With the US now in open warfare with Iran in a long-heralded conflict triggered by fear for Israel’s existence, it is worth recalling the prescience of the Israeli spy in Tehran who saw it all coming.

As the Mossad station chief in Tehran in the late 1970s, Reuven Merhav was the Israeli foreign espionage agency’s man on the ground charged with safeguarding Israel’s sensitive intelligence relationship with its closest Middle East ally, Iran under the rule of its pro-western monarch, Shah Mohammad Reza Pahlavi.

In a scenario that throws the current state of warfare between the two nations into sharp relief, Israel and Iran had fostered close ties since the Jewish state’s founding in 1948.

But a March 1978 visit to the Gulf island of Kish – then a hedonistic playground for Iran’s rich and well-connected – along with the Israeli ambassador, Uri Lubrani, convinced Merhav that the shah’s reign was crumbling and the precious strategic partnership imperiled.

With discontent rumbling and opposition protests gathering pace, the two men encountered a monarch and intelligence agents complacent and detached from the gathering storm.

The pair communicated their forebodings to their Mossad and foreign ministry bosses in Israel – only to be met by skepticism and disbelief, a feeling shared by the CIA in Washington when the same warnings were relayed to it.

Weeks later, as Merhav prepared to leave Tehran at the end of his tour of duty, he had some cautionary words for his successor.

“I’m worried that this friend of ours will soon be an enemy,” Merhav told him as described by Ronen Bergman in his 2008 book, The Secret War With Iran. “I’m giving you Iran with a time fuse.”

It was an eerily prescient forecast.

Within months, Iran was consumed with revolutionary fervor. Chaos and upheaval unfolded on the streets. As his power base quickly collapsed, the shah – so recently thought by western allies to be unassailable – fled abroad with his family in January 1979.

Weeks later, Ayatollah Ruhollah Khomeini, a radical Shia cleric, returned from Paris after years in exile, to take power and turn Iran into an Islamic state. He immediately severed all ties with Israel. With revolutionaries storming the Israeli embassy, the country’s remaining diplomats were forced to flee, lucky to escape Iran with their lives.

View image in fullscreen Ayatollah Ruhollah Khomeini in 1989. Photograph: Denis Cameron/Rex Features

Thus began a deadly ideological enmity between two countries that had been allies, shared no common border and harbored no territorial claims against each other – and which has now spiralled into open war and, last night, dragged in the United States with consequences that may transform the geopolitical map, after Donald Trump announced that the US had bombed three Iranian nuclear facilities in Fordow, Natanz and Isfahan.

With the US’s entry into the conflict on Israel’s side, Merhav’s gloomy forecast has now acquired graver dimensions than perhaps even he foresaw.

More than three decades after he first made them, I had an improbably serendipitous encounter with Merhav on a train from Hamburg in Germany to Prague, the Czech capital.

Having entered my compartment after getting on the train at Berlin, the elderly man sitting opposite noticed my reading material and asked if I was reading Arabic. I told him it was Farsi, which I was attempting to learn, in the hope that I might one day be allowed back into Iran.

I had been the Guardian’s correspondent in Tehran several years earlier, but had been forced to leave Iran in late 2007 after the authorities refused to renew my residence permit, apparently due to objections over my reporting. But the intensity of the experience had kept my interest in the country – and hopes of returning – alive.

The man replied that he spoke Farsi, in which we then attempted to converse, with limited success before reverting to English as it became clear that his fluency in the language far exceeded mine. He explained that he was Israeli and had worked in the embassy in Tehran before the revolution. He introduced himself as Reuven – at which my eyes widened in sudden recognition.

I had read Bergman’s vivid account of Merhav’s grim warnings and the subsequent evacuation of Israelis from Tehran months earlier. It left a lasting impression. Now I was in the company of the main protagonist. He sidestepped my question about the book’s description of him as Mossad’s man in Tehran, confirming only that he worked for Israel’s foreign ministry, but acknowledged knowing Bergman and his work. There was no doubt that the stranger on the train was Merhav, a fact confirmed by subsequent Google searches and one Israeli foreign ministry official I later asked.

The chance encounter added context and meaning to events I had witnessed first-hand as the Guardian’s correspondent. Much of what happened then explains why the two countries are locked in combat now. To be in Tehran then was to be a witness to what turns out to have been a long prelude to the current warfare – as tensions between Iran and Israel escalated.

Nearly every night during spring and summer, I could hear living proof of the enmity that Merhav had predicted from the balcony of my rented house in north Tehran as youthful chants of marg bar Israeel (death to Israel) and marg bar Amrika (death to America) emanated from what seemed to be a nearby training camp for the Basij, a volunteer militia run by Iran’s Islamic Revolutionary Guard Corps.

Both slogans had been absorbed into the fabric of Iran’s revolutionary landscape. They could be seen emblazoned on buildings and were a staple chant at Friday prayers. “Death to Israel” was even graffitied in a section of Isfahan’s Naqsh-e Jahan Square, a Unesco world heritage site.

Israel and Iran had been locked in shadow hostilities for years – with Tehran suspected of masterminding deadly attacks on Israeli and Jewish installations in Argentina and backing Hezbollah in Lebanon – when I arrived in Iran in early 2005. But it was after the election of Mahmoud Ahmadinejad as president in June of that year that the ante was dramatically raised.

View image in fullscreen Then Iranian president Mahmoud Ahmadinejad during the Eid-al-Fitr ceremony in Teheran on 4 November 2005. Photograph: Abedin Taherkenareh/EPA

At a conference in Tehran in October, Ahmadinejad, a populist who promoted himself as a champion of the marginalized and underprivileged, stoked international outrage with a comment that was interpreted as calling for Israel “to be wiped off the map”, although other translations offered less incendiary forms of words.

Apparently revelling in the notoriety, the president followed up by calling the Holocaust “a myth”.

Worse followed. The following year, Iran’s biggest-selling newspaper, Hamshahri, staged the results of a cartoon contest to lampoon history’s worst genocide at Tehran’s Palestine Contemporary Art Museum.

The most egregious instance of Israel-baiting came in December 2006, when the Iranian foreign ministry staged what it called a “scientific” conference purporting to prove that the murder of 6 million Jews had not happened.

The event drew a rogue’s gallery of holocaust deniers and antisemites, among them David Duke, a former imperial wizard of the Ku Klux Klan and future supporter of Donald Trump, who praised Iran for allowing “free speech” and denounced Israel as “a terrorist state”.

It was, on many levels, a sickening spectacle, both for its unsavoury catalogue of attenders and the brazen assault on truth, with some exhibits purporting to show that allied forces, rather than the Nazis, were responsible for wartime atrocities.

“The biggest turning point was Ahmadinejad being elected and then denying the Holocaust,’ said Meir Javedanfar, an Iranian-born Israeli scholar, who teaches Iran studies at Reichman University, near Tel Aviv.

“Iranians lose their mind if somebody calls the Persian Gulf the Arabian Gulf. Yet they fail to understand what kind of emotions it creates in Israelis when somebody says the Holocaust is a myth.”

The insult was compounded by growing Israeli suspicions that Iran was trying to build an atomic bomb.

The existence of a hitherto secret Iranian nuclear project, in the form of a uranium-enrichment facility at Natanz, had been disclosed by the opposition Mojahedin-e-Khalq (MEK) movement in 2002. Enrichment activities were subsequently suspended by the then reformist president, Mohammad Khatami, as a confidence-building measure as western powers exerted pressure.

Ahmadinejad ordered the program’s resumption, stridently asserting Iran’s nuclear “rights” and sending international tensions soaring while elevating Israeli fears to fever pitch.

“You have somebody who denies the Holocaust, they’re developing nuclear program, they’re supporting Hezbollah – Iran took on a new form in terms of threats,” said Javedanfar.

Israel’s preoccupation with Iran’s nuclear activities underscored how dramatically the relationship between the two one-time allies had been transformed by the fall of the shah, historians say.

“One of Iran’s most important allies in the shah’s pursuit of a nuclear program in the last decade of his rule was Israel,” said Abbas Milani, director of Iranian studies at Stanford University and a biographer of the shah. “There are published reports of cooperation between Iran, Israel and South Africa in the last decade of his rule.”

View image in fullscreen The shah of Iran, Reza Pahlevi, and his wife, Farah Diba, in Marrakesh, Morocco, in 1979. Photograph: Randy Taylor/Associated Press

The shah, said Milani, saw Israel as a “strategic ally”, a view enhanced by the two countries’ shared suspicions of Arab nationalism, as embodied by the Egyptian leader, Gamal Abdel Nasser.

“The shah was a realist. He realized Israel is there to stay and it controlled his enemies.”

Jews, meanwhile, felt relatively secure in Iran.

“Iran has had long history of very good relations with its own Jewish population,” said Milani. “It had largest Jewish population of any Muslim country at that time. There were maybe 150,000 at the time of the revolution.”

That, too, was transformed by the shah’s downfall. The onset of the Islamic regime – bringing with it a climate of fierce anti-Zionism – prompted a mass exodus of Iranian Jews, many of them eventually moving to Israel.

In the early 1980s, with Iran embroiled in a bitter attritional war with Saddam Hussein’s Iraq, Israel attempted a rapprochement. Fearing a Saddam victory at least as much as Khomeini’s regime, it sent weapons to Iranian forces, helping them to stave off defeat.

The Iranians gladly accepted the weapons – but resisted any attempts at restoring the friendship.

“I’ve spoken to Israelis who served in Iran in the embassy before the revolution and afterwards dealt with Iran,” said Javedanfar. “They bent over backwards trying to reach out to the regime. They even lobbied the Americans” – who, under President Ronald Reagan, funneled their own weapons to Iran through Israel in an attempt to win the release of US hostages in Lebanon, an initiative that ended in the Iran-Contra scandal.

“It wasn’t just for the love of Iran. They had shared interests, but they really wanted to re-establish relations with this regime. But at the end, they found out that the weapons they were giving to Iran to fire at the Iraqis were being transferred to Hezbollah and being fired at Israelis.

“Iran did not want to have any rapprochement with Israel. It was impossible.”

Yet unwillingness to mend fences may not have devolved into deadly enmity without the obduracy of Ayatollah Ali Khamenei, who became supreme leader after Khomeini’s death in 1989

Khamenei exuded a greater commitment to Israel’s destruction than his predecessor, calling the country a “cancer” on the Middle East.

At a 1991 conference, organised at a time when Israelis and the Palestine Liberation Organisation were embarking on negotiations in Madrid, the supreme leader sided with factions opposed to a peace deal with Israel – a position at odds with Iran’s president at the time, Ali Akbar Hashemi Rafsanjani.

The result was an intensification of a proxy war that Iran had previously waged haphazardly, through Hezbollah in Lebanon and Hamas. The latter group embarked on a spate of suicide bombings that killed scores of Israelis and contributed, in 1996, to the election of the hardline Benjamin Netanyahu as Israel’s prime minister, as faith waned in the Oslo peace accords signed three years earlier.

There were domestic and foreign policy reasons for Khamenei’s anti-Israel crusade.

“After the fall of the Soviet Union, he saw an opening in the Middle East for a new kind of superpower, an Islamic one, so he wanted to take that chance,” Javedanfar said.

“But it also served the economic interests of his allies, because there’s billions of dollars to be made in keeping Iran isolated. That allows them complete monopoly over all sectors of Iran’s economy, from making tomato puree to cars. And one of the ways to make sure Iran always stayed isolated and western companies did not invest was to continue the anti-Israel line.”

Milani argues that Khamenei’s animus has even deeper roots in anti-Jewish prejudice, displayed in commentary he provided to In the Shade of the Qur’an, a book by the Egyptian revolutionary and Islamic Brotherhood leader Sayyid Qutb which he translated into Farsi.

“Many people, even within the regime itself, have asked why this is in the national interest, why do you think the destruction of the state of Israel is your responsibility?” he said. “The rhetoric and the reality of the policy of anti-Zionism is part of why Khamenei does it, but I think it is also an element of antisemitism.

“Khamenei thinks Israel today is the same, or even worse than, those [Jews] who fought Muhammad at Medina. When you have that kind of antisemitic thinking guiding you, you can’t befriend Israel.”

It is a minority view among Iranians, most of whom do not view Israel as an existential enemy and would accept normalized relations, says Milani.

Soaring non-combatant casualties or massive damage to Iran’s civilian infrastructure resulting from prolonged Israeli bombardment could test such a rosy view, and instead deepen the enmity that Merhav foresaw nearly half a century ago.

For now, Milani puts the onus on Khamenei, whom he accuses of failing to live up to years of rhetorical bellicosity when it mattered.

“His devotion to the notion that ‘death to Israel’ must be part of every ritual of politics in Iran, then putting the country on the path of war, being absolutely unprepared for it when war is imminent is just criminal negligence,” he said. “To have all of this rhetoric and then to be so unprepared when Israel initially attacked – it’s just remarkable.”

Identifying potential candidate genes targeted through miRNAs is essential to discovering the function of microRNAs in gene regulatory networks. As a consequence, miRNA-related cellular processes can be determined. It can be considered a pivotal procedure to illuminate the pathogenesis of cancer. In the following, different approaches to predicting targets of miR-20b are introduced.

The miR-20b in normal physiological condition

Growing evidence has revealed the fundamental roles of miRNAs in normal physiological conditions [53]. In this regard, miRNAs act toward maintaining cell function and homeostasis through cooperation with other cellular components and plenty of effectors. It has been authenticated that miR-20b is involved in the regulatory genes network within human cells. In Table 1, we summarized the findings from related investigations around the regulatory role of miR-20b in human biological specimens and cell lines. Evidence has indicated that fundamental cellular processes such as apoptosis, proliferation, differentiation, and T-cell activation are regulated upon miR-20. On the other hand, certain levels of miR-20b in cells are associated with their required function. As a result, changes in miR-20b levels lead to dysregulation of vital biological functions, which triggers human diseases.

Evolutionarily conserved miR-20b gene and comparative biology

Comparative biology is an approach that examines the differences and similarities between organisms, especially in biology, biochemistry, genetics, and physiology [54]. Conserved intra- and inter-species genes and proteins are of interest in comparative biology as a conserved gene in one organism may have a similar function in another [55]. MiRNAs are mentioned as the conserved genes during evolution [56]. Therefore, studies on animal models provide valuable information about the gene-miRNA network, leading to a further understanding of specific miRNA functions in human physiology. According to Table 2, miR-20b can modulate biological function in different animal models of diseases. Considering affected potential targets of miR-20b in the animal models might give a clue to its mechanism of action in humans.

High-throughput analysis to predict miR-20b potential target

Nowadays, novel approaches are established to determine the miRNA-mRNA interaction and underlying mechanisms of action. A computational method is a mathematical approach to merging biology, chemistry, and computer science [57]. This high-throughput analysis can accurately describe the miRNA-related target genes in various diseases. Using computational methods, Kim et al. determined that miR-20b targets transcription factor E2F1 in breast cancer [58]. E2F1 is a transcription factor that facilitates the synthesis of DNA and cell cycle in mammalian cells [59]. Consequently, targeting E2F1 by miR-20b inhibits tumor cell division. Integrative computational algorithms can analyze dysregulated miRNA targets in autoimmune diseases to identify strong candidate genes. Significantly, miR-20b has been determined as a modulator of helper T-cells differentiation with a high score [60]. Negative and positive regulators of T-cells differentiation, such as FOXO1 (Forkhead Box O1) and STAT3 (Signal transducer and activator of transcription 3), have been defined as significant miR-20b downstream target genes.

The miR-20b and cancer

Aberrant expression patterns of miRNAs influence the critical properties of tumors, such as escaping growth inhibitors, uncontrolled growth, promoting migration and invasion, preserving proliferative signaling pathways, suppressing apoptosis, and activating angiogenesis [12]. In contrast to the tumor-promoting role of miRNAs, these non-coding RNAs can act as tumor suppressors and inhibit tumor formation and progression under certain conditions. The miRNAs exert their effects through target genes and related signaling pathways. As mentioned, miR-20b binds to various targets and regulates signaling pathways and biological processes. Over the past years, much more information has become available on the occurrence of cancer upon disruption in miR-20b function and levels (Table 3). We reviewed the role of miR-20b in different solid tumors with emphasis on either its tumor-supportive (Fig. 1) or suppressor (Fig. 2) activities.

Table 3 The biological role of miR-20b in various cancer and its targets Full size table

Fig. 1 Tumor-supportive role of miR-20b by stimulating tumor cell growth and migration while inhibiting their apoptosis Full size image

Fig. 2 Tumor-suppressor role of miR-20b by inhibiting tumor cell growth and migration Full size image

Proliferation and cell cycle

Cell division is strongly associated with tumorigenesis and counted as an essential hallmark of malignancy. At the molecular levels, extra- and intracellular signaling pathways, growth factors, and bona fide of hormones keep an equilibrium between the induction of cell division and its inhibition [12]. Abnormal activity of these regulatory pathways leads to cancerous cells with out-of-control proliferation. Various studies have assessed the efficacy of miR-20b in cell division and cell cycle. It significantly integrates into several vital cell proliferation pathways, and the improper modulation of this miRNA is responsible for preserving proliferative signaling and escaping growth inhibitors in tumor cells.

The miR-20b is overexpressed in MCF-7 and MDA-MB-231 cell lines compared to healthy cells [85]. It may refer to the potential role of miR-20b in breast cancer tumorigenesis. Olga et al. have shown that radiation increases the levels of miR-20b, which targets the Phosphatase and tensin homolog (PTEN) and Breast cancer type 1 (BRCA1) genes, and promotes the proliferation and cell cycle of breast cancer cells [86]. BRCA1 is involved in homologous recombination, a kind of DNA repair mechanism. Impairment in BRCA1 normal activity increases genomic instability and facilitates cancer cell proliferation and tumor progression. Besides, incompetent DNA repair in cancer cells yields increased proliferation. Indeed, miR-20b triggers genomic instability and elevated cell proliferation by targeting BRCA1. The growing evidence suggests that miR-20b broadly targets PTEN in multiple cancer cells. Up-regulated miR-20b negatively correlates with PTEN expression in breast cancer specimens [87]. PTEN serves as a tumor suppressor, tightly deterring transformed cell growth and division [88]. Besides, aberrant expression of miR-20b is closely related to prostate cancer [89]. MiR-20b is elevated in prostate cancer cells and triggers their proliferation. Bioinformatics analysis showed that the PTEN gene is a potential target of miR-20b, as verified by preclinical experiments [67]. Furthermore, it has been indicated that miR-20b leads to hepatocellular and colorectal cancer cell proliferation through binding PTEN and down-regulates its expression [74, 79]. Using the antagomir of miR-20b enhances PTEN levels and causes tumor regression. Antagomirs are synthetic antisense oligonucleotides that bind to the desired miRNA and reduce or block its activity [90].

Overexpression of miR-20b positively correlates with poor prognosis in lung cancer [91]. To understand the working mechanism of miR-20b in lung cancer proliferation, Xuan et al. have demonstrated that miR-20b is up-regulated in non-small cell lung cancer cells [70]. Further, molecular experiments revealed that B-cell translocation gene 3 (BTG3) reduced upon miR-20b overexpression and led to tumor cell proliferation [70]. BTG3 is an anti-proliferative protein that loses its normal function during tumorigenesis in human cancers [45]. The level of miR-20b and its function integrate into proliferation-related signaling pathways. Ectopic low-level of miR-20b is associated with tumorigenesis and progression of thyroid carcinoma [77]. It directly binds to extracellular signal-regulated kinase 2 (ERK2) and son of sevenless homolog 1 (SOS1) [77]. These proteins are member of the mitogen-activated protein kinase (MAPK) signaling pathway, which promote cellular proliferation and differentiation [92].

Despite the oncogenesis effect of miR-20b in promoting cell proliferation, miR-20b has an anti-proliferative ability in some cases. The cell cycle-regulated genes such as cyclin-dependent kinase (CDKs) and cyclin have been recognized as miR-20b target genes. In ovarian cancer, overexpression of miR-20b arrests the cell cycle in the G1 phase by reducing cyclin D1 levels [66]. The transfection of EJ cells, an invasive bladder carcinoma, with miR-20b triggers G1 phase arrest by directly targeting CDK6, CDK2, and cyclin D [76]. Furthermore, p21, a well-known inhibitor of CDKs, was indirectly increased in miR-20b-transfected cells and thus reinforced cell cycle arrest.

Metastasis

Cancerous cells acquire several properties and transform into metastatic cells, which can migrate from the original location to distant sites and form secondary tumors [93]. This advanced cancer stage is responsible for patients’ illness severity and death. Compelling evidence has indicated that various steps, such as epithelial-mesenchymal transition (EMT), migration, invasion, and angiogenesis, are involved in metastasis.

EMT

Reversible and rapid modulation of phenotype transition from epithelium to mesenchyme is termed EMT. During this peculiar process, primary tumor cells lose their surface adhesion molecules and obtain migration capabilities [94]. Many experiments have recognized the different signaling pathways, including transforming growth factor beta (TGF-β) participating in EMT [95]. In prostate cancer, the TGF-β signaling pathway significantly promotes EMT, while miR-20b mimic represses EMT by targeting the receptor of TGF- β [68]. The microRNA mimic approach has focused on using synthetic miRNA-like fragments for gene silencing [96]. Furthermore, incorporating long-noncoding RNA with miRNAs to regulate biological processes has become a field of interest for investigators. LncRNA operates as a miRNA sponge that binds to the desired miRNA with high affinity and inhibits their downstream functions [97]. H19, a long non-coding RNA, interacts with miR-20b and suppresses its biological function [84]. Mechanistically, miR-20b downregulates HIF-1 alpha, while downregulation of miR-20b activates hypoxia signaling pathways and stimulates EMT in endometrial cancer [84].

Migration and invasion

Cell migration typically occurs in embryonic development, nervous system formation, wound healing, vascular sprouting, and immune-cell trafficking [61]. The migration enables cells to change their location in either tissue or among various organs. Uncontrolled cell movement is related to pathological circumstances, including the invasion behavior of tumor cells. In invasion, malignant cells can penetrate tissue and vascular barriers into the bloodstream. Characterizing cancer cells’ migration and invasive potential and interaction with various underlying regulation mechanisms, such as miRNA, is relevant for developing therapeutic strategies against cancer.

The results of the microarray assay emphasize miR-20b key role in the miRNA-mRNA network in cervical cancer migration [69]. Cervical cancer is a prevalent malignancy in women after breast and intestine cancer [98]. Papillomavirus (HPV) infection may trigger tumorigenesis and cervical cancer progression. This virus increases miR-20b levels and leads to migration and invasion via targeting tissue inhibitor of metalloproteinases 2 (TIMP2, an inhibitor of MMP2 [69]. Metalloproteases (MMPs) such as MMP2 are complicated in the degradation of the extracellular matrix, thus enabling cell migration [99]. There is another relevance between changed miR-20b levels and MMPs in bladder cancer occurrence and progression. It has been indicated that miR-20b expression is reduced in bladder tumor cells, and elevated miRNA levels can act as a tumor suppressor [76]. MMP2 is described as the potential target of miR-20b [76]. Downregulation of miR-20b predominantly yields sustained migration and invasion of bladder tumor cells.

The miR-20b also participates in tumorigenesis and esophageal cancer progression. Indeed, miR-20b activity brings about esophageal cancer migration and invasion through the downregulation of PTEN levels [71]. The regular role of PTEN is suppressing migration through its phosphatase activity [100]. PTEN inactivates the phosphoinositide 3-kinase (PI3K) signaling pathway, which is vital to provide a front-rear gradient of molecular and chemotaxis levels for cell migration. Consequently, targeting PTEN augments PI3K signaling pathway and cancer cell migration.

Angiogenesis

Angiogenesis is the formation of blood vessels to supply cancer cells within the tumor. As a result of its unique capacity to inspire new blood vessel formation, angiogenesis plays an essential role in tumor formation and metastasis [101]. The miRNAs regulate several angiogenesis-related signaling pathways and master genes [102]. Vascular endothelial growth factor (VEGF) is secreted by various cells, such as cancer cells, to stimulate angiogenesis. Targeting VEGF either directly or indirectly through miR-20b leads to down-regulation of angiogenesis. However, the interaction between long non-coding RNAs and miR-20b leads to the induction of VEGF expression in some cases. Enhanced levels of lncCAMTA1, a long non-coding RNA, are found mainly in the MDA-MB-231 breast cancer cell line. Further analysis indicated that lncCAMTA1 binds to miR-20b, thus up-regulating VEGF level as the miR-20b target gene [62]. In addition, VEGF downstream signaling pathways such as MAPK, ERK, Janus kinase (JAK), and Signal transducer and activator of transcription (STAT) are up-regulated in MDA-MB-231 cells, and angiogenesis occurs. Interestingly, miR-20b can target the upstream regulator of the VEGF gene and trigger angiogenesis in hepatocellular carcinoma [81]. STAT3, as the miR-20b target, negatively regulates VEGF in mRNA and protein levels. Overexpression of miR-20b in hepatocellular carcinoma cells inhibits STAT3 function, leading to increased VEGF levels [81].

Cancer stem cells

Recent developments in miRNA-mRNA interaction networks have led to an interest in miR-20b in cancer stem cells (CSCs), believed to play a critical role in tumor progression and drug resistance [103]. The miR-20b may be a double-edged sword in cancer stem cell regulation.

The stemness of cancer stem cells that causes the malignancy of colorectal tumor have been explored [73]. The expression of Oct4, a stem cell marker, and MALAT1, a long non-coding RNA, are negatively related by miR-20b levels. Overexpression of miR-20b attenuates the proportion of cancer stem cells via the direct targeting of Oct4 and MALAT1, critical positive regulators of cancer stem cell stemness [73, 81, 102, 103], and finally eases tumor regression [104]. However, miR-20b could promote breast cancer stem cell proliferation and cell cycle [64]. It indirectly elevates Cyclin D1 and E2F1 mRNA levels and promotes cell proliferation in breast cancer stem cells [64]. Cyclin D1 is a crucial regulator of cell cycle progression from G1 to the S phase [104]. Overexpression of cyclin D1 and E2F1 mediates the high rate of cell cycle and division [105]. Further, an examination has revealed that miR-20b may inhibit cyclin D1 and E2F1 inhibitors, ultimately increasing cyclin D1 and E2F1 expression.

Autophagy

Autophagy is a conserved self-degradative cellular procedure to remove damaged organelles, intracellular pathogens, and misfolded proteins [106]. This fundamental process balances cell energy and response to nutrient stress [106]. The relevance of autophagy in early- and advanced cancer remains disputable. While investigations have illustrated that autophagy inhibits tumorigenesis by suppressing tumor cell growth and inducing apoptosis, it may also cause tumor cell migration and invasion [107, 108]. Depending on a wide range of regulators, including miRNAs, the role of autophagy can be different in cancer. It has been shown that miR-20b can downregulate autophagy via inhibiting RB1CC1/FIP200, autophagosome formation-related proteins [63]. Then breast cancer cells’ survival decreased. Further research should be carried out to establish closer links between miR-20b and autophagy in cancer.

Apoptosis

Apoptosis is called programmed cell death (PCD), mainly containing intrinsic and extrinsic pathways. Each pathway requires a specific initiator caspase protein which activates the downstream molecular cascade and induces apoptosis [109]. Improper apoptosis, either activation or suppression, is a key factor in human diseases like cancer [110]. A strong relationship between miR-20b and apoptosis has been reported in ovarian cancer. Increased miR-20b levels remarkably down-regulate B-cell lymphoma 2 (Bcl2) as an anti-apoptotic protein [65]. As a result, Bax protein is oligomerized on the mitochondria surface and causes induces apoptosis by releasing cytochrome c [65]. Furthermore, Increased miR-20b levels induce apoptosis in renal cell carcinoma. MAPK signaling pathway components are considered miR-20 putative targets based on the bioinformatic assay [82]. Depending on cell type, the MAPK cascade has a dual role in inducing or inhibiting apoptosis [111]. Although the MAPK signaling pathway is up-regulated in renal cancer cells and inhibits apoptosis [112], over-expression of miR-20b could inhibit this axis and induce apoptosis.

The miR-20b can inhibit apoptosis. The expression of miR-20b and its underlying mechanism of action has been investigated in cancerous thyroid cells. LncRNA- miRNA crosstalk can regulate apoptosis by affecting associated signaling pathways. double homeobox A pseudogene 8 (DUXAP8), a lncRNA, suppresses apoptosis in papillary thyroid tumor cells. Indeed, DUXAP8 binds to miR-20b and inhibits its function [78]. SOS-1 of the ERK signaling pathway, which causes cell survival, is known as the miR-20b target. Mechanistically, miR-20b inhibition by DUXAP8 improves cell proliferation while impeding apoptosis [78].

Drug resistance

Drug resistance is the main challenge in cancer treatment, primarily preventing chemotherapy’s efficacy in cancer cells and promoting cancer cell survival. MiR-20b has an essential role in chemotherapy resistance. It has been demonstrated that the activation of Syndecan-2, a transmembrane receptor, is positively correlated with chemotherapy resistance [75]. Mechanistically, miR-20b diminishes the resistance of cancer cells to chemotherapy by targeting Syndecan-2 [75]. Its capability to regulate the sensitivity of cancer cells to chemotherapy drugs through hypoxia signaling pathways has profoundly been displayed.

Interestingly, the downregulation of miR-20b in gastric cancer induces drug resistance [83]. Hypoxia-inducible factor 1 subunit alpha (HIF1A) negatively correlates with miR-20b levels in gastric cancer. As a result, increased levels of HIF1A activate downstream genes like multidrug resistance (MDR) and facilitate drug resistance [83].

An investigation of hepatocellular carcinoma cells has indicated that drug resistance negatively associates with reduced levels of miR-20b [80]. Overexpression of miR-20b leads to the sensitivity of hepatocellular cancer cells to chemotherapy drugs. From the molecular aspect, the cell division cycle 37-like 1 (CDC37L1) gene enhances drug resistance of hepatocellular cancer cells [80]. Targeting CDC37L1 by miR-20b reverses its effects. CDC37L1 is a cochaperone protein that promotes cell survival [113]. Besides, miR-20b can induce apoptosis of drug-resistance colon cancer cells by suppressing ADAM9 (A disintegrin and a metalloprotease 9), an activator of MMPs [72]. Silencing of ADAM9 expression encourages apoptosis in various tumor cells, such as ovarian and prostate cancer [114, 115].

Is ChatGPT a sign that automation is coming to film and TV writing? As far-fetched as it sounds, the arrival in November 2022 of a free prototype of the AI-powered chatbot — which has jolted observers with the sophisticated, fluid writing it can produce when prompted, even in the form of poems, essays and, yes, short scripts — has set off alarm bells about the disruption that the chatbot could wreak on the work of entertainment scribes. Still, top film and TV writers are skeptical that the technology in its current state imperils their livelihoods in any way, even as they remain cautious about the potential for future advancements.

“Do I see this in the near term replacing the kind of writing that we’re doing in writers rooms every day? No, I don’t,” says Big Fish and Aladdin writer John August, who has tested the free research preview and talked about it on the popular Scriptnotes podcast, which he co-hosts with Craig Mazin (The Last of Us). Still, he adds, “There certainly is no putting the genie back in the bottle. It’s going to be here, and we need to be thinking about how to use it in ways that advance art and don’t limit us.”

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Another prominent writer and showrunner, speaking to The Hollywood Reporter anonymously, has taken ChatGPT for several test rides and says the chatbot seems incapable of writing funny jokes or producing results that might be useful to include in a script without “substantial creative input from me.” This showrunner adds, “When people conclude that this is going to replace professional writers, I think they’re sort of swallowing an Elon Musk-style fantasy about the future that is not actually connected to the technology.”

Launched by the AI research company OpenAI, ChatGPT in its publicly available iteration can produce polished, if rote, pitches and loglines for films and television shows as well as generic outlines and scripts within seconds. The current version, trained on large quantities of text and code all predating the fourth quarter of 2021, also occasionally produces some falsehoods when answering factual queries: OpenAI CEO Sam Altman has warned that this version is “incredibly limited, but good enough at some things to create a misleading impression of greatness.” He added, “It’s a mistake to be relying on it for anything important right now. It’s a preview of progress.”

Still, several writers say the incipient technology shows promise as an ancillary creative tool. ChatGPT could help with time-consuming rote work for writers, like generating potential scene locations or character names; August suggests that non-native English speakers might use it to produce more fluid writing in the language. It can also offer plot or character ideas. Savage Grace writer and former WGA West president Howard A. Rodman compares the chatbot to “Oblique Strategies” cards that aim to re-inspire artists with prompts: He says he could use the bot “to suggest something I would never think of myself, maybe something smarter, maybe something stupider or more obvious.”

There is cause for concern, for some, about future refinements to this kind of technology. Rodman calls ChatGPT’s clear ability to improve its responses the more prompts it receives “a little chilling,” but adds, “Writers should not blind themselves to the ways in which AI technology can be useful in many ways. They should also be aware of the opportunities that it offers employers to do the thing they love best: put downward pressure on fixed costs.”

Franklin Leonard, founder and CEO of screenplay platform The Black List, doesn’t worry about ChatGPT replacing writers, but he believes it has the potential to upend the labor market and says it’s “necessary that the communities that are likely to be most affected by it, in particular writers, are the ones who are defining the guardrails around how it can be used in the context of this business.” Although he’s not privy to potential conversations, Franklin hopes that the Writers Guild is discussing this technology, as he believes the long-term financial health of the industry is “incredibly closely tied” to that of writers. (The Writers Guild of America West said in a statement, “We’re monitoring the development of ChatGPT and similar technologies in the event they require additional protections for writers.”)

Even as the technology is rapidly advancing, studios likely won’t roll the dice in exploiting works — scripts, art or otherwise — solely generated by AI just yet. There’s no copyright protection for such works because intellectual property law doesn’t recognize ownership by nonhuman creators. AI-created works would enter the public domain upon release, potentially limiting commercial interest in the format.

There’s been a push for protection of works created by AI spearheaded by Stephen Thaler, the CEO of neural network firm Imagination Engines. In 2018, he listed an AI system, the Creativity Machine, as the creator of an artwork called A Recent Entrance to Paradise, while listing himself as the owner of the copyright under the work-for-hire doctrine. The U.S. Copyright Office denied the registration, finding the work “lacks the human authorship necessary to support a copyright claim” and that “the nexus between the human mind and creative expression” is a crucial element of protection. Thaler sued the office in June over the rejection, but the office said it’s unwilling to “depart from a century of copyright jurisprudence.”

“The U.S. Copyright Office has looked at this topic through a very narrow lens,” says Joel Feldman, a lawyer whose specialty is copyright and trademark litigation. He stresses that the copyright office would be more willing to grant protection to AI-generated works as long as there’s a human co-author. “If you fed your favorite 25 songs into an AI machine, and it spit out a new work never before heard, there’s certainly a claim that your selection of those 25 songs is human authorship.”

Still, Ryan Abbott, Thaler’s lawyer and a partner at Brown Neri who specializes in the intersection of AI and copyright law, notes that the protection of works generated by AI is a question of “when” rather than “if.” “This is another instance of the law having to catch up with the state of technology,” he says. “When lawmakers were drafting copyright law, they weren’t thinking about AI.”

Whether AI programs, built on machine learning models that analyze the patterns of copyrighted works, infringe on the copyrights of artists is up in the air. Courts may find that training an AI program like OpenAI’s DALL-E 2, which generates complex images from text prompts, using copyrighted art does not fall under the fair use defense, which allows for use of protected works without permission as long as they are transformative. In November, a proposed class action was filed against Microsoft, Github and OpenAI claiming the billions of lines of computer code that their AI technology analyzes to generate its own code essentially constitute piracy. A finding of copyright infringement in the first-of-its-kind suit against AI programs that don’t acknowledge prior work would severely dampen the economic prospects of exploiting works generated by AI in Hollywood.

The prominent showrunner suggests that, if studios ever try to input their scripts into a database and use that to make a text generator in an attempt to bypass the work of a human writer, “they’re going to have tens of thousands of angry creatives on their hands.” He adds, “I’m not sure that they’re going to be interested in dynamiting all of those relationships in one fell swoop.”

Agents are also keeping a close eye on potential rights issues. CAA’s chief metaverse officer, Joanna Popper, says that the agency is endeavoring to work with cutting-edge tech companies (such as deepfake firm Deep Voodoo, from South Park’s Trey Parker and Matt Stone, which in December received $20 million in a funding round led by CAA’s Connect Ventures) to keep emerging tech talent-friendly. “I hope the industry will respond [to ChatGPT] by seeing that it’s a tool that could benefit them, while also being cognizant of making sure that their rights are honored,” she says.

_____

AI-Driven Script Pitches, as Critiqued by a Human Screenwriter

THR asked ChatGPT to generate a series of feature film elevator pitches that mix and match established hits — a time-honored industry practice — then asked Big Fish scribe John August how well they worked.

The Pitch:

Die Hard meets You’ve Got Mail

ChatGPT’s Logline:

“When a group of terrorists take over a high-end Manhattan department store on Christmas Eve, a scrappy small-business owner must team up with a tough cop to take them down and save the day, all while trading banter and flirting over email.”

John’s Notes:

“‘Small-business owner’ doesn’t bring anything helpful here. If the Meg Ryan equivalent were an anti-cop activist, there’d at least be some tension.”

The Pitch:

Fast and the Furious meets Independence Day

ChatGPT’s Logline:

“Street Warriors: When a group of skilled street racers are recruited by a top-secret government agency to fight against an alien invasion, they must use their expertise behind the wheel to save the world from destruction. As they race against time, they must push their limits and embrace their inner strength to become true heroes.”

John’s Notes:

“The second sentence is meaningless filler. We need some sense of why they’re recruited, like the drillers in Armageddon or the hero of The Last Starfighter.”

The Pitch:

The Hangover meets La La Land

ChatGPT’s Logline:

“Lost in L.A.: When a group of friends travel to Los Angeles for a wild bachelor party, they wake up to find that one of their own has gone missing. As they search the city for their missing friend, they are drawn into the vibrant world of L.A.’s music scene and must navigate their way through a series of zany mishaps and absurd adventures.”

John’s Notes:

“Weakest of the three. It’s literally just The Hangover. A true mashup might have boy and girl searching for each other after a wild night.”

John’s Overall Takeaway:

“These read like the summaries on the top sheet of script coverage. That’s not a knock; I used to be a reader at TriStar, and they’re not always easy to write. As far as the ideas themselves, they’re all taking ‘X meets Y’ far too literally. Screenwriters don’t start with this shorthand; they find their way to it to explain their take.”

A version of this story first appeared in the Jan. 11 issue of The Hollywood Reporter magazine. Click here to subscribe.

Cardiac sympathetic activation, mediated by β-adrenergic receptors (β-ARs), normally increases cardiac contraction and relaxation. Accomplishing this task requires a physiological, concerted Ca 2+ signaling, being able to increase Ca 2+ release from sarcoplasmic reticulum (SR) in systole and speed up Ca 2+ re-uptake in diastole. In heart failure (HF) myocardial β-ARs undergo desensitization/down-regulation due to sustained sympathetic adrenergic activation. β-AR desensitization/down-regulation diminishes adrenergic signaling and cardiac contractile reserve, and is conventionally considered to be detrimental in HF progression. Abnormal Ca 2+ handling, manifested as cardiac ryanodine receptor (RyR2) dysfunction and diastolic Ca 2+ leak (due to sustained adrenergic activation) also occur in HF. RyR2 dysfunction and Ca 2+ leak deplete SR Ca 2+ store, diminish Ca 2+ release in systole and elevate Ca 2+ levels in diastole, impairing both systolic and diastolic ventricular function. Moreover, elevated Ca 2+ levels in diastole promote triggered activity and arrhythmogenesis. In the presence of RyR2 dysfunction and Ca 2+ leak, further activation of the β-AR signaling in HF would worsen the existing abnormal Ca 2+ handling, exacerbating not only cardiac dysfunction, but also ventricular arrhythmogenesis and sudden cardiac death. Thus, we conclude that β-AR desensitization/down-regulation may be a self-preserving, adaptive process (acting like an intrinsic β-AR blocker) protecting the failing heart from developing lethal ventricular arrhythmias under conditions of elevated sympathetic drive and catecholamine levels in HF, rather than a conventionally considered detrimental process. This also implies that medications simply enhancing β-AR signaling (like β-AR agonists) may not be so beneficial unless they can also correct dysfunctional Ca 2+ handling in HF.

Introduction

Beta-adrenergic receptors (β-ARs) are members of a large family of cell surface receptors known as G-protein-coupled receptors (GPCRs) (1). β-ARs mediate the sympathetic adrenergic effect (the fight or flight response) on the heart. There are 3 β-AR subtypes: β1-AR, β2-AR and β3-AR in the heart (2). These receptors are predominantly found in the myocardium, vascular smooth muscle, and adipose tissue, respectively. Cardiomyocytes express all 3 subtypes, with β1-ARs being the predominant subtype, representing ~80%, whereas β2-ARs and β3-ARs comprising the remaining 17 and 3%, respectively (3, 4). In cardiomyocytes, β1-ARs are widely distributed across the cell membrane and can be activated by both norepinephrine (released from sympathetic nerve fibers) and epinephrine (from the chromaffin cells of the adrenal medulla) (5). Activation of β1-ARs and the subsequent Gs-protein coupled signaling cascade (adenylyl cyclase-cAMP-protein kinase A) leads to increased myocardial contraction (inotropy), relaxation (lusitropy), heart rate (chronotropy) and AV conduction (dromotropy), respectively (6). β2-ARs are more localized within the transverse (T)-tubules (7), have a higher affinity for epinephrine and are coupled to the Gs and Gi subunits, allowing for both stimulatory and inhibitory effects on cardiac contractile function (8). β3-ARs are expressed at low levels on cardiomyocytes, and their physiological role is less clear (9).

Heart failure (HF) is a global epidemic problem, affecting more than 37.7 million individuals globally and imposes substantial economic burden to the health care system (10). HF occurs when various assaults result in myocardial injury/loss, impairing cardiac function. In response, the neurohumoral system, particularly the sympathetic nervous system is activated. The sympathetic activation, mediated by the β-AR (mainly β1-AR) signaling cascade (as mentioned before), increases cardiac contractility, relaxation, and cardiac output. Thus, sympathetic activation acutely (or in short term) is a beneficial compensatory process. However, sustained long-term sympathetic activation in HF can lead to pathological remodeling in β-ARs, manifested as uncoupling of the β-AR signaling (desensitization) and reduced number of β-ARs (mainly β1-ARs) on cell membrane (β-AR down-regulation) (6). β-AR desensitization/down-regulation diminishes adrenergic signaling and contractile reserve, and is conventionally considered to be detrimental in HF deterioration/progression (3, 11, 12). This notion is further supported by the fact that effective treatments (e.g., beta-blockers) are associated with re-sensitization/up-regulation of the β-ARs (6). As a result, re-sensitizing/up-regulating β-ARs is currently believed to be beneficial and efforts are directed to develop medications to up-regulate/re-sensitize the β-ARs as therapeutic strategies in HF (6, 12, 13).

Abnormal Ca2+ signaling has been identified as another form of pathological remodeling in HF (14). Several critical Ca2+ handling components are dysfunctional in HF, including sarcoplasmic reticulum (SR) cardiac ryanodine receptor (RyR2, the calcium release channel) dysfunction and diastolic Ca2+ leak, reduced Ca2+ pump (the sarcoplasmic/endoplasmic reticulum Ca2+ ATPase, SERCA) activity, etc. (14). RyR2 dysfunction and Ca2+ leak could lead to depleted SR Ca2+ stores and elevated cytoplasmic Ca2+ levels in diastole, resulting in both systolic and diastolic dysfunction. In addition, by activating the inward Na+/Ca2+ exchanger (NCX) current (15), diastolic Ca2+ leak and elevated cytoplasmic Ca2+ levels could depolarize membrane potential, contributing to electrical instability, delayed afterdepolarizations, triggered arrhythmias, and sudden cardiac death (16–19). RyR2 dysfunction and Ca2+ leak in HF is caused by sustained sympathetic activation and elevated β-AR signaling (14). Thus, it is foreseeable that further activation of β-AR signaling would worsen the existing RyR2 dysfunction and Ca2+ leak, not only affecting cardiac function, but also enhancing arrhythmogenesis. Accordingly, we present our viewpoint here that in the presence of RyR2 dysfunction and Ca2+ leak, further enhancing β-AR signaling is detrimental rather than beneficial, and β-AR desensitization/down-regulation in HF may be a self-preserving, adaptive process rather than a conventionally considered detrimental process. This viewpoint will be detailed in the following discussion.

β-AR Desensitization/Down-Regulation and RyR2 Dysfunction/Ca2+ Leak Are Two Distinct Yet Related Abnormalities in HF

In HF cardiomyocytes develop 2 distinct yet related abnormalities: β-AR desensitization/down-regulation and RyR2 dysfunction/Ca2+ leak (Figure 1). Both abnormalities are allegedly caused by the sustained sympathetic activation and β-AR signaling in HF (6, 14). β-AR desensitization/down-regulation (Figure 1, abnormality 1) in HF is well-characterized, which diminishes sympathetic signaling, cardiac functional capacity and contractile reserve, and is conventionally considered to be detrimental in HF progression (3, 11, 12). On the other hand, when RyR2 dysfunction and Ca2+ leak occur (abnormality 2), further enhancing β-AR signaling would worsen the existing RyR2 dysfunction and Ca2+ leak, leading to exacerbation of cardiac dysfunction and arrhythmogenesis (Figure 1). Accordingly, we postulate that β-AR desensitization/down-regulation, acting like an intrinsic β-AR blocker and by diminishing adrenergic signaling, can limit RyR2 dysfunction and Ca2+ leak from further deteriorating under sustained sympathetic adrenergic drive with persistent high-level of catecholamines in HF. In other words, β-AR desensitization/down-regulation in HF may be a self-preserving, adaptive process (a friend) rather than a conventionally considered detrimental/pathological process (a foe). This viewpoint is supported by the following several lines of evidence.

FIGURE 1

Figure 1. Heightened adrenergic signaling in the presence of cardiac ryanodine receptor (RyR2) dysfunction and Ca2+ leak is detrimental. In heart failure (HF), sustained adrenergic activation leads to hyperphosphorylation (among other posttranslational modifications) of the RyR2 by PKA and CaMKII (through PKA-independent signaling mediated by β1-AR activation), resulting in RyR2 dysfunction and pathological Ca2+ leak. Desensitization/down-regulationof the β-AR (abnormality 1) would diminish adrenergic signaling, alleviating RyR2 dysfunction and Ca2+ leak (abnormality 2) than they would otherwise. In contrast, enhancing adrenergic signaling in HF would worsen the RyR2 dysfunction and Ca2+ leak (abnormality 2), exacerbating cardiac dysfunction and arrhythmogenesis. PKA, protein kinase A; CaMKII, Ca2+/calmodulin-dependent protein kinase II; SR, sarcoplasmic reticulum; RyR2, cardiac ryanodine receptor.

Normal Ca2+ Signaling Is Essential in Mediating Physiological Sympathetic Response

Cardiac excitation-contraction coupling (ECC) refers to the conversion from electrical excitation (in the form of an action potential) into mechanical contraction. Briefly, cardiac action potential propagates along the cell membrane, including the specialized invaginations (T-tubules) (20). Depolarization activates voltage-gated L-type Ca2+ channels (LTCCs) on the T-tubules and allows Ca2+ to enter the cell. The entering Ca2+ then binds to and activates Ca2+ release channels (RyR2) on SR, releasing Ca2+ to cytoplasm from the SR (where Ca2+ is stored at high concentrations). This process by which Ca2+ entering cardiomyocyte through LTCCs activates RyR2 is known as Ca2+ induced Ca2+ release (21). The spike of Ca2+ concentration in cytoplasm initiates actin–myosin cross-bridging, causing muscle contraction (systole). Cardiac relaxation (diastole) is initiated when RyR2 close (terminating SR Ca2+ release) and Ca2+ are pumped back into the SR by the SERCA or extruded from the cell by the Na+/Ca2+ exchanger (NCX). This coordinated Ca2+ release and reuptake in cardiomyocytes are essential for ECC.

Normal Ca2+ signaling is required in mediating physiological sympathetic response. Sympathetic activation, mediated by the β-ARs-Gs-adenylyl cyclase-cAMP-protein kinase A (PKA) signaling pathway, activates and phosphorylates LTCCs, increasing inward Ca2+ current (22). The same cascade also phosphorylates RyR2 (23), increasing Ca2+ release from SR, which in turn increases cardiac contractility (14). Adrenergic signaling also activates SERCA, speeding up Ca2+ removal from cytosol, thus improving cardiac relaxation. This normal Ca2+ signaling is essential in realizing sympathetic adrenergic effects on the heart.

Sustained Sympathetic Adrenergic Activation Leads to RyR2 Dysfunction and Ca2+ Leak, Enhancing Arrhythmogenesis in HF

It is known that ventricular arrhythmias are common in HF patients and about 50% of HF patients die due to sudden cardiac death (24). Accumulating evidence indicates that a pathogenic process of defective cardiac Ca2+ signaling plays a critical role in HF (16, 25, 26). As discussed, RyR2 open and close in a concerted manner during myocyte contraction and relaxation (27). In HF, chronic sympathetic adrenergic activation leads to unstable channels that leak Ca2+ during diastole because the channel cannot close properly (28). The RyR2 dysfunction in HF is reportedly caused by enhanced adrenergic signaling resulting in hyperphosphorylation, oxidation or S-nitrosylation of RyR2, etc., which results in Ca2+ leak by destabilizing the closed state of the channel (14). RyR2 dysfunction and Ca2+ leak can be observed in isolated cardiomyocytes as short, unsynchronized SR Ca2+ release events during diastole (29).

RyR2 dysfunction and Ca2+ leak could impair cardiac function and enhance arrhythmogenesis. Chronic Ca2+ leak via dysfunctional RyR2 depletes Ca2+ store in the SR, lowers Ca2+ transient amplitude in systole and reduces cardiac contractility (30). Ca2+ leak can lead to elevated Ca2+ levels in diastole, impairing myocyte relaxation. Thus, RyR2 dysfunction and Ca2+ leak could lead to both systolic and diastolic dysfunction. Moreover, Ca2+ leak and elevated diastolic Ca2+ levels increase Ca2+ extrusion via NCX, promoting an inward depolarizing current which is electrogenic and promotes triggered activity and arrhythmia formation (15). The detrimental effect of β-AR activation in HF is supported by evidence that treatment with β-AR agonists is associated with increased mortality in HF (31, 32), despite their short-term hemodynamic benefits. We have demonstrated recently that failing hearts are more vulnerable to develop both atrial and ventricular arrhythmias under sympathetic stimulation, despite less positive inotropy in HF (33, 34). Finally, this is also supported by the fact that β-AR blocker treatment can reduce cardiac arrhythmias and sudden cardiac death in HF (35).

Sustained Sympathetic Activation Leads to β-ARs Desensitization/Down-Regulation in HF

HF is characterized by sympathetic activation and elevated blood catecholamine levels (3). As discussed, sympathetic adrenergic activation, mediated through β-AR signaling, increases cardiac contraction and relaxation. These responses are initially beneficial in supporting the failing heart by increasing cardiac output and sustaining circulation. However, long-term overstimulation of β-ARs eventually leads to loss of responsiveness to sympathetic signaling (β-ARs desensitization) and reduced density (number) of β-ARs on cell membrane (down-regulation), resulting in diminished β-AR signaling, and loss of contractile reserve. It has been reported that β-AR desensitization/down-regulation correlates with HF severity (11). These effects underlie the pathogenesis of HF (6).

β-AR desensitization in HF is caused by phosphorylation of β-ARs themselves by PKA and G-protein coupled receptor kinases (GRKs) (6). GRK activity has been found to be augmented by prolonged β-AR activation under catecholamine exposure. GRK levels are elevated in the setting of HF and contribute to its pathogenesis (36). In addition, GRK activation and β-AR uncoupling can lead to β-ARs (mainly β1-ARs) internalization (β1-ARs shifted from the sarcolemma membrane into cytosolic compartments), contributing to β-ARs down-regulation (6). It has been reported that prolonged stimulation of β-AR signaling can induce β-AR down-regulation even without HF (37).

In the Presence of Abnormal Ca2+ Handling, Further Enhancing Adrenergic Signaling Could Be Detrimental Rather Than Beneficial in HF

RyR2 dysfunction and Ca2+ leak in HF are caused by sustained adrenergic signaling (14). It would be obvious that further activation of the adrenergic signaling can further worsen RyR2 dysfunction and Ca2+ leak (Figure 1). In HF the Ca2+ leak is usually less at rest. With enhanced adrenergic signaling during sympathetic stress, the RyR2 dysfunction and Ca2+ leak would deteriorate (14). This would exacerbate cardiac dysfunction and promote arrhythmogenesis (15). As shown in the figure, the stronger the β-AR signaling, the worse the RyR2 dysfunction and Ca2+ leak, and thus more arrhythmogenesis. It could be speculated that if there were no β-AR desensitization/down-regulation in HF, there would be an even greater risk of developing arrhythmias and sudden cardiac deaths in HF patients due to persistent sympathetic activation and elevated catecholamine levels. Our recent findings that failing hearts are more vulnerable to develop arrhythmias under sympathetic stimulation (33, 34) support this concept. In line with this, β-AR agonist treatment is associated with increased mortality in HF (31, 32) and β-AR blocker treatment can reduce cardiac arrhythmias and sudden cardiac death in HF (35).

Thus, in the presence of RyR2 dysfunction and Ca2+ leak in HF, β-ARs desensitization/down-regulation, by diminishing adrenergic signaling, helps to attenuate RyR2 dysfunction and Ca2+leak, protecting the heart from developing serious cardiac arrhythmias and sudden cardiac death. In other words, β-ARs desensitization/down-regulation in HF may act in a way similar to a β-AR blocker, attenuating adrenergic signaling, which is beneficial especially under heightened sympathetic activation with persistent high-level of catecholamines seen in HF. From this point of view, β-AR desensitization/down-regulation in HF is a self-preserving, adaptive process (a friend) rather than a detrimental, pathological process (a foe), as conventionally considered.

Conclusion

β-AR desensitization/down-regulation is one of the many characteristic abnormalities seen in HF that is conventionally considered to be detrimental in HF progression (3, 11, 12). However, emerging evidence indicates that this concept may be too simplistic and may not be correct. Despite the fact that β-AR desensitization/down-regulation in HF diminishes adrenergic signaling and cardiac contractile reserve, this process may act as an intrinsic β-AR blocker, protecting the heart from developing more severe ventricular arrhythmias and sudden cardiac death. In line with this, new medications designed to re-sensitize/up-regulate β-ARs have to also normalize Ca2+ handling to be beneficial. Otherwise, simply enhancing adrenergic signaling without correcting RyR2 dysfunction (like β-AR agonists) may exacerbate RyR2 dysfunction and Ca2+ leak, increasing the arrhythmia risk and sudden cardiac death in HF. It should be noted that β-AR blocker treatment in HF is associated with normalization of RyR2 function and Ca2+ signaling (38), besides re-sensitizing/up-regulating the β-ARs.

Data Availability Statement

The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.

Author Contributions

YZ contributed to conception of the manuscript. AM and KA wrote the first draft of the manuscript. All authors contributed to manuscript revision, read, and approved the submitted version.

Conflict of Interest

The 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.

Publisher’s Note

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

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