Increased peripheral vascular disease risk progressively constrains perfusion adaptability in the skeletal muscle microcirculation

Abstract

To determine the impact of progressive elevations in peripheral vascular disease (PVD) risk on microvascular function, we utilized eight rat models spanning "healthy" to "high PVD risk" and used a multiscale approach to interrogate microvascular function and outcomes: healthy: Sprague-Dawley rats (SDR) and lean Zucker rats (LZR); mild risk: SDR on high-salt diet (HSD) and SDR on high-fructose diet (HFD); moderate risk: reduced renal mass-hypertensive rats (RRM) and spontaneously hypertensive rats (SHR); high risk: obese Zucker rats (OZR) and Dahl salt-sensitive rats (DSS). Vascular reactivity and biochemical analyses demonstrated that even mild elevations in PVD risk severely attenuated nitric oxide (NO) bioavailability and caused progressive shifts in arachidonic acid metabolism, increasing thromboxane A2 levels. With the introduction of hypertension, arteriolar myogenic activation and adrenergic constriction were increased. However, while functional hyperemia and fatigue resistance of in situ skeletal muscle were not impacted with mild or moderate PVD risk, blood oxygen handling suggested an increasingly heterogeneous perfusion within resting and contracting skeletal muscle. Analysis of in situ networks demonstrated an increasingly stable and heterogeneous distribution of perfusion at arteriolar bifurcations with elevated PVD risk, a phenomenon that was manifested first in the distal microcirculation and evolved proximally with increasing risk. The increased perfusion distribution heterogeneity and loss of flexibility throughout the microvascular network, the result of the combined effects on NO bioavailability, arachidonic acid metabolism, myogenic activation, and adrenergic constriction, may represent the most accurate predictor of the skeletal muscle microvasculopathy and poor health outcomes associated with chronic elevations in PVD risk.

Keywords: blood flow regulation; microvascular dysfunction; peripheral vascular disease; rodent models of cardiovascular disease risk; system biology of microcirculation.

Fig. 1.

Schematic representation of the in situ cremasteric arteriolar bifurcation used for assessing “parent” and “daughter” arteriolar mechanical and hemodynamic/perfusion responses to pharmacological challenge. Open arrows represent parent or daughter arteriolar diameter in response to a specific condition; filled arrows represent parent or daughter arteriolar erythrocyte (RBC) velocity in response to a specific challenge. These data are utilized to determine both arteriolar flow volume and perfusion heterogeneity at bifurcations (γ). See text for additional details, as well as Refs. and .

Fig. 2.

Estimated cardiovascular disease (CVD) [peripheral vascular disease (PVD)] risk across animal groups used in the present study. Estimated PVD risk is determined based on the severity of obesity, impaired glycemic control, dyslipidemia, hypertension, systemic inflammation, and systemic oxidant stress in each animal group at the time of use (from data summarized in Table 1). SDR, Sprague-Dawley rats; LZR, lean Zucker rats; HSD, SDR on high-salt diet; HFD, SDR on high-fructose diet; RRM reduced renal mass-hypertensive SDR; SHR, spontaneously hypertensive rats; OZR, obese Zucker rats; DSS, Dahl salt-sensitive rats. See text for additional details.

Fig. 3.

Indexes of reactivity of ex vivo gracilis muscle resistance arterioles isolated from each group of animals used in the present study. Data are presented as the maximum bound of the acetylcholine (ACh) concentration-response relationship (A), the slope of the myogenic activation relationship, where increasing negativity reflects a more robust pressure-induced constriction (B), and the maximum bound of the phenylephrine (Phe) concentration-response relationship (C). Data (means ± SE) are presented under control conditions and after pretreatment of the isolated vessel with either the antioxidant TEMPOL or the NOS inhibitor nitro-l-arginine methyl ester (l-NAME). *P < 0.05 vs. responses in SDR or LZR. †P < 0.05 vs. responses in that group under control conditions. See text for details.

Fig. 4.

Dilator reactivity of in situ distal arterioles of the cremaster muscle within each group of animals used in the present study. Data are presented as the maximum bound of either the acetylcholine concentration-response relationship (A) or the arachidonic acid concentration-response relationship (B). Data (means ± SE) are presented under control conditions and after pretreatment of the isolated vessel with the antioxidant TEMPOL (both), the NOS inhibitor l-NAME (acetylcholine only), the PGH₂/TxA₂ receptor antagonist SQ-29548 (arachidonic acid only), or the cyclooxygenase inhibitor indomethacin (INDO). *P < 0.05 vs. responses in SDR or LZR. †P < 0.05 vs. responses in that group under control conditions. ‡P < 0.05 vs. responses in that group under TEMPOL-treated conditions. See text for details.

Fig. 5.

Vascular bioavailability of nitric oxide [NO, A; estimated from slope of the NO production vs. methacholine (Met) concentration relationship], production of PGI₂ (B; estimated from 6-keto-PGF_1α), and TxA₂ (C; estimated from 11-dehydro-TxB₂) across animal groups used in the present study. *P < 0.05 vs. responses in SDR or LZR. †P < 0.05 vs. responses in that group under control conditions. ‡P < 0.05 vs. responses in that group under TEMPOL-treated conditions. See text for details.

Fig. 6.

Vascular responses and contractile performance of in situ skeletal muscle of animal groups in the present study in response to 3 min of muscle contraction at 3 or 5 Hz (isometric twitch). Data are presented for hyperemic responses to muscle contraction (A), oxygen extraction across the gastrocnemius muscle (B), oxygen consumption across the gastrocnemius muscle (C), and % of peak force development after 3 min of the contraction bout (D). *P < 0.05 vs. responses in SDR or LZR at that contraction frequency. See text for details.

Fig. 7.

Microvascular perfusion distribution (γ) at arteriolar bifurcations within in situ cremaster muscle. Data are presented as means ± SE for each animal group spanning 1A-2A arterioles (A), 2A-3A arterioles (B), 3A-4A arterioles (C), and 4A-5A arterioles (D). *P < 0.05 vs. SDR or LZR. See text for additional details.

Fig. 8.

Predicted perfusion distributions in the distal (precapillary) microcirculation of skeletal muscle of the animal groups within the present study. Frequency distributions are calculated based on an 8-bifurcation network using the microvascular perfusion distribution coefficients (γ) determined in the in situ cremaster muscle presented in Fig. 7. A–H present the distribution of perfusion across of 256 (2⁸) parallel arterioles under each experimental condition resulting from the simulation of a dichotomous branching network. Please see text for additional details.

Fig. 9.

Data describing the cumulative changes in γ over the minute collection period in 1A-2A (A and B) and 3A-4A (C and D) arteriolar bifurcations in the animal groups of the present study. Cumulative changes in γ (presented as means ± SE) are summated either as sequential differences between successive time points (A and C) or as the sequential absolute differences between successive time points (B and D). *P < 0.05 vs. SDR or LZR. †P < 0.05 vs. HSD or HFD. ‡P < 0.05 vs. RRM or SHR. See text for additional details.

Fig. 10.

Presentation of the attractor describing the overall spatial-temporal behavior of γ at 1A-2A arteriolar bifurcations across the animal groups of the present study. The attractors are presented as iterated maps, where the respective value for γ is presented at multiple successive time points (t) within that group. A presents the attractor for 1A-2A arteriolar bifurcations in SDR (blue) and LZR (dark green); these data are grayed in B–D to facilitate comparisons. B: attractor for 1A-2A arteriolar bifurcations in HSD (light green) and HFD (cyan). C: attractor for 1A-2A arteriolar bifurcations in RRM (brown) and SHR (dark green). D: attractor for 1A-2A arteriolar bifurcations in OZR (black) and DSS (purple). See text for additional details.

Fig. 11.

Presentation of the attractor describing the overall spatial-temporal behavior of γ at 3A-4A arteriolar bifurcations across the animal groups of the present study. The attractors are presented as iterated maps, where the respective value for γ is presented at multiple successive time points within that group. A presents the attractor for 3A-4A arteriolar bifurcations in SDR (blue) and LZR (dark green); these data are grayed in B–D to facilitate comparisons. B: attractor for 3A-4A arteriolar bifurcations in HSD (light green) and HFD (cyan). C: attractor for 3A-4A arteriolar bifurcations in RRM (brown) and SHR (dark green). D: attractor for 3A-4A arteriolar bifurcations in OZR (black) and DSS (purple). See text for additional details.