Pancreatic cancer cachexia promotes cardiac dysfunction through altered adrenergic signalling in the heart

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  Figure 1. Cardiac β1‐adrenergic receptor (β1‐AR) is downregulated in mice with pancreatic ductal adenocarcinoma (PDAC) cachexia A, schematic illustrating study design. B, cumulative food intake over study duration. N = 6 male mice per group. C, heart mass normalized to initial body mass. N = 7 male sham mice and 5 male PDAC mice. D and E, quantitative PCR analysis of cardiac tissue. The eukaryotic 18S rRNA was used as the endogenous control. N = 6–12 male mice per group. F and G, Time course analysis of heart mass and Adrb1 expression in mice with PDAC. N = 5 male sham mice and 6 male PDAC mice per group. H and I, Pair‐feeding study to assess heart mass and β1‐adrenergic receptor desensitization gene signature in PDAC mice. N = 7 male mice per group. J and K, western blot analysis of β1‐AR protein (55 kDa) levels in ventricular and atrial tissue normalized to GAPDH (37 kDa). N = 7 male mice per group. L and M, western blot assessment of β‐arrestin‐1,2 (50 kDa) and GRK5 (68 kDa) in ventricular tissue of mice with PDAC cachexia. N = 7 male mice per group. Data were analysed by unpaired Student's t test (C, D, E, K and M), one‐way ANOVA (H and I) or two‐way ANOVA (B, F and G). Error bars…

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  Figure 2 Pancreatic ductal adenocarcinoma (. PDAC) desensitizes cardiac β1‐adrenergic receptor in cachectic mice A, schematic illustrating study design. B and C, assessment of heart rate response to dobutamine and propranolol treatment. N = 10 sham mice and 11 PDAC mice. One PDAC mouse was excluded from propranolol analysis due to poor injection quality. D, measurement of baseline heart rate in PDAC and sham mice. E–H, representative ECG traces and quantification of PR interval, P duration and QRS duration from baseline heart rate. I and J, assessment of baseline systolic and diastolic blood pressure. N = 4 male sham and 6 male PDAC mice. K, bulk RNA sequencing and unsupervised clustering of atrial and ventricular samples from PDAC and sham mice, focusing on genes involved in cardiac adrenergic signalling. N = 3 male mice per group. L, gene set enrichment analysis of pathways related to cardiac contraction and conduction. Data were analysed by unpaired Student's t test (B–J). Error bars denote SD for all graphs.

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  Figure 3. Neurotransmitter cycling is disrupted in pancreatic ductal adenocarcinoma (PDAC) mice A, quantitative PCR analysis of cardiac axon guidance cues. N = 6–7 male mice per group. B, quantification of sympathetic nerve fibres in cardiac tissue. N = 5 male sham and 6 male PDAC mice. Scale bar: 100 µm. C, western blot analysis of tyrosine hydroxylase protein (62 kDa) levels in stellate ganglia normalized to β‐actin (45 kDa). N = 6 male mice per group. D, high‐performance liquid chromatography (HPLC) measurement of norepinephrine levels in cardiac sympathetic nerve terminals. N = 5 male sham and 10 male PDAC mice per group. E, bulk RNA sequencing of stellate ganglia showing all differentially expressed genes in PDAC mice. N = 6 male mice per group. F, gene set enrichment analysis of stellate ganglia revealing downregulation of pathways involved in neurotransmitter cycling. G, quantitative PCR validation of select differentially expressed genes from bulk RNA sequencing in stellate ganglia. H, schematic illustrating the working hypothesis that disrupted neurotransmitter cycling contributes to increased sympathetic transmission to the heart and subsequent receptor desensitization. Red…

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  Figure 4 Pancreatic ductal adenocarcinoma (. PDAC) cachexia does not induce cholinergic transdifferentiation of sympathetic neurons in the stellate ganglia A, confocal immunofluorescence imaging of stellate ganglia stained for tyrosine hydroxylase (TH) and choline acetyltransferase (ChAT), acquired using a 40× objective. Scale bar: 50 µm. B, quantitative PCR analysis of Chat expression in stellate ganglia. Data were analysed using unpaired Student's t test. Error bars denote SD.

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  Figure 5. β1‐adrenergic receptor blockade partially attenuated cardiac adrenergic receptor desensitization in pancreatic ductal adenocarcinoma (PDAC) mice A, experimental schematic illustrating study design and continuous administration of 50 mg/kg/day of metoprolol via osmotic mini pump. B, resting heart rate measured 2 days post‐tumour and pump implantation in sham and PDAC mice. C, tumour mass at study endpoint. D, cumulative food intake measured at study endpoint. E, heart mass normalized to initial body weight. F–I, quantitative PCR (qPCR) to assess expression of β1‐adrenergic receptor (Adrb1), G‐protein receptor kinase‐5 (Grk5), β‐arrestin‐1 (Arrb1) and β‐arrestin‐2 (Arrb2) in the heart. N = 6–8 male mice per group. One of the sham control hearts was excluded from qPCR analysis due to low RNA quality. Data were analysed using two‐way ANOVA or unpaired Student's t test (C). Error bars denote SD for all graphs.

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  Figure 6. Adrenergic receptor desensitization decreases compensatory capacity in heart failure A, schematic illustrating study design. B, tumour burden in pancreatic ductal adenocarcinoma (PDAC) mice at the study endpoint. C, quantitative PCR analysis of β1‐adrenergic receptor expression in cardiac tissue. D, cardiac mass measured at study endpoint and normalized to initial body weight. E, echocardiographic measurement of left ventricular mass at study endpoint. F–H, quantitative PCR (qPCR) analysis of cardiac tissue. I, representative M‐mode echocardiographic images in parasternal short‐axis view 11 days post‐tumour and pump implantation. J–R, echocardiographic assessment of heart rate, stroke volume, cardiac output, cardiac index, ejection fraction, left ventricular end‐diastolic and end‐systolic volumes and endocardial area at end diastole and systole 11 days post‐tumour and pump implantation. N = 4–5 male mice per group. Echocardiographic assessments were completed in 4 out of 5 PDAC control mice due to time constraints during the imaging session. The eukaryotic 18S rRNA was used as the endogenous control for qPCR analyses. Data were analysed using two‐way ANOVA or unpaired Student's t…