Age-associated disruption of molecular clock expression in skeletal muscle of the spontaneously hypertensive rat

Abstract

It is well known that spontaneously hypertensive rats (SHR) develop muscle pathologies with hypertension and heart failure, though the mechanism remains poorly understood. Woon et al. (2007) linked the circadian clock gene Bmal1 to hypertension and metabolic dysfunction in the SHR. Building on these findings, we compared the expression pattern of several core-clock genes in the gastrocnemius muscle of aged SHR (80 weeks; overt heart failure) compared to aged-matched control WKY strain. Heart failure was associated with marked effects on the expression of Bmal1, Clock and Rora in addition to several non-circadian genes important in regulating skeletal muscle phenotype including Mck, Ttn and Mef2c. We next performed circadian time-course collections at a young age (8 weeks; pre-hypertensive) and adult age (22 weeks; hypertensive) to determine if clock gene expression was disrupted in gastrocnemius, heart and liver tissues prior to or after the rats became hypertensive. We found that hypertensive/hypertrophic SHR showed a dampening of peak Bmal1 and Rev-erb expression in the liver, and the clock-controlled gene Pgc1α in the gastrocnemius. In addition, the core-clock gene Clock and the muscle-specific, clock-controlled gene Myod1, no longer maintained a circadian pattern of expression in gastrocnemius from the hypertensive SHR. These findings provide a framework to suggest a mechanism whereby chronic heart failure leads to skeletal muscle pathologies; prolonged dysregulation of the molecular clock in skeletal muscle results in altered Clock, Pgc1α and Myod1 expression which in turn leads to the mis-regulation of target genes important for mechanical and metabolic function of skeletal muscle.

Figure 1. The expression of (A,D) Bmal1, (B,E) Clock, and (C,F) Per2 from the gastrocnemius and the liver of young WKY and SHR was determined by quantitative PCR.

Samples were collected every 4 hours for 40 hours. Collections were performed under red light. The dark and light bars on the graph represent extrapolated subjective day and night as defined by ZT according to the prior L∶D cycle before release into DD. A plot of amplitude with 95% confidence intervals as error bars is include next to each circadian gene expression plot.

Figure 2. Rev-erbα is a core clock gene and Dbp, and Pgc1α are clock-controlled genes.

The expression of (A,D) Rev-erb, (B,E) Dbp, and (C,F) Pgc1α in the gastrocnemius and liver of young WKY and SHR was measured by quantitative PCR. Samples were collected every 4 hours for 40 hours. Collections were performed under red light. The dark and light bars on the graph represent extrapolated subjective day and night as defined by ZT according to the prior L∶D cycle before release into DD. A plot of amplitude with 95% confidence intervals as error bars is include next to each circadian gene expression plot.

Figure 3. The expression of (A,D) Bmal1, (B,E) Clock, and (C,F) Per2 from the gastrocnemius and the liver of adult WKY and SHR was determined by quantitative PCR.

Samples were collected every 4 hours for 40 hours. Collections were performed under red light. The dark and light bars on the graph represent extrapolated subjective day and night as defined by ZT according to the prior L∶D cycle before release into DD. A plot of amplitude with 95% confidence intervals as error bars is include next to each circadian gene expression plot.

Figure 4. The expression of (A,D) Rev-erbα, (B) Dbp, and (C) Pgc1α in the gastrocnemius and liver of adult WKY and SHR was measured by quantitative PCR.

Samples were collected every 4 hours for 40 hours. Collections were performed under red light. The dark and light bars on the graph represent extrapolated subjective day and night as defined by ZT according to the prior L∶D cycle before release into DD. A plot of amplitude with 95% confidence intervals as error bars is include next to each circadian gene expression plot.

Figure 5. Myod1 is a skeletal muscle specific clock-controlled gene.

The expression of Myod1 in the gastrocnemius of (A) young and (B) adult WKY and SHR was determined by quantitative PCR. Samples were collected every 4 hours for 40 hours. Collections were performed under red light. The dark and light bars on the graph represent extrapolated subjective day and night as defined by ZT according to the prior L∶D cycle before release into DD. Note that Myod1 does not cycle in the gastrocnemius from adult SHR rats. A plot of amplitude with 95% confidence intervals as error bars is include next to each circadian gene expression plot.

Figure 6. Heart weight normalized to body weight in the WKY and SHR demonstrating the presence of cardiac hypertrophy in the SHR (A).

Gastrocnemius wet weight normalized to body weight measured from the hypertensive WKY and SHR demonstrating a loss of muscle mass in the SHR (B).