This study provides first molecular evidence for a gender-specific progression of diabetic cardiomyopathy. Using a mouse model, we measured functional, structural and molecular changes in the heart at different time points after STZ-induced diabetes. We found that the onset of cardiac dysfunction was more rapid and severe in diabetic females compared to males. This was associated with gender-specific LV dilation, which occurred earlier in diabetic females than males. We also demonstrate that early downregulation of pro-survival protein Pim-1 plays a major role in accelerating the progression of cardiomyopathy in female diabetics through upregulation of miR-1 and 208a. In vitro restoration of Pim-1 levels either through direct overexpression of Pim-1 or inhibition of miR-1 and 208a reverted this “female disadvantage” in the diabetic cardiomyocytes. Finally, molecular analysis of cardiac tissue samples from the human diabetic hearts also showed marked downregulation of Pim-1 in diabetic females.
Diastolic dysfunction is the earliest clinical sign of diabetic cardiomyopathy, followed by progression to systolic dysfunction . Female diabetic mice showed marked diastolic dysfunction within 8 weeks of STZ-induced diabetes in our study, however, this was lost at 12 weeks. Diastolic dysfunction is usually associated with a decline in the E/A ratio , but this ratio can pseudo-normalise with progression into severe diastolic dysfunction . In such cases, an increase in the end diastolic pressure can restrict the contribution of atrial contraction to LV filling . Moreover, pseudo-normal or restrictive filling patterns are related to progressive LV dilation and predict cardiac death after a first MI . Consistently, our echocardiography data showed LV dilation and thinning of the anterior LV wall associated with increased end diastolic pressure and decreased dP/dtmin in female mice at 12 weeks of STZ-induced diabetes, suggesting the exacerbation of diastolic dysfunction in female diabetic mice.
While our study demonstrated early onset of cardiomyopathy in female diabetics, Ceylan-Isik et al. reported that cardiomyocytes isolated from the female diabetic hearts were better protected than the cardiomyocytes from male diabetic hearts . Similarly, cardiomyocytes isolated from young female diabetic mice by Zhang et al. exhibited normal contractile function . Although these results are in contrast to our findings, the difference could be attributed mainly to the different experimental conditions. We used an in vivo model reflecting near clinical settings, while the above-mentioned studies used isolated cardiomyocytes. In addition, factors such as cell death and myocardial fibrosis, which play a major role in the development of cardiac dysfunction would not affect the results in in vitro settings.
We and others have shown that selected molecular alterations occur early in the diabetic heart which forms the basis for the development of structural changes [17, 18, 29]. Our earlier studies showed significant downregulation of the pro-survival protein Pim-1 in the male diabetic heart at 8 weeks after the onset of STZ-induced diabetes. Importantly, restoring the Pim-1 levels by systemic adeno-associated viral vector gene delivery halted the progression of diabetic cardiomyopathy . In the current study, we found that Pim-1 was downregulated within 4 weeks of STZ-induced diabetes in the female heart, which was associated with increased pro-apoptotic caspase-3 expression. Studies on human heart samples also confirmed significant downregulation of Pim-1 in female diabetic compared to male diabetic hearts. Although it is a difficult task to translate the findings from animal studies to humans, diabetes duration for the samples collected from human diabetic heart in our study ranged between 12 and 18 years and based on the published evidence this relates to 12 to 16 weeks of diabetes duration in mice . Of note, this was the time point when significant difference was observed between male and female STZ-induced diabetic mice in most of the functional and molecular parameters.
Pim-1 is a crucial component of the signalling machinery that counteracts cardiomyocyte apoptosis during the early phase of post-ischemic healing [15, 17, 39, 40]. This was true in our study where restoration of Pim-1 increased the survival of female diabetic cardiomyocytes. Akt is the major mediator of Pim-1. Murasaki et al demonstrated marked increase in the expression levels of Pim-1 following overexpression of cardiomyocytes, while knocking down Akt reduced Pim-1 . Interestingly, our results did not demonstrate any changes in the level of Akt at 4 weeks, suggesting that early implication of Pim-1 on survival could be Akt independent, but they could synergise later producing more apoptosis.
Our results newly show marked upregulation of miR-1 in the female diabetic heart. MiR-1 has been well demonstrated as the direct regulator of Pim-1 in the heart independent of Akt  and our earlier study showed marked improvement in the survival of male diabetic cardiomyocytes following knockdown of miR-1 . In addition to miR-1, we also found early activation of miR-208a in the female diabetic mice, which might also account for increased LV dilation early in the female diabetic heart . In support of this notion, inhibition of both miR-1 and -208a improved the survival of female diabetic cardiomyocytes. However, the miR expression study on human hearts did not reveal any significant difference between male and female diabetics although there was a trend for increased expression of miR-1 in female diabetics. This could be attributed to the long duration of diabetes (>12 years) when samples were collected from the patients. Another reason could be that all the patients underwent coronary artery bypass graft surgery for ischemic heart disease, in contrast to the isolated cardiomyopathy of the mouse model. Additional in vivo studies are necessary to understand the role of miR-1 and miR-208a in accelerating the development of cardiomyopathy in female diabetic hearts.
In summary, our results provide novel insights into the molecular mechanisms behind the rapid onset of cardiomyopathy in STZ-induced female diabetic mice, with preliminary data from human hearts supporting the pre-clinical study results. Future studies targeted on in vivo restoration of Pim-1 either by upregulation of Pim-1 or by knocking-down miR-1 will provide a platform for the development of gender specific treatment to combat the disease.
In the present study we used STZ-induced type-1 diabetes model to prove our concept. Although our earlier study has demonstrated comparable disease pattern and progression in the type-1 and type-2 diabetic model , it is essential to demonstrate if similar gender difference exhibit in type-2 diabetes. Human data shown in this study come from right atrial appendage samples, while the results from animal study are from the ventricle. Although Lamberts et al (personal communication, 2014) showed no difference in the functional properties of the trabeculae isolated from the rat atria and ventricle, further studies will be required to confirm if the same exists in the expression pattern of Pim-1 between atria and ventricle.