Role of biophysics and mechanobiology in podocyte physiology.

Podocytes form the backbone of the glomerular filtration barrier and are exposed to various mechanical forces throughout the lifetime of an individual. The highly dynamic biomechanical environment of the glomerular capillaries greatly influences the cell biology of podocytes and their pathophysiology. Throughout the past two decades, a holistic picture of podocyte cell biology has emerged, highlighting mechanobiological signalling pathways, cytoskeletal dynamics and cellular adhesion as key determinants of biomechanical resilience in podocytes. This biomechanical resilience is essential for the physiological function of podocytes, including the formation and maintenance of the glomerular filtration barrier. Podocytes integrate diverse biomechanical stimuli from their environment and adapt their biophysical properties accordingly. However, perturbations in biomechanical cues or the underlying podocyte mechanobiology can lead to glomerular dysfunction with severe clinical consequences, including proteinuria and glomerulosclerosis. As our mechanistic understanding of podocyte mechanobiology and its role in the pathogenesis of glomerular disease increases, new targets for podocyte-specific therapeutics will emerge. Treating glomerular diseases by targeting podocyte mechanobiology might improve therapeutic precision and efficacy, with potential to reduce the burden of chronic kidney disease on individuals and health-care systems alike.

The biophysical properties of TRIC-A and TRIC-B and their interactions with RyR2.

Trimeric intracellular cation channels (TRIC-A and TRIC-B) are thought to provide counter-ion currents to enable charge equilibration across the sarco/endoplasmic reticulum (SR) and nuclear membranes. However, there is also evidence that TRIC-A may interact directly with ryanodine receptor type 1 (RyR1) and 2 (RyR2) to alter RyR channel gating. It is therefore possible that the reverse is also true, where the presence of RyR channels is necessary for fully functional TRIC channels. We therefore coexpressed mouse TRIC-A or TRIC-B with mouse RyR2 in HEK293 cells to examine if after incorporating membrane vesicles from these cells into bilayers, the presence of TRIC affects RyR2 function, and to characterize the permeability and gating properties of the TRIC channels. Importantly, we used no purification techniques or detergents to minimize damage to TRIC and RyR2 proteins. We found that both TRIC-A and TRIC-B altered the gating behavior of RyR2 and its response to cytosolic Ca2+ but that TRIC-A exhibited a greater ability to stimulate the opening of RyR2. Fusing membrane vesicles containing TRIC-A or TRIC-B into bilayers caused the appearance of rapidly gating current fluctuations of multiple amplitudes. The reversal potentials of bilayers fused with high numbers of vesicles containing TRIC-A or TRIC-B revealed both Cl- and K+ fluxes, suggesting that TRIC channels are relatively non-selective ion channels. Our results indicate that the physiological roles of TRIC-A and TRIC-B may include direct, complementary regulation of RyR2 gating in addition to the provision of counter-ion currents of both cations and anions.

Multiscale model of primary motor cortex circuits predicts in vivo cell-type-specific, behavioral state-dependent dynamics.

Understanding cortical function requires studying multiple scales: molecular, cellular, circuit, and behavioral. We develop a multiscale, biophysically detailed model of mouse primary motor cortex (M1) with over 10,000 neurons and 30 million synapses. Neuron types, densities, spatial distributions, morphologies, biophysics, connectivity, and dendritic synapse locations are constrained by experimental data. The model includes long-range inputs from seven thalamic and cortical regions and noradrenergic inputs. Connectivity depends on cell class and cortical depth at sublaminar resolution. The model accurately predicts in vivo layer- and cell-type-specific responses (firing rates and LFP) associated with behavioral states (quiet wakefulness and movement) and experimental manipulations (noradrenaline receptor blockade and thalamus inactivation). We generate mechanistic hypotheses underlying the observed activity and analyzed low-dimensional population latent dynamics. This quantitative theoretical framework can be used to integrate and interpret M1 experimental data and sheds light on the cell-type-specific multiscale dynamics associated with several experimental conditions and behaviors.

Low-dimensional models of single neurons: a review.

The classical Hodgkin-Huxley (HH) point-neuron model of action potential generation is four-dimensional. It consists of four ordinary differential equations describing the dynamics of the membrane potential and three gating variables associated to a transient sodium and a delayed-rectifier potassium ionic currents. Conductance-based models of HH type are higher-dimensional extensions of the classical HH model. They include a number of supplementary state variables associated with other ionic current types, and are able to describe additional phenomena such as subthreshold oscillations, mixed-mode oscillations (subthreshold oscillations interspersed with spikes), clustering and bursting. In this manuscript we discuss biophysically plausible and phenomenological reduced models that preserve the biophysical and/or dynamic description of models of HH type and the ability to produce complex phenomena, but the number of effective dimensions (state variables) is lower. We describe several representative models. We also describe systematic and heuristic methods of deriving reduced models from models of HH type.

Acousto-holographic reconstruction of whole-cell stiffness maps.

Accurate assessment of cell stiffness distribution is essential due to the critical role of cell mechanobiology in regulation of vital cellular processes like proliferation, adhesion, migration, and motility. Stiffness provides critical information in understanding onset and progress of various diseases, including metastasis and differentiation of cancer. Atomic force microscopy and optical trapping set the gold standard in stiffness measurements. However, their widespread use has been hampered with long processing times, unreliable contact point determination, physical damage to cells, and unsuitability for multiple cell analysis. Here, we demonstrate a simple, fast, label-free, and high-resolution technique using acoustic stimulation and holographic imaging to reconstruct stiffness maps of single cells. We used this acousto-holographic method to determine stiffness maps of HCT116 and CTC-mimicking HCT116 cells and differentiate between them. Our system would enable widespread use of whole-cell stiffness measurements in clinical and research settings for cancer studies, disease modeling, drug testing, and diagnostics.

Energy efficiency of pulse shaping in electrical stimulation: the interdependence of biophysical effects and circuit design losses.

Power efficiency in electrical stimulator circuits is crucial for developing large-scale multichannel applications like bidirectional brain-computer interfaces and neuroprosthetic devices. Many state-of-the-art papers have suggested that some non-rectangular pulse shapes are more energy-efficient for exciting neural excitation than the conventional rectangular shape. However, additional losses in the stimulator circuit, which arise from employing such pulses, were not considered. In this work, we analyze the total energy efficiency of a stimulation system featuring non-rectangular stimuli, taking into account the losses in the stimulator circuit. To this end, activation current thresholds for different pulse shapes and durations in cortical neurons are modeled, and the energy required to generate the pulses from a constant voltage supply is calculated. The proposed calculation reveals an energy increase of 14%-51% for non-rectangular pulses compared to the conventional rectangular stimuli, instead of the decrease claimed in previous literature. This result indicates that a rectangular stimulation pulse is more power-efficient than the tested alternative shapes in large-scale multichannel electrical stimulation systems.

Assembly and Use of a Microfluidic Device to Study Nuclear Mechanobiology During Confined Migration.

Cancer metastasis, that is, the spreading of tumor cells from the primary tumor to distant sites, requires cancer cells to travel through pores substantially smaller than their cross section . This "confined migration" requires substantial deformation by the relatively large and rigid nucleus, which can impact nuclear compartmentalization, trigger cellular mechanotransduction pathways, and increase genomic instability. To improve our understanding of how cells perform and respond to confined migration, we developed polydimethylsiloxane (PDMS) microfluidic devices in which cells migrate through a precisely controlled "field of pillars" that closely mimic the intermittent confinement of tumor microenvironments and interstitial spaces. The devices can be designed with various densities of pillars, ranging from a very low density that does not require nuclear deformation to high densities that present microenvironment conditions with severe confinement. The devices enable assessment of cellular fitness for confined migration based on the distance traveled through the constriction area over several days. In this protocol, we present two complementary techniques to generate silicon master molds for the device fabrication: (1) SU-8 soft lithography for rapid prototyping and for devices with relatively large features; and (2) reactive ion etching (RIE) to achieve finer features and more durable molds. In addition, we describe the production, use, and validation of the devices, along with the analysis pipeline for experiments using the devices with fluorescently labeled cells. Collectively, this protocol enables the study of confined migration and is readily amendable to investigate other aspects of confined migration mechanobiology, such as nuclear pore complex function in response to nuclear deformation.

Mechanobiology-based physical therapy and rehabilitation after orthobiologic interventions: a narrative review.

PURPOSE: This review aims to summarize the evidence for the role of mechanotherapies and rehabilitation in supporting the synergy between regeneration and repair after an orthobiologic intervention. METHODS: A selective literature search was performed using Web of Science, OVID, and PubMed to review research articles that discuss the effects of combining mechanotherapy with various forms of regenerative medicine. RESULTS: Various mechanotherapies can encourage the healing process for patients at different stages. Taping, bracing, cold water immersion, and extracorporeal shockwave therapy can be used throughout the duration of acute inflammatory response. The regulation of angiogenesis can be sustained with blood flow restriction and resistance training, whereas heat therapy and tissue loading during exercise are recommended in the remodeling phase. CONCLUSION: Combining mechanotherapy with various forms of regenerative medicine has shown promise for improving treatment outcomes. However, further studies that reveal a greater volume of evidence are needed to support clinical decisions.

Selective activation of central thalamic fiber pathway facilitates behavioral performance in healthy non-human primates.

Central thalamic deep brain stimulation (CT-DBS) is an investigational therapy to treat enduring cognitive dysfunctions in structurally brain injured (SBI) patients. However, the mechanisms of CT-DBS that promote restoration of cognitive functions are unknown, and the heterogeneous etiology and recovery profiles of SBI patients contribute to variable outcomes when using conventional DBS strategies,which may result in off-target effects due to activation of multiple pathways. To disambiguate the effects of stimulation of two adjacent thalamic pathways, we modeled and experimentally compared conventional and novel 'field-shaping' methods of CT-DBS within the central thalamus of healthy non-human primates (NHP) as they performed visuomotor tasks. We show that selective activation of the medial dorsal thalamic tegmental tract (DTTm), but not of the adjacent centromedian-parafascicularis (CM-Pf) pathway, results in robust behavioral facilitation. Our predictive modeling approach in healthy NHPs directly informs ongoing and future clinical investigations of conventional and novel methods of CT-DBS for treating cognitive dysfunctions in SBI patients, for whom no therapy currently exists.

Photobiomodulation as an antioxidant substitute in post-thawing trauma of human stem cells from the apical papilla.

Cryopreservation, the most common method of preserving stem cells, requires post-processing because it produces trauma to the cells. Post-thawing trauma typically induces cell death, elevates reactive oxygen species (ROS) concentration, and lowers mitochondrial membrane potential (MMP). Although this trauma has been solved using antioxidants, we attempted to use photobiomodulation (PBM) instead of chemical treatment. We used a 950-nm near-infrared LED to create a PBM device and chose a pulsed-wave mode of 30 Hz and a 30% duty cycle. Near-infrared radiation (NIR) at 950 nm was effective in reducing cell death caused by hydrogen peroxide induced-oxidative stress. Cryodamage also leads to apoptosis of cells, which can be avoided by irradiation at 950 nm NIR. Irradiation as post-processing for cryopreservation had an antioxidant effect that reduced both cellular and mitochondrial ROS. It also increased mitochondrial mass and activated mitochondrial activity, resulting in increased MMP, ATP generation, and increased cytochrome c oxidase activity. In addition, NIR increased alkaline phosphatase (ALP) activity, a biomarker of differentiation. As a result, we identified that 950 nm NIR PBM solves cryodamage in human stem cells from the apical papilla, indicating its potential as an alternative to antioxidants for treatment of post-thawing trauma, and further estimated its mechanism.

Biomechanics of pollen pellet removal by the honey bee.

Honey bees (Apis mellifera) carry pollen back to their hive by mixing it with nectar and forming it into a pellet. The pellet must be firmly attached to their legs during flight, but also easily removable when deposited in the hive. How does the honey bee achieve these contrary aims? In this experimental study, we film honey bees removing pollen pellets and find they peel them off at speeds 2-10 times slower than their typical grooming speeds. Using a self-built pollen scraper, we find that slow removal speeds reduce the force and work required to remove the pellet under shear stress. Creep tests on individual pollen pellets revealed that pollen pellets are viscoelastic materials characterized by a Maxwell model with long relaxation times. The relaxation time enables the pellet to remain a solid during both transport and removal. We hope that this work inspires further research into viscoelastic materials in nature.

Biophysical and structural investigation of the regulation of human GTP cyclohydrolase I by its regulatory protein GFRP.

GTP Cyclohydrolase I (GCH1) catalyses the conversion of guanosine triphosphate (GTP) to dihydroneopterin triphosphate (H2NTP), the initiating step in the biosynthesis of tetrahydrobiopterin (BH4). BH4 functions as co-factor in neurotransmitter biosynthesis. BH4 homeostasis is a promising target to treat pain disorders in patients. The function of mammalian GCH1s is regulated by a metabolic sensing mechanism involving a regulator protein, GCH1 feedback regulatory protein (GFRP). Dependent on the relative cellular concentrations of effector ligands, BH4 and phenylalanine, GFRP binds GCH1 to form inhibited or activated complexes, respectively. We determined high-resolution structures of the ligand-free and -bound human GFRP and GCH1-GFRP complexes by X-ray crystallography. Highly similar binding modes of the substrate analogue 7-deaza-GTP to active and inhibited GCH1-GFRP complexes confirm a novel, dissociation rate-controlled mechanism of non-competitive inhibition to be at work. Further, analysis of all structures shows that upon binding of the effector molecules, the conformations of GCH1 or GFRP are altered and form highly complementary surfaces triggering a picomolar interaction of GFRP and GCH1 with extremely slow koff values, while GCH1-GFRP complexes rapidly disintegrate in absence of BH4 or phenylalanine. Finally, comparing behavior of full-length and N-terminally truncated GCH1 we conclude that the disordered GCH1 N-terminus does not have impact on complex formation and enzymatic activity. In summary, this comprehensive and methodologically diverse study helps to provide a better understanding of the regulation of GCH1 by GFRP and could thus stimulate research on GCH1 modulating drugs.

Improvements in Cervical Spinal Canal Diameter and Neck Disability Following Correction of Cervical Lordosis and Cervical Spondylolistheses Using Chiropractic BioPhysics Technique: A Case Series.

Cervical spondylolisthesis indicates instability of the spine and can lead to pain, radiculopathy, myelopathy and vertebral artery stenosis. Currently degenerative cervical spondylolisthesis is a wait-and-watch condition with no treatment guidelines. A literature review and discussion will be provided. 8 females presented with neck pain, disability, and history of motor vehicle collision. Radiographs revealed abnormal cervical alignment, spinal canal narrowing, and spondylolistheses. After 30 sessions of Chiropractic BioPhysics® care over 12 weeks, patients reported improved symptoms and disabilities. Radiographs revealed improvements in cervical alignment, spondylolistheses, and spinal canal diameter. Motor vehicle collision may cause instability and abnormal alignment of the cervical spine leading to cervical spondylolisthesis. Improving spinal alignment may be an effective treatment to reduce vertebral subluxation and cervical spondylolistheses and improve neck disability as a result of improved spinal alignment.

Mimicry of a biophysical pathway leads to diverse pollen-like surface patterns.

A ubiquitous structural feature in biological systems is texture in extracellular matrix that gains functions when hardened, for example, cell walls, insect scales, and diatom tests. Here, we develop patterned liquid crystal elastomer (LCE) particles by recapitulating the biophysical patterning mechanism that forms pollen grain surfaces. In pollen grains, a phase separation of extracellular material into a pattern of condensed and fluid-like phases induces undulations in the underlying elastic cell membrane to form patterns on the cell surface. In this work, LCE particles with variable surface patterns were created through a phase separation of liquid crystal oligomers (LCOs) droplet coupled to homeotropic anchoring at the droplet interface, analogously to the pollen grain wall formation. Specifically, nematically ordered polydisperse LCOs and isotropic organic solvent (dichloromethane) phase-separate at the surface of oil-in-water droplets, while, different LCO chain lengths segregate to different surface curvatures simultaneously. This phase separation, which creates a distortion in the director field, is in competition with homeotropic anchoring induced by sodium dodecyl sulfate (SDS). By tuning the polymer chemistry of the system, we are able to influence this separation process and tune the types of surface patterns in these pollen-like microparticles. Our study reveals that the energetically favorable biological mechanism can be leveraged to offer simple yet versatile approaches to synthesize microparticles for mechanosensing, tissue engineering, drug delivery, energy storage, and displays.

Mechano-Pharmacological Testing of L-Type Ca2+ Channel Modulators via Human Vascular Celldrum Model.

BACKGROUND/AIMS: This study aimed to establish a precise and well-defined working model, assessing pharmaceutical effects on vascular smooth muscle cell monolayer in-vitro. It describes various analysis techniques to determine the most suitable to measure the biomechanical impact of vasoactive agents by using CellDrum technology. METHODS: The so-called CellDrum technology was applied to analyse the biomechanical properties of confluent human aorta muscle cells (haSMC) in monolayer. The cell generated tensions deviations in the range of a few N/m² are evaluated by the CellDrum technology. This study focuses on the dilative and contractive effects of L-type Ca2+ channel agonists and antagonists, respectively. We analyzed the effects of Bay K8644, nifedipine and verapamil. Three different measurement modes were developed and applied to determine the most appropriate analysis technique for the study purpose. These three operation modes are called, particular time mode" (PTM), "long term mode" (LTM) and "real-time mode" (RTM). RESULTS: It was possible to quantify the biomechanical response of haSMCs to the addition of vasoactive agents using CellDrum technology. Due to the supplementation of 100nM Bay K8644, the tension increased approximately 10.6% from initial tension maximum, whereas, the treatment with nifedipine and verapamil caused a significant decrease in cellular tension: 10nM nifedipine decreased the biomechanical stress around 6,5% and 50nM verapamil by 2,8%, compared to the initial tension maximum. Additionally, all tested measurement modes provide similar results while focusing on different analysis parameters. CONCLUSION: The CellDrum technology allows highly sensitive biomechanical stress measurements of cultured haSMC monolayers. The mechanical stress responses evoked by the application of vasoactive calcium channel modulators were quantified functionally (N/m²). All tested operation modes resulted in equal findings, whereas each mode features operation-related data analysis.

Model-based optimized phase-deviation deep brain stimulation for Parkinson 's disease.

High-frequency deep brain stimulation (HF-DBS) of the subthalamic nucleus (STN), globus pallidus interna (GPi) and globus pallidus externa (GPe) are often considered as effective methods for the treatment of Parkinson's disease (PD). However, the stimulation of a single nucleus by HF-DBS can cause specific physical damage, produce side effects and usually consume more electrical energy. Therefore, we use a biophysically-based model of basal ganglia-thalamic circuits to explore more effective stimulation patterns to reduce adverse effects and save energy. In this paper, we computationally investigate the combined DBS of two nuclei with the phase deviation between two stimulation waveforms (CDBS). Three different stimulation combination strategies are proposed, i.e., STN and GPe CDBS (SED), STN and GPi CDBS (SID), as well as GPi and GPe CDBS (GGD). Resultantly, it is found that anti-phase CDBS is more effective in improving parkinsonian dynamical properties, including desynchronization of neurons and the recovery of the thalamus relay ability. Detailed simulation investigation shows that anti-phase SED and GGD are superior to SID. Besides, the energy consumption can be largely reduced by SED and GGD (72.5% and 65.5%), compared to HF-DBS. These results provide new insights into the optimal stimulation parameter and target choice of PD, which may be helpful for the clinical practice.

Branching morphology and biomechanics of ivy (Hedera helix) stem-branch attachments.

PREMISE: Hedera helix is a striking example of a plant with morphological traits and growth habits that vary between juvenile and adult phases. The present study focuses on its branching morphology and variations with age and change in growth habit, based on conspicuous stem-branch attachments previously described in related Araliaceae species. METHODS: We decorticated and morphologically analyzed 300 samples of ramifications from prostrate, climbing and self-supporting axes of H. helix. For bending experiments, 103 specimens with the self-supporting growth habit were collected. RESULTS: Ramifications of H. helix exhibited a so-called "finger-like" branching morphology with abaxial branch lobes and varying degrees of fusion of woody strands. Three categories of woody strand coalescence were defined. Biomechanical experiments in which the branches of stem-branch attachments were bent revealed two main modes of failure, breaking failure in (1) the attachment region and (2) the side branch. CONCLUSIONS: Coalescence of woody strands in H. helix ramifications results from accumulation of secondary xylem with age, influenced by mechanical stimuli causing specific loading situations during different growth habits. Mechanical experiments showed the tendency toward failure in the side branch with increasing fusion of woody strands, affected by the diameter ratio of the side branch to the main axis. Of specific interest is the comparison of H. helix branching with tropical Araliaceae, which do not show the described coalescence of woody strands to this extent. Fracture toughness of self-supporting H. helix axes with merged stem-branch attachment regions are comparable to other self-supporting plant species, despite anatomical and ontogenetic differences.

Experimentally-constrained biophysical models of tonic and burst firing modes in thalamocortical neurons.

Somatosensory thalamocortical (TC) neurons from the ventrobasal (VB) thalamus are central components in the flow of sensory information between the periphery and the cerebral cortex, and participate in the dynamic regulation of thalamocortical states including wakefulness and sleep. This property is reflected at the cellular level by the ability to generate action potentials in two distinct firing modes, called tonic firing and low-threshold bursting. Although the general properties of TC neurons are known, we still lack a detailed characterization of their morphological and electrical properties in the VB thalamus. The aim of this study was to build biophysically-detailed models of VB TC neurons explicitly constrained with experimental data from rats. We recorded the electrical activity of VB neurons (N = 49) and reconstructed morphologies in 3D (N = 50) by applying standardized protocols. After identifying distinct electrical types, we used a multi-objective optimization to fit single neuron electrical models (e-models), which yielded multiple solutions consistent with the experimental data. The models were tested for generalization using electrical stimuli and neuron morphologies not used during fitting. A local sensitivity analysis revealed that the e-models are robust to small parameter changes and that all the parameters were constrained by one or more features. The e-models, when tested in combination with different morphologies, showed that the electrical behavior is substantially preserved when changing dendritic structure and that the e-models were not overfit to a specific morphology. The models and their analysis show that automatic parameter search can be applied to capture complex firing behavior, such as co-existence of tonic firing and low-threshold bursting over a wide range of parameter sets and in combination with different neuron morphologies.

Electrical cortical stimulation for refractory focal epilepsy: A long-term follow-up study.

OBJECTIVE: To report the long-term seizure control and safety of open-loop electrical cortical stimulation in patients with refractory focal epilepsy of diverse etiologies. METHODS: Six patients who received a therapeutic trial of cortical stimulation were included retrospectively. The frequency of seizures was recorded before and after implantation. Surgical procedure- and stimulation-related adverse effects were also recorded. RESULTS: The mean reductions in seizures were 61% at 1 year, 68% at 2 years, and 80% at 3-7 years post-implantation. The median follow-up time was 54 months (range 36-156 months). The etiologies of epilepsy included polymicrogyria in two patients, post-traumatic in one patient, and periventricular heterotopia, post-encephalitis, and familial lateral temporal lobe epilepsy in the remaining three patients. Status epilepticus stopped immediately after stimulation in three patients with focal status epilepticus or epilepsia partialis continua at baseline, with a long-term reduction in seizures of more than 90% and improvements in conscious level. Tissue incompatibility with the connection wire was noted in one patient, which subsided after the system was removed. CONCLUSIONS: Open-loop cortical stimulation of epileptic foci improved seizure control in our patients with refractory focal epilepsy of diverse etiologies. Electrical cortical stimulation stopped epilepsia partialis continua/focal status epilepticus immediately after the intervention and exhibited a sustained effect in reducing seizures. No procedure-related complications were observed. Further case cohort studies are needed to clarify which patients respond to open-loop cortical stimulation.

Simultaneous study of mechanobiology and calcium dynamics on hESC-derived cardiomyocytes clusters.

Calcium ions act like ubiquitous second messengers in a wide amount of cellular processes. In cardiac myocytes, Ca2+ handling regulates the mechanical contraction necessary to the heart pump function. The field of intracellular and intercellular Ca2+ handling, employing in vitro models of cardiomyocytes, has become a cornerstone to understand the role and adaptation of calcium signalling in healthy and diseased hearts. Comprehensive in vitro systems and cell-based biosensors are powerful tools to enrich and speed up cardiac phenotypic and drug response evaluation. We have implemented a combined setup to measure contractility and calcium waves in human embryonic stem cells-derived cardiomyocyte 3D clusters, obtained from embryoid body differentiation. A combination of atomic force microscopy to monitor cardiac contractility, and sensitive fast scientific complementary metal-oxide-semiconductor camera for epifluorescence video recording, provided correlated signals in real time. To speed up the integrated data processing, we tested several post-processing algorithms, to improve the automatic detection of relevant functional parameters. The validation of our proposed method was assessed by caffeine stimulation (10mM) and detection/characterization of the induced cardiac response. We successfully report the first simultaneous recording of cardiac contractility and calcium waves on the described cardiac 3D models. The drug stimulation confirmed the automatic detection capabilities of the used algorithms, measuring expected physiological response, such as elongation of contraction time and Ca2+ cytosolic persistence, increased calcium basal fluorescence, and transient peaks. These results contribute to the implementation of novel, integrated, high-information, and reliable experimental systems for cardiac models and drug evaluation.