Abstract 314: Loss of Sarcospan has a Deleterious Effect on Cardiac Function in Aged Mice

Sarcospan (SSPN) has been shown to have an important role in stabilizing sarcolemmal dystrophin- and utrophin-glycoprotein adhesion complexes. Loss of sarcolemmal integrity leads to immune cell infiltration and inappropriate exchange of cellular contents with the extracellular milieu. Our laboratory has shown SSPN loss destabilizes skeletal muscle adhesion and reduces sarcolemmal dystrophin localization, whereas its complete loss due to mutation underlies development of Duchenne muscular dystrophy (DMD). Loss of dystrophin leads to cardiac dysfunction and early mortality in DMD patients. The role of SSPN in the heart is unknown. We present immunofluorescence data revealing reduction of dystrophin and the sarcoglycans with a coordinate increase of β1D integrin levels at the SSPN-null cardiac sarcolemma relative to WT. Also, SSPN loss decreases cardiac P-Akt levels, disrupting signaling promoting compensatory physiological hypertrophy. These studies suggest a fundamental role for SSPN in cardiac maintenance and function, since left ventricular mass increases with age and upon isoproterenol administration (0.8 mg/day for two wks). Aged SSPN-null mice developed hypertrophy, evidenced as increased heart/body weight ratio and left ventricular wall dimension. The SSPN-null mice lacked the characteristic initial rise in cardiac output, left ventricular ejection fraction (LvEF %), induced by chronic β-adrenergic stimulation. Functionally, aged SSPN-null hearts had an increased E/A ratio indicating restrictive ventricular filling and decreased fractional shortening F/S (%) upon isoproterenol administration. Aged untreated SSPN-null hearts had increased fibrosis compared to aged WT controls, however isoproterenol treated SSPN-null hearts displayed exacerbated fibrotic response compared to WT. To assess whether SSPN-null hearts have altered gene expression profiles during progression of pathogenesis, qRT-PCR will be utilized to measure differences in expression of fetal gene and calcium-handling proteins. In summary, we have found that SSPN has an important role in maintaining cardiac function, its loss exacerbates the hypertrophic response and localization of stabilizing adhesion complexes at the cardiac muscle sarcolemma.

Massive palmitoylation-dependent endocytosis during reoxygenation of anoxic cardiac muscle

In fibroblasts, large Ca transients activate massive endocytosis (MEND) that involves membrane protein palmitoylation subsequent to mitochondrial permeability transition pore (PTP) openings. Here, we characterize this pathway in cardiac muscle. Myocytes with increased expression of the acyl transferase, DHHC5, have decreased Na/K pump activity. In DHHC5-deficient myocytes, Na/K pump activity and surface area/volume ratios are increased, the palmitoylated regulatory protein, phospholemman (PLM), and the cardiac Na/Ca exchanger (NCX1) show greater surface membrane localization, and MEND is inhibited in four protocols. Both electrical and optical methods demonstrate that PTP-dependent MEND occurs during reoxygenation of anoxic hearts. Post-anoxia MEND is ablated in DHHC5-deficient hearts, inhibited by cyclosporine A (CsA) and adenosine, promoted by staurosporine (STS), reduced in hearts lacking PLM, and correlates with impaired post-anoxia contractile function. Thus, the MEND pathway appears to be deleterious in severe oxidative stress but may constitutively contribute to cardiac sarcolemma turnover in dependence on metabolic stress.

Abstract 070: Interaction between Mena, Connexin43, and Rac1 at the Intercalated Disc

Background: We previously reported that Mena, a member of Ena/VASP family of actin regulatory protein, is associated with heart failure (HF). Mena is localized to the intercalated disc (ICD) and interacts with ICD proteins implicated in HF. The ICD mediates force transmission and electrical coupling between cardiomyocytes. Slowed conduction in HF is associated with Cx43 remodeling, the predominant connexin isoform in the heart. Mena-/- mice develop cardiac dysfunction, conduction abnormalities, ICD disorganization, and Cx43 lateralization. We hypothesized that: 1) Mena’s interaction with the cytoskeleton is critical for ICD stabilization and maintenance of electrical function; 2) Mena may modulate signal(s) from the cardiac sarcolemma to the ICD for control of expression and localization of Cx43 through the regulation of the small GTPase Rac1. Methods and Results: Interaction of Mena with Cx43 and Rac1 was evaluated in neonatal rat ventricular myocytes (NRVM), HeLa cells, and in transgenic mice overexpressing constitutively active V12Rac1 (RacET). Pull down assay using GST-PAK1 demonstrated a direct association of Mena with active Rac1-GTP in vitro. In NRVMs, siRNA knockdown of Mena significantly increased activity of Rac1-GTPase by 2-fold (p=0.05, n=5). Western blot analysis revealed increased total Cx43 expression in Mena siRNA (1.11±0.2) compared to scramble (0.33±0.1, p<0.01, n=5). Enhanced Cx43 expression was associated with increased rate of recovery after photobleaching between cardiomyocytes by gap-FRAP analysis (Tau 153.0±4.8 vs. 73.0±1.8 sec, p<0.0001). Ongoing experiments with optical mapping of NRVM monolayers will determine whether conduction velocity may be enhanced after Mena knockdown. Confocal imaging revealed altered Cx43 trafficking and localization with Mena knockdown. Additionally in ventricles of RacET hearts, Mena expression was increased by 59% (p=0.01, n=4) compared to control, along with lateral redistribution of Cx43. Conclusions: Our results suggest that Mena is a critical regulator of the ICD at the intersection of Cx43 and Rac1. Increased Rac1 activation is associated with lateralized Cx43, and enhanced cellular coupling. Mena may modulate Cx43 localization through the regulation of Rac1 activity.

Involvement of Membrane Fluidity in Endogenous Protective Processes Running on Subcellular Membrane Systems of the Rat Heart

Membrane fluidity is a widely recognized biophysical variable that provides information about structural organization of the subcellular membranes exhibiting physical characteristics of liquid crystals. The term “fluidity” reflects in this case the tightness in packing of acyl parts of the membrane phospholipid molecules, a feature that may influence considerably the molecular mobility and via that also the sensitivity and reactivity of membrane-bound transporters, receptors and enzyme systems. Data presented in this review are aimed to demonstrate the substantial role of changes in membrane fluidity occurring in the processes associated with endogenous protection observed in cardiac sarcolemma and mitochondria in diverse pathologies, particularly in diabetes and hypertension.

Dynamic of Ion Channel Expression at the Plasma Membrane of Cardiomyocytes

Cardiac myocytes are characterized by distinct structural and functional entities involved in the generation and transmission of the action potential and the excitation-contraction coupling process. Key to their function is the specific organization of ion channels and transporters to and within distinct membrane domains, which supports the anisotropic propagation of the depolarization wave. This review addresses the current knowledge on the molecular actors regulating the distinct trafficking and targeting mechanisms of ion channels in the highly polarized cardiac myocyte. In addition to ubiquitous mechanisms shared by other excitable cells, cardiac myocytes show unique specialization, illustrated by the molecular organization of myocyte-myocyte contacts, e.g., the intercalated disc and the gap junction. Many factors contribute to the specialization of the cardiac sarcolemma and the functional expression of cardiac ion channels, including various anchoring proteins, motors, small GTPases, membrane lipids, and cholesterol. The discovery of genetic defects in some of these actors, leading to complex cardiac disorders, emphasizes the importance of trafficking and targeting of ion channels to cardiac function. A major challenge in the field is to understand how these and other actors work together in intact myocytes to fine-tune ion channel expression and control cardiac excitability.