Of Mice and Men: Toward a Better Model of Diabetic Cardiomyopathy With Application in Female Animals Free

It is becoming increasingly recognized that diabetic cardiomyopathy (DbCM) is often prevalent in individuals with pre- or early-stage type 2 diabetes (T2D), and is generally defined as ventricular dysfunction in the absence of coronary artery disease and/or hypertension (1,2). Several advancements in our understanding of the pathology of DbCM have demonstrated that it is primarily characterized by diastolic dysfunction; however, as T2D is a heterogenous disorder, DbCM can also be accompanied by systolic dysfunction or even just the latter. Although there are currently no specific therapies available for the management of DbCM, seminal findings in the field allude to a key role for oxidative stress, inflammation, fibrosis, lipotoxicity, mitochondrial dysfunction, and perturbations in substrate metabolism as critical mediators of its pathogenesis (1,2). One of the biggest challenges hindering potential advancements in medicine for DbCM remains the lack of a universally accepted preclinical model that recapitulates several of the key features that define its pathology in humans. Indeed, the most frequently used model to study insulin resistance/prediabetes, feeding rodents a high-fat diet (HFD) for several weeks to induce experimental obesity, is widely inconsistent in its ability to elicit cardiac dysfunction, in particular the aforementioned diastolic dysfunction that characterizes DbCM (3). Furthermore, female rodents are often ignored in these studies despite the incidence of cardiovascular disease being greater in women with diabetes than in men with diabetes (4,5).

In this issue of Diabetes, Karatzia et al. (6) have characterized an experimental model of T2D with DbCM in male and female mice that addresses several of the abovementioned limitations. This model incorporates 5 weeks of HFD (60.3% fat, 21.4% carbohydrate, and 18.3% protein) feeding in combination with five consecutive daily doses of anomer-equilibrated streptozotocin (EQ STZ) (40 mg/kg, vortexed for 30 s every 10 min until 90 min and then injected) from days 22 to 26 (6). From day 37 onward to the end of the study at day 60, all mice had their HFD replaced with a standard chow control diet (10.5% fat, 69.1% carbohydrate, and 20.5% protein). This model resulted in a T2D phenotype, as evidenced by elevated fed/fasting blood glucose levels, worsened glucose tolerance, and increased islet area with disrupted architecture in both males and females. Furthermore, male animals had preserved systolic function but developed diastolic dysfunction, whereas females developed both systolic and diastolic dysfunction, as determined by ultrasound echocardiography. In contrast, male mice that were fed an HFD but administered fresh STZ (injected within 15 min of dissolution) exhibited normal cardiac function, whereas female mice still developed both systolic and diastolic dysfunction. Of interest, male and female mice that received either fresh STZ or EQ STZ alone still demonstrated both systolic and diastolic dysfunction. Consistent with other studies interrogating mediators of DbCM (1,2), metabolomic profiling revealed that this HFD + EQ STZ model leads to reductions in cardiac glucose metabolism and altered redox status.

These observations offer several advantages over the HFD + low-dose STZ (single injection ranging from 75 to 90 mg/kg in mice and from 25 to 35 mg/kg in rats) mouse model, which is one of the most commonly used animal models of T2D and is characterized by a highly reproducible diastolic dysfunction (3). Most studies using this model administer freshly prepared STZ as it rapidly precipitates out of solution (7–12), although Karatzia et al. have demonstrated this concern can be bypassed by preparing EQ STZ. This aspect of STZ chemistry is often overlooked, and discrepancies in reported findings between various research groups are often attributed to animal strain and STZ dose instead. Findings from this study suggest that the equilibration of STZ anomers is another important aspect to consider when designing an experiment to ensure reproducibility and methodological rigor. While the overall cardiac phenotype of the HFD + EQ STZ T2D model is comparable with what has been observed in male rodents subjected to the HFD + low-dose STZ model of T2D, the former’s cardiac phenotype in females appears superior. Indeed, most studies have reported a lack of cardiac dysfunction in female mice with the latter model (3), which may relate to female C57BL/6J mice being resistant to the actions of STZ (13). As such, this newly proposed model could be an important tool that researchers could use to further delineate the sex differences in DbCM and develop better treatment options for female DbCM, especially considering that cardiovascular disease incidence is greater in women than men with diabetes (4,5). Furthermore, most major government-supported funding agencies have now mandated that research be carried out in both males and females (14,15), which bodes well for the utility of this model since it better represents the pathology in women with T2D.

Nonetheless, there are also important caveats to consider with the model described by Karatzia et al. In particular, the rationale for replacement of the HFD with a standard-chow low-fat diet is unclear. As T2D is often the result of underlying obesity, it is somewhat odd to remove this aspect of their model during days 37 to 60, and significant weight loss was reported whereby body weights at day 60 were comparable with those of mice solely fed the standard-chow low-fat diet. What impact this may have on the overall cardiac phenotype cannot be discerned, as a group of animals that remained on the HFD for study duration while receiving EQ STZ was not included. Another potential caveat to consider with this model relates to the use of five consecutive injections of EQ STZ (40 mg/kg) from days 22 to 26. Models of type 1 diabetes often use a protocol involving five consecutive injections of fresh STZ at 50 mg/kg (3). While the STZ dose in this particular study is slightly lower at 40 mg/kg, there may be concerns regarding whether the diabetes phenotype is more reflective of type 1 diabetes than T2D. While circulating insulin measurements confirm the presence of functional islet β-cells capable of secreting insulin, it should be noted that the insulin levels were lower in the mice that received either fresh or EQ STZ than those in mice that were solely fed an HFD for 60 days, particularly in the males.

Despite these potential concerns, no experimental animal model is perfect in its ability to reproduce human pathology, and even the best models have room for refinement. Thus, the work of Karatzia et al. to characterize a new HFD + EQ STZ mouse model of DbCM is an important advance for the field. Given the lack of animal models that fully recapitulate the pathology of DbCM in humans, especially in females, this model may play a pivotal role in progressing our understanding of T2D-related cardiovascular disease and its sex-specific differences while aiding the development of sex-specific therapies.