Diaphragm Contractile Function Research Articles

Sulforaphane improves vasodilatory reactivity in diaphragm arterioles from rats with pulmonary hypertension

Pulmonary hypertension (PH) is a chronic, progressive disease characterized by pulmonary vascular remodeling, respiratory muscle and cardiac impairments, and exercise intolerance. Specifically, the diaphragm displays compromised contractile and vascular function, precluding it from meeting the increased metabolic demands of PH-induced chronic hyperpnea. Oxidative stress has been implicated in vascular dysfunction and PH pathology, therefore our objective was to target the antioxidant mediator Nrf2 (Nuclear factor erythroid 2-related factor 2) using sulforaphane (SFN) to determine whether this could improve vascular function in the diaphragm. We hypothesized that incubation with SFN would increase vasodilatory responsiveness in diaphragm arterioles from PH rats. Methods: Female Sprague-Dawley rats were administered monocrotaline (MCT; 50 mg/kg, i.p.) to induce PH. Disease progression was monitored via echocardiography. Once animals developed PH (~3 wks), the diaphragm was removed and first-order arterioles from the medial costal diaphragm were isolated, cannulated, and pressurized at 90 cmH2O. After a 60 min equilibration period and spontaneous tone development, endothelium-dependent and -independent vasorelaxation responses to cumulative doses of acetylcholine (ACh) and sodium nitroprusside (SNP) (10−9 to 10−4 M) were determined. These vessels were subsequently incubated with SFN (10 μM, 20 min), then ACh and SNP dose-response measurements were repeated. Results: All PH rats displayed morphometric and echocardiographic criteria for disease (i.e., right ventricular hypertrophy and reduced pulmonary artery acceleration time). Incubation with SFN improved endothelium-dependent and -independent vasodilation (p<0.05). Maximal vasodilatory responses with SFN were 14% and 16% higher for ACh (30±2 vs 34±3%; P=0.02) and SNP (52±2 vs 60±2%; P=0.03), respectively. However, vasodilatory reactivity remains substantially lower than that of healthy animals. Conclusions: These findings suggest that Nrf2 activation using SFN may improve vascular function in the diaphragm with PH. Future investigations will focus on chronic administration of SFN to PH rats to determine the therapeutic potential of this compound in vivo. The results of these studies provide insight into novel pharmacological strategies to improve diaphragm vascular and contractile function, with the potential to help mitigate dyspnea and exercise intolerance and enhance life quality for PH patients. Sustained Momentum for Investigators with Laboratories Established Grants (TIM & DCP); National Institute on Aging 1R15AG078060 (BJB & DCP); Ruth L. Kirschstein National Research Service Awards 1F31HL167618-01 (AGH) and 1F31HL170643-01 (KMS). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Effects of Myocardial Infarction-induced Heart Failure on Diaphragm Arteriolar Vasorelaxation

INTRODUCTION: Diaphragm atrophy and contractile dysfunction are prevalent in heart failure (HF), diminishing physical capacity and quality of life. The delivery of blood flow and oxygen necessary to support the metabolic demand of contractions is largely governed by the resistance vasculature (i.e., arterioles) with the endothelium playing a central role in arteriolar vasorelaxation. Adequate perfusion and blood flow distribution, and thus resistance vessel vasodilation, is a major determinate of diaphragm contractile function. While the mechanistic bases of HF-induced diaphragm dysfunction continue to be elucidated, the impact of HF on diaphragm vasomotor control has not been investigated. METHODS: Adult (6 mo) Sprague-Dawley rats (n=7) were divided into two groups: 1) Healthy Control (HC; n=4) and 2) MI-induced heart failure (HF; n=3). First-order arterioles (1A) from the medial costal diaphragm were isolated, cannulated, and pressurized at 90 cmH2O and allowed to equilibrate for 60 min to develop spontaneous tone. Thereafter, endothelium-dependent and -independent vasorelaxation responses to cumulative doses of acetylcholine (ACh; 10−9 to 10−4M) and sodium nitroprusside (SNP; 10−9 to 10−4M) were determined. RESULTS: There were no differences in spontaneous development (%) or maximal diameter (μm) between HC (20 ± 4%; 178 ± 11 μm) and HF (18 ± 2%; 184 ± 29 μm) diaphragm 1As ( P > 0.05). Maximal ACh-mediated relaxation (%) was significantly reduced in HF rats compared to HC rats (52 ± 2 versus 68 ± 5%; P < 0.05). Maximal endothelial-independent vasorelaxation with SNP, was not different between HC and HF rats (71 ± 3 versus 73 ± 1%; P > 0.05). CONCLUSION: The preliminary data herein demonstrate that HF impaired endothelial function and vasomotor function in the medial costal diaphragm resistance vasculature. This suggests that, in HF, there may be a redistribution of blood flow to less effcient regions of the diaphragm (e.g., ventral costal) to sustain contractile function and compensate for the diminished medial costal vasomotor function demonstrated herein. Studies including a larger sample size are warranted to investigate the impact of HF on regional blood flow distribution and resistance vessel function in the diaphragm. Such investigations would yield novel mechanistic insights into HF-induced diaphragm dysfunction. National Institute of Aging 1R15AG078060, Ruth L. Kirschstein National Research Service Awards from the National Heart, Lung, and Blood Institute 1F31HL167618-01, and Sustained Momentum for Investigators with Laboratories Established (SMILE) Grants at Kansas State University. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

EFFECTS OF LEVOSIMENDAN ON DIAPHRAGMATIC DYSFUNCTION IN PATIENTS WITH SEPSIS.

Objective: In this study, our aim was to examine the effects of levosimendan on diaphragmatic dysfunction in patients with sepsis, as well as assess its impact on respiratory muscle contractility and the outcome of weaning. Methods: This was a single-blind, randomized, controlled trial. Patients with diaphragmatic dysfunction and failure of spontaneous breathing trials (SBTs) were randomly and equally assigned to the experimental and control groups. The experimental group received levosimendan at a loading dose of 6 μg/kg for 10 min, followed by a continuous infusion at 0.2 μg/kg/min. The control group received an equivalent dose of a placebo. The preadministration and postadministration respiratory mechanics parameters of the patients were recorded. Evaluation of the effect of levosimendan on patients with sepsis-induced diaphragm dysfunction comprised arterial blood gas analysis as well as ultrasound measurements of diaphragm excursion (DE), diaphragm thickness (DT), diaphragm thickening fraction (TFdi), and diaphragm-rapid shallow breathing index (D-RSBI). Results: Forty-four patients were enrolled in the study. We found that postadministration of levosimendan, the patients' tidal volume (GCSMV) increased, whereas the D-RSBI decreased, and the partial pressure of carbon dioxide (PACO 2 ) decreased when compared to the preadministration levels. Additionally, following levosimendan administration, patients showed increased DE and pressure support (PS) when compared to before administration (1.14 ± 0.177 vs. 1.22 ± 0.170 cm and 0.248 ± 0.03 vs. 0.284 ± 0.06, respectively) and decreased D-RSBI (22.76 ± 6.14 vs. 20.06 ± 6.04, respectively), all of which were statistically significant ( P < 0.05). In contrast, in the control group of patients, there were no statistically significant differences in the postadministration levels of DE, TFdi, and D-RSBI as compared to the preadministration period ( P > 0.05). Furthermore, in terms of weaning outcomes, we did not find any statistically significant difference in the number of patients in the two groups who eventually underwent weaning ( P = 0.545). Conclusion: In this study, we found that levosimendan enhanced diaphragm contractile function. However, further investigations are required to explore its effect on weaning outcomes in patients undergoing mechanical ventilation.

Comparative Effect of High-Frequency Nasal Cannula and Noninvasive Ventilation on the Work of Breathing and Postoperative Pulmonary Complication after Pediatric Congenital Cardiac Surgery: A Prospective Randomized Controlled Trial.

Various forms of commonly used noninvasive respiratory support strategies have considerable effect on diaphragmatic contractile function which can be evaluated using sonographic diaphragm activity parameters. To compare the magnitude of respiratory workload decreased as assessed by thickening fraction of the diaphragm and longitudinal diaphragmatic strain while using high-flow nasal cannula (HFNC) and noninvasive ventilation (NIV) modes [nasal intermittent positive pressure ventilation (NIPPV) and bilevel positive airway pressure (BiPAP)] in pediatric patients after cardiothoracic surgery. This prospective randomized controlled trial was performed at a tertiary care surgical intensive care unit in postcardiac surgery patients aged between 1 and 48 months, who were randomly allocated into three groups: 1) HFNC (with flows at 2 L/kg/min), 2) NIPPV via RAMS cannula in PSV mode (pressure support 8 cmH2O, PEEP 5 cmH2O), and 3) BiPAP in nCPAP mode (CPAP of 5 cmH2O). Measurements were recorded at baseline after extubation (R0) and subsequently every 12 hourly (R1, R2, R3, R4, R5) at 12, 24, 36, 48, and 60 hours respectively until therapy was discontinued. Sixty patients were included, with 20 patients each in the NIPPV group, HFNC group, and BiPAP group. Longitudinal strain at crura of diaphragm was lower in the BiPAP group as compared to HFNC group at R2-R4 [R2 (-4.27± -2.73 vs - 8.40± -6.40, P = 0.031), R3 (-5.32± -2.28 vs -8.44± -5.6, P = 0.015), and R4 (-3.8± -3.42 vs -12.4± -7.12, P = 0.040)]. PFR was higher in HFNC than NIPPV group at baseline and R1-R3[R0 (323 ± 114 vs 264 ± 80, P = 0.008), R1 (311 ± 114 vs 233 ± 66, P = 0.022), R2 (328 ± 116 vs 237 ± 4, P = 0.002), R3 (346 ± 112 vs 238 ± 54, P = 0.001)]. DTF and clinical parameters of increased work of breathing remain comparable between three groups. The rate of reintubation (within 48 hours of extubation or at ICU discharge) was 0.06% (1 in NIPPV, 1 in BiPAP, 2 in HFNC) and remain comparable between groups (P = 1.0). BiPAP may provide better decrease in work of breathing compared to HFNC as reflected by lower crural diaphragmatic strain pattern. HFNC may provide better oxygenation compared to NIPPV group, as reflected by higher PFR ratio. Failure rate and safety profile are similar among different methods used.

Small-Molecule Inhibition of MuRF1 Prevents Early Disuse-Induced Diaphragmatic Dysfunction and Atrophy.

In clinical conditions such as diaphragm paralysis or mechanical ventilation, disuse-induced diaphragmatic dysfunction (DIDD) is a condition that poses a threat to life. MuRF1 is a key E3-ligase involved in regulating skeletal muscle mass, function, and metabolism, which contributes to the onset of DIDD. We investigated if the small-molecule mediated inhibition of MuRF1 activity (MyoMed-205) protects against early DIDD after 12 h of unilateral diaphragm denervation. Wistar rats were used in this study to determine the compound's acute toxicity and optimal dosage. For potential DIDD treatment efficacy, diaphragm contractile function and fiber cross-sectional area (CSA) were evaluated. Western blotting investigated potential mechanisms underlying MyoMed-205's effects in early DIDD. Our results indicate 50 mg/kg bw MyoMed-205 as a suitable dosage to prevent early diaphragmatic contractile dysfunction and atrophy following 12 h of denervation without detectable signs of acute toxicity. Mechanistically, treatment did not affect disuse-induced oxidative stress (4-HNE) increase, whereas phosphorylation of (ser632) HDAC4 was normalized. MyoMed-205 also mitigated FoxO1 activation, inhibited MuRF2, and increased phospho (ser473) Akt protein levels. These findings may suggest that MuRF1 activity significantly contributes to early DIDD pathophysiology. Novel strategies targeting MuRF1 (e.g., MyoMed-205) have potential therapeutic applications for treating early DIDD.

Comparison of assessment of diaphragm function using speckle tracking between patients with successful and failed weaning: a multicentre, observational, pilot study

BackgroundDiaphragmatic ultrasound has been increasingly used to evaluate diaphragm function. However, current diaphragmatic ultrasound parameters provide indirect estimates of diaphragmatic contractile function, and the predictive value is controversial. Two-dimensional (2D) speckle tracking is an effective technology for measuring tissue deformation and can be used to measure diaphragm longitudinal strain (DLS) to assess diaphragm function. The purpose of this study was to determine the feasibility and reproducibility of DLS quantification by 2D speckle tracking and to determine whether maximal DLS could be used to predict weaning outcomes.MethodsThis study was performed in the intensive care unit of two teaching hospitals, and was divided into two studies. Study A was a prospective study to evaluate the feasibility, reliability, and repeatability of speckle tracking in assessing DLS in healthy subjects and mechanically ventilated patients. Study B was a multicentre retrospective study to assess the use of maximal DLS measured by speckle tracking in predicting weaning outcomes.ResultsTwenty-five healthy subjects and twenty mechanically ventilated patients were enrolled in Study A. Diaphragmatic speckle tracking was easily accessible. The intra- and interoperator reliability were good to excellent under conditions of eupnoea, deep breathing, and mechanical ventilation. The intraclass correlation coefficient (ICC) ranged from 0.78 to 0.95. Ninety-six patients (fifty-nine patients were successfully weaned) were included in Study B. DLS exhibited a fair linear relationship with both the diaphragmatic thickening fraction (DTF) (R2 = 0.73, p < 0.0001) and diaphragmatic excursion (DE) (R2 = 0.61, p < 0.0001). For the prediction of successful weaning, the areas under the ROC curves of DLS, diaphragmatic thickening fraction DTF, RSBI, and DE were 0.794, 0.794, 0.723, and 0.728, respectively. The best cut-off value for predicting the weaning success of DLS was less than -21%, which had the highest sensitivity of 89.19% and specificity of 64.41%.ConclusionsDiaphragmatic strain quantification using speckle tracking is easy to obtain in healthy subjects and mechanically ventilated patients and has a high predictive value for mechanical weaning. However, this method offers no advantage over RSBI. Future research should assess its value as a predictor of weaning.Trial registrationThis study was registered in the Chinese Clinical Trial Register (ChiCTR), ChiCTR2100049816. Registered 10 August 2021. http://www.chictr.org.cn/showproj.aspx?proj=131790

Diaphragm Voluntary Activation After Inspiratory Pressure Threshold Loading In Healthy Humans

Inspiratory pressure threshold loading (IPTL) elicits a reduction in diaphragm contractile function (transdiaphragmatic twitch pressure; PDI,TW). It is unknown if diaphragm fatigability after IPTL is also mediated by neural mechanisms (diaphragm voluntary activation; D-VA) or if it differs on the basis of sex. PURPOSE: We sought to determine if D-VA decreases after IPTL and whether this was different between sexes. METHODS: Healthy participants (11F; 10 M) performed IPTL to task failure with an inspired duty cycle of 0.7 at 60% PDI,MAX. Cervical magnetic stimulation of the phrenic nerves was used to assess PDI,TW and D-VA. The relationship between fatigability outcomes (PDI,TW and D-VA), time to task failure, and cumulative diaphragm work were tested to explore how each factor contributes to diaphragm fatigability. RESULTS: Main effects of time (p < 0.001) and sex (p = 0.030), along with an interaction effect (p = 0.007) were observed when examining the changes in PDI,TW after IPTL. Specifically, immediately following IPTL, the decrease in PDI,TW was greater in males compared to females (24 ± 17 vs. 13 ± 15%points; p = 0.016). D-VA was 91 ± 8%baseline in males and 88 ± 11%baseline in females immediately following IPTL. We observed main effects of time, (p < 0.001) but neither the effect of sex (p = 0.177) nor the interaction between sex and time (p = 0.304) were significant, suggesting that D-VA decreases after IPTL similarly in males and females. The decrease in PDI,TW correlated with both the total cumulative diaphragm work (R2 = 0.43; p = 0.021) and time to task failure (R2 = 0.40; p = 0.030), whereas the decrease in D-VA was significantly correlated with time to task failure (R2 = 0.24; p = 0.041) and not cumulative diaphragm work (R2 = 0.19; p = 0.079). CONCLUSION: Our results indicate that neural mechanisms contribute to diaphragm fatigability and this contribution is similar between females and males after IPTL. A decrease in contractile function can be partially explained by both the amount of work performed and the total time of the IPTL task. Decreases in D-VA appear to be influenced primarily by the total time spent performing the IPTL task rather than the amount of work performed. Moreover, these contributing factors appear to be independent of sex. Supported by the Natural Sciences and Engineering Research Council of Canada.

Characterization of skeletal muscle wasting pathways in diaphragm and limb muscles of cystic fibrosis mice.

Patients with cystic fibrosis (CF) often suffer from skeletal muscle atrophy, most often attributed to physical inactivity and nutritional factors. CF is also characterized by abnormally elevated systemic inflammation. However, it is unknown whether the lack of a functional CF transmembrane conductance regulator (CFTR) gene predisposes to exaggerated inflammation-induced muscle proteolysis. CF mice (CFTR-/-) and their wild-type (WT = CFTR+/+) littermate controls were systemically injected with Pseudomonas-derived lipopolysaccharide (LPS). After 24 h, the diaphragm and limb muscles (fast-twitch tibialis anterior, and slow-twitch soleus) were assessed for induction of inflammatory cytokines (TNFα, IL1β, and IL6), oxidative stress, canonical muscle proteolysis pathways (Calpain, Ubiquitin-Proteasome, Autophagy), muscle fiber histology, and diaphragm contractile function. At baseline, CF and WT muscles did not differ with respect to indices of inflammation, proteolysis, or contractile function. After LPS exposure, there was significantly greater induction of all proteolysis pathways (calpain activity; ubiquitin-proteasome: MuRF1 and Atrogin1; autophagy: LC3B, Gabarapl-1, and BNIP3) in CF mice for the diaphragm and tibialis anterior, but not the soleus. Proteolysis pathway upregulation and correlations with inflammatory cytokine induction were most prominent in the tibialis anterior. Diaphragm force normalized to muscle cross-sectional area was reduced by LPS to an equivalent degree in CF and WT mice. CF skeletal muscles containing a high proportion of fast-twitch fibers (diaphragm, tibialis anterior) exhibit abnormally exaggerated upregulation of multiple muscle wasting pathways after exposure to an acute inflammatory stimulus, but not under basal conditions.

Role of IL-33 receptor (ST2) deletion in diaphragm contractile and mitochondrial function in the Sugen5416/hypoxia model of pulmonary hypertension

Pulmonary arterial hypertension (PAH) is a progressive disease of the pulmonary vasculature that leads to right ventricular failure. Skeletal muscle maladaptations limit physical activity and may contribute to disease progression. The role of alarmin/inflammatory signaling in PAH respiratory muscle dysfunction is unknown. We hypothesized that diaphragm mitochondrial and contractile functions are impaired in SU5416/hypoxia-induced pulmonary hypertension due to increased systemic IL-33 signaling. We induced pulmonary hypertension in adult C57Bl/6 J (WT) and ST2 (IL1RL1) gene ablated mice by SU5416/hypoxia (SuHx). We measured diaphragm fiber mitochondrial respiration, inflammatory markers, and contractile function ex vivo. SuHx reduced coupled and uncoupled permeabilized myofiber respiration by ∼40 %. During coupled respiration with complex I substrates, ST2−/− attenuated SuHx inhibition of mitochondrial respiration (genotype × treatment interaction F[1,67] = 3.3, p = 0.07, η2 = 0.04). Flux control ratio and coupling efficiency were not affected by SuHx or genotype. A higher substrate control ratio for succinate was observed in SuHx fibers and attenuated in ST2-/- fibers (F[1,67] = 5.3, p < 0.05, η2 = 0.07). Diaphragm TNFα, but not IL-33 or NFkB, was increased in SuHx vs. DMSO in both genotypes (F[1,43] = 4.7, p < 0.05, η2 = 0.1). Diaphragm force-frequency relationships were right-shifted in SuHx vs. WT (F[3,440] = 8.4, p < 0.05, η2 = 0.0025). There was no effect of ST2−/− on the force-frequency relationship. Force decay during a fatigue protocol at 100 Hz, but not at 40 Hz, was attenuated by SuHx vs. DMSO in both genotypes (F[1,41] = 5.6, p < 0.05, η2 = 0.11). SuHx mice exhibit a modest compensation in diaphragm contractility and mitochondrial dysfunction during coupled respiration; the latter partially regulated through ST2 signaling.

Positive end-expiratory pressure affects geometry and function of the human diaphragm

Positive end-expiratory pressure (PEEP) is routinely applied in mechanically ventilated patients to improve gas exchange and respiratory mechanics by increasing end-expiratory lung volume (EELV). In a recent experimental study in rats, we demonstrated that prolonged application of PEEP causes diaphragm remodeling, especially longitudinal muscle fiber atrophy. This is of potential clinical importance, as the acute withdrawal of PEEP during ventilator weaning decreases EELV and thereby stretches the adapted, longitudinally atrophied diaphragm fibers to excessive sarcomere lengths, having a detrimental effect on force generation. Whether this series of events occurs in the human diaphragm is unknown. In the current study, we investigated if short-term application of PEEP affects diaphragm geometry and function, which are prerequisites for the development of longitudinal atrophy with prolonged PEEP application. Nineteen healthy volunteers were noninvasively ventilated with PEEP levels of 2, 5, 10, and 15 cmH2O. Magnetic resonance imaging was performed to investigate PEEP-induced changes in diaphragm geometry. Subjects were instrumented with nasogastric catheters to measure diaphragm neuromechanical efficiency (i.e., diaphragm pressure normalized to its electrical activity) during tidal breathing with different PEEP levels. We found that increasing PEEP from 2 to 15 cmH2O resulted in a caudal diaphragm displacement (19 [14-26] mm, P < 0.001), muscle shortening in the zones of apposition (20.6% anterior and 32.7% posterior, P < 0.001), increase in diaphragm thickness (36.4% [0.9%-44.1%], P < 0.001) and reduction in neuromechanical efficiency (48% [37.6%-56.6%], P < 0.001). These findings demonstrate that conditions required to develop longitudinal atrophy in the human diaphragm are present with the application of PEEP.NEW & NOTEWORTHY We demonstrate that PEEP causes changes in diaphragm geometry, especially muscle shortening, and decreases in vivo diaphragm contractile function. Thus, prerequisites for the development of diaphragm longitudinal muscle atrophy are present with the acute application of PEEP. Once confirmed in ventilated critically ill patients, this could provide a new mechanism for ventilator-induced diaphragm dysfunction and ventilator weaning failure in the intensive care unit (ICU).

Effects of Supplemental Oxygen During Mechanical Ventilation on Diaphragmatic Blood Flow

Introduction During mechanical ventilation (MV), supplemental oxygen is commonly administered to critically-ill patients to prevent hypoxemia. However, in ICU patients, hyperoxia during MV is associated with increased mortality and a decrease in ventilator free days. Hyperoxic MV can elicit a paradoxical reduction in O2 delivery to the heart, brain, and kidneys. Whereas MV in normoxia diminishes diaphragm perfusion and O2 delivery, the effect of MV with supplemental oxygen on diaphragm perfusion is unknown. Previous studies demonstrate that MV with hyperoxia substantially impairs diaphragm contractile function. Therefore, hyperoxia during MV may potentiate reductions in diaphragm perfusion with MV and predispose patients to problematic weaning. Purpose Using an established animal model of MV, we tested the following hypotheses: 1) MV supplemented with hyperoxic gas (FIO2 0.99) would reduce diaphragmatic blood flow compared to spontaneously breathing (SB) animals, and 2) hyperoxic MV would decrease diaphragmatic blood flow to a greater extent compared to MV with room air (normoxia, FIO2 0.21). Methods Using fluorescent microspheres, tissue blood flow was measured in adult female Sprague-Dawley rats (n = 4) during spontaneous breathing (SB), mechanical ventilation + hyperoxia (MV +O2), and mechanical ventilation + room air (MV + RA). Following microsphere infusion during SB, animals were intubated and immediately ventilated with supplemental hyperoxic gas for 30 min and an arterial blood sample (~100 ul) was collected to confirm hyperoxemia prior to the second microsphere infusion. Thereafter, animals were subjected to MV using room air for 30 min followed by a third infusion of microspheres. Following the final microsphere infusion, tissues were harvested for blood flow analysis. The diaphragm was sectioned into costal (ventral, medial, dorsal) and crural portions to determine blood flow distribution within the diaphragm. Results During MV + O2, average arterial PO2 (PaO2) was 370 ± 39 mmHg. Compared to SB, total diaphragm blood flow was significantly decreased with MV + O2 (50 ± 2 vs 17 ± 3 ml/min/100g; P<0.05) and MV + RA (30 ± 1 ml/min/100g; P<0.05 vs SB). Compared to SB, blood flow to the medial costal diaphragm was reduced during both MV + O2 (59 ± 5 vs 18 ± 4 ml/min/100g; P<0.05) and MV + RA (59 ± 5 vs 29 ± 6 ml/min/100g; P<0.05). During MV + RA, compared to MV + O2, blood flow to the whole diaphragm was higher (30 ± 1 vs 17 ± 3 ml/min/100g; P<0.05). Although not significant, medial costal diaphragm flow tended to be higher with MV + RA versus MV + O2 (P=0.09). Conclusion These data demonstrate that supplementing short term MV with hyperoxia versus room air reduces total diaphragmatic blood flow to a significantly greater degree, suggesting that hyperoxia during MV reduces diaphragmatic O2 delivery. This raises the likelihood that prolonged bouts of MV (i.e. 6+ h) with hyperoxia may accelerate MV-induced vascular dysfunction in the quiescent diaphragm thus exacerbating downstream contractile dysfunction.

Impact of estrogen deficiency on diaphragm and leg muscle contractile function in female mdx mice.

Female carriers of Duchenne muscular dystrophy (DMD) presenting with DMD symptomology similar to males with DMD, such as skeletal muscle weakness and cardiomyopathy, are termed manifesting carriers. There is phenotypic variability among manifesting carriers including the age of onset, which can range from the first to fourth decade of life. In females, estrogen levels typically begin to decline during the fourth decade of life and estrogen deficiency contributes to loss of muscle strength and recovery of strength following injury. Thus, we questioned whether the decline of estrogen impacts the development of DMD symptoms in females. To address this question, we studied 6–8 month-old homozygous mdx female mice randomly assigned to a sham or ovariectomy (OVX) surgical group. In vivo whole-body plethysmography assessed ventilatory function and diaphragm muscle strength was measured in vitro before and after fatigue. Anterior crural muscles were analyzed in vivo for contractile function, fatigue, and in response to eccentric contraction (ECC)-induced injury. For the latter, 50 maximal ECCs were performed by the anterior crural muscles to induce injury. Body mass, uterine mass, hypoxia-hypercapnia ventilatory response, and fatigue index were analyzed by a pooled unpaired t-test. A two-way ANOVA was used to analyze ventilatory measurements. Fatigue and ECC-injury recovery experiments were analyzed by a two-way repeated-measures ANOVA. Results show no differences between sham and OVX mdx mice in ventilatory function, strength, or recovery of strength after fatigue in the diaphragm muscle or anterior crural muscles (p ≥ 0.078). However, OVX mice had significantly greater eccentric torque loss and blunted recovery of strength after ECC-induced injury compared to sham mice (p ≤ 0.019). Although the results show that loss of estrogen has minimal impact on skeletal muscle contractile function in female mdx mice, a key finding suggests that estrogen is important in muscle recovery in female mdx mice after injury.

Role of defective calcium regulation in cardiorespiratory dysfunction in Huntington's disease.

Huntington’s disease (HD) is a progressive, autosomal dominant neurodegenerative disorder affecting striatal neurons beginning in young adults with loss of muscle coordination and cognitive decline. Less appreciated is the fact that patients with HD also exhibit cardiac and respiratory dysfunction, including pulmonary insufficiency and cardiac arrhythmias. The underlying mechanism for these symptoms is poorly understood. In the present study we provide insight into the cause of cardiorespiratory dysfunction in HD and identify a potentially novel therapeutic target. We now show that intracellular calcium (Ca2+) leak via posttranslationally modified ryanodine receptor/intracellular calcium release (RyR) channels plays an important role in HD pathology. RyR channels were oxidized, PKA phosphorylated, and leaky in brain, heart, and diaphragm both in patients with HD and in a murine model of HD (Q175). HD mice (Q175) with endoplasmic reticulum Ca2+ leak exhibited cognitive dysfunction, decreased parasympathetic tone associated with cardiac arrhythmias, and reduced diaphragmatic contractile function resulting in impaired respiratory function. Defects in cognitive, motor, and respiratory functions were ameliorated by treatment with a novel Rycal small-molecule drug (S107) that fixes leaky RyR. Thus, leaky RyRs likely play a role in neuronal, cardiac, and diaphragmatic pathophysiology in HD, and RyRs are a potential novel therapeutic target.

Reliability of diaphragm voluntary activation measurements in healthy adults

Voluntary activation can be used to assess central fatigue of the diaphragm after tasks such as exercise or inspiratory muscle loading. Cervical magnetic stimulation (CMS) of the phrenic nerves elicits an involuntary contraction, or twitch, of the diaphragm. This twitch is quantified based on a measure of transdiaphragmatic pressure and can be used to evaluate diaphragm contractile function and diaphragm voluntary activation (diaphragm-VA). The test-retest reliability of diaphragm-VA using CMS is currently unknown. Thirteen participants (4 male, 9 female; aged 25 ± 3 years) performed a series of interpolated twitch manoeuvres, which included a maximal inspiratory effort against a semi-occluded mouthpiece and 2 CMS-stimuli, 1 during the inspiratory manoeuvre and 1 after when the participant returned to functional residual capacity to quantify diaphragm-VA. Intraclass correlation coefficients (ICCs) and standard error of measurement (SEM) measured between-day and within-session reliability of diaphragm-VA, respectively. Maximal diaphragm-VA values were 91% (SD: 6; SEM: 3.9) and 92% (SD: 5; SEM: 2.2) during visits 1 and 2 (p = 0.68), respectively, and displayed "good" between-day reliability (ICC: 0.88; 95% confidence interval: 0.67-0.95; SEM: 2.7). Our results suggest that assessing diaphragm-VA using CMS is reliable in young healthy adults. Measuring diaphragm-VA may provide additional insight into the consequences and mechanisms of diaphragm fatigue. Novelty: Magnetic stimulation of the phrenic nerves can reliably measure voluntary activation of the diaphragm. Diaphragm voluntary activation can be used to provide additional insight into fatigability of the diaphragm.

Dietary nitrate supplementation increases diaphragm peak power in old mice.

Respiratory muscle function declines with ageing, contributing to breathing complications in the elderly. Here we report greater in vitro respiratory muscle contractile function in old mice receiving supplemental NaNO3 for 14days compared with age-matched controls. Myofibrillar protein phosphorylation, which enhances contractile function, did not change in our study - a finding inconsistent with the hypothesis that this post-translational modification is a mechanism for dietary nitrate to improve muscle contractile function. Nitrate supplementation did not change the abundance of calcium-handling proteins in the diaphragm of old mice, in contrast with findings from the limb muscles of young mice in previous studies. Our findings suggest that nitrate supplementation enhances myofibrillar protein function without affecting the phosphorylation status of key myofibrillar proteins. Inspiratory muscle (diaphragm) function declines with age, contributing to ventilatory dysfunction, impaired airway clearance, and overall decreased quality of life. Diaphragm isotonic and isometric contractile properties are reduced with ageing, including maximal specific force, shortening velocity and peak power. Contractile properties of limb muscle in both humans and rodents can be improved by dietary nitrate supplementation, but effects on the diaphragm and mechanisms behind these improvements remain poorly understood. One potential explanation underlying the nitrate effects on contractile properties is increased phosphorylation of myofibrillar proteins, a downstream outcome of nitrate reduction to nitrite and nitric oxide. We hypothesized that dietary nitrate supplementation would improve diaphragm contractile properties in aged mice. To test our hypothesis, we measured the diaphragm function of old (24months) mice allocated to 1mm NaNO3 in drinking water for 14days (n=8) or untreated water (n=6). The maximal rate of isometric force development (∼30%) and peak power (40%) increased with nitrate supplementation (P<0.05). There were no differences in the phosphorylation status of key myofibrillar proteins and abundance of Ca2+-release proteins in nitrate vs. control animals. In general, our study demonstrates improved diaphragm contractile function with dietary nitrate supplementation and supports the use of this strategy to improve inspiratory function in ageing populations. Additionally, our findings suggest that dietary nitrate improves diaphragm contractile properties independent of changes in abundance of Ca2+-release proteins or phosphorylation of myofibrillar proteins.

Genetic reduction of the extracellular matrix protein versican attenuates inflammatory cell infiltration and improves contractile function in dystrophic mdx diaphragm muscles

There is a persistent, aberrant accumulation of V0/V1 versican in skeletal muscles from patients with Duchenne muscular dystrophy and in diaphragm muscles from mdx mice. Versican is a provisional matrix protein implicated in fibrosis and inflammation in various disease states, yet its role in the pathogenesis of muscular dystrophy is not known. Here, female mdx and male hdf mice (haploinsufficient for the versican allele) were bred. In the resulting F1 mdx-hdf male pups, V0/V1 versican expression in diaphragm muscles was decreased by 50% compared to mdx littermates at 20–26 weeks of age. In mdx-hdf mice, spontaneous physical activity increased by 17% and there was a concomitant decrease in total energy expenditure and whole-body glucose oxidation. Versican reduction improved the ex vivo strength and endurance of diaphragm muscle strips. These changes in diaphragm contractile properties in mdx-hdf mice were associated with decreased monocyte and macrophage infiltration and a reduction in the proportion of fibres expressing the slow type I myosin heavy chain isoform. Given the high metabolic cost of inflammation in dystrophy, an attenuated inflammatory response may contribute to the effects of versican reduction on whole-body metabolism. Altogether, versican reduction ameliorates the dystrophic pathology of mdx-hdf mice as evidenced by improved diaphragm contractile function and increased physical activity.

Intrauterine growth restriction affects diaphragm function in adult female and male mice

In utero diaphragm development is critically important for postnatal respiratory function and any disturbance to fetal development may lead to diaphragm dysfunction and respiratory complications in the postnatal period. Intrauterine growth restriction (IUGR) has been shown to affect respiratory function in a sex-dependent manner; however, the effect of IUGR on diaphragm function is unknown. This study used a maternal hypoxia-induced mouse model of IUGR to investigate the impact of IUGR on diaphragm function and structure in male and female adult offspring. Pregnant BALB/c mice were housed under hypoxic conditions (10.5% O2 ) from gestational days 11 to 17.5 and then returned to normoxic conditions. Control mice were housed under normoxic conditions throughout pregnancy. At 8 weeks of age, offspring were euthanized and diaphragms isolated for functional assessment in organ bath experiments and for histological analysis. IUGR offspring were lighter at birth and remained lighter at 8 weeks of age compared to Controls. While diaphragm force (maximal or twitch) was not affected by treatment or sex, the IUGR group exhibited a longer half-relaxation time after twitch contractions compared to Control. Female offspring had a lower maximum rate of force development and higher fatigue resistance compared to males, independent of IUGR. There was no difference in the diaphragm myofibre cross-sectional area between groups or sexes. Sex and IUGR independently affect diaphragm contraction in adult mice without changes in structure. This study demonstrates that IUGR affects diaphragm contractile function in later life and could impair respiratory function if exacerbated under conditions of increased respiratory load.

Skeletal myofiber VEGF deficiency leads to mitochondrial, structural, and contractile alterations in mouse diaphragm.

Diaphragm dysfunction accompanies cardiopulmonary disease and impaired oxygen delivery. Vascular endothelial growth factor (VEGF) regulates oxygen delivery through angiogenesis, capillary maintenance, and contraction-induced perfusion. We hypothesized that myofiber-specific VEGF deficiency contributes to diaphragm weakness and fatigability. Diaphragm protein expression, capillarity and fiber morphology, mitochondrial respiration and hydrogen peroxide (H2O2) generation, and contractile function were compared between adult mice with conditional gene ablation of skeletal myofiber VEGF (SkmVEGF-/-; n = 12) and littermate controls (n = 13). Diaphragm VEGF protein was ~50% lower in SkmVEGF-/- than littermate controls (1.45 ± 0.65 vs. 3.04 ± 1.41 pg/total protein; P = 0.001). This was accompanied by an ~15% impairment in maximal isometric specific force (F[1,23] = 15.01, P = 0.001) and a trend for improved fatigue resistance (P = 0.053). Mean fiber cross-sectional area and type I fiber cross-sectional area were lower in SkmVEGF-/- by ~40% and ~25% (P < 0.05). Capillary-to-fiber ratio was also lower in SkmVEGF-/- by ~40% (P < 0.05), and thus capillary density was not different. Sarcomeric actin expression was ~30% lower in SkmVEGF-/- (P < 0.05), whereas myosin heavy chain and MAFbx were similar (measured via immunoblot). Mitochondrial respiration, citrate synthase activity, PGC-1α, and hypoxia-inducible factor 1α were not different in SkmVEGF-/- (P > 0.05). However, mitochondrial-derived reactive oxygen species (ROS) flux was lower in SkmVEGF-/- (P = 0.0003). In conclusion, myofiber-specific VEGF gene deletion resulted in a lower capillary-to-fiber ratio, type I fiber atrophy, actin loss, and contractile dysfunction in the diaphragm. In contrast, mitochondrial respiratory function was preserved alongside lower ROS generation, which may play a compensatory role to preserve fatigue resistance in the diaphragm.NEW & NOTEWORTHY Diaphragm weakness is a hallmark of diseases in which oxygen delivery is compromised. Vascular endothelial growth factor (VEGF) modulates muscle perfusion; however, it remains unclear whether VEGF deficiency contributes to the onset of diaphragm dysfunction. Conditional skeletal myofiber VEGF gene ablation impaired diaphragm contractile function and resulted in type I fiber atrophy, a lower number of capillaries per fiber, and contractile protein content. Mitochondrial function was similar and reactive oxygen species flux was lower. Diaphragm VEGF deficiency may contribute to the onset of respiratory muscle weakness.

Morphofunctional Features of The Diaphragm in Patients With Chronic Heart Failure

to study changes in the volumes of muscle, fat, and connective tissue in postmortem issue samples (autoptates) from diaphragm, right ventricle, lower limb (gastrocnemius muscle), as well as morphological changes of the diaphragm muscular structure in patients with different functional classes of heart failure (HF), and to compare them with some intravital parameters of external respiration (with maximal inspiratory pressure and its amplitude simultaneously measured by ultrasound method in particular). Autoptates of the diaphragm muscle, right ventricle, lower limb (n=39) from 20 men and 19 women (with in vivo diagnosis CHF NYHA functional class (FC) I-IV, hypertension, ischemic heart disease) were examined within 24 hours after the fatal outcome. Light optical microscopy was used to assess the percentages of muscle, connective, adipose tissue, numbers of fibroblasts, and collagen fibers. Spirometric measurements, measurement of respiratory muscles strength, and examination of the diaphragm contractile function were performed by echolocation 56.7±11.9 days before death. In patients of all NYHA FCs most pronounced changes of volume of muscle tissue were observed in the right ventricle and diaphragm, while less pronounced - in the gastrocnemius muscle. The increase in the volume of adipose tissue in patients with I-III FC CHF was most pronounced in the right ventricle and diaphragm, and less pronounced - in the gastrocnemius muscle. The greatest increase in the adipose tissue volume was recorded in the diaphragm of patients with IV FC. Changes of connective tissue volume did not follow linear dependence. The largest "leap-like" increase in the volume of connective tissue occurred in the diaphragm of patients with III FC, what significantly outstripped this process in peripheral muscles and right ventricular myocardium. There was stable relationship between structure of tissue of the diaphragm, maximal inspiratory thickness of diaphragmatic muscle, and maximal inspiratory pressure. This relation (correlation) was positive for pairs muscle tissue volume - muscle thickness and muscle tissue volume - inspiratory pressure, and negative for pairs connective tissue volume - muscle thickness, connective tissue volume - inspiratory pressure, adipose tissue volume - inspiratory pressure (r&gt;0.85, p&lt;0.01 for all these correlations). Morphofunctional changes in the diaphragm are caused by progressive decrease in the content of muscle tissue, increases of volumes of adipose and connective tissues. These changes correlate with the CHF FC, maximal inspiratory thickness of diaphragmatic muscle, and maximal inspiratory pressure. Severity of these morphological changes is maximal in patients with FC III CHF.

Sex differences in diaphragmatic fatigue and the metaboreflex following inspiratory pressure-threshold loading.

Sex differences in diaphragmatic fatigue and the metaboreflex following inspiratory pressure-threshold loading.