Thorax Online First, published on March 31, 2016 as 10.1136/thoraxjnl-2015-207559 Respiratory research ORIGINAL ARTICLE Thorax: first published as 10.1136/thoraxjnl-2015-207559 on 31 March 2016. Downloaded from Prolonged controlled mechanical ventilation in humans triggers myofibrillar contractile dysfunction and myofilament protein loss in the diaphragm Sabah N A Hussain,1,2,3 Anabelle S Cornachione,4 Céline Guichon,1 Auday Al Khunaizi,1 Felipe de Souza Leite,5 Basil J Petrof,3 Mahroo Mofarrahi,1 Nikolay Moroz,1 Benoit de Varennes,6 Peter Goldberg,1,3 Dilson E Rassier7 ▸ Additional material is ABSTRACT published online only. To view Background Prolonged controlled mechanical Key messages please visit the journal online (http://dx.doi.org/10.1136/ ventilation (CMV) in humans and experimental animals thoraxjnl-2015-207559). results in diaphragm fibre atrophy and injury. In animals, fi fi prolonged CMV also triggers signi cant declines in What is the key question? For numbered af liations see fi end of article. diaphragm myo bril contractility. In humans, the impact ▸ Does prolonged controlled mechanical of prolonged CMV on myofibril contractility remains ventilation (CMV) in humans alter the unknown. The objective of this study was to evaluate contractile performance and myofilament Correspondence to the effects of prolonged CMV on active and passive protein expression in diaphragm myofibrils? Professor Dilson Rassier, human diaphragm myofibrillar force generation and Department of Kinesiology, myofilament protein levels. What is the bottom line? McGill University, 475 Pine Ave ▸ Prolonged CMV in humans significantly West, Montréal, Québec, Methods and results Diaphragm biopsies were Canada H2W 1S4; dilson. obtained from 13 subjects undergoing cardiac surgery decreases active and passive diaphragm [email protected] (control group) and 12 brain-dead organ donors (CMV myofibrillar force generation and significantly group). Subjects in each group had been mechanically attenuates myofilament protein levels including Received 10 July 2015 – – fi those of myosin heavy chain, myosin light Revised 5 January 2016 ventilated for 2 4 and 12 74 h, respectively. Speci c Accepted 6 January 2016 force generation of diaphragm myofibrils was measured chain, troponin-C, I and T as well as titin. with atomic force cantilevers. Rates of force development Why read on? (Kact), force redevelopment after a shortening protocol ▸ fi This study implies that prolonged CMV in (Ktr) and relaxation (Krel) in fully activated myo brils fi 2+ humans elicits signi cant impairment of active (pCa =4.5) were calculated to assess myosin cross- and passive performance by diaphragm http://thorax.bmj.com/ bridge kinetics. Myofilament protein levels were fi fi myo brils as a result of increased degradation measured with immunoblotting and speci c antibodies. of essential myofilament proteins by the Prolonged CMV significantly decreased active and fi proteasome and autophagy proteolytic passive diaphragm myo brillar force generation, Kact, pathways. 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 on September 28, 2021 by guest. Protected copyright. levels were observed. relatively long periods of time, results in the devel- Conclusions Prolonged CMV in humans triggers opment of a condition known as ventilator-induced 2 significant decreases in active and passive diaphragm diaphragm dysfunction (VIDD). VIDD is asso- myofibrillar force generation. This response is mediated, ciated with diaphragm muscle fibre atrophy, ultra- in part, by impaired myosin cross-bridge kinetics and structure injury and depressed muscle decreased myofibrillar protein levels. force-generating capacity. Clinically, VIDD is a dis- tinct entity from other causes of diaphragm dys- function such as critical illness myopathy, reduced O2 delivery, electrolyte imbalance and steroid administration. For patients with acute respiratory INTRODUCTION failure, successful weaning from MV is largely Mechanical ventilation (MV) is a two-edged sword. determined by the work of breathing (dependent On one hand, it is a life-saving procedure that is on lung mechanics) and the ability of the inspira- used in intensive care units to manage patients with tory muscles in general, and the diaphragm in par- – respiratory failure and other pathologies such as ticular, to cope with the work of breathing.3 5 To cite: Hussain SNA, cardiac failure and stroke. On the other hand, MV Therefore, the development of VIDD is likely to Cornachione AS, Guichon C, is associated with numerous complications, includ- contribute to weaning failure and results in pro- et al. Thorax Published 1 Online First: [please include ing ventilator-induced lung injury. In the past longed durations of stay in intensive care units. Day Month Year] several years, many studies have indicated that use Animal studies have revealed that the severity of doi:10.1136/thoraxjnl-2015- of the controlled mode of MV (CMV), where VIDD is dependent on the mode of MV and the 207559 spontaneous diaphragm activity is inhibited for levels of spontaneous diaphragm activity. By using Hussain SNA, et al. Thorax 2016;0:1–10. doi:10.1136/thoraxjnl-2015-207559 1 Copyright Article author (or their employer) 2016. Produced by BMJ Publishing Group Ltd (& BTS) under licence. Respiratory research the assist-control mode or by allowing intermittent spontaneous To the best of our knowledge, no existing studies have Thorax: first published as 10.1136/thoraxjnl-2015-207559 on 31 March 2016. Downloaded from breathing during prolonged CMV, the severity of VIDD is miti- addressed the effects of prolonged MV on any other contractile gated.67Prolonged CMV in animals, however, results in proteins although fluctuations in myosin light chain (MyLC), decreased isometric force and endurance, muscle fibre atrophy, troponin, tropomyosin and titin levels are likely to exert a morphological anomalies of the sarcomere and various major impact on force generation. Therefore, the second object- – mitochondrial-specific dysfunctions.8 17 These pathologies have ive of this study is to test the hypothesis that MyLC, been attributed to the joint effects of increased protein degrad- troponin-C, troponin-I and troponin-T, tropomyosin and titin – ation and decreased protein synthesis.18 21 levels are all significantly decreased in the diaphragm in In brain-dead human organ donors undergoing prolonged response to prolonged CMV and to link these decreases to acti- MV, several authors have reported the development of dia- vation of several proteolytic pathways. phragm fibre atrophy, sarcomeric injuries and mitochondrial dysfunction.18 19 21 22 Despite this recent progress in human METHODS studies of VIDD, little information is as yet available regarding Experimental subjects the effects of prolonged CMV on diaphragm muscle fibre con- All protocols were approved by the appropriate Ethics tractile performance. Although it has been reported that signifi- Committees of the McGill University Health Centre. All biop- cantly lower airway occlusion pressure values are generated in sies were performed after written informed consent had been response to phrenic nerve stimulation in intensive care patients obtained. Full-thickness diaphragm biopsies from 13 normal undergoing long-term MV (>5 days) as compared with those pulmonary function subjects who were undergoing cardiac undergoing short-term MV (0.5 h),18 impaired occlusion pres- surgery (aortic or mitral valve replacement, coronary artery sure merely hints at diminished diaphragm contractility in bypass graft) constituted the control group. Biopsies were response to prolonged ventilation. obtained 30 min after MV was initiated and prior to surgery. Similarly, in vitro measurements of isometric force generation Full-thickness diaphragm biopsies from 12 brain-dead organ of permeabilised single muscle fibre samples from the dia- donors undergoing prolonged CMV constituted the CMV phragms of brain-dead organ donors who had been mechanic- group. Biopsies were obtained prior to circulatory arrest or ally ventilated for an average of 26 h were compared with those removal of any organ. All biopsies were obtained from the of control subjects who had been mechanically ventilated for anterior costal diaphragm, lateral to the insertion of the phrenic less than 2 h23 and no significant differences were observed nerve. Biopsy samples were immediately frozen in liquid nitro- between the two groups. But all these indicate that 26 h of gen, and stored at −80°C until used in immunoblotting and CMV is not a sufficient period for sarcomeric contractile dys- mRNA analyses. function to develop in humans. The question of whether longer periods of CMV might influence contractility remains untested. Diaphragm myofibrillar contractile performance Further experimentation is necessary before definitive conclu- Small muscle bundles were dissected out, tied to wooden sticks sions can be made, so the first objective of this study is to test and chemically permeabilised according to our laboratory’s – the hypothesis that human diaphragmatic sarcomere contractility standard procedures27 29 (see online supplementary file). Briefly, is negatively affected by CMV administration for periods longer muscle samples were incubated in rigour solution (pH=7.0) for http://thorax.bmj.com/ than 26 h. To test this, we used atomic force cantilevers to approximately 4 h, after which they