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Duchenne muscular dystrophy (DMD), caused by mutations in the dystrophin gene, is an X‐linked disease affecting male and rarely adult heterozygous females, resulting in death by the late 20s to early 30s. Previous studies reported depressed left ventricular function in DMD patients which may result from deranged intracellular Ca2+‐handling. To decipher the mechanism(s) underlying the depressed LV function, we tested the hypothesis that iPSC‐CMs generated from DMD patients feature blunted positive inotropic response to β‐adrenergic stimulation. To test the hypothesis, [Ca2+]i transients and contractions were recorded from healthy and DMD‐CMs. While in healthy CMs (HC) isoproterenol caused a prominent positive inotropic effect, DMD‐CMs displayed a blunted inotropic response. Next, we tested the functionality of the sarcoplasmic reticulum (SR) by measuring caffeine‐induced Ca2+ release. In contrast to HC, DMD‐CMs exhibited reduced caffeine‐induced Ca2+ signal amplitude and recovery time. In support of the depleted SR Ca2+ stores hypothesis, in DMD‐CMs the negative inotropic effects of ryanodine and cyclopiazonic acid were smaller than in HC. RNA‐seq analyses demonstrated that in DMD CMs the RNA‐expression levels of specific subunits of the L‐type calcium channel, the β1‐adrenergic receptor (ADRβ1) and adenylate cyclase were down‐regulated by 3.5‐, 2.8‐ and 3‐fold, respectively, which collectively contribute to the depressed β‐adrenergic responsiveness.

Acute Respiratory Distress Syndrome (ARDS), associated with Covid-19 infections, is characterized by diffuse lung damage, inflammation and alveolar collapse that impairs gas exchange, leading to hypoxemia and patient' mortality rates above 40%. Here, we describe the development and assessment of 100-nm liposomes that are tailored for pulmonary delivery for treating ARDS, as a model for lung diseases. The liposomal lipid composition (primarily DPPC) was optimized to mimic the lung surfactant composition, and the drug loading process of both methylprednisolone (MPS), a steroid, and N-acetyl cysteine (NAC), a mucolytic agent, reached an encapsulation efficiency of 98% and 92%, respectively. In vitro, treating lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages with the liposomes decreased TNFα and nitric oxide (NO) secretion, while NAC increased the penetration of nanoparticles through the mucus. In vivo, we used LPS-induced lung inflammation model to assess the accumulation and therapeutic efficacy of the liposomes in C57BL/6 mice, either by intravenous (IV), endotracheal (ET) or IV plus ET nanoparticles administrations. Using both administration methods, liposomes exhibited an increased accumulation profile in the inflamed lungs over 48 h. Interestingly, while IV-administrated liposomes distributed widely throughout the lung, ET liposomes were present in lungs parenchyma but were not detected at some distal regions of the lungs, possibly due to imperfect airflow regimes. Twenty hours after the different treatments, lungs were assessed for markers of inflammation. We found that the nanoparticle treatment had a superior therapeutic effect compared to free drugs in treating ARDS, reducing inflammation and TNFα, IL-6 and IL-1β cytokine secretion in bronchoalveolar lavage (BAL), and that the combined treatment, delivering nanoparticles IV and ET simultaneously, had the best outcome of all treatments. Interestingly, also the DPPC lipid component alone played a therapeutic role in reducing inflammatory markers in the lungs. Collectively, we show that therapeutic nanoparticles accumulate in inflamed lungs holding potential for treating lung disorders. SIGNIFICANCE: In this study we compare intravenous versus intratracheal delivery of nanoparticles for treating lung disorders, specifically, acute respiratory distress syndrome (ARDS). By co-loading two medications into lipid nanoparticles, we were able to reduce both inflammation and mucus secretion in the inflamed lungs. Both modes of delivery resulted in high nanoparticle accumulation in the lungs, intravenously administered nanoparticles reached lung endothelial while endotracheal delivery reached lung epithelial. Combining both delivery approaches simultaneously provided the best ARDS treatment outcome.

LMNA-related dilated cardiomyopathy is an inherited heart disease caused by mutations in the LMNA gene encoding for lamin A/C. The disease is characterized by left ventricular enlargement and impaired systolic function associated with conduction defects and ventricular arrhythmias. We hypothesized that LMNA-mutated patients’ induced Pluripotent Stem Cell-derived cardiomyocytes (iPSC-CMs) display electrophysiological abnormalities, thus constituting a suitable tool for deciphering the arrhythmogenic mechanisms of the disease, and possibly for developing novel therapeutic modalities. iPSC-CMs were generated from two related patients (father and son) carrying the same E342K mutation in the LMNA gene. Compared to control iPSC-CMs, LMNA-mutated iPSC-CMs exhibited the following electrophysiological abnormalities: (1) decreased spontaneous action potential beat rate and decreased pacemaker current (If) density, (2) prolonged action potential duration and increased L-type Ca2+ current (ICa,L) density, (3) delayed afterdepolarizations (DADs), arrhythmias and increased beat rate variability, (4) DADs, arrhythmias and cessation of spontaneous firing in response to β-adrenergic stimulation and rapid pacing. Additionally, compared to healthy control, LMNA-mutated iPSC-CMs displayed nuclear morphological irregularities and gene expression alterations. Notably, KB-R7943, a selective inhibitor of the reverse-mode of the Na+/Ca2+ exchanger, blocked the DADs in LMNA-mutated iPSC-CMs. Our findings demonstrate cellular electrophysiological mechanisms underlying the arrhythmias in LMNA-related dilated cardiomyopathy.

Introduction Familial hypertrophic cardiomyopathy (HCM) is caused by over 400 mutations affecting mostly key sarcomere components, such as titin and myosin. However, HCM also results from mutations in non-structural genes such as PRKAG2 which is involved directly in a variety of bioenergetic and metabolic pathways. When the metabolic processes fail to work properly or effectively, structural and functional aberrations resulting in cardiac dysfunction can occur. Thus, mutations in the human PRKAG2 gene lead to HCM, autosomal dominant ventricular pre-excitation - Wolff-Parkinson-White syndrome (WPW), a progressive conduction system disease and vacuolar glycogen accumulation in cardiomyocytes. Purpose To investigate the hypothesis that intervening with the bioenergetic and metabolic consequences of the R302Q mutation in the PRKAG2 gene causing inherited cardiomyopathy, will attenuate the cardiac impairments. Methods We generated mutated and isogenic induced Pluripotent Stem Cell-derived cardiomyocytes (iPSC-CMs) from a WPW patient carrying the R302Q mutation. As additional control, we used healthy volunteers' iPSC-CMs. Bioenergetic (Oxygen Consumption Rate, OCR) and metabolic status were measured using the Seahorse Flux Analyzer and LC-MS, respectively. To decipher the molecular basis underlying the bioenergetic and metabolic deficits, RNA-seq analysis was performed. Results The OCR results demonstrated in PRKAG2-mutated compared to isogenic and healthy iPSC-CMs, a 2-fold increase in maximal respiration rate and a 3.75-fold increase in spare respiratory capacity, while basal OCR parameters were similar in all groups. Importantly, when treated with the AMPK activator metformin (2.5 [mM]), all the abovementioned OCR parameters were similar in the three groups. RNA-seq analysis demonstrated that of the 553 differently expressed genes (DEGs), and of the 99 DEGs mutually differentially expressed, compared to isogenic and healthy cells, the most relevant altered pathways were glycolysis, carbon metabolism, biosynthesis of amino acids, HIF-1 signaling and fructose and mannose metabolism. These findings are consistent with the LC-MS results demonstrating in PRKAG2-mutated versus isogenic and healthy iPSC-CMs, at least a 3-fold decrease in metabolites linked to the abovementioned pathways: butyryl-carnitine, creatine, docosahexaenoic acid, GMP, IMP, myristoyl-carnitine, palmitoyl-carnitine, succinyl-Cys, UMP, UTP, UDP-GlcNAc. Conclusion PRKAG2-mutated iPSC-CMs exhibit bioenergetic and metabolic aberrations, which contribute to the cardiac pathological aspects of WPW syndrome. Importantly, treatment with the AMPK activator metformin eliminated the bioenergetic abnormalities in the mutated cells, while isogenic and healthy control cells remained unaffected. Based on these novel findings, a new therapeutic modality in WPW patients may be considered. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): Israel Science Foundation (ISF)

Background Mutations in the PRKAG2 gene encoding the γ-subunit of adenosine monophosphate kinase (AMPK) cause hypertrophic cardiomyopathy (HCM) and familial Wolff-Parkinson-White (WPW) syndrome. Patients carrying the R302Q mutation in PRKAG2 present with sinus bradycardia, escape rhythms, ventricular preexcitation, supraventricular tachycardia, and atrioventricular block. This mutation affects AMPK activity and increases glycogen storage in cardiomyocytes. The link between glycogen storage, WPW syndrome, HCM, and arrhythmias remains unknown. Objective The purpose of this study was to investigate the pathological changes caused by the PRKAG2 mutation. We tested the hypothesis that patient's induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) display clinical aspects of the disease. Methods Using clustered regularly interspaced short palindromic repeats (CRISPR) technology, we corrected the mutation and then generated isogenic iPSC-CMs. Action potentials were recorded from spontaneously firing and paced cardiomyocytes using the patch clamp technique. Using a microelectrode array setup, we recorded electrograms from iPSC-CMs clusters. Transmission electron microscopy was used to detect ultrastructural abnormalities in the mutated iPSC-CMs. Results PRKAG2-mutated iPSC-CMs exhibited abnormal firing patterns, delayed afterdepolarizations, triggered arrhythmias, and augmented beat rate variability. Importantly, CRISPR correction eliminated the electrophysiological abnormalities, the augmented glycogen, storage, and cardiomyocyte hypertrophy. Conclusion PRKAG2-mutated iPSC-CMs displayed functional and structural abnormalities, which were abolished by correcting the mutation in the patient's iPSCs using CRISPR technology.

Duchenne muscular dystrophy (DMD) is an X‐linked progressive muscle degenerative disease, caused by mutations in the dystrophin gene and resulting in death because of respiratory or cardiac failure. To investigate the cardiac cellular manifestation of DMD, we generated induced pluripotent stem cells (iPSCs) and iPSC‐derived cardiomyocytes (iPSC‐CMs) from two DMD patients: a male and female manifesting heterozygous carrier. Dystrophin mRNA and protein expression were analysed by qRT‐PCR, RNAseq, Western blot and immunofluorescence staining. For comprehensive electrophysiological analysis, current and voltage clamp were used to record transmembrane action potentials and ion currents, respectively. Microelectrode array was used to record extracellular electrograms. X‐inactive specific transcript (XIST) and dystrophin expression analyses revealed that female iPSCs underwent X chromosome reactivation (XCR) or erosion of X chromosome inactivation, which was maintained in female iPSC‐CMs displaying mixed X chromosome expression of wild type (WT) and mutated alleles. Both DMD female and male iPSC‐CMs presented low spontaneous firing rate, arrhythmias and prolonged action potential duration. DMD female iPSC‐CMs displayed increased beat rate variability (BRV). DMD male iPSC‐CMs manifested decreased I f density, and DMD female and male iPSC‐CMs showed increased I Ca,L density. Our findings demonstrate cellular mechanisms underlying electrophysiological abnormalities and cardiac arrhythmias in DMD.

Duchenne muscular dystrophy (DMD) is an X-linked progressive muscle degenerative disease caused by mutations in the dystrophin gene. We generated induced pluripotent stem cells (iPSCs) from a 13-year-old male patient carrying a deletion mutation of exons 45–50; iPSCs were subsequently differentiated into cardiomyocytes. iPSCs exhibit expression of the pluripotent markers (SOX2, NANOG, OCT4), differentiation capacity into the three germ layers, normal karyotype, genetic identity to the skin biopsy dermal fibroblasts and the patient-specific dystrophin mutation.

Aims Dilated cardiomyopathy (DCM), a myocardial disorder that can result in progressive heart failure and arrhythmias, is defined by ventricular chamber enlargement and dilatation, and systolic dysfunction. Despite extensive research, the pathological mechanisms of DCM are unclear mainly due to numerous mutations in different gene families resulting in the same outcome—decreased ventricular function. Titin (TTN)—a giant protein, expressed in cardiac and skeletal muscles, is an important part of the sarcomere, and thus TTN mutations are the most common cause of adult DCM. To decipher the basis for the cardiac pathology in titin-mutated patients, we investigated the hypothesis that induced Pluripotent Stem Cell (iPSC)-derived cardiomyocytes (iPSC-CM) generated from patients, recapitulate the disease phenotype. The hypothesis was tested by 3 Aims: (1) Investigate key features of the excitation-contraction-coupling machinery; (2) Investigate the responsiveness to positive inotropic interventions; (3) Investigate the proteome profile of the AuP cardiomyocytes using mass-spectrometry (MS). Methods and results iPSC were generated from the patients' skin fibroblasts. The major findings were: (1) Sarcomeric organization analysis in mutated iPSC-CM showed defects in assembly and maintenance of sarcomeric structure. (2) Mutated iPSC-CM exhibited diminished inotropic and lusitropic responses to β-adrenergic stimulation with isoproterenol, increased [Ca2+]out and angiotensin-II. Additionally, mutated iPSC-CM displayed prolonged recovery in response to caffeine. These findings may result from defective or lack of interactions of the sarcomeric components with titin through its kinase domain which is absent in the mutated cells. Conclusions These findings show that the mutated cardiomyocytes from DCM patients recapitulate abnormalities of the inherited cardiomyopathies, expressed as blunted inotropic response.

Mutations in SCO2 are among the most common causes of COX deficiency, resulting in reduced mitochondrial oxidative ATP production capacity, often leading to hypertrophic cardiomyopathy (HCM). To date, none of the recent pertaining reports provide deep understanding of the SCO2 disease pathophysiology. To investigate the cardiac pathology of the disease, we were the first to generate induced pluripotent stem cell (iPSC)‐derived cardiomyocytes (iPSC‐CMs) from SCO2‐mutated patients. For iPSC generation, we reprogrammed skin fibroblasts from two SCO2 patients and healthy controls. The first patient was a compound heterozygote to the common E140K mutation, and the second was homozygote for the less common G193S mutation. iPSC were differentiated into cardiomyocytes through embryoid body (EB) formation. To test the hypothesis that the SCO2 mutation is associated with mitochondrial abnormalities, and intracellular Ca2+‐overload resulting in functional derangements and arrhythmias, we investigated in SCO2‐mutated iPSC‐CMs (compared to control cardiomyocytes): (i) the ultrastructural changes; (ii) the inotropic responsiveness to β‐adrenergic stimulation, increased [Ca2+]o and angiotensin‐II (AT‐II); and (iii) the Beat Rate Variability (BRV) characteristics. In support of the hypothesis, we found in the mutated iPSC‐CMs major ultrastructural abnormalities and markedly attenuated response to the inotropic interventions and caffeine, as well as delayed afterdepolarizations (DADs) and increased BRV, suggesting impaired SR Ca2+ handling due to attenuated SERCA activity caused by ATP shortage. Our novel results show that iPSC‐CMs are useful for investigating the pathophysiological mechanisms underlying the SCO2 mutation syndrome.

PTEN-induced putative kinase 1 (PINK1) and parkin are mutated in familial forms of Parkinson's disease and are important in promoting the mitophagy of damaged mitochondria. In this study, we showed that synphilin-1 interacted with PINK1 and was recruited to the mitochondria. Once in the mitochondria, it promoted PINK1-dependent mitophagy, as revealed by Atg5 knockdown experiments and the recruitment of LC3 and Lamp1 to the mitochondria. PINK1-synphilin-1 mitophagy did not depend on PINK1-mediated phosphorylation of synphilin-1 and occurred in the absence of parkin. Synphilin-1 itself caused depolarization of the mitochondria and increased the amount of uncleaved PINK1 at the organelle. Furthermore, synphilin-1 recruited seven in absentia homolog (SIAH)-1 to the mitochondria where it promoted mitochondrial protein ubiquitination and subsequent mitophagy. Mitophagy via this pathway was impaired by synphilin-1 knockdown or by the use of a synphilin-1 mutant that is unable to recruit SIAH-1 to the mitochondria. Likewise, knockdown of SIAH-1 or the use of a catalytically inactive SIAH-1 mutant abrogated mitophagy. PINK1 disease mutants failed to recruit synphilin-1 and did not activate mitophagy, indicating that PINK1-synphilin-1-SIAH-1 represents a new parkin-independent mitophagy pathway. Drugs that activate this pathway will provide a novel strategy to promote the clearance of damaged mitochondria in Parkinson's disease.