Protein Concentration and Mitochondrial Content in the Gastrocnemius Predicts Mortality Rates in Patients with Peripheral Arterial Disease

Abstract

Objective:

This study evaluated the hypothesis that protein concentration and mitochondrial content in gastrocnemius biopsies from patients with peripheral arterial disease (PAD) predict mortality rates.

Background:

PAD patients experience advancing myopathy characterized by mitochondrial dysfunction, myofiber degradation and fibrosis in their ischemic legs, along with increased mortality rates.

Methods:

Samples from the gastrocnemius of PAD patients were used for all analyses. Protein concentration was normalized to muscle wet weight, and citrate synthase activity (standard measure of mitochondrial content in cells) was normalized to muscle wet weight and protein concentration. Protein and citrate synthase data were grouped into tertiles and 5-year, all-cause mortality for each tertile was determined with Kaplan-Meier curves and compared by the Modified Peto test. A Cox-regression model for each variable controlled for the effects of clinical characteristics.

Results:

Of the 187 study participants, 46 died during a mean follow up of 23.0 months. Five-year mortality rate was highest for patients in the lowest tertile of protein concentration. Mortality was lowest for patients in the middle tertile of citrate synthase activity when normalized to either muscle wet weight or protein concentration. The mortality hazard ratios (HR) from the Cox analysis were statistically significant for protein concentration normalized to muscle wet weight (lowest vs middle tertile; HR=2.93; p=0.008) and citrate synthase normalized to protein concentration (lowest vs. middle tertile; HR=4.68; p=0.003; and lowest vs. highest tertile; HR=2.36; p=0.027).

Conclusion:

Survival analysis of a contemporaneous population of PAD patients identifies protein and mitochondrial content of their gastrocnemius as predictors of mortality rate.

Introduction

Epidemiological studies of Peripheral Arterial Disease (PAD) estimate that its prevalence in the general population ranges from 3% to 10% ¹. In populations aged greater than 70 years, the prevalence increases to as high as 20% ¹. The mortality of symptomatic PAD patients varies between studies. Published all-cause death rates vary between 3.3 % and 7.8% per year ² and can be comparable to that of several types of cancer.

Several clinical parameters and biochemical markers have been shown to be independent predictors of mortality in the PAD population. The Ankle-brachial index (ABI) is one clinical parameter consistently correlated to mortality. The Edinburgh study examined a population of 1592 residents aged 55 to 74 years in Edinburgh, United Kingdom. At 12 years follow-up, decreasing ABI correlated with increasing all-cause mortality ³ with 63.5% of patients who had a baseline ABI of less than 0.7 being deceased compared to only 31.4% of patients with an ABI greater than 0.7 ³. The Strong Heart Study, designed to investigate cardiovascular disease in American Indians, found an adjusted mortality risk of 1.69 in those patients with ABI <0.90 and a mortality risk of 1.77 in those with ABI >1.4 compared to the reference group of patients with ABIs between 0.9 and 1.4 ⁴. The investigators found a U-shaped relationship between baseline ABI and mortality, with all-cause mortality increasing as ABI increasingly deviated from 1.04 ⁴. Other clinical comorbidities such as diabetes mellitus, hypertension, and renal dysfunction have also been shown to be predictive of mortality in patients with PAD ^{1, 5}.

Biochemical markers, particularly serum biomarkers, and their correlation to mortality have been extensively investigated in the cardiovascular literature, but studies in PAD patients are limited. Criqui and colleagues studying mortality in PAD patients, showed a correlation between highly sensitive C-reactive protein and mortality at 2 years but not at 6.6 years of follow-up ⁵. In the same study no association between mortality and several serum biomarkers (D-dimer, platelet activating factor, serum amyloid A, homocysteine, and lipoprotein-a) was found ⁵. Patients with ABI measurements <0.9 were recruited by Vidula et al who demonstrated that serum amyloid A level, C-reactive protein level, and D-Dimer level correlated with all-cause mortality at 4 years follow-up ⁶. In other studies of PAD patients, neither N-terminal pro-B-type natriuretic peptide levels ⁷ nor lipoprotein-associated phospholipase A₂ ⁸ correlated with mortality.

The latest data on the pathophysiology of PAD suggest that arterial stenoses and occlusions produce effort-induced cycles of ischemia and reperfusion in the affected limbs. These cycles appear to initiate a combination of mitochondrial dysfunction, oxidative damage, and inflammation in the affected limbs, which set in motion a cascade of injury to every structure in the leg including muscles, nerves, skin, and subcutaneous tissues ^9–11. Accumulating injury in all tissues of the leg can lead to progressive decline in muscle structure and performance (claudication) and loss of the integrity of tissues in the ischemic limb presenting as tissue loss/gangrene. Injury to the skeletal muscle of the ischemic legs (myopathy of PAD) is the better explored component of this process in PAD limbs ^{10, 11}. The importance of this myopathy to the overall health of PAD patients was recently demonstrated in a study showing that imaging and function-based biomarkers of this myopathy (calf muscle density measured via Computed Tomography scans, plantar flexion strength and knee extension power) correlate to the survival of PAD patients. More specifically, McDermott and colleagues demonstrated that lower calf muscle density and weaker lower plantar flexion strength and knee extension power were associated with increased mortality in PAD patients ¹². However, to date there have been no studies correlating biochemical biomarkers of this myopathy to mortality in patients with PAD. Skeletal muscle protein concentration and mitochondrial content are basic biochemical biomarkers of the observed myopathy. With this study we evaluate the hypothesis that protein concentration and mitochondrial content in gastrocnemius biopsies predict the 5-year mortality rate of PAD patients.

Methods

Participant Identification

This study was approved by the Institutional Review Boards of the VA Nebraska-Western Iowa Health Care System and the University of Nebraska Medical Center. Patients evaluated and diagnosed for PAD in the vascular surgery clinic of the two institutions between April 2003 and October 2010, were identified for this study. For every patient, the diagnosis of PAD was based on medical history, physical examination, significantly decreased ankle-brachial index (ABI < 0.9) and computerized or standard arteriography demonstrating stenoses and/or occlusions in the arteries supplying the lower extremities. A total of 187 patients with PAD were enrolled. Written informed consent was obtained from each patient who elected to participate.

Comorbidities

After patient enrollment, patient characteristics and comorbidities were identified using a combination of patient interview and chart review. Age, gender, race, height, weight, and Body Mass Index (BMI) were recorded. Further, the presence of Coronary Artery Disease (CAD), hypertension, dyslipidemia, diabetes mellitus (DM), tobacco use, renal dysfunction, a family history of atherosclerotic disease, and use of statins were determined for each patient. ABI measurements were gathered by standard procedures, in the vascular laboratories at the respective institution. At the time of final analysis, an assessment of mortality status was performed using a combination of chart review and telephone interview with the patient or their family.

Muscle Specimen Collection

Muscle biopsy samples were obtained from the gastrocnemius of the more symptomatic limb based on the presentation of the patient. All muscle samples were obtained from the anteromedial aspect of the gastrocnemius belly, at a level 10 cm distal to the tibial tuberosity. All biopsies were performed by, or under the supervision of, one investigator (I.I.P.) and identical operative technique was used for muscle harvest in all patients.

Protein and Citrate Synthase Determination

Assays were performed in duplicate or triplicate in skeletal muscle homogenates using a spectrophotometer (DU Series 640; Beckman Instruments, Fullerton, CA). The total protein concentration of each specimen was measured using the Pierce bicinchoninic acid (BCA) method (Thermo Fisher Scientific Inc., Rockford, IL). Protein concentration was normalized to muscle wet weight. Next, the citrate synthase activity of each specimen was measured as the increase in absorbance from the reduction of 5,5’-dithiobis-2-nitrobenzoic acid by newly formed CoA-SH. The citrate synthase activity of each specimen was expressed as specific activity referenced to total sample wet weight (grams) and total protein (milligrams).

Statistical Analysis

Baseline characteristics between decedents and survivors were compared using the nonparametric Kruskal-Wallis test for continuous variables, and the Fisher exact test for categorical variables. Tertiles of calf muscle characteristics (Protein/muscle weight, citrate synthase/muscle weight, and citrate synthase/protein) were selected, rather than quartiles or pentiles, because the number of events (deaths) was relatively low. Similarly, because of the limited sample size and limited number of events in this study, group comparisons of the Kaplan-Meier curves was chosen as the primary analysis approach, whereas the Cox multivariate regression was our secondary analysis approach. The PROC LIFETEST procedure in SAS was used to perform the primary analyses, whereas the PROC PHREG procedure was used to perform the secondary analyses. In the primary analysis, tertiles from the Kaplan-Meier curves were compared using the Modified Peto test. In the (secondary) Cox regression models, the effects of calf muscle characteristics (Protein/muscle weight, citrate synthase/muscle weight, and citrate synthase/protein) on mortality were adjusted for the covariates age, gender, race, BMI, ankle/brachial index (ABI), smoking status (Yes/No), CAD, hypertension, dyslipidemia, family history of atherosclerotic arterial disease, DM, renal dysfunction, PAD disease stage, and statins use. All statistical analyses were performed using the statistical software SAS version 9.2 (SAS Institute Inc., Cary, North Carolina).

Results

Deceased and surviving PAD patients did not differ with regards to age, sex, race, BMI, ABI, statin use, current tobacco use, or presence of: CAD, hypertension, dyslipidemia, renal insufficiency, or family history of atherosclerotic arterial disease. Using univariate analysis (Table 1), surviving patients had statistically different rates of Type II Diabetes Mellitus (p<0.009), frequency of claudication (p<0.0001), and frequency of critical limb ischemia (p<0.0001) compared to decedents. BMI was marginally significant at p=0.12 with decedents having a trend toward lower BMI than survivors.

Table 1 –

Clinical Characteristics and Biochemical Muscle Descriptors

	All (N=187)	Decedents (N=46)	Survivors (N=141)	P-value
Age (years)	64.18 (8.48)	65.00 (9.18)	63.92 (8.26)	0.70
Male (%)	97.33	100	96.45	0.34
African American (%)	7.49	8.7	7.09	0.75
BMI (kg/m2)	26.89 (6.05)	25.81 (5.60)	27.24 (6.17)	0.12
ABI	0.51 (0.40)	0.59 (0.62)	0.49 (0.29)	0.97
Current Smoker (%)	55.61	60.87	53.9	0.49
CAD (%)	42.78	52.17	39.72	0.17
HTN (%)	79.14	82.61	78.01	0.68
Dyslipidemia (%)	63.64	63.04	63.83	0.99
Family History (%)	29.41	28.26	29.79	0.99
DM (%)	39.57	56.52	34.04	0.009
Renal Failure (%)	14.44	13.04	14.89	0.99
Claudicants (%)	53.16	22.5	61.33	<0.0001
CLI (%)	46.84	77.5	38.67	<0.0001
Statin use (%)	67.91	56.52	71.63	0.069
Protein/muscle wet weight (mg/100 g)	100.59 (30.87)	79.17 (23.36)	107.57(29.84)	0.0001
Citrate Synthase/ muscle wet weight (units/100 g)	14.56 (5.92)	12.22 (4.88)	15.49 (6.02)	0.0019
Citrate Synthase/ protein (units/mg)	149.41(49.16)	159.89 (57.50)	145.99 (45.82)	0.088

Mean (SD) or %; P-values from the nonparametric Kruskal-Wallis test or the Fisher Exact test

Kaplan-Meier survival plots of tertiles for protein content normalized over muscle wet weight, citrate synthase content normalized over muscle wet weight, and citrate synthase content normalized over protein are presented in Figures 1, 2, and 3, respectively. Table 2 provides a breakdown of the lowest, middle, and highest tertile values for protein/muscle wet weight, citrate synthase/muscle wet weight, and citrate synthase /protein.

Figure 1 –

Kaplan-Meier Curves for protein concentration in gastrocnemius biopsies from PAD patients. Protein concentration was determined as protein content (mg) per muscle wet weight (g). Patients in the lowest (dashed line) tertile of protein concentration in their gastrocnemius had the worst survival with 5-year mortality of 57.0%. Patients in either the middle (continuous line) or highest (squared dot line) tertile for protein concentrations in their gastrocnemius had the best survival with 5-year mortality of 34.8% and 37.5%, respectively.

Figure 2 –

Kaplan-Meier Curves for Citrate Synthase activity normalized to muscle wet weight in gastrocnemius biopsies from PAD patients. Citrate Synthase activity was normalized to muscle wet weight (g). Patients in the middle (continuous line) tertile had the best survival with a 5-year mortality of 33.3%. This was followed by the highest (squared dot line) tertile with a 5-year mortality rate of 44.2%. Mortality at 5 years was highest (50.1%) in those patients in the lowest (dashed line) tertile.

Figure 3 –

Kaplan-Meier Curves for Citrate Synthase activity normalized to protein content in muscle biopsies from PAD patients. Citrate Synthase activity was normalized to protein content (mg). Patients in the middle (continuous line) tertile had the best survival with a 5-year mortality of 23.2%. This was followed by the highest (squared dot line) tertile with a 5-year mortality rate of 44.2%. Mortality at 5 years was highest (64.0%) in those patients in the lowest (dashed line) tertile.

Table 2 –

Tertile Breakdown for Biochemical Muscle Descriptors

Variable	Tertile	Number of patients	Mean	Standard Deviation	Range
Protein/muscle wet weight (mg/100 g)	lowest	62	66.7	16.2	(20.7, 86.2)
	middle	62	100.4	8.2	(87.1, 115.3)
	highest	63	134.1	15.5	(116.4, 187.5)
Citrate Synthase/ muscle wet weight (units/100 g)	lowest	62	8.6	2.2	(1.2, 11.4)
	middle	62	14.2	1.6	(11.5, 16.8)
	highest	63	21.2	4.3	(16.8, 34.6)
Citrate Synthase/ protein (units/mg)	lowest	62	98.4	18.6	(38.8, 123.0)
	middle	62	145.2	12	(123.5, 163.5)
	highest	63	204.3	33.1	(164.0, 315.6)

For protein concentration normalized to muscle weight, the patients in the lowest tertile of protein concentration in their gastrocnemius had the worst survival. At 5 years, the mortality in this group was 57.0%. Patients in either the middle or highest tertile for protein concentrations in their gastrocnemius had the best survival with 5-year mortality of 34.8% and 37.5%, respectively. The lowest tertile was statistically different from both the middle tertile (p=0.032) and the highest tertile (p=0.009) using the modified Peto test (Table 3).

Table 3 –

5 year Mortality

	Lowest tertile	Middle tertile	Highest tertile	P-value1
Protein/muscle wet weight (mg/100 g)	57.0%*, ‡	34.8%*	37.5%‡	* 0.032 ‡ 0.009
Citrate Synthase/ muscle wet weight (units/100 g)	50.1%*	33.3%*	44.2%	* 0.041
Citrate Synthase/ protein (units/mg)	64.0%*	23.2%*	44.2%	* 0.017

¹All comparisons performed using the Modified Peto test

When citrate synthase level was normalized to the wet weight of the muscle sample, the patients with citrate synthase level in the middle tertile had the lowest mortality (33.3%) at 5 years, pointing at a quadratic (or, U-shaped) trend. This was statistically different from the 5 year-mortality of the patient group that had citrate synthase concentration in the lowest tertile, which was 50.1%. At 5 years, these two groups were statistically different using the modified Peto test (p=0.041, Table 3). The 5 year-mortality of the patient group that had citrate synthase concentration in the highest tertile, was also higher (44.2%) than the middle tertile (33.3%) but the difference did not reach statistical significance (p=0.11, Table 3).

Citrate synthase level was also expressed relative to total protein in the muscle sample. The mortality pattern was similar to the citrate synthase/muscle wet weight data, that is, the patients with citrate synthase levels in the middle tertile once again had the best survival (5 year mortality of 23.2%), pointing at a quadratic (or, U-shaped) trend. Conversely, the mortality rate was approximately 64.0% at 5 years for the patients in the lowest tertile, and 44.2% for the patients in the highest tertile (Table 3). The middle and lowest tertiles were again found to be statistically different using the modified Peto test (p=0.017, Table 3).

Finally, in our secondary analyses, all parameters were put into a multivariate Cox regression model, controlling for all the demographics described previously in Table 1. Three clinical parameters were predictive of mortality in our model: BMI (Hazard Ratio, HR = 0.92; p = 0.005), presence of DM (HR = 2.50; p = 0.017), and presence of CAD (HR = 2.08; p = 0.035). The rest of the parameters including age, sex, race, ABI, statin use, current tobacco use, or presence of: hypertension, dyslipidemia, renal insufficiency, or family history of atherosclerotic arterial disease, clinical presentation of PAD (claudication versus CLI) and use of statin were not predictive of mortality in our cohort. Protein concentration normalized to muscle weight, and citrate synthase normalized to protein concentration were the two biochemical parameters which reached statistical significance in this model. For protein concentration, hazard ratios for the lowest, middle, and highest (reference) tertiles were, respectively, 2.93, 1.07, and 1.00. The lowest tertile of protein concentration had the highest likelihood of death and was statistically different from the middle tertile (p=0.008, Table 4). For citrate synthase normalized to protein concentration, hazard ratios for the lowest, middle (reference), and highest tertiles were, respectively, 4.96, 1.00, and 1.98. The middle tertile compared to the lowest tertile was significantly different at p=0.003 (Table 4). Similarly, the highest tertile was statistically different compared to the lowest tertile at p=0.027 (Table 4). Our data demonstrate that, over the 5-year follow-up, PAD patients that are in the middle tertile for mitochondrial density in their gastrocnemius are almost five times more likely to survive than those patients in the lowest tertile and two times more likely to survive than those patients in the highest tertile.

Table 4 –

Cox Regression Model Analysis

	Lowest tertile HR	Middle tertile HR	Highest tertile HR	P-value
Protein/muscle wet weight (mg/100 g)	2.93*, ‡	1.07*	1.00 ‡ (Reference)	* 0.008 ‡ 0.075
Citrate Synthase/ protein (units/mg)	4.68*, ‡	1.00* (Reference)	1.98‡	* 0.0033 ‡ 0.0270

HR=Hazard Ratio;

Discussion

Previous studies from our and other laboratories have established that a myopathy is present in the ischemic lower limbs of patients with PAD and it is associated with a variety of molecular and cellular abnormalities including myofiber degeneration, oxidative stress, mitochondrial dysfunction, and inflammation ^{9–11, 13–17}. In this study we chose to evaluate two basic markers of skeletal muscle biochemistry; protein concentration and mitochondrial content (citrate synthase) as possible predictors of mortality in PAD patients. Our data demonstrate that both protein concentration and mitochondrial content in the gastrocnemius of PAD patients correlate with mortality rate. PAD patients with relatively low protein concentration in their gastrocnemius have the highest mortality. Furthermore, mortality is lowest for patients in the middle tertile of citrate synthase activity when normalized to both muscle wet weight and protein concentration.

Several studies have correlated clinical parameters in a PAD patient’s history and clinical presentation with mortality. Such parameters include the presence of CLI, Diabetes Mellitus, hypertension, renal dysfunction and abnormal ABI ^{3, 5, 18–20}. These studies were completed with cohorts of several hundred to thousands of patients. In our study, using a multivariate Cox regression model, the clinical parameters that were found to be predictive of mortality were BMI, presence of DM and presence of CAD. Presence of CLI, hypertension, renal dysfunction and abnormal ABI were not statistically significant predictors in the multivariate Cox model, however, these parameters may achieve significance in a similar study with a larger sample size. Given the sample size limitations, our findings demonstrate the quality and clinical relevance of basic biochemical indices (protein concentration and mitochondrial density) in the ischemic gastrocnemius as predictors of mortality in PAD patients. Serum biomarkers such as highly sensitive C-reactive protein, serum amyloid A, and D-dimer have also been found to correlate with mortality in PAD patients ^{5, 6}. However, no mortality association was found with other serum markers including homocysteine, lipoprotein-a, N-terminal pro-B-type natriuretic peptide, platelet activating factor, and lipoprotein-associated phospholipase A₂ ^5–8. This is the first study to correlate skeletal muscle biochemical characteristics, specifically protein concentration and citrate synthase amount, with mortality. Our findings are in agreement with those of McDermott et al. In their study ¹², they investigated the relationship between calf muscle density, area and percent fat (measured by Computerized Tomographic scanning), as well as knee extension power, knee extension isometric strength and plantar flexion isometric strength with mortality in PAD patients. All measurements were performed in the legs of 434 participants with PAD, defined in this particular study as individuals with an ABI < 0.90. After adjusting for co-variates, lower calf muscle density and weaker lower plantar flexion strength and knee extension power were associated with higher all-cause mortality hazard ratios¹². The authors concluded that some of these calf muscle markers may represent the burden of atherosclerotic disease and its effect on the end organ, the muscle, and the overall health of the PAD patient.

There is a lack of studies reporting skeletal muscle protein concentration in healthy adults and the range of normal has not been established ²¹. Our data demonstrate a range of skeletal muscle protein concentration exists in patients with PAD and that its level can predict mortality rate in this patient population. Specifically, in our cohort, PAD patients with the lowest protein concentration in their gastrocnemius had more than 50% mortality. As humans age the protein synthesis and protein concentration in their skeletal muscle, as well as their muscle mass and muscle function decrease, a phenomenon known as sarcopenia ^{22, 23}. Some patients have a greater degree of sarcopenia than others, and this reflects and/or leads to greater likelihood of functional impairment and disability ^{23, 24}. Similar findings connecting poor calf skeletal muscle characteristics with decreased physical activity and functional decline have been demonstrated by McDermott and colleagues for patients with PAD ^25–27. Furthermore, a number of studies have recently demonstrated that sarcopenia is also associated with mortality rates. A recent study by the European Working Group on Sarcopenia in Older People, the ilSIRENTE trial, identified an increased mortality rate in elderly patients (age >80) with sarcopenia compared to those without, even after adjusting for possible confounders ²⁸. This study defined patients with sarcopenia as those with a gait speed of less than 0.8 m/s or low grip strength (<30kg in men and <20kg in women) plus mid-arm muscle circumference in the lower one third of all subjects ²⁸. Using this same definition, Arango-Lopera et al. confirmed higher mortality in a separate group of elderly patients with sarcopenia ²⁹. A different study used dual energy X-ray absorptiometry to determine muscle and fat mass in elderly individuals as an objective measurement of sarcopenia ³⁰. Low amount of appendicular (limb) fat-free mass, one of the sarcopenia markers in this study, was associated with increased mortality ³⁰. Finally, a study evaluating the correlation between body composition and all-cause mortality demonstrated that higher lean body mass, examined using bioelectrical impedance analyses, is predictive of improved survival ³¹. PAD is a disease of the elderly and the addition of ischemic myopathy to the sarcopenia of aging likely leads to greater reduction of skeletal muscle mass and function. We speculate, therefore, that low protein concentration in PAD calf muscles may reflect the progressive decline of the cardiovascular and neuromuscular systems of the PAD patient and it likely is one of the reasons why the patients with lowest protein in their calf muscle are at an increased likelihood of death.

Based on our findings, citrate synthase can also be used to predict mortality. As discussed with protein concentration, the normal range for citrate synthase activity has not been established ^{32, 33}. Citrate synthase is a mitochondrial matrix enzyme that catalyzes the first step of the Krebs cycle (condensation of oxaloacetate and the acetyl group of acetyl coenzyme-A) and is the most commonly used marker of mitochondrial content in cells ¹⁰. PAD patients in the mid-range of mitochondrial content have greater survival than those outside of this range. Based on our findings we speculate that in PAD patients, the extremes of mitochondrial content reflect a decline in the functional capacity of the mitochondrial energy-providing system, deterioration of the limb, and overall health of the PAD patient. It is possible that increasing mitochondrial content is the product of a switch in the phenotype of ischemic myofibers from fast-twitch, type II myofibers (low mitochondrial content) to slow-twitch, type I myofibers (high in mitochondrial content). This phenotype switch has been previously demonstrated by several groups and appears to be a marker of ischemic damage on the PAD muscle^34–37. Another possibility is that the abnormally increased mitochondrial content reflects accumulation of dysfunctional organelles ^{15, 38} possibly secondary to increased mitochondrial damage and impaired mitochondrial catabolism in the ischemic muscle. On the other hand, decreasing mitochondrial content in PAD muscle may also represent degeneration of the mitochondrial population ³⁹ which may be the result of a combination of a decrease in mitochondrial biogenesis ⁴⁰, increase in mitophagy of damaged organelles and the overall decrease of the proteome ⁴¹ of some PAD patients. In both cases the dysfunctional organelles have a reduced capacity for supporting the needs of muscle for energy, an increased susceptibility to apoptosis, and an increase in generation of reactive oxygen species ^{10, 11, 15, 42, 43}.

This is a descriptive study and therefore its principal limitation is that it cannot identify cause and effect linkages between gastrocnemius protein or mitochondrial content and mortality in PAD patients. Instead, the study demonstrated that the two markers are predictors of mortality in PAD patients, and consequently, identified the myopathy of PAD as a potential correlate to the overall health state of patients with PAD. Furthermore, our study population is fairly homogeneous, composed of primarily Caucasian males. This relative homogeneity may limit the generalizability of our findings to other populations. Additionally, the duration these patients were followed was relatively short for survival analysis, with a mean follow-up of 23.0 months. We are continuing to follow these patients to strengthen our results and increase our duration of follow-up. In this study we chose to evaluate two basic markers of skeletal muscle biochemistry; protein concentration and mitochondrial content (citrate synthase activity) as possible predictors of mortality in PAD patients. In the future more advanced markers reflecting the complexity of adaptation and damage suffered by the ischemic muscle proteome (such as levels of regulatory proteins, contractile elements, complexes of the electron transport chain, oxidatively damaged proteins, inflammatory biomarkers and cellular stress proteins) can be also evaluated as potential predictors of morbidity and mortality in this patient population. Studies of muscle from PAD limbs are crucial for our understanding of PAD pathophysiology but are difficult to be generalized mainly due to the need for obtaining a muscle biopsy. In the future, the value of surrogate markers of muscle damage such as urinary creatinine, dual-energy X-ray absorptiometry scans, as well as Computerized Tomographic and Magnetic resonance scans of the lower extremities may play a role in the measurement of skeletal muscle deterioration in PAD patients. Alterations in the neuromuscular system of the lower limbs are of central importance in the pathogenesis of PAD and future work will be critical for improving our knowledge of the mechanisms operating in the legs of PAD patients and their effects on the overall health of the patient with PAD.

Conclusions

Myopathy is a recognized component of PAD pathogenesis. Our data demonstrate that protein and mitochondrial content, two basic biochemical parameters of this myopathy, are predictors of mortality in PAD patients. Future studies are needed to determine whether interventions such as exercise therapy, revascularization, medications with neural and muscular trophic effects and medications that can optimize mitochondrial function can treat the biochemical abnormalities in PAD limbs and improve the overall health and lifespan of the patient with PAD.