Human skeletal muscle is composed of highly heterogeneous single muscle fibers (multinucleated single cells) that are commonly classified as fast or slow fiber types, yet proteoform-resolved characterization of individual human muscle fibers remains lacking. Herein, we establish a high sensitivity top-down proteomics method for the analysis of single human muscle fibers (hSMFs). Specifically, we have optimized the surfactant-free extraction protocol for analysis of chemically permeabilized ("skinned") hSMFs, a common preparation used to isolate the sarcomere prior to contractile measurements. This approach enables robust and reproducible proteoform-level coverage of key sarcomeric proteins from individual fibers using top-down LC-MS/MS. With this method, we identified extensive inter- and intra-donor fiber-to-fiber variability in isoform expression and proteoform abundance in hSMFs extracted from the heterogeneous vastus lateralis muscles. Together, these results demonstrate the capability of single-fiber top-down proteomics to resolve proteoform-level heterogeneity in human skeletal muscle and establish a methodological foundation for future studies towards elucidating skeletal muscle biology and understanding muscle-related diseases. Source data for this manuscript is available via the MassIVE repository at massive.ucsd.edu with identifier: MSV000100493.

AbstractPatients undergoing cardiopulmonary bypass procedures require inotropic support to improve hemodynamic function and cardiac output. Current inotropes such as dobutamine, can promote arrhythmias, prompting a demand for improved inotropes with little effect on intracellular Ca2+ flux. Low‐dose carbon monoxide (CO) induces inotropic effects in perfused hearts. Using the CO‐releasing pro‐drug, oCOm‐21, we investigated if this inotropic effect results from an increase in myofilament Ca2+ sensitivity. Male Sprague Dawley rat left ventricular cardiomyocytes were permeabilized, and myofilament force was measured as a function of ‐log [Ca2+] (pCa) in the range of 9.0–4.5 under five conditions: vehicle, oCOm‐21, the oCOm‐21 control BP‐21, and levosimendan, (9 cells/group). Ca2+ sensitivity was assessed by the Ca2+ concentration at which 50% of maximal force is produced (pCa50). oCOm‐21, but not BP‐21 significantly increased pCa50 compared to vehicle, respectively (pCa50 5.52 vs. 5.47 vs. 5.44; p < 0.05). No change in myofilament phosphorylation was seen after oCOm‐21 treatment. Pretreatment of cardiomyocytes with the heme scavenger hemopexin, abolished the Ca2+ sensitizing effect of oCOm‐21. These results support the hypothesis that oCOm‐21‐derived CO increases myofilament Ca2+ sensitivity through a heme‐dependent mechanism but not by phosphorylation. Further analyses will confirm if this Ca2+ sensitizing effect occurs in an intact heart.

Physical activity and several pharmacological approaches individually combat age-associated conditions and extend healthy longevity in model systems. It is tantalizing to extrapolate that combining geroprotector drugs with exercise could extend healthy longevity beyond any individual treatment. However, the current dogma suggests that taking leading geroprotector drugs on the same day as exercise may limit several health benefits. Here, we review leading candidate geroprotector drugs and their interactions with exercise and highlight salient gaps in knowledge that need to be addressed to identify if geroprotector drugs can have a harmonious relationship with exercise.

Sarcopenia is a progressive disorder characterized by age-related loss of skeletal muscle mass and function. Although significant progress has been made over the years to identify the molecular determinants of sarcopenia, the precise mechanisms underlying the age-related loss of contractile function remains unclear. Advances in "omics" technologies, including mass spectrometry-based proteomic and metabolomic analyses, offer great opportunities to better understand sarcopenia. Herein, we performed mass spectrometry-based analyses of the vastus lateralis from young, middle-aged, and older rhesus monkeys to identify molecular signatures of sarcopenia. In our proteomic analysis, we identified proteins that change with age, including those involved in adenosine triphosphate and adenosine monophosphate metabolism as well as fatty acid beta oxidation. In our untargeted metabolomic analysis, we identified metabolites that changed with age largely related to energy metabolism including fatty acid beta oxidation. Pathway analysis of age-responsive proteins and metabolites revealed changes in muscle structure and contraction as well as lipid, carbohydrate, and purine metabolism. Together, this study discovers new metabolic signatures and offers new insights into the molecular mechanisms underlying sarcopenia for the evaluation and monitoring of a therapeutic treatment of sarcopenia.

Single-cell proteomics has emerged as a powerful method to characterize cellular phenotypic heterogeneity and the cell-specific functional networks underlying biological processes. However, significant challenges remain in single-cell proteomics for the analysis of proteoforms arising from genetic mutations, alternative splicing, and post-translational modifications. Herein, we have developed a highly sensitive functionally integrated top-down proteomics method for the comprehensive analysis of proteoforms from single cells. We applied this method to single muscle fibers (SMFs) to resolve their heterogeneous functional and proteomic properties at the single cell level. Notably, we have detected single-cell heterogeneity in large proteoforms (>200 kDa) from the SMFs. Using SMFs obtained from three functionally distinct muscles, we found fiber-to-fiber heterogeneity among the sarcomeric proteoforms which can be related to the functional heterogeneity. Importantly, we reproducibly detected multiple isoforms of myosin heavy chain (~223 kDa), a motor protein that drives muscle contraction, with high mass accuracy to enable the classification of individual fiber types. This study represents the first “single-cell” top-down proteomics analysis that captures single muscle cell heterogeneity in large proteoforms and establishes a direct relationship between sarcomeric proteoforms and muscle fiber types, highlighting the potential of top-down proteomics for uncovering the molecular underpinnings of cell-to-cell variation in complex systems. Significance Statement Single-cell technologies are revolutionizing biology and molecular medicine by allowing direct investigation of the biological variability among individual cells. Top-down proteomics is uniquely capable of dissecting biological heterogeneity at the intact protein level. Herein, we develop a highly sensitive single-cell top-down proteomics method to reveal diverse molecular variations in large proteins (>200 kDa) among individual single muscle cells. Our results both reveal and characterize the differences in protein post-translational modifications and isoform expression possible between individual muscle cells. We further integrate functional properties with proteomics and accurately measure myosin isoforms for individual muscle fiber type classification. Our study highlights the potential of top-down proteomics for understanding how single-cell protein heterogeneity contributes to cellular functions.

The loss of skeletal muscle function with age, known as sarcopenia, significantly reduces independence and quality of life and can have significant metabolic consequences. Although exercise is effective in treating sarcopenia it is not always a viable option clinically, and currently, there are no pharmacological therapeutic interventions for sarcopenia. Here, we show that chronic treatment with pan-adiponectin receptor agonist AdipoRon improved muscle function in male mice by a mechanism linked to skeletal muscle metabolism and tissue remodeling. In aged mice, 6 weeks of AdipoRon treatment improved skeletal muscle functional measures in vivo and ex vivo. Improvements were linked to changes in fiber type, including an enrichment of oxidative fibers, and an increase in mitochondrial activity. In young mice, 6 weeks of AdipoRon treatment improved contractile force and activated the energy-sensing kinase AMPK and the mitochondrial regulator PGC-1a (peroxisome proliferator-activated receptor gamma coactivator one alpha). In cultured cells, the AdipoRon induced stimulation of AMPK and PGC-1a was associated with increased mitochondrial membrane potential, reorganization of mitochondrial architecture, increased respiration, and increased ATP production. Furthermore, the ability of AdipoRon to stimulate AMPK and PGC1a was conserved in nonhuman primate cultured cells. These data show that AdipoRon is an effective agent for the prevention of sarcopenia in mice and indicate that its effects translate to primates, suggesting it may also be a suitable therapeutic for sarcopenia in clinical application.

Systolic and diastolic dysfunction in diabetes have frequently been associated with abnormal calcium (Ca 2+ ) regulation. However, there is emerging evidence that Ca 2+ mishandling alone is insufficient to fully explain diabetic heart dysfunction, with focus shifting to the properties of the myofilament proteins. Our aim was to examine the effects of diabetes on myofilament Ca 2+ sensitivity and Ca 2+ handling in left ventricular tissues isolated from the same type 2 diabetic rat hearts. We measured the force-pCa relationship in skinned left ventricular cardiomyocytes isolated from 20-week-old type 2 diabetic and non-diabetic rats. Myofilament Ca 2+ sensitivity was greater in the diabetic relative to non-diabetic cardiomyocytes, and this corresponded with lower phosphorylation of cardiac troponin I (cTnI) at ser23/24 in the diabetic left ventricular tissues. Protein expression of sarco/endoplasmic reticulum Ca 2+ -ATPase (SERCA), phosphorylation of phospholamban (PLB) at Ser16, and SERCA/PLB ratio were lower in the diabetic left ventricular tissues. However, the maximum SERCA Ca 2+ uptake rate was not different between the diabetic and non-diabetic myocardium. Our data suggest that impaired contractility in the diabetic heart is not caused by SERCA Ca 2+ mishandling. This study highlights the important role of the cardiac myofilament and provides new insight on the pathophysiology of diabetic heart dysfunction.

The cardiovascular benefits of regular exercise are unequivocal, yet patients with type 2 diabetes respond poorly to exercise due to a reduced cardiac reserve. The contractile response of diabetic cardiomyocytes to β-adrenergic stimulation is attenuated, which may result in altered myofilament calcium sensitivity and posttranslational modifications of cardiac troponin I (cTnI). Treadmill running increases myofilament calcium sensitivity in nondiabetic rats, and thus we hypothesized that endurance training would increase calcium sensitivity of diabetic cardiomyocytes and alter site-specific phosphorylation of cTnI. Calcium sensitivity, or pCa50, was measured in Zucker diabetic fatty (ZDF), nondiabetic (nDM), and diabetic (DM) rat hearts after 8 wk of either a sedentary (SED) or progressive treadmill running (TR) intervention. Skinned cardiomyocytes were connected to a capacitance-gauge transducer and a torque motor to measure force as a function of pCa (-log[Ca2+]). Specific phospho-sites on cTnI and O-GlcNAcylation were quantified by immunoblot and total protein phosphorylation by fluorescent gel staining (ProQ Diamond). The novel finding in this study was that training increased pCa50 in both DM and nDM cardiomyocytes (P = 0.009). Phosphorylation of cTnI amino acid residues Ser23/24, a crucial protein kinase A site, and Threonine (Thr)144 was lower in DM hearts, but there was no effect of training on site-specific phosphorylation. In addition, total phosphorylation and O-GlcNAcylation levels were not different between SED and TR groups. These findings suggest that regular exercise may benefit the diabetic heart by specifically targeting myofilament contractile function.NEW & NOTEWORTHY We examined the effects of training on the myofilament calcium in diabetic rat hearts. After 8 wk of treadmill running, both nondiabetic and diabetic cardiomyocytes had increased myofilament calcium sensitivity compared with their sedentary counterparts, but there was no effect of training on the phosphorylation or O-GlcNAcylation status of myofilament proteins measured in this study. These data highlight one potential mechanism capable of reversing, in part, reduced cardiac reserve in the diabetic heart.

Abstract Background The diabetic heart has impaired systolic and diastolic function independent of other comorbidities. The availability of calcium is altered, but does not fully explain the cardiac dysfunction seen in the diabetic heart. Thus, we explored if myofilament protein regulation of contraction is altered. Methods Calcium sensitivity (pCa50) was measured in Zucker Diabetic Fatty (ZDF) rat hearts at the initial stage of diabetes (12-week-old) and after 8 weeks of uncontrolled hyperglycaemia (20-week-old) and in non-diabetic (nDM) littermates. Skinned cardiomyocytes were connected to a capacitance-gauge transducer and a torque motor to measure force as a function of pCa (-log[Ca2+]). Fluorescent gel stain (ProQ Diamond) was used to measure total protein phosphorylation. Specific phospho-sites on cardiac troponin I (cTnI) and total cTnI O-GlcNAcylation were quantified using immunoblot. Results pCa50 was greater in both 12- and 20-week-old diabetic (DM) rats compared to nDM littermates (p = 0.0005). Total cTnI and cTnI serine 23/24 phosphorylation were lower in DM rats (p = 0.003 & p = 0.01, respectively), but cTnI O-GlcNAc protein expression was not different. pCa50 is greater in DM rats and corresponds with an overall reduction in cTnI phosphorylation. Conclusions These findings indicate that myofilament calcium sensitivity is increased and cTnI phosphorylation is reduced in ZDF DM rats, which suggests an important role for cTnI phosphorylation in the DM heart.

New Findings What is the central question of this study? In Zucker Diabetic Fatty rats, does cardiomyocyte myofilament function change through the time course of diabetes and what are the mechanisms behind alterations in calcium sensitivity? What is the main finding and its importance? Zucker Diabetic Fatty rats had increased myofilament calcium sensitivity and reduced phosphorylation at cardiac troponin I without differential O‐GlcNAcylation. AbstractThe diabetic heart has impaired systolic and diastolic function independent of other comorbidities. The availability of calcium is altered, but does not fully explain the cardiac dysfunction seen in the diabetic heart. Thus, we explored if myofilament calcium regulation of contraction is altered while also categorizing the levels of phosphorylation and O‐GlcNAcylation in the myofilaments. Calcium sensitivity (pCa50) was measured in Zucker Diabetic Fatty (ZDF) rat hearts at the initial stage of diabetes (12 weeks old) and after 8 weeks of uncontrolled hyperglycaemia (20 weeks old) and in non‐diabetic (nDM) littermates. Skinned cardiomyocytes were connected to a capacitance‐gauge transducer and a torque motor to measure force as a function of pCa (−log[Ca2+]). Fluorescent gel stain (ProQ Diamond) was used to measure total protein phosphorylation. Specific phospho‐sites on cardiac troponin I (cTnI) and total cTnI O‐GlcNAcylation were quantified using immunoblot. pCa50 was greater in both 12‐ and 20‐week‐old diabetic (DM) rats compared to nDM littermates (P = 0.0001). Total cTnI and cTnI serine 23/24 phosphorylation were lower in DM rats (P = 0.003 and P = 0.01, respectively), but cTnI O‐GlcNAc protein expression was not different. pCa50 is greater in DM rats and corresponds with an overall reduction in cTnI phosphorylation. These findings indicate that myofilament calcium sensitivity is increased and cTnI phosphorylation is reduced in ZDF DM rats and suggests an important role for cTnI phosphorylation in the DM heart.

Cancer cachexia is the loss of lean muscle mass with or without loss of fat mass that is often highlighted by a progressive loss of skeletal muscle mass and function. The mechanisms behind the cachexia-related loss of skeletal muscle are poorly understood, including cachexia-related muscle functional impairments. Existing models have revealed some potential mechanisms, but appear limited to how the cancer develops and the type of tumors that form. We studied the C57BL6/J (B6) ApcMin/+ Tg::Fabp1-Cre TG::PIK3ca* (CANCER) mouse. In this model, mice develop highly aggressive intestinal cancers. We tested whether CANCER mice develop cancer cachexia, if muscle function is altered and if sex differences are present. Both female and male mice, B6 (CONTROL) and CANCER mice, were analyzed to determine body weight, hindlimb muscle mass, protein concentration, specific force, and fatigability. Female CANCER mice had reduced body weight and hindlimb muscle mass compared with female CONTROL mice, but lacked changes in protein concentration and specific force. Male CANCER mice had reduced protein concentration and reduced specific force, but lacked altered body weight and muscle mass. There were no changes in fatigability in either group. Our study demonstrates that CANCER mice present an early stage of cachexia, have reduced specific force in male CANCER mice and develop a sex-dependent cachexia phenotype. However, CANCER mice lack certain aspects of the syndrome seen in the human scenario and, therefore, using the CANCER mice as a preclinical model does not seem sufficient in order to maximize the translation of preclinical findings to humans.

The progressive age‐related loss of skeletal muscle mass and function, known as sarcopenia, increases the risk for impaired mobility, falls, fractures, and mortality. Currently, there are no pharmacological interventions to prevent or treat sarcopenia. Here we show that stimulation of adiponectin signaling in skeletal muscle by AdipoRon, a pan‐adiponectin receptor agonist, stimulates pathways linked to exercise and improves muscle function in old mice.Intravenous delivery of AdipoRon (1.2mg/kg bw; 3 times per week, 6 weeks) activated the AMPK‐PGC‐1a axis in skeletal muscle and induced changes in fiber type distribution in young (5 months) and old (24 months) male C57Bl6/J mice. In old mice, AdipoRon treatment improved running endurance and muscle contractile force. To explore mechanisms and enhance translational potential, the response to oral delivery through diet of AdipoRon (10, 25, or 50 mg/kg; daily feeding, 16 weeks) was investigated in young male and female C3B6F1 mice. AdipoRon enhanced insulin sensitivity and decreased body mass in females, however, these changes were not displayed in males. Increased expression of Ppargc1a and ACADM were observed in EDL of male mice in response to AdipoRon treatment and a decrease in expression of PDK4 was observed in both gastrocnemius and soleus indicating a shift toward oxidative metabolism in all muscle types. Conversely, these changes at the gene expression level were not observed in the female mice. Overall, AdipoRon treatment induced changes in the relative proportions of oxidative and glycolytic muscle fibers in EDL, gastrocnemius, and soleus; however, the skeletal muscle remodeling response to AdipoRon was muscle type and sex specific. In both sexes, the greatest response to AdipoRon was detected in EDL, which is the more glycolytic of the three muscle groups investigated. These data uncover differences in the underlying biology of skeletal muscle between males and females that may have implications for sarcopenia treatment. Taken together, our studies show that AdipoRon has potential as a therapeutic for sarcopenia in males and although sex dimorphic in its mechanism of action, is likely to also be effective as a treatment for sarcopenia in females.Support or Funding Information1I01BX003846‐01A2

Sarcomeric proteins, including myofilament and Z-disk proteins, play critical roles in regulating muscle contractile properties. A variety of isoforms and post-translational modifications (PTMs) of sarcomeric proteins have been shown to be associated with modulation of muscle functions and the occurrence of muscle diseases. Non-human primates (NHPs) are excellent research models for sarcopenia, a disease associated with alterations in sarcomeric proteins, due to their marked similarities to humans. However, the sarcomeric proteins in NHP skeletal muscle have not been well characterized. To gain a deeper understanding of sarcomeric proteins in NHP skeletal muscle, we employed top-down mass spectrometry (MS) to conduct a comprehensive analysis on isoforms and PTMs of sarcomeric proteins in rhesus macaque skeletal muscle. We identified 23 protein isoforms with 46 proteoforms of sarcomeric proteins, including 6 isoforms with 18 proteoforms from fast skeletal troponin T. Particularly, for the first time, a novel PDZ/LIM domain protein isoform, PDLIM7, was characterized with a newly identified protein sequence. Moreover, we also identified multiple PTMs on these proteins, including deamidation, methylation, acetylation, tri-methylation, phosphorylation, and S-glutathionylation. Most PTM sites were localized, including Asn13 deamidation on MLC-2S; His73 methylation on αactin; N-terminal acetylation on most identified proteins; N-terminal tri-methylation on MLC-1S, MLC-1F, MLC-2S, and MLC-2F; Ser14 phosphorylation on MLC-2S; and Ser15 and Ser16 phosphorylation on MLC-2F. In summary, a comprehensive characterization of sarcomeric proteins including multiple isoforms and PTMs in NHP skeletal muscle was achieved by analyzing intact proteins in the top-down MS approach.

Heterogeneity in skeletal muscle contraction time, peak power output, and resistance to fatigue, among others, is necessary to accommodate the wide range of functional demands imposed on the body. Underlying this functional heterogeneity are a myriad of differences in the myofilament protein isoform expression and post-translational modifications; yet, characterizing this heterogeneity remains challenging. Herein, we have utilized top-down liquid chromatography (LC)-mass spectrometry (MS)-based proteomics to characterize myofilament proteoform heterogeneity in seven rat skeletal muscle tissues including vastus lateralis, vastus medialis, vastus intermedius, rectus femoris, soleus, gastrocnemius, and plantaris. Top-down proteomics revealed that myofilament proteoforms varied greatly across the seven different rat skeletal muscle tissues. Subsequently, we quantified and characterized myofilament proteoforms using online LC-MS. We have comprehensively characterized the fast and slow skeletal troponin I isoforms, which demonstrates the ability of top-down MS to decipher isoforms with high sequence homology. Taken together, we have shown that top-down proteomics can be used as a robust and high-throughput method to characterize the molecular heterogeneity of myofilament proteoforms from various skeletal muscle tissues.

Coronary artery disease (CAD) remains the leading cause of death worldwide. The most common manifestation of CAD is myocardial infarction (MI), a result of ischemia and/or reperfusion (IR) injury of the myocardium. It is well established that exercise training improves myocardial tolerance to IR, a phenomenon known as exercise‐induced cardioprotection, but the cellular adaptations resulting in cardioprotection against IR injury are not well understood. Recent work has linked IR injury to the activation of the matrix metalloproteinase‐2 (MMP‐2). MMPs are known to be involved in the degradation of the extracellular matrix, and play a key role in scar formation following MI. While this extracellular role of MMP‐2 in MI has been well established, recent studies suggest an additional, intracellular, role of MMP activation in IR injury. MMP‐2 has been shown to have intracellular proteolytic targets in cardiomyocytes, which could contribute to the cell death seen following IR. Tissue inhibitor of metalloproteinases (TIMPs) are inhibitors of the MMPs, and TIMPs have been found in cardiomyocytes. It is thus possible that exercise provides cardioprotection against IR injury through 1) decreasing MMP‐2 protein levels, or 2) inhibiting MMP‐activation by increasing the protein levels of the TIMPs. However, no study to date has investigated the effect of exercise training on the protein levels of MMP‐2 and TIMP‐2 following IR injury. Therefore, the specific aims of this study were to determine the effects of exercise training on MMP‐2 and TIMP‐2 protein expression, and to identify regional differences in the levels of these proteins. To address these questions, we studied 3‐month old Sprague‐Dawley rats randomly assigned to sedentary (SED) or exercise (EX) groups (n=10/group). EX rats were run on a treadmill for 60 min/day, 5 days/week, for 8 weeks. IR was induced by occluding the LAD for 45 min, followed by 24 hr of reperfusion. Hearts were excised and sectioned and stained to delineate the zone at risk (ZAR) from the unaffected area (UA). Infarct size was expressed as a percentage of the ZAR. Hearts from EX rats exhibited smaller infarcts compared to SED rats (21.4% vs. 37.2%; P < 0.05). MMP‐2 and TIMP‐2 levels in ventricular homogenates were determined by Western blot. MMP‐2 protein content was increased in the ZAR compared to the UA in both EX and SED rats (P < 0.05). There was no difference in MMP‐2 protein levels between EX or SED rats. TIMP‐2 protein levels were increased in the hearts from EX rats compared to SED rats (P < 0.05). There was no difference in TIMP‐2 levels between the ZAR and UA in EX or SED rats. These results show that 1) MMP‐2 levels were significantly higher in the ZAR compared to the UA, suggesting an intracellular role of MMP‐2 in IR injury, and 2) exercise training leads to an increase in myocardial TIMP‐2 protein levels, suggesting elevated TIMP‐2 inhibition of MMP‐2 as a potential mechanism of exercise‐induced cardioprotection.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Ischemic heart disease is the leading cause of death in both males and females worldwide, with myocardial infarction due to ischemia‐reperfusion (IR) injury being the most common manifestation. Exercise is known to protect the myocardium against IR injury, but the volume of exercise sufficient to confer cardioprotection is not well understood. A single bout of exercise has been shown to confer protection against IR injury, but it is not known whether longer term exercise training (weeks) adds additional cardioprotective benefit. In addition, it is unclear to what extent sex differences contribute to cardioprotection. To address these questions, we studied female and male Sprague‐Dawley rats (3 mo of age) randomly assigned to a 5 day exercise training (EX5d) group (n=20), an 8 week exercise training (EX8w) group (n=20), or a sedentary (Sed) control group (n=10). EX5d rats ran for 5 consecutive days, while EX8w animals ran 5 days a week for 8 weeks. The speed was gradually increased until animals were running 21–23 m/min for 60 min. Maximal running capacity improved in all groups as a result of training. EX5d animals improved maximal distance run by 31.9 ± 9% (pre vs post) while EX8w animals improved by 118.5 ± 15.6% (p<0.01, EX5d vs EX8w). At least 24 h after the last training session, animals were anesthetized and the left anterior descending coronary artery was occluded via suture to induce regional ischemia for 45 min, followed by 24 h of reperfusion. After 24 hours, occlusion was reestablished and 0.5% Evans blue dye was injected into the aorta and perfused through the heart. Hearts were then sectioned, placed in a 0.1% triphenyltetrazolium chloride solution and incubated at 37° C for 10 minutes, then flash frozen in OCT. Infarct areas were quantified using ImageJ software, and infarct values calculated as a percentage of the zone at risk (ZAR). Exercise training led to a significant reduction in infarct size, with Sed, 5d, and 8w animals having infarct sizes of 37.2 ± 3, 27.5 ± 2, and 21.4 ± 1% of the ZAR, respectively (p<0.01, Sed vs EX5d and Sed vs EX8w). Hearts from Sed females had significantly smaller infarcts than those of Sed males (41.8 ± 3 and 32.6 ± 1% of the ZAR, respectively). This sex difference persisted in response to exercise training, with females exhibiting smaller infarct sizes following 5 days of exercise training compared to males (25.1 ± 1 and 29.9 ± 1% of the ZAR, in females and males respectively (p<0.05)), as well as 8 weeks of training (19.4 ± 1 and 23.5 ± 1% of the ZAR, respectively (p<0.05)). When comparing within‐sex differences in cardioprotection, 8 weeks of exercise did not lead to significant infarct size reduction compared to 5 days of exercise in males (23.5 vs 29.9% of the ZAR). However, hearts from EX8w females had significantly smaller infarct sizes when compared to EX5d females (19.4 vs 25.1% of the ZAR, respectively; p<0.01). These results show that 1) long‐term exercise training can augment infarct sparing against IR injury when compared to short duration exercise, and 2) this effect is sex‐dependent.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Sarcopenia, the age-related loss of skeletal muscle mass and strength, is a significant cause of morbidity in the elderly and is a major burden on health care systems. Unfortunately, the underlying molecular mechanisms in sarcopenia remain poorly understood. Herein, we utilized top-down proteomics to elucidate sarcopenia-related changes in the fast- and slow-twitch skeletal muscles of aging rats with a focus on the sarcomeric proteome, which includes both myofilament and Z-disc proteins-the proteins that constitute the contractile apparatuses. Top-down quantitative proteomics identified significant changes in the post-translational modifications (PTMs) of critical myofilament proteins in the fast-twitch skeletal muscles of aging rats, in accordance with the vulnerability of fast-twitch muscles to sarcopenia. Surprisingly, age-related alterations in the phosphorylation of Cypher isoforms, proteins that localize to the Z-discs in striated muscles, were also noted in the fast-twitch skeletal muscle of aging rats. This represents the first report of changes in the phosphorylation of Z-disc proteins in skeletal muscle during aging. In addition, increased glutathionylation of slow skeletal troponin I, a novel modification that may help protect against oxidative damage, was observed in slow-twitch skeletal muscles. Furthermore, we have identified and characterized novel muscle type-specific proteoforms of myofilament proteins and Z-disc proteins, including a novel isoform of the Z-disc protein Enigma. The finding that the phosphorylation of Z-disc proteins is altered in response to aging in the fast-twitch skeletal muscles of aging rats opens new avenues for the investigation of the role of Z-discs in age-related muscle dysfunction.

Premature birth affects more than 10% of live births, and is characterized by relative hyperoxia exposure in an immature host. Long-term consequences of preterm birth include decreased aerobic capacity, decreased muscular strength and endurance, and increased prevalence of metabolic diseases such as type 2 diabetes mellitus. Postnatal hyperoxia exposure in rodents is a well-established model of chronic lung disease of prematurity, and also recapitulates the pulmonary vascular, cardiovascular, and renal phenotype of premature birth. The objective of this study was to evaluate whether postnatal hyperoxia exposure in rats could recapitulate the skeletal and metabolic phenotype of premature birth, and to characterize the subcellular metabolic changes associated with postnatal hyperoxia exposure, with a secondary aim to evaluate sex differences in this model. Compared to control rats, male rats exposed to 14 days of postnatal hyperoxia then aged to 1 year demonstrated higher skeletal muscle fatigability, lower muscle mitochondrial oxidative capacity, more mitochondrial damage, and higher glycolytic enzyme expression. These differences were not present in female rats with the same postnatal hyperoxia exposure. This study demonstrates detrimental mitochondrial and muscular outcomes in the adult male rat exposed to postnatal hyperoxia. Given that young adults born premature also demonstrate skeletal muscle dysfunction, future studies are merited to determine whether this dysfunction as well as reduced aerobic capacity is due to reduced mitochondrial oxidative capacity and metabolic dysfunction.

Determining changes in protein expression and post-translational modifications (PTMs) is crucial for elucidating cellular signal transduction and disease mechanisms. Conventional antibody-based approaches have inherent problems such as the limited availability of high-quality antibodies and batch-to-batch variation. Top-down mass spectrometry (MS)-based proteomics has emerged as the most powerful method for characterization and quantification of protein modifications. Nevertheless, robust methods to simultaneously determine changes in protein expression and PTMs remain lacking. Herein, we have developed a straightforward and robust top-down liquid chromatography (LC)/MS-based targeted proteomics platform for simultaneous quantification of protein expression and PTMs with high throughput and high reproducibility. We employed this method to analyze the sarcomeric subproteome from various muscle types of different species, which successfully revealed skeletal muscle heterogeneity and cardiac developmental changes in sarcomeric protein isoform expression and PTMs. As demonstrated, this targeted top-down proteomics platform offers an excellent 'antibody-independent' alternative for the accurate quantification of sarcomeric protein expression and PTMs concurrently in complex mixtures, which is generally applicable to different species and various tissue types.

The Ca2+‐activated protease calpain has been shown to play a deleterious role in the heart during the aging process, which ultimately impacts cardiac function. Calpastatin is an endogenous inhibitor of calpain, and the ratio of calpain to calpastatin in cardiac myocytes is physiologically important because this ratio greatly impacts the ability of Ca2+ to activate calpain. It has been suggested that activation of calpain may lead to proteolytic degradation of sarcomeric proteins, which ultimately contributes to impaired contractile function in the aging heart. Exercise training has been shown to mitigate and even reverse many aspects of the aging process in the heart. However, little is known about the effects of endurance exercise training on cardiac levels of calpain I, calpain II, or calpastatin in senescent hearts. To address this question, we conducted a 3‐month endurance treadmill training study in aged rats. Exercise capacity (as measured by time to exhaustion, EX‐TIME) and VO2max were measured in 22 month old rats and then animals were divided into Sedentary (SED) vs. Exercise (EX) groups. EX animals were trained 5 days a week for 3 months, with speed and duration gradually increased to the point that animals were running at 14 m/min for 45 min. At the end of the 3‐month training period, exercise capacity and VO2max were again measured in all animals. Mean EX‐TIME and VO2max values significantly (p <0.05) declined in SED animals from Pre‐training (PRE) to Post‐training (POST). EX‐TIME declined by 58% in POST compared to PRE, while VO2max declined by 21% in POST vs. PRE. In the EX animals there were significant (p <0.05) increases in EX‐TIME (POST values 172% of PRE) and VO2max (POST values 141% of PRE). To assess cardiac function, echocardiography was used to measure Ejection Fraction (EF%) and Fractional Shortening (FS%). EX training significantly (p <0.05) improved EF% and FS% compared to SED animals (EF% was 36% greater in EX compared to SED; FS% was 26% higher in EX compared to SED). Following sacrifice, rat hearts were quickly excised, and the left ventricle was dissected and flash frozen for Western blot analysis. Endurance exercise training was associated with decreased calpain to calpastatin ratios in aged hearts compared to sedentary counterparts (p <0.05). There was no significant difference in calpain II levels between EX and SED hearts. The improved calpain/calpastatin ratio exhibited by EX animals appears to be due to a significant decrease in calpain I levels compared to SED animals (p <0.05). These results show that endurance exercise increased cardiac function in senescent rats, and that this increase was associated with a decrease in the ratio of calpain to calpastatin. Changes in the cardiac levels of calpain I appear to underlie the decrease in this ratio. Support or Funding Information Marsh Center for Research in Exercise and Movement, University of Wisconsin‐Madison