Adam G. Rokita

Differential Cardiac Remodeling in Preload Versus Afterload

Background— Hemodynamic load regulates myocardial function and gene expression. We tested the hypothesis that afterload and preload, despite similar average load, result in different phenotypes. Methods and Results— Afterload and preload were compared in mice with transverse aortic constriction (TAC) and aortocaval shunt (shunt). Compared with sham mice, 6 hours after surgery, systolic wall stress (afterload) was increased in TAC mice (+40%; P<0.05), diastolic wall stress (preload) was increased in shunt (+277%; P<0.05) and TAC mice (+74%; P<0.05), and mean total wall stress was similarly increased in TAC (69%) and shunt mice (67%) (P=NS, TAC versus shunt; each P<0.05 versus sham). At 1 week, left ventricular weight/tibia length was significantly increased by 22% in TAC and 29% in shunt mice (P=NS, TAC versus shunt). After 24 hours and 1 week, calcium/calmodulin-dependent protein kinase II signaling was increased in TAC. This resulted in altered calcium cycling, including increased L-type calcium current, calcium transients, fractional sarcoplasmic reticulum calcium release, and calcium spark frequency. In shunt mice, Akt phosphorylation was increased. TAC was associated with inflammation, fibrosis, and cardiomyocyte apoptosis. The latter was significantly reduced in calcium/calmodulin-dependent protein kinase IIΔ-knockout TAC mice. A total of 157 mRNAs and 13 microRNAs were differentially regulated in TAC versus shunt mice. After 8 weeks, fractional shortening was lower and mortality was higher in TAC versus shunt mice. Conclusions— Afterload results in maladaptive fibrotic hypertrophy with calcium/calmodulin-dependent protein kinase II–dependent altered calcium cycling and apoptosis. Preload is associated with Akt activation without fibrosis, little apoptosis, better function, and lower mortality. This indicates that different loads result in distinct phenotype differences that may require specific pharmacological interventions.

Conditional Neuronal Nitric Oxide Synthase Overexpression Impairs Myocardial Contractility

The role of the neuronal NO synthase (nNOS or NOS1) enzyme in the control of cardiac function still remains unclear. Results from nNOS−/− mice or from pharmacological inhibition of nNOS are contradictory and do not pay tribute to the fact that probably spatial confinement of the nNOS enzyme is of major importance. We hypothesize that the close proximity of nNOS and certain effector molecules like L-type Ca2+-channels has an impact on myocardial contractility. To test this, we generated a new transgenic mouse model allowing conditional, myocardial specific nNOS overexpression. Western blot analysis of transgenic nNOS overexpression showed a 6-fold increase in nNOS protein expression compared with noninduced littermates (n=12; P<0.01). Measuring of total NOS activity by conversion of [3H]-l-arginine to [3H]-l-citrulline showed a 30% increase in nNOS overexpressing mice (n=18; P<0.05). After a 2 week induction, nNOS overexpression mice showed reduced myocardial contractility. In vivo examinations of the nNOS overexpressing mice revealed a 17±3% decrease of +dp/dtmax compared with noninduced mice (P<0.05). Likewise, ejection fraction was reduced significantly (42% versus 65%; n=15; P<0.05). Interestingly, coimmunoprecipitation experiments indicated interaction of nNOS with SR Ca2+ATPase and additionally with L-type Ca2+- channels in nNOS overexpressing animals. Accordingly, in adult isolated cardiac myocytes, ICa,L density was significantly decreased in the nNOS overexpressing cells. Intracellular Ca2+-transients and fractional shortening in cardiomyocytes were also clearly impaired in nNOS overexpressing mice versus noninduced littermates. In conclusion, conditional myocardial specific overexpression of nNOS in a transgenic animal model reduced myocardial contractility. We suggest that nNOS might suppress the function of L-type Ca2+-channels and in turn reduces Ca2+-transients which accounts for the negative inotropic effect.

Overexpression of CaMKIIδc in RyR2R4496C+/- knock-in mice leads to altered intracellular Ca2+ handling and increased mortality.

OBJECTIVES We investigated whether increased Ca(2+)/calmodulin-dependent kinase II (CaMKII) activity aggravates defective excitation-contraction coupling and proarrhythmic activity in mice expressing R4496C mutated cardiac ryanodine receptors (RyR2). BACKGROUND RyR2 dysfunction is associated with arrhythmic events in inherited and acquired cardiac disease. METHODS CaMKIIδc transgenic mice were crossbred with RyR2(R4496C+/-) knock-in mice. RESULTS Heart weight-to-body weight ratio in CaMKIIδc/RyR2(R4496C) and CaMKIIδc mice was similarly increased approximately 3-fold versus wild-type mice (p < 0.05). Echocardiographic data showed comparable cardiac dilation and impaired contractility in CaMKIIδc/RyR2(R4496C) and CaMKIIδc mice. Sarcoplasmic reticulum Ca(2+) content in isolated myocytes was decreased to a similar extent in CaMKIIδc/RyR2(R4496C) and CaMKIIδc mice. However, relaxation parameters and Ca(2+) decay at 1 Hz were prolonged significantly in CaMKIIδc mice versus CaMKIIδc/RyR2(R4496C) mice. Sarcoplasmic reticulum Ca(2+) spark frequency and characteristics indicated increased sarcoplasmic reticulum Ca(2+) leak in CaMKIIδc/RyR2(R4496C) versus CaMKIIδc myocytes (p < 0.05), most likely because of increased RyR2 phosphorylation. Delayed afterdepolarizations were significantly more frequent with increased amplitudes in CaMKIIδc/RyR2(R4496C) versus CaMKIIδc mice. Increased arrhythmias in vivo (67% vs. 25%; p < 0.05) may explain the increased mortality in CaMKIIδc/RyR2(R4496C) mice, which died prematurely with only 30% alive (vs. 60% for CaMKIIδc, p < 0.05) after 14 weeks. CONCLUSIONS CaMKIIδc overexpression in RyR2(R4496C+/-) knock-in mice increases the propensity toward triggered arrhythmias, which may impair survival. CaMKII contributes to further destabilization of a mutated RyR2 receptor.

Impaired Ca2+-handling in HIF-1α+/− mice as a consequence of pressure overload

The hypoxia-inducible factor (HIF)-1 is critically involved in the cellular adaptation to a decrease in oxygen availability. The influence of HIF-1α for the development of cardiac hypertrophy and cardiac function that occurs in response to sustained pressure overload has been mainly attributed to a challenged cardiac angiogenesis and cardiac hypertrophy up to now. Hif-1α+/+ and Hif-1α+/− mice were studied regarding left ventricular hypertrophy and cardiac function after being subjected to transverse aortic constriction (TAC). After TAC, both Hif-1α+/+ and Hif-1α+/− mice developed left ventricular hypertrophy with increased posterior wall thickness, septum thickness and increased left ventricular weight to a similar extent. No significant difference in cardiac vessel density was observed between Hif-1α+/+ and Hif-1α+/− mice. However, only the Hif-1α+/− mice developed severe heart failure as revealed by a significantly reduced fractional shortening mostly due to increased end-systolic left ventricular diameter. On the single cell level this correlated with reduced myocyte shortenings, decreased intracellular Ca2+-transients and SR-Ca2+ content in myocytes of Hif-1a+/− mice. Thus, HIF-1α can be critically involved in the preservation of cardiac function after chronic pressure overload without affecting cardiac hypertrophy. This effect is mediated via HIF-dependent modulation of cardiac calcium handling and contractility.

Corrigendum: The mitochondrial uniporter controls fight or flight heart rate increases

Nature Communications 6: Article number: 6081 (2015); Published 20 January 2015; Updated 3 June 2015. In Fig. 4c of this Article, the y axis values were wrongly given as 0, 20, 40, 60 and 80 in the middle and the lower panels. The correct values should read 0, 10, 20, 30 and 40 for the middle and 0,1, 2, 3 and 4 for the lower panels.We present HI observations of the Sculptor Group starburst spiral galaxy NGC 253, obtained with the Karoo Array Telescope (KAT-7). KAT-7 is a pathfinder for the SKA precursor MeerKAT, under construction. The short baselines and low system temperature of the telescope make it very sensitive to large scale, low surface brightness emission. The KAT-7 observations detected 33% more flux than previous VLA observations, mainly in the outer parts and in the halo for a total HI mass of 2.1 ± 0.1 ×10 M . HI can be found at large distances perpendicular to the plane out to projected distances of ∼9-10 kpc away from the nucleus and ∼13-14 kpc at the edge of the disk. A novel technique, based on interactive profile fitting, was used to separate the main disk gas from the anomalous (halo) gas. The rotation curve (RC) derived for the HI disk confirms that it is declining in the outer parts, as seen in previous optical Fabry-Perot measurements. As for the anomalous component, its RC has a very shallow gradient in the inner parts and turns over at the same radius as the disk, kinematically lagging by ∼100 km/sec. The kinematics of the observed extra planar gas is compatible with an outflow due to the central starburst and galactic fountains in the outer parts. However, the gas kinematics shows no evidence for inflow. Analysis of the near-IR WISE data, shows clearly that the star formation rate (SFR) is compatible with the starburst nature of NGC 253.

Erratum: Corrigendum: The mitochondrial uniporter controls fight or flight heart rate increases

Nature Communications 6: Article number: 6081 (2015); Published 20 January 2015; Updated 3 June 2015. In Fig. 4c of this Article, the y axis values were wrongly given as 0, 20, 40, 60 and 80 in the middle and the lower panels. The correct values should read 0, 10, 20, 30 and 40 for the middle and 0,1, 2, 3 and 4 for the lower panels.We present HI observations of the Sculptor Group starburst spiral galaxy NGC 253, obtained with the Karoo Array Telescope (KAT-7). KAT-7 is a pathfinder for the SKA precursor MeerKAT, under construction. The short baselines and low system temperature of the telescope make it very sensitive to large scale, low surface brightness emission. The KAT-7 observations detected 33% more flux than previous VLA observations, mainly in the outer parts and in the halo for a total HI mass of 2.1 ± 0.1 ×10 M . HI can be found at large distances perpendicular to the plane out to projected distances of ∼9-10 kpc away from the nucleus and ∼13-14 kpc at the edge of the disk. A novel technique, based on interactive profile fitting, was used to separate the main disk gas from the anomalous (halo) gas. The rotation curve (RC) derived for the HI disk confirms that it is declining in the outer parts, as seen in previous optical Fabry-Perot measurements. As for the anomalous component, its RC has a very shallow gradient in the inner parts and turns over at the same radius as the disk, kinematically lagging by ∼100 km/sec. The kinematics of the observed extra planar gas is compatible with an outflow due to the central starburst and galactic fountains in the outer parts. However, the gas kinematics shows no evidence for inflow. Analysis of the near-IR WISE data, shows clearly that the star formation rate (SFR) is compatible with the starburst nature of NGC 253.

Diabetes increases mortality after myocardial infarction by oxidizingCaMKII

Diabetes increases oxidant stress and doubles the risk of dying after myocardial infarction, but the mechanisms underlying increased mortality are unknown. Mice with streptozotocin-induced diabetes developed profound heart rate slowing and doubled mortality compared with controls after myocardial infarction. Oxidized Ca2+/calmodulin-dependent protein kinase II (ox-CaMKII) was significantly increased in pacemaker tissues from diabetic patients compared with that in nondiabetic patients after myocardial infarction. Streptozotocin-treated mice had increased pacemaker cell ox-CaMKII and apoptosis, which were further enhanced by myocardial infarction. We developed a knockin mouse model of oxidation-resistant CaMKIIδ (MM-VV), the isoform associated with cardiovascular disease. Streptozotocin-treated MM-VV mice and WT mice infused with MitoTEMPO, a mitochondrial targeted antioxidant, expressed significantly less ox-CaMKII, exhibited increased pacemaker cell survival, maintained normal heart rates, and were resistant to diabetes-attributable mortality after myocardial infarction. Our findings suggest that activation of a mitochondrial/ox-CaMKII pathway contributes to increased sudden death in diabetic patients after myocardial infarction.

Erratum: Diabetes increases mortality after myocardial infarction by oxidizing CaMKII (Journal of Clinical Investigation (2013) 123:5 (2333) 10.1172/JCI70180)

Diabetes increases oxidant stress and doubles the risk of dying after myocardial infarction, but the mechanisms underlying increased mortality are unknown. Mice with streptozotocin-induced diabetes developed profound heart rate slowing and doubled mortality compared with controls after myocardial infarction. Oxidized Ca2+/calmodulin-dependent protein kinase II (ox-CaMKII) was significantly increased in pacemaker tissues from diabetic patients compared with that in nondiabetic patients after myocardial infarction. Streptozotocin-treated mice had increased pacemaker cell ox-CaMKII and apoptosis, which were further enhanced by myocardial infarction. We developed a knockin mouse model of oxidation-resistant CaMKIIδ (MM-VV), the isoform associated with cardiovascular disease. Streptozotocin-treated MM-VV mice and WT mice infused with MitoTEMPO, a mitochondrial targeted antioxidant, expressed significantly less ox-CaMKII, exhibited increased pacemaker cell survival, maintained normal heart rates, and were resistant to diabetes-attributable mortality after myocardial infarction. Our findings suggest that activation of a mitochondrial/ox-CaMKII pathway contributes to increased sudden death in diabetic patients after myocardial infarction.

New Therapeutic Targets in Cardiology: Arrhythmias and CaMKII

Cardiac arrhythmias are a major epidemiological and public health problem and contribute significantly to sudden cardiac death, heart failure, stroke, suffering, debilitation, and healthcare expenses. In the United States alone, sudden cardiac death is estimated to kill 250 000 to 400 000 people annually.1 Most sudden death is due to cardiac arrhythmias,2 with ventricular tachycardia and fibrillation as the most commonly (≈80%) recorded rhythms in out-of-hospital cardiac arrests.3 In patients with structural heart disease, mostly resulting from a history of myocardial infarction, arrhythmias are the main cause of death.4 Atrial fibrillation (AF) and sinus node dysfunction (SND) are the most common sustained arrhythmias. AF affects ≈2.3 million patients in the United States,5 and because the prevalence of AF increases with age, it is predicted to increase by 2.5-fold by 2050.6 Patients with AF have approximately twice the mortality rate of patients in sinus rhythm,6 and the incidence of stroke is increased by 2- to 7-fold.7 AF is a costly disease and causes a public health burden estimated at

Response to Letter Regarding Article, “Differential Cardiac Remodeling in Preload Versus Afterload”

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