Calpain inhibition protects against atrial fibrillation by mitigating diabetes-associated atrial fibrosis and calcium handling dysfunction in type 2 diabetes mice.

BACKGROUND: Diabetes mellitus (DM) is a major risk factor for atrial structural remodeling and atrial fibrillation (AF). Calpain activity is hypothesized to promote atrial remodeling and AF. OBJECTIVE: The purpose of this study was to investigate the role of calpain in diabetes-associated AF, fibrosis, and calcium handling dysfunction. METHODS: DM-associated AF was induced in wild-type (WT) mice and in mice overexpressing the calpain inhibitor calpastatin (CAST-OE) using high-fat diet feeding followed by low-dose streptozotocin injection (75 mg/kg). DM and AF outcomes were assessed by measuring blood glucose levels, fibrosis, and AF susceptibility during transesophageal atrial pacing. Intracellular Ca2+ transients, spontaneous Ca2+ release events, and intracellular T-tubule membranes were measured by in situ confocal microscopy. RESULTS: WT mice with DM had significant hyperglycemia, atrial fibrosis, and AF susceptibility with increased atrial myocyte calpain activity and Ca2+ handling dysfunction relative to control treated animals. CAST-OE mice with DM had a similar level of hyperglycemia as diabetic WT littermates but lacked significant atrial fibrosis and AF susceptibility. DM-induced atrial calpain activity and downregulation of the calpain substrate junctophilin-2 were prevented by CAST-OE. Atrial myocytes of diabetic CAST-OE mice exhibited improved T-tubule membrane organization, Ca2+ handling, and reduced spontaneous Ca2+ release events compared to littermate controls. CONCLUSION: This study confirmed that DM promotes calpain activation, atrial fibrosis, and AF in mice. CAST-OE effectively inhibits DM-induced calpain activation and reduces atrial remodeling and AF incidence through improved intracellular Ca2+ homeostasis. Our results support calpain inhibition as a potential therapy for preventing and treating AF in DM patients.

Cardiac monoamine oxidase-A inhibition protects against catecholamine-induced ventricular arrhythmias via enhanced diastolic calcium control.

AIMS: A mechanistic link between depression and risk of arrhythmias could be attributed to altered catecholamine metabolism in the heart. Monoamine oxidase-A (MAO-A), a key enzyme involved in catecholamine metabolism and longstanding antidepressant target, is highly expressed in the myocardium. The present study aimed to elucidate the functional significance and underlying mechanisms of cardiac MAO-A in arrhythmogenesis. METHODS AND RESULTS: Analysis of the TriNetX database revealed that depressed patients treated with MAO inhibitors had a lower risk of arrhythmias compared with those treated with selective serotonin reuptake inhibitors. This effect was phenocopied in mice with cardiomyocyte-specific MAO-A deficiency (cMAO-Adef), which showed a significant reduction in both incidence and duration of catecholamine stress-induced ventricular tachycardia compared with wild-type mice. Additionally, cMAO-Adef cardiomyocytes exhibited altered Ca2+ handling under catecholamine stimulation, with increased diastolic Ca2+ reuptake, reduced diastolic Ca2+ leak, and diminished systolic Ca2+ release. Mechanistically, cMAO-Adef hearts had reduced catecholamine levels under sympathetic stress, along with reduced levels of reactive oxygen species and protein carbonylation, leading to decreased oxidation of Type II PKA and CaMKII. These changes potentiated phospholamban (PLB) phosphorylation, thereby enhancing diastolic Ca2+ reuptake, while reducing ryanodine receptor 2 (RyR2) phosphorylation to decrease diastolic Ca2+ leak. Consequently, cMAO-Adef hearts exhibited lower diastolic Ca2+ levels and fewer arrhythmogenic Ca2+ waves during sympathetic overstimulation. CONCLUSION: Cardiac MAO-A inhibition exerts an anti-arrhythmic effect by enhancing diastolic Ca2+ handling under catecholamine stress.

Modulation of miR-29 influences myocardial compliance likely through coordinated regulation of calcium handling and extracellular matrix.

MicroRNAs (miRNAs) control the expression of diverse subsets of target mRNAs, and studies have found miRNA dysregulation in failing hearts. Expression of miR-29 is abundant in heart, increases with aging, and is altered in cardiomyopathies. Prior studies demonstrate that miR-29 reduction via genetic knockout or pharmacologic blockade can blunt cardiac hypertrophy and fibrosis in mice. Surprisingly, this depended on specifically blunting miR-29 actions in cardiomyocytes versus fibroblasts. To begin developing more translationally relevant vectors, we generated a novel transgene-encoded miR-29 inhibitor (TuD-29) that can be incorporated into a viral-mediated gene therapy for cardioprotection. Here, we corroborate that miR-29 expression and activity is higher in cardiomyocytes versus fibroblasts and demonstrate that TuD-29 effectively blunts hypertrophic responses in cultured cardiomyocytes and mouse hearts. Furthermore, we found that adeno-associated virus (AAV)-mediated miR-29 overexpression in mouse hearts induces early diastolic dysfunction, whereas AAV:TuD-29 treatment improves cardiac output by increasing end-diastolic and stroke volumes. The integration of RNA sequencing and miRNA-target interactomes reveals that miR-29 regulates genes involved in calcium handling, cell stress and hypertrophy, metabolism, ion transport, and extracellular matrix remodeling. These investigations support a likely versatile role for miR-29 in influencing myocardial compliance and relaxation, potentially providing a unique therapeutic avenue to improve diastolic function in heart failure patients.

Flame doping of indium ions into TiO2 nanorod arrays for enhanced photochemical water oxidation.

Indium (In) ions were diffused into a TiO2 (In-TiO2) photoelectrode via a facile and efficient flame doping method resulting in improved photo-induced carrier separation. The dopant concentration was systematically investigated, and a volcano-type relationship between the dopant concentration and photoelectrochemical (PEC) performance was observed. The optimum incident photon-to-current efficiency and photocurrent density of In-TiO2 were 38.6% and 0.70 mA cm-2 at 1.23 V, respectively, 2.1 and 11.2 times the values of pristine TiO2, respectively. In doping resulted in improved charge separation and lower surface adsorption energies for reactant molecules, as evidenced by experimental and computational methods.

Polymer Photocatalysts Containing Segregated π-Conjugation Units with Electron-Trap Activity for Efficient Natural-light-driven Bacterial Inactivation.

Development of highly efficient and metal-free photocatalysts for bacterial inactivation under natural light is a major challenge in photocatalytic antibiosis. Herein, we developed an acidizing solvent-thermal approach for inserting a non-conjugated ethylenediamine segment into the conjugated planes of 3,4,9,10-perylene tetracarboxylic anhydride to generate a photocatalyst containing segregated π-conjugation units (EDA-PTCDA). Under natural light, EDA-PTCDA achieved 99.9 % inactivation of Escherichia coli and Staphylococcus aureus (60 and 45 min), which is the highest efficiency among all the natural light antibacterial reports. The difference in the surface potential and excited charge density corroborated the possibility of a built-in electron-trap effect of the non-conjugated segments of EDA-PTCDA, thus forming a highly active EDA-PTDA/bacteria interface. In addition, EDA-PTCDA exhibited negligible toxicity and damage to normal tissue cells. This catalyst provides a new opportunity for photocatalytic antibiosis under natural light conditions.

CIB2 Is a Novel Endogenous Repressor of Atrial Remodeling.

BACKGROUND: Atrial fibrillation (AF) is a highly prevalent condition that can cause or exacerbate heart failure, is an important risk factor for stroke, and is associated with pronounced morbidity and death. Genes uniquely expressed in the atria are known to be essential for maintaining atrial structure and function. Atrial tissue remodeling contributes to arrhythmia recurrence and maintenance. However, the mechanism underlying atrial remodeling remains poorly understood. This study was designed to investigate whether other uncharacterized atrial specific genes play important roles in atrial physiology and arrhythmogenesis. METHODS: RNA-sequencing analysis was used to identify atrial myocyte specific and angiotensin II-responsive genes. Genetically modified, cardiomyocyte-specific mouse models (knockout and overexpression) were generated. In vivo and in vitro electrophysiological, histology, and biochemical analyses were performed to determine the consequences of CIB2 (calcium and integrin binding family member 2 protein) gain and loss of function in the atrium. RESULTS: Using RNA-sequencing analysis, we identified CIB2 as an atrial-enriched protein that is significantly downregulated in the left atria of patients with AF and mouse models of AF from angiotensin II infusion or pressure overload. Using cardiomyocyte-specific Cib2 knockout (Cib2-/-) and atrial myocyte-specific Cib2-overexpressing mouse models, we found that loss of Cib2 enhances AF occurrence, prolongs AF duration, and correlates with a significant increase in atrial fibrosis under stress. Conversely, Cib2 overexpression mitigates AF occurrence and atrial fibrosis triggered by angiotensin II stress. Mechanistically, we revealed that CIB2 competes with and inhibits CIB1-mediated calcineurin activation, thereby negating stress-induced structural remodeling and AF. CONCLUSIONS: Our data suggest that CIB2 represents a novel endogenous and atrial-enriched regulator that protects against atrial remodeling and AF under stress conditions. Therefore, CIB2 may represent a new potential target for treating AF.

Mitochondrial Oxidative Stress Mediates Bradyarrhythmia in Leigh Syndrome Mitochondrial Disease Mice.

Mitochondrial oxidative stress has been implicated in aging and several cardiovascular diseases, including heart failure and cardiomyopathy, ventricular tachycardia, and atrial fibrillation. The role of mitochondrial oxidative stress in bradyarrhythmia is less clear. Mice with a germline deletion of Ndufs4 subunit respiratory complex I develop severe mitochondrial encephalomyopathy resembling Leigh Syndrome (LS). Several types of cardiac bradyarrhythmia are present in LS mice, including a frequent sinus node dysfunction and episodic atrioventricular (AV) block. Treatment with the mitochondrial antioxidant Mitotempo or mitochondrial protective peptide SS31 significantly ameliorated the bradyarrhythmia and extended the lifespan of LS mice. Using an ex vivo Langendorff perfused heart with live confocal imaging of mitochondrial and total cellular reactive oxygen species (ROS), we showed increased ROS in the LS heart, which was potentiated by ischemia-reperfusion. A simultaneous ECG recording showed a sinus node dysfunction and AV block concurrent with the severity of the oxidative stress. Treatment with Mitotempo abolished ROS and restored the sinus rhythm. Our study reveals robust evidence of the direct mechanistic roles of mitochondrial and total ROS in bradyarrhythmia in the setting of LS mitochondrial cardiomyopathy. Our study also supports the potential clinical application of mitochondrial-targeted antioxidants or SS31 for the treatment of LS patients.

Metabolic rescue ameliorates mitochondrial encephalo-cardiomyopathy in murine and human iPSC models of Leigh syndrome.

BACKGROUND: Mice with deletion of complex I subunit Ndufs4 develop mitochondrial encephalomyopathy resembling Leigh syndrome (LS). The metabolic derangement and underlying mechanisms of cardio-encephalomyopathy in LS remains incompletely understood. METHODS: We performed echocardiography, electrophysiology, confocal microscopy, metabolic and molecular/morphometric analysis of the mice lacking Ndufs4. HEK293 cells, human iPS cells-derived cardiomyocytes and neurons were used to determine the mechanistic role of mitochondrial complex I deficiency. RESULTS: LS mice develop severe cardiac bradyarrhythmia and diastolic dysfunction. Human-induced pluripotent stem cell-derived cardiomyocytes (iPS-CMs) with Ndufs4 deletion recapitulate LS cardiomyopathy. Mechanistically, we demonstrate a direct link between complex I deficiency, decreased intracellular (nicotinamide adenine dinucleotide) NAD+ /NADH and bradyarrhythmia, mediated by hyperacetylation of the cardiac sodium channel NaV 1.5, particularly at K1479 site. Neuronal apoptosis in the cerebellar and midbrain regions in LS mice was associated with hyperacetylation of p53 and activation of microglia. Targeted metabolomics revealed increases in several amino acids and citric acid cycle intermediates, likely due to impairment of NAD+ -dependent dehydrogenases, and a substantial decrease in reduced Glutathione (GSH). Metabolic rescue by nicotinamide riboside (NR) supplementation increased intracellular NAD+ / NADH, restored metabolic derangement, reversed protein hyperacetylation through NAD+ -dependent Sirtuin deacetylase, and ameliorated cardiomyopathic phenotypes, concomitant with improvement of NaV 1.5 current and SERCA2a function measured by Ca2+ -transients. NR also attenuated neuronal apoptosis and microglial activation in the LS brain and human iPS-derived neurons with Ndufs4 deletion. CONCLUSIONS: Our study reveals direct mechanistic explanations of the observed cardiac bradyarrhythmia, diastolic dysfunction and neuronal apoptosis in mouse and human induced pluripotent stem cells (iPSC) models of LS.

Identifying Food Parenting Practices From Comprehensive Home Environment Survey.

OBJECTIVE: Conducting exploratory factor analysis (EFA) with the existing home environment assessment-the Comprehensive Home Environment Survey (CHES), to identify scales related to food parenting practices. METHODS: Parents of 3- to 5-year-old children (n = 172) completed the CHES surveys. After selected items from CHES were categorized into food parenting practice constructs, EFA was used to identify potential subconstructs. Internal consistency and Spearman correlation analysis were also conducted. RESULTS: The EFAs identified 4 factors within the structure, 4 within coercive control, and 2 within autonomy support. CONCLUSIONS AND IMPLICATIONS: The results provided preliminary evidence and support that the CHES can be used to measure food parenting practices. The study was limited to a small sample of non-Hispanic White and highly educated participants, less identified items within autonomy support, and lower internal consistency for several identified factors. Confirmatory factor analysis in a larger and more diverse sample is needed for future research.

Gene Therapy With the N-Terminus of Junctophilin-2 Improves Heart Failure in Mice.

BACKGROUND: Transcriptional remodeling is known to contribute to heart failure (HF). Targeting stress-dependent gene expression mechanisms may represent a clinically relevant gene therapy option. We recently uncovered a salutary mechanism in the heart whereby JP2 (junctophilin-2), an essential component of the excitation-contraction coupling apparatus, is site-specifically cleaved and releases an N-terminal fragment (JP2NT [N-terminal fragment of JP2]) that translocates into the nucleus and functions as a transcriptional repressor of HF-related genes. This study aims to determine whether JP2NT can be leveraged by gene therapy techniques for attenuating HF progression in a preclinical pressure overload model. METHODS: We intraventricularly injected adeno-associated virus (AAV) (2/9) vectors expressing eGFP (enhanced green fluorescent protein), JP2NT, or DNA-binding deficient JP2NT (JP2NTΔbNLS/ARR) into neonatal mice and induced cardiac stress by transaortic constriction (TAC) 9 weeks later. We also treated mice with established moderate HF from TAC stress with either AAV-JP2NT or AAV-eGFP. RNA-sequencing analysis was used to reveal changes in hypertrophic and HF-related gene transcription by JP2NT gene therapy after TAC. Echocardiography, confocal imaging, and histology were performed to evaluate heart function and pathological myocardial remodeling following stress. RESULTS: Mice preinjected with AAV-JP2NT exhibited ameliorated cardiac remodeling following TAC. The JP2NT DNA-binding domain is required for cardioprotection as its deletion within the AAV-JP2NT vector prevented improvement in TAC-induced cardiac dysfunction. Functional and histological data suggest that JP2NT gene therapy after the onset of cardiac dysfunction is effective at slowing the progression of HF. RNA-sequencing analysis further revealed a broad reversal of hypertrophic and HF-related gene transcription by JP2NT overexpression after TAC. CONCLUSIONS: Our prevention- and intervention-based approaches here demonstrated that AAV-mediated delivery of JP2NT into the myocardium can attenuate stress-induced transcriptional remodeling and the development of HF when administered either before or after cardiac stress initiation. Our data indicate that JP2NT gene therapy holds great potential as a novel therapeutic for treating hypertrophy and HF.

Impaired Regulatory Volume Decrease and Characterization of Underlying Volume-Activated Currents in Cystic Fibrosis Human Cholangiocyte Cell Line.

The volume-activated chloride channel (VACC) serves vital cellular functions in secretion and cell volume regulation via regulatory volume decrease (RVD) in various epithelia. Previously, we have shown that RVD in primary CF mouse cholangiocytes is impaired. Thus, the effect of CFTR defect on VACC and RVD in CF human immortalized cholangiocyte cell (HBDC) was examined in comparison with those in normal HBDC by using cell volume measurement and whole-cell patch clamp techniques, respectively. The CF HBDC had an impaired RVD, which was not further inhibited by removing the extracellular calcium or administering BAPTA-AM, NPPB, or DIDS. When exposed to a hypotonic solution, CF HBDC exhibited large, outwardly rectified currents with time-dependent inactivation at a positive potential. The amplitude of the outward currents was about three times that of the inward currents. The amplitude and reversal potential of VACC was dependent on chloride concentration. The VACC was significantly inhibited by replacing chloride with gluconate, glutamate, sucrose, or acetate in the hypotonic solution as well as by an administration of NPPB or tamoxifen, classical VACC inhibitors. Surprisingly, the VACC amplitude is greater in CF HBDC than in normal HBDC, suggesting that the channel density or open probability of VACC is increased, thus CFTR may have inhibitory effects on VACC. On the contrary, the amplitude of the volume-activated potassium current is lower in CF HBDC, suggesting the potassium channel density or open probability is decreased in CF cholangiocytes and/or CFTR may have regulatory effects on volume-activated potassium current. In conclusion, RVD is impaired in CF human cholangiocytes. The VACC of CF human cholangiocytes has similar electrophysiological characteristics as that of normal cholangiocytes but its activity is augmented in CF cholangiocytes, while volume-activated potassium current is decreased in CF human cholangiocytes, providing a fundamental underlying pathophysiologic mechanism for the impaired RVD in CF cholangiocytes.

Levofloxacin exerts broad-spectrum anticancer activity via regulation of THBS1, LAPTM5, SRD5A3, MFAP5 and P4HA1.

One cost-effective way for identifying novel cancer therapeutics is in the repositioning of available drugs for which current therapies are inadequate. Levofloxacin prevents DNA duplication in bacteria by inhibiting the activity of DNA helicase. As eukaryotic cells have similar intracellular biologic characteristics as prokaryotic cells, we speculate that antibiotics inhibiting DNA duplication in bacteria may also affect the survival of cancer cells. Here we report that levofloxacin significantly inhibited the proliferation and clone formation of cancer cells and xenograft tumor growth through cell cycle arrest at G2/M and by enhancing apoptosis. Levofloxacin significantly altered gene expression in a direction favoring anticancer activity. THBS1 and LAPTM5 were dose-dependently upregulated whereas SRD5A3, MFAP5 and P4HA1 were downregulated. Pathway analysis revealed that levofloxacin significantly regulated canonical oncogenic pathways. Specific network enrichment included a MAPK/apoptosis/cytokine-cytokine receptor interaction pathway network that associates with cell growth, differentiation, cell death, angiogenesis and development and repair processes and a bladder cancer/P53 signaling pathway network mediating the inhibition of angiogenesis and metastasis. THBS1 overlapped in 16 of the 22 enriched apoptotic pathways and the 2 pathways in the bladder cancer/P53 signaling pathway network. P4HA1 enriched in 7 of the top 10 molecular functions regulated by differential downregulated genes. Our results indicate that levofloxacin has broad-spectrum anticancer activity with the potential to benefit cancer patients already treated or requiring prophylaxis for an infectious syndrome. The efficacy we find with levofloxacin may provide insight into the discovery and the design of novel less toxic anticancer drugs.

Atrial-paced, exercise-similar heart rate envelope induces myocardial protection from ischaemic injury.

AIMS: The study investigates the role and mechanisms of clinically translatable exercise heart rate (HR) envelope effects, without dyssynchrony, on myocardial ischaemia tolerance compared to standard preconditioning methods. Since the magnitude and duration of exercise HR acceleration are tightly correlated with beneficial cardiac outcomes, it is hypothesized that a paced exercise-similar HR envelope, delivered in a maximally physiologic way that avoids the toxic effects of chamber dyssynchrony, may be more than simply a readout, but rather also a significant trigger of myocardial conditioning and stress resistance. METHODS AND RESULTS: For 8 days over 2 weeks, sedated mice were atrial-paced once daily via an oesophageal electrode to deliver an exercise-similar HR pattern with preserved atrioventricular and interventricular synchrony. Effects on cardiac calcium handling, protein expression/modification, and tolerance to ischaemia-reperfusion (IR) injury were assessed and compared to those in sham-paced mice and to the effects of exercise and ischaemic preconditioning (IPC). The paced cohort displayed improved myocardial IR injury tolerance vs. sham controls with an effect size similar to that afforded by treadmill exercise or IPC. Hearts from paced mice displayed changes in Ca2+ handling, coupled with changes in phosphorylation of calcium/calmodulin protein kinase II, phospholamban and ryanodine receptor channel, and transcriptional remodelling associated with a cardioprotective paradigm. CONCLUSIONS: The HR pattern of exercise, delivered by atrial pacing that preserves intracardiac synchrony, induces cardiac conditioning and enhances ischaemic stress resistance. This identifies the HR pattern as a signal for conditioning and suggests the potential to repurpose atrial pacing for cardioprotection.

Parenting Styles, Food Parenting Practices and Dietary Intakes of Preschoolers.

Previous evidence suggests that children's eating behaviors were largely influenced by the parent and home eating structure. This study examined the relationship between parenting styles (including authoritative, authoritarian, indulgent, and uninvolved), food parenting practices (within Structure, Coercive Control, and Autonomy Support constructs) and dietary intakes of preschoolers. Children aged 3-5 years and their parents were recruited from preschools/daycare centers and parents completed the surveys (n = 166). Dietary intakes were collected using the Harvard Service Food Frequency Questionnaire (HSFFQ), parenting style was assessed using the Parenting Dimensions Inventory-Short Version (PDI-S), and food parenting practices were measured using Comprehensive Home Environment Survey (CHES). The results showed that food parenting practices had a higher number of specific significant findings on children's nutrient and food group intakes than parenting styles. Correlation analyses showed positive parenting practices within Structure were significantly related to healthier children's intakes (e.g., vegetables, iron, and folate) and less unhealthy dietary intakes (e.g., sweets and total fats). Regression models show that children with authoritative parents consumed more fruits compared to children with authoritarian parents and indulgent parents. The results addressed the importance of parental influences for preschoolers' healthy dietary intakes, which suggested that future interventions and educational programs could enhance parenting practices to impact child diet.

A putative long noncoding RNA-encoded micropeptide maintains cellular homeostasis in pancreatic ß cells.

Micropeptides (microproteins) encoded by transcripts previously annotated as long noncoding RNAs (lncRNAs) are emerging as important mediators of fundamental biological processes in health and disease. Here, we applied two computational tools to identify putative micropeptides encoded by lncRNAs that are expressed in the human pancreas. We experimentally verified one such micropeptide encoded by a ß cell- and neural cell-enriched lncRNA TCL1 Upstream Neural Differentiation-Associated RNA (TUNAR, also known as TUNA, HI-LNC78, or LINC00617). We named this highly conserved 48-amino-acid micropeptide beta cell- and neural cell-regulin (BNLN). BNLN contains a single-pass transmembrane domain and localizes at the endoplasmic reticulum (ER) in pancreatic ß cells. Overexpression of BNLN lowered ER calcium levels, maintained ER homeostasis, and elevated glucose-stimulated insulin secretion in pancreatic ß cells. We further assessed the BNLN expression in islets from mice fed a high-fat diet and a regular diet and found that BNLN is suppressed by diet-induced obesity (DIO). Conversely, overexpression of BNLN enhanced insulin secretion in islets from lean and obese mice as well as from humans. Taken together, our study provides the first evidence that lncRNA-encoded micropeptides play a critical role in pancreatic ß cell functions and provides a foundation for future comprehensive analyses of micropeptide function and pathophysiological impact on diabetes.

Calpain-2 specifically cleaves Junctophilin-2 at the same site as Calpain-1 but with less efficacy.

Calpain proteolysis contributes to the pathogenesis of heart failure but the calpain isoforms responsible and their substrate specificities have not been rigorously defined. One substrate, Junctophilin-2 (JP2), is essential for maintaining junctional cardiac dyads and excitation-contraction coupling. We previously demonstrated that mouse JP2 is cleaved by calpain-1 (CAPN1) between Arginine 565 (R565) and Threonine 566 (T566). Recently, calpain-2 (CAPN2) was reported to cleave JP2 at a novel site between Glycine 482 (G482) and Threonine 483 (T483). We aimed to directly compare the contributions of each calpain isoform, their Ca2+ sensitivity, and their cleavage site selection for JP2. We find CAPN1, CAPN2 and their requisite CAPNS1 regulatory subunit are induced by pressure overload stress that is concurrent with JP2 cleavage. Using in vitro calpain cleavage assays, we demonstrate that CAPN1 and CAPN2 cleave JP2 into similar 75 kD N-terminal (JP2NT) and 25 kD C-terminal fragments (JP2CT) with CAPNS1 co-expression enhancing proteolysis. Deletion mutagenesis shows both CAPN1 and CAPN2 require R565/T566 but not G482/T483. When heterologously expressed, the JP2CT peptide corresponding to R565/T566 cleavage approximates the 25 kD species found during cardiac stress while the C-terminal peptide from potential cleavage at G482/T483 produces a 35 kD product. Similar results were obtained for human JP2. Finally, we show that CAPN1 has higher Ca2+ sensitivity and cleavage efficacy than CAPN2 on JP2 and other cardiac substrates including cTnT, cTnI and ß2-spectrin. We conclude that CAPN2 cleaves JP2 at the same functionally conserved R565/T566 site as CAPN1 but with less efficacy and suggest heart failure may be targeted through specific inhibition of CAPN1.

Cephalosporin antibiotics specifically and selectively target nasopharyngeal carcinoma through HMOX1-induced ferroptosis.

AIMS: Many antibiotics derived from mold metabolites have been found to possess anticarcinogenic properties. We aimed to investigate whether they may elicit anticancer activity, especially against nasopharyngeal carcinoma. MAIN METHODS: The response of nasopharyngeal and other carcinoma cell lines to cephalosporin antibiotics was evaluated in vitro and in vivo. MTT and clonogenic colony formation assays assessed the viability and proliferation of cultured cells. Flow cytometry was used to assess cell cycle parameters and apoptotic markers. Tumor growth was determined using a xenograft model in vivo. Microarray and RT-qPCR expression analyses investigate differential gene expression. Mechanistic assessment of HMOX1 in cefotaxime-mediated ferroptosis was tested with Protoporphyrin IX zinc. KEY FINDINGS: Cephalosporin antibiotics showed highly specific and selective anticancer activity on nasopharyngeal carcinoma CNE2 cells both in vitro and vivo with minimal toxicity. Cefotaxime sodium significantly regulated 11 anticancer relevant genes in CNE2 cells in a concentration-dependent manner. Pathway analyses indicate apoptotic and the ErbB-MAPK-p53 signaling pathways are significantly enriched. HMOX1 represents the top one ranked upregulated gene by COS and overlaps with 16 of 42 enriched apoptotic signaling pathways. Inhibition of HMOX1 significantly reduced the anticancer efficacy of cefotaxime in CNE2 cells. SIGNIFICANCE: Our discovery is the first to highlight the off-label potential of cephalosporin antibiotics as a specific and selective anticancer drug for nasopharyngeal carcinoma. We mechanistically show that induction of ferroptosis through HMOX1 induction mediates cefotaxime anticancer activity. Our findings provide an alternative treatment for nasopharyngeal carcinoma by showing that existing cephalosporin antibiotics are specific and selective anticancer drugs.

Oxidized CaMKII and O-GlcNAcylation cause increased atrial fibrillation in diabetic mice by distinct mechanisms.

Diabetes mellitus (DM) and atrial fibrillation (AF) are major unsolved public health problems, and diabetes is an independent risk factor for AF. However, the mechanism(s) underlying this clinical association is unknown. ROS and protein O-GlcNAcylation (OGN) are increased in diabetic hearts, and calmodulin kinase II (CaMKII) is a proarrhythmic signal that may be activated by ROS (oxidized CaMKII, ox-CaMKII) and OGN (OGN-CaMKII). We induced type 1 (T1D) and type 2 DM (T2D) in a portfolio of genetic mouse models capable of dissecting the role of ROS and OGN at CaMKII and global OGN in diabetic AF. Here, we showed that T1D and T2D significantly increased AF, and this increase required CaMKII and OGN. T1D and T2D both required ox-CaMKII to increase AF; however, we did not detect OGN-CaMKII or a role for OGN-CaMKII in diabetic AF. Collectively, our data affirm CaMKII as a critical proarrhythmic signal in diabetic AF and suggest ROS primarily promotes AF by ox-CaMKII, while OGN promotes AF by a CaMKII-independent mechanism(s). These results provide insights into the mechanisms for increased AF in DM and suggest potential benefits for future CaMKII and OGN targeted therapies.

Integrin ß1D Deficiency-Mediated RyR2 Dysfunction Contributes to Catecholamine-Sensitive Ventricular Tachycardia in Arrhythmogenic Right Ventricular Cardiomyopathy.

BACKGROUND: Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a hereditary heart disease characterized by fatty infiltration, life-threatening arrhythmias, and increased risk of sudden cardiac death. The guideline for management of ARVC in patients is to improve quality of life by reducing arrhythmic symptoms and to prevent sudden cardiac death. However, the mechanism underlying ARVC-associated cardiac arrhythmias remains poorly understood. METHODS: Using protein mass spectrometry analyses, we identified that integrin ß1 is downregulated in ARVC hearts without changes to Ca2+-handling proteins. As adult cardiomyocytes express only the ß1D isoform, we generated a cardiac specific ß1D knockout mouse model and performed functional imaging and biochemical analyses to determine the consequences of integrin ß1D loss on function in the heart in vivo and in vitro. RESULTS: Integrin ß1D deficiency and RyR2 Ser-2030 hyperphosphorylation were detected by Western blotting in left ventricular tissues from patients with ARVC but not in patients with ischemic or hypertrophic cardiomyopathy. Using lipid bilayer patch clamp single channel recordings, we found that purified integrin ß1D protein could stabilize RyR2 function by decreasing RyR2 open probability, mean open time, and increasing mean close time. Also, ß1D knockout mice exhibited normal cardiac function and morphology but presented with catecholamine-sensitive polymorphic ventricular tachycardia, consistent with increased RyR2 Ser-2030 phosphorylation and aberrant Ca2+ handling in ß1D knockout cardiomyocytes. Mechanistically, we revealed that loss of DSP (desmoplakin) induces integrin ß1D deficiency in ARVC mediated through an ERK1/2 (extracellular signal-regulated kinase 1 and 2)-fibronectin-ubiquitin/lysosome pathway. CONCLUSIONS: Our data suggest that integrin ß1D deficiency represents a novel mechanism underlying the increased risk of ventricular arrhythmias in patients with ARVC.

Cardiomyocyte PKA Ablation Enhances Basal Contractility While Eliminates Cardiac ß-Adrenergic Response Without Adverse Effects on the Heart.

RATIONALE: PKA (Protein Kinase A) is a major mediator of ß-AR (ß-adrenergic) regulation of cardiac function, but other mediators have also been suggested. Reduced PKA basal activity and activation are linked to cardiac diseases. However, how complete loss of PKA activity impacts on cardiac physiology and if it causes cardiac dysfunction have never been determined. OBJECTIVES: We set to determine how the heart adapts to the loss of cardiomyocyte PKA activity and if it elicits cardiac abnormalities. METHODS AND RESULTS: (1) Cardiac PKA activity was almost completely inhibited by expressing a PKA inhibitor peptide in cardiomyocytes (cPKAi) in mice; (2) cPKAi reduced basal phosphorylation of 2 myofilament proteins (TnI [troponin I] and cardiac myosin binding protein C), and one longitudinal SR (sarcoplasmic reticulum) protein (PLB [phospholamban]) but not of the sarcolemmal proteins (Cav1.2 α1c and PLM [phospholemman]), dyadic protein RyR2, and nuclear protein CREB (cAMP response element binding protein) at their PKA phosphorylation sites; (3) cPKAi increased the expression of CaMKII (Ca2+/calmodulin-dependent kinase II), the Cav1.2 ß subunits and current, but decreased CaMKII phosphorylation and CaMKII-mediated phosphorylation of PLB and RyR2; (4) These changes resulted in significantly enhanced myofilament Ca2+ sensitivity, prolonged contraction, slowed relaxation but increased myocyte Ca2+ transient and contraction amplitudes; (5) Isoproterenol-induced PKA and CaMKII activation and their phosphorylation of proteins were prevented by cPKAi; (6) cPKAi abolished the increases of heart rate, and cardiac and myocyte contractility by a ß-AR agonist (isoproterenol), showing an important role of PKA and a minimal role of PKA-independent ß-AR signaling in acute cardiac regulation; (7) cPKAi mice have partial exercise capability probably by enhancing vascular constriction and ventricular filling during ß-AR stimulation; and (8) cPKAi mice did not show any cardiac functional or structural abnormalities during the 1-year study period. CONCLUSIONS: PKA activity suppression induces a unique Ca2+ handling phenotype, eliminates ß-AR regulation of heart rates and cardiac contractility but does not cause cardiac abnormalities.