Biomimetic Nanomaterials for the Immunomodulation of the Cardiosplenic Axis Postmyocardial Infarction.

The spleen is an important mediator of both adaptive and innate immunity. As such, attempts to modulate the immune response provided by the spleen may be conducive to improved outcomes for numerous diseases throughout the body. Here, biomimicry is used to rationally design nanomaterials capable of splenic retention and immunomodulation for the treatment of disease in a distant organ, the postinfarct heart. Engineered senescent erythrocyte-derived nanotheranostic (eSENTs) are generated, demonstrating significant uptake by the immune cells of the spleen including T and B cells, as well as monocytes and macrophages. When loaded with suberoylanilide hydroxamic acid (SAHA), the nanoagents exhibit a potent therapeutic effect, reducing infarct size by 14% at 72 h postmyocardial infarction when given as a single intravenous dose 2 h after injury. These results are supportive of the hypothesis that RBC-derived biomimicry may provide new approaches for the targeted modulation of the pathological processes involved in myocardial infarction, thus further experiments to decisively confirm the mechanisms of action are currently underway. This novel concept may have far-reaching applicability for the treatment of a number of both acute and chronic conditions where the immune responses are either stimulated or suppressed by the splenic (auto)immune milieu.

Nanowired human cardiac organoid transplantation enables highly efficient and effective recovery of infarcted hearts.

Human cardiac organoids hold remarkable potential for cardiovascular disease modeling and human pluripotent stem cell-derived cardiomyocyte (hPSC-CM) transplantation. Here, we show cardiac organoids engineered with electrically conductive silicon nanowires (e-SiNWs) significantly enhance the therapeutic efficacy of hPSC-CMs to treat infarcted hearts. We first demonstrated the biocompatibility of e-SiNWs and their capacity to improve cardiac microtissue engraftment in healthy rat myocardium. Nanowired human cardiac organoids were then engineered with hPSC-CMs, nonmyocyte supporting cells, and e-SiNWs. Nonmyocyte supporting cells promoted greater ischemia tolerance of cardiac organoids, and e-SiNWs significantly improved electrical pacing capacity. After transplantation into ischemia/reperfusion-injured rat hearts, nanowired cardiac organoids significantly improved contractile development of engrafted hPSC-CMs, induced potent cardiac functional recovery, and reduced maladaptive left ventricular remodeling. Compared to contemporary studies with an identical injury model, greater functional recovery was achieved with a 20-fold lower dose of hPSC-CMs, revealing therapeutic synergy between conductive nanomaterials and human cardiac organoids for efficient heart repair.

Multicellular Human Cardiac Organoids Transcriptomically Model Distinct Tissue-Level Features of Adult Myocardium.

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have been widely used for disease modeling and drug cardiotoxicity screening. To this end, we recently developed human cardiac organoids (hCOs) for modeling human myocardium. Here, we perform a transcriptomic analysis of various in vitro hiPSC-CM platforms (2D iPSC-CM, 3D iPSC-CM and hCOs) to deduce the strengths and limitations of these in vitro models. We further compared iPSC-CM models to human myocardium samples. Our data show that the 3D in vitro environment of 3D hiPSC-CMs and hCOs stimulates the expression of genes associated with tissue formation. The hCOs demonstrated diverse physiologically relevant cellular functions compared to the hiPSC-CM only models. Including other cardiac cell types within hCOs led to more transcriptomic similarities to adult myocardium. hCOs lack matured cardiomyocytes and immune cells, which limits a complete replication of human adult myocardium. In conclusion, 3D hCOs are transcriptomically similar to myocardium, and future developments of engineered 3D cardiac models would benefit from diversifying cell populations, especially immune cells.

Targeting HIF-α for robust prevascularization of human cardiac organoids.

Prevascularized 3D microtissues have been shown to be an effective cell delivery vehicle for cardiac repair. To this end, our lab has explored the development of self-organizing, prevascularized human cardiac organoids by co-seeding human cardiomyocytes with cardiac fibroblasts, endothelial cells, and stromal cells into agarose microwells. We hypothesized that this prevascularization process is facilitated by the endogenous upregulation of hypoxia-inducible factor (HIF) pathway in the avascular 3D microtissues. In this study, we used Molidustat, a selective PHD (prolyl hydroxylase domain enzymes) inhibitor that stabilizes HIF-α, to treat human cardiac organoids, which resulted in 150 ± 61% improvement in endothelial expression (CD31) and 220 ± 20% improvement in the number of lumens per organoids. We hypothesized that the improved endothelial expression seen in Molidustat treated human cardiac organoids was dependent upon upregulation of VEGF, a well-known downstream target of HIF pathway. Through the use of immunofluorescent staining and ELISA assays, we determined that Molidustat treatment improved VEGF expression of non-endothelial cells and resulted in improved co-localization of supporting cell types and endothelial structures. We further demonstrated that Molidustat treated human cardiac organoids maintain cardiac functionality. Lastly, we showed that Molidustat treatment improves survival of cardiac organoids when exposed to both hypoxic and ischemic conditions in vitro. For the first time, we demonstrate that targeted HIF-α stabilization provides a robust strategy to improve endothelial expression and lumen formation in cardiac microtissues, which will provide a powerful framework for prevascularization of various microtissues in developing successful cell transplantation therapies.

Focusing Heart Failure Research on Myocardial Fibrosis to Prioritize Translation.

Heart failure (HF) has traditionally been defined by symptoms of fluid accumulation and poor perfusion, but it is now recognized that specific HF classifications hold prognostic and therapeutic relevance. Specifically, HF with reduced ejection fraction is characterized by reduced left ventricular systolic pump function and dilation and HF with preserved ejection fraction is characterized primarily by abnormal left ventricular filling (diastolic failure) with relatively preserved left ventricular systolic function. These forms of HF are distributed equally among patients with HF and likely require distinctly different strategies to mitigate the morbidity, mortality, and medical resource utilization of this disease. In particular, HF is a significant medical issue within the US Department of Veterans Affairs (VA) hospital system and constitutes a major translational research priority for the VA. Because a common underpinning of both HF with reduced ejection fraction and HF with preserved ejection fraction seems to be changes in the structure and function of the myocardial extracellular matrix, a conference was convened sponsored by the VA, entitled, "Targeting Myocardial Fibrosis in Heart Failure" to explore the extracellular matrix as a potential therapeutic target and to propose specific research directions. The conference was conceptually framed around the hypothesis that although HF with reduced ejection fraction and HF with preserved ejection fraction clearly have distinct mechanisms, they may share modifiable pathways and biological mediators in common. Inflammation and extracellular matrix were identified as major converging themes. A summary of our discussion on unmet challenges and possible solutions to move the field forward, as well as recommendations for future research opportunities, are provided.

Human cardiac organoids for the modelling of myocardial infarction and drug cardiotoxicity.

Environmental factors are the largest contributors to cardiovascular disease. Here we show that cardiac organoids that incorporate an oxygen-diffusion gradient and that are stimulated with the neurotransmitter noradrenaline model the structure of the human heart after myocardial infarction (by mimicking the infarcted, border and remote zones), and recapitulate hallmarks of myocardial infarction (in particular, pathological metabolic shifts, fibrosis and calcium handling) at the transcriptomic, structural and functional levels. We also show that the organoids can model hypoxia-enhanced doxorubicin cardiotoxicity. Human organoids that model diseases with non-genetic pathological factors could help with drug screening and development.

Regulation of metabolism by mitochondrial enzyme acetylation in cardiac ischemia-reperfusion injury.

Ischemia reperfusion injury (I/R injury) contributes significantly to morbidity and mortality following myocardial infarction (MI). Although rapid reperfusion of the ischemic myocardium was established decades ago as a highly beneficial therapy for MI, significant cell death still occurs after the onset of reperfusion. Mitochondrial dysfunction is closely associated with I/R injury, resulting in the uncontrolled production of reactive oxygen species (ROS). Considerable efforts have gone into understanding the metabolic perturbations elicited by I/R injury. Recent work has identified the critical role of reversible protein acetylation in maintaining normal mitochondrial biologic function and energy metabolism both in the normal heart and during I/R injury. Several studies have shown that modification of class I HDAC and/or Sirtuin (Sirt) activity is cardioprotective in the setting of I/R injury. A better understanding of the role of these metabolic pathways in reperfusion injury and their regulation by reversible protein acetylation presents a promising way forward in improving the treatment of cardiac reperfusion injury. Here we briefly review some of what is known about how acetylation regulates mitochondrial metabolism and how it relates to I/R injury.

HDAC inhibition helps post-MI healing by modulating macrophage polarization.

AIMS: Following an acute myocardial infarction (MI) the extracellular matrix (ECM) undergoes remodeling in order to prevent dilation of the infarct area and maintain cardiac output. Excessive and prolonged inflammation following an MI exacerbates adverse ventricular remodeling. Macrophages are an integral part of the inflammatory response that contribute to this remodeling. Treatment with histone deacetylase (HDAC) inhibitors preserves LV function and myocardial remodeling in the post-MI heart. This study tested whether inhibition of HDAC activity resulted in preserving post-MI LV function through the regulation of macrophage phenotype and early resolution of inflammation. METHODS AND RESULTS: HDAC inhibition does not affect the recruitment of CD45+ leukocytes, CD45+/CD11b+ inflammatory monocytes or CD45+/CD11b+CD86+ inflammatory macrophages for the first 3 days following infarct. Further, HDAC inhibition does not change the high expression level of the inflammatory cytokines in the first days following MI. However, by day 7, there was a significant reduction in the levels of CD45+/Cd11b+ and CD45+/CD11b+/CD86+ cells with HDAC inhibition. Remarkably, HDAC inhibition resulted in the dramatic increase in the recruitment of CD45+/CD11b+/CD206+ alternatively activated macrophages as early as 1 day which remained significantly elevated until 5 days post-MI. qRT-PCR revealed that HDAC inhibitor treatment shifts the cytokine and chemokine environment towards an M2 phenotype with upregulation of M2 markers at 1 and 5 days post-MI. Importantly, HDAC inhibition correlates with significant preservation of both LV ejection fraction and end-diastolic volume and is associated with a significant increase in micro-vessel density in the border zone at 14 days post-MI. CONCLUSION: Inhibition of HDAC activity result in the early recruitment of reparative CD45+/CD11b+/CD206+ macrophages in the post-MI heart and correlates with improved ventricular function and remodeling. This work identifies a very promising therapeutic opportunity to manage macrophage phenotype and enhance resolution of inflammation in the post-MI heart.

Angiokine Wisp-1 is increased in myocardial infarction and regulates cardiac endothelial signaling.

Myocardial infarctions (MIs) cause the loss of myocytes due to lack of sufficient oxygenation and latent revascularization. Although the administration of histone deacetylase (HDAC) inhibitors reduces the size of infarctions and improves cardiac physiology in small-animal models of MI injury, the cellular targets of the HDACs, which the drugs inhibit, are largely unspecified. Here, we show that WNT-inducible secreted protein-1 (Wisp-1), a matricellular protein that promotes angiogenesis in cancers as well as cell survival in isolated cardiac myocytes and neurons, is a target of HDACs. Further, Wisp-1 transcription is regulated by HDACs and can be modified by the HDAC inhibitor, suberanilohydroxamic acid (SAHA/vorinostat), after MI injury. We observe that, at 7 days after MI, Wisp-1 is elevated 3-fold greater in the border zone of infarction in mice that experience an MI injury and are injected daily with SAHA, relative to MI alone. Additionally, human coronary artery endothelial cells (HCAECs) produce WISP-1 and are responsive to autocrine WISP-1-mediated signaling, which functionally promotes their proangiogenic behavior. Altering endogenous expression of WISP-1 in HCAECs directly impacts their network density in vitro. Therapeutic interventions after a heart attack define the extent of infarct injury, cell survival, and overall prognosis. Our studies shown here identify a potentially novel cardiac angiokine, Wisp-1, that may contribute to beneficial post-MI treatment modalities.

HDAC1 localizes to the mitochondria of cardiac myocytes and contributes to early cardiac reperfusion injury.

RATIONALE: Recent evidence indicates that histone deacetylase enzymes (HDACs) contribute to ischemia reperfusion (I/R) injury, and pan-HDAC inhibitors have been shown to be cardioprotective when administered either before an ischemic insult or during reperfusion. We have shown previously that selective inhibition of class I HDACs provides superior cardioprotection when compared to pan-HDAC inhibition in a pretreatment model, but selective class I HDAC inhibition has not been tested during reperfusion, and specific targets of class I HDACs in I/R injury have not been identified. OBJECTIVE: We hypothesized that selective inhibition of class I HDACs with the drug MS-275 (entinostat) during reperfusion would improve recovery from I/R injury in the first hour of reperfusion. METHODS AND RESULTS: Hearts from male Sprague-Dawley rats were subjected to ex vivo I/R injury±MS-275 class I HDAC inhibition during reperfusion alone. MS-275 significantly attenuated I/R injury, as indicated by improved LV function and tissue viability at the end of reperfusion. Unexpectedly, we observed that HDAC1 is present in the mitochondria of cardiac myocytes, but not fibroblasts or endothelial cells. We then designed mitochondria-restricted and mitochondria-excluded HDAC inhibitors, and tested both in our ex vivo I/R model. The selective inhibition of mitochondrial HDAC1 attenuated I/R injury to the same extent as MS-275, whereas the mitochondrial-excluded inhibitor did not. Further assays demonstrated that these effects are attributable to a decrease in SDHA activity and subsequent metabolic ROS production in reperfusion. CONCLUSIONS: We demonstrate for the first time that HDAC1 is present within the mitochondria of cardiac myocytes, and mitochondrial HDAC1 contributes significantly to I/R injury within the first hour of reperfusion.

Inspiration from heart development: Biomimetic development of functional human cardiac organoids.

Recent progress in human organoids has provided 3D tissue systems to model human development, diseases, as well as develop cell delivery systems for regenerative therapies. While direct differentiation of human embryoid bodies holds great promise for cardiac organoid production, intramyocardial cell organization during heart development provides biological foundation to fabricate human cardiac organoids with defined cell types. Inspired by the intramyocardial organization events in coronary vasculogenesis, where a diverse, yet defined, mixture of cardiac cell types self-organizes into functional myocardium in the absence of blood flow, we have developed a defined method to produce scaffold-free human cardiac organoids that structurally and functionally resembled the lumenized vascular network in the developing myocardium, supported hiPSC-CM development and possessed fundamental cardiac tissue-level functions. In particular, this development-driven strategy offers a robust, tunable system to examine the contributions of individual cell types, matrix materials and additional factors for developmental insight, biomimetic matrix composition to advance biomaterial design, tissue/organ-level drug screening, and cell therapy for heart repair.

Cell number per spheroid and electrical conductivity of nanowires influence the function of silicon nanowired human cardiac spheroids.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) provide an unlimited cell source to treat cardiovascular diseases, the leading cause of death worldwide. However, current hiPSC-CMs retain an immature phenotype that leads to difficulties for integration with adult myocardium after transplantation. To address this, we recently utilized electrically conductive silicon nanowires (e-SiNWs) to facilitate self-assembly of hiPSC-CMs to form nanowired hiPSC cardiac spheroids. Our previous results showed addition of e-SiNWs effectively enhanced the functions of the cardiac spheroids and improved the cellular maturation of hiPSC-CMs. Here, we examined two important factors that can affect functions of the nanowired hiPSC cardiac spheroids: (1) cell number per spheroid (i.e., size of the spheroids), and (2) the electrical conductivity of the e-SiNWs. To examine the first factor, we prepared hiPSC cardiac spheroids with four different sizes by varying cell number per spheroid (∼0.5k, ∼1k, ∼3k, ∼7k cells/spheroid). Spheroids with ∼3k cells/spheroid was found to maximize the beneficial effects of the 3D spheroid microenvironment. This result was explained with a semi-quantitative theory that considers two competing factors: 1) the improved 3D cell-cell adhesion, and 2) the reduced oxygen supply to the center of spheroids with the increase of cell number. Also, the critical role of electrical conductivity of silicon nanowires has been confirmed in improving tissue function of hiPSC cardiac spheroids. These results lay down a solid foundation to develop suitable nanowired hiPSC cardiac spheroids as an innovative cell delivery system to treat cardiovascular diseases. STATEMENT OF SIGNIFICANCE: Cardiovascular disease is the leading cause of death and disability worldwide. Due to the limited regenerative capacity of adult human hearts, human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have received significant attention because they provide a patient specific cell source to regenerate damaged hearts. Despite the progress, current human hiPSC-CMs retain an immature phenotype that leads to difficulties for integration with adult myocardium after transplantation. To address this, we recently utilized electrically conductive silicon nanowires (e-SiNWs) to facilitate self-assembly of hiPSC-CMs to form nanowired hiPSC cardiac spheroids. Our previous results showed addition of e-SiNWs effectively enhanced the functions of the cardiac spheroids and improved the cellular maturation of hiPSC-CMs. In this manuscript, we examined the effects of two important factors on the functions of nanowired hiPSC cardiac spheroids: (1) cell number per spheroid (i.e., size of the spheroids), and (2) the electrical conductivity of the e-SiNWs. The results from these studies will allow for the development of suitable nanowired hiPSC cardiac spheroids to effectively deliver hiPSC-CMs for heart repair.

Nanowires and Electrical Stimulation Synergistically Improve Functions of hiPSC Cardiac Spheroids.

The advancement of human induced pluripotent stem-cell-derived cardiomyocyte (hiPSC-CM) technology has shown promising potential to provide a patient-specific, regenerative cell therapy strategy to treat cardiovascular disease. Despite the progress, the unspecific, underdeveloped phenotype of hiPSC-CMs has shown arrhythmogenic risk and limited functional improvements after transplantation. To address this, tissue engineering strategies have utilized both exogenous and endogenous stimuli to accelerate the development of hiPSC-CMs. Exogenous electrical stimulation provides a biomimetic pacemaker-like stimuli that has been shown to advance the electrical properties of tissue engineered cardiac constructs. Recently, we demonstrated that the incorporation of electrically conductive silicon nanowires to hiPSC cardiac spheroids led to advanced structural and functional development of hiPSC-CMs by improving the endogenous electrical microenvironment. Here, we reasoned that the enhanced endogenous electrical microenvironment of nanowired hiPSC cardiac spheroids would synergize with exogenous electrical stimulation to further advance the functional development of nanowired hiPSC cardiac spheroids. For the first time, we report that the combination of nanowires and electrical stimulation enhanced cell-cell junction formation, improved development of contractile machinery, and led to a significant decrease in the spontaneous beat rate of hiPSC cardiac spheroids. The advancements made here address critical challenges for the use of hiPSC-CMs in cardiac developmental and translational research and provide an advanced cell delivery vehicle for the next generation of cardiac repair.

A class of their own: exploring the nondeacetylase roles of class IIa HDACs in cardiovascular disease.

Histone deacetylases (HDACs) play integral roles in many cardiovascular biological processes ranging from transcriptional and translational regulation to protein stabilization and localization. There are 18 known HDACs categorized into 4 classes that can differ on the basis of substrate targets, subcellular localization, and regulatory binding partners. HDACs are classically known for their ability to remove acetyl groups from histone and nonhistone proteins that have lysine residues. However, despite their nomenclature and classical functions, discoveries from many research groups over the past decade have suggested that nondeacetylase roles exist for class IIa HDACs. This is not surprising given that class IIa HDACs have, for example, relatively poor deacetylase capabilities and are often shuttled in and out of nuclei upon specific pathological and nonpathological cardiac events. This review aims to consolidate and elucidate putative nondeacetylase roles for class IIa HDACs and, where possible, highlight studies that provide evidence for their noncanonical roles, especially in the context of cardiovascular maladies. There has been great interest recently in exploring the pharmacological regulators of HDACs for use in therapeutic interventions for treating cardiovascular diseases and inflammation. Thus it is of interest to earnestly consider nonenzymatic and or nondeacetylase roles of HDACs that might be key in potentiating or abrogating pathologies. These noncanonical HDAC functions may possibly yield new mechanisms and targets for drug discovery.

Evidence for a non-canonical role of HDAC5 in regulation of the cardiac Ncx1 and Bnp genes.

Class IIa histone deacetylases (HDACs) are very important for tissue specific gene regulation in development and pathology. Because class IIa HDAC catalytic activity is low, their exact molecular roles have not been fully elucidated. Studies have suggested that class IIa HDACs may serve as a scaffold to recruit the catalytically active class I HDAC complexes to their substrate. Here we directly address whether the class IIa HDAC, HDAC5 may function as a scaffold to recruit co-repressor complexes to promoters. We examined two well-characterized cardiac promoters, the sodium calcium exchanger (Ncx1) and the brain natriuretic peptide (Bnp) whose hypertrophic upregulation is mediated by both class I and IIa HDACs. Selective inhibition of class IIa HDACs did not prevent adrenergic stimulated Ncx1 upregulation, however HDAC5 knockout prevented pressure overload induced Ncx1 upregulation. Using the HDAC5((-/-)) mouse we show that HDAC5 is required for the interaction of the HDAC1/2/Sin3a co-repressor complexes with the Nkx2.5 and YY1 transcription factors and critical for recruitment of the HDAC1/Sin3a co-repressor complex to either the Ncx1 or Bnp promoter. Our novel findings support a non-canonical role of class IIa HDACs in the scaffolding of transcriptional regulatory complexes, which may be relevant for therapeutic intervention for pathologies.

HDACs Regulate miR-133a Expression in Pressure Overload-Induced Cardiac Fibrosis.

BACKGROUND: MicroRNAs (miRNAs) and histone deacetylases (HDACs) serve a significant role in the pathogenesis of a variety of cardiovascular diseases. The transcriptional regulation of miRNAs is poorly understood in cardiac hypertrophy. We investigated whether the expression of miR-133a is epigenetically regulated by class I and IIb HDACs during hypertrophic remodeling. METHODS AND RESULTS: Transverse aortic constriction (TAC) was performed in CD1 mice to induce pressure overload hypertrophy. Mice were treated with class I and IIb HDAC inhibitor (HDACi) via drinking water for 2 and 4 weeks post TAC. miRNA expression was determined by real-time polymerase chain reaction. Echocardiography was performed at baseline and post TAC end points for structural and functional assessment. Chromatin immunoprecipitation was used to identify HDACs and transcription factors associated with miR-133a promoter. miR-133a expression was downregulated by 0.7- and 0.5-fold at 2 and 4 weeks post TAC, respectively, when compared with vehicle control (P<0.05). HDAC inhibition prevented this significant decrease 2 weeks post TAC and maintained miR-133a expression near vehicle control levels, which coincided with (1) a decrease in connective tissue growth factor expression, (2) a reduction in cardiac fibrosis and left atrium diameter (marker of end-diastolic pressure), suggesting an improvement in diastolic function. Chromatin immunoprecipitation analysis revealed that HDAC1 and HDAC2 are present on the miR-133a enhancer regions. CONCLUSIONS: The results reveal that HDACs play a role in the regulation of pressure overload-induced miR-133a downregulation. This work is the first to provide insight into an epigenetic-miRNA regulatory pathway in pressure overload-induced cardiac fibrosis.

Induction and Assessment of Ischemia-reperfusion Injury in Langendorff-perfused Rat Hearts.

The biochemical events surrounding ischemia reperfusion injury in the acute setting are of great importance to furthering novel treatment options for myocardial infarction and cardiac complications of thoracic surgery. The ability of certain drugs to precondition the myocardium against ischemia reperfusion injury has led to multiple clinical trials, with little success. The isolated heart model allows acute observation of the functional effects of ischemia reperfusion injury in real time, including the effects of various pharmacological interventions administered at any time-point before or within the ischemia-reperfusion injury window. Since brief periods of ischemia can precondition the heart against ischemic injury, in situ aortic cannulation is performed to allow for functional assessment of non-preconditioned myocardium. A saline filled balloon is placed into the left ventricle to allow for real-time measurement of pressure generation. Ischemic injury is simulated by the cessation of perfusion buffer flow, followed by reperfusion. The duration of both ischemia and reperfusion can be modulated to examine biochemical events at any given time-point. Although the Langendorff isolated heart model does not allow for the consideration of systemic events affecting ischemia and reperfusion, it is an excellent model for the examination of acute functional and biochemical events within the window of ischemia reperfusion injury as well as the effect of pharmacological intervention on cardiac pre- and postconditioning. The goal of this protocol is to demonstrate how to perform in situ aortic cannulation and heart excision followed by ischemia/reperfusion injury in the Langendorff model.

Mutations in DCHS1 cause mitral valve prolapse.

Mitral valve prolapse (MVP) is a common cardiac valve disease that affects nearly 1 in 40 individuals. It can manifest as mitral regurgitation and is the leading indication for mitral valve surgery. Despite a clear heritable component, the genetic aetiology leading to non-syndromic MVP has remained elusive. Four affected individuals from a large multigenerational family segregating non-syndromic MVP underwent capture sequencing of the linked interval on chromosome 11. We report a missense mutation in the DCHS1 gene, the human homologue of the Drosophila cell polarity gene dachsous (ds), that segregates with MVP in the family. Morpholino knockdown of the zebrafish homologue dachsous1b resulted in a cardiac atrioventricular canal defect that could be rescued by wild-type human DCHS1, but not by DCHS1 messenger RNA with the familial mutation. Further genetic studies identified two additional families in which a second deleterious DCHS1 mutation segregates with MVP. Both DCHS1 mutations reduce protein stability as demonstrated in zebrafish, cultured cells and, notably, in mitral valve interstitial cells (MVICs) obtained during mitral valve repair surgery of a proband. Dchs1(+/-) mice had prolapse of thickened mitral leaflets, which could be traced back to developmental errors in valve morphogenesis. DCHS1 deficiency in MVP patient MVICs, as well as in Dchs1(+/-) mouse MVICs, result in altered migration and cellular patterning, supporting these processes as aetiological underpinnings for the disease. Understanding the role of DCHS1 in mitral valve development and MVP pathogenesis holds potential for therapeutic insights for this very common disease.

Histone Deacetylases Exert Class-Specific Roles in Conditioning the Brain and Heart Against Acute Ischemic Injury.

Ischemia-reperfusion (IR) injury comprises a significant portion of morbidity and mortality from heart and brain diseases worldwide. This enduring clinical problem has inspired myriad reports in the scientific literature of experimental interventions seeking to elucidate the pathology of IR injury. Elective cardiac surgery presents perhaps the most viable scenario for protecting the heart and brain from IR injury due to the opportunity to condition the organs prior to insult. The physiological parameters for the preconditioning of vital organs prior to insult through mechanical and pharmacological maneuvers have been heavily examined. These investigations have revealed new insights into how preconditioning alters cellular responses to IR injury. However, the promise of preconditioning remains unfulfilled at the clinical level, and research seeking to implicate cell signals essential to this protection continues. Recent discoveries in molecular biology have revealed that gene expression can be controlled through posttranslational modifications, without altering the chemical structure of the genetic code. In this scenario, gene expression is repressed by enzymes that cause chromatin compaction through catalytic removal of acetyl moieties from lysine residues on histones. These enzymes, called histone deacetylases (HDACs), can be inhibited pharmacologically, leading to the de-repression of protective genes. The discovery that HDACs can also alter the function of non-histone proteins through posttranslational deacetylation has expanded the potential impact of HDAC inhibitors for the treatment of human disease. HDAC inhibitors have been applied in a very small number of experimental models of IR. However, the scientific literature contains an increasing number of reports demonstrating that HDACs converge on preconditioning signals in the cell. This review will describe the influence of HDACs on major preconditioning signaling pathways in the heart and brain.

Silicon nanowire-induced maturation of cardiomyocytes derived from human induced pluripotent stem cells.

The current inability to derive mature cardiomyocytes from human pluripotent stem cells has been the limiting step for transitioning this powerful technology into clinical therapies. To address this, scaffold-based tissue engineering approaches have been utilized to mimic heart development in vitro and promote maturation of cardiomyocytes derived from human pluripotent stem cells. While scaffolds can provide 3D microenvironments, current scaffolds lack the matched physical/chemical/biological properties of native extracellular environments. On the other hand, scaffold-free, 3D cardiac spheroids (i.e., spherical-shaped microtissues) prepared by seeding cardiomyocytes into agarose microwells were shown to improve cardiac functions. However, cardiomyocytes within the spheroids could not assemble in a controlled manner and led to compromised, unsynchronized contractions. Here, we show, for the first time, that incorporation of a trace amount (i.e., ∼0.004% w/v) of electrically conductive silicon nanowires (e-SiNWs) in otherwise scaffold-free cardiac spheroids can form an electrically conductive network, leading to synchronized and significantly enhanced contraction (i.e., >55% increase in average contraction amplitude), resulting in significantly more advanced cellular structural and contractile maturation.