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Diabetic cardiomyopathy is a clinically distinct disease characterized by impaired cardiac function as a result of reduced contractility and hypertension-induced athero- or arteriosclerosis. This may be due either to generalized vascular disease, tissue-based injury such as focal cardiomyocyte dysmorphia, or microvascular damage manifested by myocardial capillary basement membrane (CBM) thickening. Hyperglycemia-driven increases in reactive oxygen species (ROS) have been proposed to contribute to such damage. To address this hypothesis, we utilized light (LM) and transmission electron microscopy (TEM) to demonstrate cardiomyocyte morphology and myocardial CBM thickness in the left ventricles of four mouse genotypes: FVB (background Friend virus B controls), OVE (transgenic diabetics), Mt [transgenics with targeted overexpression of the antioxidant protein metallothionein (MT) in cardiomyocytes], and OVEMt (bi-transgenic cross of OVE and Mt) animals. Mice were prepared for morphometric analysis by vascular perfusion. Focal myocardial disorganization was identified in OVE mice but not in the remaining genotypes. Not unexpectedly, myocardial CBM thickness was increased significantly in OVE relative to FVB (P < 0.05) and Mt (P < 0.05) animals (+28% and +39.5%, respectively). Remarkably, however, OVEMt myocardial CBMs showed no increase in width; rather they were ∼3% thinner than FVB controls. Although the molecular mechanisms regulating CBM width remain elusive, it seems possible that despite a significant hyperglycemic environment, MT antioxidant activity may mitigate local oxidative stress and reduce downstream excess microvascular extracellular matrix (ECM) formation. In addition, the reduction of intra- and perivascular ROS may protect against incipient endothelial damage and the CBM thickening that results from such injury. Anat Rec, 296:480–487, 2013. © 2013 Wiley Periodicals, Inc.
Classical descriptions of the chronic complications of diabetes mellitus (DM) generally include diabetic nephropathy, retinopathy and neuropathy (Nathan, 1993). More recently, it has been recognized that diabetic patients are predisposed to cardiac failure and have a particularly poor prognosis following myocardial infarction. These clinical findings together with postmortem cardiomyopathic changes in the absence of coronary artery disease, led to the first description of diabetic cardiomyopathy as a clinically distinct disease (Rubler, 1972). Subsequent studies of diabetics (Bell, 1995) have identified several left ventricular abnormalities that support the existence of diabetic cardiomyopathy as a distinct primary disease (Asghar et al., 2009).
Hyperglycemia-driven mechanisms are widely regarded as causative factors in the development of diabetic complications. These include activation of protein kinase C, (Ha et al., 2001), advanced glycation end products (AGEs) (Vlassara and Palace, 2002) elevated sorbitol (Wallner et al., 2001), increased transforming growth factor β (TGFβ) activity (Ziyadeh, 2004) and oxidative stress (Giaccco and Brownlee, 2010; Kashihara et al., 2010). Most investigators believe that a combination of these processes are involved, and though it is not known which mechanism is most dominant or pervasive, it is clear that oxidative stress plays a pivotal role in the development of chronic complications of DM (Brownlee, 2005). Overall, antioxidant status is reduced in diabetes (Wolff et al., 1991; Vijayalingam et al., 1996) and ROS are important in many diabetic complications (Baynes, 1991; Low et al., 1997).
This concept is supported by the results of several of our own studies (Liang et al., 2002; Zheng et al., 2008) in which we have described the features of a transgenic diabetic mouse (OVE26, herein referred to as OVE) that provides a remarkably accurate morphological and functional model for human DM (Zheng et al., 2008, 2004; Teiken, et al., 2011, Carlson et al., in press). In his seminal work, Liang et al. (2002) showed that breeding OVE to a second transgenic mouse (Mt) with targeted overexpression of MT in the myocardium resulted in bi-transgenic (OVEMt) progeny in which the progression to advanced diabetic cardiomyopathy was markedly reduced. These findings provided evidence for a direct role for myocardial oxidative damage in diabetic mice and demonstrated that targeted MT overexpression in cardiac myocytes resulted in significant protection against several cardiopathological consequences of DM.
Because previous studies have not examined whether targeted MT overexpression may provide protection against CBM thickening, we hypothesized that CBM width would be reduced in OVEMt bi-transgenic animals. This idea was tested with a series of LM and TEM morphometric studies in which myocardial CBM width was compared in 350-day-old (1) FVB control mice, (2) Mt mice overexpressing MT in cardiomyocytes, (3) OVE transgenic diabetic mice, and (4) bi-transgenic OVEMt mice (Mt mice bred to OVE animals).
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- MATERIALS AND METHODS
- LITERATURE CITED
The objective of the current study was to quantify myocardial CBM thickness in four age-matched mouse genotypes. We found that targeted overexpression of MT in cardiac myocytes eliminates the structural damage in these cells, a finding consistent with those demonstrated by Liang et al. (2002).
Capillary BM thickening was not measured until 1968, when Siperstein et al. demonstrated in skeletal muscle that CBM thickening was characteristic of nearly all patients (98%) with blood glucose values >140 mg dL−1 (Siperstein et al., 1968). Subsequent development of TEM techniques provided the resolution necessary to demonstrate changes in the diabetic microvasculature including thickened CBMs. It is currently widely accepted that accelerated CBM thickening is a diagnostic feature of advanced stages of DM (Viberti, 1994).
As part of an ongoing comprehensive morphometric analysis of the 350-day-old mouse heart in four different genotypes, the current study centered specifically on left ventricular myocardial CBMs. The data showed that CBM thickness in OVE diabetic animals averaged 62.18 ± 10.34 nm, while the mean of FVB controls was 48.5 ± 7.31 nm. This relatively large (28%) difference in diabetics versus controls confirmed that myocardial CBM is one of the most susceptible to hyperglycemia-driven thickening (Carlson et al., 2003). Much more remarkable, however, was our finding that CBM widths were essentially identical in FVB and OVEMt animals, indicating that myocardial CBM thickening in animals with cardiomyocyte targeted MT overexpression was not simply reduced, but rather was completely abolished.
A number of molecular mechanisms have been implicated in the pathogenesis of diabetic BM disease and a unifying hypothesis suggests that since hyperglycemia increases free radical generation, ROS may be significant mediators of diabetic tissue injury, and their increased production may represent a final common pathway toward chronic complications of the disease (Brownlee, 2005; Giaccco and Brownlee, 2010). In this regard, data in the current study strongly implicate a direct role for oxidative damage in the initiation of a pathway to myocardial injury and associated CBM thickening.
Although the molecular mechanisms responsible for diabetic CBM thickening are poorly understood and likely are multifactorial (Tsilibary, 2003), most theories invoke a form of decreased ECM turnover resulting in increased BM width. In this regard, the advanced glycation end product (AGE) hypothesis (Brownlee et al., 1984) is attractive. This concept centers on non-enzymaticglycation of proteins and other long-lived molecules, the formation of which is regulated by glucose concentration and exposure time (Vlassara and Palace, 2002) and could lead to reduced CBM turnover resulting from decreased degradation of highly cross-linked long-lived proteins such as collagen and other ECM proteins (Brownlee et al., 1984). However, formation of AGE also is believed to play a central role in increased oxidative stress and upregulated ROS (Goh and Cooper, 2008) and it is well-known that increased ROS activate various transcription factors and cytokines leading ultimately to increased expression of ECM genes, fibrosis, and thickened BMs (Mason and Wahab, 2003).
Although additional confirmation is required, current data strongly support the concept that intracellular ROS have a direct role in overproduction of ECM proteins (Ha and Lee, 2000; Brownlee, 2001). Furthermore, it has been shown that ROS activate a cascade of downstream reactions in which TGF-β and connective tissue growth factor coordinate to promote upregulation of transcription of numerous matrix genes (Nath et al., 1998; Iglesisus–de et al., 2001; Park et al., 2001), and repress that of matrix metalloproteinases—concomitant events which in vivo could lead symbiotically to decreased CBM turnover (Mason and Wahab, 2003). It is possible, therefore, that increased CBM width in diabetic animals could result either from hyperglycemia-driven ROS production and increased ECM synthesis, or decreased degradation driven by inhibited matrix metalloproteinases and inexorably increasing highly crosslinked AGE products. Alternatively, both mechanisms could be operable.
Chronic complications of diabetes exhibit a series of common morphological features including interstitial fibrosis, arteriolar and microvascular BM thickening. These structural changes occur relatively late in the course of the disease, and it seems likely that they may represent the consequences of hyperglycemia-driven oxidative stress. In a recent review, Kashihara et al. (2010) point out that in the progression to diabetic complications, numerous opportunistic macromolecules, including NAD(P)H oxidase, AGE, polyol pathway defects, uncoupled nitric oxide synthase (NOS) and oxidative phosphorylation have been implicated in the increased production of ROS and subsequent tissue damage. This interpretation is consistent with those of a number of investigators who postulate that oxidative stress is a common denominator for the pathways involved in the progression of chronic complications of diabetes (Prabhakar et al., 2007; Giacco and Brownlee, 2010; Kashihara, 2010). It follows therefore, that mechanisms leading to reduced ROS, could be expected to be protective against redundant production of ECM proteins, including those comprising myocardial CBMs.
Although MT is a group of intracellular metal-binding and cysteine-rich proteins that function primarily as metal homeostasis regulators, in the 1980s they were found to be potent antioxidants (Onosaka et al., 1988). Subsequent in vitro studies showed they reacted directly with ROS. Significantly, multidisciplinary investigations have shown that MT also function to protect against oxidative injury in vivo. It is not surprising, therefore, that the antioxidant function of MT has been explored extensively (Li et al., 2007) and in several important studies in which MT was overexpressed in the diabetic myocardium (Cai and Kang, 2001; Liang et al., 2002; Ye et al., 2003) ROS was apparently mitigated and chronic complications were effectively reduced. More recently it was shown that the MT prevention of diabetic cardiomyopathy is mainly due to its suppression of diabetes-derived nitrosative stress and damage (Cai, 2006).
Cardiac myocyte structure and function were the focus of most previous investigations of MT overexpression in the myocardium (see Li et al., 2007 for review), and it was assumed that the demonstrated MT protective function correlated closely with inhibition of ROS-induced lipid peroxidation. However, the current study centers on myocardial capillaries in similar animal genotypes and uncovers a previously unrecognized literal elimination of hyperglycemia-driven CBM thickening, the mechanism of which is not clear but appears to be directly related to ROS mitigation by MT.
In summary, the current myocardial study strongly supports an hypothesis that in response to hyperglycemia-driven oxidative stress, CBM-associated cells trigger a series of early molecular microangiopathic events leading progressively to increased ROS and subsequent sequential diabetic complications including significantly thickened myocardial CBMs. The data also indicate that MT overexpression in cardiac myocytes, and possibly other cell types is fully capable of abolishing an otherwise expected significant diabetic CBM thickening.