Jaime DeSantiago

Transgenic CaMKIIδC Overexpression Uniquely Alters Cardiac Myocyte Ca2+ Handling: Reduced SR Ca2+ Load and Activated SR Ca2+ Release

Abstract— Ca2+/calmodulin-dependent protein kinase II (CaMKII) &dgr; is the predominant cardiac isoform, and the &dgr;C splice variant is cytoplasmic. We overexpressed CaMKII&dgr;C in mouse heart and observed dilated heart failure and altered myocyte Ca2+ regulation in 3-month-old CaMKII&dgr;C transgenic mice (TG) versus wild-type littermates (WT). Heart/body weight ratio and cardiomyocyte size were increased about 2-fold in TG versus WT. At 1 Hz, twitch shortening, [Ca2+]i transient amplitude, and diastolic [Ca2+]i were all reduced by ≈50% in TG versus WT. This is explained by >50% reduction in SR Ca2+ content in TG versus WT. Peak Ca2+ current (ICa) was slightly increased, and action potential duration was prolonged in TG versus WT. Despite lower SR Ca2+ load and diastolic [Ca2+]i, fractional SR Ca2+ release was increased and resting spontaneous SR Ca2+ release events (Ca2+ sparks) were doubled in frequency in TG versus WT (with prolonged width and duration, but lower amplitude). Enhanced Ca2+ spark frequency was also seen in TG at 4 weeks (before heart failure onset). Acute CaMKII inhibition normalized Ca2+ spark frequency and ICa, consistent with direct CaMKII activation of ryanodine receptors (and ICa) in TG. The rate of [Ca2+]i decline during caffeine exposure was faster in TG, indicating enhanced Na+-Ca2+ exchange function (consistent with protein expression measurements). Enhanced diastolic SR Ca2+ leak (via sparks), reduced SR Ca2+-ATPase expression, and increased Na+-Ca2+ exchanger explain the reduced diastolic [Ca2+]i and SR Ca2+ content in TG. We conclude that CaMKII&dgr;C overexpression causes acute modulation of excitation-contraction coupling, which contributes to heart failure.

Cardiac Alternans Do Not Rely on Diastolic Sarcoplasmic Reticulum Calcium Content Fluctuations

Cardiac alternans are thought to be a precursor to life-threatening arrhythmias. Previous studies suggested that alterations in sarcoplasmic reticulum (SR) Ca2+ content are either causative or not associated with myocyte Ca2+ alternans. However, those studies used indirect measures of SR Ca2+. Here we used direct continuous measurement of intra-SR free [Ca2+] ([Ca2+]SR) (using Fluo5N) during frequency-dependent Ca2+ alternans in rabbit ventricular myocytes. We tested the hypothesis that alternating [Ca2+]SR is required for Ca2+ alternans. Amplitudes of [Ca2+]SR depletions alternated in phase with cytosolic Ca2+ transients and contractions. Some cells showed clear alternation in diastolic [Ca2+]SR during alternans, with higher [Ca2+]SR before the larger SR Ca2+ releases. However, the extent of SR Ca2+ release during the small beats was smaller than expected for the modest decrease in [Ca2+]SR. In other cells, clear Ca2+ alternans was observed without alternations in diastolic [Ca2+]SR. Additionally, alternating cells were observed, in which diastolic [Ca2+]SR fluctuations occurred interspersed by depletions in which the amplitude was unrelated to the preceding diastolic [Ca2+]SR. In all forms of alternans, the SR Ca2+ release rate was higher during large depletions than during small depletions. Although [Ca2+]SR exerts major influence on SR Ca2+ release, alternations in [Ca2+]SR are not required for Ca2+ alternans to occur. Rather, it seems likely that some other factor, such as ryanodine receptor availability after a prior beat (eg, recovery from inactivation), is of greater importance in initiating frequency-induced Ca2+ alternans. However, once such a weak SR Ca2+ release occurs, it can result in increased [Ca2+]SR and further enhance SR Ca2+ release at the next beat. In this way, diastolic [Ca2+]SR alternans can enhance frequency-induced Ca2+ alternans, even if they initiate by other means.

The IP3 Receptor Regulates Cardiac Hypertrophy in Response to Select Stimuli

Rationale: Inositol 1,4,5-trisphosphate (IP3) is a second messenger that regulates intracellular Ca2+ release through IP3 receptors located in the sarco(endo)plasmic reticulum of cardiac myocytes. Many prohypertrophic G protein–coupled receptor (GPCR) signaling events lead to IP3 liberation, although its importance in transducing the hypertrophic response has not been established in vivo. Objective: Here, we generated conditional, heart-specific transgenic mice with both gain- and loss-of-function for IP3 receptor signaling to examine its hypertrophic growth effects following pathological and physiological stimulation. Methods and Results: Overexpression of the mouse type-2 IP3 receptor (IP3R2) in the heart generated mild baseline cardiac hypertrophy at 3 months of age. Isolated myocytes from overexpressing lines showed increased Ca2+ transients and arrhythmias in response to endothelin-1 stimulation. Although low levels of IP3R2 overexpression failed to augment/synergize cardiac hypertrophy following 2 weeks of pressure-overload stimulation, such levels did enhance hypertrophy following 2 weeks of isoproterenol infusion, in response to G&agr;q overexpression, and/or in response to exercise stimulation. To inhibit IP3 signaling in vivo, we generated transgenic mice expressing an IP3 chelating protein (IP3-sponge). IP3-sponge transgenic mice abrogated cardiac hypertrophy in response to isoproterenol and angiotensin II infusion but not pressure-overload stimulation. Mechanistically, IP3R2-enhanced cardiac hypertrophy following isoproterenol infusion was significantly reduced in the calcineurin-A&bgr;–null background. Conclusion: These results indicate that IP3-mediated Ca2+ release plays a central role in regulating cardiac hypertrophy downstream of GPCR signaling, in part, through a calcineurin-dependent mechanism.

Akt regulates L-type Ca 2+ channel activity by modulating Ca vα1 protein stability

The insulin IGF-1–PI3K–Akt signaling pathway has been suggested to improve cardiac inotropism and increase Ca2+ handling through the effects of the protein kinase Akt. However, the underlying molecular mechanisms remain largely unknown. In this study, we provide evidence for an unanticipated regulatory function of Akt controlling L-type Ca2+ channel (LTCC) protein density. The pore-forming channel subunit Cavα1 contains highly conserved PEST sequences (signals for rapid protein degradation), and in-frame deletion of these PEST sequences results in increased Cavα1 protein levels. Our findings show that Akt-dependent phosphorylation of Cavβ2, the LTCC chaperone for Cavα1, antagonizes Cavα1 protein degradation by preventing Cavα1 PEST sequence recognition, leading to increased LTCC density and the consequent modulation of Ca2+ channel function. This novel mechanism by which Akt modulates LTCC stability could profoundly influence cardiac myocyte Ca2+ entry, Ca2+ handling, and contractility.

The inotropic effect of cardioactive glycosides in ventricular myocytes requires Na+–Ca2+ exchanger function

Glycoside‐induced cardiac inotropy has traditionally been attributed to direct Na+–K+‐ATPase inhibition, causing increased intracellular [Na+] and consequent Ca2+ gain via the Na+–Ca2+ exchanger (NCX). However, recent studies suggested alternative mechanisms of glycoside‐induced inotropy: (1) direct activation of sarcoplasmic reticulum Ca2+ release channels (ryanodine receptors; RyRs); (2) increased Ca2+ selectivity of Na+ channels (slip‐mode conductance); and (3) other signal transduction pathways. None of these proposed mechanisms requires NCX or an altered [Na+] gradient. Here we tested the ability of ouabain (OUA, 3 μm), digoxin (DIG, 20 μm) or acetylstrophanthidin (ACS, 4 μm) to alter Ca2+ transients in completely Na+‐free conditions in intact ferret and cat ventricular myocytes. We also tested whether OUA directly activates RyRs in permeabilized cat myocytes (measuring Ca2+ sparks by confocal microscopy). In intact ferret myocytes (stimulated at 0.2 Hz), DIG and ACS enhanced Ca2+ transients and cell shortening during twitches, as expected. However, prior depletion of [Na+]i (in Na+‐free, Ca2+‐free solution) and in Na+‐free solution (replaced by Li+) the inotropic effects of DIG and ACS were completely prevented. In voltage‐clamped cat myocytes, OUA increased Ca2+ transients by 48 ± 4% but OUA had no effect in Na+‐depleted cells (replaced by N‐methyl‐d‐glucamine). In permeabilized cat myocytes, OUA did not change Ca2+ spark frequency, amplitude or spatial spread (although spark duration was slightly prolonged). We conclude that the acute inotropic effects of DIG, ACS and OUA (and the effects on RyRs) depend on the presence of Na+ and a functional NCX in ferret and cat myocytes (rather than alternate Na+‐independent mechanisms).

Impaired contractile function and calcium handling in hearts of cardiac-specific calcineurin b1-deficient mice

To define the necessity of calcineurin (Cn) signaling for cardiac maturation and function, the postnatal phenotype of mice with cardiac-specific targeted ablation of the Cn B1 regulatory subunit (Ppp3r1) gene (csCnb1(-/-) mice) was characterized. csCnb1(-/-) mice develop a lethal cardiomyopathy, characterized by impaired postnatal growth of the heart and combined systolic and diastolic relaxation abnormalities, despite a lack of structural derangements. Notably, the csCnb1(-/-) hearts did not exhibit diastolic dilatation, despite the severe functional phenotype. Myocytes isolated from the mutant mice exhibited reduced rates of contraction/relaxation and abnormalities in calcium transients, consistent with altered sarcoplasmic reticulum loading. Levels of sarco(endo) plasmic reticulum Ca-ATPase 2a (Atp2a2) and phospholamban were normal, but phospholamban phosphorylation was markedly reduced at Ser(16) and Thr(17). In addition, levels of the Na/Ca exchanger (Slc8a1) were modestly reduced. These results define a novel mouse model of cardiac-specific Cn deficiency and demonstrate novel links between Cn signaling, postnatal growth of the heart, pathological ventricular remodeling, and excitation-contraction coupling.

Excitation-contraction coupling in ventricular myocytes is enhanced by paracrine signaling from mesenchymal stem cells

In clinical trials mesenchymal stem cells (MSCs) are transplanted into cardiac ischemic regions to decrease infarct size and improve contractility. However, the mechanism and time course of MSC-mediated cardioprotection are incompletely understood. We tested the hypothesis that paracrine signaling by MSCs promotes changes in cardiac excitation-contraction (EC) coupling that protects myocytes from cell death and enhances contractility. Isolated mouse ventricular myocytes (VMs) were treated with control tyrode, MSC conditioned-tyrode (ConT) or co-cultured with MSCs. The Ca handling properties of VMs were monitored by laser scanning confocal microscopy and whole cell voltage clamp. ConT superfusion of VMs resulted in a time dependent increase of the Ca transient amplitude (ConT(15min): ΔF/F(0)=3.52±0.38, n=14; Ctrl(15min): ΔF/F(0)=2.41±0.35, n=14) and acceleration of the Ca transient decay (τ: ConT: 269±18ms n=14; vs. Ctrl: 315±57ms, n=14). Voltage clamp recordings confirmed a ConT induced increase in I(Ca,L) (ConT: -5.9±0.5 pA/pF n=11; vs. Ctrl: -4.04±0.3 pA/pF, n=12). The change of τ resulted from increased SERCA activity. Changes in the Ca transient amplitude and τ were prevented by the PI3K inhibitors Wortmannin (100nmol/L) and LY294002 (10μmol/L) and the Akt inhibitor V (20μmol/L) indicating regulation through PI3K signal transduction and Akt activation which was confirmed by western blotting. A change in τ was also prevented in eNOS(-/-) myocytes or by inhibition of eNOS suggesting an NO mediated regulation of SERCA activity. Since paracrine signaling further resulted in increased survival of VMs we propose that the Akt induced change in Ca signaling is also a mechanism by which MSCs mediate an anti-apoptotic effect.

Ischemia/Reperfusion Injury Protection by Mesenchymal Stem Cell Derived Antioxidant Capacity

Mesenchymal stem cell (MSC) transplantation after ischemia/reperfusion (I/R) injury reduces infarct size and improves cardiac function. We used mouse ventricular myocytes (VMs) in an in vitro model of I/R to determine the mechanism by which MSCs prevent reperfusion injury by paracrine signaling. Exposure of mouse VMs to an ischemic challenge depolarized their mitochondrial membrane potential (Ψmito), increased their diastolic Ca(2+), and significantly attenuated cell shortening. Reperfusion of VMs with Ctrl tyrode or MSC-conditioned tyrode (ConT) resulted in a transient increase of the Ca(2+) transient amplitudes in all cells. ConT-reperfused cells exhibited a decreased number early after depolarization (EADs) (ConT: 6.3% vs. Ctrl: 28.4%) and prolonged survival (ConT: 58% vs. Ctrl: 33%). Ψmito rapidly recovered in Ctrl as well as ConT-treated VMs on reperfusion; however, in Ctrl solution, an exaggerated hyperpolarization of Ψmito was determined that preceded the collapse of Ψmito. The ability of ConT to attenuate the hyperpolarization of Ψmito was suppressed on inhibition of the PI3K/Akt signaling pathway or IK,ATP. However, protection of Ψmito was best mimicked by the reactive oxygen species (ROS) scavenger mitoTEMPO. Analysis of ConT revealed a significant antioxidant capacity that was linked to the presence of extracellular superoxide dismutase (SOD3) in ConT. In conclusion, MSC ConT protects VMs from simulated I/R injury by its SOD3-mediated antioxidant capacity and by delaying the recovery of Ψmito through Akt-mediated opening of IK,ATP. These changes attenuate reperfusion-induced ROS production and prevent the opening of the permeability transition pore and arrhythmic Ca(2+) release.

SR-targeted CaMKII inhibition improves SR Ca2+ handling, but accelerates cardiac remodeling in mice overexpressing CaMKIIδC

Cardiac myocyte overexpression of CaMKIIδ(C) leads to cardiac hypertrophy and heart failure (HF) possibly caused by altered myocyte Ca(2+) handling. A central defect might be the marked CaMKII-induced increase in diastolic sarcoplasmic reticulum (SR) Ca(2+) leak which decreases SR Ca(2+) load and Ca(2+) transient amplitude. We hypothesized that inhibition of CaMKII near the SR membrane would decrease the leak, improve Ca(2+) handling and prevent the development of contractile dysfunction and HF. To test this hypothesis we crossbred CaMKIIδ(C) overexpressing mice (CaMK) with mice expressing the CaMKII-inhibitor AIP targeted to the SR via a modified phospholamban (PLB)-transmembrane-domain (SR-AIP). There was a selective decrease in the amount of activated CaMKII in the microsomal (SR/membrane) fraction prepared from these double-transgenic mice (CaMK/SR-AIP) mice. In ventricular cardiomyocytes from CaMK/SR-AIP mice, SR Ca(2+) leak, assessed both as diastolic Ca(2+) shift into SR upon tetracaine in intact myocytes or integrated Ca(2+) spark release in permeabilized myocytes, was significantly reduced. The reduced leak was accompanied by enhanced SR Ca(2+) load and twitch amplitude in double-transgenic mice (vs. CaMK), without changes in SERCA expression or NCX function. However, despite the improved myocyte Ca(2+) handling, cardiac hypertrophy and remodeling was accelerated in CaMK/SR-AIP and cardiac function worsened. We conclude that while inhibition of SR localized CaMKII in CaMK mice improves Ca(2+) handling, it does not necessarily rescue the HF phenotype. This implies that a non-SR CaMKIIδ(C) exerts SR-independent effects that contribute to hypertrophy and HF, and this CaMKII pathway may be exacerbated by the global enhancement of Ca transients.

The C-terminus of the long AKAP13 isoform (AKAP-Lbc) is critical for development of compensatory cardiac hypertrophy

The objective of this study was to determine the role of A-Kinase Anchoring Protein (AKAP)-Lbc in the development of heart failure, by investigating AKAP-Lbc-protein kinase D1 (PKD1) signaling in vivo in cardiac hypertrophy. Using a gene-trap mouse expressing a truncated version of AKAP-Lbc (due to disruption of the endogenous AKAP-Lbc gene), that abolishes PKD1 interaction with AKAP-Lbc (AKAP-Lbc-ΔPKD), we studied two mouse models of pathological hypertrophy: i) angiotensin (AT-II) and phenylephrine (PE) infusion and ii) transverse aortic constriction (TAC)-induced pressure overload. Our results indicate that AKAP-Lbc-ΔPKD mice exhibit an accelerated progression to cardiac dysfunction in response to AT-II/PE treatment and TAC. AKAP-Lbc-ΔPKD mice display attenuated compensatory cardiac hypertrophy, increased collagen deposition and apoptosis, compared to wild-type (WT) control littermates. Mechanistically, reduced levels of PKD1 activation are observed in AKAP-Lbc-ΔPKD mice compared to WT mice, resulting in diminished phosphorylation of histone deacetylase 5 (HDAC5) and decreased hypertrophic gene expression. This is consistent with a reduced compensatory hypertrophy phenotype leading to progression of heart failure in AKAP-Lbc-ΔPKD mice. Overall, our data demonstrates a critical in vivo role for AKAP-Lbc-PKD1 signaling in the development of compensatory hypertrophy to enhance cardiac performance in response to TAC-induced pressure overload and neurohumoral stimulation by AT-II/PE treatment.