Mark D. McCauley

Circadian rhythms govern cardiac repolarization and arrhythmogenesis.

Sudden cardiac death exhibits diurnal variation in both acquired and hereditary forms of heart disease, but the molecular basis of this variation is unknown. A common mechanism that underlies susceptibility to ventricular arrhythmias is abnormalities in the duration (for example, short or long QT syndromes and heart failure) or pattern (for example, Brugada’s syndrome) of myocardial repolarization. Here we provide molecular evidence that links circadian rhythms to vulnerability in ventricular arrhythmias in mice. Specifically, we show that cardiac ion-channel expression and QT-interval duration (an index of myocardial repolarization) exhibit endogenous circadian rhythmicity under the control of a clock-dependent oscillator, krüppel-like factor 15 (Klf15). Klf15 transcriptionally controls rhythmic expression of Kv channel-interacting protein 2 (KChIP2), a critical subunit required for generating the transient outward potassium current. Deficiency or excess of Klf15 causes loss of rhythmic QT variation, abnormal repolarization and enhanced susceptibility to ventricular arrhythmias. These findings identify circadian transcription of ion channels as a mechanism for cardiac arrhythmogenesis.

Ryanodine Receptor Phosphorylation by Calcium/Calmodulin-Dependent Protein Kinase II Promotes Life-Threatening Ventricular Arrhythmias in Mice With Heart Failure

Background— Approximately half of patients with heart failure die suddenly as a result of ventricular arrhythmias. Although abnormal Ca2+ release from the sarcoplasmic reticulum through ryanodine receptors (RyR2) has been linked to arrhythmogenesis, the molecular mechanisms triggering release of arrhythmogenic Ca2+ remain unknown. We tested the hypothesis that increased RyR2 phosphorylation by Ca2+/calmodulin-dependent protein kinase II is both necessary and sufficient to promote lethal ventricular arrhythmias. Methods and Results— Mice in which the S2814 Ca2+/calmodulin-dependent protein kinase II site on RyR2 is constitutively activated (S2814D) develop pathological sarcoplasmic reticulum Ca2+ release events, resulting in reduced sarcoplasmic reticulum Ca2+ load on confocal microscopy. These Ca2+ release events are associated with increased RyR2 open probability in lipid bilayer preparations. At baseline, young S2814D mice have structurally and functionally normal hearts without arrhythmias; however, they develop sustained ventricular tachycardia and sudden cardiac death on catecholaminergic provocation by caffeine/epinephrine or programmed electric stimulation. Young S2814D mice have a significant predisposition to sudden arrhythmogenic death after transverse aortic constriction surgery. Finally, genetic ablation of the Ca2+/calmodulin-dependent protein kinase II site on RyR2 (S2814A) protects mutant mice from pacing-induced arrhythmias versus wild-type mice after transverse aortic constriction surgery. Conclusions— Our results suggest that Ca2+/calmodulin-dependent protein kinase II phosphorylation of RyR2 Ca2+ release channels at S2814 plays an important role in arrhythmogenesis and sudden cardiac death in mice with heart failure.

Pathogenesis of Lethal Cardiac Arrhythmias in Mecp2 Mutant Mice: Implication for Therapy in Rett Syndrome

Lethal ventricular arrhythmias in a mouse model of Rett syndrome can be prevented by phenytoin, which blocks a persistent sodium current. A Heart-Brain Connection Patients with Rett syndrome, usually girls, have many problems, including impaired brain function and cognition. One of the most unfortunate is the tendency of about 25% of these patients to die suddenly and unexpectedly, likely from cardiac problems. To get to the bottom of this, McCauley et al. examined heart physiology in mice carrying a mutation in methyl-CpG–binding protein 2 (MECP2), the gene that is defective in Rett syndrome. In addition to a Rett-like phenotype, these mice exhibited a long QT interval in their heartbeat tracings and had ventricular tachycardia, and some of the mice died of cardiac causes. Treatment with a common anticonvulsant, phenytoin, normalized these atypical heartbeats, suggesting that this treatment may help to prevent sudden death in patients with Rett syndrome. To determine the underlying cause of the dangerous heartbeats, the authors generated another strain of mice in which MECP2 was only mutated in the nervous system. These mice, which had normal heart MECP2, also showed abnormal heart beating and tachycardia, leading to the conclusion that the heart problems were actually secondary to nervous system deficits. A close look at the cardiac cells showed that even when they were removed from mice and grown in culture, an unusual persistent sodium current was apparent. This current decreased in the presence of phenytoin, consistent with its therapeutic action in the MECP2 mutant mice. The authors hypothesize that nervous system abnormalities cause remodeling of the heart in these patients, including elevation of a persistent sodium current, and suggest that sodium channel blockers such as phenytoin be tested as therapeutic agents. Rett syndrome patients often have recurrent seizures, and a similar situation may occur in patients with epilepsy from other causes. The oddly named SUDEP or “sudden unexpected death in epilepsy” is rare, but is thought also to result from frequent heartbeat abnormalities in these patients, which are similar to those in Rett patients. The connection between seizures and long QT intervals may transcend the exact nature of a patient’s disease. Rett syndrome is a neurodevelopmental disorder typically caused by mutations in methyl-CpG–binding protein 2 (MECP2) in which 26% of deaths are sudden and of unknown cause. To explore the hypothesis that these deaths may be due to cardiac dysfunction, we characterized the electrocardiograms in 379 people with Rett syndrome and found that 18.5% show prolongation of the corrected QT interval (QTc), an indication of a repolarization abnormality that can predispose to the development of an unstable fatal cardiac rhythm. Male mice lacking MeCP2 function, Mecp2Null/Y, also have prolonged QTc and show increased susceptibility to induced ventricular tachycardia. Female heterozygous null mice, Mecp2Null/+, show an age-dependent prolongation of QTc associated with ventricular tachycardia and cardiac-related death. Genetic deletion of MeCP2 function in only the nervous system was sufficient to cause long QTc and ventricular tachycardia, implicating neuronally mediated changes to cardiac electrical conduction as a potential cause of ventricular tachycardia in Rett syndrome. The standard therapy for prolonged QTc in Rett syndrome, β-adrenergic receptor blockers, did not prevent ventricular tachycardia in Mecp2Null/Y mice. To determine whether an alternative therapy would be more appropriate, we characterized cardiomyocytes from Mecp2Null/Y mice and found increased persistent sodium current, which was normalized when cells were treated with the sodium channel–blocking anti-seizure drug phenytoin. Treatment with phenytoin reduced both QTc and sustained ventricular tachycardia in Mecp2Null/Y mice. These results demonstrate that cardiac abnormalities in Rett syndrome are secondary to abnormal nervous system control, which leads to increased persistent sodium current. Our findings suggest that treatment in people with Rett syndrome would be more effective if it targeted the increased persistent sodium current to prevent lethal cardiac arrhythmias.

Animal models of arrhythmogenic cardiomyopathy

Arrhythmogenic cardiomyopathies are a heterogeneous group of pathological conditions that give rise to myocardial dysfunction with an increased risk for atrial or ventricular arrhythmias. Inherited defects in cardiomyocyte proteins in the sarcomeric contractile apparatus, the cytoskeleton and desmosomal cell-cell contact junctions are becoming recognized increasingly as major causes of sudden cardiac death in the general population. Animal models have been developed for the systematic dissection of the genetic pathways involved in the pathogenesis of arrhythmogenic cardiomyopathies. This review presents an overview of current animal models for arrhythmogenic right ventricular cardiomyopathy (ARVC), hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM) associated with cardiac arrhythmias and sudden cardiac death.

Expression and function of Kv1.1 potassium channels in human atria from patients with atrial fibrillation

Voltage-gated Kv1.1 channels encoded by the Kcna1 gene are traditionally regarded as being neural-specific with no known expression or intrinsic functional role in the heart. However, recent studies in mice reveal low-level Kv1.1 expression in heart and cardiac abnormalities associated with Kv1.1-deficiency suggesting that the channel may have a previously unrecognized cardiac role. Therefore, this study tests the hypothesis that Kv1.1 channels are associated with arrhythmogenesis and contribute to intrinsic cardiac function. In intra-atrial burst pacing experiments, Kcna1-null mice exhibited increased susceptibility to atrial fibrillation (AF). The atria of Kcna1-null mice showed minimal Kv1 family ion channel remodeling and fibrosis as measured by qRT-PCR and Masson’s trichrome histology, respectively. Using RT-PCR, immunocytochemistry, and immunoblotting, KCNA1 mRNA and protein were detected in isolated mouse cardiomyocytes and human atria for the first time. Patients with chronic AF (cAF) showed no changes in KCNA1 mRNA levels relative to controls; however, they exhibited increases in atrial Kv1.1 protein levels, not seen in paroxysmal AF patients. Patch-clamp recordings of isolated human atrial myocytes revealed significant dendrotoxin-K (DTX-K)-sensitive outward current components that were significantly increased in cAF patients, reflecting a contribution by Kv1.1 channels. The concomitant increases in Kv1.1 protein and DTX-K-sensitive currents in atria of cAF patients suggest that the channel contributes to the pathological mechanisms of persistent AF. These findings provide evidence of an intrinsic cardiac role of Kv1.1 channels and indicate that they may contribute to atrial repolarization and AF susceptibility.

Worsening renal function is not associated with response to treatment in acute heart failure

BACKGROUND About a fourth of acute decompensated heart failure (ADHF) patients develop renal dysfunction during their admission. To date, the association of ADHF treatment with the development of worsening renal function (WRF) remains contentious. Thus, we examined the association of WRF with changes in BNP levels and with mortality. METHODS We performed retrospective chart review of patients admitted with ADHF who had BNP, eGFR, creatinine and blood urea nitrogen (BUN) values measured both on admission and discharge. Survival analysis was conducted using Cox proportional hazards model and correlation was measured using Spearmans rank correlation test. RESULTS 358 patients admitted for ADHF were evaluated. WRF was defined as >20% reduction in eGFR from admission to discharge and response to treatment was assessed by ΔBNP. There was a statistically significant reduction in BNP and increase in BUN during the admission. ΔBNP did not correlate with either ΔGFR or ΔBUN. Patients who developed WRF and those who did not, had a similar reduction in BNP. On univariate survival analysis, ΔBUN, but not ΔeGFR, was associated with 1-year mortality. In multivariate Cox proportional hazards model, BUN at discharge was associated with 1-year mortality (HR: 1.02, p<0.001), but ΔeGFR and ΔBUN were not associated with the primary endpoint. CONCLUSION During ADHF treatment, ΔBNP was not associated with changes in renal function. Development of WRF during ADHF treatment was not associated with mortality. Our study suggests that development of WRF should not preclude diuresis in ADHF patients in the absence of volume depletion.

Ryanodine Receptor Phosphorylation, Calcium/Calmodulin-dependent Protein Kinase II, and Life-threatening Ventricular Arrhythmias

Ryanodine receptor (RyR2) dysfunction, which may result from a variety of mechanisms, has been implicated in the pathogenesis of cardiac arrhythmias and heart failure. In this review, we discuss the important role of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) in the regulation of RyR2-mediated Ca(2+) release. In particular, we examine how pathological activation of CaMKII can lead to an increased risk of sudden arrhythmic death. Finally, we discuss how reduction of CaMKII-mediated RyR2 hyperactivity might reduce the risk of arrhythmias and may serve as a rationale for future pharmacotherapeutic approaches.

Ambulatory ECG Recording in Mice

Telemetric ECG recording in mice is essential to understanding the mechanisms behind arrhythmias, conduction disorders, and sudden cardiac death. Although the surface ECG is utilized for short-term measurements of waveform intervals, it is not practical for long-term studies of heart rate variability or the capture of rare episodes of arrhythmias. Implantable ECG telemeters offer the advantages of simple surgical implantation, long-term recording of electrograms in ambulatory mice, and scalability with simultaneous recordings of multiple animals. Here, we present a step-by-step guide to the implantation of telemeters for ambulatory ECG recording in mice. Careful adherence to aseptic technique is required for favorable survival results with the possibility of implantation and recording from weeks to months. Thus, implantable ECG telemetry is a valuable tool for detection of critical information on cardiac electrophysiology in ambulatory animal models such as the mouse.

SPEG (Striated Muscle Preferentially Expressed Protein Kinase) Is Essential for Cardiac Function by Regulating Junctional Membrane Complex Activity

Rationale: Junctional membrane complexes (JMCs) in myocytes are critical microdomains, in which excitation–contraction coupling occurs. Structural and functional disruption of JMCs underlies contractile dysfunction in failing hearts. However, the role of newly identified JMC protein SPEG (striated muscle preferentially expressed protein kinase) remains unclear. Objective: To determine the role of SPEG in healthy and failing adult hearts. Methods and Results: Proteomic analysis of immunoprecipitated JMC proteins ryanodine receptor type 2 and junctophilin-2 (JPH2) followed by mass spectrometry identified the serine–threonine kinase SPEG as the only novel binding partner for both proteins. Real-time polymerase chain reaction revealed the downregulation of SPEG mRNA levels in failing human hearts. A novel cardiac myocyte-specific Speg conditional knockout (MCM-Spegfl/fl) model revealed that adult-onset SPEG deficiency results in heart failure (HF). Calcium (Ca2+) and transverse-tubule imaging of ventricular myocytes from MCM-Spegfl/fl mice post HF revealed both increased sarcoplasmic reticulum Ca2+ spark frequency and disrupted JMC integrity. Additional studies revealed that transverse-tubule disruption precedes the development of HF development in MCM-Spegfl/fl mice. Although total JPH2 levels were unaltered, JPH2 phosphorylation levels were found to be reduced in MCM-Spegfl/fl mice, suggesting that loss of SPEG phosphorylation of JPH2 led to transverse-tubule disruption, a precursor of HF development in SPEG-deficient mice. Conclusions: The novel JMC protein SPEG is downregulated in human failing hearts. Acute loss of SPEG in mouse hearts causes JPH2 dephosphorylation and transverse-tubule loss associated with downstream Ca2+ mishandling leading to HF. Our study suggests that SPEG could be a novel target for the treatment of HF.

Ryanodine Receptor Phosphorylation by CaMKII Promotes Life-Threatening Ventricular Arrhythmias in Mice with Heart Failure

Background— Approximately half of patients with heart failure die suddenly as a result of ventricular arrhythmias. Although abnormal Ca2+ release from the sarcoplasmic reticulum through ryanodine receptors (RyR2) has been linked to arrhythmogenesis, the molecular mechanisms triggering release of arrhythmogenic Ca2+ remain unknown. We tested the hypothesis that increased RyR2 phosphorylation by Ca2+/calmodulin-dependent protein kinase II is both necessary and sufficient to promote lethal ventricular arrhythmias. Methods and Results— Mice in which the S2814 Ca2+/calmodulin-dependent protein kinase II site on RyR2 is constitutively activated (S2814D) develop pathological sarcoplasmic reticulum Ca2+ release events, resulting in reduced sarcoplasmic reticulum Ca2+ load on confocal microscopy. These Ca2+ release events are associated with increased RyR2 open probability in lipid bilayer preparations. At baseline, young S2814D mice have structurally and functionally normal hearts without arrhythmias; however, they develop sustained ventricular tachycardia and sudden cardiac death on catecholaminergic provocation by caffeine/epinephrine or programmed electric stimulation. Young S2814D mice have a significant predisposition to sudden arrhythmogenic death after transverse aortic constriction surgery. Finally, genetic ablation of the Ca2+/calmodulin-dependent protein kinase II site on RyR2 (S2814A) protects mutant mice from pacing-induced arrhythmias versus wild-type mice after transverse aortic constriction surgery. Conclusions— Our results suggest that Ca2+/calmodulin-dependent protein kinase II phosphorylation of RyR2 Ca2+ release channels at S2814 plays an important role in arrhythmogenesis and sudden cardiac death in mice with heart failure.