Abstract MP101: Functional Maintenance of the Sarcomere Requires Bag3-dependent Autophagy

Bcl2-associated athanogene-3 (BAG3) is a pro-autophagy co-chaperone highly expressed in the heart. Clinical studies show BAG3 haploinsufficiency and mutations are associated with heart failure (HF). However, these studies are largely observational and fail to go beyond the observed phenotype and study mechanism. One study in neonatal myocytes suggests a role for BAG3 in structural maintenance of the sarcomere. However, the structural and functional significance of BAG3 in adult myocytes is not known. We found that myofilament BAG3 expression decreases in human heart failure and is associated with impaired myofilament force-generating capacity (F max ). To assess whether rescuing BAG3 levels could restore function, we used a mouse model of HF and treated with BAG3 gene therapy via AAV9. Myofilament function was assessed in skinned cardiomyocytes by force-calcium relationship. HF mice experienced a reduction in F max , but this was fully restored to sham levels by BAG3 gene therapy.To explore mechanism, we used mass spectrometry to identify the BAG3-interactome at the myofilament and found heat shock proteins (HSP) 70 and B8 among the top hits. Immunofluorescence further showed that HSP70, B8, and BAG3 each localized to the sarcomere z-disk. This BAG3-Hsp complex had previously been described to promote ubiquitin-dependent autophagy in skeletal muscle. Notably, in both human HF samples and in the mouse HF model, myofilament ubiquitin levels increased significantly. However, BAG3 gene therapy in HF reduces ubiquitin levels and restores autophagy flux. This suggests that BAG3 serves a role in proteostasis for the sarcomere, which may explain the functional effect of BAG3 gene therapy.To further explore the impact of the BAG3/HSP complex on myofilament function, we used a mouse model with the P209L BAG3 mutation, which had previously been described to disrupt client processing by the complex. We found cardiomyocytes from P209L mice had significantly reduced F max and elevated myofilament ubiquitin levels, suggesting BAG3-dependent autophagy is required to maintain function. Together, our data identify a functional role for BAG3 at the sarcomere and indicate BAG3-mediated autophagy is an important mechanism for maintaining myofilament proteostasis.

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Abstract 16485: The Nitroxyl Donor 1-Nitrosocyclohexyl Acetate Limits Cardiac Superoxide Upregulation and Diastolic Dysfunction in a Mouse Model of Diabetic Cardiomyopathy

Circulation ◽

10.1161/circ.130.suppl_2.16485 ◽

2014 ◽

Vol 130 (suppl_2) ◽

Author(s):

Rebecca H Ritchie ◽

Nga Cao ◽

Yung George Wong ◽

Sarah Rosli ◽

Helen Kiriazis ◽

...

Keyword(s):

Heart Failure ◽

Mouse Model ◽

Diastolic Dysfunction ◽

Diabetic Cardiomyopathy ◽

Ex Vivo ◽

Systolic Function ◽

Left Ventricular ◽

Cardiomyocyte Hypertrophy ◽

Lv Diastolic Dysfunction ◽

The Impact

Nitroxyl (HNO), a redox congener of NO•, is a novel regulator of cardiovascular function combining vasodilator and positive inotropic properties. Our previous studies have demonstrated these properties occur concomitantly in the intact heart; HNO moreover also exhibits antihypertrophic and superoxide-suppressing actions. HNO donors may thus offer favorable actions in heart failure. The impact of chronic HNO donor administration has however yet to be reported in this context. We tested the hypothesis that the HNO donor 1-nitrosocyclohexyl acetate (1-NCA) limits cardiomyocyte hypertrophy and left ventricular (LV) diastolic dysfunction in a mouse model of diabetic cardiomyopathy in vivo. Male 6 week-old FVB/N mice received either streptozotocin (55 mg/kg/day i.p. for 5 days, n=17), to induce type 1 diabetes, or citrate vehicle (n=16). After 4 weeks of hyperglycemia, mice were allocated to 1-NCA therapy (83mg/kg/day i.p.) or vehicle, and followed for a further 4 weeks. As shown in the table, blood glucose was unaffected by 1-NCA. LV diastolic dysfunction was evident in diabetic mice, measured as echocardiography-derived A wave velocity, deceleration time and E:A ratio; LV systolic function was preserved. Diabetes-induced diastolic dysfunction was accompanied by increased LV cardiomyocyte size, hypertrophic and pro-fibrotic gene expression, and upregulation of LV superoxide. These characteristics of diabetic cardiomyopathy were largely prevented by 1-NCA treatment. Selectivity of 1-NCA as a donor of HNO versus NO• was demonstrated by the sensitivity of the coronary vasodilation response of 1-NCA to the HNO scavenger L-cysteine (4mM), but not to the NO• scavenger hydroxocobalamin (50μM), in the normal rat heart ex vivo (n=3-7). Collectively, our studies provide the first evidence that HNO donors may represent a promising new strategy for the treatment of diabetic cardiomyopathy, and implies their therapeutic efficacy in settings of chronic heart failure.

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Intracellular Dyssynchrony of Diastolic Cytosolic [Ca 2+ ] Decay in Ventricular Cardiomyocytes in Cardiac Remodeling and Human Heart Failure

Circulation Research ◽

10.1161/circresaha.113.300895 ◽

2013 ◽

Vol 113 (5) ◽

pp. 527-538 ◽

Cited By ~ 39

Author(s):

Felix Hohendanner ◽

Senka Ljubojević ◽

Niall MacQuaide ◽

Michael Sacherer ◽

Simon Sedej ◽

...

Keyword(s):

Heart Failure ◽

Cardiac Remodeling ◽

Human Heart ◽

Cyclopiazonic Acid ◽

Cardiomyocyte Hypertrophy ◽

Human Heart Failure ◽

Mitochondrial Density ◽

Human Cardiomyocytes ◽

End Stage ◽

The Impact

Rationale : Synchronized release of Ca 2+ into the cytosol during each cardiac cycle determines cardiomyocyte contraction. Objective: We investigated synchrony of cytosolic [Ca 2+ ] decay during diastole and the impact of cardiac remodeling. Methods and Results: Local cytosolic [Ca 2+ ] transients (1-µm intervals) were recorded in murine, porcine, and human ventricular single cardiomyocytes. We identified intracellular regions of slow (slowCaR) and fast (fastCaR) [Ca 2+ ] decay based on the local time constants of decay (TAU local ). The SD of TAU local as a measure of dyssynchrony was not related to the amplitude or the timing of local Ca 2+ release. Stimulation of sarcoplasmic reticulum Ca 2+ ATPase with forskolin or istaroxime accelerated and its inhibition with cyclopiazonic acid slowed TAU local significantly more in slowCaR, thus altering the relationship between SD of TAU local and global [Ca 2+ ] decay (TAU global ). Na + /Ca 2+ exchanger inhibitor SEA0400 prolonged TAU local similarly in slowCaR and fastCaR. FastCaR were associated with increased mitochondrial density and were more sensitive to the mitochondrial Ca 2+ uniporter blocker Ru360. Variation in TAU local was higher in pig and human cardiomyocytes and higher with increased stimulation frequency (2 Hz). TAU local correlated with local sarcomere relengthening. In mice with myocardial hypertrophy after transverse aortic constriction, in pigs with chronic myocardial ischemia, and in end-stage human heart failure, variation in TAU local was increased and related to cardiomyocyte hypertrophy and increased mitochondrial density. Conclusions: In cardiomyocytes, cytosolic [Ca 2+ ] decay is regulated locally and related to local sarcomere relengthening. Dyssynchronous intracellular [Ca 2+ ] decay in cardiac remodeling and end-stage heart failure suggests a novel mechanism of cellular contractile dysfunction.

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Sympathetic neural responses in heart failure during exercise and after exercise training

Clinical Science ◽

10.1042/cs20201306 ◽

2021 ◽

Vol 135 (4) ◽

pp. 651-669

Author(s):

Catherine F. Notarius ◽

John S. Floras

Keyword(s):

Heart Failure ◽

Exercise Training ◽

Sympathetic Nerve ◽

Cardiovascular Response ◽

Future Research ◽

Human Heart Failure ◽

Reduced Ejection Fraction ◽

Direct Measurements ◽

Response To Exercise ◽

The Impact

Abstract The sympathetic nervous system coordinates the cardiovascular response to exercise. This regulation is impaired in both experimental and human heart failure with reduced ejection fraction (HFrEF), resulting in a state of sympathoexcitation which limits exercise capacity and contributes to adverse outcome. Exercise training can moderate sympathetic excess at rest. Recording sympathetic nerve firing during exercise is more challenging. Hence, data acquired during exercise are scant and results vary according to exercise modality. In this review we will: (1) describe sympathetic activity during various exercise modes in both experimental and human HFrEF and consider factors which influence these responses; and (2) summarise the effect of exercise training on sympathetic outflow both at rest and during exercise in both animal models and human HFrEF. We will particularly highlight studies in humans which report direct measurements of efferent sympathetic nerve traffic using intraneural recordings. Future research is required to clarify the neural afferent mechanisms which contribute to efferent sympathetic activation during exercise in HFrEF, how this may be altered by exercise training, and the impact of such attenuation on cardiac and renal function.

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Abstract 85: Cardiac Pathology Is Worsened by Loss of the Cell Junction Protein Claudin-5 and Improved by Claudin-5 Gene Therapy

Circulation Research ◽

10.1161/res.111.suppl_1.a85 ◽

2012 ◽

Vol 111 (suppl_1) ◽

Author(s):

Dawn A Delfín ◽

Kevin E Schill ◽

Ying Xu ◽

Sarah Swager ◽

Paul M L Janssen ◽

...

Keyword(s):

Heart Failure ◽

Gene Therapy ◽

Preliminary Evidence ◽

Cell Junction ◽

Human Heart Failure ◽

Adeno Associated Virus ◽

Cardiac Pathology ◽

Adhesion Protein ◽

Junction Protein ◽

A Cell

Claudin-5 is a cell-cell adhesion protein that plays established roles in the blood-brain barrier and cancer. We were the first to demonstrate that it plays a role in the heart with implications for heart failure and cardiomyopathy. Several animal models with cardiac pathology show reduced cardiac claudin-5 levels. Importantly, 60% of human heart failure patients show reduced levels of cardiac claudin-5, independent of changes in other cell junction proteins in the heart, supporting that the loss of cardiac claudin-5 has clinical relevance. To test our hypothesis that claudin-5 plays an important role in protecting the heart, we used mouse models to demonstrate whether circumventing reductions in cardiac claudin-5 is able to improve cardiac histology and physiology, and whether ablation of claudin-5 is able to induce cardiac pathology. We now show that maintaining cardiac claudin-5 levels using gene therapy (cldn5 transduction using an adeno-associated virus vector) significantly reduced cardiac pathology in mice which otherwise demonstrate heart failure and cardiomyopathy indicators. We also present preliminary evidence of the effects of knocking out the cldn5 gene specifically in cardiomyocytes using the inducible cre-loxP system.

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Cardiac Gene Therapy with Phosphodiesterases PDE2A and PDE4B Ameliorates Cardiac Function in a Mouse Model of Heart Failure Induced by Chronic Isoproterenol Infusion

Biophysical Journal ◽

10.1016/j.bpj.2016.11.2873 ◽

2017 ◽

Vol 112 (3) ◽

pp. 531a

Author(s):

Jerome Leroy

Keyword(s):

Heart Failure ◽

Gene Therapy ◽

Mouse Model ◽

Cardiac Function ◽

Cardiac Gene ◽

Isoproterenol Infusion

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The role of heat shock proteins and co-chaperones in heart failure

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.2016.0530 ◽

2017 ◽

Vol 373 (1738) ◽

pp. 20160530 ◽

Cited By ~ 27

Author(s):

Mark J. Ranek ◽

Marisa J. Stachowski ◽

Jonathan A. Kirk ◽

Monte S. Willis

Keyword(s):

Heart Failure ◽

Quality Control ◽

Heat Shock ◽

Heat Shock Proteins ◽

Structural Integrity ◽

Protein Quality ◽

Protein Quality Control ◽

Experimental Models ◽

Human Heart Failure ◽

Shock Proteins

The ongoing contractile and metabolic demands of the heart require a tight control over protein quality control, including the maintenance of protein folding, turnover and synthesis. In heart disease, increases in mechanical and oxidative stresses, post-translational modifications (e.g., phosphorylation), for example, decrease protein stability to favour misfolding in myocardial infarction, heart failure or ageing. These misfolded proteins are toxic to cardiomyocytes, directly contributing to the common accumulation found in human heart failure. One of the critical class of proteins involved in protecting the heart against these threats are molecular chaperones, including the heat shock protein70 (HSP70), HSP90 and co-chaperones CHIP (carboxy terminus of Hsp70-interacting protein, encoded by the Stub1 gene) and BAG-3 (BCL2-associated athanogene 3). Here, we review their emerging roles in the maintenance of cardiomyocytes in human and experimental models of heart failure, including their roles in facilitating the removal of misfolded and degraded proteins, inhibiting apoptosis and maintaining the structural integrity of the sarcomere and regulation of nuclear receptors. Furthermore, we discuss emerging evidence of increased expression of extracellular HSP70, HSP90 and BAG-3 in heart failure, with complementary independent roles from intracellular functions with important therapeutic and diagnostic considerations. While our understanding of these major HSPs in heart failure is incomplete, there is a clear potential role for therapeutic modulation of HSPs in heart failure with important contextual considerations to counteract the imbalance of protein damage and endogenous protein quality control systems. This article is part of the theme issue ‘Heat shock proteins as modulators and therapeutic targets of chronic disease: an integrated perspective’.

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