Wolfgang A. Linke

Myocardial Structure and Function Differ in Systolic and Diastolic Heart Failure

Background— To support the clinical distinction between systolic heart failure (SHF) and diastolic heart failure (DHF), left ventricular (LV) myocardial structure and function were compared in LV endomyocardial biopsy samples of patients with systolic and diastolic heart failure. Methods and Results— Patients hospitalized for worsening heart failure were classified as having SHF (n=22; LV ejection fraction (EF) 34±2%) or DHF (n=22; LVEF 62±2%). No patient had coronary artery disease or biopsy evidence of infiltrative or inflammatory myocardial disease. More DHF patients had a history of arterial hypertension and were obese. Biopsy samples were analyzed with histomorphometry and electron microscopy. Single cardiomyocytes were isolated from the samples, stretched to a sarcomere length of 2.2 &mgr;m to measure passive force (Fpassive), and activated with calcium-containing solutions to measure total force. Cardiomyocyte diameter was higher in DHF (20.3±0.6 versus 15.1±0.4 &mgr;m, P<0.001), but collagen volume fraction was equally elevated. Myofibrillar density was lower in SHF (36±2% versus 46±2%, P<0.001). Cardiomyocytes of DHF patients had higher Fpassive (7.1±0.6 versus 5.3±0.3 kN/m2; P<0.01), but their total force was comparable. After administration of protein kinase A to the cardiomyocytes, the drop in Fpassive was larger (P<0.01) in DHF than in SHF. Conclusions— LV myocardial structure and function differ in SHF and DHF because of distinct cardiomyocyte abnormalities. These findings support the clinical separation of heart failure patients into SHF and DHF phenotypes.

Reverse engineering of the giant muscle protein titin

Through the study of single molecules it has become possible to explain the function of many of the complex molecular assemblies found in cells. The protein titin provides muscle with its passive elasticity. Each titin molecule extends over half a sarcomere, and its extensibility has been studied both in situ and at the level of single molecules. These studies suggested that titin is not a simple entropic spring but has a complex structure-dependent elasticity. Here we use protein engineering and single-molecule atomic force microscopy to examine the mechanical components that form the elastic region of human cardiac titin. We show that when these mechanical elements are combined, they explain the macroscopic behaviour of titin in intact muscle. Our studies show the functional reconstitution of a protein from the sum of its parts.

Titin Isoform Switch in Ischemic Human Heart Disease

Background—Ischemia-induced cardiomyopathy usually is accompanied by elevated left ventricular end-diastolic pressure, which follows from increased myocardial stiffness resulting from upregulated collagen expression. In addition to collagen, a main determinant of stiffness is titin, whose role in ischemia-induced left ventricular stiffening was studied here. Human heart sarcomeres coexpress 2 principal titin isoforms, a more compliant N2BA isoform and a stiffer N2B isoform. In comparison, normal rat hearts express almost no N2BA titin. Methods and Results—Gel electrophoresis and immunoblotting were used to determine the N2BA-to-N2B titin isoform ratio in nonischemic human hearts and nonnecrotic left ventricle of coronary artery disease (CAD) patients. The average N2BA-to-N2B ratio was 47:53 in severely diseased CAD transplanted hearts and 32:68 in nonischemic transplants. In normal donor hearts and donor hearts with CAD background, relative N2BA titin content was ≈30%. The titin isoform shift in CAD transplant hearts coincided with a high degree of modifications of cardiac troponin I, probably indicating increased preload. Immunofluorescence microscopy on CAD transplant specimens showed a regular cross-striated arrangement of titin and increased expression of collagen and desmin. Force measurements on isolated myofibrils revealed reduced passive-tension levels in sarcomeres of CAD hearts with high left ventricular end-diastolic pressure compared with sarcomeres of normal hearts. In a rat model of ischemia-induced myocardial infarction (left anterior descending coronary artery ligature), 43% of animals, but only 14% of sham-operated animals, showed a distinct N2BA titin band on gels. Conclusions—A titin isoform switch was observed in chronically ischemic human hearts showing extensive remodeling, which necessitated cardiac transplantation. The shift, also confirmed in rat hearts, caused reduced titin-derived myofibrillar stiffness. Titin modifications in long-term ischemic myocardium could impair the ability of the heart to use the Frank-Starling mechanism.

Protein Kinase G Modulates Human Myocardial Passive Stiffness by Phosphorylation of the Titin Springs

The sarcomeric titin springs influence myocardial distensibility and passive stiffness. Titin isoform composition and protein kinase (PK)A-dependent titin phosphorylation are variables contributing to diastolic heart function. However, diastolic tone, relaxation speed, and left ventricular extensibility are also altered by PKG activation. We used back-phosphorylation assays to determine whether PKG can phosphorylate titin and affect titin-based stiffness in skinned myofibers and isolated myofibrils. PKG in the presence of 8-pCPT-cGMP (cGMP) phosphorylated the 2 main cardiac titin isoforms, N2BA and N2B, in human and canine left ventricles. In human myofibers/myofibrils dephosphorylated before mechanical analysis, passive stiffness dropped 10% to 20% on application of cGMP-PKG. Autoradiography and anti-phosphoserine blotting of recombinant human I-band titin domains established that PKG phosphorylates the N2-B and N2-A domains of titin. Using site-directed mutagenesis, serine residue S469 near the COOH terminus of the cardiac N2-B–unique sequence (N2-Bus) was identified as a PKG and PKA phosphorylation site. To address the mechanism of the PKG effect on titin stiffness, single-molecule atomic force microscopy force–extension experiments were performed on engineered N2-Bus–containing constructs. The presence of cGMP-PKG increased the bending rigidity of the N2-Bus to a degree that explained the overall PKG-mediated decrease in cardiomyofibrillar stiffness. Thus, the mechanically relevant site of PKG-induced titin phosphorylation is most likely in the N2-Bus; phosphorylation of other titin sites could affect protein–protein interactions. The results suggest that reducing titin stiffness by PKG-dependent phosphorylation of the N2-Bus can benefit diastolic function. Failing human hearts revealed a deficit for basal titin phosphorylation compared to donor hearts, which may contribute to diastolic dysfunction in heart failure.

Passive Stiffness Changes Caused by Upregulation of Compliant Titin Isoforms in Human Dilated Cardiomyopathy Hearts

In the pathogenesis of dilated cardiomyopathy, cytoskeletal proteins play an important role. In this study, we analyzed titin expression in left ventricles of 19 control human donors and 9 severely diseased (nonischemic) dilated cardiomyopathy (DCM) transplant-patients, using gel-electrophoresis, immunoblotting, and quantitative RT-PCR. Both human-heart groups coexpressed smaller (≈3 MDa) N2B-isoform and longer (3.20 to 3.35 MDa) N2BA-isoforms, but the average N2BA:N2B-protein ratio was shifted from ≈30:70 in controls to 42:58 in DCM hearts, due mainly to increased expression of N2BA-isoforms >3.30 MDa. Titin per unit tissue was decreased in some DCM hearts. The titin-binding protein obscurin also underwent isoform-shifting in DCM. Quantitative RT-PCR revealed a 47% reduction in total-titin mRNA levels in DCM compared with control hearts, but no differences in N2B, all-N2BA, and individual-N2BA transcripts. The reduction in total-titin transcripts followed from a decreased area occupied by myocytes and increased connective tissue in DCM hearts, as detected by histological analysis. Force measurements on isolated cardiomyofibrils showed that sarcomeric passive tension was reduced on average by 25% to 30% in DCM, a reduction readily predictable with a model of wormlike-chain titin elasticity. Passive-tension measurements on human-heart fiber bundles, before and after titin proteolysis, revealed a much-reduced relative contribution of titin to total passive stiffness in DCM. Results suggested that the titin-isoform shift in DCM depresses the proportion of titin-based stiffness by ≈10%. We conclude that a lower-than-normal proportion of titin-based stiffness in end-stage failing hearts results partly from loss of titin and increased fibrosis, partly from titin-isoform shift. The titin-isoform shift may be beneficial for myocardial diastolic function, but could impair the contractile performance in systole.

The Giant Protein Titin Emerging Roles in Physiology and Pathophysiology

Titin is a giant protein of vertebrate striated muscles (M(r), > or = 3000 kD). Its molecules are of filamentous shape and span from the Z disk to the M line, thereby forming a third filament system of the sarcomere. This filament system is important for both the structural integrity of the myofibril and the passive tension response of a stretched muscle fiber. The determination of the cDNA sequence of human cardiac titin has shown that the cardiac titin filament is formed by a single, giant. 27,000-residue-long polypeptide chain. The titin strand has a modular structure, and different modular arrangements are expressed in different muscle tissue types by differential splicing. In the A band, the titin modules provide regular arrays of binding sites for other sarcomeric proteins, thereby contributing to a precise assembly of myofibrillar proteins in vivo. In the I band, two specific motif families, tandem-immunoglobulin domains and PEVK-rich sequences, confer extensibility to the titin filament. Expression of muscle tissue-specific length variants of the PEVK region by alternative splicing may explain the differences in the passive tension properties between various striated muscle types. Apart from the titin sequences with apparent functions for muscle structure and elasticity, the titin molecule contains a class of unique sequence insertions. Among these sequences are phosphorylation sites, a serine/threonine kinase domain, and binding sites for muscle-specific calpain proteases. Thus, it is likely that the titin filament also plays a role in myofibrillar signal transduction pathways.

Isoform Diversity of Giant Proteins in Relation to Passive and Active Contractile Properties of Rabbit Skeletal Muscles

The active and passive contractile performance of skeletal muscle fibers largely depends on the myosin heavy chain (MHC) isoform and the stiffness of the titin spring, respectively. Open questions concern the relationship between titin-based stiffness and active contractile parameters, and titins importance for total passive muscle stiffness. Here, a large set of adult rabbit muscles (n = 37) was studied for titin size diversity, passive mechanical properties, and possible correlations with the fiber/MHC composition. Titin isoform analyses showed sizes between ∼3300 and 3700 kD; 31 muscles contained a single isoform, six muscles coexpressed two isoforms, including the psoas, where individual fibers expressed similar isoform ratios of 30:70 (3.4:3.3 MD). Gel electrophoresis and Western blotting of two other giant muscle proteins, nebulin and obscurin, demonstrated muscle type–dependent size differences of ≤70 kD. Single fiber and single myofibril mechanics performed on a subset of muscles showed inverse relationships between titin size and titin-borne tension. Force measurements on muscle strips suggested that titin-based stiffness is not correlated with total passive stiffness, which is largely determined also by extramyofibrillar structures, particularly collagen. Some muscles have low titin-based stiffness but high total passive stiffness, whereas the opposite is true for other muscles. Plots of titin size versus percentage of fiber type or MHC isoform (I-IIB-IIA-IID) determined by myofibrillar ATPase staining and gel electrophoresis revealed modest correlations with the type I fiber and MHC-I proportions. No relationships were found with the proportions of the different type II fiber/MHC-II subtypes. Titin-based stiffness decreased with the slow fiber/MHC percentage, whereas neither extramyofibrillar nor total passive stiffness depended on the fiber/MHC composition. In conclusion, a low correlation exists between the active and passive mechanical properties of skeletal muscle fibers. Slow muscles usually express long titin(s), predominantly fast muscles can express either short or long titin(s), giving rise to low titin-based stiffness in slow muscles and highly variable stiffness in fast muscles. Titin contributes substantially to total passive stiffness, but this contribution varies greatly among muscles.

Titin Mutations in iPS cells Define Sarcomere Insufficiency as a Cause of Dilated Cardiomyopathy

A giant disruption of the heart Certain forms of heart failure originate from genetic mutations. Understanding how the culprit mutant proteins alter normal heart function could lead to more effective treatments. One candidate is the giant protein tintin, which is mutated in a subset of patients with dilated cardiomyopathy. Through a combination of patient-derived stem cells, tissue engineering, and gene editing, Hinson et al. found that disease-associated titin mutations disrupt the function of the contractile unit in heart muscle. As a result, the heart does not respond properly to mechanical and other forms of stress. Science, this issue p. 982 Mutations in titin cause heart disease by disrupting the sarcomere, which normally helps the heart adapt to stress. Human mutations that truncate the massive sarcomere protein titin [TTN-truncating variants (TTNtvs)] are the most common genetic cause for dilated cardiomyopathy (DCM), a major cause of heart failure and premature death. Here we show that cardiac microtissues engineered from human induced pluripotent stem (iPS) cells are a powerful system for evaluating the pathogenicity of titin gene variants. We found that certain missense mutations, like TTNtvs, diminish contractile performance and are pathogenic. By combining functional analyses with RNA sequencing, we explain why truncations in the A-band domain of TTN cause DCM, whereas truncations in the I band are better tolerated. Finally, we demonstrate that mutant titin protein in iPS cell–derived cardiomyocytes results in sarcomere insufficiency, impaired responses to mechanical and β-adrenergic stress, and attenuated growth factor and cell signaling activation. Our findings indicate that titin mutations cause DCM by disrupting critical linkages between sarcomerogenesis and adaptive remodeling.

Passive and active tension in single cardiac myofibrils

Single myofibrils were isolated from chemically skinned rabbit heart and mounted in an apparatus described previously (Fearn et al., 1993; Linke et al., 1993). We measured the passive length-tension relation and active isometric force, both normalized to cross sectional area. Myofibrillar cross sectional area was calculated based on measurements of myofibril diameter from both phase-contrast images and electron micrographs. Passive tension values up to sarcomere lengths of approximately 2.2 microns were similar to those reported in larger cardiac muscle specimens. Thus, the element responsible for most, if not all, passive force of cardiac muscle at physiological sarcomere lengths appears to reside within the myofibrils. Above 2.2 microns, passive tension continued to rise, but not as steeply as reported in multicellular preparations. Apparently, structures other than the myofibrils become increasingly important in determining the magnitude of passive tension at these stretched lengths. Knowing the myofibrillar component of passive tension allowed us to infer the stress-strain relation of titin, the polypeptide thought to support passive force in the sarcomere. The elastic modulus of titin is 3.5 x 10(6) dyn cm-2, a value similar to that reported for elastin. Maximum active isometric tension in the single myofibril at sarcomere lengths of 2.1-2.3 microns was 145 +/- 35 mN/mm2 (mean +/- SD; n = 15). This value is comparable with that measured in fixed-end contractions of larger cardiac specimens, when the amount of nonmyofibrillar space in those preparations is considered. However, it is about 4 times lower than the maximum active tension previously measured in single skeletal myofibrils under similar conditions (Bartoo et al., 1993).

Unfolding of Titin Domains Explains the Viscoelastic Behavior of Skeletal Myofibrils

The elastic section of the giant muscle protein titin contains many immunoglobulin-like domains, which have been shown by single-molecule mechanical studies to unfold and refold upon stretch-release. Here we asked whether the mechanical properties of Ig domains and/or other titin regions could be responsible for the viscoelasticity of nonactivated skeletal-muscle sarcomeres, particularly for stress relaxation and force hysteresis. We show that isolated psoas myofibrils respond to a stretch-hold protocol with a characteristic force decay that becomes more pronounced following stretch to above 2.6-microm sarcomere length. The force decay was readily reproducible by a Monte Carlo simulation taking into account both the kinetics of Ig-domain unfolding and the worm-like-chain model of entropic elasticity used to describe titins elastic behavior. The modeling indicated that the force decay is explainable by the unfolding of only a very small number of Ig domains per titin molecule. The simulation also predicted that a unique sequence in titin, the PEVK domain, may undergo minor structural changes during sarcomere extension. Myofibrils subjected to 1-Hz cycles of stretch-release exhibited distinct hysteresis that persisted during repetitive measurements. Quick stretch-release protocols, in which variable pauses were introduced after the release, revealed a two-exponential time course of hysteresis recovery. The rate constants of recovery compared well with the refolding rates of Ig-like or fibronectin-like domains measured by single-protein mechanical analysis. These findings suggest that in the sarcomere, titins Ig-domain regions may act as entropic springs capable of adjusting their contour length in response to a stretch.