Methanol gel electrophoresis: Separation of human fast and slow myosin light chain 1 and other myofibrillar protein isoforms on a single gel format.

This report describes a novel sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) resolving gel format that consistently yields the electrophoretic separation of the fast and slow isoforms of human sarcomeric myosin light chain 1 (MLC1). The inclusion of methanol as a constituent of the resolving gel impacted the electrophoretic mobility of proteins across a broad range of molecular masses. There was greater separation of the fast and slow isoforms of human MLC1, as well as separation and high resolution of fast and slow isoforms of the three myosin heavy chain isoforms that are expressed in human skeletal muscle on the same gel format. Furthermore, the same resolving gel format substantially altered the electrophoretic mobility of at least one isoform of tropomyosin in human striated muscle. It is possible that the inclusion of methanol in SDS-PAGE resolving gels could improve the separation of other proteins that are expressed in muscle and in other tissues and cell types.

Lesion level-dependent systemic muscle wasting after spinal cord injury is mediated by glucocorticoid signaling in mice.

An incomplete mechanistic understanding of skeletal muscle wasting early after spinal cord injury (SCI) precludes targeted molecular interventions. Here, we demonstrated systemic wasting that also affected innervated nonparalyzed (supralesional) muscles and emerged within 1 week after experimental SCI in mice. Systemic muscle wasting caused muscle weakness, affected fast type 2 myofibers preferentially, and became exacerbated after high (T3) compared with low (T9) thoracic paraplegia, indicating lesion level-dependent ("neurogenic") mechanisms. The wasting of nonparalyzed muscle and its rapid onset and severity beyond what can be explained by disuse implied unknown systemic drivers. Muscle transcriptome and biochemical analysis revealed a glucocorticoid-mediated catabolic signature early after T3 SCI. SCI-induced systemic muscle wasting was mitigated by (i) endogenous glucocorticoid ablation (adrenalectomy) and (ii) pharmacological glucocorticoid receptor (GR) blockade and was (iii) completely prevented after T3 relative to T9 SCI by genetic muscle-specific GR deletion. These results suggest that neurogenic hypercortisolism contributes to a rapid systemic and functionally relevant muscle wasting syndrome early after paraplegic SCI in mice.

Targeting Troponin C with Small Molecules Containing Diphenyl Moieties: Calcium Sensitivity Effects on Striated Muscles and Structure-Activity Relationship.

Despite large investments from academia and industry, heart failure, which results from a disruption of the contractile apparatus, remains a leading cause of death. Cardiac muscle contraction is a calcium-dependent mechanism, which is regulated by the troponin protein complex (cTn) and specifically by the N-terminal domain of its calcium-binding subunit (cNTnC). There is an increasing need for the development of small molecules that increase calcium sensitivity without altering the systolic calcium concentration, thereby strengthening the cardiac function. Here, we examined the effect of our previously identified calcium-sensitizing small molecule, ChemBridge compound 7930079, in the context of several homologous muscle systems. The effect of this molecule on force generation in isolated cardiac trabeculae and slow skeletal muscle fibers was measured. Furthermore, we explored the use of Gaussian accelerated molecular dynamics in sampling highly predictive receptor conformations based on NMR-derived starting structures. Additionally, we took a rational computational approach for lead optimization based on lipophilic diphenyl moieties. This integrated structural-biochemical-physiological approach led to the identification of three novel low-affinity binders, which had similar binding affinities to the known positive inotrope trifluoperazine. The most potent identified calcium sensitizer was compound 16 with an apparent affinity of 117 ± 17 µM.

Association between skeletal muscle mitochondrial dysfunction and insulin resistance in patients with rheumatoid arthritis: a case-control study.

BACKGROUND: Insulin resistance affects a substantial proportion of patients with rheumatoid arthritis (RA). Skeletal muscle mitochondrial dysfunction results in the accumulation of lipid intermediates that interfere with insulin signaling. We therefore sought to determine if lower oxidative phosphorylation and muscle mitochondrial content are associated with insulin resistance in patients with RA. METHODS: This was a cross-sectional prospective study of RA patients. Matsuda index from the glucose tolerance test was used to estimate insulin sensitivity. Mitochondrial content was measured by citrate synthase (CS) activity in snap-frozen muscle samples. Mitochondrial function was measured by using high-resolution respirometry of permeabilized muscle fibers and electron transport chain complex IV enzyme kinetics in isolated mitochondrial subpopulations. RESULTS: RA participants demonstrated lower insulin sensitivity as measured by the Matsuda index compared to controls [median 3.95 IQR (2.33, 5.64) vs. 7.17 (5.83, 7.75), p = 0.02]. There was lower muscle mitochondrial content among RA vs. controls [median 60 mU/mg IQR (45, 80) vs. 79 mU/mg (65, 97), p = 0.03]. Notably, OxPhos normalized to mitochondrial content was higher among RA vs. controls [mean difference (95% CI) = 0.14 (0.02, 0.26), p = 0.03], indicating a possible compensatory mechanism for lower mitochondrial content or lipid overload. Among RA participants, the activity of muscle CS activity was not correlated with the Matsuda index (ρ = - 0.05, p = 0.84), but it was positively correlated with self-reported (IPAQ) total MET-minutes/week (ρ = 0.44, p = 0.03) and Actigraph-measured time on physical activity (MET rate) (ρ = 0.47, p = 0.03). CONCLUSIONS: Mitochondrial content and function were not associated with insulin sensitivity among participants with RA. However, our study demonstrates a significant association between muscle mitochondrial content and physical activity level, highlighting the potential for future exercise interventions that enhance mitochondrial efficiency in RA patients.

Human and African ape myosin heavy chain content and the evolution of hominin skeletal muscle.

Humans are unique among terrestrial mammals in our manner of walking and running, reflecting 7 to 8 Ma of musculoskeletal evolution since diverging with the genus Pan. One component of this is a shift in our skeletal muscle biology towards a predominance of myosin heavy chain (MyHC) I isoforms (i.e. slow fibers) across our pelvis and lower limbs, which distinguishes us from chimpanzees. Here, new MyHC data from 35 pelvis and hind limb muscles of a Western gorilla (Gorilla gorilla) are presented. These data are combined with a similar chimpanzee dataset to assess the MyHC I content of humans in comparison to African apes (chimpanzees and gorillas) and other terrestrial mammals. The responsiveness of human skeletal muscle to behavioral interventions is also compared to the human-African ape differential. Humans are distinct from African apes and among a small group of terrestrial mammals whose pelvis and lower limb muscle is slow fiber dominant, on average. Behavioral interventions, including immobilization, bed rest, spaceflight and exercise, can induce modest decreases and increases in human MyHC I content (i.e. -9.3% to 2.3%, n = 2033 subjects), but these shifts are much smaller than the mean human-African ape differential (i.e. 31%). Taken together, these results indicate muscle fiber content is likely an evolvable trait under selection in the hominin lineage. As such, we highlight potential targets of selection in the genome (e.g. regions that regulate MyHC content) that may play an important role in hominin skeletal muscle evolution.

Targeting Troponin C with Small Molecules Containing Diphenyl Moieties: Calcium Sensitivity Effects on Striated Muscle and Structure Activity Relationship.

Despite large investments from academia and industry, heart failure, which results from a disruption of the contractile apparatus, remains a leading cause of death. Cardiac muscle contraction is a calcium-dependent mechanism, which is regulated by the troponin protein complex (cTn) and specifically by the N-terminal domain of its calcium binding subunit (cNTnC). There is an increasing need for the development of small molecules that increase calcium sensitivity without altering systolic calcium concentration, thereby strengthening cardiac function. Here, we examined the effect of our previously identified calcium sensitizing small molecule, ChemBridge compound 7930079, in the context of several homologous muscle systems. The effect of this molecule on force generation in isolated cardiac trabeculae and slow skeletal muscle fibers was measured. Furthermore, we explored the use of Gaussian accelerated molecular dynamics in sampling highly predictive receptor conformations based on NMR derived starting structures. Additionally, we took a rational computational approach for lead optimization based on lipophilic diphenyl moieties. This led to the identification of three novel low affinity binders, which had similar binding affinities to known positive inotrope trifluoperazine. The most potent identified calcium sensitizer was compound 16 with an apparent affinity of 117 ± 17 µM .

Small Molecule RPI-194 Stabilizes Activated Troponin to Increase the Calcium Sensitivity of Striated Muscle Contraction.

Small molecule cardiac troponin activators could potentially enhance cardiac muscle contraction in the treatment of systolic heart failure. We designed a small molecule, RPI-194, to bind cardiac/slow skeletal muscle troponin (Cardiac muscle and slow skeletal muscle share a common isoform of the troponin C subunit.) Using solution NMR and stopped flow fluorescence spectroscopy, we determined that RPI-194 binds to cardiac troponin with a dissociation constant KD of 6-24 µM, stabilizing the activated complex between troponin C and the switch region of troponin I. The interaction between RPI-194 and troponin C is weak (KD 311 µM) in the absence of the switch region. RPI-194 acts as a calcium sensitizer, shifting the pCa50 of isometric contraction from 6.28 to 6.99 in mouse slow skeletal muscle fibers and from 5.68 to 5.96 in skinned cardiac trabeculae at 100 µM concentration. There is also some cross-reactivity with fast skeletal muscle fibers (pCa50 increases from 6.27 to 6.52). In the slack test performed on the same skinned skeletal muscle fibers, RPI-194 slowed the velocity of unloaded shortening at saturating calcium concentrations, suggesting that it slows the rate of actin-myosin cross-bridge cycling under these conditions. However, RPI-194 had no effect on the ATPase activity of purified actin-myosin. In isolated unloaded mouse cardiomyocytes, RPI-194 markedly decreased the velocity and amplitude of contractions. In contrast, cardiac function was preserved in mouse isolated perfused working hearts. In summary, the novel troponin activator RPI-194 acts as a calcium sensitizer in all striated muscle types. Surprisingly, it also slows the velocity of unloaded contraction, but the cause and significance of this is uncertain at this time. RPI-194 represents a new class of non-specific troponin activator that could potentially be used either to enhance cardiac muscle contractility in the setting of systolic heart failure or to enhance skeletal muscle contraction in neuromuscular disorders.

Links between muscle phenotype and life history: differentiation of myosin heavy chain composition and muscle biochemistry in precocial and altricial pinniped pups.

In marine mammals, muscular development has been identified as a rate-limiting factor in achieving adult dive capacities. This study investigates the rate that myosin heavy chain (MHC) composition matures in a postural and locomotor skeletal muscle for four pinniped species with different lactation lengths: hooded seals, Cystophora cristata; harp seals, Pagophilus groenlandicus; northern fur seals, Callorhinus ursinus, and Steller sea lions, Eumetopias jubatus. The ontogeny of MHC isoform expression was compared with developmental rates of myoglobin concentrations, and aerobic (citrate synthase, ß-hydroxyacyl-CoA dehydrogenase) and anaerobic (lactate dehydrogenase) enzyme activities. Within taxonomic families, species with shorter lactation periods had more mature muscles biochemically at birth, and fiber types differentiated earlier during ontogeny (Phocidae: hooded > harp seals, Otariidae: northern fur seals > Steller sea lions). Northern fur seal neonates had the most phenotypically-mature muscles in this study, with no immature MHC isoforms. The relationship between muscle biochemistry and MHC composition became more pronounced with age, and developed to reflect swimming mode and activity levels. In adults, phocids had more slow-twitch oxidative protein in their primary locomotor muscle, the Longissimus dorsi (LD), than otariids which likely reflects oxygen-sparing strategies for the phocids' longer dives. Conversely, northern fur seal muscles had higher proportions of fast-twitch MHCs in the Pectoralis and LD, likely indicative of this species' smaller size and higher mass-specific metabolic rates. Thus, muscle phenotype is linked with species life history, and a mismatch between muscle biochemistry and MHC composition at weaning has important implications for the first year of independent foraging in pinniped pups.

Modeling Human Cancer-induced Cachexia.

Cachexia is a wasting syndrome characterized by pronounced skeletal muscle loss. In cancer, cachexia is associated with increased morbidity and mortality and decreased treatment tolerance. Although advances have been made in understanding the mechanisms of cachexia, translating these advances to the clinic has been challenging. One reason for this shortcoming may be the current animal models, which fail to fully recapitulate the etiology of human cancer-induced tissue wasting. Because pancreatic ductal adenocarcinoma (PDA) presents with a high incidence of cachexia, we engineered a mouse model of PDA that we named KPP. KPP mice, similar to PDA patients, progressively lose skeletal and adipose mass as a consequence of their tumors. In addition, KPP muscles exhibit a similar gene ontology as cachectic patients. We envision that the KPP model will be a useful resource for advancing our mechanistic understanding and ability to treat cancer cachexia.

3-Chlorodiphenylamine activates cardiac troponin by a mechanism distinct from bepridil or TFP.

Despite extensive efforts spanning multiple decades, the development of highly effective Ca2+ sensitizers for the heart remains an elusive goal. Existing Ca2+ sensitizers have other targets in addition to cardiac troponin (cTn), which can lead to adverse side effects, such as hypotension or arrhythmias. Thus, there is a need to design Ca2+-sensitizing drugs with higher affinity and selectivity for cTn. Previously, we determined that many compounds based on diphenylamine (DPA) were able to bind to a cTnC-cTnI chimera with moderate affinity (Kd ∼10-120 µM). Of these compounds, 3-chlorodiphenylamine (3-Cl-DPA) bound most tightly (Kd of 10 µM). Here, we investigate 3-Cl-DPA further and find that it increases the Ca2+ sensitivity of force development in skinned cardiac muscle. Using NMR, we show that, like the known Ca2+ sensitizers, trifluoperazine (TFP) and bepridil, 3-Cl-DPA is able to bind to the isolated N-terminal domain (N-domain) of cTnC (Kd of 6 µM). However, while the bulky molecules of TFP and bepridil stabilize the open state of the N-domain of cTnC, the small and flexible 3-Cl-DPA molecule is able to bind without stabilizing this open state. Thus, unlike TFP, which drastically slows the rate of Ca2+ dissociation from the N-domain of isolated cTnC in a dose-dependent manner, 3-Cl-DPA has no effect on the rate of Ca2+ dissociation. On the other hand, the affinity of 3-Cl-DPA for a cTnC-TnI chimera is at least an order of magnitude higher than that of TFP or bepridil, likely because 3-Cl-DPA is less disruptive of cTnI binding to cTnC. Therefore, 3-Cl-DPA has a bigger effect on the rate of Ca2+ dissociation from the entire cTn complex than TFP and bepridil. Our data suggest that 3-Cl-DPA activates the cTn complex via a unique mechanism and could be a suitable scaffold for the development of novel treatments for systolic heart failure.

Current understanding of conventional and novel co-expression patterns of mammalian sarcomeric myosin heavy chains and light chains.

A central tenet of muscle physiology that has accrued from several decades of intense investigations is that myosin, and the vast set of isoforms that constitute its six subunits, is a major regulator of contractile properties of smooth, cardiac and skeletal muscle. Two frequent questions are (1) how many myosin heavy chain (MyHC) isoforms and myosin light chain (MLC) isoforms are expressed in mammalian striated muscles and (2) which isoforms of MyHC and MLC are expressed, at the protein level, with each other - that is, what patterns of co-expression exist in single striated muscle fibers? The answer to the former question is straightforward: eleven MyHC isoforms and nine MLC isoforms, are expressed in a developmentally-regulated and muscle-specific manner. The answer to the latter question, on the other hand, is not clear-cut. The observed number of MyHC and MLC isoform combinations among single fibers is far less than the total number of potential permutations, indicating strict regulation of expression in individual muscle cells. This article provides a review of the current and still evolving understanding of the complexity of muscle fiber types defined on the basis of expression patterns of MyHC and MLC isoforms that constitute an intact functioning molecule.

Desensitizing mouse cardiac troponin C to calcium converts slow muscle towards a fast muscle phenotype.

KEY POINTS: The Ca2+ -desensitizing D73N mutation in slow skeletal/cardiac troponin C caused dilatated cardiomyopathy in mice, but the consequences of this mutation in skeletal muscle were not known. The D73N mutation led to a rightward shift in the force versus pCa (-log [Ca]) relationship in slow-twitch mouse fibres. The D73N mutation led to a rightward shift in the force-stimulation frequency relationship and reduced fatigue resistance of mouse soleus muscle. The D73N mutation led to reduced cross-sectional area of slow-twitch fibres in mouse soleus muscle without affecting fibre type composition of the muscle. The D73N mutation resulted in significantly shorter times to peak force and to relaxation during isometric twitches and tetani in mouse soleus muscle. The D73N mutation led to major changes in physiological properties of mouse soleus muscle, converting slow muscle toward a fast muscle phenotype. ABSTRACT: The missense mutation, D73N, in mouse cardiac troponin C has a profound impact on cardiac function, mediated by a decreased myofilament Ca2+ sensitivity. Mammalian cardiac muscle and slow skeletal muscle normally share expression of the same troponin C isoform. Therefore, the objective of this study was to determine the consequences of the D73N mutation in skeletal muscle, as a potential mechanism that contributes to the morbidity associated with heart failure or other conditions in which Ca2+ sensitivity might be altered. Effects of the D73N mutation on physiological properties of mouse soleus muscle, in which slow-twitch fibres are prevalent, were examined. The mutation resulted in a rightward shift of the force-stimulation frequency relationship, and significantly faster kinetics of isometric twitches and tetani in isolated soleus muscle. Furthermore, soleus muscles from D73N mice underwent a significantly greater reduction in force during a fatigue test. The mutation significantly reduced slow fibre mean cross-sectional area without affecting soleus fibre type composition. The effects of the mutation on Ca2+ sensitivity of force development in soleus skinned slow and fast fibres were also examined. As expected, the D73N mutation did not affect the Ca2+ sensitivity of force development in fast fibres but resulted in substantially decreased Ca2+ sensitivity in slow fibres. The results demonstrate that a point mutation in a single constituent of myofilaments (slow/cardiac troponin C) led to major changes in physiological properties of skeletal muscle and converted slow muscle toward a fast muscle phenotype with reduced fatigue resistance and Ca2+ sensitivity of force generation.

Chimpanzee super strength and human skeletal muscle evolution.

Since at least the 1920s, it has been reported that common chimpanzees (Pan troglodytes) differ from humans in being capable of exceptional feats of "super strength," both in the wild and in captive environments. A mix of anecdotal and more controlled studies provides some support for this view; however, a critical review of available data suggests that chimpanzee mass-specific muscular performance is a more modest 1.5 times greater than humans on average. Hypotheses for the muscular basis of this performance differential have included greater isometric force-generating capabilities, faster maximum shortening velocities, and/or a difference in myosin heavy chain (MHC) isoform content in chimpanzee relative to human skeletal muscle. Here, we show that chimpanzee muscle is similar to human muscle in its single-fiber contractile properties, but exhibits a much higher fraction of MHC II isoforms. Unlike humans, chimpanzee muscle is composed of ∼67% fast-twitch fibers (MHC IIa+IId). Computer simulations of species-specific whole-muscle models indicate that maximum dynamic force and power output is 1.35 times higher in a chimpanzee muscle than a human muscle of similar size. Thus, the superior mass-specific muscular performance of chimpanzees does not stem from differences in isometric force-generating capabilities or maximum shortening velocities-as has long been suggested-but rather is due in part to differences in MHC isoform content and fiber length. We propose that the hominin lineage experienced a decline in maximum dynamic force and power output during the past 7-8 million years in response to selection for repetitive, low-cost contractile behavior.

Increased hypoxia-inducible factor-1α in striated muscle of tumor-bearing mice.

Cancer cachexia is a progressive wasting disease resulting in significant effects on the quality of life and high mortality. Most studies on cancer cachexia have focused on skeletal muscle; however, the heart is now recognized as a major site of cachexia-related effects. To elucidate possible mechanisms, a proteomic study was performed on the left ventricles of colon-26 (C26) adenocarcinoma tumor-bearing mice. The results revealed several changes in proteins involved in metabolism. An integrated pathway analysis of the results revealed a common mediator in hypoxia-inducible factor-1α (HIF-1α). Work by other laboratories has shown that extensive metabolic restructuring in the C26 mouse model causes changes in gene expression that may be affected directly by HIF-1α, such as glucose metabolic genes. M-mode echocardiography showed progressive decline in heart function by day 19, exhibited by significantly decreased ejection fraction and fractional shortening, along with posterior wall thickness. Using Western blot analysis, we confirmed that HIF-1α is significantly upregulated in the heart, whereas there were no changes in its regulatory proteins, prolyl hydroxylase domain-containing protein 2 (PHD2) and von Hippel-Lindau protein (VHL). PHD2 requires both oxygen and iron as cofactors for the hydroxylation of HIF-1α, marking it for ubiquination via VHL and subsequent destruction by the proteasome complex. We examined venous blood gas values in the tumor-bearing mice and found significantly lower oxygen concentration compared with control animals in the third week after tumor inoculation. We also examined select skeletal muscles to determine whether they are similarly affected. In the diaphragm, extensor digitorum longus, and soleus, we found significantly increased HIF-1α in tumor-bearing mice, indicating a hypoxic response, not only in the heart, but also in skeletal muscle. These results indicate that HIF-1α may contribute, in part, to the metabolic changes that occur during cancer cachexia.NEW & NOTEWORTHY We used proteomics and metadata analysis software to identify contributors to metabolic changes in striated muscle during cancer cachexia. We found increased expression of hypoxia-inducible factor-1α in the heart and skeletal muscle, suggesting a potential target for the therapeutic treatment of cancer cachexia.

Functional Segregation within the Muscles of Aquatic Propulsion in the Asiatic Water Monitor (Varanus salvator).

Water monitor lizards (Varanus salvator) swim using sinusoidal oscillations generated at the base of their long (50% of total body length) tail. In an effort to determine which level of the structural/organizational hierarchy of muscle is associated with functional segregation between the muscles of the tail base, an array of muscle features-myosin heavy chain profiles, enzymatic fiber types, twitch and tetanic force production, rates of fatigue, muscle compliance, and electrical activity patterns-were quantitated. The two examined axial muscles, longissimus, and iliocaudalis, were generally similar at the molecular, biochemical, and physiological levels, but differed at the biomechanics level and in their activation pattern. The appendicular muscle examined, caudofemoralis, differed from the axial muscles particularly at the molecular and physiological levels, and it exhibited a unique compliance profile and pattern of electrical activation. There were some apparent contradictions between the different structural/organizational levels examined. These contradictions, coupled with a unique myosin heavy chain profile, lead to the hypothesis that there are previously un-described molecular/biochemical specializations within varanid skeletal muscles.

Ibuprofen ameliorates fatigue- and depressive-like behavior in tumor-bearing mice.

AIMS: Cancer-related fatigue (CRF) is often accompanied by depressed mood, both of which reduce functional status and quality of life. Research suggests that increased expression of pro-inflammatory cytokines is associated with skeletal muscle wasting and depressive- and fatigue-like behaviors in rodents and cancer patients. We have previously shown that treatment with ibuprofen, a nonsteroidal anti-inflammatory drug, preserved muscle mass in tumor-bearing mice. Therefore, the purpose of the present study was to determine the behavioral effects of ibuprofen in a mouse model of CRF. MAIN METHODS: Mice were injected with colon-26 adenocarcinoma cells and treated with ibuprofen (10mg/kg) in the drinking water. Depressive-like behavior was determined using the forced swim test (FST). Fatigue-like behaviors were determined using voluntary wheel running activity (VWRA) and grip strength. The hippocampus, gastrocnemius muscle, and serum were collected for cytokine analysis. KEY FINDINGS: Tumor-bearing mice showed depressive-like behavior in the FST, which was not observed in mice treated with ibuprofen. VWRA and grip strength declined in tumor-bearing mice, and ibuprofen attenuated this decline. Tumor-bearing mice had decreased gastrocnemius muscle mass and increased expression of IL-6, MAFBx and MuRF mRNA, biomarkers of protein degradation, in the muscle. Expression of IL-1ß and IL-6 was also increased in the hippocampus. Treatment with ibuprofen improved muscle mass and reduced cytokine expression in both the muscle and hippocampus of tumor-bearing mice. SIGNIFICANCE: Ibuprofen treatment reduced skeletal muscle wasting, inflammation in the brain, and fatigue- and depressive-like behavior in tumor-bearing mice. Therefore, ibuprofen warrants evaluation as an adjuvant treatment for CRF.

Knock-in mice harboring a Ca(2+) desensitizing mutation in cardiac troponin C develop early onset dilated cardiomyopathy.

The physiological consequences of aberrant Ca(2+) binding and exchange with cardiac myofilaments are not clearly understood. In order to examine the effect of decreasing Ca(2+) sensitivity of cTnC on cardiac function, we generated knock-in mice carrying a D73N mutation (not known to be associated with heart disease in human patients) in cTnC. The D73N mutation was engineered into the regulatory N-domain of cTnC in order to reduce Ca(2+) sensitivity of reconstituted thin filaments by increasing the rate of Ca(2+) dissociation. In addition, the D73N mutation drastically blunted the extent of Ca(2+) desensitization of reconstituted thin filaments induced by cTnI pseudo-phosphorylation. Compared to wild-type mice, heterozygous knock-in mice carrying the D73N mutation exhibited a substantially decreased Ca(2+) sensitivity of force development in skinned ventricular trabeculae. Kaplan-Meier survival analysis revealed that median survival time for knock-in mice was 12 weeks. Echocardiographic analysis revealed that knock-in mice exhibited increased left ventricular dimensions with thinner walls. Echocardiographic analysis also revealed that measures of systolic function, such as ejection fraction (EF) and fractional shortening (FS), were dramatically reduced in knock-in mice. In addition, knock-in mice displayed electrophysiological abnormalities, namely prolonged QRS and QT intervals. Furthermore, ventricular myocytes isolated from knock-in mice did not respond to ß-adrenergic stimulation. Thus, knock-in mice developed pathological features similar to those observed in human patients with dilated cardiomyopathy (DCM). In conclusion, our results suggest that decreasing Ca(2+) sensitivity of the regulatory N-domain of cTnC is sufficient to trigger the development of DCM.

Nucleotide and protein sequences for dog masticatory tropomyosin identify a novel Tpm4 gene product.

Jaw-closing muscles of several vertebrate species, including members of Carnivora, express a unique, "masticatory", isoform of myosin heavy chain, along with isoforms of other myofibrillar proteins that are not expressed in most other muscles. It is generally believed that the complement of myofibrillar isoforms in these muscles serves high force generation for capturing live prey, breaking down tough plant material and defensive biting. A unique isoform of tropomyosin (Tpm) was reported to be expressed in cat jaw-closing muscle, based upon two-dimensional gel mobility, peptide mapping, and immunohistochemistry. The objective of this study was to obtain protein and gene sequence information for this unique Tpm isoform. Samples of masseter (a jaw-closing muscle), tibialis (predominantly fast-twitch fibers), and the deep lateral gastrocnemius (predominantly slow-twitch fibers) were obtained from adult dogs. Expressed Tpm isoforms were cloned and sequencing yielded cDNAs that were identical to genomic predicted striated muscle Tpm1.1St(a,b,b,a) (historically referred to as αTpm), Tpm2.2St(a,b,b,a) (ßTpm) and Tpm3.12St(a,b,b,a) (γTpm) isoforms (nomenclature reflects predominant tissue expression ("St"-striated muscle) and exon splicing pattern), as well as a novel 284 amino acid isoform observed in jaw-closing muscle that is identical to a genomic predicted product of the Tpm4 gene (δTpm) family. The novel isoform is designated as Tpm4.3St(a,b,b,a). The myofibrillar Tpm isoform expressed in dog masseter exhibits a unique electrophoretic mobility on gels containing 6 M urea, compared to other skeletal Tpm isoforms. To validate that the cloned Tpm4.3 isoform is the Tpm expressed in dog masseter, E. coli-expressed Tpm4.3 was electrophoresed in the presence of urea. Results demonstrate that Tpm4.3 has identical electrophoretic mobility to the unique dog masseter Tpm isoform and is of different mobility from that of muscle Tpm1.1, Tpm2.2 and Tpm3.12 isoforms. We conclude that the unique Tpm isoform in dog masseter is a product of the Tpm4 gene and that the 284 amino acid protein product of this gene represents a novel myofibrillar Tpm isoform never before observed to be expressed in striated muscle.