Tg Hearts Research Articles

Diastolic dysfunction in Alzheimer’s disease model mice is associated with Aβ-amyloid aggregate formation and mitochondrial dysfunction

Alzheimer's Disease (AD) is a progressive neurodegenerative disease caused by the deposition of Aβ aggregates or neurofibrillary tangles. AD patients are primarily diagnosed with the concurrent development of several cardiovascular dysfunctions. While few studies have indicated the presence of intramyocardial Aβ aggregates, none of the studies have performed detailed analyses for pathomechanism of cardiac dysfunction in AD patients. This manuscript used aged APPSWE/PS1 Tg and littermate age-matched wildtype (Wt) mice to characterize cardiac dysfunction and analyze associated pathophysiology. Detailed assessment of cardiac functional parameters demonstrated the development of diastolic dysfunction in APPSWE/PS1 Tg hearts compared to Wt hearts. Muscle function evaluation showed functional impairment (decreased exercise tolerance and muscle strength) in APPSWE/PS1 Tg mice. Biochemical and histochemical analysis revealed Aβ aggregate accumulation in APPSWE/PS1 Tg mice myocardium. APPSWE/PS1 Tg mice hearts also demonstrated histopathological remodeling (increased collagen deposition and myocyte cross-sectional area). Additionally, APPSWE/PS1 Tg hearts showed altered mitochondrial dynamics, reduced antioxidant protein levels, and impaired mitochondrial proteostasis compared to Wt mice. APPSWE/PS1 Tg hearts also developed mitochondrial dysfunction with decreased OXPHOS and PDH protein complex expressions, altered ETC complex dynamics, decreased complex activities, and reduced mitochondrial respiration. Our results indicated that Aβ aggregates in APPSWE/PS1 Tg hearts are associated with defects in mitochondrial respiration and complex activities, which may collectively lead to cardiac diastolic dysfunction and myocardial pathological remodeling.

Exercise mitigates reductive stress-induced cardiac remodeling in mice

The endoplasmic reticulum (ER) regulates protein folding and maintains proteostasis in cells. We observed that the ER transcriptome is impaired during chronic reductive stress (RS) in cardiomyocytes. Here, we hypothesized that a prolonged moderate treadmill exercise mitigates the RS-induced ER dysfunction and cardiac remodeling in cardiac-specific constitutively active Nrf2 mice (CaNrf2-TG). RNA sequencing showed notable alterations in the ER transcriptome of TG hearts at 4, 12, and 24 weeks (16, 28, and 35 genes, respectively). Notably, the downregulation of ER genes was significant at 12 weeks, and further pronounced at 24 weeks, at which the cardiac pathology is evident. We also observed increased levels of ubiquitinated proteins in CaNrf2-TG hearts across all ages, along with VCP, a marker of ERAD function, at 24 weeks. These findings indicate that constitutive Nrf2 activation and RS impair protein-folding activity and augments ERAD function over time. Exercise intervention for 20 weeks (beginning at 6 weeks of age), reduced cardiomyocyte hypertrophy (from 448 μm2 to 280 μm2) in TG mice, through adaptive remodeling, and preserved the cardiac function. However, while exercise did not influence antioxidants or ER stress protein levels, it significantly improved ERAD function and autophagy flux (LC-I to LC-II) in the TG-EXE hearts. Collectively, our findings underscore the prophylactic potential of exercise in mitigating RS-associated pathology, highlighting its essential role in maintaining cellular proteostasis through ER-independent mechanisms.

Reduction in Insulin Uncovers a Novel Effect of VEGFB on Cardiac Substrate Utilization.

The heart relies heavily on external fatty acid (FA) for energy production. VEGFB (vascular endothelial growth factor B) has been shown to promote endothelial FA uptake by upregulating FA transporters. However, its impact on LPL (lipoprotein lipase)-mediated lipolysis of lipoproteins, a major source of FA for cardiac use, is unknown. VEGFB transgenic (Tg) rats were generated by using the α-myosin heavy chain promoter to drive cardiomyocyte-specific overexpression. To measure coronary LPL activity, Langendorff hearts were perfused with heparin. In vivo positron emission tomography imaging with [18F]-triglyceride-fluoro-6-thia-heptadecanoic acid and [11C]-palmitate was used to determine cardiac FA uptake. Mitochondrial FA oxidation was evaluated by high-resolution respirometry. Streptozotocin was used to induce diabetes, and cardiac function was monitored using echocardiography. In Tg hearts, the vectorial transfer of LPL to the vascular lumen is obstructed, resulting in LPL buildup within cardiomyocytes, an effect likely due to coronary vascular development with its associated augmentation of insulin action. With insulin insufficiency following fasting, VEGFB acted unimpeded to facilitate LPL movement and increase its activity at the coronary lumen. In vivo PET imaging following fasting confirmed that VEGFB induced a greater FA uptake to the heart from circulating lipoproteins as compared with plasma-free FAs. As this was associated with augmented mitochondrial oxidation, lipid accumulation in the heart was prevented. We further examined whether this property of VEGFB on cardiac metabolism could be useful following diabetes and its associated cardiac dysfunction, with attendant loss of metabolic flexibility. In Tg hearts, diabetes inhibited myocyte VEGFB gene expression and protein secretion together with its downstream receptor signaling, effects that could explain its lack of cardioprotection. Our study highlights the novel role of VEGFB in LPL-derived FA supply and utilization. In diabetes, loss of VEGFB action may contribute toward metabolic inflexibility, lipotoxicity, and development of diabetic cardiomyopathy.

Hypercontractile cardiac phenotype in mice overexpressing the regulatory subunit PR72 of protein phosphatase 2A.

The activity, localization, and substrate specificity of the protein phosphatase 2A (PP2A) heterotrimer are controlled by various regulatory B subunits. PR72 belongs to the B'' gene family and has been shown to be upregulated in human heart failure. However, little is known about the functions of PR72 in the myocardium. To address this issue, we generated a transgenic mouse model with heart-specific overexpression of PP2A-PR72. Biochemical and physiological methods were used to determine contractility, Ca2+ cycling parameters, and protein phosphorylation. A 2.5-fold increase in PR72 expression resulted in moderate cardiac hypertrophy. Maximal ventricular pressure was increased in catheterized transgenic mice (TG) compared to wild-type (WT) littermates. This was accompanied by an increased shortening of sarcomere length and faster relaxation at the single-cell level in TG. In parallel with these findings, the peak amplitude of Ca2+ transients was increased, and the decay in intracellular Ca2+ levels was shortened in TG compared to WT. The changes in Ca2+ cycling in TG were also evident from an increase in the full duration and width at half maximum of Ca2+ sparks. Consistent with the contractile data, phosphorylation of phospholamban at threonine-17 was higher in TG hearts. The lower expression of the Na+/Ca2+ exchanger may also contribute to the hypercontractile state in transgenic myocardium. Our results suggest that PP2A-PR72 plays an important role in regulating cardiac contractile function and Ca2+ cycling, indicating that the upregulation of PR72 in heart failure is an attempt to compensate functionally.

Abstract P2019: Diastolic Dysfunction In Alzheimer's Disease Is Associated With aβ- Amyloid Aggregate Formation And Mitochondrial Dysfunction

Background: Alzheimer's disease (AD) is a neurodegenerative disease with memory impairment caused by an accumulation of toxic protein aggregates (Aβ- amyloid plaques and tau-derived neurofibrillary tangles). Several studies reported cardiovascular abnormalities with diastolic dysfunction in AD patients; however, the direct mechanism of cardiac dysfunction in AD patients remains unexplored. Therefore, we investigated the pathophysiological mechanism of cardiac dysfunction in the mice model of AD. Hypothesis: Two most commonly used mice models of AD was thoroughly evaluated for cardiac dysfunction, histopathology, and biochemical changes over aging. Methods and Result: To determine the mechanism of AD-associated cardiac dysfunction, we used two different AD mice models: double transgenic mice expressing a chimeric mouse/human amyloid precursor protein and a mutant human presenilin 1 (APP/PS1 Tg), and transgenic mice that is homozygous for a Psen1 mutation and the co-injected APPSwe and tauP301L transgenes (3xTg-AD). Cardiac function measurement using non-invasive echocardiography in aged Ntg and AD mice models showed no difference in systolic cardiac functional parameters. However, diastolic function assessment using Doppler echocardiographic measurements showed ventricular diastolic dysfunction with reduced diastolic filling in APP/PS1 Tg and 3xTg-AD mice. Western blot analysis showed Aβ- amyloid protein expression in APP/PS1 Tg hearts. Immunohistochemical and immunofluorescence staining of the APP/PS1 Tg heart sections further confirmed the Aβ- amyloid protein expression and Aβ- amyloid aggregate formation in the cardiomyocytes. Further evaluation of APP/PS1 Tg hearts demonstrated increased collagen deposition (Picro-Sirius red staining) and increased cardiomyocyte cross-sectional area. Interestingly, APP/PS1 Tg hearts also displayed mitochondrial dysfunction with decreased mitochondrial bioenergetics and significantly reduced mitochondrial function regulatory parameters. Conclusion: Overall, we found diastolic dysfunction development with Aβ- amyloid aggregate formation, fibrotic remodeling, increased cardiomyocyte cross-sectional area, and mitochondrial dysfunction in the AD mice hearts.

Abstract P2061: The Intersection Of O-GlcNAc And Yap - A Novel Molecular Axis In Heart Failure

Introduction: Heart failure (HF) and pathological myocardial hypertrophy continue to grow world-wide and are strongly tied to obesity, hyperglycemia, and ischemia. These stressors lead to increased levels of O-beta-N-Acetylglucosamine modified proteins (OGN) in the heart. OGN levels are determined by the net activity of two enzymes, OGT and OGA. Our recent work showed excessive OGN was sufficient to cause HF and pathological myocardial hypertrophy. Here we test the relationship between OGN and Yes Associated Protein (YAP), a known promoter of cardiac hypertrophy. Objectives: Test if OGN modifies YAP in the heart and evaluate the role of OGN modified YAP in the development of pathologic cardiac hypertrophy and heart failure. Methods: We have shown that mouse models with myocardial OGT overexpression (OGT TG) developed pathological cardiac hypertrophy and HF. We found these animals have increased YAP activity. Myocardial OGA overexpression (OGA TG) attenuated OGN following transaortic constriction(TAC) and were protected against cardiac hypertrophy and HF. Interbreeding OGT TG with OGA TG rescued animals from pathological hypertrophy, HF and normalized YAP activity . We quantified levels of activated YAP protein/gene expression in hearts from WT, OGT, OGA and OGT x OGA mice. We evaluated YAP/TEAD1 target gene expression levels in OGA TG mice protected against TAC. We used an adenoviral YAP/TEAD expression reporter to quantify YAP/TEAD1 activity in cardiac myocytes, in the presence and absence of increased O-GlcNAcylation. Results: We found WT levels of activated (nuclear) YAP in OGA TG, increased active YAP in OGT TG and normal levels in OGT x OGA mice. YAP/TEAD1 target genes are upregulated in OGT TG hearts but interbred OGT x OGA animals have normal cardiac function and YAP/TEAD1 target gene expression. OGA TG mice who are protected against TAC induced heart failure/hypertrophy and have decreased myocardial levels of YAP/TEAD1 gene targets involved in pathological hypertrophy. Increased O-GlcNAcylation in cardiac myocytes activates YAP/TEAD activity and YAP/TEAD downstream gene expression. Conclusion: These preliminary data suggest a novel pathway for promoting pathological hypertrophy and HF via activation of the O-GlcNAc -YAP/TEAD1 axis.

23-OR: Reduction in Insulin Uncovers a Novel Effect of VEGFB on Cardiac Substrate Utilization

VEGFB is known to up-regulate FA delivery through its effect on EC FA transporters. In its absence, intracellular lipid accumulation was reduced. Paradoxically, cardiac-specific overexpression of VEGFB demonstrated a lowered heparin-releasable lipoprotein lipase (LPL) activity with reduced FA utilization in vivo. Currently, the mechanism behind this in vivo metabolic effect of VEGFB on cardiac LPL is unclear. In transgenic (Tg) hearts, the vectorial transfer of LPL from myocytes to the vascular lumen is obstructed, resulting in LPL buildup within cardiomyocytes. Given that insulin is known to inhibit LPL translocation and thus FA metabolism in the heart, this observation is likely a secondary effect of VEGFB on vascular development with its associated augmentation of insulin delivery. We tested whether lowering of insulin by fasting would validate our hypothesis. WT fasted hearts enhanced their heparin-releasable LPL activity in an attempt to switch substrate utilization to FA. Intriguingly, this increase in LPL activity following fasting was even more dramatic in Tg hearts. Thus, with insulin deficiency following fasting, VEGFB works unimpeded to facilitate LPL movement. This effect occurred through the activation of p38 MAPK. PET imaging using 18FTHA revealed augmented LPL-derived FA and impaired FDG glucose uptake in the Tg fasted heart. Additionally, this increased delivery of LPL-derived FA correlated to augmented mitochondrial oxidation determined by high resolution oxygraphy. We examined whether this property of VEGFB on FA uptake and oxidation could be useful following diabetes, with its attendant loss of metabolic flexibility. Unexpectedly, in Tg hearts, diabetes inhibited myocyte VEGFB gene expression and protein secretion together with its downstream receptor signaling. Our data suggests that following diabetes, loss of VEGFB action may contribute towards the metabolic inflexibility, lipotoxicity and diabetic cardiomyopathy. Disclosure R.Shang: None. C.Lee: None. B.Hussein: None. B.Rodrigues: None. Funding Canadian Institutes of Health Research (PJT180558); Diabetes Canada (OG3215585BR)

VEGF-B hypertrophy predisposes to transition from diastolic to systolic heart failure in hypertensive rats.

Cardiac energy metabolism is centrally involved in heart failure (HF), although the direction of the metabolic alterations is complex and likely dependent on the particular stage of HF progression. Vascular endothelial growth factor B (VEGF-B) has been shown to modulate metabolic processes and to induce physiological cardiac hypertrophy; thus, it could be cardioprotective in the failing myocardium. This study investigates the role of VEGF-B in cardiac proteomic and metabolic adaptation in HF during aldosterone and high-salt hypertensive challenges. Male rats overexpressing the cardiac-specific VEGF-B transgene (VEGF-B TG) were treated for 3 or 6 weeks with deoxycorticosterone-acetate combined with a high-salt (HS) diet (DOCA + HS) to induce hypertension and cardiac damage. Extensive longitudinal echocardiographic studies of HF progression were conducted, starting at baseline. Sham-treated rats served as controls. To evaluate the metabolic alterations associated with HF, cardiac proteomics by mass spectrometry was performed. Hypertrophic non-treated VEGF-B TG hearts demonstrated high oxygen and adenosine triphosphate (ATP) demand with early onset of diastolic dysfunction. Administration of DOCA + HS to VEGF-B TG rats for 6 weeks amplified the progression from cardiac hypertrophy to HF, with a drastic drop in heart ATP concentration. Dobutamine stress echocardiographic analyses uncovered a significantly impaired systolic reserve. Mechanistically, the hallmark of the failing TG heart was an abnormal energy metabolism with decreased mitochondrial ATP, preceding the attenuated cardiac performance and leading to systolic HF. This study shows that the VEGF-B TG accelerates metabolic maladaptation which precedes structural cardiomyopathy in experimental hypertension and ultimately leads to systolic HF.

Transgenic Expression of Nrf2 Induces a Pro-Reductive Stress and Adaptive Cardiac Remodeling in the Mouse.

Nuclear factor, erythroid 2 like 2 (Nfe2l2 or Nrf2), is a transcription factor that protects cells by maintaining a homeostatic redox state during stress. The constitutive expression of Nrf2 (CaNrf2-TG) was previously shown to be pathological to the heart over time. We tested a hypothesis that the cardiac-specific expression of full length Nrf2 (mNrf2-TG) would moderately increase the basal antioxidant defense, triggering a pro-reductive environment leading to adaptive cardiac remodeling. Transgenic and non-transgenic (NTG) mice at 7–8 months of age were used to analyze the myocardial transcriptome, structure, and function. Next generation sequencing (NGS) for RNA profiling and qPCR-based validation of the NGS data, myocardial redox levels, and imaging (echocardiography) were performed. Transcriptomic analysis revealed that out of 14,665 identified mRNAs, 680 were differently expressed (DEG) in TG hearts. Of 680 DEGs, 429 were upregulated and 251 were downregulated significantly (FC > 2.0, p < 0.05). Gene set enrichment analysis revealed that the top altered pathways were (a) Nrf2 signaling, (b) glutathione metabolism and (c) ROS scavenging. A comparative analysis of the glutathione redox state in the hearts demonstrated significant differences between pro-reductive vs. hyper-reductive conditions (233 ± 36.7 and 380 ± 68.7 vs. 139 ± 8.6 µM/mg protein in mNrf2-TG and CaNrf2-TG vs. NTG). Genes involved in fetal development, hypertrophy, cytoskeletal rearrangement, histone deacetylases (HDACs), and GATA transcription factors were moderately increased in mNrf2-TG compared to CaNrf2-TG. Non-invasive echocardiography analysis revealed an increase in systolic function (ejection fraction) in mNrf2-TG, suggesting an adaptation, as opposed to pathological remodeling in CaNrf2-TG mice experiencing a hyper-reductive stress, leading to reduced survival (40% at 60 weeks). The effects of excess Nrf2-driven antioxidant transcriptome revealed a pro-reductive condition in the myocardium leading to an adaptive cardiac remodeling. While pre-conditioning the myocardial redox with excess antioxidants (i.e., pro-reductive state) could be beneficial against oxidative stress, a chronic pro-reductive environment in the myocardium might transition the adaptation to pathological remodeling.

Abstract P2045: Transcriptome Versus Proteomic Discrepancy Is Associated With Structural-functional Dys-synchrony In Reductive Stress Myocardium

Background: A real-time action of Nrf2 in response to a stress is vital to preserve the redox homeostasis, but its activation under unstressed settings tilt the redox balance towards the reductive arm, leading to reductive stress (RS). In this study, we identified that &gt;50% mismatch between mRNA and protein levels in RS myocardium undergoing structural/functional dys-synchrony. Methods: NGS-RNA Seq (for RNA), Tandem Mass Tagging (TMT) Mass spectrometry (for proteins) and speckle tracking strain echocardiography (STE) using Vevo2100 system was performed in the cardiac-specific constitutively active Nrf2 transgenic (CaNrf2-TG/TG) and non-transgenic (NTG) mice (n=4/group) at ~6-7 months of age. Results: Comparison of NGS and TMT data in TG vs. NTG hearts showed surprising results. Out of 104 commonly found gene products in NGS and proteins at a FC≥1.5, the levels of 50 proteins are not consistent with their RNA expression. The proteins that are steadily expressed according to their respective mRNA levels are classified as “transcription-sync proteins (T-SP)” and the ones that do not match are termed as “transcription-nonsync proteins. While changes in Nrf2-driven antioxidant genes ( Nqo1, Gsr, Gsta1-4, and Gstm1 ) were grouped under “T-SP”, other transcripts including Atp5pb, Ces1d, Cmbl, Cox7a1, Cpt1b, Fah and Gstk1 did not quantitatively match with their translated products in TG hearts. Further, STE demonstrated an impaired wall motion (diastolic and systolic velocity), increased fractional shortening (20% vs 50%; p&lt;0.0001), decreased global longitudinal strain (-10% vs -25%, p&lt;0.0002), increased IVRT (6.46 vs 13.8 ms; p&lt;0.0001) and IVCT (8.06 vs 12.5 ms; p&lt;0.0005) in TG vs. NTG mice. Changes in the systolic and diastolic functions with structural abnormalities demonstrate cardiac dys-synchronicity in TG hearts with chronic RS exhibiting impaired transcriptome and proteome profiles. Conclusions: Our comparative analysis of RNA and protein in RS hearts demonstrated that 50% of the proteins do not match with the quantitative changes of their transcripts. We highlight that the RS could be a novel mechanism for disproportionate transcription vs translation, which leads to structural and functional dys-synchrony in the TG hearts.

Abstract P3091: Transgenic Expression Of Nrf2 Induces A Pro-reductive Stress And Adaptive Cardiac Remodeling In Mice

Background: NFE2L2, nuclear factor, erythroid 2 like 2 (Nrf2), is a transcription factor that protects cells through maintaining a homeostatic redox state during stress. Constitutive expression of Nrf2 (CaNrf2-TG) was previously shown to be pathological to the heart over time. We tested a hypothesis that the cardiac-specific expression of full length Nrf2 (mNrf2-TG) would moderately increase the basal antioxidant defense, triggering a pro-reductive environment leading to adaptive cardiac remodeling. Methods: Cardiac-specific full-length Nrf2 transgenic (mNrf2-TG) and non-transgenic (NTG) mice hearts on the C57/BL6 background at 7-8 months of age were used to analyze the transcriptome and cardiac structure/function. Next generation sequencing (NGS) for RNA profiling (n=3/group) and qPCR-based validation (n=5/group) of the NGS data were accomplished. Myocardial redox levels and imaging (echocardiography) were performed. Results: Transcriptomic analysis revealed that out of 14665 identified mRNAs, 679 were differently expressed (DEG) in TG hearts. Of 679 DEGs, 429 were upregulated and 251 were downregulated significantly (FC&gt;2, P&lt;0.05). Gene Set Enrichment Analysis revealed that the top altered pathways were (a) Nrf2 signaling, (b) glutathione metabolism and (c) ROS scavenging. A comparative analysis of glutathione redox state in the hearts demonstrated significant differences between pro-reductive vs. hyper-reductive conditions (233 ± 36.7 and 380 ± 68.7 vs. 139 ± 8.6 μM/mg protein in mNrf2-TG and CaNrf2-TG vs NTG). Expression of fetal genes and hypertrophic biomarkers, cytoskeletal genes, histone deacetylases (HDACs), and GATA transcription factors were moderately increased in mNrf2-TG compared to CaNrf2-TG hearts. Non-invasive echocardiography analysis revealed an increase in systolic function (Ejection Fraction) in mNrf2-TG, suggesting an adaption, as opposed to pathological remodeling in CaNrf2-TG mice experiencing a hyper-reductive stress, leading to reduced survival (40% at 60 weeks). Conclusion: The effects of excess Nrf2-driven antioxidant transcriptome revealed a pro-reductive condition in the myocardium leading to an adaptive cardiac remodeling. While pre-conditioning the myocardial redox with excess antioxidants (i.e. pro-reductive state) could be beneficial against oxidative stress, a chronic pro-reductive environment in the myocardium might transition the adaptation to pathological remodeling.

Abstract P3041: DWORF Overexpression In The Heart Results In Enhanced Mitochondrial Function

Background: Calcium (Ca 2+ ) plays a pivotal role in regulating physiological processes in the heart including excitation-contraction coupling, Ca 2+ signaling, and mitochondrial function. It is well established that both Ca 2+ dysregulation and altered cardiac energy metabolism are universal features of heart failure (HF). Dwarf open reading frame (DWORF), a newly discovered microprotein, increases SERCA2a activity in part by relieving SERCA2a inhibition by phospholamban (PLN). DWORF overexpression via cardiac specific transgene or gene therapy is cardioprotective in genetic and experimental HF mouse models. In addition to activation of SERCA2a by PLN displacement, preliminary data indicates that enhanced mitochondrial function contributes to the cardioprotective effects of DWORF. Here we tested the effects of DWORF overexpression on cardiac mitochondrial function and Ca 2+ dynamics. Methods and Results: We isolated mitochondria from wildtype (WT) and DWORF transgenic (Tg) mouse hearts and measured O 2 consumption rates using a Seahorse mito stress test. We found that DWORF Tg mice have increased respiratory capacity compared to WT (DWORF Tg 1150.0 pmol/min, WT 799.4 pmol/min +/-146.2) and enhanced spare respiratory capacity (DWORF Tg 489.1 pmol/min, WT 229.4 pmol/min +/- 102.1). To measure mitochondrial Ca 2+ dynamics, we performed Ca 2+ retention capacity assays and found that DWORF Tg mice have increased Ca 2+ uptake rates compared to WT (DWORF Tg 32.32 s, WT 46.46 s +/- 4.39). Western blot analysis of the mitochondrial Ca 2+ uniporter (MCU) in mito extracts from WT and DWORF Tg hearts indicated elevated levels of MCU. However, electron transport chain (ETC) protein expression indicated no significant differences, indicating the enhanced mitochondrial activity observed in DWORF Tg mice was not due to increased ETC levels. Conclusions: DWORF Tg mouse heart mitochondria exhibit increased maximal respiration rates and enhanced spare respiratory capacity. However, ETC protein levels are unchanged, indicating altered enzyme activity rather than expression. Additionally, mitochondria from DWORF Tg hearts uptake Ca 2+ faster into the mitochondria through the increased levels of MCU, suggesting enhanced mitochondrial Ca 2+ dynamics.

Tandem Mass Tagging Based Identification of Proteome Signatures for Reductive Stress Cardiomyopathy.

Nuclear factor erythroid 2-related factor 2 (NRF2), a redox sensor, is vital for cellular redox homeostasis. We reported that transgenic mice expressing constitutively active Nrf2 (CaNrf2-TG) exhibit reductive stress (RS). In this study, we identified novel protein signature for RS-induced cardiomyopathy using Tandem Mass Tag (TMT) proteomic analysis in heart tissues of TG (CaNrf2-TG) mice at 6–7 months of age. A total of 1,105 proteins were extracted from 22,544 spectra. About 560 proteins were differentially expressed in TG vs. NTg hearts, indicating a global impact of RS on the myocardial proteome. Over 32 proteins were significantly altered in response to RS -20 were upregulated and 12 were downregulated in the hearts of TG vs. NTg mice, suggesting that these proteins could be putative signatures of RS. Scaffold analysis revealed a clear distinction between TG vs. NTg hearts. The majority of the differentially expressed proteins (DEPs) that were significantly altered in RS mice were found to be involved in stress related pathways such as antioxidants, NADPH, protein quality control, etc. Interestingly, proteins that were involved in mitochondrial respiration, lipophagy and cardiac rhythm were dramatically decreased in TG hearts. Of note, we identified the glutathione family of proteins as the significantly changed subset of the proteome in TG heart. Surprisingly, our comparative analysis of NGS based transcriptome and TMT-proteome indicated that ~50% of the altered proteins in TG myocardium was found to be negatively correlated with their transcript levels. In association with the altered proteome the TG mice displayed pathological cardiac remodeling.

Activation of PKC results in improved contractile effects and Ca2+ cycling by inhibition of PP2A-B56α.

Protein phosphatase 2A (PP2A) represents a heterotrimer that is responsible for the dephosphorylation of important regulatory myocardial proteins. This study was aimed to test whether the phosphorylation of PP2A-B56α at Ser41 by PKC is involved in the regulation of myocyte Ca2+ cycling and contraction. For this purpose, heart preparations of wild-type (WT) and transgenic mice overexpressing the nonphosphorylatable S41A mutant form (TG) were stimulated by administration of the direct PKC activator phorbol 12-myristate 13-acetate (PMA), and functional effects were studied. PKC activation was accompanied by the inhibition of PP2A activity in WT cardiomyocytes, whereas this effect was absent in TG. Consistently, the increase in the sarcomere length shortening and the peak amplitude of Ca2+ transients after PMA administration in WT cardiomyocytes was attenuated in TG. However, the costimulation with 1 µM isoprenaline was able to offset these functional deficits. Moreover, TG hearts did not show an increase in the phosphorylation of the myosin-binding protein C after administration of PMA but was detected in corresponding WT. PMA modulated voltage-dependent activation of the L-type Ca2+ channel (LTCC) differently in the two genotypes, shifting V1/2a by +1.5 mV in TG and by -2.4 mV in WT. In the presence of PMA, ICaL inactivation remained unchanged in TG, whereas it was slower in corresponding WT. Our data suggest that PKC-activated enhancement of myocyte contraction and intracellular Ca2+ signaling is mediated by phosphorylation of B56α at Ser41, leading to a decrease in PP2A activity.NEW & NOTEWORTHY The importance of the serine-41 phosphorylation site on B56α in reducing PP2A activity was demonstrated for the first time using a transgenic mutation model. Direct activation of PKC inhibits PP2A, leading to increased phosphorylation of MyBP-C in cardiomyocytes. The increased phosphorylation of contractile proteins is influenced by the PKC-phosphoB56α-PP2A signaling cascade resulting in improved intracellular Ca2+ handling and enhanced contractility and relaxation. PKC-mediated inhibition of PP2A also leads to modulation of the LTCC activation and inactivation kinetics.

Cardiac expression of microRNA-7 is associated with adverse cardiac remodeling

Although microRNA-7 (miRNA-7) is known to regulate proliferation of cancer cells by targeting Epidermal growth factor receptor (EGFR/ERBB) family, less is known about its role in cardiac physiology. Transgenic (Tg) mouse with cardiomyocyte-specific overexpression of miRNA-7 was generated to determine its role in cardiac physiology and pathology. Echocardiography on the miRNA-7 Tg mice showed cardiac dilation instead of age-associated physiological cardiac hypertrophy observed in non-Tg control mice. Subjecting miRNA-7 Tg mice to transverse aortic constriction (TAC) resulted in cardiac dilation associated with increased fibrosis bypassing the adaptive cardiac hypertrophic response to TAC. miRNA-7 expression in cardiomyocytes resulted in significant loss of ERBB2 expression with no changes in ERBB1 (EGFR). Cardiac proteomics in the miRNA-7 Tg mice showed significant reduction in mitochondrial membrane structural proteins compared to NTg reflecting role of miRNA-7 beyond the regulation of EGFR/ERRB in mediating cardiac dilation. Consistently, electron microscopy showed that miRNA-7 Tg hearts had disorganized rounded mitochondria that was associated with mitochondrial dysfunction. These findings show that expression of miRNA-7 in the cardiomyocytes results in cardiac dilation instead of adaptive hypertrophic response during aging or to TAC providing insights on yet to be understood role of miRNA-7 in cardiac function.

Ectopic Expression of miR‐424(322)/503 Triggers Acute and Reversible Dilated Cardiomyopathy

Background Cardiovascular disease is the topmost cause of death worldwide before cancer, chronic respiratory diseases, Alzheimer's and kidney diseases. Dilated cardiomyopathy (DCM) is one of the most common cardiovascular diseases with an estimated prevalence of 1 in 2500 individuals. DCM is clinically characterized by ventricular chamber dilation and systolic dysfunction eventually leading to heart failure. MicroRNAs have been shown to play an integral role in regulating the progression of cardiomyopathy along with heart failure and as a result have become novel targets for therapy. Previously, our lab has shown that the miR-424(322)/503 is highly enriched in embryonic heart during early cardiac development but its expression drops to very low level after birth. Interestingly, other studies have revealed the upregulation of miR-424(322)/503 in failing human hearts. However, the relation between miR-424(322)/503 and heart failure is not yet known. Methods and results To understand the role of miR-424(322)/503 in adult heart, we created tetracycline-controlled cardiomyocyte-specific miR-424(322)/503 expressing transgenic mice (Tg). miR-424(322)/503 was induced in Tg and wildtype (Wt) mice (8 weeks) using doxycycline containing chow continuously for a month. Cardiac function was monitored every week using transthoracic echocardiography. The hearts were then harvested for molecular and histological analyses. Echocardiographic analysis revealed continuous deterioration of cardiac function in Tg mice starting from 2 weeks of doxycycline induction. This was evidenced by significant decrease in ejection fraction and fractional shortening from 46±1.9% and 22±1.09% at week 2 to 15±2.7% and 6±1.2% at week 4, respectively. Cardiac failure markers (ANP and BNP) were also found to be upregulated by 8-10 folds in Tg as compared to Wt hearts. Further analysis with tissue staining in Tg hearts exhibited dilated chambers with thin ventricular wall, presence of fibrosis and disorganized myocytes; a phenotype consistent with DCM. Next, to further see the causative role of miR-424(322)/503 and determine if miR-424(322)/503 is required for heart failure progression, mice were fed with doxycycline chow for 2 weeks, which is the time-point when the heart failure started to occur, and then continued with normal chow. We observed that the heart function had recovered as indicated by improvement in ejection fraction and fractional shortening from 31.94±7.55% and 14.98±3.95% in week 3 to 51.27±5.02% and 25.79±2.92% in week 12, respectively. ANP and BNP levels dropped significantly in week 12 compared to weeks 2-3. This suggests that miR-424(322)/503 plays a role in DCM progression. Conclusion Overall, this is the first study showing that upregulated miR-424(322)/503 in the heart is sufficient to lead to DCM and miR-424(322)/503 induced heart failure is reversible. Thus, this study supports miR-424(322)/503 as a novel potential therapeutic target for DCM.

Endothelial NADPH oxidase 4 protects against angiotensin II-induced cardiac fibrosis and inflammation.

AimsEndothelial activation and inflammatory cell infiltration have important roles in the development of cardiac fibrosis induced by renin–angiotensin system activation. NADPH oxidases (Nox proteins) are expressed in endothelial cells (ECs) and alter their function. Previous studies indicated that Nox2 in ECs contributes to angiotensin II (AngII)‐induced cardiac fibrosis. However, the effects of EC Nox4 on cardiac fibrosis are unknown.Methods and resultsTransgenic (TG) mice overexpressing endothelial‐restricted Nox4 were studied alongside wild‐type (WT) littermates as controls. At baseline, Nox4 TG mice had significantly enlarged hearts compared with WT, with elongated cardiomyocytes (increased by 18.5%, P < 0.01) and eccentric hypertrophy but well‐preserved cardiac function by echocardiography and in vivo pressure–volume analysis. Animals were subjected to a chronic AngII infusion (AngII, 1.1 mg/kg/day) for 14 days. Whereas WT/AngII developed a 2.1‐fold increase in interstitial cardiac fibrosis as compared with WT/saline controls (P < 0.01), TG/AngII mice developed significant less fibrosis (1.4‐fold increase, P > 0.05), but there were no differences in cardiac hypertrophy or contractile function between the two groups. TG hearts displayed significantly decreased inflammatory cell infiltration with reduced levels of vascular cell adhesion molecule 1 in both the vasculature and myocardium compared with WT after AngII treatment. TG microvascular ECs stimulated with AngII in vitro supported significantly less leukocyte adhesion than WT ECs.ConclusionsA chronic increase in endothelial Nox4 stimulates physiological cardiac hypertrophy and protects against AngII‐induced cardiac fibrosis by inhibiting EC activation and the recruitment of inflammatory cells.

Abstract 470: Reactivation of Fetal MicroRNAs Contribute to Dilated Cardiomyopathy

Background: Dilated cardiomyopathy (DCM) is the most common cardiomyopathies that often leads to heart failure worldwide. MicroRNAs have emerged as an integral player in regulating diverse biological processes including those in the cardiovascular system. Previously, our lab has shown that the miR-424(322)/503 cluster is highly enriched during early cardiac fate determination and drives cardiomyocyte specification. However, the expression level of miR-424(322)/503 gradually decreases in the postnatal heart. Other studies have revealed the upregulation of miR-424(322)/503 in failing human hearts. Nevertheless, the association between miR-424(322)/503 and heart disease has not been studied yet. Methods and Results: In this study, we created transgenic mice (TG) with tetracycline-controlled cardiomyocyte-specific miR-424(322)/503 expression. The TG mice and control mice (8 weeks) were fed with doxycycline-containing chow for up to a month. During this period, the weight of the mice and cardiac function were monitored. The weight of TG mice dropped significantly while controls grew normally. Echocardiography analysis showed continuous deterioration of cardiac function starting from 2 weeks. Ejection fraction and fractional shortening (n=8) decreased to 46±1.9% and 22±1.09% at 2 weeks and 15±2.7% and 6±1.2% at 4 weeks, respectively. In addition, left ventricular internal diameter (LVID,d and LVID,s) increased from 3.64±0.098 and 2.5±0.085 to 4.2±0.2 and 3.9±0.18 respectively. Morphologically, we found the hearts of TG mice to be larger than WT mice. H and E staining revealed dilated ventricular chambers and thinning of ventricular walls in TG. Likewise, fibrosis was observed in TG hearts through Masson trichrome stain. In addition, both atrial natriuretic peptides and brain natriuretic peptides increased by &gt;8 folds in TG hearts (n=5). The level of β-MHC was higher while α-MHC was lower in TG hearts vs control. These indicate that TG phenotype is consistent with DCM. Conclusion: Overall, this is the first study showing that upregulated miR-424(322)/503 in the heart is sufficient to lead to DCM. This study can potentially help establish miR-424(322)/503 as a novel therapeutic target to ameliorate heart failure progression.

Abstract 249: ERBB2 and beta-1-Adrenergic Receptor Cross-talk Underlies Cardiac Dysfunction

ERBB2 is a member of the EGFR growth factor receptor family and a primary target for anti-cancer treatment in patients diagnosed with breast cancer. ERBB2 targeting is known to cause cardiotoxicity, resulting in cardiac dilation while, less is understood about the underlying mechanisms. Studies have shown a direct co-relationship between beta2-adrenergic receptors (β2ARs) and ERBB2 in the cardiac hypertrophy wherein increased ERBB2 expression is associated with elevated β2ARs. Given the key role of β1ARs in cardiac function, we assessed potential cross-talk between ERBB2 and β1ARs. Towards this end, we have used a transgenic mouse model of cardiomyocyte-specific overexpression of microRNA-7 (miR-7 Tg) that abrogates ERBB2 expression in the adult cardiomyocytes and associated with cardiac dilation. Radio-ligand binding in miR-7 Tg hearts showed selective increase in β1AR density with no changes in the β2ARs. In contrast, cardiomyocyte-specific overexpression of ERBB2 results cardiac hypertrophy and is associated with significant increase in β2AR density with no changes in β1ARs. These observations show that ERBB2 expression may differentially regulate β1AR versus β2AR providing a unique role for ERBB2 in cardiac physiology. Further, ERBB2 expression is elevated in an age-dependent manner in C57BL6 mice that may account for hypertrophic response. Consistently, miR-7 Tg mice have age-dependent cardiac dilation consistent with the loss in the ERBB2 expression. To test for such a role, C57BL6 mice were administered vehicle, AG1875 (EGFR inhibitor) or AG825 (ERBB2 inhibitor) for two weeks to assess for changes in cardiac function. Administration of AG825 resulted significant cardiac dysfunction and dilation compared to AG1875 or vehicle controls, conferring the idea that inhibition of ERBB2 and not EGFR that mediates deleterious remodeling. Proteomic analysis showed dramatic differences in the associated proteins following AG825 compared to vehicle or AG1478 and our presentation will elucidate the results on how homeostatic ERBB2 signaling and expression is key for cardiac function and how chemotherapeutic inhibition of ERBB2 underlies deleterious pathways leading to cardiotoxicity.

Abstract 160: Atg7-Dependent Activation of Mitochondrial Autophagy in Cardiomyocytes

Introduction: Autophagy-related protein 7 (Atg7) has been studied for its role in cellular macroautophagy induction and regulation in different cell systems. We have previously reported that Atg7 regulates cardiac macroautophagy. However, whether Atg7 plays any role in the initiation and regulation of mitochondrial autophagy (mitophagy) in cardiomyocytes remains elusive. Objective: We investigated subcellular localization and molecular function of Atg7 in the regulation of mitophagy through a series of in vitro and in vivo experiments. Methods and Results: We isolated mitochondria from mouse hearts by subcellular fractionation and conducted biochemical experiments which revealed localization of Atg7 on the mitochondrial membrane. Next, we co-stained for Atg7 in isolated heart mitochondria stained with Mitotracker dye and found Atg7 localizes to mitochondrial membrane. To elucidate molecular function of Atg7 on mitophagy, adenovirus-mediated Atg7 overexpressed cultured neonatal rat cardiomyocytes (NRCs) were treated with mitophagy inducer carbonyl cyanide 3-chlorophenylhydrazone (CCCP) in vitro and stained with organelle-specific mitochondria and lysosome dyes to count mitophagy fluorescent dots by confocal microscopy. Atg7 overexpressed NRCs exhibited an increase in mitophagy dots than β-gal treated NRCs following CCCP induction. Further, we have treated cardiac specific Atg7 overexpressed (Atg7 Tg) mice and littermate non-transgenic (Ntg) mice with CCCP to assess mitophagy induction in vivo . Immunoblotting confirmed enhanced accumulation of mature autophagolysosomal marker LC3-II protein in mitochondrial fraction from Atg7 Tg hearts. Conclusions: Our in vitro and in vivo findings indicate that Atg7 localizes to the mitochondrial membrane, regulates recruitment and initiation of mitophagy machinery, and facilitates mitophagy in cardiomyocytes.