Investigating cardiac responses to ischemia/reperfusion injury: insights into mineralocorticoid receptor and sex-specific responses

Abstract

This thesis is presented in the form of one peer reviewed publication and one chapter presented in the form of a submitted manuscript, abstracted below. Publication abstract (Chapter 2): Loss of mineralocorticoid receptor signaling selectively in cardiomyocytes can ameliorate cardiac fibrotic and inflammatory responses caused by excess mineralocorticoids. The aim of this study was to characterize the role of cardiomyocyte mineralocorticoid receptor signaling in ischemia–reperfusion injury and recovery and to identify a role of mineralocorticoid receptor modulation of cardiac function. Wild-type and cardiomyocyte mineralocorticoid receptor knockout mice (8 weeks) were uninephrectomized and maintained on (1) high salt (0.9% NaCl, 0.4% KCl) or (2) high salt plus deoxycorticosterone pellet (0.3 mg/d, 0.9% NaCl, 0.4% KCl). After 8 weeks of treatment, hearts were isolated and subjected to 20 minutes of global ischemia plus 45 minutes of reperfusion. Mineralocorticoid excess increased peak contracture during ischemia regardless of genotype. Recovery of left ventricular developed pressure and rates of contraction and relaxation post ischemia–reperfusion were greater in knockout versus wild-type hearts. The incidence of arrhythmic activity during early reperfusion was significantly higher in wild-type than in knockout hearts. Levels of autophosphorylated Ca2+/calmodulin protein kinase II (Thr287) were elevated in hearts from wild-type versus knockout mice and associated with increased sodium hydrogen exchanger-1 expression. These findings demonstrate that cardiomyocyte-specific mineralocorticoid receptor–dependent signaling contributes to electromechanical vulnerability in acute ischemia–reperfusion via a mechanism involving Ca2+/calmodulin protein kinase II activation in association with upstream alteration in expression regulation of the sodium hydrogen exchanger-1.

Chapter abstract (Chapter 3): Nitric oxide (NO) is an important regulator of cardiac function and plays a key role in ischemic cardioprotection. The role of chronic NO deficiency in coordinating ischemic vulnerability in female myocardium has not been established. The aim of this study was to determine the influence of chronic in vivo NO synthase inhibition in modulating ex vivo ischemia-reperfusion responses in female hearts (relative to males). Mice were subjected to L-NAME (L-NG-Nitroarginine-methyl-ester) treatment in vivo for 8 weeks. Cardiac fibrotic, inflammatory and cardiomyocyte Ca2+ handling related gene expression changes were assessed. Hearts were Langendorff-perfused, subjected to 20 minutes global ischemia with 45 minutes reperfusion. In response to this moderate ex vivo ischemic insult, hearts derived from L-NAME treated female animals exhibited increased incidence of reperfusion arrhythmias, diastolic abnormality and reduced contractile recovery in reperfusion. This differential response was observed even though baseline performance of hearts from L-NAME treated animals was not different to vehicle controls, myocardial inflammatory and fibrotic indices were similar in males and females and the systolic blood pressure effect of L-NAME administration was equivalent in both sexes. To examine underlying pre-disposing mechanisms, expression of a panel of candidate genes encoding proteins involved in electromechanical homeostasis (particularly relevant to ischemic challenge) was evaluated in normoxic myocardial tissues from the L-NAME- and vehicle-treated animals. Analysis revealed that L-NAME treatment in females selectively regulated expression of genes related directly and indirectly to cardiomyocyte Ca2+ handling in a manner consistent with destabilization of Ca2+ homeostasis and arrhythmogenesis. Our investigation provides new insight into the role of sustained decrease in NO bioavailability in determining distinctive female cardiac vulnerability to ischemic challenge.