BIN1, Myotubularin, and Dynamin-2 Coordinate T-Tubule Growth in Cardiomyocytes.

BACKGROUND: Transverse tubules (t-tubules) form gradually in the developing heart, critically enabling maturation of cardiomyocyte Ca2+ homeostasis. The membrane bending and scaffolding protein BIN1 (bridging integrator 1) has been implicated in this process. However, it is unclear which of the various reported BIN1 isoforms are involved, and whether BIN1 function is regulated by its putative binding partners MTM1 (myotubularin), a phosphoinositide 3'-phosphatase, and DNM2 (dynamin-2), a GTPase believed to mediate membrane fission. METHODS: We investigated the roles of BIN1, MTM1, and DNM2 in t-tubule formation in developing mouse cardiomyocytes, and in gene-modified HL-1 and human-induced pluripotent stem cell-derived cardiomyocytes. T-tubules and proteins of interest were imaged by confocal and Airyscan microscopy, and expression patterns were examined by RT-qPCR and Western blotting. Ca2+ release was recorded using Fluo-4. RESULTS: We observed that in the postnatal mouse heart, BIN1 localizes along Z-lines from early developmental stages, consistent with roles in initial budding and scaffolding of t-tubules. T-tubule proliferation and organization were linked to a progressive and parallel increase in 4 detected BIN1 isoforms. All isoforms were observed to induce tubulation in cardiomyocytes but produced t-tubules with differing geometries. BIN1-induced tubulations contained the L-type Ca2+ channel, were colocalized with caveolin-3 and the ryanodine receptor, and effectively triggered Ca2+ release. BIN1 upregulation during development was paralleled by increasing expression of MTM1. Despite no direct binding between MTM1 and murine cardiac BIN1 isoforms, which lack exon 11, high MTM1 levels were necessary for BIN1-induced tubulation, indicating a central role of phosphoinositide homeostasis. In contrast, the developing heart exhibited declining levels of DNM2. Indeed, we observed that high levels of DNM2 are inhibitory for t-tubule formation, although this protein colocalizes with BIN1 along Z-lines, and binds all 4 isoforms. CONCLUSIONS: These findings indicate that BIN1, MTM1, and DNM2 have balanced and collaborative roles in controlling t-tubule growth in cardiomyocytes.

Regulation of cardiomyocyte t-tubule structure by preload and afterload: Roles in cardiac compensation and decompensation.

Mechanical load is a potent regulator of cardiac structure and function. Although high workload during heart failure is associated with disruption of cardiomyocyte t-tubules and Ca2+ homeostasis, it remains unclear whether changes in preload and afterload may promote adaptive t-tubule remodelling. We examined this issue by first investigating isolated effects of stepwise increases in load in cultured rat papillary muscles. Both preload and afterload increases produced a biphasic response, with the highest t-tubule densities observed at moderate loads, whereas excessively low and high loads resulted in low t-tubule levels. To determine the baseline position of the heart on this bell-shaped curve, mice were subjected to mildly elevated preload or afterload (1 week of aortic shunt or banding). Both interventions resulted in compensated cardiac function linked to increased t-tubule density, consistent with ascension up the rising limb of the curve. Similar t-tubule proliferation was observed in human patients with moderately increased preload or afterload (mitral valve regurgitation, aortic stenosis). T-tubule growth was associated with larger Ca2+ transients, linked to upregulation of L-type Ca2+ channels, Na+-Ca2+ exchanger, mechanosensors and regulators of t-tubule structure. By contrast, marked elevation of cardiac load in rodents and patients advanced the heart down the declining limb of the t-tubule-load relationship. This bell-shaped relationship was lost in the absence of electrical stimulation, indicating a key role of systolic stress in controlling t-tubule plasticity. In conclusion, modest augmentation of workload promotes compensatory increases in t-tubule density and Ca2+ cycling, whereas this adaptation is reversed in overloaded hearts during heart failure progression. KEY POINTS: Excised papillary muscle experiments demonstrated a bell-shaped relationship between cardiomyocyte t-tubule density and workload (preload or afterload), which was only present when muscles were electrically stimulated. The in vivo heart at baseline is positioned on the rising phase of this curve because moderate increases in preload (mice with brief aortic shunt surgery, patients with mitral valve regurgitation) resulted in t-tubule growth. Moderate increases in afterload (mice and patients with mild aortic banding/stenosis) similarly increased t-tubule density. T-tubule proliferation was associated with larger Ca2+ transients, with upregulation of the L-type Ca2+ channel, Na+-Ca2+ exchanger, mechanosensors and regulators of t-tubule structure. By contrast, marked elevation of cardiac load in rodents and patients placed the heart on the declining phase of the t-tubule-load relationship, promoting heart failure progression. The dependence of t-tubule structure on preload and afterload thus enables both compensatory and maladaptive remodelling, in rodents and humans.

Direct regulation of the cardiac ryanodine receptor (RyR2) by O-GlcNAcylation.

BACKGROUND: O-GlcNAcylation is the enzymatic addition of a sugar, O-linked ß-N-Acetylglucosamine, to the serine and threonine residues of proteins, and is abundant in diabetic conditions. We have previously shown that O-GlcNAcylation can trigger arrhythmias by indirectly increasing pathological Ca2+ leak through the cardiac ryanodine receptor (RyR2) via Ca2+/calmodulin-dependent kinase II (CaMKII). However, RyR2 is well known to be directly regulated by other forms of serine and threonine modification, therefore, this study aimed to determine whether RyR2 is directly modified by O-GlcNAcylation and if this also alters the function of RyR2 and Ca2+ leak. METHODS: O-GlcNAcylation of RyR2 in diabetic human and animal hearts was determined using western blotting. O-GlcNAcylation of RyR2 was pharmacologically controlled and the propensity for Ca2+ leak was determined using single cell imaging. The site of O-GlcNAcylation within RyR2 was determined using site-directed mutagenesis of RyR2. RESULTS: We found that RyR2 is modified by O-GlcNAcylation in human, animal and HEK293 cell models. Under hyperglycaemic conditions O-GlcNAcylation was associated with an increase in Ca2+ leak through RyR2 which persisted after CaMKII inhibition. Conversion of serine-2808 to alanine prevented an O-GlcNAcylation induced increase in Ca2+ leak. CONCLUSIONS: These data suggest that the function of RyR2 can be directly regulated by O-GlcNAcylation and requires the presence of serine-2808.

Depressed HCN4 function in the type 2 diabetic sinoatrial node.

Diabetic patients often have impaired heart rate (HR) control. HR is regulated both intrinsically within the sinoatrial node (SAN) and via neuronal input. Previously, we found lower ex vivo HR in type 2 diabetic rat hearts, suggesting impaired HR generation within the SAN. The major driver of pacemaking within the SAN is the activity of hyperpolarisation-activated cyclic nucleotide-gated 4 (HCN(4)) channels. This study aimed to investigate whether the lower intrinsic HR in the type 2 diabetic heart is due to changes in HCN4 function, protein expression and/ or distribution. The intrinsic HR response to HCN4 blockade was determined in isolated Langendorff-perfused hearts of Zucker type 2 Diabetic Fatty (ZDF) rats (DM) and their non-diabetic ZDF littermates (nDM). HCN4 protein expression and membrane localisation were determined using western blot and immunofluorescence, respectively. We found that the intrinsic HR was lower in DM compared to nDM hearts. The change in intrinsic HR in response to HCN4 blockade with ivabradine was diminished in DM hearts, which normalised the intrinsic HR between the groups. HCN4 protein expression was decreased in the SAN of DM compared to nDM controls with no change in the fraction of HCN4 localised to the membrane of SAN cardiomyocytes. The lower intrinsic HR in DM is likely due to decreased HCN4 expression and depressed HCN4 function. Our study provides a novel understanding into the intrinsic mechanisms underlying altered HR control in type 2 diabetes.

Long-chain acylcarnitine 18:1 acutely increases human atrial myocardial contractility and arrhythmia susceptibility.

Long-chain acylcarnitines (LCACs) are known to directly alter cardiac contractility and electrophysiology. However, the acute effect of LCACs on human cardiac function is unknown. We aimed to determine the effect of LCAC 18:1, which has been associated with cardiovascular disease, on the contractility and arrhythmia susceptibility of human atrial myocardium. Additionally, we aimed to assess how LCAC 18:1 alters Ca2+ influx and spontaneous Ca2+ release in vitro. Human right atrial trabeculae (n = 32) stimulated at 1 Hz were treated with LCAC 18:1 at a range of concentrations (1-25 µM) for a 45-min period. Exposure to the LCAC induced a dose-dependent positive inotropic effect on myocardial contractility (maximal 1.5-fold increase vs. control). At the 25 µM dose (n = 8), this was paralleled by an enhanced propensity for spontaneous contractions (50% increase). Furthermore, all LCAC 18:1 effects on myocardial function were reversed following LCAC 18:1 washout. In fluo-4-AM-loaded HEK293 cells, LCAC 18:1 dose dependently increased cytosolic Ca2+ influx relative to vehicle controls and the short-chain acylcarnitine C3. In HEK293 cells expressing ryanodine receptor (RyR2), this increased Ca2+ influx was linked to an increased propensity for RyR2-mediated spontaneous Ca2+ release events. Our study is the first to show that LCAC 18:1 directly and acutely alters human myocardial function and in vitro Ca2+ handling. The metabolite promotes proarrhythmic muscle contractions and increases contractility. The exploratory findings in vitro suggest that LCAC 18:1 increases proarrhythmic RyR2-mediated spontaneous Ca2+ release propensity. The direct effects of metabolites on human myocardial function are essential to understand cardiometabolic dysfunction.NEW & NOTEWORTHY For the first time, the fatty acid metabolite, long-chain acylcarnitine 18:1, is shown to acutely increase the arrhythmia susceptibility and contractility of human atrial myocardium. In vitro, this was linked to an influx of Ca2+ and an enhanced propensity for spontaneous RyR2-mediated Ca2+ release.

Docosahexaenoic acid prevents palmitate-induced insulin-dependent impairments of neuronal health.

The importance of fatty acids (FAs) for healthy brain development and function has become more evident in the past decades. However, most studies focus on the hypothalamus as an important FA-sensing brain region involved in energy homeostasis. Less work has been done to evaluate the effects of FAs on brain regions such as the hippocampus or cortex, two important centres of learning, memory formation, and cognition. Furthermore, the mechanisms of how FAs modulate the neuronal development and function are incompletely understood. Therefore, this study examined the effects of the saturated FA palmitic acid (PA) and the polyunsaturated FA docosahexaenoic acid (DHA) on primary hippocampal and cortical cultures isolated from P0/P1 Sprague Dawley rat pups. Exposure to PA, but not DHA, resulted in severe morphological changes in primary neurons such as cell body swelling, axonal and dendritic blebbing, and a reduction in synaptic innervation, compromising healthy cell function and excitability. Pharmacological assessment revealed that the PA-mediated alterations were caused by overactivation of neuronal insulin signaling, demonstrated by insulin stimulation and phosphoinositide 3-kinase inhibition. Remarkably, co-exposure to DHA prevented all PA-induced morphological changes. This work provides new insights into how FAs can affect the cytoskeletal rearrangements and neuronal function via modulation of insulin signaling.

Inotropic and lusitropic, but not arrhythmogenic, effects of adipocytokine resistin on human atrial myocardium.

The adipocytokine resistin is released from epicardial adipose tissue (EAT). Plasma resistin and EAT deposition are independently associated with atrial fibrillation. The EAT secretome enhances arrhythmia susceptibility and inotropy of human myocardium. Therefore, we aimed to determine the effect of resistin on the function of human myocardium and how resistin contributes to the proarrhythmic effect of EAT. EAT biopsies were obtained from 25 cardiac surgery patients. Resistin levels were measured by ELISA in 24-h EAT culture media (n = 8). The secretome resistin concentrations increased over the culture period to a maximal level of 5.9 ± 1.2 ng/mL. Coculture with ß-adrenergic agonists isoproterenol (n = 4) and BRL37344 (n = 13) had no effect on EAT resistin release. Addition of resistin (7, 12, 20 ng/mL) did not significantly increase the spontaneous contraction propensity of human atrial trabeculae (n = 10) when given alone or in combination with isoproterenol. Resistin dose-dependently increased trabecula-developed force (maximal 2.9-fold increase, P < 0.0001), as well as the maximal rates of contraction (2.6-fold increase, P = 0.002) and relaxation (1.8-fold increase, P = 0.007). Additionally, the postrest potentiation capacity of human trabeculae was reduced at all resistin doses, suggesting that the inotropic effect induced by resistin might be due to altered sarcoplasmic reticulum Ca2+ handling. EAT resistin release is not modulated by common arrhythmia triggers. Furthermore, exogenous resistin does not promote arrhythmic behavior in human atrial trabeculae. Resistin does, however, induce an acute dose-dependent positive inotropic and lusitropic effect.

Acute interaction between human epicardial adipose tissue and human atrial myocardium induces arrhythmic susceptibility.

Epicardial adipose tissue (EAT) deposition has a strong clinical association with atrial arrhythmias; however, whether a direct functional interaction exists between EAT and the myocardium to induce atrial arrhythmias is unknown. Therefore, we aimed to determine whether human EAT can be an acute trigger for arrhythmias in human atrial myocardium. Human trabeculae were obtained from right atrial appendages of patients who have had cardiac surgery (n = 89). The propensity of spontaneous contractions (SCs) in the trabeculae (proxy for arrhythmias) was determined under physiological conditions and during known triggers of SCs (high Ca2+, ß-adrenergic stimulation). To determine whether EAT could trigger SCs, trabeculae were exposed to superfusate of fresh human EAT, and medium of 24 h-cultured human EAT treated with ß1/2 (isoproterenol) or ß3 (BRL37344) adrenergic agonists. Without exposure to EAT, high Ca2+ and ß1/2-adrenergic stimulation acutely triggered SCs in, respectively, 47% and 55% of the trabeculae that previously were not spontaneously active. Acute ß3-adrenergic stimulation did not trigger SCs. Exposure of trabeculae to either superfusate of fresh human EAT or untreated medium of 24 h-cultured human EAT did not induce SCs; however, specific ß3-adrenergic stimulation of EAT did trigger SCs in the trabeculae, either when applied to fresh (31%) or cultured (50%) EAT. Additionally, fresh EAT increased trabecular contraction and relaxation, whereas media of cultured EAT only increased function when treated with the ß3-adrenergic agonist. An acute functional interaction between human EAT and human atrial myocardium exists that increases the propensity for atrial arrhythmias, which depends on ß3-adrenergic rather than ß1/2-adrenergic stimulation of EAT.

Quadruply Stranded Metallo-Supramolecular Helicate [Pd2(hextrz)4]4+ Acts as a Molecular Mimic of Cytolytic Peptides.

[Pd2(hextrz)4]4+ is a quadruply stranded helicate, a novel bioinorganic complex designed to mimic the structure and function of proteins due to its high stability and supramolecular size. We have previously reported that [Pd2(hextrz)4]4+ exhibited cytotoxicity toward a range of cell lines, with IC50 values ranging from 3 to 10 µM. Here we demonstrate that [Pd2(hextrz)4]4+ kills cells by forming pores within the cell membrane, a mechanism of cell death analogous to the naturally occurring cytolytic peptides. [Pd2(hextrz)4]4+ induced cell death is characterized by an initial influx of Ca2+, followed by nuclear condensation and mitochondrial swelling. This is accompanied by progressive cell membrane damage that results in the formation of large blebs at the cell surface. This allows the efflux of molecules from the cell leading to loss of cell viability. These data suggest that it may be possible to design metallo-supramolecular complexes to mimic the cytotoxic action of pore forming proteins and peptides and so provide a new class of drug to treat cancer, autoimmune disorders, and microbial infection.

Inhibition of calcium/calmodulin-dependent kinase II restores contraction and relaxation in isolated cardiac muscle from type 2 diabetic rats.

BACKGROUND: Calcium/calmodulin-dependent kinase II-delta (CaMKIIδ) activity is enhanced during hyperglycemia and has been shown to alter intracellular calcium handling in cardiomyocytes, ultimately leading to reduced cardiac performance. However, the effects of CaMKIIδ on cardiac contractility during type 2 diabetes are undefined. METHODS: We examined the expression and activation of CaMKIIδ in right atrial appendages from non-diabetic and type 2 diabetic patients (n = 7 patients per group) with preserved ejection fraction, and also in right ventricular tissue from Zucker Diabetic Fatty rats (ZDF) (n = 5-10 animals per group) during early diabetic cardiac dysfunction, using immunoblot. We also measured whole heart function of ZDF and control rats using echocardiography. Then we measured contraction and relaxation parameters of isolated trabeculae from ZDF to control rats in the presence and absence of CaMKII inhibitors. RESULTS: CaMKIIδ phosphorylation (at Thr287) was increased in both the diabetic human and animal tissue, indicating increased CaMKIIδ activation in the type 2 diabetic heart. Basal cardiac contractility and relaxation were impaired in the cardiac muscles from the diabetic rats, and CaMKII inhibition with KN93 partially restored contractility and relaxation. Autocamtide-2-related-inhibitor peptide (AIP), another CaMKII inhibitor that acts via a different mechanism than KN93, fully restored cardiac contractility and relaxation. CONCLUSIONS: Our results indicate that CaMKIIδ plays a key role in modulating performance of the diabetic heart, and moreover, suggest a potential therapeutic role for CaMKII inhibitors in improving myocardial function during type 2 diabetes.

Enhanced Cytosolic Ca2+ Activation Underlies a Common Defect of Central Domain Cardiac Ryanodine Receptor Mutations Linked to Arrhythmias.

Recent three-dimensional structural studies reveal that the central domain of ryanodine receptor (RyR) serves as a transducer that converts long-range conformational changes into the gating of the channel pore. Interestingly, the central domain encompasses one of the mutation hotspots (corresponding to amino acid residues 3778-4201) that contains a number of cardiac RyR (RyR2) mutations associated with catecholaminergic polymorphic ventricular tachycardia (CPVT) and atrial fibrillation (AF). However, the functional consequences of these central domain RyR2 mutations are not well understood. To gain insights into the impact of the mutation and the role of the central domain in channel function, we generated and characterized eight disease-associated RyR2 mutations in the central domain. We found that all eight central domain RyR2 mutations enhanced the Ca2+-dependent activation of [3H]ryanodine binding, increased cytosolic Ca2+-induced fractional Ca2+ release, and reduced the activation and termination thresholds for spontaneous Ca2+ release in HEK293 cells. We also showed that racemic carvedilol and the non-beta-blocking carvedilol enantiomer, (R)-carvedilol, suppressed spontaneous Ca2+ oscillations in HEK293 cells expressing the central domain RyR2 mutations associated with CPVT and AF. These data indicate that the central domain is an important determinant of cytosolic Ca2+ activation of RyR2. These results also suggest that altered cytosolic Ca2+ activation of RyR2 represents a common defect of RyR2 mutations associated with CPVT and AF, which could potentially be suppressed by carvedilol or (R)-carvedilol.

FKBPs facilitate the termination of spontaneous Ca2+ release in wild-type RyR2 but not CPVT mutant RyR2.

FK506-binding proteins 12.6 (FKBP12.6) and 12 (FKBP12) tightly associate with the cardiac ryanodine receptor (RyR2). Studies suggest that dissociation of FKBP12.6 from mutant forms of RyR2 contributes to store overload-induced Ca(2+) release (SOICR) and Ca(2+)-triggered arrhythmias. However, these findings are controversial. Previous studies focused on the effect of FKBP12.6 on the initiation of SOICR and did not explore changes in the termination of Ca(2+) release. Less is known about FKBP12. We aimed to determine the effect of FKBP12.6 and FKBP12 on the termination of SOICR. Using single-cell imaging, in cells expressing wild-type RyR2, we found that FKBP12.6 and FKBP12 significantly increase the termination threshold of SOICR without changing the activation threshold of SOICR. This effect, dependent on the association of each FKBP with RyR2, reduced the magnitude of Ca(2+) release but had no effect on the propensity for SOICR. In contrast, neither FKBP12.6 nor FKBP12 was able to regulate an arrhythmogenic variant of RyR2, despite a conserved protein interaction. Our results suggest that both FKBP12.6 and FKBP12 play critical roles in regulating RyR2 function by facilitating the termination of SOICR. The inability of FKBPs to mediate a similar effect on the mutant RyR2 represents a novel mechanism by which mutations within RyR2 lead to arrhythmia.

Oxidation of RyR2 Has a Biphasic Effect on the Threshold for Store Overload-Induced Calcium Release.

At the single-channel level, oxidation of the cardiac ryanodine receptor (RyR2) is known to activate and inhibit the channel depending on the level of oxidation. However, the mechanisms through which these changes alter the activity of RyR2 in a cellular setting are poorly understood. In this study, we determined the effect of oxidation on a common form of RyR2 regulation; store overload-induced Ca(2+) release (SOICR). We found that oxidation resulted in concentration and time-dependent changes in the activation threshold for SOICR. Low concentrations of the oxidant H2O2 resulted in a decrease in the threshold for SOICR, which led to an increase in SOICR events. However, higher concentrations of H2O2, or prolonged exposure, reversed these changes and led to an increase in the threshold for SOICR. This increase in the threshold for SOICR in most cells was to such an extent that it led to the complete inhibition of SOICR. Acute exposure to high concentrations of H2O2 led to an initial decrease and then increase in the threshold for SOICR. In the majority of cells the increased threshold could not be reversed by the application of the reducing agent dithiothreitol. Therefore, our data suggest that low levels of RyR2 oxidation increase the channel activity by decreasing the threshold for SOICR, whereas high levels of RyR2 oxidation irreversibly increase the threshold for SOICR leading to an inhibition of RyR2. Combined, this indicates that oxidation regulates RyR2 by the same mechanism as phosphorylation, methylxanthines, and mutations, via changes in the threshold for SOICR.

Roles of the NH2-terminal domains of cardiac ryanodine receptor in Ca2+ release activation and termination.

The NH2-terminal region (residues 1-543) of the cardiac ryanodine receptor (RyR2) harbors a large number of mutations associated with cardiac arrhythmias and cardiomyopathies. Functional studies have revealed that the NH2-terminal region is involved in the activation and termination of Ca(2+) release. The three-dimensional structure of the NH2-terminal region has recently been solved. It is composed of three domains (A, B, and C). However, the roles of these individual domains in Ca(2+) release activation and termination are largely unknown. To understand the functional significance of each of these NH2-terminal domains, we systematically deleted these domains and assessed their impact on caffeine- or Ca(2+)-induced Ca(2+) release and store overload-induced Ca(2+) release (SOICR) in HEK293 cells. We found that all deletion mutants were capable of forming caffeine- and ryanodine-sensitive functional channels, indicating that the NH2-terminal region is not essential for channel gating. Ca(2+) release measurements revealed that deleting domain A markedly reduced the threshold for SOICR termination but had no effect on caffeine or Ca(2+) activation or the threshold for SOICR activation, whereas deleting domain B substantially enhanced caffeine and Ca(2+) activation and lowered the threshold for SOICR activation and termination. Conversely, deleting domain C suppressed caffeine activation, abolished Ca(2+) activation and SOICR, and diminished protein expression. These results suggest that domain A is involved in channel termination, domain B is involved in channel suppression, and domain C is critical for channel activation and expression. Our data shed new insights into the structure-function relationship of the NH2-terminal domains of RyR2 and the action of NH2-terminal disease mutations.

Role of Cys³6°² in the function and regulation of the cardiac ryanodine receptor.

The cardiac Ca²âº release channel [ryanodine receptor type 2 (RyR2)] is modulated by thiol reactive agents, but the molecular basis of RyR2 modulation by thiol reagents is poorly understood. Cys³6³5 in the skeletal muscle RyR1 is one of the most hyper-reactive thiols and is important for the redox and calmodulin (CaM) regulation of the RyR1 channel. However, little is known about the role of the corresponding cysteine residue in RyR2 (Cys³6°²) in the function and regulation of the RyR2 channel. In the present study, we assessed the impact of mutating Cys³6°² (C³6°²A) on store overload-induced Ca²âº release (SOICR) and the regulation of RyR2 by thiol reagents and CaM. We found that the C³6°²A mutation suppressed SOICR by raising the activation threshold and delayed the termination of Ca²âº release by reducing the termination threshold. As a result, C³6°²A markedly increased the fractional Ca²âº release. Furthermore, the C³6°²A mutation diminished the inhibitory effect of N-ethylmaleimide on Ca²âº release, but it had no effect on the stimulatory action of 4,4'-dithiodipyridine (DTDP) on Ca²âº release. In addition, Cys³6°² mutations (C³6°²A or C³6°²R) did not abolish the effect of CaM on Ca²âº-release termination. Therefore, RyR2-Cys³6°² is a major site mediating the action of thiol alkylating agent N-ethylmaleimide, but not the action of the oxidant DTDP. Our data also indicate that residue Cys³6°² plays an important role in the activation and termination of Ca²âº release, but it is not essential for CaM regulation of RyR2.

Chronic bilateral renal denervation reduces cardiac hypertrophic remodelling but not ß-adrenergic responsiveness in hypertensive type 1 diabetic rats.

NEW FINDINGS: What is the central question of this study? Can bilateral renal denervation, an effective antihypertensive treatment in clinical and experimental studies, improve cardiac ß-adrenoceptor responsiveness in a diabetic model with underlying hypertension? What is the main finding and its importance? Bilateral renal denervation did not affect ß-adrenergic responsiveness in the diabetic hypertensive rat heart, but denervation reduced the hypertension-induced concentric hypertrophic remodelling. This suggests that the positive haemodynamic changes induced by renal denervation are most likely to reflect an attenuation of sympathetic effects on the systemic vasculature and/or the renal function rather than direct sympathetic modulation of the heart. Bilateral renal denervation (BRD) has been shown to normalise blood pressure in clinical and experimental studies of hypertension by reducing systemic sympathetic output. This study determined the effect of BRD on cardiac ß-adrenoceptor (AR) responsiveness in a diabetic model with underlying hypertension using the transgenic (mRen-2)27 rats. Bilateral renal denervation or sham surgeries were conducted repeatedly at 3, 6 and 9 weeks in Ren-2 rats with or without streptozotocin (STZ)-induced diabetes (4 × n = 7); Sprague-Dawley rats (n = 6) served as control animals. Cardiac function was determined in isolated hearts at 18 weeks of age. Normalised left ventricular developed pressure and relaxation was recorded in response to incremental concentrations of the ß-AR agonist isoprenaline (from 10-10 to 10-7 m) or the ß3 -AR agonist BRL37344 (from 10(-13) to 10(-6 ) m). Expression levels of ß1 -AR were determined by Western blot. Both inotropic and lusitropic ß-AR responsiveness was reduced in the hypertensive diabetic hearts, but these responses were unaltered after BRD. Expression levels of ß1 -AR were increased after BRD (Sham, 0.85 ± 0.11 versus 1.01 ± 0.05 a.u.; BRD, 1.45 ± 0.11 versus 1.46 ± 0.07 a.u.; Ren-2 versus Ren-2 STZ, P < 0.05 versus Sham). No effect of ß3 -AR agonist stimulation with BRL37344 was observed. Interestingly, BRD increased left ventricular diastolic volume in both the Ren-2 and the Ren-2 STZ groups. Bilateral renal denervation did not restore the attenuated cardiac ß-AR responsiveness in the diabetic hypertensive rats, but it reduced the extent of hypertension-induced concentric hypertrophic remodelling. Thus, the haemodynamic protection offered by renal denervation appears to reflect an attenuated sympathetic innervation of the systemic vasculature and/or kidney rather than a direct cardiac effect.

Phospholamban knockout breaks arrhythmogenic Ca²âº waves and suppresses catecholaminergic polymorphic ventricular tachycardia in mice.

RATIONALE: Phospholamban (PLN) is an inhibitor of cardiac sarco(endo)plasmic reticulum Ca²âº ATPase. PLN knockout (PLN-KO) enhances sarcoplasmic reticulum Ca²âº load and Ca²âº leak. Conversely, PLN-KO accelerates Ca²âº sequestration and aborts arrhythmogenic spontaneous Ca²âº waves (SCWs). An important question is whether these seemingly paradoxical effects of PLN-KO exacerbate or protect against Ca²âº-triggered arrhythmias. OBJECTIVE: We investigate the impact of PLN-KO on SCWs, triggered activities, and stress-induced ventricular tachyarrhythmias (VTs) in a mouse model of cardiac ryanodine-receptor (RyR2)-linked catecholaminergic polymorphic VT. METHODS AND RESULTS: We generated a PLN-deficient, RyR2-mutant mouse model (PLN-/-/RyR2-R4496C+/-) by crossbreeding PLN-KO mice with catecholaminergic polymorphic VT-associated RyR2-R4496C mutant mice. Ca²âº imaging and patch-clamp recording revealed cell-wide propagating SCWs and triggered activities in RyR2-R4496C+/- ventricular myocytes during sarcoplasmic reticulum Ca²âº overload. PLN-KO fragmented these cell-wide SCWs into mini-waves and Ca²âº sparks and suppressed the triggered activities evoked by sarcoplasmic reticulum Ca²âº overload. Importantly, these effects of PLN-KO were reverted by partially inhibiting sarco(endo)plasmic reticulum Ca²âº ATPase with 2,5-di-tert-butylhydroquinone. However, Bay K, caffeine, or Li⁺ failed to convert mini-waves to cell-wide SCWs in PLN-/-/RyR2-R4496C+/- ventricular myocytes. Furthermore, ECG analysis showed that PLN-KO mice are not susceptible to stress-induced VTs. On the contrary, PLN-KO protected RyR2-R4496C mutant mice from stress-induced VTs. CONCLUSIONS: Our results demonstrate that despite severe sarcoplasmic reticulum Ca²âº leak, PLN-KO suppresses triggered activities and stress-induced VTs in a mouse model of catecholaminergic polymorphic VT. These data suggest that breaking up cell-wide propagating SCWs by enhancing Ca²âº sequestration represents an effective approach for suppressing Ca²âº-triggered arrhythmias.

Regulation of RYR2 by sarcoplasmic reticulum Ca(2+).

Ca(2+) is arguably the most important ion involved in the contraction of the heart. The cardiac ryanodine receptor (RyR2), the major Ca(2+) release channel located in the sarcoplasmic reticulum (SR) membrane, is responsible for releasing the bulk of Ca(2+) required for contraction. Moreover, RyR2 is also crucial for maintaining SR Ca(2+) homeostasis by releasing Ca(2+) from the SR when it becomes overloaded with Ca(2+) . During normal contraction, RyR2 is activated by cytosolic Ca(2+) , whereas during store overload conditions, the opening of RyR2 is governed by SR Ca(2+) . Although the process of the cytosolic control of RyR2 is well established, the molecular mechanism by which SR luminal Ca(2+) regulates RyR2 has only recently been elucidated and remains controversial. In addition to the activation of RyR2, SR luminal Ca(2+) also determines when the RyR2 channel closes. RyR2-mediated Ca(2+) release from the SR does not continue until the SR is completely depleted. Rather, it ceases when SR luminal Ca(2+) falls below a certain level. Given the importance of SR Ca(2+) , it is not surprising that the SR luminal Ca(2+) level is tightly controlled by SR Ca(2+) -buffering proteins. Consequently, the opening and closing of RyR2 is heavily influenced by the presence of such proteins, particularly those associated with RyR2, such as calsequestrin and the histidine-rich Ca(2+) -binding protein. These proteins appear to indirectly alter RyR2 activity by modifying the microdomain SR Ca(2+) level surrounding RyR2.