Diabetes-Induced Cardiomyocyte Passive Stiffening Is Caused by Impaired Insulin-Dependent Titin Modification and Can Be Modulated by Neuregulin-1.

RATIONALE: Increased titin-dependent cardiomyocyte tension is a hallmark of heart failure with preserved ejection fraction associated with type-2 diabetes mellitus. However, the insulin-related signaling pathways that modify titin-based cardiomyocyte tension, thereby contributing to modulation of diastolic function, are largely unknown. OBJECTIVE: We aimed to determine how impaired insulin signaling affects titin expression and phosphorylation and thus increases passive cardiomyocyte tension, and whether metformin or neuregulin-1 (NRG-1) can correct disturbed titin modifications and increased titin-based stiffness. METHODS AND RESULTS: We used cardiac biopsies from human diabetic (n=23) and nondiabetic patients (n=19), cultured rat cardiomyocytes, left ventricular tissue from apolipoprotein E-deficient mice with streptozotocin-induced diabetes mellitus (n=12-22), and ZSF1 (obese diabetic Zucker fatty/spontaneously hypertensive heart failure F1 hybrid) rats (n=5-6) and analyzed insulin-dependent signaling pathways that modulate titin phosphorylation. Titin-based passive tension was measured using permeabilized cardiomyocytes. In human diabetic hearts, we detected titin hypophosphorylation at S4099 and hyperphosphorylation at S11878, suggesting altered activity of protein kinases; cardiomyocyte passive tension was significantly increased. When applied to cultured cardiomyocytes, insulin and metformin increased titin phosphorylation at S4010, S4099, and S11878 via enhanced ERK1/2 (extracellular signal regulated kinase 1/2) and PKCα (protein kinase Cα) activity; NRG-1 application enhanced ERK1/2 activity but reduced PKCα activity. In apolipoprotein E-deficient mice, chronic treatment of streptozotocin-induced diabetes mellitus with NRG-1 corrected titin phosphorylation via increased PKG (protein kinase G) and ERK1/2 activity and reduced PKCα activity, which reversed the diabetes mellitus-associated changes in titin-based passive tension. Acute application of NRG-1 to obese ZSF1 rats with type-2 diabetes mellitus reduced end-diastolic pressure. CONCLUSIONS: Mechanistically, we found that impaired cGMP-PKG signaling and elevated PKCα activity are key modulators of titin-based cardiomyocyte stiffening in diabetic hearts. We conclude that by restoring normal kinase activities of PKG, ERK1/2, and PKCα, and by reducing cardiomyocyte passive tension, chronic NRG-1 application is a promising approach to modulate titin properties in heart failure with preserved ejection fraction associated with type-2 diabetes mellitus.

Severe blunt muscle trauma in rats: only marginal hypoxia in the injured area.

BACKGROUND: After severe muscle trauma, hypoxia due to microvascular perfusion failure is generally believed to further increase local injury and to impair healing. However, detailed analysis of hypoxia at the cellular level is missing. Therefore, in the present work, spectroscopic measurements of microvascular blood flow and O2 supply were combined with immunological detection of hypoxic cells to estimate O2 conditions within the injured muscle area. MATERIALS AND METHODS: Severe blunt muscle trauma was induced in the right Musculus gastrocnemius of male Wistar rats by a standardized "weight-drop" device. Microvascular blood flow, relative hemoglobin amount, and hemoglobin O2 saturation were determined by laser Doppler and white-light spectroscopy. Hypoxic cells were detected by histologic evaluation of covalent binding of pimonidazole and expression of HIF-1α. RESULTS: Directly after trauma and until the end of experiment (480 minutes), microvascular blood flow and relative hemoglobin amount were clearly increased. In contrast to blood flow and relative hemoglobin amount, there was no immediate but a delayed increase of microvascular hemoglobin O2 saturation. Pimonidazole immunostaining revealed a hypoxic fraction (percentage area of pimonidazole-labelled muscle cells within the injured area) between 8 to 3%. There was almost no HIF-1α expression detectable in the muscle cells under each condition studied. CONCLUSIONS: In the early phase (up to 8 hours) after severe blunt muscle trauma, the overall microvascular perfusion of the injured area and thus its O2 supply is clearly increased. This increased O2 supply is obviously sufficient to ensure normoxic (or even hyperoxic) conditions in the vast majority of the cells.

Myeloid hypoxia-inducible factor-1α is essential for skeletal muscle regeneration in mice.

The outstanding regeneration ability of skeletal muscle is based on stem cells that become activated and develop to myoblasts after myotrauma. Proliferation and growth of myoblasts result in self-renewal of skeletal muscle. In this article, we show that myotrauma causes a hypoxic microenvironment leading to accumulation of the transcription factor hypoxia-inducible factor-1 (HIF-1) in skeletal muscle cells, as well as invading myeloid cells. To evaluate the impact of HIF-1 in skeletal muscle injury and repair, we examined mice with a conditional HIF-1α knockout targeted to skeletal muscle or myeloid cells in a model of soft tissue trauma. No differences in acute trauma size were detected between control and HIF-1α knockout mice. However, muscles of myeloid HIF-1α knockout mice showed a significant delay in myoblast proliferation and growth of regenerating myofibers, in association with decreased expression of cyclooxygenase-2 in HIF-1α-deficient myeloid cells. Moreover, the removal of necrotic cell debris and the regeneration of endothelial cell structure were impaired in myeloid HIF-1α knockout mice that showed delayed invasion of macrophages to the injury site. Our findings for the first time, to our knowledge, demonstrate that myeloid HIF-1α is required for adequate skeletal muscle regeneration.

Transforming growth factor beta mediates apoptosis in the ganglion cell layer during all programmed cell death periods of the developing murine retina.

Transforming growth factor beta (TGF-beta) is an extracellular signaling molecule known to mediate programmed cell death (PCD) in the developing retina. In the present study, we investigated the expression profiles and activity levels of TGF-beta ligand and TGF-beta receptors (TbetaR) during the successive physiological PCD periods of the developing postnatal mouse retina. The peak of TbetaR expression levels--revealed by Western Blots and MLEC assays--coincided with the main periods of postnatal (P) retinal murine PCD at P2, P9, and P15. Immunocytochemical studies showed that the localization of the TbetaRs is restricted to the ganglion cell layer. Application of a neutralizing anti-TGF-beta antibody to E15 and P9 retinal cultures resulted in a significant decrease in the number of TUNEL-positive neurons specifically in the ganglion cell or prospective ganglion cell layer. Treatment of P2 and P15 organotypic murine retinal wholemount cultures with exogenous recombinant TGF-beta significantly increased cell death levels. In the P15 retina, where PCD affects ganglion cells and photoreceptors, TGF-beta induced cell death of large retinal ganglion cells, whereas small ganglion cells and photoreceptor neurons remained unaffected. Our data indicate that TGF-beta mediated apoptosis during all postnatal retinal PCD phases specifically affects the fate of retinal ganglion cells.