Small-hairpin RNA and pharmacological targeting of neutral sphingomyelinase prevent diaphragm weakness in rats with heart failure and reduced ejection fraction

Heart failure with reduced ejection fraction (HFREF) increases neutral sphingomyelinase (NSMase) activity and mitochondrial reactive oxygen species (ROS) emission and causes diaphragm weakness. We tested whether a systemic pharmacological NSMase inhibitor or short-hairpin RNA (shRNA) targeting NSMase isoform 3 (NSMase3) would prevent diaphragm abnormalities induced by HFREF caused by myocardial infarction. In the pharmacological intervention, we used intraperitoneal injection of GW4869 or vehicle. In the genetic intervention, we injected adeno-associated virus serotype 9 (AAV9) containing shRNA targeting NSMase3 or a scrambled sequence directly into the diaphragm. We also studied acid sphingomyelinase-knockout mice. GW4869 prevented the increase in diaphragm ceramide content, weakness, and tachypnea caused by HFREF. For example, maximal specific forces (in N/cm 2 ) were vehicle [sham 31 ± 2 and HFREF 26 ± 2 ( P < 0.05)] and GW4869 (sham 31 ± 2 and HFREF 31 ± 1). Respiratory rates were (in breaths/min) vehicle [sham 61 ± 3 and HFREF 84 ± 11 ( P < 0.05)] and GW4869 (sham 66 ± 2 and HFREF 72 ± 2). AAV9-NSMase3 shRNA prevented heightening of diaphragm mitochondrial ROS and weakness [in N/cm 2 , AAV9-scrambled shRNA: sham 31 ± 2 and HFREF 27 ± 2 ( P < 0.05); AAV9-NSMase3 shRNA: sham 30 ± 1 and HFREF 30 ± 1] but displayed tachypnea. Both wild-type and ASMase-knockout mice with HFREF displayed diaphragm weakness. Our study suggests that activation of NSMase3 causes diaphragm weakness in HFREF, presumably through accumulation of ceramide and elevation in mitochondrial ROS. Our data also reveal a novel inhibitory effect of GW4869 on tachypnea in HFREF likely mediated by changes in neural control of breathing.