Matthew S. Sulkin

3D multifunctional integumentary membranes for spatiotemporal cardiac measurements and stimulation across the entire epicardium

Means for high-density multiparametric physiological mapping and stimulation are critically important in both basic and clinical cardiology. Current conformal electronic systems are essentially 2D sheets, which cannot cover the full epicardial surface or maintain reliable contact for chronic use without sutures or adhesives. Here we create 3D elastic membranes shaped precisely to match the epicardium of the heart via the use of 3D printing, as a platform for deformable arrays of multifunctional sensors, electronic and optoelectronic components. Such integumentary devices completely envelop the heart, in a form-fitting manner, and possess inherent elasticity, providing a mechanically stable biotic/abiotic interface during normal cardiac cycles. Component examples range from actuators for electrical, thermal and optical stimulation, to sensors for pH, temperature and mechanical strain. The semiconductor materials include silicon, gallium arsenide and gallium nitride, co-integrated with metals, metal oxides and polymers, to provide these and other operational capabilities. Ex vivo physiological experiments demonstrate various functions and methodological possibilities for cardiac research and therapy.

Processing and analysis of cardiac optical mapping data obtained with potentiometric dyes

Optical mapping has become an increasingly important tool to study cardiac electrophysiology in the past 20 years. Multiple methods are used to process and analyze cardiac optical mapping data, and no consensus currently exists regarding the optimum methods. The specific methods chosen to process optical mapping data are important because inappropriate data processing can affect the content of the data and thus alter the conclusions of the studies. Details of the different steps in processing optical imaging data, including image segmentation, spatial filtering, temporal filtering, and baseline drift removal, are provided in this review. We also provide descriptions of the common analyses performed on data obtained from cardiac optical imaging, including activation mapping, action potential duration mapping, repolarization mapping, conduction velocity measurements, and optical action potential upstroke analysis. Optical mapping is often used to study complex arrhythmias, and we also discuss dominant frequency analysis and phase mapping techniques used for the analysis of cardiac fibrillation.

Stretchable, multiplexed pH sensors with demonstrations on rabbit and human hearts undergoing ischemia

Stable pH is an established biomarker of health, relevant to all tissues of the body, including the heart. Clinical monitoring of pH in a practical manner, with high spatiotemporal resolution, is particularly difficult in organs such as the heart due to its soft mechanics, curvilinear geometry, heterogeneous surfaces, and continuous, complex rhythmic motion. The results presented here illustrate that advanced strategies in materials assembly and electrochemical growth can yield interconnected arrays of miniaturized IrOx pH sensors encapsulated in thin, low-modulus elastomers to yield conformal monitoring systems capable of noninvasive measurements on the surface of the beating heart. A thirty channel custom data acquisition system enables spatiotemporal pH mapping with a single potentiostat. In vitro testing reveals super-Nernstian sensitivity with excellent uniformity (69.9 ± 2.2 mV/pH), linear response to temperature (-1.6 mV °C(-1) ), and minimal influence of extracellular ions (<3.5 mV). Device examples include sensor arrays on balloon catheters and on skin-like stretchable membranes. Real-time measurement of pH on the surfaces of explanted rabbit hearts and a donated human heart during protocols of ischemia-reperfusion illustrate some of the capabilities. Envisioned applications range from devices for biological research, to surgical tools and long-term implants.

Mitochondrial Dysfunction Causing Cardiac Sodium Channel Downregulation in Cardiomyopathy

Cardiomyopathy is associated with cardiac Na(+) channel downregulation that may contribute to arrhythmias. Previously, we have shown that elevated intracellular NADH causes a decrease in cardiac Na(+) current (I(Na)) signaled by an increase in mitochondrial reactive oxygen species (ROS). In this study, we tested whether the NADH-mitochondria ROS pathway was involved in the reduction of I(Na) in a nonischemic cardiomyopathic model and correlated the findings with myopathic human hearts. Nonischemic cardiomyopathy was induced in C57BL/6 mice by hypertension after unilateral nephrectomy, deoxycorticosterone acetate (DOCA) pellet implantation, and salt water substitution. Sham operated mice were used as controls. After six weeks, heart tissue and ventricular myocytes isolated from mice were utilized for whole cell patch clamp recording, NADH/NAD(+) level measurements, and mitochondrial ROS monitoring with confocal microscopy. Human explanted hearts were studied using optical mapping. Compared to the sham mice, the arterial blood pressure was higher, the left ventricular volume was significantly enlarged (104.7±3.9 vs. 87.9±6.1 μL, P<0.05), and the ejection fraction was reduced (37.1±1.8% vs. 49.4±3.7%, P<0.05) in DOCA mice. Both the whole cell and cytosolic NADH level were increased (279±70% and 123±2% of sham, respectively, P<0.01), I(Na) was decreased (60±10% of sham, P<0.01), and mitochondrial ROS overproduction was observed (2.9±0.3-fold of sham, P<0.01) in heart tissue and myocytes of myopathic mice vs. sham. Treatment of myocytes with NAD(+) (500 μM), mitoTEMPO (10 μM), chelerythrine (50 μM), or forskolin (5 μM) restored I(Na) back to the level of sham. Injection of NAD(+) (100mg/kg) or mitoTEMPO (0.7 mg/kg) twice (at 24h and 1h before myocyte isolation) to animals also restored I(Na). All treatments simultaneously reduced mitochondrial ROS levels to that of controls. CD38 was found to transduce the extracellular NAD(+) signal. Correlating with the mouse model, failing human hearts showed a reduction in conduction velocity that improved with NAD(+). Nonischemic cardiomyopathy was associated with elevated NADH level, PKC activation, mitochondrial ROS overproduction, and a concomitant decrease in I(Na). Reducing mitochondrial ROS by application of NAD(+), mitoTEMPO, PKC inhibitors, or PKA activators, restored I(Na). NAD(+) improved conduction velocity in human myopathic hearts.

Optical Mapping of Action Potentials and Calcium Transients in the Mouse Heart

The mouse heart is a popular model for cardiovascular studies due to the existence of low cost technology for genetic engineering in this species. Cardiovascular physiological phenotyping of the mouse heart can be easily done using fluorescence imaging employing various probes for transmembrane potential (Vm), calcium transients (CaT), and other parameters. Excitation-contraction coupling is characterized by action potential and intracellular calcium dynamics; therefore, it is critically important to map both Vm and CaT simultaneously from the same location on the heart1-4. Simultaneous optical mapping from Langendorff perfused mouse hearts has the potential to elucidate mechanisms underlying heart failure, arrhythmias, metabolic disease, and other heart diseases. Visualization of activation, conduction velocity, action potential duration, and other parameters at a myriad of sites cannot be achieved from cellular level investigation but is well solved by optical mapping1,5,6. In this paper we present the instrumentation setup and experimental conditions for simultaneous optical mapping of Vm and CaT in mouse hearts with high spatio-temporal resolution using state-of-the-art CMOS imaging technology. Consistent optical recordings obtained with this method illustrate that simultaneous optical mapping of Langendorff perfused mouse hearts is both feasible and reliable.

Patient-specific flexible and stretchable devices for cardiac diagnostics and therapy

Advances in material science techniques and pioneering circuit designs have led to the development of electronic membranes that can form intimate contacts with biological tissues. In this review, we present the range of geometries, sensors, and actuators available for custom configurations of electronic membranes in cardiac applications. Additionally, we highlight the desirable mechanics achieved by such devices that allow the circuits and substrates to deform with the beating heart. These devices unlock opportunities to collect continuous data on the electrical, metabolic, and mechanical state of the heart as well as a platform on which to develop high definition therapeutics.

Tuning the electrical properties of the heart by differential trafficking of KATP ion channel complexes

ABSTRACT The copy number of membrane proteins at the cell surface is tightly regulated. Many ion channels and receptors present retrieval motifs to COPI vesicle coats and are retained in the early secretory pathway. In some cases, the interaction with COPI is prevented by binding to 14-3-3 proteins. However, the functional significance of this antagonism between COPI and 14-3-3 in terminally differentiated cells is unknown. Here, we show that ATP-sensitive K+ (KATP) channels, which are composed of Kir6.2 and SUR1 subunits, are stalled in the Golgi complex of ventricular, but not atrial, cardiomyocytes. Upon sustained &bgr;-adrenergic stimulation, which leads to activation of protein kinase A (PKA), SUR1-containing channels reach the plasma membrane of ventricular cells. We show that PKA-dependent phosphorylation of the C-terminus of Kir6.2 decreases binding to COPI and, thereby, silences the arginine-based retrieval signal. Thus, activation of the sympathetic nervous system releases this population of KATP channels from storage in the Golgi and, hence, might facilitate the adaptive response to metabolic challenges.

Transmural APD gradient synchronizes repolarization in the human left ventricular wall

AIMS The duration and morphology of the T wave predict risk for ventricular fibrillation. A transmural gradient in action potential duration (APD) in the ventricular wall has been suggested to underlie the T wave in humans. We hypothesize that the transmural gradient in APD compensates for the normal endocardium-to-epicardium activation sequence and synchronizes repolarization in the human ventricular wall. METHODS AND RESULTS We made left ventricular wedge preparations from 10 human donor hearts and measured transmural activation and repolarization patterns by optical mapping, while simultaneously recording a pseudo-ECG. We also studied the relation between local timings of repolarization with the T wave in silico. During endocardial pacing (1 Hz), APD was longer at the subendocardium than at the subepicardium (360 ± 17 vs. 317 ± 20 ms, P < 0.05). The transmural activation time was 32 ± 4 ms and resulted in final repolarization of the subepicardium at 349 ± 18 ms. The overall transmural dispersion in repolarization time was smaller than that of APD. During epicardial pacing, the dispersion in repolarization time increased, whereas that of APD remained similar. The morphology of the T wave did not differ between endocardial and epicardial stimulation. Simulations explained the constant T wave morphology without transmural APD gradients. CONCLUSION The intrinsic transmural difference in APD compensates for the normal cardiac activation sequence, resulting in more homogeneous repolarization of the left ventricular wall. Our data suggest that the transmural repolarization differences do not fully explain the genesis of the T wave.

CD36 Protein Influences Myocardial Ca2+ Homeostasis and Phospholipid Metabolism CONDUCTION ANOMALIES IN CD36-DEFICIENT MICE DURING FASTING

Background: Myocardial function during fasting was examined in wild-type and CD36−/− mice. Results: CD36−/− mice have abnormalities of myocardial Ca2+, phospholipid composition, and cAMP levels and manifest electrical anomalies during fasting. Conclusion: CD36 influences myocardial adaptation by impacting Ca2+ dynamics and phospholipid metabolism. Significance: Inter-dependence of lipid metabolism and Ca2+ homeostasis can contribute to cardiac dysfunction during metabolic stress. Sarcolemmal CD36 facilitates myocardial fatty acid (FA) uptake, which is markedly reduced in CD36-deficient rodents and humans. CD36 also mediates signal transduction events involving a number of cellular pathways. In taste cells and macrophages, CD36 signaling was recently shown to regulate store-responsive Ca2+ flux and activation of Ca2+-dependent phospholipases A2 that cycle polyunsaturated FA into phospholipids. It is unknown whether CD36 deficiency influences myocardial Ca2+ handling and phospholipid metabolism, which could compromise the heart, typically during stresses. Myocardial function was examined in fed or fasted (18–22 h) CD36−/− and WT mice. Echocardiography and telemetry identified conduction anomalies that were associated with the incidence of sudden death in fasted CD36−/− mice. No anomalies or death occurred in WT mice during fasting. Optical imaging of perfused hearts from fasted CD36−/− mice documented prolongation of Ca2+ transients. Consistent with this, knockdown of CD36 in cardiomyocytes delayed clearance of cytosolic Ca2+. Hearts of CD36−/− mice (fed or fasted) had 3-fold higher SERCA2a and 40% lower phospholamban levels. Phospholamban phosphorylation by protein kinase A (PKA) was enhanced after fasting reflecting increased PKA activity and cAMP levels in CD36−/− hearts. Abnormal Ca2+ homeostasis in the CD36−/− myocardium associated with increased lysophospholipid content and a higher proportion of 22:6 FA in phospholipids suggests altered phospholipase A2 activity and changes in membrane dynamics. The data support the role of CD36 in coordinating Ca2+ homeostasis and lipid metabolism and the importance of this role during myocardial adaptation to fasting. Potential relevance of the findings to CD36-deficient humans would need to be determined.

Three-dimensional printing physiology laboratory technology

Since its inception in 19th-century Germany, the physiology laboratory has been a complex and expensive research enterprise involving experts in various fields of science and engineering. Physiology research has been critically dependent on cutting-edge technological support of mechanical, electrical, optical, and more recently computer engineers. Evolution of modern experimental equipment is constrained by lack of direct communication between the physiological community and industry producing this equipment. Fortunately, recent advances in open source technologies, including three-dimensional printing, open source hardware and software, present an exciting opportunity to bring the design and development of research instrumentation to the end user, i.e., life scientists. Here we provide an overview on how to develop customized, cost-effective experimental equipment for physiology laboratories.