Actin Myosin Interaction

Nanomedicine and Nanotechnology for Heart Failure Research, Diagnosis, and Treatment

Kevin O. Maher , in Heart Failure in the Child and Young Adult, 2018

Nanotechnology Used in the Study of Myocellular Contractility

Actin–myosin interaction and force generation are key to myocardial function and central to the pathophysiology of heart failure. A nano approach to investigation of actin–myosin physiology allows for research to be done at the level of the individual molecule, with the potential to increase the understanding of both normal physiology and the diseased state of the myocardium. Hariadi and colleagues developed a DNA nanoscaffold for studying actin–myosin behavior. Direct visualization of actin filament gliding, recognizing that changes in cross-bridge compliance that influences gliding speed, with myosin ensembles serving as energy reservoirs (Fig. 60.5) [16].

Figure 60.5. (A) Schematic of nanotube filament motility assay for actin–myosin model of contraction. (B) Time-lapsed images of actin filaments in green moving along human cardiac myosin labeled nanotubes in red.

Liu and colleagues evaluated the nanomechanical features of living, single cardiomyocytes, quantitatively measuring torsions, contractions, and calcium intensity using an atomic force cantilever, part of atomic force microscopy [9]. The atomic force microscope (Fig. 60.1) was a milestone in nanotechnology development that occurred in the 1980's as the scanning tunneling instrument was also being invented. Drug-induced changes in contractility and force generation at the nanonewton level were determined for the individual myocyte. This type of nano research will allow a greater understanding of force generation within the myocyte, the pathophysiology of heart failure, and can serve as a model for in vitro drug development and testing.

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The Cytoskeletal Network of the Trabecular Meshwork*

B. Tian , ... B. Geiger , in Encyclopedia of the Eye, 2010

Actomyosin-Modulating Gene Therapy

Modulating proteins that negatively regulate actin–myosin interactions can also induce TM relaxation. Caldesmon is such a protein, whose function is the regulation of actomyosin contractility. When caldesmon is overexpressed, actin becomes uncoupled from myosin, which can affect both actomyosin-driven contractility and actin polymerization. In addition, exoenzyme C3 transferase may also affect actin–myosin interactions. Rho GTPases are the preferred intracellular targets of exoenzyme C3 transferase. The latter specifically inhibits Rho-GTP at the beginning of the Rho activation cascade, thereby blocking the whole Rho cascade. Adenovirus-delivered exoenzyme C3 transferase (C3-toxin) complementary DNA (cDNA) and nonmuscle caldesmon cDNA have been successfully expressed in cultured human TM cells. Perfusions in organ-cultured human or monkey eyes following overexpression of these genes have shown significant increases in outflow facility. Specific inhibition of Rho-kinase activity in the TM by dominant-negative Rho expression also increases outflow facility in organ-cultured anterior segments of postmortem human eyes. All these suggest that, similar to pharmacological approaches, gene therapies may also inhibit actomyosin system in the TM and in turn increase trabecular outflow facility through blocking the Rho activation pathway and overexpressing modulating proteins.

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Inherited Cardiomyopathies

Polakit Teekakirikul , ... Christine E. Seidman , in Emery and Rimoin's Principles and Practice of Medical Genetics (Sixth Edition), 2013

47.2.5 Animal Models of HCM

Mutations in sarcomere proteins may alter actin-myosin interaction, intracellular calcium cycling, force generation or the transmission of force. However, the heterogeneity of HCM in the human population has challenged the dissection of the precise molecular mechanisms that lead from inherited gene defect to clinical phenotype.

To evaluate the consequences of specific mutations experimental cell and in vivo models of HCM have been developed. Genetically modified animals have been developed incorporating different human mutations in MYH7, MYBPC3, and TNNT, and TNNI3. Rodents carrying these gene mutations develop a phenotype that recapitulates human HCM, including age-dependent myocardial hypertrophy, fibrosis, and mycoyte disarray. Interrogation of the biophsyical properties of sarcomeres with particular mutations with myocardial contractility, myocyte biochemistry and transcriptional responses has helped to clarify the molecular events associated with specific HCM mutations.

An HCM model (denoted as α-MHC403/+) that expresses the human myosin heavy chain mutation, Arg403Gln (88) has been extensively studied. The cardiac phenotype of α-MHC403/+ mice closely mirrors human HCM histopathology. Although general life expectancy is not significantly altered, heterozygous mice demonstrate increased arrhythmogenicity and risk for exercise-induced sudden death as compared with their wild-type littermates (89). Enhanced systolic contractile performance (dominant-activating effect) was identified by examining single mutant myosin molecules from α-MHC403/+ myocytes as well as intact heart preparations (90–92). Diastolic function has been shown to be significantly impaired even prior to the development of hypertrophy (93).

Why sarcomere protein mutations in cardiac myocytes that enhance biophysical properties lead to cardiac hypertrophy became an important mechanistic question. Biochemical studies highlighted the crucial role of intracellular Ca2+ handling plays in linking alterations of muscle contraction to myocyte hypertrophy. α-MHC403/+ myocytes have diminished SR release of calcium in response to caffeine and decreased levels of calcium binding proteins calsequestrin, ryanidine receptor, triadin, and junctin (94). These biochemical alterations may worsen cardiac diastolic dysfunction in HCM. Abnormal calcium signaling has been demonstrated in other mouse models harboring a human mutation in troponin T (95) and regulatory myosin light chains (96) and has been proposed to be a remarkable characteristic of other cardiomyopathies (97,98).

The biochemical defects identified in HCM models has raised the possibility that diminishing calcium signals might be beneficial in HCM. To test this, the L-type calcium channel blocker, diltiazem was administered to young α-MHC403/+ mice (99) prior to the development of histologic or morphologic abnormalities (prehypertrophic phase: age 6–8 weeks). Diltiazem treatment was associated with a decreased degree of fibrosis, and hypertrophy, both at the gross and histologic levels. Aberrant myocyte biochemistry was also improved with normalization of levels of calcium binding proteins (Figure 47-5) (94,100). One consequence of the Arg403Gln mutation may be abnormal sequestration of calcium by the mutant sarcomere with ultimate depletion of SR Ca2+ levels. Diltiazem may serve to blunt this effect by restoring more appropriate calcium cycling between the SR and the cytoplasm and thereby interrupting the signals leading to hypertrophic remodeling. Fundamental dysregulation of calcium handling caused by myosin mutations may in part mediate the hypertrophic response of the myocyte (Figure 47-4).

FIGURE 47-5. Normalization of abnormal Ca2+ improves histopathology in a mouse model of HCM. Left panel. Histopathology of diltiazem-treated wild-type and α-MHC403/+ mice as compared with untreated mice. Whole heart sections (top; magnification = 2.5×) stained with Masson trichrome reveals fibrosis (blue staining) in untreated HCM mice. Histology (bottom; magnification = 100×) shows a reduction in myocyte hypertrophy, disarray, and fibrosis in diltiazem-treated animals compared with untreated animals. Right panel. Cardiac myocyte Ca2+-related protein expression is normalized by diltiazem treatment. Western blot analysis of calsequestrin (CSQ), ryanodine receptor (RyR2), triadin, and junctin shows a reduction in the amount of protein expression in untreated α-MHC403/+ mice. Diltiazem treatment for 8 days (CSQ) or 30 weeks (triadin, junctin, RyR2, CSQ) leads to normalization of the expression levels of these proteins. Coomassie staining is shown to indicate sample loading in each lane.

 (Adapted from Semsarian, C., Ahmad, I., Giewat, M., et al. The L-type Calcium Channel Inhibitor Diltiazem Prevents Cardiomyopathy in a Mouse Model. J. Clin. Invest. 2002, 109, 1013, with permission of The American Society for Clinical Investigation.)

Calcium cycling abnormalities may enhance the probability for after depolarization in cardiac myocytes, a cellular substrate for cardiac arrhythmias, a rampant complication in HCM (101). Electrophysiologic studies in MHC403/+ mice showed more ventricular arrhythmias than in mice with low-risk mutations in the myosin binding protein C (102,103). MHC403/+ mice have also been used to asses the correlation between HCM histopathology and arrhythmic events. In human HCM, myocardial fibrosis has been recognized in postmortem hearts from HCM patients who succumbed to SCD (104) and myocardial fibrosis has been proposed to be a significant contributor to life-threatening arrhythmias (105,106). However, analyses of arrhythmia susceptibility in α-MHC403/+ mice in the context of myocardial fibrosis, hypertrophy and myocyte disarray demonstrated a significant correlation only between inducible ventribular arrhythmias and the degree of hypertrophy (107). Consistent with these experimental data, substantial LVH (LV wall thickness ≥30mm) is established risk factor for sudden death in human patients (108). Given these findings, the strategies that prevent hypertrophy in HCM would potentially reduce the risk for arrhythmia development and sudden cardiac death. Alteration of biophysical properties and intracellular Ca2+ in HCM myocytes as well as higher energy demands impose great stress on cardiac myocytes. Molecular studies in the α-MHC403/+ mouse model also indicate that fetal cardiac genes typically repressed following embryonic development re-expressed with myocyte stress (109), a finding similar to the studies in human HCM hearts (110). Recent investigations have also uncovered a relationship between the early onset of calcium cycling defect in stressed myocytes and the premature myocyte demise and myocardial scarring (interstitial matrix expansion and focal replacement fibrosis) in HCM mouse models (111). The myocyte enhancer factor-2 (Mef2) family of transcription factors is activated in several myocardial pathologies that are marked by cardiac hypertrophy and myocardial fibrosis (112) and Mef2 expression identifies stressed myocytes. Mef2 activity has been assessed in α-MHC403/+ mice, using a Mef2-LacZ (β-galactosidase) transgene. Mef2 activity in HCM hearts was strikingly inhomogeneous and closely associated with foci of myocyte necrosis and scarring (Figure 47-6). In homozygous mutant (α-MHC403/403) mice that have marked and relentless pathologic remodeling until death at day 10, Mef2 activation was substantially accentuated and evident shortly before rampant myocyte demise, implicating Mef2 activation in HCM as a molecular marker of stressed myocytes that are destined to die (111). Abnormal calcium signaling can lead to Mef2 activation via calcium/calmodulin dependent protein kinase II (CaMKII) phosphorylation (113,114), a pathway that may link aberrant calcium signaling to premature myocyte death and focal myocardial scarring in HCM hearts.

FIGURE 47-6. Mef2 activation in HCM myocytes. A. Colocalization of Mef2-dependent reporter activity with fibrosis (left, Gomori trichrome staining) and necrosis (right, von Kossa staining) in 30-week-old αMHC403/+/Mef2-LacZ hearts. B. βMHC re-expression (green, identified by YFP) occurred in regions of myocardial scars (red, identified by WGA) in 30-week-old αMHC403/+/Mef2-LacZ/βMHC-YFP hearts. Right shows magnified view of inset. C. Cross-sectional areas of myocytes from 30-week-old WT/Mef2-LacZ/βMHC-YFP hearts (WT cells; open bar) and αMHC403/+/Mef2-LacZ/βMHC-YFP hearts (403/+ cells; black bars) irrespective of their fluorescence. D. Cross-sectional areas of the 403/+ cells grouped according to their YFP fluorescence (YFP− and YFP+, respectively). E. Increased phosphorylation of HDAC5 and Mef2-dependent reporter activity in αMHC403/403 hearts. Western blot analyses of protein lysates from 6-day-old WT, αMHC403/+(403/+), and αMHC403/403(403/403) hearts, reacted with antibodies specific Phospho-HDAC5, total HDAC5, and GAPDH.

 (Adapted from Konno, T.; Chen, D.; Wang, L., et al. Heterogeneous Myocyte Enhancer Factor-2 (Mef2) Activation in Myocytes Predicts Focal Scarring in Hypertrophic Cardiomyopathy. PNAS 2010, 107, 18097–18102.)

HCM mice have also been studied to define the early transcriptional changes that occur in response to a sarcomere gene mutation (109,115). All expressed RNAs in mutant myocyte and non-myocyte cells from the hearts of young prehypertrophic mice were compared with RNAs found in myocytes and non-myocytes from wild-type mice. These experiments revealed a striking increase in the expression of pro-fibrotic molecules within the transforming growth factor (Tgf) signaling pathway (including Tgf-β1, Tgf-β2, periostin and connective tissue growth factor) and cell-cycle proteins in non-myocyte cells of prehypertrophic hearts. BrdU labeling and Ki617 immunostaining confirmed transcriptional profiling data and revealed increased proliferation of non-myocytes.

To assess whether silencing of these profibrotic molecules could prevent the emergence of HCM pathology, anti-Tgf-β neutralizing antibody was administered to young prehypertrophic mutant mice, a treatment that markedly diminished the emergence of myocardial fibrosis and hypertrophy (Figure 47-7) (115). To extend these studies, losartan, an angiotensin II (type 1) receptor antagonist was also used to inhibit Tgf-β pathway signals. Similar to the neutral antibodies, chronic administration of losartan to prehypertrophic mutant mice prevented the emergence of LV hypertrophy and HCM pathologic features (Figure 47-7). Taken together, these data define Tgf-β signaling as a pivotal mechanism for myocardial fibrosis development and a potentially important contributor to diastolic dysfunction and heart failure (Figure 47-8). Preemptive pharmacologic antagonism of Tgf-β signals warrants clinical study in asymptomatic patients with sarcomere gene mutations.

FIGURE 47-7. Treatment with anti-TGF-β neutralizing antibody (TGF-β NAb) or losartan prevents the development of cardiac hypertrophy and fibrosis. Upper panel. Whole heart sections from mice treated with control IgG (Rb IgG) shows fibrosis (blue, Mason trichrome stain), unlike mice treated with TGF-β NAb or losartan. Lower panel. Histologic sections stained with wheat-germ agglutinin (green) to demonstrate fibrosis and DAPI (blue) from BrdU-treated mice show nonmyocyte cell proliferation (magenta) in Rb IgG-treated mice that is reduced by TGF-β NAb or losartan treatment.

 (Adapted with permission from Teekakirikul, P.; Eminaga, S.; Toka, O., et al. Cardiac Fibrosis in Mice with Hypertrophic Cardiomyopathy Is Mediated by Non-myocyte Proliferation and Requires Tgf-β. J. Clin. Invest. 2010, 120, 3520–3529, with permission of The American Society for Clinical Investigation.)

FIGURE 47-8. A model for fibrosis and diastolic dysfunction from HCM sarcomere gene mutations. Mutant myocytes have increased biophysical properties and abnormal Ca2+ homeostasis, factors that trigger mechanical and/or biochemical signals that activate gene transcription, e.g. Tgf-β. Tgf-β then stimulates nonmyocyte proliferation and expression of profibrotic molecules. Activated nonmyocyte cells secrete profibrotic factors that expand the interstitium, generate more stresses imposed on mutant myocytes, and promote myocyte death with resultant focal scarring. Tgf-β-mediated increased interstitial and focal fibrosis may contribute to diastolic dysfunction in HCM hearts. Antagonism of Tgf-β signaling by a neutralizing antibody (NAb) or losartan attenuates nonmyocyte proliferation and profibrotic gene expression, thereby preventing cardiac fibrosis.

 (Adapted with permission from Teekakirikul, P., Eminaga, S., Toka, O., et al. Cardiac Fibrosis in Mice with Hypertrophic Cardiomyopathy Is Mediated by Non-myocyte Proliferation and Requires Tgf-β. J. Clin. Invest. 2010, 120, 3520–3529, with permission of The American Society for Clinical Investigation.)

In addition to the α-MHC mouse model, a variety of other genetically modified mice, rats, and rabbits have been developed, incorporating mutations in β-myosin heavy chain, cMyBPC, α-tropomyosin, and cardiac troponin T (93,116–120). These models typically show myocyte disarray, variable degrees of hypertrophy, systolic and diastolic dysfunction. The β-MHC403/+ transgenic rabbit model (117) is an attractive model, as the β-isoform of MHC is predominant in rabbit and adult human hearts, whereas the α-isoform predominates in mice and rats. These rabbits show significant hypertrophy, fibrosis, diastolic dysfunction and an increased risk of sudden death (117). As with the α-MHC403/+ mouse model, impaired myocardial relaxation develops in advance of hypertrophy (121). This finding has recently been replicated and extended to human patients with HCM (122).

Pharmacologic studies have also been performed to target fibrosis pathways rather than intracellular calcium handling and to reverse myocardial fibrosis in some other animal models of HCM. For examples, angiotensin II receptor type 1 blockade was associated with the reversal of interstitial fibrosis and decreased expression of collagen Ia and Tgf-β1 in mice with a cardiac troponin T mutation (123) who received losartan treatment (124). β-MHC403/+ transgenic rabbits were administered the HMG-CoA reductase inhibitor, simvastatin, for 12 weeks. As compared with placebo-treated transgenic animals, simvastatin therapy was associated with regression of hypertrophy and fibrosis as well as improved cardiac function and decreased intracardiac filling pressures. In addition, levels of activated stress-responsive signaling kinases (ERK 1/2) were reduced, suggesting that these pathways that trigger myocardial fibrosis and dysfunction may be modulated to improve disease expression (123).

Elevated myocardial aldosterone and aldosterone synthase mRNA levels have been demonstrated in both humans with HCM and in cTnT-Q (123) mice. Treatment with the mineralocorticoid receptor antagonist, spironolactone, reversed interstitial fibrosis, attenuated myocyte disarray, and improved diastolic function (125). These promising results in animal studies highlight the importance of ongoing collaborative basic science and human clinical investigation to further our understanding of the pathophysiology of HCM and to allow for the development of novel treatment strategies for human disease.

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Diagnostic Tests in Cardio-oncology

Gina Biasillo MD , ... W. Gregory Hundley MD , in Clinical Cardio-Oncology, 2016

Troponins

The troponin complex, involved in the actin-myosin interaction in the cardiomyocytes, thus cardiac contraction and relaxation, is composed of 3 subunits, troponin C, troponin T, and troponin I (Fig. 16.1). It is mostly found in the sarcomeres of myocardial cells and in small quantities in the cytoplasm. With cardiomyocyte necrosis, there is a rapid depletion of the cytoplasmic pool, followed by a larger release of troponins into circulation after breakdown of the contractile apparatus. Accordingly, detection of cardiac troponins (cTns) in peripheral blood is indicative of cardiomyocyte necrosis. CTns have high tissue specificity and an evolving sensitivity for the detection of (even small amounts of) myocardial necrosis with the new highly sensitive (HS) assays. Historically, only one assay has been patented for cardiac troponin T (cTnT), but multiple assays are available for cardiac troponin I (cTnI). These assays will be collectively referred to as cTns unless otherwise specified.

Initially, cTns emerged as very useful markers in the diagnosis and risk stratification of patients with suspected and proven acute coronary syndromes. 7 However, their use has been extended to detect cardiac damage in other clinical settings, such as cardiac hypertrophy, heart failure, acute pulmonary embolism, blunt trauma, sepsis, stroke, and renal insufficiency. 8 Moreover, in the 2 last decades, cTns have also been evaluated in oncologic settings, in particular to detect cardiac toxicity associated with anticancer drugs (Table 16.1). 21

The role of cTns as indicators of anthracycline-induced cardiotoxicity was first studied in animal models. CTnT elevation correlated with administered drug dose and with the degree of cardiac damage, as demonstrated by histology. 22 A large number of studies suggest the translation of these findings to patients receiving potentially cardiotoxic therapies. In one of the first larger-scale studies, Lipshultz and colleagues 9 observed an increase in the plasma concentrations of cTns in about 30% of children treated with doxorubicin, thus confirming that cTns are sensitive and specific markers of drug-induced myocardial injury, even months before its clinical recognition by symptoms or decline in ejection fraction. 9 Data from the European Institute of Oncology (EIO) group confirmed these data in the adult population (Fig. 16.2) and the role of cTns in predicting cardiac dysfunction in adult patients undergoing a high dose of anticancer drugs for different malignancies (Fig. 16.3). Importantly, this was not confined to high-dose chemotherapy regimens with anthracyclines (Fig. 16.4).

In these studies, cTnI increase predicted the development of LV dysfunction in the month following the completion of chemotherapy as well as its severity (Fig. 16.5). 10,11,14 The magnitude of cTnI elevation was on the order of 0.5 to 2.0 ng/mL (ie, 1–4 times the upper limit of reference), which is on the order of what is seen, for instance, with nonsevere myocarditis and small myocardial infarctions.

Troponin measurements are useful not only for the determination of cardiac toxicity induced by high-dose chemotherapy but also in prediction and monitoring of cardiac damage induced by standard dose chemotherapy. 12,15 In addition, increases in cTns are also observed in patients receiving newer anticancer drugs such as trastuzumab (Fig. 16.6). However, it has been debated whether this is due to trastuzumab itself or the interaction and unmasking of prior chemotherapy-induced myocardial injury as these drugs are often given in sequence in clinical practice. In agreement, most of the elevations are noted at baseline or with the first cycle. Still, even in these patients treated with different newer targeted cancer therapies, cTn elevation is predictive of development of late cardiac dysfunction and occurrence of cardiac events. 17 Data from the EIO group, in particular, suggest that cTnI identifies patients at risk of developing LV dysfunction (see Fig. 16.6). Moreover, cTnI helps to identify those patients who will less likely recover from toxicity despite heart failure treatments (see Fig. 16.6). 16 Interestingly, cTns provide important information also in the absence of detectable levels, helping to identify patients at low risk; in fact, the negative predictive value of cTns is in excess of 90% (Fig. 16.7). Finally, repeat assessment of cTn levels 1 month after chemotherapy allows for risk restratification of patients with cTn elevation during cancer therapy: those with persistent elevation represent the highest risk group, whereas those with normalization cTn levels by 1 month are at intermediate risk (Fig. 16.8).

Based on these data, cTns were given a central role in the European Society for Medical Oncology (ESMO) 2012 guidelines on the surveillance of patients undergoing chemotherapy with cardiotoxic agents (Fig. 16.9). Determination of cTnI at baseline and periodically during anticancer therapy gives the opportunity to schedule a strength surveillance of cardiac function in selected high-risk patients. On the other hand, the aforementioned high negative predictive value allows to safely identify patients at low risk, who could be excluded from the programs of long-term cardiac monitoring, with a respective lowering of costs. 23

Currently, measurement of troponin levels only immediately before and immediately after each cycle seems to be effective and is transferable from research to clinical practice. Indeed, repeated troponin measurements and early treatment with cardioprotective agents, in patients showing increased levels, are very effective in preventing cardiac dysfunction and associated adverse events Indeed, in a randomized trial including 114 patients, showing persistent elevation of cTnI after chemotherapy, a treatment with enalapril, an angiotensin-converting enzyme inhibitor, prevented the development of cardiac dysfunction in all patients and significantly reduced the incidence of major adverse cardiac events 24 (Fig. 16.10).

However, there are still some limitations for the general use of cTns in clinical practice. Some studies failed to detect changes in troponins during or after anticancer treatments. 25,26 This could be related to different factors: various anticancer protocols used, varying times of sampling associated with different drugs administration schedule, lack of standardization of different assays, cardiac end-points definition and follow-up length taken into consideration, and imprecision in the estimation of LVEF. 27 Furthermore, one has to consider the possibility of cTn elevations not related to the myocardial injury caused by chemotherapy but other factors. Accordingly, patients with cancer may still have cTn elevation because of myocardial ischemia or pulmonary embolism or may have so-called false elevations because of liver function impairment. The development of HS assays, able to detect very low amounts of cTns in the systemic circulation, may be more problematic in this regard as most patients with cancer have, in fact, cTn levels just slightly above the cutoff values of upper limit of normal with the current levels. In the first study to use these new assays, neither an advantage nor a disadvantage was noted. Decrease in peak longitudinal strain and increase in HS-troponin I levels were predictive of development of LV dysfunction. 19 Whether both parameters need to be obtained in routine clinical practice remains debatable. Furthermore, standardization of cTn assessment is needed; both are important areas for research in the future.

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The Contractile Machinery of Skeletal Muscle

Clara Franzini-Armstrong , H. Lee Sweeney , in Muscle, 2012

The Z-Line is Involved in Signaling

Given that it transmits the force generated by the actin–myosin interaction, the Z-line is an ideal place to locate force sensors that can signal adaptation in muscle in response to load. The Z-line proteins that are thought to have potential signaling as well as structural roles include three families of proteins: myotilin, FATZ, and enigma (76). The myotilin family includes myotilin, paladin, and myopalladin. These proteins have immunoglobulin domains and bind α-actinin, filamin, and FATZ. The FATZ family includes FATZ (also called casarcin and myozenin) and their binding partners include myotilin, filamin, telethonin, α-actinin, and ZASP. FATZ-1 and FATZ-3 occur in fast-twitch muscles and FATZ-2 occurs in slow-twitch and cardiac muscles. Proteins of the enigma family have an amino-terminal PDZ domain and 0-3 LIM domains at the COOH terminal. Cypher/ZASP/Oracle is the most studied enigma member (77–79). Cypher/ZASP may serve as a linker-strut by binding to α-actinin via its PDZ domain and may be involved in signaling as it binds protein kinase C via its LIM domains. Cypher is essential for maintaining Z-band structure and muscle integrity (79). Mutations in Cypher lead to dilated cardiomyopathy and skeletal muscle myopathies that are termed zaspopathies.

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Skeletal Muscle: Structure, Chemistry, and Function

JAMES S. LIEBERMAN , ... DAVID D. KILMER , in The Physiological Basis of Rehabilitation Medicine (Second Edition), 1994

Calsequestrin

Calcium ions released from the SR are essential in initiating the actin-myosin interaction. Normal Ca2+ concentration in myofibrillar cytosol is 10−7 M, too small to initiate the actin-myosin interaction. Maximal muscle contraction requires a calcium concentration of 10−5 M. There is an active calcium pump that concentrates Ca2+ in the sarcoplasmic reticulum to 10−4 M. Calsequestrin is a sarcoplasmic reticulum protein that provides another 40-fold increase in calcium storage. Through these mechanisms the myofibrillar cytosol is essentially depleted of Ca2+ during muscle relaxation. Excitation of the T tubules in the sarcoplasmic reticular system releases Ca2+ to the myofibrillar fluid in concentrations as high as 2 × 10−4 M. Ca2+ release involves structurally complex proteins at loci that are dihydropyridine-sensitive receptors. 43 The receptor peptide has a molecular weight of 212 kd, binds dihydropyridine, is phosphorylated by ATP, and is similar to the large peptides of voltage-sensitive sodium channels.

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KINETIC MODEL OF MUSCLE CONTRACTION

V.I. Descherevsky , in Biological and Biochemical Oscillators, 1973

Publisher Summary

This chapter describes the kinetic model of muscle contraction. The myosin-actin interaction is the necessary condition for striated muscle contraction. These muscle proteins are located in the two systems of protofibrils, which are able to make contact with each other by means of myosin cross-bridges at certain discrete points only. According to the sliding filament concept, it is the interaction of myosin bridges with the active sites of the actin protofibrils that provides the moving force of the contracting muscle. Each bridge must act cyclically because the filament length does not change considerably during contraction. The mechanical properties of the contracting muscle are determined at each moment by the distribution of its myosin bridges among the stages of an elementary working bridge cycle.

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Cellular Physiology of Gastrointestinal Smooth Muscle

Khalil N. Bitar , ... Sita Somara , in Physiology of the Gastrointestinal Tract (Fifth Edition), 2012

17.2.1.3.1 Tropomyosin

TM regulates smooth muscle contraction by blocking the myosin binding sites on actin, preventing actin–myosin interaction. Phosphorylated HSP27 aids in the exposure of the myosin binding sites on actin for actin–myosin interaction by stabilizing the displaced TM on actin during contraction. 69,82 ACh induced an increased association of TM with phosphorylated HSP27 in smooth muscle cells, 82 and in vitro studies demonstrated strong direct interaction of GST tagged phosphorylated HSP27 with TM. 82 Direct interaction of phosphorylated HSP27 with TM is critical for maintaining the displaced TM on actin to expose the myosin binding site.

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Practice of Toxicologic Pathology

Graham S. Smith , ... Robin M. Walker , in Haschek and Rousseaux's Handbook of Toxicologic Pathology (Third Edition), 2013

Troponins

Cardiac troponins (cTns) are globular contractile proteins found in striated muscle that form a complex that regulates the actin–myosin interaction required for muscle contraction. They are released from myocardium in proportion to the degree of tissue injury and disruption of myocyte membranes. There are three different troponins. The troponin (Tn) complex binds to the thin actin myofilament via tropomyosin (TnT), and mediates both calcium activation (TnC) and inhibition (TnI) of thick and thin myofilament sliding to produce contraction. There are cardiac and skeletal muscle forms of TnI and TnT, but not for TnC.

Troponins are not found to any significant extent in other tissues. Cardiac troponins I and T (cTnI and cTnT) have high sensitivity and specificity for damage to heart muscle from various causes in humans and across a wide range of animal species. These can be effectively used in mice, rats, rabbits, and dogs, with less published information available regarding use in non-human primates (NHPs), as well as relatively few publications regarding application in chronic studies in animals. There is a need to use appropriate blood collection methods for cTn determinations that avoid cardiac injury due to cardiac puncture, and also to avoid cardiac ischemia such as occurs with blood collection post-mortem or during euthanasia using carbon dioxide.

Commercial cTn assays developed for humans do not have the same immunoreactivity and functional sensitivity (i.e., level at which a change from background can be reproducibly detected) in the common non-clinical testing species; therefore, it is important to demonstrate that the assay chosen is appropriate for the non-clinical species being assessed. Each assay has a different lower limit of detection with large variation across species and platforms tested. Cardiac troponins have low baseline levels supporting sensitive detection. The newer assays detect cTns in the low pg/mL range, thus increasing the sensitivity and precision of detection of baseline cTn concentrations in healthy control populations at levels that were previously undetectable using earlier generation commercial assays, and thus potentially increasing the ability to detect test article-induced myocardial injury.

Use of an ultrasensitive cTnI assay has demonstrated variability in baseline measurements among/between strains, sexes, and surgical alterations (i.e., castration or ovariectomization) as well as different ages in rats. While experimentally induced cardiac lesions are associated with increased serum cTn in animals, cTns can increase in the absence of apparent alterations in myocyte morphology. Differences between stocks of Sprague-Dawley rats in mortality, histopathologic cardiac injury, and increases in cTnI have been demonstrated following administration of the cardiotoxicant isoproterenol. The amount of cTnI release is affected by tissue content. Partial depletion of tissue cTnI such as with inanition, weight loss, and heart failure has been reported in rats.

The time of peak cTn response and duration of response depend on the mechanism of cardiac injury, dose, and frequency of test article-administration. In many situations, cTn release can occur within minutes of injury. Increased serum cTnI levels have been demonstrated by 1 hour after a single dose of the cardiotoxicant isoproterenol, peaking as early as 2–4 hours, and decreasing with a half-life of 6 hours in rats, thus rendering the parameter potentially useful for both early detection of acute injury and reversibility.

Faster kinetics of clearance occurs in animals compared with humans. To formalize how cTns should be used in regulatory studies, a Request for Qualification by FDA of Cardiac Troponin as a Blood Biomarker for Non-clinical Toxicology Studies was submitted to the FDA in 2008.

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Calcium Channel Blockers in Hypertension

Alberto Zanchetti , in Hypertension, 2007

Calcium Channels in the Cardiovascular System

Calcium is a ubiquitous intracellular messenger coupling membrane-mediated stimuli with cellular responses. 2, 3 In the cardiovascular system, increased intracellular calcium triggers the actin-myosin interaction and the subsequent contraction of myocytes and vascular smooth muscle. Because essential hypertension is characterized by enhanced vasoconstrictive tone, transmembrane calcium exchange in vascular smooth muscle plays an important role in hypertension and is an obvious target for antihypertensive compounds. 3 Physiologically, the extracellular to intracellular calcium ion concentration gradient is positive. Numerous membrane mechanisms maintain this gradient, thus allowing entrance of calcium ions necessary for contraction, but avoiding excess intracellular calcium leading to cell injury. Calcium extrusion from the cell is regulated by the calcium-sodium exchange mechanism, in which one calcium ion is transported out of the cell in exchange for three sodium ions entering the cell, and by an adenosine triphosphate (ATP)–dependent calcium pump, which extrudes calcium with the conversion of ATP to adenosine diphosphate. Calcium inflow occurs through two main sets of channels, the receptor-operated and the voltage-gated calcium channels, as well as through a leak pathway. In addition, at the intracellular level, calcium-binding proteins (including calmodulin) and mechanisms regulating calcium exchange into and out of the sarcoplasmic reticulum and the mitochondria play essential roles. 2

Receptor-operated channels are often components of messenger-responsive receptors, but the major targets of pharmacologic actions are the voltage-gated channels. Five major subtypes of this family are known: T, L, N, P/Q, and R. Only T and L channels are known to occur in cardiovascular tissue. The T class is activated and inactivated at low membrane potentials, whereas the L-type channel is activated at high membrane potentials. The L-type channel is the dominant one, functionally, in the cardiovascular system, although some role has been ascribed to the T channel as well, particularly in the physiology of sinus node cells. Figure 22-1 shows that the L-type voltage-gated calcium channel is made up of four subunits, α1 and α2-δ, β, and γ, 2 but the α1-subunit appears to be the dominant one at least in cardiovascular tissue, and it is known to be coded by at least 10 different genes.

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