Rodent Model Of Heart Failure Research Articles

NAD+ precursors prolong survival and improve cardiac phenotypes in a mouse model of Friedreich's Ataxia.

Friedreich's ataxia (FRDA) is a progressive disorder caused by insufficient expression of frataxin, which plays a critical role in assembly of iron-sulfur centers in mitochondria. Individuals are cognitively normal but display a loss of motor coordination and cardiac abnormalities. Many ultimately develop heart failure. Administration of nicotinamide adenine dinucleotide-positive (NAD+) precursors has shown promise in human mitochondrial myopathy and rodent models of heart failure, including mice lacking frataxin in cardiomyocytes. We studied mice with systemic knockdown of frataxin (shFxn), which display motor deficits and early mortality with cardiac hypertrophy. Hearts in these mice do not "fail" per se but become hyperdynamic with small chamber sizes. Data from an ongoing natural history study indicate that hyperdynamic hearts are observed in young individuals with FRDA, suggesting that the mouse model could reflect early pathology. Administering nicotinamide mononucleotide or riboside to shFxn mice increases survival, modestly improves cardiac hypertrophy, and limits increases in ejection fraction. Mechanistically, most of the transcriptional and metabolic changes induced by frataxin knockdown are insensitive to NAD+ precursor administration, but glutathione levels are increased, suggesting improved antioxidant capacity. Overall, our findings indicate that NAD+ precursors are modestly cardioprotective in this model of FRDA and warrant further investigation.

Rodent Models of Dilated Cardiomyopathy and Heart Failure for Translational Investigations and Therapeutic Discovery.

Even with modern therapy, patients with heart failure only have a 50% five-year survival rate. To improve the development of new therapeutic strategies, preclinical models of disease are needed to properly emulate the human condition. Determining the most appropriate model represents the first key step for reliable and translatable experimental research. Rodent models of heart failure provide a strategic compromise between human in vivo similarity and the ability to perform a larger number of experiments and explore many therapeutic candidates. We herein review the currently available rodent models of heart failure, summarizing their physiopathological basis, the timeline of the development of ventricular failure, and their specific clinical features. In order to facilitate the future planning of investigations in the field of heart failure, a detailed overview of the advantages and possible drawbacks of each model is provided.

AW-NI-3: EFFECTS OF RENAL DENERVATION ON LEFT AND RIGHT VENTRICULAR FUNCTION IN HYPERTENSIVE RATS WITH HEART FAILURE INDUCED BY VOLUME OVERLOAD

Objective: Clinical studies showed that renal denervation (RDN) is effective in blood pressure lowering in hypertension. Previously, we found that RDN prolongs survival in a hypertensive rodent model of heart failure due to volume overload from aorto-caval fistula (ACF). Whether it is due to afterload removal, or due to intrinsic effects of RDN on the right (RV) or left (LV) ventricular function, is currently unknown and was the focus of our investigation. Design and method: 8-week-old Ren-2 transgenic male rats (model of angiotensin II-dependent hypertension) underwent ACF surgery to induce volume overload. After one week, animals underwent bilateral RDN (mechanical and chemical by topical phenol application) from laparotomy. Two weeks after RDN, the functions of both ventricles were measured by simultaneous biventricular pressure-volume analysis, and the animals were examined using echocardiography each week. Results: RDN in ACF rats only slightly reduced maximum pressure in RV but did not affect maximum pressure in LV. Yet, RDN in ACF animals significantly reduced hypertrophy and dilatation of both ventricles and decreased lung and liver congestion. RDN in ACF rats improved load-independent measures of contractility of both ventricles (end-systolic elastance and preload recruitable stroke work). RDN in ACF rats reduced renal levels of norepinephrine (NE) but restored depleted NE both in RV and LV. At the gene expression level, RDN in ACF led to favorable remodeling which was reflected as increased transcription of beta-1 adrenergic receptors in LV and beta-2 adrenergic receptors in RV and LV, decreased transcription of natriuretic peptide A gene (NPPA), collagen 1, and monoaminoxidase A (MAOA). These changes were relatively more pronounced in RV than LV. Interestingly, RDN in ACF rats also significantly inhibited the activity of neprilysin in the kidney. Conclusions: Besides the effects on pressure, RDN favorably affected load-independent measures of chamber function, markers of myocardial remodeling, and restored myocardial norepinephrine levels. These data indicate that RDN effects extend beyond pressure lowering and could be a promising method in the treatment of both right and left ventricular heart failure.

Exogenous ketone supplementation: an emerging tool for physiologists with potential as a metabolic therapy.

What is the topic of this review? The integrative physiological response to exogenous ketone supplementation. What advances does it highlight? The physiological effects and therapeutic potential of exogenous ketones on metabolic health, cardiovascular function, cognitive processing, and modulation of inflammatory pathways and immune function. Also highlighted are current challenges and future directions of the field. Exogenous oral ketone supplements, primarily in form of ketone salts or esters, have emerged as a useful research tool for manipulating metabolism with potential therapeutic application targeting various aspects of several common chronic diseases. Recent literature has investigated the effects of exogenously induced ketosis on metabolic health, cardiovascular function, cognitive processing, and modulation of inflammatory pathways and immune function. This narrative review provides an overview of the integrative physiological effects of exogenous ketone supplementation and highlights current challenges and future research directions. Much of the existing research on therapeutic applications - particularly mechanistic studies - has involved pre-clinical rodent and/or cellular models, requiring further validation in human clinical studies. Existing human studies report that exogenous ketones can lower blood glucose and improve some aspects of cognitive function, highlighting the potential therapeutic application of exogenous ketones for type 2 diabetes and neurological diseases. There is also support for the ability of exogenous ketosis to improve cardiac metabolism in rodent models of heart failure with supporting human studies emerging; long-terms effects of exogenous ketone supplementation on the human cardiovascular system and lipid profiles are needed. An important avenue for future work is provided by research accelerating technologies that enable continuous ketone monitoring and/or the development of more palatable ketone mixtures that optimize plasma ketone kinetics to enable sustained ketosis. Lastly, research exploring the physiological interactions between exogenous ketones and varying metabolic states (e.g., exercise, fasting, metabolic disease) should yield important insights that can be used to maximize the health benefits of exogenous ketosis.

Searching for an experimental rodent model of heart failure with preserved ejection fraction: Re-visited.

Heart failure with preserved ejection fraction (HFpEF) currently accounts for over 50% of all heart failure cases. It displays a large number of comorbidities, and the pathophysiological mechanisms underlying the disease remain uncertain and treatment options are limited. The heterogeneity and complexity of the disease, and its specific comorbidities, can limit the number of animal models that could ideally mimic it. The current study describes and compares the efficacy of the most popular approaches from a quantitative point of view. A review and meta-analysis of more than 500 experimental protocols was performed with special attention to these models created to induce heart failure by the most common comorbidities associated with human HFpEF, e.g., hypertension, diabetes, obesity and aging. The analysis included a wide spectrum of outcomes (alterations in body weight, lung and left ventricle weights, laboratory, hemodynamic, echocardiographic, and histopathological data as well as animal mortality) and possible covariates that could determine the utility of the particular model, such as animal age, species, experimental period and genetic modification. A wide range of systemic hypertension as well as diabetes (obesity) - related animal models are used for pre-clinical studies on heart failure, but some of them fail to replicate HFpEF. Future studies should include an evaluation of other features besides diastolic dysfunction that confirm that this is an HFpEF model, or the potential to progress to heart failure with reduced ejection fraction (HFrEF).

Abstract 334: Gut Microbiome Derived Trimethyl-n-oxide Exacerbates Inflammation And Induces Cardiac Fibrosis In Human Cardiac Fibroblasts And In Rodent Model Of Cardiorenal Disease

Trimethlyamine N-oxide (TMAO), a gut-microbiome derived metabolite of dietary choline and other trimethylamine containing nutrients, has been consistently associated with poor prognosis with cardiovascular diseases (CVD) in recent years. Clinical studies have demonstrated that systemic circulating TMAO levels are independently associated with adverse CVD events such as MI, heart failure (HF), stroke, CKD and death. Previous studies have reported elevated TMAO both in mice models of atherosclerosis and in patients with atherosclerosis, and association between increased TMAO levels with adverse outcomes in HF patients, with significant correlation between increased TMAO levels and heart failure risk. The role and underlying mechanisms of TMAO in mediating disease pathophysiology remains elusive. Here, we use primary human cardiac fibroblasts and rodent models of heart failure with TMAO challenge to study its role in the heart and in cardiac fibroblasts. C57BL/6 mice were administered with TMAO (0.2%) in the drinking water for 2, 4 and 8 weeks. Heart tissues were collected at the end of the study to evaluate cardiac fibrosis, and blood samples were evaluated for TMAO levels. For heart failure studies, ZSF1 rats that underwent uninephrectomy surgery were fed high salt diet and were used as a model for heart failure with preserved ejection fracture (HFpEF), and were followed for 12 weeks. ZSF1 rats were also fed with high choline diet and cardiac function was measured using echocardiography. We demonstrated an elevation in blood TMAO levels, with a concomitant increase in circulating pro-inflammatory cytokine levels and upregulation in fibrosis markers in hearts from mice administered TMAO. In a ZSF1 rat model of HFpEF, we showed that increased TMAO levels correlated with deteriorating cardiac dysfunction. In human cardiac fibroblasts, stimulation with TMAO promoted upregulation of key fibrosis markers such as TGF-β1 in a dose-dependent manner. Inflammation, cardiac function, and cardiac fibrosis were exacerbated in mice administered with TMAO in their drinking water. Furthermore, in ZSF1 rat model for HFpEF, there was a significant association of high TMAO levels to worsening of cardiac and renal dysfunction.

KLF2 regulates neutrophil activation and thrombosis in cardiac hypertrophy and heart failure progression.

It is widely recognized that inflammation plays a critical role in cardiac hypertrophy and heart failure. However, clinical trials targeting cytokines have shown equivocal effects, indicating the need for a deeper understanding of the precise role of inflammation and inflammatory cells in heart failure. Leukocytes from human subjects and a rodent model of heart failure were characterized by a marked reduction in expression of Klf2 mRNA. Using a mouse model of angiotensin II–induced nonischemic cardiac dysfunction, we showed that neutrophils played an essential role in the pathogenesis and progression of heart failure. Mechanistically, chronic angiotensin II infusion activated a neutrophil KLF2/NETosis pathway that triggered sporadic thrombosis in small myocardial vessels, leading to myocardial hypoxia, cell death, and hypertrophy. Conversely, targeting neutrophils, neutrophil extracellular traps (NETs), or thrombosis ameliorated these pathological changes and preserved cardiac dysfunction. KLF2 regulated neutrophil activation in response to angiotensin II at the molecular level, partly through crosstalk with HIF1 signaling. Taken together, our data implicate neutrophil-mediated immunothrombotic dysregulation as a critical pathogenic mechanism leading to cardiac hypertrophy and heart failure. This neutrophil KLF2-NETosis-thrombosis mechanism underlying chronic heart failure can be exploited for therapeutic gain by therapies targeting neutrophils, NETosis, or thrombosis.

Sigmar1's Subcellular Localization and Function in the Heart

Background Sigma-1 receptor (Sigmar1) is a widely expressed molecular chaperone protein in mammalian cell systems. Accumulating research demonstrated the cardioprotective roles of Sigmar1 targeted ligands in clinical and preclinical rodent models of heart failure. However, Extensive biochemical and immuno-electron microscopic research demonstrated Sigmar1's sub-cellular localization largely depends on cell and organ types. Despite extensive studies, Sigmar1's subcellular localization and transmembrane topology in cardiomyocytes remain elusive. Objective We determined Sigmar1's subcellular localization, transmembrane topology, and functionality using complementary microscopy, biochemical, and functional assays in cardiomyocytes. Methods and Results Subcellular fractionation of heart cell lysates showed Sigmar1's localization in purified mitochondria fraction. Sigmar1's mitochondrial localization in isolated adult mouse cardiomyocytes by immunocytochemistry was confirmed by Sigmar1 co-localization with the mitochondrial indicator OXPHOS. We performed quantum dots in transmission electron microscopy and observed Sigmar1 leveled quantum dots on the mitochondrial membranes. We also confirmed Sigmar1 localization confocal microscopy using isolated mitochondria stained with Mito-Tracker red and Sigmar1 with the anti-Sigmar1 antibody. We confirmed Sigmar1 is an integral mitochondrial membrane protein by performing a series of biochemical experiments, including alkaline extraction and proteinase K treatment of purified heart mitochondria. We determined the Sigmar1's structural requirement for mitochondrial localization by expressing Sigmar1 fragments in cells. Immunostaining with anti-FLAG antibody showed that full-length Sigmar1 and Sigmar1's C terminal-deletion mutants (amino acid (aa) 1-223 and aa1-101) is localized in the mitochondrial membrane (mitochondria stained with Mito-Tracker red) whereas N- terminal deletion mutants (aa 102-223) were unable to incorporate into the mitochondria. Finally, we determined the functional role of Sigmar1 in cardiomyocytes using high-resolution Respirometry (Oroboros O2K) in intact cardiomyocytes. Genetic loss-of-function studies demonstrated that siRNA knockdown of Sigmar1 causes a significant reduction of oxygen flux in the presence of Complex I substrates and mitochondrial respiration un-coupler, under ADP supported OXPHOS state in isolated neonatal cardiomyocytes. Conclusions Overall, our studies revealed that Sigmar1 localizes to mitochondrial membranes and indispensable for maintaining mitochondrial respiratory homeostasis in cardiac myocytes.

Divergent and Overlapping Roles for Selected Phytochemicals in the Regulation of Pathological Cardiac Hypertrophy.

Plant-based foods, like fruits, vegetables, whole grains, legumes, nuts, seeds and other foodstuffs, have been deemed as heart healthy. The chemicals within these plant-based foods, i.e., phytochemicals, are credited with protecting the heart. However, the mechanistic actions of phytochemicals, which prevent clinical endpoints, such as pathological cardiac hypertrophy, are still being elucidated. We sought to characterize the overlapping and divergent mechanisms by which 18 selected phytochemicals prevent phenylephrine- and phorbol 12-myristate 13-acetate-mediated cardiomyocyte enlargement. Of the tested 18 compounds, six attenuated PE- and PMA-mediated enlargement of neonatal rat ventricular myocytes. Cell viability assays showed that apigenin, baicalein, berberine hydrochloride, emodin, luteolin and quercetin dihydrate did not reduce cell size through cytotoxicity. Four of the six phytochemicals, apigenin, baicalein, berberine hydrochloride and emodin, robustly inhibited stress-induced hypertrophy and were analyzed further against intracellular signaling and genome-wide changes in mRNA expression. The four phytochemicals differentially regulated mitogen-activated protein kinases and protein kinase D. RNA-sequencing further showed divergence in gene regulation, while pathway analysis demonstrated overlap in the regulation of inflammatory pathways. Combined, this study provided a comprehensive analysis of cardioprotective phytochemicals. These data highlight two defining observations: (1) that these compounds predominantly target divergent gene pathways within cardiac myocytes and (2) that regulation of overlapping signaling and gene pathways may be of particular importance for the anti-hypertrophic actions of these phytochemicals. Despite these new findings, future works investigating rodent models of heart failure are still needed to understand the roles for these compounds in the heart.

Novel Cardiac Troponin I S43/45N Substitution does not Cause Aberrant Cardiac Remodeling or Dysfunction in Mice

Elevated protein kinase C (PKC) expression and activity, along with downstream phosphorylation of cardiac troponin I (cTnI) are linked to cardiac dysfunction. Specifically, elevated cTnI S43/45 phosphorylation is reported in human and rodent models of heart failure, but the exact role of phosphorylation at this site is still debated. Transgenic (tg) mice expressing cardiac-specific phospho-mimetic S43/45D develop contractile dysfunction, followed by cardiac hypertrophy, fibrosis, and progressive heart failure. Thus, S43/45 phosphorylation is postulated to be a sarcomeric node for modulating contractile function in the heart. To further test this idea, it is critical to show that a phospho-null substitution at this site does not alter wild-type function. Multiple groups have used the conventional nonpolar alanine (A) phospho-null substitution at S43/45 and report diminished myocyte and contractile function, which resembles the outcome after phosphorylation. We postulate the nonpolar properties of A potentially alter interactions between cTnI, troponin T (TnT) and C (TnC). As a result, the amide R side chain in asparagine (N), with a polarity somewhat similar to the hydroxyl group in S, could make N a reasonable non-phosphorylatable residue without overt effects on wild-type function. Previously, adult rat myocytes virally transduced with S43/45N (SN)in our laboratory did not display overt changes in contractile function compared to wild-type myocytes. In ongoing experiments, generation of tg mice expressing a cardiac-specific cTnISN also show no changes in in vivo cardiac contractile function or evidence of remodeling in mice up to at least four months of age. These findings suggest the novel N substitution acts as a “functionally conservative” phospho-null substitution at cTnIS43/45 in myocardium.

Ketone Ester Treatment Improves Cardiac Function and Reduces Pathologic Remodeling in Preclinical Models of Heart Failure.

Accumulating evidence suggests that the failing heart reprograms fuel metabolism toward increased utilization of ketone bodies and that increasing cardiac ketone delivery ameliorates cardiac dysfunction. As an initial step toward development of ketone therapies, we investigated the effect of chronic oral ketone ester (KE) supplementation as a prevention or treatment strategy in rodent heart failure models. Two independent rodent heart failure models were used for the studies: transverse aortic constriction/myocardial infarction (MI) in mice and post-MI remodeling in rats. Seventy-five mice underwent a prevention treatment strategy with a KE comprised of hexanoyl-hexyl-3-hydroxybutyrate KE (KE-1) diet, and 77 rats were treated in either a prevention or treatment regimen using a commercially available β-hydroxybutyrate-(R)-1,3-butanediol monoester (DeltaG; KE-2) diet. The KE-1 diet in mice elevated β-hydroxybutyrate levels during nocturnal feeding, whereas the KE-2 diet in rats induced ketonemia throughout a 24-hour period. The KE-1 diet preventive strategy attenuated development of left ventricular dysfunction and remodeling post-transverse aortic constriction/MI (left ventricular ejection fraction±SD, 36±8 in vehicle versus 45±11 in KE-1; P=0.016). The KE-2 diet therapeutic approach also attenuated left ventricular dysfunction and remodeling post-MI (left ventricular ejection fraction, 41±11 in MI-vehicle versus 61±7 in MI-KE-2; P<0.001). In addition, ventricular weight, cardiomyocyte cross-sectional area, and the expression of ANP (atrial natriuretic peptide) were significantly attenuated in the KE-2-treated MI group. However, treatment with KE-2 did not influence cardiac fibrosis post-MI. The myocardial expression of the ketone transporter and 2 ketolytic enzymes was significantly increased in rats fed KE-2 diet along with normalization of myocardial ATP levels to sham values. Chronic oral supplementation with KE was effective in both prevention and treatment of heart failure in 2 preclinical animal models. In addition, our results indicate that treatment with KE reprogrammed the expression of genes involved in ketone body utilization and normalized myocardial ATP production following MI, consistent with provision of an auxiliary fuel. These findings provide rationale for the assessment of KEs as a treatment for patients with heart failure.

Endothelial Mesenchymal Transition and Cardiorenal Syndrome in a Rodent Model of Heart Failure with a Preserved Ejection Fraction

Background Effective clinical management of heart failure, particularly Heart Failure with a preserved Ejection Fraction (HFpEF), is often challenged by concurrent renal dysfunction, known as the cardiorenal syndrome (CRS). Combined with the notable lack of evidence-based therapies for HFpEF, it is crucial to elucidate mechanisms of disease progression and targetable pathways in HFpEF with and without CRS. Using an established murine model, we investigated the presence of pathophysiological remodeling in the kidney in HFpEF. Methods Uninephrectomized wild-type mice were infused with d-aldosterone (HFpEF, n = 11) or saline (Sham, n = 10) and all received 1% NaCl drinking water. After 4 weeks, once HFpEF developed, mice were euthanized, and organ weights measured. Histology and RNA gene analysis were performed on the kidneys. Results This well-characterized HFpEF model features moderate hypertension, lung congestion, and exercise intolerance accompanied by cardiac hypertrophy, diastolic dysfunction and cardiac fibrosis. Renal hypertrophy was evident as shown by a 46% increase of kidney-to-body weight mass in HFpEF (10.8±0.3 mg/g) vs. Sham (7.4±0.3 mg/g; P Conclusion This rodent model of HFpEF aptly mirrors the major hallmarks of clinical HFpEF, with evidence of concurrent renal hypertrophy and fibrosis. Activation of Endo-MT markers suggests this may mediate the presence of the cardiorenal syndrome in HFpEF. Further studies are warranted to investigate these findings in patients with HFpEF and elucidate potential therapeutic avenues for CRS in HFpEF patient populations.

Effects of empagliflozin and target-organ damage in a novel rodent model of heart failure induced by combined hypertension and diabetes

Type 2 diabetes mellitus and hypertension are two major risk factors leading to heart failure and cardiovascular damage. Lowering blood sugar by the sodium-glucose co-transporter 2 inhibitor empagliflozin provides cardiac protection. We established a new rat model that develops both inducible diabetes and genetic hypertension and investigated the effect of empagliflozin treatment to test the hypothesis if empagliflozin will be protective in a heart failure model which is not based on a primary vascular event. The transgenic Tet29 rat model for inducible diabetes was crossed with the mRen27 hypertensive rat to create a novel model for heart failure with two stressors. The diabetic, hypertensive heart failure rat (mRen27/tetO-shIR) were treated with empagliflozin (10 mg/kg/d) or vehicle for 4 weeks. Cardiovascular alterations were monitored by advanced speckle tracking echocardiography, gene expression analysis and immunohistological staining. The novel model with increased blood pressure und higher blood sugar levels had a reduced survival compared to controls. The rats develop heart failure with reduced ejection fraction. Empagliflozin lowered blood sugar levels compared to vehicle treated animals (182.3 ± 10.4 mg/dl vs. 359.4 ± 35.8 mg/dl) but not blood pressure (135.7 ± 10.3 mmHg vs. 128.2 ± 3.8 mmHg). The cardiac function was improved in all three global strains (global longitudinal strain − 8.5 ± 0.5% vs. − 5.5 ± 0.6%, global radial strain 20.4 ± 2.7% vs. 8.8 ± 1.1%, global circumferential strain − 11.0 ± 0.7% vs. − 7.6 ± 0.8%) and by increased ejection fraction (42.8 ± 4.0% vs. 28.2 ± 3.0%). In addition, infiltration of macrophages was decreased by treatment (22.4 ± 1.7 vs. 32.3 ± 2.3 per field of view), despite mortality was not improved. Empagliflozin showed beneficial effects on cardiovascular dysfunction. In this novel rat model of combined hypertension and diabetes, the improvement in systolic and diastolic function was not secondary to a reduction in left ventricular mass or through modulation of the afterload, since blood pressure was not changed. The mRen27/tetO-shIR strain should provide utility in separating blood sugar from blood pressure-related treatment effects.

Empagliflozin Blunts Worsening Cardiac Dysfunction Associated With Reduced NLRP3 (Nucleotide-Binding Domain-Like Receptor Protein 3) Inflammasome Activation in Heart Failure.

Although empagliflozin was shown to profoundly reduce cardiovascular events in diabetic patients and blunt the decline in cardiac function in nondiabetic mice with established heart failure (HF), the mechanism of action remains unknown. We treated 2 rodent models of HF with 10 mg/kg per day empagliflozin and measured activation of the NLRP3 (nucleotide-binding domain-like receptor protein 3) inflammasome in the heart. We show for the first time that beneficial effects of empagliflozin in HF with reduced ejection fraction (HF with reduced ejection fraction [HFrEF]; n=30-34) occur in the absence of changes in circulating ketone bodies, cardiac ketone oxidation, or increased cardiac ATP production. Of note, empagliflozin attenuated activation of the NLRP3 inflammasome and expression of associated markers of sterile inflammation in hearts from mice with HFrEF, implicating reduced cardiac inflammation as a mechanism of empagliflozin that contributes to sustained function in HFrEF in the absence of diabetes mellitus. In addition, we validate that the beneficial cardiac effects of empagliflozin in HF with preserved ejection fraction (HFpEF; n=9-10) are similarly associated with reduced activation of the NLRP3 inflammasome. Lastly, the ability of empagliflozin to reduce inflammation was completely blunted by a calcium (Ca2+) ionophore, suggesting that empagliflozin exerts its benefit upon restoring optimal cytoplasmic Ca2+ levels in the heart. These data provide evidence that the beneficial cardiac effects of empagliflozin are associated with reduced cardiac inflammation via blunting activation of the NLRP3 inflammasome in a Ca2+-dependent manner and hence may be beneficial in treating HF even in the absence of diabetes mellitus.

Cognitive impairment in heart failure is associated with altered Wnt signaling in the hippocampus.

Age represents the highest risk factor for death due to cardiovascular disease. Heart failure (HF) is the most common cardiovascular disease in elder population and it is associated with cognitive impairment (CI), diminishing learning and memory process affecting life quality and mortality in these patients. In HF, CI has been associated with inadequate O2 supply to the brain; however, an important subset of HF patients displays CI with almost no alteration in cerebral blood flow. Importantly, nothing is known about the pathophysiological mechanisms underpinning CI in HF with no change in brain tissue perfusion. Here, we aimed to study memory performance and learning function in a rodent model of HF that shows no change in blood flow going to the brain. We found that HF rats presented learning impairments and memory loss. In addition, HF rats displayed a decreased level of Wnt/β-catenin signaling downstream elements in the hippocampus, one pathway implicated largely in aging diseases. Taken together, our results suggest that in HF rats CI is associated with dysfunction of the Wnt/β-catenin signaling pathway. The mechanisms involved in the alterations of Wnt/β-catenin signaling in HF and its contribution to the development/maintenance of CI deserves future investigations.

Empagliflozin Improves Diastolic Function in a Nondiabetic Rodent Model of Heart Failure With Preserved Ejection Fraction

Recent studies send an unambiguous signal that the class of agents known as sodium-glucose-linked co-transporter-2inhibitors (SGLT2i) prevent heart failure hospitalization in patients with type 2 diabetes. However, the mechanisms remain unclear. Herein the authors utilize a rodent model of heart failure with preserved ejection fraction (HFpEF), and demonstrate that treatment with the SGLT2i empagliflozin, reduces left ventricular mass, improving both wall stress and diastolic function. These findings extend the observation that the main mechanism of action of empagliflozin involves improved hemodynamics (i.e., reduction in preload and afterload) and provide a rationale for upcoming trials in patients with HFpEF irrespective of glycemic status.

Mitochondrial Morphology, Dynamics, and Function in Human Pressure Overload or Ischemic Heart Disease With Preserved or Reduced Ejection Fraction.

The FOXO3a (forkhead box O3a)-BNIP3 (B-cell lymphoma 2/adenovirus E1B 19kDa interacting protein 3) pathway modulates mitochondrial dynamics and function and contributes to myocardial remodeling in rodent models of heart failure. We sought to investigate the expression of this pathway along with the expression of mitochondrial biogenesis (PGC-1α [peroxisome proliferator-activated receptor-γ coactivator-1α]), dynamics (DRP-1 [dynamin-related protein 1], OPA-1 [optic atrophy 1], and MFN 2 [mitofusin 2]), and oxidative phosphorylation (citrate synthase and electron transport chain complexes) markers and COX IV (cytochrome C oxidase) activity in myocardium from patients with valvular or ischemic heart disease and heart failure with preserved ejection fraction (HFpEF) or heart failure with reduced ejection fraction (HFrEF). Subepicardial left ventricular biopsies (10×1×1 mm3) were obtained at aortic valve replacement (HFpEFAVR, n=5; and HFrEFAVR, n=4), coronary artery bypass grafting (HFpEFCABG, n=5; and HFrEFCABG, n=5), or left ventricular assist device implantation (HFrEFLVAD, n=4). Subepicardial biopsies from patients with normal left ventricular function (n=2) and from donor hearts (n=3) served as controls (normal). Relative to normal, mitochondrial fragmentation and cristae destruction were evident, and mitochondrial area was decreased in HFpEF; 1.00±0.09 versus 0.71±0.08; P=0.016. These mitochondrial morphological changes were more pronounced in HFrEF (0.54±0.06); P=0.002 HFpEF versus HFrEF. BNIP3 (monomer+dimer) expression was increased in HFpEF (3.99±2.44) and in HFrEF (5.19±1.70) relative to normal; P=0.004 and P<0.001, respectively. However, BNIP3 monomer was increased in HFrEF (4.32±1.43) compared with normal (0.99±0.06) and HFpEF (1.97±0.90); P=0.001 and 0.004, respectively. The HFrEF group uniquely showed increase in DRP-1 expression (1.94±0.38) and decreases in PGC-1α expression (0.61±0.07) and COX IV activity (0.70±0.10) relative to normal; P=0.013, P<0.001, and P<0.001, respectively, with no significant change in electron transport chain complexes expression. These findings in human myocardium confirm studies in rodents where contractile dysfunction is associated with activation of the FOXO3a-BNIP3 pathway and altered mitochondrial dynamics, biogenesis, and function.

Targeting Calpain for Heart Failure Therapy: Implications From Multiple Murine Models

Heart failure remains a major cause of morbidity and mortality in developed countries. There is still a strong need to devise new mechanism-based treatments for heart failure. Numerous studies have suggested the importance of the Ca2+-dependent protease calpain in cardiac physiology and pathology. However, no drugs are currently under development or testing in human patients to target calpain for heart failure treatment. Herein the data demonstrate that inhibition of calpain activity protects against deleterious ultrastructural remodeling and cardiac dysfunction in multiple rodent models of heart failure, providing compelling evidence that calpain inhibition is a promising therapeutic strategy for heart failure treatment.

Abstract 21011: Crispr/ndcas9 Adenoviral Vector System as a Tool for Promoting Mitochondrial Biogenesis in Cardiomyocytes

Mitochondrial dysfunction contributes to heart failure. Our recent work revealed that genes in mitochondrial biogenesis and quality control, such as the peroxisome proliferator activated receptor gamma coactivator-1α(PGC-1α) and mitochondrial transcription factor A(TFAM), are downregulated in a rodent model of heart failure. Here, we apply CRISPR/Cas9 technology as a transcriptional activator to target sequences upstream of the start site to promote transcription of PGC-1α or TFAM. Since cardiomyocytes are difficult to transduce, we developed an adenoviral gene delivery system for expression of the CRISPR/Cas9 activating complex and a targeting gRNA to modulate expression of any gene of interest in differentiated cardiomyocytes, with our initial focus on activating the metabolic gene program. We use nuclease-dead Cas9 (ndCas9) fused to an activation domain VP64 and guide RNAs for activation of target genes. Proof-of-principle evidence was obtained by activating a cyclic AMP response element (CRE)-driven luciferase reporter gene and activation of endogenous CRE-regulable genes (PCK1 and PGC-1α). Next, we constructed ndCas9-gRNA adenoviral vectors to target PGC-1α and TFAM in the rat genome to determine if transcription of these genes can be increased. Functional effects of viral gene transduction in H9C2 cells and neonatal rat ventricular myocytes are assessed by determining mitochondrial DNA copy number (mtDNA)/genomic DNA ratio, and mtDNA quantity by picogreen labeling. Results: We have successfully upregulated the luciferase gene and endogenous CRE-regulated gene PCK1 in the same reporter cell line with ndCas9-VP64, as evidenced by significant increases in mRNA and protein expression levels. We have constructed and delivered adenoviral vectors with ndCas9 that are efficiently expressed by the host cells (NRVMs and H9c2 cells) and showed increased expression of PGC1a and TFAM in H9c2 cells. Importantly, an increase in mtDNA copy number was evoked by TFAM activation. For the first time, we show that the CRISPR/ndCas9 system can be used as a powerful tool for modulating transcription of native genes involved in mitochondrial biogenesis in cardiac cells, opening the door to developing novel therapeutic approaches for heart failure

Renal Sympathetic Denervation Reverses Diastolic Dysfunction in a Rodent Model of Heart Failure with Preserved Ejection Fraction (HFpEF)

Background: Heart failure with preserved ejection fraction (HFpEF) accounts for 50% of heart failure and has generally poor prognosis. However, there are no approved therapies available for reducing mortality or hospitalization rates in these patients. Renal sympathetic denervation (RDN) is under investigation for the treatment of resistant hypertension and primarily acts primarily by modulating afferent and efferent renal sympathetic nerves activity. We have previously reported the cardioprotective effects of RDN in myocardial ischemia-reperfusion injury. The aim of this study is to investigate the therapeutic potential of RF-RDN in a rodent model of HFpEF. Methods: 4-week-old male Sprague-Dawley rats were subjected to supracoronary aortic banding (SAB) for 14 weeks followed by administration of L-NAME (2.6mg/kg/24h) for 8 weeks to develop HFpEF. At 22-weeks after SAB, bilateral radiofrequency (RF)-RDN was performed in combination with the application of phenol on the renal arteries. Echocardiography was performed to assess systolic and diastolic function at 4 week intervals throughout the experimental protocol. Results: Twenty-two weeks following SAB and L-NAME, echocardiography revealed left ventricular (LV) hypertrophy with preserved EF and diastolic dysfunction [attenuated E/A ratio and prolonged deceleration time (DcT)]. Following RF-RDN, E/A significantly increased (1.10 ± 0.05 vs. 1.63 ± 0.06, p < 0.01) and DcT significantly shortened (38.7 ± 0.9 ms vs. 31.8 ± 0.8 ms, p < 0.01) compared to those of prior to RF-RDN. There were no significant changes in LV ejection fraction throughout the study. Conclusion: RF-RDN reversed diastolic dysfunction in a rodent model of HFpEF without affecting LV systolic function. Our preliminary results suggest that RF-RDN may have therapeutic potential in the setting of HFpEF.