Reduced Calcium Sensitivity Research Articles

Cytosolic domain regulates the calcium sensitivity and surface expression of BEST1 channels in the HEK293 cells

BEST family is a class of Ca2+-activated Cl- channels evolutionary well conserved from bacteria to human. The human BEST paralogs (BEST1-BEST4) share significant amino acid sequence homology in the N-terminal region, which forms the transmembrane helicases and contains the direct calcium-binding site, Ca2+-clasp. But the cytosolic C-terminal region is less conserved in the paralogs. Interestingly, this domain-specific sequence conservation is also found in the BEST1 orthologs. However, the functional role of the C-terminal region in the BEST channels is still poorly understood. Thus, we aimed to understand the functional role of the C-terminal region in the human and mouse BEST1 channels by using electrophysiological recordings. We found that the calcium-dependent activation of BEST1 channels can be modulated by the C-terminal region. The C-terminal deletion hBEST1 reduced the Ca2+-dependent current activation and the hBEST1-mBEST1 chimera showed a significantly reduced calcium sensitivity to hBEST1 in the HEK293 cells. And the C-terminal domain could regulate cellular expression and plasma membrane targeting of BEST1 channels. Our results can provide a basis for understanding the C-terminal roles in the structure-function of BEST family proteins. [BMB Reports 2023; 56(3): 172-177].

Glycogen synthase kinase 3β localizes to the Z-disc to maintain length dependent activation

Glycogen synthase kinase 3β (GSK-3β) can modulate myofilament function in vitro. However, it’s in vivo role, mechanism, and translational relevance are unknown, which we investigated using inducible cardiomyocyte-specific GSK-3β KO mice. Compared to tamoxifen-treated GSK-3βfl/fl/Cre-mice (Con), skinned myocytes from KO mice had reduced calcium sensitivity at long sarcomere lengths (SL = 2.2 μm), but there were no differences at short SL (∼1.9 μm). Thus, myocytes from KO mice did not sensitize to calcium with stretch, a mechanism called length dependent activation (LDA) underlying the organ-level Frank-Starling law.

Using novel small molecules to alter cardiac thin filament function

Inherited cardiomyopathies such as hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM) have a high population prevalence of 1 in 500 and 1 in 250 individuals, respectively, in the US and Europe. Disease-related HCM and DCM effects range enormously from being asymptomatic to leading to heart failure and a need for heart transplantation. HCM is characterized by left ventricular (LV) hypertrophy, typically with increased calcium sensitivity of thin filament activation, while DCM exhibits LV dilatation and reduced calcium sensitivity.

The ACTC A331P hypertrophic cardiomyopathy mutation enhances basal contractile activity and causes resting muscle dysfunction

The ACTC A331P HCM mutation is located on a subdomain 3, surface strand of actin that is vital for troponin-tropomyosin-based regulation of contraction. Previous studies found A331P ACTC mouse models were indistinguishable from control, and data generated with reconstituted bovine trabeculae suggested the variant reduced calcium sensitivity and tension, results at odds with those found for other HCM mutations. Here we employed a multidisciplinary approach to further examine the effects of the mutation across numerous levels of organization to better understand disease pathogenesis.

Abstract 11592: Glycogen Synthase Kinase 3β Localizes to the Z-Disc to Maintain Length Dependent Activation

Glycogen Synthase Kinase 3β (GSK-3β) can modulate myofilament function in vitro . However, it’s in vivo role, mechanism, and translational relevance are unknown, which we investigated here using inducible cardiomyocyte specific GSK-3β KO mice. Compared to tamoxifen-treated GSK-3β fl/fl Cre- mice (Con), skinned myocytes from KO mice had reduced calcium sensitivity at long sarcomere lengths (SL = 2.2 μm), but there were no differences at short SL (~1.9 μm). Thus, myocytes from KO mice did not sensitize to calcium with stretch, a mechanism called length dependent activation (LDA) that underlies the organ-level Frank-Starling law. LDA has been attributed to (1) phosphorylation of myofilament proteins, (2) altered lattice-spacing, and (3) changes to titin’s elastic properties. Using mass spectrometry and small energy x-ray diffraction we ruled out the first two mechanisms, however we did find that GSK-3β KO myocytes had decreased passive tension – indicating the loss of LDA was due to loss of titin as a length sensor. Interestingly, immunofluorescence showed that GSK-3β localized to the z-disc when phosphorylated at Y216 and, via mass spectrometry, phosphorylated primarily z-disc proteins including several sites on the structural protein Ablim-1, which we showed also localizes to the z-disc. These data suggested that GSK-3β’s effect on titin is likely through altering its ability to anchor to the z-disc through targeting of these z-disc proteins. To provide further evidence that GSK-3β is specifically altering LDA, we genetically removed a downstream effecter of LDA, cardiac myosin binding protein-C (cMyBP-C). In vitro treatment with exogenous GSK-3β was able to increase calcium sensitivity at long SLs in both KO and WT mice but had no effect on cMyBPC KO mice or mice lacking the c-terminal domains of cMyBPC that are known to be important for LDA. Lastly, we found that human heart failure patients had less myofilament GSK-3β compared to non-failing patients, and that these same samples had a depressed LDA. This work has identified a novel mechanism by which GSK-3β localizes to the myofilament to modulate LDA and indicates that z-disc localized GSK-3β may be a possible therapeutic target to restore the Frank-Starling mechanism in heart failure patients.

Mechanisms in Tridax procumbens leaf extract reversal of paroxetine-induced erectile dysfunction in corpus cavernosum of male Wistar rats

Introduction: Aqueous leave extract of Tridax procumbens (AETPL) is reported to improve erectile functions; however, the mechanism is unclear. This study investigates the mechanism involved in the contractile activity of the corpus cavernosum after AETPL treatment of paroxetine-induced erectile dysfunctional adult male Wistar rats.
 Methods: A total of 20 male Wistar rats were categorized into four groups of five and treated orally for four weeks: Group 1 (distilled water), Group 2 (paroxetine 10 mg/kg), Group 3 (paroxetine + AETPL 100 mg/kg), and Group 4 (paroxetine + Viagra 0.5 mg/kg). Contractile responses of excised corpus cavernosum strips (CS) were determined in response to acetylcholine (ACh), phenylephrine (PHE), potassium chloride (KCl), and calcium chloride (CaCl2), and after incubation in L-NAME, indomethacin, nifedipine, adenosine, caffeine, nicorandil, and acetovanillone.
 Results: The relaxation response (%) of CS to ACh was significantly inhibited in the paroxetine group compared to the AETPL- and the Viagra-co-treated group. Pre-incubation in L-NAME considerably enhanced the percentage relaxation in groups co-treated with AETPL and Viagra. Groups co-treated with AETPL and Viagra significantly inhibited contraction in response to cumulative doses of CaCl2. Contractile responses of CS to cumulative doses of PHE after incubation in caffeine and adenosine were considerably inhibited in groups co-treated with AETPL and Viagra. Similarly, nicorandil (10-4 M) enhanced the percentage relaxation to cumulative doses of ACh (10-9 — 10-5 M) in groups co-treated with AETPL and Viagra. The pre-incubation of CS with acetovanillone (10-4 M) enhanced the percentage relaxation to ACh across groups.
 Conclusion: Erectile dysfunction was reversed by AETPL-induced antioxidant/NADPH oxidase inhibitor activity, reduced calcium sensitivity, activation of ATP-sensitive K+ channel, and endothelial Nitric Oxide (NO) release.

Abstract 343: Glycogen Synthase Kinase 3β Localizes to the Cardiac Myofilament via Py216 and Dynamically Modulates Calcium Sensitivity

Localization of kinases to sub-cellular compartments allows them dynamic control over specific subsets of their targets. We previously found GSK-3β could modulate myofilament Ca 2+ sensitivity. However, whether GSK-3β modulates Ca 2+ sensitivity in vivo , if it localizes to the myofilament, and the consequences, are unknown. In myofilament enriched LV tissue from human non-failing and heart failure (HF) patients (n = 66) we found GSK-3β does localize to the myofilament and is altered by sex, age, and HF. To determine its in vivo functional role, we used myocyte specific inducible GSK-3β knock-out mice for skinned myocyte force-calcium experiments and found that GSK-3β reduction reduced calcium sensitivity. Further, we measured function in human samples and found myofilament GSK-3β levels directly correlated to Ca 2+ sensitivity. To establish how GSK-3β binds to the myofilament, we performed co-IP and IHC with phosphorylated forms of GSK-3β. GSK-3β phosphorylated at Y216 had a high affinity for the myofilament and localized to the z-disc. Mutating Y216 to a phospho-mimetic increased binding to the myofilament while mutating it to a phospho-null ablated binding.To identify GSK-3β’s myofilament targets, we performed mass spectrometry on myofilament phospho-enriched samples from GSK-3β KO and WT mice. As GSK-3β modulated Ca 2+ sensitivity, we expected to detect thin filament proteins. However, in agreement with its localization to the z-disc, GSK-3β primarily phosphorylated z-disc proteins.In the GSK-3β KO mice, strain analysis revealed the posterior wall contracted significantly earlier than the anterior wall, indicating baseline mechanical dyssynchrony. The WT mice had synchronous contraction, and interestingly there was significantly higher myofilament GSK-3β in the anterior wall compared to the posterior wall, a difference we hypothesize maintains synchrony. Thus, losing this fine control over Ca 2+ sensitivity as in the GSK-3β KO mice would induce mechanical dyssynchrony. Overall, these findings reveal that GSK-3β dynamically localizes to the myofilament to modulate Ca 2+ sensitivity through Y216 phosphorylation. The consequence of this “fine tuning” maintains chamber level mechanical synchrony.

Right atrial contractile dynamics are impaired in patients with postcapillary pulmonary hypertension.

Left ventricular (LV) dysfunction in conjunction with postcapillary pulmonary hypertension (PH) is frequently associated with right ventricular (RV) dysfunction, determining the patient prognosis. Compensatory mechanisms for RV dysfunction have not been previously evaluated in detail. Since calcium dependent right atrial (RA) dynamics are a surrogate for RA contractile properties, the present study examined the calcium dependency of RA tissue obtained from patients with or without postcapillary PH. In total, 15 patients with PH (PH group; mean age, 70.7±7.2 years) and 10 patients without postcapillary PH (non-PH group; mean age, 55.7±11.8 years) who were scheduled to undergo elective left heart valve surgery were included in the current study. Calcium concentration (pCa; shown as the negative log10) against force curves were generated, while LV and RV function was evaluated by echocardiography. Echocardiography data revealed a significantly reduced LV function in the PH group, while the RV function was preserved in the two groups, precluding overt RV dysfunction. In the PH group, significantly reduced force values were detected at high pCa values when compared with the non-PH group force, indicating impaired RA function. Furthermore, reduced calcium sensitivity was observed (which was determined as the pCa at half maximal activation) in the PH group, and the presence of a compensatory mechanism for reduced force capacity was hypothesized. In conclusion, the preliminary results of the current study showed impaired RA contractile properties in postcapillary hypertension with preserved RV function. The diminished RA compensatory mechanisms may lead to accelerated RV dysfunction in the clinical course of postcapillary PH.

Physiologic force-frequency response in engineered heart muscle by electromechanical stimulation

A hallmark of mature mammalian ventricular myocardium is a positive force-frequency relationship (FFR). Despite evidence of organotypic structural and molecular maturation, a positive FFR has not been observed in mammalian tissue engineered heart muscle. We hypothesized that concurrent mechanical and electrical stimulation at frequencies matching physiological heart rate will result in functional maturation. We investigated the role of biomimetic mechanical and electrical stimulation in functional maturation in engineered heart muscle (EHM). Following tissue consolidation, EHM were subjected to electrical field stimulation at 0, 2, 4, or 6 Hz for 5 days, while strained on flexible poles to facilitate auxotonic contractions. EHM stimulated at 2 and 4 Hz displayed a similarly enhanced inotropic reserve, but a clearly diverging FFR. The positive FFR in 4 Hz stimulated EHM was associated with reduced calcium sensitivity, frequency-dependent acceleration of relaxation, and enhanced post-rest potentiation. This was paralleled on the cellular level with improved calcium storage and release capacity of the sarcoplasmic reticulum and enhanced T-tubulation. We conclude that electro-mechanical stimulation at a physiological frequency supports functional maturation in mammalian EHM. The observed positive FFR in EHM has important implications for the applicability of EHM in cardiovascular research.

Regulatory role of the second gelsolin‐like domain of Caenorhabditis elegans gelsolin‐like protein 1 (GSNL‐1) in its calcium‐dependent conformation and actin‐regulatory activities

Caenorhabditis elegans gelsolin-like protein-1 (GSNL-1) is an unconventional member of the gelsolin family of actin-regulatory proteins. Unlike typical gelsolin-related proteins with three or six G domains, GSNL-1 has four gelsolin-like (G) domains (G1-G4) and exhibits calcium-dependent actin filament severing and capping activities. The first G domain (G1) of GSNL-1 is necessary for its actin-regulatory activities. However, how other domains in GSNL-1 participate in regulation of its functions is not understood. Here, we report biochemical evidence that the second G domain (G2) of GSNL-1 has a regulatory role in its calcium-dependent conformation and actin-regulatory activities. Comparison of the sequences of gelsolin-related proteins from various species indicates that sequences of G2 are highly conserved. Among the conserved residues in G2, we focused on D162 of GSNL-1, since equivalent residues in gelsolin and severin are part of the calcium-binding sites and is a pathogenic mutation site in human gelsolin causing familial amyloidosis, Finish-type. The D162N mutation does not alter the inactive and fully calcium-activated states of GSNL-1 for actin filament severing (at 20 nM GSNL-1) and capping activities (at 50 nM GSNL-1). However, under these conditions, the mutant shows reduced calcium sensitivity for activation. By contrast, the D162N mutation strongly enhances susceptibility of GSNL-1 to chymotrypsin digestion only at high calcium concentrations but not at low calcium concentrations. The mutation also reduces affinity of GSNL-1 with actin monomers. These results suggest that G2 of GSNL-1 functions as a regulatory domain for its calcium-dependent actin-regulatory activities by mediating conformational changes of the GSNL-1 molecule.

Complexin facilitates exocytosis and synchronizes vesicle release in two secretory model systems

Complexins (Cplxs) are small, SNARE-associated proteins believed to regulate fast, calcium-triggered exocytosis. However, studies have pointed to either an inhibitory and/or facilitatory role in exocytosis, and the role of Cplxs in synchronizing exocytosis is relatively unexplored. Here, we compare the function of two types of complexin, Cplx 1 and 2, in two model systems of calcium-dependent exocytosis. In mouse neuromuscular junctions (NMJs), we find that lack of Cplx 1 significantly reduces and desynchronizes calcium-triggered synaptic transmission; furthermore, high-frequency stimulation elicits synaptic facilitation, instead of normal synaptic depression, and the degree of facilitation is highly sensitive to the amount of cytoplasmic calcium buffering. In Cplx 2-null adrenal chromaffin cells, we also find decreased and desynchronized evoked release, and identify a significant reduction in the vesicle pool close to the calcium channels (immediately releasable pool, IRP). Viral transduction with either Cplx 1 or 2 rescues both the size of the evoked response and the synchronicity of release, and it restores the IRP size. Our findings in two model systems are mutually compatible and indicate a role of Cplx 1 and 2 in facilitating vesicle priming, and also lead to the new hypothesis that Cplxs may synchronize vesicle release by promoting coupling between secretory vesicles and calcium channels.

A Novel Mouse Model of Nebulin-Based Nemaline Myopathy

Nebulin - a giant sarcomeric protein - plays a pivotal role in skeletal muscle contractility by regulating thin filament length and function. Although mutations in the gene encoding nebulin (NEB) are a frequent cause of nemaline myopathy (NM), the most common non-dystrophic congenital myopathy, the mechanisms by which mutations in NEB cause muscle weakness remain largely unknown. To better understand these mechanisms, we have generated a mouse model in which NEB exon 55 is deleted (NEBΔex55), a mutation known to frequently occur in NM patients.NEBΔex55 mice are born close to Mendelian ratio's, but show growth retardation after birth. Electronmicroscopy shows nemaline rods - a hallmark feature of NM - in muscle fibers from NEBΔex55 mice. Western blotting studies with nebulin-specific antibodies reveal much reduced nebulin levels in muscle from NEBΔex55 mice. Immunofluorescence confocal microscopy studies with tropomodulin antibodies and phalloidin reveal that thin filament length is reduced in muscle fibers from NEBΔex55 mice. In line with reduced thin filament length, the maximal force generating capacity of skinned muscle fibers is reduced in NEBΔex55 mice with a more pronounced reduction at longer sarcomere lengths. Finally, in NEBΔex55 mice the regulation of contraction is impaired, as evidenced by marked changes in cross bridge cycling kinetics and by a reduction of the calcium sensitivity of force generation. This reduced calcium sensitivity was observed only at short sarcomere lengths, suggesting that nebulin might play a role in the length dependence of activation.In conclusion, we have generated the first nebulin-based NM model. Our data indicate that the phenotype of NEBΔex55 mice closely recapitulates that observed previously by us in patients harboring this particular mutation.

Inflammation and cardiac dysfunction during sepsis, muscular dystrophy, and myocarditis.

Inflammation plays an important role in cardiac dysfunction under different situations. Acute systemic inflammation occurring in patients with severe burns, trauma, and inflammatory diseases causes cardiac dysfunction, which is one of the leading causes of mortality in these patients. Acute sepsis decreases cardiac contractility and impairs myocardial compliance. Chronic inflammation such as that occurring in Duchenne muscular dystropshy and myocarditis may cause adverse cardiac remodeling including myocyte hypertrophy and death, fibrosis, and altered myocyte function. However, the underlying cellular and molecular mechanisms for inflammatory cardiomyopathy are still controversial probably due to multiple factors involved. Potential mechanisms include the change in circulating blood volume; a direct inhibition of myocyte contractility by cytokines (tumor necrosis factor (TNF)-α, interleukin (IL)-1β); abnormal nitric oxide and reactive oxygen species (ROS) signaling; mitochondrial dysfunction; abnormal excitation-contraction coupling; and reduced calcium sensitivity at the myofibrillar level and blunted β-adrenergic signaling. This review will summarize recent advances in diagnostic technology, mechanisms, and potential therapeutic strategies for inflammation-induced cardiac dysfunction.

TNF‐α mediated regulation of myosin light chain 20 phosphorylation in lymphatic muscle

We have shown that mesenteric collecting lymphatic vessels isolated from rats with a high fructose induced metabolic syndrome (MetSyn) exhibit a 50% reduction in force generating capacity and reduced calcium sensitivity. We hypothesized that the elevated level of the inflammatory cytokine TNF‐α associated with MetSyn contributes to the mesenteric lymphatic force reduction. Rat mesenteric lymphatic muscle cells (RMLCs) were cultured in the presence of varying amounts of TNF‐α for 1, 24, and 96 hours, and the basal phosphorylation of myosin light chain 20 (MLC20), an indicator for lymphatic muscle contraction was assessed. We found that chronic TNF‐α (20ng/ml) stimulation significantly reduced phospho‐MLC20 to MLC20 ratio in the basal state at 96 hrs while they were unchanged in 1 and 24 hour treated cells. Furthermore chronic stimulation of RMLCs with lipopolysaccharide recapitulated the results from chronic TNF‐ α exposure. We propose that lymphatic muscle force generation capacity reduced in MetSyn animals is partially due to inflammation‐mediated MLC20 phosphorylation regulation. Our data suggest a potential mechanism whereby elevated endotoxin translocation associated with an increase in TNF‐α contribute to the reduction in force production and calcium sensitivity in mesenteric lymphatic vessels of MetSyn animals. Supported by NIH HL86650 to MM; AHA09POST2280005 to SC.

Impaired myogenic tone in mesenteric arteries from overweight rats

BackgroundRats fed high fat (HFD) or high sucrose (HSD) diets develop increased adiposity as well as impaired vasodilatory responsiveness stemming from oxidative stress. Moreover, HFD rats become hypertensive compared to either control (Chow) or HSD fed rats, suggesting elevated vascular tone. We hypothesized that rats with increased adiposity and oxidative stress demonstrate augmented pressure-induced vasoconstriction (i.e. myogenic tone) that could account for the hypertensive state.MethodsMale Sprague-Dawley rats were fed Chow, HFD or HSD for 6 weeks. The effects of oxidative stress and endogenous nitric oxide on myogenic responses were examined in small mesenteric arteries by exposing the arteries to incremental intraluminal pressure steps in the presence of antioxidants or an inhibitor of nitric oxide synthase, LNNA (100 μM).ResultsContrary to the hypothesis, rats fed either HSD or HFD had significantly impaired myogenic responses despite similar vascular morphology and passive diameter responses to increasing pressures. Vascular smooth muscle (VSM) calcium levels were normal in HFD arteries suggesting that diminished calcium sensitivity was responsible for the impaired myogenic response. In contrast, VSM calcium levels were reduced in HSD arteries but were increased with pre-exposure of arteries to the antioxidants tiron (10 mM) and catalase (1200 U/mL), also resulting in enhanced myogenic tone. These findings show that oxidative stress impairs myogenic tone in arteries from HSD rats by decreasing VSM calcium. Similarly, VSM calcium responses were increased in arteries from HFD rats following treatment with tiron and catalase, but this did not result in improved myogenic tone. Nitric oxide is involved in the impaired myogenic response in HFD, but not HSD, rats since inhibition with LNNA resulted in maximal myogenic responses at lower intraluminal pressures and VSM calcium levels, further implicating reduced calcium sensitivity in the impaired response.ConclusionThe impaired myogenic responses observed in isolated arteries from HSD and HFD rats are attributed to changes in VSM calcium signaling.

Levosimendan improves calcium sensitivity of diaphragm muscle fibres from a rat model of heart failure

Diaphragm muscle weakness occurs in patients with heart failure (HF) and is associated with exercise intolerance and increased mortality. Reduced sensitivity of diaphragm fibres to calcium contributes to diaphragm weakness in HF. Here we have investigated the ability of the calcium sensitizer levosimendan to restore the reduced calcium sensitivity of diaphragm fibres from rats with HF. Coronary artery ligation in rats was used as an animal model for HF. Sham-operated rats served as controls. Fifteen weeks after induction of HF or sham operations animals were killed and muscle fibres were isolated from the diaphragm. Diaphragm fibres were skinned and activated with solutions containing incremental calcium concentrations and 10 µM levosimendan or vehicle (0.02% DMSO). Developed force was measured at each calcium concentration, and force-calcium concentration relationships were plotted. Calcium sensitivity of force generation was reduced in diaphragm muscle fibres from HF rats, compared with fibres from control rats (P < 0.01). Maximal force generation was ∼25% lower in HF diaphragm fibres than in control fibres (P < 0.05). Levosimendan significantly increased calcium sensitivity of force generation in diaphragm fibres from HF and control rats, without affecting maximal force generation. Levosimendan enhanced the force generating capacity of diaphragm fibres from HF rats by increasing the sensitivity of force generation to calcium concentration. These results provide strong support for testing the effect of calcium sensitizers on diaphragm muscle weakness in patients with HF.

TOLL-LIKE RECEPTOR 4 MODULATES CALCIUM SENSITIZATION IN RESISTANCE ARTERIES: 3D.07

Objectives: Inflammation is associated with hypertension and cardiovascular disease. The role of the innate immune system on microvascular smooth muscle function remains unclear. We tested whether Toll-like receptors (TLRs), key players of the innate immune system, sensitize microvascular myosin light chain phosphatase (MLCP), thus calcium sensitization, and thereby the contractile apparatus. Methods: Mesenteric resistance arteries (RA) of TLR4-/−, TLR2−/−, MyD88−/− and wildtype mice (C57/Bl6) were investigated by wire myography. After viability testing calcium sensitivity was assessed in depolarized RA (120 mol/L K+) exposed to stepwise increases in extracellular calcium (0 to 3 mmol/L). Calcium sensitivity was determined before and after application of 10 μmol/L sodium nitroprusside (SNP), the soluble guanylate cyclase (sGC) inhibitor ODQ (1 μmol/L), the MLCP inhibitor Calyculin A (120 nmol/L) or in presence of the Rho kinase inhibitor Y27632 (1 μmol/L). Wall-to-Lumen ratio (W/L) was assessed by histology. Blood pressure was assessed by radiotelemetry. Results: TLR4−/−, but not TLR2−/− or MyD88−/− mice were characterized by a reduced calcium sensitivity as compared to wildtype. Calyculin A and ODQ, but not Y27632 or the supplementation of NO abolished this difference in TLR4−/−. W/L was reduced in TLR4−/−, whereas blood pressure was similar between TLR4−/− and wildtype mice. Conclusions: The MyD88-independent TLR4 signaling pathway is characterized by attenuated vascular contractility under resting conditions. This involves attenuated calcium desensitization based on a cGMP-dependent activation of the MLCP in TLR4−/−.

Altered myofilament function depresses force generation in patients with nebulin-based nemaline myopathy (NEM2)

Nemaline myopathy (NM), the most common non-dystrophic congenital myopathy, is clinically characterized by muscle weakness. However, the mechanisms underlying this weakness are poorly understood. Here, we studied the contractile phenotype of skeletal muscle from NM patients with nebulin mutations (NEM2). SDS-PAGE and Western blotting studies revealed markedly reduced nebulin protein levels in muscle from NM patients, whereas levels of other thin filament-based proteins were not significantly altered. Muscle mechanics studies indicated significantly reduced calcium sensitivity of force generation in NM muscle fibers compared to control fibers. In addition, we found slower rate constant of force redevelopment, as well as increased tension cost, in NM compared to control fibers, indicating that in NM muscle the rate of cross-bridge attachment is reduced, whereas the rate of cross-bridge detachment is increased. The resulting reduced fraction of force generating cross-bridges is expected to greatly impair the force generating capacity of muscle from NM patients. Thus, the present study provides important novel insights into the pathogenesis of muscle weakness in nebulin-based NM.

Frequency- and afterload-dependent cardiac modulation in vivo by troponin I with constitutively active protein kinase A phosphorylation sites.

Acute beta-adrenergic stimulation enhances cardiac contractility, accelerates muscle relaxation, and amplifies the inotropic and lusitropic response to increased stimulation frequency. These effects are modulated by phosphorylation of calcium handling and myofilament proteins such as troponin I (TnI) by protein kinase A (PKA). To more directly delineate the role of TnI PKA phosphorylation, transgenic mice were generated that overexpress cardiac TnI in which the serine residues normally targeted by PKA are mutated to aspartic acid to mimic constitutive phosphorylation (TnIDD22,23). Native cardiac TnI was near completely replaced in one transgenic line as assessed by in vitro phosphorylation, and this led to reduced calcium sensitivity of myofibrillar MgATPase, as expected. TnIDD22,23 mice had mildly enhanced basal systolic and diastolic function, and displayed marked augmentation of frequency-dependent inotropy and relaxation, with a peak frequency response 2-fold greater in mutants than controls (P<0.005). Increasing afterload prolonged relaxation more in nontransgenic than TnIDD22,23 (P<0.02), whereas contractile responses to afterload were similar between these strains. Isoproterenol treatment eliminated the differential force-frequency and afterload response between TnIDD22,23 and controls. In contrast to in vivo studies, isolated isometric trabeculae from nontransgenic and TnIDD22,23 mice had similar basal, isoproterenol-, and frequency-stimulated function, suggesting that muscle shortening may be important to TnI PKA effects. These results support a novel role for cardiac TnI PKA phosphorylation in the rate-dependent enhancement of systolic and diastolic function in vivo and afterload sensitivity of relaxation. These results have implications for cardiac failure in which force-frequency modulation is blunted and afterload relaxation sensitivity increased in association with diminished PKA TnI phosphorylation.

Interplay of Troponin- and Myosin-Based Pathways of Calcium Activation in Skeletal and Cardiac Muscle: The Use of W7 as an Inhibitor of Thin Filament Activation

To investigate the interplay between the thin and thick filaments during calcium activation in striated muscle, we employed n-(6-aminohexyl) 5-chloro-1-napthalenesulfonamide (W7) as an inhibitor of troponin C and compared its effects with that of the myosin-specific inhibitor, 2,3-butanedione 2-monoxime (BDM). In both skeletal and cardiac fibers, W7 reversibly inhibited ATPase and tension over the full range of calcium activation between pCa 8.0 and 4.5, resulting in reduced calcium sensitivity and cooperativity of ATPase and tension activations. At maximal activation in skeletal fibers, the W7 concentrations for half-maximal inhibition ( K I) were 70–80 μM for ATPase and 20–30 μM for tension, nearly >200-fold lower than BDM (20 mM and 5–8 mM, respectively). When W7 (50 μM) and BDM (20 mM) were combined in skeletal fibers, the ATPase and tension-pCa curves exhibited lower apparent cooperativity and maxima and higher calcium sensitivity than expected from two independent activation pathways, suggesting that the interplay between the thin and thick filaments varies with the level of activation. Significantly, the inhibition of W7 increased the ATPase/tension ratio during activation in both muscle types. W7 holds much promise as a potent and reversible inhibitor of thin filament-mediated calcium activation of skeletal and cardiac muscle contraction.