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.

A systematic review of the incidence of hypersensitivity reactions and post-contrast acute kidney injury after ioversol in more than 57,000 patients: part 1-intravenous administration.

OBJECTIVES: To evaluate the incidence of adverse drug reactions (ADRs), including hypersensitivity reactions (HSRs) and post-contrast acute kidney injury (PC-AKI), after intravenous (IV) administration of ioversol. MATERIALS AND METHODS: A systematic literature search (1980-2021) of studies documenting IV use of ioversol and presence or absence of ADRs, HSRs, or PC-AKI was performed. Key information including patients' characteristics, indication and dose of ioversol, safety outcome incidence, intensity and seriousness were extracted. RESULTS: Thirty-one studies (> 57,000 patients) were selected, including 4 pediatric studies. The incidence of ADRs in adults was reported in 12 studies from ioversol clinical development with a median (range) of 1.65% (0-33.3%), and 3 other studies with an incidence between 0.13 and 0.28%. The incidence of HSRs (reported in 2 studies) ranged from 0.20 to 0.66%, and acute events (4 studies) from 0.23 to 1.80%. Severe reactions were rare with a median (range) of 0 (0-4%), and none were reported among pediatric patients. The incidence of ADRs and HSRs with ioversol, especially those of severe intensity, was among the lowest in studies comparing different iodinated contrast media (ICM) of the same class. PC-AKI incidence was variable (1-42% in 5 studies); however, ioversol exposure per se did not increase the incidence. CONCLUSIONS: When administered by the IV route, ioversol has a good safety profile comparable to that of other ICM within the same class, with a low incidence of severe/serious ADRs overall, and particularly HSRs. PC-AKI incidence does not seem to be increased compared to patients who did not receive ioversol. Further well-designed studies are warranted to confirm these results. KEY POINTS: • Ioversol has a good safety profile in adult and pediatric patients when IV administered. • ADR and HSR incidence with ioversol, especially those of severe intensity, was among the lowest compared to other ICM. • IV administration of ioversol per se did not increase PC-AKI incidence.

A systematic review of the incidence of hypersensitivity reactions and post-contrast acute kidney injury after ioversol: part 2-intra-arterial administration.

OBJECTIVES: To evaluate the incidence of adverse drug reactions (ADRs), including hypersensitivity reactions (HSRs) and post-contrast acute kidney injury (PC-AKI), after intra-arterial (IA) administration of ioversol. METHODS AND MATERIALS: A systematic literature search was performed (1980-2021) and studies documenting IA use of ioversol, and reporting safety outcomes were selected. Key information on study design, patients' characteristics, indication, dose, and type of safety outcome were extracted. RESULTS: Twenty-eight studies (including two pediatric studies) with 8373 patients exposed to IA ioversol were selected. Studies were highly heterogenous in terms of design, PC-AKI definition, and studied population. PC-AKI incidence after coronary angiography was 7.5-21.9% in a general population, 4.0-26.4% in diabetic patients, and 5.5-28.9% in patients with chronic kidney disease (CKD). PC-AKI requiring dialysis was rare and reported mainly in patients with severe CKD. No significant differences in PC-AKI rates were shown in studies comparing different iodinated contrast media (ICM). Based on seven studies of ioversol clinical development, the overall ADR incidence was 1.6%, comparable to that reported with other non-ionic ICM. Pediatric data were scarce with only one study reporting on PC-AKI incidence (12%), and one reporting on ADR incidence (0.09%), both after coronary angiography. CONCLUSIONS: After ioversol IA administration, PC-AKI incidence was highly variable between studies, likely reflecting the heterogeneity of the included study populations, and appeared comparable to that reported with other ICM. The rate of other ADRs appears to be low. Well-designed studies are needed for a better comparison with other ICM. KEY POINTS: • PC-AKI incidence after IA administration of ioversol appears to be comparable to that of other ICM, despite the high variability between studies. • The need for dialysis after IA administration of ioversol is rare. • No obvious difference was found regarding the safety profile of ioversol between IA and IV administration.

Cardiac Overexpression of PDE4B Blunts ß-Adrenergic Response and Maladaptive Remodeling in Heart Failure.

BACKGROUND: The cyclic AMP (adenosine monophosphate; cAMP)-hydrolyzing protein PDE4B (phosphodiesterase 4B) is a key negative regulator of cardiac ß-adrenergic receptor stimulation. PDE4B deficiency leads to abnormal Ca2+ handling and PDE4B is decreased in pressure overload hypertrophy, suggesting that increasing PDE4B in the heart is beneficial in heart failure. METHODS: We measured PDE4B expression in human cardiac tissues and developed 2 transgenic mouse lines with cardiomyocyte-specific overexpression of PDE4B and an adeno-associated virus serotype 9 encoding PDE4B. Myocardial structure and function were evaluated by echocardiography, ECG, and in Langendorff-perfused hearts. Also, cAMP and PKA (cAMP dependent protein kinase) activity were monitored by Förster resonance energy transfer, L-type Ca2+ current by whole-cell patch-clamp, and cardiomyocyte shortening and Ca2+ transients with an Ionoptix system. Heart failure was induced by 2 weeks infusion of isoproterenol or transverse aortic constriction. Cardiac remodeling was evaluated by serial echocardiography, morphometric analysis, and histology. RESULTS: PDE4B protein was decreased in human failing hearts. The first PDE4B-transgenic mouse line (TG15) had a ≈15-fold increase in cardiac cAMP-PDE activity and a ≈30% decrease in cAMP content and fractional shortening associated with a mild cardiac hypertrophy that resorbed with age. Basal ex vivo myocardial function was unchanged, but ß-adrenergic receptor stimulation of cardiac inotropy, cAMP, PKA, L-type Ca2+ current, Ca2+ transients, and cell contraction were blunted. Endurance capacity and life expectancy were normal. Moreover, these mice were protected from systolic dysfunction, hypertrophy, lung congestion, and fibrosis induced by chronic isoproterenol treatment. In the second PDE4B-transgenic mouse line (TG50), markedly higher PDE4B overexpression, resulting in a ≈50-fold increase in cardiac cAMP-PDE activity caused a ≈50% decrease in fractional shortening, hypertrophy, dilatation, and premature death. In contrast, mice injected with adeno-associated virus serotype 9 encoding PDE4B (1012 viral particles/mouse) had a ≈50% increase in cardiac cAMP-PDE activity, which did not modify basal cardiac function but efficiently prevented systolic dysfunction, apoptosis, and fibrosis, while attenuating hypertrophy induced by chronic isoproterenol infusion. Similarly, adeno-associated virus serotype 9 encoding PDE4B slowed contractile deterioration, attenuated hypertrophy and lung congestion, and prevented apoptosis and fibrotic remodeling in transverse aortic constriction. CONCLUSIONS: Our results indicate that a moderate increase in PDE4B is cardioprotective and suggest that cardiac gene therapy with PDE4B might constitute a new promising approach to treat heart failure.

Imipramine as an alternative to formamide to detubulate rat ventricular cardiomyocytes.

NEW FINDINGS: What is the central question of this study? Can imipramine, an antidepressant agent that is a cationic amphiphilic drug that interferes with the phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2 ) interactions with proteins maintaining the tubular system, be validated as a new detubulating tool? What is the main finding and its importance? Imipramine was validated as a more efficient and less toxic detubulating agent of cardiomyocytes than formamide. New insights are provided on how PI(4,5)P2 is crucial to maintaining T-tubule attachment to the cell surface and on the cardiotoxic effects of imipramine overdoses. ABSTRACT: Cardiac T-tubules are membrane invaginations essential for excitation-contraction coupling (ECC). Imipramine, like other cationic amphiphilic drugs, interferes with phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2 ) interactions with proteins maintaining the tubular system connected to the cell surface. Our main purpose was to validate imipramine as a new detubulating agent in cardiomyocytes. Staining adult rat ventricular myocytes (ARVMs) with di-4-ANEPPS, we showed that unlike formamide, imipramine induces a complete detubulation with no impact on cell viability. Using the patch-clamp technique, we observed a ∼40% decrease in cell capacitance after imipramine pretreatment and a reduction of ICa,L amplitude by ∼72%. These parameters were not affected in atrial cells, excluding direct side effects of imipramine. ß-Adrenergic receptor (ß-AR) stimulation of the remaining ICa,L with isoproterenol (Iso) was still effective. ECC was investigated in ARVMs loaded with Fura-2 and paced at 1 Hz, allowing simultaneous measurement of the Ca2+ transient (CaT) and sarcomere shortening (SS). Amplitude of both CaT and SS was decreased by imipramine and partially restored by Iso. Furthermore, detubulated cells exhibited Ca2+ homeostasis perturbations. Real-time cAMP variations induced by Iso using a Förster resonance energy transfer biosensor revealed ∼27% decreased cAMP elevation upon ß-AR stimulation. To conclude, we validated a new cardiomyocyte detubulation method using imipramine, which is more efficient and less toxic than formamide. This antidepressant agent induces the hallmark effects of detubulation on ECC and its ß-AR stimulation. Besides, we provide new insights on how an imipramine overdose may affect cardiac function and suggest that PI(4,5)P2 is crucial for maintaining T-tubule structure.

Investigating cardiac ß-adrenergic nuclear signaling with FRET-based biosensors.

By activating membrane ß-adrenergic receptors (ß-AR), noradrenaline and adrenaline are the most powerful stimulators of cardiac function. ß-ARs are coupled to the synthesis of cAMP, which activates the cAMP-dependent protein kinase (PKA). PKA regulates the key proteins of excitation-contraction coupling but also gene expression. While an acute activation of the cAMP/PKA pathway allows adaptation of cardiac output to exercise, its chronic activation is deleterious by promoting pathological remodeling of the heart. The use of probes based on fluorescence resonance energy transfer (FRET) and located specifically at the level of the cytoplasm or the nucleus make it possible to highlight the differential mechanisms by which ß-ARs control PKA activation in these two compartments. The characterization of these mechanisms is important in order to better understand the deleterious effects of chronic activation of the ß-adrenergic pathway in the heart.

PDE4 and mAKAPß are nodal organizers of ß2-ARs nuclear PKA signalling in cardiac myocytes.

Aims: ß1- and ß2-adrenergic receptors (ß-ARs) produce different acute contractile effects on the heart partly because they impact on different cytosolic pools of cAMP-dependent protein kinase (PKA). They also exert different effects on gene expression but the underlying mechanisms remain unknown. The aim of this study was to understand the mechanisms by which ß1- and ß2-ARs regulate nuclear PKA activity in cardiomyocytes. Methods and results: We used cytoplasmic and nuclear targeted biosensors to examine cAMP signals and PKA activity in adult rat ventricular myocytes upon selective ß1- or ß2-ARs stimulation. Both ß1- and ß2-AR stimulation increased cAMP and activated PKA in the cytoplasm. Although the two receptors also increased cAMP in the nucleus, only ß1-ARs increased nuclear PKA activity and up-regulated the PKA target gene and pro-apoptotic factor, inducible cAMP early repressor (ICER). Inhibition of phosphodiesterase (PDE)4, but not Gi, PDE3, GRK2 nor caveolae disruption disclosed nuclear PKA activation and ICER induction by ß2-ARs. Both nuclear and cytoplasmic PKI prevented nuclear PKA activation and ICER induction by ß1-ARs, indicating that PKA activation outside the nucleus is required for subsequent nuclear PKA activation and ICER mRNA expression. Cytoplasmic PKI also blocked ICER induction by ß2-AR stimulation (with concomitant PDE4 inhibition). However, in this case nuclear PKI decreased ICER up-regulation by only 30%, indicating that other mechanisms are involved. Down-regulation of mAKAPß partially inhibited nuclear PKA activation upon ß1-AR stimulation, and drastically decreased nuclear PKA activation upon ß2-AR stimulation in the presence of PDE4 inhibition. Conclusions: ß1- and ß2-ARs differentially regulate nuclear PKA activity and ICER expression in cardiomyocytes. PDE4 insulates a mAKAPß-targeted PKA pool at the nuclear envelope that prevents nuclear PKA activation upon ß2-AR stimulation.

Cyclic nucleotide phosphodiesterases in heart and vessels: A therapeutic perspective.

Cyclic nucleotide phosphodiesterases (PDEs) degrade the second messengers cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), thereby regulating multiple aspects of cardiac and vascular muscle functions. This highly diverse class of enzymes encoded by 21 genes encompasses 11 families that are not only responsible for the termination of cyclic nucleotide signalling, but are also involved in the generation of dynamic microdomains of cAMP and cGMP, controlling specific cell functions in response to various neurohormonal stimuli. In the myocardium and vascular smooth muscle, the PDE3 and PDE4 families predominate, degrading cAMP and thereby regulating cardiac excitation-contraction coupling and smooth muscle contractile tone. PDE3 inhibitors are positive inotropes and vasodilators in humans, but their use is limited to acute heart failure and intermittent claudication. PDE5 is particularly important for the degradation of cGMP in vascular smooth muscle, and PDE5 inhibitors are used to treat erectile dysfunction and pulmonary hypertension. There is experimental evidence that these PDEs, as well as other PDE families, including PDE1, PDE2 and PDE9, may play important roles in cardiac diseases, such as hypertrophy and heart failure, as well as several vascular diseases. After a brief presentation of the cyclic nucleotide pathways in cardiac and vascular cells, and the major characteristics of the PDE superfamily, this review will focus on the current use of PDE inhibitors in cardiovascular diseases, and the recent research developments that could lead to better exploitation of the therapeutic potential of these enzymes in the future.

[Cyclic nucleotide phosphodiesterases: role in the heart and therapeutic perspectives]. / Phosphodiestérases des nucléotides cycliques : rôle dans le cœur et potentiel thérapeutique.

Cyclic nucleotide phosphodiesterases (PDEs) degrade the second messengers cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), thereby regulating multiple aspects of cardiac function. This highly diverse class of enzymes encoded by 21 genes encompasses 11 families that are not only responsible for the termination of cyclic nucleotide signalling, but are also involved in the generation of dynamic microdomains of cAMP and cGMP, controlling specific cell functions in response to various neurohormonal stimuli. In the myocardium, the PDE3 and PDE4 families predominate, degrading cAMP and thereby regulating cardiac excitation-contraction coupling. PDE3 inhibitors are positive inotropes and vasodilators in humans, but their use is limited to acute heart failure and intermittent claudication. PDE5 inhibitors, which are used with success to treat erectile dysfunction and pulmonary hypertension, do not seem efficient in heart failure with preserved ejection fraction. There is experimental evidence however that these PDE, as well as other PDE families including PDE1, PDE2 and PDE9, may play important roles in cardiac diseases, such as hypertrophy and heart failure (HF). After a brief presentation of the cyclic nucleotide pathways in cardiac myocytes and the major characteristics of the PDE superfamily, this review will focus on the potential use of PDE inhibitors in HF, and the recent research developments that could lead to a better exploitation of the therapeutic potential of these enzymes in the future.

Control of cytoplasmic and nuclear protein kinase A by phosphodiesterases and phosphatases in cardiac myocytes.

AIMS: The cAMP-dependent protein kinase (PKA) mediates ß-adrenoceptor (ß-AR) regulation of cardiac contraction and gene expression. Whereas PKA activity is well characterized in various subcellular compartments of adult cardiomyocytes, its regulation in the nucleus remains largely unknown. The aim of the present study was to compare the modalities of PKA regulation in the cytoplasm and nucleus of cardiomyocytes. METHODS AND RESULTS: Cytoplasmic and nuclear cAMP and PKA activity were measured with targeted fluorescence resonance energy transfer probes in adult rat ventricular myocytes. ß-AR stimulation with isoprenaline (Iso) led to fast cAMP elevation in both compartments, whereas PKA activity was fast in the cytoplasm but markedly slower in the nucleus. Iso was also more potent and efficient in activating cytoplasmic than nuclear PKA. Similar slow kinetics of nuclear PKA activation was observed upon adenylyl cyclase activation with L-858051 or phosphodiesterase (PDE) inhibition with 3-isobutyl-1-methylxantine. Consistently, pulse stimulation with Iso (15 s) maximally induced PKA and myosin-binding protein C phosphorylation in the cytoplasm, but marginally activated PKA and cAMP response element-binding protein phosphorylation in the nucleus. Inhibition of PDE4 or ablation of the Pde4d gene in mice prolonged cytoplasmic PKA activation and enhanced nuclear PKA responses. In the cytoplasm, phosphatase 1 (PP1) and 2A (PP2A) contributed to the termination of PKA responses, whereas only PP1 played a role in the nucleus. CONCLUSION: Our study reveals a differential integration of cytoplasmic and nuclear PKA responses to ß-AR stimulation in cardiac myocytes. This may have important implications in the physiological and pathological hypertrophic response to ß-AR stimulation.