Lack of collagen VIII reduces fibrosis and promotes early mortality and cardiac dilatation in pressure overload in mice.

AIMS: In pressure overload, left ventricular (LV) dilatation is a key step in transition to heart failure (HF). We recently found that collagen VIII (colVIII), a non-fibrillar collagen and extracellular matrix constituent, was reduced in hearts of mice with HF and correlated to degree of dilatation. A reduction in colVIII might be involved in LV dilatation, and we here examined the role of reduced colVIII in pressure overload-induced remodelling using colVIII knock-out (col8KO) mice. METHODS AND RESULTS: Col8KO mice exhibited increased mortality 3-9 days after aortic banding (AB) and increased LV dilatation from day one after AB, compared with wild type (WT). LV dilatation remained increased over 56 days. Forty-eight hours after AB, LV expression of main structural collagens (I and III) was three-fold increased in WT mice, but these collagens were unaltered in the LV of col8KO mice together with reduced expression of the pro-fibrotic cytokine TGF-ß, SMAD2 signalling, and the myofibroblast markers Pxn, α-SMA, and SM22. Six weeks after AB, LV collagen mRNA expression and protein were increased in col8KO mice, although less pronounced than in WT. In vitro, neonatal cardiac fibroblasts from col8KO mice showed lower expression of TGF-ß, Pxn, α-SMA, and SM22 and reduced migratory ability possibly due to increased RhoA activity and reduced MMP2 expression. Stimulation with recombinant colVIIIα1 increased TGF-ß expression and fibroblast migration. CONCLUSION: Lack of colVIII reduces myofibroblast differentiation and fibrosis and promotes early mortality and LV dilatation in response to pressure overload in mice.

ROS-mediated decline in maximum Ca2+-activated force in rat skeletal muscle fibers following in vitro and in vivo stimulation.

We hypothesised that normal skeletal muscle stimulated intensely either in vitro or in situ would exhibit reactive oxygen species (ROS)-mediated contractile apparatus changes common to many pathophysiological conditions. Isolated soleus (SOL) and extensor digitorum longus (EDL) muscles of the rat were bubbled with 95% O(2) and stimulated in vitro at 31°C to give isometric tetani (50 Hz for 0.5 s every 2 s) until maximum force declined to ≤30%. Skinned superficial slow-twitch fibers from the SOL muscles displayed a large reduction (∼41%) in maximum Ca(2+)-activated specific force (F(max)), with Ca(2+)-sensitivity unchanged. Fibers from EDL muscles were less affected. The decrease in F(max) in SOL fibers was evidently due to oxidation effects on cysteine residues because it was reversed if the reducing agent DTT was applied prior to activating the fiber. The GSH:GSSG ratio was ∼3-fold lower in the cytoplasm of superficial fibers from stimulated muscle compared to control, confirming increased oxidant levels. The presence of Tempol and L-NAME during in vitro stimulation prevented reduction in F(max). Skinned fibers from SOL muscles stimulated in vivo at 37°C with intact blood supply also displayed reduction in F(max), though to a much smaller extent (∼12%). Thus, fibers from muscles stimulated even with putatively adequate O(2) supply display a reversible oxidation-induced decrease in F(max) without change in Ca(2+)-sensitivity, consistent with action of peroxynitrite (or possibly superoxide) on cysteine residues of the contractile apparatus. Significantly, the changes closely resemble the contractile deficits observed in a range of pathophysiological conditions. These findings highlight how readily muscle experiences ROS-related deficits, and also point to potential difficulties when defining muscle performance and fatigue.

Temporary fatigue and altered extracellular matrix in skeletal muscle during progression of heart failure in rats.

Patients with congestive heart failure (CHF) experience increased skeletal muscle fatigue. The mechanism underlying this phenomenon is unknown, but a deranged extracellular matrix (ECM) might be a contributing factor. Hence, we examined ECM components and regulators in a rat postinfarction model of CHF. At various time points during a 3.5 mo-period after induction of CHF in rats by left coronary artery ligation, blood, interstitial fluid (IF), and muscles were sampled. Isoflurane anesthesia was employed during all surgical procedures. IF was extracted by wicks inserted intermuscularly in a hind limb. We measured cytokines in plasma and IF, whereas matrix metalloproteinase (MMP) activity and collagen content, as well as the level of glycosaminoglycans and hyaluronan were determined in hind limb muscle. In vivo fatigue protocols of the soleus muscle were performed at 42 and 112 days after induction of heart failure. We found that the MMP activity and collagen content in the skeletal muscles increased significantly at 42 days after induction of CHF, and these changes were time related to increased skeletal muscle fatigability. These parameters returned to sham levels at 112 days. VEGF in IF was significantly lower in CHF compared with sham-operated rats at 3 and 10 days, but no difference was observed at 112 days. We conclude that temporary alterations in the ECM, possibly triggered by VEGF, are related to a transient development of skeletal muscle fatigue in CHF.

Enhanced matrix metalloproteinase activity in skeletal muscles of rats with congestive heart failure.

Patients with congestive heart failure (CHF) are prone to increased skeletal muscle fatigue. Elevated circulatory concentrations of tumor necrosis factor (TNF)-alpha and monocyte chemoattractant protein-1, which may stimulate matrix metalloproteinase (MMP) activity and, thereby, contribute to skeletal muscle dysfunction, are frequently found in CHF. However, whether skeletal muscle MMP activity is altered in CHF is unknown. Hence, we have used a gelatinase assay to assess the activity of MMP and tissue inhibitors of MMP in single skeletal muscles of rats with CHF 6 wk after induction of myocardial infarction. Sham-operated (Sham) rats were used as controls. We also measured the gene expression and protein contents of MMP-2 and MMP-9 in skeletal muscles of these rats. Plasma MMP activity was nearly seven times higher (P < 0.05) in CHF than in Sham rats. Concomitantly, the MMP activity within single slow- and fast-twitch skeletal muscles of CHF rats increased two- to fourfold compared with Sham animals, whereas tissue inhibitor of MMP activity did not differ (P > 0.05). Preformed MMP-2 and MMP-9 were probably activated in CHF, because neither their gene expression nor protein levels were altered (P > 0.05). Serum concentrations of TNF-alpha and monocyte chemoattractant protein-1 remained unchanged (P > 0.05) between CHF and Sham rats during the 6-wk observation period. We conclude that development of CHF in rats enhances MMP activity, which in turn may distort the normal contractile function of skeletal muscle, thereby contributing to increased skeletal muscle fatigue.

Reduced level of serine(16) phosphorylated phospholamban in the failing rat myocardium: a major contributor to reduced SERCA2 activity.

OBJECTIVE: Heart failure is associated with alterations in contractile parameters and accompanied by abnormalities in intracellular calcium homeostasis. Sarcoplasmic reticulum Ca(2+) ATPase (SERCA2) and phospholamban (PLB) are important in intracellular calcium cycling. The aim of the present study was to examine mechanisms causing reductions in SERCA2 activity in the failing heart. METHODS: Myocardial infarction (MI) was induced in male Wistar rats, and animals with congestive heart failure were examined 6 weeks after the primary operation. RESULTS: Serine(16) monomeric and pentameric phosphorylated PLB were significantly downregulated (50 and 55%, respectively), whereas threonine(17) phosphorylated PLB was unchanged in failing compared to sham hearts. Protein phosphatases 1 and 2A were significantly upregulated (26 and 42%, respectively) and phosphatase 2C significantly downregulated (29%), whereas the level of protein kinase A regulatory subunit II remained unchanged during heart failure. Increasing PLB phosphorylation by forskolin in isolated cardiomyocytes after inhibition of the Na(+)-Ca(2+) exchanger activity had significantly greater effect on SERCA2 activity in failing than in sham cells (49 and 20% faster transient decline, respectively). Decreasing PLB phosphorylation by the protein kinase A inhibitor H89 had significantly less effect on SERCA2 activity in failing compared to sham cardiomyocytes (20 and 75% slower transient decline, respectively). CONCLUSION: The observed changes in SERCA2 activity after increasing and decreasing serine(16) PLB phosphorylation in cardiomyocytes from sham and failing hearts, suggest that the observed reduction in serine(16) PLB phosphorylation is one major factor determining the reduced SERCA2 activity in heart failure after MI.