Targeting calcium regulators as therapy for heart failure: focus on the sarcoplasmic reticulum Ca-ATPase pump.

Impaired myocardial Ca2+ cycling is a critical contributor to the development of heart failure (HF), causing changes in the contractile function and structure remodeling of the heart. Within cardiomyocytes, the regulation of sarcoplasmic reticulum (SR) Ca2+ storage and release is largely dependent on Ca2+ handling proteins, such as the SR Ca2+ ATPase (SERCA2a) pump. During the relaxation phase of the cardiac cycle (diastole), SERCA2a plays a critical role in transporting cytosolic Ca2+ back to the SR, which helps to restore both cytosolic Ca2+ levels to their resting state and SR Ca2+ content for the next contraction. However, decreased SERCA2a expression and/or pump activity are key features in HF. As a result, there is a growing interest in developing therapeutic approaches to target SERCA2a. This review provides an overview of the regulatory mechanisms of the SERCA2a pump and explores potential strategies for SERCA2a-targeted therapy, which are being investigated in both preclinical and clinical studies.

Identification and Characterization of p300-Mediated Lysine Residues in Cardiac SERCA2a.

Impaired calcium uptake resulting from reduced expression and activity of the cardiac sarco-endoplasmic reticulum Ca2+ ATPase (SERCA2a) is a hallmark of heart failure (HF). Recently, new mechanisms of SERCA2a regulation, including post-translational modifications (PTMs), have emerged. Our latest analysis of SERCA2a PTMs has identified lysine acetylation as another PTM which might play a significant role in regulating SERCA2a activity. SERCA2a is acetylated, and that acetylation is more prominent in failing human hearts. In this study, we confirmed that p300 interacts with and acetylates SERCA2a in cardiac tissues. Several lysine residues in SERCA2a modulated by p300 were identified using in vitro acetylation assay. Analysis of in vitro acetylated SERCA2a revealed several lysine residues in SERCA2a susceptible to acetylation by p300. Among them, SERCA2a Lys514 (K514) was confirmed to be essential for SERCA2a activity and stability using an acetylated mimicking mutant. Finally, the reintroduction of an acetyl-mimicking mutant of SERCA2a (K514Q) into SERCA2 knockout cardiomyocytes resulted in deteriorated cardiomyocyte function. Taken together, our data demonstrated that p300-mediated acetylation of SERCA2a is a critical PTM that decreases the pump's function and contributes to cardiac impairment in HF. SERCA2a acetylation can be targeted for therapeutic aims for the treatment of HF.

Tough Decoy-Mediated Cardiac Gene Suppression.

MicroRNA (miRNA) is a small, non-coding RNA molecule (~22 nucleotides) that acts as a post-transcriptional gene regulator, primarily by inhibiting the translation of target mRNA transcripts or affecting cell mRNA stability. Since miRNAs are comprehensively involved in gene regulation, their abnormalities are associated with various human diseases, including cardiovascular disease. Additionally, targeted inhibition of disease-related miRNAs and their targets should have therapeutic potential. Therefore, this chapter describes the experimental steps for targeted inhibition of specific miRNAs using adenoviral vectorized tough decoys that efficiently silence miRNA function in cardiac cells.

MicroRNAs in Dystrophinopathy.

Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD), which represent the range of dystrophinopathies, account for nearly 80% of muscle dystrophy. DMD and BMD result from the loss of a functional dystrophin protein, and the leading cause of death in these patients is cardiac remodeling and heart failure. The pathogenesis and progression of the more severe form of DMD have been extensively studied and are controlled by many determinants, including microRNAs (miRNAs). The regulatory role of miRNAs in muscle function and the differential miRNA expression in muscular dystrophy indicate the clinical significance of miRNAs. This review discusses the relevant microRNAs as potential biomarkers and therapeutic targets for DMD and DMD cardiomyopathy as examples of dystrophinopathies.

SUMOylation does not affect cardiac troponin I stability but alters indirectly the development of force in response to Ca2.

Post-translational modification of the myofilament protein troponin I by phosphorylation is known to trigger functional changes that support enhanced contraction and relaxation of the heart. We report for the first time that human troponin I can also be modified by SUMOylation at lysine 177. Functionally, TnI SUMOylation is not a factor in the development of passive and maximal force generation in response to calcium, however this modification seems to act indirectly by preventing SUMOylation of other myofilament proteins to alter calcium sensitivity and cooperativity of myofilaments. Utilising a novel, custom SUMO site-specific antibody that recognises only the SUMOylated form of troponin I, we verify that this modification occurs in human heart and that it is upregulated during disease.

Ubiquitin and Ubiquitin-like Proteins in Cancer, Neurodegenerative Disorders, and Heart Diseases.

Post-translational modification (PTM) is an essential mechanism for enhancing the functional diversity of proteins and adjusting their signaling networks. The reversible conjugation of ubiquitin (Ub) and ubiquitin-like proteins (Ubls) to cellular proteins is among the most prevalent PTM, which modulates various cellular and physiological processes by altering the activity, stability, localization, trafficking, or interaction networks of its target molecules. The Ub/Ubl modification is tightly regulated as a multi-step enzymatic process by enzymes specific to this family. There is growing evidence that the dysregulation of Ub/Ubl modifications is associated with various diseases, providing new targets for drug development. In this review, we summarize the recent progress in understanding the roles and therapeutic targets of the Ub and Ubl systems in the onset and progression of human diseases, including cancer, neurodegenerative disorders, and heart diseases.

MicroRNAs and Calcium Signaling in Heart Disease.

In hearts, calcium (Ca2+) signaling is a crucial regulatory mechanism of muscle contraction and electrical signals that determine heart rhythm and control cell growth. Ca2+ signals must be tightly controlled for a healthy heart, and the impairment of Ca2+ handling proteins is a key hallmark of heart disease. The discovery of microRNA (miRNAs) as a new class of gene regulators has greatly expanded our understanding of the controlling module of cardiac Ca2+ cycling. Furthermore, many studies have explored the involvement of miRNAs in heart diseases. In this review, we aim to summarize cardiac Ca2+ signaling and Ca2+-related miRNAs in pathological conditions, including cardiac hypertrophy, heart failure, myocardial infarction, and atrial fibrillation. We also discuss the therapeutic potential of Ca2+-related miRNAs as a new target for the treatment of heart diseases.

Apoptin gene delivery by a PAMAM dendrimer modified with a nuclear localization signal peptide as a gene carrier for brain cancer therapy.

In this study, we aimed to synthesize PAMAMG3 derivatives (PAMAMG3-KRRR and PAMAMG3-HKRRR), using KRRR peptides as a nuclear localization signal and introduced histidine residues into the KRRR-grafted PAMAMG3 for delivering a therapeutic, carcinoma cell-selective apoptosis gene, apoptin into human primary glioma (GBL-14) cells and human dermal fibroblasts. We examined their cytotoxicity and gene expression using luciferase activity and enhanced green fluorescent protein PAMAMG3 derivatives in both cell lines. We treated cells with PAMAMG3 derivative/apoptin complexes and investigated their intracellular distribution using confocal microscopy. The PAMAMG3-KRRR and PAMAMG3-HKRRR dendrimers were found to escape from endolysosomes into the cytosol. The JC-1 assay, glutathione levels, and Annexin V staining results showed that apoptin triggered cell death in GBL-14 cells. Overall, these findings indicated that the PAMAMG3-HKRRR/apoptin complex is a potential candidate for an effective nonviral gene delivery system for brain tumor therapy in vitro.

Analysis of extracellular vesicle miRNA profiles in heart failure.

Extracellular vesicles (EVs) have recently emerged as an important carrier for various genetic materials including microRNAs (miRs). Growing evidences suggested that several miRs transported by EVs were particularly involved in modulating cardiac function. However, it has remained unclear what miRs are enriched in EVs and play an important role in the pathological condition. Therefore, we established the miR expression profiles in EVs from murine normal and failing hearts and consecutively identified substantially altered miRs. In addition, we have performed bioinformatics approach to predict potential cardiac outcomes through the identification of miR targets. Conclusively, we observed approximately 63% of predicted targets were validated with previous reports. Notably, the predicted targets by this approach were often involved in both beneficial and malicious signalling pathways, which may reflect heterogeneous cellular origins of EVs in tissues. Lastly, there has been an active debate on U6 whether it is a proper control. Through further analysis of EV miR profiles, miR-676 was identified as a superior reference control due to its consistent and abundant expressions. In summary, our results contribute to identifying specific EV miRs for the potential therapeutic targets in heart failure and suggest that miR-676 as a new reference control for the EV miR studies.

Pkm2 Regulates Cardiomyocyte Cell Cycle and Promotes Cardiac Regeneration.

BACKGROUND: The adult mammalian heart has limited regenerative capacity, mostly attributable to postnatal cardiomyocyte cell cycle arrest. In the last 2 decades, numerous studies have explored cardiomyocyte cell cycle regulatory mechanisms to enhance myocardial regeneration after myocardial infarction. Pkm2 (Pyruvate kinase muscle isoenzyme 2) is an isoenzyme of the glycolytic enzyme pyruvate kinase. The role of Pkm2 in cardiomyocyte proliferation, heart development, and cardiac regeneration is unknown. METHODS: We investigated the effect of Pkm2 in cardiomyocytes through models of loss (cardiomyocyte-specific Pkm2 deletion during cardiac development) or gain using cardiomyocyte-specific Pkm2 modified mRNA to evaluate Pkm2 function and regenerative affects after acute or chronic myocardial infarction in mice. RESULTS: Here, we identify Pkm2 as an important regulator of the cardiomyocyte cell cycle. We show that Pkm2 is expressed in cardiomyocytes during development and immediately after birth but not during adulthood. Loss of function studies show that cardiomyocyte-specific Pkm2 deletion during cardiac development resulted in significantly reduced cardiomyocyte cell cycle, cardiomyocyte numbers, and myocardial size. In addition, using cardiomyocyte-specific Pkm2 modified RNA, our novel cardiomyocyte-targeted strategy, after acute or chronic myocardial infarction, resulted in increased cardiomyocyte cell division, enhanced cardiac function, and improved long-term survival. We mechanistically show that Pkm2 regulates the cardiomyocyte cell cycle and reduces oxidative stress damage through anabolic pathways and ß-catenin. CONCLUSIONS: We demonstrate that Pkm2 is an important intrinsic regulator of the cardiomyocyte cell cycle and oxidative stress, and highlight its therapeutic potential using cardiomyocyte-specific Pkm2 modified RNA as a gene delivery platform.

Generation of Ventricular-Like HiPSC-Derived Cardiomyocytes and High-Quality Cell Preparations for Calcium Handling Characterization.

Human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) provide a valuable human source for studying the basic science of calcium (Ca2+) handling and signaling pathways as well as high-throughput drug screening and toxicity assays. Herein, we provide a detailed description of the methodologies used to generate high-quality iPSC-CMs that can consistently reproduce molecular and functional characteristics across different cell lines. Additionally, a method is described to reliably assess their functional characterization through the evaluation of Ca2+ handling properties. Low oxygen (O2) conditions, lactate selection, and prolonged time in culture produce high-purity and high-quality ventricular-like cardiomyocytes. Similar to isolated adult rat cardiomyocytes (ARCMs), 3-month-old iPSC-CMs exhibit higher Ca2+ amplitude, faster rate of Ca2+ reuptake (decay-tau), and a positive lusitropic response to ß-adrenergic stimulation compared to day 30 iPSC-CMs. The strategy is technically simple, cost-effective, and reproducible. It provides a robust platform to model cardiac disease and for the large-scale drug screening to target Ca2+ handling proteins.

Cardioprotective role of APIP in myocardial infarction through ADORA2B.

In ischemic human hearts, the induction of adenosine receptor A2B (ADORA2B) is associated with cardioprotection against ischemic heart damage, but the mechanism underlying this association remains unclear. Apaf-1-interacting protein (APIP) and ADORA2B transcript levels in human hearts are substantially higher in patients with heart failure than in controls. Interestingly, the APIP and ADORA2B mRNA levels are highly correlated with each other (R = 0.912). APIP expression was significantly increased in primary neonatal cardiomyocytes under hypoxic conditions and this induction reduced myocardial cell death via the activation of the AKT-HIF1α pathway. Accordingly, infarct sizes of APIP transgenic mice after left anterior descending artery ligation were significantly reduced compared to those of wild-type mice. Strikingly, knockdown of APIP expression impaired the cytoprotective effects of ADORA2B during hypoxic damage. Immunoprecipitation and proximity ligation assays revealed that APIP interacts with ADORA2B, leading to the stabilization of both proteins by interfering with lysosomal degradation, and to the activation of the downstream PKA-CREB signaling pathways. ADORA2B levels in the hearts of APIPTg/Tg, APIPTg/+, and Apip+/- mice were proportionally downregulated. In addition, ADORA2B D296G derived from the rs200741295 polymorphism failed to bind to APIP and did not exert cardioprotective activity during hypoxia. Moreover, Adora2b D296G knock-in mice were more vulnerable than control mice to myocardial infarction and intentional increases in APIP levels overcame the defective protection of the ADORA2B SNP against ischemic injury. Collectively, APIP is crucial for cardioprotection against myocardial infarction by virtue of binding to and stabilizing ADORA2B, thereby dampening ischemic heart injury.

Role of SIRT1 in Modulating Acetylation of the Sarco-Endoplasmic Reticulum Ca2+-ATPase in Heart Failure.

RATIONALE: SERCA2a, sarco-endoplasmic reticulum Ca2+-ATPase, is a critical determinant of cardiac function. Reduced level and activity of SERCA2a are major features of heart failure. Accordingly, intensive efforts have been made to develop efficient modalities for SERCA2a activation. We showed that the activity of SERCA2a is enhanced by post-translational modification with SUMO1 (small ubiquitin-like modifier 1). However, the roles of other post-translational modifications on SERCA2a are still unknown. OBJECTIVE: In this study, we aim to assess the role of lysine acetylation on SERCA2a function and determine whether inhibition of lysine acetylation can improve cardiac function in the setting of heart failure. METHODS AND RESULTS: The acetylation of SERCA2a was significantly increased in failing hearts of humans, mice, and pigs, which is associated with the reduced level of SIRT1 (sirtuin 1), a class III histone deacetylase. Downregulation of SIRT1 increased the SERCA2a acetylation, which in turn led to SERCA2a dysfunction and cardiac defects at baseline. In contrast, pharmacological activation of SIRT1 reduced the SERCA2a acetylation, which was accompanied by recovery of SERCA2a function and cardiac defects in failing hearts. Lysine 492 (K492) was of critical importance for the regulation of SERCA2a activity via acetylation. Acetylation at K492 significantly reduced the SERCA2a activity, presumably through interfering with the binding of ATP to SERCA2a. In failing hearts, acetylation at K492 appeared to be mediated by p300 (histone acetyltransferase p300), a histone acetyltransferase. CONCLUSIONS: These results indicate that acetylation/deacetylation at K492, which is regulated by SIRT1 and p300, is critical for the regulation of SERCA2a activity in hearts. Pharmacological activation of SIRT1 can restore SERCA2a activity through deacetylation at K492. These findings might provide a novel strategy for the treatment of heart failure.

Role of the PRC2-Six1-miR-25 signaling axis in heart failure.

The reduced expression of cardiac sarco-endoplasmic reticulum Ca2+ ATPase (SERCA2a) is a hallmark of heart failure. We previously showed that miR-25 is a crucial transcriptional regulator of SERCA2a in the heart. However, the precise mechanism of cardiac miR-25 regulation is largely unknown. Literatures suggested that miR-25 is regulated by the transcriptional co-factor, sine oculis homeobox homolog 1 (Six1), which in turn is epigenetically regulated by polycomb repressive complex 2 (PRC 2) in cardiac progenitor cells. Therefore, we aimed to investigate whether Six1 and PRC2 are indeed involved in the regulation of the miR-25 level in the setting of heart failure. Six1 was up-regulated in the failing hearts of humans and mice. Overexpression of Six1 led to adverse cardiac remodeling, whereas knock-down of Six1 attenuated pressure overload-induced cardiac dysfunction. The adverse effects of Six1 were ameliorated by knock-down of miR-25. The epigenetic repression on the Six1 promoter by PRC2 was significantly reduced in failing hearts. Epigenetic repression of Six1 is relieved through a reduction of PRC2 activity in heart failure. Six1 up-regulates miR-25, which is followed by reduction of cardiac SERCA2a expression. Collectively, these data showed that the PRC2-Six1-miR-25 signaling axis is involved in heart failure. Our finding introduces new insight into potential treatments of heart failure.

Experimental models of cardiac physiology and pathology.

Experimental models of cardiac disease play a key role in understanding the pathophysiology of the disease and developing new therapies. The features of the experimental models should reflect the clinical phenotype, which can have a wide spectrum of underlying mechanisms. We review characteristics of commonly used experimental models of cardiac physiology and pathophysiology in all translational steps including in vitro, small animal, and large animal models. Understanding their characteristics and relevance to clinical disease is the key for successful translation to effective therapies.

miR-146a Suppresses SUMO1 Expression and Induces Cardiac Dysfunction in Maladaptive Hypertrophy.

RATIONALE: Abnormal SUMOylation has emerged as a characteristic of heart failure (HF) pathology. Previously, we found reduced SUMO1 (small ubiquitin-like modifier 1) expression and SERCA2a (sarcoplasmic reticulum Ca2+-ATPase) SUMOylation in human and animal HF models. SUMO1 gene delivery or small molecule activation of SUMOylation restored SERCA2a SUMOylation and cardiac function in HF models. Despite the critical role of SUMO1 in HF, the regulatory mechanisms underlying SUMO1 expression are largely unknown. OBJECTIVE: To examine miR-146a-mediated SUMO1 regulation and its consequent effects on cardiac morphology and function. METHODS AND RESULTS: In this study, miR-146a was identified as a SUMO1-targeting microRNA in the heart. A strong correlation was observed between miR-146a and SUMO1 expression in failing mouse and human hearts. miR-146a was manipulated in cardiomyocytes through AAV9 (adeno-associated virus serotype 9)-mediated gene delivery, and cardiac morphology and function were analyzed by echocardiography and hemodynamics. Overexpression of miR-146a reduced SUMO1 expression, SERCA2a SUMOylation, and cardiac contractility in vitro and in vivo. The effects of miR-146a inhibition on HF pathophysiology were examined by transducing a tough decoy of miR-146a into mice subjected to transverse aortic constriction. miR-146a inhibition improved cardiac contractile function and normalized SUMO1 expression. The regulatory mechanisms of miR-146a upregulation were elucidated by examining the major miR-146a-producing cell types and transfer mechanisms. Notably, transdifferentiation of fibroblasts triggered miR-146a overexpression and secretion through extracellular vesicles, and the extracellular vesicle-associated miR-146a transfer was identified as the causative mechanism of miR-146a upregulation in failing cardiomyocytes. Finally, extracellular vesicles isolated from failing hearts were shown to contain high levels of miR-146a and exerted negative effects on the SUMO1/SERCA2a signaling axis and hence cardiomyocyte contractility. CONCLUSIONS: Taken together, our results show that miR-146a is a novel regulator of the SUMOylation machinery in the heart, which can be targeted for therapeutic intervention.

Acute Left Ventricular Unloading Reduces Atrial Stretch and Inhibits Atrial Arrhythmias.

BACKGROUND: Left atrium (LA) physiology is influenced by changes in left ventricular (LV) performance and load. OBJECTIVES: The purpose of this study was to define the effect of acute changes in LV loading conditions on LA physiology in subacute myocardial infarction (MI). METHODS: MI was percutaneously induced in 19 Yorkshire pigs. One to 2 weeks after MI, 14 pigs underwent acute LV unloading using a percutaneous LV assist device, Impella. The remaining 5 pigs underwent acute LV loading by percutaneous induction of aortic regurgitation. A pressure-volume catheter was inserted into the LA using a percutaneous transseptal approach, and LA pressure-volume loops were continuously monitored. Atrial arrhythmia inducibility was examined by burst-pacing of the right atrium. Nicotinamide adenine dinucleotide phosphate oxidase (NOX) levels and ryanodine receptor phosphorylation were examined in LA tissues to study the potential effect of stretch-dependent oxidative stress. RESULTS: MI resulted in reduced LV ejection fraction and increased LV end-diastolic pressure with concomitant increase in LA pressure and volumes. Acute LV unloading resulted in a reduction of LV end-diastolic pressure, which led to proportional decreases in mean LA pressure and maximum LA volume. LA pressure-volume loops exhibited a pump flow-dependent, left-downward shift. This was associated with reduced LA passive stiffness, suggesting the alleviation of the LA stretch that was present after MI. Prior to acute unloading of the LV, 71% of the pigs were arrhythmia-inducible; LV unloading reduced this to 29% (p = 0.02). Time to spontaneous termination of atrial arrhythmias was decreased from median 55 s (range 5 to 300 s) to 3 s (range 0 to 59 s). In contrast, acute LV loading with aortic regurgitation increased LA pressure without a significant effect on arrhythmogenicity. Molecular analysis of LA tissue revealed that NOX2 expression was increased after MI, whereas acute LV unloading reduced NOX2 levels and diminished ryanodine receptor phosphorylation. CONCLUSIONS: Acute LV unloading relieves LA stretch and reduces atrial arrhythmogenicity in subacute MI.