Patients with Duchenne muscular dystrophy (DMD) have a progressive dilated cardiomyopathy associated with fatal cardiac arrhythmias. Electrical and functional abnormalities have been attributed to cardiac fibrosis; however, electrical abnormalities may occur in the absence of overt cardiac histopathology. Here we show that structural and functional remodeling of the cardiac sarcoplasmic reticulum (SR) Ca ²⁺ release channel/ryanodine receptor (RyR2) occurs in the mdx mouse model of DMD. RyR2 from mdx hearts were S-nitrosylated and depleted of calstabin2 (FKBP12.6), resulting in “leaky” RyR2 channels and a diastolic SR Ca ²⁺ leak. Inhibiting the depletion of calstabin2 from the RyR2 complex with the Ca ²⁺ channel stabilizer S107 (“rycal”) inhibited the SR Ca ²⁺ leak, inhibited aberrant depolarization in isolated cardiomyocytes, and prevented arrhythmias in vivo. This suggests that diastolic SR Ca ²⁺ leak via RyR2 due to S-nitrosylation of the channel and calstabin2 depletion from the channel complex likely triggers cardiac arrhythmias. Normalization of the RyR2-mediated diastolic SR Ca ²⁺ leak prevents fatal sudden cardiac arrhythmias in DMD.

The recent geographical expansion of phlebotomine vectors of Leishmania infantum in the Mediterranean subregion has been attributed to ongoing climate changes. At these latitudes, the activity of sand flies is typically seasonal; because seasonal phenomena are also sensitive to general variations in climate, current phenological data sets can provide a baseline for continuing investigations on sand fly population dynamics that may impact on future scenarios of leishmaniasis transmission. With this aim, in 2011–2013 a consortium of partners from eight Mediterranean countries carried out entomological investigations in sites where L. infantum transmission was recently reported. A common protocol for sand fly collection included monthly captures by CDC light traps, complemented by sticky traps in most of the sites. Collections were replicated for more than one season in order to reduce the effects of local weather events. In each site, the trapping effort was left unchanged throughout the survey to legitimate inter-seasonal comparisons. Data from 99,000 collected specimens were analyzed, resulting in the description of seasonal dynamics of 56,000 sand flies belonging to L. infantum vector species throughout a wide geographical area, namely P. perniciosus (Portugal, Spain and Italy), P. ariasi (France), P. neglectus (Greece), P. tobbi (Cyprus and Turkey), P. balcanicus and P. kandelakii (Georgia). Time of sand fly appearance/disappearance in collections differed between sites, and seasonal densities showed variations in each site. Significant correlations were found between latitude/mean annual temperature of sites and i) the first month of sand fly appearance, that ranged from early April to the first half of June; ii) the type of density trend, varying from a single peak in July/August to multiple peaks increasing in magnitude from May through September. A 3-modal trend, recorded for P. tobbi in Cyprus, represents a novel finding for a L. infantum vector. Adults ended the activity starting from mid September through November, without significant correlation with latitude/mean annual temperature of sites. The period of potential exposure to L.infantum in the Mediterranean subregion, as inferred by adult densities calculated from 3 years, 37 sites and 6 competent vector species, was associated to a regular bell-shaped density curve having a wide peak center encompassing the July-September period, and falling between early May to late October for more than 99% of values. Apparently no risk for leishmaniasis transmission took place from December through March in the years considered. We found a common pattern of nocturnal females activity, whose density peaked between 11 pm and 2 am. Despite annual variations, multiple collections performed over consecutive years provided homogeneous patterns of the potential behavior of leishmaniasis vectors in selected sites, which we propose may represent sentinel areas for future monitoring. In the investigated years, higher potential risk for L. infantum transmission in the Mediterranean was identified in the June-October period (97% relative vector density), however such risk was not equally distributed throughout the region, since density waves of adults occurred earlier and were more frequent in southern territories. Recent projections on global warming indicate a constant rise of temperatures in the Mediterranean subregion in the near-mid future. While this phenomenon already caused geographical expansion of several arthropod-borne diseases, it is likely to affect also temporal parameters of seasonally transmitted diseases such as leishmaniasis, a protozoan infection spread by the bite of phlebotomine sand flies. Phenology of sand flies consists in the periods of emergence of adults and their disappearance from collections following an activity period during warm months, which can be characterized by peaks of abundance. Current phenological observations can be important for continuing investigations on sand fly dynamics that may impact on leishmaniasis transmission in the future. With this aim, partners from eight Mediterranean countries identified sites with documented Leishmania infantum transmission by six different vector species and performed multiannual trappings. From the high number of 56,000 specimens collected throughout an area spanning from Portugal at west to Georgia at east, the current seasonal dynamics of Mediterranean vectors was obtained. Both, period of adults emergence and type of density trend were found to be significantly correlated with latitude or mean annual temperature of trapping sites. At the southernmost latitudes, vector activity started as early as begin of April and ended by late November, showing that the no-risk period of potential exposure to L. infantum lasted only 4 months.

Myocardial ischemic disease is the major cause of death worldwide. After myocardial infarction, reperfusion of infracted heart has been an important objective of strategies to improve outcomes. However, cardiac ischemia/reperfusion (I/R) is characterized by inflammation, arrhythmias, cardiomyocyte damage, and, at the cellular level, disturbance in Ca ²⁺ and redox homeostasis. In this study, we sought to determine how acute inflammatory response contributes to reperfusion injury and Ca ²⁺ homeostasis disturbance after acute ischemia. Using a rat model of I/R, we show that circulating levels of TNF-α and cardiac caspase-8 activity were increased within 6 h of reperfusion, leading to myocardial nitric oxide and mitochondrial ROS production. At 1 and 15 d after reperfusion, caspase-8 activation resulted in S-nitrosylation of the RyR2 and depletion of calstabin2 from the RyR2 complex, resulting in diastolic sarcoplasmic reticulum (SR) Ca ²⁺ leak. Pharmacological inhibition of caspase-8 before reperfusion with Q-LETD-OPh or prevention of calstabin2 depletion from the RyR2 complex with the Ca ²⁺ channel stabilizer S107 (“rycal”) inhibited the SR Ca ²⁺ leak, reduced ventricular arrhythmias, infarct size, and left ventricular remodeling after 15 d of reperfusion. TNF-α–induced caspase-8 activation leads to leaky RyR2 channels that contribute to myocardial remodeling after I/R. Thus, early prevention of SR Ca ²⁺ leak trough normalization of RyR2 function is cardioprotective.

TRPM4 is a non-selective Ca²⁺-activated cation channel expressed in the heart, particularly in the atria or conduction tissue. Mutations in the Trpm4 gene were recently associated with several human conduction disorders such as Brugada syndrome. TRPM4 channel has also been implicated at the ventricular level, in inotropism or in arrhythmia genesis due to stresses such as ß-adrenergic stimulation, ischemia-reperfusion, and hypoxia re-oxygenation. However, the physiological role of the TRPM4 channel in the healthy heart remains unclear. We aimed to investigate the role of the TRPM4 channel on whole cardiac function with a Trpm4 gene knock-out mouse (Trpm4^-/-) model. Morpho-functional analysis revealed left ventricular (LV) eccentric hypertrophy in Trpm4^-/- mice, with an increase in both wall thickness and chamber size in the adult mouse (aged 32 weeks) when compared to Trpm4^+/+ littermate controls. Immunofluorescence on frozen heart cryosections and qPCR analysis showed no fibrosis or cellular hypertrophy. Instead, cardiomyocytes in Trpm4^-/- mice were smaller than Trpm4^+/+with a higher density. Immunofluorescent labeling for phospho-histone H3, a mitosis marker, showed that the number of mitotic myocytes was increased 3-fold in the Trpm4^-/-neonatal stage, suggesting hyperplasia. Adult Trpm4^-/- mice presented multilevel conduction blocks, as attested by PR and QRS lengthening in surface ECGs and confirmed by intracardiac exploration. Trpm4^-/-mice also exhibited Luciani-Wenckebach atrioventricular blocks, which were reduced following atropine infusion, suggesting paroxysmal parasympathetic overdrive. In addition, Trpm4^-/- mice exhibited shorter action potentials in atrial cells. This shortening was unrelated to modifications of the voltage-gated Ca²⁺ or K⁺ currents involved in the repolarizing phase. TRPM4 has pleiotropic roles in the heart, including the regulation of conduction and cellular electrical activity which impact heart development.

Pulmonary hypertension provokes right heart failure and arrhythmias. Better understanding of the mechanisms underlying these arrhythmias is needed to facilitate new therapeutic approaches for the hypertensive, failing right ventricle (RV). The aim of our study was to identify the mechanisms generating arrhythmias in a model of RV failure induced by pulmonary hypertension. Rats were injected with monocrotaline to induce either RV hypertrophy or failure or with saline (control). ECGs were measured in conscious, unrestrained animals by telemetry. In isolated hearts, electrical activity was measured by optical mapping and myofiber orientation by diffusion tensor-MRI. Sarcoplasmic reticular Ca²⁺ handling was studied in single myocytes. Compared with control animals, the T-wave of the ECG was prolonged and in three of seven heart failure animals, prominent T-wave alternans occurred. Discordant action potential (AP) alternans occurred in isolated failing hearts and Ca²⁺ transient alternans in failing myocytes. In failing hearts, AP duration and dispersion were increased; conduction velocity and AP restitution were steeper. The latter was intrinsic to failing single myocytes. Failing hearts had greater fiber angle disarray; this correlated with AP duration. Failing myocytes had reduced sarco(endo)plasmic reticular Ca²⁺-ATPase activity, increased sarcoplasmic reticular Ca²⁺-release fraction, and increased Ca²⁺ spark leak. In hypertrophied hearts and myocytes, dysfunctional adaptation had begun, but alternans did not develop. We conclude that increased electrical and structural heterogeneity and dysfunctional sarcoplasmic reticular Ca²⁺ handling increased the probability of alternans, a proarrhythmic predictor of sudden cardiac death. These mechanisms are potential therapeutic targets for the correction of arrhythmias in hypertensive, failing RVs.