Helix B surface peptide attenuates diabetic cardiomyopathy via AMPK-dependent autophagy

doi:10.1016/j.bbrc.2016.11.091

Biochemical and Biophysical Research Communications

Volume 482, Issue 4, 22 January 2017, Pages 665-671

https://doi.org/10.1016/j.bbrc.2016.11.091 Get rights and content

Highlights

•
HBSP could improve cardiac function in mice with diabetic cardiomyopathy.
•
HBSP exerted protective effects through restoration of autophagy.
•
Activation of AMPK might be involved.

Abstract

Background

Erythropoietin (EPO) has been reported to exert protective effects on a host of damaged tissues. However, the erythropoietic effect of this hormone can result in high risks of thrombosis, stroke, and hypertension, remarkably limiting the clinical use of EPO. Helix B surface peptide (HBSP) is a small peptide derived from the helix-B domain of EPO. Surprisingly, HBSP retains the tissue protective properties of EPO without altering the hematocrit. Thus, we evaluated the possible role of HBSP on diabetic cardiomyopathy.

Methods

Diabetes was induced in mice by intraperitoneal injections of streptozocin (STZ). Mice were randomly treated with normal saline or HBSP. Cardiac function, fibrosis, apoptosis, and myocardial mitochondrial morphology were examined. For in vitro experiments, H9C2 myoblast cells were randomly grouped as normal glucose (NG, 5 mM), NG+HBSP (100 nM), high glucose (HG, 33 mM), HG+HBSP (100 nM), HG+HBSP+3-methyladenine (3-MA, 10 mM), HG+rapamycin (Rapa, 100 nM), and HG+HBSP+Compound C (CC, 10 mM). Autophagosomes, LC3 dots, apoptosis and mitochondria membrane potential (MMP) of H9C2 cells were examined.The expressions of LC3, p62, p-AMPK (Thr172) and p-mTOR (Ser2448) were examined by Western blot.

Results

HBSP markedly improved cardiac function, attenuated cardiac interstitial fibrosis, inhibited myocardial apoptosis, and ameliorated mitochondrial ultrastructure in mice with diabetic cardiomyopathy. HG reduced autophagy in H9C2 cells. HBSP enhanced autophagy in HG-treated H9C2 cells. HBSP reduced the apoptosis index of HG-treated H9C2 cells. HBSP increased the MMP of HG-treated H9C2 cells. HBSP increased the levels of p-AMPK (Thr172), and reduced p-mTOR (Ser2448) in HG-treated H9C2 cells, and the increase of p-AMPK (Thr172) was accompanied by the stimulation of autophagy. Autophagy inhibitor 3-MA and AMPK inhibitor CC mitigated HBSP-induced beneficial effect, whereas autophagy inducer Rapa alleviated the HG-induced cell apoptosis.

Conclusions

HBSP attenuates diabetic cardiomyopathy via autophagy mediated by AMPK-dependent pathway. HBSP may be a potential therapeutic intervention for diabetic cardiomyopathy.

Introduction

Cardiovascular complications are the major causes of morbidity and mortality in diabetic patients. Diabetic cardiomyopathy (DCM) is a ventricular dysfunction independent of coronary artery disease and hypertension [1]. Various molecular mechanisms for the development of DCM are associated with inflammation, myocardial fibrosis, mitochondrial damage, myocardial apoptosis and autophagy [2], [3], [4], [5]. However, the underlying mechanisms of DCM remain ambiguous, and no effective strategy has been established to prevent the progression of heart failure in diabetic patients.

Erythropoietin (EPO) is an endogenous protein that is clinically used to treat anemia caused by chronic kidney disease, human immunodeficiency virus, and chemotherapy. This hormone is also used to limit the number of blood transfusions for surgery [6], [7]. EPO was found to exert tissue-protective effects on many organs [8]. However, considerably high doses of EPO are needed to achieve tissue protection, resulting in severe side effects, such as thrombosis, stroke, and hypertension [9]. Helix B surface peptide (HBSP), an 11-amino acid sequence derived from the aqueous surface of the helix B of EPO, was recently shown to display potent tissue protective property [10]. Long-term administration of HBSP ameliorated the progression of postmyocardial infarction-dilated cardiomyopathy [11]. Treatment with HBSP protected against diabetic retinopathy in a rodent streptozocin (STZ)-induced type 1 diabetes model [12]. Therefore, HBSP is of great interest as a potential therapeutic intervention for DCM.

Autophagy is a highly conserved catabolic process that plays a housekeeping role in maintaining cellular homeostasis [13]. The AMP-activated protein kinase (AMPK) is a serine/threonine kinase that senses energy status and regulates cellular energy homeostasis [14]. As a key initiator of autophagic progress, AMPK is essential to cardiac survival and function. Nutrient starvation leads to elevated cellular AMP/ATP ratio, which activates AMPK. This process promotes autophagy through multiple pathways, one of which is the inhibition of mTOR. Increased phosphorylation of AMPK at Thr172 determines the phosphorylation of tuberus sclerosis complex 2 which leads to the decreased phosphorylation of mTOR at Ser2448. Induction of autophagy degrades proteins and damaged organelles, generating numerous amino acids and fatty acids for energy production, which improves myocardial stress tolerance [14]. High glucose treatment reduced AMPK phosphorylation and induced mammalian target of rapamycin (mTOR) [15]. Furthermore, convincing evidence suggested that restoration of AMPK-induced autophagy significantly improved cardiac function in type 1 diabetes [16], [17], [18]. Therefore, we hypothesize that HBSP could attenuate DCM by activating autophagy mediated by AMPK-dependent pathway.

Section snippets

Animals preparation for in vivo experiments

Male C57BL/6 mice 8 weeks-old (weight, 25–30 g) received human care in adherence with the Care and Use of Laboratory Animals published by the US National Institutes of Health. HBSP (HPLC content $\geq$ 98%) was purchased from Science Peptide Biological Technology (Shanghai, China) and dissolved in normal saline. The experimental mice received intraperitoneal injections of streptozotocin (STZ, Sigma, St. Louis, MO) dissolved in 0.1 ml citrate buffer (pH 4.5) at 50 mg/kg for 5 consecutive days to

HBSP improves cardiac function and alleviates myocardial interstitial fibrosis in mice with diabetic cardiomyopathy

LVEF and LVSF were decreased in the DCM group compared with the CON or CON+HBSP group. LVEF and LVSF in the DCM+HBSP group were elevated compared with the DCM group. LVEDV and LVESV were increased in the DCM group compared with the CON or CON+HBSP group, whereas HBSP inhibited the increase in LVEDV and LVESV in the DCM mice (Fig. 1 A-1 E). The DCM group showed enhanced interstitial fibrosis compared with the CON or CON+HBSP group. HBSP attenuated the level of interstitial fibrosis caused by

Discussion

EPO stimulates red blood cell progenitors by binding to an EPO receptor (EPOR) homodimer (EPOR)₂, thereby stimulating the erythropoietic activity [27]. The tissue-protective effect of EPO is achieved by EPO binding to a heterodimer receptor complex composed of EPOR and β common receptor (βCR) subunits. This complex is also known as the innate repair receptor (IRR or EPOR-βCR), which activates critical cell surviving pathways [28]. Previous studies demonstrated that EPO protected pancreatic

Conflict of interest

None.

Acknowledgements

This work was supported by National Natural Science Foundation of China (No. 81200100).

References (39)

J.G. Duncan
Mitochondrial dysfunction in diabetic cardiomyopathy
Biochim. Biophys. Acta
(2011)
J. Asbun et al.
The pathogenesis of myocardial fibrosis in the setting of diabetic cardiomyopathy
J. Am. Coll. Cardiol.
(2006)
Y. Guo et al.
A novel protective mechanism for mitochondrial aldehyde dehydrogenase (ALDH2) in type i diabetes-induced cardiac dysfunction: role of AMPK-regulated autophagy
Biochim. Biophys. Acta
(2015)
G. Zhou et al.
Metallothionein suppresses angiotensin II-induced nicotinamide adenine dinucleotide phosphate oxidase activation, nitrosative stress, apoptosis, and pathological remodeling in the diabetic heart
J. Am. Coll. Cardiol.
(2008)
M. Collino et al.
Flipping the molecular switch for innate protection and repair of tissues: long-lasting effects of a non-erythropoietic small peptide engineered from erythropoietin
Pharmacol. Ther.
(2015)
C. Yang et al.
A novel proteolysis-resistant cyclic helix B peptide ameliorates kidney ischemia reperfusion injury
Biochim. Biophys. Acta
(2014)
S. Sun et al.
Lin28a protects against diabetic cardiomyopathy via the PKA/ROCK2 pathway
Biochem. Biophys. Res. Commun.
(2016)
W. Martinet et al.
Autophagy in cardiovascular disease
Trends Mol. Med.
(2007)
W.B. Kannel et al.
Diabetes and cardiovascular disease. The Framingham study
Jama
(1979)
Y. Mano et al.
Overexpression of human C-reactive protein exacerbates left ventricular remodeling in diabetic cardiomyopathy
Circ. J.
(2011)

J. Wang et al.

Inhibition of aldehyde dehydrogenase 2 by oxidative stress is associated with cardiac dysfunction in diabetic rats

Mol. Med.

(2011)

A. Palazzuoli et al.

The role of erythropoietin stimulating agents in anemic patients with heart failure: solved and unresolved questions

Ther. Clin. Risk. Manag.

(2014)

F. Li et al.

Erythropoietin on a tightrope: balancing neuronal and vascular protection between intrinsic and extrinsic pathways

Neurosignals

(2004)

M. Gassmann et al.

Non-erythroid functions of erythropoietin

Adv. Exp. Med. Biol.

(2003)

H. Ehrenreich et al.

Recombinant human erythropoietin in the treatment of acute ischemic stroke

Stroke

(2009)

M. Brines et al.

Nonerythropoietic, tissue-protective peptides derived from the tertiary structure of erythropoietin

Proc. Natl. Acad. Sci. U. S. A.

(2008)

I. Ahmet et al.

Chronic administration of small nonerythropoietic peptide sequence of erythropoietin effectively ameliorates the progression of postmyocardial infarction-dilated cardiomyopathy

J. Pharmacol. Exp. Ther.

(2013)

C.M. McVicar et al.

Intervention with an erythropoietin-derived peptide protects against neuroglial and vascular degeneration during diabetic retinopathy

Diabetes

(2011)

M. Taneike et al.

Inhibition of autophagy in the heart induces age-related cardiomyopathy

Autophagy

(2010)

Cited by (37)

Inhibiting mir-34a-5p regulates doxorubicin-induced autophagy disorder and alleviates myocardial pyroptosis by targeting Sirt3-AMPK pathway
2023, Biomedicine and Pharmacotherapy
Doxorubicin (DOX) is a commonly used chemotherapy drug widely applied in various cancers such as breast cancer, leukemia, and sarcomas. However, its usage is limited by cardiotoxicity. Additionally, the cardiac toxicity of DOX accumulates with dose and duration, making it imperative to identify therapeutic targets for DOX-induced cardiomyopathy (DIC). It has been reported that miRNAs are involved in the progression of DIC. Mir-34a-5p has been identified as an early diagnostic marker for DIC. While studies have shown the involvement of mir-34a-5p in DIC apoptosis, it has not been validated in animal models, nor has the potential improvement of DIC by inhibiting mir-34a-5p been confirmed. Autophagy and pyroptosis are key factors in the development of DIC and can serve as therapeutic targets for its treatment. In this study, we found that mir-34a-5p was upregulated in the heart after DOX treatment and that the inhibition of mir-34–5p reduced autophagy and pyroptosis in DIC. We also found that the inhibition of mir-34a-5p inhibited pyroptosis by regulating autophagy and reducing mitochondrial reactive oxygen species. Moreover, we identified Sirtuin3 (Sirt3) as a target gene of mir-34a-5p using a double-luciferase reporter assay. overexpression Sirt3 reduced pyroptosis by alleviating autophagy. Our research findings suggest that inhibiting mir-34a-5p has a beneficial role in alleviating autophagy and pyroptosis in DIC. This provides therapeutic prospects for treating DIC.
Cyclic helix B peptide alleviates proinflammatory cell death and improves functional recovery after traumatic spinal cord injury
2023, Redox Biology
Necroptosis and pyroptosis, two types of proinflammatory programmed cell death, were recently found to play important roles in spinal cord injury (SCI). Moreover, cyclic helix B peptide (CHBP) was designed to maintain erythropoietin (EPO) activity and protect tissue against the adverse effects of EPO. However, the protective mechanism of CHBP following SCI is still unknown. This research explored the necroptosis- and pyroptosis-related mechanism underlying the neuroprotective effect of CHBP after SCI.
Gene Expression Omnibus (GEO) datasets and RNA sequencing were used to identify the molecular mechanisms of CHBP for SCI. A mouse model of contusion SCI was constructed, and HE staining, Nissl staining, Masson staining, footprint analysis and the Basso Mouse Scale (BMS) were applied for histological and behavioural analyses. qPCR, Western blot analysis, immunoprecipitation and immunofluorescence were utilized to analyse the levels of necroptosis, pyroptosis, autophagy and molecules associated with the AMPK signalling pathway.
The results revealed that CHBP significantly improved functional restoration, elevated autophagy, suppressed pyroptosis, and mitigated necroptosis after SCI. 3-Methyladenine (3-MA), an autophagy inhibitor, attenuated these beneficial effects of CHBP. Furthermore, CHBP-triggered elevation of autophagy was mediated by the dephosphorylation and nuclear translocation of TFEB, and this effect was due to stimulation of the AMPK-FOXO3a-SPK2-CARM1 and AMPK-mTOR signalling pathways.
CHBP acts as a powerful regulator of autophagy that improves functional recovery by alleviating proinflammatory cell death after SCI and thus might be a prospective therapeutic agent for clinical application.
Autophagy-nutrient sensing pathways in diabetic complications
2022, Pharmacological Research
The incidence of diabetes has been increasing in recent decades which is affecting the population of both, developed and developing countries. Diabetes is associated with micro and macrovascular complications which predominantly result from hyperglycemia and disrupted metabolic pathways. Persistent hyperglycemia leads to increased reactive oxygen species (ROS) generation, formation of misfolded and abnormal proteins, and disruption of normal cellular functioning. The inability to maintain metabolic homeostasis under excessive energy and nutrient input, which induces insulin resistance, is a crucial feature during the transition from obesity to diabetes. According to various study reports, redox alterations, intracellular stress and chronic inflammation responses have all been linked to dysregulated energy metabolism and insulin resistance. Autophagy has been considered a cleansing mechanism to prevent these anomalies and restore cellular homeostasis. However, disrupted autophagy has been linked to the pathogenesis of metabolic disorders such as obesity and diabetes. Recent studies have reported that the regulation of autophagy has a beneficial role against these conditions. When there is plenty of food, nutrient-sensing pathways activate anabolism and storage, but the shortage of food activates homeostatic mechanisms like autophagy, which mobilises internal stockpiles. These nutrient-sensing pathways are well conserved in eukaryotes and are involved in the regulation of autophagy which includes SIRT1, mTOR and AMPK. The current review focuses on the role of SIRT1, mTOR and AMPK in regulating autophagy and suggests autophagy along with these nutrient-sensing pathways as potential therapeutic targets in reducing the progression of various diabetic complications.
Therapeutic effects of Chinese herbal medicines and their extracts on diabetes
2021, Biomedicine and Pharmacotherapy
With the improvement of people's living standards and changes in the environment, the incidence of diabetes has increased rapidly. It has gradually become one of the main diseases threatening the health and life of modern people, bringing a great burden to the society. Although the existing treatment methods can effectively control the symptoms of diabetes and delay its progression, they have not brought satisfactory improvement in the quality of life and treatment of patients. Traditional Chinese herbal medicines and their extracts combine thousands of years of experience and the scientific basis provided by modern experimental research, which is expected to bring a qualitative leap in the clinical management of diabetes. Therefore, this article systematically reviews studies on the effects of Chinese herbal medicine and its extracts on diabetes and its complications, and aims to bring new ideas and options for the clinical treatment of diabetes.
Autophagy in the diabetic heart: A potential pharmacotherapeutic target in diabetic cardiomyopathy
2021, Ageing Research Reviews
Association of diabetes with an elevated risk of cardiac failure has been clinically evident. Diabetes potentiates diastolic and systolic cardiac failure following the myocardial infarction that produces the cardiac muscle-specific microvascular complication, clinically termed as diabetic cardiomyopathy. Elevated susceptibility of diabetic cardiomyopathy is primarily caused by the generation of free radicals in the hyperglycemic milieu, compromising the myocardial contractility and normal cardiac functions with increasing redox insult, impaired mitochondria, damaged organelles, apoptosis, and cardiomyocytes fibrosis. Autophagy is essentially involved in the recycling/clearing the damaged organelles, cytoplasmic contents, and aggregates, which are frequently produced in cardiomyocytes. Although autophagy plays a vital role in maintaining the cellular homeostasis in diligent cardiac tissues, this process is frequently impaired in the diabetic heart. Given its clinical significance, accumulating evidence largely showed the functional aspects of autophagy in diabetic cardiomyopathy, elucidating its intricate protective and pathogenic outcomes. However, etiology and molecular readouts of these contrary autophagy activities in diabetic cardiomyopathy are not yet comprehensively assessed and translated. In this review, we attempted to assess the role of autophagy and its adaptations in the diabetic heart. To delineate the molecular consequences of these events, we provided detailed insights into the autophagy regulation pieces of machinery including the mTOR/AMPK, TFEB/ZNSCAN3, FOXOs, SIRTs, PINK1/Parkin, Nrf2, miRNAs, and others in the diabetic cardiomyopathy. Given the clinical significance of autophagy in the diabetic heart, we further discussed the potential pharmacotherapeutic strategies towards targeting autophagy. Taken together, the present report meticulously assessed autophagy, its adaptations, and molecular regulations in diabetic cardiomyopathy and reviewed the current autophagy-targeting strategies.
Trehalose alleviates apoptosis by protecting the autophagy-lysosomal system in alveolar macrophages during human silicosis
2020, Life Sciences
Citation Excerpt :
Two autophagic signaling pathways exist in the cell: a mammalian target of rapamycin (mTOR)-dependent mechanism and a mTOR-independent mechanism. The former mechanism involves the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mTOR signaling pathway [10–11], and the latter mechanism includes the Bcl-2 and Beclin-1 signaling pathway [12], the p53 signaling pathway [13], and others. Notably, our previous study revealed a blockage of autophagic degradation in AMs during human silicosis.
Alveolar macrophages (AMs) are the primary targets of silicosis. Blockade of autophagy may aggravate the apoptosis of AMs. Trehalose (Tre), a transcription factor EB (TFEB) activator, may impact the autophagy-lysosomal system in AMs during silicosis. However, the mechanism by which Tre acts upon AMs in silicosis is unknown.
We collected AMs from twenty male workers exposed to silica and divided them into observer and silicosis patient groups. AMs from the two groups were then exposed to Tre. Western blot was used to measure the expression of autophagy-associated proteins. Lysosomal-associated membrane protein 1 (LAMP1) expression was observed using immunofluorescence and western blot. Apoptosis of the AMs was detected by TUNEL assay and western blot.
Tre induced localization of TFEB to the nucleus in the AMs of both groups. After Tre exposure, LAMP1 levels increased and LC3 levels decreased in the AMs of both groups, suggesting that Tre may increase the function of the autophagy-lysosomal system. The LC3-II/I ratio in the Tre-exposed AMs was lower than in the AMs not exposed to Tre. The LC3-II/I ratio in AMs subjected to Tre plus Bafilomycin (Baf) was higher than the ratio in cells exposed to Tre or Baf individually. Additionally, p62 levels decreased after Tre stimulation in the AMs of both groups. This indicates that Tre may accelerate the process of autophagic degradation. We also found decreased levels of cleaved caspase-3 after Tre treatment in the AMs of both groups. However, p-mTOR (Ser2448) and p-mTOR (Ser2481) levels did not change significantly after Tre treatment, suggesting that the mTOR signaling pathway was not affected by Tre treatment.
Our findings suggest that the restoration of autophagy-lysosomal function by Tre may be a potential protective strategy against silicosis.

View all citing articles on Scopus

View full text

Helix B surface peptide attenuates diabetic cardiomyopathy via AMPK-dependent autophagy

Highlights

Abstract

Background

Methods

Results

Conclusions

Introduction

Section snippets

Animals preparation for in vivo experiments

HBSP improves cardiac function and alleviates myocardial interstitial fibrosis in mice with diabetic cardiomyopathy

Discussion

Conflict of interest

Acknowledgements

Biochim. Biophys. Acta

J. Am. Coll. Cardiol.

Biochim. Biophys. Acta

J. Am. Coll. Cardiol.

Pharmacol. Ther.

Biochim. Biophys. Acta

Biochem. Biophys. Res. Commun.

Trends Mol. Med.

Diabetes and cardiovascular disease. The Framingham study

Jama

Overexpression of human C-reactive protein exacerbates left ventricular remodeling in diabetic cardiomyopathy

Circ. J.

Inhibition of aldehyde dehydrogenase 2 by oxidative stress is associated with cardiac dysfunction in diabetic rats

Mol. Med.

The role of erythropoietin stimulating agents in anemic patients with heart failure: solved and unresolved questions

Ther. Clin. Risk. Manag.

Erythropoietin on a tightrope: balancing neuronal and vascular protection between intrinsic and extrinsic pathways

Neurosignals

Non-erythroid functions of erythropoietin

Adv. Exp. Med. Biol.

Recombinant human erythropoietin in the treatment of acute ischemic stroke

Stroke

Nonerythropoietic, tissue-protective peptides derived from the tertiary structure of erythropoietin

Proc. Natl. Acad. Sci. U. S. A.

Chronic administration of small nonerythropoietic peptide sequence of erythropoietin effectively ameliorates the progression of postmyocardial infarction-dilated cardiomyopathy

J. Pharmacol. Exp. Ther.

Intervention with an erythropoietin-derived peptide protects against neuroglial and vascular degeneration during diabetic retinopathy

Diabetes

Inhibition of autophagy in the heart induces age-related cardiomyopathy

Autophagy