Cytoskeletal disarray increases arrhythmogenic vulnerability during sympathetic stimulation in a model of hypertrophic cardiomyopathy.

Familial hypertrophic cardiomyopathy (FHC) patients are advised to avoid strenuous exercise due to increased risk of arrhythmias. Mice expressing the human FHC-causing mutation R403Q in the myosin heavy chain gene (MYH6) recapitulate the human phenotype, including cytoskeletal disarray and increased arrhythmia susceptibility. Following in vivo administration of isoproterenol, mutant mice exhibited tachyarrhythmias, poor recovery and fatigue. Arrhythmias were attenuated with the ß-blocker atenolol and protein kinase A inhibitor PKI. Mutant cardiac myocytes had significantly prolonged action potentials and triggered automaticity due to reduced repolarization reserve and connexin 43 expression. Isoproterenol shortened cycle length, and escalated electrical instability. Surprisingly isoproterenol did not increase CaV1.2 current. We found alterations in CaV1.2-ß1 adrenergic receptor colocalization assessed using super-resolution nanoscopy, and increased CaV1.2 phosphorylation in mutant hearts. Our results reveal for the first time that altered ion channel expression, co-localization and ß-adrenergic receptor signaling associated with myocyte disarray contribute to electrical instability in the R403Q mutant heart.

Haemoconcentration, not decreased blood temperature, increases blood viscosity during cold water immersion.

INTRODUCTION: Prolonged cold-water immersion (CWI) has the potential to cause significant hypothermia and haemoconcentration; both of which have previously been shown to independently increase blood viscosity in vitro. The purpose of this study was to determine the effect of CWI on blood viscosity and examine the relative contribution of decreased blood temperature and haemoconcentration. METHODS: Ten healthy volunteers were immersed to mid-sternum in 10°C water for 90 minutes. Gastrointestinal (GI) temperature, haematocrit (Hct), and blood viscosity were measured pre- and post-CWI. RESULTS: CWI caused mean (SD) GI temperature to decrease from 37.5 (0.3)°C to 36.2 (0.7)°C (P < 0.05). CWI also caused mean Hct to increase from 40.0 (3.5)% to 45.0 (2.9)% (P < 0.05). As a result of the haemoconcentration and decreased GI temperature during CWI the mean blood viscosity increased by 19% from 2.80 (0.28) mPa·s⁻¹ to 3.33 (0.42) mPa·s⁻¹ (P < 0.05). However, when the pre-CWI blood sample was measured at the post-CWI GI temperature (36.2°C) there was no significant difference in the blood viscosity when compared to the pre-CWI (37.5°C) blood sample (2.82 (0.20) mPa·s-1 and 2.80 (0.28) mPa·s-1 respectively). Furthermore, the changes in Hct and blood viscosity during CWI were significantly correlated with an r = 0.84. CONCLUSION: The results of the current study show that prolonged, severe CWI causes a significant 19% increase in blood viscosity. In addition, the results strongly suggest that almost all of the increased blood viscosity seen following CWI is the result of haemoconcentration, not decreased blood temperature.

ß-adrenergic-mediated dynamic augmentation of sarcolemmal CaV 1.2 clustering and co-operativity in ventricular myocytes.

KEY POINTS: Prevailing dogma holds that activation of the ß-adrenergic receptor/cAMP/protein kinase A signalling pathway leads to enhanced L-type CaV 1.2 channel activity, resulting in increased Ca2+ influx into ventricular myocytes and a positive inotropic response. However, the full mechanistic and molecular details underlying this phenomenon are incompletely understood. CaV 1.2 channel clusters decorate T-tubule sarcolemmas of ventricular myocytes. Within clusters, nanometer proximity between channels permits Ca2+ -dependent co-operative gating behaviour mediated by physical interactions between adjacent channel C-terminal tails. We report that stimulation of cardiomyocytes with isoproterenol, evokes dynamic, protein kinase A-dependent augmentation of CaV 1.2 channel abundance along cardiomyocyte T-tubules, resulting in the appearance of channel 'super-clusters', and enhanced channel co-operativity that amplifies Ca2+ influx. On the basis of these data, we suggest a new model in which a sub-sarcolemmal pool of pre-synthesized CaV 1.2 channels resides in cardiomyocytes and can be mobilized to the membrane in times of high haemodynamic or metabolic demand, to tune excitation-contraction coupling. ABSTRACT: Voltage-dependent L-type CaV 1.2 channels play an indispensable role in cardiac excitation-contraction coupling. Activation of the ß-adrenergic receptor (ßAR)/cAMP/protein kinase A (PKA) signalling pathway leads to enhanced CaV 1.2 activity, resulting in increased Ca2+ influx into ventricular myocytes and a positive inotropic response. CaV 1.2 channels exhibit a clustered distribution along the T-tubule sarcolemma of ventricular myocytes where nanometer proximity between channels permits Ca2+ -dependent co-operative gating behaviour mediated by dynamic, physical, allosteric interactions between adjacent channel C-terminal tails. This amplifies Ca2+ influx and augments myocyte Ca2+ transient and contraction amplitudes. We investigated whether ßAR signalling could alter CaV 1.2 channel clustering to facilitate co-operative channel interactions and elevate Ca2+ influx in ventricular myocytes. Bimolecular fluorescence complementation experiments reveal that the ßAR agonist, isoproterenol (ISO), promotes enhanced CaV 1.2-CaV 1.2 physical interactions. Super-resolution nanoscopy and dynamic channel tracking indicate that these interactions are expedited by enhanced spatial proximity between channels, resulting in the appearance of CaV 1.2 'super-clusters' along the z-lines of ISO-stimulated cardiomyocytes. The mechanism that leads to super-cluster formation involves rapid, dynamic augmentation of sarcolemmal CaV 1.2 channel abundance after ISO application. Optical and electrophysiological single channel recordings confirm that these newly inserted channels are functional and contribute to overt co-operative gating behaviour of CaV 1.2 channels in ISO stimulated myocytes. The results of the present study reveal a new facet of ßAR-mediated regulation of CaV 1.2 channels in the heart and support the novel concept that a pre-synthesized pool of sub-sarcolemmal CaV 1.2 channel-containing vesicles/endosomes resides in cardiomyocytes and can be mobilized to the sarcolemma to tune excitation-contraction coupling to meet metabolic and/or haemodynamic demands.

Cold Acclimation Does Not Alter Physiological or Perceptual Responses During Subsequent Exercise in the Heat.

INTRODUCTION: Warfighters often train and conduct operations in cold environments. Specifically, military trainees and divers that are repeatedly exposed to cold water may experience inadvertent cold acclimatization, which results in body heat retention. These same warfighters can quickly switch between environments (cold to hot or hot to cold) given the nature of their work. This may present a risk of early onset of hyperthermia when cold-acclimatized warfighters are subsequently exposed to physiological insults that increase body temperature, such as exercise and heat stress. However, there is currently no evidence that suggests this is the case. The purpose of this work, therefore, is to determine what impact, if any, repeated immersion in cold water has on subsequent exercise in the heat. MATERIALS AND METHODS: Twelve healthy subjects (values in mean ± SD: age, 25.6 ± 5.2 years; height, 174.0 ± 8.9 cm; weight, 75.6 ± 13.1 kg) voluntarily provided written informed consent in accordance with the San Diego State University Institutional Review Board. They first completed 120 minutes of moderate treadmill walking in 40°C and 40% relative humidity. During this trial, subjects' physiological and perceptual responses were recorded. Twenty-four hours later, subjects began a cold acclimation protocol, which consisted of seven, 90-minute immersions in cold water (10°C, water level to chest). Each immersion was also separated by 24 hours. Subjects then repeated a subsequent trial of exercise in the heat 24 hours after the final immersion of the cold acclimation protocol. RESULTS: Results from cold acclimation revealed no change in core temperature, a decrease in skin temperature, and attenuated shivering and lactate responses, which supports a successful insulative-hypothermic cold acclimation response. This type of cold acclimation response primarily results in heat retention with associated energy conservation. Findings for heat trials (pre-cold acclimation and post-cold acclimation) revealed no differences between trials for all measurements, suggesting that cold acclimation did not influence physiological or perceptual responses during exercise in the heat. CONCLUSION: Our findings indicate that military divers or trainees that are frequently exposed to cold water, and hence have the ability to experience cold acclimatization, will likely not be at greater risk of increased thermal strain when subsequently exposed to physical activity in hot environments. In this study, no physiological or perceptual differences were observed between trials before and after cold acclimation, suggesting that cold acclimation does not present a greater hyperthermia risk during subsequent exercise in the heat.