This study investigated the association of type 2 diabetes and the MetS with myocardial fibrosis (assessed by picrosirius red staining and immunostaining for collagen I and III), cardiomyocyte size, capillary length density, diffusion radius, arteriolar dimensions, and myocardial expression of CML and RAGE. We confirmed the impaired diastolic function of type 2 diabetic and MetS patients [2, 34], as shown by their increased pulmonary capillary wedge pressure and reduced mitral septal E'. Our data demonstrate that the impaired diastolic function of type 2 diabetic and MetS patients was not dependent on increased myocardial fibrosis, cardiomyocyte hypertrophy, alteration of the myocardial microvascular structure, or increased myocardial expression of CML or RAGE. These data suggest that the increased myocardial fibrosis and CML expression reported by previous studies of diabetic patients with heart failure [3, 12, 21, 22, 33] were a consequence, rather than an initiating cause, of impaired cardiac function.
Our findings for myocardial fibrosis and CML expression are in agreement with a recent study by van Heerebeek et al.  who studied endomyocardial LV biopsies from patients hospitalized with worsening heart failure. They found no increase in fibrosis or CML expression in LV biopsies of diabetic subjects with normal LV ejection fraction, but increased fibrosis and CML expression in patients with reduced LV ejection fraction . The present study extends these findings to a much earlier stage in the evolution of diabetic cardiomyopathy by study of epicardial LV biopsies from diabetic and MetS patients with increased LV filling pressures without symptomatic heart failure. Although the diabetic patients had reduced mitral septal E', we found no differences between the three patient groups for Doppler measures of left ventricular filling or left atrial size, suggesting that the impairment of diastolic function was much less than that which occurs in heart failure with preserved ejection fraction.
Cardiomyocyte size has been reported to be increased [3, 13, 17], normal , and reduced in diabetic patients . Our finding of no increase in cardiomyocyte width of diabetic and MetS patients is in agreement with reports of similar cardiac mass for diabetic and non-diabetic individuals , and the association between increased insulin levels and cardiac hypertrophy is largely accounted for by associations with body size . Moreover, our finding of no effect of diabetes and the MetS on arteriolar dimensions is in agreement with a previous autopsy study of myocardial arteries . Our failure to detect hypertrophy of myocardial arterioles similar to that reported for small arteries in subcutaneous tissue of patients with type 2 diabetes  may be because the arterioles examined in our study were much smaller in size, and it is also possible that the effects of diabetes on vascular hypertrophy are different for different vascular beds. The similar capillary length density and arteriolar dimensions of diabetic and non-diabetic patients in this study is in agreement with the similar myocardial perfusion of diabetic and non-diabetic subjects in the fasting state [38, 39], although it does not exclude an effect of diabetes and the MetS on microvascular function.
Our finding that RAGE expression was predominantly localized to vascular endothelium and capillaries is in agreement with previous studies , although, to our knowledge, this is the first comparison of myocardial RAGE expression in control, diabetic, and MetS patients. Whereas cell-surface-bound RAGE plays an essential role in mediating the effects of AGEs, soluble RAGE may act as an AGE inhibitor, by preventing the binding of AGEs to the cell-surface-bound RAGE receptor . Our finding of similar circulating levels of soluble RAGE and cardiac expression of RAGE suggest RAGE did not participate in the impaired diastolic function of diabetic and MetS patients in our study. The similar plasma CML and LMWF levels of diabetic patients to those of control patients is in agreement with our previous study , and likely reflects their well-controlled diabetes.
Differences between human and animal models of diabetes
Our study highlights possible differences between diabetic humans and animal models of diabetes. In addition to the lack of increased myocardial fibrosis, we found no evidence for the cardiomyocyte and arteriolar hypertrophy and reduced myocardial capillary density reported in animal models of diabetes [7, 9–11], which may be related to poorer diabetic control in animal models.
Alternative mechanisms of impaired diastolic function of diabetes and the MetS
Many mechanisms other than increased fibrosis, cardiomyocyte hypertrophy and alteration in myocardial vasculature have been proposed to explain the diastolic dysfunction of diabetes and the MetS. These include alterations in cardiomyocyte metabolism, abnormal calcium homeostasis and contractile mechanisms, oxidative stress, direct glucotoxicity, and inflammation [1, 2, 5, 8, 22, 43, 44]. Our data are compatible with a role for both insulin resistance and hyperglycemia in the pathogenesis of the diastolic dysfunction of diabetes and the MetS .
Our study had a number of limitations. These included a limited sample size because of the need for myocardial biopsies from each patient. However, in a separate study of 11 control, 7 diabetic, and 6 MetS patients with aortic stenosis we similarly found no association of diabetes or the MetS with increased myocardial fibrosis or altered cardiomyocyte width, capillary length density, diffusion radius, or arteriolar dimensions, although, in comparison with the patients in this study, aortic stenosis patients had more interstitial fibrosis, greater cardiomyocyte width, lower capillary length density, and greater diffusion radius (unpublished data). Another limitation was the inherent selection bias caused by the sampling of patients presenting for coronary artery bypass graft surgery. However, in a comparison of aortic stenosis patients with and without coronary artery disease we found no effect of coronary artery disease on myocardial fibrosis, cardiomyocyte width, capillary length density, diffusion radius, or arteriolar dimensions (unpublished data). Patients with coronary artery disease were an important patient group to study because of the high prevalence of coronary artery disease in diabetic patients [45, 46], and coronary artery disease patients with diabetes are twice as likely as those without diabetes to develop heart failure . Although the frequent use of antihypertensive therapy, including inhibitors of the renin angiotensin system and statin therapy, may have attenuated the effect of MetS and diabetes on the myocardium, the MetS and diabetic patients in this study had evidence of diastolic dysfunction. Moreover, diabetes remains a predictor of heart failure when these therapies are included in multivariate analysis . To avoid the effect of coronary stenoses on myocardial structure and the microvasculature we took particular care to collect biopsies from the same epicardial region of the LV myocardium without evidence of ischemia or wall motion abnormality, which was proximal to significant flow-limiting coronary stenoses and collaterals. However, we do not know if the data obtained apply to other regions of the myocardium, such as myocardium distal to significant flow-limiting coronary stenoses. It is also unknown whether our findings are relevant to the right ventricle, which may also contribute to diabetic cardiomyopathy . Moreover, the generalizability of our findings is limited by the exclusion of women. Although we had echocardiographic data for only a limited number of patients we had hemodynamic data, including ventriculograms and pulmonary capillary wedge pressures, which provided a direct measure of LV filling pressure, for all patients.