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Nebulin rules thin-filament length in fast skeletal muscle but collaborates with leiomodin-2 in muscles optimized for efficiency., Regulating the thin-filament length in muscle is crucial for controlling the number of myosin motors that generate power. The giant protein nebulin forms a long slender filament that associates along the length of the thin filament in skeletal muscle with functions that remain largely obscure. Here nebulin’s role in thin-filament length regulation was investigated by targeting entire super-repeats in the Neb gene; nebulin was either shortened or lengthened by 115 nm. Its effect on thin-filament length was studied using high-resolution structural and functional techniques. Results revealed that thin-filament length is strictly regulated by the length of nebulin in fast muscles. Nebulin’s control is less tight in slow muscle types where a distal nebulin-free thin-filament segment exists, the length of which was found to be regulated by leiomodin-2 (Lmod2). We propose that strict length control by nebulin promotes high-speed shortening and that dual-regulation by nebulin/Lmod2 enhances contraction efficiency.

Background: Low myocardial cGMP-PKG (cyclic guanosine monophosphate-protein kinase G) activity has been associated with increased cardiomyocyte diastolic stiffness in heart failure with preserved ejection fraction. Cyclic guanosine monophosphate is mainly hydrolyzed by PDE (phosphodiesterases) 5a and 9a. Importantly, PDE9a expression has been reported to be upregulated in human heart failure with preserved ejection fraction myocardium and chronic administration of a PDE9a inhibitor reverses preestablished cardiac hypertrophy and systolic dysfunction in mice subjected to transverse aortic constriction (TAC). We hypothesized that inhibiting PDE9a activity ameliorates diastolic dysfunction. Methods: To examine the effect of chronic PDE9a inhibition, 2 diastolic dysfunction mouse models were studied: (1) TAC-deoxycorticosterone acetate and (2) Lepr db/db . PDE9a inhibitor (5 and 8 mg/kg per day) was administered to the mice via subcutaneously implanted osmotic minipumps for 28 days. The effect of acute PDE9a inhibition was investigated in intact cardiomyocytes isolated from TAC-deoxycorticosterone acetate mice. Atrial natriuretic peptide together with PDE9a inhibitor were administered to the isolated intact cardiomyocytes through the cell perfusate. Results: For acute inhibition, no cellular stiffness reduction was found, whereas chronic PDE9a inhibition resulted in reduced left ventricular chamber stiffness in TAC-deoxycorticosterone acetate, but not in Lepr db/db mice. Passive cardiomyocyte stiffness was reduced by chronic PDE9a inhibition, with no differences in myocardial fibrosis or cardiac morphometry. PDE9a inhibition increased the ventricular-arterial coupling ratio, reflecting impaired systolic function. Conclusions: Chronic PDE9a inhibition lowers left ventricular chamber stiffness in TAC-deoxycorticosterone acetate mice. However, the usefulness of PDE9a inhibition to treat high-diastolic stiffness may be limited as the required PDE9a inhibitor dose also impairs systolic function, observed as a decline in ventricular-arterial coordination, in this model.

Titin, the largest protein known, forms an elastic myofilament in the striated muscle sarcomere. To establish titin’s contribution to skeletal muscle passive stiffness, relative to that of the extracellular matrix, a mouse model was created in which titin’s molecular spring region was shortened by deleting 47 exons, the TtnΔ112-158 model. RNA sequencing and super-resolution microscopy predicts a much stiffer titin molecule. Mechanical studies with this novel mouse model support that titin is the main determinant of skeletal muscle passive stiffness. Unexpectedly, the in vivo sarcomere length working range was shifted to shorter lengths in TtnΔ112-158 mice, due to a ~ 30% increase in the number of sarcomeres in series (longitudinal hypertrophy). The expected effect of this shift on active force generation was minimized through a shortening of thin filaments that was discovered in TtnΔ112-158 mice. Thus, skeletal muscle titin is the dominant determinant of physiological passive stiffness and drives longitudinal hypertrophy.Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter).

RATIONALE: Diaphragm weakness in critically ill patients prolongs ventilator dependency and duration of hospital stay, and increases mortality and health care costs. The mechanisms underlying diaphragm weakness include cross-sectional fiber atrophy and contractile protein dysfunction, but whether additional mechanisms are at play is unknown.OBJECTIVES: To test the hypothesis that mechanical ventilation with positive end-expiratory pressure (PEEP) induces longitudinal atrophy by displacing the diaphragm in caudal direction and reducing the length of fibers.METHODS: We studied structure and function of diaphragm fibers of mechanically ventilated critically ill patients, and mechanically ventilated rats with normal and increased titin compliance.MEASUREMENTS AND MAIN RESULTS: (1) PEEP causes a caudal movement of the diaphragm, both in critically ill patients and in rats, and this caudal movement reduces fiber length; (2) diaphragm fibers of 18h mechanically ventilated rats (PEEP: 2.5 cmH2O) adapt to the reduced length by absorbing serially-linked sarcomeres, the smallest contractile units in muscle (i.e. longitudinal atrophy); (3) increasing the compliance of titin molecules reduces longitudinal atrophy.CONCLUSION: Mechanical ventilation with PEEP results in longitudinal atrophy of diaphragm fibers, a response which is modulated by the elasticity of the giant sarcomeric protein titin. We postulate that longitudinal atrophy, in concert with the aforementioned cross-sectional atrophy, hampers spontaneous breathing trials in critically ill patients: during these efforts end-expiratory lung volume is reduced, and the shortened diaphragm fibers are stretched to excessive sarcomere lengths. At these lengths, muscle fibers generate less force and diaphragm weakness ensues.

Recently, linkage analysis of two large unrelated multigenerational families identified a novel dilated cardiomyopathy (DCM)-linked mutation in the gene coding for alpha-tropomyosin (TPM1) resulting in the substitution of an aspartic acid for an asparagine (at residue 230). To determine how a single amino acid mutation in α-tropomyosin (Tm) can lead to a highly penetrant DCM we generated a novel transgenic mouse model carrying the D230N mutation. The resultant mouse model strongly phenocopied the early onset of cardiomyopathic remodeling observed in patients as significant systolic dysfunction was observed by 2 months of age. To determine the precise cellular mechanism(s) leading to the observed cardiac pathology we examined the effect of the mutation on Ca2+ handling in isolated myocytes and myofilament activation in vitro. D230N-Tm filaments exhibited a reduced Ca2+ sensitivity of sliding velocity. This decrease in sensitivity was coupled to increase in the peak amplitude of Ca2+ transients. While significant, and consistent with other DCMs, these measurements are comprised of complex inputs and did not provide sufficient experimental resolution. We then assessed the primary structural effects of D230N-Tm. Measurements of the thermal unfolding of D230N-Tm vs WT-Tm revealed an increase in stability primarily affecting the C-terminus of the Tm coiled-coil. We conclude that the D230N-Tm mutation induces a decrease in flexibility of the C-terminus via propagation through the helical structure of the protein, thus decreasing the flexibility of the Tm overlap and impairing its ability to regulate contraction. Understanding this unique structural mechanism could provide novel targets for eventual therapeutic interventions in patients with Tm-linked cardiomyopathies.

Purpose Dodecafluoropentane emulsion (DDFPe) is a perfluorocarbon with high oxygen dissolving, transport, and delivery capacity that may offer the potential to limit ischemic injury prior to clinical reperfusion. Here we investigated the cardiac protective potential of DDFPe in a mouse model of myocardial infarction. Methods Myocardial infarction was initiated by permanent ligation of the left anterior descending (LAD) coronary artery. Mice were administered vehicle or 5-hydroxydecanoate (5-HD) intravenously 10 min before LAD occlusion followed by a single intravenous administration of vehicle or DDFPe immediately after occlusion. Heart tissue and serum samples were collected 24 after LAD occlusion for measurement of infarct size and cardiac troponin I (cTnI) levels, respectively. Results DDFPe treatment reduced infarct size by approximately 72 % (36.9 ± 4.2 % for vehicle vs 10.4 ± 2.3 % for DDFPe; p