Contractile dysfunction in hypertrophied hearts with deficient insulin receptor signaling: possible role of reduced capillary density.

Diabetics have worse outcomes than nondiabetics after a variety of cardiac insults. We tested the hypothesis that impaired insulin receptor signaling in myocytes worsens cardiac remodeling and function following injury, even in the absence of hyperglycemia. Mice with cardiomyocyte-restricted knock out of the insulin receptor (CIRKO) and wild type (WT) mice were treated with isoproterenol (ISO) for 2 or 5 days. Heart rates and cardiac mass increased comparably following ISO in WT and CIRKO mice. After 5 days, WT hearts were hyperdynamic by echocardiographic and left ventricular pressure measurements. However, CIRKO hearts had a blunted increase in contractility and relaxation following ISO. Interestingly, single myocytes isolated from both CIRKO ISO and WT ISO hearts had increased cellular shortening with prolonged time to peak shortening vs. respective shams. Thus, loss of myocytes or extramyocyte factors, rather than intrinsic dysfunction of surviving myocytes, caused the blunted inotropic response in ISO treated CIRKO hearts. Indeed, CIRKO ISO mice had increased troponin release after 2 days and greater interstitial and sub-endocardial fibrosis at 5 days than did ISO WT. Apoptosis assessed by TUNEL and caspase staining was increased in CIRKO ISO compared to WT ISO hearts; however, very few of the apoptotic nuclei were clearly in cardiac myocytes. After 5 days of ISO treatment, VEGF expression was increased in WT but not in CIRKO hearts. In keeping with this finding, capillary density was reduced in CIRKO ISO relative to WT ISO. Basal expression of hypoxia-inducible factor-1alpha was lower in CIRKO vs. WT hearts and may explain the blunted VEGF response. Thus, absence of insulin receptor signaling in the cardiac myocyte worsens catecholamine-mediated myocardial injury, at least in part, via mechanisms that tend to impair myocardial blood flow and increase ischemic injury.

Reduced cardiac efficiency and altered substrate metabolism precedes the onset of hyperglycemia and contractile dysfunction in two mouse models of insulin resistance and obesity.

Hyperglycemia is associated with altered myocardial substrate use, a condition that has been hypothesized to contribute to impaired cardiac performance. The goals of this study were to determine whether changes in cardiac metabolism, gene expression, and function precede or follow the onset of hyperglycemia in two mouse models of obesity, insulin resistance, and diabetes (ob/ob and db/db mice). Ob/ob and db/db mice were studied at 4, 8, and 15 wk of age. Four-week-old mice of both strains were normoglycemic but hyperinsulinemic. Hyperglycemia develops in db/db mice between 4 and 8 wk of age and in ob/ob mice between 8 and 15 wk. In isolated working hearts, rates of glucose oxidation were reduced by 28-37% at 4 wk and declined no further at 15 wk in both strains. Fatty acid oxidation rates and myocardial oxygen consumption were increased in 4-wk-old mice of both strains. Fatty acid oxidation rates progressively increased in db/db mice in parallel with the earlier onset and greater duration of hyperglycemia. In vivo, cardiac catheterization revealed significantly increased left ventricular contractility and relaxation (positive and negative dP/dt) in both strains at 4 wk of age. dP/dt declined over time in db/db mice but remained elevated in ob/ob mice at 15 wk of age. Increased beta-myosin heavy chain isoform expression was present in 4-wk-old mice and persisted in 15-wk-old mice. Increased expression of peroxisomal proliferator-activated receptor-alpha regulated genes was observed only at 15 wk in both strains. These data indicate that altered myocardial substrate use and reduced myocardial efficiency are early abnormalities in the hearts of obese mice and precede the onset of hyperglycemia. Obesity per se does not cause contractile dysfunction in vivo, but loss of the hypercontractile phenotype of obesity and up-regulation of peroxisomal proliferator-activated receptor-alpha regulated genes occur later and are most pronounced in the presence of longstanding hyperglycemia.

Gonadotropin-mediated regulation of the murine VEGF expression in MA-10 Leydig cells.

Presence of vascular endothelial growth factor (VEGF) is not only limited to cells directly involved in angiogenesis but has also been demonstrated in steroidogenic cells like testicular Leydig cells. Because Leydig cells are subjected to regulation by gonadotropic hormones and produce steroid hormones, we have investigated here the effects of human chorionic gonadotropin (hCG) or steroid hormones on VEGF expression in cultured mouse tumor Leydig cells (MA-10 cells) and have then analyzed the underlying molecular mechanisms. Northern blot analysis and enzyme-linked immunosorbent assays revealed increases in VEGF mRNA and protein levels, respectively, over 3-20 hours in MA-10 cells after stimulation with hCG or 8-Br-cAMP. Although MA-10 cells lack the classical progesterone receptor, progesterone was able to stimulate VEGF expression. Promoter analyses and antibody supershift experiments suggested that the proximal region is able to constitutively bind the transcription factors Sp1 and Sp3. Mutations of 2 potential Sp1 binding sites in the proximal region showed the requirement of these motifs for stimulation of VEGF by hCG and 8-Br-cAMP. The distal cytosine-rich sequence interacts with so far-unidentified faster migrating factors. Following stimulation with hCG or 8-Br-cAMP, the binding of these proteins was increased in the complexes formed in the proximal and distal regions. VEGF expression in Leydig cells is regulated by gonadotropin via a cAMP-dependent mechanism, and the transcription factors Sp1 and Sp3 appear to be involved in the activation of the promoter. Progesterone also appears to play a role in the regulation of VEGF, acting presumably via a nonconventional receptor that remains to be characterized yet.

Minimally invasive aortic banding in mice: effects of altered cardiomyocyte insulin signaling during pressure overload.

We developed a minimally invasive method for producing left ventricular (LV) pressure overload in mice. With the use of this technique, we quickly and reproducibly banded the transverse aorta with low surgical morbidity and mortality. Minimally invasive transverse aortic banding (MTAB) acutely and chronically increased LV systolic pressure, increased heart weight-to-body weight ratio, and induced myocardial fibrosis. We used this technique to determine whether reduced insulin signaling in the heart altered the cardiac response to pressure overload. Mice with cardiac myocyte-restricted knockout of the insulin receptor (CIRKO) have smaller hearts than wild-type (WT) controls. Four weeks after MTAB, WT and CIRKO mice had comparably increased LV systolic pressure, increased cardiac mass, and induction of mRNA for beta-myosin heavy chain and atrial natriuretic factor. However, CIRKO hearts were more dilated, had depressed LV systolic function by echocardiography, and had greater interstitial fibrosis than WT mice. Expression of connective tissue growth factor was increased in banded CIRKO hearts compared with WT hearts. Thus lack of insulin signaling in the heart accelerates the transition to a more decompensated state during cardiac pressure overload. The use of the MTAB approach should facilitate the study of the pathophysiology and treatment of pressure-overload hypertrophy.

Stimulatory effect of progesterone on the expression of steroidogenic acute regulatory protein in MA-10 Leydig cells.

The steroidogenic acute regulatory protein (StAR), by virtue of its ability to facilitate the intramitochondrial transport of cholesterol, plays an important role in regulating steroid hormone biosynthesis in steroidogenic cells. In agreement with published data, both StAR expression and progesterone production in MA-10 mouse Leydig tumor cells could be stimulated with hCG and 8Br-cAMP. Addition of aminoglutethimide, an inhibitor of cholesterol side chain cleavage (P450(scc)) enzyme, not only resulted in a drastic inhibition of progesterone production but also in an attenuation of StAR expression in response to either hCG or 8-Br-cAMP. Therefore, we addressed the question of whether progesterone, the end product of the steroidogenic cascade in these cells, could be in a position to regulate the StAR gene expression. In MA-10 cells, we report here that progesterone in microgram amounts can induce StAR gene expression in a time- and dose-dependent manner. StAR expression in response to a maximally effective concentration of progesterone of 10 microg/ml was highest at 6 h and started decreasing thereafter. The effect of progesterone on StAR protein and StAR mRNA induction was mimicked by its synthetic analog, progestin R5020, but not by other steroids, including dexamethasone, estradiol, testosterone, and dihydrotestosterone. Dexamethasone, in contrast, was able to inhibit StAR expression in MA-10 cells. Surprisingly, RU486, a potent antagonist of progesterone and glucocorticoid action, had a stimulatory effect on StAR mRNA levels. Reverse transcription-polymerase chain reaction analysis demonstrated the absence of the classical form of progesterone receptor in MA-10 cells. Thus, for the first time, a direct stimulatory effect of a steroid on StAR gene expression has been demonstrated. Furthermore, these results provide a new insight, indicating that progesterone mediates the activation of StAR expression exerted presumably through a novel, nonclassical progesterone receptor in mouse Leydig cells.