Bone morphogenetic protein 1.3 inhibition decreases scar formation and supports cardiomyocyte survival after myocardial infarction

Despite the high prevalence of ischemic heart diseases worldwide, no antibody-based treatment currently exists. Starting from the evidence that a specific isoform of the Bone Morphogenetic Protein 1 (BMP1.3) is particularly elevated in both patients and animal models of myocardial infarction, here we assess whether its inhibition by a specific monoclonal antibody reduces cardiac fibrosis. We find that this treatment reduces collagen deposition and cross-linking, paralleled by enhanced cardiomyocyte survival, both in vivo and in primary cultures of cardiac cells. Mechanistically, we show that the anti-BMP1.3 monoclonal antibody inhibits Transforming Growth Factor β pathway, thus reducing myofibroblast activation and inducing cardioprotection through BMP5. Collectively, these data support the therapeutic use of anti-BMP1.3 antibodies to prevent cardiomyocyte apoptosis, reduce collagen deposition and preserve cardiac function after ischemia.

GSK3β Serine 389 Phosphorylation Modulates Cardiomyocyte Hypertrophy and Ischemic Injury

Prior studies show that glycogen synthase kinase 3β (GSK3β) contributes to cardiac ischemic injury and cardiac hypertrophy. GSK3β is constitutionally active and phosphorylation of GSK3β at serine 9 (S9) inactivates the kinase and promotes cellular growth. GSK3β is also phosphorylated at serine 389 (S389), but the significance of this phosphorylation in the heart is not known. We analyzed GSK3β S389 phosphorylation in diseased hearts and utilized overexpression of GSK3β carrying ser→ala mutations at S9 (S9A) and S389 (S389A) to study the biological function of constitutively active GSK3β in primary cardiomyocytes. We found that phosphorylation of GSK3β at S389 was increased in left ventricular samples from patients with dilated cardiomyopathy and ischemic cardiomyopathy, and in hearts of mice subjected to thoracic aortic constriction. Overexpression of either GSK3β S9A or S389A reduced the viability of cardiomyocytes subjected to hypoxia–reoxygenation. Overexpression of double GSK3β mutant (S9A/S389A) further reduced cardiomyocyte viability. Determination of protein synthesis showed that overexpression of GSK3β S389A or GSK3β S9A/S389A increased both basal and agonist-induced cardiomyocyte growth. Mechanistically, GSK3β S389A mutation was associated with activation of mTOR complex 1 signaling. In conclusion, our data suggest that phosphorylation of GSK3β at S389 enhances cardiomyocyte survival and protects from cardiomyocyte hypertrophy.

Abstract P336: Endogenous Alpha-1a Adrenergic Receptors Promote Cardiomyocyte Survival Through Suppression Of Rip Kinase-mediated Necroptosis

Our previous work has demonstrated essential protective roles for the endogenous cardiomyocyte alpha-1A adrenergic receptor (α1A-AR) subtype in mouse models of heart failure. However, the underlying mechanism of this protective phenotype is unclear. To address this gap in knowledge, we bred a mouse line lacking α1A-ARs on cardiomyocytes by crossing αMHC-cre mice with floxed α1A mice (CMKO= cre+ fl/fl, CMWT= cre- fl/fl), and subjected males to permanent LAD ligation. CMKO mice had increased serum HMGB1 level, larger infarcts and higher mortality. We found that RIP1/3-mediated programmed necrosis (necroptosis), but not apoptosis was exaggerated in CMKO mice 3 days after ligation. We then tested whether RIP1 inhibition with Nec-1s could mitigate this injury. Mice were given Nec-1s (1.65 mg/kg) or vehicle 10 mins prior to LAD ligation, followed by daily IV injection. Nec-1s treatment diminished post-ligation RIP1 (0.62±0.02 vs. 0.78±0.23 A.U., p=NS) and RIP3 expression (0.33±0.1 vs. 0.26±0.10 A.U., p=NS) in CMWT and CMKO mice respectively. Serum level of HMGB1 on D3 was markedly reduced in both CMWT (45.1%) and CMKO (61.1 %) after Nec-1s treatment. There was no difference between Nec-1s treated CMWT and CMKO mice (147±53 vs. 174±37 pg/mL, p=NS), indicating that blocking the RIP kinase pathway abrogates the exaggerated cell death in CMKO mice after ligation. Likewise, Nec-1s-treated CMKO mice had similar infarct areas to CMWT controls (16.2±4.5 vs. 19.9±4.6%, p=NS), further confirming that targeting necroptosis abrogates pathological damage. Collectively these Nec-1s data suggest that RIP-mediated necroptosis may account for larger infarcts in CMKO mice. Interestingly, expression of the apoptosis markers c-caspase-3 and PARP was similar between CMWT and CMKO mice, suggesting that the α1A-AR specifically regulates necroptosis. In sum, our data demonstrate that RIP kinase-mediated necroptosis contributes to susceptibility to injury in mice lacking cardiomyocyte α1A-ARs.

Mitofusin-2 Enhances Mitochondrial Contact With the Endoplasmic Reticulum and Promotes Diabetic Cardiomyopathy

Diabetic cardiomyopathy has been associated with mitochondrial damage. Mitochondria–endoplasmic reticulum (ER) contact is an important determinant of mitochondrial function and ER homeostasis. We therefore investigated whether hyperglycemia can damage the mitochondria by increasing their contact with the ER in cardiomyocytes. We found that hyperglycemia induced mitochondria–ER contact in cardiomyocytes, as evidenced by the increased MMM1, MDM34, and BAP31 expressions. Interestingly, the silencing of Mfn2 reduced the cooperation between the mitochondria and the ER in cardiomyocytes. Mfn2 silencing improved cardiomyocyte viability and function under hyperglycemic conditions. Additionally, the silencing of Mfn2 markedly attenuated the release of calcium from the ER to the mitochondria, thereby preserving mitochondrial metabolism in cardiomyocytes under hyperglycemic conditions. Mfn2 silencing reduced mitochondrial reactive oxygen species production, which reduced mitochondria-dependent apoptosis in hyperglycemia-treated cardiomyocytes. Finally, Mfn2 silencing attenuated ER stress in cardiomyocytes subjected to high-glucose stress. These results demonstrate that Mfn2 promotes mitochondria–ER contact in hyperglycemia-treated cardiomyocytes. The silencing of Mfn2 sustained mitochondrial function, suppressed mitochondrial calcium overload, prevented mitochondrial apoptosis, and reduced ER stress, thereby enhancing cardiomyocyte survival under hyperglycemic conditions.

Molecular Mechanisms And Clinical Implications Of Multiple Forms Of Mitophagy In The Heart

Mitochondria, the primary ATP-producing organelles, are highly abundant in cardiomyocytes. Mitochondrial function readily deteriorates in the presence of stress and, thus, maintenance of mitochondrial quality is essential for sustaining pump function in the heart. Cardiomyocytes under stress attempt to maintain mitochondrial quality primarily through dynamic changes in their morphology, namely fission and fusion, degradation and biogenesis. Mitophagy, a mitochondria-specific form of autophagy, is a major mechanism of degradation. The level of mitophagy is altered in stress conditions, which, in turn, significantly affects mitochondrial function, cardiomyocyte survival and death and cardiac function. Thus, mitophagy has been emerging as a promising target for treatment of cardiac conditions. To develop specific interventions modulating the activity of mitophagy in the heart, understanding how mitochondria are degraded in a given condition is important. Increasing lines of evidence suggest that there are multiple mechanisms by which mitochondria are degraded through mitophagy in the heart. For example, in addition to the well-established mechanism commonly utilized by general autophagy, involving Atg7 and LC3, recent evidence suggests that an alternative mechanism, independent of Atg7 and LC3, also mediates mitophagy in the heart. Here we describe molecular mechanisms through which mitochondria are degraded in the heart and discuss their functional significance. We also discuss molecular interventions to modulate the activity of mitophagy and their potential applications for cardiac conditions.

Identification and Comparison of Hyperglycemia-Induced Extracellular Vesicle Transcriptome in Different Mouse Stem Cells

Extracellular vesicles (EVs) derived from stem /progenitor cells harbor immense potential to promote cardiomyocyte survival and neovascularization, and to mitigate ischemic injury. However, EVs’ parental stem/progenitor cells showed modest benefits in clinical trials, suggesting autologous stem cell/EV quality might have been altered by stimuli associated with the co-morbidities such as hyperglycemia associated with diabetes. Hyperglycemia is a characteristic of diabetes and a major driving factor in cardiovascular disease. The functional role of stem/progenitor cell-derived EVs and the molecular signature of their secreted EV cargo under hyperglycemic conditions remain elusive. Therefore, we hypothesized that hyperglycemic stress causes transcriptome changes in stem/progenitor cell-derived EVs that may compromise their reparative function. In this study, we performed an unbiased analysis of EV transcriptome signatures from 3 different stem/progenitor cell types by RNA sequencing. The analysis revealed differential expression of a variety of RNA species in EVs. Specifically, we identified 241 common-dysregulated mRNAs, 21 ncRNAs, and 16 miRNAs in three stem cell-derived EVs. Gene Ontology revealed that potential function of common mRNAs mostly involved in metabolism and transcriptional regulation. This study provides potential candidates for preventing the adverse effects of hyperglycemia-induced stem/progenitor cell-derived EV dysfunction, and reference data for future biological studies and application of stem/progenitor cell-derived EVs.

PIM1 Promotes Survival of Cardiomyocytes by Upregulating c-Kit Protein Expression

Enhancing cardiomyocyte survival is crucial to blunt deterioration of myocardial structure and function following pathological damage. PIM1 (Proviral Insertion site in Murine leukemia virus (PIM) kinase 1) is a cardioprotective serine threonine kinase that promotes cardiomyocyte survival and antagonizes senescence through multiple concurrent molecular signaling cascades. In hematopoietic stem cells, PIM1 interacts with the receptor tyrosine kinase c-Kit upstream of the ERK (Extracellular signal-Regulated Kinase) and Akt signaling pathways involved in cell proliferation and survival. The relationship between PIM1 and c-Kit activity has not been explored in the myocardial context. This study delineates the interaction between PIM1 and c-Kit leading to enhanced protection of cardiomyocytes from stress. Elevated c-Kit expression is induced in isolated cardiomyocytes from mice with cardiac-specific overexpression of PIM1. Co-immunoprecipitation and proximity ligation assay reveal protein–protein interaction between PIM1 and c-Kit. Following treatment with Stem Cell Factor, PIM1-overexpressing cardiomyocytes display elevated ERK activity consistent with c-Kit receptor activation. Functionally, elevated c-Kit expression confers enhanced protection against oxidative stress in vitro. This study identifies the mechanistic relationship between PIM1 and c-Kit in cardiomyocytes, demonstrating another facet of cardioprotection regulated by PIM1 kinase.

Abstract 507: Activation of the Classically Pro-Apoptotic Death Receptor 5 Promotes Physiological Hypertrophy and Cardiomyocyte Survival

Heart failure is hallmarked by a combination of cardiomyocyte hypertrophy and death. Apoptosis, one of the primary mechanisms of cell death, occurs through finely tuned extrinsic or intrinsic pathways. Of the mediators involved in extrinsic apoptotic signaling, some have been extensively studied, such as tumor necrosis factor ((TNF)-α), while others have been relatively untouched. One such receptor is Death Receptor 5 (DR5) which, along with its ligand TNF-Related Apoptosis Inducing Ligand (TRAIL), have recently been implicated as a biomarker in determining the progression and outcome in patients following multiple heart failure etiologies, suggesting a novel role of DR5 signaling in the heart. These studies suggest a potentially protective role for DR5 in the heart; however, the function of TRAIL/DR5 in the heart has been virtually unstudied. Our goal was to explore the role of TRAIL/DR5 in cardiomyocyte hypertrophy and survival with the hypothesis that DR5 promotes cardiomyocyte survival and growth through non-canonical mechanisms. Mice treated with the DR5 agonist bioymifi or a DR5 agonist antibody, MD5-1, were absent of cell death, while an increase in hypertrophy was observed without a decline in cardiac function. In isolated cardiomyocytes, this pro-hypertrophic phenotype was determined to operate through MMP-dependent cleavage of HB-EGFR, leading to transactivation of EGFR and ERK1/2 signaling. To determine the role of DR5 in heart failure, a chronic catecholamine administration model was used and DR5 activation was found to decrease cardiomyocyte death and cardiac fibrosis. ERK1/2, a well characterized pro-survival, pro-hypertrophic kinase is activated in the heart with DR5 agonist administration and may represent the mechanistic link through which DR5 is imparting cardioprotection. In summary, DR5 activation promotes cardiomyocyte hypertrophy and survival and prevents cardiac fibrosis via a non-canonical MMP-EGFR-ERK1/2 pathway. Taken together, these studies identify a previously undetermined role for DR5 in the heart and identify novel therapeutic target for the treatment of heart failure.

A Novel Molecular Mechanism of IKKε-Mediated Akt/mTOR Inhibition in the Cardiomyocyte Autophagy after Myocardial Infarction

Autophagy of cardiomyocytes after myocardial infarction (MI) is an important factor affecting the prognosis of MI. Excessive autophagy can lead to massive death of cardiomyocytes, which will seriously affect cardiac function. IKKε plays a crucial role in the occurrence of autophagy, but the functional role in MI remains largely unknown. To evaluate the impact of IKKε on the autophagy of cardiomyocytes after MI, MI was induced by surgical left anterior descending coronary artery ligation in IKKε knockout (KO) mice and wild-type (WT) mice. Starvation of H9c2 cells with IKKε siRNA and rescued with IKKε overexpressed afterwards to test the mechanism of IKKε in autophagy in vitro. Our results demonstrated that the expression of IKKε was upregulated in mice myocardial tissues which were consistent with cardiomyocyte autophagy after MI. Significantly, the IKKε KO mice showed increased infarct size, decreased viable cardiomyocytes, and exacerbated cardiac dysfunction when compared with the wild-type mice. Western blot and electron micrography analysis also revealed that loss of IKKε induces excessive cardiomyocyte autophagy and reduced the expression of p-Akt and p-mTOR. Similar results were observed in IKKε siRNA H9c2 cells in vitro which were under starvation injury. Notably, the levels of p-Akt and p-mTOR can restore in IKKε rescued cells. In conclusion, our results indicated that IKKε protects cardiomyocyte survival by reduced autophagy following MI via regulation of the Akt/mTOR signaling pathway. Thus, our study suggests that IKKε might represent a potential therapeutic target for the treatment of MI.