Defective mitochondrial fusion, altered respiratory function, and distorted cristae structure in skin fibroblasts with heterozygous OPA1 mutations.

Deleterious consequences of heterozygous OPA1 mutations responsible for autosomal dominant optic atrophy remain a matter of debate. Primary skin fibroblasts derived from patients have shown diverse mitochondrial alterations that were however difficult to resolve in a unifying scheme. To address the potential use of these cells as disease model, we undertook parallel and quantitative analyses of the diverse reported alterations in four fibroblast lines harboring different OPA1 mutations, nonsense or missense, in the guanosine triphosphatase or the C-terminal coiled-coil domains. We tackled several factors potentially underlying discordant reports and showed that fibroblasts with heterozygous OPA1 mutations present with several mitochondrial alterations. These included defective mitochondrial fusion during pharmacological challenge with the protonophore carbonyl cyanide m-chlorophenyl hydrazone, significant mitochondrial elongation with decreased OPA1 and DRP1 proteins, and abnormal mitochondrial fragmentation during glycolysis shortage or exogenous oxidative stress. Respiratory complex IV activity and subunits steady-state were decreased without alteration of the mitochondrial deoxyribonucleic acid size, amount or transcription. Physical link between OPA1 protein and oxidative phosphorylation was shown by reciprocal immunoprecipitation. Altered cristae structure coexisted with normal response to pro-apoptotic stimuli and expression of Bax or Bcl2 proteins. Skin fibroblasts with heterozygous OPA1 mutations thus share significant mitochondrial remodeling, and may therefore be useful for analyzing disease pathophysiology. Identifying whether the observed alterations are also present in ganglion retinal cells, and which of them underlies their degeneration process remains however an essential goal for therapeutic strategy.

Cardiac Myosin-binding protein C modulates the tuning of the molecular motor in the heart.

Cardiac myosin binding protein C (cMyBP-C) is an important regulator of cardiac contractility. Its precise effect on myosin cross-bridges (CBs) remains unclear. Using a cMyBP-C(-/-) mouse model, we determined how cMyBP-C modulates the cyclic interaction of CBs with actin. From papillary muscle mechanics, CB characteristics were provided using A. F. Huxley's equations. The probability of myosin being weakly bound to actin was higher in cMyBP-C(-/-) than in cMyBP-C(+/+). However, the number of CBs in strongly bound, high-force generated state and the force generated per CB were lower in cMyBP-C(-/-). Overall CB cycling and the velocity of CB tilting were accelerated in cMyBP-C(-/-). Taking advantage of the presence of cMyBP-C in cMyBP-C(+/+) myosin solution but not in cMyBP-C(-/-), we also analyzed the effects of cMyBP-C on the myosin-based sliding velocity of actin filaments. At baseline, sliding velocity and the relative isometric CB force, as determined by the amount of alpha-actinin required to arrest thin filament motility, were lower in cMyBP-C(-/-) than in cMyBP-C(+/+). cAMP-dependent protein kinase-mediated cMyBP-C phosphorylation further increased the force produced by CBs. We conclude that cMyBP-C prevents inefficient, weak binding of the myosin CB to actin and has a critical effect on the power-stroke step of the myosin molecular motor.

Parallel up-regulation of FGF-2 and hyaluronan during development of cardiac hypertrophy in rat.

The importance of glycosaminoglycan hyaluronan (HA) and its receptor CD44 in cell proliferation is becoming increasingly evident. Expression of the genes coding for hyaluronan synthase 1 (HAS1), HAS2, HAS3, CD44, fibroblast growth factor-2 (FGF-2), and FGF receptor-1 (FGFR-1) and the histological evidence for increases of HA and CD44 were investigated in an experimental rat model of cardiac hypertrophy. The abdominal aorta was ligated to induce cardiac hypertrophy, and mRNAs prepared from heart tissue were analyzed after 1, 6, and 42 days. The total concentration of HA was quantified, and HA and CD44 were studied histochemically. The expression of HAS1, HAS2, CD44, and FGF-2 was considerably up-regulated at days 1 and 6 and returned to basal levels after 42 days. FGFR-1 was up-regulated at day 1 but at basal levels once more at days 6 and 42. The concentration of HA significantly increased in aorta-ligated rats. Histochemical analysis showed increased expression of CD44 in hypertrophied myocardium mainly in and around the coronary arteries. These results agree well with other studies of tissue growth (malignancies and wound healing). The increase of HA, its synthases, and receptor in parallel with FGF-2 and its receptor illustrates their complicated interplay in the development of cardiac hypertrophy. The up-regulation of both HAS1 and HAS2 indicates the importance of HA production in the hypertrophic process and the possibility that HA is needed for two different purposes for the heart to be able to adapt to the increased afterload caused by aortic ligature.

Aldosterone-induced coronary dysfunction in transgenic mice involves the calcium-activated potassium (BKCa) channels of vascular smooth muscle cells.

BACKGROUND: Cardiomyocyte-specific overexpression of aldosterone synthase in male (MAS) mice induces a nitric oxide-independent coronary dysfunction. Because calcium-activated potassium (BKCa) channels are essential for vascular smooth muscle cell (VSMC) relaxation, we hypothesized that aldosterone alters their expression and/or function in VSMCs. METHODS AND RESULTS: Left coronary artery segments were isolated from MAS or male wild-type mice and mounted in a wire myograph. Responses to acetylcholine were assessed (in the presence of a nitric oxide synthase inhibitor) without or with the cyclooxygenase inhibitor diclofenac, the KCa inhibitors charybdotoxin plus apamin, or the BKCa inhibitor iberiotoxin. Expression of BKCa was quantified in hearts by real-time quantitative polymerase chain reaction and Western blot and in isolated coronary arteries by polymerase chain reaction. The effect of aldosterone on BKCa expression also was studied in cultured rat aortic VSMCs. Acetylcholine-mediated coronary relaxation was markedly decreased in MAS mice and was prevented by spironolactone. Diclofenac did not affect the MAS-induced impairment in the responses to acetylcholine, whereas charybdotoxin plus apamin virtually abolished the relaxation in both male wild-type and MAS mice. After iberiotoxin, relaxation to acetylcholine was decreased to a larger extent in male wild-type than in MAS, leading to similar levels of relaxation. BKCa-alpha and -beta1 subunit expressions were significantly decreased in MAS heart and coronary arteries. In cultured VSMCs, aldosterone induced a concentration-dependent decrease in BKCa expression, which was prevented by spironolactone. CONCLUSIONS: Aldosterone overexpression altered VSMC BKCa expression and coronary BKCa-dependent relaxation. The resulting alteration of relaxing responses may contribute to the deleterious effects of aldosterone in cardiovascular diseases. BKCa channels may therefore be useful therapeutic targets in cardiovascular diseases.

Effects of sex differences on constitutive nitric oxide synthase expression and activity in response to pressure overload in rats.

Clinical studies have documented sex differences in left ventricular (LV) hypertrophy patterns, but the mechanisms are so far poorly defined. This study aimed to determine whether 1) severe pressure overload altered expression and/or activity of cardiac constitutive nitric oxide synthase (NOS1 and NOS3) and 2) these changes were modulated according to sex. Analyses were performed 0.4-20 wk after thoracic aortic constriction (TAC) in male and female Wistar rats. Male rats with TAC exhibited early signs of cardiac dysfunction, as shown by echocardiographic and LV end-diastolic pressure measurements, whereas females with TAC exhibited higher LV hypertrophy (+96% vs. males at 20 wk; P < 0.05). After TAC, cardiac NOS1 expression was rapidly induced (0.4 wk) and stable afterward in males (P < 0.05 vs. sham groups), whereas it was delayed in females. Accordingly, specific NOS1 activity was increased by 2 wk in male rats with TAC (+122%; P < 0.001 vs. sham groups) and only by 20 wk in females (+220%; P < 0.001 vs. sham groups). NOS1 activity was correlated with NOS1 level. Regarding cardiac NOS3, expression was unaffected by TAC, and the decrease in activity observed at early and late times in male and female rats with TAC, respectively, is shown to be related to NOS3 allosteric regulator caveolin-1 level. The data demonstrated a unique sex-dependent regulation of the constitutive NOSs in response to TAC in rats; such a difference might play a role in the sex-dependent adaptability of the heart in response to pressure overload.

17beta-estradiol regulates constitutive nitric oxide synthase expression differentially in the myocardium in response to pressure overload.

Estrogens [E(2)] exert direct and indirect effects that can modulate the development of cardiac disease. However, the precise mechanisms that are involved remain undefined. Our objective was to investigate whether E(2) affected the activity and expression of constitutive nitric oxide synthase (NOS) isoforms (NOS3 and NOS1) in cardiac hypertrophy induced by thoracic aortic constriction (TAC). Ovariectomized (Ovx) and nonovariectomized Wistar rats were subjected to TAC. Ovx animals received E(2) or placebo 3 wk after surgery for 11 wk. Afterward cardiac function and degree of left ventricular hypertrophy were assessed by echocardiography. NOS activity and expression were studied by biochemical techniques. TAC led to significant left ventricular hypertrophy (>90%) irrespective of hormonal status. Cardiac performance declined more in TAC+Ovx (-20%, P < 0.015) than in the two other TAC groups [TAC and TAC+Ovx+E(2)]. Total NOS activity decreased significantly in the Ovx groups. In response to TAC, total NOS activity increased whatever the E(2) status. Specific NOS3 activity dramatically decreased in the Ovx groups (-55%, P < 0.009) and was unaltered by TAC. By using coimmunoprecipitation assays, we showed that NOS3/caveolin-1 complexes negatively regulated NOS3 activity as a function of E(2) status. On the other hand, NOS1 expression and activity were markedly increased in hypertrophied myocardium (P < 0.003), irrespective of E(2) status. This study demonstrates a differential regulation of NOS expression and activity in response to pressure overload and E(2) status, the former being mainly involved in the induction of NOS1, whereas the latter regulated NOS3 activity and in turn cardiac function.

ANG II type 1 receptor antagonist irbesartan inhibits coronary angiogenesis stimulated by chronic intermittent hypoxia in neonatal rats.

Chronic hypoxia has been shown to stimulate myocardial microvascular growth and improve cardiac ischemic tolerance in young and adult rats. The aim of this study was to determine whether the ANG II type 1 receptor (AT(1)) pathway was involved in these processes. Newborn Wistar rats, exposed to chronic intermittent hypoxia (8 h/day) for 10 days, were simultaneously treated with AT(1) receptor blocker irbesartan and compared with untreated animals. The major finding is that chronic hypoxia increased the capillary supply of myocardial tissue, which was even more pronounced in hypertrophied right ventricle, whereas increased arteriolar supply was found only in the left ventricle. This angiogenic response was completely prevented by irbesartan. Moreover, chronic hypoxia improved the postischemic recovery of cardiac contractile function during reperfusion, and this protective effect was also completely abolished by irbesartan. Chronic hypoxia increased the myocardial density of AT(1) but not of ANG II type 2 receptor subtypes, whereas the effect of irbesartan was not significant. The expression of caveolin-1alpha markedly increased in response to chronic hypoxia, and irbesartan prevented this effect. Neither hypoxia nor irbesartan treatment altered the expression of nitric oxide synthase 3, heat shock protein 90, and VEGF. It is concluded that the AT(1) receptor pathway plays an important role in coronary angiogenesis and improved cardiac ischemic tolerance induced in neonatal rats by chronic hypoxia.

Expression and localization of caveolins during postnatal development in rat heart: implication of thyroid hormone.

Caveolins modulate signaling pathways involved in cardiac development. Caveolin-1 exists in two isoforms: the beta-isoform derivates from an alternative translational start site that creates a protein truncated by 31 amino acids, mainly expressed in endothelial cells, whereas caveolin-3 is present in muscle cells. Our aim was to define caveolin distribution and expression during cardiac postnatal development using immunofluorescence and Western blotting. Caveolin-3 sarcolemmal labeling appeared as dotted lines from days 1 to 5 and as continuous lines after 14 days of age. Caveolin-3 expression, low at birth, increased (4-fold) to reach a maximum (P < 0.05) by day 5 and then decreased to stabilize in adults. Total caveolin-1 and its alpha-isoform were codistributed at birth in endothelial and smooth muscle cells; afterward, only the caveolin-1alpha labeling became limited to endothelium. Quantitative analysis indicated a similar temporal pattern of both total caveolin-1 and caveolin-1alpha expression, suggesting that caveolin-1alpha and -1beta are coregulated; the caveolin-1alpha levels increased fourfold by day 5 to reach a maximum by day 14 (P < 0.05). Tyrosine-14-caveolin-1 phosphorylation, low at birth, increased suddenly around day 14 (8-fold vs. day 1) and returning afterward to basal level. Because the T3/T4 level is maximal by day 14, caveolin-1 expression/phosphorylation profiles were analyzed in hypothyroid heart. The levels of caveolin-1alpha and consequently tyrosine-14-caveolin-1 phosphorylation, but not that of caveolin-3, decreased (50%) in hypothyroid 14-day-old rats. Our data demonstrate that, during postnatal cardiac growth, 1) caveolins are distinctly regulated, and 2) thyroid hormones are involved in caveolin-1alpha expression.

Up-regulation of cardiac nitric oxide synthase 1-derived nitric oxide after myocardial infarction in senescent rats.

Nitric oxide (NO) has been implicated in the development of heart failure, although the source, significance, and functional role of the different NO synthase (NOS) isoforms in this pathology are controversial. The presence of a neuronal-type NOS isoform (NOS1) in the cardiac sarcoplasmic reticulum has been recently discovered, leading to the hypothesis that NOS1-derived NO may notably alter myocardial inotropy. However, the regulation and role(s) of NOS1 in cardiac diseases remain to be determined. Using an experimental model of myocardial infarction (MI) in senescent rats, we demonstrated a significant increase in cardiac NOS1 expression and activity in MI, coupled with the translocation of this enzyme to the sarcolemma through interactions with caveolin-3. The enhanced NOS1 activity counteracts the decrease in cardiac NOS3 expression and activity observed in heart failure. We demonstrated an increased interaction between NOS1 and its regulatory protein HSP90 in post-MI hearts, a potential mechanism for the higher NOS1 activity in this setting. Finally, preferential in vivo inhibition of NOS1 activity enhanced basal post-MI left ventricular dysfunction in senescent rats. These results provide the first evidence that increased NOS1-derived NO production may play a significant role in the autocrine regulation of myocardial contractility after MI in aging rats.

Caveolin-1 and -3 dissociations from caveolae to cytosol in the heart during aging and after myocardial infarction in rat.

OBJECTIVE: Caveolins, the structural proteins of caveolae, modulate numerous signaling pathways including Nitric Oxide (NO) production. Among the caveolin family, caveolin-1 and -3 are mainly expressed in endothelial and muscle cells, respectively. In this study, we investigate whether (i) changes in caveolin abundance and/or distribution occur during cardiac aging and failure in rat, and (ii) the process could influence NO synthase (NOS) activity. METHODS: Using immunohistolabelling and Western blot approaches, expression and distribution of caveolins were analysed in adult (Ad), senescent (S-Sh) and myocardial infarction-induced failing (S-MI) hearts. NOS3/caveolin-1 interactions were evaluated by immunoprecipitation assays. RESULTS: At the microscope level, caveolin-1 distribution in the endothelial cells was unchanged between the groups. Conversely the typical distribution of caveolin-3 in myocyte sarcolemma was dramatically altered in S-MI rats, resulting in a heterogeneous pattern throughout the septum. Total abundance of caveolin-1 and -3 remained stable whatever the group. In the fractions free of caveolae (Triton X-100 soluble), the levels of caveolin-1 alpha and -3 increased with aging (+20%, and +104%, P<0.05 versus Ad, respectively) and were further enhanced in S-MI (+25%, +30%, P<0.05, P<0.001 versus S-Sh respectively). In these fractions, NOS3/caveolin-1 alpha complexes increased as well. In addition, NOS activity was negatively correlated to caveolin-1 level in the cytosolic fractions. CONCLUSIONS: We demonstrate that dissociation of caveolin from caveolae is associated with aging and heart failure, the process being related to the decreased NOS activity.

Cardiac modulations of ANG II receptor expression in rats with hypoxic pulmonary hypertension.

Right ventricular myocardial hypertrophy during hypoxic pulmonary hypertension is associated with local renin-angiotensin system activation. The expression of angiotensin II type 1 (AT(1)) and type 2 (AT(2)) receptors in this setting has never been investigated. We have therefore examined the chronic hypoxia pattern of AT(1) and AT(2) expression in the right and left cardiac ventricles, using in situ binding and RT-PCR assays. Hypoxia produced right, but not left, ventricular hypertrophy after 7, 14, and 21 days, respectively. Hypoxia for 2 days was associated in each ventricle with a simultaneous and transient increase (P < 0.05) in AT(1) binding and AT(1) mRNA levels in the absence of any significant change in AT(2) expression level. Only after 14 days of hypoxia, AT(2) binding increased (P < 0.05) in the two ventricles, concomitantly with a right ventricular decrease (P < 0.05) in AT(2) mRNA. Along these data, AT(1) and AT(2) binding remained unchanged in both the left and hypertrophied right ventricles from rats treated with monocrotaline for 30 days. These results indicate that chronic hypoxia induces modulations of AT(1) and AT(2) receptors in both cardiac ventricles probably through direct and indirect mechanisms, respectively, which modulations may participate in myogenic (at the level of smooth or striated myocytes) rather than in the growth response of the heart to hypoxia.

Angiotensin II AT(2) receptor inhibits smooth muscle cell migration via fibronectin cell production and binding.

To explore the vascular function of the angiotensin II (ANG II) AT(2) receptor subtype (AT(2)R), we generated a vascular smooth muscle cell (SMC) line expressing the AT(2)R (SMC-vAT(2)). The involvement of AT(2)R in the motility response of SMCs was examined in SMC-vAT(2) cells and their controls (SMC-v) cultured on either laminin or fibronectin matrix proteins with the agarose drop technique. All experiments were conducted in the presence of a saturating concentration of losartan to inactivate the AT(1)R subtype. Under basal conditions, both cell lines migrated outside drops, but on laminin only. Treatment with ANG II significantly inhibited the migration of SMC-vAT(2) but not SMC-v cells, and this effect was prevented by the AT(2)R antagonist CGP-42112A. The decreased migration of SMC-vAT(2) was not associated with changes in cell growth, cytoskeleton stiffness, or smooth muscle actin, desmin, and tenascin expression. However, it was correlated with increased synthesis and binding of fibronectin. Both responses were prevented by incubation with selective AT(2)R antagonists. Addition of GRGDTP peptide, which prevents cell attachment of fibronectin, reversed the AT(2)R inhibitory effect on SMC-vAT(2) migration. These results suggest that activated ANG II AT(2)R inhibits SMC migration via cellular fibronectin synthesis and associated cell binding.