Cardiovascular diseases (CVD) are the leading cause of hospitalization and death globally, and their incidence is continuously increasing ^[1][2][1,2]. CVD is a broader term which includes disturbances of the heart rhythm, cardiac valve pathologies, genetically driven malformations and, ultimately, peripheral or coronary artery diseases (PAD and CAD), which may culminate, respectively, in critical limb ischemia (CLI) and heart failure (HF).

The use of cells with stem/progenitor characteristics in PAD and CLI has shown a success in clinical translation to a certain extent, given the ability of the chosen cells (e.g., derived from bone marrow, peripheral blood, or cord blood) to promote de novo vasculogenesis by a robust “paracrine effect” [3]. By contrast, the use of a similar setting to regenerate the contractile mass of the heart to compensate the loss of myocytes due to acute/chronic ischemia and/or inflammation has been largely unsuccessful and controversial, due to the absence of resident stem cells that could be activated in situ and/or expanded in vitro prior to being reinjected into the failing hearts [4]. Alternatives to this deficiency have been sought in the use of induced pluripotent stem cells (iPSCs), whose derived cardiomyocytes (CMs) have been employed in preclinical models in small and large animals ^[5][6][5,6], and even in pioneering studies in humans [7]. Although scaled-up systems to produce therapeutic quantities of these cells with enhanced purity have been set, anticipating industrial production [5], several caveats have been expressed due to risks of arrhythmogenicity, incomplete maturation, potential tumor formation, and (at least for allogenic use) immune reactions [8].

Given the lack of endogenous regenerative capacity of the myocardium, the consequence of acute/chronic cardiac ischemia is still considered an irreparable damage leading to progressive replacement of the contractile cells with a stiff, fibrotic scar. Under these conditions, the heart undergoes a series of morphological transformations (e.g., rearrangement of the contractile apparatus and modification of the geometry [9]), changes in mechanical characteristics [10] and reduction of the pumping efficiency [11], representing signs of HF.

With the advent of the tissue engineering [12], the introduction of biological fabrication methods combined with refined systems for cellular genetic manipulation [13] and decryption of mechano-sensitive cues [14] has enabled new strategies to enhance the efficacy of cardiac cell therapy and the elaboration of disease modeling systems using 3D biology approaches [15]. This renews the hope that after the disappointment arising from the failure of the “classical” cell therapy approaches, it will be possible to reach a condition to regenerate the human heart, which still represents the “holy grail” of cardiology.

2. Xenotransplantation and Cardiac Repair

Despite advances with pharmacotherapies and devices, the only curative option for end-stage heart failure is orthotopic heart transplant (OHT) ^[16][122]. However, due to the restricted number of human organs available for transplantation, only few patients can receive cardiac allotransplant each year ^[17][123]. The imbalance between the number of hearts necessary for transplantation and the availability of transplantable organs is continuously growing, and this makes the elaboration of alternative solutions extremely urgent. In this respect, xenotransplantation of animal-derived (e.g., pig) hearts may provide an ethical and unlimited resource. Heart xenotransplantation has been so far addressed in several animal studies, including on non-human primates as the closest species to humans ^[18][124], even if recently a gene-edited porcine heart was transplanted in a patient with an end-stage heart failure patient ^[19][20][125,126]. The recipient patient lived for two months receiving high doses of immunosuppressive agents, hence becoming the longest-living human survivor of a cardiac xenotransplant. Importantly, the technical success of this intervention warrants future clinical studies to refine immunosuppression protocols, monitor the possible introduction of infectious pathogens, evaluate the graft function, and assess the physiological performance of the xenotransplant.

2.1. Immunological Challenges

The major challenge with organ xenotransplantation is related to the expression of peculiar tissue antigens that cause acute rejection in humans. One of the most well-known of these antigens is the oligosaccharide galactose-α1,3-galactose (αGal), which is present in all mammals with the exception of humans and non-human primates of the old world ^[21][127]. Antibodies recognizing the αGal antigen rapidly activate the complement-mediated immune response, resulting in acute rejection of the transplanted organs due to a tissue degeneration process occurring within minutes to hours by primarily targeting the vascular endothelial cells ^[22][23][128,129]. Graft failure and thrombotic microangiopathy can be also induced in the transplanted organ due to activation of vascular endothelial cells by low levels of anti-αGal antibodies or abnormal coagulation due to incompatibilities in the coagulation/anticoagulation factors ^[24][25][130,131]. Concerning cellular xenotransplantation (e.g., pig-derived cardiac cells in human hearts), although the hyperacute xenograft rejection does not occur, xenogeneic cells can still trigger immune responses, such as the activation of T-cells through direct and indirect pathways. This direct activation is elicited by the binding of T-cell receptors of the recipient to swine leukocyte antigen class I and class II on porcine donor antigen-presenting cells, such as dendritic cells or endothelial cells constitutively expressing CD80/86 ^[26][132]. An indirect activation of the immune system is also initiated by the recognition of porcine-specific antigens by major histocompatibility complex (MHC) class II of the recipients. The subsequent T-cell stimulation results in B-cell activation and antibody production, thus mediating humoral xenograft rejection ^[27][133]. Several strategies have been elaborated to circumvent the problem of xenoreactive T-cells, such as the repetitive administration of CTLA4Ig (abatacept) and anti-CD40 mAb, as well as anti-CD154 mAb via CD28 ^[27][133]. This is, so far, the most successful approach, with positive results for porcine skin ^[28][29][134,135], pancreas ^[29][135], and heart transplantation ^[30][136].

2.2. Genetic Engineering Strategies of Animals and Cells to Ensure Immunological Compatibility

A breakthrough in xenotransplantation has been provided by the generation of α1,3-galactosyltransferase gene-knockout (GTKO) pigs by cloning ^[31][137], or nuclear transfer of spontaneously null mutant cells ^[32][138], thus opening the way to the transplantation of xenoantigen-free organs. Unfortunately, despite that organs and cells from the GTKO pigs demonstrated prolonged graft survival, xenograft rejection still occurred and was associated with the activation of the innate immune system and coagulation ^[33][139]. Therefore, to further improve the outcomes, GTKO pig cells were engineered to express human complement-regulatory proteins, as investigated in vitro ^[34][140] and in vivo ^[35][141]. In the meantime, other porcine antigens, e.g., the N-glycolylneuraminic acid (NeuGc), were found to induce natural antibodies in humans, thereby explaining the suboptimal results obtained with the heterotropic transplantation of GTKO pig tissues ^[36][142]. The disappointing results of single-knockout animals were at least in part corrected by the generation of pigs with multiple knockouts. For example, in 2015 double-knockout pigs lacking the genes for the N-glycolylneuraminic acid and galactose α-1,3-galactose to prevent the adverse effects of the antibody–antigen interactions were generated ^[37][143]. The addition of a further mutation in the β1,4 N-acetylgalactosaminyltransferase (Sda) (β4GalNT2) gene to the previous double-knockout background further increased compatibility ^[38][144]. In another study, pigs overexpressing pCTLA4-Ig were produced both from a wild-type and GTKO background to address T cell-mediated immune response. However, these pigs exhibited reduced humoral immunity, which made necessary the use of antibiotics to maintain their health ^[39][145]. An alternative way to that of generating mutant animals for combinations of xenoantigens involves, finally, the overexpression of immune regulatory ligands. For example, by overexpressing the human programmed cell death ligand 1 (PD-L1), known to suppress the proliferation of human CD4 T-cells and enhance regulatory T-cells coupled expansion with increased interleukin-10 production, a focus was made on the potential of using human transgenes to foster tolerance of other cell and tissue xenotransplants ^[40][146]. Taken together, these results suggest that an ideal candidate as a xenogenic donor of hearts for human transplantation might be a pig carrying multiple xenoantigen knockout mutations, and engineered to expresses low levels of human complement regulatory proteins and human coagulation regulatory proteins. In this respect, the expression of human transgenes, such as heme oxygenase 1 and CD47, might be valuable for improving graft survival due to general anti-inflammatory effects and the suppressive effect on monocyte and macrophage function ^[41][147].

3. Harnessing Cell Mechano-Sensation to Repair/Regenerate the Heart

Cells are continuously exposed to mechanical stimuli deriving from the surrounding ECM or from neighboring cells. The term “mechano-transduction” indicates the ability of cells to convert these physical signals into intracellular signaling and biological responses affecting cell phenotype and functions. This mechanism requires the involvement of specific molecules expressed at the cell membrane that act as mechano-sensors, such as integrins, stretch-activated ion channels or G protein-coupled receptors ^[42][148]. Due to their phenotypic control in differentiated cells and cells with progenitor characteristics [14], the integration of mechanical cues in the current design of advanced cardiac engineering is becoming a crucial component.

3.1. From Force Decryption to Intracellular Signaling

The force sensing starts from the focal adhesion, a large complex which contains several specialized cytoplasmic proteins that communicate directly with the cytoskeleton. In particular, in mature focal adhesions, the transmembrane proteins integrins are connected with ECM proteins through the extracellular head domains and with actin cytoskeleton through the activation of talin and vinculin ^[43][149]. Additionally, several actin-regulating proteins are involved in integrin-dependent force transmission ^[44][150]. During the initial adhesion formation, integrins are linked to the cytoskeleton by talin, then α-actinin competes with talin for the binding to integrin tails. At this point, α-actinin links actin to integrins and transmits forces to the ECM to complete adhesion maturation. The transduction of the signal subsequently occurs through contraction/alteration of the cytoskeleton that activates downstream signaling. In particular, it has been demonstrated that the integrin-focal adhesions complex modulates the Hippo pathway and its nuclear transducers, YAP (Yes-associated protein) and TAZ (transcriptional coactivator with PDZ-binding motif, also known as WWTR1) ^[44][150]. The activity of the YAP/TAZ complex is related to the mechanical status of the cells and it is controlled by the geometric constraints of the cells, or stiffness of the adhesion substrate. In particular, the YAP/TAZ duo is translocated from the cytoplasm (where it is transcriptionally inactive) to the nucleus (where it is active) depending on mechanically activated phosphorylation status controlled by the Hippo kinase pathway ^[45][151]. Increased substrate stiffness or cell spreading shift the equilibrium toward a nuclear localization of the complex, which is then free to interact with TEA domain (TEAD) DNA binding proteins to regulate gene transcription ^[46][152]. By contrast, in cells subjected to low forces such as by adhering onto substrates with low stiffness, or in the occurrence of less spread shapes, YAP/TAZ are phosphorylated by Hippo kinases and this causes an increase in proteasomal degradation and consequent reduction of the transcription factor in the nucleus ^[46][152]. In addition to the reversible control of the degradation by the kinase pathway, the YAP/TAZ duo can be forced to enter the nucleus by the stress-fibers-dependent physical deformation of the nuclear lamina and the consequent opening of the nuclear pores ^[46][152]. Transmission of forces from cytoskeleton to the nucleus is particularly relevant, not only to allow the trafficking in and out of transcription factors, but also to alter the topology of the nucleus by affecting the activation status of the chromatin and promoting gene transcription ^[47][153]. Indeed, interfering with actin cytoskeleton polymerization using pharmacological inhibitors not only affects YAP nuclear localization, but also determines a relaxation of the chromatin resulting in a decrease nuclear stiffness and a reduction of canonical YAP targets transcription ^[48][154]. The case of the YAP/TAZ complex is not unique; in fact, other transcription factors involved in cardiac biology are also mechanically regulated. One example is the pathway controlled by the serum response factor (SRF) transcriptional activator and the co-activator myocardin-related transcription factor (MRTF), which requires Rho-dependent actin polymerization to translocate into the nucleus. MRTF interact with SRF controlling the transcription of genes related to ECM production and smooth muscle differentiation ^[49][155]. Opposite to the YAP/TAZ-dependent pathway, interfering with MRTF nuclear localization using actin cytoskeleton inhibitors prevents cell differentiation and the onset of pro-pathological conditions ^[50][156]. Mechanical stimulation controls, finally, the transcriptional activity of β-catenin by promoting the phosphorylation of the protein at a specific tyrosine residue implicated in the interaction with E-cadherin. The consequent reduction in the interaction with the adhesion molecule favors the phosphorylated β-catenin nucleus translocation where, in turn, it activates the transcription of WNT-responsive genes ^[50][156].

3.2. Mechanotransduction in Heart Physiology and Pathology

Mechanotransduction plays an important role in driving cardiac morphogenesis. Changes in ECM stiffness in the heart primordium promote the initial beating of the embryonic myocytes by opening the mechanosensitive Ca²⁺ channels, before the onset of electromechanical coupling ^[51][158]. The stiffness of the surrounding ECM also plays a role in the switch between fetal proliferative and early postnatal hypertrophic growth. In fact, the stiffening of the ECM surrounding the proliferating myocytes promotes sarcomere organization, allowing cardiomyocytes to contract with a higher force ^[52][159]. By contrast, the softening of the cardiac matrix by treating with collagenases suppresses beating by altering cytoskeletal conformation, preventing sarcomere assembly into cardiomyocytes ^[53][160], and extending the cardiomyocyte proliferation phase in vivo ^[54][161]. At early postnatal and adult stages, cardiomyocytes do not proliferate and cardiac growth is supported by the expansion of cellular size (hypertrophy). This event is characterized by an apparently irreversible cardiomyocyte differentiation due to the inability to complete cytokinesis. While the inhibition of cell division is essential to prevent uncontrolled growth of the myocardium, it also limits the ability of the cardiomyocytes to re-enter the cell cycle and regenerate the injured heart. The fine modulation of ECM rigidity could play a major role in this. This evidence is supported by experiments in zebrafish, in which a transient softening of the ECM allows the dedifferentiation of cardiomyocytes sarcomere structure, and thus the regeneration of cardiac tissue by reactivation of myocytes growth ^[55][162]. The relationship between matrix stiffness and the reversible differentiation of cardiomyocytes has been further demonstrated by experiments performed on low-stiffness gels where a higher proliferation was maintained ^[56][163]. This finding has an interesting readout in patients implanted with left ventricle assist devices, where the mechanical unloading of the ventricular tissue seems to be accompanied by the restoration of myocytes’ proliferation ^[57][164]. Altogether, these demonstrations suggest that a fine tuning of mechanical and viscoelastic characteristics of the extracellular environment could be crucial to promote re-entry of the myocytes into the cell cycle, thus giving rise to an authentic cardiac regeneration program ^[57][164].

3.3. Mechanical-Dependent Pathologic Signaling

Besides instructing the correct differentiation of stem and progenitor cells for engineering cardiac tissues, mechanical proprieties of the matrix have also been shown to drive the differentiation of cardiac-resident cells towards pro-pathological cell fates ^[58][165]. This is relevant for progression of the maladaptive myocardial remodeling (one of the processes predisposing to HF) due to inflammation and an excessive production of ECM proteins by the so-called “myofibroblasts” ^[59][166]. Mechanical cues are emerging as a major driver of cardiac myofibroblast activation. In a recent work ^[59][166], for example, reswearchers have shown that after myocardial infarction, cardiac stromal cells are directly exposed to incremental strain/compression forces that induce the activation of the YAP-dependent transcriptional pathway, leading to myofibroblast activation and abundant collagen deposition. Blockading of the YAP/TAZ complex using verteporfin, a drug that interferes with the binding of YAP to TEADs, attenuates cardiac fibrosis and remodeling. It was interesting to note that inhibition of the mechanical pathway by treating cells with the drug overrode the TGFβ-dependent myofibroblasts’ activation ^[60][167]. While this revealed a crosstalk between mechanical cues and the humoral control of fibrosis, it also showed that mechanical cues are prevalent in the pathological vs. physiological differentiation of cardiac fibroblasts. The cooperation between the Hippo/YAP pathway and TGFβ signaling was also revealed by another study published by reusearchers. The researchers. In this study, we showed that interfering with the YAP/TAZ transcriptional function was sufficient to revert the matrix compaction ability of human cardiac fibroblasts primed with TGFβ ^[61][168], again confirming the relevance of mechanical cues for pathology progression.