Ca²⁺ acts as the second messenger and participates in multiple biological processes in cells. Ca²⁺ plays a decisive role in the systolic function of the heart. Generally, the intracellular Ca²⁺ content is much lower than that in extracellular (7). In normal physiological conditions, intracellular Ca²⁺ content is usually lower than 0.05 μmol/L (8). Ca²⁺ in cardiomyocytes mainly comes from extracellular and sarcoplasmic reticulum (SR) (9). When the myocardial cell membrane is depolarization, the L-type Ca²⁺ channels (LTCC) on the cell membrane are open, issues in the flow of extracellular Ca²⁺ into the cells. When intracellular Ca²⁺ concentration is higher than 10 μmol/L, ryanodine receptors (RyR) in the SR are activated, Ca²⁺ in SR flow to the cytoplasm, which is called Ca²⁺-induced Ca²⁺ release (10). Ca²⁺-induced Ca²⁺ release is the core of the calcium signal transduction mechanism (11). In general, the increase of Ca²⁺ will lead to myocardial contraction, and then Ca²⁺ in the myocardial diastolic phase is transported back to the SR through SR Ca²⁺ ATPase (SERCA) or transferer to extracellular through Na⁺/Ca²⁺ exchanger (NCX) in the plasma membrane. However, during ischemia, ATP deficiency inhibited Na⁺/K⁺-ATPase and reduced Na⁺ excretion, anaerobic glycolysis led to cytoplasmic acidification, and the increased H⁺ was discharged through Na⁺/H⁺ exchanger (NHX) to promote extracellular Na⁺ influx. Together with Na⁺/HCO₃⁻ cotransporter and persistent Na⁺ channels, all mechanisms increase intracellular Na⁺ content (12). Abnormally elevated intracellular Na⁺ changed the membrane potential leading to NCX reverse exchange, which increased intracellular Ca²⁺ concentration (13). Moreover, the reperfusion blood flow took away the extracellular H⁺, which led to increased gradient difference of H⁺ between intracellular and extracellular. The combined effects were strengthened, and the intracellular Ca²⁺ concentration would further increase (6). In addition, studies have shown that Ca²⁺-sensitive RyR is disrupted during myocardial I/R (14). The leakage of Ca²⁺ in the SR is also the cause of calcium overload.

Calpain is an intracellular non-lysosomal cysteine protease, one of the few proteases that the second messenger Ca²⁺ can directly activate. Due to the proteolysis ability of calpain being finite, calpain mainly plays a role in regulating rather than removing substrates in cells (15). Up to now, 15 types of calpain isoforms have been identified in the human body (The distribution of calpain in the human body is shown in Table 1) (15–17), among which calpain-1 (μ-calpain) and calpain-2 (m-calpain) are the most common, they are activated by micromolar and millimolar concentrations of Ca²⁺ in vitro experiments, respectively (18). Usually, intracellular Ca²⁺ concentration can only activate calpain-1 in physiological conditions. However, in pathological conditions, such as ischemia-reperfusion, intracellular Ca²⁺ concentration is higher than that in normal conditions. Abnormally elevated Ca²⁺ are combined with other mechanisms, such as phospholipids or phosphorylation, to activate calpain-2 (19). Calpain is usually in the form of an inactive enzyme that binds to calpastatin, an endogenous calpain inhibitor, to form an isomerase dimer, which can be located in the cytoplasm, endoplasmic reticulum, or mitochondria (20). Calpastatin inhibits calpain activation by binding to calpain through its calpain inhibitor domain (21). When the intracellular Ca²⁺ concentration was increased, calpain was separated from calpastatin and allowed to shift to the membrane (22). Calpain transfers to the cell membrane, binding to phospholipids to reduce the required Ca²⁺ concentration for activation or directly to the vicinity of Ca²⁺ channels. The activation of calpain is also affected by oxidative stress (23, 24). The reactive oxygen species produced by oxidative stress can directly inhibit ATPase, reduce the outflow of Ca²⁺ and destroy the calcium channels on the SR, resulting in the Ca²⁺ in the SR flowing into the cytoplasm, thereby activating calpain. After reaching the threshold of Ca²⁺ concentration for activation, the inactive enzyme was self-melted into the active form (18), stayed on the membrane, or was released into the cytoplasm to hydrolyze substrate proteins. Intracellular acidification significantly inhibits the activation of calpain (25). Therefore, calpain usually has the highest activity at the reperfusion stage after the pH value returns to normal. Calpastatin is another major factor affecting the activation of calpain in addition to the concentration of Ca²⁺ (26). Calpastatin plays a role in preventing calpain's excessive activation with the increased Ca²⁺ concentration. In contrast, calpastatin will be degraded by activated calpain or proteasome during myocardial ischemia-reperfusion (25, 27), issue in increased activation of calpain. Due to the activation of calpain being associated with Ca²⁺, which is an uneven distribution in cells, the subcellular localization of calpain may be related to its activation. Recent studies have found an interaction between the integrated membrane protein tiny tim50 (Ttm50) and calpain. This interaction promotes the directional movement of calpain to the Golgi body or endoplasmic reticulum, which is responsible for calcium storage. Moreover, Ttm50 increases the sensitivity of calpain to Ca²⁺ and enhances the activation of calpain (28). The possible mechanism of Ttm50 promoting calpain activation is: calpain is guided to move to a relatively high calcium environment in cells, and the conformation of calpain is changed to increase its affinity to Ca²⁺ so that calpain can be effectively activated in physiological conditions (19). Of course, there are other mechanisms to reduce the dependence of calpain activation on Ca²⁺ concentration, such as calpain phosphorylation and phospholipid-mediated calpain autolysis. Several phosphatidylinositols have been found to increase the autolysis of calpain on the cell membrane (29). ERK directly or indirectly activates calpain through phosphorylation of calpain or reduced Ca²⁺ concentration required for activation (30, 31). Up to now, the activation and regulation mechanism of calpain has not been fully understood, which needs further exploration (The activation mechanism of calpain during I/R is detailed in Figure 1).

Apoptosis is a highly controlled process of cell death, independently carried out by entirely healthy or sublethally injured cells in response to physiological or pathological stimulation (64). Several experiments have shown that inhibition of apoptosis during I/R can reduce myocardial infarction size in mice (65–67). Apoptosis signal can be transmitted through the cell surface death receptor pathway (external pathway) and mitochondrial pathway (internal pathway). Caspase plays a vital role in apoptosis (68). The caspase family mainly has three subclasses: promoter (caspase-8, 9, 10), effector (caspase-3, 6, 7), and inflammatory factors (caspase-1, 4, 5). Calpain can directly act on caspase family proteins to influence apoptosis—for example, calpain cleavages caspase-3 and caspase-7 to activate apoptosis (39–41). Calpain also can cleavage caspase-12 to trigger apoptosis during endoplasmic reticulum stress (42). Caspase-3 also cleavages endogenous calpastatin after activation, enhancing calpain activity (69). Calpain can cleavage inhibitor apoptosis protein (IAP), such as cellular IAP2 (cIAP2), and X chromosome-linked IAP (XIAP), to increase the expression of caspase-3 and promote the occurrence of apoptosis (44). IAP acts as a caspase inhibitor in cells (70) and binds to caspase-3,7 and 9 through its baculovirus IAP repeat (BIR) domain to block caspase activation and negatively regulate intrinsic and extrinsic apoptosis pathways (71).

Calpain can affect apoptosis not only by directly interfering with caspases but also by cutting essential BCL-2 family proteins in the mitochondrial apoptotic pathway (45, 72, 73). BCL-2 family proteins are divided into three subtypes: pro-survival BCL-2 family members, such as BCL-2 and BCL-XL; the multi-BH-domain pro-apoptotic members, such as BAX, BAK, and BOK; the pro-apoptotic “BH3-only” proteins, such as BIM, BID, and BAD (74). The three subtypes interact to regulate the permeability of the mitochondrial outer membrane (75). The main process of mitochondrial apoptotic is BIM, BID activated by intracellular death signal, activated BIM, BID stimulates BAX, BAK and inactivates pro-survival BCL-2 protein. BAX or BAK can form a channel on the outer membrane of mitochondria. Cytochrome C, apoptotic peptidase activating factor 1 (APAF1), and deoxyribonucleotide complexes enter the cytoplasm through the channel and assemble into apoptotic body complexes (76), which activate caspase-9, and its downstream caspase-3 induces apoptosis (77). Under the stimulation of ischemia-reperfusion injury, calpain-1 cuts BID into t-BID to activate it (47). T-BID can induce apoptosis by affecting mitochondrial outer membrane permeability in the following three ways: (i) Mediated BAX and BAK oligomerization; (ii) Self-forming homodimer; (iii) Induced mitochondrial permeability transition pore (mPTP) opening and remolded mitochondrial inner membrane (78). Decreasing calpain-1 expression by specific siRNA can significantly decrease isoproterenol-induced expression of Bax, t-Bid and cytochrome C release in cardiomyocytes (79). In addition, calpain can also cleave the N-terminal of BAX into an 18 kDa fragment to activate BAX and promote cytochrome C release from mitochondria to induce apoptosis (45).

However, some researchers have shown that calpain-2 can be used as a negative regulator of caspase activation and apoptosis. Activation of calpain-2 not only failed to cleave and activate caspase-3 but also inhibited it, which was mainly related to the inactivation of caspase-9 by calpain cleaving, blocking the activation of caspase-3 by dATP and cytochrome C (40). In addition, calpain can hydrolyze P53 protein to inhibit apoptosis induced by DNA damage (48). The content of P53 protein increased rapidly during myocardial I/R. Inhibition of P53 significantly attenuated the I/R injury (80). In summary, there may be two effects of calpain on apoptosis induced by myocardial I/R, and different calpain subtypes may have different effects on caspase (The mechanism of calpain affecting apoptosis is shown in Figure 2).

Mitochondrial-mediated necrosis is caused by intracellular microenvironment disorders, such as severe oxidative stress and intracellular Ca²⁺ overload, usually presented as necrosis (81). The critical event of mitochondrial-mediated necrosis is the opening of mitochondrial permeability transition pore (mPTP) that is located on the mitochondrial inner membrane. mPTP opening is mainly mediated by Ca²⁺ concentration and mitochondrial membrane potential. An increase in Ca²⁺ concentration and depolarization of mitochondrial membrane potential will promote mPTP opening. The mPTP opening leads to increased permeability of the mitochondrial inner membrane that results in a dissipation of the proton gradient across the inner membrane. Water entered the mitochondrial matrix along the permeability gradient, resulting in mitochondrial swelling. Since the area of the mitochondrial inner membrane is much larger than that of the outer membrane, the expansion of the inner membrane is often accompanied by the loss of the integrity of the outer membrane. BAK and BAX mediate the formation of large enough pores in the outer membrane to allow the extrusion of the inner membrane. Moreover, proton leakage leads to the disappearance of mitochondrial membrane potential and the inhibition of ATP synthase leads to oxidative phosphorylation uncoupling, which lead to mitochondrial dysfunction. Dysfunctional mitochondria cannot produce enough energy to close mPTP, eventually leading to necrosis (82). The specific mechanism of necrosis caused by the opening of mPTP is not precise, which may be due to the increase of glycolysis rate cannot offset the depletion of cell energy and leads to the acidification of the cytoplasm. ATP deficiency damages the enzymes that pump Na⁺, H⁺, and Ca²⁺ out of the cell and the enzymes that pump Ca²⁺ into the endoplasmic reticulum. The sharp loss of Na⁺ and Ca²⁺ gradient inside and outside the cell causes cell membrane collapse and cell death with necrosis characteristics (82). Compared with apoptosis, the process of intracellular energy consumption (such as DNA repair and protein translation) during necrosis does not terminate. On the contrary, energy consumption has been intensified, and the intracellular energy contradiction has been aggravated. Therefore, a sharp decrease in ATP is also a sign of necrosis (5). Calpain exists not only in the cytoplasm but also in the mitochondria. Studies have shown that calpain activity in cardiac mitochondria increases during I/R (83). Ca²⁺ and ROS are considered to be two key activators for mPTP opening (84). Calpain induces Ca²⁺-induced mPTP opening through cleavage of complex I subunits (ND6 and NDUFC2) on the electron transport chain (85), The destruction of complex 1 leads to sensitization of mPTP (86). In addition, the increased calpain activity in mitochondrial can impair mitochondrial respiratory chain complex I (49, 50) and ATP synthase, such as ATP synthase subunit α (ATP5A1), these will cause mitochondrial dysfunction and increase the production of reactive oxygen species (ROS) (51, 52). Recent experiments have proven that overexpression of calpain inhibitors can maintain the activities of ATP5A1 and ATP synthase, prevent mitochondrial ROS production and reduce necrosis after hypoxia and reoxygenation (34). In summary, calpain not only promotes the opening of mPTP but also acts on ATP synthase in the process of mitochondrial-mediated necrosis, affecting mitochondrial energy production, aggravating intracellular energy contradiction, and promoting cell necrosis (The mechanism of calpain involving mitochondrial-mediated necrosis is shown in Figure 2).

Autophagy-dependent cell death is a PCD that depends on autophagy and its components (81). Autophagy, a function of conservative evolution, transports endogenous or exogenous substances in the cytoplasm to lysosomes for degradation. It plays a crucial role in quality control, stress alleviation, and recovery of cell homeostasis. It is usually used as a means of survival (87). However, in some pathophysiological environments, autophagy also can cause cell death, known as autophagy-dependent cell death (88). It is unclear whether autophagy-dependent cell death is caused by the high autophagy rate (excessive catabolism of intracellular components) or the change in the nature of the autophagy process itself. Therefore, this paper mainly introduces the role of calpain in autophagy-related molecules. Autophagy is mainly divided into macroautophagy, microautophagy, and chaperone-mediated autophagy. Autophagy usually refers to macroautophagy (88). Autophagy occurs mainly through two steps: autophagosome formation and transfer of autophagic substrates to lysosome (89). Beclin-1, part of the class III phosphatidylinositol-3-kinase (PI3K) complex, is responsible for phosphatidylinositol (PI) phosphorylation to form autophagosome precursors, which is indispensable in the formation of autophagosomes (90). Autophagy during reperfusion is closely related to Beclin-1. Autophagy is attenuated in Beclin-1 knockout mice during myocardial reperfusion (91). Calpain also plays a particular role in autophagy (92). Calpain can lyse Beclin-1 to reduce autophagy activity (53). Calpain also cleaves autophagy-related proteins (ATGs) to influence autophagy (54), ATGs are closely related to autophagosome maturation and autophagy (93), and ATGs play different roles in different stages of autophagy. For example, ATG-5 is involved in forming autophagosomes that encapsulate proteins or organelles. ATG-5 is the cutting substrate of calpain. After being cut, ATG-5 not only inhibits the formation of autophagosomes but also translocates to mitochondria and binds with BCL-XL, resulting in the release of cytochrome C (94). In addition, calpain affects autophagy by cleaving apoptosis-related molecules such as BAX-interacting factor 1 (BIF-1) (55). Although most studies have shown that calpain affects autophagy, most experiments only focus on the effect of calpain on the autophagy mechanism. Autophagy and autophagy-dependent cell death and the relationship between calpain and autophagy-dependent cell death need further research (The mechanism of calpain affecting autophagy-dependent cell death is shown in Figure 2).

Lysosome-dependent cell death is a PCD caused by the permeability of the lysosome membrane; proteolytic enzymes are released. The upstream mechanism of lysosomal membrane permeabilization (LMP) has not been fully understood. Previous studies have indicated that LMP mainly occurs after mitochondrial outer membrane permeabilization, which constitutes an intrinsic apoptosis-associated phenomenon. However, under certain experimental conditions, lysosomal membrane permeabilization can precede mitochondrial permeability, thereby mediating lysosomal-dependent cell death. The mechanism may be related to the recruitment of BAX to the lysosome membrane for pore formation (81). The stability of the lysosome membrane is mainly associated with the cholesterol content of the lysosome and member 1A of heat shock protein family A (HSPA1A; best known as HSP70) (95). Calpain affects lysosomal membrane permeability through various mechanisms (96). Calpain can cleave lysosomal associated membrane protein 2 (LAMP2) on the lysosomal membrane. High glycosylation of LAMP2 can prevent the lysosomal membrane from being dissolved by lysosomal enzyme (97). When LAMP2 is cleaved by calpain, lysosomal membrane permeabilization and proteolytic enzyme release lead to lysosome-dependent cell death (56, 57). Heat shock protein 70 (HSP70) plays a crucial role in maintaining lysosome stability (98). It can increase the activity of acid sphingomyelinase (ASM) by binding to the lysosomal anionic phospholipid bis monoacylglycerol phosphate (BMP) and promote the conversion of sphingomyelin from lysosome membrane to ceramide. Ceramide is one of the sphingolipids, these lipids serve structural roles in biomembranes (99). On the lysosomal membrane, ceramide enhances membrane acyl chain order and increases lateral packing of lipids in vitro (100). The content of ceramide affects the spatial conformation of lysosomal membrane, increases the fusion ability of lysosomal membrane with intracellular vesicles and cell membrane, and thus affects the composition and volume of lysosomal membrane, which is helpful to maintain the stability of lysosome (101). Interestingly, HSP70 is a calpain substrate in vivo and vitro (98, 102). HSP70 is more likely to be cleaved by calpain after carbonylation. Hsp70 is the regulator of calpain-mediated lysosomal permeabilization or rupture after I/R injury (58). Although there is no direct evidence to prove that lysosome-dependent cell death mediates the cell death of cardiomyocytes during ischemia-reperfusion injury, it may be hidden in autophagy-dependent cell death or apoptosis due to its close relationship with autophagy. Therefore, it is still described here (The mechanism of calpain affecting lysosome-dependent cell death is shown in Figure 2).

Parthanatos is a PCD mediated by poly ADP-ribose polymerase 1 (PARP-1) and is mainly activated by oxidative stress-induced DNA damage and chromatin decomposition (103). PARP-1 is a chromatin-related nuclear protein that plays a vital role in the repair of DNA single-strand or double-strand breaks. PARP-1 promotes DNA repair by identifying DNA ends and uses nicotinamide adenine dinucleotide (NAD⁺) and ATP to form poly (ADP-ribose)-sylation to change chromatin structure. However, DNA base modification affected by oxidative stress, hypoxia, and inflammatory stimuli can lead to excessive activation of PARP-1 (104). Over-activated PARP-1 consumes much NAD⁺ and ATP, leading to mitochondrial transmembrane potential dissipation. Over-consumption of intracellular energy leads to energy collapse and amplification of necrosis (105). Some scholars believe that parthanatos is a step before cell death. It is unclear whether specific inhibition of parthanatos can benefit myocardial I/R injury. Apoptosis-inducing factor mitochondria-associated 1 (AIFM1, also known as AIF) is necessary for parthanatos to perform. Over-activated PARP1 binds to AIFM1, leading to the transfer of AIFM1 from mitochondria to the nucleus and causing lethal chromatin dissolution (106). Calpain is directly involved in the occurrence of parthanatos in cells. PARP-1 is activated after DNA damage and is transferred to the mitochondrial membrane after activation. Calpain-1 in the mitochondrial intermembrane space is used to shear and activate AIF (107). The activated AIF is transferred to the nucleus, decomposed DNA into large fragments with histone H2AX and acyclic proteins (59). The release of t-AIF from mitochondria also requires calpain cleavage of BID to activate BAX formation channels (108). In addition, HSP70 can increase the stability of AIF and reduce nuclear transfer (60). Calpain can promote the transport of AIF from mitochondria to cytoplasm or nucleus by degrading HSP70 (61). Therefore, calpain may be synergistic in the induction of parthanatos during ischemia-reperfusion injury (109) (The mechanism of calpain affecting parthanatos is shown in Figure 2).

In the past, necrosis was believed to be an accidental or passive type of cell death. However, with the deepening of research, it was found that cell death factors such as tumor necrosis factor-α (TNF-α) could cause a kind of “programmed” necrosis, namely necroptosis (110). After the binding and activating of cell death factors with corresponding receptors, receptor-interacting protein kinase 1 (RIPK1) and receptor-interacting protein kinase 3 (RIPK3) were recruited into the cells to form a “necrosome.” Subsequently, the mixed line kinase domain like (MLKL) was activated (5) and shifted to the cell membrane to create a channel leading to membrane permeabilization (111). Studies have shown that calpain regulates necroptosis (62). Calpain can degrade c-Jun-N-terminal kinase 1 (JNK1) specific inhibitor, JNK-interacting protein-1 (JIP-1), induce JNK activation, and increase the expression of RIPK1 (62). Calpain also can increase the expression of RIPK3 by affecting the phosphorylation of signal transducer and activator of transcription 3 (STAT3), promote the phosphorylation of MLKL, and induce cardiomyocyte necroptosis (63). Inhibition of calpain can reduce the number of activated AIF and the occurrence of necroptosis (112) (The mechanism of calpain affecting necroptosis is shown in Figure 2).

Pyroptosis is a form of PCD closely related to innate immune response, characterized by cell membrane permeabilization and extracellular release of inflammatory factors. The central part of Pyroptosis is the activation of gasdermin D (GSDMD). GSDMD has an inhibitory -COOH terminal and death-promoting -NH₂ terminal, which interacts to maintain the inactivation of GSDMD (113). Cells form inflammatory bodies after identifying various stressors (such as DAMPs and PAMPs), which recruit and activate inflammatory caspases (108). Inflammatory caspases can cut and activate GSDMD. After GSDMD activation, pores are formed on the cell membrane, releasing cellular contents, such as inflammatory factors, and ultimately Pyroptosis. Research has shown that inhibition of GSDMD activation during myocardial ischemia-reperfusion can reduce the occurrence of Pyroptosis and myocardial injury (114). The occurrence of Pyroptosis requires the involvement of inflammatory caspases such as caspase-1 and inflammatory bodies (115, 116). In myocardial I/R, calpain can up-regulate the expression of NLRP-3, making it form more inflammatory bodies with ASC to activate caspase-1 and induce Pyroptosis, causing myocardial injury (38) (The mechanism of calpain affecting Pyroptosis is shown in Figure 2).