Ion channel macromolecular complexes in cardiomyocytes: roles in sudden cardiac death.

The movement of ions across specific channels embedded on the membrane of individual cardiomyocytes is crucial for the generation and propagation of the cardiac electric impulse. Emerging evidence over the past 20 years strongly suggests that the normal electric function of the heart is the result of dynamic interactions of membrane ion channels working in an orchestrated fashion as part of complex molecular networks. Such networks work together with exquisite temporal precision to generate each action potential and contraction. Macromolecular complexes play crucial roles in transcription, translation, oligomerization, trafficking, membrane retention, glycosylation, post-translational modification, turnover, function, and degradation of all cardiac ion channels known to date. In addition, the accurate timing of each cardiac beat and contraction demands, a comparable precision on the assembly and organizations of sodium, calcium, and potassium channel complexes within specific subcellular microdomains, where physical proximity allows for prompt and efficient interaction. This review article, part of the Compendium on Sudden Cardiac Death, discusses the major issues related to the role of ion channel macromolecular assemblies in normal cardiac electric function and the mechanisms of arrhythmias leading to sudden cardiac death. It provides an idea of how these issues are being addressed in the laboratory and in the clinic, which important questions remain unanswered, and what future research will be needed to improve knowledge and advance therapy.

NMR structural analysis of cardiac troponin C: Monitoring conformational changes induced by binding calcium, troponin I, and calcium -sensitizing drugs

Contraction of cardiac muscle is regulated through the Ca2+ dependent protein-protein interactions of the troponin complex (Tn). The critical role cardiac troponin C (cTnC) plays as the Ca2+ receptor in this complex makes it an attractive target for positive inotropic compounds. In this study, the ten Met methyl groups in cTnC, [98% 13C ϵ]-Met cTnC, are used as structural markers to monitor conformational changes in cTnC and identify sites of interaction between cTnC and cardiac troponin I (cTnI) responsible for the Ca2+ dependent interactions. In addition the structural consequences that a number of Ca2+-sensitizing compounds have on free cTnC and the cTnC·cTnI complex were characterized. Using heteronuclear NMR experiments and monitoring chemical shift changes in the ten Met methyl 1H-13C correlations in 3Ca2+ cTnC when bound to cTnI revealed an anti-parallel arrangement for the two proteins such that the N-domain of cTnI interacts with the C-domain of cTnC. The large chemical shifts in Mets-81, -120, and -157 identified points of contact between the proteins that include the C-domain hydrophobic surface in cTnC and the A, B, and D helical interface located in the regulatory N-domain of cTnC. TnI association [cTnI(33–80), cTnI(86–211), or cTnI(33–211)] was found also to dramatically reduce flexibility in the D/E central linker of cTnC as monitored by line broadening in the Met 1H- 13C correlations of cTnC induced by a nitroxide spin label, MTSSL, covalently attached to cTnC at Cys 84. TnI association resulted in an extended cTnC that is unlike the compact structure observed for free cTnC. The Met 1H-13C correlations also allowed the binding characteristics of bepridil, TFP, levosimendan, and EMD 57033 to the apo, 2Ca2+, and Ca2+ saturated forms of cTnC to be determined. In addition, the location of drug binding on the 3Ca2+cTnC·cTnI complex was identified for bepridil and TFP. Use of a novel spin-labeled phenothiazine, and detection of isotope filtered NOEs, allowed identification of drug binding sites in the shallow hydrophobic cup in the C-terminal domain, and on two hydrophobic surfaces on N-regulatory domain in free 3Ca2+ cTnC. In contrast, only one N-domain drug binding site exists in 3Ca2+ cTnC·cTnI complex. The methyl groups of Met 45, 60 and 80, which are grouped in a hydrophobic patch near site II in cTnC, showed the greatest change upon titration with bepridil or TFP, suggesting that this is a critical site of drug binding in both free cTnC and when associated with cTnI. The strongest NOEs were seen for Met-60 and -80, which are located on helices C and D, respectively, of Ca2+ binding site II. These results support the conclusion that the small hydrophobic patch which includes Met-45, -60, and -80 constitutes a drug binding site, and that binding drugs to this site will lead to an increase in Ca2+ binding affinity of site II while preserving maximal cTnC activity. Thus, the subregion in cTnC makes a likely target against which to design new and selective Ca2+-sensitizing compounds. ^

Bepridil opens the regulatory N-terminal lobe of cardiac troponin C

Cardiac troponin C (cTnC) is the calcium-dependent switch for contraction in heart muscle and a potential target for drugs in the therapy of congestive heart failure. This calmodulin-like protein consists of two lobes connected by a central linker; each lobe contains two EF-hand domains. The regulatory N-terminal lobe of cTnC, unlike that of skeletal troponin C (sTnC), contains only one functional EF-hand and does not open fully upon the binding of Ca2+. We have determined the crystal structure of cTnC, with three bound Ca2+ ions, complexed with the calcium-sensitizer bepridil, to 2.15-Å resolution. In contrast to apo- and 3Ca2+-cTnC, the drug-bound complex displays a fully open N-terminal lobe similar to the N-terminal lobes of 4Ca2+-sTnC and cTnC bound to a C-terminal fragment of cardiac troponin I (residues 147–163). The closing of the lobe is sterically hindered by one of the three bound bepridils. Our results provide a structural basis for the Ca2+-sensitizing effect of bepridil and reveal the details of a distinctive two-stage mechanism for Ca2+ regulation by troponin C in cardiac muscle.

Regulation of fatty acid homeostasis in cells: Novel role of leptin

It is proposed that an important function of leptin is to confine the storage of triglycerides (TG) to the adipocytes, while limiting TG storage in nonadipocytes, thus protecting them from lipotoxicity. The fact that TG content in nonadipocytes normally remains within a narrow range, while that of adipocytes varies enormously with food intake, is consistent with a system of TG homeostasis in normal nonadipocytes. The facts that when leptin receptors are dysfunctional, TG content in nonadipocytes such as islets can increase 100-fold, and that constitutively expressed ectopic hyperleptinemia depletes TG, suggest that leptin controls the homeostatic system for intracellular TG. The fact that the function and viability of nonadipocytes is compromised when their TG content rises above or falls below the normal range suggests that normal homeostasis of their intracellular TG is critical for optimal function and to prevent lipoapoptosis. Thus far, lipotoxic diabetes of fa/fa Zucker diabetic fatty rats is the only proven lipodegenerative disease, but the possibility of lipotoxic disease of skeletal and/or cardiac muscle may require investigation, as does the possible influence of the intracellular TG content on autoimmune and neoplastic processes.

Radial and longitudinal diffusion of myoglobin in single living heart and skeletal muscle cells

We have used a fluorescence recovery after photobleaching (FRAP) technique to measure radial diffusion of myoglobin and other proteins in single skeletal and cardiac muscle cells. We compare the radial diffusivities, Dr (i.e., diffusion perpendicular to the long fiber axis), with longitudinal ones, Dl (i.e., parallel to the long fiber axis), both measured by the same technique, for myoglobin (17 kDa), lactalbumin (14 kDa), and ovalbumin (45 kDa). At 22°C, Dl for myoglobin is 1.2 × 10−7 cm2/s in soleus fibers and 1.1 × 10−7 cm2/s in cardiomyocytes. Dl for lactalbumin is similar in both cell types. Dr for myoglobin is 1.2 × 10−7 cm2/s in soleus fibers and 1.1 × 10−7 cm2/s in cardiomyocytes and, again, similar for lactalbumin. Dl and Dr for ovalbumin are 0.5 × 10−7 cm2/s. In the case of myoglobin, both Dl and Dr at 37°C are about 80% higher than at 22°C. We conclude that intracellular diffusivity of myoglobin and other proteins (i) is very low in striated muscle cells, ≈1/10 of the value in dilute protein solution, (ii) is not markedly different in longitudinal and radial direction, and (iii) is identical in heart and skeletal muscle. A Krogh cylinder model calculation holding for steady-state tissue oxygenation predicts that, based on these myoglobin diffusivities, myoglobin-facilitated oxygen diffusion contributes 4% to the overall intracellular oxygen transport of maximally exercising skeletal muscle and less than 2% to that of heart under conditions of high work load.

Unravelling the complexity of muscle impairment in chronic neck pain

Exercise interventions are deemed essential for the effective management of patients with neck pain. However, there has been a lack of consensus on optimal exercise prescription, which has resulted from a paucity of studies to quantify the precise nature of muscle impairment, in people with neck pain. This masterclass will present recent research from our laboratory, which has utilized surface electromyography to investigate cervical flexor muscle impairment in patients with chronic neck pain. This research has identified deficits in the motor control of the deep and superficial cervical flexor muscles in people with chronic neck pain, characterized by a delay in onset of neck muscle contraction associated with movement of the upper limb. In addition, people with neck pain demonstrate an altered pattern of muscle activation, which is characterized by reduced deep cervical flexor muscle activity during a low load cognitive task and increased activity of the superficial cervical flexor muscles during both cognitive tasks and functional activities. The results have demonstrated the complex, multifaceted nature of cervical muscle impairment, which exists in people with a history of neck pain. In turn, this has considerable implications for the rehabilitation of muscle function in people with neck pain disorders. (C) 2004 Elsevier Ltd. All rights reserved.

Neck flexor muscle fatigue is side specific in patients with unilateral neck pain

Despite the evidence of greater fatigability of the cervical flexor muscles in neck pain patients, the effect of unilaterality of neck pain on muscle fatigue has not been investigated. This study compared myoelectric manifestations of sternocleidomastoid (SCM) and anterior scalene (AS) muscle fatigue between the painful and non-painful sides in patients with chronic unilateral neck pain. Myoelectric signals were recorded from the sternal head of SCM and the AS muscles bilaterally during sub-maximal isometric cervical flexion contractions at 25% and 50% of the maximum voluntary contraction (MVC). The time course of the mean power frequency, average rectified value and conduction velocity of the electromyographic signals were calculated to quantify myoelectric manifestations of muscle fatigue. Results revealed greater estimates of the initial value and slope of the mean frequency for both the SCM and AS muscles on the side of the patient's neck pain at 25% and 50% of MVC. These results indicate greater myoelectric manifestations of muscle fatigue of the superficial cervical flexor muscles ipsilateral to the side of pain. This suggests a specificity of the effect of pain on muscle function and hence the need for specificity of therapeutic exercise in the management of neck pain patients. (C) 2003 European Federation of Chapters of the International Association for the Study of Pain. Published by Elsevier Ltd. All rights reserved.

Investigation of signaling pathways that mediate the inotropic effect of urotensin-II in human heart

Objective: This study investigated signaling pathways that may contribute to the potent positive inotropic effect of human urotensin-II (hU-II) in human isolated right atrial trabeculae obtained from patients with coronary artery disease. Methods: Trabeculae were set up in tissue baths and stimulated to contract at 1 Hz. Tissues were incubated with 20 nM hU-II with or without phorbol 12-myristate 13-acetate (PMA, 10 muM) to desensitize PKC, the PKC inhibitor chelerythrine (10 muM), 10 muM 4alpha-phorbol that does not desensitize PKC, the myosin light chain kinase inhibitor wortmannin (50 nM, 10 muM), or the Rho kinase inhibitor Y-27632 (0.1 - 10 muM). Activated RhoA was determined by affinity immunoprecipitation, and phosphorylation of signaling proteins was determined by SDS-PAGE. Results: hU-II caused a potent positive inotropic response in atrial trabeculae, and this was concomitant with increased phosphorylation of regulatory myosin light chain (MLC-2, 1.8 +/- 0.4-fold, P < 0.05, n = 6) and PKCalpha/betaII (1.4 +/- 0.2-fold compared to non-stimulated controls, P < 0.05, n = 7). Pretreatment of tissues with PMA caused a marked reduction in the inotropic effect of hU-II, but did not affect hU-II-mediated phosphorylation of MLC-2. The inotropic response was inhibited by chelerythrine, but not 4alpha-phorbol or wortmannin. Although Y-27632 also reduced the positive inotropic response to hU-II, this was associated with a marked reduction in basal force of contraction. RhoA. GTP was immunoprecipitated in tissues pretreated with or without hU-II, with findings showing no detectable activation of RhoA in the agonist stimulated tissues. Conclusions: The findings indicated that hU-II increased force of contraction in human heart via a PKC-dependent mechanism and increased phosphorylation of MLC-2, although this was independent of PKC. The positive inotropic effect was independent of myosin light chain kinase and RhoA-Rho kinase signaling pathways. (C) 2004 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.

Resistance training for the older adult: Manipulating training variables to enhance muscle strength

Resistance training has been shown to reliably and substantially enhance muscle function in older adults and these improvements can be accompanied by improved functional performance. Training variables should be manipulated to enhance muscle strength and minimize injury risks in this population.

Detection of elevated protein modification in human cerebellum in alcoholic cerebellar degeneration

Modification of proteins by reactive ethanol metabolites has been known for some time to occur in the liver, the main site of ethanol metabolism. In more recent studies of laboratory animals, similar modifications have been detected in organs with lesser ability to metabolize ethanol, such as skeletal and cardiac muscle and brain. Such modification may alter protein function or form a neoantigen, making it a target for immune attack. We now report an analysis of protein modification derived from ethanol metabolites in human brain tissue by ELISA using adduct-specific antibodies. We obtained autopsy cerebellum samples from 10 alcoholic cerebellar degeneration cases and 10 matched controls under informed written consent from the next of kin and clearance from the UQ Human Ethics Committee. Elevated levels of protein modifications derived from acetaldehyde (unreduced-acetaldehyde and acetaldehyde-advanced glycation end-product adducts), from malondialdehyde (malondialdehyde adducts) and from combined adducts (malondialdehydeacetaldehyde (MAA) adducts) were detected in alcoholic cerebellar degeneration samples when compared to controls. Other adduct types found in liver samples, such as reduced-acetaldehyde and those derived from hydroxyethyl radicals, were not detected in brain samples. This may reflect the different routes of ethanol metabolism in the two tissues. This is the first report of elevated protein modification in alcoholic cerebellar degeneration, and suggests that such modification may play a role in the pathogenesis of brain injury. Supported by NIAAA under grant NIH AA12404 and the NHMRC (Australia) under grant #981723.

Detection of elevated protein modification in alcoholic cerebellar degeneration

Modification of proteins by reactive ethanol metabolites has been known, for some time, to occur in the liver, the main site of ethanol metabolism. More recently, similar modifi cation has been detected in organs with lesser ability to metabolise ethanol such as skeletal and cardiac muscle, and brain. Such modifi cation may alter protein function or form a neoantigen, making it a target for immune attack.

Improved estimation of postmortem interval with Cardiac Troponin T and improved analytical methods

Accurate knowledge of the time since death, or postmortem interval (PMI), has enormous legal, criminological, and psychological impact. In this study, an investigation was made to determine whether the relationship between the degradation of the human cardiac structure protein Cardiac Troponin T and PMI could be used as an indicator of time since death, thus providing a rapid, high resolution, sensitive, and automated methodology for the determination of PMI. ^ The use of Cardiac Troponin T (cTnT), a protein found in heart tissue, as a selective marker for cardiac muscle damage has shown great promise in the determination of PMI. An optimized conventional immunoassay method was developed to quantify intact and fragmented cTnT. A small sample of cardiac tissue, which is less affected than other tissues by external factors, was taken, homogenized, extracted with magnetic microparticles, separated by SDS-PAGE, and visualized with Western blot by probing with monoclonal antibody against cTnT. This step was followed by labeling and available scanners. This conventional immunoassay provides a proper detection and quantitation of cTnT protein in cardiac tissue as a complex matrix; however, this method does not provide the analyst with immediate results. Therefore, a competitive separation method using capillary electrophoresis with laser-induced fluorescence (CE-LIF) was developed to study the interaction between human cTnT protein and monoclonal anti-TroponinT antibody. ^ Analysis of the results revealed a linear relationship between the percent of degraded cTnT and the log of the PMI, indicating that intact cTnT could be detected in human heart tissue up to 10 days postmortem at room temperature and beyond two weeks at 4C. The data presented demonstrates that this technique can provide an extended time range during which PMI can be more accurately estimated as compared to currently used methods. The data demonstrates that this technique represents a major advance in time of death determination through a fast and reliable, semi-quantitative measurement of a biochemical marker from an organ protected from outside factors. ^

Engineering Highly-functional, Self-regenerative Skeletal Muscle Tissues with Enhanced Vascularization and Survival in Vivo

Tissue engineering of biomimetic skeletal muscle may lead to development of new therapies for myogenic repair and generation of improved in vitro models for studies of muscle function, regeneration, and disease. For the optimal therapeutic and in vitro results, engineered muscle should recreate the force-generating and regenerative capacities of native muscle, enabled respectively by its two main cellular constituents, the mature myofibers and satellite cells (SCs). Still, after 20 years of research, engineered muscle tissues fall short of mimicking contractile function and self-repair capacity of native skeletal muscle. To overcome this limitation, we set the thesis goals to: 1) generate a highly functional, self-regenerative engineered skeletal muscle and 2) explore mechanisms governing its formation and regeneration in vitro and survival and vascularization in vivo.

By studying myogenic progenitors isolated from neonatal rats, we first discovered advantages of using an adherent cell fraction for engineering of skeletal muscles with robust structure and function and the formation of a SC pool. Specifically, when synergized with dynamic culture conditions, the use of adherent cells yielded muscle constructs capable of replicating the contractile output of native neonatal muscle, generating >40 mN/mm2 of specific force. Moreover, tissue structure and cellular heterogeneity of engineered muscle constructs closely resembled those of native muscle, consisting of aligned, striated myofibers embedded in a matrix of basal lamina proteins and SCs that resided in native-like niches. Importantly, we identified rapid formation of myofibers early during engineered muscle culture as a critical condition leading to SC homing and conversion to a quiescent, non-proliferative state. The SCs retained natural regenerative capacity and activated, proliferated, and differentiated to rebuild damaged myofibers and recover contractile function within 10 days after the muscle was injured by cardiotoxin (CTX). The resulting regenerative response was directly dependent on the abundance of SCs in the engineered muscle that we varied by expanding starting cell population under different levels of basic fibroblast growth factor (bFGF), an inhibitor of myogenic differentiation. Using a dorsal skinfold window chamber model in nude mice, we further demonstrated that within 2 weeks after implantation, initially avascular engineered muscle underwent robust vascularization and perfusion and exhibited improved structure and contractile function beyond what was achievable in vitro.

To enhance translational value of our approach, we transitioned to use of adult rat myogenic cells, but found that despite similar function to that of neonatal constructs, adult-derived muscle lacked regenerative capacity. Using a novel platform for live monitoring of calcium transients during construct culture, we rapidly screened for potential enhancers of regeneration to establish that many known pro-regenerative soluble factors were ineffective in stimulating in vitro engineered muscle recovery from CTX injury. This led us to introduce bone marrow-derived macrophages (BMDMs), an established non-myogenic contributor to muscle repair, to the adult-derived constructs and to demonstrate remarkable recovery of force generation (>80%) and muscle mass (>70%) following CTX injury. Mechanistically, while similar patterns of early SC activation and proliferation upon injury were observed in engineered muscles with and without BMDMs, a significant decrease in injury-induced apoptosis occurred only in the presence of BMDMs. The importance of preventing apoptosis was further demonstrated by showing that application of caspase inhibitor (Q-VD-OPh) yielded myofiber regrowth and functional recovery post-injury. Gene expression analysis suggested muscle-secreted tumor necrosis factor-α (TNFα) as a potential inducer of apoptosis as common for muscle degeneration in diseases and aging in vivo. Finally, we showed that BMDM incorporation in engineered muscle enhanced its growth, angiogenesis, and function following implantation in the dorsal window chambers in nude mice.

In summary, this thesis describes novel strategies to engineer highly contractile and regenerative skeletal muscle tissues starting from neonatal or adult rat myogenic cells. We find that age-dependent differences of myogenic cells distinctly affect the self-repair capacity but not contractile function of engineered muscle. Adult, but not neonatal, myogenic progenitors appear to require co-culture with other cells, such as bone marrow-derived macrophages, to allow robust muscle regeneration in vitro and rapid vascularization in vivo. Regarding the established roles of immune system cells in the repair of various muscle and non-muscle tissues, we expect that our work will stimulate the future applications of immune cells as pro-regenerative or anti-inflammatory constituents of engineered tissue grafts. Furthermore, we expect that rodent studies in this thesis will inspire successful engineering of biomimetic human muscle tissues for use in regenerative therapy and drug discovery applications.

Poly(3-hydroxyoctanoate), a promising new material for cardiac tissue engineering

Cardiac tissue engineering (CTE) is currently a prime focus of research due to an enormous clinical need. In this work, a novel functional material, Poly(3-hydroxyoctanoate), P(3HO), a medium chain length polyhydroxyalkanoate (PHA), produced using bacterial fermentation, was studied as a new potential material for CTE. Engineered constructs with improved mechanical properties, crucial for supporting the organ during new tissue regeneration, and enhanced surface topography, to allow efficient cell adhesion and proliferation, were fabricated. Our results showed that the mechanical properties of the final patches were close to that of cardiac muscle. Biocompatibility of the P(3HO) neat patches, assessed using Neonatal ventricular rat myocytes (NVRM), showed that the polymer was as good as collagen in terms of cell viability, proliferation and adhesion. Enhanced cell adhesion and proliferation properties were observed when porous and fibrous structures were incorporated to the patches. Also, no deleterious effect was observed on the adults cardiomyocytes’ contraction when cardiomyocytes were seeded on the P(3HO) patches. Hence, P(3HO) based multifunctional cardiac patches are promising constructs for efficient CTE. This work will provide a positive impact on the development of P(3HO) and other PHAs as a novel new family of biodegradable functional materials with huge potential in a range of different biomedical applications, particularly CTE, leading to further interest and exploitation of these materials.