Silvia Guatimosim

Calmodulin kinase II inhibition protects against structural heart disease

β-Adrenergic receptor (βAR) stimulation increases cytosolic Ca2+ to physiologically augment cardiac contraction, whereas excessive βAR activation causes adverse cardiac remodeling, including myocardial hypertrophy, dilation and dysfunction, in individuals with myocardial infarction. The Ca2+-calmodulin–dependent protein kinase II (CaMKII) is a recently identified downstream element of the βAR-initiated signaling cascade that is linked to pathological myocardial remodeling and to regulation of key proteins involved in cardiac excitation-contraction coupling. We developed a genetic mouse model of cardiac CaMKII inhibition to test the role of CaMKII in βAR signaling in vivo. Here we show CaMKII inhibition substantially prevented maladaptive remodeling from excessive βAR stimulation and myocardial infarction, and induced balanced changes in excitation-contraction coupling that preserved baseline and βAR-stimulated physiological increases in cardiac function. These findings mark CaMKII as a determinant of clinically important heart disease phenotypes, and suggest CaMKII inhibition can be a highly selective approach for targeting adverse myocardial remodeling linked to βAR signaling.

Molecular Mechanisms Involved in the Angiotensin-(1-7)/Mas Signaling Pathway in Cardiomyocytes

Recently there has been growing evidence suggesting that beneficial effects of angiotensin-(1-7) [Ang-(1-7)] in the heart are mediated by its receptor Mas. However, the signaling pathways involved in these effects in cardiomyocytes are unknown. Here, we investigated the involvement of the Ang-(1-7)/Mas axis in NO generation and Ca2+ handling in adult ventricular myocytes using a combination of molecular biology, intracellular Ca2+ imaging, and confocal microscopy. Acute Ang-(1-7) treatment (10 nmol/L) leads to NO production and activates endothelial NO synthase and Akt in cardiomyocytes. Ang-(1-7)–dependent NO raise was abolished by pretreatment with A-779 (1 &mgr;mol/L). To confirm that Ang-(1-7) action is mediated by Mas, we used cardiomyocytes isolated from Mas-deficient mice. In Mas-deficient cardiomyocytes, Ang-(1-7) failed to increase NO levels. Moreover, Mas-ablation was accompanied by significant alterations in the proteins involved in the regulation of endothelial NO synthase activity, indicating that endothelial NO synthase and its binding partners are important effectors of the Mas-mediated pathway in cardiomyocytes. We then investigated the role of the Ang-(1-7)/Mas axis on Ca2+ signaling. Cardiomyocytes treated with 10 nmol/L of Ang-(1-7) did not show changes in Ca2+-transient parameters such as peak Ca2+ transients and kinetics of decay. Nevertheless, cardiomyocytes from Mas-deficient mice presented reduced peak and slower [Ca2+]i transients when compared with wild-type cardiomyocytes. Lower Ca2+ ATPase of the sarcoplasmic reticulum expression levels accompanied the reduced Ca2+ transient in Mas-deficient cardiomyocytes. Therefore, chronic Mas-deficiency leads to impaired Ca2+ handling in cardiomyocytes. Collectively, these observations reveal a key role for the Ang-(1-7)/Mas axis as a modulator of cardiomyocyte function.

Highly efficient siRNA delivery system into human and murine cells using single-wall carbon nanotubes

Development of RNA interference (RNAi) technology utilizing short interfering RNA sequences (siRNA) has focused on creating methods for delivering siRNAs to cells and for enhancing siRNA stability in vitro and in vivo. Here, we describe a novel approach for siRNA cellular delivery using siRNA coiling into carboxyl-functionalized single-wall carbon nanotubes (SWCNTs). The CNT-siRNA delivery system successfully demonstrates nonspecific toxicity and transfection efficiency greater than 95%. This approach offers the potential for siRNA delivery into different types of cells, including hard-to-transfect cells, such as neuronal cells and cardiomyocytes. We also tested the CNT-siRNA system in a non-metastatic human hepatocellular carcinoma cell line (SKHep1). In all types of cells used in this work the CNT-siRNA delivery system showed high efficiency and apparent no side effects for various in vitro applications.

Nuclear Ca2+ regulates cardiomyocyte function

In the heart, cytosolic Ca(2+) signals are well-characterized events that participate in the activation of cell contraction. In contrast, nuclear Ca(2+) contribution to cardiomyocyte function remains elusive. Here, we examined functional consequences of buffering nuclear Ca(2+) in neonatal cardiomyocytes. We report that cardiomyocytes contain a nucleoplasmic reticulum, which expresses both ryanodine receptor (RyR) and inositol 1,4,5-trisphosphate receptor (InsP(3)R), providing a possible way for active regulation of nuclear Ca(2+). Adenovirus constructs encoding the Ca(2+) buffer protein parvalbumin were targeted to the nucleus with a nuclear localization signal (Ad-PV-NLS) or to the cytoplasm with a nuclear exclusion signal (Ad-PV-NES). A decrease in the amplitude of global Ca(2+) transients and RyR-II expression, as well as an increase in cell beating rate were observed in Ad-PV-NES and Ad-PV-NLS cells. When nuclear Ca(2+) buffering was imposed nuclear enlargement, increased calcineurin expression, NFAT translocation to the nucleus and subcellular redistribution of atrial natriuretic peptide were observed. Furthermore, prolongation of action potential duration occurred in adult ventricular myocytes. These results suggest that nuclear Ca(2+) levels underlie the regulation of specific protein targets and thereby modulate cardiomyocyte function. The local nuclear Ca(2+) signaling and the structures that control it constitute a novel regulatory motif in the heart.

Carbon nanotube interaction with extracellular matrix proteins producing scaffolds for tissue engineering

In recent years, significant progress has been made in organ transplantation, surgical reconstruction, and the use of artificial prostheses to treat the loss or failure of an organ or bone tissue. In recent years, considerable attention has been given to carbon nanotubes and collagen composite materials and their applications in the field of tissue engineering due to their minimal foreign-body reactions, an intrinsic antibacterial nature, biocompatibility, biodegradability, and the ability to be molded into various geometries and forms such as porous structures, suitable for cell ingrowth, proliferation, and differentiation. Recently, grafted collagen and some other natural and synthetic polymers with carbon nanotubes have been incorporated to increase the mechanical strength of these composites. Carbon nanotube composites are thus emerging as potential materials for artificial bone and bone regeneration in tissue engineering.

Influence of spontaneous calcium events on cell-cycle progression in embryonal carcinoma and adult stem cells

Spontaneous Ca(2+) events have been observed in diverse stem cell lines, including carcinoma and mesenchymal stem cells. Interestingly, during cell cycle progression, cells exhibit Ca(2+) transients during the G(1) to S transition, suggesting that these oscillations may play a role in cell cycle progression. We aimed to study the influence of promoting and blocking calcium oscillations in cell proliferation and cell cycle progression, both in neural progenitor and undifferentiated cells. We also identified which calcium stores are required for maintaining these oscillations. Both in neural progenitor and undifferentiated cells calcium oscillations were restricted to the G1/S transition, suggesting a role for these events in progression of the cell cycle. Maintenance of the oscillations required calcium influx only through inositol 1,4,5-triphosphate receptors (IP(3)Rs) and L-type channels in undifferentiated cells, while neural progenitor cells also utilized ryanodine-sensitive stores. Interestingly, promoting calcium oscillations through IP(3)R agonists increased both proliferation and levels of cell cycle regulators such as cyclins A and E. Conversely, blocking calcium events with IP(3)R antagonists had the opposite effect in both undifferentiated and neural progenitor cells. This suggests that calcium events created by IP(3)Rs may be involved in cell cycle progression and proliferation, possibly due to regulation of cyclin levels, both in undifferentiated cells and in neural progenitor cells.

Paradoxical Cellular Ca2+ Signaling in Severe but Compensated Canine Left Ventricular Hypertrophy

In conscious dogs with severe left ventricular (LV) hypertrophy (H) (doubling of LV/body weight), which developed gradually over 1 to 2 years after aortic banding, baseline LV function was well compensated. The LV was able to generate twice the LV systolic pressure without an increase in LV end-diastolic pressure, or decrease in LV dP/dt or LV wall thickening. However, LV myocytes isolated from LVH dogs exhibited impaired contraction at baseline and in response to Ca2+. There was no change in L-type Ca2+ channel current (ICa) density but the ability of ICa to trigger Ca2+ release from the sarcoplasmic reticulum (SR) was reduced. Immunoblot analysis revealed a 68% decrease in SERCA2a, and a 35% decrease in the number of ryanodine receptors (RyR2), with no changes in protein level of calsequestrin, Na+/Ca2+ exchanger or phospholamban (PLB), but with both RyR2 and PLB hyperphosphorylated. Spontaneous Ca2+ sparks in LVH cells were found to have prolonged duration but similar intensities despite the reduced SR Ca2+ load. A higher Ca2+ spark rate was observed in LVH cells, but this is inconsistent with the reduced SR Ca2+ content. However, Ca2+ waves were found to be less frequent, slower and were more likely to be aborted in Ca2+-challenged LVH cells. These paradoxical observations could be accounted for by a nonuniform SR Ca2+ distribution, RyR2 hyperphosphorylation in the presence of decreased global SR Ca2+ load. We conclude that severe LVH with compensation masks cellular and subcellular Ca2+ defects that remain likely contributors to the limited contractile reserve of LVH.

Calcium Biology of the Transverse Tubules in Heart

Abstract: Ca2+ sparks in heart muscle are activated on depolarization by the influx of Ca2+ through dihydropyridine receptors in the sarcolemmal (SL) and transverse tubule (TT) membranes. The cardiac action potential is thus able to synchronize the [Ca2+]i transient as Ca2+ release is activated throughout the cell. Increases in the amount of Ca2+ within the sarcoplasmic reticulum (SR) underlie augmented Ca2+ release globally and an increase in the sensitivity of the ryanodine receptors (RyRs) to be triggered by the local [Ca2+]i. In a similar manner, phosphorylation of the RyRs by protein kinase A (PKA) increases the sensitivity of the RyRs to be activated by local [Ca2+]i. Heart failure and other cardiac diseases are associated with changes in SR Ca2+ content, phosphorylation state of the RyRs, [Ca2+]i signaling defects and arrhythmias. Additional changes in transverse tubules and nearby junctional SR may contribute to alterations in local Ca2+ signaling. Here we briefly discuss how TT organization can influence Ca2+ signaling and how changes in SR Ca2+ release triggering can influence excitation‐contraction (EC) coupling. High speed imaging methods are used in combination with single cell patch clamp experiments to investigate how abnormal Ca2+ signaling may be regulated in health and disease. Three issues are examined in this presentation: (1) normal Ca2+‐induced Ca2+ release and Ca2+ sparks, (2) abnormal SR Ca2+ release in disease, and (3) the triggering and propagation of waves of elevated [Ca2+]i.

Dysautonomia Due to Reduced Cholinergic Neurotransmission Causes Cardiac Remodeling and Heart Failure

ABSTRACT Overwhelming evidence supports the importance of the sympathetic nervous system in heart failure. In contrast, much less is known about the role of failing cholinergic neurotransmission in cardiac disease. By using a unique genetically modified mouse line with reduced expression of the vesicular acetylcholine transporter (VAChT) and consequently decreased release of acetylcholine, we investigated the consequences of altered cholinergic tone for cardiac function. M-mode echocardiography, hemodynamic experiments, analysis of isolated perfused hearts, and measurements of cardiomyocyte contraction indicated that VAChT mutant mice have decreased left ventricle function associated with altered calcium handling. Gene expression was analyzed by quantitative reverse transcriptase PCR and Western blotting, and the results indicated that VAChT mutant mice have profound cardiac remodeling and reactivation of the fetal gene program. This phenotype was attributable to reduced cholinergic tone, since administration of the cholinesterase inhibitor pyridostigmine for 2 weeks reversed the cardiac phenotype in mutant mice. Our findings provide direct evidence that decreased cholinergic neurotransmission and underlying autonomic imbalance cause plastic alterations that contribute to heart dysfunction.

Cardiac anti‐remodelling effect of aerobic training is associated with a reduction in the calcineurin/NFAT signalling pathway in heart failure mice

Cardiomyocyte hypertrophy occurs in response to a variety of physiological and pathological stimuli. While pathological hypertrophy in heart failure is usually coupled with depressed contractile function, physiological hypertrophy associates with increased contractility. In the present study, we explored whether 8 weeks of moderate intensity exercise training would lead to a cardiac anti‐remodelling effect in an experimental model of heart failure associated with a deactivation of a pathological (calcineurin/NFAT, CaMKII/HDAC) or activation of a physiological (Akt–mTOR) hypertrophy signalling pathway. The cardiac dysfunction, exercise intolerance, left ventricle dilatation, increased heart weight and cardiomyocyte hypertrophy from mice lacking α2A and α2C adrenoceptors (α2A/α2CARKO mice) were associated with sympathetic hyperactivity induced heart failure. The relative contribution of Ca2+–calmodulin high‐affinity (calcineurin/NFAT) and low‐affinity (CaMKII/HDAC) targets to pathological hypertrophy of α2A/α2CARKO mice was verified. While nuclear calcineurin B, NFATc3 and GATA‐4 translocation were significantly increased in α2A/α2CARKO mice, no changes were observed in CaMKII/HDAC activation. As expected, cyclosporine treatment decreased nuclear translocation of calcineurin/NFAT in α2A/α2CARKO mice, which was associated with improved ventricular function and a pronounced anti‐remodelling effect. The Akt/mTOR signalling pathway was not activated in α2A/α2CARKO mice. Exercise training improved cardiac function and exercise capacity in α2A/α2CARKO mice and decreased heart weight and cardiomyocyte width paralleled by diminished nuclear NFATc3 and GATA‐4 translocation as well as GATA‐4 expression levels. When combined, these findings support the notion that deactivation of calcineurin/NFAT pathway‐induced pathological hypertrophy is a preferential mechanism by which exercise training leads to the cardiac anti‐remodelling effect in heart failure.