Martina Krüger

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.

Developmental changes in contractility and sarcomeric proteins from the early embryonic to the adult stage in the mouse heart

Developmental changes in force‐generating capacity and Ca2+ sensitivity of contraction in murine hearts were correlated with changes in myosin heavy chain (MHC) and troponin (Tn) isoform expression, using Triton‐skinned fibres. The maximum Ca2+‐activated isometric force normalized to the cross‐sectional area (FCSA) increased mainly during embryogenesis and continued to increase at a slower rate until adulthood. During prenatal development, FCSA increased about 5‐fold from embryonic day (E)10.5 to E19.5, while the amount of MHC normalized to the amount of total protein remained constant (from E13.5 to E19.5). This suggests that the development of structural organization of the myofilaments during the embryonic and the fetal period may play an important role for the improvement of force generation. There was an overall decrease of 0.5 pCa units in the Ca2+ sensitivity of force generation from E13.5 to the adult, of which the main decrease (0.3 pCa units) occurred within a short time interval, between E19.5 and 7 days after birth (7 days pn). Densitometric analysis of SDS‐PAGE and Western blots revealed that the major switches between troponin T (TnT) isoforms occur before E16.5, whereas the transition points of slow skeletal troponin I (ssTnI) to cardiac TnI (cTnI) and of β‐MHC to α‐MHC both occur around birth, in temporal correlation with the main decrease in Ca2+ sensitivity. To test whether the changes in Ca2+ sensitivity are solely based on Tn, the native Tn complex was replaced in fibres from E19.5 and adult hearts with fast skeletal Tn complex (fsTn) purified from rabbit skeletal muscle. The difference in pre‐replacement values of pCa50 (−log([Ca2+]m−1)) required for half‐maximum force development) between E19.5 (6.05 ± 0.01) and adult fibres (5.64 ± 0.04) was fully abolished after replacement with the exogenous skeletal Tn complex (pCa50= 6.12 ± 0.05 for both stages). This suggests that the major developmental changes in Ca2+ sensitivity of skinned murine myocardium originate primarily from the switch of ssTnI to cTnI.

Force Kinetics and Individual Sarcomere Dynamics in Cardiac Myofibrils after Rapid Ca2+ Changes

Kinetics of force development and relaxation after rapid application and removal of Ca(2+) were measured by atomic force cantilevers on subcellular bundles of myofibrils prepared from guinea pig left ventricles. Changes in the structure of individual sarcomeres were simultaneously recorded by video microscopy. Upon Ca(2+) application, force developed with an exponential rate constant k(ACT) almost identical to k(TR), the rate constant of force redevelopment measured during steady-state Ca(2+) activation; this indicates that k(ACT) reflects isometric cross-bridge turnover kinetics. The kinetics of force relaxation after sudden Ca(2+) removal were markedly biphasic. An initial slow linear decline (rate constant k(LIN)) lasting for a time t(LIN) was abruptly followed by an ~20 times faster exponential decay (rate constant k(REL)). k(LIN) is similar to k(TR) measured at low activating [Ca(2+)], indicating that k(LIN) reflects isometric cross-bridge turnover kinetics under relaxed-like conditions (see also. Biophys. J. 83:2142-2151). Video microscopy revealed the following: invariably at t(LIN) a single sarcomere suddenly lengthened and returned to a relaxed-type structure. Originating from this sarcomere, structural relaxation propagated from one sarcomere to the next. Propagated sarcomeric relaxation, along with effects of stretch and P(i) on relaxation kinetics, supports an intersarcomeric chemomechanical coupling mechanism for rapid striated muscle relaxation in which cross-bridges conserve chemical energy by strain-induced rebinding of P(i).

The Giant Protein Titin as an Integrator of Myocyte Signaling Pathways

The giant muscle protein titin, the backbone of the sarcomere, harbors a complex molecular spring whose stiffness is variably tuned in health and disease. Titin is increasingly recognized as a crucial integrator of diverse myocyte signaling pathways. The titin-associated signalosome includes hotspots of protein-protein interactions important for the regulation of protein quality-control mechanisms, hypertrophic gene activation, and mechanosensing.

Thyroid Hormone Regulates Developmental Titin Isoform Transitions via the Phosphatidylinositol-3-Kinase/ AKT Pathway

Titins, giant sarcomere proteins with major mechanical/signaling functions, are expressed in 2 main isoform classes in the mammalian heart: N2B (3000 kDa) and N2BA (>3200 kDa). A dramatic isoform switch occurs during cardiac development, from fetal N2BA titin (3700 kDa) expressed before birth to a mix of smaller N2BA/N2B isoforms found postnatally; adult rat hearts almost exclusively have N2B titin. The isoform switch, which can be reversed in chronic human heart failure, alters myocardial distensibility and mechanosignaling. Here we determined factors regulating this switch using, as a model system, primary cardiomyocyte cultures prepared from embryonic rats. In standard culture, the mean N2B percentage initially was 14% and increased by ≈60% within 1 week, resembling the in vivo switching. The titin isoform transition was independent of endothelin-1–induced myocyte hypertrophy and was not altered by pacing, contractile arrest, or cell stretch; however, it was modestly impaired by decreasing substrate rigidity and strongly dependent on serum components. Angiotensin II significantly promoted the transition. The mean N2B proportion in 1-week-old cultures dropped 20% to 25% in hormone-reduced medium, but addition of 3,5,3′-triiodo-l-thyronine (T3) nearly restored the proportion to that found in standard culture. This T3 effect was not prevented by bisphenol A, a specific inhibitor of the classic genomic pathway of T3 action. In contrast, the titin switch could be stalled by the phosphatidylinositol 3-kinase inhibitor LY294002, which decreased the proportion of N2B mRNA transcripts within hours and suppressed a rapid T3-induced increase in Akt phosphorylation. Also, angiotensin II, but not endothelin-1 or cell stretch, enhanced Akt phosphorylation. Thus, although matrix stiffness modulates developmental titin isoform transitions, these transitions are mainly regulated through phosphatidylinositol 3-kinase/Akt-dependent signaling triggered particularly by T3 via a rapid action pathway.

Differential changes in titin domain phosphorylation increase myofilament stiffness in failing human hearts

AIMSnTitin-based myofilament stiffness is defined by the expression levels of the cardiac titin-isoforms, N2B and N2BA, and by phosphorylation of the elastic titin domains N2-B unique sequence (N2-Bus) and PEVK. Phosphorylation of the N2-Bus by cGMP-dependent protein kinase (PKG) or cAMP-dependent protein kinase (PKA) decreases titin stiffness, whereas phosphorylation of the PEVK-domain by PKC increases it. We aimed to identify specific sites within the N2-Bus phosphorylated by PKA and PKG and to determine whether differential changes in titin domain phosphorylation could affect passive stiffness in human failing hearts.nnnMETHODS AND RESULTSnUsing mass spectrometry, we identified seven partly conserved PKA/PKG-targeted phosphorylation motifs in human and rat N2-Bus. Polyclonal antibodies to pSer4185, pSer4010, and pSer4099 in the N2-Bus, and to pSer11878 in the PEVK-region were used to quantify titin-domain phosphorylation by western blot analyses of a set of human donor and failing hearts with similar titin-isoform composition. Passive tension determined in skinned human myocardial fibre preparations was significantly increased in failing compared with donor hearts, notably at shorter sarcomere lengths where titin contributes most to total passive tension. Phosphorylation of Ser4185, Ser4010, and Ser4099 in the N2-Bus was significantly reduced in failing hearts, whereas phosphorylation of Ser11878 in the PEVK-region was increased compared with donor hearts.nnnCONCLUSIONnWe conclude that hypo-phosphorylation of the N2-Bus and hyper-phosphorylation of the PEVK domain can act complementary to elevate passive tension in failing human hearts. Differential changes in titin-domain phosphorylation may be important to fine-tune passive myocardial stiffness and diastolic function of the heart.

Titin, a Central Mediator for Hypertrophic Signaling, Exercise-Induced Mechanosignaling and Skeletal Muscle Remodeling.

Titin is a giant scaffold protein with multiple functions in striated muscle physiology. Due to the elastic I-band domains and the filament-like integration in the half-sarcomere titin is an important factor for sarcomere assembly and serves as an adaptable molecular spring that determines myofilament distensibility. Protein-interactions e.g., with muscle ankyrin repeat proteins or muscle LIM-protein link titin to hypertrophic signaling and via p62 and Muscle Ring Finger proteins to mechanisms that control protein quality control. This review summarizes our current knowledge on titin as a central node for exercise-induced mechanosignaling and remodeling and further highlights the pathophysiological implications.

Expression and purification of human cardiac troponin subunits and their functional incorporation into isolated cardiac mouse myofibrils.

The three subunits of the human cardiac troponin complex (hcTnC, hcTnI, hcTnT) were overexpressed in E. coli, purified and reconstituted to form the hcTn complex. This complex was then incorporated into subcellular bundles of mouse cardiac myofibrils whereby the native mcTn complex was replaced. On thus exchanged myofibrils, isometric force kinetics following sudden changes in free Ca(2+) concentration were measured using atomic force cantilevers. Following the exchange, the myofibrillar force remained fully Ca(2+) regulated, i.e. myofibrils were completely relaxed at pCa 7.5 and developed the same maximum Ca(2+)-activated isometric force upon increasing the pCa to 4.5 as unexchanged myofibrils. The replacement of endogenous mcTn by wild-type hcTn neither altered the kinetics of Ca(2+)-induced force development of the mouse myofibrils nor the kinetics of force relaxation induced by the sudden, complete removal of Ca(2+). Preparations of functional Tn reconstituted myofibrils provide a promising model to study the role of Tn in kinetic mechanisms of cardiac myofibrillar contraction and relaxation.

Titin: central player of hypertrophic signaling and sarcomeric protein quality control

Abstract The giant sarcomeric protein titin has multiple important functions in striated muscle cells. Due to its gigantic size, its central position in the sarcomere and its elastic I-band domains, titin is a scaffold protein that is important for sarcomere assembly, and serves as a molecular spring that defines myofilament distensibility. This review focuses on the emerging role of titin in mechanosensing and hypertrophic signaling, and further highlights recent evidence that links titin to sarcomeric protein turnover.

Acute exercise modifies titin phosphorylation and increases cardiac myofilament stiffness.

Titin-based myofilament stiffness is largely modulated by phosphorylation of its elastic I-band regions N2-Bus (decreases passive stiffness, PT) and PEVK (increases PT). Here, we tested the hypothesis that acute exercise changes titin phosphorylation and modifies myofilament stiffness. Adult rats were exercised on a treadmill for 15 min, untrained animals served as controls. Titin phosphorylation was determined by Western blot analysis using phosphospecific antibodies to Ser4099 and Ser4010 in the N2-Bus region (PKG and PKA-dependent. respectively), and to Ser11878 and Ser 12022 in the PEVK region (PKCα and CaMKIIδ-dependent, respectively). Passive tension was determined by step-wise stretching of isolated skinned cardiomyocytes to sarcomere length (SL) ranging from 1.9 to 2.4 μm and showed a significantly increased PT from exercised samples, compared to controls. In cardiac samples titin N2-Bus phosphorylation was significantly decreased by 40% at Ser4099, however, no significant changes were observed at Ser4010. PEVK phosphorylation at Ser11878 was significantly increased, which is probably mediated by the observed exercise-induced increase in PKCα activity. Interestingly, relative phosphorylation of Ser12022 was substantially decreased in the exercised samples. Surprisingly, in skeletal samples from acutely exercised animals we detected a significant decrease in PEVK phosphorylation at Ser11878 and an increase in Ser12022 phosphorylation; however, PKCα activity remained unchanged. In summary, our data show that a single exercise bout of 15 min affects titin domain phosphorylation and titin-based myocyte stiffness with obviously divergent effects in cardiac and skeletal muscle tissues. The observed changes in titin stiffness could play an important role in adapting the passive and active properties of the myocardium and the skeletal muscle to increased physical activity.