Clinical use of the chemotherapeutic doxorubicin (DOX) promotes skeletal muscle atrophy and weakness, adversely affecting patient mobility and strength. Although the mechanisms responsible for DOX-induced skeletal muscle dysfunction remain unclear, studies implicate the significant production of reactive oxygen species (ROS) in this pathology. Supraphysiological ROS levels can enhance protein degradation via autophagy, and it is established that DOX upregulates autophagic signaling in skeletal muscle. To determine the precise contribution of accelerated autophagy to DOX-induced skeletal muscle dysfunction, we inhibited autophagy in the soleus via transduction of a dominant negative mutation of the autophagy related 5 (ATG5) protein. Targeted inhibition of autophagy prevented soleus muscle atrophy and contractile dysfunction acutely following DOX administration, which was associated with a reduction in mitochondrial ROS and maintenance of mitochondrial respiratory capacity. These beneficial modifications were potentially the result of enhanced transcription of antioxidant response element-related genes and increased antioxidant capacity. Specifically, our results showed significant upregulation of peroxisome proliferator-activated receptor gamma co-activator 1-alpha, nuclear respiratory factor-1, nuclear factor erythroid-2-related factor-2, nicotinamide-adenine dinucleotide phosphate quinone dehydrogenase-1, and catalase in the soleus with DOX treatment when autophagy was inhibited. These findings establish a significant role of autophagy in the development of oxidative stress and skeletal muscle weakness following DOX administration.

The importance of endothelial cell (EC) autophagy to vascular homeostasis in the context of health and disease is evolving. Earlier, we reported that intact EC autophagy is requisite to maintain shear-stress-induced nitric oxide (NO) generation via glycolysis-dependent purinergic signalling to endothelial NO synthase (eNOS). Here, we illustrate the translational and functional significance of these findings. First, we assessed translational relevance using older male humans and mice that exhibit blunted EC autophagy and impaired arterial function vs. adult controls. Active hyperaemia evoked by rhythmic handgrip exercise-elevated radial artery shear-rate similarly from baseline in adult and older subjects for 60 min. Compared with baseline, indexes of autophagy initiation, p-eNOS^S1177 activation, and NO generation, occurred in radial artery ECs obtained from adult but not older volunteers. Regarding mice, indexes of autophagy and p-eNOS^S1177 activation were robust in ECs from adult but not older animals that completed 60-min treadmill-running. Furthermore, 20 dyne • cm² laminar shear stress × 45-min increased autophagic flux, glycolysis, ATP production, and p-eNOS^S1177 in primary arterial ECs obtained from adult but not older mice. Concerning functional relevance, we next questioned whether the inability to initiate EC autophagy, glycolysis, and p-eNOS^S1177 in vitro precipitates arterial dysfunction ex vivo. Compromised intraluminal flow-mediated vasodilation displayed by arteries from older vs. adult mice was recapitulated in vessels from adult mice by (i) NO synthase inhibition; (ii) acute autophagy impairment using 3-methyladenine (3-MA); (iii) EC Atg3 depletion (iecAtg3KO mice); (iv) purinergic 2Y₁-receptor (P2Y₁-R) blockade; and (v) germline depletion of P2Y₁-Rs. Importantly, P2Y₁-R activation using 2-methylthio-ADP (2-Me-ADP) improved vasodilatory capacity in arteries from (i) adult mice treated with 3-MA; (ii) adult iecAtg3KO mice; and (iii) older animals with repressed EC autophagy. Arterial dysfunction concurrent with pharmacological, genetic, and age-associated EC autophagy compromise is improved by activating P2Y₁-Rs.

Doxorubicin (DOX) is a highly effective chemotherapeutic agent used in the treatment of various cancer types. Nevertheless, it is well known that DOX promotes the development of severe cardiovascular complications. Therefore, investigation into the underlying mechanisms that drive DOX-induced cardiotoxicity is necessary to develop therapeutic countermeasures. In this regard, autophagy is a complex catabolic process that is increased in the heart following DOX exposure. However, conflicting evidence exists regarding the role of autophagy dysregulation in the etiology of DOX-induced cardiac dysfunction. This study aimed to clarify the contribution of autophagy to DOX-induced cardiotoxicity by specifically inhibiting autophagosome formation using a dominant negative autophagy gene 5 (ATG5) adeno-associated virus construct (rAAV-dnATG5). Acute (2-day) and delayed (9-day) effects of DOX (20 mg/kg intraperitoneal injection (i.p.)) on the hearts of female Sprague–Dawley rats were assessed. Our data confirm established detrimental effects of DOX on left ventricular function, redox balance and mitochondrial function. Interestingly, targeted inhibition of autophagy in the heart via rAAV-dnATG5 in DOX-treated rats ameliorated the increase in mitochondrial reactive oxygen species emission and the attenuation of cardiac and mitochondrial function, but only at the acute timepoint. Deviation in the effects of autophagy inhibition at the 2- and 9-day timepoints appeared related to differences in ATG5–ATG12 conjugation, as this marker of autophagosome formation was significantly elevated 2 days following DOX exposure but returned to baseline at day 9. DOX exposure may transiently upregulate autophagy signaling in the rat heart; thus, long-term inhibition of autophagy may result in pathological consequences.

Introduction: Despite rigorous rehabilitation aimed at restoring muscle health, anterior cruciate ligament (ACL) injury is often hallmarked by significant long-term quadriceps muscle weakness. Derangements in mitochondrial function are a common feature of various atrophying conditions, yet it is unclear to what extent mitochondria are involved in the detrimental sequela of quadriceps dysfunction after ACL injury. Using a preclinical, non-invasive ACL injury rodent model, our objective was to explore the direct effect of an isolated ACL injury on mitochondrial function, muscle atrophy, and muscle phenotypic transitions.Methods: A total of 40 male and female, Long Evans rats (16-week-old) were exposed to non-invasive ACL injury, while 8 additional rats served as controls. Rats were euthanized at 3, 7, 14, 28, and 56 days after ACL injury, and vastus lateralis muscles were extracted to measure the mitochondrial respiratory control ratio (RCR; state 3 respiration/state 4 respiration), mitochondrial reactive oxygen species (ROS) production, fiber cross sectional area (CSA), and fiber phenotyping. Alterations in mitochondrial function and ROS production were detected using two-way (sex:group) analyses of variance. To determine if mitochondrial characteristics were related to fiber atrophy, individual linear mixed effect models were run by sex.Results: Mitochondria-derived ROS increased from days 7 to 56 after ACL injury (30–100%, P < 0.05), concomitant with a twofold reduction in RCR (P < 0.05). Post-injury, male rats displayed decreases in fiber CSA (days 7, 14, 56; P < 0.05), loss of IIa fibers (day 7; P < 0.05), and an increase in IIb fibers (day 7; P < 0.05), while females displayed no changes in CSA or phenotyping (P > 0.05). Males displayed a positive relationship between state 3 respiration and CSA at days 14 and 56 (P < 0.05), while females only displayed a similar trend at day 14 (P = 0.05).Conclusion: Long-lasting impairments in quadriceps mitochondrial health are present after ACL injury and play a key role in the dysregulation of quadriceps muscle size and composition. Our preclinical data indicate that using mitoprotective therapies may be a potential therapeutic strategy to mitigate alterations in muscle size and characteristic after ACL injury.