Compromised muscle mitochondrial metabolism is a hallmark of peripheral arterial disease, especially in patients with the most severe clinical manifestation — critical limb ischemia (CLI). We asked whether inflexibility in metabolism is critical for the development of myopathy in ischemic limb muscles. Using Polg mtDNA mutator (D257A) mice, we reveal remarkable protection from hind limb ischemia (HLI) due to a unique and beneficial adaptive enhancement of glycolytic metabolism and elevated ischemic muscle PFKFB3. Similar to the relationship between mitochondria from CLI and claudicating patient muscles, BALB/c muscle mitochondria are uniquely dysfunctional after HLI onset as compared with the C57BL/6 (BL6) parental strain. AAV-mediated overexpression of PFKFB3 in BALB/c limb muscles improved muscle contractile function and limb blood flow following HLI. Enrichment analysis of RNA sequencing data on muscle from CLI patients revealed a unique deficit in the glucose metabolism Reactome. Muscles from these patients express lower PFKFB3 protein, and their muscle progenitor cells possess decreased glycolytic flux capacity in vitro. Here, we show supplementary glycolytic flux as sufficient to protect against ischemic myopathy in instances where reduced blood flow–related mitochondrial function is compromised preclinically. Additionally, our data reveal reduced glycolytic flux as a common characteristic of the failing CLI patient limb skeletal muscle.
Biochem. J. (2019) 476 1521–1537, DOI: 10.1042/BCJ20190182The authors would like to include an additional acknowledgement for their article. The authors acknowledge the assistance of Dr Deborah M. Muoio from Duke University who helped develop the biochemical assay platform used in this article and whose intellectual discussions contributed to the initial idea for this study. The authors apologize for the omission of Dr Muoio from the article.
Fatigue and muscle weakness are common symptoms in chronic heart failure that significantly compromise patients' quality of life. Skeletal muscle abnormality is an important contributor to these symptoms, independent of cardiac function. Heart failure with reduced ejection fraction (HFrEF) causes histological changes in limb muscles, including changes in muscle fiber type distribution and fiber atrophy. Metabolic alterations often accompany these changes, and several reports show shifts from oxidative to glycolytic fiber phenotype. There are conflicting reports on HFrEF‐related abnormalities in limb muscle. There are approximately 3–4 million patients with HFrEF in the United States, and while the disease is more prevalent in males, it affects a large number of females. Animal studies have largely focused on effects of HFrEF in males whereas studies in humans have included mostly males with mild or moderate HFrEF. We conducted the current study to determine whether the effects of HFrEF on limb muscle abnormalities are dependent on sex and disease severity. We performed myocardial infarction to induce HFrEF or Sham surgery in 8–9 week old male and female Sprague Dawley rats. Animals that underwent MI surgery were grouped into either moderate or severe HFrEF; severe HFrEF was determined by 1) transmural infarct ≥35% of left ventricle and septal area and 2) right ventricle hypertrophy. We have used SMASH code (MatLab) to analyze fiber type distribution and cross‐sectional area (CSA) in cryogenic sections of tibialis anterior of males and females Sham, moderate, and severe HFrEF rats (n=3–4 per group). Compared to Sham, type IIb/x CSA decreased in males with moderate (28% ± 11%) and severe HFrEF (39% ± 5%) (P < 0.05). Female rats with moderate and severe HFrEF had fiber CSA similar to Sham. Fiber type distribution did not change for either males or females animals with CHF compared to Sham. Overall, our initial data show that HFrEF causes muscle atrophy in males independently of disease severity, but not in females. However, we cannot exclude the possibility that limb muscle atrophy occurs in post‐menopausal females with HFrEF. Support or Funding Information R01‐HL130318 to LFF This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .
The most severe manifestation of peripheral arterial disease (PAD) is critical limb ischemia (CLI). CLI patients suffer high rates of amputation and mortality; accordingly, there remains a clear need both to better understand CLI and to develop more effective treatments. Gastrocnemius muscle was obtained from 32 older (51–84 years) non-PAD controls, 27 claudicating PAD patients (ankle-brachial index [ABI] 0.65 ± 0.21 SD), and 19 CLI patients (ABI 0.35 ± 0.30 SD) for whole transcriptome sequencing and comprehensive mitochondrial phenotyping. Comparable permeabilized myofiber mitochondrial function was paralleled by both similar mitochondrial content and related mRNA expression profiles in non-PAD control and claudicating patient tissues. Tissues from CLI patients, despite being histologically intact and harboring equivalent mitochondrial content, presented a unique bioenergetic signature. This signature was defined by deficits in permeabilized myofiber mitochondrial function and a unique pattern of both nuclear and mitochondrial encoded gene suppression. Moreover, isolated muscle progenitor cells retained both mitochondrial functional deficits and gene suppression observed in the tissue. These findings indicate that muscle tissues from claudicating patients and non-PAD controls were similar in both their bioenergetics profile and mitochondrial phenotypes. In contrast, CLI patient limb skeletal muscles harbor a unique skeletal muscle mitochondriopathy that represents a potentially novel therapeutic site for intervention.
Abstract The various functions of skeletal muscle (movement, respiration, thermogenesis, etc.) require the presence of oxygen (O2). Inadequate O2 bioavailability (ie, hypoxia) is detrimental to muscle function and, in chronic cases, can result in muscle wasting. Current therapeutic interventions have proven largely ineffective to rescue skeletal muscle from hypoxic damage. However, our lab has identified a mammalian skeletal muscle that maintains proper physiological function in an environment depleted of O2. Using mouse models of in vivo hindlimb ischemia and ex vivo anoxia exposure, we observed the preservation of force production in the flexor digitorum brevis (FDB), while in contrast the extensor digitorum longus (EDL) and soleus muscles suffered loss of force output. Unlike other muscles, we found that the FDB phenotype is not dependent on mitochondria, which partially explains the hypoxia resistance. Muscle proteomes were interrogated using a discovery-based approach, which identified significantly greater expression of the transmembrane glucose transporter GLUT1 in the FDB as compared to the EDL and soleus. Through loss-and-gain-of-function approaches, we determined that GLUT1 is necessary for the FDB to survive hypoxia, but overexpression of GLUT1 was insufficient to rescue other skeletal muscles from hypoxic damage. Collectively, the data demonstrate that the FDB is uniquely resistant to hypoxic insults. Defining the mechanisms that explain the phenotype may provide insight towards developing approaches for preventing hypoxia-induced tissue damage.
Alterations to branched-chain keto acid (BCKA) oxidation have been implicated in a wide variety of human diseases, ranging from diabetes to cancer. Although global shifts in BCKA metabolism-evident by gene transcription, metabolite profiling, and in vivo flux analyses have been documented across various pathological conditions, the underlying biochemical mechanism(s) within the mitochondrion remain largely unknown. In vitro experiments using isolated mitochondria represent a powerful biochemical tool for elucidating the role of the mitochondrion in driving disease. Such analyses have routinely been utilized across disciplines to shed valuable insight into mitochondrial-linked pathologies. That said, few studies have attempted to model in vitro BCKA oxidation in isolated organelles. The impetus for the present study stemmed from the knowledge that complete oxidation of each of the three BCKAs involves a reaction dependent upon bicarbonate and ATP, both of which are not typically included in respiration experiments. Based on this, it was hypothesized that the inclusion of exogenous bicarbonate and stimulation of respiration using physiological shifts in ATP-free energy, rather than excess ADP, would allow for maximal BCKA-supported respiratory flux in isolated mitochondria. This hypothesis was confirmed in mitochondria from several mouse tissues, including heart, liver and skeletal muscle. What follows is a thorough characterization and validation of a novel biochemical tool for investigating BCKA metabolism in isolated mitochondria.
