Ahf-Caltide, a Novel Polypeptide Derived from Calpastatin, Protects against Oxidative Stress Injury by Stabilizing the Expression of CaV1.2 Calcium Channel

Reperfusion after ischemia would cause massive myocardial injury, which leads to oxidative stress (OS). Calcium homeostasis imbalance plays an essential role in myocardial OS injury. CaV1.2 calcium channel mediates calcium influx into cardiomyocytes, and its activity is modulated by a region of calpastatin (CAST) domain L, CSL54-64. In this study, the effect of Ahf-caltide, derived from CSL54-64, on myocardial OS injury was investigated. Ahf-caltide decreased the levels of LDH, MDA and ROS and increased heart rate, coronary flow, cell survival and SOD activity during OS. In addition, Ahf-caltide permeated into H9c2 cells and increased CaV1.2, CaVβ2 and CAST levels by inhibiting protein degradation. At different Ca2+ concentrations (25 nM, 10 μM, 1 mM), the binding of CSL to the IQ motif in the C terminus of the CaV1.2 channel was increased in a H2O2 concentration-dependent manner. CSL54-64 was predicted to be responsible for the binding of CSL to CaV1.2. In conclusion, Ahf-caltide exerted a cardioprotective effect on myocardial OS injury by stabilizing CaV1.2 protein expression. Our study, for the first time, proposed that restoring calcium homeostasis by targeting the CaV1.2 calcium channel and its regulating factor CAST could be a novel treatment for myocardial OS injury.


Introduction
Acute myocardial infarction is the leading cause of death and disability worldwide.The essential remedy for ischemia is to restore blood flow timely.However, reperfusion after ischemia can cause additional cellular damage, termed as ischemia-reperfusion (IR) injury, which leads to cardiac dysfunction such as myocardial stunning, reperfusion arrhythmia and myocyte death [1][2][3].Among the complex system networks involved in the pathological mechanisms of IR injury, oxidative stress (OS) is one of the most important pathological mechanisms [1].OS refers to the imbalance between reactive oxygen species (ROS) production and cellular antioxidant capacity [4].Elevated ROS induces the influx of extracellular Ca 2+ via the voltage-sensitive Ca 2+ channel opening, which is related with myocardial OS injury [1,5].
Ca V 1.2 calcium channel is the main route for Ca 2+ influx into cardiomyocytes, which triggers the release of intracellular Ca 2+ for excitation-contraction coupling.The Ca V 1.2 channel is a heterotetramer composed of α 1 , α 2 δ, β 2 and γ subunits.The α 1 subunit consists of four transmembrane regions, three intramembrane loop regions, N terminus (NT) and C terminus (CT) [6,7].The dysregulation of intracellular Ca 2+ homeostasis due to Ca V 1.2 channels plays an important role in myocardial OS injury, and Ca V 1.2 channel activity was altered after exposure to ROS [6,8].Accordingly, the calcium homeostasis imbalance during OS may be related with myocardial injury.The alternations in the density or activity of Ca V 1.2 channel and elevated ROS level have been reported to contribute to myocardial OS injury [1,8].Therefore, restoring calcium homeostasis by regulating the Ca V 1.2 channel may benefit in alleviating myocardial OS injury.
Calpastatin (CAST) is a calcium-dependent cysteine protease, composed of a unique Nterminal region (domain L, CS L ) and four homologous C-terminal regions (domains 1-4) [9][10][11].CS L partially reactivates the Ca V 1.2 channel, and this is mediated by the region containing amino acid (a.a.) residues 54-64 (EGKPKEHTEPK, CS L 54-64) [9,[12][13][14].Subsequent studies have confirmed that CS L binds to PreIQ and IQ motifs on the CT of the α 1 subunit of the Ca V 1.2 channel [15,16].CAST localizes in the area of the Z-disk, around which Ca V 1.2 channels in the T-tubular system are also localized [15].Thus, it is reasonable to think that the concentration of CAST is high enough to affect the activity of the Ca V 1.2 channel, which may be related with myocardial OS injury.
The α 1 subunit of the Ca V 1.2 channel is a therapeutic target in OS-induced myocardial injury [17][18][19][20][21]. Peptides and peptidomimetics have been widely used for the prevention, diagnosis and treatment of cardiovascular diseases [22,23].In this study, with the target being the α 1 subunit of the Ca V 1.2 channel, Ahf-caltide, a polypeptide derived from CS L 54-64, was investigated.Ahf-caltide significantly alleviated myocardial injury induced by OS, followed by the upregulation of the Ca V 1.2 channel α 1 subunit (Ca V 1.2) and β2 subunit (Ca V β2) levels.In addition, with the increase in H 2 O 2 concentration, the binding of CS L to IQ motif was gradually increased and CS L 54-64 was responsible for CS L binding to Ca V 1.2.Therefore, Ahf-caltide might be a promising therapeutic target in the prevention of myocardial OS injury.

Protective Effect of Ahf-Caltide on H 2 O 2 -Induced Oxidative Stress Injury
In H 2 O 2 -induced OS injury in rats, compared with the control group, the heart rate and coronary flow were decreased (Figure 1B,C, p < 0.05).However, compared with the H 2 O 2 group, the heart rate and coronary flow increased with the treatment of Ahf-caltide (p < 0.01, Figure 1B,C).In addition, LDH content significantly increased after H 2 O 2 injury, which was decreased after Ahf-caltide treatment (Figure 1D).Furthermore, compared with the H 2 O 2 group, BNP level was markedly decreased after Ahf-caltide treatment (Figure 1E,F).
The Ca V 1.2 channel is significantly glutathionylated by ROS, which contributes to the development of cardiac pathology and is a therapeutic target in heart diseases [16][17][18][19][20]. CS L 54-64 was reported to activate the Ca V 1.2 channel [9].Therefore, the protective mechanism of Ahf-caltide against H 2 O 2 -induced OS injury was studied from the perspective of the Ca V 1.2 channel.The specificity of Ca V 1.2 antibody was verified, which is shown in Figure S1.It was notable that the Ca V 1.2 protein level was decreased to 51% of the control with H 2 O 2 treatment, which was significantly increased by Ahf-caltide (Figure 1E,F).There was no significant difference in the Ca V β2 level among groups.The expression of CAST was higher in the H 2 O 2 group or Ahf-caltide group than that in the control group.However, the CAST level showed no difference between the H 2 O 2 group and Ahf-caltide group.The above results suggested that Ahf-caltide had a protective effect on H 2 O 2 -induced myocardial OS injury by up-regulating the Ca V 1.2 level.

Protective Effects of Ahf-Caltide on Cardiac IR Injury
In the in vitro IR model, similar results were detected.Ahf-caltide relieved the changes in heart rate, coronary flow and LDH activity induced by IR injury in a dosedependent manner (Figure 2B-D).SOD activity was increased and MDA level was decreased in the 10 µM Ahf-caltide group (Figure 2E,F).These results indicated that Ahfcaltide might have a protective effect on IR injury by enhancing the endogenous cellular anti-oxidant capacity.There was no significant difference in CaV1.2 and CaVβ2 protein levels among groups (Figure 2G,H).CAST level decreased to 73.40 ± 8.67% (n = 5) of the control levels in IR injury hearts, while Ahf-caltide increased the level of CAST protein.These results suggested that the protective effect of Ahf-caltide might be mediated via upregulation of the CAST level.

Protective Effects of Ahf-Caltide on Cardiac IR Injury
In the in vitro IR model, similar results were detected.Ahf-caltide relieved the changes in heart rate, coronary flow and LDH activity induced by IR injury in a dose-dependent manner (Figure 2B-D).SOD activity was increased and MDA level was decreased in the 10 µM Ahf-caltide group (Figure 2E,F).These results indicated that Ahf-caltide might have a protective effect on IR injury by enhancing the endogenous cellular anti-oxidant capacity.There was no significant difference in Ca V 1.2 and Ca V β2 protein levels among groups (Figure 2G,H).CAST level decreased to 73.40 ± 8.67% (n = 5) of the control levels in IR injury hearts, while Ahf-caltide increased the level of CAST protein.These results suggested that the protective effect of Ahf-caltide might be mediated via up-regulation of the CAST level.

Ahf-Caltide Alleviated Oxidative Stress Injury in H9c2 Cells by Inhibiting Protein Degradation
Ahf-caltide conjugated with rodamine was used to examine the permeability of the peptide across the membrane and, if so, its cellular localization.As a result, immunofluorescence analysis identified that Ahf-caltide was localized in the cytoplasm of H9c2 cells evenly (Figure 3A).In addition, as shown in Figure 3B,C, the cell viability was significantly decreased in the CoCl 2 group compared to the control group and was increased by Ahf-caltide at a concentration of 0.3, 1, 3 or 10 µM (p < 0.05).LDH activity was significantly increased to two-to three-fold following exposure to CoCl 2 compared to that in the control group, which was markedly reduced in the H9c2 cells pretreated with Ahf-caltide (Figure 3B,C).Meanwhile, compared to the control group, the ROS level was significantly increased in the CoCl 2 group, which decreased with the treatment of Ahf-caltide (Figure 3D,E).
The levels of Ca V 1.2, Ca V β2 and CAST proteins were further explored.They were down-regulated in the CoCl 2 group, which were reversed by Ahf-caltide (Figure 3F,G).To examine whether the changes in the expression of the Ca V 1.2 channel were due to de novo synthesis, cycloheximide (CHX), an inhibitor of protein synthesis, was employed in the presence or absence of 10 µM Ahf-caltide for up to 36 h.Following the treatment of CHX, Ca V 1.2, Ca V β2 and CAST levels showed a time-dependent decrease in normal cells.However, Ahf-caltide increased the protein levels of Ca V 1.2, Ca V β2 and CAST in CHX-exposed cells (Figure 3H,I), suggesting that protein synthesis was not involved in the regulation of Ahf-caltide.It was reported that the ubiquitin-proteasome system and the autophagy-lysosomal pathway were involved in Ca V 1.2 channel degradation [24].Thus, the proteasome inhibitor MG-132 (10 µM) and lysosomal inhibitor leupeptin (10 µM) were applied.It was found that CAST, Ca V 1.2 and Ca V β2 levels were increased by MG-132, which were further increased by Ahf-caltide.In contrast, there was no difference in Ca V 1.2 and Ca V β2 levels between the leupeptin group and CoCl 2 group (Figure 3J,K).However, CAST level was increased by leupeptin and then further increased under Ahfcaltide treatment (Figure 3J,K).These data strongly supported that proteasome inhibition was involved in the increase in CAST, Ca V 1.2 and Ca V β2 mediated by Ahf-caltide, and lysosomal inhibition also was involved in the increase in CAST.

Protective Effects of Ahf-Caltide on Cardiac IR Injury
In the in vitro IR model, similar results were detected.Ahf-caltide relieve changes in heart rate, coronary flow and LDH activity induced by IR injury in a dependent manner (Figure 2B-D).SOD activity was increased and MDA level wa creased in the 10 µM Ahf-caltide group (Figure 2E,F).These results indicated tha caltide might have a protective effect on IR injury by enhancing the endogenous ce anti-oxidant capacity.There was no significant difference in CaV1.2 and CaVβ2 protei els among groups (Figure 2G,H).CAST level decreased to 73.40 ± 8.67% (n = 5) control levels in IR injury hearts, while Ahf-caltide increased the level of CAST pr These results suggested that the protective effect of Ahf-caltide might be mediated v regulation of the CAST level.

Ahf-Caltide Alleviated Oxidative Stress Injury in H9c2 Cells by Inhibiting Protein Degradation
Ahf-caltide conjugated with rodamine was used to examine the permeability of the peptide across the membrane and, if so, its cellular localization.As a result, immunofluorescence analysis identified that Ahf-caltide was localized in the cytoplasm of H9c2 cells evenly (Figure 3A).In addition, as shown in Figure 3B,C, the cell viability was significantly decreased in the CoCl2 group compared to the control group and was increased by Ahfcaltide at a concentration of 0.3, 1, 3 or 10 µM (p < 0.05).LDH activity was significantly increased to two-to three-fold following exposure to CoCl2 compared to that in the control group, which was markedly reduced in the H9c2 cells pretreated with Ahf-caltide (Figure 3B,C).Meanwhile, compared to the control group, the ROS level was significantly increased in the CoCl2 group, which decreased with the treatment of Ahf-caltide (Figure 3D,E).
The levels of CaV1.2, CaVβ2 and CAST proteins were further explored.They were down-regulated in the CoCl2 group, which were reversed by Ahf-caltide (Figure 3F,G).To examine whether the changes in the expression of the CaV1.2 channel were due to de novo synthesis, cycloheximide (CHX), an inhibitor of protein synthesis, was employed in the presence or absence of 10 µM Ahf-caltide for up to 36 h.Following the treatment of CHX, CaV1.2, CaVβ2 and CAST levels showed a time-dependent decrease in normal cells.However, Ahf-caltide increased the protein levels of CaV1.2, CaVβ2 and CAST in CHX-exposed cells (Figure 3H,I), suggesting that protein synthesis was not involved in the regulation of Ahf-caltide.It was reported that the ubiquitin-proteasome system and the autophagylysosomal pathway were involved in CaV1.2 channel degradation [24].Thus, the proteasome inhibitor MG-132 (10 µM) and lysosomal inhibitor leupeptin (10 µM) were applied.It was found that CAST, CaV1.2 and CaVβ2 levels were increased by MG-132, which were further increased by Ahf-caltide.In contrast, there was no difference in CaV1.2 and CaVβ2 levels between the leupeptin group and CoCl2 group (Figure 3J,K).However, CAST level was increased by leupeptin and then further increased under Ahf-caltide treatment (Figure 3J,K).These data strongly supported that proteasome inhibition was involved in the increase in CAST, CaV1.2 and CaVβ2 mediated by Ahf-caltide, and lysosomal inhibition also was involved in the increase in CAST.

The Binding Characteristics of CSL to CaV1.2 Were Affected by Oxidative Stress
In our previous study, it was shown that CSL could bind to the CT1 of CaV1.2 [15,16,25].But it was not known whether the binding characteristics changed during OS.In the present study, GST pull-down assay was used to examine the effect of OS on the binding of CSL to CaV1.2.
As was shown in Figure 4A,B, 10 µΜ CSL with various concentrations of H2O2 (0 to 10 -1 M) treatment was incubated with CT1 at [Ca 2+ ] of 25 nM, 10 µΜ and 1 mM.The binding densities of CSL to CT1 were increased with increasing concentrations of H2O2 (0 to10 −2 M) at any [Ca 2+ ] and increased with increasing [Ca 2+ ] at fixed H2O2 concentration including the H2O2-free condition.Notably, at [Ca 2+ ] of 25 nM, 10 µM and 1 mM, compared with the 10 −2 M H2O2 group, the CSL binding to CT1 was decreased by 10 −1 M H2O2.Moreover, the CSL bound to CT1 in the SDS-PAGE gel appeared in double bands with 10 −2 M H2O2 treatment and was shifted up in the 10 −1 M H2O2 group related to the treatment of H2O2 (0 to10 −2 M).Then, the effects of H2O2 on the CSL binding to PreIQ or IQ were examined.In 10 µM [Ca 2+ ], the amount of CSL binding to PreIQ was not significantly different compared to the control (Figure 4C,D, Table 1).However, the effect of H2O2 on the CSL binding to IQ was similar to that to CT1 (Figure 4E,F, Table 1).

Were Affected by Oxidative Stress
In our previous study, it was shown that CS L could bind to the CT1 of Ca V 1.2 [15,16,25].But it was not known whether the binding characteristics changed during OS.In the present study, GST pull-down assay was used to examine the effect of OS on the binding of CS L to Ca As was shown in Figure 4A,B, 10 µM CS L with various concentrations of H 2 O 2 (0 to 10 −1 M) treatment was incubated with CT1 at [Ca 2+ ] of 25 nM, 10 µM and 1 mM.The binding densities of CS L to CT1 were increased with increasing concentrations of H 2 O 2 (0 to10 −2 M) at any [Ca 2+ ] and increased with increasing [Ca 2+ ] at fixed H 2 O 2 concentration including the H 2 O 2 -free condition.Notably, at [Ca 2+ ] of 25 nM, 10 µM and 1 mM, compared with the 10 −2 M H 2 O 2 group, the CS L binding to CT1 was decreased by 10 −1 M H 2 O 2 .Moreover, the CS L bound to CT1 in the SDS-PAGE gel appeared in double bands with 10 −2 M H 2 O 2 treatment and was shifted up in the 10 −1 M H 2 O 2 group related to the treatment of H 2 O 2 (0 to10 −2 M).Then, the effects of H 2 O 2 on the CS L binding to PreIQ or IQ were examined.In 10 µM [Ca 2+ ], the amount of CS L binding to PreIQ was not significantly different compared to the control (Figure 4C,D, Table 1).However, the effect of H 2 O 2 on the CS L binding to IQ was similar to that to CT1 (Figure 4E,F, Table 1).NT contained calmodulin (CaM) binding sites, which was reported to be involved in the regulation of CaV1.2 channel activity [26].But it is still unknown whether CSL could bind to NT.Thus, the interaction of CSL with NT was examined using pull-down assay.Our results showed that CSL bound to NT in a [CSL]-dependent manner (Figure 4G,H).However, the concentration of Ca 2+ has no effect on the binding of CSL to GST-NT (Figure 4I,J).The maximal binding (Bmax) of CSL to NT were 0.59, 0.69 and 0.65 mol/mol at 25 nM, 10 µM and 1 mM [Ca 2+ ], respectively, which suggested that the interaction between CSL  NT contained calmodulin (CaM) binding sites, which was reported to be involved in the regulation of Ca V 1.2 channel activity [26].But it is still unknown whether CS L could bind to NT.Thus, the interaction of CS L with NT was examined using pull-down assay.Our results showed that CS L bound to NT in a [CS L ]-dependent manner (Figure 4G,H).However, the concentration of Ca 2+ has no effect on the binding of CS L to GST-NT (Figure 4I,J).The maximal binding (B max ) of CS L to NT were 0.59, 0.69 and 0.65 mol/mol at 25 nM, 10 µM and 1 mM [Ca 2+ ], respectively, which suggested that the interaction between CS L and NT appeared to follow the one-site model of Hill's equation (Table 2).Then, the effect of H 2 O 2 on the binding of CS L to NT was examined.The binding of CS L to NT was not affected by 10 −6 -10 −1 M H 2 O 2 (Figure 4K,L, Table 1).All these results showed that the binding of CS L to the IQ domain, but not PreIQ or NT, was affected by H 2 O 2 treatment, suggesting that the IQ domain was involved in the regulation of OS on the interaction of CS L with Ca V 1.2.Molecular docking was used to investigate the binding properties of CS L to Ca V 1.2.The results of epitope analysis are shown in Table 3, and the count represented the amount of referenced docking pose clusters.The 47, 26 and 10 docked poses of the interaction of CS L with PreIQ/IQ/NT were displayed and the most likely binding postures with the highest score among the five clusters were drawn in blue (Figure 5(A1,B1,C1)), and the corresponding fingerprints are shown in Figure 5(A2,B2,C2), respectively.PreIQ/IQ/NT were closely bound to CS L with different positions and the binding region on CS L was limited to a fixed area, which is the most obvious in Figure 5(A3,B3,C3).Furthermore, while binding to PreIQ/IQ/NT, the most likely residues of CS L were all located in CS L 54-64 (Figure 5(A5,B5,C5)).And the binding free energy of NT and CS L 54-64 was significantly higher than that of PreIQ/IQ, indicating that the binding affinity of NT to CS L 54-64 was the lowest.The binding free energies of amino acids Glu 1612 and Lys 1620 on PreIQ, Glu 1655 on IQ and Cys 136 on NT to CS L 54-64 were minimal, suggesting that the amino acids were the main sites responsible for the binding of Ca V 1.2 to CS L 54-64 (Table 4).These results indicated that the binding region of CS L to Ca V 1.2 was located on Glu 54-Lys64.ing to PreIQ/IQ/NT, the most likely residues of CSL were all located in CSL54-64 (Figure 5(A5,B5,C5)).And the binding free energy of NT and CSL54-64 was significantly higher than that of PreIQ/IQ, indicating that the binding affinity of NT to CSL54-64 was the lowest.The binding free energies of amino acids Glu 1612 and Lys 1620 on PreIQ, Glu 1655 on IQ and Cys 136 on NT to CSL54-64 were minimal, suggesting that the amino acids were the main sites responsible for the binding of CaV1.2 to CSL54-64 (Table 4).These results indicated that the binding region of CSL to CaV1.2 was located on Glu 54-Lys64.

Discussion
In this study, the effect and mechanism of Ahf-caltide, a polypeptide derived from CS L 54-64, on myocardial OS injury were investigated.Ahf-caltide alleviated myocardial OS injury, assessed with decreased LDH content, MDA content, BNP level, ROS level and increased heart rate, coronary flow, cell survival and SOD activity.Ahf-caltide permeabilized into H9c2 cells and increased Ca V 1.2, Ca V β2 and CAST levels by inhibiting protein degradation.CS L 54-64 was predicted to be responsible for the binding of CS L to Ca V 1.2.At different [Ca 2+ ], the binding of CS L to CT1/IQ motifs on the α 1 subunit of Ca V 1.2 was increased in a H 2 O 2 concentration-dependent manner.These results suggested that Ahf-caltide exerts a cardioprotective role in myocardial OS damage by upregulating Ca V 1.2 channel expression.
In our study, the changes in the expression of α 1 subunit and β 2 subunit of Ca V 1.2 channel were observed in three OS models.In CoCl 2 and H 2 O 2 models, the α 1 subunit protein level was decreased, which was consistent with the previous studies.The density of Ca V 1.2 channel estimated by dihydropyridine binding assay was decreased at 3 and 8 weeks after myocardial infarction, respectively [27,28].In contrast, in the IR model, α 1 subunit expression did not change, consistent with the report of Zucchi R et al. [29].In addition, β subunit is an important regulator of Ca V 1.2 channel, with the function of enhancing the channel surface expression by inhibiting α 1 subunit degradation and supporting voltage gating [30,31].In the CoCl 2 model, β subunit expression was decreased, which was in line with the previous report that β subunit expression was downregulated in patients with atrial fibrillation [30].Interestingly, in H 2 O 2 models, unlike the decrease in Ca V 1.2 level, Ca V β2 level was not altered.Cell death has been demonstrated to be a fundamental process in cardiovascular diseases [2,4].However, the different changes in α 1 and β subunit expression might not be simply attributed to the loss of cardiomyocytes that occurred in ischemia and reperfusion.
During OS, Ca 2+ influx into cardiomyocytes mediated by the Ca V 1.2 calcium channel results in Ca 2+ overload and eventually leads to cell injury [6,32].Current therapeutic strategies for myocardial IR injury focus on the inhibition of Ca V 1.2 channel activity.However, it cannot be ignored that clinically, heart failure with reduced ejection fraction and atrial fibrillation commonly coexist, and most calcium channel blockers are not recommended in heart failure with reduced ejection fraction [33,34].Inhibition of the Ca V 1.2 channel activity would cause a shortening of action potential duration and refractoriness, exacerbating the development of atrial fibrillation [35].In addition, the negative inotropic effect of Ca V 1.2 channel blockers results in impaired systolic function, which limits its use in the clinic [34,36,37].The negative inotropic effect is mediated by downregulation of the Ca V 1.2 channel activity [38].Therefore, from a long-term perspective, some patients with heart failure might not benefit from calcium channel blockade [33].The Ca V 1.2 channel plays an important role in regulating signaling pathways, and Ca 2+ influx through the channel triggers the expression of a group of Ca 2+ -regulated genes, which are necessary for cell survival [39,40].Studies have shown that the activation of Ca V 1.2 channel reduced neuronal cell death after and decreased OS in cortical neurons [41,42].Therefore, we believed that opening the Ca V 1.2 calcium channel, rather than blocking the channel, may be beneficial in alleviating myocardial OS injury.In the present study, Ahf-caltide exerted a myocardial protective role by upregulating the expression of the Ca V 1.2 channel α 1 and β subunits.To our knowledge, this is the first report that states that the upregulation of the Ca V 1.2 channel could alleviate myocardial OS injury.
In addition, the mechanism by which Ahf-caltide increased Ca V 1.2 channel expression was studied, and it was shown that Ahf-caltide penetrated into H9c2 cells and was located in the cytoplasm.Ahf-caltide with amino acid residues, 3Glu, 1Gl, 3Ly, 2Pro, 1His and 1Thr exhibited strong hydrophilicity, which promotes phospholipid scrambling and membrane leakage [43].Moreover, Lys and Glu are involved in the generation of locally polar regions inside the bilayers, which reduces the activation energy for flip-flop, therefore altering phospholipids passing through the hydrophobic passage [44].In the presence of protein synthesis inhibitors, Ca V 1.2 and Ca V β2 levels were increased by Ahf-caltide, indicating that Ahf-caltide inhibited protein degradation.Intracellular protein degradation occurs via three mechanisms, including the ubiquitin-proteasome system, lysosomal and autophagy pathway and calpain system [24].In the present study, Ca V 1.2 and Ca V β2 levels were increased in the presence of MG-132, rather than leupeptin, indicating that the proteasome pathway was involved in the degradation of the channel, which is consistent with the previous report [45].Ca V 1.2 and Ca V β2 levels were further increased by Ahf-caltide in the presence of MG-132, suggesting that the upregulation of Ca V 1.2 channel expression by Ahf-caltide was due to its inhibition on protein degradation mediated by proteasome (Figure 6).
channel activity [38].Therefore, from a long-term perspective, some patients with heart failure might not benefit from calcium channel blockade [33].The CaV1.2 channel plays an important role in regulating signaling pathways, and Ca 2+ influx through the channel triggers the expression of a group of Ca 2+ -regulated genes, which are necessary for cell survival [39,40].Studies have shown that the activation of CaV1.2 channel reduced neuronal cell death after ischemia and decreased OS in cortical neurons [41,42].Therefore, we believed that opening the CaV1.2 calcium channel, rather than blocking the channel, may be beneficial in alleviating myocardial OS injury.In the present study, Ahf-caltide exerted a myocardial protective role by upregulating the expression of the CaV1.2 channel α1 and β subunits.To our knowledge, this is the first report that states that the upregulation of the CaV1.2 channel could alleviate myocardial OS injury.
In addition, the mechanism by which Ahf-caltide increased CaV1.2 channel expression was studied, and it was shown that Ahf-caltide penetrated into H9c2 cells and was located in the cytoplasm.Ahf-caltide with amino acid residues, 3Glu, 1Gl, 3Ly, 2Pro, 1His and 1Thr exhibited strong hydrophilicity, which promotes phospholipid scrambling and membrane leakage [43].Moreover, Lys and Glu are involved in the generation of locally polar regions inside the bilayers, which reduces the activation energy for flip-flop, therefore altering phospholipids passing through the hydrophobic passage [44].In the presence of protein synthesis inhibitors, CaV1.2 and CaVβ2 levels were increased by Ahf-caltide, indicating that Ahf-caltide inhibited protein degradation.Intracellular protein degradation occurs via three mechanisms, including the ubiquitin-proteasome system, lysosomal and autophagy pathway and calpain system [24].In the present study, CaV1.2 and CaVβ2 levels were increased in the presence of MG-132, rather than leupeptin, indicating that the proteasome pathway was involved in the degradation of the channel, which is consistent with the previous report [45].CaV1.2 and CaVβ2 levels were further increased by Ahf-caltide in the presence of MG-132, suggesting that the upregulation of CaV1.2 channel expression by Ahf-caltide was due to its inhibition on protein degradation mediated by proteasome (Figure 6).The calpain system consists of a family of calpain proteases and the endogenous calpain inhibitor CAST [24].Studies have shown that in IR injury, after CAST activity was inhibited, calpain was activated and the Ca V 1.2 channel protein was cleaved, resulting in an irreversible run-down of the channel [46].An increase in the intracellular Ca 2+ and calpain activity also result in CAST proteolysis [47].In this respect, CAST is not only a downstream effector of the Ca V 1.2 channel, but also an upstream of the Ca V 1.2 channel.Therefore, CAST expression was also examined in this study.Consistent with previous studies, in IR and CoCl 2 models, CAST level was decreased [47,48].However, in the H 2 O 2 model, CAST level was upregulated.The difference in CAST expression among OS models might be related to the interaction and functional cross-talk between CAST and the Ca V 1.2 channel.In addition, under MG-132 or leupeptin treatment, CAST level was upregulated, indicating that both the lysosomal pathway and proteasome pathway were involved in the proteolysis of CAST, which was inhibited after Ahf-caltide treatment.Therefore, Ahf-caltide might increase the CAST level by inhibiting the lysosomal pathway and proteasome pathway, thereby elevating the Ca V 1.2 level (Figure 6).
It was reported that the CT of α 1 subunit was sensitive to proteolysis [49].Ahf-caltide was designed to target the α 1 subunit, so the interaction between Ahf-caltide and the α 1 subunit was explored during OS.Considering that Ahf-caltide was small and difficult to detect, the binding of CS L to the α 1 subunit was examined instead.The pathological level of H 2 O 2 (>10 −7 M) leads to OS [50,51].With the increase in [Ca 2+ ] from 25 nM (low [Ca 2+ ] level) to 10 µM (high [Ca 2+ ] level) and then to 1 mM (a saturating [Ca 2+ ] level), the binding of CS L to CT1 was enhanced by H 2 O 2 (10 −6 -10 −2 M) [50][51][52][53][54][55].These results suggested that the binding of CS L to CT1 was enhanced with the increase in OS at pathological [Ca 2+ ].In addition, under the 10 −1 M H 2 O 2 treatment, the binding of CS L to CT1 was slightly decreased and the position of the bound CS L significantly shifted up, which might be due to the structure of CS L altered by the high concentration of H 2 O 2 .Moreover, IQ, not PreIQ or NT, was involved in the effect of H 2 O 2 on the binding of CS L to Ca V 1.2.
In this study, CS L could bind to CT1/PreIQ/IQ and the binding of CS L to CT1 was in a [Ca 2+ ] dependent manner, which were consistent with our previous findings [15].NT regulates the inactivation of the Ca V 1.2 channel [26,56].CS L was found to bind to NT, suggesting that NT might be one of the targets of CS L in the regulation of the Ca V 1.2 channel activity.Next, molecular docking was used to predict the binding properties of CS L to Ca V 1.2, and the binding region was found to be mainly located in CS L 54-64.The binding affinity of CS L 54-64 to NT was significantly lower than that to CT, suggesting that CT was the main target for CS L in the regulation of the Ca V 1.2 channel activity.In addition, amino acids Glu 1612 on PreIQ and Glu 1655 on IQ were found to be the main binding sites of CS L to Ca V 1.2.In the previous studies, both CS L and CaM were found to bind to CT1 [15,16].CS L suppressed CaM binding to IQ and did not directly interact with CaM, suggesting that CS L and CaM might share the binding sites on the CT of Ca V 1.2 [16,27].Glu 1612 on PreIQ and Glu 1655 on IQ are involved in the binding of CaM to Ca V 1.2 [24,57].The predicted sites on Ca V 1.2 of CS L in our study were consistent with the previous studies [15,16,25].Combined with pull-down assay results, the inhibition of Ca V 1.2 proteolysis mediated by Ahf-caltide during OS was most likely due to the interaction between the peptide and CT.

Animal Model Preparation
Healthy male sprague-Dawley rats, weighing 200-250 g, were purchased from Beijing Huafukang Biotechnology Co., Ltd.(Beijing, China).and the production license number was SCXK (Jing) 2019-0008.The rats were raised in the Experimental Animal Center of China Medical University and housed for three days under standard laboratory conditions.All experimental procedures were conducted in accordance with the Animal Protection and Use Committee of China Medical and the application approval number was CMU2020175.
To investigate the effects of Ahf-caltide on hearts subjected to H 2 O 2 , the rats were randomly divided into three groups: control group, H 2 O 2 group and Ahf-caltide group, with six rats in each group.The rats were anaesthetized by intraperitoneal injection of 1% pentobarbital sodium (40 mg/kg).Then, the hearts were mounted on a Langendorff apparatus and perfused at a constant flow (10 mL/min) with Krebs-Henseleit (K-H) buffer containing (in mmol/L) 11.10 glucose, 118.50 NaCl, 25.00 NaHCO 3 , 4.70 KCl, 1.20 KH 2 PO 4 , 2.50 CaCl 2 and 1.20 MgSO 4 .The perfusate was gassed with 95% O 2 and 5% CO 2 (pH 7.4).In the control group, the hearts were perfused with K-H buffer for 60 min.In the H 2 O 2 group, the hearts were perfused with K-H buffer for 15 min, followed by 200 µM H 2 O 2 for 60 min.In the Ahf-caltide group, the hearts were perfused with 200 µM H 2 O 2 and 10 µM Ahf-caltide for 60 min (Figure 1A).
To further investigate the effects of Ahf-caltide on hearts subjected to IR, the rats were randomly divided into five groups: control group, IR group and high-dose, medium-dose and low-dose of Ahf-caltide groups.In the control group, the hearts were perfused with K-H buffer purely.In the IR group, the hearts were perfused with K-H buffer for 15 min, followed by 45 min of no-flow ischemia and 60 min of reperfusion.In the peptide treatment groups, the hearts were perfused with Ahf-caltide (0.3, 1 or 10 µM) for 10 min before ischemia and during reperfusion (Figure 2A).

Cell Culture and OS Model Preparation
H9c2 cells were cultured in the DMEM medium supplemented with 10% fetal bovine serum and 1% streptomycin/penicillin in a humidified incubator at 37 °C with 5% CO 2 .The cells were randomly divided into six groups: control group, CoCl 2 group (600 µM, 24 h) and Ahf-caltide (0.3, 1, 3 or 10 µM) groups.In the Ahf-caltide groups, cells were pretreated with Ahf-caltide for 0.5 h, followed by the exposure to 600 µM CoCl 2 for 24 h.

Immunofluorescence
The immunofluorescence assay was carried out according to the instruction.Briefly, when the cells were approximately 80-90% confluent, H9c2 cells were fixed with 1% formaldehyde at 37 • C for 15 min.Then, the cells were treated with 10 µM Ahf-caltide conjugated with Rodamine (GL Biochem Ltd., Shanghai, China) and incubated for 24 h.Then, Hoechst 33342 solution was added to each well for cell staining and the cells were incubated for 20-30 min.Then, the cells were washed with PBS 2-3 times.Finally, fluorescence microscopy (Olympus, Tokyo, Japan) was used for immunofluorescence-labeled sections.

Cell Viability Assay
Cell viability was determined using a CCK-8 kit.In brief, in the 96-well plate, H9c2 cells at a density of 1 × 10 4 cells/well were incubated with Ahf-caltide (0.3, 1, 3 or 10 µM) or 600 µM CoCl 2 .Then, 10 µL of CCK-8 solution was added to each well and the cells were incubated for 1 h.The absorbance was measured with a microplate reader at 450 nm.

Detection of LDH, MDA and SOD
In the 24-well plate, H9c2 cells at a density of 1 × 10 5 cells/well were incubated with Ahf-caltide or CoCl 2 .Myocardial cell damage was assessed by detecting the LDH released into the culture medium or coronary effluent according to the instructions of the kit.The MDA content and SOD activity were detected at the end of perfusion time to evaluate the OS damage following the manufacturer's instructions, respectively.

Determination of ROS
H9c2 cells (2 × 10 5 /well) were separately seeded into 35 mm cell culture dishes.After treatment with Ahf-caltide or CoCl 2 , the cells were cultured with serum-free medium and incubated with 10 µM DCFH-DA diluted using serum-free and phenol red-free DMEM at 37 °C for 20 min.Then the cells were washed three times with serum-free and phenol red-free DMEM.The membrane-permeable and non-fluorescent 2 ,7 -dichlorofluorescin diacetate (DCFH-DA) was catalyzed by intracellular esterase and then oxidized into fluorescent DCF, which was detected by a confocal laser scanning microscope (Nikon, Shanghai, China) at an excitation wavelength of 488 nm and emission wavelength of 525 nm.

Western Blot Assay
After the end of experiments, the total proteins of the left ventricle or cultured cells were extracted using RIPA lysis buffer containing the protease inhibitor cocktail.The proteins were separated using SDS-PAGE and transferred onto a PVDF membrane.The membranes were blocked with 5% non-fat milk for 2 h in Tris-buffer saline containing 0.1% Tween 20 at room temperature and then were incubated with anti-BNP antibody (1:2000), anti-CAST antibody (1:1200), anti-Ca V 1.2 antibody (1:500), anti-Ca V β2 antibody (1:1000) and anti-GAPDH antibody (1:1500) at 4 °C overnight, respectively.After washing with Trisbuffer saline containing 0.1% Tween 20 three times, the membranes were incubated with the corresponding secondary antibody at room temperature for 2 h.The blots were detected using an enhanced chemiluminescence detection solution, and then, protein abundance was quantified by chemiluminescence system.
The binding curve of CS L to was fitted with the one-site model of Hill's equation in SigmaPlot 10.0 software.The bound CS L (y) was expressed as follows: y = B max •X/(K d + X), where B max is the maximum binding, X is the concentration of the free ligand and K d is the apparent dissociation constant.

Molecular Docking
The three-dimensional structures of CS L (PDB:3DF0), PreIQ (PDB: 3G43) and IQ (PDB: 2BE6) were downloaded from the Protein Data Bank.The coding sequences of NT were obtained from the National Center for Biotechnology Information, and then, I-TASSER homology modeling server (https://zhanglab.ccmb.med.umich.edu/I-TASSER/,(accessed on 20 March 2021) was used to model the structure of the NT protein [59].
The binding of CS L or CS L 54-64 to NT/PreIQ/IQ was simulated by the proteinprotein docking tool of the Molecular Operating Environment (MOE, Chemical Computing Group Inc., Montreal, QC, Canada).Epitope analysis and protein-ligand interaction fingerprints analysis were used to calculate the active binding sites of CS L to NT/PreIQ/IQ.The fingerprint was built on the interactions between CS L and NT/PreIQ/IQ, such as hydrogen bonds, ionic interactions and surface contacts and so on, as shown in Figure 5(A2,A3,B2,B3,C2,C3) [11].The binding sites with the highest binding score were chosen based on the lowest binding free energy of the protein-protein complex.

Statistical Analysis
Data were expressed as the mean ± standard deviation.The Student's t-test was used for comparisons between two groups, and one-way ANOVA with the Dunnett's test was used for multiple data comparisons.p < 0.05 was considered statistically significant.

Conclusions
In conclusion, Ahf-caltide has a protective effect against myocardial injury induced by OS.Ahf-caltide increased the Ca V 1.2 channel α 1 subunit, β subunit and CAST levels by inhibiting protein degradation during OS.CS L 54-64 was responsible for the binding of CS L to the Ca V 1.2 channel IQ motif.In addition, with the increase in OS, the binding affinity of CS L to IQ, rather than PreIQ or NT, also increased.Therefore, Ahf-caltide is a promising therapeutic polypeptide to restore calcium homeostasis, and the present study provides a new direction and strategy for the treatment of myocardial OS injury.This perspective of increasing Ca V 1.2 expression is clinically important, since it offers a new direction to treat patients with heart failure, who were discharged on a contraindicated calcium channel blocker.

Figure 1 .
Figure 1.The protection of Ahf-caltide on the Langerdoff-perfused rat heart subjected to H2O2 treatment.(A) Schematic diagram of the experimental protocol.(B,C) Time course of changes in heart rate (B) and coronary flow (C) among groups.(D) LDH activity of coronary effluent in groups.(E,F) Representatives immunoblots (E) and quantitative analysis (F) of CaV1.2, CaVβ2, CAST and BNP levels in hearts of groups.* p < 0.05, ** p < 0.01 and *** p < 0.001 vs. control group; # p < 0.05 and ## p < 0.01 vs. H2O2 group.NS, not significant.Results represent at least five independent experiments.

Figure 2 .
Figure 2. The protection of Ahf-caltide on the Langerdoff-perfused rat hearts subjected to IR injury.(A) Schematic diagram of the experimental protocol.(B,C) Time course of changes in heart rate (B)

Figure 1 .
Figure 1.The protection of Ahf-caltide on the Langerdoff-perfused rat heart subjected to H 2 O 2 treatment.(A) Schematic diagram of the experimental protocol.(B,C) Time course of changes in heart rate (B) and coronary flow (C) among groups.(D) LDH activity of coronary effluent in groups.(E,F) Representatives immunoblots (E) and quantitative analysis (F) of Ca V 1.2, Ca V β2, CAST and BNP levels in hearts of groups.* p < 0.05, ** p < 0.01 and *** p < 0.001 vs. control group; # p < 0.05 and ## p < 0.01 vs. H 2 O 2 group.NS, not significant.Results represent at least five independent experiments.

Figure 1 .
Figure 1.The protection of Ahf-caltide on the Langerdoff-perfused rat heart subjected to H2O ment.(A) Schematic diagram of the experimental protocol.(B,C) Time course of changes in rate (B) and coronary flow (C) among groups.(D) LDH activity of coronary effluent in groups Representatives immunoblots (E) and quantitative analysis (F) of CaV1.2, CaVβ2, CAST and levels in hearts of groups.* p < 0.05, ** p < 0.01 and *** p < 0.001 vs. control group; # p < 0.05 an < 0.01 vs. H2O2 group.NS, not significant.Results represent at least five independent experim

Figure 2 .
Figure 2. The protection of Ahf-caltide on the Langerdoff-perfused rat hearts subjected to IR (A) Schematic diagram of the experimental protocol.(B,C) Time course of changes in heart r

Figure 2 .
Figure 2. The protection of Ahf-caltide on the Langerdoff-perfused rat hearts subjected to IR injury.(A) Schematic diagram of the experimental protocol.(B,C) Time course of changes in heart rate (B) and coronary flow (C) among groups.(D) LDH activity of coronary effluent in groups.(E,F) The SOD activity (E) and MDA content (F) in the heart tissues.(G,H) Representative images (G) and quantitative analysis (H) of Ca V 1.2, Ca V β2 and CAST in hearts among groups.* p < 0.05 and *** p < 0.001 vs. control group; # p < 0.05, ## p < 0.01 and ### p < 0.001 vs. IR group.NS, not significant.Results represent at least five independent experiments.

Figure 3 .
Figure 3.The protection by Ahf-caltide on H9c2 cell injury induced by CoCl 2 treatment.(A) The localization of Ahf-caltide in H9c2 cells examined by fluorescence microscopy (scale bars, 100 µm).(B) The cell viability was detected by CCK-8.(C) LDH activity of supernatant in groups.(D,E) ROS level in groups (scale bars, 100 µm).(F,G) Representative immunoblots (F) and quantitative analysis (G) of expression levels of Ca V 1.2, Ca V β2, CAST in groups.(H,I) Representative immunoblots (H) and quantitative analysis (I) of the effects of cycloheximide (CHX) on the expression of Ca V 1.2, Ca V β2, CAST in groups.(J,K) Representative immunoblots (J) and quantitative analysis (K) of the effects of MG-132/Leupeptin on the levels of Ca V 1.2, Ca V β2, CAST of groups.* p < 0.05, ** p < 0.01 and *** p < 0.001 vs. control group or as indicated on the figure; # p < 0.05, ## p < 0.01 and ### p < 0.001 vs. CoCl 2 group.NS, no significance.Results represent at least three independent experiments.

Figure 4 .
Figure 4. Concentration-dependent effect of H2O2 on CSL binding to N-and C-terminus of CaV1.2 channel α1 subunit.GST pull-down assay for the effect of H2O2 (10 −1 -10 −6 M) on the binding of CSL to CT1 (A) or PreIQ (C) or IQ (E) or NT (K).Plots of the effect of H2O2 with fi ed curves on the CSL binding to CT1 (B) or PreIQ (D) or IQ (F) or NT (L).Pull-down assay (G) and binding curves (H) for CSL binding to NT. Pull-down assay for the effect of [Ca 2+ ] (I) and quantitative analysis (J) on the binding of CSL to NT. Symbols are as indicated in each graph.NS, no significance.Protein bands were visualized using Coomassie brilliant blue staining and indicated by arrows.Results represent at least four independent experiments.

Figure 4 .
Figure 4. Concentration-dependent effect of H 2 O 2 on CS L binding to N-and C-terminus of Ca V 1.2 channel α 1 subunit.GST pull-down assay for the effect of H 2 O 2 (10 −1 -10 −6 M) on the binding of CS L to CT1 (A) or PreIQ (C) or IQ (E) or NT (K).Plots of the effect of H 2 O 2 with fitted curves on the CS L binding to CT1 (B) or PreIQ (D) or IQ (F) or NT (L).Pull-down assay (G) and binding curves (H) for CS L binding to NT. Pull-down assay for the effect of [Ca 2+ ] (I) and quantitative analysis (J) on the binding of CS L to NT. Symbols are as indicated in each graph.NS, no significance.Protein bands were visualized using Coomassie brilliant blue staining and indicated by arrows.Results represent at least four independent experiments.

Figure 5 .
Figure 5. Molecular docking analysis of CS L or CS L 54-64 binding to N-and C-terminus of Ca V 1.2 channel α 1 subunit.(A-C) Improved five groups of epitope clusters in Table 3 corresponding to the totally docked poses of (A1) CS L /PreIQ, (B1) CS L /IQ, (C1) CS L /NT; the fingerprints for the interaction obtained from the five groups' docked poses between CS L and (A2) PreIQ, (B2) IQ, (C2) NT; the fingerprints for the interaction from the first group pose between CS L and (A3) PreIQ, (B3) IQ, (C3) NT; the docked poses of CS L 54-64 binding to (A4) PreIQ, (B4) IQ, (C4) NT; the interaction diagram of Lys 64, Lys 56 and Glu 54 on CS L 54-64 with PreIQ (A5), IQ (B5) and NT (C5), respectively.The diagrams in the first group with the highest score are represented by blue "spaghetti" and the others with a translucent "spaghetti".Blue structures represent PreIQ/IQ/NT, red structures represent CS L or CS L 54-64.

Figure 6 .
Figure 6.Mechanism scheme for the protection of Ahf-caltide on myocardial OS injury.The left panel is the recapitulative scheme for myocardial OS injury.The right panel is the recapitulative scheme for the effect of Ahf-caltide during OS.

Figure 6 .
Figure 6.Mechanism scheme for the protection of Ahf-caltide on myocardial OS injury.The left panel is the recapitulative scheme for myocardial OS injury.The right panel is the recapitulative scheme for the effect of Ahf-caltide during OS.

Table 1 .
The effect of H2O2 on the binding of CSL to GST-CT1/PreIQ/IQ/NT.

Table 1 .
The effect of H 2 O 2 on the binding of CS L to GST-CT1/PreIQ/IQ/NT.

Table 2 .
Parameters for binding of CS L to GST-NT.d , apparent dissociation constant, B max , maximum binding, R 2 , coefficient of determination.2.5.CS L 54-64 Was the Binding Region of CSL to Ca V 1.2 K

Table 3 .
Epitope Analysis for CS L as a receptor binding to PreIQ/IQ/NT.

Table 3 .
Epitope Analysis for CSL as a receptor binding to PreIQ/IQ/NT.

Table 4 .
Predicted binding sites of CS L 54-65 to Ca V 1.2 channels.