Kathy Zimmerman

A Dynamic Pathway for Calcium-Independent Activation of CaMKII by Methionine Oxidation

Calcium/calmodulin (Ca2+/CaM)-dependent protein kinase II (CaMKII) couples increases in cellular Ca2+ to fundamental responses in excitable cells. CaMKII was identified over 20 years ago by activation dependence on Ca2+/CaM, but recent evidence shows that CaMKII activity is also enhanced by pro-oxidant conditions. Here we show that oxidation of paired regulatory domain methionine residues sustains CaMKII activity in the absence of Ca2+/CaM. CaMKII is activated by angiotensin II (AngII)-induced oxidation, leading to apoptosis in cardiomyocytes both in vitro and in vivo. CaMKII oxidation is reversed by methionine sulfoxide reductase A (MsrA), and MsrA-/- mice show exaggerated CaMKII oxidation and myocardial apoptosis, impaired cardiac function, and increased mortality after myocardial infarction. Our data demonstrate a dynamic mechanism for CaMKII activation by oxidation and highlight the critical importance of oxidation-dependent CaMKII activation to AngII and ischemic myocardial apoptosis.

Cardiac Hypertrophy Is Not a Required Compensatory Response to Short-Term Pressure Overload

BACKGROUND Cardiac hypertrophy is considered a necessary compensatory response to sustained elevations of left ventricular (LV) wall stress. METHODS AND RESULTS To test this, we inhibited calcineurin with cyclosporine (CsA) in the setting of surgically induced pressure overload in mice and examined in vivo parameters of ventricular volume and function using echocardiography. Normalized heart mass increased 45% by 5 weeks after thoracic aortic banding (TAB; heart weight/body weight, 8.3+/-0.9 mg/g [mean+/-SEM] versus 5. 7+/-0.1 mg/g unbanded, P<0.05). Similar increases were documented in the cell-surface area of isolated LV myocytes. In mice subjected to TAB+CsA treatment, we observed complete inhibition of hypertrophy (heart weight/body weight, 5.2+/-0.3 mg/g at 5 weeks) and myocyte surface area (endocardial and epicardial fractions). The mice tolerated abolition of hypertrophy with no signs of cardiovascular compromise, and 5-week mortality was not different from that of banded mice injected with vehicle (TAB+Veh). Despite abolition of hypertrophy by CsA (LV mass by echo, 83+/-5 mg versus 83+/-2 mg unbanded), chamber size (end-diastolic volume, 33+/-6 microL versus 37+/-1 microL unbanded), and systolic ejection performance (ejection fraction, 97+/-2% versus 97+/-1% unbanded) were normal. LV mass differed significantly in TAB+Veh animals (103+/-5 mg, P<0.05), but chamber volume (end-diastolic volume, 44+/-6 microL), ejection fraction (92+/-2%), and transstenotic pressure gradients (70+/-14 mm Hg in TAB+Veh versus 77+/-11 mm Hg in TAB+CsA) were not different. CONCLUSIONS In this experimental setting, calcineurin blockade with CsA prevented LV hypertrophy due to pressure overload. TAB mice treated with CsA maintain normal LV size and systolic function.

T-Tubule Remodeling During Transition From Hypertrophy to Heart Failure

Rationale: The transverse tubule (T-tubule) system is the ultrastructural substrate for excitation–contraction coupling in ventricular myocytes; T-tubule disorganization and loss are linked to decreased contractility in end stage heart failure (HF). Objective: We sought to examine (1) whether pathological T-tubule remodeling occurs early in compensated hypertrophy and, if so, how it evolves during the transition from hypertrophy to HF; and (2) the role of junctophilin-2 in T-tubule remodeling. Methods and Results: We investigated T-tubule remodeling in relation to ventricular function during HF progression using state-of-the-art confocal imaging of T-tubules in intact hearts, using a thoracic aortic banding rat HF model. We developed a quantitative T-tubule power (TTpower) index to represent the integrity of T-tubule structure. We found that discrete local loss and global reorganization of the T-tubule system (leftward shift of TTpower histogram) started early in compensated hypertrophy in left ventricular (LV) myocytes, before LV dysfunction, as detected by echocardiography. With progression from compensated hypertrophy to early and late HF, T-tubule remodeling spread from the LV to the right ventricle, and TTpower histograms of both ventricles gradually shifted leftward. The mean LV TTpower showed a strong correlation with ejection fraction and heart weight to body weight ratio. Over the progression to HF, we observed a gradual reduction in the expression of a junctophilin protein (JP-2) implicated in the formation of T-tubule/sarcoplasmic reticulum junctions. Furthermore, we found that JP-2 knockdown by gene silencing reduced T-tubule structure integrity in cultured adult ventricular myocytes. Conclusions: T-tubule remodeling in response to thoracic aortic banding stress begins before echocardiographically detectable LV dysfunction and progresses over the development of overt structural heart disease. LV T-tubule remodeling is closely associated with the severity of cardiac hypertrophy and predicts LV function. Thus, T-tubule remodeling may constitute a key mechanism underlying the transition from compensated hypertrophy to HF.

Oxidation of CaMKII determines the cardiotoxic effects of aldosterone

Excessive activation of the β-adrenergic, angiotensin II (Ang II) and aldosterone signaling pathways promotes mortality after myocardial infarction, and antagonists targeting these pathways are core therapies for treating this condition. Catecholamines and Ang II activate the multifunctional Ca2+/calmodulin-dependent protein kinase II (CaMKII), the inhibition of which prevents isoproterenol-mediated and Ang II–mediated cardiomyopathy. Here we show that aldosterone exerts direct toxic actions on myocardium by oxidative activation of CaMKII, causing cardiac rupture and increased mortality in mice after myocardial infarction. Aldosterone induces CaMKII oxidation by recruiting NADPH oxidase, and this oxidized and activated CaMKII promotes matrix metalloproteinase 9 (MMP9) expression in cardiomyocytes. Myocardial CaMKII inhibition, overexpression of methionine sulfoxide reductase A (an enzyme that reduces oxidized CaMKII) or NADPH oxidase deficiency prevented aldosterone-enhanced cardiac rupture after myocardial infarction. These findings show that oxidized myocardial CaMKII mediates the cardiotoxic effects of aldosterone on the cardiac matrix and establish CaMKII as a nodal signal for the neurohumoral pathways associated with poor outcomes after myocardial infarction.

Lowering Plasma Cholesterol Levels Halts Progression of Aortic Valve Disease in Mice

Background— Treatment of hyperlipidemia produces functional and structural improvements in atherosclerotic vessels. However, the effects of treating hyperlipidemia on the structure and function of the aortic valve have been controversial, and any effects could be confounded by pleiotropic effects of hypolipidemic treatment. The goal of this study was to determine whether reducing elevated plasma lipid levels with a “genetic switch” in Reversa mice (Ldlr−/−/Apob100/100/Mttpfl/fl/Mx1-Cre+/+) reduces oxidative stress, reduces pro-osteogenic signaling, and retards the progression of aortic valve disease. Methods and Results— After 6 months of hypercholesterolemia, Reversa mice exhibited increases in superoxide, lipid deposition, myofibroblast activation, calcium deposition, and pro-osteogenic protein expression in the aortic valve. Maximum aortic valve cusp separation, as judged by echocardiography, was not altered. During an additional 6 months of hypercholesterolemia, superoxide levels, valvular lipid deposition, and myofibroblast activation remained elevated. Furthermore, calcium deposition and pro-osteogenic gene expression became more pronounced, and the aortic cusp separation decreased from 0.85±0.04 to 0.70±0.04 mm (mean±SE; P<0.05). Rapid normalization of cholesterol levels at 6 months of age (by inducing expression of Cre recombinase) normalized aortic valve superoxide levels, decreased myofibroblast activation, reduced valvular calcium burden, suppressed pro-osteogenic signaling cascades, and prevented reductions in aortic valve cusp separation. Conclusions— Collectively, these data indicate that reducing plasma lipid levels by genetic inactivation of the mttp gene in hypercholesterolemic mice with early aortic valve disease normalizes oxidative stress, reduces pro-osteogenic signaling, and halts the progression of aortic valve stenosis.

Sildenafil prevents and reverses transverse-tubule remodeling and Ca(2+) handling dysfunction in right ventricle failure induced by pulmonary artery hypertension.

Right ventricular (RV) failure (RVF) is the main cause of death in patients with pulmonary artery hypertension (PAH). Sildenafil, a phosphodiesterase type 5 inhibitor, was approved recently for treatment of PAH patients. However, the mechanisms underlying RV contractile malfunction and the benefits of sildenafil on RV function are not well understood. We aimed to investigate the following: (1) the ultrastructural and excitation-contraction coupling alterations underlying PAH-induced RVF; (2) whether the ultrastructural changes are reversible; and (3) the mechanisms underlying the therapeutic benefits of sildenafil in PAH-RVF. We used a single injection of monocrotaline in Wistar rats to induce pulmonary vascular proliferation, which led to PAH and RVF. RV myocytes displayed severe transverse (T)-tubule loss and disorganization, as well as blunted and dys-synchronous sarcoplasmic reticulum Ca2+ release. Sildenafil prevented and reversed the monocrotaline-induced PAH and LV filling impairment. Early intervention with sildenafil prevented RV hypertrophy and the development of RVF, T-tubule remodeling, and Ca2+ handling dysfunction. Although late treatment with sildenafil did not reverse RV hypertrophy in animals with established RVF, RV systolic function was improved. Furthermore, late intervention partially reversed both the impairment of myocyte T-tubule integrity and Ca2+ handling protein and sarcoplasmic reticulum Ca2+ release function in monocrotaline-treated rats. In conclusion, PAH-induced increase in RV afterload causes severe T-tubule remodeling and Ca2+ handling dysfunction in RV myocytes, leading to RV contractile failure. Sildenafil prevents and partially reverses ultrastructural, molecular, and functional remodeling of failing RV myocytes. Reversal of pathological T-tubule remodeling, although incomplete, is achievable without the regression of RV hypertrophy.

Evidence for active regulation of pro-osteogenic signaling in advanced aortic valve disease.

Objective—To test the hypothesis that valvular calcium deposition, pro-osteogenic signaling, and function can be altered in mice with advanced aortic valve disease. Methods and Results—“Reversa” mice were given a Western-type diet for 12 months and screened for the presence of aortic valve stenosis. Mice with advanced valve disease were assigned to 1 of 2 groups: (1) those with continued progression for 2 months and (2) those with regression for 2 months, in which lipid lowering was accomplished by a genetic switch. Control mice were normocholesterolemic for 14 months. Mice with advanced valve disease had massive valvular calcification that was associated with increases in bone morphogenetic protein signaling, Wnt/&bgr;-catenin signaling, and markers of osteoblastlike cell differentiation. Remarkably, reducing plasma lipids with a genetic switch dramatically reduced markers of pro-osteogenic signaling and significantly reduced valvular calcium deposition. Nevertheless, despite a marked reduction in valvular calcium deposition, valve function remained markedly impaired. Phosphorylated Smad2 levels and myofibroblast activation (indexes of profibrotic signaling) remained elevated. Conclusion—Molecular processes that contribute to valvular calcification and osteogenesis remain remarkably labile during the end stages of aortic valve stenosis. Although reductions in valvular calcium deposition were not sufficient to improve valvular function in the animals studied, these findings demonstrate that aortic valve calcification is a remarkably dynamic process that can be modified therapeutically, even in the presence of advanced aortic valve disease.

Microtubule-Mediated Defects in Junctophilin-2 Trafficking Contribute to Myocyte Transverse-Tubule Remodeling and Ca2+ Handling Dysfunction in Heart Failure

Background— Cardiac dysfunction in failing hearts of human patients and animal models is associated with both microtubule densification and transverse-tubule (T-tubule) remodeling. Our objective was to investigate whether microtubule densification contributes to T-tubule remodeling and excitation–contraction coupling dysfunction in heart disease. Methods and Results— In a mouse model of pressure overload–induced cardiomyopathy by transaortic banding, colchicine, a microtubule depolymerizer, significantly ameliorated T-tubule remodeling and cardiac dysfunction. In cultured cardiomyocytes, microtubule depolymerization with nocodazole or colchicine profoundly attenuated T-tubule impairment, whereas microtubule polymerization/stabilization with taxol accelerated T-tubule remodeling. In situ immunofluorescence of heart tissue sections demonstrated significant disorganization of junctophilin-2 (JP2), a protein that bridges the T-tubule and sarcoplasmic reticulum membranes, in transaortic banded hearts as well as in human failing hearts, whereas colchicine injection significantly preserved the distribution of JP2 in transaortic banded hearts. In isolated mouse cardiomyocytes, prolonged culture or treatment with taxol resulted in pronounced redistribution of JP2 from T-tubules to the peripheral plasma membrane, without changing total JP2 expression. Nocodazole treatment antagonized JP2 redistribution. Moreover, overexpression of a dominant-negative mutant of kinesin 1, a microtubule motor protein responsible for anterograde trafficking of proteins, protected against JP2 redistribution and T-tubule remodeling in culture. Finally, nocodazole treatment improved Ca2+ handling in cultured myocytes by increasing the amplitude of Ca2+ transients and reducing the frequency of Ca2+ sparks. Conclusion— Our data identify a mechanistic link between microtubule densification and T-tubule remodeling and reveal microtubule-mediated JP2 redistribution as a novel mechanism for T-tubule disruption, loss of excitation–contraction coupling, and heart failure.

β-Adrenergic receptor antagonists ameliorate myocyte T-tubule remodeling following myocardial infarction

β‐Adrenergic receptor (AR) blockers provide substantial clinical benefits, including improving overall survival and left ventricular (LV) function following myocardial infarction (MI), though the mechanisms remain incompletely defined. The transverse‐tubule (T‐tubule) system of ventricular myocytes is an important determinant of cardiac excitation‐contraction function. T‐tubule remodeling occurs early during LV failure. We hypothesized that β‐AR blockers prevent T‐tubule remodeling and thereby provide therapeutic benefits. A murine model of MI was utilized to examine the effect of β‐AR blockers on T‐tubule remodeling following LV MI. We applied the in situ imaging of T‐tubule structure from Langendorff‐perfused intact hearts with laser scanning confocal microscopy. We found that MI caused remarkable T‐tubule remodeling near the infarction border zone and moderate LV remodeling remote from the MI. Metoprolol and carvedilol administered 6 d after MI for 4 wk each increased the T‐tubule integrity at the remote and border zones. At the molecular level, both β‐AR blockers restored border and remote zone expression of junctophilin‐2 (JP‐2), which is involved in T‐tubule organization and formation of the T‐tubule/sarcoplasmic reticulum junctions. In contrast, β‐AR blockers had no significant effects on caveolin‐3 expression. In summary, our data show that β‐AR antagonists can protect against T‐tubule remodeling after MI, suggesting a novel therapeutic mechanism of action for this drug class. Preservation of JP‐2 expression may contribute to the beneficial effects of metoprolol and carvedilol on T‐tubule remodeling.— Chen, B., Li, Y., Jiang, S., Xie, Y.‐P., Guo, A., Kutschke, W., Zimmerman, K., Weiss, R. M., Miller, F. J., Anderson, M. E., Song, L.‐S. β‐Adrenergic receptor antagonists ameliorate myocyte T‐tubule remodeling following myocardial infarction. FASEB J. 26, 2531‐2537 (2012). www.fasebj.org

Critical roles of junctophilin-2 in T-tubule and excitation–contraction coupling maturation during postnatal development

AIMS Emerging evidence indicates a critical role for junctophilin-2 (JP2) in T-tubule integrity and assembly of cardiac dyads in adult ventricular myocytes. In the postnatal stage, one of the critical features of myocyte maturation is development of the T-tubule system, though the mechanisms remain poorly understood. In this study, we aim to determine whether JP2 is required for normal cardiac T-tubule maturation. METHODS AND RESULTS Using in situ confocal imaging of intact murine hearts, we found T-tubules were absent in both left- and right-ventricular myocytes at postnatal Day 8 and did not appear until Day 10. Quantification of T-tubule structural integrity using the T-tubule power (TT(power)) index revealed a progressive increase in TT(power) between postnatal Days 10 and 19. By postnatal Day 19, TT(power) was similar to that in adult murine cardiomyocytes, indicative of a nearly matured T-tubule network. JP2 levels increased dramatically during development, reaching levels observed in adult hearts by postnatal Day 14. Deficiency of JP2, using a mouse model in which a JP2-specific shRNA is expressed during embryonic development, severely impaired T-tubule maturation, with equivalent decreases in the left- and right-ventricular TT(power). We also detected a gradual increase in the density of transverse but not longitudinal tubules during development, and JP2 deficiency abolished the increase in the density of transverse elements. Alterations in T-tubules caused significant reduction in Ca(2+) transient amplitude and marked increase in Ca(2+) release dyssynchrony, Ca(2+) alternans, and spontaneous Ca(2+) waves, leading to contractile failure. CONCLUSION Our data identify a critical role for JP2 in T-tubule and excitation-contraction coupling maturation during development.