ADAMTS13 Deficiency Shortens the Life Span of Mice With Experimental Diabetes

ADAMTS13 Deficiency Does Not Affect Major Metabolic Parameters, Even Though It Causes Premature Death in Diabetic Mice

Adamts13^−/− mice with STZ-induced diabetes died before their diabetic WT littermates. Log-rank analysis showed significant (P = 0.0022) differences between the survival curves of the two groups of diabetic mice (Fig. 1A). All diabetic Adamts13^−/− mice had died by 6 months after diabetes induction, whereas 53% of the diabetic WT mice had died. No mortality was recorded in two groups of age-matched nondiabetic Adamts13^−/− and nondiabetic WT mice studied in parallel. Monitoring of relevant metabolic parameters did not show signs of worsening diabetes that could predict premature death in Adamts13^−/− mice. As expected, induction of diabetes with STZ was associated with a reduction in body weight (30), which was comparable in Adamts13^−/− and WT mice (Table 1). The two groups of diabetic mice had higher levels of blood glucose and plasma triglycerides than those in the corresponding nondiabetic control mice. No differences in plasma cholesterol levels were observed among the experimental groups (Table 1). Measurement of serum ADAMTS13 activity showed similar levels in nondiabetic (10 ± 2.3%) and diabetic (8.7 ± 1.3%) WT mice at 4 months.

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Figure 1

Adamts13 deficiency impairs survival of diabetic mice. A: Kaplan-Meier curves comparing survival of STZ-induced diabetic WT (n = 19) and Adamts13^−/− (n = 20) mice and their corresponding nondiabetic controls (n = 9 or 10 mice/group) throughout the study period. The P value represents the result of the log-rank test. Original magnification ×200. B: Urinary albumin-to-creatinine ratio (Ualb/Ucreat) measured at 4 months in WT and Adamts13^−/− mice with diabetes and in age-matched nondiabetic mice. C: Representative images (left panel) and quantification (score; right panel) of mesangial matrix accumulation at 4 months in kidney tissue stained with periodic acid Schiff. Original magnification ×630. Scale bars = 50 μm. Data for B and C are the mean ± SEM (n = 7 or 8 mice/group) and were analyzed by ANOVA with the Tukey post hoc test.

To evaluate whether diabetes could drive the prothrombotic phenotype of Adamts13^−/− mice to the point of causing their death, we compared the ability of whole blood taken from ad hoc groups of Adamts13^−/− and WT mice (with and without diabetes) to form platelet thrombi. Mice were studied 4 months after diabetes induction, when 60% mortality had already been reached among Adamts13^−/− mice with diabetes. As shown in Fig. 2A, no difference was observed in the area covered by thrombi between diabetic and nondiabetic WT mice. As expected, Adamts13 deficiency was associated with increased capability to form thrombi (P < 0.01 vs. WT mice), which was not, however, heightened by diabetes.

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Figure 2

Diabetes does not exacerbate the prothrombotic status of Adamts13^−/− mice. A: Representative images (left panels) and experimental design (right top panel) of the assay for ex vivo evaluation of blood thrombotic activity. Blood was taken from nondiabetic and diabetic WT and Adamts13^−/− mice at 4 months, and its ability to form platelet thrombi was tested on immobilized collagen under shear stress. The graph (right bottom panel) shows the percentage of the surface of immobilized collagen occupied by thrombi. Data are the mean ± SEM (n = 3 mice/group) and were analyzed by ANOVA with the Tukey post hoc test. B: Representative immunofluorescence images (left panel) and quantification (score; right panel) of VWF accumulation in the heart at 4 months. Original magnification ×400. C: Representative immunofluorescence images (left panel) and quantification (right panel) of capillary density (number of capillaries per 100 cardiomyocytes) at 4 months. Original magnification ×400. Data are the mean ± SEM (n = 5–8 mice/group) and were analyzed by ANOVA with the Tukey post hoc test. Scale bars = 50 μm.

Diabetic Adamts13^−/− and diabetic WT mice had developed albuminuria to a similar extent 4 months after disease induction (Fig. 1B). In this early phase of the disease, morphological analysis of the kidney showed the presence of mild glomerular mesangial expansion in both diabetic Adamts13^−/− and diabetic WT mice (Fig. 1C). VWF deposits had similarly increased in diabetic mice of both genotypes (Supplementary Fig. 1A). Fibrinogen deposits were rare or absent within glomeruli in both groups of diabetic mice (Supplementary Fig. 1B). Furthermore, ultrastructural analysis of the kidney did not reveal the presence of platelet thrombi or clumps in the glomeruli of diabetic mice (data not shown).

ADAMTS13 Deficiency Increases Propensity for Arrhythmias in Diabetic Mice

We next evaluated whether cardiac abnormalities could be the cause of the premature death of diabetic Adamts13^−/− mice. Analysis of cardiac tissue from all experimental groups, through the use of hematoxylin-eosin staining, did not reveal signs of ischemia or thrombosis. Moreover, glycogen deposition, a marker of ischemic injury (31), was similar in all the experimental groups (Supplementary Fig. 2). Consistent with previous observations (32), VWF levels were higher in the hearts of diabetic WT mice than in the hearts of nondiabetic WT mice, and the lack of ADAMTS13 did not result in a further increase in VWF staining (Fig. 2B). Cardiac fibrinogen staining was similar in all the experimental groups (Supplementary Fig. 3). Cardiac microvascular density was significantly lower in WT mice with diabetes than in WT mice without diabetes (Fig. 2C). The number of capillaries in the myocardia of diabetic Adamts13^−/− mice was similarly lower than that in nondiabetic Adamts13^−/− mice (Fig. 2C).

We then evaluated the impact of ADAMTS13 deficiency on myocyte hypertrophy, a common feature of cardiac remodeling and dysfunction in diabetes. We observed a marked enlargement of the cardiomyocyte area in diabetic compared with nondiabetic WT mice (Fig. 3A). The lack of Adamts13 per se was associated with increased cardiomyocyte hypertrophy compared with that in WT mice, which was not further exacerbated by diabetes. The total number of cardiomyocytes in the left ventricle of diabetic WT mice was significantly lower (by 20%) than the number in nondiabetic WT mice. Myocyte numbers in nondiabetic and diabetic Adamts13^−/− mice were similar to those observed in diabetic WT mice (Fig. 3B). The expression of nitrotyrosine, a marker of oxidative damage, was significantly increased in cardiomyocytes and vessels of diabetic WT mice (Fig. 3C and D). High nitrotyrosine staining was present in cardiomyocytes of diabetic Adamts13^−/− mice (P = 0.05 vs. nondiabetic Adamts13^−/− mice) (Fig. 3C). In vessels, oxidative damage was already detected in nondiabetic Adamts13^−/− mice and remained at similar levels in diabetic mice (Fig. 3D).

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Figure 3

Cardiac tissue analyses. Cardiac hypertrophy is expressed as the area of cardiomyocytes (A) and the number of cardiomyocytes (B) in diabetic WT and diabetic Adamts13^−/− mice and in age-matched nondiabetic WT and nondiabetic Adamts13^−/− mice at 4 months. Oxidative stress at 4 months was quantified (scored) on the basis of nitrotyrosine staining in cardiomyocytes (C) and vessels of cardiac tissue (D). E: Representative images of cardiac interstitial collagen by Sirius red staining (left panel) and quantification of deposition area (square micrometers; right panel). Original magnification ×400. Scale bars = 50 μm. F: Representative images of cardiac TSP1 staining by immunoperoxidase (left panel) and its quantification (score; right panel). Original magnification ×630. Scale bars = 20 μm. Data are the mean ± SEM (n = 5–8 mice/group) and were analyzed by ANOVA with the Tukey post hoc test.

The hearts of diabetic mice of both genotypes showed a similar increase in interstitial collagen deposition when compared with deposition in nondiabetic mice (Fig. 3E). Similar to fibrosis, significant TSP1 upregulation was observed in diabetic mice of both genotypes (Fig. 2F). Staining for TSP1 localized in perivascular and interstitial areas, whereas it was not detectable in cardiomyocytes (Fig. 2F).

We created acute stress in mice by administering a low dose of dobutamine during echocardiography and ECG monitoring to evaluate contractile reserve, an effective predictor of survival. As shown in Fig. 4, global cardiac function at rest was similar in each experimental group. Although the HR increased upon administration of the dobutamine dose in both groups of WT mice (Fig. 4A), dobutamine increased LVFS and LVESWT exclusively in nondiabetic WT mice (Fig. 4B and C). It is interesting that acute dobutamine-induced stress triggered increases in HR, LVFS, and LVESWT in nondiabetic and diabetic Adamts13^−/− mice, although differences in HR and LVFS in nondiabetic Adamts13^−/− mice did not reach statistical significance (Fig. 4A–C). Even though arrhythmic events were undetectable before the dobutamine injection, we observed premature ventricular contractions and ventricular salvos in diabetic Adamts13^−/− mice during dobutamine-induced stress, which were undetectable in the other groups (Fig. 4D and E). The arrythmia score did indeed show that the absence of ADAMTS13 drastically increased susceptibility to ventricular arrhythmias after dobutamine challenge in diabetic mice (P < 0.05 vs. diabetic WT mice). Of note, a similar dose of dobutamine did not elicit premature ventricular contractions and ventricular salvos in nondiabetic and diabetic WT mice, nor in nondiabetic Adamts13^−/− mice (Fig. 4D).

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Figure 4

Adamts13 deficiency preserves the myocardial contractile reserve and increases the propensity for arrhythmic events during dobutamine challenge in diabetic mice. HR (A), percentage LVFS (B), and percentage LVESWT (C) were evaluated at rest (pre) and at peak after intraperitoneal injection of a single bolus of dobutamine (1.5 μg/g body wt) (post) at 4 months. D: Arrhythmias (arrhythmia score) were evaluated at peak after dobutamine injection in all experimental groups. E: Representative ECG traces coupled to corresponding M-mode images at the peak of dobutamine action during stress echocardiography. Data are the mean ± SEM (n = 3–5 mice/group). Statistical analyses were performed with the paired Student t test (A–C) and ANOVA with the Tukey post hoc test (D).

ADAMTS13 Deficiency Is Associated With Phosphorylation and Dislocation of Cx43 in Cardiomyocytes of Diabetic Mice

Cx43 is the major structural protein of the ventricular gap junctions localized in the intercalated disc that enables appropriate electrical coupling in healthy hearts (33). Phosphorylation of Cx43 causes pathological remodeling, which may lead to sudden arrhythmia and death (34). We evaluated, using Western blotting, the extent of Ser368 phosphorylation of Cx43 in cardiac tissue at 4 months. Levels of phosphorylated Cx43 showed a trend toward an increase in diabetic compared with nondiabetic WT mice (Fig. 5A). Lack of ADAMTS13 in diabetic mice significantly enhanced Cx43 phosphorylation (P < 0.01 vs. nondiabetic Adamts13^−/− mice). We then used immunohistochemistry to assess the expression and localization of Cx43. In nondiabetic WT and nondiabetic Adamts13^−/− mice, Cx43 was organized and aligned with myocardial architecture at intercellular junctions (Fig. 5B). Higher levels of Cx43 were detected in hearts of diabetic WT mice (P < 0.05 vs. nondiabetic WT mice) (Fig. 5C); in these hearts, however, Cx43 localization was still restricted within myocyte gap junctions (Fig. 5B and D). Cx43 expression further increased in cardiomyocytes of diabetic Adamts13^−/− mice (P < 0.01 vs. nondiabetic Adamts13^−/− mice), and its distribution was highly disorganized: Cx43 staining became wider and less linear, with more prevalence in the mid–myocytic regions (Fig. 5B and D). Cx43 was highly disorganized in cardiac tissue of diabetic WT mice that were sacrificed at the end of the study (i.e., at 6 months, when 60% mortality was reached in this group; Cx43 dislocation: 49.3 ± 6.8%) to an extent similar to that in diabetic Adamts13^−/− mice at 4 months (Supplementary Fig. 4). This finding reveals that the cardiac abnormalities observed in Adamts13^−/− diabetic mice, which may be responsible for animal death, develop later in diabetic WT mice. To demonstrate that cardiac arrythmia changes in diabetic Adamts13^−/− mice specifically depend on Cx43 remodeling, we analyzed the expression and distribution of desmin, an intermediate filament protein expressed by cardiac muscle cells, whose alteration is also associated with arrhythmogenic cardiomyopathy (35). Immunoperoxidase staining did not show differences in desmin levels among the experimental groups (Supplementary Fig. 5).

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Figure 5

Adamts13 deficiency associates with increased and displaced Cx43 expression in cardiomyocytes of diabetic mice. A: Representative Western blotting (left panel) and densitometric analysis (right panel) of Ser368-phosphorylated Cx43 (p368-Cx43) protein levels in heart tissue from nondiabetic and diabetic WT and Adamts13^−/− mice at 4 months. GAPDH was used as the loading control. B: Representative images of immunoperoxidase staining of Cx43 on heart specimens from nondiabetic and diabetic WT and Adamts13^−/− mice. Scale bars = 50 μm. Original magnification ×400. C: Quantification of Cx43 staining expressed as the percentage of mean positive area per high-power field. D: Quantification of dislocated Cx43 staining expressed as the percentage of positive fields on total fields. Data are the mean ± SEM (n = 4–8 mice/group) and were analyzed by ANOVA with the Tukey post hoc test.

Cx43 Remodeling in Diabetic Adamts13^−/− Mice Is Associated With CaMKII Phosphorylation

In search of intracellular pathways implicated in Cx43 phosphorylation, we focused on CaMKII, a multifunctional serine-threonine protein kinase that is abundant in the heart and has been implicated in diabetes-associated arrhythmias (15,36). CaMKII phosphorylation sites have been identified in the carboxyl terminus of Cx43 (37), and CaMKII has been recognized as an important signal for controlling Cx43 localization and conduction velocity in the myocardium (38). CaMKIIδ is the predominant isoform within the myocardium, but expression of the CaMKIIγ isoform also has been detected (39,40). We explored the activation of CaMKII by assessing expression levels and the extent of CaMKII phosphorylation in the left ventricles of mice in the nondiabetic and diabetic groups. Western blotting demonstrated the presence of the CaMKII isoforms CaMKIIδ_C (56 kDa), CaMKIIδ_B (plus CaMKIIδ₉, which differs by only three amino acids; 58 KDa), and CaMKIIγ (60 kDa) (Fig. 6A) in all groups of mice. No differences were observed in the levels of total CaMKII expression (Supplementary Fig. 6A). It is notable that increases in the phosphorylation of all the CaMKII isoforms were observed in diabetic Adamts13^−/− mice (P < 0.001 vs. diabetic WT mice), as indicated by the ratio of phosphorylated to total CaMKII expression (Fig. 6C).

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Figure 6

Adamts13 deficiency associates with CaMKII activation and Ca²⁺ handling protein disturbance in cardiomyocytes of diabetic mice. A: Representative Western blotting of phosphorylated and total CaMKII. The three major bands seen for each sample represent the isoforms CaMKIIγ (62 kDa), CaMKIIδ_b plus δ₉ (58 kDa), and CaMKIIδ_c (56 kDa) (left panel). The graph (right panel) quantifies this with the use of densitometric analysis of CaMKII activation, expressed as the ratio of phosphorylated to total CaMKII. Representative Western blotting show phosphorylated Thr17-PLN (CaMKII site) (B, left panel) and phosphorylated Ser16-PLN (PKA site) (C, left panel), and the graphs quantify this through the use of densitometric analysis of phosphorylated Thr17-PLN (B, right panel) and Ser16-PLN (C, right panel), expressed as the ratio of phosphorylated to total PLN. D and E: Representative Western blotting (left panels) and quantification (right panels) of Serca-2a (D) and NCX protein (E). GAPDH was used as the loading control. Protein expression was evaluated in heart tissue from nondiabetic and diabetic WT and Adamts13^−/− mice at 4 months. Data are expressed as the mean ± SEM (n = 4 or 5 mice/group) and were analyzed by ANOVA with the Tukey post hoc test.

CaMKII Phosphorylation in Adamts13^−/− Diabetic Mice Disturbs Ca²⁺ Handling Proteins

The finding that CaMKII activity was enhanced in cardiac tissue of diabetic Adamts13^−/− mice prompted us to assess Ca²⁺ handling proteins that are regulated directly by CaMKII and contribute to the triggered arrhythmia (14). Western blotting showed more phosphorylation of a specific CaMKII target site on PLN (pThr17-PLN) in diabetic Adamts13^−/− than in diabetic WT mice (Fig. 6B). The hearts of diabetic Adamts13^−/− mice had reduced expression of Serca-2a (Fig. 6D), which transports calcium to the sarcoplasmic reticulum (14). We then evaluated the expression of NCX, which is considered to be the dominant Ca²⁺ efflux mechanism in cardiomyocytes and, together with Serca-2a, is responsible for reducing the amount of intracellular Ca²⁺ (41). Results showed a significant reduction of NCX levels in diabetic compared with nondiabetic Adamts13^−/− mice (Fig. 6E). The aforementioned alterations in Ca²⁺ handling proteins we observed in diabetic Adamts13^−/− mice were associated with lower gene expression of the Ca^{2 +} channel β subunit (Ca_Vβ2) (Supplementary Fig. 7), a strong modulator of l-type calcium currents in adult cardiomyocytes (42).

Next, given that the activation of the β-ADR signaling pathway has been implicated in Cx43 synthesis (43,44) and CaMKII activation (45) in cardiomyocytes, we investigated β-ADR expression and activity in the hearts. Western blotting did not show differences in β1-ADR protein levels among the experimental groups (Supplementary Fig. 8). As shown in Fig. 6C, phosphorylation of PLN at Ser16, a target of cAMP-dependent protein kinase (PKA) (14), the predominant regulator of β-ADR–mediated enhancement of cardiac contractility, was not statistically different among the experimental groups, which rules out the possibility that β-ADR signaling has a role in dictating CaMKII activation in our setting.

ADAMTS13 Affects CaMKII Phosphorylation in Cultured Cardiomyocytes

Evidence shows that, in cardiomyocytes, the TSP1-CD47 axis promotes CaMKII activation, which in turn is responsible for left ventricular heart failure (18,19). In vitro analyses showed that TSP1 and ADAMTS13 form protein complexes (22). We hypothesized that in cardiomyocytes of diabetic mice, which are exposed to higher TSP1 levels (21), circulating ADAMTS13 could bind TSP1 and hamper TSP1-CD47 signaling, thereby limiting CaMKII activation. To verify this hypothesis in vitro, we used murine HL-1 cardiomyocytes, an established model of contractile adult cardiomyocytes (46). Cardiomyocytes were exposed to TSP1 (0.2 nmol/L), rhADAMTS13 (0.3 μg/mL), or both combined for 6 h. Western blotting showed more phosphorylated CaMKII after stimulation with either TSP1 or rhADAMTS13 than in unstimulated (control) HL-1 cells. This effect disappeared when TSP1 and rhADAMTS13 were added together, as indicated by densitometric analyses (Fig. 7). To investigate whether hyperglycemia directly affects TSP1 expression and CaMKII activity in cardiomyocytes, HL-1 cells were exposed to a low or high glucose concentration. High glucose did not affect the release of TSP1 in the cell supernatant (Supplementary Fig. 9), and as a consequence, no changes in CaMKII phosphorylation were observed with respect to low glucose (Supplementary Fig. 10).

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Figure 7

ADAMTS13 and TSP1 combined limit CaMKII phosphorylation in cultured cardiomyocytes. A: Representative Western blotting of phosphorylated and total CaMKII protein expression in HL-1 cells exposed for 6 h to rhADAMTS13 (0.3 µg/mL), TSP1 (0.2 nmol/L), or both. GAPDH was used as the loading control. B: Quantification by densitometric analysis of total CaMKII (T-CaMKII) expression. C: CaMKII activation expressed as the ratio of phosphorylated CaMKII (p-CaMKII) to T-CaMKII. Data are the mean ± SEM (n = 4 samples/group) and were analyzed by ANOVA with the Tukey post hoc test.