Tissue-embedded stretchable nanoelectronics reveal endothelial cell-mediated electrical maturation of human 3D cardiac microtissues.

Clinical translation of stem cell therapies for heart disease requires electrical integration of transplanted cardiomyocytes. Generation of electrically matured human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) is critical for electrical integration. Here, we found that hiPSC-derived endothelial cells (hiPSC-ECs) promoted the expression of selected maturation markers in hiPSC-CMs. Using tissue-embedded stretchable mesh nanoelectronics, we achieved a long-term stable map of human three-dimensional (3D) cardiac microtissue electrical activity. The results revealed that hiPSC-ECs accelerated the electrical maturation of hiPSC-CMs in 3D cardiac microtissues. Machine learning-based pseudotime trajectory inference of cardiomyocyte electrical signals further revealed the electrical phenotypic transition path during development. Guided by the electrical recording data, single-cell RNA sequencing identified that hiPSC-ECs promoted cardiomyocyte subpopulations with a more mature phenotype, and multiple ligand-receptor interactions were up-regulated between hiPSC-ECs and hiPSC-CMs, revealing a coordinated multifactorial mechanism of hiPSC-CM electrical maturation. Collectively, these findings show that hiPSC-ECs drive hiPSC-CM electrical maturation via multiple intercellular pathways.

Heart regeneration: 20 years of progress and renewed optimism.

Cardiovascular disease is a leading cause of death worldwide, and thus there remains great interest in regenerative approaches to treat heart failure. In the past 20 years, the field of heart regeneration has entered a renaissance period with remarkable progress in the understanding of endogenous heart regeneration, stem cell differentiation for exogenous cell therapy, and cell-delivery methods. In this review, we highlight how this new understanding can lead to viable strategies for human therapy. For the near term, drugs, electrical and mechanical devices, and heart transplantation will remain mainstays of cardiac therapies, but eventually regenerative therapies based on fundamental regenerative biology may offer more permanent solutions for patients with heart failure.

Senescence mechanisms and targets in the heart.

Cellular senescence is a state of irreversible cell cycle arrest associated with ageing. Senescence of different cardiac cell types can direct the pathophysiology of cardiovascular diseases (CVDs) such as atherosclerosis, myocardial infarction, and cardiac fibrosis. While age-related telomere shortening represents a major cause of replicative senescence, the senescent state can also be induced by oxidative stress, metabolic dysfunction, and epigenetic regulation, among other stressors. It is critical that we understand the molecular pathways that lead to cellular senescence and the consequences of cellular senescence in order to develop new therapeutic approaches to treat CVD. In this review, we discuss molecular mechanisms of cellular senescence, explore how cellular senescence of different cardiac cell types (including cardiomyocytes, cardiac endothelial cells, cardiac fibroblasts, vascular smooth muscle cells, and valve interstitial cells) can lead to CVD, and highlight potential therapeutic approaches that target molecular mechanisms of cellular senescence to prevent or treat CVD.

Mitochondria and metabolic transitions in cardiomyocytes: lessons from development for stem cell-derived cardiomyocytes.

Current methods to differentiate cardiomyocytes from human pluripotent stem cells (PSCs) inadequately recapitulate complete development and result in PSC-derived cardiomyocytes (PSC-CMs) with an immature or fetal-like phenotype. Embryonic and fetal development are highly dynamic periods during which the developing embryo or fetus is exposed to changing nutrient, oxygen, and hormone levels until birth. It is becoming increasingly apparent that these metabolic changes initiate developmental processes to mature cardiomyocytes. Mitochondria are central to these changes, responding to these metabolic changes and transitioning from small, fragmented mitochondria to large organelles capable of producing enough ATP to support the contractile function of the heart. These changes in mitochondria may not simply be a response to cardiomyocyte maturation; the metabolic signals that occur throughout development may actually be central to the maturation process in cardiomyocytes. Here, we review methods to enhance maturation of PSC-CMs and highlight evidence from development indicating the key roles that mitochondria play during cardiomyocyte maturation. We evaluate metabolic transitions that occur during development and how these affect molecular nutrient sensors, discuss how regulation of nutrient sensing pathways affect mitochondrial dynamics and function, and explore how changes in mitochondrial function can affect metabolite production, the cell cycle, and epigenetics to influence maturation of cardiomyocytes.

Pluripotent stem cell-derived cardiomyocytes for treatment of cardiomyopathic damage: Current concepts and future directions.

Today, cell replacement therapy using pluripotent stem cell-derived cardiomyocytes (PSC-CMs) remains a research endeavor, with several hurdles that must be overcome before delivery of PSC-CMs can become a therapeutic reality. In this review, we highlight major findings to date from pre-clinical studies involving delivery of PSC-CMs and consider remaining challenges that must be addressed for successful clinical translation. Our goal is to provide an overview of the current status of cardiomyocyte replacement therapy and what challenges must be addressed before successful clinical translation of such therapies will be possible.

Sustained Activation of AMPK Enhances Differentiation of Human iPSC-Derived Cardiomyocytes via Sirtuin Activation.

Recent studies suggest that metabolic regulation may improve differentiation of cardiomyocytes derived from induced pluripotent stem cells (iPSCs). AMP-activated protein kinase (AMPK) is a master regulator of metabolic activities. We investigated whether AMPK participates in iPSC-derived cardiomyocyte differentiation. We observed that AMPK phosphorylation at Thr172 increased at day 9 but then decreased after day 11 of differentiation to cardiomyocytes. Inhibition of AMPK with compound C significantly reduced mRNA and protein expression of cardiac troponins TNNT2 and TNNI3. Moreover, sustained AMPK activation using AICAR from days 9 to 14 of differentiation increased mRNA and protein expression of both TNNT2 and TNNI3. AICAR decreased acetylation of histone 3 at Lys9 and 56 and histone 4 at Lys16 (known target sites for nuclear-localized sirtuins [SIRT1, SIRT6]), suggesting that AMPK activation enhances sirtuin activity. Sustained AMPK activation during days 9-14 of differentiation induces sirtuin-mediated histone deacetylation and may enhance cardiomyocyte differentiation from iPSCs.

Association of Clinical Rejection Versus Rejection on Protocol Biopsy With Cardiac Allograft Vasculopathy in Pediatric Heart Transplant Recipients.

BACKGROUND: Two or more early rejections (<1 y) or any late acute rejection (>1 y) have been associated with coronary artery vasculopathy (CAV) in pediatric heart transplant (HT) recipients. We hypothesized that clinical rejection defined by concurrent new-onset heart failure or left ventricular systolic dysfunction is more strongly associated with future CAV than rejection diagnosed on protocol biopsy. METHODS: We identified all subjects <21 years old who received first HT at Boston Children's Hospital during 1986-2015 with at least 1 post-HT coronary angiogram. CAV was diagnosed using 2010 International Society for Heart and Lung Transplantation guidelines. Time to CAV diagnosis was assessed using a Cox model with occurrence of clinical rejection analyzed as a time-varying covariate. RESULTS: Of 228 study subjects, 106 remained rejection-free, 77 had rejection diagnosed only on protocol biopsy (≥2R cellular or antibody-mediated), and 45 had a clinical rejection. Subjects with rejection diagnosed only on protocol biopsy were not at higher risk of CAV (hazard ratio [HR] 1.09, 95% confidence interval [CI]: 0.54-2.09). In contrast, clinical rejection was significantly associated with risk of CAV (HR 4.84, 95% CI: 2.99-7.83). Late rejection was associated with a higher risk of CAV (HR 4.27, 95% CI: 2.42-7.51) if it was clinical rejection but not if it was diagnosed on protocol biopsy (HR 0.83, 95% CI: 0.51-1.37). CONCLUSIONS: Clinical rejection poses a far greater risk for future CAV than rejection on protocol biopsy in pediatric HT recipients. Preventing CAV should therefore become the focus of medical management after initial treatment and resolution of clinical rejection.

Inhibition of mTOR Signaling Enhances Maturation of Cardiomyocytes Derived From Human-Induced Pluripotent Stem Cells via p53-Induced Quiescence.

BACKGROUND: Current differentiation protocols to produce cardiomyocytes from human induced pluripotent stem cells (iPSCs) are capable of generating highly pure cardiomyocyte populations as determined by expression of cardiac troponin T. However, these cardiomyocytes remain immature, more closely resembling the fetal state, with a lower maximum contractile force, slower upstroke velocity, and immature mitochondrial function compared with adult cardiomyocytes. Immaturity of iPSC-derived cardiomyocytes may be a significant barrier to clinical translation of cardiomyocyte cell therapies for heart disease. During development, cardiomyocytes undergo a shift from a proliferative state in the fetus to a more mature but quiescent state after birth. The mechanistic target of rapamycin (mTOR)-signaling pathway plays a key role in nutrient sensing and growth. We hypothesized that transient inhibition of the mTOR-signaling pathway could lead cardiomyocytes to a quiescent state and enhance cardiomyocyte maturation. METHODS: Cardiomyocytes were differentiated from 3 human iPSC lines using small molecules to modulate the Wnt pathway. Torin1 (0 to 200 nmol/L) was used to inhibit the mTOR pathway at various time points. We quantified contractile, metabolic, and electrophysiological properties of matured iPSC-derived cardiomyocytes. We utilized the small molecule inhibitor, pifithrin-α, to inhibit p53 signaling, and nutlin-3a, a small molecule inhibitor of MDM2 (mouse double minute 2 homolog) to upregulate and increase activation of p53. RESULTS: Torin1 (200 nmol/L) increased the percentage of quiescent cells (G0 phase) from 24% to 48% compared with vehicle control (P<0.05). Torin1 significantly increased expression of selected sarcomere proteins (including TNNI3 [troponin I, cardiac muscle]) and ion channels (including Kir2.1) in a dose-dependent manner when Torin1 was initiated after onset of cardiomyocyte beating. Torin1-treated cells had an increased relative maximum force of contraction, increased maximum oxygen consumption rate, decreased peak rise time, and increased downstroke velocity. Torin1 treatment increased protein expression of p53, and these effects were inhibited by pifithrin-α. In contrast, nutlin-3a independently upregulated p53, led to an increase in TNNI3 expression and worked synergistically with Torin1 to further increase expression of both p53 and TNNI3. CONCLUSIONS: Transient treatment of human iPSC-derived cardiomyocytes with Torin1 shifts cells to a quiescent state and enhances cardiomyocyte maturity.

Steady-state and regenerative hematopoiesis occurs normally in mice in the absence of GDF11.

Tightly regulated production of mature blood cells is essential for health and survival in vertebrates and dependent on discrete populations of blood-forming (hematopoietic) stem and progenitor cells. Prior studies suggested that inhibition of growth differentiation factor 11 (GDF11) through soluble activin receptor type II (ActRII) ligand traps or neutralizing antibodies promotes erythroid precursor cell maturation and red blood cell formation in contexts of homeostasis and anemia. As Gdf11 is expressed by mature hematopoietic cells, and erythroid precursor cell expression of Gdf11 has been implicated in regulating erythropoiesis, we hypothesized that genetic disruption of Gdf11 in blood cells might perturb normal hematopoiesis or recovery from hematopoietic insult. Contrary to these predictions, we found that deletion of Gdf11 in the hematopoietic lineage in mice does not alter erythropoiesis or erythroid precursor cell frequency under normal conditions or during hematopoietic recovery after irradiation and transplantation. In addition, although hematopoietic cell-derived Gdf11 may contribute to the pool of circulating GDF11 protein during adult homeostasis, loss of Gdf11 specifically in the blood system does not impair hematopoietic stem cell function or induce overt pathological consequences. Taken together, these results reveal that hematopoietic cell-derived Gdf11 is largely dispensable for native and transplant-induced blood formation.

Nanoscale Technologies for Prevention and Treatment of Heart Failure: Challenges and Opportunities.

The adult myocardium has a limited regenerative capacity following heart injury, and the lost cells are primarily replaced by fibrotic scar tissue. Suboptimal efficiency of current clinical therapies to resurrect the infarcted heart results in injured heart enlargement and remodeling to maintain its physiological functions. These remodeling processes ultimately leads to ischemic cardiomyopathy and heart failure (HF). Recent therapeutic approaches (e.g., regenerative and nanomedicine) have shown promise to prevent HF postmyocardial infarction in animal models. However, these preclinical, clinical, and technological advancements have yet to yield substantial enhancements in the survival rate and quality of life of patients with severe ischemic injuries. This could be attributed largely to the considerable gap in knowledge between clinicians and nanobioengineers. Development of highly effective cardiac regenerative therapies requires connecting and coordinating multiple fields, including cardiology, cellular and molecular biology, biochemistry and chemistry, and mechanical and materials sciences, among others. This review is particularly intended to bridge the knowledge gap between cardiologists and regenerative nanomedicine experts. Establishing this multidisciplinary knowledge base may help pave the way for developing novel, safer, and more effective approaches that will enable the medical community to reduce morbidity and mortality in HF patients.

Dysregulation of IL-33/ST2 signaling and myocardial periarteriolar fibrosis.

Microvascular dysfunction in the heart and its association with periarteriolar fibrosis may contribute to the diastolic dysfunction seen in heart failure with preserved ejection fraction. Interleukin-33 (IL-33) prevents global myocardial fibrosis in a pressure overloaded left ventricle by acting via its receptor, ST2 (encoded by the gene, Il1rl1); however, whether this cytokine can also modulate periarteriolar fibrosis remains unclear. We utilized two approaches to explore the role of IL-33/ST2 in periarteriolar fibrosis. First, we studied young and old wild type mice to test the hypothesis that IL-33 and ST2 expression change with age. Second, we produced pressure overload in mice deficient in IL-33 or ST2 by transverse aortic constriction (TAC). With age, IL-33 expression increased and ST2 expression decreased. These alterations accompanied increased periarteriolar fibrosis in aged mice. Mice deficient in ST2 but not IL-33 had a significant increase in periarteriolar fibrosis following TAC compared to wild type mice. Thus, loss of ST2 signaling rather than changes in IL-33 expression may contribute to periarteriolar fibrosis during aging or pressure overload, but manipulating this pathway alone may not prevent or reverse fibrosis.

Is Myocarditis an Independent Risk Factor for Post-Transplant Mortality in Pediatric Heart Transplant Recipients?

BACKGROUND: Previous studies suggest that children with myocarditis who receive heart transplantation (HT) may be at higher risk of post-transplant mortality compared with children who are transplanted for idiopathic dilated cardiomyopathy. We hypothesized that these differences are because of more severe heart failure at HT in children with myocarditis. METHODS AND RESULTS: We identified 221 children with myocarditis and 1583 with idiopathic dilated cardiomyopathy who were <18 years old and listed for HT in the United States between July 2004 and December 2013 using the Organ Procurement and Transplant Network database. We compared baseline characteristics at listing and at HT and used Cox models to determine whether myocarditis is independently associated with wait-list mortality (or becoming too sick to transplant) or post-transplant graft loss (death/re-HT). Children with myocarditis were more likely to be listed while on assisted ventilation, mechanical circulatory support and with renal dysfunction. Overall, 137 children with myocarditis and 1249 with idiopathic dilated cardiomyopathy received HT. In unadjusted analysis, children with myocarditis were at higher risk of wait-list mortality (hazard ratio 2.1; 95% confidence interval 1.5-3.0) and showed a trend toward increased risk of post-transplant graft loss (hazard ratio 1.4; 95% confidence interval 1.0-2.2). However, in adjusted analysis, myocarditis was not associated with wait-list mortality (hazard ratio 1.3, 95% confidence interval 0.9-1.9) or post-transplant graft loss (hazard ratio 1.3, 95% confidence interval 0.9-2.0). CONCLUSIONS: Among children listed for HT, those with myocarditis have more severe heart failure than children with idiopathic dilated cardiomyopathy. After adjustment for severity of illness, myocarditis does not confer additional risk for wait-list or post-transplant mortality.

Cardiac stem cell therapy and the promise of heart regeneration.

Stem cell therapy for cardiac disease is an exciting but highly controversial research area. Strategies such as cell transplantation and reprogramming have demonstrated both intriguing and sobering results. Yet as clinical trials proceed, our incomplete understanding of stem cell behavior is made evident by numerous unresolved matters, such as the mechanisms of cardiomyocyte turnover or the optimal therapeutic strategies to achieve clinical efficacy. In this Perspective, we consider how cardiac stem cell biology has led us into clinical trials, and we suggest that achieving true cardiac regeneration in patients may ultimately require resolution of critical controversies in experimental cardiac regeneration.

Model systems for cardiovascular regenerative biology.

There is an urgent clinical need to develop new therapeutic approaches to treat heart failure, but the biology of cardiovascular regeneration is complex. Model systems are required to advance our understanding of biological mechanisms of cardiac regeneration as well as to test therapeutic approaches to regenerate tissue and restore cardiac function following injury. An ideal model system should be inexpensive, easily manipulated, easily reproducible, physiologically representative of human disease, and ethically sound. In this review, we discuss computational, cell-based, tissue, and animal models that have been used to elucidate mechanisms of cardiovascular regenerative biology or to test proposed therapeutic methods to restore cardiac function following disease or injury.

Delivery of basic fibroblast growth factor with a pH-responsive, injectable hydrogel to improve angiogenesis in infarcted myocardium.

A pH- and temperature-responsive, injectable hydrogel has been designed to take advantage of the acidic microenvironment of ischemic myocardium. This system can improve therapeutic angiogenesis methods by providing spatio-temporal control of angiogenic growth factor delivery. The pH- and temperature-responsive random copolymer, poly(N-isopropylacrylamide-co-propylacrylic acid-co-butyl acrylate) (p[NIPAAm-co-PAA-co-BA]), was synthesized by reversible addition fragmentation chain transfer polymerization. This polymer was a liquid at pH 7.4 and 37 °C but formed a physical gel at pH 6.8 and 37 °C. Retention of biotinylated basic fibroblast growth factor (bFGF) between 0 and 7 days after injection into infarcted rat myocardium was 10-fold higher with hydrogel delivery versus saline. Following 28 days of treatment in vivo, capillary and arteriolar densities were increased 30-40% by polymer + bFGF treatment versus saline + bFGF or polymer-only controls. Treatment with polymer + bFGF for 28 days resulted in a 2-fold improvement in relative blood flow to the infarct region versus day 0, whereas saline + bFGF or polymer-only had no effect. Fractional shortening determined by echocardiography was significantly higher following treatment with polymer + bFGF (30 ± 1.4%) versus saline (25 ± 1.2%) and polymer alone (25 ± 1.8%). By responding to local changes in pH- and temperature in an animal model of ischemia, this hydrogel system provided sustained, local delivery of bFGF, improved angiogenesis, and achieved therapeutic effects in regional blood flow and cardiac function.

Injectable pH- and temperature-responsive poly(N-isopropylacrylamide-co-propylacrylic acid) copolymers for delivery of angiogenic growth factors.

A new sharply pH- and temperature-responsive hydrogel system was designed for delivering drugs to regions of local acidosis, as found in wound healing, tumor sites, or sites of ischemia. The reversible addition-fragmentation chain transfer (RAFT) polymerization technique was used to synthesize copolymers of N-isopropylacrylamide (NIPAAM) and propylacrylic acid (PAA) with feed ratios of PAA between 0 and 20 mol %. The pH-responsive viscoelastic properties of these materials as a function of pH and temperature were quantified by rheometry. At physiologic pH (7.4) and 5 wt %, the polymer did not form gels but rather remained soluble at temperatures as high as 50 degrees C. At lower pH values (pH ca. 5.5 and below), the polymer was liquid at 20 degrees C, but exhibited a sol-gel phase transformation with increasing temperature and existed as a physical gel at 37 degrees C. Incorporation of the hydrophobic monomer, butyl acrylate, into the random copolymer raised the pH of gel formation to greater than 6.0 at 37 degrees C. Drug loading studies demonstrated that p(NIPAAm-co-PAA) hydrogels are able to maintain the bioactivity of basic fibroblast growth factor following storage in hydrogel for 40 h and can provide sustained pH-dependent release of vascular endothelial growth factor over a period of at least three weeks. This hydrogel system will thus gel at controllable acidic pH values upon injection, and is designed to undergo gradual dissolution as it performs its drug delivery function and the ischemic site returns to physiological pH.

Vascular oxidative stress precedes high blood pressure in spontaneously hypertensive rats.

This study examines whether longitudinal antioxidant treatment initiated in prehypertensive spontaneously hypertensive rats (SHR) can attenuate vascular oxidant stress and prevent blood pressure elevation during development. Male SHR and age-matched Wistar-Kyoto rats (WKY) were treated from 6 to 11 weeks of age with Tempol (4-hydroxy-2,2,6,6-tetramethylpiperidinoxyl) (1 mmol/l in drinking water), a membrane-permeable superoxide dismutase mimetic. Mean systolic blood pressures (SBPs) were measured by tail-cuff Agonist-induced and basal O2- production was measured in thoracic aortas of 6- and 11-week-old SHR and WKY by lucigenin-derived chemiluminescence and oxidative fluorescent microscopy, respectively. SBP of 6-week-old SHR (131 +/- 5 mmHg) and WKY (130 +/- 4 mmHg) were not different; however, 11-week-old SHR SBP (171 +/- 4 mmHg) was significantly greater (p = .0001) than 11-week-old WKY SBP (143 +/- 5 mmHg). Tempol treatment completely, but reversibly, prevented this age-related rise in SHR SBP (SHR + Tempol: 137 +/- 4 mmHg; p < .0001 versus untreated SHR). Agonist-induced vascular O2- was increased in 6- (p = .03) and 11-week-old SHR (p < .0001) and 11-week-old WKY (p = .03) but not in 6-week-old WKY. Long-term Tempol treatment significantly lowered O2- production in both strains. Basal O2- measurements in both 6- and 11-week-old SHR were qualitatively increased compared with age-matched WKY; this increase in SHR was inhibited with in vitro Tempol treatment. These data show that antioxidant treatment to reduce oxidative stress prevents the age-related development of high blood pressure in an animal model of genetic hypertension.

Locally enhanced angiogenesis promotes transplanted cell survival.

A developing therapy for complete or partial loss of function in various tissues and organs involves transplanting an appropriate cell population, capable of compensating for the existing deficiencies. Clinical application of this type of strategy is currently limited by the death or dedifferentiation of the transplanted cells after delivery to the recipient. A delay in thorough vascularization of the implant area creates an environment low in oxygen and other nutrients, and likely contributes to the initial death of transplanted cells. We have addressed this problem by sustained delivery of vascular endothelial growth factor (VEGF), an initiator of angiogenesis, from a porous polymer matrix utilized simultaneously for cell delivery. As expected from previous studies, VEGF delivered from these constructs elicited an enhanced angiogenic response over a 2-week period when implanted subcutaneously in SCID mice. Hepatocytes implanted using VEGF-containing matrices demonstrated significantly greater survival after 1 week in vivo as compared with cells implanted on matrices without growth factor. The results of this study therefore indicate that enhancing vascularization in the location of transplanted cells promotes their survival. In addition, this delivery system may be used in future studies to directly promote cell survival and function by also providing growth factors specific to the transplanted cells.