Aortocaval Shunt Research Articles

DNA Methylation Analysis Identifies Novel Epigenetic Loci in Dilated Murine Heart upon Exposure to Volume Overload

Left ventricular (LV) dilatation, a prominent risk factor for heart failure (HF), precedes functional deterioration and is used to stratify patients at risk for arrhythmias and cardiac mortality. Aberrant DNA methylation contributes to maladaptive cardiac remodeling and HF progression following pressure overload and ischemic cardiac insults. However, no study has examined cardiac DNA methylation upon exposure to volume overload (VO) despite being relatively common among HF patients. We carried out global methylome analysis of LV harvested at a decompensated HF stage following exposure to VO induced by aortocaval shunt. VO resulted in pathological cardiac remodeling, characterized by massive LV dilatation and contractile dysfunction at 16 weeks after shunt. Although methylated DNA was not markedly altered globally, 25 differentially methylated promoter regions (DMRs) were identified in shunt vs. sham hearts (20 hypermethylated and 5 hypomethylated regions). The validated hypermethylated loci in Junctophilin-2 (Jph2), Signal peptidase complex subunit 3 (Spcs3), Vesicle-associated membrane protein-associated protein B (Vapb), and Inositol polyphosphate multikinase (Ipmk) were associated with the respective downregulated expression and were consistently observed in dilated LV early after shunt at 1 week after shunt, before functional deterioration starts to manifest. These hypermethylated loci were also detected peripherally in the blood of the shunt mice. Altogether, we have identified conserved DMRs that could be novel epigenetic biomarkers in dilated LV upon VO exposure.

Abstract 10270: High Shear Stress Decreases ERG Causing Endothelial to Mesenchymal Transition and Pulmonary Arterial Hypertension

Introduction: Computational modeling studies indicated that pathological high shear stress (HSS) of 100 dynes/cm 2 is generated in pulmonary arteries (PA) (100-500μM) in patients with a ventricular septal defect or idiopathic pulmonary arterial hypertension (PAH) and occlusive vascular remodeling. Endothelial-to-mesenchymal transition (EndMT) is a feature of PAH. Hypothesis: Pathological HSS induces EndMT, which contributes to the initiation and progression of PAH. Methods: We apply the Ibidi perfusion system to human PA endothelial cells (EC), to determine whether HSS (100 dynes/cm 2 ) induces EndMT, when compared to normal laminar shear stress (LSS) (15 dynes/cm 2 ). We assessed the mechanism and targeted it to prevent PAH in a mouse with HSS resulting from an aortocaval (AV) shunt. Results: HSS induced EndMT, as assessed by an increase in transcription factors, SNAI1 and SNAI2, reduced BMPR2 (previously shown to inhibit EndMT), decreased EC markers PECAM1 and CDH5, and increased mesenchymal markers, ACTA2 and FSP-1. While the flow-induced transcription factors, KLF2 and KLF4 were similar in LSS and HSS, the co-transcription factor ERG was reduced with HSS. Indeed ERG siRNA under LSS caused EndMT whereas under HSS, transfection of ERG prevented EndMT. To address the significance of our observations in an animal model we created an AV shunt in mice and compared PAH in those sham operated vs transfected with an adeno-associated viral (AAV2- ESGHGYF) vector selectively targeting PAEC with a luciferase (control) or an ERG expressing construct (N=10 per group). Eight weeks after AV shunt, right ventricular systolic pressures was 21.9 ±0.6 mmHg in sham, 37.2±1.0 mmHg in AV shunt with luciferase vector (p<0.01 vs sham) and 29.2±0.8 mmHg in ERG-vector (p<0.01 vs both sham and luciferase). Muscularization of peripheral PA was similarly reduced in ERG-vector (p<0.01 vs luciferase) but not normalized (p<0.01 vs sham) as was immunohistochemical evidence of EndMT (p<0.01 vs luciferase). Conclusions: Our study is the first to document pathological HSS as an inducer of EndMT and PAH and to show that this results from reduced PAEC ERG. Agents that upregulate ERG could reverse HSS mediated PAH and occlusive vascular remodeling as a consequence of high flow or narrowed PAs.

Hemodynamic stress‐induced cardiac remodelling is not modulated by ablation of phosphodiesterase 4D interacting protein

Adrenergic stimulation in the heart activates the protein kinase A (PKA), which phosphorylates key proteins involved in intracellular Ca2+ handling. PKA is held in proximity to its substrates by protein scaffolds, the A kinase anchoring proteins (AKAPs). We have previously identified the transcript of phosphodiesterase 4D interacting protein (Pde4dip; also known as myomegalin), one of the sarcomeric AKAPs, as being differentially expressed following hemodynamic overload, a condition inducing hyperadrenergic state in the heart. Here, we addressed whether PDE4DIP is involved in the adverse cardiac remodelling following hemodynamic stress. Homozygous Pde4dip knockout (KO) mice, generated by CRISPR‐Cas9 technology, and wild‐type (WT) littermates were exposed to aortocaval shunt (shunt) or transthoracic aortic constriction (TAC) to induce hemodynamic volume overload (VO) or pressure overload (PO), respectively. The mortality, cardiac structure, function and pathological cardiac remodelling were followed up after hemodynamic injuries. The PDE4DIP protein level was markedly downregulated in volume‐overloaded‐ but upregulated in pressure‐overloaded‐WT hearts. Following shunt or TAC, mortality rates were comparably increased in both genotypes. Twelve weeks after shunt or TAC, Pde4dip‐KO animals showed a similar degree of cardiac hypertrophy, dilatation and dysfunction as WT mice. Cardiomyocyte hypertrophy, myocardial fibrosis, reactivation of cardiac stress genes and downregulation of ATPase, Ca2+ transporting, cardiac muscle, slow twitch 2 transcript did not differ between WT and Pde4dip‐KO hearts following shunt or TAC. In summary, despite a differential expression of PDE4DIP protein in remodelled WT hearts, Pde4dip deficiency does not modulate adverse cardiac remodelling after hemodynamic VO or PO.

Pirfenidone ameliorates pulmonary arterial pressure and neointimal remodeling in experimental pulmonary arterial hypertension by suppressing NLRP3 inflammasome activation.

Pulmonary arterial hypertension (PAH) is a fatal disease characterized by increased pulmonary arterial pressure, inflammation, and neointimal remodeling of pulmonary arterioles. Serum levels of interleukin (IL)‐1β and IL‐18 are elevated in PAH patients and may enhance proinflammatory neointimal remodeling. NLRP3 inflammasome activation induces cleavage of the cytokines IL‐1β and IL‐18, required for their secretion. Pirfenidone (PFD), an antifibrotic and anti‐inflammatory drug, has been suggested to inhibit NLRP3 inflammasome activation. We hypothesized that PFD delays the progression of PAH by suppressing NLRP3 inflammasome activation. We assessed the effects of PFD treatment in a rat model for neointimal PAH induced by monocrotaline and aortocaval shunt using echocardiographic, hemodynamic, and vascular remodeling parameters. We measured inflammasome activation by NLRP3 immunostaining, Western blots for caspase‐1, IL‐1β, and IL‐18 cleavage, and macrophage IL‐1β secretion. PFD treatment ameliorated pulmonary arterial pressure, pulmonary vascular resistance, and pulmonary vascular remodeling in PAH rats. In PAH rats, immunostaining of NLRP3 in pulmonary arterioles and caspase‐1, IL‐1β, and IL‐18 cleavage in lung homogenates were increased compared to controls, reflecting NLRP3 inflammasome activation in vivo. PFD decreased IL‐1β and IL‐18 cleavage, as well as macrophage IL‐1β secretion in vitro. Our studies show that PFD ameliorates pulmonary hemodynamics and vascular remodeling in experimental PAH. Although PFD did not affect all NLRP3 inflammasome parameters, it decreased IL‐1β and IL‐18 cleavage, the products of NLRP3 inflammasome activation that are key to its downstream effects. Our findings thus suggest a therapeutic benefit of PFD in PAH via suppression of NLRP3 inflammasome activation.

Pirfenidon Prevents Experimental Flow-Associated PAH by Suppression of the NLRP3 Inflammasome

<h3>Purpose</h3> Pulmonary arterial hypertension (PAH) is a fatal disease with characteristic vascular remodeling, including fibrosis, within an inflammatory milieu in the distal pulmonary vascular bed. Interleukin (IL)-1β and IL-18 levels, two potent proinflammatory cytokines, are elevated in patient serum and animal models of PAH. The release of these cytokines is regulated by the activation of the NLRP3 inlfammasome, hallmarked by caspase-1 cleavage. We hypothesized that pirfenidone (PFD) an antifibrotic and antinflammatory agent delays the progression of PAH by suppressing NLRP3 inflammasome activation. <h3>Methods</h3> We investigated the effect of preventive PFD treatment in the rat model for neointimal PAH induced by monocrotaline and aortocaval shunt. The disease severity was assessed by pulmonary hemodynamics and histology. The NLRP3 inflammasome activation was assessed by immunohistochemisry and immunoblotting on lung homogenate. For proof of concept, the effect of PFD on NLRP3 inflammasome was additionally tested, by qPCR and ELISA, on bone marrow derived macrophages (BMDMs) after LPS/nigericin stimulation. <h3>Results</h3> Inflammasome activation was increased in the model compared to control as assessed by cleavage the ratio of caspase-1 (145%, p=0.023), IL-1β (142%, p=0.002) and IL-18 (210%, p=0.006). PFD treatment led to reduction of mPAP and occlusion (mPAP:28 ±8 mmHg vs 36 ±4mmHg, p=0.004; occlusion 28 ±4% vs 18 ±10%, p=0.008). So did the level of IL-1β and IL-18 activation when compared to the PAH untreated group (35%, p=0.027; 68%, p=0.005). The perivascular collagen content correlated with mPAP (p=0.025) and occlusion (p<0.001) regarding fibrosis. On BMDMs, both the inflammasome related gene expression relative to control and the IL-1β excretion were reduced by PFD (NLRP3: 40% p<0.001; IL-1β: 61% p<0.001; IL-18: 50% p<0.001 & IL-1β in medium: 44% p<0.001). <h3>Conclusion</h3> Inflammasome is activated in experimental flow-associated PAH, based on the cleavage of caspase-1 and its products IL-1β and IL-18. Its activation correlates with pulmonary hemodynamics and vascular remodeling. PFD suppresses NLRP3 inflammasome activation and ameliorates PAH, assessed by pulmonary hemodynamics and vascular remodeling.

Iatrogenic aortocaval shunt treated by stent graft placement into the inferior vena cava

Fifty six year old male sustained an injury to the abdominal aorta during his 1992 lumbar disc surgery with subsequent aortocaval shunt formation. In order to close the shunt, his abdominal aorta and both common iliac arteries were ligated and bilateral aortoiliac bypass was implanted. Postoperatively the patient remained asymptomatic for several years. Subsequently he developed arterial hypertension, his exercise capacity diminished and he complained of exertional dyspnea. Intraarterial DSA demonstrated filling of the occluded aortic segment from intercostal and lumbar arteries as well and a shunt between this segment and the inferior vena cava. The hemodynamic significance of the shunt was confirmed using catheterization. Attempted reduction of the shunt flow by intercostal arteries embolization was not successful. We were successful in closing the fistula using a custom-made stent graft. CTA performed 18 years after the procedure confirmed absence of the shunt and patent inferior vena cava. The patient is now asymptomatic 23 years after the original surgery.

Effects of Trandolapril on Structural, Contractile and Electrophysiological Remodeling in Experimental Volume Overload Heart Failure.

Chronic volume overload induces multiple cardiac remodeling processes that finally result in eccentric cardiac hypertrophy and heart failure. We have hypothesized that chronic angiotensin-converting enzyme (ACE) inhibition by trandolapril might affect various remodeling processes differentially, thus allowing their dissociation. Cardiac remodeling due to chronic volume overload and the effects of trandolapril were investigated in rats with an aortocaval fistula (ACF rats). The aortocaval shunt was created using a needle technique and progression of cardiac remodeling to heart failure was followed for 24 weeks. In ACF rats, pronounced eccentric cardiac hypertrophy and contractile and proarrhythmic electrical remodeling were associated with increased mortality. Trandolapril substantially reduced the electrical proarrhythmic remodeling and mortality, whereas the effect on cardiac hypertrophy was less pronounced and significant eccentric hypertrophy was preserved. Effective suppression of electrical proarrhythmic remodeling and mortality but not hypertrophy indicates that the beneficial therapeutic effects of ACE inhibitor trandolapril in volume overload heart failure might be dissociated from pure antihypertrophic effects.

Volume Load-Induced Right Ventricular Failure in Rats Is Not Associated With Myocardial Fibrosis.

BackgroundRight ventricular (RV) function and failure are key determinants of morbidity and mortality in various cardiovascular diseases. Myocardial fibrosis is regarded as a contributing factor to heart failure, but its importance in RV failure has been challenged. This study aims to assess whether myocardial fibrosis drives the transition from compensated to decompensated volume load-induced RV dysfunction.MethodsWistar rats were subjected to aorto-caval shunt (ACS, n = 23) or sham (control, n = 15) surgery, and sacrificed after 1 month, 3 months, or 6 months. Echocardiography, RV pressure-volume analysis, assessment of gene expression and cardiac histology were performed.ResultsAt 6 months, 6/8 ACS-rats (75%) showed clinical signs of RV failure (pleural effusion, ascites and/or liver edema), whereas at 1 month and 3 months, no signs of RV failure had developed yet. Cardiac output has increased two- to threefold and biventricular dilatation occurred, while LV ejection fraction gradually decreased. At 1 month and 3 months, RV end-systolic elastance (Ees) remained unaltered, but at 6 months, RV Ees had decreased substantially. In the RV, no oxidative stress, inflammation, pro-fibrotic signaling (TGFβ1 and pSMAD2/3), or fibrosis were present at any time point.ConclusionsIn the ACS rat model, long-term volume load was initially well tolerated at 1 month and 3 months, but induced overt clinical signs of end-stage RV failure at 6 months. However, no myocardial fibrosis or increased pro-fibrotic signaling had developed. These findings indicate that myocardial fibrosis is not involved in the transition from compensated to decompensated RV dysfunction in this model.

Cyclic stretching boosts microRNA-499 to regulate Bcl-2 via microRNA-208a in atrial fibroblasts.

MicroRNAs that modulate transcription can regulate other microRNAs and are also up‐regulated under pathological stress. MicroRNA‐499 (miR‐499), microRNA‐208a (miR‐208a) and B‐cell lymphoma 2 (Bcl‐2) play roles in cardiovascular diseases, such as direct reprogramming of cardiac fibroblast into cardiomyocyte and cardiomyocyte apoptosis. Whether miR208a, miR499 and Bcl‐2 were critical regulators in cardiac fibroblast apoptosis under mechanical stretching conditions in human cardiac fibroblasts‐adult atrial (HCF‐aa) was investigated. Using negative pressure, HCF‐aa grown on a flexible membrane base were cyclically stretched to 20% of their maximum elongation. In adult rats, an aortocaval shunt was used to create an in vivo model of volume overload. MiR208a was up‐regulated early by stretching and returned to normal levels with longer stretching cycles, whereas the expression of miR499 and Bcl‐2 was up‐regulated by longer stretching times. Pre‐treatment with antagomir‐499 reversed the miR‐208a down‐regulation, whereas Bcl‐2 expression could be suppressed by miR‐208a overexpression. In the HCF‐aa under stretching for 1 h, miR‐499 overexpression decreased pri‐miR‐208a luciferase activity; this inhibition of pri‐miR‐208a luciferase activity with stretching was reversed when the miR‐499‐5p binding site in pri‐miR‐208a was mutated. The addition of antagomir‐208a reversed the Bcl‐2‐3′UTR suppression from stretching for 1 h. Flow cytometric analysis revealed that pre‐treatment with miR‐499 or antagomir‐208a inhibited cellular apoptosis in stretched HCF‐aa. In hearts with volume overload, miR‐499 overexpression inhibited myocardial miR‐208a expression, whereas Bcl‐2 expression could be suppressed by the addition of miR‐208a. In conclusion, miR‐208a mediated the regulation of miR‐499 on Bcl‐2 expression in stretched HCF‐aa and hearts with volume overload.

TMEM16A Regulates Pulmonary Arterial Smooth Muscle Cells Proliferation via p38MAPK/ERK Pathway in High Pulmonary Blood Flow-Induced Pulmonary Arterial Hypertension

Objective: Pulmonary arterial hypertension (PAH) is a complex disease of the small pulmonary arteries that is mainly characterized by vascular remodeling. It has been demonstrated that excessive proliferation of pulmonary arterial smooth muscle cells (PASMCs) plays a pivotal role in vascular remodeling during PAH. The present study was undertaken to explore the role of TMEM16A in regulating PASMCs proliferation in high pulmonary blood flow-induced PAH. Methods: Aortocaval shunt surgery was undertaken to establish an animal model. Pulmonary artery pressure and pulmonary vascular structure remodeling (PVSR) were tested. Immunohistochemical staining and Western blot were performed to investigate the expression of TMEM16A. The proliferation of PASMCs was tested by the MTT assay. After treating PASMCs with TMEM16A-siRNA, the expression of proliferating cell nuclear antigen (PCNA), phosphorylated p38 mitogen-activated protein kinase (p-p38MAPK), and phosphorylated extracellular signal-regulated kinase (p-ERK) signaling in PASMCs were tested. Results: PAH and PVSR developed 11 weeks postoperation. Elevated expression of TMEM16A accompanied by high expression of PCNA in pulmonary arteries of the shunt group was observed. The increased proliferation of PASMCs and increased expression of TMEM16A and PCNA, along with activated p-p38MAPK and p-ERK signaling in PASMCs of the shunt group, were all attenuated by siRNA-specific TMEM16A knockdown. Conclusion: TMEM16A regulates PASMCs proliferation in high pulmonary blood flow-induced PAH, and the p38MAPK/ERK signaling pathway is probably involved.

Abstract 14952: Hemodynamic Comparison of Left Ventricular Function in Pressure Overload- versus Volume Overload-Induced Heart Failure Models by Invasive Pressure-Volume Analysis

Introduction: Both sustained left ventricular (LV) pressure overload (PO) and volume overload (VO) induces LV remodeling and eventually development of heart failure (HF). Using rat models, the present study aimed to provide a detailed comparison of distinct aspects of LV function in PO- and VO-induced HF. Methods: PO and VO was induced by transverse aortic constriction (TAC, n=12) and aortocaval shunt (AV-shunt, n=12) creation respectively. Controls underwent corresponding sham operations (n=11). LV remodeling was characterized by echocardiography, histology, qRT PCR, and western blot. LV function was assessed by invasive pressure-volume (P-V) analysis. Results: Both sustained PO and VO resulted in the development of HF, as evidenced by increased LV BNP mRNA expression, pulmonary edema, and characteristic symptoms. While the extent of LV hypertrophy was comparable between the HF models, PO induced concentric while VO evoked eccentric LV remodeling. P-V analysis revealed impaired systolic function in both HF models. Accordingly, decreased ejection fraction and impaired ventriculo-arterial coupling (calculated as the ratio of arterial elastance/LV contractility [VAC]: 0.38±0.05 vs. 1.30±0.13, ShamTAC vs. TAC and 0.52±0.08 vs. 1.17±0.13, ShamAV-Shunt vs. AV-shunt; p&lt;0.05) was detected in both HF models. However, in case of VO the severely reduced LV contractility (slope of end-systolic P-V relationship: 1.79±0.19 vs. 0.52±0.06, ShamAV-Shunt vs. AV-shunt, p&lt;0.05 and 2.14±0.28 vs. 2.03±0.21, ShamTAC vs. TAC p&gt;0.05) underpinned the contractility-afterload mismatch, while in case of PO the increased afterload (arterial elastance: 0.77±0.07 vs. 2.64±0.28, ShamTAC vs. TAC and 0.80±0.07 vs. 0.54±0.05, ShamAV-Shunt vs. AV-shunt; p&lt;0.05) was the main determinant. Furthermore, prolongation of active relaxation occurred to a greater extent in case of PO. In addition, increased myocardial stiffness was only observed in PO-induced HF. Conclusion: Systolic function was reduced in both HF models. However, different factors underpinned the impaired VAC in case of VO (reduced LV contractility) and PO (increased arterial elastance). Furthermore, although diastolic function deteriorated in both models, it occurred to a greater extent in case of PO.

866 Percutaneous Closure of Large Aortocaval Shunts

866 Percutaneous Closure of Large Aortocaval Shunts

Effect of atorvastatin on cardiomyocyte hypertrophy through suppressing MURC induced by volume overload and cyclic stretch.

MURC (muscle‐restricted coiled‐coil protein) is a hypertrophy‐related gene. Hypertrophy can be induced by mechanical stress. The purpose of this research was to investigate the hypothesis that MURC mediates hypertrophy in cardiomyocytes under mechanical stress. We used the in vivo model of an aortocaval shunt (AV shunt) in adult Wistar rats to induce myocardial hypertrophy. We also used the in vitro model of cyclic stretch in rat neonatal cardiomyocytes to clarify MURC expression and the molecular regulation mechanism. The flexible membrane culture plate seeding with cardiomyocytes Cardiomyocytes seeded on a flexible membrane culture plate were stretched by vacuum pressure to 20% of maximum elongation at 60 cycles/min. AV shunt induction enhanced MURC protein expression in the left ventricular myocardium. Treatment with atorvastatin inhibited the hypertrophy induced by the AV shunt. Cyclic stretch markedly enhanced MURC protein and mRNA expression in cardiomyocytes. Addition of extracellular‐signal‐regulated kinase (ERK) inhibitor PD98059, ERK small interfering RNA (siRNA), angiotensin II (Ang II) antibody and atorvastatin before stretch, abolished the induction of MURC protein. An electrophoretic mobility shift assay showed that stretch enhanced the DNA binding activity of serum response factor. Stretch increased but MURC mutant plasmid, ERK siRNA, Ang II antibody and atorvastatin reversed the transcriptional activity of MURC induced by stretch. Adding Ang II to the cardiomyocytes also induced MURC protein expression. MURC siRNA and atorvastatin inhibited the hypertrophic marker and protein synthesis induced by stretch. Treatment with atorvastatin reversed MURC expression and hypertrophy under volume overload and cyclic stretch.

Abstract 16647: Loss of Reversibility in Flow-Induced Pulmonary Arterial Hypertension is Associated With a Senescent Vascular Phenotype and Can Be Rescued by Targeted Senolysis

Introduction: Patients with congenital heart disease (CHD) are at risk of developing progressive pulmonary arterial hypertension (PAH). Timely hemodynamic unloading (HU) by surgical shunt correction usually reverses the arteriopathy, but the timepoint when or reason why PAH becomes irreversible remains elusive. Methods: PAH was created in syngeneic rats by monocrotalin (MCT) injection and an aorto-caval shunt. For HU at different PAH stages, the left donor lung with PAH was transplanted into healthy recipients at t14/t21/t28. Recipients were sacrificed 21days after transplantation. Results: MCT+Shunt induced medial neomuscularization from t14, and neointimal lesions from t21, causing arteriolar occlusion and PAH. HU normalized the vascular morphology in all t14 rats and in 7/10 t21 rats, whereas no reversal or progressive occlusion occurred in 3/10 t21 and all t28 rats. RNA-sequencing revealed that activated pro-proliferative/regenerative, pro-apoptotic and DNA-repair pathways were associated with reversal after HU. This was confirmed immunohistochemically by increased vascular-specific Ki67, Caspase3 and Rad51 protein expression. In contrast, irreversibility was associated with deactivation of latter pathways and activated pro-survival, pro-senescence and pro-inflammatory pathways, as confirmed by increased Survivin, p16 ink4A , p21 cip1 , TNFα, IL6 and MMP2. These findings were also confirmed in human PAH-CHD explant tissue. Finally, we report that the senolytic BCL2 antagonist ABT263 selectively targets rat endothelial cells made senescent by irradiation. Moreover, low-dose ABT263 treatment from t25 to t32 in MCT+Shunt rats significantly improved pulmonary hemodynamics and relieved PAH symptoms. Conclusions: Similar to clinical observations in PAH-CHD, this study showed that HU reverses PAH only in an early stage, and describes a zone of no return. We characterized the mechanistic basis for (loss of) reversibility, showing a distinct switch from increased proliferation/regeneration, apoptosis and DNA-repair, to a growth arrested, apoptosis-resistant and intrinsically pro-inflammatory phenotype, suggesting a role for senescence. Reversal of this phenotype by targeted senolysis also reversed PAH beyond the zone of no return.

Acetylation contributes to hypertrophy-caused maturational delay of cardiac energy metabolism.

A dramatic increase in cardiac fatty acid oxidation occurs following birth. However, cardiac hypertrophy secondary to congenital heart diseases (CHDs) delays this process, thereby decreasing cardiac energetic capacity and function. Cardiac lysine acetylation is involved in modulating fatty acid oxidation. We thus investigated what effect cardiac hypertrophy has on protein acetylation during maturation. Eighty-four right ventricular biopsies were collected from CHD patients and stratified according to age and the absence (n = 44) or presence of hypertrophy (n = 40). A maturational increase in protein acetylation was evident in nonhypertrophied hearts but not in hypertrophied hearts. The fatty acid β-oxidation enzymes, long-chain acyl CoA dehydrogenase (LCAD) and β-hydroxyacyl CoA dehydrogenase (βHAD), were hyperacetylated and their activities positively correlated with their acetylation after birth in nonhypertrophied hearts but not hypertrophied hearts. In line with this, decreased cardiac fatty acid oxidation and reduced acetylation of LCAD and βHAD occurred in newborn rabbits subjected to cardiac hypertrophy due to an aortocaval shunt. Silencing the mRNA of general control of amino acid synthesis 5-like protein 1 reduced acetylation of LCAD and βHAD as well as fatty acid oxidation rates in cardiomyocytes. Thus, hypertrophy in CHDs prevents the postnatal increase in myocardial acetylation, resulting in a delayed maturation of cardiac fatty acid oxidation.

Characteristics of Pulmonary Vascular Remodeling in a Novel Model of Shunt-Associated Pulmonary Arterial Hypertension.

BackgroundEstablishing a shunt-induced pulmonary arterial hypertension (PAH) model in mice would be of great scientific value, but no such models have been reported to date. Here, we established a shunt-associated PAH in mice to investigate the characteristics of pulmonary vascular remodeling, which provides a new platform for the in-depth study of PAH associated with congenital heart disease (CHD).Material/MethodsEighty mice were randomly divided into the heavy shunt group (n=32), the small shunt group (n=32), the sham operation group (n=8), and the control group (n=8). The septum of the abdominal aorta and inferior vena cava was cut directly to create a heavy abdominal aortocaval shunt. Pulmonary artery pressure, right ventricular hypertrophy index, and lung tissue morphology were evaluated in the 4th, 6th, 8th, and 12th weeks in the shunt groups.ResultsShunt-associated PAH by abdominal aortocaval shunt in mice was successfully established. The shunt patency rate was significantly higher in the heavy shunt group. Significant differences were observed between the heavy shunt group and other groups in terms of pulmonary artery pressure and the right ventricular hypertrophy index. Tissue sections revealed a thickened pulmonary intimal layer and muscular layer and stenosis of the lumen in the shunt groups. Immunofluorescent assay results showed significant proliferations of PAH smooth muscle cells and endothelial cells, consistent with the clinical pulmonary vascular remodeling seen in human patients with severe PAH.ConclusionsShunt-associated PAH established by directly cutting the septum between the abdominal aorta and inferior vena cava is a stable and reliable model for research on PAH associated with CHD.

Cardiomyocyte proliferation prevents failure in pressure overload but not volume overload.

Induction of the cell cycle is emerging as an intervention to treat heart failure. Here, we tested the hypothesis that enhanced cardiomyocyte renewal in transgenic mice expressing cyclin D2 would be beneficial during hemodynamic overload. We induced pressure overload by transthoracic aortic constriction (TAC) or volume overload by aortocaval shunt in cyclin D2-expressing and WT mice. Although cyclin D2 expression dramatically improved survival following TAC, it did not confer a survival advantage to mice following aortocaval shunt. Cardiac function decreased following TAC in WT mice, but was preserved in cyclin D2-expressing mice. On the other hand, cardiac structure and function were compromised in response to aortocaval shunt in both WT and cyclin D2-expressing mice. The preserved function and improved survival in cyclin D2-expressing mice after TAC was associated with an approximately 50% increase in cardiomyocyte number and exaggerated cardiac hypertrophy, as indicated by increased septum thickness. Aortocaval shunt did not further impact cardiomyocyte number in mice expressing cyclin D2. Following TAC, cyclin D2 expression attenuated cardiomyocyte hypertrophy, reduced cardiomyocyte apoptosis, fibrosis, calcium/calmodulin-dependent protein kinase IIδ phosphorylation, brain natriuretic peptide expression, and sustained capillarization. Thus, we show that cyclin D2-induced cardiomyocyte renewal reduced myocardial remodeling and dysfunction after pressure overload but not after volume overload.

Echocardiographic evaluation of diastolic function in mouse models of heart disease

BackgroundMouse models of heart disease are extensively employed. The echocardiographic characterization of contractile function is usually focused on systolic function with fewer studies assessing diastolic function. Furthermore, the applicability of diverse echocardiographic parameters of diastolic function that are commonly used in humans has not been extensively evaluated in different pathophysiological models in mice. Methods and resultsWe used high resolution echocardiography to evaluate parameters of diastolic function in mouse models of chronic pressure overload (aortic constriction), volume overload (aorto-caval shunt), heart failure with preserved ejection fraction (HFpEF; DOCA-salt hypertension), and acute sarcoplasmic reticulum dysfunction induced by thapsigargin - all known to exhibit diastolic dysfunction. Left atrial area increased in all three chronic models while mitral E/A was difficult to quantify at high heart rates. Isovolumic relaxation time (IVRT) and Doppler E/E′ increased significantly and the peak longitudinal strain rate during early filling (peak reverse longitudinal strain rate) decreased significantly after aortic constriction, with the changes being proportional to the magnitude of hypertrophy. In the HFpEF model, reverse longitudinal strain rate decreased significantly but changes in IVRT and E/E′ were non-significant, consistent with less severe dysfunction. With volume overload, there was a significant increase in reverse longitudinal strain rate and decrease in IVRT, indicating a restrictive physiology. Acute thapsigargin treatment caused significant prolongation of IVRT and decrease in reverse longitudinal strain rate. ConclusionThese results indicate that the combined measurement of left atrial area plus reverse longitudinal strain rate and/or IVRT provide an excellent overall assessment of diastolic function in the diseased mouse heart, allowing distinction between different types of pathophysiology.

Shunt Surgery, Right Heart Catheterization, and Vascular Morphometry in a Rat Model for Flow-induced Pulmonary Arterial Hypertension.

In this protocol, PAH is induced by combining a 60 mg/kg monocrotalin (MCT) injection with increased pulmonary blood flow through an aorto-caval shunt (MCT+Flow). The shunt is created by inserting an 18-G needle from the abdominal aorta into the adjacent caval vein. Increased pulmonary flow has been demonstrated as an essential trigger for a severe form of PAH with distinct phases of disease progression, characterized by early medial hypertrophy followed by neointimal lesions and the progressive occlusion of the small pulmonary vessels. To measure the right heart and pulmonary hemodynamics in this model, right heart catheterization is performed by inserting a rigid cannula containing a flexible ball-tip catheter via the right jugular vein into the right ventricle. The catheter is then advanced into the main and the more distal pulmonary arteries. The histopathology of the pulmonary vasculature is assessed qualitatively, by scoring the pre- and intra-acinar vessels on the degree of muscularization and the presence of a neointima, and quantitatively, by measuring the wall thickness, the wall-lumen ratios, and the occlusion score.

Impact of aortocaval shunt flow on cardiac and renal function in unilateral nephrectomized rats.

We previously reported significantly enhanced cardiac remodeling post aortocaval fistula (AV) in unilateral nephrectomized (UNX) rats. However, the relationship between the size of the AV and the cardiorenal effects in UNX rats remains unknown. In the present study, AV was induced by 20, 18 and 16 gauge needles in UNX rats to see if larger shunt would definitely induce heavier cardiac and renal damage in UNX rats. Our results demonstrated that bigger shunt size is linked with proportional more significant cardiorenal remodeling and dysfunction in UNX rats. Expression of inflammatory biomarkers including CRP, TNF-α, IL-6, IL-1β, TGF-β and MCP-1 in left kidney and heart was significantly increased in all UNX + AV groups compared to Sham rats. Inflammation might thus participate in the worsening cardiorenal functions and remodeling processes in this model.