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Patients with heart failure (HF) have augmented vascular tone, which increases cardiac workload, impairing ventricular output and promoting further myocardial dysfunction. The molecular mechanisms underlying the maladaptive vascular responses observed in HF are not fully understood. Vascular smooth muscle cells (VSMCs) control vasoconstriction via a [Ca.sup.2+]-dependent process, in which the type 1 inositol 1,4,5-trisphosphate receptor (IP3R1) on the sarcoplasmic reticulum (SR) plays a major role. To dissect the mechanistic contribution of intracellular [Ca.sup.2+] release to the increased vascular tone observed in HF, we analyzed the remodeling of IP3R1 in aortic tissues from patients with HF and from controls. VSMC IP3R1 channels from patients with HF and HF mice were hyperphosphorylated by both serine and tyrosine kinases. VSMCs isolated from [IP3R1.sup.VSMC-/-] mice exhibited blunted [Ca.sup.2+] responses to angiotensin II (ATII) and norepinephrine compared with control VSMCs. [IP3R1.sup.VSMC-/-] mice displayed significantly reduced responses to ATII, both in vivo and ex vivo. HF [IP3R1.sup.VSMC-/-] mice developed significantly less afterload compared with HF [IP3R1.sup.fl/fl] mice and exhibited significantly attenuated progression toward decompensated HF and reduced interstitial fibrosis. [Ca.sup.2+]-dependent phosphorylation of the MLC by MLCK activated VSMC contraction. MLC phosphorylation was markedly increased in VSMCs from patients with HF and HF mice but reduced in VSMCs from HF [IP3R1.sup.VSMC-/-] mice and HF WT mice treated with ML-7. Taken together, our data indicate that VSMC IP3R1 is a major effector of increased vascular tone, which contributes to increased cardiac afterload and decompensation in HF., Introduction Heart failure (HF) is a complex clinical syndrome that remains a leading cause of mortality despite decades of intense efforts to develop novel therapeutics (1, 2). Beginning early in [...]

The type 2 ryanodine receptor (RyR2) is a [Ca.sup.2] release channel on the endoplasmic reticulum (ER) of several types of cells, including cardiomyocytes and pancreatic [beta] cells. In cardiomyocytes, RyR2-dependent [Ca.sup.2] release is critical for excitation-contraction coupling; however, a functional role for RyR2 in [beta] cell insulin secretion and diabetes mellitus remains controversial. Here, we took advantage of rare RyR2 mutations that were identified in patients with a genetic form of exercise-induced sudden death (catecholaminergic polymorphic ventricular tachycardia [CPVT]). As these mutations result in a 'leaky' RyR2 channel, we exploited them to assess RyR2 channel function in [beta] cell dynamics. We discovered that CPVT patients with mutant leaky RyR2 present with glucose intolerance, which was heretofore unappreciated. In mice, transgenic expression of CPVT-associated RyR2 resulted in impaired glucose homeostasis, and an in-depth evaluation of pancreatic islets and [beta] cells from these animals revealed intracellular [Ca.sup.2] leak via oxidized and nitrosylated RyR2 channels, activated ER stress response, mitochondrial dysfunction, and decreased fuel-stimulated insulin release. Additionally, we verified the effects of the pharmacological inhibition of intracellular [Ca.sup.2] leak in CPVT-associated RyR2-expressing mice, in human islets from diabetic patients, and in an established murine model of type 2 diabetes mellitus. Taken together, our data indicate that RyR2 channels play a crucial role in the regulation of insulin secretion and glucose homeostasis., Introduction The molecular mechanisms underlying insulin secretion and glucose metabolism have not been fully elucidated (1-6). Numerous studies have demonstrated that calcium ([Ca.sup.2+]) plays a pivotal role in insulin secretion [...]

Antagonists of L-type [Ca.sup.2+] channels (LTCCs) have been used to treat human cardiovascular diseases for decades. However, these inhibitors can have untoward effects in patients with heart failure, and their overall therapeutic profile remains nebulous given differential effects in the vasculature when compared with those in cardiomyocytes. To investigate this issue, we examined mice heterozygous for the gene encoding the pore-forming subunit of LTCC (calcium channel, voltage-dependent, L type, α1C subunit [Cacna1c mice; referred to herein as α1[C.sup.+/-] mice]) and mice in which this gene was loxP targeted to achieve graded heart-specific gene deletion (termed herein α1C-loxP mice). Adult cardiomyocytes from the hearts of α1[C.sup.-/+] mice at 10 weeks of age showed a decrease in LTCC current and a modest decrease in cardiac function, which we initially hypothesized would be cardioprotective. However, α1[C.sup.+/-] mice subjected to pressure overload stimulation, isoproterenol infusion, and swimming showed greater cardiac hypertrophy, greater reductions in ventricular performance, and greater ventricular dilation than α1[C.sup.+/-] controls. The same detrimental effects were observed in α1C-loxP animals with a cardiomyocytespecific deletion of one allele. More severe reductions in α1C protein levels with combinatorial deleted alleles produced spontaneous cardiac hypertrophy before 3 months of age, with early adulthood lethality. Mechanisti-cally, our data suggest that a reduction in LTCC current leads to neuroendocrine stress, with sensitized and leaky sarcoplasmic reticulum [Ca.sup.2+] release as a compensatory mechanism to preserve contractility. This state results in calcineurin/nuclear factor of activated T cells signaling that promotes hypertrophy and disease., Introduction Voltage-gated L-type [Ca.sup.2+] channels (LTCCs) are the primary source of [Ca.sup.2+] influx to initiate cardiac excitation-contraction coupling (ECC) (1, 2). The molecular composition of the LTCC in cardiomyocytes includes [...]

During the classic 'fight-or-flight' stress response, sympathetic nervous system activation leads to catechol-amine release, which increases heart rate and contractility, resulting in enhanced cardiac output. Catecholamines bind to β-adrenergic receptors, causing cAMP generation and activation of PKA, which phosphorylates multiple targets in cardiac muscle, including the cardiac ryanodine receptor/calcium release channel (RyR2) required for muscle contraction. PKA phosphorylation of RyR2 enhances channel activity by sensitizing the channel to cytosolic calcium ([Ca.sup.2+]). Here, we found that mice harboring RyR2 channels that cannot be PKA phosphorylated (referred to herein as RyR2-[S2808A.sup.+/+] mice) exhibited blunted heart rate and cardiac contractile responses to catecholamines (isoproterenol). The isoproterenol-induced enhancement of ventricular myocyte [Ca.sup.2+] transients and fractional shortening (contraction) and the spontaneous beating rate of sinoatrial nodal cells were all blunted in RyR2-[S2808A.sup.+/+] mice. The blunted cardiac response to catecholamines in RyR2-[S2808A.sup.+/+] mice resulted in impaired exercise capacity. RyR2-[S2808A.sup.+/+] mice were protected against chronic catecholaminergic-induced cardiac dysfunction. These studies identify what we believe to be new roles for PKA phosphorylation of RyR2 in both the heart rate and contractile responses to acute catecholaminergic stimulation., Introduction During exercise, heart rate (chronotropy) and cardiac contractility (inotropy) increase to meet the metabolic demands of the organs. Stress-induced activation of the sympathetic nervous system (SNS) results in catecholamine [...]

Increased sarcoplasmic reticulum (SR) [Ca.sup.2+] leak via the cardiac ryanodine receptor/calcium release channel (RyR2) is thought to play a role in heart failure (HF) progression. Inhibition of this leak is an emerging therapeutic strategy. To explore the role of chronic PKA phosphorylation of RyR2 in HF pathogenesis and treatment, we generated a knockin mouse with aspartic acid replacing serine 2808 (mice are referred to herein as RyR2-[S2808D.sup.+/+] mice). This mutation mimics constitutive PKA hyperphosphorylation of RyR2, which causes depletion of the stabilizing subunit FKBP12.6 (also known as calstabin2), resulting in leaky RyR2. RyR2-[S2808D.sup.+/+] mice developed age-dependent cardiomyopathy, elevated RyR2 oxidation and nitrosylation, reduced SR [Ca.sup.2+] store content, and increased diastolic SR [Ca.sup.2+] leak. After myocardial infarction, RyR2-[S2808D.sup.+/+] mice exhibited increased mortality compared with WT littermates. Treatment with S107, a 1,4-benzothiazepine derivative that stabilizes RyR2-calstabin2 interactions, inhibited the RyR2-mediated diastolic SR [Ca.sup.2+] leak and reduced HF progression in WT and RyR2-[S2808D.sup.+/+] mice. In contrast, β-adrenergic receptor blockers improved cardiac function in WT but not in RyR2-[S2808D.sup.+/+] mice.Thus, chronic PKA hyperphosphorylation of RyR2 results in a diastolic leak that causes cardiac dysfunction. Reversing PKA hyperphosphorylation of RyR2 is an important mechanism underlying the therapeutic action of β-blocker therapy in HF., Introduction Heart failure (HF), a syndrome characterized by progressive deterioration of cardiac function, represents a major public health burden and is a leading cause of morbidity and mortality in the [...]

The [Ca.sup.2+] release channel ryanodine receptor 2 (RyR2) is required for excitation-contraction coupling in the heart and is also present in the brain. Mutations in RyR2 have been linked to exercise-induced sudden cardiac death (catecholaminergic polymorphic ventricular tachycardia [CPVT]). CPVT-associated RyR2 mutations result in "leaky" RyR2 channels due to the decreased binding of the calstabin2 (FKBP12.6) subunit, which stabilizes the closed state of the channel. We found that mice heterozygous for the R2474S mutation in Ryr2 (Ryr2-R2474S mice) exhibited spontaneous generalized tonic-clonic seizures (which occurred in the absence of cardiac arrhythmias), exercise-induced ventricular arrhythmias, and sudden cardiac death. Treatment with a novel RyR2-specific compound (S107) that enhances the binding of calstabin2 to the mutant Ryr2-R2474S channel inhibited the channel leak and prevented cardiac arrhythmias and raised the seizure threshold. Thus, CPVT-associated mutant leaky Ryr2-R2474S channels in the brain can cause seizures in mice, independent of cardiac arrhythmias. Based on these data, we propose that CPVT is a combined neurocardiac disorder in which leaky RyR2 channels in the brain cause epilepsy, and the same leaky channels in the heart cause exercise-induced sudden cardiac death., Introduction Pharmacological seizure models have implicated abnormalities in intracellular [Ca.sup.2+] cycling of inhibitory interneurons and/or astrocytes as a mechanism of seizure generation (1), (2), and the inositol 1,4,5-trisphosphate receptor (IP3R), [...]

Antagonists of L-type Ca2+ channels (LTCCs) have been used to treat human cardiovascular diseases for decades. However, these inhibitors can have untoward effects in patients with heart failure, and their overall therapeutic profile remains nebulous given differential effects in the vasculature when compared with those in cardiomyocytes. To investigate this issue, we examined mice heterozygous for the gene encoding the pore-forming subunit of LTCC (calcium channel, voltage-dependent, L type, α1C subunit [Cacna1c mice; referred to herein as α1C-/+ mice]) and mice in which this gene was loxP targeted to achieve graded heart-specific gene deletion (termed herein α1C-loxP mice). Adult cardiomyocytes from the hearts of α1C-/+ mice at 10 weeks of age showed a decrease in LTCC current and a modest decrease in cardiac function, which we initially hypothesized would be cardioprotective. However, α1C-/+ mice subjected to pressure overload stimulation, isoproterenol infusion, and swimming showed greater cardiac hypertrophy, greater reductions in ventricular performance, and greater ventricular dilation than α1C+/+ controls. The same detrimental effects were observed in α1C-loxP animals with a cardiomyocytespecific deletion of one allele. More severe reductions in α1C protein levels with combinatorial deleted alleles produced spontaneous cardiac hypertrophy before 3 months of age, with early adulthood lethality. Mechanistically, our data suggest that a reduction in LTCC current leads to neuroendocrine stress, with sensitized and leaky sarcoplasmic reticulum Ca2+ release as a compensatory mechanism to preserve contractility. This state results in calcineurin/nuclear factor of activated T cells signaling that promotes hypertrophy and disease. [ABSTRACT FROM AUTHOR]

Increased sarcoplasmic reticulum (SR) Ca2+ leak via the cardiac ryanodine receptor/calcium release channel (RyR2) is thought to play a role in heart failure (HF) progression. Inhibition of this leak is an emerging therapeutic strategy. To explore the role of chronic PKA phosphorylation of RyR2 in HF pathogenesis and treatment, we generated a knockin mouse with aspartic acid replacing serine 2808 (mice are referred to herein as RyR2-S2808D+/+ mice). This mutation mimics constitutive PKA hyperphosphorylation of RyR2, which causes depletion of the stabilizing subunit FKBP12.6 (also known as calstabin2), resulting in leaky RyR2. RyR2-S2808D+/+ mice developed age-dependent cardiomyopathy, elevated RyR2 oxidation and nitrosylation, reduced SR Ca2+ store content, and increased diastolic SR Ca2+ leak. After myocardial infarction, RyR2-S2808D+/+ mice exhibited increased mortality compared with WT littermates. Treatment with S107, a 1,4-benzothiazepine derivative that stabilizes RyR2-calstabin2 interactions, inhibited the RyR2-mediated diastolic SR Ca2+ leak and reduced HF progression in WT and RyR2-S2808D+/+ mice. In contrast, β-adrenergic receptor blockers improved cardiac function in WT but not in RyR2-S2808D+/+ mice.Thus, chronic PKA hyperphosphorylation of RyR2 results in a diastolic leak that causes cardiac dysfunction. Reversing PKA hyperphosphorylation of RyR2 is an important mechanism underlying the therapeutic action of β-blocker therapy in HF. [ABSTRACT FROM AUTHOR]

During the classic "fight-or-flight" stress response, sympathetic nervous system activation leads to catecholamine release, which increases heart rate and contractility, resulting in enhanced cardiac output. Catecholamines bind to β-adrenergic receptors, causing cAMP generation and activation of PKA, which phosphorylates multiple targets in cardiac muscle, including the cardiac ryanodine receptor/calcium release channel (RyR2) required for muscle contraction. PKA phosphorylation of RyR2 enhances channel activity by sensitizing the channel to cytosolic calcium (Ca²+). Here, we found that mice harboring RyR2 channels that cannot be PKA phosphorylated (referred to herein as RyR2-S2808A+/+ mice) exhibited blunted heart rate and cardiac contractile responses to catecholamines (isoproterenol). The isoproterenol-induced enhancement of ventricular myocyte Ca²+ transients and fractional shortening (contraction) and the spontaneous beating rate of sinoatrial nodal cells were all blunted in RyR2-S2808A+/+ mice. The blunted cardiac response to catecholamines in RyR2-S2808A+/+ mice resulted in impaired exercise capacity. RyR2-S2808A+/+ mice were protected against chronic catecholaminergic-induced cardiac dysfunction. These studies identify what we believe to be new roles for PKA phosphorylation of RyR2 in both the heart rate and contractile responses to acute catecholaminergic stimulation. [ABSTRACT FROM AUTHOR]