Long-term exercise induces physiological cardiac adaptation, a condition referred to as athlete's heart. Exercise tolerance is known to be associated with decreased cardiac passive stiffness. Passive stiffness of the heart muscle is determined by the giant elastic protein titin. The adult cardiac muscle contains two titin isoforms: the more compliant N2BA and the stiffer N2B. Titin-based passive stiffness may be controlled by altering the expression of the different isoforms or via post-translational modifications such as phosphorylation. Currently, there is very limited knowledge about titin's role in cardiac adaptation during long-term exercise. Our aim was to determine the N2BA/N2B ratio and post-translational phosphorylation of titin in the left ventricle and to correlate the changes with the structure and transverse stiffness of cardiac sarcomeres in a rat model of an athlete's heart. The athlete's heart was induced by a 12-week-long swim-based training. In the exercised myocardium the N2BA/N2B ratio was significantly increased, Ser11878 of the PEVK domain was hypophosphorlyated, and the sarcomeric transverse elastic modulus was reduced. Thus, the reduced passive stiffness in the athlete's heart is likely caused by a shift towards the expression of the longer cardiac titin isoform and a phosphorylation-induced softening of the PEVK domain which is manifested in a mechanical rearrangement locally, within the cardiac sarcomere.

Desmin intermediate filaments (IFs) play an important role in maintaining the structural and functional integrity of muscle cells. They connect contractile myofibrils to plasma membrane, nuclei, and mitochondria. Disturbance of their network due to desmin mutations or deficiency leads to an infringement of myofibril organization and to a deterioration of mitochondrial distribution, morphology, and functions. The nature of the interaction of desmin IFs with mitochondria is not clear. To elucidate the possibility that desmin can directly bind to mitochondria, we have undertaken the study of their interaction in vitro. Using desmin mutant Des(Y122L) that forms unit-length filaments (ULFs) but is incapable of forming long filaments and, therefore, could be effectively separated from mitochondria by centrifugation through sucrose gradient, we probed the interaction of recombinant human desmin with mitochondria isolated from rat liver. Our data show that desmin can directly bind to mitochondria, and this binding depends on its N-terminal domain. We have found that mitochondrial cysteine protease can disrupt this interaction by cleavage of desmin at its N-terminus.

Muscle biomechanics relies on active motor protein assembly and passive strain transmission through cytoskeletal structures. The desmin filament network aligns myofibrils at the z-discs, provides nuclear-sarcolemmal anchorage and may also serve as memory for muscle repositioning following large strains. Our previous analyses of R349P desmin knock-in mice, an animal model for the human R350P desminopathy, already depicted pre-clinical changes in myofibrillar arrangement and increased fiber bundle stiffness. As the effect of R349P desmin on axial biomechanics in fully differentiated single muscle fibers is unknown, we used our MyoRobot to compare passive visco-elasticity and active contractile biomechanics in single fibers from fast- and slow-twitch muscles from adult to senile mice, hetero- or homozygous for the R349P desmin mutation with wild type littermates. We demonstrate that R349P desmin presence predominantly increased axial stiffness in both muscle types with a pre-aged phenotype over wild type fibers. Axial viscosity and Ca2+-mediated force were largely unaffected. Mutant single fibers showed tendencies towards faster unloaded shortening over wild type fibers. Effects of aging seen in the wild type appeared earlier in the mutant desmin fibers. Our single-fiber experiments, free of extracellular matrix, suggest that compromised muscle biomechanics is not exclusively attributed to fibrosis but also originates from an impaired intermediate filament network.

Mechanical ventilation (MV) is essential for patients with sepsis-related respiratory failure but can cause ventilator-induced diaphragm dysfunction (VIDD), which involves diaphragmatic myofiber atrophy and contractile inactivity. Mitochondrial DNA, oxidative stress, mitochondrial dynamics, and biogenesis are associated with VIDD. Hypoxia-inducible factor 1α (HIF-1α) is crucial in the modulation of diaphragm immune responses. The mechanism through which HIF-1α and mitochondria affect sepsis-related diaphragm injury is unknown. We hypothesized that MV with or without endotoxin administration would aggravate diaphragmatic and mitochondrial injuries through HIF-1α. C57BL/6 mice, either wild-type or HIF-1α-deficient, were exposed to MV with or without endotoxemia for 8 h. MV with endotoxemia augmented VIDD and mitochondrial damage, which presented as increased oxidative loads, dynamin-related protein 1 level, mitochondrial DNA level, and the expressions of HIF-1α and light chain 3-II. Furthermore, disarrayed myofibrils; disorganized mitochondria; increased autophagosome numbers; and substantially decreased diaphragm contractility, electron transport chain activities, mitofusin 2, mitochondrial transcription factor A, peroxisome proliferator activated receptor-γ coactivator-1α, and prolyl hydroxylase domain 2 were observed (p < 0.05). Endotoxin-stimulated VIDD and mitochondrial injuries were alleviated in HIF-1α-deficient mice (p < 0.05). Our data revealed that endotoxin aggravated MV-induced diaphragmatic dysfunction and mitochondrial damages, partially through the HIF-1α signaling pathway.

Mechanical ventilation (MV) is required to maintain life for patients with sepsis-related acute lung injury but can cause diaphragmatic myotrauma with muscle damage and weakness, known as ventilator-induced diaphragm dysfunction (VIDD). Hypoxia-inducible factor 1α (HIF-1α) plays a crucial role in inducing inflammation and apoptosis. Low-molecular-weight heparin (LMWH) was proven to have anti-inflammatory properties. However, HIF-1α and LMWH affect sepsis-related diaphragm injury has not been investigated. We hypothesized that LMWH would reduce endotoxin-augmented VIDD through HIF-1α. C57BL/6 mice, either wild-type or HIF-1α-deficient, were exposed to MV with or without endotoxemia for 8 h. Enoxaparin (4 mg/kg) was administered subcutaneously 30 min before MV. MV with endotoxemia aggravated VIDD, as demonstrated by increased interleukin-6 and macrophage inflammatory protein-2 levels, oxidative loads, and the expression of HIF-1α, calpain, caspase-3, atrogin-1, muscle ring finger-1, and microtubule-associated protein light chain 3-II. Disorganized myofibrils, disrupted mitochondria, increased numbers of autophagic and apoptotic mediators, substantial apoptosis of diaphragm muscle fibers, and decreased diaphragm function were also observed (p < 0.05). Endotoxin-exacerbated VIDD and myonuclear apoptosis were attenuated by pharmacologic inhibition by LMWH and in HIF-1α-deficient mice (p < 0.05). Our data indicate that enoxaparin reduces endotoxin-augmented MV-induced diaphragmatic injury, partially through HIF-1α pathway inhibition.

Small Ankyrins (sAnk1) are muscle-specific isoforms generated by the Ank1 gene that participate in the organization of the sarcoplasmic reticulum (SR) of striated muscles. Accordingly, the volume of SR tubules localized around the myofibrils is strongly reduced in skeletal muscle fibers of 4- and 10-month-old sAnk1 knockout (KO) mice, while additional structural alterations only develop with aging. To verify whether the lack of sAnk1 also alters intracellular Ca2+ handling, cytosolic Ca2+ levels were analyzed in stimulated skeletal muscle fibers from 4- and 10-month-old sAnk1 KO mice. The SR Ca2+ content was reduced in sAnk1 KO mice regardless of age. The amplitude of the Ca2+ transients induced by depolarizing pulses was decreased in myofibers of sAnk1 KO with respect to wild type (WT) fibers, while their voltage dependence was not affected. Furthermore, analysis of spontaneous Ca2+ release events (sparks) on saponin-permeabilized muscle fibers indicated that the frequency of sparks was significantly lower in fibers from 4-month-old KO mice compared to WT. Furthermore, both the amplitude and spatial spread of sparks were significantly smaller in muscle fibers from both 4- and 10-month-old KO mice compared to WT. These data suggest that the absence of sAnk1 results in an impairment of SR Ca2+ release, likely as a consequence of a decreased Ca2+ store due to the reduction of the SR volume in sAnk1 KO muscle fibers.

Ageing is associated with an increase in the incidence of heart failure, even if the existence of a real age-related cardiomyopathy remains controversial. Effective contraction and relaxation of cardiomyocytes depend on efficient production of ATP (handled by mitochondria) and on proper Ca2+ supply to myofibrils during excitation-contraction (EC) coupling (handled by Ca2+ release units, CRUs). Here, we analyzed mitochondria and CRUs in hearts of adult (4 months old) and aged (≥24 months old) mice. Analysis by confocal and electron microscopy (CM and EM, respectively) revealed an age-related loss of proper organization and disposition of both mitochondria and EC coupling units: (a) mitochondria are improperly disposed and often damaged (percentage of severely damaged mitochondria: adults 3.5 ± 1.1%; aged 16.5 ± 3.5%); (b) CRUs that are often misoriented (longitudinal) and/or misplaced from the correct position at the Z line. Immunolabeling with antibodies that mark either the SR or T-tubules indicates that in aged cardiomyocytes the sarcotubular system displays an extensive disarray. This disarray could be in part caused by the decreased expression of Cav-3 and JP-2 detected by western blot (WB), two proteins involved in formation of T-tubules and in docking SR to T-tubules in dyads. By WB analysis, we also detected increased levels of 3-NT in whole hearts homogenates of aged mice, a product of nitration of protein tyrosine residues, recognized as marker of oxidative stress. Finally, a detailed EM analysis of CRUs (formed by association of SR with T-tubules) points to ultrastructural modifications, i.e., a decrease in their frequency (adult: 5.1 ± 0.5; aged: 3.9 ± 0.4 n./50 μm2) and size (adult: 362 ± 40 nm; aged: 254 ± 60 nm). The changes in morphology and disposition of mitochondria and CRUs highlighted by our results may underlie an inefficient supply of Ca2+ ions and ATP to the contractile elements, and possibly contribute to cardiac dysfunction in ageing.