Stacey Brickson

Role of myosin heavy chain composition in the stretch activation response of rat myocardium

The speed and force of myocardial contraction during systolic ejection is largely dependent on the intrinsic contractile properties of cardiac myocytes. As the myosin heavy chain (MHC) isoform of cardiac muscle is an important determinant of the contractile properties of individual myocytes, we studied the effects of altered MHC isoform expression in rat myocardium on the mechanical properties of skinned ventricular preparations. Skinned myocardium from thyroidectomized rats expressing only the β MHC isoform displayed rates of force redevelopment that were about 2.5‐fold slower than in myocardium from hyperthyroid rats expressing only the α MHC isoform, but the amount of force generated at a given level of Ca2+ activation was not different. Because recent studies suggest that the stretch activation response in myocardium has an important role in systolic function, we also examined the effect of MHC isoform expression on the stretch activation response by applying a rapid stretch (1% of muscle length) to an otherwise isometrically contracting muscle fibre. Sudden stretch of myocardium resulted in a concomitant increase in force that quickly decayed to a minimum and was followed by a delayed redevelopment of force (i.e. stretch activation) to levels greater than prestretch force. β MHC expression dramatically slowed the overall rate of the stretch activation response compared to expression of α MHC isoform; specifically, the rate of force decay was ∼2‐fold slower and the rate of delayed force development was ∼2.5‐fold slower. In contrast, MHC isoform had no effect on the amplitude of the stretch activation response. Collectively, these data show that expression of β MHC in myocardium dramatically slows rates of cross‐bridge recruitment and detachment which would be expected to decrease power output and contribute to depressed systolic function in end‐stage heart failure.

Oxidant production and immune response after stretch injury in skeletal muscle.

PURPOSE This study investigated oxidant production and associated immune response after acute muscle stretch injury. METHODS A standardized single stretch injury was performed on the tibialis anterior (TA) muscle of 36 male New Zealand white rabbits while contralateral control limbs underwent a sham surgery. Animals were sacrificed 0, 4, 12, 24, 48, and 72 h after injury. Potential sites of oxidant production, measured with a dichlorofluorescein (DCF) probe, were evaluated using two separate buffers. RESULTS Nonmitochondrial oxidant production measured under basal buffer conditions (0.1 M potassium phosphate) was increased in both injured and control limbs at 24 h (P < 0.01) and was greater in the injured limb at 12 and 48 h (P < 0.01). There was also an interaction of time and injury (P < 0.05). Maximum oxidant production by neutrophils and macrophages, stimulated by the induced buffer (including 1.7 mM ADP, 0.1 mM NADPH, 0.1 mM FeCl3), was increased in both injured and control limbs at 4 h (P < 0.01) and was greater in the injured limb at 48 h (P < 0.01). Myeloperoxidase (MPO) activity, indicating the presence of activated neutrophils, was higher in the injured limb at 4 and 48 h (P < 0.01). The activities of superoxide radical producing and quenching enzymes, xanthine oxidase (XO) and superoxide dismutase (SOD), were elevated at 24 (P < 0.01) and 4 h (P < 0.05), respectively, but showed no difference between injured and control limbs. CONCLUSION We conclude that acute muscle stretch injury and the required surgeries to generate the injury result in a biphasic increase in oxidant production in both injured and control limbs, suggesting a systemic immune response. The increase in oxidant production at 4 h may be caused by an increase in activated neutrophils, whereas XO activity may contribute to oxidant generation at 24 h.

The Influence of Macrophage Depletion on Ligament Healing

Despite a complex cascade of cellular events to reconstruct damaged extracellular matrix (ECM), ligament healing results in a mechanically inferior, scar-like tissue. During normal healing, the number of macrophages significantly increases within the wound site. Then, granulation tissue expands into any residual, normal ligamentous tissue (creeping substitution), resulting in a larger region of healing, greater mechanical compromise, and an inefficient repair process. To study the effects of macrophages on the repair process, bilateral, surgical rupture of their medial collateral ligaments (MCLs) was done on rats. Treatment animals received liposome-encapsulated clodronate, 2 days before rupture to ablate phagocytosing macrophages. Ligaments were then collected at days 5, 11, and 28 for immunohistochemistry (IHC) and/or mechanical testing. Clodronate treatment reduced both the M1 and M2 macrophages at day 5 and altered early healing. However, the macrophages effectively returned to control levels after day 5 and reinitiated a wound-healing response. Our results suggest that an early macrophage response, which is necessary for debridement of damaged tissue in the wound, is also important for cytokine release to mediate normal repair processes. Additionally, nonspecific inhibition of macrophages (without regard to specific macrophage populations) can control excessive granulation tissue formation but is detrimental to early matrix formation and ligament strength.

Interleukin expression after injury and the effects of interleukin-1 receptor antagonist.

Ligament healing follows a series of complex coordinated events involving various cell types, cytokines, as well as other factors, producing a mechanically inferior tissue more scar-like than native tissue. Macrophages provide an ongoing source of cytokines to modulate inflammatory cell adhesion and migration as well as fibroblast proliferation. Studying interleukins inherent to ligament healing during peak macrophage activation and angiogenesis may elucidate inflammatory mediators involved in subsequent scar formation. Herein, we used a rat healing model assayed after surgical transection of their medial collateral ligaments (MCLs). On days 3 and 7 post-injury, ligaments were collected and used for microarray analysis. Of the 12 significantly modified interleukins, components of the interleukin-1 family were significantly up-regulated. We therefore examined the influence of interleukin-1 receptor antagonist (IL-1Ra) on MCL healing. Transected rat MCLs received PBS or IL-1Ra at the time of surgery. Inhibition of IL-1 activation decreased pro-inflammatory cytokines (IL-1α, IL-1β, IL-12, IL-2, and IFN-γ), myofibroblasts, and proliferating cells, as well as increased anti-inflammatory cytokines (IL-10), endothelial cells/blood vessel lumen, M2 macrophages, and granulation tissue size without compromising the mechanical properties. These results support the concept that IL-1Ra modulates MCL-localized granulation tissue components and cytokine production to create a transient environment that is less inflammatory. Overall, IL-1Ra may have therapeutic potential early in the healing cascade by stimulating the M2 macrophages and altering the granulation tissue components. However, the single dose of IL-1Ra used in this study was insufficient to maintain the more regenerative early response. Due to the transient influence on most of the healing components tested, IL-1Ra may have greater therapeutic potential with sustained delivery.

The Influence of Interleukin-4 on Ligament Healing

Despite a complex cascade of cellular events to reconstruct the damaged extracellular matrix, ligament healing results in a mechanically inferior scarred ligament. During normal healing, granulation tissue expands into any residual normal ligamentous tissue (creeping substitution), resulting in a larger region of healing, greater mechanical compromise and an inefficient repair process. To control creeping substitution and possibly enhance the repair process, the antiinflammatory cytokine, interleukin‐4 (IL‐4), was administered to rats before and after rupture of their medial collateral ligaments. In vitro experiments showed a time‐dependent effect on fibroblast proliferation after IL‐4 treatment. In vivo treatments with IL‐4 (100 ng/mL IV) for 5 days resulted in decreased wound size and type III collagen and increased type I procollagen, indicating a more regenerative early healing in response to the IL‐4 treatment. However, continued treatment of IL‐4 to day 11 antagonized this early benefit and slowed healing. Together, these results suggest that IL‐4 not only influences the macrophages and T lymphocytes but also stimulates fibroblasts associated with the proliferative phase of healing in a dose‐, cell‐, and time‐dependent manner. Although treatment significantly influenced healing in the first week after injury, IL‐4 alone was unable to maintain this early regenerative response.

CD11b+ neutrophils predominate over RAM11+ macrophages in stretch-injured muscle

The purpose of this study was to determine whether both neutrophils and macrophages infiltrate the hematoma site of stretch‐injured rabbit tibialis anterior muscle. The Mab.198 antibody was used to detect CD11b+ neutrophils or macrophages. Neutrophils were identified specifically by using the RPN3/57 antibody. The RAM11 antibody was used to detect macrophages. The histological characteristics of the hematoma site, torn fibers or inflammatory cells, were present primarily at 4 and 24 h, but not at 48 and 72 h after injury. A difference in the Mab.198+ cellular concentration was detected over time between uninjured and injured muscles (P = 0.03). The injured‐uninjured difference in the RPN3/57+ or RAM11+ cellular concentrations approached significance (P = 0.07) or else was deemed insignificant (P = 0.13), respectively. Therefore, neutrophils may predominate over RAM11+ macrophages in stretch‐injured muscle. These findings may influence the antiinflammatory strategies used to treat stretch injuries.

Enhanced medial collateral ligament healing using mesenchymal stem cells: dosage effects on cellular response and cytokine profile.

Mesenchymal stem cells (MSCs) have potential therapeutic applications for musculoskeletal injuries due to their ability to differentiate into several tissue cell types and modulate immune and inflammatory responses. These immune-modulatory properties were examined in vivo during early stage rat medial collateral ligament healing. Two different cell doses (low dose 1 × 106 or high dose 4 × 106 MSCs) were administered at the time of injury and compared with normal ligament healing at days 5 and 14 post-injury. At both times, the high dose MSC group demonstrated a significant decrease in M2 macrophages compared to controls. At day 14, fewer M1 macrophages were detected in the low dose group compared to the high dose group. These results, along with significant changes in procollagen I, proliferating cells, and endothelialization suggest that MSCs can alter the cellular response during healing in a dose-dependent manner. The higher dose ligaments also had increased expression of several pro-inflammatory cytokines at day 5 (IL-1β, IFNγ, IL-2) and increased expression of IL-12 at day 14. Mechanical testing at day 14 revealed increased failure strength and stiffness in low dose ligaments compared to controls. Based on these improved mechanical properties, MSCs enhanced functional healing when applied at a lower dose. Different doses of MSCs uniquely affected the cellular response and cytokine expression in healing ligaments. Interestingly, the lower dose of cells proved to be most effective in improving functional properties.

Interleukin-1 Receptor Antagonist Modulates Inflammation and Scarring after Ligament Injury

Abstract Ligaments have limited regenerative potential and as a consequence, repair is protracted and results in a mechanically inferior tissue more scar-like than native ligament. We previously reported that a single injection of interleukin-1 receptor antagonist (IL-1Ra) delivered at the time of injury, decreased the number of M2 macrophage-associated inflammatory cytokines. Based on these results, we hypothesized that IL-1Ra administered after injury and closer to peak inflammation (as would occur clinically), would more effectively decrease inflammation and thereby improve healing. Since IL-1Ra has a short half-life, we also investigated the effect of multiple injections. The objective of this study was to elucidate healing of a medial collateral ligament (MCL) with either a single IL-1Ra injection delivered one day after injury or with multiple injections of IL-1Ra on days 1, 2, 3, and 4. One day after MCL injury, rats received either single or multiple injections of IL-1Ra or PBS. Tissue was then collected at days 5 and 11. Both single and multiple IL-1Ra injections reduced inflammatory cytokines, but did not change mechanical behavior. A single injection of IL-1Ra also reduced the number of myofibroblasts and increased type I procollagen. Multiple IL-1Ra doses provided no additive response and, in fact, reduced the M2 macrophages. Based on these results, a single dose of IL-1Ra was better at reducing the MCL-derived inflammatory cytokines compared to multiple injections. The changes in type I procollagen and myofibroblasts further suggest a single injection of IL-1Ra enhanced repair of the ligament but not sufficiently to improve functional behavior.

Subjective well-being and training load predict in-season injury and illness risk in female youth soccer players

Background The purpose of this study was to evaluate the effects of training load (TL) and well-being on injury and illness risk in youth soccer players. Methods Throughout a 20-week season, 75 female adolescent soccer players reported mood, fatigue, stress, soreness, sleep quality, sleep hours, TL, injuries and illnesses. Well-being measures were recorded from −3 (worst) to +3 (best). TL was expressed as daily, weekly and monthly, as well as an acute:chronic workload ratio (weekly divided by monthly). Variables were compared between days with and without an injury, and with or without an illness. Poisson regression models were developed to predict daily injuries and illnesses using well-being and TL (z-scores) as predictors. Results 36 injuries and 52 illnesses were recorded. Days with an injury had lower (worse) daily mood (1.24±0.2 vs 1.16±0.1, p=0.012) and higher daily TL (517±138 vs 440±158, p=0.010). Average monthly TL was higher preceding days with an illness (12 442 ±409 vs 12 627 ±403, p=0.043), while no differences were found with respect to other measures of TL or well-being. Worse daily mood (p=0.011, OR=0.012), higher daily TL (p<0.001, OR=1.98), and higher prior day TL (p=0.040, OR=1.34) were independent predictors of injury, while weekly (p=0.005, OR=1.50) and monthly TL (p=0.007, OR=1.54) were predictors of illness. Conclusions Lower mood and higher acute TL are associated with increased injury risk, while higher chronic TL increases the risk of illness. Monitoring well-being and TL may facilitate intervention to reduce in-season injury and illness.

Short-Term Heart Rate Recovery is Related to Aerobic Fitness in Elite Intermittent Sport Athletes.

Abstract Watson, AM, Brickson, SL, Prawda, ER, and Sanfilippo, JL. Short-term heart rate recovery is related to aerobic fitness in elite intermittent sport athletes. J Strength Cond Res 31(4): 1055–1061, 2017—Although heart rate recovery (HRR) has been suggested as a measure of fitness, minimal data exist among athletes. The purpose of this study was to determine if HRR is related to aerobic fitness in elite athletes and whether this relationship is influenced by sex or body composition. Eighty-four collegiate athletes (45 male athletes) underwent body fat percentage (BF%) determination by dual-energy x-ray absorptiometry and maximal treadmill testing followed by 5 minutes of recovery. V[Combining Dot Above]O2max and heart rate (HRmax) were determined, and HRR was calculated as a percentage of HRmax at 10 seconds, 30 seconds, and 1, 2, 3, 4, and 5 minutes after test completion. After stratifying by sex, participants were grouped as high fit or low fit based on V[Combining Dot Above]O2max median split. Heart rate recovery was compared between sexes and fitness level at each time point. Multivariable regression analysis was used to identify independent predictors of HRR using V[Combining Dot Above]O2max, BF%, and sex as covariates. Heart rate recovery did not differ significantly between sexes and was faster among high-fit participants at 10 and 30 seconds, but at no other time. V[Combining Dot Above]O2max was significantly correlated with HRR at 10 and 30 seconds (r = −0.34, p < 0.001 and r = −0.28, p = 0.008) only. After controlling for BF% and sex, V[Combining Dot Above]O2max remained significantly associated with HRR at 10 seconds (p = 0.007) but not at 30 seconds (p = 0.067) or any time thereafter. Aerobic capacity is related to faster HRR during the first 30 seconds only, suggesting that only very short term HRR should be used as a measure of aerobic fitness in intermittent sport athletes.