Heart beat relies on coordinated calcium regulation through RyR2 and SERCA2a activity, as well as impulse propagation mediated by NaV1.5 activity. Here, we present small molecules combining a 1,4-benzothia- or 1,4-benzoxazepine scaffold and a 3-(3,4-dihydroxyphenyl)-2-propenoic acid residue of which five compounds (4, 9, 10, 12, and 13) reduced cardiac RyR2 activity and stimulated SERCA2a activity in our cell systems. The acceleration of SERCA2 activity was particularly enhanced by cyanoborane derivatives 11-13. The substantial impact on SERCA2 activation relates to the presence of polar (zwitterionic) N-B fragments, which can increase the binding affinity to SERCA2 and result in a more efficient activation. Compounds 12 and 13 at a concentration of 10 μM additionally reduced NaV1.5 activity in Chinese hamster ovary (CHO) cells, indicating a multitargeted drug property. We propose that the multitargeted actions of these novel compounds have the potential to enhance heart failure therapy, particularly in personalized treatment approaches.

In the heart, genetic or acquired mishandling of diastolic [Ca2+] by ryanodine receptor type 2 (RyR2) overactivity correlates with risks of arrhythmia and sudden cardiac death. Strategies to avoid these risks include decrease of Ca2+ release by drugs modulating RyR2 activity or increase in Ca2+ uptake by drugs modulating SR Ca2+ ATPase (SERCA2a) activity. Here, we combine these strategies by developing experimental compounds that act simultaneously on both processes. Our screening efforts identified the new 1,4-benzothiazepine derivative GM1869 as a promising compound. Consequently, we comparatively studied the effects of the known RyR2 modulators Dantrolene and S36 together with GM1869 on RyR2 and SERCA2a activity in cardiomyocytes from wild type and arrhythmia-susceptible RyR2R2474S/+ mice by confocal live-cell imaging. All drugs reduced RyR2-mediated Ca2+ spark frequency but only GM1869 accelerated SERCA2a-mediated decay of Ca2+ transients in murine and human cardiomyocytes. Our data indicate that S36 and GM1869 are more suitable than dantrolene to directly modulate RyR2 activity, especially in RyR2R2474S/+ mice. Remarkably, GM1869 may represent a new dual-acting lead compound for maintenance of diastolic [Ca2+].

Our study identified a first multi-targeted compound (GM1869) inhibiting RyR2-mediated Ca 2+ leak together with activating SERCA2a function that may represent a critical breakthrough for treatment of diastolic Ca 2+ leak in heart failure. In the heart, genetic or acquired mishandling of diastolic [Ca 2+ ] by ryanodine receptor type 2 (RyR2) overactivity correlates with risks of arrhythmia and sudden cardiac death. Strategies to avoid these risks include decrease of Ca 2+ release by drugs modulating RyR2 activity or increase in Ca 2+ uptake by drugs modulating SR Ca 2+ ATPase (SERCA2a) activity. Here, we combine these strategies by developing experimental compounds that act simultaneously on both processes. Our screening efforts identified the new 1,4-benzothiazepine derivative GM1869 as a promising compound. Consequently, we comparatively studied the effects of the known RyR2 modulators Dantrolene and S36 together with GM1869 on RyR2 and SERCA2a activity in cardiomyocytes from wild type and arrhythmia-susceptible RyR2 R2474S/+ mice by confocal live-cell imaging. All drugs reduced RyR2-mediated Ca 2+ spark frequency but only GM1869 accelerated SERCA2a-mediated decay of Ca 2+ transients in murine and human cardiomyocytes. Our data indicate that S36 and GM1869 are more suitable than dantrolene to directly modulate RyR2 activity, especially in RyR2 R2474S/+ mice. Remarkably, GM1869 may represent a new dual-acting lead compound for maintenance of diastolic [Ca 2+ ].

AIMS:Cardiac arrhythmias are a major factor in the occurrence of morbidity and sudden death in patients with cardiovascular disease. Disturbances of Ca2+ homeostasis in the heart contribute to the initiation and maintenance of cardiac arrhythmias. Extrasystolic increases in intracellular Ca2+ lead to delayed afterdepolarizations and triggered activity, which can result in heart rhythm abnormalities. It is being suggested that the Ca2+-activated nonselective cation channel TRPM4 is involved in the aetiology of triggered activity, but the exact contribution and in vivo significance are still unclear.METHODS AND RESULTS:In vitro electrophysiological and calcium imaging technique as well as in vivo intracardiac and telemetric electrocardiogram measurements in physiological and pathophysiological conditions were performed. In two distinct Ca2+-dependent proarrhythmic models, freely moving Trpm4-/- mice displayed a reduced burden of cardiac arrhythmias. Looking further into the specific contribution of TRPM4 to the cellular mechanism of arrhythmias, TRPM4 was found to contribute to a long-lasting Ca2+ overload-induced background current, thereby regulating cell excitability in Ca2+ overload conditions. To expand these results, a compound screening revealed meclofenamate as a potent antagonist of TRPM4. In line with the findings from Trpm4-/- mice, 10 µM meclofenamate inhibited the Ca2+ overload-induced background current in ventricular cardiomyocytes and 15 mg/kg meclofenamate suppressed catecholaminergic polymorphic ventricular tachycardia-associated arrhythmias in a TRPM4-dependent manner.CONCLUSION:The presented data establish that TRPM4 represents a novel target in the prevention and treatment of Ca2+-dependent triggered arrhythmias.

Axial tubule junctions with the sarcoplasmic reticulum control the rapid intracellular Ca2+-induced Ca2+ release that initiates atrial contraction. In atrial myocytes we previously identified a constitutively increased ryanodine receptor (RyR2) phosphorylation at junctional Ca2+ release sites, whereas non-junctional RyR2 clusters were phosphorylated acutely following β-adrenergic stimulation. Here, we hypothesized that the baseline synthesis of 3',5'-cyclic adenosine monophosphate (cAMP) is constitutively augmented in the axial tubule junctional compartments of atrial myocytes. Confocal immunofluorescence imaging of atrial myocytes revealed that junctin, binding to RyR2 in the sarcoplasmic reticulum, was densely clustered at axial tubule junctions. Interestingly, a new transgenic junctin-targeted FRET cAMP biosensor was exclusively co-clustered in the junctional compartment, and hence allowed to monitor cAMP selectively in the vicinity of junctional RyR2 channels. To dissect local cAMP levels at axial tubule junctions versus subsurface Ca2+ release sites, we developed a confocal FRET imaging technique for living atrial myocytes. A constitutively high adenylyl cyclase activity sustained increased local cAMP levels at axial tubule junctions, whereas β-adrenergic stimulation overcame this cAMP compartmentation resulting in additional phosphorylation of non-junctional RyR2 clusters. Adenylyl cyclase inhibition, however, abolished the junctional RyR2 phosphorylation and decreased L-type Ca2+ channel currents, while FRET imaging showed a rapid cAMP decrease. In conclusion, FRET biosensor imaging identified compartmentalized, constitutively augmented cAMP levels in junctional dyads, driving both the locally increased phosphorylation of RyR2 clusters and larger L-type Ca2+ current density in atrial myocytes. This cell-specific cAMP nanodomain is maintained by a constitutively increased adenylyl cyclase activity, contributing to the rapid junctional Ca2+-induced Ca2+ release, whereas β-adrenergic stimulation overcomes the junctional cAMP compartmentation through cell-wide activation of non-junctional RyR2 clusters.

Rationale Cyclic adenosine monophosphate (cAMP) is a ubiquitous second messenger which, upon β-adrenergic receptor (β-AR) stimulation, acts in microdomains to regulate cardiac excitation-contraction coupling by activating the cAMP-dependent protein kinase (PKA) phosphorylation of calcium handling proteins. One crucial microdomain is in vicinity of the cardiac ryanodine receptor type 2 (RyR2) which is associated with arrhythmogenic diastolic calcium leak from the sarcoplasmic reticulum (SR) often occurring upon RyR2 hyperphosphorylation by PKA and calcium/calmodulin-dependent kinase. Objective We sought to establish a real time approach capable of directly visualizing cAMP and its pathological changes in the vicinity of RyR2 by generating a proper targeted biosensor and transgenic mouse model to express it in adult cardiomyocytes. Methods and Results We generated transgenic mice expressing a novel targeted fluorescent biosensor for RyR2-associated cAMP in adult mouse cardiomyocytes. In healthy cardiomyocytes, β 1 -AR but not β 2 -AR stimulation strongly increased local RyR2-associated cAMP levels. However, in cardiac hypertrophy induced by aortic banding, there was a marked subcellular redistribution of phosphodiesterases (PDEs) 2, 3 and 4, which included a dramatic loss of the local pool of PDE4. This was also accompanied by measurable β 2 -AR-induced cAMP signals, increased SR calcium leak and arrhythmia susceptibility. Conclusions Our new targeted biosensor expressed in transgenic mice can visualize cAMP levels in the vicinity of cardiac RyR2 in healthy and diseased cardiomyocytes. In the future, this novel biosensor can be used to better understand alterations of RyR2-associated cAMP in cardiovascular diseases and local actions of new therapies.

Signaling via cGMP-dependent protein kinase I (cGKI) and canonical transient receptor potential (TRPC) channels appears to be involved in the regulation of cardiac hypertrophy. Recent evidence suggests that TRPC channels are targets for cGKI, and phosphorylation of these channels may mediate the antihypertrophic effects of cGMP signaling. We tested this concept by investigating the role of cGMP/cGKI signaling on angiotensin II (A II)-induced cardiac hypertrophy using a control group (Ctr), trpc6−/−, trpc3−/−, trpc3−/−/6−/−, βRM mice, and trpc3−/−/6−/− × βRM mice. βRM mice express cGKIβ only in the smooth muscle on a cGKI−/− background. The control group was composed of littermate mice that contained at least one wild type gene of the respective genotype. A II was infused by minipumps (7 days; 2 mg/kg/day) in Ctr, trpc6−/−, trpc3−/−, trpc3−/−/6−/−, βRM, and trpc3−/−/6−/− × βRM mice. Hypertrophy was assessed by measuring heart weight per tibia length (HW/TL) and fibrosis by staining of heart slices. A II-induced increase in HW/TL and fibrosis was absent in trpc3 −/− mice, whereas an increase in HW/TL and fibrosis was evident in Ctr and trpc6−/−, minimal or absent in trpc3−/−, moderate in βRM, and dramatic in trpc3−/−/6−/− βRM mice. These results suggest that TRPC3 may be necessary for A II-induced cardiac hypertrophy. On the other hand, hypertrophy and fibrosis were massively increased in βRM mice on a TRPC3/6 × cGKI−/−KO background, indicating an “additive” coupling between both signaling pathways.

Signaling via cGMP-dependent protein kinase is the major pathway of the NO/cGMP cascade in vascular smooth muscle (SM), heart, CNS, and other cells. cGMP-dependent protein kinases (cGK) are serine/threonine kinases that are widely distributed in eukaryotes. Two genes – prkg1 and prkg2 - code for cGKs, namely cGKI and cGKII. In mammals, two isozymes, cGKIα and cGKIβ, are generated from the prkg1 gene. The cGKI isozymes are prominent in all types of smooth muscle, platelets and specific neuronal areas such as cerebellar Purkinje cells, hippocampal neurons, and the lateral amygdala. The cGKII prevails in the secretory epithelium of the small intestine, the juxta-glomerular cells, the adrenal cortex, the chondrocytes, and in the nucleus suprachiasmaticus. Both cGKs are major down- stream effectors of many, but not all signalling events of the NO/cGMP and the ANP/BNP/CNP/cGMP pathways. cGKI relaxes smooth muscle tone and prevents platelet aggregation, whereas cGKII inhibits renin secretion, chloride/water secretion in the small intestine, the resetting of the clock during early night and endochondreal bone growth. cGKs are involved in cardiovascular and non-cardiovascular processes and are modulators of cell growth and many other functions. Recent evidence suggest that vascular smooth muscle relaxation is not affected by cGK-dependent phosphorylation of smooth muscle expressed TRPC channels, but by cGK-dependent inhibition of TRPC-6 in endothelial cells and attenuation of endothelial release of smooth muscle relaxing factors. Angiotensin increases blood pressure and induces cardiac hypertrophy. Hypertrophy is reduced in sildenafil treated wild type mice but not in beta rescue mice. This suggests that the sildenafil effects are mediated by inhibition of a cGMP hydrolysing phosphodiesterase. Restricted evidence supports the notion that sildenafil affects cGMP levels in cardiomyofibroblasts and/or in cardiac endothelial cells. It will be shown that cGK are essential modulators of many regulatory processes.

Background Signaling via cGMP-dependent protein kinase I (cGKI) is the major pathway in vascular smooth muscle (SM), by which endothelial NO regulates vascular tone. Recent evidence suggests that canonical transient receptor potential (TRPC) channels are targets of cGKI in SM and mediate the relaxant effects of cGMP signaling. We tested this concept by investigating the role of cGMP/cGKI signaling on vascular tone and peripheral resistance using Trpc6, Trpc3, Trpc3/6, Trpc1/3/6, and SM-specific cGKI (sm-cGKI) mice.

Cardiac CaV1.2 channels play a critical role in cardiac function. It has been proposed that the carboxyl-terminal intracellular tail of the CaV1.2 channel is the target of Ca(2+)-dependent and Ca(2+)-independent regulation of the channel. Recent studies on C-terminal truncated forms of the CaV1.2 channel reported neonatal death, reduced CaV1.2 current, and failure of β-adrenergic stimulation of these channels in ventricular cardiomyocytes (CMs). Here, we used atrial CMs at embryonic day 18.5 that expressed a C-terminal truncated form of the CaV1.2 channel (Stop/Stop). Surprisingly, the atrial CMs showed robust L-type Ca(2+) currents which could be stimulated by forskolin, an activator of adenylyl cyclase. These currents exhibited a left-ward shift in the voltage-dependent activation curve and a reduced sensitivity to the Ca(2+) channel blocker isradipine as compared to currents in wild-type atrial CMs. RT-PCR analysis revealed normal levels of mRNA for the CaV1.2 channel but a twofold increase in the level of mRNA for the CaV1.3 channel in the Stop/Stop atrium as compared to wild-type atrium. A Western blot analysis indicated an increase of CaV1.3 protein in the Stop/Stop atrium. We suggest that, in contrast to Stop/Stop ventricular CMs, Stop/Stop atrial CMs can compensate the functional loss of the truncated CaV1.2 channel with an upregulation of the CaV1.3 channel.

Thyroid hormones are known to influence various processes of cell differentiation. Recently, it was reported that hypothyroidism reduces the sensitivity to Ca2+-channel antagonists in the rat uterus. We examined the sensitivity to dihydropyridines of the uterus from mice that had reduced thyroid hormone levels. Isradipine relaxed with the same potency precontracted uterine muscle strips from control and hypothyroid mice, independently from a pseudo-pregnant state. These results demonstrate that hypothyroidism does not change dihydropyridine sensitivity (i.e., the pattern of Ca2+-channel expression) in the murine uterus.

Background Signaling by intracellular cGMP and cGMP-dependent protein kinase I (cGKI) is the major pathway in vascular smooth muscle, by which endothelial NO regulates vascular tone. The most important targets of cGKI include the myosin-interacting subunit of myosin phosphatase 1, the regulator of G-protein signaling 2, the inositol receptor associated cGKI-substrate, and the BK channel. Recent evidence suggest that TrpC channels are also targets of cGKI in smooth muscle and mediate, at least partially, the relaxant effects of cGMP.

Muscarinic stimulation of urinary bladder induces contraction via an increase in intracellular Ca2+ concentration that results from Ca2+ influx through Ca2+ channels and/or IP3-mediated Ca2+ release controlled by phospholipase C (PLC) signalling. The significance of PLC/IP3 signalling in this cascade has recently been questioned because PLC inhibitors were without effect on carbachol-induced contractions in detrusor muscle strips. However, PLC/IP3-mediated Ca2+ release was clearly observed in recordings of Ca2+ signals in isolated myocytes. Therefore, we investigated the presence of PLC/IP3-dependent Ca2+ release by directly monitoring Ca2+ signals in intact detrusor muscle strips. Concomitant Ca2+ signals from Ca2+ channel activity were eliminated by the Ca2+ channel antagonist isradipine (3 µM) or by the use of muscles from Cav1.2 channel-deficient (SMACKO) mice. In absence of Ca2+ channel activity, carbachol elicited contractions and Ca2+ signals in muscles from wild type and SMACKO mice that were inhibited by the PLC inhibitor U73122 (10 µM). The results show that PLC/IP3-dependent Ca2+ release is activated by stimulation with carbachol in urinary bladder smooth muscle but has a minor contribution to overall carbachol-induced Ca2+ signals.