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A prominent feature of striated muscle is the regular lateral alignment of adjacent sarcomeres. An important intermyofibrillar linking protein is the intermediate filament protein desmin, and based on biochemical and structural studies in primary cultures of myocytes it has been proposed that desmin interacts with the sarcomeric protein nebulin. Here we tested whether nebulin is part of a novel biomechanical linker complex, by using a recently developed nebulin knockout (KO) mouse model and measuring Z-disk displacement in adjacent myofibrils of both extensor digitorum longus (EDL) and soleus muscle. Z-disk displacement increased as sarcomere length (SL) was increased and the increase was significantly larger in KO fibers than in wild-type (WT) fibers; results in 3-day-old and 10-day-old mice were similar. Immunoelectron microscopy revealed reduced levels of desmin in intermyofibrillar spaces adjacent to Z-disks in KO fibers compared with WT fibers. We also performed siRNA knockdown of nebulin and expressed modules within the Z-disk portion of nebulin (M160-M170) in quail myotubes and found that this prevented the mature Z-disk localization of desmin filaments. Combined, these data suggest a model in which desmin attaches to the Z-disk through an interaction with nebulin. Finally, because nebulin has been proposed to play a role in specifying Z-disk width, we also measured Z-disk width in nebulin KO mice. Results show that most Z-disks of KO mice were modestly increased in width (approximately 80 nm in soleus and approximately 40 nm in EDL fibers) whereas a small subset had severely increased widths (up to approximately 1 microm) and resembled nemaline rod bodies. In summary, structural studies on a nebulin KO mouse show that in the absence of nebulin, Z-disks are significantly wider and that myofibrils are misaligned. Thus the functional roles of nebulin extend beyond thin filament length regulation and include roles in maintaining physiological Z-disk widths and myofibrillar connectivity.

Previous work using the retrogradely transported immunotoxin, saporin (SAP) conjugated to a monoclonal antibody against dopamine-β-hydroxylase (DBH; DSAP), to selectively lesion norepinephrine (NE) and epinephrine (E) neurons projecting to the medial hypothalamus, demonstrated the essential role of these neurons for appetitive ingestive responses to glucoprivation. Here, we again utilized this lesion to assess the importance of these same neurons for the consummatory phase of glucoprivic feeding. To test consummatory responses, milk was infused intraorally through a chronic cheek fistula until rejected. Appetitive responses were tested in the same rats using pelleted food. Feeding responses to insulin-induced hypoglycemia, 2-deoxy- d -glucose (2DG)-induced blockade of glucose utilization, mercaptoacetate (MA)-induced blockade of fatty acid oxidation, 0.9% saline, and 18-h food deprivation were assessed. Unlike unconjugated SAP controls, the DSAP rats did not increase their food intake in response to glucoprivic challenges in either the pelleted food or the intraoral feeding tests. However, the DSAP rats did not differ from SAPs in their ingestive responses to food deprivation and blockade of fatty acid oxidation. The selective impairment of glucoprivic feeding responses indicates that DSAP did not impair the underlying circuitry required for either appetitive or consummatory ingestive responding but eliminated the mechanism for control of this circuitry specifically by glucoprivation. Results suggest that both appetitive and consummatory responses to glucoprivation are controlled and coordinated by multilevel terminations of the same catecholamine neurons.

Rationale: Mechanisms underlying diastolic dysfunction need to be better understood. Objective: To study the role of titin in diastolic dysfunction using a mouse model of experimental heart failure induced by transverse aortic constriction. Methods and Results: Eight weeks after transverse aortic constriction surgery, mice were divided into heart failure (HF) and congestive heart failure (CHF) groups. Mechanical studies on skinned left ventricle myocardium measured total and titin-based and extracellular matrix-based passive stiffness. Total passive stiffness was increased in both HF and CHF mice, and this was attributable to increases in both extracellular matrix-based and titin-based passive stiffness, with titin being dominant. Protein expression and titin exon microarray analysis revealed increased expression of the more compliant N2BA isoform at the expense of the stiff N2B isoform in HF and CHF mice. These changes are predicted to lower titin-based stiffness. Because the stiffness of titin is also sensitive to titin phosphorylation by protein kinase A and protein kinase C, back phosphorylation and Western blot assays with novel phospho-specific antibodies were performed. HF and CHF mice showed hyperphosphorylation of protein kinase A sites and the proline glutamate valine lysine (PEVK) S26 protein kinase C sites, but hypophosphorylation of the PEVK S170 protein kinase C site. Protein phosphatase I abolished differences in phosphorylation levels and normalized titin-based passive stiffness levels between control and HF myocardium. Conclusion: Transverse aortic constriction-induced HF results in increased extracellular matrix-based and titin-based passive stiffness. Changes in titin splicing occur, which lower passive stiffness, but this effect is offset by hyperphosphorylation of residues in titin spring elements, particularly of PEVK S26. Thus, complex changes in titin occur that combined are a major factor in the increased passive myocardial stiffness in HF.

The increase in Ca2+ sensitivity that occurs when sarcomere length (SL) is increased (LDA), is considered the cellular basis of the Frank-Starling law of the heart. Recent findings show increased LDA in a mouse model in which the N2B spring element has been excised (N2B KO) (Lee et al., 2010). Here we investigated LDA in the presence of 3% Dextran (Dex), the effect of PKA phosphorylation on LDA, and structural changes of myofilaments by using low-angle X-ray diffraction. Fiber bundles of skinned papillary muscle from left ventricular of N2B KO and WT mice were used. The passive tension, SL and X-ray diffraction patterns were collected at various SLs from 1.95 to 2.3µm. From X-ray diffraction patterns, myofilament lattice spacing (LS; d10) and the position of the myosin head relative to the thin filaments (intensity ratio; I11/I10) were obtained. We also performed mechanical studies to characterize Ca2+ sensitivity and LDA. For X-ray diffraction study, d10 was significantly decreased with SL regardless of genotype and Dex (p-values

Heart Failure is characterized by an impaired ability of the left ventricle to fill or eject blood and is typically accompanied with cardiac hypertrophy. Furthermore, sustained cardiac hypertrophy transitions into maladaptive hypertrophy, with left ventricular dilation, decrease contractile properties, increased myocardial stiffness (through alterations in titin and collagen), and reduced cardiac output. The current aim was to investigate alterations in heart function, titin isoform shifts, and myocardial stiffness following transverse aortic constriction-induced heart failure in mouse. Echocardiology showed increased aortic velocity (0.5 vs 4.5m/s), diastolic (55 vs 90ul) and systolic (20 vs 53ul) ventricular volume, LV mass (133 vs 220mg) and decreased ejection fraction (65% vs 35%) and mitral valve deceleration time (23 vs 18ms) (indicating a significant increase in myocardial stiffness). Additionally, E/A ratios dramatically increased from 1.5 to 5, signifying extensive heart failure. In order to further characterize myocardial stiffness, skinned LV muscle bundle fibers were mechanically stretched and titin and collagen forces were measured. At sarcomere length 2.15, data showed an increase in total passive tension (35%), total collagen force (120%), and total titin force (15%). Protein gels were ran to help identify the source of increased titin tension. Gels showed an increase in the long N2BA as related to the short N2B titin isoform (0.20 vs 0.37) indicating that titin passive tension should actually decrease, not increase. However, western blots using phospho-specific antibodies for titin's S11878 and S12022 (phosphorylated by PKCα resulting in increased passive tension) indicate a 2.5 fold increase in phosphorylation in the end-stage heart failure mice. In conclusion, TAC-induced heart failure in mice resulted in an increased diastolic stiffness partially through PKCα phosphorylation-mediated increases in titin-based tension.

Background— Titin is the largest mammalian (≈3000 to 4000 kDa) and myofilament protein that acts as a molecular spring in the cardiac sarcomere and determines systolic and diastolic function. Loss of titin in ischemic hearts has been reported, but the mechanism of titin degradation is not well understood. Matrix metalloproteinase-2 (MMP-2) is localized to the cardiac sarcomere and, on activation in ischemia/reperfusion injury, proteolyzes specific myofilament proteins. Here we determine whether titin is an intracellular substrate for MMP-2 and if its degradation during ischemia/reperfusion contributes to cardiac contractile dysfunction. Methods and Results— Immunohistochemistry and confocal microscopy in rat and human hearts showed discrete colocalization between MMP-2 and titin in the Z-disk region of titin and that MMP-2 is localized mainly to titin near the Z disk of the cardiac sarcomere. Both purified titin and titin in skinned cardiomyocytes were proteolyzed when incubated with MMP-2 in a concentration-dependent manner, and this was prevented by MMP inhibitors. Isolated rat hearts subjected to ischemia/reperfusion injury showed cleavage of titin in ventricular extracts by gel electrophoresis, which was confirmed by reduced titin immunostaining in tissue sections. Inhibition of MMP activity with ONO-4817 prevented ischemia/reperfusion-induced titin degradation and improved the recovery of myocardial contractile function. Titin degradation was also reduced in hearts from MMP-2 knockout mice subjected to ischemia/reperfusion in vivo compared with wild-type controls. Conclusion— MMP-2 localizes to titin at the Z-disk region of the cardiac sarcomere and contributes to titin degradation in myocardial ischemia/reperfusion injury.

Protein kinase C-alpha (PKCalpha) was recently reported to increase myocardial stiffness, an effect that was proposed to be due to phosphorylation of two highly conserved sites (S11878 and S12022) within the proline-glutamic acid-valine-lysine (PEVK) rich spring element of titin. To test this proposal we investigated the effect of PKCalpha on phosphorylation and passive stiffness in a mouse model lacking the titin exons that contain these two phosphorylation sites, the PEVK knockout (KO). We used skinned, gelsolin-extracted, left ventricular myocardium from wildtype and PEVK KO mice. Consistent with previous work we found that PKCalpha increased passive stiffness in the WT myocardium by 27+/-6%. Importantly, this effect was completely abolished in KO myocardium. In addition, increases in the elastic and viscous moduli at a wide range of frequencies (properties important in diastolic filling) following PKCalpha incubation (27+/-3% and 20+/-4%, respectively) were also ablated in the KO. Back phosphorylation assays showed that titin phosphorylation following incubation with PKCalpha was significantly reduced by 36+/-12% in skinned PEVK KO myocardial tissues. The remaining phosphorylation in the KO suggests that PKCalpha sites exist in the titin molecule outside the PEVK region; these sites are not involved in increasing passive stiffness. Our results firmly support that the PEVK region of cardiac titin is phosphorylated by PKCalpha and that this increases passive tension. Thus, the PEVK spring element is the critical site of PKCalpha's involvement in passive myocardial stiffness.

Mutations in a variety of myofibrillar genes cause dilated cardiomyopathy (DCM) in humans, usually with dominant inheritance and incomplete penetrance. Here, we sought to clarify the functional effects of the previously identified DCM-causing TTN 2-bp insertion mutation (c.43628insAT) and generated a titin knock-in mouse model mimicking the c.43628insAT allele. Mutant embryos homozygous for the Ttn knock-in mutation developed defects in sarcomere formation and consequently died before E9.5. Heterozygous mice were viable and demonstrated normal cardiac morphology, function and muscle mechanics. mRNA and protein expression studies on heterozygous hearts demonstrated elevated wild-type titin mRNA under resting conditions, suggesting that up-regulation of the wild-type titin allele compensates for the unstable mutated titin under these conditions. When chronically exposed to angiotensin II or isoproterenol, heterozygous mice developed marked left ventricular dilatation (p

We recently published a PEVK KO mouse model in which all exons that constitute the PEVK region of cardiac titin (N2B cardiac isoform), exons 219-225, had been excised, and reported cardiac hypertrophy and increased passive stress (Granzier et al., 2009 Circ Res., 11, 557). Here we investigated the phenotype of EDL (fast twitch) and soleus (slow twitch) skeletal muscle of wildtype and homozygous PEVK KO mice. Muscle mass was significantly increased in both soleus (51±5%) and EDL (21±6%) muscles; we are currently studying whether the underlying hypertrophy mechanisms are similar to those previously found in the heart. Because the excised exons make up a small portion of the PEVK segment of skeletal muscle titins we expected modest differences, if any at all, in passive stress. Unexpectedly, passive stress was significantly increased in soleus and EDL muscles, both when measurements (SL 3.0μm) were made at the whole muscle level (40±7% and 67±16%, respectively) and at the fiber bundle level (50±14% and 81±1%). Gel electrophoresis revealed, in both EDL and soleus, expression of a single titin isoform in wildtype muscle, but surprisingly, co-expression of two isoforms in the KO muscles. The larger isoform co-migrated with the isoform expressed in wt muscle and thus is likely to represent KO titin (exons 219-225 represent ∼30kDa, which is insufficient to cause a mobility difference). The smaller isoform, found in both EDL and soleus KO muscle, is likely to represent a splicing adaption that correlates with increased passive tension. We are currently investigating the identity of these exons using titin exon microarray analysis. In summary, excision of titin exons 219-225 in skeletal muscle results in elevated passive stress with the underlying cause likely being due to an adaption in titin splicing.

Phosphorylation of cardiac titin spring elements by serine/threonine (S/T) protein kinases affects the diastolic function of the heart. The detection and identification of these phosphorylation sites in different species is critical to our understanding of this important biological process. β-adrenergic and cGMP activation of PKA and PKG, respectively, plays critical roles in the functional regulation of the heart and cardiovascular system; the PKA/PKG target domain is the N2B spring element. The current aim is to determine differences in N2B phosphorylation between mouse and human using autoradiography and mass spectrometry. For autoradiography, mouse and human N2B recombinant fragments were incubated with PKA or PKG, separated by SDS-PAGE (4-20% gradient), and Coomassie blue stained. Autoradiography results indicate that human N2B is a stronger substrate for PKA and PKG than mouse N2B. To understand the molecular basis of these differences we carried out alignment analysis of human and mouse N2B sequences (53% homology) and determined phosphorylation sites of the human N2B by MS. For mass spectrometry, the human N2B protein band was excised, digested with trypsin, and the products analyzed by nano-LC-MS/MS. The MS analysis of human N2B detected 10 residues phosphorylated by PKA (2S/8T) and 7 by PKG (6S/1T). Comparing MS data alignment of the mouse and human N2B sequences showed that mouse N2B has less possible phosphorylation sites for PKA/PKG compared to human N2B; only S478, phosphorylated by both PKA and PKG in human, is present in mouse N2B. Also, S348 in human is substituted by a T in mouse. In conclusion, the data suggests that β-adrenergic and cGMP activation may be evolutionarily less important in mouse heart function than in human.

Titin (3-4MDa) and nebulin (700-900kDa) are large sarcomeric proteins of skeletal muscle. Both proteins contain sites of differential splicing throughout their length. To study splicing during development we used a home-made exon microarray representing all of titin's and nebulin's exons and compared exon expression between mice of different ages in numerous muscle types. We found that a gradual reduction in titin's transcript size in exons encoding the elastic PEVK segment located in the I-band correlates with a reduced titin protein (typically ∼0.2 MDa smaller in adults) and stiffer sarcomere in all skeletal muscles studied (soleus, m. gastrocnemius, tibialis cranialis, extensor digitorum longus, and quadriceps). Nebulin undergoes less dramatic differential splicing than titin, but it was found that exons encoding the M177/M178 repeat in the z-disc are upregulated in the adult versus embryonic samples. Additionally, the soleus muscle exhibited a 4-fold increase in expression level of the SH3 domain of nebulin and a 3-fold increase in the serine rich exons. These findings correlate with structural changes in the z-disc which are wider in the adult (133nm) versus the neonatal soleus samples (121nm, pvalue 0.04). Thus titin and nebulin transcripts are tuned during development with changes in titin altering the I-band properties and changes in nebulin affecting the Z-disc.

Rationale : Protein kinase C (PKC) regulates contractility of cardiac muscle cells by phosphorylating thin- and thick- filament-based proteins. Myocardial sarcomeres also contain a third myofilament, titin, and it is unknown whether titin can be phosphorylated by PKC and whether it affects passive tension. Objective : The purpose of this study was to examine the effect of PKC on titin phosphorylation and titin-based passive tension. Methods and Results : Phosphorylation assays with PKCα revealed that titin is phosphorylated in skinned myocardial tissues; this effect is exacerbated by pretreating with protein phosphatase 1. In vitro phosphorylation of recombinant protein representing titin’s spring elements showed that PKCα targets the proline – glutamate – valine – lysine (PEVK) spring element. Furthermore, mass spectrometry in combination with site-directed mutagenesis identified 2 highly conserved sites in the PEVK region that are phosphorylated by PKCα (S11878 and S12022); when these 2 sites are mutated to alanine, phosphorylation is effectively abolished. Mechanical experiments with skinned left ventricular myocardium revealed that PKCα significantly increases titin-based passive tension, an effect that is reversed by protein phosphatase 1. Single molecule force-extension curves show that PKCα decreases the PEVK persistence length (from 1.20 nm to 0.55 nm), without altering the contour length, and using a serially-linked wormlike chain model we show that this increases titin-based passive force with a sarcomere length dependence that is similar to that measured in skinned myocardium after PKCα phosphorylation. Conclusions : PKC phosphorylation of titin is a novel and conserved pathway that links myocardial signaling and myocardial stiffness.