Felipe de Souza Leite

Effects of controlled mechanical ventilation on sepsis-induced diaphragm dysfunction in rats.

Objectives:Diaphragm dysfunction develops during severe sepsis as a consequence of hemodynamic, metabolic, and intrinsic abnormalities. Similarly, 12 hours of controlled mechanical ventilation also promotes diaphragm dysfunction. Importantly, patients with sepsis are often treated with mechanical ventilation for several days. It is unknown if controlled mechanical ventilation exacerbates sepsis-induced diaphragm dysfunction, and this forms the basis for these experiments. We investigate the effects of 12-hour controlled mechanical ventilation on contractile function, fiber dimension, cytokine production, proteolysis, autophagy, and oxidative stress in the diaphragm of septic rats. Design:Randomized controlled experiment. Setting:Animal research laboratory. Subjects:Adult male Wistar rats. Interventions:Treatment with a single intraperitoneal injection of either saline or Escherichia coli lipopolysaccharide (5 mg/kg). After 12 hours, the saline-treated animals (controlled mechanical ventilation) and half of the septic animals (lipopolysaccharide + controlled mechanical ventilation) were submitted to 12 hours of controlled mechanical ventilation while the remaining septic animals (lipopolysaccharide) were breathing spontaneously for 12 hours. They were compared to a control group. All animals were studied 24 hours after saline or lipopolysaccharide administration. Measurements and Main Results:Twenty-four hours after saline or lipopolysaccharide administration, diaphragm contractility was measured in vitro. We also measured diaphragm muscle fiber dimensions from stained cross sections, and inflammatory cytokines were determined by proteome array. Activities of calpain, caspase-3, and proteasome, expression of 20S-proteasome &agr; subunits, E2 conjugases, E3 ligases, and autophagy were measured with immunoblotting and quantitative polymerase chain reaction. Lipopolysaccharide and/or controlled mechanical ventilation independently decreased diaphragm contractility and fiber dimensions and increased diaphragm interleukin-6 production, protein ubiquitination, expression of Atrogin-1 and Murf-1, calpain and caspase-3 activities, autophagy, and protein oxidation. Compared with lipopolysaccharide alone, lipopolysaccharide + controlled mechanical ventilation worsened diaphragm contractile dysfunction, augmented diaphragm interleukin-6 levels, autophagy, and protein oxidation, but exerted no exacerbating effects on diaphragm fiber dimensions, calpain, caspase-3, or proteasome activation. Conclusions:Twelve hours of controlled mechanical ventilation potentiates sepsis-induced diaphragm dysfunction, possibly due to increased proinflammatory cytokine production and autophagy and worsening of oxidative stress.

Non-crossbridge forces in activated striated muscles: a titin dependent mechanism of regulation?

When skeletal muscles are stretched during activation in the absence of myosin-actin interactions, the force increases significantly. The force remains elevated throughout the activation period. The mechanism behind this non-crossbridge force, referred to as static tension, is unknown and generates debate in the literature. It has been suggested that the static tension is caused by Ca2+-induced changes in the properties of titin molecules that happens during activation and stretch, but a comprehensive evaluation of such possibility is still lacking. This paper reviews the general characteristics of the static tension, and evaluates the proposed mechanism by which titin may change the force upon stretch. Evidence is presented suggesting that an increase in intracellular Ca2+ concentration leads to Ca2+ binding to the PEVK region of titin. Such binding increases titin stiffness, which increases the overall sarcomere stiffness and causes the static tension. If this form of Ca2+-induced increase in titin stiffness is confirmed in future studies, it may have large implications for understating of the basic mechanisms of muscle contraction.

The increase in non-cross-bridge forces after stretch of activated striated muscle is related to titin isoforms.

Skeletal muscles present a non-cross-bridge increase in sarcomere stiffness and tension on Ca(2+) activation, referred to as static stiffness and static tension, respectively. It has been hypothesized that this increase in tension is caused by Ca(2+)-dependent changes in the properties of titin molecules. To verify this hypothesis, we investigated the static tension in muscles containing different titin isoforms. Permeabilized myofibrils were isolated from the psoas, soleus, and heart ventricle from the rabbit, and tested in pCa 9.0 and pCa 4.5, before and after extraction of troponin C, thin filaments, and treatment with the actomyosin inhibitor blebbistatin. The myofibrils were tested with stretches of different amplitudes in sarcomere lengths varying between 1.93 and 3.37 μm for the psoas, 2.68 and 4.21 μm for the soleus, and 1.51 and 2.86 μm for the ventricle. Using gel electrophoresis, we confirmed that the three muscles tested have different titin isoforms. The static tension was present in psoas and soleus myofibrils, but not in ventricle myofibrils, and higher in psoas myofibrils than in soleus myofibrils. These results suggest that the increase in the static tension is directly associated with Ca(2+)-dependent change in titin properties and not associated with changes in titin-actin interactions.

Arginylation of Myosin Heavy Chain Regulates Skeletal Muscle Strength

Protein arginylation is a posttranslational modification with an emerging global role in the regulation of actin cytoskeleton. To test the role of arginylation in the skeletal muscle, we generated a mouse model with Ate1 deletion driven by the skeletal muscle-specific creatine kinase (Ckmm) promoter. Ckmm-Ate1 mice were viable and outwardly normal; however, their skeletal muscle strength was significantly reduced in comparison to controls. Mass spectrometry of isolated skeletal myofibrils showed a limited set of proteins, including myosin heavy chain, arginylated on specific sites. Atomic force microscopy measurements of contractile strength in individual myofibrils and isolated myosin filaments from these mice showed a significant reduction of contractile forces, which, in the case of myosin filaments, could be fully rescued by rearginylation with purified Ate1. Our results demonstrate that arginylation regulates force production in muscle and exerts a direct effect on muscle strength through arginylation of myosin.

Prolonged controlled mechanical ventilation in humans triggers myofibrillar contractile dysfunction and myofilament protein loss in the diaphragm

Background Prolonged controlled mechanical ventilation (CMV) in humans and experimental animals results in diaphragm fibre atrophy and injury. In animals, prolonged CMV also triggers significant declines in diaphragm myofibril contractility. In humans, the impact of prolonged CMV on myofibril contractility remains unknown. The objective of this study was to evaluate the effects of prolonged CMV on active and passive human diaphragm myofibrillar force generation and myofilament protein levels. Methods and results Diaphragm biopsies were obtained from 13 subjects undergoing cardiac surgery (control group) and 12 brain-dead organ donors (CMV group). Subjects in each group had been mechanically ventilated for 2–4 and 12–74 h, respectively. Specific force generation of diaphragm myofibrils was measured with atomic force cantilevers. Rates of force development (Kact), force redevelopment after a shortening protocol (Ktr) and relaxation (Krel) in fully activated myofibrils (pCa2+=4.5) were calculated to assess myosin cross-bridge kinetics. Myofilament protein levels were measured with immunoblotting and specific antibodies. Prolonged CMV significantly decreased active and passive diaphragm myofibrillar force generation, Kact, Ktr and Krel. Myosin heavy chain (slow), troponin-C, troponin-I, troponin-T, tropomyosin and titin protein levels significantly decreased in response to prolonged CMV, but no effects on α-actin, α-actinin or nebulin levels were observed. Conclusions Prolonged CMV in humans triggers significant decreases in active and passive diaphragm myofibrillar force generation. This response is mediated, in part, by impaired myosin cross-bridge kinetics and decreased myofibrillar protein levels.

Reduced passive force in skeletal muscles lacking protein arginylation

Arginylation is a posttranslational modification that plays a global role in mammals. Mice lacking the enzyme arginyltransferase in skeletal muscles exhibit reduced contractile forces that have been linked to a reduction in myosin cross-bridge formation. The role of arginylation in passive skeletal myofibril forces has never been investigated. In this study, we used single sarcomere and myofibril measurements and observed that lack of arginylation leads to a pronounced reduction in passive forces in skeletal muscles. Mass spectrometry indicated that skeletal muscle titin, the protein primarily linked to passive force generation, is arginylated on five sites located within the A band, an important area for protein-protein interactions. We propose a mechanism for passive force regulation by arginylation through modulation of protein-protein binding between the titin molecule and the thick filament. Key points are as follows: 1) active and passive forces were decreased in myofibrils and single sarcomeres isolated from muscles lacking arginyl-tRNA-protein transferase (ATE1). 2) Mass spectrometry revealed five sites for arginylation within titin molecules. All sites are located within the A-band portion of titin, an important region for protein-protein interactions. 3) Our data suggest that arginylation of titin is required for proper passive force development in skeletal muscles.

Residual force enhancement is regulated by titin in skeletal and cardiac myofibrils

When a skeletal muscle is stretched while it contracts, the muscle produces a relatively higher force than the force from an isometric contraction at the same length: a phenomenon referred to as residual force enhancement. Residual force enhancement is puzzling because it cannot be directly explained by the classical force–length relationship and the sliding filament theory of contraction, the main paradigms in the muscle field. We used custom‐built instruments to measure residual force enhancement in skeletal myofibrils, and, for the first time, in cardiac myofibrils. Our data report that residual force enhancement is present in skeletal muscles, but not cardiac muscles, and is regulated by the different isoforms of the titin protein filaments.

Residual force depression in single sarcomeres is abolished by MgADP-induced activation

The mechanisms behind the shortening-induced force depression commonly observed in skeletal muscles remain unclear, but have been associated with sarcomere length non-uniformity and/or crossbridge inhibition. The purpose of this study was twofold: (i) to evaluate if force depression is present in isolated single sarcomeres, a preparation that eliminates sarcomere length non-uniformities and (ii) to evaluate if force depression is inhibited when single sarcomeres are activated with MgADP, which biases crossbridges into a strongly-bound state. Single sarcomeres (n = 16) were isolated from rabbit psoas myofibrils using two micro-needles (one compliant, one rigid), piercing the sarcomere externally adjacent to the Z-lines. The sarcomeres were contracted isometrically and subsequently shortened, in both Ca2+- and MgADP-activating solutions. Shortening in Ca2+-activated samples resulted in a 27.44 ± 9.04% force depression when compared to isometric contractions produced at similar final sarcomere lengths (P < 0.001). There was no force depression in MgADP-activated sarcomeres (force depression = −1.79 ± 9.69%, P =  0.435). These results suggest that force depression is a sarcomeric property, and that is associated with an inhibition of myosin-actin interactions.

Prolonged force depression after mechanically demanding contractions is largely independent of Ca2+ and reactive oxygen species

Increased production of reactive oxygen/nitrogen species (ROS) and impaired cellular Ca2+ handling are implicated in the prolonged low‐frequency force depression (PLFFD) observed in skeletal muscle after both metabolically and mechanically demanding exercise. Metabolically demanding high‐intensity exercise can induce PLFFD accompanied by ROS‐dependent fragmentation of the sarcoplasmic reticulum Ca2+ release channels, the ryanodine receptor 1s (RyR1s). We tested whether similar changes occur after mechanically demanding eccentric contractions. Human subjects performed 100 repeated drop jumps, which require eccentric knee extensor contractions upon landing. This exercise caused a major PLFFD, such that maximum voluntary and electrically evoked forces did not recover within 24 h. Drop jumps induced only minor signs of increased ROS, and RyR1 fragmentation was observed in only 3 of 7 elderly subjects. Also, isolated mouse muscle preparations exposed to drop‐jump–mimicking eccentric contractions showed neither signs of increased ROS nor RyR1 fragmentation. Still, the free cytosolic [Ca2+] during tetanic contractions was decreased by ~15% 1 h after contractions, which can explain the exaggerated force decrease at low‐stimulation frequencies but not the major frequency‐independent force depression. In conclusion, PLFFD caused by mechanically demanding eccentric contractions does not involve any major increase in ROS or RyR1 fragmentation.—Kamandulis, S., de Souza Leite, F., Hernandez, A., Katz, A., Brazaitis, M., Bruton, J. D., Venckunas, T., Masiulis, N., Mickeviciene, D., Eimantas, N., Subocius, A., Rassier, D. E., Skurvydas, A., Ivarsson, N., Westerblad, H. Prolonged force depression after mechanically demanding contractions is largely independent of Ca2+ and reactive oxygen species. FASEB J. 31, 4809–4820 (2017). www.fasebj.org

Microfluidic perfusion shows intersarcomere dynamics within single skeletal muscle myofibrils

Significance The sarcomere contains a variety of molecules responsible for force generation in striated muscles. During muscle contraction, many sarcomeres work cooperatively to produce force. The mechanisms behind the interaction among sarcomeres during muscle activation have puzzled scientists for decades. To investigate intersarcomere dynamics, we used microfluidic perfusions to activate a single sarcomere within isolated myofibrils. Using computational sarcomere tracking, we observed that the contraction of one sarcomere affects other sarcomeres in series. Studying the interactions between sarcomeres is crucial, because sarcomere nonuniformity has been long associated with several phenomena in muscle contraction that cannot be easily understood. The sarcomere is the smallest functional unit of myofibrils in striated muscles. Sarcomeres are connected in series through a network of elastic and structural proteins. During myofibril activation, sarcomeres develop forces that are regulated through complex dynamics among their structures. The mechanisms that regulate intersarcomere dynamics are unclear, which limits our understanding of fundamental muscle features. Such dynamics are associated with the loss in forces caused by mechanical instability encountered in muscle diseases and cardiomyopathy and may underlie potential target treatments for such conditions. In this study, we developed a microfluidic perfusion system to control one sarcomere within a myofibril, while measuring the individual behavior of all sarcomeres. We found that the force from one sarcomere leads to adjustments of adjacent sarcomeres in a mechanism that is dependent on the sarcomere length and the myofibril stiffness. We concluded that the cooperative work of the contractile and the elastic elements within a myofibril rules the intersarcomere dynamics, with important consequences for muscle contraction.