Autophagy protein 5 controls flow-dependent endothelial functions.

Dysregulated autophagy is associated with cardiovascular and metabolic diseases, where impaired flow-mediated endothelial cell responses promote cardiovascular risk. The mechanism by which the autophagy machinery regulates endothelial functions is complex. We applied multi-omics approaches and in vitro and in vivo functional assays to decipher the diverse roles of autophagy in endothelial cells. We demonstrate that autophagy regulates VEGF-dependent VEGFR signaling and VEGFR-mediated and flow-mediated eNOS activation. Endothelial ATG5 deficiency in vivo results in selective loss of flow-induced vasodilation in mesenteric arteries and kidneys and increased cerebral and renal vascular resistance in vivo. We found a crucial pathophysiological role for autophagy in endothelial cells in flow-mediated outward arterial remodeling, prevention of neointima formation following wire injury, and recovery after myocardial infarction. Together, these findings unravel a fundamental role of autophagy in endothelial function, linking cell proteostasis to mechanosensing.

Novel Cardiokine GDF3 Predicts Adverse Fibrotic Remodeling After Myocardial Infarction.

BACKGROUND: Myocardial infarction (MI) induces a repair response that ultimately generates a stable fibrotic scar. Although the scar prevents cardiac rupture, an excessive profibrotic response impairs optimal recovery by promoting the development of noncontractile fibrotic areas. The mechanisms that lead to cardiac fibrosis are diverse and incompletely characterized. We explored whether the expansion of cardiac fibroblasts after MI can be regulated through a paracrine action of cardiac stromal cells. METHODS: We performed a bioinformatic secretome analysis of cardiac stromal PW1+ cells isolated from normal and post-MI mouse hearts to identify novel secreted proteins. Functional assays were used to screen secreted proteins that promote fibroblast proliferation. The expressions of candidates were subsequently analyzed in mouse and human hearts and plasmas. The relationship between levels of circulating protein candidates and adverse post-MI cardiac remodeling was examined in a cohort of 80 patients with a first ST-segment-elevation MI and serial cardiac magnetic resonance imaging evaluations. RESULTS: Cardiac stromal PW1+ cells undergo a change in paracrine behavior after MI, and the conditioned media from these cells induced a significant increase in the proliferation of fibroblasts. We identified a total of 12 candidates as secreted proteins overexpressed by cardiac PW1+ cells after MI. Among these factors, GDF3 (growth differentiation factor 3), a member of the TGF-ß (transforming growth factor-ß) family, was markedly upregulated in the ischemic hearts. Conditioned media specifically enriched with GDF3 induced fibroblast proliferation at a high level by stimulation of activin-receptor-like kinases. In line with the secretory nature of this protein, we next found that GDF3 can be detected in mice and human plasma samples, with a significant increase in the days after MI. In humans, higher GDF3 circulating levels (measured in the plasma at day 4 after MI) were significantly associated with an increased risk of adverse remodeling 6 months after MI (adjusted odds ratio, 1.76 [1.03-3.00]; P=0.037), including lower left ventricular ejection fraction and a higher proportion of akinetic segments. CONCLUSIONS: Our findings define a mechanism for the profibrotic action of cardiac stromal cells through secreted cardiokines, such as GDF3, a candidate marker of adverse fibrotic remodeling after MI. REGISTRATION: URL: https://www. CLINICALTRIALS: gov; Unique identifier: NCT01113268.

Preclinical evaluation of targeted therapies in Sdhb-mutated tumors.

Therapies for metastatic SDHB-dependent pheochromocytoma and paraganglioma (PPGL) are limited and poorly efficient. New targeted therapies and identification of early non-invasive biomarkers of response are thus urgently needed for these patients. We characterized an in vivo allograft model of spontaneously immortalized murine chromaffin cells (imCC) with inactivation of the Sdhb gene by dynamic contrast-enhanced MRI (DCE-MRI) and 18FDG-PET. We evaluated the response to several therapies: IACS-010759 (mitochondrial respiratory chain complex I inhibitor), sunitinib (tyrosine kinase inhibitor with anti-angiogenic activity), talazoparib (poly ADP ribose polymerase (PARP) inhibitor) combined or not to temozolomide (alkylating agent), pharmacological inhibitors of HIF2a (PT2385 and PT2977 (belzutifan)) and molecular inactivation of HIF2a (imCC Sdhb-/- shHIF2a). Multimodal imaging was performed, including magnetic resonance spectroscopy (1H-MRS) to monitor the level of succinate in vivo. The allografted model of Sdhb-/- imCC reflected SDHB-deficient tumors, with increased angiogenesis and a particular avidity for 18FDG. After 14 days of treatment, IACS-010759, sunitinib and talazoparib at high doses allowed a significant reduction of the tumor volumes. In contrast to the tumor growth inhibition observed in Sdhb-/- shHIF2a imCC tumors, pharmacological inhibitors of HIF2a (PT2385 and belzutifan) showed no antitumor action in this model, alone or in combination with sunitinib. 1H-MRS, but not DCE-MRI, enabled the monitoring response to sunitinib, which was the best treatment in this study, promoting a decrease in succinate levels detected in vivo. This study paves the way for new therapeutic options and reveals a potential new early biomarker of response to treatment in SDHB-dependent PPGL.

Carbonic anhydrase 9 immunohistochemistry as a tool to predict or validate germline and somatic VHL mutations in pheochromocytoma and paraganglioma-a retrospective and prospective study.

The development of pheochromocytomas and paragangliomas is strongly linked to the presence of germline mutations in more than 15 predisposing genes. Among them, germline and somatic VHL mutations account for ~10% of all cases. In contrast with SDHA and SDHB immunohistochemistries that are routinely used to validate SDHx gene mutations, there is no such tool available for VHL mutations. The aim of this study was to evaluate whether CA9 immunostaining could be used as a tool to predict the presence or validate the pathogenicity of VHL gene mutations in paraganglioma. Immunohistochemistry for CA9 was performed on 207 tumors. A retrospective series of 100 paragangliomas with known mutation status for paraganglioma susceptibility genes was first investigated. Then, a prospective series of 107 paragangliomas was investigated for CA9 immunostaining followed by germline and/or somatic genetic testing of all paraganglioma susceptibility genes by next-generation sequencing. Cytosolic CA9 protein expression was heterogeneous in the different samples. However, we observed that a membranous CA9 staining was almost exclusively observed in VHL-related cases. Forty two of 48 (88%) VHL-mutated samples showed a CA9 membranous immunostaining. Positive cells were either isolated, varying from 1 or 2 cells (5% of cases) to 10-20 cells per tumor block (35% of cases), grouped in areas of focal positivity representing between 1 and 20% of the tissue section (35% of cases), or widely distributed on 80-100% of the tumor sections (25% of samples). In contrast, 142/159 (91%) of non-VHL-mutated tumors presented no membrane CA9 localization. Our results demonstrate that VHL gene mutations can be predicted or validated reliably by an easy-to-perform and low-cost immunohistochemical procedure. CA9 immunohistochemistry on paragangliomas will improve the diagnosis of VHL-related disease, which is important for the surveillance and therapeutic management of paraganglioma patients, and in case of germline mutation, their family members.

Telomerase Activation and ATRX Mutations Are Independent Risk Factors for Metastatic Pheochromocytoma and Paraganglioma.

PURPOSE: Pheochromocytomas and paragangliomas (PPGLs) are rare neuroendocrine tumors. Whereas most PPGLs are benign, up to 20% may become metastatic with SDHB- and FH-mutated tumors showing the higher risk. We aimed at determining the contribution of immortalization mechanisms to metastatic progression.Experimental Design: Immortalization mechanisms were investigated in 200 tumors. To identify telomerase (+) tumors, we analyzed genomic alterations leading to transcriptional activation of TERT comprising promoter mutations, hypermethylation and gain copy number. To identify tumors that activated the alternative lengthening of telomere (ALT) mechanism, we combined analyses of telomere length by slot blot, telomere heterogeneity by telomere FISH, and ATRX mutations by next-generation sequencing. Univariate/multivariate and metastasis-free survival (MFS) and overall survival (OS) analyses were carried out for assessment of risk factors and clinical outcomes. RESULTS: Only 37 of 200 (18.5%) tumors achieved immortalization. Telomerase activation occurred in 12 metastatic tumors and was prevalent in SDHB-mutated paragangliomas (P = 2.42e-09). ALT features were present in 25 tumors, mostly pheochromocytomas, regardless of metastatic status or molecular group (P = 0.169), yet ATRX mutations were found preferentially in SDHB/FH-mutated metastatic tumors (P = 0.0014). Telomerase activation and ATRX mutations were independent factors of poor prognosis: MFS (hazard ratio, 48.2 and 33.1; P = 6.50E-07 and 1.90E-07, respectively); OS (hazard ratio, 97.4 and 44.1; P = 4.30E-03 and 2.00E-03, respectively) and were associated with worse MFS and OS (log-rank tests P < 0.0001). CONCLUSIONS: Assessment of telomerase activation and ATRX mutations could be used to identify metastatic PPGLs, particularly in tumors at high risk of progression.

Establishment of a mouse xenograft model of metastatic adrenocortical carcinoma.

Adrenocortical carcinoma is a rare neoplasm with a poor prognosis. Very important advances have been made in the identification of the genetic determinants of adrenocortical carcinoma pathogenesis but our understanding is still limited about the mechanisms that determine cancer spread and metastasis. One major problem hindering preclinical experimentation for new therapies for adrenocortical carcinoma is represented by the lack of suitable animal models for metastatic disease. With the aim to overcome these limitations, in this study we tested several protocols in order to establish a mouse xenograft model of metastatic adrenocortical carcinoma. The most efficient method, based upon intrasplenic injection followed by splenectomy, produced metastases with high efficiency, whose development could be followed over time by bioluminescence measurements. We expect that the availability of this model will greatly improve the possibilities for preclinical testing of new treatments for advanced-stage disease.

Dosage-dependent regulation of VAV2 expression by steroidogenic factor-1 drives adrenocortical carcinoma cell invasion.

Adrenocortical carcinoma (ACC) is a rare endocrine malignancy with a dismal prognosis. Genomic studies have enabled progress in our understanding of the molecular bases of ACC, but factors that influence its prognosis are lacking. Amplification of the gene encoding the transcription factor steroidogenic factor-1 (SF-1; also known as NR5A1) is one of the genetic alterations common in ACC. We identified a transcriptional regulatory mechanism involving increased abundance of VAV2, a guanine nucleotide exchange factor for small GTPases that control the cytoskeleton, driven by increased expression of the gene encoding SF-1 in ACC. Manipulating SF-1 and VAV2 abundance in cultured ACC cells revealed that VAV2 was a critical factor for SF-1-induced cytoskeletal remodeling and invasion in culture (Matrigel) and in vivo (chicken chorioallantoic membrane) models. Analysis of ACC patient cohorts indicated that greater VAV2 abundance robustly correlated with poor prognosis in ACC patients. Because VAV2 is a druggable target, our findings suggest that blocking VAV2 may be a new therapeutic approach to inhibit metastatic progression in ACC patients.

In Vivo Detection of Succinate by Magnetic Resonance Spectroscopy as a Hallmark of SDHx Mutations in Paraganglioma.

PURPOSE: Germline mutations in genes encoding mitochondrial succinate dehydrogenase (SDH) are found in patients with paragangliomas, pheochromocytomas, gastrointestinal stromal tumors, and renal cancers. SDH inactivation leads to a massive accumulation of succinate, acting as an oncometabolite and which levels, assessed on surgically resected tissue are a highly specific biomarker of SDHx-mutated tumors. The aim of this study was to address the feasibility of detecting succinate in vivo by magnetic resonance spectroscopy. EXPERIMENTAL DESIGN: A pulsed proton magnetic resonance spectroscopy ((1)H-MRS) sequence was developed, optimized, and applied to image nude mice grafted with Sdhb(-/-) or wild-type chromaffin cells. The method was then applied to patients with paraganglioma carrying (n = 5) or not (n = 4) an SDHx gene mutation. Following surgery, succinate was measured using gas chromatography/mass spectrometry, and SDH protein expression was assessed by immunohistochemistry in resected tumors. RESULTS: A succinate peak was observed at 2.44 ppm by (1)H-MRS in all Sdhb(-/-)-derived tumors in mice and in all paragangliomas of patients carrying an SDHx gene mutation, but neither in wild-type mouse tumors nor in patients exempt of SDHx mutation. In one patient, (1)H-MRS results led to the identification of an unsuspected SDHA gene mutation. In another case, it helped define the pathogenicity of a variant of unknown significance in the SDHB gene. CONCLUSIONS: Detection of succinate by (1)H-MRS is a highly specific and sensitive hallmark of SDHx mutations. This noninvasive approach is a simple and robust method allowing in vivo detection of the major biomarker of SDHx-mutated tumors.

Long-term functional benefits of human embryonic stem cell-derived cardiac progenitors embedded into a fibrin scaffold.

BACKGROUND: Cardiac-committed cells and biomimetic scaffolds independently improve the therapeutic efficacy of stem cells. In this study we tested the long-term effects of their combination. METHODS: Eighty immune-deficient rats underwent permanent coronary artery ligation. Five to 7 weeks later, those with an echocardiographically measured ejection fraction (EF) ≤55% were re-operated on and randomly allocated to receive a cell-free fibrin patch (n = 25), a fibrin patch loaded with 700,000 human embryonic stem cells (ESC) pre-treated to promote early cardiac differentiation (SSEA-1(+) progenitors [n = 30]), or to serve as sham-operated animals (n = 25). Left ventricular function was assessed by echocardiography at baseline and every month thereafter until 4 months. Hearts were then processed for assessment of fibrosis and angiogenesis and a 5-component heart failure score was constructed by integrating the absolute change in left ventricular end-systolic volume (LVESV) between 4 months and baseline, and the quantitative polymerase chain reaction (qPCR)-based expression of natriuretic peptides A and B, myosin heavy chain 7 and periostin. All data were recorded and analyzed in a blinded manner. RESULTS: The cell-treated group consistently yielded better functional outcomes than the sham-operated group (p = 0.002 for EF; p = 0.01 for LVESV). Angiogenesis in the border zone was also significantly greater in the cell-fibrin group (p = 0.006), which yielded the lowest heart failure score (p = 0.04 vs sham). Engrafted progenitors were only detected shortly after transplantation; no grafted cells were identified after 4 months. There was no teratoma identified. CONCLUSIONS: A fibrin scaffold loaded with ESC-derived cardiac progenitors resulted in sustained improvement in contractility and attenuation of remodeling without sustained donor cell engraftment. A paracrine effect, possibly on innate reparative responses, is a possible mechanism for this enduring effect.

Angiotensin II promotes thoracic aortic dissections and ruptures in Col3a1 haploinsufficient mice.

Vascular Ehlers-Danlos syndrome is a dramatic inherited disease caused by mutations of type III collagen (COL3A1) gene, associated with early-onset occurrence of arterial ruptures. Col3a1(+/-) heterozygous mice, the only vascular Ehlers-Danlos syndrome model available to date, have no spontaneous early vascular phenotype. Our objective was to determine the susceptibility of Col3a1(+/-) mice to develop arterial ruptures under high blood pressure (BP) conditions induced by a 4-week infusion of angiotensin II (AngII). AngII (1 µg/kg per minute) significantly and comparably increased systolic BP in Col3a1(+/-) and Col3a1(+/+) mice but led to a higher premature mortality rate in Col3a1(+/-) mice compared with Col3a1(+/+) mice (73% versus 36%; P=0.03), particularly during the first-week infusion (55% versus 0%). Echocardiography and histological analysis evidenced that early deaths were caused by thoracic aortic ruptures preceded by dissections and associated with low aortic collagen fibrils content. Remarkably, lowering the dose of AngII (0.5 µg/kg per minute) rescued the first-week premature deaths of Col3a1(+/-) mice while decreasing the rises in systolic BP (P=0.05 compared with the high-dose AngII), resulting in similar mortality rates in both groups of mice at the end of the 4-week period (30% versus 50% in Col3a1(+/-) and Col3a1(+/+) mice; P=0.30). Finally, norepinephrine infusion (3.9 µg/kg per minute) did not induced significant mortality in both groups, whereas it significantly increased systolic BP, comparably with the high and with the low dose of AngII in Col3a1(+/-) mice (P=0.53 and P=1.00, respectively). Our findings demonstrated the extreme sensitivity of Col3a1 insufficient mice to prematurely develop thoracic aortic ruptures in response to AngII and its associated high levels in BP.

Cardiomyocyte overexpression of neuronal nitric oxide synthase delays transition toward heart failure in response to pressure overload by preserving calcium cycling.

BACKGROUND: Defects in cardiomyocyte Ca(2+) cycling are a signature feature of heart failure (HF) that occurs in response to sustained hemodynamic overload, and they largely account for contractile dysfunction. Neuronal nitric oxide synthase (NOS1) influences myocyte excitation-contraction coupling through modulation of Ca(2+) cycling, but the potential relevance of this in HF is unknown. METHODS AND RESULTS: We generated a transgenic mouse with conditional, cardiomyocyte-specific NOS1 overexpression (double-transgenic [DT]) and studied cardiac remodeling, myocardial Ca(2+) handling, and contractility in DT and control mice subjected to transverse aortic constriction (TAC). After TAC, control mice developed eccentric hypertrophy with evolution toward HF as revealed by a significantly reduced fractional shortening. In contrast, DT mice developed a greater increase in wall thickness (P<0.0001 versus control+TAC) and less left ventricular dilatation than control+TAC mice (P<0.0001 for both end-systolic and end-diastolic dimensions). Thus, DT mice displayed concentric hypertrophy with fully preserved fractional shortening (43.7+/-0.6% versus 30.3+/-2.6% in control+TAC mice, P<0.05). Isolated cardiomyocytes from DT+TAC mice had greater shortening, intracellular Ca(2+) transients, and sarcoplasmic reticulum Ca(2+) load (P<0.05 versus control+TAC for all parameters). These effects could be explained, at least in part, through modulation of phospholamban phosphorylation status. CONCLUSIONS: Cardiomyocyte NOS1 may be a useful target against cardiac deterioration during chronic pressure-overload-induced HF through modulation of calcium cycling.

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.

Myocyte apoptosis during acute myocardial infarction in rats is related to early sarcolemmal translocation of annexin A5 in border zone.

Annexin A5 is a Ca2+-dependent phospholipid binding protein well known for its high phosphatidylserine affinity. In vitro, translocation to sarcolemma and externalization of endogenous annexin A5 in the cardiomyocyte has recently been demonstrated to exert a proapoptotic effect. To determine whether these in vitro findings occurred in vivo, we performed myocardial infarction (MI) and studied the time course of apoptosis and annexin A5 localization (0.5 to 8 h) in the border zone around the infarcted area. This zone that was defined as Evans blue unstained and triphenyltetrazolium chloride (TTC) stained, represented 42.3 +/- 5.5% of the area at risk and showed apoptotic characteristics (significant increases in caspase 3 activity 2.3-fold at 0.5 h; P < 0.05), transferase-mediated dUTP nick-end labeling-positive cardiomyocytes (15.8 +/- 0.8% at 8 h), and DNA ladder. When compared with sham-operated rats, we found that in this area, annexin A5 was translocated to the sarcolemma as early as 0.5 h after MI and that translocation increased with time. Moreover, the amount of annexin A5 was unchanged in the border zone and decreased in the infarcted area after 1 h (77.1 +/- 4.8%; P < 0.01 vs. perfused area), suggesting a release in the latter but not in the former. In conclusion, we demonstrated that annexin A5 translocation is an early and rapid event of the whole border zone, likely due to Ca2+ increase. Part of this translocation occurred in areas where apoptosis was later detected and suggests that in vivo as in vitro annexin A5 might be involved in the regulation of early apoptotic events during cardiac pathological situations.

Spironolactone reduces fibrosis of dilated atria during heart failure in rats with myocardial infarction.

AIMS: Congestive heart failure (CHF) is associated with severe structural changes of atria, contributing to impaired atrial function and the risk of arrhythmia. This study investigated the effects of CHF treatments on atrial remodelling. METHODS AND RESULTS: Three months after myocardial infarction (MI), rats were treated for 1 month with spironolactone, lisinopril, or atenolol alone or in combination. Echocardiography-Doppler tissue imaging, haemodynamic measurements, and 24-h Holter monitoring were used to characterize the cardiomyopathy. Atrial fibrosis was quantified with Picrosirius Red staining. Left atrial diameter was increased (5.8+/-0.6 mm in MI vs. 3.6+/-0.3 mm in sham; P<0.0001), as was atrial fibrosis (26.7+/-3.8% in MI vs. 10.5+/-2.2% in sham; P<0.0001), which correlated with left ventricular (LV) dysfunction after 3 months of MI. P-wave duration was also increased and premature atrial beats were frequent on the 24-h electrocardiogram. Similar improvements in LV dysfunction were observed after 1 month of spironolactone, ACE-inhibitor, or beta-blocker therapy alone or in combination. Atrial hyperexcitability was reduced by all the treatments, but only spironolactone attenuated atrial fibrosis and reduced P-wave duration. CONCLUSION: Atrial fibrosis caused by chronic CHF is reduced by spironolactone.

Spironolactone improves carotid artery fibrosis and distensibility in rat post-ischaemic heart failure.

Myocardial infarction causes neurohormonal activation involving aldosterone and angiotensin II (AngII). These hormones may increase arterial stiffness, an independent cardiovascular risk factor contributing to progression of congestive heart failure (CHF). This study aimed to determine the effect of aldosterone and AngII blockade on carotid artery distensibility and collagen density in adult Wistar rats with MI-induced CHF. Five groups were studied: Sham-operated, CHF, CHF + spironolactone, CHF + lisinopril, CHF + Spironolactone + Lisinopril. After echocardiography, in vitro isobaric carotid distensibility (echo-tracking technique) and collagen density were measured, and the incremental elastic modulus (Einc) calculated. In the CHF group, intra-ventricular pressure and cardiac weight were increased; carotid distensibility was reduced (CHF: 0.42 +/- 0.30 per mmHg(3) versus sham: 1.75 +/- 0.50 per mmHg(3); P < 0.001), and collagen content increased by 87% when compared to sham. All treatments reduced intra-ventricular pressure, carotid distensibility and fibrosis when compared to CHF but did not change cardiac weight. However, carotid distensibility and intra-ventricular pressure were not completely restored towards sham values and were significantly and inversely related. Spironolactone, which did not decrease significantly blood pressure, was the only drug reducing Einc independently of wall stress (WS). Thus, MI-induced CHF was associated with carotid artery remodeling. This vascular change, which may contribute to maintain cardiac hypertrophy and CHF, is largely prevented by AngII and aldosterone blockade. Only spironolactone reduced the stiffness of carotid wall material independently of blood pressure and WS.

Role of myocardial neuronal nitric oxide synthase-derived nitric oxide in beta-adrenergic hyporesponsiveness after myocardial infarction-induced heart failure in rat.

BACKGROUND: An emerging concept is that a neuronal isoform of nitric oxide synthase (NOS1) may regulate myocardial contractility. However, a role for NOS1-derived nitric oxide (NO) in heart failure (HF) has not been defined. METHODS AND RESULTS: Using a model of myocardial infarction-induced HF, we demonstrated that cardiac NOS1 expression and activity increased in HF rats (P<0.05 and P<0.001 versus shams, respectively). This was associated with translocation of NOS1 from the ryanodine receptor to the sarcolemma through interactions with caveolin-3 in HF hearts. With ex vivo and in vivo pressure-volume analysis, cardiac NOS1-derived NO was found to be negatively inotropic in shams but not HF hearts. Ventricular elastance (E(es)) was significantly reduced in HF rats (P<0.05), and tau, the time constant of left ventricular relaxation, was prolonged (both P<0.05). Acute NOS1 inhibition significantly increased E(es) by 33+/-3% and tau by 17+/-2% (P<0.05) in shams, although these effects were significantly attenuated in HF hearts. beta-Adrenergic stimulation induced a marked increase in systolic performance in sham hearts, with the responses being significantly blunted in HF hearts. E(es) increased by 163+/-42% (P<0.01) in sham hearts and 56+/-9% in HF hearts, and LV +dP/dt increased by 97+/-9% (P<0.01) in shams and 37+/-7% (P<0.05) in the HF group. Interestingly, preferential NOS1 inhibition enhanced the blunted responses of LV +dP/dt and E(es) to beta-adrenergic stimulation in HF rats but had no effect in shams. CONCLUSIONS: These results provide the first evidence that increased NOS1-derived NO production may play a role in the autocrine regulation of myocardial contractility in HF.

Comparison of human skeletal myoblasts and bone marrow-derived CD133+ progenitors for the repair of infarcted myocardium.

OBJECTIVES: The present study was designed as a face-to-face functional comparison of human skeletal myoblasts (SMs) and CD133(+) bone marrow-derived hematopoietic progenitors in an animal model of semichronic myocardial infarction. BACKGROUND: Compared with SMs, bone marrow-derived cells have the advantage of plasticity and might more effectively regenerate ischemic cardiac tissue. However, few data exist on the comparative efficacy of these two cell types in semichronic infarcts. METHODS: A myocardial infarction was created by coronary ligation in 32 nude rats. Ten days later, rats received in-scar injections of human SMs, CD133(+) progenitors, or culture medium. Left ventricular function was assessed before and one month after transplantation by echocardiography and pressure-volume loops. Immunofluorescence, polymerase chain reaction, and in situ hybridization were used to detect cells grafted in the hearts. RESULTS: One month after transplantation, left ventricular ejection fraction decreased by 8 +/- 4% in controls, whereas it increased by 7 +/- 3% in CD133(+)-grafted hearts (p = 0.0015 vs. controls) and further by 15 +/- 5% in SM-treated hearts (p = 0.008 vs. controls). Systolic indices yielded by pressure-volume loops paralleled these data. Engrafted myotubes were identified in all SM-treated hearts by immunofluorescence, whereas in CD133(+)-grafted hearts, few human cells were only detected by polymerase chain reaction. CONCLUSIONS: In the setting of postinfarction scars, the transplantation of bone marrow-derived CD133(+) progenitors improves cardiac function, but this benefit is not superior to that afforded by myogenic cells.

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.