The Use of Bumper Wire Technique and Intravascular Ultrasound for Precise Aorto-Ostial Stenting.

Background: Aorto-ostial interventions are challenging due to the limitations of contemporary equipment, imprecise ostial demarcation, and problematic ostial lesion characteristics. Suboptimal stent placement is common and portends worse clinical outcomes. Procedural and long-term outcomes of the bumper wire technique with intravascular ultrasound (IVUS) assessment have not been investigated. Methods: A single-center retrospective study was conducted. Patients who underwent ostial lesion percutaneous coronary intervention (PCI) with the bumper wire technique between January 2019 and September 2020 were identified. The primary endpoint was to determine the geographic miss rate defined by inadequate ostial coverage or excess stent protrusion of > 2 mm by IVUS or angiography. The secondary endpoint was target lesion failure (TLF) at 6 months after PCI, defined as the composite of cardiovascular death, target vessel myocardial infarction (MI), and target lesion revascularization. Results: In total, 45 patients were identified. The average age was 71.7 years old, and 85.4% were men. Indication for PCI was acute coronary syndrome in about a third of patients. Twenty-six patients had left main ostial lesions and 19 patients had right coronary artery ostial lesions. Geographic miss was detected in two patients (4.4%): one patient (2.2%) had excess proximal stent protrusion and one patient (2.2%) had an ostial miss. Six patients were lost to follow-up. TLF, stroke, or major bleeding were not observed in any of the patients. Conclusion: The bumper wire technique is safe and efficient with low rates of geographic miss or adverse clinical outcomes. This is the first study to confirm precise aorto-ostial stent implantation with the bumper wire technique using IVUS confirmation.

Altered Expression of Zonula occludens-1 Affects Cardiac Na+ Channels and Increases Susceptibility to Ventricular Arrhythmias.

Zonula occludens-1 (ZO-1) is an intracellular scaffolding protein that orchestrates the anchoring of membrane proteins to the cytoskeleton in epithelial and specialized tissue including the heart. There is clear evidence to support the central role of intracellular auxiliary proteins in arrhythmogenesis and previous studies have found altered ZO-1 expression associated with atrioventricular conduction abnormalities. Here, using human cardiac tissues, we identified all three isoforms of ZO-1, canonical (Transcript Variant 1, TV1), CRA_e (Transcript Variant 4, TV4), and an additionally expressed (Transcript Variant 3, TV3) in non-failing myocardium. To investigate the role of ZO-1 on ventricular arrhythmogenesis, we generated a haploinsufficient ZO-1 mouse model (ZO-1+/-). ZO-1+/- mice exhibited dysregulated connexin-43 protein expression and localization at the intercalated disc. While ZO-1+/- mice did not display abnormal cardiac function at baseline, adrenergic challenge resulted in rhythm abnormalities, including premature ventricular contractions and bigeminy. At baseline, ventricular myocytes from the ZO-1+/- mice displayed prolonged action potential duration and spontaneous depolarizations, with ZO-1+/- cells displaying frequent unsolicited (non-paced) diastolic depolarizations leading to spontaneous activity with multiple early afterdepolarizations (EADs). Mechanistically, ZO-1 deficient myocytes displayed a reduction in sodium current density (INa) and an increased sensitivity to isoproterenol stimulation. Further, ZO-1 deficient myocytes displayed remodeling in ICa current, likely a compensatory change. Taken together, our data suggest that ZO-1 deficiency results in myocardial substrate susceptible to triggered arrhythmias.

Calmodulin kinase II regulates atrial myocyte late sodium current, calcium handling, and atrial arrhythmia.

BACKGROUND: Atrial fibrillation (AF) is the most common type of arrhythmia. Abnormal atrial myocyte Ca2+ handling promotes aberrant membrane excitability and remodeling that are important for atrial arrhythmogenesis. The sequence of molecular events leading to loss of normal atrial myocyte Ca2+ homeostasis is not established. Late Na+ current (INa,L) is increased in atrial myocytes from AF patients together with an increase in activity of Ca2+/calmodulin-dependent kinase II (CaMKII). OBJECTIVE: The purpose of this study was to determine whether CaMKII-dependent phosphorylation at Ser571 on NaV1.5 increases atrial INa,L, leading to aberrant atrial Ca2+ cycling, altered electrophysiology, and increased AF risk. METHODS: Atrial myocyte electrophysiology, Ca2+ handling, and arrhythmia susceptibility were studied in wild-type and Scn5a knock-in mice expressing phosphomimetic (S571E) or phosphoresistant (S571A) NaV1.5 at Ser571. RESULTS: Atrial myocytes from S571E but not S571A mice displayed an increase in INa,L and action potential duration, and with adrenergic stress have increased delayed afterdepolarizations. Frequency of Ca2+ sparks and waves was increased in S571E atrial myocytes compared to wild type. S571E mice showed an increase in atrial events induced by adrenergic stress and AF inducibility in vivo. Isolated S571E atria were more susceptible to spontaneous atrial events, which were abrogated by inhibiting sarcoplasmic reticulum Ca2+ release, CaMKII, or the Na+/Ca2+ exchanger. Expression of phospho-NaV1.5 at Ser571 and autophosphorylated CaMKII were increased in atrial samples from human AF patients. CONCLUSION: This study identified CaMKII-dependent regulation of NaV1.5 as an important upstream event in Ca2+ handling defects and abnormal impulse generation in the setting of AF.

Defining new mechanistic roles for αII spectrin in cardiac function.

Spectrins are cytoskeletal proteins essential for membrane biogenesis and regulation and serve critical roles in protein targeting and cellular signaling. αII spectrin (SPTAN1) is one of two α spectrin genes and αII spectrin dysfunction is linked to alterations in axon initial segment formation, cortical lamination, and neuronal excitability. Furthermore, human αII spectrin loss-of-function variants cause neurological disease. As global αII spectrin knockout mice are embryonic lethal, the in vivo roles of αII spectrin in adult heart are unknown and untested. Here, based on pronounced alterations in αII spectrin regulation in human heart failure we tested the in vivo roles of αII spectrin in the vertebrate heart. We created a mouse model of cardiomyocyte-selective αII spectrin-deficiency (cKO) and used this model to define the roles of αII spectrin in cardiac function. αII spectrin cKO mice displayed significant structural, cellular, and electrical phenotypes that resulted in accelerated structural remodeling, fibrosis, arrhythmia, and mortality in response to stress. At the molecular level, we demonstrate that αII spectrin plays a nodal role for global cardiac spectrin regulation, as αII spectrin cKO hearts exhibited remodeling of αI spectrin and altered ß-spectrin expression and localization. At the cellular level, αII spectrin deficiency resulted in altered expression, targeting, and regulation of cardiac ion channels NaV1.5 and KV4.3. In summary, our findings define critical and unexpected roles for the multifunctional αII spectrin protein in the heart. Furthermore, our work provides a new in vivo animal model to study the roles of αII spectrin in the cardiomyocyte.

Protein Phosphatase 2A Regulates Cardiac Na+ Channels.

RATIONALE: Voltage-gated Na+ channel ( INa) function is critical for normal cardiac excitability. However, the Na+ channel late component ( INa,L) is directly associated with potentially fatal forms of congenital and acquired human arrhythmia. CaMKII (Ca2+/calmodulin-dependent kinase II) enhances INa,L in response to increased adrenergic tone. However, the pathways that negatively regulate the CaMKII/Nav1.5 axis are unknown and essential for the design of new therapies to regulate the pathogenic INa,L. OBJECTIVE: To define phosphatase pathways that regulate INa,L in vivo. METHODS AND RESULTS: A mouse model lacking a key regulatory subunit (B56α) of the PP (protein phosphatase) 2A holoenzyme displayed aberrant action potentials after adrenergic stimulation. Unbiased computational modeling of B56α KO (knockout) mouse myocyte action potentials revealed an unexpected role of PP2A in INa,L regulation that was confirmed by direct INa,L recordings from B56α KO myocytes. Further, B56α KO myocytes display decreased sensitivity to isoproterenol-induced induction of arrhythmogenic INa,L, and reduced CaMKII-dependent phosphorylation of Nav1.5. At the molecular level, PP2A/B56α complex was found to localize and coimmunoprecipitate with the primary cardiac Nav channel, Nav1.5. CONCLUSIONS: PP2A regulates Nav1.5 activity in mouse cardiomyocytes. This regulation is critical for pathogenic Nav1.5 late current and requires PP2A-B56α. Our study supports B56α as a novel target for the treatment of arrhythmia.

Concerted regulation of skeletal muscle contractility by oxygen tension and endogenous nitric oxide.

It is generally accepted that inhibition of nitric oxide synthase (NOS) facilitates, and thus nitric oxide (NO) inhibits, contractility of skeletal muscle. However, standard assessments of contractility are carried out at a nonphysiological oxygen tension [partial pressure of oxygen (pO2)] that can interfere with NO signaling (95% O2). We therefore examined, in normal and neuronal NOS (nNOS)-deficient mice, the influence of pO2 on whole-muscle contractility and on myocyte calcium flux and sarcomere shortening. Here, we demonstrate a significant enhancement of these measures of muscle performance at low physiological pO2 and an inhibitory influence at higher physiological pO2, which depend on endogenous nNOS. At 95% O2 (which produces oxidative stress; muscle core pO2 approximately 400 mmHg), force production is enhanced but control of contractility by NO/nitrosylation is greatly attenuated. In addition, responsivity to pO2 is altered significantly in nNOS mutant muscle. These results reveal a fundamental role for the concerted action of NO and O2 in physiological regulation of skeletal muscle contractility, and suggest novel molecular aspects of myopathic disease. They suggest further that the role of NO in some cellular systems may require reexamination.

Nitric oxide regulation of myocardial contractility and calcium cycling: independent impact of neuronal and endothelial nitric oxide synthases.

The mechanisms by which nitric oxide (NO) influences myocardial Ca2+ cycling remain controversial. Because NO synthases (NOS) have specific spatial localization in cardiac myocytes, we hypothesized that neuronal NOS (NOS1) found in cardiac sarcoplasmic reticulum (SR) preferentially regulates SR Ca2+ release and reuptake resulting in potentiation of the cardiac force-frequency response (FFR). Transesophageal pacing (660 to 840 bpm) in intact C57Bl/6 mice (WT) stimulated both contractility (dP/dtmax normalized to end-diastolic volume; dP/dt-EDV) by 51+/-5% (P<0.001) and lusitropy (tau; tau) by 20.3+/-2.0% (P<0.05). These responses were markedly attenuated in mice lacking NOS1 (NOS1-/-) (15+/-2% increase in dP/dt-EDV; P<0.001 versus WT; and no change in tau; P<0.01 versus WT). Isolated myocytes from NOS1-/- (approximately 2 months of age) also exhibited suppressed frequency-dependent sarcomere shortening and Ca2+ transients ([Ca2+]i) compared with WT. SR Ca2+ stores, a primary determinant of the FFR, increased at higher frequencies in WT (caffeine-induced [Ca2+]i at 4 Hz increased 107+/-23% above 1 Hz response) but not in NOS1-/- (13+/-26%; P<0.01 versus WT). In contrast, mice lacking NOS3 (NOS3-/-) had preserved FFR in vivo, as well as in isolated myocytes with parallel increases in sarcomere shortening, [Ca2+]i, and SR Ca2+ stores. NOS1-/- had increased SR Ca2+ ATPase and decreased phospholamban protein abundance, suggesting compensatory increases in SR reuptake mechanisms. Together these data demonstrate that NOS1 selectively regulates the cardiac FFR via influences over SR Ca2+ cycling. Thus, there is NOS isoform-specific regulation of different facets of rate-dependent excitation-contraction coupling; inactivation of NOS1 has the potential to contribute to the pathophysiology of states characterized by diminished frequency-dependent inotropic responses.

Role of nitric oxide in the pathophysiology of heart failure.

Nitric oxide (NO) plays critical roles in the regulation of integrated cardiac and vascular function and homeostasis. An understanding of the physiologic role and relative contribution of the three NO synthase isoforms (neuronal--NOS1, inducible--NOS2, and endothelial--NOS3) is imperative to comprehend derangements of the NO signaling pathway in the failing cardiovascular system. Several theories of NO and its regulation have developed as explanations for the divergent observations from studies in health and disease states. Here we review the physiologic and pathophysiologic influence of NO on cardiac function, in a framework that considers several theories of altered NO signaling in heart failure. We discuss the notion of spatial compartmentalization of NO signaling within the myocyte in an effort to reconcile many controversies about derangements in the influences of NO in the heart and vasculature.

Nitric oxide regulates the heart by spatial confinement of nitric oxide synthase isoforms.

Subcellular localization of nitric oxide (NO) synthases with effector molecules is an important regulatory mechanism for NO signalling. In the heart, NO inhibits L-type Ca2+ channels but stimulates sarcoplasmic reticulum (SR) Ca2+ release, leading to variable effects on myocardial contractility. Here we show that spatial confinement of specific NO synthase isoforms regulates this process. Endothelial NO synthase (NOS3) localizes to caveolae, where compartmentalization with beta-adrenergic receptors and L-type Ca2+ channels allows NO to inhibit beta-adrenergic-induced inotropy. Neuronal NO synthase (NOS1), however, is targeted to cardiac SR. NO stimulation of SR Ca2+ release via the ryanodine receptor (RyR) in vitro, suggests that NOS1 has an opposite, facilitative effect on contractility. We demonstrate that NOS1-deficient mice have suppressed inotropic response, whereas NOS3-deficient mice have enhanced contractility, owing to corresponding changes in SR Ca2+ release. Both NOS1-/- and NOS3-/- mice develop age-related hypertrophy, although only NOS3-/- mice are hypertensive. NOS1/3-/- double knockout mice have suppressed beta-adrenergic responses and an additive phenotype of marked ventricular remodelling. Thus, NOS1 and NOS3 mediate independent, and in some cases opposite, effects on cardiac structure and function.

Imbalance between xanthine oxidase and nitric oxide synthase signaling pathways underlies mechanoenergetic uncoupling in the failing heart.

Inhibition of xanthine oxidase (XO) in failing hearts improves cardiac efficiency by an unknown mechanism. We hypothesized that this energetic effect is due to reduced oxidative stress and critically depends on nitric oxide synthase (NOS) activity, reflecting a balance between generation of nitric oxide (NO) and reactive oxygen species. In dogs with pacing-induced heart failure (HF), ascorbate (1000 mg) mimicked the beneficial energetic effects of allopurinol, increasing both contractility and efficiency, suggesting an antioxidant mechanism. Allopurinol had no additive effect beyond that of ascorbate. Crosstalk between XO and NOS signaling was assessed. NOS inhibition with N(G)-monomethyl-L-arginine (L-NMMA; 20 mg/kg) had no effect on basal contractility or efficiency in HF, but prevented the +26.2+/-3.5% and +66.5+/-17% enhancements of contractility and efficiency, respectively, observed with allopurinol alone. Similarly, improvements in contractility and energetics due to ascorbate were also inhibited by L-NMMA. Because of the observed NOS-XO crosstalk, we predicted that in normal hearts NOS inhibition would uncover a depression of energetics caused by XO activity. In normal conscious dogs, L-NMMA increased myocardial oxygen consumption (MVO2) while lowering left ventricular external work, reducing efficiency by 31.1+/-3.8% (P<0.005). Lowered efficiency was reversed by XO inhibition (allopurinol, 200 mg) or by ascorbate without affecting cardiac load or systemic hemodynamics. Single-cell immunofluorescence detected XO protein in cardiac myocytes that was enhanced in HF, consistent with autocrine signaling. These data show that both NOS and XO signaling systems participate in the regulation of myocardial mechanical efficiency and that upregulation of XO relative to NOS contributes to mechanoenergetic uncoupling in heart failure.