Starch is a major storage carbohydrate in plants. It is an important source of calories in the human and animal diet. Also, it is widely used in various industries. Native starch consists of water-insoluble semicrystalline granules formed by natural glucose polymers amylose and amylopectin. The physicochemical properties of starch are determined by the amylose:amylopectin ratio in the granule and degrees of their polymerization and phosphorylation. Potato Solanum tuberosum L. is one of the main starch-producing crops. Growing industrial needs necessitate the breeding of plant varieties with increased starch content and specified starch properties. This task demands detailed information on starch metabolism in the producing plant. It is a complex process, requiring the orchestrated work of many enzymes, transporter and targeting proteins, transcription factors, and other regulators. Two types of starch are recognized with regard to their biological functions. Transitory starch is synthesized in chloroplasts of photosynthetic organs and degraded in the absence of light, providing carbohydrates for cell needs. Storage starch is synthesized and stored in amyloplasts of storage organs: grains and tubers. The main enzymatic reactions of starch biosynthesis and degradation, as well as carbohydrate transport and metabolism, are well known in the case of transitory starch of the model plant Arabidopsis thaliana. Less is known about features of starch metabolism in storage organs, in particular, potato tubers. Several issues remain obscure: the roles of enzyme isoforms and different regulatory factors in tissues at various plant developmental stages and under different environmental conditions; alternative enzymatic processes; targeting and transport proteins. In this review, the key enzymatic reactions of plant carbohydrate metabolism, transitory and storage starch biosynthesis,and starch degradation are discussed, and features specific for potato are outlined. Attention is also paid to theknown regulatory factors affecting starch metabolism Starch is the main storage carbohydrate in plants. Itconstitutes up to 85 % of the dry matter of their edibleparts: cereal grains , potato tubers Solanum tuberosum L., edible roots Lam., and yam Dioscorea alata L.),sago palm stems Metroxylon sagu Rottb., plantain fruitMusa spp. . Starch provides a great portion of calories forhuman and animal nutrition. In addition, it is a naturalreproducible and biodegradable material used in nonfoodindustry, e. g., in the production of fabric, paper,drugs, and plasticsChemically, starch is a mixture of amylose and amylopectin.These natural glucose polymers form waterinsolublesemicrystalline granules. Amylopectin consistsof highly branched glucan molecules, the linear regionsof which are formed by α-1,4-glycosidic bonds, whereasthe branching points are formed by α-1,6-bonds. Amyloseis a practically linear polymer with few branches.Amylopectin molecules constitute about 75–80 % ofstarch weight. They form the structural framework of thegranule, consisting of repetitive amorphous and semicrystallinelamellae. Amylose molecules are dispersed inthe semicrystalline amylopectin matrix .The amylose:amylopectin ratio determines the dietetic,physicochemical, and functional starch propertiesessential for particular industries. The starch presentin food is classified into glycemic and resistant. Beingreadily digestible in the small intestine, amylopectinincreases the glycemic potential of starch. In contrast,higher amylose contents make starch more resistant. Resistant starch is less degradable byamylases in the small intestine. It serves as substrate formicrobes in the large intestine to produce short-chainfatty acids, which exert local antiinflammatory and antitumoreffects . Also, physicochemical(gelatinization and retrogradation) and functional (swellingand viscosity) properties are taken into considerationin certain applications. These properties are determined genetically: by size and morphology of starch granules,amylose:amylopectin ratio, glucan branching, and glucanphosphorylation .Potato (Solanum tuberosum L.) ranks fourth amongstarch-producing crops in the world, next to maize,cassava, and wheat. Potato starch differs from cerealstarches in a variety of important features. Potato amyloseand amylopectin have higher degrees of polymerizationand phosphorylation; therefore, potato starchis more suitable for bioplastic production . In response to theincreasing commercial demand, the global production ofpotato starch steadily increases: 3.7 million tons in 2018and 3.9 million tons in 2020 . To obtain native starches withspecified properties and to increase the overall amountof starch per plant are topical tasks in potato breeding.The key enzymes in starch biosynthesis (see the Table)and their genes have been studied in detail in modelplants  and in crops, includingpotato .Starch is produced by the polymerization of ADPglucose,catalyzed by granule-bound (GBSS) and soluble(SS) starch synthases. Other enzymes involved arethe starch branching enzyme (SBE) and the debranchingenzyme (DBE).Starch metabolism is a component of general carbohydratemetabolism. It is essential for plant functions:growth, development, and stress response. The enzymesand genes associated with the metabolism of starch andother plant carbohydrates have been extensively studiedfor three decades. A considerable body of data on the locationof these genes in plant genomes and their expressionhas been accumulated. Seventy-five S. tuberosumgenes have been mapped on the reference genome,and the expression patterns of 64 genes in leaves andtubers have been studied . It has been shown that starchmetabolism genes experienced numerous duplicationsand produced paralogs by sub- and neofunctionalization during evolution. The paralogous genes, whichencode different enzyme isoforms, show tissue- and/orstage-specific expression patterns . Variousfactors affect the expression of starch metabolismgenes: circadian rhythms, photoperiod, levels of planthormones and sugars, and stressing factors (drought andcold) . Although the keyenzymatic reactions in starch biosynthesis and degradationare well known, there are many unclear pointsconcerning alternative enzymatic processes and theirlocalization inside the cell, roles of particular isozymesin starch metabolism in different organs at different developmentalstages, and the influence of regulatory factors.By now, functions of many proteins involved instarch metabolism in potato have been identified (seethe Table). Most enzymes have isoforms with partiallyoverlapping functions .Translocator proteins essential for transporting metabolitesthrough plastid membranes are important for starchmetabolism as well. They also have multiple forms:adenylate translocators NTT1 and NTT2 , glucose transporter pGlcT1 ,glucose 6-phosphate translocator  , maltose transporter MEX1 , and triosephosphate translocator  .The amylose:amylopectin ratio in plants can bemodified by raising lines carrying certain alleles ofgenes involved in starch synthesis. Such accessionswere obtained in cereals; for instance, in maize Z. mays.The amylose extender (ae−) mutation is associated withthe loss of the activity of the starch-branching enzymeSBEIIb. Starch in plants with the ae− phenotype isenriched with amylose, and its amylopectin chains arelonger . The maize phenotype whosestarch has practically no amylose is named waxy, andit is determined by a mutation in the gene for granuleboundstarch synthase GBSSI. Its endosperm is gluey.However, the breeding of potato varieties with specifiedstarch properties is complicated by its autotetraploidgenome. The only amylose-free potato cultivar Elianewas obtained by mutation-assisted breeding . Genetic engineering and genome editingaimed at the modification of key starch biosynthesisgenes produced plants with expected phenotypes:amylose-free starch (knockout and knockdown of theGBSS1 gene), amylose-enriched starch (knockdown of both SBE1 and SBE2), and starch with modified amylopectinproperties (editing of SBE1 and/or SBE2) .In some cases, plants with a desired phenotype acquiredadditional traits. For example, the increase inamylose content obtained by antisense suppression of thegenes for starch-branching enzymes SBE1 and SBE2 wasaccompanied by a decrease in starch content, formationof smaller granules, and larger tubers . Apparently, changes in certain starch metabolismsteps may affect the overall carbohydrate metabolismin the plant. An association study revealed genetic lociassociated with starch content and productivity (tuberweight), whereas the functions of some detected geneswere unknown at all, and some other genes were involvedin signaling and regulation: transcriptional andposttranscriptional . Part of thedetected SNPs exerted antagonistic effects on potatoproductivity and starch content .Thus, the investigation of regulation pathways of starchmetabolism genes is important for improving potatoquality and productivity.Two starch forms are recognized with regard to biologicfunction: transitory and storage. Transitory starch issynthesized and accumulated in chloroplasts of photosyntheticorgans (leaves) in the daytime and degraded indarkness to provide nutrients for the cell. Storage starchis synthesized in amyloplasts (nonphotosynthetic plastids)of storage organs  and storedthere for a long time to be utilized in the preparation forsprouting .The main difference in carbohydrate metabolism betweencells of leaves and storage organs is in the sourcesof carbohydrates and ATP required for starch synthesisand enzymatic reactions. In leaves, they can form in thesame cells that produce transitory starch, and in storageorgans, they are imported from photosynthetic ones.During photosynthesis, chloroplasts produce ATP andfix atmospheric carbon dioxide by the Calvin–Bensoncycle, in which triose phosphate is produced as an intermediate. Part of triose phosphatemolecules remain in the chloroplast stroma to serve ascarbohydrate material for transitory starch synthesis.The rest is transported to cytosol by triose phosphatetranslocator TPT . In chloroplasts,a series of enzymatic reactions converts triose phosphateto glucose-1-phosphate (G1P). Then ADP-glucosepyrophosphorylase (AGPase) converts G1P to ADPglucose,the main substrate for starch synthesis .As triose phosphate molecules are exported to cytosol,they are converted to sucrose, which is then deliveredto storage organs through phloem and apoplast to be acarbohydrate material for storage starch synthesis. Sucroseis transported into cells of storage organs either bysucrose transporter proteins or, after being hydrolyzed byinvertase to glucose and fructose, by hexose transporters. There are two pathways to cleave sucrosein cell cytoplasm: saccharolytic, catalyzed by sucrosesynthase SuSy, or hydrolytic, catalyzed by invertase Inv.Invertase irreversibly cleaves sucrose to glucose andfructose, and SuSy catalyzes the reversible cleavage tofructose and UDP-glucose . Thepredominant pathway depends on the tuber developmentstage. At the beginning of growth, at a high cell divisionrate, the hydrolytic pathway prevails, and the saccharolyticpathway steps forward at the starch accumulationstage . The SuSy-catalyzedpathway is important for the rate of starch accumulationin potato. It has been shown that a decrease in SuSy activityreduces starch content in mature tubers . Seven SuSyisoforms have been predicted in potato, and the SuSy4gene had tissue-specific expression in growing tubers. It is likely that somesucrose synthase isoforms are involved in sucrosetransport through phloem, as observed in A. thaliana.The subsequent steps, catalyzed by fructokinase FK,hexokinase HK, and UDP-glucose pyrophosphorylase,produce a pool of phosphorylated hexoses in cytosol.They can be reversibly interconverted by cytosolicisoforms of phosphoglucoisomerase PGI2 and phosphoglucomutasePGM2 . One of the phosphorylated hexoses, glucose-6-phosphate, is transported to amyloplasts by membraneglucose-6-phosphate/phosphate-translocator GPT . In amyloplasts, G6P is utilized forADP-glucose production. The rate of starch biosynthesisin potato tubers directly depends on G6P transport .ATP is also imported to storage tissues from photosyntheticones. It is delivered inside amyloplasts by plastidadenylate translocator NTT. It is known that even a slightdecrease in NTT activity reduces the overall starch contentin potato tubers , whereas thecombination of NTT and GPT overexpression increasesit . There may be an alternativeroute of hexose transport to amyloplasts: direct importof glucose-1-phosphate (G1P) and its utilization inADP-glucose synthesis. There is evidence that this routeacts in potato, although the corresponding transporter protein is not known yet . Two candidateG1P transporters through plasma membrane wererecently found in A. thaliana .ADP-glucose acts as the substrate for starch biosynthesis.It is produced in a reversible reaction catalyzedby ADP-glucose pyrophosphorylase (AGPase) in thestroma of chloroplasts and amyloplasts. AGPase synthesizesADP-glucose and pyrophosphate (PPi) fromG1P and ATP. AGPase is a heterotetramer consisting oftwo large and two small subunits, AGPL and AGPS. Itsactivity is essential for starch synthesis in potato tubers. Inorganic pyrophosphatase(PPase) degrades pyrophosphate to orthophosphate. The plastid PPase isoform contributes much to starch accumulation in potato tubers.Lines knocked down for the StpsPPase gene had lowercontents of starch, in particular, amylose, and smallergranules. That study also recorded elevated amounts ofstarch biosynthesis intermediates: pyrophosphate, glucose,fructose, hexose phosphates, and, unexpectedly,ADP-glucose. The increase in ADP-glucose contentindicates that pyrophosphate does not affect the directionof the AGPase-catalyzed reaction in potato. Thus,the mechanism by which PPase participates in starchsynthesis in potato tubers is still to be understood .An alternative cytosolic pathway of ADP-glucosesynthesis, catalyzed by SuSy and UGPase, acts in cereals(monocots). It is essential for grain growth. ADPglucoseis transported to amyloplast by the Brittle1-liketransporter protein (BT1) . Thehomolog protein of BT1 (StBT1) has been found in S. tuberosumplant. However, there is no evidence for ADPglucosetransport through the amyloplast membrane.The StBT1 protein performs unidirectional transport ofAMP, ADP, and ATP .Transitory starch synthesis in leaf chloroplasts and storagestarch synthesis in tuber amyloplasts follow basicallythe same route. Amylose and amylopectin synthesis isperformed by 16 key enzymes belonging to the followinggroups: starch synthases, starch-branching enzymes,and starch-debranching enzymes (see the Table). Mostenzymes exist as isoforms, the functions of which maypartly overlap .Starch synthases catalyze the formation of glycosidicbonds by transferring the glucose residue of ADP-glucoseto the nonreducing end of the glucose polymer.They are subdivided into granule-bound (GBSS) andsoluble (SS) starch synthases. The former synthesizelong chains, mainly in amylose, and long chain fragmentsin amylopectin. The latter include a series ofisoforms: SS1, SS2, SS3, SS4, SS5, and SS6. Of them,SS1, SS2, and SS3 synthesize chains of various lengthsin amylopectin . The SS4 isoformperforms a special function among starch synthases, as itinitiates starch granule formation .One or two large starch granules instead of five to sevenwild-type small ones were found in A. thaliana plantswith the knocked out ss4 gene . Thefunctions of SS5 and SS6 are still vague. The C end ofthe SS5 protein lacks the conservative fragment characteristicof starch synthases, which has catalytic domainGT1, although the protein has the conservative glucanbindingsite. Probably, SS5 is involved in starch granule initiation, as it has been shown that the loss of SS5 fromA. thaliana reduces the granule number in leaves . The SS6 isoform and its gene were found inpotato in recent years , andthe role of this enzyme is unknown. It may participatein granule growth, as it is directly bound to it; in addition,it bears conservative motifs XXGGL and KXGGL,characteristic of glycosyl transferase domains of starchsynthases GT1 and GT5, respectively .Starch granule initiation was an obscure issue fora long time. The studies reported by now concern transitorystarch initiation in the model plant A. thaliana,but it seems that our notion of some key steps in granuleformation may be extended to other plant species. As mentioned above, SS4 is themain granule-initiating enzyme, and SS5 and SS6 alsotake part in the process. Maltooligosaccharides, probablyforming in the degradation of starch polyglucansby amylases, are the substrate .The steric interaction of starch synthases, substratemolecules, and the growing granule is driven by thePTST2 and PTST1 proteins, targeting to starch. Theyare associated with starch synthases SS4 and GBSS1,respectively. Also, they contain a carbohydrate-bindingdomain . Note that PTST2 isnot found in potato tubers, and this fact indicates thatthe starch granule initiation processes in A. thaliana andpotato differ . A heteromultimericcomplex of isoamylases ISA1 and ISA2 has been shownto influence starch granule initiation in potato tubers.By all appearances, isoamylases suppress the formationof new starch granules by disrupting the formation ofsoluble glucan molecules in chloroplast stroma .As the chains of amylose and amylopectin moleculesare elongated by starch synthases SS1, SS2, and SS3,starch-branching enzymes SBE attach side branches tothem . Starch-branching enzymescleave α-1,4-glycosidic bonds of polyglucans, synthesizedby starch synthases, and attach short chains to theso-called acceptor chain by forming α-1,6-glycosidicbonds. The starch-branching enzymes of potato havethree isoforms: SBE1.1, SBE1.2, SBE2, and SBE3(formerly designated as SBE1) (see the Table) . Thus, forms referred to in otherpapers as SBE1 and SBE2 are designated as SBE3 andSBE2 according to the notation of Van Harsselaar et al.SBE3 produces mainly long side chains, and SBE2 producesshort amylopectin chains .The roles of SBE1.1 and SBE1.2 in starch productionare unknown, but studies of A. thaliana demonstratea pleiotropic effect of SBE1 on plant growth and development.Transformants overexpressing SBE1 were white-colored and low. They had a longer life cycle andproduced fewer seeds than control plants . The joint action of different isoforms affectsamylopectin structure. Experiments with potato plantswith the knocked out genes sbe3 and/or sbe2 (designatedby the experimenters as sbe1 and sbe2) demonstrate thatsbe3 inactivity results in the formation of starch withlonger amylopectin chains and lower branching level.The knockout of sbe2 with active sbe3 did not affect theamylopectin structure much, but the number of starchgranules in potato tubers increased and size decreased.Debranching enzymes (DBE) are another group ofenzymes involved in the formation of the amylopectinstructure (see the Table). They reconstruct branchedglucans into easier crystallizable forms, which is essentialfor granule formation .Debranching enzymes include isoamylases (ISA), whichcatalyze the hydrolysis of α-1,6-glycosidic amylopectinbonds and remove excessive branching. Potato isoamylasesinclude isoforms ISA1, ISA2, and ISA3. The ISA1and ISA2 proteins can form heteromultimers, capableof more efficient removal of long outer chains of amylopectin. The ISA3 isoenzymeis important for starch degradation, as it cleaves shortouter chains of glucans . Transgenicpotato plants with lower expression of the isa1, isa2,and isa3 genes had significantly less starch in developingtubers, whereas the starch contents in leaves did notchange. The plants also had fewer and larger granulesand higher sucrose contents, probably resulting from theincrease in the overall granule surface and easier accessfor degrading enzymes .In addition to starch synthases, branching and debranchingenzymes, the synthesis of starch granulesinvolves α-glucan phosphorylases. Their plastid (PHO1)and cytoplasmic (PHO2) isoforms catalyze the reversibletransfer of the glycosylic group of glucose-1-phosphateto the nonreducing end of the chain of an α-1,4-boundglucan . The PHO2 enzyme isinvolved in carbohydrate metabolism in cytoplasm, andPHO1 contributes to starch synthesis and degradationin plastids. It has been shown that at lower temperaturesstarch synthesis in potato tubers can also followthe phosphorylase pathway with G1P as the substrate.Degradation is an intrinsic part of the metabolism ofstarch and carbohydrates in general, although it hasbeen studied much poorer than starch biosynthesis.The degradation pathways of transitory starch havebeen investigated in most detail in leaves of the model plant A. thaliana. The knowledge of starch degradationin potato tubers is limited to cold-induced sweeteningand sprouting. The main steps of starch degradationare the release of soluble glucan from starch granules,glucan conversion to linear forms ,maltooligosaccharide hydrolysis to maltose,and subsequent maltose metabolism in the cell. Starchdegradation is performed by a broad range of enzymes:α- and β-amylases, isoamylase, α-glucan water dikinase(GWD), phosphoglucan water dikinase (PWD),α-glucan phosphorylase, phosphoglucan phosphatase,and 4-α-glucanotransferase (see the Table).Starch granule degradation is initiated by GWD andPWD. They phosphorylate glucans at positions C6 andC3 of glucose residues, making them more hydrophilicand allow α-, β-, and isoamylases access to them (seethe Table) . Thephosphorylation by GWD seems to play the key role instarch degradation in potato tubers and leaves . Tubers oftransgenic potato plants with lower expression of theStGWD gene were less prone to starch degradation atlow temperatures .The next step of starch granule degradation is glucanhydrolysis by amylases. Potato α- and β-amylasesinclude many isoforms, and functions of some of themare not known in detail (see the Table) . By extrapolating data on A. thaliana, wesuppose that β-amylases BAM1 and BAM3 hydrolyzelinear fragments of amylose and amylopectin, andthe degradation of branched fragments demands thedebranching enzyme DBE (ISA3 in potato) .With knocked down StBAM3, starch content in potatoleaves was higher than in the wild genotype . Cold-induced sweetening in potato tubersis also affected by some amylase species: α-amylaseAMY2 (AMY23) and β-amylases BAM1 and BAM9.The supposed function of BAM1 and BAM9 is starchdegradation in plastids, and AMY2 is likely to degradephytoglycogen in cytosol . An alternativepathway of starch degradation is observed inA. thaliana. It is initiated by α-amylase AMY3, whichreleases linear and branched glucans from starch granules,and these glucans are then hydrolyzed by β- andisoamylases  .Alongside starch glucan hydrolysis by amylases,the glucans are dephosphorylated by phosphoglucanphosphatases SEX4 (Starch Excess) and LSF2 (LIKESEX FOUR2), first described in A. thaliana. Theseprocesses are interrelated: phosphorylation by dikinasesincreases granule solubility and makes them accessiblefor amylases, whereas phosphate moieties may hamper hydrolysis .Reduction of SEX4 or LSF2 activities in potato inhibitedstarch degradation in leaves. Starch content in tubersremained unchanged, and granules were smaller andless phosphorylated .The cooperation of dikinases, amylases, and phosphatasesproduces a pool of soluble maltooligosaccharides(linear glucans). Maltooligosaccharides are degradedby two pathways: hydrolytic, by β-amylases, or phosphorolytic,by α-glucan phosphorylase PHO1 . The end product of thephosphorolytic pathway is G1P, which can be utilizedin metabolism inside the plastid. Also, glucose can beproduced by 4-α-glucanotransferase DPE1 (DisProportionatingEnzyme) and exported to cytosol by glucosetransporter pGlcT1 . Knockdown of the chloroplast enzyme DPE slowsdown starch degradation in potato leaves in the cold andinduces maltooligosaccharide accumulation, althoughthese effects are not observed in tubers . Cold-induced sweetening in potato tubers is accompaniedby increasing β-amylase activity and highermaltose content .Maltose, which is the predominant product of hydrolyticstarch degradation, is exported to cytosol by thetransmembrane transporter MEX1 . Incytosol, maltose is processed by 4-α-glucanotransferaseDPE2 or phosphorylase PHO2 to glucose or G1P, whichare then converted to sucrose by the joint action of PGI2,PGM2, HK, UGPase, SPS (sucrose phosphate synthase),and SPP (sucrose phosphate phosphatase) .Sucrose is exported from leaf cells to storage organs;also, it is used in cell metabolism. In potato tubers,sucrose is used as a source of nutrients in sprouting,and its level controls dormancy release .To delay sprouting, potato tubers are stored at lowtemperatures, 2–5 °C, and these conditions initiatecold-induced sweetening. This process involves sucrosehydrolysis by vacuolar acid invertase AcInv, encodedby the Pain-1 gene, and the accumulation of reducingsugars (glucose and fructose) in tubers . Knockout of Pain-1 resulted inlower contents of reducing sugars .One of the key regulators of cold-induced sweeteningis invertase inhibitor SbAI, which inhibits AcInv. It has been shown that SbAIcan also inhibit α- and β-amylases , in potato tubers, thereby influencing therate of starch degradation in cold-induced sweetening.Starch metabolism requires orchestrated work of manyenzymes, transporters, and targeting proteins, whichimplies many regulation levels: gene expression, posttranscriptionalregulation, and the posttranslationalregulation of enzymatic activity. The expression patternsof genes for key enzymes involved in starch metabolismare well known in various plant species, but much lessis known about expression-regulating factors . The difficulty is that the starchmetabolizingenzymes exist as numerous isoforms,which are encoded by the corresponding number ofparalogous genes. The expression patterns of these genesdepend on tissue  and developmental stage, as shown in A. thalianaand maize . In potato,the tissue-specific mode of expression has been shownfor SuSy4, SS5, SBE3, APL3, PHO1a, PHO1b, GPT1.1,GPT2.1, SEX4, and NTT2 in tubers and for AMY1.1,APL1, and BAM3.1 in leaves . A number of external and internal factors affectthe expression of starch biosynthesis genes: circadianrhythms, photoperiod, and sugar content . It is known that the expressionrates of GBSSI, LSF1, LSF2, SEX4, and BAM3 inA. thaliana leaves are governed by transcription factorsdepending on circadian rhythms and photoperiod, so thatthe demand for energy is rapidly met in response to ambientchanges . Theexpression rates of the genes GBSS, SuSy, and AGPaserespond to photoperiod in growing potato tubers, beinghighest in the end of the light time and lowest in thebeginning. This variation is determined by the influx ofphotoassimilates from leaves .The formation of the storage organ, tuber, from thestolon is an important step in potato plant development.It includes intense starch production, the formation ofstarch granules, and increase in metabolite flux. Tuberformation is a complex process, influenced by environmentalfactors (photoperiod) and a variety of signals:biochemical, hormonal, and molecular, mediated bymicroRNAs and transcription factors . The investigation of tuberformation contributed much to the understanding ofmechanisms that regulate starch metabolism in potatotubers.Plant hormones are an important factor influencingthe expression of genes involved in starch metabolism,and their effect on tuber formation has been studied insufficient detail. The level of abscisic acid correlateswith starch accumulation in potato tubers . Treatment of stolons with indole acetic acid increased starch content in growing tubers, buta twofold increase in concentration caused the oppositeeffect . A correlation between thetranscription rates of the genes for auxin, on the onehand, and starch biosynthesis , on the other hand, was observed in theformation of cassava storage roots .Sugars  are anothergroup of signaling molecules influencing the expressionof starch metabolism genes. Sucrose increases theexpression of the SuSy and AGPase genes in potato.The rates of SuSy and AGPase expression are high ingrowing tubers, but they decrease rapidly after the separationof the tuber from the plant and, correspondingly,cease of sucrose import from photosynthesizing organs.The differential expression of starch biosynthesisgenes was detected at various tuber development stages. Theexpression rate of the SS4 gene was elevated at the stolonstage, and it lowered with tuber growth, confirming therole of this starch synthase in granule initiation . Also, tuber growth was accompanied byan increase in the expression rate of sucrose synthaseSuSy4 and decrease in the expression of cell wall invertasecw-Inv. These changes point to transition to thesucrose synthase-mediated pathway of sucrose degradation.Genes for glucose-6-phosphate translocator GPT,adenylate translocator NTT, ADP-glucose pyrophosphorylase(AGPase), starch synthases, and starch-branchingenzymes increased their expression with tuber growth.Of this group, the isogenes SuSy4, SBE3, and GPT2.1demonstrated just tuber-specific expression . Coexpressionanalysis was employed to investigate the mechanismsof molecular regulation of gene activity, and transcriptionfactors LOB, TIFY5a, and WRKY4 were foundto be associated with the expression of the SuSy4 andGPT2.1 genes . Analysisof coexpression networks for starch biosynthesis genesof seven plant species  revealed the involvementof 24 transcription factors .Little is known about mechanisms regulating starchmetabolism genes at the posttranscriptional step. Posttranscriptionalregulation involves a variety of factors,including RNA-binding proteins (RBPs), microRNAs,and alternative splicing, so that plants can rapidly reprogramtheir transcriptomes in response to externaland internal factors. Photoperiod significantly influences microRNA expression patterns in the growth anddevelopment of potato tubers. It has been shown thatdifferentially expressing microRNAs are targeted togenes coding for transcription factors and RNA-bindingregulatory proteins StGRAS, StTCP2/4, and StPTB6.Posttranslational regulation is the next step of proteinactivity control. It is mediated by allosteric regulation, inwhich an effector molecule is bound to a noncatalytic siteof the enzyme, altering its conformation, catalytic properties,and, thereby, its specificity and interaction withother proteins . Allosteric regulationinvolves protein phosphorylation and the formationof multimeric complexes and disulfide bridges . Many starch-metabolizingenzymes assume the phosphorylated state: PGI,PGM1, AGPase, SS3, GWD1, GWD2, DPE2, AMY3,BAM1, BAM3, LDA, pGlcT, and MEX1 . ADP-glucose pyrophosphorylase (AGPase)is a clear example of allosterically regulated potatoenzyme. It is activated by 3-phosphoglyceric acid andinhibited by inorganic phosphate . Depending on the redox state in the cell, AGPasecan be reversibly inactivated by the formation of disulfidebridges between small subunits of the heterotetramer.Enzymes can aggregate into complexes known asmetabolons . Complexesformed by starch biosynthesis enzymes were found in theendosperm of growing cereal seeds; in particular, SSIII,SSIIa, SBEIIa, and SBEIIb form a protein complex . Protein complexes of PTST2 and SS4form in the initiation of starch granules in A. thalianaleaves . Potato isoamylasesISA1 and ISA2 form a heterotetrameric complex, whichcontrols starch granule formation .The investigation of starch metabolism in potato plants,particularly, starch biosynthesis and degradation in tubers,is topical in connection with the growing demandfor potato starch in industry. A large body of informationon key enzymes for starch and carbohydrate metabolismin various crops and the model species A. thaliana hasbeen accumulated in the past three decades. Althoughthe starch biosynthesis scheme is basically the samein different species, there are significant variationsassociated with different sets of isozymes, features oftheir functions, metabolite transport pathways , and the existence of intricate and multileveledregulation, governed by external (photoperiod andtemperature) and internal  factors. Isogenesencoding six starch synthase isoforms, seven sucrosesynthases, nine β-amylases, and three to five for eachof the starch-branching and other enzymes were identifiedin the potato genome .The functions of many isoforms, including the majorityof α- and β-amylases, are still unknown. Someisogenes  demonstrate tuberspecificexpression and activity variation at variousstages of tuber formation . Isoenzymes AMY23, BAM1,BAM9 are specifically involved in starch degradationand carbohydrate metabolism in cold-induced sweetening. Also, the action of various factorson starch accumulation during tuber development hasbeen shown: transcription factors LOB, TIFY5a, andWRKY4; plant hormones (auxin and abscisic acid);sugars; and microRNAs, the contents of which maymediate the effect of photoperiod. However, the functionsof many isoenzymes and proteins involved in theregulatory and directing functions in starch metabolismin potato plants are poorly explored. To resolve this issue,modern methods are proposed: combined analysisof the metabolome and transcriptome inside a singlecell or tissue . Bottom-upproteomics also seems promising in search for new components. For example, the analysisof 36 proteins associated with potato starch granulesrevealed, in addition to already known starch metabolismenzymes, targeting and regulatory proteins describedin A. thaliana: PTST1 (Protein Targeting to Starch),ESV1 (Early StarVation1), and LESV (Like ESV). Also,Kunitz-type proteinase inhibitor and enzymes involvedin redox regulation (thioredoxin TRX and glutathioneperoxidase GPX) were found . Detailedinformation on all components involved in starchmetabolism and on their interactions, including theirbehavior under varying ambient conditions, is essentialfor raising potato varieties with high performance andspecified starch properties.The authors declare no conflict of interest.Abt M., Pfister B., Sharma M., Eicke S., Bürgy L., Neale I., Seung D.,Zeeman S.C. STARH SYNTHASE5, a noncanonical starch synthase-like protein, promotes starch granule initiation in Arabidopsis.Plant Cell. 2020;32(8):2543-2565. DOI 10.1105/tpc.19.00946.Andersson M., Melander M., Pojmark P., Larsson H., Bülow L.,Hofvander P. Targeted gene suppression by RNA interference: anefficient method for production of high-amylose potato lines. J. Biotechnol.2006;123:137-148. 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DOI 10.1104/pp.123.1.319.Yu T.S., Zeeman S.C., Thorneycroft D., Fulton D.C., Dunstan H.,Lue W.L., Hegemann B., Tung S.Y., Umemoto T., Chapple A.,Tsai D.L., Wang S.M., Smith A.M., Chen J., Smith S.M. α-Amylaseis not required for breakdown of transitory starch in Arabidopsisleaves. J. Biol. Chem. 2005;280(11):9773-9779. DOI 10.1074/jbc.M413638200.Zeeman S.C., Kossmann J., Smith A.M. Starch: its metabolism,evolution, and biotechnological modification in plants. Annu.Rev. Plant Biol. 2010;61:209-234. DOI 10.1146/annurevarplant-042809-112301.Zhang H., Liu J., Hou J., Yao Y., Lin Y., Ou Y., Song B., Xie C. The potatoamylase inhibitor gene SbAI regulates cold-induced sweeteningin potato tubers by modulating amylase activity. Plant Biotechnol. J.2014;12(7):984-993. DOI 10.1111/pbi.12221.Zhang L., Häusler R.E., Greiten C., Hajirezaei M.R., HaferkampI., Neuhaus H.E., Flügge U.I., Ludewig F. Overridingthe co-limiting import of carbon and energy into tuber amyloplastsincreases the starch content and yield of transgenic potatoplants. Plant Biotechnol. J. 2008;6(5):453-464. DOI 10.1111/j.1467-7652.2008.00332.x.Zrenner R., Salanoubat M., Willmitzer L., Sonnewald U. Evidence ofthe crucial role of sucrose synthase for sink strength using transgenicpotato plants (Solanum tuberosum L.). Plant J. 1995;7(1):97-107.DOI 10.1046/j.1365-313x.1995.07010097.x.


Osteopathic manipulative treatment (OMT) is used in both inpatient and outpatient settings. Evidence suggests that OMT can reduce both patients’ recovery time and the financial cost of their acute medical treatment and rehabilitation. Multiple studies from neonatal intensive care units (NICUs) are presented in this article that demonstrate infants treated with OMT recover faster, are discharged earlier, and have lower healthcare costs than their non-OMT-treated counterparts. Data clearly show that adjunctive OMT facilitates feeding coordination in newborns, such as latching, suckling, swallowing, and breathing, and increases long-term weight gain and maintenance, which reduces hospital length of stay (LOS). Osteopathic techniques, such as soft tissue manipulation, balanced ligamentous tension, myofascial release, and osteopathic cranial manipulation (OCM), can reduce regurgitation, vomiting, milky bilious, or bloody discharge and decrease the need for constipation treatment. OMT can also be effective in reducing the complications of pneumonia in premature babies. Studies show the use of OCM and lymphatic pump technique (LPT) reduces the occurrence of both aspiration and environmentally acquired pneumonia, resulting in significantly lower morbidity and mortality in infants. Based on published findings, it is determined that OMT is clinically effective, cost efficient, a less invasive alternative to surgery, and a less toxic choice to pharmacologic drugs. Therefore, routine incorporation of OMT in the NICU can be of great benefit in infants with multiple disorders. Future OMT research should aim to initiate clinical trial designs that include randomized controlled trials with larger cohorts of infants admitted to the NICU. Furthermore, a streamlined and concerted effort to elucidate the underlying molecular mechanisms associated with the beneficial effects of OMT will aid in understanding the significant value of incorporating OMT into optimal patient care. Osteopathic manipulative medicine (OMM) is an evidenced-based comprehensive approach to healthcare in which osteopathic physicians apply osteopathic philosophy, structural assessment, and the use of osteopathic manipulative treatment (OMT) in the diagnosis and treatment of patients by emphasizing that structure and function are integrated in health and disease states. Indeed, the science and art of OMM is becoming increasingly popular in the healthcare field, primarily due to its clinical effectiveness and minimally invasive nature ,2,3. OstThe use of OMT in neonatal intensive care units (NICUs) has demonstrated tangible successes in the treatment of pneumonia, ankyloglossia, feeding dysfunction, and gastrointestinal distress in preterm babies as well as in assisting with weight gain and maintenance of newborns after discharge. A recent prospective multicenter observational study (OSTINF Study) found that osteopathic treatment is associated with major positive changes in the severity of health complaints in neonates and infants . TherefoThe primary basis of osteopathic medicine is the belief that somatic dysfunctions can result in associated conditions impacting peripheral nerves, blood flow, immune system efficacy, and organ function. In general, the goal of osteopathic treatment is to realign the body and motivate self-healing at both a superficial and deeper level by manipulating the fascia ,7. FasciFurthermore, because fascia is part of the interstitial space, which is a primary source of lymph and a major fluid compartment in the body, it is most likely that the effects of osteopathic manipulations on the fascia also affect the fluid dynamics at the applied tissue sites and reduce the edematous condition and inflammatory activity . In addiIn the following sections, a brief overview of common OMT techniques used in the NICU setting are provided, followed by clinical findings of their benefits from around the globe. These findings demonstrate the effectiveness of OMT in treating neonates and infants and provide evidence that adjunctive OMT can significantly decrease the cost of medical care and the length of hospital stay (LOS). The following five OMT techniques are demonstrated to be safe and effective in neonates and infants admitted to the NICU: (A) lymphatic pump technique (LPT); (B) diaphragm treatment (DPT); (C) balanced ligamentous tension (BLT); (D) myofascial release (MFR); and (E) osteopathic cranial manipulation (OCM) .The lymphatic system, which is essential for fluid balance, fatty acid absorption, and immune system regulation, plays a crucial role in collecting and directing tissue fluid, immune cells, antigens, pathogens, and proteins from tissue interstitial space and delivering them to regional lymph nodes . InternaDiaphragm, the main inspiratory muscle, generates craniocaudal movement of its dome during contraction and relaxation . The thoThis is a category of treatments involving steady pressure, stroking, stretching, kneading, and separation of tissue origin and insertion, which is achieved by targeting the myofascia using different manipulative treatments. Indeed, a clear identification of myofascial dysfunction requires complete patient history and physical evaluation based on the presenting complaint. Studies show that a series of osteopathic treatments, whether soft tissue, myofascial release, or cranial techniques, can significantly improve motor function in children with moderate to severe spastic cerebral palsy . TherefoLigaments connect bone to bone and, as such, span the joints of the body. Balanced ligamentous tension (BLT) is an indirect and passive technique that targets strain within ligamentous structures. BLT can target joints of the pelvis, spine, shoulders, knees, elbows, etc. In general, BLT technique uses the principle that disengagement of the diagnosed somatic dysfunctions can relieve underlying pain and morbidity . In addiMyofascial release (MFR) can be performed both as a direct technique, by engaging restrictive barriers, and as an indirect technique, by moving away from barriers, with balanced tension. The MFR technique manipulates the myofascial complex to release binding tissues that are impeding normal muscle function. This will help stimulate the body’s healing processes, which facilitates improvements in restriction, restores function, and decreases patient discomfort, all of which improves overall health . AlthougIn addition to dysfunctions within the peripheral nervous system, osteopathic physicians are well trained to provide balance to the central nervous system (CNS) through osteopathic cranial manipulation (OCM). The benefits are clearly evident in neonates, especially those manifesting plagiocephaly, or misshapen head, which results from abnormal distortion of the cranial bones that can occur in utero, during birth, or afterward. Plagiocephaly and feeding difficulty are two of the most commonly treated presentations in the NICU. Indeed, one of the most important uses of OCM is to correct plagiocephaly ,28. CranThe reliability of diagnostic procedures for early brain dysfunction and the efficacy of OCM in correcting them in the long term has also been shown recently . OCM canFollowing delivery, several factors contribute to the need for extended length of stay (LOS) in the NICU ,32. HistInfants and elderly are at the highest risk of respiratory tract infections and disease progression toward lung consolidation and pneumonia. Indeed, respiratory infections are the leading infectious cause of hospitalization and death among elderly and a leading cause of hospitalization in children . This inIn this respect, the application of OMT can be of significant benefit for the prevention of both hospital-acquired  and community-acquired pneumonia (CAP) . AdjunctBoth ankyloglossia and cranial nerve dysfunctions, and the associated restrictions in tongue movements, are common in pediatric patients. Ankyloglossia is the result of an unusually short frenulum  or accumulation of excessive tissue in the same region ,60. The The ability of infants to regulate their temperature, stabilize breathing, and gain weight is the most common criterion for NICU discharge . Consequp = 0.042) was documented in the osteopathic group compared to the control group, with a greater effect observed in infants with very low birth weight [One of the most significant factors that prolongs LOS is delayed transition from gavage to nipple feeding, which has been shown to decrease in premature infants who received OMT ,67. Cerrh weight . Therefon = 162) and limited demographic information, which may have an impact on overall external validity. Stress-induced damage to the gastric mucosa occurs frequently in neonates . Numeroup < 0.001). Cicchitti et al. (2020) [In another multicenter, randomized, controlled trial, newborns were assigned to either standard prenatal care (control group) or OMT (treatment group), and the effects on LOS were documented . The pri. (2020)  also dem. (2020) ,68,70.In 2007, the economic burden of preterm infant care in the NICU was approximately $5.8 billion , accountPublished findings from NICU clinics around the world clearly support the fact that OMT has the potential to reduce overall cost of neonatal health services by shortening the need for infants to continue to stay in the NICU. Cumulative data from numerous studies demonstrate the ability of OMT to expedite NICU discharge and are discussed more in depth in the following sections, and the comparative data analysis from three of these publications is provided in a bar graph .p < 0.001) for the OMT study group and an associated $1816.22 (€1586.01) (p < 0.001) cost reduction per infant. Altogether, OMT saved a total of $572,575.00  over the 14 months the study was active [Substantive evidence for a positive impact of OMT in reducing the need for hospitalization comes from Cerritelli et al. (2015), which involved the inclusion of OMT into treatment plans in preterm infants admitted to the NICU . This sts active . It is tn = 652) and a control group who received only standard care (n = 597). Nearly 49% of the babies were preterm infants . The findings showed that the experimental group had undergone a marked weight gain of 83 g vs. 35 g for the control group (p < 0.001). The findings also showed that infants in the full-term group required less OMT sessions versus the preterm infant subgroup . Further breakdown of the preterm subgroup showed that those born at 27–31 weeks of gestation required seven sessions on average in contrast to four sessions for babies born at 32–33 weeks. Additional study findings included a reduction in LOS between premature babies  in the OMT group as compared to those in the control group. Overall, the preterm infants who received OMT had a mean LOS of only 27.5 days versus as much as 61.3 days in the non-OMT group (control). However, a similar trend was not observed in term infants [A recent study by Cicchitti et al. (2020), included another large cohort of NICU admissions and utilized retrospective data collected from 2008 to 2016 at the Santo Spirito Hospital in Pescara, Italy . The stu infants . Pizzolorusso et al. (2014) demonstrated that the application of two osteopathic treatments to moderate preterm and late preterm infants (32–36 weeks gestation) resulted in average savings of $777.11 (€740.00) per day per infant by reducing the duration of their hospitalization . Additiop < 0.3). This equated to 6 days of hospital stay reduction for preterm infants treated with OMT. With respect to cost, the experimental group had net savings of $3387.80 (€2958.39) per preterm per length of stay as their total cost was $5143.68 (€4491.71) for the OMT group vs. $8531.47 (€7450.09) for the control group. By the end of the 15-month study period, the experimental group had saved an estimated $159,226.58  [Similar reductions in hospital stay have been observed by Cerritelli et al. (2013) who recruited eight osteopathic physicians to perform routine 20 min osteopathic examinations and treatments to NICU patients at Santo Spirito Hospital in Pescara, Italy . Infants,044.30) . It is noteworthy that there are significant limitations in the above studies, especially the small effect sizes and lack of heterogenous population. Furthermore, most of the afore-mentioned studies were performed within single hospital settings, often within the same country. Only a small number of these studies on OMT in the NICU used data from multiple hospitals or care centers. Therefore, future research should seek participation from a variety of institutions to enable larger population sizes and more diverse demographics. Such criterion-compliant research can also help substantiate the claim that OMT significantly benefits neonates and allows for shorter and more affordable hospital stays. In addition, the reader is encouraged to refer to another of our published article in this Special Issue of Medicines, which provides a more comprehensive review of various OMT approaches and their therapeutic uses .This review article discusses data from multiple clinical studies that clearly demonstrate the therapeutic benefits of OMT in decreasing both recovery time and the financial cost of medical treatment and rehabilitation in the NICU. Studies show that adjunctive OMT helps feeding coordination in newborns and decreases the need for constipation treatment. Furthermore, OMT can be highly effective in reducing the complications of both aspiration and environmentally acquired pneumonia in premature babies. Five OMT techniques are most frequently used in the NICU setting, namely, lymphatic pump techniques, diaphragm treatment, balanced ligamentous tension, myofascial release, and osteopathic cranial manipulation, which have been discussed at length. The multi-institutional and randomized OSTINF Study found that OMT is associated with significant reductions in the severity of health complaints in neonates and infants . HoweverFurthermore, development of both in vitro and in vivo models to study the beneficial effects of OMT and the myofascial changes occurring post-OMT would be of significant translational value . Current


Pathological cardiac remodeling as an aftermath of a severe cardiac injury can lead to ventricular dysfunction and subsequent heart failure. Adamts4, a metalloproteinase, and disintegrin with thrombospondin-like motif, involved in the turnover of certain extracellular matrix molecules and pathogenesis of osteoarthritis, also plays a role in cardiac remodeling although little is presently known about its expression and function in the heart. Here, we have investigated the dynamic expression pattern of Adamts4 during cardiogenesis and also in the adult heart. To our surprise, adult cardiac injury reactivated Adamts4 expression concomitant with fibrosis induction. To better understand the mechanism, cultured H9c2 cardiomyocyte cells were subjected to ROS injury and Hypoxia. Moreover, through combinatorial treatment with SB431542 (an inhibitor of Tgf-β1), and Adamts4 siRNA mediated gene knockdown, we were able to decipher a regulatory hierarchy to the signal cascade being at the heart of Tgf-β regulation. Besides the hallmark expression of Adamts4 and Tgf-β1, expression of other fibrosis-related markers like Collagen-III, alpha-SMA and Periostin were also assessed. Finally, increased levels of Adamts4 and alpha-SMA proteins in cardiac patients also resonated well with our animal and cell culture studies. Overall, in this study, we highlight, Adamts4 as a novel biomarker of adult cardiac injury. By 2016, at least 30 and 50 million cases of cardiovascular diseases (CVD) were recorded in the US and India respectively with a mortality rate of more than 70% in India3. Extracellular matrix (ECM) remodeling in heart is one of the contributing factors of pathogenesis in cardiovascular diseases5. Collagen-I forms the major component of matrix interstitium of the myocardium, the other components of ECM include Collagen-III, fibronectin, proteoglycans, matrix metalloproteinases (MMPs), and tissue inhibitors of matrix metalloproteinases (TIMPs). The proportion and biochemistry of the ECM change due to underlying pressure overload, cardiac injury, myocardial infarction (MI), and/or ischemia–reperfusion (I/R) injury that leads to extracellular matrix reorganization which in turn is modulated by changes in turnover of matrix proteins6. A typical cardiac remodeling post-cardiac injury undergoes, three major phases namely inflammatory, proliferative and maturation leading to a mature scar formation7.Heart failure remains a global cause of mortality and morbidity and an issue of public health concern with over 20 million people diagnosed with at least first-time heart failure across the world10.Although the initial stages of ECM remodeling are essential as it prevents rupture of the ventricular wall and prevents ventricular dilatation, however, extensive and unregulated ECM remodeling leads to progressive fibrosis in the heart and impairment of cardiac functioning11. An important MMP is the Adamts family which apart from inhabiting the functions of an MMP also acts as disintegrins. Adamts4 a member of the Adamts family is a metalloproteinase and a disintegrin with thrombospondin like motifs12. Adamts4 has been known to bind to the ECM proteins and executes cleavage of ECM proteoglycans like aggrecan, versican and brevican apart from regulating collagen turnover. There is not sufficient information in the context of cardiac remodeling with a focus on the involvement of Adamts4. So far it is known that Adamts4 knockout in animal models leads to a reduction in plaques in cases of high fat induced atherosclerosis13 and also, inhibition of Adamts4 with pentosan polysulfate following aortic banding improves cardiac functioning6. However, there is not much literature about Adamts4 expression and function in the developing and adult heart at the basal level and also in post cardiac injury. Only recently studies have shown that Adamts4 along with Adamts1 levels remain elevated in patients with acute aortic dissection and also in patients with coronary artery disease15.The MMPs are a family of zinc-dependent proteases involved in the turnover of collagen20. Finally and most importantly, we also validated our hypothesis in human clinical samples and demonstrated the induced expression of ADAMTS4 in patients with indicated cardiac ailments.Here in our present study, for the first time, we aim to identify Adamts4 as a novel biomarker of adult cardiac injury under stress conditions. We have detected a strong expression of Adamts4 protein in the developing cardiac chamber myocardium in utero compared to very restricted expression in the adult murine hearts. To our surprise, we have also detected the expression of Adamts4 in vivo murine model of myocardial infarction (MI), localized in adult cardiomyocytes. To better understand the molecular insights of Adamts4 induction and associated affected signaling pathway activation, we have used H9c2, a rat ventricular myoblast cell line for several in vitro assays. Likewise, Adamts4 expression was induced in H9c2 cells, subjected to hypoxia (Hyp) and ROS injury inductions in vitro. Moreover, we manipulated the expression of Adamts4 with siRNA-mediated loss of function and TGF-β inhibitor studies in H9c2 cells to evaluate its regulation and dependency on TGF-β signaling since TGF-β has been long known to be a characteristic marker for inflammatory and fibrotic responses following pathological stress including MI, ischemia and reperfusion (I/R) injury22.) throughout the LV of chamber myocardium.Immunohistochemistry (IHC) with anti-Adamts4 antibody shows Adamts4 protein expresses strongly in developing murine cardiac chambers but the expression significantly wanes in adult murine hearts. In contrast to the adult heart Fig. e where AWhile Adamts4 expression in adult murine hearts significantly waned in comparison to developing hearts, it is found that MI-induced adult murine heart shows significant reactivation of Adamts4 protein. IHC with anti-Adamts4 antibody shows a strong expression of Adamts4 in the infarct and border zone Fig. a followi2O2 and Hypoxia (Hyp) treatment. Figure 25 and Hif-1α26 respectively validated by real-time qPCR, showing upregulation after injury for both the markers. Also, Adamts4 and Collagen-III28 expression at mRNA levels shows elevation after both stress treatments  expression at protein levels assessed by western blot (WB) shows significant upregulation following H2O2 and Hypoxia treatments . Interestingly, this elevated expression significantly reduced for both Adamts4 and Tgf-β1 in presence of ALKI pre-treatment. Interesting to note, a non-specific nuclear presence of Adamts4 is observed irrespective of treatment  staining with anti-Adamts4 and anti-Tgf-β1 antibody shows an elevated expression of both the markers following H35 IF staining show a reduction in expression of both markers in H2O2 and Hyp groups pre-treated with ALKI after a significant upregulation in the expression of both markers observed in the injury groups; H2O2 and Hyp as measured by assessing fluorescence intensities of Col-III and α-SMA  and α-SMA2O2 and Hyp treatments group in comparison to the control set but this elevation is significantly reduced in ALKI + H2O2 and ALKI + Hyp groups respectively in comparison to the control group  and hypoxia (ATSsi + Hyp) treatment show a significant reduction in the levels of Adamts4 expression as shown by Adamts4 IF staining  was done, shown by Collagen-III and α-SMA IF staining  or inferior wall MI (IWMI) Fig. a–c Furth13 and patients with acute coronary syndrome15. Importantly, Adamts4 being a secretory protein in nature, it resides both intracellularly and is also secreted from cardiomyocytes out into the ECM where its function is to regulate the turnover of other proteins by directly affecting other resident cells including but not limited to cardiac fibroblasts, endothelial and smooth muscle cells in the same ECM milieu. However, injury induced function of Adamts4 in other adult cardiac resident cell types is unknown. The expression of Adamts4 significantly decreased from being widespread in the chamber myocardium of developing hearts to very restricted expression in normal adult hearts  but to state whether Adamts4 and Periostin directly interact, requires further experimentation to establish any direct association between these two ECM molecules. α-SMA has been known to induce fibroblast contractility and is highly expressed in infarct myofibroblasts to prevent extreme remodeling and thereafter cardiac rupture46. As our data shows the hallmark expression of Adamts4 in cardiac stress conditions, it can be considered as a novel biomarker for cardiac related injuries leading way for therapeutics to manipulate its expression following cardiac injury for improvement of cardiac functioning. Finally, our work hypothesises that Adamts4 being an ECM secretory protein, after being secreted from chamber myocardium regulates ECM and its remodeling, the details of this mechanism require further depth, understanding and experimentations.To conclude, Adamts4 is upregulated in response to cardiac stress in 24.All experiments involving animals were carried out with the experimental protocols and procedures reviewed and approved by the Cincinnati Children’s Hospital Medical Center Biohazard Safety Committee and Institutional Animal Care and Use Committee. The study was performed in accordance with ARRIVE guidelines and all animal experiments were conducted in compliance with the relevant guidelines. Timed-matings were established, with the morning of an observed copulation plug set at E0.5. For embryonic studies, whole embryos or hearts were harvested on E10.5, E12.5, E14.5, and E18.5 days and proceeded with IHC. For MI induction, 8–10 weeks old adult male Swiss albino mice were used. MI was performed as described previouslyWhole embryos and embryonic or adult hearts were harvested, washed in 1X PBS, fixed in 4% paraformaldehyde, dehydrated, and embedded in paraffin. Later the tissue sectioning (5–7 μm) was done using a microtome (Leica Biosystems). For immunohistochemistry, the tissue sections were deparaffinized and hydrated after which antigen retrieval was done using citrate buffer (pH 6) washed in 1X PBS and thereafter, blocking solution (2% BSA with 0.1% Tween20) was added to the sections for 1 h. Later after blocking, the sections were incubated overnight at 4 °C. The following day, the incubated sections were washed in PBS and incubated with secondary antibodies against their respective primary antibodies. The sections were mounted using Vectashied (Vector Labs). The primary antibodies used were Adamts4 , MF20 , and respective secondary antibodies [anti-rabbit/mouse Alexa 488  or 594  fluorescent-conjugated; Molecular Probes, 1:100). Topro3  was used as a nuclear stain. Fluorescent images were acquired using a Zeiss LSM 510 confocal microscope and LSM version 3.2 SP2 software.2. Blood collection timing varied between 12 and 36 h of onset or hospitalisation. Control group consisted of blood collected from healthy volunteers who were not known to be diagnosed with any cardiac ailments or any other lifestyle disease for the record. The characteristics of the patient samples are provided in the following Table A total of 30 affected clinical samples were used for this study. Venous blood samples were used for the study. The approval for the collection was ethically approved by the Institutional ethics committee of IPGME&R, Kolkata (IPGME&R/IEC/2019/517). All experiments were approved and performed according to the institutional guidelines of IPGME&R, Kolkata. Informed consent was obtained from all participants. Two broad categories of cardiac conditions were selected namely, Dilated cardiomyopathy (DCM), Myocardial Infarction . MI patients routinely given Statin, high intensity, usually 80 mg of Atorvastatin. Loading doses of Aspirin 300 mg and Clopidogrel 300 mg along with Angiotensin Receptor Blocker (ARB) or Angiotensin Converting Enzyme Inhibitor (ACEI), β-Blocker, nitroglycerin, tranquilizer. DCM cases were given ACEI/ARB/Angiotensin Receptor Neprilysin Inhibitor (ARNI), SGLT2 inhibitors, β-blocker, diuretic, Mineralocorticoid Receptor Antagonist (MRA) and supportive therapy like Oin-vitro experiments were performed on H9c2, rat ventricular cardiomyoblast cell line. The cells were cultured in Dulbecco’s modified Eagle’s medium  supplemented with 10% FBS  and 1% penicillin/streptomycin (Pen-Strep) cocktail  maintained at a sterile humidified CO2 incubator at 5% levels and 37 °C23. The cells were used for further experiments at about 75–80% confluency. For experimental treatments, serum-free DMEM was used. For ROS generation, the cells were treated with H2O2 (100 μM) for 1 h25. For hypoxia induction, the cells were put in an anaerobic chamber with an anerogas pack  and anaero indicator tablet  according to the manufacturer’s instructions. The anaerobic chamber with cells was incubated in the CO2 incubator for 12 h, the indicator color change confirmed hypoxia induction in the chamber47.All the 2O2 and hypoxia induction as earlier mentioned20. For knockdown of Adamts4, cells were transfected with Adamts4 siRNA (50 pmol)  and Lipofectamine RNAiMax reagent  when the cells were at least 70% confluent for 48 h following manufacturer’s protocol after overnight serum starvation. For negative control, scrambled siRNA (silence select negative control no 1 siRNA)  was used similarly. After 48 h of Adamts4 siRNA treatment, the cells were subjected to H2O2 and hypoxia treatment as previously described.For TGF-β inhibition, the cells were pre-treated with a potent ALK inhibitor, SB431542 (10 μM)  for 30 min following which the cells were exposed to HTotal RNA was isolated from control and treated H9c2 cells with Trizol  following the Trizol-Chloroform method. Taking 1 μg of total RNA isolated, cDNA was synthesized using Biorad cDNA synthesis kit  in 20 μl of total volume according to manufacturer’s supplied protocol. The cDNA prepared was used for primer optimization and standardization using DNA Taq polymerase . The PCRs were performed for 35 cycles using 20 pmol of the rodent primer pairs: Adamts4 (F): 5′-TCATGAACTGGGCCATGTCT-3′ and (R): 5′-GTCAGTGATGAATCGGGGCAC-3′; Hif-1α (F): 5′-CCAGCAGACCCAGTTACAGA-3′ and (R): 5′-TTCCTGCTCTGTCTGGTGAG-3′; β-actin (F): 5′-TCTTCCAGCCCTTCCTTCCTG-3′ and (R): 5′-CACACAGAGTACTTGCGCTC-3′ and Catalase (F): 5′-CCTCGTTCAGGATGTGGTTT-3′ and (R): 5′-TCTGGTGATATCGTGGGTGA-3′, Tgf-β1 (F): 5′-CTGAACCAAGGAGACGGAATAC-3′ and (R): 5′-CTCTGTGGAGCTGAAGCAATAG-3′ and Collagen-III (F): 5′-CTGGTCCTGTTGGTCCATCT-3′ and (R): 5′-ACCTTTGTCACCTCGTGGAC-3′. These primer pairs were further used for real-time PCR studies. Real-time PCR was performed using Bio-Rad real-time PCR kit . The gene expression of the mentioned genes was normalized with β-actin.48. The immunoblots developed using Clarity™ Western ECL substrate  and scanned using ChemiDocMP (Bio-Rad) as previously described49. The antibodies used were Adamts4  used at a concentration of 2 μg/ml, α-SMA  used at a dilution of 1:500, Tgf-β1  used at a concentration of 1 μg/ml and Vimentin  at a dilution of 1:1000. After overnight incubation at 4 °C with these primary antibodies, the corresponding secondary antibodies-HRP conjugated secondary goat anti-rabbit  and HRP conjugated goat anti-mouse secondary antibodies  were added against their respective primary antibodies at a dilution of 1:3000. The SDS gels were also stained with 2.5% coomassie  and destained with coomassie de-stainer to obtain total protein intensities. The coomassie stained gels were scanned using ChemiDoc MP mentioned earlier. Quantification of intensities was done using ImageJ software (NIH). Figures for unprocessed blots and gels are also included in Supplementary Figs. Protein isolation was done using ice-cold mammalian lysis buffer  containing protease inhibitor cocktail (Genetix GX-2811AR) and phosphatase inhibitor (Genetix GX-0211AR). The proteins were stored in small aliquots at − 80 °C until further use for Western Blotting (WB). Western blotting was performed as previously described25 except for the omission of permeabilization steps for staining with antibodies. Adamts4  used as mentioned earlier, Tgf-β1  used at a concentration of 2 μg/ml, α-SMA  used at 1:400 dilution, collagen III  at a dilution of 1:1000 and Periostin  used at a dilution of 1:500. After overnight incubation at 4 °C with these primary antibodies, the corresponding secondary antibodies Alexa flour 488 goat anti-rabbit secondary antibody  and Alexa flour 594 goat anti-mouse secondary antibody  were added against their respective primary antibodies. All cell nuclei were stained with DAPI . Images were acquired using Leica confocal microscope and LasX software. The fluorescence intensities were measured and quantified with the help of ImageJ software (NIH).Immunostaining was performed in H9c2 cells following the protocol previously describedELISA kit  was used to perform ELISA. Serum proteins were diluted with sample buffer in the ratio of 1:10 and samples were loaded onto the wells in duplicates. ELISA was performed as stated by the manufacturer’s protocol.P < 0.05.All the results are mostly represented as mean ± Standard Deviation of mean (SD) or as median, quartiles and range Fig. . StatistSupplementary Information.