Franz J. Baudenbacher

Myofilament Ca2+ sensitization causes susceptibility to cardiac arrhythmia in mice

In human cardiomyopathy, anatomical abnormalities such as hypertrophy and fibrosis contribute to the risk of ventricular arrhythmias and sudden death. Here we have shown that increased myofilament Ca2+ sensitivity, also a common feature in both inherited and acquired human cardiomyopathies, created arrhythmia susceptibility in mice, even in the absence of anatomical abnormalities. In mice expressing troponin T mutants that cause hypertrophic cardiomyopathy in humans, the risk of developing ventricular tachycardia was directly proportional to the degree of Ca2+ sensitization caused by the troponin T mutation. Arrhythmia susceptibility was reproduced with the Ca2+-sensitizing agent EMD 57033 and prevented by myofilament Ca2+ desensitization with blebbistatin. Ca2+ sensitization markedly changed the shape of ventricular action potentials, resulting in shorter effective refractory periods, greater beat-to-beat variability of action potential durations, and increased dispersion of ventricular conduction velocities at fast heart rates. Together these effects created an arrhythmogenic substrate. Thus, myofilament Ca2+ sensitization represents a heretofore unrecognized arrhythmia mechanism. The protective effect of blebbistatin provides what we believe to be the first direct evidence that reduction of Ca2+ sensitivity in myofilaments is antiarrhythmic and might be beneficial to individuals with hypertrophic cardiomyopathy.

Records of an ancient Martian magnetic field in ALH84001

Although Mars does not presently appear to have a global dynamo magnetic field, strong crustal fields have recently been detected by the Mars Global Surveyor above surfaces formed ∼3 or more Ga. We present magnetic and textural studies of Martian meteorite ALH84001 demonstrating that 4 Ga carbonates containing magnetite and pyrrhotite carry a stable natural remanent magnetization. Because ^(40)Ar/^(39)Ar thermochronology demonstrates that most ALH84001 carbonates have probably been well below the Curie point of magnetite since near the time of their formation [Weiss et al., Earth Planet. Sci. Lett. (2002) this issue], their magnetization originated at 3.9–4.1 Ga on Mars. This magnetization is at least 500 million years (Myr) older than that known in any other planetary rock, and its strong intensity suggests that Mars had generated a geodynamo and global magnetic field within 450–650 Myr of its formation. The intensity of this field was roughly within an order of magnitude of that at the surface of the present-day Earth, sufficient for magnetotaxis by the bacteria whose magnetofossils have been reported in ALH84001 and possibly for the production of the strong crustal anomalies. Chromite in ALH84001 may retain even older records of Martian magnetic fields, possibly extending back to near the time of planetary formation.

Microfluidic platform for real-time signaling analysis of multiple single T cells in parallel.

Deciphering the signaling pathways that govern stimulation of naïve CD4+ T helper cells by antigen-presenting cells via formation of the immunological synapse is key to a fundamental understanding of the progression of successful adaptive immune response. The study of T cell-APC interactions in vitro is challenging, however, due to the difficulty of tracking individual, non-adherent cell pairs over time. Studying single cell dynamics over time reveals rare, but critical, signaling events that might be averaged out in bulk experiments, but these less common events are undoubtedly important for an integrated understanding of a cellular response to its microenvironment. We describe a novel application of microfluidic technology that overcomes many limitations of conventional cell culture and enables the study of hundreds of passively sequestered hematopoietic cells for extended periods of time. This microfluidic cell trap device consists of 440 18 micromx18 micromx10 microm PDMS, bucket-like structures opposing the direction of flow which serve as corrals for cells as they pass through the cell trap region. Cell viability analysis revealed that more than 70% of naïve CD4+ T cells (TN), held in place using only hydrodynamic forces, subsequently remain viable for 24 hours. Cytosolic calcium transients were successfully induced in TN cells following introduction of chemical, antibody, or cellular forms of stimulation. Statistical analysis of TN cells from a single stimulation experiment reveals the power of this platform to distinguish different calcium response patterns, an ability that might be utilized to characterize T cell signaling states in a given population. Finally, we investigate in real time contact- and non-contact-based interactions between primary T cells and dendritic cells, two main participants in the formation of the immunological synapse. Utilizing the microfluidic traps in a daisy-chain configuration allowed us to observe calcium transients in TN cells exposed only to media conditioned by secretions of lipopolysaccharide-matured dendritic cells, an event which is easily missed in conventional cell culture where large media-to-cell ratios dilute cellular products. Further investigation into this intercellular signaling event indicated that LPS-matured dendritic cells, in the absence of antigenic stimulation, secrete chemical signals that induce calcium transients in T(N) cells. While the stimulating factor(s) produced by the mature dendritic cells remains to be identified, this report illustrates the utility of these microfluidic cell traps for analyzing arrays of individual suspension cells over time and probing both contact-based and intercellular signaling events between one or more cell populations.

Myofilament Ca sensitization increases cytosolic Ca binding affinity, alters intracellular Ca homeostasis, and causes pause-dependent Ca-triggered arrhythmia.

Rationale: Ca binding to the troponin complex represents a major portion of cytosolic Ca buffering. Troponin mutations that increase myofilament Ca sensitivity are associated with familial hypertrophic cardiomyopathy and confer a high risk for sudden death. In mice, Ca sensitization causes ventricular arrhythmias, but the underlying mechanisms remain unclear. Objective: To test the hypothesis that myofilament Ca sensitization increases cytosolic Ca buffering and to determine the resulting arrhythmogenic changes in Ca homeostasis in the intact mouse heart. Methods and Results: Using cardiomyocytes isolated from mice expressing troponin T (TnT) mutants (TnT-I79N, TnT-F110I, TnT-R278C), we found that increasing myofilament Ca sensitivity produced a proportional increase in cytosolic Ca binding. The underlying cause was an increase in the cytosolic Ca binding affinity, whereas maximal Ca binding capacity was unchanged. The effect was sufficiently large to alter Ca handling in intact mouse hearts at physiological heart rates, resulting in increased end-diastolic [Ca] at fast pacing rates, and enhanced sarcoplasmic reticulum Ca content and release after pauses. Accordingly, action potential (AP) regulation was altered, with postpause action potential prolongation, afterdepolarizations, and triggered activity. Acute Ca sensitization with EMD 57033 mimicked the effects of Ca-sensitizing TnT mutants and produced pause-dependent ventricular ectopy and sustained ventricular tachycardia after acute myocardial infarction. Conclusions: Myofilament Ca sensitization increases cytosolic Ca binding affinity. A major proarrhythmic consequence is a pause-dependent potentiation of Ca release, action potential prolongation, and triggered activity. Increased cytosolic Ca binding represents a novel mechanism of pause-dependent arrhythmia that may be relevant for inherited and acquired cardiomyopathies.

High-resolution room-temperature sample scanning superconducting quantum interference device microscope configurable for geological and biomagnetic applications

We have developed a scanning superconducting quantum interference device (SQUID) microscope system with interchangeable sensor configurations for imaging magnetic fields of room-temperature (RT) samples with submillimeter resolution. The low-critical-temperature (Tc) niobium-based monolithic SQUID sensors are mounted on the tip of a sapphire and thermally anchored to the helium reservoir. A 25μm sapphire window separates the vacuum space from the RT sample. A positioning mechanism allows us to adjust the sample-to-sensor spacing from the top of the Dewar. We achieved a sensor-to-sample spacing of 100μm, which could be maintained for periods of up to four weeks. Different SQUID sensor designs are necessary to achieve the best combination of spatial resolution and field sensitivity for a given source configuration. For imaging thin sections of geological samples, we used a custom-designed monolithic low-Tc niobium bare SQUID sensor, with an effective diameter of 80μm, and achieved a field sensitivity of 1.5...

Investigating impact demagnetization through laser impacts and SQUID microscopy

Understanding demagnetization by hypervelocity impacts is crucial for the interpreta- tion of planetary magnetic anomalies and remanent magnetization in meteorites. We de- scribe an innovative approach for investigating the effects of impacts on the remanent magnetization of geologic materials. It consists of the combination of pulsed laser impacts and Superconducting Quantum Interference Device (SQUID) microscopy. Laser impacts are nondestructive, create shocks with peak pressures as high as several hundred GPa, and allow well-calibrated modeling of shock wave propagation within the impacted sam- ples. High-resolution SQUID microscopy quantitatively maps the magnetic field of room- temperature samples with an unprecedented spatial resolution of ;100 mm. We present shock modeling and magnetic field data obtained for two laser impacts on a magnetite- bearing basalt sample. Magnetic measurements show a demagnetized area at the impact locations. We also show that high-resolution magnetic measurements combined with im- pact modeling provide a continuous relation between the demagnetization intensity and the peak pressure undergone by the sample. This promising technique will allow for the investigation of the demagnetization behavior of a variety of geological materials upon impacts, with implications for our understanding of the magnetization of extraterrestrial materials and of terrestrial impact structures.

High resolution low-temperature superconductivity superconducting quantum interference device microscope for imaging magnetic fields of samples at room temperatures

We have developed a microscope to image weak magnetic fields using submillimeter pickup coils made from conventional low-temperature superconducting niobium wire coupled to the input circuit of a superconducting quantum interference device (SQUID). The pickup coil and the SQUID sensor are mounted in the vacuum space of the cryostat and are thermally anchored to the liquid helium reservoir. A 25 μm thick sapphire window separates the room temperature (RT) sample and the vacuum space. The spacing between the pickup coil and RT sample was typically less than 130 μm. The spatial resolution is limited by the diameter of the pickup coil. The pickup coils are easily interchangeable, allowing us to adapt the SQUID microscope to a variety of different measurements. We have achieved a spatial resolution of 250 μm with a magnetic field sensitivity of 850 fT/Hz1/2 or a spatial resolution of 500 μm with a magnetic field sensitivity of 330 fT/Hz1/2. We have used this instrument to measure various biomagnetic and paleom...

Modification of the Cytosensor™ microphysiometer to simultaneously measure extracellular acidification and oxygen consumption rates

Abstract The Cytosensor™ microphysiometer has been modified by incorporating several platinum electrodes into the plunger head. The electrodes have been used to measure simultaneously the change in extracellular oxygen consumption rates and extracellular acidification rates during addition of various non-physiologic agents: 2-deoxy- d -glucose, sodium fluoride, paraoxon-ethyl, and antimycin A. Measurement of two parameters, oxygen and acidification, enables investigation of the aerobic and anaerobic metabolic consequences of these agents. Nafion membranes cast onto the platinum electrodes were useful in minimizing adsorption of cell-related interferents.

Combinatorial Polymer Electrospun Matrices Promote Physiologically-Relevant Cardiomyogenic Stem Cell Differentiation

Myocardial infarction results in extensive cardiomyocyte death which can lead to fatal arrhythmias or congestive heart failure. Delivery of stem cells to repopulate damaged cardiac tissue may be an attractive and innovative solution for repairing the damaged heart. Instructive polymer scaffolds with a wide range of properties have been used extensively to direct the differentiation of stem cells. In this study, we have optimized the chemical and mechanical properties of an electrospun polymer mesh for directed differentiation of embryonic stem cells (ESCs) towards a cardiomyogenic lineage. A combinatorial polymer library was prepared by copolymerizing three distinct subunits at varying molar ratios to tune the physicochemical properties of the resulting polymer: hydrophilic polyethylene glycol (PEG), hydrophobic poly(ε-caprolactone) (PCL), and negatively-charged, carboxylated PCL (CPCL). Murine ESCs were cultured on electrospun polymeric scaffolds and their differentiation to cardiomyocytes was assessed through measurements of viability, intracellular reactive oxygen species (ROS), α-myosin heavy chain expression (α-MHC), and intracellular Ca2+ signaling dynamics. Interestingly, ESCs on the most compliant substrate, 4%PEG-86%PCL-10%CPCL, exhibited the highest α-MHC expression as well as the most mature Ca2+ signaling dynamics. To investigate the role of scaffold modulus in ESC differentiation, the scaffold fiber density was reduced by altering the electrospinning parameters. The reduced modulus was found to enhance α-MHC gene expression, and promote maturation of myocyte Ca2+ handling. These data indicate that ESC-derived cardiomyocyte differentiation and maturation can be promoted by tuning the mechanical and chemical properties of polymer scaffold via copolymerization and electrospinning techniques.

Heat conduction calorimeter for massively parallel high throughput measurements with picoliter sample volumes

We have developed a bulk micromachined calorimeter with a sensitivity of 1.5nW∕Hz1∕2 and a 1ms time constant using a thin film thermopile as the sensing element. The thermopile consists of seven titanium and bismuth thermocouples with a total Seebeck coefficient of 574μV∕K. The device is capable of measuring enthalpies in chemical or biological reactions in volumes as small as a few picoliters. The device can be fabricated and operated in a massively parallel fashion in combination with ink-jet printing technologies in air and at room temperature, making it ideally suited for biological and biochemical experiments.