Nkx2-5 Loss of Function in the His-Purkinje System Hampers Its Maturation and Leads to Mechanical Dysfunction.

The ventricular conduction or His-Purkinje system (VCS) mediates the rapid propagation and precise delivery of electrical activity essential for the synchronization of heartbeats. Mutations in the transcription factor Nkx2-5 have been implicated in a high prevalence of developing ventricular conduction defects or arrhythmias with age. Nkx2-5 heterozygous mutant mice reproduce human phenotypes associated with a hypoplastic His-Purkinje system resulting from defective patterning of the Purkinje fiber network during development. Here, we investigated the role of Nkx2-5 in the mature VCS and the consequences of its loss on cardiac function. Neonatal deletion of Nkx2-5 in the VCS using a Cx40-CreERT2 mouse line provoked apical hypoplasia and maturation defects of the Purkinje fiber network. Genetic tracing analysis demonstrated that neonatal Cx40-positive cells fail to maintain a conductive phenotype after Nkx2-5 deletion. Moreover, we observed a progressive loss of expression of fast-conduction markers in persistent Purkinje fibers. Consequently, Nkx2-5-deleted mice developed conduction defects with progressively reduced QRS amplitude and RSR' complex associated with higher duration. Cardiac function recorded by MRI revealed a reduction in the ejection fraction in the absence of morphological changes. With age, these mice develop a ventricular diastolic dysfunction associated with dyssynchrony and wall-motion abnormalities without indication of fibrosis. These results highlight the requirement of postnatal expression of Nkx2-5 in the maturation and maintenance of a functional Purkinje fiber network to preserve contraction synchrony and cardiac function.

Tocotrienols-enriched Self-nanoemulsifying Drug Delivery System Enhances the Antileukemic Activity of All-trans Retinoic Acid but not Electrocardiogram Alterations Evoked by Its Combination with Arsenic Trioxide.

All-trans retinoic acid and arsenic trioxide are the leading choices for the treatment of acute promyelocytic leukemia. Notwithstanding the impressive differentiative properties of all-trans retinoic acid and the apoptotic properties of arsenic trioxide, some problems still occur in acute promyelocytic leukemia treatment. These problems are due to patients' relapses, mainly related to changes in the ligand-binding domain of RARα (retinoic acid receptor α) and the cardiotoxic effects caused by arsenic trioxide. We previously developed a self-nanoemulsifying drug delivery system enriched with tocotrienols to deliver all-trans retinoic acid (SNEDDS-TRF-ATRA). Herein, we have evaluated if tocotrienols can help revert ATRA resistance in an APL cell line (NB4-R2 compared to sensitive NB4 cells) and mitigate the cardiotoxic effects of arsenic trioxide in a murine model. SNEDDS-TRF-ATRA enhanced all-trans retinoic acid cytotoxicity in NB4-R2 (resistant) cells but not in NB4 (sensitive) cells. Moreover, SNEDDS-TRF-ATRA did not significantly change the differentiative properties of all-trans retinoic acid in both NB4 and NB4-R2 cells. Combined administration of SNEDDS-TRF-ATRA and arsenic trioxide could revert QTc interval prolongation caused by ATO but evoked other electrocardiogram alterations in mice, such as T wave flattening. Therefore, SNEDDS-TRF-ATRA may enhance the antileukemic properties of all-trans retinoic acid but may influence ECG changes caused by arsenic trioxide administration. SNEDDS-TRF-ATRA presents cytotoxicity in resistant APL cells (NB4-R2). Combined administration of ATO and SNEDDS-TRF-ATRA in mice prevented the prolongation of the QTc interval caused by ATO but evoked ECG abnormalities such as T wave flattening.

Spironolactone as a Potential New Treatment to Prevent Arrhythmias in Arrhythmogenic Cardiomyopathy Cell Model.

Arrhythmogenic cardiomyopathy (ACM) is a rare genetic disease associated with ventricular arrhythmias in patients. The occurrence of these arrhythmias is due to direct electrophysiological remodeling of the cardiomyocytes, namely a reduction in the action potential duration (APD) and a disturbance of Ca2+ homeostasis. Interestingly, spironolactone (SP), a mineralocorticoid receptor antagonist, is known to block K+ channels and may reduce arrhythmias. Here, we assess the direct effect of SP and its metabolite canrenoic acid (CA) in cardiomyocytes derived from human-induced pluripotent stem cells (hiPSC-CMs) of a patient bearing a missense mutation (c.394C>T) in the DSC2 gene coding for desmocollin 2 and for the amino acid replacement of arginine by cysteine at position 132 (R132C). SP and CA corrected the APD in the muted cells (vs. the control) in linking to a normalization of the hERG and KCNQ1 K+ channel currents. In addition, SP and CA had a direct cellular effect on Ca2+ homeostasis. They reduced the amplitude and aberrant Ca2+ events. In conclusion, we show the direct beneficial effects of SP on the AP and Ca2+ homeostasis of DSC2-specific hiPSC-CMs. These results provide a rationale for a new therapeutical approach to tackle mechanical and electrical burdens in patients suffering from ACM.

Ephrin-B1 regulates the adult diastolic function through a late postnatal maturation of cardiomyocyte surface crests.

The rod-shaped adult cardiomyocyte (CM) harbors a unique architecture of its lateral surface with periodic crests, relying on the presence of subsarcolemmal mitochondria (SSM) with unknown role. Here, we investigated the development and functional role of CM crests during the postnatal period. We found in rodents that CM crest maturation occurs late between postnatal day 20 (P20) and P60 through both SSM biogenesis, swelling and crest-crest lateral interactions between adjacent CM, promoting tissue compaction. At the functional level, we showed that the P20-P60 period is dedicated to the improvement of relaxation. Interestingly, crest maturation specifically contributes to an atypical CM hypertrophy of its short axis, without myofibril addition, but relying on CM lateral stretching. Mechanistically, using constitutive and conditional CM-specific knock-out mice, we identified ephrin-B1, a lateral membrane stabilizer, as a molecular determinant of P20-P60 crest maturation, governing both the CM lateral stretch and the diastolic function, thus highly suggesting a link between crest maturity and diastole. Remarkably, while young adult CM-specific Efnb1 KO mice essentially exhibit an impairment of the ventricular diastole with preserved ejection fraction and exercise intolerance, they progressively switch toward systolic heart failure with 100% KO mice dying after 13 months, indicative of a critical role of CM-ephrin-B1 in the adult heart function. This study highlights the molecular determinants and the biological implication of a new late P20-P60 postnatal developmental stage of the heart in rodents during which, in part, ephrin-B1 specifically regulates the maturation of the CM surface crests and of the diastolic function.

Neurovascular hypoxia after mild traumatic brain injury in juvenile mice correlates with heart-brain dysfunctions in adulthood.

AIM: Retrospective studies suggest that mild traumatic brain injury (mTBI) in pediatric patients may lead to an increased risk of cardiac events. However, the exact functional and temporal dynamics and the associations between heart and brain pathophysiological trajectories are not understood. METHODS: A single impact to the left somatosensory cortical area of the intact skull was performed on juvenile mice (17 days postnatal). Cerebral 3D photoacoustic imaging was used to measure the oxygen saturation (sO2 ) in the impacted area 4 h after mTBI followed by 2D and 4D echocardiography at days 7, 30, 90, and 190 post-impact. At 8 months, we performed a dobutamine stress test to evaluate cardiac function. Lastly, behavioral analyses were conducted 1 year after initial injury. RESULTS: We report a rapid and transient decrease in cerebrovascular sO2 and increased hemoglobin in the impacted left brain cortex. Cardiac analyses showed long-term diastolic dysfunction and a diminished systolic strain response under stress in the mTBI group. At the molecular level, cardiac T-p38MAPK and troponin I expression was pathologic modified post-mTBI. We found linear correlations between brain sO2 measured immediately post-mTBI and long-term cardiac strain after 8 months. We report that initial cerebrovascular hypoxia and chronic cardiac dysfunction correlated with long-term behavioral changes hinting at anxiety-like and memory maladaptation. CONCLUSION: Experimental juvenile mTBI induces time-dependent cardiac dysfunction that corresponds to the initial neurovascular sO2 dip and is associated with long-term behavioral modifications. These imaging biomarkers of the heart-brain axis could be applied to improve clinical pediatric mTBI management.

Involvement of Vasopressin in Tissue Hypoperfusion during Cardiogenic Shock Complicating Acute Myocardial Infarction in Rats.

Acute heart failure (AHF) due to acute myocardial infarction (AMI) is likely to involve cardiogenic shock (CS), with neuro-hormonal activation. A relationship between AHF, CS and vasopressin response is suspected. This study aimed to investigate the implication of vasopressin on hemodynamic parameters and tissue perfusion at the early phase of CS complicating AMI. Experiments were performed on male Wistar rats submitted or not to left coronary artery ligation (AMI and Sham). Six groups were studied Sham and AMI treated or not with either a vasopressin antagonist SR-49059 (Sham-SR, AMI-SR) or agonist terlipressin (Sham-TLP, AMI-TLP). Animals were sacrificed one day after surgery (D1) and after hemodynamic parameters determination. Vascular responses to vasopressin were evaluated, ex vivo, on aorta. AHF was defined by a left ventricular ejection fraction below 40%. CS was defined by AHF plus tissue hypoperfusion evidenced by elevated serum lactate level or low mesenteric oxygen saturation (SmO2) at D1. Mortality rates were 40% in AMI, 0% in AMI-SR and 33% in AMI-TLP. Immediately after surgery, a sharp decrease in SmO2 was observed in all groups. At D1, SmO2 recovered in Sham and in SR-treated animals while it remained low in AMI and further decreased in TLP-treated groups. The incidence of CS among AHF animals was 72% in AMI or AMI-TLP while it was reduced to 25% in AMI-SR. Plasma copeptin level was increased by AMI. Maximal contractile response to vasopressin was decreased in AMI (32%) as in TLP- and SR- treated groups regardless of ligation. Increased vasopressin secretion occurring in the early phase of AMI may be responsible of mesenteric hypoperfusion resulting in tissue hypoxia. Treatment with a vasopressin antagonist enhanced mesenteric perfusion and improve survival. This could be an interesting therapeutic strategy to prevent progression to cardiogenic shock.

Identification of Cx43 variants predisposing to ventricular fibrillation in the acute phase of ST-elevation myocardial infarction.

AIMS: Ventricular fibrillation (VF) occurring in the acute phase of ST-elevation myocardial infarction (STEMI) is the leading cause of sudden cardiac death worldwide. Several studies showed that reduced connexin 43 (Cx43) expression and reduced conduction velocity increase the risk of VF in acute myocardial infarction (MI). Furthermore, genetic background might predispose individuals to primary VF (PVF). The primary objective was to evaluate the presence of GJA1 variants in STEMI patients. The secondary objective was to evaluate the arrhythmogenic impact of GJA1 variants in STEMI patients with VF. METHODS AND RESULTS: The MAP-IDM prospective cohort study included 966 STEMI patients and was designed to identify genetic predisposition to VF. A total of 483 (50.0%) STEMI patients with PVF were included. The presence of GJA1 variants increased the risk of VF in STEMI patients [from 49.1 to 70.8%, P = 0.0423; odds ratio (OR): 0.40; 95% confidence interval: 0.16-0.97; P = 0.04]. The risk of PVF decreased with beta-blocker intake (from 53.5 to 44.8%, P = 0.0085), atrial fibrillation (from 50.7 to 26.4%, P = 0.0022), and with left ventricular ejection fraction >50% (from 60.2 to 41.4%, P < 0.0001). Among 16 GJA1 variants, three novel heterozygous missense variants were identified in three patients: V236I, H248R, and I327M. In vitro studies of these variants showed altered Cx43 localization and decreased cellular communication, mainly during acidosis. CONCLUSION: Connexin 43 variants are associated with increased VF susceptibility in STEMI patients. Restoring Cx43 function may be a potential therapeutic target to prevent PVF in patients with acute MI. CLINICAL TRIAL REGISTRATION: Clinical Trial Registration: https://clinicaltrials.gov/ct2/show/NCT00859300.

Polyester Nanocapsules for Intravenous Delivery of Artemether: Formulation Development, Antimalarial Efficacy, and Cardioprotective Effects In Vivo.

Artemether (ATM) is an effective antimalarial drug that also has a short half-life in the blood. Furthermore, ATM is also cardiotoxic and is associated with pro-arrhythmogenic risks. We aimed to develop a delivery system enabling the prolonged release of ATM into the blood coupled with reduced cardiotoxicity. To achieve this, we prepared polymeric nanocapsules (NCs) from different biodegradable polyesters, namely poly(D,L-lactide) (PLA), poly-ε-caprolactone (PCL), and surface-modified NCs, using a monomethoxi-polyethylene glycol-block-poly(D,L-lactide) (PEG5kDa-PLA45kDa) polymer. Using this approach, we were able to encapsulate high yields of ATM (>85%, 0−4 mg/mL) within the oily core of the NCs. The PCL-NCs exhibited the highest percentage of ATM loading as well as a slow release rate. Atomic force microscopy showed nanometric and spherical particles with a narrow size dispersion. We used the PCL NCs loaded with ATM for biological evaluation following IV administration. As with free-ATM, the ATM-PCL-NCs formulation exhibited potent antimalarial efficacy using either the "Four-day test" protocol (ATM total at the end of the 4 daily doses: 40 and 80 mg/kg) in Swiss mice infected with P. berghei or a single low dose (20 mg/kg) of ATM in mice with higher parasitemia (15%). In healthy rats, IV administration of single doses of free-ATM (40 or 80 mg/kg) prolonged cardiac QT and QTc intervals and induced both bradycardia and hypotension. Repeated IV administration of free-ATM (four IV doses at 20 mg/kg every 12 h for 48 h) also prolonged the QT and QTc intervals but, paradoxically, induced tachycardia and hypertension. Remarkably, the incorporation of ATM in ATM-PCL-NCs reduced all adverse effects. In conclusion, the encapsulation of ATM in biodegradable polyester NCs reduces its cardiovascular toxicity without affecting its antimalarial efficacy.

Stabilizing Ryanodine Receptors Improves Left Ventricular Function in Juvenile Dogs With Duchenne Muscular Dystrophy.

BACKGROUND: Duchenne muscular dystrophy is associated with progressive deterioration in left ventricular (LV) function. The golden retriever muscular dystrophy (GRMD) dog model recapitulates the pathology and clinical manifestations of Duchenne muscular dystrophy. Importantly, they develop progressive LV dysfunction starting at early age. OBJECTIVES: The authors tested the cardioprotective effect of chronic administration of the ARM036, a small molecule that stabilizes the closed conformation of the cardiac sarcoplasmic reticulum ryanodine receptor/calcium release channel (RyR2) in young GRMD-dogs. METHODS: Two-month-old GRMD-dogs were treated with ARM036 or placebo for 4 months. Healthy-dogs of the same genetic background served as controls. Cardiac function was evaluated by conventional and 2-dimensional speckle-tracking echocardiography. Cardiac cellular and molecular analyses were performed at 6 months old. RESULTS: Conventional echocardiography showed normal LV dimensions and ejection fraction in 6-month-old GRMD dogs. Interestingly, 2-dimensional speckle-tracking echocardiography revealed decreased global longitudinal strain and the presence of hypokinetic segments in placebo-treated GRMD dogs. Single-channel measurements revealed higher RyR2 open probability at low resting Ca2+ in GRMD cardiomyocytes than in controls. ARM036 prevented those in vivo and in vitro dysfunctions in GRMD dogs. Myofilament Ca2+-sensitivity was increased in permeabilized GRMD cardiomyocytes at short sarcomere length. ARM036 had no effect on this parameter. Cross-bridge cycling kinetics were altered in GRMD myocytes and recovered with ARM036 treatment, which coincided with the level of myosin binding protein-C-S glutathionylation. CONCLUSIONS: GRMD-dogs exhibit early LV dysfunction associated with altered myofilament contractile properties. These abnormalities were prevented pharmacologically by stabilizing RyR2 with ARM036.

pH-sensitive doxorubicin-tocopherol succinate prodrug encapsulated in docosahexaenoic acid-based nanostructured lipid carriers: An effective strategy to improve pharmacokinetics and reduce toxic effects.

Side effects often limit the use of doxorubicin (DOX) in cancer treatment. We have recently developed a nanostructured lipid carrier (NLC) formulation for synergistic chemotherapy, encapsulating DOX and the anticancer adjuvants docosahexaenoic acid (DHA) and α-tocopherol succinate (TS). Hydrophobic ion-pairing with TS allowed a high DOX entrapment in the nanocarrier. In this work, we investigated the pharmacokinetics of this formulation after intravenous administration in mice. The first data obtained led us to propose synthesizing covalent DOX-TS conjugates to increase DOX retention in the NLC. We successfully conjugated DOX to TS via an amide or hydrazone bond. In vitro studies in 4T1 tumor cells indicated low cytotoxicity of the amide derivative, while the hydrazone conjugate was effective in killing cancer cells. We encapsulated the hydrazone derivative in a DHA-based nanocarrier (DOX-hyd-TS/NLC), which had reduced particle size and high drug encapsulation efficiency. The pH-sensitive hydrazone bond allowed controlled DOX release from the NLC, with increased drug release at acidic conditions. In vivo studies revealed that DOX-hyd-TS/NLC had a better pharmacokinetic profile than free DOX and attenuated the short-term cardiotoxic effects caused by DOX, such as QT prolongation and impaired left ventricular systolic function. Moreover, this formulation showed excellent therapeutic performance by reducing tumor growth in 4T1 tumor-bearing mice and decreasing DOX-induced toxicity to the heart and liver, demonstrated by hematologic, biochemical, and histologic analyses. These results indicate that DOX-hyd-TS/NLC may be a promising nanocarrier for breast cancer treatment.

Sarcoplasmic Reticulum Calcium Release Is Required for Arrhythmogenesis in the Mouse.

Dysfunctional sarcoplasmic reticulum Ca2+ handling is commonly observed in heart failure, and thought to contribute to arrhythmogenesis through several mechanisms. Some time ago we developed a cardiomyocyte-specific inducible SERCA2 knockout mouse, which is remarkable in the degree to which major adaptations to sarcolemmal Ca2+ entry and efflux overcome the deficit in SR reuptake to permit relatively normal contractile function. Conventionally, those adaptations would also be expected to dramatically increase arrhythmia susceptibility. However, that susceptibility has never been tested, and it is possible that the very rapid repolarization of the murine action potential (AP) allows for large changes in sarcolemmal Ca2+ transport without substantially disrupting electrophysiologic stability. We investigated this hypothesis through telemetric ECG recording in the SERCA2-KO mouse, and patch-clamp electrophysiology, Ca2+ imaging, and mathematical modeling of isolated SERCA2-KO myocytes. While the SERCA2-KO animals exhibit major (and unique) electrophysiologic adaptations at both the organ and cell levels, they remain resistant to arrhythmia. A marked increase in peak L-type calcium (I CaL) current and slowed I CaL decay elicited pronounced prolongation of initial repolarization, but faster late repolarization normalizes overall AP duration. Early afterdepolarizations were seldom observed in KO animals, and those that were observed exhibited a mechanism intermediate between murine and large mammal dynamical properties. As expected, spontaneous SR Ca2+ sparks and waves were virtually absent. Together these findings suggest that intact SR Ca2+ handling is an absolute requirement for triggered arrhythmia in the mouse, and that in its absence, dramatic changes to the major inward currents can be resisted by the substantial K+ current reserve, even at end-stage disease.

Could a Multi-Marker and Machine Learning Approach Help Stratify Patients with Heart Failure?

Half of the patients with heart failure (HF) have preserved ejection fraction (HFpEF). To date, there are no specific markers to distinguish this subgroup. The main objective of this work was to stratify HF patients using current biochemical markers coupled with clinical data. The cohort study included HFpEF (n = 24) and heart failure with reduced ejection fraction (HFrEF) (n = 34) patients as usually considered in clinical practice based on cardiac imaging (EF ≥ 50% for HFpEF; EF < 50% for HFrEF). Routine blood tests consisted of measuring biomarkers of renal and heart functions, inflammation, and iron metabolism. A multi-test approach and analysis of peripheral blood samples aimed to establish a computerized Machine Learning strategy to provide a blood signature to distinguish HFpEF and HFrEF. Based on logistic regression, demographic characteristics and clinical biomarkers showed no statistical significance to differentiate the HFpEF and HFrEF patient subgroups. Hence a multivariate factorial discriminant analysis, performed blindly using the data set, allowed us to stratify the two HF groups. Consequently, a Machine Learning (ML) strategy was developed using the same variables in a genetic algorithm approach. ML provided very encouraging explorative results when considering the small size of the samples applied. The accuracy and the sensitivity were high for both validation and test groups (69% and 100%, 64% and 75%, respectively). Sensitivity was 100% for the validation and 75% for the test group, whereas specificity was 44% and 55% for the validation and test groups because of the small number of samples. Lastly, the precision was acceptable, with 58% in the validation and 60% in the test group. Combining biochemical and clinical markers is an excellent entry to develop a computer classification tool to diagnose HFpEF. This translational approach is a springboard for improving new personalized treatment methods and identifying "high-yield" populations for clinical trials.

Polylactide Nanocapsules Attenuate Adverse Cardiac Cellular Effects of Lyso-7, a Pan-PPAR Agonist/Anti-Inflammatory New Thiazolidinedione.

Lyso-7 is a novel synthetic thiazolidinedione, which is a receptor (pan) agonist of PPAR α,ß/δ,γ with anti-inflammatory activity. We investigated the cardiotoxicity of free Lyso-7 in vitro (4.5-450 nM), and Lyso-7 loaded in polylactic acid nanocapsules (NC) in vivo (Lyso-7-NC, 1.6 mg/kg). In previous work, we characterized Lyso-7-NC. We administered intravenously Lyso-7, Lyso-7-NC, control, and blank-NC once a day for seven days in mice. We assessed cell contraction and intracellular Ca2+ transients on single mice cardiomyocytes enzymatically isolated. Lyso-7 reduced cell contraction and accelerated relaxation while lowering diastolic Ca2+ and reducing Ca2+ transient amplitude. Lyso-7 also promoted abnormal ectopic diastolic Ca2+ events, which isoproterenol dramatically enhanced. Incorporation of Lyso-7 in NC attenuated drug effects on cell contraction and prevented its impact on relaxation, diastolic Ca2+, Ca2+ transient amplitude, Ca2+ transient decay kinetics, and promotion of diastolic Ca2+ events. Acute effects of Lyso-7 on cardiomyocytes in vitro at high concentrations (450 nM) were globally similar to those observed after repeated administration in vivo. In conclusion, we show evidence for off-target effects of Lyso-7, seen during acute exposure of cardiomyocytes to high concentrations and after repeated treatment in mice. Nano-encapsulation of Lyso-7 in polymeric NC attenuated the unwanted effects, particularly ectopic Ca2+ events known to support life-threatening arrhythmias favored by stress or exercise.

Nanomedicine in Oncocardiology: Contribution and Perspectives of Preclinical Studies.

Cancer and cardiovascular diseases are the leading causes of death and morbidity worldwide. Strikingly, cardiovascular disorders are more common and more severe in cancer patients than in the general population, increasing incidence rates. In this context, it is vital to consider the anticancer efficacy of a treatment and the devastating heart complications it could potentially cause. Oncocardiology has emerged as a promising medical and scientific field addressing these aspects from different angles. Interestingly, nanomedicine appears to have great promise in reducing the cardiotoxicity of anticancer drugs, maintaining or even enhancing their efficacy. Several studies have shown the benefits of nanocarriers, although with some flaws when considering the concept of oncocardiology. Herein, we discuss how preclinical studies should be designed as closely as possible to clinical protocols, considering various parameters intrinsic to the animal models used and the experimental protocols. The sex and age of the animals, the size and location of the tumors, the doses of the nanoformulations administered, and the acute vs. the long-term effects of treatments are essential aspects. We also discuss the perspectives offered by non-invasive imaging techniques to simultaneously assess both the anticancer effects of treatment and its potential impact on the heart. The overall objective is to accelerate the development and validation of nanoformulations through high-quality preclinical studies reproducing the clinical conditions.

Dystrophin Deficiency Causes Progressive Depletion of Cardiovascular Progenitor Cells in the Heart.

Duchenne muscular dystrophy (DMD) is a devastating condition shortening the lifespan of young men. DMD patients suffer from age-related dilated cardiomyopathy (DCM) that leads to heart failure. Several molecular mechanisms leading to cardiomyocyte death in DMD have been described. However, the pathological progression of DMD-associated DCM remains unclear. In skeletal muscle, a dramatic decrease in stem cells, so-called satellite cells, has been shown in DMD patients. Whether similar dysfunction occurs with cardiac muscle cardiovascular progenitor cells (CVPCs) in DMD remains to be explored. We hypothesized that the number of CVPCs decreases in the dystrophin-deficient heart with age and disease state, contributing to DCM progression. We used the dystrophin-deficient mouse model (mdx) to investigate age-dependent CVPC properties. Using quantitative PCR, flow cytometry, speckle tracking echocardiography, and immunofluorescence, we revealed that young mdx mice exhibit elevated CVPCs. We observed a rapid age-related CVPC depletion, coinciding with the progressive onset of cardiac dysfunction. Moreover, mdx CVPCs displayed increased DNA damage, suggesting impaired cardiac muscle homeostasis. Overall, our results identify the early recruitment of CVPCs in dystrophic hearts and their fast depletion with ageing. This latter depletion may participate in the fibrosis development and the acceleration onset of the cardiomyopathy.

Hypoxic Conditions Promote Rhythmic Contractile Oscillations Mediated by Voltage-Gated Sodium Channels Activation in Human Arteries.

Arterial smooth muscle exhibits rhythmic oscillatory contractions called vasomotion and believed to be a protective mechanism against tissue hypoperfusion or hypoxia. Oscillations of vascular tone depend on voltage and follow oscillations of the membrane potential. Voltage-gated sodium channels (Nav), responsible for the initiation and propagation of action potentials in excitable cells, have also been evidenced both in animal and human vascular smooth muscle cells (SMCs). For example, they contribute to arterial contraction in rats, but their physiopathological relevance has not been established in human vessels. In the present study, we investigated the functional role of Nav in the human artery. Experiments were performed on human uterine arteries obtained after hysterectomy and on SMCs dissociated from these arteries. In SMCs, we recorded a tetrodotoxin (TTX)-sensitive and fast inactivating voltage-dependent INa current. Various Nav genes, encoding α-subunit isoforms sensitive (Nav 1.2; 1.3; 1.7) and resistant (Nav 1.5) to TTX, were detected both in arterial tissue and in SMCs. Nav channels immunostaining showed uniform distribution in SMCs and endothelial cells. On arterial tissue, we recorded variations of isometric tension, ex vivo, in response to various agonists and antagonists. In arterial rings placed under hypoxic conditions, the depolarizing agent KCl and veratridine, a specific Nav channels agonist, both induced a sustained contraction overlaid with rhythmic oscillations of tension. After suppression of sympathetic control either by blocking the release of catecholamine or by antagonizing the target adrenergic response, rhythmic activity persisted while the sustained contraction was abolished. This rhythmic activity of the arteries was suppressed by TTX but, in contrast, only attenuated by antagonists of calcium channels, Na+/Ca2+ exchanger, Na+/K+-ATPase and the cardiac Nav channel. These results highlight the role of Nav as a novel key element in the vasomotion of human arteries. Hypoxia promotes activation of Nav channels involved in the initiation of rhythmic oscillatory contractile activity.

Deciphering DSC2 arrhythmogenic cardiomyopathy electrical instability: From ion channels to ECG and tailored drug therapy.

BACKGROUND: Severe ventricular rhythm disturbances are the hallmark of arrhythmogenic cardiomyopathy (ACM), and are often explained by structural conduction abnormalities. However, comprehensive investigations of ACM cell electrical instability are lacking. This study aimed to elucidate early electrical myogenic signature of ACM. METHODS: We investigated a 41-year-old ACM patient with a missense mutation (c.394C>T) in the DSC2 gene, which encodes desmocollin 2. Pathogenicity of this variant was confirmed using a zebrafish DSC2 model system. Control and DSC2 patient-derived pluripotent stem cells were reprogrammed and differentiated into cardiomyocytes (hiPSC-CM) to examine the specific electromechanical phenotype and its modulation by antiarrhythmic drugs (AADs). Samples of the patient's heart and hiPSC-CM were examined to identify molecular and cellular alterations. RESULTS: A shortened action potential duration was associated with reduced Ca2+ current density and increased K+ current density. This finding led to the elucidation of previously unknown abnormal repolarization dynamics in ACM patients. Moreover, the Ca2+ mobilised during transients was decreased, and the Ca2+ sparks frequency was increased. AAD testing revealed the following: (1) flecainide normalised Ca2+ transients and significantly decreased Ca2+ spark occurrence and (2) sotalol significantly lengthened the action potential and normalised the cells' contractile properties. CONCLUSIONS: Thorough analysis of hiPSC-CM derived from the DSC2 patient revealed abnormal repolarization dynamics, prompting the discovery of a short QT interval in some ACM patients. Overall, these results confirm a myogenic origin of ACM electrical instability and provide a rationale for prescribing class 1 and 3 AADs in ACM patients with increased ventricular repolarization reserve.