Fast skeletal myosin binding protein-C expression exacerbates dysfunction in heart failure.

During heart failure, gene and protein expression profiles undergo extensive compensatory and pathological remodeling. We previously observed that fast skeletal myosin binding protein-C (fMyBP-C) is upregulated in diseased mouse hearts. While fMyBP-C shares significant homology with its cardiac paralog, cardiac myosin binding protein-C (cMyBP-C), there are key differences that may affect cardiac function. However, it is unknown if the expression of fMyBP-C expression in the heart is a pathological or compensatory response. We aim to elucidate the cardiac consequence of either increased or knockout of fMyBP-C expression. To determine the sufficiency of fMyBP-C to cause cardiac dysfunction, we generated cardiac-specific fMyBP-C over-expression mice. These mice were further crossed into a cMyBP-C null model to assess the effect of fMyBP-C in the heart in the complete absence of cMyBP-C. Finally, fMyBP-C null mice underwent transverse aortic constriction (TAC) to define the requirement of fMyBP-C during heart failure development. We confirmed the upregulation of fMyBP-C in several models of cardiac disease, including the use of lineage tracing. Low levels of fMyBP-C caused mild cardiac remodeling and sarcomere dysfunction. Exclusive expression of fMyBP-C in a heart failure model further exacerbated cardiac pathology. Following 8 weeks of TAC, fMyBP-C null mice demonstrated greater protection against heart failure development. Mechanistically, this may be due to the differential regulation of the myosin super-relaxed state. These findings suggest that the elevated expression of fMyBP-C in diseased hearts is a pathological response. Targeted therapies to prevent upregulation of fMyBP-C may prove beneficial in the treatment of heart failure. Significance Statement: Recently, the sarcomere - the machinery that controls heart and muscle contraction - has emerged as a central target for development of cardiac therapeutics. However, there remains much to understand about how the sarcomere is modified in response to disease. We recently discovered that a protein normally expressed in skeletal muscle, is present in the heart in certain settings of heart disease. How this skeletal muscle protein affects the function of the heart remained unknown. Using genetically engineered mouse models to modulate expression of this skeletal muscle protein, we determined that expression of this skeletal muscle protein in the heart negatively affects cardiac performance. Importantly, deletion of this protein from the heart could improve heart function suggesting a possible therapeutic avenue.

Mechanoregulation and function of calponin and transgelin.

It is well known that chemical energy can be converted to mechanical force in biological systems by motor proteins such as myosin ATPase. It is also broadly observed that constant/static mechanical signals potently induce cellular responses. However, the mechanisms that cells sense and convert the mechanical force into biochemical signals are not well understood. Calponin and transgelin are a family of homologous proteins that participate in the regulation of actin-activated myosin motor activity. An isoform of calponin, calponin 2, has been shown to regulate cytoskeleton-based cell motility functions under mechanical signaling. The expression of the calponin 2 gene and the turnover of calponin 2 protein are both under mechanoregulation. The regulation and function of calponin 2 has physiological and pathological significance, as shown in platelet adhesion, inflammatory arthritis, arterial atherosclerosis, calcific aortic valve disease, post-surgical fibrotic peritoneal adhesion, chronic proteinuria, ovarian insufficiency, and tumor metastasis. The levels of calponin 2 vary in different cell types, reflecting adaptations to specific tissue environments and functional states. The present review focuses on the mechanoregulation of calponin and transgelin family proteins to explore how cells sense steady tension and convert the force signal to biochemical activities. Our objective is to present a current knowledge basis for further investigations to establish the function and mechanisms of calponin and transgelin in cellular mechanoregulation.

Evolution and function of calponin and transgelin.

Calponin and transgelin (originally named SM22) are homologous cytoskeleton proteins that regulate actin-activated myosin motor functions in smooth muscle contraction and non-muscle cell motility during adhesion, migration, proliferation, phagocytosis, wound healing, and inflammatory responses. They are abundant cytoskeleton proteins present in multiple cell types whereas their physiological functions remain to be fully established. This focused review summarizes the evolution of genes encoding calponin and transgelin and their isoforms and discusses the structural similarity and divergence in vertebrate and invertebrate species in the context of functions in regulating cell motility. As the first literature review focusing on the evolution of the calponin-transgelin family of proteins in relevance to their structure-function relationship, the goal is to outline a foundation of current knowledge for continued investigations to understand the biological functions of calponin and transgelin in various cell types during physiological and pathological processes.

Early Divergence of the C-Terminal Variable Region of Troponin T Via a Pair of Mutually Exclusive Alternatively Spliced Exons Followed by a Selective Fixation in Vertebrate Heart.

Troponin T (TnT) is the thin filament anchoring subunit of troponin complex and plays an organizer role in the Ca2+-regulation of striated muscle contraction. From an ancestral gene emerged ~ 700 million years ago in Bilateria, three homologous genes have evolved in vertebrates to encode muscle type-specific isoforms of TnT. Alternative splicing variants of TnT are present in vertebrate and invertebrate muscles to add functional diversity. While the C-terminal region of TnT is largely conserved, it contains an alternatively spliced segment emerged early in C. elegans, which has evolved into a pair of mutually exclusive exons in arthropods (10A and 10B of Drosophila TpnT gene) and vertebrates (16 and 17 of fast skeletal muscle Tnnt3 gene). The C-terminal alternatively spliced segment of TnT interfaces with the other two subunits of troponin with functional significance. The vertebrate cardiac TnT gene that emerged from duplication of the fast TnT gene has eliminated this alternative splicing by the fixation of an exon 17-like constitutive exon, indicating a functional value in slower and rhythmic contractions. The vertebrate slow skeletal muscle TnT gene that emerged from duplication of the cardiac TnT gene has the exon 17-like structure conserved, indicating its further function in sustained and fatigue resistant contractions. This functionality-based evolution is consistent with the finding that exon 10B-encoded segment of Drosophila TnT homologous to the exon 17-encoded segment of vertebrate fast TnT is selectively expressed in insect heart and leg muscles. The evolution of the C-terminal variable region of TnT demonstrates a submolecular mechanism in modifying striated muscle contractility and for the treatment of muscle and heart diseases.

Evolution of the N-Terminal Regulation of Cardiac Troponin I for Heart Function of Tetrapods: Lungfish Presents an Example of the Emergence of Novel Submolecular Structure to Lead the Capacity of Adaptation.

Troponin-based Ca2+ regulation of striated muscle contraction emerged approximately 700 million years ago with largely conserved functions during evolution. Troponin I (TnI) is the inhibitory subunit of troponin and has evolved into three muscle type-specific isoforms in vertebrates. Cardiac TnI is specifically expressed in the adult heart and has a unique N-terminal extension implicating a specific value during natural selection. The N-terminal extension of cardiac TnI in higher vertebrates contains ß-adrenergic-regulated protein kinase A (PKA) phosphorylation sites as a mechanism to enhance cardiac muscle relaxation and facilitate ventricular filling. Phylogenic studies showed that the N-terminal extension of cardiac TnI first emerged in the genomes of early tetrapods as well as primordial lobe-finned fishes such as the coelacanth whereas it is absent in ray-finned fish. This apparently rapid evolution of ß-adrenergic regulation of cardiac function suggests a high selection value for the heart of vertebrate animals on land to work under higher metabolic demands. Sequencing and PKA phosphorylation data showed that lungfish cardiac TnI has evolved with an amphibian-like N-terminal extension with prototype PKA phosphorylation sites while its overall structure remained fish like. The data demonstrate that the submolecular structure of TnI may evolve ahead of the whole protein for cardiac muscle contractility to adapt to new environmental conditions. Understanding the evolution of the ß-adrenergic regulation of TnI and cardiac adaptation to the increased energetic demands of life on land adds knowledge for the treatment of human heart diseases and failure.

A rapid degradation of calponin 2 is required for cytokinesis.

Calponin 2 is an actin cytoskeleton-associated protein and plays a role in regulating cell motility-related functions such as phagocytosis, migration, and division. We previously reported that overexpression of calponin 2 inhibits the rate of cell proliferation. To investigate the underlying mechanism, our present study found that the levels of endogenous calponin 2 in NIH3T3 and HEK293 cells rapidly decreased before cell division characterized by an absence at the actin contractile ring. In cells lacking endogenous calponin 2, transfective expression of GFP-fusion calponin 2 inhibited cell proliferation similar to that of nonfusion calponin 2. Fluorescent imaging studies of mitotic cells indicated that a proper level of calponin 2 expression and effective degradation during cytokinesis are necessary for normal cell division. Computer-assisted dynamic image analysis of dividing cells revealed that overexpression of calponin 2 significantly affects motility and shape behaviors of cells only on the interval from the start of anaphase to the start of cytokinesis, i.e., the pre-cytokinesis phase, but not on the interval from the start of cytokinesis to 50% completion of cytokinesis. The pre-cytokinesis degradation of calponin 2 was attenuated by MG132 inhibition of the ubiquitin proteasome and inhibitor of protein kinase C (PKC), suggesting that PKC phosphorylation-triggered degradation of calponin 2 could determine the rate of cytokinesis. The novel role of calponin 2 in regulating the rate of cytokinesis may be targeted for therapeutic applications such as in an inhibition of malignant tumor growth.

The muscle-relaxing C-terminal peptide from troponin I populates a nascent helix, facilitating binding to tropomyosin with a potent therapeutic effect.

The conserved C-terminal end segment of troponin I (TnI) plays a critical role in regulating muscle relaxation. This function is retained in the isolated C-terminal 27 amino acid peptide (residues 184-210) of human cardiac TnI (HcTnI-C27): When added to skinned muscle fibers, HcTnI-C27 reduces the Ca2+-sensitivity of activated myofibrils and facilitates relaxation without decreasing the maximum force production. However, the underlying mechanism of HcTnI-C27 function is unknown. We studied the conformational preferences of HcTnI-C27 and a myopathic mutant, Arg192His, (HcTnI-C27-H). Both peptides were mainly disordered in aqueous solution with a nascent helix involving residues from Trp191 to Ile195, as shown by NMR analysis and molecular dynamics simulations. The population of nascent helix was smaller in HcTnI-C27-H than in HcTnI-C27, as shown by circular dichroism (CD) titrations. Fluorescence and isothermal titration calorimetry (ITC) showed that both peptides bound tropomyosin (αTm), with a detectably higher affinity (∼10 µM) of HcTnI-C27 than that of HcTnI-C27-H (∼15 µM), consistent with an impaired Ca2+-desensitization effect of the mutant peptide on skinned muscle strips. Upon binding to αTm, HcTnI-C27 acquired a weakly stable helix-like conformation involving residues near Trp191, as shown by transferred nuclear Overhauser effect spectroscopy and hydrogen/deuterium exchange experiments. With the potent Ca2+-desensitization effect of HcTnI-C27 on skinned cardiac muscle from a mouse model of hypertrophic cardiomyopathy, the data support that the C-terminal end domain of TnI can function as an isolated peptide with the intrinsic capacity of binding tropomyosin, providing a promising therapeutic approach to selectively improve diastolic function of the heart.

Evolution of Flight Muscle Contractility and Energetic Efficiency.

The powered flight of animals requires efficient and sustainable contractions of the wing muscles of various flying species. Despite their high degree of phylogenetic divergence, flight muscles in insects and vertebrates are striated muscles with similarly specialized sarcomeric structure and basic mechanisms of contraction and relaxation. Comparative studies examining flight muscles together with other striated muscles can provide valuable insights into the fundamental mechanisms of muscle contraction and energetic efficiency. Here, we conducted a literature review and data mining to investigate the independent emergence and evolution of flight muscles in insects, birds, and bats, and the likely molecular basis of their contractile features and energetic efficiency. Bird and bat flight muscles have different metabolic rates that reflect differences in energetic efficiencies while having similar contractile machinery that is under the selection of similar natural environments. The significantly lower efficiency of insect flight muscles along with minimized energy expenditure in Ca2+ handling is discussed as a potential mechanism to increase the efficiency of mammalian striated muscles. A better understanding of the molecular evolution of myofilament proteins in the context of physiological functions of invertebrate and vertebrate flight muscles can help explore novel approaches to enhance the performance and efficiency of skeletal and cardiac muscles for the improvement of human health.

High efficiency preparation of skinned mouse cardiac muscle strips from cryosections for contractility studies.

NEW FINDINGS: What is the central question of this study? Can frozen cardiac papillary muscles and cryosectioning be used to reliably obtain uniform cardiac muscle strips with high yields? What is the main finding and its importance? A new method was developed using frozen cardiac papillary muscles and cryosectioning to reliably obtain uniform cardiac muscle strips with high yields. Experimental results demonstrate that this new methodology significantly increases the efficiency and application of quantitative biomechanical studies using skinned muscle fibres with an additional advantage of no need for transferring live animals. ABSTRACT: Skinned cardiac muscle preparations are widely used to study contractile function of myofilament proteins and pathophysiological changes. The current methods applied in these biomechanical studies include detergent permeabilization of freshly isolated papillary muscle, ventricular trabeculae, surgically dissected ventricular muscle strips, mechanically blended cardiac muscle bundles or myocytes, and enzymatically isolated single cardiomyocytes. To facilitate and expand the skinned cardiac muscle approach, we have developed an efficient and readily practical method for mechanical studies of skinned mouse cardiac papillary muscle strips prepared from cryosections. Longitudinal papillary muscle strips of 120-150 µm width cut from 35-70 µm-thick cryosections are mounted to a force transducer and chemically skinned for the studies of force-pCa and sarcomere length-tension relationship and rate of tension redevelopment. In addition to more effective skinning and perfusion than with whole papillary muscle and much higher yield of useful preparations than that from trabeculae, this new methodology has two more major advantages. One is to allow for the use of frozen cardiac muscle in storage to maximize the value of muscle samples, facilitating resource sharing among research institutions without the need of transferring live animals or fresh biopsies. The other is that the integrity of the muscle strips is well preserved during the preparation and mechanical studies, allowing coupled characterization of myofilament proteins. The combined power of biomechanics and protein biochemistry can provide novel insights into integrative physiological and pathophysiological mechanisms of cardiac muscle contraction while the high yield of high-quality muscle strips also provides an efficient platform for development of therapeutic reagents.

Rats genetically selected for low and high aerobic capacity exhibit altered soleus muscle myofilament functions.

Aerobic exercise capacity is critical to bodily health. As a model to investigate the mechanisms that determine health and disease, we employed low (LCR) and high (HCR) capacity running rat models selectively bred to concentrate the genes responsible for divergent aerobic running capacity. To investigate the skeletal muscle contribution to this innate difference in running capacity we employed an approach combining examination of the myofilament protein composition and contractile properties of the fast fiber extensor digitorum longus (EDL) and slow fiber soleus (SOL) muscles from LCR and HCR rats. Intact muscle force experiments demonstrate that SOL, but not EDL, muscles from LCR rats exhibit a three times greater decrease in fatigued force. To investigate the mechanism of this increased fatigability in the LCR SOL muscle, we determined the myofilament protein composition and functional properties. Force-Ca2+ measurements demonstrate decreased Ca2+ sensitivity of single skinned SOL muscle fibers from LCR compared with that of HCR rats. Segregating SOL fibers into fast and slow types demonstrates that the decreased Ca2+ sensitivity in LCR SOL results from a specific decrease in slow-type SOL fiber Ca2+ sensitivity such that it was similar to that of fast-type fibers. These results identify that the altered myofilament contractile properties of LCR SOL slow-type fibers result in a fast muscle type Ca2+ sensitivity and the LCR muscle phenotype. Overall our findings demonstrate alterations of the myofilament proteins could contribute to fatigability of the SOL muscle and the decreased innate aerobic running performance of LCR compared with HCR rats.

The evolutionarily conserved C-terminal peptide of troponin I is an independently configured regulatory structure to function as a myofilament Ca2+-desensitizer.

The C-terminal end segment of troponin subunit I (TnI) is a structure highly conserved among the three muscle type-specific isoforms and across vertebrate species. Partial deletion or point mutation in this segment impairs cardiac muscle relaxation. In the present study, we characterized the C-terminal 27 amino acid peptide of human cardiac TnI (HcTnI-C27) for its role in modulating muscle contractility. Biologically or chemically synthesized HcTnI-C27 peptide retains an epitope structure in physiological solutions similarly to that in intact TnI as recognized by an anti-TnI C-terminus monoclonal antibody (mAb TnI-1). Protein binding studies found that HcTnI-C27 retains the binding affinity for tropomyosin as previously shown with intact cardiac TnI. A restrictive cardiomyopathy mutation R192H in this segment abolishes the bindings to mAb TnI-1 and tropomyosin, demonstrating a pathogenic loss of function. Contractility studies using skinned muscle preparations demonstrated that addition of HcTnI-C27 peptide reduces the Ca2+-sensitivity of myofibrils without decreasing maximum force production. The results indicate that the C-terminal end segment of TnI is a regulatory element of troponin, which retains the native configuration in the form of free peptide to confer an effect on myofilament Ca2+-desensitization. Without negative inotropic impact, this short peptide may be developed into a novel reagent to selectively facilitate cardiac muscle relaxation at the activated state as a potential treatment for heart failure.

Transgenic expression of carbonic anhydrase III in cardiac muscle demonstrates a mechanism to tolerate acidosis.

Carbonic anhydrase III (CAIII) is abundant in liver, adipocytes, and skeletal muscles, but not heart. A cytosolic enzyme that catalyzes conversions between CO2 and HCO3- in the regulation of intracellular pH, its physiological role in myocytes is not fully understood. Mouse skeletal muscles lacking CAIII showed lower intracellular pH during fatigue, suggesting its function in stress tolerance. We created transgenic mice expressing CAIII in cardiomyocytes that lack endogenous CAIII. The transgenic mice showed normal cardiac development and life span under nonstress conditions. Studies of ex vivo working hearts under normal and acidotic conditions demonstrated that the transgenic and wild-type mouse hearts had similar pumping functions under normal pH. At acidotic pH, however, CAIII transgenic mouse hearts showed significantly less decrease in cardiac function than that of wild-type control as shown by higher ventricular pressure development, systolic and diastolic velocities, and stroke volume via elongating the time of diastolic ejection. In addition to the effect of introducing CAIII into cardiomyocytes on maintaining homeostasis to counter acidotic stress, the results demonstrate the role of carbonic anhydrases in maintaining intracellular pH in muscle cells as a potential mechanism to treat heart failure.

Downregulation of calponin 2 contributes to the quiescence of lung macrophages.

Calponin 2 is an actin cytoskeleton-associated regulatory protein that inhibits the activity of myosin-ATPase and cytoskeleton dynamics. Recent studies have demonstrated that deletion of calponin 2 restricts the proinflammatory activation of macrophages in atherosclerosis and arthritis to attenuate the disease progression in mice. Here we demonstrate that the levels of calponin 2 vary among different macrophage populations, which may reflect their adaptation to specific tissue microenvironment corresponding to specific functional states. Interestingly, lung resident macrophages express significantly lower calponin 2 than peritoneal resident macrophages, which correlates with decreased substrate adhesion and reduced expression of proinflammatory cytokines and a proresolution phenotype. Deletion of calponin 2 in peritoneal macrophages also decreased substrate adhesion and downregulated the expression of proinflammatory cytokines. Providing the first line of defense against microbial invasion while receiving constant exposure to extrinsic antigens, lung macrophages need to maintain a necessary level of activity while limiting exaggerated inflammatory reaction. Therefore, their low level of calponin 2 may reflect an important physiological adaption. Downregulation of calponin 2 in macrophages may be targeted as a cytoskeleton-based novel mechanism, possibly via endoplasmic reticulum stress altering the processing and secretion of cytokines, to regulate immune response and promote quiescence for the treatment of inflammatory diseases.

Double deletion of calponin 1 and calponin 2 in mice decreases systemic blood pressure with blunted length-tension response of aortic smooth muscle.

Calponin is a family of actin filament-associated regulatory proteins. Among its three isoforms, calponin 1 is smooth muscle specific and calponin 2 is expressed in smooth muscle and certain non-muscle cells. Previous studies showed that calponin 1 knockout mice had detectable changes in the contractility of urogenital smooth muscle whereas other smooth muscles were less affected. To investigate the possibility that calponins 1 and 2 have overlapping functions in smooth muscle, we examined the effect of double knockout of calponin 1 and calponin 2 genes (Cnn1 and Cnn2) on smooth muscle functions. The results showed for the first time that calponin 1 and calponin 2 double knockout in mice does not cause lethality. The double knockout mice showed decreased systemic blood pressure, decreased force development and blunted length tension response in endothelial-removed aortic rings. A compensatory increase of calponin 1 was found in smooth muscle of Cnn2-/- mice but not vice versa. Cnn1-/- and Cnn2-/- double knockout aortic smooth muscle exhibits faster relaxation than that of wild type control. Double deletion or co-suppression of calponin 1 and calponin 2 in vascular smooth muscle to blunt myogenic response may present a novel approach to develop new treatment for hypertension.

Deletion of calponin 2 attenuates the development of calcific aortic valve disease in ApoE-/- mice.

Calcific aortic valve disease (CAVD) is a leading cause of cardiovascular mortality and lacks non-surgical treatment. The pathogenesis of CAVD involves perturbation of valvular cells by mechanical stimuli, including shear stress, pressure load and leaflet stretch, of which the molecular mechanism requires further elucidation. We recently demonstrated that knockout (KO) of Cnn2 gene that encodes calponin isoform 2, a mechanoregulated cytoskeleton protein, attenuates atherosclerosis in ApoE KO mice. Here we report that Cnn2 KO also decreased calcification of the aortic valve in ApoE KO mice, an established model of CAVD. Although myeloid cell-specific Cnn2 KO highly effectively attenuated vascular atherosclerosis that shares many pathogenic processes with CAVD, it did not reduce aortic valve calcification in ApoE KO mice. Indicating a function in the pathogenesis of CAVD, calponin 2 participates in myofibroblast differentiation that is a leading step in the development of CAVD. The aortic valves of ApoE KO mice exhibited increased expression of calponin 2 and smooth muscle actin (SMA), a hallmark of myofibroblasts. The expression of calponin 2 increased during myofibroblast-like differentiation of primary sheep aortic valve interstitial cells and during the osteogenic differentiation of mouse myofibroblasts. Cnn2 KO attenuated TGFß1-induced differentiation of myofibroblasts in culture as shown by the lower expression of SMA and less calcification than that of wild type (WT) cells. These findings present calponin 2 as a novel molecular target for the treatment and prevention of CAVD.

TNNT1 nemaline myopathy: natural history and therapeutic frontier.

We describe the natural history of 'Amish' nemaline myopathy (ANM), an infantile-onset, lethal disease linked to a pathogenic c.505G>T nonsense mutation of TNNT1, which encodes the slow fiber isoform of troponin T (TNNT1; a.k.a. TnT). The TNNT1 c.505G>T allele has a carrier frequency of 6.5% within Old Order Amish settlements of North America. We collected natural history data for 106 ANM patients born between 1923 and 2017. Over the last two decades, mean age of molecular diagnosis was 16 ± 27 days. TNNT1 c.505G>T homozygotes were normal weight at birth but failed to thrive by age 9 months. Presenting neonatal signs were axial hypotonia, hip and shoulder stiffness, and tremors, followed by progressive muscle weakness, atrophy and contractures. Affected children developed thoracic rigidity, pectus carinatum and restrictive lung disease during infancy, and all succumbed to respiratory failure by 6 years of age (median survival 18 months, range 0.2-66 months). Muscle histology from two affected children showed marked fiber size variation owing to both Type 1 myofiber smallness (hypotrophy) and Type 2 fiber hypertrophy, with evidence of nemaline rods, myofibrillar disarray and vacuolar pathology in both fiber types. The truncated slow TNNT1 (TnT) fragment (p.Glu180Ter) was undetectable in ANM muscle, reflecting its rapid proteolysis and clearance from sarcoplasm. Similar functional and histological phenotypes were observed in other human cohorts and two transgenic murine models (Tnnt1-/- and Tnnt1 c.505G>T). These findings have implications for emerging molecular therapies, including the suitably of TNNT1 gene replacement for newborns with ANM or other TNNT1-associated myopathies.

Aerobic Exercise Preconception and During Pregnancy Enhances Oxidative Capacity in the Hindlimb Muscles of Mice Offspring.

Liu, J, Lee, I, Feng, H-Z, Galen, SS, Hüttemann, PP, Perkins, GA, Jin, J-P, Hüttemann, M, and Malek, MH. Aerobic exercise preconception and during pregnancy enhances oxidative capacity in the hindlimb muscles of mice offspring. J Strength Cond Res 32(5): 1391-1403, 2018-Little is known about the effect of maternal exercise on offspring skeletal muscle health. The purpose of this study, therefore, was to determine whether maternal exercise (preconception and during pregnancy) alters offspring skeletal muscle capillarity and mitochondrial biogenesis. We hypothesized that offspring from exercised dams would have higher capillarity and mitochondrial density in the hindlimb muscles compared with offspring from sedentary dams. Female mice in the exercise condition had access to a running wheel in their individual cage 30 days before mating and throughout pregnancy, whereas the sedentary group did not have access to the running wheel before mating and during pregnancy. Male offspring from both groups were killed when they were 2 months old, and their tissues were analyzed. The results indicated no significant (p > 0.05) mean differences for capillarity density, capillarity-to-fiber ratio, or regulators of angiogenesis such as VEGF-A and TSP-1. Compared with offspring from sedentary dams, however, offspring from exercised dams had an increase in protein expression of myosin heavy chain type I (MHC I) (∼134%; p = 0.009), but no change in MHC II. For mitochondrial morphology, we found significant (all p-values ≤ 0.0124) increases in mitochondrial volume density (∼55%) and length (∼18%) as well as mitochondria per unit area (∼19%). For mitochondrial enzymes, there were also significant (all p-values ≤ 0.0058) increases in basal citrate synthase (∼79%) and cytochrome c oxidase activity (∼67%) in the nonoxidative muscle fibers as well as increases in basal (ATP) (∼52%). Last, there were also significant mean differences in protein expression for regulators (FIS1, Lon protease, and TFAM) of mitochondrial biogenesis. These findings suggest that maternal exercise before and during pregnancy enhances offspring skeletal muscle mitochondria functionality, but not capillarity.

Antibody Epitope Analysis to Investigate Folded Structure, Allosteric Conformation, and Evolutionary Lineage of Proteins.

BACKGROUND: Study of folded structure of proteins provides insights into their biological functions, conformational dynamics and molecular evolution. Current methods of elucidating folded structure of proteins are laborious, low-throughput, and constrained by various limitations. Arising from these methods is the need for a sensitive, quantitative, rapid and high-throughput method not only analysing the folded structure of proteins, but also to monitor dynamic changes under physiological or experimental conditions. OBJECTIVES: In this focused review, we outline the foundation and limitations of current protein structure-determination methods prior to discussing the advantages of an emerging antibody epitope analysis for applications in structural, conformational and evolutionary studies of proteins. METHODS: We discuss the application of this method using representative examples in monitoring allosteric conformation of regulatory proteins and the determination of the evolutionary lineage of related proteins and protein isoforms. RESULTS: The versatility of the method described herein is validated by the ability to modulate a variety of assay parameters to meet the needs of the user in order to monitor protein conformation. Furthermore, the assay has been used to clarify the lineage of troponin isoforms beyond what has been depicted by sequence homology alone, demonstrating the nonlinear evolutionary relationship between primary structure and tertiary structure of proteins. CONCLUSION: The antibody epitope analysis method is a highly adaptable technique of protein conformation elucidation, which can be easily applied without the need for specialized equipment or technical expertise. When applied in a systematic and strategic manner, this method has the potential to reveal novel and biomedically meaningful information for structure-function relationship and evolutionary lineage of proteins.

Carbonic Anhydrase III Is Expressed in Mouse Skeletal Muscles Independent of Fiber Type-Specific Myofilament Protein Isoforms and Plays a Role in Fatigue Resistance.

Carbonic anhydrase III (CAIII) is a metabolic enzyme and a regulator for intracellular pH. CAIII has been reported with high level expression in slow twitch skeletal muscles. Here we demonstrate that CAIII is expressed in multiple slow and fast twitch muscles of adult mouse independent of the expression of myosin isoforms. Expressing similar fast type of myofilament proteins, CAIII-positive tibial anterior (TA) muscle exhibits higher tolerance to fatigue than that of CAIII-negative fast twitch extensor digitorum longus (EDL) muscle in in situ contractility studies. We further studied the muscles of CAIII knockout (Car3-KO) mice. The loss of CAIII in soleus and TA muscles in Car3-KO mice did not change muscle mass, sarcomere protein isoform contents, and the baseline twitch and tetanic contractility as compared with age-matched wild type (WT) controls. On the other hand, Car3-KO TA muscle showed faster force reduction at the beginning but higher resistance at the end during a fatigue test, followed by slower post fatigue recovery than that of WT TA muscle. Superfused Car3-KO soleus muscle also had faster total force reduction during fatigue test than that of WT soleus. However, it showed a less elevation of resting tension followed by a better post fatigue recovery under acidotic stress. CAIII was detected in neonatal TA and EDL muscle, downregulated during development, and then re-expressed in adult TA but not EDL muscles. The expression of CAIII in Tnnt1-KO myopathy mouse soleus muscle that has diminished slow fiber contents due to the loss of slow troponin T remained high. Car3-KO EDL, TA, and soleus muscles showed no change in the expression of mitochondria biomarker proteins. The data suggest a fiber type independent expression of CAIII with a role in the regulation of intracellular pH in skeletal muscle and may be explored as a target for improving fatigue resistance and for the treatment of TNNT1 myopathies.

Protein Structure-Function Relationship at Work: Learning from Myopathy Mutations of the Slow Skeletal Muscle Isoform of Troponin T.

Troponin T (TnT) is the sarcomeric thin filament anchoring subunit of the troponin complex in striated muscles. A nonsense mutation in exon 11 of the slow skeletal muscle isoform of TnT (ssTnT) gene (TNNT1) was found in the Amish populations in Pennsylvania and Ohio. This single nucleotide substitution causes a truncation of the ssTnT protein at Glu180 and the loss of the C-terminal tropomyosin (Tm)-binding site 2. As a consequence, it abolishes the myofilament integration of ssTnT and the loss of function causes an autosomal recessive nemaline myopathy (NM). More TNNT1 mutations have recently been reported in non-Amish ethnic groups with similar recessive NM phenotypes. A nonsense mutation in exon 9 truncates ssTnT at Ser108, deleting Tm-binding site 2 and a part of the middle region Tm-binding site 1. Two splicing site mutations result in truncation of ssTnT at Leu203 or deletion of the exon 14-encoded C-terminal end segment. Another splicing mutation causes an internal deletion of the 39 amino acids encoded by exon 8, partially damaging Tm-binding site 1. The three splicing mutations of TNNT1 all preserve the high affinity Tm-binding site 2 but still present recessive NM phenotypes. The molecular mechanisms for these mutations to cause myopathy provide interesting models to study and understand the structure-function relationship of TnT. This focused review summarizes the current knowledge of TnT isoform regulation, structure-function relationship of TnT and how various ssTnT mutations cause recessive NM, in order to promote in depth studies for further understanding the pathogenesis and pathophysiology of TNNT1 myopathies toward the development of effective treatments.