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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 detected multiple isoforms of myosin heavy chain (~223 kDa), a motor protein that drives muscle contraction, with high reproducibility to enable the classification of individual fiber types. This study reveals 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.

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 StatementSingle-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 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 pCa

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.

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.

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. ABSTRACT 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 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 mechanisms behind the progressive loss of skeletal muscle mass and function seen in patients with cancer cachexia are still largely unknown, thus we tested whether a new murine model of colorectal cancer could better represent the human condition. We concluded that although the tumor‐bearing model tested had reductions in body weight, muscle mass, specific force and a sex‐specific phenotype, the model tested had a limited lifespan and ultimately impaired the ability to translate findings to the human condition.

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.

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.

Sarcopenia, the loss of skeletal muscle mass and function with advancing age, is a significant cause of disability and loss of independence in the elderly and thus, represents a formidable challenge for the aging population. Nevertheless, the molecular mechanism(s) underlying sarcopenia-associated muscle dysfunction remain poorly understood. In this study, we employed an integrated approach combining top-down targeted proteomics with mechanical measurements to dissect the molecular mechanism(s) in age-related muscle dysfunction. Top-down targeted proteomic analysis uncovered a progressive age-related decline in the phosphorylation of myosin regulatory light chain (RLC), a critical protein involved in the modulation of muscle contractility, in the skeletal muscle of aging rats. Top-down tandem mass spectrometry analysis identified a previously unreported bis-phosphorylated proteoform of fast skeletal RLC and localized the sites of decreasing phosphorylation to Ser14/15. Of these sites, Ser14 phosphorylation represents a previously unidentified site of phosphorylation in RLC from fast-twitch skeletal muscle. Subsequent mechanical analysis of single fast-twitch fibers isolated from the muscles of rats of different ages revealed that the observed decline in RLC phosphorylation can account for age-related decreases in the contractile properties of sarcopenic fast-twitch muscles. These results strongly support a role for decreasing RLC phosphorylation in sarcopenia-associated muscle dysfunction and suggest that therapeutic modulation of RLC phosphorylation may represent a new avenue for the treatment of sarcopenia.

Aged skeletal muscle demonstrates declines in muscle mass and deterioration of mitochondrial content and function. Peroxisome proliferator-activated receptor γ co-activator 1α (PGC-1α) plays an important role in promoting muscle mitochondrial biogenesis in response to exercise training, but its role in senescent muscle is not clear. In the present study we hypothesize that a downregulation of the PGC-1α signaling pathway contributes to mitochondrial deterioration in aged muscle whereas endurance training ameliorates the deficits. Three groups of Fischer 344/BNF1 rats were used: young, sedentary (Y, 4 months); old, sedentary (O, 22 months); and old trained (OT, 22 months), subjected to treadmill running at 17.5 m/min, 10% grade for 45 min/day, 5 days/week for 12-weeks. PGC-1α mRNA and nuclear PGC-1α protein content in the soleus muscle were both decreased in O vs. Y rats, whereas OT rats showed a 2.3 and 1.8-fold higher PGC-1α content than O and Y rats, respectively (P

Tendon mechanical properties are thought to degrade during aging but improve with exercise. A remaining question is whether exercise in aged animals provides sufficient regenerative, systemic stimulus to restore younger mechanical behaviors. Herein we address that question with tail tendons from aged and exercised rats, which would be subject to systemic effects but not direct loading from the exercise regimen. Twenty-four month old rats underwent one of three treadmill exercise training protocols for 12 months: sedentary (walking at 0° incline for 5 min/day), moderate (running at 0° incline for 30 min/day), or high (running at 4° incline for 30 min/day). A group of 9 month old rats were used to provide an adult control, while a group of 3 month old rats provided a young control. Tendons were harvested at sacrifice and mechanically tested. Results show significant age-dependent differences in modulus, ultimate stress, relaxation rate, and percent relaxation. Relaxation rate was strain-dependent, consistent with nonlinear superposition or Schapery models but not with quasilinear viscoelasticity (QLV). Trends in exercise data suggest that with exercise, tendons assume the elastic character of younger rats (lower elastic modulus and ultimate stress).

Aging is associated with a decline in cardiac function. Exercise intervention has been suggested as a way to improve this decrement. Age-related decline in cardiac function is associated with decreases in fatty acid oxidation, mitochondrial function and AMP-activated protein kinase (AMPK) activity. The molecular mechanisms involved with age-related changes in mitochondrial function and substrate metabolism are poorly understood. We determined gene expression differences in hearts of Young (6 mo), Old (33 mo), and old exercise trained (Old + EXE) (34 mo) FBN rats, using Qiagen PCR arrays for Glucose, Fatty acid, and Mitochondrial metabolism. Old rats demonstrated decreased (p < 0.05) expression for key genes in fatty acid oxidation, mitochondrial function, and AMPK signaling. There were no differences in the expression of genes involved in glucose metabolism with age. These gene expression changes occurred prior to altered protein translation as we found no differences in the protein content of peroxisome proliferator activated receptor gamma, coactivators 1 alpha (PGC-1α), peroxisome proliferator activated receptor alpha (PPARα), and AMPKα2 between young and old hearts. Four months of exercise training did not attenuate the decline in the gene expression in aged hearts. Despite this lack of change in gene expression, exercise-trained rats demonstrated increased exercise capacity compared to their sedentary counterparts. Taken together, our results show that differential expression of genes associated with fatty acid metabolism, AMPK signaling and mitochondrial function are superfluous and decrease in the aging heart which may play a role in age-related declines in fatty acid oxidation, AMPK activity and mitochondrial function in the heart.

Aging is generally associated with a decline in several indices of cardiac function. The cellular mechanisms for this decline are not completely understood. The ability of the myocardium to perform external work (power output) is a critical aspect of ventricular function. The purpose of this study was to determine the effect of aging on loaded shortening and power output properties. We measured force–velocity properties in permeabilized (skinned) myocytes from the hearts of 9-, 24-, and 33-month-old male Fisher 344 × Brown Norway F1 hybrid rats (F344BN) during loaded contractions using a force-clamp technique. Power output was calculated by multiplying force and shortening velocity values. We found that peak power output normalized to maximal force was significantly decreased by 18% and 31% in myocytes from 24- and 33-month-old group, respectively, compared with 9-month group (p < .05). These results suggest that aging is associated with a significant decrease in the ability of the myocardium to do work.

Similar to other subdisciplines in kinesiology, exercise physiology (EP) as a field is facing challenges in both research (creation and dissemination of new knowledge) and education (classroom instruction and student mentoring). In the current communication, we will learn from the history, analyze the current status of the field, and provide some perspectives based primarily on our knowledge and experience working as faculty members at the University of Wisconsin–Madison.

Substrate utilization favors fatty acid oxidation in normal healthy hearts. Aging is associated with attenuated mitochondrial biogenesis and fatty acid metabolism in the heart, but the mechanism of...

Aging is associated with declining cardiac contractile function as well as changes in substrate metabolism and mitochondrial function. The relationship between age-related changes in cardiac metabo...

Mechanisms underlying sarcopenia in skeletal muscle have not been fully elucidated, but mitochondrial dysfunction appears to play a role. The PGC-1α mediated pathway plays an important role in prom...

Myocardial function is enhanced by endurance exercise training, but the cellular mechanisms underlying this improved function remain unclear. A number of studies have shown that the characteristics of cardiac myocytes vary across the width of the ventricular wall. We have previously shown that endurance exercise training alters the Ca2+sensitivity of tension as well as contractile protein isoform expression in rat cardiac myocytes. We tested the hypothesis that these effects of training are not uniform across the ventricular wall but are more pronounced in the subendocardial (Endo) region of the myocardium. Female Sprague-Dawley rats were divided into sedentary control (C) and exercise trained (T) groups. T rats underwent 11 wk of progressive treadmill exercise. Myocytes were isolated from the Endo region of the myocardium and from the subepicardial (Epi) region of both T and C hearts. We found an increase in the Ca2+sensitivity of tension in T cells compared with C cells, but this difference was larger in the Endo cells than in the Epi cells. In addition, we found a training-induced increase in atrial myosin light chain 1 (aMLC1) expression that was larger in the Endo compared with Epi samples. We conclude that effects of exercise training on myocyte contractile and biochemical properties are greater in myocytes from the Endo region of the myocardium than those from the Epi region. In addition, these results provide evidence that the increase in aMLC1expression may be responsible for some of the training-induced increase in myocyte Ca2+sensitivity of tension.

Myocardial function is enhanced by endurance exercise training, but the cellular mechanisms underlying this improved function remain unclear. Exercise training increases the sensitivity of rat cardiac myocytes to activation by Ca2+, and this Ca2+ sensitivity has been shown to be highly dependent on sarcomere length. We tested the hypothesis that exercise training increases this length dependence in cardiac myocytes. Female Sprague-Dawley rats were divided into sedentary control (C) and exercise-trained (T) groups. The T rats underwent 11 wk of progressive treadmill exercise. Heart weight increased by 14% in T compared with C rats, and plantaris muscle citrate synthase activity showed a 39% increase with training. Steady-state tension was determined in permeabilized myocytes by using solutions of various Ca2+concentration (pCa), and tension-pCa curves were generated at two different sarcomere lengths for each myocyte (1.9 and 2.3 μm). We found an increased sarcomere length dependence of both maximal tension and pCa50 (the Ca2+ concentration giving 50% of maximal tension) in T compared with C myocytes. The ΔpCa50 between the long and short sarcomere length was 0.084 ± 0.023 (mean ± SD) in myocytes from C hearts compared with 0.132 ± 0.014 in myocytes from T hearts ( n = 50 myocytes per group). The Δmaximal tension was 5.11 ± 1.42 kN/m2 in C myocytes and 9.01 ± 1.28 in T myocytes. We conclude that exercise training increases the length dependence of maximal and submaximal tension in cardiac myocytes, and this change may underlie, at least in part, training-induced enhancement of myocardial function.

Cachexia is commonly seen in cancer and is characterized by severe muscle wasting, but little is known about the effect of cancer cachexia on expression of contractile protein isoforms such as myosin. Other causes of muscle atrophy shift expression of myosin isoforms toward increased fast (type II) isoform expression. We injected mice with murine C-26 adenocarcinoma cells, a tumor cell line that has been shown to cause muscle wasting. Mice were killed 21 days after tumor injection, and hindlimb muscles were removed. Myosin heavy chain (MHC) and myosin light chain (MLC) content was determined in muscle homogenates by SDS-PAGE. Body weight was significantly lower in tumor-bearing (T) mice. There was a significant decrease in muscle mass in all three muscles tested compared with control, with the largest decrease occurring in the soleus. Although no type IIb MHC was detected in the soleus samples from control mice, type IIb comprised 19% of the total MHC in T soleus. Type I MHC was significantly decreased in T vs. control soleus muscle. MHC isoform content was not significantly different from control in plantaris and gastrocnemius muscles. These data are the first to show a change in myosin isoform expression accompanying muscle atrophy during cancer cachexia.

Aging is associated with declining cardiac contractile function as well as changes in metabolism and mitochondrial function. The relationship between age‐related changes in cardiac metabolism and declining cardiac contractile function has not been determined. In order to define the role energetics play in changes in contractile function, we measured mitochondrial NADH, [NADH] m , during continuous contractions of isolated left ventricular myocytes from young (Y) and old (O) FBN rats. Second, we explored the role of metabolic disruption with rotenone and increased workload with isoproterenol (ISO) had on age‐related changes in myocytes shortening. Single, intact myocytes were stimulated for 10 min of continuous contraction at either 2 Hz or 4 Hz while being perfused with Ringer9s solution. Properties of shortening (peak shortening and rate of shortening) were measured at the onset (T0) and after 10 min (T10) of continuous contraction, and the decline in shortening over time (T10/T0) was determined. Although young and old myocytes had similar contractile function under resting conditions, old myocytes demonstrated decrements in [NADH] m during continuous stimulation, while young myocytes maintained constant [NADH]m over this time. In addition, old myocytes exhibited impaired contractile function to a workload (ISO) and metabolic (rotenone) stress compared to young myocytes. Taken together, these results demonstrated that old myocytes are susceptible to stress‐induced contractile dysfunction which may be related to altered cellular energetics.

Aging is characterized by a progressive decline in cardiac function, but endurance exercise training has been shown to retard a number of deleterious effects of aging. However, underlying mechanisms by which exercise training improves age-related decrements in myocardial contractile function are not well understood. The purpose of this study was to determine the effects of exercise training on power output properties in permeablized (skinned) myocytes of old rats. Thirty-month-old rats were divided into sedentary control (C) and groups undergoing 11 weeks of treadmill exercise training at moderate intensity (MI) and at high intensity (HI). Peak power output normalized to maximal force was significantly increased in MI but not in HI compared to C with significant increases in atrial myosin light chain 1 in ventricle. These results suggest that MI exercise training is beneficial as a significant increase was seen in the ability of the myocardium to do work, but this effect was not seen with HI training.

Using an intact rat model we studied the role of alpha and beta adrenergic agonists in recruiting intrapulmonary arteriovenous anastamoses (IPAVs). Exercise and hypoxia, which recruit IPAVs, activa...

It is recognized that with ageing of the rodent heart there is a shift in myosin heavy chain (MHC) isoform from α-MHC to β-MHC. Previous studies have shown evidence of significant regional differen...

Prolonged selective breeding of mice (Mus musculus) for high levels of voluntary wheel running has favoured an unusual phenotype (“mini muscles”), apparently caused by a single Mendelian recessive allele, in which most hind-limb muscles are markedly reduced in mass, but have increased mass-specific activities of mitochondrial enzymes. We examined whether these changes reflect changes in fibre size, number or ultrastructure in normal and “mini-muscle” mice within the two (of four) selectively bred lines (lab designations L3 and L6) that exhibit the phenotype at generations 26 and 27. In both lines, the gastrocnemius and plantaris muscles are smaller in mass (by > 50% and 20%, respectively) in affected individuals. The mass-specific activities of mitochondrial enzymes in the gastrocnemius and plantaris muscles were increased in the mini phenotype in both lines, with stronger effects in the gastrocnemius muscle. In the gastrocnemius, the % myosin heavy chain (MHC) IIb was reduced by 50% in L3 and by 30% in L6, whereas the % MHC IIa and I were higher, particularly in L3. Fibre number in the plantaris muscle did not significantly differ between mini and normal muscles, although muscle mass was a significant positive correlate of fibre number. Small fibres were more abundant in mini than normal muscles in L3. Mitochondrial volume density was significantly higher in mini than normal muscle fibres in L3, but not in L6. Microscopy revealed a surprising attribute of the mini muscles: an abundance of small, minimally differentiated, myofibril-containing cells positioned in a disorderly fashion, particularly in the surface layer. We hypothesise that these unusual cells may be satellite cells or type IIb fibres that did not complete their differentiation. Together, these observations suggest that mice with the mini phenotype have reduced numbers of type IIb fibres in many of their hind-limb muscles, leading to a decrease in mass and an increase in mass-specific aerobic capacity in muscles that typically have a high proportion of type IIb fibres. Moreover, the several statistically significant interactions between muscle phenotype and line indicate that the effect of the underlying allele is altered by genetic background.

Recent work suggests that chronic exercise induces alterations in the contractile properties of cardiac myocytes. These alterations include increased sensitivity to activation by Ca, changes in the force-length relationship, and increased power output. A recently observed shift in expression of myosin light chain 1 subunit isoforms induced by training may provide a molecular mechanism for these contractile alterations.

Myocardial function is enhanced by endurance exercise training, but the cellular mechanisms underlying this improved function remain unclear. The ability of the myocardium to perform external work is a critical aspect of ventricular function, but previous studies of myocardial adaptation to exercise training have been limited to measurements of isometric tension or unloaded shortening velocity, conditions in which work output is zero. We measured force-velocity properties in single permeabilized myocyte preparations to determine the effect of exercise training on loaded shortening and power output. Female Sprague-Dawley rats were divided into sedentary control (C) and exercise trained (T) groups. T rats underwent 11 wk of progressive treadmill exercise. Myocytes were isolated from T and C hearts, chemically skinned, and attached to a force transducer. Shortening velocity was determined during loaded contractions at 15°C by using a force-clamp technique. Power output was calculated by multiplying force times velocity values. We found that unloaded shortening velocity was not significantly different in T vs. C myocytes (T = 1.43 muscle lengths/s, n = 46 myocytes; C = 1.12 muscle lengths/s, n = 43 myocytes). Training increased the velocity of loaded shortening and increased peak power output (peak power = 0.16 P/Po × muscle length/s for T myocytes; peak power = 0.10 P/Po× muscle length/s for C myocytes, where P/Po is relative tension). We found no effect of training on myosin heavy chain isoform content. These results suggest that training alters power output properties of single cardiac myocytes and that this adaptation may improve the work capacity of the myocardium.

Previous studies have shown that endurance exercise training increases myocardial contractility. We have previously described training-induced alterations in myocardial contractile function at the cellular level, including an increase in the Ca2+ sensitivity of tension. To determine the molecular mechanism(s) of these changes, oligonucleotide microarrays were used to analyze the gene expression profile in ventricles from endurance-trained rats. We used an 11-wk treadmill training protocol that we have previously shown results in increased contractility in cardiac myocytes. After the training, the hearts were removed and RNA was isolated from the ventricles of nine trained and nine control rats. With the use of an Affymetrix Rat Genome U34A Array, we detected altered expression of 27 genes. Several genes previously found to have increased expression in hypertrophied myocardium, such as atrial natriuretic factor and skeletal α-actin, were decreased with training in this study. From the standpoint of altered contractile performance, the most significant finding was an increase in the expression of atrial myosin light chain 1 (aMLC-1) in the trained ventricular tissue. We confirmed microarray results for aMLC-1 using RT-PCR and also confirmed a training-induced increase in aMLC-1 protein using two-dimensional gel electrophoresis. aMLC-1 content has been previously shown to be increased in human cardiac hypertrophy and has been associated with increased Ca2+sensitivity of tension and increased power output. These results suggest that increased expression of aMLC-1 in response to training may be responsible, at least in part, for previously observed training-induced enhancement of contractile function.

The heart is known to respond to a program of chronic exercise in ways that enhance cardiac function. However, the cellular mechanisms involved in training-induced improvements in the contractile function of the myocardium are not known. In this study we tested the hypothesis that increased contractility of the myocardium associated with exercise training is due, in part, to increases in the Ca2+ sensitivity of steady-state tension. Female Sprague-Dawley rats were randomly divided into sedentary control (C) and exercise-trained (T) groups. The T rats underwent 11 wk of progressive treadmill exercise (1 h/day, 5 days/wk, 26 m/min, 20% grade). Evidence of training effect included a 5.9% increase in heart mass, increases in heart weight-to-body weight ratio, and a 60% increase in skeletal muscle citrate synthase activity in T rats compared with C rats. After the training program, cardiac myocytes were isolated from T and C hearts. Myocytes were chemically skinned (i.e., the sarcolemma was removed) and attached to a force transducer, and steady-state tension was determined in solutions of various Ca2+ concentrations ([Ca2+]). Myocytes isolated from the hearts of T rats showed a significantly ( P < 0.01) increased sensitivity of tension to [Ca2+]. The [Ca2+] giving 50% of maximal tension (pCa50) was 5.90 ± 0.033 and 5.82 ± 0.023 (SD) in T and C myocytes, respectively ( n = 70 myocytes/group). This result suggests that exercise training affects the myofibrillar proteins, such that Ca2+ sensitivity is increased, and that this may be the mechanism that underlies, at least in part, the effect of training to increase myocardial contractility.