Human Cardiac Muscle Research Articles

Immunohistochemical distribution of desmin in the human fetal heart.

Desmin is a member of the intermediate filaments, which play crucial roles in the maturation, maintenance and recovery of muscle fibers. Its expression has been examined in human cardiac muscle, rat and chicken, but its spatial distribution in the human fetal heart has not been described. The present study investigated desmin expression in the human fetal heart and associated great vessels in 14 mid-term fetuses from 9 to 18 weeks of gestation. Immunoreactivity for myosin heavy chain (MHC) and alpha smooth muscle actin (α-SMA), as well as neuron-specific enolase (NSE), was also examined. Increased expression of desmin from 9 to 18 weeks was clearly localized in the atrial wall, the proximal portions of the pulmonary vein and vena cava, and around the atrioventricular node. Desmin-positive structures were also positive for MHC. Meanwhile, the great vessels were also positive for α-SMA. The distribution of desmin exhibited a pattern quite different from that described in previous studies of rat and chicken. Thus, desmin in the human fetal heart does not seem to play a general role in myocardial differentiation but rather a specific role closely related to the maturation of the α-isozyme of MHC. Desmin expression in the developing fetal heart also appeared to be induced by mechanical stress due to the involvement of venous walls against the atrium.

BAG3 Directly Interacts with Mutated alphaB-Crystallin to Suppress Its Aggregation and Toxicity

A homozygous disruption or genetic mutation of the bag3 gene causes progressive myofibrillar myopathy in mouse and human skeletal and cardiac muscle disorder while mutations in the small heat shock protein αB-crystallin gene (CRYAB) are reported to be responsible for myofibrillar myopathy. Here, we demonstrate that BAG3 directly binds to wild-type αB-crystallin and the αB-crystallin mutant R120G, via the intermediate domain of BAG3. Peptides that inhibit this interaction in an in vitro binding assay indicate that two conserved Ile-Pro-Val regions of BAG3 are involved in the interaction with αB-crystallin, which is similar to results showing BAG3 binding to HspB8 and HspB6. BAG3 overexpression increased αB-crystallin R120G solubility and inhibited its intracellular aggregation in HEK293 cells. BAG3 suppressed cell death induced by αB-crystallin R120G overexpression in differentiating C2C12 mouse myoblast cells. Our findings indicate a novel function for BAG3 in inhibiting protein aggregation caused by the genetic mutation of CRYAB responsible for human myofibrillar myopathy.

Spontaneous Oscillatory Contraction (SPOC): Quantifying the Contractile Performance of Human Cardiomyocytes

SPontaneous Oscillatory Contraction (SPOC) describes repetitive cycles of rapid lengthening (relaxation) and slow shortening (contraction) observed in small bundles of striated muscle cells (myocytes). SPOCs can be induced in both skeletal and cardiac muscles. Here we describe SPOCs in human cardiac muscle, and in particular focus on SPOCS from left ventricles. All samples were frozen in liquid N either within minutes of the loss of the blood supply to the heart (i.e. soon after the aorta was clamped), or within four hours of surgical removal of non-failing hearts that were perfused with cardioplegic solution, thus reducing post-mortem damage to inhibit spontaneous contractions. SPOC is best considered as a third state that is intermediate to contraction and relaxation. SPOC frequency (SPOC period) and shortening velocities correlate well body size in various animals thus, SPOC is likely to reflect the physiology of the heart as it functioned in life. Tiny samples (<1 mg) of human immobilized cardiomyocytes are exposed to precise ionic conditions to induce SPOC. SPOC cycles are recorded at 25 frames per second in bright field yielding high spatial and temporal resolution information. Measurements of the mean rates of shortening (systole) and lengthening (diastole), and of the SPOC periods enable us to determine if SPOC parameters change with age in non-failing human heart samples. We have examined non-failing heart left ventricular samples using donors from ages 3 weeks to 65 years. These hearts exhibit a range of heart rates that correlate with donor age. We also used the SPOC technique to identify functional defects in samples from patients diagnosed with hypertrophic cardiomyopathy (HCM) who had undergone cardiomyectomy of the enlarged interventricular septum. We only examined patients with known genetic mutations in cardiac sarcomeric genes.

The Importance of Being Profiled: Improving Drug Candidate Safety and Efficacy Using Ion Channel Profiling

Profiling of putative lead compounds against a representative panel of relevant enzymes, receptors, ion channels, and transporters is a pragmatic approach to establish a preliminary view of potential issues that might later hamper development. An early idea of which off-target activities must be minimized can save valuable time and money during the preclinical lead optimization phase if pivotal questions are asked beyond the usual profiling at hERG. The best data for critical evaluation of activity at ion channels is obtained using functional assays, since binding assays cannot detect all interactions and do not provide information on whether the interaction is that of an agonist, antagonist, or allosteric modulator. For ion channels present in human cardiac muscle, depending on the required throughput, manual-, or automated-patch-clamp methodologies can be easily used to evaluate compounds individually to accurately reveal any potential liabilities. The issue of expanding screening capacity against a cardiac panel has recently been addressed by developing a series of robust, high-throughput, cell-based counter-screening assays employing fluorescence-based readouts. Similar assay development approaches can be used to configure panels of efficacy assays that can be used to assess selectivity within a family of related ion channels, such as Nav1.X channels. This overview discusses the benefits of in vitro assays, specific decision points where profiling can be of immediate benefit, and highlights the development and validation of patch-clamp and fluorescence-based profiling assays for ion channels (for examples of fluorescence-based assays, see Bhave et al., 2010; and for high-throughput patch-clamp assays see Mathes, 2006; Schrøder et al., 2008).

Enhancing effects of salicylate on quinidine-induced block of human wild type and LQT3 related mutant cardiac Na+ channels

It is unknown whether salicylate enhances the action of antiarrhythmic agents on human Na+ channels with state dependency and tissue specificity. We therefore investigated effects of salicylate on quinidine-induced block of human cardiac and skeletal muscle Na+ channels. Human cardiac wild-type (hH1), LQT3-related mutant (ΔKPQ), and skeletal muscle (hSkM1) Na+ channel α subunits were expressed in COS7 cells. Effects of salicylate on quinidine-induced tonic and use-dependent block of Na+ channel currents were examined by the whole-cell patch-clamp technique. Salicylate enhanced the quinidine-induced tonic and use-dependent block of both hH1 and hSkM1 currents at a holding potential (HP) of -100 mV but not at -140 mV. Salicylate decreased the IC50 value for the quinidine-induced tonic block of hH1 at an HP of -100 mV, and produced a negative shift in the steady-state inactivation curve of hH1 in the presence of quinidine. According to the modulated receptor theory, it is probable that salicylate decreases the dissociation constant for quinidine binding to inactivated-state channels. Furthermore, salicylate significantly enhanced the quinidine-induced tonic and use-dependent block of the peak and steady-state ΔKPQ channel currents. The results suggest that salicylate enhances quinidine-induced block of Na+ channels via increasing the affinity of quinidine to inactivated state channels.

Cystic fibrosis transmembrane conductance regulator in human muscle: Dysfunction causes abnormal metabolic recovery in exercise

Individuals with cystic fibrosis (CF) have exercise intolerance and skeletal muscle weakness not solely attributable to physical inactivity or pulmonary function abnormalities. CF transmembrane conductance regulator (CFTR) has been demonstrated in human bronchial smooth and cardiac muscle. Using (31)P-magnetic resonance spectroscopy of skeletal muscle, we showed CF patients to have lower resting muscle adenosine triphosphate and delayed phosphocreatine recovery times after high-intensity exercise, suggesting abnormal muscle aerobic metabolism; and higher end-exercise pH values, suggesting altered bicarbonate transport. Our objective was to study CFTR expression in human skeletal muscle. We studied CFTR expression in human skeletal muscle by Western blot with anti-CFTR antibody (Ab) L12B4 and demonstrated a single band with expected molecular weight of 168kDa. We isolated the cDNA by reverse transcription polymerase chain reaction and directly sequenced a 975bp segment (c. 3,600-4,575) that was identical to the human CFTR sequence. We showed punctate staining of CFTR in sarcoplasm and sarcolemma by immunofluorescence microscopy with L12B4 Ab and secondary Alexa 488-labeled Ab. We confirmed CFTR expression in the sarcotubular network and sarcolemma by electron microscopy, using immunogold-labeled anti-CFTR Ab. We observed activation of CFTR Cl(-) channels with iodide efflux, on addition of forskolin, 3-isobutyl-1-methyl-xanthine, and 8-chlorphenylthio-cyclic adenosine monophosphate, in wild-type C57BL/6J isolated muscle fibers in contrast to no efflux from mutant F508del-CFTR muscle. We speculate that a defect in sarcoplasmic reticulum CFTR Cl(-) channels could alter the electrochemical gradient, causing dysregulation of Ca(2+) homeostasis, for example, ryanodine receptor or sarco(endo)plasmic reticulum Ca(2+) adenosine triphosphatases essential to excitation-contraction coupling leading to exercise intolerance and muscle weakness in CF.

Developmental and Pathological Changes in the Human Cardiac Muscle Mitochondrial DNA Organization, Replication and Copy Number

Adult human heart mitochondrial DNA (mtDNA) has recently been shown to have a complex organization with abundant dimeric molecules, branched structures and four-way junctions. In order to understand the physiological significance of the heart-specific mtDNA maintenance mode and to find conditions that modify human heart mtDNA structure and replication, we analyzed healthy human heart of various ages as well as several different heart diseases, including ischemic heart disease, dilated as well as hypertrophic cardiomyopathies, and several mitochondrial disorders. By using one- and two-dimensional agarose gel electrophoresis, various enzymatic treatments and quantitative PCR we found that in human newborns heart mtDNA has a simple organization, lacking junctional forms and dimers. The adult-type branched forms are acquired in the early childhood, correlating with an increase in mtDNA copy number. Mitochondrial disorders involving either mutations in the mtDNA polymerase γ (PolGα) or mtDNA helicase Twinkle, while having no obvious cardiac manifestation, show distinct mtDNA maintenance phenotypes, which are not seen in various types of diseased heart or in mitochondrial disorders caused by point mutations or large-scale deletions of mtDNA. The findings suggest a link between cardiac muscle development, mtDNA copy number, replication mode and topological organization. Additionally, we show that Twinkle might have a direct role in the maintenance of four-way junctions in human heart mtDNA.

Distribution of costameric proteins in normal human ventricular and atrial cardiac muscle.

In the mature heart, the intercalated disc and costameres provide the cell-cell and cell-matrix junctions respectively. Intercalated disc is situated at the bipolar ends of the cardiomyocytes and the myofibrils are anchored at this structure. The costameres mediate integration with the extracellular matrix that covers individual cardiomyocytes laterally. Costameres are considered as "proteic machinery" that appears to comprise two protein complexes: the dystrophin-glycoprotein complex (DGC) and the vinculin-talin-integrin system. There are structural differences between atrial and ventricular myocytes, but there have been relatively few studies that have analyzed costameres and focal adhesion function in cardiac cells. Our previous study carried out only on atrial myocytes, demonstrated that the DGC and talin-vinculin-integrin complexes had a costameric distribution that, unlike skeletal muscle, it localized only on the I band. We performed a further immunohistochemical analysis extending also the evaluation to the normal human cardiac muscle fibers obtained from ventricle and interventricular septum, in order to define the distribution and the spatial relationship between the proteins of the two complexes also in the other heart districts. Immunoconfocal microscopy of cardiac tissue revealed the costameric distribution of DGC and of vinculin-talin-integrin system, the association of all tested proteins in intercalated disks, in disagreement with other Authors, and in T-tubule with irregular spokelike extensions penetrating toward the center of the cell. Moreover, our data showed that all tested proteins colocalize between each other.

S165F mutation of junctophilin 2 affects Ca2+ signalling in skeletal muscle

JPs (junctophilins) contribute to the formation of junctional membrane complexes in muscle cells by physically linking the t-tubule (transverse-tubule) and SR (sarcoplasmic reticulum) membranes. In humans with HCM (hypertrophic cardiomyopathy), mutations in JP2 are linked to altered Ca2+ signalling in cardiomyocytes; however, the effects of these mutations on skeletal muscle function have not been examined. In the present study, we investigated the role of the dominant-negative JP2-S165F mutation (which is associated with human HCM) in skeletal muscle. Consistent with the hypertrophy observed in human cardiac muscle, overexpression of JP2-S165F in primary mouse skeletal myotubes led to a significant increase in myotube diameter and resting cytosolic Ca2+ concentration. Single myotube Ca2+ imaging experiments showed reductions in both the excitation-contraction coupling gain and RyR (ryanodine receptor) 1-mediated Ca2+ release from the SR. Immunoprecipitation assays revealed defects in the PKC (protein kinase C)-mediated phosphorylation of the JP2-S165F mutant protein at Ser165 and in binding of JP2-S165F to the Ca2+ channel TRPC3 (transient receptor potential cation canonical-type channel 3) on the t-tubule membrane. Therefore both the hypertrophy and altered intracellular Ca2+ signalling in the JP2-S165F-expressing skeletal myotubes can be linked to altered phosphorylation of JP2 and/or altered cross-talk among Ca2+ channels on the t-tubule and SR membranes.

Engineering cardiac tissue in vivo from human adipose-derived stem cells

Cardiac tissue engineering offers promise as a surgical approach to cardiac repair, but requires an adequate source of cardiomyocytes. Here we evaluate the potential for generating human cardiac muscle cells in vivo from adipose-derived stem cells (ASC) by co-implanting in a vascularised tissue engineering chamber with inducing rat cardiomyocytes (rCM). Co-implantation (ASC–rCM) was compared with rCM or ASC controls alone after 6 weeks. Immunostaining using human nucleus specific antibody and cardiac markers revealed several fates for ASC in the chamber; (1) differentiation into cardiomyocytes and integration with co-implanted rCM; (2) differentiation into smooth muscle cells and recruitment into vascular structures; (3) adipogenic differentiation. ASC–rCM and ASC groups grew larger tissue constructs than rCM alone (212 ± 25 μl, 171 ± 16 μl vs. 137 ± 15 μl). ASC–rCM and rCM groups contracted spontaneously at up to 140 bpm and generated a 10–15-fold larger volume of cardiac muscle (14.5 ± 4.8 μl and 18.5 ± 2.6 μl) than ASC alone group (1.3 ± 0.5 μl). Vascular volume in ASC–rCM group was twice that of the rCM group (28.7 ± 5.0 μl vs. 14.8 ± 1.8 μl). The cardiac tissue engineered by co-implanting human ASC with neonatal rCM showed in vivo plasticity of ASC and their cardiomyogenic potential in tissue engineering. ASC contribution to vascularisation also promoted the growth of engineered tissue, confirming their utility in this setting.

Transplantation of a Tissue-Engineered Human Vascularized Cardiac Muscle

Myocardial regeneration strategies have been hampered by the lack of sources for human cardiomyocytes (CMs) and by the significant donor cell loss following transplantation. We assessed the ability of a three-dimensional tissue-engineered human vascularized cardiac muscle to engraft in the in vivo rat heart and to promote functional vascularization. Human embryonic stem cell-derived CMs alone or with human endothelial cells (human umbilical vein endothelial cells) and embryonic fibroblasts (triculture constructs) were seeded onto biodegradable porous scaffolds. The resulting tissue constructs were transplanted to the in vivo rat heart and formed cardiac tissue grafts. Immunostaining studies for human-specific CD31 and alpha-smooth muscle actin demonstrated the formation of both donor (human) and host (rat)-derived vasculature within the engrafted triculture tissue constructs. Intraventricular injection of fluorescent microspheres or lectin resulted in their incorporation by human-derived vessels, confirming their functional integration with host coronary vasculature. Finally, the number of blood vessels was significantly greater in the triculture tissue constructs (60.3 +/- 8/mm(3), p < 0.05) when compared with scaffolds containing only CMs (39.0 +/- 14.4/mm(3)). In conclusion, a tissue-engineered human vascularized cardiac muscle can be established ex vivo and transplanted in vivo to form stable grafts. By utilizing a multicellular preparation we were able to increase biograft vascularization and to show that the preexisting human vessels can become functional and contribute to tissue perfusion.

Muscle Lim Protein Interacts with Cofilin 2 and Regulates F-Actin Dynamics in Cardiac and Skeletal Muscle

The muscle LIM protein (MLP) and cofilin 2 (CFL2) are important regulators of striated myocyte function. Mutations in the corresponding genes have been directly associated with severe human cardiac and skeletal myopathies, and aberrant expression patterns have often been observed in affected muscles. Herein, we have investigated whether MLP and CFL2 are involved in common molecular mechanisms, which would promote our understanding of disease pathogenesis. We have shown for the first time, using a range of biochemical and immunohistochemical methods, that MLP binds directly to CFL2 in human cardiac and skeletal muscles. The interaction involves the inter-LIM domain, amino acids 94 to 105, of MLP and the amino-terminal domain, amino acids 1 to 105, of CFL2, which includes part of the actin depolymerization domain. The MLP/CFL2 complex is stronger in moderately acidic (pH 6.8) environments and upon CFL2 phosphorylation, while it is independent of Ca(2+) levels. This interaction has direct implications in actin cytoskeleton dynamics in regulating CFL2-dependent F-actin depolymerization, with maximal depolymerization enhancement at an MLP/CFL2 molecular ratio of 2:1. Deregulation of this interaction by intracellular pH variations, CFL2 phosphorylation, MLP or CFL2 gene mutations, or expression changes, as observed in a range of cardiac and skeletal myopathies, could impair F-actin depolymerization, leading to sarcomere dysfunction and disease.

Ca 2+ ‐independent positive molecular inotropy for failing rabbit and human cardiac muscle by α‐myosin motor gene transfer

Current inotropic therapies used to increase cardiac contractility of the failing heart center on increasing the amount of calcium available for contraction, but their long-term use is associated with increased mortality due to fatal arrhythmias. Thus, there is a need to develop and explore novel inotropic therapies that can act via calcium-independent mechanisms. The purpose of this study was to determine whether fast alpha-myosin molecular motor gene transfer can confer calcium-independent positive inotropy in slow beta-myosin-dominant rabbit and human failing ventricular myocytes. To this end, we generated a recombinant adenovirus (AdMYH6) to deliver the full-length human alpha-myosin gene to adult rabbit and human cardiac myocytes in vitro. Fast alpha-myosin motor expression was determined by Western blotting and immunocytochemical analysis and confocal imaging. In experiments using electrically stimulated myocytes from ischemic failing hearts, AdMYH6 increased the contractile amplitude of failing human [23.9+/-7.8 nm (n=10) vs. AdMYH6 amplitude 78.4+/-16.5 nm (n=6)] and rabbit myocytes. The intracellular calcium transient amplitude was not altered. Control experiments included the use of a green fluorescent protein or a beta-myosin heavy chain adenovirus. Our data provide evidence for a novel form of calcium-independent positive inotropy in failing cardiac myocytes by fast alpha-myosin motor protein gene transfer.

Physiological function and transplantation of scaffold-free and vascularized human cardiac muscle tissue

Success of human myocardial tissue engineering for cardiac repair has been limited by adverse effects of scaffold materials, necrosis at the tissue core, and poor survival after transplantation due to ischemic injury. Here, we report the development of scaffold-free prevascularized human heart tissue that survives in vivo transplantation and integrates with the host coronary circulation. Human embryonic stem cells (hESCs) were differentiated to cardiomyocytes by using activin A and BMP-4 and then placed into suspension on a rotating orbital shaker to create human cardiac tissue patches. Optimization of patch culture medium significantly increased cardiomyocyte viability in patch centers. These patches, composed only of enriched cardiomyocytes, did not survive to form significant grafts after implantation in vivo. To test the hypothesis that ischemic injury after transplantation would be attenuated by accelerated angiogenesis, we created "second-generation," prevascularized, and entirely human patches from cardiomyocytes, endothelial cells (both human umbilical vein and hESC-derived endothelial cells), and fibroblasts. Functionally, vascularized patches actively contracted, could be electrically paced, and exhibited passive mechanics more similar to myocardium than patches comprising only cardiomyocytes. Implantation of these patches resulted in 10-fold larger cell grafts compared with patches composed only of cardiomyocytes. Moreover, the preformed human microvessels anastomosed with the rat host coronary circulation and delivered blood to the grafts. Thus, inclusion of vascular and stromal elements enhanced the in vitro performance of engineered human myocardium and markedly improved viability after transplantation. These studies demonstrate the importance of including vascular and stromal elements when designing human tissues for regenerative therapies.

Turnover After the Fallout

Human cardiac muscle cells slowly renew into adulthood, a potential that may be therapeutically exploited.

Cardiac Myosin Binding Protein-C Phosphorylation in a β-Myosin Heavy Chain Background

Cardiac myosin binding protein-C (cMyBP-C) phosphorylation modulates cardiac contractility. When expressed in cMyBP-C-null (cMyBP-C((t/t))) hearts, a cMyBP-C phosphomimetic (cMyBP-C(AllP+)) rescued cardiac dysfunction and protected the hearts from ischemia/reperfusion injury. However, cMyBP-C function may be dependent on the myosin isoform type. Because these replacements were performed in the mouse heart, which contains predominantly alpha-myosin heavy chain (alpha-MyHC), the applicability of the data to humans, whose cardiomyocytes contain predominantly beta-MyHC, is unclear. We determined the effect(s) of cMyBP-C phosphorylation in a beta-MyHC transgenic mouse heart in which >80% of the alpha-MyHC was replaced by beta-MyHC, which is the predominant myosin isoform in human cardiac muscle. To determine the effects of cMyBP-C phosphorylation in a beta-MyHC background, transgenic mice expressing normal cMyBP-C (cMyBP-C(WT)), nonphosphorylatable cMyBP-C (cMyBP-C(AllP)(-)), or cMyBP-C(AllP+) were bred into the beta-MyHC background (beta). These mice were then crossed into the cMyBP-C((t/t)) background to ensure the absence of endogenous cMyBP-C. cMyBP-C((t/t)/beta) and cMyBP-C(AllP)(-)(:(t/t)/beta) mice died prematurely because of heart failure, confirming that cMyBP-C phosphorylation is essential in the beta-MyHC background. cMyBP-C(AllP+:(t/t)/beta) and cMyBP-C(WT:(t/t)/beta) hearts showed no morbidity and mortality, and cMyBP-C(AllP+:(t/t)/beta) hearts were significantly cardioprotected from ischemia/reperfusion injury. cMyBP-C phosphorylation is necessary for basal myocardial function in the beta-MyHC background and can preserve function after ischemia/reperfusion injury. Our studies justify exploration of cMyBP-C phosphorylation as a therapeutic target in the human heart.

Quantitative Assay of Skeletal Muscle alpha-actin Expression In Normal and Pathological Human and Mouse Hearts

We have developed a polyclonal antibody specific to skeletal muscle actin (ACTA1) in the presence of cardiac actin (ACTC) and have used it to quantify the skeletal actin content of human and mouse cardiac muscle. Heart muscle myofibrils were separated by SDS-PAGE and Western blotted. The membrane was stained first with MemCode total protein stain and then probed in with the anti-skeletal muscle actin antibody, visualised by ECL. The ECL band signal was normalised to MemCode-stained actin band. Skeletal muscle myofibrils were used as a 100% skeletal actin standard. For the negative control we used myofibrils from skeletal muscle of a skeletal actin knockout mouse crossed with a transgenic mouse over-expressing cardiac actin in skeletal muscle. There was no detectable signal from the skeletal actin antibody in the pure cardiac actin control.Human non-failing donor heart muscle contained 21±2% skeletal actin (n=9). This is comparable to previous estimates using N-terminal sequencing or Mass spectroscopy. In both end-stage failing heart muscle and in myectomy samples from HCM muscle the skeletal actin content was much higher (58±5% in both cases, n=12, 11). The increase in skeletal actin content of myopathic muscle was highly significant (p< 0.001). Mouse heart muscle (C57BL/6 strain) contains 26±3% skeletal actin (n=7). This is similar to human heart. ACTC DCM mutation E361G expressed at 50% in mouse heart has 16±3% (n=8) skeletal actin but ACTC HCM mutation E99K expressed at 50% is not significantly different from NTG 24±2% (n=5). We conclude that in human heart, acquired heart failure or failure secondary to HCM is associated with an increased content of skeletal muscle actin. In contrast, in mouse genetic models of HCM and DCM skeletal muscle actin content may be lower than normal.

Composition of titin isoforms of skeletal and cardiac muscles in pathologies

An electophoretic study of changes in composition of titin isoforms in human and rat skeletal and cardiac muscles is carried out. A more considerable decrease in the content of intact titin isoforms was observed than in the content of N2A-titin in the dorsal muscle of patients with the “stiff-person syndrome” and in m. soleus of humans and rats during development of “muscle hypogravity syndrome” and than in the content of N2BA- and N2B-titins in hypertrophic heart of spontaneously hypertensive rats. The relation between reduction of titin content in m. soleus and the increase of time the rats were in conditions of simulated microgravity is revealed. On electrophoregrams of left ventricle myocardium of patients with terminal stage of dilated cardiomyopathy the intact titin and N2BA-titin were absent and a considerable decrease in the content of N2B-titin was observed. This could be the consequence of the terminal stage of pathology. It follows that development of the diseases is accompanied by a greater destruction of intact titin than of its other forms which may be important for diagnostics of pathological processes.

Requiem for a Heavyweight

The development of rapid, automated, and accurate laboratory testing for creatine kinase MB (CK-MB) revolutionized the treatment of patients with acute cardiac events in the 1970s and 1980s.1 To clinicians, CK-MB values augmented a thorough history, physical, and ECG findings, and elevations rapidly became the gold standard for identifying cardiac injury.1 CK-MB allowed earlier diagnosis of acute myocardial infarction (AMI), and detection of reinfarction, and measurements could be used to provide a facile clinical estimate of infarct size. Elevations of CK-MB were never intended to be synonymous with myocardial infarction, only indicative of cardiac injury.1 However, because of the relative insensitivity of measurements, increased concentrations occurred predominantly with larger insults such those associated with acute ischemic heart disease. For that reason, AMI was rarely diagnosed, assuming appropriate timing of the samples, in the absence of a CK-MB elevation.2,3 CK-MB assays initially relied on the measurement of enzyme activity, but over time, improved accuracy and ease of use were established by the use of mass assays. Mass assays allowed earlier detection of abnormal values and improved both clinical sensitivity and specificity. However, mass assays unmasked an increased frequency of CK-MB elevations due primarily to skeletal muscle injury because of their increased sensitivity.4–6 Clinical use of the percent relative index was then initiated. This approach improved the specificity of elevations for cardiac muscle injury but was insensitive when concurrent cardiac injury and skeletal muscle injury were present because elevations from skeletal muscle often are of a large magnitude.7–10 A large number of analytical confounds such as macrokinases and interfering substances also were substantial problems with these assays.10,11 Attempts to standardize assays12 have been partially successful, but differences still exist between manufacturers and even between the same testing antibodies used on different analytical platforms (ie, …

Atomic force microscope observation of branching in single transcript molecules derivedfrom human cardiac muscle

We have used an atomic force microscope to examine a clinically derived sample ofsingle-molecule gene transcripts, in the form of double-stranded cDNA, (c: complementary)obtained from human cardiac muscle without the use of polymerase chain reaction (PCR)amplification. We observed a log-normal distribution of transcript sizes, withmost molecules being in the range of 0.4–7.0 kilobase pairs (kb) or 130–2300 nmin contour length, in accordance with the expected distribution of mRNA (m:messenger) sizes in mammalian cells. We observed novel branching structures notpreviously known to exist in cDNA, and which could have profound negative effects ontraditional analysis of cDNA samples through cloning, PCR and DNA sequencing.