Mitochondrial-specific autophagy linked to mitochondrial dysfunction following traumatic freeze injury in mice

The objective of this study was to interrogate the link between mitochondrial dysfunction and mitochondrial-specific autophagy in skeletal muscle. C57BL/6J mice were used to establish a time course of mitochondrial function and autophagy induction after fatigue ( n = 12), eccentric contraction-induced injury ( n = 20), or traumatic freeze injury (FI, n = 28); only FI resulted in a combination of mitochondrial dysfunction, i.e., decreased mitochondrial respiration, and autophagy induction. Moving forward, we tested the hypothesis that mitochondrial-specific autophagy is important for the timely recovery of mitochondrial function after FI. Following FI, there is a significant increase in several mitochondrial-specific autophagy-related protein contents including dynamin-related protein 1 (Drp1), BCL1 interacting protein (BNIP3), Pink1, and Parkin (~2-fold, P < 0.02). Also, mitochondrial-enriched fractions from FI muscles showed microtubule-associated protein light chain B1 (LC3)II colocalization suggesting autophagosome assembly around the damaged mitochondrial. Unc-51 like autophagy activating kinase (Ulk1) is considered necessary for mitochondrial-specific autophagy and herein we utilized a mouse model with Ulk1 deficiency in adult skeletal muscle ( myogenin-Cre). While Ulk1 knockouts had contractile weakness compared with littermate controls (−27%, P < 0.02), the recovery of mitochondrial function was not different, and this may be due in part to a partial rescue of Ulk1 protein content within the regenerating muscle tissue of knockouts from differentiated satellite cells in which Ulk1 was not genetically altered via myogenin-Cre. Lastly, autophagy flux was significantly less in injured versus uninjured muscles (−26%, P < 0.02) despite the increase in autophagy-related protein content. This suggests autophagy flux is not upregulated to match increases in autophagy machinery after injury and represents a potential bottleneck in the clearance of damaged mitochondria by autophagy.

Defective angiogenesis in CXCL12 mutant mice impairs skeletal muscle regeneration

Background During muscle regeneration, the chemokine CXCL12 (SDF-1) and the synthesis of some specific heparan sulfates (HS) have been shown to be critical. CXCL12 activity has been shown to be heavily influenced by its binding to extracellular glycosaminoglycans (GAG) by modulating its presentation to its receptors and by generating haptotactic gradients. Although CXCL12 has been implicated in several phases of tissue repair, the influence of GAG binding under HS influencing conditions such as acute tissue destruction remains understudied. Methods To investigate the role of the CXCL12/HS proteoglycan interactions in the pathophysiology of muscle regeneration, we performed two models of muscle injuries (notexin and freeze injury) in mutant CXCL12Gagtm/Gagtm mice, where the CXCL12 gene having been selectively mutated in critical binding sites of CXCL12 to interact with HS. Histological, cytometric, functional transcriptomic, and ultrastructure analysis focusing on the satellite cell behavior and the vessels were conducted on muscles before and after injuries. Unless specified, statistical analysis was performed with the Mann-Whitney test. Results We showed that despite normal histology of the resting muscle and normal muscle stem cell behavior in the mutant mice, endothelial cells displayed an increase in the angiogenic response in resting muscle despite the downregulated transcriptomic changes induced by the CXCL12 mutation. The regenerative capacity of the CXCL12-mutated mice was only delayed after a notexin injury, but a severe damage by freeze injury revealed a persistent defect in the muscle regeneration of CXCL12 mutant mice associated with vascular defect and fibroadipose deposition with persistent immune cell infiltration. Conclusion The present study shows that CXCL12 is crucial for proper muscle regeneration. We highlight that this homing molecule could play an important role in drastic muscle injuries and that the regeneration defect could be due to an impairment of angiogenesis, associated with a long-lasting fibro-adipogenic scar.

An In Vitro Model for Assessing Corneal Keratocyte Spreading and Migration on Aligned Fibrillar Collagen

Background: Corneal stromal cells (keratocytes) are responsible for developing and maintaining normal corneal structure and transparency, and for repairing the tissue after injury. Corneal keratocytes reside between highly aligned collagen lamellae in vivo. In addition to growth factors and other soluble biochemical factors, feedback from the extracellular matrix (ECM) itself has been shown to modulate corneal keratocyte behavior. Methods: In this study, we fabricate aligned collagen substrates using a microfluidics approach and assess their impact on corneal keratocyte morphology, cytoskeletal organization, and patterning after stimulation with platelet derived growth factor (PDGF) or transforming growth factor beta 1 (TGFβ). We also use time-lapse imaging to visualize the dynamic interactions between cells and fibrillar collagen during wound repopulation following an in vitro freeze injury. Results: Significant co-alignment between keratocytes and aligned collagen fibrils was detected, and the degree of cell/ECM co-alignment further increased in the presence of PDGF or TGFβ. Freeze injury produced an area of cell death without disrupting the collagen. High magnification, time-lapse differential interference contrast (DIC) imaging allowed cell movement and subcellular interactions with the underlying collagen fibrils to be directly visualized. Conclusions: With continued development, this experimental model could be an important tool for accessing how the integration of multiple biophysical and biochemical signals regulate corneal keratocyte differentiation.

Regional Influences of Mean Temperature and Variance Changes on Freeze Risk in Apples

Both mean temperature and daily temperature variance affect freeze risk in apples. Freeze damage to blossoms was assessed using a sequential model. In the model, once the chilling requirement was reached, growing degree days were accumulated and phenological stages determined based on growing degree thresholds derived from historical phenological observations. Critical temperatures for each stage were obtained from the literature and used to identify the occurrence freeze injury based on minimum temperature occurrence. In New York, temperature variance was the dominant climatological factor controlling freeze risk. A small <5% increase in variance counteracted mean temperature increases of up to 5.5 °C leading to increased freeze risk despite warming temperatures. In other apple-growing regions in the northwestern and southeastern United States, changes in freeze risk were dominated by changes in mean temperature. This demonstrates that in some regions the risk of freeze injury under future climate conditions may be more sensitive to changes in temperature variance. Variance is currently not well simulated by climate models. Because freeze risk also increases when the chill requirement is reduced, adaptation decisions to transition to lower chill requirement cultivars may be ill-advised in northern climates similar to New York as even the highest chill requirements were satisfied under the conditions with the greatest warming. This was not the case in other regions where the adoption of lower chill requirement cultivars may be warranted.

Assessment of Freeze Injury of Grapevine Green Tissues in Response to Cultivars and a Cryoprotectant Product

Spring frosts and subsequent crop losses threaten the economic sustainability of fruit crop producers all over the world. This study used a controlled-freezing technique to impose a post-budburst freezing stress to grapevine shoots forced from one-node cuttings [‘Albariño’, ‘Cabernet Franc’, ‘Cabernet Sauvignon’, and ‘Pinot Grigio’ (Vitis vinifera)] and whole plants [‘Noiret’ (Vitis hybrid)]. Our goal was to investigate the incidence of freeze injury among cultivars, stage of phenological development, and a potassium salt-based fertilizer (KDL) with potential cryoprotectant activity. Among the V. vinifera cultivars, the incidence of mortality of shoots exposed to −3.5 °C was highest for ‘Albariño’ (71%) and lowest for ‘Cabernet Sauvignon’ (51%). Cuttings sprayed with KDL 24 hours before cold temperature exposure exhibited 16% lower shoot mortality and lower osmotic potential (Ψs) (−0.92 MPa) than the unsprayed cuttings (−0.77 MPa). However, application of KDL did not impact shoot mortality for whole ‘Noiret’ vines. Mortality for ‘Noiret’ shoots greatly increased with the advancement of phenological development, ranging from 10% in wooly buds to 78% in shoots ≈10-cm long. The practical significance of KDL remains questionable; cultivar selection still appears to be a more reliable method for avoiding spring frost, by planting late bursting cultivars in more frost-prone areas.