Diversity of cells and signals in the cardiovascular system.

This white paper is the outcome of the seventh UC Davis Cardiovascular Research Symposium on Systems Approach to Understanding Cardiovascular Disease and Arrhythmia. This biannual meeting aims to bring together leading experts in subfields of cardiovascular biomedicine to focus on topics of importance to the field. The theme of the 2022 Symposium was 'Cell Diversity in the Cardiovascular System, cell-autonomous and cell-cell signalling'. Experts in the field contributed their experimental and mathematical modelling perspectives and discussed emerging questions, controversies, and challenges in examining cell and signal diversity, co-ordination and interrelationships involved in cardiovascular function. This paper originates from the topics of formal presentations and informal discussions from the Symposium, which aimed to develop a holistic view of how the multiple cell types in the cardiovascular system integrate to influence cardiovascular function, disease progression and therapeutic strategies. The first section describes the major cell types (e.g. cardiomyocytes, vascular smooth muscle and endothelial cells, fibroblasts, neurons, immune cells, etc.) and the signals involved in cardiovascular function. The second section emphasizes the complexity at the subcellular, cellular and system levels in the context of cardiovascular development, ageing and disease. Finally, the third section surveys the technological innovations that allow the interrogation of this diversity and advancing our understanding of the integrated cardiovascular function and dysfunction.

Guidelines for assessment of cardiac electrophysiology and arrhythmias in small animals.

Cardiac arrhythmias are a major cause of morbidity and mortality worldwide. Although recent advances in cell-based models, including human-induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM), are contributing to our understanding of electrophysiology and arrhythmia mechanisms, preclinical animal studies of cardiovascular disease remain a mainstay. Over the past several decades, animal models of cardiovascular disease have advanced our understanding of pathological remodeling, arrhythmia mechanisms, and drug effects and have led to major improvements in pacing and defibrillation therapies. There exist a variety of methodological approaches for the assessment of cardiac electrophysiology and a plethora of parameters may be assessed with each approach. This guidelines article will provide an overview of the strengths and limitations of several common techniques used to assess electrophysiology and arrhythmia mechanisms at the whole animal, whole heart, and tissue level with a focus on small animal models. We also define key electrophysiological parameters that should be assessed, along with their physiological underpinnings, and the best methods with which to assess these parameters.

Functional response of the isolated, perfused normoxic heart to pyruvate dehydrogenase activation by dichloroacetate and pyruvate.

Dichloroacetate (DCA) and pyruvate activate pyruvate dehydrogenase (PDH), a key enzyme that modulates glucose oxidation and mitochondrial NADH production. Both compounds improve recovery after ischemia in isolated hearts. However, the action of DCA and pyruvate in normoxic myocardium is incompletely understood. We measured the effect of DCA and pyruvate on contraction, mitochondrial redox state, and intracellular calcium cycling in isolated rat hearts during normoxic perfusion. Normalized epicardial NADH fluorescence (nNADH) and left ventricular developed pressure (LVDP) were measured before and after administering DCA (5 mM) or pyruvate (5 mM). Optical mapping of Rhod-2AM was used to measure cytosolic calcium kinetics. DCA maximally activated PDH, increasing the ratio of active to total PDH from 0.48 ± 0.03 to 1.03 ± 0.03. Pyruvate sub-maximally activated PDH to a ratio of 0.75 ± 0.02. DCA and pyruvate increased LVDP. When glucose was the only exogenous fuel, pyruvate increased nNADH by 21.4 ± 2.9 % while DCA reduced nNADH by 21.4 ± 6.1 % and elevated the incidence of premature ventricular contractions (PVCs). When lactate, pyruvate, and glucose were provided together as exogenous fuels, nNADH increased with DCA, indicating that PDH activation with glucose as the only exogenous fuel depletes PDH substrate. Calcium transient time-to-peak was shortened by DCA and pyruvate and SR calcium re-uptake was 30 % longer. DCA and pyruvate increased SR calcium load in myocyte monolayers. Overall, during normoxia when glucose is the only exogenous fuel, DCA elevates SR calcium, increases LVDP and contractility, and diminishes mitochondrial NADH. Administering DCA with plasma levels of lactate and pyruvate mitigates the drop in mitochondrial NADH and prevents PVCs.

Properties of blebbistatin for cardiac optical mapping and other imaging applications.

Blebbistatin is a recently discovered myosin II inhibitor. It is rapidly becoming a compound of choice to reduce motion artifacts during cardiac optical mapping, as well as to study cell motility and cell invasion. Although blebbistatin has a number of advantages over other electromechanical uncouplers, many of its properties have yet to be addressed. Here we describe several methodological issues associated with the use of blebbistatin, including its spectral properties, reversibility, and its effect on tissue metabolic state. We show that if precautions are not taken, perfusion with blebbistatin may result in blebbistatin precipitate that accumulates in the vasculature. Although such precipitate is fluorescent, it is not detectable within wavelength bands that are typically used for transmembrane voltage fluorescence imaging (i.e., emission wavelengths >600 nm). Therefore, blockage of the microcirculation by blebbistatin may cause data misinterpretation in studies that use voltage-sensitive dyes. Blebbistatin may also impact imaging of green fluorophores due to the spectral shift it causes in endogenous tissue fluorescence. 3D excitation-emission matrices of blebbistatin in precipitate form and in various solutions (DMSO, water, and 1 % aqueous albumin) revealed significant changes in the fluorescence of this molecule in different environments. Finally, we examined the reversibility of blebbistatin's uncoupling effect on cardiac contraction. Our findings provide important new information about the properties of this myosin II inhibitor, which will aid in the proper design and interpretation of studies that use this compound.

KairoSight: Open-Source Software for the Analysis of Cardiac Optical Data Collected From Multiple Species.

Cardiac optical mapping, also known as optocardiography, employs parameter-sensitive fluorescence dye(s) to image cardiac tissue and resolve the electrical and calcium oscillations that underly cardiac function. This technique is increasingly being used in conjunction with, or even as a replacement for, traditional electrocardiography. Over the last several decades, optical mapping has matured into a "gold standard" for cardiac research applications, yet the analysis of optical signals can be challenging. Despite the refinement of software tools and algorithms, significant programming expertise is often required to analyze large optical data sets, and data analysis can be laborious and time-consuming. To address this challenge, we developed an accessible, open-source software script that is untethered from any subscription-based programming language. The described software, written in python, is aptly named "KairoSight" in reference to the Greek word for "opportune time" (Kairos) and the ability to "see" voltage and calcium signals acquired from cardiac tissue. To demonstrate analysis features and highlight species differences, we employed experimental datasets collected from mammalian hearts (Langendorff-perfused rat, guinea pig, and swine) dyed with RH237 (transmembrane voltage) and Rhod-2, AM (intracellular calcium), as well as human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) dyed with FluoVolt (membrane potential), and Fluo-4, AM (calcium indicator). We also demonstrate cardiac responsiveness to ryanodine (ryanodine receptor modulator) and isoproterenol (beta-adrenergic agonist) and highlight regional differences after an ablation injury. KairoSight can be employed by both basic and clinical scientists to analyze complex cardiac optical mapping datasets without requiring dedicated computer science expertise or proprietary software.

Downregulation of beta-microglobulin to diminish T-lymphocyte lysis of non-syngeneic cell sources of engineered heart tissue constructs.

The presence of non-autologous major histocompatibility complex class I (MHC-I) molecules on the surface of the grafted cells is one of the main reasons for their rejection in non-syngeneic hosts. We present a straightforward strategy to decrease the presence of MHC-I by shRNA inhibition of beta-2-microglobulin (B2M), a conservative light chain of MHC-I, on the surface of two main cell types that are used to engineer heart tissue constructs. Engineered heart tissue constructs can be generated by combining mouse WT19 fibroblasts and mouse embryonic stem cell-derived cardiac myocytes (mESC-CM). WT19 fibroblasts were stably transduced with an anti-B2M shRNA, which yielded a cell line with dramatically reduced B2M expression levels (16 ± 11% of mock treated control cell line). Interferon gamma treatment increased the levels of B2M expression by >3-fold in both control and transduced fibroblasts; yet, B2M expression levels still remained very low in the transduced cells. When compared with their unmodified counterparts, transduced fibroblasts caused 5.7-fold lesser activation of cognate T-cells. B2M depletion in mESC-CM was achieved by 72 h transduction with anti-B2M shRNA lentiviral particles. Transduced mESC-CM exhibited regular beating and expressed classical cardiac markers. When compared with their unmodified counterparts, transduced mESC-CM caused 2.5-fold lesser activation of cognate T-cells. In vivo assessment of B2M downregulation was performed by analyzing the preferential survival of B2M-downregulated cells in the intraperitoneal cavity of allogeneic mice. Both B2M-downregulated fibroblasts and B2M-downregulated myocytes survived significantly better when compared to their unmodified counterparts (2.01 ± 0.4 and 5.07 ± 1.6 fold increase in survival, respectively). In contrast, when modified WT19 fibroblasts were injected into the intraperitoneal cavity of syngeneic C57Bl/6 mice, no significant survival advantage was observed. Notably, the preferential survival of B2M-downregulated cells persisted in allogeneic hosts with normal levels of natural killer cells, although the effect was lesser in magnitude. Use of shRNA against beta-2-microglobulin offers a simple and effective approach to minimize immunogenicity of the main cellular components of cardiac tissue constructs in non-syngeneic recipients.

HLA Class I Depleted hESC as a Source of Hypoimmunogenic Cells for Tissue Engineering Applications.

BACKGROUND: Rapidly improving protocols for the derivation of autologous cells from stem cell sources is a welcome development. However, there are many circumstances when off-the-shelf universally immunocompatible cells may be needed. Embryonic stem cells (ESCs) provide a unique opportunity to modify the original source of differentiated cells to minimize their rejection by nonautologous hosts. HYPOTHESIS: Immune rejection of nonautologous human embryonic stem cell (hESC) derivatives can be reduced by downregulating human leukocyte antigen (HLA) class I molecules, without affecting the ability of these cells to differentiate into specific lineages. METHODS AND RESULTS: Beta-2-microglobulin (B2M) expression was decreased by lentiviral transduction using human anti-HLA class I light-chain B2M short hairpin RNA. mRNA levels of B2M were decreased by 90% in a RUES2-modified hESC line, as determined by quantitative real time-polymerase chain reaction analysis. The transduced cells were selected under puromycin pressure and maintained in an undifferentiated state. The latter was confirmed by Oct4 and Nanog expression, and by the formation of characteristic round-shaped colonies. B2M downregulation led to diminished HLA-I expression on the cell surface, as determined by flow cytometry. When used as target cells in a mixed lymphocyte reaction assay, transduced hESCs and their differentiated derivatives did not stimulate allogeneic T-cell proliferation. Using a cardiac differentiation protocol, transduced hESCs formed a confluent layer of cardiac myocytes and maintained a low level of B2M expression. Transduced hESCs were also successfully differentiated into a hepatic lineage, validating their capacity to differentiate into multiple lineages. CONCLUSIONS: HLA-I depletion does not preclude hESC differentiation into cardiac or hepatic lineages. This methodology can be used to engineer tissue from nonautologous hESC sources with improved immunocompatibility.