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Rodent diabetic models, used to understand the pathophysiology of diabetic cardiomyopathy (DCM), remain several limitations. Engineered cardiac tissues (ECTs) have emerged as robust 3D in vitro models to investigate structure–function relationships as well as cardiac injury and repair. Advanced glycation end-products (AGEs), produced through glycation of proteins or lipids in response to hyperglycemia, are important pathogenic factor for the development of DCM. In the current study, we developed a murine-based ECT model to investigate cardiac injury produced by AGEs. We treated ECTs composed of neonatal murine cardiac cells with AGEs and observed AGE-related functional, cellular, and molecular alterations: (1) AGEs (150 µg/mL) did not cause acute cytotoxicity, which displayed as necrosis detected by medium LDH release or apoptosis detected by cleaved caspase 3 and TUNEL staining, but negatively impacted ECT function on treatment day 9; (2) AGEs treatment significantly increased the markers of fibrosis (TGF-β, α-SMA, Ctgf, Collagen I-α1, Collagen III-α1, and Fn1) and hypertrophy (Nppa and Myh7); (3) AGEs treatment significantly increased ECT oxidative stress markers (3-NT, 4-HNE, HO-1, CAT, and SOD2) and inflammation response markers (PAI-1, TNF-α, NF-κB, and ICAM-1); and (4) AGE-induced pathogenic responses were all attenuated by pre-application of AGE receptor antagonist FPS-ZM1 (20 µM) or the antioxidant glutathione precursor N-acetylcysteine (5 mM). Therefore, AGEs-treated murine ECTs recapitulate the key features of DCM’s functional, cellular and molecular pathogenesis, and may serve as a robust in vitro model to investigate cellular structure-function relationships, signaling pathways relevant to DCM and pharmaceutical intervention strategies.

Cadmium (Cd) is an important environmental pollutant and long-term Cd exposure is closely related to autoimmune diseases, cancer, cardiovascular diseases (CVD), and hepatic dysfunction. Zinc (Zn) is an essential metal that plays key roles in protein structure, catalysis, and regulation of their function. Numerous studies have shown that Zn can reduce Cd toxicity; however, the underlying mechanisms have not been extensively explored. Preclinical studies have revealed direct competition for sarcolemmal uptake between these two metals. Multiple sarcolemmal transporters participate in Cd uptake, including Zn transporters, calcium channels, and DMT1 (divalent metal transporter 1). Zn also induces several protective mechanisms, including MT (metallothionein) induction and favorable redox homeostasis. This review summarizes current knowledge related to the role of Zn and metal transporters in reducing Cd toxicity and discusses potential future directions of related research.

The pathogenesis and clinical features of diabetic cardiomyopathy have been well-studied in the past decade, but effective approaches to prevent and treat this disease are limited. Diabetic cardiomyopathy occurs as a result of the dysregulated glucose and lipid metabolism associated with diabetes mellitus, which leads to increased oxidative stress and the activation of multiple inflammatory pathways that mediate cellular and extracellular injury, pathological cardiac remodelling, and diastolic and systolic dysfunction. Preclinical studies in animal models of diabetes have identified multiple intracellular pathways involved in the pathogenesis of diabetic cardiomyopathy and potential cardioprotective strategies to prevent and treat the disease, including antifibrotic agents, anti-inflammatory agents and antioxidants. Some of these interventions have been tested in clinical trials and have shown favourable initial results. In this Review, we discuss the mechanisms underlying the development of diabetic cardiomyopathy and heart failure in type 1 and type 2 diabetes mellitus, and we summarize the evidence from preclinical and clinical studies that might provide guidance for the development of targeted strategies. We also highlight some of the novel pharmacological therapeutic strategies for the treatment and prevention of diabetic cardiomyopathy.

Cadmium (Cd) is a nonessential heavy metal and a prevalent environmental toxin that has been shown to induce significant cardiomyocyte apoptosis in neonatal murine engineered cardiac tissues (ECTs). In contrast, zinc (Zn) is a potent metallothionein (MT) inducer, which plays an important role in protection against Cd toxicity. In this study, we investigated the protective effects of Zn against Cd toxicity in ECTs and explore the underlying mechanisms. ECTs were constructed from neonatal ventricular cells of wild-type (WT) mice and mice with global MT gene deletion (MT-KO). In WT-ECTs, Cd (5−20 μM) caused a dose-dependent toxicity that was detected within 8 h evidenced by suppressed beating, apoptosis, and LDH release; Zn (50−200 μM) dose-dependently induced MT expression in ECTs without causing ECT toxicity; co-treatment of ECT with Zn (50 µM) prevented Cd-induced toxicity. In MT-KO ECTs, Cd toxicity was enhanced; but unexpectedly, cotreatment with Zn provided partial protection against Cd toxicity. Furthermore, Cd, but not Zn, significantly activated Nrf2 and its downstream targets, including HO-1; inhibition of HO-1 by a specific HO-1 inhibitor, ZnPP (10 µM), significantly increased Cd-induced toxicity, but did not inhibit Zn protection against Cd injury, suggesting that Nrf2-mediated HO-1 activation was not required for Zn protective effect. Finally, the ability of Zn to reduce Cd uptake provided an additional MT-independent mechanism for reducing Cd toxicity. Thus, Zn exerts protective effects against Cd toxicity for murine ECTs that are partially MT-mediated. Further studies are required to translate these findings towards clinical trials.

Little is known about the effectiveness of treprostinil in higher-risk paediatric patients with various pulmonary arterial hypertension genotypes. This study was designed to investigate the prognosis of higher-risk paediatric patients with idiopathic or heritable pulmonary arterial hypertension (IPAH/HPAH) after treprostinil therapy.

Compression of left main coronary artery is a severe complication in PAH and the treatment strategy is tricky and should be individualized. We presented a patient with large atrial septal defect (ASD) and non-Eisenmenger pulmonary arterial hypertension (PAH). Right heart catheterization (RHC) showed the defect was non-correctable due to a pulmonary vascular resistance (PVR) of 9.49 Wood Units. EKG suggested myocardial ischemia and coronary angiography and coronary CTA confirmed proximal left main coronary artery was compressed by extremely dilated pulmonary arterial trunk, which is named left main coronary compression syndrome (LMCS). Percutaneous coronary intervention (PCI) using drug-eluting stent was performed and combined PAH-specific medications was prescribed simultaneously. At 6-month following the PCI procedure, PVR had decreased to 3.43 Wood Units, and percutaneous ASD closure procedure was performed. A 6-month follow-up the patient was asymptomatic and had significantly improved pulmonary hemodynamics. LMCS is rare among patients with ASD-PAH. PCI combined with PAH-specific medications allowed the closure of ASD, resulting in clinical improvement.

Background: Although transthoracic echocardiography (TTE) routinely establishes the diagnosis of double outlet right ventricle (DORV), it can be suboptimal for depicting exact ventricular septal defect (VSD) position, especially with respect to the outflow tracts. Advanced imaging with computed tomography angiography (CTA) can help visualize structures and relationships not easily seen by echo. Using computer-aided design, we have the ability to create three-dimensional (3D) models of the intracardiac anatomy, which can be helpful for better depicting the overall anatomy to assist surgical planning.Methods: Patients with a diagnosis of DORV were retrospectively reviewed at our institution from October 2013 to April 2015. Patients who preoperatively underwent both TTE and CTA with 3D reconstruction of the intracardiac anatomy were included. Computed tomography angiography findings with 3D intracardiac model creation were compared to the surgical findings.Results: Twenty-five patients underwent surgical repair of DORV during the study period. Five patients had CTA with 3D reconstruction, in addition to the standard TTE images, and were included in the study. In all five cases, CTA with 3D reconstruction of the intracardiac anatomy accurately depicted the VSD position relative to important adjacent structures, including the outflow tracts.Conclusion: Three-dimensional reconstruction of the intracardiac anatomy using CTA data can provide accurate data for presurgical planning of DORV repair and has the potential for being especially useful in patients for whom intracardiac anatomy and VSD position cannot be well seen by TTE. A larger prospective analysis is warranted to help validate this approach.

Despite many improvements in the field of renal transplantation, the key problem that persists is the lack of organs for all the patients who need kidneys. This problem continues despite the addition of extended criteria donors and donation after cardiac death. Compounding this issue is the high discard rate and there are no good means to truly predict renal function using current pretransplantation testing parameters. In an isolated renal perfusion model using porcine kidneys, we tested the proof of principle that a Vevo 2100 high-frequency high-resolution ultrasound system (Fujifilm VisualSonics, Inc., Toronto, Canada) could assess renal parenchymal perfusion and flow in the central renal vessels which could not assess by conventional ultrasound. Images and velocities were easily obtained during these studies. High-frequency ultrasound imaging may be a feasible and reproducible method for assessing renal parenchymal integrity and function pretransplantation. Further studies are required to determine the sensitivity and specificity of this approach in comparison with traditional renal biopsy pretransplantation with the goal of increasing the identification and use of donated kidneys for transplantation.

The goal of cellular cardiomyoplasty is to replace damaged myocardium by healthy myocardium achieved by host myocardial regeneration and/or transplantation of donor cardiomyocytes (CMs). In the case of CM transplantation, studies suggest that immature CMs may be the optimal cell type to survive and functionally integrate into damaged myocardium. In the present study, we tested the hypothesis that active proliferation of immature CMs contributes graft survival and functional recovery of recipient myocardium. We constructed engineered cardiac tissue from gestational day 14 rat fetal cardiac cells (EFCT) or day 3 neonatal cardiac cells (ENCT). Culture day 7 EFCTs or ENCTs were implanted onto the postinfarct adult left ventricle (LV). CM proliferation rate of EFCT was significantly higher than that of ENCT at 3 days and 8 weeks after the graft implantation, whereas CM apoptosis rate remained the same in both groups. Echocardiogram showed that ENCT implantation sustained LV contraction, whereas EFCT implantation significantly increased the LV contraction at 8 weeks versus sham group (p< 0.05, analysis of variance). These results suggest that active CM proliferation may play a critical role in immature donor CM survival and the functional recovery of damaged recipient myocardium.

The relative contributions of inflammation and ischemia to the pathogenesis of periventricular leukomalacia (PVL) have not been elucidated. We hypothesized that fetal cardiovascular function and cerebral blood flow velocity (BFV) would be decreased in a rat model of chorioamnionitis. We also tested whether placental inflammation was related to proximity to the cervix in our model of chorioamnionitis intracervical lipopolysaccharide (LPS) or vehicle (PBS) injection. On embryonic d 15, Sprague-Dawley rats underwent baseline maternal and fetal echocardiography, followed by LPS or PBS injection, then serial echocardiographic evaluations until embryonic day (ED) 21. One hour after birth, pups had middle cerebral artery (MCA) BFV measured. Placentas of LPS-exposed pups exhibited uniform, higher inflammation grades (p< 0.001). All fetal BFVs increased with advancing GA (p< 0.001) whereas resistance index (RI) decreased (p< 0.001). There was no difference in fetal BFV between the groups other than a reduced gestation-related increase in descending aorta BFV in LPS-exposed rats (p< 0.05). Newborn pups exposed to LPS had lower heart rate (p0.006) and MCA BFV (p0.024) and higher RI (p0.003) and pulsatility index (PI; p0.004). In conclusion, intracervical LPS injection produces an inflammation independent of placental position, a blunted increase in gestation-related aortic BFV, and a decrease in MCA BFV in newborn pups.

The relative contributions of inflammation and ischemia to the pathogenesis of periventricular leukomalacia (PVL) have not been elucidated. We hypothesized that fetal cardiovascular function and cerebral blood flow velocity (BFV) would be decreased in a rat model of chorioamnionitis. We also tested whether placental inflammation was related to proximity to the cervix in our model of chorioamnionitis [intracervical lipopolysaccharide (LPS) or vehicle (PBS) injection]. On embryonic d 15, Sprague-Dawley rats underwent baseline maternal and fetal echocardiography, followed by LPS or PBS injection, then serial echocardiographic evaluations until embryonic day (ED) 21. One hour after birth, pups had middle cerebral artery (MCA) BFV measured. Placentas of LPS-exposed pups exhibited uniform, higher inflammation grades (p < 0.001). All fetal BFVs increased with advancing GA (p < 0.001) whereas resistance index (RI) decreased (p < 0.001). There was no difference in fetal BFV between the groups other than a reduced gestation-related increase in descending aorta BFV in LPS-exposed rats (p < 0.05). Newborn pups exposed to LPS had lower heart rate (p = 0.006) and MCA BFV (p = 0.024) and higher RI (p = 0.003) and pulsatility index (PI; p = 0.004). In conclusion, intracervical LPS injection produces an inflammation independent of placental position, a blunted increase in gestation-related aortic BFV, and a decrease in MCA BFV in newborn pups.

Skeletal muscle–derived stem cells (MDSCs) are able to differentiate into cardiomyocytes (CMs). However, it remains to be investigated whether differentiated CMs contract similar to native CMs. Here, we developed a three-dimensional collagen gel bioreactor (3DGB) that induces a working CM phenotype from MDSCs, and the contractile properties are directly measured as an engineered cardiac tissue. Neonate rat MDSCs were isolated from hind-leg muscles via the preplate technique. Isolated MDSCs were approximately 60% positive to Sca-1 and negative to CD34, CD45, or c-kit antigens. We sorted Sca-1(−) MDSCs and constructed MDSC-3DGBs by mixing MDSCs with acid soluble rat tail collagen type-I and matrix factors. MDSC-3DGB exhibited spontaneous cyclic contraction by culture day 7. MDSC-3DGB expressed cardiac-specific genes and proteins. Histological assessment revealed that cardiac-specific troponin-T and -I expressed in a typical striation pattern and connexin-43 was expressed similar to the native fetal ventricular papillary muscle. β-Adrenergic stimulation increased MDSC-3DGB spontaneous beat frequency. MDSC-3DGB generated contractile force and intracellular calcium ion transients similar to engineered cardiac tissue from native cardiac cells. Results suggest that MDSC-3DGB induces a working CM phenotype in MDSCs and is a useful 3D culture system to directly assess the contractile properties of differentiated CMs in vitro.

We have previously shown that populations of skeletal muscle-derived stem cells (MDSCs) exhibit sex-based differences for skeletal muscle and bone repair, with female cells demonstrating superior engrafting abilities to males in skeletal muscle while male cells differentiating more robustly toward the osteogenic and chondrogenic lineages. In this study, we tested the hypothesis that the therapeutic capacity of MDSCs transplanted into myocardium is influenced by sex of donor MDSCs or recipient. Male and female MDSCs isolated from the skeletal muscle of 3-week-old mice were transplanted into recipient male or female dystrophin-deficient (mdx) hearts or into the hearts of male SCID mice following acute myocardial infarction. In the mdxmodel, no difference was seen in engraftment or blood vessel formation based on donor cell or recipient sex. In the infarction model, MDSC-transplanted hearts showed higher postinfarction angiogenesis, less myocardial scar formation, and improved cardiac function compared to vehicle controls. However, sex of donor MDSCs had no significant effects on engraftment, angiogenesis, and cardiac function. VEGF expression, a potent angiogenic factor, was similar between male and female MDSCs. Our results suggest that donor MDSC or recipient sex has no significant effect on the efficiency of MDSC-triggered myocardial engraftment or regeneration following cardiac injury. The ability of the MDSCs to improve cardiac regeneration and repair through promotion of angiogenesis without differentiation into the cardiac lineage may have contributed to the lack of sex difference observed in these models.

Cardiomyocyte (CM) transplantation is one therapeutic option for cardiac repair. Studies suggest that fetal CMs display the best cell type for cardiac repair, which can finitely proliferate, integrate with injured host myocardium, and restore cardiac function. We have recently developed an engineered early embryonic cardiac tissue (EEECT) using embryonic cardiac cells and have shown that EEECT contractile properties and cellular proliferative response to cyclic mechanical stretch stimulation mimic developing fetal myocardium. However, it remains unknown whether cyclic mechanical stretch–mediated high cellular proliferation activity within EEECT reflects CM or non-CM population. Studies have shown that p38-mitogen-activated protein kinase (p38MAPK) plays an important role in both cyclic mechanical stretch stimulation and cellular proliferation. Therefore, in the present study, we tested the hypothesis that cyclic mechanical stretch (0.5 Hz, 5% strain for 48 h) specifically increases EEECT CM proliferation mediated by p38MAPK activity. Cyclic mechanical stretch increased CM, but not non-CM, proliferation and increased p38MAPK phosphorylation. Treatment of EEECT with the p38MAPK inhibitor, SB202190, reduced CM proliferation. The negative CM proliferation effects of SB202190 were not reversed by concurrent stretch stimulation. Results suggest that immature CM proliferation within EEECT can be positively regulated by mechanical stretch and negatively regulated by p38MAPK inhibition.

Stem cells contained in the amniotic membrane may be useful for cellular repair of the damaged heart. Previously, we showed that amnion-derived cells (ADCs) express embryonic stem cell surface markers and pluripotent stem cell-specific transcription factor genes. These ADCs also possess the potential for mesoderm (cardiac) lineage differentiation. In the present study we investigated whether untreated naive ADC transplantation into the injured left ventricular (LV) myocardium is beneficial as a cell-based cardiac repair strategy in a rat model. ADCs were isolated from Lewis rat embryonic day 14 amniotic membranes. FACS analysis revealed that freshly isolated ADCs contained stage-specific embryonic antigen-1 (SSEA-1), Oct-4-positive cells, and mesenchymal stromal cells, while hematopoietic stem cell marker positive cells were absent. Reverse transcription-PCR revealed that naive ADCs expressed cardiac and vascular specific genes. We injected freshly isolated ADCs (2 × 106cells suspended in PBS, ADC group) into acutely infarcted LV myocardium produced by proximal left coronary ligation. PBS was injected in postinfarction controls (PBS group). Cardiac function was assessed at 2 and 6 weeks after injection. ADC treatment attenuated LV dilatation and sustained LV contractile function at 2 and 6 weeks in comparison to PBS controls (p< 0.05, ANOVA). LV peak systolic pressure and maximum dP/dt of ADC-treated heart were higher and LV end-diastolic pressure and negative dP/dt were lower than in PBS controls (p< 0.05). Histological assessment revealed that infarcted myocardium of the ADC-treated group had less fibrosis, thicker ventricular walls, and increased capillary density (p< 0.05). The fate of injected ADCs was confirmed using ADCs derived from EGFP(+) transgenic rats. Immunohistochemistry at 6 weeks revealed that EGFP(+) cells colocalized with von Willebrand factor, α-smooth muscle actin, or cardiac troponin-I. Our results suggest that naive ADCs are a potential cell source for cellular cardiomyoplasty.

Stem cells are classically defined by their multipotent, long-term proliferation, and self-renewal capabilities. Here, we show that increased antioxidant capacity represents an additional functional characteristic of muscle-derived stem cells (MDSCs). Seeking to understand the superior regenerative capacity of MDSCs compared with myoblasts in cardiac and skeletal muscle transplantation, our group hypothesized that survival of the oxidative and inflammatory stress inherent to transplantation may play an important role. Evidence of increased enzymatic and nonenzymatic antioxidant capacity of MDSCs were observed in terms of higher levels of superoxide dismutase and glutathione, which appears to confer a differentiation and survival advantage. Further when glutathione levels of the MDSCs are lowered to that of myoblasts, the transplantation advantage of MDSCs over myoblasts is lost when transplanted into both skeletal and cardiac muscles. These findings elucidate an important cause for the superior regenerative capacity of MDSCs, and provide functional evidence for the emerging role of antioxidant capacity as a critical property for MDSC survival post-transplantation.

Stem cells are classically defined by their multipotent, long-term proliferation, and self-renewal capabilities. Here, we show that increased antioxidant capacity represents an additional functional characteristic of muscle-derived stem cells (MDSCs). Seeking to understand the superior regenerative capacity of MDSCs compared with myoblasts in cardiac and skeletal muscle transplantation, our group hypothesized that survival of the oxidative and inflammatory stress inherent to transplantation may play an important role. Evidence of increased enzymatic and nonenzymatic antioxidant capacity of MDSCs were observed in terms of higher levels of superoxide dismutase and glutathione, which appears to confer a differentiation and survival advantage. Further when glutathione levels of the MDSCs are lowered to that of myoblasts, the transplantation advantage of MDSCs over myoblasts is lost when transplanted into both skeletal and cardiac muscles. These findings elucidate an important cause for the superior regenerative capacity of MDSCs, and provide functional evidence for the emerging role of antioxidant capacity as a critical property for MDSC survival post-transplantation.

Newborn animals are more resistant to anoxia than older animals, partly due to an increased tolerance of the immature heart to anoxia. Newborn animals also have a more robust preterminal gasp. We investigated the relationship between gasping and cardiac function in immature and maturing rats exposed to anoxia. Immature postnatal day 7 (PND7) rats (n13) and maturing PND17 rats (n13) were exposed to 100% nitrogen (anoxia) for 10 min. Echocardiography was used to calculate cardiac contractility (CC) by left ventricular shortening fraction and cardiac output (CO) from Doppler velocity recordings of pulmonary artery blood flow. In a separate group of PND7 rats, CC and CO were recorded after the paralytic agent pancuronium was used to prevent gasping. Anoxia decreased CC and CO in PND7 and PND17 rats, followed by a partial and transient recovery. Gasping preceded recovery of CO and was required to sustain CO. Gasping in PND7 rats lasted longer (541 s versus351 s, p< 0.01) and resulted in a greater recovery of CC and CO. Anoxia-induced gasping and the associated recovery of cardiac function were abolished by paralysis. Thus, anoxia-induced gasping transiently improves cardiac function, and more robust gasping in immature rats is associated with increased cardiac anoxic tolerance.

Newborn animals are more resistant to anoxia than older animals, partly due to an increased tolerance of the immature heart to anoxia. Newborn animals also have a more robust preterminal gasp. We investigated the relationship between gasping and cardiac function in immature and maturing rats exposed to anoxia. Immature postnatal day 7 (PND7) rats (n = 13) and maturing PND17 rats (n = 13) were exposed to 100% nitrogen (anoxia) for 10 min. Echocardiography was used to calculate cardiac contractility (CC) by left ventricular shortening fraction and cardiac output (CO) from Doppler velocity recordings of pulmonary artery blood flow. In a separate group of PND7 rats, CC and CO were recorded after the paralytic agent pancuronium was used to prevent gasping. Anoxia decreased CC and CO in PND7 and PND17 rats, followed by a partial and transient recovery. Gasping preceded recovery of CO and was required to sustain CO. Gasping in PND7 rats lasted longer (541 s versus 351 s, p < 0.01) and resulted in a greater recovery of CC and CO. Anoxia-induced gasping and the associated recovery of cardiac function were abolished by paralysis. Thus, anoxia-induced gasping transiently improves cardiac function, and more robust gasping in immature rats is associated with increased cardiac anoxic tolerance.

Oxygen tension is a critical factor for appropriate embryonic and fetal development. Chronic hypoxia exposure alters cardiovascular (CV) function and structure in the late fetus and newborn, yet the immature myocardium is considered to be less sensitive to hypoxia than the mature heart. We tested the hypothesis that hypoxia during the period of primary CV morphogenesis impairs immature embryonic CV function and embryo growth. We incubated fertile white Leghorn chick embryos in 15% oxygen (hypoxia) or 21% oxygen (control) until Hamburger-Hamilton stage 21 (3.5 d). We assessed in ovo viability and dysmorphic features and then measured ventricular pressure and dimensions and dorsal aortic arterial impedance at stage 21. Chronic hypoxia decreased viability and embryonic wet weight. Chronic hypoxia did not alter heart rate or the ventricular diastolic indices of end-diastolic pressure, maximum ventricular –dP/dt, or tau. Chronic hypoxia decreased maximum ventricular +dP/dt and peak pressure, increased ventricular end-systolic volume, and decreased ventricular ejection fraction, consistent with depressed systolic function. Arterial afterload (peripheral resistance) increased and both dorsal aortic SV and steady-state hydraulic power decreased in response to hypoxia. Thus, reduced oxygen tension during early cardiac development depresses ventricular function, increases ventricular impedance (afterload), delays growth, and decreases embryo survival, suggesting that a critical threshold of oxygen tension is required to support morphogenesis and cardiovascular function in the early embryo.

Oxygen tension is a critical factor for appropriate embryonic and fetal development. Chronic hypoxia exposure alters cardiovascular (CV) function and structure in the late fetus and newborn, yet the immature myocardium is considered to be less sensitive to hypoxia than the mature heart. We tested the hypothesis that hypoxia during the period of primary CV morphogenesis impairs immature embryonic CV function and embryo growth. We incubated fertile white Leghorn chick embryos in 15% oxygen (hypoxia) or 21% oxygen (control) until Hamburger-Hamilton stage 21 (3.5 d). We assessed in ovoviability and dysmorphic features and then measured ventricular pressure and dimensions and dorsal aortic arterial impedance at stage 21. Chronic hypoxia decreased viability and embryonic wet weight. Chronic hypoxia did not alter heart rate or the ventricular diastolic indices of end-diastolic pressure, maximum ventricular –dP/dt, or tau. Chronic hypoxia decreased maximum ventricular dP/dt and peak pressure, increased ventricular end-systolic volume, and decreased ventricular ejection fraction, consistent with depressed systolic function. Arterial afterload (peripheral resistance) increased and both dorsal aortic SV and steady-state hydraulic power decreased in response to hypoxia. Thus, reduced oxygen tension during early cardiac development depresses ventricular function, increases ventricular impedance (afterload), delays growth, and decreases embryo survival, suggesting that a critical threshold of oxygen tension is required to support morphogenesis and cardiovascular function in the early embryo.

Mechanical load influences embryonic ventricular growth, morphogenesis, and function. To date, little is known regarding how the embryonic left ventricular (LV) myocardium acquires a three-dimensional (3D) fiber architecture distribution or how altered mechanical load influences local myofiber architecture. We tested the hypothesis that altered mechanical load changes the maturation process of local 3D fiber architecture of the developing embryonic LV compact myocardium. We measured transmural myofiber angle distribution in the LV compact myocardium in Hamburger-Hamilton stages 21, 27, 31, and 36 chick embryos during normal development or following either left atrial ligation (LAL; LV hypoplasia model) or conotruncal banding (CTB; LV hyperplasia model). The embryonic LV was stained with f-actin and then z-serial optical sectioning was performed using a laser confocal scanning microscope. We reconstructed local 3D myofiber images and computed local transmural myofiber angle distribution. Transmural myofiber angles in compact myocardium (in LV sagittal sections) were oriented in a circumferential direction until stage 27 (-10 to 10°). Myofibers in the outer side of compact myocardium shifted to a more longitudinal direction by stage 36 (10 to 40°), producing a transmural gradient in myofiber orientation. Developmental changes in transmural myofiber angle distribution were significantly delayed following LAL, while the changes in angle distribution were accelerated following CTB. Results suggest that mechanical load modulates the maturation process of myofiber architecture distribution in the developing LV compact myocardium. © 2005 Wiley-Liss, Inc.

Hypoplastic left heart syndrome (HLHS) is a rare but deadly congenital malformation, which can be created experimentally in the chick embryo by left atrial ligation (LAL). The goal of this study was to examine the cellular changes leading to the profound remodeling of ventricular myocardial architecture that occurs in this model. Hypoplasia of left heart structures was produced after 3H-thymidine prelabeling by partial LAL at stage 24, thereby reducing its volume, and redistributing blood preferentially to the developing right ventricle (RV). Controls included both sham-operated and intact stage-matched embryos. Survivors were studied 4 days after the ligation, when the heart organogenesis was essentially complete. Paraffin sections of the hearts were subjected to autoradiography and immunohistochemistry to detect changes in history of cell proliferation and expression of myosin, and growth factors implicated in cardiomyocyte proliferation. Sampling for apoptosis detection using TUNEL assay was done at stages 29 and 34. LAL resulted in decreased levels of proliferation in the left ventricular compact layer and trabeculae. The right ventricular compact layer also showed a slight decrease, but the trabeculae showed no differences. Anti-myosin staining was significantly reduced in all compartments. The expression levels of growth factors were altered as well. Apoptosis was increased in the right atrioventricular mesenchyme, with no changes in the working myocardium. These data suggest that changes in cardiomyocyte proliferation play a significant role in the pathogenesis of HLHS. Anat Rec 267:137–145, 2002. © 2002 Wiley-Liss, Inc.

Hypoplastic left heart syndrome (HLHS) is a rare but deadly congenital malformation, which can be created experimentally in the chick embryo by left atrial ligation (LAL). The goal of this study was to examine the cellular changes leading to the profound remodeling of ventricular myocardial architecture that occurs in this model. Hypoplasia of left heart structures was produced after 3H‐thymidine prelabeling by partial LAL at stage 24, thereby reducing its volume, and redistributing blood preferentially to the developing right ventricle (RV). Controls included both sham‐operated and intact stage‐matched embryos. Survivors were studied 4 days after the ligation, when the heart organogenesis was essentially complete. Paraffin sections of the hearts were subjected to autoradiography and immunohistochemistry to detect changes in history of cell proliferation and expression of myosin, and growth factors implicated in cardiomyocyte proliferation. Sampling for apoptosis detection using TUNEL assay was done at stages 29 and 34. LAL resulted in decreased levels of proliferation in the left ventricular compact layer and trabeculae. The right ventricular compact layer also showed a slight decrease, but the trabeculae showed no differences. Anti‐myosin staining was significantly reduced in all compartments. The expression levels of growth factors were altered as well. Apoptosis was increased in the right atrioventricular mesenchyme, with no changes in the working myocardium. These data suggest that changes in cardiomyocyte proliferation play a significant role in the pathogenesis of HLHS. Anat Rec 267:137–145, 2002. © 2002 Wiley‐Liss, Inc.

1.The aim of this study was to define from umbilical artery flow velocity waveforms absolute peak systolic and time-averaged velocity, fetal heart rate, fetal heart rate variability and flow velocity variability, and the relation between fetal heart rate and velocity variables in early pregnancy. 2.A total of 108 women presenting with a normal pregnancy from 10 to 20 weeks of gestation consented to participate in a cross-sectional study design. Doppler ultrasound recordings were made from the free-floating loop of the umbilical cord. 3.Umbilical artery peak systolic and time-averaged velocity increased at 10–20 weeks, whereas fetal heart rate decreased at 10–15 weeks of gestation and plateaued thereafter. Umbilical artery peak systolic velocity variability and fetal heart rate variability increased at 10–20 and 15–20 weeks respectively. 4.The inverse relationship between umbilical artery flow velocity and fetal heart rate at 10–15 weeks of gestation suggests that the Frank–Starling mechanism regulates cardiovascular control as early as the late first and early second trimesters of pregnancy. A different underlying mechanism is suggested for the observed variability profiles in heart rate and umbilical artery peak systolic velocity. It is speculated that heart rate variability is mediated by maturation of the parasympathetic nervous system, whereas peak systolic velocity variability reflects the activation of a haemodynamic feedback mechanism.

Cardiac output is affected by the diastolic filling characteristics of the ventricle. We hypothesized that the relative contributions of passive and active filling change as the ventricle develops from a smooth-walled tube to a trabeculated four-chamber heart. In stage 12 to 27 white Leghorn chick embryos, we simultaneously measured ventricular pressure with a servo-null micropressure system and dorsal aortic and atrioventricular velocities with a 20-MHz pulsed-Doppler velocity meter. The analog waveforms were sampled at 500 Hz and converted to digital format via an analog/digital board. We partitioned diastole into passive and active components. The passive phase began with the return of the pressure curve to baseline and extended to the onset of the a-wave. The active phase began with the upstroke of the atrial velocity curve and extended to the upstroke of the ventricular pressure curve at end-diastole. Data are presented as mean ± SEM (n± 6 at each stage) and analyzed by analysis of variance and regression analysis. At similar cycle lengths ranging from 480 to 600 ms (p> 0.05), end-diastolic pressure increased from 0.24 ± 0.02 mm Hg at stage 12 to 0.55 ± 0.01 mm Hg at stage 27. Passive and active filling volumes were 92 (0.0038 ± 0.0005 mm3) and 8 (0.0004 ± 0.0002 mm3), respectively, at stage 12 and changed to 24 (0.23 ± 0.08 mm3) and 76 (0.62 ± 0.08 mm3), respectively, at stage 27. The ratio of passive to active filling volume decreased from 7.89 to 0.35. Thus, active ventricular filling became dominant as the trabeculae formed in the embryonic ventricle. These observations define the diastolic filling characteristics of the embryonic heart during primary cardiac morphogenesis. (Pediatr Res29: 334–337, 1991)

ABSTRACT: Cardiac output is affected by the diastolic filling characteristics of the ventricle. We hypothesized that the relative contributions of passive and active filling change as the ventricle develops from a smooth-walled tube to a trabeculated four-chamber heart. In stage 12 to 27 white Leghorn chick embryos, we simultaneously measured ventricular pressure with a servo-null micropressure system and dorsal aortic and atrioventricular velocities with a 20-MHz pulsed-Doppler velocity meter. The analog waveforms were sampled at 500 Hz and converted to digital format via an analog/digital board. We partitioned diastole into passive and active components. The passive phase began with the return of the pressure curve to baseline and extended to the onset of the a-wave. The active phase began with the upstroke of the atrial velocity curve and extended to the upstroke of the ventricular pressure curve at end-diastole. Data are presented as mean ± SEM (n = 6 at each stage) and analyzed by analysis of variance and regression analysis. At similar cycle lengths ranging from 480 to 600 ms (p > 0.05), end-diastolic pressure increased from 0.24 ± 0.02 mm Hg at stage 12 to 0.55 ± 0.01 mm Hg at stage 27. Passive and active filling volumes were 92 (0.0038 ± 0.0005 mm3) and 8% (0.0004 ± 0.0002 mm3), respectively, at stage 12 and changed to 24 (0.23 ± 0.08 mm3) and 76% (0.62 ± 0.08 mm3), respectively, at stage 27. The ratio of passive to active filling volume decreased from 7.89 to 0.35. Thus, active ventricular filling became dominant as the trabeculae formed in the embryonic ventricle. These observations define the diastolic filling characteristics of the embryonic heart during primary cardiac morphogenesis.

Although atrial morphologic changes are well documented, the description of early atrial function is limited. We used videomicroscopic methods to define the function of the contracting atrium in stage 16 to 24 white Leghorn chick embryos. We exposed the embryo in ovo (right side up) and imaged the ventricle, then repositioned the embryo (left side up) and imaged the atrium (n≥ 8 per stage). We traced the atrial endocardial border and then measured atrial perimeter (mm) and cross-sectional area (mm2). A 20-MHz pulsed Doppler velocity meter was used to measure atrioventricular blood velocity during atrial imaging in an additional six stage 21 embryos. Data were tested by analysis of variance and regression analysis. Mean heart rate change after repositioning was −4 ± 1. Atrial maximum and minimum area increased linearly versus embryo stage (y= 0.10x− 1.41, r= 0.89, p< 0.05 and y = 0.05x− 0.67, r= 0.82, p< 0.05, respectively). Shortening fraction (percentage of reduction) of atrial perimeter and area decreased from 32.3 ± 2.0 to 27.5 ± 1.8 (p< 0.05) and 56.2 ± 3.0 to 47.7 ± 2.0 (p< 0.05), respectively, from stage 16 to 24. During atrial contraction, the velocity of circumferential wall shortening increased linearly with stage (y= 0.22x− 2.08, r= 0.81, p< 0.01); however, the velocity of lengthening was similar between stages (p= 0.45). Simultaneous atrial imaging and pulsed Doppler velocity measurement showed that passive atrioventricular flow occurred late in atrial lengthening and active atrioventricular flow occurred during atrial contraction. Thus, atrial function increases in parallel with morphogenesis during early cardiac development, and measures of atrial function can now be incorporated into a physiologic model of the developing cardiovascular system. (Pediatr Res 32: 333–337, 1992)

Atrial natriuretic peptide (ANP) exerts hemodynamic effects by direct venodilation in the chick embryo. We hypothesized that ANP-induced venodilation affects ventricular diastolic filling resulting in reduced ventricular preload. Chick ANP (0.1 μg in 10 μL of normal saline) was suffused onto the vitelline vascular bed in stage 21 (3 1/2 d) chick embryos. Equivalent aliquots of normal saline were suffused as sham controls, and normal embryos received no suffusion. We measured simultaneously dorsal aortic blood velocity and atrioventricular blood velocity with a 20-MHz pulsed-Doppler velocity meter. Analog wave forms were digitally sampled at 500 Hz, and the dorsal aortic cross-sectional area was used to calculate dorsal aortic blood flow. Passive ventricular filling volume equaled dorsal aortic stroke volume multiplied by the fraction of passive area; active filling volume equaled dorsal aortic stroke volume multiplied by the fraction of active area. Data were summarized as mean ± SEM (n≥ 7 per group) and analyzed by analysis of variance. Cycle lengths were similar in ANP-suffused, sham control, and normal embryos. Dorsal aortic blood flow decreased from 0.49 ± 0.04 mm3/s at baseline to 0.27 ± 0.05 mm3/s at 4 min post-ANP suffusion (p< 0.05) and was unchanged in sham control and normal embryos (p> 0.05). Passive ventricular filling was reduced by ANP suffusion, whereas active filling was unaffected, resulting in a decreased passive/active filling ratio from 0.64 ± 0.07 at baseline to 0.32 ± 0.08 at 4 min in ANP-suffused embryos (p< 0.05). Passive/active ratio was unchanged in sham control and normal embryos. Thus, ANP-mediated vasodilation reduces cardiac output via decreased passive ventricular filling in the embryonic heart.

The objective of this study was to identify the best method for reconstructing blood-flow velocities from the early human umbilical artery to determine the physiological changes in fetal blood-flow velocity and heart rate. Pulsed Doppler recordings from the umbilical artery with a duration of approximately 7 s were made at 10–20 weeks of gestation. For reconstruction of the blood-flow velocity from the Doppler audio signal, the maximum (envelope), mean and mode frequency reconstruction methods were used. For the assessment of variability in blood-flow velocity and heart rate in the umbilical artery, the maximum velocity reconstruction method is preferred because it is relatively insensitive to noise, nonuniform insonation, and wall filter settings.

Cardiovascular defects represent the largest cause of childhood mortality. Of the 25,000 infants diagnosed with cardiovascular abnormalities each year, 5000 (20) of those infants will die in the first year of life despite the best medical care. Thus, the broad spectrum of research to define cardiovascular structural and functional maturation includes human embryo, fetal, and neonatal studies, as well as experiments using animal, organ, cell, and subcellular protocols in chick embryo, rat, rabbit, dog, pig, and lamb models. New information further details the maturation of myocyte sarcolemmal and sarcoplasmic calcium transport, the composition of the regulatory thin filament, and the distribution of α-actins, β-myosins, and components of the extracellular matrix in the developing and diseased heart. Coronary angiogenesis in the developing and hypertrophied myocardium is more uniform than found in the mature heart. New quantitative data are available on myocardial growth in the embryonic and neonatal myocardium, and following heart transplantation. Investigation of embryonic, fetal, and neonatal cardiovascular function further highlights the dynamic interdependence of the maturing heart and systemic and pulmonary vascular beds. Exciting information continues to accumulate on the role of the endothelium in the regulation of pulmonary vascular tone.

ABSTRACT: Atrial natriuretic peptide (ANP) exerts hemodynamic effects by direct venodilation in the chick embryo. We hypothesized that ANP-induced venodilation affects ventricular diastolic filling resulting in reduced ventricular preload. Chick ANP (0.1 µg in 10 µL of normal saline) was suffused onto the vitelline vascular bed in stage 21 (3 1/2 d) chick embryos. Equivalent aliquots of normal saline were suffused as sham controls, and normal embryos received no suffusion. We measured simultaneously dorsal aortic blood velocity and atrioventricular blood velocity with a 20-MHz pulsed-Doppler velocity meter. Analog wave forms were digitally sampled at 500 Hz, and the dorsal aortic cross-sectional area was used to calculate dorsal aortic blood flow. Passive ventricular filling volume equaled dorsal aortic stroke volume multiplied by the fraction of passive area; active filling volume equaled dorsal aortic stroke volume multiplied by the fraction of active area. Data were summarized as mean ± SEM (n= 7 per group) and analyzed by analysis of variance. Cycle lengths were similar in ANP-suffused, sham control, and normal embryos. Dorsal aortic blood flow decreased from 0.49 ± 0.04 mm3/s at baseline to 0.27 ± 0.05 mm3/s at 4 min post-ANP suffusion (p< 0.05) and was unchanged in sham control and normal embryos (p> 0.05). Passive ventricular filling was reduced by ANP suffusion, whereas active filling was unaffected, resulting in a decreased passive/active filling ratio from 0.64 ± 0.07 at baseline to 0.32 ± 0.08 at 4 min in ANP-suffused embryos (p< 0.05). Passive/active ratio was unchanged in sham control and normal embryos. Thus, ANP-mediated vasodilation reduces cardiac output via decreased passive ventricular filling in the embryonic heart.

ABSTRACT: Although atrial morphologic changes are well documented, the description of early atrial function is limited. We used videomicroscopic methods to define the function of the contracting atrium in stage 16 to 24 white Leghorn chick embryos. We exposed the embryo in ovo (right side up) and imaged the ventricle, then repositioned the embryo (left side up) and imaged the atrium (n = 8 per stage). We traced the atrial endocardial border and then measured atrial perimeter (mm) and cross-sectional area (mm2). A 20-MHz pulsed Doppler velocity meter was used to measure atrioventricular blood velocity during atrial imaging in an additional six stage 21 embryos. Data were tested by analysis of variance and regression analysis. Mean heart rate change after repositioning was -4 ± 1%. Atrial maximum and minimum area increased linearly versus embryo stage (y = 0.10x - 1.41, r = 0.89, p < 0.05 and y = 0.05x - 0.67, r = 0.82, p < 0.05, respectively). Shortening fraction (percentage of reduction) of atrial perimeter and area decreased from 32.3 ± 2.0% to 27.5 ± 1.8% (p < 0.05) and 56.2 ± 3.0% to 47.7 ± 2.0% (p < 0.05), respectively, from stage 16 to 24. During atrial contraction, the velocity of circumferential wall shortening increased linearly with stage (y = 0.22x - 2.08, r = 0.81, p < 0.01); however, the velocity of lengthening was similar between stages (p = 0.45). Simultaneous atrial imaging and pulsed Doppler velocity measurement showed that passive atrioventricular flow occurred late in atrial lengthening and active atrioventricular flow occurred during atrial contraction. Thus, atrial function increases in parallel with morphogenesis during early cardiac development, and measures of atrial function can now be incorporated into a physiologic model of the developing cardiovascular system.

Due to substantial progress of medications to treat pulmonary arterial hypertension (PAH), clinicians are now reconsidering interventional procedures for congenital heart disease (CHD) that were previously considered to be contraindicated or high-risk. This study provides a retrospective evaluation of our institutional approach to treat PAH, proceed to CHD defect closure, and then continue PAH treatment, termed “treat-repair-treat”.