Distinct Types of Cell Death and the Implication in Diabetic Cardiomyopathy

1Diabetic cardiomyopathy (DCM) is manifested as specific abnormalities of myocardial structure and function in diabetic patients without hypertension or coronary artery disease in clinic. DCM is regarded as the one of the most common complications of diabetes to increase the risk of heart failure. Hyperglycemia, insulin resistance, fatty acids, oxidative stress, inflammation, myocardial fibrosis and hypertrophy, mitochondrial dysfunction, endoplasmic reticulum stress, and endothelial dysfunction are possible molecular foundations for DCM. Reactive oxygen species (ROS) produced by fatty-acid oxidation or nicotinamide adenine dinucleotide phosphate (NADPH) oxidase induce cell death or tissue damage. Essentially, cell death is considered to be the terminal pathway of cardiomyocytes during DCM. Experimental models of DCM show that metabolic dysfunction, cardiac structural, or functional abnormalities are similar to the pathology of human DCM. Streptozotocin (STZ) is the most common agent to induce type 1 diabetes mellitus (T1DM). Zucker diabetic fatty rat, leptin receptor deficiency mice (db/db), and leptin deficiency (ob/ob) mice are useful models to study type 2 DM (T2DM). Recently, echocardiography, computed tomography, magnetic resonance imaging, and single-photon emission computed tomography are used for the diagnosis of DCM. In vitro, DCM is often imitated in rat embryonic heart derived H9C2 cells, neonatal cardiomyocytes or cardiac fibroblasts with high glucose (HG) or advance glycation end product (AGE) stimulation.

2A large number of studies have shown several distinct types of cardiomyocyte death. Morphologically, cell death can be classified into four different forms. (1) Type I or apoptosis, featuring with cytoplasmic shrinkage, nuclear pyknosis, karyorrhexis, DNA fragmentation, and plasma membrane blebbing, and eventually apoptotic body formation. (2) Type II, or autophagy, showing extensive cytoplasmic vacuolization to form autophagosome, phagocytosis and subsequent lysosomal degradation. (3) Type III or necrosis, manifesting with distinctive morphology, is different from type I and type II cell death. Its morphological changes contain organelle and plasma membrane rupture, finalizing with disposal of cell corpses absent of evident phagocytic and lysosomal involvement. (4) Type IV or entosis, displaying “cell-in-cell” cytological features in which cell winners engulf and kill cell losers (Figure 1). Biopsy showed that the apoptosis of a diabetic heart was 85 times higher than that of the nondiabetic heart, indicating that cardiomyocytes in diabetes were sensitive to suffer cell death. To understand the detailed mechanism of cell death in cardiomyocytes is beneficial to propose novel and ideal strategies for DCM.

3Apoptosis is also called programmed cell death (PCD), which is the fastest form of cell death. Proteolytic cascade induced by caspases is the key biochemical characteristic of apoptosis. Caspases with an inactive proenzyme form are widely expressed in cardiomyocytes. Immediately after activation, a protease cascade is initiated by other procaspases depending on their proteolytic activity. This proteolytic cascade amplifies apoptotic pathways and finally results in rapid and irreversible cell death. There are two main pathways, including extrinsic apoptotic pathway and intrinsic apoptotic pathway, involved in the mechanisms of apoptosis. The extrinsic apoptotic pathway is also known as death receptor pathway, during which multiple death ligands, such as tumor necrosis factor-α (TNF-α) and Fas, bind to their homologous receptors to trigger cell death. Then, caspase 8 and caspase 9 are initiated to subsequently activate executioner caspases (caspase 3, caspase 6, and caspase 7), and finally result in apoptosis. The intrinsic pathway is known as mitochondrial pathway. During intrinsic pathway, a group of proteins called the complex permeability transition pore (CPTP) form a megapore and cover the inner and outer membranes of the mitochondria. Mitochondrial proteins such as cytochrome C (Cyto C), high temperature requirement protein A2 (HtrA2)/Omi, and second mitochondria-derived activator of caspases/direct inhibitors of apoptosis-binding protein with low pI (Smac/Diablo) are released into the cytosol. Then, Cyto C combines with apoptosis protease activating factor-1 (Apaf-1) to form a complex named “apoptosome”, which serves as a platform for the cleavage and activation of downstream caspases. Activation of caspases ultimately leads to cell dismantling (Figure 2).

4A great number of studies have demonstrated that long-term hyperglycemia in diabetic patients induces cardiomyocyte apoptosis. Cardiomyocyte apoptosis leads to cell loss to decrease cardiac contractile function and to ultimately promote cardiac remodeling. It has been proven that astragalus polysaccharides (APS) protected HG induced-H9C2 cell apoptosis by reducing Cyto C release and inhibiting caspase activity. That is to say APS has the potential ability to attenuate DCM through suppressing both extrinsic and intrinsic apoptotic pathways. One study reported that nicorandil decreased TdT-mediated DUTP nick end labeling (TUNEL)-positive cells, B-cell lymphoma-2 (bcl-2)-associated X (bax) as well as cleaved caspase 3 expression, while it increased bcl-2 expression in the heart of diabetic rats. Moreover, 5-HD, the competitive antagonist of nicorandil, increased apoptosis but reduced the phosphorylation of phosphatidylinositol 3-kinase (P13K), protein kinase B (AKT), endothelial nitric oxide synthase (eNOS), and mammalian target of rapamycin (mTOR) in H9C2 cardiomyocyte with high glucose stimulation. These data demonstrated that nicorandil alleviated hyperglycemia-caused cardiomyocyte apoptosis. Li et al. found that long noncoding RNAs H19/MicroRNA-675 axis was involved in HG-induced cardiomyocyte apoptosis by downregulation of voltage-dependent anion channel 1 (VDAC1), which was required for the mitochondria-mediated apoptosis. Recent studies also demonstrated that miR-186-5p overexpression suppressed apoptosis in HG-treated cardiomyocytes. Chronic and severe endoplasmic reticulum (ER) stress also resulted in cell apoptosis. APS provided cardioprotective effects on DCM by inhibiting cardiomyocytes apoptosis via suppressing protein kinase RNA-like ER kinase (PERK) and activating transcription factor 6 (ATF6)-related pathway of ER stress. Matrine induced caspase 3 and caspase 9 down-regulation along with bcl-2 and p53 up-regulation. In addition, matrine administration inhibited the transforming growth factor beta (TGF-β) induced PERK signal pathway activation, which was involved in ER stress-induced apoptosis. Together, these events suggest that apoptosis promotes cardiomyocyte damage during DCM through multiple upstream signal pathways. Thus, compounds or molecules inhibiting apoptosis may serve as potential therapeutic agents for DCM.

5Autophagy is initially described as a degradation process to eliminate damaged proteins and dysfunctional organelles. Autophagy is usually induced by nutrient deprivation, hypoxia, oxidative stress, genotoxic stress, or high glucose. Autophagy commences by autophagosome formation, a double-membrane vesicle swallowing up cytoplasmic materials. Autophagy is tightly regulated by autophagy-related (ATG) proteins, which is encoded by a family of highly conserved genes. Multiple signaling pathways including adenosine 5′-monophosphate (AMP)-activated protein kinase (AMPK), mTOR, unc-51-like kinase 1 (ULK1), PI3K/AKT, GTPases, calcium, and protein synthesis are involved in autophagy.

6The real role of autophagy on DCM is quite conflicting. Various studies have demonstrated the cardioprotective effect of autophagy in the heart of diabetic animals. In a study that explored the effect of aldehyde dehydrogenase 2 (ALDH2) on diabetes-induced myocardial dysfunction, there was decreased ratio of microtubule-associated protein 1 light chain 3-II (LC3II)-to-LC3I, reduced autophagy related 7 (ATG7) expression but increased sequestosome 1 (p62) levels, suggesting depressed autophagy in diabetes. ALDH2 overexpression or its agonist Alda-1 improved autophagy to reverse diabetes or high glucose-induced dysfunctions via AMPK and forkhead box O3a (FOXO3a). Sirtuin3 (Sirt3) deficiency decreased LC3 puncta, autophagosomes, and LC3II level, via acetylated-Foxo3A enhancement and parkin inhibition, indicating that Sirt3 deficiency exacerbates diabetic cardiac dysfunction. Activating cannabinoid receptor 2 (CB2) produced a cardioprotective effect in DCM as well as cardiomyocytes under HG challenge through inducing AMPK-mTOR-p70S6K signaling-mediated autophagy. Surprisingly, another study suggested that diminished autophagy gave adaptive response to limit diabetic cardiac injury in type 1 diabetic mice. Consistently, diabetes-associated myocardial mitochondrial dysfunction was associated with enhanced autophagy in the myocardium of diabetic Goto-Kakizaki rats. Hence, determining whether autophagy is adaptive or maladaptive is critical for the execution of efficient therapeutic treatment for DCM.

7Necrotic cell death contains a great diversity of cell death processes. Necrosis may occur in the case of extensive damage such as high temperature and mechanical stress, leading to cell integrity destruction. In this case, necrosis is passive and does not require any specific signaling pathways. Another type of necrosis, called secondary necrosis, occurs at the late stage of apoptosis or autophagy when dead cells can't be removed by phagocytosis. Secondary necrosis is regarded as independent of initial signal events such as apoptosis or autophagy. However, necrosis can also be a result of signaling cascade. Early study showed that diabetes increased necrosis by fourfold in cardiomyocytes.

8Necroptosis is the best-characterized form of programmed necrosis, showing features of both apoptosis and necrosis. The pathway of necroptosis activation after high glucose stimulation is mediated by ligand to death receptors such as tumor necrosis factor receptor 1 (TNFR1) and Fas receptors. Damage-associated molecular patterns (DAMPs), nucleotide-binding, and oligomerization domain (NOD)-like receptors (NLRs) ripoptosome, and protein kinase R (PKR) complexes also trigger necroptosis. TNFR1-mediated necroptosis is the most thoroughly studied pathway to activate necroptosis. After TNF-α binds to TNFR1, there are three possible divergent functions including cell survival, apoptosis, or necroptosis via different signaling complexes. Complex I is prosurvival and Complex IIa is proapoptotic. Complex IIb formation results in necroptosis. Receptor-interacting protein 3 (RIP3) is regarded as a critical regulator of necroptosis. RIP3 regulates necroptosis on a RIP1-dependent manner in diabetes. Once recruited by RIP1 after HG exposure, RIP3 is activated by auto-phosphorylation to promote mixed lineage kinase domain like protein (MLKL) phosphorylation. Then, MLKL oligomerizes and translocates to the cell membrane, interacts with phosphatidylinositol lipids and cadiolipin to result in membrane permeabilization. Calmodulin-dependent protein kinase II (CaMKII) is a newly found RIP3 substrate to induce necroptosis. CaMKII is abundant in the myocardium and is inactivated under normal conditions. CaMKII phosphorylation by intracellular Ca2+ or RIP1 facilitates necroptosis. Meanwhile, HG also increases ROS to activate CaMKII by oxidation. Altogether, the above pathological changes trigger the opening of mitochondrial permeability transition pore (mPTP), which is involved in the final pathway of necroptosis (Figure 3). Some research found that knockdown of cyclophilin D (CypD), a protein that increases mPTP opening probability, protected against RIP3-induced cardiomyocyte necrosis. RIP3 up-regulation increased phosphoglycerate mutase 5 (PGAM5) expression, enhanced CypD phosphorylation, and finally resulted in mPTP opening. Excessive opening of the mPTP also amplified the death signal and ultimately led to necroptosis in the endothelial cells.

9It was observed that when H9C2 cells were exposed to high glucose, cell viability, the activity of ALDH2 mRNA or protein expression were suppressed, while ROS levels as well as RIP1, RIP3, and MLKL expression were enhanced. Necrostain-1 (Nec-1, a specific inhibitor of necroptosis) or Alda-1 (the ALDH2 activator) attenuated HG-induced down-regulation of ALDH2 and enhancement of RIP1, RIP3 and MLKL. In general, ALDH2 activation protected against HG-induced H9C2 cell injury, partly by inhibiting necroptosis. Another study showed that HG markedly increased the expression of Toll-like receptor 4 (TLR4), which was attenuated by NAC, a ROS scavenger. HG also significantly increased the expression of RIP3, which was ameliorated by TAK-242 (an inhibitor of TLR4) or Nec-1. In addition, NAC mitigated HG-induced RIP3 expression up-regulation. More importantly, DZ (a mitochondrial KATP channel opener) and Pin (a non-selective KATP channel opener) reduced the increased levels of TLR4 and RIP3 induced by HG. Taken together, the opening of KATP channels inhibited necroptosis through ROS-TLR4 pathway to protect H9C2 cells against HG-induced damage. Our recent research revealed that inhibitor 1 of protein phosphatase 1 (I1PP1) over-expression alleviated CaMKIIδ alternative splicing disorder, suppressed ROS overproduction, inhibited CaMKII oxidation, suppressed necroptosis, and ultimately alleviated high glucose-induced cardiomyocyte injury.

10Pyroptosis is another novel form of programmed necrosis. It is responsible for cellular lysis and extracellular release of proinflammatory cytokines such as interleukin-1β (IL-1β) and interleukin-18 (IL-18) by HG stimulation. There are two different pathways including the canonical pathway and noncanonical pathway in pyroptosis. In the canonical pathway, cytoplasmic multiprotein complexes, named inflammasome, consist of the nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) family (including NLRP3, NLRP1, NLRC4, NLRP9 and NLRP6), the pyrin and HIN domain (PYHIN) protein families (absent in melanoma 2, AIM2), and pyrin proteins. Distinct inflammasomes are recognized by pathogen-associated molecular patterns (PAMPs) or DAMPs to activate caspase 1, leading to pyroptosis. In the noncanonical pathway, bacterial lipopolysaccharide (LPS) is delivered to the cytosol to activate caspase 11. Activated caspase 11 directly induces pyroptosis. The cleaved caspase 11 also activates the Gasdermin-D (GSDMD) to form a membrane pore. Meanwhile, the NLRP3 inflammasome is activated by the N-terminal fragment of GSDMD (GSDMD-N) to trigger pyroptosis via the canonical pathway.

11Emerging evidence has verified that pyroptosis is involved in the progression of DCM. Jeyabal et al. found that NLRP3, caspase 1, and ELAV-like RNA binding protein 1 (ELAVL1, also called Hu-Antigen, HuR) expression in both human diabetic heart and HG-exposed human ventricular cardiomyocytes were augmented. Further study identified HuR as a direct target of miR-9. MiR-9 mimic transfection reduced HuR, caspase 1, and IL-1β expression to inhibit HG-induced pyroptosis in human cardiomyocytes. This study highlighted that targeting miR-9/HuR would play a therapeutic role in cell death during DCM. Recently, Wang et al. found that absent in melanoma 2 (AIM2) inflammasome, caspase-1, IL-1β, and GSDMD were elevated in the myocardium of diabetes mellitus. ROS was also increased in H9C2 cells with HG stimulation. AIM2-shRNA reduced caspase 1 and IL-1β expression, suppressed GSDMD-N level and attenuated HG-induced pyroptosis. Furthermore, AIM2 level dropped significantly if ROS was inhibited. This study indicated that AIM2 inhibition is beneficial to attenuate diabetic cardiomyopathy via alleviating GSDMD-N-related pyroptosis (Figure 4). These findings confirmed the distinctive role of pyroptosis in DCM. To inhibit the pyroptosis signaling pathways is to capably expand the potential therapeutic targets for DCM treatment.

12Entosis seems fundamentally different from most typical forms of cell death due to the requirement of engulfing cells to execute. Entosis is mediated by cellular engulfment via E-cadherin, α-catenin, Rho family of GTPases, and rho-associated kinase (ROCK). Entosis is identified as an essential mechanism of embryo implantation, which plays pro- and antitumorigenic roles in cancer. However, no data regarding the role of entosis in cardiovascular diseases has been found until now, and the possible mechanisms were incompletely defined.

13Ferroptosis is an iron- and lipotoxicity-dependent form of regulated cell death (RCD). It is characterized by small mitochondria with reduced crista and condensed or ruptured outer membrane. Acyl-CoA synthetase long-chain family member 4 (ACSL4), lysophosphatidylcholine acyltransferase 3 (LPCAT3), and arachidonate lipoxygenase (ALOXs) pathways mediate the oxidation of polyunsaturated fatty acids, which is necessary for the lipid toxicity of ferroptosis. ACSL4 up-regulation is the marker of ferroptosis. Decreasing antioxidant glutathione or inhibiting glutathione peroxidases inevitably enhanced ROS formation by erastin, a cell-permeable ferroptosis activator. Ferroptosis has been reported to be regulated during drug therapy; whether it participates in the pathology of DCM is unclear. In consideration of ROS formation promoting ferroptosis, there is a serious possibility that ferroptosis is involved in DCM.

14Parthanatos is a poly ADP-ribose (PAR) polymerase-1 (PARP-1)-dependent type of cell death, which is activated by oxidative stress-induced DNA damage. Neither apoptotic body formation nor DNA fragmentation is observed during parthanatos. Parthanatos does not produce cell swelling or lysosomal degradation. Energy exhaustion and apoptosis-inducing factor (AIF) release from the mitochondria mediated by PAR or calpain are possibly the main mechanisms of parthanatos. Parthanatos has been reported to be involved in cardiovascular diseases, renal diseases, diabetes, and neurodegeneration. PARP inhibitor L-2286 prevented cardiac remodeling, improved systolic function, and delayed the development of heart failure. Thus, PARP inhibition may have the potential ability to attenuate DCM.

15Netotic cell death is a form of RCD driven by neutrophil extracellular traps (NETs), which is regulated by NADPH oxidase-mediated ROS production and histone citrullination. NET formation and release, or NETosis relies on ROS production, autophagy, granular enzyme release, and translocation from the cytosol to the nucleus. Histone citrullination eventually leads to chromatin depletion, nuclear membrane destruction, and chromatin fiber release. Efforts could be endeavored to explore the mechanisms of NETosis underlying DCM, thereby providing new therapeutic policies for DCM.

16Lysosomal-dependent cell death (LCD), also known as lysosomal cell death, is a form of regulated cell death mediated by intralysosomal components or iron translocation resulting from lysosomal membrane permeabilization (LMP) to amplify or initiate cell death during apoptosis, autophagy, and ferroptosis. Although the relation between LCD and DCM was easy to be deduced, further studies are still indispensable.

17Alkaliptosis is driven by intracellular alkalization. It is known that down-regulation of inhibitor of nuclear factor kappa B kinase subunit beta (IKBKB) and nuclear factor-κB (NF-κB) induces alkaliptosis. The pathological significance of alkaliptosis in human diseases remains complicated, and the main signals of allkaliptosis need to be illuminated in the future.

18Oxeiptosis is a novel caspase-independent RCD induced by ROS, which is mediated through Kelch-like ECH-associated protein 1 (KEAP1)/PGAM5/apoptosis-inducing factor 1 mitochondrial (AIFM1) pathway. Since ROS accumulation links to multiple physiological and pathological processes, it is very likely that oxeiptosis promotes the development of several diseases including DCM.

19Different types of cell death are mediated by distinct but overlapping central pathways. One study found that autophagy was inhibited but apoptosis was amplified in diabetic heart. Elevated bax/bcl-2 ratio, increased cleaved caspase 3 level, augmented NLRP3, cleaved caspase 1, IL-1β, and cleaved GSDMD expression along with increased ROS production were detected in the myocardium of db/db mice, indicating the involvement of apoptosis and pyroptosis in DCM. Altogether, several types of cell death may coexist in the occurrence and development of DCM. Moreover, apoptosis, necrosis, necroptosis, pyroptosis, autophagy, or ferroptosis might act simultaneously during DCM. Inhibition of one kind of cell death may possibly promote other types of cell death and cause compensation, which eventually increases the complexity of pathogenesis about DCM and enhance difficulty of treatment for DCM.

20Current therapeutic strategies for DCM involve insulin and insulin-secreting agents, oral antihyperglycemic medication, β-blockers, angiotensin converting enzyme inhibitors, angiotensin II receptor antagonists, calciumion channel antagonists, and hydroxymethylglutaryl CoA reductase inhibitors. However, the above synthetic drugs have many adverse effects including gastrointestinal disturbances, hepatotoxicity, abdominal pain, genitourinary tract infection, and so on. As cell death is a determinant pathological fate in DCM, the executors of cell death signal pathways are obviously potential therapeutic targets for DCM. Many drugs or compounds are being used in or under clinical trials. Take some examples: Metformin, a common antidiabetic drug, activates AMPK and improves autophagy via inhibiting the mTOR pathway and alleviating pyroptosis. Crocin, a carotenoid extracted from saffron, improves the deteriorated cardiac function in diabetic animals by inhibiting apoptosis and normalizing autophagy. Protein phosphatase 2A (PP2A) is a central cardiac phosphatase that regulates the functions of diverse myocytes through target molecules. Okadaic acid (OA), an inhibitor of PP2A, inhibited apoptosis of experimental diabetic mellitus-related cardiomyopathy. These researches will improve the understanding of cell death in DCM and set the stage for novel therapies in future. Several drug or compounds targeting the signals in cell death have been used in clinics or experiments for DCM (Table 1).

21Taken together, cell death is the terminal pathway of cardiomyocytes during DCM. Although upstream signal regulation seems to attenuate cell death effectively, it may produce a series of nonspecific effects. Targeting cell death directly may be more practicable than its upstream signaling pathway.

22YC researched documents and wrote the manuscript. YH, XL, IA, and WZ revised the manuscript. GM designed the study and reviewed the manuscript. All authors approved the final version of the manuscript.

23The work was funded by grants (81770279, 81670243 and 81873470) from the National Natural Science Foundation of China, a major project of Natural Science Research in Jiangsu Higher Education Institutions (18KJA310005), the Six Talent Peaks Project in Jiangsu Province (2018-WSN-062), grants from the China Postdoctoral Science Foundation (2017M610342 and 2019T120449), a Jiangsu Planned Project for Postdoctoral Research Funds (1701050A), a Research and Innovation Project of Graduate Students in Jiangsu Province (KYCX18_2401), a Science and Technology Project of Taicang City (TC2019KJFZ02), and the Nantong University Cooperative Innovation Program of Small Molecular Compound R&D (NTU2016-1).

24The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.