SPRR1A is a key downstream effector of MiR-150 during both maladaptive cardiac remodeling in mice and human cardiac fibroblast activation.

MicroRNA-150 (miR-150) is conserved between rodents and humans, is significantly downregulated during heart failure (HF), and correlates with patient outcomes. We previously reported that miR-150 is protective during myocardial infarction (MI) in part by decreasing cardiomyocyte (CM) apoptosis and that proapoptotic small proline-rich protein 1a (Sprr1a) is a direct CM target of miR-150. We also showed that Sprr1a knockdown in mice improves cardiac dysfunction and fibrosis post-MI and that Sprr1a is upregulated in pathological mouse cardiac fibroblasts (CFs) from ischemic myocardium. However, the direct functional relationship between miR-150 and SPRR1A during both post-MI remodeling in mice and human CF (HCF) activation was not established. Here, using a novel miR-150 knockout;Sprr1a-hypomorphic (Sprr1ahypo/hypo) mouse model, we demonstrate that Sprr1a knockdown blunts adverse post-MI effects caused by miR-150 loss. Moreover, HCF studies reveal that SPRR1A is upregulated in hypoxia/reoxygenation-treated HCFs and is downregulated in HCFs exposed to the cardioprotective ß-blocker carvedilol, which is inversely associated with miR-150 expression. Significantly, we show that the protective roles of miR-150 in HCFs are directly mediated by functional repression of profibrotic SPRR1A. These findings delineate a pivotal functional interaction between miR-150 and SPRR1A as a novel regulatory mechanism pertinent to CF activation and ischemic HF.

Financial Impact of the Indiana University Student-Run Free Clinic.

Student-run free clinics (SRFCs) serve as an important public safety-net for un- and under-insured patients. Few studies have investigated their financial impact or return of investment to the community. The aim of this study was to estimate the financial impact of the Indiana University Student Outreach Clinic (IUSOC) using national market values of medical visits and lab services in 2021.From internal triage classification records maintained from March to December 2021, Current Procedural Terminology (CPT) codes were assigned corresponding to "New Patient," "Returning Quick or Long" and "Fast Track" visits. Total Work Relative Value Units (wRVUs) were calculated to then estimate the total monetary value for medical services. Similarly, the collection of laboratory services rendered from March to December 2021 were assigned costs using the 2021 Center for Medicare and Medicaid Services (CMS) fee schedule.There were a total of 1475 medical visits identified in this study under the triage categories of interest, with 440 New Patient visits, 1032 Returning Quick and Long visits, and 3 Fast Track visits. From the calculated total wRVUs, the estimated monetary value for medical services was $95,413.79. Additionally, there were 3633 eligible lab tests ordered; almost half of these (1523, 41.9%) were categorized as routine lab tests (CMP, BMP, CBC, lipid panel). The estimated value of lab services was $56,296.81.Our estimates reveal that the IUSOC provided nearly $150,000 worth of free healthcare to the community in 2021. Further, most patients were seen for return visits, revealing the opportunity to streamline logistics.

Noncoding RNAs as Key Regulators for Cardiac Development and Cardiovascular Diseases.

Noncoding RNAs (ncRNAs) play fundamental roles in cardiac development and cardiovascular diseases (CVDs), which are a major cause of morbidity and mortality. With advances in RNA sequencing technology, the focus of recent research has transitioned from studies of specific candidates to whole transcriptome analyses. Thanks to these types of studies, new ncRNAs have been identified for their implication in cardiac development and CVDs. In this review, we briefly describe the classification of ncRNAs into microRNAs, long ncRNAs, and circular RNAs. We then discuss their critical roles in cardiac development and CVDs by citing the most up-to-date research articles. More specifically, we summarize the roles of ncRNAs in the formation of the heart tube and cardiac morphogenesis, cardiac mesoderm specification, and embryonic cardiomyocytes and cardiac progenitor cells. We also highlight ncRNAs that have recently emerged as key regulators in CVDs by focusing on six of them. We believe that this review concisely addresses perhaps not all but certainly the major aspects of current progress in ncRNA research in cardiac development and CVDs. Thus, this review would be beneficial for readers to obtain a recent picture of key ncRNAs and their mechanisms of action in cardiac development and CVDs.

MiR-150 blunts cardiac dysfunction in mice with cardiomyocyte loss of ß1-adrenergic receptor/ß-arrestin signaling and controls a unique transcriptome.

The ß1-adrenergic receptor (ß1AR) is found primarily in hearts (mainly in cardiomyocytes [CMs]) and ß-arrestin-mediated ß1AR signaling elicits cardioprotection through CM survival. We showed that microRNA-150 (miR-150) is upregulated by ß-arrestin-mediated ß1AR signaling and that CM miR-150 inhibits maladaptive remodeling post-myocardial infarction. Here, we investigate whether miR-150 rescues cardiac dysfunction in mice bearing CM-specific abrogation of ß-arrestin-mediated ß1AR signaling. Using CM-specific transgenic (TG) mice expressing a mutant ß1AR (G protein-coupled receptor kinase [GRK]-ß1AR that exhibits impairment in ß-arrestin-mediated ß1AR signaling), we first generate a novel double TG mouse line overexpressing miR-150. We demonstrate that miR-150 is sufficient to improve cardiac dysfunction in CM-specific GRK-ß1AR TG mice following chronic catecholamine stimulation. Our genome-wide circular RNA, long noncoding RNA (lncRNA), and mRNA profiling analyses unveil a subset of cardiac ncRNAs and genes as heretofore unrecognized mechanisms for beneficial actions of ß1AR/ß-arrestin signaling or miR-150. We further show that lncRNA Gm41664 and GDAP1L1 are direct novel upstream and downstream regulators of miR-150. Lastly, CM protective actions of miR-150 are attributed to repressing pro-apoptotic GDAP1L1 and are mitigated by pro-apoptotic Gm41664. Our findings support the idea that miR-150 contributes significantly to ß1AR/ß-arrestin-mediated cardioprotection by regulating unique ncRNA and gene signatures in CMs.

MiR-150 Attenuates Maladaptive Cardiac Remodeling Mediated by Long Noncoding RNA MIAT and Directly Represses Profibrotic Hoxa4.

BACKGROUND: MicroRNA-150 (miR-150) plays a protective role in heart failure (HF). Long noncoding RNA, myocardial infarction-associated transcript (MIAT) regulates miR-150 function in vitro by direct interaction. Concurrent with miR-150 downregulation, MIAT is upregulated in failing hearts, and gain-of-function single-nucleotide polymorphisms in MIAT are associated with increased risk of myocardial infarction (MI) in humans. Despite the correlative relationship between MIAT and miR-150 in HF, their in vivo functional relationship has never been established, and molecular mechanisms by which these 2 noncoding RNAs regulate cardiac protection remain elusive. METHODS: We use MIAT KO (knockout), Hoxa4 (homeobox a4) KO, MIAT TG (transgenic), and miR-150 TG mice. We also develop DTG (double TG) mice overexpressing MIAT and miR-150. We then use a mouse model of MI followed by cardiac functional, structural, and mechanistic studies by echocardiography, immunohistochemistry, transcriptome profiling, Western blotting, and quantitative real-time reverse transcription-polymerase chain reaction. Moreover, we perform expression analyses in hearts from patients with HF. Lastly, we investigate cardiac fibroblast activation using primary adult human cardiac fibroblasts and in vitro assays to define the conserved MIAT/miR-150/HOXA4 axis. RESULTS: Using novel mouse models, we demonstrate that genetic overexpression of MIAT worsens cardiac remodeling, while genetic deletion of MIAT protects hearts against MI. Importantly, miR-150 overexpression attenuates the detrimental post-MI effects caused by MIAT. Genome-wide transcriptomic analysis of MIAT null mouse hearts identifies Hoxa4 as a novel downstream target of the MIAT/miR-150 axis. Hoxa4 is upregulated in cardiac fibroblasts isolated from ischemic myocardium and subjected to hypoxia/reoxygenation. HOXA4 is also upregulated in patients with HF. Moreover, Hoxa4 deficiency in mice protects the heart from MI. Lastly, protective actions of cardiac fibroblast miR-150 are partially attributed to the direct and functional repression of profibrotic Hoxa4. CONCLUSIONS: Our findings delineate a pivotal functional interaction among MIAT, miR-150, and Hoxa4 as a novel regulatory mechanism pertinent to ischemic HF.

Interactions between noncoding RNAs as epigenetic regulatory mechanisms in cardiovascular diseases.

Cardiovascular diseases (CVDs) represent the foremost cause of mortality in the United States and worldwide. It is estimated that CVDs account for approximately 17.8 million deaths each year. Despite the advances made in understanding cellular mechanisms and gene mutations governing the pathophysiology of CVDs, they remain a significant cause of mortality and morbidity. A major segment of mammalian genomes encodes for genes that are not further translated into proteins. The roles of the majority of such noncoding ribonucleic acids (RNAs) have been puzzling for a long time. However, it is becoming increasingly clear that noncoding RNAs (ncRNAs) are dynamically expressed in different cell types and have a comprehensive selection of regulatory roles at almost every step involved in DNAs, RNAs and proteins. Indeed, ncRNAs regulate gene expression through epigenetic interactions, through direct binding to target sequences, or by acting as competing endogenous RNAs. The profusion of ncRNAs in the cardiovascular system suggests that they may modulate complex regulatory networks that govern cardiac physiology and pathology. In this review, we summarize various functions of ncRNAs and highlight the recent literature on interactions between ncRNAs with an emphasis on cardiovascular disease regulation. Furthermore, as the broad-spectrum of ncRNAs potentially establishes new avenues for therapeutic development targeting CVDs, we discuss the innovative prospects of ncRNAs as therapeutic targets for CVDs.

Cardiomyocyte microRNA-150 confers cardiac protection and directly represses proapoptotic small proline-rich protein 1A.

MicroRNA-150 (miR-150) is downregulated in patients with multiple cardiovascular diseases and in diverse mouse models of heart failure (HF). miR-150 is significantly associated with HF severity and outcome in humans. We previously reported that miR-150 is activated by ß-blocker carvedilol (Carv) and plays a protective role in the heart using a systemic miR-150 KO mouse model. However, mechanisms that regulate cell-specific miR-150 expression and function in HF are unknown. Here, we demonstrate that potentially novel conditional cardiomyocyte-specific (CM-specific) miR-150 KO (miR-150 cKO) in mice worsens maladaptive cardiac remodeling after myocardial infarction (MI). Genome-wide transcriptomic analysis in miR-150 cKO mouse hearts identifies small proline-rich protein 1a (Sprr1a) as a potentially novel target of miR-150. Our studies further reveal that Sprr1a expression is upregulated in CMs isolated from ischemic myocardium and subjected to simulated ischemia/reperfusion, while its expression is downregulated in hearts and CMs by Carv. We also show that left ventricular SPRR1A is upregulated in patients with HF and that Sprr1a knockdown in mice prevents maladaptive post-MI remodeling. Lastly, protective roles of CM miR-150 are, in part, attributed to the direct and functional repression of proapoptotic Sprr1a. Our findings suggest a crucial role for the miR-150/SPRR1A axis in regulating CM function post-MI.

CaMKII-dependent ryanodine receptor phosphorylation mediates sepsis-induced cardiomyocyte apoptosis.

Sepsis is associated with cardiac dysfunction, which is at least in part due to cardiomyocyte apoptosis. However, the underlying mechanisms are far from being understood. Using the colon ascendens stent peritonitis mouse model of sepsis (CASP), we examined the subcellular mechanisms that mediate sepsis-induced apoptosis. Wild-type (WT) CASP mice hearts showed an increase in apoptosis respect to WT-Sham. CASP transgenic mice expressing a CaMKII inhibitory peptide (AC3-I) were protected against sepsis-induced apoptosis. Dantrolene, used to reduce ryanodine receptor (RyR) diastolic sarcoplasmic reticulum (SR) Ca2+ release, prevented apoptosis in WT-CASP. To examine whether CaMKII-dependent RyR2 phosphorylation mediates diastolic Ca2+ release and apoptosis in sepsis, we evaluated apoptosis in mutant mice hearts that have the CaMKII phosphorylation site of RyR2 (Serine 2814) mutated to Alanine (S2814A). S2814A CASP mice did not show increased apoptosis. Consistent with RyR2 phosphorylation-dependent enhancement in diastolic SR Ca2+ release leading to mitochondrial Ca2+ overload, mitochondrial Ca2+ retention capacity was reduced in mitochondria isolated from WT-CASP compared to Sham and this reduction was absent in mitochondria from CASP S2814A or dantrolene-treated mice. We conclude that in sepsis, CaMKII-dependent RyR2 phosphorylation results in diastolic Ca2+ release from SR which leads to mitochondrial Ca2+ overload and apoptosis.

High Thyrotropin Is Critical for Cardiac Electrical Remodeling and Arrhythmia Vulnerability in Hypothyroidism.

Background: Hypothyroidism, the most common endocrine disease, induces cardiac electrical remodeling that creates a substrate for ventricular arrhythmias. Recent studies report that high thyrotropin (TSH) levels are related to cardiac electrical abnormalities and increased mortality rates. The aim of the present work was to investigate the direct effects of TSH on the heart and its possible causative role in the increased incidence of arrhythmia in hypothyroidism. Methods: A new rat model of central hypothyroidism (low TSH levels) was created and characterized together with the classical propylthiouracil-induced primary hypothyroidism model (high TSH levels). Electrocardiograms were recorded in vivo, and ionic currents were recorded from isolated ventricular myocytes in vitro by the patch-clamp technique. Protein and mRNA were measured by Western blot and quantitative reverse transcription polymerase chain reaction in rat and human cardiac myocytes. Adult human action potentials were simulated in silico to incorporate the experimentally observed changes. Results: Both primary and central hypothyroidism models increased the L-type Ca2+ current (ICa-L) and decreased the ultra-rapid delayed rectifier K+ current (IKur) densities. However, only primary but not central hypothyroidism showed electrocardiographic repolarization abnormalities and increased ventricular arrhythmia incidence during caffeine/dobutamine challenge. These changes were paralleled by a decrease in the density of the transient outward K+ current (Ito) in cardiomyocytes from animals with primary but not central hypothyroidism. In vitro treatment with TSH for 24 hours enhanced isoproterenol-induced spontaneous activity in control ventricular cells and diminished Ito density in cardiomyocytes from control and central but not primary hypothyroidism animals. In human myocytes, TSH decreased the expression of KCND3 and KCNQ1, Ito, and the delayed rectifier K+ current (IKs) encoding proteins in a protein kinase A-dependent way. Transposing the changes produced by hypothyroidism and TSH to a computer model of human ventricular action potential resulted in enhanced occurrence of early afterdepolarizations and arrhythmia mostly in primary hypothyroidism, especially under ß-adrenergic stimulation. Conclusions: The results suggest that suppression of repolarizing K+ currents by TSH underlies most of the electrical remodeling observed in hypothyroidism. This work demonstrates that the activation of the TSH-receptor/protein kinase A pathway in the heart is responsible for the cardiac electrical remodeling and arrhythmia generation seen in hypothyroidism.

Non-ß-Blocking Carvedilol Analog, VK-II-86, Prevents Ouabain-Induced Cardiotoxicity.

BACKGROUND: It has been shown that carvedilol and its non ß-blocking analog, VK-II-86, inhibit spontaneous Ca2+ release from the sarcoplasmic reticulum (SR). The aim of this study is to determine whether carvedilol and VK-II-86 suppress ouabain-induced arrhythmogenic Ca2+ waves and apoptosis in cardiac myocytes. Methods and Results: Rat cardiac myocytes were exposed to toxic doses of ouabain (50 µmol/L). Cell length (contraction) was monitored in electrically stimulated and non-stimulated conditions. Ouabain treatment increased contractility, frequency of spontaneous contractions and apoptosis compared to control cells. Carvedilol (1 µmol/L) or VK-II-86 (1 µmol/L) did not affect ouabain-induced inotropy, but significantly reduced the frequency of Ca2+ waves, spontaneous contractions and cell death evoked by ouabain treatment. This antiarrhythmic effect was not associated with a reduction in Ca2+ calmodulin-dependent protein kinase II (CaMKII) activity, phospholamban and ryanodine receptor phosphorylation or SR Ca2+ load. Similar results could be replicated in human cardiomyocytes derived from stem cells and in a mathematical model of human myocytes. CONCLUSIONS: Carvedilol and VK-II-86 are effective to prevent ouabain-induced apoptosis and spontaneous contractions indicative of arrhythmogenic activity without affecting inotropy and demonstrated to be effective in human models, thus emerging as a therapeutic tool for the prevention of digitalis-induced arrhythmias and cardiac toxicity.

Toll-Like Receptor 4 and NLRP3 Caspase 1- Interleukin-1ß-Axis are Not Involved in Colon Ascendens Stent Peritonitis-Associated Heart Disease.

Hemodynamic collapse and myocardial dysfunction are among the major causes of death in severe sepsis. The purpose of this study was to assess the role played by toll-like receptor 4 and by the NLRP3 inflammasome in the cardiac dysfunction that occurs after high-grade polymicrobial sepsis. We performed the colon ascendens stent peritonitis (CASP) surgery in Tlr4, Nlrp3, and caspase-1 mice. We also assessed for the first time the electrical heart function in the colon ascendens stent peritonitis (CASP) model. The QJ interval was increased in wild-type C57BL/6J mice after CASP when compared with sham controls, a result paralleled by an increase in the cardiac action potential (AP) duration (APD). The decreases in ejection fraction (EF), left ventricle end diastolic volume, stroke volume, and cardiac output found after CASP were similar among all groups of mice. Similar heart response was found when Nlrp3 mice were submitted to high-grade cecal ligation and puncture. Despite developing cardiac dysfunction similar to wild types after CASP, Nlrp3 mice had reduced circulating levels of interleukin (IL)-1ß, IL-6 and tumor necrosis factor-α. Our results demonstrate that the genetic ablation of Tlr4, Nlrp3, and caspase-1 does not prevent the cardiac dysfunction, despite preventing the increase in pro-inflammatory cytokines, indicating that these are not feasible targets to therapy in high-grade sepsis.

The reduced myofilament responsiveness to calcium contributes to the negative force-frequency relationship in rat cardiomyocytes: role of reactive oxygen species and p-38 map kinase.

The force-frequency relationship (FFR) is an important intrinsic regulatory mechanism of cardiac contractility. However, a decrease (negative FFR) or no effect (flat FFR) on contractile force in response to an elevation of heart rate is present in the normal rat or in human heart failure. Reactive oxygen species (ROS) can act as intracellular signaling molecules activating diverse kinases as calcium-calmodulin-dependent protein kinase II (CaMKII) and p-38 MAP kinase (p-38K). Our aim was to elucidate the intracellular molecules implicated in the FFR of isolated rat ventricular myocytes. The myocytes were field-stimulated via two-platinum electrodes. Sarcomere length was recorded with a video camera. Ca2+ transients and intracellular pHi were recorded by epifluorescence. Increasing frequency from 0.5 to 3 Hz decreased cell shortening without changes in pHi. This negative FFR was changed to positive FFR when the myocytes were pre-incubated with the ROS scavenger MPG, the NADPH oxidase blocker apocynin, or by inhibiting mitochondrial ROS production with 5-HD. Similar results were obtained when the cells were pre-incubated with the CaMKII blocker, KN-93, or the p-38K inhibitor, SB-202190. Consistently, the levels of phosphorylation of p-38K and the oxidation of CaMKII were significantly higher at 2 Hz than at 0.5 Hz. Despite the presence of positive inotropic effect during stimulation frequency enhancement, Ca2+ transient amplitudes were reduced in MPG- and SB-202190-treated myocytes. In conclusion, our results indicate that the activation of the intracellular pathway involving ROS-CaMKII-p-38K contributes to the negative FFR of rat cardiomyocytes, likely by desensitizing the response of contractile myofilaments to Ca2+.

Calcium/Calmodulin Protein Kinase II-Dependent Ryanodine Receptor Phosphorylation Mediates Cardiac Contractile Dysfunction Associated With Sepsis.

OBJECTIVES: Sepsis is associated with cardiac contractile dysfunction attributed to alterations in Ca handling. We examined the subcellular mechanisms involved in sarcoplasmic reticulum Ca loss that mediate altered Ca handling and contractile dysfunction associated with sepsis. DESIGN: Randomized controlled trial. SETTING: Research laboratorySUBJECTS:: Male wild type and transgenic miceINTERVENTIONS:: We induced sepsis in mice using the colon ascendens stent peritonitis model. MEASUREMENTS AND MAIN RESULTS: Twenty-four hours after colon ascendens stent peritonitis surgery, we observed that wild type mice had significantly elevated proinflammatory cytokine levels, reduced ejection fraction, and fractional shortening (ejection fraction %, 54.76 ± 0.67; fractional shortening %, 27.53 ± 0.50) compared with sham controls (ejection fraction %, 73.57 ± 0.20; fractional shortening %, 46.75 ± 0.38). At the cardiac myocyte level, colon ascendens stent peritonitis cells showed reduced cell shortening, Ca transient amplitude and sarcoplasmic reticulum Ca content compared with sham cardiomyocytes. Colon ascendens stent peritonitis hearts showed a significant increase in oxidation-dependent calcium and calmodulin-dependent protein kinase II activity, which could be prevented by pretreating animals with the antioxidant tempol. Pharmacologic inhibition of calcium and calmodulin-dependent protein kinase II with 2.5 µM of KN93 prevented the decrease in cell shortening, Ca transient amplitude, and sarcoplasmic reticulum Ca content in colon ascendens stent peritonitis myocytes. Contractile function was also preserved in colon ascendens stent peritonitis myocytes isolated from transgenic mice expressing a calcium and calmodulin-dependent protein kinase II inhibitory peptide (AC3-I) and in colon ascendens stent peritonitis myocytes isolated from mutant mice that have the ryanodine receptor 2 calcium and calmodulin-dependent protein kinase II-dependent phosphorylation site (serine 2814) mutated to alanine (S2814A). Furthermore, colon ascendens stent peritonitis S2814A mice showed preserved ejection fraction and fractional shortening (ejection fraction %, 73.06 ± 6.31; fractional shortening %, 42.33 ± 5.70) compared with sham S2814A mice (ejection fraction %, 71.60 ± 4.02; fractional shortening %, 39.63 ± 3.23). CONCLUSIONS: Results indicate that oxidation and subsequent activation of calcium and calmodulin-dependent protein kinase II has a causal role in the contractile dysfunction associated with sepsis. Calcium and calmodulin-dependent protein kinase II, through phosphorylation of the ryanodine receptor would lead to Ca leak from the sarcoplasmic reticulum, reducing sarcoplasmic reticulum Ca content, Ca transient amplitude and contractility. Development of organ-specific calcium and calmodulin-dependent protein kinase II inhibitors may result in a beneficial therapeutic strategy to ameliorate contractile dysfunction associated with sepsis.

Macrophage-dependent IL-1ß production induces cardiac arrhythmias in diabetic mice.

Diabetes mellitus (DM) encompasses a multitude of secondary disorders, including heart disease. One of the most frequent and potentially life threatening disorders of DM-induced heart disease is ventricular tachycardia (VT). Here we show that toll-like receptor 2 (TLR2) and NLRP3 inflammasome activation in cardiac macrophages mediate the production of IL-1ß in DM mice. IL-1ß causes prolongation of the action potential duration, induces a decrease in potassium current and an increase in calcium sparks in cardiomyocytes, which are changes that underlie arrhythmia propensity. IL-1ß-induced spontaneous contractile events are associated with CaMKII oxidation and phosphorylation. We further show that DM-induced arrhythmias can be successfully treated by inhibiting the IL-1ß axis with either IL-1 receptor antagonist or by inhibiting the NLRP3 inflammasome. Our results establish IL-1ß as an inflammatory connection between metabolic dysfunction and arrhythmias in DM.

Role of CaMKII and ROS in rapid pacing-induced apoptosis.

Tachycardia promotes cell death and cardiac remodeling, leading to congestive heart failure. However, the underlying mechanism of tachycardia- or rapid pacing (RP)-induced cell death remains unknown. Myocyte loss by apoptosis is recognized as a critical factor in the progression to heart failure and simulation of tachycardia by RP has been shown to increase the intracellular levels of at least two potentially proapoptotic molecules, Ca(2+) and reactive oxygen species (ROS). However, whether these molecules mediate tachycardia- or RP-induced cell death has yet to be determined. The aim of this study was to examine the subcellular mechanisms underlying RP-induced apoptosis. For this purpose rat ventricular myocytes were maintained quiescent or paced at 0.5, 5 and 8Hz for 1hr. RP at 5 and 8Hz decreased myocyte viability by 58±3% and 75±6% (n=24), respectively, compared to cells maintained at 0.5Hz, and increased caspase-3 activity and Bax/Bcl-2 ratio, indicative of apoptosis. RP-induced cell death and apoptosis were prevented when pacing protocols were conducted in the presence of either the ROS scavenger, MPG, or nifedipine to reduce Ca(2+) entry or the CaMKII inhibitors, KN93 and AIP. Consistently, myocytes from transgenic mice expressing a CaMKII inhibitory peptide (AC3-I) were protected against RP-induced cell death. Interestingly, tetracaine and carvedilol used to reduce ryanodine receptor (RyR) diastolic Ca(2+) release, and ruthenium red used to prevent Ca(2+) entry into the mitochondria prevented RP-induced cell death, whereas PI3K inhibition with Wortmannin exacerbated pacing-induced cell mortality. We conclude that CaMKII activation and ROS production are involved in RP-induced apoptosis. Particularly, our results suggest that CaMKII-dependent posttranslational modifications of the cardiac ryanodine receptor (RyR) leading to enhanced diastolic Ca(2+) release and mitochondrial Ca(2+) overload could be the underlying mechanism involved. We further show that RP simultaneously activates a protective cascade involving PI3K/AKT signaling which is however, insufficient to completely suppress apoptosis.

Calcium-calmodulin kinase II mediates digitalis-induced arrhythmias.

BACKGROUND: Digitalis-induced Na(+) accumulation results in an increase in Ca(2+)(i) via the Na(+)/Ca(2+) exchanger, leading to enhanced sarcoplasmic reticulum (SR) Ca(2+) load, responsible for the positive inotropic and toxic arrhythmogenic effects of glycosides. A digitalis-induced increase in Ca(2+)(i) could also activate calcium-calmodulin kinase II (CaMKII), which has been shown to have proarrhythmic effects. Here, we investigate whether CaMKII underlies digitalis-induced arrhythmias and the subcellular mechanisms involved. METHODS AND RESULTS: In paced rat ventricular myocytes (0.5 Hz), 50 µmol/L ouabain increased contraction amplitude by 160 ± 5%. In the absence of electric stimulation, ouabain promoted spontaneous contractile activity and Ca(2+) waves. Ouabain activated CaMKII (p-CaMKII), which phosphorylated its downstream targets, phospholamban (PLN) (Thr17) and ryanodine receptor (RyR) (Ser2814). Ouabain-induced spontaneous activity was prevented by inhibiting CaMKII with 2.5 µmol/L KN93 but not by 2.5 µmol/L of the inactive analog, KN92. Similar results were obtained using the CaMKII inhibitor, autocamtide-2 related inhibitory peptide (AIP) (1 to 2.5 µmol/L), and in myocytes from transgenic mice expressing SR-targeted AIP. Consistently, CaMKII overexpression exacerbated ouabain-induced spontaneous contractile activity. Ouabain was associated with an increase in SR Ca(2+) content and Ca(2+) spark frequency, indicative of enhanced SR Ca(2+) leak. KN93 suppressed the ouabain-induced increase in Ca(2+) spark frequency without affecting SR Ca(2+) content. Similar results were obtained with digoxin. In vivo, ouabain-induced arrhythmias were prevented by KN93 and absent in SR-AIP mice. CONCLUSIONS: These results show for the first time that CaMKII mediates ouabain-induced arrhythmic/toxic effects. We suggest that CaMKII-dependent phosphorylation of the RyR, resulting in Ca(2+) leak from the SR, is the underlying mechanism involved.