Decreased SERCA2a Expression Research Articles

Abstract 14399: Novel Mechanisms of Diastolic Dysfunction in Cardiac Aging

Aging is associated with a progressive decline in cardiac diastolic function. Membranous α-klotho, predominantly produced by the kidney, can be cleaved and form circulating α-klotho, which acts as an anti-aging hormone. Echocardiography showed klotho deficiency in 20-24-month-old heterozygous klotho (KL +/- ) mice aggravates left ventricular hypertrophy (LVH) and diastolic dysfunction (DD) compared with old wild type (OWT) littermates. Supplementation with α-Klotho (0.1mg/kg/day. i.p) for 10 weeks significantly reduced LV mass and ameliorated DD in both OWT and OKL. The improvement in DD was confirmed by Millar catheter measurement of -dP/dt. To gain mechanistic insights, we performed unbiased acetylomics using Acetyl-lysine antibody enrichment, followed by Tandem mass tag proteomics analysis. We identified 1215 lysine acetylation sites (K-Ac) on >1000 cardiac proteins, of which 220 K-Ac sites were significantly hyperacetylated in OWT hearts. There were 63 Ac-K sites >2-fold hyperacetylated in aged hearts (OWT vs. YWT). The comparison of OKL to YWT augmented the number of Ac-K sites to 82. These sites include redox proteins, several metabolic and mitochondrial proteins involved in fatty acid and amino acid metabolism, and the TCA cycle. Several isoforms of myosin heavy chains (MHC) were also hyperacetylated at multiple sites. Some hyperacetylated MHC (e.g. K941) are predicted to alter the molecular structure and relaxation kinetics by Molecular Dynamic Simulation. In addition to delayed Ca 2+ reuptake by myocardial sarcoplasmic reticulum ATPase (SERCA2a), decreased SERCA2a expression and increased acetylated SERCA2 in aged hearts, which was all corrected upon klotho supplementation, we found potential novel mechanisms of DD with hyperacetylation of MHC, concomitant with a decrease in Sirt1 deacetylase, in OWT mice that was aggravated in OKL, which was replenished upon klotho supplementation. Collectively, we demonstrated klotho deficiency aggravates cardiac aging phenotypes, associated with hyperacetylation of several cardiac proteins due to diminished Sirt1. Klotho supplementation rejuvenates several cardiac aging phenotypes, including regression of LVH and improvement of DD, in parallel with reversal of protein acetylation.

Abstract 530: Mechanisms Underlying Phospholamban L39 Stop (PLN L39X) Cardiomyopathy

Background: Defective calcium (Ca++) handling is a hallmark of HF across species. Together with the Sarco/Endoplasmic Reticulum Ca-ATPase (SERCA2a), Phospholamban (PLN) has emerged as a critical regulator of Ca++homeostasis. Worldwide, PLN mutations are identified with increasing frequency in patients with dilated, hypertrophic and arrhythmogenic cardiomyopathy (CMPs) but the causative defects leading to the CMP remain incompletely understood. While preclinical studies have unequivocally shown that absence of PLN (PLNKO) is therapeutic in rodent HF models, the discovery of a human pathogenic mutation (L39X) presumed to be the human equivalent of the PLNKO lead to the conclusion that PLN ablation was lethal in human, mitigating any enthusiasm in targeting PLN inhibition in PLN-associated disease or HF treatment. The objective of this proposal is to levarge the use of “induced-pluripotent” stem cells (iPSCs) derived cardiomyocytes (CM) from homozygote L39X carriers to elucidate the role of PLN (L39) in human pathophysiology. Methodology and Results: We obtained mononuclear cells from Homozygotes (Hom) L39 carriers and generated 11 iPSC clones. To derive CMs, we used the direct differentiation method temporally modulating the Wnt/β-catenin signaling. Immunocytostaining revealed positive expression of cardiac troponin T as well as PLN. In Hom L39 derived CMs, PLN showed an abnormal cytoplasmic distribution, formed intracellular aggregates and there was loss of perinuclear localization when compared to matched WT iPSC-CMs. Using fura-2AM, we observed decreased calcium transient amplitude in iPSC-CMs from L39 compared to WT with prolongation of the time constant of relaxation and early after depolarization (EAD). Lastly, we saw a 70% and 50% reduction in the protein and mRNA expression of PLN and SERCA2a respectively. Conclusions: Our data suggest that the L39 PLN mutant is expressed but mis-located within the cardiomyocytes. The mis-location of PLN was associated to decreased SERCA2a expression impaired Ca++ handling and increased arrhythmogenicity. Further studies will be required to fully elucidate the impact of the mutation in HF pathophysiology

Roles of sarcoplasmic reticulum Ca2+ ATPase pump in the impairments of lymphatic contractile activity in a metabolic syndrome rat model

The intrinsic lymphatic contractile activity is necessary for proper lymph transport. Mesenteric lymphatic vessels from high-fructose diet-induced metabolic syndrome (MetSyn) rats exhibited impairments in its intrinsic phasic contractile activity; however, the molecular mechanisms responsible for the weaker lymphatic pumping activity in MetSyn conditions are unknown. Several metabolic disease models have shown that dysregulation of sarcoplasmic reticulum Ca2+ ATPase (SERCA) pump is one of the key determinants of the phenotypes seen in various muscle tissues. Hence, we hypothesized that a decrease in SERCA pump expression and/or activity in lymphatic muscle influences the diminished lymphatic vessel contractions in MetSyn animals. Results demonstrated that SERCA inhibitor, thapsigargin, significantly reduced lymphatic phasic contractile frequency and amplitude in control vessels, whereas, the reduced MetSyn lymphatic contractile activity was not further diminished by thapsigargin. While SERCA2a expression was significantly decreased in MetSyn lymphatic vessels, myosin light chain 20, MLC20 phosphorylation was increased in these vessels. Additionally, insulin resistant lymphatic muscle cells exhibited elevated intracellular calcium and decreased SERCA2a expression and activity. The SERCA activator, CDN 1163 partially restored lymphatic contractile activity in MetSyn lymphatic vessel by increasing phasic contractile frequency. Thus, our data provide the first evidence that SERCA2a modulates the lymphatic pumping activity by regulating phasic contractile amplitude and frequency, but not the lymphatic tone. Diminished lymphatic contractile activity in the vessels from the MetSyn animal is associated with the decreased SERCA2a expression and impaired SERCA2 activity in lymphatic muscle.

Loss of myocardial retinoic acid receptor α induces diastolic dysfunction by promoting intracellular oxidative stress and calcium mishandling in adult mice

Retinoic acid receptor (RAR) has been implicated in pathological stimuli-induced cardiac remodeling. To determine whether the impairment of RARα signaling directly contributes to the development of heart dysfunction and the involved mechanisms, tamoxifen-induced myocardial specific RARα deletion (RARαKO) mice were utilized. Echocardiographic and cardiac catheterization studies showed significant diastolic dysfunction after 16wks of gene deletion. However, no significant differences were observed in left ventricular ejection fraction (LVEF), between RARαKO and wild type (WT) control mice. DHE staining showed increased intracellular reactive oxygen species (ROS) generation in the hearts of RARαKO mice. Significantly increased NOX2 (NADPH oxidase 2) and NOX4 levels and decreased SOD1 and SOD2 levels were observed in RARαKO mouse hearts, which were rescued by overexpression of RARα in cardiomyocytes. Decreased SERCA2a expression and phosphorylation of phospholamban (PLB), along with decreased phosphorylation of Akt and Ca2+/calmodulin-dependent protein kinase II δ (CaMKII δ) was observed in RARαKO mouse hearts. Ca2+ reuptake and cardiomyocyte relaxation were delayed by RARα deletion. Overexpression of RARα or inhibition of ROS generation or NOX activation prevented RARα deletion-induced decrease in SERCA2a expression/activation and delayed Ca2+ reuptake. Moreover, the gene and protein expression of RARα was significantly decreased in aged or metabolic stressed mouse hearts. RARα deletion accelerated the development of diastolic dysfunction in streptozotocin (STZ)-induced type 1 diabetic mice or in high fat diet fed mice. In conclusion, myocardial RARα deletion promoted diastolic dysfunction, with a relative preserved LVEF. Increased oxidative stress have an important role in the decreased expression/activation of SERCA2a and Ca2+ mishandling in RARαKO mice, which are major contributing factors in the development of diastolic dysfunction. These data suggest that impairment of cardiac RARα signaling may be a novel mechanism that is directly linked to pathological stimuli-induced diastolic dysfunction.

Phospholamban-Independent Adrenergic Reserve in SERCA2 Ablated Hearts

Normal cardiac contraction and relaxation requires efficient operation of the SR Ca2+ ATPase, SERCA2a. In failing hearts, decreased SERCA2a expression and slowed SR Ca2+ reuptake are thought to significantly underlie contractile dysfunction. We have studied the relationship between decreased SERCA2a expression and cardiac function using a mouse model of conditional cardiac Serca2 ablation, the Serca2fl/fl mouse. In this model, Cre activation by tamoxifen injection efficiently deletes Serca2 from the heart; four weeks post-knockout, cardiac SERCA2a protein content is below 5% of normal levels. Serca2 KO mice survive 7-10 weeks post knockout with only mild in vivo impairment prior to this time, suggesting that this loss of SERCA2a protein can be temporarily compensated. We found that isolated SERCA2a KO hearts retained the ability to respond to s-adrenergic stimulation ex vivo despite the loss of most SERCA2a protein. Because the protein regulator of SERCA2a, phospholamban (PLN), is thought to be a major component of the cardiac s-adrenergic response, this finding was unexpected and warranted further detailed investigation. To identify the mechanism underlying this preserved s-adrenergic response in SERCA2-depleted hearts, we bred Serca2fl/fl and PLN-/- mice to generate an inducible Serca2 knockout mouse line lacking PLN. Serca2fl/fl;PLN-/- mice were injected with tamoxifen to induce Serca2 gene disruption, and isolated hearts from Serca2KO;PLN-/- (“DKO”) mice were evaluated by Langendorff perfusion 4-5 weeks after Serca2 knockout. Control PLN-/- hearts show little response to s-adrenergic stimulation, and DKO hearts have impaired contractility under baseline conditions. DKO hearts, however, show improved systolic and diastolic function when perfused with 50 nM isoproterenol, indicating that targets of s-adrenergic signaling beyond PLN are able to support a significant inotropic and lusitropic response when cardiac contractility is impaired. We will discuss how these findings support new mechanisms underlying heart performance during stress and disease.

Sarcoplasmic Reticulum-Independent Contractile Function in Serca2 Ablated Hearts

Effective contraction and relaxation of the heart requires the robust transport of Ca2+ to and from the sarcoplasmic reticulum (SR). The cardiac SR Ca-ATPase, SERCA2a, is responsible for sequestering Ca2+ to the SR during the contractile cycle, and has diminished expression and function in failing hearts. We have used a model of inducible cardiac-specific Serca2 gene disruption, the Serca2fl/fl mouse, to probe the relationship between decreased SERCA2a expression and cardiac dysfunction. 4 weeks after gene disruption, SERCA2a protein content in KO hearts is less than 5% of control. Despite severely impaired ex vivo function of isolated hearts and cardiomyocytes at this time, in vivo heart function is mildly impaired and KO mice survive until 7-10 weeks post-knockout, indicating that loss of SERCA2a is temporarily compensated. This finding of preserved cardiac function in vivo in the context of severe SERCA2a depletion is surprising and warrants a detailed dissection of potential compensatory mechanisms. Published studies have found that sarcolemmal Ca2+ transport mechanisms such as L-type Ca2+ current, Na-Ca exchange, and plasma membrane Ca-ATPase activity are increased following this loss of SERCA2a protein. These observations prompt the hypothesis that sarcolemmal Ca2+ transport remodeling may partially compensate for diminished SR Ca2+ handling in KO hearts. We have begun to investigate this hypothesis by perfusing isolated Serca2 KO hearts with caffeine to evaluate SR-independent contractile function. During caffeine perfusion, left ventricular (LV) end-diastolic pressures were lower in KO hearts than controls consistent with the reduced SR Ca2+ stores in KO myocytes. Interestingly, during prolonged caffeine perfusion, systolic performance was similar in control and KO hearts. This is functional evidence that trans-sarcolemmal transient adaptations in KO hearts are not of sufficient magnitude to differentiate KO from WT when SR function is disabled by caffeine.

Increasing Glutamate Promotes Ischemia-Reperfusion-Induced Ventricular Arrhythmias in Rats in vivo

Background: Glutamate mediates cerebral ischemia injury via N-methyl-<smlcap>D</smlcap>-aspartate (NMDA) receptor-coupled ion channels, but the activities of glutamate in the heart remain unclear. Aims: To investigate whether or not glutamate contributes to ischemia- and reperfusion (IR)-induced arrhythmias. Methods: Myocardial IR was induced by occlusion of the left anterior descending coronary artery for 30 min and reperfusion for another 30 min. A score system was used to quantify arrhythmias. MK801 (a noncompetitive NMDA receptor antagonist), dihydrokainate (DHK, a glutamate transporter inhibitor) and gabapentin (GBP, a glutamate release inhibitor) were used before ischemia. Serum glutamate levels, Ca²⁺-ATPase activity, SERCA2a protein expression and myocardial mitochondrial Ca²⁺ content were assayed. Results: Myocardial IR caused a significant increase in serum glutamate and high incidences of ventricular arrhythmias. GBP and MK801 significantly ameliorated ventricular arrhythmias, improved SERCA2a expression and sarcoplasmic reticulum Ca²⁺-ATPase activity and reduced Ca²⁺ accumulated in mitochondria. By contrast, DHK significantly exacerbated reperfusion-related arrhythmias and mitochondrial Ca²⁺ overload while it decreased SERCA2a expression and activity. Conclusion: This study showed that glutamate mediates reperfusion arrhythmias, and the corresponding mechanism may be associated with Ca²⁺ overload via the NMDA receptor. Reperfusion arrhythmias may be prevented by inhibiting the release of glutamate or by antagonizing NMDA receptors.

Carbon Monoxide Pollution Promotes Cardiac Remodeling and Ventricular Arrhythmia in Healthy Rats

Epidemiologic studies associate atmospheric carbon monoxide (CO) pollution with adverse cardiovascular outcomes and increased cardiac mortality risk. However, there is a lack of data regarding cellular mechanisms in healthy individuals. To investigate the chronic effects of environmentally relevant CO levels on cardiac function in a well-standardized healthy animal model. Wistar rats were exposed for 4 weeks to filtered air (CO < 1 ppm) or air enriched with CO (30 ppm with five peaks of 100 ppm per 24-h period), consistent with urban pollution. Myocardial function was assessed by echocardiography and analysis of surface ECG and in vitro by measuring the excitation-contraction coupling of single left ventricular cardiomyocytes. Chronic CO pollution promoted left ventricular interstitial and perivascular fibrosis, with no change in cardiomyocyte size, and had weak, yet significant, effects on in vivo cardiac function. However, both contraction and relaxation of single cardiomyocytes were markedly altered. Several changes occurred, including decreased Ca(2+) transient amplitude and Ca(2+) sensitivity of myofilaments and increased diastolic intracellular Ca(2+) subsequent to decreased SERCA-2a expression and impaired Ca(2+) reuptake. CO pollution increased the number of arrhythmic events. Hyperphosphorylation of Ca(2+)-handling and sarcomeric proteins, and reduced responses to beta-adrenergic challenge were obtained, suggestive of moderate CO-induced hyperadrenergic state. Chronic CO exposure promotes a pathological phenotype of cardiomyocytes in the absence of underlying cardiomyopathy. The less severe phenotype in vivo suggests a role for compensatory mechanisms. Arrhythmia propensity may derive from intracellular Ca(2+) overload.

Deletion of the Inducible 70-kDa Heat Shock Protein Genes in Mice Impairs Cardiac Contractile Function and Calcium Handling Associated With Hypertrophy

Hspa1a and Hspa1b genes encode stress-inducible 70-kDa heat shock proteins (Hsp70) that protect cells from insults such as ischemia. Mice with null mutations of both genes (KO) were generated, and their cardiac phenotype was explored. Heart rate and blood pressures were normal in the KO mice. Hearts from KO mice were more susceptible to both functional and cellular damage by ischemia/reperfusion. Cardiac hypertrophy developed in Hsp70-KO mice. Ca2+ transients in cardiomyocytes of KO mice showed a delayed (120%) calcium decline and decreased sarcoplasmic reticulum calcium content. Cell shortening was decreased by 35%, and rates of contraction and relaxation were slower by 40%. These alterations can be attributed to the absence of Hsp70 because viral expression of Hsp70 in KO cultured cardiomyocytes restored these parameters. One mechanism underlying myocyte dysfunction could be decreased SERCA2a expression. This hypothesis was supported by a prolonged calcium decline and decreased SERCA2a protein. Viral SERCA2a expression restored contractility and Ca2+ transients. We examined the involvement of Jun N-terminal kinase (JNK), p38-mitogen-activated protein kinase (p38-MAPK), Raf-1, and extracellular signal-regulated kinase (ERK) in SERCA2a downregulation and the cardiac phenotype of KO mice. Levels of phosphorylated JNK, p38-MAPK, Raf-1, and ERK were elevated in KO hearts. Activation of the Raf-1-ERK pathway in normal cardiomyocytes resulted in decreased SERCA2a. Absence of Hsp70 leads to dysfunctional cardiomyocytes and impaired stress response of Hsp70-KO hearts against ischemia/reperfusion. In addition, deletion of Hsp70 genes might induce cardiac dysfunction and development of cardiac hypertrophy through the activation of JNK, p38-MAPK, Raf-1, and ERK.

Diastolic Dysfunction in Human Cardiac Allografts is Related with Reduced SERCA2a Gene Expression

Previous studies demonstrated that impaired left ventricular (LV) relaxation in cardiac allografts limits exercise tolerance post-transplant despite preserved systolic ejection fraction (EF). This study tested in human cardiac allografts whether the isovolumic relaxation time (IVRT), which provides the basis for most of diastolic LV filling, relates with gene expression of regulatory proteins of calcium homeostasis or cardiac matrix proteins. Gene expression was studied in 31 heart transplant recipients (25 male, 6 female) 13-83 months post-transplant with LVEF >50%, LV end-diastolic pressure <20 mmHg, normal LV mass index and without allograft rejection or significant cardiac pathology. IVRT related with the other diastolic parameters e-wave velocity (r = -0.46; p = 0.01), e/a-wave ratio (r = -0.5; p < 0.01) but not with heart frequency (r = -0.16; p = 0.4). No relation of IVRT was observed for immunosuppression, mean rejection grade or other medication. IVRT was not related with gene expression of desmin, collagen I, phospholamban, the Na+-Ca2+ exchanger, the ryanodine receptor or interstitial fibrosis but correlated inversely with SERCA2a (r = -0.48; p = 0.02). Prolonged IVRT is associated with decreased SERCA2a expression in cardiac allografts without significant other pathology. Similar observations in non-transplanted patients with diastolic failure suggest that decreased SERCA2a expression is an important common pathomechanism.

Inhibition of maladaptive cardiac hypertrophy by the Ras effector protein Rlf

The monomeric G-protein Ras has been shown to have an obligatory role in the cardiac hypertrophic response by coordinating the activity of the mitogen activated protein kinases (MAPKs). Ras activation of ERK is associated with an adaptive hypertrophic response while JNK activation is associated with maladaptive hypertrophy. We identified the Ral guanine nucleotide dissociation stimulator like factor (Rlf) as a protein that binds to and interacts with Ras in cardiomyocytes. To understand the role of Rlf in regulating cardiomyocyte signaling, we assessed MAPK activation in neonatal rat ventricular myocytes (NRVMs) infected with adenoviruses encoding Rlf, or co-infected with constitutively active (V12-Ras) and Rlf. Rlf overexpression in NRVMs decreased agonist (endothelin-1 or phenylephrine) and V12-Ras mediated JNK activation without significantly altering ERK activation. To determine the effect of Rlf on cardiac hypertrophy in vivo, we generated transgenic mice with cardiac-specific overexpression of Rlf. Transgenic and nontransgenic male mice were treated with isoproteronol (ISO; 30 mg/kg/day) for 14 days. ISO treatment increased heart weight in transgenic and nontransgenic littermates to a similar extent. However, the hearts of Rlf transgenic mice treated with ISO displayed decreased fibrosis and myocardial cell loss compared to nontransgenic mice. Furthermore, Rlf transgenic mice were protected from ISO-mediated decreased SERCA2a expression. Overall, our results demonstrate that Rlf attenuates Ras-mediated activation of the JNK pathway and protects from maladaptive cardiac hypertrophy. (Supported by grants from the NIH and AHA)