Mayilvahanan Shanmugam

Angiotensin II induces afterdepolarizations via reactive oxygen species and calmodulin kinase II signaling

Renin-angiotensin system inhibitors significantly reduce the incidence of arrhythmias. However, the underlying mechanism(s) is not well understood. We aim to test the hypothesis that angiotensin II (Ang II) induces early afterdepolarizations (EADs) and triggered activities (TAs) via the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-ROS-calmodulin kinase II (CaMKII) pathway. ROS production was analyzed in the isolated rabbit myocytes loaded with ROS dye. Ang II (1-2 μM) increased ROS fluorescence in myocytes, which was abolished by Ang II type 1 receptor blocker losartan, NADPH oxidase inhibitor apocynin, and antioxidant MnTMPyP, respectively. Action potentials were recorded using the perforated patch-clamp technique. EADs emerged in 27 out of 41 (66%) cells at 15.8 ± 1.6 min after Ang II (1-2 μM) perfusion. Ang II-induced EADs were eliminated by losartan, apocynin, or trolox. The CaMKII inhibitor KN-93 (n=6) and inhibitory peptide (AIP) (n=4) also suppressed Ang II-induced EADs, whereas the inactive analogue KN-92 did not. Nifedipine, a blocker of L-type Ca current (I(Ca)(2+)(,L)), or ranolazine, an inhibitor of late Na current (I(Na)(+)), abolished Ang II-induced EADs. The effects of Ang II on major membrane currents were evaluated using voltage clamp. While Ang II at same concentrations had no significant effect on total outward K(+) current, it enhanced I(Ca.L) and late I(Na), which were attenuated by losartan, apocynin, trolox, or KN-93. We conclude that Ang II induces EADs via intracellular ROS production through NADPH oxidase, activation of CaMKII, and enhancement of I(Ca,L) and late I(Na). These results provide evidence supporting a link between renin-angiotensin system and cardiac arrhythmias.

Decreased sarcolipin protein expression and enhanced sarco(endo)plasmic reticulum Ca2+ uptake in human atrial fibrillation.

Sarcolipin (SLN), a key regulator of cardiac sarco(endo)plasmic reticulum (SR) Ca(2+) ATPase, is predominantly expressed in atria and mediates β-adrenergic responses. Studies have shown that SLN mRNA expression is decreased in human chronic atrial fibrillation (AF) and in aortic banded mouse atria; however, SLN protein expression in human atrial pathology and its role in atrial SR Ca(2+) uptake are not yet elucidated. In the present study, we determined the expression of major SR Ca(2+) handling proteins in atria of human AF patients and in human and in a mouse model of heart failure (HF). We found that the expression of SR Ca(2+) uptake and Ca(2+) release channel proteins are significantly decreased in atria but not in the ventricles of pressure-overload induced HF in mice. In human AF and HF, the expression of SLN protein was significantly decreased; whereas the expressions of other major SR Ca(2+) handling proteins were not altered. Further, we found that the SR Ca(2+) uptake was significantly increased in human AF. The selective downregulation of SLN and enhanced SR Ca(2+) uptake in human AF suggest that SLN downregulation could play an important role in abnormal intracellular Ca(2+) cycling in atrial pathology.

Increased sarcolipin expression and decreased sarco(endo)plasmic reticulum Ca2+ uptake in skeletal muscles of mouse models of Duchenne muscular dystrophy

Abnormal intracellular Ca2+ handling is an important factor in the progressive functional decline of dystrophic muscle. In the present study, we investigated the function of sarco(endo)plasmic reticulum (SR) Ca2+ ATPase (SERCA) in various dystrophic muscles of mouse models of Duchenne muscular dystrophy. Our studies show that the protein expression of sarcolipin, a key regulator of the SERCA pump is abnormally high and correlates with decreased maximum velocity of SR Ca2+ uptake in the soleus, diaphragm and quadriceps of mild (mdx) and severe (mdx:utr−/−) dystrophic mice. These changes are more pronounced in the muscles of mdx:utr−/− mice. We also found increased expression of SERCA2a and calsequestrin specifically in the dystrophic quadriceps. Immunostaining analysis further showed that SERCA2a expression is associated both with fibers expressing slow-type myosin and regenerating fibers expressing embryonic myosin. Together, our data suggest that sarcolipin upregulation is a common secondary alteration in all dystrophic muscles and contributes to the abnormal elevation of intracellular Ca2+ concentration via SERCA inhibition.

Ablation of phospholamban and sarcolipin results in cardiac hypertrophy and decreased cardiac contractility

AIMS Improving the sarco(endo)plasmic reticulum (SR) Ca(2+)-ATPase (SERCA) function has clinical implications in treating heart failure. The present study aimed to determine the effect of constitutive activation of the SERCA pump on cardiac contractility in normal mice and during pressure-overload-induced cardiac hypertrophy. METHODS AND RESULTS The SERCA pump was constitutively activated in both atrial and ventricular chambers of the mouse heart by ablating its key regulators, phospholamban (PLN) and sarcolipin (SLN). The double-knockout (dKO) mice for PLN and SLN showed increased SERCA pump activity, Ca(2+) transients and SR Ca(2+) load, and developed cardiac hypertrophy. Echocardiographic measurements showed that the basal cardiac function was not affected in the young dKO mice. However, the cardiac function worsened upon ageing and when subjected to pressure overload. CONCLUSION Our studies suggest that the constitutive activation of the SERCA pump is detrimental to cardiac function. Our findings also emphasize the need for dynamic regulation of the SERCA pump by PLN and/or SLN to maintain cardiac contractility in normal conditions and during pathophysiological states.

Ablation of sarcolipin results in atrial remodeling.

Sarcolipin (SLN) is a key regulator of sarco(endo)plasmic reticulum (SR) Ca(2+)-ATPase (SERCA), and its expression is altered in diseased atrial myocardium. To determine the precise role of SLN in atrial Ca(2+) homeostasis, we developed a SLN knockout (sln-/-) mouse model and demonstrated that ablation of SLN enhances atrial SERCA pump activity. The present study is designed to determine the long-term effects of enhanced SERCA activity on atrial remodeling in the sln-/- mice. Calcium transient measurements show an increase in atrial SR Ca(2+) load and twitch Ca(2+) transients. Patch-clamping experiments demonstrate activation of the forward mode of sodium/calcium exchanger, increased L-type Ca(2+) channel activity, and prolongation of action potential duration at 90% repolarization in the atrial myocytes of sln-/- mice. Spontaneous Ca(2+) waves, delayed afterdepolarization, and triggered activities are frequent in the atrial myocytes of sln-/- mice. Furthermore, loss of SLN in atria is associated with increased interstitial fibrosis and altered expression of genes encoding collagen and other extracellular matrix proteins. Our results also show that the sln-/- mice are susceptible to atrial arrhythmias upon aging. Together, these findings indicate that ablation of SLN results in increased SERCA activity and SR Ca(2+) load, which, in turn, could cause abnormal intracellular Ca(2+) handling and atrial remodeling.

Increased Sarcolipin Expression and Adrenergic Drive in Humans With Preserved Left Ventricular Ejection Fraction and Chronic Isolated Mitral Regurgitation

Background— There is currently no therapy proven to attenuate left ventricular (LV) dilatation and dysfunction in volume overload induced by isolated mitral regurgitation (MR). To better understand molecular signatures underlying isolated MR, we performed LV gene expression analyses and overlaid regulated genes into ingenuity pathway analysis in patients with isolated MR. Methods and Results— Gene arrays from LV tissue of 35 patients, taken at the time of surgical repair for isolated MR, were compared with 13 normal controls. Cine-MRI was performed in 31 patients before surgery to measure LV function and volume from serial short-axis summation. LV end-diastolic volume was 2-fold (P=0.005) higher in MR patients than in normal controls, and LV ejection fraction was 64±7% (50%–79%) in MR patients. Ingenuity pathway analysis identified significant activation of pathways involved in &bgr;-adrenergic, cAMP, and G-protein–coupled signaling, whereas there was downregulation of pathways associated with complement activation and acute phase response. SERCA2a and phospholamban protein were unchanged in MR versus control left ventricles. However, mRNA and protein levels of the sarcoplasmic reticulum Ca2+ ATPase (SERCA) regulatory protein sarcolipin, which is predominantly expressed in normal atria, were increased 12- and 6-fold, respectively. Immunofluorescence analysis confirmed the absence of sarcolipin in normal left ventricles and its marked upregulation in MR left ventricles. Conclusions— These results demonstrate alterations in multiple pathways associated with &bgr;-adrenergic signaling and sarcolipin in the left ventricles of patients with isolated MR and LV ejection fraction >50%, suggesting a beneficial role for &bgr;-adrenergic blockade in isolated MR.

Xanthine oxidase inhibition preserves left ventricular systolic but not diastolic function in cardiac volume overload

Xanthine oxidase (XO) is increased in human and rat left ventricular (LV) myocytes with volume overload (VO) of mitral regurgitation and aortocaval fistula (ACF). In the setting of increased ATP demand, XO-mediated ROS can decrease mitochondrial respiration and contractile function. Thus, we tested the hypothesis that XO inhibition improves cardiomyocyte bioenergetics and LV function in chronic ACF in the rat. Sprague-Dawley rats were randomized to either sham or ACF ± allopurinol (100 mg·kg(-1)·day(-1), n ≥7 rats/group). Echocardiography at 8 wk demonstrated a similar 37% increase in LV end-diastolic dimension (P < 0.001), a twofold increase in LV end-diastolic pressure/wall stress (P < 0.05), and a twofold increase in lung weight (P < 0.05) in treated and untreated ACF groups versus the sham group. LV ejection fraction, velocity of circumferential shortening, maximal systolic elastance, and contractile efficiency were significantly depressed in ACF and significantly improved in ACF + allopurinol rats, all of which occurred in the absence of changes in the maximum O2 consumption rate measured in isolated cardiomyocytes using the extracellular flux analyzer. However, the improvement in contractile function is not paralleled by any attenuation in LV dilatation, LV end-diastolic pressure/wall stress, and lung weight. In conclusion, allopurinol improves LV contractile function and efficiency possibly by diminishing the known XO-mediated ROS effects on myofilament Ca(2+) sensitivity. However, LV remodeling and diastolic properties are not improved, which may explain the failure of XO inhibition to improve symptoms and hospitalizations in patients with severe heart failure.

Role of Exopolysaccharide in Aggregatibacter actinomycetemcomitans–Induced Bone Resorption in a Rat Model for Periodontal Disease

Aggregatibacter actinomycetemcomitans a causative agent of periodontal disease in humans, forms biofilm on biotic and abiotic surfaces. A. actinomycetemcomitans biofilm is heterogeneous in nature and is composed of proteins, extracellular DNA and exopolysaccharide. To explore the role played by the exopolysaccharide in the colonization and disease progression, we employed genetic reduction approach using our rat model of A. actinomycetemcomitans-induced periodontitis. To this end, a genetically modified strain of A. actinomycetemcomitans lacking the pga operon was compared with the wild-type strain in the rat infection model. The parent and mutant strains were primarily evaluated for bone resorption and disease. Our study showed that colonization, bone resorption/disease and antibody response were all elevated in the wild-type fed rats. The bone resorption/disease caused by the pga mutant strain, lacking the exopolysaccharide, was significantly less (P < 0.05) than the bone resorption/disease caused by the wild-type strain. Further analysis of the expression levels of selected virulence genes through RT-PCR showed that the decrease in colonization, bone resorption and antibody titer in the absence of the exopolysaccharide might be due to attenuated levels of colonization genes, flp-1, apiA and aae in the mutant strain. This study demonstrates that the effect exerted by the exopolysaccharide in A. actinomycetemcomitans-induced bone resorption has hitherto not been recognized and underscores the role played by the exopolysaccharide in A. actinomycetemcomitans-induced disease.

Cardiac specific expression of threonine 5 to alanine mutant sarcolipin results in structural remodeling and diastolic dysfunction.

The functional importance of threonine 5 (T5) in modulating the activity of sarcolipin (SLN), a key regulator of sarco/endoplasmic reticulum (SR) Ca2+ ATPase (SERCA) pump was studied using a transgenic mouse model with cardiac specific expression of threonine 5 to alanine mutant SLN (SLNT5A). In these transgenic mice, the SLNT5A protein replaces the endogenous SLN in atria, while maintaining the total SLN content. The cardiac specific expression of SLNT5A results in severe cardiac structural remodeling accompanied by bi-atrial enlargement. Biochemical analyses reveal a selective downregulation of SR Ca2+ handling proteins and a reduced SR Ca2+ uptake both in atria and in the ventricles. Optical mapping analysis shows slower action potential propagation in the transgenic mice atria. Doppler echocardiography and hemodynamic measurements demonstrate a reduced atrial contractility and an impaired diastolic function. Together, these findings suggest that threonine 5 plays an important role in modulating SLN function in the heart. Furthermore, our studies suggest that alteration in SLN function can cause abnormal Ca2+ handling and subsequent cardiac remodeling and dysfunction.

Transcriptome Profiling of Wild-Type and pga-Knockout Mutant Strains Reveal the Role of Exopolysaccharide in Aggregatibacter actinomycetemcomitans.

Exopolysaccharides have a diverse set of functions in most bacteria including a mechanistic role in protecting bacteria against environmental stresses. Among the many functions attributed to the exopolysaccharides, biofilm formation, antibiotic resistance, immune evasion and colonization have been studied most extensively. The exopolysaccharide produced by many Gram positive as well as Gram negative bacteria including the oral pathogen Aggregatibacter actinomycetemcomitans is the homopolymer of β(1,6)-linked N-acetylglucosamine. Recently, we reported that the PGA-deficient mutant of A. actinomycetemcomitans failed to colonize or induce bone resorption in a rat model of periodontal disease, and the colonization genes, apiA and aae, were significantly down regulated in the mutant strain. To understand the role of exopolysaccharide and the pga locus in the global expression of A. actinomycetemcomitans, we have used comparative transcriptome profiling to identify differentially expressed genes in the wild-type strain in relation to the PGA-deficient strain. Transcriptome analysis revealed that about 50% of the genes are differently expressed (P < 0.05 and fold change >1.5). Our study demonstrated that the absence of the pga locus affects the genes involved in peptidoglycan recycling, glycogen storage, and virulence. Further, using confocal microscopy and plating assays, we show that the viability of pga mutant strain is significantly reduced during biofilm growth. Thus, this study highlights the importance of pga genes and the exopolysaccharide in the virulence of A. actinomycetemcomitans.