Diaphragm weakness is a significant health problem in chronic heart failure (CHF) patients because: a) compromises their ability to sustain ventilation and limits physical activity due to dyspnea, b) triggers sympathetic activation that ca lead to cardiac arrhythmias and death or induces vasoconstriction and fatigue in limb muscles, and c) impairs airway clearance, predisposing patients to pneumonia. These observations highlight the importance of understanding the mechanisms underlying diaphragm abnormalities and the need to identify biological targets for prevention of weakness in CHF. CHF diaphragm weakness is predominantly caused by loss of specific force (i.e. contractile dysfunction) due to alterations in myofibrillar proteins, and reactive oxygen species (ROS) have been implicated in this process. The mitochondria electron transport chain has been considered the main source of ROS in muscle. However, our preliminary data from human diaphragm biopsies and animal models suggest that NADPH oxidases (Nox) localized predominantly in diaphragm sarcolemma (Nox2) and mitochondria (Nox4) are involved in the heightened ROS and diaphragm weakness in CHF. Moreover, our pilot studies suggest that myofibrillar protein thiol oxidation is a key molecular event in CHF induced diaphragm weakness. Based on our data, we propose to complete three specific aims: 1) to determine the role of diaphragmatic Nox2 complex on excess ROS and weakness in CHF, 2) to determine whether Nox4 and mitochondrial ROS are mediators of diaphragm weakness in CHF, 3) to define the mechanism of ROS-induced diaphragm contractile dysfunction in CHF. To address these aims, we will use inducible skeletal muscle specific knockout mice and intra-pleural injection of recombinant adeno-associated virus with shRNA or expression plasmids under control of a skeletal muscle-specific promoter to target diaphragm fibers in Sham and CHF mice. We will also isolate diaphragm single fibers from sham and CHF mice and perform experiments to determine if specific force deficits can be rescued in vitro. Finally, we will use global label free proteomics and differential Cysteine labeling to determine the abundance of proteins and redox status of specific Cysteine residues in diaphragm from Sham and CHF animals. Our focus on ROS sources, molecular species and targets, and reversibility is critical to understand the pathophysiology and set the stage for nove therapies to treat diaphragm weakness that contributes to the morbidity and mortality in CHF patients.

Public Health Relevance

Chronic Heart Failure (CHF) affects millions of people and diaphragm weakness is a significant health problem in CHF patients because it contributes to morbidity and mortality. Our goal is to understand the mechanisms underlying diaphragm abnormalities to identify biological targets for prevention of weakness in CHF. Guided by on our preliminary studies, we will test the sources, cellular mechanisms, and molecular targets of oxidants that mediate diaphragm weakness in CHF. The biology we will investigate is novel and driven by consistent findings in human diaphragm biopsies and animal models.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL130318-03
Application #
9402638
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Adhikari, Bishow B
Project Start
2016-01-01
Project End
2020-12-31
Budget Start
2018-01-01
Budget End
2018-12-31
Support Year
3
Fiscal Year
2018
Total Cost
Indirect Cost
Name
University of Florida
Department
Physiology
Type
Sch Allied Health Professions
DUNS #
969663814
City
Gainesville
State
FL
Country
United States
Zip Code
32611
Ferreira, Leonardo F (2018) Mitochondrial basis for sex-differences in metabolism and exercise performance. Am J Physiol Regul Integr Comp Physiol 314:R848-R849
Kelley, Rachel C; Ferreira, Leonardo F (2017) Diaphragm abnormalities in heart failure and aging: mechanisms and integration of cardiovascular and respiratory pathophysiology. Heart Fail Rev 22:191-207
Ferreira, Leonardo F; Laitano, Orlando (2016) Regulation of NADPH oxidases in skeletal muscle. Free Radic Biol Med 98:18-28