Slow-twitch Skeletal Muscle Fibers Research Articles

The sodium/ascorbic acid co-transporter SVCT2 distributes in a striated membrane-enriched domain at the M-band level in slow-twitch skeletal muscle fibers.

Vitamin C plays key roles in cellular homeostasis, functioning as a potent antioxidant and a positive regulator of cell differentiation. In skeletal muscle, the vitamin C/sodium co-transporter SVCT2 is preferentially expressed in oxidative slow fibers. SVCT2 is up-regulated during the early fusion of primary myoblasts and decreases during initial myotube growth, indicating the relevance of vitamin C uptake via SVCT2 for early skeletal muscle differentiation and fiber-type definition. However, our understanding of SVCT2 expression and function in adult skeletal muscles is still limited. In this study, we demonstrate that SVCT2 exhibits an intracellular distribution in chicken slow skeletal muscles, following a highly organized striated pattern. A similar distribution was observed in human muscle samples, chicken cultured myotubes, and isolated mouse myofibers. Immunohistochemical analyses, combined with biochemical cell fractionation experiments, reveal a strong co-localization of SVCT2 with intracellular detergent-soluble membrane fractions at the central sarcomeric M-band, where it co-solubilizes with sarcoplasmic reticulum proteins. Remarkably, electrical stimulation of cultured myofibers induces the redistribution of SVCT2 into a vesicular pattern. Our results provide novel insights into the dynamic roles of SVCT2 in different intracellular compartments in response to functional demands.

Pulsatile permeabilized slow-twitch skeletal muscle fibers from knock-in mice carrying a sMyBP-C E248K pathogenic variant: Potential for a myofilament pacemaker that initiates myogenic tremors

Pulsatile permeabilized slow-twitch skeletal muscle fibers from knock-in mice carrying a sMyBP-C E248K pathogenic variant: Potential for a myofilament pacemaker that initiates myogenic tremors

CHCHD4-TRIAP1 regulation of innate immune signaling mediates skeletal muscle adaptation to exercise

Exercise training can stimulate the formation of fatty-acid-oxidizing slow-twitch skeletal muscle fibers, which are inversely correlated with obesity, but the molecular mechanism underlying this transformation requires further elucidation. Here, we report that the downregulation of the mitochondrial disulfide relay carrier CHCHD4 by exercise training decreases the import of TP53-regulated inhibitor of apoptosis 1 (TRIAP1) into mitochondria, which can reduce cardiolipin levels and promote VDAC oligomerization in skeletal muscle. VDAC oligomerization, known to facilitate mtDNA release, can activate cGAS-STING/NFKB innate immune signaling and downregulate MyoD in skeletal muscle, thereby promoting the formation of oxidative slow-twitch fibers. In mice, CHCHD4 haploinsufficiency is sufficient to activate this pathway, leading to increased oxidative muscle fibers and decreased fat accumulation with aging. The identification of a specific mediator regulating muscle fiber transformation provides an opportunity to understand further the molecular underpinnings of complex metabolic conditions such as obesity and could have therapeutic implications.

Thin filament regulation of cardiac muscle power output: Implications for targets to improve human failing hearts.

The heart's pumping capacity is determined by myofilament power generation. Power is work done per unit time and measured as the product of force and velocity. At a sarcomere level, these contractile properties are linked to the number of attached cross-bridges and their cycling rate, and many signaling pathways modulate one or both factors. We previously showed that power is increased in rodent permeabilized cardiac myocytes following PKA-mediated phosphorylation of myofibrillar proteins. The current study found that that PKA increased power by ∼30% in permeabilized cardiac myocyte preparations (n = 8) from human failing hearts. To address myofilament molecular specificity of PKA effects, mechanical properties were measured in rat permeabilized slow-twitch skeletal muscle fibers before and after exchange of endogenous slow skeletal troponin with recombinant human Tn complex that contains cardiac (c)TnT, cTnC and either wildtype (WT) cTnI or pseudo-phosphorylated cTnI at sites Ser23/24Asp, Tyr26Glu, or the combinatorial Ser23/24Asp and Tyr26Glu. We found that cTnI Ser23/24Asp, Tyr26Glu, and combinatorial Ser23/24Asp and Tyr26Glu were sufficient to increase power by ∼20%. Next, we determined whether pseudo-phosphorylated cTnI at Ser23/24 was sufficient to increase power in cardiac myocytes from human failing hearts. Following cTn exchange that included cTnI Ser23/24Asp, power output increased ∼20% in permeabilized cardiac myocyte preparations (n = 6) from the left ventricle of human failing hearts. These results implicate cTnI N-terminal phosphorylation as a molecular regulator of myocyte power and could serve as a regional target for small molecule therapy to unmask myocyte power reserve capacity in human failing hearts.

Thin filament regulation of striated muscle power output: implications for targets to improve failing human hearts.

The heart's pumping capacity is determined by myofilament power generation. Power is work done per unit time and measured as the product of force and velocity. At a sarcomere level, these contractile properties are linked to the number of attached cross-bridges and their cycling rate; and many signaling pathways modulate one or both of these factors. We previously showed that rodent permeabilized cardiac myocyte power is increased following PKA-mediated phosphorylation of myofibrillar proteins (Herron, Korte, McDonald, Circ. Res. 2001). This increase in power demonstrates a capacity for improved myofilament function and this therapeutic potential adds significance for identification of the molecular specificity underlying enhanced power output. To address molecular specificity at the thin filament level, myofilament mechanical properties were measured before and after exchange of endogenous troponin with recombinant human Tn complex, which contained cardiac (c)TnT, cTnC and either WT cTnI or pseudo-phosphorylated cTnI at sites Ser23/24Asp, Y26E, or the combinatorial Ser23/24Asp and Y26E in single permeabilized rat slow-twitch skeletal muscle fibers. We found that cTnI Ser23/24Asp, Y26E, and combinatorial Ser23/24Asp and Y26E were sufficient for a ∼20% increase in power. We then tested the power reserve capacity in myofilaments from failing human hearts. We found that PKA increased power by ∼35% in permeabilized cardiac myocyte preparations from human failing hearts. Next, we determined if pseudo-phosphorylated cTnI at Ser23/24 was sufficient to increase power in cardiac myocytes from human failing hearts. Following cTn exchange that included cTnI Ser23/24Asp, power output increased ∼20% in permeabilized cardiac myocyte preparations from the left ventricle of human failing hearts. These results implicate cTnI N-terminal phosphorylation is a molecular regulator of myocyte power and could serve as a target to augment myocyte power reserve capacity in failing human hearts.

Molecular regulation of stretch activation.

Stretch activation is defined as a delayed increase in force after rapid stretches. Although there is considerable evidence for stretch activation in isolated cardiac myofibrillar preparations, few studies have measured mechanisms of stretch activation in mammalian skeletal muscle fibers. We measured stretch activation following rapid step stretches [∼1%-4% sarcomere length (SL)] during submaximal Ca2+ activations of rat permeabilized slow-twitch skeletal muscle fibers before and after protein kinase A (PKA), which phosphorylates slow myosin binding protein-C. PKA significantly increased stretch activation during low (∼25%) Ca2+ activation and accelerated rates of delayed force development (kef) during both low and half-maximal Ca2+ activation. Following the step stretches and subsequent force development, fibers were rapidly shortened to original sarcomere length, which often elicited a shortening-induced transient force overshoot. After PKA, step shortening-induced transient force overshoot increased ∼10-fold following an ∼4% SL shortening during low Ca2+ activation levels. kdf following step shortening also increased after PKA during low and half-maximal Ca2+ activations. We next investigated thin filament regulation of stretch activation. We tested the interplay between cardiac troponin I (cTnI) phosphorylation at the canonical PKA and novel tyrosine kinase sites on stretch activation. Native slow-skeletal Tn complexes were exchanged with recombinant human cTn complex with different human cTnI N-terminal pseudo-phosphorylation molecules: 1) nonphosphorylated wild type (WT), 2) the canonical S22/23D PKA sites, 3) the tyrosine kinase Y26E site, and 4) the combinatorial S22/23D + Y26E cTnI. All three pseudo-phosphorylated cTnIs elicited greater stretch activation than WT. Following stretch activation, a new, elevated stretch-induced steady-state force was reached with pseudo-phosphorylated cTnI. Combinatorial S22/23D + Y26E pseudo-phosphorylated cTnI increased kdf. These results suggest that slow-skeletal myosin binding protein-C (sMyBP-C) phosphorylation modulates stretch activation by a combination of cross-bridge recruitment and faster cycling kinetics, whereas cTnI phosphorylation regulates stretch activation by both redundant and synergistic mechanisms; and, taken together, these sarcomere phosphoproteins offer precision targets for enhanced contractility.

Cardiac Troponin I N-Terminal Pseudo-Phosphorylation Regulates Power Output and Stretch Activation

Beat-to-beat heart function is modulated by β-adrenergic stimulation, which elicits PKA phosphorylation of several cardiac myocyte proteins including cardiac troponin I (cTnI). PKA phosphorylation of the cTnI N-terminal region is known to regulate myofilament Ca2+ sensitivity of force. Our lab has also observed PKA phosphorylation increases cardiac myofilament power and augments transient force overshoot. However, it is unknown if cTnI N-terminal phosphorylation per se affects myofilament power and stretch activation. We tested the hypothesis that cTnI N-terminal pseudo-phosphorylation is sufficient to increase power and stretch activation. Molecular sufficiency was investigated by measuring power and stretch activation after cTn exchange into rat permeabilized slow-twitch skeletal muscle fibers, since slow-skeletal (ss)TnI lacks N-terminal phosphorylation sites. Four different human cTnI N-terminal pseudo-phosphorylation states were tested: (i) non-phosphorylated WT, (ii) the canonical S22/23D PKA sites, (iii) the tyrosine kinase Y26E site, also known to modulate myofilament Ca2+ sensitivity, and (iv) the combinatorial S22/23D + Y26E cTnI. Following S22/23D cTnI exchange, fiber power increased ∼18% compared to WT cTn exchange where power decreased ∼3% (p < 0.05). Following Y26E cTnI exchange, power also increased (∼6%), albeit to a less degree than S22/23D. Exchange of combined S22/23D + Y26E cTnI resulted in a similar power increase (∼17%) as S22/23D alone, indicating S22/23D cTnI pseudo-phosphorylation is sufficient to regulate myofilament power. Exchange with S22/23D, Y26E, and S22/23D + Y26E cTnI all augmented stretch activation to a similar amount, implicating redundant site-specific stretch regulation. Finally, only the combinatorial S22/23D + Y26E cTnI increased stretch-activated rates of force development, implicating synergistic regulation of stretch-activated force kinetics. Overall, cTnI N-terminal pseudo-phosphorylation regulates myofilament power and stretch activation by a combination of dominant, redundant and synergistic biophysical mechanisms, which may complement each other to tune beat-to-beat myocardial output demand.

Calsequestrin, a key protein in striated muscle health and disease.

Calsequestrin (CASQ) is the most abundant Ca2+ binding protein localized in the sarcoplasmic reticulum (SR)of skeletal and cardiac muscle. The genome of vertebrates contains two genes, CASQ1 and CASQ2. CASQ1 and CASQ2 have a high level of homology, but show specific patterns of expression. Fast-twitch skeletal muscle fibers express only CASQ1, both CASQ1 and CASQ2 are present in slow-twitch skeletal musclefibers, while CASQ2 is the only protein present in cardiomyocytes. Depending on the intraluminal SR Ca2+ levels, CASQ monomers assemble to form large polymers, which increase their Ca2+ binding ability. CASQ interacts with triadin and junctin, two additional SR proteins which contribute to localize CASQ to the junctional region of the SR (j-SR) and also modulate CASQ ability to polymerize into large macromolecular complexes. In addition to its ability to bind Ca2+ in the SR, CASQ appears also to be able to contribute to regulation of Ca2+ homeostasis in muscle cells. Both CASQ1 and CASQ2 are able to either activate and inhibit the ryanodine receptors (RyRs) calcium release channels, likely through their interactions with junctin and triadin. Additional evidence indicates that CASQ1 contributes to regulate the mechanism of store operated calcium entry in skeletal muscle via a direct interaction with the Stromal Interaction Molecule 1 (STIM1). Mutations in CASQ2 and CASQ1 have been identified, respectively, in patients with catecholamine-induced polymorphic ventricular tachycardia and in patients with some forms of myopathy. This review will highlight recent developments in understanding CASQ1 and CASQ2 in health and diseases.

What a drag!: Skeletal myosin binding protein-C affects sarcomeric shortening.

Colson discusses a recent investigation of the functional effect of slow myosin binding protein-C in slow-twitch skeletal muscle fibers.

MYBP-C Phosphorylation Accelerates Sarcomere Shortening as Thin Filaments Slide through the C-Zone

Myosin Binding Protein-C (MyBP-C) is a 140-150 kDa thick filament protein located in the middle third of each sarcomere, i.e., the C-zone, and is likely a key regulator of contraction. Phosphorylation of MyBP-C occurs at 4 residues within its N-terminus and PKA phosphorylation of MyBP-C in hearts is associated with faster cardiac muscle twitch dynamics (Tong et. al. Circ. Res. 2008). Here we investigated potential mechanisms by which MyBP-C phosphorylation regulates myofilament function. We utilized slow-twitch skeletal muscle fibers since PKA phosphorylates slow skeletal (ss)MyBP-C but not (ss)troponin I. Permeabilized rat slow-twitch fibers were mounted between a force transducer and motor, and calcium activated to elicit ∼30% maximal force. We measured rates of force development and transient force overshoots following a slack re-stretch maneuver and monitored sarcomere shortening during load-clamps. Following PKA, force development rates increased ∼40% and there was a near-doubling in the magnitude of transient force overshoot. We also observed faster shortening velocities at all loads following PKA treatment; this may arise from (i) faster cross-bridge cycling rates, (ii) greater number of cross-bridges during load clamps, and/or (iii) attenuation of internal drag imposed by binding of MyBP-C to actin. To address these mechanisms, we quantified sarcomere length shortening as thin filaments traversed into the C-zone. Before PKA, there tended to be a slowdown as sarcomeres shortened from ∼3.05 to 2.90 µm. After PKA, sarcomeres shortened a considerably greater distance during similar load clamps and, at times, shortening even accelerated as thin filaments slid through the C-zone. Overall these results suggest MyBP-C phosphorylation speeds sarcomere shortening and power output by a combination of faster cross-bridge cycling kinetics, recruitment of cross-bridges, and reduction in drag forces as thin filaments slide through the C-zone.

Exercise induces muscle fiber type switching via transient receptor potential melastatin 2-dependent Ca2+ signaling.

The aim of the present study was to examine whether transient receptor potential melastatin 2 (TRPM2) plays a role in muscle fiber-type transition during exercise. Mice were trained at a speed of 12 m/min at a slope of 0° for 60 min for 5 consecutive days/wk for 4 wk. Exhaustion tests were performed on the treadmill (the speed was set at 6 m/min at a slope of 0° and increased at a rate of 1 m/min every 6 min). Isolated primary skeletal muscle cells from TRPM2-knockout (KO) mice showed lower amplitudes of electrical stimuli (ES)-induced Ca2+ signals when compared with wild-type (WT) mice due to a defect in Ca2+ influx. Moreover, TRPM2-KO mice had a higher proportion of fast-twitch skeletal muscle fibers and a lower proportion of slow-twitch muscle fibers before exercise than WT mice. After exercise, the expression of slow-twitch skeletal muscle fibers was increased only in WT mice but not in TRPM2-KO mice. ES-induced nuclear translocation of the Ca2+-dependent transcription factor NFATc1 was significantly lower in TRPM2-KO mice than in WT mice. TRPM2-KO mice also showed decreased mitochondrial Ca2+ and membrane potential. Lactate levels were higher in the skeletal muscle cells of TRPM2-KO mice before and after ES compared with WT mice. Collectively, these data indicate that TRPM2-mediated Ca2+ signaling plays a critical role in the regulation of fiber-type switching and mitochondrial function in skeletal muscle. NEW & NOTEWORTHY TRPM2 has been shown to play an important role in a variety of cellular functions. However, the role of TRPM2 in skeletal muscle remains poorly understood. Here, we provide evidence that TRPM2-mediated Ca2+ signaling is required for training-induced improvement in skeletal muscle mitochondrial function and fiber type transition.

Molecule specific effects of PKA-mediated phosphorylation on rat isolated heart and cardiac myofibrillar function

Increased cardiac myocyte contractility by the β-adrenergic system is an important mechanism to elevate cardiac output to meet hemodynamic demands and this process is depressed in failing hearts. While increased contractility involves augmented myoplasmic calcium transients, the myofilaments also adapt to boost the transduction of the calcium signal. Accordingly, ventricular contractility was found to be tightly correlated with PKA-mediated phosphorylation of two myofibrillar proteins, cardiac myosin binding protein-C (cMyBP-C) and cardiac troponin I (cTnI), implicating these two proteins as important transducers of hemodynamics to the cardiac sarcomere. Consistent with this, we have previously found that phosphorylation of myofilament proteins by PKA (a downstream signaling molecule of the beta-adrenergic system) increased force, slowed force development rates, sped loaded shortening, and increased power output in rat skinned cardiac myocyte preparations. Here, we sought to define molecule-specific mechanisms by which PKA-mediated phosphorylation regulates these contractile properties. Regarding cTnI, the incorporation of thin filaments with unphosphorylated cTnI decreased isometric force production and these changes were reversed by PKA-mediated phosphorylation in skinned cardiac myocytes. Further, incorporation of unphosphorylated cTnI sped rates of force development, which suggests less cooperative thin filament activation and reduced recruitment of non-cycling cross-bridges into the pool of cycling cross-bridges, a process that would tend to depress both myocyte force and power. Regarding MyBP-C, PKA treatment of slow-twitch skeletal muscle fibers caused phosphorylation of MyBP-C (but not slow skeletal TnI (ssTnI)) and yielded faster loaded shortening velocity and ∼30% increase in power output. These results add novel insight into the molecular specificity by which the β-adrenergic system regulates myofibrillar contractility and how attenuation of PKA-induced phosphorylation of cMyBP-C and cTnI may contribute to ventricular pump failure.

Perilipin 4 in human skeletal muscle: localization and effect of physical activity.

Perilipins (PLINs) coat the surface of lipid droplets and are important for the regulation of lipid turnover. Knowledge about the physiological role of the individual PLINs in skeletal muscle is limited although lipid metabolism is very important for muscle contraction. To determine the effect of long-term exercise on PLINs expression, 26 middle-aged, sedentary men underwent 12 weeks combined endurance and strength training intervention. Muscle biopsies from m. vastus lateralis and subcutaneous adipose tissue were taken before and after the intervention and total gene expression was measured with deep mRNA sequencing. PLIN4 mRNA exhibited the highest expression of all five PLINs in both tissues, and the expression was significantly reduced after long-term exercise in skeletal muscle. Moreover, PLIN4 mRNA expression levels in muscle correlated with the expression of genes involved in de novo phospholipid biosynthesis, with muscular content of phosphatidylethanolamine and phosphatidylcholine, and with the content of subsarcolemmal lipid droplets. The PLIN4 protein was mainly located at the periphery of skeletal muscle fibers, with higher levels in slow-twitch as compared to fast-twitch skeletal muscle fibers. In summary, we report reduced expression of PLIN4 after long-term physical activity, and preferential slow-twitch skeletal muscle fibers and plasma membrane-associated PLIN4 location.

Effect of levosimendan on the contractility of muscle fibers from nemaline myopathy patients with mutations in the nebulin gene

BackgroundNemaline myopathy (NM), the most common non-dystrophic congenital myopathy, is characterized by generalized skeletal muscle weakness, often from birth. To date, no therapy exists that enhances the contractile strength of muscles of NM patients. Mutations in NEB, encoding the giant protein nebulin, are the most common cause of NM. The pathophysiology of muscle weakness in NM patients with NEB mutations (NEB-NM) includes a lower calcium-sensitivity of force generation. We propose that the lower calcium-sensitivity of force generation in NEB-NM offers a therapeutic target. Levosimendan is a calcium sensitizer that is approved for use in humans and has been developed to target cardiac muscle fibers. It exerts its effect through binding to slow skeletal/cardiac troponin C. As slow skeletal/cardiac troponin C is also the dominant troponin C isoform in slow-twitch skeletal muscle fibers, we hypothesized that levosimendan improves slow-twitch muscle fiber strength at submaximal levels of activation in patients with NEB-NM.MethodsTo test whether levosimendan affects force production, permeabilized slow-twitch muscle fibers isolated from biopsies of NEB-NM patients and controls were exposed to levosimendan and the force response was measured.ResultsNo effect of levosimendan on muscle fiber force in NEB-NM and control skeletal muscle fibers was found, both at a submaximal calcium level using incremental levosimendan concentrations, and at incremental calcium concentrations in the presence of levosimendan. In contrast, levosimendan did significantly increase the calcium-sensitivity of force in human single cardiomyocytes. Protein analysis confirmed that the slow skeletal/cardiac troponin C isoform was present in the skeletal muscle fibers tested.ConclusionsThese findings indicate that levosimendan does not improve the contractility in human skeletal muscle fibers, and do not provide rationale for using levosimendan as a therapeutic to restore muscle weakness in NEB-NM patients. We stress the importance of searching for compounds that improve the calcium-sensitivity of force generation of slow-twitch muscle fibers. Such compounds provide an appealing approach to restore muscle force in patients with NEB-NM, and also in patients with other neuromuscular disorders.Electronic supplementary materialThe online version of this article (doi:10.1186/s13395-015-0037-7) contains supplementary material, which is available to authorized users.

Molecule Specific Effects of PKA-Mediated Phosphorylation on Myofibrillar Function

Increased cardiac myocyte contractility by the beta-adrenergic system is an important mechanism to elevate cardiac output to meet greater hemodynamic load and this process is often depressed in failing hearts. While increased contractility involves augmented myoplasmic calcium transients, the myofilaments also adapt to boost the transduction of the calcium signal. Accordingly, ventricular contractility is tightly correlated with PKA-mediated phosphorylation of two myofibrillar proteins, cardiac myosin binding protein-C (cMyBP-C) and cardiac troponin I (cTnI), implicating these two proteins as important transducers of hemodynamics to the cardiac sarcomere. Consistent with this, we have previously found that phosphorylation of myofilament proteins by PKA (a downstream signaling molecule of the beta-adrenergic system) increased force, slowed force development rates, sped loaded shortening, and increased power output in rat skinned cardiac myocyte preparations. Here, we sought to define molecule-specific mechanisms by which PKA-mediated phosphorylation modulates these contractile properties. Regarding cTnI, the incorporation of thin filaments with a majority of unphosphorylated cTnI (as observed in some models of late stage heart failure) decreased isometric force production at any given activator [Ca2+] and these changes were reversed by PKA-mediated phosphorylation in skinned cardiac myocytes. In addition, incorporation of unphosphorylated cTnI sped rates of force development, which suggests less cooperative thin filament activation and recruitment of non-cycling cross-bridges into the pool of cycling cross-bridges, a process that would tend to depress myocyte force and power. Regarding cMyBP-C, PKA treatment of slow-twitch skeletal muscle fibers caused phosphorylation of MyBP-C (but not TnI) and yielded faster loaded shortening velocity and an ∼30% increase in power output. These results add novel insight into the molecular specificity by which the beta-adrenergic system controls myofibrillar contractility and how attenuation of PKA-induced phosphorylation of cMyBP-C and cTnI may contribute to ventricular pump failure.

Differentiation of the intracellular structure of slow- versus fast-twitch muscle fibers through evaluation of the dielectric properties of tissue

Slow-twitch (type 1) skeletal muscle fibers have markedly greater mitochondrial content than fast-twitch (type 2) fibers. Accordingly, we sought to determine whether the dielectric properties of these two fiber types differed, consistent with their distinct intracellular morphologies. The longitudinal and transverse dielectric spectrum of the ex vivo rat soleus (a predominantly type 1 muscle) and the superficial layers of rat gastrocnemius (predominantly type 2) (n = 15) were measured in the 1 kHz–10 MHz frequency range and modeled to a resistivity Cole–Cole function. Major differences were especially apparent in the dielectric spectrum in the 1 to 10 MHz range. Specifically, the gastrocnemius demonstrated a well-defined, higher center frequency than the soleus muscle, whereas the soleus muscle showed a greater difference in the modeled zero and infinite resistivities than the gastrocnemius. These findings are consistent with the fact that soleus tissue has larger and more numerous mitochondria than gastrocnemius. Evaluation of tissue at high frequency could provide a novel approach for assessing intracellular structure in health and disease.

Length Dependence of Force Generation is Controlled by Phosphorylation of cTnI at Serines 23/24

The steepness of the Frank-Starling relationship is modulated by numerous physiological factors including beta-adrenergic stimulation, which steepens the relationship. This arises in part from increased myofibrillar length dependence of force and power by PKA, a downstream signaling molecule of the beta-adrenergic system. Since PKA has multiple myofibrillar substrates including titin, myosin binding protein-C (MyBP-C), and cardiac troponin I (cTnI), we sought to define if phosphorylation of one of these molecules was sufficient to control length-tension relationships. We focused on cTnI since (i) we previously observed a relationship between cTnI phosphorylation and the steepness of ventricular function curves in rat working hearts, (ii) 2D-DIGE indicated a distribution of cTnI phosphorylation states consistent with our previous observation of two populations of length-tension relationships (one shallow the other steep) in rat skinned cardiac myocytes, and (iii) troponin can be more readily exchanged in permeabilized striated muscle cell preparations. To test if PKA-mediated phosphorylation of cTnI is sufficient to mediate length dependence of force we used skinned rat slow-twitch skeletal muscle fibers, which exhibit shallow length-tension relationships that are unaltered by PKA. A sarcomere length-tension relationship was measured during submaximal Ca2+ activation, then the fiber was incubated in a troponin (Tn) exchange solution, which replaced ∼80% of the endogenous slow-skeletal Tn with cTn. After cTn incorporation the length-tension relationship remained shallow as predicted since the purified cTnI lacks phosphate incorporation. Next, the fiber was incubated in PKA, which caused the length-tension relationship to shift from shallow to steep. In addition, exchange with cTn having cTnI serines 23/24 modified to aspartic acids to mimic phosphorylation steepened the length-tension relationship. These results indicate that phosphorylation of cTnI serines 23/24 is sufficient to control length dependence of force generation in striated muscle.

Capillaries are Embedded in the Sarcolemma of Murine Slow Twitch Skeletal Muscle Fibers

The purpose of this study was to evaluate the in vivo, 3D spatial relationship between capillaries (CAP), mitochondria (MITO), and three fiber types within murine Tibialis anterior (TA) muscle. Two photon excitation microscopy was utilized for in vivo imaging of MITO (NADH autofluorescence), vasculature, and interstitial space using injected dyes. A custom program was used to trace fibers and determine contact and volume ratios between CAP and 127 muscle fibers. Fiber type (slow (ST), intermediate (IT), fast‐twitch (FT)) was determined from relative NADH intensity and fiber size. Microscopy revealed that half of ST fiber CAP (53.2 ± 4.0%) had at least 50% of their circumference embedded in a groove in the sarcolemma. Embedded CAP were tightly associated with dense MITO populations lateral to the CAP grooves but nearly absent below the groove where fibrils dominated. CAP embedding was significantly lower in IT (29.5 ± 4.2%) and FT (14.2 ± 1.8%). Electron microscopy confirmed ST embedded CAP and MITO localization. A specialized sarcolemma/CAP groove is present in murine ST fibers resulting in CAP embedding in a MITO rich region. This structure optimizes CAP O2 delivery to highly aerobic ST fibers and selectively restricts O2 delivery to FT fibers. Localized contact of CAP with ST plasma membranes suggests receptors or transporters for blood borne signaling or substrate molecules may also be concentrated in these regions.

Length Dependence of Cardiac Myocyte Force Generation is Mediated by CTnI Phosphorylation

According to the Frank-Starling relationship, greater ventricular volume increases ventricular output. The Frank-Starling relationship is based partly on the length-tension relationship in cardiac myocytes. Recently, we identified two populations of length-tension relationships in mammalian cardiac myocytes, which was dependent on PKA-induced myofibrillar phosphorylation. We also observed a relationship between cardiac troponin I (cTnI) phosphorylation and steepness of ventricular function curves in rat working hearts. Thus, we tested the hypothesis that phosphorylation of cTnI is sufficient and/or necessary to control the length dependence of force generation. Our approach took advantage of the fact that troponin can be readily exchanged in permeabilized striated muscle cell preparations. We first used permeabilized rat slow-twitch skeletal muscle fibers, which exhibit shallow length-tension relationships that are unaltered by PKA. A sarcomere length-tension relationship was measured during submaximal Ca2+ activation then the fiber was incubated in a troponin (Tn) exchange solution, which replaced nearly all the endogenous slow Tn with cTn. After cTn incorporation the length-tension relationship remained shallow as predicted since the purified cTnI lacks phosphate incorporation. Next, the fiber was incubated in PKA and a final sarcomere length-tension relationship measured. Contrary to our prediction, the length-tension relationship remained shallow after PKA phosphorylation of exogeneous cTnI. We next tested whether cTnI phosphorylation is necessary to steepen length-tension relationships in a cardiac myofibrillar background. For these experiments, we exchanged unphosphorylated cTn into a PKA treated myocyte that displayed a steep length-tension relationship. In this case cTn exchange shifted the length-tension relationship from a steep to a shallow relation. These results indicate that cTnI phosphorylation is not sufficient to alter length dependence of force generation in slow-twitch skeletal muscle fibers but appears necessary to convert the length-tension relationship from steep to shallow in cardiac myocytes.

Absolute amounts and diffusibility of HSP72, HSP25, and αB-crystallin in fast- and slow-twitch skeletal muscle fibers of rat

Heat shock proteins (HSPs) are essential for normal cellular stress responses. Absolute amounts of HSP72, HSP25, and αB-crystallin in rat extensor digitorum longus (EDL) and soleus (SOL) muscle were ascertained by quantitative Western blotting to better understand their respective capabilities and limitations. HSP72 content of EDL and SOL muscle was only ∼1.1 and 4.6 μmol/kg wet wt, respectively, and HSP25 content approximately twofold greater (∼3.4 and ∼8.9 μmol/kg, respectively). αB-crystallin content of EDL muscle was ∼4.9 μmol/kg but in SOL muscle was ∼30-fold higher (∼140 μmol/kg). To examine fiber heterogeneity, HSP content was also assessed in individual fiber segments; every EDL type II fiber had less of each HSP than any SOL type I fiber, whereas the two SOL type II fibers examined were indistinguishable from the EDL type II fibers. Sarcolemma removal (fiber skinning) demonstrated that 10-20% of HSP25 and αB-crystallin was sarcolemma-associated in SOL fibers. HSP diffusibility was assessed from the extent and rate of diffusion out of skinned fiber segments. In unstressed SOL fibers, 70-90% of each HSP was readily diffusible, whereas ∼95% remained tightly bound in fibers from SOL muscles heated to 45°C. Membrane disruption with Triton X-100 allowed dispersion of HSP72 and sarco(endo)plasmic reticulum Ca(2+)-ATPase pumps but did not alter binding of HSP25 or αB-crystallin. The amount of HSP72 in unstressed EDL muscle is much less than the number of its putative binding sites, whereas SOL type I fibers contain large amounts of αB-crystallin, suggesting its importance in normal cellular function without upregulation.