Keita Uchida

Distinct macrophage lineages contribute to disparate patterns of cardiac recovery and remodeling in the neonatal and adult heart

Significance This study addresses a fundamentally important and widely debated issue in the field of inflammation, which is why inflammation can be simultaneously deleterious after injury and yet is essential for tissue repair. Recently, an important new paradigm has emerged in the macrophage field: Organs are replete with resident macrophages of embryonic origin, distinct from monocyte-derived macrophages. In this article, we use a new model of cardiac injury and show that distinct macrophage populations derived from embryonic and adult lineages are important determinants of tissue repair and inflammation, respectively. Our data suggest that therapeutics, which inhibit monocyte-derived macrophages and/or selectively harness the function of embryonic-derived macrophages, may serve as novel treatments for heart failure. The mechanistic basis for why inflammation is simultaneously both deleterious and essential for tissue repair is not fully understood. Recently, a new paradigm has emerged: Organs are replete with resident macrophages of embryonic origin distinct from monocyte-derived macrophages. This added complexity raises the question of whether distinct immune cells drive inflammatory and reparative activities after injury. Previous work has demonstrated that the neonatal heart has a remarkable capacity for tissue repair compared with the adult heart, offering an ideal context to examine these concepts. We hypothesized that unrecognized differences in macrophage composition is a key determinant of cardiac tissue repair. Using a genetic model of cardiomyocyte ablation, we demonstrated that neonatal mice expand a population of embryonic-derived resident cardiac macrophages, which generate minimal inflammation and promote cardiac recovery through cardiomyocyte proliferation and angiogenesis. During homeostasis, the adult heart contains embryonic-derived macrophages with similar properties. However, after injury, these cells were replaced by monocyte-derived macrophages that are proinflammatory and lacked reparative activities. Inhibition of monocyte recruitment to the adult heart preserved embryonic-derived macrophage subsets, reduced inflammation, and enhanced tissue repair. These findings indicate that embryonic-derived macrophages are key mediators of cardiac recovery and suggest that therapeutics targeting distinct macrophage lineages may serve as novel treatments for heart failure.

Hypotension Due to Kir6.1 Gain‐of‐Function in Vascular Smooth Muscle

Background KATP channels, assembled from pore‐forming (Kir6.1 or Kir6.2) and regulatory (SUR1 or SUR2) subunits, link metabolism to excitability. Loss of Kir6.2 results in hypoglycemia and hyperinsulinemia, whereas loss of Kir6.1 causes Prinzmetal angina–like symptoms in mice. Conversely, overactivity of Kir6.2 induces neonatal diabetes in mice and humans, but consequences of Kir6.1 overactivity are unknown. Methods and Results We generated transgenic mice expressing wild‐type (WT), ATP‐insensitive Kir6.1 [Gly343Asp] (GD), and ATP‐insensitive Kir6.1 [Gly343Asp,Gln53Arg] (GD‐QR) subunits, under Cre‐recombinase control. Expression was induced in smooth muscle cells by crossing with smooth muscle myosin heavy chain promoter–driven tamoxifen‐inducible Cre‐recombinase (SMMHC‐Cre‐ER) mice. Three weeks after tamoxifen induction, we assessed blood pressure in anesthetized and conscious animals, as well as contractility of mesenteric artery smooth muscle and KATP currents in isolated mesenteric artery myocytes. Both systolic and diastolic blood pressures were significantly reduced in GD and GD‐QR mice but normal in mice expressing the WT transgene and elevated in Kir6.1 knockout mice as well as in mice expressing dominant‐negative Kir6.1 [AAA] in smooth muscle. Contractile response of isolated GD‐QR mesenteric arteries was blunted relative to WT controls, but nitroprusside relaxation was unaffected. Basal KATP conductance and pinacidil‐activated conductance were elevated in GD but not in WT myocytes. Conclusions KATP overactivity in vascular muscle can lead directly to reduced vascular contractility and lower blood pressure. We predict that gain of vascular KATP function in humans would lead to a chronic vasodilatory phenotype, as indeed has recently been demonstrated in Cantu syndrome.

Tuning the electrical properties of the heart by differential trafficking of KATP ion channel complexes

ABSTRACT The copy number of membrane proteins at the cell surface is tightly regulated. Many ion channels and receptors present retrieval motifs to COPI vesicle coats and are retained in the early secretory pathway. In some cases, the interaction with COPI is prevented by binding to 14-3-3 proteins. However, the functional significance of this antagonism between COPI and 14-3-3 in terminally differentiated cells is unknown. Here, we show that ATP-sensitive K+ (KATP) channels, which are composed of Kir6.2 and SUR1 subunits, are stalled in the Golgi complex of ventricular, but not atrial, cardiomyocytes. Upon sustained &bgr;-adrenergic stimulation, which leads to activation of protein kinase A (PKA), SUR1-containing channels reach the plasma membrane of ventricular cells. We show that PKA-dependent phosphorylation of the C-terminus of Kir6.2 decreases binding to COPI and, thereby, silences the arginine-based retrieval signal. Thus, activation of the sympathetic nervous system releases this population of KATP channels from storage in the Golgi and, hence, might facilitate the adaptive response to metabolic challenges.

Fibroblast Growth Factor Receptor 1 Signaling in Adult Cardiomyocytes Increases Contractility and Results in a Hypertrophic Cardiomyopathy

Fibroblast growth factors (FGFs) and their receptors are highly conserved signaling molecules that have been implicated in postnatal cardiac remodeling. However, it is not known whether cardiomyocyte-expressed FGF receptors are necessary or sufficient for ventricular remodeling in the adult heart. To determine whether cardiomyocytes were competent to respond to an activated FGF receptor, and to determine if this signal would result in the development of hypertrophy, we engineered a doxycycline (DOX)-inducible, cardiomyocyte-specific, constitutively active FGF receptor mouse model (αMHC-rtTA, TRE-caFgfr1-myc). Echocardiographic and hemodynamic analysis indicated that acute expression of caFGFR1 rapidly and directly increased cardiac contractility, while chronic expression resulted in significant hypertrophy with preservation of systolic function. Subsequent histologic analysis showed increased cardiomyocyte cross-sectional area and regions of myocyte disarray and fibrosis, classic features of hypertrophic cardiomyopathy (HCM). Analysis of downstream pathways revealed a lack of clear activation of classical FGF-mediated signaling pathways, but did demonstrate a reduction in Serca2 expression and troponin I phosphorylation. Isolated ventricular myocytes showed enhanced contractility and reduced relaxation, an effect that was partially reversed by inhibition of actin-myosin interactions. We conclude that adult cardiomyocytes are competent to transduce FGF signaling and that FGF signaling is sufficient to promote increased cardiomyocyte contractility in vitro and in vivo through enhanced intrinsic actin-myosin interactions. Long-term, FGFR overexpression results in HCM with a dynamic outflow tract obstruction, and may serve as a unique model of HCM.

KATP channel gain-of-function leads to increased myocardial L-type Ca2+ current and contractility in Cantu syndrome

Significance ATP-sensitive potassium (KATP) channels are present in cardiac and smooth muscle; when activated, they relax blood vessels and decrease cardiac action potential duration, reducing cardiac contractility. Cantu syndrome (CS) is caused by mutations in KATP genes that result in overactive channels. Contrary to prediction, we show that the myocardium in both CS patients and in animal models with overactive KATP channels is hypercontractile. We also show that this results from a compensatory increase in calcium channel activity, paralleled by specific alterations in phosphorylation of the calcium channel itself. These findings have implications for the way the heart compensates for decreased excitability and volume load in general and for the basis of, and potential therapies for, CS specifically. Cantu syndrome (CS) is caused by gain-of-function (GOF) mutations in genes encoding pore-forming (Kir6.1, KCNJ8) and accessory (SUR2, ABCC9) KATP channel subunits. We show that patients with CS, as well as mice with constitutive (cGOF) or tamoxifen-induced (icGOF) cardiac-specific Kir6.1 GOF subunit expression, have enlarged hearts, with increased ejection fraction and increased contractility. Whole-cell voltage-clamp recordings from cGOF or icGOF ventricular myocytes (VM) show increased basal L-type Ca2+ current (LTCC), comparable to that seen in WT VM treated with isoproterenol. Mice with vascular-specific expression (vGOF) show left ventricular dilation as well as less-markedly increased LTCC. Increased LTCC in KATP GOF models is paralleled by changes in phosphorylation of the pore-forming α1 subunit of the cardiac voltage-gated calcium channel Cav1.2 at Ser1928, suggesting enhanced protein kinase activity as a potential link between increased KATP current and CS cardiac pathophysiology.

Electrophysiologic consequences of KATP gain of function in the heart: Conduction abnormalities in Cantu syndrome

BACKGROUND Gain-of-function (GOF) mutations in the KATP channel subunits Kir6.1 and SUR2 cause Cantu syndrome (CS), a disease characterized by multiple cardiovascular abnormalities. OBJECTIVE The purpose of this study was to better determine the electrophysiologic consequences of such GOF mutations in the heart. METHODS We generated transgenic mice (Kir6.1-GOF) expressing ATP-insensitive Kir6.1[G343D] subunits under α-myosin heavy chain (α-MHC) promoter control, to target gene expression specifically in cardiomyocytes, and performed patch-clamp experiments on isolated ventricular myocytes and invasive electrophysiology on anesthetized mice. RESULTS In Kir6.1-GOF ventricular myocytes, KATP channels showed decreased ATP sensitivity but no significant change in current density. Ambulatory ECG recordings on Kir6.1-GOF mice revealed AV nodal conduction abnormalities and junctional rhythm. Invasive electrophysiologic analyses revealed slowing of conduction and conduction failure through the AV node but no increase in susceptibility to atrial or ventricular ectopic activity. Surface ECGs recorded from CS patients also demonstrated first-degree AV block and fascicular block. CONCLUSION The primary electrophysiologic consequence of cardiac KATP GOF is on the conduction system, particularly the AV node, resulting in conduction abnormalities in CS patients who carry KATP GOF mutations.

Functional roles of KATP channel subunits in metabolic inhibition

ATP-sensitive potassium channel (KATP) activation can drastically shorten action potential duration (APD) in metabolically compromised myocytes. We showed previously that SUR1 with Kir6.2 forms the functional channel in mouse atria while Kir6.2 and SUR2A predominate in ventricles. SUR1 is more sensitive to metabolic stress than SUR2A, raising the possibility that KATP in atria and ventricles may respond differently to metabolic stress. Action potential duration (APD) and calcium transient duration (CaTD) were measured simultaneously in both atria and ventricles by optical mapping of the posterior surface of Langendorff-perfused hearts from C57BL wild-type (WT; n=11), Kir6.2(-/-) (n=5), and SUR1(-/-) (n=6) mice during metabolic inhibition (MI, 0mM glucose+2mM sodium cyanide). After variable delay, MI led to significant shortening of APD in WT hearts. On average, atrial APD shortened by 60.5 ± 2.7% at 13.1 ± 2.1 min (n=6, p<0.01) after onset of MI. Ventricular APD shortening (56.4 ± 10.0% shortening at 18.2 ± 1.8 min) followed atrial APD shortening. In SUR1(-/-) hearts (n=6), atrial APD shortening was abolished, but ventricular shortening (65.0 ± 15.4% at 25.33 ± 4.48 min, p<0.01) was unaffected. In Kir6.2(-/-) hearts, two disparate responses to MI were observed; 3 of 5 hearts displayed slight shortening of APD in the ventricles (24 ± 3%, p<0.05) and atria (39.0 ± 1.9%, p<0.05) but this shortening occurred later and to much less extent than in WT (p<0.05). Marked prolongation of ventricular APD was observed in the remaining hearts (327% and 489% prolongation) and was associated with occurrence of ventricular tachyarrhythmias. The results confirm that Kir6.2 contributes to APD shortening in both atria and ventricle during metabolic stress, and that SUR1 is required for atrial APD shortening while SUR2A is required for ventricular APD shortening. Importantly, the results show that the presence of SUR1-dependent KATP in the atria results in the action potential being more susceptible to metabolically driven shortening than the ventricle.

DMSO Protects against Stress-Induced Sealing of Cardiac T-Tubules

Cardiac t-tubules are critical for efficient excitation-contraction coupling and undergo significant remodeling during various experimental and clinical stress conditions including heart failure. Recently, we have shown that recovery of ventricular myocytes after a brief hyposmotic shock is associated with significant sealing of t-tubules. Dimethyl Sulfoxide (DMSO) is a commonly used solvent and has been reported to have cardioprotective properties although the mechanisms underlying these protective effects remain largely unknown. Therefore, we tested whether the cardioprotective effects of DMSO are mediated by its action on t-tubular remodeling. We found that application of DMSO at the time of resolution of hyposmotic stress, when t-tubule sealing occurs, reduced the amount of fluorescent dextran trapped within sealed t-tubules. The effect of DMSO displayed sharp biphasic concentration dependence with 1% being the most effective dose (∼4-fold reduction of dextran trapping; p<0.01) while 10% DMSO was ineffective in preventing t-tubule sealing. The data were corroborated by measuring IK1 tail current which reflects the amount of ttubular membrane. IK1 tail was recorded in cardiomyocytes that were previously detubulated using the hyposmotic stress protocol. Consistent with reduced dextran trapping in the presence of 1% DMSO, normalized IK1 tail was significantly larger in cardiomyocytes that had undergone detubulation with 1% DMSO (19.8 ± 2.4 % vs 7.9 ± 2.4 %, p<0.01). Image analysis of cardiomyocyte dimensions during the osmotic stress protocol did not reveal any significant differences in cells treated with 1% DMSO upon resolution of hyposmotic stress. In particular, cell dimensions returned fully to pre-stress values in both control and DMSO groups. The data suggest that DMSO prevents t-tubule remodeling independent of its osmotic effects.

Sulfonylurea Receptor Subunit Composition of KATP Channels in Dog and Human Hearts

ATP sensitive potassium (KATP) channels are assembled by four pore-forming inward rectifier subunits (Kir6.1 or Kir6.2), each associated with one sulfonylurea subunit (SUR1 or SUR2). The differential subunit composition defines distinct KATP channel properties and tissue-specific function. Although the consensus has been that cardiac KATP channels are SUR2A-based channels, our recent studies indicate that mouse cardiac KATP channels are chamber-specific: SUR1-based channels in atria vs. SUR2A-based channels in ventricles. To test whether chamber specificity is an universal property of cardiac KATP channels in other species, whole-cell and inside-out excised patch-clamp techniques were applied to examine the effects of SUR- specific channel openers (pinacidil acts on SUR2A and diazoxide on SUR1) on isolated dog and human cardiomyocytes. In dog atria, pinacidil- and diazoxide-induced whole-cell currents were 40 ± 4 pA/pF (n = 14) and 13 ± 6 pA/pF (n = 11, p 0.05), respectively; while the ventricular currents were 117 ± 75 pA (n = 3) and 92 (n = 1), respectively. The results indicate that SUR2A-based channels are predominant in dog heart, while abundant SUR1- and SUR2A-based channels co-exist in human heart, in both atria and ventricles. Therefore, there is marked variability between all three species studied, which should be considered carefully when using either mice or dogs to model the effects of human channels in either normal or diseased human hearts.