Synchronous force and Ca2+ measurements for repeated characterization of excitation-contraction coupling in human myocardium.

Dysfunctional Ca2+ signaling affects the myocardial systole and diastole, may trigger arrhythmia and cause transcriptomic and proteomic modifications in heart failure. Thus, synchronous real-time measurement of Ca2+ and force is essential to investigate the relationship between contractility and Ca2+ signaling and the alteration of excitation-contraction coupling (ECC) in human failing myocardium. Here, we present a method for synchronized acquisition of intracellular Ca2+ and contraction force in long-term cultivated slices of human failing myocardium. Synchronous time series of contraction force and intracellular Ca2+ were used to calculate force-calcium loops and to analyze the dynamic alterations of ECC in response to various pacing frequencies, post-pause potentiation, high mechanical preload and pharmacological interventions in human failing myocardium. We provide an approach to simultaneously and repeatedly investigate alterations of contractility and Ca2+ signals in long-term cultured myocardium, which will allow detecting the effects of electrophysiological or pharmacological interventions on human myocardial ECC.

DNMT3A clonal hematopoiesis-driver mutations induce cardiac fibrosis by paracrine activation of fibroblasts.

Hematopoietic mutations in epigenetic regulators like DNA methyltransferase 3 alpha (DNMT3A), play a pivotal role in driving clonal hematopoiesis of indeterminate potential (CHIP), and are associated with unfavorable outcomes in patients suffering from heart failure (HF). However, the precise interactions between CHIP-mutated cells and other cardiac cell types remain unknown. Here, we identify fibroblasts as potential partners in interactions with CHIP-mutated monocytes. We used combined transcriptomic data derived from peripheral blood mononuclear cells of HF patients, both with and without CHIP, and cardiac tissue. We demonstrate that inactivation of DNMT3A in macrophages intensifies interactions with cardiac fibroblasts and increases cardiac fibrosis. DNMT3A inactivation amplifies the release of heparin-binding epidermal growth factor-like growth factor, thereby facilitating activation of cardiac fibroblasts. These findings identify a potential pathway of DNMT3A CHIP-driver mutations to the initiation and progression of HF and may also provide a compelling basis for the development of innovative anti-fibrotic strategies.

Contractility measurements for cardiotoxicity screening with ventricular myocardial slices of pigs.

AIMS: Cardiotoxicity is one major reason why drugs do not enter or are withdrawn from the market. Thus, approaches are required to predict cardiotoxicity with high specificity and sensitivity. Ideally, such methods should be performed within intact cardiac tissue with high relevance for humans and detect acute and chronic side effects on electrophysiological behaviour, contractility, and tissue structure in an unbiased manner. Herein, we evaluate healthy pig myocardial slices and biomimetic cultivation setups (BMCS) as a new cardiotoxicity screening approach. METHODS AND RESULTS: Pig left ventricular samples were cut into slices and spanned into BMCS with continuous electrical pacing and online force recording. Automated stimulation protocols were established to determine the force-frequency relationship (FFR), frequency dependence of contraction duration, effective refractory period (ERP), and pacing threshold. Slices generated 1.3 ± 0.14 mN/mm2 force at 0.5 Hz electrical pacing and showed a positive FFR and a shortening of contraction duration with increasing pacing rates. Approximately 62% of slices were able to contract for at least 6 days while showing stable ERP, contraction duration-frequency relationship, and preserved cardiac structure confirmed by confocal imaging and X-ray diffraction analysis. We used specific blockers of the most important cardiac ion channels to determine which analysis parameters are influenced. To validate our approach, we tested five drug candidates selected from the Comprehensive in vitro Proarrhythmia Assay list as well as acetylsalicylic acid and DMSO as controls in a blinded manner in three independent laboratories. We were able to detect all arrhythmic drugs and their respective mode of action on cardiac tissue including inhibition of Na+, Ca2+, and hERG channels as well as Na+/Ca2+ exchanger. CONCLUSION: We systematically evaluate this approach for cardiotoxicity screening, which is of high relevance for humans and can be upscaled to medium-throughput screening. Thus, our approach will improve the predictive value and efficiency of preclinical cardiotoxicity screening.

Ex vivo Culture and Contractile Force Measurements of Non-human Primate Heart Slices.

Cardiovascular diseases are the leading cause of death and morbidity worldwide. Patient mortality has been successfully reduced by nearly half in the last four decades, mainly due to advances in minimally invasive surgery techniques and interventional cardiology methods. However, a major hurdle is still the translational gap between preclinical findings and the conversion into effective therapies, which is partly due to the use of model systems that fail to recapitulate key aspects of human physiology and disease. Large animal models such as pigs and non-human primates are highly valuable because they closely resemble humans but are costly and time intensive. Here, we provide a method for long-term ex vivo culture of non-human primate (NHP) myocardial tissue that offers a powerful alternative for a wide range of applications including electrophysiology studies, drug screening, and gene function analyses.

Optimized Conditions for the Long-Term Maintenance of Precision-Cut Murine Myocardium in Biomimetic Tissue Culture.

Organotypic heart slices from mice might provide a promising in vitro model for cardiac research because of the vast availability of genetically modified specimens, combined with the unrestricted feasibility of experimental interventions. However, murine heart slices undergo rapid degeneration in culture. Therefore, we developed optimal conditions to preserve their structure and function in culture. Mouse ventricular heart samples were transversely cut into 300 µm thick slices. Slices were then cultured under various conditions of diastolic preload, systolic compliance and medium agitation. Continuous stimulation was performed either by optical stimulation or by electrical field stimulation. Contractility was continuously measured, and cellular survival, structure and gene expression were analyzed. Significant improvements in viability and function were achieved by elastic fixation with the appropriate diastolic preload and the rapid shaking of a ß-mercaptoethanol-supplemented medium. At 1 Hz pacing, mouse heart slices maintained stable contractility for up to 48 h under optogenetic pacing and for one week under electrical pacing. In cultured slices, the native myofibril structure was well preserved, and the mRNAs of myosin light chain, titin and connexin 43 were constantly expressed. Conclusions: Adult murine heart slices can be preserved for one week and provide a new opportunity to study cardiac functions.

Preparation of Human Myocardial Tissue for Long-Term Cultivation.

Cardiomyocyte cultivation has seen a vast number of developments, ranging from two-dimensional (2D) cell cultivation to iPSC derived organoids. In 2019, an ex vivo way to cultivate myocardial slices obtained from human heart samples was demonstrated, while approaching in vivo condition of myocardial contraction. These samples originate mostly from heart transplantations or left-ventricular assist device placements. Using a vibratome and a specially developed cultivation system, 300 µm thick slices are placed between a fixed and a spring wire, allowing for stable and reproducible cultivation for several weeks. During cultivation, the slices are continuously stimulated according to individual settings. Contractions can be displayed and recorded in real-time, and pharmacological agents can be readily applied. User-defined stimulation protocols can be scheduled and performed to assess vital contraction parameters like post-pause-potentiation, stimulation threshold, force-frequency relation, and refractory period. Furthermore, the system enables a variable pre- and afterload setting for a more physiological cultivation. Here, we present a step-by-step guide on how to generate a successful long-term cultivation of human left ventricular myocardial slices, using a commercial biomimetic cultivation solution.

Migratory and anti-fibrotic programmes define the regenerative potential of human cardiac progenitors.

Heart regeneration is an unmet clinical need, hampered by limited renewal of adult cardiomyocytes and fibrotic scarring. Pluripotent stem cell-based strategies are emerging, but unravelling cellular dynamics of host-graft crosstalk remains elusive. Here, by combining lineage tracing and single-cell transcriptomics in injured non-human primate heart biomimics, we uncover the coordinated action modes of human progenitor-mediated muscle repair. Chemoattraction via CXCL12/CXCR4 directs cellular migration to injury sites. Activated fibroblast repulsion targets fibrosis by SLIT2/ROBO1 guidance in organizing cytoskeletal dynamics. Ultimately, differentiation and electromechanical integration lead to functional restoration of damaged heart muscle. In vivo transplantation into acutely and chronically injured porcine hearts illustrated CXCR4-dependent homing, de novo formation of heart muscle, scar-volume reduction and prevention of heart failure progression. Concurrent endothelial differentiation contributed to graft neovascularization. Our study demonstrates that inherent developmental programmes within cardiac progenitors are sequentially activated in disease, enabling the cells to sense and counteract acute and chronic injury.

MicroRNA-365 regulates human cardiac action potential duration.

Abnormalities of ventricular action potential cause malignant cardiac arrhythmias and sudden cardiac death. Here, we aim to identify microRNAs that regulate the human cardiac action potential and ask whether their manipulation allows for therapeutic modulation of action potential abnormalities. Quantitative analysis of the microRNA targetomes in human cardiac myocytes identifies miR-365 as a primary microRNA to regulate repolarizing ion channels. Action potential recordings in patient-specific induced pluripotent stem cell-derived cardiac myocytes show that elevation of miR-365 significantly prolongs action potential duration in myocytes derived from a Short-QT syndrome patient, whereas specific inhibition of miR-365 normalizes pathologically prolonged action potential in Long-QT syndrome myocytes. Transcriptome analyses in these cells at bulk and single-cell level corroborate the key cardiac repolarizing channels as direct targets of miR-365, together with functionally synergistic regulation of additional action potential-regulating genes by this microRNA. Whole-cell patch-clamp experiments confirm miR-365-dependent regulation of repolarizing ionic current Iks. Finally, refractory period measurements in human myocardial slices substantiate the regulatory effect of miR-365 on action potential in adult human myocardial tissue. Our results delineate miR-365 to regulate human cardiac action potential duration by targeting key factors of cardiac repolarization.

Animal models and animal-free innovations for cardiovascular research: current status and routes to be explored. Consensus document of the ESC Working Group on Myocardial Function and the ESC Working Group on Cellular Biology of the Heart.

Cardiovascular diseases represent a major cause of morbidity and mortality, necessitating research to improve diagnostics, and to discover and test novel preventive and curative therapies, all of which warrant experimental models that recapitulate human disease. The translation of basic science results to clinical practice is a challenging task, in particular for complex conditions such as cardiovascular diseases, which often result from multiple risk factors and comorbidities. This difficulty might lead some individuals to question the value of animal research, citing the translational 'valley of death', which largely reflects the fact that studies in rodents are difficult to translate to humans. This is also influenced by the fact that new, human-derived in vitro models can recapitulate aspects of disease processes. However, it would be a mistake to think that animal models do not represent a vital step in the translational pathway as they do provide important pathophysiological insights into disease mechanisms particularly on an organ and systemic level. While stem cell-derived human models have the potential to become key in testing toxicity and effectiveness of new drugs, we need to be realistic, and carefully validate all new human-like disease models. In this position paper, we highlight recent advances in trying to reduce the number of animals for cardiovascular research ranging from stem cell-derived models to in situ modelling of heart properties, bioinformatic models based on large datasets, and state-of-the-art animal models, which show clinically relevant characteristics observed in patients with a cardiovascular disease. We aim to provide a guide to help researchers in their experimental design to translate bench findings to clinical routine taking the replacement, reduction, and refinement (3R) as a guiding concept.

Sequential Defects in Cardiac Lineage Commitment and Maturation Cause Hypoplastic Left Heart Syndrome.

BACKGROUND: Complex molecular programs in specific cell lineages govern human heart development. Hypoplastic left heart syndrome (HLHS) is the most common and severe manifestation within the spectrum of left ventricular outflow tract obstruction defects occurring in association with ventricular hypoplasia. The pathogenesis of HLHS is unknown, but hemodynamic disturbances are assumed to play a prominent role. METHODS: To identify perturbations in gene programs controlling ventricular muscle lineage development in HLHS, we performed whole-exome sequencing of 87 HLHS parent-offspring trios, nuclear transcriptomics of cardiomyocytes from ventricles of 4 patients with HLHS and 15 controls at different stages of heart development, single cell RNA sequencing, and 3D modeling in induced pluripotent stem cells from 3 patients with HLHS and 3 controls. RESULTS: Gene set enrichment and protein network analyses of damaging de novo mutations and dysregulated genes from ventricles of patients with HLHS suggested alterations in specific gene programs and cellular processes critical during fetal ventricular cardiogenesis, including cell cycle and cardiomyocyte maturation. Single-cell and 3D modeling with induced pluripotent stem cells demonstrated intrinsic defects in the cell cycle/unfolded protein response/autophagy hub resulting in disrupted differentiation of early cardiac progenitor lineages leading to defective cardiomyocyte subtype differentiation/maturation in HLHS. Premature cell cycle exit of ventricular cardiomyocytes from patients with HLHS prevented normal tissue responses to developmental signals for growth, leading to multinucleation/polyploidy, accumulation of DNA damage, and exacerbated apoptosis, all potential drivers of left ventricular hypoplasia in absence of hemodynamic cues. CONCLUSIONS: Our results highlight that despite genetic heterogeneity in HLHS, many mutations converge on sequential cellular processes primarily driving cardiac myogenesis, suggesting novel therapeutic approaches.

Increased susceptibility of human endothelial cells to infections by SARS-CoV-2 variants.

Coronavirus disease 2019 (COVID-19) spawned a global health crisis in late 2019 and is caused by the novel coronavirus SARS-CoV-2. SARS-CoV-2 infection can lead to elevated markers of endothelial dysfunction associated with higher risk of mortality. It is unclear whether endothelial dysfunction is caused by direct infection of endothelial cells or is mainly secondary to inflammation. Here, we investigate whether different types of endothelial cells are susceptible to SARS-CoV-2. Human endothelial cells from different vascular beds including umbilical vein endothelial cells, coronary artery endothelial cells (HCAEC), cardiac and lung microvascular endothelial cells, or pulmonary arterial cells were inoculated in vitro with SARS-CoV-2. Viral spike protein was only detected in HCAECs after SARS-CoV-2 infection but not in the other endothelial cells tested. Consistently, only HCAEC expressed the SARS-CoV-2 receptor angiotensin-converting enzyme 2 (ACE2), required for virus infection. Infection with the SARS-CoV-2 variants B.1.1.7, B.1.351, and P.2 resulted in significantly higher levels of viral spike protein. Despite this, no intracellular double-stranded viral RNA was detected and the supernatant did not contain infectious virus. Analysis of the cellular distribution of the spike protein revealed that it co-localized with endosomal calnexin. SARS-CoV-2 infection did induce the ER stress gene EDEM1, which is responsible for clearance of misfolded proteins from the ER. Whereas the wild type of SARS-CoV-2 did not induce cytotoxic or pro-inflammatory effects, the variant B.1.1.7 reduced the HCAEC cell number. Of the different tested endothelial cells, HCAECs showed highest viral uptake but did not promote virus replication. Effects on cell number were only observed after infection with the variant B.1.1.7, suggesting that endothelial protection may be particularly important in patients infected with this variant.

Cardiac-specific deletion of voltage dependent anion channel 2 leads to dilated cardiomyopathy by altering calcium homeostasis.

Voltage dependent anion channel 2 (VDAC2) is an outer mitochondrial membrane porin known to play a significant role in apoptosis and calcium signaling. Abnormalities in calcium homeostasis often leads to electrical and contractile dysfunction and can cause dilated cardiomyopathy and heart failure. However, the specific role of VDAC2 in intracellular calcium dynamics and cardiac function is not well understood. To elucidate the role of VDAC2 in calcium homeostasis, we generated a cardiac ventricular myocyte-specific developmental deletion of Vdac2 in mice. Our results indicate that loss of VDAC2 in the myocardium causes severe impairment in excitation-contraction coupling by altering both intracellular and mitochondrial calcium signaling. We also observed adverse cardiac remodeling which progressed to severe cardiomyopathy and death. Reintroduction of VDAC2 in 6-week-old knock-out mice partially rescued the cardiomyopathy phenotype. Activation of VDAC2 by efsevin increased cardiac contractile force in a mouse model of pressure-overload induced heart failure. In conclusion, our findings demonstrate that VDAC2 plays a crucial role in cardiac function by influencing cellular calcium signaling. Through this unique role in cellular calcium dynamics and excitation-contraction coupling VDAC2 emerges as a plausible therapeutic target for heart failure.

Progressive stretch enhances growth and maturation of 3D stem-cell-derived myocardium.

Bio-engineered myocardium has great potential to substitute damaged myocardium and for studies of myocardial physiology and disease, but structural and functional immaturity still implies limitations. Current protocols of engineered heart tissue (EHT) generation fall short of simulating the conditions of postnatal myocardial growth, which are characterized by tissue expansion and increased mechanical load. To investigate whether these two parameters can improve EHT maturation, we developed a new approach for the generation of cardiac tissues based on biomimetic stimulation under application of continuously increasing stretch. Methods: EHTs were generated by assembling cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CM) at high cell density in a low collagen hydrogel. Maturation and growth of the EHTs were induced in a custom-made biomimetic tissue culture system that provided continuous electrical stimulation and medium agitation along with progressive stretch at four different increments. Tissues were characterized after a three week conditioning period. Results: The highest rate of stretch (S3 = 0.32 mm/day) increased force development by 5.1-fold compared to tissue with a fixed length, reaching contractility of 11.28 mN/mm². Importantly, intensely stretched EHTs developed physiological length-dependencies of active and passive forces (systolic/diastolic ratio = 9.47 ± 0.84), and a positive force-frequency relationship (1.25-fold contractility at 180 min-1). Functional markers of stretch-dependent maturation included enhanced and more rapid Ca2+ transients, higher amplitude and upstroke velocity of action potentials, and pronounced adrenergic responses. Stretch conditioned hiPSC-CMs displayed structural improvements in cellular volume, linear alignment, and sarcomere length (2.19 ± 0.1 µm), and an overall upregulation of genes that are specifically expressed in adult cardiomyocytes. Conclusions: With the intention to simulate postnatal heart development, we have established techniques of tissue assembly and biomimetic culture that avoid tissue shrinkage and yield muscle fibers with contractility and compliance approaching the properties of adult myocardium. This study demonstrates that cultivation under progressive stretch is a feasible way to induce growth and maturation of stem cell-derived myocardium. The novel tissue-engineering approach fulfills important requirements of disease modelling and therapeutic tissue replacement.

SARS-CoV-2 infects and induces cytotoxic effects in human cardiomyocytes.

AIMS: Coronavirus disease 2019 is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and has emerged as a global pandemic. SARS-CoV-2 infection can lead to elevated markers of cardiac injury associated with higher risk of mortality. It is unclear whether cardiac injury is caused by direct infection of cardiomyocytes or is mainly secondary to lung injury and inflammation. Here, we investigate whether cardiomyocytes are permissive for SARS-CoV-2 infection. METHODS AND RESULTS: Two strains of SARS-CoV-2 infected human induced pluripotent stem cell-derived cardiomyocytes as demonstrated by detection of intracellular double-stranded viral RNA and viral spike glycoprotein expression. Increasing concentrations of viral RNA are detected in supernatants of infected cardiomyocytes, which induced infections in Caco-2 cell lines, documenting productive infections. SARS-CoV-2 infection and induced cytotoxic and proapoptotic effects associated with it abolished cardiomyocyte beating. RNA sequencing confirmed a transcriptional response to viral infection as demonstrated by the up-regulation of genes associated with pathways related to viral response and interferon signalling, apoptosis, and reactive oxygen stress. SARS-CoV-2 infection and cardiotoxicity was confirmed in a 3D cardiosphere tissue model. Importantly, viral spike protein and viral particles were detected in living human heart slices after infection with SARS-CoV-2. Coronavirus particles were further observed in cardiomyocytes of a patient with coronavirus disease 2019. Infection of induced pluripotent stem cell-derived cardiomyocytes was dependent on cathepsins and angiotensin-converting enzyme 2, and was blocked by remdesivir. CONCLUSION: This study demonstrates that SARS-CoV-2 infects cardiomyocytes in vitro in an angiotensin-converting enzyme 2- and cathepsin-dependent manner. SARS-CoV-2 infection of cardiomyocytes is inhibited by the antiviral drug remdesivir.

The Degree of t-System Remodeling Predicts Negative Force-Frequency Relationship and Prolonged Relaxation Time in Failing Human Myocardium.

The normally positive cardiac force-frequency relationship (FFR) becomes flat or negative in chronic heart failure (HF). Here we explored if remodeling of the cardiomyocyte transverse tubular system (t-system) is associated with alterations in FFR and contractile kinetics in failing human myocardium. Left-ventricular myocardial slices from 13 failing human hearts were mounted into a biomimetic culture setup. Maximum twitch force (F), 90% contraction duration (CD90), time to peak force (TTP) and time to relaxation (TTR) were determined at 37°C and 0.2-2 Hz pacing frequency. F1 Hz/F0.5 Hz and F2 Hz/F0.5 Hz served as measures of FFR, intracellular cardiomyocyte t-tubule distance (ΔTT) as measure of t-system remodeling. Protein levels of SERCA2, NCX1, and PLB were quantified by immunoblotting. F1 Hz/F0.5 Hz (R 2 = 0.82) and F2 Hz/F0.5 Hz (R 2 = 0.5) correlated negatively with ΔTT, i.e., samples with severe t-system loss exhibited a negative FFR and reduced myocardial wall tension at high pacing rates. PLB levels also predicted F1 Hz/F0.5 Hz, but to a lesser degree (R 2 = 0.49), whereas NCX1 was not correlated (R 2 = 0.02). CD90 correlated positively with ΔTT (R 2 = 0.39) and negatively with SERCA2/PLB (R 2 = 0.42), indicating that both the t-system and SERCA activity are important for contraction kinetics. Surprisingly, ΔTT was not associated with TTP (R 2 = 0) but rather with TTR (R 2 = 0.5). This became even more pronounced when interaction with NCX1 expression was added to the model (R 2 = 0.79), suggesting that t-system loss impairs myocardial relaxation especially when NCX1 expression is low. The degree of t-system remodeling predicts FFR inversion and contraction slowing in failing human myocardium. Moreover, together with NCX, the t-system may be important for myocardial relaxation.

Long-term functional and structural preservation of precision-cut human myocardium under continuous electromechanical stimulation in vitro.

In vitro models incorporating the complexity and function of adult human tissues are highly desired for translational research. Whilst vital slices of human myocardium approach these demands, their rapid degeneration in tissue culture precludes long-term experimentation. Here, we report preservation of structure and performance of human myocardium under conditions of physiological preload, compliance, and continuous excitation. In biomimetic culture, tissue slices prepared from explanted failing human hearts attain a stable state of contractility that can be monitored for up to 4 months or 2000000 beats in vitro. Cultured myocardium undergoes particular alterations in biomechanics, structure, and mRNA expression. The suitability of the model for drug safety evaluation is exemplified by repeated assessment of refractory period that permits sensitive analysis of repolarization impairment induced by the multimodal hERG-inhibitor pentamidine. Biomimetic tissue culture will provide new opportunities to study drug targets, gene functions, and cellular plasticity in adult human myocardium.

Publisher Correction: Long-term functional and structural preservation of precision-cut human myocardium under continuous electromechanical stimulation in vitro.

The original version of this Article incorrectly acknowledged Elisabeth Reiser and Rene Schramm as a corresponding author. This has now been corrected in both the PDF and HTML versions of the Article.

The CYCLOCALYX study: Ovulatory cycle affects circulating compartments of the endothelial glycocalyx in blood.

PROBLEM: The endothelial glycocalyx (EGX) plays an important role in vascular integrity. Recently, increased levels of EGX components were detected in the circulating blood of healthy pregnant women and were related to the increased tendency to edema formation during gestation. However, the EGX has not yet been systematically studied in non-pregnant women during ovulatory cycles. METHOD OF STUDY: Serum levels of EGX components syndecan-1, heparan sulfate, and hyaluronan in healthy women (n = 16) at 3 phases of the ovulatory cycle (early follicular phase, at ovulation, and mid-luteal phase) were compared with a control group of healthy men (n = 10). Using immunofluorescence microscopy in cultured human umbilical vein endothelial cells, the effects of progesterone and estrogen on the EGX were measured. RESULTS: Syndecan-1 increased from 11.1 ± 2.4 ng/mL at ovulation to 12.6 ± 2.3 ng/mL in mid-luteal phase (P = .031) and of heparan sulfate from 663 ± 35 ng/mL to 782 ± 55 ng/mL (P = .011). In contrast to estrogen, there was a detrimental effect of progesterone on the EGX in HUVECs. CONCLUSION: The relationship between the natural menstrual cycle and the EGX as an indicator of vascular permeability may provide a new explanation for premenstrual edema in healthy women. This may be an attendant phenomenon of a regular physiological process, the hormonal downregulation of the vascular barrier during pregnancy.

Diabetes Mellitus-Induced Microvascular Destabilization in the Myocardium.

BACKGROUND: Diabetes mellitus causes microcirculatory rarefaction and may impair the responsiveness of ischemic myocardium to proangiogenic factors. OBJECTIVES: This study sought to determine whether microvascular destabilization affects organ function and therapeutic neovascularization in diabetes mellitus. METHODS: The authors obtained myocardial samples from patients with end-stage heart failure at time of transplant, with or without diabetes mellitus. Diabetic (db) and wild-type (wt) pigs were used to analyze myocardial vascularization and function. Chronic ischemia was induced percutaneously (day 0) in the circumflex artery. At day 28, recombinant adeno-associated virus (rAAV) (5 × 1012 viral particles encoding vascular endothelial growth factor-A [VEGF-A] or thymosin beta 4 [Tß4]) was applied regionally. CD31+ capillaries per high power field (c/hpf) and NG2+ pericyte coverage were analyzed. Global myocardial function (ejection fraction [EF] and left ventricular end-diastolic pressure) was assessed at days 28 and 56. RESULTS: Diabetic human myocardial explants revealed capillary rarefaction and pericyte loss compared to nondiabetic explants. Hyperglycemia in db pigs, even without ischemia, induced capillary rarefaction in the myocardium (163 ± 14 c/hpf in db vs. 234 ± 8 c/hpf in wt hearts; p < 0.005), concomitant with a distinct loss of EF (44.9% vs. 53.4% in nondiabetic controls; p < 0.05). Capillary density further decreased in chronic ischemic hearts, as did EF (both p < 0.05). Treatment with rAAV.Tß4 enhanced capillary density and maturation in db hearts less efficiently than in wt hearts, similar to collateral growth. rAAV.VEGF-A, though stimulating angiogenesis, induced neither pericyte recruitment nor collateral growth. As a result, rAAV.Tß4 but not rAAV.VEGF-A improved EF in db hearts (34.5 ± 1.4%), but less so than in wt hearts (44.8 ± 1.5%). CONCLUSIONS: Diabetes mellitus destabilized microvascular vessels of the heart, affecting the amplitude of therapeutic neovascularization via rAAV.Tß4 in a translational large animal model of hibernating myocardium.

Impaired endothelial shear stress induces podosome assembly via VEGF up-regulation.

Podosomes are dynamic cytoskeletal membrane structures with local adhesive and proteolytic activity. They are critically involved in angiogenesis and vascular adaptive growth. Here, we studied in HUVECs and murine small vessels whether shear stress controls podosome assembly and local proteolytic activity. Podosomes were characterized by immunohistochemistry, and their proteolytic activity was assessed as degradation imprints in fluorescent gelatin that was used as growth substrate. Compared with controls (10 dyn/cm(2)), the number of podosomes formed per time was doubled when cells were exposed to low shear stress (0.3 dyn/cm(2)) or even increased 5-fold under static conditions. This was a result of an enhanced expression of VEGF after reduction of shear stress. Consequently, enhanced podosome formation could be prevented by a VEGF receptor antagonist as well by interruption of VEGF signaling via inhibition of PI3K, Src, or p38. Increase of podosome assembly went along with significantly augmented cell motility. In vivo experiments in mouse arteries confirmed increased endothelial podosome numbers when shear stress was abolished by vessel occlusion. We conclude that shear stress, by reducing VEGF release, inhibits podosome assembly. Hence, endothelial cell-mediated matrix proteolysis and migratory activity are inhibited, thereby stabilizing the structure of the vessel wall.-Fey, T., Schubert, K. M., Schneider, H., Fein, E., Kleinert, E., Pohl, U., Dendorfer, A. Impaired endothelial shear stress induces podosome assembly via VEGF up-regulation.