Chromatin architecture and its modification is involved in gene regulation as well as DNA repair, recombination, and replication. In the cell nucleus, DNA is packaged by histones to form structured nucleosome units. Chromatin remodeling is governed by two categories of enzymes: histone modifiers and ATP-driven nucleosome repositioners.

Transcriptional controllers encompass:

HDACs and HMTs are associated with certain cardiac pathologies (Tables 1.1 and 1.2). Mutations in genes encoding HMTs may contribute to congenital heart disease.

Class-1 nuclear histone deacetylases (HDAC1–HDAC3 and HDAC8), class-2 HDACs, which shuttle between the cytosol and nucleus and can further be subdivided into subclass-2a (HDAC4, HDAC5, HDAC7, and HDAC9) and -2b (HDAC6 and HDAC10), and class-4 HDACs (HDAC11), which localize to the nucleus and cytosol and depend on zinc. Class-2a HDACs repress cardiac hypertrophy. On the other hand, class-3 HDACs, or sirtuins (SIRT1–SIRT7), rely on nicotinamide adenine dinucleotide (NAD). They reside in both nucleus and cytoplasm.

The class-2b cytoplasmic histone deacetylase HDAC6, which lodges in striated (cardiac and skeletal) myocytes and deacetylates cytoskeletal proteins, is involved in adverse remodeling induced by chronically signaling angiotensin-2 [2]. HDAC6 mice develop cardiac hypertrophy and fibrosis, but without cardiac function alteration, in response to angiotensin-2 administration during up to 8 weeks, whereas wild-type mice develop systolic dysfunction. Inhibition of HDAC6 preserves systolic function.

Histone acetylation and methylation that result from developmental programs of the heart dictate cardiac gene expression. Histone Lys methyltransferase KMT6³ and paired box (Pax) transactivation domain-interacting protein PaxIP1 (or PTIP) are two HMTs involved in chromatin condensation. The former participates in cardiac myosin heavy chain (MHC) gene regulation, as it interacts with a long noncoding RNA in chromatin [1].

Trimethylation H3Kme is typically associated with transcriptional elongation and DNA methylation that cooperate to regulate the expression of double homeobox Dux4 gene in the failing human heart [1].

Histone-3 trimethylation at Lys4 (e.g., H3Kme) and some types of acetylation (H3Kac and H4Kac) are generally correlated with permissive gene transcription, as they weaken the connection of histone proteins and DNA.

In particular, H3Kme enables the expression of the KCNIP2 gene that encodes K channel-interacting protein KChIP2 involved in repolarization [1]. Expression of the KCNIP2 gene associated with reduced H3Kme at its promoter falls in heart failure. cAMP-responsive element-binding protein (CREB)-binding protein (CBP) and P300 are two HATs detected on activated genes in the left and right ventricles of the heart. Binding of cardiac transcription factors, such as SRF and GATA4, may be mediated at least partly by P300 acetyltransferase [1].

The histone modifiers , additional sex comb-like protein ASxL2 and histone Lys methyltransferase KMT6, interact at MHC promoters [1].

On the other hand, methylation of H3K and H3Kme and deacetylation are correlated with gene transcription suppression, as they cause chromatin condensation (i.e., transcriptionally repressive chromatin conformation) [1]. In mouse models, ischemic reperfusion provokes H3Kme at the Sirt1 promoter, hence suppressing the Sirt1 gene transcription.

However, whereas deacetylation of histone tails is linked to transcriptional suppression, deacetylation of nonhistone proteins often activates gene expression.

The Polycomb repressive complexes PRC1 and PRC2 cause gene silencing via intrinsic HMT activity. The PRC2 holoenzyme is a complex of HMTs, which leads to H3Kme modification.

The regulation of gene expression involves the coordinated actions of the transcriptional machinery and regulators (i.e., activators or repressors at gene promoters). Gene transcription can result from a loss of inhibitory methylation and a gain of stimulatory acetylation in a given Lys residue (e.g., K) of histone H3.

At a given station, the velocity and possibly the direction of blood flow change (e.g., in limb arteries, but not in cerebral arteries) during the cardiac cycle, in addition to spatial variations along the vascular circuit. The endothelium is sensitive to hemodynamic shear stresses applied at its luminal surface (Vol. 5, Chap. 9. Vascular Endothelium).

Blood flow via hemodynamic stress exerted on the wall-wetted surface as well as within the wall can promote expression of genes that encode antiatherogenic, antithrombotic, and anti-inflammatory factors [3].

The stress response relies on the hypothalamic–pituitary–adrenal axis. This activated axis triggers the release of glucocorticoids from the adrenal gland. Glucocorticoids have potent anti-inflammatory and immunosuppressive effects. These stress hormones connect to the glucocorticoid receptor (GR), a member of the nuclear receptor superfamily of ligand-dependent transcription factors (NR3c1).

Expression of genes involved in GR-deficient hearts include [6]: (1) the maintenance of cardiac contractility (DMD [dystrophin] and RYR2 [cardiac ryanodine receptor]); (2) repression of adverse cardiac hypertrophy (Klf15 [Krüppel-like factor-15 and TRIM63 [Tripartite motif-containing protein-63; also known as Muscle-specific RING finger ubiquitin–protein ligase (MURF1)]); (3) promotion of cardiomyocyte survival (Ptgds [prostaglandin-D synthase]); and (4) inhibition of inflammation decreases (LCN2 [lipocalin-2], and Zfp36 [Zinc finger protein-36 or tristetraprolin]). The dysregulated glucocorticoid-responsive target genes (lowered expression) encode many transcription factors, plasmalemmal receptors, and effectors of G protein-coupled receptor (GPCR) and mitogen-activated protein kinase (MAPK) signaling [6]. A glucocorticoid signaling deficiency to cardiomyocyte thus causes maladaptive cardiac hypertrophy, heart failure, and death [6].

In addition to GR, cardiomyocytes express the mineralocorticoid receptor (MR; NR3c2) that binds aldosterone and glucocorticoids with similar high affinity. Glucocorticoids typically circulate at much higher levels than aldosterone, and cardiac-specific MR may be primarily occupied by glucocorticoids [6]. However, the contribution of MR is negligible.

Patients who bear a single nucleotide polymorphism (A3669G) in the GR gene are characterized by an increased expression of the dominant-negative receptor variant GRβ and concomitant glucocorticoid resistance. They have an augmented risk of coronary artery disease, adverse cardiac hypertrophy, systolic dysfunction, and heart failure [6]. In addition, patients with Addison’s disease identified by a decreased production of glucocorticoids have heart failure.

The heart is the first organ built during embryo- and fetogenesis. During the fetal life, differentiated cardiomyocytes enter the cell cycle. The primitive heart formed in the early stages of cardiac specification mostly consists of quiescent cells. As development progresses, cardiomyocytes at the outer curvature resume proliferation to form the future chambers. On the other hand, cardiomyocytes at the inner curvature do not enter the cell cycle and will form the conduction system.

Embryonic and neonatal cardiomyocytes are capable of synthesizing, assembling, and disassembling their contractile proteins via the assembly of contractile structures for chromosome segregation and cytokinesis when they proliferate [7].

Proliferation and differentiation are not mutually exclusive in the developing as well as adult heart in some animal species that can regenerate their cardiac pump. For example, the damaged zebrafish heart reactivates a developmental program involving the expansion of GATA4+ cardiomyocytes to achieve regeneration [7].

However, the cardiomyocyte proliferation is limited in mammals. In humans, the cardiomyocyte stops dividing early after birth, possibly due to intracellular activation of the cyclin-dependent kinase inhibitors CKI1b and CKI2c, when breathing brings a sudden and marked increase in oxygenation ( the arterial oxygen partial pressure ranges from 2.4 to 3.7 kPa [18–28 mmHg] which, during the fetal life, rises to 13.3 kPa [100 mmHg]) [7]. At birth, oxygen free radicals and reactive oxygen species (ROS) may stop cardiomyocytes proliferation and favor their terminal differentiation.

The neonatal cardiomyocytes lack a developed T-tubular network (microscopic invaginated segments of the sarcolemma); the myofibrils are located near the sarcolemma. In growing cardiomyocytes, T-tubules develop within a few days after birth. Caveolin-3 localized to the nanoscopic invaginations of the sarcolemma, caveolae, transiently associates with primitive T-tubules. The location of various K, Na1, and Ca1.2 channels relies on caveolae.

Bridging integrator BIn1 (or amphiphysin-2) is a major agent of T-tubule genesis and transfer of Ca handling proteins in T-tubules. This membrane scaffold initiates T-tubule genesis in striated myocytes, asit induces membrane curvature. In addition, it determines the delivery of microtubule-transported membrane proteins. In particular, it forms BIn1–Ca1.2 clusters and tethers microtubules to membrane, thereby supporting Ca1.2 delivery to the T-tubular membrane [8]. It especially anchors microtubules and Ca1.2 at junctional membrane complexes. These specialized nanodomains connect ion channel clusters between transverse tubule and sarcoplasmic reticulum that are involved in excitation–contraction coupling that links membrane depolarization to mechanical contraction. A small entry of extracellular Ca through Ca1.2a causes a large Ca release from its major intracellular store through RyR2 channels. This Ca-induced Ca release (CICR) inactivates Ca1.2a for repolarization. Altered splicing and mutations of Bin1 are involved in myopathies.

In cardiomyocytes, junctophilin-2 encoded by the JPH2 gene is a major structural protein in T-tubules that forms junctional membrane complexes. Junctophilin-2 spans the junctional distance (∼12 nm cleft) and tethers the plasma membrane to that of the sarcoplasmic reticulum to facilitate Ca handling [9]. Its N-terminus is attached to the T-tubular sarcolemma and its C-terminal transmembrane domain is embedded in the sarcoplasmic reticulum. Junctophilin-2 is necessary for the development of postnatal T-tubules and stabilization of junctional membrane complexes in mature cardiomyocytes. Junctophilin-2 also interacts with caveolin-3 [9]. Its deficiency by mutations or expression downregulation is linked to hypertrophic cardiomyopathy, arrhythmias, and heart failure.

The epicardium, which can be considered as a cardiac stem cell niche, is subjected to a relative hypoxia that yet enables the proliferation of cardiac progenitor cells. Epicardial progenitor cells are activated during embryogenesis and generate cardiomyocytes, endotheliocytes, and vascular smooth myocytes, as well as in adulthood after injury, once they are stimulated by thymosin-β4 [7]. The latter activates epicardial progenitors by upregulating antioxidative enzymes, thereby attenuating oxidative stress in these cells. In addition, a low oxygen partial pressure promotes the proliferation of human cardiomyocyte progenitors [7].

In addition, cardiomyocytes are subjected to an increased mechanical load after birth. Cells of the inner wall surface, which sense shear in addition to stretch, are the first to exit cell cycle [7]. Cardiomyocytes are equipped with stress and strain sensors in Z disks and titin filaments that can modify the transcriptional activity. Stretch can provoke the production of growth factors and cytokines, such as IL6 and IGF1, which serve as paracrine controllers of cardiomyocyte proliferation [7].⁴ Mechanical strain increases the intracellular level of ROS that promote cardiomyocyte differentiation and block their division.

Neighboring fibroblasts, endotheliocytes, smooth myocytes, adipocytes, and inflammatory cells influence cardiomyocyte proliferation, as they release paracrine messengers, in particular growth factors and cytokines.

In neonatal cardiomyocytes, fibroblast growth factor FGF2 promotes DNA synthesis via activated protein kinase-C [7]. In the adult myocardium, the FGF2 activity is potently inhibited by FGF16, which is preferentially expressed in the postnatal heart.

In neonatal cardiomyocyte cultures, platelet-derived growth factor supports cell proliferation, as it represses the G1-phase inhibitor CKI1b, activates PKB, and inhibits GSK3β [7].

Inflammatory meukocytes secrete the TNFSF12 cytokine that helps cardiomyocyte proliferation upon binding to TNFRSF12a receptor expressed by neonatal cardiomyocytes and downregulated in adulthood, except in pathological conditions [7].

Interleukin-6, which is abundantly secreted in the heart by both epicardiocytes and hypoxic adipose stromal cells, augments the proliferation rate of cultured slow-dividing neonatal cardiomyocytes [7].

Neuregulin-1, a member of the EGF family, is an inducer of adult cardiomyocyte proliferation via HER4 receptor [7]. Neuregulin-1 is first expressed by the endocardial endothelium of neonatal hearts, where it participates in trabeculation and cushion formation, and later by endotheliocytes. Periostin is another secreted molecule that may increase proliferation of both neonatal and adult cardiomyocytes via integrins and the PI3K pathway [7].

The composition of the extracellular matrix contributes to the control of the rate of cardiomyocyte proliferation before and after birth. The cardiomyocyte division in the developing heart correlates with the regulated synthesis of matrix proteic constituents and integrins. In the fetal myocardium, the extracellular matrix that is enriched in fibronectin supports cardiomyocyte proliferation [7].

On the other hand, an elevated concentration of collagen-1 impedes cardiomyocyte proliferation after birth [7]. Focal adhesion kinase (FAK) that assists fibronectin binding to integrins facilitates cardiomyocyte proliferation during cardiogenesis heart development. FAK-related nonkinase (FRNK), the noncatalytic FAK C-terminus, is transiently expressed in the postnatal heart with peak levels occurring just prior to the withdrawal of cardiomyocytes from the cell cycle [7]. Therefore, FRNK may dampen FAK-dependent cardiomyocyte cell cycle progression and facilitate cell cycle exit in the postnatal myocardium.

Cardiogenesis as well as cardiac homeostasis in adulthood is controlled by several different microRNAs [7]. These genome-encoded small RNAs mediate posttranscriptional silencing, thereby preventing unwanted gene expression.

The expression of microRNA-195, a member of the MIR15 family, is induced when cardiomyocytes exit the cell cycle. MiR29a and miR133 also suppress cardiomyocyte proliferation.

On the other hand, miR199a-3p and miR590-3p are able to cause cell cycle reentry and cytokinesis of cultured adult cardiomyocytes [7]. In vivo, these miRs specifically promoted cardiomyocyte (but not fibroblast) proliferation in both neonatal and adult hearts after myocardial infarction.

MicroRNA-499 promotes neonatal cardiomyocyte proliferation via Sox6 and cyclin-D1 [7]. The microRNA-17–92 cluster is also involved in cardiomyocyte proliferation, as its overexpression engenders cardiomyocyte proliferation in embryonic, postnatal, and adult hearts.

The cardiomyocyte proliferation can be stimulated by different types of extracellular molecules. In the mouse adult heart, a slow-rate cardiomyocyte renewal is sustained by the division of preformed cardiomyocytes [7].

The exogenous administration of selected growth factors is capable of inducing neonatal and, in some instances, also adult cardiomyocyte proliferation [7]. The cardiac regeneration in the adult heart may also be achieved by reactivating the cardiogenesis program.

In addition, certain diffusible factors can regulate the proliferation and cardiac commitment of endogenous or implanted stem cells. The type of cardiomyocytes (nodal, atrial, or ventricular) can be identified by electrophysiological properties (e.g., shape of action potential waveform and duration and amplitude of generated action potentials). The characterization of action potentials enables automated categorization of cardiomyocyte populations from embryoid bodies (i.e., aggregates of pluripotent stem cells formed from embryonic or induced pluripotent stem cells by reprogramming somatic cells) for optimal implantation [10]. The Notch pathway operates between adjacent cells expressing Notch ligands (Jag1 and Jag2 and DLL1 and DLL3–DLL4) and receptors (Notch-1–Notch-4; Vol. 3, Chap. 10. Morphogen Receptors). In the developing heart, Notch modulates cardiomyocyte survival, cardiac stem cell differentiation, and angiogenesis and regulates trabeculation, cardiomyocyte proliferation, and valve formation [11]. In the postnatal heart, the Notch axis controls cardiac precursor expansion and differentiation, thereby sustaining cardiomyocyte proliferation.

However, in adult mice hearts, reactivation of the Notch pathway does not assist cardiac regeneration after myocardial infarction [7]. Therefore, at least in adult rodent hearts, cardiac regeneration does not necessarily mimic that in embryonic hearts.

On the other hand, in the adult heart of mice overexpressing the Notch ligand Jagged-1 on cardiomyocytes, activated Notch limits adverse cardiac remodeling [11]. Notch actually impedes cardiomyocyte apoptosis as well as cardiac hypertrophy in adult mouse hearts subjected to pressure overload and fibrosis mediated by transforming growth factor-β and its effector connective tissue growth factor [11]. Hence, the Notch pathway acts on cardiac mesenchymal stromal cells (cardiac fibroblasts and precursor cells). It reduces the proliferation of myofibroblasts. It also stimulates the expansion of SCA1+ and NKx2-5+ cardiac precursor cells [11].

Overexpression of cyclin-A2 that enables the cell cycle progression through the G2–M checkpoint (Vol. 2, Chap. 2. Cell Growth and Proliferation) can engender cardiomyocyte proliferation [7]. Cyclin-D1 causes a sustained DNA synthesis, but abnormal patterns of multinucleation. Overexpressed cyclin-D2 supports regenerative proliferation.

In transgenic mice, overexpression of CDK2 kinase also transiently raises cardiomyocyte proliferation [7]. Less-differentiated and mononucleated cardiomyocytes abound.

The transcription factor and regulator of cardiogenesis Meis homeodomain-containing protein Meis1 acts as a modulator of cell proliferation after birth.

The genetic Meis1 deletion extends cardiomyocyte proliferation after birth and reactivates cell division in adult hearts [7].

The postnatal cardiomyocyte proliferation is controlled by many signaling cascades, such as the Hippo pathway, and modulated by microRNAs.

The Hippo kinase cassette intervenes in myogenesis, organ size control, and regeneration of the heart [12]. Kinases and scaffold proteins of the Hippo axis suppress cell proliferation. The Hippo pathway regulates the activity of TEA domain-containing transcription factors (TEAD1–TEAD4) mainly via phosphorylation of the transcriptional coactivators YAP1 and WWTR1 that activate TEAD factors (Vol. 4, Chap. 10. Other Major Types of Signaling Mediators).

The cardiomyocyte proliferation involves the activation of IGF1 receptor, subsequent PKB activation, and inactivation of GSK3β, which stabilizes β-catenin, a promoter cardiac development [7].

The main effectors of the Hippo pathway are the transcriptional coactivators YAP1 and YAP2 that derepress the genes activated by the Wnt–β Ctnn pathway. Genetic deletion of the upstream inactivating effectors (STK4, WW45 [Sav1], and LaTS1/2) causes cardiac hyperplasia [7]. On the other hand, transgenic mice overexpressing STK4, LaTS1, or LaTS2 die postnatally with dilated cardiomyopathy without compensatory ventricular myocyte hypertrophy.

Various types of adult progenitor cells have been injected into damaged hearts or into coronary circulation, such as mesenchymal stromal cells, hematopoietic stem cells, endothelial progenitor cells, skeletal myoblasts, and cardiac progenitor cells to ensure cardiac protection, metabolism, contractility, proper remodeling, and neovascularization via stem cell paracrine signaling using growth factors, cytokines, and chemokines (FGF1–FGF2, HGF, IGF1, PDGF, VEGF, Nrg1, angiopoietin-1, IL6, and CXCL12), as well as differentiation and repopulation.

The member HSPa12b of the HSP70 family is predominately expressed in endotheliocytes of the heart, brain, lung, kidney, and adipose tissue, among others, but not in liver sinusoids [13]. In the heart and brain, the vascular endothelium of vessels of all sizes produces HSPa12b, whereas in lungs and adipose tissue, it is mainly synthesized in capillaries. It is required for angiogenesis, specifically in adhesion, migration, and tube formation.

The heat shock protein HSPa12b possesses 22 client proteins such as aryl hydrocarbon receptor nuclear translocator (ARNT). Among them, A-kinase-anchoring protein AKAP12 and podocalyxin-like protein PodxL are implicated in cell adhesion.

HSPa12b protects endotheliocytes against lipopolysaccharide-induced dysfunction and inflammation, as well as cerebral ischemia–reperfusion injury via the PI3K–PKB pathway possibly stimulated by angiopoietin-1 [13].

Transgenic mice expressing human HSPa12b specifically in endotheliocytes (without change in expression of other HSPs [i.e., HSPb5, HSP25, HSP60, HSP72, and HSP90]) have an improved cardiac function and remodeling assessed by left ventricular enlargement, wall thinning, and fibrosis, with a lower rate of cardiomyocyte apoptosis and higher capillary and arteriolar densities, from 1 to 4 weeks after myocardial infarction compared with wild-type mice [14]. The HSPa12b chaperone yields a protective and reparative response to ischemic insult in the heart.

Under ischemia, levels of proteins involved in cardiomyocyte survival and angiogenesis, such as NOS3 (without significant change of NOS2), Ang1, VEGF, and BCL2, are further elevated [14]. Angiopoietin-1 has an anti-inflammatory and protective actions on endotheliocytes via the PI3K–PKB axis, a decreased expression of cell adhesion molecules ICAM1 and VCAM1 and hence immunocyte recruitment, and an increased expression of antiapoptotic proteins such as BCL2. The VEGF factor augments NOS3 expression, thereby enhancing nitric oxide synthesis. The latter messenger acts against oxidative stress, inflammation, hypertrophy, and fibrosis, thereby reducing adverse cardiac remodeling. Protected cardiomyocytes, in turn, secrete VEGF and paracrine factors that protect endotheliocytes and support angiogenesis.

The function of HSP90 and HSP70 is tightly controlled by cochaperones that regulate the specificity and dynamics of a chaperone reaction, as they recognize specific target proteins and control ATP binding and hydrolysis. Cochaperones also participate in the decision toward degradation of misfolded proteins.

For example, HSP70 binds to its substrate assisted by its cochaperone HSP40. The additional cochaperone HSP70/HSP90-organizing protein homolog (HOP)⁵ promotes the transfer of the substrate to HSP90 that enables conformational changes of the substrate. Other additional cochaperones, such as the HSP90 cochaperone P23, which is the cytosolic prostaglandin-E synthase PGeS3, and prolyl isomerase, regulate HSP90 ATPase activity and protein folding [15].

Small chaperones (sHsp or HSPb) constitute a family of 11 members that can homo- and hetero-oligomerize. This polymerization can be regulated by transient phosphorylation in response to stress [15].

Small chaperones are devoid of ATPase activity. Nonetheless, they can prevent misfolded protein aggregation and regulate intracellular signaling, sometimes, in cooperation with HSP70 and HSP90.

The ER of cardiomyocytes consists of a vesicular compartment mainly in the perinuclear region involved in protein synthesis, and another compartment organized around myofibrils and T-tubules, which controls excitation–contraction coupling via calcium cycling. The two networks are interconnected.

Hypoxia, blood pressure overload, and drug-induced insults activate the ER chaperone machinery that maintains the proper balance between protein folding and degradation in the ER.

The ER chaperone machinery that assists protein folding and catalyzes disulphide bond formation includes:

These molecular chaperones act in concert with three ER membrane complexes, PERK, IRE1α, and ATF6, which activate the ER stress response [15]. In unstressed conditions, PERK (or eIF2α K3 kinase), IRN1 (or inositol-requiring Ser/Thr protein kinase and endoribonuclease IRe1 or IRe1α), and ATF6 bind to HSPa5 in the ER lumen, which inhibits their function. Upon ER stress, HSPa5 connects to misfolded proteins in the ER lumen, dissociating from PERK, IRN1, and ATF6 that become activated. The PERK (pancreatic eIF2α ER kinase) represses protein synthesis, as it phosphorylates the eukaryotic initiation factor-2α. Endoplasmic reticulum-to-nucleus signaling protein ERN1 generates a spliced XBP1 mRNA encoding a potent transcription factor that activates the expression of ER stress response genes. X-box-binding protein XBP1 upregulates genes involved in ubiquitin-dependent protein proteasomal degradation. Activating transcription factor ATF6 translocates to the Golgi body upon activation and undergoes cleavage by specific peptidases, yielding a soluble cytosolic transcription factor that enters the nucleus and induces further transcription of ER stress response genes.

Prolonged or extreme ER stress induces the expression of the transcription factor CHOP that triggers synthesis of proapoptotic proteins and ER-mediated cell death.

C-terminus of HSP70-interacting protein (CHIP) is a ubiquitin ligase highly expressed in the heart that, in cooperation with HSP70 and BCL2-associated athanogene (BAG) family molecular chaperone regulator BAG1, controls degradation of sarcomeric proteins by the proteasome [15].

In the absence of CHIP, ischemic damage leads to an increased infarct size. When misfolded proteins accumulate, proteic aggregates form that can activate autophagy by lysosomal hydrolases. Autophagy is observed in heart failure and ischemic heart disease [15].

The cochaperones BAG3 and HSPb8 operate in autophagic clearance in the heart (chaperone-assisted selective autophagy) [15]. Mutations of the Bag3 gene cause dilated cardiomyopathy.

Chaperones and scaffold proteins promote the formation of proteic complexes and stabilize the interaction of signaling components. The HSP90 substrates include the kinases Src, Raf, PKB, PI3K, and PDK1, as well as NOS3 [15]. Interaction of HSP90 with kinases involves the cochaperone protein Cdc37 (also called HSP90 chaperone protein kinase-targeting subunit).

Most signaling mediators are degraded upon inhibition of HSP90. The latter operates in VEGF-dependent NOS3 activation in endotheliocytes that leads to the disruption of the caveolin–NOS3 complex and promotes the NOS3–HSP90 association. The NOS3–HSP90 dimer recruits PKB that phosphorylates NOS3 (Ser1177) [15].

In addition to HSP90, sHSPs and cochaperones (e.g., HSPb6, HSPb8, melusin, and BAG3) regulate signaling pathways that control cardiomyocyte survival and calcium cycling. The small HSPb6 chaperone promotes cardiomyocyte survival via PKB and inhibition of the MAP3K5–JNK/P38MAPK modules. XS in response to β-adrenergic stimuli, HspB6 that binds to PP1 phosphatase, hence increasing phospholamban phosphorylation, is phosphorylated by PKA (Ser16), thereby activating SERCA2a pump [15].

Morgana and Melusin cooperate with HSP90 to regulate signal transduction in pathophysiological processes. Morgana is ubiquitously expressed, whereas Melusin is a striated muscle-specific chaperone [16]. Morgana controls genomic stability by regulating the centrosome cycle via the RoCK2 kinase. Melusin, or integrin-β-binding protein Itgβ1BP2 organizes ERK signaling in cardiomyocytes and regulates cardiac hypertrophy in response to pressure overload.

Melusin, in association with HSP90, promotes the organization of the Melusin signalosome that regulates ERK1, ERK2, and PKB signalings in response to different stresses. The Melusin signalosome comprises the scaffold and RhoGAPIQGAP1 that binds to FAK as well as the kinases of MAPK modules cRaf, MAP2K1, MAP2K2, ERK1, and ERK2.

Chaperones can tether to polypeptide variants encoded by mutated genes and support their refolding, hence restoring their function. Moreover, HSP90 can prevent transposon-induced genetic variations via piRNAs involved in transposon silencing [15].

The Hspb5 gene is mutated (R120G) in certain types of cardiomyopathies. The R157H mutation that affects the interaction of HSPb5 with titin can be detected in dilated cardiomyopathy. Conversely, overexpression of HSPb5 prevents formation of proteic aggregates of desmin variants that cause cardiomyopathies. A heterozygous Hspb6 point mutation (P20L) can also be identified in some cases of dilated cardiomyopathy.

The mitochondrial chaperone HSP40 contributes to the prevention of dilated cardiomyopathy. Overexpression of HSPb1 ensures protection against pacing-induced cardiomyocyte damage, as it preserves sarcomeric constituents from proteolysis by calpain [15]. HSPb6–HSPb8 also protect against tachycardia-induced atriomyocyte remodeling.

In cardiomyocytes, stretch and hence contraction trigger the production of reactive oxygen and nitrogen species that target Ca-signaling proteins ( mechanochemotransduction). This process involves nitric oxide synthases (NOS1 more than NOS3), NADPH oxidase NOx2, and Ca-calmodulin-dependent kinase CamK2 [17].

The NOS1 subtype is situated closer to the Ca release sites, that is, to ryanodine receptors. The NOS1 isoform, but not NOS3, operates in afterload-induced Ca sparks via elevated ryanodine receptor sensitivity, thereby enhancing contractility to counter mechanical load [17].

On the other hand, spontaneous Ca sparks during diastole can be arrhythmogenic. Both CamK2 and NOx2 also contribute to afterload-induced Ca sparks. However, mechanotransduction launched by NOS1 and CamK2 does not depend on NOx2, as inhibition of NOS1 and CamK2, but not NOx2, in cardiomyocytes eliminates Ca sparks, at least in a mouse model of familial hypertrophic cardiomyopathy with enhanced mechanotransduction [17].

Cycle of Ca influx and outflux from the cytosol relies on Ca store refilling. The latter process is carried out by Ca reuptake from the cytosol and activation of Ca influx using the slow store-operated Ca entry (SOCE) mechanism. The latter is delayed by more than 30 s after store depletion.

In cardiomyocytes, SOCE coexists with Ca-induced Ca release (CICR) from the ER mediated by the couple formed by plasmalemmal voltage-gated Ca channels and ryanodine receptors on the apposed ER membrane.

In response to binding of angiotensin-2 and endothelin-1 to their respective GPCRs, the newly produced inositol trisphosphate (IP) binds to its receptor (IPR) on the ER membrane, causing Ca release from its main store.

Circulating sphingosine 1-phosphate signals via RhoA GTPase that promotes cardiomyocyte survival and PLCε to prime the phosphorylation (activation) of protein kinase-D1, thereby ensuring cardioprotection against ischemia–reperfusion injury [29]. The PKD1 kinase phosphorylates (inhibits) the cofilin phosphatase slingshot homolog SSH1, thereby impeding cofilin-2 translocation to mitochondria in response to oxidative stress or ischemia–reperfusion injury. Cofilin-2 links to the proapoptotic protein BAX. Therefore, S1P preserves mitochondrial integrity upon oxidative stress and supports cardiomyocyte survival.

The member of the forkhead box family of transcription factors FoxO1 is involved in oxidative stress response, immunity, pluripotency in embryonic stem cells, and regulation of cell metabolism, proliferation, and death (Vol. 4, Chap. 10. Other Major Types of Signaling Mediators).

The FoxO1 factor promotes gluconeogenesis, as it stimulates synthesis of hepatic glucose 6-phosphatase and phosphoenolpyruvate carboxykinase [30]. In skeletal muscle, FoxO1 raises expression of pyruvate dehydrogenase kinase, thereby decreasing glucose oxidation. In the heart, FoxO1 regulates glucose and fatty acid metabolism, as it targets the genes that encode pyruvate dehydrogenase kinase PDK4 and nitiric oxide synthase NOS2 [30].

In the heart, cellular insulin-stimulated glucose uptake through the GluT1 (basal glucose uptake) and mainly GluT4 (stimulated glucose uptake) and glycolysis leads to the formation of pyruvate that undergoes oxidation in the mitochondria to yield ATP. In cardiomyocytes, insulin resistance is linked to PDK4 overexpression, hence with lowered glucose and heightened fatty acid oxidation due to nuclear FoxO1 action [30].

The FoxO1 factor also contributes to the regulation of the supply and oxidation of fatty acids. Dietary fatty acids are delivered to the heart from lipoprotein lipase-mediated lipolysis of triglyceride-rich lipoproteins as well as by albumin-bound fatty acids. The uptake of fatty acids by cardiomyocytes across the plasma membrane is mediated by three protein-fatty acid translocases: plasmalemmal and mitochondrial glutamate oxaloacetate transaminase GOT2, fatty acid transport protein (FATP), and the ScaRb3 receptor. AcylCoA synthase enhances fatty acid uptake. The FoxO1 agent is implicated in lipid supply and storage, as its nuclear localization is correlated with an elevated plasmalemmal density of ScaRb3.

The FoxO1 factor is involved in the regulation of the oxidative stress response. It increases expression of antioxidant genes such as those that encode superoxide dismutase and the stress sensor GADD45, thereby promoting ROS scavenging and preventing DNA damage [30]. It collaborates with SIRT1, hence activating expression of antioxidant genes such as those that encode SOD and catalase.

In embryonic stem cells, FoxO1 can maintain their pluripotency, a site acts on promoters of the OCT4 and SOX2 genes.

β-adrenoceptor regulation of cardiac contraction and gene expression is mediated by protein kinase-A. The PKA activity is also controlled by several other messengers acting via Gs-coupled receptors. The velocity and specificity of β-AR signaling are determined by the localization of the PKA holoenzyme and associated A-kinase anchoring proteins as well as the spatiotemporal pattern of cAMP signaling.

The second messenger cAMP is small diffusible molecule. The cAMP–PKA couple is organized into signaling complexes organized by A-kinase anchoring proteins that tether inactive PKA holoenzymes and other signaling molecules and distributed in discrete compartments in cardiomyocytes [31]. From these cellular foci, specific cAMP signals propagate to determined subcellular loci. In cardiomyocytes, AKAP6 anchors PKA to multiple subcellular compartments, including the nucleus and nuclear membrane [32]. In the nucleus, AKAP6 also connects to PDE4d3 that controls the cAMP level.

On the sarcoplasmic reticulum in cardiomyocytes, PKA regulates the calcium cycling for excitation–contraction coupling that is dysfunctional in heart diseases. The direct activation of adenylate cyclases or application of a cAMP analog provokes a much slower and smaller increase in PKA activity than stimulated adrenergic receptors [31]. The signaling primed by adrenoceptors has a preferential access to the sarcoplasmic reticulum, the major calcium store. Calcium ions are released through ryanodine receptors and taken up through sarco(endo)plasmic reticulum Ca ATPase. The triggered cAMP–PKA signaling leads to phosphorylation of these ion carriers and/or associated regulatory proteins, thereby raising Ca flux through these channels to adapt cardiac contraction.

In the absence of cAMP, the PKA holoenzyme is a heterotetramer consisting of two catalytic subunits bound and inhibited by a dimer of regulatory subunits. Activation of adenylate cyclases increases intracellular cAMP concentration. This second messenger dissociates (activates) the catalytic subunits.

Then, PKA phosphorylates many substrates in various subcellular compartments, such as the ion channels involved in excitation–contraction coupling, sarcolemmal Ca1.2a and sarcoplasmic reticulum RyR2, the SERCA inhibitor phospholamban (Pln), and the sarcomeric components cardiac myosin-binding protein-C (cMyBPc) and troponin-I (cTnnI; Sect. 4.​1.​1).

In the nucleus of cardiomyocytes, PKA targets the transcription factors of the CREB family and class-2 histone deacetylases HDAC4 and HDAC5.

The cAMP concentration depends on its production by adenylate cyclases and degradation by phosphodiesterases (PDE1–PDE4 and PDE8) in cardiomyocytes. Members of the cAMP-specific PDE4 family (PDE4a–PDE4b and PDE4d encoded by three genes) are major inhibitors of the cAMP messenger. At the nuclear envelope, PDE4d complexes with AKAP6. It is involved in the regulation of cardiomyocyte hypertrophy. The loss of PDE4d can cause dilated cardiomyopathy, at least in mice. AKAP-anchored PDE4 controls the extent of PKA released upon βAR stimulation and transferred into the nucleus from a cytosolic pool [32].

In the cytoplasm, phosphatases PP1 and PP2 contribute to the termination of PKA action, whereas only PP1 acts in the nucleus [32].

In adult rat ventriculomyocytes, β-adrenoceptor stimulation raises quickly the cytoplasmic and nuclear cAMP concentration, whereas PKA activity elevation is prompt in the cytoplasm, but slower in the nucleus [32]. Similar slow kinetics of nuclear PKA activation is observed upon adenylate cyclase activation or phosphodiesterase inhibition. Pulse stimulation causes maximal PKA activation and phosphorylation of myosin-binding protein-C in the cytoplasm, but has a slight impact on PKA activation and phosphorylation of cAMP response element-binding protein in the nucleus.

The nuclear PKA activity is temporally dissociated from that of nuclear cAMP elevation and cytoplasmic PKA activation. Once PKA holoenzyme is activated in the cytoplasm, its catalytic subunit translocates to the nucleus [32].

In cardiomyocytes, the upstream inhibitors PDE3 and PDE4 and downstream inhibitors PP1, PP2, and PP3 can participate in the cytoplasmic and nuclear PKA response to βAR stimulation.

The PDE3 and PDE4 enzymes represent the major PDE activity in cardiomyocyte nuclei. They primarily localize at the nuclear envelope. The PDE4d subtype controls the nuclear as well as cytosolic PKA activity, whereas PDE3 has no effect [32].

The PP1 and PP2 phosphatases contribute differently to PKA target sites in the cytoplasm and nucleus. They counteract the action of the βAR–cAMP–PKA axis, especially the phosphorylation of mediators of the electromechanical coupling, but PP3 has no effect [32]. The PP3 phosphatase may play a minor role when PP1 and PP2 are active. However, PP3 intervenes in pathogenesis, especially cardiac adverse hypertrophy resulting from pressure overload. In particular, PP3 dampens β AR effect on Pln in spontaneously hypertensive rats. Moreover, it is detected in the nucleus of cardiomyocytes in the diseased myocardium, but not in the case of healthy hearts [32].

In addition, cAMP can also activate GEFs (RapGEF3–RapGEF4) at the plasma membrane and nuclear and perinuclear compartment area in cardiomyocytes [32]. These effectors increase nuclear Ca concentration as well as CamK2 dependent nuclear export of HDAC5 and subsequent derepression of the MEF2 transcription factor. Unlike CamK2- and PKD-mediated phosphorylation, PKA phosphorylates HDAC5 in the nucleus, thereby causing its nuclear retention and the repression of the hypertrophic gene program.

A sustained βAR activation, as that occurring during intense and prolonged physical exercise or after myocardial injury, increases nuclear PKA activity and CREB phosphorylation [32]. Transcription factors of the CREB family can contribute to βAR-dependent maladaptive remodeling.

After cardiac injury, multiple signaling pathways, such as the Notch, Wnt, TGFβ, and BMP axes, are activated.

The Notch-primed signaling exerts a negative feedback on the Wnt–β Ctnn pathway; the intracellular domain of Notch receptor can bind to Wnt signaling effectors such as Disheveled, thereby inhibiting Wnt signaling.

Calcium ions are major second messengers in both the cardiac pump and vasculature. Localized changes in Ca ions encompass waves, oscillations, sparks, sparklets, puffs, transients, and flashes, which can correspond to different triggering events.