-Atrial fibrillation (AF) is frequently associated with enhanced inflammatory response. The "NACHT, LRR and PYD domain containing protein 3" (NLRP3)-inflammasome mediates caspase-1 activation and interleukin-1β release in immune cells, but is not known to play a role in cardiomyocytes (CMs). Here, we assessed the role of CM NLRP3-inflammasome in AF. -NLRP3-inflammasome activation was assessed by immunoblot in atrial whole-tissue lysates and CMs from patients with paroxysmal (pAF) or long-standing persistent (chronic) AF (cAF). To determine whether CM-specific activation of NLPR3 is sufficient to promote AF, a CM-specific knock-in mouse model expressing constitutively active NLRP3 (CM-KI) was established. In vivo electrophysiology was used to assess atrial arrhythmia vulnerability. To evaluate the mechanism of AF, electrical activation pattern, Ca spark frequency (CaSF), atrial effective refractory period (AERP), and morphology of atria were evaluated in CM-KI mice and WT littermates. -NLRP3-inflammasome activity was increased in atrial CMs of pAF and cAF patients. CM-KI mice developed spontaneous premature atrial contractions and inducible AF, which was attenuated by a specific NLRP3-inflammasome inhibitor, MCC950. CM-KI mice exhibited ectopic activity, abnormal sarcoplasmic-reticulum Ca-release, AERP shortening and atrial hypertrophy. Adeno-associated virus subtype-9 mediated CM-specific knockdown of suppressed AF development in CM-KI mice. Finally, genetic inhibition of prevented AF development in CREM transgenic mice, a well-characterized mouse model of spontaneous AF. -Our study establishes a novel pathophysiological role for CM NLRP3-inflammasome signaling with a mechanistic link to the pathogenesis of AF, and establishes inhibition of NLRP3 as a potential novel AF-therapy approach.

Background— Abnormal Ca 2+ handling may contribute to impaired atrial contractility and arrhythmogenesis in human chronic atrial fibrillation (cAF). Here, we assessed the phosphorylation levels of key proteins involved in altered Ca 2+ handling and contractility in cAF patients. Methods and Results— Total and phosphorylation levels of Ca 2+ -handling and myofilament proteins were analyzed by Western blotting in right atrial appendages of 49 patients in sinus rhythm and 52 cAF patients. We found a higher total activity of type 1 (PP1) and type 2A phosphatases in cAF, which was associated with inhomogeneous changes of protein phosphorylation in the cellular compartments, ie, lower protein kinase A (PKA) phosphorylation of myosin binding protein-C (Ser-282 site) at the thick myofilaments but preserved PKA phosphorylation of troponin I at the thin myofilaments and enhanced PKA (Ser-16 site) and Ca 2+ -calmodulin protein kinase (Thr-17 site) phosphorylation of phospholamban. PP1 activity at sarcoplasmic reticulum is controlled by inhibitor-1 (I-1), which blocks PP1 in its PKA-phosphorylated form only. In cAF, the ratio of Thr-35–phosphorylated to total I-1 was 10-fold higher, which suggests that the enhanced phosphorylation of phospholamban may result from a stronger PP1 inhibition by PKA-hyperphosphorylated (activated) I-1. Conclusions— Altered Ca 2+ handling in cAF is associated with impaired phosphorylation of myosin binding protein-C, which may contribute to the contractile dysfunction after cardioversion. The hyperphosphorylation of phospholamban probably results from enhanced inhibition of sarcoplasmic PP1 by hyperphosphorylated I-1 and may reinforce the leakiness of ryanodine channels in cAF. Restoration of sarcoplasmic reticulum–associated PP1 function may represent a new therapeutic option for treatment of atrial fibrillation.

Rationale: Heart failure (HF) is known to be associated with increased Ca 2؉ /calmodulin-dependent protein kinase (CaMK)II expression and activity. There is still controversial discussion about the functional role of CaMKII in HF. Moreover, CaMKII inhibition has never been investigated in human myocardium.Objective: We sought to investigate detailed CaMKII␦ expression in end-stage failing human hearts (dilated and ischemic cardiomyopathy) and the functional effects of CaMKII inhibition on contractility. Methods and Results:Expression analysis revealed that CaMKII␦, both cytosolic ␦ C and nuclear ␦ B splice variants, were significantly increased in both right and left ventricles from patients with dilated or ischemic cardiomyopathy versus nonfailing. Experiments with isometrically twitching trabeculae revealed significantly improved force frequency relationships in the presence of CaMKII inhibitors (KN-93 and AIP). Increased postrest twitches after CaMKII inhibition indicated an improved sarcoplasmic reticulum (SR) Ca 2؉ loading. This was confirmed in isolated myocytes by a reduced SR Ca 2؉ spark frequency and hence SR Ca 2؉ leak, resulting in increased SR Ca 2؉ load when inhibiting CaMKII. Ryanodine receptor type 2 phosphorylation at Ser2815, which is known to be phosphorylated by CaMKII thereby contributing to SR Ca 2؉ leak, was found to be markedly reduced in KN-93-treated trabeculae. Interestingly, CaMKII inhibition did not influence contractility in nonfailing sheep trabeculae.Conclusions: The present study shows for the first time that CaMKII inhibition acutely improves contractility in human HF where CaMKII␦ expression is increased. The mechanism proposed consists of a reduced SR Ca Key Words: Ca 2ϩ /calmodulin-dependent kinase II Ⅲ heart failure Ⅲ contractility Ⅲ calcium Ⅲ sarcoplasmic reticulum Ca 2ϩ leak Ⅲ ryanodine receptor H eart failure (HF) is accompanied by systolic and diastolic contractile dysfunction caused by abnormalities in intracellular Ca 2ϩ handling and structural remodeling. Several targets associated with the remodeling processes have been identified. The sarcoplasmic reticulum (SR) Ca 2ϩ -ATPase (SERCA) protein levels have been reported to be downregulated and paralleled by a reduced SR Ca 2ϩ uptake capacity in the human failing heart. [1][2][3] In contrast, the sarcolemmal Na ϩ /Ca 2ϩ exchanger protein expression and activity were found to be increased thereby even more effectively competing with the reduced SERCA activity for cytosolic Ca 2ϩ -removal. 3,4 The net effect is an impaired SR Ca 2ϩ loading, which leads to smaller intracellular Ca 2ϩ transients and elevated diastolic Ca 2ϩ levels in HF. 5 Thus, impaired contractility with reduced contractile force and diastolic dysfunction are well-accepted determinants in HF. 6 Intracellular Ca 2ϩ homeostasis of cardiac myocytes is also regulated by phosphorylation of several key Ca 2ϩ -handling proteins. An important regulatory kinase is the Ca 2ϩ /calmodulin-dependent protein kinase (CaMK)II. 7 It is a serine/ threonine protein kinase t...

COVID-19 is a rapidly spreading outbreak globally. Emerging evidence demonstrates that older individuals and people with underlying metabolic conditions of diabetes mellitus, hypertension, and hyperlipidemia are at higher risk of morbidity and mortality. The SARS-CoV-2 infects humans through the angiotensin converting enzyme (ACE-2) receptor. The ACE-2 receptor is a part of the dual system renin-angiotensin-system (RAS) consisting of ACE-Ang-II-AT1R axis and ACE-2-Ang-(1–7)-Mas axis. In metabolic disorders and with increased age, it is known that there is an upregulation of ACE-Ang-II-AT1R axis with a downregulation of ACE-2-Ang-(1–7)-Mas axis. The activated ACE-Ang-II-AT1R axis leads to pro-inflammatory and pro-fibrotic effects in respiratory system, vascular dysfunction, myocardial fibrosis, nephropathy, and insulin secretory defects with increased insulin resistance. On the other hand, the ACE-2-Ang-(1–7)-Mas axis has anti-inflammatory and antifibrotic effects on the respiratory system and anti-inflammatory, antioxidative stress, and protective effects on vascular function, protects against myocardial fibrosis, nephropathy, pancreatitis, and insulin resistance. In effect, the balance between these two axes may determine the prognosis. The already strained ACE-2-Ang-(1–7)-Mas in metabolic disorders is further stressed due to the use of the ACE-2 by the virus for entry, which affects the prognosis in terms of respiratory compromise. Further evidence needs to be gathered on whether modulation of the renin angiotensin system would be advantageous due to upregulation of Mas activation or harmful due to the concomitant ACE-2 receptor upregulation in the acute management of COVID-19.

The sympathetic nervous system provides the most powerful stimulation of cardiac function, brought about via norepinephrine and epinephrine and their postsynaptic beta-adrenergic receptors. More than 30 years after the first use of practolol in patients with heart failure beta blockers are now the mainstay of the pharmacological treatment of chronic heart failure. Many aspects of their mechanism of action are well understood, but others remain unresolved. This review focuses on a number of questions that are key to further developments in the field. What accounts for and what is the role of beta-adrenergic desensitization, a hallmark of the failing heart? Is part of this adaptation predominantly beneficial and should therefore be reinforced, another part mainly maladaptive and therefore a target for antagonists? Which lessons can be drawn from studies in genetically engineered mice, which from (pharmaco) genetic studies? Finally, what are promising targets downstream of beta-adrenergic receptors that go beyond the current neurohumoral blockade?

A hallmark of mature mammalian ventricular myocardium is a positive force-frequency relationship (FFR). Despite evidence of organotypic structural and molecular maturation, a positive FFR has not been observed in mammalian tissue engineered heart muscle. We hypothesized that concurrent mechanical and electrical stimulation at frequencies matching physiological heart rate will result in functional maturation. To this end, we investigated the role of such biomimetic mechanical and electrical stimulation in functional maturation in engineered heart muscle (EHM) comprising collagen type I and neonatal rat heart cells. Following tissue consolidation (8 days), EHM were subjected to electrical field stimulation at 0, 2, 4, or 6 Hz for 5 days, while strained on flexible poles to facilitate auxotonic contractions. EHM stimulated at 2 and 4 Hz displayed a similarly enhanced inotropic reserve, but a clearly diverging FFR. The positive FFR in 4 Hz stimulated EHM was associated with reduced calcium sensitivity, frequency-dependent acceleration of relaxation, and enhanced post-rest potentiation. This was paralleled on the cellular level with improved calcium storage and release capacity of the sarcoplasmic reticulum, increased amounts of SERCA2a and RyR2 protein, and enhanced T-tubulation. We demonstrate that electromechanical stimulation at a frequency matching closely the physiological heart rate supports functional maturation in mammalian EHM. The observed positive FFR in EHM has important implications for the applicability of EHM in cardiovascular research and drug testing.

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Rationale: Post-operative atrial fibrillation (POAF) is a common and troublesome complication of cardiac surgery. POAF is generally believed to occur when post-operative triggers act on a pre-existing vulnerable substrate, but the underlying cellular and molecular mechanisms are largely unknown. Objective: To identify cellular POAF-mechanisms in right-atrial samples from patients without a history of atrial fibrillation undergoing open-heart surgery. Methods and Results: Multicellular action potentials, membrane ion-currents (perforated patch-clamp) or simultaneous membrane-current (ruptured patch-clamp) and [Ca 2+ ]i-recordings in atrial cardiomyocytes, along with protein-expression levels in tissue homogenates or cardiomyocytes, were assessed in 265 atrial samples from patients without or with POAF. No indices of electrical, profibrotic, or connexin remodeling were noted in POAF, but Ca 2+ -transient amplitude was smaller while spontaneous sarcoplasmic-reticulum (SR) Ca 2+ -release events and L-type Ca 2+ -current alternans occurred more frequently. Ca 2+ /calmodulin-dependent protein kinase-II (CaMKII) protein-expression, CaMKII-dependent phosphorylation of the cardiac ryanodine-receptor channel type-2 (RyR2) and RyR2 single-channel open-probability were significantly increased in POAF. SR Ca 2+ -content was unchanged in POAF despite greater SR Ca 2+ -leak, with a trend towards increased SR Ca 2+ -ATPase activity. POAF patients also showed stronger expression of activated components of the NLRP3-inflammasome system in atrial whole-tissue homogenates and cardiomyocytes. Acute application of interleukin-1beta caused NLRP3-signaling activation and CaMKII-dependent RyR2/phospholamban hyperphosphorylation in HL-1-cardiomyocytes and enhanced spontaneous SR Ca 2+ -release events in both POAF-cardiomyocytes and HL-1-cardiomyocytes. Computational modeling showed that RyR2-dysfunction and increased SR Ca 2+ -uptake are sufficient to reproduce the Ca 2+ -handling phenotype and indicated an increased risk of proarrhythmic delayed afterdepolarizations in POAF-subjects in response to interleukin-1beta. Conclusions: Pre-existing Ca 2+ -handling abnormalities and activation of NLRP3-inflammasome/CaMKII signaling are evident in atrial cardiomyocytes from patients who subsequently develop POAF. These molecular substrates sensitize cardiomyocytes to spontaneous Ca 2+ -releases and arrhythmogenic afterdepolarizations, particularly upon exposure to inflammatory mediators. Our data reveal a potential cellular and molecular substrate for this important clinical problem.