Myocardiocyte autophagy in the context of myocardiocytes regeneration: a potential novel therapeutic strategy

Abstract

Background

The regeneration strategy involves several aspects, such as reprogramming aspects, targeting pathophysiological processes, and inducing the physiological one. Autophagy targeting is a potential physiological/pathogenetic strategy to enhance myocardiocytes' function. Myocardiocytes' injury-related death remains to be the highest in our era. Unfortunately, myocardiocytes have a limited proliferation capacity to compensate for what was lost by infarction. However, partially injured myocardiocytes can be preserved by improving the autophagy process of myocardiocytes.

Main text

Autophagy induction involved controlling the cellular and subcellular environment as well as gene expression. Autophagy is well known to prolong the longevity of cell and human life. Inhibition of the mTOR receptor, proapoptotic gene Bnip3, IP3, and lysosome inhibitors, inhibition of microRNA-22 and overexpression of microRNA-99a, modulators of activated protein kinase with adenosine monophosphate, resveratrol, sirtuin activators, Longevinex and calcium lowering agents can promote physiological myocardiocyte autophagy and improve post-myocardial modulation and recovery speed. The paper aimed to assess autophagy role in myocardiocytes regeneration modulation.

Conclusions

The autophagy strategy can be applied to infarcted myocardiocytes, as well as heart failure. However, cell self-eating is not the preferred therapy for preserving injured myocardiocytes or causing regeneration.

Introduction

The misery of myocardial infarction-associated death remains the most frequent despite the remarkable advances in molecular biopathophysiology. Current censuses showed that more than 4 hundred thousand individuals globally suffer from cardiovascular issues. Furthermore, the misery continues to involve approximately 18 million death annually [1].

The impaired myocardiocytes include ischemic heart, infarcted heart, and chronic heart failure. The pathogenesis is quite similar and involved autophagy impairment or complete cessation. Restoration of impaired myocardiocyte function remains a challenge for patients with injured myocardiocytes. Despite the current therapies to enhance antioxidant defense mechanisms (induced by autophagy) of myocardiocytes against oxidative stress, it does still not reach the required goal [2,3,4]. Perhaps a better intervention would be to modify the natural detoxification system that includes autophagy and an antioxidant defense system simultaneously. However, autophagy is part of the antioxidant defense system; therefore, autophagy induction probably replaces antioxidant therapy.

Autophagy term returns to 1963 when C de Duve's firstly used this term and later discoveries continued by Yoshinori Ohsumi [5]. Induction of myocardiocytes autophagy involved regulation of gene expression, cytoplasmic and cellular environment modulations. Self-eating at hunger seems not a good idea, but for survival reasons, the cells are adjusted to degrade themselves in hope of rescue.

More recently, studies have supported the hypothesis that autophagy improved myocardiocyte regeneration through several mechanisms, including special microRNAs (microRNA-22 inhibition, MicroRNA-421 overexpression, and microRNA-99a overexpression) and the expression of damage-regulated autophagy modulator 1 (DRAM1) [6,7,8,9,10,11,12,13,14,15,16,17,18] (Fig. 1) DRAM1 induces autophagy through inhibition of phosphoinositide 3-kinase–Akt–mTOR-ribosomal protein S6 axis [19].

Fig. 1

Autophagy in the context of myocardiocyte regeneration and preservation of partially impaired myocardiocytes. Induction of myocardiocyte autophagy ameliorates regeneration strategies and increases myocardiocyte longevity. The regeneration and autophagy induction mechanisms involved the activation of several common genes, transcription factors, microRNAs (all the mentioned microRNAs must be overexpressed, not necessarily simultaneously), and epigenetics that are required for both processes. Autophagy activation induces the expression of Atg proteins that probably interfere with the proliferation signaling pathway and activate it. However, it remains to be elucidated since the signaling machinery is not fully understood. The role of autophagy in maintaining myocardiocytes' mass and function is through resolving oxidative stress, stress of the endoplasmic reticulum (ER), and reducing the lactic acid and calcium paradoxes (these functions indicated by asterisks) in the basal metabolite and ischemia as well as reperfusion. Subsequently, autophagy improves the intracellular homeostasis including contractile function and regeneration capacity of the myocardiocytes. GLP-1 group of anti-diabetic medications in addition to other factors can affect autophagy. Oxidative stress and endoplasmic reticulum stress are critical to maintaining autophagy and myocardiocyte mass from apoptosis and cell death, which are necessary to maintain cardiac function. IL interleukin, GLP-1 glucagon-like peptide-1, ROS reactive oxygen species

Targeted microRNAs can be recruited as a potential inducer for autophagy. However, several microRNAs act as autophagy inhibitors such as MIR23A, MIR29A, and MIR138-5p [20].

However, autophagy can be activated through hyperthermia and hypoxia mediated by IGF-1, food deprivation, inhibition of mTOR signaling, chaperones, and FOXO transcription factors. In addition, autophagy can be promoted through resveratrol, LY294002 (LY), metformin, and Velcade [5, 21,22,23,24,25,26,27]. Whereas 3-methyladenine (3-MA) and bafilomycin A1 are well-known inhibitors of autophagy [5].

Three main signaling transduction groups regulate autophagy: first includes pathways that directly involve core autophagy-related proteins (ATG) such as PI3K–AKT-, AMPK-pathways, and mTOR-; second includes signaling pathways that regulate ATG proteins activity such as cAMP-, Ca²⁺-, and RTK/Rap1/Ras/MAPK-pathways; and finally, ATG proteins transcription regulation by mainly NF-κB-, HIF-1-, TFEB, TFE3, and FOXO-pathways as well as their subregulators such as NR1I2 and ESR1[28].

Therapeutic applications of autophagy are not limited to myocardiocytes but can be recruited to cure cancer, neurodegenerative diseases, infections, and promote the function of impaired cells. Autophagy impairment is a hallmark of the pathophysiology of these diseases. The self-eating of microorganisms, cancer cells, and the enhancement of impaired function of neurons and myocardiocytes make autophagy a physiological and pathological catabolic process, but its regulation can be effectively used as a therapeutic strategy.

The paper sought to assess the potential mechanism of autophagy in myocardiocyte organelles and the role of autophagy in regeneration enhancement and preserving of the partially impaired myocardiocytes.

Main text

Myocardiocyte autophagy: potential mechanism

Three well-known mechanisms are responsible for the degradation of intracellular debridements. According to the signaling markers that flag debridements, microautophagy, macroautophagy, and chaperone-mediated autophagy are present. Recent advances in molecular medicine have revealed that several autophagy-related proteins are involved in autophagy regulation, such as systems that produce modified complexes Atg8-PE and Atg5-Atg12-Atg16 [29]. Approximately 35 autophagy-related protein (Atg) genes have been identified, 17 of which are indispensable. The molecular mechanism of myocardiocyte autophagy involved several complex signaling pathways and structures such as Beclin1-Atg14L-Vps15-Vps34 proteins. Phagophore is the first phase of autophagosome formation. Two primary complexes are involved in autophagosome formation, each of which has its role in norm and pathology [30, 31]. The first complex consists of III PI3 K Vps34, Atg6/Beclin1, Atg14, and Vps15/p150.73. Whereas the second complex mainly involved the serine/threonine kinase Atg1 signaling axis that its functionality depends on the presence of LC3 (microtubule-associated protein light chain 3), GATE-16 (Golgi-associated ATPase enhancer of 16 KDa), and GABARAP (G-amino butyric acid type A receptor-associated protein) proteins [29].

The second phase of autophagy is elongation that involved ubiquitin-like conjugation pathways; Atg8/MAP-LC3/GABAP/GATE-16 and Atg12 systems. Atg8 protein undergoes cleavage for its carboxyl-terminal by cysteine protease Atg4. Then, Atg4 undergoes a long complexed signaling cascade involving activation of Atg4 by Atg7 and Atg3 that culminate in activation of Atg8 and it is binding with the LC3 –II of the lysosome and formation of Atg8/LC3–II complex. The Atg8/LC3 –II complex bind with autophagosome in a covalent bound unless Atg4 cleaves it to be recycled and degraded by the phagolysosome [29] (Fig. 2).

Fig. 2

Schematic presentation of the autophagy mechanism. Two primary complexes are involved in autophagosome formation, the first includes III PI3 K Vps34, Atg6/Beclin1, Atg14, and Vps15/p150.73, whereas the second includes the serine/threonine kinase Atg1 signaling axis. In addition, autophagy is done in two phases; the first phase includes phagophore formation, and the second phase includes elongation that involved ubiquitin-like conjugation pathways; Atg8/MAP-LC3/GABAP/GATE-16 and Atg12 systems, in the center of the figure. Inducing autophagy can be done in various ways, indicated by a plus sign. During the second phase, Atg8 protein undergoes cleavage for its carboxyl-terminal by cysteine protease Atg4. Then, Atg4 undergoes a long complexed signaling cascade involving activation of Atg4 by Atg7 and Atg3 that culminate in activation of Atg8 and it is binding with the LC3–II of the lysosome and formation of Atg8/LC3–II complex. The Atg8/LC3–II complex bind with autophagosome in a covalent bound unless Atg4 cleaves it to be recycled and degraded by the phagolysosome. Not necessarily, the presented modulators can target all these pathways and signaling steps

The direction of autophagosome and lysosome is strictly regulated by transcription and post-transcription modulations, as well as epigenetics. Furthermore, the formation of an autolysosome is crucial and involved genetic/epigenetic/transcriptional/posttranscriptional modifications [32]. TFEB, MiT, FOXO, forkhead box, E2F1, and NF-kB transcription factors are involved in the regulation of autophagy. Epigenetics is also involved, as histone post-translational modifications include methylation, acetylation, and deacetylation.

Induction of myocardiocyte autophagy occurs physiologically under hypoxic conditions. Several mechanistic pathways are potentially possible to take place, Inhibition of mTOR receptor, proapoptotic gene Bnip3, IP3, and lysosome inhibitors depending on the hypoxia severity. During anoxia, hypoxia-inducible factor 1, α subunit (HIF1A) is activated. HIF1A induces the expression of the Bnip3 gene (encode for BH3-only protein) which is required for disrupting the BCL2–BECN1 interaction by competitive binding of BCL2 (B-cell CLL/lymphoma 2) [33]. Bnip3 is an important gene for activating autophagy during hypoxic and non-hypoxic states.

Autophagy on the road of organelles

Autophagy of subcellular components is crucial for maintaining healthy cells. Furthermore, organelle product autophagy is involved in maintaining the longevity of myocardiocytes. Interestingly, autophagy not only maintains the health of cells; therefore, the idea of living longer is now combined with living healthy [34, 35]. Organelles' autophagy involved the degradation of impaired mitochondria (mitophagy), impaired endoplasmic reticulum and peroxisomes (pexophagy), nucleus segments (micronucleophagy), ribosomes (ribophagy) [36]. The impaired organelles autophagy is considered a macroautophagy type. However, microautophagy also occurs under specific conditions that are not discussed here.

Mitophagy in myocardiocytes

Mitochondria are the dome of energy in living cells. Energy is required almost for each cellular process. The impairment of mitochondrial functions and the reduction of precursor energy production sources contribute as a primary pathogenetic clue to most pathologies, including myocardiocyte infarction [37, 38]. Mitochondrial function correction involves mitochondrial autophagy, which is an optimal therapeutic target to eliminate the sequelae of mitochondrial dysfunction (ATP deficiency, reactive oxygen species, and cell apoptosis). The first sign of mitochondrial injury is mitochondrial DNA damage caused by free radicals and inflammatory cytokines [39]. Mitochondria, like any other organelles, are sensitive to ischemias and hypoxemias, as well as ischemic reperfusion injuries. Preserving partially injured mitochondria involves correction of physiological activity by autophagy and reduction of the production of free radicals. Furthermore, the NLRP3 inflammasome is activated during ischemia and starvation conditions [40, 41]. The complete signaling mechanism involved in the correction of mitochondrial function by autophagy is not yet fully understood and requires additional research. However, the statistical analysis of several studies concluded that giving autophagy agonists reduce the oxidative stress of healthy and ischemic myocardiocyte [23, 42].

Mitophagy and pexophagy involve Slt2 and Hog1 signal transduction pathways, each of which has its role in the pathogenesis of myocardiocytes function impairment [43]. However, in yeast MAPK signaling cascade plays a key role in determining selective autophagy [44].

Autophagy of the endoplasmic reticulum of myocardiocytes

Two stages are in front of the endoplasmic reticulum to pass the infarction; first, to preserve from ischemia and second, to rescue from ischemic reperfusion injury [45]. When the endoplasmic reticulum is in a deficit of energy, it begins to unfold proteins to produce the unfolded protein response [46]. Usually, these proteins aggregate in the cytoplasm of the myocardiocytes to form inclusion around the nucleus and cell membrane. Degradation of such pathological proteins is crucial to release the myocardiocytes from the endoplasmic reticulum stress state. Failure to degrade unfolded proteins results in activation of endoplasmic reticulum autophagy mechanisms. However, isolated autophagy of the proliferative endoplasmic reticulum can be autophagy.

The smooth endoplasmic reticulum is also involved in the pathogenesis of myocardiocyte damage during infarction. Well known, the smooth endoplasmic reticulum is a storage for cations primarily calcium. During attacks of ischemia, the calcium content of the intraendoplasmic reticulum will be released into the cytoplasm, leading to persistent contraction of poorly oxygenated myocardiocytes [47]. Furthermore, after reperfusion, a large amount of oxygen and calcium reenters the myocardiocyte from the extracellular space, resulting in a worse condition of calcium content and oxygen in the myocardiocyte, which consequently leads to the formation of oxygen free radicals and a large amount of intracellular calcium and subsequently, damage to the membranes of the membraned structures especially which have a high lipid ratio [48, 49].

Endoplasmic reticulum autophagy enhances the metabolic outputs of the rough endoplasmic reticulum and improves the cations controlled by the smooth endoplasmic reticulum. The signaling mechanism in which autophagy induces its effects on the endoplasmic reticulum remains an area for future research. However, a more recent study showed that the use of a highly selective agonist of α2-adrenergic receptors, dexmedetomidine improves myocardiocyte ischemic/reperfusion injury by down-regulation of the endoplasmic reticulum stress signaling pathway that includes glucose-regulated protein 78 (GRP78), protein kinase R-like endoplasmic reticulum kinase (PERK), homologous protein C/EBP (CHOP), inositol-requiring protein 1 (IRE1), and activating transcription factor 6 (ATF6) [50].

Pexophagy in myocardiocyte

Starvation and other unfavorable conditions (e.g., in Saccharomyces cerevisiae glucose-containing medium without a nitrogen source) induce activation of the self-eating pathophysiological mechanisms to involve peroxisomes autophagy. Selective autophagy of peroxisomes can be as micro- and macroautophagy. The mechanism involved in pexophagy includes the formation of phagophores from a particular cytosolic phagophore assembly site that later engulfs the impaired peroxisome to form an autophagosome. The molecular mechanism that initiates, directs cargo, fusion of autophagosome with lysosome to form autolysosome, and degrades cargo content (peroxisome) is identical in other organelles [29]. The phagophore assembly site, where the double membrane vacuole is synthesized, is believed to be initiated by the core autophagy-related protein ATG9L1 [51]. ATG9L1 functionality depends on Atg17, Atg29, and Atg31 proteins in addition to the dispensable Atg11 protein [52]. However, debate remains on the mechanism that involved the synthesis and the primary place of the phagophore assembly site for each organelle. Understanding of the possible mechanisms would be recruited to target organelles autophagy on the subcellular level which will have the highest drug target selectivity.

Autophagy in the context of myocardiocyte regeneration

Regeneration strategy of impaired myocardiocytes involves proliferation/differentiation of already mature myocardiocytes, but unfortunately, adult myocardiocytes regenerate approximately 1% per year [53]. Therefore, a surrogate strategy is required such as preserving the partially impaired myocardiocytes through autophagy induction simultaneously with regeneration. Currently, regeneration can be through transdifferentiation of mesoderm derived cells/local cardiac stem cell into ventricular/atrial myocardiocytes through epigenetic modifications (e.g., histone methylation, acetylation, deacetylation)/cardiac transcription factors (e.g., GATA5, Mef2c, Tbx5, Nkx2.5)/small molecules (e.g., CHIR99021, A83-0,1BIX01294, AS8351, Y27632, OAC2, SU16F, SC1, JNJ10198409)/cardiac non-coding microRNAs (e.g., miR15, miR-128, miR-101a, and miR-133). Also regeneration can be through differentiation of induced/pluripotent stem cells from various origins such as cardiac or skin fibroblast by Yamanaka factors (Oct4, Sox2, Klf4, C-myc) into myocardiocytes via cardiac transcription factors (e.g., GATA5, Mef2c, Tbx5, Nkx2.5) and via special microenvironment [53].

Our hypothesis of autophagy-based myocardiocyte regeneration endorses the cross-activation of a specific common mechanism between autophagy and myocardiocyte regeneration (for example, by transcription factors). For better understanding, carrying research on special cardiac microRNAs, cardiac transcription factors, and epigenetics is required, which is our current project. However, it remains difficult to control for other factors' role in the regeneration strategy such as oxidative stress and endoplasmic stress which each of them plays a key role in the pathophysiology and regeneration aspects of myocardiocytes. Each of these pathophysiological events required extra investigation to determine their role in regeneration that accordingly, we may regulate their activity.

In vitro, more recent findings addressed leptin b an important role in myocardiocytes proliferation capacity after controlled myocardiocyte injury [54].

Currently used medications in the context of autophagy enhancement

Unfortunately, currently, the use of drugs to induce autophagy is still a poorly developed concept. However, several medications that are used for the treatment of several other common diseases have been identified to induce autophagy of cells of the whole organism without organelle selectivity. Approximately 225 autophagy modulators have been identified, of which 174 are autophagy inducers, 31 are suppressors, while 20 have dual effects depending on the biological context (e.g., melatonin in normal cells induces autophagy, but in tumor cells reduces autophagy) [55] (Table 1) Autophagy modulators have more than 1005 targets, every modulator has multiple targets and usually targets not the autophagy proteins directly but their regulators.

Table 1 Group of medications with potentiality of autophagy enhancement, not limited to this list

Metformin is the most commonly used medication to manage type 2 diabetes [56]. Metformin was found to induce beta-cell insulin production as well as reduce the resistance of insulin-sensitive cells to insulin. Moreover, recent findings suggested that metformin is involved in inducing autophagy as well ferroptosis [57]. The molecular mechanism involved activation of the AMPK-MTOR/PGC-1alpha signaling pathway [58]. Probably, activation of apoptosis genes by metformin is a protective mechanism for cells from transdifferentiation to another cell linage such as fibroblast (e.g., in post-myocardial infarction modulation scar tissue) [15].

More recently, in vivo findings have suggested that metformin administration after myocardial infarction increases heart wall rupture incidence [58].

Discussion

Autophagy is the only mechanism that can degrade long-lived proteins and organelles, unlike the ubiquitin proteasome system. Since autophagy is involved in the regulation of cell differentiation, it is likely that it can be recruited to optimize current myocardiocyte regeneration strategies, such as transcription factors in pluripotent stem cell differentiation. However, the regeneration mechanism remains in the womb stage and requires additional elucidation. But gene expression analysis demonstrated that knockdown Atg5 protein, which is involved in the autophagy regulation, results in a reduction of myocardiocytes regeneration and later fibrosis and hypertrophy as well as low ejection fraction. Regeneration of myocardiocytes involved proliferation of neo cells and angiogenesis. In part, these processes are regulated by the same genetic, epigenetic, transcription factors and non-coding microRNAs that regulate autophagy [62]. Therefore, experimental studies have found an increase in myocardiocytes regeneration during induction of autophagy [63].

Cell differentiation and proliferation are partially regulated by common molecular pathways that involved crosstalk between autophagy and Wnt catenin signaling pathways. Ischemic conditions induced autophagy of the β-catenin and Disheveled proteins by the LC3 component of autophagy. Not only that, but also the GSK3β signaling pathway, another part of the Wnt catenin signaling pathways, can induce autophagy by phosphorylating the TSC complex [64]. Markers of successful myocardiocyte autophagy include LC3-I/II and Beclin1 levels [65].

Interestingly, recent findings by Cui et al. have addressed the crucial role of lysosomal membrane permeability (LMP) in the regulation of cell death. The study demonstrated that overexpression of lysosomal membrane-associated protein 2 (Lamp2) results in induction of autophagy flow and reduction of myocardiocyte death from ischemia [66]. Transinfection of glucose, oxygen, and serum-deprived myocardiocytes with an adenovirus vector containing the Lamp2 gene resulted in a remarkable improvement in deprived cell autophagy and subsequently preserved ischemic myocardiocytes. At the molecular level, Lamp2 induces its effect through upregulation of cathepsin B and cathepsin D [67,68,69]. Dysregulation of Lamp2 protein results in a rare X-linked cardiomyopathy disease called Danon disease [70].

Current challenges and future prospects

So far, autophagy is well-known inducer for cellular antioxidant defense system which is necessary for protection against myocardial ischemic attacks/reperfusion injury [59]. However, recent bio-molecular advances didn’t explain the potential role of autophagy in heart regeneration and especially myocardiocytes [60]. We suggest that molecular and cellular processes of autophagy involve the activation of a common signaling pathway for both autophagy and regeneration. Therefore, during unfavorable conditions such as low myocardiocytes perfusion (ischemia) and hypoxia, autophagy is activated which is probably also required for successful regeneration strategy.

We also suggest that an optimal level of autophagy is required for successful heart regeneration in all potential regeneration strategies. Transcriptionally, autophagy is regulated by MITF and FOXO, as the transcription families well as CREB and ATF transcription families [32, 61]. This gives a better opportunity for overregulation to target autophagy, enhance or suppress autophagy.

Conclusions

Autophagy is a natural subcellular catabolic detoxification process that involves the degradation of altered organelles and cytoplasmic components, such as proteins, to maintain cell homeostasis [71]. Autophagy-induced cell physiology can be applied to mechanically healthy and injured cells. Maintaining a high level of autophagy involved epigenetic regulation of autophagy gene expression. And with age, these epigenetic markers are lost. Therefore, cells age and impair function, including myocardiocytes. Suggesting, myocardiocytes death is primarily due to autophagy mechanisms dysregulation. Probably, a combination of reperfusion of myocardiocytes and inducing autophagy optimizes current therapy for myocardial infarction. Since autophagy reduces oxidative stress, lactic acidosis, and Ca⁺² paradoxes (reperfusion injury).

Autophagy plays a key role in the term of myocardiocytes' pathogenesis and dysfunction. The basal myocardiocytes' metabolic state requires autophagy to catabolize the toxic end-products of intracellular reactions. Autophagy behavior regulation is critical in health and disease since its involvement in the basal metabolic state and pathogenesis of diseases. Therefore, induction of autophagy through a safe and effective strategy is a potential cure for myocardiocytes dysfunction.