Remotely Triggered LAD Occlusion Using a Balloon Catheter in Spontaneously Breathing Mice.

Acute myocardial infarction (AMI) is a prevalent and high-mortality cardiovascular condition. Despite advancements in revascularization strategies for AMI, it frequently leads to myocardial ischemia-reperfusion injury (IRI), amplifying cardiac damage. Murine models serve as vital tools for investigating both acute injury and chronic myocardial remodeling in vivo. This study presents a unique closed-chest technique for remotely inducing myocardial IRI in mice, enabling the investigation of the very early phase of occlusion and reperfusion using in-vivo imaging such as MRI or PET. The protocol utilizes a remote occlusion method, allowing precise control over ischemia initiation after chest closure. It reduces surgical trauma, enables spontaneous breathing, and enhances experimental consistency. What sets this technique apart is its potential for simultaneous noninvasive imaging, including ultrasound and magnetic resonance imaging (MRI), during occlusion and reperfusion events. It offers a unique opportunity to analyze tissue responses in almost real-time, providing critical insights into processes during ischemia and reperfusion. Extensive systematic testing of this innovative approach was conducted, measuring cardiac necrosis markers for infarction, assessing the area at risk using contrast-enhanced MRI, and staining infarcts at the scar maturation stage. Through these investigations, emphasis was placed on the value of the proposed tool in advancing research approaches to myocardial ischemia-reperfusion injury and accelerating the development of targeted interventions. Preliminary findings demonstrating the feasibility of combining the proposed innovative experimental protocol with noninvasive imaging techniques are presented herein. These initial results highlight the benefit of utilizing the purpose-built animal cradle to remotely induce myocardial ischemia while simultaneously conducting MRI scans.

Alginate sulfate-nanoparticles loaded with hepatocyte growth factor and insulin-like growth factor-1 improve left ventricular repair in a porcine model of myocardial ischemia reperfusion injury.

Nanomedicine offers great potential for the treatment of cardiovascular disease and particulate systems have the capacity to markedly improve bioavailability of therapeutics. The delivery of pro-angiogenic hepatocyte growth factor (HGF) and pro-survival and pro-myogenic insulin-like growth factor (IGF-1) encapsulated in Alginate-Sulfate nanoparticles (AlgS-NP) might improve left ventricular (LV) functional recovery after myocardial infarction (MI). In a porcine ischemia-reperfusion model, MI is induced by 75 min balloon occlusion of the mid-left anterior descending coronary artery followed by reperfusion. After 1 week, pigs (n = 12) with marked LV-dysfunction (LV ejection fraction, LVEF < 45%) are randomized to fusion imaging-guided intramyocardial injections of 8 mg AlgS-NP prepared with 200 µg HGF and IGF-1 (HGF/IGF1-NP) or PBS (Control). Intramyocardial injection is safe and pharmacokinetic studies of Cy5-labeled NP confirm superior cardiac retention compared to intracoronary infusion. Seven weeks after intramyocardial-injection of HGF/IGF1-NP, infarct size, measured using magnetic resonance imaging, is significantly smaller than in controls and is associated with increased coronary flow reserve. Importantly, HGF/IGF1-NP-treated pigs show significantly increased LVEF accompanied by improved myocardial remodeling. These findings demonstrate the feasibility and efficacy of using AlgS-NP as a delivery system for growth factors and offer the prospect of innovative treatment for refractory ischemic cardiomyopathy.

Arteriovenous Fistulae in Chronic Kidney Disease and the Heart: Physiological, Histological, and Transcriptomic Characterization of a Novel Rat Model.

Background Arteriovenous fistulae (AVFs) are the gold standard for vascular access in those requiring hemodialysis but may put an extra hemodynamic stress on the cardiovascular system. The complex interactions between the heart, kidney, and AVFs remain incompletely understood. Methods and Results We characterized a novel rat model of five-sixths partial nephrectomy (NX) and AVFs. NX induced increases in urea, creatinine, and hippuric acid. The addition of an AVF (AVF+NX) further increased urea and a number of uremic toxins such as trimethylamine N-oxide and led to increases in cardiac index, left and right ventricular volumes, and right ventricular mass. Plasma levels of uremic toxins correlated well with ventricular morphology and function. Heart transcriptomes identified altered expression of 8 genes following NX and 894 genes following AVF+NX, whereas 290 and 1431 genes were altered in the kidney transcriptomes, respectively. Gene ontology and Kyoto Encyclopedia of Genes and Genomes analysis revealed gene expression changes related to cell division and immune activation in both organs, suppression of ribosomes and transcriptional activity in the heart, and altered renin-angiotensin signaling as well as chronodisruption in the kidney. All except the latter were worsened in AVF+NX compared with NX. Conclusions Inflammation and organ dysfunction in chronic kidney disease are exacerbated following AVF creation. Furthermore, our study provides important information for the discovery of novel biomarkers and therapeutic targets in the management of cardiorenal syndrome.

Clec4e-Receptor Signaling in Myocardial Repair After Ischemia-Reperfusion Injury.

The bacterial C-type lectin domain family 4 member E (CLEC4E) has an important role in sterile inflammation, but its role in myocardial repair is unknown. Using complementary approaches in porcine, murine, and human samples, we show that CLEC4E expression levels in the myocardium and in blood correlate with the extent of myocardial injury and left ventricular (LV) functional impairment. CLEC4E expression is markedly increased in the vasculature, cardiac myocytes, and infiltrating leukocytes in the ischemic heart. Loss of Clec4e signaling is associated with reduced acute cardiac injury, neutrophil infiltration, and infarct size. Reduced myocardial injury in Clec4e -/- translates into significantly improved LV structural and functional remodeling at 4 weeks' follow-up. The early transcriptome of LV tissue from Clec4e -/- mice versus wild-type mice reveals significant upregulation of transcripts involved in myocardial metabolism, radical scavenging, angiogenesis, and extracellular matrix organization. Therefore, targeting CLEC4E in the early phase of ischemia-reperfusion injury is a promising therapeutic strategy to modulate myocardial inflammation and enhance repair after ischemia-reperfusion injury.

Cardiac Microvascular Endothelial Cells in Pressure Overload-Induced Heart Disease.

BACKGROUND: Chronic pressure overload predisposes to heart failure, but the pathogenic role of microvascular endothelial cells (MiVEC) remains unknown. We characterized transcriptional, metabolic, and functional adaptation of cardiac MiVEC to pressure overload in mice and patients with aortic stenosis (AS). METHODS: In Tie2-Gfp mice subjected to transverse aortic constriction or sham surgery, we performed RNA sequencing of isolated cardiac Gfp+-MiVEC and validated the signature in freshly isolated MiVEC from left ventricle outflow tract and right atrium of patients with AS. We next compared their angiogenic and metabolic profiles and finally correlated molecular and pathological signatures with clinical phenotypes of 42 patients with AS (50% women). RESULTS: In mice, transverse aortic constriction induced progressive systolic dysfunction, fibrosis, and reduced microvascular density. After 10 weeks, 25 genes predominantly involved in matrix-regulation were >2-fold upregulated in isolated MiVEC. Increased transcript levels of Cartilage Intermediate Layer Protein (Cilp), Thrombospondin-4, Adamtsl-2, and Collagen1a1 were confirmed by quantitative reverse transcription polymerase chain reaction and recapitulated in left ventricle outflow tract-derived MiVEC of AS (P<0.05 versus right atrium-MiVEC). Fatty acid oxidation increased >2-fold in left ventricle outflow tract-MiVEC, proline content by 130% (median, IQR, 58%-474%; P=0.008) and procollagen secretion by 85% (mean [95% CI, 16%-154%]; P<0.05 versus right atrium-MiVEC for all). The altered transcriptome in left ventricle outflow tract-MiVEC was associated with impaired 2-dimensional-vascular network formation and 3-dimensional-spheroid sprouting (P<0.05 versus right atrium-MiVEC), profibrotic ultrastructural changes, and impaired diastolic left ventricle function, capillary density and functional status, especially in female AS. CONCLUSIONS: Pressure overload induces major transcriptional and metabolic adaptations in cardiac MiVEC resulting in excess interstitial fibrosis and impaired angiogenesis. Molecular rewiring of MiVEC is worse in women, compromises functional status, and identifies novel targets for intervention.

Peripheral Blood RNA Levels of QSOX1 and PLBD1 Are New Independent Predictors of Left Ventricular Dysfunction After Acute Myocardial Infarction.

BACKGROUND: The identification of patients with acute myocardial infarction (MI) at risk of subsequent left ventricular (LV) dysfunction remains challenging, but it is important to optimize therapies. The aim of this study was to determine the unbiased RNA profile in peripheral blood of patients with acute MI and to identify and validate new prognostic markers of LV dysfunction. METHODS: We prospectively enrolled a discovery cohort with acute MI (n=143) and performed whole-blood RNA profiling at different time points. We then selected transcripts on admission that related to LV dysfunction at follow-up and validated them by quantitative polymerase chain reaction in the discovery cohort, in an external validation cohort (n=449), and in a representative porcine MI model with cardiac magnetic resonance-based measurements of infarct size and postmortem myocardial pathology (n=33). RESULTS: RNA profiling in the discovery cohort showed upregulation of genes involved in chemotaxis, IL (interleukin)-6, and NF-κB (nuclear factor-κB) signaling in the acute phase of MI. Expression levels of the majority of these transcripts paralleled the rise in cardiac troponin T and decayed at 30 days. RNA levels of QSOX1, PLBD1, and S100A8 on admission with MI correlated with LV dysfunction at follow-up. Using quantitative polymerase chain reaction, we confirmed that QSOX1 and PLBD1 predicted LV dysfunction (odds ratio, 2.6 [95% CI, 1.1-6.1] and 3.2 [95% CI, 1.4-7.4]), whereas S100A8 did not. In the external validation cohort, we confirmed QSOX1 and PLBD1 as new independent markers of LV dysfunction (odds ratio, 1.41 [95% CI, 1.06-1.88] and 1.43 [95% CI, 1.08-1.89]). QSOX1 had an incremental predictive value in a model consisting of clinical variables and cardiac biomarkers (including NT-proBNP [N-terminal pro-B-type natriuretic peptide]). In the porcine MI model, whole-blood levels of QSOX1 and PLBD1 related to neutrophil infiltration in the ischemic myocardium in an infarct size-independent manner. CONCLUSIONS: Peripheral blood QSOX1 and PLBD1 in acute MI are new independent markers of LV dysfunction post-MI.

C-reactive protein during and after myocardial infarction in relation to cardiac injury and left ventricular function at follow-up.

BACKGROUND: Acute myocardial infarction (MI) invokes a large inflammatory response, which contributes to myocardial repair. HYPOTHESIS: We investigated whether C-reactive protein (CRP) measured during MI vs at 1 month follow-up improves the prediction of left ventricular (LV) function. METHODS: We prospectively enrolled 131 consecutive patients with acute MI and without non-cardiovascular causes of inflammation. We correlated admission and peak levels of CRP during hospitalization and high-sensitivity (hs) CRP at 1 month follow-up with markers of cardiac injury. Clinical follow-up and echocardiography for LV function were performed at a mean of 17 months. RESULTS: Median CRP levels were 1.89 mg/L on admission with MI, peaked to 12.10 mg/L during hospitalization and dropped to 1.24 mg/L at 1 month. Although admission CRP levels only weakly correlated with ejection fraction in the acute phase of MI (coefficient -0.164, P = 0.094), peak CRP was significantly related to ejection fraction (coefficient -0.4, P < 0.001), hsTroponin T (0.389, P < 0.001), and white blood cell count (0.389, P < 0.001). hsCRP at 1 month was not related to the extent of acute cardiac injury. These findings were replicated in an independent cohort of 57 patients. Peak CRP predicted LV dysfunction at follow-up (OR 11.0, 3.1-39.5 per log CRP, P < 0.001), persisting after adjustment for infarct size (OR 5.1, 1.1-23.6, P = 0.037), while hsCRP at 1 month was unrelated to LV function at follow-up. CONCLUSIONS: hsCRP 1 month post-MI does not relate to acute cardiac injury or LV function at follow-up, but we confirm that peak CRP is an independent predictor of LV dysfunction at follow-up.

Molecular signature of progenitor cells isolated from young and adult human hearts.

The loss of endogenous cardiac regenerative capacity within the first week of postnatal life has intensified clinical trials to induce cardiac regeneration in the adult mammalian heart using different progenitor cell types. We hypothesized that donor age-related phenotypic and functional characteristics of cardiac progenitor cells (CPC) account for mixed results of cell-based cardiac repair. We compared expression profiles and cell turnover rates of human heart-derived c-kitpos progenitors (c-kitpos CPC) and cardiosphere-derived cells (CDC) from young and adult donor origin and studied their in vitro angiogenic and cardiac differentiation potential, which can be relevant for cardiac repair. We report that 3-dimensional CDC expansion recapitulates a conducive environment for growth factor and cytokine release from adult donor cells (aCDC) that optimally supports vascular tube formation and vessel sprouting. Transdifferentiation capacity of c-kitpos CPCs and CDCs towards cardiomyocyte-like cells was modest, however, most notable in young c-kitpos cells and adult CDCs. Progenitors isolated with different methods thus show cell- and donor-specific characteristics that may account for variable contributions in functional myocardial recovery.

Sustained Placental Growth Factor-2 Treatment Does Not Aggravate Advanced Atherosclerosis in Ischemic Cardiomyopathy.

Angiogenic growth factor therapy for ischemic cardiovascular disease carries a risk of stimulating atherosclerotic plaque growth. We evaluated risk benefit ratio of sustained administration of recombinant human placental growth factor (rhPlGF)-2 in mice with advanced atherosclerosis and chronic ischemic cardiomyopathy. We maintained apolipoprotein E-deficient mice on a high cholesterol diet and induced myocardial infarction by transient ligation at 4 weeks. At 8 weeks, we assessed left ventricular (LV) function and randomized mice to receive rhPlGF-2 or vehicle (VEH) subcutaneously for 28 days. Administration of rhPlGF-2 significantly increased PlGF plasma levels without adverse hemodynamic or systemic inflammatory effects. RhPlGF-2 did not increase plaque area, composition, or vulnerability in the aortic arch. RhPlGF-2 significantly improved contractile function and reduced LV end-systolic and end-diastolic volume indices with a concomitant increase in capillary and arteriolar density in ischemic myocardium. RhPlGF-2 may represent a promising therapeutic strategy in chronic ischemic cardiomyopathy.

Right ventricular and pulmonary vascular reserve in asymptomatic BMPR2 mutation carriers.

BACKGROUND: Non-invasive estimates have suggested that asymptomatic BMPR2 mutation carriers may have an abnormal pulmonary vascular response to exercise and hypoxia. However, this has not been assessed with "gold standard" invasive measures. METHODS: Eight controls and 8 asymptomatic BMPR2 mutation carriers underwent cardiac magnetic resonance imaging with simultaneous invasive pressure recording during bicycle exercise in normoxia, hypoxia and after sildenafil administration. Abnormal pulmonary vascular reserve was defined as an increase in mean pulmonary artery pressure relative to cardiac output (P/Q slope) >3 mm Hg/liter/min. RESULTS: During normoxic exercise, BMPR2 mutation carriers had a similar P/Q slope when compared with healthy subjects. Only 1 of 8 BMPR2 mutation carriers had a P/Q slope >3 mm Hg/liter/min. During exercise in hypoxia, 3 of 8 BMPR2 mutation carriers had P/Q slopes >3 mm Hg/liter/min compared with none of the controls. Sildenafil decreased the P/Q slope both in controls and BMPR2 mutation carriers. The exercise-induced increase in right ventricular ejection fraction was similar between groups. None of the BMPR2 mutation carriers developed pulmonary arterial hypertension within 2 (range 1.3 to 2.8) years. CONCLUSIONS: The presence of a BMPR2 mutation, per se, is not associated with an abnormal pulmonary vascular and right ventricular functional response to exercise in asymptomatic individuals. Longer follow-up will be required to determine whether a P/Q slope of >3 mm Hg/liter/min during exercise in normoxia or hypoxia is a sign of pre-clinical disease expression.

Decreased Soluble Guanylate Cyclase Contributes to Cardiac Dysfunction Induced by Chronic Doxorubicin Treatment in Mice.

AIMS: The use of doxorubicin, a potent chemotherapeutic agent, is limited by cardiotoxicity. We tested the hypothesis that decreased soluble guanylate cyclase (sGC) enzyme activity contributes to the development of doxorubicin-induced cardiotoxicity. RESULTS: Doxorubicin administration (20 mg/kg, intraperitoneally [IP]) reduced cardiac sGC activity in wild-type (WT) mice. To investigate whether decreased sGC activity contributes to doxorubicin-induced cardiotoxicity, we studied mice with cardiomyocyte-specific deficiency of the sGC α1-subunit (mice with cardiomyocyte-specific deletion of exon 6 of the sGCα1 allele [sGCα1-/-CM]). After 12 weeks of doxorubicin administration (2 mg/kg/week IP), left ventricular (LV) systolic dysfunction was greater in sGCα1-/-CM than WT mice. To further assess whether reduced sGC activity plays a pathogenic role in doxorubicin-induced cardiotoxicity, we studied a mouse model in which decreased cardiac sGC activity was induced by cardiomyocyte-specific expression of a dominant negative sGCα1 mutant (DNsGCα1) upon doxycycline removal (Tet-off). After 8 weeks of doxorubicin administration, DNsGCα1tg/+, but not WT, mice displayed LV systolic dysfunction and dilatation. The difference in cardiac function and remodeling between DNsGCα1tg/+ and WT mice was even more pronounced after 12 weeks of treatment. Further impairment of cardiac function was attenuated when DNsGCα1 gene expression was inhibited (beginning at 8 weeks of doxorubicin treatment) by administering doxycycline. Furthermore, doxorubicin-associated reactive oxygen species generation was higher in sGCα1-deficient than WT hearts. Innovation and Conclusion: These data demonstrate that a reduction in cardiac sGC activity worsens doxorubicin-induced cardiotoxicity in mice and identify sGC as a potential therapeutic target. Various pharmacological sGC agonists are in clinical development or use and may represent a promising approach to limit doxorubicin-associated cardiotoxicity. Antioxid. Redox Signal. 26, 153-164.

Neovascularization Potential of Blood Outgrowth Endothelial Cells From Patients With Stable Ischemic Heart Failure Is Preserved.

BACKGROUND: Blood outgrowth endothelial cells (BOECs) mediate therapeutic neovascularization in experimental models, but outgrowth characteristics and functionality of BOECs from patients with ischemic cardiomyopathy (ICMP) are unknown. We compared outgrowth efficiency and in vitro and in vivo functionality of BOECs derived from ICMP with BOECs from age-matched (ACON) and healthy young (CON) controls. METHODS AND RESULTS: We isolated 3.6±0.6 BOEC colonies/100×10(6) mononuclear cells (MNCs) from 60-mL blood samples of ICMP patients (n=45; age: 66±1 years; LVEF: 31±2%) versus 3.5±0.9 colonies/100×10(6) MNCs in ACON (n=32; age: 60±1 years) and 2.6±0.4 colonies/100×10(6) MNCs in CON (n=55; age: 34±1 years), P=0.29. Endothelial lineage (VEGFR2(+)/CD31(+)/CD146(+)) and progenitor (CD34(+)/CD133(-)) marker expression was comparable in ICMP and CON. Growth kinetics were similar between groups (P=0.38) and not affected by left ventricular systolic dysfunction, maladaptive remodeling, or presence of cardiovascular risk factors in ICMP patients. In vitro neovascularization potential, assessed by network remodeling on Matrigel and three-dimensional spheroid sprouting, did not differ in ICMP from (A)CON. Secretome analysis showed a marked proangiogenic profile, with highest release of angiopoietin-2 (1.4±0.3×10(5) pg/10(6) ICMP-BOECs) and placental growth factor (5.8±1.5×10(3) pg/10(6) ICMP BOECs), independent of age or ischemic disease. Senescence-associated ß-galactosidase staining showed comparable senescence in BOECs from ICMP (5.8±2.1%; n=17), ACON (3.9±1.1%; n=7), and CON (9.0±2.8%; n=13), P=0.19. High-resolution microcomputed tomography analysis in the ischemic hindlimb of nude mice confirmed increased arteriogenesis in the thigh region after intramuscular injections of BOECs from ICMP (P=0.025; n=8) and CON (P=0.048; n=5) over vehicle control (n=8), both to a similar extent (P=0.831). CONCLUSIONS: BOECs can be successfully culture-expanded from patients with ICMP. In contrast to impaired functionality of ICMP-derived bone marrow MNCs, BOECs retain a robust proangiogenic profile, both in vitro and in vivo, with therapeutic potential for targeting ischemic disease.

Placental growth factor 2--A potential therapeutic strategy for chronic myocardial ischemia.

OBJECTIVES: We investigated whether sustained infusion of recombinant human placental growth factor-2 (rhPlGF-2) improves myocardial perfusion and left ventricular (LV) function in a porcine model of ischemic cardiomyopathy (ICM). METHODS: We induced myocardial ischemia using a flow-limiting stent in the LAD. Four weeks later, we randomized pigs with confirmed myocardial dysfunction to blinded rhPlGF-2 administration (PlGF2, 15 µg/kg/day, 14 days) or PBS (CON). At 8 weeks, we measured hemodynamics, contractile function and regional perfusion at rest and during stress using MRI and microspheres. We evaluated neovascularization post mortem. RESULTS: RhPlGF-2 administration increased PlGF serum levels more than 63-fold (83 3 ± 361 versus 11 ± 5 pg/ml CON, P<0.05) without adverse effects. After 4weeks, rhPlGF-2 significantly enhanced perfusion in the ischemic region at rest (0.83 ± 0.32 versus 0.58 ± 0.21 ml/min/g CON, P<0.05) and during hyperemia (1.50 ± 0.50 versus 1.02 ± 0.46 ml/min/g CON, P<0.05). Consequently, regional contractile function in rhPlGF-2-treated pigs improved at rest (37 ± 15% versus 23 ± 9% CON, P<0.05) and during high dose dobutamine stress (53 ± 31% versus 27 ± 16% CON, P<0.05). Enhanced perfusion translated into a greater improvement in LV ejection fraction and in preload-recruitable stroke work in rhPlGF-2-treated animals than in CON (52 ± 11 versus 41 ± 9%, and 76 ± 24 versus 41 ± 21 mmHg, respectively, P<0.05 for both), which was associated with significantly greater vascular density in the ischemic region. CONCLUSIONS: In chronic ICM, systemic rhPlGF-2 administration significantly enhances regional myocardial perfusion, contractile function at rest and during stress, and induces a prominent recovery of global cardiac function. PlGF-2 protein infusion is safe and may represent a promising therapy in chronic ICM.

Concomitant Phosphodiesterase 5 Inhibition Enhances Myocardial Protection by Inhaled Nitric Oxide in Ischemia-Reperfusion Injury.

Enhanced cyclic guanosine monophosphate (cGMP) signaling may attenuate myocardial ischemia-reperfusion injury (I/R) and improve left ventricular (LV) functional recovery after myocardial infarction (MI). We investigated the cardioprotection afforded by inhaled NO (iNO), the phosphodiesterase 5 (PDE5)-specific inhibitor tadalafil (TAD), or their combination (iNO+TAD) in C57Bl6J mice subjected to 6-minute left anterior descending artery ligation followed by reperfusion. We measured plasma and cardiac concentrations of cGMP during early reperfusion, quantified myocardial necrosis and inflammation by serial troponin-I (TnI) and myeloperoxidase-positive cell infiltration at day 3, and evaluated LV function and remodeling after 4 weeks using echocardiography and pressure-conductance catheterization. Administration of iNO, TAD, or both during I/R was safe and hemodynamically well tolerated. Compared with untreated mice (CON), only iNO+TAD increased plasma and cardiac-cGMP levels during early reperfusion (80 ± 12 versus 36 ± 6 pmol/ml and 0.15 ± 0.02 versus 0.05 ± 0.01 pmol/mg protein, P < 0.05 for both). Moreover, iNO+TAD reduced TnI at 4 hours to a greater extent (P < 0.001 versus CON) than either alone (P < 0.05 versus CON) and was associated with significantly less myocardial inflammatory cell infiltration at day 3. After 4 weeks and compared with CON, iNO+TAD was associated with increased fractional shortening (43 ± 1 versus 33 ± 2%, P < 0.01), larger stroke volumes (14.9 ± 1.2 versus 10.2 ± 0.9 µl, P < 0.05), enhanced septal and posterior wall thickening (P < 0.05 and P < 0.001, respectively), and attenuated LV dilatation (P < 0.001), whereas iNO or TAD alone conferred less benefit. Thus, iNO+TAD has superior efficacy to limit early reperfusion injury and attenuate adverse LV remodeling. Combination of inhaled NO with a long-acting PDE5 inhibitor may represent a promising strategy to reduce ischemic damage following reperfusion and better preserve LV function.

Sildenafil improves exercise hemodynamics in Fontan patients.

BACKGROUND: Patients with Fontan circulation have reduced exercise capacity. The absence of a presystemic pump may limit flow through the pulmonary circulation, restricting ventricular filling and cardiac output. We evaluated exercise hemodynamics and the effect of sildenafil on exercise hemodynamics in Fontan patients. METHODS AND RESULTS: Ten Fontan patients (6 men, 20±4 years) underwent cardiac magnetic resonance imaging at rest and during supine bicycle exercise before and after sildenafil. Systemic ventricular volumes were obtained at rest and during low- (34±15 W), moderate- (69±29 W), and high-intensity (97±36 W) exercise using an ungated, free-breathing cardiac magnetic resonance sequence and analyzed correcting for cardiac phase and respiratory translation. Radial and pulmonary artery pressures and cGMP were measured. Before sildenafil, cardiac index increased throughout exercise (4.0±0.9, 5.9±1.1, 7.0±1.6, 7.4±1.7 L/(min·m(2)); P<0.0001) with 106±49% increase in heart rate. Stroke volume index (P=0.015) and end-diastolic volume index (P=0.001) decreased during exercise. End-systolic volume index remained unchanged (P=0.8). Total pulmonary resistance index (P=0.005) increased, whereas systemic vascular resistance index decreased during exercise (P<0.0001). Sildenafil increased cardiac index (P<0.0001) and stroke volume index (P=0.003), especially at high-intensity exercise (interaction P=0.004 and P=0.003, respectively). Systemic vascular resistance index was reduced (P<0.0001-interaction P=0.1), whereas total pulmonary resistance index was reduced at rest and reduced further during exercise (P=0.008-interaction P=0.029). cGMP remained unchanged before sildenafil (P=0.9), whereas it increased significantly after sildenafil (P=0.019). CONCLUSIONS: In Fontan patients, sildenafil improved cardiac index during exercise with a decrease in total pulmonary resistance index and an increase in stroke volume index. This implies that pulmonary vasculature represents a physiological limitation, which can be attenuated by sildenafil, the clinical significance of which warrants further study.

Increased cardiac myocyte PDE5 levels in human and murine pressure overload hypertrophy contribute to adverse LV remodeling.

BACKGROUND: The intracellular second messenger cGMP protects the heart under pathological conditions. We examined expression of phosphodiesterase 5 (PDE5), an enzyme that hydrolyzes cGMP, in human and mouse hearts subjected to sustained left ventricular (LV) pressure overload. We also determined the role of cardiac myocyte-specific PDE5 expression in adverse LV remodeling in mice after transverse aortic constriction (TAC). METHODOLOGY/PRINCIPAL FINDINGS: In patients with severe aortic stenosis (AS) undergoing valve replacement, we detected greater myocardial PDE5 expression than in control hearts. We observed robust expression in scattered cardiac myocytes of those AS patients with higher LV filling pressures and BNP serum levels. Following TAC, we detected similar, focal PDE5 expression in cardiac myocytes of C57BL/6NTac mice exhibiting the most pronounced LV remodeling. To examine the effect of cell-specific PDE5 expression, we subjected transgenic mice with cardiac myocyte-specific PDE5 overexpression (PDE5-TG) to TAC. LV hypertrophy and fibrosis were similar as in WT, but PDE5-TG had increased cardiac dimensions, and decreased dP/dtmax and dP/dtmin with prolonged tau (P<0.05 for all). Greater cardiac dysfunction in PDE5-TG was associated with reduced myocardial cGMP and SERCA2 levels, and higher passive force in cardiac myocytes in vitro. CONCLUSIONS/SIGNIFICANCE: Myocardial PDE5 expression is increased in the hearts of humans and mice with chronic pressure overload. Increased cardiac myocyte-specific PDE5 expression is a molecular hallmark in hypertrophic hearts with contractile failure, and represents an important therapeutic target.

Differential effects of progenitor cell populations on left ventricular remodeling and myocardial neovascularization after myocardial infarction.

OBJECTIVES: We compared biological repair after acute myocardial infarction (AMI) with selected porcine progenitor cell populations. BACKGROUND: Cell types and mechanisms responsible for myocardial repair after AMI remain uncertain. METHODS: In a blinded, randomized study, we infused autologous late-outgrowth endothelial progenitor cells (EPC) (n = 10, 34 +/- 22 x 10(6) CD29-31-positive, capable of tube formation), allogeneic green fluorescent peptide-labeled mesenchymal stem cells (MSC) (n = 11, 10 +/- 2 x 10(6) CD29-44-90-positive, capable of adipogenic and osteogenic differentiation), or vehicle (CON) (n = 12) in the circumflex artery 1 week after AMI. Systolic function (ejection fraction), left ventricular (LV) end-diastolic and end-systolic volumes, and infarct size were assessed with magnetic resonance imaging at 1 week and 7 weeks. Cell engraftment and vascular density were evaluated on postmortem sections. RESULTS: Recovery of LV ejection fraction from 1 to 7 weeks was similar between groups, but LV remodeling markedly differed with a greater increase of LV end-systolic volume in MSC and CON (+11 +/- 12 ml/m(2) and +7 +/- 8 ml/m(2) vs. -3 +/- 11 ml/m(2) in EPC, respectively, p = 0.04), and a similar trend was noted for LV end-diastolic volume (p = 0.09). After EPC, infarct size decreased more in segments with >50% infarct transmurality (p = 0.02 vs. MSC and CON) and was associated with a greater vascular density (p = 0.01). Late outgrowth EPCs secrete higher levels of the pro-angiogenic placental growth factor (733 [277 to 1,214] pg/10(6) vs. 59 [34 to 88] pg/10(6) cells in MSC, p = 0.03) and incorporate in neovessels in vivo. CONCLUSIONS: Infusion of late-outgrowth EPCs after AMI improves myocardial infarction remodeling via enhanced neovascularization but does not mediate cardiomyogenesis. Endothelial progenitor cell transfer might hold promise for heart failure prevention via pro-angiogenic or paracrine matrix-modulating effects.

Cardioselective nitric oxide synthase 3 gene transfer protects against myocardial reperfusion injury.

Nitric oxide modulates the severity of myocardial ischemia-reperfusion (I/R) injury. We investigated whether cardioselective nitric oxide synthase 3 (NOS3) gene transfer could confer myocardial protection against I/R injury in pigs and examined potential molecular mechanisms. I/R injury was induced by balloon occlusion of the left anterior descending artery for 45 min followed by 4 or 72 h reperfusion. Hemodynamic and pathological changes were measured in pigs in the absence (n = 11) or presence of prior intracoronary retroinfusion of human NOS3 (AdNOS3, 5 x 10(10) PFU, n = 13) or control vector (AdRR5, 5 x 10(10) PFU, n = 11). Retrograde NOS3 gene transfer selectively increased NOS3 expression and NO bioavailability in the area at risk (AAR) without changing endogenous NOS isoform expression. At 4 h R, LV systolic (dP/dt(max)) and diastolic (dP/dt(min)) function was better preserved in AdNOS3- than in AdRR5-injected pigs (2,539 +/- 165 vs. 1,829 +/- 156 mmHg/s, and -2,781 +/- 340 vs. -2,062 +/- 292 mmHg/s, respectively, P < 0.05 for both). Myocardial infarct size (% AAR) was significantly smaller in AdNOS3 than in control and AdRR5 and associated with a significantly greater reduction in cardiac myeloperoxidase activity, a marker of neutrophil infiltration. The latter effects were sustained at 72 h R in a subset of pigs (n = 7). In the AAR, intercellular endothelial adhesion molecule-1 expression and cardiomyocyte apoptosis were significantly lower in AdNOS3. In conclusion, single myocardial NOS3 retroinfusion attenuates I/R injury, and causes a sustained reduction in myocardial infarct size and inflammatory cell infiltration. Gene-based strategies to increase NO bioavailability may have therapeutic potential in myocardial I/R.

Gender-specific modulation of the response to arterial injury by soluble guanylate cyclase α1.

OBJECTIVE: Soluble guanylate cyclase (sGC), a heterodimer composed of alpha and beta subunits, synthesizes cGMP in response to nitric oxide (NO). NO modulates vascular tone and structure but the relative contributions of cGMP-dependent versus cGMP-independent mechanisms remain uncertain. We studied the response to vascular injury in male (M) and female (F) mice with targeted deletion of exon 6 of the sGCα1 subunit (sGCα1(-/-)), resulting in a non-functional heterodimer. METHODS: We measured aortic cGMP levels and mRNA transcripts encoding sGC α1, α2, and ß1 subunits in wild type (WT) and sGCa1(-/-) mice. To study the response to vascular injury, BrdU-incorporation and neointima formation (maximum intima to media (I/M) ratio) were determined 5 and 28 days after carotid artery ligation, respectively. RESULTS: Aortic cGMP levels were 4-fold higher in F than in M mice in both genotypes, and, within each gender, 4-fold higher in WT than in sGCa1(-/-). In contrast, sGCα1, sGCα2, and sGCß1 mRNA expression did not differ between groups. ³H-thymidine incorporation in cultured sGCa1(-/-) smooth muscle cells (SMC) was 27%±12% lower than in WT SMC and BrdU-incorporation in carotid arteries 5 days after ligation was significantly less in sGCa1(-/-) M than in WT M. Neointima area and I/M 28 days after ligation were 65% and 62% lower in sGCa1(-/-) M than in WT M mice (p<0,05 for both) but were not different in F mice. CONCLUSION: Functional deletion of sGCa1 resulted in reduced cGMP levels in male sGCa1(-/-) mice and a gender-specific effect on the adaptive response to vascular injury.

Ventricular phosphodiesterase-5 expression is increased in patients with advanced heart failure and contributes to adverse ventricular remodeling after myocardial infarction in mice.

BACKGROUND: Ventricular expression of phosphodiesterase-5 (PDE5), an enzyme responsible for cGMP catabolism, is increased in human right ventricular hypertrophy, but its role in left ventricular (LV) failure remains incompletely understood. We therefore measured LV PDE5 expression in patients with advanced systolic heart failure and characterized LV remodeling after myocardial infarction in transgenic mice with cardiomyocyte-specific overexpression of PDE5 (PDE5-TG). METHODS AND RESULTS: Immunoblot and immunohistochemistry techniques revealed that PDE5 expression was greater in explanted LVs from patients with dilated and ischemic cardiomyopathy than in control hearts. To evaluate the impact of increased ventricular PDE5 levels on cardiac function, PDE5-TG mice were generated. Confocal and immunoelectron microscopy revealed increased PDE5 expression in cardiomyocytes, predominantly localized to Z-bands. At baseline, myocardial cGMP levels, cell shortening, and calcium handling in isolated cardiomyocytes and LV hemodynamic measurements were similar in PDE5-TG and wild-type littermates. Ten days after myocardial infarction, LV cGMP levels had increased to a greater extent in wild-type mice than in PDE5-TG mice (P<0.05). Ten weeks after myocardial infarction, LV end-systolic and end-diastolic volumes were larger in PDE5-TG than in wild-type mice (57+/-5 versus 39+/-4 and 65+/-6 versus 48+/-4 muL, respectively; P<0.01 for both). LV systolic dysfunction and diastolic dysfunction were more marked in PDE5-TG than in wild-type mice, associated with enhanced hypertrophy and reduced contractile function in isolated cardiomyocytes from remote myocardium. CONCLUSIONS: Increased PDE5 expression predisposes mice to adverse LV remodeling after myocardial infarction. Increased myocardial PDE5 expression in patients with advanced cardiomyopathy may contribute to the development of heart failure and represents an important therapeutic target.