEDICAL SCIENCES
Targeted deletion of titin N2B region leads to diastolic dysfunction and cardiac atrophy

Michael H. Radke*, Jun Peng{dagger}, Yiming Wu{dagger}, Mark McNabb{dagger}, O. Lynne Nelson{ddagger}, Henk Granzier{dagger}, and Michael Gotthardt*,{dagger},§

*Department of Neuromuscular and Cardiovascular Cell Biology, Max-Delbrück-Center for Molecular Medicine, D-13122 Berlin-Buch, Germany; and {dagger}Department of Veterinary and Comparative Anatomy, Pharmacology, and Physiology, and {ddagger}Department of Veterinary Clinical Sciences, Washington State University, Pullman, WA 99164

Edited by Christine E. Seidman, Harvard Medical School, Boston, MA, and approved December 20, 2006 (received for review September 27, 2006)


   Abstract
Top
 Abstract
Results
 Discussion
 Methods
 Acknowledgements
 References
 
Titin is a giant protein that is in charge of the assembly and passive mechanical properties of the sarcomere. Cardiac titin contains a unique N2B region, which has been proposed to modulate elasticity of the titin filament and to be important for hypertrophy signaling and the ischemic stress response through its binding proteins FHL2 and {alpha}B-crystallin, respectively. To study the role of the titin N2B region in systole and diastole of the heart, we generated a knockout (KO) mouse deleting only the N2B exon 49 and leaving the remainder of the titin gene intact. The resulting mice survived to adulthood and were fertile. Although KO hearts were small, they produced normal ejection volumes because of an increased ejection fraction. FHL2 protein levels were significantly reduced in the KO mice, a finding consistent with the reduced size of KO hearts. Ultrastructural analysis revealed an increased extension of the remaining spring elements of titin (tandem Ig segments and the PEVK region), which, together with the reduced sarcomere length and increased passive tension derived from skinned cardiomyocyte experiments, translates to diastolic dysfunction as documented by echocardiography. We conclude from our work that the titin N2B region is dispensable for cardiac development and systolic properties but is important to integrate trophic and elastic functions of the heart. The N2B-KO mouse is the first titin-based model of diastolic dysfunction and, considering the high prevalence of diastolic heart failure, it could provide future mechanistic insights into the disease process.

cardiac muscle | hypertrophy | mechanics | cardiology | disease

Titin forms a continuous filament along the myofibril that determines the elastic properties of cardiac myocytes (for review, see ref. 1). The extensible region of titin is found in the I-band region of the sarcomere and comprises tandemly arranged Ig-like domains and the so-called PEVK region (2). In addition, cardiac titin contains a third extensible region, the N2B element (2), which is absent in skeletal muscle. The N2B region extends greatly toward the upper limit of the physiological sarcomere length of cardiac muscle (3, 4). It has been suggested that this extension reduces the steepness of the passive force–sarcomere length relation, decreasing the likelihood of the unfolding of Ig domains (3). Mutations in the N2B region can lead to dilated or hypertrophic cardiomyopathy, apparently through altered affinity to FHL2, a heart-specific member of the LIM domain gene family (5). To understand the role of the titin N2B region in cardiac function and disease, we have eliminated exon 49, which encodes the N2B region, and investigated its effect on the mechanical and trophical properties of the knockout (KO) heart.


   Results
Top
 Abstract
 Results
Discussion
 Methods
 Acknowledgements
 References
 
N2B-Deficient Titin Integrates Properly into the Sarcomere. Using homologous recombination we replaced titin exon 49 with an Flp recombinase target (FRT)-flanked neomycin resistance cassette that was subsequently removed by germ line expression of the Flp recombinase (Fig. 1A). Homozygous KO mice survive to adulthood and are fertile, with no obvious abnormalities. PCR, Southern blotting, and protein gels confirmed the deletion of the N2B region (Fig. 1 B–D). We studied titin expression in knockout mice by Western blotting and found that upon excision of the N2B region, the reading frame is maintained and that the C-terminal M-line region is included in the truncated protein (Fig. 2A). The N2B-deficient protein is integrated properly into the sarcomere as shown by immunofluorescence labeling with the anti-M-line-titin antibody (Fig. 2 B and C). Overall, there was no phenotypic change in the initial molecular characterization of the N2B-KO aside from a minor but significant shift in titin isoform expression from the smaller (stiffer) N2B to the larger (more compliant) N2BA isoform [see supporting information (SI) Fig. 7].


Figure 1
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  		Fig. 1. Generation of titin N2B-deficient animals. (A) Targeting strategy. The neomycin resistance cassette in the targeting vector replaces the N2B region (exon 49) in the WT allele. The exon encodes three Ig domains and a unique sequence. The Flp recombinase was used to remove the neomycin cassette. The Southern probe is shown as a black box. Restriction sites for Southern blot analysis are EcoRI (E) and BamHI (B). The FRT sites are depicted as gray arrowheads, and primer-binding sites for PCR genotyping are shown as black arrows. (B) PCR genotyping. PCR products derived from the WT allele (+) are 376 bp; targeted allele (neo), 210 bp; KO allele (–), 252 bp. (C) Genomic Southern blot of genomic DNA from the same mice analyzed in B. Digestion of DNA from WT (+/+), heterozygous (neo/+ or +/–), and homozygous (–/–) KO animals with EcoRI and BamHI produces bands of the expected sizes (+, 5.2 kb; neo, 8.2 kb; –, 6.4 kb). (D) Coomassie blue-stained 2% agarose gel with ventricular lysates from +/+, +/–, and –/– animals. N2BA, N2B isoforms, and T2 degradation products are indicated. In the heterozygote, the WT and the mutant proteins (higher mobility) are expressed. The KO expresses only truncated N2B and N2BA titin (tN2BA and tN2B).


Figure 2
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  		Fig. 2. The exon 49-KO affects exclusively the N2B region with proper integration of the truncated protein into the sarcomere. (A) Western blot. India ink staining shows equal loading for WT (+/+) and KO (–/–). Z1-Z2 antibody recognizes the N terminus of titin; the N2B-US antibody, the unique N2B sequence; and the M8/M9 antibody, the C terminus of titin. In KO mice, only the N2B region is deleted, whereas the N- and C-terminal parts of the titin protein are unaffected. (B) Confocal microscopy of double-immunofluorescence-stained cardiomyocytes. Anti-{alpha}-actinin (red), labels the Z-disk, and anti-N2B (green) localizes at the I-band close to the Z-disk in WT animals (+/+). In KO mice, the N2B staining is absent. (C) Costaining with anti-{alpha}-actinin (red) and the anti-M8/M9 (green) antibody directed against the M-band region of titin shows proper integration of N2B-deficient titin into the sarcomere (alternating actinin/M-band titin staining). (Scale bar, 5 µm.)

Reduced Heart Size and Impaired Diastolic Function in the Absence of the Titin N2B Region. Functional analysis of the N2B-knockout animal with echocardiography revealed that KO mice had significantly reduced ventricular dimensions (reduced internal diameter, diastolic volume, and calculated LV/body weight ratio); see Table 1. Interestingly, fractional shortening was increased in the KO mice (by 22% relative to littermate controls), resulting in a stroke volume that was not significantly different from that of wild-type mice (WT). The reduced heart to body weight ratio calculated from echo data was consistent with actual weight measurements (SI Fig. 8).


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  		Table 1. Echocardiography of N2B-KO hearts

 
Diastolic function was evaluated by using Doppler imaging of mitral inflow (Table 2). In KO animals, we found a significant reduction in deceleration time (MV DT) and an increased in the E/A ratio, indicating a restrictive filling pattern.


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  		Table 2. Doppler of N2B-KO hearts

 
Diastolic Wall Stress Is Increased in N2B-KO Hearts. To study diastolic and systolic function under controlled conditions, we performed isolated heart experiments. Representative wall stress–volume diagrams from WT and KO animals are provided in Fig. 3A. Although developed wall stress does not change significantly (circles), the diastolic wall stress (squares) is increased in the KO, especially at high volumes. Statistical analysis demonstrates a robust increase in diastolic wall stress in KO versus WT mice (P < 0.006, n = 18) (Fig. 3B Left). Similar results were found in the presence of dobutamine or propranolol (Fig. 3B Center and Right). Systolic function was normal in KO mice, and no significant differences were found in developed wall stress of WT and KO mice at all volumes tested (Fig. 3 C and D). Dobutamine increased developed wall stress of hearts of all genotypes with a trend toward a larger increase in the KO (Fig. 3D; P = 0.08, n = 18).


Figure 3
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  		Fig. 3. N2B-deficient hearts have increased diastolic wall stress. (A) Examples of {sigma} – V relation in WT (Left) and KO mice (Right). Diastolic stress is shown by squares and developed stress by circles. (B) Mean ± SEM of WT (light gray) and KO (dark gray) mice (six animals each). Results are shown at Veq + 15%, under baseline conditions (BL) and in the presence of either dobutamine (Dob) or propranolol (Prop). Under all conditions, diastolic wall stress is increased in KO mice (P < 0.01). (C and D) Developed wall stress at Veq and Veq + 15%. Developed wall stress is largely unchanged between the genotypes. There is a trend toward higher developed {sigma} in the presence of dobutamine, which did not reach statistical significance (P = 0.08). The corresponding pressures are provided in SI Fig. 9.

Altered Structure and Mechanical Properties of the N2B-Deficient Sarcomere. To investigate the molecular mechanism underlying altered diastolic function in N2B-KO animals, we studied the mechanical and structural properties of skinned cardiomyocytes (Fig. 4). Although the myocyte width, length, cross-sectional area, and maximal active tension were unchanged in myocytes of N2B-KO mice, slack sarcomere length was reduced significantly (Fig. 4E). Furthermore, passive tension of cardiac myocytes, which is known to be largely the result of titin (6), was increased significantly (Fig. 4F).


Figure 4
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  		Fig. 4. Slack sarcomere length (SL) is reduced, and passive tension is increased in skinned cardiomyocytes of N2B-KO mice. Myocyte length (A), width (B), cross-sectional area (C), and maximal force produced (D; tested at two sarcomere lengths) are unchanged in N2B-KO (–/–) versus WT (+/+) control animals. (E) Slack sarcomere length is significantly reduced in KO versus WT cardiomyocytes (n = 30 each). (F) Passive tension is increased in KO versus WT at sarcomere length >2 µm. Myocytes were derived from age-matched KO (n = 11) and littermate control WT animals (n = 10) at 6 months of age.

To understand the structural basis of increased titin-based passive tension, we investigated how the absence of the N2B element affects the extensibility of the remaining spring elements of titin (Fig. 5). We demarcated those elements with antibodies by using immunoelectron microscopy (compare Fig. 5B). The distance of titin epitopes from the Z-disk was plotted against sarcomere length (Fig. 5C) to deduce the extension of the proximal tandem Ig segment (T-12 to UN), the N2B-US sequence only present in WT mice (UC to UN), the PEVK (UC-I84), and the distal tandem Ig segment (I84 to MIR). The segment lengths at a sarcomere length of 2.3 µm revealed that excision of the N2B element results in an increased extension of the tandem Ig segments (both proximal and distal) as well as the PEVK region (Fig. 5D). The PEVK region, although shortest, extends the most in response to the loss of N2B elasticity, followed by the proximal Ig region and the distal Ig (Fig. 5E).


Figure 5
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  		Fig. 5. Compensation for loss of the N2B element by the PEVK and tandem Ig regions as determined by immunoelectron microscopy. (A) WT (+/+) and KO (–/–) LVs were colabeled with anti-N2B and anti-MIR antibody. The N2B labeling is only observed in the +/+ and absent in the –/– animal. (B) Antibody-binding sites are shown along the titin I-band region. (C) Measurement of epitope distance from the Z-line at various sarcomere lengths tested on +/+ and –/– muscle strips. (D) Segment extension at sarcomere length 2.3 µm. Tandem Ig and PEVK segments are increased in length in KO mice. (E) Increased extension of tandem Ig and PEVK segments in KO mice as a percentage of extension of I-band titin. Loss of the N2B element results in the largest extension of PEVK segment followed by the proximal tandem Ig and the distal tandem Ig region.

Overall, the differences in sarcomere slack length, extensibility, and increase in passive tension of N2B-deficient cardiomyocytes provide the biomechanical framework to explain the impaired diastolic function and increased diastolic wall stress in the KO animals.

Reduced Cardiac Growth Is Associated with Decreased FHL2 Protein Levels. To address the molecular basis of the reduced size of KO hearts, we determined protein levels for the N2B-binding proteins {alpha}B-crystallin and FHL2. Only the latter has been linked to atrophy/hypertrophy signaling (5, 7), and as expected, we found {alpha}B-crystallin unchanged but FHL2 protein down-regulated in the KO (Fig. 6 A–C). Although RNA levels were similar in KO and WT animals (Fig. 6D), FHL2 protein is reduced in N2B-KO mice. Finally, because trophic changes in the FHL2-KO are accompanied by an increased mRNA level of the hypertrophy marker atrial natriuretic peptide (ANP) (7), we also measured ANP expression in N2B-KO mice. Indeed, we found that ANP was also up-regulated in the N2B-KO (Fig. 6E).


Figure 6
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  		Fig. 6. FHL2 protein level is decreased in the N2B-KO, {alpha}B-crystallin is unchanged, and ANP expression is increased. (A) The titin N2B-binding proteins FHL2 and {alpha}B-crystallin were quantified by Western blot analysis of WT (+/+) and KO (–/–) LVs (n = 3 each). Although {alpha}B-crystallin is unchanged, FHL2 levels are reduced significantly in KOs (–/–). (B and C) Quantification of FHL2 and {alpha}B-crystallin normalized to actin levels and (+/+) set to 100%. The difference is significant at P < 0.01 (n = 6 per genotype). (D and E) FHL2 RNA levels as determined by real-time quantitative RT-PCR from left ventricle are unchanged (D), but the hypertrophy marker ANP (E) is up-regulated in KO animals (n = 3 per genotype).


   Discussion
Top
 Abstract
 Results
 Discussion
Methods
 Acknowledgements
 References
 
The cardiac-specific N2B element integrates mechanical and signaling functions through its large extensible region and its interaction with {alpha}B-crystallin and FHL2. Analysis of the N2B-KO mice allowed us to test the hypothesis that the N2B unique sequence eliminates the necessity for unfolding of Ig domains under physiological conditions (3). In absence of the N2B element, we found that the extension of PEVK and tandem Ig segments is increased (Fig. 5). At the upper limit of the physiological sarcomere length range, the increased extension is the highest for the PEVK region, suggesting that at this sarcomere length the PEVK is most compliant. Interestingly, the extra extension of the tandem Ig segment is distributed unevenly between the proximal and distal tandem Ig segment, highest in the proximal Ig segment (Fig. 5D), which leads to the conclusion that the proximal tandem Ig segment is more compliant than the distal tandem Ig, consistent with in vitro studies (3). Thus, our work supports the view that the extensibility provided by the N2B unique sequence obviates the need for the unfolding of Ig domains toward the upper limit of the physiological sarcomere length range.

The passive force of titin is entropic in nature, with force increasing with the titin fractional extension (8). Thus, the increased extension of the tandem Ig segment and PEVK region of the N2B-KO mice will result in a larger fractional extension and hence a higher passive force, consistent with our measurements made on cardiac myocytes (Fig. 4). Considering that within the physiological sarcomere length range titin is the main contributor to passive tension of the myocardium (9), increased passive tension of cardiac myocytes is a likely explanation of the elevated diastolic LV wall stress that was found in the isolated heart experiments (Fig. 3). The reduced slack length of cardiac myocytes of KO mice might also relate to the excision of one of the spring elements of titin. The mean square end-to-end distance of a flexible chain at zero external force (titin in slack sarcomeres) is a function of the contour length of the chain. Thus, a reduction in contour length (N2B-KO) will result in a reduction of the end-to-end distance and hence a reduction in slack sarcomere length. It has been speculated for more than a decade that titin is a determinant of slack length (10), and our work provides direct evidence thereof.

Increased diastolic wall stress explains the diastolic dysfunction of KO mice that was revealed by echocardiography. KO mice had a reduced deceleration time of the E wave (early rapid filling phase) and a restrictive filling pattern as revealed by the reduced ratio of the peak of E wave and peak of A wave (late filling because of atrial systole). Because MV DT is inversely correlated with LV stiffness in both animals (11) and humans (12), the reduction in MV DT of N2B-KO mice can be ascribed to their increased LV stiffness. Atrial contraction will contribute less to the filling of a stiff ventricle explaining the reduced E:A ratio of KO mice. Thus, echocardiography reveals that N2B-KO mice have diastolic dysfunction as a result of a stiff ventricle. The slight increase in expression of N2BA titin N2B-KO mice (SI Fig. 7) may be viewed as an attempt to improve diastolic function [the N2BA isoform is less stiff than N2B titin (ref. 13)]. However, the ensuing reduction in passive tension is small ({approx}5%) and apparently insufficient to offset the large increase in stiffness that results from the excision of the N2B element.

In the N2B-KO mouse we did not find major changes in systolic function. Maximal active tension of skinned cardiac myocytes was not different in KO mice (Fig. 4D), and the isolated heart experiments did not reveal significant systolic changes (Fig. 3 C and D). There was a trend toward an increased dobutamine response in the N2B-KO (Fig. 3D), but it did not reach statistical significance (P = 0.08). Interestingly, the echocardiography study revealed an increased fractional shortening in KO mice (Table 1). Titin-based passive tension has been suggested to increase calcium sensitivity (1), but this effect only takes place at long sarcomere lengths and, thus, is unlikely to be involved in prolonging the ejection phase (where sarcomeres are short). Future studies are required to establish the molecular mechanism underlying increased fractional shortening in KO mice. Assuming that the intrinsic myocardial tension during systole is indeed not altered in the KO mice, their smaller ventricles could result in a higher ventricular pressure, which might help explain their increased ejection fraction, although other factors could be involved as well.

Previous work on the N2B region in zebrafish by using both a genetic mutant (pickm171) and a morpholino approach to disrupt expression of the N2B exon resulted in sarcomere disassembly and impaired systolic function (14). This outcome is in contrast to the N2B-KO mice, which survive to adulthood with no detectable defect in sarcomere assembly or systolic function. Because the pickm171 mutation generates a stop codon, the resulting truncated protein lacks not only a functional N2B region but also the M-line region. Our previous work on titin in sarcomere assembly indicated a critical role for the M-line region of titin (15, 16). Thus, the lack of the M-line region in pickm171 might explain their severe phenotype. The morpholino approach resulted in the same phenotype as the pickm171 mutant. It remains to be examined whether this outcome is because of a lack of exon specificity (i.e., whether the M-line region is absent) or whether there is a true species difference. In the mouse, the N2B region does not appear to play a major role in systolic function.

The N2B region of titin not only determines the elastic properties of the sarcomere, it also relates to the ischemic stress and cardiac hypertrophy responses through its binding proteins {alpha}B-crystallin (17) and FHL2 (18), respectively. We did not find changes in {alpha}B-crystallin expression in KO mice, suggesting that interaction between {alpha}B-crystallin and the N2B region of titin is not required for normal cardiac function.

The previously published FHL2-KO (7) and our N2B-KO share the impaired balance of hypertrophy and atrophy. We found that FHL2 protein was down-regulated upon loss of its binding site within the N2B region of titin. This down-regulation occurs at the posttranslational level because mRNA levels are unchanged (Fig. 6). Both altered FHL2 protein levels and the relation of FHL2 and N2B to cardiac growth make it tempting to speculate that the interaction of N2B and FHL2 provides a novel regulatory pathway to control the balance of hypertrophy and atrophy in the heart. Consistent with this proposal is the recent finding that patients with a mutation in the titin N2B region (S3799Y) develop hypertrophic cardiomyopathy associated with an increased binding of FHL2 to titin (5).

In summary, we used a KO approach to establish the N2B region as a critical determinant of the elastic properties of the heart and cardiac growth. The N2B-KO mouse holds the potential for studying interaction between titin and FHL2 and as a tool to develop therapeutic strategies to combat diastolic dysfunction.


   Methods
Top
 Abstract
 Results
 Discussion
 Methods
Acknowledgements
 References
 
Generation of Titin N2B-KO Mice (Titin N2B–/–). A targeting construct was assembled to replace exon 49 (N2B exon) with a FRT-flanked neomycin expression cassette. Titin N2B (+/neo) animals were generated from two independent ES cell clones with subsequent removal of the neomycin cassette by using the Flp deleter stain (19). For more details on generation and genotyping of KO mice, see SI Methods.

All experiments involving animals were carried out following institutional and National Institutes of Health guidelines (20).

Echocardiography. For echocardiography we used the Vevo 770 system (VisualSonics Inc., Toronto, ON, Canada) with a 45-MHz transducer mounted on an integrated rail system. Standard imaging planes, M-mode, Doppler, and functional calculations were obtained according to American Society of Echocardiography guidelines. The LV parasternal long-axis four-chamber view was used to derive fractional shortening, ejection fraction, and ventricular dimensions and volumes. The subcostal long-axis view from the left apex was used for Doppler imaging of mitral inflow and aortic ejection profiles. An extended description of the procedure is available in SI Methods.

Isolated Heart Experiments. The developed and passive wall stress to volume relationship ({sigma} – V) was determined by using the isolated heart preparation with a single-beat Frank–Starling protocol. The Frank–Starling protocol was run first in normal Tyrode solution, then the response to beta-adrenergic stimulation (0.2 µM dobutamine) and beta-adrenergic blockade (0.1 µM propranolol) was determined. Details are available in SI Methods.

Myocyte Mechanics. Cardiac myocytes were isolated from mouse LV as described previously (6). Passive tension–sarcomere length curves were obtained as detailed in ref. 21. Briefly, myocytes were added to a temperature-controlled flow-through chamber mounted on the stage of a phase-contrast microscope. One end of a single cell was glued to a motor. The free end was then bent with a micromanipulator so that the myocyte axis aligned with the microscope optical axis, and the myocyte cross-sectional area was measured. Finally, the free end of the myocyte was glued to a force transducer. Sarcomere length was measured by laser diffraction. Cells were activated with a pCa 4.5 (pCa adjusted with CaCO3) activating solution to verify cell quality, and then passive tension was measured in relaxing solution (6). Experiments were conducted at room temperature.

Gel Electrophoresis and Western Blot Analysis. Protein samples from LVs were prepared by homogenization in liquid N2 and lysis in 8 M urea/50% (vol/vol) glycerol/DTT (80 mM final)/protease inhibitors (leupeptin, E-64, PMSF). Titin isoforms were separated by using an SDS/agarose gel electrophoresis system followed by Coomassie blue staining (22). FHL2 and {alpha}B-crystallin were separated on 12% SDS/polyacrylamide gels. For Western blot analysis, samples were transferred to nitrocellulose membranes (GE Healthcare, Piscataway, NJ) for titin or Hybond P PVDF membranes (GE Healthcare). Membranes were blocked with 5% skim milk in PBS-Tween 20 (PBS-T) followed by incubation with antibodies: anti-Z1-Z2, UN, N2B Us, UC, and M8-M9 (all generous gifts from S. Labeit), and Vectastain (Vector Laboratories, Burlingame, CA) avidin/biotin–AP complex 1:10,000 was used for detec-tion. For loading, control membranes were stained with India ink. Commercial antibodies against FHL2 (mouse monoclonal; MBL, Naga-Ku, Nagoya, Japan) or {alpha}B-crystallin (rabbit polyclonal; Calbiochem, San Diego, CA) were used according to the manufacturer's instructions and detected by using horseradish peroxidase-conjugated secondary antibodies and chemiluminescence staining with ECL (Supersignal West Pico Chemiluminescent Substrate; Pierce, Rockford, IL).

Real-Time PCR. RNA from three individual ventricles was isolated and amplified with TaqMan probes for ANP, FHL2, and 18S RNA (for normalization) as described previously (16).

Immunofluorescence Staining and Immunoelectron Microscopy. Cardiomyocytes were seeded to laminin-coated glass coverslips (4 h) and fixed with cold methanol. Primary antibodies were: mouse monoclonal anti-sarcomeric {alpha}-actinin (1:500; EA53; Sigma–Aldrich, St. Louis, MO) and the rabbit polyclonal antibodies anti-titin N2B (1:200) and anti-titin M8/M9 (1:200) (both gifts from Siegfried Labeit, Universitätsklinikum Mannheim, Germany). Secondary fluorescent-conjugated secondary antibodies were: Alexa Fluor 488 goat anti-rabbit (Molecular Probes Jackson ImmunoResearch, West Grove, PA). Stained cells were analyzed on a confocal scanning laser microscope (LSM 5 Pascal version 3.0 SP2; Karl Zeiss, Jena, Germany) with a PLAN-NEOFLUAR 100x lens (1.3 NA) (Karl Zeiss). Immunoelectron microscopy to localize titin epitopes at different sarcomere lengths has been described previously (3). SI Methods contains a detailed version including references to the antibodies used.

Statistical Analysis. For statistical analysis, SPSS 11.0 software was used. All results are expressed as means ± SEM. An unpaired two-tailed t test was performed to assess differences between two groups.


   Acknowledgements
Top
 Abstract
 Results
 Discussion
 Methods
 Acknowledgements
References
 
We thank Beate Goldbrich, Gemaine Wright, Joe Popper, and Martin Taube for expert technical assistance and Arnd Heuser for support with the Vevo System. This work was supported by American Heart Association Grant GIA 0450195Z (to M.G.), Deutsche Forschungsgemeinschaft Grant GO 865/3 (to M.G.), the Sofja–Kovalevskaya program of the Alexander von Humboldt Foundation (to M.G.), and by National Institutes of Health Grant HL61487/HL62881 (to H.G.).


   Footnotes
 

Abbreviations: ANP, atrial natriuretic peptide; FRT, Flp recombinase target; KO, knockout; LV, left ventricular; MV DT, mitral valve deceleration time.

§To whom correspondence should be addressed at: Department of Veterinary and Comparative Anatomy, Pharmacology, and Physiology, Washington State University, Wegner Hall, Room 205, Pullman, WA 99164-6520 or Max-Delbrück-Center for Molecular Medicine Berlin-Buch, Robert Rössle Strasse 10, 13122 Berlin, Germany. E-mail: gotthard{at}vetmed.wsu.edu or gotthardt{at}mdc-berlin.de

Author contributions: M.H.R., J.P., H.G., and M.G. designed research; M.H.R., J.P., Y.W., M.M., and O.L.N. performed research; M.H.R., J.P., Y.W., M.M., O.L.N., H.G., and M.G. analyzed data; and H.G. and M.G. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS direct submission.

This article contains supporting information online at www.pnas.org/cgi/content/full/0608543104/DC1.

© 2007 by The National Academy of Sciences of the USA


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 Methods
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W. A. Linke
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