Diastolic Heart Failure Model Research Articles

Depressed myocardial cross-bridge cycling kinetics in a female guinea pig model of diastolic heart failure.

Cardiac hypertrophy is associated with diastolic heart failure (DHF), a syndrome in which systolic function is preserved but cardiac filling dynamics are depressed. The molecular mechanisms underlying DHF and the potential role of altered cross-bridge cycling are poorly understood. Accordingly, chronic pressure overload was induced by surgically banding the thoracic ascending aorta (AOB) in ∼400 g female Dunkin Hartley guinea pigs (AOB); Sham-operated age-matched animals served as controls. Guinea pigs were chosen to avoid the confounding impacts of altered myosin heavy chain (MHC) isoform expression seen in other small rodent models. In vivo cardiac function was assessed by echocardiography; cardiac hypertrophy was confirmed by morphometric analysis. AOB resulted in left ventricle (LV) hypertrophy and compromised diastolic function with normal systolic function. Biochemical analysis revealed exclusive expression of β-MHC isoform in both sham control and AOB LVs. Myofilament function was assessed in skinned multicellular preparations, skinned single myocyte fragments, and single myofibrils prepared from frozen (liquid N2) LVs. The rates of force-dependent ATP consumption (tension-cost) and force redevelopment (Ktr), as well as myofibril relaxation time (Timelin) were significantly blunted in AOB, indicating reduced cross-bridge cycling kinetics. Maximum Ca2+ activated force development was significantly reduced in AOB myocytes, while no change in myofilament Ca2+ sensitivity was observed. Our results indicate blunted cross-bridge cycle in a β-MHC small animal DHF model. Reduced cross-bridge cycling kinetics may contribute, at least in part, to the development of DHF in larger mammals, including humans.

Left Atrial Circulatory Assistance in Simulated Diastolic Heart Failure Model: First in Vitro and in Vivo

We are developing a left atrial assist device (LAAD) that is implanted at the mitral position to treat diastolic heart failure (DHF) represented by heart failure with preserved ejection fraction. The LAAD was tested at 3 pump speeds on a pulsatile mock loop with a pneumatic pump that simulated DHF conditions by adjusting the diastolic drive. The LAAD was implanted in 6 calves, and the hemodynamics were assessed. In 3 cases, DHF conditions were induced by using a balloon inserted into the left ventricle, and in 2 cases, mitral valve replacement was also performed after the second aortic cross-clamp. DHF conditions were successfully induced in the in vitro study. With LAAD support, cardiac output, aortic pressure and left atrial pressure recovered to normal values, whereas pulsatility was maintained for both in vivo and in vitro studies. Echocardiography showed no left ventricular outflow tract obstruction, and the LAAD was successfully replaced by a mechanical prosthetic valve. These initial in vitro and in vivo results support our hypothesis that use of the LAAD increases cardiac output and aortic pressure and decreases left atrial pressure, while maintaining arterial pulsatility.

Left atrial assist device for heart failure with preserved ejection fraction: initial results with torque control mode in diastolic heart failure model.

A novel pump, the left atrial assist device (LAAD), is a device specifically for the treatment of heart failure with preserved ejection fraction (HFpEF). The LAAD is a mixed-flow pump that is implanted in the mitral position and delivers blood from the left atrium to the left ventricle. During the development process, we aimed to explore whether device activation in torque control (TC) mode would improve the function of the LAAD. The TC mode causes adjustment of the pump speed automatically during each cardiac cycle in order to maintain a specified torque. In this study, we tested four different TC settings (TC modes 0.9, 1.0, 1.25, and 1.5) using an in vitro mock circulatory loop. Mild, moderate, and severe diastolic heart failure (DHF) conditions, as well as normal heart condition, were simulated with the four TC modes. Also, we evaluated the LAAD in vivo with three calves. The LAAD was implanted at the mitral position with four TC settings (TC modes 0.9, 1.0, 1.1, 1.2). With LAAD support, the in vitro cardiac output and aortic pressure recovered to normal heart levels at TC 1.25 and 1.5 even under severe DHF conditions with little pump regurgitation. The TC mode tested in vivo with three calves, and it also showed favorable result without elevating the left ventricular end-diastolic pressure. These initial in vitro and in vivo results suggest that the TC mode could be potentially effective, and the LAAD could be a treatment option for HFpEF patients.

PLK2 is a novel regulator of osteopontin-driven fibrosis and diastolic dysfunction in permanent atrial fibrillation

Abstract Background and aim Atrial fibrillation (AF) is frequently accompanied by cardiac fibrosis and diastolic heart failure. Due to the heterogeneous nature and complexity of fibrosis, the knowledge of the underlying pathomechanisms is limited. Thus, effective antifibrotic pharmacotherapy is missing. The objective of this study was to decipher the role of polo-like kinase 2 (PLK2) in the pathogenesis of cardiac fibrosis and left ventricular diastolic dysfunction. We put particular emphasis on the identification of profibrotic downstream targets of PLK2, which can serve as therapeutic targets. Methods and results This study was based on human atrial tissue biopsies and peripheral blood samples, a PLK2 knockout mouse model, a canine tachy-pacing model and specific pharmacological interventions on cardiac fibroblasts. In human atrial AF tissue samples, PLK2 was 50% downregulated by hypoxia-induced promoter methylation compared to sinus rhythm (SR) control. Confirmatory analysis of a canine tachy-pacing model showed PLK2 downregulation exclusively in the atria but not in the ventricles. Specific pharmacological inhibition as well as genetic deletion of PLK2 led to a striking myofibroblast phenotype. Discovery proteomics revealed that the global knockout of PLK2 resulted in de novo secretion of the inflammatory cytokine osteopontin (OPN) in cardiac fibroblasts and concomitant ventricular fibrosis in the PLK2 knockout mouse model. An ELISA analysis of peripheral blood samples of AF patients with electrophysiologically proven fibrosis, confirmed significantly increased OPN plasma concentrations compared to SR and non-fibrosis AF controls. Consequently, echocardiography on PLK2 KO mice revealed left ventricular diastolic dysfunction, tachycardia and fibrosis-typical surface ECG anomalies (PQ and QRS prolongation). Mechanistically, we identified the ERK1/2 signaling pathway as the molecular link between reduced expression of PLK2 and elevated osteopontin transcription. In a reverse translational attempt, we successfully tested the capability of 5-amino-salicylic acid (5-ASA) to inhibit osteopontin transcription and to reverse a TGF-β-induced myofibroblast phenotype in vitro. Currently the long-term administration of 5-ASA is tested in PLK2 knockout mice to evaluate the therapeutic potential to prevent cardiac fibrosis and diastolic heart failure development. Conclusion and clinical impact We identified PLK2 as an epigenetically regulated kinase involved in the pathophysiology of fibrosis in AF. PLK2 knockout mice can serve as a model of diastolic heart failure wherein OPN is a promising therapeutic target. Our results strengthen the current hypothesis that atrial fibrillation is not only an ion channel disease but a complex systemic disorder. Restoration of physiological PLK2 expression and blockade of osteopontin release with 5-ASA may constitute valuable new drug targets for the prevention and treatment of fibrosis and diastolic heart failure in AF. Funding Acknowledgement Type of funding source: Public Institution(s). Main funding source(s): Faculty of Medicine, Carl Gustav Carus, Dresden, “MeDDrive Start” Grant

Abstract 946: Aberrant Activation of Nitric Oxide Synthase 1 in the Heart Accelerates Diastolic Dysfunction Though Protein S-nitrosylation

Heart failure refers to an inappropriate blood supply to the periphery. Among subtype of heart failure, pathophysiology and effective therapeutic strategy of systolic heart failure is well established. However, molecular mechanism and specific regimens for diastolic heart failure (DHF) still remains unclear. Here we propose nitric oxide synthase (NOS) 1 mediated exacerbation of DHF and therapeutic potential for novel therapeutics. We screened alteration of posttranslational modifications in DHF which were induced either by SAUNA (SAlty drinking water/Unilateral Nephrectomy/Aldosterone) model or transverse aortic constriction (TAC) model and observed that protein S-nitrosylation was dramatically increased. Total RNA sequencing revealed that transcription amount of NOS1 was aberrantly increased in DHF, whereas NOS2 or NOS3 was not changed. Either non-selective NOS inhibitor or specific NOS1 inhibitor effectively blocked diastolic dysfunction. To elucidate the molecular target of S-nitrosylation, we performed biotin switch assay and requested protein sequencing which were newly appeared in the presence of GSNO. Among novel signal, we identified and confirmed that Histone deacetylase (HDAC) 2 was target molecule of NOS1. HDAC2 cysteine 262 and cysteine 274 were responsible residue of NOS1-mediated S-nitrosylation. We generated knock-in mice carrying S-nitrosylation defect HDAC2 mutant, HDAC2 C262A/C274A (2CA). HDAC2 2CA knock-in mice underwent SAUNA surgery or TAC and diastolic function of those mice was measured by early diastole (E) and mitral valve annulus movement (E’). When compared to wild type littermates, the diastolic function assessed by E/E’ ratio in HDAC2 2CA knock-in mice were relatively well conserved both in SAUNA- and TAC-induced DHF model. Survival rate after TAC operation was also dramatically ameliorated in HDAC2 2CA knock-in mice. Taken together, we proposed novel molecular axis, NOS1/S-NO HDAC2, accelerating diastolic dysfunction and which implicated notable molecular target for treatment of DHF patients.

Adeno-associated virus 9-mediated RNA interference targeting SOCS3 alleviates diastolic heart failure in rats

ObjectiveTo explore the effect of adeno-associated virus 9-mediated RNA interference targeting SOCS3 (AAV9-SOCS3 siRNA) on the treatment of diastolic heart failure (DHF). MethodA rat DHF model was established, and cardiac function and hemodynamic changes were measured. HE, Sirius red and TUNEL staining were applied to observe the pathological changes in the myocardium. Immunoblotting and immunohistochemical staining were utilized to detect SOCS3 expression. The expression levels of various factors, including fibrosis-related factors (collagen I, collagen II, α-SMA and TGF-β), inflammatory-related factors (IL-1β, IL-6, TNF-α, p-p65 and ICAM-1) and factors related to the JAK/STAT signal pathway were analyzed by immunoblotting and/or qPCR. The serum levels of IL-1β, IL-6, and TNF-α were measured using ELISA. ResultsSOCS3 expression was significantly downregulated in the DHF rat model by SOCS3 siRNA delivery. In the successfully established DHF rat model, cardiac function was clearly decreased, and cardiomyocyte apoptosis and myocardial fibrosis were significantly increased. These changes were ameliorated by treatment with AAV9-SOCS3 siRNA. The expression levels of p-JAK2 and p-STAT3 were significantly upregulated in the AAV9-SOCS3 siRNA group compared with the sham and AAV9-siRNA control groups, indicating that SOCS3 is a negative regulator of this signaling pathway. The expression levels of collagen I/III, α-SMA and TGF-β were also decreased at both the mRNA and protein levels. In addition, the serum and myocardial tissue expression levels of inflammatory-related factors, such as IL-6, IL-1β, and TNF-α, were also reduced by the administration of AAV9-SOCS3 siRNA compared with the AAV9-siRNA control. ConclusionsSOCS3 gene silencing by AAV9-SOCS3 siRNA administration in a DHF rat model significantly reduced myocardial fibrosis and the inflammatory response and improved heart function. Therefore, this treatment is a potential therapeutic method for treating DHF.

Effects of coupling meglumine cyclic adenylate and the human granulocyte colony-ostimulating factor on rat with diastolic heart failure

Objectives To explore the clinical effect of coupling meglumine cyclic adenylate(MCA)and the human granulocyte colony-ostimulating factor(G-CSF)on rat with diastolic heart failure(DHF). Methods Totally 60 rats of DHF model were evenly divided into 4 groups according to random number: Control group(n=15, control), Model group(n=15, DHF model), MCA group(n=15, treated with MCA)and MCA+ G-CSF group(n=15, treated with MCA plus G-CSF). MCA group were administered by intragastric injection of MCA 30 mg/kg/d for 15 d, MCA+ G-CSF group were administered by intragastric injection of MCA 30 mg/kg/d and plus G-CSF 100 μg/kg/d for 15 d, while Control group and Model group were given same volume of saline solution.BIOPAC SYSTEM was used to analyze the model establishment.The mRNA levels of GATA-4 and Cx43 were measured by RT-PCR.The protein expressions of GATA-4, Cx43, cTNI and c-kit were measured with western blotting.ELISA and flow cytometry were used to detect cAMP and differentiation rate of bone marrow mesenchymal stem cells(BMSCs), respectively. Results Compared with MCA group, the denaturation degree of myocardial tissues in DHF rat was significantly improved than in MCA+ G-CSF group.Moreover, the level of GATA-4(1.62±0.09), Cx43(1.02±0.07), cTNI(1.42±0.12), c-kit(0.65±0.02), cAMP(283.67±18.09)nmol/L and BMSCs cell differentiation rate(38.62±1.52)% in MCA+ G-CSF group were significantly promoted(all P<0.05)than in MCA group, GATA-4(0.82±0.07), Cx43(0.52±0.05), cTNI(0.86±0.13), c-kit(0.48±0.03), cAMP(198.83±16.03)nmol/L and BMSCs cell differentiation rate(19.82±0.89)%. Conclusions The combination of MCA with G-CSF is significantly improved DHF than single MAC treatment, which may regulate BMSCs differentiation though cAMP/PKA signaling pathways. Key words: Adenosine cyclophosphate meglumine; Granulocyte colony stimulating factor; Diastolic heart failure; Bone marrow mesenchymal stem cells

Toll-Like Receptor 9 Promotes Survival in SERCA2a KO Heart Failure Mice.

Aim. Inflammation is important in heart failure (HF). The role of the immune receptor toll-like receptor 9 (TLR9) in HF is not understood and not investigated in diastolic HF. We investigated the role of TLR9 in a murine diastolic HF model caused by cardiomyocyte SERCA2a excision. Methods and Results. We crossed SERCA2a KO and TLR9 KO mice to generate four mouse lines. Tamoxifen-induced cardiomyocyte SERCA2a gene excision was carried out in mice, causing diastolic HF. After 7.6 weeks, cardiac functions and dimensions were analyzed by echocardiography and heart tissues were processed. HF mice depleted of TLR9 demonstrated reduced survival compared to SERC2a KO mice, with a median life expectancy of 58 days compared to 63 days. Both HF groups displayed increased left atrium size, lung weight, fetal gene expressions, monocyte/macrophage infiltration, and fibrosis. However, there were no significant differences between the groups. Conclusion. In mice with SERCA2a KO-induced diastolic HF, the absence of TLR9 reduced median life expectancy. The cause remains elusive, as all investigated HF parameters were unaltered. Still, these findings support a salutary role of TLR9 in some subsets of HF conditions and underline the importance for future studies on the mechanisms of TLR9 in diastolic HF.

The sGC stimulator BAY 41-8543 in a rat model of hypertension-induced heart failure

Hypertension with left ventricular hypertrophy is a major cause of diastolic heart failure (DHF). Due to its high prevalence and high rate of mortality, DHF represents a major challenge in today's cardiovascular medicine; with limited therapeutic options. Soluble guanylate cyclase (sGC) stimulation is emerging as a promising treatment option in DHF, and is currently under investigation in preclinical and clinical studies. The present study investigates the effect of the sGC stimulator BAY 41-8543 in a transgenic rat model of hypertension-induced heart failure. We used 4 week-old male double transgenic rats expression both human renin and angiotensinogen genes (dTGRs). At 7 weeks of age, dTGRs exhibit striking cardiac hypertrophy with fibrosis and inflammation, ventricular arrhythmias and heart failure, which is accompanied with high mortality. We compared vehicle-treated dTGR (receiving 10% transcutol, 20% cremophor, 70% water) to those receiving 3 mg/kg/d BAY 41-8543, and vehicle-treated SD control rats (single oral dose per day for 3 weeks). We performed in vivo echocardiography, hemodynamic monitoring, cardiac electrophysiology studies and blood pressure measurements. Endothelial function was measured in isolated mesenteric arteries. Transcriptional analyses in cardiac tissue were performed using qRT-PCR and gene-microarray. Cardiac tissue was analyzed using histology. Treatment of dTGRs with BAY 41-8543 resulted in 100% survival at week 7, whereas only 24% of vehicle-treated dTGRs survived. Mean arterial pressure in dTGRs was significantly by BAY 41-8543 reduced (197 ± 11 mmHg vehicle vs 133 ± 4 mmHg BAY 41-8543). In addition, BAY 41-8543 significantly decreased in vivo total peripheral resistance and improved endothelium-dependent vasorelaxation of isolated mesenteric arteries. Furthermore BAY 41-8543 prevented fibrosis and inflammation of cardiac tissue. Echocardiography and invasive hemodynamic monitoring revealed BAY 41-8543 significantly increased ejection fraction and cardiac output in dTGR, whereas vehicle-treated had preserved systolic function but reduced diastolic function. In addition, diastolic compliance was significantly enhanced by BAY 41-8543, as shown by myocardial strain analysis and end-diastolic pressure volume relationship (EDPVR); indicative of an improved diastolic function. In vivo programmed electrical stimulation revealed a high ventricular tachycardia induction rate in vehicle-treated dTGRs (46%), which was significantly reduced in BAY 41-8543-treated dTGR (11%). Myocardial gene-microarray analysis showed a reversal of dysregulated genes in dTGR by BAY 41-8543 treatment. Our data demonstrate that BAY 41-8543 improves survival and cardiac performance in a transgenic rat model of hypertension-induced DHF. We postulate that treatment of DHF with sGC stimulators offers a novel therapeutic potential for humans.

Abstract P070: Exercise Prevents Cardiac Diastolic Dysfunction in a Female Model of Over-Nutrition Induced Obesity by Reducing Oxidant Stress and Fibrosis without Lowering Bodyweight

Obesity is being classified as a global epidemic by both the WHO and the CDC. Sedentary lifestyle and consumption of a high-fat/high-fructose Western diet (WD) are implicated in this epidemic. Women are particularly vulnerable to obesity related cardiovascular disease. Obese women suffer higher rates of hypertension, insulin resistance and heart failure, especially diastolic heart failure early in their lives. There are no evidence based treatments for diastolic heart failure. We hypothesized that voluntary daily exercise would prevent WD induced diastolic dysfunction by reducing oxidant stress, fibrosis and inflammation. To test this hypothesis, we developed a diastolic heart failure model by subjecting C57BL6/J female mice to a solely WD fed regimen for 16 weeks. We treated a parallel cohort with daily exercise, via voluntary wheel running, for the entire 16 weeks of WD feeding alongside control diet (CD) groups (n=7 for each group). We monitored food consumption, running activity and body weight. After 16 weeks, we assessed diastolic function by cardiac MRI and echocardiography. Detailed myocardial staining and Western blotting were done for cardiac oxidant stress, fibrosis and inflammatory markers. Both imaging modalities revealed diastolic dysfunction with WD feeding that was normalized by voluntary exercise. Mice ran similarly high intensities on both CD (6.5 km/d) and WD (7.1km/d). While WD feeding increased bodyweight, there was no reduction in weight with exercise. Body composition analysis showed WD fed mice treated with voluntary exercise had increases in visceral fat weight similar to sedentary WD fed mice. There was a notable increase in lean body mass with exercise. WD feeding resulted in insulin resistance that was prevented by exercise. Finally, while WD feeding markedly increased oxidant stress and fibrosis in sedentary mice, exercise prevented myocardial oxidant stress and fibrosis. Our work provides seminal evidence that diastolic dysfunction of over-nutrition induced obesity can be prevented by exercise. Surprisingly, our study suggests that the mechanisms behind the amelioration of diastolic dysfunction are predominantly through reductions in oxidant stress and fibrosis without reductions in body weight or visceral fat.

Abstract 18739: Acute Effect of the Na+/ca2+ Exchanger (ncx) Inhibitor Sea0400 on Diastolic Function in vivo and in vitro in a Model of Heart Failure With Preserved Ejection Fraction

Background: Heart failure with preserved ejection fraction (HFPEF) is increasingly common but underlying cellular mechanisms are not well understood. We investigated cardiomyocyte function and the role of SEA0400, in vivo and in vitro in a rat model of diastolic heart failure. Methods: Young male Wistar rats were subjected to subtotal nephrectomy (NXT) or sham operation (SOP). After 24 weeks in vivo (pressure-volume loops) and in vitro characterization (cardiomyocyte function (Ca2+ transients, sarcoplasmic reticulum (SR) diastolic Ca2+ leak (Ca2+ sparks) and SR Ca2+ content)) as well as NCX function (caffeine-induced Ca2+ transient, TAU) and protein expression) were determined without an with the NCX inhibitor SEA0400 (in vivo: 1 mg/kgKG for 30 minutes/ in vitro: 300nM for 5 minutes incubation). Results: NXT rats showed stable compensated renal impairment and signs and symptoms of HFPEF (hypertrophied left ventricle (LV), left- and upward shift of end diastolic pressure (EDP) volume relationship, increased lung weight/body weight ratio indicating pulmonary congestion and preserved LV systolic function (EF, dP/dt)). In LV cardiomyocytes from NXT Ca2+ transient amplitude was unchanged but time for early (50%) decay was significantly prolonged at 24 weeks and correlated with diastolic dysfunction (EDP) in vivo. TAU of the caffeine transient was significantly prolonged at 24 weeks indicating reduced NCX forward mode activity, while NCX protein expression was up-regulated, suggesting increased NCX reverse mode activity. Ca2+ spark frequency was increased and SR Ca2+ content was decreased in NXT (p&lt;0.05). SEA0400 significantly accelerated Ca2+ transient decay and reduced Ca2+ spark frequency in NXT. Acute in vivo treatment with SEA0400 significantly enhanced active relaxation (isovolumetric relaxation constant) of the LV in NXT. Conclusion: This model of HFPEF is associated with prolonged cytosolic Ca2+ decay and increased diastolic Ca2+ leak in LV cardiomyocytes. Acute treatment with NCX inhibitor SEA0400 normalized cytosolic Ca2+ transients, improved net transsarcolemmal Ca2+ export and decreased SR Ca2+ leak in NXT, in line with a role for reverse mode NCX activity in HFPEF. In vivo active relaxation was improved after acute SEA0400 treatment.

Abstract 606: Soluble Guanylate Cyclase (sGC) Stimulator Reduces The Cardiac And Renal Damage In Angiotensin II (Ang II)-induced Diastolic Heart Failure

Diastolic heart failure (DHF) is estimated to account for approximately 40% of heart failure cases. Due to the high prevalence and high mortality rates, diastolic heart failure represents a major challenge in cardiovascular medicine. The novel therapeutic alternative with NO-independent direct stimulators of sGC has recently been approved for the treatment of pulmonary hypertension. We analyzed the role of the sGC stimulator BAY 41-8543 in a transgenic rat model of hypertension-induced diastolic heart failure. We used 4 week-old male double transgenic rats harboring both human renin and angiotensinogen genes (dTGRs). At age 7 weeks, dTGR show striking cardiac hypertrophy with fibrosis, severe diastolic dysfunction but preserved systolic function, albuminuria, and renal failure. We compared vehicle-treated dTGR receiving 10% transcutol, 20% cremophor, 70% water, dTGR receiving 3 mg/kg/d BAY 41-8543, and vehicle-treated SD control rats (single oral dose per day for 3 weeks). Systolic blood pressure increased progressively in vehicle-treated dTGRs compared with SD rats. BAY 41-8543 significantly reduced the blood pressure (197 ± 11 mmHg vehicle vs 133 ± 4 mmHg BAY 41-8543). Treatment with sGC stimulator ameliorated albuminuria (0.3 ± 0.06 mg/8h at week 7) compared with vehicle-treated dTGRs (12.4 ± 2.6 mg/8h at week 7). In addition BAY 41-8543 prevented fibrosis and inflammation in kidney and heart. Cardiac hypertrophy or myocyte size were not reduced by BAY 41-8543 treatment. Cardiac echocardiography and hemodynamic investigations showed that BAY 41-8543 increased ejection fraction and improved diastolic function including speckle tracking and tissue doppler imaging. Programmed electrical stimulation showed a high non-sustained and sustained ventricular tachycardia induction rate in vehicle-treated dTGRs (46%), which was significantly reduced in BAY 41-8543-treated dTGR (11%). Finally, BAY 41-8543 treatment resulted in 100% survival at week 7, whereas 76% of vehicle-treated dTGRs died. Our data demonstrate that sGC stimulators ameliorate cardiac and renal end-organ damage including electrical remodeling in a transgenic rat model of hypertension-induced DHF. Treatment of DHF with sGC stimulators offers a novel therapeutic potential.

Genetics of diastolic heart failure

Heart failure explains a large portion of heart diseases. Molecular mechanisms determining cardiac function, by inference dysfunction in heart failure, are incompletely understood, especially in the common (or congestive) systolic (SHF) and diastolic heart failure (DHF). Limited genome-wide association studies (GWASs) in humans are reported on SHF and no GWAS has been performed on DHF. Genetic analyses in a rodent model of true DHF, Dahl salt-sensitive (DSS) rats, have begun to unravel the genetic components determining diastolic function. Diastolic dysfunction of DSS rats can be ameliorated or even normalized by distinct quantitative trait loci (QTLs), designated as diastolic function/blood pressure QTLs (DF/BP QTLs), which also affect blood pressure (BP). However, an improvement in diastolic dysfunction is merely transitory from a single DF/BP QTL, despite a permanent lowering of BP. A long-term protection against diastolic dysfunction can be realized only through combining specific DF/BP QTLs. Moreover, the worsening diastolic dysfunction with age can also be reversed in a different combination of DF/BP QTLs. Thus, distinct genes in combinations must be involved in the physiological mechanisms ameliorating or reversing diastolic dysfunction. As not all the QTLs that influence BP can affect diastolic function, it is not BP reduction itself that restores diastolic function, but rather specific genes that are uniquely integrated into the pathways of blood pressure homeostasis as well as diastolic function. Thus, the elucidation of pathophysiological mechanisms causal to hypertensive diastolic dysfunction will not only provide new diagnostic tools, but also novel therapeutic targets and strategies in reducing, curing, and even reversing DHF.

Compensated Cardiomyocyte Ca2+ Homeostasis in a Rat Model of Diastolic Heart Failure

In approximately half of heart failure patients, systolic function is near normal while diastolic function is impaired. This condition, called diastolic heart failure (DHF), or heart failure with preserved ejection fraction (HFPEF), has been reported to result from reduced passive distensibility of the ventricle, which impairs filling. However, the contribution of altered Ca2+ handling to impaired cardiomyocyte relaxation in this condition remains largely uninvestigated. We examined this issue in a rat model of DHF following aortic stenosis. 6 weeks after aortic banding, cardiac function was characterized by echocardiography, and rats exhibiting systolic heart failure (left atrial diameter >5mm, left ventricular dilation and reduced fractional shortening) were excluded. Echo Doppler measurements revealed decreased peak early diastolic tissue velocity in DHF rats compared to sham-operated controls (64±5 vs 85±5 mm/s, P<0.05). Both heart and lung weights were increased in DHF animals, indicating hypertrophy and congestion. In isolated cardiomyocytes, Ca2+ transients recorded by whole-cell fluorescence (fluo 4-AM) during field stimulation were of larger magnitude in DHF than control cells, across a range of frequencies (0.5-6 Hz). Ca2+ transients also decayed more rapidly in DHF, and at high frequencies exhibited elevated SR content and a smaller increase in diastolic [Ca2+]. Such augmentation of Ca2+ homeostasis was consistent with an increase in both sarcoplasmic reticulum Ca2+ reuptake and Ca2+ extrusion in DHF cells. Confocal imaging of cells stained with di-8-ANEPPs indicated maintained t-tubule organization, consistent with an unaltered time to peak of the Ca2+ transients. Thus, contrary to impaired Ca2+ homeostasis widely reported in systolic heart failure, our results suggest that DHF develops despite improved Ca2+ handling. We postulate that other alterations such as increased passive ventricular stiffness or increased myofilament Ca2+ sensitivity must impair diastolic function in this condition.

Abstract 19068: Connective Tissue Growth Factor in Human Diastolic Heart Failure from the Taiwan Diastolic Heart Failure Registry and a Canine Model of Diastolic Dysfunction

BACKGROUND: Diastolic heart failure (DHF) is characterized by myocardial interstitial fibrosis and left ventricular hypertrophy. The study investigated the association between connective tissue growth factor (CTGF) and DHF from animal model to clinical indices. METHODS: The participants of current study were enrolled from the Taiwan Diastolic Heart Failure Registry (TDHFR). 1284 patients were screened and a total of 125 patients with a diagnosis of DHF confirmed by echocardiography were recruited. The severity of DHF was decided according to tissue doppler imaging (E/e’). Soluble plasma levels of CTGF were measured in all subjects and the associations with diastolic function parameters were calculated. Canine model of DHF was induced by aortic banding. Hemodynamic and echocardiographic data were obtained before and after pressure loading (at baseline and after 2 weeks). Myocardium tissues were collected, and western blotting was used to detect the protein expression of CTGF for each dog. The correlation between CTGF, the severity of diastolic dysfunction and hemodynamic changes was then calculated. RESULTS: Patients with severe DHF presented significantly higher plasma CTGF levels than the controls. Significant correlation were found for plasma CTGF and E/e’ (r = 0.80, p&lt;0.001) in advanced DHF patients (E/e’ &gt;15). After 2 weeks in canine models, the protein expression of CTGF from LV myocardial tissue was significantly increased (p&lt;0.01) compared with the controls (shame dogs). Moreover, the expression of CTGF paralleled the severity of LV diastolic dysfunction parameters (r= 0.70, p= 0.03 for E/e’) and hemodynamic changes (r= 0.75, p= 0.02). After adjusting the possible confounding factors for diastolic dysfunction, CTGF levels were associated with diastolic parameters both in human and canine models (Plasma CTGF, B=0.064, p&lt;0.001; tissue CTGF, B= 9.87, p = 0.05). CONCLUSIONS: Both Plasma and myocardium CTGF levels had significant correlations to the severity of DHF. Our study offered the evidence to apply novel therapies for DHF patients aim to down-regulate the overexpression of CTGF.

Myofilament Phosphorylation and Function in Diastolic Heart Failure

This study aimed to compare alterations in phosphorylation and function of sarcomeric proteins in diastolic heart failure (DHF) vs. normal myocardium under two conditions of tissue procurement: beating-heart biopsy and postmortem tissue sampling. Left ventricular tissue samples were procured from normal (CTRL) or old dogs made hypertensive by renal wrapping (DHF), by either taking biopsies of the beating heart (n=7) or excising tissue postmortem (n=8). Isolated permeabilized cardiomyocytes were attached to a force transducer and passive tension (Fpassive) was measured between 1.8 and 2.4 μm, calcium sensitivity (pCa50) at 2.2 μm sarcomere length. Phosphorylation of myofilament proteins was assessed using the SYPRO-Ruby (total protein) / Pro-Q Diamond (phosphoprotein) system. Expression of total or phosphorylated protein, including titin-PEVK, cTnI, and PKCα was also quantified by immunoblot. Postmortem tissues and biopsy samples showed similar changes in DHF vs. CTRL, including increased phospho-PEVK, phospho-PKCα, and Fpassive, but decreased titin N2BA:N2B isoform ratio and reduced phosphorylation of total titin, cMyBPC, cTnT, cTnI and cTnI (S23/24). Differences were apparent in terms of lowered pCa50 in postmortem DHF, but increased pCa50 in biopsy DHF. Reduced MLC2 phosphorylation in DHF vs. CTRL was observed in biopsies, but not in postmortem tissues. A degradation form of titin (T2) was more abundant in postmortem tissues than in biopsies. We conclude that this DHF model is characterized by titin-isoform shift towards the stiff N2B isoform, a deficit in total titin phosphorylation, but increases in PKCα-dependent phosphorylation of titin-PEVK-domain and cardiomyocyte Fpassive, as well as altered pCa50. For a few parameters, postmortem and beating-heart samples differed in the direction of change in DHF vs. CTRL, particularly regarding Ca2+-sensitivity. However, most parameters changed in the same direction, suggesting the cellular events associated with death alter sample properties to a modest degree.

Differences in Sarcoplasmic Reticulum Ca(superscript 2+) Leak Characteristics between Systolic and Diastolic Heart Failure Rabbit Models

Diastolic heart failure (DHF) and systolic heart failure (SHF) are two clinical subsets of chronic heart failure (CHF). Sarcoplasmic reticulum (SR) Ca²⁺ leak has been measured in SHF and might contribute to contractile dysfunction and arrhythmogenesis. However, no study has investigated a similar phenomenon in DHF. Thus, we established DHF and SHF rabbit models and compared the differences in Ca²⁺ leak between these models. New Zealand white rabbits were randomly divided into three groups (n = 8 in each group): sham operation (SO) group, DHF group and SHF group. Cardiac functions were determined by echocardiography and hemodynamic assays. The SR Ca²⁺ leak was measured with a calcium-imaging device and the expression and activities of related proteins were evaluated with Western blots and autophosphorylation. In the DHF group, there was significantly increased ventricular wall thickness and stiffness, reduced diastolic function, and total amount of FK506 binding protein 12.6 (FKBP12.6), increased expression and activity of protein kinase A (PKA) and phosphorylation site (P2809) in the ryanodine receptor (RyR2), but no prominent Ca²⁺ leak. In the SHF group, there was significantly increased ventricular cavity size, reduced systolic function, increased SR Ca²⁺ leak, reduced total amount of FKBP12.6 and FKBP12.6-RyR2 association, increased expression and activity of PKA and Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) and their RyR2 phosphorylation sites with unchanged P2030. Our results suggest that a prominent SR Ca²⁺ leak was not observed in the DHF model, which may provide a new idea for the reasons in preserved systolic function, and CaMKII possibly plays a more important role in SR Ca²⁺ leak.

Evaluation of left ventricular twist in rabbit model with heart failure by speckle tracking imaging

Objective To assess left ventricular twist and untwist in rabbit models with diastolic heart failure(DHF) and systolic heart failure(SHF) by speckle tracking imaging(STI).Methods Thirtythree male New Zealand rabbits were divided into three groups:one group was used to establish DHF model by suprarenal abdominal aortic constriction(n = 12) and the other was used to establish SHF model by abdominal aortic constriction subdiaphragmly above celiac artery origin(n = 11),another was control group established by sham operation(n = 10).After two weeks,left ventricular twist-related parameters were measured.by STE.Finallly,left ventricular catheterization and myocardial pathological examination were performed.Results Compared with control group,peak twist angle (Ptw) and peak twisting velocity(PTV)remained unchanged in DHF group (P ＞ 0.05) and decreased in SHF group (P ＜ 0.05).Isovolumic untwisting rate (IUR) decreased,untwisting half-time(UHT) was prolonged,time to peak twist (tPtw) and peak untwisting velocity (tPUTV) were delayed in SHF and DHF groups.In additon,Peak untwisting velocity(PUTV) decreased in SHF group,but increased in DHF group (P ＜0.05).Ptw correlated correlated positively with LVEF,+ dp/dtmax significantly in both groups (DHF group:r = 0.897,P =0.002; r = 0.719,P = 0.044;SHF group:r = 0.727,P = 0.041; r = 0.780,P = 0.022).A significantly positive correlation was observed between PUTV and - dp/dtmax Tau,LVEDP(r = 0.801,P = 0.017; r =0.814,P = 0.014; r = 0.875,P = 0.004) in SHF group.Conclusions STI is useful in evaluating left ventricular twist and untwist in rabbit model of DHF and SHF. Key words: Echocardiography; Heart failure,congestive; Torsion; Speckle tracking imaging

Sildenafil Preserves Lung Endothelial Function and Prevents Pulmonary Vascular Remodeling in a Rat Model of Diastolic Heart Failure

Pulmonary hypertension as a frequent complication of left heart disease (PH-LHD) is characterized by lung endothelial dysfunction and vascular remodeling. Although PH-LHD contributes to morbidity and mortality in heart failure, established therapies for PH-LHD are lacking. We tested the effect of chronic sildenafil treatment in an experimental model of PH-LHD. In Sprague-Dawley rats, PH-LHD was induced by supracoronary aortic banding. Oral sildenafil treatment (60 mg/kg daily) was initiated after 7 days, and lung endothelial function (n=5), vascular remodeling, and right ventricular function (n=11 each) were analyzed 9 weeks after banding. As compared with sham-operated controls, aortic banding induced pulmonary hypertension and lung endothelial dysfunction evident as lack of endothelial nitric oxide production and endothelium-dependent vasodilation. These changes were associated with an increased pulmonary vascular resistance, medial thickening, and biventricular cardiac hypertrophy. Sildenafil treatment largely attenuated these pathological changes and was not associated with detectable adverse effects pertinent to lung vascular barrier function, edema formation, or systemic hemodynamics. Our data identify sildenafil as a promising therapy for PH-LHD. In light of its documented protective effects at the myocardial level in heart failure, sildenafil presents a particularly attractive strategy in that it simultaneously targets cardiac remodeling and secondary PH-LHD.

Abstract 5082: Interferon- γ Plays a Pivotal Role in Hypertension-Induced Cardiac Remodeling and Diastolic Heart Failure

Background: Diastolic heart failure (DHF) is clinical conundrum and accounts for ~50% of all HF admissions. There remains a paucity of therapies. Hypertension is the major cause of DHF and is associated with LV hypertrophy (LVH). Inflammatory cytokines are increased in hypertension but it is unknown if they mediate hypertension-induced cardiac remodeling. We tested the hypothesis that interferon- γ plays a pivotal role in the progression from hypertension-induced LVH to DHF. Methods: Twelve-week old BALB/c (WT) and interferon- γ deficient (IFN γ KO) mice underwent either saline (saline; n=10) or aldosterone-infusion (ALDO; n=20). All underwent uninephrectomy and drank 1% salt water for 4-weeks. Four-weeks after surgery, tail-cuff blood pressure (BP), echocardiogram and gene/protein analyses were performed. Isolated adult rat ventricular myocytes (ARVM) were treated with either interferon- γ (500nM) and/or aldosterone (50nM) for 30min. Results: BP was increased in WT-ALDO vs. WT-saline mice (P&lt;0.01). Interestingly BP was not as increased in IFN γ KO-ALDO vs. WT-ALDO mice (127±5 vs. 136±4mmHg, P=NS). There was LVH in WT-ALDO vs. WT-saline mice (P&lt;0.05). LVH was further increased in IFN γ KO-ALDO vs. WT-ALDO mice (HW/BW ratio: 4.9±0.1 vs. 4.3±0.1mg/g; P&lt;0.01). LV end diastolic dimension was no different between IFNã KO-ALDO vs. WT-ALDO hearts. However, LV end systolic dimension (LVESD) was significantly smaller in IFNã KO-ALDO vs. IFNã KO-saline and WT-ALDO mice (1.12±0.1 vs. 1.4±0.1, and 2.1±0.3, respectively; P &lt;0.05 for both). LVH was associated with an increase in myocardial ANP gene and protein expression in WT-ALDO hearts (P&lt;0.05 vs. WT-sham). Intriguingly ANP gene and protein expression was not increased in IFNã KO-ALDO vs. WT-ALDO hearts. In ARVM pretreatment with interferon-ã prior to aldosterone significantly inhibited aldosterone-induced ERK1/2 phosphorylation, another marker of cardiomyocyte hypertrophy (P&lt;0.05). Conclusions: Interferon- γ modulates cardiomyocyte hypertrophy partly independently of BP. There is a propensity to adverse cardiac remodeling with interferon- γ deficiency in this model of hypertension and DHF. Our findings suggest that interferon- γ may represent a novel target for hypertensive heart disease and DHF.