Patency of anterior circulation branch vessels after Pipeline embolization: longer-term results from 82 aneurysm cases.

OBJECTIVE The Pipeline Embolization Device (PED) has become increasingly used for the treatment of intracranial aneurysms. Given its high metal surface area coverage, there is concern for the patency of branch vessels that become covered by the device. Limited data exist regarding the patency of branch vessels adjacent to aneurysms that are covered by PEDs. The authors assessed the rate of intracranial internal carotid artery, anterior circulation branch vessel patency following PED placement at their institution. METHODS The authors retrospectively reviewed the records of 82 patients who underwent PED treatment between 2009 and 2014 and in whom the PED was identified to cover branch vessels. Patency of the anterior cerebral, posterior communicating, anterior choroidal, and ophthalmic arteries was evaluated using digital subtraction angiography preoperatively and postoperatively after PED deployment and at longer-term follow-up. RESULTS Of the 127 arterial branches covered by PEDs, there were no immediate postoperative occlusions. At angiographic follow-up (mean 10 months, range 3-34.7 months), arterial side branches were occluded in 13 (15.8%) of 82 aneurysm cases and included 2 anterior cerebral arteries, 8 ophthalmic arteries, and 3 posterior communicating arteries. No cases of anterior choroidal artery occlusion were observed. Patients with branch occlusion did not experience any neurological symptoms. CONCLUSIONS In this large series, the longer-term rate of radiographic side branch arterial occlusion after coverage by a flow diverter was 15.8%. Terminal branch vessels, such as the anterior choroidal artery, remained patent in this series. The authors' series suggests that branch vessel occlusions are clinically silent and should not deter aneurysm treatment with flow diversion.

Ruptured mycotic cerebral aneurysm development from pseudoocclusion due to septic embolism.

BACKGROUND: Cerebral mycotic aneurysms are rare sequelae of systemic infections that can cause profound morbidity and mortality with rupture. Direct bacterial extension and vessel integrity compromise from septic emboli have been implicated as mechanisms for formation of these lesions. We report the 5-day development of a ruptured mycotic aneurysm arising from a septic embolism that caused a focal M1 pseudoocclusion. CASE DESCRIPTION: A 14-year-old girl developed acute left-sided hemiparesis while hospitalized for subacute bacterial endocarditis that was found after she presented with a 2-week history of fever, myalgia, shortness of breath, and lethargy. Mitral valve vegetations were confirmed in the setting of hemophilus bacteremia. Brain magnetic resonance (MR) imaging and angiography confirmed middle cerebral artery infarct with focal pseudoocclusion of the distal M1 segment. Given that further middle cerebral artery territory was at risk, a trial of heparin was attempted for revascularization but required discontinuation owing to hemorrhagic conversion. Decline of the patient's mental status necessitated craniectomy for decompression. Postoperatively, her mental status improved with residual left hemiparesis. On the third postoperative day (5 days after MR angiography), the patient's neurologic condition acutely declined, with development of right-sided mydriasis. Computed tomography (CT) angiography revealed a ruptured 19 × 16 mm pseudoaneurysm arising from the M1 site of the previous occlusion. Emergent coiling of aneurysm and parent vessel followed by hematoma evacuation ensued. At discharge, the patient had residual left hemiparesis but intact speech and cognition. CONCLUSION: Focal occlusions due to septic emboli should be considered high-risk for mycotic aneurysm formation, prompting aggressive monitoring with neuroimaging and treatment when indicated.

Cardiac G-protein-coupled receptor kinase 2 ablation induces a novel Ca2+ handling phenotype resistant to adverse alterations and remodeling after myocardial infarction.

BACKGROUND: G-protein-coupled receptor kinase 2 (GRK2) is a primary regulator of ß-adrenergic signaling in the heart. G-protein-coupled receptor kinase 2 ablation impedes heart failure development, but elucidation of the cellular mechanisms has not been achieved, and such elucidation is the aim of this study. METHODS AND RESULTS: Myocyte contractility, Ca(2+) handling and excitation-contraction coupling were studied in isolated cardiomyocytes from wild-type and GRK2 knockout (GRK2KO) mice without (sham) or with myocardial infarction (MI). In cardiac myocytes isolated from unstressed wild-type and GRK2KO hearts, myocyte contractions and Ca(2+) transients were similar, but GRK2KO myocytes had lower sarcoplasmic reticulum (SR) Ca(2+) content because of increased sodium-Ca(2+) exchanger activity and inhibited SR Ca(2+) ATPase by local protein kinase A-mediated activation of phosphodiesterase 4 resulting in hypophosphorylated phospholamban. This Ca(2+) handling phenotype is explained by a higher fractional SR Ca(2+) release induced by increased L-type Ca(2+) channel currents. After ß-adrenergic stimulation, GRK2KO myocytes revealed significant increases in contractility and Ca(2+) transients, which were not mediated through cardiac L-type Ca(2+) channels but through an increased SR Ca(2+). Interestingly, post-MI GRK2KO mice showed better cardiac function than post-MI control mice, which is explained by an improved Ca(2+) handling phenotype. The SR Ca(2+) content was better maintained in post-MI GRK2KO myocytes than in post-MI control myocytes because of better-maintained L-type Ca(2+) channel current density and no increase in sodium-Ca(2+) exchanger in GRK2KO myocytes. An L-type Ca(2+) channel blocker, verapamil, reversed some beneficial effects of GRK2KO. CONCLUSIONS: These data argue for novel differential regulation of L-type Ca(2+) channel currents and SR load by GRK2. G-protein-coupled receptor kinase 2 ablation represents a novel beneficial Ca(2+) handling phenotype resisting adverse remodeling after MI.

Increasing cardiac contractility after myocardial infarction exacerbates cardiac injury and pump dysfunction.

RATIONALE: Myocardial infarction (MI) leads to heart failure (HF) and premature death. The respective roles of myocyte death and depressed myocyte contractility in the induction of HF after MI have not been clearly defined and are the focus of this study. OBJECTIVES: We developed a mouse model in which we could prevent depressed myocyte contractility after MI and used it to test the idea that preventing depression of myocyte Ca(2+)-handling defects could avert post-MI cardiac pump dysfunction. METHODS AND RESULTS: MI was produced in mice with inducible, cardiac-specific expression of the ß2a subunit of the L-type Ca(2+) channel. Myocyte and cardiac function were compared in control and ß2a animals before and after MI. ß2a myocytes had increased Ca(2+) current; sarcoplasmic reticulum Ca(2+) load, contraction and Ca(2+) transients (versus controls), and ß2a hearts had increased performance before MI. After MI, cardiac function decreased. However, ventricular dilation, myocyte hypertrophy and death, and depressed cardiac pump function were greater in ß2a versus control hearts after MI. ß2a animals also had poorer survival after MI. Myocytes isolated from ß2a hearts after MI did not develop depressed Ca(2+) handling, and Ca(2+) current, contractions, and Ca(2+) transients were still above control levels (before MI). CONCLUSIONS: Maintaining myocyte contractility after MI, by increasing Ca(2+) influx, depresses rather than improves cardiac pump function after MI by reducing myocyte number.

CaMKII negatively regulates calcineurin-NFAT signaling in cardiac myocytes.

RATIONALE: Pathological cardiac myocyte hypertrophy is thought to be induced by the persistent increases in intracellular Ca(2+) needed to maintain cardiac function when systolic wall stress is increased. Hypertrophic Ca(2+) binds to calmodulin (CaM) and activates the phosphatase calcineurin (Cn) and CaM kinase (CaMK)II. Cn dephosphorylates cytoplasmic NFAT (nuclear factor of activated T cells), inducing its translocation to the nucleus where it activates antiapoptotic and hypertrophic target genes. Cytoplasmic CaMKII regulates Ca(2+) handling proteins but whether or not it is directly involved in hypertrophic and survival signaling is not known. OBJECTIVE: This study explored the hypothesis that cytoplasmic CaMKII reduces NFAT nuclear translocation by inhibiting the phosphatase activity of Cn. METHODS AND RESULTS: Green fluorescent protein-tagged NFATc3 was used to determine the cellular location of NFAT in cultured neonatal rat ventricular myocytes (NRVMs) and adult feline ventricular myocytes. Constitutively active (CaMKII-CA) or dominant negative (CaMKII-DN) mutants of cytoplasmic targeted CaMKII(deltac) were used to activate and inhibit cytoplasmic CaMKII activity. In NRVM CaMKII-DN (48.5+/-3%, P<0.01 versus control) increased, whereas CaMKII-CA decreased (5.9+/-1%, P<0.01 versus control) NFAT nuclear translocation (Control: 12.3+/-1%). Cn inhibitors were used to show that these effects were caused by modulation of Cn activity. Increasing Ca(2+) increased Cn-dependent NFAT translocation (to 71.7+/-7%, P<0.01) and CaMKII-CA reduced this effect (to 17.6+/-4%). CaMKII-CA increased TUNEL and caspase-3 activity (P<0.05). CaMKII directly phosphorylated Cn at Ser197 in CaMKII-CA infected NRVMs and in hypertrophied feline hearts. CONCLUSION: These data show that activation of cytoplasmic CaMKII inhibits NFAT nuclear translocation by phosphorylation and subsequent inhibition of Cn.

c-Kit+ bone marrow stem cells differentiate into functional cardiac myocytes.

The utility of bone marrow cells (BMCs) to regenerate cardiac myocytes is controversial. The present study examined the capacity of different types of BMCs to generate functional cardiac myocytes. Isolated c-kit(+) BMCs (BMSCs), c-kit(+) and crude BMCs from the adult feline femur were membrane stained with PKH26 dye or infected with a control enhanced green fluorescence protein transcript (EGFP)-adenovirus prior to co-culture upon neonatal rat ventricular myocytes (NRVM). Co-cultured cells were immuno-stained for c-kit, alpha-tropomyosin, alpha-actinin, connexin 43 (Cx43) and Ki67 and analyzed with confocal microscopy. Electrophysiology of BMSC derived myocytes were compared to NRVMs within the same culture dish. Gap junction function was analyzed by fluorescence recovery after photo-bleaching (FRAP). BMCs proliferated and differentiated into cardiac myocytes during the first 48 hours of co-culturing. These newly formed cardiac myocytes were able to contract spontaneously or synchronously with neighboring NRVMs. The myogenic rate of c-kit(+) BMSCs was significantly greater than c-kit(+) and crude BMCs (41.2 +/- 2.1, 6.1 +/- 1.2, and 17.1 +/- 1.5%, respectively). The newly formed cardiac myocytes exhibited an immature electrophysiological phenotype until they became electrically coupled to NRVMs through functional gap junctions. BMSCs did not become functional myocytes in the absence of NRVMs. In conclusion, c-kit(+) BMSCs have the ability to transdifferentiate into functional cardiac myocytes.

Ca2+ influx through T- and L-type Ca2+ channels have different effects on myocyte contractility and induce unique cardiac phenotypes.

T-type Ca(2+) channels (TTCCs) are expressed in the developing heart, are not present in the adult ventricle, and are reexpressed in cardiac diseases involving cardiac dysfunction and premature, arrhythmogenic death. The goal of this study was to determine the functional role of increased Ca(2+) influx through reexpressed TTCCs in the adult heart. A mouse line with cardiac-specific, conditional expression of the alpha1G-TTCC was used to increase Ca(2+) influx through TTCCs. alpha1G hearts had mild increases in contractility but no cardiac histopathology or premature death. This contrasts with the pathological phenotype of a previously studied mouse with increased Ca(2+) influx through the L-type Ca(2+) channel (LTCC) secondary to overexpression of its beta2a subunit. Although alpha1G and beta2a myocytes had similar increases in Ca(2+) influx, alpha1G myocytes had smaller increases in contraction magnitude, and, unlike beta2a myocytes, there were no increases in sarcoplasmic reticulum Ca(2+) loading. Ca(2+) influx through TTCCs also did not induce normal sarcoplasmic reticulum Ca(2+) release. alpha1G myocytes had changes in LTCC, SERCA2a, and phospholamban abundance, which appear to be adaptations that help maintain Ca(2+) homeostasis. Immunostaining suggested that the majority of alpha1G-TTCCs were on the surface membrane. Osmotic shock, which selectively eliminates T-tubules, induced a greater reduction in L- versus TTCC currents. These studies suggest that T- and LTCCs are in different portions of the sarcolemma (surface membrane versus T-tubules) and that Ca(2+) influx through these channels induce different effects on myocyte contractility and lead to distinct cardiac phenotypes.

Increased cardiac myocyte progenitors in failing human hearts.

BACKGROUND: Increasing evidence, derived mainly from animal models, supports the existence of endogenous cardiac renewal and repair mechanisms in adult mammalian hearts that could contribute to normal homeostasis and the responses to pathological insults. METHODS AND RESULTS: Translating these results, we isolated small c-kit+ cells from 36 of 37 human hearts using primary cell isolation techniques and magnetic cell sorting techniques. The abundance of these cardiac progenitor cells was increased nearly 4-fold in patients with heart failure requiring transplantation compared with nonfailing controls. Polychromatic flow cytometry of primary cell isolates (<30 microm) without antecedent c-kit enrichment confirmed the increased abundance of c-kit+ cells in failing hearts and demonstrated frequent coexpression of CD45 in these cells. Immunocytochemical characterization of freshly isolated, c-kit-enriched human cardiac progenitor cells confirmed frequent coexpression of c-kit and CD45. Primary cardiac progenitor cells formed new human cardiac myocytes at a relatively high frequency after coculture with neonatal rat ventricular myocytes. These contracting new cardiac myocytes exhibited an immature phenotype and frequent electric coupling with the rat myocytes that induced their myogenic differentiation. CONCLUSIONS: Despite the increased abundance and cardiac myogenic capacity of cardiac progenitor cells in failing human hearts, the need to replace these organs via transplantation implies that adverse features of the local myocardial environment overwhelm endogenous cardiac repair capacity. Developing strategies to improve the success of endogenous cardiac regenerative processes may permit therapeutic myocardial repair without cell delivery per se.

Ca2+- and mitochondrial-dependent cardiomyocyte necrosis as a primary mediator of heart failure.

Loss of cardiac myocytes in heart failure is thought to occur largely through an apoptotic process. Here we show that heart failure can also be precipitated through myocyte necrosis associated with Ca2+ overload. Inducible transgenic mice with enhanced sarcolemmal L-type Ca2+ channel (LTCC) activity showed progressive myocyte necrosis that led to pump dysfunction and premature death, effects that were dramatically enhanced by acute stimulation of beta-adrenergic receptors. Enhanced Ca2+ influx-induced cellular necrosis and cardiomyopathy was prevented with either LTCC blockers or beta-adrenergic receptor antagonists, demonstrating a proximal relationship among beta-adrenergic receptor function, Ca2+ handling, and heart failure progression through necrotic cell loss. Mechanistically, loss of cyclophilin D, a regulator of the mitochondrial permeability transition pore that underpins necrosis, blocked Ca2+ influx-induced necrosis of myocytes, heart failure, and isoproterenol-induced premature death. In contrast, overexpression of the antiapoptotic factor Bcl-2 was ineffective in mitigating heart failure and death associated with excess Ca2+ influx and acute beta-adrenergic receptor stimulation. This paradigm of mitochondrial- and necrosis-dependent heart failure was also observed in other mouse models of disease, which supports the concept that heart failure is a pleiotropic disorder that involves not only apoptosis, but also necrotic loss of myocytes in association with dysregulated Ca2+ handling and beta-adrenergic receptor signaling.

Adolescent feline heart contains a population of small, proliferative ventricular myocytes with immature physiological properties.

Recent studies suggest that rather than being terminally differentiated, the adult heart is a self-renewing organ with the capacity to generate new myocytes from cardiac stem/progenitor cells (CS/PCs). This study examined the hypotheses that new myocytes are generated during adolescent growth, to increase myocyte number, and these newly formed myocytes are initially small, mononucleated, proliferation competent, and have immature properties. Ventricular myocytes (VMs) and cKit(+) (stem cell receptor) CS/PCs were isolated from 11- and 22-week feline hearts. Bromodeoxyuridine incorporation (in vivo) and p16(INK4a) immunostaining were measured to assess myocyte cell cycle activity and senescence, respectively. Telomerase activity, contractions, Ca(2+) transients, and electrophysiology were compared in small mononucleated (SMMs) and large binucleated (LBMs) myocytes. Heart mass increased by 101% during adolescent growth, but left ventricular myocyte volume only increased by 77%. Most VMs were binucleated (87% versus 12% mononucleated) and larger than mononucleated myocytes. A greater percentage of SMMs was bromodeoxyuridine positive (SMMs versus LBMs: 3.1% versus 0.8%; P<0.05), and p16(INK4a) negative and small myocytes had greater telomerase activity than large myocytes. Contractions and Ca(2+) transients were prolonged in SMMs versus LBMs and Ca(2+) release was disorganized in SMMs with reduced transient outward current and T-tubule density. The T-type Ca(2+) current, usually seen in fetal/neonatal VMs, was found exclusively in SMMs and in myocytes derived from CS/PC. Myocyte number increases during adolescent cardiac growth. These new myocytes are initially small and functionally immature, with patterns of ion channel expression normally found in the fetal/neonatal period.

Mutations and polymorphisms in the human ornithine transcarbamylase gene.

Ornithine transcarbamylase (OTC) deficiency, an X-linked, semidominant disorder, is the most common inherited defect in ureagenesis resulting in hyperammonemia. The previous two mutation updates for the OTC gene were published in 1993 and 1995 and included 36 and 30 mutations respectively. This comprehensive update contains a compilation of 244 mutations including 13 polymorphisms. Twenty-four of the mutations are reported here for the first time. Forty-two percent of the disease-causing mutations are associated with acute neonatal hyperammonemia; 21% were found in patients with late onset disease and approximately 37% were found in manifesting heterozygous females, most of which are presumed to confer a neonatal phenotype in hemizygous males. Also included are residual enzyme activities and residual incorporation of ammonium nitrogen into urea and results of expression studies for a small proportion of the mutations. Most mutations in the OTC gene are "private" and are distributed throughout the gene with paucity of mutation in the sequence encoding the leader peptide (exon 1 and beginning of exon 2) and in exon 7. Almost all mutations in consensus splicing sites confer a neonatal phenotype. Thirteen polymorphisms have been found, several of which are useful for allele tracking in patients in whom the mutation can't be found. Even with sequencing of the entire reading frame and exon/intron boundaries, only about 80% of the mutations are detected in patients with proven OTC deficiency. The remaining probably occur within the introns or in regulatory domains.