Essential Role of the RIα Subunit of cAMP-Dependent Protein Kinase in Regulating Cardiac Contractility and Heart Failure Development.

BACKGROUND: The heart expresses 2 main subtypes of cAMP-dependent protein kinase (PKA; type I and II) that differ in their regulatory subunits, RIα and RIIα. Embryonic lethality of RIα knockout mice limits the current understanding of type I PKA function in the myocardium. The objective of this study was to test the role of RIα in adult heart contractility and pathological remodeling. METHODS: We measured PKA subunit expression in human heart and developed a conditional mouse model with cardiomyocyte-specific knockout of RIα (RIα-icKO). Myocardial structure and function were evaluated by echocardiography, histology, and ECG and in Langendorff-perfused hearts. PKA activity and cAMP levels were determined by immunoassay, and phosphorylation of PKA targets was assessed by Western blot. L-type Ca2+ current (ICa,L), sarcomere shortening, Ca2+ transients, Ca2+ sparks and waves, and subcellular cAMP were recorded in isolated ventricular myocytes (VMs). RESULTS: RIα protein was decreased by 50% in failing human heart with ischemic cardiomyopathy and by 75% in the ventricles and in VMs from RIα-icKO mice but not in atria or sinoatrial node. Basal PKA activity was increased ≈3-fold in RIα-icKO VMs. In young RIα-icKO mice, left ventricular ejection fraction was increased and the negative inotropic effect of propranolol was prevented, whereas heart rate and the negative chronotropic effect of propranolol were not modified. Phosphorylation of phospholamban, ryanodine receptor, troponin I, and cardiac myosin-binding protein C at PKA sites was increased in propranolol-treated RIα-icKO mice. Hearts from RIα-icKO mice were hypercontractile, associated with increased ICa,L, and [Ca2+]i transients and sarcomere shortening in VMs. These effects were suppressed by the PKA inhibitor, H89. Global cAMP content was decreased in RIα-icKO hearts, whereas local cAMP at the phospholamban/sarcoplasmic reticulum Ca2+ ATPase complex was unchanged in RIα-icKO VMs. RIα-icKO VMs had an increased frequency of Ca2+ sparks and proarrhythmic Ca2+ waves, and RIα-icKO mice had an increased susceptibility to ventricular tachycardia. On aging, RIα-icKO mice showed progressive contractile dysfunction, cardiac hypertrophy, and fibrosis, culminating in congestive heart failure with reduced ejection fraction that caused 50% mortality at 1 year. CONCLUSIONS: These results identify RIα as a key negative regulator of cardiac contractile function, arrhythmia, and pathological remodeling.

Dual effect of cardiac FKBP12.6 overexpression on excitation-contraction coupling and the incidence of ventricular arrhythmia depending on its expression level.

FKBP12.6, a binding protein to the immunosuppressant FK506, which also binds the ryanodine receptor (RyR2) in the heart, has been proposed to regulate RyR2 function and to have antiarrhythmic properties. However, the level of FKBP12.6 expression in normal hearts remains elusive and some controversies still persist regarding its effects, both in basal conditions and during ß-adrenergic stimulation. We quantified FKBP12.6 in the left ventricles (LV) of WT (wild-type) mice and in two novel transgenic models expressing distinct levels of FKBP12.6, using a custom-made specific anti-FKBP12.6 antibody and a recombinant protein. FKBP12.6 level in WT LV was very low (0.16 ± 0.02 nmol/g of LV), indicating that <15% RyR2 monomers are bound to the protein. Mice with 14.1 ± 0.2 nmol of FKBP12.6 per g of LV (TG1) had mild cardiac hypertrophy and normal function and were protected against epinephrine/caffeine-evoked arrhythmias. The ventricular myocytes showed higher [Ca2+]i transient amplitudes than WT myocytes and normal SR-Ca2+ load, while fewer myocytes showed Ca2+ sparks. TG1 cardiomyocytes responded to 50 nM Isoproterenol increasing these [Ca2+]i parameters and producing RyR2-Ser2808 phosphorylation. Mice with more than twice the TG1 FKBP12.6 value (TG2) showed marked cardiac hypertrophy with calcineurin activation and more arrhythmias than WT mice during ß-adrenergic stimulation, challenging the protective potential of high FKBP12.6. RyR2R420Q CPVT mice overexpressing FKBP12.6 showed fewer proarrhythmic events and decreased incidence and duration of stress-induced bidirectional ventricular tachycardia. Our study, therefore, quantifies for the first time endogenous FKBP12.6 in the mouse heart, questioning its physiological relevance, at least at rest due its low level. By contrast, our work demonstrates that with caution FKBP12.6 remains an interesting target for the development of new antiarrhythmic therapies.

HIV-Tat induces a decrease in IKr and IKsvia reduction in phosphatidylinositol-(4,5)-bisphosphate availability.

Patients with HIV present with a higher prevalence of QT prolongation, of which molecular bases are still not clear. Among HIV proteins, Tat serves as a transactivator that stimulates viral genes expression and is required for efficient HIV replication. Tat is actively secreted into the blood by infected T-cells and affects organs such as the heart. Tat has been shown to alter cardiac repolarization in animal models but how this is mediated and whether this is also the case in human cells is unknown. In the present study, we show that Tat transfection in heterologous expression systems led to a decrease in hERG (underlying cardiac IKr) and human KCNE1-KCNQ1 (underlying cardiac IKs) currents and to an acceleration of their deactivation. This is consistent with a decrease in available phosphatidylinositol-(4,5)-bisphosphate (PIP2). A mutant Tat, unable to bind PIP2, did not reproduce the observed effects. In addition, WT-Tat had no effect on a mutant KCNQ1 which is PIP2-insensitive, further confirming the hypothesis. Twenty-four-hour incubation of human induced pluripotent stem cells-derived cardiomyocytes with Wild-type Tat reduced IKr and accelerated its deactivation. Concordantly, this Tat incubation led to a prolongation of the action potential (AP) duration. Events of AP alternans were also recorded in the presence of Tat, and were exacerbated at a low pacing cycle length. Altogether, these data obtained on human K+ channels both in heterologous expression systems and in human cardiomyocytes suggest that Tat sequesters PIP2, leading to a reduction of IKr and IKs, and provide a molecular mechanism for QT prolongation in HIV-infected patients.

Effect of the 1-h bundle on mortality in patients with suspected sepsis in the emergency department: a stepped wedge cluster randomized clinical trial.

PURPOSE: The efficacy of the 1-h bundle for emergency department (ED) patients with suspected sepsis, which includes lactate measurement, blood culture, broad-spectrum antibiotics administration, administration of 30 mL/kg crystalloid fluid for hypotension or lactate ≥ 4 mmol/L, remains controversial. METHODS: We carried out a pragmatic stepped-wedge cluster-randomized trial in 23 EDs in France and Spain. Adult patients with Sepsis-3 criteria or a quick sequential organ failure assessment (SOFA) score ≥ 2 or a lactate > 2 mmol/L were eligible. The intervention was the implementation of the 1-h sepsis bundle. The primary outcome was in-hospital mortality truncated at 28 days. Secondary outcomes included volume of fluid resuscitation at 24 h, acute heart failure at 24 h, SOFA score at 72 h, intensive care unit (ICU) length of stay, number of days on mechanical ventilation or renal replacement therapy, vasopressor free days, unnecessary antibiotic administration, and mortality at 28 days. 1148 patients were planned to be analysed; the study period ended after 873 patients were included. RESULTS: 872 patients (mean age 66, 42% female) were analyzed: 387 (44.4%) in the intervention group and 485 (55.6%) in the control group. Median SOFA score was 3 [1-5]. Median time to antibiotic administration was 40 min in the intervention group vs 113 min in the control group (difference - 73 [95% confidence interval (CI) - 93 to - 53]). There was a significantly higher rate, volume, and shorter time to fluid resuscitation within 3 h in the intervention group. There were 47 (12.1%) in-hospital deaths in the intervention group compared to 61 (12.6%) in the control group (difference in percentage - 0.4 [95% CI - 5.1 to 4.2], adjusted relative risk (aRR) 0.81 [95% CI 0.48 to 1.39]). There were no differences between groups for other secondary endpoints. CONCLUSIONS: Among patients with suspected sepsis in the ED, the implementation of the 1-h sepsis bundle was not associated with significant difference in in-hospital mortality. However, this study may be underpowered to report a statistically significant difference between groups.

Current dilemmas in the diagnosis and management of follicular thyroid tumors.

Follicular carcinoma (FTC) is a relatively uncommon type of differentiated thyroid carcinoma. Guidelines have often dealt with FTC and papillary thyroid cancer as a single disease. Over the last decade, however, a better understanding of these two types of thyroid cancer indicates that they cannot be analysed together. Neither ultrasonography nor fine-needle aspiration cytology can provide a clear distinction between FTC and follicular adenoma. New molecular diagnostic techniques may be used to identify a subpopulation of follicular neoplasms with a low probability of being malignant. Diagnostic surgery-usually hemithyroidectomy- is recommended for most thyroid follicular lesions without a certain preoperative diagnosis. If FTC is diagnosed most-perhaps not all- patients will require a completion thyroidectomy. While widely invasive FTC usually does not pose diagnostic or therapeutic doubts, consensus on the diagnosis of non-invasive follicular lesions is still lacking. Prognosis of FTC is mostly dependent on local invasion and distant metastasis that, in turn, correlate with tumor size.

Distribution of Borrelia burgdorferi sensu lato in Ixodes ricinus populations across central Britain.

Lyme borreliosis is rapidly emerging in the United Kingdom, with over 1000 cases per annum now reported. Lyme borreliosis is caused by the Borrelia burgdorferi sensu lato (s.l.) group of spirochetes, which are transmitted by ixodid ticks. In the United Kingdom, Ixodes ricinus is recognized as the principal vector of the spirochetes, and this tick species is widely distributed across the country. However, as yet, it is unclear whether the distribution of B. burgdorferi essentially mirrors that of its vector, or if there are marked differences between the 2. The aim of this survey was to investigate the prevalence of B. burgdorferi in I. ricinus populations across northern England, north Wales, and the Scottish Border region. We surveyed for questing I. ricinus nymphs and adults at 17 sites, encountering ticks at 12. At 8 sites, large numbers (>160) ticks were collected, and at 3 of these sites B. burgdorferi infection prevalence was significantly higher (>7.5%) than the other 5 (<1.0%). Habitat type was associated with B. burgdorferi prevalence, with ticks from deciduous and mixed woodland being significantly more likely to be infected than those from other habitat types. Identification of infecting Borrelia species indicated that Borrelia valaisiana was the most common and widespread species encountered. B. garinii was common at sites where infection prevalence was high, and B. afzelii was also occasionally encountered at these sites. The survey revealed a surprisingly uneven distribution of B. burgdorferi s.l. across the region, thereby indicating that the presence of ticks does not necessarily mean the presence of the pathogen. Indeed, the spirochete appears to be absent or very rare at some sites where ticks are abundant.

Sleeve gastrectomy and Roux-en-Y gastric bypass are equally effective in correcting insulin resistance.

BACKGROUND: Laparoscopic Roux-en-Y gastric bypass (LRYGB) and laparoscopic sleeve gastrectomy (LSG) are associated with glucose metabolism improvement although data on insulin resistance remission rates after these procedures are lacking. AIMS: Primary aim was to compare insulin resistance remission rates achieved after LRYGB and LSG, using population-specific HOMA-IR cut-off points. Secondary objectives were to analyze factors associated with type 2 diabetes mellitus (T2DM) complete remission according to the new American Diabetes Association criteria and to examine changes in HOMA-B during follow-up. METHODS: Non-randomized, prospective cohort study of patients undergoing LRYGB or LSG with a minimal follow-up of 24 months. Patients on insulin therapy were excluded. RESULTS: At baseline, 56 (48.7%) of the 115 LRYGB group and 48 (61.5%) of the 78 LSG group had insulin resistance, and 29 (25.2%) and 20 (25.6%) T2DM, respectively. No differences were detected in insulin resistance remission rate (92.9% LRYGB and 87.5% LSG, p = 0.355) nor in T2DM complete remission at 2 years (62.1 vs 60% respectively, p = 0.992). Factors independently associated with T2DM complete remission were diabetes treatment and a greater decrease in 3-month HOMA-IR index. The HOMA-B index showed a progressive decline during follow-up. CONCLUSION: Both surgical techniques are equally effective in achieving insulin resistance normalization in the majority of severely obese patients. Three-month HOMA-IR reduction after surgery was the main predictor of T2DM complete remission.