SGLT2 Inhibitor, Canagliflozin, Attenuates Myocardial Infarction in the Diabetic and Nondiabetic Heart

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SUMMARY
The authors hypothesized that despite similar cardiovascular event rates, the improved cardiovascular survival from sodium glucose transporter 2 (SGLT2) inhibition, seen clinically, could be via a direct cytoprotective effect, including protection against myocardial ischemia/reperfusion injury. Langendorff-perfused hearts, from diabetic and nondiabetic rats, fed long-term for 4 weeks with canagliflozin, had lower infarct sizes; this being the first demonstration of canagliflozin's cardioprotective effect against ischemia/reperfusion injury in both diabetic and nondiabetic animals. By contrast, direct treatment of isolated nondiabetic rat hearts with canagliflozin, solubilized in the isolated Langendorff perfusion buffer, had no impact on infarct size. This latter study demonstrates that the infarct-sparing effect of long-term treatment with canagliflozin results from either a glucose-independent effect or up-regulation of cardiac prosurvival pathways. These results further suggest that SGLT2 inhibitors could be repurposed as novel cardioprotective interventions in high-risk with a significant reduction of cardiovascular mortality. Equally remarkably, this reduction in cardiovascular mortality was seen notably early-within 1 to 2 months-following the introduction of the respective SGLT2 inhibitor. The mechanism underlying the reduction in cardiovascular mortality is not clear and has been subject to much conjecture: seemingly, improvements in blood sugar control were comparatively minor and improvements in terms of diuresis, weight loss, and blood pressure reduction inadequate to fully explain the differences observed. Indeed, many, including ourselves, have speculated a potential pleiotropic beneficial effect for this class of glucose-lowering therapy (3)(4)(5).
The hypothesis that SGLT2 inhibitors may have pleiotropic effects appears to be supported by other observations from the clinical trial data, not least that SGLT2 inhibition appears to have minimal impact upon the cardiovascular event rate-be it myocardial infarction or stroke, admissions with unstable angina or the need for a coronary revascularization procedure (1,2). As such, there appears to be minimal impact of SGLT2 inhibition upon macrovascular (arterial atheromatous) disease-but overall, despite experiencing the same frequency of cardiovascular events, survival nonetheless appears to be better in those taking SGLT2 inhibitors, a benefit that strikingly manifests within the first few months of treatment.   Finally, we wished to ascertain whether the SGLT2 inhibitor would have a direct, cardioprotective effect in the isolated heart, and to this end, we undertook a further group of experiments with "acute" exposure to the SGLT2 inhibitor, with the drug added to the Langendorff perfusate throughout the perfusion protocol.
Using the SGLT2 inhibitor, canagliflozin, in a reverse-translational study, we found that long-term pre-administration over 4 weeks led to a significant

METHODS
For a detailed description of all methods, see the Supplemental Appendix. In brief, ZL and ZDF rats were monitored weekly with random blood glucose (A) Body weight index. The diabetic ZDF rats were significantly larger than the nondiabetic ZL rats. Canagliflozin administration in the ZL led to a significant reduction in body mass index that was absent in the ZDF diabetic rats. n ¼ 8 to 10 per group. (B) Random glucose concentration on day of experiment. As expected, ZDF diabetic rats had significantly higher blood glucose concentrations compared to the nondiabetic ZL controls (p < 0.0001; n ¼ 6 to 9 per group). Canagliflozin had no impact upon blood glucose in the ZL group (p ¼ NS; n ¼ 9 to 10 per group), but significantly reduced glucose in the diabetic ZDF rats (p < 0.0001; n ¼ 6 to 9 per group). (C to E) Renal manifestations of diabetes in the ZDF rats. (C) Urine glucose, measured by urinalysis strip test. No glucosuria was detectable in the control ZL rats, but there was significant glucosuria in ZL rats on canagliflozin. As expected, significant glucosuria was found in both ZDF control and canagliflozintreated groups. (D) Blood urea nitrogen was significantly higher in the ZDF rats compared with the nondiabetic ZL: 11 AE 2 mg/dl versus 19 AE 2 mg/dl (p ¼ 0.006, n ¼ 6 per group). (E) A similar pattern was observed in the urine albumin/creatinine ratio-the diabetic ZDF rats demonstrating a significantly higher albumin excretion compared with the nondiabetic ZL rat: 160 AE 39 mg/g versus 3,319 AE 577 mg/g     Figure 1C). In addition, the ZDF rats were found to have evidence of end-organ manifestations of their diabetes, as represented by abnormal renal function and albuminuria ( Figures 1C and 1D). We are therefore confident that the ZDF represents a reasonable approximation of the human obese type 2 diabetic phenotype with significant and established diabetes at the time of experimentation.

CHARACTERIZATION
Unexpectedly, we found that diabetic rats treated with canagliflozin were heavier than untreated We found a small, but significant, difference between myocardial infarct size in the control arms of the diabetic ZDF and the nondiabetic ZL rat heart groups (p ¼ 0.04) ( Figure 4A, Supplemental Figure 2).
This difference is expected in Langendorff-perfused hearts where glucose is the sole energy substrate (see review [8]). We found that canagliflozin, mirroring the important data by Andreadou et al. (9) in the mouse, significantly reduced myocardial infarct size in diabetic ZDF rats. Infarct size relative to the control chow-fed ZDF rats was significantly attenuated, from 37 AE 3% to 20 AE 2% of the area at risk (p ¼ 0.001) ( Figure 4A). Importantly, canagliflozin also significantly abrogated myocardial injury in the nondiabetic ZL rats, reducing infarct size from   Figure 5A). There was no difference in the area at risk between any of the groups ( Figure 5B).

DISCUSSION
Our study provides the first evidence to our knowledge that long-term oral administration of canagli-  washout before ischemia, suggests a mechanism that imbues a "memory," potentially through the recruitment of signaling pathways. And if a signaling pathway, it is a pathway whose efficacy, unlike that of ischemic conditioning (11), is seemingly not affected by the presence of significant diabetes (the severity of the diabetic phenotype confirmed by evidence of the development of nephropathy). One such mechanism may be through a Jak-STAT3 pathway, as suggested by Iliodromitis's group However, with 40 min of washout before induction of ischemia, it seems somewhat unlikely that significant quantities of canagliflozin would remain within the heart. Our data would therefore appear to suggest that the observed protection from long-term administration of canagliflozin is less likely to be mediated through NHE inhibition, but perhaps through another pleiotropic pathway capable of triggering a "memory" effect through activation of signaling cascades.
Already identified candidate pathways include the aforementioned Jak/STAT3 pathway (9) that may also help attenuate oxidative stress and fibrotic myocardial remodeling (22) or perhaps through AMPK (23) (also found in kidney to reduce ischemia/reperfusion injury [24]), although these are not hypotheses that we have yet tested.
Finally, SGLT2 inhibitors have been found to imbue significant protection in the vasculature of diabetic ZDF rats, with preservation of endothelial function. This endothelial protection appears to be mediated through attenuation of long-term glucotoxicity and amelioration of oxidative stress (25). This could translate into myocardial protection ex vivo, but we did not find significant differences in coronary flow in our model between canagliflozin-treated versus control-treated animals (data shown in the   TRANSLATIONAL OUTLOOK 2: Our data suggest that infarct limitation is also seen in nondiabetic animals, raising the tantalizing potential for repurposing these drugs to improve cardiovascular outcomes in all high-risk cardiovascular patients, irrespective of diabetic status.
Lim et al.