Tristan W. Harding

Cardiac CaMKIIδ splice variants exhibit target signaling specificity and confer sex-selective arrhythmogenic actions in the ischemic-reperfused heart

BACKGROUND Ischemia-related arrhythmic incidence is generally lower in females (vs males), though risk is selectively increased in women with underlying cardiopathology. Ca(2+)/calmodulin dependent kinase II (CaMKII) has been implicated in ischemia/reperfusion arrhythmias, yet the role of CaMKII in the ischemic female heart has not been determined. The aim of this study was to define the role and molecular mechanism of CaMKII activation in reperfusion arrhythmias in male/female hearts. METHODS AND RESULTS Male and female rat hearts and cardiomyocytes were subjected to multiple arrhythmogenic challenges. An increased capacity to upregulate autophosphorylated CaMKII (P-CaMKII) in Ca(2+)-challenged female hearts was associated with an enhanced ability to maintain diastolic function. In ischemia/reperfusion, female hearts (vs male) exhibited less arrhythmias (59 ± 18 vs 548 ± 9, s, p<0.05), yet had augmented P-CaMKII (2.69 ± 0.30 vs 1.50 ± 0.14, rel. units, p<0.05) and downstream phosphorylation of phospholamban (1.71 ± 0.42 vs 0.90 ± 0.10, p<0.05). In contrast, hypertrophic female hearts had more reperfusion arrhythmias and lower phospholamban phosphorylation. Isolated myocyte experiments (fura-2) confirmed Ca(2+)-handling arrhythmogenic involvement. Molecular analysis showed target specificity of CaMKII was determined by post-translational modification, with CaMKIIδB and CaMKIIδC splice variants selectively co-localized with autophosphorylation and oxidative modifications of CaMKII respectively. CONCLUSIONS This study provides new mechanistic evidence that CaMKIIδ splice variants are selectively susceptible to autophosphorylation/oxidation, and that augmented generation of P-CaMKIIδB(Thr287) is associated with arrhythmia suppression in the female heart. Collectively these findings indicate that therapeutic approaches based on selective CaMKII splice form targeting may have potential benefit, and that sex-selective CaMKII intervention strategies may be valid.

Cardiomyocyte Functional Etiology in Heart Failure With Preserved Ejection Fraction Is Distinctive—A New Preclinical Model

Background Among the growing numbers of patients with heart failure, up to one half have heart failure with preserved ejection fraction (HFpEF). The lack of effective treatments for HFpEF is a substantial and escalating unmet clinical need—and the lack of HFpEF‐specific animal models represents a major preclinical barrier in advancing understanding of HFpEF. As established treatments for heart failure with reduced ejection fraction (HFrEF) have proven ineffective for HFpEF, the contention that the intrinsic cardiomyocyte phenotype is distinct in these 2 conditions requires consideration. Our goal was to validate and characterize a new rodent model of HFpEF, undertaking longitudinal investigations to delineate the associated cardiac and cardiomyocyte pathophysiology. Methods and Results The selectively inbred Hypertrophic Heart Rat (HHR) strain exhibits adult cardiac enlargement (without hypertension) and premature death (40% mortality at 50 weeks) compared to its control strain, the normal heart rat. Hypertrophy was characterized in vivo by maintained systolic parameters (ejection fraction at 85%–90% control) with marked diastolic dysfunction (increased E/E′). Surprisingly, HHR cardiomyocytes were hypercontractile, exhibiting high Ca2+ operational levels and markedly increased L‐type Ca2+ channel current. In HHR, prominent regions of reparative fibrosis in the left ventricle free wall adjacent to the interventricular septum were observed. Conclusions Thus, the cardiomyocyte remodeling process in the etiology of this HFpEF model contrasts dramatically with the suppressed Ca2+ cycling state that typifies heart failure with reduced ejection fraction. These findings may explain clinical observations, that treatments considered appropriate for heart failure with reduced ejection fraction are of little benefit for HFpEF—and suggest a basis for new therapeutic strategies.

1062 INCREASED ARRHYTHMOGENESIS IN HYPERTROPHIC FEMALE CARDIOMYOCYTES – IS THERE A CAUSATIVE ROLE FOR CaMKII?

Background: Relative mortality risk associated with hypertrophy (and hypertension) is accentuated in women, yet knowledge of specific causes/mechanisms is lacking. Ca2+/calmodulin-dependent protein kinase II (CaMKII) can mediate cardiac hypertrophy and ischemia/reperfusion arrhythmias in males. Despite marked sex differences in cardiomyocyte Ca2+-handling, the sex-specificity of CaMKII involvement remains undefined. The aim of this study was to discern the role of CaMKII in female ischemic myocardium, and assess the impact of primary cardiac hypertrophy on female arrhythmogenic vulnerability. Methods: Control rat hearts (male/female) were Langendorff-perfused and subjected to global ischemia/reperfusion (25mins/10mins; n = 8). Hearts were treated with a CaMKII inhibitor (KN93, 0.5 &mgr;M) 10mins immediately before/after ischemia (n = 7-9), and left ventricular function measured (intraventricular balloon). Isolated cardiomyocytes from female Hypertrophic Heart Rat (HHR) and Normal Heart Rat (NHR) hearts were superfused (4 Hz, 2.0 mM Ca2+, 37C) and treated with 10 nM isoproterenol (5mins). Pacing was ceased and spontaneous contraction measured for 30secs. Results: Arrhythmogenesis (ventricular tachycardia/fibrillation) was reduced in reperfused female hearts, and was unresponsive to CaMKII inhibition – contrasting with males (untreated vs KN93, duration secs; male 553 ± 11 vs 305 ± 72*, female 96 ± 40* vs 117 ± 51*; p < 0.05 vs male). Female arrhythmogenesis was increased despite augmented phospholamban Thr17 (CaMKII-specific residue) phosphorylation and CaMKII Thr287 auto-phosphorylation in females. HHR-derived isolated cardiomyocytes exhibited more frequent (HHR vs NHR, beats; 13.4 + 1.9 vs 8.5 + 1.2, p < 0.05) and earlier occurring (1.6 + 0.4 vs 3.7 + 0.9, p < 0.05) spontaneous activity compared with NHR when isoproterenol challenged. Conclusions: These data indicate CaMKII activation/actions differ in female/male myocardium, and may be further modulated when cells have an underlying genetic hypertrophic pathology.

422 DIFFERENTIAL Ca2+ DYSREGULATION AND CONTRACTILITY IN AGED MALE AND FEMALE HYPERTROPHIC CARDIOMYOCYTES

Background: Prior to menopause, females are less likely to develop cardiac hypertrophy than males. Post menopause the incidence of hypertrophy is increased with females displaying overall worse outcomes than males. The aim of this study was to characterize cardiomyocyte contractility and intracellular Ca2+ differences in adult and aged male and female rats exhibiting a genetically determined cardiac enlargement which predisposes for premature mortality. Design and Methods: Cardiomyocytes isolated by collagenase digestion of adult (12–16 weeks) and aged (52–60 weeks) HHR (Hypertrophic Heart Rat) and NHR (Normal Heart Rat) were fura 2-AM loaded. Single myocyte contractility and [Ca2+]i were measured by edge-detection and microfluorimetry under basal physiologic conditions (4 Hz, 2.0 mM Ca2+, 37°C). Results: Male and female HHR myocytes were significantly longer than NHR myocytes in both young adult (M:127.6 ± 7 vs 116.1 ± 4; F:131.0 ± 5 vs 101.4 ± 3 &mgr;m) and aged groups (M:136.3 ± 5 vs 111.6 ± 4; F:139.6 ± 5 vs 115.9 ± 6 &mgr;m). Basal contractility was higher in male and female adult HHR myocytes compared with NHR (M: 65% increase; F: 61% increase). In aged animals hypercontractility was apparent in the HHR males but not females (M 132% increase; F 4% increase). Similarly, Ca2+ transient amplitude was increased in adult HHR male and female myocytes compared with NHR (M: 153% increase; F: 80% increase) but not in aged females. Conclusions: These findings indicate that genetic cardiac hypertrophy is associated with hypercontractility and altered Ca2+ homeostasis in young male and female adult rats. In aged females hypercontractility is not sustained suggesting earlier decompensation and failure in hypertrophy.