Shu Nakao

The Effect of Pimobendan on Left Atrial Pressure in Dogs with Mitral Valve Regurgitation

BACKGROUND The effects of pimobendan on left atrial pressure (LAP) in dogs with mitral valve disease (MR) have not been documented in a quantitative manner. OBJECTIVE The objective was to document and study the short-term effects of pimobendan on LAP and echocardiographic parameters in MR dogs. ANIMALS Eight healthy Beagle dogs weighing 10.0-14.7 kg (3 males and 5 females; aged 2 years) were used. METHODS Experimental, cross-over, and interventional study. Dogs with surgically induced MR received pimobendan at either 0.25 mg/kg or 0.50 mg/kg p.o. q12h for 7 days and then, after a 7-day wash-out period, the other dosage. LAP was measured for 30 minutes at baseline and again on days 1, 2, 4, and 7 of pimobendan administration. RESULTS Mean LAP was significantly decreased after the administration of 0.25 mg/kg (15.81 ± 5.44 mmHg to 12.67 ± 5.71 mmHg, P < .001) and 0.50 mg/kg (15.76 ± 5.45 mmHg to 10.77 ± 5.23 mmHg, P < .001). Also, the 0.50 mg/kg group led to a significantly lower LAP (P < .01) compared with the 0.25 mg/kg group. Significant reduction was seen for the first time 4 days after the administration of 0.25 mg/kg and a day after the administration of 0.50 mg/kg. CONCLUSIONS AND CLINICAL IMPORTANCE Pimobendan decreased LAP in a dose-dependent manner in dogs with acute MR caused by experimental chordal rupture. This study did not evaluate adverse effects of high-dose pimobendan, and additional studies in clinical patients are warranted.

Stimulus-dependent regulation of nuclear Ca2+ signaling in cardiomyocytes: a role of neuronal calcium sensor-1.

In cardiomyocytes, intracellular calcium (Ca2+) transients are elicited by electrical and receptor stimulations, leading to muscle contraction and gene expression, respectively. Although such elevations of Ca2+levels ([Ca2+]) also occur in the nucleus, the precise mechanism of nuclear [Ca2+] regulation during different kinds of stimuli, and its relationship with cytoplasmic [Ca2+] regulation are not fully understood. To address these issues, we used a new region-specific fluorescent protein-based Ca2+ indicator, GECO, together with the conventional probe Fluo-4 AM. We confirmed that nuclear Ca2+ transients were elicited by both electrical and receptor stimulations in neonatal mouse ventricular myocytes. Kinetic analysis revealed that electrical stimulation-elicited nuclear Ca2+ transients are slower than cytoplasmic Ca2+ transients, and chelating cytoplasmic Ca2+ abolished nuclear Ca2+ transients, suggesting that nuclear Ca2+ are mainly derived from the cytoplasm during electrical stimulation. On the other hand, receptor stimulation such as with insulin-like growth factor-1 (IGF-1) preferentially increased nuclear [Ca2+] compared to cytoplasmic [Ca2+]. Experiments using inhibitors revealed that electrical and receptor stimulation-elicited Ca2+ transients were mainly mediated by ryanodine receptors and inositol 1,4,5-trisphosphate receptors (IP3Rs), respectively, suggesting different mechanisms for the two signals. Furthermore, IGF-1-elicited nuclear Ca2+ transient amplitude was significantly lower in myocytes lacking neuronal Ca2+ sensor-1 (NCS-1), a Ca2+ binding protein implicated in IP3R-mediated pathway in the heart. Moreover, IGF-1 strengthened the interaction between NCS-1 and IP3R. These results suggest a novel mechanism for receptor stimulation-induced nuclear [Ca2+] regulation mediated by IP3R and NCS-1 that may further fine-tune cardiac Ca2+ signal regulation.

Targeting miR-423-5p Reverses Exercise Training–Induced HCN4 Channel Remodeling and Sinus Bradycardia

Rationale: Downregulation of the pacemaking ion channel, HCN4 (hyperpolarization-activated cyclic nucleotide gated channel 4), and the corresponding ionic current, If, underlies exercise training–induced sinus bradycardia in rodents. If this occurs in humans, it could explain the increased incidence of bradyarrhythmias in veteran athletes, and it will be important to understand the underlying processes. Objective: To test the role of HCN4 in the training-induced bradycardia in human athletes and investigate the role of microRNAs (miRs) in the repression of HCN4. Methods and Results: As in rodents, the intrinsic heart rate was significantly lower in human athletes than in nonathletes, and in all subjects, the rate-lowering effect of the HCN selective blocker, ivabradine, was significantly correlated with the intrinsic heart rate, consistent with HCN repression in athletes. Next-generation sequencing and quantitative real-time reverse transcription polymerase chain reaction showed remodeling of miRs in the sinus node of swim-trained mice. Computational predictions highlighted a prominent role for miR-423-5p. Interaction between miR-423-5p and HCN4 was confirmed by a dose-dependent reduction in HCN4 3′-untranslated region luciferase reporter activity on cotransfection with precursor miR-423-5p (abolished by mutation of predicted recognition elements). Knockdown of miR-423-5p with anti-miR-423-5p reversed training-induced bradycardia via rescue of HCN4 and If. Further experiments showed that in the sinus node of swim-trained mice, upregulation of miR-423-5p (intronic miR) and its host gene, NSRP1, is driven by an upregulation of the transcription factor Nkx2.5. Conclusions: HCN remodeling likely occurs in human athletes, as well as in rodent models. miR-423-5p contributes to training-induced bradycardia by targeting HCN4. This work presents the first evidence of miR control of HCN4 and heart rate. miR-423-5p could be a therapeutic target for pathological sinus node dysfunction in veteran athletes.

Cor triatriatum sinister with incomplete atrioventricular septal defect in a cat.

An 11-month-old, 3 kg, female domestic shorthair cat was referred to evaluate cardiac structure and function. Echocardiography revealed the membrane dividing the left atrium into two chambers, a large defect in the lower part of the atrial septum, and turbulent blood flow from the distal left atrium into the right atrium. These findings suggested cor triatriatum sinister (CTS) with incomplete atrioventricular septal defect (AVSD). The cat was treated with medications for management of congestive heart failure. In the end, she died from right-sided heart failure 17 months after the initial presentation. At necropsy, a fibromuscular membrane with a round orifice in the left atrium and an ostium primum defect were confirmed, and the definitive diagnosis of CTS with incomplete AVSD was made. To our knowledge, this study presents the first case report of CTS with incomplete AVSD in a cat.

Possible signaling pathways mediating neuronal calcium sensor-1-dependent spatial learning and memory in Mice

Intracellular Ca2+ signaling regulates diverse functions of the nervous system. Many of these neuronal functions, including learning and memory, are regulated by neuronal calcium sensor-1 (NCS-1). However, the pathways by which NCS-1 regulates these functions remain poorly understood. Consistent with the findings of previous reports, we revealed that NCS-1 deficient (Ncs1-/-) mice exhibit impaired spatial learning and memory function in the Morris water maze test, although there was little change in their exercise activity, as determined via treadmill-analysis. Expression of brain-derived neurotrophic factor (BDNF; a key regulator of memory function) and dopamine was significantly reduced in the Ncs1-/- mouse brain, without changes in the levels of glial cell-line derived neurotrophic factor or nerve growth factor. Although there were no gross structural abnormalities in the hippocampi of Ncs1-/- mice, electron microscopy analysis revealed that the density of large dense core vesicles in CA1 presynaptic neurons, which release BDNF and dopamine, was decreased. Phosphorylation of Ca2+/calmodulin-dependent protein kinase II-α (CaMKII-α, which is known to trigger long-term potentiation and increase BDNF levels, was significantly reduced in the Ncs1-/- mouse brain. Furthermore, high voltage electric potential stimulation, which increases the levels of BDNF and promotes spatial learning, significantly increased the levels of NCS-1 concomitant with phosphorylated CaMKII-α in the hippocampus; suggesting a close relationship between NCS-1 and CaMKII-α. Our findings indicate that NCS-1 may regulate spatial learning and memory function at least in part through activation of CaMKII-α signaling, which may directly or indirectly increase BDNF production.

The anatomical basis of bradycardia-tachycardia syndrome in elderly dogs with chronic degenerative valvular disease.

The hearts of seven elderly dogs in which bradycardia-tachycardia syndrome (BTS) had been diagnosed electrocardiographically were examined post mortem. The clinical basis of the underlying heart disease was invariably mitral or mitral and tricuspid regurgitation. Microscopical examination of the sinoatrial (SA) node and the SA junctional region consistently revealed depletion of SA nodal cells, with a corresponding increase in fibrous or fibro-fatty tissue that interrupted contiguity between the SA node and the surrounding atrial myocardium. The left and right atrial walls showed an increased amount of fibrous tissue in the myocardium and disruption of the muscle bundle architecture (interstitial myocardial fibrosis) to varying degrees. Qualitatively, these changes in the SA node and the SA node region resembled those associated with ageing in elderly people with or without BTS. Thus, it is possible that the pathological process affecting the SA node in these dogs was fundamentally related to ageing and may have caused BTS, in combination with atrial myocardial lesions caused by mitral and tricuspid regurgitation.

Point: Exercise training-induced bradycardia is caused by changes in intrinsic sinus node function

it is well known that athletes have a resting sinus bradycardia—their resting heart rate can be half the normal value ([7][1])— and this is normally attributed to high vagal tone ([3][2]). This is a logical assumption, because we have known since 1921 from the work of the physiologist Otto Loewi

Neuronal Ca(2+) sensor-1 contributes to stress tolerance in cardiomyocytes via activation of mitochondrial detoxification pathways.

Identification of the molecules involved in cell death/survival pathways is important for understanding the mechanisms of cell loss in cardiac disease, and thus is clinically relevant. Ca2+-dependent signals are often involved in these pathways. Here, we found that neuronal Ca2+-sensor-1 (NCS-1), a Ca2+-binding protein, has an important role in cardiac survival during stress. Cardiomyocytes derived from NCS-1-deficient (Ncs1-/-) mice were more susceptible to oxidative and metabolic stress than wild-type (WT) myocytes. Cellular ATP levels and mitochondrial respiration rates, as well as the levels of mitochondrial marker proteins, were lower in Ncs1-/- myocytes. Although oxidative stress elevated mitochondrial proton leak, which exerts a protective effect by inhibiting the production of reactive oxygen species in WT myocytes, this response was considerably diminished in Ncs1-/- cardiomyocytes, and this would be a major reason for cell death. Consistently, H2O2-induced loss of mitochondrial membrane potential, a critical early event in cell death, was accelerated in Ncs1-/- myocytes. Furthermore, NCS-1 was upregulated in hearts subjected to ischemia-reperfusion, and ischemia-reperfusion injury was more severe in Ncs1-/- hearts. Activation of stress-induced Ca2+-dependent survival pathways, such as Akt and PGC-1α (which promotes mitochondrial biogenesis and function), was diminished in Ncs1-/- hearts. Overall, these data demonstrate that NCS-1 contributes to stress tolerance in cardiomyocytes at least in part by activating certain Ca2+-dependent survival pathways that promote mitochondrial biosynthesis/function and detoxification pathways.

Overexpression of heart-specific small subunit of myosin light chain phosphatase results in heart failure and conduction disturbance

Mutations in genes encoding components of the sarcomere cause cardiomyopathy, which is often associated with abnormal Ca2+ sensitivity of muscle contraction. We have previously shown that a heart-specific myosin light chain phosphatase small subunit (hHS-M21) increases the Ca2+ sensitivity of muscle contraction. The aim of the present study was to investigate the function of hHS-M21 in vivo and the causative role of abnormal Ca2+ sensitivity in cardiomyopathy. We generated transgenic mice with cardiac-specific overexpression of hHS-M21. We confirmed that hHS-M21 increased the Ca2+ sensitivity of cardiac muscle contraction in vivo, which was not followed by an increased phosphorylation of myosin light chain 2 isoforms. hHS-M21 transgenic mice developed severe systolic dysfunction with myocardial fibrosis and degeneration of cardiomyocytes in association with sinus bradycardia and atrioventricular conduction defect. The contractile dysfunction and cardiac fibrosis were improved by treatment with the Rho kinase inhibitor fasudil. Our findings suggested that the overexpression of hHS-M21 results in cardiac dysfunction and conduction disturbance via non-myosin light chain 2 phosphorylation-dependent regulation. NEW & NOTEWORTHY The present study is the first to develop mice with transgenic overexpression of a heart-specific myosin light chain phosphatase small subunit (hHS-M21) and to examine the effects of hHS-M21 on cardiac function. Elevation of hHS-M21 induced heart failure with myocardial fibrosis and degeneration of cardiomyocytes accompanied by supraventricular arrhythmias.

Rebuttal from Boyett et al.

Karl Popper ([7][1]) maintained that the job of the scientist is to erect hypotheses and then attempt to destroy them by testing, and this is what we have been doing. However, the problem is that the concept of high vagal tone in the athlete (and also at night time—another story) and the use of