1. HFD-induced diabetic cardiomyopathy is blunted by ASMase inhibitor
To explore whether ASMase was correlated with cardiomyopathy in animal models of diabetes, we induced type 2 diabetes (T2D) in C57BL/6 mice, which were fed on high-fat diet for 16 weeks. These mice gained weight faster than control mice fed on chow, had higher plasma fasting glucose levels (Fig. 1b), impaired glucose tolerance (Fig. 1a), and hypertriglyceridemia (Fig. 1c). However, plasma cholesterol levels were not significantly different between HFD and Chow mice without imipramine (Fig. 1d). To our surprise, imipramine significantly increased plasma cholesterol level in HFD mice.
Heart weight was increased in HFD mice compared with Chow mice (Fig. 1Q) along with a significantly increase in the cross-sectional area of the cardiomyocytes (Fig. 1S). Furthermore, the echocardiographic assessment showed that LV dimension volume at systole (Volume;s, Fig. 1m) and diastole(Volume;d, Fig. 1n), LV dimension diameters at systole (Diameters;s, Fig. 1o) were markedly increased in the HFD treated mice, while left ventricular anterior wall and posterior wall thickness (LVAW and LVPW, Fig. 1i-l) showed an increasing trend. Cardiac output (CO, Fig. 1h) was decreased in HFD mice, even though ejection fraction (EF%, Fig. 1f) and fractional shortening (FS%, Fig. 1g) just showed a trend of reduction. What’s more, morphometric and histological analysis were performed at 16 weeks after HFD and showed disordered arrangement of myocardial fibers and a significant deposition of fibrotic tissue in the left ventricle of HFD animals. Staining of heart tissues with Sirius Red and Masson’s Trichrome indicated excessive fibrosis in HFD mice (Fig. 1s). Treatment with imipramine reverted all above parameters, CO and fibrosis, back to values like those observed in chow animals.
2. Cardiac ASMase is activated to promote NADPH oxidases activity and apoptosis in HFD mice
We have shown that multiple abnormal parameters involving lipid metabolism and cardiac function during HFD treatment are ablated by ASMase inhibitor, imipramine. We next asked whether ASMase and its metabolites, ceramide (CER) were served as direct causative roles to trigger DCM and regulate downstream molecular mechanism. We observed that protein expression of cardiac ASMase was significantly increased after 16 weeks HFD feeding, concomitant with highly elevated ASMase activity (Fig. 2a-c). As expected, activated ASMase dramatically motivated ceramide (CER) accumulation in hearts of HFD animals (Fig. 2d). Meanwhile, tissue sections of HFD hearts revealed obvious lipid accumulation, evidenced by Oil Red O staining, compared with chow hearts (Fig. 2e), consistent with changes of plasma lipid parameters. In contrast, imipramine administration clearly diminished cardiac ASMase activity and protected against the gain of ceramide and lipid content in HFD-treated animals.These data support the notion that attenuation of cardiac dysfunction and fibrosis in imipramine treated HFD mice was due, in part, to blockade of ASMase activity.
Oxidative stress caused by ceramide accumulation have demonstrated to implicate in obesity and diabetes-associated cardiac dysfunction pathogenesis.However, direct in vivo evidence of ASMase participation is missing.To address this question, we examined NADPH oxidase enzymes (NOXs), whose primary function is to generate reactive oxygen species (ROS), and its oxidative product 4-HNE contents. The HFD mice exhibited higher NOX2 and NOX4 protein expressions, as well as increased 4-HNE protein levels, which were reversed by ASMase inhibitor (Fig. 2f-i). Furthermore, apoptosis occurrence was examined by TUNEL assay and high cleaved caspase-3 expression, which also reversed by ASMase inhibitor, imipramine (Fig. 2j-k). Together, this analysis reveals that ASMase is activated and may promotes HFD-induced cardiomyopathy via NADPH oxidases-mediated oxidative stress and apoptosis.
3. High glucose, palmitate acid and ceramide positively activate ASMase and induce oxidative stress and apoptosis in H9c2 cells
We next sought to delineate whether HFD-induced cardiomyopathy was directly caused by ASMase. H9c2 cardiomyocytes were treated with high glucose and palmitate acid (HG + PA) or exogeneous CER for 24h to mimic the hyperglycemia and hyperlipemia in vivo. We found that both HG + PA and exogeneous CER resulted in obvious upregulation of ASMase protein expressions (Fig. 3a-b). HG + PA treatment remarkably caused cardiomyocytes hypertrophy as evidenced by increased Nppb and Myh7 gene expressions. However, cardiac hypertrophy was not caused by addition of CER (Fig. 3d-e). Imipramine prevented against HG + PA and CER induced ASMase protein expression and restored cardiac hypertrophy.
Next, we went on to assess whether HG + PA and CER mediated ASMase activation was involved in NADPH oxidases activation and apoptosis. We verified that HG + PA and CER treatments led to approximately 2 or 3-fold of NOX4 protein expressions (Fig. 3a-b), while NOX2 expression was not affected. What is noteworthy is that mRNA levels of NOXs were not changed by HG + PA and CER administration (Fig. 3c). ROS production, indicated by fluorescence, were sharply elevated 12h after treatment with HG + PA and CER (Fig. 3f-g). These effects were also antagonized by imipramine. Oxidative stress can induce cell apoptosis. Therefore, TUNEL assay was performed. As anticipated, HG + PA and CER caused cell apoptosis, which is consistent with the observation in upregulation of cleaved caspase-3 protein (Fig. 3h-j). Application of imipramine restored cell viability and alleviated cleaved caspase-3 expressions, which were served as both oxidative damage and apoptosis indicators. Together, these data demonstrate that high glucose, palmitate acid and ceramide can activate ASMase, induce oxidative stress and apoptosis in H9c2 cells.
4. ASMase knockdown dampens high glucose, palmitate acid and ceramide induced oxidative stress and apoptosis in H9c2 cells
We next tested whether HG + PA and CER-triggered oxidative stress and apoptosis was ASMase-dependent. ASMase knocked down in H9c2 cells was performed by using siRNA. Three different siRNA sequences were designed and used in present study. As shown in Fig. 4a, sequence C caused a marked decline in ASMase protein expression and was used for the following experiments. Relative to the vehicle group, the protein expression of NOX4 was significantly increased in the HG + PA or CER group, which was reversed in the ASMase-siRNA group. However, NOX2 expression level had no changed regardless of HG + PA or CER treatment with or without ASMase-siRNA (Fig. 4b-c). Unexpectedly, the transcriptional levels of NOX4, NOX2 and NOX1 were not altered by HG + PA and CER, nor was it altered by ASMase-siRNA treatment (Fig. 4d), suggesting a crucial role of ASMase in regulating NOX4 protein expression.
We also tested the potential for ASMase to modulate ROS generation and apoptosis in vitro. Incubation with HG + PA or CER augmented the ROS generation and cell apoptosis, as well as marked elevated cleaved-caspase-3 expression, whereas in the presence of ASMase-siRNA, these responses were partially ameliorated (Fig. 4g-k). Intriguingly, although we observed a similar effect in both HG + PA and CER on H9c2 cells, it was worth noting that CER could not induce cardiomyocyte hypertrophy, which was different from HG + PA. These results suggested that oxidative stress, cardiac hypertrophy, and apoptosis were enhanced by high glucose, palmitate acid or ceramide treatment and restored by ASMase knockdown.
5. NOX4 activation is required for ASMase-mediated cardiac hypertrophy and apoptosis
We next investigated whether NADPH oxidases were necessary for ASMase-mediated oxidative stress, apoptosis, and hypertrophic growth of cardiomyocytes. We took approach to suppress NADPH oxidases by apocynin, a commonly used inhibitor for enzymes. The H9c2 cells were pre-incubated with 100µMapocynin for 30 mins and then were exposed to HG + PA and CER for 24 h respectively. The protein expressions of ASMase and NOX4 were substantially promoted by HG + PA and CER as noted above. Apocynin, served as an NADPH oxidases inhibitor, significantly suppressed NOX 4 expression. However, apocynin did not affect ASMase protein expression (Fig. 5a-b). Meanwhile, we found not statistically significant of mRNA levels of NADPH oxidases 4, 2 and 1(Fig. 5c). Furthermore, a decline in ROS generation and apoptosis was observed when NOX4 activity were abolished by addition of apocynin (Fig. 5f-j). Apocynin also exerted an anti-hypertrophy effect as shown by inhibition of Nppb gene expression that caused by HG + PA (Fig. 5d-e). These data enabled us to visualize that NOX4 functioned as a downstream target of ASMase and was required for ASMase. Inhibition NADPH oxidases would rescue cardiac function under ASMase activation.
6. ASMaseMyh6KO mice are protected from HFD-induced cardiomyopathy
To explore the therapeutic potential of ASMase in HFD-induced cardiomyopathy and exclude the systemic influence of drug, we generated ASMaseMyh6KO mice by CRISPR/Cas9 genome-editing technology confirmed ASMase depletion in vivo. ASMaseMyh6KO mice had not significantly effects on blood glucose levels and glucose tolerance (Fig. 6a-b). However, results from echocardiographic measurements showed that LVEF, fractional shortening (FS), the ratio of the early to late diastolic mitral inflow velocities (E/A) were all dramatically increased whereas LVESD and LVEDD were decreased in ASMaseMyh6KO+HFD mice compared to those in HFD group at end of 20 weeks (Fig. 6c-n). Morphologically, mice of HFD treatment exhibited significantly myocardial disarray under H&E staining, which reversed by ASMase depletion. Heart sections were also stained with Masson to evaluate the degrees of fibrosis. The results revealed that cardiac fibrosis was improved in ASMase deficiency mice compared with control. Cardiac sections stained with wheat germ agglutinin (WGA) showed that ASMase deficiency decreased cardiomyocyte cross-sectional area (Fig. 6p-q). These results suggested that ASMase deficiency single attenuated HFD-induced cardiac dysfunction, hypertrophy, and fibrosis. These improvements on heart seemed independent of hypoglycemic effects.
7. ASMaseMyh6KO mice reduces NAPDH oxidase activity and apoptosis in HFD mice
Given our observation that ASMaseMyh6KO mice are protected from experimental cardiomyopathy and demonstrated in cellular experiments that ASMase was responsive to cardiac hypertrophy through NADPH oxidases activations and apoptosis. We hypothesized that the protective effects of ASMase deficiency were attributed to blockade of oxidative stress and apoptosis mediated by NAPDH oxidases. To verify this hypothesis, we accessed NADPH oxidase 2 and 4 expressions and apoptosis in ASMase-deficient mice induced by HFD. We observed that HFD-induced ASMase upregulation was significantly blocked by ASMase knockout, like the findings invitro using siRNA (Fig. 7a-b).In agreement with the inhibition of ASMase, the NOX2 and NOX4 expressions elicited by HFD were both significantly decreased in the hearts from ASMase-deficient mice (Fig. 7a, c-d).Again, the immumohistochemical staining data mirrored that ceramide content tended to decrease in HFD ASMase-knockdown hearts, however, the decrease did not reach statistical significant and higher than control groups (Fig. 7e), suggesting that ceramide is not too affected by ASMase knockdown. However, the deficiency of ASMase still blocked myocardial apoptosis in HFD-treated mouse heart (Fig. 7f-g), indicating ASMase is independent of ceramide. Together, our results suggest strongly that therapeutic strategies targeted to ASMase-mediated activation might be beneficial in attenuating oxidative stress, apoptosis and cardiomyopathy associated with obesity, type 2 diabetes, and insulin resistance.