C ardiomyocyte excitation-contraction coupling (ECC) is the crucial process that links calcium (C... more C ardiomyocyte excitation-contraction coupling (ECC) is the crucial process that links calcium (Ca 2+ ) trafficking to active force generation and subsequent relaxation of the cardiomyocyte. After suprathreshold myocyte excitation, Ca 2+ rapidly influxes into the myocyte cytosol through depolarization-activated inward Ca 2+ channels. This Ca 2+ influx induces Ca 2+ release from the sarcoplasmic reticulum; together, these mechanisms raise free cytosolic Ca 2+ concentration, increasing the probability of Ca 2+ -troponin C binding and thus activating the contractile machinery. In normal physiology, a fine-tuned percentage of cytosolic Ca 2+ is then pumped back into the sarcoplasmic reticulum reservoir for future calcium-induced calcium release. 1 A simplified overview of this highly orchestrated ECC process is shown in Figure . Sympathetic β-adrenergic signaling is a well-established rapid inotropic 2 and lusitropic 3 modulator of ECC. 1 Briefly, βadrenergic receptor stimulation of Gs type G-protein-coupled receptors activates adenylyl cyclase to form cAMP. cAMP subsequently activates protein kinases that phosphorylate various proteins essential to electrophysiology, calcium handling, and, thus, ECC, including the L-type calcium channel, 4,5 phospholamban, 6 ryanodine receptor, 7 troponin I, 8 and myosin-binding protein C 9 (Figure ). A comprehensive review of sympathetic activation effects on ECC is available elsewhere. Cardiomyocyte ECC and associated β-adrenergic signaling can be further modulated by factors secreted by both distant and nearby cells. For example, chemokine CXCL12, also known as stromal cell-derived factor, can activate its receptor CXCR4 to attenuate β-adrenergic-mediated increases in adult
Atomic force microscopy (AFM) is used to study mechanical properties of biological materials at s... more Atomic force microscopy (AFM) is used to study mechanical properties of biological materials at submicron length scales. However, such samples are often structurally heterogeneous even at the local level, with different regions having distinct mechanical properties. Physical or chemical disruption can isolate individual structural elements but may alter the properties being measured. Therefore, to determine the micromechanical properties of intact heterogeneous multilayered samples indented by AFM, we propose the Hybrid Eshelby Decomposition (HED) analysis, which combines a modified homogenization theory and finite element modeling to extract layer-specific elastic moduli of composite structures from single indentations, utilizing knowledge of the component distribution to achieve solution uniqueness. Using finite element model-simulated indentation of layered samples with micron-scale thickness dimensions, biologically relevant elastic properties for incompressible soft tissues, and layer-specific heterogeneity of an order of magnitude or less, HED analysis recovered the prescribed modulus values typically within 10% error. Experimental validation using bilayer spin-coated polydimethylsiloxane samples also yielded selfconsistent layer-specific modulus values whether arranged as stiff layer on soft substrate or soft layer on stiff substrate. We further examined a biophysical application by characterizing layer-specific microelastic properties of full-thickness mouse aortic wall tissue, demonstrating that the HED-extracted modulus of the tunica media was more than fivefold stiffer than the intima and not significantly different from direct indentation of exposed media tissue. Our results show that the elastic properties of surface and subsurface layers of microscale synthetic and biological samples can be simultaneously extracted from the composite material response to AFM indentation. HED analysis offers a robust approach to studying regional micromechanics of heterogeneous multilayered samples without destructively separating individual components before testing.
Marfan syndrome (MFS) is an autosomal dominant disease of the connective tissue due to mutations ... more Marfan syndrome (MFS) is an autosomal dominant disease of the connective tissue due to mutations in the fibrillin-1 gene (FBN1). This study aimed at characterizing microelastic properties of the ascending aorta wall and lung parenchyma tissues from wild type (WT) and agematched Fbn1 hypomorphic mice (Fbn1 mgR/mgR mice) to identify tissue-specific biomechanical effects of aging and disease in MFS. Atomic force microscopy (AFM) was used to indent lung parenchyma and aortic wall tissues, using Hybrid Eshelby Decomposition analysis to extract layerspecific properties of the intima and media. The intima stiffened with age and was not different between WT and Fbn1 mgR/mgR tissues, whereas the media layer of mutant aortas showed progressive structural and mechanical degradation with a modulus that was 50% softer than WT by 3.5 months of age. Similarly, mutant mice displayed progressive structural and mechanical deterioration of lung tissue, which was over 85% softer than WT by 3.5 months of age. Chronic treatment with the angiotensin type I receptor antagonist, losartan, attenuated the aorta and lung tissue degradation, resulting in structural and mechanical properties not significantly different from age-matched WT controls. By revealing micromechanical softening of elastin-rich aorta and lung tissues with disease progression in fibrillin-1 deficient mice, our findings support the use of losartan as a prophylactic treatment that may abrogate the life-threatening symptoms of MFS.
Human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) have the ability of... more Human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) have the ability of differentiating into functional cardiomyocytes (CMs) for cell replacement therapy, tissue engineering, drug discovery and toxicity screening. From a scale-free, co-expression network analysis of transcriptomic data that distinguished gene expression profiles of undifferentiated hESC, hESC-, fetaland adult-ventricular(V) CM, two candidate chromatin remodeling proteins, SMYD1 and SMARCD1 were found to be differentially expressed. Using lentiviral transduction, SMYD1 and SMARCD1 were over-expressed and suppressed, respectively, in single hESC-VCMs as well as the 3D constructs Cardiac Micro tissues (CMT) and tissue strips (CTS) to mirror the endogenous patterns, followed by dissection of their roles in controlling cardiac gene expression, contractility, Ca 2+ -handling, electrophysiological functions and in vitro maturation. Interestingly, compared to independent single transductions, simultaneous SMYD1 overexpression and SMARCD1 suppression in hESC-VCMs synergistically interacted to increase the contractile forces of CMts and Ctss with up-regulated transcripts for cardiac contractile, Ca 2+ -handing, and ion channel proteins. Certain effects that were not detected at the single-cell level could be unleashed under 3D environments. The two chromatin remodelers SMYD1 and SMARCD1 play distinct roles in cardiac development and maturation, consistent with the notion that epigenetic priming requires triggering signals such as 3D environmental cues for pro-maturation effects. Human adult cardiomyocytes (CMs) are terminally differentiated and non-regenerative. Using in vitro differentiation methods, ventricular (V) CMs derived from human embryonic stem cells (hESCs) or induced pluripotent stem cells (iPSCs) are a valuable source of genuine heart VCMs for cell-based transplantation therapies and as
An area of active research in the field of cardiac gene therapy aims to achieve high transfection... more An area of active research in the field of cardiac gene therapy aims to achieve high transfection efficiency without eliciting immune or inflammatory reactions. Nanomedicine offers an attractive alternative to traditional viral delivery vehicles because nanoparticle technology can enable safer and more controlled delivery of therapeutic agents. Here we describe the use of lipidoid nanoparticles for delivery of modified mRNA (modRNA) to the myocardium in vivo, with a focus on rodent models that represent a first step toward preclinical studies. Three major procedures are discussed in this chapter: 1) preparation of lipid modRNA nanoparticles, 2) intramyocardial delivery of the lipid modRNA nanoparticles by direct injection with an open chest technique in rats, and 3) intracoronary delivery of the lipid modRNA nanoparticles with open chest and temporary aortic cross clamping in rats.
Background: Myocardial delivery of non-excitable cells-namely human mesenchymal stem cells (hMSCs... more Background: Myocardial delivery of non-excitable cells-namely human mesenchymal stem cells (hMSCs) and c-kit + cardiac interstitial cells (hCICs)remains a promising approach for treating the failing heart. Recent empirical studies attempt to improve such therapies by genetically engineering cells to express specific ion channels, or by creating hybrid cells with combined channel expression. This study uses a computational modeling approach to test the hypothesis that custom hypothetical cells can be rationally designed to restore a healthy phenotype when coupled to human heart failure (HF) cardiomyocytes. Methods: Candidate custom cells were simulated with a combination of ion channels from non-excitable cells and healthy human cardiomyocytes (hCMs). Using a genetic algorithm-based optimization approach, candidate cells were accepted if a root mean square error (RMSE) of less than 50% relative to healthy hCM was achieved for both action potential and calcium transient waveforms for the cell-treated HF cardiomyocyte, normalized to the untreated HF cardiomyocyte. Results: Custom cells expressing only non-excitable ion channels were inadequate to restore a healthy cardiac phenotype when coupled to either fibrotic or nonfibrotic HF cardiomyocytes. In contrast, custom cells also expressing cardiac ion channels led to acceptable restoration of a healthy cardiomyocyte phenotype when coupled to fibrotic, but not non-fibrotic, HF cardiomyocytes. Incorporating the cardiomyocyte inward rectifier K + channel was critical to accomplishing this phenotypic rescue while also improving single-cell action potential metrics associated with arrhythmias, namely resting membrane potential and action potential duration. The computational approach also provided insight into the rescue mechanisms, whereby heterocellular coupling enhanced cardiomyocyte L-type calcium current and promoted calcium-induced calcium release. Finally, as a therapeutically translatable strategy, we simulated delivery of hMSCs and hCICs genetically engineered to express the cardiomyocyte inward rectifier K + channel,
SUMMARYPluripotent stem cell-derived cardiomyocytes (PSC-CMs) provide an unprecedented opportunit... more SUMMARYPluripotent stem cell-derived cardiomyocytes (PSC-CMs) provide an unprecedented opportunity to study human heart development and disease. A major caveat however is that they remain functionally and structurally immature in culture, limiting their potential for disease modeling and regenerative approaches. Here, we address the question of how different metabolic pathways can be modulated in order to induce efficient hPSC-CM maturation. We show that PPAR signaling acts in an isoform-specific manner to balance glycolysis and fatty acid oxidation (FAO). PPARD activation or inhibition results in efficient respective up- or down-regulation of the gene regulatory networks underlying FAO in hPSC-CMs. PPARD induction further increases mitochondrial and peroxisome content, enhances mitochondrial cristae formation and augments FAO flux. Lastly PPARD activation results in enhanced myofibril organization and improved contractility. Transient lactate exposure, commonly used in hPSC-CM puri...
The lack of biomimetic in vitro models of the human heart has posed a critical barrier to progres... more The lack of biomimetic in vitro models of the human heart has posed a critical barrier to progress in the field of modeling cardiac disease. Human engineered cardiac tissues (hECTs)autonomous, beating structures that recapitulate key aspects of native cardiac muscle physiologyoffer an attractive alternative to traditional in vitro models. Here we describe the use of hECTs to advance our understanding and modeling of cardiac diseases in order to test therapeutic interventions, with a focus on contractile dysfunction in the setting of inherited and acquired forms of cardiomyopathies. Four major procedures are discussed in this chapter: (1) preparation of hECTs from human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) on single-tissue and multitissue bioreactors; (2) data acquisition of hECT contractile function on both of these platforms; (3) hECT modeling of hereditary phospholamban-R14 deletion-dilated cardiomyopathy; and (4) cryo-injury and doxorubicin-induced hECT models of acquired cardiomyopathy.
Myocardial delivery of human c-kit+ cardiac interstitial cells (hCICs) and human mesenchymal stem... more Myocardial delivery of human c-kit+ cardiac interstitial cells (hCICs) and human mesenchymal stem cells (hMSCs), an emerging approach for treating the failing heart, has been limited by an incomplete understanding of the effects on host myocardium. This computational study aims to model hCIC and hMSC effects on electrophysiology and calcium cycling of healthy and diseased human cardiomyocytes (hCM), and reveals a possible cardiotherapeutic benefit independent of putative regeneration processes. First, we developed an original hCIC mathematical model with an electrical profile comprised of distinct experimentally identified ion currents. Next, we verified the model by confirming it is representative of published experiments on hCIC whole-cell electrophysiology and on hCIC co-cultures with rodent cardiomyocytes. We then used our model to compare electrophysiological effects of hCICs to other non-excitable cells, as well as clinically relevant hCIC-hMSC combination therapies and fused ...
Journal of Pharmacological and Toxicological Methods, 2021
Since January 2020 Elsevier has created a COVID-19 resource centre with free information in Engli... more Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre -including this research content -immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active.
Reliable and predictive human-specific in vitro heart models can revolutionize drug discovery and... more Reliable and predictive human-specific in vitro heart models can revolutionize drug discovery and development. With the advent of pluripotent stem cell technologies, human cardiomyocytes can now be readily produced in large quantities. Using tissue engineering techniques, they can be further assembled into cardiac tissues of specific 2D and 3D configurations, to create models that behave and function like the native human heart. Novoheart (BC, Canada) uniquely offers the MyHeartTM Platform of bioengineered human heart constructs, designed to provide researchers with effective models of either healthy or diseased human hearts. As in vitro, human-based assays become more widely accepted, the next decade could witness a shift away from animal testing towards more accurate and scalable human assays like the MyHeartTM Platform.
Background Delivery of stem cells to the failing heart is a promising therapeutic strategy. Howev... more Background Delivery of stem cells to the failing heart is a promising therapeutic strategy. However, the improvement in cardiac function in animal studies has not fully translated to humans. To help bridge the gap between species, we investigated the effects of adult human cardiac stem cells (hCSCs) on contractile function of human engineered cardiac tissues (hECTs) as a species-specific model of the human myocardium. Methods Human induced pluripotent stem cell-derived cardiomyoctes (hCMs) were mixed with Collagen/Matrigel to fabricate control hECTs, with an experimental group of hCSC-supplemented hECT fabricated using a 9:1 ratio of hCM to hCSC. Functional testing was performed starting on culture day 6, under spontaneous conditions and also during electrical pacing from 0.25 to 1.0 Hz, measurements repeated at days 8 and 10. hECTs were then frozen and processed for gene analysis using a Nanostring assay with a cardiac targeted custom panel. Results The hCSC-supplemented hECTs disp...
Background: Friedreich's ataxia (FRDA) is an autosomal recessive disease caused by a non-coding m... more Background: Friedreich's ataxia (FRDA) is an autosomal recessive disease caused by a non-coding mutation in the first intron of the frataxin (FXN) gene that suppresses its expression. Compensatory hypertrophic cardiomyopathy, dilated cardiomyopathy, and conduction system abnormalities in FRDA lead to cardiomyocyte (CM) death and fibrosis, consequently resulting in heart failure and arrhythmias. Murine models have been developed to study disease pathology in the past two decades; however, differences between human and mouse physiology and metabolism have limited the relevance of animal studies in cardiac disease conditions. To bridge this gap, we aimed to generate species-specific, functional in vitro experimental models of FRDA using 2-dimensional (2D) and 3-dimensional (3D) engineered cardiac tissues from FXN-deficient human pluripotent stem cell-derived ventricular cardiomyocytes (hPSC-hvCMs) and to compare their contractile and electrophysiological properties with healthy tissue constructs. Methods: Healthy control and FRDA patient-specific hPSC-hvCMs were derived by directed differentiation using a small molecule-based protocol reported previously. We engineered the hvCMs into our established human ventricular cardiac tissue strip (hvCTS) and human ventricular cardiac anisotropic sheet (hvCAS) models, and functional assays were performed on days 7-17 post-tissue fabrication to assess the electrophysiology and contractility of FRDA patient-derived and FXN-knockdown engineered tissues, in comparison with healthy controls. To further validate the disease model, forced expression of FXN was induced in FXN-deficient tissues to test if disease phenotypes could be rescued.
Human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) have the ability of... more Human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) have the ability of differentiating into functional cardiomyocytes (CMs) for cell replacement therapy, tissue engineering, drug discovery and toxicity screening. From a scale-free, co-expression network analysis of transcriptomic data that distinguished gene expression profiles of undifferentiated hESC, hESC-, fetal- and adult-ventricular(V) CM, two candidate chromatin remodeling proteins, SMYD1 and SMARCD1 were found to be differentially expressed. Using lentiviral transduction, SMYD1 and SMARCD1 were over-expressed and suppressed, respectively, in single hESC-VCMs as well as the 3D constructs Cardiac Micro Tissues (CMT) and Tissue Strips (CTS) to mirror the endogenous patterns, followed by dissection of their roles in controlling cardiac gene expression, contractility, Ca2+-handling, electrophysiological functions and in vitro maturation. Interestingly, compared to independent single transductions, simul...
The promising clinical benefits of delivering human mesenchymal stem cells (hMSCs) for treating h... more The promising clinical benefits of delivering human mesenchymal stem cells (hMSCs) for treating heart disease warrant a better understanding of underlying mechanisms of action. hMSC exosomes increase myocardial contractility; however, the exosomal cargo responsible for these effects remains unresolved. This study aims to identify lead cardioactive hMSC exosomal microRNAs to provide a mechanistic basis for optimizing future stem cell-based cardiotherapies. Integrating systems biology and human engineered cardiac tissue (hECT) technologies, partial least squares regression analysis of exosomal microRNA profiling data predicted microRNA-21-5p (miR-21-5p) levels positively correlate with contractile force and calcium handling gene expression responses in hECTs treated with conditioned media from multiple cell types. Furthermore, miR-21-5p levels were significantly elevated in hECTs treated with the exosome-enriched fraction of the hMSC secretome (hMSC-exo) versus untreated controls. Thi...
Cardiac excitation-contraction coupling (ECC) is the orchestrated process of initial myocyte elec... more Cardiac excitation-contraction coupling (ECC) is the orchestrated process of initial myocyte electrical excitation, which leads to calcium entry, intracellular trafficking, and subsequent sarcomere shortening and myofibrillar contraction. Neurohumoral β-adrenergic signaling is a well-established mediator of ECC; other signaling mechanisms, such as paracrine signaling, have also demonstrated significant impact on ECC but are less well understood. For example, resident heart endothelial cells are well-known physiological paracrine modulators of cardiac myocyte ECC mainly via NO and endothelin-1. Moreover, recent studies have demonstrated other resident noncardiomyocyte heart cells (eg, physiological fibroblasts and pathological myofibroblasts), and even experimental cardiotherapeutic cells (eg, mesenchymal stem cells) are also capable of altering cardiomyocyte ECC through paracrine mechanisms. In this review, we first focus on the paracrine-mediated effects of resident and therapeutic...
Methods in molecular biology (Clifton, N.J.), 2017
An area of active research in the field of cardiac gene therapy aims to achieve high transfection... more An area of active research in the field of cardiac gene therapy aims to achieve high transfection efficiency without eliciting immune or inflammatory reactions. Nanomedicine offers an attractive alternative to traditional viral delivery vehicles because nanoparticle technology can enable safer and more controlled delivery of therapeutic agents. Here we describe the use of lipidoid nanoparticles for delivery of modified mRNA (modRNA) to the myocardium in vivo, with a focus on rodent models that represent a first step toward preclinical studies. Three major procedures are discussed in this chapter: (1) preparation of lipid modRNA nanoparticles, (2) intramyocardial delivery of the lipid modRNA nanoparticles by direct injection with an open chest technique in rats, and (3) intracoronary delivery of the lipid modRNA nanoparticles with open chest and temporary aortic cross clamping in rats.
Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) provide a unique tool to e... more Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) provide a unique tool to evaluate cellular properties in disease models, investigate underlying mechanisms and optimize cell-based therapies. We recently reported that after plating 30 day old single hiPSC-CMs on undiluted Matrigel ''Mattress'' (thickness ~0.5 mm) for 5 days, mattress hiPSC-CMs developed a rod-like cell shape, aligned myofilament structure, increased sarcomere length and exhibited robust cell shortening. Here, we compared the electrophysiological properties of mattress hiPSC-CMs to hiPSC-CMs cultured on standard substrates (control) and acutely-isolated adult rabbit CMs. The action potentials of mattress hiPSC-CMs were comparable to rabbit CMs. Compared to control, mattress hiPSC-CMs had significantly increased the action potential upstroke velocity due to twofold increase in sodium current (INa) (À68.61511.63 pA$pF-1 in mattress vs. À29.6657.50 pA$pF-1 in control at À30mV). The inward rectifier current (IK1) is responsible for the maintenance of resting membrane potential and contributes to phase 3 of the action potential. Although resting potential and action potential duration was not different from rabbit CMs, IK1 density in mattress hiPSC-CMs was nine-time smaller than in rabbit CMs, but there was no difference between mattress and control hiPSC-CMs (À4.8650.66 pA$pF-1 in mattress vs. À43.7554.13 pA$pF-1 in rabbit vs. À3.9850.83 pA$pF-1 in control at À120 mV). The funny current (If) is an inward current activating at hyperpolarized membrane potential, which is detectable in the developing heart but absent in the adult ventricle and contributes to the spontaneous beating of hiPSC-CMs. Mattress hiPSC-CMs exhibit robust If that was not significantly different from control (À6.2251.31 pA$pF-1 in mattress vs.-5.6051.43 pA$pF-1 in control at À120 mV). In summary, the matrigel mattress method enables the rapid generation of robustly contracting single hiPSC-CMs and increases INa, with negligible effect on IK1 or If.
Marfan syndrome (MFS) is an autosomal dominant disease of the connective tissue due to mutations ... more Marfan syndrome (MFS) is an autosomal dominant disease of the connective tissue due to mutations in the fibrillin-1 gene (FBN1). This study aimed at characterizing microelastic properties of the ascending aortic wall and lung parenchyma tissues from wild type (WT) and age-matched Fbn1 hypomorphic mice (Fbn1(mgR/mgR) mice) to identify tissue-specific biomechanical effects of aging and disease in MFS. Atomic force microscopy was used to indent lung parenchyma and aortic wall tissues, using Hybrid Eshelby Decomposition analysis to extract layer-specific properties of the intima and media. The intima stiffened with age and was not different between WT and Fbn1(mgR/mgR) tissues, whereas the media layer of MFS aortas showed progressive structural and mechanical degradation with a modulus that was 50% softer than WT by 3.5 months of age. Similarly, MFS mice displayed progressive structural and mechanical deterioration of lung tissue, which was over 85% softer than WT by 3.5 months of age. ...
Uploads
Papers by Kevin Costa