Paradoxical effects of ZIM3, a CRISPRi effector, on human induced pluripotent stem-cell-derived cardiomyocyte electrophysiology.

We show that zinc finger imprinted 3 (Zim3), when used as Zim3-KRAB-dCas9 effector in interference CRISPR, without any guide RNAs, paradoxically up-regulates key cardiac ion channel genes in human-induced pluripotent stem-cell-derived cardiomyocytes (iPSC-CMs), responsible for healthy resting membrane potential, repolarization of the action potential, and electrical transmission of signals. These were found to yield expected functional enhancements consistent with a more mature iPSC-CM phenotype, with potentially desirable properties.

Synaptic wiring motifs in posterior parietal cortex support decision-making.

The posterior parietal cortex exhibits choice-selective activity during perceptual decision-making tasks1-10. However, it is not known how this selective activity arises from the underlying synaptic connectivity. Here we combined virtual-reality behaviour, two-photon calcium imaging, high-throughput electron microscopy and circuit modelling to analyse how synaptic connectivity between neurons in the posterior parietal cortex relates to their selective activity. We found that excitatory pyramidal neurons preferentially target inhibitory interneurons with the same selectivity. In turn, inhibitory interneurons preferentially target pyramidal neurons with opposite selectivity, forming an opponent inhibition motif. This motif was present even between neurons with activity peaks in different task epochs. We developed neural-circuit models of the computations performed by these motifs, and found that opponent inhibition between neural populations with opposite selectivity amplifies selective inputs, thereby improving the encoding of trial-type information. The models also predict that opponent inhibition between neurons with activity peaks in different task epochs contributes to creating choice-specific sequential activity. These results provide evidence for how synaptic connectivity in cortical circuits supports a learned decision-making task.

CRISPRi gene modulation and all-optical electrophysiology in post-differentiated human iPSC-cardiomyocytes.

Uncovering gene-phenotype relationships can be enabled by precise gene modulation in human induced pluripotent stem-cell-derived cardiomyocytes (iPSC-CMs) and follow up phenotyping using scalable all-optical electrophysiology platforms. Such efforts towards human functional genomics can be aided by recent CRISPR-derived technologies for reversible gene inhibition or activation (CRISPRi/a). We set out to characterize the performance of CRISPRi in post-differentiated iPSC-CMs, targeting key cardiac ion channel genes, KCNH2, KCNJ2, and GJA1, and providing a multiparametric quantification of the effects on cardiac repolarization, stability of the resting membrane potential and conduction properties using all-optical tools. More potent CRISPRi effectors, e.g., Zim3, and optimized viral delivery led to improved performance on par with the use of CRISPRi iPSC lines. Confirmed mild yet specific phenotype changes when CRISPRi is deployed in non-dividing differentiated heart cells is an important step towards more holistic pre-clinical cardiotoxicity testing and for future therapeutic use in vivo.

OptoDyCE-plate as an affordable high throughput imager for all optical cardiac electrophysiology.

We present a simple low-cost system for comprehensive functional characterization of cardiac function under spontaneous and paced conditions, in standard 96 and 384-well plates. This full-plate actuator/imager, OptoDyCE-plate, uses optogenetic stimulation and optical readouts of voltage and calcium (parallel recordings from up to 100 wells in 384-well plates are demonstrated). The system is validated with syncytia of human induced pluripotent stem cell derived cardiomyocytes, iPSC-CMs, grown as monolayers, or in quasi-3D isotropic and anisotropic constructs using electrospun matrices, in 96 and 384-well format. Genetic modifications, e.g. interference CRISPR (CRISPRi), and nine compounds of acute and chronic action were tested, including five histone deacetylase inhibitors (HDACis). Their effects on voltage and calcium were compared across growth conditions and pacing rates. We also demonstrated optogenetic point pacing via cell spheroids to study conduction in 96-well format, as well as temporal multiplexing to register voltage and calcium simultaneously on a single camera. Opto-DyCE-plate showed excellent performance even in the small samples in 384-well plates. Anisotropic structured constructs may provide some benefits in drug testing, although drug responses were consistent across tested configurations. Differential voltage vs. calcium responses were seen for some drugs, especially for non-traditional modulators of cardiac function, e.g. HDACi, and pacing rate was a powerful modulator of drug response, highlighting the need for comprehensive multiparametric assessment, as offered by OptoDyCE-plate. Increasing throughput and speed and reducing cost of screening can help stratify potential compounds early in the drug development process and accelerate the development of safer drugs.

OptoDyCE-plate as an affordable high throughput imager for all optical cardiac electrophysiology.

We present a simple low-cost system for comprehensive functional characterization of cardiac function under spontaneous and paced conditions, in standard 96 and 384-well plates. This full-plate actuator/imager, OptoDyCE-plate, uses optogenetic stimulation and optical readouts of voltage and calcium from all wells in parallel. The system is validated with syncytia of human induced pluripotent stem cell derived cardiomyocytes, iPSC-CMs, grown as monolayers, or in quasi-3D isotropic and anisotropic constructs using electrospun matrices, in 96 and 394-well format. Genetic modifications, e.g. interference CRISPR (CRISPRi), and nine compounds of acute and chronic action were tested, including five histone deacetylase inhibitors (HDACis). Their effects on voltage and calcium were compared across growth conditions and pacing rates. We also demonstrated deployment of optogenetic cell spheroids for point pacing to study conduction in 96-well format, and the use of temporal multiplexing to register voltage and calcium simultaneously on a single camera in this stand-alone platform. Opto-DyCE-plate showed excellent performance even in the small samples in 384-well plates, in the various configurations. Anisotropic structured constructs may provide some benefits in drug testing, although drug responses were consistent across tested configurations. Differential voltage vs. calcium responses were seen for some drugs, especially for non-traditional modulators of cardiac function, e.g. HDACi, and pacing rate was a powerful modulator of drug response, highlighting the need for comprehensive multiparametric assessment, as offered by OptoDyCE-plate. Increasing throughput and speed and reducing cost of screening can help stratify potential compounds early in the drug development process and accelerate the development of safer drugs.

Chronic Lower Back Pain in Weight Lifters: Epidemiology, Evaluation, and Management.

¼ Chronic lower back pain (LBP) is common in both nonathletes and weight lifters, but the diagnosis and treatment should be approached differently in these 2 populations based on the unique movement patterns causing the pain.¼ Injury rates of weight lifters are far less than those of contact sports, ranging from 1.0 to 4.4 injuries per 1,000 workout hours. However, the lower back was consistently one of the top 2 injury sites for weight lifters, accounting for anywhere from 23% to 59% of all injuries. LBP was most often associated with the squat or deadlift.¼ Guidelines for evaluating general LBP are applicable to weight lifters, including a thorough history and physical examination. However, the differential diagnosis will change based on the patient's lifting history. Of the many etiologies of back pain, weight lifters are most likely to be diagnosed with muscle strain or ligamentous sprain, degenerative disk disease, disk herniation, spondylolysis, spondylolisthesis, or lumbar facet syndrome.¼ Traditional recommended therapies include nonsteroidal anti-inflammatory drugs, physical therapy, and activity modification, which are often insufficient to resolve pain and prevent injury recurrence. Because most athletes will want to continue to lift weights, lifting-specific behavior modifications focused on improved technique and correcting mobility and muscular imbalances are important aspects of management in this patient population.

CRISPRi Gene Modulation and All-Optical Electrophysiology in Post-Differentiated Human iPSC-Cardiomyocytes.

Uncovering gene-phenotype relationships can be enabled by precise gene modulation in human induced pluripotent stem-cell-derived cardiomyocytes (iPSC-CMs) and follow up phenotyping using scalable all-optical electrophysiology platforms. Such efforts towards human functional genomics can be aided by recent CRISPR-derived technologies for reversible gene inhibition or activation (CRISPRi/a). We set out to characterize the performance of CRISPRi in post-differentiated iPSC-CMs, targeting key cardiac ion channel genes, KCNH2, KCNJ2, and GJA1, and providing a multiparametric quantification of the effects on cardiac repolarization, stability of the resting membrane potential and conduction properties using all-optical tools. More potent CRISPRi effectors, e.g. Zim3, and optimized viral delivery led to improved performance on par with the use of CRISPRi iPSC lines. Confirmed mild yet specific phenotype changes when CRISPRi is deployed in non-dividing differentiated heart cells is an important step towards more holistic pre-clinical cardiotoxicity testing and for future therapeutic use in vivo.

Simultaneous Widefield Voltage and Dye-Free Optical Mapping Quantifies Electromechanical Waves in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

Coupled electromechanical waves define a heart's function in health and diseases. Optical mapping of electrical waves using fluorescent labels offers mechanistic insights into cardiac conduction abnormalities. Dye-free/label-free mapping of mechanical waves presents an attractive non-invasive alternative. In this study, we developed a simultaneous widefield voltage and interferometric dye-free optical imaging methodology that was used as follows: (1) to validate dye-free optical mapping for quantification of cardiac wave properties in human iPSC-cardiomyocytes (CMs); (2) to demonstrate low-cost optical mapping of electromechanical waves in hiPSC-CMs using recent near-infrared (NIR) voltage sensors and orders of magnitude cheaper miniature industrial CMOS cameras; (3) to uncover previously underexplored frequency- and space-varying parameters of cardiac electromechanical waves in hiPSC-CMs. We find similarity in the frequency-dependent responses of electrical (NIR fluorescence-imaged) and mechanical (dye-free-imaged) waves, with the latter being more sensitive to faster rates and showing steeper restitution and earlier appearance of wavefront tortuosity. During regular pacing, the dye-free-imaged conduction velocity and electrical wave velocity are correlated; both modalities are sensitive to pharmacological uncoupling and dependent on gap-junctional protein (connexins) determinants of wave propagation. We uncover the strong frequency dependence of the electromechanical delay (EMD) locally and globally in hiPSC-CMs on a rigid substrate. The presented framework and results offer new means to track the functional responses of hiPSC-CMs inexpensively and non-invasively for counteracting heart disease and aiding cardiotoxicity testing and drug development.

Gene Modulation with CRISPR-based Tools in Human iPSC-Cardiomyocytes.

Precise control of gene expression (knock-out, knock-in, knockdown or overexpression) is at the heart of functional genomics - an approach to dissect the contribution of a gene/protein to the system's function. The development of a human in vitro system that can be patient-specific, induced pluripotent stem cells, iPSC, and the ability to obtain various cell types of interest, have empowered human disease modeling and therapeutic development. Scalable tools have been deployed for gene modulation in these cells and derivatives, including pharmacological means, DNA-based RNA interference and standard RNA interference (shRNA/siRNA). The CRISPR/Cas9 gene editing system, borrowed from bacteria and adopted for use in mammalian cells a decade ago, offers cell-specific genetic targeting and versatility. Outside genome editing, more subtle, time-resolved gene modulation is possible by using a catalytically "dead" Cas9 enzyme linked to an effector of gene transcription in combination with a guide RNA. The CRISPRi / CRISPRa (interference/activation) system evolved over the last decade as a scalable technology for performing functional genomics with libraries of gRNAs. Here, we review key developments of these approaches and their deployment in cardiovascular research. We discuss specific use with iPSC-cardiomyocytes and the challenges in further translation of these techniques.

Portable low-cost macroscopic mapping system for all-optical cardiac electrophysiology.

Significance: All-optical cardiac electrophysiology enables the visualization and control of key parameters relevant to the detection of cardiac arrhythmias. Mapping such responses in human induced pluripotent stem-cell-derived cardiomyocytes (hiPSC-CMs) is of great interest for cardiotoxicity and personalized medicine applications. Aim: We introduce and validate a very low-cost compact mapping system for macroscopic all-optical electrophysiology in layers of hiPSC-CMs. Approach: The system uses oblique transillumination, low-cost cameras, light-emitting diodes, and off-the-shelf components (total < $ 15 , 000 ) to capture voltage, calcium, and mechanical waves under electrical or optical stimulation. Results: Our results corroborate the equivalency of electrical and optogenetic stimulation of hiPSC-CMs, and V m - [ Ca 2 + ] i similarity in conduction under pacing. Green-excitable optical sensors are combinable with blue optogenetic actuators (chanelrhodopsin2) only under very low green light ( < 0.05 mW / mm 2 ). Measurements in warmer culture medium yield larger spread of action potential duration and higher conduction velocities compared to Tyrode's solution at room temperature. Conclusions: As multiple optical sensors and actuators are combined, our results can help handle the "spectral congestion" and avoid parameter distortion. We illustrate the utility of the system for uncovering the action of cellular uncoupling agents and show extensibility to an epi-illumination mode for future imaging of thicker native or engineered tissues.

Protein and mRNA Quantification in Small Samples of Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes in 96-Well Microplates.

We describe a method for protein quantification and for mRNA quantification in small sample quantities of human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Demonstrated here is how the capillary-based protein detection system Wes™ by ProteinSimple and the Power SYBR™ Green Cells-to-CT™ Kit by Invitrogen can be applied to individual samples in a 96-well microplate format and thus made compatible with high-throughput (HT) cardiomyocyte assays. As an example of the usage, we illustrate that Cx43 protein and GJA1 mRNA levels in hiPSC-CMs are enhanced when the optogenetic actuator, channelrodopsin-2 (ChR2), is genetically expressed in them. Instructions are presented for cell culture and lysate preparations from hiPSC-CMs, along with optimized parameter settings and experimental protocol steps. Strategies to optimize primary antibody concentrations as well as ways for signal normalization are discussed, i.e., antibody multiplexing and total protein assay. The sensitivity of both the Wes and Cells-to-CT kit enables protein and mRNA quantification in a HT format, which is important when dealing with precious small samples. In addition to being able to handle small cardiomyocyte samples, these streamlined and semi-automated processes enable quick mechanistic analysis.

Altered propionate metabolism contributes to tumour progression and aggressiveness.

The alteration of metabolic pathways is a critical strategy for cancer cells to attain the traits necessary for metastasis in disease progression. Here, we find that dysregulation of propionate metabolism produces a pro-aggressive signature in breast and lung cancer cells, increasing their metastatic potential. This occurs through the downregulation of methylmalonyl coenzyme A epimerase (MCEE), mediated by an extracellular signal-regulated kinase 2-driven transcription factor Sp1/early growth response protein 1 transcriptional switch driven by metastatic signalling at its promoter level. The loss of MCEE results in reduced propionate-driven anaplerotic flux and intracellular and intratumoral accumulation of methylmalonic acid, a by-product of propionate metabolism that promotes cancer cell invasiveness. Altogether, we present a previously uncharacterized dysregulation of propionate metabolism as an important contributor to cancer and a valuable potential target in the therapeutic treatment of metastatic carcinomas.

Integration of Engineered "Spark-Cell" Spheroids for Optical Pacing of Cardiac Tissue.

Optogenetic methods for pacing of cardiac tissue can be realized by direct genetic modification of the cardiomyocytes to express light-sensitive actuators, such as channelrhodopsin-2, ChR2, or by introduction of light-sensitized non-myocytes that couple to the cardiac cells and yield responsiveness to optical pacing. In this study, we engineer three-dimensional "spark cells" spheroids, composed of ChR2-expressing human embryonic kidney cells (from 100 to 100,000 cells per spheroid), and characterize their morphology as function of cell density and time. These "spark-cell" spheroids are then deployed to demonstrate site-specific optical pacing of human stem-cell-derived cardiomyocytes (hiPSC-CMs) in 96-well format using non-localized light application and all-optical electrophysiology with voltage and calcium small-molecule dyes or genetically encoded sensors. We show that the spheroids can be handled using liquid pipetting and can confer optical responsiveness of cardiac tissue earlier than direct viral or liposomal genetic modification of the cardiomyocytes, with 24% providing reliable stimulation of the iPSC-CMs within 6 h and >80% within 24 h. Moreover, our data show that the spheroids can be frozen in liquid nitrogen for long-term storage and transportation, after which they can be deployed as a reagent on site for optical cardiac pacing. In all cases, optical stimulation was achieved at relatively low light levels (<0.15 mW/mm2) when 5 ms or longer pulses were used. Our results demonstrate a scalable, cost-effective method with a cryopreservable reagent to achieve contactless optical stimulation of cardiac cell constructs without genetically modifying the myocytes, that can be integrated in a robotics-amenable workflow for high-throughput drug testing.

Musculoskeletal Considerations for Exercise and Sport: Before, During, and After Pregnancy.

There is little written in the orthopaedic literature regarding common musculoskeletal problems that women encounter in relation to pregnancy and their clinical and surgical management. Exercise and other physical activity are generally recommended for most women before, during, and after pregnancy. Unfortunately, a variety of musculoskeletal issues may keep women from starting, continuing, or resuming a healthy exercise regimen throughout a notable portion of their reproductive years. Untreated and undertreated orthopaedic conditions in female athletes may therefore have further unintended negative effects on maternal and fetal health. This article reviews the existing literature on musculoskeletal health considerations before, during, and after pregnancy to provide practical information to orthopaedic surgeons who treat women of all ages and athletic abilities.

IDseq-An open source cloud-based pipeline and analysis service for metagenomic pathogen detection and monitoring.

BACKGROUND: Metagenomic next-generation sequencing (mNGS) has enabled the rapid, unbiased detection and identification of microbes without pathogen-specific reagents, culturing, or a priori knowledge of the microbial landscape. mNGS data analysis requires a series of computationally intensive processing steps to accurately determine the microbial composition of a sample. Existing mNGS data analysis tools typically require bioinformatics expertise and access to local server-class hardware resources. For many research laboratories, this presents an obstacle, especially in resource-limited environments. FINDINGS: We present IDseq, an open source cloud-based metagenomics pipeline and service for global pathogen detection and monitoring (https://idseq.net). The IDseq Portal accepts raw mNGS data, performs host and quality filtration steps, then executes an assembly-based alignment pipeline, which results in the assignment of reads and contigs to taxonomic categories. The taxonomic relative abundances are reported and visualized in an easy-to-use web application to facilitate data interpretation and hypothesis generation. Furthermore, IDseq supports environmental background model generation and automatic internal spike-in control recognition, providing statistics that are critical for data interpretation. IDseq was designed with the specific intent of detecting novel pathogens. Here, we benchmark novel virus detection capability using both synthetically evolved viral sequences and real-world samples, including IDseq analysis of a nasopharyngeal swab sample acquired and processed locally in Cambodia from a tourist from Wuhan, China, infected with the recently emergent SARS-CoV-2. CONCLUSION: The IDseq Portal reduces the barrier to entry for mNGS data analysis and enables bench scientists, clinicians, and bioinformaticians to gain insight from mNGS datasets for both known and novel pathogens.

Syncytium cell growth increases Kir2.1 contribution in human iPSC-cardiomyocytes.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) enable cardiotoxicity testing and personalized medicine. However, their maturity is of concern, including relatively depolarized resting membrane potential and more spontaneous activity compared with adult cardiomyocytes, implicating low or lacking inward rectifier potassium current (Ik1). Here, protein quantification confirms Kir2.1 expression in hiPSC-CM syncytia, albeit several times lower than in adult heart tissue. We find that hiPSC-CM culture density influences Kir2.1 expression at the mRNA level (potassium inwardly rectifying channel subfamily J member 2) and at the protein level and its associated electrophysiology phenotype. Namely, all-optical cardiac electrophysiology and pharmacological treatments reveal reduction of spontaneous and irregular activity and increase in action potential upstroke in denser cultures. Blocking Ik1-like currents with BaCl2 increased spontaneous frequency and blunted action potential upstrokes during pacing in a dose-dependent manner only in the highest-density cultures, in line with Ik1's role in regulating the resting membrane potential. Our results emphasize the importance of syncytial growth of hiPSC-CMs for more physiologically relevant phenotype and the power of all-optical electrophysiology to study cardiomyocytes in their multicellular setting.NEW & NOTEWORTHY We identify cell culture density and cell-cell contact as an important factor in determining the expression of a key ion channel at the transcriptional and the protein levels, KCNJ2/Kir2.1, and its contribution to the electrophysiology of human induced pluripotent stem cell-derived cardiomyocytes. Our results indicate that studies on isolated cells, out of tissue context, may underestimate the cellular ion channel properties being characterized.

Dense neuronal reconstruction through X-ray holographic nano-tomography.

Imaging neuronal networks provides a foundation for understanding the nervous system, but resolving dense nanometer-scale structures over large volumes remains challenging for light microscopy (LM) and electron microscopy (EM). Here we show that X-ray holographic nano-tomography (XNH) can image millimeter-scale volumes with sub-100-nm resolution, enabling reconstruction of dense wiring in Drosophila melanogaster and mouse nervous tissue. We performed correlative XNH and EM to reconstruct hundreds of cortical pyramidal cells and show that more superficial cells receive stronger synaptic inhibition on their apical dendrites. By combining multiple XNH scans, we imaged an adult Drosophila leg with sufficient resolution to comprehensively catalog mechanosensory neurons and trace individual motor axons from muscles to the central nervous system. To accelerate neuronal reconstructions, we trained a convolutional neural network to automatically segment neurons from XNH volumes. Thus, XNH bridges a key gap between LM and EM, providing a new avenue for neural circuit discovery.

Age-induced accumulation of methylmalonic acid promotes tumour progression.

The risk of cancer and associated mortality increases substantially in humans from the age of 65 years onwards1-6. Nonetheless, our understanding of the complex relationship between age and cancer is still in its infancy2,3,7,8. For decades, this link has largely been attributed to increased exposure time to mutagens in older individuals. However, this view does not account for the established role of diet, exercise and small molecules that target the pace of metabolic ageing9-12. Here we show that metabolic alterations that occur with age can produce a systemic environment that favours the progression and aggressiveness of tumours. Specifically, we show that methylmalonic acid (MMA), a by-product of propionate metabolism, is upregulated in the serum of older people and functions as a mediator of tumour progression. We traced this to the ability of MMA to induce SOX4 expression and consequently to elicit transcriptional reprogramming that can endow cancer cells with aggressive properties. Thus, the accumulation of MMA represents a link between ageing and cancer progression, suggesting that MMA is a promising therapeutic target for advanced carcinomas.

Dynamic Incorporation of Histone H3 Variants into Chromatin Is Essential for Acquisition of Aggressive Traits and Metastatic Colonization.

Metastasis is the leading cause of cancer mortality. Chromatin remodeling provides the foundation for the cellular reprogramming necessary to drive metastasis. However, little is known about the nature of this remodeling and its regulation. Here, we show that metastasis-inducing pathways regulate histone chaperones to reduce canonical histone incorporation into chromatin, triggering deposition of H3.3 variant at the promoters of poor-prognosis genes and metastasis-inducing transcription factors. This specific incorporation of H3.3 into chromatin is both necessary and sufficient for the induction of aggressive traits that allow for metastasis formation. Together, our data clearly show incorporation of histone variant H3.3 into chromatin as a major regulator of cell fate during tumorigenesis, and histone chaperones as valuable therapeutic targets for invasive carcinomas.

Adeno-Associated Virus Mediated Gene Delivery: Implications for Scalable in vitro and in vivo Cardiac Optogenetic Models.

Adeno-associated viruses (AAVs) provide advantages in long-term, cardiac-specific gene expression. However, AAV serotype specificity data is lacking in experimental models relevant to cardiac electrophysiology and cardiac optogenetics. We aimed to identify the optimal AAV serotype (1, 6, or 9) in pursuit of scalable rodent and human models using genetic modifications in cardiac electrophysiology and optogenetics, in particular, as well as to elucidate the mechanism of virus uptake. In vitro syncytia of primary neonatal rat ventricular cardiomyocytes (NRVMs) and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were infected with AAVs 1, 6, and 9 containing the transgene for eGFP or channelrhodopsin-2 (ChR2) fused to mCherry. In vivo adult rats were intravenously injected with AAV1 and 9 containing ChR2-mCherry. Transgene expression profiles of rat and human cells in vitro revealed that AAV1 and 6 significantly outperformed AAV9. In contrast, systemic delivery of AAV9 in adult rat hearts yielded significantly higher levels of ChR2-mCherry expression and optogenetic responsiveness. We tracked the mechanism of virus uptake to purported receptor-mediators for AAV1/6 (cell surface sialic acid) and AAV9 (37/67 kDa laminin receptor, LamR). In vitro desialylation of NRVMs and hiPSC-CMs with neuraminidase (NM) significantly decreased AAV1,6-mediated gene expression, but interestingly, desialylation of hiPSC-CMs increased AAV9-mediated expression. In fact, only very high viral doses of AAV9-ChR2-mCherry, combined with NM treatment, yielded consistent optogenetic responsiveness in hiPSC-CMs. Differences between the in vitro and in vivo performance of AAV9 could be correlated to robust LamR expression in the intact heart (neonatal rat hearts as well as adult human and rat hearts), but no expression in vitro in cultured cells (primary rat cells and hiPS-CMs). The dynamic nature of LamR expression and its dependence on environmental factors was further corroborated in intact adult human ventricular tissue. The combined transgene expression and cell surface receptor data may explain the preferential efficiency of AAV1/6 in vitro and AAV9 in vivo for cardiac delivery and mechanistic knowledge of their action can help guide cardiac optogenetic efforts. More broadly, these findings are relevant to future efforts in gene therapy for cardiac electrophysiology abnormalities in vivo as well as for genetic modifications of cardiomyocytes by viral means in vitro applications such as disease modeling or high-throughput drug testing.