Hypertension is a major public health concern and poses a significant risk for sudden cardiac death (SCD). However, the characterisation of human tissues tends to be macroscopic, with little appreciation for the quantification of the pathological remodelling responsible for the advancement of the disease. While the components of hypertensive remodelling are well established, the timeline and comparative quantification of pathological changes in hypertension have not been shown before. Here, we sought to identify the phasing of cardiac remodelling with hypertension using post-mortem tissue from SCD patients with early and advanced hypertensive heart disease (HHD). In order to study and quantify the progression of phenotypic changes, human specimens were contrasted to a well-described angiotensin-II-mediated hypertensive mouse model. While cardiomyocyte hypertrophy is an early adaptive response in the mouse that stabilises in established hypertension and declines as the disease progresses, this finding did not translate to the human setting. In contrast, optimising fibrosis quantification methods and applying them to each setting identified perivascular fibrosis as the prevailing possible cause for overall disease progression. Indeed, assessing myocardial inflammation highlights CD45+ inflammatory cell infiltration that precedes fibrosis and is an early-phase event in response to elevated arterial pressures that may underscore perivascular remodelling. Along with aetiology insight, we highlight cross-species comparison for quantification of cardiac remodelling in human hypertension. As such, this platform could assist with the development of therapies specific to the disease phase rather than targeting global components of hypertension, such as blood pressure lowering.

Introduction: The ERK1/2 cascade, activated by RAF kinases, is a key pathway for cardiac remodelling and cytoprotection. Due to activating oncogenic mutations in BRAF, small molecule RAF inhibitors have been developed. However, owing to inhibitor resistance or paradoxical ERK1/2 pathway activation, a new generation of ‘paradox breaking’ drugs are currently in clinical trials. Our previous work characterised RAF targeting in hypertension, where it is instrumental in driving cardiac remodelling. Here we explored whether use of the RAF paradox breakers (e.g. PLX8394) are viable modalities for targeting RAF in hypertension. Hypothesis: We hypothesize that RAF ‘paradox breakers’ will protect cardiac function by inhibiting maladaptive hypertensive remodelling. Methods & Results: To assess the role of RAF inhibition in cardiac adaptation to hypertension in vivo , C57Bl/6J mice were treated with angiotensin-II (AngII; 0.8mg/kg/d, 7d n=8;) without/with 5mg/kg/d PLX8394 (n=11). PLX8394 alone had no detrimental effect on cardiac function or remodelling. AngII promoted cardiac hypertrophy, by increasing left ventricular (LV) wall thickness (WT) and decreasing LV internal diameter (ID; assessed by echocardiography). PLX8394 had no impact on AngII-induced hypertrophy (WT:ID, p=0.45), or abundance of AngII-induced hypertrophic Nppa (p=0.77) or Nppb (p=0.49) mRNAs. Additionally, PLX8394 did not affect AngII-induced perivascular fibrosis (p=0.69; picrosirius red histology) or moderate Col1a1 (p=0.22) and Col3a1 (p=0.82) mRNAs. However, PLX8394 use in AngII-hypertension was pro-inflammatory, with increased pro-inflammatory cytokines ( Il1b , p=0.02; Il6 , p=0.008) and the inflammatory cell marker CD45 (p=0.001) mRNAs; a response echoed in hearts labelled by immunohistochemistry for CD45 (p=0.02). Moreover, western blotting revealed elevated PARP cleavage (p=0.005), indicating increased apoptosis with PLX8394 use. Conclusion: Despite no adverse effect on cardiac function or global hypertensive remodelling at 7 days, RAF inhibition by PLX8394 appears to be pro-inflammatory and pro-apoptotic. Thus, long-term use of RAF ‘paradox breakers’ in hypertensive individuals may be cardiotoxic and promote adverse cardiac events.