Acto-Myosin Cross-Bridge Stiffness Depends on the Nucleotide State of Myosin II.

How various myosin isoforms fulfill the diverse physiological requirements of distinct muscle types remain unclear. Myosin II isoforms expressed in skeletal muscles determine the mechanical performance of the specific muscles. Here, we employed a single-molecule optical trapping method and compared the chemomechanical properties of slow and fast muscle myosin II isoforms. Stiffness of the myosin motor is key to its force-generating ability during muscle contraction. We found that acto-myosin (AM) cross-bridge stiffness depends on its nucleotide state as the myosin progresses through the ATPase cycle. The strong actin bound "AM.ADP" state exhibited >2 fold lower stiffness than "AM rigor" state. The two myosin isoforms displayed similar "rigor" stiffness. We conclude that the time-averaged stiffness of the slow myosin is lower due to prolonged duration of the AM.ADP state, which determines the force-generating potential and contraction speed of the muscle, elucidating the basis for functional diversity among myosins.

Transformation of the Nonprocessive Fast Skeletal Myosin II into a Processive Motor.

Myosin family motors play diverse cellular roles. Precise insights into how the light chains contribute to the functional variabilities among myosin motors, however, remain unresolved. Here, it is demonstrated that the fast skeletal muscle myosin II isoform myosin heavy chain (MHC-IID) can be transformed into a processive motor, by simply replacing the native regulatory light chain MLC2f with the regulatory light chain variant MLC2v from the slow muscle myosin II. Single molecule kinetic analyses and optical trapping measurements of the hybrid motor reveal marked changes such as increased association rate of myosin toward adenosine triphosphate (ATP) and actin by more than twofold. The direct consequence of high adenosine diphosphate (ADP) affinity and increased actin rebinding is the altered overall actomyosin association time during the cross-bridge cycle. The data indicate that the MLC2v influences the duty ratio in the hybrid motor, suggestive of promoting interhead communication and enabling processive movement. This finding establishes that the regulatory light chain fine-tunes the motor's mechanical output that may have important implications under physiological conditions. Furthermore, the success of this approach paves the way to engineer motors from a known motor protein element to assemble highly specialized biohybrid machines for potential applications in nano-biomedicine and engineering.

Intrinsic MYH7 expression regulation contributes to tissue level allelic imbalance in hypertrophic cardiomyopathy.

HCM, the most common inherited cardiac disease, is mainly caused by mutations in sarcomeric genes. More than a third of the patients are heterozygous for mutations in the MYH7 gene encoding for the ß-myosin heavy chain. In HCM-patients, expression of the mutant and the wildtype allele can be unequal, thus leading to fractions of mutant and wildtype mRNA and protein which deviate from 1:1. This so-called allelic imbalance was detected in whole tissue samples but also in individual cells. There is evidence that the severity of HCM not only depends on the functional effect of the mutation itself, but also on the fraction of mutant protein in the myocardial tissue. Allelic imbalance has been shown to occur in a broad range of genes. Therefore, we aimed to examine whether the MYH7-alleles are intrinsically expressed imbalanced or whether the allelic imbalance is solely associated with the disease. We compared the expression of MYH7-alleles in non-HCM donors and in HCM-patients with different MYH7-missense mutations. In the HCM-patients, we identified imbalanced as well as equal expression of both alleles. Also at the protein level, allelic imbalance was determined. Most interestingly, we also discovered allelic imbalance and balance in non-HCM donors. Our findings therefore strongly indicate that apart from mutation-specific mechanisms, also non-HCM associated allelic-mRNA expression regulation may account for the allelic imbalance of the MYH7 gene in HCM-patients. Since the relative amount of mutant mRNA and protein or the extent of allelic imbalance has been associated with the severity of HCM, individual analysis of the MYH7-allelic expression may provide valuable information for the prognosis of each patient.

ATP turnover by individual myosin molecules hints at two conformers of the myosin active site.

Coupling of ATP hydrolysis to structural changes in the motor domain is fundamental to the driving of motile functions by myosins. Current understanding of this chemomechanical coupling is primarily based on ensemble average measurements in solution and muscle fibers. Although important, the averaging could potentially mask essential details of the chemomechanical coupling, particularly for mixed populations of molecules. Here, we demonstrate the potential of studying individual myosin molecules, one by one, for unique insights into established systems and to dissect mixed populations of molecules where separation can be particularly challenging. We measured ATP turnover by individual myosin molecules, monitoring appearance and disappearance of fluorescent spots upon binding/dissociation of a fluorescent nucleotide to/from the active site of myosin. Surprisingly, for all myosins tested, we found two populations of fluorescence lifetimes for individual myosin molecules, suggesting that termination of fluorescence occurred by two different paths, unexpected from standard kinetic schemes of myosin ATPase. In addition, molecules of the same myosin isoform showed substantial intermolecular variability in fluorescence lifetimes. From kinetic modeling of our two fluorescence lifetime populations and earlier solution data, we propose two conformers of the active site of myosin, one that allows the complete ATPase cycle and one that dissociates ATP uncleaved. Statistical analysis and Monte Carlo simulations showed that the intermolecular variability in our studies is essentially due to the stochastic behavior of enzyme kinetics and the limited number of ATP binding events detectable from an individual myosin molecule with little room for static variation among individual molecules, previously described for other enzymes.

[Environmental health relevance of airborne microorganisms in ambient and indoor air]. / Umweltmedizinische Relevanz von luftgetragenen Mikroorganismen im Außen- und Innenbereich.

Airborne microorganisms occur ubiquitously in the ambient air. Besides allergic and irritative-toxic effects, they can cause infections after inhalation. Occupational studies have shown that an increased incidence of respiratory diseases is found in adequately exposed workers. In addition to respiratory diseases, severe systemic infections can also occur in particular cases, such as in the case of a hantavirus infection that is recognized as an occupational disease. In studies from environmental medicine, respiratory diseases have also been observed in residents living in the vicinity of livestock facilities and evaporative cooling towers. In the latter case, an infection risk may be caused by inhalation of legionella-contaminated aerosol from the exhaust air of such systems.Currently, there are no health-related exposure limits for airborne microorganisms released from such facilities. Environmental risk assessment can be carried out on the basis of the guideline VDI 4250 part 1, which relies on an excess of natural background concentration by facility-specific emissions. For the approval practice, the LAI-Leitfaden Bioaerosole is a uniform, standardized method for the determination and assessment of bioaerosol exposure.In indoor spaces, only a few mold types, such as Aspergillus fumigatus are able to trigger infections by local or systemic infection of the human organism. In particular, persons with an immune deficiency or allergies must be informed about the risks of mold exposure in indoor air. In general, mold growth in indoor spaces is a hygienic problem and must not be accepted as a matter of principle.

Stiff matrix induces switch to pure ß-cardiac myosin heavy chain expression in human ESC-derived cardiomyocytes.

Human pluripotent stem cell (hPSC)-derived cardiomyocytes hold great potential for in vitro modeling of diseases like cardiomyopathies. Yet, knowledge about expression and functional impact of sarcomeric protein isoforms like the myosin heavy chain (MyHC) in hPSC-cardiomyocytes is scarce. We hypothesized that ventricular ß-MyHC expression alters contraction and calcium kinetics and drives morphological and electrophysiological differentiation towards ventricular-like cardiomyocytes. To address this, we (1) generated human embryonic stem cell-derived cardiomyocytes (hESC-CMs) that switched towards exclusive ß-MyHC, and (2) functionally and morphologically characterized these hESC-CMs at the single-cell level. MyHC-isoforms and functional properties were investigated during prolonged in vitro culture of cardiomyocytes in floating cardiac bodies (soft conditions) vs. culture on a stiff matrix. Using a specific anti-ß-MyHC and a newly generated anti-α-MyHC-antibody, we found individual cardiomyocytes grown in cardiac bodies to mostly express both α- and ß-MyHC-protein isoforms. Yet, 35 and 75 days of cultivation on laminin-coated glass switched 66 and 87 % of all cardiomyocytes to exclusively express ß-MyHC, respectively. Twitch contraction and calcium transients were faster for CMs on laminin-glass. Surprisingly, both parameters were only little affected by the MyHC-isoform, although hESC-CMs with only ß-MyHC had much lower ATP-turnover and tension cost, just as in human ventricular cardiomyocytes. Spontaneous contractions and no strict coupling of ß-MyHC to ventricular-like action potentials suggest that MyHC-isoform expression does not fully determine the hESC-CM differentiation status. Stiff substrate-induced pure ß-MyHC-protein expression in hESC-CMs, with several contractile parameters close to ventricular cardiomyocytes, provides a well-defined in vitro system for modeling of cardiomyopathies and drug screening approaches.

Two independent switches regulate cytoplasmic dynein's processivity and directionality.

Cytoplasmic dynein is a microtubule-based molecular motor that participates in a multitude of cell activities, from cell division to organelle transport. Unlike kinesin and myosin, where different tasks are performed by highly specialized members of these superfamilies, a single form of the dynein heavy chain is utilized for different functions. This versatility demands an extensive regulation of motor function. Using an improved application of an optical trap, we were now able to demonstrate that cytoplasmic dynein can generate a discrete power stroke as well as a processive walk in either direction; i.e., towards the plus- or towards the minus-end of a microtubule. Thus, dynein's motor functions can be described by four basic modes of motion: processive and nonprocessive movement, and movement in the forward and reverse directions. Importantly, these four modes of movement can be controlled by two switches. One switch, based on phosphate, determines the directionality of movement. The second switch, depending on magnesium, converts cytoplasmic dynein from a nonprocessive to a processive motor. The two switches can be triggered separately or jointly by changing concentrations of phosphate and magnesium in the local environment. The control of four modes of movement by two switches has major implications for our understanding of the cellular functions and regulation of cytoplasmic dynein. Based on recent studies of dynein's structure we are able to draw new conclusions on cytoplasmic dynein's stepping mechanism.

Familial hypertrophic cardiomyopathy: functional effects of myosin mutation R723G in cardiomyocytes.

Familial Hypertrophic Cardiomyopathy (FHC) is frequently caused by mutations in the ß-cardiac myosin heavy chain (ß-MyHC). To identify changes in sarcomeric function triggered by such mutations, distinguishing mutation effects from other functional alterations of the myocardium is essential. We previously identified a direct effect of mutation R723G (MyHC723) on myosin function in slow Musculus soleus fibers. Here we investigate contractile features of left ventricular cardiomyocytes of FHC-patients with the same MyHC723-mutation and compare these to the soleus data. In mechanically isolated, triton-permeabilized MyHC723-cardiomyocytes, maximum force was significantly lower but calcium-sensitivity was unchanged compared to donor. Conversely, MyHC723-soleus fibers showed significantly higher maximum force and reduced calcium-sensitivity compared to controls. Protein phosphorylation, a potential myocardium specific modifying mechanism, might account for differences compared to soleus fibers. Analysis revealed reduced phosphorylation of troponin I and T, myosin-binding-protein C, and myosin-light-chain 2 in MyHC723-myocardium compared to donor. Saturation of protein-kinaseA phospho-sites led to comparable, i.e., reduced MyHC723-calcium-sensitivity in cardiomyocytes as in M. soleus fibers, while maximum force remained reduced. Myofibrillar disarray and lower density of myofibrils, however, largely account for reduced maximum force in MyHC723-cardiomyocytes. The changes seen when phosphorylation of sarcomeric proteins in myocardium of affected patients is matched to control tissue suggest that the R723G mutation causes reduced Ca(++)-sensitivity in both cardiomyocytes and M. soleus fibers. In MyHC723-myocardium, however, hypophosphorylation can compensate for the reduced calcium-sensitivity, while maximum force generation, lowered by myofibrillar deficiency and disarray, remains impaired, and may only be compensated by hypertrophy.

Tau protein diffuses along the microtubule lattice.

Current models for the intracellular transport of Tau protein suggest motor protein-dependent co-transport with microtubule fragments and diffusion of Tau in the cytoplasm, whereas Tau is believed to be stationary while bound to microtubules and in equilibrium with free diffusion in the cytosol. Observations that members of the microtubule-dependent kinesin family show Brownian motion along microtubules led us to hypothesize that diffusion along microtubules could also be relevant in the case of Tau. We used single-molecule total internal reflection fluorescence microscopy to probe for diffusion of individual fluorescently labeled Tau molecules along microtubules. This allowed us to avoid the problem that microtubule-dependent diffusion could be masked by excess of labeled Tau in solution that might occur in in vivo overexpression experiments. We found that approximately half of the individually detected Tau molecules moved bidirectionally along microtubules over distances up to several micrometers. Diffusion parameters such as diffusion coefficient, interaction time, and scanned microtubule length did not change with Tau concentration. Tau binding and diffusion along the microtubule lattice, however, were sensitive to ionic strength and pH and drastically reduced upon enzymatic removal of the negatively charged C termini of tubulin. We propose one-dimensional Tau diffusion guided by the microtubule lattice as one possible additional mechanism for Tau distribution. By such one-dimensional microtubule lattice diffusion, Tau could be guided to both microtubule ends, i.e. the sites where Tau is needed during microtubule polymerization, independently of directed motor-dependent transport. This could be important in conditions where active transport along microtubules might be compromised.

Mechanical and kinetic properties of ß-cardiac/slow skeletal muscle myosin.

We aimed to establish reference parameters to identify functional effects of familial hypertrophic cardiomyopathy-related point mutations in the ß-cardiac/slow skeletal muscle myosin heavy chain (ß-cardiac/MyHC-1). We determined mechanical and kinetic parameters of the ß-cardiac/MyHC-1 using human soleus muscle fibers that express the same myosin heavy chain (MyHC-1) as ventricular myocardium (ß-cardiac). The observed parameters are compared to previously reported data for rabbit psoas muscle fibers. We found all of the examined kinetic parameters to be slower in soleus fibers than in rabbit psoas muscle. Somewhat surprisingly, however, we also found that the stiffness of the ß-cardiac/MyHC-1 head domain is more than 3-fold lower than the stiffness of the fast isoform of psoas fibers. Furthermore, and different from rabbit psoas muscle, in human soleus fibers both the occupancy of force-generating cross-bridge states as well as the elastic extension of force-generating heads increase with temperature. Thus, a myosin head in the force generating states makes an increasing contribution to force with temperature. We support some of our fiber data by data from in vitro motility and optical trapping assays. Initial findings with FHC-related point mutations in the converter imply that the differences in stiffness of the head domain between the slow and fast isoform may well be due to particular differences in the amino acid sequence of the converter. We show that the slower kinetics may be linked to a larger flexibility of the ß-cardiac/MyHC-1 isoform compared to fast MyHC isoforms.

Unequal allelic expression of wild-type and mutated ß-myosin in familial hypertrophic cardiomyopathy.

Familial hypertrophic cardiomyopathy (FHC) is an autosomal dominant disease, which in about 30% of the patients is caused by missense mutations in one allele of the ß-myosin heavy chain (ß-MHC) gene (MYH7). To address potential molecular mechanisms underlying the family-specific prognosis, we determined the relative expression of mutant versus wild-type MYH7-mRNA. We found a hitherto unknown mutation-dependent unequal expression of mutant to wild-type MYH7-mRNA, which is paralleled by similar unequal expression of ß-MHC at the protein level. Relative abundance of mutated versus wild-type MYH7-mRNA was determined by a specific restriction digest approach and by real-time PCR (RT-qPCR). Fourteen samples from M. soleus and myocardium of 12 genotyped and clinically well-characterized FHC patients were analyzed. The fraction of mutated MYH7-mRNA in five patients with mutation R723G averaged to 66 and 68% of total MYH7-mRNA in soleus and myocardium, respectively. For mutations I736T, R719W and V606M, fractions of mutated MYH7-mRNA in M. soleus were 39, 57 and 29%, respectively. For all mutations, unequal abundance was similar at the protein level. Importantly, fractions of mutated transcripts were comparable among siblings, in younger relatives and unrelated carriers of the same mutation. Hence, the extent of unequal expression of mutated versus wild-type transcript and protein is characteristic for each mutation, implying cis-acting regulatory mechanisms. Bioinformatics suggest mRNA stability or splicing effectors to be affected by certain mutations. Intriguingly, we observed a correlation between disease expression and fraction of mutated mRNA and protein. This strongly suggests that mutation-specific allelic imbalance represents a new pathogenic factor for FHC.

Cytoplasmic dynein is not a conventional processive motor.

Cytoplasmic dynein is a microtubule-based molecular motor with a multitude of functions from cell division to organelle transport. Cargo transport is often achieved as a co-complex with dynactin and it is believed that this co-complex enhances the processive translocation of cargo along the microtubule tracks (King and Schroer, 2000; Culver-Hanlon et al., 2006). Single molecule studies have revealed that dynein on its own can also act as a processive motor (Reck-Peterson et al., 2006; Toba et al., 2006). However, these studies did not allow the detection of a non-processive motor function. Previous studies based on the transport of vesicles or liposomes indicated that processive transport could only be achieved by an ensemble of motor molecules (Schroer & Sheetz, 1991; Wang and Sheetz, 2000; Muresan et al., 2001). Here we use the three bead dumbbell assay to show for the first time, that cytoplasmic dynein is a non-processive motor at low ATP concentrations. Processivity can be restored even in the absence of dynactin by increasing the ATP concentration to 100muM. We propose that an altered occupancy of the different ATP binding sites (AAA1-4) acts as a modulator between processive and non-processive stepping.

Cycling Cross-Bridges Contribute to Thin Filament Activation in Human Slow-Twitch Fibers.

It has been shown that not only calcium but also strong binding myosin heads contribute to thin filament activation in isometrically contracting animal fast-twitch and cardiac muscle preparations. This behavior has not been studied in human muscle fibers or animal slow-twitch fibers. Human slow-twitch fibers are interesting since they contain the same myosin heavy chain isoform as the human heart. To explore myosin-induced activation of the thin filament in isometrically contracting human slow-twitch fibers, the endogenous troponin complex was exchanged for a well-characterized fast-twitch skeletal troponin complex labeled with the fluorescent dye N-((2-(Iodoacetoxy)ethyl)-N-methyl)amino-7-nitrobenz-2-oxa-1,3-diazole (fsTn-IANBD). The exchange was ≈70% complete (n = 8). The relative contributions of calcium and strong binding cross-bridges to thin filament activation were dissected by increasing the concentration of calcium from relaxing (pCa 7.5) to saturating levels (pCa 4.5) before and after incubating the exchanged fibers in the myosin inhibitor para-aminoblebbistatin (AmBleb). At pCa 4.5, the relative contributions of calcium and strong binding cross-bridges to thin filament activation were ≈69 and ≈31%, respectively. Additionally, switching from isometric to isotonic contraction at pCa 4.5 revealed that strong binding cross-bridges contributed ≈29% to thin filament activation (i.e., virtually the same magnitude obtained with AmBleb). Thus, we showed through two different approaches that lowering the number of strong binding cross-bridges, at saturating calcium, significantly reduced the activation of the thin filament in human slow-twitch fibers. The contribution of myosin to activation resembled that which was previously reported in rat cardiac and rabbit fast-twitch muscle preparations. This method could be applied to slow-twitch human fibers obtained from the soleus muscle of cardiomyopathy patients. Such studies could lead to a better understanding of the effect of point mutations of the cardiac myosin head on the regulation of muscle contraction and could lead to better management by pharmacological approaches.

Advanced Single-Cell Mapping Reveals that in hESC Cardiomyocytes Contraction Kinetics and Action Potential Are Independent of Myosin Isoform.

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) represent an attractive model to investigate CM function and disease mechanisms. One characteristic marker of ventricular specificity of human CMs is expression of the ventricular, slow ß-myosin heavy chain (MyHC), as opposed to the atrial, fast α-MyHC. The main aim of this study was to investigate at the single-cell level whether contraction kinetics and electrical activity of hESC-CMs are influenced by the relative expression of α-MyHC versus ß-MyHC. For effective assignment of functional parameters to the expression of both MyHC isoforms at protein and mRNA levels in the very same hESC-CMs, we developed a single-cell mapping technique. Surprisingly, α- versus ß-MyHC was not related to specific contractile or electrophysiological properties of the same cells. The multiparametric cell-by-cell analysis suggests that in hESC-CMs the expression of genes associated with electrical activity, contraction, calcium handling, and MyHCs is independently regulated.

Mg2+ -free ATP regulates the processivity of native cytoplasmic dynein.

Cytoplasmic dynein, a microtubule-based motor protein, is responsible for many cellular functions ranging from cargo transport to cell division. The various functions are carried out by a single isoform of cytoplasmic dynein, thus requiring different forms of motor regulation. A possible pathway to regulate motor function was revealed in optical trap experiments. Switching motor function from single steps to processive runs could be achieved by changing Mg2+ and ATP concentrations. Here, we confirm by single molecule total internal reflection fluorescence microscopy that a native cytoplasmic dynein dimer is able to switch to processive runs of more than 680 consecutive steps or 5.5 µm. We also identified the ratio of Mg2+ -free ATP to Mg.ATP as the regulating factor and propose a model for dynein processive stepping.

Efficient Knock-in of a Point Mutation in Porcine Fibroblasts Using the CRISPR/Cas9-GMNN Fusion Gene.

During CRISPR/Cas9 mediated genome editing, site-specific double strand breaks are introduced and repaired either unspecific by non-homologous end joining (NHEJ) or sequence dependent by homology directed repair (HDR). Whereas NHEJ-based generation of gene knock-out is widely performed, the HDR-based knock-in of specific mutations remains a bottleneck. Especially in primary cell lines that are essential for the generation of cell culture and animal models of inherited human diseases, knock-in efficacy is insufficient and needs significant improvement. Here, we tested two different approaches to increase the knock-in frequency of a specific point mutation into the MYH7-gene in porcine fetal fibroblasts. We added a small molecule inhibitor of NHEJ, SCR7 (5,6-bis((E)-benzylideneamino)-2-mercaptopyrimidin-4-ol), during genome editing and screened cell cultures for the point mutation. However, this approach did not yield increased knock-in rates. In an alternative approach, we fused humanized Cas9 (hCas9) to the N-terminal peptide of the Geminin gene (GMNN). The fusion protein is degraded in NHEJ-dominated cell cycle phases, which should increase HDR-rates. Using hCas9-GMNN and point mutation-specific real time PCR screening, we found a two-fold increase in genome edited cell cultures. This increase of HDR by hCas9-GMNN provides a promising way to enrich specific knock-in in porcine fibroblast cultures for somatic cloning approaches.

Burst-Like Transcription of Mutant and Wildtype MYH7-Alleles as Possible Origin of Cell-to-Cell Contractile Imbalance in Hypertrophic Cardiomyopathy.

Hypertrophic Cardiomyopathy (HCM) has been related to many different mutations in more than 20 different, mostly sarcomeric proteins. While development of the HCM-phenotype is thought to be triggered by the different mutations, a common mechanism remains elusive. Studying missense-mutations in the ventricular beta-myosin heavy chain (ß-MyHC, MYH7) we hypothesized that significant contractile heterogeneity exists among individual cardiomyocytes of HCM-patients that results from cell-to-cell variation in relative expression of mutated vs. wildtype ß-MyHC. To test this hypothesis, we measured force-calcium-relationships of cardiomyocytes isolated from myocardium of heterozygous HCM-patients with either ß-MyHC-mutation Arg723Gly or Arg200Val, and from healthy controls. From the myocardial samples of the HCM-patients we also obtained cryo-sections, and laser-microdissected single cardiomyocytes for quantification of mutated vs. wildtype MYH7-mRNA using a single cell RT-qPCR and restriction digest approach. We characterized gene transcription by visualizing active transcription sites by fluorescence in situ hybridization of intronic and exonic sequences of MYH7-pre-mRNA. For both mutations, cardiomyocytes showed large cell-to-cell variation in Ca++-sensitivity. Interestingly, some cardiomyocytes were essentially indistinguishable from controls what might indicate that they had no mutant ß-MyHC while others had highly reduced Ca++-sensitivity suggesting substantial fractions of mutant ß-MyHC. Single-cell MYH7-mRNA-quantification in cardiomyocytes of the same patients revealed high cell-to-cell variability of mutated vs. wildtype mRNA, ranging from essentially pure mutant to essentially pure wildtype MYH7-mRNA. We found 27% of nuclei without active transcription sites which is inconsistent with continuous gene transcription but suggests burst-like transcription of MYH7. Model simulations indicated that burst-like, stochastic on/off-switching of MYH7 transcription, which is independent for mutant and wildtype alleles, could generate the observed cell-to-cell variation in the fraction of mutant vs. wildtype MYH7-mRNA, a similar variation in ß-MyHC-protein, and highly heterogeneous Ca++-sensitivity of individual cardiomyocytes. In the long run, such contractile imbalance in the myocardium may well induce progressive structural distortions like cellular and myofibrillar disarray and interstitial fibrosis, as they are typically observed in HCM.

Differences in Contractile Function of Myofibrils within Human Embryonic Stem Cell-Derived Cardiomyocytes vs. Adult Ventricular Myofibrils Are Related to Distinct Sarcomeric Protein Isoforms.

Characterizing the contractile function of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) is key for advancing their utility for cellular disease models, promoting cell based heart repair, or developing novel pharmacological interventions targeting cardiac diseases. The aim of the present study was to understand whether steady-state and kinetic force parameters of ß-myosin heavy chain (ßMyHC) isoform-expressing myofibrils within human embryonic stem cell-derived cardiomyocytes (hESC-CMs) differentiated in vitro resemble those of human ventricular myofibrils (hvMFs) isolated from adult donor hearts. Contractile parameters were determined using the same micromechanical method and experimental conditions for both types of myofibrils. We identified isoforms and phosphorylation of main sarcomeric proteins involved in the modulation of force generation of both, chemically demembranated hESC-CMs (d-hESC-CMs) and hvMFs. Our results indicate that at saturating Ca2+ concentration, both human-derived contractile systems developed forces with similar rate constants (0.66 and 0.68 s-1), reaching maximum isometric force that was significantly smaller for d-hESC-CMs (42 kPa) than for hvMFs (94 kPa). At submaximal Ca2+-activation, where intact cardiomyocytes normally operate, contractile parameters of d-hESC-CMs and hvMFs exhibited differences. Ca2+ sensitivity of force was higher for d-hESC-CMs (pCa50 = 6.04) than for hvMFs (pCa50 = 5.80). At half-maximum activation, the rate constant for force redevelopment was significantly faster for d-hESC-CMs (0.51 s-1) than for hvMFs (0.28 s-1). During myofibril relaxation, kinetics of the slow force decay phase were significantly faster for d-hESC-CMs (0.26 s-1) than for hvMFs (0.21 s-1), while kinetics of the fast force decay were similar and ~20x faster. Protein analysis revealed that hESC-CMs had essentially no cardiac troponin-I, and partially non-ventricular isoforms of some other sarcomeric proteins, explaining the functional discrepancies. The sarcomeric protein isoform pattern of hESC-CMs had features of human cardiomyocytes at an early developmental stage. The study indicates that morphological and ultrastructural maturation of ßMyHC isoform-expressing hESC-CMs is not necessarily accompanied by ventricular-like expression of all sarcomeric proteins. Our data suggest that hPSC-CMs could provide useful tools for investigating inherited cardiac diseases affecting contractile function during early developmental stages.

Detection of Legionella-contaminated aerosols in the vicinity of a bio-trickling filter of a breeding sow facility - A pilot study.

The urbanization of agricultural areas results in a reduction of distances between residential buildings and livestock farms. In the public debate, livestock farming is increasingly criticized due to environmental disturbance and odor nuisance originating from such facilities. One method to reduce odor and ammonia is by exhaust air treatment, for example, by biological exhaust air purification processes with bio-trickling filters filled with tap water. Higher temperatures in the summer time and the generation of biofilms are ideal growth conditions for Legionella. However, there are no studies on the presence of Legionella in the water of bio-trickling filters and the release of Legionella-containing aerosols. Therefore, the aim of this study was to investigate Legionella in wash water and emitted bioaerosols of a bio-trickling filter system of a breeding sow facility. For this purpose, measurements were carried out using a cyclone sampler. In addition, samples of wash water were taken. Legionella were not found by culture methods. However, using molecular biological methods, Legionella spp. could be detected in wash water as well as in bioaerosol samples. With antibody-based methods, Legionella pneumophila were identified. Further studies are needed to investigate the environmental health relevance of Legionella-containing aerosols emitted by such exhaust air purification systems.