Genetically Encoded Biosensors Reveal PKA Hyperphosphorylation on the Myofilaments in Rabbit Heart Failure.

RATIONALE: In heart failure, myofilament proteins display abnormal phosphorylation, which contributes to contractile dysfunction. The mechanisms underlying the dysregulation of protein phosphorylation on myofilaments is not clear. OBJECTIVE: This study aims to understand the mechanisms underlying altered phosphorylation of myofilament proteins in heart failure. METHODS AND RESULTS: We generate a novel genetically encoded protein kinase A (PKA) biosensor anchored onto the myofilaments in rabbit cardiac myocytes to examine PKA activity at the myofilaments in responses to adrenergic stimulation. We show that PKA activity is shifted from the sarcolemma to the myofilaments in hypertrophic failing rabbit myocytes. In particular, the increased PKA activity on the myofilaments is because of an enhanced ß2 adrenergic receptor signal selectively directed to the myofilaments together with a reduced phosphodiesterase activity associated with the myofibrils. Mechanistically, the enhanced PKA activity on the myofilaments is associated with downregulation of caveolin-3 in the hypertrophic failing rabbit myocytes. Reintroduction of caveolin-3 in the failing myocytes is able to normalize the distribution of ß2 adrenergic receptor signal by preventing PKA signal access to the myofilaments and to restore contractile response to adrenergic stimulation. CONCLUSIONS: In hypertrophic rabbit myocytes, selectively enhanced ß2 adrenergic receptor signaling toward the myofilaments contributes to elevated PKA activity and PKA phosphorylation of myofilament proteins. Reintroduction of caveolin-3 is able to confine ß2 adrenergic receptor signaling and restore myocyte contractility in response to ß adrenergic stimulation.

A novel method for monitoring troponin T fragment from rabbit skeletal muscle during aging using quartz crystal microbalance.

BACKGROUND: Troponin T (TnT) is degraded during aging of meat. The proteolytic fragment of TnT, especially the 30 kDa fragment, is used as one of indices for estimating aging of meat. We have tried to use quartz crystal microbalance (QCM), which is widely used to analyze interaction among macromolecules, to detect proteolytic fragments of TnT during aging of meat. RESULT: The frequency of the QCM sensor with immobilized anti-TnT antibody in high-salt solution extracts of both myofibrils and whole meat decreased with time of aging. The staining intensity of the bands, including a 30 kDa fragment bound to anti-TnT antibody, also increased with time of aging in western blotting. These results confirm that TnT is degraded during aging and released from thin filaments, and QCM analysis is sufficiently sensitive to detect the TnT fragments. CONCLUSION: The QCM analysis of muscle and myofibrillar extracts using anti-TnT antibody-immobilized sensor can be used as a convenient tool for monitoring the extent of aging of meat. © 2015 Society of Chemical Industry.

Skeletal muscle mitochondria exhibit decreased pyruvate oxidation capacity and increased ROS emission during surgery-induced acute insulin resistance.

Development of acute insulin resistance represents a negative factor after surgery, but the underlying mechanisms are not fully understood. We investigated the postoperative changes in insulin sensitivity, mitochondrial function, enzyme activities, and release of reactive oxygen species (ROS) in skeletal muscle and liver in pigs on the 2nd postoperative day after major abdominal surgery. Peripheral and hepatic insulin sensitivity were assessed by D-[6,6-²H2]glucose infusion and hyperinsulinemic euglycemic step clamping. Surgical trauma elicited a decline in peripheral insulin sensitivity (∼34%, P<0.01), whereas hepatic insulin sensitivity remained unchanged. Intramyofibrillar (IFM) and subsarcolemma mitochondria (SSM) isolated from skeletal muscle showed a postoperative decline in ADP-stimulated respiration (V(ADP)) for pyruvate (∼61%, P<0.05, and ∼40%, P<0.001, respectively), whereas V(ADP) for glutamate and palmitoyl-L-carnitine (PC) was unchanged. Mitochondrial leak respiration with PC was increased in SSM (1.9-fold, P<0.05) and IFM (2.5-fold, P<0.05), indicating FFA-induced uncoupling. The activity of the pyruvate dehydrogenase complex (PDC) was reduced (∼32%, P<0.01) and positively correlated to the decline in peripheral insulin sensitivity (r=0.748, P<0.05). All other mitochondrial enzyme activities were unchanged. No changes in mitochondrial function in liver were observed. Mitochondrial H2O2 and O2·â» emission was measured spectrofluorometrically, and H2O2 was increased in SSM, IFM, and liver mitochondria (∼2.3-, ∼2.5-, and ∼2.3-fold, respectively, all P<0.05). We conclude that an impairment in skeletal muscle mitochondrial PDC activity and pyruvate oxidation capacity arises in the postoperative phase along with increased ROS emission, suggesting a link between mitochondrial function and development of acute postoperative insulin resistance.

Mitochondrial respiratory capacity and coupling control decline with age in human skeletal muscle.

Mitochondrial health is critical to physiological function, particularly in tissues with high ATP turnover, such as striated muscle. It has been postulated that derangements in skeletal muscle mitochondrial function contribute to impaired physical function in older adults. Here, we determined mitochondrial respiratory capacity and coupling control in skeletal muscle biopsies obtained from young and older adults. Twenty-four young (28 ± 7 yr) and thirty-one older (62 ± 8 yr) adults were studied. Mitochondrial respiration was determined in permeabilized myofibers from the vastus lateralis after the addition of substrates oligomycin and CCCP. Thereafter, mitochondrial coupling control was calculated. Maximal coupled respiration (respiration linked to ATP production) was lower in muscle from older vs. young subjects (P < 0.01), as was maximal uncoupled respiration (P = 0.06). Coupling control in response to the ATP synthase inhibitor oligomycin was lower in older adults (P < 0.05), as was the mitochondria flux control ratio, coupled respiration normalized to maximal uncoupled respiration (P < 0.05). Calculation of respiratory function revealed lower respiration linked to ATP production (P < 0.001) and greater reserve respiration (P < 0.01); i.e., respiratory capacity not used for phosphorylation in muscle from older adults. We conclude that skeletal muscle mitochondrial respiratory capacity and coupling control decline with age. Lower respiratory capacity and coupling efficiency result in a reduced capacity for ATP production in skeletal muscle of older adults.

The kinase domains of obscurin interact with intercellular adhesion proteins.

Obscurins comprise a family of giant (~870- to 600-kDa) and small (~250- to 55-kDa) proteins that play important roles in myofibrillogenesis, cytoskeletal organization, and cell adhesion and are implicated in hypertrophic cardiomyopathy and tumorigenesis. Giant obscurins are composed of tandem structural and signaling motifs, including 2 serine/threonine kinase domains, SK1 and SK2, present at the COOH terminus of giant obscurin-B. Using biochemical and cellular approaches, we show for the first time that both SK1 and SK2 possess enzymatic activities and undergo autophosphorylation. SK2 can phosphorylate the cytoplasmic domain of N-cadherin, a major component of adherens junctions, and SK1 can interact with the extracellular domain of the ß1-subunit of the Na(+)/K(+)-ATPase, which also resides in adherens junctions. Immunostaining of nonpermeabilized myofibers and cardiocytes revealed that some obscurin kinase isoforms localize extracellularly. Quantification of the exofacial expression of obscurin kinase proteins indicated that they occupy ~16 and ~5% of the sarcolemmal surface in myofibers and cardiocytes, respectively. Treatment of heart lysates with peptide-N-glycosidase F revealed that while giant obscurin-B localizes intracellularly, possessing dual kinase activity, a small obscurin kinase isoform that contains SK1 localizes extracellularly, where it undergoes N-glycosylation. Collectively, our studies demonstrate that the obscurin kinase domains are enzymatically active and may be involved in the regulation of cell adhesion.

Engineering of protein glutaminase for highly efficient modification of fish myofibrillar protein through structure-based and computational-aided strategy.

Protein glutaminase (PG; EC 3.5.1.44) is a class of food-grade enzyme with the potential to significantly improve protein functionality. However, its low catalytic activity and stability greatly hindered industrial application. In this study, we employed structural-based engineering and computational-aided design strategies to target the engineering of protein glutaminase PG5, which led to the development of a combinatorial mutant, MT8, exhibiting a specific activity of 31.1 U/mg and a half-life of 216.2 min at 55 °C. The results indicated that the flexible region in MT8 shifted from the C-terminus to the N-terminus, with increased N-terminal flexibility positively correlating with its catalytic activity. Additionally, MT8 notably boosted fish myofibrillar proteins (MPs) solubility under the absence of NaCl conditions and enhanced their foaming and emulsifying properties. Key residues like Asp31, Ser72, Asn121, Asp471, and Glu485 were crucial for maintaining PG5-myosin interaction, with Ser72 and Asn121 making significant energy contributions.

Histone demethylase KDM5 regulates cardiomyocyte maturation by promoting fatty acid oxidation, oxidative phosphorylation, and myofibrillar organization.

AIMS: Human pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) provide a platform to identify and characterize factors that regulate the maturation of CMs. The transition from an immature foetal to an adult CM state entails coordinated regulation of the expression of genes involved in myofibril formation and oxidative phosphorylation (OXPHOS) among others. Lysine demethylase 5 (KDM5) specifically demethylates H3K4me1/2/3 and has emerged as potential regulators of expression of genes involved in cardiac development and mitochondrial function. The purpose of this study is to determine the role of KDM5 in iPSC-CM maturation. METHODS AND RESULTS: KDM5A, B, and C proteins were mainly expressed in the early post-natal stages, and their expressions were progressively downregulated in the post-natal CMs and were absent in adult hearts and CMs. In contrast, KDM5 proteins were persistently expressed in the iPSC-CMs up to 60 days after the induction of myogenic differentiation, consistent with the immaturity of these cells. Inhibition of KDM5 by KDM5-C70 -a pan-KDM5 inhibitor, induced differential expression of 2372 genes, including upregulation of genes involved in fatty acid oxidation (FAO), OXPHOS, and myogenesis in the iPSC-CMs. Likewise, genome-wide profiling of H3K4me3 binding sites by the cleavage under targets and release using nuclease assay showed enriched of the H3K4me3 peaks at the promoter regions of genes encoding FAO, OXPHOS, and sarcomere proteins. Consistent with the chromatin and gene expression data, KDM5 inhibition increased the expression of multiple sarcomere proteins and enhanced myofibrillar organization. Furthermore, inhibition of KDM5 increased H3K4me3 deposits at the promoter region of the ESRRA gene and increased its RNA and protein levels. Knockdown of ESRRA in KDM5-C70-treated iPSC-CM suppressed expression of a subset of the KDM5 targets. In conjunction with changes in gene expression, KDM5 inhibition increased oxygen consumption rate and contractility in iPSC-CMs. CONCLUSION: KDM5 inhibition enhances maturation of iPSC-CMs by epigenetically upregulating the expressions of OXPHOS, FAO, and sarcomere genes and enhancing myofibril organization and mitochondrial function.

Myofilament Ca2+ desensitization mediates positive lusitropic effect of neuronal nitric oxide synthase in left ventricular myocytes from murine hypertensive heart.

Neuronal nitric oxide synthase (NOS1 or nNOS) exerts negative inotropic and positive lusitropic effects through Ca(2+) handling processes in cardiac myocytes from healthy hearts. However, underlying mechanisms of NOS1 in diseased hearts remain unclear. The present study aims to investigate this question in angiotensin II (Ang II)-induced hypertensive rat hearts (HP). Our results showed that the systolic function of left ventricle (LV) was reduced and diastolic function was unaltered (echocardiographic assessment) in HP compared to those in shams. In isolated LV myocytes, contraction was unchanged but peak [Ca(2+)]i transient was increased in HP. Concomitantly, relaxation and time constant of [Ca(2+)]i decay (tau) were faster and the phosphorylated fraction of phospholamban (PLN-Ser(16)/PLN) was greater. NOS1 protein expression and activity were increased in LV myocyte homogenates from HP. Surprisingly, inhibition of NOS1 did not affect contraction but reduced peak [Ca(2+)]i transient; prevented faster relaxation without affecting the tau of [Ca(2+)]i transient or PLN-Ser(16)/PLN in HP, suggesting myofilament Ca(2+) desensitization by NOS1. Indeed, relaxation phase of the sarcomere length-[Ca(2+)]i relationship of LV myocytes shifted to the right and increased [Ca(2+)]i for 50% of sarcomere shortening (EC50) in HP. Phosphorylations of cardiac myosin binding protein-C (cMyBP-C(282) and cMyBP-C(273)) were increased and cardiac troponin I (cTnI(23/24)) was reduced in HP. Importantly, NOS1 or PKG inhibition reduced cMyBP-C(273) and cTnI(23/24) and reversed myofilament Ca(2+) sensitivity. These results reveal that NOS1 is up-regulated in LV myocytes from HP and exerts positive lusitropic effect by modulating myofilament Ca(2+) sensitivity through phosphorylation of key regulators in sarcomere.

Tetrahydrobiopterin improves diastolic dysfunction by reversing changes in myofilament properties.

Despite the increasing prevalence of heart failure with preserved left ventricular function, there are no specific treatments, partially because the mechanism of impaired relaxation is incompletely understood. Evidence indicates that cardiac relaxation may depend on nitric oxide (NO), generated by NO synthase (NOS) requiring the co-factor tetrahydrobiopterin (BH(4)). Recently, we reported that hypertension-induced diastolic dysfunction was accompanied by cardiac BH(4) depletion, NOS uncoupling, a depression in myofilament cross-bridge kinetics, and S-glutathionylation of myosin binding protein C (MyBP-C). We hypothesized that the mechanism by which BH(4) ameliorates diastolic dysfunction is by preventing glutathionylation of MyBP-C and thus reversing changes of myofilament properties that occur during diastolic dysfunction. We used the deoxycorticosterone acetate (DOCA)-salt mouse model, which demonstrates mild hypertension, myocardial oxidative stress, and diastolic dysfunction. Mice were divided into two groups that received control diet and two groups that received BH(4) supplement for 7days after developing diastolic dysfunction at post-operative day 11. Mice were assessed by echocardiography. Left ventricular papillary detergent-extracted fiber bundles were isolated for simultaneous determination of force and ATPase activity. Sarcomeric protein glutathionylation was assessed by immunoblotting. DOCA-salt mice exhibited diastolic dysfunction that was reversed after BH(4) treatment. Diastolic sarcomere length (DOCA-salt 1.70±0.01 vs. DOCA-salt+BH(4) 1.77±0.01µm, P<0.001) and relengthening (relaxation constant, τ, DOCA-salt 0.28±0.02 vs. DOCA-salt+BH(4) 0.08±0.01, P<0.001) were also restored to control by BH(4) treatment. pCa(50) for tension increased in DOCA-salt compared to sham but reverted to sham levels after BH(4) treatment. Maximum ATPase rate and tension cost (ΔATPase/ΔTension) decreased in DOCA-salt compared to sham, but increased after BH(4) treatment. Cardiac MyBP-C glutathionylation increased in DOCA-salt compared to sham, but decreased with BH(4) treatment. MyBP-C glutathionylation correlated with the presence of diastolic dysfunction. Our results suggest that by depressing S-glutathionylation of MyBP-C, BH(4) ameliorates diastolic dysfunction by reversing a decrease in cross-bridge turnover kinetics. These data provide evidence for modulation of cardiac relaxation by post-translational modification of myofilament proteins.

Myofibrillar distribution of succinate dehydrogenase activity and lipid stores differs in skeletal muscle tissue of paraplegic subjects.

Lack of physical activity has been related to an increased risk of developing insulin resistance. This study aimed to assess the impact of chronic muscle deconditioning on whole body insulin sensitivity, muscle oxidative capacity, and intramyocellular lipid (IMCL) content in subjects with paraplegia. Nine subjects with paraplegia and nine able-bodied, lean controls were recruited. An oral glucose tolerance test was performed to assess whole body insulin sensitivity. IMCL content was determined both in vivo and in vitro using (1)H-magnetic resonance spectroscopy and fluorescence microscopy, respectively. Muscle biopsy samples were stained for succinate dehydrogenase (SDH) activity to measure muscle fiber oxidative capacity. Subcellular distributions of IMCL and SDH activity were determined by defining subsarcolemmal and intermyofibrillar areas on histological samples. SDH activity was 57 ± 14% lower in muscle fibers derived from subjects with paraplegia when compared with controls (P < 0.05), but IMCL content and whole body insulin sensitivity did not differ between groups. In muscle fibers taken from controls, both SDH activity and IMCL content were higher in the subsarcolemmal region than in the intermyofibrillar area. This typical subcellular SDH and IMCL distribution pattern was lost in muscle fibers collected from subjects with paraplegia and had changed toward a more uniform distribution. In conclusion, the lower metabolic demand in deconditioned muscle of subjects with paraplegia results in a significant decline in muscle fiber oxidative capacity and is accompanied by changes in the subcellular distribution patterns of SDH activity and IMCL. However, loss of muscle activity due to paraplegia is not associated with substantial lipid accumulation in skeletal muscle tissue.

Protein kinase D increases maximal Ca2+-activated tension of cardiomyocyte contraction by phosphorylation of cMyBP-C-Ser315.

Cardiac myosin-binding protein C (cMyBP-C) is involved in the regulation of cardiac myofilament contraction. Recent evidence showed that protein kinase D (PKD) is one of the kinases that phosphorylate cMyBP-C. However, the mechanism by which PKD-induced cMyBP-C phosphorylation affects cardiac contractile responses is not known. Using immunoprecipitation, we showed that, in contracting cardiomyocytes, PKD binds to cMyBP-C and phosphorylates it at Ser(315). The effect of PKD-mediated phosphorylation of cMyBP-C on cardiac myofilament function was investigated in permeabilized ventricular myocytes, isolated from wild-type (WT) and from cMyBP-C knockout (KO) mice, incubated in the presence of full-length active PKD. In WT myocytes, PKD increased both myofilament Ca(2+) sensitivity (pCa(50)) and maximal Ca(2+)-activated tension of contraction (T(max)). In cMyBP-C KO skinned myocytes, PKD increased pCa(50) but did not alter T(max). This suggests that cMyBP-C is not involved in PKD-mediated sensitization of myofilaments to Ca(2+) but is essential for PKD-induced increase in T(max). Furthermore, the phosphorylation of both PKD-Ser(916) and cMyBP-C-Ser(315) was contraction frequency-dependent, suggesting that PKD-mediated cMyBP-C phosphorylation is operational primarily during periods of increased contractile activity. Thus, during high contraction frequency, PKD facilitates contraction of cardiomyocytes by increasing Ca(2+) sensitivity and by an increased T(max) through phosphorylation of cMyBP-C.

Propofol attenuates angiotensin II-induced vasoconstriction by inhibiting Ca2+-dependent and PKC-mediated Ca 2+ sensitization mechanisms.

PURPOSE: Angiotensin II (Ang II)-induced vascular contraction is mediated by Ca(2+)-dependent mechanisms and Ca(2+) sensitization mechanisms. The phosphorylation of protein kinase C (PKC) regulates myofilament Ca(2+) sensitivity. We have previously demonstrated that sevoflurane inhibits Ang II-induced vasoconstriction by inhibiting PKC phosphorylation, whereas isoflurane inhibits Ang II-induced vasoconstriction by decreasing intracellular Ca(2+) concentration ([Ca(2+)](i)) in vascular smooth muscle. Propofol also induces vasodilation; however, the effect of propofol on PKC-mediated myofilament Ca(2+) sensitivity is poorly understood. The aim of this study is to determine the mechanisms by which propofol inhibits Ang II-induced vascular contraction in rat aortic smooth muscle. METHODS: An isometric force transducer was used to investigate the effect of propofol on vasoconstriction, a fluorometer was used to investigate the change in [Ca(2+)](i), and Western blot testing was used to analyze Ang II-induced PKC phosphorylation. RESULTS: Ang II (10(-7) M) elicited a transient contraction of rat aortic smooth muscle, which was associated with an elevation of [Ca(2+)](i). Propofol (10(-6 )M) inhibited Ang II-induced vascular contraction (P < 0.01) and increase in [Ca(2+)](i) (P < 0.05) in rat aortic smooth muscle. Ang II also induced a rapid increase in [Ca(2+)](i) in cultured vascular smooth muscle cells, which was suppressed by propofol (P < 0.05). Propofol (10(-6) M) attenuated Ang II-stimulated PKC phosphorylation (P < 0.05). CONCLUSION: These results suggest that the inhibitory effect of propofol on Ang II-induced vascular contraction is mediated by the attenuation of a Ca(2+)-dependent pathway and Ca(2+) sensitivity through the PKC signaling pathway.

A novel protein found in the I bands of myofibrils is produced by alternative splicing of the DLST gene.

BACKGROUND: It is not known if the dihydrolipoamide succinyltransferase (DLST) gene, a mitochondrial protein, undergoes alternative splicing. We identified an uncharacterized protein reacting with an anti-DLST antibody in the I bands of myofibrils in rat skeletal muscle. METHODS: Immunocytochemical staining with an anti-DLST antibody, the purification and amino acid sequence analysis of the protein, and the isolation and sequencing of the protein's cDNA were carried out to clarify the properties of the protein and its relationship to the DLST gene. RESULTS: A pyrophosphate concentration >10 mM was necessary to extract the protein from myofibrils in the presence of salt with a higher concentration than 0.6 M, at an alkaline pH of 7.5-8.0. The protein corresponded to the amino acid sequence of the C-terminal side of DLST. The cDNAs for this protein were splicing variants of the DLST gene, with deletions of both exons 2 and 3, or only exon 2 or 3. These variants possessed an open reading frame from an initiation codon in exon 8 of the DLST gene to a termination codon in exon 15, generating a protein with a molecular weight of 30 kDa. CONCLUSIONS: The DLST gene undergoes alternative splicing, generating the protein isolated from the I bands of myofibrils. GENERAL SIGNIFICANCE: The DLST gene produces two different proteins with quite different functions via alternative splicing.

The ubiquitin ligase Ozz decreases the replacement rate of embryonic myosin in myofibrils.

Myosin, the most abundant myofibrillar protein in skeletal muscle, functions as a motor protein in muscle contraction. Myosin polymerizes into the thick filaments in the sarcomere where approximately 50% of embryonic myosin (Myh3) are replaced within 3 h (Ojima K, Ichimura E, Yasukawa Y, Wakamatsu J, Nishimura T, Am J Physiol Cell Physiol 309: C669-C679, 2015). The sarcomere structure including the thick filament is maintained by a balance between protein biosynthesis and degradation. However, the involvement of a protein degradation system in the myosin replacement process remains unclear. Here, we show that the muscle-specific ubiquitin ligase Ozz regulates replacement rate of Myh3. To examine the direct effect of Ozz on myosin replacement, eGFP-Myh3 replacement rate was measured in myotubes overexpressing Ozz by fluorescence recovery after photobleaching. Ozz overexpression significantly decreased the replacement rate of eGFP-Myh3 in the myofibrils, whereas it had no effect on other myosin isoforms. It is likely that ectopic Ozz promoted myosin degradation through increment of ubiquitinated myosin, and decreased myosin supply for replacement, thereby reducing myosin replacement rate. Intriguingly, treatment with a proteasome inhibitor MG132 also decreased myosin replacement rate, although MG132 enhanced the accumulation of ubiquitinated myosin in the cytosol where replaceable myosin is pooled, suggesting that ubiquitinated myosin is not replaced by myosin in the myofibril. Collectively, our findings showed that Myh3 replacement rate was reduced in the presence of overexpressed Ozz probably through enhanced ubiquitination and degradation of Myh3 by Ozz.

Inhibition of prostaglandin-degrading enzyme 15-PGDH rejuvenates aged muscle mass and strength.

Treatments are lacking for sarcopenia, a debilitating age-related skeletal muscle wasting syndrome. We identifed increased amounts of 15-hydroxyprostaglandin dehydrogenase (15-PGDH), the prostaglandin E2 (PGE2)-degrading enzyme, as a hallmark of aged tissues, including skeletal muscle. The consequent reduction in PGE2 signaling contributed to muscle atrophy in aged mice and results from 15-PGDH-expressing myofibers and interstitial cells, such as macrophages, within muscle. Overexpression of 15-PGDH in young muscles induced atrophy. Inhibition of 15-PGDH, by targeted genetic depletion or a small-molecule inhibitor, increased aged muscle mass, strength, and exercise performance. These benefits arise from a physiological increase in PGE2 concentrations, which augmented mitochondrial function and autophagy and decreased transforming growth factor-ß signaling and activity of ubiquitin-proteasome pathways. Thus, PGE2 signaling ameliorates muscle atrophy and rejuvenates muscle function, and 15-PGDH may be a suitable therapeutic target for countering sarcopenia.

The scaling of myofibrillar actomyosin ATPase activity in apid bee flight muscle in relation to hovering flight energetics.

For all types of locomotion, the overall efficiency with which chemical energy is converted into mechanical work increases with increasing body size. In order to gain insight into the determinants of the scaling of overall efficiency, we measured the scaling of the rate of ATP utilisation during cyclical contractions using glycerinated fibres from the dorsolongitudinal flight muscle of several species of apid bees, covering a ninefold range in body mass. The efficiency of ATP utilisation by the crossbridges is one of the stages that determines the overall efficiency of locomotion. The mechanochemical coefficient was calculated from the ratio of the net power output to the rate of ATP hydrolysis and ranged from 6.5 to 9.7 kJ mol(-1) ATP. The corresponding gross myofibrillar efficiency was 15-23%, increasing concomitantly with body mass (M(b)) and decreasing with increasing wingbeat frequency (n) and scaling as M(b)(0.184) and n(-1.168) in bumblebees and as M(b)(0.153) and n(-0.482) in euglossine bees. Overall efficiency of hovering in bumblebees and euglossine bees was calculated using previously published metabolic power data and revised estimates of the mechanical power output to take into account the drag due to the leading edge vortex that has not been included in previous models. The scaling of overall efficiency of hovering flight in apid bees was not as pronounced as the scaling of myofibrillar efficiency. Therefore the scaling of myofibrillar efficiency with body mass (or frequency) only explained part of the scaling of overall efficiency, and it is likely that the efficiency of other steps in the transduction of chemical energy into mechanical work (e.g. the efficiency of mitochondrial oxidative recovery) may also scale with body mass.

Acceleration of crossbridge kinetics by protein kinase A phosphorylation of cardiac myosin binding protein C modulates cardiac function.

Normal cardiac function requires dynamic modulation of contraction. beta1-adrenergic-induced protein kinase (PK)A phosphorylation of cardiac myosin binding protein (cMyBP)-C may regulate crossbridge kinetics to modulate contraction. We tested this idea with mechanical measurements and echocardiography in a mouse model lacking 3 PKA sites on cMyBP-C, ie, cMyBP-C(t3SA). We developed the model by transgenic expression of mutant cMyBP-C with Ser-to-Ala mutations on the cMyBP-C knockout background. Western blots, immunofluorescence, and in vitro phosphorylation combined to show that non-PKA-phosphorylatable cMyBP-C expressed at 74% compared to normal wild-type (WT) and was correctly positioned in the sarcomeres. Similar expression of WT cMyBP-C at 72% served as control, ie, cMyBP-C(tWT). Skinned myocardium responded to stretch with an immediate increase in force, followed by a transient relaxation of force and finally a delayed development of force, ie, stretch activation. The rate constants of relaxation, k(rel) (s-1), and delayed force development, k(df) (s-1), in the stretch activation response are indicators of crossbridge cycling kinetics. cMyBP-C(t3SA) myocardium had baseline k(rel) and k(df) similar to WT myocardium, but, unlike WT, k(rel) and k(df) were not accelerated by PKA treatment. Reduced dobutamine augmentation of systolic function in cMyBP-C(t3SA) hearts during echocardiography corroborated the stretch activation findings. Furthermore, cMyBP-C(t3SA) hearts exhibited basal echocardiographic findings of systolic dysfunction, diastolic dysfunction, and hypertrophy. Conversely, cMyBP-C(tWT) hearts performed similar to WT. Thus, PKA phosphorylation of cMyBP-C accelerates crossbridge kinetics and loss of this regulation leads to cardiac dysfunction.

Functional aberration of myofibrils by cardiomyopathy-causing mutations in the coiled-coil region of the troponin-core domain.

Two cardiomyopathy-causing mutations, E244D and K247R, in human cardiac troponin T (TnT) are located in the coiled-coil region of the Tn-core domain. To elucidate effects of mutations in this region on the regulatory function of Tn, we measured Ca(2+)-dependent ATPase activity of myofibrils containing various mutants of TnT at these residues. The results confirmed that the mutant E244D increases the maximum ATPase activity without changing the Ca(2+)-sensitivity. The mutant K247R was shown for the first time to have the effect similar to the mutant E244D. Furthermore, various TnT mutants (E244D, E244M, E244A, E244K, K247R, K247E, and K247A) showed various effects on the maximum ATPase activity while the Ca(2+)-sensitivity was unchanged. Molecular dynamics simulations of the Tn-core containing these TnT mutants suggested that the hydrogen-bond network formed by the side chains of neighboring residues around residues 244 and 247 is important for Tn to function properly.

Atypical fast SERCA1a protein expression in slow myofibers and differential S-nitrosylation prevented by exercise during long term bed rest.

We monitored changes in SERCA isoform specific expression and S-nitrosylation in myofibers of lower limb soleus (SOL) and vastus lateralis (VL) muscle biopsies before and after 60 days of voluntary long term bed rest (BR) without (BR-CTRL group, n = 8) and with exercise countermeasure (BR-EX group, n = 8). Before BR, a typical myofiber type-specific distribution of fast and slow SERCA1/2a isoforms was seen. After BR, a subpopulation (approx. 15%) of slow myofibers in BR-CTRL additionally expressed the fast SERCA1a isoform which was not seen in BR-EX. After BR, SERCA1a S-nitrosylation patterns analyzed by the biotin-switch assay decreased in disused SOL only but increased in both muscles following exercise. Differential SERCA1a S-nitrosylation and SERCA1a/2a co-expression in subsets of slow myofibers should be considered as signs of an altered cytosolic Ca(2+) homeostasis following chronic muscle disuse. Exercise preserved myofiber type-specific SERCA1a expression and S-nitrosylation in VL and SOL in a different way, suggesting muscle-specific responses to the countermeasure protocol applied during bed rest.

Localization of creatine kinase isoenzymes in myofibrils. II. Chicken heart muscle.

Chicken heart muscle contains almost exclusively the BB isoenzyme of creatine kinase (CK), its myofibrils, moreover, lack an M-line. This tissue thus provides an interesting contrast to skeletal muscle, in which some of the MM-CK present as predominant CK isoenzyme is bound at the myofibrillar M-line. Approx. 2% of the total CK activity in a chicken heart homogenate remains bound to the myofibrillar fraction after repeated washing cycles; both the fraction and the absolute amount of CK bound are about threefold lower than in skeletal muscle. Almost all of the bound enzyme is located within the Z-line region of each sarcomere, as revealed by indirect fluorescent-antibody staining with antiserum against purified chicken BB-CK. After incubation with exogenous purified MM-CK, positive immunofluorescent staining for M-type CK at the H-region of heart myofibrils was observed, along with weaker fluorescence in the Z-line region. Chicken heart myofibrils may thus possess binding sites for both M and B forms of CK.