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/cm2) 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/cm2, 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.

Pharmacological targeting of mitochondrial reactive oxygen species counteracts diaphragm weakness in chronic heart failure.

Diaphragm muscle weakness in chronic heart failure (CHF) is caused by elevated oxidants and exacerbates breathing abnormalities, exercise intolerance, and dyspnea. However, the specific source of oxidants that cause diaphragm weakness is unknown. We examined whether mitochondrial reactive oxygen species (ROS) cause diaphragm weakness in CHF by testing the hypothesis that CHF animals treated with a mitochondria-targeted antioxidant have normal diaphragm function. Rats underwent CHF or sham surgery. Eight weeks after surgeries, we administered a mitochondrial-targeted antioxidant (MitoTEMPO; 1 mg·kg(-1)·day(-1)) or sterile saline (Vehicle). Left ventricular dysfunction (echocardiography) pre- and posttreatment and morphological abnormalities were consistent with the presence of CHF. CHF elicited a threefold (P < 0.05) increase in diaphragm mitochondrial H2O2 emission, decreased diaphragm glutathione content by 23%, and also depressed twitch and maximal tetanic force by ∼20% in Vehicle-treated animals compared with Sham (P < 0.05 for all comparisons). Diaphragm mitochondrial H2O2 emission, glutathione content, and twitch and maximal tetanic force were normal in CHF animals receiving MitoTEMPO. Neither CHF nor MitoTEMPO altered the diaphragm protein levels of antioxidant enzymes: superoxide dismutases (CuZn-SOD or MnSOD), glutathione peroxidase, and catalase. In both Vehicle and MitoTEMPO groups, CHF elicited a ∼30% increase in cytochrome c oxidase activity, whereas there were no changes in citrate synthase activity. Our data suggest that elevated mitochondrial H2O2 emission causes diaphragm weakness in CHF. Moreover, changes in protein levels of antioxidant enzymes or mitochondrial content do not seem to mediate the increase in mitochondria H2O2 emission in CHF and protective effects of MitoTEMPO.

Diaphragm Abnormalities in Patients with End-Stage Heart Failure: NADPH Oxidase Upregulation and Protein Oxidation.

Patients with heart failure (HF) have diaphragm abnormalities that contribute to disease morbidity and mortality. Studies in animals suggest that reactive oxygen species (ROS) cause diaphragm abnormalities in HF. However, the effects of HF on ROS sources, antioxidant enzymes, and protein oxidation in the diaphragm of humans is unknown. NAD(P)H oxidase, especially the Nox2 isoform, is an important source of ROS in the diaphragm. Our main hypothesis was that diaphragm from patients with HF have heightened Nox2 expression and p47phox phosphorylation (marker of enzyme activation) that is associated with elevated protein oxidation. We collected diaphragm biopsies from patients with HF and brain-dead organ donors (controls). Diaphragm mRNA levels of Nox2 subunits were increased 2.5-4.6-fold over controls (p < 0.05). Patients also had increased protein levels of Nox2 subunits (p47phox, p22phox, and p67phox) and total p47phox phosphorylation, while phospho-to-total p47phox levels were unchanged. The antioxidant enzyme catalase was increased in patients, whereas glutathione peroxidase and superoxide dismutases were unchanged. Among markers of protein oxidation, carbonyls were increased by ~40% (p < 0.05) and 4-hydroxynonenal and 3-nitrotyrosines were unchanged in patients with HF. Overall, our findings suggest that Nox2 is an important source of ROS in the diaphragm of patients with HF and increases in levels of antioxidant enzymes are not sufficient to maintain normal redox homeostasis. The net outcome is elevated diaphragm protein oxidation that has been shown to cause weakness in animals.